Molecular Analysis of a Multi-Resistant Bovine Pasteurella Multocida Strain from the U.S.A

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): ICE Pmu1 , an integrative conjugative element (ICE) of Pasteurella multocida : analysis of the regions that comprise 12 antimicrobial resistance genes. Journal of Antimicrobial Chemotherapy 67, 84 - 90

MICHAEL, G. B., K. KADLEC, M. T. SWEENEY, E. BRZUSZKIEWICZ, H. LIESEGANG, R. DANIEL, R. W. MURRAY, J. L. WATTS and S. SCHWARZ (2012): ICE Pmu1 , an integrative conjugative element (ICE) of Pasteurella multocida : structure and transfer. Journal of Antimicrobial Chemotherapy 67, 91 - 100

MICHAEL, G. B.*, C. EIDAM*, K. KADLEC, K. MEYER, M. T. SWEENEY, R. W. MURRAY, J. L. WATTS and S. SCHWARZ (2012): Increased MICs of gamithromycin and tildipirosin in the presence of the genes erm (42) and msr (E)-mph (E) for bovine Pasteurella multocida and Mannheimia haemolytica. Journal of Antimicrobial Chemotherapy 67, 1555 – 1557 * both authors contributed equally to this study

MICHAEL, G. B., C. FREITAG, S. WENDLANDT, C. EIDAM, A. T. FEßLER, G. V. LOPES, K. KADLEC and S. SCHWARZ (2015): Emerging issues in antimicrobial resistance of bacteria from food-producing animals. Future Microbiology 10, 427 - 443

Further aspects have been presented at national or international conferences as oral presentation or as posters:

MICHAEL, G. B., K. KADLEC, M. T. SWEENEY, E. BRZUSZKIEWICZ, H. LIESEGANG, R. DANIEL, J. L. WATTS and S. SCHWARZ* (2011): Molecular analysis of emerging antimicrobial resistance properties among bovine Pasteurella multocida . Proceedings of the 4th Symposium on Antimicrobial Resistance in Animals and the Environment (ARAE), 27.-29.06.2011 in Tours, France. *Oral presentation

MICHAEL, G. B.*, K. KADLEC, M. T. SWEENEY, E. BRZUSZKIEWICZ, H. LIESEGANG, R. DANIEL, J. L. WATTS and S. SCHWARZ (2011): Whole genome sequencing of the multi-resistant Pasteurella multocida strain 36950. Proceedings of the International Pasteurellaceae Conference (IPC), 24.-27.08.2011 in Elsinore, Denmark. *Oral Presentation

SCHWARZ, S.*, G. B. MICHAEL, K. KADLEC, M. T. SWEENEY, E. BRZUSZKIEWICZ, H. LIESEGANG, R. DANIEL, and J. L. WATTS (2011): Acquisition of antimicrobial resistance genes and mutations in Pasteurella multocida : insights from the analysis of a multi-resistant strain. Proceedings of the International Pasteurellaceae Conference (IPC), 24.-27.08.2011 in Elsinore, Denmark. *Oral presentation

MICHAEL, G. B., K. KADLEC, M. T. SWEENEY, E. BRZUSZKIEWICZ, H. LIESEGANG, R. DANIEL, J. L. WATTS and S. SCHWARZ (2011): Genetic basis of fluoroquinolone resistance in a bovine Pasteurella multocida isolate. Proceedings of the International Pasteurellaceae Conference (IPC), 24.-27.08.2011 in Elsinore, Denmark. Poster 3

MICHAEL, G. B., K. KADLEC, M. T. SWEENEY, E. BRZUSZKIEWICZ, H. LIESEGANG, R. DANIEL, J. L. WATTS and S. SCHWARZ (2011):

Genetic relatedness of bovine Pasteurella multocida and Mannheimia haemolytica isolates carrying the resistance genes erm (42) and/or msr (E)-mph (E). Proceedings of the International Pasteurellaceae Conference (IPC), 24.-27.08.2011 in Elsinore, Denmark. Poster 5

KADLEC, K., G. B. MICHAEL, M. T. SWEENEY, E. BRZUSZKIEWICZ, H. LIESEGANG, R. DANIEL, J. L. WATTS and S. SCHWARZ (2011): Identification of resistance gene cassettes in bovine Pasteurella multocida . Proceedings of the International Pasteurellaceae Conference (IPC), 24.-27.08.2011 in Elsinore, Denmark. Poster 2

KADLEC, K., G. B. MICHAEL, M. T. SWEENEY, E. BRZUSZKIEWICZ, H. LIESEGANG, R. DANIEL, J. L. WATTS and S. SCHWARZ (2011): Genetic basis of macrolide, triamilide and lincosamide resistance in a bovine Pasteurella multocida isolate. Proceedings of the International Pasteurellaceae Conference (IPC), 24.-27.08.2011 in Elsinore, Denmark. Poster 4

MICHAEL, G. B., K. KADLEC, M. T. SWEENEY, E. BRZUSZKIEWICZ, H. LIESEGANG, R. DANIEL, J. L. WATTS and S. SCHWARZ (2011): Identification of an integrative and conjugative element (ICE) carrying twelve resistance genes in Pasteurella multocida . Proceedings of the 51 st 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.

54 th Annual meeting of American Association of Veterinary Laboratory Diagnosticians (AAVLD)/ United States Animal Health Association (USAHA), 28.09.-05.10.2011 in Buffalo, NY, USA. *Oral presentation

MICHAEL, G. B.*, K. KADLEC, M. T. SWEENEY, E. BRZUSZKIEWICZ, H. LIESEGANG, R. DANIEL, R. W. MURRAY, J. L. WATTS and S. SCHWARZ (2012): Identification and characterization of the integrative and conjugative element ICE Pmu1 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 ICE Pmu1 from bovine Pasteurella multocida and Mannheimia haemolytica . Proceedings of the 7 th International Conference on Antimicrobial Agents in Veterinary Medicine (AAVM), 16.-19.09.2014 in Berlin, Germany. Poster p. 98

MICHAEL, G. B., C. EIDAM, M. T. SWEENEY, R. W. MURRAY, A. POEHLEIN, A. LEIMBACH, H. LIESEGANG, R. DANIEL, J. L. WATTS, S. SCHWARZ and K. KADLEC* (2014): Integrative and conjugative elements (ICEs) conferring multi-resistance in bovine Pasteurella multocida and Mannheimia haemolytica . Proceedings of the 7 th International Conference on Antimicrobial Agents in Veterinary Medicine (AAVM), 16.-19.09.2014 in Berlin, Germany. *Oral presentation

CONTENTS

Page Chapter 1 Introduction . 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 .. 22 1.4. Mobile genetic elements ... 25 1.5. Aims of the present doctoral thesis . 29 References .. 31 Chapter 2 Molecular basis of macrolide, triamilide, and lincosamide resistance in Pasteurella multocida from bovine respiratory disease . 41 Chapter 3 ICE Pmu1 , an integrative conjugative element (ICE) of Pasteurella multocida : analysis of the regions that comprise 12 antimicrobial resistance genes . 45

Chapter 4 ICE Pmu1 , an integrative conjugative element (ICE) of Pasteurella multocida : structure and transfer . 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 .. 57 Chapter 7 General discussion . 61 7.1. Molecular mechanisms of macrolide-triamilide resistance in P. multocida 36950 .... 63 7.2. Multi-resistance genotype of P. multocida 36950 . 65 7.2.1. Resistance gene region 1 ..... 66 7.2.2. Resistance gene region 2 ..... 68 7.2.3. Resistance mediating mutations in P. multocida 36950 ... 72

7.3. Multi-resistance mobile genetic element ICE Pmu1 .. 74 7.3.1. Identification and general characteristics of ICE Pmu1 74 7.3.2. Transfer of ICE Pmu1 .... 78 7.3.3. ICE Pmu1 -related elements .. 80 7.4. Additional features of the genome of P. multocida 36950 .. 82 7.4.1. General characteristics of the genome and genomic comparison . 82 7.4.2. Putative virulence factors . 84 7.4.3. CRISPR systems in P. multocida 36950 ... 84 7.5. Concluding remarks .. 85 References . 88 Chapter 8 Summary .. 101 Chapter 9 Zusammenfassung . 107 Acknowledgements 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 23 Table 2: Antimicrobial resistance genes, resistance-mediating mutations and their associated resistance phenotypes in P. multocida 36950 73

