molecular analysis of a multi-resistant bovine pasteurella
TRANSCRIPT
University of Veterinary Medicine Hannover
Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut
Molecular analysis of a
multi-resistant bovine Pasteurella multocida
strain from the U.S.A.
THESIS
Submitted in partial fulfilment of the requirements for the degree of a
Doctor of Natural Sciences
- Doctor rerum naturalium -
(Dr. rer. nat.)
by
Geovana Brenner Michael, PhD
Ijuí, Brazil
Hannover, 2015
Supervisor: Apl. Prof. Dr. med. vet. Stefan Schwarz
1. Examiner: Apl. Prof. Dr. med. vet. Stefan Schwarz
Friedrich-Loeffler-Institut (FLI), Institut of Farm Animal
Genetics
2. Examiner: Apl. Prof. Dr. rer. nat. Ute Radespiel
Institute of Zoology, University of Veterinary Medicine
Hannover, Foundation
Date of oral examination: May 13, 2015
Geovana Brenner Michael, PhD was supported by the Gesellschaft der Freunde der
Tierärztlichen Hochschule Hannover e.V.
to
Tom element
“Foi muito bom:
temeremos menos,
compreenderemos mais e
se Deus for servido,
amaremos mais.”
João Ubaldo Ribeiro, Um Brasileiro em Berlin
Parts of this thesis have already been published:
KADLEC, K., G. B. MICHAEL, M. T. SWEENEY, E. BRZUSZKIEWICZ, H.
LIESEGANG, R. DANIEL, J .L. WATTS and S. SCHWARZ (2011):
Molecular basis of macrolide, triamilide, and lincosamide resistance in Pasteurella
multocida from bovine respiratory disease.
Antimicrobial Agents of Chemotherapy 55, 2475 - 2477
MICHAEL, G. B., K. KADLEC, M. T. SWEENEY, E. BRZUSZKIEWICZ, H.
LIESEGANG, R. DANIEL, R. W. MURRAY, J. L. WATTS and S. SCHWARZ (2012):
ICEPmu1, an integrative conjugative element (ICE) of Pasteurella multocida: analysis
of the regions that comprise 12 antimicrobial resistance genes.
Journal of Antimicrobial Chemotherapy 67, 84 - 90
MICHAEL, G. B., K. KADLEC, M. T. SWEENEY, E. BRZUSZKIEWICZ, H.
LIESEGANG, R. DANIEL, R. W. MURRAY, J. L. WATTS and S. SCHWARZ (2012):
ICEPmu1, an integrative conjugative element (ICE) of Pasteurella multocida:
structure and transfer.
Journal of Antimicrobial Chemotherapy 67, 91 - 100
MICHAEL, G. B.*, C. EIDAM*, K. KADLEC, K. MEYER, M. T. SWEENEY, R. W.
MURRAY, J. L. WATTS and S. SCHWARZ (2012):
Increased MICs of gamithromycin and tildipirosin in the presence of the genes
erm(42) and msr(E)-mph(E) for bovine Pasteurella multocida and Mannheimia
haemolytica.
Journal of Antimicrobial Chemotherapy 67, 1555 – 1557
* both authors contributed equally to this study
MICHAEL, G. B., C. FREITAG, S. WENDLANDT, C. EIDAM, A. T. FEßLER, G. V.
LOPES, K. KADLEC and S. SCHWARZ (2015):
Emerging issues in antimicrobial resistance of bacteria from food-producing animals.
Future Microbiology 10, 427 - 443
Further aspects have been presented at national or international
conferences as oral presentation or as posters:
MICHAEL, G. B., K. KADLEC, M. T. SWEENEY, E. BRZUSZKIEWICZ, H.
LIESEGANG, R. DANIEL, J. L. WATTS and S. SCHWARZ* (2011):
Molecular analysis of emerging antimicrobial resistance properties among bovine
Pasteurella multocida.
Proceedings of the 4th Symposium on Antimicrobial Resistance in Animals and the
Environment (ARAE), 27.-29.06.2011 in Tours, France. *Oral presentation
MICHAEL, G. B.*, K. KADLEC, M. T. SWEENEY, E. BRZUSZKIEWICZ, H.
LIESEGANG, R. DANIEL, J. L. WATTS and S. SCHWARZ (2011):
Whole genome sequencing of the multi-resistant Pasteurella multocida strain 36950.
Proceedings of the International Pasteurellaceae Conference (IPC), 24.-27.08.2011
in Elsinore, Denmark. *Oral Presentation
SCHWARZ, S.*, G. B. MICHAEL, K. KADLEC, M. T. SWEENEY, E.
BRZUSZKIEWICZ, H. LIESEGANG, R. DANIEL, and J. L. WATTS (2011):
Acquisition of antimicrobial resistance genes and mutations in Pasteurella multocida:
insights from the analysis of a multi-resistant strain.
Proceedings of the International Pasteurellaceae Conference (IPC), 24.-27.08.2011
in Elsinore, Denmark. *Oral presentation
MICHAEL, G. B., K. KADLEC, M. T. SWEENEY, E. BRZUSZKIEWICZ, H.
LIESEGANG, R. DANIEL, J. L. WATTS and S. SCHWARZ (2011):
Genetic basis of fluoroquinolone resistance in a bovine Pasteurella multocida isolate.
Proceedings of the International Pasteurellaceae Conference (IPC), 24.-27.08.2011
in Elsinore, Denmark. Poster 3
MICHAEL, G. B., K. KADLEC, M. T. SWEENEY, E. BRZUSZKIEWICZ, H.
LIESEGANG, R. DANIEL, J. L. WATTS and S. SCHWARZ (2011):
Genetic relatedness of bovine Pasteurella multocida and Mannheimia haemolytica
isolates carrying the resistance genes erm(42) and/or msr(E)-mph(E).
Proceedings of the International Pasteurellaceae Conference (IPC), 24.-27.08.2011
in Elsinore, Denmark. Poster 5
KADLEC, K., G. B. MICHAEL, M. T. SWEENEY, E. BRZUSZKIEWICZ, H.
LIESEGANG, R. DANIEL, J. L. WATTS and S. SCHWARZ (2011):
Identification of resistance gene cassettes in bovine Pasteurella multocida.
Proceedings of the International Pasteurellaceae Conference (IPC), 24.-27.08.2011
in Elsinore, Denmark. Poster 2
KADLEC, K., G. B. MICHAEL, M. T. SWEENEY, E. BRZUSZKIEWICZ, H.
LIESEGANG, R. DANIEL, J. L. WATTS and S. SCHWARZ (2011):
Genetic basis of macrolide, triamilide and lincosamide resistance in a bovine
Pasteurella multocida isolate.
Proceedings of the International Pasteurellaceae Conference (IPC), 24.-27.08.2011
in Elsinore, Denmark. Poster 4
MICHAEL, G. B., K. KADLEC, M. T. SWEENEY, E. BRZUSZKIEWICZ, H.
LIESEGANG, R. DANIEL, J. L. WATTS and S. SCHWARZ (2011):
Identification of an integrative and conjugative element (ICE) carrying twelve
resistance genes in Pasteurella multocida.
Proceedings of the 51st Interscience Conference on Antimicrobial Agents and
Chemotherapy (ICAAC), 17.-20.09.2011 in Chicago, USA. Poster C1-622
MURRAY, R. W. *, E. S. PORTIS, L. JOHANSEN, S. F. KOTARSKI, K. KADLEC,
G. B. MICHAEL, J. L. WATTS, and S. SCHWARZ (2011):
Genotypic characterization of selected resistant Mannheimia haemolytica and
Pasteurella multocida associated with bovine respiratory disease from the Pfizer
Animal Health Susceptibility Surveillance Program 1999-2007.
54th Annual meeting of American Association of Veterinary Laboratory Diagnosticians
(AAVLD)/ United States Animal Health Association (USAHA), 28.09.-05.10.2011 in
Buffalo, NY, USA. *Oral presentation
MICHAEL, G. B.*, K. KADLEC, M. T. SWEENEY, E. BRZUSZKIEWICZ, H.
LIESEGANG, R. DANIEL, R. W. MURRAY, J. L. WATTS and S. SCHWARZ (2012):
Identification and characterization of the integrative and conjugative element
ICEPmu1 from bovine Pasteurella multocida which carries and transfers 12
resistance genes.
Proceedings of the 3rd ASM Conference on Antimicrobial Resistance in Zoonotic
Bacteria and Foodborne Pathogens in Animals, Humans and the Environment, 26.-
29.06.2012 in Aix-en-Provence, France. *Oral presentation
EIDAM, C., G. B. MICHAEL, K. KADLEC, M. T. SWEENEY, R.W. MURRAY J. L.
WATTS and S. SCHWARZ (2012):
Elevated minimum inhibitory concentrations of tildipirosin and gamithromycin among
bovine Pasteurella multocida and Mannheimia haemolytica that carry the genes
erm(42) and/or msr(E)-mph(E).
Proceedings of the 3rd ASM Conference on Antimicrobial Resistance in Zoonotic
Bacteria and Foodborne Pathogens in Animals, Humans and the Environment, 26.-
29.06.2012 in Aix-en-Provence, France. Poster pp. 79 - 80
MICHAEL, G. B., M. T. SWEENEY, R. W. MURRAY, J. L. WATTS, S. SCHWARZ
and K. KADLEC (2014):
Structural variations in the resistance gene regions of the integrative and conjugative
element ICEPmu1 from bovine Pasteurella multocida and Mannheimia haemolytica.
Proceedings of the 7th International Conference on Antimicrobial Agents in Veterinary
Medicine (AAVM), 16.-19.09.2014 in Berlin, Germany. Poster p. 98
MICHAEL, G. B., C. EIDAM, M. T. SWEENEY, R. W. MURRAY, A. POEHLEIN, A.
LEIMBACH, H. LIESEGANG, R. DANIEL, J. L. WATTS, S. SCHWARZ and K.
KADLEC* (2014):
Integrative and conjugative elements (ICEs) conferring multi-resistance in bovine
Pasteurella multocida and Mannheimia haemolytica.
Proceedings of the 7th International Conference on Antimicrobial Agents in Veterinary
Medicine (AAVM), 16.-19.09.2014 in Berlin, Germany. *Oral presentation
MICHAEL, G. B., M. T. SWEENEY, R. W. MURRAY, J. L. WATTS, S. SCHWARZ
and K. KADLEC (2014):
Structural variations in the resistance gene regions of the integrative and conjugative
element ICEPmu1 from bovine Pasteurella multocida and Mannheimia haemolytica.
Proceedings of the 7th International Conference on Antimicrobial Agents in Veterinary
Medicine (AAVM), 16.-19.09.2014 in Berlin, Germany. Poster pp. 98 - 99
CONTENTS
Page
Chapter 1 Introduction .HHHHHHHHHHHHHHHHHHHHHHH 17
1.1. General characteristics of Pasteurella multocida $$$$$. 20
1.2. Diseases associated with P. multocida $$$$$$$$$.. 21
1.3. Antimicrobial resistance of P. multocida isolates HHHHH.. 22
1.4. Mobile genetic elements HHHHHHHHHHHHHHH... 25
1.5. Aims of the present doctoral thesis HHHHHHHHHHH. 29
References HHHHHHHHHHHHHHHHHHHHHHH.. 31
Chapter 2 Molecular basis of macrolide, triamilide, and lincosamide
resistance in Pasteurella multocida from bovine respiratory
disease HHHHHHHHHHHHHHHHHHHHHHHHH.
41
Chapter 3 ICEPmu1, an integrative conjugative element (ICE) of Pasteurella
multocida: analysis of the regions that comprise 12 antimicrobial
resistance genes HHHHHHHHHHHHHHHHHHHHH.
45
Chapter 4 ICEPmu1, an integrative conjugative element (ICE) of Pasteurella
multocida: structure and transfer HHHHHHHHHHHHHH.
49
Chapter 5 Increased MICs of gamithromycin and tildipirosin in the presence
of the genes erm(42) and msr(E)-mph(E) for bovine Pasteurella
multocida and Mannheimia haemolytica $$$$$$$$$$$.
53
Chapter 6 Emerging issues in antimicrobial resistance of bacteria from food-
producing animals HHHHHHHHHHHHHHHHHHHH..
57
Chapter 7 General discussion HHHHHHHHHHHHHHHHHHHH. 61
7.1. Molecular mechanisms of macrolide-triamilide resistance in
P. multocida 36950 HHHHH...HHHHHHHHHH.HH
63
7.2. Multi-resistance genotype of P. multocida 36950 HHHH.H
7.2.1. Resistance gene region 1 HHHHHHHHHHH.....
7.2.2. Resistance gene region 2 HHH..HHHHHHHH...
7.2.3. Resistance mediating mutations in P. multocida
36950 HHHHHHHHHHHHHHHHHHHH...
65
66
68
72
7.3. Multi-resistance mobile genetic element ICEPmu1 HHH.H.
7.3.1. Identification and general characteristics of ICEPmu1
7.3.2. Transfer of ICEPmu1 HHHHHHHHH..HHHH..
7.3.3. ICEPmu1-related elements HHHHHHHH..HHH
74
74
78
80
7.4. Additional features of the genome of P. multocida 36950 H..
7.4.1. General characteristics of the genome and genomic
comparison HHHHHHHHHHHHHHHHHH.
