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By Max Aravena-Román BScAppSci FASM This thesis is presented for the degree of Doctor of Philosophy School of Pathology and Laboratory Medicine of Western Australia 2015 Classification, antimicrobial susceptibility and virulence factors of Aeromonas species in Western Australia

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Page 1: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

Phylogeny, antimicrobial susceptibility and

virulence factors of Western Australian

By

Max Aravena-Román BScAppSci FASM

This thesis is presented for the degree of Doctor of

Philosophy

School of Pathology and Laboratory Medicine of

Western Australia

2015

Classification, antimicrobial susceptibility and virulence factors of Aeromonas species in

Western Australia

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TABLE OF CONTENTS TABLE OF CONTENTS.................................................................................................i

SUMMARY.....................................................................................................................ix

DECLARATION............................................................................................................xi

ACKNOWLEDGEMENTS.........................................................................................xiii

THESIS STRUCTURE.................................................................................................xv

ABBREVIATIONS.......................................................................................................xvi

LIST OF TABLETS......................................................................................................xx

LIST OF FIGURES...................................................................................................xxiii

CHAPTER 1: LITERATURE REVIEW..........................................................1

1.1. GENERAL INTRODUCTION.......................................................................1

1.2. HISTORY........................................................................................................1

1.3. TAXONOMY..................................................................................................2

1.3.1. Early taxonomy..........................................................................................2

1.3.2. Current taxonomy......................................................................................3

1.3.3. Controversial taxonomic issues.................................................................3

1.3.3.1. Aeromonas allosaccharophila..............................................................4

1.3.3.2. Aeromonas spp. HG 11........................................................................8

1.3.3.3. Aeromonas culicicola...........................................................................8

1.4. LABORATORY IDENTIFICATION...........................................................9

1.4.1. Isolation.....................................................................................................9

1.4.2. Identification by phenotypic methods.....................................................12

1.4.3. Identification by commercial systems.....................................................13

1.4.4. Additional phenotypic methods..............................................................15

1.4.5. Semi-automated systems.........................................................................15

1.4.6. Identification by molecular methods.......................................................16

1.4.6.1. Typing methods………………………………………......................16

1.4.6.2. Identificartion based on 16S-23S rRNA gene sequence……………17

1.4.6.3. Identification based on housekeeping gene sequence........................18

1.4.6.4. Specific genes used as identification targets......................................18

1.4.6.5. Restriction enzyme-based methods....................................................19

1.4.6.6. PCR-based methods............................................................................20

1.4.6.7. Disadvantages of molecular methods.................................................20

1.5. SEROTYPING............................................................................................21

1.6. ECOLOGY..................................................................................................22

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1.6.1. Aquatic environments.............................................................................22

1.6.1.1. Distribution in water...............................................................................22

1.6.1.2. Water quality...........................................................................................23

1.6.1.3. Effects of temperature on growth and toxin production.........................24

1.6.1.4. Aeromonas in drinking water..................................................................25

1.6.2. Aeromonas in foods.................................................................................28

1.6.2.1 Distribution of Aeromonas spp. in foods............................................28

1.7. EPIDEMIOLOGY AND PUBLIC HEALTH ISSUES............................30

1.7.1. Water-associated infections.....................................................................31

1.7.2. Foods-associated infections.....................................................................32

1.7.3. Aeromonas and fish infections................................................................32

1.8. BIOREMEDIAL AND BIODEGRADABLE PROPERTIES....................33

1.9. VIRULENCE FACTORS...........................................................................34

1.9.1. Adherence................................................................................................35

1.9.2. Pili............................................................................................................36

1.9.3. Invasins....................................................................................................39

1.9.4. S-layer......................................................................................................40

1.9.4.1. Structural arrangements......................................................................41

1.9.4.2. Binding properties..............................................................................41

1.9.4.3. Genes involved in S-layer synthesis...................................................41

1.9.4.4. S-layer and virulence..........................................................................42

1.9.5. The lipopolysaccharide (LPS).................................................................42

1.9.5.1. Functions of the LPS.........................................................................42

1.9.5.2. Immunological and antigenic properties of the LPS.........................43

1.9.5.3. Genes involved in LPS synthesis.......................................................43

1.9.6. Outer membrane proteins (OMP)............................................................44

1.9.7. Flagella....................................................................................................45

1.9.7.1. Synthesis, regulation and expression of flagella................................45

1.9.7.2. Functions associated with flagella......................................................46

1.9.8. Secretion systems....................................................................................46

1.9.8.1. Type II secretion systems (T2SS).......................................................48

1.9.8.2. Type III secretion systems (T3SS).....................................................48

1.9.8.3. Type IV secretion systems (T4SS).....................................................49

1.9.8.4. Type VI secretion systems (T6SS).....................................................50

1.9.9. Exotoxins................................................................................................51

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1.9.9.1. Aerolysin...........................................................................................52

1.9.9.1.1. Action on host tissue..................................................................53

1.9.9.1.2. Molecular characteristics and prevalence...................................53

1.9.9.2. Cytotoxic enterotoxin (Act)...............................................................54

1.9.9.3. Haemolysins.......................................................................................55

1.9.9.4. Enterotoxins.......................................................................................56

1.10. Additional extracellular products............................................................59

1.10.1. Proteases.............................................................................................59

1.10.2. Lipases................................................................................................60

1.10.3. Nucleases (DNases)............................................................................61

1.10.4. Chitinases............................................................................................62

1.11. Iron uptake...............................................................................................63

1.12. Quorum sensing (QS)..............................................................................64

1.13. Biofilm formation....................................................................................65

1.14. Additional virulence factors....................................................................66

1.15. INFECTIONS CAUSED BY AEROMONAS SPECIES........................67

1.15.1. Gastroenteritis.....................................................................................68

1.15.1.1. Disease presentation....................................................................68

1.15.1.2. Evidence against Aeromonas as an enteric pathogen..................69

1.15.1.3. Evidence supporting Aeromonas as an enteric pathogen............71

1.15.1.4. Species involved..........................................................................72

1.15.2. Skin and soft-tissue infections (SSTIs)..............................................72

1.15.3. Septicaemia.........................................................................................73

1.15.4 Respiratory tract infections.................................................................76

1.15.5. Urogenital tract infections..................................................................76

1.15.6. Intra-abdominal infections..................................................................77

1.15.7. Infections due to medicial leech therapy............................................78

1.15.8. Meningitis...........................................................................................79

1.15.9. Zoonotic infections.............................................................................79

1.15.10. Burns...................................................................................................80

1.15.11. Eye infections.....................................................................................80

1.15.12. Osteomyelitis and suppuratives arthritis.............................................81

1.16. ANTIMICROBIAL SUSCEPTIBILITIES.............................................81

1.16.1. -Lactamases......................................................................................83

1.16.2. Extended-spectrum (ESBL) -lactamas production...........................86

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1.16.3. Plasmid-mediated resistance..............................................................88

1.16.4. Quinolones..........................................................................................89

1.16.5. Genes encoding for antimicrobial resistance......................................89

1.16.6. Antimicrobial usage: recommendations............................................90

1.17. CONCLUSIONS.....................................................................................91

CHAPTER 2: MATERIALS AND METHODS..............................................95

2.1. MATERIALS................................................................................................95

2.1.1. Chemical and reagents.............................................................................95

2.1.2. Solutions..................................................................................................95

2.1.2.1. DepC-treated water.............................................................................95

2.1.2.2. Ethidium bromide (10 mg/ml)............................................................95

2.1.2.3. Chemical lysis stock solution.............................................................95

2.1.2.4. HCCA matrix solution........................................................................95

2.1.3. Bacteriological media..............................................................................95

2.1.4. Gas chromatography................................................................................96

2.1.5. Antimicrobials.........................................................................................96

2.1.6. Bacterial strains.......................................................................................96

2.1.7. Primers.....................................................................................................96

2.2. METHODS.................................................................................................115

2.2.1. Bacterial culture methods......................................................................115

2.2.2. Acid production from carbohydrates.....................................................112

2.2.3. Hydrolysis of aesculin...........................................................................115

2.2.4. Alkylsulfatase activity...........................................................................115

2.2.5. Detection of a CAMP-like factor...........................................................116

2.2.6. Catalase activity.....................................................................................116

2.2.7. DNase activity.......................................................................................116

2.2.8. Elastase activity.....................................................................................116

2.2.9. Gas from glucose...................................................................................117

2.2.10. Gelatin hydrolysis..................................................................................117

2.2.11. Oxidation of potassium gluconate.........................................................117

2.2.12. Ability to grow on TCBS medium........................................................117

2.2.13. -Haemolysis activity...........................................................................118

2.2.14. Production of hydrohen sulphide from cysteine...................................118

2.2.15. Production of indole from tryptophan…………...................................118

2.2.15.1. Rapid spot method....................................................................118

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2.2.15.2. Kovacs’ method........................................................................118

2.2.16. Jordan’s tartrate test..............................................................................118

2.2.17. Lipase activity.......................................................................................119

2.2.18. Utilization of malonate..........................................................................119

2.2.19. Amino acid degradation........................................................................119

2.2.20. Motility..................................................................................................120

2.2.20.1. Wet mount method....................................................................120

2.2.20.2. Motility medium method...........................................................120

2.2.21. ONPG activity.......................................................................................120

2.2.22. Oxidase activity.....................................................................................120

2.2.23. Phenylalanine deaminase activity..........................................................121

2.2.24. Pyrazinamidase activity.........................................................................121

2.2.25. Pyrrolidonyl--naphthylamide activity.................................................121

2.2.26. Salt tolerance.........................................................................................121

2.2.27. Stapholysin activity...............................................................................122

2.2.28. Hydrolysis of starch...............................................................................122

2.2.29. Hydrolysis of tyrosine...........................................................................122

2.2.30. Urease activity.......................................................................................122

2.2.31. Utilization of DL-lactate, acetate and urocanic acid.............................123

2.2.32. Utilization of citrate (Simmon’s method).............................................123

2.2.33. Voges-Proskauer test.............................................................................123

2.3. AMPLIFICATION OF GYRB AND RPOD GENES.................................123

2.3.1 Preparation of template DNA................................................................123

2.3.2. Polymerase chain reaction (PCR)..........................................................124

2.3.3. DNA sequencing....................................................................................124

2.3.4. Detection of virulence gene products by Bioanalyzer...........................125

2.4. METHODS USED IN THE CHARACTERIZATION OF AEROMONAS

AUSTRALIENSIS SP. NOV........................................................................................126

2.4.1. Phenotypic characterization..................................................................126

2.4.2. Antimicrobial susceptibility testing......................................................127

2.4.3. Fatty acid methyl ester (FAME) analysis..............................................127

2.4.3.1. Inoculation of TSBA plates..............................................................128

2.4.3.2. Harvesting.........................................................................................128

2.4.3.3. Saponification...................................................................................128

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2.4.3.4. Methylation.......................................................................................128

2.4.3.5. Extraction..........................................................................................128

2.4.3.6. Washing........................................................................................... 129

2.4.3.7. Interpretation of results.....................................................................129

2.4.4. Protein analysis by MALDI-TOF..........................................................129

2.4.4.1. Sample preparation...........................................................................129

2.4.5. Genotypic characterization....................................................................130

2.4.5.1. PCR and sequence analysis..............................................................130

2.5. ANTIMICROBIAL SUSCEPTIBILITY TESTING..................................131

2.5.1. Agar dilution..........................................................................................131

2.5.2. Disk diffusion........................................................................................132

2.5.3. Minimum inhibitory concentration testing: E-strip method..................132

2.6. ELECTRON MICROSCOPY ANALYSIS................................................133

2.7. STATISTICAL ANALYSIS.......................................................................133

CHAPTER 3: PHENOTYPIC CHARACTERIZATION OF AEROMONAS

SPECIES.......................................................................................................................136

3.1. INTRODUCTION.......................................................................................136

3.2. Bacterial strains.....................................................................................136

3.3. RESULTS....................................................................................................137

3.3.1. Biochemical characteristics of type and reference strains.....................137

3.3.2. Overall classification.............................................................................137

3.3.3. Clinical isolates......................................................................................137

3.3.4. Environmental isolates..........................................................................138

3.3.5. Distribution of Aeromonas spp. in clinical samples..............................138

3.3.6. Distribution of Aeromonas spp. in environmental samples...................138

3.3.7. General phenotypic characteristics........................................................138

3.3.8. Susceptibility to colistin........................................................................154

3.3.9. Production of pyrrolidonyl--naphthylamide........................................154

3.3.10. Susceptibility to deferoxamine (DEF)...................................................154

3.3.11. Production of a CAMP-like factor.........................................................154

3.3.12. Utilization of citrate: Simmon’s vs. Hänninen’s medium.....................155

3.3.13. Susceptibility to the vibriostatic agent O/129........................................155

3.3.14. Growth on thiosulfate salt sucrose agar (TCBS)...................................155

3.4. DISCUSSION.............................................................................................155

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CHAPTER 4: GENOTYPIC CHARACTERIZATION OF AEROMONAS

SPECIES.......................................................................................................................158

4.1. INTRODUCTION.......................................................................................158

4.2. Bacterial strains.....................................................................................159

4.3. RESULTS....................................................................................................159

4.3.1. Overall distribution of species following genotypic identification.......159

4.3.2. Distribution of Aeromonas spp. in clinical specimens..........................159

4.3.3. Distribution of Aeromonas spp. in environmental specimens...............159

4.3.4. Phenotypic differentiation of Aeromonas dhakensis from other major

Aeromonas species........................................................................................................174

4.3.5. Intra- and inter-species dissimilarities...................................................174

4.4. DISCUSSION.............................................................................................174

CHAPTER 5: ANTIMICROBIAL SUSCEPTIBILITIES..........................179

5.1. INTRODUCTION.......................................................................................179

5.2. Bacterial strains.....................................................................................179

5.3. Antimicrobial agents..............................................................................179

5.4. RESULTS...................................................................................................180

5.5. DISCUSSION............................................................................................181

CHAPTER 6: DESCRIPTION OF AEROMONAS AUSTRALIENSIS SP.

NOV..............................................................................................................................187

6.1. INTRODUCTION.......................................................................................187

6.2. Bacterial strains.....................................................................................187

6.3. RESULTS....................................................................................................188

6.3.1. Phenotypic characteristics.....................................................................188

6.3.2. FAME profiles.......................................................................................189

6.3.3. Protein profile........................................................................................189

6.3.4. Genotypic characteristics.......................................................................189

6.3.5. Antimicrobial susceptibilities................................................................190

6.4. DISCUSSION.............................................................................................213

6.4.1 Formal description of Aeromonas australiensis sp. nov……………..215

CHAPTER 7: VIRULENCE GENES PRESENT IN WESTERN

AUSTRALIAN AEROMONAS SPECIES.................................................................217

7.1. INTRODUCTION.......................................................................................217

7.2. Bacterial strains.....................................................................................218

7.3. RESULTS....................................................................................................218

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7.3.1. Overall distribution of virulence genes.................................................218

7.3.2. Distribution of virulence genes in stool specimens...............................218

7.3.3. Distribution of virulence genes in extra-intestinal specimens...............219

7.3.4. Distribution of virulence genes among environmental specimens........219

7.3.5. Additional features................................................................................219

7.3.6. Percentage identity of nucleotide sequences of positive products from

this study compared to sequences deposited in GenBank.....................220

7.4. DISCUSSION..............................................................................................220

CHAPTER 8: GENERAL DISCUSSION......................................................247

REFERENCES............................................................................................................257

ATTACHED CD-ROM...............................................................................................312

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SUMMARY

Members of the genus Aeromonas are Gram-negative rods globally distributed in

aquatic and soil environments. For over one hundred years they have been associated

with infections in humans, other mammals and cold-blooded species. Infections in fish

and snails have resulted in serious financial losses to the aquaculture and French snail

farming industry.

Before the advent of molecular techniques, classification of Aeromonas was based

solely on the different phenotypic characteristics associated with each individual

species. However, the heterogeneous nature of motile and mesophilic Aeromonas

species has led to an unreliable and unstable taxonomy and schemes designed for the

identification of this group have not always been suitable for the identification of non-

motile, psychrophilic species.

The aims of this research were:

1. To characterize a collection of clinical and environmental Aeromonas isolates

from the state of Western Australia using phenotypic and genotypic methods in

order to determine the prevalence of species in this region.

2. To investigate the taxonomic position of isolates as determined by phylogenetic

trees.

3. To determine the antimicrobial susceptibility patterns of clinical and

environmental Aeromonas spp. to antibacterial agents currently in use in clinical

practice.

4. To assess the presence of virulence factors of Aeromonas species in order to

determine the presence of pathogenic strains currently circulating within the WA

community and its environment.

Aeromonas isolates were collected from rural and metropolitan areas of Western

Australia, the largest state in Australia covering an area of 2.5 million km2, for a period

of over 20 years. Phenotypic characterization of isolates was performed by a

conventional biochemical method that included more than 35 tests and by which

approximately 93% of the isolates were identified to the species level. Aeromonas

hydrophila was by far the most predominant aeromonad isolated from clinical and

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environmental samples and represented more than 50% of the species. These results

suggested that phenotypic identification was inadequate since 7% of the strains could

not be assigned to any known taxa.

Genotypic identification was based on the molecular sequences of the gyrB and rpoD

housekeeping genes by a PCR-based method. Phylogenetic trees generated from the

nucleotide sequences of the isolates tested indicated that A. dhakensis and not A.

hydrophila was the most frequently isolated aeromonad. Genotypic classification

resulted in the assignation of 99% of the strains to a species suggesting that accurate

identification of Aeromonas must involve a molecular method.

The antimicrobial susceptibility pattern of each isolate was assessed against 26

antimicrobials representing all classes currently in-use in clinical practice. Susceptibility

of each isolate was determined by the agar dilution and E-strip methods. Antibiotic

profiles indicated that the level of antimicrobial resistance in Western Australian

aeromonads is generally very low although antimicrobial susceptibility testing should

be performed in all strains isolated from human clinical material.

Phylogenetic trees derived from the nucleotide sequences of the gyrB and rpoD

housekeeping genes showed that the position of strain 266 isolated from irrigation water

in rural Western Australia did not cluster with any of the current validated Aeromonas

species. Extensive polyphasic testing that included multilocus phylogenetic analysis,

cellular fatty acid, protein profiles and DNA-DNA hybridization confirmed that strain

266 represented a novel Aeromonas species for which the name A. australiensis species

novo was proposed.

The distribution and prevalence of 13 virulence genes and the activity of four

extracellular enzymes was examined among 130 Aeromonas strains comprising 11

different species. Detection of virulence genes was performed by a PCR-based method

while enzyme activity was evaluated by biological assays. Results indicated that clinical

and environmental strains of A. hydrophila and A. dhakensis are more likely to carry

multiple virulence genes compared to strains of A. veronii and A. caviae. However, the

pathogenic potential of Aeromonas may be strain rather than species dependent, thus

under certain conditions which include host predisposition, a range of aeromonads may

be able to cause infection.

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DECLARATION

_______________________________________

All work presented in this thesis was performed by me and contributions made by

others are duly stated. Identification of Aeromonas by phenotypic methods and

antimicrobial susceptibility testing was performed entirely by me. Identification by

molecular methods and detection of virulence genes was performed by me except for

the preparation of gels and sequencing that was performed by staff from the PCR

Laboratory at PathWest, Nedlands. Polyphasic identification of Aeromonas

australiensis was 50% performed by me and 50% by Dr. R. Beaz-Hidalgo, Facultat de

Cience i Medicina de la Salut, University Rovira i Virgili, Reus, Spain.

Electronmicrograph of bacterial cells of A. australiensis was performed by Prof. Maria

Jose Figueras, Facultat de Cience i Medicina de la Salut, University Rovira i Virgili,

Reus, Spain.

This thesis contains a series of published work that has been co-authored. The following

journal articles constitute the individual chapters of this thesis:

Aravena-Román, M., B. J. Chang, T. R. Riley, and T. J. J. Inglis (2011a). Phenotypic

characteristics of human clinical and environmental Aeromonas in Western Australia.

Pathology 43: 350-356 (Chapter 3).

Aravena-Román, M., G. B. Harnett, T. V. Riley, T. J. J. Inglis and B. J. Chang (2011b).

Aeromonas aquariorum is widely distributed in clinical and environmental specimens

and can be misidentified as Aeromonas hydrophila. Journal of Clinical Microbiology

49: 3006-3008 (Chapter 4).

Aravena-Román, M., T. J. J. Inglis, B. Henderson, T. V. Riley, and B. J. Chang (2012).

Antimicrobial susceptibilities of Aeromonas strains isolated from clinical and

environmental sources to 26 antimicrobial agents. Antimicrobial Agents and

Chemotherapy 56: 1110-1112 (Chapter 5).

Aravena-Román, M., R. Beaz-Hidalgo, T. J. J. Inglis, T. V. Riley, A. J. Martínez-

Murcia, B. J. Chang and M. J. Figueras (2013). Aeromonas australiensis sp. nov.

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isolated from irrigation water in Western Australia. International Journal of

Evolutionary and Systematic Microbiology 63: 2270-2276 (Chapter 6)

Aravena-Román, M., T. J. J. Inglis, T. V. Riley and B. J. Chang (2014). Distribution of

13 virulence genes among clinical and environmental Aeromonas species in Western

Australia European Journal of Clinical Microbiology and Infectious Diseases 33: 1889-

1895 (Chapter 7).

Except for the work performed in the description of the new species A. australiensis

(50% of experimental work), all experimental work (100%) and initial manuscripts

preparation (100%) was performed by me. Editorial advice and guidance for the

manuscripts’ submissions and final corrected versions were provided by my supervisors

Professor Barbara Chang (40% editorial), Professor Timothy Inglis (30% editorial) and

Professor Thomas Riley (30% editorial). Other co-authors provided access to laboratory

equipment and facilities.

Max Aravena-Román

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ACKNOWLEDGEMENTS

I am indebted to my supervisors B. Chang, T. Inglis and T. Riley for their continuous

support, encouragement and guidance.

I would like to thank the staff of the Microbiology Division at PathWest, Nedlands

campus who provided bacterial isolates and access to equipment and reagents. To Rod

Bowman for making the necessary funds available to finance this project.

Thanks to Dr. Nicky Buller, Bacteriology Laboratory, Agriculture Department of

Western Australia, South Perth; Mr. Steve Munyard, Division of Microbiology and

Infectious Diseases, PathWest, Nedlands campus; Mr. Neil Stingemore, Department of

Microbiology, Fremantle Hospital, PathWest, Fremantle campus; Mr. Peter Campbell,

Department of Microbiology, Princess Margaret Hospital, Subiaco, Perth; Professor

Peter Käempfer, Institut für Angewandte Mikrobiologie, Justus-Liebig Universität,

Giessen, Germany; Professor Silvia Kirov, Department of Pathology, University of

Tasmania, Hobart, Tasmania, Australia; Dr. J. Michael Janda, Microbial Diseases

Laboratory, State of California, USA; and Dr. David Miñana-Galbis, Facultat de

Farmacia, Unitat de Microbiologia, Universitat de Barcelona, Barcelona, Spain for

kindly providing bacterial isolates.

To my colleagues, Glenys Chidlow, Gerry Harnett, Adam Merritt, Nikki Foster, Avram

Levy, and Barbara Henderson for their advice, guidance and support. A special thanks

to Diane Bleasdale for her excellent librarian services, to John Boehm from Excel,

PathWest, for providing me with special media and reagents and to my Spanish

colleagues, Professor María Jose Figueras and Dr. Roxana Beaz-Hidalgo from the

Facultat de Cience and Medicina de la Salut, University of Rovira i Virgili, Reus, Spain

and Dr. Antonio Martínez-Murcia from the Departamento de Produccion Vegetal y

Microbiología, EPSO, Universidad Miguel Hernández, Orihuela Alicante, Spain for

their invaluable training, advice and for their generosity in sharing bacterial isolates.

Thank you to Dr. Eduardo Alvarez from ICBM, Programa de Microbiología y

Micología, Facultad de Medicina, University of Chile, Santiago, Chile who provided

much training in sequence analysis and other computer issues and to Cati Nuñez from

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the Facultat de Cience and Medicina de la Salut, University of Rovira i Virgili, Reus,

Spain for her invaluable technical support.

Finally, thanks to my wonderful wife Naomi for her unconditional love and support.

To my beloved Mum Carmen Román Díaz (RIP)

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THESIS STRUCTURE

The body of this research is preceded by an extensive review of the literature in Chapter

1 in which historical, taxonomical issues, antimicrobial susceptibility and the relation of

Aeromonas to human disease are presented. All materials and methods described in

Chapters 3 to 7 are outlined in Chapter 2. Chapters 3 to 7 of this thesis are based on

material published by the candidate and peer reviewed.

Chapter 3 describes the characterization of isolates by phenotypic methods followed by

classification by genotypic methods as presented in Chapter 4. The antimicrobial

susceptibility pattern of 193 strains constitutes Chapter 5. The discovery and proposal

of a novel Aeromonas species is described in Chapter 6. In Chapter 7, the virulence

potential based on the detection and distribution of virulence genes and enzyme activity

is examined in a selected group of strains. Final discussion addressing the results and

conclusions obtained from all other chapters is presented in Chapter 8.

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ABBREVIATIONS ACN acetonitrile ADA ampicillin dextrin agar ADWA Agriculture Department of Western Australia AFLP amplified fragment length polymorphism AMX amoxicillin AMC amoxicillin-clavulanate AMK amikacin AMP ampicillin AnO2 anaerobic APW alkaline peptone water Aq. soln. aqueous solution ATCC American Type Culture Collection ATM aztreonam BAA blood ampicillin agar bv biovar BOC British Oxygen Company bp base pair(s) BSA bovine serum albumin cm centimetre C degrees Celsius CCUG Culture Collection of the University of Göteborg CFA cellular fatty acid CFU colony forming unit(s) CAMP Christie-Atkins-Munch-Peterson CAPD continuous ambulatory peritoneal dialysis CAZ ceftazidime CDC Center for Disease Control CECT Coleccion Española de Cultivos Tipo CEF cephalothin CFZ cefazolin CHO Chinese hamster ovary CIN cefsulodin irgasan novobiocin CIP Collection Bactérienne de l’Institute Pasteur CIP ciprofloxacin CLED cysteine lactose electrolyte deficient CLSI Clinical Laboratory Standard Institute CNA colistin nalidixic acid COL colistin CRO ceftriaxone CSF cerebral spinal fluid d day(s) Da Dalton DAA Difco ampicillin agar DEF deferoxamine DepC diethyl procarbonate DDH DNA-DNA hybridization DNA deoxyribonucleic acid DNAT deoxyribonucleic acid agar plus toluidine blue

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DNase deoxyribonuclease dNTP deoxyribonuclease triphosphate(s) DOX doxycycline DSM Deutsche Sammlung von Mikroorganismen und Zelkuturen ERIC enterobacterial repetitive intergenic consensus ESBL extended-spectrum -lactamase FA formic acid FAME fatty acid methyl ester(s) FEP cefepime FH Fremantle hospital FOX cefoxitin g gram(s) g relative centrifugal force G + C guanine plus cytosine GC gas chromatograph GCAT glycerophospholipid-cholesterol acyltransferase GCF gelatine-cysteine-thiosulfate GEN gentamicin GMP guanosine monophosphate GSP glutamate starch phenol h hour(s) HBA horse blood agar HCCA -cyano-4-hydroxycinnamic acid HG hybridization group HIA heart infusion agar HIB heart infusion broth HPLC high performance liquid chromatography HUS haemolytic uraemic syndrome I intermediate IM intramuscular IP intraperitoneal IBB inositol bile salts brilliant green kb kilobases(s) Km2 square kilometre L litre LBA Luria Bertoni agar LDC lysine decarboxylase LMG Culture Collection of the Laboratorium voor Microbiologie Gent LPS lipopolysaccharide LT labile toxin M molar M mole(s) MALDI-TOF matrix assistedlaser-desorption/ionization mass spectrometry time-of

flight MEM meropenem mg milligram(s) MHA Mueller-Hinton agar MIC minimum inhibitory concentration MIC50 MIC required to inhibit the growth of 50% of organisms MIC90 MIC required to inhibit the growth of 90% of organisms min minute(s) ml millilitre(s)

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MLCK myosin light chain kinase MLPA multilocus phylogenetic analysis MLST multilocus sequence analysis mm millimetre(s) mM millimole(s) MTCC Microbial Type Culture Collection and GeneBank MWA Metropolitan Water Authority MW molecular weight MXF moxifloxacin NaCl sodium chloride NA nutrient agar NAL nalidixic acid N/A not applicable NaOH sodium hydroxide NCIMB National Collection of Industrial and Marine Bacteria NCTC National Collection of Type Cultures ND not detected NIT nitrofurantoin nm nanometre(s) No. number NOR norfloxacin NSW New South Wales nt nucleotide(s) O2 oxygen O/129 2,4-diamino-6,7-diisopropylpteridine ONPG o-nitrophenyl--D-galactopyranoside O/F oxidation/fermentation o/v overnight PCR polymerase chain reaction PFGE pulse field gel electrophoresis pH concentration of hydrogen ions PMH Princess Margaret Hospital PPA phenylalanine deaminase psi pounds per square inch PYR pyrrolidonyl--naphthylamide PYZ pyrazinamidase QE II Queen Elizabeth II R resistant RAPD randomly amplified polymorphic DNA RBC red blood cells RILs rabbit ileal loops RNA ribonucleic acid rpm revolutions per minute s second(s) S susceptible SAA starch ampicillin agar SBA sheep blood agar SCGH Sir Charles Gairdner Hospital SDS sodium dodecyl sulphate SDH Swan District Hospital SF summed feature SI similarity index

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spp. species sp. nov. species novo ssp. subspecies SSSD Salmonella Shigella agar plus sodium desoxycholate ST stable toxin SXT trimethoprim-sulfamethoxazole TCBS thiosulfate citrate bile sucrose TFA trifluoroacetic acid TGC tigecycline TIM ticarcillin-clavulanate TMP trimethoprim TOB tobramycin TSA trypticase soy agar TSB trypticase soy broth TSBA trypticase soy broth agar TZP pipercillin-tazobactam U unit(s) micron(s) g microgram(s) l microlitre(s) m micrometre(s) M micromole(s) UPW ultrapure water w/v weight to volume WA Western Australia XLDA xylose lysine desoxycholate agar XDCA sylose desoxycholate citrate agar + positive negative

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LIST OF TABLETS Table 1.1 Current Aeromonas species p. 5

Table 1.2 Examples of media used in the isolation of Aeromonas

from different sources

p. 14

Table 1.3 Distribution of Aeromonas in water sourcesfrom different locations

p. 26

Table 1.4 Enumeration of Aeromonas in different foodstuffs

p. 29

Table 1.5 Characteristics of pili described in Aeromonas species

p. 37

Table 1.6 Selected effector proteins associated with different secretion systems

p. 47

Table 1.7 Toxins secreted by Aeromonas p. 57

Table 1.8 Clinical characteristics of patients with HUS-associated Aeromonas

p. 70

Table 1.9 Major categories of Aeromonas septicaemia disease presentation

p. 75

Table 1.10 -lactamases produced by Aeromonas species p. 84

Table 1.11 ESBL-producing Aeromonas species p. 87

Table 2.1 Chemicals and reagents used in this project p. 97

Table 2.2 Bacteriological media used in this project p. 99

Table 2.3 Antimicrobial agents used in this project p. 101

Table 2.4 Type and reference strains used in this project p. 102

Table 2.5 Type strains used as positive and negative controls p. 105

Table 2.6 Clinical strains used in this project p. 106

Table 2.7 Environmental strains used in this project p. 109

Table 2.8 Primers used in this project p. 111

Table 2.9 Aeromonas strains used in virulence studies p. 113

Table 2.10 Interpretation of disk diffusion results p. 134

Table 2.11 Interpretation of E-strip MIC values p. 135

Table 3.1 Biochemical characteristics of type and reference Aeromonas strains

p. 139

Table 3.2 Biochemical characteristics of Aeromonas isolated from human clinical material

p. 145

Table 3.3 Biochemical characteristics of Aeromonas isolated from environmental sources

p. 149

Table 3.4 Distribution of Aeromonas spp. among clinical and p. 153

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environmental samples after phenotypic characterization

Table 4.1 Type and reference strains GenBank accession numbers p. 160

Table 4.2 GenBank accession numbers of wild strains for rpoD and gyrB gene sequences

p. 162

Table 4.3 Distribution of Aeromonas spp. among clinical and environmental samples following genotypic characterization

p. 173

Table 4.4 Biochemical characteristics of Aeromonas after genotypic identification

p. 175

Table 4.5 Evolutionary distances based on the percentage sequence dissimilarities of all current Aeromonas spp. and 60 isolates identified as A. aquariorum using Clustal_W and Mega 5 software

CD-ROM

Table 5.1 Antimicrobial susceptibilities determined for different Aeromonas spp.

p. 182

Table 5.2 Antibiotic susceptibilities of Aeromonas spp. by source of isolation

p. 184

Table 6.1 Key tests for the phenotypic identification of strain 266T

from other Aeromonas spp.

p. 192

Table 6.2 Key tests used to differentiate strain 266T from other D-mannitol non-fermentative Aeromonas

p. 197

Table 6.3 Cellular fatty acid profiles of strain 266T and current Aeromonas spp.

p. 198

Table 6.4 Evolutionary distances based on the percentage sequence dissimilarities of current Aeromonas and strain 266T using Clustal_W and Mega 4 software

CD-ROM

Table 6.5 DNA-DNA hybridization values between strain 266T and closely related Aeromonas spp.

p. 204

Table 7.1 Distribution of virulence genes among Western Australian Aeromonas species

p. 221

Table 7.2 Distribution of virulence genes in Aeromonas spp. isolated from stools

p. 223

Table 7.3 Distribution of virulence genes in Aeromonas spp. isolated from blood

p. 225

Table 7.4 Distribution of virulence genes in Aeromonas spp. isolated from wounds

p. 227

Table 7.5 Distribution of virulence genes in Aeromonas spp. isolated p. 230

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from miscellaneous specimens

Table 7.6 Distribution of virulence genes in Aeromonas spp. isolated from environmental sources

p. 232

Table 7.7 Additional features p. 234

Table 7.8 Percentage identity of gene product sequences from this study compared with sequences deposited in GenBank

p. 235

Table 7.9 Accession numbers of sequences derived from virulence genes and deposited in GenBank

p. 237

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LIST OF FIGURES

Figure 1.1 Unrooted neighbour-joining phylogenetic tree derived from gyrB sequences showing subspecies and biovars

p. 7

Figure 1.2 Unrooted neighbour-joining phylogenetic tree derived from gyrB sequences showing current Aeromonas species

p. 10

Figure 1.3 Unrooted neighbour-joining phylogenetic tree derived from rpoD sequences showing current Aeromonas species

p. 11

Figure 4.1 Concatenated neighbour-joining phylogenetic tree showing the position of A. dhakensis strains derived from the rpoD and gyrB sequences

p. 169

Figure 4.2 Concatenated neighbour-joining phylogenetic tree showing the position of A. caviae strains derived from the rpoD and gyrB genes sequences

p. 170

Figure 4.3 Concatenated neighbour-joining phylogenetic tree showing the position of A. hydrophila strains derived from the rpoD and gyrB genes sequences

p. 171

Figure 4.4 Concatenated neighbour-joining phylogenetic tree derived from the rpoD and gyrB genes sequences showing the position of A. veronii bv. sobria and other species including strain 266

p. 172

Figure 6.1 Electron microscopy images of strain 266T p. 191

Figure 6.2 Protein spectrum of strain 266T p. 203

Figure 6.3 Unrooted neighbour-joining phylogenetic tree derived from the 16S rRNA gene sequences showing the relationships of strain 266T with all other Aeromonas species

p. 205

Figure 6.4 Unrooted neighbour-joining phylogenetic tree derived from dnaJ sequences showing the relationships of strain 266T with the type strains of all other Aeromonas species

p. 206

Figure 6.5 Unrooted neighbour-joining phylogenetic tree derived from dnaX sequences showing the relationships of strain 266T with the type strains of all other Aeromonas species

p. 207

Figure 6.6 Unrooted neighbour-joining phylogenetic tree derived from gyrA sequences showing the relationships of strain 266T with the type strains of all other Aeromonas species

p. 208

Figure 6.7 Unrooted neighbour-joining phylogenetic tree derived from gyrB sequences showing the relationships of strain 266T with the type strains of all other Aeromonas species

p. 209

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Figure 6.8 Unrooted neighbour-joining phylogenetic tree derived from recA sequences showing the relationships of strain 266T with the type strains of all other Aeromonas species

p. 210

Figure 6.9 Unrooted neighbour-joining phylogenetic tree derived from rpoD sequences showing the relationships of strain 266T with the type strains of all other Aeromonas species

p. 211

Figure 6.10 Unrooted neighbour-joining phylogenetic tree derived from the MLPA of concatenated sequences of six housekeeping genes sequences showing the relationships of strain 266T with several strains of all other Aeromonas species

p. 212

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CHAPTER 1: LITERATURE REVIEW

1.1. GENERAL INTRODUCTION

Aeromonas species are authoctonous inhabitants of aquatic environments that can be

frequently isolated from human clinical material, environmental and food sources

(Janda and Abbott 2010). Infections due to Aeromonas occur in amphibians, reptiles

and snails, where the latter infections are a significant problem for the snail industry in

France (Kodjo et al. 1997). In humans, aeromonads have been associated with serious

infections in both immunocompromised and healthy individuals while infections in fish

represent a serious threat to the aquaculture industry resulting in significant financial

loss.

Once considered organisms of doubtful clinical significance the interest in Aeromonas

has grown considerably over the past three decades as reflected by a sixfold increase in

research publications (Janda and Abbott 2010). In the tsunami that devastated parts of

Asia in 2004 Aeromonas species were the predominant (22.6%) Gram-negative isolated

from wounds of victims (Hiransuthikul et al. 2005). This led to the recommendation

that assessment of wound infections in tsunami survivors, empirical antimicrobial

therapy should always include agents with activity against Aeromonas (Lim 2005).

Similarly, Aeromonas was present in high concentration in water samples following the

hurricane Katrina disaster that affected New Orleans (Presley et al. 2006). This review

discusses current taxonomic classification and identification methods. Secondly,

description of putative virulence factors and their association with Aeromonas

infections is examined. Finally, the response of Aeromonas to antimicrobial agents is

reviewed.

1.2. HISTORY

Infections due to Aeromonas species have been described for more than a hundred years

and several reviews have credited the first reports to the work of Zimmerman and

Sanarelli in the late 1880s (Abeyta et al. 1988; Altwegg and Geiss 1989; Joseph and

Carnahan 1994). These cases were followed by other reports of Aeromonas-like bacteria

including the water-borne bacterium, Bacillus hydrophilus, isolated from water and

diseased frogs (Chester 1901) and Proteus melanovogenes implicated as the cause of

black rot in eggs and also isolated from human faeces (Miles and Halnan 1937).

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-- 2 --

According to Joseph and Carnahan (1994), the first report of human infection caused by

aeromonads was by Hill et al. (1954) who described a case of fulminant septicaemia

and metastatic myositis caused by an unknown bacterium. The microorganism that was

recovered from multiple organs and in pure form from cerebral spinal fluid was

considered an undescribed member of the family Pseudomonadaceae, tribe Spirilleae

and genus Vibrio.

The genus Aeromonas was first proposed by Kluyver and van Niel (1936) who

recommended that the species Acetobacter liquefaciens be renamed Aeromonas

liquefaciens, then the only species and type species of the genus. The newly proposed

genus was formally accepted in the seventh edition of Bergey’s Manual of

Determinative Bacteriology (Snieszko 1957). The type species of the genus, A.

hydrophila, was later proposed by Stanier (1943) based on the phenotypic

characteristics of Proteus hydrophilus, a fermentative, polar flagellated bacterium. Since

their discovery, Aeromonas or Aeromonas-like bacteria have been assigned to several

genera including Aerobacter, Bacillus, Pseudomonas, Proteus and Vibrio (Joseph and

Carnahan 1994).

1.3. TAXONOMY

Due to the heterogeneous nature of the genus the taxonomy of Aeromonas has been

considered complex and confusing (Schubert 1974; Popoff and Veron 1976; Joseph and

Carnahan 1994; Wahli et al. 2005). The inability to separate genospecies using

biochemical methods (Altwegg et al. 1990) and the poor correlation that existed

between genotypic and phenotypic methods (Austin et al. 1998; Martínez-Murcia et al.

2000) led to an unstable nomenclature (Popoff and Veron 1976; Abbot et al. 1992; Vila

et al. 2002; Ørmen et al. 2005) resulting in conflicting data (de la Morena et al. 1993;

Huys et al. 1997a; Valera and Esteve 2002; Huys et al. 2005).

1.3.1. Early taxonomy

Prior to the 1980s, classification of Aeromonas was based solely on differential

phenotypic characteristics such as growth temperature and motility (Popoff and Veron

1976). Thus, Aeromonas was classified into two major groups: a large group that

comprised the motile, mesophilic and heterogenous species that also included potential

human pathogens; and a second smaller group of homogenous species represented by A.

salmonicida, a non-motile, psychrophilic species primarily considered fish pathogens

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-- 3 --

(McNicol et al. 1980; Janda et al. 1984; Kasai et al. 1998; Pidiyar et al. 2002; Martin-

Carnahan and Joseph 2005).

In 1981, Popoff and colleagues used DNA-DNA hybridization (DDH) to classify 55

motile aeromonads. Results revealed that A. hydrophila, A. caviae and A. sobria were

well differentiated but each species contained more than one hybridization group (HG),

a term used to refer to DNA groups that could not be differentiated phenotypically. As a

consequence, investigators began to use DDH values to determine hybridization groups

(HGs), which were defined as having at least 70% DNA homology with the designated

type strain (Wayne et al. 1987). The use of the term “hybridization group” dropped out

of use over the last decade. The last hybridization group was DNA HG 18 assigned to

A. culicicola (Pidiyar et al. 2002). Instead, the term “genomic species” or “genospecies”

followed by a reference number has been recommended to describe unnamed groups

(Janda and Abbott 2010).

1.3.2. Current taxonomy

The genus Aeromonas resides in the family Aeromonadaceae (Colwell et al. 1986)

within the subclass Gammaproteobacteria (Saavedra et al. 2007). There are currently

27 recognized species and six subspecies (Table 1.1), and two biovars (Fig. 1.1). The

complete genome of all type strains representing all species and selected reference

strains have now been sequenced (Seshadri et al. 2006; Colston et al. 2014).

In recent years, the classification of Aeromonas has been based on the nucleotide

sequences of housekeeping genes which have the ability to reliably discriminate

between all species in the genus (Yañez et al. 2003; Soler et al. 2004; Thompson et al.

2004; Nhung et al. 2007; Adekambi et al. 2008; Miñana-Galbis et al. 2009). As a

consequence, 15 new Aeromonas species have been described since 2000, with the

majority recovered from environmental sources.

1.3.3. Controversial taxonomic issues

Controversial taxonomic issues discussed in previous reviews (Janda and Abbott 2010)

can now be considered partly or completely resolved. Extensive genotypic and

phenotypic evidence confirmed that: A. trota was identical to A. enteropelogenes

(Schubert et al. 1990a; Carnahan et al. 1991a; Carnahan 1993; Collins et al. 1993; Huys

et al. 1996b; 2002b) and A. ichthiosmia should be considered a junior synonym of A.

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-- 4 --

veronii (Fanning et al. 1985; Schubert et al. 1990b; Collins et al. 1993; Huys et al.

1996a; 2001). The unnamed Aeromonas group 501 (Hickman-Brenner et al. 1988) has

been reclassified as A. diversa sp. nov. (Miñana-Galbis et al. 2010) and A. hydrophila

ssp. anaerogenes has been included in the species A. caviae (Miñana-Galbis et al.

2013).

Phylogenetic evidence indicated that strains of A. hydrophila ssp. dhakensis belonged to

the species A. aquariorum (Martínez-Murcia et al. 2008; 2009). Previously, the species

A. hydrophila consisted of three subspecies including ssp. hydrophila and ssp. ranae

(Huys et al. 2003). Recently, Beaz-Hidalgo et al. (2013) combined A. hydrophila ssp.

dhakensis (Huys et al. 2002a) and A. aquariorum (Martínez-Murcia et al. 2008) and

proposed the creation of A. dhakensis sp. nov. comb. nov. Due to inconsistent genotypic

and phenotypic feature, “A. sharmana” (Saha and Chakrabarti 2006) has not been

included in the genus (Martínez-Murcia et al. 2007; Lamy et al. 2010).

1.3.3.1. Aeromonas allosaccharophila

This species was proposed by Martínez-Murcia et al. (1992a) based on two strains

recovered from diseased elvers (Anguilla anguilla) and one from human stools.

Evidence against A. allosaccharophila representing a separate species derived from

discrepancies reported in the biochemical profiles of the original strains (Martínez-

Murcia et al. 1992a; Esteve et al. 1995b; Huys et al. 1996a; 2001); amplified fragment

length polymorphism (AFLP) and fluorescent amplified fragment length polymorphism

(FAFLP) patterns identical to those of A. veronii (Huys et al. 1996b; Huys and Swings

1999); the nucleotide sequences of several housekeeping genes showed A.

allosaccharophila in close proximity to A. veronii and not sufficiently distant to

confidently separate the two species (Nhung et al. 2007; Miñana-Galbis et al. 2009;

Lamy et al. 2010). Evidence supporting A. allosaccharophila as a separate species

derived from i) its unique 16S rDNA sequence composition that clearly differentiated

this species from most other members of the genus including A. veronii (Martínez-

Murcia et al. 1992a); ii) the nucleotide sequences of the rpoD and gyrB housekeeping

genes (Yañez et al. 2003; Soler et al. 2004; Saavedra et al. 2006) (Figs. 1.2 and 1.3); iii)

multilocus sequence analysis showed that A. allosaccharophila and A. veronii were

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-- 5

--

Tab

le 1

.1

Cur

rent

Aer

omon

as sp

ecie

s

Spec

ies

HG

So

urce

of t

ype

stra

in

Ref

eren

ce

A. h

ydro

phila

ssp.

hyd

roph

ila

1 Ti

n of

milk

with

fish

y od

our

Stan

ier (

1943

)

A. sa

lmon

icid

a ss

p. a

chro

mog

enes

3

Fish

(Sal

mo

trut

ta)

Smith

(196

3)

A. sa

lmon

icid

a ss

p. sa

lmon

icid

a

Salm

on (S

alm

o sa

lar)

Sc

hube

rt (1

967b

)

A. sa

lmon

icid

a ss

p. m

asou

cida

Fish

blo

od (O

ncor

hync

hus m

asou

) K

imur

a (1

969)

A. so

bria

7

Fish

Po

poff

and

Ver

on (1

976)

A. m

edia

5

Riv

er w

ater

A

llen

et a

l. (1

983)

A. c

avia

e 4

Gui

nea-

pig

Popo

ff (1

984)

A. v

eron

ii

8/10

Fr

og re

d le

g/sp

utum

H

ickm

an-B

renn

er e

t al.

(198

7)

Aero

mon

as ss

p.

11

Ank

le su

ture

H

ickm

an-B

renn

er e

t al.

(198

7)

A. sc

hube

rtii

12

Fore

head

abs

cess

H

ickm

an-B

renn

er e

t al.

(198

8)

A. e

ucre

noph

ila

6 C

arp

Schu

bert

and

Heg

azi (

1988

)

A. sa

lmon

icid

a ss

p. sm

ithia

Fish

A

ustin

et a

l. (1

989)

A. tr

ota

14

Hum

an fa

eces

C

arna

han

et a

l. (1

991a

)

A. ja

ndae

i 9

Faec

es

Car

naha

n et

al.

(199

1c)

A. a

llosa

ccha

roph

ila

15

Dis

ease

d el

vers

/hum

an fa

eces

M

artín

ez-M

urci

a et

al.

(199

2a)

A. e

nche

leia

16

Eu

rope

an e

els

Este

ve e

t al.

(199

5a)

A. b

estia

rum

2

Infe

cted

fish

A

li et

al.

(199

6)

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-- 6

--

Tab

le 1

.1

Con

tinue

d.

Spec

ies

HG

So

urce

of t

ype

stra

in

Ref

eren

ce

A. p

opof

fii

17

Drin

king

wat

er p

rodu

ctio

n pl

ant

Huy

s et a

l. (1

997b

)

A. sa

lmon

icid

a ss

p. p

ectin

olyt

ica

W

ater

from

cis

tern

Pa

van

et a

l. (2

000)

A. h

ydro

phila

ssp.

rana

e

Farm

ed fr

og

Huy

s et a

l. (2

003)

A. si

mia

e

Mon

key

faec

es

Har

f-M

onte

il et

al.

(200

4)

A. m

ollu

scor

um

W

edge

-she

lls (D

onax

trun

culu

s)

Miñ

ana-

Gal

bis e

t al.

(200

4a)

A. b

ival

vium

Coc

kles

(Car

dium

spp.

) M

iñan

a-G

albi

s et a

l. (2

007)

A. te

cta

St

ool o

f a c

hild

with

dia

rrho

ea

Dem

arta

et a

l. (2

008)

A. p

isci

cola

Dis

ease

d fis

h B

eaz-

Hid

algo

et a

l. (2

009)

A. fl

uvia

lis

R

iver

wat

er

Alp

eri e

t al.

(201

0a)

A. d

iver

saa

13

Hum

an le

g w

ound

M

iñan

a-G

albi

s et a

l. (2

010)

A. sa

nare

llii

H

uman

wou

nd

Alp

eri e

t al.

(201

0b)

A. ta

iwan

enes

is

B

urn

wou

nd

Alp

eri e

t al.

(201

0b)

A. ri

vuli

Fr

eshw

ater

Fi

guer

as e

t al.

(201

1a)

A. a

ustr

alie

nsis

Trea

ted

efflu

ent w

ater

A

rave

na-R

omán

et a

l. (2

013)

A. d

hake

nsis

b

Chi

ldre

n w

ith d

iarr

hoea

B

eaz-

Hid

algo

et a

l. (2

013)

A. c

aver

nico

la

Is

olat

ed fr

om w

ater

bro

ok

Mar

tínez

-Mur

cia

et a

l. (2

013)

a p

revi

ousl

y cl

assi

fied

as A

erom

onas

gro

up 5

01 (H

ickm

an-B

renn

er e

t al.

1988

); b co

mbi

ned

from

A. h

ydro

phila

ssp.

dha

kens

is (H

uys e

t al.

2002

a) a

nd A

. aqu

ario

rum

(Mar

tínez

-Mur

cia

et a

l. 20

08)

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-7-

Figure 1.1 Unrooted neighbour-joining phylogenetic tree derived from gyrB nucleotide

sequences showing subspecies and biovars. The phylogenetic tree was constructed with

530 nt. Numbers at the nodes indicate bootstrap values. Bar, 0.005 estimated

substitutions per site.

A. salmonicida spp. salmonicida (CECT 894T)

A. salmonicida ssp. smithia (CIP 104757)

A. salmonicida spp. masoucida (CECT 896)

A. salmonicida spp. pectinolytica (34mel)

A. salmonicida ssp. achromogenes (CECT 895)

A. veronii bv. sobria (ATCC 9071)

A. veronii bv. veronii (DSM 7386T)

A. hydrophila spp. dhakensis (LMG 19562T)

A. hydrophila ssp. hydrophila (ATCC 7966T)

A. hydrophila spp. ranae (LMG 19707T)

97

84

100

0.005

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-8-

located in different phylogenetic lines and exhibited a high degree of nucleotide

diversity (Martino et al. 2011).

1.3.3.2. Aeromonas spp. HG 11

This unnamed Aeromonas derived from two strains that could not be included in the

original description of A. veronii (Hickman-Brenner et al. 1987). Evidence that

supported the inclusion of Aeromonas HG11 into A. encheleia was based on AFLP

(Huys et al. 1996b) and 16S-23S rDNA-RFLP patterns (Laganowska and Kaznowski

2004); high DDH values (84-87%) between Aeromonas HG11 strains and the type

strain of A. encheleia LMG 16330T (Huys et al. 1997a); and divergent values for gyrB

(2.1-2.2%), rpoD (1.4-1.7%), dnaJ (1.3%), cpn60UT (0.7%) and rpoB (0.9%) (Yañez et

al. 2003; Soler et al. 2004; Nhung et al. 2007; Miñana-Galbis et al. 2009; Lamy et al.

2010). In contrast, phenotypic profiles (Valera and Esteve 2002) and different tRNA

patterns suggested that these two species represent distinct taxa (Laganowska and

Kaznowski 2005). Moreover, the 16S rRNA sequence of A. encheleia and Aeromonas

sp. HG11 differed by eight nucleotides at hypervariable positions 457 to 476 (Martínez-

Murcia 1999), a significant feature considering that in Aeromonas the 16S rRNA gene

similarities range from 96.9 to 100% (Martínez-Murcia 1992a).

1.3.3.3. Aeromonas culicicola

This species originated from strains isolated from the midgut of the mosquito species

Culex quinquefasciatus and Aedes aegyptii (Pidiyar et al. 2002). Evidence that A.

culicicola represents a heterotypic synonym of A. veronii derived from the low

interspecies nucleotide substitution rates for several housekeeping genes (Soler et al.

2004; Nhung et al. 2007; Miñana-Galbis et al. 2009; Lamy et al. 2010); similar

phenotypic and cellular fatty acid profiles (Huys et al. 2005); DDH values well above

70% between A. culicicola MTCC 3249T and A. veronii ATCC 35624T (Huys et al.

2005; Nhung et al. 2007) compared to 44% by the initial report (Pidiyar et al. 2002);

16S rRNA RFLP profiles similar to those of A. veronii (Lamy et al. 2010). In contrast,

16S DNA-RFLP patterns reported by two studies showed that A. culicicola differed

sufficiently from all other members of the genus (Figueras et al. 2005; Kaznowski and

Konecka 2005). Moreover, gyrB gene sequence placed A. culicicola in a separate line of

descent where it differed from A. jandaei by 56 nucleotides (Yañez et al. 2003)

compared to a single nucleotide difference using 16S rDNA (Pidiyar et al. 2003).

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1.4. LABORATORY IDENTIFICATION

Aeromonas species are non-fastidious, catalase and oxidase positive, facultatively

anaerobic Gram-negative fermentative bacilli (Janda 1985). The majority of the species

produce -haemolysis on horse and sheep blood agar and most can produce indole from

tryptophan. Although the optimal temperature for growth is 28C, aeromonads can grow

at temperatures ranging from 1 to 42C (Mateos et al. 1993; Hänninen et al. 1995c) and

can adapt and survive in highly acidic (pH 3.5) environments (Karem et al. 1994).

Traditionally, susceptibility to the vibriostatic agent 2, 4-diamino-6, 7-

diisopropylpteridine (O/129; 150 g disk) and the inability of aeromonads to grow on

thiosulfate citrate bile salts sucrose agar (TCBS) and on 6% NaCl have been used as

preliminary tests to differentiate Aeromonas from closely related Vibrios and

Plesiomonas species. In general, the close phenotypic similarity of aeromonads and

poorly equipped laboratories hampers the identification of aeromonads to species level.

Thus, small laboratories should confine identification to the genus level and significant

clinical or environmental strains should be sent to reference centres for further work

(Abbott et al. 1992).

1.4.1. Isolation

Aeromonas species can grow on most solid media including MacConkey, Hektoen

enteric and xylose lysine desoxycholate (XLDA) agars, although colony size and

plating efficiency differences have been observed (Desmond and Janda 1986; Janda and

Abbott 1999). Plating efficiency appeared to be strain rather than species dependent

(Desmond and Janda 1986). The concentration of salt is critical since Aeromonas do not

usually grow in media containing greater than 3% NaCl (Abbott et al. 2003).

Occasionally, strains of A. trota have been reported to withstand concentrations close to

4% (0.68 M) NaCl (Delamare et al. 2000). An optimal Aeromonas-medium should

contain substrates that do not interfere with the oxidase test (Moulsdale 1983) or include

lactose in its composition as this carbohydrate is highly unsatisfactory for primary

isolation (Millership et al. 1983). The number of Aeromonas species recovered from

different samples has been attributed to variations in technique and media employed to

isolate these organisms (Nazer et al. 1986). A variety of media or variations of well-

established formulae have been developed to isolate and quantify aeromonads from

food, water and human faecal specimens based on biological properties such as

production of amylase and starch activity or the natural tolerance of the majority of

these organisms to ampicillin (Table 1.2).

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-10-

Figure 1.2 Unrooted neighbour-joining phylogenetic tree derived from gyrB sequences

showing current Aeromonas species. The phylogenetic tree was constructed with 530 nt.

Numbers at the nodes indicate bootstrap values. Bar, 0.01 estimated substitutions per

site.

A. popoffii (CIP 105493T)

A. bestiarum (ATCC 51108T)

A. piscicola (CECT 7443T)

A. salmonicida (CECT 894T)

A. molluscorum (DSM 17090T)

A. eucrenophila (ATCC 23309T)

A. encheleia (DSM 11577T)

A. tecta (CECT 7083T)

A. rivuli (CECT 7518T)

A. caviae (ATCC 23212)

A. media (ATCC 33907T)

A. bivalvium (CECT 7113T)

A. sanarellii (CECT 7402T)

A. cavernicola (CECT 7862T)

A. dhakensis (LMG 19562T)

A. hydrophila (ATCC 7966T)

A. jandaei (CECT 4228T)

A. fluvialis (CECT 7401T)

A. sobria (CIP 7433T)

A. veronii (ATCC 9071)

A. australiensis (CECT 8023T)

A. allosaccharophila (DSM 11576T)

A. trota (ATCC 49657T)

A. taiwanensis (CECT 7403T)

A. simiae (DSM 16559T)

A. schubertii (ATCC 43700T)

A. diversa (CECT 4254T) 100

99

96

90

85

79

73

0.01

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-11-

Figure 1.3 Unrooted neighbour-joining phylogenetic tree derived from rpoD sequences

showing all Aeromonas species. The phylogenetic tree was constructed with 653 nt.

Numbers at the nodes indicate bootstrap values. Bar, 0.02 estimated substitutions per

site.

A. taiwanensis (CECT 7403T)

A. sanarellii (CECT 7402T)

A. caviae (ATCC 13136T)

A. dhakensis (LMG 7862T)

A. hydrophila (ATCC 7966T)

A. eucrenophila (ATCC 23309T)

A. tecta (CECT 7082T)

A. media (ATCC 33907T)

A. encheleia (DSM 11577T)

A. diversa (CECT 4254T)

A. simiae (DSM 16559T)

A. schubertii (CECT 4240T)

A. bivalvium (CECT 7113T)

A. molluscorum (DSM 17090T)

A. rivuli (CECT 7518T)

A. jandaei (ATCC 49568T)

A. trota (ATCC 49657T)

A. australiensis (CECT 8023T)

A. fluvialis (CECT 7401T)

A. veronii (ATCC 9071)

A. allosaccharophila (DSM 11576T)

A. sobria (CIP 7433T)

A. cavernicola (CECT 7862T)

A. salmonicida (CECT 894T)

A. popoffii (CIP 105493T)

A. bestiarum (ATCC 51108T)

A. piscicola (CECT 7443T)

100

100

100

98

92

99

90

97

93

86

93

93

0.02

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-12-

Huddleston et al. (2007) recommended that ampicillin should not be used as a selective

agent in isolation medium for Aeromonas when a complete analysis of Aeromonas

diversity and density is desired. These authors argued that media containing ampicillin

was likely to inhibit the growth of ampicillin-susceptible strains resulting in an

underestimation of densities and species diversity.

1.4.2. Identification by phenotypic methods

Identification of Aeromonas by phenotypic methods has been based on the ability of

these bacteria to ferment carbohydrates with vigorous gas production (Kluyver and van

Neil 1936; Stanier 1943; Schubert 1968). However, identification based on biochemical

tests is often unable to accurately identify Aeromonas beyond genus level as phenotypic

features are unstable and vary within the species (Davin-Regli et al. 1998; Martínez-

Murcia et al. 2000; Figueras et al. 2005; Wahli et al. 2005). Moreover, biochemical

analyses depend on the transcription and translation of proteins which in turn are

influenced by environmental factors such as temperature or carbohydrate repression

potentially affecting production of proteins (Knochel 1989; 1990).

Phenotypic identification is also influenced by the number and type of tests and testing

conditions (Valera and Esteve 2002; Esteve et al. 2003; Demarta et al. 2004),

geographical source (Kaznowski et al. 1989) and interpretation of data and

reproducibility of results (Abbott et al. 2003; Ørmen et al. 2005). Inaccurate

identification is further compromised by those species in which only a handful of strains

have been described (Abbott et al. 1992), by the application of schemes designed to

identify clinical isolates to classify strains isolated from environmental and fish sources

(Wakabayashi et al. 1981; Kaznowski et al. 1989; Ashbolt et al. 1995; Borrell et al.

1998; Ørmen et al. 2005). Furthermore, many of the biochemical schemes used in

clinical laboratories predate the description of new taxa leading some authors to

question whether the efficiency of older biochemical schemes are suitable to identify

more recently described species (Edberg et al. 2007).

An identification scheme, the Aerokey II (Carnahan et al. 1991b; Joseph and Carnahan

1994) based on a small subset of highly discriminatory biochemical tests and the

AeroMat-1/AsalMat-1 designed exclusively for the identification of A. salmonicida to

species and subspecies levels, respectively, were developed (Higgins et al. 2007).

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-13-

However, Aerokey II has not been generally adopted by laboratories due to the

inconsistent biochemical profiles expressed by some species, costs and long incubation

times required (Abbott et al. 1992; Janda and Abbott 1998). Furthermore, Aerokey II

may be unsuitable for those regions harbouring strains with unique phenotypic profiles

or due to the heterogeneous character of some species (Altwegg et al. 1990) while a

lack of congruence between Aerokey II and genotypic identification has been reported

(Noterdaeme et al. 1996).

Other potential identifying markers proposed to differentiate Aeromonas species

included susceptibility to cephalexin (Janda and Motyl 1985), induced colistin

resistance (Fosse et al. 2003b), production of a CAMP-like factor (Figura and

Guglielmetti 1987) and maximum growth temperature determined with a temperature-

gradient incubator (Havelaar et al. 1992; Hӓnninen 1994). The production of acetic acid

in glucose-containing media is a peculiar characteristic displayed by certain species

whereby some aeromonads become unviable (“the suicide phenomenon”). This test was

designed as an identification marker to separate A. caviae (Namdari and Cabelli 1989).

1.4.3. Identification by commercial systems

A plethora of commercial systems such as Vitek, API, MicroScan Walk/Away, BBL

Crystal Enteric/Non-fermenter, Biolog and the Phoenix 100 ID/AST contain selected

Aeromonas species in their databases (Hӓnninen 1994; Park et al. 2003; Soler et al.

2003b; Huddleston et al. 2006; O’Hara 2006). Unfortunately, identification of

Aeromonas by these systems is inadequate resulting in major errors (Janda and Abbott

2010). Among the major identification problems encountered with these systems are:

misidentification of Aeromonas species as V. cholerae and V. damsela (Abbott et al.

1998), partly attributed to the lower salt concentration (0.45% NaCl) recommended by

the manufacturer in the preparation of the inoculum in the Vitek identification system

(Park et al. 2003); production of acid by the API 20E is temperature-dependent

resulting in false-negative results if the strip is incubated at 37C (Hӓnninen 1994); the

percentage of correct identifications for MicroScan Walk/Away (14.5%) and BBL

Crystal Enteric/Non-fermenter (20.3%) systems is low (Soler et al. 2003b) while the

Phoenix 100 ID/AST identified only 60% of Aeromonas (O’Hara 2006).

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-14-

Tab

le 1

.2

Exam

ples

of m

edia

use

d in

the

isol

atio

n of

Aer

omon

as fr

om d

iffer

ent s

ourc

es

Med

ia

So

urce

/pur

pose

R

efer

ence

Glu

tam

ate

star

ch p

heno

l (G

SP);

Red

aga

r (Ps

eudo

mon

as-

Aero

mon

as-s

elec

tive

agar

) Fo

ods o

f ani

mal

or

igin

/env

ironm

enta

l sou

rces

U

llman

et a

l. (2

005)

; Yuc

el a

nd

Erdo

gan

(201

0)

Blo

od a

mpi

cilli

n ag

ar (B

AA

); O

xoid

Aer

omon

as a

gar;

Star

ch

amip

icill

in a

gar (

SAA

) Se

afoo

d R

obin

son

et a

l. (1

984)

; Pal

umbo

et

al. (

1985

); Pi

n et

al.

(199

4); T

sai

and

Che

n (1

996)

B

lood

aga

r con

tain

ing

p-ni

troph

enol

gly

cerin

e Fa

ecal

sam

ples

B

urke

et a

l. (1

983)

; Rob

inso

n et

al.

(198

6)

Car

y-B

lair

med

ium

Tr

ansp

ort m

ediu

m

Moy

er (1

987)

Difc

o Ae

rom

onas

aga

r (D

AA

); am

pici

llin

bloo

d ag

ar (A

BA

); xy

lose

des

oxyc

hola

te c

itrat

e ag

ar (X

DC

A) a

nd a

lkal

ine

pept

one

wat

er (A

PW)

Chi

ldre

n st

ools

/ car

riage

rate

W

ilcox

et a

l. (1

992)

Am

pici

llin-

Dex

trin

Aga

r (A

DA

) R

aw, p

roce

ssed

and

read

y-to

-ea

t foo

ds sa

mpl

es

Kin

gom

e et

al.

(200

4)

XD

CA

, DN

A to

luid

ine

agar

(DN

AT)

; Sal

mon

ella

-Shi

gella

so

dium

des

oxyc

hola

te (S

SSD

) aga

r Fa

ecal

car

riage

rate

M

iller

ship

et a

l. (1

983)

; von

G

raev

enitz

and

Zin

terh

ofer

(197

0);

Wau

ters

(197

3); F

igur

a (1

985)

in

osito

l-bile

-sal

ts-b

rillia

nt g

reen

(IB

B) a

nd c

efsu

lodi

n-irg

asan

-no

vobi

ocin

aga

r (C

IN);

BA

A

Fa

ecal

/abi

lity

to g

row

on

thes

e m

edia

A

ltorf

er e

t al.

(198

5); M

oyer

et a

l. (1

991)

Page 41: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-15-

1.4.4. Additional phenotypic methods

Many non-biochemical methods have been employed as alternatives to biochemical

identification for typing or identification purposes, or both. Some, such as the use of

core oligosaccharides from the endotoxins have not been readily adopted as routine

identification methods (Shaw and Hodder 1978). Isoenzyme analysis has been used as

both a screening method to investigate the epidemiology of hospital infections and as an

identification tool (Picard and Goullet 1987; Altwegg et al. 1988). Multi-loccus enzyme

electrophoresis (MLEE) has been considered useful as a sole method for species

identification and shows good correlation with taxonomic groupings as determined by

DDH (Altwegg et al. 1991c; Miñana-Galbis et al. 2004b). In contrast, phage typing,

although specific to the genus Aeromonas, may be over-sensitive (Altwegg et al. 1988).

The use of outer membrane protein (OMP) composition as a typing method is

cumbersome and time consuming and OMP profiles are influenced by temperature and

the air-supply available to the bacterial cultures (Küijper et al. 1989a). Methods such as

radiolabelled cell proteins (radioPAGE) profiles are difficult to interpret and prone to

subjective bias (Stephenson et al. 1987) while conflicting data have been reported with

whole-protein fingerprinting (Millership and Want 1993; Alavandi et al. 2001; Szczuka

and Kaznowski 2007).

1.4.5. Semiautomated systems

Two semi-automated systems based on the analysis of cellular fatty acid methyl esters

by gas-liquid chromatography (GLC-FAMEs) and by measuring the differences in

protein mass generated by the matrix-assisted laser-desorption/ionization mass

spectrometry time-of flight (MALDI-MS-TOF) are widely used in identification of

Aeromonas. Both methods are expensive and in the case of GLC-FAME require highly

trained personnel. The systems can be used for the rapid identification of bacteria

(Rahman et al. 2002) or as a typing tool (Osterhout et al. 1991; von Graevenitz et al.

1991; Huys et al. 1994, 1995; Donohue et al. 2006, 2007).

The reproducibility of the GLC-FAMEs system depends greatly on media, temperature

of incubation, sets of strains, GC model used to analyse cellular fatty acid patterns and

previous exposure to antibiotics (Canonica and Pisano 1988; Huys et al. 1994;

Kӓempfer et al. 1994). A high identification rate of Aeromonas to species level has been

reported by MALDI-TOF users making this system the most accurate for identification

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-16-

of these bacteria (Lamy et al. 2011). For those laboratories that can afford it, the

MALDI-TOF has largely superseded most automated identification systems. Although

the instrument is expensive, consumables and operational costs are lower than those

incurred by the MIDI system, the most commonly used system used to detect FAME. It

also requires less laboratory space than the MIDI system.

1.4.6. Identification by molecular methods

Practically every known molecular technique, each with its own strengths and

weaknesess, has been used in the classification and typing of aeromonads since Popoff

et al. (1981) placed them into DNA hybridization groups. In Aeromonas, the use of a

single typing method to determine interrelationship between species may not be

adequate as the potential for discrimination increases by combining different molecular

methods (Altwegg et al. 1988; Davin-Regli et al. 1998; Soler et al. 2003a; Morandi et

al. 2005). The application of these methods has been useful in establishing the

epidemiological relationships between aeromonads recovered from very different

sources (Villari et al. 2003). However, a situation similar to phenotypic identification

exists where a lack of congruence between different molecular methods has been

recognized (Hӓnninen and Siitonen 1995; Graf 1999a; Martínez-Murcia 1999; Figueras

et al. 2000b; Yañez et al. 2003; Laganowska and Kasnowski 2005; Saavedra et al.

