Living Together in a World of Extended-Spectrum Cephalosporin Resistance: Molecular Snapshots of a Complex Epidemiology

Living together in a world of extended-spectrum cephalosporin resistance: molecular snapshots of a complex epidemiology

Apostolos Liakopoulos Living together in a world of extended-spectrum cephalosporin resistance: molecular snapshots of a complex epidemiology

Printing of this thesis was financially supported by the Wageningen Bioveterinary Research and the Department of Infectious Diseases and Immunology, Utrecht University

PhD Thesis, Utrecht University, The Netherlands

ISBN: 978-94-6233-787-9

Cover: Apostolos Liakopoulos & Bo Derks Layout and printing: Gildeprint, Enschede Living together in a world of extended-spectrum cephalosporin resistance: molecular snapshots of a complex epidemiology

Samenleven in een wereld van extended-spectrum cefalosporine resistentie: moleculaire snapshots van een complexe epidemiologie.

(met een samenvatting in het Nederlands)

Proefschrift

ter verkrijging van de graad van doctor aan de Universiteit Utrecht op gezag van de rector mag- nificus, prof.dr. G.J. van der Zwaan, ingevolge het besluit van het college voor promoties in het openbaar te verdedigen op donderdag 30 november 2017 des middags te 12.45 uur

door

Apostolos Liakopoulos geboren op 29 December 1984 te Athene, Griekenland Promotor: Prof.dr. D.J Mevius

Copromotoren: Dr. H. Smith Dr. M.S.M. Brouwer

This thesis was accomplished with financial support from Dutch Ministry of Economic Affairs through the 1Health4Food (1H4F) project under the ESBL Attribution (ESBLAT) consortium (project number: TKI-AF-12067). To my beloved niece Sotiria and nephew Dimitrio.

Contents

Chapter 1 General introduction 9

Chapter 2A Extended-spectrum cephalosporin-resistant Salmonella enterica 39 serovar Heidelberg strains, the Netherlands

Chapter 2B Molecular characterization of extended-spectrum cephalosporin- 55 resistant Enterobacteriaceae from wild kelp gulls in South America

Chapter 3A High prevalence of intra-familial co-colonization by extended- 65 spectrum cephalosporin resistant Enterobacteriaceae in preschool children and their parents in Dutch households

Chapter 3B Molecular characterization of ESBL-producing Escherichia coli in 89 pigs and pig farmers – a longitudinal study

Chapter 4A A review of SHV extended-spectrum β-lactamases: neglected yet 99 ubiquitous

Chapter 4B Plasmid epidemiology of SHV-12-producing Escherichia coli from 163 human and animal origin: X factor(s) of an emerging plasmid family

Chapter 5 General discussion 187

Appendix Summary in Dutch 205 Summary in English 209 Acknowledgments 213 About the author 217 List of publications 219

CHAPTER 1

General introduction

General introduction 1 The introduction of antibiotics to clinical therapy has revolutionized modern medicine since their discovery at the beginning of the twentieth century by Gerhardt Domagk (sulfamidochrysoidine) and Alexander Flemming (penicillin)1,2. Antibiotics constitute one of the most successful forms of therapy due to their bactericidal or bacteriostatic activity without damaging host cells and tissues, enabling the control of infectious diseases and the morbidity and mortality that accompany them. As a result they rank amongst the most commonly prescribed drugs, with more than 70 billion clinical doses administered globally in 20103. However, their efficacy is gradually compromised by the emergence and dissemination of antibiotic resistance4. It is estimated that antibiotic resistance will result in 10 million deaths every year by 2050, followed by a reduction in Gross Domestic Product (GDP) ranging between 2% and 3.5%5. In the Communique of the G20 Summit held in Hangzhou, China, in 2016, the threat of antibiotic resistance was listed among the five factors with great impact on world economy, while the UN General Assembly High-Level Meeting of Heads of State agreed on a global collaboration, highlighting the worldwide attention that this issue receives6.

Although antibiotic resistance pre-dates human antibiotic use7, the introduction of antibiotics in clinical and agricultural settings led to the accumulation and development of resistance mechanisms by bacteria and subsequently to the emergence of multidrug resistant bacterial infections, resulting in a post-antibiotic era (Figure 1)8. Genes conferring antibiotic resistance have been identified using function-based or sequence-based screening of metagenomic libraries of natural and host-associated environments, underscoring that antibiotic resistance constitutes not only a public health threat but also an ecological issue with respect to the environment, animals and humans9,10.

β-lactam antibiotics including penicillins, cephalosporins, monobactams and carbapenems constitute the most important family of antimicrobial agents, in terms of both the large number of compounds available and prescription volume11. The firstβ -lactam analogue (benzyl-penicillin) poorly penetrated Gram-negative bacteria and selected for penicillin-resistant penicillinase-producing staphylococci, especially Staphylococcus aureus12. In response, semi-synthetic penicillins that can either penetrate Gram-negative bacteria (e.g. ampicillin and carbenicillin) or that are able to evade hydrolysis from penicillinases (methicillin and oxacillins) were developed in the early 1960s11. The usage of the β-lactam analogues against Gram-negative bacteria was compromised by the selection and dissemination of plasmid-mediated penicillinases (e.g. TEM-1 and SHV-1) among Enterobacteriaceae, imposing the development of second-, third- and fourth-generation cephalosporins (e.g. cefuroxime, cefotaxime, ceftriaxone, ceftazidime and cefepime) and of β-lactamase inhibitors (e.g. clavulanic acid)13-15. In the subsequent years, cephalosporins have been extensively

General introduction | 11 Figure 1. The timeline of key events in the emergence of antibiotic resistance (adapted from Ventola)16. Bactericidal and bacteriostatic antibiotics are color-coded blue and green respectively. Dates are based upon early reports of resistance in the literature. In the case of pan-drug-resistant Acinetobacter and Pseudomonas, the date is based upon reports of health care transmission or outbreaks. Note: penicillin was in limited use prior to widespread population usage in 1943. R: resistant, XDR: extensively drug-resistant and PDR: pan-drug-resistant.

12 | Chapter 1 used worldwide as primary therapy of infections, such as pneumonia and intra-abdominal sepsis, resulting in the emergence of cephalosporin resistance17,18. As a consequence, the use of carbape- 1 nems (last resort antibiotics) has been increased19, leading to the subsequent rise of carbapenem resistance and almost untreatable bacterial infections (Figure 1)20.

β-Lactam resistance mechanisms

Target modification The targets for β-lactam antibiotics are cell wall-synthetizing enzymes21, known as penicillin-binding proteins (PBPs), consisting of transpeptidase or carboxypeptidase domains, which are involved in the terminal steps of peptidoglycan cross-linking in both Gram-negative and Gram-positive bacteria22,23. β-lactams mimic the substrate of penicillin-binding proteins, occupying their active site where a serine residue attacks the carbonyl of the β-lactam ring, resulting in the opening of the ring and the formation of a covalent acyl-enzyme complex23. The hydrolysis of this complex is very slow, effectively preventing peptidoglycan synthesis and eventually leading to bacterial cell death24. However, the alteration of endogenous PBPs to exhibit low-affinity for β-lactam antibiotics by either point mutations or homologous recombination, the acquisition of an additional low- affinity PBP, the overexpression of an endogenous low-affinity PBP or a combination of the above have been employed by bacteria in order to confer resistance to β-lactams24. Clinical resistance to β-lactams in Enterobacteriaceae is not commonly associated with target modification, yet resistance conferred by point mutations in PBPs has been documented25,26.

Decreased uptake Hydrophilic antibiotics, such as β-lactams, must diffuse across the outer membrane of the bacterial cell through water-filled channels formed by a set of specialized outer membrane proteins called porins27, and then cross the periplasm before reaching their targets proteins, known as penicillin- binding proteins (PBPs) which lie on the outer surface of the cytoplasmic membrane28. Therefore, mutations causing (most commonly) porin loss, a modification of the size or conductance of the porin channel or a lower expression level of a porin have a direct impact on the uptake of β-lactams, reducing their steady-state periplasmic concentration leading to a reduction in PBP inactivation29. Although the involvement of porins in antibiotic resistance has been described in many bacterial species, one of the earliest examples is the involvement of the OmpF porin from Escherichia coli in β-lactam resistance30.

General introduction | 13 To regulate their internal environment by preventing the intracellular accumulation of toxic compounds, bacteria have evolved energy-dependent systems to pump such molecules out of the cell (multidrug efflux pumps) in a process that does not involve the alteration or degradation of these molecules31. The occurrence of mutational events leading to an increased expression of a given efflux pump or its efficiency towards a particular compound, results subsequently in the decreased periplasmic accumulation of this compound32. One of the best-studied examples is the AcrAB-TolC RND family pump of E. coli, known to transport several compounds against their concentration gradient including β-lactams33,34. Upregulated efflux pumps may work synergistically with porin modifications, augmenting dramatically the discharge of antibiotics35.

Enzymatic inactivation The most significant mechanism by which resistance to β-lactams arises, is the production of β-lactam hydrolysing enzymes, known as β-lactamases36. β-lactamases probably participate in cell- wall metabolism, as evidenced by the fact that the expression of inducible AmpC β-lactamases and peptidoglycan recycling enzymes are closely linked37. However, they are able to bind, acylate, and use a strategically located water molecule to hydrolyse and subsequently inactivate the β-lactam antibiotics before they reach their PBP target38. Two major classification schemes exist to categorize β-lactamase enzymes (Table 1) based on their amino acid sequence homology (Ambler classes A through D) or their substrate and inhibitor profile (Bush-Jacoby-Medeiros groups 1 through 4)39-41. β-lactamases can also be loosely identified based on their substrate specificities as penicillinases, AmpC-type cephalosporinases (AmpCs), extended-spectrum β-lactamases (ESBLs) and carbapenemases (Table 1)42. The enzymatic inactivation of extended-spectrum cephalosporins (ESCs) is the focus of the present thesis and it will be described more thoroughly in the next paragraphs.

Extended-spectrum cephalosporin resistance: an overview

Extended-spectrum cephalosporins, such as cefoperazone, cefotaxime, ceftazidime, ceftriaxone (third-generation) are potent and safe antibiotics with broad-spectrum activity and favourable pharmacokinetic/ pharmacodynamic characteristics43. ESCs rank among the most frequently prescribed antibiotics for both mild-to-serious community- and healthcare-associated infections44. The selection pressure conferred by the use of ESCs in both human and veterinary medicine, and in agriculture practice resulted in a significant increase of resistance, followed by prolonged treatments, increased morbidity and mortality, as well as inflating health care costs45. The prevalence of ESC-resistance varies depending upon geographic location and patient-specific risk factors, however resistance to ESCs in Gram-negative bacteria (especially Enterobacteriaceae) constitutes an urgent public health threat nationally and internationally17,18,46,47.

14 | Chapter 1 Table 1. Classification schemes for bacterialβ -lactamases (adapted from Bush & Jacoby)41. Inhibited by 1 Bush- Molecular Repre- Distinctive CA or Jacoby class EDTA Defining characteristic(s) sentative substrate(s) TZB* group (subclass) enzyme(s) 1 C Cephalosporins No No Greater hydrolysis of cephalosporins E. coli AmpC, than benzylpenicillin; hydrolyses P99, ACT-1, cephamycins CMY-2, FOX-1, MIR-1 1e C Cephalosporins No No Increased hydrolysis of ceftazidime GC1, CMY-37 and often other oxyimino-β-lactams 2a A Penicillins Yes No Greater hydrolysis of benzylpenicillin PC1 than cephalosporins 2b A Penicillins, early Yes No Similar hydrolysis of benzylpenicillin TEM-1, TEM-2, cephalosporins and cephalosporins SHV-1 2be A Extended-spectrum Yes No Increased hydrolysis of oxyimino-β- TEM-3, SHV-2, cephalosporins, lactams (cefotaxime, ceftazidime, CTX-M-15, monobactams ceftriaxone, cefepime, aztreonam) PER-1, VEB-1 2br A Penicillins No No Resistance to clavulanic acid, sul- TEM-30, SHV- bactam, and tazobactam 10 2ber A Extended-spectrum No No Increased hydrolysis of oxyimino-β- TEM-50 cephalosporins, lactams combined with resistance monobactams to clavulanic acid, sulbactam, and tazobactam 2c A Carbenicillin Yes No Increased hydrolysis of carbenicillin PSE-1, CARB-3 2ce A Carbenicillin, Yes No Increased hydrolysis of carbenicillin, RTG-4 cefepime cefepime, and cefpirome 2d D Cloxacillin Variable No Increased hydrolysis of cloxacillin or OXA-1, OXA- oxacillin 10 2de D Extended-spectrum Variable No Hydrolyses cloxacillin or oxacillin and OXA-11, cephalosporins oxyimino-β-lactams OXA-15 2df D Carbapenems Variable No Hydrolyses cloxacillin or oxacillin and OXA-23, carbapenems OXA-48 2e A Extended-spectrum Yes No Hydrolyses cephalosporins. Inhibited CepA cephalosporins by clavulanic acid but not aztreonam 2f A Carbapenems Variable No Increased hydrolysis of carbape- KPC-2, IMI-1, nems, oxyimino-β-lactams, cepha- SME-1 mycins 3a B (B1) Carbapenems No Yes Broad-spectrum hydrolysis including IMP-1, VIM-1, B (B2) carbapenems but not monobactams CcrA, IND-1

L1, CAU-1, GOB-1, FEZ-1 3b B (B3) Carbapenems No Yes CphA, Sfh-1 *CA: clavulanic acid and TZB: tazobactam

General introduction | 15 The emergence of ESC-resistance is greatly attributed to the production of two types of cephalosporinases (ESBLs and AmpCs)17,18, yet additional resistance mechanisms have emerged including low-affinity PBPs24, efflux pumps48 and repressed porin expression29. The ESBL type consists of enzymes that hydrolyse their substrates by forming an acyl enzyme through an active site serine, conferring resistance to penicillins, first-, second- and third-generation cephalosporins, as well as monobactams (but not cephamycins or carbapenems), while they are inhibited by β-lactamase inhibitors17. According to the β-lactamases classification schemes, these enzymes belong either to class A and subgroup 2be or to class D and subgroup 2de41. The AmpC type consists of serine-β- lactamases exhibiting similar hydrolytic spectrum with ESBLs, but β-lactamase inhibitors have much less effect on AmpC β-lactamases, although some are inhibited by tazobactam or sulbactam18. In addition, they can hydrolyse cephamycins and in a low rate carbapenems18. These β-lactamases, based on the classification schemes, belong to class C and the subgroups 1 or 1e41.

Genes conferring ESC-resistance are commonly located on plasmids, hence the ability to classify these plasmids based on their phylogenetic relatedness in homogeneous plasmid families (inc/rep- types) is useful in order to track the transmission and the evolutionary origin of extended-spectrum cephalosporinase (ESCase) genes49. As a consequence, a molecular classification scheme based on plasmid replicons (replicase gene and cis-acting regulatory elements) present in Enterobacteria- ceae has been proposed (PCR-based inc/rep typing)50. Several of these families consist of plasmids with propensity to acquire ESCase genes and rapidly disseminate among Enterobacteriaceae, underscoring the existence of epidemic plasmid families51, for some of which further subtyping schemes have been proposed52-56. Acquisition of ESCase genes by epidemic plasmids contributes to their emergence and dissemination among different bacterial species and/or diverse reservoirs.

Forces shaping the complex epidemiology of ESC-resistant Enterobacteriaceae

Plethora of acquired extended-spectrum cephalosporinase genes More than 350 ESBL genes have been described up until today, mostly belonging to four β-lactamase gene families, namely CTX-M, OXA, SHV and TEM (https://www.lahey.org/Studies/). The CTX-M β-lactamases are closely related to genes found in the chromosome of several Kluy- vera spp57-62, while their name reflects their hydrolytic activity at least against cefotaxime17. These β-lactamases belong to a heterogeneous family that consists of at least five groups designated as CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-9 and CTX-M-25 (named after the archetypal enzymes of each group), that differ from each other by ≥10% amino acid residues63. The CTX-M β-lactamases exhibit 40% or less identity with ESBLs of the OXA, SHV and TEM families17. In contrast with CTX-M

16 | Chapter 1 β-lactamases, the majority of enzymes belonging to OXA, SHV and TEM families are not able to hydrolyse extended-spectrum cephalosporins, however, derivative enzymes within these families 1 with ESCase phenotypes have evolved from parental enzymes with narrow spectra by accumulating amino acid substitutions17. A variety of distinct ESBL gene families have been described, including BES, GES, IBC, PER, SFO, TLA and VEB, but their prevalence is very low among Enterobacteriaceae17. In addition to the high number of known ESBL genes, approximately 250 ESCase genes of the AmpC type have been identified (https://www.lahey.org/Studies/). The majority of these genes belong to the CMY family, whereas several other less prevalent families include ACC, ACT, DHA, FOX, LAT, MIR and MOX18. However, only minor amino acid differences discriminate these fami- lies18.

This enormous number of acquired resistance genes able to confer ESC-resistance phenotype greatly influences the complexity of the epidemiology of the Enterobacteriaceae.

Mobility of extended-spectrum cephalosporinase genes among Enterobacteriaceae Extended-spectrum cephalosporinase genes predominantly reported in Enterobacteriaceae have been associated with mobile genetic elements (MGEs), namely insertion sequences (ISs), transposons, plasmids and phage-related elements which are pivotal in their persistence and dissemination within bacterial species and reservoirs64. The diversity of these genetic elements and their involvement in intra- and inter-species dissemination of ESCase genes enhance the complex epidemiology of ESC-resistant Enterobacteriaceae. The most important combinations of mobile elements with ESCase genes, with special emphasis on plasmids, are discussed in the following paragraphs.

Mobile genetic elements as carriers of extended-spectrum β-lactamase genes

Genes encoding enzymes of the CTX-M family (blaCTX-M) have been mobilized from the chromosome of different Kluyvera spp57-62, owing to the involvement of several genetic elements such as the ISEcp1 and ISCR1, as well as phage-related elements65-68. ISEcp1-like elements (IS1380 family) have been identified upstream of genes belonging to the blaCTX-M-1, blaCTX-M-2, blaCTX-M-9 and 69-74 blaCTX-M-25 gene groups . A single copy of ISEcp1 is sufficient to mobilise the adjacent sequences by transposition, after recognition of a variety of DNA sequences as right inverted repeats (IRRs) and enhance the expression of these genes by providing a promoter sequence75. Another mobile

76 element found in association with blaCTX-M genes, especially of blaCTX-M-2 and blaCTX-M-9 groups , is ISCR1 (IS91 family) that is able to mobilise the nearby sequence by rolling-circle transposition77.

The IS-mediated transposition of blaCTX-M genes on conjugative plasmids has been documented in several cases. E. coli harbouring blaCTX-M-1 gene on IncI1α/γ plasmids was identified in poultry faecal samples collected from 10 slaughterhouses in France78, and subsequently similar E. coli isolates

General introduction | 17 were recovered from human patients in different parts of France79, highlighting a potential link between animals and humans for the dissemination of this gene and plasmid combination. Since then, IncI1α/γ plasmids encoding the blaCTX-M-1 gene have been widely reported in Enterobacte- 80-93 riaceae from human and animal origin . In addition to IncI1α/γ plasmids, blaCTX-M-1 gene has been documented on IncN plasmids in isolates recovered from both humans and animals80,81,94-100, suggesting the cross-transmission of IncN plasmids between reservoirs.

The spread of blaCTX-M-2 gene in Enterobacteriaceae is largely attributed to IncHI2 plasmids, where 53,71,101-105 blaCTX-M-2 is mainly associated with ISCR1 embedded in complex class-1 integrons . Pla- smids belonging to the IncL/M family are responsible for the dissemination of blaCTX-M-3 among Enterobacteriaceae in Algeria, Belgium, Bulgaria, China, Croatia, France, Korea and Poland, usually co-residing with armA, a 16S rRNA methylase conferring pan-aminoglycoside resistance106-115.

Similarly to blaCTX-M-2, plasmids of the IncHI2 family are also associated with maintenance and dif- 71,92,116-120 fusion of blaCTX-M-9 in Enterobacteriaceae, particularly in Salmonella species .

In addition to the previous blaCTX-M genes, a 1-year longitudinal study of a dairy farm in the United

Kingdom indicated the emergence of genetically unrelated E. coli encoding blaCTX-M-14 on an IncK 121 plasmid . Since then, IncK plasmids encoding blaCTX-M-14 in association with ISEcp1 have been observed in E. coli isolates of human and animal origin from different countries (Australia, China, Denmark, France, Germany, Korea, Netherlands, Portugal and Spain) underscoring an epidemic

79,122-131 dissemination of IncK plasmids encoding blaCTX-M-14 within human and animal reservoirs .