7.4.2. Putative virulence factors HHHHHHHHHHHH.
7.4.3. CRISPR systems in P. multocida 36950 HHHHH...
82
82
84
84
7.5. Concluding remarks HHHHHHHHHHHHHHHHH.. 85
References HHHHHHHHHHHHHHHHHHHHHHH. 88
Chapter 8 Summary HHHHHHHHHHHHHHHHHHHHHHHH.. 101
Chapter 9 Zusammenfassung HHHHHHHHHHHHHHHHHHHH. 107
Acknowledgements HHHHHHHHHHHHHHHHHHHHHHHHH 113
LIST OF ABBREVIATIONS
(cited in Chapters 1 and 7)
A. pleuropneumoniae Actinobacillus pleuropneumoniae
BRD bovine respiratory diseases
CDS coding sequence
CRISPR clustered regularly interspaced short palindromic repeats
CLSI Clinical and Laboratory Standards Institute
E. coli Escherichia coli
HGT horizontal gene transfer
H. somni Histophilus somni
ICE integrative and conjugative element
IS insertion sequence
LPS lipopolysaccharide
M. haemolytica Mannheimia haemolytica
MGE mobile genetic element
MIC minimal inhibitory concentration
NGS next-generation sequencing
P. multocida Pasteurella multocida
PMT Pasteurella multocida toxin
QRDR quinolone-resistance determining region
RefSeq reference sequence
Tn transposon
V. cholera Vibrio cholera
WGS whole genome shotgun sequencing
LIST OF TABLES AND FIGURES
(showed in Chapters 1 and 7)
Page
Table 1: Antimicrobial resistance genes identified in P. multocida HH 23
Table 2: Antimicrobial resistance genes, resistance-mediating
mutations and their associated resistance phenotypes in P.
multocida 36950 HHHHHHHHHHHHHHHHHHHH
73
Page
Figure 1: Comparative analysis of the resistance gene region 1 of P.
multocida 36950 HHHHHHHHHHHHHHHHHHHH
68
Figure 2: Comparative analysis of the resistance gene region 2 of P.
multocida 36950 HHHHHHHHHHHHHHHHHHHH
71
Figure 3: Circular plot of the genome of P. multocida 36950 HHHHH.. 75
Figure 4: Organization of ICEPmu1 $$$$$$$$$$$$$$$$ 76
Figure 5: Site-specific recombination of ICEPmu1 into the tRNALeu of
different strains HHHHHHHHHHHHHHHHHHHH..
79
Introduction Chapter 1
19
1. INTRODUCTION
Antimicrobial resistance is an ever evolving field in which the development and the
use of new antimicrobial agents is usually followed sooner or later by the occurrence
of bacteria that exhibit resistance to these antimicrobial agents. This applies not only
to antimicrobial agents that are used in human or veterinary medicine, but also to
those used in horticulture and aquaculture.
The introduction of newer antimicrobial agents, such as ceftiofur, florfenicol,
tilmicosin, tulathromycin and most recently tildipirosin and gamithromycin, during the
past two decades has dramatically improved the treatment options in bovine
respiratory disease (BRD). During the same time period, the implementation of
standardized susceptibility test methods and BRD-specific interpretive criteria has
substantially improved the ability to detect clinical resistance in the BRD pathogens.
Although overall levels of resistance to these newer antimicrobial agents are low in
Europe (HENDRIKSEN et al. 2008), recent data from the U.S.A. and Canada have
indicated the potential for emergence and dissemination of antimicrobial multi-
resistance in Pasteurella (P.) multocida and Mannheimia (M.) haemolytica from
cases of BRD in cattle (PORTIS et al. 2012). These data indicate the need for long-
term surveillance of antimicrobial resistance in the BRD pathogens and a better
understanding of the epidemiology of antimicrobial resistance in these pathogens.
Since the genetic basis of antimicrobial multi-resistance in the aforementioned
P. multocida isolates was unknown, this doctoral thesis project was conducted to
identify the resistance genes and resistance-mediating mutations in one
representative multi-resistant P. multocida strain by using whole genome sequencing
followed by functional cloning and expression of the newly identified resistance
genes as well as analysis of their transferability and association with a mobile genetic
element.
Chapter 1 Introduction
20
1.1. General characteristics of Pasteurella multocida
Pasteurella multocida was named after Louis Pasteur who identified this
bacterium in 1881 as the cause of fowl cholera and “multocida” in Latin means many
killing, i.e., pathogenic for many species (http://www.bacterio.net/pasteurella.html). It
is a Gram-negative, nonmotile, facultatively anaerobic bacterium that belongs to the
family Pasteurellaceae. P. multocida is a coccobacillus of 0.3 – 1.2 µm length that
does not form spores. It is oxidase-positive and catalase-positive, and can ferment
various carbohydrates. A typical bipolar staining with methylene blue can be seen in
smears taken from wounds or tissues rather than from cultures (HAGAN et al. 1988).
The species P. multocida is subdivided into the four subspecies multocida, gallicida,
septica and the recently described tigris (CAPITINI et al. 2002; HARPER et al. 2006).
Based on their capsular types, P. multocida isolates are currently classified into the
five serogroups A, B, D, E, and F (CARTER 1967; RIMLER and RHOADES 1987;
HARPER et al. 2012). Their further classification into 16 serotypes (1–16) is based
mainly on lipopolysaccharide (LPS) antigens using the Heddleston scheme
(CARTER 1955; HEDDLESTON et al.1972; HARPER et al. 2006).
P. multocida isolates possess a number of virulence factors including the
polysaccharide capsule and the variable carbohydrate surface molecule LPS. There
is a well documented association of the capsule type with particular hosts and
diseases. Fowl cholera is most commonly associated with P. multocida type A
strains, while haemorrhagic septicemia is caused only by P. multocida types B and E.
P. multocida from cases of atrophic rhinitis usually belong to type D (HARPER et al.
2012). P. multocida of capsular type F have been found in turkeys and other animals
(SHEWEN and RICE CONLON 1993; CATRY et al. 2005). In strains belonging to
serogroups A and B, the capsule has been shown to help resist phagocytosis by host
immune cells. In addition, capsule type A has also been shown to help resist
complement-mediated lysis (BOYCE and ADLER, 2000; CHUNG et al. 2001). A
study in a serovar 1 strain showed that a full-length LPS molecule was essential for
the bacteria to be fully virulent in chickens (HARPER et al. 2004). Strains that cause
atrophic rhinitis in pigs express the P. multocida toxin (PMT), the gene of which is
Introduction Chapter 1
21
located on a bacteriophage (PULLINGER et al. 2004). PMT is responsible for the
twisted snouts observed in infected pigs.
1.2. Diseases associated with P. multocida
P. multocida is considered as a zoonotic pathogen. Human infections are
commonly associated with bites, scratches, or licks of dogs and cats, more rarely
with bites of pigs. However, infections without epidemiologic evidence of animal
contact may also occur in humans. P. multocida is commonly found as a commensal
in the oropharyngeal microbiota of cats and dogs, but also in that of other animals.
As such, P. multocida is frequently isolated from cat bite abscesses in both cats and
humans (FRESHWATER 2008). One study on “bacteriological warfare among cats”
(LOVE et al. 2000) described the role of P. multocida and other bacteria in bite-
associated infections in cats. Another study reported that in 50 % of dog bites and in
75 % of cat bites the wound was contaminated with P. multocida (TALAN et al.
1999).
In animals, P. multocida is remarkable for the number and range of specific
disease syndromes with which it is associated, and for the wide range of host
species that are affected (WILKIE et al. 2012). P. multocida can act as a primary or
as a secondary pathogen in various animal species. As a primary pathogen – or at
least a pathogen that has the principal role in the disease process – P. multocida
causes haemorrhagic septicaemia in cattle and water buffaloes, septicaemia in other
ungulates, fowl cholera in poultry, atrophic rhinitis in pigs and snuffles in rabbits. As a
secondary pathogen, it is involved in a variety of diseases, in which P. multocida
makes a major contribution, although it requires other factors for the disease
condition to develop (WILKIE et al. 2012). Such diseases mainly include lower
respiratory tract diseases in ungulates, such as cattle and pigs, which then are
referred to as bovine respiratory disease (BRD) or swine respiratory disease (SRD)
(KEHRENBERG et al. 2006; SCHWARZ 2008; WILKIE et al. 2012).
BRD is one of the economically most important diseases in cattle. Global losses
of the feedlot industry due to BRD are estimated to be over $ 3 billion per year
Chapter 1 Introduction
22
(WATTS and SWEENEY 2010). BRD is a multi-factorial and multi-agent disease
which is often also called ‘shipping fever’. This designation refers to some of the
factors that play a relevant role in the development of the disease. Transportation
over long distances, often associated with exhaustion, starvation, dehydration,
chilling or overheating, serves as an important stress factor. Additional stress factors
include passage through auction markets, commingling of animals from different
herds, dusty environmental conditions in the feedlot and nutritional stress associated
with changes in diet. Initial viral infections may pave the way for subsequent bacterial
infections, in which besides P. multocida, also M. haemolytica, and Histophilus
somni, are important pathogens (DABO et al. 2007).
1.3. Antimicrobial resistance in P. multocida isolates
Antimicrobial agents are commonly used to combat P. multocida involved in
BRD and other infections. As a consequence, P. multocida has developed and or
acquired resistance to a wide range of antimicrobial agents. A summary of what has
been known in terms of antimicrobial resistance genes in P. multocida has been
published by KEHRENBERG et al. (2006). A further update was published by
SCHWARZ (2008) (Table 1).
Table 1 provides an overview about the antimicrobial resistance genes
identified in P. multocida, that confer resistance to the various classes of
antimicrobial agents. This overview presents the situation prior to the start of the
present doctoral thesis. Moreover, the location of the different genes as well as the
mechanism of resistance specified by them is listed. As can be seen from Table 1,
numerous antimicrobial resistance genes have been identified in P. multocida.
However, no genes conferring resistance to macrolides, such as tilmicosin or
tulathromycin, had been identified. In addition, no gentamicin resistance genes were
known in P. multocida. Finally, naturally occurring P. multocida isolates that exhibited
resistance to fluoroquinolones had also not been detected.
Introduction Chapter 1
23
Table 1: Antimicrobial resistance genes identified in P. multocida (Schwarz 2008)
Antimicrobial agents
Resistance mechanism
Resistance gene(s)
Location on MGEs
1 or
chromosomal DNA
Reference
Penicillins enzymatic inactivation blaROB-1 unknown Philippon et al. 1986
blaTEM-1 pFAB-1 Naas et al. 2001
blaPSE-1 pJR2 Wu et al. 2003
Tetracyclines active efflux (Major Facilitator Superfamily)
tet(H) pVM111; Tn5706
Hansen et al. 1993; Kehrenberg et al. 1998
tet(B) chromosomal Kehrenberg and Schwarz 2001a
tet(G) pJR1 Wu et al. 2003
tet(L) chromosomal Kehrenberg et al. 2005a
Tetracyclines target site protection (ribosome protective protein)
tet(M) chromosomal Chaslus-Dancla et al. 1995; Hansen et al. 1996
non-fluorinated phenicols
enzymatic inactivation (acetylation)
catA1 Plasmid 2 Vassort-Bruneau et al. 1996
catA3 Plasmid 2 Vassort-Bruneau et al. 1996
catB2 pJR2 Wu et al. 2003
all phenicols active efflux (Major Facilitator Superfamily)
floR pCCK381 Kehrenberg and Schwarz 2005b
kanamycin, neomycin
enzymatic inactivation (phosphorylation)
aphA1 pCCK3152 Kehrenberg and Schwarz 2005c
aphA3 pCCK411 Kehrenberg and Schwarz 2005c
Streptomycin enzymatic inactivation (adenylation)
strA-strB pPMSS1 Kehrenberg and
Schwarz 2001b
streptomycin/ spectinomycin
enzymatic inactivation (adenylation)
aadA1 pJR2 Wu et al. 2003
aadA14 pCCK647 Kehrenberg et al. 2005d
Trimethoprim target replacement (trimethoprim-resistant dihydrofolate reductase)
dfrA20 pCCK154 Kehrenberg and Schwarz 2005e
Sulfonamides target replacement (sulfonamide-resistant dihydropteroate synthase)
sul2 pPMSS1 Kehrenberg and Schwarz 2001b
1 MGEs: mobile genetic elements 2 not further specified plasmid
Most of the antimicrobial resistance genes detected in P. multocida were
located on plasmids or transposons. Usually, small non-conjugative plasmids were
detected which carried one or more antimicrobial resistance genes. Most often the
Chapter 1 Introduction
24
streptomycin resistance genes strA-strB were found together with the sulphonamide
resistance gene sul2. However, in plasmid pVM111, a Tn5706-like tetR-tet(H)
segment responsible for tetracycline resistance was found to be inserted between
sul2 and strA via illegitimate recombination and resulted in a sul2–tetR–tet(H)–strA–
strB multi-resistance gene cluster (KEHRENBERG et al. 2003).
Detailed structural analysis of the resistance plasmids showed that they were
composed of segments previously found in other bacteria. As such, the first
florfenicol resistance plasmid identified in P. multocida, pCCK381, harboured a floR
gene known from Enterobacteriaceae while its plasmid replication and mobilisation
genes corresponded to those on the Dichelobacter nodosus plasmid pDN1, whereas
other segments of pCCK381 were higly similar to the Vibrio salmonicida plasmid
pRVS1 (KEHRENBERG and SCHWARZ, 2005b). These findings suggested that
plasmid pCCK381 is the product of interplasmid recombination events.
Only a minority of the resistance genes identified in P. multocida seem to be
indigenous to this species. Among them are the trimethoprim resistance gene dfrA20
(KEHRENBERG and SCHWARZ, 2005e) and the streptomycin/spectinomycin
resistance gene aadA14 (KEHRENBERG et al. 2005d). These two resistance genes
have so far exclusively been found in P. multocida. In contrast, most of the
antimicrobial resistance genes found in P. multocida, such as sul2, strA, strB, floR,
catA1, catA3, aphA1, aphA3 or aadA1, have also been detected in a wide range of
other bacteria. This observation confirmed that P. multocida exchanges antimicrobial
resistance genes with other bacteria within and beyond the family Pasteurellaceae. In
the case of the tetracycline resistance gene tet(L), even an exchange with Gram-
positive bacteria has been assumed as the gene tet(L) is widely disseminated among
staphylococci, streptococci and enterococci (KEHRENBERG et al. 2005a).
Plasmids and transposons, that carry resistance genes, play a crucial role in
horizontal transfer events with P. multocida acting either as donor or as recipient of
antimicrobial resistance genes.
Introduction Chapter 1
25
1.4. Mobile genetic elements
Mobile genetic elements (MGEs) are DNA segments, considered as “natural
genetic engineers”, which code for at least proteins involved in their movement.
(HALL and COLLIS 1995; TOUSSAINT and MERLIN 2002; FROST et al. 2005).
MGEs are autonomous transposable elements and according to a revised
nomenclature may be defined as “specific DNA segments that can repeatedly insert
into one or more sites in one or more genomes” (ROBERTS et al. 2008). They may
be considered as selfish genetic elements in cases in which they promote their
spread without necessarily increasing their host’s fitness, but they may confer
beneficial or negative effects on their bacterial hosts. Their movement may be
restricted to the host genome (intracellular mobility) or occur between bacterial cells
(intercellular mobility). The intercellular mobility may involve different horizontal gene
transfer (HGT) mechanisms (e.g., transduction, conjugation or mobilization).