2006). Methods employed in the characterization and typing of aeromonads included

those based on restriction enzymes used to digest genomic DNA [ribotyping, amplified

fragment length polymorphism (AFLP), fluorescence amplified fragment length

polymorphism (FAFLP), restriction fragment length polymorphism (RFLP)]; PCR-

based methods [randomly amplified polymorphic DNA (RAPD), enterobacterial

repetitive intergenic consensus (ERIC), repetitive extragenic palindromic (REP)] and

PCR followed by DNA sequencing targeting single or multiple genes (MLST/MLSA).

In the case of AFLP and FAFLP, digestion of DNA with restriction enzymes was

followed by PCR. Other methods used included pulse field gel-electrophoresis (PFGE)

and plasmid profiles.

1.4.6.1 Typing methods

Although some of the methods mentioned in the previous section can be used for both

identification and typing purposes some are more suitable as typing methods for the

determination of strain relatedness. The use of plasmid profiles was reported to be

relatively unstable and not useful in genomic typing (Altwegg et al. 1988) while others

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-17-

are more suitable for fingerprinting at strain level (Chang and Janda 2005). The poor

discriminatory patterns precluded PFGE to be used as an identification method. Instead,

PFGE offers an effective alternative as a typing method (Bonadonna et al. 2001;

Abdullah et al. 2003). The most satisfactory methods used in Aeromonas typing include

RFLP, RAPD, ERIC and AFLP and can be applied to determine the relatedness of

isolates in recurrent infections, the linkage of infections to environmental sources and

pseudo-outbreaks of disease (Janda and Abbott 2010).

1.4.6.2. Identification based on 16S-23S rRNA gene sequence

The most common target used in bacterial identification in laboratories world-wide is

the 16S rRNA gene (Stackebrandt and Goebels 1994; Petti et al. 2005; Boudewijns et

al. 2006; Janda and Abbott 2007). In aeromonads, 16S rRNA gene sequence signature

regions that differentiate some species from all other members in the genus have been

described (Demarta et al. 1999; Figueras et al. 2000b; Martínez-Murcia et al. 2000). As

a consequence, 16S rRNA-based probes designed to identify individual species directly

from samples have been developed (Ash et al. 1993a/b; Dorsch et al. 1994; Khan and

Cerniglia 1997; Demarta et al. 1999). Genus specific primers based on the 16S-23S

rRNA intergenic spacer region (ISR) have been designed to confirm the identity of

aeromonads following initial morphological and biochemical tests (Kong et al. 1999).

Overall, 16S rRNA sequencing has been found unsuitable to accurately differentiate

Aeromonas species (Martínez-Murcia et al. 2000) as the resolution power of the 16S

rRNA gene is limited when used to differentiate organisms that have identical or similar

sequences (Fox et al. 1992; Martínez-Murcia et al. 1992b; Thompson et al. 2004;

Morandi et al. 2005). For example, the DNA relatedness between A. caviae and A. trota

is 30% although their 16S rRNA sequences differ by only three nucleotides. On the

other hand, A. veronii and A. sobria differ by 14 nucleotides while they are 60 to 65%

related in DNA pairing studies (Martínez-Murcia et al. 1992b).

Secondly, the intragenomic heterogeneity of most Aeromonas based on rrn operon

nucleotide polymorphisms showed values ranging from 0.06 to 1.5%. The latter value

reported in A. veronii, a species known to possess up to six copies of the 16S rRNA

gene (Morandi et al. 2005; Alperi et al. 2008). Roger et al. (2012a) showed that

aeromonads harboured 8 to 11 rrn operons with 10 operons being observed in more than

92% of the strains studied. Although the use of the 16S rRNA gene as an identification

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-18-

tool for aeromonads has been found useful by some (Figueras et al. 2005; Al-Benwan et

al. 2007), it should not be the only gene used for Aeromonas species identification and

delineation. This method has now been superseded by the use of housekeeping genes

sequencing (Husslein et al. 1992; Cascón et al. 1996; Khan and Cerniglia 1997; Yañez

et al. 2003; Soler et al. 2004; Nhung et al. 2007; Adekambi et al. 2008; Miñana-Galbis

et al. 2009).

1.4.6.3. Identification based on housekeeping gene sequence

Housekeeping genes are universally distributed among bacterial species and are rarely

predisposed to horizontal transfer as may be the case with 16S rRNA (Yañez et al.

2003; Morandi et al. 2005). The sequence divergence of housekeeping genes is usually

greater than that of 16S rRNA and in some cases the mean substitution rate is four to six

times higher (Yamamoto and Harayama 1996; Yañez et al. 2003; Soler et al. 2004;

Küpfer et al. 2006; Saavedra et al. 2006; Nhung et al. 2007; Beaz-Hidalgo et al. 2009;

Figueras et al. 2011b). Housekeeping genes provide better targets for Aeromonas

delineation (Yañez et al. 2003) with the added advantage that these methods are less

laborious to perform than DDH.

The sequences of the housekeeping genes recA, rpoB, dnaJ and cpn60 UT were

comparable with gyrB and rpoD and superior to 16S rRNA for the differentiation of

Aeromonas species (Küpfer et al. 2006; Nhung et al. 2007; Miñana-Galbis et al. 2009;

Lamy et al. 2010). The combined sequence of several housekeeping genes, multiloccus

sequence typing (MLST) also known as multilocus sequence analysis (MLSA) is a

powerful tool that can be used to determine the microbial diversity and classification of

these organisms (Beaz-Hidalgo et al. 2009; Figueras et al. 2011a; Martino et al. 2011).

1.4.6.4. Specific genes used as identification targets

Primers designed to detect virulence genes that allow the direct identification of specific

species included the aerolysin gene of A. trota (Husslein et al. 1992; Khan et al. 1999),

the lip gene of A. hydrophila (Cascón et al. 1996) and a 421 bp sequence from the 3’

region of the surface array protein (vapA) gene of A. salmonicida (Gustafson et al.

1992). The latter assay doubles as a non-invasive method to monitor A. salmonicida in

carrier fish and as a virulence marker (Gustafson et al. 1992). The glycerophospholipid-

cholesterol acyltransferase (GCAT) gene is universally present in Aeromonas (Chacón

et al. 2002) and has been used as a target to identify aeromonads to the genus level

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(Puthucheary et al. 2012). Multiple-PCR (mPCR) assays capable of identifying up to

98% of Aeromonas species by detecting the presence of virulence genes have been

developed (Sen 2005; Chang et al. 2008). The m-PCR assay based on oligonucleotide

primers directed to the AHCYTOENT gene was designed for the rapid and specific

detection of A. hydrophila in diseased fish including viable but non-culturable cells

(Chu and Lu 2005a). Multiple-PCR assays are less expensive to run than RFLP and

almost complete agreement with identification by biochemical methods has been

reported (Sen 2005).

1.4.6.5. Restriction enzyme-based methods

Several enzyme-based methods such as ribotyping, AFLP and restriction endonuclease

analysis have been used alone, or in combination, as identification and typing tools in

the classification of aeromonads. Of these, ribotyping is a useful method for species

identification. It requires minimal DNA for testing and several strains can be tested

simultaneously (Rautelin et al. 1995b; Martínez-Murcia et al. 2000; Soler et al. 2003a).

Ribotyping has been the method of choice to demonstrate familial transmission and

long-term colonization by A. caviae (Moyer et al. 1992; Rautelin et al. 1995b) and to

determine routes of furunculosis in Finnish salmon caused by A. salmonicida ssp.

salmonicida (Hӓnninen et al. 1995b). Ribotyping was found to be more sensitive than

MLEE (Altwegg et al. 1991b) and superior to PFGE in an epidemiological study of A.

salmonicida ssp. salmonicida (Hӓnninen and Hirvela-Koski 1997). PFGE patterns from

mesophilic aeromonads revealed a high level of genetic heterogeneity (Talon et al.

1996; Hӓnninen and Hirvelӓ-Koski 1997; Villari et al. 2000). In contrast, PFGE patterns

for A. salmonicida ssp. salmonicida confirmed the genetic homogeneity of this species

(Hӓnninen and Hirvelӓ-Koski 1997; Miyata et al. 1995).

Bauab et al. (2003) suggested that ribotyping was a useful epidemiological tool suitable

for the study of Aeromonas infections. However, ribotyping was found to be less

discriminatory than ERIC-PCR (Soler et al. 2003a) and due to the genetically

homogeneous nature of A. salmonicida (Hӓnninen et al. 1995b), unsuitable for typing

these species (Altwegg and Luthi-Hottenstein 1991).

As mentioned in section 1.4.6.1 above, one of the most established molecular methods

used as a typing and identification tool for Aeromonas is RFLP (East and Collins 1993;

Borrell et al. 1997; Nagpal et al. 1998; Graf 1999a; Figueras et al. 2000b; Martínez-

Murcia et al. 2000; Soler et al. 2003a; Laganowska and Kaznowski 2004; Kaznowski

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and Konecka 2005; Ghatak et al. 2007a). On the other hand, patterns generated by

AFLP allow clear differentiation of strains within a given species and correlate well

with DDH data suggesting that AFLP can be used for subtyping of aeromonads (Janssen

et al. 1996). Both AFLP and FAFLP have been proposed for epidemiological and

evolutionary studies (Huys et al. 1996b, 1997a, 2001; Janssen et al. 1996; Huys and

Swings 1999).

1.4.6.6. PCR-based methods

PCR-based methods used in the study of aeromonads include RAPD, AFLP and ERIC.

RAPD requires small amounts of genomic DNA (Miyata et al. 1995) while ERIC-PCR

has generally been used in combination with other methods as a typing or differential

tool (Davin-Regli et al. 1998; Sechi et al. 2002; Soler et al. 2003a; Szczuka and

Kaznowski 2004). Both ERIC and RAPD are considered superior to REP-PCR for

distinguishing Aeromonas species clones and for epidemiological investigation (Davin-

Regli et al. 1998; Szczuka and Kaznowski 2004). As a sole testing method, ERIC-PCR

was found more discriminatory for aeromonads than RFLP and REP (Soler et al.

2003a).

1.4.6.7. Disadvantages of molecular methods

In general, most molecular-based methods are time consuming, expensive and labour

intensive and do not always provide reliable and rapid results (Talon et al. 1996; Davin-

Regli et al. 1998; Figueras et al. 2000b; Sen 2005). Some methods are limited in their

applicability because they require materials not readily available in routine laboratories

while others cannot reliably discriminate between strains (Moyer et al. 1992). Other

methods, due to the type of results produced are more suitable for typing purposes than

for species identification (Taçao et al. 2005b). In addition, ribotyping, RFLP and AFLP

patterns can be difficult to interpret (Martínez-Murcia et al. 2000; Morandi et al. 2005;

Sen 2005) while RFLP is highly dependent on the type and number of endonucleases

used (Huys et al. 1996b; Graf 1999a; Figueras et al. 2000b; Kaznowski and Konecka

2005; Ghatak et al. 2007b). Atypical RFLP patterns have been recognized in clinical

strains (Alperi et al. 2008; Puthucheary et al. 2012) more often than in environmental

isolates (p < 0.01) due to microheterogeneities in the 16S rRNA gene (Alperi et al.

2008). The presence of microheterogeneities compromises accurate identification

(Morandi et al. 2005). The taxonomic value of AFLP as a reliable identification tool has

not yet been demonstrated (Martínez-Murcia 1999). Variations in DNA concentrations

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can affect reproducibility of RAPD (Davin-Regli et al. 1995). This method is also

primer dependent (Oakey et al. 1995; 1996a) while interpretation of RAPD-PCR

fingerprints may be affected by co-migration of DNA fragments due to electrophoretic

resolution (Oakey et al. 1998). Due to a lack of standardization and with the exception

of AFLP and ribotyping, results obtained from most methods are often difficult to

compare (Tindall et al. 2010).

1.5. SEROTYPING

Serotyping was considered a promising tool to rapidly differentiate Aeromonas from

other oxidase-positive bacteria (Joseph and Carnahan 1994; Korbsrisate et al. 2002).

However, the variable typability rate of Aeromonas and antisera availability has

hampered the use of serology as a routine identification method in clinical laboratories

(Havelaar et al. 1992; Millership and Want 1993; Bonadonna et al. 2001). As a

consequence, serotyping of Aeromonas has been confined to a few specialized

laboratories.

No absolute association has been described between serotypes and certain phenotypes

as Aeromonas species are serologically heterogeneous, and no serogroup has been

uniquely associated with a single species (Havelaar et al. 1992; Millership and Want

1993; Bauab et al. 2003). The most dominant serogroups O:11, O:16, O:18, O:34 and

O:83 have been associated with gastroenteritis and septicaemia (Kokka et al. 1991;

Merino et al. 1993; Bauab et al. 2003). These serotypes can be present in up to 50% of

the typable strains isolated from human clinical material (Korbsrisate et al. 2002). The

loss of the O:34 antigen lipopolysaccharide due to mutation of the gne gene can affect

motility despite complete flagellar biogenesis as the absence of O:34 antigen affects

both swarming and swimming motilities (Canals et al. 2006a). Strains with the O:34

antigen have been found to have a high level of adhesion when grown at 20 but not at

37C. Thus, the O:34 antigen acts as an adhesion (Merino et al. 1996a).

Serotypes O:11 and O:34 have the capacity to produce a capsule when grown in

glucose-rich medium (Martínez et al. 1995). Group IIA capsules have been found in A.

hydrophila serotypes O:18 and O:34, while group IIB capsules are found in the O:21

and O:27 serogroups (Zhang et al. 2003). Serotype O:11 strains are known to possess an

S-layer that can confer resistance to the bactericidal activity of normal serum (Kokka et

al. 1991) in addition to being associated with invasive infections in an animal model

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system (Paula et al. 1988). S-layers have also been described in serogroups O:14 and

O:81 of A. hydrophila which possessed S-layer proteins different from A. hydrophila

TF7 and A. salmonicida A450 (Esteve et al. 2004). Clinical strains have been found to

be less amenable to serotyping than environmental isolates (Millership and Want 1993).

A differential serological test that determines the presence of A. salmonicida while

ruling out A. hydrophila as the cause of furunculosis in Californian trout (Oncorhynchus

Mykiss, Walbaum, 1792) was developed by Markovic et al. (2007).

1.6. ECOLOGY

The ubiquitous nature of Aeromonas is reflected by the isolation of these organisms

from every environmental niche capable of sustaining bacterial growth. Although,

compared to other aquatic organisms like Pseudomonas species, Aeromonas are less

able to degrade simple compounds to be used as carbon sources (Schubert 1987). In

earlier ecological studies, laboratory personnel were confronted with isolation

procedures and identification schemes which, at the time, were based on phenotypic

testing only (Schubert 1987).

1.6.1. Aquatic environments

Aeromonas species have been recovered from surface water, fish ponds, brooks, sewage

in various stages of treatment, untreated and treated drinking water, rivers, lakes,

groundwater, wastewater, activated sludge, seawater (estuaries), spring, and stagnant

water (Freij 1984; Ørmen and Østensvik 2001). Despite the ubiquitous nature of these

micro-organisms in aquatic environments, their natural reservoir is still unknown.

Several possible niches have been proposed including the flora of plankton and seawater

(Simidu et al. 1971); as natural inhabitans of chironomid egg masses, a feature also

shared by V. cholerae (Senderovich et al. 2008); as inhabitans of duckweed, a potential

reservoir for infections of humans consuming contaminated fish (Rahman et al. 2007a);

and the ability to survive inside Acanthamoeba and remained viable during the

encystment process while exhibiting high levels of recovery from mature cysts (Yousuf

et al. 2013).

1.6.1.1. Distribution in water

The distribution of Aeromonas in water supplies varies depending on the levels of

pollution, geographical region, methods and media used in the identification of

aeromonads and the type of sample analysed (Araujo et al. 1991; Huys et al. 1995;

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Kühn et al. 1997a/b; Sechi et al. 2002; Pablos et al. 2011). Furthermore, the diversity,

density and overall composition of aeromonads vary depending on the time of the year

(Kühn et al. 1997b; Rahman et al. 2007a). Aeromonads have been found to persist for

prolonged periods of time in different water systems (Kühn et al. 1992, 1997c; Rahman

et al. 2007a). Multiple clones have survived and multiplied in raw surface water after

the treatment process (Kühn et al. 1997b) while phenotypically and genotypically stable

clones could persist in treatment systems over long periods of time. As a result, clones

may have spread from hospitalized children with diarrhoea to fish farmed for human

consumption through the sewage water treatment system (Rahman et al. 2007a).

Bacterial populations can increase from 103 to 106 CFU ml after bottling (Hunter 1993)

to 2.7 x 106 CFU/ml and 1.9 x 106 CFU/ml in sediment sewage water and in duckweed

aquaculture-based hospital sewage water treatment plant, respectively (Rahman et al.

2007a).

The distribution of Aeromonas species varies according to the type of water analysed.

Both A. veronii bv. sobria and A. caviae have been predominant in sediment sewage

water and treated sewage effluents (Ashbolt et al. 1995; Rahman et al. 2007a). The high

incidence of A. caviae in sewage and wastewater suggests that this species may have a

role as a potential indicator of water pollution (Araujo et al. 1991; Ramteke et al. 1993).

In general, data from most studies implicate A. hydrophila as the most prominent

species isolated from water samples. Minor species such as A. culicicola and A. popoffii

have also been recovered from raw and treated waste water (Table 1.3) while A.

eucrenophila was isolated from water and infected fish (Singh and Sanyal 1999;

Figueira et al. 2011).

1.6.1.2. Water quality

The presence of Aeromonas in water depends primarily on the organic material content

of the water, water temperature, the length of time in the distribution network and the

presence of chlorine residues (Seidler et al. 1980; Kaper et al. 1981; Hird et al. 1983;

van der Kooij and Hijnen 1988; Borrell et al. 1998; Korzeniewska et al. 2005). The

survival rate of A. hydrophila in mineral water depended largely on the concentrations

of dissolved solid and organic matter and not on temperature of storage (Korzeniewska

et al. 2005). A significant correlation between organic matter content and total numbers

of mesophilic aeromonads in waters has been reported (Araujo et al. 1989;

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Korzeniewska et al. 2005). In polluted water, a correlation also exists between the

numbers of aeromonads, faecal coliforms and the concentration of organic matter as

measured by biological oxygen demand (Araujo et al. 1991). The isolation of

Aeromonas from chlorinated water suggests a high organic loading as a result of

inadequate chlorination (Abbott et al. 1992). Although polluted waters rich in nutrients

readily support the growth of aeromonads, the presence of low molecular weight fatty

acids, amino acids or carbohydrates in low concentrations can also promote growth of

these organisms in less polluted waters (van der Kooij and Hijnen 1988). Indeed, A.

hydrophila could survive for considerable periods of time in filtered-autoclaved fresh

water or in filtered-autoclaved nutrient-poor water in the absence of natural microflora

(Kersters et al. 1996; Korzeniewska et al. 2005). In some regions, aeromonads have

been found to be more numerous than total coliforms in drinking (Schubert 1987) and

fresh water, and their presence may be an indicator of water quality (Knochel and

Jeppesen 1990).

1.6.1.3. Effects of temperature on growth and toxin production

The incidence of Aeromonas is usually low during winter compared to summer

(Millership and Chattopadhyay 1985; Chauret et al. 2001). The ability of aeromonads to

grow at low temperatures (5C) is a serious public health concern (Callister and Agger

1987; Nishikawa and Kishi 1988; Tsai and Chen 1996; Chang et al. 2008).

Environmental isolates are adapted to competitive growth at lower temperatures than

clinical isolates (Callister and Agger 1987). Toxin production is not necessarily

inhibited at low temperatures (Eley et al. 1993) and enterotoxigenic A. hydrophila

strains have been recovered from oysters stored for 18 months at 72C (Abeyta et al.

1986). Maalej et al. (2004) demonstrated that A. hydrophila enter a viable-but-not-

culturable (VBNC) state when exposed to nutritionally-deficient natural seawater at low

temperatures. Changes in temperature from 5 to 23C allowed multiple biological

activities such as adherence and haemolytic activity to be restored. The ability to enter

this VBNC state may explain the persistence of A. hydrophila in water systems during

winter (Maalej et al. 2004).

The ability of bacteria to enter a VBNC state permits the survival of microorganisms

when confronted with adverse environmental conditions. In this state, bacteria fail to

grow on routine microbiological media although they remain viable and retain virulence

(Fakruddin et al. 2013). Ramamurthy et al. (2014) stated that the VBNC had important

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-25-

implication in several fields, including environmental monitoring, food technology, and

infectious disease management. These authors suggested that it was important to

investigate the association of bacterial pathogens under VBNC state and the

water/foodborne outbreaks. Studies have shown that A. hydrophila in a VBNC state

may not be as virulent to goldfish compared to normal culturable bacteria (Rahman et

al. 2001). However, from the public health point of view, culture-negative food,

environmental and clinical samples may not necessarily be an indication of a pathogen-

free status. Moreover, low grade infections may be due to the presence of VBNC in

water and food and in some instances incorrectly attributed to viruses when no bacteria

have been detected (Fakkrudin et al. 2013).

1.6.1.4. Aeromonas in drinking water

The incidence of Aeromonas in drinking water from distribution systems is generally

low (Le Chevalier et al. 1982). However, the affinity of A. hydrophila for low

molecular weight substrates indicates that this organism can readily grow if these

compounds are available in drinking water supplies (van der Kooij and Hijnen 1988). In

Denmark, Aeromonas species constituted 28% of the bacterial load in drinking water

with A. hydrophila as the dominant species (Knochel and Jeppesen 1990). The presence

of these organisms in drinking water is undesirable because Aeromonas strains have

been associated with a broad spectrum of human diseases (Gracey et al. 1982a; Burke et

al.1984b; Villari et al. 2003). The relatively high presence of Aeromonas in public

water systems in the USA was attributed to the inability of these systems to maintain an

adequate concentration of residual chlorine throughout the distribution system (Egorov

et al. 2011). The association of aeromonads in drinking water supplies with human

infections and ability to grow in distribution system biofilms, led to the inclusion of

Aeromonas in the first and second editions of the Contaminant Candidate List (CCL)

issued by the United States Environmental Protection Agency (USEPA 1998) and also

in the list of opportunistic bacterial pathogens among the major pathogens and parasites

of health concern (Bitton 2014). Moreover, the presence of Aeromonas in food and

water represents a vehicle for Aeromonas infections (Ottaviani et al. 2011).

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Tab

le 1

.3

Dis

tribu

tion

of A

erom

onas

spp.

in w

ater

sour

ces f

rom

diff

eren

t loc

atio

ns

Spec

ies (

%)

Loc

atio

n T

ype

of w

ater

R

efer

ence

A.

cavi

ae

(55%

); A.

hy

drop

hila

(3

4%);

A.

sobr

ia

(6%

); Ae

rom

onas

spp.

(5%

) N

orth

ern

Spai

n

Sew

age,

rive

r, se

a A

rauj

o et

al.

(199

1)

A. h

ydro

phila

(51

%);1 A

. ca

viae

(26

%);1 A

. ve

roni

i (1

1%);

Unk

now

n sp

p. (1

1%)

Finl

and

Fres

h, d

rinki

ng

Hän

nine

n an

d Si

itone

n (1

995)

A. h

ydro

phila

(39%

);1 A. c

avia

e (2

3%);

A. so

bria

(17%

) B

elgi

um

Drin

king

, raw

/trea

ted

surf

ace

and

phre

atic

gr

ound

wat

er

Huy

s et a

l. (1

995)

A. so

bria

(14%

); A.

cav

iae

(11%

); A.

hyd

roph

ila (9

.5%

) In

dia

Met

ropo

litan

wat

er

supp

ly, b

ore,

drin

king

A

lava

ndi e

t al.

(199

9)

A. so

bria

(70%

); A.

pop

offii

(30%

) R

ussi

a D

rinki

ng

Ivan

ova

et a

l. (2

001)

A. h

ydro

phila

(67%

); A.

salm

onic

ida

(26%

); A.

sobr

ia (1

1%)

Sard

inia

, Ita

ly

Coa

stal

mar

ine

wat

ers

Sech

i et a

l. (2

002)

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-27-

Tab

le 1

.3

Con

tinue

d.

Spec

ies (

%)

Loc

atio

n

Typ

e of

wat

er

Ref

eren

ce

A. h

ydro

phila

2 ; A. v

eron

ii (b

oth

biov

ars)

2 In

dia

Riv

er

Shar

ma

et a

l. (2

005)

A. c

ulic

icol

a (4

5%);

A. v

eron

ii (3

6%);

A. s

alm

onic

ida

(8%

); A.

hyd

roph

ila (7

%)

Spai

n D

rinki

ng

Figu

eras

et a

l. (2

005)

A. h

ydro

phila

(25%

) In

dia

Su

rfac

e B

how

mik

et a

l. (2

009)

A. m

edia

(~67

%);

A. c

avia

e (3

3%)

Leon

, Spa

in

Drin

king

Pa

blos

et a

l. (2

010)

A. d

hake

nsis

3 (55%

); A.

ver

onii

bv. s

obria

(27%

);

A. h

ydro

phila

(9%

) A

ustra

lia

Irrig

atio

n, re

serv

oir,

tre

ated

, bor

e,

chlo

rinat

ed

Ara

vena

-Rom

án e

t al.

(201

1b)

1 Iden

tifie

d as

com

plex

; 2 Perc

enta

ges n

ot g

iven

; 3 Prev

ious

ly c

lass

ified

as A

. aqu

ario

rum

.

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-28-

1.6.2. Aeromonas in foods

Reports of Aeromonas-associated foodborne outbreaks began to appear frequently from

the late-1970s reaching a peak in the 1980s (Abeyta et al. 1986; Isonhood and Drake

2002). Aeromonas species are not unusually resistant to traditional food processing

techniques but are regularly isolated in variable numbers from vegetables, minced beef,

pork, chicken, seafood, milk, cheese, fish, cream (Callister and Agger 1987; Nishikawa

and Kishi 1988; Palumbo et al. 1989; Knochel and Jeppesen 1990; Kirov et al. 1993;

Szabo et al. 2000; Villari et al. 2000; Castro-Escarpuli et al. 2003). This may explain

the presence of Aeromonas in the stools of healthy humans since this represents

transient colonization probably due to consumption of contaminated foods or drinking

water.

The concentration of aeromonads varies depending on the food analysed and the

location (Table 1.4). The incidence can vary from no aeromonads found in vegetables in

Sweden (Krovacek et al. 1992) to large concentrations detected in raw food samples in

Switzerland (Gobat and Jemmi 1993). Although food industries supplied with

inadequately treated water may allow the spread of highly toxic strains and cause

diarrhoeal illness (Abbott et al. 1992), contamination of food samples does not always

originate from water (Hänninen and Siitonen 1995). A significant higher incidence of

pathogenic aeromonads has been detected in raw food than in processed and ready-to-

eat food samples (Kingome et al. 2004). Furthermore, the ability of aeromonads to grow

in refrigerated grocery store produce, milk and meat implicates these bacteria as

potential food pathogens, and these products may represent an important vehile of

transmission (Kirov et al. 1993). Aeromonads harbouring virulence factors have been

isolated world-wide from a variety of foods (Martins et al. 2002; Awan et al. 2006;

Rodríguez-Calleja et al. 2006; Yucel and Erdogan 2010).

1.6.2.1. Distribution of Aeromonas spp. in foods

Overall, the most frequently isolated species from foods world-wide is A. hydrophila.

This species has been recovered from fish, seafood, raw milk, poultry and red meats

Nishikawa and Kishi 1988; Palumbo et al. 1989; Knochel and Jeppesen 1990; Hudson

and De Lacy 1991; Gobat and Jemmi 1993; Tsai and Chen 1996; Kingome et al. 2004;

Rodríguez-Calleja et al. 2006; Yucel and Erdogan 2010). The frequent isolation of A.

hydrophila from oysters suggests that oysters may offer a better environment for growth

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-29-

Tab

le 1

.4

Enum

erat

ion

of A

erom

onas

spp.

in d

iffer

ent f

oods

tuff

s

Sour

ce

Inci

denc

e

Loc

atio

n R

efer

ence

Ret

ail

groc

ery

stor

e pr

oduc

e1 1

x 10

2 to

2.3

x 1

04 /g

USA

C

allis

ter a

nd A

gger

(198

7)

Var

ious

pro

duct

s2 10

2 to

104 /g

N

ew Z

elan

d H

udso

n an

d D

e La

cy (1

991)

Veg

etab

les a

nd ra

w m

ilk

Aero

mon

as n

o is

olat

ed

Swed

en

Kro

vace

k et

al.

(199

2)

Raw

food

sam

ples

6

x 10

6 C

FU/g

Sw

itzer

land

G

obat

and

Jem

mi (

1993

)

Lettu

ce

105

to 1

07 C

FU/g

A

ustra

lia

Szab

o et

al.

(200

0)

Var

ious

pro

duct

s3 10

4 to

105

CFU

/g

Italy

V

illar

i et a

l. (2

000)

Org

anic

veg

etab

les

Not

est

imat

ed

Nor

ther

n Ir

elan

d M

cMah

on a

nd W

ilson

(200

1)

Seaf

ood

104 b

acte

ria/g

G

erm

any

Ullm

an e

t al.

(200

5)

1 Initi

al c

once

ntra

tion

of a

erom

onad

s es

timat

ed a

t the

tim

e of

pur

chas

e. G

row

th in

crea

sed

10 to

100

0 fo

ld a

fter

14 d

ays

incu

batio

n at

5C

. 2 In

clud

ed r

eady

to

eat

mea

t, po

ultry

, sh

ellfi

sh,

fish,

mea

ts,

sala

ds.

Enum

erat

ion

of a

erom

onad

s w

as d

eter

min

ed b

y di

rect

pla

ting

out.

3 Prod

ucts

incl

uded

veg

etab

les,

chee

ses,

mea

ts a

nd ic

e cr

eam

s.

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-30-

than other seafood (Tsai and Chen 1996). In Spain, A. hydrophila has been found as the

predominant species in rabbit meat in addition to Y. enterocolitica, Listeria spp. and S.

aureus (Rodríguez-Calleja et al. 2006). Potentially pathogenic species such as A. sobria,

A. trota and A. veronii bv. veronii, occasionally associated with gastroenteritis, have

been isolated from a variety of foods (Nishikawa and Kishi 1988; Granum et al 1998;

Merino et al. 1995; Janda and Abbott 1998). In Northern Ireland, A. schubertii (21%)

was the most common aeromonad isolated from organic vegetables (McMahon and

Wilson 2001) while an investigation of frozen fish samples in Mexico reported that A.

salmonicida (67.5%) and A. bestiarum (20.9%) accounted for the majority (88.3%) of

the isolates (Castro-Escarpulli et al. 2003). There is evidence that species such as A.

trota are able to grow in 0.68M NaCl, the concentration used as food preservative

(Delamare et al. 2000).

1.7. EPIDEMIOLOGY AND PUBLIC HEALTH ISSUES

Unlike other recognized pathogens such as N. meningitidis, N. gonorrhoeae, and S.

Typhi, Aeromonas is not a reportable organism. In the United Kingdom (UK)

Aeromonas bacteraemia is a voluntarily reportable condition while in the state of

California, USA, the practice of reporting infections with aeromonads was discontinued

(Janda and Abbott 2010). The incidence of aeromonads in healthy humans has been

estimated to vary between 1 and 3.5% compared to 10.8% in faeces from diarrhoeic

patients (von Graevenitz and Mench 1968; Goodwin et al. 1983; Edberg et al. 2007;

Rahman et al. 2007a; Suarez et al. 2008). The combined incidence of Aeromonas

septicaemia in the USA and the UK has been estimated to be 1.5 per million (Janda and

Abbott 2010). Colonization of humans with aeromonads begins very early in life. A

survey of 52 cesarean-borne Peruvian children showed that 23% of the infants

harboured Aeromonas during the first week of life without developing clinical

symptons. Colonization of the infants was attributed to the hospital water (Pazzaglia et

al. 1990a).

Although Aeromonas are present in most foods and aquatic environments, the global

incidence of infections caused by these microbes is unknown (Hӓnninen and Siitonen

1995). Asymptomatic human carriers could serve as vectors for the organism, in

particular, individuals working as food handlers (Abeyta and Wekell 1988). The

presence of virulence factors in water isolates of A. hydrophila (Bondi et al. 2000)

reinforces the notion that from the public health perspective, the isolation of

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-31-

aeromonads from water and foods is associated with intestinal and extraintestinal

infections. Episodes of diarrhoea in children and adults after consumption of

contaminated food and drinking water have been described (Freij 1984; Lehane &

Rawlin 2000). This is particularly important in developing countries such as India

where river water contaminated with aeromonads species is used for drinking and

recreational activities (Sharma et al. 2005). Indeed, accidents in water-related

recreational activities have resulted in serious infections with these organisms (Bossi-

Küpfer et al. 2007). Less commonly, infections with aeromonads due to animal bites

have been reported. These infections have been attributed to the disruption of the natural

environment of animals due to expansion of urban areas into rural regions (Angel et al.

2002; Kunimoto et al. 2004).

1.7.1. Water-associated infections

The presence of multiple virulence factors in Aeromonas isolated from water including

chlorinated water represents a serious public health concern (Alavandi et al. 1999;

Figueras et al. 2005; Snowden et al. 2006; Rahman et al. 2007a; Bhowmik et al. 2009).

The proportion of aeromonads strains carrying putative virulence factors varies from 36

to 71% (Seidler et al. 1980; Kaper et al. 1981; Kühn et al. 1997b). In humans,

gastrointestinal and soft tissue infections are the result of exposure to or ingestion of

contaminated water supplies (von Graevenitz and Mench 1968; Washington 1972;

Joseph et al. 1979; Seidler et al. 1980). Experiments on mice have shown that the

ability to cause damage by Aeromonas isolated from clinical and water sources is

comparable to toxigenic V. cholerae (Bhowmik et al. 2009).

Raw waters prepared for human consumption from sewage-polluted surface waters

loaded with pathogenic Aeromonas represent a potentially greater health risk to the

human population than the use of underground water (Schubert 1991a). Polluted waters

represent a health hazard to the human population in general but to the military,

commercial divers and people involved in aquatic sports in particular (Berg et al. 2011).

Several Aeromonas species possessing a variety of cytotoxins and other virulence

factors have been isolated from drinking, river, sea and fresh water (Ashbolt et al. 1995;

Ivanova et al. 2001; Balaji et al. 2004; Sharma et al. 2005; Khan et al. 2008; Berg et al.

2011). Evidence for the water-borne origin of infections caused by Aeromonas in

humans derived from several studies (Picard and Goullet 1987; Khajanchi et al. 2010;

Pablos et al. 2010; Lye 2011). In drinking and mineral water, Aeromonas can persist for

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-32-

a long time due to biofilm formation (Dorsh et al. 1994; Kühn et al. 1997b; Chauret et

al. 2001; Villari et al. 2003). Thus, Aeromonas counts have been proposed as an

additional indicator of water quality in the United States (US) and other countries

(Villari et al. 2003). In addition, the US Environmental Protection Agency’s

Contaminant Candidate List has included A. hydrophila as an emerging pathogen in

drinking water (Borchardt et al. 2003). Surprinsingly, a 2011 study recommended that

Aeromonas should not be included in further editions of the CCL concluding that these

microbes do not represent a significant public health hazard (Egorov et al. 2011).

1.7.2. Food-associated infections

Like water, contaminated fish, meats and poultry also represent a health hazard to

humans as these products are an integral food source of the human diet (Kirov 1993;

Hӓnninen and Siitonen 1995; Rahman et al. 2007a). However, despite that most foods

can be contaminated with aeromonads as described in section 1.5.2, only a few reports

have implicated aeromonads as the cause of food-poisoning outbreaks (Abeyta et al.

1986; Todd et al. 1989; Kirov 1997). The most compelling evidence to date derived

from the consumption of ready-to-eat shrimp cocktail by a 38 year-old man who

developed gastroenteritis. Ribotyping patterns revealed that the patient’s stools and the

shrimp contained identical Aeromonas spp. (Altwegg et al. 1991a). In Sweden, 24

people developed food-poisoning symptoms including severe acute diarrhoea,

abdominal pain, headache, fever, and vomiting after consuming food contaminated with

a highly virulent A. hydrophila strain (Krovacek et al. 1995). Consumers are regularly

exposed to toxin-producing strains without registering signs of malaise although

theoretically, food-poisoning could result from colonization and less likely, by

intoxication due to the elaboration of preformed toxins (Knochel and Jeppesen 1990;

Kirov 1993; Villari et al. 2000). In humans, food poisoning due to consumption of

contaminated fish can lead to septicaemia (Ketover et al. 1973) or contact with fish can

cause serious infections particularly in immunocompromised individuals (Lehane and

Rawlin 2000). Thus, recommendations designed to control and limit the growth of these

and other potentially pathogenic bacteria have been proposed (Szabo et al. 2000).

1.7.3. Aeromonas and fish infections

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Aeromonas species are part of the normal microbial flora of fishes and other aquatic

animals and plants (Simidu et al. 1971; Trust and Sparrow 1974). Among freshwater

fish, Aeromonas and Vibrio species predominate (Trust and Sparrow 1974).

Aeromonas-related disease in fish is of high economic significance and has become a

major problem to fish aquaculture (Austin and Austin 1987; Nash et al. 2006; In-

Young-and Joh 2007; Zmyslowska et al. 2009; Pridgeon et al. 2011). In order to control

aeromonasis, aquaculture farmers rely primarily on the use of antimicrobials. However,

this practice is expensive and a potential risk to the environment and human health

(Harikhrishnan et al. 2010a). Recent studies have proposed the bacteriolytic properties

of the predator bacterium Bdellovibrio and the antibacterial properties of the

extracellular products of Bacillus amyloliquefaciens for the control of pathogenic A.

hydrophila (Cao et al. 2011, Cao et al. 2012). The latter is considered a promising

probiotic for the biocontrol of A. hydrophila infections in the eel A. anguilla (Cao et al.

2011) while Bdellovibrio strain F16 significantly reduced the cell density of A.

hydrophila exhibiting 100% lysis activity against this pathogen (Cao et al. 2012). A

recent method that increases the effectiveness of solar disinfection via a thin-film fixed

bed reactor has been developed for the solar photocatalytic inactivation of A. hydrophila

(Khan et al. 2012).

The species most often associated with fish infections are A. salmonicida, A. hydrophila

and A. veronii although re-identification of a group of aeromonads isolated from

diseased fish revealed that other species including A. sobria, A. salmonicida, A.

bestiarum, A. hydrophila, A. piscicola and a strain of A. tecta prevailed (Beaz-Hidalgo

et al. 2009). Furunculosis in fish is typically caused by A. salmonicida while A.

hydrophila, the primary aetiological agent in red-sore disease (Hazen 1979), can also

cause furunculosis and septicaemia in various fish species leading to severe losses in

farm production (Wakabayashi et al. 1981; Nash et al. 2006). Usually, infection by

these bacteria is manifested as an acute form involving septicaemia and episodes of

haemorrhage at the bases of the fins, loss of appetite and melanosis. Subacute to chronic

forms of the disease are usually observed in older fish accompanied by lethargy, slight

exophthalmia and haemorrhaging muscle and internal organs (Joseph and Carnahan

1994; Austin and Adams 1996).

1.8. BIOREMEDIAL AND BIODEGRADABLE PROPERTIES

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-34-

Aeromonas may play significant environmental roles or express unsual properties

including the detoxification or removal of environmental toxins in groundwaters,

industrial affluents and contaminated soils; maintaining the balance of carbon and

nitrogen elements in the aquatic biosphere by virtue of their chitinolytic activity; the

ability of some strains to generate electricity; the degradation of polypectate by A.

salmonicida ssp. pectinolytica; the removal of pesticides (Pavan et al. 2000; Pham et al.

2003; López et al. 2005; Lan et al. 2008).

Bioremedial properties associated with Aeromonas include the removal of selenite from

contaminated groundwaters (Hunter and Kuykendall 2006); assimilation of seleniferous

compounds present in agricultural drainage (Rael and Frankenberger 1996); reduction

of arsenate to arsenite (Anderson and Cook 2004); production of a biosurfactant (Ilori et

al. 2005) and the ability to decolorize triarylmethane dyes (Ogugbue and Sawidis 2011).

These properties can have a significant impact on the environment, as triarylmethane

dyes can exert toxic effects in plants and their disposal on land, may have a direct

impact on soil fertility and possibly agricultural productivity (Ogugbue and Sawidis

2011). Surfactants have important bioremedial properties with environmental and

biotechnological applications that can be applied in the food and pharmaceutical

industries (Ilori et al. 2005). On the negative side, the ability of Aeromonas to reduce

sulphite to H2S, ferric to ferrous iron and oxidise cathodic hydrogen are properties

strongly associated with microbial influenced corrosion, one of the most destructive

modes of metal corrosion (McLeod et al. 1998).

1.9. VIRULENCE FACTORS

Assessing virulence in Aeromonas has been difficult due to the variety of hosts that

different species appear to infect and differences in growth requirements (Froquet et al.

2007). Virulence in Aeromonas has been investigated primarily through animal lethality

studies (Daily et al. 1981; Wong et al. 1996), using immunocompromised or septic

mice (Lye et al. 2007; Khajanchi et al. 2011) and healthy animals (Janda et al. 1985).

Other models proposed include the tropical fish blue gourami (Fock et al. 2001) and

zebrafish (Rodríguez et al. 2008); the free-living protozoan Tetrahymena (Pang et al.

2012) and the unicellular amoebae Dictyostelium (Froquet et al. 2007). The worm

Caenorhabditis elegans has also served has a model after infection with A. hydrophila

and the production of toxic symptoms (Couillault and Eubank 2002). A mouse model

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-35-

was also developed to determine the gastrointestinal colonization rate among

environmental Aeromonas isolates (Lye 2009).

However, the medicinal leech model of Graf (2000) has been recognized as a promising

model to assess virulence in Aeromonas (Janda and Abbott 2010). Several genes

involved in a multitude of activities have been identified in Aeromonas residing in the

leech digestive tract (Silver et al. 2007a). Moreover, the potential for discovering other

genes and their products makes the medicinal leech an exciting model to determine the

virulence of Aeromonas strains. A recent study used comparative genomic and

functional analyses of virulence genes to assess virulence of two A. hydrophila strains

isolated from a human wound (Grim et al. 2013). This is probably the most promising

method to date, it can be easily reproduced and a library of well-characterized

Aeromonas pathotypes can be created.

1.9.1. Adherence

The attachment of bacteria to host cells allows a close interaction with tissue and body

fluids and for maximal effect of any toxins that aeromonads may produce (Atkinson and

Trust 1980, Atkinson et al. 1987). Adhesion may be mediated by pili, flagella,

filamentous networks and possibly the lipopolysaccharide (LPS) O-antigen. Non-

filamentous adhesins in the form of a polysaccharide capsule or outer membrane

proteins may also be involved (Atkinson and Trust 1980; Carrello et al. 1988; Hokama

and Iwanaga 1991; Merino et al. 1996a; Gryllos et al. 2001; Zhang et al. 2003; Fang et

al. 2004). Adhesion to cell lines has been used as a model for intestinal infection which

has been correlated with enteropathogenicity (Kirov et al. 1995a). The ability of

aeromonads to adhere to cell lines may depend significantly on the temperature, source

of isolation, species, and the type of cell line (Neves et al. 1994; Kirov et al. 1995a;

Snowden et al. 2006). Further, probiotic bacteria inhibit the ability of Aeromonas to

adhere to human epithelium and traslocate due to competition for adhesion sites (Hatje

et al. 2011).

Several mechanisms that recognise different binding sites on erythrocytes, buccal

epithelium and other cells have been described in A. hydrophila (Atkinson and Trust

1980; Ascensio et al. 1991). Studies have shown that A. hydrophila can bind to sialic

acid-rich glycoproteins, lactoferrin, collagen and laminin via a lectin-like mechanism

(Ascensio et al. 1991) while the interaction of A. caviae, A. hydrophila and A. sobria

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with mucins has also been investigated (Ascension et al. 1998). In A. caviae, the

ability to attach to inert surfaces such as glass has been associated with hyperpiliation of

the cells through the presence of type IV pili (Béchet and Blondeau 2003).

1.9.2. Pili

Pili are cell associated structures often involved in adhesion and some of which act as

haemagglutinins (Carrello et al. 1988; Hokama and Iwanaga 1991; Kirov et al. 1995b).

Type IV pili have been purified from A. veronii bv. sobria (Carrello et al. 1988;

Hokama and Iwanaga 1991, 1992; Iwagana and Hokama 1992; Kirov and Sanderson

1996), A. hydrophila (Atkinson and Trust 1980; Carrello et al. 1988; Hokama et al.

1990; Honma and Nakasone 1990; Ho et al. 1990), A. caviae (Carrello et al. 1988;

Kirov et al. 1998) and A. trota (Nakasone et al. 1996). Morphologically, pili appear as a

thin, long flexible structure, usually present in small numbers (type-L) and a more

numerous, shorter, thicker and straight pilus (S-pili). Occasionally, Aeromonas pili can

form rope-like bundles also known as bundle-forming pili (Bfp) that are usually present

in 5 to 10% of cells (Kirov and Sanderson 1996). The molecular masses of the subunit

proteins range from 4 to 23 kDa and despite similar morphology pili from different

strains can be biochemically and immunologically unrelated (Ho et al. 1990; Hokama

and Iwanaga 1991; Iwanaga and Hokama 1992; Kirov and Sanderson 1996) (Table 1.5).

Expression of pili depends on the culture medium and temperature of incubation.

Growth in liquid medium favours the production of both pilus types particularly at

lower (22ºC) temperatures (Carrello et al. 1988; Hokama and Iwanaga 1991; Kirov et

al. 1995b; Kirov and Sanderson 1996). The S-type pili can be expressed under different

conditions of growth although in purified form the haemagglutinating function may be

lost (Ho et al. 1990). Genes involved in pilus biogenesis have been characterized in A.

hydrophila and A veronii bv. sobria. There are at least two distinct families of type IV

pilus, Tap and Bfp (Barnett et al. 1997). In A. hydrophila, a type IV pilin subunit is

encoded by the tapA gene, one of four genes comprising the tap cluster (Pepe et al.

1996). The remaining genes tapB and tapC also have a role in pilus biogenesis while the

tapD gene has been associated with the production of a type IV leader peptidase/N-

methyltransferase involved in extracellular secretion of aerolysin and protease. The

proteins encoded by these four genes are closely related to the products of the pilABCD

gene cluster described in P. aeruginosa (Pepe et al. 1996). The tap cluster is also

expressed by A. veronii bv. sobria and other Aeromonas species although differences in

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-37-

Tab

le 1

.5

Cha

ract

eris

tics o

f pili

des

crib

ed in

Aer

omon

as sp

ecie

s

Spec

ies/

stra

in

Pilu

s typ

e A

gglu

tinat

ion

of

eryt

hroc

ytes

A

dher

e to

: M

W

(kD

a)

Dia

met

er

(nm

)

Sour

ce

Ref

eren

ce

A. h

ydro

phila

A6

Not

des

crib

ed

Hum

an G

r O

Buc

cal

epith

eliu

m

Faec

al

Atk

inso

n &

Tru

st

(198

0)

A. h

ydro

phila

/cav

iae/

sobr

ia

L-pi

li (f

lexi

ble)

H

uman

Gr O

H

Ep-2

cel

ls

2.5

Faec

al,

wat

er

Car

rello

et a

l. (1

988)

A. h

ydro

phila

/cav

iae/

sobr

ia

S-pi

li (s

traig

ht)

Hum

an G

r O

Hep

-2 c

ells

5

Faec

al,

wat

er

Car

rello

et a

l. (1

988)

A. h

ydro

phila

Ae6

W

W-p

ili

(fle

xibl

e)

Hum

an, r

abbi

t H

uman

/rabb

it in

test

ine

21.0

7

Faec

al

Hok

ama

et a

l. (1

990)

A. h

ydro

phila

Ae6

R

-pili

(s

traig

ht)

No

aggl

utin

atio

n hu

man

/GP

Fa

iled

to

adhe

re

18.0

9

Faec

al

Hon

ma

& N

akas

one

(199

0)

A. h

ydro

phila

AH

26

Stra

ight

N

o ag

glut

inat

ion

hum

an/G

P

17

.0

7 to

9

Faec

al

Ho

et a

l. (1

990)

A. so

bria

Ae1

Fl

exib

le

Hum

an/G

P/ov

ine/

bovi

ne/a

vian

4.

0 7

to 9

Fa

ecal

H

o et

al.

(199

0)

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-38-

Tab

le 1

.5

Con

tinue

d.

Spec

ies/

Stra

in

Pilu

s typ

e A

gglu

tinat

ion

of

eryt

hroc

ytes

A

dher

e to

: M

W

(kD

a)

Dia

met

er

(nm

)5 So

urce

R

efer

ence

A. so

bria

Ae1

Fl

exib

le

Hum

an G

r A

/rabb

it H

uman

/rabb

it in

test

ine

23.0

7

Faec

al

Hok

ama

& Iw

anag

a (1

991)

A.

sobr

ia T

AP1

3 Fl

exib

le

No

aggl

utin

atio

n hu

man

/rabb

it/sh

eep

Rab

bit i

ntes

tine

23.0

7

Faec

al

Iwan

aga

& H

okam

a (1

992)

A.

sobr

ia A

e24

Flex

ible

/ w

avy

Hum

an/ra

bbit

Rab

bit i

ntes

tine

19.0

7

Faec

al

Hok

ama

& Iw

anag

a (1

992)

A.

ver

onii

bv. s

obria

Fl

exib

le/

bund

les

No

aggl

utin

atio

n hu

man

Gr O

21

.0

Blo

ody

stoo

ls

Kiro

v an

d Sa

nder

son

(199

6)

A. tr

ota

Flex

ible

N

o ag

glut

inat

ion

hum

an/ra

bbit

R

abbi

t int

estin

e 20

.0

7 Su

rfac

e w

ater

N

akas

one

et a

l. (1

996)

A. c

avia

e

Flex

ible

/ bu

ndle

s

H

Ep-2

23

.0

Faec

al

Kiro

v et

al.

(199

8)

GP,

Gui

nea

pig;

, n

ot d

eter

min

ed; G

r, gr

oup

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-39-

the predicted N-terminal amino acid sequence between the cloned TapA pilins and

purified Bfp pilin have been observed (Barnett et al. 1997; Kirov et al. 1998, 2000).

Recently, a 22-kb locus comprising 17 pilus-related genes similar to the mannose-

sensitive hemagglutinin of Vibrio cholerae and responsible for encoding the bundle-

forming pilus was characterized in A. veronii bv. sobria (Hadi et al. 2012).

Bfp plays an important role in the pathogenesis of gastrointestinal infection caused by

Aeromonas by promoting colonization and forming bacterium-to-bacterium linkages

(Kirov et al. 1999). The removal of Bfp can decrease adhesion by up to 80% (Kirov et

al. 1999) while mutation of the major Bfp pilin gene mshA greatly reduces the

bacterium's ability to adhere and form biofilms (Hadi et al. 2012). By contrast,

mutagenesis experiments showed that inactivation of tapA had no effect on bacterial

adherence to Hep-2, Henle 407 and human intestinal cells suggesting that the Tap pili

may not be as significant as Bfp pili for Aeromonas intestinal colonization (Kirov et al.

2000).

1.9.3. Invasins

Studies on invasins are sparse in Aeromonas considering that invasion is a recognized

virulence factor (Chu and Lu 2005b). Although not as invasive as some E. coli strains

which have invasion in vitro levels 200 times greater than most Aeromonas, the ability

of Aeromonas to penetrate and replicate may have significant clinical implications as

dysenteric symptoms have been associated with invasive species (Lawson et al. 1985;

Watson et al. 1985; Gray et al. 1990; Nishikawa et al. 1994). Several studies have

shown that A. hydrophila, A. caviae and A. sobria strains isolated from human and non-

human sources were able to invade HEp-2 and Caco-2 cells (Lawson et al. 1985;

Watson et al. 1985; Nishikawa et al. 1994; Shaw et al. 1995). On the other hand, strains

of A. hydrophila and A. sobria isolated from fish and hare showed greater ability to

invade HEp-2 cells compared to environmental aeromonads (Krovacek et al. 1991).

The mechanism of invasion involves components of both bacterium and host cells

including bacterial outer membrane proteins, cell membrane receptors, signal

transductions and cytoskeletal rearrangement (Chu and Lu 2005b). Extracellular

products may play a very minor role in the morphological changes that occur during the

invasion process (Leung et al. 1996). Brush border and microvilli disruption have been

associated with adhesion and invasion of Caco-2 cells but no actin accumulation that is

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associated with the attaching and effacing process in enteropathogenic E. coli

(Nishikawa et al. 1994).

The invasive ability of A. hydrophila has been investigated using different fish cell lines

and virulent and avirulent isolates. Only virulent strains were able to multiply and cause

cytopathic changes within the affected cells (Leung et al. 1996; Low et al. 1998). Low

et al. (1998) showed that cytopathic changes occurred concomitantly with

rearrangements of microfilaments (MFs) in a process involving three stages of

infection. In stage I, cells detach and elongate; in stage II, cells connect to neighbouring

cells by tubular cytoplasmic extensions resulting in less confluent monolayers with a

satellite-like organization; in stage III, bacteria are abundantly present in cells and

vacuoles resulting in eventual detachment and lysis. Moreover, the F-actin

rearrangement process involves the formation of an actin cloud immediately after the

bacterium becomes in contact with the cell (first phase) followed by reorganization

(depolymerisation) of actin fibres (second phase).

Chu and Lu (2005b) showed that polymerization of MFs was inhibited by cytochalasin

in a dose dependent manner, resulting in inhibition of invasion by A. hydrophila Ahj-1

into epithelioma papillosum cells of carp (EPC). By contrast, pretreatment of EPC cells

with colchicines and nocodazole, inhibitors of microtubule (MT) formation, had no

effect on the process of invasion. Thus, MFs but not MTs are required for the

internalization of A. hydrophila into EPC cells (Low et al. 1998; Chu and Lu 2005b).

These results indicate that actin polymerization is involved in the invasion process of

Aeromonas. Invasion by Aeromonas can lead to mucosal damage similar to that

produced by Shigella species as shown in a rabbit model although some strains may still

invade without causing extensive destruction. This divergence has been attributed to the

different routes of entry employed by bacterial cells such as Peyer’s patches of the

ileum, lymphatics and passage through the mucosa via other mechanisms (Pazzaglia et

al. 1990b). Gavin et al. (2003) showed that the introduction of lafA into lafA mutants

enhanced invasion of HEp-2 cells and biofilm formation in vitro.

1.9.4. S-layer In Aeromonas, the S-layer (originally called the A-layer), is considered a primary

virulence factor due to its extraordinary binding capabilities (Kay et al. 1981; Trust et

al. 1983; Chu et al. 1991). S-layer has been described in A. salmonicida, A. hydrophila

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and A. sobria. Isolates carrying the O:11 somatic antigen and a S-layer have been

implicated mostly in invasive rather than localized infections in humans (Janda et al.

1987b; Paula et al. 1988; Merino et al. 1995; Kirov 1997). S-layers conferred protection

to the bacterium from the serum killing activity of the host and from proteases by acting

as a physical barrier to the lytic complement components (Munn et al. 1982) and by

facilitating the entry into macrophages (Trust et al. 1983).

1.9.4.1. Structural arrangements

S-layers are regular, two-dimensional assemblies of protein monomers that often

constitute the outermost layer of the cell envelope of many bacteria (Sletyr and Messner

1983). The spatial arrangements of the S-layer in Aeromonas vary from hexagonal,

tetragonal to linear oblique arrays with a lattice constant of 12.0-12.5 nm (Dooley et al.

1989). The S-layer of A. salmonicida consists of regular, two-dimensional protein

monomers with MWs between 49.0 and 52.0 kDa (Belland and Trust 1987; Chu et al.

1991). The S-layer contributes to the physical properties of the A. salmonicida cell

envelope. Loss of the S-layer can lead to changes in the physical properties allowing the

organism to grow at higher than usual temperature (Ishiguro et al. 1981).

1.9.4.2. Binding properties

S-layer can bind to host basement membranes molecules such as fibronectin, laminin

and collagen-IV (Kay and Trust 1991). The S-layer of A. salmonicida can specifically

bind to porphyrins, other heme analogues (Kay et al. 1985), and immunoglobulins

(Phipps and Kay 1988) allowing the organism to survive in vivo by avoiding

phagocytosis (Kay et al. 1985; Dooley and Trust 1988).

1.9.4.3. Genes involved in S-layer synthesis

At the genetic level, little or no homology exists between the S-layer gene of A.

salmonicida (vapA) and that of A. hydrophila (ashA) (Belland and Trust 1987).

Although the gene is always present in A. salmonicida, the failure of a strain to produce

S-layer is probably due to either deletion or rearrangement of the entire gene or parts of

it or alterations in the expression of vapA (Gustafson et al. 1992). Secretion of the AhsA

protein in A. hydrophila (Thomas and Trust 1995b) is mediated by the spsD gene while

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a protein encoded by the apsE gene of A. salmonicida may provide the necessary energy

to the secretory apparatus (Noonan and Trust 1995). Loss of expression of the S-layer

due to growth at 30°C results in genetic rearrangement in which N-terminal sequences

of the A protein are lost by gene deletion (Belland and Trust 1987).

1.9.4.4. S-layer and virulence

Because of the different types of diseases caused by A. salmonicida and A. hydrophila

each organism may use its S-layer in a different manner despite morphological

similarity (Dooley and Trust 1988; Murray et al. 1988). In A. hydrophila the S-layer

may not be the principal virulence factor in fish as it is in A. salmonicida (Thomas et al.

1997). In contrast, mutagenesis experiments have shown that in A. salmonicida S-layer

deficient mutants virulence can decrease up to >105 fold when the organism is incubated

at higher than normal temperatures (Ishiguro et al. 1981) or that binding of the S-layer

to IgG can only take effect when the A-protein is intact (Phipps and Kay 1988).

1.9.5. The lipopolysaccharide (LPS)

The endotoxin component of LPS produced by Aeromonas is similar to that of other

Gram-negative bacteria. A combination of hexose and heptose monosaccharide residues

constitutes the core region of the LPS in motile aeromonads (Shaw and Hodder 1978).

1.9.5.1. Functions of the LPS

The LPS confers protection to the bacterium against the bactericidal effects of normal

serum. The loss of the O-antigenic polysaccharide chains allows access of complement

components to their target producing bactericidal effects. The LPS also acts as an

adhesin in human epithelial and HEp-2 cells (Gryllos et al. 2001; Vilches et al. 2007),

particularly in strains from serogroup O:34 (Merino et al. 1996a). The ability of A.

sobria to adhere to HEp-2 cells was found to correlate with the level of LPS expression

and growth phase (Paula et al. 1988; Francki and Chang 1994). Other functions include

a role in the assembly and maintenance of the S-layer of A. salmonicida and excretion

of exotoxins. Strains lacking the O-antigen (rough strains) excrete less toxin than those

strains with abundant O-antigen LPS (smooth strains) (Chart et al. 1984).

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Temperature plays a significant role in the production of smooth and rough LPS.

Smooth LPS is produced when strains are grown at 20C which correlates with the

ability of O:34 to colonize the germfree chicken gut at this temperature but not at 37C

(Merino et al. 1992; 1996a/b). In addition, mutagenesis experiments revealed that

smooth strains are more virulent when grown at lower temperatures (Gryllos et al.

2001). The type of LPS produced is also influenced by salt concentration. At high

osmolarity, smooth LPS is produced despite incubation at 37C. By contrast, cells

cultivated at low osmolarity produced rough LPS (Aguilar et al. 1997). Aguilar et al.

(1997) showed that cells grown at high osmolarity were more virulent for fish and mice,

had increased extracellular activities, enhanced adhesion to HEp-2 cells and were

resistant to the bactericidal activity of non-immune serum.

1.9.5.2. Immunological and antigenic properties of LPS

In A. salmonicida strains, the O-polysaccharide chains are very homogeneous with

respect to cell length, strongly immunogenic and antigenically cross-reactive (Chart et

al. 1984). The O-polysaccharide chains can traverse the surface protein array of virulent

strains of A. salmonicida becoming exposed on the cell surface. These properties and

the considerable antigenic conservation of A. salmonicida have been proposed as a

potential target in the design of an effective vaccine (Chart et al. 1984). Antiserum

raised against A. hydrophila LPS decreased the mortality of suckling mice from 100 to

30% (Wong et al. 1996). LPS with similar properties to those described by Chart et al.

(1984) for A. salmonicida were reported in two A. hydrophila strains, although A.

hydrophila can produce a LPS with O-polysaccharide chains of heterogeneous as well

as homogeneous lengths (Dooley et al. 1985).

1.9.5.3. Genes involved in LPS synthesis

The gaIU gene encodes GaIU, a UDP-glucose pyrophosphorylase responsible for the

synthesis of UDP-glucose from glucose-1-phosphate (Vilches et al. 2007). The gaIU

gene is distributed in all mesophilic aeromonads. Mutations of the gene may affect the

survival of Aeromonas in serum, decrease adhesion ability and reduce virulence of O:34

strains as shown by a septicaemic model with fish and mice (Vilches et al. 2007).

Mutations in the galU gene of A. hydrophila AH-3 (O:34) result in the production of

two bands compared to one in the wild type which corresponds to two types of LPS

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(Vilches et al. 2007). The flm cluster (consisting of five different genes flmA, flmB,

flmD, neuA and nueB) of A. caviae Sch3N is involved in LPS O-antigen biosynthesis

and possibly in flagellum assembly (Gryllos et al. 2001).

1.9.6. Outer membrane proteins (OMP)

Outer membrane proteins have been associated with the transport of ions and molecules

across the outer membrane, cell architecture allowing the passage of toxins (Howard

and Buckley 1985), and ability to act as an adhesin (Atkinson and Trust 1980).

Haemagglutination has been shown to correlate with the presence of a 43 kDa OMP

(Atkinson and Trust 1980) while the carbohydrate-reactive-OMP (CROMP) of A.

hydrophila A6 may act as an adhesin able to attach to erythrocytes or intestinal

epithelium via a fucose site (Quinn et al. 1993). The outer surface of A. hydrophila is

carbohydrate-reactive and the ability to adhere to human red cells and human colonic

cancer cells depends on ligands expressed on its external surfaces (Quinn et al. 1994).

However, these carbohydrate-reactive proteins may not be uniformly distributed among

all Aeromonas species (Küijper et al. 1989a).

The gene encoding for the 43 kDa OMP of A. hydrophila has been cloned and

expressed in E. coli resulting in a recombinant adhesin with the ability to confer up to

87.5% protection in blue gourami against homologous A. hydrophila challenge (Fang et

al. 2004). Results suggested that the 43 kDa OMP is a conserved protein found in A.

hydrophila and A. sobria and may share similar antigenic characteristics with V.

anguillarum and E. tarda (Fang et al. 2004). Jeanteur et al. (1992) showed that A.

hydrophila Ah65 shares similar N-terminal sequences and channel-forming properties

with other Gram-negative species particularly E. coli. Further, different porin types have

been described in various A. hydrophila strains including protein VI, which shares the

same molecular mass and almost identical amino terminus with the OmpW of V.

cholerae (Jeanteur et al. 1992; Quinn et al. 1994).

A vaccine based on the antigenic properties of OMPs was developed to control A.

hydrophila in fish. The survival of the vaccinated fish improved 50% compared to

unvaccinated controls (Thangaviji et al. 2012). Another vaccine candidate, based on the

recombinant A. hydrophila OMP48, increased the survival of fish immunized when

challenged with virulent A. hydrophila and Edwarsiella tarda. The gene coding OMP48

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had high similarity to LamB porin genes of A. hydrophila, A. salmonicida and V.

parahaemolyticus (Khushiramani et al. 2012).

1.9.7. Flagella

Flagella are complex bacterial organelles associated with multiple roles in bacteria-host

interactions. Two distinct flagellar systems are expressed in Aeromonas, a polar

flagellum (Fla) for swimming in liquid media and multiple lateral flagella (Laf) for

swarming on solid surfaces or viscous conditions (Rabaan et al. 2001; Gavin et al.

2002, 2003; Kirov et al. 2004). Basically, flagella are helical propellers that consist of a

filament made up of polymerized protein subunits, attached by a hook structure to the

basal body (Macnab and DeRossier 1988).

1.9.7.1. Synthesis, regulation and expression of flagella

Synthesis of flagella represents a high metabolic cost for the bacterium in terms of

resources and energy. Expression of both flagella is highly regulated by environmental

factors and other regulators (Kirov 2003; Merino et al. 2006). Lateral flagella are

usually present on 50 to 60% of the bacterial cells when the bacterium is grown in high

viscosity medium but are absent in liquid medium (Kirov et al. 2002; Wilhems et al.

2011). Synthesis of lateral flagella is under the control of the polar flagellar system

although mutations in the polar fla genes do not prevent expression of the lateral

flagella (Gavin et al. 2002; Santos et al. 2010).

Regulation of flagellum biogenesis involves a combination of transcriptional,

translational, and post-translational regulation (Aldridge and Hughes 2002; Soutourina

and Bertin 2003). These genes have been divided in three categories: early genes

encoding regulatory proteins, middle genes encoding structural units and the late genes

involved in the chemo-sensor machinery (Aldridge and Hughes 2002). The polar and

lateral flagellar systems of A. hydrophila AH-3 consist of more than 55 and 38 genes

distributed in five regions, and a single chromosomal region, respectively (Canals et al.

2006a/b). In A. caviae, several polar flagella genes responsible for encoding different

components of the flagella machinery have been identified (Rabaan et al. 2001; Gavin

et al. 2002; Kirov et al. 2002). In A. hydrophila, expression of polar flagellum appears

to be organized in four transcriptional levels (classes I to IV), where each level serves as

the activator for the next transcriptional level. Thus, transcription of polar flagellum

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genes in this organism operates in a hierarchichal sequence similar, but not identical, to

the transcriptional hierarchies of V. cholerae and P. aeruginosa (Wilhems et al. 2011).

The alternative sigma factor 54 (rpoN) of AH-3 is another important flagellar

regulatory protein essential for transcription of both polar and lateral flagellar gene

systems (Canals et al. 2006b). The Fla genes, flaA and flab are widely distributed in

mesophilic Aeromonas (Rabaan et al. 2001). The percentage of the Laf genes lafA1 and

lafA2 range between 60 and 100% (Gavin et al. 2002, 2003; Kirov et al. 2004). In

aeromonads associated with diarrhoeal illness, Laf genes are usually present in 50 to

60% of the strains (Kirov et al. 2002; Aguilera-Arreola et al. 2007). The flmA and flmB

of the flm gene cluster also involved in lateral flagella synthesis are found in all

mesophilic Aeromonas (Gryllos et al. 2001).

1.9.7.2. Functions associated with flagella

In addition to providing a means of locomotion to the bacterium, flagella have multi-

functional roles in pathogenesis. Mutations of some or most of the genes encoding both

flagellar types can result in complete loss of motility, LPS O-antigen and flagellin

expression leading to reduction in adherence, invasion of epithelial cells and biofilm

formation (Whitby et al. 1992; Merino et al. 1997; Gryllos et al. 2001; Rabaan et al.

2001; Gavin et al. 2002, 2003; Kirov et al. 2002, 2004; Canals et al. 2006ab; Santos et

al. 2010). Moreover, the presence of lateral and polar flagella in combination with other

virulence factors such as a T3SS-like apparatus and secretion of enterotoxins is strongly

associated with virulence (Kirov 2003; Sen and Lye 2007).

1.9.8. Secretion systems

Gram-negative bacteria possess systems that secrete and inject pathogenic proteins into

the cytosol of eukaryotic cells via needle-like structures disrupting cell function and

arquitecture (Table 1.6) (Burr et al. 2003; Sha et al. 2005; Bingle et al. 2008). There are

currently six types of secretion transport systems recognized (types I to VI) and all

utilize adenosine triphosphate (ATP) as the energy source to drive transport of

macromolecules (Christie 2001). Of these, types II to IV are large multi-protein

complexes that can span the entire cell envelope (Bingle et al. 2008). In Aeromonas,

four (II, III, IV and VI) secretion systems have been described.

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Table 1.6 Selected effector proteins associated with different secretion systems

Protein

Putative function

Secrected

by:

References

Act Cytotoxic enterotoxin T2SS Chopra et al. (2000)

AexT Cytolytic enterotoxin T3SS Braun et al. (2002)

AopP Inhibits the NF-B signalling pathway T3SS Fehr et al. (2006)

AopH Aeromonas outer protein T3SS Dacanay et al. (2006)

AopO Aeromonas outer protein T3SS Dacanay et al. (2006)

AscC Outer membrane pore of T3SS T3SS Dacanay et al. (2006)

AopB Formation of the T3SS translocon T3SS Sha et al. (2005)

AexU ADP-ribosyltransferase T3SS

Sha et al. (2007)

AcTra Several roles including pilus assembly and core components

T4SS Rangrez et al. (2006)

Hcp Inhibits phagocytosis T6SS Suarez et al. (2010a)

VgrG1 Actin ADP-ribosylating activity T6SS Suarez et al. (2010b)

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1.9.8.1. Type II Secretion System (T2SS)

The T2SS of Aeromonas is highly homologous to systems found in other Gram-

negative bacteria such as V. cholerae and P. aeruginosa (Schoenhofen et al. 1998).

Important proteins are secreted by Aeromonas through the T2SS including the lipolytic

enzyme GCAT (Brumlik et al. 1997), the cytotoxic enterotoxin Act (Chopra et al. 2000)

and proaerolysin, the inactive precursor of the channel-forming toxin aerolysin (Howard

and Buckley 1986; Jiang and Howard 1992). Proaerolysin concentrates in the periplasm

then passess through it on its way out of the cell (Burr et al. 2001). In A. hydrophila, an

85 kDa complex containing the ExeA and ExeB proteins is involved in the secretion of

aerolysin (Schoenhofen et al. 1998). The genes exeC-N and exeAB encode a T2SS in A.

hydrophila (Jiang and Howard 1992; Pepe et al. 1996).

