detection of extended spectrum beta- lactamase and …
TRANSCRIPT
DETECTION OF EXTENDED SPECTRUM BETA-LACTAMASE AND AmpC BETA-LACTAMASE-PRODUCING
ENTEROBACTERIACEAE CLINICAL ISOLATES AND TESTING THEIR SUSCEPTIBILITY TO NOVEL
ANTIBIOTICS
Thesis
Submitted for partial Fulfillment of the Master Degree in Medical Microbiology and Immunology
By
Noha Yousry Elsayed (M.B.B.CH.)
Supervised by
Prof. Dr. Magda Ibrahim Ayoub
Professor of Medical Microbiology and Immunology Faculty of Medicine, Cairo University
Prof. Dr. Maisa Mohamed Omar
Professor of Medical Microbiology and Immunology Theodor Bilharz Research Institute
Dr. Alaa Mohamed Reda
Lecturer of Medical Microbiology and Immunology, Faculty of Medicine, Cairo University
Faculty of Medicine Cairo University
2015
To My Family
ABSTRACT
Extended spectrum β-lactamases (ESBLs) and AmpC β-lactamase are enzymes produced by a variety of Gram-negative bacteria which confer an increased
resistance to commonly used antibiotics and represent a substantial clinical threat. There are currently no standard diagnostic tests available for detection of AmpC. A
total of 100 Enterobacteriaceae isolates were collected. ESBL production was screened by using the disc diffusion test and was confirmed by the combination disc diffusion test, screening of AmpC production was done by cefoxitin disc test, disc
approximation test and confirmation was done by AmpC disc test. Isolates screened positive for ESBL were investigated for their susceptibility to novel antibiotics
(Temocillin, Tigecycline, Colistin and Doripenem) by E-test. Results showed that among 100 Enterobacteriaceae isolates, 45 were screened positive for ESBL-
production using the disc diffusion test and 36 were confirmed by the combination disc test. Nine isolates were screened for AmpC-production using the cefoxitin disc
test and 5 isolates were confirmed as AmpC producers by AmpC disc test Using MAST D68C set, 35 isolates were ESBL producers, 2 were AmpC producers, one isolate was both ESBL and AmpC producers. All isolates were found sensitive to
Tigecycline and Doripenem. Forty-three isolates were sensitive to Colistin, while for Temocillin, thirty-seven isolates were found sensitive. MAST D68C test appears to be a promising way to detect isolates producing ESBL and/or AmpC. Tigecycline, Doripenem, Temocillin and Colistin revealed excellent activity against ESBL an
KEY WORDS DETECTION OF EXTENDED SPECTRUM BETA-LACTAMASE AND AMPC BETA-LACTAMASE-PRODUCING ENTEROBACTERIACEAE CLINICAL ISOLATES AND TESTING THEIR SUSCEPTIBILITY TO NOVEL ANTIBIOTICS
To My Family
Acknowledgement First and foremost, I wish to express my sincerest thanks to
ALLAH for his care and generosity throughout my life
I wish to express my sincere appreciation and deepest gratitude to
Prof. Dr. Magda Ibrahim Ayoub, Professor of Medical Microbiology
and Immunology, Faculty of Medicine, Cairo University, for her
supervision, illuminating guidance, valuable instructions as well as her
support throughout this work.
I am deeply grateful to Prof. Dr. Maisa Mohamed Omar, Professor
of Medical Microbiology and Immunology, Theodor Bilharz Research
Institute, for her continuous help, support and direct supervision of the
work and for her fruitful thinking which was behind the progress of the
work.
My sincere thanks are due to Dr. Alaa Mohamed Reda, Lecturer of
Medical Microbiology and Immunology, Faculty of Medicine, Cairo
University, for her supervision, help and cooperation throughout this
work.
I am deeply indebted to Dr. Doaa Gamal Desouky, Lecturer of
Medical Microbiology and Immunology, Theodor Bilharz Research
Institute, for her kind help, encouragement, precious advice and great
support throughout the whole work.
Special thanks and gratitude to Dr. Inas El Defrawy, current head of
Microbiology Department, Theodor Bilharz Research Institute, for her
inspiring enthusiasm and sincere advice that guided me through my research.