Insertion sequence (IS) elements are MGEs commonly found in the
chromosome or on plasmids. They are the smallest and most simple transposition
modules with sizes of ca. 0.6 – 2.5 kb and code only for proteins involved in their own
mobility. The ends of IS elements are characterized by short perfect or imperfect
inverted repeats of different lengths. The transposition occurs directly from one site to
another in the host genome (intracellular mobility) and there is no independent form,
as in case of bacteriophages or plasmids. IS elements have different degrees of
target-site specificity and may mediate insertions (e.g. the integration of plasmids into
the host chromosome), deletions, inversions and translocations in the host DNA. In
mobilization events, IS elements may also capture genes or regions of the host
chromosome and insert them into plasmids (PARTRIDGE 2011). Additionally, IS
elements may also have complete or partial promoter sequences which may drive
the expression of mobilized (or adjacent) genes, as the β-lactamase blaCTX-M-15 gene
overexpressed by the ISEcp1 element (PARTRIDGE 2011; TOLEMAN and WALSH
2011; CANTÓN et al. 2012). After the mobilization events, a composite structure
comprising the IS elements and the captured genes or regions is generated (IS-
mobilized DNA segment-IS) which is named composite transposon (formerly
Chapter 1 Introduction
26
transposons type I). The transposition of these composite transposons will be then
performed by one or both IS elements.
In contrast to IS elements, the elements named as transposons (Tn) code for
additional proteins that are not involved in their transposition. As mentioned before,
they may be a composite dependent of IS-type transposition modules (composite
transposon) or independent of IS (e.g. Tn3). These latter elements are considered as
units (also called unit transposons, formerly transposon type II/Tn3 family) which
carry genes for transposition and accessory genes (ROBERTS et al. 2008). They are
usually larger transposons (at least ca. 5 kb in size), have closely related terminal
inverted repeats and move by replicative transposition in the host genome
(intracellular mobility). In this transposition event, the following proteins are usually
involved: a transposase (encoded by a tnpA gene), a resolvase (encoded by a tnpR
gene) and a site of resolution (res site). As accessory genes, they commonly carry
antimicrobial resistance genes, e.g. the β-lactamase blaTEM gene encoded by Tn3
(HEFFRON et al. 1979).
Bacteriophages (phages) are virus-like organisms that infect bacteria and are
considered the most common microorganism in the biosphere. Noteworthy, phages
play an important role as MGEs, especially in the transfer of antimicrobial resistance
genes. The genome of phages may vary from ca. 2 kb to > 250 kb. The most
common phage particles contain a capsid (protein head) which surrounds the double-
stranded DNA and is attached to a tail (HATFULL and HENDRIX 2011). Phages may
undergo (i) a lysogenic cycle in which they integrate into the host genome (existing
as a prophage) by transposition or site-specific recombination (CAMPBELL 1992)
and replicate passively together with the host DNA, or (ii) a lytic cycle as an
autonomous form which will then be released by cell lysis. These released phage
particles may infect other cells by injecting their DNA into them (intercellular mobility).
This phage-mediated transfer of genetic information between a donor and a recipient
cell, without a direct contact between the cells, is named transduction, which – due
to the protection of the DNA by the capsid – is nuclease-resistant. In some cases,
host DNA (any sort of bacterial DNA, as chromosome fragments, plasmids,
transposons and IS elements) may be incorporated into the capsid of the phages and
Introduction Chapter 1
27
then be transferred from one host to another host cell (generalized transduction) or
both, phage DNA and fragments of bacterial DNA, may be incorporated into the
capsid (specialized transduction) (THOMAS and NIELSEN 2005).
Another process of intercellular mobility of DNA is the conjugation. However
for this, a direct contact (mating) between donor (F+) and a recipient cell (F-) and the
formation of a pore (mating pore) for the passage of the conjugative element are
necessary. Although there are also conjugative transposons, conjugation is the most
important process for the transfer of plasmids between bacteria under natural
conditions.
Plasmids are MGEs which are able to perform self-replication (independent
from the host chromosome) and may exist within the bacterial cell in an autonomous
form (extrachromosomal DNA). All plasmids have at least one origin of replication
and code for the proteins involved in the process of replication. The size of plasmids
may range from < 1 kb to several hundred kb. They may be inserted in part or
completely into the host chromosome, mostly by either homologous or site-specific
recombination. Some plasmids, the conjugative plasmids, are able to mediate their
own transfer from one cell to another. For this, they carry also genes directly involved
in their transfer and in the maintenance/stabilization of the contact between the
mating bacteria (SMILLIE et al. 2010). Many naturally occurring plasmids are either
conjugative (self-transmissible) or mobilizable (HALL and COLLIS 1995). The
transfer of a plasmid by mobilization may occur whether additional functions
necessary for the mating are present. Nevertheless, if the size of a plasmid is
compatible with the capsid size of a phage, this plasmid may be also transferred by
transduction. Beyond conjugation and transduction, the mechanism of
transformation may also play a role in the uptake of a plasmid by a recipient cell. In
contrast to conjugation, the transfer of DNA by transformation is not done by cell-to-
cell contact. Instead, transformation means the uptake of DNA that has been
released in the extracellular environment (naked DNA). Moreover, for the efficient
uptake of a plasmid or other free extracellular DNA, a recipient cell has to be in a
physiological state of competence (THOMAS and NIELSEN 2005).
Chapter 1 Introduction
28
These general descriptions and classification of MGEs as transposon, phages
or plasmids and their correlation with HGT mechanisms are important for a better
understanding of the biology of MGEs. However, this classification cannot be easily
applied to all MGEs, e.g. mobilizable (MTn) or conjugative transposons (CTn) in
which plasmid-related mobilisation or transfer functions are found. Since they are not
self-replicating, they cannot be classified as plasmids. For definition, plasmids are
maintained by their replication, but transposons by their integration in the host
genome and subsequent vertical dissemination during division of the host cell
(vertical transfer) (BURRUS et al. 2002a).
Tn916 from Enterococcus faecalis was the first element described as a
conjugative transposon, due to its ability to perform intracellular transposition and
conjugation. The intracellular transposition of this element is supported by the
mechanism of site-specific excision and a low specificity of integration (BURRUS et
al. 2002a). Other elements have been identified and some of them showed higher
target-site specificity than Tn916 or proved to be site-specific (site-specific integrative
and conjugative elements). In most cases, these elements (conjugative transposons
and site-specific integrative and conjugative elements) are able to integrate into a
unique site, e.g. genes encoding tRNAs, and cannot perform transposition to other
sites within the host genome. In this way, the site-specific integration systems of
these elements show more similarities to prophages than to transposons. For these
reasons, BURRUS and colleagues (2002a) proposed a new class of MGEs, named
as integrative and conjugate elements (ICEs), which includes “all elements that
excise by site-specific recombination into a circular form, self-transfer by conjugation
and integrate into the host genome, whatever the specificity and the mechanism of
integration and conjugation is. These elements would also be able to replicate during
the conjugation event, but this replication should not be involved in their
maintenance” (BURRUS et al. 2002a). According to this nomenclature, conjugative
transposons are ICEs able to transpose within the host genome. In the same way,
genomic islands may be also included into the ICE nomenclature and those that are
non-mobile may be considered as truncated or defective ICEs (WOZNIAK and
WALDOR 2010). Additional studies have suggested that some ICEs may be able to
Introduction Chapter 1
29
perform self-replication which has imposed more complexity on the nomenclature of
MGEs (WOZNIAK and WALDOR 2010).
In ICEs as well as in phages, the genes involved in the same function are
grouped in regions which are named as modules (e.g. the backbone modules:
recombination, conjugation and regulation) conferring a mosaic structure to the
elements. It has been suggested that genetic events involving insertion of MGEs into
another and deletions may lead to the acquisition or exchange of some modules and
may drive the modular evolution of ICEs. For example, the exchange or acquisition of
a transfer module may alter the host specificity of an ICE (OSBORN and BÖLTNER
2002; BURRUS et al. 2002b). ICEs are composed of core genes and accessory
genes (or cargo genes). While the core genes are more important for the spreading
and maintenance of the ICEs, the accessory genes, which may include genes for
antimicrobial, heavy metal or phage resistance but also metabolic activities, are
relevant for the fitness of the host and its survival under specific conditions. ICEs
have been considered an important driving force of bacterial evolution (MOHD-ZAIN
et al. 2004; SETH-SMITH and CROUCHER 2009; ROCHE et al 2010).
1.5. Aims of the present doctoral thesis
During recent years, multi-resistant P. multocida and M. haemolytica isolates
have been detected in the U.S.A. and Canada. These isolates also exhibited
resistance to florfenicol, macrolides, triamilides and fluoroquinolones – resistance
properties that had not been seen so far in these bacteria. Neither the genetic basis
of resistance to macrolides, triamilides and fluoroquinolones was known nor whether
these resistance properties were transferable.
The aims of the present doctoral thesis were
1. to perform the gap closure of the whole genome sequence of the
representative P. multocida strain 36950 and analyse the sequence to
identify the molecular mechanisms of the expanded multi-resistance
Chapter 1 Introduction
30
phenotype with particular reference to the macrolide-triamilide and
fluoroquinolone resistance, and
2. to characterize the genetic environment of resistance genes, with particular
reference to the macrolide-triamilide resistance, in order to:
a. identify a linkage between these genes and other resistance genes
which may enable the co-selection of macrolide-triamilide resistance
even in the absence of a direct selection pressure,
b. determine whether the resistance genes identified in P. multocida
36950 are located on mobile genetic elements, and
c. investigate the potential of dissemination of such resistance genes
located on mobile genetic elements (horizontal gene transfer).
3. to evaluate the in vitro activities of new macrolide antimicrobial agents
against P. multocida isolates
To investigate the role of putative genes in macrolide resistance, the whole
genome sequence of the representative P. multocida 36950 was determined and
analysed. Putative resistance genes were identified and cloning and expression
experiments were performed [Chapter 2].
To identify additional antimicrobial resistance genes, their physical linkage, and
resistance-mediating mutations responsible for the multi-resistance phenotype of this
strain, further sequence analysis and genomic comparisons of the whole genome of
P. multocida 36950 were carried out [Chapter 3]. Such analysis allowed also the
characterization of the genetic environment of all antimicrobial resistance genes
identified in P. multocida 36950 [Chapter 3].
To determine the transfer ability of the resistance determinates, conjugation
experiments were performed. Moreover, the functional activity of the resistance
genes in different recipient strains was also tested [Chapter 4].
In 2011, the 15-membered macrolide gamithromycin (Zactran®) and the 16-
membered macrolide tildipirosin (Zuprevo®) were approved for the treatment of BRD.
The newly identified macrolide and triamilide resistance genes erm(42) and/or
Introduction Chapter 1
31
msr(E)-mph(E) [Chapter 2] were investigated for their ability to also confer resistance
to gamithromycin and tildipirosin [Chapter 5].
In order to underline the importance of the new findings concerning the
molecular mechanism of resistance, especially of macrolide resistance, in P.
multocida and M. haemolytica from BRD, some of the issues discussed in chapters 2
– 6 were emphasised in a review [Chapter 6].
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41
Chapter 2
Molecular basis of macrolide, triamilide, and
lincosamide resistance in Pasteurella multocida
from bovine respiratory disease
Kristina Kadlec, Geovana Brenner Michael, Michael T.
Sweeney, Elzbieta Brzuszkiewicz, Heiko Liesegang, Rolf
Daniel, Jeffrey L. Watts and Stefan Schwarz
Antimicrobial Agents of Chemotherapy 55, 2475 - 2477 (2011)
doi: 10.1093/jac/dku385
http://jac.oxfordjournals.org/content/70/2/420.long
Chapter 2 Molecular basis of macrolide, triamilide, and lincosamide resistance
42
CONTRIBUTION TO THE ARTICLE
The extent of Geovana Brenner Michael’s contribution to the article is evaluated
according to the following scale:
A. has contributed to collaboration (0-33%).
B. has contributed significantly (34-66%).
C. has essentially performed this study independently (67-100%).
1. Design of the project including design of individual experiments: B
2. Performing of the experimental part of the study: B
3. Analysis of the experiments: B
4. Presentation and discussion of the study in article form: B
Molecular basis of macrolide, triamilide, and lincosamide resistance Chapter 2
43
ABSTRACT
The mechanism of macrolide-triamilide resistance in Pasteurella multocida has been
unknown. During whole-genome sequencing of a multiresistant bovine P. multocida
isolate, three new resistance genes, the rRNA methylase gene erm(42), the
macrolide transporter gene msr(E), and the macrolide phosphotransferase gene
mph(E), were detected. The three genes were PCR amplified, cloned into suitable
plasmid vectors, and shown to confer either macrolide-lincosamide resistance
[erm(42)] or macrolide-triamilide resistance [msr(E)-mph(E)] in macrolide-susceptible
Escherichia coli and P. multocida hosts.
45
Chapter 3
ICEPmu1, an integrative conjugative element (ICE) of
Pasteurella multocida: analysis of the regions that
comprise 12 antimicrobial resistance genes
Geovana B. Michael, Kristina Kadlec, Michael T. Sweeney,
Elzbieta Brzuszkiewicz, Heiko Liesegang, Rolf Daniel, Robert
W. Murray, Jeffrey L. Watts and Stefan Schwarz
Journal of Antimicrobial Chemotherapy 67, 84 - 90 (2012)
doi: 10.1093/jac/dkr406
http://jac.oxfordjournals. org/content/67/1/84.long
Chapter 3 ICEPmu1: analysis of the resistance gene regions
46
CONTRIBUTION TO THE ARTICLE
The extent of Geovana Brenner Michael’s contribution to the article is evaluated
according to the following scale:
A. has contributed to collaboration (0-33%).
B. has contributed significantly (34-66%).
C. has essentially performed this study independently (67-100%).
1. Design of the project including design of individual experiments: B
2. Performing of the experimental part of the study: C
3. Analysis of the experiments: C
4. Presentation and discussion of the study in article form: C
ICEPmu1: analysis of the resistance gene regions Chapter 3
47
ABSTRACT
Background: In recent years, multiresistant Pasteurella multocida isolates from
bovine respiratory tract infections have been identified. These isolates have exhibited
resistance to most classes of antimicrobial agents commonly used in veterinary
medicine, the genetic basis of which, however, is largely unknown.