1.9.8.2. Type III Secretion System (T3SS)

The cytotoxic effect of Aeromonas towards cell lines is dependent upon a functional

T3SS (Chacón et al. 2003, 2004) which plays an important role in the virulence of

several species (Burr et al. 2001, 2003; Yu et al. 2004; Sha et al. 2005, 2007; Dacanay

et al. 2006; Sierra et al. 2007; Du and Galan 2009). T3SSs have been identified in A.

hydrophila and A. salmonicida strains isolated from clinical and fish sources,

respectively (Burr et al. 2003; Sha et al. 2007) while the T3SS of a pathogenic A. sobria

strain was associated with causing disease in farmed perch (Perca fluviatilis) (Wahli et

al. 2005). Expression of T3SS is affected by environmental factors, particularly calcium

depletion and a high Mg2+ concentration. Recent evidence suggests that a complex

interconnection between the expression of the T3SS and other virulence factors such as

the LPS, the PhoPQ two-component system and the ahyIR quorum sensing system exist

(Vilches et al. 2009). The T3SS has been found in approximately 50% of Aeromonas

strains world wide (Chacón et al. 2004) and in one study, genes encoding the T3SS

were higher in clinical (56%) than in environmental (26%) strains (Vilches et al. 2004).

In A. hydrophila, the genetic organization of the T3SS shares great similarity to the

T3SS of both Yersinia species and P. aeruginosa (Yu et al. 2004). T3SS-encoding

genes can be located on the chromosome, as in A. hydrophila AH-1 (Yu et al. 2004) or

spread on plasmids and the chromosome, as in A. salmonicida ssp. salmonicida (Burr et

al. 2002; Stuber et al. 2003; Fehr et al. 2006). The distribution of T3SS-encoding genes

varies within the species. In A. caviae the incidence is usually low compared to the high

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frequency found in extraintestinal isolates of A. veronii and A. hydrophila (Chacón et al.

2004).

T3SS mediates translocation of cytotoxins to host cells affecting several biological

functions (Burr et al. 2003; Sierra et al. 2007). Deletions or mutations of T3SS genes

encoding effector proteins result in reduction of cytotoxicity (Burr et al. 2003; Vilches

et al. 2004; Yu et al. 2004), inflammatory cytokines and chemokines levels (Fadl et al.

2006) and phagocytosis (Vilches et al. 2004; Yu et al. 2004). Many effector and

putative proteins with diverse biological activities have been associated with T3SSs

(Vilches et al. 2004). The most common and well-characterized T3SS effector proteins

included AexT, AopP, AopH and AopO of A. salmonicida (Burr et al. 2002, Dacanay et

al. 2006, Fehr et al. 2006) and the AexU protein of A. hydrophila (Sha et al. 2007)

(Table 1.6). Most effector proteins present in aeromonads are the equivalent of effector

proteins found in other pathogenic bacteria. The AexT toxin, an extracellular ADP-

ribosyltransferase found in A. salmonicida ssp. salmonicida is highly similar to the

ExoS and ExoT toxins secreted by the T3SS of P. aeruginosa (Braun et al. 2002). The

AopP potein found equally in typical and atypical A. salmonicida strains, shares

sequence homology with the YopJ protein of Y. enterocolitica (Fehr et al. 2006).

Although the biological activity of most effector proteins may differ, the final outcome

usually results in cell changes and lysis. Morphological changes and cell lysis caused by

the AexT toxin of A. salmonicida ssp. salmonicida requires contact with host cells

(Braun et al. 2002); AopP inhibits the NF-B signalling pathway blocking cytokine

production promoting apoptosis in host cells (Fehr et al. 2006); the full-length and NH2-

terminal domain of the protein AexU causes changes in cell morphology due to actin

filament organization (Sierra et al. 2007). Sha et al. (2005) reported a positive

correlation between T3SS, the cytotoxic enterotoxin (Act) and quorum sensing (QS).

1.9.8.3. Type IV Secretion System (T4SS)

T4SSs are macromolecular transfer systems present in Gram negative and Gram

positive bacteria that translocate proteins and nucleoprotein complexes (Cao and Saier

2001; Schröder and Lanka 2005). The T4SS of Agrobacterium tumefaciens has been

used as a model to predict the structure and function of this secretory system (Cao and

Saier 2001; Christie and Cascales 2005). Moreover, the sequences and structure of

T4SSs are homologous to those of conjugative transfer systems of naturally occurring

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plasmids (Ding et al. 2003). The structural constituent of the secretory apparatus is

provided by VirB2-VirB11, a group of proteins which may also play a direct role in

translocation (Cao and Saier 2001; Schröder and Lanka 2005). T4SS has been described

in A. culicicola (Rangrez et al. 2006) and in an A. caviae strain isolated from a hospital

effluent (Rhodes et al. 2004). The T4SS of A. culicicola resembles that of plasmids

RP721, pAc3249A and pKM101 of E. coli, the VirB-D4 systems of A. tumefaciens, B.

henselae, A. caviae and the Ptl system of B. pertussis. Furthermore, the high homology

observed between the A. culicicola and pAc3249A suggests that A. culicicola may have

acquired the plasmid through lateral transfer while residing in the mosquito gut, the site

of isolation of A. culicicola (Rangrez et al. 2006). The plasmid pRA1 was found in an

A. hydrophila strain pathogenic to fish. The sequence of pRA1, a member of the IncA/C

family, featured a T4-like conjugative plasmid transfer system that carried multidrug

resistance genes and a hipAB-related gene cluster. In addition to drug resistance, hipAB

a toxin-antitoxin module may be involved in biofilm formation (Fricke et al. 2009).

Rangrez et al. (2010) described three ATPases from a new T4SS of Aeromonas veronii

plasmid pAC3249A and showed that these ATPases could bind and hydrolyze ATP.

1.9.8.4. Type VI Secretion System (T6SS)

The T6SS is widely spread in nature and has been reported in many pathogenic and

non-pathogenic bacteria where it is involved in a variety of roles (Williams et al. 1996;

Suarez et al. 2008; Bingle et al. 2008; Pukatzki et al. 2009). The T6SS is independent

of the T3SS and the flagellar secretion system (Suarez et al. 2008). In A. hydrophila

SSU the T6SS gene cluster is located in the chromosome which is regulated by the σ54

activator encoded by the vasH gene (Suarez et al. 2008). Two main classes of proteins

are secreted by T6SS, the haemolysin coregulated protein (Hcp) and the valine-glycine

repeat G (VgrG). Hcp is secreted by all bacteria with a functional T6SS, plays a role in

the transport of proteins out of the bacterial cell and into the cytosol of infected host

cells or into the extracellular space (Pukatzki et al. 2009). Hcp binds to macrophages

inducing the production of IL-10 and transforming growth factor (TGF)-, affecting the

activation and maturation of macrophages and recruitment of other cellular immune

components. Expression and translocation of the hcp gene are associated with the vasH

and vasK genes (Suarez et al. 2008; 2010a). Deletion of vasH in A. hydrophila SSU

impaired expression of hcp while deletion of vasK allowed expression and translocation

of Hcp, but not its secretion into the extracellular milieu (Suarez et al. 2008). As a

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consequence, SSU mutants were readily phagocytosed by murine macrophages

suggesting that the secreted form of Hcp played a role in the evasion of the host

immune system by inhibiting phagocytosis and promoting the spread of the bacterium

in the host (Suarez et al. 2010a).

VgrG shares structural features with the cell-puncturing device of T4 bacteriophage

(Kanamaru et al. 2002). In addition, the C-terminal extensions of some VgrG carry

functional domains that may serve as effector-domains or “evolved VgrGs” (Pukatzki et

al. 2007). Pathogenic bacteria may have up to 10 VgrGs of which three paralogues are

present in A. hydrophila (Pukatzki et al. 2009). The T6SS-associated proteins have been

implicated in a variety of biological functions including cross-linking of host actin,

degradation of the peptidoglycan layer, ADP-ribosylation of host proteins inducing

apoptosis and inhibiting phagocytic activity in macrophages (Pukatzki et al. 2009;

Suarez et al. 2010b). Both, Hcp and VgrG play dual roles as structural components and

effector proteins of T6SS (Cascales 2008). Recently, mutagenesis experiments showed

that paralogues of Hcp and VgrG also influenced bacterial motility, protease production

and biofilm formation. Moreover, these paralogues were required for optimal bacterial

virulence and dissemination to mouse peripheral organs (Sha et al. 2013).

1.9.9. Exotoxins

Aeromonas species produce a wide range of extracellular toxins and enzymes that are

associated with cytotoxicity, haemolytic and enterotoxic effects in host tissue

(Wadström et al. 1976; Janda 1985; Shotts et al. 1985; Vadivelu et al. 1991; Mateos et

al. 1993; Pemberton et al. 1997; Chopra et al. 2000; Kirov et al. 2002; Krzyminska et

al. 2006). Many distinct and unrelated exotoxins have been described in these bacteria

over the years reflecting the diversity that exists among Aeromonas strains (Table 1.7)

(Notermans et al. 1986; Todd et al. 1989; Vadivelu et al. 1991; Granum et al. 1998).

The production of exotoxins in vitro is influenced by the type of media, culture

conditions, growth temperature and variations in osmotic stress (Ljungh and Kronevi

1982; Asao et al. 1986; Mateos et al. 1993; Granum et al. 1998).

Under iron limitation, there is a pronounced increase in toxin production which is

repressed in the presence of glucose (Thornley et al. 1997). Although production of

exotoxins has been reported equally in both, non-enteric and enteric isolates, the

production of enterotoxin and a cholera-toxin factor have been more prominent among

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enteric isolates (Vadivelu et al. 1991). Thus, enterotoxins appear to play an essential

role in Aeromonas-associated gastroenteritis (Krzyminska et al. 2006). Exotoxins can

induce cytoplasmic vacuolation and cell death in different cell lines including those

derived from the intestinal mucosa (Barer et al. 1986; Vadivelu et al. 1991; Di Pietro et

al. 2005; Ghatak et al. 2006). However, the use of tissue cultures as a rapid assay has

not always been found suitable as a screening test (Chakraborty et al. 1984).

1.9.9.1. Aerolysin

Aerolysin is one of the most studied toxins produced by Aeromonas. Also referred to as

-haemolysin, cytotoxic enterotoxin or cytolytic enterotoxin, aerolysin has generated

enormous interest for the last 40 years (Bernheimer and Avigad 1974). Intially purified

by Buckley et al. (1982), the action of aerolysin on various cell lines and rabbit ileal

loop test to demonstrate cytotoxic and enterotoxic activity, respectively, is well

documented (Asao et al. 1986; Chopra et al. 1993; Ljungh and Wadström 1983;

Scheffer et al. 1988; Ferguson et al. 1997). Although aerolysin can be expressed in E.

coli differences in the mechanisms involved in secretion and excretion between E. coli

and A. trota have been observed (Khan et al. 1998). Various mechanisms involved in

the secretion of aerolysin have been described. The notion that a 23 kDa peptide signal

sequence was involved in the translocation of the pro-aerolysin followed by proteolytic

cleavage activation by serine protease was proposed but not universally accepted

(Howard and Buckley 1986; Husslein et al. 1991; Chopra et al. 1993). Pepe et al.

(1996) showed that the tapD gene of A. hydrophila encodes a type IV leader

peptidase/N-methyltransferase essential for extracellular secretion of aerolysin and

protease. Another mechanism of secretion involved the binding of pro-aerolysin to

glycosylphosphatidylinositol-anchored proteins on target cells to integrate into the

plasma membrane (Brodsky et al. 1999).

Salient features of aerolysin include resistance to proteases, lack of inhibition by lipids

or inactivation by gangliosides and reducing agents. The toxin readily binds to

erythrocytes at 37, but not at 4C (Ferguson et al. 1997). Variation in haemolytic

activity has been associated with different receptor affinities of the aerolysin molecule

for a particular erythrocyte type (Husslein et al. 1991; Ferguson et al. 1997).

Similarities between the physical and biological properties of aerolysin with the

exotoxin of P. aeruginosa and a haemolysin secreted by V. parahaemolyticus,

respectively, have been reported (Ljungh et al. 1981; Ljungh and Wadström 1983).

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1.9.9.1.1. Action on host tissue

Aerolysin can form weakly ion-permeable channels similar to those produced by the α-

toxin of S. aureus (Chakraborty et al. 1990). Aerolysin causes changes in the membrane

permeability leading to osmotic lysis in erythrocytes (Bernheimer and Avigad 1974; Di

Pietro et al. 2005) and cross-reacts with cholera toxin (Chopra et al. 1993; Thornley et

al. 1997). The toxin can be lethal to mice at low (0.1 μg) concentrations (Bernheimer &

Avigad 1974; Chakraborty et al. 1987; Chopra et al. 1993). The action of aerolysin on

the epithelial barrier has been described in several studies using a variety of cell lines

(Abrami et al. 2003; Epple et al. 2004; Bücker et al. 2011). Bücker et al. (2011) used

human colonic epithelial cells (HT-29/B6 cells) to describe the mechanisms involved in

epithelial barrier dysfunction caused by aerolysin during Aeromonas infection. The

action of aerolysin on HT-29/B6 cells resulted in transcellular and paracellular

resistance by inducing chloride secretion and tight junction redistribution, respectively.

Therefore, diarrhoea caused by aeromonads appears to be mediated by two mechanisms,

transcellular secretion and paracellular leak flux (Bücker et al. 2011). The impairement

of epithelial integrity may also affect wound closure contributing to the necrotizing

process observed in wound infections and intestinal epithelial lesions.

1.9.9.1.2. Molecular characteristics and prevalence

The complete nucleotide sequences of the aerolysin toxin in A. hydrophila and A. trota

have been described. In the case of A. hydrophila the sequences were independently

described revealing inconsistent results (Chakraborty et al. 1986; Howard et al. 1987;

Husslein et al. 1988; Khan et al. 1998). Similarly, a phylogenetic tree based on the

deduced amino acid sequences of the aerolysin genes from several Aeromonas species

revealed the presence of three groups of genes (Khan et al. 1998). Aerolysin shares

sequence similarities with the α-toxin of S. aureus. Both are very hydrophilic and

contain an almost identical string of 10 amino acids (Howard et al. 1987; Murray et al.

1988). Although aerolysin is unique to the genus Aeromonas and it is present in most

species (Husslein et al. 1991, 1992; Ørmen and Østensvik 2001; Ottaviani et al. 2011)

the prevalence of the encoding gene varies greatly depending on the geographical region

and source of isolation (Chacón et al. 2003).

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1.9.9.2. Cytotoxic enterotoxin (Act)

The differentiation of the cytotoxic enterotoxin Act as a separate protein from aerolysin

has been controversial. Aerolysin and Act share similar amino acid sequence and most

biological properties (Buckley and Howard 1999). Despite the degree of homology

(93% amino acid identity) that exists between these two proteins (Chopra et al. 1993),

Act can now be distinguished from aerolysin by monoclonal antibodies neutralization

and receptor specificity (Ferguson et al. 1997; Chopra and Houston 1999a). In order to

avoid further confusion, Act is here described as a different toxin from aerolysin.

The properties of Act have been the source of many studies and a multitude of

biological activities have been described for this protein (Chopra et al. 2000; Ribardo et

al. 2002; Galindo et al. 2004). Basically, the Act protein acts as an early signaling

molecule by rapidly releasing calcium from intracellular stores leading to the production

of prostaglandin (PGE2) and tumor necrosis factor alpha (TNFα) while at the same time

down-regulating activation transcription factor NF-κB (Chopra et al. 2000; Ribardo et

al. 2002). In murine macrophages and human intestinal epithelial cells, Act activates the

kinase cascade increasing reduction/oxidative stress factors and production of reactive

oxygen species. Act can lyse erythrocytes, destroy tissue culture cell lines, induce a

fluid secretory response in ligated intestinal loop models and is lethal to mice (Chopra

and Houston 1999b; Chopra et al. 2000). These effects can lead to an extensive

inflammatory response and intestinal tissue damage including Act-induced apoptosis

leading to cell death (Xu et al. 1998; Chopra et al. 2000; Ribardo et al. 2002; Galindo et

al. 2004). Act is an essential contributor to Aeromonas-mediated gastroenteritis

followed by Alt and Ast, respectively (Xu et al. 1998; Sha et al. 2002).

At the genetic level, the multiple biological activities of the Act toxin may be the

function of different molecular regions. The Act protein is encoded by the act gene

(Albert et al. 2000) which in A. hydrophila is optimally expressed at 37C and at a pH

7.0. The act promoter is repressed by glucose and in A. hydrophila the activity of the act

gene increases in the presence of Ca2+ while expression of the act gene is regulated by

iron (Sha et al. 2001). Microarray analyses show that Act can induce many genes

including those involved in apoptosis of T84 cells, caspase-3-cleavage, immune-related

genes, transcription factors, phosphorylation or activation of signaling molecules,

adhesion molecules, Ca2+ mobilization and cytokines (Galindo et al. 2003; 2005). The

functional domain of the cytolytic enterotoxin produced by A. hydrophila SSU shared

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some amino acid homology with Clostridium perfringens type A enterotoxin and the

listeriolysin produced by Listeria monocytogenes suggesting that the genes may have

derived from a common ancestor (Chopra et al. 1993).

1.9.9.3. Haemolysins

The haemolysin toxin is different from the aerolysin protein and although both toxins

are activated by trypsin, the export pathway and haemolytic activity of these two

proteins are different (Asao et al. 1986; Hirono and Aoki 1991; Hirono et al. 1992). The

term hlyA was proposed to differentiate the haemolysin gene from aerA to denote

aerolysin (Wong et al. 1998). The haemolytic activity of Aeromonas has been closely

related to cytotoxicity (Honda et al. 1985; Kozaki et al. 1987; Wang et al. 1996, 2003)

and haemolysins are considered one of the important virulence factors produced by

Aeromonas. In A. hydrophila, the interaction of haemolysin with erythrocyte

membranes is influenced by temperature and growth (Ljungh et al. 1981; Asao et al.

1984, 1986; Titball and Munn 1985; Kosazi et al. 1987; Knochel 1989). Many

haemolysins with different MWs and biological functions have been purified and

characterized in Aeromonas (Table 1.7). The haemolysin protein is probably bound

intracellularly as an inactive precursor that is formed during the late logarithmic phase

of growth and released by lysis. Haemolysin can cause diarrhoea by induction of HCO

ion via the cystic fibrosis transmembrane conductance regulator (Takahashi et al. 2006).

The gene hlyA is widely dispersed among Aeromonas species (Hirono and Aoki 1991)

and it is possible that haemolysin genes evolved from a single ancestral gene (Hirono et

al. 1992). The amino acid composition of haemolysins produced by some strains varies

compared to the amino acid composition of aerolysin (Wong et al. 1998) suggesting

that the origin of the haemolysin genes may be different from that of aerolysin (Hirono

and Aoki 1991, 1993). Aeromonas haemolysins have been reported to contain regions

homologous to the Vibrio vulnificus and Vibrio cholerae cytolysin-haemolysin (Hirono

and Aoki 1993) while a high level of homology (96%) has been reported between

different aeromonad strains (Erova et al. 2007). Among the major Aeromonas species,

hlyA has been detected in A. caviae (Wang et al. 1996; Heuzenroeder et al. 1999; Pablos

et al. 2010) and it is practically ubiquitous in A. hydrophila (Heuzenroeder et al. 1999;

Wu et al. 2007). In A. veronii the prevalence of hlyA ranges from 0 to 77% (Wang et al.

1996; 2003; Wu et al. 2007; Pablos et al. 2010). Recently, a diarrhogenic strain of A.

trota 701 was found to produce both haemolysin and protease (Takahashi et al. 2014).

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1.9.9.4. Enterotoxins

Enterotoxins may play significant roles in the pathology of Aeromonas-induced

gastrointestinal disease. The two more frequently studied cytotonic enterotoxins are the

heat-labile (Alt) and the heat-stable (Ast) toxins although other enterotoxins have been

described in Aeromonas (Table 1.7). These enterotoxins are biologically and genetically

unrelated to the LT and ST of E. coli and the cholera-toxin (CT) (Kaper et al. 1981)

although using synthetic oligonucleotide DNA probes, Schultz and McCardell (1988)

demonstrated that regions of A. hydrophila DNA were homologous with the CT probes.

Most cytotonic enterotoxins share common mechanisms of action including elongation

of Chinese hampster ovarian (CHO) cells, increasing levels of cAMP or PGE2 in tissue

culture cells, changes in adrenal YI cells, fluid accumulation in the rabbit ligated

intestinal loops without mucosal injury and accumulation of intestinal fluid in infant

mice (Ljungh and Wadström 1979, 1983; Chakraborty et al. 1984, 1987; Chopra et al.

1992b, 1996; McCardell et al. 1995).

Alt and Ast are encoded by the alt and ast gene, respectively (James et al. 1982; Chopra

et al. 1996; Albert et al. 2000; Krzyminska et al. 2003). Mutagenesis experiments

showed that Alt and Ast in combination with Act can induce gastroenteritis in a mouse

model (Sha et al. 2002). In gastrointestinal infection, production of more than one toxin

appears to correlate with the type of stools and severity of the diarrhoeal episode. The

presence of both alt and ast has been associated with severe watery diarrhoea in A.

hydrophila-induced infection while isolates positive for alt have only been associated

with loose stools (Albet et al. 2000). Unlike the cytotoxic enterotoxin that causes

extensive damage to epithelium, the cytotonic enterotoxins do not cause degeneration of

crypts and villi of the small intestine (Chopra and Houston 1999b). The alt and ast

genes have been detected in clinical and environmental isolates world-wide. However,

the prevalence of these genes varies considerably depending on the source, species,

geographical location and the number of isolates tested (Potomski et al. 1987b; Borrell

et al. 1998; Trower et al. 2000; Sen and Rodgers 2004; Aguilera-Arreola et al. 2007;

Wu et al. 2007; Pablos et al. 2010).

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Tab

le 1

.7

Toxi

ns se

cret

ed b

y Ae

rom

onas

Gen

e/pr

otei

n O

rgan

ism

(sou

rce)

M

W

(kD

a)

% Id

entit

y w

ith

othe

r pr

otei

ns

Ref

eren

ce

Hae

mol

ysin

A.

hyd

roph

ila B

3646

W

retli

nd a

nd H

eden

(197

3)

Aer

olys

in

A. h

ydro

phila

38

(hum

an is

olat

e?)

50-5

3

B

ernh

eim

er &

Avi

gad

(197

4)

-H

aem

olys

in (a

erol

ysin

) A.

hyd

roph

ila K

140/

K14

4 (h

uman

dia

rrho

eal i

sola

tes)

65

Lj

ungh

et a

l. (1

981)

-H

aem

olys

in

A. h

ydro

phila

K14

0/K

144

(hum

an d

iarr

hoea

l iso

late

s)

50

Ljun

gh e

t al.

(198

1)

Ente

roto

xin

A. h

ydro

phila

K14

0/K

144

(hum

an d

iarr

hoea

l iso

late

s)

15

Ljun

gh e

t al.

(198

1)

Ente

roto

xin

A. h

ydro

phila

AH

2 an

d A

H11

33 (h

uman

dia

rrho

eal

isol

ates

)

C

hakr

abor

ty e

t al.

(198

4)

Hae

mol

ysin

A.

hyd

roph

ila A

H-1

(hum

an d

iarr

hoea

l iso

late

) 48

-50

Asa

o et

al.

(198

4)

CT-

toxi

n re

late

d fa

ctor

A.

hyd

roph

ila/A

. sob

ria

(hum

an d

iarr

hoea

l iso

late

s)

Hon

da e

t al.

(198

5)

Hae

mol

ysin

(H-ly

sin)

A.

salm

onic

ida

(fis

h is

olat

e)

25.9

Ti

tbal

l & M

unn

(198

5)

Hae

mol

ysin

A.

hyd

roph

ila (h

uman

and

drin

king

wat

er is

olat

es)

Not

erm

ans e

t al.

(198

6)

Hae

mol

ysin

A.

hyd

roph

ila C

A-1

1 (e

nviro

nmen

tal i

sola

te)

50

Asa

o et

al.

(198

6)

Aer

olys

in

A. h

ydro

phila

(rai

nbow

trou

t) 53

.8

Orig

inal

aer

olys

in

How

ard

et a

l. (1

987)

aerA

A.

trot

a A

B3

(hum

an d

iarr

hoea

l iso

late

) 54

.4

77%

with

aer

olys

in

Hus

slei

n et

al.

(198

8)

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-58-

Tab

le 1

.7

C

ontin

ued.

Gen

e/pr

otei

n O

rgan

ism

(sou

rce)

M

W

(kD

a)

% Id

entit

y w

ith o

ther

pr

otei

ns

Ref

eren

ce

Hae

mol

ysin

A.

sobr

ia 3

3 (h

uman

isol

ate)

49

K

ozak

i et a

l. (1

989)

AH

H1

A. h

ydro

phila

ATC

C 7

966

(tinn

ed m

ilk is

olat

e)

63.6

50

% w

ith V

. cho

lera

e H

lyA

H

irono

& A

oki (

1991

)

AH

H3

A. h

ydro

phila

28S

A (e

el is

olat

e)

54.7

94

% w

ith a

erol

ysin

H

irono

et a

l. (1

992)

AH

H5

A. h

ydro

phila

AH

-1 (h

uman

isol

ate)

53

.7

92%

with

aer

olys

in

Hiro

no e

t al.

(199

2)

ASA

1 A.

sobr

ia 3

3 (h

uman

isol

ate)

53

.9

66%

with

aer

olys

in

Hiro

no e

t al.

(199

2)

ASH

3 A.

salm

onic

ida

17-2

(fis

h is

olat

e)

54.2

66

% w

ith a

erol

ysin

H

irono

& A

oki (

1993

)

ASH

4 A.

salm

onic

ida

17-2

(fis

h is

olat

e)

63.4

45

% w

ith V

. cho

lera

e H

lyA

H

irono

& A

oki (

1993

)

Cyt

olyt

ic e

nter

otox

in (A

ct)

A. h

ydro

phila

SSU

(hum

an d

iarr

hoea

l iso

late

) 54

.5

93%

with

aer

olys

in

Cho

pra

et a

l. (1

993)

Cyt

oton

ic e

nter

otox

in (A

st)

A. h

ydro

phila

SSU

(hum

an d

iarr

hoea

l iso

late

) 35

C

hopr

a et

al.

(199

4)

Cyt

oton

ic e

nter

otox

in (A

lt)

A. h

ydro

phila

SSU

(hum

an d

iarr

hoea

l iso

late

) 44

45

-51%

with

ph

osph

olip

ase/

lipas

e

Cho

pra

et a

l. (1

996)

Hly

A

A. h

ydro

phila

A6

(hum

an d

iarr

hoea

l iso

late

) 69

51

% w

ith V

. cho

lera

e H

lyA

W

ong

et a

l. (1

998)

Cyt

otox

ic e

nter

otox

in

A. v

eron

ii bv

. sob

ria (i

sola

ted

from

lam

b ki

dney

) 40

Tr

ower

et a

l. (2

000)

hlyA

A.

hyd

roph

ila S

SU (h

uman

dia

rrho

eal i

sola

te)

49

96%

with

A. h

ydro

phila

A

TCC

796

6 ha

emol

ysin

Er

ova

et a

l. (2

007)

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1.10. Additional extracellular products

Aeromonas species can secrete a plethora of degradative enzymes with the ability to

hydrolyse a wide range of substrates (Shotts et al. 1985). In the past, the

characterization of extracellular products (ECP) was based on the purification of

individual proteins from selected Aeromonas strains. The new approach involves the

construction of extracellular proteome maps which determine the major extracellular

products involved in the virulence of A. hydrophila AH-1 (Yu et al. 2007).

1.10.1. Proteases

Proteases may play a critical role in the early stages of infection by protecting the

bacterial cell against complement-mediated killing, causing tissue damage and by

protecting the bacterium from host defences while providing nutrients for cell

proliferation (Shieh 1987; Leung and Stevenson 1988a/b; Pemberton et al. 1997; Khan

et al. 2008). Protease production is temperature-dependent as production can decrease

significantly at 37C (Mateos et al. 1993; Swift et al. 1999b; Yu et al. 2007). In A.

sobria, the concentration of salt was found to influence the production of serine

protease into the milieu (Khan et al. 2007).

The genes encoding for serine protease are highly conserved in Aeromonas species

(Chacón et al. 2003). Extracellular secretion of protease has been linked with the tapD

gene (Pepe et al. 1996). Despite the competitive advantage that production of serine

proteases confers to aeromonads, deletion mutation has shown that in A. salmonicida

and A. hydrophila proteases are not essential for the virulence of these species in the

models used (Vipond et al. 1998; Cascón et al. 2000a). However, Liu et al. (2010)

showed that a purified protease was lethal to rainbow trout while the combined effects

of proteases and haemolysins have been detrimental to fish (Fyfe et al. 1988; Rodríguez

et al. 1992).

The most common proteases are serine and metallopreoteases. Serine proteases with

different MWs have been described in A. hydrophila (Cho et al. 2003), A. sobria

(Kobayashi et al. 2006), A. trota (Husslein et al. 1991), A. caviae (Nakasone et al.

2004) and A. salmonicida (Gudmundsdottir et al. 2003). Serine proteases participate in

the activation of aerolysin (Abrami et al. 1998), the extracellular toxin GCAT,

haemolysin and possibly other ECPs (Lee and Ellis 1990; Eggset et al. 1994; Vipond et

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al. 1998; Yu et al. 2007). The metallo-protease TagA identified in A. hydrophila SSU is

widely distributed in Aeromonas species and has been reported in isolates from patients

with wound infections and gastroenteritis (Pillai et al. 2006). TagA has been associated

with haemolytic-uraemic syndrome where it potentiates the activity of C1-INH

inhibiting the classical complement-mediated lysis of erythrocytes and increasing serum

resistance (Pillai et al. 2006).

Proteases with affinity for specific substrates such as elastin, casein and gelatin have

been identified (Cascón et al. 2000ab; Esteve and Birkbeck 2004; Han et al. 2008;

Meng et al. 2009; Zacaria et al. 2010). Others proteases can induce intense vacuolation

in Vero cells including cellular death by apoptosis (Martins et al. 2007). A kexin-like

serine protease in A. sobria 288 (ASP) possesses a unique occluding region which may

serve as a potential target for antisepsis drugs (Kobayashi et al. 2009a/b). ASP acts by

enhancing vascular permeability in rat skin supporting the notion that a correlation

between ASP production and soft-tissue lesions exists (Yokoyama et al. 2002). Other

features associated with ASP include reduction of blood pressure by activating the

kallikrein/kinin system (Imamura et al. 2006), promoting human plasma coagulation

through activation of prothrombin (Nitta et al. 2007) and the formation of pus and

oedema through the action of anaphylatoxin C5a (Nitta et al. 2008). All these

observations have led to the conclusion that ASP mediates the induction of

disseminated intravascular coagulation through -thrombin production, a common and

lethal consequence of sepsis (Nitta et al. 2007).

1.10.2. Lipases

Lipases, like proteases, are important for bacterial nutrition (Pemberton et al. 1997) and

several roles in microbe metabolism have been associated with these compounds

(Anguita et al. 1993). The most studied lipase to date is GCAT which is present in all

members of the Vibrionaceae with the exception of Plesiomonas shigelloides

(MacIntyre et al. 1979). GCAT can use cholesterol as an acyl acceptor, has a molecular

mass of approximately 25 kDa and possesses haemolysin, leukocytolysin and cytotoxic

activities (Eggset et al. 1994; Nerland 1996; Vipond et al. 1998). GCAT shares many

properties with the mammalian lecithin:cholesterol acyltransferase enzyme (Thorton et

al. 1988). When combined with the LPS (the GCAT-LPS complex, MW = 2000 kDa),

the specific haemolytic activity and lethal toxicity of GCAT-LPS is stronger than the

native GCAT resulting in complete lysis of erythrocytes (Lee and Ellis 1990; Hirono

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and Aoki 1993; Eggset et al. 1994; Nerland 1996; Bricknell et al. 1997; Thornley et al.

1997). Differences in MWs between GCAT produced by A. hydrophila and A.

salmonicida have been reported (Thornton et al. 1988), while polyclonal antibody

prepared against A. salmonicida GCAT does not cross-react with A. hydrophila GCAT

despite the similar amino acid termini of these proteins. Norwhistanding the pathology

associated with GCAT, its role as a virulence factor in humans is still controversial

(Chopra and Houston 1999b). The virulence of GCAT and serine protease mutants was

shown to be similar to the effects caused by wild strains of A. salmonicida after IP

injection of Atlantic salmon smots (Vipond et al. (1998).

Although some similarities exist between other lipases produced by A. hydrophila, they

are not identical. Some lipases are membrane-bound while others are present in the

periplasmic space (Anguita et al. 1993; Chuang et al. 1997). The characteristics of some

lipases depend on the encoding genes which are distributed in all Aeromonas species

(Chacón et al. 2003). Those encoded by the lip and lipH3 genes have esterase but not

phospholipase activities (Anguita et al. 1993; Chuang et al. 1997); the apl-1 gene

encode a non-haemolytic lipase with phospholipase C activity (Ingham and Pemberton

1995) while the pla gene encodes a non-haemolytic, non-cytotoxic and non-enterotoxic

lipoprotein with phospholipase A1 activity (Merino et al. 1999). Other significant

differences include the number of amino acid residues, optimal temperature and pH,

thermal stability and substrate specificity (Anguita et al. 1993; Ingham and Pemberton

1995; Chuang et al. 1997; Merino et al. 1999). At the amino acid level, sequence

similarities have been reported between the lip, lipH3, apl-1, pla and alt gene products

(Chopra et al. 1996; Merino et al. 1999) while the putative lipase substrate-binding

domain V-H-F-L-G-H-S-L-G-A is shared by several species particularly those

belonging to serogroups O:11, O:16 and O:34 (Watanabe et al. 2004). Lipases may act

by altering the plasma membrane of host cells affecting permeability and raising

accessibility to toxins (Soler et al. 2002; Mendes-Marquez et al. 2012).

1.10.3. Nucleases

The role of extracellular nucleases as a virulence factor contributing to disease has not

been supported by experimental work. The most probable roles of nucleases are

primarily nutritional, due to their ability to degrade nucleic acids, and protective, as

nucleases provide a barrier to the entry of foreign DNA into the host (Pemberton et al.

1997). Few genes encoding these enzymes have been cloned (Dodd and Pemberton

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1996; Pemberton et al. 1997; Nam et al. 2004) and, of those examined, DNAse genes

have been found more frequently in clinical than in environmental isolates (Chacón et

al. 2003). Genes encoding DNases with different MWs have been identified in various

A. hydrophila strains. The dns gene of strain CHC-1 encodes a 25 kDa protein (Chang

et al. 1992) while dnsH and nucH present in strain JMP636 encode proteins of

approximately 27.4 and 114 kDa, respectively (Dodd and Pemberton 1996, 1999).

The deduced amino acid sequence of Dns is highly homologous with the DNase

produced by V. cholerae (Chang et al. 1992). By contrast, the NucH has no known

homologue on the basis of its nucleotide or predicted protein sequence (Dodd and

Pemberton 1996). DnsH and Dns are identical in size (210 amino acids) and contain

92% similarity and 89% amino acid identity, respectively (Dodd and Pemberton 1999).

Dns is an extracellular enzyme (Chang et al. 1992) that accumulates equally in both the

periplamic and cytoplasmic space suggesting that DnsH is not secreted (Dodd and

Pemberton 1999). A 25 kDa protein with endo- and exonuclease activity was identified

in A. hydrophila ATCC 14715. The nuclease was capable of complete (100%)

degradation of double-stranded DNA but only partial (70%) degradation of single-

stranded DNA. The ability to possess endo- and exonuclease activity by an intracellular

nuclease is considered rare among prokaryotes (Nam et al. 2004).

1.10.4. Chitinases

Chitin is one of the most abundant biopolymers present in the aquatic biosphere. It is

found in the exoskeleton of insects, molluscs, crustaceans and the cell wall of fungi.

Chitin is a source of food for Aeromonas and provides access to carbon, nitrogen and

energy supplies (Pemberton et al. 1997), thus, contributing to the survival of chitin-

hydrolyzing organisms (Roffey and Pemberton 1990). The degradation of chitin occurs

in two successive steps mediated by different chitinolytic enzymes (Lan et al. 2004).

More specifically, chitinases catalize the hydrolysis of the -1-4 linkage of N-acetyl-D-

glucosamine polymers of chitin (Chen et al. 1991). Several genes encoding for -N-

acetylglucosaminidases have been identified in A. hydrophila resulting in the expression

of proteins with different MWs and distinct biological and kinetic properties (Lan et al.

2004, 2006, 2008). Chitin-degrading enzymes with distinct MWs and biological

properties have also been described in other Aeromonas species (Yabuki et al. 1986;

Roffey and Pemberton 1990; Ueda and Arain 1992; Sitrit et al. 1995; Ueda et al. 1995;

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Hiraga et al. 1997; Lin et al. 1997; Wu et al. 2001; Lan et al. 2004). Mehmood et al.

(2010) described four chitinases from A. caviae CB101 which were encoded by a single

gene chi1. The location of the enzyme in the cell, whether present in the periplasmic or

cytoplasmic space, appears to influence its role in chitin metabolism (Lan et al. 2008).

1.11. Iron uptake

Siderophores, a virulence factor in pathogenic bacteria, provide bacteria with iron from

the host during infection (Byers et al. 1991; Chopra and Houston 1999b). Two types of

siderophores are produced by Aeromonas, enterobactins and amonabactins. Aeromonas-

producing amonabactins can obtain iron from host transferrin and lactoferrin

(Barghouthi et al. 1989, 1991; Byers et al. 1991; Stintzi and Raymond 2000). In

contrast, enterobactin producers do not utilise transferrin in serum but rely exclusively

on host heme iron (Byers et al. 1991). Statistically, amonabactin producers are more

resistant to complement lysis than enterobactin-producing strains (Massad et al. 1991).

Two biologically active forms of amonobactin were described in A. hydrophila 495A2,

amonabactin T which contains lysine, glycine or tryptophan, and amonabactin P which

contains phenylalanine (Barghouthi et al. 1989).

In A. hydrophila, amonobactin is encoded by the amo gene which resembles the entC

gene of E. coli. The nucleotide sequences of amoA and entC suggest that these genes

may share a common ancestor. The biosynthesis of enterobactin involves several genes,

aebA, B, C and E, also functionally related to the E. coli genes. In Aeromonas, synthesis

of 2, 3-dihydroxybenzoic acid (2, 3-DHB) is encoded by the gene amo, in the

aminobactin-producers and by aeb, found among enterobactin-producers (Massad et al.

1994). Suppression of the amoA gene impaired excretion of 2, 3-DHB and amonabactin

resulting in mutants that were more sensitive to growth inhibition by iron restriction

compared to the wild strain (Barghouthi et al. 1991). The iron siderophore receptor gene

fstA of A. salmonicida is homologous with the fstA of other pathogenic Gram-negative

species suggesting that this gene is widely dispersed in these bacteria (Pemberton et al.

1997). Although siderophore production is a common trait in Aeromonas not all strains

are able to produce siderophores (Barghouthi et al. 1989; Zywno et al. 1992; Santos et

al. 1999). Through a siderophore-independent process, most isolates can also use

various heme compounds as sole iron sources (Massad et al. 1991). The combination of

siderophores and phenotypic characteristics was proposed as a taxonomic criterion to

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separate between different genospecies and to evaluate the pathogenic potential of some

species (Zywno et al. 1992).

1.12. Quorum Sensing (QS) Quorum sensing (QS) is a chemical signalling system that regulates gene expression

when bacteria reach a critical cell population density (Swift et al. 1997). Many Gram-

negative bacteria utilize acyl-homoserine lactone (AHL) which are low-molecular-mass

signalling molecules of different chain-lengths (Swift et al. 1999b; Jangid et al. 2007).

A-layer, protease, lipase and pigment production, cytotoxicity of ECP cells and a low

LD50 in A. salmonicida are regulated by quorum sensing (Rasch et al. 2007; Schwenteit

et al. 2010). However, production of virulence factors does not always correlate with

the production and accumulation of AHLs which are encoded by the luxRI (AI-1system)

genes that are universally present in Aeromonas (Jangid et al. 2007). From the

taxonomic view point, sequence analysis of luxRI shows that the genus Aeromonas

forms a distinct lineage from other genera in the class Proteobacteria (Jangid et al.

2007). The close homology of luxRI with the iciA gene of E. coli suggests that in

Aeromonas an association between QS and cell division may exist as iciA is involved in

chromosomal replication (Swift et al. 1997; Chopra and Houston 1999b). Mutations of

these genes can lead to alteration or inactivation of several activities including

exoenzyme activity (Swift et al. 1999a; Bi et al. 2007), biofilm formation (Lynch et al.

2002), changes in the OMP profiles and biochemical characteristics, reduction of

butanediol fermentation, protease activity, adherence, attenuation of cytotoxicity on

epithelial carp cells and LD50 and inability to produce a detectable S-layer (Swift et al.

1999b; Vivas et al. 2004; Bi et al. 2007; Van Houdt et al. 2007). Mutation in the luxS

gene in SSU impaired the secretion of effector proteins of the T6SS but not of T3SS

(Khajanchi et al. 2009). However, mutations in the ahyI and ahyR genes have not

always resulted in alterations in the virulence potential of aeromonads (Defoirdt et al.

2005).

Two other quorum sensing systems including a LuxS-based (AI-2) and the QseBC (AI-

3) two-component system have been described in A. hydrophila SSU (Kozlova et al.

2008; Khajanchi et al. 2009; 2012). The AI-1 and AI-2 QS sytems are positive and

negative regulators of virulence, respectively, while deletion of A1-3 in SSU was shown

to affect attenuation of A. hydrophila in a septicaemic mouse model of infection,

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bacterial motility and biofilm formation (Khajanchi et al. 2012; Kozlova and Pekala

2012). It is also possible that the QseBC (A1-3) system may be linked to AI-1 and AI-2

QS systems in modulating bacterial virulence possibly through the cyclic diguanosine

monophosphate (Khajanchi et al. 2012).

Sulphur-containing AHLs act as QS inhibitors reducing protease production.

Interference with AHL-mediated quorum sensing is considered a promising target for

the development of a new generation of antimicrobial therapeutics and may represent an

important tool as a bacterial disease control measure in the aquaculture industry

(Defoirdt et al. 2005; Rasch et al. 2007; Khajanchi et al. 2009; Schwenteit et al. 2010).

Recently, a thermostable N-acyl homoserine lactonase derived from Bacillus strain

AI96 successufully attenuated A. hydrophila infection reducing zebrafish mortality (Cao

et al. 2012).

1.14. Biofilm formation

Aeromonas are efficient colonisers of surfaces and are an important constituent of

bacterial biofilms in both water distribution systems and food processing environments

(Chauret et al. 2001). The control of biofilm formation is of significant interest to the

industrial, public health and medical sectors. The ability of Aeromonas to form biofilms

may contribute to the persistence of these organisms in environmental reservoirs where

they exhibit increased resistance to normal bactericidal treatments (Lynch et al. 2002;

Rahman et al. 2007b). This is particularly significant in the food industry and

individuals residing along rivers where the presence of biofilm producing Aeromonas

spp. poses a serious danger to public health (Van Houdt and Michiels 2010; Odeyemi et al.

2012). Biofilm formation in food-processing environments has the potential to act as a

persistent source of microbial contamination leading to food spoilage or transmission of

disease (Chavant et al. 2007; Van Houdt and Michiels 2010). As a result, some studies

have been designed to demonstrate biofilm formation by Aeromonas on food produce

(Elhariry 2011) while others aimed to find products that can eliminate microbial

biofilms and their effective control in food industries (Farias Millezi et al. 2013).

Furthermore, the biofilm forming potential of these bacteria may pose a challenge

during treatment of infections associated with antimicrobial-resistant Aeromonas

species (Igbinosa et al. 2014).

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As mentioned in Section 1.13, biofilm formation is one of several virulence factors

regulated by quorum sensing in particular by the C4-HSL QS molecules (Lynch et al.

2002; Defoirdt et al. 2005). In addition to quorum sensing, biofilm formation has been

associated with hyperpiliation of the cells involving the type IV pili in A. caviae (Bechet

and Blodeau 2003), with the bundle-forming pilus in A. veronii bv. sobria (Hadi et al.

2012) and with the presence of both polar and lateral (the flaA+/lafA+ genotype) flagella

(Kirov 2003; Santos et al. 2010). Rahman et al. (2007b) showed that in A. veronii bv.

sobria the signalling molecule c-di-GMP is influenced by the GGDEF and EAL domain

proteins AdrA and YhjH, respectively. The GGDEF domain protein AdrA also

influenced the level of the C4-HSL QS molecule. Alterations in the c-di-GMP levels by

the GGDEF domain protein AdrA regulate the multicellular behaviour, biofilm

formation and adherence to plant and animal surfaces. Overproduction of c-di-GMP was

shown to modulate transcriptional levels of genes involved in biofilm formation and

motility phenotype in A. hydrophila SSU in a QS-dependent manner, involving both AI-

1 and AI-2 systems (Kozlova et al. 2012). A recent finding suggests that the T6SS

effector protein VgrG, discussed in section 1.8.8.4, is essential for biofilm formation in

A. hydrophila (Sha et al. 2013).

1.14. Additional virulence factors

A plethora of other virulence factors which may or may not contribute to the

pathogenesis of Aeromonas have been described in these organisms. Immunophilin-like

proteins encoded by the ilpA and fkpA genes in A. hydrophila have no known functions

and express proteins with no obvious virulent effects as shown in an animal model

(Wong et al. 1997). By contrast, over-expression of the dam gene in A. hydrophila SSU

influences the virulence of this organism by altering the expression of T3SS and T2SS-

associated Act protein, as well as affecting motility and proteinase production (Erova et

al. 2006). The sodA and sodB genes in A. hydrophila ATCC 7966T code for a Mn-SOD

(superoxide dismutase) and a Fe-SOD, respectively. Fe-SOD is essential for the aerobic

viability of the organism and prevents damage to DNA while Mn-SOD protects the

bacterial cells against environmental superoxide (Leclère et al. 2004). The collagenase

gene acg enhances the adhesive, invasive and cytotoxic ability of A. veronii RY001 on

ECP cells (Han et al. 2008). The glycolytic enzyme enolase identified in the diarrhoeal

strain A. hydrophila SSU was associated with its surface expression and its ability to

bind plasminogen. Moreover, the enolase gene could play a potentially important role in

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the viability of SSU (Sha et al. 2003, 2009). The synthesis of heat shock proteins is a

mechanism by which Aeromonas respond to thermal stress and confers protection to

aeromonads present in foods and food processing environments (Osman et al. 2011).

Although Aeromonas are generally considered non-capsulated organisms, the presence

of a capsule has been demonstrated in A. salmonicida and A. hydrophila serotypes O:11

and O:34 when grown in a glucose-rich medium (Martínez et al. 1995) and a capsule

gene cluster was identified in the whole genome of A. hydrophila PPD134/91 (Yu et al.

2005). The presence of group II capsules in A. hydrophila strongly correlates with the

serum and phagocytic survival activities of the organism in a fish model of infection

(Zhang et al. 2003). Finally, the role of cathepsin K in goldfish following A. hydrophila

infections has yet to be elucidated (Harikrishnan et al. 2010).

1.15. INFECTIONS CAUSED BY AEROMONAS SPP.

Human infections caused by Aeromonas species have been reported with increasing

frequency for the past 40 years although the exact prevalence of Aeromonas infections

on a global scale is unknown (Figueras 2005; Senderovich et al. 2012). The presence of

Aeromonas in the midgut of mosquitoes and the common housefly (Musca domestica)

represents a possible source of infection in cases where there is no exposure to

contaminated water, soil or foods (Nayduch 2001, 2002; Pidiyar et al. 2002). Although

gastroenteritis is the main condition associated with these organisms, many cases of

extraintestinal infections involving aeromonads have been described (Figueras 2005;

Parker and Shaw 2011). The rate of monomicrobial infections involving aeromonads

varies from 16 to 50% (Kelly et al. 1993; Tena et al. 2007). However, it has been

difficult to assess the role played by aeromonads in polymicrobial infections particularly

in cases where other recognized pathogens are concomitantly isolated.

Infections caused by Aeromonas can be serious and occasionally fatal in

immunocompromised patients (Harris et al. 1985; González-Barca et al. 1997).

Aeromonas-induced infections have been divided into four categories: (i) cellulitis or

wound infections associated with exposure to water or soil; (ii) septicaemia, usually

associated with hepatic, biliary or pancreatic disease or with malignancy; (iii) acute-

onset diarrheal disease of short duration; (iv) miscellaneous infections not associated

with any discernible physiological condition or environmental event (von Graevenitz

and Mensch 1968). The most important form of Aeromonas infection is sepsis as

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reflected by the large number of publications compiled on Aeromonas septicaemia

(Janda and Abbott 2010).

1.15.1. Gastroenteritis

The most common infection associated with Aeromonas species in humans is

gastroenteritis. The isolation rate for Aeromonas varies from <2 to 6.9% (von

Graevenitz and Mensch 1968; Rautelin et al. 1995b; Chan and Ng 2004; Pokhrel and

Thapa 2004). In tropical environments, the intestinal carriage can reach up to 30%

(Pitarangsi et al. 1982). During diarrhoeal disease the intestinal tract may be colonised

simultaneously with different Aeromonas strains (Kuijper et al. 1989b; Moyer et al.

1992).

After surviving the acid environment of the stomach and the small intestine (Karem et

al. 1994) Aeromonas must compete with the normal flora and survive the by-products

of metabolism and other compounds (Janda and Abbott 2010). Attachment to intestinal

epithelium is essential and bacterial flagella and pili play important roles in this step.

After attachment, the pathology involved depends on the elaboration of enterotoxins

causing enteritis, and dysentery or colitis if invasion of the gastrointestinal epithelium

has occurred (Janda and Abbott 2010). The diarrhoeal episode that follows is due to

exposure to the enterotoxins produced by Aeromonas, described in Section 1.9.9.4.

1.15.1.1. Disease presentation

The most common presentation observed in Aeromonas-induced intestinal infection is

watery diarrhoea (Figueras 2005). Patients experience fever, vomiting, abdominal

cramps/pain, dehydration and blood in the stools (Janda et al. 1983a). In 50% of the

cases, diarrhoea persists for more than 10 days and up to 30% require hospitalization.

Rarely, the clinical presentation is suggestive of ulcerative colitis (Gracey et al.

1982ab). Dysentery-like syndrome associated with Aeromonas has been sporadically

reported and often requires hospitalization (Rahman and Willoughby 1980; Vila et al.

2003). Abdominal cramps and pain, mucus and blood in the stools are common

symptoms of dysentery-like enteritis (Janda and Duffey 1988).

Much rarer is a cholera-like disease linked to Aeromonas (Shimada et al. 1984; Sawle et

al. 1986; Janda and Duffey 1988). The most compelling case of a cholera-like disease

involving aeromonads was described by Champsaur et al. (1982) in a Thai woman

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admitted to a Paris hospital. Among the clinical features observed included lethargy,

thirst, vomiting, dry mucous membranes, muscle cramps and rice-water diarrhoea. The

culpable organism, a strain of A. sobria (possibly A. veronii according to current

taxonomy), produced enterotoxin, cytolysin, proteolysin, haemolysin, and a cell-

rounding factor (Champsaur et al. 1982). Recently, A. caviae was recovered from the

stools of a 2 year old girl with a cholera-like illness in India (Jagadish Kumar and

Vijaya Kumar 2013).

Aeromonas is one of several micro-organisms implicated in travellers’ diarrhoea (TD), a

common health problem affecting travellers after visiting developing countries (Vila et

al. 2003; Gascón 2006). TD occurs globally and affects children as well as adults

(Gracey et al. 1984; Gascón et al. 1993; Hӓnninen et al. 1995a; Rautelin et al. 1995a;

Vila et al. 2003). In rare occasions, TD can be fatal (Sawle et al. 1986). Symptoms

associated with TD include watery and inflammatory diarrhoea, abdominal cramps and

fever (Vila et al. 2003). Severe atypical presentations following Aeromonas-induced

infection have been described including ulcerative and segmental colitis, ileal

ulceration, intra-mural intestinal haemorrhage with small bowel obstruction and

refractory inflammatory disease (Janda and Abbott 2010).

Haemolytic uraemic syndrome (HUS), a serious disease characterized by haemolytic

anaemia, acute kidney failure and thrombocytopaenia has been associated with

Aeromonas-induced enteritis (Bogdanović et al. 1991; Figueras et al. 2007a). Only a

few cases have been reported to date (Table 1.8). Clinical evidence indicates that

Aeromonas-related HUS is more responsive to treatment with antimicrobials compared

to HUS induced by enterohaemorrhagic E. coli EHEC (Fang et al. 1999). There are

reports that Aeromonas may contain Shiga toxin genes, typical of EHEC (Haque et al.

1996; Alperi and Figueras 2010).

1.15.1.2. Evidence against Aeromonas as an enteric pathogen

Despite the overwhelming data accumulated in the last 40 years, Aeromonas has yet to

be universally accepted as a bona fide enteric pathogen (Chu et al. 2006). The evidence

associating aeromonads with diarrhoea is circumstantial (Nishikawa and Kishi 1988;

Szabo et al. 2000). Aeromonas can be isolated from human faecal material in the

absence of diarrhoeal symptoms unlike other enteric pathogens (Küijper et al. 1987);

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Tab

le 1

.8

Clin

ical

cha

ract

eris

tics o

f pat

ient

s with

HU

S-as

soci

ated

Aer

omon

as

Age

/Sex

D

iarr

hoea

pr

odro

me/

bloo

d

Spec

ies

Site

of

isol

atio

n So

urce

of

isol

atio

n T

reat

men

t O

utco

me

Ref

eren

ces

NS

NS

A. so

bria

Fa

eces

N

S N

S Su

rviv

al

San

Joaq

uin

and

Pick

ett

(198

8)

23 m

/F

6d/y

esa

A. h

ydro

phila

Fa

eces

N

S Pe

riton

eal d

ialy

sis,

antih

yper

tens

ive

drug

s, pa

cked

re

d ce

ll tra

nsfu

sion

s

Surv

ival

B

ogda

novi

c et

al.

(199

1)

NS

NS

A. h

ydro

phila

Fa

eces

N

S N

S N

S R

obso

n et

al.

(199

2)

6 m

/F

7 d/

yesb

A. so

bria

Fa

eces

aq

uariu

m

wat

er

Hae

mod

ialy

sis,

rena

l tra

nspl

ant

Surv

ival

Fi

ller e

t al.

(200

0)

36 y

/M

2 m

ths/

yesb

A. h

ydro

phila

B

lood

se

afoo

d R

egul

ar h

aem

odia

lysi

s, an

tihyp

erte

nsiv

e dr

ugs,

cefti

zoxi

me

Surv

ival

Fa

ng e

t al.

(199

9)

40 y

/F

8 d/

no

A. v

eron

ii bv

. so

bria

Fa

eces

N

S C

ortic

oste

roid

s, fr

esh

froz

en

plas

ma,

cip

roflo

xaci

n Su

rviv

al

Figu

eras

et a

l. (2

007a

)

Mod

ified

from

Fig

uera

s et a

l. (2

007a

); a W

ater

y di

arrh

oea

beca

me

bloo

dy o

n da

y 7;

b W

ater

y di

arrh

oea

final

ly b

ecam

e bl

oody

; NS,

not

spec

ified

.

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there is no animal model for Aeromonas gastroenteritis and attempts to induce diarrhoea

in human volunteers and primates have, so far, been unsuccessful or inconclusive

(Pitarangsi et al. 1982; Morgan et al. 1985; Kirov 1993); the Henle-Koch postulates

have not been fulfilled including molecular postulates (Evans 1976; Falkow 2004); in

one study, no significant difference in the prevalence of virulence factors between

strains from diarrhoeic patients and controls were observed (Figura et al. 1986).

1.15.1.3. Evidence supporting Aeromonas as an enteric pathogen

Epidemiological evidence is strongly indicative that aeromonads are capable of causing

gastroenteritis. Aeromonas species have been recovered from diarrhoeal stools more

frequently than from control subjects (Holmberg and Farmer 1984; Agger et al. 1985;

Nishikawa and Kishi 1988; Deodhar et al. 1991; Rautelin et al. 1995b; Bravo et al.

2012). Enterotoxigenic strains have been isolated from children with diarrhoea (10.2%)

more often than those without (0.6%) diarrhoeal symptoms (Gracey et al. 1982b).

Isolation rates for aeromonads from diarrhoeic patients have been reported to be similar

to those of Salmonella enterica (Senderovich et al. 2012).

Despite assertions often made that there are no documented outbreaks due to

aeromonads (Nishikawa and Kishi 1988; Szabo et al. 2000), outbreaks involving

Aeromonas have been reported from several locations including enteritis due to A.

hydrophila in a neonatal intensive care unit in Germany (cited in Agger 1986), in a

pediatric hematology-oncology unit in Northen India involving A. sobria (Taneja et al.

2004), several Aeromonas species were associated with diarrhoeal disease in two

Brazilian studies (Guerra et al. 2007; Mendez-Marquez et al. 2012), a food poisoning

outbreak due to A. hydrophila in Sweden (Krovacek et al. 1995) and a small outbreak of

diarrhoeal infections occurred in Scotland (Nathwani et al. 1991) plus two outbreaks in

day care centres in the USA (de la Morena et al. 1993).

An immunological response from a healthy patient after severe Aeromonas-induced

diarrhoea strongly suggests that Aeromonas can behave as an enteric pathogen

(Palfreeman et al. 1983). The minimum inoculum necessary to induce diarrhoea by

Aeromonas was estimated at 104 cells ranking third behind Shigella (10-100) and

Campylobacter species (500-1000), respectively (Gascón 2006). The most likely

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scenario to date is that acute enteritis caused by aeromonads is strain-dependent (von

Graevenitz 2007).

1.15.1.4. Species involved

The most frequently isolated species from human faecal material are A. hydrophila, A.

caviae, and A. veronii (both biovars). Of these, A. caviae has been the most predominant

species reported by a number of studies (Altwegg 1985; Travis and Washington 1985;

Mégraud 1986; Kuijper et al. 1987; Wilcox et al. 1992; de la Morena et al. 1993;

Rautelin et al. 1995b; Bravo et al. 2012; Senderovich et al. 2012). Other species

including A. bestiarum, A. jandaei, A. media, A. schubertii, A. taiwanensis and A. trota

are sporadically isolated (Hӓnninen and Siitonen 1995; Pablos et al. 2010; Bravo et al.

2012; Senderovich et al. 2012). However, the enterotoxigenic potential of Aeromonas is

not species-specific (Singh and Sanyal 1992a) and Aeromonas-induced gastroenteritis is

not confined to a single genomospecies or biotype/genotype within a single taxon

(Albert et al. 2000).

1.15.2. Skin and soft-tissue infections (SSTIs)

Skin, soft tissue, muscle, and bone infections represent the second most common type

of infections caused by Aeromonas species (Janda and Abbott 2010). A high percentage

(60%) of infections involving aeromonads is polymicrobial (McCraken and Barkley

1972; Smith 1980a; Gold and Salit 1993) and although rare, infections involving more

than one Aeromonas species have been documented (Joseph et al. 1979, 1991). In many

cases, A. hydrophila is usually the most prevalent species (Gold and Salit 1993; Wu et

al. 2011; Chao et al. 2013). A recent study found that the clinical presentation between

patients with monomicrobial infection differed markedly from those with polymicrobial

SSTIs (Chao et al. 2013). Previously, Harris et al. (1985) reported no significant

differences between the clinical presentation, severity of disease, or outcome of patients

with either monomicrobial or polymicrobial infections. Usually, SSTIs are the result of

exposure to contaminated water or soil (von Gravenitz and Mensch 1968; Vally et al.

2004) and can affect both immunocompromised and healthy individuals (McCraken and

Barkley 1972; Smith 1980a; Lynch et al. 1981; Heckerling et al. 1983). Most

documented cases are the result of community-acquired infection, but nosocomially-

acquired infections particularly after surgery do occur (Lynch et al. 1981; Gold and

Salit 1993).

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Gold and Salit (1993) reported 11 cases of SSTIs caused by A. hydrophila and reviewed

the literature covering a period of 20 years (1973-1993). The clinical spectrum of

infections due to this organism included several forms of cellulitis, myonecrosis,

ecthyma gangrenosum, furunculosis, localized soft-tissue abscesses and skin nodules

suggesting that SSTIs involving Aeromonas can manifest in a wide range of clinical

presentations. Although wound infections caused by Aeromonas are not always fatal

(Gold and Salit 1993), in some cases, infection has resulted in serious complications

including death or amputation of affected limbs (Blatz 1979; Vally et al. 2004;

Abuhammour et al. 2006). Necrotizing fasciitis due to Aeromonas is rarely seen in

healthy individuals but has been reported in individuals with liver disease or

malignancy (Cui et al. 2007; Lee et al. 2008) and in patients with no prior contact with

aquatic animals or contaminated water (Ko et al. 2000).

1.15.3. Septicaemia

The most important form of Aeromonas infection is sepsis (Davis et al. 1978). Although

most infections caused by aeromonads are the result of exposure or ingestion of

contaminated soil, water, or food, in many cases the source of infection is unknown

(Harris et al. 1985; Roberts et al. 2006; Morinaga et al. 2011). A rare and severe case of

sepsis caused by A. hydrophila was reported in a patient with arthritis being treated with

the anti-arthritic agent tocilizumab (Okumura et al. 2011). The clinical manifestations

of Aeromonas septicaemia are similar to other Gram-negative bacilli including Vibrios

(Sirinavin et al. 1984; Park et al. 2011). Ko et al. (2005) compared the pathogenicity of

two bactaraemic isolates and showed that a strain of A. hydrophila Ah-2743 was more

pathogenic than Klebsiella pneumoniae p-129. The occurrence and 30-d fatality rate of

Aeromonas in patients with severe underlying conditions resembled those of P.

aeruginosa (Llopis et al. 2004). The mortality rate associated with Aeromonas

septicaemia in children and adults with and without underlying malignancies varied

from 55 to 75% (Ketover et al. 1973; Sirinavin et al. 1984; Janda and Duffy 1988).

Polymicrobial infections can range from 24 to 56% (Ko and Chuang 1995; Llopis et al.

2004; Tsai et al. 2006).

Janda and Abott (2010) categorized Aeromonas septicaemia into four groups (Table

1.9). Invariably in the majority of the cases patients have an underlying condition

involving the hepatobiliary system or malignancy (Janda and Brenden 1987; Ko and

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Chuang 1995; Janda and Abbott 1998; Llopis et al. 2004). In a retrospective study

involving 41 Taiwanese patients, the predominant haematological malignancies

associated with Aeromonas bacteraemia were acute myelogenous leukaemia (37.8%),

myelodysplastic syndrome (26.7%) and non-Hodgkin lymphoma (17.8%). Most patients

experienced fever (88.9%), septic shock (40%) and altered consciousness (26.7%). On

average, a fatal outcome was observed less than four days between the collection of

blood samples and death (Tsai et al. 2006).

Community-acquired Aeromonas septicaemia constitutes the majority of the cases

compared to nosocomial infection (Ko et al. 2000). Nosocomial infections can occur in

patients with no history of water exposure or cross-contamination by hospital

environment and health care workers (Ko et al. 2000). In most cases, the suspected

source is the patient’s own gastrointestinal tract (Harris et al. 1985; Roberts et al. 2006),

probably from injury due to antineoplastic chemotherapies or gastrointestinal

colonization (Sherlock et al. 1987; DePauw and Verweij 2005). In patients with

cirrhosis of the liver, spontaneous bacterial peritonitis, hypotension, diabetes mellitus

and high Pugh scores usually predict a fatal outcome (Ko and Chuang 1995). Patients

with a concomitant infectious focus and a high severity score at onset tend to perform

poorly and have a worse prognosis (Ko et al. 2000). In general, males tend to be more

affected than females and children (Sirinavin et al. 1984; Janda and Brenden 1987; Ko

et al. 2000; Llopis et al. 2004; Chuang et al. 2011).

The most predominant species recovered from blood are A. hydrophila, A. veronii bv.

sobria and A. caviae (Ketover et al. 1973; Sirinavin et al. 1984; Janda and Brenden

1987; Ko and Chuang 1995; Ko et al. 2000; Llopis et al. 2004). This is consistent with a

seven year retrospective Taiwanese study were 56% of the isolates belonged to A.

hydrophila, 29% to A. veronii bv. sobria and 14% to A. caviae. Furthermore, mortality

rates and acute physiology and chronic health evaluation II (APACHE II) scores

suggested that A. veronii bv. sobria and A. hydrophila bacteraemia was more severe

than bacteraemia due to A. caviae (Chuang et al. 2011).

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Tab

le 1

.9

Maj

or c

ateg

orie

s of A

erom

onas

sept

icae

mia

dis

ease

pre

sent

atio

n

Cat

egor

y G

roup

U

nder

lyin

g ri

sk

fact

ors

Prec

ipita

ting

even

ts

Port

al o

f ent

ry

Mor

talit

y (%

)

I Im

mun

ocom

prom

ised

pe

rson

s H

epat

obili

ary

dise

ase,

mal

igna

ncy

Rec

ent a

ntin

eopl

astic

ch

emot

hera

py, n

eutro

poen

ia

Gas

troin

test

inal

trac

t, so

ft tis

sue,

intra

-abd

omin

al ro

ute,

co

ntam

inan

ted

indw

ellin

g de

vice

s

32-5

5

II

Trau

ma

patie

nts

Can

var

y fr

om n

one

to m

ultip

le

cond

ition

s, in

clud

ing

diab

etes

Cru

sh in

jury

, pen

etra

ting

inju

ries,

near

-dro

wni

ng

even

ts, b

urns

Cut

aneo

us-s

ubcu

tane

ous

tissu

es, r

espi

rato

ry tr

act

60

III

Hea

lthy

pers

ons

Non

e ap

pare

nt

at

time

of p

rese

ntat

ion

Non

e no

ted

Unk

now

n <2

0

IV

Rec

onst

ruct

ion

surg

ery

patie

nts

Mal

igna

ncy,

tra

umat

ic in

jury

re

sulti

ng in

am

puta

tion

Med

icin

al le

ech

ther

apy

Tiss

ue fl

ap

<5

Mod

ified

from

Jand

a an

d A

bbot

t (20

10).

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1.15.4. Respiratory tract infections

Respiratory infections caused by Aeromonas are rare and the isolation of these bacteria

from the respiratory tract is usually considered a transient occurrence (Gonçalves et al.

1992; Takano et al. 1996; Janda and Abbott 1998; Bravo et al. 2003; Kao et al. 2003).

As in most infections associated with aeromonads, there is often a predisposing

underlying condition (Reines and Cook 1981; Baddour and Baselski 1988; Takano et al.

1996; Bravo et al. 2003). However, although rare, fulminant pneumonia has been

reported in healthy individuals as young as five-years old with no history of exposure or

consumption of water (Scott et al. 1978; Gonçalves et al. 1992; Kao et al. 2003; Nagata

et al. 2011). Haemoptysis is present in one half of cases (Scott et al. 1978; Reines and

Cook 1981; Gonçalves et al. 1992; Takano et al. 1996; Miyake et al. 2000). Infections

can be monomicrobial or polymicrobial and may be community or nosocomially-

acquired (Baddour and Baselski 1988). The mortality rate associated with aeromonads

has been estimated between 50 to 83% (Takano et al. 1996; Janda and Abbott 2010).

Many cases of Aeromonas-induced pneumonia were preceded by near-drowning events

(Reines and Cook 1981; Ender and Dolan 1997; Miyake et al. 2000; Mukhopadyay et

al. 2003; Bossi-Küpfer et al. 2007). Mortality rates as high as 60% have been reported

for aeromonad-related pneumonia in these cases (Ender and Dolan 1997). Both A.

veronii bv. sobria and A. hydrophila have been isolated from fatal cases (Mellersh et al.

1984; Miyake et al. 2000; Bossi-Küpfer et al. 2007). The latter species was isolated

from 19 specimens including 14 respiratory tract specimens at a hospital in Sheffield,

England (Mellersh et al. 1984) and also in pure culture from the pharyngeal exudate of a

59 year-old diabetic female with anaemia and pharyngitis (Tena et al. 2007). More

recently, a multiresistant strain of A. caviae was thought to be the caused of severe

pneumonia in a cancer patient (Yu et al. 2010).

1.15.5. Urogenital tract infections

Aeromonas species are rarely associated with urinary tract infections (UTIs) and very

few cases have been described (Filler et al. 2000; Hua et al. 2004; Al-Benwan et al.

2007; Figueras et al. 2007a). To date, the most cited cases of Aeromonas-induced UTIs

have involved young children. Both A. hydrophila and A. popoffii were isolated from a

neonate (Bartolome et al. 1989) and a 13 year-old boy suffering from spina bifida,

respectively (Hua et al. 2004). A serious case was described in a six-month old girl with

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diarrhoea who developed acute renal failure requiring dialysis and subsequently a renal

transplant. The source of the culpable bacterium, a haemolytic and cytotoxic A. sobria

strain was probably aquarium water or the bathtub (Filler et al. 2000). In adults, A.

caviae was isolated at 105 cells/ ml of urine in a 39 year-old male with symptoms of

cystitis and a history of frequency, dysuria, haematuria, and weight loss (Al-Benwan et

al. 2007). A 69 year-old diabetic male with chronic hepatitis and an indwelling device

developed UTI with A. veronii bv. sobria and bacteraemia with A. veronii bv. veronii.