Currently, blaCTX-M-15 is the most widespread and prevalent gene invading all human and animal compartments as well as the environment all over the world132. This gene is mainly associated with ISEcp1 that contributes to its expression and mobilization from the Kluyvera genome to IncF

133,134 plasmids . However, it has been documented that IncF plasmids encoding blaCTX-M-15 gene vary in size (50 to 200 kb), carry the repFII replicon alone or in combination with repFIA and/or repFIB, and show different antisense RNA sequence variants in the repFII replicon49,135,136; as a consequence IncF plasmids form a rather heterogeneous group. IncF plasmids encoding blaCTX-M-15 have been recorded among Enterobacteriaceae of diverse origin worldwide including Belgium, Canada, China, France, India, Kuwait, Portugal, Spain, Sweden, Switzerland, and United Kingdom51,137.

The majority of the blaTEM genes has been shown to be carried by Tn3 transposons, but also by Tn1 and Tn2 transposons138. These are three of the earliest described bacterial transposons and exhibit high identity with each other containing a transposase (tnpA), a resolvase (tnpR) gene and

138 a resolution site (res) . The association of blaTEM ESBL genes (blaTEM-3, blaTEM-21, and blaTEM-24) with

18 | Chapter 1 Tn3 transposons, which apart from the usual transposition processes are able to move via TnpA- dependent, TnpR- and recA-independent one-ended transposition139, resulted in their mobilization 1 49 to plasmids, mostly of the IncA/C family . In contrast, the blaTEM-52 gene that constitutes the most prevalent ESCase gene of the TEM family, was first identified onIncI1 α/γ plasmids in various Sal- monella enterica serotypes from Belgium and France140. This finding was followed by reports of

IncI1α/γ plasmids encoding blaTEM-52 mostly in E. coli of human and animal origin from Belgium, Denmark, France, Germany, Netherlands and Portugal79,91,99,102,141-143.

Although only few studies have investigated the genetic support of ESCase genes of the SHV family, analysis of the flanking sequences of several of these genes bla( SHV-2, blaSHV-2a, blaSHV-5 and 144 blaSHV-12) among Enterobacteriaceae, has revealed their association mostly with the IS26 element , reflecting its involvement in the mobilization of blaSHV ancestor gene from the chromosome of Kleb- siella pneumoniae145. This element contributes to their expression by supplying a hybrid promoter composed of a -35 box located into the IR of IS26 and a -10 box located in the vicinity of the blaSHV 146,147 genes . The IS26-mediated mobilization of blaSHV-12 has been documented predominantly on IncI1α/γ plasmids among Enterobacteriaceae of diverse origin in Bulgaria, Ethiopia, France, Italy, Poland, Portugal, Spain and Taiwan79,80,89,108,148-154.

Mobile genetic elements as carriers of AmpC β-lactamase genes Although several genetic elements have been shown to be involved in the mobilization of AmpC genes onto plasmids155-158, only the most relevant are discussed here. ISEcp1 has been identified in association with different blaCMY genes, especially the predominant blaCMY-2, as well as blaACC-1 and 158-165 blaACC-4 . Other AmpC genes (blaCMY-1, blaCMY-8, blaCMY-9, blaCMY-10, blaCMY-11, blaCMY-19, blaMOX-1 and 77 blaDHA-1) have been identified downstream of ISCR1 embedded in complex sul1-type integrons , 166 and blaCMY-13 was described as flanked by two IS26 elements in the same orientation . ISEcp1- mediated transposition potentially resulted in the association of blaCMY-2 mainly with plasmids belonging to IncA/C, IncI1α/γ and IncK families among Enterobacteriaceae (especially E. coli and Salmonella) recovered from humans and animals worldwide49,89,93,136,167-169.

Interspecies high-risk sequence types Although different members of Enterobacteriaceae exhibiting ESC-resistance have been responsible for community- and hospital-acquired infections, ESC-resistant E. coli is considered the major threat because of its metabolic versatility resulting in the ability to inhabit diverse reservoirs and form part of the intestinal flora of humans and animals19,170. Phylo-typing and multilocus sequence typing have revealed that E. coli has a diverse population structure171,172. This overall diversity of E. coli reflects on the structure of the ESC-resistant subpopulation of the species, with various

General introduction | 19 sequence types (STs) assigned to several phylo-groups being able to inhabit both human and non-human reservoirs, contributing to the transmission of the ESC-resistant phenotype and subsequently complicating the epidemiology. The most important of these E. coli STs are discussed below.

E. coli ST10 The E. coli ST10, belonging to phylo-group A, is considered low-virulent human intestinal coloniser exhibiting antibiotic susceptibility173,174. However, this ST has been associated with ESBL-producing isolates recovered from human community- and hospital-acquired infections175,176, as well as

177,178 livestock and meat products . For instance, E. coli ST10 isolates encoding blaCTX-M-1 have been recovered from human bloodstream infections and poultry, and isolates encoding blaTEM-52 from human urinary tract infections (UTIs) and poultry91. Similarly, ESBL-producing E. coli ST10 isolates have been documented among retail meat products, including chicken and pork meat, livestock and human infections and colonization179,180.

E. coli ST69 E. coli isolates designated as ST69 (phylo-group D), were initially reported during an outbreak of extra-intestinal infections in Berkeley, California, accounting for 11% of all UTIs but 52% of antibiotic-resistant UTI isolates181. Since then, E. coli ST69 isolates have been associated with approximately 10% to 20% of all human E. coli infections, often exhibiting a multidrug resistant phenotype182-187. E. coli isolates recovered from meat products, such as beef, chicken and pork, as well as raw sewage and river water have been assigned to this ST188-191. Apart from encoding several virulence genes185,188, experimental studies have proven that isolates of this ST from human and non-human reservoirs are equally able to cause UTI in a mouse model192.

E. coli ST95 E. coli belonging to ST95 (phylo-group B2) are the cause of 6% of human extra-intestinal infec- tions173, mostly recovered from blood and ascitic fluid193,194. Apart from human infections, ST95 has been reported in poultry with well-documented avian pathogenicity195-197. E. coli ST95 isolates recovered from human neonatal meningitis are able to cause colisepticemia in poultry198, while it is proven that isolates of the same ST recovered from avian colibacillosis are able to cause neonatal

199 meningitis in a rat model . Although E. coli ST95 isolates encoding blaCTX-M genes have been reported193, this ST exhibits relative low frequency of multidrug resistance173,200.

20 | Chapter 1 E. coli ST117 E. coli ST117 belongs to phylo-group D and constitutes a known avian pathogenic E. coli, yet it has 1 been reported to cause human infections such as UTIs or sepsis and bovine mastitis191,201,202. In the Netherlands, isolates of this ST producing ESBL have been documented from human and poultry reservoirs91,180. In the subsequent years, ESCase-producing isolates of ST117 have been reported among humans, livestock and wild animals56,202-207. Interestingly, E. coli ST117 isolates have been recovered from both human, food and animal sources encoding virulence genes common in extraintestinal pathogenic STs201,208-210.

E. coli ST131 Initially reported in 2008, E. coli ST131 (phylo-group B2) is a globally emerging ST of E. coli211-213. Although it has been identified primarily in human infections, accounting for a large fraction of cases214, non-human intestinal carriage of ST131 has been reported for wild, companion and livestock animals215,216. E. coli ST131 exhibits a multidrug resistance phenotype, which usually includes fluoroquinolone resistance and ESBL production, specifically CTX-M-15, accounting for up to 88% of antibiotic-resistant infections in a variety of clinical settings, depending on specific

185,215 resistance phenotype . However, this ST has been reported to encode a variety of blaCTX-M genes depending on the geographic location of isolation186,211,215,217,218. E. coli ST131 is considered a major public health threat and is currently under intense investigation212,215,219-226.

Miscellaneous STs In addition to the aforementioned E. coli STs, K. pneumoniae ST15 has been widely identified in humans, but also in companion animals and horses227,228 and the prevalent K. pneumoniae ST101 from humans in Italy has been also found among companion animals116,229. Finally, the recently reported human high-risk international Enterobacter cloacae ST114 has been also found to pre- dominate among companion animals and horses230,231.

Nexus and interplay of reservoirs Until recently the human clinical environment was considered to be the only significant reservoir of ESCase genes, but the rise of community-acquired infections of ESC-resistant Enterobacteria- ceae has fuelled interest in the occurrence of these isolates in alternative environments47. Soon after, ESC-resistant Enterobacteriaceae, especially E. coli, have been documented in livestock and companion animals232, wildlife233, insects234-236 and the environment237, suggesting that their reservoirs is wider than expected. As a consequence, there are several routes through which humans can be exposed to ESCase genes and/or ESC-resistant Enterobacteriaceae, such as livestock and companion animals carrying ESC-resistant isolates in their intestinal flora237, contaminated meat

General introduction | 21 products, crops exposed to contaminated sludge, manure and slurry237, drinking water originated for contaminated groundwater or surface water237, coastal waters used for recreation or shellfish production237, inhalation of contaminated dust and air238 and houseflies carrying ESC-resistant isolates234,235.

The abundance of potential reservoirs of ESC-resistant Enterobacteriaceae, cross-transmission of these microorganisms within them, and gene dissemination by mobile elements among species depict a complicated scenario for ESC-resistant Enterobacteriaceae epidemiology.

Figure 2. Extended-spectrum cephalosporinase gene flow within the ecosystem (adapted from Wellington. et al)237.

22 | Chapter 1 Aim and outline of the thesis 1 Extended-spectrum cephalosporinase-producing Enterobacteriaceae from human and animal origin have emerged worldwide during the last decades17,18,46,47,232,239. Although studies documenting direct transmission between humans and animals are rare, the existence of shared reservoirs of extended-spectrum cephalosporinase genes, plasmids and/or STs suggests cross-transmissions and raises the concern of a possible zoonotic source of ESBL/AmpC-producers for humans98,240-246. The aim of this thesis is to explore the molecular relatedness of extended-spectrum cephalosporin- resistant Enterobacteriaceae of human and animal origin and assess their cross-transmission and epidemiology from a “One health” perspective.

Chapter 2 focuses on the emergence and the molecular characteristics of extended-spectrum cephalosporin-resistant Salmonella enterica serotype Heidelberg isolates in the Netherlands. Their recent emergence was attributed to food-producing animals and poultry products imported from Brazil, while no human infections linked to these contaminated animals and products have been yet documented in the Netherlands (Chapter 2a). In addition, the potential contribution of Kelp gulls of the southern hemisphere in the dissemination of ESC-resistant S. Heidelberg and other Enterobacteriaceae is presented (Chapter 2b).

In Chapter 3, Enterobacteriaceae transmission among humans and between humans and animals is discussed. A cross-sectional study among Dutch preschool children and their parents is presented (Chapter 3a), describing the molecular characteristics of the recovered ESC-resistant Enterobac- teriaceae and the frequency of intra-familial colonization with identical isolates. The longitudinal presence and molecular diversity of ESC-resistant E. coli from humans and pigs within the same pig farms are presented, confirming transmission events between farmers and their pigs (Chapter 3b). In Chapter 4, SHV extended-spectrum β-lactamases are discussed. The global epidemiology of Enterobacteriaceae encoding SHV ESCases is reviewed in Chapter 4a, highlighting the ubiquity of these extended-spectrum cephalosporinases. Finally, the recent association of blaSHV-12 with IncX3 plasmids among E. coli isolates in the Netherlands, as well as the genetic and functional characteristics of these plasmids contributing to blaSHV-12 emergence are reported (Chapter 4b).

General introduction | 23 References

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28 | Chapter 1 106 Golebiewski, M. et al. Complete nucleotide ducing CTX-M-3 beta-lactamase in a cancer sequence of the pCTX-M3 plasmid and its hospital in Bulgaria. J Chemother 20, 593-599, involvement in spread of the extended-spectrum doi:10.1179/joc.2008.20.5.593 (2008). 1 beta-lactamase gene blaCTX-M-3. Antimicro- 115 Literacka, E. et al. blaCTX-M genes in esche- bial agents and chemotherapy 51, 3789-3795, richia coli strains from Croatian Hospitals are doi:10.1128/AAC.00457-07 (2007). located in new (blaCTX-M-3a) and widely spread 107 Galimand, A., Sabtcheva, S., Courvalin, P. & (blaCTX-M-3a and blaCTX-M-15) genetic struc- Lambert, T. Worldwide disseminated armA ami- tures. Antimicrobial agents and chemotherapy noglycoside resistance methylase gene is borne 53, 1630-1635, doi:10.1128/AAC.01431-08 by composite transposon Tn1548. Antimicro- (2009). bial agents and chemotherapy 49, 2949-2953, 116 Donati, V. et al. Extended-Spectrum-Beta-Lac- doi:10.1128/Aac.49.7.2949-2953.2005 (2005). tamases, AmpC Beta-Lactamases and Plasmid 108 Markovska, R., Schneider, I., Ivanova, D., Mitov, Mediated Quinolone Resistance in Klebsiella I. & Bauernfeind, A. Predominance of IncL/M and spp. from Companion Animals in Italy. PloS IncF plasmid types among CTX-M-ESBL-produc- one 9, doi:ARTN e9056410.1371/journal. ing Escherichia coli and Klebsiella pneumoniae pone.0090564 (2014). in Bulgarian hospitals. APMIS : acta pathologica, 117 Garcia, A. et al. Acquisition and diffusion of microbiologica, et immunologica Scandinavica bla CTX-M-9 gene by R478-IncHI2 derivative 122, 608-615, doi:10.1111/apm.12204 (2014). plasmids. Fems Microbiol Lett 271, 71-77, 109 Markovska, R. D., Stoeva, T. J., Bojkova, K. doi:10.1111/j.1574-6968.2007.00695.x (2007). D. & Mitov, I. G. Epidemiology and Molecu- 118 Herrera-Leon, S., Gonzalez-Sanz, R., Rodriguez, lar Characterization of Extended-Spectrum I., Rodicio, M. R. & Echeita, M. A. Spread of a Beta-Lactamase-Producing Enterobacter spp., multiresistant CTX-M-9-producing Salmonella Pantoea agglomerans, and Serratia marcescens enterica serotype Virchow phage type 19 in Isolates from a Bulgarian Hospital. Microbial Spain. European journal of clinical microbiology drug resistance 20, 131-137, doi:10.1089/ & infectious diseases : official publication of mdr.2013.0102 (2014). the European Society of Clinical Microbiology 110 Alouache, S. et al. Characterization of ESBLs 29, 901-905, doi:10.1007/s10096-010-0939-6 and Associated Quinolone Resistance in Esch- (2010). erichia coli and Klebsiella pneumoniae Isolates 119 Coelho, A. et al. Role of IncHI2 plasmids har- from an Urban Wastewater Treatment Plant in bouring blaVIM-1, blaCTX-M-9, aac(6’)-Ib and Algeria. Microbial drug resistance 20, 30-38, qnrA genes in the spread of multiresistant doi:10.1089/mdr.2012.0264 (2014). Enterobacter cloacae and Klebsiella pneu- 111 Zhu, W. H. et al. Complete Nucleotide Sequence moniae strains in different units at Hospital of pCTX-M360, an Intermediate Plasmid Vall d’Hebron, Barcelona, Spain. International between pEL60 and pCTX-M3, from a Multi- journal of antimicrobial agents 39, 514-517, drug-Resistant Klebsiella pneumoniae Strain doi:10.1016/j.ijantimicag.2012.01.006 (2012). Isolated in China. Antimicrobial agents and 120 Novais, A. et al. Dissemination and persistence chemotherapy 53, 5291-5293, doi:10.1128/ of blaCTX-M-9 are linked to class 1 integrons Aac.00032-09 (2009). containing CR1 associated with defective trans- 112 Kang, H. Y. et al. Characterization of conjuga- poson derivatives from Tn402 located in early tive plasmids carrying antibiotic resistance genes antibiotic resistance plasmids of IncHI2, IncP1-al- encoding 16S rRNA methylase, extended-spec- pha, and IncFI groups. Antimicrobial agents and trum beta-lactamase, and/or plasmid-mediated chemotherapy 50, 2741-2750, doi:10.1128/ AmpC beta-lactamase. Journal of microbiology AAC.00274-06 (2006). 47, 68-75, doi:10.1007/s12275-008-0158-3 121 Liebana, E. et al. Longitudinal farm study of (2009). extended-spectrum beta-lactamase-mediated 113 Bogaerts, P. et al. Emergence of ArmA and RmtB resistance. Journal of clinical microbiology aminoglycoside resistance 16S rRNA methylases 44, 1630-1634, doi:10.1128/JCM.44.5.1630- in Belgium. J Antimicrob Chemoth 59, 459-464, 1634.2006 (2006). doi:10.1093/jac/dkl527 (2007). 122 Valverde, A. et al. Spread of bla(CTX-M-14) Is 114 Sabtcheva, S. et al. Nosocomial spread of Driven Mainly by IncK Plasmids Disseminated armA-mediated high-level aminoglycoside among Escherichia coli Phylogroups A, B1, and resistance in Enterobacteriaceae isolates pro- D in Spain. Antimicrobial agents and chemother-

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34 | Chapter 1 richia coli clonal group by multilocus sequence C. & Madec, J. Y. Veterinary hospital-acquired typing. Journal of clinical microbiology 43, infections in pets with a ciprofloxacin-resistant 5860-5864, doi:10.1128/JCM.43.12.5860- CTX-M-15-producing Klebsiella pneumoniae 1 5864.2005 (2005). ST15 clone. The Journal of antimicrobial chemo- 218 Banerjee, R. & Johnson, J. R. Escherichia therapy 67, 770-771, doi:10.1093/jac/dkr527 coli ST131: variations on a theme of clonal (2012). expansion. Enfermedades infecciosas y micro- 229 Mammina, C. et al. Sequence type 101 (ST101) biologia clinica 31, 355-356, doi:10.1016/j. as the predominant carbapenem-non-sus- eimc.2013.01.004 (2013). ceptible Klebsiella pneumoniae clone in an 219 Pitout, J. D. & DeVinney, R. Escherichia coli acute general hospital in Italy. International ST131: a multidrug-resistant clone primed journal of antimicrobial agents 39, 543-545, for global domination. F1000Research 6, doi:10.1016/j.ijantimicag.2012.02.012 (2012). doi:10.12688/f1000research.10609.1 (2017). 230 Izdebski, R. et al. MLST reveals potentially high- 220 Nicolas-Chanoine, M. H., Bertrand, X. & Madec, risk international clones of Enterobacter cloacae. J. Y. Escherichia coli ST131, an intriguing The Journal of antimicrobial chemotherapy 70, clonal group. Clin Microbiol Rev 27, 543-574, 48-56, doi:10.1093/jac/dku359 (2015). doi:10.1128/CMR.00125-13 (2014). 231 Haenni, M. et al. High prevalence of interna- 221 Banerjee, R. & Johnson, J. R. A new clone tional ESBL CTX-M-15-producing Enterobacter sweeps clean: the enigmatic emergence of cloacae ST114 clone in animals. The Journal of Escherichia coli sequence type 131. Antimicro- antimicrobial chemotherapy 71, 1497-1500, bial agents and chemotherapy 58, 4997-5004, doi:10.1093/jac/dkw006 (2016). doi:10.1128/AAC.02824-14 (2014). 232 Ewers, C., Bethe, A., Semmler, T., Guenther, 222 Riley, L. W. Pandemic lineages of extraintestinal S. & Wieler, L. H. Extended-spectrum beta-lac- pathogenic Escherichia coli. Clinical microbi- tamase-producing and AmpC-producing ology and infection : the official publication Escherichia coli from livestock and companion of the European Society of Clinical Microbi- animals, and their putative impact on public ology and Infectious Diseases 20, 380-390, health: a global perspective. Clinical microbiol- doi:10.1111/1469-0691.12646 (2014). ogy and infection : the official publication of the 223 Qureshi, Z. A. & Doi, Y. Escherichia coli sequence European Society of Clinical Microbiology and type 131: epidemiology and challenges in treat- Infectious Diseases 18, 646-655, doi:10.1111/ ment. Expert review of anti-infective therapy 12, j.1469-0691.2012.03850.x (2012). 597-609, doi:10.1586/14787210.2014.899901 233 Guenther, S., Ewers, C. & Wieler, L. H. Extend- (2014). ed-Spectrum Beta-Lactamases Producing E. coli 224 Mathers, A. J., Peirano, G. & Pitout, J. D. Esche- in Wildlife, yet Another Form of Environmen- richia coli ST131: The quintessential example of tal Pollution? Frontiers in microbiology 2, 246, an international multiresistant high-risk clone. doi:10.3389/fmicb.2011.00246 (2011). Advances in applied microbiology 90, 109-154, 234 Usui, M. et al. The Role of Flies in Spreading the doi:10.1016/bs.aambs.2014.09.002 (2015). Extended-Spectrum beta-lactamase Gene from 225 Schembri, M. A. et al. Molecular Characteri- Cattle. Microbial drug resistance 19, 415-420, zation of the Multidrug Resistant Escherichia doi:10.1089/mdr.2012.0251 (2013). coli ST131 Clone. Pathogens 4, 422-430, 235 Blaak, H. et al. Detection of Extended-Spectrum doi:10.3390/pathogens4030422 (2015). Beta-Lactamase (ESBL)-Producing Escherichia 226 Dautzenberg, M. J., Haverkate, M. R., coli on Flies at Poultry Farms. Applied and Bonten, M. J. & Bootsma, M. C. Epidemic environmental microbiology 80, 239-246, potential of Escherichia coli ST131 and Kleb- doi:10.1128/Aem.02616-13 (2014). siella pneumoniae ST258: a systematic review 236 Schaumburg, F. et al. A geospatial analysis of and meta-analysis. BMJ open 6, e009971, flies and the spread of antimicrobial resistant doi:10.1136/bmjopen-2015-009971 (2016). bacteria. Int J Med Microbiol 306, 566-571, 227 Ewers, C. et al. Clonal spread of highly suc- doi:10.1016/j.ijmm.2016.06.002 (2016). cessful ST15-CTX-M-15 Klebsiella pneumoniae 237 Wellington, E. M. et al. The role of the natu- in companion animals and horses. The Journal ral environment in the emergence of antibiotic of antimicrobial chemotherapy 69, 2676-2680, resistance in gram-negative bacteria. The Lancet. doi:10.1093/jac/dku217 (2014). Infectious diseases 13, 155-165, doi:10.1016/ 228 Haenni, M., Ponsin, C., Metayer, V., Medaille, S1473-3099(12)70317-1 (2013).