Page

Figure 1: Comparative analysis of the resistance gene region 1 of P. multocida 36950 68 Figure 2: Comparative analysis of the resistance gene region 2 of P. multocida 36950 71 Figure 3: Circular plot of the genome of P. multocida 36950 .. 75 Figure 4: Organization of ICE Pmu1 76 Figure 5: Site-specific recombination of ICE Pmu1 into the tRNA Leu of different strains .. 79

Chapter 1

Introduction

Introduction Chapter 1

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

1.1. General characteristics of Pasteurella multocida Pasteurella multocida was named after Louis Pasteur who identified this bacterium in 1881 as the cause of fowl cholera and “ multocida ” in Latin means many killing, i.e., pathogenic for many species (http://www.bacterio.net/pasteurella.html). It is a Gram-negative, nonmotile, facultatively anaerobic bacterium that belongs to the family Pasteurellaceae . P. multocida is a coccobacillus of 0.3 – 1.2 µm length that does not form spores. It is oxidase-positive and catalase-positive, and can ferment various carbohydrates. A typical bipolar staining with methylene blue can be seen in smears taken from wounds or tissues rather than from cultures (HAGAN et al . 1988). The species P. multocida is subdivided into the four subspecies multocida , gallicida , septica and the recently described tigris (CAPITINI et al . 2002; HARPER et al . 2006). Based on their capsular types, P. multocida isolates are currently classified into the five serogroups A, B, D, E, and F (CARTER 1967; RIMLER and RHOADES 1987; HARPER et al . 2012). Their further classification into 16 serotypes (1–16) is based mainly on lipopolysaccharide (LPS) antigens using the Heddleston scheme (CARTER 1955; HEDDLESTON et al .1972; HARPER et al . 2006). P. multocida isolates possess a number of virulence factors including the polysaccharide capsule and the variable carbohydrate surface molecule LPS. There is a well documented association of the capsule type with particular hosts and diseases. Fowl cholera is most commonly associated with P. multocida type A strains, while haemorrhagic septicemia is caused only by P. multocida types B and E. P. multocida from cases of atrophic rhinitis usually belong to type D (HARPER et al . 2012). P. multocida of capsular type F have been found in turkeys and other animals (SHEWEN and RICE CONLON 1993; CATRY et al . 2005). In strains belonging to serogroups A and B, the capsule has been shown to help resist phagocytosis by host immune cells. In addition, capsule type A has also been shown to help resist complement-mediated lysis (BOYCE and ADLER, 2000; CHUNG et al . 2001). A study in a serovar 1 strain showed that a full-length LPS molecule was essential for the bacteria to be fully virulent in chickens (HARPER et al . 2004). Strains that cause atrophic rhinitis in pigs express the P. multocida toxin (PMT), the gene of which is

Introduction Chapter 1

located on a bacteriophage (PULLINGER et al . 2004). PMT is responsible for the twisted snouts observed in infected pigs.

1.2. Diseases associated with P. multocida P. multocida is considered as a zoonotic pathogen. Human infections are commonly associated with bites, scratches, or licks of dogs and cats, more rarely with bites of pigs. However, infections without epidemiologic evidence of animal contact may also occur in humans. P. multocida is commonly found as a commensal in the oropharyngeal microbiota of cats and dogs, but also in that of other animals. As such, P. multocida is frequently isolated from cat bite abscesses in both cats and humans (FRESHWATER 2008). One study on “bacteriological warfare among cats” (LOVE et al . 2000) described the role of P. multocida and other bacteria in bite- associated infections in cats. Another study reported that in 50 % of dog bites and in 75 % of cat bites the wound was contaminated with P. multocida (TALAN et al . 1999). In animals, P. multocida is remarkable for the number and range of specific disease syndromes with which it is associated, and for the wide range of host species that are affected (WILKIE et al . 2012). P. multocida can act as a primary or as a secondary pathogen in various animal species. As a primary pathogen – or at least a pathogen that has the principal role in the disease process – P. multocida causes haemorrhagic septicaemia in cattle and water buffaloes, septicaemia in other ungulates, fowl cholera in poultry, atrophic rhinitis in pigs and snuffles in rabbits. As a secondary pathogen, it is involved in a variety of diseases, in which P. multocida makes a major contribution, although it requires other factors for the disease condition to develop (WILKIE et al . 2012). Such diseases mainly include lower respiratory tract diseases in ungulates, such as cattle and pigs, which then are referred to as bovine respiratory disease (BRD) or swine respiratory disease (SRD) (KEHRENBERG et al . 2006; SCHWARZ 2008; WILKIE et al . 2012). BRD is one of the economically most important diseases in cattle. Global losses of the feedlot industry due to BRD are estimated to be over $ 3 billion per year

Chapter 1 Introduction

(WATTS and SWEENEY 2010). BRD is a multi-factorial and multi-agent disease which is often also called ‘shipping fever’. This designation refers to some of the factors that play a relevant role in the development of the disease. Transportation over long distances, often associated with exhaustion, starvation, dehydration, chilling or overheating, serves as an important stress factor. Additional stress factors include passage through auction markets, commingling of animals from different herds, dusty environmental conditions in the feedlot and nutritional stress associated with changes in diet. Initial viral infections may pave the way for subsequent bacterial infections, in which besides P. multocida , also M. haemolytica , and Histophilus somni , are important pathogens (DABO et al . 2007).

1.3. Antimicrobial resistance in P. multocida isolates Antimicrobial agents are commonly used to combat P. multocida involved in BRD and other infections. As a consequence, P. multocida has developed and or acquired resistance to a wide range of antimicrobial agents. A summary of what has been known in terms of antimicrobial resistance genes in P. multocida has been published by KEHRENBERG et al . (2006). A further update was published by SCHWARZ (2008) (Table 1). Table 1 provides an overview about the antimicrobial resistance genes identified in P. multocida , that confer resistance to the various classes of antimicrobial agents. This overview presents the situation prior to the start of the present doctoral thesis. Moreover, the location of the different genes as well as the mechanism of resistance specified by them is listed. As can be seen from Table 1, numerous antimicrobial resistance genes have been identified in P. multocida . However, no genes conferring resistance to macrolides, such as tilmicosin or tulathromycin, had been identified. In addition, no gentamicin resistance genes were known in P. multocida . Finally, naturally occurring P. multocida isolates that exhibited resistance to fluoroquinolones had also not been detected.

Introduction Chapter 1

Table 1: Antimicrobial resistance genes identified in P. multocida (Schwarz 2008)

Antimicrobial Resistance Resistance Location on Reference agents mechanism gene(s) MGEs 1 or chromosomal DNA

Penicillins enzymatic inactivation bla ROB-1 unknown Philippon et al . 1986

bla TEM-1 pFAB-1 Naas et al . 2001

bla PSE-1 pJR2 Wu et al . 2003 Tetracyclines active efflux (Major tet (H) pVM111; Hansen et al . 1993; Facilitator Superfamily) Tn 5706 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 tet (M) chromosomal Chaslus-Dancla et al . 1995; (ribosome protective Hansen et al . 1996 protein) 2 non-fluorinated enzymatic inactivation catA1 Plasmid Vassort-Bruneau et al . 1996 phenicols (acetylation) catA3 Plasmid 2 Vassort-Bruneau et al . 1996 catB2 pJR2 Wu et al . 2003 all phenicols active efflux (Major floR pCCK381 Kehrenberg and Schwarz Facilitator Superfamily) 2005b kanamycin, enzymatic inactivation aphA1 pCCK3152 Kehrenberg and Schwarz neomycin (phosphorylation) 2005c aphA3 pCCK411 Kehrenberg and Schwarz 2005c Streptomycin enzymatic inactivation strA-strB pPMSS1 Kehrenberg and (adenylation) Schwarz 2001b streptomycin/ enzymatic inactivation aadA1 pJR2 Wu et al . 2003 spectinomycin (adenylation) aadA14 pCCK647 Kehrenberg et al . 2005d Trimethoprim target replacement dfrA20 pCCK154 Kehrenberg and Schwarz (trimethoprim-resistant 2005e dihydrofolate reductase) Sulfonamides target replacement sul2 pPMSS1 Kehrenberg and Schwarz (sulfonamide-resistant 2001b dihydropteroate synthase)

1 MGEs: mobile genetic elements 2 not further specified plasmid

Most of the antimicrobial resistance genes detected in P. multocida were located on plasmids or transposons. Usually, small non-conjugative plasmids were detected which carried one or more antimicrobial resistance genes. Most often the