After successful treatment with IV ceftriaxone the patient was discharged but re-

admitted a few weeks later with necrotizing fasciitis due to A. veronii bv. veronii

(Hsueh et al. 1998).

1.15.6. Intra-abdominal infections

Intra-abdominal infection is a broad term used to describe many different types of

infections such as peritonitis, pancreatitis, acute cholangitis and hepatic abscesses

(Janda and Abbott 2010). The majority of cases are community-acquired and males are

usually more affected than females (2:1 ratio) (Clark and Chenoweth 2003). In serious

Aeromonas infections such as liver abscesses the prognosis is usually poor in

immunocompromised individuals as a result of the underlying conditions (Colaco 1982;

Clark and Chenoweth 2003).

Peritonitis in adults is not uncommon and many cases have been reported for the last 30

years (Freij 1985; Khardori and Fainstein 1988; Muñoz et al. 1994; Ruíz de González et

al. 1994; Elcuaz et al. 1995; Ko and Chuang 1995). Peritonitis is usually a secondary

sequela to primary Aeromonas infection and in 75% of the cases has been associated

with bacteraemia (Muñoz et al. 1994; Ruíz de González et al. 1994; Elcuaz et al. 1995;

Ko and Chuang 1995). Cases of peritonitis have been described in children including

those with ruptured appendixes (Freij 1985; Khardori and Fainstein 1988) and in

patients undergoing continuous ambulatory peritoneal dialysis (Solaro and Michael

1986; Chang et al. 2005; Yang et al. 2008). The presence of Aeromonas as a cause of

peritonitis or liver abscess should alert clinicians to the potential presence of underlying

malignancies that may otherwise not been detected, a situation similar to the isolation of

Streptococcus bovis group and Clostridium septicum from blood as these organisms are

strongly associated with bowel malignancy (Bailey and Scott 1994).

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Huang et al. (2006) reviewed 49 cases of primary and secondary Aeromonas peritonitis

in a nine year period. Data from this study indicated that primary peritonitis occurred

more often in individuals (97%) with liver disease in which half of the cases were

accompanied by bacteraemia. In secondary peritonitis, 44% of the cases were health-

care associated infections. Most peritoneal cultures (85%) were polymicrobial in nature

usually involving members of the Enterobacteriaceae (Huang et al. 2006). The

mortality rates attributed to primary and secondary peritonitis in this series were 23 and

15%, respectively (Huang et al. 2006). These values differed from the 60% overall

mortality rate for aeromonad peritonitis reported by Muñoz et al. (1994). The gross

mortality rate for spontaneous bacterial peritonitis caused by A. hydrophila or A. veronii

was estimated at 56% (Wu et al. 2009).

A review of 39 cases indicated that hepatobiliary infections occurred in 71% of the

patients with cholangitis and 22% with cholecystitis. Complications in the latter group

included empyema or gangrene of the gallbladder while nine (22%) patients developed

liver abscesses (Clark and Chenoweth 2003). The overall mortality (24%) of this series

was higher than the 10% overall mortality reported by others (Chan et al. 2000).

Immunosuppression, malignancy and bile duct stones are the major predisposing

underlying conditions of intra-abodominal infections (Chan et al. 2000; Clark and

Chenoweth 2003). The consumption of freshwater fish and transmural migration from

the gastrointestinal tract has been identified as possible sources of infection (Solaro and

Michael 1986; Yang et al. 2008). The three major species A. hydrophila, A. caviae and

A. veronii have been frequently isolated while A. salmonicida was identified in a patient

with peritonitis undergoing continuous ambulatory peritoneal dialysis (Yang et al.

2008).

1.15.7. Infections due to medicinal leech therapy

Medicinal leeches (Hirudo medicinalis) are used to relieve venous congestion after

plastic and reconstructive surgery. Wound discharge is a common feature and most

infections respond to either antimicrobial therapy and/or debridement (Mercer et al.

1987). A retrospective study of 47 cases in Belgium showed that soft-tissue infections

after medicinal leech therapy were largely polymicrobial (Bauters et al. 2007).

Aeromonas hydrophila has frequently been isolated from wound samples following

leech therapy (Whitlock et al. 1983; Dickson et al. 1984; Mercer et al. 1987; Snower et

al. 1989; Fenollar et al. 1999), although cases involving A. caviae (Bauters et al. 2007)

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and A. sobria (Fenollar et al. 1999) have been described. Graf (1999b) used

biochemical and molecular methods to characterize Aeromonas isolated from the gut of

the leech revealing that A. veronii bv. sobria was the main bacterium present in the

digestive tract of the parasite. In a recent study, Laufer et al. (2008) isolated two distinct

Aeromonas species in Hirudo orientalis, namely, A. veronii and A. jandaei. The study

also revealed that these species could colonize the species Hirudo verbena.

The presence of bacteria other than aeromonas has been attributed to the leech being fed

contaminated blood or the failure to properly decontaminate the surface of the parasite

(Graf 1999b). Prophylactic therapy with an appropriate antimicrobial to protect patients

from infections caused by Aeromonas should be considered for individuals undergoing

medicinal leech treatment, in particular, immunocompromised patients (Bauters et al.

2007).

1.15.8. Meningitis

Cases of meningitis caused by Aeromonas species are very rare (Seetha et al. 2004).

Although most patients have a predisposing condition, meninigitis due to cranial

surgery (Qadri et al. 1976), as complication of medicinal leech therapy (Ouderkirk et

al. 2004) and cranial injury have been described (Pampín et al. 2012). However, in

some meninigits cases no obvious predisposing condition has been reported (Sirinavin

et al. 1984; Seetha et al. 2004). Most cases have been attributed to A. hydrophila

followed by A. veronii bv. sobria and in the majority of cases the organism was

recovered in cerebral spinal fluid. A fatal outcome was observed in 33% of the patients

(Parras et al. 1993).

1.15.9. Zoonotic infections

Aeromonas infections due to animal bites are an infrequent event. Most infections are

polymicrobial in nature and the contribution of aeromonads to these infections is not

totally clear (Janda and Abbott 2010). Aeromonas form part of the oropharyngeal flora

of reptiles and have been recovered from the mouth, fangs and venom of snakes (Jorge

et al. 1998; Janda and Abbott 2010). To date, A. hydrophila has been the species most

commonly isolated alone or in combination with other bacteria, from animal-related

infections including snake bites (Jorge et al. 1998), bear attack (Kunimoto et al. 2004),

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catfish (Murphy et al. 1992), alligator and crocodile bites (Raynor et al. 1983, Flandry

et al. 1989, Mekisic and Wardill 1992) and infections after shark attacks (Royle et al.

1997). Unusual cases of zoonotic infection include the isolation of A. hydrophila and

Peptostreptococcus species from a wound on the foot of an 11 year-old boy after

stepping on a stingray in a muddy river in Argentina (Pollack et al. 1998); abundant

growth of A. hydrophila was recovered from the wound of a 17 year old boy with cyclic

neutropoenia after being bitten by his pet piranha (Revord et al. 1988).

1.15.10. Burns

Aeromonas species are occasionally the cause of bacteraemia in burn patients and more

than 20 cases of Aeromonas infection following burn accidents have been recorded

since the 1980s including monomicrobial and polymicrobial infections (Ampel and

Peter 1981; Barillo et al. 1996; Purdue and Hunt 1996; Kienzle et al. 2000; Wilcox et

al. 2000; Chim and Song 2007; Lai et al. 2007). Burn injury may predispose to

immunosuppression making the host more susceptible to Aeromonas infections (Barillo

et al. 1996). Possible sources of contamination include extinction of a fire with dirty

water or by rolling on dirt (Purdue and Hunt 1996) and by immersion in water

immediately post burn (Kienzle et al. 2000). Exposure to water, however, has not

always been the source of these infections (Barillo et al. 1996). Infection with

Aeromonas following an electrical burn was described by Wilcox et al. (2000). All three

major species, A. hydrophila, A. caviae and A. veronii bv. sobria have been recovered

from burn patients. Infections harbouring more than one Aeromonas species have been

described (Kienzle et al. 2000). Irrespective of the species isolated, the mortality rate

associated with Aeromonas bacteraemia in burn patients is high (Lai et al. 2007).

1.15.11. Eye infections

Eye infections are extremely rare and usually occur as a result of injury or trauma

(Smith 1980ab, Cohen et al. 1983; Washington 1972, 1973) and to a lesser extent, by

wearing contaminated soft contact lenses (Pinna et al. 2004; Hondur et al. 2008). The

clinical manifestations associated with these infections include corneal ulceration,

endophthalmitis, conjunctivitis and keratitis (Feaster et al. 1978; Cohen et al. 1983; Puri

et al. 2003; Pinna et al. 2004; Khan et al. 2007; Sohn et al. 2007). Although Aeromonas

species have been involved in serious eye infections, the isolation of these organisms

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from eye specimens does not always indicate infection and may represent colonization.

Conjunctival colonization with A. hydrophila and H. influenzae without any evidence of

infection was reported in a 7 year-old boy after sustaining a penetrating injury to his eye

with a safety pin (Smith 1980ab). To date, all cases of endophthalmitis involving

aeromonads have been polymicrobial. Both A. hydrophila and P. shigelloides were

isolated from the anterior chamber fluid of an 8 year-old boy following a penetrating

injury by a fish hook (Cohen et al. 1983). In another case, A. hydrophila was isolated

with C. perfringens, Bacillus species, and coryneform bacteria after perforation of the

eye as a result of a dynamite explosion (Washington 1972).

1.15.12. Osteomyelitis and suppurative arthritis

Although rare, oesteomyelitis and suppurative arthritis involving Aeromonas have been

reported in both immunocompromised (López et al. 1968; Chmel and Armstrong 1976)

and healthy individuals (Blatz 1979; Karam et al. 1983). In immunocompromised

patients, the outcome of these infections can be fatal despite appropriate antimicrobial

therapy (Dean and Post 1967).

1.16. ANTIMICROBIAL SUSCEPTIBILITIES

Data on the antimicrobial susceptibility of Aeromonas has derived primarily from A.

hydrophila, A. caviae and A. veronii isolates (Ko et al. 1996; González-Barca et al.

1997). More recently, the antimicrobial susceptibility of less frequently isolated species

such as0 A. allosaccharophila, A. jandaei, A. schubertii, A. trota, A. popoffii and A.

veronii bv. veronii has been determined (Overman and Janda 1999; Soler et al. 2002;

Fosse et al. 2003a; Girlich et al. 2010). The agar dilution test has been the preferred

method and a good correlation between agar dilution and disk diffusion has been

reported (Koehler and Ashdown 1993). Previously, interpretative criteria for Aeromonas

were based on guidelines established for Pseudomonas, Acinetobacter or

Enterobacteriaceae (Koehler and Ashdown 1993; Overman and Janda 1999; Lupiola-

Gomez et al. 2003). New guidelines and interpretative criteria for Aeromonas are now

available for a handful of species (CLSI 2006; Jorgensen and Hindler 2007).

The antimicrobial susceptibility of Aeromonas species is predictable in most parts of the

world (Jones and Wilcox 1995). However, assessing the susceptibility patterns of

clinically significant isolates is highly recommended. Antimicrobial resistance may be

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strain-dependent and fatal outcomes have been associated with resistant strains

(González-Barca et al. 1997). Moreover, antimicrobials to which aeromonads are

intrinsically resistant have been administered in up to 20% of infections involving these

bacteria (Scott et al. 1978; Vila et al. 2002; Bravo et al. 2003; Figueras 2005).

Consequently, empirical antimicrobial therapy is usually inappropriate and

recrudescence of infections due to ineffective early treatment compromises or delays

patient’s recovery (Mellersh et al. 1984; Revord et al. 1988; Kelly et al. 1993; Al-

Benwan et al. 2007).

The isolation of multi-resistant strains from food, aquaculture, and other environs is of

clinical concern since these are potential sources of Aeromonas-induced infections

(Goñi-Urriza et al. 2000; Rhodes et al. 2000; Nawaz et al. 2010). Resistance patterns

have been reported in Aeromonas isolated from intestinal specimens, vegetables and

water sources (Pokhrel and Thapa 2004; Palu et al. 2006). Resistance observed in

environmental aeromonads may be related to the amount of pollution associated with

these environments since heavily polluted waters may contain multiple resistance

plasmids (Huddleston et al. 2006). In clinical isolates, antibiotic resistance has been

associated with heavily populated areas probably reflecting local antibiotic usage (Goñi-

Urriza et al. 2002).

Aeromonas can rapidly become resistant to multiple antibiotics, particularly to -

lactams, when exposed to substrates that allow for selection of mutant strains (Bakken

et al. 1988). With the exception of Asian isolates, world-wide resistance to tetracycline

and chloramphenicol in clinical isolates has been consistently low (Reinhard and

George 1985; Gosling 1986; Burgos et al. 1990; Koehler and Ashdown 1993). Asian

strains also tend to be less susceptible to cefamandole, cotrimoxazole, pipercillin,

imipenem and third generation cephalosporins (Chang and Bolton 1987; Ko and

Chuang 1995; Ko et al. 1996, 2000; Chan et al. 2000).

Aeromonas are universally resistant to penicillin, carbenicillin, erythromycin,

streptomycin and clindamycin (Jones and Wilcox 1995). Except for A. trota,

Aeromonas are intrinsically resistant to ampicillin although ampicillin-susceptible

strains belonging to species other than A. trota have been reported (Carnahan et al.

1991a; Abbott et al. 2003; Chan and Ng 2004; Huddleston et al. 2007; Figueira et al.

2011; Aravena-Román et al. 2012). Resistance to the aminoglycosides is low and while

most isolates are susceptible to gentamicin and amikacin tolerance to tobramycin has

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been increasingly recognized (Singh and Sanyal 1992a; Koehler and Ashdown 1993;

Kirov et al. 1995a; Ko et al. 1996; Overman and Janda 1999; Goñi-Urriza et al. 2000;

Vila et al. 2002; Aravena-Román et al. 2012).

1.16.1. -lactamases

Production of -lactamases is the most common mechanism of antimicrobial resistance

in Aeromonas. The secretion of multiple -lactamases by some strains is not unusual

(Zemelman et al. 1984; Bakken et al. 1988; Iaconis and Sanders 1990; Walsh et al.

1997; Fosse et al. 2004). Inducible chromosomally-mediated -lactamases with action

against penicillins, cephalosporins and carbapenems (Ambler class B, C and D) are

produced by Aeromonas (Table 1.10) (Iaconis and Sanders 1990; Segatore et al. 1993;

Rasmussen et al. 1994; Walsh et al. 1996; Yang and Bush 1996; Zhiyong et al. 2002;

Fosse et al. 2003a). For the most part, the biochemical, genetic and enzymatic

properties of Aeromonas-induced -lactamases derived from cloning experiments using

E. coli as a recipient (Hedges et al. 1985; Rasmussen et al. 1994; Rossolini et al. 1996;

Marchandin et al. 2003; Neuwirth et al. 2007). Significantly, conventional in vitro

susceptibility tests may sometimes fail to detect these β-lactamases compromising

therapeutic challenge (Chen et al. 2012).

-lactamases produced by Aeromonas can be susceptible to potassium clavulanate

although the combination of this inhibitor and ampicillin does not always result in lower

MICs to ampicillin. This is probably due to the intrinsic resistance of aeromonads to

ampicillin (Zemelman et al. 1984; Bakken et al. 1988). Further, exposure to ampicillin-

clavulanate has been associated with overproduction of chromosomal cephalosporinase

and imipenem resistance suggesting that the combination may induce multiresistance

(Sánchez-Céspedes et al. 2009). Changes in peptidoglycan composition (Tayler et al.

2010) and single point mutations alter -lactamase expression or induction in A.

hydrophila while derepression can lead to overexpression of multiple enzymes (Walsh

et al. 1997). Aeromonas species are considered the natural reservoir of class C

cephalosporinases and transposition genes that could be readily transfered to members

of the Enterobacteriaceae (Fosse et al. 2003c).

Metallo--lactamases (MBLs) are -lactamases active against carbapenems (Rasmussen

and Bush 1997). The first carbapenemase was detected in A. hydrophila (Shannon et al.

1986) and later found in A. veronii (both biovars) (Bakken et al. 1988).

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-84-

Tab

le 1

.10

-la

ctam

ases

pro

duce

d by

Aer

omon

as sp

ecie

s G

roup

C

lass

ifica

tion1

Fam

ily

Nam

e L

ocat

ion

Spec

ies

Ref

eren

ce

SBL

A

Car

beni

cilla

se

AER

-1

C

A. h

ydro

phila

H

edge

s et a

l. (1

985)

C

C

epha

losp

orin

ase

A1

C

A. h

ydro

phila

, A. s

obri

a Ia

coni

s and

San

ders

(199

0)

C

A

mpC

A

sbA

1 C

A.

jand

aei2

Ras

mus

sen

et a

l. (1

994)

D

O

XA

A

sbB

1 C

A.

jand

aei2

Ras

mus

sen

et a

l. (1

994)

D

Pe

nici

llina

se

Am

pH, A

mpS

C

A.

cav

iae,

A. v

eron

ii bv

. sob

ria,

A. h

ydro

phila

Fo

sse

et a

l. (2

003a

)

C

A

mpC

(FO

X-1

) C

AV

1 C

A.

cav

iae

Foss

e et

al.

(200

3c)

C

A

mpC

C

epS,

Cep

H

C

A. c

avia

e, A

. ver

onii

bv. s

obria

, A.

hyd

roph

ila

Foss

e et

al.

(200

3a)

A

TE

M

TEM

-1-li

ke,

TEM

-24

P A.

hyd

roph

ila, A

. cav

iae

Foss

e et

al.

(200

4)

Mar

chan

din

et a

l. (2

003)

A

C/P

A.

hyd

roph

ila, A

. cav

iae

Wu

et a

l. (2

011)

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-85-

Tab

le 1

.10

Con

tinue

d.

Gro

up

Cla

ssifi

catio

n1 Fa

mily

N

ame

Loc

atio

n Sp

ecie

s R

efer

ence

MB

L B

C

arba

pena

mas

es

A2h

, A2s

C

A.

hyd

roph

ila, A

. sob

ria

Iaco

nis a

nd S

ande

rs (1

990)

B

C

arba

pena

mas

es

Asb

M1

C

A. ja

ndae

i R

asm

usse

n et

al.

(199

4)

B

C

arba

pena

mas

es

Cph

A

C

A. h

ydro

phila

, A. v

eron

ii (b

oth

biov

ars)

, A. j

anda

ei

Foss

e et

al.

(200

3a)

B

C

arba

pena

mas

es

ImiS

C

A.

ver

onii

bv. s

obria

W

alsh

et a

l. (1

998)

B

IM

P IM

P-19

P

A. c

avia

e N

euw

irth

et a

l. (2

007)

B

V

IM

VIM

I

A. h

ydro

phila

Li

bisc

h et

al.

(200

8)

1 Am

bler

cla

ss; 2

Prev

ious

ly n

amed

as

A. s

obri

a; C

, chr

omos

omal

; P, p

lasm

id; I

, int

egro

n; A

sb, A

erom

onas

sob

ria

-lact

amas

es; C

AV

, fou

nd in

A.

cavi

ae; C

ep, c

hrom

osom

al c

epha

losp

orin

ase;

Cph

A, c

arba

pene

m h

ydro

lyzi

ng A

. hyd

roph

ila; I

miS

, im

ipen

emas

e fr

om A

. ver

onii

bv. s

obria

; IM

P,

activ

e on

imip

enem

; TEM

, nam

ed f

or p

atie

nt T

emon

eira

; VIM

, ver

ona

inte

gron

-enc

oded

met

allo

--la

ctam

ase;

SB

L, S

erin

e

-lact

amas

es; M

BL,

M

etal

lo-

-lact

amas

es; M

odifi

ed fr

om Ja

nda

and

Abb

ott (

2010

).

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-86-

MBLs have now been described in A. dhakensis, A. caviae, A. hydrophila, A. jandaei, A.

sobria and A. salmonicida (Rasmussen and Bush 1997; Wu et al. 2012). The most

common MBL found in aeromonads is CphA (Wu et al. 2012). Production of MBLs in

A. caviae is rare even in derepressed mutants suggesting that imipenem, aztreonam and

ceftazidime can be administered as an alternative therapy for infections caused by this

species (Walsh et al. 1997; Lupiola-Gómez et al. 2003). Imipenem resistance has been

reported in several Aeromonas species (González-Barca et al. 1997; Tena et al. 2007;

Wu et al. 2012) particularly A. jandaei and A. veronii (Overman and Janda 1999;

Sánchez-Céspedes et al. 2009; Figueira et al. 2011). Carbapenemase-producing strains

can only be detected when the inoculum size is increased since most isolates will be

susceptible to imipenem if a conventional inoculum is used (Shannon et al. 1986:

Rossolini et al. 1995; Wu et al. 2012). Although meropenem is largely more active than

imipenem against aeromonads (Clark 1992), the latter is recommended against

infections caused by strains overexpressing group-1 -lactamases (Lupiola-Gómez et al.

2003).

1.16.2. Extended-spectrum -lactamase (ESBL) production

The incidence of ESBLs in Aeromonas species is low. The first ESBL was described in

an A. caviae strain isolated from the diarrhoeaic stools of a 76 year-old man with

intestinal ischaemia in France (Marchandin et al. 2003). Fosse et al. (2004) described

the isolation of A. hydrophila recovered from the wound of an 87 year-old female with

necrotizing fasciitis that simultaneously produced class A, B, C and D -lactamases.

Surprinsingly, in both cases, the aeromonads were concomitantly isolated with an E.

aerogenes strain that harboured a 180 kb TEM-24 plasmid. ESBLs were also detected

in A. hydrophila isolated from the blood of a three year-old boy with bacteraemia and

diarrhoea (Rodríguez et al. 2005) and from A. caviae recovered from the sputum of a 68

year-old male with oesophageal cancer (Ye et al. 2010).

ESBL-producing aeromonads have been recovered from clinical, environmental and

mussel isolates with one fatal case reported among those isolated from human clinical

material. Nosocomially-acquired infection was diagnosed in four patients, two from the

community while the source of the remaining two was unknown. The majority of the

patients had an underlying malignancy (Table 1.11). The isolation of ESBL-producer

aeromonads from blood represents a serious clinical concern and the presence of these

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-87-

Tab

le 1

.11

ESB

L-pr

oduc

ing

Aero

mon

as sp

ecie

s

Clin

ical

Spec

ies

Spec

imen

G

ende

r/ag

e (y

ears

) C

linic

al d

iagn

osis

Ref

eren

ce

A. c

avia

e St

ool

M/7

8 In

test

inal

isch

aem

ia

Mar

chan

din

et a

l. (2

003)

A. h

ydro

phila

W

ound

F/

87

Nec

rtizi

ng fa

sciit

is

Foss

e et

al.

(200

4)

A. h

ydro

phila

B

lood

M

/3

Bac

tera

emia

, pne

umon

ia

Rod

rigue

z et

al.

(200

5)

A. c

avia

e Sp

utum

M

/68

Oes

opha

geal

can

cer

Ye

et a

l. (2

010)

A. h

ydro

phila

B

lood

F/

70

Bac

tera

emia

, han

d ph

lebi

tis

Wu

et a

l. (2

011)

A. c

avia

e

Blo

od

M/5

5 Pr

imar

y ba

cter

aem

ia

Wu

et a

l. (2

011)

A. c

avia

e B

lood

M

/52

Prim

ary

bact

erae

mia

W

u et

al.

(201

1)

A. c

avia

e B

lood

F/

65

Bac

tera

emia

, han

d ph

lebi

tis

Wu

et a

l. (2

011)

Env

iron

men

tal/O

ther

A. m

edia

A

ctiv

e sl

udge

Fou

nd in

:

S

witz

erla

nd

Picä

o et

al.

(200

8)

A. a

llosa

ccha

roph

ila

Riv

er w

ater

Fr

ance

G

irlic

h et

al.

(201

0)

A. h

ydro

phila

R

iver

sedi

men

t

C

hina

Lu

et a

l. (2

010)

A. h

ydro

phila

/A. c

avia

e M

usse

l

C

roat

ia

Mar

avić

et a

l. (2

013)

Mod

ified

from

Wu

et a

l. (2

011)

; M, m

ale;

F, f

emal

e.

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-88-

enzymes should be excluded from isolates with a cefotaxime-resistant profile (Wu et al.

2011). ESBL-encoding genes were recently detected in 21 (14%) (13 A. caviae and

eight A. hydrophila) isolates, with bla (CTX-M-15) identified in 19 and bla (SHV-12) in

12 isolates in Aeromonas isolated from wild-growing Mediterranean mussel (Mytilus

galloprovincialis) in the eastern coast of the Adriatic Sea, Croatia. Of these, bla (CTX-

M-15) was located on conjugative IncFIB-type plasmids in A. caviae isolates (Maravić

et al. 2013).

The detection of ESBLs in Aeromonas prompted examination of environmental sources

resulting in the screening for multi-drug resistance bacteria in different water

environments. These findings suggest that Aeromonas can act as either recipient or

vectors of resistant elements from other Gram-negative bacteria particularly from the

Enterobacteriaceae (Fosse et al. 2003c). The current procedure used to detect the

presence ESBLs is based on the clavulanate-based synergy (double-disk) technique

usually applied to the Enterobacteriaceae (Fosse et al. 2004; Rodríguez et al. 2005).

Genotypic confirmation of the presence of ESBLs can also be determined by PCR or

ESBL sequencing (Maravić et al. 2013).

1.16.3. Plasmid-mediated resistance

Although antimicrobial resistance in Aeromonas is largely chromosomally mediated

(Ianconis and Sanders 1990; Lupiola-Gomez et al. 2003), plasmids harbouring

resistance genes have been described in several species (Rhodes et al. 2000, 2004;

Cattoir et al. 2008). Plasmids of variable MWs with the ability to confer resistance to

both antimicrobials and metals have been recovered from Aeromonas isolated from

water, food and human sources (Huddlestone et al. 2006; Palu et al. 2006). Plasmids

encoding resistance genes can be disseminated between different bacterial species under

natural conditions (Rhodes et al. 2000). Class I and 2 integrons have been reported in

strains associated with beef cattle in Australia. Thus, it is possible that the environment

is likely to act as reservoir and disseminator of integron-containing bacteria in beef

(Barlow and Gobiuos 2009). Class I integrons have been detected in A. veronii isolated

from catfish suggesting that this species may act as the reservoir for integrons and

putative pathogenic genes (Nawaz et al. 2010). Class 1 integrons have also been

detected in environmental members of the Enterobacteriaceae. Quinolone resistance

due to plasmid-mediated genes found in the Enterobacteriaceae have been identified in

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Aeromonas isolated from environmental and clinical sources (Cattoir et al. 2008;

Sánchez-Céspedes et al. 2008).

1.16.4. Quinolones

Quinolones are among the most effective antimicrobial agents used against Aeromonas

infections and fluoroquinolones have shown excellent in vitro activity against most

species (Reinhard and George 1985; Ko et al. 1996, 2003). Among the

fluoroquinolones, ciprofloxacin proved to be the most effective against murine A.

hydrophila infections (Ko et al. 2003). Resistance to quinolones has been linked with

mutations of the gyrA gene and is associated with reduced susceptibility to nalidixic

acid (Chang and Bolton 1987; Goñi-Urriza et al. 2002; Vila et al. 2002). Rhodes et al.

(2000) showed that resistance to nalidixic acid was source-dependent. Human-derived

strains were more resistant than aquaculture strains while in waste water isolates

resistance was nearly five times more prevalent than surface water isolates. Ozonation

of water may reduce quinolone resistance and increase production of metallo--

lactamase (Figueira et al. 2011).

Elevated MIC values due to quinolone resistance in A. hydrophila, A. caviae and A.

sobria have been associated with mutations in type II toposisomerases of gyrA, gyrB,

parC and parE genes which contain a quinolone resistance-determining region (Goñi-

Urriza et al. 2002; Sinha et al. 2004). Mutations in these genes are attributed to double-

or single-amino acid substitutions conferring a high resistance to fluoroquinolones

(Sinha et al. 2004). Other quinolone-resistant mechanisms described in Aeromonas

include a reduction on the level of uptake or an active efflux system (Poole 2000).

1.16.5. Genes encoding for antimicrobial resistance

The distribution of resistance genes in Aeromonas varies among the species. The cepS

gene is almost universally present in A. veronii, A. hydrophila and A. caviae while the

frequency of the amps gene appears to be strain-dependent (Walsh et al. 1997). The

asbA1 and asbB1 genes which encode class C and D -lactamases, respectively, have

been detected in A. jandaei (Rasmussen et al. 1994). CphA is encoded by cphA and has

been detected in A. dhakensis, A. veronii (both biovars), A. hydrophila, A. jandaei and

A. salmonicida (ssp. salmonicida and achromogenes) but not in A. caviae, A. trota or A.

schubertii (Rossolini et al. 1996; Walsh et al. 1997; Wu et al. 2012). Despite

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harbouring the cphA gene, some species are unable to express MBL activity suggesting

that genetic modifications capable of silencing the gene exist (Rossolini et al. 1995; Wu

et al. 2012). Two integron-borne MBLs have been identified in Aeromonas isolated

from stools in France and Hungary. The genes blaIMP-19 gene and blaVIM-4 encoding for

IMP-19 and VIM were detected in A. caviae and A. hydrophila, respectively (Neuwirth

et al. 2007; Libisch et al. 2008). VIM which confers resistance to all -lactam

antibiotics except aztreonam could not be detected by the MBL Etest and only disks

with and without EDTA or PCR would demonstrate the presence of the enzyme and

gene respectively (Libisch et al. 2008).

ESBLs encoded by the blaTEM , blaPER , blaCTX-M, and blaSHV genes can be found in both

chromosomes or plasmids. Of these, blaPER-3, which is rarely described in Aeromonas,

has been detected in three strains (Picȁo et al. 2008; Girlich et al. 2010; Wu et al. 2011)

and in a single isolate also harbouring the blaCTX-M-15 gene in mussel (Maravić et al.

2013). Quinolone resistance is mediated by the qnrS2 gene and has been detected in A.

punctata (A. caviae) and A. veronii strains isolated from water and clinical sources,

respectively (Cattoir et al. 2008; Sánchez-Céspedes et al. 2008). QnrS2 can be

transferred from Aeromonas species to E. coli TPO10 with the consequent increase of

MICs of quinolones and fluoroquinolones (Cattoir et al. 2008).

1.16.6. Antimicrobial usage: recommendations

Several recommendations regarding the usage or testing of antimicrobials directed

against aeromonads have been proposed. Fluoroquinolones should not be used in

treating paediatrics patients (Overman and Janda 1999) or in infections caused by

Aeromonas resistant to nalidixic acid (Vila et al. 2002). Tobramycin, imipenem and

cefoxitin should be omitted as alternative therapies due to the high resistance shown by

certain species to these antimicrobials (Overman and Janda 1999). A lack of clinical

usefulness precludes testing susceptibility to ampicillin, carbenicillin and cephalothin

(Overman and Seabolt 1983). Aeromonas species carrying carbapenemase-encoding

genes should be considered resistant to this antimicrobial class until confirmation is

performed (Rossolini et al. 1996; Wu et al. 2012). Multi-resistant A. hydrophila strains

isolated from children with acute diarrhoea were reported from India. However, these

results were misleading as some antibiotics tested (vancomycin, bacitracin, methicillin

and novobiocin) are not those usually used to treat Aeromonas in routine clinical

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settings (Subashkumar et al. 2006). Finally, resistance to quinolones is possibly due to

over-prescription of this antimicrobial class in some locations (Sinha et al. 2004).

1.17. CONCLUSIONS

With the advent of molecular methods, the taxonomy of aeromonads has progressed

considerably in the last two decades. The nomenclature issues, a conflict that has

besieged bacteriologists for some time, could be simply resolved by acknowledging

both senior and junior nomenclature in final reports. For example, any isolate identified

as A. caviae or A. trota should be reported as A. punctata (jun. syn. A. caviae), and A.

enteropelogenes (jun. syn. A. trota), respectively. In this way, all nomenclatures would

be simultaneously recognized without compromising clinical information. More than

100 cases of Aeromonas infections involving both immunocompromised and

immunocompetent individuals of all age groups have been published since 1999

(Chopra and Houston 1999b; Figueras 2005). This continuously growing evidence

supports the notion that Aeromonas can no longer be considered merely opportunistic

pathogens despite the failure to reproduce disease in an animal model.

Despite these drawbacks, evidence strongly suggests that infections caused by

Aeromonas may be strain dependent and that some species may contain more virulent

strains than others. This is perhaps the most important concept associated with

Aeromonas pathogenicity in the long history of the genus. Thus, unlike other recognized

and well-established pathogens such as S. pyogenes or S. Typhi where every strain can

cause infection and a disease state, in Aeromonas only certain strains appear to do so.

This is particularly evident in A. caviae and its association with infant gastroenteritis,

which suggests that this species should be considered a human pathogen. Figueras

(2005) stated that the use of commercial identification systems incorrectly contributed

to the establishment of A. hydrophila as the cause of most cases of infections involving

Aeromonas. This is consistent with data from recent studies which reveal that A.

hydrophila is not one of the most predominant species when identification of isolates is

based on molecular methods, and that the prevalence of other species is beginning to

emerge (Aravena-Román et al. 2011b; Puthucheary et al. 2012). Data from future

surveys will determine more accurately the real prevalence of pathogenic species, and

Aeromonas species in general, if genotypic methods are used in the identification of

these organisms. In contrast, in the aquaculture industry, Aeromonas species are

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recognized bona fide pathogens of many fish species, as supported by the blue gourami

and zebrafish models of infection and every effort is made to prevent or decrease the

impact of these organisms by the fish industry.

Definitive identification of these organisms should be confined to those isolates deemed

to be significant or the source of an outbreak, while isolates not fitting these criteria

should be reported to genus level only (Abbott et al. 2003; Figueras 2005). Genotypic

identification allows detection of infrequently isolated Aeromonas species and

molecular sequences of several housekeeping genes of strains representing all type and

many reference strains have been deposited in GenBank. The case described by Fontes

et al. (2010) provided the perfect example of how an Aeromonas species that had not

been isolated since its original description was found within five years of its discovery.

In 2004, Harf-Monteil et al. proposed A. simiae based on two strains isolated from the

faeces of healthy monkeys. Fontes et al. (2010) isolated this species from a pig sample

while determining the prevalence of Aeromonas in pig slaughterhouses in Portugal. The

isolate was identified on the basis of 16s rRNA, gyrB and rpoD sequencing.

Janda and Duffey (1988) recommended that identification of mesophilic aeromonads

must become more standardised before meaningful comparisons can be made between

studies carried out at a various locations throughout the world. This remains an

important recommendation and ideally, a set of guidelines that include selected

phenotypic and genotypic tests, standard incubation conditions and media should be

globally adopted. Moreover, primers and testing conditions should be adhered to

eliminate or keep laboratory variations to a minimum (Ørmen et al. 2005).

Thornley et al. (1997) stated that from a diagnostic point of view, it would be highly

desirable to be able to recognize pathogenic strains of Aeromonas from non-pathogenic

ones, an idealistic concept that, at the present time, is not yet feasible. This statement is

supported by a recommendation to establish a collection of well-defined strains

representing all known clinically relevant Aeromonas species including strains of

known pathogenicity in different animal models (Janda and Abbott 2010). This

recommendation merits consideration and should be supported.

Finally, the ubiquitous nature of aeromonads, the widespread presence of virulence

factors and antimicrobial resistance genes, and the potential for severe symptoms to

occur, reinforce the notion that aeromonads recovered from human clinical material

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should be treated as potential pathogens. This is particularly relevant in

immunocompromised individuals and those at extreme ages. At the present time, the

significance of Aeromonas species isolated from human specimens can only be assessed

on clinical grounds.

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CHAPTER 2: MATERIALS AND METHODS

2.1. MATERIALS

2.1.1. Chemicals and reagents

All standard laboratory chemicals and reagents and their suppliers are listed in Table

2.1.

2.1.2. Solutions

Sterile distilled water and physiological saline (0.85% NaCl) solutions were obtained

from Excel (Perth, Australia). Ultrapure distilled water used in the preparation of PCR

master mixtures was obtained from Fisher-Biotec (Perth, Australia). Deionized water

was prepared in-house by the Hepatitis Laboratory, PathWest, Nedlands, using a MilliQ

filter system (Millipore ®, Australia).

2.1.2.1. DepC-treated water

Four hundred microlitres of a 0.1 % (v/v) diethyl pyrocarbonate (depC) solution was

added to 400 ml high purity water, stirred and incubated o/v at 37C. The mixture was

then autoclaved at 15 psi for 60 min.

2.1.2.2. Ethidium bromide (10 mg/ml)

One gram of ethidium bromide was added to 100 ml of water and stir for several hours

until dissolved. The bottle containing the solution was wrapped in aluminium foil and

kept in stored at 2 to 8C.

2.1.2.3. Chemical lysis stock solution

This solution was used to extract DNA and was prepared in-house by staff from the

PCR Laboratory (PathWest, Nedlands). The solution consisted of 50 ml 0.5M NaOH;

12.5 ml 10% SDS and 437.5 ml high pure water and was stored in 50 l aliquots at

room temperature.

2.1.2.4. HCCA matrix solution

The HCCA solution was prepared fresh by mixing 475 l of UPW, 500 l ACN and 25

l of 100% TFA in an Eppendorf tube and thoroughly vortex to produce a volume of 1

ml.

2.1.3. Bacteriological media

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Bacteriological media used in this study were manufactured by Excel, Perth (Australia)

and are listed in Table 2.2.

2.1.4. Gas chromatography

All gases used in the detection of FAMEs were obtained from BOC (Victoria,

Australia) and included instrumental air, and ultra high purity (99.999% purity)

hydrogen and nitrogen.

2.1.5. Antimicrobials

Antimicrobial used in this study and their suppliers are listed in Table 2.3.

2.1.6. Bacterial strains

Reference and type strains including culture collection designation and provider are

listed in Table 2.4. Strains used as positive and negative controls in biochemical tests

are listed in Table 2.5. Clinical and environmental isolates used in this research

including location and source of isolation are listed in Tables 2.6 and 2.7, respectively.

Clinical isolates were collected from 1988 to 2008 while environmental isolates were

collected from 1998 to 2008 from rural and metropolitan regions of Western Australia,

the largest state in Australia covering an area of 2.5 Km2.

Isolates used in virulence studies were collected between 2002 and 2008 and were

isolated from rural and metropolitan areas of Western Australia. Clinical isolates were

collected from 46 males and 43 females while the gender of 9 patients was not

available. The age of the patients ranged from 5 months to 89 years. Isolates used in

virulence studies were randomly selected which were previously identified by extensive

conventional biochemical testing and a selection of genotypic targets namely 16S rRNA

and housekeeping genes sequences.

2.1.7. Primers

Primers used in this research are listed in Table 2.8. All primers were manufactured by

Fisher Biotec (Perth, Australia).

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-97-

Table 2.1 Chemicals and reagents used in this project

Chemical/Reagent

Supplier

Acetonitrile Bruker Daltonik

Adonitol Sigma

Agarose Scientifix

Amygdalin Sigma

Andrade’s indicator Sigma

L-arabinose Sigma

Bovine serum albumin Sigma

PE buffer II Applied Biosystems

Cellobiose Sigma

Clinitest tablet Bayer Diagnostics

p- dimethylaminocinnamaldehyde Sigma-Aldrich

Deoxynucleoside triphosphate Applied Biosystems

Diethyl pyrocarbonate Sigma

Ethanol (HPLC grade) BDH

Ethidium bromide Sigma-Aldrich

10% Ferric chloride aq. soln. Excel

Ferrous ammonium sulphate (1% w/v aq. soln.) Excel

Formic acid Bruker

Glucose Sigma

Glucose-1-phosphate Sigma

Glucose-6-phosphate Sigma

Glycerol Sigma

Hydrochloric acid (HCl) (6N) Mallinkrodt

Hexane (HPLC grade) Merck

Hydrogen peroxide Ajax Finechem

m-Inositol Sigma

Indole (Spot) Excel

Indole (Kovacs’) Excel

DL-lactate Excel

Lactose Sigma

Lactulose Sigma

Page 124: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-98-

Table 2.1 Continued.

Chemical/Reagent

Supplier

Lugol’s iodine Amber Scientific

Magnesium chloride (MgCl2) Applied Biosystems

Maltose Sigma

D-mannitol Sigma

D-mannose Sigma

Melibiose Sigma

Methanol (HPLC grade) Merck

-Methyl-D-glucoside Sigma

Methyl-tert butyl ether (HPLC grade) Mallinkrodt

-Nitrophenyl--D-galactopyranoside Rosco

Pyrrolidonyl--naphthalimadase Remel

Raffinose Sigma

L-rhamnose Sigma

Salicin Sigma

Sodium dodecyl sulfate Bio-Rad

Sodium hydroxide pellets (NaOH) (ACS grade) Merck

Sodium hydroxide (bacterial lysis solution) Thermal Fisher

D-sorbitol Sigma

Sucrose Sigma

Taq polymerase Applied Biosystems

Tetramethyl-p-phenylenediamine dihydrochloride Becton Dickinson

Trifluoroacetic acid Bruker Daltonik

Voges-Proskauer reagent1 Excel

Voges-Proskauer reagent2 Excel

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-99-

Table 2.2 Bacteriological media used in this project

Media

Supplier

acetate slant Excel

aesculin Excel

arginine broth Excel

citrate (Simmon’s) Excel

citrate (Hänninen’s) Excel

CLED agar Excel

CNA agar Excel

enriched lauryl sulphate agar (50 mm) Excel

DNA agar Excel

gelatine cysteine thiosulfate Excel

gelatine Excel

gluconate Excel

heart infusion agar Excel

heart infusion broth Excel

horse blood agar Excel

Jordan’s tartrate Excel

DL-lactate Excel

lipase Excel

lysine broth Excel

malonate Excel

motility medium Excel

Mueller-Hinton agar Excel

nutrient agar Excel

nutrient agar plus heat-killed S. aureus cells Excel

nutrient agar plus 0.2 % NaCl and 0.1% SDS Excel

nutrient agar plus 0.33% w/v elastin Excel

NaCl broth (0 and 3%) Excel

ornithine broth Excel

peptone water Excel

peptone water (¼ strength) Excel

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-100-

Table 2.2 Continued.

Media

Supplier

phenylalanine deaminase Excel

pyrazinamidase slants Excel

sheep blood agar Excel

sterile distilled water Excel

sterile saline (0.85% NaCl) Excel

starch agar Excel

trypicase soy broth Excel

TSBA agar Excel

tyrosine Excel

urea (Christensen’s) Excel

urocanic acid Excel

VP medium Excel

Page 127: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-101-

Table 2.3 Antimicrobial agents used in this project

Antimicrobial Supplier

amikacin Sigma

amoxicillin GlaxoSmithKline

amoxicillin-clavulanate GlaxoSmithKline

ampicillin (E-strip) BioMérieux

aztreonam Bristol-Myers Squibb

cefazolin Sigma

cefepime OmegaPharm

cefoxitin Sigma

ceftazidime Sigma

ceftriaxone Sigma

cephalothin Sigma

ciprofloxacin MP Biomedicals

colistin sulphate (polymyxin E) Fluka

colistin sulphate (polymyxin E) (E-strip) BioMérieux

deferoxamine Rosco

2,4-diamino-6,7-diisopropylpteridine Oxoid

doxycycline AB Biodisk

gentamicin Pfizer

meropenem (E-strip) BioMérieux

meropenem AstraZeneca

moxifloxacin Bayer

nalidixic acid Fluka

nitrofurantoin Sigma

norfloxacin Sigma

pipercillin-tazobactam Sigma

tetracycline MP Biomedicals

ticarcillin-clavulanate GlaxoSmithKline

tigecycline (E-strip) BioMérieux

tobramycin MP Biomedicals

trimethoprim MP Biomedicals

sulfamethoxazole Sigma

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-102

-

Tab

le 2

.4

Type

and

refe

renc

e st

rain

s use

d in

this

pro

ject

Spec

ies

St

rain

no.

O

ther

des

igna

tion(

s)

A. a

llosa

ccha

roph

ila

ATC

C 5

1208

T C

ECT

4199

T , LMG

140

59T , C

CU

G 3

1218

T

A. d

hake

nsis

* C

ECT

7289

T D

SM 1

8362

T

A. a

ustr

alie

nsis

C

ECT

8023

T LM

G 2

6707

T

A. b

estia

rum

A

TCC

511

08T

CD

C 9

533-

76T , C

ECT

4227

T , LM

G 1

3444

T , Pop

off 2

18T

A. b

ival

vium

C

ECT

7113

T LM

G 2

3376

T

A. c

avia

e A

TCC

154

68T

CEC

T 83

8T , LM

G 3

775T , P

opof

f 545

T

A. c

avia

e A

TCC

131

36T

CEC

T 42

26T , P

opof

f 267

T

A. c

ulic

icol

a C

ECT

5761

T M

TCC

324

9T , DSM

176

76T , C

IP 1

0776

3T

A. d

iver

sa

CEC

T 42

54T

ATC

C 4

3946

T , CD

C 2

478-

85T ; L

MG

173

21T

A. e

nche

leia

D

SM 1

1577

T C

ECT

4342

T , ATC

C 5

1929

T , NC

IMB

134

42T , L

MG

163

30T

A. e

ucre

noph

ila

ATC

C 2

3309

T C

ECT

4224

T , LM

G 3

774T , N

CIM

B 7

4T , Pop

off 5

46T

A. fl

uvia

lis

CEC

T 74

01T

LMG

246

81T

*Pre

viou

sly

clas

sifie

d as

A. a

quar

ioru

m

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-103

-

Tab

le 2

.4

C

ontin

ued.

Spec

ies

St

rain

no.

O

ther

des

igna

tion(

s)

A. h

ydro

phila

subs

p. h

ydro

phila

A

TCC

7966

T C

ECT

839T , D

SM 3

0187

T , Pop

off 5

43T

A. h

ydro

phila

subs

p. d

hake

nsis

LM

G 1

9562

T C

CU

G 4

5377

T , DSM

176

89T

A. h

ydro

phila

subs

p. ra

nae

LMG

197

07T

CC

UG

462

11T , D

SM 1

7695

T

A. ja

ndae

i A

TCC

495

68T

CEC

T 42

28T , A

1642

T , LM

G 1

2221

T

A. m

edia

A

TCC

339

07T

CEC

T 42

32T , L

MG

907

3T , NC

IMB

223

7T

A. m

ollu

scor

um

DSM

170

90T

CEC

T 58

64T , L

MG

222

14T

A. p

isci

cola

C

ECT

7443

T LM

G 2

4783

T

A. p

opof

fii

CIP

105

493T

CEC

T 51

76T , L

MG

175

41T , C

CU

G 3

9350

T , ATC

C B

AA

-243

A. ri

vuli

C

ECT

7518

T D

SM 2

2539

T

A. sa

lmon

icid

a sp

p. sa

lmon

icid

a C

ECT

894T

ATC

C 3

3658

T , CIP

103

209T , L

MG

378

0T

A. sa

lmon

icid

a ss

p. a

chro

mog

enes

C

ECT

895T

ATC

C 3

3659

T , LM

G 1

4900

T , NC

IMB

111

0T

A. sa

lmon

icid

a ss

p. m

asou

cida

C

ECT

896T

ATC

C 2

7013

T , CIP

103

210T , L

MG

378

2T

A. sa

lmon

icid

a ss

p. p

ectin

olyt

ica

DSM

126

09T

34 m

elT

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-104

-

Tab

le 2

.4

C

ontin

ued.

Spec

ies

Stra

in n

o.

Oth

er d

esig

natio

n(s)

A. sa

lmon

icid

a ss

p. sm

ithia

C

IP 1

0475

7T NC

IM A

TCC

493

93T , C

ECT

5179

T

A. sa

nare

llii

CEC

T 74

02T

CIP

110

203T , L

MG

246

82T

A. sc

hube

rtii

ATC

C 4

3700

T C

ECT

4240

T , LM

G 9

074T , C

DC

244

6-81

T

A. si

mia

e D

SM 1

6559

T C

IP 1

0779

8T , CC

UG

473

78T

A. so

bria

C

IP 7

433T

CEC

T 42

45T , P

opof

f 208

T , ATC

C 4

3979

T , LM

G 3

783T , C

DC

953

8-76

T

A. so

bria

C

DC

954

0-76

LM

G 1

3469

A. ta

iwan

ensi

s C

ECT

7403

T LM

G 2

4683

T

A. te

cta

CEC

T 70

82T

DSM

173

00T

A. tr

ota

ATC

C 4

9657

T C

ECT

4255

T , A16

46T , L

MG

122

23T

A. v

eron

ii bi

ovar

sobr

ia

ATC

C 9

071

CEC

T 42

46, N

CIM

B 3

7, L

MG

378

5

A. v

eron

ii bi

ovar

ver

onii

DSM

738

6T A

TCC

356

24T , C

ECT

4257

T

Aero

mon

as sp

p. H

G11

C

ECT

4253

A

TCC

359

41, N

CIM

B 1

3014

Page 131: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-105-

Table 2.5 Type and reference strains used as positive and negative controls

Species

Designation

Aeromonas hydrophila ATCC 7966T

Bacillus subtilis ATCC 6633

Moraxella catarrhalis ATCC 25238T

Corynebacterium xerosis ATCC 9016

Enterococcus faecalis ATCC 29212

Escherichia coli ATCC 25922

Escherichia coli K12

Klebsiella pneumoniae ATCC 700603

Proteus mirabilis ATCC 12453

Proteus vulgaris NCTC 4635

Pseudomonas aeruginosa PA01

Pseudomonas aeruginosa ATCC 27853

Salmonella paratyphi ATCC 9150

Staphylococcus aureus ATCC 25923

Streptococcus agalactiae ATCC 12386

Vibrio parahaemolyticus ATCC 43996

Yersinia enterocolitica ATCC 27729

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-

Tab

le 2

.6

Clin

ical

stra

ins u

sed

in th

is p

roje

ct

Stra

in

Sour

ce

Loc

atio

n St

rain

So

urce

L

ocat

ion

Stra

in

Sour

ce

Loc

atio

n

21

Unk

now

n PM

H

75

Blo

od

SCG

H

102

Stoo

l SC

GH

23

W

ound

PM

H

77

Wou

nd

Roc

king

ham

10

3 St

ool

Car

narv

on

24

Wou

nd

PMH

78

C

APD

flui

d SC

GH

10

4 W

ound

D

erby

25

Sh

unt

PMH

79

W

ound

A

lban

y 10

5 St

ool

SCG

H

26

Unk

now

n PM

H

80

Blo

od

Roc

king

ham

10

6 B

lood

SD

H

27

App

endi

x PM

H

81

Blo

od

SCG

H

107

Wou

nd

Car

narv

on

28

Stoo

l SC

HG

83

Sp

utum

SC

GH

10

8 St

ool

Arm

adal

e 47

Sp

utum

SC

HG

84

B

lood

SD

H

109

Blo

od

Arm

adal

e 56

B

one

chip

s M

andu

rah

85

Blo

od

SCG

H

110

Blo

od

SCG

H

57

Blo

od

SCG

H

86

Wou

nd

Pinj

arra

11

1 B

lood

SC

GH

58

B

lood

SC

GH

87

B

lood

K

algo

orlie

11

2 W

ound

SC

GH

59

B

lood

SC

GH

88

W

ound

SC

GH

11

3 D

rain

flui

d SC

GH

60

B

lood

SD

H

89

Bile

SC

GH

11

4 St

ool

Arm

adal

e 61

B

iliar

y st

ent

SCG

H

90

Wou

nd

SCG

H

115

Stoo

l A

lban

y 62

T-

tube

tip

SCG

H

91

Wou

nd

Ger

aldt

on

116

Wou

nd

Arm

adal

e 65

B

lood

SC

GH

92

C

yst

SCG

H

117

Wou

nd

Arm

adal

e 66

W

ound

SC

GH

93

U

rine

New

man

11

8 Sp

utum

SC

GH

67

W

ound

A

rmad

ale

94

Stoo

l N

arro

gin

120

Stoo

l A

rmad

ale

68

Blo

od

SCG

H

95

Wou

nd

SDH

12

1 W

ound

SD

H

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-107

-

Tab

le 2

.6

Con

tinue

.

Stra

in

Sour

ce

Loc

atio

n St

rain

So

urce

L

ocat

ion

Stra

in

Sour

ce

Loc

atio

n

69

Wou

nd

Bus

selto

n 96

B

lood

SC

GH

12

3 A

bsce

ss

SCG

H

70

Blo

od

Arm

adal

e 97

St

ool

SCG

H

124

Wou

nd

Ger

aldt

on

71

Wou

nd

Col

lie

98

Pus

Arm

adal

e 12

5 B

lood

Po

rt H

edla

nd

72

Blo

od

Bun

bury

99

St

ool

SCG

H

126

Wou

nd

Kun

unur

ra

73

Wou

nd

SCG

H

100

Stoo

l SD

H

127

Wou

nd

Col

lie

74

Wou

nd

SCG

H

101

Ulc

er

Col

lie

128

Wou

nd

Byf

ord

129

Wou

nd

Ger

aldt

on

156

Stoo

l PM

H

187

Stoo

l M

arga

ret R

iver

13

0 W

ound

A

rmad

ale

158

Stoo

l D

enm

ark

188

Bile

SC

GH

13

1 B

lood

A

rmad

ale

159

Wou

nd

Exm

outh

18

9 St

ool

Hal

ls C

reek

13

3 M

ortu

ary

SCG

H

163

Wou

nd

Bus

selto

n 19

0 W

ound

M

anjim

up

134

Ear

New

man

16

4 Sp

utum

SC

GH

20

0 B

lood

PM

H

135

Blo

od

Car

narv

on

165

Unk

now

n N

orse

man

21

1 Ea

r B

ridge

tow

n 13

6 St

ool

Bus

selto

n 16

6 St

ool

Bas

send

ean

212

Wou

nd

New

man

13

7 St

ool

SCG

H

167

Wou

nd

Bus

selto

n 21

3 W

ound

B

oddi

ngto

n 13

8 Tr

ache

a

SCG

H

168

Wou

nd

SCG

H

214

Faec

es

Nor

tham

13

9 St

ool

SDH

16

9 St

ool

Stirl

ing

215

Faec

es

Swan

Vie

w

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-108

-

Tab

le 2

.6

Con

tinue

d.

Stra

in

Sour

ce

Loc

atio

n St

rain

So

urce

L

ocat

ion

Stra

in

Sour

ce

Loc

atio

n

140

Perit

onea

l flu

id

SCG

H

171

Sput

um

SCG

H

216

Faec

es

Kat

anni

ng

141

Toe

nail

Exm

outh

17

2 U

rine

N

ewm

an

217

Faec

es

Osb

orne

Par

k 14

2 St

ool

PMH

17

4 W

ound

Ex

mou

th

218

Blo

od

PMH

14

3 B

urn

PMH

17

5 St

ool

Arm

adal

e 21

9 Fa

eces

W

athe

roo

144

Blis

ter

FH

176

Wou

nd

SCG

H

220

Wou

nd

P. H

edla

nd

145

Blo

od

FH

177

Wou

nd

SCG

H

221

Blo

od

SCG

H

146

Wou

nd

FH

178

Bile

SC

GH

26

9 B

lood

SC

GH

14

7 B

urn

PMH

17

9 St

ool

Unk

now

n 27

0 W

ound

To

m P

rice

148

Ulc

er

FH

180

Stoo

l M

anjim

up

278

Wou

nd

Der

by

149

Blo

od

FH

181

Stoo

l SC

GH

27

9 W

ound

D

erby

15

0 Ti

ssue

FH

18

2 W

ound

C

ollie

151

Blo

od

FH

183

Stoo

l B

oddi

ngto

n

152

Blo

od

FH

184

Stoo

l SD

H

15

3 St

ool

Col

lie

185

Wou

nd

SCG

H

15

4 B

lood

C

ollie

18

6 W

ound

D

erby

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-109

-

Tab

le 2

.7

Envi

ronm

enta

l stra

ins u

sed

in th

is p

roje

ct

Stra

in

So

urce

L

ocat

ion

Stra

in

Sour

ce

Loc

atio

n

29

Mul

let f

ish

Path

Wes

t 24

1 W

ater

U

nkno

wn

30

Bar

ram

undi

A

DW

A

242

Wat

er

Unk

now

n 31

G

oura

mi

AD

WA

24

3 W

ater

U

nkno

wn

32

Fish

A

DW

A

244

Wat

er

Unk

now

n 33

K

oi

AD

WA

24

5 W

ater

U

nkno

wn

34

Fish

A

DW

A

246

Wat

er

Unk

now

n 35

Fi

sh le

sion

A

DW

A

247

Wat

er

Unk

now

n 19

9 C

rab

Car

narv

on

250

Wat

er

Unk

now

n 22

2 C

horin

ated

wat

er

Serp

entin

e su

pply

MW

A

251

Wat

er

Unk

now

n 22

3 W

ater

U

nkno

wn

252

Wat

er

Unk

now

n 22

4 B

orew

ater

W

anne

roo

25

3 W

ater

U

nkno

wn

225

Res

ervo

ir ra

w

Sout

h D

anda

lup

254

Wat

er

Unk

now

n 22

6 W

ater

N

olla

mus

a 25

5 W

ater

U

nkno

wn

227

Res

ervo

ir ra

w

Nor

th D

anda

lup

256

Wat

er

Unk

now

n

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-110

-

Tab

le 2

.7

Con

tinue

d.

Stra

in

So

urce

L

ocat

ion

Stra

in

Sour

ce

Loc

atio

n

228

Trea

ted

wat

er

Salte

r Poi

nt

257

Wat

er

Unk

now

n 22

9 Tr

eate

d w

ater

A

pple

cros

s 25

8 Ir

rigat

ion

wat

er

Unk

now

n 23

0 W

ater

M

WA

John

Wal

listo

n 25

9 W

ater

U

nkno

wn

231

Sche

me

wat

er

City

of M

elvi

lle

260

Wat

er

Unk

now

n 23

2 W

ater

Th

omps

on re

serv

oir

261

Irrig

atio

n w

ater

U

nkno

wn

233

Wat

er

Unk

now

n 26

2 W

ater

U

nkno

wn

234

Ret

icul

atio

n M

undi

jong

26

3 Ir

rigat

ion

wat

er

Unk

now

n 23

5 W

ater

U

nkno

wn

264

Irrig

atio

n w

ater

U

nkno

wn

236

Wat

er

Unk

now

n 26

5 Ir

rigat

ion

wat

er

Unk

now

n 23

7 W

ater

U

nkno

wn

266

Irrig

atio

n w

ater

D

alw

allin

u 23

8 W

ater

U

nkno

wn

267

Irrig

atio

n W

ater

U

nkno

wn

239

Wat

er

Unk

now

n 26

8 Ir

rigat

ion

wat

er

Unk

now

n 24

0 W

ater

U

nkno

wn

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-111

-

Tab

le 2

.8

Prim

ers u

sed

in th

is p

roje

ct

Gen

e Pr

imer

sequ

ence

(5’ →

3’)

Pr

oduc

t siz

e (b

p)

Ref

eren

ce

gyrB

7F

: GG

GG

TCTA

CTG

CTT

CA

CC

AA

14

R: T

TGTC

CG

GG

TTG

TAC

TCG

TC

960

- 110

0 Y

añez

et a

l. (2

003)

rpoD

70

Fs: A

CG

AC

TGA

CC

CG

GTA

CG

CA

TGTA

70

Rs:

ATA

GA

AA

TAA

CC

AG

AC

GTA

AG

TT

820

Sole

r et a

l. (2

004)

aerA

/hae

m

F: C

CTA

TGG

CC

TGA

GC

GA

GA

AG

R

: CC

AG

TTC

CA

GTC

CC

AC

CA

CT

431

Sole

r et a

l. (2

002)

aexT

F:

GG

CG

CTT

GG

GC

TCTA

CA

C

R: G

AG

CC

CG

CG

CA

TCTT

CA

G

535

Bra

un e

t al.

(200

2)

alt

F: A

AA

GC

GTC

TGA

CA

GC

GA

AG

T R

: AG

CG

CA

TAG

GC

GTT

CTC

TT

320

Agu

ilera

-Arr

eola

et a

l. (2

005)

ascV

F:

ATG

GA

CG

GC

GC

CA

TGA

AG

TT

R: T

ATT

CG

CC

TTC

AC

CC

ATC

CC

71

0 C

hacó

n et

al.

(200

4)

aspA

F:

CA

CC

GA

AG

TATT

GG

GTC

AG

G

R: G

GC

TCA

TGC

GTA

AC

TCTG

GT

350

Sole

r et a

l. (2

002)

ast

F: A

TCG

TCA

GC

GA

CA

GC

TTC

TT

R: C

TCA

TCC

CTT

GG

CTT

GTT

GT

504

Agu

ilera

-Arr

eola

et a

l. (2

005)

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-112

-

Tab

le 2

.8

Con

tinue

d.

Gen

e Pr

imer

sequ

ence

(5’ →

3’)

Pr

oduc

t siz

e (b

p)

Ref

eren

ce

BfpA

F:

CC

GC

AG

GTG

TGA

TGTT

TTA

C

R: T

GC

GG

TGTT

ATT

GTT

TGC

T 25

1 Se

chi e

t al.

(200

2)

BfpG

F:

ATG

CC

AA

AG

CTG

AC

TGG

TCT

R

: GA

CA

TGA

TTC

CC

GTT

ATA

AA

23

3 Se

chi e

t al.

(200

2)

flaA

F: T

CC

AA

CC

GTY

TGA

CC

TC

R: G

MY

TGG

TTG

CG

RA

TGG

T 60

8 Se

n an

d R

odge

rs (2

004)

lafA

F:

CC

AA

CTT

(T/C

)GC

(C/T

)TC

(T/C

)(C

/A)T

GA

CC

R

: TC

TTG

GTC

AT(

G/A

)TTG

GTG

CT(

C/T

)

737

Agu

ilera

-Arr

eola

et a

l. (2

005)

stx-

1 F:

ATA

AA

TTG

CC

ATT

CG

TTG

AC

TAC

R

: AG

AA

CG

CC

CA

CTG

AG

ATC

ATC

180

Pato

n an

d Pa

ton

(199

8)

stx-

2 F:

GG

CA

CTG

CTT

GA

AA

CTG

CTC

C

R: T

CG

CC

AG

TTA

TCTG

AC

ATT

CTG

255

Pato

n an

d Pa

ton

(199

8)

vasH

F:

CTC

TAG

AC

CG

GTG

AA

CC

CA

TCA

AG

CG

CG

TCC

AC

T R

: TC

CC

CC

CG

GG

CTG

GTG

GC

CA

GC

AG

CA

GA

GG

CA

ATA

16

52

Suar

ez e

t al.

(200

8)

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- 113 -

Table 2.9 Aeromonas strains used in virulence studies

Species No. of strains

Source Strain number

A. allosaccharophila 1 Stool 100 A. dhakensis 31 Wound 67, 71, 73, 79, 91, 95, 104, 107, 141,

176, 220, 279 Blood 60, 70, 154 Stool 169, 180, 183, Sputum 47 Fish 31, 32 Bone chips 56 Urine 93 Water 223, 229, 230, 235, 241, 242, 256, 257 A. australiensis 1 Irrigation water 266 A. bestiarum 1 Blood 68 A. caviae 27 Wound 143, 163, 270 Blood 57, 58, 65, 75, 80, 96, 106, 109, 110, 200 T-tube tip 62 Stool 94, 102, 103, 156, 158, 187, 216 CAPD fluid 78 Bile 178, 188 Peritoneal fluid 140 Water 264 Fish 30 A. hydrophila 29 Wound 23, 69, 90, 98, 101, 112, 117, 126, 128,

148 Blood 59, 84, 149, 151, 152 Stool 133 Biliary stent 61 Fish 34 Sputum 83, 118 Drain fluid 113 Bile 89 Tissue 150 Water 231, 243, 245, 260, 261, Pancreas cyst 92 A. jandaei 3 Fish 35 Water 253, 262

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-- 114 --

Table 2.9 Continued.

Species No. of strains

Source Strain number

A. media 2 Blood 85 Stool 179 A. salmonicida 2 Wound 190 Crab 199 A. schubertii 1 Wound 186 A. veronii bv. sobria 31 Wound 24, 66, 129, 147, 174, Blood 72, 81, 111, 125, 131, 218, 221, 269 Stool 99, 137, 166, 184, 189, 215, 219 Shunt 25 Appendix 27 Sputum 171 Fish 33 Water 224, 237, 247, 254, 259, 265, 268

Total 129

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-- 115 --

2.2. METHODS

2.2.1. Bacterial culture methods

All isolates were stored at 70C in 5% serum-glycerol medium. Working cultures for

identification purposes were subcultured onto HBA and incubated at 35C in air.

Isolates were subcultured three times before they were used for biochemical testing.

Working cultures for antimicrobial susceptibility testing and detection of virulence

genes were subcultured onto HBA only once and incubated at 35C. Broth cultures

were prepared by inoculating one single colony into a 10 ml TSB or HIB tube followed

by o/v at 35C without shaking.

2.2.2. Acid production from carbohydrates Carbohydrates used in this project are listed in Table 2.1. Carbohydrate fermentation

reactions were performed in a peptone water base (Oxoid, Basingstoke, UK) containing

1% (w/v) of the desired sugar and 1% (v/v) Andrade’s indicator. Sugars were obtained

from Sigma (St. Louis, Mo. USA). Carbohydrate-containing broths were inoculated

with a drop from an overnight culture and incubated at 35C in air for up to seven days.

A change in the colour of the broth from blue to yellow denoted acid production

(Abbott et al. 2003).

2.2.3. Hydrolysis of aesculin Aesculin hydrolysis was determined by inoculating a broth containing aesculin that was

incubated at 35C for up to seven days in air. A blackening of the broth was considered

a positive reaction (Cowan and Steel 1993).

Positive control: Enterococcus faecalis ATCC 29212

Negative control: Streptococcus agalactiae ATCC 12386

2.2.4. Alkylsulfatase activity

Alkylsulfatase activity was determined by spot inoculating a nutrient agar plate

containing 0.2% NaCl and 0.1% SDS with an overnight culture. The plate was

incubated in air at 35ºC for up to seven days. A turbid halo surrounding the growth was

indicative of alkylsulfatase activity (Abbott et al. 2003).

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2.2.5. Detection of a CAMP-like factor

Detection of a CAMP-like factor was determined by inoculating two sheep blood agar

plates with a single, diametric streak of S. aureus ATCC 25923 (-toxin-producing

strain). Tests strains were streak-inoculated at right-angles to but not touching the

staphylococcal inoculum. The plates were incubated aerobically and anaerobically at

35C overnight. A positive result was indicated by production and diffusion of a

completely clear area shaped like an arrow head in the zone of discolouration caused by

the -toxin (Figura and Guglielmetti 1987).

Positive control: Streptococcus agalactiae ATCC 12386

Negative control: Enterococcus faecalis ATCC 29212

2.2.6. Catalase activity

Catalase activity was determined by emulsifying a 24 colony grown in nutrient agar, in

3% hydrogen peroxide on a glass slide and observing for gas production (MacFaddin

1976). Immediate bubbling was considered a positive reaction (Cowan and Steel 1993).

Positive control: Staphylococcus aureus ATCC 25923

Negative control: Streptococcus pyogenes ATCC 19615

2.2.7. DNase activity DNase activity was determined by inoculating a plate containing 0.2% DNA and 0.01%

Toluidine Blue O with an overnight culture that was incubated at 35ºC for up to 7 days.

A clear pink zone around the inoculum indicated the production of extracellular

deoxyribonuclease (Schreier 1969).

Positive control: Moraxella catarrhalis ATCC 25238

Negative control: Escherichia coli ATCC 25922

2.2.8. Elastase activity

Elastase activity was determined by spot inoculating a plate containing 0.33% (w/v)

elastin with an overnight culture that was incubated in air at 35ºC for two days. If no

clear zone was detected after 48 h incubation which indicated a positive reaction, the

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-- 117 --

plates were further incubated at room temperature for up to seven days (Rust et al.

1994).

Negative Control: Escherichia coli K12

Positive Control: Pseudomonas aeruginosa PAO1

2.2.9. Gas from glucose A broth containing 1% glucose and fitted with a Durham tube was inoculated with a

drop from an overnight broth culture and incubated in air at 35ºC for 24 h. When gas

was produced it was trapped at the top of the Durham tube forming a bubble. Glucose

was fermented when the broth turned from green to yellow after overnight incubation

(Abbott et al. 2003).

2.2.10. Gelatin hydrolysis

Gelatin hydrolysis was determined by inoculating tubes containing gelatin with a heavy

inoculum from an overnight culture. Tubes were incubated at 35C in air for up to seven

days. Gelatin hydrolysis was indicated by the development of a pink to red colour

(Pickett et al. 1991).

2.2.11. Oxidation of potassium gluconate

Oxidation of potassium gluconate was determined by inoculating a tube containing

gluconic acid with a drop from an overnight culture. Tubes were incubated at 35C for

48 h. After the incubation period a Clinitest tablet (Bayer Diagnostics, Bridgend, UK)

was added. A positive reaction was denoted by a light-green to rusty-yellow colour. A

negative reaction was indicated by a deep blue colour (Pickett and Pedersen 1970).

2.2.12. Ability to grow on TCBS medium

A TCBS plate was inoculated with a drop from a HIB broth and incubated in air at 35C

for 24 h (Bailey and Scott’s 1994). Any growth was considered a positive result.