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36 | Chapter 1

CHAPTER 2 A

Extended-spectrum cephalosporin- resistant Salmonella enterica serovar Heidelberg strains, the Netherlands

Apostolos Liakopoulos1, Yvon Geurts1, Cindy M. Dierikx1, Michael S.M. Brouwer1, Arie Kant1, Ben Wit2, Raymond Heymans2, Wilfrid van Pelt3 and Dik J. Mevius1, 4

1Department of Bacteriology and Epidemiology, CVI of Wageningen University, Lelystad, the Netherlands; 2Netherlands Food and Consumer Product Safety Authority (NVWA), Utrecht, the Netherlands; 3Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands; 4Faculty of Veterinary Medicine, Department of Infectious Diseases and Immunology, Utrecht University, Utrecht, the Netherlands.

Published in Emerging Infectious Diseases 2016 Jul;22(7):1257-61 Abstract

Extended-spectrum cephalosporin-resistant Salmonella enterica serovar Heidelberg strains (JF6X01.0022/ XbaI.0251, JF6X01.0326/ XbaI.1966, JF6X01.0258/ XbaI.1968, and JF6X01.0045/ XbaI.1970) have been identified in the United States with pulsed-field gel electrophoresis. Our examination of isolates showed introduction of these strains in the Netherlands and highlight the need for active surveillance and intervention strategies by public health organizations.

40 | Chapter 2A Introduction

Salmonella enterica serovar Heidelberg is among the most prevalent causes of human salmonellosis in the United States and Canada but has been reported infrequently in Europe1-3. Although most nontyphoidal Salmonella infections are self-limiting and resolve within a few days, Salmonella ser. 2A Heidelberg tends to provoke invasive infections (e.g., myocarditis and bacteremia) that require antimicrobial drug therapy4. To treat systemic nontyphoidal Salmonella infections, third-generation cephalosporins are preferred drugs for children or for adults with fluoroquinolone contraindicati- ons5. Resistance to third-generation cephalosporins is increasing in S. enterica infections, mainly because of production of plasmid-mediated extended-spectrum or AmpC β-lactamases6.

Resistance to extended-spectrum cephalosporins (ESCs) among Salmonella Heidelberg strains found in human infections, food-producing animals, and poultry meat indicates zoonotic and foodborne transmission of these strains and potential effects on public health7, 8. Unlike in Canada and the United States, few ESC-resistant Salmonella Heidelberg strains have been documented in Europe9-13. However, increased occurrence of ESC resistance in S. enterica infections and decreased susceptibility to fluoroquinolones compromise the use of these drugs and constitute a serious public health threat6, 14.

Few data are available regarding prevalence of ESC-resistant Salmonella Heidelberg isolates in Europe, their underlying antimicrobial drug resistance gene content, and genetic platforms (i.e., plasmids and insertion sequence [IS] elements) associated with resistance genes. We attempted to determine the occurrence and molecular characteristics of Salmonella Heidelberg isolates recovered from human patients, food-producing animals, and poultry meat in the Netherlands during 1999–2013.

The Study

During 1999–2013, the Netherlands National Institute of Public Health and the Environment collected 437 Salmonella Heidelberg isolates from human infections (n = 77 [17.6%]), food-producing animals (n = 138 [31.6%]), poultry meat (n = 170 [38.9%]), and other sources (n = 52 [11.9%]). From this collection, we selected 200 epidemiologically unrelated isolates for further analysis (Table; Technical Appendix).

Extended-spectrum cephalosporin-resistant Salmonella enterica serovar Heidelberg strains, the Netherlands | 41 MICs for antimicrobial agents were determined with the broth microdilution method (online Technical Appendix) and showed a higher frequency of multidrug non–wild-type susceptibility phenotype in isolates from poultry meat (n = 44 [68.8%]) than in isolates from food-producing animals (n = 14 [31.8%]) and human infections (n = 16 [19.5%]). Most human infections exhibited wild-type MICs to most antimicrobial agents tested (Table).

Of the 200 Salmonella Heidelberg isolates in the study, 47 (23.5%) were ESC resistant. ESC resistance in Salmonella Heidelberg isolates increased from 33.3% in 2011 to 60.0% in 2012 to 75.0% in 2013, after which Salmonella Heidelberg was the predominant serotype in ESC-resistant Salmonella isolates in the Netherlands (Figure 1).

These isolates showed MICs for cefotaxime and ceftazidime of 2 to >4 mg/L and 4 to >16 mg/L, respectively; non–wild-type susceptibility to fluoroquinolones was 87.2%. The emergence of isolates with decreased susceptibility to these first-line antimicrobial drugs limits effective treatment options for potential human infections.

Figure 1. Occurrence of extended-spectrum cephalosporin-resistant Salmonella enterica serovar Heidelberg isolates, the Netherlands, 1999–2013.

ESC typing of the 47 isolates, performed by microarray analysis followed by PCR and sequencing

(Technical Appendix), revealed the presence of the blaCMY-2 gene in 41 ESC-resistant Salmonella Heidelberg isolates that exhibited an AmpC β-lactamase phenotype. The other 6 isolates exhibited an extended-spectrum β-lactamase phenotype and encoded blaCTX-M-2 (n = 4), blaCTX-M-1 (n = 1), or blaCTX-M-14 (n = 1) genes (Figure 2).

42 | Chapter 2A We assessed the genetic relatedness of the 47 cephalosporin-resistant Salmonella Heidelberg isolates by using the standardized XbaI–pulsed-field gel electrophoresis (PFGE) (online Techni- cal Appendix), which identified 2 major PFGE types: XbaI.1968 and XbaI.1973 (PFGE numbers assigned by the European Centre for Disease Prevention and Control, Solna, Sweden). Of the 47 isolates, 26 (55.3%) belonged to XbaI.1968 and 5 (10.6%) belonged to XbaI.1973. Forty-one of 2A the isolates were blaCMY-2 carriers, 31 (75.6%) of which belonged to these 2 PFGE types; 10 (24.4%) were distributed equally among other PFGE types. Six of the 47 isolates were blaCTX-M carriers associated with 5 PFGE types (Figure 2). Comparing these isolates with those in the PulseNet database (http://www.cdc.gov/pulsenet/index.html) revealed the introduction of 4 epidemic clones of ESC- resistant Salmonella Heidelberg strains in the Netherlands (JF6X01.0022/XbaI.0251, JF6X01.0326/ XbaI.1966, JF6X01.0258/XbaI.1968, and JF6X01.0045/XbaI.1970). To raise awareness and determine whether related ESC-resistant Salmonella Heidelberg isolates had been observed in other European countries, the Epidemic Intelligence Information System (European Centre for Disease Prevention and Control) issued an alert on September 18, 2014.

We successfully transferred plasmids carrying extended-spectrum or AmpC β-lactamases from ESC- resistant Salmonella Heidelberg isolates to the recipient E. coli DH10B strain (Technical Appendix). PCR-based Inc/Rep typing and multilocus or double-locus sequence typing (ST) of the plasmids revealed that the blaCMY-2 or blaCTX-M genes were located on plasmids for 46 (97.8%) of the 47 isolates. ESC-resistant Salmonella Heidelberg isolates encoding blaCMY-2 on IncI1/ST12 plasmids were associated predominantly with the XbaI.1968 (n = 26 [78.8%]) PFGE type; those encoding blaCMY-2 on IncA/C plasmids were associated with XbaI.1973 (n = 5 [71.4%]). Isolates encoding blaCTX-M-2 on

IncHI2P/ST2, blaCTX-M-1 on IncI1/ST49, and blaCTX-M-14 on IncI1/ST80 plasmids were associated with XbaI.1964, XbaI.1963, and XbaI.1966, respectively (Figure 2).

The blaCMY-2 gene was present in 12 different PFGE types and was carried on plasmids of 2 different incompatibility groups (IncI1/ST12 and IncA/C) or on the chromosome. This gene’s diverse genetic background suggests that emergence of the blaCMY-2–producing Salmonella Heidelberg strain in the Netherlands results not only from expansion of a single clone but from multiclonal dissemination of the strain and horizontal transfer of plasmids encoding the blaCMY-2 gene. IncI1/

ST12 and IncA/C plasmids have been associated with the blaCMY-2 gene in Salmonella Heidelberg isolates in the United States and Canada8, 15.

We analyzed a subset of ESC-resistant Salmonella Heidelberg isolates to determine the size and conjugation frequency of plasmids carrying extended-spectrum and AmpC β-lactamases. We also assessed a subset of Salmonella Heidelberg isolates (n = 17) for each PFGE type, including isolates

Extended-spectrum cephalosporin-resistant Salmonella enterica serovar Heidelberg strains, the Netherlands | 43 2011-2013 15 Col (1) Str (3) (2) TetSmxStr (1) TetSmxTmp WT (8) 0 13 AmpCol (1) (1) AmpFotTazTetSmx (4) AmpFotTazTetSmxNalCip Col (2) (2) TetSmxNalCip (1) TetSmxNalCipGenStrKan WT (2) 5 40 2011-2013 (26) AmpFotTazTetSmxNalCip (1) AmpFotTazTetSmxNalCipCol (1) AmpFotTazTetSmxNalCipGenStrKan (6) AmpFotTazTetSmxNalCipStr (1) AmpFotTazTetSmxTmpNalCipChl Col (2) (1) TetSmxNalCip (1) TetSmxNalCipGenStr (1) TetSmxNalCipStr 35 4 (1) AmpFotTazTetSmxNalCip NalCipCol (1) Str (1) (1) TetSmxNalCipGenStr 1 2008-2010 23 ChlCol (1) Col (10) Str (1) StrCol (5) (1) TetCol (1) TetNalCip (1) TetSmxTmpStrCol (1) TetStrKanCol (1) TetStrSmxCol WT (1) 0 7 AmpCol (1) (1) AmpFotTazNalCip (1) AmpFotTazTetSmxGenStrKanCol Col (4) 2 6 2008-2010 (1) AmpFotTaz (1) AmpFotTazSmxTmpChlStrCol (1) AmpFotTazStrCol Col (2) NalCipCol (1) 3 6 Col (2) NalCipCol (1) Str (1) StrCol (2) 0 2005-2007 22 (1) AmpFotTazStr AmpSmxTmpNalCip (1) (1) AmpTet NalCip (2) SmxStr (1) (1) Tet (1) TetSmxNalCip WT (14) 1 5 AmpTetSmxTmpNal- Cip (1) WT (4) 0 15 2005-2007 NalCip (3) SmxCipGen (1) SmxGen (1) SmxTmpNalCip (1) (1) TetSmxTmp WT (8) 0 0 0 2002-2004 10 AmpSmxStr (1) (1) AmpTetSmx SmxStr (3) Str (1) (1) TetSmxStr WT (3) 0 16 Amp (3) AmpSmxTmpNalCipStr (2) AmpStr (2) NalCip (5) SmxStrTmp (1) WT (3) 0 3 2002-2004 AmpSmxStr (1) WT (2) 0 1 WT (1) 0 serovar Heidelberg isolates included in the study: Amp, ampicillin; Cip, ciprofloxacin; Chl, chloramphenicol; Col, colistin; Fot, cefotaxime; Heidelberg isolates included in the study: Amp, ampicillin; Cip, ciprofloxacin; serovar serovar Heidelberg isolates recovered from human infections, food-producing animals, and poultry meat, the Netherlands, 1999–2013*. human infections, food-producing from Heidelberg isolates recovered serovar enterica

1999-2001 13 Amp (1) AmpCol (1) AmpSmxTmpStr (1) AmpTetSmxTmpStr (1) SmxStr (1) Str (5) (1) TetSmxTmpStr (2) W.T 0 5 NalCip (1) (4) W.T 0 3 1999-2001 AmpTetSmxTmpNal- CipStr (1) SmxTmpStr (1) (1) W.T 0 0 0 Salmonella Salmonella enterica Characteristics of Continued. Table. Source Human infections No. isolates studied Resistance phenotypes (no.)* isolates No. ESC-resistant animals Food-producing No. isolates studied Resistance phenotypes (no.) isolates No. ESC-resistant Poultry meat No. isolates studied Table. Source Resistance phenotypes (no.) isolates No. ESC-resistant Other No. isolates studied Resistance phenotypes (no.) isolates No. ESC-resistant *Resistance phenotypes of the extended- wild type; ESC-resistant, tetracycline; Tmp, trimethoprim; WT, ceftazidime; Tet, Taz, streptomycin; Gen, gentamicin; Kan, kanamycin; Nal, nalidixic acid; Smx, sulfamethoxazole; Str, spectrum cephalosporin-resistant.

44 | Chapter 2A 2A 2011-2013 15 Col (1) Str (3) (2) TetSmxStr (1) TetSmxTmp WT (8) 0 13 AmpCol (1) (1) AmpFotTazTetSmx (4) AmpFotTazTetSmxNalCip Col (2) (2) TetSmxNalCip (1) TetSmxNalCipGenStrKan WT (2) 5 40 2011-2013 (26) AmpFotTazTetSmxNalCip (1) AmpFotTazTetSmxNalCipCol (1) AmpFotTazTetSmxNalCipGenStrKan (6) AmpFotTazTetSmxNalCipStr (1) AmpFotTazTetSmxTmpNalCipChl Col (2) (1) TetSmxNalCip (1) TetSmxNalCipGenStr (1) TetSmxNalCipStr 35 4 (1) AmpFotTazTetSmxNalCip NalCipCol (1) Str (1) (1) TetSmxNalCipGenStr 1 2008-2010 23 ChlCol (1) Col (10) Str (1) StrCol (5) (1) TetCol (1) TetNalCip (1) TetSmxTmpStrCol (1) TetStrKanCol (1) TetStrSmxCol WT (1) 0 7 AmpCol (1) (1) AmpFotTazNalCip (1) AmpFotTazTetSmxGenStrKanCol Col (4) 2 6 2008-2010 (1) AmpFotTaz (1) AmpFotTazSmxTmpChlStrCol (1) AmpFotTazStrCol Col (2) NalCipCol (1) 3 6 Col (2) NalCipCol (1) Str (1) StrCol (2) 0 2005-2007 22 (1) AmpFotTazStr AmpSmxTmpNalCip (1) (1) AmpTet NalCip (2) SmxStr (1) (1) Tet (1) TetSmxNalCip WT (14) 1 5 AmpTetSmxTmpNal- Cip (1) WT (4) 0 15 2005-2007 NalCip (3) SmxCipGen (1) SmxGen (1) SmxTmpNalCip (1) (1) TetSmxTmp WT (8) 0 0 0 2002-2004 10 AmpSmxStr (1) (1) AmpTetSmx SmxStr (3) Str (1) (1) TetSmxStr WT (3) 0 16 Amp (3) AmpSmxTmpNalCipStr (2) AmpStr (2) NalCip (5) SmxStrTmp (1) WT (3) 0 3 2002-2004 AmpSmxStr (1) WT (2) 0 1 WT (1) 0 serovar Heidelberg isolates included in the study: Amp, ampicillin; Cip, ciprofloxacin; Chl, chloramphenicol; Col, colistin; Fot, cefotaxime; Heidelberg isolates included in the study: Amp, ampicillin; Cip, ciprofloxacin; serovar serovar Heidelberg isolates recovered from human infections, food-producing animals, and poultry meat, the Netherlands, 1999–2013*. human infections, food-producing from Heidelberg isolates recovered serovar enterica

1999-2001 13 Amp (1) AmpCol (1) AmpSmxTmpStr (1) AmpTetSmxTmpStr (1) SmxStr (1) Str (5) (1) TetSmxTmpStr (2) W.T 0 5 NalCip (1) (4) W.T 0 3 1999-2001 AmpTetSmxTmpNal- CipStr (1) SmxTmpStr (1) (1) W.T 0 0 0 Salmonella Salmonella enterica Characteristics of Continued. Table. Source Human infections No. isolates studied Resistance phenotypes (no.)* isolates No. ESC-resistant animals Food-producing No. isolates studied Resistance phenotypes (no.) isolates No. ESC-resistant Poultry meat No. isolates studied Table. Source Resistance phenotypes (no.) isolates No. ESC-resistant Other No. isolates studied Resistance phenotypes (no.) isolates No. ESC-resistant *Resistance phenotypes of the Gen, gentamicin; Kan, kanamycin; Nal, nalidixic acid; Smx, sulfamethoxazole; Str, streptomycin; Taz, ceftazidime; Tet, tetracycline; Tmp, trimethoprim; WT, wild type; ESC-resistant, extended- wild type; ESC-resistant, tetracycline; Tmp, trimethoprim; WT, ceftazidime; Tet, Taz, streptomycin; Gen, gentamicin; Kan, kanamycin; Nal, nalidixic acid; Smx, sulfamethoxazole; Str, spectrum cephalosporin-resistant.

Extended-spectrum cephalosporin-resistant Salmonella enterica serovar Heidelberg strains, the Netherlands | 45 for each type if they showed variation in extended-spectrum and AmpC β-lactamase genes or in gene location. This assessment sought to detect the upstream presence of resistance genes

(blaCTX-M and blaCMY) of frequently encountered insertion sequences (ISEcp1, ISCR1, and IS26) (Figure 2; Technical Appendix).

We attribute the increase of ESC-resistant Salmonella Heidelberg isolates in the Netherlands to the frequent occurrence of isolates carrying IncI1/ST12 plasmids encoding blaCMY-2 in food-producing animals and poultry products imported from Brazil. Isolates from imported poultry products are associated predominantly with PFGE types XbaI.1968 and XbaI.1973 (Figure 2). A similar introduction of ESC-resistant Salmonella Heidelberg strains in Ireland was associated with imported poultry meat from Brazil (R. Slowey, pers. comm.). Although ESC-resistant Salmonella Heidelberg strains are rarely reported in Europe, their introduction through imported poultry meat could pose a public health risk; Brazil is among the world’s leading countries for exporting poultry meat.

Conclusions Most ESC-resistant Salmonella Heidelberg isolates in our study had profiles (XbaI.0251, XbaI.1966, XbaI.1968, and XbaI.1970) indistinguishable from those of previous epidemic types (JF6X01.0022, JF6X01.0326, JF6X01.0258, and JF6X01.0045) that caused outbreaks and showed potency for bloodstream infections16. Our identification of clonal clusters shared by ESC-resistantSalmonella Heidelberg strains in food-producing animals or poultry meat that can cause human infections underscores the risk for potential zoonotic or foodborne transmission of these strains to humans. Although we observed a frequent occurrence of ESC-resistant Salmonella Heidelberg isolates in poultry products, no human infections linked to these contaminated products have been yet documented in the Netherlands. Nevertheless, the risk of potential zoonotic or foodborne transmission of ESC-resistant Salmonella Heidelberg strains highlights the necessity for active surveillance and intervention strategies by public health organizations.