Chapter 1 Introduction

streptomycin resistance genes strA -strB were found together with the sulphonamide resistance gene sul2 . However, in plasmid pVM111, a Tn 5706 -like tetR-tet( H) segment responsible for tetracycline resistance was found to be inserted between sul2 and strA via illegitimate recombination and resulted in a sul2–tetR–tet( H)–strA– strB multi-resistance gene cluster (KEHRENBERG et al . 2003). Detailed structural analysis of the resistance plasmids showed that they were composed of segments previously found in other bacteria. As such, the first florfenicol resistance plasmid identified in P. multocida , pCCK381, harboured a floR gene known from Enterobacteriaceae while its plasmid replication and mobilisation genes corresponded to those on the Dichelobacter nodosus plasmid pDN1, whereas other segments of pCCK381 were higly similar to the Vibrio salmonicida plasmid pRVS1 (KEHRENBERG and SCHWARZ, 2005b). These findings suggested that plasmid pCCK381 is the product of interplasmid recombination events. Only a minority of the resistance genes identified in P. multocida seem to be indigenous to this species. Among them are the trimethoprim resistance gene dfrA20 (KEHRENBERG and SCHWARZ, 2005e) and the streptomycin/spectinomycin resistance gene aadA14 (KEHRENBERG et al . 2005d). These two resistance genes have so far exclusively been found in P. multocida . In contrast, most of the antimicrobial resistance genes found in P. multocida , such as sul2 , strA , strB , floR , catA1 , catA3 , aphA1 , aphA3 or aadA1 , have also been detected in a wide range of other bacteria. This observation confirmed that P. multocida exchanges antimicrobial resistance genes with other bacteria within and beyond the family Pasteurellaceae. In the case of the tetracycline resistance gene tet (L), even an exchange with Gram- positive bacteria has been assumed as the gene tet (L) is widely disseminated among staphylococci, streptococci and enterococci (KEHRENBERG et al . 2005a). Plasmids and transposons, that carry resistance genes, play a crucial role in horizontal transfer events with P. multocida acting either as donor or as recipient of antimicrobial resistance genes.

Introduction Chapter 1

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 bla CTX-M-15 gene overexpressed by the IS Ecp1 element (PARTRIDGE 2011; TOLEMAN and WALSH 2011; CANTÓN et al . 2012). After the mobilization events, a composite structure comprising the IS elements and the captured genes or regions is generated (IS- mobilized DNA segment-IS) which is named composite transposon (formerly

Chapter 1 Introduction

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. Tn 3). These latter elements are considered as units (also called unit transposons , formerly transposon type II/Tn 3 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 bla TEM gene encoded by Tn 3 (HEFFRON et al . 1979). Bacteriophages (phages) are virus-like organisms that infect bacteria and are considered the most common microorganism in the biosphere. Noteworthy, phages play an important role as MGEs, especially in the transfer of antimicrobial resistance genes. The genome of phages may vary from ca. 2 kb to > 250 kb. The most common phage particles contain a capsid (protein head) which surrounds the double- stranded DNA and is attached to a tail (HATFULL and HENDRIX 2011). Phages may undergo (i) a lysogenic cycle in which they integrate into the host genome (existing as a prophage) by transposition or site-specific recombination (CAMPBELL 1992) and replicate passively together with the host DNA, or (ii) a lytic cycle as an autonomous form which will then be released by cell lysis. These released phage particles may infect other cells by injecting their DNA into them (intercellular mobility). This phage-mediated transfer of genetic information between a donor and a recipient cell, without a direct contact between the cells, is named transduction , which – due to the protection of the DNA by the capsid – is nuclease-resistant. In some cases, host DNA (any sort of bacterial DNA, as chromosome fragments, plasmids, transposons and IS elements) may be incorporated into the capsid of the phages and

Introduction Chapter 1

then be transferred from one host to another host cell (generalized transduction) or both, phage DNA and fragments of bacterial DNA, may be incorporated into the capsid (specialized transduction) (THOMAS and NIELSEN 2005). Another process of intercellular mobility of DNA is the conjugation . However for this, a direct contact (mating) between donor (F +) and a recipient cell (F -) and the formation of a pore (mating pore) for the passage of the conjugative element are necessary. Although there are also conjugative transposons, conjugation is the most important process for the transfer of plasmids between bacteria under natural conditions. Plasmids are MGEs which are able to perform self-replication (independent from the host chromosome) and may exist within the bacterial cell in an autonomous form (extrachromosomal DNA). All plasmids have at least one origin of replication and code for the proteins involved in the process of replication. The size of plasmids may range from < 1 kb to several hundred kb. They may be inserted in part or completely into the host chromosome, mostly by either homologous or site-specific recombination. Some plasmids, the conjugative plasmids, are able to mediate their own transfer from one cell to another. For this, they carry also genes directly involved in their transfer and in the maintenance/stabilization of the contact between the mating bacteria (SMILLIE et al . 2010). Many naturally occurring plasmids are either conjugative (self-transmissible) or mobilizable (HALL and COLLIS 1995). The transfer of a plasmid by mobilization may occur whether additional functions necessary for the mating are present. Nevertheless, if the size of a plasmid is compatible with the capsid size of a phage, this plasmid may be also transferred by transduction. Beyond conjugation and transduction, the mechanism of transformation may also play a role in the uptake of a plasmid by a recipient cell. In contrast to conjugation, the transfer of DNA by transformation is not done by cell-to- cell contact. Instead, transformation means the uptake of DNA that has been released in the extracellular environment (naked DNA). Moreover, for the efficient uptake of a plasmid or other free extracellular DNA, a recipient cell has to be in a physiological state of competence (THOMAS and NIELSEN 2005).

Chapter 1 Introduction

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). Tn 916 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 Tn 916 or proved to be site-specific (site-specific integrative and conjugative elements). In most cases, these elements (conjugative transposons and site-specific integrative and conjugative elements) are able to integrate into a unique site, e.g. genes encoding tRNAs, and cannot perform transposition to other sites within the host genome. In this way, the site-specific integration systems of these elements show more similarities to prophages than to transposons. For these reasons, BURRUS and colleagues (2002a) proposed a new class of MGEs, named as integrative and conjugate elements (ICEs), which includes “all elements that excise by site-specific recombination into a circular form, self-transfer by conjugation and integrate into the host genome, whatever the specificity and the mechanism of integration and conjugation is. These elements would also be able to replicate during the conjugation event, but this replication should not be involved in their maintenance” (BURRUS et al . 2002a). According to this nomenclature, conjugative transposons are ICEs able to transpose within the host genome. In the same way, genomic islands may be also included into the ICE nomenclature and those that are non-mobile may be considered as truncated or defective ICEs (WOZNIAK and WALDOR 2010). Additional studies have suggested that some ICEs may be able to

Introduction Chapter 1

perform self-replication which has imposed more complexity on the nomenclature of MGEs (WOZNIAK and WALDOR 2010). In ICEs as well as in phages, the genes involved in the same function are grouped in regions which are named as modules (e.g. the backbone modules: recombination, conjugation and regulation) conferring a mosaic structure to the elements. It has been suggested that genetic events involving insertion of MGEs into another and deletions may lead to the acquisition or exchange of some modules and may drive the modular evolution of ICEs. For example, the exchange or acquisition of a transfer module may alter the host specificity of an ICE (OSBORN and BÖLTNER 2002; BURRUS et al . 2002b). ICEs are composed of core genes and accessory genes (or cargo genes). While the core genes are more important for the spreading and maintenance of the ICEs, the accessory genes, which may include genes for antimicrobial, heavy metal or phage resistance but also metabolic activities, are relevant for the fitness of the host and its survival under specific conditions. ICEs have been considered an important driving force of bacterial evolution (MOHD-ZAIN et al . 2004; SETH-SMITH and CROUCHER 2009; ROCHE et al 2010).

1.5. Aims of the present doctoral thesis During recent years, multi-resistant P. multocida and M. haemolytica isolates have been detected in the U.S.A. and Canada. These isolates also exhibited resistance to florfenicol, macrolides, triamilides and fluoroquinolones – resistance properties that had not been seen so far in these bacteria. Neither the genetic basis of resistance to macrolides, triamilides and fluoroquinolones was known nor whether these resistance properties were transferable.