Methods: Genomic DNA of a representative P. multocida isolate was subjected to
whole genome sequencing. Genes have been predicted by the YACOP program,
compared with the SWISSProt/EMBL databases and manually curated using the
annotation software ERGO. Susceptibility testing was performed by broth
microdilution according to CLSI recommendations.
Results: The analysis of one representative P. multocida isolate identified an 82 kb
integrative and conjugative element (ICE) integrated into the chromosomal DNA. This
ICE, designated ICEPmu1, harboured 11 resistance genes, which confer resistance
to streptomycin/spectinomycin (aadA25), streptomycin (strA and strB), gentamicin
(aadB), kanamycin/neomycin (aphA1), tetracycline [tetR-tet(H)], chloramphenicol/
florfenicol (floR), sulphonamides (sul2), tilmicosin/clindamycin [erm(42)] or tilmicosin/
tulathromycin [msr(E)-mph(E)]. In addition, a complete blaOXA-2 gene was detected,
which, however, appeared to be functionally inactive in P. multocida. These
resistance genes were organized in two regions of approximately 15.7 and 9.8 kb.
Based on the sequences obtained, it is likely that plasmids, gene cassettes and
insertion sequences have played a role in the development of the two resistance
gene regions within this ICE.
Conclusions: The observation that 12 resistance genes, organized in two resistance
gene regions, represent part of an ICE in P. multocida underlines the risk of
simultaneous acquisition of multiple resistance genes via a single horizontal gene
transfer event.
49
Chapter 4
ICEPmu1, an integrative conjugative element (ICE) of
Pasteurella multocida: structure and transfer
Geovana B. Michael, Kristina Kadlec, Michael T. Sweeney,
Elzbieta Brzuszkiewicz, Heiko Liesegang, Rolf Daniel, Robert
W. Murray, Jeffrey L. Watts and Stefan Schwarz
Journal of Antimicrobial Chemotherapy 67, 91 - 100 (2012)
doi: 10.1093/jac/dkr411
http://jac.oxfordjournals.org/content/67/1/91.long
Chapter 4 ICEPmu1: structure and transfer
50
CONTRIBUTION TO THE ARTICLE
The extent of Geovana Brenner Michael’s contribution to the article is evaluated
according to the following scale:
A. has contributed to collaboration (0-33%).
B. has contributed significantly (34-66%).
C. has essentially performed this study independently (67-100%).
1. Design of the project including design of individual experiments: C
2. Performing of the experimental part of the study: C
3. Analysis of the experiments: C
4. Presentation and discussion of the study in article form: C
ICEPmu1: structure and transfer Chapter 4
51
ABSTRACT
Background: Integrative and conjugative elements (ICEs) have not been detected in
Pasteurella multocida. In this study the multiresistance ICEPmu1 from bovine P.
multocida was analysed for its core genes and its ability to conjugatively transfer into
strains of the same and different genera.
Methods: ICEPmu1 was identified during whole genome sequencing. Coding
sequences were predicted by bioinformatic tools and manually curated using the
annotation software ERGO. Conjugation into P. multocida, Mannheimia haemolytica
and Escherichia coli recipients was performed by mating assays. The presence of
ICEPmu1 and its circular intermediate in the recipient strains was confirmed by PCR
and sequence analysis. Integration sites were sequenced. Susceptibility testing of
the ICEPmu1-carrying recipients was conducted by broth microdilution.
Results: The 82214 bp ICEPmu1 harbours 88 genes. The core genes of ICEPmu1,
which are involved in excision/integration and conjugative transfer, resemble those
found in a 66641 bp ICE from Histophilus somni. ICEPmu1 integrates into a tRNALeu
and is flanked by 13 bp direct repeats. It is able to conjugatively transfer to P.
multocida, M. haemolytica and E. coli, where it also uses a tRNALeu for integration
and produces closely related 13 bp direct repeats. PCR assays and susceptibility
testing confirmed the presence and the functional activity of the ICEPmu1-associated
resistance genes in the recipient strains.
Conclusions: The observation that the multiresistance ICEPmu1 is present in a
bovine P. multocida and can easily spread across strain and genus boundaries
underlines the risk of a rapid dissemination of multiple resistance genes, which will
distinctly decrease the therapeutic options.
53
Chapter 5
Increased MICs of gamithromycin and tildipirosin in
the presence of the genes erm(42) and msr(E)-
mph(E) for bovine Pasteurella multocida and
Mannheimia haemolytica
Geovana B. Michael*, Christopher Eidam*, Kristina Kadlec,
Kerstin Meyer, Michael T. Sweeney, Robert W. Murray, Jeffrey
L. Watts and Stefan Schwarz
Journal of Antimicrobial Chemotherapy 67, 1555 - 1557 (2012)
doi: 10.1093/jac/dks076
http://jac.oxfordjournals.org/content/67/6/1555.long
* both authors contributed equally to this study
Chapter 5 Increased MICs of gamithromycin and tildipirosin
54
CONTRIBUTION TO THE ARTICLE
The extent of Geovana Brenner Michael’s contribution to the article is evaluated
according to the following scale:
A. has contributed to collaboration (0-33%).
B. has contributed significantly (34-66%).
C. has essentially performed this study independently (67-100%).
1. Design of the project including design of individual experiments: B
2. Performing of the experimental part of the study: C
3. Analysis of the experiments: C
4. Presentation and discussion of the study in article form: C
Increased MICs of gamithromycin and tildipirosin Chapter 5
55
ABSTRACT
Background: Two new macrolides, gamithromycin and tildipirosin, have been
approved for the treatment of bovine respiratory disease (BRD) in 2011. The aim of
this study was to determine whether the recently identified ICEPmu1-associated
macrolide resistance genes erm(42) and msr(E)-mph(E) have an effect on minimum
inhibitory concentrations (MICs) of these two new macrolides.
Methods: Clones carrying the genes erm(42) and msr(E)-mph(E) and naturally
occurring Pasteurella multocida (n=32) and Mannheimia haemolytica isolates (n=22)
from BRD cases which carry the genes erm(42) and/or msr(E)-mph(E) were tested
for their MIC values of gamithromycin and tildipirosin.
Results: In the clone carrying erm(42), the MIC of tildipirosin increased 128-fold to
32 mg/L while that of gamithromycin increased only 16-fold to 4 mg/L. In the clone
carrying msr(E)-mph(E), an opposite observation was made: the MIC of tildipirosin
increased only 8-fold to 2 mg/L while that of gamithromycin increased 256-fold to 64
mg/L. P. multocida field isolates that carried all three genes showed MIC values of
16-64 mg/L for gamithromycin and 16-32 mg/L for tildipirosin while similar MIC values
of 32-64 mg/L for both macrolides were seen among the M. haemolytica field isolates
carrying all three resistance genes. The ten P. multocida isolates that carried only
erm(42) exhibited low MICs of 2-4 mg/L for gamithromycin but had higher MICs of
16-32 mg/L for tildipirosin. The single M. haemolytica that harboured only erm(42)
showed MIC values of 4 mg/L and 32 mg/L for gamithromycin and tildipirosin,
respectively. The two P. multocida isolates that carried only msr(E)-mph(E) exhibited
a high MIC of 32 mg/L for gamithromycin and a low MIC of 2 mg/L for tildipirosin.
Conclusions: The analysis of P. multocida and M. haemolytica field isolates from
BRD cases confirmed the results obtained with the cloned erm(42) and msr(E)-
mph(E) amplicons. Pronounced increases in the gamithromycin MIC values were
seen in the presence of msr(E)-mph(E) whereas distinct increases in the tildipirosin
MICs were detected in the presence of erm(42). Isolates that carry all three genes
showed elevated MICs to both new macrolides.
57
Chapter 6
Emerging issues in antimicrobial resistance of
bacteria from food-producing animals
Geovana B. Michael, Christin Freitag, Sarah Wendlandt,
Christopher Eidam, Andrea T. Feßler, Graciela Volz Lopes,
Kristina Kadlec and Stefan Schwarz
Future Microbiology 10, 427 - 443 (2015)
doi: 10.2217/fmb.14.93
http://www.futuremedicine.com/doi/abs/10.2217/fmb.14.93
Chapter 6 Emerging issues in antimicrobial resistance
58
CONTRIBUTION TO THE ARTICLE
The extent of Geovana Brenner Michael’s contribution to the article is evaluated
according to the following scale:
A. has contributed to collaboration (0-33%).
B. has contributed significantly (34-66%).
C. has essentially performed this study independently (67-100%).
1. Design of the project including design of individual experiments: B
2. Performing of the experimental part of the study: B
3. Analysis of the experiments: B
4. Presentation and discussion of the study in article form: B
Emerging issues in antimicrobial resistance Chapter 6
59
ABSTRACT
During the last decade, antimicrobial resistance in bacteria from food-producing
animals has become a major research topic. In this review, different emerging
resistance properties related to bacteria of food-producing animals are highlighted.
These include (i) extended-spectrum β-lactamase-producing Enterobacteriaceae, (ii)
carbapenemase-producing bacteria, (iii) bovine respiratory tract pathogens, such as
Pasteurella multocida and Mannheimia haemolytica, which harbor the multiresistance
mediating integrative and conjugative element ICEPmu1, (iv) Gram-positive and
Gram-negative bacteria that carry the multiresistance gene cfr; and (v) the
occurrence of numerous novel antimicrobial resistance genes in livestock-associated
methicillin-resistant Staphylococcus aureus. The emergence of the aforementioned
resistance properties is mainly based on the exchange of mobile genetic elements
that carry the respective resistance genes.
General discussion Chapter 7
63
7. GENERAL DISCUSSION
This doctoral study has initially investigated the molecular mechanisms of the multi-
resistance phenotype of a bovine P. multocida 36950 isolated from a case of BRD in
a Nebraska feedlot.
The molecular basis of the expanded multi-resistance phenotype of P.
multocida 36950, conferred by 12 antimicrobial resistance genes and three
resistance-mediating mutations, was identified by the sequence analysis of the
genome of this strain and revealed diverse resistance mechanisms, as:
1. enzymatic drug inactivation by hydrolysis (via OXA-2 enzyme) and group
transfer [via AadA25, AadB, AphA1, Mph(E) and StrA-StrB enzymes],
2. drug target modification by mutation (mutations in the quinolone resistance
determining regions of gyrA and parC genes), methylation [via Erm(42)
enzyme] and replacement of sensitive enzymes by resistant enzymes (via
resistant Sul2 enzyme) and
3. active efflux of drugs [via FloR, Msr(E) and Tet(H) exporters]
7.1. Molecular mechanisms of macrolide-triamilide resistance in
P. multocida 36950
The study described in Chapter 2 is a good example of the impact of next-
generation sequencing (NGS) technology in the identification of novel antimicrobial
resistance genes. As repeated transformation experiments proved unsuccessful, it
was assumed that the genes responsible for macrolide-triamilide resistance in P.
multocida 36950 were located in the chromosomal DNA. The molecular basis of the
macrolide-triamilide resistance in P. multocida 36950 was solely revealed by the
whole genome sequencing analysis. Due to a low similarity of the rRNA methylase
gene erm(42) to the known macrolide or lincosamide resistance genes, this gene
was not detected by PCR assays designed to detected any of the until then known
erm genes. This novel erm(42) gene, which codes for an rRNA methylase that
Chapter 7 General discussion
64
chemically modifies the ribosomal target site for macrolides and lincosamides,
proved to confer resistance to the 14- and 16-membered macrolides used in
veterinary medicine, such as erythromycin and tilmicosin, as well as to lincosamides,
such as clindamycin. The two additionally detected genes msr(E)-mph(E) confer
resistance not only to 14- and 16-membered macrolides, but also to the triamilide
tulathromycin. The genes msr(E)-mph(E) code for an ABC transporter and a
macrolide phosphotransferase, respectively. As such, three different genes, each
representing one of the three major resistance mechanisms – target site modification,
active efflux and enzymatic inactivation – have been identified to account for the
high-level macrolide-triamilide resistance in P. multocida 36950.
This study [Chapter 2], along with the report by DESMOLAIZE and colleagues
(2011a) on erm(42), which was published independently and in another journal but
almost at the same time, were the first reports on the genetics of macrolide,
triamilide, and lincosamide resistance in P. multocida. However, none of these two
reports could explain the exact mechanism(s) by which these resistance genes have
become integrated into the chromosomal DNA of P. multocida strains. In the case of
P. multocida 36950, it was understood after further sequence analysis and
experiments, as published in the studies discussed in Chapters 3, 4 and 6.
After the approval of the 16-membered macrolide tilmicosin (Micotil®) in 1992
and the 15-membered triamilide tulathromycin (Draxxin®) in 2005 for use in BRD,
two new macrolides have been approved during the year 2011 for the treatment of
BRD pathogens. These are the 15-membered macrolide gamithromycin (Zactran®)
and the 16-membered macrolide tildipirosin (Zuprevo®). To determine whether
erm(42) and msr(E)-mph(E) also confer resistance to these two new macrolides, we
first tested P. multocida B130 clones that carried either erm(42) or msr(E)-mph(E)
[CHAPTER 2] for their minimal inhibitory concentration (MICs) of gamithromycin and
tildipirosin by broth macrodilution according to Clinical and Laboratory Standards
Institute (CLSI, 2013) recommendations. The recipient strain P. multocida B130
showed 8-fold lower MICs of 0.25 mg/L to both, gamithromycin and tildipirosin, as
compared to tulathromycin (2 mg/L). In the presence of erm(42), the MIC of
tildipirosin increased 128-fold to 32 mg/L while that of gamithromycin increased only
General discussion Chapter 7
65
16-fold to 4 mg/L. In the presence of msr(E)-mph(E), an opposite observation was
made: the MIC of tildipirosin increased only 8-fold to 2 mg/L while that of
gamithromycin increased 256-fold to 64 mg/L. Based on these increases in the MIC
values, it appears as if erm(42) has mainly an effect on the tildipirosin MIC whereas
msr(E)-mph(E) increases preferentially the gamithromycin MIC in P. multocida B130
[CHAPTER 5].