Figure 2. Characteristics of extended-spectrum cephalosporin-resistant Salmonella enterica serovar Heidelberg isolates, the Netherlands, 1999–2013. The dendrogram was generated by using BioNumerics version 6.6 (Applied Maths, Sint-Martens-Latem, Belgium) and indicates results of a cluster analysis on the basis of XbaI–pulsed-field gel electrophoresis (PFGE) fingerprinting. Similarity between the profiles was calculated with the Dice similarity coefficient and used 1% optimization and 1% band tolerance as position tolerance settings. The dendrogram was constructed with the UPGMA method based on the resulting similarity matrix. Amp, ampicillin; Cip, ciprofloxacin; Chl, chloramphenicol; Col, colistin; Fot, cefotaxime; FPA, food-producing animals; Gen, gentamicin; HI, human infection; Kan, kanamycin; Nal, nalidixic acid; ND, not determined (i.e., refers to isolates recovered in the Netherlands but with unknown origin of the sample); pCC, plasmid clonal complex; PM, poultry meat; pST, plasmid sequence type; Smx, sulfamethoxazole; Str, streptomycin; Taz, ceftazidime; Tet, tetracycline; Tmp, trimethoprim. *Pat- tern numbers assigned by The European Surveillance System molecular surveillance service of the European Centre for Disease Prevention and Control database and corresponding pattern numbers from the PulseNet database (http://www.cdc.gov/pulsenet/ index.html). †Results refer to the conjugation frequencies during filter-mating experiments. ‡Chromosomal location confirmed by I-CeuI PFGE of total bacterial DNA, followed by Southern blot hybridization. §No transconjugants were obtained after liquid and filter-mating experiments, suggesting the presence of nonconjugative plasmids or conjugation frequencies below detection limits. ¶Insertion sequences ISEcp1, ISCR1, or IS26 were not found upstream of the extended-spectrum β-lactamase genes for these PFGE types. #This PFGE fingerprint was not submitted to The European Surveillance System molecular surveillance service of the European Centre for Disease Prevention and Control database for name assignment.

46 | Chapter 2A 2A (footnote on page 46). Figure 2 Figure

Extended-spectrum cephalosporin-resistant Salmonella enterica serovar Heidelberg strains, the Netherlands | 47 Materials and Methods

Bacterial Strains and Identification During 1999–2013, the Dutch National Institute of Public Health (RIVM) collected 30,472 Salmo- nella isolates from various surveillance programs on patients with salmonellosis and from farms, slaughterhouses, and retail markets. The isolates originated from human infections (n = 17,363), food-producing animals (n = 6,136), poultry meat (n = 1,260) and other sources (n = 5,713). Using micronitration, RIVM performed serotyping based on somatic (O) and flagellar (H) antigens according to the latest version of the White-Kaufmann-Le Minor scheme17. Recovered Salmonella isolates were stored at –80°C in Peptone Broth supplemented with 30% (v/v) glycerol for further analysis.

We selected 200 isolates from the 437 Salmonella enterica serovar Heidelberg isolates received at RIVM during 1999–2013. Only the first isolate per patient was included, and to avoid epidemiologically clustered isolates in the selection, only 1 isolate was included per sample type (i.e., human infection, food-producing animal, poultry meat, or others) and origin (i.e., hospital, institute, laboratory, farm, company, or surveillance program) per 14-day period.

Antimicrobial Susceptibility Testing The susceptibility of the isolates to antimicrobial agents was assessed by broth microdilution, as described by the International Standard Organization (standard 20776–1:2006), by using micro- titer trays with a custom-designed, dehydrated panel of antimicrobial drugs (EUMVS, Sensititre, Thermo Fischer, Basingstoke, UK). The antimicrobial agents tested included ampicillin, cefotaxime, ceftazidime, ciprofloxacin, chloramphenicol, colistin, florfenicol, gentamicin, kanamycin, nalidixic acid, streptomycin, sulfamethoxazole, trimethoprim, and tetracycline. Escherichia coli strain ATCC 25922 and Enterococcus faecalis strain ATCC 29212 were used as quality controls. For interpretation, we used epidemiologic cutoff values recommended by the European Committee on Antimicrobial Susceptibility Testing (http://mic.eucast.org). Multidrug non–wild-type phenotype was defined as non–wild-type MICs to ≥1 antimicrobial agents from ≥3 antimicrobial classes. Production of extended-spectrum or AmpC β-lactamases was evaluated by a combined disc test that used discs of cefotaxime and ceftazidime with (30/10 μg) and without clavulanic acid (30 μg) and a disc of cefoxitin (30 μg) for all isolates; this process satisfied the phenotypic criteria indicative of extended-spectrum cephalosporinase production, as recommended by the European Centre for Disease Prevention Control1.

48 | Chapter 2A Characterization of Resistance Determinants We assessed the presence of genes conferring the extended-spectrum cephalosporinase-resistant phenotype. DNA was extracted by using the DNeasy Blood and Tissue kit (QIAGEN, Hilden, Germany) according to the manufacturer’s recommendations. All isolates putatively producing extended-spectrum or AmpC β-lactamases were screened for a broad spectrum of extended- 2A spectrum and AmpC β-lactamase gene families by using the Check-MDR CT-101 array platform (Check-Points, Wageningen, the Netherlands) according to the manufacturer’s recommendations. The presence of extended-spectrum or AmpC β-lactamase genes was confirmed by PCR and subsequent sequencing as described18. The nucleotide and deduced amino acid sequences were compared with sequences in the Lahey clinic database (http://www.lahey.org/Studies).

Clonal Analysis All isolates carrying extended-spectrum or AmpC β-lactamases (n = 47) and isolates randomly selected on the basis of year and source of isolation (n = 64) were analyzed for genetic relatedness by pulsed-field gel electrophoresis (PFGE) ofXbaI -digested genomic DNA by using a CHEF DR-III apparatus (Bio-Rad Laboratories, Hercules, CA, USA), according to the standardized protocol of PulseNet19. XbaI-digested genomic DNA from S. enterica ser. Braenderup strain H9812 was used as a molecular reference marker20. Image normalization and construction of similarity matrices were carried out by using BioNumerics, version 6.6 (Applied Maths, Sint-Martens-Latem, Belgium). Bands were assigned manually, and dendrograms were generated by employing the Unweighted Pair Group Method with Arithmetic mean based on the Dice similarity index, by using 1% optimization and 1% band tolerance as position tolerance settings.

PFGE fingerprints of the isolates were submitted to The European Surveillance System molecular surveillance service of the European Centre for Disease Prevention and Control database, which assigned pattern names. PFGE fingerprints were subsequently compared with those from the PulseNet database. An alert was issued on September 18, 2014, through the European Epidemic Intelligence Information System for the Food and Waterborne Diseases and Zoonoses network to raise awareness and determine whether related extended-spectrum cephalosporinase-resistant S. enterica ser. Heidelberg isolates had been observed in other member countries of the European Epidemic Intelligence Information System.

Extended-spectrum cephalosporin-resistant Salmonella enterica serovar Heidelberg strains, the Netherlands | 49 Plasmid Analysis The replicon types were characterized for all plasmids carrying extended-spectrum or AmpC β-lactamases. Purified plasmid DNA was transformed into DH10B cells by electroporation (Invi- trogen, Van Allen Way, CA USA) under the following conditions: 1.25 kV/cm, 200 Ω, 25 μFar21. Transformants were selected on Luria-Bertani agar plates supplemented with cefotaxime (1 mg/L). PCR-based replicon typing was conducted on the transformants to determine the replicon type of the plasmid by using the PBRT KIT—PCR-based replicon typing (DIATHEVA, Fano, Italy); plasmid multilocus or double-locus sequence typing (pMLST or pDLST) were used to further subtype IncI1α/γ and IncHI2 plasmids as previously described22, 23. A subset of transformants (n = 16) was selected according to PFGE profile of the parental strain, replicon type of the plasmid, and antimicrobial-resistance determinant. These plasmids were subjected to S1-PFGE for accurate determination of molecular sizes24. If no transformants were obtained, the chromosomal location of the extended-spectrum or AmpC β-lactamase genes was confirmed by I-CeuI PFGE of total bacterial DNA, followed by Southern blot hybridization, as described25.

Conjugation Experiments The transferability of the extended-spectrum cephalosporinase-resistant phenotype by conjugation was assessed for the subset of S. enterica ser. Heidelberg isolates described above. Plasmid-free rifampicin-resistant E. coli E3110 was used as a recipient strain for liquid-mating assays in a ratio of 1:1. Filter-mating assays were attempted for strains for which no transconjugants were obtained by liquid mating. For both liquid-and filter-mating assays, the donor and recipient strains in mid- exponential phase were co-incubated for 4 hours without agitation at 37oC. Transconjugants were selected on MacConkey agar supplemented with a combination of rifampicin (100 mg/L) and cefotaxime (1 mg/L). Positive transconjugants were confirmed by PCR amplification for the resistance determinant. All mating assays were conducted in triplicate. The conjugation frequency was calculated as the number of transconjugants per donor cell.

Analysis of Regions Upstream of Resistance Determinants

The association of blaCTX-M and blaCMY genes with frequently encountered insertion sequences (ISEcp1, ISCR1, and IS26) was assessed for a subset of S. enterica ser. Heidelberg isolates (n = 17) representing each unique PFGE profile and the variation in extended-spectrum or AmpC β-lactamase gene type and its location (chromosome or plasmid replicon type). This association was investigated with PCR by using forward primers specific for ISEcp1, ISCR1, or IS26 and a reverse

26 primer for blaCTX-M or blaCMY genes, as described . Subsequently, sequence analysis confirmed the amplicons obtained.

50 | Chapter 2A Acknowledgements

The authors gratefully acknowledge Johanna Takkinen, Ivo van Walle and the curators of The European Surveillance System (TESSy) molecular surveillance service (MSS) of the European Centre for Disease Prevention and Control (ECDC) database for assigning reference type/pattern names 2A to our PFGE types. We are also grateful to Patrick McDermott and Jason Abbott for helping with the comparison of our PFGE types with those from PulseNet database, as well as John Egan and Rosemarie Slowey for providing information about the ESC-resistant Salmonella Heidelberg strains detected in Ireland.

Funding

This work was supported by the Dutch Ministry of Economic Affairs (BO-22.04-008-001).

Extended-spectrum cephalosporin-resistant Salmonella enterica serovar Heidelberg strains, the Netherlands | 51 References

1. European Centre for Disease Prevention and from 2001 to 2005. The Journal of antimicrobial Control. EU protocol for harmonised monitoring chemotherapy 2009; 64: 1181-6. of antimicrobial resistance in human Salmonella 12. Batchelor M, Hopkins KL, Threlfall EJ et al. and Campylobacter isolates. Stockholm, 2014. Characterization of AmpC-mediated resistance 2. Public Health Agency of Canada. National in clinical Salmonella isolates recovered from Enteric Surveillance Program (NESP), Annual humans during the period 1992 to 2003 in Summary 2012. Ottawa, Canada, 2014. England and Wales. Journal of clinical microbi- 3. CDC. National Antimicrobial Resistance Mon- ology 2005; 43: 2261-5. itoring System for Enteric Bacteria (NARMS): 13. Burke L, Hopkins KL, Meunier D et al. Resistance Human Isolates Final Report, 2012. Atlanta, to third-generation cephalosporins in human Georgia: U.S: Department of Health and Human non-typhoidal Salmonella enterica isolates from Services, 2014. England and Wales, 2010-12. The Journal of 4. Hoffmann M, Zhao S, Pettengill J et al. Compar- antimicrobial chemotherapy 2014; 69: 977-81. ative genomic analysis and virulence differences 14. Piddock LJ. Fluoroquinolone resistance in Salmo- in closely related salmonella enterica serotype nella serovars isolated from humans and food heidelberg isolates from humans, retail meats, animals. FEMS microbiology reviews 2002; 26: and animals. Genome biology and evolution 3-16. 2014; 6: 1046-68. 15. Andrysiak AK, Olson AB, Tracz DM et al. Genetic 5. Hohmann EL. Nontyphoidal salmonellosis. Clini- characterization of clinical and agri-food isolates cal infectious diseases : an official publication of of multi drug resistant Salmonella enterica sero- the Infectious Diseases Society of America 2001; var Heidelberg from Canada. BMC microbiology 32: 263-9. 2008; 8: 89. 6. Miriagou V, Tassios PT, Legakis NJ et al. Expand- 16. Centers for Disease Control and Prevention. ed-spectrum cephalosporin resistance in Investigation update: multistate outbreak of non-typhoid Salmonella. International journal human Salmonella Heidelberg infections linked of antimicrobial agents 2004; 23: 547-55. to “kosher broiled chicken livers” from Schreiber 7. Dutil L, Irwin R, Finley R et al. Ceftiofur resistance Processing Corporation. Atlanta, GA: Centers in Salmonella enterica serovar Heidelberg from for Disease Control and Prevention, 2011. chicken meat and humans, Canada. Emerging 17. Grimont P, Weil F. Antigenic formulae of the Sal- infectious diseases 2010; 16: 48-54. monella serovars. Paris, France: Pasteur Institute, 8. Folster JP, Pecic G, Singh A et al. Characterization 2007. of extended-spectrum cephalosporin-resistant 18. Dierikx CM, van Duijkeren E, Schoormans Salmonella enterica serovar Heidelberg isolated AH et al. Occurrence and characteristics of from food animals, retail meat, and humans in extended-spectrum-beta-lactamase- and the United States 2009. Foodborne pathogens AmpC-producing clinical isolates derived from and disease 2012; 9: 638-45. companion animals and horses. The Journal of 9. Aarestrup FM, Hasman H, Olsen I et al. Inter- antimicrobial chemotherapy 2012; 67: 1368-74. national spread of bla(CMY-2)-mediated 19. Ribot EM, Fair MA, Gautom R et al. Standardiza- cephalosporin resistance in a multiresistant tion of pulsed-field gel electrophoresis protocols Salmonella enterica serovar Heidelberg isolate for the subtyping of Escherichia coli O157:H7, stemming from the importation of a boar by Salmonella, and Shigella for PulseNet. Food- Denmark from Canada. Antimicrobial agents borne pathogens and disease 2006; 3: 59-67. and chemotherapy 2004; 48: 1916-7. 20. Hunter SB, Vauterin P, Lambert-Fair MA et al. 10. Miriagou V, Filip R, Coman G et al. Expand- Establishment of a universal size standard strain ed-spectrum cephalosporin-resistant salmonella for use with the PulseNet standardized pulsed- strains in Romania. Journal of clinical microbiol- field gel electrophoresis protocols: converting ogy 2002; 40: 4334-6. the national databases to the new size standard. 11. Gonzalez-Sanz R, Herrera-Leon S, de la Fuente Journal of clinical microbiology 2005; 43: 1045- M et al. Emergence of extended-spectrum 50. beta-lactamases and AmpC-type beta-lact- 21. Hordijk J, Wagenaar JA, Kant A et al. amases in human Salmonella isolated in Spain Cross-sectional study on prevalence and molec-

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Extended-spectrum cephalosporin-resistant Salmonella enterica serovar Heidelberg strains, the Netherlands | 53

CHAPTER 2 B

Molecular characterization of extended-spectrum cephalosporin- resistant Enterobacteriaceae from wild kelp gulls in South America

Apostolos Liakopoulos1, Björn Olsen2,3, Yvon Geurts1, Karin Artursson4, Charlotte Berg5, Dik J. Mevius1,6 and Jonas Bonnedahl2,7

1Department of Bacteriology and Epidemiology, CVI of Wageningen University, Lelystad, the Netherlands; 2Section of Clinical Microbiology and Infectious Diseases, Department of Medical Sciences, Uppsala University, Uppsala, Sweden; 3Zoonosis Science Center, IMBIM, Uppsala University, Uppsala, Sweden; 4National Veterinary Institute, Uppsala, Sweden; 5Department of Animal Environment and Health, Swedish University of Agricultural Sciences, Uppsala, Sweden; 6Faculty of Veterinary Medicine, Department of Infectious Diseases and Immunology, Utrecht University, Utrecht, the Netherlands; 7Department of Infectious Diseases, Kalmar County Hospital, Kalmar, Sweden.

Published in Antimicrobial Agents and Chemotherapy 2016 Oct 21;60(11):6924-6927 Abstract

Extended-spectrum-cephalosporin-resistant Enterobacteriaceae are a public health concern due to limited treatment options. Here, we report on the occurrence and the molecular characteristics of extended-spectrum-cephalosporin-resistant Enterobacteriaceae recovered from wild birds (kelp gulls). Our results revealed kelp gulls as a reservoir of various extended-spectrum cephalosporinase genes associated with different genetic platforms. In addition, we report for the first time the presence of a known epidemic clone of Salmonella enterica serotype Heidelberg (JF6X01.0326/ XbaI.1966) among wild birds.

56 | Chapter 2B Extended-spectrum cephalosporinase-producing Enterobacteriaceae have been reported worldwide amongst isolates obtained from humans, food-producing and companion animals, as well as environmental sources1. In spite of limited number of studies regarding the occurrence of antibiotic resistance in natural environments, where animals do not naturally come into contact with antibiotics, the occurrence of extended-spectrum cephalosporin (ESC)-resistant Enterobac- teriaceae has been detected in wild birds lately, especially in gull populations (Laridae)2-7. The Kelp gull (Larus dominicanus) is a large gull species distributed in coastal areas through much of the southern hemisphere and is the only gull species inhabiting the Antarctic continent. It is known 2B to be a food generalist, regularly feeding on food resulting from human activities (abattoirs, gar- bage, sewage outfalls, etc)8. This behaviour makes it an interesting sentinel species when studying environmental spread of antibiotic resistant bacteria. Our aim was to determine the occurrence and the molecular characteristics of ESC-resistant Enterobacteriaceae isolates recovered from Kelp gulls, as this species could favour the dissemination of ESC-resistant Enterobacteriaceae in human populations and the pristine Antarctic environment.

During November 2012, fresh faecal specimens (n=50) were collected from a flock of approximately 500 Kelp gulls on a sandy beach where they were roosting in Ushuaia, in Argentina. All samples were enriched either in brain heart infusion broth (Becton–Dickinson, Franklin Lakes, NJ, USA), supplemented with 16 mg/L vancomycin, or buffered peptone water (SVA, Uppsala, Sweden) for 18–24 h in 37 °C, and subsequently inoculated on ChromID™ ESBL (bioMérieux, Solna, Sweden) for the selective isolation of ESBL-producing Enterobacteriaceae, or modified semisolid Rappaport Vassiliadis agar for the selective isolation of Salmonella species (SVA, Uppsala, Sweden), respectively. Presumptive extended-spectrum cephalosporinase producing isolates were identified using MALDI-TOF Mass spectrometry (Brucker, Coventry, UK), while Salmonella isolates were further serotyped by the microtitration method. Antibiotic susceptibility of the isolates was assessed by broth microdilution and interpreted according the epidemiologic cut-off values recommended by the European Committee on Antimicrobial Susceptibility Testing (http://mic.eucast.org), whereas ESBL and/or AmpC production was evaluated by a combined disc test, as previously described9. Genes conferring ESC-resistant phenotype were sought and their genetic location on either the chromosome or a plasmid was determined as previously described9. Standard methods (PCR-based replicon typing, pMLST/pDLST/RST and S1 nuclease PFGE) were applied for further plasmid analysis, while the conjugal transferability of the extended-spectrum cephalosporinase genes and the presence of known insertion sequences upstream of them was examined9. Genetic relatedness among Escherichia coli and Salmonella enterica serotype Heidelberg (S. Heidelberg) isolates was assessed by MLST and XbaI-PFGE typing respectively, as previously described9, 10.

Molecular characterization of ESC-resistant Enterobacteriaceae from wild kelp gulls in South America | 57 Overall, we recovered 37 non-duplicate ESC-resistant Enterobacteriaceae isolates from 34 of the faecal samples included in the study. Among them, 91.9% (n=34) were identified as E. coli and 8.1% (n=3) as S. Heidelberg. The co-presence of ESC-resistant E. coli and S. Heidelberg was documented in three faecal samples. The recovered isolates exhibited non-wild-type MICs mainly for ciprofloxacin (n=27; 73.0%), nalidixic acid (n=25; 67.6%), tetracycline (n=22; 59.5%), sulfamethoxazole (n=20; 54.0%) and chloramphenicol (n=15; 40.5%). All isolates were susceptible to meropenem and tigecycline, whereas they exhibited non-wild-type MICs for the remaining tested agents varying from

5.4 to 27.0%. All E. coli isolates exhibited ESBL phenotype and carried blaCTX-M-2 (n=14; 41.2%), blaCTX-M-14 (n=11; 32.3%), blaSHV-2 (n=4; 11.8%), blaSHV-2A (n=4; 11.8%) and blaCTX-M-15 (n=1; 2.9%) genes, whereas all S. Heidelberg exhibited AmpC phenotype and carried the blaCMY-2 gene.

The broad-host-range IncI1α/γ (n=21; 67.7%) and narrow-host-range IncF (n=7; 22.6%) plasmids were by far the most common rep-types accounted for the ESC-resistant phenotype among the recovered isolates. The blaCTX-M-2 gene was found mainly on the chromosome (n=6; 42.9%) or on plasmids of different replicon types, including IncF plasmids with fused FIB-FII replicons (n=5; 35.7%), IncHI2 (n=1; 7.1%), IncA/C (n=1; 7.1%) and non typeable (n=1; 7.14%) ones. The blaCTX-M-14 and blaCTX-M-15 genes were identified exclusively onIncI1 /ST80 and IncFIA-FIB plasmids, respectively. The blaSHV-2 was associated with IncI1/ST12 and blaSHV-2A with IncI1/ST187 and IncFIA-

FIB plasmids, whereas the blaCMY-2 gene was located on IncI1/ST12 plasmids. Detailed results regarding the subtyping, the size and the transferability of the plasmids are summarized in Table 1.