The aims of the present doctoral thesis were 1. to perform the gap closure of the whole genome sequence of the representative P. multocida strain 36950 and analyse the sequence to identify the molecular mechanisms of the expanded multi-resistance

Chapter 1 Introduction

phenotype with particular reference to the macrolide-triamilide and fluoroquinolone resistance, and 2. to characterize the genetic environment of resistance genes, with particular reference to the macrolide-triamilide resistance, in order to: a. identify a linkage between these genes and other resistance genes which may enable the co-selection of macrolide-triamilide resistance even in the absence of a direct selection pressure, b. determine whether the resistance genes identified in P. multocida 36950 are located on mobile genetic elements, and c. investigate the potential of dissemination of such resistance genes located on mobile genetic elements (horizontal gene transfer). 3. to evaluate the in vitro activities of new macrolide antimicrobial agents against P. multocida isolates

To investigate the role of putative genes in macrolide resistance, the whole genome sequence of the representative P. multocida 36950 was determined and analysed. Putative resistance genes were identified and cloning and expression experiments were performed [ Chapter 2 ]. To identify additional antimicrobial resistance genes, their physical linkage, and resistance-mediating mutations responsible for the multi-resistance phenotype of this strain, further sequence analysis and genomic comparisons of the whole genome of P. multocida 36950 were carried out [Chapter 3 ]. Such analysis allowed also the characterization of the genetic environment of all antimicrobial resistance genes identified in P. multocida 36950 [Chapter 3 ]. To determine the transfer ability of the resistance determinates, conjugation experiments were performed. Moreover, the functional activity of the resistance genes in different recipient strains was also tested [Chapter 4 ]. In 2011, the 15-membered macrolide gamithromycin (Zactran ®) and the 16- membered macrolide tildipirosin (Zuprevo ®) were approved for the treatment of BRD. The newly identified macrolide and triamilide resistance genes erm (42) and/or

Introduction Chapter 1

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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Chapter 1 Introduction

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Chapter 1 Introduction

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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

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

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.

Chapter 3

ICE Pmu1 , 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 ICE Pmu1 : analysis of the resistance gene regions

CONTRIBUTION TO THE ARTICLE

The extent of Geovana Brenner Michael’s contribution to the article is evaluated according to the following scale:

A. has contributed to collaboration (0-33%).

B. has contributed significantly (34-66%).

C. has essentially performed this study independently (67-100%).

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

2. Performing of the experimental part of the study: C

3. Analysis of the experiments: C

4. Presentation and discussion of the study in article form: C

ICE Pmu1 : analysis of the resistance gene regions Chapter 3

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 ICE Pmu1 , 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 bla OXA-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.

Chapter 4

ICE Pmu1 , an integrative conjugative element (ICE) of Pasteurella multocida : structure and transfer

Geovana B. Michael, Kristina Kadlec, Michael T. Sweeney, Elzbieta Brzuszkiewicz, Heiko Liesegang, Rolf Daniel, Robert W. Murray, Jeffrey L. Watts and Stefan Schwarz

Journal of Antimicrobial Chemotherapy 67, 91 - 100 (2012) doi: 10.1093/jac/dkr411 http://jac.oxfordjournals.org/content/67/1/91.long

Chapter 4 ICE Pmu1 : structure and transfer

CONTRIBUTION TO THE ARTICLE

The extent of Geovana Brenner Michael’s contribution to the article is evaluated according to the following scale:

A. has contributed to collaboration (0-33%).

B. has contributed significantly (34-66%).

C. has essentially performed this study independently (67-100%).

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

2. Performing of the experimental part of the study: C

3. Analysis of the experiments: C

4. Presentation and discussion of the study in article form: C

ICE Pmu1 : structure and transfer Chapter 4

ABSTRACT

Background: Integrative and conjugative elements (ICEs) have not been detected in Pasteurella multocida . In this study the multiresistance ICE Pmu1 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: ICE Pmu1 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 ICE Pmu1 and its circular intermediate in the recipient strains was confirmed by PCR and sequence analysis. Integration sites were sequenced. Susceptibility testing of the ICE Pmu1 -carrying recipients was conducted by broth microdilution. Results: The 82214 bp ICE Pmu1 harbours 88 genes. The core genes of ICE Pmu1 , which are involved in excision/integration and conjugative transfer, resemble those found in a 66641 bp ICE from Histophilus somni . ICE Pmu1 integrates into a tRNA Leu 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 tRNA Leu for integration and produces closely related 13 bp direct repeats. PCR assays and susceptibility testing confirmed the presence and the functional activity of the ICE Pmu1 -associated resistance genes in the recipient strains. Conclusions: The observation that the multiresistance ICE Pmu1 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.

Chapter 5

Increased MICs of gamithromycin and tildipirosin in the presence of the genes erm (42) and msr (E)- mph (E) for bovine Pasteurella multocida and Mannheimia haemolytica

Geovana B. Michael*, Christopher Eidam*, Kristina Kadlec, Kerstin Meyer, Michael T. Sweeney, Robert W. Murray, Jeffrey L. Watts and Stefan Schwarz

Journal of Antimicrobial Chemotherapy 67, 1555 - 1557 (2012) doi: 10.1093/jac/dks076 http://jac.oxfordjournals.org/content/67/6/1555.long * both authors contributed equally to this study

Chapter 5 Increased MICs of gamithromycin and tildipirosin

CONTRIBUTION TO THE ARTICLE

The extent of Geovana Brenner Michael’s contribution to the article is evaluated according to the following scale:

A. has contributed to collaboration (0-33%).

B. has contributed significantly (34-66%).

C. has essentially performed this study independently (67-100%).

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

2. Performing of the experimental part of the study: C

3. Analysis of the experiments: C

4. Presentation and discussion of the study in article form: C

Increased MICs of gamithromycin and tildipirosin Chapter 5

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 ICE Pmu1 -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.

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

CONTRIBUTION TO THE ARTICLE

The extent of Geovana Brenner Michael’s contribution to the article is evaluated according to the following scale:

A. has contributed to collaboration (0-33%).

B. has contributed significantly (34-66%).

C. has essentially performed this study independently (67-100%).

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

2. Performing of the experimental part of the study: B

3. Analysis of the experiments: B

4. Presentation and discussion of the study in article form: B

Emerging issues in antimicrobial resistance Chapter 6

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 ICE Pmu1 , (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.

Chapter 7

General discussion

General discussion Chapter 7

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

chemically modifies the ribosomal target site for macrolides and lincosamides, proved to confer resistance to the 14- and 16-membered macrolides used in veterinary medicine, such as erythromycin and tilmicosin, as well as to lincosamides, such as clindamycin. The two additionally detected genes msr (E)-mph (E) confer resistance not only to 14- and 16-membered macrolides, but also to the triamilide tulathromycin. The genes msr (E)-mph (E) code for an ABC transporter and a macrolide phosphotransferase, respectively. As such, three different genes, each representing one of the three major resistance mechanisms – target site modification, active efflux and enzymatic inactivation – have been identified to account for the high-level macrolide-triamilide resistance in P. multocida 36950. This study [Chapter 2 ], along with the report by DESMOLAIZE and colleagues (2011a) on erm (42), which was published independently and in another journal but almost at the same time, were the first reports on the genetics of macrolide, triamilide, and lincosamide resistance in P. multocida . However, none of these two reports could explain the exact mechanism(s) by which these resistance genes have become integrated into the chromosomal DNA of P. multocida strains. In the case of P. multocida 36950, it was understood after further sequence analysis and experiments, as published in the studies discussed in Chapters 3 , 4 and 6. After the approval of the 16-membered macrolide tilmicosin (Micotil®) in 1992 and the 15-membered triamilide tulathromycin (Draxxin®) in 2005 for use in BRD, two new macrolides have been approved during the year 2011 for the treatment of BRD pathogens. These are the 15-membered macrolide gamithromycin (Zactran®) and the 16-membered macrolide tildipirosin (Zuprevo®). To determine whether erm (42) and msr (E)-mph (E) also confer resistance to these two new macrolides, we first tested P. multocida B130 clones that carried either erm (42) or msr (E)-mph (E) [CHAPTER 2 ] for their minimal inhibitory concentration (MICs) of gamithromycin and tildipirosin by broth macrodilution according to Clinical and Laboratory Standards Institute (CLSI, 2013) recommendations. The recipient strain P. multocida B130 showed 8-fold lower MICs of 0.25 mg/L to both, gamithromycin and tildipirosin, as compared to tulathromycin (2 mg/L). In the presence of erm (42), the MIC of tildipirosin increased 128-fold to 32 mg/L while that of gamithromycin increased only