This observation was confirmed by testing a total of 69 naturally occurring P.
multocida (n=40) and M. haemolytica (n=29) isolates from BRD cases, which carry
the genes erm(42) and/or msr(E)-mph(E). These isolates were collected in the Pfizer
Animal Health Susceptibility Surveillance Program for bovine respiratory disease
between 1999 and 2007 from various states in the U.S.A. If all three genes were
present, the 21 P. multocida isolates showed MIC values of 16 – 64 mg/L for
gamithromycin and 16 – 32 mg/L for tildipirosin whereas similar MIC values of 32 –
64 mg/L for both macrolides were seen among the corresponding 20 M. haemolytica
isolates. The ten P. multocida isolates that carried only erm(42) exhibited low MICs of
2 – 4 mg/L for gamithromycin, but had higher MICs of 16 – 32 mg/L for tildipirosin.
The single M. haemolytica that harboured only erm(42) showed MIC values of 4 mg/L
and 32 mg/L for gamithromycin and tildipirosin, respectively. Finally, the two P.
multocida isolates that carried only the msr(E)-mph(E) operon exhibited a high MIC
of 32 mg/L for gamithromycin and a low MIC of 2 mg/L for tildipirosin [CHAPTER 5].
Similar observations for gamithromycin were also published by Desmolaize and co-
workers (DESMOLAIZE et al. 2011b; ROSE et al. 2012)
7.2. Multi-resistance genotype of P. multocida 36950
P. multocida 36950 exhibited resistance to most antimicrobial agents approved
for the control of bovine respiratory diseases. This included resistance to
tetracyclines (32 mg/L), chloramphenicol (16 mg/L), sulphonamides (≥512 mg/L) and
spectinomycin (≥512 mg/L), but also to enrofloxacin (2 mg/L), florfenicol (8 mg/L),
tilmicosin (≥128 mg/L) and tulathromycin (≥128 mg/L). Moreover, high minimum
inhibitory concentrations of the aminoglycosides streptomycin (≥64 mg/L), gentamicin
Chapter 7 General discussion
66
(128 mg/L), kanamycin and neomycin (≥32 mg/L each) and the lincosamide
clindamycin (≥128 mg/L) were detected. Whole genome sequencing revealed that all
resistance genes found in P. multocida 36950 were located in two resistance gene
regions 1 and 2 which were located 42,526 bp apart from each other [CHAPTER 3].
7.2.1. Resistance gene region 1
The resistance gene region 1 is 15,711 bp in size and contains a total of six
antimicrobial resistance genes in addition to insertion sequences and a regulatory
gene [CHAPTER 3] (Fig. 1). The resistance gene region 1 is bracketed by copies of
the insertion element ISApl1 originally identified in the chromosomal DNA of the
porcine respiratory tract pathogen Actinobacillus pleuropneumoniae (TEGETMEYER
et al. 2008). Upon inspection of the sequences immediately up- and downstream of
each of the two copies of ISApl1, the repeated sequence GT was detected upstream
of the right-handed copy and downstream of the left-handed copy of ISApl1. This
might suggest that the entire resistance gene region 1 was inserted via an ISApl1-
mediated integration or recombination process. Almost in the middle of the resistance
gene region 1, a novel ISCR element designated ISCR21, was detected. ISCR21 is
1751 bp in size and has a single reading frame for a 430-aa transposase which is
next related (83.5 % identity and 89.1 % homology) to the recently described
transposase of ISCR20 from Escherichia coli (BERÇOT et al. 2010).
Upstream of ISCR2, the four resistance genes sul2, strA, strB and aphA1, all
oriented in the same direction, were identified. The gene sul2 codes for a
dihydropteroate synthase of 281 aa that confers sulfonamide resistance. It should be
noted that the start codon and the adjacent ten codons in the 5’ terminus of the gene
differed completely from the sequences of any other known sul2 gene. The aa
sequence deduced from codons 12-281 was indistinguishable from that of the 271-aa
Sul2 proteins commonly found among Pasteurellaceae and other organisms
(SCHWARZ 2008). A 168-bp spacer separated the sul2 gene from the strA gene. An
identical spacer sequence was seen in plasmids pB1003 from P. multocida (SAN
MILLAN et al. 2009), pPASS1 from Pasteurella aerogenes (KEHRENBERG and
SCHWARZ 2001), and pMS260 from A. pleuropneumoniae (ITO et al. 2004). The
General discussion Chapter 7
67
gene strA codes for a 267-aa aminoglycoside 3’’-phosphotransferase. The gene strB
codes for a 278-aa aminoglycoside 6-phosphotransferase. Both genes are involved
in streptomycin resistance. The deduced StrA and StrB amino acid sequences were
indistinguishable from those found in a wide variety of bacteria. Another 335 bp
downstream of strB, a third aminoglycoside resistance gene, aphA1, was detected.
This gene codes for a different type of aminoglycoside 3'-phosphotransferase which
confers resistance to kanamycin and neomycin. The 271-aa AphA1 protein showed
99.6 - 100 % identity to the corresponding proteins of Avibacterium paragallinarum
and A. pleuropneumoniae (HSU et al. 2007; KANG et al. 2009). The sul2-strA-strB-
aphA1 segment showed 99.8 % nucleotide sequence identity to the corresponding
sequence of the IncQ-like plasmid pIE1130 from an uncultured eubacterium
(accession no. AJ271879). A segment carrying these antimicrobial resistance genes
has also been found on plasmids in Enterobacteriaceae (KEHRENBERG et al. 2003;
CAIN and HALL 2012) and seems to be – at least in part - derived from transposons
(CAIN and HALL 2012). These reports and the fact that many of the aforementioned
genes have been commonly found in Enterobacteriaceae suggest the occurrence of
genetic exchanges between isolates of this family and Pasteurellaceae [Chapter 3].
Downstream of ISCR21, the terminal 257 bp of an ISCR2-associated
transposase gene as well as the adjacent 234 bp of the ISCR2 element were
detected. Downstream of this ISCR2 relic, the gene floR for a 404-aa phenicol-
specific exporter protein of the Major Facilitator Superfamily (MFS) was located. The
FloR protein differed by 1–4 aa from the FloR proteins previously described,
including those found in P. multocida (KEHRENBERG et al. 2008; KEHRENBERG
and SCHWARZ, 2005), Bibersteinia trehalosi (KEHRENBERG et al. 2006) and Vibrio
cholera (HOCHHUT et al. 2001). The floR gene was followed by a gene for a 101-aa
LysR transcriptional regulator protein whose reading frame overlapped by 6 bp with
the sequence of a complete ISCR2 element of 1845 bp. Another 185 bp downstream
of ISCR2, the rRNA methylase gene erm(42) for resistance to 14- and 16-membered
macrolides and lincosamides was detected [CHAPTER 2]. Database searches
revealed that the 301-aa Erm(42) protein is only distantly related (<30 % identity) to
other Erm proteins, but shows 99.3 % identity to an erythromycin resistance protein
Chapter 7 General discussion
68
of 303 aa from plasmid pPDP9106b (accession no. AB601890) of a fish-pathogenic
Photobacterium damselae subsp. piscicida strain [formerly known as Pasteurella
piscicida]. The entire floR-lysR-ISCR2-erm(42) region showed 96.2 % sequence
identity to that of plasmid pPDP9106b. Moreover, the sul2-strA-strB segment and the
∆ISCR2-floR-lysR-ISCR2 segment were present in the SXT element of V. cholerae
(HOCHHUT et al. 2001) even if in different orientations and not interrupted by an
ISCR21 element [CHAPTER 3].
Fig. 1: Comparative analysis of the resistance gene region 1 of P. multocida 36950
7.2.2. Resistance gene region 2
The resistance region 2 is 9,789 bp in size and comprises also six different
resistance genes in addition to regulatory genes and insertion sequences [CHAPTER
3] (Fig. 2). The left-handed part of resistance gene region 2 is characterized by a
largely truncated transposon Tn5706 (KEHRENBERG et al. 1998) of which only the
0 2 4 6 8 10 12 14
GTGT
ISApl1 ISApl1aphA1 strB strA sul2 ISCR21∆
ISCR2 floR lysR ISCR2 erm(42)
0 2 4 6
10 8 6
catA3
P. damselae
pPDP9106b
unculturedbacteriumpIE1130
P. multocida
36950
10 12 14 16∆ISCR2 ∆ISCR2
V. cholerae
MO10
0 2 4 6 8 10 12 14
GTGT
ISApl1 ISApl1aphA1 strB strA sul2 ISCR21∆
ISCR2 floR lysR ISCR2 erm(42)
0 2 4 6
10 8 6
catA3
P. damselae
pPDP9106b
unculturedbacteriumpIE1130
P. multocida
36950
10 12 14 1610 12 14 16∆ISCR2 ∆ISCR2
V. cholerae
MO10
General discussion Chapter 7
69
repressor gene tetR including 95 bp of the downstream region and 133 bp in the
upstream region remained. These 133 bp, however, included the spacer region
between tetR and the tetracycline resistance gene tet(H) with the promoters required
for tetR and tet(H) transcription as well as the 5’ end of the tet(H) reading frame.
Detailed analysis revealed that a recombination between the initial part of the
tet(H) gene and the att1 site of a class 1 integron has occurred [CHAPTER 3]. Thus,
the three resistance gene cassettes present in this class 1 integron also became
integrated into the chromosomal DNA of P. multocida 36950. The first gene cassette
is 591 bp in size, has a 59-base element of 60 bp and contains an aadB gene for a
177-aa aminoglycoside 2’’-O-adenyltransferase which confers gentamicin resistance.
The AadB protein was indistinguishable from a wide variety of AadB proteins from
Gram-negative bacteria deposited in the databases. However, to the best of our
knowledge, this is the first report of a gentamicin resistance gene in P. multocida.
The second gene cassette is 856 bp in size, also has a 59-base element of 60 bp
and harbours a novel aadA gene variant, designated aadA25, for combined
resistance to streptomycin and spectinomycin. The deduced sequence of the 259-aa
AadA25 protein differed by five amino acid exchanges from the next related variants
AadA21 or AadA3c (ANTUNES et al. 2007; PAN et al. 2008). The third gene cassette
is 876 bp in size, has a 59-base element of 70 bp and contains the gene blaOXA-2
which codes for a narrow-spectrum β-lactamase of 275 aa. While database searches
identified blaOXA-2 genes indistinguishable from that of P. multocida 36950 mainly in
Enterobacteriaceae and Pseudomonas aeruginosa, this gene has not been seen
before in P. multocida. However, it has been described, as part of a plasmid-borne
gene cassette, in the porcine respiratory tract pathogen Bordetella bronchiseptica
(KADLEC et al. 2007). Although sequence analysis does not give a hint towards
functional inactivity, this blaOXA-2 gene obviously does not confer resistance to β-
lactam antibiotics in P. multocida 36950.
Immediately downstream of the 59-base element of the blaOXA-2 gene cassette,
a 4,386-bp segment was found which consisted of the genes msr(E)-mph(E)
bracketed by two IS26 elements located in the same orientation. Insertion sequences
of the type IS26 are widespread among Enterobacteriaceae, but have rarely been
Chapter 7 General discussion
70
seen in Pasteurellaceae (KEHRENBERG et al. 2006). IS26 is 859 bp in size, exhibits
14-bp terminal perfect inverted repeats and produces 8-bp direct repeats at its
integration site (MOLLET et al. 1983). The msr(E) gene codes for an ABC transporter
protein of 491 aa while the mph(E) gene codes for a macrolide phosphotransferase
protein of 294 aa. These two genes are organized in an operon-like structure and are
separated by a non-coding spacer sequence of 55 bp. Database searches identified
these genes on plasmids in Klebsiella pneumoniae and other Enterobacteriaceae
(GOLEBIEWSKI et al. 2007; GONZALEZ-ZORN et al. 2005; SHEN et al. 2009) as
well as in Acinetobacter baumannii (POIREL et al. 2008; ZARRILLI et al. 2008),
where they have been referred to as mel or mef(E) and mph or mph2. No direct
repeats were detectable, neither up- and downstream of each of the two IS26 copies,
nor upstream of the left IS26 copy and downstream of the right IS26 copy.
The sixth resistance gene in region 2, the tetracycline resistance gene tet(H)
accompanied by its repressor gene tetR, was located in another truncated Tn5706
element which was found 106 bp downstream of the right-hand IS26. Both terminal
insertion sequences IS1596 and IS1597 present in the composite transposon
Tn5706 (KEHRENBERG et al. 1998) were absent. The Tn5706-homologous
sequence in the part downstream of tetR stopped exactly at the position where
otherwise the IS1596 sequence was found. In the part downstream of tet(H), the
Tn5706-homologous sequence stopped 65 bp after the translational stop codon of
tet(H). Immediately thereafter, perfect nucleotide sequence identity to the whole
genome sequence of Mannheimia succiniciproducens MBEL55E was observed. The
tet(H) gene found in P. multocida 36950 codes for a 400-aa tetracycline efflux protein
of the Major Facilitator Superfamily. It differed by a single homologous aa exchange,
N258H, from the Tet(H) protein of Tn5706 [CHAPTER 3]. Interestingly, the gene
tet(H) was first identified in an avian P. multocida isolate (HANSEN et al. 1993). This
first report occurred in the early 1990s and five years later, the location of tet(H) gene
as part of Tn5706 was shown. The location of tet(H) on a transposon may explain the
wide dissemination of this tet gene among Pasteurellaceae members and its
occurrence on plasmids and in the chromosomal DNA (KEHRENBERG et al. 1998).
General discussion Chapter 7
71
Fig. 2: Comparative analysis of the resistance gene region 2 of P. multocida 36950
In summary, the two resistance gene regions contained a total of twelve
different resistance genes, some of which, e.g. erm(42), msr(E), mph(E) as well as
the cassette-borne genes aadB, aadA25 and blaOXA-2, are novel genes in P.
multocida. The structural comparisons as shown in Figures 1 and 2 strongly suggest
that both resistance gene regions have developed as a result of integration and
recombination processes in which insertion sequences and ISCR elements seemed
to have played a key role. Moreover, the analysis of the two resistance gene regions
clearly showed that P. multocida is able to acquire resistance genes from other
Gram-negative bacteria, to incorporate them into its chromosomal DNA, and to use
these genes to gain resistance against the respective antimicrobial agents.
Chapter 7 General discussion
72
7.2.3. Resistance mediating mutations in P. multocida 36950
Besides the resistance genes identified in the resistance regions 1 and 2, P.
multocida 36950 exhibited resistance to antimicrobial agents, such as the
fluoroquinolone enrofloxacin, for which resistance is often based on mutations in
specific target genes. Fluoroquinolone resistance in P. multocida and other bovine
respiratory tract pathogens has very rarely – if at all – been observed [Chapter 3]. As
in many other bacteria, quinolone/fluoroquinolone resistance is most likely due to
mutations in the genes gyrA and parC coding for DNA gyrase and topoisomerase IV
(CÁRDENAS et al. 2001).