Three insertion sequence elements previously associated with the mobilization and support of extended-spectrum cephalosporinase genes were identified. Briefly, in all isolates encoding blaCTX-

M-2, the gene was accompanied upstream by a copy of the ISCR1 in the same orientation as the resistance gene, regardless of the plasmid replicon type or if the gene was chromosomally located

(Table 1). Similarly, ISEcp1 was found upstream of the blaCTX-M-15 and blaCMY-2 genes, while IS26 was found upstream of the blaSHV-2 and blaSHV-2A genes. Neither ISEcp1, ISCR1 nor IS26 insertion sequences were found upstream of blaCTX-M-14 gene (Table 1).

High diversity of genotypes was observed amongst the E. coli isolates resulting in 17 different sequence types, each comprised of one to six isolates. The most predominant genotypes were ST744 (n=6; 17.6%), ST617 (n=5; 14.7%), ST57 (n=3; 8.8%), ST93 (n=3; 8.8%) and ST4038 (n=3; 8.8%), while isolates belonging to ST10, ST69, ST88, ST101, ST117, ST212, ST359, ST1011, ST1193, ST2485, STNew1 and STNew2 were also identified. AllS. Heidelberg isolates belonged to the epidemic clone JF6X01.0326/XbaI.1966 (PulseNet database). Different ESBL determinants were found among isolates with the same genotype, conversely, different genotypes carrying the

58 | Chapter 2B same ESBL determinants were identified (Table 1).

Several studies have documented the occurrence of ESC-resistant Enterobacteriaceae isolates among wild birds ranging from 0 to 37%4, 11-15. However, our study revealed higher occurrence among Kelp gulls in accordance with studies regarding Brown-headed and Franklin’s gulls16, 17. Although the resistance gene families described in this study are similar to those reported pre-

14-23 viously , we documented for the first time the presence of blaSHV-2A and the predominance of B blaCTX-M-2 among wild birds. The latter mirrors the situation observed for nosocomial infections in 2 24, 25 Argentinian hospitals , confirming the endemicity ofbla CTX-M-2 within this area and its potential transmission from humans to wild birds and/or vice versa. Of note was the association of blaCTX-M-2 gene with ISCR1 on four different plasmid replicon types associated with six different E. coli STs and on the chromosome of five other differentE. coli STs, underscoring that ISCR1 has probably played a significant role in the capture of this gene by conjugative plasmids, and in its further inter-replicon and inter-clone dissemination. Moreover, our data suggest the horizontal transfer of a conjugative IncI1/ST80 plasmid (105 Kb) encoding blaCTX-M-14 among five different E. coli STs, underscoring the dissemination of this gene owing to a successful plasmid-gene combination.

Among the 17 different STs detected here, we identified several, namely ST10, ST69, ST101, ST117, ST167, ST617 and ST744 that have been previously reported from ESC-resistant E. coli isolates of human and animal origin1, 15, 16, 19. Interestingly, some of the identified STs (ST10, ST117, ST157, ST359, ST617 and ST744) have been previously reported among wild birds as well, but they have been found to harbour different extended-spectrum cephalosporinase genes, suggesting that avian commensal E. coli strains play a role in the maintenance and dissemination of these genes1,

15, 16, 19 . In contrast with the solely ESC-resistant S. Heidelberg isolate encoding blaCMY-2 on a 97Kb IncN plasmid reported previously from an Argentinian adult inpatient26, here we documented for the first time the presence in wild birds of a known epidemic ESC-resistant S. Heidelberg clone

(JF6X01.0326/XbaI.1966), encoding blaCMY-2 on a 110Kb IncI1/ST12 plasmid. This PFGE-type circulating in USA and recently being introduced to Europe9, has been documented to cause outbreaks and exhibit potency for bloodstream infections27.

In conclusion, although there are few studies on the presence of resistance genes conferring ESC-resistant phenotype amongst Enterobacteriaceae from wild birds, to our knowledge this is the first report on the detailed characterisation of ESC-resistant Enterobacteriaceae, including the underlying antibiotic resistance gene content and its genetic support (plasmids and IS elements). Our data implies that Kelp gulls act as reservoirs of a variety of extended-spectrum cephalosporinase genes associated with different genetic platforms that could facilitate their horizontal

Molecular characterization of ESC-resistant Enterobacteriaceae from wild kelp gulls in South America | 59 R1 R1 R1 R1 R1 R1 R1 R1 R1 R1 R1 Ecp1 Ecp1 d d d d d Up- stream region IS IS26 IS26 ISC ISC ISC ISC ISC IS ISC IS26 IS26 - - - - - ISC ISC ISC ISC ISC c c c c c c c ). -9 Transferability Conjugative Conjugative Conjugative Conjugative Conjugative Conjugative Conjugative Conjugative Conjugative Conjugative Non conjugative Conjugative Conjugative Non conjugative Non conjugative Non conjugative Non conjugative Conjugative Conjugative Non conjugative Conjugative Non conjugative Size (Kb) 110 145 121 125 70 105 105 105 105 105 87 208 202 NA NA NA 100 132 170 NA 205 NA http://mlst.warwick.ac.uk/mlst/dbs/Ecoli). ) in Ushuaia, Argentina, 2012. Plasmid characteristics Plasmid type/subtype (no.) IncI1/ST12 (3) IncI1/ST12 (3) IncI1/ST12 (1) IncI1/ST187 (3) IncF/F1:A1:B1 (1) IncI1/ST80 (6) IncI1/ST80 (1) IncI1/ST80 (2) IncI1/ST80 (1) IncI1/ST80 (1) IncF/F1:A1:B1 (1) IncF/F18:A-:B1 (3) IncHI2/ST2 (1) NA NA NA IncA/C (1) NT (1) IncF/F24:A-:B1 (1) NA IncF/F24:A-:B10 (1) NA Location (no.) Plasmid (3) Plasmid (3) Plasmid (1) Plasmid (4) Plasmid (6) Plasmid (1) Plasmid (2) Plasmid (1) Plasmid (1) Plasmid (1) Plasmid (3) Plasmid (1) (2) Chromosome (1) Chromosome (1) Chromosome Plasmid (1) Plasmid (1) Plasmid (1) (1) Chromosome Plasmid (1) (1) Chromosome Larus dominicanus (6) (1) (2) (1) (1) (1) (3) (3) (1) (1) (1) (1) (1) (1) (1) (1) (4) (3) (3) (1) CTX-M-14 SHV-2A CTX-M-14 CTX-M-2 CTX-M-2 SHV-2 CTX-M-14 CTX-M-14 CTX-M-2 CTX-M-2 CTX-M-14 CTX-M-2 CTX-M-2 SHV-2 CTX-M-2 CTX-M-2 CTX-M-15 CTX-M-2 CTX-M-2 CMY-2 bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla ESBL/AmpC gene (no.) bla AMP,CAZ,CTX,TET (1) AMP,CAZ,CTX,TET (1) AMP,CHL,CIP,CTX (1) AMP,CAZ,CHL,CIP,CTX,NAL,SMX,TET AMP,CAZ,CTX (3) AMP,CAZ,CTX (1) AMP,CAZ,CTX,SMX,TET (1) AMP,CTX,TET (1) AMP,CAZ,CIP,CTX,NAL,SMX (1) AMP,CAZ,CIP,CTX,NAL,TET (1) AMP,CAZ,CHL,CTX,SMX,TET AMP,CAZ,CIP,CTX,NAL,TET (1) AMP,CAZ,CIP,CTX,NAL,TET AMP,CAZ,CHL,CIP,CTX,NAL,TET (4) AMP,CAZ,CHL,CIP,CTX,NAL,TET (3) AMP,CAZ,CIP,CTX,GEN,NAL,SMX,TET,TMP (1) AMP,CAZ,CIP,CST,CTX,NAL,SMX,TET AMP,CAZ,CIP,CTX,NAL (1) AMP,CAZ,CIP,CTX,NAL (1) AMP,CAZ,CHL,CIP,CTX,GEN,NAL,SMX (1) AMP,CAZ,CIP,CTX,NAL,SMX (3) AMP,CAZ,CIP,CTX,NAL AMP,CAZ,CHL,CTX,SMX,TMP (1) AMP,CAZ,CHL,CTX,SMX,TMP (1) AMP,CAZ,CIP,CTX,GEN,SMX (1) AMP,CAZ,CTX,SMX,TET (1) AMP,CAZ,CTX (1) AMP,CAZ,CIP,CTX,GEN,NAL,SMX Resistance phenotypes (no.) (1) AMP,CAZ,CHL,CIP,CST,CTX,NAL,SMX,TET,TMP (2) AMP,AZM,CAZ,CHL,CIP,CTX,NAL,SMX,TET,TMP (3) AMP,CHL,CIP,CST,CTX,NAL,SMX,TET,TMP b a a ST10 (2) ST359 (1) ST4038 (3) ST69 (1) ST88 (1) ST117 (1) ST1193 (1) ST2485 (1) ST617 (5) ST57 (3) STNew1 (2) ST101 (1) ST93 (3) ST212 (1) ST1011 (1) STNew2 (1) ST/PFGE-type (no.) ST744 (6) JF6X01.0326 (3) or IS26 insertion sequences were not found upstream of the ESBL-genes for these STs. not found upstream or IS26 insertion sequences were CR1 , IS Characteristics of ESC-resistant Enterobacteriaceae isolates recovered from Kelp gulls ( from isolates recovered Enterobacteriaceae Characteristics of ESC-resistant Ecp1 (34) . Heidelberg (3) PFGE patternto the PulseNet database. numbers corresponding The IS Assignment to specific ST could not be performed as uploading new sequences and STs based on AB1 files is no longer supported by MLST database ( Assignment to specific ST could not be performed as uploading new sequences and STs No transconjugants were obtained either after liquid mating experiments suggesting either non conjugative plasmids or conjugation frequencies below the detection limit (≤1x10 obtained either after liquid mating experiments suggesting non conjugative plasmids or conjugation frequencies No transconjugants were Table 1. Table Species (no.) E. coli NAL: colistin, CTX: cefotaxime, GEN: gentamicin, MEM: meropenem, CHL: chloramphenicol, CST: CAZ: ceftazidime, CIP: ciprofloxacin, sequence type, AMP: ampicillin, AZM: azithromycin, ST: tetracycline, TGC: tigecycline, TMP: trimethoprim and NA: not applicable. nalidixic acid, SMX: sulfamethoxazole, TET: a b c d S

60 | Chapter 2B transfer. In addition, our findings underscore the potential role of Kelp gulls as a bridge species for ESC-resistant Enterobacteriaceae between humans and wildlife and as a spreader of these isolates among human populations and naturally antibiotic-resistant bacteria free environments (Antarctic continent) via their movement and migration.

Acknowledgements

The authors gratefully acknowledge Dr Kees Veldman, Joop Testerink and Marga Japing for the 2B antimicrobial susceptibility testing of the isolates and Quark Expeditions for supporting the fieldtrip. Preliminary results from this study were presented as an oral presentation at the 26th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), 9–12 April 2016, Amsterdam, The Netherlands.

Funding

This work was supported by the Dutch Ministry of Economic Affairs (BO-22.04-008-001).

Molecular characterization of ESC-resistant Enterobacteriaceae from wild kelp gulls in South America | 61 References

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Molecular characterization of ESC-resistant Enterobacteriaceae from wild kelp gulls in South America | 63

CHAPTER 3 A

High prevalence of intra-familial co-colonization by extended- spectrum cephalosporin resistant Enterobacteriaceae in preschool children and their parents in Dutch households

Apostolos Liakopoulos1, Gerrita van den Bunt2,3, Yvon Geurts1, Martin C. J. Bootsma2,4, Mark Toleman5, Daniela Ceccarelli1, Wilfrid van Pelt3, Dik J. Mevius1,6

1Department of Bacteriology and Epidemiology, Wageningen Bioveterinary Research (WBVR), Lelystad, the Netherlands; 2Julius Center for Health Sciences and Primary Care, University Medical Center Utrecht (UMCU), Utrecht, the Netherlands; 3Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, the Netherlands; 4Department of Mathematics, Faculty of Science, Utrecht University, Utrecht, the Netherlands; 5Division of Infection and Immunity, School of Medicine, Cardiff University, Cardiff, UK; 6Faculty of Veterinary Medicine, Department of Infectious Diseases and Immunology, Utrecht University, Utrecht, the Netherlands.

Submitted Abstract

Extended-spectrum cephalosporin (ESC)-resistant Enterobacteriaceae pose a serious infection control challenge for public health. The emergence of the ESC-resistant phenotype is mostly facilitated by plasmid-mediated horizontal ESBL and AmpC gene transfer within Enterobacteriaceae. Current data regarding the plasmid contribution to this emergence within the Dutch human population is limited. Hence, the aim of this study was to gain insight into the role of plasmids in the dissemination of ESBL/AmpC genes inside Dutch households with preschool children and precisely delineate co-colonization.

In 87 ESC-resistant Enterobacteriaceae from faecal samples of parents and preschool children within 66 Dutch households, genomic localization, plasmid type and insertion sequences linked to ESBL/AmpC genes were determined. An epidemiologically relevant subset of the isolates based on household co-carriage, was assessed for genetic relatedness by MLST and XbaI-PFGE. Chromo- somal location of specific ESBL/AmpC genes was confirmed when necessary.

The narrow-host range I1α and F plasmids were found to be the major facilitators of ESBL/AmpC- gene dissemination. Interestingly, we documented a relatively high occurrence of chromosomal integration of typically plasmid-encoded ESBL/AmpC-genes. A high diversity of non-epidemic Escherichia coli sequence types (STs) was revealed; however, the predominant STs belonged to the pandemic lineages of extraintestinal pathogenic E. coli (ExPEC) ST131 and ST69. In 7 of the 14 households with intra-familial co-colonization we documented ESC-resistant Enterobacteriaceae, potentially because of the child to/from parent clonal transmission.

Our findings underscore that typing beyond the ESBL/AmpC gene level is essential to trace transmission events even within epidemiologically linked cases. Intra-familial co-colonization in Dutch household with identical and with unrelated ESC-resistant Enterobacteriaceae occur both more often than expected based on chance.

66 | Chapter 3A Introduction

Extended-spectrum cephalosporin-resistant (ESC-resistant) Enterobacteriaceae have emerged worldwide as a significant cause of hospital-, health care- and community-associated infections 1-4. The increase in prevalence of ESC-resistant phenotype, which has been observed in the last decades, is mainly due to the production of extended-spectrum β-lactamases (ESBLs) and to a lesser degree to acquired AmpC β-lactamases 1-4. ESBL and AmpC β-lactamases belong to different structural and functional classification groups 5,6, resulting in differences in their hydrolytic spectrums. Yet, they are both able to hydrolyse the oxyimino-β-cephalosporins commonly used in clinical practice, such as cefotaxime and ceftazidime 1,3. The emergence of the ESC-resistant phenotype is facilitated mostly by the plasmid-mediated horizontal ESBL and AmpC gene transfer within 3A Enterobacteriaceae 7.

Transmission of ESC-resistant Enterobacteriaceae within households and subsequently amongst the community has been documented to occur in multiple ways: from patients with community- acquired infections 8, patients recently discharged or cared for in a hospital 9-13, infants colonized after neonatal ICU admission 14, adopted children that originated from countries with high prevalence of ESC-resistant Enterobacteriaceae 15 and international travellers to their household contacts 16.

Several studies have reported the predominance of blaCTX-M genes in ESC-resistant Enterobacteri- aceae, mostly Escherichia coli, in the Netherlands among hospital and primary care patients 17-19, nursing home residents 20 and humans in the community 21,22. Among them, E. coli belonging to sequence types (ST)10, ST38, ST69 and ST131 recovered from Dutch individuals have been recently associated with ESC-resistant phenotype 17-21,23-30. However, data regarding the genetic support, plasmid replicon types and Insertion Sequence (IS) of ESBL/AmpC genes and subsequently their plasmid-mediated dissemination among the Dutch human population have not been fully investigated.

We previously reported the results of a two-year cross-sectional study on the intestinal colonization with ESC-resistant Enterobacteriaceae in Dutch households with preschool children 22. In this study co-colonization with an identical ESC-resistant Enterobacteriaceae between children and their parents was solely defined based on the presence of same ESBL/AmpC gene(s). In order to elucidate the role of plasmids in the dissemination of ESBL/AmpC genes, refine the transmission analysis and delineate co-colonization inside the households more precisely, we extended the analysis to gene localization, plasmid and strain typing of the ESC-resistant Enterobacteriaceae collected during the cross-sectional study. Results and new intra-familial co-colonization data are reported here.

High prevalence of intra-familial co-colonization by ESC-resistant Enterobacteriaceae in preschool children and parents | 67 Results

Plasmid, insertion sequence and ESBL/AmpC gene association Results of genomic localization of ESBL/AmpC genes found in 87 ESC-resistant Enterobacteriaceae are summarized in Table S1. Overall, most of the genes were plasmid located, independent on source of isolation (child or parent) and bacterial species.

Among ESBL/AmpC genes from preschool children isolates, the majority (75.7%; 28 out of 37) was encoded on plasmids belonging to 10 different replicon types or non typeable ones. Each replicon type was associated with one to four different ESBL/AmpC genes. The most predominant was IncI1α (35.7%; n=10) associated with blaSHV-12 (n=5), blaCTX-M-1 (n=2), blaCMY-2 (n=2) or blaTEM-52c

(n=1), followed by IncF (21.4%; n=6) associated with blaCTX-M-15 (n=3), blaCTX-M-3 (n=1), blaCTX-M-14b

(n=1) or blaCTX-M-27 (n=1) genes. Genes blaCTX-M-3, blaCTX-M-15, blaCTX-M-14, blaCTX-M-14b, and blaCMY-2 were also located on the chromosome in 24.3% (n=9) of the cases.

Similarly for the preschool children isolates, the majority of the ESBL/AmpC genes (86.0%; 43 out of 50) derived from parent isolates were encoded on plasmids belonging to 11 different replicon types or non typeable ones (Table S1). Plasmids assigned to IncI1α replicon type were the most prevalent (39.5%; n=17) in association with blaCTX-M-1 (n=8), blaCTX-M-15 (n=4), blaSHV-12 (n=2), blaCTX-

M-3 (n=1), blaTEM-52c (n=1), blaCMY-2 (n=1) or blaDHA-1 (n=1). The second most represented replicon type, IncF (30.2%; n=13), was associated with blaCTX-M-15 (n=7), blaCTX-M-27 (n=3), blaCTX-M-3 (n=1), blaCTX-M-14 (n=1) or blaCTX-M-14var (n=1) genes. A small proportion of genes (14.0%; n=7), namely blaCTX-M-15, blaCTX-M-14, and blaCTX-M-14b was encoded on the chromosome.

Subtyping of IncI1α/γ plasmids from both parents and children isolates revealed the presence of 28 plasmid sequence types (pST) (27 among IncI1α plasmids and one among IncI1γ plasmids), each encoding one to two ESBL/AmpC genes. The most predominant types were pST3 (20.7%; n=6) and pST9 (10.3%; n=3), mostly associated with blaCTX-M-1 and blaSHV-12. (Figure 1a). Subtyping of IncF plasmids assigned them to 12 different replicon sequence types (RST) encoding one to three ESBL/ AmpC genes (Figure 1b). The most prevalent ones were F1:A2:B20 (15.0%; n=3), F2:A-:B- (15.0%; n=3) and F2:A4:B1 (15.0%; n=3) in association with blaCTX-M-27, blaCTX-M-14, blaCTX-M-3 and blaCTX-M-15.

Eighty-one (93%) of the ESBL/AmpC genes under investigation were associated with insertion elements ISEcp1 (n=62), IS26 (n=17) or ISCR1 (n=2) (Table S1). ISEcp1 was mostly associated with ESBL genes belonging to CTX-M-1-group in both children (n=16) and parents (n=25), as well as with genes of the CTX-M-9-group (n=7 and n=8, respectively) and blaCMY-2 (n=4 and n=2, respec-

68 | Chapter 3A tively). IS26 was 100% associated with blaSHV-12 both in children (n=5) and parents (n=3), while

ISCR1 was detected only in 2 E. coli isolates from parents in association with blaCTX-M-2 or blaCTX-M-15.

E. coli isolates recovered from the same faecal sample could either belong to different STs holding the same IncI1α plasmid carrying blaCTX-M-1 (households 12 and 21) or be clonal isolates (household 43).

Variability in plasmid and IS distribution for fifty-five isolates recovered either from parent or child in a household are reported in Table 1.

Figure 1. Association between ESBL/AmpC genes with (A) IncI1α/γ plasmid STs and (B) IncF replicon sequence types.