I am also grateful to my colleagues in the Microbiology Department at
Theodor Bilharz Research Institute, for their kind help and support.
I would like to thank my beloved mother, father and my sister for their
support, love and faith in me throughout my life.
My warmest appreciation goes to my dear husband for his continuous
encouragement, sacrifices and for being there for me.
Special thanks to my little baby Candy.
Noha Yousry
2015
I
LIST OF CONTENTS
Pages
LIST OF ABBREVIATIONS I
LIST OF TABLES II
LIST OF FIGURES III
INTRODUCTION AND AIM OF THE WORK 1
REVIEW OF LITERATURE
CHAPTER I: BETA-LACTAM ANTIBIOTICS
CHAPTER II: BETA-LACTAMASES
CHAPTER III: EXTENDED SPECTRUM BETA-LACTAMASE
CHAPTER IV: AmpC BETA-LACTAMASES
CHAPTER V:ESBL AND AmpC CO-PRODUCTION
Chapter VI: INFECTION CONTROL MEASURES TO OVERCOME ESBL AND/OR AmpC-PRODUCING ORGANISMS
Chapter VII: NOVEL ANTIBIOTICS IN TREATING ESBL AND AmpC PRODUCING ENTEROBACTERIACEAE
MATERIALS AND METHODS
RESULTS
DISCUSSION
SUMMARY
CONCLUSIONS
RECOMMENDATIONS
REFERENCES
ARABIC SUMMARY
I
LIST OF ABBREVIATIONS
AK Amikacin
AmpC-R AmpC-mediated resistance
AmpR AmpC regulator
ASC Advanced spectrum cephalosporins
ATCC American type culture collection
ATM Aztreonam
BA Boronic acid
BES Brazilian extended-spectrum cephalosporinase
BD Becton Dickinson
BIL Bilal
BLI(s) β-lactamase inhibitor(s)
CA Clavulanic acid
CAD Cloxacillin agar dilution
CAM Cefoxitin-agar medium
CAMHB Cation-adjusted Mueller-Hinton broth
CAZ Ceftazidime
CIP Ciprofloxacin
CLSI Clinical Laboratory Standards Institute
Clox-DDST Cloxacillin double disk synergy test
CMY Cephamycin
CN E test with cefotetan
CNI E test with cefotetan +cloxacillin
CSB Cell Suspension buffer
CTX Cefotaxime
DDST Double-disk synergy test
DHA Dhahran hospital
DPT Disk-potentiation test
ELISA Enzyme-linked immunosorbent assay
ESBL(s) Extended-spectrum β-lactamase(s)
II
ESC Extended spectrum cephalosporins
FEP Cefepime
FDA Food and Drug Administration
FOX Cefoxitin
ICU Intensive care unit
IPM Imipenem
IRT Inhibitor resistant TEM
KB Kirby-Bauer
MBL(s) Metallo-β-lactamase(s)
MDDST Modified double disk synergy test
MHA Muller-Hinton agar
MIC(s) Minimum inhibitory concentration(s)
MIR Miriam Hospital
MOX Moxalactam
NaCl Sodium chloride
PABL(s) Plasmid-mediated AmpC β-lactamases enzyme(s)
pAmpC Plasmid-mediated AmpC
PBP(s) Penicillin-binding protein(s)
TBRI Theodor Bilharz Research Institute
TDT Three-dimensional test
TE Tris EDTA
TZP Piperacillin-tazobactam
UTI Urinary tract infection
VEB Vietnam extended-spectrum ß-lactamase
β-lactam Beta-lactam
β-lactamases Beta-lactamases
III
LIST OF TABLES
Table No. Table Title Page
Table 1: Groups and examples of β-lactam antimicrobial agents 6
Table 2: Classification schemes for bacterial β-lactamases 12
Table 3: Screening and confirmatory tests for ESBLs in Klebsiella pneumonia, Escherichia coli and Proteus mirabilis
20-
21
Table 4: Distribution of Enterobacteriaceae isolates recovered from
different clinical specimens.