Analysis of the quinolone resistance determining regions (QRDR) within the
genes gyrA and parC identified in P. multocida 36950 showed two bp exchanges in
the QRDR of gyrA which resulted in amino acid alterations: GGT → AGT (Gly75-to-
Ser75) and AGC → AGA (Ser83-to-Arg83). In addition, a single bp exchange in the
QRDR of parC, TCA → TTA, which resulted in a Ser80-to-Leu80 exchange, was also
seen in P. multocida 36950. While single amino acid exchanges within the QRDR of
GyrA are usually only associated with resistance to the quinolone nalidixic acid, two
and more amino acid exchanges in the QRDRs of GyrA and ParC accompany
resistance to fluoroquinolones such as enrofloxacin. While alterations at codon 75 in
gyrA have rarely been detected (PREISLER et al. 2006), alterations at codon 83 in
gyrA and at codon 80 in parC have frequently been described in connection with
fluoroquinolone resistance in other bacteria (HOOPER 2001; GIBELLO et al. 2004;
PIDDOCK 2002). In P. multocida, only a single gyrA mutation AGC → ATC which
results in a Ser83-to-Ile83 exchange has been described to be associated high level
resistance to nalidixic acid (MIC >256 mg/L), but susceptibility to ciprofloxacin (MIC
0.12 mg/L) (CÁRDENAS et al. 2001). The mutations detected in gyrA and parC of P.
multocida 36950 are to the best of our knowledge the first examples of
fluoroquinolone resistance-mediating mutations in P. multocida.
Table 2 shows a summary of all resistance genes and resistance-mediating
mutations found in P. multocida 36950 including their associate resistance
phenotypes [Chapter 3].
General discussion Chapter 7
73
Table 2: Antimicrobial Resistance genes, resistance-mediating mutations and their associated resistance phenotypes in P. multocida 36950
Antimicrobial agents MIC (mg/L) Resistance genes / mutations1
Tetracycline 32 tetR-tet(H)
Chloramphenicol / Florfenicol
16 / 8 floR
Sulfonamides ≥ 512 sul2
Streptomycin ≥ 64 strA, strB, aadA25
Kanamycin / Neomycin
≥ 32 / ≥ 32 aphA1
Gentamicin 128 aadB
Spectinomycin ≥ 512 aadA25
Nalidixic acid / Enrofloxacin
≥ 256 / 2 G75S, S83R (GyrA); S80L (ParC)
Tulathromycin ≥ 128 msr(E)-mph(E), [erm(42)] 2
Gamithromycin 32 msr(E)-mph(E), [erm(42)] 2
Tilmicosin ≥ 128 erm(42), msr(E)-mph(E)
Tildipirosin 16 erm(42), [msr(E)-mph(E)] 2
Clindamycin ≥ 128 erm(42)
1 The β-lactamase gene blaOXA-2 is not functionally active in P. multocida 36950 for
unknown reasons
2 The genes in square brackets play only an additional role in resistance to the respective
antimicrobial agents
Chapter 7 General discussion
74
7.3. Multi-resistance mobile genetic element ICEPmu1
7.3.1. Identification and general characteristics of ICEPmu1
Comparisons of the whole genome sequence of P. multocida 36950 with the
genome sequence of P. multocida Pm70 identified an integrative and conjugative
element of 82-kb, which was present in P. multocida 36950 but absent in P. multocida
Pm70 and most other members of the family Pasteurellaceae (Fig. 3) [Chapter 4].
The designation of this ICE based mainly on the nomenclature proposal by
Burrus and co-workers (2002). This proposal suggested to use the initials of the
name of the bacterium from which it was isolated and a number, which may identify
the strain or correspond to the rank of the discovery of the element (BURRUS et al.
2002). Since there has already an ICE described in Proteus mirabilis and named
ICEPm1 (FLANNERY et al. 2011), the ICE from P. multocida 36950 received the
designation ICEPmu1, as it is the first ICE detected in P. multocida.
ICEPmu1 is 82,214 bp in size and was found to be integrated into the second of
six genomic copies of a tRNALeu. A copy of an integral tRNALeu (Pmu_3620) proved
to be part of the ICEPmu1 and was located close to the right terminus. As a result of
the integration, it is flanked by 13-bp perfect direct repeats (5'-GATTTTGAATCAA-3').
ICEPmu1 included the resistance gene region 1 at its left terminus and the resistance
region 2 close to its right terminus. ICEPmu1 showed a G + C content (41.9 %)
different from that of the genome of its host (40.4 %). The higher G + C content of
ICEPmu1 resulted from the higher G + C content of the sequences present in the two
resistance gene regions. Within ICEPmu1, a total of 88 open reading frames were
identified among which a function was predicted by sequence comparisons or – in
the case of the resistance genes – confirmed phenotypically for 56 of them (Fig. 4).
A comparison between ICEPmu1 and the 66,641 bp ICE from Histophilus somni
strain 2336 (GenBank accession no. NC_010519.1) (MOHD-ZAIN et al. 2004)
revealed that 66 of the 88 genes found in ICEPmu1 are also present in the ICE from
H. somni. However, the ICE from H. somni lacks most of the two ICEPmu1-
associated resistance gene regions. Of resistance gene region 1, only one copy of
the insertion sequence ISApl1 and of the resistance gene region 2, only one copy of
General discussion Chapter 7
75
the tetracycline repressor gene tetR and the tetracycline resistance gene tet(H) were
present in the ICE of H. somni.
Fig. 3: Circular plot of the genome of P. multocida 36950. The blue rings 1 and 2 represent the coding sequences (CDS) on the leading and lagging strand, respectively. The rings 3–16 show the orthologous CDS according to the Needleman–Wunsch algorithm in the following organisms in the order of appearance (outside to inside): P. multocida Pm70, H. influenzae R2866, H. somni 129PT, H. influenzae 86-028NP, H. somni 2336, H. influenzae Rd KW20, M. succiniproducens MBEL55E, A. succinogenes 130Z, H. influenzae PittEE, H. influenzae PittGG, A. pleuropneumoniae JL03, H. ducreyi 35000HP, H. parasuis SH0165 and H. influenzae R2846. The red bars represent the coding sequences of the different strains with the best conformity to the respective coding sequences of P. multocida 36950 and the grey bars show the CDS of strain 36950 with no orthologues in the respective other organisms. The colours from red to grey illustrate the value of the algorithm. ICEPmu1 is indicated by black lines.
Ch
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Ge
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Fig. 4: Organization of ICEPmu1. The regions in grey represent the flanking regions of this ICE when inserted into the genome of P. multocida 36950. The different genes are depicted and regions or genes of particular relevance are indicated. The resistance gene regions 1 and 2 are shown as boxes. Numbers above the various genes are in agreement with the database entry of the P. multocida 36950 whole genome sequence (GenBank accession no. CP003022).
General discussion Chapter 7
77
Analysis of the coding sequences of ICEPmu1 revealed the presence of the
essential genes for the functionality of an ICE, like the genes involved in
excision/integration and conjugative transfer. Two genes coding for phage integrases
were identified close to the left attachment site (attL). The first integrase gene
(Pmu_2700) was located 2,319 bp and the second (Pmu_2880) 19,284 bp from the
left terminus. Both integrase proteins harboured in the C-terminus the three strongly
conserved residues, the arginine residues in BOX A and BOX B (major clusters of
similarity) and the active site tyrosine residue in BOX C (ESPOSITO and SCOCCA
1997; NUNES-DÜBY et al. 1998) [Chapter 4]
A relaxase gene (Pmu_2890) was found downstream of the second integrase
gene in the central region. This region harboured most of the core genes which
encode the proteins involved in DNA cleavage [putative type I restriction-modification
system methyltransferase subunit, (Pmu_2900)], proteins necessary for a
conjugative transfer [a protein for the formation of type IV pilus (Pmu_3230), TraD-
(Pmu_3190), TraG- (Pmu_3040), TraC-like (Pmu_3070) proteins], and a protein
involved in DNA replication [DNA topoisomerase III (Pmu_3290)]. Moreover, genes
for a protein with a lysozyme-like domain (Pmu_3210), a multicopper oxidase protein
(Pmu_3360) and two other genes for enzymes potentially involved in the metabolism
of alcohol as well as aldehydes and ketones were detected (Pmu_3370 and
Pmu_3330).
Downstream of the resistance gene region 2, genes coding for proteins involved
in DNA replication, such as the single-stranded DNA-binding protein (Pmu_3540)
and an ATPase involved in chromosome partitioning (Pmu_3610) were found. The
analysis of this right-hand terminal region revealed also the presence of the gene
dnaB (Pmu_3600) coding for the DNA helicase DnaB and a gene for a ParB family
protein (Pmu_3590) with a predicted DNA nuclease function. This final core gene-
containing region has been reported as the most conserved region among diverse
proteobacterial ICEs (MOHD-ZAIN et al. 2004).
Chapter 7 General discussion
78
7.3.2. Transfer of ICEPmu1
The ability of ICEPmu1 to transfer to P. multocida strain E348-08, M.
haemolytica 39229 and E. coli HK225 strains by conjugation was confirmed
experimentally. Similar transfer frequencies to the different hosts ranging from 1.4 x
10-4 to 2.9 x 10-6 were observed [Chapter 4].
The screening of the transconjugants by susceptibility testing and PCR assays
confirmed the transfer of all resistance genes. Moreover, the higher MIC values seen
with the E. coli transconjugant, especially for chloramphenicol (32-fold), florfenicol
(64-fold) and ampicillin (16-fold), point towards a better functional activity of the floR
and blaOXA-2 genes in the E. coli host. In this regard, it should be noted that most of
the resistance genes found in ICEPmu1 are not indigenous Pasteurellaceae genes,
but have been found in various members of the Enterobacteriaceae (SCHWARZ
2008).
When ICEs move from one bacterial cell to another, they (i) mediate their
excision from a host genome by site-specific recombination, (ii) form a circular
intermediate and transfer themselves as this circular intermediate by conjugation,
and (iii) insert into a new host genome (BURRUS et al. 2002). The detection of this
circular intermediate is a proof that the respective ICE is mobile. In the case of
ICEPmu1, the detection of this intermediate form was conducted by inverse standard
or nested PCR approaches. The standard or nested PCRs assays for the circular
form of ICEPmu1 were positive for all transconjugants and the donor strain 36950,
and – as expected – negative for the original recipient cells. The nested PCR was
developed to overcome the lower specificity of the left outward primer as recognized
when the standard PCR was performed with E. coli transconjugant. In this case, the
left outward primer annealed also with the right-hand flanking region of the ICE in the
E. coli genome. Analysis of the sequences of the specific amplicons identified the
sequence of the recircularization point (5'-GATTTTGAATCAA-3'), which was in
agreement with the sequence of the direct repeats found immediately up- and
downstream of the termini of ICEPmu1 (Fig. 5).
G
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Ch
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Fig. 5: Site-specific recombination of ICEPmu1 into the tRNALeu of different strains. The sequences of the tRNALeu are shown in the orientation that matches the orientation of the ICEPmu1 sequence. The left attachment sites (attL) and the right attachment sites (attR), the sequences involved in the crossover and the resulting direct repeats located on the left termini (DR-L) and on the right termini (DR-R) of the inserted ICEPmu1 are also shown. Due to the presence of at least part of a second ICEPmu1 copy in the same site, the true attL site was not identified in the M. haemolytica transconjugants.
Chapter 7 General discussion
80
Sequence analysis of the amplicons obtained by standard and inverse PCRs
proved that the insertion point of the ICEPmu1 in all transconjugants was located in a
tRNALeu. The tRNALeu, in which the ICE was inserted in the P. multocida E348-08
transconjugant, showed the same sequence as the one in P. multocida 36950. In the
E. coli HK225 transconjugant, the ICE was inserted into the tRNALeuX, between the
genes intB [coding for a putative prophage P4 integrase] and yjgB [coding for a flavin
mononucleotide (FMN) phosphatase]. In the M. haemolytica 39229 transconjugant,
the sequence of the inverse PCR from the right-hand flanking region showed 100 %
identity with the sequence found in the contigs 83 – 31 (Ctg83_Ctg31 – GenBank
accession no. AASA01000058.1) from M. haemolytica PHL213. This region
contained a partial tRNALeu and the xseA gene [coding for the large subunit of the
exodeoxyribonuclease VII]. Analysis of the sequence of these contigs showed that
this strain also harboured at least part of an ICE related to ICEPmu1. Analysis of the
sequences around the integration site showed an exchange of two consecutive
adenines for a cytosine and a guanine (5'-GATTTTGAATCCG-3') in the direct repeat
at the right terminus of the M. haemolytica 39229 transconjugant. The analysis of the
region flanking the right terminus of ICEPmu1 in the M. haemolytica 39229
transconjugant revealed the presence of at least the terminal part of a second
ICEPmu1 copy [Chapter 4].
7.3.3. ICEPmu1-related elements
As described before, the macrolide resistance genes in P. multocida 36950
were found to be located in the accessory gene regions of the ICEPmu1. However, in
the study published by DESMOLAIZE and colleagues (2011b), no ICE was detected
in the isolates which carried the macrolide resistance genes. The authors have only
characterized a fragment of 10,539 bp (accession no. JF769133) of the bovine P.
multocida strain 3361 which was also isolated in the United States. This 10,539-bp
fragment corresponds to part of the second accessory gene region of ICEPmu1. In
this way, the studies of chapter 2, 3 and 4 revealed a more comprehensive
characterization of the genetic environment of the macrolide resistance genes and
identified ten additional antimicrobial resistance genes.
General discussion Chapter 7
81
After the publication of these studies [Chapters 2-4], ICEPmu1-related
elements were found in P. multocida, M. haemolytica and H. somni isolated from
cases of BRD in Nebraska feedlots, USA (KLIMA et al. 2014). These ICEs were not
fully sequenced. The authors screened the respective isolates for the presence of
antimicrobial resistance genes and for ICE-associated genes originating from
ICEPmu1 and tested them for their transfer abilities. In this study, variants of
ICEPmu1 were detected, which harboured some or all 12 ICEPmu1-associated
antimicrobial resistance genes.