General discussion Chapter 7

16-fold to 4 mg/L. In the presence of msr (E)-mph (E), an opposite observation was made: the MIC of tildipirosin increased only 8-fold to 2 mg/L while that of gamithromycin increased 256-fold to 64 mg/L. Based on these increases in the MIC values, it appears as if erm (42) has mainly an effect on the tildipirosin MIC whereas msr (E)-mph (E) increases preferentially the gamithromycin MIC in P. multocida B130 [CHAPTER 5]. This observation was confirmed by testing a total of 69 naturally occurring P. multocida (n=40) and M. haemolytica (n=29) isolates from BRD cases, which carry the genes erm (42) and/or msr (E)-mph (E). These isolates were collected in the Pfizer Animal Health Susceptibility Surveillance Program for bovine respiratory disease between 1999 and 2007 from various states in the U.S.A. If all three genes were present, the 21 P. multocida isolates showed MIC values of 16 – 64 mg/L for gamithromycin and 16 – 32 mg/L for tildipirosin whereas similar MIC values of 32 – 64 mg/L for both macrolides were seen among the corresponding 20 M. haemolytica isolates. The ten P. multocida isolates that carried only erm (42) exhibited low MICs of 2 – 4 mg/L for gamithromycin, but had higher MICs of 16 – 32 mg/L for tildipirosin. The single M. haemolytica that harboured only erm (42) showed MIC values of 4 mg/L and 32 mg/L for gamithromycin and tildipirosin, respectively. Finally, the two P. multocida isolates that carried only the msr (E)-mph (E) operon exhibited a high MIC of 32 mg/L for gamithromycin and a low MIC of 2 mg/L for tildipirosin [CHAPTER 5 ]. Similar observations for gamithromycin were also published by Desmolaize and co- workers (DESMOLAIZE et al . 2011b; ROSE et al . 2012)

7.2. Multi-resistance genotype of P. multocida 36950

P. multocida 36950 exhibited resistance to most antimicrobial agents approved for the control of bovine respiratory diseases. This included resistance to tetracyclines (32 mg/L), chloramphenicol (16 mg/L), sulphonamides (≥512 mg/L) and spectinomycin (≥512 mg/L), but also to enrofloxacin (2 mg/L), florfenicol (8 mg/L), tilmicosin (≥128 mg/L) and tulathromycin (≥128 mg/L). Moreover, high minimum inhibitory concentrations of the aminoglycosides streptomycin (≥64 mg/L), gentamicin

Chapter 7 General discussion

(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 IS Apl1 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 IS Apl1 , the repeated sequence GT was detected upstream of the right-handed copy and downstream of the left-handed copy of IS Apl1. This might suggest that the entire resistance gene region 1 was inserted via an IS Apl1 - mediated integration or recombination process. Almost in the middle of the resistance gene region 1, a novel IS CR element designated IS CR21 , was detected. IS CR21 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 IS CR20 from Escherichia coli (BERÇOT et al . 2010) . Upstream of IS CR2 , the four resistance genes sul2 , strA , strB and aphA1 , all oriented in the same direction, were identified. The gene sul2 codes for a dihydropteroate synthase of 281 aa that confers sulfonamide resistance. It should be noted that the start codon and the adjacent ten codons in the 5’ terminus of the gene differed completely from the sequences of any other known sul2 gene. The aa sequence deduced from codons 12-281 was indistinguishable from that of the 271-aa Sul2 proteins commonly found among Pasteurellaceae and other organisms (SCHWARZ 2008). A 168-bp spacer separated the sul2 gene from the strA gene. An identical spacer sequence was seen in plasmids pB1003 from P. multocida (SAN MILLAN et al . 2009), pPASS1 from Pasteurella aerogenes (KEHRENBERG and SCHWARZ 2001), and pMS260 from A. pleuropneumoniae (ITO et al . 2004). The

General discussion Chapter 7

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 IS CR21 , the terminal 257 bp of an IS CR2 -associated transposase gene as well as the adjacent 234 bp of the IS CR2 element were detected. Downstream of this IS CR2 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 IS CR2 element of 1845 bp. Another 185 bp downstream of IS CR2 , the rRNA methylase gene erm (42) for resistance to 14- and 16-membered macrolides and lincosamides was detected [ CHAPTER 2 ]. Database searches revealed that the 301-aa Erm(42) protein is only distantly related (<30 % identity) to other Erm proteins, but shows 99.3 % identity to an erythromycin resistance protein

Chapter 7 General discussion

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 -IS CR2 -erm (42) region showed 96.2 % sequence identity to that of plasmid pPDP9106b. Moreover, the sul2 -strA -strB segment and the ∆IS CR2 -floR-lysR -IS CR2 segment were present in the SXT element of V. cholerae (HOCHHUT et al . 2001) even if in different orientations and not interrupted by an IS CR21 element [CHAPTER 3].

catA3 uncultured bacterium pIE1130

10 8 6 P. damselae pPDP9106b

0 2 4 6

GT GT P. multocida 36950

0 2 4 6 8∆ 10 12 14 IS Apl1 aphA1 strBstrAsul2 IS CR21 IS CR2 floR lysR IS CR2 erm (42) IS Apl1

V. cholerae MO10

10 12 14 16 ∆IS CR2 ∆IS CR2 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 Tn 5706 (KEHRENBERG et al . 1998) of which only the

General discussion Chapter 7

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 bla OXA-2 which codes for a narrow-spectrum β-lactamase of 275 aa. While database searches identified bla OXA-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 bla OXA-2 gene obviously does not confer resistance to β- lactam antibiotics in P. multocida 36950.

Immediately downstream of the 59-base element of the bla OXA-2 gene cassette, a 4,386-bp segment was found which consisted of the genes msr (E)-mph (E) bracketed by two IS 26 elements located in the same orientation. Insertion sequences of the type IS 26 are widespread among Enterobacteriaceae, but have rarely been

Chapter 7 General discussion

seen in Pasteurellaceae (KEHRENBERG et al . 2006). IS 26 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 IS 26 copies, nor upstream of the left IS 26 copy and downstream of the right IS 26 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 Tn 5706 element which was found 106 bp downstream of the right-hand IS 26 . Both terminal insertion sequences IS 1596 and IS 1597 present in the composite transposon Tn 5706 (KEHRENBERG et al . 1998) were absent. The Tn 5706 -homologous sequence in the part downstream of tetR stopped exactly at the position where otherwise the IS 1596 sequence was found. In the part downstream of tet (H), the Tn 5706 -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 Tn 5706 [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 Tn 5706 was shown. The location of tet (H) on a transposon may explain the wide dissemination of this tet gene among Pasteurellaceae members and its occurrence on plasmids and in the chromosomal DNA (KEHRENBERG et al. 1998).

General discussion Chapter 7

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 bla OXA-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 IS CR elements seemed to have played a key role. Moreover, the analysis of the two resistance gene regions clearly showed that P. multocida is able to acquire resistance genes from other Gram-negative bacteria, to incorporate them into its chromosomal DNA, and to use these genes to gain resistance against the respective antimicrobial agents.

Chapter 7 General discussion

7.2.3. Resistance mediating mutations in P. multocida 36950 Besides the resistance genes identified in the resistance regions 1 and 2, P. multocida 36950 exhibited resistance to antimicrobial agents, such as the fluoroquinolone enrofloxacin, for which resistance is often based on mutations in specific target genes. Fluoroquinolone resistance in P. multocida and other bovine respiratory tract pathogens has very rarely – if at all – been observed [Chapter 3 ]. As in many other bacteria, quinolone/fluoroquinolone resistance is most likely due to mutations in the genes gyrA and parC coding for DNA gyrase and topoisomerase IV (CÁRDENAS et al . 2001) . Analysis of the quinolone resistance determining regions (QRDR) within the genes gyrA and parC identified in P. multocida 36950 showed two bp exchanges in the QRDR of gyrA which resulted in amino acid alterations: GGT → AGT (Gly75-to- Ser75) and AGC → AGA (Ser83-to-Arg83). In addition, a single bp exchange in the QRDR of parC , TCA → TTA, which resulted in a Ser80-to-Leu80 exchange, was also seen in P. multocida 36950. While single amino acid exchanges within the QRDR of GyrA are usually only associated with resistance to the quinolone nalidixic acid, two and more amino acid exchanges in the QRDRs of GyrA and ParC accompany resistance to fluoroquinolones such as enrofloxacin. While alterations at codon 75 in gyrA have rarely been detected (PREISLER et al . 2006), alterations at codon 83 in gyrA and at codon 80 in parC have frequently been described in connection with fluoroquinolone resistance in other bacteria (HOOPER 2001; GIBELLO et al . 2004; PIDDOCK 2002). In P. multocida , only a single gyrA mutation AGC → ATC which results in a Ser83-to-Ile83 exchange has been described to be associated high level resistance to nalidixic acid (MIC >256 mg/L), but susceptibility to ciprofloxacin (MIC 0.12 mg/L) ( CÁRDENAS et al . 2001) . The mutations detected in gyrA and parC of P. multocida 36950 are to the best of our knowledge the first examples of fluoroquinolone resistance-mediating mutations in P. multocida . Table 2 shows a summary of all resistance genes and resistance-mediating mutations found in P. multocida 36950 including their associate resistance phenotypes [Chapter 3 ].