High prevalence of intra-familial co-colonization by ESC-resistant Enterobacteriaceae in preschool children and parents | 69 Table 1. ESC-resistant Enterobacteriaceae recovered from parents or children in Dutch households. Household Isolate* Bacterial ST/CC ESBL/AmpC Location Plasmid rep/ Plasmid Insertion species genes^ inc-type subtype Sequence

1 C-12371 E. coli ND blaTEM-52var Plasmid X1 NA IS26

2 C-13309 E. coli ND blaSHV-12 Plasmid I1α pST3/ pCC3 IS26

3 C-13311 E. coli ND blaCMY-2 Plasmid I1α pST43 ISEcp1

4 C-20046 E. coli ND blaCTX-M-15 Plasmid F F2:A4:B1 ISEcp1

5 C-20895 E. coli ND blaCMY-2 Plasmid I1γ pST189 ISEcp1

6 C-21281a E. coli ND blaCTX-M-14 Chromosome - - ISEcp1

7 C-24053 E. cloacae ND blaCTX-M-3 Chromosome - - ISEcp1

8 C-24900 E. coli ND blaCTX-M-15 Chromosome - - ISEcp1

9 C-25932 E. coli ND blaCTX-M-15 Plasmid NT NP

10 C-26971 E. coli ND blaSHV-12 Plasmid I1α pST95 IS26

11 C-29568 E. coli ND blaCTX-M-15 Plasmid HI2 pST3 ISEcp1

12 C-29929a E. coli 345 blaCTX-M-1 Plasmid I1α pST35 ISEcp1

13 C-29929b E. coli 131/131 blaCTX-M-1 Plasmid I1α pST35 ISEcp1

C-29945 E.coli ND blaSHV-12 Plasmid I1α pST227 IS26

14 C-31162 E. coli ND blaCTX-M-24 Plasmid N pST3 ISEcp1

15 C-42978 E. coli ND blaCMY-2 Plasmid I1α pST2/ pCC2 ISEcp1

16 C-43494 E. coli ND blaCMY-2 Chromosome - NA ISEcp1

17 C-44647 E. cloacae ND blaCTX-M-15 Chromosome - NA ISEcp1

18 C-45577 E. coli ND blaCTX-M-1 Plasmid N ST1 ISEcp1

19 C-50954 E. coli ND blaCTX-M-14b Chromosome - - ISEcp1

20 C-51026 E. coli ND blaCTX-M-1 Plasmid K NA ISEcp1

21 P-12356a E. coli 1775 blaCTX-M-1 Plasmid I1α pST3/ pCC3 ISEcp1

22 P-12356b E. coli 648/648 blaCTX-M-1 Plasmid I1α pST7/ pCC7 ISEcp1

P-13127 E. coli ND blaCTX-M-1 Plasmid I1α pST58/ pCC58 ISEcp1

23 P-13277 E. coli ND blaCTX-M-27 Plasmid F F2:A-:B- IS26

24 P-14152 E. coli ND blaCTX-M-14var Plasmid F F24:A-:B1 ISEcp1

25 P-14808 E. coli ND blaCTX-M-1 Plasmid I1α pST3/ pCC3 ISEcp1

26 P-15052a E. coli ND blaCTX-M-15 Plasmid F F2:A4:B1 ISEcp1

27 P-15274 E. coli ND blaCTX-M-1 Plasmid I1α pST3/ pCC3 ISEcp1

28 P-16235 E. coli ND blaCTX-M-1 Plasmid X1 NA IS26

29 P-16817 E. coli ND blaCTX-M-15 Plasmid F F1:A1:B1 IS26

30 P-18176 E. coli ND blaCTX-M-15 Chromosome - - IS26

31 P-18216 E. coli ND blaCTX-M-2 Plasmid HI1 NA ISCR1

32 P-19659 E. coli ND blaSHV-12 Plasmid I1α pST3/ pCC3 IS26

33 P-20005 E. coli ND blaCTX-M-15 Plasmid F F1:A4:B1 ISEcp1

34 P-20371 E. coli ND blaCTX-M-15 Plasmid I1α pST188 ISEcp1

35 P-21458 E. coli ND blaCTX-M-1 Plasmid I1α pST58/ pCC58 ISEcp1

36 P-23698 E. coli ND blaSHV-12 Plasmid N pST1 IS26

70 | Chapter 3A Table 1. Continued Household Isolate* Bacterial ST/CC ESBL/AmpC Location Plasmid rep/ Plasmid Insertion species genes^ inc-type subtype Sequence

37 P-23883 E. coli ND blaCTX-M-15 Chromosome - - ISEcp1

38 P-25030 E. coli ND blaDHA-1 Plasmid NT NP

39 P-26355 E. coli ND blaCTX-M-14 Plasmid F F2:A-:B- NP

40 P-26492 E. coli ND blaCMY-2 Plasmid I1α pST12/ pCC12 ISEcp1

41 P-26517 E. coli ND blaCTX-M-14 Plasmid B/O NA ISEcp1

42 P-28847 E. coli ND blaCTX-M-27 Plasmid F-R F1:A2:B20 IS26

43 P-29344a E. coli 10/10 blaCTX-M-15 Plasmid F F4:A-:B- ISEcp1

P-29344b E. coli 10/10 blaCTX-M-15 Plasmid F F2:A1:B1 ISEcp1

44 P-29754 E. coli ND blaCTX-M-1 Plasmid I1α pST3/ pCC3 ISEcp1 45 P-30462a E. coli ND Plasmid pST31/ pCC31 ISEcp1 blaCTX-M-15 I1α 3A

46 P-30656 E. coli ND blaCTX-M-14 Plasmid B/O NA ISEcp1 47 P-41705 K. pneu- ND Plasmid HIB-M NA ISEcp1 bla moniae CTX-M-15

48 P-44471 E. coli ND blaCTX-M-15 Chromosome - - ISEcp1

49 P-45037 E. coli ND blaCTX-M-15 Plasmid I1α pST1 ISEcp1

50 P-45865 E. coli ND blaCTX-M-15 Plasmid K NA ISEcp1

51 P-45995 E. coli ND blaCTX-M-15 Plasmid F F2:A4:B1 ISEcp1

52 P-50908 E. coli ND blaCMY-2 Plasmid K NA ISEcp1 *Letters a and b indicate isolates with distinct colony morphotypes originated from the same faecal sample ^Gene typing performed in (van den Bunt et al., 2016)22 C: child, P: parent, ST/CC: sequence type/clonal complex, pST/pCC: plasmid sequence type/plasmid clonal complex, ND: not determined, NA: not available, NP: not present and NT: non typeable plasmid.

Child-parent pairs Thirty-two ESC-resistant isolates, mostly E. coli, recovered from parent and child within the same household (n=14), including isolates of the same species recovered from the same faecal sample as distinctive colonial morphotypes (a and b) were further characterised based on their genetic relatedness, plasmid replicon and IS type (Table 2). Based on these additional criteria, intra-familial colonization was revisited.

ESC-resistant E. coli recovered from Dutch children and parents were found to belong to 14 partially overlapping STs (Table 2). Each of the 11 STs among children was comprised of one to five isolates and associated with one to four different ESBL/AmpC genes; among parents different STs comprised one to four isolates and one to three ESBL/AmpC genes. Six common STs were identified between children and parents, namely, ST10, ST38, ST69, ST131, ST301 and ST665. The most predominant ST among children was ST131 (n=4) associated with blaCTX-M-3 (n=2), blaCTX-M-15 (n=1) or blaTEM-52c (n=1). Among parents the most predominant STs was ST131 (n=3) associated with blaCTX-M-3 (n=2) or blaTEM-52c

(n=1), followed by ST69 (n=4) associated with blaCTX-M-27 (n=2), blaCTX-M-14 (n=1) or blaCTX-M-15 (n=1).

High prevalence of intra-familial co-colonization by ESC-resistant Enterobacteriaceae in preschool children and parents | 71 In 12 of 14 households we documented the colonization of both the child and the parent by either the same ESC-resistant bacterial species or different ESC-resistant Enterobacteriaceae encoding the same acquired ESBL/AmpC gene (Table 2); and only 10 were colonized by the same bacterial species encoding the same acquired ESBL/AmpC gene.

Seven child-parent pairs were found to carry E. coli of the same STs encoding the same ESBL/AmpC gene on the same genetic location, either chromosome (household 62) or plasmid belonging to the same replicon type and subtype (households 54, 56, 61, 63, 65, 66; Table 2). PFGE analysis revealed identical XbaI-PFGE profiles among paired child and parent isolates belonging to the same STs, confirming the genetic relatedness observed by MLST.

Participants belonging to these 7 households had a Dutch nationality, and the households contained either one (n=2; 28.6%) or two children (n=5; 71.4%). In 5 of the households companion animals were present, and in 6 households one child attended day-care, which was not necessarily the screened child. Median ages were 33 years [interquartile range (IQR) 30-35] in parents, and 25 months in children (IQR 12-36). From the 7 ESBL-positive parents, 5 were female, which is comparable to the overall female-male-distribution of participating parents in the study, and one of these parents used antibiotics in the 6 months previous to sampling, whereas only 3.2% of the 983 parents used antibiotics in the past 6 months. From the 7 children, 2 were female, which is lower as compared to the overall female-male-distribution in children (50.2%) and 5 attended day-care, in line with the percentage among total children (52.0%). None of the 7 children used antibiotics in the past 6 months, compared to 7.6% of the 983 children.

Table 2. Molecular characteristics of the ESC-resistant Enterobacteriaceae recovered from child-parent pairs in 14 Dutch households. Bacterial ESBL/AmpC Plasmid rep/ Plasmid Insertion Household Isolate* ST/ CC Location species gene^ inc-type subtype Sequence

53 C1a E. coli 1380 blaCTX-M-15 Plasmid K NA NP

C1b E. coli 34/10 blaCTX-M-15 Plasmid F F1:A1:B16 ISEcp1

# P1 E. coli 3036 blaCTX-M-15; Plasmid I1α pST68/ IS26

blaDHA-1 pCC31

54 C2 E. coli 131/131 blaTEM-52c Plasmid I1α pST36/ pCC5 NP

P2 E. coli 131/131 blaTEM-52c Plasmid I1α pST36/ pCC5 NP

55 C3a E. coli 38/38 blaCTX-M-14b Plasmid F F29:A4:B10 ISEcp1

C3b E. coli 93/168 blaCTX-M-15 Plasmid F ND ISEcp1

P3a E. coli 10/10 blaCTX-M-3 Plasmid I1α pST57 ISEcp1

P3b E. coli 3610 blaCTX-M-14b Chromosome - - ISEcp1

56 C4 E. coli 301/165 blaCTX-M-14 Plasmid K NA ISEcp1

72 | Chapter 3A Table 2. Continued. Household Isolate* Bacterial ST/ CC ESBL/AmpC Location Plasmid rep/ Plasmid Insertion species gene^ inc-type subtype Sequence

P4 E. coli 301/165 blaCTX-M-14 Plasmid K NA ISEcp1

57 C5 E. coli 131/131 blaCTX-M-15 Chromosome - - ISEcp1

SLV P5 K. pneu- 570 blaCTX-M-15 Plasmid F ND ISEcp1 moniae

58 C6 E. coli 131/131 blaCTX-M-3 Plasmid F F2:A-:B- ISEcp1

P6 E. coli 131/131 blaCTX-M-3 Plasmid F F29:A-:B- ISEcp1

59 C7 K. pneu- 48 blaCTX-M-15 Chromosome - - ISEcp1 moniae

P7 E. coli 69/69 blaCTX-M-15 Chromosome - - ISEcp1 60 C8 E. coli 1312 bla Plasmid colE NA ISEcp1 CTX-M-15 3A P8 E. coli 23 blaCTX-M-1 Plasmid I1α pST190 ISEcp1

61 C9 E. coli 131/131 blaCTX-M-3 Plasmid Y NA ISEcp1

P9 E. coli 131/131 blaCTX-M-3 Plasmid Y NA ISEcp1

62 C10 E. coli 38/38 blaCTX-M-14 Chromosome - - ISEcp1

P10a E. coli 38/38 blaCTX-M-14 Chromosome - - ISEcp1

P10b E. coli 38/38 blaCTX-M-14 Chromosome - - ISEcp1

63 C11 E. coli 665 blaSHV-12 Plasmid I1α pST95 IS26

P11 E. coli 665 blaSHV-12 Plasmid I1α pST95 IS26

64 C12 E. coli 10/10 blaSHV-12 Plasmid I1α pST228 IS26

P12 E. coli 69/69 blaCTX-M-27 Plasmid F F95:A-:B1 IS26

65 C13 E. coli 69/69 blaCTX-M-14 Plasmid B/O NA ISEcp1

P13 E. coli 69/69 blaCTX-M-14 Plasmid B/O NA ISEcp1

66 C14 E. coli 69/69 blaCTX-M-27 Plasmid F F1:A2:B20 IS26

P14 E. coli 69/69 blaCTX-M-27 Plasmid F F1:A2:B20 IS26 *Letters a and b indicate isolates with distinct colony morphotypes originated from the same faecal sample. ^Gene typing performed in (van den Bunt et al., 2016)22. # IncI1α carries blaCTX-M15 only. Transformants carrying blaDHA-1 were not recovered (performed in duplicate). C: child, P: parent, ST/CC: sequence type/clonal complex, SLV: single locus variant, pST/pCC: plasmid sequence type/plasmid clonal complex, ND: not determined, NA: not available and NP: not present.

By comparing the households with parent-child pairs co-colonized by non-identical to the ones with identical ESC-resistant Enterobacteriaceae (Table 3), we observed within the latter that there were more households with 2 children (71.4% versus 42.9%), more children in the household attended day-care (85.7% versus 57.1), more often companion animals were present in the household (71.4 versus 28.6) and more parents worked (healthcare-related) with children (28.6% versus 0.0%). None of the differences we observed between the two groups were statistically significant.

High prevalence of intra-familial co-colonization by ESC-resistant Enterobacteriaceae in preschool children and parents | 73 Table 3. Comparison of households with identical and non-identical ESC-resistant Enterobacteriaceae. Variable* Household Identical^ (n=7) Non-identical (n=7) n (%) n (%) Age of the child <=12 months 2 (28.6) 0 (0.0) 13-36 months 4 (57.1) 5 (71.4) 37-48 months 1 (14.3) 2 (28.6) Age of the parent <=34 5 (71.4) 2 (28.6) >34 2 (28.6) 5 (71.4) Gender of the child (male) 5 (71.4) 1 (14.3) Gender of the parent (male) 2 (28.6) 3 (42.9) Dutch nationality of the household 7 (100.0) 6 (85.7) Number of children in the household 1 child 2 (28.6) 4 (57.1) 2 children 5 (71.4) 3 (42.9) Participating child attending day-care 5 (71.4) 4 (57.1) A child in the household attends day-care 6 (85.7) 4 (57.1) Animals in the household 5 (71.4) 2 (28.6) Child uses antimicrobials 0 (0.0) 0 (0.0) Parent uses antimicrobials 1 (14.3) 0 (0.0) Parent works with children (healthcare related) 2 (28.6) 0 (0.0) *Fisher exact test was performed to test differences between households where child and parent were colonized with the identical and non-identical ESC-resistant Enterobacteriaceae , and none of the variables were statistically significant different. ^The term identical is used to define ESC-resistant Enterobacteriaceae assigned to the same ST/PFGE-pattern within the species carrying an identical ESBL/AmpC-gene on the same genetic location [plasmid type (and subtype) or the chromosome].

Intra-familial co-colonization Among 66 Dutch households in which at least one individual was colonized with an ESC-resistant Enterobacteriaceae, only from 14 (21.2%) we recovered an ESC-resistant isolate from both child and parent (Table 2). Given the 983 households and the observed prevalence in children and parents (ref Gerrita), the expected prevalence of finding co-colonization within a household in a one-to-one relationship purely based on chance was calculated to be 0.16%. This corresponds to an expected 1.6 out of 983 households, in which both parent and child are ESBL/AmpC carriers. Further molecular characterization of the recovered ESC-resistant isolates, revealed 7 households with identical STs/PFGE-patterns within the species carrying the same ESBL/AmpC gene on the same genetic location. The observed co-colonization (n=7) was significantly higher than the expected one (n=1.6) (P<0.002).

74 | Chapter 3A The probability of carrying identical STs/PFGE-patterns, ESBL/AmpC-gene type and genetic location of the ESBL/AmpC-gene in children and parents were 0.87, 0.046 and 0.14, respectively. To allow for correlations between the presence of certain bacterial species, certain ESBL/AmpC genes and genetic location [plasmid types (and subtypes) or chromosome], we only used the entity with the highest diversity, i.e., the genetic location of the ESBL/AmpC gene for further calculations. We observed 50% (n=7) of the household shared the same location of the ESBL/AmpC gene, which is significantly higher (P<0.001) than the expected number (0.046*14=0.65).

Discussion 3A Compared to our previous study 22, we here refined the molecular analysis of ESC-resistant Ente- robacteriaceae isolated from Dutch preschool children and their parents to gain insight into the co-carriage of ESBL/AmpC genes within the same household.

The majority of the ESBL/AmpC genes was encoded on plasmids assigned to the narrow-host range I1α/γ and F replicon types, confirming the importance of these plasmid families in the dissemination of ESBL/AmpC genes within the Dutch human population 19,21,25,30,44,45. These plasmid families were shown to encode the genetic machinery (addiction and partitioning systems) for their efficient maintenance and stability within bacterial hosts, even without the positive selective pressure exerted by antibiotics 46,47. Interestingly, replicon types with known wide range of hosts were not identified among the ESBL/AmpC-encoding plasmids, suggesting the limited potential diffusion of these plasmids to genera other than Enterobacteriaceae in the enteric cavity.

The presence of ISEcp1 and IS26 upstream of most of blaCTX-M and blaSHV-12 genes, respectively, possibly provides strong promoter sequences for high-level expression 48-50 and reflects their involvement in the mobilization of these resistance genes from the chromosome of Kluyvera and K. pneumoniae, respectively 51,52. The mobilization of ESBL/AmpC genes on known conjugative plasmids could facilitate their subsequent intra- and inter-species diffusion. The relative frequent occurrence of chromosomal integration of typically plasmid-encoded blaCTX-M genes may have been facilitated by the presence of ISEcp1, in an attempt of the bacterial host to maintain the beneficialbla CTX-M genes by lowering the fitness cost derived from harbouring an entire plasmid 53. This is supported by the recent identification of chromosomalbla CTX-M genes mostly in E. coli isolates (including ST38 and ST131, as observed here), which were associated with ISEcp1-mediated transposition to the bacterial chromosome 54-61. Further information on the chromosomal integration site and genetic context of the integrated blaCTX-M genes might be obtained by whole genome sequencing of the isolates.

High prevalence of intra-familial co-colonization by ESC-resistant Enterobacteriaceae in preschool children and parents | 75 The diversity of E. coli STs found within Dutch preschool children and parents suggests that commensal E. coli act as reservoir of ESBL/AmpC genes. The most prevalent STs among ESC-resistant E. coli were found to be the pandemic lineages of extraintestinal pathogenic E. coli (ExPEC) ST131 and ST69, known to cause infections of a great range of severity, including urinary and bloodstream infections 62. These STs have been associated with a competitive advantage over other E. coli STs owing to a combination of antimicrobial resistance and virulence determinants, subsequently promoting their clonal expansion and prevalence over less virulent and/or more susceptible E. coli lineages 62,63. As a result of this combination of antimicrobial resistance and virulence determinants, these E. coli STs could result in higher morbidity and mortality in case of infections.

Intra-familial transmission has been documented for different bacterial pathogens, including ESBL-producing Enterobacteriaceae 9,12,14,15,64. In our study, we clearly observed a cluster of ESC- resistant Enterobacteriaceae carriers within Dutch households, since we documented co-carriage with identical ESC-resistant Enterobacteriaceae (n=7) within 983 households more often than expected based on purely chance. We hypothesize clonal transmission occurred between children and parents within these households. Although the ESC-resistant E. coli strains recovered from these households belonged to human-related STs, we cannot rule out the exposure of each child and parent pair to a common source, as we did not investigate the presence of ESC-resistant Enterobacteriaceae from other sources, such as food or companion animals. For the remaining households, the high diversity of ESBL/AmpC genes, plasmid replicon types (and subtypes) and/or STs between colonized children and parents of the same households, hints to unrelated acquisitions and individuals sharing the same risk factors (e.g., travelling, improper hand hygiene) rather than sharing the same source (e.g., either a household member or same contaminated food source). Although the large sample size of the study (n=983 households), there was not enough power to detect differences in characteristics between the households where individuals were sharing and the ones not sharing identical ESC-resistant isolates.

