Molecular analysis of a multi-resistant bovine Pasteurella multocida · 2019. 1. 10. · Molecular...

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



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:


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.


in Elsinore, Denmark. *Oral presentation



Genetic basis of fluoroquinolone resistance in a bovine Pasteurella multocida isolate.


in Elsinore, Denmark. Poster 3



Genetic relatedness of bovine Pasteurella multocida and Mannheimia haemolytica

isolates carrying the resistance genes erm(42) and/or msr(E)-mph(E).





Identification of resistance gene cassettes in bovine Pasteurella multocida.





Genetic basis of macrolide, triamilide and lincosamide resistance in a bovine

Pasteurella multocida isolate.





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.


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.


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.


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.


68

Figure 2: Comparative analysis of the resistance gene region 2 of P.


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

17

Chapter 1

Introduction

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


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


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.


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


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.


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


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


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).


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


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


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


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








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


doi: 10.1093/jac/dkr411


Chapter 4 ICEPmu1: structure and transfer

50







1. Design of the project including design of individual experiments: 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


doi: 10.1093/jac/dks076


* both authors contributed equally to this study

Chapter 5 Increased MICs of gamithromycin and tildipirosin

54











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








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.

61

Chapter 7

General discussion

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


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


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


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


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


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


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).


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.


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].


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


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


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

ap

ter 7

Ge

ne

ral d

iscussio

n

76

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).


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).


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

en

era

l discu

ssion

Ch

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79

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.


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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.


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-


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,


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.


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


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


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


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


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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101

Chapter 8

Summary

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].

107

Chapter 9

Zusammenfassung

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!

Molecular analysis of a multi-resistant bovine Pasteurella multocida · 2019. 1. 10. · Molecular...

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