Positive control: V. parahaemolyticus ATCC 43996

Negative control: Escherichia coli ATCC 25922

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-- 118 --

2.2.13. -Haemolysis activity

-haemolytic activity was determined by streaking a portion of a 5% (v/v) sheep blood

agar plate and incubating at 35ºC overnight in air. Clearing around the inoculum was

evidence of red cell haemolysis (Bailey and Scott’s 1994).

2.2.14. Production of hydrogen sulfide from cysteine

The medium designed by Veron and Gasser (1963) was used to detect the production of

hydrogen sulfide from cysteine. Tubes were inoculated from an 18-24 h TSA culture

and then incubate at 35ºC for up to seven days in air. A positive reaction was indicated

by a diffuse blackening of the medium radiating from the stab line.

2.2.15. Production of indole from tryptophan

2.2.15.1. Rapid spot indole method

A portion of a colony was spread onto a filter impregnated with p-

dimethylaminocinnamaldehyde and incubated at room temperature for 2 min. A blue

colour indicated a positive result (MacFaddin 1976).

2.2.15.2. Kovacs’method

A peptone water broth was inoculated with a drop from an overnight culture and

incubated at 35C for 48 h. A drop of Kovacs’ reagent was added and the tube shaked

slightly. The development of a red colour denoted a positive reaction (Cowan and Steel

1993).

Positive control: Escherichia coli ATCC 25922

Negative control: Klebsiella pneumoniae ATCC 700603

2.2.16. Jordan’s Tartrate test

A well-isolated colony from a pure, 18-24 h culture growing on HBA was stabbed

deeply to about one-fourth inch from the bottom of the tube. Tubes were incubated

aerobically, with caps loosened, at 35ºC for up to 72 h in air (Edwards and Ewing

1972). A positive result occurred when a yellow colour developed in the lower portion

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-- 119 --

of the tube while the surface zone remained red. Negative test: no colour change; the

medium remained alkaline with a red colour throughout the tube.

Positive Control: Escherichia coli ATCC 25922

Negative Control: Salmonella paratyphi ATCC 9150

2.2.17. Lipase activity

Lipase activity was determined by using corn oil as substrate based on the recipe by

Hugo and Beveridge (1962). Using a young agar HIA slant culture as a source of

inoculum, a line of inoculation was made from the bottom to the top of the slant. The

tubes were incubated at 35ºC in air and observed daily for seven days. Positive reactions

were indicated by the development of a dark blue colour in the medium, in the growth

or both.

2.2.18. Utilization of malonate

Utilization of malonate was determined by inoculating a broth containing malonate with

an overnight culture that was incubated at 35C in air for up to two days. A blue colour

indicated a positive reaction (Cowan and Steel 1993).

Negative control: Escherichia coli ATCC 25922

Positive control: Klebsiella pneumoniae ATCC 700603

2.2.19. Amino acid degradation

The Moeller’s method was used to determine lysine and ornithine decarboxylase and

arginine dehydrolase activity. Tubes containing these amino acids and a control tube

without any amino acid were inoculated with an overnight broth culture grown at 35C

without shaking, sealed with paraffin oil and incubated for up to four days before

discarding. The media first became yellow due to acid production from the glucose;

later, if decarboxylation or dehydroxylation occurred, the medium became purple

indicating a positive reaction. The control tube remained yellow (Cowan and Steel

1993).

Negative control: Proteus vulgaris NCTC 4635

Positive control: Aeromonas hydrophila ATCC 7699

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-- 120 --

2.2.20. Motility

2.2.20.1. Wet mount method

A drop from a trypticase soy broth incubated o/v at 35C was placed onto a clean glass

slide, covered and observed under phase contrast. Displacement of the bacterial cells in

the medium was considered a positive reaction (Cowan and Steel 1993).

2.2.20.2. Motility medium method

Motility was determined by inoculating motility medium by slightly stabbing the

surface of the agar to a depth no greater than 5 –7 mm. The tube was incubated for up to

seven days before discarding. Growth radiating away from the site of inoculum and

spreading throughout the tube was indicative of motility (Cowan and Steel 1993).

Positive control: Pseudomonas aeruginosa ATCC 27853

Negative control: Klebsiella pneumoniae ATCC 700603

2.2.21. ONPG activity

Detection of the enzyme -nitrophenyl--D-galactopyranoside was determined by

preparing a dense bacterial suspension (4 MacFarland) in 0.25 ml sterile saline. A

tablet containing the substrate (Rosco Diagnostics, Taastrup, Denmark) was added and

the tube sealed. After 4 h incubation at 35C in air, a positive reaction was indicated by

the development of a deep yellow colour as per manufacturer’s instructions (Rosco,

Taastrup, Denmark).

Positive control: Escherichia coli ATCC 25922

Negative control: Proteus mirabilis ATCC 12453

2.2.22. Oxidase activity

Oxidase activity was determined by rubbing a 24 colony onto the surface of a filter

paper impregnated with fresh tetramethyl-p-phenylenediamine dihydrochloride. The

appearance of a purple colour within 5 seconds denoted a positive reaction (Isenberg

1992).

Positive control: Pseudomonas aeruginosa ATCC 27853

Negative control: Escherichia coli ATCC 25922

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-- 121 --

2.2.23. Phenylalanine deaminase activity

A slant containing the amino acid phenylalanine was inoculated with a colony from an

overnight culture and incubated at 35C for 24 h in air. After o/v incubation a few

drops of 0.2 ml 10% (aq. soln.) ferric chloride were added. A strong green colour that

developed within one minute was considered a positive reaction (Cowan and Steel

1993).

Negative control: Escherichia coli ATCC 25922

Positive control: Proteus mirabilis ATCC 12453

2.2.24. Pyrazinamidase activity Tubes containing pyrazinamide were inoculated from an overnight culture and

incubated at 35ºC for two days in air. The slopes were flooded with freshly made 1%

(w/v) aqueous ferrous ammonium sulfate and examined for the presence of pyrazoic

acid. Positive pyrazinamidase activity was indicated by a pinkish rusty colour. Lack of

activity resulted in a colourless reaction after 15 minutes (Carnahan et al. 1990).

Negative control: Yersinia enterocolitica ATCC 27729

Positive control: Corynebacterium xerosis ATCC 9016

2.2.25. Pyrrolidonyl--naphthylamide activity

Commercially obtained filter paper discs were impregnated with L-pyrrolidonyl--

naphthylamide (Remel, Lenexa, KS, USA) which served as a substrate for the detection

of pyrrolidonyl arylamidase. A large colony from an 18-24 h culture was rubbed onto a

moisten disk with a sterile loop and allowed to incubate at room temperature for 2 min

before one drop of colour developer was added. A positive result was indicated by the

development of a pink to red colour within one minute of adding the colour developer.

Negative result showed cream, yellow, or no colour within one minute of adding colour

developer (Facklam et al. 1982).

Negative control: Streptococcus agalactiae ATCC 12386

Positive control: Enterococcus faecalis ATCC 29212

2.2.26. Salt tolerance

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-- 122 --

Growth in 0 and 3% NaCl broths was determined by inoculating the tubes with a drop

from an o/v culture followed by incubation at 35ºC in air for up to seven days. A change

from clear to turbid indicated growth (Abbott et al. 2003).

2.2.27. Stapholysin activity

Bacteriolytic activity was determined by spot inoculating a plate containing heat-killed

cells of S. aureus ATCC 25923 with tests strains that were incubated for five days at

35ºC. A positive reaction was denoted as a clearing (lysis) of the opaque medium

around the inoculated aeromonads (Satta et al. 1977).

2.2.28. Hydrolysis of starch

Starch agar plates were spot inoculated with an o/v culture and incubated at 30C for

five days. Plates were flooded with Lugol’s iodine solution at the end of the incubation

period. A clear colourless zone around the inoculum indicated that starch was

hydrolysed (Cowan and Steel 1993).

Positive control: Bacillus subtilis ATCC 6633

Negative control: Escherichia coli ATCC 25922

2.2.29. Hydrolysis of tyrosine

Hydrolysis of tyrosine was determined by spot inoculating a plate containing 0.5% L-

tyrosine crystals in brain heart infusion agar. Plates were incubated at 35ºC in air for up

to seven days. Clearing around the zone of inoculum indicated that tyrosine was

hydrolysed (Abbott et al. 2003).

2.2.30. Urease activity

A urea agar slant (Christensen’s medium) was heavily inoculated with an overnight

culture and incubated at 35C for up to 7 days. Hydrolysis of urea was indicated by the

development of a pink to red colour (Cowan and Steel 1993).

Positive control: Proteus mirabilis ATCC 12453

Negative control: Escherichia coli ATCC 25922

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-- 123 --

2.2.31. Utilization of DL-lactate, acetate and urocanic acid

The slants of tubes containing the appropriate substrate were inoculated with a heavy

inoculum from an o/v HBA with sterile loop. Growth and a bright blue colour indicated

a positive reaction. Tubes were incubated for four days before discarding (Hänninen

1994).

2.2.32. Utilization of citrate (Simmon’s method)

A plate containing citrate was spot inoculated from an o/v culture and incubated at 35C

in air and examined daily for seven days for growth and colour change. Growth and a

bright blue colour indicated a positive reaction (Cowan and Steel 1993).

Negative control: Escherichia coli ATCC 25922

Positive control: Klebsiella pneumoniae ATCC 700603

2.2.33. Voges-Proskauer test

Acetylmethylcarbinol production (VP test) was determined by inoculating a semi-solid

medium with an o/v culture and incubated for three days at 35C in air. A positive

reaction indicating production of acetylmethylcarbinol was denoted by the development

of a red colour after addition of VP1 and VP2 reagents (Cowan and Steel 1993).

Negative control: Escherichia coli ATCC 25922

Positive control: Klebsiella pneumoniae ATCC 700603

2.3. AMPLIFICATION OF gyrB AND rpoD GENES

2.3.1. Preparation of template DNA

DNA was extracted by the method of Coenye and LiPuma (2002) and used to amplify

genes involved in identification and virulence. Three to four large isolated colonies

grown from an overnight culture on HBA were suspended in 50 l of bacterial lysing

solution prepared in-house by the PCR Laboratory, PathWest (Nedlands) and heated at

100C for 15 min in a dry heating block. The suspension was then diluted with 950 l

of depC water and vortex followed by centrifugation for 5 min at 15000 g to pellet solid

material. This stock solution was kept at 70C. Working solution was prepared by

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-- 124 --

diluting 20 l of the stock solution with 180 l of UPW (1:10) and kept at 4C during

testing.

2.3.2. Polymerase chain reaction (PCR)

Primers were diluted to a concentration of 500 M with UPW. Amplification of DNA

was performed by using 20 l of a PCR mixture containing 8 l of template DNA, 2 l

of 10x PE buffer II (Applied Biosystems, Calif. USA), 0.1 l of 2% BSA (0.01% final

concentration; Sigma, NSW, Australia), 2 l of a 25 mM MgCl2 solution (2.5 mM final

concentration; Applied Biosystems, California. USA), 0.16 l deoxynucleoside

triphosphate at 25 mM each (0.2 mM final concentration; (Applied Biosystems, Calif.

USA), 0.1 l of PE TaqGold at 5 U/l (0.75 U final concentration; Applied Biosystems,

Calif. USA); 0.008 l of a 500 mM solution of each primer (0.2 mM final

concentration; Fisher Biotec, Australia) and 7.624 l of UPW to make the final volume

of 12 l. The PCR mixture was prepared in a large volume to produce 200 tubes

(virulence genes) and 400 (gyrB and rpoD genes) tubes of 12 l each under a sterile,

class II bio-safety cabinet. Tubes containing all of the ingredients except the template

DNA were stored at 20C.

Amplification was carried out on a Gene Amp® PCR System 2720 thermal cycler

(Applied Biosystems). The protocol used for the gyrB and rpoD genes consisted of 1

cycle at 95C for 10 min (denaturation); 45 cycles of 94C for 30 s (melting); 55C for

30 s (annealing) and 72C for 1 min (elongation) and a final extension round at 72C for

7 min followed by cooling at 4C. The protocol for amplication of virulence genes was

similar except that the annealing temperature ranged from 50 to 65C appropriate for

each primer pair as reported by other researchers (Table 2.8) and a shorter elongation

time (72C for 45 s). Separation of amplicons and sequencing of the gyrB and rpoD

genes was performed by the staff of the PCR Laboratory (PathWest Nedlands). The

PCR amplicons were separated by electrophoresis using 2% agarose and visualized

using ethidium bromide.

2.3.3. DNA Sequencing

Purification of the PCR product preceded sequencing and was performed using

ExoSAP-IT (USB Corporation, Cleveland, USA) according to the manufacturer’s

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instructions, to remove excess dNTPs and oligonucleotide primers. The nucleotide

sequences on both strands of the DNA were determined with template-specific primers

using fluorescence-based cycle sequencing reactions (BigDye Terminator v3.1 Cycle

Sequencing Kit, AB, Foster City, USA). Cost-saving modifications to the

manufacturer’s protocol included reducing the reaction premix volume by 25% and

adding extra BigDye sequencing buffer to maintain volume. All incubation steps were

completed in thermal cyclers (AB2720 thermal cycler, AB, Foster City, USA).

Unincorporated dye terminators were removed from sequencing reactions using gel-

filtration following the manufacturer’s protocol (DyeEx 2.0 Spin Kit, Qiagen, GmbH,

Germany). The final product was heated for 5 mins at 94°C with 2× volume of

formamide (Hi-Di formamide, AB, Foster City, USA). Capillary electrophoresis was

performed on a 16-capillary genetic analyzer (3130xl Genetic Analyzer, AB, Foster

City, USA) using POP-6 separation matrix (AB, Foster City, USA).

The ChromasPro V1.41 was used to edit the sequence data. Forward and reverse

sequences of gyrB and rpoD genes were independently aligned using the Clustal_X

version 1.8 as described by Thompson et al. (1997) and accessed via BioEdit Sequence

Aligment Editor V7.0.5.2. Genetic distances were obtained using Kimura’s (1980) two-

parameter model and concatenated trees were constructed by the neighbour-joining

method of Saitou and Nei (1987) with the MEGA version 2 program devised by Kumar

et al. (2001). The Basic Local Alignment Search Tool (BLAST) was used to analyze

DNA homologies via the National Center for Biotechnology Information (NCBI) server

at the National Library of Medicine (Bethesda, MD, USA).

Evolutionary distances and sequence dissimilarity percentages were calculated using the

Clustal_W (Thompson et al. 1994) and MEGA version 5.05 software (Tamura et al.

2011). The rpoD and gyrB nucleotide sequences of type, reference and wild strains were

deposited in GenBank and accession numbers are listed in Tables 4.1 and 4.2,

respectively.

2.3.4. Detection of virulence gene products by Bioanalyzer

Following amplification, the PCR amplicons were separated by loading the tubes

containing the 20 l mixture in a QIAxcel analyzer (Qiagen, Hilden, Germany) using a

DNA Screening cartridge (Qiagen). A 4 l of an appropriate molecular size marker (QX

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Size Markers 50-800 bp or 15 to 2.5 kb, Qiagen) diluted with six l of QX DNA

dilution buffer (Qiagen) and a row of 12 tubes containing 15 l of a 15bp to 1Kb

alignment marker (Qiagen) sealed with one drop of paraffin oil was included in each

run. Detection of positive products was visualized as per the Qiagen program and

manual (Qiagen). Selected strains showing a product with a size expected for each gene

were sequenced as described in 2.3.3 and their sequences compared with those

deposited in GenBank using the Basic Local Alignment Search Tool (BLAST)

(Altschul et al. 1990).

2.4. METHODS USED IN THE CHARACTERIZATION OF AEROMONAS

AUSTRALIENSIS SP. NOV.

Strain 266T was isolated from an enriched lauryl sulphate agar (50 mm) plate after water

from a treated effluent used for irrigation at a sports-ground in the South-West of WA

was tested for total coliform count by membrane filtration. Initial phenotypic and

genotypic (rpoD and gyrB gene sequences) analyses, CFA profiles and MALDI-TOF

spectra determined from strain 266 were performed by the author. Cell size,

morphology and the presence of flagella were determined by electron microscopy

following procedures described previously (Collado et al. 2009). Electron micrographs

for strain 266 were prepared by Professor M. J. Figueras (Unitat de Microbilogia,

Department de Ciènces Mèdiques Básiques, Facultat de Medicina i Ciènces de la Salut,

IISPV, Universitat Rovira i Virgili, Reus Spain).

2.4.1. Phenotypic characterization

Biochemical and physiological tests used for the characterization of strain 266T were

performed at 30 and 35C. All strains of type species belonging to the genus Aeromonas

were tested in parallel under identical conditions in laboratories in Australia by the

author and in Spain by Dr. R. Beaz-Hidalgo (Unitat de Microbilogia, Department de

Ciènces Mèdiques Básiques, Facultat de Medicina i Ciènces de la Salut, IISPV,

Universitat Rovira i Virgili, Reus Spain). A total of 36 phenotypic tests were selected

from those performed by Abbott et al. (2003) outlined in section 2.2 following the

procedure described by Alperi et al. (2010b). Strain 266T was tested for citrate

utilization by the method of Hänninen (1994) and Simmon’s (Cowan and Steel 1993);

oxidation of potassium gluconate, production of lipase, urease, Jordan’s tartrate,

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malonate utilization, phenylalanine deaminase (PPA) activity, nitrate reduction

(MacFaddin 1976) and bacteriolytic activity (expression of stapholysin) (Satta et al.

1977).

Acid production from carbohydrate was performed in broth at a final concentration of

1% (w/v) of the desired sugar and 1% (v/v) Andrade’s indicator (Excel, Perth,

Australia) as well as by the method described in Alperi et al. (2010b). The following

carbohydrates were used: adonitol, amygdalin, L-arabinose, cellobiose, dulcitol,

fructose, galactose, glucose, glucose-1-phosphate, glucose-6-phosphate, glycerol, myo-

inositol, lactose, lactulose, maltose, mannose, D-mannitol, melibiose, -methyl-D-

glucoside, raffinose, L-rhamnose, ribose, salicin, D-sorbitol, saccharose (sucrose), and

trehalose. Additional carbohydrate fermentation was investigated with the API 20E and

API CH50 systems (bioMérieux, Marcy l’Etoile, France).

Ability to grow at different temperatures was assayed on TSA supplemented with sheep

blood at 4, 25, 30, 35 and 44C. Acid production from carbohydrates, hydrolysis of

aesculin, urea, DNA and production of hydrogen sulphide from cysteine were observed

for seven days. Other tests were read as described by Abbott et al. (2003). Appropriate

positive and negative controls were included.

2.4.2. Antimicrobial susceptibility testing

The antimicrobial susceptibility of strain 266T was determined by the agar dilution

method according to CLSI standards (CLSI 2011). Antimicrobial agents used in this

project included the following: amikacin, amoxicillin, amoxicillin-clavulanate,

aztreonam, cephalothin, cefazolin, cefepime, cefoxitin, ceftazidime, ceftriaxone,

ciprofloxacin, colistin, gentamicin, meropenem, moxifloxacin, nalidixic acid,

nitrofurantoin, norfloxacin, pipercillin-tazobactam, tetracycline, ticarcillin-clavulanate,

tobramycin, trimethoprim, and trimethoprim-sulfamethozaxole. Interpretative criteria

were in accordance with the CLSI (CLSI 2006).

2.4.3 Fatty acid methyl ester (FAME) analysis

Determination and identification of CFA composition was performed by the protocol

described in the Sherlock® version 6.0 Microbial Identification System software

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package (MIDI-Inc, Newark, Delaware). Bacterial cultures, preparation of reagents and

extraction procedures were according to the MIDI-Inc instructions manual (Paisley

1999). CFAs were analysed by fine capillary column GC chromatography using a

Hewett-Packard GC model 6890 as described by Osterhout et al. (1991).

2.4.3.1. Inoculation of TSBA plates

TSBA plates were prepared by Excel (Perth, WA) according to the guidelines provided

by the MIDI-Inc manual. Inoculation was performed by streaking the plates into four

quadrants with a sterile loop from an HBA from an o/v culture. Plates were incubated at

28C for 48h.

2.4.3.2. Harvesting

A bacterial mass of approximately 20 mg was harvested from the third quadrant of the

TSBA plate with a sterile, disposable bacteriology loop and smeared around the lower 2

cm of a borosilicate Wheaton tube (MIDI-Inc, Del. USA). All cultures had similar

physiological age when they were harvested and CFA analyses were performed in

triplicate.

2.4.3.3. Saponification

Bacteria were saponified by adding 1 ml of Reagent 1 at 100C, vortex after 5 min for

20s followed by further 25 min incubation in a waterbath (Grant Instruments,

Cambridge, UK). Reagent 1 consisted of 45 g of NaOH (ACS grade) dissolved in 150

ml methanol (HPLC grade) and 150 ml of sterile distilled water.

2.4.3.4. Methylation

Methylation was performed by adding 2 ml of Reagent 2 to each tube, vortex for 5 to

10s and transferring the tubes to an 80C waterbath (Grant Instruments, Cambridge,

UK) for 10 min, followed by rapid cooling. Reagent 2 was prepared by mixing 275 ml

of ethanol (HPLC grade) with 325 6N HCl.

2.4.3.5. Extraction

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1.5 ml of Reagent 3 were added to each suspension and inverted for 10 min followed by

removal of the bottom phase. Reagent 3 consisted of 200 ml hexane (HPLC grade) and

200 ml methyl-tert butyl ether (HPLC grade).

2.4.3.6. Washing

Three ml of Reagent 4 were added to each suspension and mixed by inversion for five

min. The top third of the organic phase was removed and place in a testing vial. Reagent

4 consisted of 10.8 g NaOH pellets (ACS grade) dissolved in 900 ml of sterile distilled

water. Extracted FAME preparations were run in batches with a calibration control

immediately after extraction.

2.4.3.7. Interpretation of results

FAMEs analyses were interpreted according to Huys et al. (1994). Results were

automatically issued by the system and included a chromatograph with the identification

of the organism associated with a similarity index (SI). Any SI value > 0.500 indicated a

good identification provided the difference with a second organism was > 0.100; SI

values of 0.300 and 0.500 suggested that if the difference between the organism named

first was > 0.100 from the second organism, the identification was good but it

represented an atypical strain; SI values < 0.300 indicated that the organism may not be

in the database (Paisley 1999).

2.4.4. Protein analysis by MALDI-TOF

The protein analysis of strain 266T was performed using a Bruker Microflex LT

MALDI-TOF mass spectrometer (Bruker Daltonik, GmhH, Germany). Sample

preparation using formic acid extraction method was performed as per manufacturer’s

instructions (Eisentraut, TechNote FormicAcidMethod.doc, version 1.0; 2009 Bruker

Daltonik). All strains were tested six times.

2.4.4.1. Sample preparation

The contents of a 1l loopful from an HBA o/v culture were transfered into an

Eppendorf tube containing 300 l deionized water. The mixture was vortex for one

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minute to generate a homogeneous suspension. To this suspension, 900 l of pure

ethanol were added and vortex for one min. The suspension was twice centrifuged for 2

min at 13000 rpm and the supernatant discarded to completely remove all residual

ethanol. To the pellet, 50 l of 70% aqueous FA (prepared by mixing 30 l water and

70 l 100% FA) were added and vigorously mixed by pipetting up and down followed

by vortexing. A further 50 l ACN were added and mixed as before followed by

centrifugation for 2 min at 13000 rpm. One l of the microorganism extract supernatant

was placed on a clean MALDI target, dried in a laminar airflow cabinet followed by

addition of 1 l HCCA matrix solution. The MALDI target was then inserted into the

MALDI-TOF mass specetrometer. Identification of isolates and interpretation of

spectral patterns were as per manufacturer’s instructions (Bruker Daltonik).

2.4.5. Genotypic characterization

The initial taxonomic position of strain 266T was determined from the nucleotide

sequences of the gyrB and rpoD genes by the author. Further multilococcus

phylogenetic analysis based on the molecular sequences of the 16S rRNA, gyrB, rpoD,

recA, dnaJ, gyrA and dnaX genes and DDH studies were performed by Dr. R. Beaz-

Hidalgo (Unitat de Microbilogia, Department de Ciènces Mèdiques Básiques, Facultat

de Medicina i Ciènces de la Salut, IISPV, Universitat Rovira i Virgili, Reus Spain).

2.4.5.1. PCR and sequence analysis

DNA extraction and conditions for amplifying the 16S rRNA, gyrB, rpoD, recA, dnaJ,

gyrA and dnaX genes were performed as described by Martínez-Murcia et al. (1992b,

2011). DNA extraction for PCR and DDH studies was performed using the Easy DNA

(Invitrogen) kit. Purified PCR products were prepared for sequencing by using the

BigDye Terminator V.1.1 cycle sequencing kit (Applied Biosystems) and sequencing

was performed with an ABI PRISM 310 and ABI 3130XL genetic analyser (Applied

Biosystems). Using the Clustal_X program, version 1.8 (Thompson et al. 1997), the

sequences obtained were independently aligned with sequences of the type and

reference strains of all members of the genus Aeromonas taken from in-house data base

(Martínez-Murcia et al. 2011) and some 16S rRNA sequences retrieved from the

GenBank.

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Genetic distance and clustering were determined using Kimura’s two parameter model

method (Kimura 1980) and evolutionary trees were constructed by the neighbour-

joining method (Saitou and Nei 1987) using the Mega4 program (Tamura et al. 2007).

Stability of the relationship was assessed by the bootstrap method (1000 replications).

DDH experiments were conducted using the methods described by Ziemke et al. (1998)

and Urdiain et al. (2008). Re-association was performed under optimal conditions at

70C, single- and double-stranded DNA molecules were separated by the use of

hydroxyapatite. Colour development was measured at 405 nm using a Biotek Power

Wave XS2 microplate reader (Biotek® Instrument Inc.). Reported mean DNA-DNA

relatedness values (%) and standard deviations were based on a minimum of three

hybridizations for both, direct and reciprocal reactions. DDH studies were performed

between strain 266T and the type strains of A. veronii (CECT 4257T), A.

allosaccharophila (CECT 4199T) and A. fluvialis (CECT 7401T) as these were the

phylogenetically closest species both in the 16S rRNA gene and the MLPA.

2.5. ANTIMICROBIAL SUSCEPTIBILITY TESTING Antimicrobial susceptibility testing was performed by the agar dilution breakpoint and

disk diffusion methods as described by the CLSI (2006). The E-strip method was used

to determine the MIC for ampicillin, colistin, doxycycline and tigecycline.

2.5.1. Agar dilution

Plates used in agar dilution testing were obtained from Excel (Perth, WA). Plates

containing amoxicillin-clavulanate, timentin and pipercillin-tazobactam were used

within 24 h after preparation. All plates were pre-dried with lids off for 30 min at 35ºC

before inoculation. A TSB tube was inoculated with three to four individual colonies

from a HBA plate incubated o/v at 35 ºC and shaken for 2 h at the same temperature to

achieve log phase. Each log phase broth was standardised to the equivalent of 0.5

McFarland with sterile saline. This suspension was further diluted 1 in 10 with ¼

strength peptone water (Excel, Perth) which was used to inoculate wells in the replica

tray (Mast Laboratories Ltd. England). Plates were inoculated within 15 min of filling

the wells and incubated in air at 35ºC for 24 h.

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A CLED and CNA plates were included at the beginning and at the end of each run to

check for Gram-positive contaminants and cross contamination of wells. The following

reference strains were included in each run Klebsiella pneumoniae ATCC 700603,

Escherichia coli ATCC 25922, Escherichia coli ATCC 35218, Pseudomonas

aeruginosa ATCC 27853, and Enterococcus faecalis ATCC 29212. Susceptibility was

defined as absence of growth on solid medium after 24 h incubation. Presence of growth

indicated non-susceptibility.

2.5.2. Disk diffusion

A 0.5 McFarland suspension was prepared in sterile saline from bacteria cultured on

HBA after incubation o/v at 35ºC. Mueller-Hinton plates were lawn-inoculated with this

suspension and appropriate antimicrobial disks placed on the surface of the agar. Plates

with AMP, CEF and O/129 disks were incubated at 35ºC those with DEF disks were

incubated at 30ºC. After 24 h incubation, zone sizes were measured and interpreted

according to the following; values for O129 were obtained from the Oxoid Manual

(1998); DEF values as per coagulase negative staphylococci from the Rosco Manual

(2000); AMP and CEF from CLSI (2006). Interpretation of results is given in Table

2.10.

2.5.3. Minimum inhibitory concentration testing: E-strip method

E-strips stored at 20C were allowed to equilibrate to room temperature for 20 min

before opening. A 0.5 McFarland suspension was prepared in sterile saline from

bacteria cultured on HBA after incubation o/v at 35ºC. The suspension was dispensed

with a sterile pipette to cover the entire surface of a Mueller-Hinton plate and allowed to

dry for 10 min. An E-strip containing a gradient of the appropriate antimicrobial was

placed onto the plate and incubated at 35C for 24 h.

MICs were read and recorded independently by two individuals. Interpretative criteria

for tigecycline and ampicillin were derived from those described for the

Enterobacteriaceae by the Food and Drug Administration (bioMérieux 2010) and those

for doxycycline were derived from the CLSI (2011) document as outlined in Table 1 of

the E-strip pacakage insert. MIC values for colistin were obtained from Fosse et al.

(2003b) and shown in Table 2.11. Escherichia coli ATCC 25922 was used as a quality

control.

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2.6. ELECTRON MICROSCOPY ANALYSIS

Cell size, morphology and the presence of flagella were determined by electron

microscopy following procedures previously described by Collado et al. (2009).

2.7. STATISTICAL ANALYSIS

Chapters 3, 5 and 7

Statistical analyses were conducted with Fisher’s exact method of contingency table

analysis using statistical software (Prism version 5.0; GraphPad, Inc., San Diego, CA).

Chapter 6

Statistical analyses were based on the 2Yc with Yates Corrections for relative small

numbers (Yates 1934).

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Table 2.10 Interpretation of disk diffusion results (zones sizes in mm)

Category

Antimicrobials (concentration in g)

O/129 (150)

AMP (10)

CEF (30)

DEF (250)

R No Zone 13 > 18 14

I 14-16 15-17

S Any zone 17 14 16

R, resistant; I, intermediate; S, susceptible; AMP, ampicillin; CEF, cephalexin; DEF, deferoxamine.

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Table 2.11 Interpretation of E-strip MIC values

Category

MIC (concentration in g/ml)

AMP COL DOX TGC

R 32 2 16 8

I 16 8 4

S 8 < 2 4 2

R, resistant; I, intermediate; S, susceptible; AMP, ampicillin; COL, colistin; DOX, doxycycline; TGC, tigercillin.

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CHAPTER 3: PHENOTYPIC CHARACTERIZATION OF

AEROMONAS SPECIES

3.1. INTRODUCTION

The genus Aeromonas comprises facultatively anaerobic, glucose fermenting, oxidase

positive, Gram-negative rods found globally in water and soil environments (Janda and

Abbott 1998). The need for accurately identifying Aeromonas is based on the notion

that only a few species are considered pathogenic to humans (Carnahan et al. 1991b).

However, the taxonomy of Aeromonas has been described as difficult and confusing

(Harris et al. 1985). This has been partly due to a lack of definitive phenotypic markers,

different testing methodologies and an increasing number of new species (Miñana-

Galbis et al. 2002). Indeed, several new species have been proposed in the last decade in

addition to the 17 DNA hybridization groups described in the most recent edition of

Bergey’s Manual of Systematic Bacteriology (Martin-Carnahan and Joseph 2005).

Previously, classification of Aeromonas species was primarily based on two

characteristics: motility and growth temperature. Psychrophylic and non-motile species

were represented by A. salmonicida while mesophilic and motile species included all

the remaining aeromonads. The vigorous metabolic activity of most Aeromonas species

particularly those of the mesophilic group, formed the basis for the classification of

these organisms (Schubert 1968). The ability to ferment many carbohydrates and other

substrates has been utilized by several authors in the quest to find suitable differential

characteristics (George et al. 1986; Käempfer and Altwegg 1992; Valera and Esteve

2002).

The aim of this Chapter was to characterize a collection of clinical and environmental

Aeromonas based on the scheme designed by Abbott et al. (2003). Minor modifications

from the original scheme included the omission of production of pectinase and ability to

grow in potassium cyanide medium. The former test allowed differentiation between

subsets of A. salmonicida, a species that was not considered in the study while the latter

was omitted due to the hazardous nature of the substrate. Furthermore, to complement

Abbott’s scheme, several novel tests were introduced while previously described

phenotypic tests were revisited in order to find new phenotypic markers.

3.2. Bacterial strains

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Bacterial isolates used in this study are listed in Tables 2.4, 2.6 and 2.7. Organisms used

as positive and negative control are listed in Table 2.5. Clinical isolates were collected

from intestinal and extra-intestinal sites over a period of 20 years (1988 to 2008). Of

these, 46 (32%) were recovered from human clinical material from patients residing

outside the Perth metropolitan area. Environmental and animal isolates were collected

over a 10 year period (1998 to 2008) from all regions of Western Australia, the largest

state of Australia covering an area of approximately 2.5 million square kilometres. All

isolates were considered mesophilic in nature.

3.3. RESULTS

3.3.1. Biochemical characteristics of type and reference strains

The biochemical characteristics of 15 reference strains were in agreement with those

described by Abbott et al. (2003). In contrast to the original descriptions, biochemical

differences were observed for the following type strains; A. bivalvium CECT 7113T

produced acid from salicin (Miñana-Galbis et al. 2007); A. molluscorum DSM 17090T

produced acid from D-lactose and hydrolysed aesculin (Miñana-Galbis et al. 2004a); A.

simiae DSM 16559T produced gas from glucose, -haemolysis on SBA and acid from

D-lactose and salicin (Harf-Monteil et al. 2004) (Table 3.1).

3.3.2. Overall classification

Overall, 185 (92.9%) isolates were identified to species level. Of these, eight (4%)

resembled members of the A. hydrophila complex and six (3%) could not be assigned to

any taxon due to conflicting biochemical profiles. Members of the A. hydrophila

complex included A. hydrophila, A.bestiarum and A. salmonicida.

3.3.3. Clinical isolates

Eighty (54.8%) isolates were identified as A. hydrophila; 36 (24.7%) as A. caviae and

18 (12.3%) as A. veronii bv. sobria. Three isolates were identified as A. eucrenophila A.

jandaei and A. schubertii each constituting 0.7% of the total respectively. Four isolates

(2.7%) could not be assigned to any taxon and five (3.4%) were identified as members

of the A. hydrophila complex (Table 3.2).

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3.3.4. Environmental isolates

Twenty-four isolates (45.27%) were identified as A. hydrophila; 13 (24.5%) as A.

veronii bv. sobria; seven (13.2%) as A. bestiarum; two (3.7%) as A. caviae; two (3.7%)

as A.jandaei and one (1.9%) strain each as A. salmonicida and A. schubertii. In addition,

two (3.7%) could not be identified to species level and three (5.6%) were classified as

members of the A. hydrophila complex (Table 3.3).

3.3.5. Distribution of Aeromonas spp. in clinical samples

Aeromonas hydrophila was the most prevalent aeromonad in wound (69.0%), sterile

sites (45.5%), blood (45.5%) and stool specimens (41.2%) followed by A. caviae wound

(12.1%), sterile sites (36.4%), blood (36.4) and stools (35.3%). Isolates identified as A.

veronii bv. sobria were present in wound (10.3%), sterile sites (18.1%), blood (12.1%)

and stool specimens (11.8%). Aeromonas hydrophila (60%) and A. veronii bv. sobria

(40%) were the only species isolated from sputum samples (Table 3.4).

3.3.6. Distribution of Aeromonas spp. in environmental samples

In water samples, A. hydrophila (46.7%) was the most frequently recovered species

followed by A. veronii bv. sobria (22.2%) and A. bestiarum (15.6%), respectively. Other

species isolated from water included, A. hydrophila complex (6.7%) and single (2.2%)

strains were identified as A. jandaei and A. schubertii. Aeromonas hydrophila (42.9%)

and A. caviae (28.6%) were predominant in fish samples followed by one strain (14.3%)

each of A. jandaei and A. veronii bv. sobria. Two (4.4%) unidentified isolates were

isolated from water and a single isolate from crab was identified as A. salmonicida

(Table 3.4).

3.3.7. General phenotypic characteristics

Overall, the majority of the strains were positive for the following tests: motility,

oxidase, catalase, ONPG, arginine dehydrolase, gelatinase, DNase and lipase activity,

acid production from glycerol, maltose, D-mannitol, mannose glucose-1-phosphate and

glucose-6-phosphate, hydrogen sulphide from cysteine, growth in 0 and 3% NaCl broth.

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- 139

- Tab

le 3

.1

Bio

chem

ical

cha

ract

eris

tics o

f typ

e an

d re

fere

nce

Aero

mon

as st

rain

s

Cha

ract

er

A. a

llosa

ccha

roph

ila

AT

CC

512

08T

A

. bes

tiaru

m

AT

CC

147

15T

A

. biv

alvi

um

CE

CT

711

3T A

. cav

iae

AT

CC

154

68T

A

. cul

icic

ola

CE

CT

576

1T A

. enc

hele

ia

DSM

157

7T

Indo

le

+ +

+ +

+ +

Citr

ate

+ +

+

A

ceta

te

+

M

alon

ate

VP

+

+

LDC

+

+ +

+

PP

A

+

+

Gas

from

glu

cose

+

+

+

Aci

d pr

oduc

tion

from

:

L-a

rabi

nose

+

+ +

+

Cel

lobi

ose

+

+

L

acto

se

+

Mel

ibio

se

-met

hyl-D

-glu

cosi

de

L

-rha

mno

se

+ +

+

Sal

icin

+

+

Suc

rose

+

+ +

+ +

+ A

escu

lin h

ydro

lysi

s +

+ +

+

H

2S fr

om c

yste

ine

+ +

+

+

+

Page 166: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-- 1

40 --

Tab

le 3

.1

Con

tinue

d.

Cha

ract

er

A. a

llosa

ccha

roph

ila

AT

CC

512

08T

A

. bes

tiaru

m

AT

CC

147

15T

A

. biv

alvi

um

CE

CT

711

3T A

. cav

iae

AT

CC

154

68T

A

. cul

icic

ola

CE

CT

576

1T A

. enc

hele

ia

DSM

157

7T

Glu

cona

te

+

D

L-la

ctat

e

+

Uro

cani

c ac

id

+ +

+ +

Jord

an’s

tartr

ate

+

PZ

A

+

+

+

+

-h

aem

olys

is

+

+

Alk

ylsu

lfata

se

+

El

asta

se

+

Ty

rosi

ne

+

+

Am

pici

llinR

R

R

R

R

R

R

C

epha

loth

inR

S R

S

R

S S

Star

ch

+ +

+

PY

R

+

+

Def

erox

amin

eR S

R

R

R

R

R

O/1

29R

R

R

R

R

R

R

Gro

wth

in T

CB

S

+

CA

MP

(aer

obic

)

+

+

+

CA

MP

(ana

erob

ic)

+

C

olis

tinR

S R

S

R

R

S

Page 167: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-- 1

41 --

Tab

le 3

.1

Con

tinue

d.

Cha

ract

er

A. e

ucre

noph

ila

AT

CC

233

09T

A

. hyd

roph

ila

AT

CC

796

6T

A. j

anda

ei

AT

CC

495

68T

A

. med

ia

AT

CC

339

07T

A

. mol

lusc

orum

D

SM 1

7090

T

A. p

opof

fii

CIP

105

493T

In

dole

+

+ +

+

C

itrat

e

+

+ A

ceta

te

Mal

onat

e

+

VP

+ +

+ LD

C

+ +

PPA

+

+

+

Gas

from

glu

cose

+

+

+

Aci

d pr

oduc

tion

from

:

L-a

rabi

nose

+

+

+

+ +

C

ello

bios

e +

+ +

L

acto

se

+

+

M

elib

iose

+

-met

hyl-D

-glu

cosi

de

+

+

L

-rha

mno

se

S

alic

in

+

Suc

rose

+

+

+

+

A

escu

lin h

ydro

lysi

s +

+

+

H2S

from

cys

tein

e

+

+

Page 168: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-- 1

42 --

Tab

le

3.1

Con

tinue

d.

Cha

ract

er

A. e

ucre

noph

ila

AT

CC

233

09T

A

. hyd

roph

ila

AT

CC

796

6T

A. j

anda

ei

AT

CC

495

68T

A

. med

ia

AT

CC

339

07T

A

. mol

lusc

orum

D

SM 1

7090

T

A. p

opof

fii

CIP

105

493T

G

luco

nate

+

DL-

lact

ate

+

U

roca

nic

acid

+

+

Jo

rdan

’s ta

rtrat

e +

+

PY

Z +

+

+

+

-hae

mol

ysis

+

+

A

lkyl

sulfa

tase

+

+

El

asta

se

+

Ty

rosi

ne

+

+

+

A

mpi

cilli

nR

R

R

R

S R

R

C

epha

loth

inR

R

R

I R

S

R

Star

ch

+ +

+

PY

R

+

D

efer

oxam

ineR

R

R

R

R

R

R

O/1

29R

R

R

R

R

R

S G

row

th in

TC

BS

+ +

CA

MP

(aer

obic

) +

+ +

CA

MP

(ana

erob

ic)

+ +

+

+

Col

istin

R S

S R

S

S S

Page 169: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-- 1

43 --

Tab

le 3

.1

Con

tinue

d.

Cha

ract

er

A. s

chub

ertii

A

TC

C 4

3700

T

A. s

imia

e D

SM 1

6559

T

A. s

obri

a C

IP 7

433T

A

. tro

ta

AT

CC

496

57T

A

. ver

onii

bv

. sob

ria

AT

CC

907

1T

A. v

eron

ii b

v. v

eron

ii D

SM 7

386T

In

dole

+

+ +

+ C

itrat

e

+

+ A

ceta

te

+

+

Mal

onat

e

V

P +

+ +

LDC

+

+ +

+ +

PPA

G

as fr

om g

luco

se

+

+

+ +

Aci

d pr

oduc

tion

from

:

L-a

rabi

nose

+

+

Cel

lobi

ose

+ +

+

+

L

acto

se

+

+

M

elib

iose

-m

ethy

l-D-g

luco

side

+

+

L-r

ham

nose

Sal

icin

+

+

Suc

rose

+

+

+

+ A

escu

lin h

ydro

lysi

s

+

+

+

H2S

from

cys

tein

e

+

+

Page 170: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-- 1

44 --

Tab

le 3

.1

Con

tinue

d.

Cha

ract

er

A. s

chub

ertii

A

TC

C 4

3700

T

A. s

imia

e D

SM 1

6559

T

A. s

obri

a C

IP 7

433T

A

. tro

ta

AT

CC

496

57T

A

. ver

onii

bv

. sob

ria

AT

CC

907

1T

A. v

eron

ii b

v. v

eron

ii D

SM 7

386T

G

luco

nate

+

+ D

L-la

ctat

e +

+

U

roca

nic

acid

+

+ +

Jord

an’s

tartr

ate

+ +

PYZ

-h

aem

olys

is

+ +

+ +

+ A

lkyl

sulfa

tase

+

+ El

asta

se

Tyro

sine

A

mpi

cilli

nR

R

R

S S

R

R

Cep

halo

thin

R R

S

S R

S

S St

arch

+

+ +

PYR

+

Def

erox

amin

eR R

R

S

R

R

R

O/1

29R

S R

R

S

R

R

Gro

wth

in T

CB

S +

+

+

+ C

AM

P (a

erob

ic)

+

C

AM

P (a

naer

obic

)

C

olis

tinR

S S

S S

R

R

Page 171: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

- 145

-

Tab

le 3

.2

Bio

chem

ical

cha

ract

eris

tics o

f Aer

omon

as is

olat

ed fr

om h

uman

clin

ical

mat

eria

l (%

pos

itive

)

Cha

ract

er

A. h

ydro

phila

com

plex

n

= 8

A. h

ydro

phila

n

= 10

4 A

. cav

iae

n =

38

A. b

estia

rum

n

= 7

A. v

eron

ii bv

. sob

ria

n =

29

Indo

le

75

99

95

86

100

Citr

ate

63

72

77

57

93

Ace

tate

10

0 90

79

29

93

M

alon

ate

38

36

16

14

45

VP

75

97

0 71

90

LD

C

88

98

0 10

0 10

0 PP

A

13

22

21

29

41

Gas

from

glu

cose

75

93

0

86

90

Aci

d pr

oduc

tion

from

:

L

-ara

bino

se

38

37

100

43

0

Cel

lobi

ose

38

15

87

0 24

Lac

tose

25

12

95

0

41

M

elib

iose

13

1

0 0

10

-met

hyl-D

-glu

cosi

de

88

85

0 71

17

Raf

finos

e 0

1 24

0

7

L-r

ham

nose

0

6 0

0 0

S

alic

in

88

88

97

86

3

D-s

orbi

tol

25

0 3

0 0

S

ucro

se

100

93

100

100

97

Aes

culin

hyd

roly

sis

100

98

97

100

0 H

2S fr

om c

yste

ine

100

96

21

71

97

Page 172: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-- 1

46 --

Tab

le 3

.2

Con

tinue

d.

Cha

ract

er

A. h

ydro

phila

com

plex

n

= 8

A. h

ydro

phila

n

= 10

4 A

. cav

iae

n =

38

A. b

estia

rum

n

= 7

A. v

eron

ii bv

. sob

ria

n =

29

Glu

cona

te

38

73

0 14

93

D

L-la

ctat

e 63

69

74

0

0 U

roca

nic

acid

10

0 83

79

86

76

Jo

rdan

’s ta

rtrat

e 13

19

34

29

21

PY

Z 50

37

89

29

41

-hae

mol

ysis

88

85

24

57

86

St

apho

lysi

n 38

75

0

43

0 A

lkyl

sulfa

tase

38

42

3

14

24

Elas

tase

75

85

0

42

0 Ty

rosi

ne

88

61

18

0 28

A

mpi

cilli

nR

100

100

100

86

97

Cep

halo

thin

R 63

82

97

29

14

St

arch

50

16

84

57

38

PY

R

13

0 0

0 0

Def

erox

amin

eR 88

99

10

0 10

0 97

O

/129

R 88

95

10

0 86

86

G

row

th in

TC

BS

50

51

84

29

66

CA

MP

(aer

obic

) 50

66

0

71

34

CA

MP

(ana

erob

ic)

63

75

0 71

17

C

olis

tinR

50

75

13

0 59

Page 173: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-- 1

47 --

Tab

le 3

.2

Con

tinue

d.

Cha

ract

er

Aer

omon

as sp

p.

n =

6 A

. jan

daei

n

= 3

A. e

ucre

noph

ila

n =

1 A

. sal

mon

icid

a n

= 1

A. s

chub

ertii

n

= 2

Indo

le

33

100

100

100

100

Citr

ate

0 10

0 0

100

0 A

ceta

te

66

100

100

100

100

Mal

onat

e 0

33

0 0

50

VP

16

100

0 10

0 10

0 LD

C

66

100

0 0

100

PPA

50

66

0

100

0 G

as fr

om g

luco

se

83

100

0 10

0 0

Aci

d pr

oduc

tion

from

:

L

-ara

bino

se

50

0 10

0 10

0 0

C

ello

bios

e 66

0

100

100

0

Lac

tose

33

0

100

100

0

Mel

ibio

se

33

66

0 0

0

-m

ethy

l-D-g

luco

side

33

33

0

100

0

Raf

finos

e 50

0

0 0

0

L-r

ham

nose

16

0

0 0

0

Sal

icin

33

0

100

100

0

D-s

orbi

tol

0 0

0 10

0 0

S

ucro

se

100

0 10

0 10

0 0

Aes

culin

hyd

roly

sis

50

0 10

0 10

0 0

H2S

from

cys

tein

e 66

10

0 10

0 10

0 50

Page 174: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-- 1

48 --

Tab

le 3

.2

Con

tinue

d.

Cha

ract

er

Aer

omon

as sp

p.

n =

6 A

. jan

daei

n

= 3

A. e

ucre

noph

ila

n =

1 A

. sal

mon

icid

a n

= 1

A. s

chub

ertii

n

= 2

Glu

cona

te

50

100

0 10

0 50

D

L-la

ctat

e 16

0

0 0

50

Uro

cani

c ac

id

66

100

0 0

100

Jord

an’s

tartr

ate

16

33

100

0 0

PYZ

50

0

100

0 0

-h

aem

olys

is

50

100

100

100

100

Stap

holy

sin

0 0

0 10

0 0

Alk

ylsu

lfata

se

33

33

0 0

50

Elas

tase

0

0 0

100

0 Ty

rosi

ne

16

66

0 0

0 A

mpi

cilli

nR

100

100

100

100

100

Cep

halo

thin

R 0

66

100

100

0 St

arch

50

33

0

0 50

PY

R

50

66

0 0

0 D

efer

oxam

ineR

83

100

100

100

100

O12

9R

83

100

100

100

50

Gro

wth

in T

CB

S 50

33

0

100

50

CA

MP

(aer

obic

) 0

0 0

0 50

C

AM

P (a

naer

obic

) 0

33

0 10

0 0

Col

istin

R 50

10

0 0

0 50

Page 175: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-- 1

49 --

Tab

le 3

.3

B

ioch

emic

al c

hara

cter

istic

s of A

erom

onas

isol

ated

from

env

ironm

enta

l sou

rces

(% p

ositi

ve)

Cha

ract

eris

tics

A. h

ydro

phila

n

= 24

A

. ver

onii

bv. s

obri

a n

= 11

A

. bes

tiaru

m

n =

7 A

. cav

iae

n =

2 A

. jan

daei

n

= 2

Indo

le

96

100

86

100

100

Citr

ate

92

91

57

50

100

Ace

tate

96

82

29

10

0 10

0 M

alon

ate

4 0

14

0 0

VP

92

91

71

0 10

0 LD

C

96

100

100

0 10

0 PP

A

33

36

28

0 50

G

as fr

om g

luco

se

88

73

86

0 10

0 A

cid

prod

uctio

n fr

om:

L-

Ara

bino

se

13

0 43

10

0 0

Cel

lobi

ose

0 18

0

100

0 La

ctos

e 0

27

0 10

0 0

Mel

ibio

se

0 9

0 0

100

-M

ethy

l-D-g

luco

side

88

18

71

0

50

Raf

finos

e 0

0 0

0 0

L-R

ham

nose

4

0 0

0 0

Salic

in

88

0 86

10

0 0

D-S

orbi

tol

0 0

0 0

0 Su

cros

e 10

0 92

10

0 10

0 0

Aes

culin

hyd

roly

sis

96

0 10

0 10

0 0

Page 176: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-- 1

50 --

T

able

3.3

Con

tinue

d.

Cha

ract

eris

tics

A. h

ydro

phila

n

= 24

A

. ver

onii

bv. s

obri

a n

= 11

A

. bes

tiaru

m

n =

7 A

. cav

iae

n =

2 A

. jan

daei

n

= 2

H2S

from

cys

tein

e 96

10

0 71

10

0 10

0 G

luco

nate

25

91

14

0

100

DL-

Lact

ate

75

0 0

0 0

Jord

an’s

tartr

ate

25

36

29

0 50

PY

Z 54

9

29

100

0

-Hae

mol

ysis

96

91

57

0

100

Stap

holy

sin

79

0 43

0

0 A

lkyl

sulfa

tase

63

45

14

0

0 El

asta

se

100

0 43

0

0 Ty

rosi

ne

13

27

0 0

50

Am

pici

llinR

10

0 91

86

10

0 10

0 C

epha

loth

inR

71

27

29

100

50

Star

ch

17

64

57

100

50

PYR

0

0 0

0 10

0 D

efer

oxam

ineR

100

100

100

100

100

O12

9R

100

54

86

100

100

Gro

wth

in T

CB

S 21

45

29

0

0 C

AM

P O

88

18

71

0

0 C

AM

P A

nO

96

18

71

0 0

Col

istin

R 63

33

0

100

100

Page 177: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-- 1

51 --

Tab

le 3

.3

Con

tinue

d.

Cha

ract

eris

tics

Aer

omon

as sp

p.

n=2

A. h

ydro

phila

com

plex

n=

3 A

. sal

mon

icid

a n=

1 A

. sch

uber

tii

n=1

Indo

le

0 33

10

0 10

0 C

itrat

e 0

100

100

0 A

ceta

te

50

100

100

100

Mal

onat

e 0

0 0

0 V

P 0

33

100

100

LDC

10

0 10

0 0

100

PPA

10

0 0

100

0 G

as fr

om g

luco

se

100

100

100

0 A

cid

prod

uctio

n fr

om:

L-A

rabi

nose

0

33

100

0 C

ello

bios

e 50

0

100

0 La

ctos

e 0

0 10

0 0

Mel

ibio

se

50

0 0

0

-Met

hyl-D

-glu

cosi

de

100

67

100

0 R

affin

ose

50

0 0

0 L-

Rha

mno

se

100

0 0

0 Sa

licin

0

100

100

0 D

-Sor

bito

l 0

0 10

0 0

Sucr

ose

100

100

100

100

Aes

culin

hyd

roly

sis

0 10

0 10

0 0

Page 178: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-- 1

52 --

Tab

le 3

.3

Con

tinue

d.

Cha

ract

eris

tics

Aer

omon

as sp

p.

n=2

A. h

ydro

phila

com

plex

n=

3 A

. sal

mon

icid

a n=

1 A

. sch

uber

tii

n=1

H2S

from

cys

tein

e 10

0 10

0 10

0 0

Glu

cona

te

100

33

100

100

DL-

Lact

ate

0 10

0 0

0 U

roca

nic

acid

50

10

0 0

100

Jord

an’s

tartr

ate

50

0 0

0 PY

Z 50

33

0

0

-Hae

mol

ysis

10

0 10

0 10

0 10

0 St

apho

lysi

n 0

67

100

0 A

lkyl

sulfa

tase

50

67

0

0 El

asta

se

0 10

0 10

0 0

Tyro

sine

50

10

0 0

0 A

mpi

cilli

nR

100

100

100

100

Cep

halo

thin

R 0

67

100

0 St

arch

10

0 67

0

100

PYR

50

0

0 0

Def

erox

amin

eR 10

0 10

0 10

0 10

0 O

129R

10

0 10

0 10

0 0

Gro

wth

in T

CB

S 50

33

10

0 10

0 C

AM

P O

0

100

0 0

CA

MP

AnO

0

100

100

0 C

olis

tinR

50

67

0 0

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- 153

- Tab

le 3

.4

Dis

tribu

tion

of A

erom

onas

spp.

am

ong

clin

ical

and

env

ironm

enta

l sam

ples

afte

r phe

noty

pic

char

acte

rizat

ion

C

linic

al

Env

iron

men

tal

Spec

ies

No.

isol

ated

/ (

%)

Wou

nd

Sput

um

Ster

ile

site

B

lood

U

rine

St

ool

Unk

now

n W

ater

Fi

sh

Cra

b

Aero

mon

as sp

p.

6

(3.0

) 1

(1.7

)

2

(5.9

) 1

(33.

3)

2 (4

.4)

A. b

estia

rum

7 (3

.5)

7

(15.

6)

A. c

avia

e 3

8 (1

9.0)

7

(12.

1)

4

(36.

4)

12 (3

6.4)

12 (3

5.3)

1

(33.

3)

2

(28.

6)

A. e

ucre

noph

ila

1

(0.5

)

1 (2

.9)

A. h

ydro

phila

10

4 (5

2.2)

40

(69.

0)

3 (6

0.0)

5

(45.

5)

15 (4

5.5)

2

14 (4

1.2)

1

(33.

3)

21 (4

6.7)

3

(42.

9)

A. h

ydro

phila

co

mpl

ex

8

(4.0

)

3 (5

.2)

1 (3

.0)

1

(2.9

) 3

(6.7

)

A. ja

ndae

i

3 (1

.5)

1

(3.0

)

1

(2.2

) 1

(14.

3)

A. sa

lmon

icid

a

1 (0

.5)

1

A. sc

hube

rtii

2

(1.0

) 1

(1.7

)

1

(2.2

)

A. v

eron

ii bv

. so

bria

2

9 (1

4.5)

6

(10.

3)

2 (4

0.0)

2

(18.

1)

4 (1

2.1)

4 (1

1.8)

10 (2

2.2)

1

(14.

3)

Tot

al

199

58

5 11

33

2

34

3 45

7

1

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- 154 -

Most strains were uniformly negative for ornithine decarboxylase, acid production from

adonitol, amygdalin and m-inositol and production of a diffusible brown pigment. Weak

urease activity was detected in three clinical isolates only identified as A. hydrophila, A.

caviae and A. veronii bv. sobria. The remaining isolates were negative for this test.

3.3.8. Susceptibility to colistin

Resistance to colistin was observed in A. hydrophila (75%), A. veronii bv. sobria (59%);

A. hydrophila complex (50%) and A. jandaei (100%) and less frequently in A. caviae

(13.9%). Among the colistin resistant species, strains identified as A. jandaei produced

much higher MIC values than the other resistant species (results not shown).

3.3.9. Production of pyrrolidonyl--naphthylamide

PYR activity was detected in seven (3.5%) strains comprising four (2.7%) clinical and

three (5.7%) environmental isolates and in the type strains of A. sobria CIP 7433T, A.

bivalvium CECT 7113T, A. allosaccharophila ATCC 51208T and A. jandaei ATCC

49568T. No PYR activity was detected on the remaining isolates. Clinical isolates

showing PYR activity were identified as A. hydrophila complex (strain 221),

Aeromonas spp. (strains 100 and 114) and A. hydrophila (strain 189). PYR+

environmental strains belonged to A. jandaei (strains 35 and 262) and Aeromonas spp.

(strain 265).

3.3.10. Susceptibility to deferoxamine (DEF)

Most isolates were resistant to DEF except for four (2.7%) clinical isolates and the type

strains of A. allosaccharophila ATCC 51208T and A. sobria CIP 7433T. Isolates

susceptible to DEF were identified as A. hydrophila complex (strain 221), A. hydrophila

(strain 184), A. veronii bv. sobria (strain 211) and Aeromonas spp. (strain 100).

3.3.11. Production of a CAMP-like factor

The production of a CAMP-like factor, under aerobic and anaerobic conditions was

observed in the following species: A. bestiarum (71% O2; 71% AnO2); A. hydrophila

(66% O2; 75% AnO2); A. hydrophila complex (50% O2; 63% AnO2) and A. veronii bv.

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-- 155 --

sobria (34% O2; 17% AnO2). CAMP-like factor was detected under aerobic conditions

in one strain (50%) of A. schubertii and under anaerobic conditions in single strains of

A. jandaei (33%) and A. salmonicida (100%).

3.3.12. Utilization of citrate: Simmon’s vs Hänninen’s medium

Fifty-seven (28.6%) strains were able to utilize citrate using Simmon’s medium but not

Hänninen’s. Seven (4.6%) were positive in Hänninen’s medium alone; 52 (26.1%)

produced a positive result in both media, while 36 (23.7%) strains failed to utilized this

substrate.

3.3.13. Susceptibility to the vibriostatic agent O/129

Susceptibility to O/129 was observed in 16 (8%) strains from different species and

included eight (5.5%) clinical and eight (15.1%) environmental strains.

3.3.14. Growth on thiosulfate salt bile sucrose agar (TCBS)

The ability to grow on TCBS agar was observed in 116 (58.3%) strains which included

16 (30.2%) environmental and 100 (68.5%) clinical isolates.

3.4. DISCUSSION

A conventional biochemical scheme was employed to identify a collection of

Aeromonas strains recovered from clinical and environmental sources in Western

Australia. Data from this study showed that A. hydrophila, A. caviae and A. veronii bv.

sobria were the most frequently isolated species (92.9%) a result consistent with

previous studies (Altwegg and Geiss 1989; Hänninen and Siitonen 1995; Abbott et al.

2003). In contrast to other reports, no significant differences between the biochemical

profiles of clinical and environmental Aeromonas were found (Aguilera-Arreola et al.

2005; Ørmen et al. 2005). This may be partly attributed to the low number of

environmental strains tested. However, phenotypic differences were observed between

strains examined in this study with those reported elsewhere (Hänninen 1994; Valera

and Esteve 2002; Abbott et al. 2003).

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-- 156 --

Biochemically, strains of A. hydrophila were less likely to produce acid from L-

arabinose (37%) compared to previous reports where >80% of strains were positive for

this test (Käempfer and Altwegg 1992; Abbott et al. 2003). Utilization of urocanic acid

and hydrolysis of tyrosine was observed in 83 and 61% of A. hydrophila strains,

respectively. In contrast, Abbott et al. (2003) reported that only a handful of strains

(12%) utilized urocanic acid while no strain hydrolysed tyrosine. These results

concurred with previous observations which highlight the heterogeneity of A.

hydrophila (Kirov 1993; Hänninen 1994; Abbott et al. 2003), probably reflecting

geographical differences between the strains (Altwegg et al. 1990). Other differences

were observed in A. caviae strains were the majority were able to produce acid from

salicin (98.5%) and lactose (95.0%). In contrast, Valera and Esteve (2002) reported that

only 33% of the A. caviae strains produced acid from salicin while Käempfer and

Altwegg (1992) found that 64% of A. caviae strains produced acid from lactose.

The ability of A. veronii bv. sobria to produce a CAMP-like factor under aerobic and

anaerobic conditions was consistent with the observations by Carnahan et al. (1991b)

and Altwegg et al. (1990) but not with those of Figura and Guglielmetti (1987). The

biochemical profiles of A. veronii bv. sobria were consistent with the study by Ashbolt

et al. (1995). Phenotypically, this species appeared more stable than A. hydrophila and

A. caviae, although Esteve et al. (2003) suggested that A. veronii bv. sobria constituted

a heterogenous taxon that required further revision. Variations in phenotype may have

clinical significance as an association between biotype and enterotoxin production has

been suggested (Turnbull et al. 1984) but not universally supported (George et al.

1986). Traditionally, Aeromonas are considered resistant to the vibriostatic agent O/129

and should not grow on TCBS agar, characteristics that allow members of this genus to

be differentiated from Plesiomonas and Vibrios (Cowan and Steel 1993; Esteve et al.

2003). However, results from the present study indicate that these tests are no longer

reliable suggesting that the ability to grow on this selective medium and resistance to

O/129 is strain dependent.

Assigning strains to a particularly taxon proved to be difficult in cases where: (i) the

range allocated for a positive result varied from 16 to 84%; (ii) the percentage positive

for a test was no greater than 60 or 70%; (iii) the end points for positive reactions could

not be reliably determined for tests such as Jordan’s tartrate, production of

phenylalanine deaminase and pyrazinamidase, despite the use of positive and negative

controls. The inclusion of tests with a positive rate of nearly 100% [-galactoside

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-- 157 --

production (ONPG), gelatin hydrolysis or nearly 100% negative (hydrolysis of urea,

inability to produce acid from m-inositol, adonitol and amygdalin) did not contribute to

the overall differentiation of aeromonads.

The choice of media and methods used to determine phenotypic traits can affect the

biochemical identification of Aeromonas (Carnahan et al. 1991b; Esteve et al. 2003).

For example, significantly (p = 0.0004) more strains were able to utilize citrate as a

carbon source when Simmons’ medium was used than with the medium described by

Hänninen (1994). Similarly, the use of Kovacs medium to determine indole production

was significantly (p = 0.0001) more sensitive than the spot indole test. The quest to find

new phenotypic markers to reliably identify Aeromonas to species level continues to be

a difficult task. Previously described and novel tests introduced in this study did not

improve the discriminatory power of the scheme and did not contribute to the

phenotypic classification of these organisms. The PYR+ activity detected in less

frequently isolated species such as A. allosaccharophila, A. bivalvium, A. jandaei and A.

sobria is a promising phenotypic marker that can be used to rapidly and reliably

differentiate these organims from PYR species but more strains need to be tested to

confirm the validity of this test.

In this Chapter we have described the phenotypic characteristics of 199 Aeromonas

isolates and determined the current distribution of species among clinical and

environmental sources in Western Australia. Despite the unreliable nature of phenotypic

identification, biochemical differentiation is still the only identification method

available in some laboratories. Furthermore, biochemical differentiation remains a

requisite when describing novel species. Janda and Duffey (1988) suggested that

identification of mesophilic Aeromonas species must become more standardised before

more meaningful comparisons can be made between studies carried out at various

regions throughout the world, a suggestion supported by this study. Results from this

study indicate that accurate identification of Aeromonas must involve the use of

molecular methods and the nucleotide sequences of several housekeeping genes have

been proposed for this purpose (Soler et al. 2004; Nhung et al. 2007) and this is

presented in the next chapter.

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-- 158 --

CHAPTER 4: GENOTYPIC CHARACTERIZATION OF

AEROMONAS SPECIES

4.1. INTRODUCTION

The genus Aeromonas has long been recognized to contain strains that are difficult to

differentiate from one another, particularly when identification is based on phenotypic

methods alone (Abbott et al. 2003). However, advances in molecular methods and the

development of novel molecular targets have significantly improved the discrimination

of bacteria not usually amenable to identification by conventional biochemical methods

(Yamamoto and Harayama 1996).

In the last decade, the nucleotide sequences of several housekeeping genes have been

used to characterize members of the genus Aeromonas (Yañez et al. 2003; Soler et al.

2004; Küpfer et al. 2006; Nhung et al. 2007; Miñana-Galbis et al. 2009; Lamy et al.

2010). Housekeeping genes perform essential functions in bacteria and, unlike the 16S

rRNA gene, are single-copy genes where horizontal transfer seldom occurs (Yañez et al.

2003; Soler et al. 2004).

The primary aim of this Chapter was to re-classify Aeromonas strains previously

characterized by phenotypic methods as described in Chapter 3, inferred by the rpoD

and gyrB genes. The rpoD gene encodes one of the sigma (σ70) factors that confer

promoter-specific transcription initiation on RNA polymerase while gyrB encodes the

B-subunit of the DNA gyrase, a type II DNA topoisomerase (Yañez et al. 2003; Soler et

al. 2004). Both genes have similar substitution rates (<2%) and a similar number of

variable positions (34% for rpoD and 32% for gyrB). These genes have, individually or

simultaneously, been used for the analysis of Aeromonas (Yañez et al. 2003; Soler et al.

2004). When combined, rpoD and gyrB have shown to be a reliable tool in the

differentiation of these bacteria. Individually, gyrB allows the differentiation of closely

related taxa such as Aeromonas sp. HG 11/A. encheleia and A. veronii/A. culicicola/A.

allosaccharophila whereas rpoD differentiates A. salmonicida from A. bestiarum (Soler

et al. 2004).

A second aim was to show how classification by a molecular method affects the

distribution of species within clinical and environmental sources.

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-- 159 --

4.2. Bacterial strains

Bacterial strains used in this Chapter are listed in Tables 2.4, 2.6 and 2.7. Clinical

samples were isolated from wound (54 samples), stool (33 samples), blood (33

samples), and 23 from miscellaneous specimens. Environmental specimens were

collected from water (44 samples), fish (7 samples), and crab (1 sample). The nucleotide

sequences of the gyrB and rpoD genes obtained from wild and reference strains used in

this study were deposited in GenBank/EMBL and accession numbers are listed in

Tables 4.1 and 4.2.

4.3. RESULTS

4.3.1 Overall distribution of species following genetic identification

Sixty (30.7%) isolates clustered around the type strain of A. dhakensis LMG 19562T

(Fig. 4.1), 36 (18.4%) around A. caviae ATCC 13136T (Fig. 4.2), 38 (19.4%) around A.

hydrophila ATCC 7966T (Fig. 4.3) and 49 (25.1%) around A. veronii bv. sobria ATCC

9071T (Fig. 4.4).

4.3.2. Distribution of Aeromonas spp. in clinical specimens

The most prevalent species was A. veronii bv. sobria (25.1%) followed by A. dhakensis

and A. caviae (both at 23.8%) and A. hydrophila (23.0%). The prevalence of A.

dhakensis was wounds (40.7%), faeces (12.1%) and blood (9.0%). Most isolates

recovered from blood samples were identified as A. caviae (32.2%) and A. veronii bv.

sobria (30.3%) followed by A. hydrophila (21.2%). Other species isolated from human

clinical material included: A. allosaccharophila (strain 100 from stool); A. bestiarum

(strain 68 from blood); A. media (strains 85 from blood and 179 from stool); A.

salmonicida (strain 190 from wound) and A. schubertii (strain 186 from wound) (Table

4.3).

4.3.3. Distribution of Aeromonas spp. in environmental specimens

Overall, A. dhakensis (50.0%) was the most frequently identified species followed by A.

veronii bv. sobria (25.0%). Both species were the most frequently identified Aeromonas

in water samples 54.5 and 27.2%, respectively while six different species were

identified in fish samples (Table 4.3).

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-160

-

Tab

le 4

.1

Type

and

refe

renc

e st

rain

s Gen

bank

acc

essi

on n

umbe

rs

Spec

ies

Cul

ture

col

lect

ion

no.

rpoD

gy

rB

A.

allo

sacc

haro

phila

D

SM 1

1576

FN

7733

42

FN81

3470

A.

aqu

ario

rum

(rec

lass

ified

as A

. dha

kens

is)

CEC

T 72

89

FN77

3316

FN

6917

67

A. a

ustr

alie

nsis

C

ECT

8023

FN

7733

35

FN69

1773

A.

bes

tiaru

m

ATC

C 5

1108

FN

7733

17

FN70

6556

A.

biv

alvi

um

CEC

T 71

13

FN77

3318

FN

6917

68

A. c

aver

nico

la

CEC

T 78

62

H

Q44

2702

A.

cav

iae

ATC

C 1

3136

FN

7733

19

FN69

1769

A.

cul

icic

ola

CEC

T 57

61

FR87

2757

FN

6917

69

A. d

iver

sa

CEC

T 42

54

AY

1693

45

AY

1018

06

A. e

nche

leia

D

SM 1

1577

FN

7733

20

FN79

6740

A.

euc

reno

phila

A

TCC

233

09

FN77

3321

FN

7065

57

A. fl

uvia

lis

CEC

T 74

01

FJ60

3453

FJ

6034

55

A. h

ydro

phila

ssp.

hyd

roph

ila

ATC

C 7

966

FN77

3322

FN

7065

58

A. h

ydro

phila

ssp.

dha

kens

is

LMG

195

62

HQ

4428

00

HQ

4427

11

A. h

ydro

phila

ssp.

rana

e LM

G 1

9707

HE9

6566

9 A.

jand

aei

ATC

C 4

9568

FN

7733

23

FN70

6559

A.

med

ia

ATC

C 3

3907

FN

7733

24

FN70

6560

A.

mol

lusc

orum

D

SM 7

090

FN77

3325

FN

7065

61

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-161

-

Tab

le 4

.1

Con

tinue

d.