General discussion Chapter 7

Table 2: Antimicrobial Resistance genes, resistance-mediating mutations and their associated resistance phenotypes in P. multocida 36950

Antimicrobial agents MIC (mg/L) Resistance genes / mutations 1

Tetracycline 32 tetR-tet (H) Chloramphenicol / 16 / 8 floR Florfenicol Sulfonamides ≥ 512 sul2

Streptomycin ≥ 64 strA , strB , aadA25

Kanamycin / ≥ 32 / ≥ 32 aphA1 Neomycin Gentamicin 128 aadB

Spectinomycin ≥ 512 aadA25

Nalidixic acid / ≥ 256 / 2 G75S, S83R (GyrA); S80L (ParC) Enrofloxacin 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 bla OXA-2 is not functionally active in P. multocida 36950 for unknown reasons

2 The genes in square brackets play only an additional role in resistance to the respective antimicrobial agents

Chapter 7 General discussion

7.3. Multi-resistance mobile genetic element ICE Pmu1

7.3.1. Identification and general characteristics of ICE Pmu1 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 ICE Pmu1 , as it is the first ICE detected in P. multocida . ICE Pmu1 is 82,214 bp in size and was found to be integrated into the second of six genomic copies of a tRNA Leu . A copy of an integral tRNA Leu (Pmu_3620) proved to be part of the ICE Pmu1 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'). ICE Pmu1 included the resistance gene region 1 at its left terminus and the resistance region 2 close to its right terminus. ICE Pmu1 showed a G + C content (41.9 %) different from that of the genome of its host (40.4 %). The higher G + C content of ICE Pmu1 resulted from the higher G + C content of the sequences present in the two resistance gene regions. Within ICE Pmu1 , 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 ICE Pmu1 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 ICE Pmu1 are also present in the ICE from H. somni . However, the ICE from H. somni lacks most of the two ICE Pmu1 - associated resistance gene regions. Of resistance gene region 1, only one copy of the insertion sequence IS Apl1 and of the resistance gene region 2, only one copy of

General discussion Chapter 7

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. ICE Pmu1 is indicated by black lines.

Chapter 7 General discussion

ICE when inserted into the or genes of particular relevance are as boxes. Numbers above the various genes are in 36950 whole genome sequence (GenBank accession no. P. multocida e regions in grey represent the flanking regions of this . Th Pmu1 36950. The different genes are depicted and regions

P. P. multocida . ) Organization ICE of

indicated. The resistance gene regions 1 agreement and with 2 the are database entry shown of the Fig. 4: genome of CP003022

General discussion Chapter 7

Analysis of the coding sequences of ICE Pmu1 revealed the presence of the essential genes for the functionality of an ICE, like the genes involved in excision/integration and conjugative transfer. Two genes coding for phage integrases were identified close to the left attachment site ( attL ). The first integrase gene (Pmu_2700) was located 2,319 bp and the second (Pmu_2880) 19,284 bp from the left terminus. Both integrase proteins harboured in the C-terminus the three strongly conserved residues, the arginine residues in BOX A and BOX B (major clusters of similarity) and the active site tyrosine residue in BOX C (ESPOSITO and SCOCCA 1997; NUNES-DÜBY et al . 1998) [Chapter 4 ] A relaxase gene (Pmu_2890) was found downstream of the second integrase gene in the central region. This region harboured most of the core genes which encode the proteins involved in DNA cleavage [putative type I restriction-modification system methyltransferase subunit, (Pmu_2900)], proteins necessary for a conjugative transfer [a protein for the formation of type IV pilus (Pmu_3230), TraD- (Pmu_3190), TraG- (Pmu_3040), TraC-like (Pmu_3070) proteins], and a protein involved in DNA replication [DNA topoisomerase III (Pmu_3290)]. Moreover, genes for a protein with a lysozyme-like domain (Pmu_3210), a multicopper oxidase protein (Pmu_3360) and two other genes for enzymes potentially involved in the metabolism of alcohol as well as aldehydes and ketones were detected (Pmu_3370 and Pmu_3330). Downstream of the resistance gene region 2, genes coding for proteins involved in DNA replication, such as the single-stranded DNA-binding protein (Pmu_3540) and an ATPase involved in chromosome partitioning (Pmu_3610) were found. The analysis of this right-hand terminal region revealed also the presence of the gene dnaB (Pmu_3600) coding for the DNA helicase DnaB and a gene for a ParB family protein (Pmu_3590) with a predicted DNA nuclease function. This final core gene- containing region has been reported as the most conserved region among diverse proteobacterial ICEs (MOHD-ZAIN et al . 2004).

Chapter 7 General discussion

7.3.2. Transfer of ICE Pmu1 The ability of ICE Pmu1 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 bla OXA-2 genes in the E. coli host. In this regard, it should be noted that most of the resistance genes found in ICE Pmu1 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 ICE Pmu1 , 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 ICE Pmu1 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 ICE Pmu1 (Fig. 5).

General discussion Chapter 7

Leu g direct site was not are also shown. are also shown. attL Pmu1 quences of the tRNA sequence. The left attachment sites strains. The se

Pmu1 of different

copy in the same site, the true Leu Pmu1 tion tion of the ICE e right termini (DR-R) e oftermini (DR-R) right the inserted ICE into the tRNA

), ), the sequences involved in the crossover and the resultin Pmu1 attR transconjugants. M. haemolytica M. specific recombination of ICE - Site

) ) and the right attachment sites ( attL identified in the Fig. 5: are shown in the orientation that matches the orienta ( repeats onlocated the left and termini (DR-L) on th Due to the presence of at least part of a second ICE

Chapter 7 General discussion

Sequence analysis of the amplicons obtained by standard and inverse PCRs proved that the insertion point of the ICE Pmu1 in all transconjugants was located in a tRNA Leu . The tRNA Leu , 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 tRNA LeuX , 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 tRNA Leu 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 ICE Pmu1 . 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 ICE Pmu1 in the M. haemolytica 39229 transconjugant revealed the presence of at least the terminal part of a second ICE Pmu1 copy [Chapter 4 ].

7.3.3. ICE Pmu1 -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 ICE Pmu1 . 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 ICE Pmu1 . In this way, the studies of chapter 2 , 3 and 4 revealed a more comprehensive characterization of the genetic environment of the macrolide resistance genes and identified ten additional antimicrobial resistance genes.

General discussion Chapter 7

After the publication of these studies [Chapters 2-4], ICE Pmu1 -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 ICE Pmu1 and tested them for their transfer abilities. In this study, variants of ICE Pmu1 were detected, which harboured some or all 12 ICE Pmu1 -associated antimicrobial resistance genes. The complete sequence of an ICE Pmu1-related element, the ICE Mh1 , 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). ICE Mh1 may have evolved by a recombination event between ICE Pmu1 and a second ICE, possibly the putative ICE of M. haemolytica USDA-ARS-USMARC- 183 isolated in Kansas, USA. Five out of 12 ICE Pmu1 -associated antimicrobial resistance genes were found in ICE Mh1 , 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 ICE Pmu1 -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 ICE Pmu1-related elements in isolates from Nebraska feedlots, but not from those in Alberta, Canada. According to the authors, the same antimicrobial use protocol for BRD control and treatment has been used in in Nebraska and Alberta. However, calves with low weight and feedlots with high-density of animals were seen in Nebraska. The authors speculate that in the Nebraska those low-weight calves were likely to be submitted to metaphylactic treated upon arrival, due to the high risks of BRD acquisition, and that in a high-density production system higher amounts of antimicrobial agents are necessary for the prevention of diseases. Since the multi-

Chapter 7 General discussion

resistance ICE Pmu1 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 ICE Pmu1, which comprises 88 CDSs, was absent in these four genomes. According to the data provided by the National Center for Biotechnology Information (NCBI) the size of the genomes, the average GC content and the number of CDSs varied as 2.2 – 2.4 Mb,

General discussion Chapter 7

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 ICE Pmu1 was also not found in these drafted genomes. Phylogenetic analysis using 7,931 single nucleotide polymorphisms (SNPs) of common positions in all P. multocida strains revealed a very close relationship even among such unrelated strains from different geographic regions, serotypes, animal hosts and disease conditions (BOYCE et al . 2012). Additional, fully closed genomes are necessary for a better understanding of the pathogenic mechanisms and the host specificity of P. multocida strains.