The complete sequence of an ICEPmu1-related element, the ICEMh1, was
revealed by the whole genome sequence analysis of M. haemolytica 42548 which
was obtained from a case of BRD in a Pennsylvania feedlot, USA (EIDAM et al.
2015). ICEMh1 may have evolved by a recombination event between ICEPmu1 and
a second ICE, possibly the putative ICE of M. haemolytica USDA-ARS-USMARC-
183 isolated in Kansas, USA. Five out of 12 ICEPmu1-associated antimicrobial
resistance genes were found in ICEMh1, the genes strA, strB, aphA1, tetR-tet(H) and
sul2. Interestingly, in these aforementioned studies (KLIMA et al. 2014; EIDAM et al.
2015), the ICEs were transferred by conjugation from M. haemolytica into P.
multocida, but not from M. haemolytica to E. coli recipient cells. However, as
described in Chapter 4, KLIMA and colleagues (2014) were also able to transfer the
ICEPmu1-related elements from P. multocida to E. coli. In this way, P. multocida may
play an important role in the dissemination of ICEs among bacteria of different
families, such as Pasteurellaceae and Enterobacteriaceae (KLIMA et al. 2014).
It is important to note that KLIMA and colleagues (2014) were able to identify
ICEPmu1-related elements in isolates from Nebraska feedlots, but not from those in
Alberta, Canada. According to the authors, the same antimicrobial use protocol for
BRD control and treatment has been used in in Nebraska and Alberta. However,
calves with low weight and feedlots with high-density of animals were seen in
Nebraska. The authors speculate that in the Nebraska those low-weight calves were
likely to be submitted to metaphylactic treated upon arrival, due to the high risks of
BRD acquisition, and that in a high-density production system higher amounts of
antimicrobial agents are necessary for the prevention of diseases. Since the multi-
Chapter 7 General discussion
82
resistance ICEPmu1 and its related variants were found among the major pathogens
(P. multocida, M. haemolytica and H. somni) involved in BRD from feedlots in the
United States, it may be suggested that these elements are the result of
recombination processes prompted by the selection pressure within the feedlot
system in this country (KLIMA et al. 2014).
7.4. Additional features of the genome of P. multocida 36950
7.4.1. General characteristics of the genome and genomic comparison
According to the analysis of the genome sequence of P. multocida 36950
(Reference Sequence no. NC_016808.1) comprises a genome with 2,349,518 bp,
which contains 2,064 predicted coding sequences (CDS) and has an average GC
content of 40.4 %. A total of six rRNA operons and 54 tRNAs were identified.
Moreover, the sequence analysis of the genome has confirmed the results of the
PCR assay performed to determine the capsular type, P. multocida 36950 belongs to
the capsular type A. Further analysis revealed that it belongs to serotype 3 (A:3).
A comparison of the bovine P. multocida strain 36950 with the avian strain
Pm70 (RefSeq no. NC_002663.1), which was at the beginning of this study the only
other completely assembled genome of P. multocida, revealed that a total of 118
CDSs (5.7 %) are unique to strain 36950. Meanwhile, there are another four
completely assembled genomes of P. multocida deposited in the GenBank
(http://www.ncbi.nlm.nih.gov/ genome/genomes/912?, last accessed: 2015/03/28).
However, three of them, the strains HN06 (RefSeq no. NC_017027.1), 3480 (RefSeq
no. NC_017764.1) and HB03 (RefSeq no. NZ_CP003328.1) were isolated from
diseased pigs and the last one is a P. multocida ATCC43137. Solely the genome of
strain HN06 contained a plasmid (RefSeq no. NC_017035.1), a 5360-bp plasmid
which carries the antimicrobial resistance genes strA and sul2. The ICEPmu1, which
comprises 88 CDSs, was absent in these four genomes. According to the data
provided by the National Center for Biotechnology Information (NCBI) the size of the
genomes, the average GC content and the number of CDSs varied as 2.2 – 2.4 Mb,
General discussion Chapter 7
83
40.2 – 40.4 %, and 2091 – 2293 CDSs, respectively. Additionally, there are 19
genomes (including three Pasteurella multocida subsp. gallicida) currently
represented as drafts in the GenBank (http://www.ncbi.nlm.nih.gov/genome/
genomes/912?, last accessed: 2015/03/28).
For over a decade the genome of P. multocida strain Pm70 (data of release:
2000/10/24) remained as the only whole genome sequence of a P. multocida strain
available in the GenBank. In contrast, in the last three years, 24 genomes of P.
multocida strains (including that of strain 36950) were released. It is clear that the
NGS technologies have contributed to this increase in available genome sequences.
In this way, draft genomes have been easily generated by NGS technologies.
However, the closure of gaps, improvement and finishing of a genome – time-
consuming and laborious – are missing in many sequencing projects. Such draft
sequences may have quality limitations that impose difficulties for the analysis of
data and for the use of them to determine the physical localization of the genes in the
genome and in comparative studies (PETTERSSON et al. 2009; Zhang et al. 2011).
Considering these limitations, BOYCE and colleagues (2012) have compared
the genomes of P. multocida strain 36950 and strain Pm70 with drafted genomes of
avian P. multocida strains X73 (RefSeq no. NZ_CM001580.1), caprine Anand1_goat
[whole genome shotgun sequencing (WGS) project no. AFRS01], avian VP161,
bovine M1404 and porcine P903 and P3480. For the last four strains, there are no
WGS projects available in the GenBank. Moreover, the authors compared these
genomes with the genome of Pasteurella multocida subsp. gallicida str.
Anand1_poultry (WGS project no. AFRR01). Depending on the quality and coverage
of the drafted genomes used for the comparison, the authors have found that they
may share from 1,100 to 1,786 CDSs. The ICEPmu1 was also not found in these
drafted genomes. Phylogenetic analysis using 7,931 single nucleotide
polymorphisms (SNPs) of common positions in all P. multocida strains revealed a
very close relationship even among such unrelated strains from different geographic
regions, serotypes, animal hosts and disease conditions (BOYCE et al. 2012).
Additional, fully closed genomes are necessary for a better understanding of the
pathogenic mechanisms and the host specificity of P. multocida strains.
Chapter 7 General discussion
84
7.4.2. Putative virulence factors
The molecular basis of the pathogenicity of P. multocida is still not well
understood and some processes are completely unknown. However, some factors
have been recognized as putative virulence factors due to their potential association
with pathogenic mechanisms (CHALLACOMBE and INZANA 2008; BOYCE et al.
2012; HARPER et at. 2012; WILKIE et al. 2012). In P. multocida strain 36950 some
of the identified factors (beyond those factors involved in capsule formation) were
proteins involved in:
1. adherence and colonization: PtfA (type 4 fimbriae), ZnuA (periplasmic
zinc uptake system/adhesin B precursor), Hsf (surface fibril protein),
TadD (non-specific tight adherence protein D), NanB (neuraminidase or
siliadase B),
2. secretion mechanisms: OmpA and OmpH (outer membrane proteins A
and H),
3. lipopolysaccharide synthesis: GalE (UDP-glucose 4-epimerase),
4. iron utilization: ExbB and ExD (accessory proteins, Ton-dependent
transport of iron compounds), TonB (iron transporter), HgbA
(hemoglobin-binding protein A), Fur (ferric uptake regulation protein).
The genome of P. multocida 36950 lacks the gene toxA, which encodes a
dermonecrotoxin, also named as P. multocida toxin (PMT). PMT is considered a
major virulence factor associated with porcine atrophic rhinitis and is more commonly
found in isolates of serogroup D (PULLINGER et al. 2004). Moreover, one of the two
Pasteurella filamentous hemagglutinin genes, the gene pfhB1, proved to be
truncated due to a frameshift mutation. It has been shown that the genes pfhB1 and
pfhB2 show homology to the virulence-associated filamentous hemagglutinin genes
of Bordetella pertussis, fhaB1 and fhaB2, which are involved in the adherence of
bacteria to the host cells (RELMAN et al. 1989; MAY et al. 2001).
7.4.3. CRISPR systems in P. multocida 36950
Clustered regularly interspaced short palindromic repeat (CRISPR) systems
have a defence function, they confer resistance against infection by
General discussion Chapter 7
85
extrachromosomal agents like phages and plasmids, depending on the sequences
present in the spacers. In this way, they may limit transduction and conjugation, two
major routes of HGT (HAFT et al. 2005; POURCEL et al. 2005).
In the genome of P. multocida 36950, a CRISPR/Cas Ypest-subtype (Fig. 4)
and its CRISPR-associated module were found located approximately 11 kb away
from the right terminus of the ICEPmu1 [Chapter 4]. Moreover, a second CRISPR
locus was found with 130 direct repeats and 129 spacers. However, neither CRISPR-
associated cas genes nor the CRISPR-associated module were present. According
to a search in the databank CRISPRdb (http://crispr.u-psud.fr/crispr/, last accessed
2015/03/28) (GRISSA et al. 2007), the 28-bp direct repeats (5’-TTTCTAAGCTGCC
TATACGGCAGTTAAC-3’) of this second locus were the same found in one CRISPR
of P. multocida strains Pm70, HN06 and 3480, but no identity was found among the
spacers. The CRISPR-associated cas genes and the CRISPR-associated module
were also absent in these strains. The sequence of some CRISPR spacers found in
P. multocida 36950 showed high identity to the genome of bacteriophage F108 (93 -
100 %) and P2 and L-413C (96 %). Phage F108 is a temperate transducing
Pasteurella phage of ca. 30 kb (double-stranded DNA). It has been shown that the
phage F108 is able to infect P. multocida and integrate its genome at tRNALeu
(CAMPOY et al. 2006). Phage P2 (temperate double-stranded DNA) is part of an
environmentally widespread family, Myoviridae. The phage L-413C and P2 differ
solely by the lysogeny-related genes. Phages morphologically identical with
coliphage P2 have been identified in P. multocida (ACKERMANN and KARAIVANOV
1984). In bovine M. haemolytica phages belonging to P2 phage family were also
identified (HIGHLANDER et al. 2006).
7.5. Concluding remarks
The ICEPmu1 described in this doctoral thesis project is to the best of our
knowledge the first ICE identified in P. multocida. It is closely related in its core genes
to a family of diverse proteobacterial ICEs, but also harboured two regions of
accessory genes which consisted mainly of insertion sequences and antimicrobial
Chapter 7 General discussion
86
resistance genes [Chapters 3 and 4]. A matter of concern is the high similarity
among ICEPmu1 found in P. multocida 36950, the ICE in H. somni 2336 (MOHD-
ZAIN et al. 2004), the ICE segment available in the incomplete M. haemolytica
PHL23 genome sequence (strain ATCC BAA-410) (GIOIA et al. 2006), but also the
most recently described ICEMh1 of M. haemolytica 42548 (EIDAM et al. 2015). P.
multocida, M. haemolytica and H. somni represent the major pathogens involved in
bovine respiratory disease (DABO et al. 2007; WATTS and SWEENEY 2010) and
the aforementioned four strains were all isolated from cases of respiratory tract
infections in cattle. These observations corroborate the results of our in vitro transfer
experiments and show that horizontal intergenus transfer of closely related ICEs has
obviously already happened in vivo. Since ICEs are among the most important
elements mediating horizontal gene transfer between a wide range of bacterial hosts,
the spreading of multi-resistance ICEs, such as ICEPmu1, may seriously decrease
the therapeutic options for bovine respiratory disease. Moreover, the particular
structure of the resistance gene regions may allow the incorporation of further
cassette-borne resistance genes but also the acquisition of resistance genes via
insertion sequence-mediated recombination processes.
Since no new classes of antimicrobial agents for use in livestock animals are to
be expected in the near future, the superior aim of all people, who prescribe and
apply antimicrobial agents, must be to preserve the efficacy of the currently available
antimicrobial agents for as long as possible [CHAPTER 6]. This includes measures
to counteract the emergence of antimicrobial (multi-)resistance among bacteria from
livestock animals. There is no fast and easy solution to the problem. More likely, it
will be a joint approach that includes on one side (i) improved preventive measures
such as vaccination, (ii) improved farm management accompanied by a tendency to
implement integrated farming systems, (iii) improved hygiene on farms, and (iv)
prudent and judicious use of antimicrobial agents. On the other side, more emphasis
must be put on research to identify emerging resistance genes, the mobile genetic
elements with which they are associated and the modes of spreading of these
elements. Understanding the mechanism(s) of resistance and knowing the conditions
of optimized horizontal gene transfer are important first steps to develop means and
General discussion Chapter 7
87
ways to inhibit the resistance mechanism (SCHWARZ and KEHRENBERG 2006)
and to counteract resistance gene dissemination. Especially the knowledge about co-
located resistance genes, which allow co-selection and persistence of resistance
genes even in the absence of a direct selection pressure, is indispensable to predict
the success or failure of measures such as the ban or the limitation of use of a
certain antimicrobial agent in order to reduce resistance rates. Another issue is the
non-therapeutic use of antimicrobial agents for growth promotion (reviewed by
MARSHALL and LEVY 2011). Although antimicrobial growth promoters have been
banned in 2006 from use in food-producing animals in the European Union, they are
still used in many non-EU countries. The amount of antimicrobial agents used for
growth promotion may be equal or even superior to the amount used in therapy
(MARSHALL and LEVY 2011). It would be an option to consider a global ban of
antimicrobial growth promoters in food animal production, especially since there are
examples which showed that the ban of antimicrobial growth promoters had no
negative impact on health and productivity of food-producing animals (WIERUP
2001; AARESTRUP et al. 2010).
Since bacteria live in polymicrobial environments on the skin and the mucosal
surfaces of humans and animals, there will always be partners for the exchange of
genetic material. Therefore, it is impossible to prevent the dissemination of plasmids,
transposons or ICEs within bacterial populations. However, using correct dosage
schemes and choosing the most promising antimicrobial agent based on the results
of in vitro susceptibility testing will minimize the spread of resistant bacteria and
resistance genes. Commercial large-scale rearing of livestock without using
antimicrobial agents is not possible to date. Although the use of antimicrobial agents
is considered an important factor driving antimicrobial resistance, very limited
detailed information on the use of antimicrobial agents in animals is currently
available (SILLEY et al. 2012; BOS et al. 2013). However, it is necessary to
understand which antimicrobial agents are used at which quantities for which
purpose in which animal species. Factors like co-location of resistance genes on the
same mobile genetic element, co-transfer of these resistance genes during spread of
the element as well as co-selection and persistence of resistance genes during direct
Chapter 7 General discussion
88
or indirect selection pressure play an important role in the interplay between
antimicrobial agents and bacteria. It is important to understand that antimicrobial
resistance is an evolutionary principle by which bacteria try to adapt to changed
environmental conditions, i.e. survival in the presence of antimicrobial agents. As
such, it is impossible to stop antimicrobial resistance. However, it is possible to slow
down the development and dissemination of antimicrobial resistance by reduction of
the selection pressure and prudent and judicious therapeutic use of the available
antimicrobial agents.