Taking into consideration the close contact between children and their parents, our findings highlight the high prevalence of intra-familial co-colonization by ESC-resistant Enterobacteriaceae in Dutch households with preschool children. Still, we cannot exclude the possibility of underes- timating this prevalence owing to the fact that we have only sampled one of the parents of each household. In addition, we calculated that even within epidemiologically linked cases, considering only ESC-resistant isolates encoding the same ESBL/AmpC gene as an indication of co-colonization, a statistically significant overestimation of the prevalence of true co-colonization was observed. We therefore argue that a potential transmission event from a preschool child to its parent or vice versa can only be assumed if at least the ESBL/AmpC gene and the encoding plasmid replicon type

76 | Chapter 3A (and subtype) are identical between the Enterobacteriaceae recovered from both parent and child. In conclusion, our findings suggest that typing beyond the ESBL/AmpC gene level is essential to better tracing transmission events. The prevalence of ESC-resistant Enterobacteriaceae within the Dutch general population is relatively low, but clearly there are clusters within households, and intra-familial co-colonization seems to occur more often than expected based on chance.

Materials and Methods

Bacterial isolates Eighty-seven ESC-resistant Enterobacteriaceae (E. coli, Klebsiella pneumoniae and Enterobacter cloa- 3A cae) previously isolated from faecal samples of parents and preschool children within 66 households 22 were included in this study. Previously uncharacterized isolates of the same species recovered from the same faecal sample as distinctive colonial morphotypes were also included in this study (isolates a and b, Table 1 and 2). Pure isolates were stored at -80°C in Peptone Broth supplemented with 30% (v/v) glycerol. Isolates recovered from child and parent were designated as C and P, respectively.

Plasmid typing and insertion sequence analysis Total bacterial DNA was extracted using the DNeasy Blood and Tissue kit (QIAGEN, Hilden, Germany) according to manufacturer’s recommendations. Plasmid DNA was extracted using the alkaline lysis method and transformed into DH10B cells via electroporation (Invitrogen, Van Allen Way, CA USA)31.

Transformants (Trfs) were selected on LB agar plates supplemented with 1 mg/L cefotaxime and tested for the presence of the ESBL/AmpC gene of the corresponding donor isolate by PCR, as previously described 22. Replicon typing of each ESBL/AmpC-encoding plasmid was determined by the PBRT KIT (DIATHEVA, Fano, Italy) according to manufacturer’s recommendations, with the addition of single PCRs for IncX4 and ColE plasmids, as previously described 32,33. Subtyping of plasmids belonging to replicon types for which subtyping schemes are available (F, HI2, I1α/γ and N), was performed as previously described 34-37. When necessary, chromosomal location of the ESBL/AmpC genes was confirmed by I-Ceu-I-PFGE of total DNA followed by Southern blot hybridization with intragenic β-lactamase and 16S rDNA and/or S1-PFGE with intragenic β-lactamase probes, as previously described 38,39.

The presence of frequent insertion sequences (IS) ISCR1, ISEcp1 and IS26 in the immediate upstream region of the ESBL/AmpC genes was determined for all ESC-resistant isolates by PCR using combinations of primers for IS and ESBL/AmpC genes, as previously described 31.

High prevalence of intra-familial co-colonization by ESC-resistant Enterobacteriaceae in preschool children and parents | 77 Clonal analysis E. coli and K. pneumoniae isolates were characterized by multi-locus sequence typing (MLST), as previously described 40,41. Genetic relatedness of E. coli isolates belonging to the same sequence type (ST) and recovered from child and parent isolates from the same household, was assessed by PFGE of XbaI-digested genomic DNA using a CHEF DR-III apparatus (Bio-Rad Laboratories, Hercules, CA, USA) following the standardized protocol of PulseNet 42. XbaI-digested genomic DNA from Salmonella enterica serotype Braenderup strain H9812 was used as a molecular reference marker 43. Cluster analysis was performed using BioNumerics, version 6.6 (Applied Maths, Sint-Martens- Latem, Belgium) as previously described 31.

Intra-familial co-colonization Based on the observed prevalence of ESC-resistant Enterobacteriaceae in children and parents, we determined the probability that both the parent and the child in a household are colonized if we assume colonization in children and their parents is uncorrelated. We compared this to the observed frequency of co-colonization by binomial probability testing.

The plasmid and strain typing was performed to shed more insight into intra-familial transmission. Family members exposed to the same source are expected to be colonized by identical ESC- resistant Enterobacteriaceae, defined as isolated belonging to the same ST/PFGE-pattern within the bacterial species carrying an identical ESBL/AmpC-gene on the same genetic location [plasmid type (and subtype) or the chromosome]. Family members sharing risk factors are less likely to be colonized by an identical ESC-resistant Enterobacteriaceae. We therefore determined the probability that both parent and child carry an identical ESBL/AmpC-gene type on the same genetic location of their ESBL/AmpC genes [plasmid types (and subtypes) or chromosome]. The expected number of co-colonized households was compared to the observed number of co-colonization by binomial probability testing as well. See Appendix in supplementary material for more information on the calculations.

In addition, characteristics of the households with identical ESBL/AmpC-gene type were compared to the non-identical households by Fisher’s exact test. Binomial probability testing and Fisher’s exact tests were performed in STATA 13 (StataCorp LP, College Station, TX, USA) and P-values <0.05 were considered as statistically significant.

78 | Chapter 3A Acknowledgments

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This work was partially presented at the 25th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID), 25–28 April 2015, Copenhagen, Denmark (Poster number P0958).

Funding

This work was supported by the Dutch Ministry of Economic Affairs through the 1Health4Food 3A (1H4F) project under the ESBL Attribution (ESBLAT) consortium (project number: TKI-AF-12067).

High prevalence of intra-familial co-colonization by ESC-resistant Enterobacteriaceae in preschool children and parents | 79

1 3 2 1 2 2 1 1 1 4 5 Grand Total 1 37 34 2 2 2 6 6 1 1 1 1 1 1 10

TEM-52c bla

1 1 1 1

TEM-52var bla

1 1 1 1

SHV12 bla

5 5 5 5

DHA-1 bla

CTX-M3 bla

1 1 1 2 3

CTX-M27 bla

1 1

CTX-M24 bla

1 1 1 1

CTX-M2 bla

CTX-M15 bla

1 1 1 1 1 9 2 2 1 1 1 1 11

CTX-M14var bla

CTX-M14b bla

2 1 1 2

CTX-M14 bla

1 1 4 2 2 1 1 4

CTX-M1 bla

1 1 1 1 2 4 2 4

CMY-2 bla 1 1 2 1 1 4 2 4 Ecp1 Ecp1 Ecp1 26 Ecp1 26 Ecp1 Ecp1 Ecp1 Ecp1 Ecp1 γ α IS NP IS IS NP IS IS IS IS IS IS IS IS Plasmid, Insertion Sequence and ESBL/AmpC genes distribution per origin (child or parent), per bacterial species in entire dataset. per bacterial species in entire Plasmid, Insertion Sequence and ESBL/AmpC genes distribution per origin (child or parent), IncK IncN IncX1 IncI1 chromosome chromosome ColE IncB/O IncHI2 IncI1 E. cloacae E. coli Table S1. Table Count of ESBL/ pAmpC genes

80 | Chapter 3A

1 13 3 3 1 1 1 48 7 1 6 3 3 17 2 1 50 Grand Total 1 1 1 1 1 1 1 6 1 5

TEM-52c bla

1 1 1 1

TEM-52var bla

SHV12 bla

1 1 3 2 2 3

DHA-1 bla 1 1

CTX-M3 bla

1 1 3 3 1 1 1 1

CTX-M27 bla

4 4 1 1

CTX-M24 bla

CTX-M2 bla

1 1

CTX-M15 bla

3 1 1 4 1 3 4 1 15 17 1 1 1 1 1 3 3

CTX-M14var bla

1 1

CTX-M14b bla

1 1 1 1 1 1

CTX-M14 bla

1 1 2 2 3 3 7 7

CTX-M1 bla

8 1 8 9 9

CMY-2 bla 1 1 1 1 2 2 Ecp1 26 26 Ecp1 Ecp1 26 CR1 Ecp1 Ecp1 Ecp1 26 Ecp1 α NP IS IS IS IS IS IS IS IS IS IS NP IS IS Continued. IncK IncN IncX1 chromosome IncB/O IncI1 chromosome IncY NT IncF E. coli K. pneumoniae Table S1. Table Count of ESBL/ pAmpC genes Parent

High prevalence of intra-familial co-colonization by ESC-resistant Enterobacteriaceae in preschool children and parents | 81

87 1 1 2 1 1 1 1 7 1 1 1 1 12 4 Grand Total 1 1 1 1

TEM-52c bla

TEM-52var bla

SHV12 bla

DHA-1 bla

1 1 1

CTX-M3 bla

6 1 1 1 1

CTX-M27 bla

5 1 1 3 3

CTX-M24 bla

CTX-M2 bla

1 1 1

CTX-M15 bla

28 2 1 1 1 1 5 6 1

CTX-M14var bla

1 1 1

CTX-M14b bla

CTX-M14 bla

11 1 1

CTX-M1 bla

13 1

CMY-2 bla 6 CR1 Ecp1 Ecp1 26 26 Ecp1 26 Ecp1 IS IS IS NP IS IS IS IS IS NP Continued. IncF HIB-M IncF-R IncHI1 IncF IncY NT K. pneumoniae NT: not typable plasmid NT: NP: IS not present Grand Total Table S1. Table Count of ESBL/ pAmpC genes

82 | Chapter 3A References

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86 | Chapter 3A

CHAPTER 3 B

Molecular characterization of ESBL- producing Escherichia coli in pigs and pig farmers – a longitudinal study

Wietske Dohmen1*, Apostolos Liakopoulos2*, Marc Bonten3, Dik Mevius2,4, Dick Heederik1 * These authors contributed equally

1Division of Environmental Epidemiology, Institute for Risk Assessment Sciences, Utrecht University, Utrecht, the Netherlands; 2Department of Bacteriology and Epidemiology, Wageningen Bioveterinary Research, Lelystad, the Netherlands; 3Department of Medical Microbiology, University Medical Center Utrecht, Utrecht, the Netherlands; 4Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands.

In preparation Abstract

ESBL-producing E. coli represent a public health issue due to limited therapeutic options for treatment of infections. Although, earlier published cross-sectional data had revealed that direct contact with pigs is an important risk factor for human ESBL-producing E. coli carriage, longitudinal data on within-pig farm epidemiology are limited. Here, we report on the presence and molecular diversity of ESBL-producing E. coli from humans and pigs longitudinally within the same pig farms, confirming transmission events between pig farmers and their pigs.

90 | Chapter 3B Livestock, including pigs, can carry extended-spectrum beta-lactamase (ESBL)-producing Entero- bacteriaceae1. ESBL-producing Enterobacteriaceae can be transmitted from pigs to humans living and/or working on farms through direct contact with pigs2-5. We determined the presence and molecular aspects of ESBL-producing E. coli on pig farms longitudinally, including the similarity and persistence of ESBL-producing E. coli in pig farmers that were repeated carriers.

In the Netherlands, 40 conventional pig farms were enrolled between 2011 and 2013 in a longitudinal study regarding the presence of ESBL-producing E. coli in pigs and humans. However, one farm (Farm 18) dropped out from the study after the first sampling round, while only 34 of these farms provided with human fecal samples. During four repeated sampling moments with a six-month interval, rectal swabs from 60 pigs on all farms were collected by veterinarians. Pig rectal swabs were pooled into 10 pools of 6 pigs each per farm2. Fecal samples were possibly to be obtained from 147 farmers, their family members and employees only from 34 of these farms. Both human and pooled pig samples were analysed for the presence of ESBL-producing E. coli by 3B selective plating2,6. Presumptive ESBL-producing isolates were identified using MALDI-TOF Mass spectrometry (Brucker, Coventry, UK). ESBL genes were identified and characterized by means of PCR, microarray and gene sequencing for each distinctive colonial morphotype (1 to 4 per sample). Isolates from pig farmers who carried the same ESBL gene at more than one sampling moment and 3 to 5 (similar ESBL gene harbouring) isolates of their pigs were selected for further molecular analysis. Plasmids carrying the ESBL genes were determined using a transformation-based approach. PCR-based replicon typing (PBRT), plasmid multilocus sequence typing (pMLST), and multilocus sequence typing (MLST) were performed to check for similarities on the ESBL encoding plasmid and harbouring strain level respectively2,6.

ESBL genes were detected in E. coli isolates from 17 participants living or working in 14 pig farms (mostly farmers) in one or more sampling moment(s) (Table 1). Overall human prevalence was 4% and ranged from 6% in the first sampling moment to 2% in the last sampling moment, whereas overall farm prevalence was 34% and ranged from 45% to 28% between the first and last sampling moments. Out of 39 farms with available longitudinal data, in 18 (46%) farms positive for ESBL-producing E. coli pigs were found at one or more sampling moment(s), of which 9 (23%) farms at all four sampling moments. In the remaining 21 (54%) farms pigs were negative for ESBL-producing isolates at all sampling moments. Overall, ESBL-producing E. coli was more frequently found in pigs than in humans living and/or working on pig farms (Table 1).

Molecular characterization of ESBL-producing Escherichia coli in pigs and pig farmers – a longitudinal study | 91 Table 1. ESBL genes in human and pig isolates on pig farms.

ESBL genes

Farm Origin 0 months1 6 months 12 months 18 months

1* Human NP blaCTX-M-1 (2) blaCTX-M-1 (1) blaCTX-M-1 (3)

2 Pigs blaCTX-M-1 (16) blaCTX-M-1 (12) blaCTX-M-1 (4) blaCTX-M-1 (10)

2 Pigs blaCTX-M-1 (1) NP NP NP

3 Pigs blaCTX-M-1 (1) blaCTX-M-2 (2) blaCTX-M-1 (1) blaCTX-M-2 (1)

blaCTX-M-2 (4) blaTEM-52 (3) blaTEM-52 (4) blaTEM-52 (8)

blaTEM-52 (6)

4* Human A blaCTX-M-1 (1) NP NP NP

Human B blaCTX-M-1 (1) blaCTX-M-1 (1) NP NP

Pigs blaCTX-M-1 (7) blaCTX-M-1 (6) blaCTX-M-1 (3) NP

5* Human NP blaCTX-M-1 (2) NP blaCTX-M-14 (1)

Pigs blaCTX-M-1 (1) blaCTX-M-1 (3) blaCTX-M-1 (1) blaCTX-M-1 (2)

blaCTX-M-14 (10) blaCTX-M-14 (2) blaCTX-M-14 (2) blaCTX-M-14 (3)

blaTEM-52 (1)

6 Pigs blaCTX-M-1 (4) NP NP NP

7 Pigs blaCTX-M-14 (1) NP NP NP

8 Pigs blaCTX-M-1 (16) blaCTX-M-1 (13) blaCTX-M-1 (13) blaCTX-M-1 (8)

9* Human blaCTX-M-1 (1) blaCTX-M-15 (1) NP NS

Pigs blaCTX-M-1 (13) blaCTX-M-1 (7) blaCTX-M-1 (9) blaCTX-M-1 (3)

blaCTX-M-15 (1)

10* Human NP NP blaCTX-M-1 (2) NP

Pigs NP NP blaCTX-M-1 (9) blaCTX-M-1 (12)

11 Human NP blaCTX-M-15 (1) NP NP

12 Pigs blaTEM-52 (1) blaCTX-M-1 (1) NP blaTEM-52 (5)

blaTEM-52 (1)

13 Pigs blaCTX-M-15 (11) blaCTX-M-1 (1) NP NP

NP blaCTX-M-15 (2) NP NP

14 Human NP blaCTX-M-1 (2) NP NS

15 Human NP NP NP blaCTX-M-2 (1)

16* Human blaCTX-M-1 (1)

Pigs blaCTX-M-1 (6) blaCTX-M-1 (9) blaCTX-M-1 (8) blaCTX-M-1 (6)

17 Human NP NP blaCTX-M-1 (1) NP

92 | Chapter 3B Table 1. Continued.

ESBL genes

Farm Origin 0 months1 6 months 12 months 18 months

18** Pigs blaTEM-52 (9) NS NS NS

19* Human A NP NP blaTEM-52 (1) NP

Human B blaTEM-52 (2) NP NP NP

Pigs blaCTX-M-1 (1) blaCTX-M-1 (1) blaCTX-M-1 (1) blaTEM-52 (4)

blaTEM-52 (14) blaTEM-52 (11) blaTEM-52 (10)

20* Human A blaCTX-M-1 (1) NP blaCTX-M-1 (2) NP

Human B blaCTX-M-1 (1) NP NP NP

Pigs blaCTX-M-1 (10) blaCTX-M-1 (6) blaCTX-M-1 (5) blaCTX-M-1 (2)

blaCTX-M-32 (1) 21 Pigs bla (11) bla (6) bla (7) bla (5) CTX-M-1 CTX-M-1 CTX-M-1 CTX-M-1 3B

blaTEM-52 (2) blaTEM-52 (1) blaTEM-52 (2)

22 Pigs blaTEM-52 (1) NP NP NP

23* Human blaCTX-M-14 (2) NP NP NP

Pigs blaCTX-M-14 (7) blaCTX-M-1 (1) blaCTX-M-14 (1) NP bla (1) CTX-M-14 NP

24 Human blaCTX-M-1 (3) NP NP NP * Within these farms the presence of the same ESBL genes was established in both pig farmers and their pigs in one or more sampling moment(s). ** This farm dropped out from the study after the first sampling round. 1 All human and pig blaCTX-M-1 isolates collected in T0 were not tested for the presence of additional genes. 2 The number indicates the total number of distinctive colonial morphotypes tested that carried the ESBL gene. NP: not ESBL-producing E. coli present and NS: no sample(s) were obtained.

The blaCTX-M-1 gene was predominant among the recovered ESBL-producing isolates, detected in 25 out of 34 human (74%) and 262 out of 393 pig isolates (67%). Other frequently found ESBL genes were blaTEM-52 [n=83 (21%) and n=3 (9%) isolates in pigs and humans, respectively], blaCTX-M-14

[n=27 (7%) and n=3 (9%)] and blaCTX-M-15 [n=14 (4%) and n=2 (6%)]. On several farms different ESBL gene types were found within the same sampling moment and/or the present gene types could differ over time within the same farm, underscoring the complex and dynamic epidemiology of ESBL-producing E. coli within pig farms.

In contrast with the low carriage of blaCTX-M-1 gene among the general population in the Nether- lands, 12 out of the 17 (71%) participants positive for the presence of ESBL-producing E. coli

Molecular characterization of ESBL-producing Escherichia coli in pigs and pig farmers – a longitudinal study | 93 carried the blaCTX-M-1 gene at least once. In previous studies on 1695 residents of Amsterdam and 2432 residents living in the vicinity of livestock farms, 26 out of 145 (18%) and 13 out of 109

7,8 (12%) ESBL-producing isolate positive participants carried the blaCTX-M-1 gene respectively . In addition, these studies revealed that the human related blaCTX-M-15 gene was the most frequent gene among the Dutch general population, whereas in our study this gene was found in only 2 participants (Farms 9 and 11; Table 1), underscoring that pig farmers and the general population differ on their ESBL gene type carriage. In most cases, the ESBL gene type found in pig farmers was similar to the predominantly or exclusively detected ESBL gene type in pig isolates on the same farm (Table 1), suggesting transmission between farmers and their animals. An epidemiological association between the presence of ESBL-producing E. coli in humans and pigs was already

2 shown by previous work . Considering that blaCTX-M-1 is frequently found in livestock (mainly in pigs), transmission is likely to occur from animals to humans. However, given the co-existence of blaCTX-M-15 in both a human and pooled pig sample within one farm, it cannot be excluded that transmission is bi-directional. Detailed data regarding the gene types in pig and human isolates are listed in Table 1.

Out of 134 participants with at least 3 analysed samples, 3 pig farmers from 3 different farms carried the same ESBL gene (blaCTX-M-1) more than once. From these 3 farms with repeated human carriers, 11 E. coli isolates from pig farmers and 33 E. coli isolates from pigs were subjected to further analysis. All of them harboured the blaCTX-M-1 gene on plasmids of three IncI1α/γ subtypes, namely pST7/pCC7 [n=21 (48%)], pST3/pCC3 [n=17 (39%)] and pST38/pCC3 [n=6 (14%)]. E. coli isolates were distributed into 14 different sequence types (STs), each comprised of one to 11 isolates, with ST453/CC86 (n=11) and ST58/CC155 (n=7) being the predominant ones, while isolates belonging to ST10, ST48, ST88, ST218, ST227, ST398, ST410, ST711, ST1486, ST1670, ST1952 and ST3321 were also identified. Half of the STs from human origin (ST10, ST48, ST227, ST453, ST711, ST1486, ST1670) E. coli present has been previously associated with E. coli from porcine origin, whereas they have incidentally been described in E. coli from humans9-11.