Spec

ies

Cul

ture

col

lect

ion

no.

rpoD

gy

rB

A.

pis

cico

la

CEC

T 74

43

FM99

9969

FM

9999

63

A. p

opof

fii

CIP

105

493

FN77

3336

FN

7065

62

A. ri

vuli

CEC

T 75

18

FJ96

9433

FJ

9694

34

A. sa

lmon

icid

a ss

p. sa

lmon

icid

a C

ECT

894

AY

1693

27

AY

1017

73

A. sa

lmon

icid

a ss

p. a

chro

mog

enes

C

ECT

895

AY

1693

29

AY

1017

84

A. sa

lmon

icid

a ss

p. m

asou

cida

C

ECT

896

AY

1693

30

AY

1017

90

A. sa

lmon

icid

a ss

p. p

ectin

olyt

ica

DSM

126

09

AY

1693

24

AY

1017

85

A. sa

lmon

icid

a ss

p. sm

ithia

C

IP 1

0475

7

AM

2621

59

A. sa

nare

lli

CEC

T 74

02

FJ47

2929

FJ

6072

77

A. sc

hube

rtii

CEC

T 42

40

AY

1693

36

AY

1017

72

A. si

mia

e D

SM 1

6559

D

Q41

159

FN70

6563

A.

sobr

ia

CD

C 9

540-

76

FN77

3345

FN

7065

64

A. ta

iwan

ensi

s C

ECT

7403

FJ

4749

28

FJ80

7272

A.

tect

a C

ECT

7082

FN

7733

37

FN79

6745

A.

trot

a

ATC

C 4

9657

FN

7733

39

FN79

6746

A.

ver

onii

bv. s

obria

A

TCC

907

1 FN

7733

40

FN79

6747

A.

ver

onii

bv. v

eron

ii D

SM 7

386

FN77

3341

FN

7967

48

Aero

mon

as sp

p. H

G11

C

ECT

4253

A

Y16

9343

A

J964

951

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-162

-

Tab

le 4

.2

Gen

Ban

k ac

cess

ion

num

bers

of w

ild st

rain

s for

rpoD

and

gyr

B ge

ne se

quen

ces

A. d

hake

nsis

(pre

viou

sly c

lass

ified

as A

. aqu

ario

rum

)

Stra

in n

o.

Sour

ce

rpoD

gy

rB

Stra

in n

o.

Sour

ce

rpoD

gy

rB

31

Fish

FN

7733

34

FN69

1766

10

7 W

ound

FR

6758

38

FR67

5869

32

Fi

sh

FN79

6726

FN

7065

55

121

Wou

nd

FR67

5839

FR

6758

70

47

Sput

um

FR67

5826

FR

8659

66

123

Wou

nd

FR67

5841

FR

6758

71

56

Bon

e ch

ips

FN77

3333

FN

7967

34

124

Wou

nd

FR67

5840

FR

6758

72

60

Blo

od

FR67

5827

FR

6758

58

139

Stoo

l FR

6758

42

FR67

6941

67

W

ound

FR

6758

28

FR67

5859

14

1 W

ound

FR

6758

43

FR67

6942

70

B

lood

FN

7967

24

FN79

6735

15

4 B

lood

FR

6758

44

FN79

6752

71

W

ound

FR

6758

29

FR67

5860

16

5 U

nkno

wn

FR67

5845

FR

6769

43

73

Wou

nd

FR67

5830

FR

6758

61

168

Wou

nd

FR67

5846

FR

6769

44

74

Wou

nd

FR67

5831

FR

6758

62

169

Stoo

l FR

6758

47

FR67

6945

79

W

ound

FR

6758

32

FR67

5863

17

2 U

rine

FR67

5886

FR

6769

46

88

Wou

nd

FR67

5833

FR

6758

64

176

Wou

nd

FR67

5887

FR

6769

47

91

Wou

nd

FR67

5834

FR

6758

65

180

Stoo

l FR

6758

88

FR67

6948

93

U

rine

FR67

5835

FR

6758

66

182

Wou

nd

FR67

5889

FR

6769

49

95

Wou

nd

FR67

5836

FR

6758

67

183

Stoo

l FR

6758

90

FR67

6950

10

4 W

ound

FR

6758

37

FR67

5868

21

2 W

ound

FR

6758

91

FR67

6951

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- 163

- T

able

4.2

C

ontin

ued.

A. d

hake

nsis

(pre

viou

sly c

lass

ified

as A

. aqu

ario

rum

)

Stra

in n

o.

Sour

ce

rpoD

gy

rB

Stra

in n

o.

Sour

ce

rpoD

gy

rB

213

Wou

nd

FR67

5892

FR

6769

52

240

Wat

er

FR67

5853

FR

6815

77

220

Wou

nd

FN80

8215

FR

6769

53

241

Wat

er

FR67

5854

FR

6815

78

222

Wat

er

FR68

2782

FR

6769

54

242

Wat

er

FR67

5855

FR

6815

79

223

Wat

er

FN80

8216

FR

6769

55

244

Wat

er

FR67

5856

FR

6815

80

226

Wat

er

FR67

5893

FR

6769

56

246

Wat

er

FR68

1589

FR

6815

81

227

Wat

er

FR67

5894

FR

6769

57

250

Wat

er

FR68

1590

FR

6815

82

228

Wat

er

FR67

5848

FR

6769

58

251

Wat

er

FR68

1591

FR

6815

83

229

Wat

er

FN79

6725

FN

7967

36

255

Wat

er

FR68

1592

FR

6815

84

230

Wat

er

FN80

8217

FR

6769

59

256

Wat

er

FN79

6728

FN

7967

38

232

Wat

er

FR67

5849

FR

6769

60

257

Wat

er

FN79

6733

FN

7967

39

234

Wat

er

FR67

5850

FR

6815

74

258

Wat

er

FR68

1593

FR

6815

85

235

Wat

er

FN79

6727

FN

7967

37

263

Wat

er

FR68

1594

FR

6815

86

236

Wat

er

FR67

5851

FR

6815

75

278

Wou

nd

FR68

1595

FR

6815

87

239

Wat

er

FR67

5852

FR

6815

76

279

Wou

nd

FR68

1596

FR

6815

88

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-- 1

64 --

Tab

le 4

.2

Con

tinue

d.

A. h

ydro

phila

Stra

in N

o.

Sour

ce

rpoD

gy

rB

Stra

in N

o.

Sour

ce

rpoD

gy

rB

23

Wou

nd

FR68

1805

FR

6815

97

118

Sput

um

FR68

1875

FR

6817

44

34

Fish

FR

6818

06

FR68

1598

12

6 W

ound

FR

6818

76

FR68

1745

59

B

lood

FR

6818

07

FR68

1599

12

7 W

ound

FR

6818

77

FR68

1746

61

B

iliar

y st

ent

FN79

5730

FN

7967

41

128

Wou

nd

FR68

1878

FR

6817

47

69

Wou

nd

FR68

1808

FR

6816

00

130

Wou

nd

FR68

1879

FR

6817

48

77

Wou

nd

FR68

1809

FR

6816

01

133

Mor

tuar

y FR

6818

80

FR68

1749

83

Sp

utum

FR

6818

10

FR68

1602

14

4 W

ound

FR

6818

81

FR68

1750

84

B

lood

FR

6818

11

FR68

1603

14

5 B

lood

FR

6818

82

FR68

1751

89

B

ile

FR68

1865

FR

6816

05

148

Wou

nd

FR68

1883

FR

6817

52

90

Wou

nd

FR68

1866

FR

6816

06

149

Blo

od

FR68

1884

FR

6817

53

98

Blo

od

FR68

1867

FR

6817

36

150

Tiss

ue

FR68

1885

FR

6817

54

101

Wou

nd

FR68

1868

FR

6817

37

151

Blo

od

FR68

1886

FR

6817

55

105

Stoo

l FR

6818

69

FR68

1738

15

2 B

lood

FR

6818

87

FR68

1756

11

2 W

ound

FR

6818

70

FR68

1739

18

5 W

ound

FR

6818

88

FR68

1757

11

3 D

rain

flui

d FR

6818

71

FR68

1740

23

1 W

ater

FR

6818

89

FR68

1758

11

5 St

ool

FR68

1872

FR

6817

41

243

Wat

er

FR68

1890

FR

6817

59

116

Wou

nd

FR68

1873

FR

6817

42

245

Wat

er

FR68

1891

FR

6817

60

117

Wou

nd

FR68

1874

FR

6817

43

260

Wat

er

FR68

1892

FR

6817

61

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-- 1

65 --

Tab

le 4

.2

Con

tinue

d.

A. c

avia

e

Stra

in N

o.

Sour

ce

rpoD

gy

rB

Stra

in N

o.

Sour

ce

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gy

rB

21

Unk

now

n FR

6819

06

FR68

2025

10

9 B

lood

FR

6819

23

FR68

2039

26

U

nkno

wn

FR68

1907

FR

6820

26

110

Blo

od

FR68

1924

FR

6821

32

30

Fish

FR

6819

08

FR68

2027

14

0 Pe

riton

eal f

luid

FR

6819

25

FR68

2133

57

B

lood

FR

6819

09

FR68

2028

14

2 St

ool

FR68

2022

FR

6820

40

58

Blo

od

FR68

1910

FR

6820

29

143

Wou

nd

FR68

2023

FR

6820

41

62

T-tu

be ti

p FR

6819

11

FR68

2030

15

3 St

ool

FR68

2011

FR

6820

43

65

Blo

od

FR68

1912

FR

6820

31

156

Stoo

l FR

6820

12

FR68

2044

75

B

lood

FR

6819

13

FR86

5963

15

8 St

ool

FR68

2013

FR

6821

34

78

CA

PD fl

uid

FR68

1914

FR

6820

32

163

Wou

nd

FR68

2014

FR

8659

64

80

Blo

od

FR68

1915

FR

6820

33

167

Wou

nd

FR68

2015

FR

6821

35

87

Blo

od

FR68

1916

FR

6820

34

178

Bile

FR

6820

16

FR68

2136

94

St

ool

FR68

1917

FR

6820

35

187

Stoo

l FR

6820

17

FR68

2137

96

B

lood

FR

6819

18

FR68

2504

18

8 B

ile

FR68

2018

FR

6821

38

102

Stoo

l FR

6819

19

FR68

2036

20

0 B

lood

FR

6820

19

FR68

2139

10

3 St

ool

FR68

1920

FR

6820

37

216

Stoo

l FR

6820

20

FR68

2140

10

6 B

lood

FR

6819

21

FR68

2038

21

7 St

ool

FN79

6729

FR

6825

05

108

Stoo

l FR

6819

22

FR68

2131

27

0 W

ound

FR

6820

21

FR68

2507

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-- 1

66 --

T

able

4.2

Con

tinue

d.

A. v

eron

ii bv

. sob

ria

St

rain

No.

So

urce

rp

oD

gyrB

St

rain

No.

So

urce

rp

oD

gyrB

24

W

ound

FR

6820

24

FR68

2508

13

1 B

lood

FR

6827

65

FR68

2522

25

W

ound

FR

6825

72

FR68

2509

13

4 W

ound

FR

6827

66

FR68

2523

27

A

ppen

dix

FR68

2573

FN

7967

49

135

Blo

od

FR68

2767

FR

6825

24

28

Stoo

l FR

6825

74

FR68

2510

13

6 St

ool

FR68

2768

FR

6825

25

33

Fish

FR

6825

75

FR68

2511

14

7 W

ound

FR

6827

69

FR68

2527

66

B

lood

FN

7967

31

FR68

2512

15

9 W

ound

FR

6827

70

FR68

2528

72

B

lood

FR

6825

76

FR68

2513

16

4 Sp

utum

FR

6827

71

FR68

2529

81

B

lood

FR

6825

77

FR68

2514

16

6 St

ool

FR68

2772

FR

6825

30

97

Stoo

l FR

6825

78

FR68

2515

17

1 Sp

utum

FR

6827

73

FR68

2531

99

St

ool

FR68

2579

FR

6825

16

174

Wou

nd

FR68

2774

FR

6825

32

111

Blo

od

FR68

2580

FR

6825

17

175

Stoo

l FR

6827

75

FR68

2533

11

4 St

ool

FR68

2581

FR

6825

18

177

Wou

nd

FR68

2776

FR

6825

34

120

Stoo

l FR

6827

62

FR68

2519

18

4 St

ool

FR68

2777

FR

6825

35

125

Blo

od

FR68

2763

FR

6825

20

211

Wou

nd

FR68

2778

FR

6825

37

129

Wou

nd

FR68

2764

FR

6825

21

214

Stoo

l FR

6827

79

FR68

2538

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-- 1

67 --

Tab

le 4

.2

Con

tinue

d.

A. v

eron

ii bv

. sob

ria

St

rain

No.

So

urce

rp

oD

gyrB

St

rain

No.

So

urce

rp

oD

gyrB

21

5 St

ool

FR68

2780

FR

6825

39

247

Wat

er

FR68

2790

FR

6825

48

218

Blo

od

FR68

2781

FR

6825

40

252

Wat

er

FR68

2791

FR

6825

49

219

Stoo

l FR

6827

83

FR68

2541

25

4 W

ater

FR

6827

92

FR68

2550

22

1 B

lood

FR

6827

84

FR68

2542

25

9 W

ater

FR

6827

93

FR68

2551

22

4 W

ater

FR

6827

85

FR68

2543

26

5 W

ater

FR

6827

94

FR68

2552

22

5 W

ater

FR

6827

86

FR68

2544

26

7 W

ater

FN

7967

32

FN79

6750

23

3 W

ater

FR

6827

87

FR68

2545

26

8 W

ater

FR

6827

96

FR68

2553

23

7 W

ater

FR

6827

88

FR68

2546

26

9 B

lood

FR

6827

97

FR68

2554

238

Wat

er

FR68

2789

FR

6825

47

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-- 1

68 --

Tab

le 4

.2

Con

tinue

d.

Mis

cella

neou

s spe

cies

Spec

ies

Stra

in n

o.

Sour

ce

rpoD

gy

rB

A. j

anda

ei

35

Fish

lesi

on

FR68

2798

FN

7967

42

25

3 W

ater

FN

7733

26

FR68

2555

26

2 W

ater

FN

7733

27

FN79

6743

A. s

alm

onic

ida

190

Wou

nd

FN77

3330

FR

6828

01

19

9 C

rab

FN77

3331

FN

7967

44

A. m

edia

29

Fi

sh

FN77

3332

FN

6917

72

85

B

lood

FR

6827

99

FR68

2802

17

9 St

ool

FR68

2800

FR

6828

03

A, s

chub

ertii

18

6 W

ound

FR

8659

67

FN69

1774

A. b

estia

rum

68

B

lood

FN

7733

43

FN69

1771

A. a

llosa

ccha

roph

ila

100

Stoo

l FN

7733

44

FN69

1770

A. a

ustr

alie

nsis

stra

in 2

66T

Isol

ated

from

wat

er

dnaJ

H

E611

954

dnaX

H

E611

951

gyrA

H

E611

952

gyrB

FN

6917

73

recA

H

E611

953

rpoD

FN

7733

35

16S

rRN

A

HE6

1195

5

Page 195: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

- 169 -

Figure 4.1 Concatenated neighbour-joining phylogenetic tree showing the position of

A. dhakensis strains derived from the rpoD and gyrB sequences (1339 nt).

Isolates (93 172) Isolates (222 257)

Isolate 213 Isolate 258

Isolate 226 Isolates (235 250) Isolate 71 Isolate 107

Isolate 31 Isolate 227

Isolate 228 Isolate 241

Isolate 255 Isolate 141

Isolate 67 Isolates (73 74)

Isolate 176 Isolate 256 Isolate 242

Isolate 279 Isolates (47 95 139 165)

Isolate 183 Isolate 32

Isolates (230 236 249) Isolate 263

Isolate 121 A. dhakensis (LMG 19562T)) Isolates (223 232 240)

Isolate 168 Isolate 278

Isolate 182 Isolate 180

Isolate 104 Isolates (56 220)

Isolate 239 Isolate 60 Isolate 79

Isolate 91 Isolates (123 124)

Isolates (229 234) Isolate 251

Isolate 154 Isolate 212

Isolate 70 Isolate 88

Isolate 169 Isolate 244

A. hydrophila (ATCC 7966T) A. caviae (ATCC 13136)

A. taiwanensis (CECT 7403T) A. sanarellii (CECT 7402T)

A. jandaei (ATCC 49568T) A. trota (ATCC 49657T)

A. sobria (CDC 9540-76) A. fluvialis (CECT 7401T)

A. allosaccharophila (DSM 11576T) A. veronii bv. sobria (ATCC 9071)

A. bestiarum (ATCC 51108T) A. popoffii (CIP 105493T))

A. piscicola (CECT 7443T) A. salmonicida (CECT 894T)

A. molluscorum (DSM 17090T) A. rivuli (CECT 7518T)

A. bivalvium (CECT 7113T) A. media (ATCC 39907T)

A. eucrenophila (ATCC 23309T) A. tecta (CECT 7082T)

A. encheleia (DSM 11577T) Aeromonas spp. HG 11 (CECT 4253)

A. simiae (DSM 16559T) A. diversa (CECT 4254T)

A. schubertii (ATCC 43700T) 100 100

100

100

99

75

97

99

98

96

88

75

100

78

85

85

70

71

72

100

0.02

Page 196: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-- 170 --

Figure 4.2 Concatenated neighbour-joining phylogenetic tree showing the position of

A. caviae strains derived from the rpoD and gyrB genes sequences (1330 nt).

Isolate 58 Isolate 156

Isolate 80 Isolate 87 Isolate 75

Isolate 110 Isolate 108

Isolates (146 163 167) Isolate 62 Isolate 103

Isolate 102 Isolate 158 Isolate 78 Isolate 216

Isolate 21 Isolate 30

Isolate 142 Isolate 140 Isolate 96

Isolates (57 270) Isolate 153

Isolate 106 A. caviae (ATCC 13136)

Isolate 65 Isolate 26 Isolate 264

Isolate 188 Isolate 143

Isolate 178 Isolate 109

Isolate 217 Isolate 94

Isolate 187 Isolate 200

A. taiwanensis (CECT 7403T) A. sanarellii (CECT 7402T)

A. media (ATCC 39907T) A. dhakensis (CECT 7289T)

A. hydrophila (ATCC 7966T) A. trota (ATCC 49657T)

A. jandaei (ATCC 49568T) A. sobria (CDC 9540-76)

A. fluvialis (CECT 7401T)) A. allosaccharophila (DSM 11576T)

A. veronii bv. sobria (ATCC 9071) A. encheleia (DSM 11577T)

Aeromonas spp. HG11 (CECT 4253) A. eucrenophila (ATCC 23309T)

A. tecta (CECT 7082T) A. molluscorum (DSM 17090T)

A. rivuli (CECT 7518T) A. bivalvium (CECT 7113T)

A. salmonicida (CECT 894T) A. piscicola (CECT 7443T)

A. bestiarum (ATCC 51108T) A. popoffii (CIP 105493T)

A. simiae (DSM 16559T) A. diversa (CECT 4254T)

A. schubertii (ATCC 43700T) 99 99

99

97

99 98

99

86 94

87

99

73

81

97 99

98

99

99

80

0.02

Page 197: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-- 171 --

Figure 4.3 Concatenated neighbour-joining phylogenetic tree showing the position of

A. hydrophila strains derived from the rpoD and gyrB genes sequences (1331 nt).

Isolate 34 Isolate 77

Isolate 59 Isolate 146

Isolates (90 115) Isolate 130

Isolate 89 Isolate 127 Isolate 86

Isolate 116 Isolates (117 118 260)

Isolate 83 Isolate 61

Isolate 113 Isolate 98

Isolate 185 Isolate 23

Isolate 84 Isolate 243

Isolate 69 Isolate 133

Isolates (144 145 148 150 151 152) A. hydrophila (ATCC 7966T Isolate 112 Isolate 101

Isolates (126 245) Isolate 128

Isolate 105 Isolate 231

Isolate 261 A. dhakensis (CECT 7289T)

A. salmonicida (CECT 894T) A. piscicola (CECT 7443T)

A. bestiarum (ATCC 51108T) A. popoffii (CIP 105493T)

A. trota (ATCC 49657T) A. jandaei (ATCC 49568T)

A. sobria (CDC 9540-76) A. fluvialis (CECT 7401T)

A. allosaccharophila (DSM 11576T) A. veronii bv. sobria (ATCC 9071)

A. taiwanensis (CECT 7403T) A. sanarellii (CECT 7402T)

A. caviae (ATCC 13136) A. molluscorum (DSM 17090T)

A. rivuli (CECT 7518T) A. bivalvium (CECT 7113T)

A. media (ATCC 39907T) A. encheleia (DSM 11577T)

Aeromonas spp. HG11 (CECT 4253) A. eucrenophila (ATCC 23309T)

A. tecta (CECT 7082T) A. simiae (DSM 16559T)

A. diversa (CECT 4254T) A. schubertii (ATCC 43700T) 99 99

99

99

93

92

96

97

98

99

71

99 99

99

84

72

99

0.02

Page 198: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-- 172 --

Figure 4.4 Concatenated neighbour-joining phylogenetic tree derived from the rpoD

and gyrB genes sequences (1341 nt) showing the position of A. veronii bv. sobria and

other species including strain 266.

Isolate 111 Isolate 114

Isolate 159 Isolate 174

Isolate 177 Isolate 259

Isolate 269 Isolates (28 166)

Isolate 211 Isolate 125

Isolate 134 Isolates (224 225)

Isolates (164 171) Isolates (24 25)

Isolate 136 Isolate 268

Isolates (131 135) Isolates (247 252)

Isolate 265 Isolate 129

Isolate 33 Isolate 97

Isolates (99 120) Isolate 72

Isolates (175 233) Isolate 184

Isolate 237 Isolate 238

Isolate 254 A. veronii bv. sobria (ATCC 9071)

Isolate 27 Isolate 66

Isolate 218 Isolate 147

Isolate 214 Isolate 215

Isolates (81 219) Isolate 221

Isolate 267 Isolate 100

A. allosaccharophila (DSM 11576T) Isolate 266

A. fluvialis (CECT 7401T) A. sobria (CDC 9540-76)

Isolate 253 Isolate 262

Isolate 35 A. jandaei (ATCC 49568T)

A. trota (ATCC 49657T) Isolate 199 A. salmonicida (CECT 894T)

Isolate 190 A. popoffii (CIP 105493T)

A. piscicola (CECT 7443T) Isolate 68 A. bestiarum (ATCC 51108T)

A. dhakensis (CECT 7289T) A. hydrophila (ATCC 7966T)

Isolate 85 Isolate 179

Isolate 29 A. media (ATCC 39907T)

A. encheleia (DSM 11577T) Aeromonas spp.HG 11 (CECT 4253)

A. eucrenophila (ATCC 23309T) A. tecta (CECT 7082T)

A. taiwanensis (CECT 7403T) A. caviae (ATCC 13136)

A. sanarellii (CECT 7402T) A. bivalvium (CECT 7113T)

A. molluscorum (DSM 17090T) A. rivuli (CECT 7518T) A. simiae (DSM 16559T)

A. diversa (CECT 4254T) Isolate 186

A. schubertii (ATCC 43700T) 99 99

99

99

99

99

99

99

98

99

99

99

99

70

99

74

83

99 96

79

99

99

97

79

92

79

0.02

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- 173

-

Tab

le 4

.3 D

istri

butio

n of

Aer

omon

as sp

p. a

mon

g cl

inic

al a

nd e

nviro

nmen

tal s

ampl

es fo

llow

ing

geno

typi

c ch

arac

teriz

atio

n

Clin

ical

E

nvir

onm

enta

l

Spec

ies

No.

isol

ated

(%

) W

ound

St

ool

Blo

od

Mis

cella

neou

s T

otal

W

ater

Fi

sh

Cra

b T

otal

A. a

llosa

ccha

roph

ila

1 (0

.5)

1 (3

.0)

1

(0.6

)

A. d

hake

nsis

60

(30.

7)

22 (4

0.7)

4 (1

2.1)

3 (9

.0)

5 (2

7.1)

34

(23.

8)

24 (5

4.5)

2

(28.

5)

26

(50.

0)

A. b

estia

rum

1

(0.5

)

1 (3

.0)

1 (0

.6)

A. c

avia

e 36

(18.

4)

5

(9.2

) 11

(33.

3)

11 (3

2.2)

7

(30.

4)

34 (2

3.8)

1 (2

.2)

1 (1

4.2)

2 (3

.8)

A. h

ydro

phila

38

(19

.4)

16 (2

9.6)

2 (6

.0)

7 (2

1.2)

8

(34.

7)

33 (2

3.0)

4 (9

.0)

1 (1

4.2)

5

(9.6

)

A. ja

ndae

i 3

(1.5

)

2

(4.5

) 1

(14.

2)

3 (5

.7)

A. m

edia

3

(1.5

)

1

(3.0

)

1 (3

.0)

2 (1

.3)

1

(14.

2)

1 (1

.9)

A. sa

lmon

icid

a 2

(1.0

)

1 (1

.8)

1

(0.6

)

1

1(

1.9)

A. sc

hube

rtii

1 (0

.5)

1

(1.8

)

1 (0

.6)

A. v

eron

ii bv

. sob

ria

49 (2

5.1)

9

(16.

7)

14 (4

2.4)

10

(30.

3)

3 (1

3.0)

36

(25.

1)

12 (2

7.2)

1

(14.

2)

13

(25.

0)

Aero

mon

as sp

p.

1 (0

.5)

1 (2

.2)

1

(1.9

)

Tot

al

195

54

33

33

23

143

44

7 1

52

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- 174 -

4.3.4. Phenotypic differentiation of Aeromonas dhakensis from other major spp.

Biochemically, A. dhakensis could be differentiated from A. hydrophila by its inability

to produce acid from L-arabinose, ability to utilize citrate (93%) and produce

alkylsulfatase (73%). In contrast, all A. hydrophila strains produced acid from L-

arabinose but were less likely to utilize citrate (26%) as a carbon source or produced

alkylsulfatase (3%); from A. caviae by a positive Vogues-Proskauer reaction (95%

positive), production of elastase (93%), stapholysin (82%) and LDC (95%) while A.

caviae was usually negative in all these tests; from A. veronii bv. sobria by its ability to

utilize DL-lactate (78 versus 2%) and production of stapholysin (82 versus 0%) (Table

4.4).

4.3.5. Intra- and inter-species dissimilarities

The intra-species dissimilarity derived from the combination of the rpoD and gyrB

(approximately 1,294 bp) ranged from 0.4 to 3.5% between the type species and the

wild strains identified as A. dhakensis. Interspecies dissimilarity ranged from 19.1%

between A. molluscorum and A. diversa to 1.4% between A. encheleia and Aeromonas

spp. HG11 (Table 4.5 in the CD ROM attached).

4.4. DISCUSSION

The distribution of A. dhakensis strains in clinical and water samples found in the

present study contradicts the long-standing notion that A. caviae, A. hydrophila, and A.

veronii bv. sobria represent the most frequently isolated aeromonads (Altwegg and

Geiss 1989; Janda and Abbott 1998; Ørmen et al. 2005). The number of A. hydrophila

strains reclassified into several different species after genotypic characterization

indicates that accurate identification of aeromonads requires molecular methods.

Furthermore, these results concurred with those of Soler et al. (2004) who suggested

that the combined analysis of more than one target improved the resolving power and

the ability to differentiate between closely related species.

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- 175

- T

able

4.4

B

ioch

emic

al c

hara

cter

istic

s of A

erom

onas

afte

r gen

otyp

ic id

entif

icat

ion

(% p

ositi

ve)

A

dh

Ahy

A

ca

Avs

Am

e A

ja

Asa

A

al

Abe

A

sc

N

o. o

f str

ains

Cha

ract

eris

tics

60

38

36

49

3 3

2 1

1 1

Indo

le

95

100

92

96

100

100

100

+ +

Citr

ate

93

26

78

84

0

100

100

VP

95

100

0 90

0

67

100

+ LD

C

95

100

3 10

0 0

100

50

+ +

+ G

as fr

om g

luco

se

90

95

3 88

0

100

50

+ +

Aci

d fr

om:

L-A

rabi

nose

0

100

100

4 10

0 0

100

+

C

ello

bios

e 0

13

81

40

100

0 10

0 +

Lact

ose

3 16

92

30

10

0 0

100

Man

nito

l 10

0 10

0 10

0 98

10

0 10

0 10

0 +

+

-M-D

-glu

cosi

de

93

82

0 38

0

33

100

Salic

in

95

95

97

20

100

0 10

0

Su

cros

e 10

0 82

10

0 98

10

0 0

100

+ +

G-1

-P/G

-6-P

98

10

0 8

100

100

100

100

+ +

+ A

escu

lin h

ydro

lysi

s 97

10

0 97

42

10

0 0

100

+ +

Glu

cona

te

60

74

0 75

0

100

50

-H

aem

olys

is

95

66

31

92

33

100

100

+ +

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-- 1

76 --

Tab

le 4

.4

Con

tinue

d.

A

dh

Ahy

A

ca

Avs

Am

e A

ja

Asa

A

al

Abe

A

sc

N

o. o

f str

ains

Cha

ract

eris

tics

60

38

36

49

3 3

2 1

1 1

Util

izat

ion

of:

DL

Lact

ate

78

76

72

0 0

0 0

+ U

roca

nic

83

87

78

78

33

100

50

+

+

PZA

43

32

94

16

67

0

0

St

apho

lysi

n 82

89

0

2 0

0 50

A

lkyl

sulfa

tase

73

3

3 26

0

33

0 +

+ El

asta

se

93

100

0 0

0 0

100

+

Ty

rosi

ne

53

66

19

40

33

67

50

+ C

epha

loth

inR

93

95

92

86

100

67

100

R

S R

PY

R

0 0

0 8

0 67

0

+

D

efer

oxam

ineR

100

100

100

94

100

100

100

S R

R

O

129R

98

92

10

0 78

10

0 10

0 10

0 +

+ +

Gro

wth

in T

CB

S 57

39

89

62

0

33

50

CA

MP

aero

bic

88

55

0 22

0

0 50

+

CA

MP

anae

robi

c 90

68

0

24

0 0

100

Col

istin

R 77

76

17

64

0

100

100

R

S S

Adh,

A. d

hake

nsis

; Ahy

, A. h

ydro

phila

; Aca

, A. c

avia

e; A

vs, A

. ver

onii

bv. s

obria

; Am

e, A

. med

ia; A

ja, A

. jan

daei

; Asa

, A. s

alm

onic

ida;

Aal

, A.

allo

sacc

haro

phila

; Abe

, A. b

estia

rum

; Asc

, A. s

chub

ertii

; PZA

, pyr

azin

amid

ase

activ

ity; P

YR

, py

rrol

idon

yl-

-nap

hthy

lam

ide

activ

ity;

TCB

S, th

iosu

lpha

te c

itrat

e bi

le su

cros

e ag

ar.

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-177-

The results presented in this chapter revealed that, when used independently, sequences

of both genes led to comparable identification, suggesting that gyrB and rpoD were

equivalent markers for the taxonomic discrimination of Aeromonas spp. Phylogenetic

trees generated from the rpoD and gyrB sequences (Figs. 4.1 to 4.4) were comparable to

the one derived from a partial rpoB gene sequence (Lamy et al. 2010) except that the

former sequences consistently placed A. molluscorum well within the centre of the trees,

while it was placed in a more distant position when the tree was constructed from the

rpoB sequences alone.

Data presented here may also help to explain the biochemical and genotypic

heterogeneity previously observed in A. hydrophila (Miyata et al. 1995; Janda and

Abbott 1998). Results from this study suggest that a lack of congruence between

phenotypic and genotypic identification exists consistent with a previous study (Beaz-

Hidalgo et al. 2010). Correct identification occurred in only 35 (33.6%) out of 104

strains phenotypically identified as A. hydrophila, while the remaining strains were re-

identified as A. dhakensis (54 strains, 51.9%), A. veronii bv. sobria (14, 13.4%), and A.

bestiarum (one strain, 1.2%) by molecular analysis.

There are several reasons for this to occur. Firstly, the usefulness of many tests used in

here and also observed by others (Abbott et al. 2003) reveals that Aeromonas lack

reliable biochemical markers. Secondly, the discriminatory value of some tests ranging

from 16 to 75% is not optimal. Thirdly, the true phenotypic profiles of the minor or less

frequently isolated species remains unknown. This situation may eventually be

resolved, at least for the major species, by determining the phenotypic characteristics of

genotypically identified strains. For example, production of elastase was observed only

in strains of A. hydrophila (100%), A. salmonicida (100%) and A. dhakensis (93%).

Similarly, DL lactate was utilized by A. hydrophila (76%), A. dhakensis (78%) and A.

caviae (72%) while stapholysin production was observed mainly in A. hydrophila

(89%), A. salmonicida (50%) and A. dhakensis (82%). Also among the major species,

acid from L-arabinose was produced by all A. hydrophila, A. caviae and A. salmonicida

but not by A. dhakensis (0%) strains. Similarly, acid production from cellobiose was

observed in the majority of A. caviae (81%) and all A. salmonicida (100%) but not in A.

dhakensis (0%) and rarely in A. hydrophila (4%) strains. These results suggest that these

phenotypic characteristics may be considered validated in genetically identified

aeromonads.

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-178-

The interspecies dissimilarity values obtained between A. encheleia and Aeromonas sp.

HG11, confirms the close relationship that exists between these species (Table 4.5 CD-

ROM). The sequence divergence of specific isolates ranged from 0.4% for strains 223,

232 and 240 to 3.5% for strain 213. This result is consistent with the positions of these

isolates as shown in Fig. 4.1 suggesting also that the taxonomic position of strain 213

requires further investigation. Similarly, the position of isolate 266 indicates that this

strain forms a separate line of descent from other species in the genus with A.

allosaccharophila DSM 11576T and A. fluvialis CECT 7401T as its closest relatives and

requires further investigation (Fig. 4.4).

In this chapter, WA clinical and environmental Aeromonas isolates previously classified

by a phenotypic scheme were re-identified by determining the sequences of the gyrB

and rpoD housesekeeping genes. Thus, results in this chapter revealed that accurate

identification of these bacteria is compromised when only a phenotypic method is used.

Hence, distribution of the species is also compromised and in the case of A. dhakensis

(formerly A. aquariorum) the study shows that this species is globally distributed and

can be misidentified as A. hydrophila consistent with previous observations (Figueras et

al. 2009).

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-179-

CHAPTER 5: ANTIMICROBIAL SUSCEPTIBILITIES

5.1. INTRODUCTION

Antimicrobial resistance in these organisms is usually chromosomally mediated, but -

lactamases produced by aeromonads may occasionally be encoded by plasmids (Fosse

et al. 2004; Sánchez-Céspedes et al. 2008) or integrons (Barlow and Gobius 2009).

These enzymes have activity against most -lactam antimicrobial agents, including

cefepime and other extended-spectrum cephalosporins. Antimicrobial susceptibility

reporting for Aeromonas generally followed guidelines for the Enterobacteriaceae until

the Clinical and Laboratory Standards Institute (CLSI) recently published

recommendations (CLSI 2011).

The objective of this chapter was to determine the antimicrobial susceptibility profiles

of a collection of Aeromonas strains against 26 antimicrobial agents by the agar dilution

breakpoint and E-strip methods. The strains were previously characterized by extensive

phenotypic and genotypic methods and were isolated from clinical, fish, and

environmental sources.

5.2. Bacterial strains

Bacterial strains used in this project are listed in Tables 2.6 and 2.7. A total of 193

strains were examined, of these 144 were isolated from clinical specimens including 54

from wound, 33 from blood, 34 from stools and 23 from miscellaneous sources (Table

2.6). Environmental isolates included a total of 49 strains comprising 43 from water,

five from fish and one from crab meat (Table 2.7). All strains were previously

characterized by extensive biochemical testing (Aravena-Román et al. 2011a) and their

identities confirmed genotypically from their gyrB and rpoD gene sequences (Aravena-

Román et al. 2011b). Ten Aeromonas spp. were represented including A. dhakensis (58

strains); A. veronii bv. sobria (49 strains); A. hydrophila (39 strains); A. caviae (36

strains); A. jandaei (three strains); A. media (three strains); A. salmonicida (two strains),

and one strain each of A. allosaccharophila, A. bestiarum and A. schubertii.

5.3. Antimicrobial agents

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-180-

Antimicrobial agents tested included amikacin, amoxicillin, amoxicillin-clavulanate,

cephalothin, cefazolin, cefepime, cefoxitin, ceftadizime, ceftriaxone, ciprofloxacin,

gentamicin, meropenem, moxifloxacin, nalidixic acid, nitrofurantoin, norfloxacin,

pipercillin-tazobactam, tetracycline, ticarcillin-clavulanate, tobramycin, trimethoprim,

and trimethoprim-sulfamethoxazole. E-strips containing doxycycline (AB Biodisk,

Solna, Sweden), ampicillin, tigecycline, meropenem and colistin (BioMérieux,

Marcyl’Etoile, France) were used to determine MICs.

Interpretative criteria for tigecycline, meropenem and ampicillin were derived from

those described for the Enterobacteriaceae by the Food and Drug Administration

(BioMérieux 2010), and those for doxycycline were derived from guidelines described

by the CLSI (2011), as outlined in Table 1 of the E-strip package insert. Interpretative

criteria for colistin were from Fosse et al. (2003b) (Table 2.9). Interpretative criteria for

the reminding antimicrobials were in accordance with the CLSI (CLSI 2006).

5.4. RESULTS

All isolates were inhibited by amikacin, cefepime (8 g/ml), ciprofloxacin, meropenem,

norfloxacin, and tigecycline. Three (1.6%) strains were inhibited by amoxicillin as

shown by the agar dilution and confirmed by the E-strip method. The MIC values were

8 g/ml for all three isolates which included one clinical and one environmental A.

veronii bv. sobria and one environmental A. dhakensis isolate (Table 5.1). Thirty-two

isolates (16.5%) failed to grow in the presence of amoxicillin-clavulanate, while 17

(8.8%) were non-susceptible to ticarcillin-clavulanate (16/2 g/ml). Of these, eight

(4.4%) were also non-susceptible to the higher concentration of ticarcillin-clavulanate

(64/2 g/ml).

Susceptibility to cephalothin and cefazolin was observed in 53 (27.4%) and 40 (20.7%)

isolates, respectively. A moderate level of susceptibility was detected with cefoxitin

(126 isolates, 65.2%) and colistin (86 isolates, 44.5%). The majority of the isolates were

susceptible to the remaining antimicrobial agents. The MICs for doxycycline ranged

from 0.064 to 24.0 g/ml, those for tigecycline ranged from 0.064 to 3.0 g/ml, and

those for colistin ranged from 0.094 to >256 g/ml. Susceptibility to doxycycline and

tigecycline was high in clinical strains, at 97.2 and 100%, respectively.

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-181-

There was no statistically significant difference in antimicrobial susceptibility between

clinical and environmental isolates of A. dhakensis. In contrast, clinical isolates of A.

veronii bv. sobria were less susceptible than environmental strains (p = 0.0226). Other

statistically significant differences were observed for amoxicillin-clavulanate between

A. dhakensis and A. hydrophila (p = 0.0036) (A. dhakensis was less susceptible than A.

hydrophila) and between A. dhakensis and A. veronii bv. sobria (p = 0.0053) (A. veronii

bv. sobria was less susceptible than A. dhakensis) but not between A. dhakensis and A.

caviae. Further, susceptibility to cephalothin was significantly higher in A. veronii bv.

sobria than in A. dhakensis, A. caviae, and A. hydrophila (p = 0.0001) (Table 5.1).

Nine clinical isolates (6.2%) were able to grow in agar plates containing 4 g/ml of

tobramycin, including seven (14.2%) A. veronii bv. sobria, one (2.7%) A. caviae, and

one (33.3%) A. media isolate. Multidrug non-susceptible patterns were observed in

three (1.5%) isolates. Of these, A. caviae strain 138 was less susceptible to most -

lactams, including aztreonam. A. veronii bv. sobria strain 189 was the only isolate to

grow in the presence of both gentamicin and tobramycin. Among the minor species, the

single A. allosaccharophila strain exhibited a multidrug resistance profile including

resistance to both fluoroquinolones and trimethoprim/sulfamethoxazole (Table 5.1).

Susceptibility to colistin was recorded in 57 (39.05%) clinical and 29 (59.1%)

environmental isolates. Aeromonas caviae was the most susceptible species (83.7%),

next to A. dhakensis (31.0%). Most environmental isolates were susceptible to

tetracycline (81.6%) and nalidixic acid (93.8%). Moderate susceptibility was observed

with amoxicillin-clavulanate (46.9%), cephalothin (46.9%), and cefoxitin (63.2%),

while only five (10.2%) isolates were susceptible to cefazolin (Table 5.2).

5.5. DISCUSSION

In general, growth of Aeromonas was inhibited by most antimicrobial agents, with few

isolates showing a multidrug non-susceptible profile. Susceptibility to tetracycline was

high (94.3%), consistent with previous reports from Australia and the United States

(Koehler and Ashdown 1993). In contrast, tetracycline resistance in up to 49% of

isolates has been reported in studies from the Asian region (Chang and Bolton 1987; Ko

et al. 1996).

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-182

-

Tab

le 5

.1

Ant

imic

robi

al su

scep

tibili

ties d

eter

min

ed fo

r diff

eren

t Aer

omon

as sp

p. (p

erce

ntag

e/nu

mbe

r of s

train

s sus

cept

ible

)

Ant

imic

robi

al a

gent

Breakpoint(s) (g/ml)

A. caviae (n = 36)

A. dhakensis (n = 58)

A. hydrophila (n = 38)

A. veronii bv. sobria (n = 49)

A. jandaei (n = 3)

A. media (n = 3)

A. salmonicida (n = 2)

A. allosaccharophila (n = 1)

A. bestiarum (n = 1)

A. schubertii (n = 1)

Am

oxic

illin

8

0 1.

7 (1

) 0

4.0

(2)

0 0

0 R

R

R

A

mox

icill

in-c

lavu

lana

te

8/4

13.9

(5)

24.1

(14)

2.

6 (1

) 16

.3 (8

) 66

.7 (2

) 0

0 R

S

S N

orflo

xaci

n 4

100

100

100

100

100

10

0 10

0 R

S

S C

ipro

floxa

cin

1 10

0 10

0 10

0 10

0 10

0 10

0 10

0 R

S

S N

itrof

uran

toin

32

97

.2 (3

5)

100

100

100

100

100

100

S S

S Tr

imet

hopr

im

8 86

.1 (3

1)

96.5

(56)

94

.7 (3

6)

97.9

(1)

100

100

100

R

S S

Cep

halo

thin

8

8.3

(3)

22.4

(13)

5.

2 (2

) 77

.5 (3

8)

0 0

0 R

R

S

Mer

open

em

0.25

97

.2 (3

5)

100

97.3

(37)

95

.9 (4

7)

100

100

100

R

S S

1

97.2

(35)

10

0 97

.3 (3

7)

100

100

100

100

S S

S

4 10

0 10

0 97

.3 (3

7)

100

100

100

100

S S

S G

enta

mic

in

4 10

0 10

0 10

0 97

.9 (4

8)

100

100

100

S S

S To

bram

ycin

4

94.4

(34)

10

0 10

0 87

.7 (4

3)

100

66.7

(2)

100

S S

S A

mik

acin

16

10

0 10

0 10

0 10

0 10

0 10

0 10

0 S

S S

Cef

triax

one

1 97

.2 (3

5)

96.5

(56)

94

.7 (3

6)

100

100

100

100

S S

S C

efta

zidi

me

0.5

94.4

(34)

98

.2 (5

7)

94.7

(36)

10

0 10

0 66

.7 (2

) 10

0 R

S

S

4 97

.2 (3

5)

100

100

100

100

100

100

S S

S A

ztre

onam

4

100

100

100

100

100

100

100

S S

S

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-183

-

Tab

le 5

.1

Con

tinue

d.

Ant

imic

robi

al a

gent

Breakpoint(s) (g/ml)

A. caviae (n = 36)

A. dhakensis (n = 58)

A. hydrophila (n = 38)

A. veronii bv. sobria (n = 49)

A. jandaei (n = 3)

A. media (n = 3)

A. samonicida (n = 2)

A. allosaccharophila (n = 1)

A. bestiarum (n = 1)

A. schubertii (n = 1)

Tica

rcill

in-c

lavu

lana

te

16/2

94

.4 (3

4)

96.5

(56)

86

.8 (3

3)

87.7

(43)

10

0 33

.3 (1

) 10

0 R

S

S

64

/2

100

98.2

(57)

97

.3 (3

7)

93.8

(46)

10

0 66

.7 (2

) 10

0 S

S

S Tr

imet

h/su

lfam

etho

xazo

le

2/38

94

.4 (3

4)

100

100

100

100

100

100

R

S

S C

efep

ime

0.5

97.2

(35)

98

.2 (5

7)

100

100

100

100

100

S S

S

8

100

100

100

100

100

100

100

S S

S

Nal

idix

ic a

cid

16

97.2

(35)

94

.2 (5

5)

100

95.9

(47)

10

0 10

0 10

0 R

S

S

Cef

oxiti

n 8

69.4

(25)

20

.6 (1

2)

86.8

(33)

97

.9 (4

8)

100

66.7

(2)

100

R

R

S

Pipe

rcill

in-ta

zoba

ctam

16

/4

97.2

(35)

98

.2 (5

7)

97.3

(37)

95

.9 (4

7)

100

100

100

S S

S

64

/4

97.2

(35)

10

0 10

0 95

.9 (4

7)

100

100

100

S S

S

Mox

iflox

acin

1

97.2

(35)

98

.2 (5

7)

100

100

100

100

100

S S

S

Tetra

cycl

ine

4 91

.6 (3

3)

93.1

(54)

97

.3 (3

7)

97.9

(48)

10

0 10

0 10

0 R

S

S

Cef

azol

in

2 0*

0

3.2

(1)*

* 5.

5 (2

)^

0 0#

0

R

NT

R

D

oxyc

yclin

e S,

4;

I, 8

; R,

16

86.1

(31)

93

.1 (5

4)

94.7

(36)

97

.9 (4

8)

100

100

100

S S

S

Tige

cycl

ine

S,

2; I,

4; R

,8

100

100

100

100

100

100

100

S S

S

Col

istin

S,

<2

91.6

(33)

24

.1 (1

4)

28.9

(11)

87

.7 (4

3)

0 10

0 10

0 S

R

S

*15

stra

ins t

este

d; *

*31

stra

ins t

este

d; ^

36 st

rain

s tes

ted;

#on

ly o

ne st

rain

test

ed; N

T, n

ot te

sted

; R, r

esis

tant

; S, s

usce

ptib

le; I

, int

erm

edia

te

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-184-

Table 5.2 Antimicrobial susceptibilities of Aeromonas spp. by source of isolation

MIC

Percentage (no. of strains susceptible)

Antimicrobial agent

Breakpoint(s)

(g/ml)

All isolates (n = 193)

Clinical (n = 144)

Environmental (n = 49)

Amoxicillin 8 1.6 (3) 0.7 (1) 4.0 (2) Amoxicillin-clavulanate 8/4 16.5 (32) 6.25 (9) 46.9 (23) Norfloxacin 4 100 100 100 Ciprofloxacin 1 100 100 100 Nitrofurantoin 32 99.5 (192) 99.3 (143) 100 Trimethoprim 8 92.7 (179) 91.0 (131) 97.9 (48) Cephalothin 8 27.4 (53) 20.8 (30) 46.9 (23) Meropenem 0.25 100 100 100 1 100 100 100 4 100 100 100 Gentamicin 4 99.5 (192) 99.3 (143) 100 Tobramycin 4 95.3 (184) 93.8 (135) 100 Amikacin 16 100 100 100 Ceftriaxone 1 96.9 (187) 95.8 (138) 100 Ceftazidime 0.5 97.4 (188) 96.5 (139) 100 4 99.5 (192) 99.3 (143) 100 Aztreonam 4 99.5 (192) 99.3 (143) 100 Ticarcillin-clavulanate 16/2 91.2 (176) 88.9 (128) 97.9 (48) 64/2 95.9 (185) 95.1 (137) 97.9 (48) Trimethoprim-sulfamethoxazole

2/38 98.9 (191) 98.6 (142) 100

Cefepime 0.5 98.9 (191) 98.6 (142) 100 8 100 100 100 Nalidixic acid 16 96.9 (187) 97.9 (141) 93.8 (46) Cefoxitin 8 65.2 (126) 65.9 (95) 63.2 (31) Pipercillin-tazobactam 16/4 97.4 (188) 96.5 (139) 100 64/4 98.9 (191) 98.6 (142) 100 Moxifloxacin 1 98.9 (191) 99.3 (143) 97.9 (48) Tetracycline 4 94.3 (182) 95.1 (137) 81.6 (40) Cefazolin 2 20.7 (40) 8.2 (9)a 10.2 (5) Doxycycline S, 4; I, 8; R,16 97.9 (189) 97.2 (140) 100 Tigecycline S, 2; I, 4; R,8 100 100 100 Colistin S, <2 44.5 (86) 39.5 (57) 59.1 (29)

a109 strains tested; MIC, minimum inhibitory concentration; S, susceptible; R, resistant; I, intermediate

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-185-

The three amoxicillin-susceptible isolates described here confirm that amoxicillin-

susceptible strains other than A. trota (Carnahan et al. 1991a) occur, as previously

reported (Abbott et al. 2003; Huddlestone et al. 2007), and that their growth may be

suppressed by amoxicillin-containing media. Susceptibility to cepalothin was high in A.

veronii bv. sobria, a feature that has been reported by others and used as a phenotypic

marker to differentiate this species from other aeromonads (Koehler and Ashdown

1993). Similarly, susceptibility to colistin was proposed as an identifying marker for

Aeromonas (Fosse et al. 2003b). Results for colistin were consistent with those obtained

by Fosse et al. (2003b) for A. hydrophila (61.7% resistance in this research, versus

85.8%) and A. jandaei (100% resistance in both studies). However, MIC results

presented here differed from the previous study for A. veronii bv. sobria (61.7% versus

2.5%) and for A. caviae (16.2% versus 2.1%).

The number of isolates susceptible to pipercillin-tazobactam (97.4 and 98.9%) and

ticarcillin-clavulanate (91.2 and 95.9%) were much higher than those susceptible to

amoxicillin-clavulanate (16.5%), suggesting that the former two antimicrobials could be

considered for the treatment of infections caused by Aeromonas. Zemelman et al.

(1984) reported that, depending on the strain, the MIC to amoxicillin decreased from

two to eight fold in combination with clavulanate, thus increasing the activity of this

agent. However, prolonged use of amoxicillin-clavulanate to treat infections caused by

A. veronii bv. sobria has resulted in overexpression of carbapenemases and

cephalosporinases (Sánchez-Céspedes et al. 2009).

All isolates were susceptible to meropenem. A single A. hydrophila isolate that grew in

all three agar dilution concentrations was susceptible by the E-strip method using two

different inocula, 1.5 x 108 CFU/ml and 3.0 x 108 CFU/ml. A large inoculum (3.0 x 108

CFU/ml) has been recommended to detect carbapenemase production before antibiotic

therapy using carbapenems is considered as conventional in vitro susceptibility testing

may fail to detect the presence of carbapenemases in otherwise carbapenemase-

susceptible phenotypes (Rossolini et al. 1996).

Differences in antimicrobial susceptibility between clinical and environmental strains

have been previously described. The resistance observed in environmental aeromonads

has been associated with heavily polluted waters as the source of multiple resistance

plasmids (Huddlestone et al. 2006). In contrast, data from this study suggest that (i)

environmental strains are not the principal source of resistance but that antibiotic

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-186-

resistance in clinical isolates may be due to the selective pressure to which these

organisms may have been exposed, (ii) water sources are less polluted in Western

Australia than other regions, and (iii) environmental strains may have acquired

resistance determinants from clinical strains.

Empirical treatment in some cases does not include cover for Aeromonas species

particularly in infections where the antimicrobials employed are directed toward

microorganims such as staphylococci and streptococci. Inappropriate antimicrobial

therapy has been administered in 20% of infections involving aeromonads (Scott et al.

1978; Vila et al. 2002; Bravo et al. 2003; Figueras 2005) with the potential to increase

morbidity and mortality of affected individuals.

No visible resistance patterns were detected among the major species with the exception

of a few tobramycin-resistant A. caviae and A. veronii bv. sobria strains while most

isolates were highly susceptible to the fluoroquinolones, aminoglycosides,

trimethoprim/sulfamethoxazole, meropenem and third and fourth generation

cephalosporins.

In this chapter, the antimicrobial susceptibility patterns of 193 WA clinical and

environmental Aeromonas isolates were tested against 26 antimicrobial agents. Results

showed that the number of multidrug non-susceptible Aeromonas species in WA

remains low thus, providing clinicians with a wide choice of antimicrobial agents to

treat infections with these bacteria, consistent with other reports (Ko et al. 1996;

Zhiyong et al. 2002). However, antimicrobial susceptibility testing for clinically

significant strains is highly recommended, as resistance to antibacterial agents may be

strain dependent.

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-187-

CHAPTER 6: DESCRIPTION OF AEROMONAS

AUSTRALIENSIS SP. NOV.

6.1. INTRODUCTION

Currently, the genus Aeromonas consists of 27 validated species, seven subspecies and

two biovars. However, some represent synonyms of other species as is the case for A.

trota (Carnahan et al. 1991a), junior synonym of A. enteropelogenes while A.

ichthiosmia (Schubert et al. 1990a) is a junior synonym of A. veronii (Huys et al. 2001).

The position of Aeromonas group HG11 is still uncertain while A. aquariorum and A.

hydrophila ssp. dhakensis have been combined to form A. dhakensis comb. nov. sp. nov

(Beaz-Hidalgo et al. 2013). Further, A. hydrophila ssp. anaerogenes has been

reclassified as A. caviae (Miñana-Galbis et al. 2013) while the validity of A. culicicola

(Pidiyar et al. 2002) and the recognition of A. punctata (Schubert 1967ab) as a senior

synonym of A. caviae have been a source of controversy among microbiologists.

In recent years, the use of 16S rRNA gene sequence to differentiate between Aeromonas

species has been superseded by the use of single-copy genes (Yañez et al. 2003; Soler et

al. 2004; Küpfer et al. 2006; Nhung et al. 2007; Sepe et al. 2008; Miñana-Galbis et al.

2009). Sequences derived from rpoD and gyrB were used for the first time in the

definition of the species A. tecta and A. dhakensis (previously A. aquariorum) by

Demarta et al. 2008 and Martínez-Murcia et al. 2008, respectively, while four

housekeeping genes were used in the description of A. piscicola and A. diversa (Beaz-

Hidalgo et al. 2009; Miñana-Galbis et al. 2010).

During the course of this study, a Gram-negative, facultatively anaerobic bacillus,

designated strain 266T was isolated from an irrigation water sample collected in the

South-West of Western Australia. Initial phenotypic and genotypic testing suggested

that strain 266 may represent a novel Aeromonas spp. The purpose of this chapter was

to use a polyphasic approach to investigate the true taxonomic position of strain 266T.

6.2. Bacterial strains

Bacterial strains used here are listed in Table 2.4. GenBank accession numbers

deposited for strain 266T are listed in Table 4.2.

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-188-

6.3. RESULTS

6.3.1. Phenotypic characteristics

Strain 266T consisted of motile rods with the presence of a polar flagellum. Cells stained

Gram-negative, showing straight, non-spore forming and non-encapsulated rods, 0.6-0.9

m wide and 1.8-2.7 m long (Fig. 6.1), oxidase and catalase positive, reduced nitrate

to nitrite and were susceptible to O/129 (150 g). Colonies on TSA plus sheep blood

were 1.5 to 2.0 mm in diameter, glossy, circular and beige in colour after 24 h at 35C.

No brown diffusible pigment was produced on TSA at 35C. Growth occurred at 25, 30

and 35C, but not at 4 or 44C after 24 h on TSA plus sheep blood. -haemolysis was

observed on sheep (5%) blood agar. Strain 266T grew on MacConkey and TCBS agars

and on nutrient broth in 0 and 3% NaCl but not in 6% NaCl broth. Indole was produced

from tryptophan. The ONPG reaction was positive when tested by disk (Rosco,

Taastrup, Denmark) but not with the API 20E strip (BioMérieux).

Strain 266T did not utilize citrate (Simmon’s and Hänninen’s methods), malonate or

produced gas from glucose but a positive citrate reaction was observed with the API

20E strip. DL-lactate was utilized at 30 but not at 35C. Hydrogen sulphide, urease and

elastase were not produced and aesculin was not hydrolysed. Clearing of tyrosine-

containing medium was not observed but starch was hydrolysed after five days

incubation. DNase and lipase activity were detected and potassium gluconate was

oxidised. Strain 266T utilized acetate, and arginine was dehydrolased, lysine was

decarboxylated but not ornithine. A positive reaction was observed for VP, gelatin and

urocanic acid. No activity was detected for stapholysin, phenylalanine deaminase,

alkylsulfatase, pyrazynamidase and Jordan’s tartrate was negative.

Acid was produced from the following carbohydrates: fructose, galactose, glucose,

glycerol, glucose-1-phosphate, glucose-6-phosphate, maltose, mannose, N-acetyl-

glucosamine, ribose, saccharose and trehalose but not from adonitol, amygdalin, L-

arabinose, cellobiose, dulcitol, myo-inositol, lactose, lactulose, D-mannitol, melibiose,

-methyl-D-glucoside, raffinose, L-rhamnose, salicin and D-sorbitol. Acid production

was observed for the following carbohydrates with the API 50C strip (BioMérieux):

glycerol, D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, N-acetyl-

glucosamine, D-maltose, D-saccharose, D-trehalose, starch, glycogen and potassium

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-189-

gluconate was oxidized. Key biochemical characteristics used to differentiate strain

266T from all other Aeromonas spp.are presented in Table 6.1. Phenotypically, Strain

266T can be differentiated from other D-mannitol negative species by several

biochemical and physiological tests (Table 6.2).

6.3.2. FAME profile

The CFA composition of strain 266T contained 28.7% sum in Feature 3 (C16:1 w7c or C16:1

w6c), 11.4% sum in Feature 8 (C18:1 w7c or C18:1 w6c), 11.3% C16:0, 7.2% C16:1 w7c alcohol,

6.0% C12:0, 5.6% sum in Feature 2 (C12:0 aldehyde? or C16:1 iso I or C14:0 3OH), 3.7% sum in

Feature 9 (C16:0 10 methyl or C17:1 iso w9c), 3.5% iso-C15:0, 3.3% C17:1 w8c, 3.2% iso-C17:0,

2.5% C14:0 and 1.7% C16:0 N alcohol (Table 6.3).

6.3.3. Protein profile

The mass spectra of strain 266T ranged from 2000 to 11300 Da and differed from the

closes related species A. allosaccharophila, A. fluvialis and A. veronii (Fig. 6.2).

6.3.4. Genotypic characteristics

Analysis of the 16S rRNA gene (1503 bp) confirmed that strain 266T belonged to the

genus Aeromonas and showed the highest 16S rRNA gene sequence similarity with the

type strains of A. fluvialis (99.6%) followed by A. allosaccharophila and A. veronii both

with a similarity of 99.5%, these also being the closest neighbours in the phylogenetic

tree (Fig. 6.3). Strain 266T showed the minimum interspecies similarity with A. veronii

(3.2%), which was higher than those obtained between A. piscicola and A. bestiarum

(approximately 2.1%) or A. allosacchorophila and A. veronii (approximately 2.9%) as

reported by Martínez-Murcia et al. (2011) and shown in Table 6.4 in the CD ROM

attached. The DDH results between strain 266T and the type strains of A.

allosacccharophila, A. veronii and A. fluvialis were 65.3, 63.7 and 52.2%, respectively,

all below the 70% limit for species delineation (Wayne et al. 1987; Stackebrandt and

Goebel 1994) (Table 6.5). Analysis of gyrB and rpoD genes suggested that strain 266T

formed a phylogenetic line independent of other species in the genus. The sequences of

six housekeeping genes (gyrB, rpoD, recA, danJ, gyrA, and dnaX) were aligned with

those of strain 266 culminating with a concatenated tree (MLPA) derived from all six

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-190-

genes confiming that genetically, strain 266 formed a separate line of descent and that

A. veronii and A. allosaccharophila were the nearest relatives (Figs 6.4 to 6.10).

6.3.5. Antimicrobial susceptibilities

Strain 266T was resistant to amoxicillin and cefazolin and was susceptible to amikacin,

amoxicillin-clavulanate, aztreonam, cephalothin, cefepime, cefoxitin, ceftazidime,

ceftriaxone, ciprofloxacin, colistin, gentamicin, meropenem, moxifloxacin, nalidixic

acid, nitrofurantoin, norfloxacin, pipercillin-tazobactam, tetracycline, ticarcillin-

clavulanate, tobramycin, trimethoprim and trimethoprim-sulfamethoxazole.

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-191-

Figure 6.1 Electron microscopy images of strain 266T. A. Scanning electron

microscope (Bar 4 μm). B. Transmission electron microscope, negative stain (Bar 500

nm).

A

B

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-192

-

T

able

6.1

Key

test

s for

the

phen

otyp

ic id

entif

icat

ion

of st

rain

266

T fr

om o

ther

Aer

omon

as sp

p.

Cha

ract

eris

tics

Strain 266

T

A. allosaccharophila

A. dhakensis

§

A. bestiarum

A. bivalvium

A. caviae

A. diversa

Φ

A. encheleia

-h

aem

olys

is

+ V

()

n(+)

+(

+)

(

) V

()

+(+)

V

(+)

Vog

ues P

rosk

auer

reac

tion

+

()

+(+)

V

(+)

(

)

()

V(+

)

()

LDC

+

+(+)

+(

+)

V(+

) +(

+)

(

)

()

(

) G

luco

se (g

as)

+(+)

+(

+)

V(+

)

()

(

)

()

V(+

) A

escu

lin h

ydro

lysi

s

V

(+)

+(+)

V

(+)

+(+)

V

(+)

(

) V

(+)

Aci

d fr

om:

L-ar

abin

ose

V(+

)

()

+(+)

+(

+)

+(+)

()

(

) Sa

licin

()

+(+)

V

(+)

+(+)

V

(+)

(

)

(+)

D-m

anni

tol

+(+)

+(

+)

+(+)

+(

+)

+(+)

()

+(+)

U

tiliz

atio

n of

:

C

itrat

e

V

()

nd(+

)

()

+(+)

+(

+)

nd(

)

()

DL-

lact

ate

++

()

(

)

()

+(+)

+(

) nd

()

(

)

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-193

-

T

able

6.1

Con

tinue

d.

Cha

ract

eris

tics

Strain 266

T

A. eucrenophila

A. fluvialis

#

A. hydrophila

A. jandaei

A. media

A. molluscorum

-h

aem

olys

is

+ +(

+)

(

) +(

+)

+(+)

V

()

V(

) V

ogue

s Pro

skau

er re

actio

n +

(

)

()

+(+)

+(

+)

(

)

()

LDC

+

(

)

()

+(+)

+(

+)

(

)

()

Glu

cose

(gas

)

V

(+)

+(+)

+(

+)

+(+)

()

(

) A

escu

lin h

ydro

lysi

s

V

(+)

(

) +(

+)

(

) V

(+)

+(+)

A

cid

from

:

L-ar

abin

ose

V(+

)

()

V(+

)

()

+(+)

+(

+)

Salic

in

V(

) +(

+)

V(

)

()

V(+

) nd

(+)

D-m

anni

tol

+(+)

n(

+)

+(+)

+(

+)

+(+)

+(

+)

Util

izat

ion

of:

C

itrat

e

()

+(+)

+(

+)

+(+)

V

(+)

+(+)

D

L-la

ctat

e ++

(

) nd

()

V(

)

()

V(+

) V

()

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-194

-

T

able

6.1

Con

tinue

d.

Cha

ract

eris

tics

Strain 266

T

A. popoffii

A. piscicola

¦

A. rivuli

Ø

A. salmonicida

A. sanarellii

¥

A. schubertii

A. simiae

*

-h

aem

olys

is

+

()

+(+)

+(

+)

V(+

)

()

V(+

)

()

Vog

ues P

rosk

auer

reac

tion

+ +(

+)

+(+)

V

(+)

V(

)

()

V(+

)

()

LDC

+

(

) +(

+)

(

) V

(+)

(

) V

(+)

+(+)

G

luco

se (g

as)

+(+)

+(

+)

(

) V

()

(

)

()

(

) A

escu

lin h

ydro

lysi

s

()

+(+)

()

+(+)

+(

+)

(

) V

()

Aci

d fr

om:

L-ar

abin

ose

V(+

)

()

(

) +(

+)

+(+)

()

(

) Sa

licin

()

+(+)

()

V(

) +(

+)

(

)

()

D-m

anni

tol

+(+)

+(

+)

(

) +(

+)

+(+)

()

(

) U

tiliz

atio

n of

:

C

itrat

e

+(

+)

nd

nd(

) +(

)

()

V(+

) nd

()

DL-

lact

ate

++ V

(+)

(

) nd

()

(

) nd

(+)

V(+

+ ) nd

(++ )

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-195

-

T

able

6.1

Con

tinue

d.

Cha

ract

eris

tics

Strain 266

T

A. sobria

A. taiwanensis

¥

A. tecta

A. trota

A. veronii bv. sobria

A. veronii bv. veronii

-h

aem

olys

is

+ V

(+)

(

) +(

+)

V(+

) +(

+)

+(+)

V

ogue

s Pro

skau

er re

actio

n +

+(

)

()

V(+

)

()

+(+)

V

()

LDC

+

+(+)

()

V(+

) +(

+)

(

) +(

+)

Glu

cose

(gas

)

+(

)

()

+(+)

V

(+)

+(+)

+(

+)

Aes

culin

hyd

roly

sis

V(

) +(

+)

V(+

)

()

(

) +(

+)

Aci

d fr

om:

L-

arab

inos

e

V

()

+(+)

()

(

)

(+)

(

) Sa

licin

V

()

+(+)

V

()

(

)

()

+(+)

D

-man

nito

l

+(

+)

+(+)

+(

+)

+(+)

+(

+)

+(+)

U

tiliz

atio

n of

:

Citr

ate

+(+)

+(

+)

(+

) +(

+)

V(

) +(

+)

DL-

lact

ate

++

()

nd(+

)

()

+(+)

(+)

(+

)

Page 222: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-196

-

Abb

revi

atio

ns:

+, 8

5-10

0% o

f st

rain

s po

sitiv

e;

, 0

to 1

5% o

f st

rain

s po

sitiv

e; V

, 16

-84%

of

stra

ins

posi

tive.

All

test

s ha

ve b

een

perf

orm

ed fo

r typ

e st

rain

s of

the

diff

eren

t spe

cies

and

resu

lts a

re e

xpre

ssed

as

in b

rack

ets

as (+

) or (

); nd

, no

data

ava

ilabl

e. T

ests

for

stra

in 2

66T w

ere

perf

orm

ed a

t 30

and

35ºC

. Dat

a fr

om s

peci

es 1

-15

wer

e ob

tain

ed fr

om A

bbot

t et a

l. (2

003)

with

the

exce

ptio

n of

test

s

indi

cate

d as

nd,

thes

e au

thor

s pe

rfor

med

test

s at

35º

C w

ith th

e ex

cept

ion

of A

. pop

offii

and

A. s

obria

whi

ch w

ere

test

ed a

t 25º

C. O

ther

test

s w

ere

perf

orm

ed a

s fo

llow

s: * H

arf-

Mon

teil

et a

l. (2

004)

(30º

C);

† Miñ

ana-

Gal

bis

et a

l. (2

004a

) (25

ºC);

‡ Miñ

ana-

Gal

bis

et a

l. (2

007)

(30º

C);

§ Mar

tínez

Mur

cia

et a

l. (2

008)

( 25

ºC);

¶ Dem

arta

et a

l. (2

008)

(30º

C);

# Alp

eri e

t al.

(201

0a) (

30ºC

); ¦ B

eaz-

Hid

algo

et a

l. (2

009)

(25º

C); ¥

Alp

eri e

t al.

(201

0b) (

30º

C);

ØFi

guer

as e

t al.

(201

1a) (

30ºC

); ΦM

iñan

a-G

albi

s et

al.

(201

0) (3

0ºC

). + Po

sitiv

e at

30

C b

ut n

ot a

t

35C

.

Page 223: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-197

-

Tab

le 6

.2

Key

test

s use

d to

diff

eren

tiate

stra

in 2

66T

from

oth

er D

-man

nito

l non

-fer

men

tativ

e Ae

rom

onas

spp.