Chapter 7 General discussion

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) s ystems have a defence function, they confer resistance against infection by

General discussion Chapter 7

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 ICE Pmu1 [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 tRNA Leu (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 ICE Pmu1 described in this doctoral thesis project is to the best of our knowledge the first ICE identified in P. multocida . It is closely related in its core genes to a family of diverse proteobacterial ICEs, but also harboured two regions of accessory genes which consisted mainly of insertion sequences and antimicrobial

Chapter 7 General discussion

resistance genes [Chapters 3 and 4 ]. A matter of concern is the high similarity among ICE Pmu1 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 ICE Mh1 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 ICE Pmu1 , may seriously decrease the therapeutic options for bovine respiratory disease. Moreover, the particular structure of the resistance gene regions may allow the incorporation of further cassette-borne resistance genes but also the acquisition of resistance genes via insertion sequence-mediated recombination processes. Since no new classes of antimicrobial agents for use in livestock animals are to be expected in the near future, the superior aim of all people, who prescribe and apply antimicrobial agents, must be to preserve the efficacy of the currently available antimicrobial agents for as long as possible [CHAPTER 6 ]. This includes measures to counteract the emergence of antimicrobial (multi-)resistance among bacteria from livestock animals. There is no fast and easy solution to the problem. More likely, it will be a joint approach that includes on one side (i) improved preventive measures such as vaccination, (ii) improved farm management accompanied by a tendency to implement integrated farming systems, (iii) improved hygiene on farms, and (iv) prudent and judicious use of antimicrobial agents. On the other side, more emphasis must be put on research to identify emerging resistance genes, the mobile genetic elements with which they are associated and the modes of spreading of these elements. Understanding the mechanism(s) of resistance and knowing the conditions of optimized horizontal gene transfer are important first steps to develop means and

General discussion Chapter 7

ways to inhibit the resistance mechanism (SCHWARZ and KEHRENBERG 2006) and to counteract resistance gene dissemination. Especially the knowledge about co- located resistance genes, which allow co-selection and persistence of resistance genes even in the absence of a direct selection pressure, is indispensable to predict the success or failure of measures such as the ban or the limitation of use of a certain antimicrobial agent in order to reduce resistance rates. Another issue is the non-therapeutic use of antimicrobial agents for growth promotion (reviewed by MARSHALL and LEVY 2011). Although antimicrobial growth promoters have been banned in 2006 from use in food-producing animals in the European Union, they are still used in many non-EU countries. The amount of antimicrobial agents used for growth promotion may be equal or even superior to the amount used in therapy (MARSHALL and LEVY 2011). It would be an option to consider a global ban of antimicrobial growth promoters in food animal production, especially since there are examples which showed that the ban of antimicrobial growth promoters had no negative impact on health and productivity of food-producing animals (WIERUP 2001; AARESTRUP et al . 2010). Since bacteria live in polymicrobial environments on the skin and the mucosal surfaces of humans and animals, there will always be partners for the exchange of genetic material. Therefore, it is impossible to prevent the dissemination of plasmids, transposons or ICEs within bacterial populations. However, using correct dosage schemes and choosing the most promising antimicrobial agent based on the results of in vitro susceptibility testing will minimize the spread of resistant bacteria and resistance genes. Commercial large-scale rearing of livestock without using antimicrobial agents is not possible to date. Although the use of antimicrobial agents is considered an important factor driving antimicrobial resistance, very limited detailed information on the use of antimicrobial agents in animals is currently available (SILLEY et al . 2012; BOS et al . 2013). However, it is necessary to understand which antimicrobial agents are used at which quantities for which purpose in which animal species. Factors like co-location of resistance genes on the same mobile genetic element, co-transfer of these resistance genes during spread of the element as well as co-selection and persistence of resistance genes during direct

Chapter 7 General discussion

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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HAFT, D.H., J. SELENGUT, E. F. MONGODIN and K. E. NELSON (2005): A guild of 45 CRISPR-associated (Cas) protein families and multiple CRISPR/Cas subtypes exist in prokaryotic genomes. PLoS Comput. Biol. 1, 474 - 483

HANSEN, L. M., L. M. MCMURRY, S. B. LEVY and D. C. HIRSH (1993): A new tetracycline resistance determinant, TetH, from Pasteurella multocida specifying active efflux of tetracycline. Antimicrob. Agents Chemother. 37, 2699 - 2705

HARPER, M., J. D. BOYCE and B. ADLER (2012): The key surface components of Pasteurella multocida : capsule and lipopolysaccharide. Curr. Top. Microbiol. Immunol. 361, 39 – 51

General discussion Chapter 7

HIGHLANDER, S. K., S. WEISSENBERGER, L. E. ALVAREZ, G. M. WEINSTOCK and P. B. BERGET (2006): Complete nucleotide sequence of a P2 family lysogenic bacteriophage, φMhaA1- PHL101, from Mannheimia haemolytica serotype A1. Virology 350, 79 - 89

HOCHHUT, B., Y. LOTFI, D. MAZEL, S. M. FARUQUE, R. WOODGATE and M. K. WALDOR (2001): Molecular analysis of antibiotic resistance gene clusters in Vibrio cholerae O139 and O1 SXT constins. Antimicrob. Agents Chemother. 45, 2991 - 3000

HOOPER, D. C. (2001): Mechanisms of action of antimicrobials: focus on fluoroquinolones. Clin. Infect. Dis. 32, 9 - 15

HSU, Y. M., H. K. SHIEH, W. H. CHEN, T. Y. SUN and J. H. SHIANG (2007): Antimicrobial susceptibility, plasmid profiles and haemocin activities of Avibacterium paragallinarum strains. Vet. Microbiol. 124: 209 - 218

ITO, H., H. ISHII and M. AKIBA (2004): Analysis of the complete nucleotide sequence of an Actinobacillus pleuropneumoniae streptomycin-sulfonamide resistance plasmid, pMS260. Plasmid 51: 41 - 47

KADLEC, K., I. WIEGAND, C. KEHRENBERG and S. SCHWARZ (2007): Studies on the mechanisms of β-lactam resistance in Bordetella bronchiseptica . J. Antimicrob. Chemother. 59, 396 - 402

Chapter 7 General discussion

KANG, M., R. ZHOU, L. LIU, P. R. LANGFORD and H. CHEN (2009): Analysis of an Actinobacillus pleuropneumoniae multi-resistance plasmid, pHB0503. Plasmid 61, 135 - 139

KEHRENBERG, C., C. WERCKENTHIN and S. SCHWARZ (1998): Tn 5706 , a transposon-like element from Pasteurella multocida mediating tetracycline resistance. Antimicrob. Agents Chemother. 42, 2116 - 2118

KEHRENBERG, C., and S. SCHWARZ (2001): Occurrence and linkage of genes coding for resistance to sulfonamides, streptomycin and chloramphenicol in bacteria of the genera Pasteurella and Mannheimia . FEMS Microbiol. Lett. 205, 283 - 290

KEHRENBERG, C., N. T. THAM and S. SCHWARZ (2003): New plasmid-borne antibiotic resistance gene cluster in Pasteurella multocida . Antimicrob. Agents Chemother. 47, 2978 - 2980

KEHRENBERG, C., and S. SCHWARZ (2005): Plasmid-borne florfenicol resistance in Pasteurella multocida . J. Antimicrob. Chemother. 55, 773 - 775

KEHRENBERG, C, D. MEUNIER, H. TARGAN, T A. CLOECKAERT, S. SCHWARZ and J. Y. MADEC (2006): Plasmid-mediated florfenicol resistance in Pasteurella trehalosi . J. Antimicrob. Chemother. 58: 13 - 17

KEHRENBERG, C., J. WALLMANN and S. SCHWARZ (2008): Molecular analysis of florfenicol-resistant Pasteurella multocida isolates in Germany. J. Antimicrob. Chemother. 62, 951 - 955

General discussion Chapter 7

KLIMA, C. L., R. ZAHEER, S. R. COOK C. W. BOOKER, S. HENDRICK, T. W. ALEXANDER and T. A. MCALLISTER (2014): Pathogens of bovine respiratory disease in North American feedlots conferring multidrug resistance via integrative conjugative elements. J. Clin. Microbiol. 52, 438 - 448

MARSHALL, B. M., and S. B. LEVY (2011): Food animals and antimicrobials: impacts on human health. Clin. Microbiol. Rev. 24, 718 – 733