60
Table 5: Results of screening and confirmatory tests used for
detection of ESBL-producing Enterobacteriaceae isolates in
relation to type of isolate
62
Table 6: Results of different methods used for detection of AmpC-
producing Enterobacteriaceae isolates in relation to type of
isolate.
65
Table 7: Results of different methods used for detection of ESBL-
producing Enterobacteriaceae isolates in relation to type of
isolate.
66
Table 8: Result of Mast D68C ESBL and AmpC detection set in
relation to type of isolate.
67
Table 9: Performance of Mast D68C set in detection of ESBL
production in relation to combined disc diffusion method in
the 45 isolates screened positive for ESBL
68
Table 10: Performance of Mast D68C set in detection of AmpC
production in relation to AmpC disc test in 9 isolates
screened positive for AmpC
69
Table 11: Antibiotic Susceptibility Patterns of bacterial isolates 71
Table 12: E-test result of novel antibiotics 74
IV
LIST OF FIGURES
Fig. No. Figure title Page
Fig. 1: Penicillin and most other β-lactam antibiotics act by
inhibiting penicillin-binding proteins, which normally catalyze
cross-linking of bacterial cell walls
7
Fig. 2: Mechanism of action of β-lactamases 13
Fig. 3: Metallo beta lactamase mechanism 13
Fig. 4: Scheme of AmpC induction process 26
Fig. 5: Three-dimensional extract test. Positive results in the form
of distortion in the cefoxitin inhibition zone near the agar
slits that contain extracts of the AmpC positive test isolate
36
Fig. 6: AmpC disk test results 37
Fig. 7: CAM assay: AmpC-positive extracts produce a zone of
growth around wells
38
Fig. 8: APB-DPT showing an increase in zone diameter in CAZ+APB
compared to CAZ only
40
Fig. 9: APB-broth microdilution test: an eightfold or greater
decrease in the MIC of CAZ with the addition of APB is
indicative of the production of class C β-Lactamases
41
Fig. 10: DDST showing synergy between cloxacillin containing disk
and both CAZ and CTX disks
42
Fig. 11: An AmpC positive isolate with cefotetan (CN) MIC 4,
cefotetan + cloxacillin CNI) MIC 0.5 (i.e. 8 folds reduction
in MIC)
39
Fig. 12: Screening for ESBL production by the disc diffusion test: 61
Fig. 13: Confirmation of ESBL production by the combined disc
method
62
Fig. 14: Screening for AmpC production by the cefoxitin disc test. 63
Fig. 15: Screening for AmpC production by disc approximation test. 64
Fig. 16: Positive AmpC disc test confirming AmpC production 64
Fig. 17: Modified double disc synergy test showing positive result
for ESBL production
65
V
Fig. No. Figure title Page
Fig. 18: Result of Mast D68C ESBL and AmpC detection set 67
Fig. 19: Detection of ESBL production by Mast D68C set showing
ESBL positive result
68
Fig. 20: Detection of AmpC production by the Mast D68C set
showing AmpC positive result
69
Fig. 21: Detection of ESBL and AmpC Co- production by the Mast
D68C set
70
Fig. 22: Tigecycline E-test showing a sensitive isolate 72
Fig. 23: Doripenem E-test showing a sensitive isolate 72
Fig. 24: Colistin E-test showing a sensitive isolate 73
Fig. 25: Temocillin E-test showing a resistant isolate 73
Introduction
1
INTRODUCTION
Enterobacteriaceae are major pathogens responsible for various
nosocomial and community acquired infections (Fraser et al., 2013).
Beta-lactam (β-Lactam) antibiotics are considered the cornerstone in
treating Enterobacteriaceae infections, this is due to their broad spectrum of
activity and safety profile (Jacoby and Munoz-Price, 2005).
The spread of extended spectrum beta-lactamases (ESBLs) and AmpC
among isolated Enterobacteriaceae from both community and health-care
settings reduces the reliability and effectiveness of β-lactam antibiotics as
options in therapy (Falagas and Karageorgopoulos, 2009).