Tes

t

Stra

in 2

66T

A. s

chub

ertii

A

. sim

iae

A. d

iver

sa

-h

aem

olys

is

+ +

+

Indo

le

+

+

VP

+ +

+

LDC

+

+ +

Glu

cose

(gas

)

Hyd

roly

sis o

f:

Aes

culin

+

Star

ch*

+ +

+ +

Aci

d fr

om:

D-s

acch

aros

e +

+

L-ar

abin

ose

salic

in

Util

izat

ion

of:

citra

te

+

DL-

lact

ate

* +

+ +

*St

arch

hyd

roly

sis a

nd u

tiliz

atio

n of

DL-

lact

ate

tube

wer

e in

cuba

ted

at 3

0C

. All

othe

r tes

ts w

ere

perf

orm

ed a

t 35

C

Page 224: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

- 198

-

Tab

le 6

.3

Cel

lula

r fat

ty a

cid

prof

iles o

f stra

in 2

66T a

nd c

urre

nt A

erom

onas

spp.

C

ellu

lar

fatt

y ac

id (%

)

Spec

ies

12:0

13

:0 i

13:0

14

:0

SF1

15:0

i 16

:1 w

7c

alco

hol

SF2

16:0

N

alco

hol

Stra

in 2

66T

8.88

1.

28

t 3.

01

t 3.

38

5.80

9.

44

1.03

A. a

llosa

ccha

roph

ila D

SM 1

1576

T 5.

77

t t

2.72

t

1.53

5.

18

8.14

1.

21

A. d

hake

nsis

CEC

T 72

89T

5.72

t

t 6.

30

1.09

1.

53

3.91

12

.35

1.04

A. b

estia

rum

ATC

C 5

1108

T 6.

62

t t

4.11

t

1.25

6.

52

9.05

1.

11

A. b

ival

vium

CEC

T 71

13T

7.41

1.

20

t 2.

79

ND

1.

88

ND

10

.90

ND

A. c

avia

e A

TCC

131

36T

7.48

t

t 4.

18

ND

1.

59

4.99

9.

81

1.31

A. c

ulic

icol

a C

ECT

5761

T 6.

56

1.67

t

3.94

N

D

2.84

6.

18

8.30

t

A. d

iver

sa C

ECT

4254

T 7.

07

1.21

t

4.71

t

1.34

5.

81

9.14

2.

42

A. e

nche

leia

DSM

115

77T

6.81

t

ND

3.

08

ND

2.

34

ND

9.

67

ND

A. e

ucre

noph

ila A

TCC

233

09T

7.61

2.

11

t 3.

05

ND

6.

38

ND

8.

54

ND

A. fl

uvia

lis C

ECT

7401

T 7.

37

2.66

t

3.21

N

D

3.06

2.

56

9.29

1.

94

A. h

ydro

phila

ATC

C 7

966T

6.67

t

t 5.

60

ND

1.

58

5.02

9.

74

1.67

A. ja

ndae

i ATC

C 4

9658

T 4.

90

t 1.

09

5.63

t

2.64

4.

52

5.69

2.

65

A. m

edia

ATC

C 3

3907

T 6.

84

1.09

N

D

2.34

N

D

1.94

N

D

7.87

N

D

A. m

ollu

scor

um D

SM 1

7090

T 6.

50

t t

3.39

t

1.27

t

12.2

1 t

Page 225: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-- 1

99 --

T

able

6.3

Con

tinue

d.

Cel

lula

r fa

tty

acid

(%)

Spec

ies

12:0

13

:0 i

13:0

14

:0

SF1

15:0

i 16

:1 w

7c

alco

hol

SF2

16:0

N

alco

hol

Stra

in 2

66T

8.88

1.

28

t 3.

01

t 3.

38

5.80

9.

44

1.03

A. p

isci

cola

CEC

T 74

43T

7.34

1.

04

t 3.

17

ND

2.

98

4.10

9.

03

t

A. p

opof

fii C

IP 1

0549

3T 7.

46

1.18

t

3.51

N

D

1.99

6.

28

10.4

6 1.

62

A. ri

vuli

CEC

T 75

18T

8.55

t

t 3.

36

ND

1.

84

t 13

.34

ND

A. sa

lmon

icid

a C

ECT

894T

11.6

4 N

D

1.14

1.

70

2.51

N

D

t 19

.80

t

A. sa

nare

llii C

ECT

7402

T 8.

33

2.09

t

2.62

N

D

2.95

N

D

10.1

4 N

D

A. sc

hube

rtii

ATC

C 4

3700

T 8.

33

2.10

1.

90

4.26

1.

43

2.63

1.

43

9.76

3.

02

A. si

mia

e D

SM 1

6559

T 4.

98

t t

4.33

N

D

2.89

N

D

10.0

8 N

D

A. so

bria

CIP

743

3T 5.

66

1.10

3.

60

4.44

3.

16

2.49

2.

45

8.23

2.

16

A. ta

iwan

ensi

s CEC

T 74

03T

10.3

9 1.

57

t 3.

44

ND

2.

80

ND

12

.06

ND

A. te

cta

CEC

T 70

82T

8.60

1.

13

t 3.

23

ND

3.

29

ND

16

.68

ND

A. tr

ota

ATC

C 4

9657

T 5.

52

1.55

t

3.91

N

D

3.40

1.

44

7.43

1.

00

A. v

eron

ii bv

. sob

ria A

TCC

907

1T 6.

57

2.09

t

3.80

t

3.85

5.

54

8.35

1.

11

A. v

eron

ii bv

. ver

onii

DSM

738

6T 5.

16

t t

3,32

t

1.52

6.

00

7.69

1.

02

Page 226: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-- 2

00 --

T

able

6.3

Con

tinue

d.

C

ellu

lar

fatt

y ac

id (%

)

Spec

ies

16:0

i SF

3 16

:0

15:0

i 3O

H

SF9

17:0

i 17

:1

8wc

17:0

SF

8

Stra

in 2

66T

t 32

.37

7.55

4.

82

3.55

1.

68

1.98

t

7.70

A. a

llosa

ccha

roph

ila D

SM 1

1576

T t

38.1

1 17

.34

2.34

2.

35

1.91

t

t 8.

25

A. d

hake

nsis

CEC

T 72

89T

t 32

.76

15.3

3 2.

14

1.26

1.

05

1.49

t

7.59

A. b

estia

rum

ATC

C 5

1108

T t

39.2

0 14

.96

1.33

1.

13

t 1.

17

t 7.

58

A. b

ival

vium

CEC

T 71

13T

t 36

.80

16.9

9 3.

20

2.57

2.

49

1.15

t

8.47

A. c

avia

e A

TCC

131

36T

t 40

.67

16.2

5 1.

99

1.72

1.

09

t t

8.49

A. c

ulic

icol

a C

ECT

5761

T t

34.0

2 12

.06

3.46

3.

06

1.86

1.

23

t 8.

69

A. d

iver

sa C

ECT

4254

T t

34.2

8 16

.64

1.73

1.

35

t t

t 8.

19

A. e

nche

leia

DSM

115

77T

t 43

.99

17.2

8 3.

04

2.28

2.

17

t t

6.85

A. e

ucre

noph

ila A

TCC

233

09T

t 37

.94

13.5

1 5.

21

5.13

2.

83

t t

5.45

A. fl

uvia

lis C

ECT

7401

T 1.

15

27.3

0 9.

66

6.95

3.

67

4.08

1.

45

t 10

.34

A. h

ydro

phila

ATC

C 7

966T

t 36

.55

18.2

8 1.

10

1.40

t

t t

7.61

A. ja

ndae

i ATC

C 4

9658

T t

27.2

9 10

.99

1.69

2.

75

1.45

2.

76

1.36

7.

95

A. m

edia

ATC

C 3

3907

T t

38.4

2 17

.36

2.67

4.

55

4.22

t

t 10

.61

A. m

ollu

scor

um D

SM 1

7090

T t

37.8

3 13

.80

3.43

2.

05

2.30

1.

27

t 8.

40

Page 227: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-- 2

01 --

Tab

le 6

.3

C

ontin

ued.

Cel

lula

r fa

tty

acid

(%)

Spec

ies

16:0

i SF

3 16

:0

15:0

i 3O

H

SF9

17:0

i 17

:1

8wc

17:0

SF

8

Stra

in 2

66T

t 32

.37

7.55

4.

82

3.55

1.

68

1.98

t

7.70

A. p

isci

cola

CEC

T 74

43T

3.16

36

.44

13.9

4 3.

04

3.48

2.

84

t t

4.35

A. p

opof

fii C

IP 1

0549

3T t

40.5

5 13

.68

3.40

2.

10

1.75

t

t 5.

10

A. ri

vuli

CEC

T 75

18T

t 39

.04

17.1

4 2.

80

1.28

1.

84

t t

4.58

A. sa

lmon

icid

a C

ECT

894T

t 31

.31

14.5

1 t

t t

2.88

2.

14

7.14

A. sa

nare

llii C

ECT

7402

T t

32.9

3 16

.93

4.48

3.

33

3.05

t

t 9.

61

A. sc

hube

rtii

ATC

C 4

3700

T t

29.1

9 10

.78

3.38

2.

11

1.25

1.

86

t 5.

72

A. si

mia

e D

SM 1

6559

T t

33.5

0 17

.04

2.52

5.

16

4.27

t

t 12

.74

A. so

bria

CIP

743

3T t

31.1

9 10

.75

2.68

2.

09

1.78

5.

95

2.77

6.

02

A. ta

iwan

ensi

s CEC

T 74

03T

t 35

.50

16.0

9 3.

48

2.17

1.

31

t t

8.60

A. te

cta

CEC

T 70

82T

ND

38

.80

11.2

2 7.

98

2.29

1.

48

t N

D

4.01

A. tr

ota

ATC

C 4

9657

T t

33.9

2 15

.45

3.80

4.

83

3.16

1.

06

t 9.

16

A. v

eron

ii bv

. sob

ria A

TCC

907

1T t

29.9

0 12

.28

4.40

5.

97

3.65

1.

42

t 6.

54

A. v

eron

ii bv

. ver

onii

DSM

738

6T t

34.2

0 14

.29

2.10

2.

39

1.53

2.

54

1.22

11

.29

Page 228: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-- 2

02 --

Abb

revi

atio

ns. *

Sum

in F

eatu

res (

SF) c

onta

ined

cel

lula

r fat

ty a

cids

that

can

not b

e se

para

ted

by th

is sy

stem

;

SF1

(C15

:1 is

o H

/13:

0 3O

H o

r C13

:0 3

OH

/15:

1 is

o H);

SF2

(C12

:0 a

ldeh

yde?

or C

16:1

iso

I or C

14:0

3O

H);

SF3

(C16

:1 w

7c o

r C16

:1 w

6c);

SF8

(C18

:1 w

7c o

r C18

:1 w

6c);

SF 9

(C16

:0 1

0-m

ethy

l or C

17:1

iso

w9c

);

SF7

(C19

:1w

7c o

r 19:

1 w

6c) d

etec

ted

in A

. riv

uli (

2.4%

);

14:0

iso

3OH

was

det

ecte

d in

A. p

isci

cola

(1.2

5%);

15:0

3O

H (1

.20%

) and

17:

1 w

6c (1

.02%

) wer

e de

tect

ed in

A. s

obri

a

ND

, not

det

ecte

d; i,

iso;

t, tr

ace

(val

ues <

1%

not

show

n)

Page 229: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-- 2

03 --

Figu

re 6

.2 P

rote

in sp

ectru

m fo

r stra

in 2

66T

(Bru

ker m

icro

flex

LT M

ALD

I-TO

F m

ass s

pect

rom

eter

, Bru

ker D

alto

nik,

Gm

bH, G

erm

any)

.

Page 230: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-- 2

04 --

Tab

le 6

.5

DN

A-D

NA

hyb

ridiz

atio

n va

lues

bet

wee

n st

rain

266

T and

clo

sely

rela

ted

Aero

mon

as sp

ecie

s

(Res

ults

are

exp

ress

ed a

s the

mea

n of

thre

e de

term

inat

ions

. Sta

ndar

d de

viat

ions

are

incl

uded

in p

aren

thes

is)

Lab

elle

d D

NA

Stra

in

266T

A. f

luvi

alis

CE

CT

740

1T

A. v

eron

ii

CE

CT

425

7T

A. a

llosa

ccha

roph

ila

CE

CT

419

9T

266T

100

56.8

(9.2

) 47

.6 (9

.2)

65.0

(0.5

)

A. f

luvi

alis

CE

CT

740

1T 62

.6 (3

.2)

100

A. v

eron

ii C

EC

T 4

257T

65.8

(3.2

)

10

0

A. a

llosa

ccha

roph

ila C

ECT

419

9T 65

.5 (0

.5)

100

Page 231: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

- 205 -

Figure 6.3 Unrooted neighbour-joining phylogenetic tree derived from the 16S rRNA

gene sequences showing the relationships of strain 266T with all other Aeromonas

species. The phylogenetic tree was constructed with 1322 nt. Numbers at the nodes

indicate bootstrap values. Bar, 0.002 estimated substitutions per site.

A. salmonicida NCIMB1102T (X60405) A. bestiarum CECT 4227T (NR 026089) A. piscicola CECT7 443T (FM999971)

A. molluscorum CECT 5864T (AY532691) A. encheleia CECT 4342T (AJ224309)

A. tecta CECT 7082T (AJ458403) A.eucrenophila ATCC 23309T (X74675)

A. bivalvium CECT7113T (DQ504429) A. popoffii LMG 317541T(AJ224308)

A. rivuli DSM 22539T (FJ976900) A. sobria NCIMB 12065T (X60412) A. media ATCC 33907T (X74679)

A. hydrophila ATCC 7966T (X60404) A. sanarellii CECT 7402T (FJ230076)

A. taiwanensis CECT 7403T (A2-50) (FJ230077) A. aquariorum CECT 7289T (MDC47T) (EU085557) A. hydrophila dhakensis LMG 19562T (AJ508765)

A. trota ATCC 49657T (X60415) A. caviae NCIMB 13016T (X60408)

A. allosaccharophila CECT 4199T (S39232) A. fluvialis CECT 7401T (FJ230078)

266T A. jandaei ATCC 49568T (X60413)

A. veronii ATCC 35624T (X60414) A. simiae IBSS6874T (AJ536821)

A. diversa CECT 4254T (GQ365710) A. schubertii ATCC 43700T (X60416) 88

94

85

63

62 58

47

99

98

48

43

59

66

92 70

39

0.002

A

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Figure 6.4 Unrooted neighbour-joining phylogenetic tree derived from dnaJ sequences

showing the relationships of strain 266T with the type strains of all other Aeromonas

species. The phylogenetic tree was constructed with 596 nt. Numbers at the nodes

indicate bootstrap values. Bar, 0.02 estimated substitutions per site.

A. allosaccharophila CECT 4199T (HQ443058) A. veronii CECT 4257 T (HQ443060)

266T (HE611954) A. fluvialis 717T (FJ603454)

A. jandaei CECT 4228T (HQ443074) A. sobria CECT 4245T (HQ443076)

A. hydrophila CECT 839T (HQ443048) A. dhakensis CECT 7289T (HQ443050)

A. trota CECT 4255T (HQ443038) A. bivalvium CECT 7113T (HQ443036)

A. salmonicida CECT 894T (HQ442979) A. popoffii CECT 5176T (HQ442995)

A. piscicola CECT 7443T (HQ442992) A. bestiarum CECT 4227T (HQ442988)

A. molluscorum CECT 5864T (HQ443000) A. rivuli DSM 22539T (FJ969432)

A. media CECT 4232T (HQ443012) A. encheleia CECT 4342T (HQ443025)

A. eucrenophila CECT 4224T (HQ443015) A. tecta CECT 7082T (HQ443020)

A. taiwanensis A2-50T (FJ807270) A. caviae CECT 838T (HQ443008)

A. sanarellii A2-67T (FJ807279) A. simiae CIP 107798T (HQ443081)

A. schubertii CECT 4240T (HQ443088) A. diversa CECT 4254T (HQ443084) 99

100

99

77

75 88

56

95

49

84

85

45 97

51 70

93

64

90

76

67

55

68

64

0.02

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A. veronii CECT 4257T (HQ442469) A. fluvialis CECT 7401T (HQ442464)

266T (HE611951) A. allosaccharophila CECT 4199T (HQ442457) A. sobria CECT 4245T (HQ442447)

A. jandaei CECT 4228T (HQ442455) A. hydrophila CECT 839T (HQ442472)

A. dhakensis CECT 7289T (HQ442483) A. trota CECT 4255T (HQ442490)

A. taiwanensis CECT 7403T (HQ442491) A. salmonicida CECT 894T (HQ442441)

A. popoffii CECT 5176T (HQ442437) A. piscicola CECT 7443T (HQ442434)

A. bestiarum CECT 4227T (HQ442429) A. molluscorum CECT 5864T (HQ442519) A. rivuli DSM 22539T (HQ442524)

A. bivalvium CECT 7113T (HQ442527) A. eucrenophila CECT 4224T (HQ442509)

A. tecta CECT 7082T (HQ442502) A. encheleia CECT 4342T (HQ442495)

A. caviae CECT 838T (HQ442422 A. sanarellii CECT 7402T (HQ442508)

A. media CECT 4232T (HQ442507) A. simiae CIP 107798T (HQ442528)

A. schubertii CECT 4240T (HQ442533) A. diversa CECT 4254T (HQ442534)

95

99

92

92

54 85

53 48

31 74

73

56 61

25

53

44

0.01

Figure 6.5 Unrooted neighbour-joining phylogenetic tree derived from dnaX sequences showing

the relationships of strain 266T with the type strains of all other Aeromonas species.

The phylogenetic tree was constructed with 493 nt. Numbers at the nodes indicate

bootstrap values. Bar, 0.01 estimated substitutions per site.

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Figure 6.6 Unrooted neighbour-joining phylogenetic tree derived from gyrA sequences

showing the relationships of strain 266T with the type strains of all other Aeromonas

species. The phylogenetic tree was constructed with 707 nt. Numbers at the nodes

indicate bootstrap values. Bar, 0.01 estimated substitutions per site.

A. veronii CECT 4257T (HQ443160) 266T (HE611952)

A. jandaei CECT 4228T (HQ443185) A. fluvialis 717T (FJ603456) A. allosaccharophila CECT 4199T (HQ443156)

A. sobria CECT 4245T (HQ443148) A. trota CECT 4255T (HQ443187)

A. hydrophila CECT 839T (HQ443174) A. dhakensis CECT 7289T (HQ443166)

A. eucrenophila CECT 4224T (HQ443115) A. tecta CECT 7082T (HQ443122)

A. molluscorum CECT 5864T (HQ443110) A. rivuli DSM 22539T (FJ969436) A. salmonicida CECT 894T (HQ443089)

A. popoffii CECT 5176T (HQ443108) A. piscicola CECT 7443T (HQ443100) A. bestiarum CECT 4227T (HQ443097)

A. media CECT 4232T (HQ443134) A. encheleia CECT 4342T (HQ443139)

A. bivalvium CECT 7113T (HQ443141) A. sanarellii A2-67T (FJ807276)

A. taiwanensis A2-50T (FJ807274) A. caviae CECT 838T (HQ443146) A. simiae CIP 107798T (HQ443191)

A. diversa CECT 4254T (HQ443194) A. schubertii CECT 4240T (HQ443198) 99

99

99

87

98

97

82 95

66

86

85

58

45

44

38

42

43

35

99

0.01

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Figure 6.7 Unrooted neighbour-joining phylogenetic tree derived from gyrB sequences

showing the relationships of strain 266T with the type strains of all other Aeromonas

species. The phylogenetic tree was constructed with 545 nt. Numbers at the nodes

indicate bootstrap values. Bar, 0.01 estimated substitutions per site.

A. piscicola CECT 7443T (HQ442690) A. bestiarum CECT 4227T (HQ442683)

A. salmonicida CECT 894T (HQ442680) A. popoffii CECT 5176T (HQ442693)

A. eucrenophila CECT 4224T (HQ442657) A. tecta CECT 7082T (HQ442662)

A. encheleia CECT 4342T (HQ442655) A. molluscorum CECT 5864T (HQ442671)

A. rivuli DSM 22539T (FJ969434) A. media CECT 4232T (HQ442709)

A. bivalvium CECT 7113T (HQ442703) A. caviae CECT 838T (HQ442748)

A. sanarellii A2-67T (FJ807277) A. hydrophila CECT 839T (HQ442746)

A. dhakensis CECT 7289T (HQ442712) A. jandaei CECT 4228T (HQ442736) A. allosaccharophila CECT 4199T (HQ442733)

266T (FN691773) A. veronii CECT 4257T (HQ442728)

A. sobria CECT 4245T (HQ442698) A. fluvialis 717T (FJ603455)

A. trota CECT 4255T (HQ442718) A. taiwanensis A2-50T (FJ807272)

A. simiae CIP 107798T (HQ442758) A. diversa CECT 4254T (HQ442756)

A. schubertii CECT 4240T (HQ442755) 99

99

92

33 86

44 43

48

40

85

72

38

47

31

38

26 61

59

25

0.01

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Figure 6.8 Unrooted neighbour-joining phylogenetic tree derived from recA sequences

showing the relationships of strain 266T with the type strains of all other Aeromonas

species. The phylogenetic tree was constructed with 598 nt. Numbers at the nodes

indicate bootstrap values. Bar, 0.01 estimated substitutions per site.

A. piscicola CECT 7443T (HQ442954) A. bestiarum CECT 4227T (HQ442949)

A. salmonicida CECT 894T (HQ442955) A. popoffii CECT 5176T (HQ442941

A. sobria CECT 4245T (HQ442940) A. allosaccharophila CECT 4199T (HQ442961) A. fluvialis 717T (FJ603457) A. veronii CECT 4257T (HQ442970)

266T (HE611953) A. hydrophila CECT 839T (HQ442926)

A. trota CECT 4255T (HQ442933) A. caviae CECT 838T (HQ442921)

A. jandaei CECT 4228T (HQ442915) A. dhakensis CECT 7289T (HQ442908)

A. media CECT 4232T (HQ442972) A. tecta CECT 7082T (HQ442895)

A. eucrenophila CECT 4224T (HQ442892) A. encheleia CECT 4342T (HQ442884)

A. sanarellii A2-67T (FJ807278) A. taiwanensis A2-50T (FJ807273)

A. molluscorum CECT 5864T (HQ442877) A. rivuli DSM 22539T (FJ969435)

A. bivalvium CECT 7113T (HQ442882) A. simiae CIP 107798T (HQ442869)

A. diversa CECT 4254T (HQ442872) A. schubertii CECT 4240T (HQ442876)

99

75

96

93

79

66

47

57

42

56

55

90

80

71

65

53 36

36

0.01

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Figure 6.9 Unrooted neighbour-joining phylogenetic tree derived from rpoD sequences

showing the relationships of strain 266T with the type strains of all other Aeromonas

species. The phylogenetic tree was constructed with 667 nt. Numbers at the nodes

indicate bootstrap values. Bar, 0.02 estimated substitutions per site.

A. allosaccharophila CECT 4199T (HQ442825) A. veronii CECT 4257T (HQ442833)

A. fluvialis 717T (FJ603453) 266T (FN773335)

A. sobria CECT 4245T (HQ442867) A. trota CECT 4255T (HQ442822)

A. jandaei CECT 4228T (HQ442840) A. hydrophila CECT 839T (HQ442791)

A. dhakensis CECT 7289T (HQ442798) A. salmonicida CECT 894T (HQ442843)

A. bestiarum CECT 4227T (HQ442854) A. piscicola CECT 7443T (HQ442859) A. popoffii CECT 5176T (HQ442853)

A. sanarellii A2-67T (FJ807275) A. taiwanensis A2-50T (FJ807271)

A. caviae CECT 838T (HQ442790) A. media CECT 4232T (HQ442785)

A. encheleia CECT 4342T (HQ442778) A. eucrenophila CECT 4224T (HQ442770)

A. tecta CECT 7082T (HQ442762) A. bivalvium CECT 7113T (HQ442817)

A. molluscorum CECT 5864T (HQ442812) A. rivuli DSM 22539T (FJ969433)

A. simiae CIP 107798T (HQ442811) A. diversa CECT 4254T (HQ442805)

A. schubertii CECT 4240T (HQ442809) 100 100

100

45 99

94

100

60

98

99

92

86

96

91

54

35

51

48

40

33

0.02

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Figure 6.10 Unrooted neighbour-joining phylogenetic tree derived from the MLPA of

concatenated sequences of six housekeeping genes (gyrB, rpoD, recA, dnaJ, gyrA and

dnaX) sequences showing the relationships of strain 266T with several strains of all

other Aeromonas species. The phylogenetic tree was constructed with 4204 nt. Numbers

at the nodes indicate bootstrap values. Accession numbers for all Aeromonas strains are

provided in Martínez-Murcia et al. (2011). Bar, 0.01 estimated substitutions per site.

A. veronii CECT 5761T

A. australiensis 266T A. allosaccharophila CECT 4199T

A. fluvialis CECT 7401T

A. sobria CECT 5254T

A. jandaei CECT 4228T

A. hydrophila CECT 839T

A. dhakensis CECT 7289T

A. trota CECT 4255T

A. taiwanensis CECT 7402T

A. caviae CECT 838T

A. sanarellii CECT 7403T

A. eucrenophila CECT 4224T

A. tecta CECT 7082T

A. encheleia CECT 4342T

A. media CECT 4232T

A. molluscorum CECT 5864T

A. rivuli DSM 22539T

A. bivalvium CECT 7113T

A. salmonicida CECT 894T

A. popoffii CECT 5176T

A. piscicola CECT 7443T

A. bestiarum CECT 4227T

A. simiae CIP 107798T

A. diversa CECT 4254T

A. schubertii CECT 4240T 100 100

100

99 100

81

100

89

98

99

95

100

71 100

99

93

85

69

59

0.01

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6.4. DISCUSSION

The phenotypic and genotypic characteristics of strain 266T were investigated using a

polyphasic approach in order to determine its taxonomic position as initial classification

inferred by the nucleotide sequences of the rpoD and gyrB genes suggested that strain

266T occupied a phylogenetic branch separate from all current Aeromonas species. A

small zone of inhibition (~10.8 mm in diameter) with a 150 g disk containing the

vibriostatic agent O/129 was observed after o/v incubation on blood agar. Susceptibility

to O/129 is uncommon in the genus and was previously reported for two strains of A.

eucrenophila and one of A. veronii by Abbott et al. (2003), and recently for the newly

proposed species A. cavernicola (Martínez-Murcia et al. 2013).

The inability of strain 266T to produce acid from D-mannitol is a significant phenotypic

marker as the majority of the species in the genus can produce acid from this

carbohydrate with the exception of A. schubertii, A. simiae, A. diversa, and some strains

of A. trota. Strain 266T can be differentiated from A. schubertii by producing indole

from tryptophan and acid from D-saccharose; from A. simiae by being haemolytic

(strain 266T exhibited -haemolysis while A. simiae did not) and positive for VP and

indole reactions; from A. diversa by its ability to decarboxylate lysine and produce acid

from D-saccharose and from A. trota by being positive for VP but negative for the

utilization of citrate.

The CFA composition of strain 266T suggested that subtle differences exist between

strain 266T and other D-mannitol negative Aeromonas (Table 6.2). Moreover, based on

CFAs profiles, Aeromonas species can be divided into two groups, those that produce

C16:1 w7c alcohol and C16:0 N alcohols, and those that do not. However, identification of

bacteria by analysis of their FAMEs is more suitable for slow-growing bacteria such as

non-fermenters (Osterhout et al. 1991) and, in agreement with the comments by

Käempfer et al. (1994), fatty acid patterns show a limited resolution to split Aeromonas

species. The Similarity Index (SI) values obtained for strain 266T varied between 0.200

and <0.300 and in most instances, named A. schubertii as a possible match. According

to this system, SI values of <0.300 may represent an atypical strain of the species named

first in the chromatogram. This identification was consistent with the fact that A.

schubertii shared similar biochemical features with strain 266T. The FAME

compositions of the Aeromonas strains analysed in this project differed significantly

from previous reports (Lambert et al. 1983; Huys et al. 1994; Käempfer et al. 1994).

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Consistent with previous observations, variations in FAME data can be attributed to

differences in culture conditions, sets of strains used, the type of media and equipment

employed to analyse results (Huys et al. 1994).

At the genotypic level, strain 266T showed a separate line of descent from all other

Aeromonas species as depicted in phylogenetic trees constructed with nucleotide

sequences of six individual housekeeping genes (Figs. 6.4 to 6.9) and the concatenated

tree derived from the MLPA (Fig. 6.10). According to the ad hoc committee for the re-

evaluation of the species definition in bacteriology, a minimum of five housekeeping

genes are recommended to define a species (Stackebrandt et al. 2002). The species A.

fluvialis, A. taiwanensis, A. sanarellii and A. rivuli have all been defined with the

concatenated sequences of five genes (rpoD, gyrB, dnaJ, recA and gyrA) (Alperi et al.

2010a/b; Figueras et al. 2011a).

Recently, a MLPA of the genus Aeromonas based on the information derived from

seven concatenated genes (rpoD, gyrB, dnaJ, recA, gyrA, dnaX, and atpD)

demonstrated concordance with the species delineation based on the DDH results

(Martínez-Murcia et al. 2011). Almost the same phylogenetic conclusions were recently

inferred by Roger et al. (2012b) using MLPA based also on seven housekeeping genes

(dnaK, gltA, gyrB, radA, rpoB, tsf and zipA) of which six were different from the ones

employed by Martínez-Murcia et al. (2011).

According to Käempfer and Glaeser (2012) and Martínez-Murcia et al. (2011), a critical

comparison of the different tree topologies based on single genes is important to

determine genes that may be affected by lateral gene transfer or subsequent

recombination events. The trees constructed with the six individual genes showed that

in all of them strain 266T formed a clear distinctive branch but always clustered near the

species A. veronii, A. fluvialis and A. allosaccharophila. This finding is further

supported by the DNA relatedness values below the 70% limit for species delineation

determined between strain 266T and the type strains of A. allosacccharophila, A. veronii

and A. fluvialis (Wayne et al. 1987; Stackebrandt and Goebel 1994). The MLPA

showed once more a perfect agreement with DDH results, because both demonstrated

that strain 266T represents a new Aeromonas species.

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6.4.1. Formal description of Aeromonas australiensis sp. nov.

Motile rods with polar flagella (Figure 6.1b). Cells are Gram-negative, straight, non-

spore-forming and non-encapsulated rods, 0.6-0.9 μm wide and 1.8-2.7 μm long,

oxidase- and catalase-positive, reduce nitrate to nitrite and are susceptible to the

vibriostatic agent O/129 (150 μg). Colonies on TSA plus sheep blood are 1.5-2.0 mm in

diameter, glossy, circular and beige in clolour after 24 h at 35C. No brown diffusible

pigment is produced on TSA at 35C. Growth occurs at 25, 30 and 35C, but not at 4 or

44C after 24 h on TSA plus sheep blood. -Haemolysis is observed on sheep (5%)

blood agar. Grows on MacConkey (Difco) and thiosulfate-citrate-bile-sucrose agar

(Difco) and in nutrient broth in 0 and 3% NaCl, but not at 6% NaCl. Indole is produced

from tryptophan. The ONPG reaction is positive when tested by disc (Rosco) but not in

the API 20E strip. Does not utilize citrate (Simmon’s and Hänninen’s methods) or

malonate or produce gas from glucose, but a positive citrate reaction is observed with

the API 20E strip. DL-Lactate is utilized at 30C but not at 35C.

Hydrogen sulphide, urease and elastase are not produced and it does not hydrolyse

aesculin. No clearing of tyrosine-containing medium, but starch hydrolysis is positive

after 5 days. Produces DNase and lipase and oxidizes potassium gluconate.

Dehydrolyses arginine and lysine is decarboxylated, but not ornithine. Utilizes acetate

and urocanic acid and it is positive for the Voges-Proskauer reaction and hydrolysis of

gelatin. No bacteriolytic activity (stapholysin) is detected. Negative for phenylalanine

deaminase, alkylsulfatase, pyrazinamidase and Jordan’s tartrate.

Acid is produced from the following carbohydrates: fructose, galactose, glucose,

glycerol, glycogen, glucose-1-phosphate, glucose-6-phosphate, maltose, mannose, N-

acetylglucosamine, ribose, sucrose and trehalose, but not from adonitol, amygdalin, L-

arabinose, cellobiose, dulcitol, myo-inositol, lactose, lactulose, D-mannitol, melibiose,

methyl--D-glucoside, raffinose, L-rhamnose, salicin or D-sorbitol. Acid production is

observed for the following carbohydrates with the API 50CH strip: glycerol, D-ribose,

D-galactose, D-glucose, D-fructose, D-mannose, N-acetylglucosamine, maltose,

sucrose, trehalose, starch, glycogen and potassium gluconate. The type strain is 266T

(=CECT 8023T = LMG 26707T), isolated from treated effluent in the south-west region

of Western Australia.

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In this chapter, the taxonomic position of a previously unknown Aeromonas isolate has

been determined using extensive phenotypic and genotypic testing which confirmed that

strain 266T represents a novel Aeromonas species for which the name Aeromonas

australiensis (aus.tra.li.en’sis. N. L. fem. Adj. australiensis, of or belonging to

Australia) sp. nov. has been proposed.

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CHAPTER 7: VIRULENCE GENES PRESENT IN

WESTERN AUSTRALIAN AEROMONAS SPP.

7.1. INTRODUCTION

Aeromonas species are ubiquitous Gram-negative bacilli found in aquatic environments,

many types of foods and in vertebrate and invertebrate organisms. In humans,

Aeromonas species have been isolated practically from every body site and are the

aetiological agents of serious human infections including bacteraemia and meningitis.

Aeromonas sepsis, frequently fatal in humans, is usually associated with malignancies

or other chronic underlying illnesses although infections are occasionally reported in

immunocompetent individuals (Janda and Abbott 2010). Although the genus

Aeromonas currently comprises 27 species only a few are considered pathogenic to

humans. Of these, A. hydrophila, A. veronii bv. sobria and A. caviae are of clinical

significance (Janda and Abbott 2010). However, by virtue of its previous isolation from

human clinical material either as A. aquariorum or A. hydrophila ssp. dhakensis, the

newly combined species A. dhakensis sp. nov. comb. nov. (Beaz-Hidalgo et al. 2013;

Puah et al. 2013) should be considered one of the major Aeromonas species.

Many putative virulence factors have been identified in these organisms including

exotoxins, surface structures and secretory systems (Yu et al. 2004; Sen and Lye 2007;

Chopra et al. 2009). The detection of virulence genes is considered a practical method

of screening a large number of Aeromonas isolates for potential virulence (Sen and

Rodgers 2004). Attempts to reproduce disease with aeromonads in laboratory animals

and human volunteers have failed to build a robust case for causality based on Koch’s

postulates (Janda and Abbott 2010). As a consequence, a plethora of alternative models

of infection including the unicellular amoeba Dictyostelium and the medicinal leech

Hirudo medicinalis among others have been proposed to assess the virulence potential

of aeromonads (Janda and Abbott 2010). One of the drawbacks of these models is that

the complex patho-physiology of the in-vivo infection may not be fully reproducible in

non-mammalian models.

The aim of this chapter was to determine the presence of 13 virulence genes among

genotypically-characterized clinical and environmental strains as described in Chapter

4. A PCR-based method was used to detect the genes coding for aerolysin/haemolysin

(aerA/haem), serine protease (aspA), heat-labile (alt) and heat-stable (ast) cytotoxins,

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components of the type 3 (aexT and ascV) and type 6 (vasH) secretion systems, lateral

(lafA) and polar (flaA) flagella, bundle-forming pilus (BfpA and BfpG) and a Shiga-like

toxin (stx-1 and stx-2). This is the first study of this kind in Australia.

7.2. Bacterial strains

Bacterial strains used in this investigation and their source of isolation are listed in

Table 2.9. Primers used in this study are listed in Table 2.8.

7.3. RESULTS

7.3.1. Overall distribution of virulence genes

The overall distribution of nine virulence genes in all Aeromonas isolates tested is

shown in Table 7.1. The most prevalent genes were aerA/haem 77% (100/129), alt 53%

(69/129) and lafA 51% (67/129) while ascV 16% (16/129) and aexT 13% (13/129) were

the least frequently detected. The genes coding for a bundle-forming pilus (BfpA and

BfpG) and a Shiga-like toxin (stx-1 and stx-2) could not be detected in any isolate

(results not shown). Virulence genes more prevalent in environmental than in clinical

isolates were aexT (26 vs. 9%); (p = 0.0295), ascV (39 vs. 8%) (p = 0.0004), aspA (61

vs. 19%) (p = 0.0001), and vasH (48 vs. 19%) (p = 0.0023), respectively. By contrast,

lafA (59 vs. 29%) (p < 0.0040) was present more often in clinical than in environmental

strains. Among the major species, the most prevalent virulence genes were: A. caviae,

lafA 55% (15/27) and aerA/haem 52% (14/27); A. dhakensis, alt 81% (25/31),

aerA/haem 74% (23/31), flaA and lafA both 64% (20/31) and vasH 61% (19/31); A.

hydrophila, ast 93% (27/29), alt 86% (25/29), aerA/haem 79% (23/29), lafA 69%

(20/29) and aspA 52% (15/31); A. veronii bv. sobria, aerA/haem 100% (31/31), ascV

32% (10/31) and 26% (8/31) for both alt and aexT.

7. 3.2. Distribution of virulence genes in stool isolates

The prevalence of virulence genes in stool specimens is shown in Table 7.2. The

aerA/haem and lafA genes were equally distributed in 55% (11/20) of the total isolates

followed by ast 45% (9/20) and alt 40% (8/20). The flaA+/lafA+ genotype was present in

20% (4/20) of total isolates while 35% (7/35) had both alt and ast. Ten% (2/20) of the

strains harboured more than five virulence genes.

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7.3.3. Distribution of virulence genes in extra-intestinal isolates

The prevalence of virulence genes in extra-intestinal specimens is shown in Table 7.3

(blood), Table 7.4 (wounds) and Table 7.5 (miscellaneous specimens). Overall,

aerA/haem, lafA and alt were the most prevalent genes in these specimens. The

distribution of these genes in blood was 86% (24/28), 46% (13/28), 46% (13/28); in

wounds 91% (29/32), 69% (22/32) and 56% (18/32), and in miscellaneous specimens

83% (15/18), 67% (12/18) and 56% (10/18), respectively. Five or more virulence genes

were detected in 28% (9/32) wound isolates, 25% (7/28) in blood, and 22% (4/18)

miscellaneous specimens. The flaA+/lafA+ genotype was present in 28% (9/32) wound,

17% (3/18) miscellaneous specimen and 7% (2/28) blood isolates. Both alt and ast were

present in 33% (6/18) miscellaneous specimens, 29% (8/28) blood and 25% (8/32)

wound isolates.

7.3.4. Distribution of virulence genes among environmental isolates

The prevalence of virulence genes in Aeromonas isolated from environmental samples

is shown in Table 7.6. The most prevalent genes were aerA/haem 68% (21/31), alt 61%

(19/31), aspA 61% (19/31) and vasH 48% (15/31). The flaA+/lafA+ genotype was

present in 9.6% (3/31) of total isolates while 29% (9/31) harboured both alt and ast

genes. Individually, flaA was distributed in 39% (12/31); lafA in 29% (9/31), alt in 61%

(19/31) and ast in 39% (12/31) of total isolates.

7.3.5. Additional features

Overall, 27% (35/129) of the total isolates harboured five or more virulence genes

including 22% (22/98) in clinical and 42% (13/31) in environmental isolates. Five or

more virulence genes were detected in 100% (3/3) A. jandaei, 48% (14/29) A.

hydrophila, 42% (13/31), A. dhakensis, 19% (6/31) A. veronii bv. sobria and the single

strains of A. allosaccharophila and A. australiensis but not in A. bestiarum, A. caviae,

A. media, A. salmonicida and A. schubertii. Among the major species, the average

number of virulence genes detected was: A. dhakensis 4.3, A. hydrophila 4.3, A. veronii

bv. sobria 2.7 and A. caviae 1.7. The flaA+lafA+ genotype was present in 39% (12/31) A.

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dhakensis, 21% (6/29) A. hydrophila, 4% (1/25) A. caviae, 3% (1/31) A. veronii bv.

sobria and in both A. media isolates (Tables 7.7).

7.3.6. Percentage identity of nucleotide sequences of positive products from

this study compared to sequences deposited in GenBank

The nucleotide sequences of gene products from selected strains were compared with

sequences deposited in GenBank and shown in Table 7.8. Accesion numbers for these

sequences are shown in Table 7.9. Unspecific amplification products were detected for

the vasH gene. The percentage of nucleotide identity for aerA/haem ranged from 71.2 to

96.5% over a 323 bp length; alt 90.9 to 93.8% over 244 bp; ast 94.7% over 265 bp;

aexT 88.0 to 94.1% over 510 bp; ascV 83.8% over 500 bp; aspA 71.2 to 93.7% over 306

bp; flaA 71.0 to 90.5% over 326 to 328 bp; lafA 69.3 to 83.0% over 555 to 580 bp and

vasH 86.0% over 572 bp. These results were not included in the original publication.

7.4. DISCUSSION

The distribution of 13 virulence genes assayed among 129 Aeromonas isolates was

determined in order to evaluate the pathogenic potential of these bacteria. The majority

(96%; 124/129) of the strains contained at least one virulence gene. The frequency of alt

and ast in stool isolates was 40% and 45%, respectively. In other studies, the frequency

for alt ranged from 16 to 35% and for ast 6 to 97% (Albert et al. 2000; Aguilera-

Arreola et al. 2005, 2007; Senderovich et al. 2012). In A. hydrophila, ast has been

detected between 30 and 91% of the isolates tested while has been absent in A. caviae

and A. veronii (Sen and Rodgers 2004; Aguilera-Arreola et al. 2007). In another study,

alt was almost exclusively detected in diarrhoeic isolates (Aguilera-Arreola et al. 2005).

The wide variations in the distribution of enterotoxin genes lend support to the

observations by Chopra et al. (2009) who stated that the prevalence of virulence genes

may depend on the strains examined at the time of testing.

The aerA/haem gene was detected in 77% of the total isolates consistent with other

reports where the prevalence of this gene ranged from 72 to 89% (Aguilera-Arreola et

al. 2007; Chacón et al. 2003; Puthucheary et al. 2012).

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-

Tab

le 7

.1 D

istri

butio

n of

viru

lenc

e ge

nes a

mon

g W

este

rn A

ustra

lian

Aero

mon

as sp

ecie

s

Gen

e fr

eque

ncy

(%)

Spec

ies

No.

test

ed

aerA

/hae

m

aexT

al

t as

cV

aspA

as

t fla

A

lafA

va

sH

A. a

llosa

ccha

roph

ila

1c

+

+

+ +

+

A. a

ustr

alie

nsis

1e

+

+ +

+

A. b

estia

rum

1c

+

+

+

A. c

avia

e 25

c 14

(56)

1(

4)

4 (1

6)

5 (2

0)

3 (1

2)

15 (6

0)

2 (8

)

2

e

1

(50)

1

(50)

T

otal

27

14 (5

2)

1 (4

) 5

(18)

1

(4)

5 (1

8)

3 (1

1)

15 (5

5)

2 (7

)

A. d

hake

nsis

21

c 17

(81)

1

(5)

15 (7

1)

2 (9

) 3

(14)

5

(34)

13

(62)

17

(81)

11

(52)

10

e 6

(60)

5

(50)

10

(100

) 3

(30)

6

(60)

4

(40)

7

(70)

3

(30)

8

(80)

T

otal

31

23 (7

4)

6 (1

9)

25 (8

1)

5 (1

6)

9 (2

9)

9 (2

9)

20 (6

4)

20 (6

4)

19 (6

1)

A. h

ydro

phila

23

c 20

(87)

1

(4)

20 (8

7)

10 (4

3)

21 (9

1)

6 (2

6)

16 (6

9)

3 (1

3)

6

e 3

(50

5 (8

3)

5 (8

3)

6 (1

00)

3 (5

0)

4 (6

7)

2 (3

3)

T

otal

29

23 (7

9)

1 (3

) 25

(86)

15

(52)

27

(93)

9

(31)

20

(69)

5

(17)

A. ja

ndae

i 3e

2

(67

2 (6

7)

2 (6

7)

2 (6

7)

1 (3

3)

2 (6

7)

2 (6

7)

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-

Tab

le 7

.1

Con

tinue

d.

Gen

e fr

eque

ncy

(%)

Spec

ies

No.

test

ed

aerA

/hae

m

aexT

al

t as

cV

aspA

as

t fla

A

lafA

va

sH

A. m

edia

2c

1

(50)

1

(50)

1

(50)

1

(50)

2

(100

) 2

(100

)

A. sa

lmon

icid

a 1c

+

+

+

+

1e

+

+ +

+

A. sc

hube

rtii

1c

+

+

A. v

eron

ii bv

. sob

ria

23c

23 (1

00)

5 (2

2)

7 (3

0)

6 (2

6)

3 (1

3)

5 (2

2)

3 (1

3)

6 (2

6)

3 (1

3)

8

e 8

(100

) 3

(37)

1

(12)

4

(50)

2

(25)

1

(12)

1

(12)

3

(37)

T

otal

31

31 (1

00)

8 (2

6)

8 (2

6)

10 (3

2)

5 (1

6)

6 (1

9)

4 (1

3)

6 (1

9)

6 (1

9)

Tota

l clin

ical

98

79

(81)

a 9

(9)b

49 (5

0)c

9 (9

)d 19

(19)

e 38

(39)

f 29

(29)

g 58

(59)

h 19

(19)

i

Tota

l env

ironm

enta

l 31

21

(68)

a 8

(26)

b 20

(64)

c 12

(39)

d 19

(61)

e 12

(39)

f 12

(39)

g 9

(29)

h 15

(48)

i

Gra

nd to

tal

129

100

(77)

17

(13)

69

(53)

21

(16)

38

(29)

50

(39)

41

(32)

67

(51)

34

(26)

, n

ot d

etec

ted;

+, d

etec

ted;

c, c

linic

al; e

, env

ironm

enta

l; a p

= 0.

1453

; b p

= 0.

0295

; c p =

0.21

52; d

p =

0.00

04; e p

< 0.

0001

; f p =

1.00

00; g

p =

0.37

97; h p

= 0

.004

0; i p

= 0

.002

3

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-223

-

Tab

le 7

.2

Dis

tribu

tion

of v

irule

nce

gene

s in

Aero

mon

as sp

p. is

olat

ed fr

om st

ools

(n =

20)

Gen

es d

etec

ted

Spec

ies

Stra

in

no.

a/h

alt

ast

lafA

fla

A

ascV

ae

xT

aspA

va

sH

Stool1

Age

Gender

Clin

ical

dat

a

A. a

llosa

ccha

roph

ila

100

+

+ +

+

+

74

F C

ampy

loba

cter

je

juni

als

o is

olat

ed2

A. d

hake

nsis

16

9

+ +

+ +

L 35

M

In

fect

ed C

hron

s2

18

0

+ +

+

+

W

80

F Pe

rsis

tent

di

arrh

oea;

di

verti

culit

is

18

3

+ +

+ +

L 63

F

Dia

rrho

ea

A. c

avia

e 94

+

+

L 74

M

N

/D

10

2 +

W

71

F D

iarr

hoea

pos

t ch

emot

hera

py2

10

3 +

+ +

+

L 57

F

Dia

rrho

ea fo

r 2

wee

ks

15

6

+ +

+

+

L 5

m

F R

ecen

t tra

vel

15

8

+

W

63

M

Rec

ent t

rave

l

187

+

+ L

44

F Pr

em m

enop

ause

216

+ +

+

SF

74

F

N/D

N

/D, n

o da

ta; 1 St

ool c

onsi

sten

cy, A

. allo

sacc

haro

phila

was

isol

ated

from

a c

olos

tom

y sp

ecim

en; L

, loo

se, W

, wat

ery,

SF,

sem

i-for

med

; 2 le

ucoc

ytes

det

ecte

d in

stoo

ls; a

/h, a

erA/

haem

; M, m

ale;

F, f

emal

e.

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-224

-

T

able

7.2

Con

tinue

d.

Gen

es d

etec

ted

Spec

ies

Stra

in

no.

a/h

alt

ast

lafA

fla

A

ascV

ae

xT

aspA

va

sH

Stool1

Age

Gender

Clin

ical

dat

a

A. h

ydro

phila

13

3

+ +

+ +

+

31

M

Po

st/m

orte

m

spec

imen

A.

med

ia

179

+

+ +

+

L

74

M

Prol

onge

d in

trave

nous

an

tibio

tics

A. v

eron

ii bv

sobr

ia

99

+

W

78

F

N/D

2

137

+ +

W

33

M

N

/D

16

6 +

+

+

L 70

F

Dia

rrho

ea

18

4 +

W

78

M

N/D

2

189

+

+ W

67

F

Dia

rrho

ea,

mae

lena

, Tr

icho

mon

as

hom

inis

+

21

5 +

+

+

SF

89

F N

/D

21

9 +

W

61

F D

iarr

hoea

for 1

w

eek2

Tota

l no.

11

8

9 11

7

1 1

3 5

%

55

40

45

55

35

5

5 15

25

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-225

-

Tab

le 7

.3 D

istri

butio

n of

viru

lenc

e ge

nes i

n Ae

rom

onas

spp.

isol

ated

from

blo

od (n

= 2

8)

Gen

es d

etec

ted

Spec

ies

Stra

in

no.

a/h

alt

ast

flaA

la

fA

aspA

ae

xT

ascV

va

sH

Age

Gender

Clin

ical

dat

a

A. d

hake

nsis

60

+

+

+

75

M

Acu

te re

nal f

ailu

re

70

+

+

70

M

Abd

omin

al se

psis

154

+ +

+

+

+ N

/D

N/D

N

/D

A. b

estia

rum

68

+

+

+

+

46

F N

/D; p

olym

icro

bial

A.

cav

iae

57

+

+

48

M

N

/D

58

83

F N

/D

65

+

82

M

Cho

lang

eo c

arci

nom

a;

poly

mic

robi

al

75

+

53

F H

ickm

an c

athe

ter

colle

cted

blo

od; N

/D

80

+

80

F N

/D

96

+

+

72

M

N/D

106

72

M

Se

ptic

109

+

+

70

F

Epig

astri

c pa

in;

poly

mic

robi

al

11

0 +

+

50

M

N/D

; pol

ymic

robi

al

20

0

+

+

N/D

N

/D

N/D

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-226

-

Tab

le 7

.3

Con

tinue

d.

Gen

es d

etec

ted

Sp

ecie

s St

rain

no

. a/

h al

t as

t fla

A

lafA

as

pA

aexT

as

cV

vasH

Age

Gender

Clin

ical

dat

a

A. h

ydro

phila

59

+

+ +

+

+

65

M

Febr

ile n

eutro

peni

c

84

+ +

+

+ +

81

F

N/D

; Sta

phyl

ococ

cus a

ureu

s als

o is

olat

ed

14

9 +

+ +

+

+

68

M

N/D

151

+ +

+ +

73

M

N

/D

15

2 +

+ +

73

M

N/D

A.

med

ia

85

+

+

+

+

17

F

Prol

onge

d vi

ral-l

ike

illne

ss;

poly

mic

robi

al

A. v

eron

ii bv

sobr

ia

72

+

56

M

C

ance

r/pan

crea

s; p

olym

icro

bial

81

+

+

89

F

Sept

ic sh

ock

11

1 +

+

+

88

F Li

ver c

ance

r; po

lym

icro

bial

125

+ +

+ +

+

88

F

Vom

iting

131

+

+ +

69

M

Le

ukae

mia

; On

chem

othe

rapy

218

+ +

+

+ +

<1 5

N/D

N

/D

22

1 +

+

47

F Fe

ver;

brea

st c

ance

r; po

lym

icro

bial

269

+ +

+

+

+ +

81

M

Fe

ver;

AM

L To

tal n

o.

24

13

8 7

13

5 5

3 2

%

86

46

29

25

46

18

18

11

7

a/h,

aer

A/ha

em; A

ML,

acu

te m

yelo

blas

tic le

ukae

mia

; N/D

, no

data

; M, m

ale;

F, f

emal

e

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-

Tab

le 7

.4

Dis

tribu

tion

of v

irule

nce

gene

s in

Aero

mon

as sp

p. is

olat

ed fr

om w

ound

s (n

= 32

)

Gen

es d

etec

ted

Spec

ies

Stra

in

no.

a/h

alt

ast

flaA

la

fA

aspA

ae

xT

ascV

va

sH

Age

Gender

Clin

ical

dat

a

A. d

hake

nsis

67

+

+

+ +

+

20

M

Cel

lulit

is

71

+

+

+ +

+

+

16

F In

fect

ed la

cera

tion

of fo

ot (r

iver

wat

er)

73

+

+

+ +

28

M

App

endi

citis

79

+

+

+

60

M

Infe

cted

fing

er

91

+

+

+

78

F Le

g w

ound

95

+

+

+ +

+

50

M

Wou

nd in

fect

ed p

ost e

lbow

surg

ery;

on

kefle

x

104

+ +

+

+ 60

F

Non

-hea

ling

shin

; on

flucl

oxic

illin

10

7 +

+

+ +

+

39

M

Sept

ic w

ound

righ

t-han

d; o

n flu

coxi

cilli

n

14

1 +

+ +

+

N/D

U

N

/D

17

6 +

+ +

+

26

M

Puru

lent

wou

nd o

oze

right

-leg

22

0 +

+

+ +

+

+

N/D

U

U

lcer

27

9 +

+

+

14

M

Ost

eom

yelit

is le

ft th

umb;

Sta

phyl

ococ

cus

aure

us a

lso

isol

ated

.

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-228

-

Tab

le 7

.4

Con

tinue

d.

Spec

ies

Stra

in

no.

a/h

alt

ast

flaA

la

fA

aspA

ae

xT

ascV

va

sH

Age

Gender

Clin

ical

dat

a

A. c

avia

e 14

3 +

+

+

N/D

U

N

/D

16

3

50

M

Han

d w

ound

27

0 +

+

+

37

F In

fect

ed w

ound

; Sta

phyl

ococ

cus

aure

us a

lso

isol

ated

A.

hyd

roph

ila

23

+

+

N

/D

U

N/D

69

+

+ +

+

+

18

M

Infe

cted

subu

ngua

l hae

mat

oma;

po

lym

icro

bial

90

+ +

+

36

F

Ulc

er; S

taph

yloc

occu

s aur

eus a

nd

anae

robe

s als

o is

olat

ed

98

+

+ +

+

35

M

Infe

cted

left

hand

10

1 +

+ +

+

76

F M

ultip

le u

lcer

s

11

2 +

+ +

+ +

+

66

F Po

st/la

para

tom

y an

d w

ound

br

eakd

own;

pol

ymic

robi

al

11

7

+ +

+

+

+

54

F N

/D; p

olym

icro

bial

12

6

+

22

M

Dirt

y pu

rule

nt a

quat

ic w

ound

12

8 +

+ +

+

13

M

Stap

hylo

cocc

us a

ureu

s als

o is

olat

ed

14

8 +

+

+

73

M

N/D

Page 255: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-229

-

Tab

le 7

.4

Con

tinue

d.

Gen

es d

etec

ted

Spec

ies

Stra

in

no.

a/h

alt

ast

flaA

la

fA

aspA

ae

xT

ascV

va

sH

Age

Gender

Clin

ical

dat

a

A. sa

lmon

icid

a 19

0 +

+

+

+

65

M

Off

ensi

ve sm

elly

pur

ulen

t di

scha

rge

of le

ft-po

int f

inge

r A.

schu

bert

ii 18

6 +

+

42

M

Pus f

rom

infe

cted

wou

nd in

foot

A. v

eron

ii bv

. sob

ria

24

+

<1

5 U

N

/D

66

+

+

+

+

+

47

F R

ight

-low

er le

g

12

9 +

+

+

48

M

Infe

cted

wou

nd ri

ght-a

nkle

14

7 +

+ +

<15

M

N/D

17

4 +

+

71

M

Infe

cted

thum

b na

il; M

ixed

an

aero

bes a

lso

isol

ated

To

tal n

o.

29

18

13

10

22

6 3

3 11

%

91

56

41

31

69

19

9

9 34

N/D

, no

data

; M, m

ale,

F, f

emal

e, U

, unk

now

n; a

/h, a

erA/

haem

.

Page 256: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-230

-

Tab

le 7

.5

Dis

tribu

tion

of v

irule

nce

gene

s in

Aero

mon

as sp

p. is

olat

ed fr

om m

isce

llane

ous s

peci

men

s (n

= 18

)

Gen

es d

etec

ted

Spec

ies

Stra

in

no.

a/h

alt

ast

flaA

la

fA

aspA

as

cV

vasH

A

ge

Gen

der

Sour

ce

C

linic

a da

ta

A. d

hake

nsis

47

+

+

+

81

M

sp

utum

Le

u 3+

;Abu

ndan

t gr

owth

; N/D

56

+

+ +

35

M

bone

chi

ps

Infe

cted

frac

ture

93

+ +

+

35

M

urin

e U

rinar

y tra

ct

infe

ctio

n A.

cav

iae

62

+

+

47

M

cath

eter

s Li

ver t

rans

plan

t; po

lym

icro

bial

78

+

75

M

dial

ysis

flu

id

Perit

oniti

s

14

0

57

F

dial

ysis

flu

id

Perit

onea

l dia

lysi

s;

poly

mic

robi

al

17

8

+ +

34

F

bile

B

iliar

y ob

stru

ctio

n;

poly

mic

robi

al

18

8

+

+

68

F

bile

A

cute

cho

lecy

stiti

s;

poly

mic

robi

al

A. h

ydro

phila

61

+

+ +

+

62

M

ca

thet

ers

Bili

ary

seps

is;

poly

mic

robi

al

83

+

+ +

+

53

F

sput

um

Leu

3+; N

/D;

89

+

+ +

+

46

F

bile

C

hola

ngiti

s;

poly

mic

robi

al

Page 257: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-231

-

T

able

7.5

C

ontin

ued.

Gen

es d

etec

ted

Spec

ies

Stra

in

no.

a/h

alt

ast

flaA

la

fA

aspA

as

cV

vasH

A

ge

Gen

der

Sour

ce

Clin

ical

dat

a

A. h

ydro

phila

92

+

+

+ +

+

66

F

tissu

e Pa

ncre

atic

ne

cros

is;

poly

mic

robi

al

11

3 +

+ +

+ +

+

+ 35

M

dr

ain

fluid

N

/D; p

olym

icro

bial

11

8 +

+ +

+

+

30

F

sput

um

Exac

erba

tion

of

CF;

pol

ymic

robi

al;

15

0 +

+ +

73

M

tis

sue

Foot

infe

ctio

n

A. v

eron

ii bv

sobr

ia

25

+

<15

N/D

ca

thet

ers

N/D

27

+

+

+

+ +

<1

5 N

/D

tissu

e N

/D

17

1 +

83

F

sput

um

Asp

iratio

n pn

eum

onia

; po

lym

icro

bial

To

tal n

o.

15

10

7 3

12

5 2

1

%

83

56

39

17

67

28

11

6

M, m

ale;

F, f

emal

e; N

/D, n

o da

ta; a

/h, a

erA/

haem

; CF,

cyt

isc

fibro

sis;

Leu

3+,

man

y le

ucoc

ytes

seen

on

mic

rosc

opy.

Page 258: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-232

-

Tab

le 7

.6

Dis

tribu

tion

of v

irule

nce

gene

s am

ong

Aero

mon

as sp

p. is

olat

ed fr

om e

nviro

nmen

tal s

ourc

es (n

= 3

1)

Gen

es d

etec

ted

Spec

ies

Stra

in

no.

a/h

alt

ast

flaA

la

fA

aspA

ae

xT

ascV

va

sH

Sour

ce

Loc

atio

n

A. a

ustr

alie

nsis

26

6 +

+

+ +

+

IW

Rur

al

A. d

hake

nsis

31

+

+

+

Fish

A

DW

A

32

+

+

+

+

Fish

A

DW

A

22

3

+ +

+

+ +

+ +

Unk

now

n U

nkno

wn

22

9 +

+

+ +

+

+

TW

Unk

now

n

230

+ +

+

+

W

ater

M

etro

polit

an

23

5 +

+ +

+

+

Wat

er

Unk

now

n

241

+ +

+

+ +

W

ater

U

nkno

wn

24

2

+

+

+

+

Wat

er

Unk

now

n

256

+ +

+ +

+ +

Wat

er

Unk

now

n

257

+ +

+

+ +

+

+ W

ater

U

nkno

wn

A. c

avia

e 30

Fish

A

DW

A

26

4

+

+

IW

Unk

now

n A.

hyd

roph

ila

34

+

+

+ +

Fi

sh

AD

WA

231

+

+ +

+

SW

M

etro

polit

an

24

3 +

+

+

+

+

Wat

er

Unk

now

n

Page 259: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-233

-

Tab

le 7

.6

C

ontin

ued.

Gen

es d

etec

ted

Spec

ies

Stra

in

no.

a/h

alt

ast

flaA

la

fA

aspA

ae

xT

ascV

va

sH

Sour

ce

Loc

atio

n

A. h

ydro

phila

24

5

+ +

+

+

Wat

er

Unk

now

n

260

+

+

+

+ W

ater

U

nkno

wn

26

1 +

+ +

+ +

+

IW

Unk

now

n A.

jand

aei

35

+

+ +

+

+ Fi

sh le

sion

A

DW

A

25

3 +

+

+

+ +

Wat

er

Unk

now

n

262

+

+

+ +

+

W

ater

U

nkno

wn

A. sa

lmon

icid

a 19

9 +

+

+

+

Cra

b R

ural

A.

ver

onii

bv. s

obria

33

+

+

+ +

Fish

A

DW

A

22

4 +

+

BW

M

etro

polit

an

23

7 +

+

+ +

+

Wat

er

Unk

now

n

247

+ +

+

W

ater

U

nkno

wn

25

4 +

Wat

er

Unk

now

n

259

+

+

+

+ W

ater

U

nkno

wn

26

5 +

+

IW

Unk

now

n

268

+

+ IW

U

nkno

wn

Tota

l no.

21

19

12

12

9

19

8 12

15

%

68

61

39

39

29

61

26

39

48

a/h

, aer

A/ha

em; I

W, i

rrig

atio

n w

ater

; TW

, tre

ated

wat

er; B

W, b

ore

wat

er; A

DW

A, A

gric

ultu

re D

epar

tmen

t of W

este

rn A

ustra

lia

Page 260: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-234

-

Tab

le 7

.7

Add

ition

al fe

atur

es

Spec

ies (

no. s

trai

ns)

Ave

rage

no.

gen

es p

er st

rain

fla

A+ la

fA+ g

enot

ype

(%)

>5 v

irul

ence

gen

es (%

)

C

lin

Env

T

otal

C

lin

Env

T

otal

C

lin

Env

T

otal

A. c

avia

e (2

7)

1.8

1.0

1.7

4 0

~4

0 0

0

A. d

hake

nsis

(31)

4.

0 5.

1 4.

3 48

a 20

a 39

33

c 60

c 42

A. h

ydro

phila

(29)

4.

2 4.

6 4.

3 22

b 17

b 21

48

d 50

d 48

A. v

eron

ii bv

. sob

ria (3

1)

2.6

2.8

2.7

3 0

4

22e

12e

19

Tot

al

3.1

3.4

3.2

19

9 17

26

30

27

A. a

llosa

ccha

roph

ila (1

) 5

0

10

0

A. a

ustr

alie

nsis

(1)

4

0

10

0

A. b

estia

rum

(1)

4

0

0

A. ja

ndae

i (3)

5

0

100

A. m

edia

(2)

4

10

0

0

A. sa

lmon

icid

a (2

) 4

4 4

0 0

0 0

0 0

A. sc

hube

rtii

(1)

2

0

0

a Pe

rcen

tage

diff

eren

ces

are

stat

istic

ally

sig

nific

ant

(p <

0.0

001)

; b Pe

rcen

tage

diff

eren

ces

are

not

stat

istic

ally

sig

nific

ant

(p =

0.4

75);

c Perc

enta

ge d

iffer

ence

s ar

e st

atis

tical

ly s

igni

fican

t (p

= 0

.000

2);

d Per

cent

age

diff

eren

ces

are

not

stat

istic

ally

sig

nific

ant

(p =

0.8

87);

e Perc

enta

ge d

iffer

ence

s are

not

stat

istic

ally

sign

ifica

nt (p

= 0

.889

2); C

lin, c

linic

al is

olat

es; E

nv, e

nviro

nmen

tal i

sola

tes.

Page 261: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

- 235

-

Tab

le 7

.8 P

erce

ntag

e id

entit

y of

gen

e pr

oduc

t seq

uenc

es fr

om th

is st

udy

com

pare

d w

ith se

quen

ces d

epos

ited

in G

enB

ank

Gen

e Sp

ecie

s/st

rain

no.

%

L

engt

h (b

p)

Spec

ies

Acc

esio

n no

.

aerA

/hae

m

A. d

hake

nsis

60,

73, 2

56, 2

57, 2

79

71.2

32

3 A.

ver

onii

bv. s

obria

A.

hyd

roph

ila

A. h

ydro

phila

AB

1090

93

AY

6110

33

AF4

1046

6

A.

aus

tral

iens

is 2

66

90.4

32

3

A. b

estia

rum

68

78.3

32

3

A. c

avia

e 27

0 96

.5

323

A.

jand

aei 3

5 90

.4

323

A.

ver

onii

bv. s

obria

125

, 215

, 221

, 237

, 259

, 269

93

.4

323

A.

ver

onii

bv. s

obria

125

, 215

, 221

, 237

, 259

, 269

93

.1

323

A. sa

lmon

icid

a

X65

048

alt

A. d

hake

nsis

31,

32,

60,

67,

180

, 183

, 223

, 229

, 235

, 241

, 242

, 256

90

.9

244

A. h

ydro

phila

A.

hyd

roph

ila

A. h

ydro

phila

A.

hyd

roph

ila

JBN

1302

JQ

0031

97

L775

73

JX48

9379

A.

aus

tral

iens

is 2

66

96.3

24

4

A. c

avia

e 10

3, 1

88, 2

00, 2

64

93.0

24

4

A. h

ydro

phila

34,

59,

61

94.2

24

4

A. ja

ndae

i 253

, 262

94

.7

244

A.

ver

onii

bv. s

obria

111

, 218

, 247

, 269

93

.8

244

ast

A. d

hake

nsis

154

, 169

, 183

A. c

avia

e 10

3, 1

43, 1

56

94.7

26

5 A.

hyd

roph

ila

JQ00

3211

A. h

ydro

phila

23,

34,

59,

83,

113

, 117

, 243

, 261

A. m

edia

179

; A. v

eron

ii bv

. sob

ria 2

7, 6

6, 1

25, 2

69

aexT

A.

dha

kens

is 3

1, 3

2, 2

20, 2

30; A

.med

ia 8

5 94

.1

510

A. v

eron

ii EF

0260

79

Page 262: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

-- 2

36 --

Tab

le 7

.8

Con

tinue

d.

Gen

e Sp

ecie

s/st

rain

no.

%

L

engt

h (b

p)

Spec

ies

Acc

esio

n no

.

aexT

A.

ver

onii

bv. s

obria

33,

66,

218

, 224

, 269

;

A. d

hake

nsis

31,

32,

220

, 230

; A.m

edia

85

88.0

51

0 A.

salm

onic

ida

AF2

8836

6

A. v

eron

ii bv

. sob

ria 3

3, 6

6, 2

18, 2

24, 2

69;

ascV

A.

allo

sacc

haro

phila

100

; A. d

hake

nsis

47,

220

; 83

.8

500

A. v

eron

ii bv

. ver

onii

HM

5845

87

A.

aus

tral

iens

is 2

66; A

. ver

onii

bv. s

obria

66,

218

, 221

, 269

as

pA

A. a

ustr

alie

nsis

266

85

.6

306

A. h

ydro

phila

A

F126

213

A.

aus

tral

iens

is 2

66

86.2

30

6 A.

sobr

ia

AF2

5347

1

A. h

ydro

phila

34,

69,

84,

92,

149

, 261

71

.2

306

A. h

ydro

phila

A

F126

213

A.

jand

aei 2

62

92.4

30

6 A.

sobr

ia

AF2

5347

1

A. v

eron

iii b

v. so

bria

27

93.7

30

6 A.

sobr

ia

AF2

5347

1

A. sa

lmon

icid

a 19

9 90

.1

306

A. sa

lmon

icid

a

X67

043

flaA

A. d

hake

nsis

60,

67,

176

, 183

, 229

84

.6

326

A. h

ydro

phila

JQ

0032

17

A.

dha

kens

is 6

0, 6

7, 1

76, 1

83, 2

29

90.5

32

7 A.

hyd

roph

ila

AY

4243

58

A.

hyd

roph

ila 9

2, 1

51, 2

61

78.8

32

7 A.

salm

onic

ida

EU

4103

42

A.

bes

tiaru

m 6

8 71

.0

328

A. sa

lmon

icid

a

EU41

0342

la

fA

A. h

ydro

phila

133

83

.0

555

A. h

ydro

phila

D

Q12

4694

A. h

ydro

phila

260

78

.9

580

A. h

ydro

phila

D

Q12

4694

A. m

edia

179

72

.7

580

A. p

unct

ata

A

F348

135

A.

dha

kens

is 9

5 74

.3

580

A. p

unct

ata

A

F348

135

A.

dha

kens

is 9

5 69

.3

580

A. ja

ndae

i A

Y22

8331

va

sH

A. d

hake

nsis

31

86.0

57

2 A.

hyd

roph

ila

GQ

3597

79

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-- 2

37 --

Tab

le 7

.9

Acc

essi

on n

umbe

rs o

f seq

uenc

es d

eriv

ed fr

om v

irule

nce

gene

s and

dep

osite

d in

Gen

Ban

k

Gen

e Sp

ecie

s St

rain

no.

A

cces

sion

no.

Sp

ecie

s St

rain

no.