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Summary Chapter 8
103
8. SUMMARY
Geovana Brenner Michael, PhD: Molecular analysis of a multi-resistant bovine
Pasteurella multocida strain from the U.S.A.
The present doctoral thesis aimed at investigating a multi-resistant Pasteurella
multocida strain obtained from a case of bovine respiratory disease (BRD) in the
U.S.A. for its genomic structure and the genetic basis of multi-resistance. Particular
emphasis was put on the identification of novel genes that confer resistance to
macrolides and triamilides as members from these classes are frequently used to
combat BRD and the genetics of resistance to macrolides in BRD pathogens,
including P. multocida, were largely unknown.
For this doctoral thesis, the representative P. multocida strain 36950 was
chosen. Since PCR-directed searches for known erm genes as well as repeated
transformation attempts were unsuccessful, P. multocida 36950 was subjected to
whole genome sequencing. Contigs obtained from the draft genome led to the
identification of a novel rRNA methylase gene erm(42), the macrolide transporter
gene msr(E), and the macrolide phosphotransferase gene mph(E). Functional
cloning and expression of these genes in a macrolide susceptible P. multocida
recipient strain confirmed that erm(42) was mainly responsible for resistance to
tilmicosin and lincosamides such as clindamycin and only slightly increased the
minimal inhibitory concentrations (MICs) of tulathormycin. In contrast, msr(E)-mph(E)
were responsible for resistance to tilmicosin and tulathromycin, but had no effect on
the MICs of lincosamides. The results of this study described for the first time the
molecular basis of macrolide, triamilide, and lincosamide resistance in P. multocida
[Chapter 2].
Further analysis of P. multocida 36950 genome and genomic comparisons
revealed that these three genes were located on a mobile genetic element, an
integrative and conjugative element (ICE), designated ICEPmu1. It was also the first
report of an ICE in P. multocida [Chapter 3]. In addition to the three macrolide/triami-
lide resistance genes, another nine antimicrobial resistance genes were found to be
Chapter 8 Summary
104
part of ICEPmu1. Eleven of the 12 resistance genes conferred resistance to
streptomycin (strA and strB), streptomycin/spectinomycin (aadA25), gentamicin
(aadB), kanamycin/neomycin (aphA1), tetracycline [tetR-tet(H)], chloramphenicol/
florfenicol (floR), sulphonamides (sul2), tilmicosin/clindamycin [erm(42)] or
tilmicosin/tulathromycin [msr(E)-mph(E)]. In addition, a complete β-lactamase gene
blaOXA-2 was detected, which, however, appeared to be functionally inactive in P.
multocida. These resistance genes were organized in two regions of approximately
15.7 and 9.8 kb. Furthermore, resistance to nalidixic acid and enrofloxacin was due
to point mutations within the quinolone-resistance determining region (QRDR) of the
genes gyrA and parC [Chapter 3]. Such an expanded multi-resistance phenotype
has very rarely been observed in P. multocida and other bovine respiratory tract
pathogens. And the resistance genes and resistance-mediating mutations detected
could fully explain this multi-resistance phenotype [Chapter 3].
The 82,214 bp ICEPmu1 harbours 88 genes. The core genes of ICEPmu1,
which are involved in excision/integration and conjugative transfer, resemble those
found in a 66,641 bp ICE from Histophilus somni. ICEPmu1 integrates into a tRNALeu
and is flanked by 13 bp direct repeats. It is able to transfer by conjugation to P.
multocida, M. haemolytica and E. coli, where it also uses a tRNALeu for integration
and produces closely related 13 bp direct repeats at the integration site. The
presence of ICEPmu1 and its circular intermediate in the transconjugands was
confirmed by PCR and sequence analysis. PCR assays and susceptibility testing
confirmed the presence and the functional activity of the ICEPmu1-associated
resistance genes in the transconjugands. The gene blaOXA-2 proved to be inactive in
P. multocida and M. haemolytica recipients, but was functionally active in the E. coli
recipient strain [Chapter 4].
The novel macrolide and triamilide resistance genes were tested for their ability
to confer resistance to gamithromycin and tildipirosin, two novel macrolides approved
during the course of this doctoral thesis. Based on the observed increases in the MIC
values in P. multocida B130 carrying the cloned erm(42) or msr(E)-mph(E), it
appears as if erm(42) has mainly an effect on the tildipirosin MIC (128-fold increase)
whereas msr(E)-mph(E) increases the gamithromycin MIC 256-fold in P. multocida
Summary Chapter 8
105
B130. These observations were confirmed with P. multocida and M. haemolytica field
isolates that carried the three genes in different combinations [CHAPTER 5].
ICEPmu1 proved to move across species and genus boundaries and since it
carries 12 resistance genes, some of which confer resistance to the most recently
approved antimicrobial agents for treatment of BRD, its dissemination drastically
limits the treatment options. As such, the spread of ICEPmu1 is considered an
emerging issue in antimicrobial resistance of food-producing animals [Chapter 6].
Zusammenfassung Chapter 9
109
9. ZUSAMMENFASSUNG
Geovana Brenner Michael, PhD: Molekulare Analyse eines multi-resistenten
Pasteurella multocida-Stammes boviner Herkunft
aus den U.S.A.
In der vorliegenden Dissertation wurde ein multi-resistenter Pasteurella multocida-
Stamm von einem an einer Atemwegsinfektion erkrankten Rind aus den U.S.A.
hinsichtlich seiner Genomstruktur und den genetischen Grundlagen der Multi-
Resistenz untersucht. Ein besonderer Schwerpunkt der Arbeiten war die
Identifizierung neuer Gene für Resistenz gegenüber Makroliden und Triamiliden.
Vertreter dieser beiden Wirkstoffklassen werden häufig bei Atemwegsinfektionen von
Rindern eingesetzt und die Grundlagen der Resistenz gegenüber Makroliden und
Triamiliden bei entsprechenden Erregern waren weitgehend unbekannt.
Für diese Dissertation wurde der repräsentative P. multocida-Stamm 36950
ausgewählt. Da PCR-basierte Suchen nach bekannten erm-Genen sowie
Transformationsexperiemnte keine Erfolge zeigten, wurde P. multocida 36950 einer
Gesamtgenomsequenzierung unterzogen. Die Untersuchung der dabei erhaltenen
Contigs führte zur Identifizierung des neuen rRNA-Methylase-Gens erm(42), des
Makrolid-Transportergens msr(E) und des Makrolid-Phosphotransferasegens
mph(E). Funktionelle Klonierung und Expression dieser Gene in einem
makrolidempfindlichen P. multocida-Empfängerstamm bestätigten, dass erm(42) in
erster Linie Resistenz gegenüber Tilmicosin und Linkosamiden wie Clindamycin
vermittelte, aber die minimale Hemmkonzentration (MHK) für Tulathormycin nur leicht
erhöhte. Im Gegensatz dazu vermittelten die Gene msr(E)-mph(E) Resistenz
gegenüber Tilmicosin und Tulathromycin, hatten aber keinen Effekt auf die MHK-
Werte für Linkosamide. Die Ergebnisse dieser Untersuchungen klärten erstmalig die
genetischen Grundlagen der Resistenz gegenüber Makroliden, Triamiliden und
Linkosamiden bei P. multocida [Chapter 2].
Weitere Untersuchungen der Genomsequenz von P. multocida 36950 sowie
Vergleiche mit anderen Genomen zeigten, dass die drei vorab identifizierten
Chapter 9 Zusammenfassung
110
Resistenzgene Bestandteil eines mobilen genetischen Elements, des integrativen
und konjugativen Elements ICEPmu1, waren. ICEPmu1 ist das erste bei P. multocida
jemals beschriebene ICE [Chapter 3]. Zusätzlich zu den drei Makrolid/Triamilid-
Resistenzgenen trägt ICEPmu1 noch weitere neun Resistenzgene. Elf der insgesamt
12 Resistenzgene vermitteln Resistenz gegenüber Streptomycin (strA und strB),
Streptomycin/Spectinomycin (aadA25), Gentamicin (aadB), Kanamycin/Neomycin
(aphA1), Tetrazyklin [tetR-tet(H)], Chloramphenicol/Florfenicol (floR), Sulphonamiden
(sul2), Tilmicosin/Clindamycin [erm(42)] oder Tilmicosin/Tulathromycin [msr(E)-
mph(E)]. Zusätzlich wurde ein komplettes β-Laktamasegen, blaOXA-2, nachgewiesen,
welches aber bei P. multocida funktionell inaktiv zu sein scheint. Alle diese
Resistenzgene waren in zwei Regionen von etwa 15.7 und 9.8 kb Größe organisiert.
Resistenz gegenüber dem Chinolon Nalidixinsäure und dem Fluorchinolon
Enrofloxacin basierte auf Punktmutationen in der Chinlonresistenz-vermittelten
Region der Gene gyrA und parC [Chapter 3]. Solch ein umfassender Multi-
Resistenzphänotyp wurde bislang selten bei P. multocida und anderen bovinen
Atemwegsinfektionserregern beobachtet. Die nachgewiesenen Resistenzgene und
resistenzvermittelnden Mutationen erklären vollständig den nachgewiesenen Multi-
Resistenzphänotyp [Chapter 3].
Das 82.214 bp große ICEPmu1 besitzt insgesamt 88 Gene. Die Gene von
ICEPmu1, deren Genprodukte in Prozesse wie Exzision/Integration und konjugativer
Transfer beteiligt sind, ähneln denen, die bei einem 66.641 bp großen ICE von
Histophilus somni gefunden wurden. ICEPmu1 integriert in eine tRNALeu und wird
von 13 bp großen direkten Sequenzwiederholungen flankiert. ICEPmu1 überträgt
sich durch Konjugation in andere Bakterien wie P. multocida, M. haemolytica und E.
coli, wo es auch eine tRNALeu zur Integration nutzt und eng verwandte 13 bp große
direkte Sequenzwiederholungen an der Integrationsstelle produziert. ICEPmu1 und
seine zirkuläre Zwischenform wurden in den Transkonjuganden mittels PCR- und
Sequenzanalysen bestätigt. PCR-Analysen und Empfindlichkeitsprüfungen zeigten
dass die ICEPmu1-assoziierten Resistenzgene in den Transkonjuganden funktionell
aktiv waren. Lediglich das Gen blaOXA-2 war in den P. multocida- und M. haemolytica-
Transkonjuganden inaktiv, in dem E. coli-Transkonjugand jedoch aktiv [Chapter 4].
Zusammenfassung Chapter 9
111
Die neuen Makrolid/Triamilid-Resistenzgene wurden hinsichtlich ihrer Fähigkeit
getestet, auch Resistenz gegenüber Gamithromycin und Tildipirosin, zwei neuen
Makroliden, die im Laufe dieses Dissertationsprojektes zugelassen wurden, zu
vermitteln. Basierend auf den beobachteten Steigerungen der MHK-Werte für P.
multocida B130-Klone, die die klonierten Gene erm(42) oder msr(E)-mph(E) trugen,
vermittelt erm(42) in erster Linie Resistenz gegenüber Tildipirosin MIC (128-facher
Anstieg des MHK-Werts) während in Gegenwart von msr(E)-mph(E) ein 256-facher
Anstieg des MHK-Werts für Gamithromycin bei P. multocida B130 zu verzeichnen
war. Diese Beobachtungen wurden durch die Untersuchung von P. multocida- und
M. haemolytica-Feldisolate bestätigt, die die drei Resistenzgene in unterschiedlichen
Kombinationen enthielten [CHAPTER 5].
ICEPmu1 ist in der Lage, sich über Stamm-, Spezies- und Genusgrenzen
auszubreiten. Da es über 12 Resistenzgene verfügt, die zum Teil auch Resistenz
gegenüber den neusten, für die Behandlung boviner Atemwegsinfektionen
zugelassenen Wirkstoffen vermitteln, reduziert die Ausbreitung dieses ICEs drastisch
die therapeutischen Optionen. Die Verbreitung von ICEPmu1 bei bovinen
Atemwegsinfektionserregern wird als besondere Bedrohung in Bezug auf
antimikrobielle Resistenz bei Infektionserregern Lebensmittel liefernder Tiere
angesehen [Chapter 6].
113
ACKNOWLEDGMENTS
Background: In the beginning was the unknown antimicrobial resistance
mechanismH Thank you Prof. Dr. Stefan Schwarz for giving me the opportunity to
work on this exciting project and for your guidance through all these years! I also
thank Prof. Dr. Heiner Niemann and Prof. Dr. Dr. h.c. Thomas C. Mettenleiter for their
continuous support and interest in this project.
Methods: I am deeply grateful to all collaborators - Jeff Watts, PhD, Michael T.
Sweeney, MSc and Robert W. Murray, MSc for providing the P. multocida strain
36950, a P. multoRESISTANTcida, and Prof. Dr. Rolf Daniel, Dr. Heiko Liesegang,
Dr. Elzbieta Brzuszkiewicz and Dr. Anja Poehlein (Anja in Portuguese means Angel!)
for all your efforts in helping me to close the GAPS of my knowledge in sequence
analysis – and the members of the research group “Molecular Microbiology and
Antibiotic Resistance”: (i) Kristina Kadlec, PhD, Andrea T. Feßler, PhD., Dr.
Christopher Eidam and Sarah Wendlandt, PhD for helpful discussions and culinary
specialties and (ii) Roswitha Becker, Regina Ronge, Vivian Hensel, Ute Beermann,
Marita Meurer and especially the former member Kerstin Meyer for their invaluable
technical assistance and for the pleasant time. I thank the “Gesellschaft der Freunde
der Tierärztlichen Hochschule Hannover e.V.” for the financial support.
Results: Many gaps were closedH Manuscripts were writtenH and I’m so glad
reading papers in which the studies of this doctoral thesis are not only included in the
references, but have also inspired the work of other people!
Conclusions: If you have support, it doesn’t matter how tricky a situation may be.
Family and friends, thank you SO MUCH! TOM você é o ton da minha vida!