Extended spectrum β-lactamase producing organisms have enzymes that
hydrolyze the β-lactam ring of β-lactam antibiotics like penicillins,
cephalosporins and monobactams but not cephamycins (cefoxitin and
cefotetan) and carbapenems rendering them ineffective. However, they are
inhibited by β-lactamase inhibitors (clavulanic acid, sulbactam and
tazobactam). The ESBLs are frequently plasmid encoded. Plasmids responsible
for ESBL production frequently carry genes encoding resistance to other
classes of antimicrobial drugs such as fluroquinolones, co-trimoxazole,
tetracyclines and aminoglycosides. Therefore, antibiotic options in the
treatment of ESBL-producing organisms are extremely limited (Paterson and
Bonomo, 2005).
Class C β-lactamases (AmpC) are cephalosporinases whose encoding
genes were presumed to be chromosomally-mediated. They have been
described in many bacterial species e.g. Citrobacter freundii, Enterobacter
spp., Hafnia alvei, Morganella morganii, Pseudomonas aeruginosa and
Serratia marcescens. In these genera, AmpC is inducible via a system
Introduction
2
involving 3 cellular proteins (AmpD, AmpG and AmpR). Escherichia coli also
possess AmpC gene which is normally expressed at a low level and is non-
inducible since AmpR is missing (Moland, 2008).
Since the late 1980s AmpC has disseminated on plasmids. Plasmid-
mediated AmpC represent a substantial clinical threat as their presence renders
the bacteria resistant to most β-lactams including cephamycins and β-lactam/β-
lactamase inhibitors combinations (Decré et al., 2002).
Transmissible plasmids have acquired genes for AmpC enzymes, which
consequently can now appear in bacteria poorly expressing a chromosomal
blaAmpC gene, such as E. coli, or lacking AmpC gene as K. pneumoniae, P.
mirabilis and Salmonella spp..
Techniques to identify AmpC β-lactamase production are still evolving
and are not yet optimized for the clinical laboratory, which probably now
underestimates the prevalence of this resistance mechanism (Jacoby, 2009).
Detection of AmpC in a strain with a coexisting ESBL is even more
challenging (Philippon et al., 2002). Also the presence of an ESBL can be
masked by the expression of an AmpC. So the co-existence of AmpCs and
ESBLs in the same strain may result in false negative tests for the detection of
ESBLs by the current CLSI criteria (Thomson, 2001).
Infections caused by such resistant organisms can increase the length of
hospital stay and result in intensive care unit (ICU) admission. Also
inappropriate treatment of these complex infections can increase mortality and
morbidity. Whereas, rapid detection of these enzymes allows for de-escalation
to more targeted therapy and it is also an important infection control issue;
therefore there is a requirement for a simple and reliable diagnostic test for
confirmation of AmpC and ESBL production (Naveen et al., 2013).
Introduction
3
Carbapenems are a class of antimicrobials that are structurally related to
penicillin They can usually be used to treat infections due to AmpC-producing
bacteria, but carbapenem resistance can arise in some organisms by mutations
that reduce influx (outer membrane porin loss) or enhance efflux (efflux pump
activation) (Jacoby, 2009).
Doripenem, the newest addition to the carbapenem class of antibiotics,
was approved by the Food and Drug Administration (FDA) to treat intra-
abdominal infections and urinary tract infections caused by ESBL and AmpC-
producing Enterobacteriaceae (Shanthi and Sekar, 2011).
Tigecycline had good activity against most ESBL-producing and AmpC-
hyperproducing Enterobacteriaceae, and may be a therapeutic alternative to
carbapenems in some infections caused by ESBL and AmpC-producing
isolates, many of which are also multiresistant to quinolones, aminoglycosides
and classical tetracyclines (Meagher et al., 2005).
Temocillin (the 6-α-methoxy derivative of ticarcillin) has been re-
launched in the UK. This modification increases stability to β-lactamases,
including AmpC and extended-spectrum types. It is a potential alternative to
carbapenems against the growing number of infections, particularly those of the
urinary tract, that are caused by ESBL producers and other cephalosporin-
resistant strains (Livermore et al., 2006).
Colistin, an intravenous formulation of a polymyxin, has fairly reliable
in vitro activity against the ESBL and AmpC β-lactamase–producing
Enterobacteriaceae, and it might be useful in the treatment of co-infection with
these organisms (Zohreh et al. 2013).