A

cces

sion

no.

aerA

/hae

m

A. a

ustr

alie

nsis

26

6 H

G97

7017

A.

bes

tiaru

m

68

HG

9770

18

A.

dha

kens

is

73

HG

9770

19

A. d

hake

nsis

27

9 H

G97

7020

A.

hyd

roph

ila

59

HG

9770

21

A. h

ydro

phila

14

8 H

G97

7022

A.

jand

aei

35

HG

9770

23

A. v

eron

ii bv

. sob

ria

215

HG

9770

24

A.

ver

onii

bv. s

obria

23

7 H

G97

7025

A.

ver

onii

bv. s

obria

12

5 H

G97

7026

A.

ver

onii

bv. s

obria

22

1 H

G97

7027

A.

ver

onii

bv. s

obria

26

9 H

G97

7028

A.

cav

iae

270

HG

9770

29

A. ja

ndae

i 25

3 H

G97

7030

A.

hyd

roph

ila

151

HG

9770

31

A. d

hake

nsis

60

H

G97

7032

A.

dha

kens

is

256

HG

9770

33

A. d

hake

nsis

25

7 H

G97

7034

A.

ver

onii

bv. s

obria

25

9 H

G97

7035

aexT

A.

dha

kens

is

220

HG

9770

36

A. v

eron

ii bv

. sob

ria

269

HG

9770

37

A.

ver

onii

bv. s

obria

33

H

G97

7038

A.

ver

onii

bv. s

obria

66

H

G97

7039

A.

ver

onii

bv. s

obria

13

1 H

G97

7040

A.

ver

onii

bv. s

obria

21

8 H

G97

7041

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-- 2

38 --

Tab

le 7

.9

Con

tinue

d.

Gen

e Sp

ecie

s St

rain

no.

A

cces

sion

no.

Sp

ecie

s St

rain

no.

A

cces

sion

no.

aexT

A.

ver

onii

bv. s

obria

22

4 H

G97

7042

A.

dha

kens

is

31

HG

9770

43

A.

dha

kens

is

230

HG

9770

44

A. d

hake

nsis

32

H

G97

7045

A.

med

ia

85

HG

9770

46

A. v

eron

ii bv

. sob

ria

237

HG

9770

47

alt

A. a

ustr

alie

nsis

26

6 H

G97

7048

A.

cav

iae

10

3 H

G97

7049

A.

cav

iae

18

8 H

G97

7050

A.

cav

iae

26

4 H

G97

7051

A.

cav

iae

20

0 H

G97

7052

A.

dha

kens

is

31

HG

9770

53

A.

dha

kens

is

67

HG

9770

54

A. d

hake

nsis

60

H

G97

7055

A.

dha

kens

is

183

HG

9770

56

A. d

hake

nsis

18

0 H

G97

7057

A.

dha

kens

is

32

HG

9770

58

A. d

hake

nsis

25

6 H

G97

7059

A.

dha

kens

is

223

HG

9770

60

A. d

hake

nsis

22

9 H

G97

7061

A.

dha

kens

is

235

HG

9770

62

A. d

hake

nsis

24

1 H

G97

7063

A.

dha

kens

is

242

HG

9770

64

A. h

ydro

phila

34

H

G97

7065

A.

hyd

roph

ila

61

HG

9770

66

A. h

ydro

phila

59

H

G97

7067

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-- 2

39 --

Tab

le 7

.9

Con

tinue

d.

Gen

e Sp

ecie

s St

rain

no.

A

cces

sion

no.

Sp

ecie

s St

rain

no.

A

cces

sion

no.

alt

A. ja

ndae

i 25

3 H

G97

7068

A.

jand

aei

262

HG

9770

69

A.

ver

onii

bv. s

obria

26

9 H

G97

7070

A.

ver

onii

bv. s

obria

24

7 H

G97

7071

A.

ver

onii

bv. s

obria

11

1 H

G97

7072

A.

ver

onii

bv. s

obria

21

8 H

G97

7073

ascV

A.

allo

sacc

haro

phila

10

0 H

G97

7074

A.

aus

tral

iens

is

266

HG

9770

75

A.

dha

kens

is

256

HG

9770

76

A. d

hake

nsis

22

3 H

G97

7077

A.

dha

kens

is

220

HG

9770

78

A. v

eron

ii bv

. sob

ria

27

HG

9770

79

A.

ver

onii

bv. s

obria

24

7 H

G97

7080

A.

ver

onii

bv. s

obria

13

1 H

G97

7081

ast

A. h

ydro

phila

23

H

G97

7082

A.

hyd

roph

ila

149

HG

9770

83

A.

hyd

roph

ila

243

HG

9770

84

A. v

eron

ii bv

. sob

ria

27

HG

9770

85

A.

cav

iae

103

HG

9770

86

A. c

avia

e 21

6 H

G97

7087

A.

cav

iae

270

HG

9770

88

A. m

edia

17

9 H

G97

7089

A.

jand

aei

262

HG

9770

90

A. v

eron

ii bv

. sob

ria

269

HG

9770

91

A.

ver

onii

bv. s

obria

12

5 H

G97

7092

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-- 2

40 --

Tab

le 7

.9

Con

tinue

d.

Gen

e Sp

ecie

s St

rain

no.

A

cces

sion

no.

Sp

ecie

s St

rain

no.

A

cces

sion

no.

flaA

A. b

estia

rum

68

H

G97

7093

A.

dha

kens

is

60

HG

9770

94

A.

dha

kens

is

67

HG

9770

95

A. d

hake

nsis

18

3 H

G97

7096

A.

dha

kens

is

176

HG

9770

97

A. d

hake

nsis

22

9 H

G97

7098

A.

hyd

roph

ila

92

HG

9770

99

A. h

ydro

phila

69

H

G97

7100

A.

hyd

roph

ila

261

HG

9771

01

A. h

ydro

phila

15

1 H

G97

7102

A.

hyd

roph

ila

231

HG

9771

03

A. c

avia

e 96

H

G97

7104

A.

ver

onii

bv. s

obria

23

7 H

G97

7105

A.

ver

onii

bv. s

obria

21

5 H

G97

7106

lafA

A.

dha

kens

is

95

HG

9771

07

A. d

hake

nsis

10

4 H

G97

7108

A.

dha

kens

is

220

HG

9771

09

A. m

edia

17

9 H

G97

7110

A.

ver

onii

bv. s

obria

26

9 H

G97

7111

A.

ver

onii

bv. s

obria

12

5 H

G97

7112

A.

ver

onii

bv. s

obria

66

H

G97

7113

A.

cav

iae

158

HG

9771

14

A.

cav

iae

109

HG

9771

15

A. c

avia

e 14

3 H

G97

7116

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-- 2

41 --

Tab

le 7

.9

Con

tinue

d.

Gen

e Sp

ecie

s St

rain

no.

A

cces

sion

no.

Sp

ecie

s St

rain

no.

A

cces

sion

no.

lafA

A.

hyd

roph

ila

101

HG

9771

17

A. h

ydro

phila

34

H

G97

7118

A.

hyd

roph

ila

260

HG

9771

19

A. h

ydro

phila

13

3 H

G97

7120

aspA

A.

aus

tral

iens

is

266

HG

9771

21

A. d

hake

nsis

56

H

G97

7122

A.

dha

kens

is

230

HG

9771

23

A. d

hake

nsis

10

7 H

G97

7124

A.

hyd

roph

ila

34

HG

9771

25

A. h

ydro

phila

26

1 H

G97

7126

A.

hyd

roph

ila

69

HG

9771

27

A. h

ydro

phila

84

H

G97

7128

A.

hyd

roph

ila

149

HG

9771

29

A. h

ydro

phila

92

H

G97

7130

A.

jand

aei

262

HG

9771

31

A. sa

lmon

icid

a 19

9 H

G97

7132

A.

ver

onii

bv. s

obria

27

H

G97

7133

A.

ver

onii

bv. s

obria

25

9 H

G97

7134

A.

ver

onii

bv. s

obria

21

8 H

G97

7135

vasH

A.

dha

kens

is

31

HG

9771

36

A. d

hake

nsis

15

4 H

G97

7137

A.

dha

kens

is

70

HG

9771

38

A. d

hake

nsis

67

H

G97

7139

A.

jand

aei

35

HG

9771

40

A. ja

ndae

i 25

3 H

G97

7141

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- 242 -

The primers used in the detection of aerA/haem can amplify several related genes which

encode toxins with a variety of names including aerolysin, aerolysin-haemolysin,

haemolysin-aerolysin, haemolysin, and cytolytic enterotoxin, hence the generic term

aerolysin-haemolysin genes (Soler et al. 2002). The prevalence of aerA/haem in A.

veronii bv. sobria detected in all (100%) isolates tested was also reported by Aguilera-

Arreola et al. (2007).

The prevalence of the ascV (16%) and aexT (13%) genes was low, consistent with other

reports (Aguilera-Arreola et al. 2005; Puthucheary et al. 2012; Senderovich et al. 2012).

In this study, these genes were more often detected in environmental than in clinical

isolates (ascV (39 vs. 8%; p < 0.0004; aexT 26 vs. 9%; p < 0.0295). Braun et al. (2002),

exclusively detected aexT in A. salmonicida ssp. salmonicida but not in other

Aeromonas spp. while Chacón et al. (2004) detected ascV and aexT in all intestinal and

extra-intestinal A. hydrophila and A. veronii isolates but only in a few extra-intestinal A.

caviae isolates. Based on these results, it appears that the distribution patterns of the

T3SS genes are strain and source dependent. The prevalence of the aspA gene (29%)

was low compared with the high frequency (75 to 77%), reported by Chacón et al.

(2003) and Puthucheary et al. (2012) who evaluated the distribution of virulence genes

and molecular characterization of Aeromonas species from Spain and Malaysia,

respectively. However, the prevalence of aspA in A. hydrophila (52%) isolates was

similar (58%) to the study by Aguilera-Arreola et al. (2005).

Lateral flagella (lafA) play an important role in cell adherence, invasion and biofilm

formation (Gavin et al. 2003). The presence of both genes (the flaA+lafA+ genotype) has

been associated with intense biofilm formation (Santos et al. 2010), a characteristic

feature of persistent infections. The frequency of the lafA gene (51%) was similar to the

overall frequency (60%) reported in mesophilic aeromonads by Gavin et al. (2003). In

other studies (Aguilera-Arreola et al. 2005, 2007; Senderovich et al. 2012), the

frequency of the lafA gene ranged from 37 to 41% although in one study (Aguilera-

Arreola et al. 2005), lafA was detected in 84% of A. hydrophila isolates. On the other

hand, the prevalence of the flaA (32%) gene was low compared to the range 59 to 74%

reported by others (Sen and Rodgers 2004; Puthucheary et al. 2012; Senderovich et al.

2012). In a recent study, flaA (94%) and lafA (71%) were highly prevalent in A. caviae

isolated from water, food and human faeces (Santos et al. 2010).

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-- 243 --

No amplification products were detected for the virulence genes BfpA, BfpG, stx-1, and

stx-2. These virulence genes are rarely investigated and their prevalence among

Aeromonas from other locations needs to be evaluated. Sechi et al. (2002) detected the

BfpG gene in four out of 46 A. hydrophila isolates collected from water samples in

Sardinia, Italy. By contrast, BfpA was not detected in any isolate, consistent with results

from this study. There have been few reports of Aeromonas strains producing a Shiga-

like toxin or carrying the encoding genes (Haque et al. 1996; Alperi and Figueras 2010).

One such gene, stx-l, is plasmid-mediated and it is possible that in this study, strains

carrying the stx-1 may have been lost during storage. It is also possible that due to the

fact that primer design is based on the nucleotide sequence of one species, species-

specific variations in the gene sequences of the species evaluated resulted in failure to

amplify providing false negative results.

The vasH (Sigma 54-dependent transcriptional regulator) gene is a relatively recent

addition to the arsenal of virulence factors described in Aeromonas spp. Together with

vasK the gene is a component and/or is essential for expression of the T6SS. These

genes were found in the T6SS of the diarrhoeal isolate A. hydrophila SSU and in A.

hydrophila ATCC 7966T (Suarez et al. 2008). In the present study, vasH was detected

primarily among environmental (48%, 15/31) rather than in clinical (19%, 19/98)

strains.

Results from this study reveal that among the major species, A. hydrophila and A.

dhakensis contain more strains that possess multiple virulence genes compared to other

clinically relevant species like A. caviae and A. veronii bv. sobria. On the other hand,

strains from A. allosaccharophila and A. jandaei also harbour many virulence genes

suggesting that in Aeromonas the pathogenic potential may be strain rather than species

related. In the present study not many virulence genes were detected in A. caviae.

However, other studies suggest that this species should be considered an enteric

pathogen capable of harbouring several virulence determinants including the production

of a cholera-like and a Shiga-like toxin (Haque et al. 1996; Mokracka et al. 2001;

Alperi and Figueras 2010). It is also possible that variations in gene sequences are

responsible for lack of amplification in A. caviae.

This raises the question of how many and what virulence genes are essential for an

Aeromonas strain to cause infection. In general, pathogens should possess the necessary

virulence genes to gain entry, adhere, colonize, causing damage in host tissue while

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-- 244 --

evading the host defence mechanisms, and in some cases spread, leading to systemic

infection. In Aeromonas, multiple virulence factors most likely work in concert (Yu et

al. 2005) where the product of one gene may facilitate the action of other genes or act

synergistically (Albert et al. 2000). Some authors observed that combinations or subsets

of virulence factors can be found among different isolates responsible for a wide range

of infections (Sen and Rodgers 2004; Puthucheary et al. 2012). Virulence genes such as

aerA, hlyA, alt, ast, act are thought to contribute to enteritis-related virulence (Janda and

Abbott 2010) while the severity of the diarrhoea has been associated with the number

and type of enterotoxin genes present (Albert et al. 2000; Chopra et al. 2009).

Enterotoxigenic aeromonads possessing both the alt and ast genes may represent true

diarrhoeal pathogens (Albert et al. 2000) although this hypothesis has not been

supported by others (Aguilera-Arreola et al. 2007) who suggested that aerolysin-

haemolysin may be sufficient to cause diarrhoea particularly in patients colonized with

A. caviae or A. veronii. The latter would explain the production of diarrhoea found

among patients from the present study infected with these species and lacking either alt

or ast. Moreover, aerA/haem and lafA are among the most predominant virulence genes

present in isolates from intestinal specimens suggesting that these genes may play an

essential role in the pathogenesis of aeromonads isolated from these sites. In this study,

with the exception of two cases, Aeromonas was the only recognized enteric pathogen

and no parasitic or mixed infections were recorded (Table 7.2).

The variable percentage identity found between the sequences of selected strains

compared to sequences deposited in GenBank for the nine genes has been previously

reported by others. The ASA1 protein secreted by the A. sobria 33, a human isolate and

the ASH3 produced by the fish isolate A. salmonicida 17-2 were found to be 66%

identical with aerolysin (Table 1.8) (Hirono et al. 1992; Hirono & Aoki 1993). On the

other hand, the cytotonic enterotoxin (Alt) produced by the human diarrhoeal isolate A.

hydrophila SSU showed 45 to 51% identity with phospholipase/lipase (Chopra et al.

1996). These results suggest that Aeromonas can produce a variety of extracellular

products that may be unique to specific strains. This is not surprinsing considering that

some Aeromonas strains can produce several enzymes with different biological

properties (Wretlind and Heden 1973; Honda et al. 1985; Howard and Buckley 1985;

Kozaki et al. 1987).

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-- 245 --

The virulence genes investigated in this study represent a subset of the many virulence

factors described in Aeromonas, and the roles of only some of these genes have been

defined in the pathogenesis of aeromonads (Chopra et al. 2009). In this study, the only

gene found to be significantly more common in clinical than in environmental isolates

was lafA. Recently, Grim et al. (2013) used a combination of whole genome-sequence

and phenotypic assays to compare the virulence potential between two A. hydrophila

strains isolated from a patient with a polymicrobial wound infection. The more virulent

isolate harboured genes encoding for act, T3SS, flagella, haemolysins, capsule and a

homolog of exotoxin A found in Pseudomonas aeruginosa. The isolate was also lethal

to mice injected with a dose of 1 x 107 CFU. Thus a virulent pathotype of A. hydrophila

has now been identified and further genomic analysis is likely to reveal more distinct

pathotypes within the genus.

In this Chapter, 129 genotypically-characterized WA Aeromonas isolates of clinical and

environmental origin were examined for 13 putative virulence determinants to add to

the current body of knowledge on virulence-associated characteristics of Aeromonas.

This is the first study of this kind in Australia. Results from this study showed that the

distribution of these genes varies from strain to strain irrespective of the species and

source of isolation. Furthermore, this study reinforces the clinical relevance previously

attributed to A. dhakensis (as A. aquariorum or A. hydrophila ssp. dhakensis), a species

known to possess many virulence genes (Figueras et al. 2009; Sedláček, et al. 2012;

Puthucheary et al. 2012). Moreover, although clinical isolates belonging to A.

hydrophila and A. dhakensis can harbour many virulence genes, not all strains do so.

Genomic comparisons combined with phenotypic studies appear to be a suitable and

practical approach for the identification of virulent pathotypes in Aeromonas.

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-- 246 --

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-- 247 --

CHAPTER 8: GENERAL DISCUSSION

This thesis consists of several peer-reviewed publications in which the phenotypic,

genotypic, antimicrobial susceptibility profiles and the presence of several virulence

factors were investigated in a collection of Aeromonas isolated from human clinical

material, various water sources and fish samples. In addition, the taxonomic position of

an isolate recovered from irrigation water was investigated by extensive phenotypic and

genotypic testing leading to the proposal of a novel Aeromonas species.

Despite the ubiquitous nature of Aeromonas, a genus that has been associated with

infections in warm and cold-blooded animals including humans for more than a hundred

years, the lack of an animal model of infection has undermined the significance of this

genus as a true human pathogen. The failure of aeromonads to fulfil Koch’s postulates

has led bacteriologists to consider these bacteria opportunistic microorganisms rather

than recognized bona fide pathogens. This is highly surprising considering the

devastating impact that infection with these bacteria has caused to the aquaculture and

other related industries resulting in enormous financial loss (Kodjo et al. 1997; Nash et

al. 2006). In the past, the complex taxonomy of the genus undermined an understanding

of the potential pathogenic significance of Aeromonas, and their distribution. However,

the introduction of molecular methods has facilitated a more accurate differentiation of

the species. As a consequence, the real distribution of Aeromonas in all environments is

starting to emerge.

Therefore, the aims of this thesis were:

1. To determine the identity and distribution of local clinical and environmental

Aeromonas isolates by phenotypic and genotypic methods.

2. To introduce novel phenotypic methods and revisit older ones with the aim

to find new biochemical markers.

3. To examine the antimicrobial susceptibilities of local clinical and

environmental isolates.

4. To identify isolates with uncertain taxonomic positions

5. To investigate the presence of selected virulence genes among local clinical

and environmental isolates.

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-- 248 --

Classification of Aeromonas isolates

This study began with the phenotypic classification of 199 Aeromonas isolates from

various clinical and environmental sources. Identification was based on a scheme

comprising more than 60 biochemical and physiological assays (Abbott et al. 2003).

Novel tests were introduced to find additional biochemical markers for an improved

identification. Overall, most isolates (93%) were identified to species level. Among the

clinical isolates, A. hydrophila (52.2%), A. caviae (19.0%) and A. veronii bv. sobria

(14.5%) accounted for 92% of the total isolates. This is in accordance with other studies

where together these species usually account for > 85% of the clinical isolates for this

genus (Altwegg and Geiss 1989; Abbott et al. 2003). Among water isolates, A.

hydrophila (46%) was the most common species followed by A. veronii bv. sobria

(22%). The high frequency of isolation of A. hydrophila supports the notion that the

frequency with which various species occur in clinical and environmental specimens,

are probably due to differences in the virulence potential of the strains (Janda et al.

1984; Barer et al. 1986; Kuijper et al. 1989b). It may also explain the reason why this

species has been the most studied aeromonad (Figueras 2005).

Earlier studies used numerical taxonomic techniques in combination with a large

number of biochemical characters to identify Aeromonas. However, no study was able

to characterize every isolate tested (Bryant et al. 1986a; Renaud et al. 1988; Kaznowski

et al. 1989; Käempfer and Altwegg 1992) reflecting the phenotypic homogeneity within

the genus. Nevertheless, in some studies, phenotypic identification in combination with

numerical taxonomy was able to produce discrete phenotypic clusters allowing the

recognition of two novel species (Miñana-Galbis et al. 2004, 2007). In this study,

identification of Aeromonas to the species level using biochemical methods was fraught

with difficulties including the low positivity rate of some tests, interpretation of end-

points, and the low number of strains representing environmental and infrequently

isolated species. Moreover, the introduction of novel tests in this study failed to provide

useful phenotypic markers further confirming that the identification of Aeromonas by

phenotypic methods is unreliable and some isolates are likely to be misidentified or

cannot be assigned to any definitive taxon (Figueras et al. 2007b; Ghatak et al. 2007b).

Following phenotypic classification, the genetic relationships of all isolates were

determined from gyrB and rpoD gene sequences. As a result, 99.5% of the strains re-

identified were placed in a taxon compared to 93% by the previous method. The new

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-- 249 --

distribution indicated that in WA A. caviae, A. dhakensis, A. hydrophila and A. veronii

bv. sobria were the most prevalent species in clinical specimens accounting for 96% of

the total isolates. Moreover, the frequency of these species among human clinical

material was very similar with A. veronii bv. sobria (25%) slightly more prevalent than

A. caviae and A. dhakensis (both at 23.8%) and A. hydrophila (23%), respectively.

Thus, the difference in the frequency of isolation of A. hydrophila from clinical and

environmental specimens fell significantly from 52 to 19% (p < 0.0001) after genotypic

identification. These results provide strong evidence that the distribution of Aeromonas

species largely correlates with the identification method employed. The high prevalence

of A. dhakensis in this study has been reported in recent studies suggesting that this

species is globally distributed in clinical specimens (Figueras 2005; Puthucheary et al.

2012; Wu et al. 2012).

Misidentification of isolates may also explain the phenotypic heterogeneity previously

associated with A. hydrophila, A. caviae and A. veronii (Miyata et al. 1995; Graf 1999a;

Korbsrisate et al. 2002; Abbott et al. 2003). It is also possible that among Aeromonas

species different ecotypes capable of exploiting a specific ecological niche exist.

Ecotypes have been described among strain that exhibit higher than 99% average

nucleotide identity (ANI) although the gene content of strains of the same species can

vary up to 30%. This difference begs the question of whether these strains should

belong to the same species (Konstantinidis and Tiedje 2005). Future studies designed to

compare the gene content between clinical and environmental isolates using ANI as a

tool may be forthcoming. Thus, this study contributes to an important knowledge about

the frequency of Aeromonas species in WA indicating that a more accurate distribution

of the genus is beginning to emerge.

The description of Aeromonas australiensis sp. nov.

In Chapter 4, the position of strain 266 inferred from the gyrB and rpoD gene sequences

showed that this isolate formed a separate line of descent from all other species in the

genus. Furthermore, the inability of the strain to produce acid from D-mannitol was

significant as most species in the genus do so. Subsequent extensive phenotypic and

genotypic testing confirmed that strain 266T indeed represented a novel Aeromonas

species (Aravena-Román et al. 2013). Proposing new species based on a single strain

has been a source of controversy among bacteriologists. This situation has led some

authors to recommend that the Bacteriological Code be revised and that a minimum

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number of standard tests and strains should be included in the description of new

species (Christensen et al. 2001; Janda and Abbott 2002) of which genotypic methods

should be mandatory (Figueras et al. 2006). However, there are a few drawbacks with

these recommendations. Firstly, it may take a very long time to collect the minimum

number of strains recommended from geographically and epidemiologically unrelated

areas. Secondly, strains may be lost in storage or simply forgotten in culture collections.

Thirdly, sequences from nearly every bacterial species have been placed on GenBank

and are readily available for comparison. The latter point is reinforced by the recent

isolation of A. simiae following a survey to determine the prevalence of Aeromonas in

slaughterhouses in northern Portugal. The strain was isolated among 703 isolates and

was identified on the basis of 16S rDNA, gyrB and rpoD sequencing (Fontes et al.

2010). Aeromonas simiae was first described on the basis of two strains isolated from

faeces of healthy monkeys (M. fascicularis) from Mauritius (Harf-Monteil et al. 2004).

A second study recently reported that A. taiwanensis constituted 6% of the Aeromonas

species isolated from diarrhoeal stools in Israel. In this study, identification of the

isolates was based on the sequences of the rpoD gene (Senderovich et al. 2012). The

original description of A. taiwanensis was based in a single strain recovered from an

infected burn wound of a 40 year-old male from Taiwan (Alperi et al. 2010b).

These findings suggest that A. australiensis may be isolated by others in future studies.

Isolation of A. australiensis outside Australia would indicate a global distribution of the

species while isolation within Australia would suggest that the species is indigenous to

this region only. The discovery of A. australiensis from irrigation water is a significant

contribution to the understanding of the global distribution of this genus and adds to the

list of new aeromonads described in the last 14 years. This increasing number of new

Aeromonas species also coincided with the rapid increase of new bacterial species

described over the same period of time (Janda and Abbott 2010). Furthermore, the

recognition of a novel species reinforces the notion that accurate identification of these

bacteria must include a molecular approach.

Antimicrobial susceptibility

The antimicrobial susceptibility patterns of Aeromonas determined in this study indicate

that the number of multi-drug resistance strains found locally is extremely low. In

contrast to other reports, no Aeromonas strain isolated in WA was found to carry

resistance mechanisms such as ESBLs or the presence of MBLs (Rasmussen and Bush

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1997; Neuwirth et al. 2007; Libisch et al. 2008; Wu et al. 2012). All isolates tested in

this study were exquisitely susceptible to the fluoroquinolones ciprofloxacin and

norfloxacin (100%) while resistance to nalidixic acid was very low (3.1%). The latter

result is in sharp contrast with the high rates of resistance to nalidixic acid reported by

Rhodes et al. (2000) who observed resistance to nalidixic acid in 94% of human derived

and 52% of aquaculture aeromonads. Similarly, Figueira et al. (2011) reported

resistance to this antimicrobial agent in 90.6% of waste water and 17.6% of surface

water isolates. In Taiwan, resistant to fluoroquinolones is emerging where up to 14% of

Aeromonas showed tolerance to this antimicrobial class (Wu et al. 2007). On the other

hand, resistance to tetracycline in WA aeromonads is low (<6%) whereas reports from

Asia suggest that up to 49% of the isolates can be resistant to this antimicrobial class

(Chang and Bolton 1987; Ko et al. 1996).

Based on the low antimicrobial resistance exhibited by environmental aeromonads

consisting primarily of strains isolated from water samples it is safe to suggest that

water is not an ecological niche for resistance mechanisms in WA. By contrast, reports

from several locations reveal that multi-resistant Aeromonas strains can be found among

water and foods sources (Goñi-Urriza et al. 2000; Rhodes et al. 2000; Nawaz et al.

2010; Esteve et al. 2012). In one study, consumption of contaminated water was

implicated in serious infections caused by ESBL-producing Aeromonas (Rodríguez et

al. 2005). Furthermore, the high susceptibility nature of environmental strains to most

antimicrobial classes reported in this study suggests that clinical strains may act as a

potential reservoir for resistance mechanisms. This is consistent with previous

observations that suggested that resistant strains isolated from clinical samples may

release compounds into the environment and provide a source of constant selection that

maintains pressure for populations of resistant strains (Davies and Davies 2010). Thus,

results from this and other studies confirm that variations in the antimicrobial profiles

exist in Aeromonas strains isolated from different locations.

From the clinical point of view, the presence of aeromonads in human clinical material

may impact patient management as incorrect empirically therapy has been administered

in a significant number of cases involving Aeromonas (Scott et al. 1978; Vila et al.

2002; Bravo et al. 2003; Figueras 2005). The overall susceptibility profile of

Aeromonas was deemed to be stable during the decade mid-1980s to mid-1990s (Janda

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and Abbott 2010), a trend that appears to continue in this region as indicated by this

study.

There were 11 isolates with a multi-resistant profile. One was isolated from water, two

from diseased fish and the rest from human clinical material. Of these, only one

exhibited resistance to the aminoglycosides, 3rd generation chephalosporins, lower

concentration cefepime but was susceptible to meropenem, fluoroquinolones, amikacin

and high concentration of cefepime. The remaining resistant isolates were invariably

susceptible to the fluoroquinolones while the majority were also susceptible to the

aminoglycosides, meropenem and 3rd and 4th generation cephalosporins. From the

clinical point of view, clinicians still have more than one choice of antimicrobials at

their disposal to treat these resistant isolates. In conclusion, this research provides

significant information about the antimicrobial resistance patterns of local clinical

Aeromonas species and may guide clinicians to implement correct antimicrobial

therapy. That is, if Aeromonas spp. are suspected or proven, then antimicrobials such as

fluoroquinolones, aminoglycosides, carbapenems, 3rd and 4th generation cephalosporins

can be safely administered.

Distribution and significance of virulence genes

In this study, the pathogenic potential of 129 genotypically-characterized isolates

comprising 11 Aeromonas species was evaluated by detecting the presence of 13

virulence genes using a PCR-based method. Of these, 98 isolates were of clinical origin

and 31 derived primarily from water and fish samples. Aeromonas was the sole

aetiological agent in 60% of the cases while the remining 40% were isolated with

another pathogen or as part of a polymicrobial bacterial population. The majority (17,

85%) of the isolates recovered from stools were from symptomatic patients who had

watery diarrhoea or loose faeces and in some cases blood and leucocytes were present in

the specimen. These parameters are usually associated with gastroenteritis. Although no

clinical data was obtained in 31% of the clinical cases, Aeromonas was the only

microorganism isolated in most (26, 84%) while 5 (16%) cases were polymicrobial.

Strains isolated from fish derived mainly from diseased animals.

Overall, the majority of the isolates (96%) harboured at least one virulence gene

compared to 65% of the total isolates from another study (Kingome et al. 1999). The

number of virulence genes found in multidrug resistant isolates ranged from 1 to 4 with

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one isolate not included in the virulence study. These isolates were no more pathogenic,

in terms of virulence genes detected, than others in the study. Therefore, there was not a

relation between the most virulent strains and their antibiotic profile found. Results

from this and other studies from locations as diverse as Mexico, Spain, Bangladesh,

Italy, USA and Israel (Albert et al. 2000; Sechi et al. 2002; Sen and Rodgers 2004;

Aguilera-Arreola et al. 2007; Senderovich et al. 2012) indicate that the distribution of

virulence genes among the species is highly variable. Comparison between studies is

difficult due to the number of isolates tested, source of isolation, identification method

used to characterize isolates and choice of virulence genes (Sechi et al. 2002; Chacón et

al. 2003; Wu et al. 2007). Some studies were designed to evaluate the virulence

potential of different strains of the same species (Soler et al. 2002; Yu et al. 2005) while

others targeted the detection of a single virulence gene from several species (Chacón et

al. 2003; Yu et al. 2004). A recent study from Malaysia evaluated the pathogenic

potential of 94 genotypically-characterized clinical isolates comprising five species by

detecting the prevalence of 10 virulence genes (Puthucheary et al. 2012). Of these, only

six (aerA, alt, ast, flaA, aspA and aexT) virulence genes were common with those used

in this study. The prevalence of aerA and alt within the major species was remarkable

similar with the present study while the prevalence of the remaining four genes differed

significantly depending on the gene and the species.

In this study, aerA/haem was highly prevalent in WA isolates while the remaining

virulence genes were randomly distributed among the species. And although many

isolates harboured multiple virulence genes, not a single strain carried the full

complement of the 13 virulence genes. Several virulence genes including alt, aspA,

vasH, ascV and aexT were more prevalent in environmental rather than in clinical

isolates. These differences were statistically significant and suggested that

environmental isolates may represent a reservoir of potentially pathogenic strains. Any

discernible virulence pattern present is tenuous and evidence from this study does not

support that each species carried distinct sets of genes as reported by others (Kirov et al.

2002; Aguilera-Arreola et al. 2007; Puthucheary et al. 2012). In addition to A.

dhakensis and A. hydrophila, strains from A. allosaccharophila and A. jandaei also

harboured multiple virulence genes. The presence of multiple virulence genes or other

virulence factors in less frequently isolated species suggest that strains from these

species are potentially pathogen (Soler et al. 2002; Chacón et al. 2003; Senderovich et

al. 2012).

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Although no single or combination of virulence factors has been unequivocally

correlated to virulence in Aeromonas (Aguilera-Arreola et al. 2007), the presence of

T3SS and toxin genes in clinical strains would elevate Aeromonas to the same category

as the primary pathogens Y. enterocolitica, Salmonella enterica, enteropathogenic E.

coli and Shigella flexnery (Chacón et al. 2004). The high prevalence of aerA/haem

(81%) in clinical isolates suggests that strains possessing this virulence gene are

potentially pathogenic and may be diarrhoeagenic in vivo (Janda and Abbott 2010) as

both aerA and hlyA are considered virulence markers for Aeromonas (Heuzenroeder et

al. 1999; González-Serrano et al. 2002). Thus, despite the low number of virulence

genes detected among A. veronii bv. sobria isolates, the pathogenic potential previously

attributed to this species (Daily et al. 1981; Janda et al. 1985; Janda and Kokka 1991;

Kirov and Hayward 1993; Lye et al. 2007) should be maintained as every strain (100%)

harboured the aerA/haem gene. It is also possible that the action of this toxin alone may

account for the infectious process associated with strains harbouring aerA/haem in this

study. Similarly, while the frequency of isolation and clinical relevance previously

attributed to A. hydrophila has been overestimated (Figueras et al. 2009), isolation of

this species from serious human infections continuous to grow. In a recent report, A.

hydrophila was recovered from a posttraumatic brain abscess following a head injury

and was described as an aggressive pathogen (Mahabeer et al. 2014). Unfortunately, the

isolate was identified by a commercial system without further confirmation by a

molecular method. Nevertheless, this case reinforces the pathogenic potential attributed

to this species in particular and to Aeromonas in general.

The low number of virulence genes detected in A. caviae was consistent with previous

reports and has been one of the main reasons to consider this species less pathogenic

than A. veronii and A. hydrophila (Honda et al. 1985; Kirov et al. 1986; Majeed et al.

1990; Eley et al 1993; Martins et al. 2002). A lack of virulence genes is contrary to the

notion that the presence of high number of virulence genes is associated with a high

pathogenic potential among Aeromonas strains (Nawaz et al. 2010). However, growing

evidence suggests that A. caviae should be considered a bona fide pathogen. Firstly, A.

caviae strains can possess virulence factors considered to be significant in the

pathogenesis of Aeromonas-associated infections (Callister and Agger 1987; Gray et al.

1990; Namdari and Bottone 1990b; Deodhar et al. 1991; Singh and Sanyal 1992b;

Kirov and Hayward 1993; Shaw et al. 1995; Wang et al. 1996; Mokracka et al. 2001;

Ghatak et al. 2006; Krzymińska et al. 2003, 2011). Secondly, the pathogenic potential

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-- 255 --

of A. caviae is enhanced by animal passage suggesting that expression of virulence

genes may be reactivated in genes that were previously repressed (Singh and Sanyal

1992c; Krzymińska et al. 2003). Thirdly, the predominance of A. caviae in diarrhoeal

stools from neonates and children with gastroenteritis is further evidence that A. caviae

should be considered a true enteric pathogen (Altwegg and Geiss 1989; Namdari and

Bottone 1990b; Pazzaglia et al. 1990a; Moyer et al. 1991; Wilcox et al. 1992; Albert et

al. 2000; Rabaan et al. 2001; Bravo et al. 2012; Senderovich et al. 2012). Evidence now

exists for water-to-human transmission by members of the A. caviae-A. media group

(Khajanchi et al. 2010). Fourthly, A. caviae has been implicated in serious human

infections affecting immunocompetent individuals (Kumar et al. 2012). This would also

support the notion that to date, there is no consensus as to which virulence factor(s) is

the most critical for human infections (Chakraborty et al. 1987) and that a hierarchical

classification of virulence factors for Aeromonas does not exist or cannot, at this stage,

be established (Aguilera-Arreola et al. 2007).

Predicting virulence of Aeromonas isolates based on changes in transcription of c-jun

and c-fos in human tissue culture cells has been recently proposed (Hayes et al. 2009)

and although detection of virulence genes can be used to determine the pathogenic

potential of Aeromonas, this method only demonstrates that some virulence genes are

present in some strains but not in others. Instead, the study by Grim et al. (2013)

demonstrated that genotypic differences correlated with functional virulence factor

assays and allowed to identify a virulent pathotype of A. hydrophila capable of causing

wound infections in humans. This study offers several advantages over the detection of

virulence genes or the detection of virulence products by bioassays alone. Taken

together, these observations suggest that the combinations of methods used by Grim et

al. (2013) should be considered the standard method to evaluate the pathogenic

potential of Aeromonas species and that a library of truly pathogenic strains should be

created as previously proposed (Janda and Abbott et al. 2010).

CONCLUSIONS

The characterization of a large collection of clinical and environmental isolates indicate

that in Western Australia the species A. veronii bv. sobria, A. dhakensis, A. caviae and

A. hydrophila are the most prevalent. Characterization of isolates by genotypic methods

is also likely to identify less frequently isolated species including A. allosaccharophila,

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A. salmonicida, A. bestiarum, A. jandaei, A.media, A. schubertii and uncover potentially

novel species. From the clinical point of view, the antimicrobial susceptibilities

determined in this study provide clinicians with several choices of antimicrobials to

empirically initiate therapy if Aeromonas are suspected to be present. The detection of

clinical and environmental isolates harbouring multiple virulence genes among several

Aeromonas species contributes to the current knowledge on the virulence of these

bacteria. Finally, data from this and other studies suggest that the pathogenic potential

in Aeromonas is probably strain- rather than species-dependent.

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Evolutionary distances based on the percentage sequence dissimilarities of all current Aeromonas species and 60 isolates identified as A. dhakensis (A. aquariorum) using Clustal W and Mega 5 software (combined gyrB and rpoD dissimilarities). Numbers in brackets indicate strains with similar nucleotide sequences.

A. allosaccharophila (DSM 11576)A. aquariorum (CECT 7289) 9.8A. bestiarum (ATCC 51108) 10.0 9.2A. bivalvium (CECT 7113) 12.8 11.2 11.4A. caviae (ATCC 13136) 10.6 8.7 11.1 11.1A. encheleia (DSM 11577) 11.3 10.0 9.9 10.7 9.6A. eucrenophila (ATCC 23309) 10.8 8.5 10.4 11.0 9.0 7.0A. hydrophila (ATCC 7966) 9.4 4.6 9.2 12.7 8.8 10.0 8.9A. jandaei (ATCC 49568) 7.2 7.9 9.8 11.4 9.6 9.3 8.4 9.3A. media (ATCC 39907) 8.7 9.2 8.3 10.1 7.5 6.9 6.5 8.8 8.7A. molluscorum (DSM 17090) 15.3 12.8 12.7 10.8 13.4 11.0 11.7 13.5 13.6 12.0A. popoffii (CIP 105493) 10.7 9.5 4.3 11.5 12.6 10.8 10.2 10.4 10.5 10.1 11.7A. simiae (DSM 16559) 17.1 15.9 16.2 17.6 16.0 16.0 16.6 16.1 14.7 16.0 17.4 17.1A. sobria (CDC 9540-76) 8.2 10.2 10.7 13.2 11.4 12.0 11.2 10.1 8.8 11.0 13.5 11.4 17.7A. tecta (CECT 7082) 10.6 9.9 9.6 11.5 9.4 7.0 5.7 9.8 9.4 7.0 12.3 10.7 16.1 10.5A. trota (ATCC 49657) 8.9 10.2 10.6 12.2 9.4 11.8 10.4 10.3 8.9 10.0 13.6 11.4 16.0 10.6 11.0A. veronii bv. sobria (ATCC 9071) 3.5 10.3 10.6 12.5 10.2 10.2 10.7 9.8 7.4 9.5 14.6 11.3 17.0 8.4 10.3 8.6A. salmonicida ssp. salmonicida (CEC 10.6 10.4 6.9 11.8 11.7 10.3 10.0 10.0 10.7 10.2 12.5 8.0 17.7 10.5 9.6 11.5 10.5Aeromonas spp. HG11 (CECT 4253) 10.8 9.6 8.9 10.2 9.2 1.4 6.4 9.6 8.8 6.5 11.2 10.1 15.9 11.1 6.2 11.2 10.2 9.8A. piscicola (CECT 7443) 10.7 10.0 4.5 12.8 11.7 10.1 10.7 10.1 10.8 10.0 12.6 5.2 17.7 11.6 10.4 10.8 11.7 6.3 9.8A. rivuli (CECT 7518) 14.6 12.3 11.6 10.3 12.2 10.2 10.7 13.0 13.3 11.0 5.4 11.4 18.1 12.5 11.5 13.2 14.1 13.3 10.4 12.3A. fluvialis (CECT 7401) 5.3 9.5 10.3 12.8 11.0 10.6 11.1 9.8 8.4 10.3 15.0 11.5 17.6 7.7 10.6 9.4 5.5 11.3 10.4 11.8 13.6A. taiwanensis (CECT 7403) 11.6 10.8 12.8 11.5 6.6 10.6 9.9 10.6 10.8 8.5 13.6 12.8 16.7 12.4 10.8 10.3 11.3 13.2 10.2 13.4 13.0 12.3A. sanarellii (CECT 7402) 11.6 9.5 11.8 10.8 5.7 10.8 10.2 9.9 10.6 7.7 14.4 12.8 16.9 12.9 11.1 11.1 10.4 13.3 10.8 12.6 12.7 11.5 7.0A. diversa (CECT 2478) 16.6 16.0 16.9 18.0 15.5 16.4 16.7 15.5 16.6 15.5 19.1 18.5 11.1 18.1 17.4 17.4 17.7 17.6 15.9 18.0 18.3 17.6 15.9 16.8Isolate 31 9.6 1.4 9.3 11.2 8.8 10.2 8.6 5.0 8.0 9.3 12.9 9.9 16.0 10.0 10.1 9.8 10.2 10.5 9.6 10.6 12.0 9.6 11.3 10.0 16.4Isolate 32 9.9 1.2 9.3 11.0 8.5 9.4 8.7 5.0 8.7 8.7 13.0 9.8 16.1 10.3 10.0 10.2 10.4 10.1 9.3 10.0 12.0 9.6 10.4 9.5 16.3 1.6Isolates (47 95 139 165) 10.5 1.8 9.5 11.7 9.3 10.3 8.3 6.1 8.5 9.4 12.4 9.6 16.0 10.7 9.6 10.3 11.1 10.4 9.5 10.0 12.4 10.5 10.8 10.5 16.8 2.0 2.0Isolates (56 220) 9.5 1.8 9.2 11.5 8.7 9.9 8.6 4.4 8.4 9.1 13.2 10.2 16.8 10.1 9.8 10.2 10.2 10.3 9.6 9.8 12.0 9.4 10.7 9.8 16.6 2.0 1.8 2.6Isolate 60 9.2 1.2 9.4 11.0 8.3 10.2 8.2 4.4 8.2 8.9 13.0 10.0 16.2 9.6 9.4 9.9 10.0 10.1 9.9 10.2 11.8 9.4 10.3 9.4 16.1 1.7 1.6 2.3 1.1Isolate 67 9.9 1.1 9.8 11.7 8.3 10.4 8.4 4.5 8.6 9.3 13.6 10.3 16.0 10.4 9.6 10.0 10.6 10.5 10.0 10.3 12.6 9.9 10.6 9.5 16.1 1.6 1.5 2.4 1.8 1.1Isolate 70 10.6 1.7 10.1 11.9 9.4 10.5 9.4 4.9 9.1 9.6 13.9 10.8 16.8 10.8 10.5 10.8 10.8 11.0 10.5 10.8 12.9 10.4 11.5 10.1 17.0 2.3 1.7 2.8 1.7 1.7 1.8Isolate 71 9.3 1.6 9.3 11.6 8.9 10.5 8.6 5.2 8.4 9.5 13.2 9.9 16.1 10.3 10.1 10.4 10.4 11.0 9.9 10.5 12.3 9.4 11.4 10.2 16.6 1.0 2.0 2.2 1.8 1.7 1.4 2.3Isolates (73 74) 9.9 1.1 9.6 11.6 8.7 10.3 8.7 4.7 8.5 9.3 13.7 10.2 16.3 10.5 9.9 10.3 10.6 10.4 10.2 10.2 12.5 9.9 11.0 9.9 16.4 1.6 1.1 2.4 1.4 1.1 0.6 1.4 1.6Isolate 79 9.3 1.3 9.5 11.2 8.4 10.3 8.3 4.5 8.3 9.0 13.3 10.1 16.3 10.0 9.5 9.8 10.1 10.2 10.0 10.3 12.0 9.5 10.4 9.5 16.2 1.6 1.7 2.4 1.2 0.3 1.2 2.0 1.8 1.2Isolate 88 11.0 2.0 10.4 12.0 9.4 11.1 8.9 5.2 9.4 9.5 14.3 11.2 17.0 11.0 10.4 11.0 11.4 11.7 10.8 11.4 13.0 10.7 11.7 9.9 17.4 2.6 2.4 3.1 2.4 2.0 1.9 0.9 2.6 2.1 2.1Isolate 91 9.4 1.2 9.2 11.1 8.3 10.4 8.2 4.4 8.2 8.9 13.2 9.8 16.0 10.1 9.4 9.6 10.2 10.3 9.9 10.2 11.7 9.4 10.3 9.4 15.9 1.7 1.6 2.1 1.1 0.4 1.1 1.9 1.7 1.1 0.5 2.2Isolates (93 172) 9.9 2.0 9.6 11.7 9.4 10.3 8.9 5.2 8.7 10.1 12.7 10.2 16.8 10.8 10.2 10.7 10.6 10.7 9.6 10.3 12.3 9.8 11.9 10.5 16.8 2.0 2.6 2.5 1.6 1.9 2.4 2.3 1.4 2.4 2.0 2.6 1.9Isolate 104 10.0 1.7 9.3 11.7 8.7 10.3 8.6 4.7 8.4 9.5 13.2 10.1 16.3 10.4 10.0 10.4 10.3 10.7 9.5 10.1 12.3 9.9 10.8 9.9 16.3 2.3 2.5 2.5 0.9 1.6 1.9 2.4 1.9 2.1 1.7 2.7 1.4 1.5Isolate 107 9.8 1.7 9.5 11.8 8.9 10.7 8.4 5.3 8.6 9.8 13.4 10.1 16.3 10.6 10.1 10.1 10.6 11.1 9.9 10.6 12.3 9.6 11.3 10.2 16.4 1.3 2.1 2.1 1.9 1.8 1.3 2.4 0.5 1.7 1.7 2.5 1.6 1.5 1.8Isolate 121 10.0 1.3 9.8 11.6 9.0 10.3 8.9 4.8 8.8 9.5 13.4 10.1 16.8 10.3 10.3 10.4 10.5 10.7 10.2 10.6 12.6 9.8 10.7 10.1 16.4 1.7 1.7 2.7 2.1 1.7 1.6 2.2 1.9 1.6 1.6 2.1 1.7 2.9 2.4 2.2Isolates (123 124) 9.5 1.1 9.5 11.2 8.5 10.3 8.3 4.7 8.3 9.4 13.0 9.9 16.1 10.2 9.8 10.3 10.3 10.2 10.0 10.1 12.3 9.5 10.8 9.6 16.2 2.0 1.9 2.2 1.4 0.7 1.2 2.2 2.0 1.4 0.8 2.3 0.7 1.6 1.5 1.9 2.0Isolate 141 9.6 1.1 9.5 11.4 8.7 10.3 8.4 4.7 8.2 9.3 13.2 10.1 16.1 10.3 9.9 10.2 10.4 10.3 10.0 10.1 12.3 9.6 11.0 9.9 16.2 1.0 1.1 1.8 1.2 1.1 1.0 1.4 1.0 0.6 1.2 2.1 1.1 1.8 1.9 1.1 1.6 1.4Isolate 154 10.1 1.8 9.8 11.5 8.8 10.6 8.7 4.6 8.8 9.4 13.3 10.5 16.4 10.2 10.1 10.2 10.5 11.0 10.1 10.8 12.2 9.9 11.1 9.9 16.3 2.3 2.2 2.9 1.4 1.0 1.5 1.3 2.1 1.9 1.3 1.4 1.2 1.8 1.7 2.1 2.2 1.3 1.9Isolate 168 10.2 1.3 9.5 11.7 8.9 9.6 8.6 4.9 8.3 9.4 12.8 10.1 15.8 10.6 10.0 10.8 10.6 10.8 9.2 10.3 12.2 10.2 10.3 10.2 16.0 1.9 1.9 2.3 1.9 2.0 1.9 2.2 2.3 1.9 2.1 2.5 1.8 2.1 1.6 2.2 2.0 1.9 1.9 2.3Isolate 169 10.4 1.3 9.6 11.7 9.4 10.5 9.0 4.9 8.7 9.6 13.3 10.2 15.9 10.6 10.3 10.4 10.8 11.0 10.1 10.6 12.5 10.4 11.3 10.3 16.3 2.3 2.1 2.4 2.1 1.7 1.8 0.8 2.3 1.8 2.0 1.1 1.7 1.9 1.8 2.2 2.2 1.8 1.8 1.3 1.6Isolate 176 9.4 0.7 9.3 11.3 8.2 10.2 8.4 4.3 8.2 9.1 13.4 9.9 15.8 10.0 9.6 10.1 10.2 10.3 9.8 10.1 12.2 9.4 10.4 9.4 15.9 1.2 1.1 2.0 1.4 0.7 0.4 1.4 1.0 0.6 0.8 1.7 0.7 2.0 1.5 1.1 1.2 1.0 0.6 1.3 1.5 1.4Isolate 180 9.9 1.0 9.2 11.8 9.0 9.5 8.7 4.6 8.0 9.6 13.0 10.0 16.3 10.4 9.9 10.5 10.5 10.6 9.1 9.8 12.2 9.8 11.1 10.1 16.4 1.6 1.8 2.2 1.0 1.7 1.6 1.9 1.6 1.6 1.8 2.2 1.7 1.4 0.9 1.7 1.9 1.6 1.4 2.0 1.1 1.5 1.2Isolate 182 10.5 1.8 10.0 12.2 8.9 10.5 8.7 4.8 9.3 9.4 13.0 10.7 16.1 10.8 10.1 10.3 11.2 11.1 10.0 10.7 12.4 10.4 10.5 10.0 16.0 2.2 2.2 2.8 1.8 1.9 1.6 2.3 2.0 2.0 2.0 2.6 1.7 2.4 1.7 1.9 2.1 2.2 2.0 2.0 1.3 2.1 1.4 1.8Isolate 183 10.1 1.0 9.6 11.4 9.1 10.6 8.5 5.2 8.4 9.6 13.3 10.0 16.2 10.4 10.2 10.3 10.5 10.5 9.9 10.3 12.5 10.0 11.1 9.9 16.6 1.4 1.6 1.8 2.0 1.7 1.4 2.1 1.8 1.6 1.6 2.2 1.5 2.0 1.7 1.5 1.7 1.4 1.4 2.2 1.3 1.7 1.2 1.2 2.0Isolate 212 10.1 1.3 9.8 11.8 8.9 10.3 8.8 4.5 8.6 9.3 13.6 10.5 16.4 10.2 10.0 10.3 10.5 10.7 9.8 10.7 12.6 10.1 10.8 10.0 16.3 1.9 1.7 2.6 1.7 1.1 1.2 0.8 1.7 1.4 1.4 1.1 1.5 1.9 1.8 1.8 1.8 1.8 1.4 0.7 1.8 0.8 0.8 1.5 1.7 1.9Isolate 213 11.9 3.5 11.6 14.1 11.0 12.4 10.5 7.0 10.7 11.8 15.0 12.4 18.7 12.7 12.0 12.2 12.7 12.9 11.6 12.5 14.2 11.8 13.5 11.9 18.5 3.3 3.9 4.2 3.5 3.8 3.1 3.6 2.5 3.5 3.9 3.7 3.8 2.5 3.6 2.4 4.2 3.9 3.1 3.3 3.8 3.6 3.1 3.1 3.7 3.5 3.0Isolates (222 257) 10.3 2.1 10.1 12.3 9.6 10.7 9.1 5.3 9.1 10.3 13.4 10.6 17.1 11.1 10.4 10.8 11.1 11.3 10.1 11.1 12.7 10.2 11.9 10.5 17.4 1.7 2.5 2.8 2.1 2.4 2.3 2.2 1.1 2.3 2.5 2.3 2.4 1.1 2.4 1.4 2.0 2.7 1.7 2.2 2.4 2.2 1.9 1.7 2.5 2.3 1.8 2.2Isolates (223 232 240) 9.5 0.4 9.2 11.2 8.7 9.8 8.5 4.6 7.9 9.2 13.0 9.5 16.0 10.0 9.9 10.2 10.1 10.2 9.4 10.0 12.5 9.3 10.8 9.8 16.0 1.4 1.2 1.8 1.8 1.2 1.1 1.7 1.6 1.1 1.3 2.0 1.2 1.8 1.7 1.7 1.1 0.9 1.1 1.8 1.3 1.3 0.7 1.2 1.8 1.0 1.3 3.7 2.3Isolate 226 10.0 1.9 9.5 12.0 8.7 10.4 9.0 4.9 8.6 9.8 13.4 10.5 16.4 10.6 10.2 10.7 10.5 11.0 9.6 10.5 12.6 9.6 11.7 9.9 16.4 1.7 2.3 2.5 1.7 2.0 1.7 2.2 0.7 1.9 2.1 2.5 2.0 1.3 1.4 0.8 2.4 2.3 1.3 2.1 2.2 2.2 1.3 1.5 1.9 2.1 1.6 2.4 1.2 1.9Isolate 227 10.0 1.2 9.6 11.3 8.8 10.7 8.0 4.8 8.6 8.9 13.3 10.3 16.6 10.4 10.3 10.2 10.5 11.0 10.4 10.7 12.4 9.6 11.1 9.3 16.3 1.4 1.6 2.2 2.0 1.4 1.3 2.1 1.6 1.5 1.5 1.8 1.4 2.2 2.3 1.5 1.7 1.5 1.1 1.8 2.1 2.1 1.1 2.0 2.0 1.6 1.7 3.5 2.3 1.2 1.9Isolate 228 10.2 1.4 10.0 11.7 8.6 10.7 8.5 4.6 8.8 9.4 13.3 10.5 16.3 10.6 10.3 9.5 10.7 10.8 10.4 10.7 12.6 10.0 10.6 9.9 16.1 1.4 1.8 2.4 2.2 1.6 1.3 2.3 1.8 1.7 1.5 2.4 1.6 2.4 2.5 1.5 1.9 1.7 1.3 2.0 2.3 2.3 1.3 2.2 2.0 1.8 1.9 3.7 2.5 1.4 2.1 1.0Isolates (229 234) 9.3 1.2 9.2 11.1 8.2 10.2 8.2 4.4 8.2 9.1 13.2 9.8 16.2 9.9 9.6 10.0 10.0 10.4 9.9 10.1 11.7 9.2 10.5 9.3 16.1 1.6 1.6 2.2 0.8 0.5 1.0 1.9 1.4 1.2 0.6 2.0 0.5 1.6 1.3 1.3 1.7 0.6 1.2 1.0 1.9 1.9 0.8 1.4 1.8 1.4 1.5 3.3 2.1 1.2 1.7 1.2 1.4Isolates (230 236 249) 9.6 0.7 9.2 11.5 8.8 10.4 8.4 4.9 8.2 8.9 13.3 9.5 16.0 10.0 10.3 10.4 10.5 10.5 10.0 10.3 12.6 9.8 11.0 9.8 16.3 1.3 1.5 2.3 2.1 1.5 1.2 2.0 1.3 1.4 1.6 2.3 1.5 2.5 2.0 1.8 1.4 1.6 1.4 1.8 1.8 1.8 0.8 1.7 1.9 1.5 1.4 3.6 2.2 0.9 2.0 1.5 1.7 1.5Isolates (235 250) 9.3 1.5 9.3 11.6 8.7 10.3 8.6 4.9 8.2 9.5 13.2 10.1 16.3 10.3 9.9 10.4 10.4 10.6 9.8 10.2 12.3 9.2 11.4 9.9 16.4 1.3 1.9 2.0 1.7 1.6 1.5 2.2 0.5 1.5 1.7 2.5 1.6 1.3 2.0 0.6 2.0 1.9 0.9 2.3 2.2 2.2 1.1 1.5 2.1 1.7 1.8 2.6 1.2 1.5 0.6 1.5 1.7 1.3 1.8Isolate 239 9.3 1.5 8.9 11.3 8.5 9.9 8.4 4.1 8.2 9.1 12.9 9.9 16.6 9.9 9.5 10.0 10.0 10.3 9.4 9.6 11.8 9.2 10.5 9.5 16.3 1.7 1.9 2.5 0.3 1.0 1.5 1.8 1.5 1.5 1.1 2.1 1.0 1.3 0.6 1.6 1.8 1.3 1.3 1.3 1.6 1.8 1.1 0.7 1.5 1.7 1.4 3.2 1.8 1.5 1.4 1.7 1.9 0.7 1.8 1.4Isolate 241 9.9 1.6 9.8 11.5 8.6 10.6 8.0 4.8 8.5 9.2 13.5 10.3 16.4 10.6 10.0 10.3 10.7 10.7 10.1 10.4 12.4 10.0 11.1 9.5 16.6 1.4 2.0 2.1 1.8 1.7 1.4 2.3 1.2 1.6 1.8 2.0 1.7 2.0 2.1 1.3 2.1 2.0 1.0 2.4 2.3 2.3 1.2 1.6 2.2 1.8 1.9 3.3 1.9 1.6 1.5 1.2 1.4 1.4 1.9 0.9 1.5Isolate 242 10.0 1.4 9.8 11.3 8.6 10.8 8.0 4.8 8.4 9.4 13.3 9.8 16.8 10.4 9.6 9.9 10.5 10.6 10.3 10.7 12.3 9.6 10.8 9.6 16.9 1.8 1.8 2.6 2.0 1.6 1.3 2.1 1.8 1.5 1.7 2.0 1.4 2.6 2.3 1.7 1.7 1.5 1.3 2.0 2.3 2.1 1.1 1.8 2.2 1.6 1.7 3.7 2.3 1.4 2.1 1.6 1.8 1.4 1.7 1.7 1.7 1.6Isolate 244 10.3 1.1 9.8 11.8 9.2 10.3 8.6 4.7 8.4 9.3 13.4 10.3 16.2 10.5 10.0 10.3 10.7 10.7 9.6 10.7 12.6 10.3 11.0 10.2 16.3 2.1 1.9 2.2 1.9 1.3 1.6 1.0 2.1 1.6 1.6 1.3 1.5 1.7 1.6 2.0 2.0 1.6 1.6 1.1 1.4 0.4 1.2 1.3 1.9 1.5 0.4 3.4 2.0 1.1 2.0 1.9 2.1 1.7 1.6 2.0 1.6 2.1 1.9Isolate 251 9.8 1.2 9.0 11.2 8.6 10.6 8.3 4.5 8.6 8.8 13.3 9.5 16.4 10.2 9.9 9.9 10.3 10.4 10.1 10.2 12.0 9.5 10.6 9.6 16.2 1.8 1.6 2.4 1.6 1.0 1.3 2.1 2.0 1.5 0.9 2.0 0.8 2.2 1.7 1.7 1.5 1.1 1.5 1.4 1.9 1.9 1.1 2.0 1.8 1.2 1.7 3.9 2.7 1.2 2.3 1.1 1.4 0.8 1.5 1.9 1.3 2.0 1.6 1.7Isolate 255 9.8 0.9 9.2 11.4 8.5 10.2 8.4 4.5 8.3 9.3 12.7 9.8 16.0 10.3 9.6 9.8 10.4 10.2 9.6 9.9 12.3 9.6 10.7 9.9 16.0 0.9 1.5 1.7 1.7 1.4 1.1 2.0 1.1 1.3 1.5 2.3 1.4 1.9 2.0 1.2 1.6 1.7 0.7 2.1 1.8 2.0 0.9 1.3 1.7 1.3 1.6 3.2 1.8 1.1 1.4 1.1 0.9 1.3 1.4 1.0 1.4 0.7 1.5 1.8 1.5Isolate 256 9.9 1.5 9.4 11.6 8.5 10.6 9.0 5.1 8.3 9.9 13.9 10.2 15.9 10.3 10.0 10.6 10.2 11.1 10.2 10.5 12.7 9.8 11.0 9.6 16.6 1.7 1.9 2.5 1.7 1.6 1.3 1.8 1.3 1.5 1.7 2.1 1.6 2.3 1.4 1.4 2.0 1.9 1.3 2.1 2.2 1.8 0.9 1.5 2.1 1.7 1.6 3.4 2.2 1.5 1.2 1.9 2.1 1.3 1.6 1.4 1.4 1.5 1.7 2.0 1.9 1.4Isolate 258 10.1 2.2 10.1 12.4 9.4 11.1 9.1 5.6 8.9 10.3 13.8 10.8 17.1 11.0 10.4 11.0 11.2 11.4 10.5 11.0 12.9 10.0 12.2 10.6 17.3 2.0 2.6 2.7 2.4 2.3 2.0 2.9 1.2 2.0 2.4 3.2 2.3 2.0 2.7 1.3 2.7 2.6 1.6 3.0 2.9 2.9 1.8 2.2 2.8 2.4 2.5 2.1 1.9 2.2 1.3 2.2 2.4 2.0 2.5 0.7 2.1 1.6 2.4 2.7 2.6 1.7 2.1Isolate 263 9.9 1.3 9.4 11.6 8.7 10.3 8.7 4.7 8.5 9.1 13.2 10.0 15.5 10.3 10.2 10.0 10.6 10.7 10.1 10.5 12.5 9.9 10.7 10.0 16.2 1.1 1.7 2.3 2.3 1.7 1.2 1.8 1.1 1.6 1.8 2.1 1.7 2.5 2.4 1.6 1.6 2.0 1.2 2.0 2.2 1.8 1.0 2.1 1.9 1.9 1.6 3.6 2.2 1.3 1.8 1.3 1.1 1.7 1.0 1.6 2.0 1.3 1.9 2.0 1.7 0.8 1.4 2.3Isolate 278 10.2 1.5 9.8 11.7 8.9 9.5 8.8 4.9 8.7 9.3 13.2 10.5 16.4 10.6 10.2 10.8 10.6 10.6 9.5 10.3 12.2 10.1 10.5 10.2 16.4 1.7 1.3 2.7 1.3 1.8 1.7 1.6 2.1 1.3 1.9 2.3 1.8 2.3 2.0 2.2 1.8 2.1 1.3 2.1 0.6 2.0 1.3 1.3 1.3 1.7 1.6 3.6 2.2 1.5 2.0 1.9 2.1 1.7 1.8 2.0 1.4 2.1 2.1 1.8 1.9 1.6 2.0 2.7 2.0Isolate 279 10.0 1.4 9.6 11.5 8.6 11.0 8.3 4.8 8.5 9.4 13.7 10.0 16.6 10.6 9.8 10.0 10.5 10.6 10.2 10.6 12.5 10.0 10.8 9.8 16.7 1.5 1.8 2.3 1.9 1.4 1.3 2.1 1.7 1.3 1.1 2.0 1.0 2.5 1.8 1.4 1.5 1.7 1.1 2.2 2.0 1.9 0.9 1.7 2.1 1.3 1.7 3.8 2.2 1.4 1.8 1.8 1.8 1.5 1.7 1.6 1.6 1.5 1.0 1.7 1.4 1.4 1.4 2.3 1.9 2.0A. schubertii (ATCC 43700) 16.7 15.5 16.4 17.1 15.8 16.6 16.4 15.4 15.6 15.4 19.3 17.6 11.2 18.1 17.0 17.0 17.7 17.5 15.8 17.4 18.2 17.5 15.5 16.8 3.6 16.0 15.9 16.3 16.3 15.6 15.6 16.7 16.1 16.0 15.8 17.0 15.4 16.6 16.1 16.0 16.2 15.5 15.8 16.1 15.8 16.0 15.4 16.2 15.5 16.1 16.1 18.4 17.0 15.8 16.2 15.9 15.4 15.6 15.9 16.0 16.1 15.9 16.2 16.1 15.8 15.3 16.0 16.8 15.4 16.2 16.2

Page 339: Phylogeny, antimicrobial susceptibility and Classification ... · Phylogeny, antimicrobial susceptibility and virulence factors of Western Australian. By . Max Aravena-Román BScAppSci

Evolutionary distances based on the percentage sequence dissimilarities of current Aeromonas and strain 266T using Clustal_W and Mega 4 software (combined gyrB and rpoD dissimilarities)

A. piscicola (CECT_7443) A. rivuli (CECT 7518) 12.0A. bestiarum (ATCC 51108) 4.3 11.5A. popoffii (CIP 105493) 4.9 11.6 4.1A_salmonicida ssp. salmonicida (CECT 894) 6.1 13.0 6.6 7.6A. molluscorum (DSM 17090) 12.4 5.4 12.4 11.8 12.4A. media (ATCC 39907) 10.3 11.0 8.6 10.3 10.2 11.9A. encheleia (DSM 11577) 10.0 10.1 9.7 10.9 10.0 10.9 6.7A. eucrenophila (ATCC 23309) 10.8 11.0 10.4 10.4 9.5 11.8 6.3 6.8A. trota (ATCC 49657) 11.0 13.0 10.5 11.2 11.2 13.5 10.0 11.8 10.4A. sobria (CDC 9540-76) 11.2 12.4 10.3 11.2 10.2 13.2 10.8 12.0 11.4 10.3A. jandaei (ATCC 49568) 10.7 13.1 9.5 10.4 10.7 13.3 8.4 9.4 8.3 8.7 9.0A. allosaccharophila (DSM 11576) 10.5 14.3 9.5 10.2 10.1 14.8 8.2 11.2 10.4 8.8 7.6 6.8A. caviae (ATCC 13136) 12.2 12.2 11.5 12.9 11.9 13.2 7.7 9.6 9.0 9.5 11.2 9.4 10.3A. schubertii (ATCC 43700) 17.7 18.3 16.7 18.2 17.7 19.3 15.8 17.1 16.8 16.7 18.7 16.1 16.6 16.2A. simiae (DSM 16559) 17.9 18.0 16.6 17.3 17.9 17.1 16.1 16.1 16.6 15.8 18.2 15.0 16.8 16.1 11.7A. tecta (CECT 7082) 10.3 11.4 9.5 10.7 9.2 12.4 6.5 6.9 5.3 11.1 10.1 9.0 9.9 9.1 17.2 15.8A. bivalvium (CECT 7113) 12.4 9.9 11.1 11.2 11.2 10.3 9.7 10.5 10.7 11.6 13.1 11.1 12.3 10.7 16.9 17.4 11.1A. hydrophila (ATCC 7966) 10.1 12.9 9.2 10.3 9.6 13.1 8.8 10.2 9.0 10.4 10.1 9.2 9.4 8.8 15.6 16.3 9.5 12.5A. dhakensis (A. aquariorum) (CECT 7289) 10.1 12.4 9.3 9.6 10.2 12.8 9.4 10.2 8.7 10.2 10.4 7.9 9.7 8.8 15.7 16.2 9.9 11.1 4.8A. veronii bv. sobria (ATCC 9071) 11.6 13.7 10.2 10.8 10.3 14.4 9.1 10.3 10.3 8.7 7.8 7.2 3.3 9.9 17.7 16.7 10.0 12.2 9.7 10.3A. veronii bv.veronii (DSM 7386) 10.9 13.9 10.1 10.2 10.0 14.5 8.5 9.9 9.9 8.5 7.7 6.7 3.1 10.2 17.6 17.2 9.3 12.0 9.3 9.6 1.7A. taiwanensis (CECT 7403) 13.5 12.9 13.0 12.9 13.1 13.3 8.5 10.5 9.5 10.2 11.9 10.5 11.2 6.4 15.7 16.6 10.4 11.1 10.4 10.8 11.1 10.9A. sanarellii (CECT 7402) 12.6 12.4 11.8 12.8 13.2 13.9 7.5 10.8 9.9 11.2 12.8 10.4 11.5 5.5 17.1 17.1 10.4 10.3 10.1 9.9 10.2 10.8 6.9Strain 266 11.7 14.2 11.0 11.9 11.0 15.0 9.9 11.9 10.4 8.4 8.8 8.7 5.6 10.8 17.9 17.7 11.1 13.1 10.4 11.1 5.5 5.4 11.1 11.7A. diversa (CECT 4254) 13.1 12.8 11.7 13.2 12.2 13.9 8.5 5.5 9.6 12.6 13.1 10.7 12.0 10.8 12.8 16.5 9.2 12.4 11.0 11.2 12.0 11.9 11.5 12.3 13.2A. fluvialis (CECT 7401) 11.6 13.3 10.0 11.1 10.9 14.5 10.0 10.7 10.8 9.6 7.3 8.2 5.4 10.8 17.6 17.4 10.0 12.4 9.9 9.6 5.4 5.0 12.2 11.6 7.5 12.5Aeromonas spp. HG11 (CECT 4253) 9.6 10.3 8.7 10.1 9.4 11.1 6.3 1.5 6.2 11.1 11.0 8.8 10.8 9.2 16.2 16.0 6.1 10.0 9.9 9.9 10.3 9.9 10.1 10.8 11.9 4.2 10.4A. culicicola (CECT 5761) 11.4 13.8 10.5 11.1 10.2 14.8 9.2 10.7 10.2 9.2 8.1 7.2 3.7 10.2 18.0 17.7 10.3 13.2 10.0 10.8 1.7 2.3 11.5 10.8 5.1 12.4 5.6 10.7