MAY, B. J., Q. ZHANG, L. L. LI, M. L. PAUSTIAN, T. S. WHITTAM and V. KAPUR (2001): Complete genomic sequence of Pasteurella multocida , Pm70. Proc. Natl. Acad. Sci. U S A. 98, 3460 - 3465

MOLLET, B., S. IIDA, J. SHEPHERD and W. ARBER (1983): Nucleotide sequence of IS 26 , a new prokaryotic mobile genetic element. Nucleic Acids Res. 11, 6319 - 6330

NUNES-DÜBY, S. E., H. J. KWON, R. S. TIRUMALAI, T. ELLENBERGER and A. LANDY (1998): Similarities and differences among 105 members of the Int family of site-specific recombinases. Nucleic Acids Res. 26, 391 - 406

Chapter 7 General discussion

PAN, J. C., R. YE, H. Q. WANG, H. Q. W. XIANG, ZHANG, X. F. YU, D. M. MENG and Z. S. HE (2008): Vibrio cholerae O139 multiple-drug resistance mediated by Yersinia pestis pIP1202- like conjugative plasmids. Antimicrob. Agents Chemother. 52, 3829 - 3836

PETTERSSON, E., J. LUNDEBERG and A. AHMADIAN (2009): Generations of sequencing technologies. Genomics. 93, 105 – 111

PIDDOCK, L. J. (2002): Fluoroquinolone resistance in Salmonella serovars isolated from humans and food animals. FEMS Microbiol. Rev. 26, 3 - 16

POIREL, L., W. MANSOUR, O. BOUALLEGUE and P. NORDMANN (2008): Carbapenem-resistant Acinetobacter baumannii isolates from Tunisia producing the OXA-58-like carbapenem-hydrolyzing oxacillinase OXA-97. Antimicrob. Agents Chemother. 52, 1613 - 1617

POURCEL, C., G. SALVIGNOL and G.VERGNAUD (2005): CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of bacteriophage DNA, and provide additional tools for evolutionary studies. Microbiology 151, 653 - 63

PREISLER, A., M. A. MRAHEIL and P. HEISIG (2006): Role of novel gyrA mutations in the suppression of the fluoroquinolone resistance genotype of vaccine strain Salmonella Typhimurium vacT (GyrA D87G). J. Antimicrob. Chemother. 57, 430 - 436

General discussion Chapter 7

PULLINGER, G. D., T. BEVIR and A. J. LAX (2004): The Pasteurella multocida toxin is encoded within a lysogenic bacteriophage. Mol. Microbiol. 51, 255 - 269

RELMAN, D. A., M. DOMENIGHINI, E. TUOMANEN, R. RAPPUOLI and S. FALKOW (1989): Filamentous hemagglutinin of Bordetella pertussis : nucleotide sequence and crucial role in adherence. Proc. Natl. Acad. Sci. USA. 6, 2637 - 2641

ROSE, S., B. DESMOLAIZE, P. JAJU, C. WILHELM, R. WARRASS and S. DOUTHWAITE (2012): Multiplex PCR to identify macrolide resistance determinants in Mannheimia haemolytica and Pasteurella multocida . Antimicrob. Agents Chemother. 56, 3664 - 3669

SAN MILLAN, A., J. A. ESCUDERO, B. GUTIERREZ, L. HIDALGO, N. GARCIA, M. LLAGOSTERA, L. DOMINGUEZ and B. GONZALEZ-ZORN (2009): Multiresistance in Pasteurella multocida is mediated by coexistence of small plasmids. Antimicrob. Agents Chemother. 53, 3399 - 3404

Chapter 7 General discussion

SCHWARZ, S., and C. KEHRENBERG (2006): Old dogs that learn new tricks: modified antimicrobial agents that escape pre-existing resistance mechanisms. Int. J. Med. Microbiol. 296 Suppl 41, 45 - 49

SHEN, P., Z. WEI, Y. JIANG, X. DU, S. JI, Y. YU and L. LI (2009): Novel genetic environment of the carbapenem-hydrolyzing β-lactamase KPC-2 among Enterobacteriaceae in China. Antimicrob. Agents Chemother. 53, 4333 - 4338

SILLEY, P., S. SIMJEE and S. SCHWARZ (2012): Surveillance and monitoring of antimicrobial resistance and antibiotic consumption in humans and animals. Rev. Sci. Tech. Off. Int. Epiz. 31, 105 - 120

TEGETMEYER, H. E., S. C. JONES, P. R. LANGFORD and N. BALTES (2008): IS Apl1 , a novel insertion element of Actinobacillus pleuropneumoniae , prevents ApxIV-based serological detection of serotype 7 strain AP76. Vet. Microbiol. 128, 342 - 353

ZHANG, J., R. CHIODINI, A. BADR and G. ZHANG (2011): The impact of next-generation sequencing on genomics. J. Genet. Genomics 38, 95 - 109

WATTS, J. L., and M. T. SWEENEY (2010): Antimicrobial resistance in bovine respiratory disease pathogens: measures, trends, and impact on efficacy. Vet. Clin. North Am. Food Anim. Pract. 26, 79 - 88

General discussion Chapter 7

WIERUP, M. (2001): The Swedish experience of the 1986 year ban of antimicrobial growth promoters, with special reference to animal health, disease prevention, productivity, and usage of antimicrobials. Microb. Drug Resist. 7, 183 - 190

WILKIE, I. W., M. HARPER, J. D. BOYCE and B. ADLER (2012): Pasteurella multocida : diseases and pathogenesis. Curr. Top. Microbiol. Immunol. 361, 1 - 22

ZARRILLI, R., D. VITALE, A. DI POPOLO, M. BAGATTINI, Z. DAOUD, A. U. KHAN, C. AFIF and M. TRIASSI (2008):

A plasmid-borne bla OXA-58 gene confers imipenem resistance to Acinetobacter baumannii isolates from a Lebanese hospital. Antimicrob. Agents Chemother. 52, 4115 - 4120

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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 ICE Pmu1 . It was also the first report of an ICE in P. multocida [Chapter 3 ]. In addition to the three macrolide/triamilide resistance genes, another nine antimicrobial resistance genes were found to be

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part of ICE Pmu1. 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 bla OXA-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 ICE Pmu1 harbours 88 genes. The core genes of ICE Pmu1 , which are involved in excision/integration and conjugative transfer, resemble those found in a 66,641 bp ICE from Histophilus somni . ICE Pmu1 integrates into a tRNA Leu 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 tRNA Leu for integration and produces closely related 13 bp direct repeats at the integration site. The presence of ICE Pmu1 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 ICE Pmu1 -associated resistance genes in the transconjugands. The gene bla OXA-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

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B130. These observations were confirmed with P. multocida and M. haemolytica field isolates that carried the three genes in different combinations [CHAPTER 5 ]. ICE Pmu1 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 ].

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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

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Resistenzgene Bestandteil eines mobilen genetischen Elements, des integrativen und konjugativen Elements ICE Pmu1 , waren. ICE Pmu1 ist das erste bei P. multocida jemals beschriebene ICE [Chapter 3]. Zusätzlich zu den drei Makrolid/Triamilid- Resistenzgenen trägt ICE Pmu1 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, bla OXA-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 ICE Pmu1 besitzt insgesamt 88 Gene. Die Gene von ICE Pmu1 , 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. ICE Pmu1 integriert in eine tRNA Leu und wird von 13 bp großen direkten Sequenzwiederholungen flankiert. ICE Pmu1 überträgt sich durch Konjugation in andere Bakterien wie P. multocida , M. haemolytica und E. coli , wo es auch eine tRNA Leu zur Integration nutzt und eng verwandte 13 bp große direkte Sequenzwiederholungen an der Integrationsstelle produziert. ICE Pmu1 und seine zirkuläre Zwischenform wurden in den Transkonjuganden mittels PCR- und Sequenzanalysen bestätigt. PCR-Analysen und Empfindlichkeitsprüfungen zeigten dass die ICE Pmu1 -assoziierten Resistenzgene in den Transkonjuganden funktionell aktiv waren. Lediglich das Gen bla OXA-2 war in den P. multocida - und M. haemolytica - Transkonjuganden inaktiv, in dem E. coli -Transkonjugand jedoch aktiv [Chapter 4 ].

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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 ]. ICE Pmu1 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 ].

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ACKNOWLEDGMENTS

Background: In the beginning was the unknown antimicrobial resistance mechanism 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. multo RESISTANT cida , 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 closed Manuscripts were written 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!

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