In two pig farmers, isolates belonged to identical STs and encoded blaCTX-M-1 on identical plasmid subtypes in different sampling moments, possibly due to persistent ESBL carriage or to repeated- transmission from their pigs (Table 2). For the remaining pig farmer, only the blaCTX-M-1 carrying plasmid subtype was identical over time. On all three farms some of the human and pig isolates within the same farm were similar in both plasmid subtype and strain ST, suggesting clonal transmission which was confirmed on isolates from farm 4 in a previous study3. On farm 20, plasmid subtype pST7/pCC7 was exclusively identified, indicating the possibility of horizontal gene transfer as well (Table 2). Isolates obtained from pigs and the pig farmer on farm 1 showed the greatest

94 | Chapter 3B diversity in plasmid subtype and strain STs, illustrating that both clonal transmission and plasmid transfer might have occurred. Farm 1 was an open farm, i.e. imported new pigs from external farms, in contrast to farm 4 and 20. This could be of influence on the diversity seen on farm 1 compared to the other two farms, because of the constant influx of ESBL encoding plasmids and strains. The molecular characteristics of the isolates of repeated human carriers and their pigs are listed in Table 2.

Table 2. Plasmid IncI1α/γ subtypes and E. coli sequence types on 3 farms with repeated blaCTX-M-1 human carriers. Sampling moment 0 months* 6 months 12 months 18 months Origin Plasmid MLST Plasmid MLST Plasmid MLST Plasmid MLST type (ST) type (ST) type (ST) type (ST) (pST/pCC) (pST/pCC) (pST/pCC) (pST/pCC) Human NP NP 7/7 48 (1) 3/3 10 (1) 3/3 1486 (1) 7/7 1670 (1) 7/7 48 (1) 7/7 1670 (1) 3B

Pigs NP NP 3/3 58 (3) 3/3 58 (1) 3/3 1486 (1)

Farm 1 3/3 398 (1) 3/3 398 (1) 3/3 1952 (1) 3/3 410 (1) 38/3 58 (1) 7/7 58 (1) 7/7 398 (1) 38/3 1486 (1)

Human 3/3 453 (1) 3/3 453 (1) NP NP NP NP

Pigs 3/3 453 (3) 3/3 453 (1) NP NP NP NP 7/7 58 38/3 453 (4)

Farm 4 7/7 453

Human 7/7 711 (1) NP NP 7/7 227 (2) NP NP

Pigs 7/7 10 (1) NP NP 7/7 88 (1) NP NP 7/7 227 (2) 7/7 218 (1)

Farm 20 7/7 711 (1) 7/7 227 (1) 7/7 3321 7/7 711 (2) (1)

* Plasmid subtype and MLST was constructed from WGS in one human and two pig isolates from farm 4 and one human and one pig isolate from farm 20 during the first sampling moment (3). NP: not ESBL-producing E.coli present.

This is the first study that provides molecular characteristics of ESBL-producingE. coli in pigs and pig farmers repeatedly over time. Great diversity was seen at the level of strain, gene and plasmid, which complicates interpretation. We documented both clonal dissemination and horizontal gene

Molecular characterization of ESBL-producing Escherichia coli in pigs and pig farmers – a longitudinal study | 95 transfer of blaCTX-M-1 gene between pigs and pig farmers. Clonal transmission of ESBL is not unlikely to occur in an epidemiologically linked setting, such as farmers having frequent direct contact with animals2-5,12. Only 2 pig farmers of 134 people living and/or working on a pig farm carried identical isolates over time, persistent carriage or repeated transmission events due to ongoing exposure to ESBL in pigs can both be an explanation for these observations.

Within-pig farm epidemiology of ESBL-producing E. coli is mostly facilitated by animal related STs with potentially low capability of colonization and maintenance in human enteric cavity, leading to low prevalence and low percentage of persistence among pig farmers in spite of their close contact with their pigs.

Acknowledgments

Results from this study were partially presented in a poster presentation at the 13th International Society for Veterinary Epidemiology and Economics Conference in Maastricht, The Netherlands, 2012 (abstract 12787), in a poster presentation at the 23rd European Congress of Clinical Micro- biology and Infectious Diseases in Berlin, Germany, 2013 (P1471), in an oral presentation at the 23rd Conference on Epidemiology in Occupational Health in Utrecht, The Netherlands, 2013 (abstract 328), in a poster presentation at the 53th Interscience Conference on Antimicrobial Agents and Chemotherapy in Denver, USA, 2013 (C2-1610), in an oral presentation at the 3rd International One Health Congress in Amsterdam, The Netherlands, 2015 (abstract 148), and in an poster presentation at the 28th annual International Society for Environmental Epidemiology conference in Rome, Italy, 2016 (P3-247). The authors would like to thank all farmers, family members, and employees for participation and all field workers, laboratory workers, and veterinarians for assistance. The authors would further like to thank ZLTO, LTO-Noord, the veterinarians and PorQ for recruitment of the farms.

Funding

This work was supported by Senter Novem (Contract no. FND07003); Product Boards for Livestock and Meat (Contract no. 13.31.001); Netherlands Organization for Research and Development ZonMw (Contract no. 50-51700-98-053); and 1Health4Food (1H4F) project under the ESBLAT consortium (TKI-AF-12067). No funding sources had any involvement other than financial support of this research.

96 | Chapter 3B References

1 Ewers, C., Bethe, A., Semmler, T., Guenther, infectious diseases 22, 1257-1261, doi:10.3201/ S. & Wieler, L. H. Extended-spectrum beta-lac- eid2207.151377 (2016). tamase-producing and AmpC-producing 7 Wielders, C. C. et al. Extended-spectrum Escherichia coli from livestock and companion beta-lactamase- and pAmpC-producing Entero- animals, and their putative impact on public bacteriaceae among the general population in health: a global perspective. Clinical microbiol- a livestock-dense area. Clinical microbiology ogy and infection : the official publication of the and infection : the official publication of the European Society of Clinical Microbiology and European Society of Clinical Microbiology and Infectious Diseases 18, 646-655, doi:10.1111/ Infectious Diseases 23, 120 e121-120 e128, j.1469-0691.2012.03850.x (2012). doi:10.1016/j.cmi.2016.10.013 (2017). 2 Dohmen, W. et al. Carriage of extended-spec- 8 Reuland, E. A. et al. Prevalence and risk factors trum beta-lactamases in pig farmers is associated for carriage of ESBL-producing Enterobacteria- with occurrence in pigs. Clinical microbiology ceae in Amsterdam. The Journal of antimicrobial and infection : the official publication of the chemotherapy 71, 1076-1082, doi:10.1093/jac/ European Society of Clinical Microbiology and dkv441 (2016). Infectious Diseases 21, 917-923, doi:10.1016/j. 9 Ramos, S. et al. Clonal Diversity of ESBL-Produc- cmi.2015.05.032 (2015). ing Escherichia coli in Pigs at Slaughter Level in 3 de Been, M. et al. Dissemination of cephalo- Portugal. Foodborne pathogens and disease 10, 3B sporin resistance genes between Escherichia 74-79, doi:10.1089/fpd.2012.1173 (2013). coli strains from farm animals and humans by 10 Herrero-Fresno, A., Larsen, I. & Olsen, J. E. specific plasmid lineages.PLoS genetics 10, Genetic relatedness of commensal Escherichia e1004776, doi:10.1371/journal.pgen.1004776 coli from nursery pigs in intensive pig production (2014). in Denmark and molecular characterization of 4 Hammerum, A. M. et al. Characterization of genetically different strains. Journal of applied extended-spectrum beta-lactamase (ESBL)-pro- microbiology 119, 342-353, doi:10.1111/ ducing Escherichia coli obtained from Danish jam.12840 (2015). pigs, pig farmers and their families from farms 11 Garcia-Cobos, S. et al. Molecular Typing of with high or no consumption of third-or Enterobacteriaceae from Pig Holdings in fourth-generation cephalosporins. J Antimicrob North-Western Germany Reveals Extend- Chemoth 69, 2650-2657, doi:10.1093/jac/ ed-Spectrum and AmpC beta-Lactamases dku180 (2014). Producing but no Carbapenem Resistant Ones. 5 Moodley, A. & Guardabassi, L. Transmission of PloS one 10, doi:ARTN e013453310.1371/jour- IncN Plasmids Carrying bla(CTX-M-1) between nal.pone.0134533 (2015). Commensal Escherichia coli in Pigs and Farm 12 Dierikx, C. et al. Extended-spectrum-beta-lac- Workers. Antimicrobial agents and chemother- tamase- and AmpC-beta-lactamase-producing apy 53, 1709-1711, doi:10.1128/Aac.01014-08 Escherichia coli in Dutch broilers and broiler (2009). farmers. The Journal of antimicrobial chemo- 6 Liakopoulos, A. et al. Extended-Spectrum Ceph- therapy 68, 60-67, doi:10.1093/jac/dks349 alosporin-Resistant Salmonella enterica serovar (2013). Heidelberg Strains, the Netherlands. Emerging

Molecular characterization of ESBL-producing Escherichia coli in pigs and pig farmers – a longitudinal study | 97

CHAPTER 4 A

A review of SHV extended-spectrum β-lactamases: neglected yet ubiquitous

Apostolos Liakopoulos1, Dik Mevius1,2, Daniela Ceccarelli1

1Department of Bacteriology and Epidemiology, CVI of Wageningen University, Lelystad, Netherlands; 2Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands.

Published in Frontiers in Microbiology 2016 Sep; 5(7):1374 Abstract

β-lactamases are the primary cause of resistance to β-lactams among members of the family Enterobacteriaceae. SHV enzymes have emerged in Enterobacteriaceae causing infections in health care in the last decades of the 20st century, and they are now observed in isolates in different epidemiological settings both in human, animal and the environment. Likely originated from a chromosomal penicillinase of Klebsiella pneumoniae, SHV β-lactamases currently encompass a large number of allelic variants including extended-spectrum β-lactamases (ESBL), non-ESBL and several not classified variants. SHV enzymes have evolved from a narrow- to an extended-spectrum of hydrolyzing activity, including monobactams and carbapenems, as a result of amino acid changes that altered the configuration around the active site of theβ -lactamases. SHV-ESBLs are usually encoded by self-transmissible plasmids that frequently carry resistance genes to other drug classes and have become widespread throughout the world in several Enterobacteriaceae, emphasizing their clinical significance.

100 | Chapter 4A Introduction

Thanks to their ability to inhibit cell wall biosynthesis, β-lactams remained the first-line defense against bacterial infections for over 20 years, before resistant bacteria appeared in clinical practice. Resistance to this class of drugs can be the result of antibiotic target site alteration, prevention of antibiotic access by altered permeability or forced efflux, or antibiotic degradation1 . The latter, represents the primary resistance mechanism in Gram-negative bacteria producing β-lactamase enzymes able to covalently bind the carbonyl moiety of the β-lactam ring and hydrolyze its amide bond 2. Naturally occurring chromosomally located β-lactamases are quite common in Gram-negative bacteria; likely evolved from penicillin-binding proteins, when produced in small quantity they do not significantly contribute to antibiotic resistance. It was the appearance of the first plasmid-mediated β-lactamase TEM-1 3 to designate the beginning of an unstoppable phenomenon in the 1960s. Ever since, the introduction of new natural or synthetic drugs to replace old ones in an attempt to limit the insurgence of antibiotic resistant bacteria triggered a chain reaction providing bacteria with a constant selective pressure driving the expansion of different resistance mechanisms 4. 4A In recent years β-lactamases have extensively diversified in response to the clinical use of new generations of β-lactams (penicillin, cephalosporins, carbapenems, and monobactams) leading to the need of classification schemes. Based on primary structure 5, enzymatic properties and biochemical attributes 6, and the increasingly available amino acid sequences 7 four major classes (A, B, C, D) can be acknowledged. Serine β-lactamases belonging to class A are the most abundant 8, with more than 500 enzymes, including the most clinically significant extended spectrumβ -lactamases (ESBL) variants, i.e. CTX-M-, TEM- and SHV-type enzymes 9.

Although SHV enzymes did not undergo the explosive dissemination observed for CTX-M-type variants 10, in recent years they have been found in several Enterobacteriaceae outside of the typical clinical hosts Klebsiella pneumoniae and Escherichia coli, with a rising allele variability (http:// www.lahey.org/studies), and in different environmental niches. Many admirable works describing the biochemistry, the genetics and the evolution of SHV β-lactamases have appeared over the last years. The aim of this review is to provide the readers with an updated overview on SHV β-lactamases, their amino acid variants and spectrum of activity, and to describe the occurrence of plasmid-associated SHV enzymes in Enterobacteriaceae and their epidemiological significance.

A review of SHV extended-spectrum β- lactamases: neglected yet ubiquitous | 101 Origin and diversity of the SHV family

11 The firstbla SHV-1 gene was identified in the 1970s inE. coli . The encoded enzyme SHV-1 (sulfhy- dryl reagent variable) proved its activity against penicillins and first generation cephalosporins12 and was confirmed part of the conjugative plasmid p45313 (Table 1). The most likely ancestor of the plasmid-mediated SHV-1 is a chromosomal species-specific penicillinase detected in faecalK. pneumoniae isolates from neonates 14. The enzyme showed a typical antibiogram with penicillin rather than cephalosporin resistance and a marked inhibition by clavulanic acid. How blaSHV-1 moved from the chromosome to the plasmid does not have a conclusive explanation since the proposed association with a transposable element 15 has not been confirmed.

As of today, 1891 SHV allelic variants have been described, having developed resistance to 3rd generation cephalosporin 16, monobactam and carbapenems 17. Only a small proportion is bio- chemically and/or genetically characterized (http://www.lahey.org/studies). SHV β-lactamases can be divided into three subgroups on the basis of molecular characteristics or functional properties: i) subgroup 2b (n=37), able to hydrolyze penicillins and early cephalosporins (cephaloridine and cephalothin) and strongly inhibited by clavulanic acid and tazobactam; ii) subgroup 2br (n=7), broad-spectrum β-lactamases that acquired resistance to clavulanic acid; and iii) subgroup 2be (n=46), comprises ESBLs that can also hydrolyze one or more oxyimino β-lactams (cefotaxime, ceftazidime, and aztreonam). More than half of these variants (n=99) has not been classified yet due to absence of biochemical characterization.

Figure 1 illustrates a phylogenetic analysis of 149 out of the 189 SHV β-lactamase variants whose amino acid sequences were available online (http://www.lahey.org/studies), as of July 2016. Unlike other β-lactamase families 18,19, there is no clear clustering of the different subgroups, as also mirrored by gene-based analysis (Supplementary Figure S1). Among the majority of unclassified variants, subgroup 2b and the few 2br variants are scattered all over the tree. Subgroup 2be showed clustering of most of the ESBL variants (including SHV-2a, SHV-5 and SHV-12), together with few non-classified enzymes (SHV-29, SHV-152, SHV-153, SHV-160 and SHV-165). It has been proposed that SHV β-lactamases descended from an unidentified ancestor holding an extended spectrum phenotype (2be) and that subgroup 2b derived from it 20. Our analysis showed that several of SHV ESBL variants were scattered along the tree with short branch lengths with neighboring 2b or unknown variants within the SHV phylogeny (i.e. SHV-40, SHV-11 and SHV-35; Figure 1),

1. Of the 194 variants available on line (http://www.lahey.org/studies), 5 were withdrawn or invalidated as only partial sequence.

102 | Chapter 4A supporting the hypothesis that they evolved from multiple variants, probably within the antibiotic era. Among the non-ESBL variants, blaSHV-11 represents one of the most successful and, together with blaSHV-1, the likely source of evolution for the existing SHV ESBL variants. blaSHV-11 was first identified as plasmid-encoded in clinical K. pneumoniae from Switzerland 21 and ever since has been isolated worldwide.

Figure 1. Maximum likelihood amino acid tree of 149 SHV-type β-lactamases. Variants whose sequence has not been released in GenBank as of July 2016, that show partial sequence or are identical to others (http://www.lahey.org/studies/) were not included in the analysis. SHV-180 and SHV-181 share the same sequence as well as SHV-121 and SHV-136. The tree was implemented in Mega version 6.06 22. Solid circles represent: red, extended-spectrum β-lactamases (2be; n=46); green, broad-spectrum β-lactamases (2br, n=5); and blue, penicillinases (2b, n=30). Unclassified alleles are reported in black (n=68).

A review of SHV extended-spectrum β- lactamases: neglected yet ubiquitous | 103 Although nearly displaced, together with TEM, by CTX-M enzymes over the years 10, 46 ESBL blaSHV genes have been described so far (Table 1). The first report of SHV-mediated resistance to third-generation cephalosporins was in 1983 with the isolation and characterization of blaSHV-2, encoded by plasmid pBP60 in a German clinical isolate of Klebsiella ozaenae and showing only a

23 few nucleotide mismatches with blaSHV-1 . In a few years four other ESBL variants were identified as plasmid-encoded in clinical K. pneumoniae, showing variable gene homologies with the blaSHV-1 24 and blaSHV-2 sequences (50-90%): blaSHV-2a encoded by conjugative plasmid pZMP1 ; blaSHV-3 on 25 pUD18 ; blaSHV-4, widely disseminated from France as a result of a single K. pneumoniae clone 26,27 diffusion ; and blaSHV-5 able to hydrolyze broad-spectrum cephalosporins and monobactams 28 . Of these first variants, the most epidemiologically successful werebla SHV-2a and blaSHV-5, which will be further discussed, together with blaSHV-2 and blaSHV-12, in a dedicated paragraph (section 5).

Interestingly, blaSHV-3 and blaSHV-4 have been only sporadically detected since their first description. blaSHV-3 seems to be geographically restricted to the USA where it was detected in E. coli of animal origin, associated with other antibiotic resistance genes such as blaCTX-M-15, blaCTX-M-24, blaCMY-2, 29 and/or blaTEM-1 . blaSHV-4 was identified also inEnterobacter aerogenes and Citrobacter diversus in different countries 30-32.

The last two decades witnessed the appearance of several new variants (blaSHV-7, blaSHV-8, blaSHV-9, blaSHV-31, blaSHV-38, blaSHV-40, blaSHV-41, and blaSHV-42) whose dissemination was restricted to limited cases

(Supplementary Table S1). A few variants seem to be geographically constrained: i) blaSHV-106, only 33 described in Portuguese isolates of K. pneumoniae together with blaTEM-1, and/or blaCTX-M-32 ; ii) 34,35 36 blaSHV-55, in Portugal and recently in Brazil ; and iii) blaSHV-57, in E. coli isolates from Taiwan and 37,38 39 China . A variant worth to mention is blaSHV-27 , that has been detected on different plasmids in E. coli, K. pneumoniae and Enterobacter cloacae, associated with a vast array of antibiotic resistance genes (blaDHA-1, blaTEM-1, blaTEM-1b, blaCMY-2, blaIMP, blaCTX-M-14, blaCTX-M-15, blaSHV-12, blaSHV-27, 40-44 blaOXA-1, dfrA5, ereA2) .

Most of SHV ESBLs (25 out of 46) are unique cases, with only one report so far. Seventeen variants are exclusively found in clinical K. pneumoniae: blaSHV-6, blaSHV-13, blaSHV-16, blaSHV-18, blaSHV-23, blaSHV-45, blaSHV-64, blaSHV-66, blaSHV-86, blaSHV-90, blaSHV-91, blaSHV-98, blaSHV-99, blaSHV-100, blaSHV-104, blaSHV-105, and blaSHV-134. These variants have been described worldwide (Brazil, Portugal, Algeria, USA, Tunisia, Netherlands, France, South Africa, Colombia, and China) and are mostly associated to plasmids (Table 1). Some of these variants are sporadically accompanied by other antibiotic resistance genes like in the case

36 of: i) blaSHV-45 encoded by an IncA/C plasmid together with blaCTX-M-2 and blaSHV-27 ; ii) blaSHV-134 45 encoded by an IncFIIA plasmid accompanied by a second plasmid carrying blaVIM-1 ; iii) and blaSHV-

105, conferring reduced susceptibility to ceftazidime, ceftriaxone, and aztreonam together with

104 | Chapter 4A 46 blaSHV-1 and blaSHV-5 . One of the oldest variants, blaSHV-6, was only described in France in 1991 in 47 a K. pneumoniae clinical case . It might be speculated that the 180 kb plasmid encoding blaSHV-6 and conferring decreased susceptibility to ceftazidime and aztreonam was not stable or it reduced bacterial strain fitness preventing a successful dissemination.

Four variants have been described only in clinical E. coli: i) blaSHV-15, described together with blaCMY-2 in a strain imported from India into the United Kingdom (http://www.lahey.org/studies/); ii) blaSHV-24, identified in Japan on a transferable 150-kb plasmid conferring high-level resistance to ceftazidime but not cefotaxime and cefazolin 56; emergence of SHV-24 might have been driven by the extensive use of ceftazidime in Japan, enabling bacterial survival in high concentrations of this

66 drug; iii) blaSHV-102, recovered in a Spanish hospital and hydrolyzing cefotaxime and ceftazidime ; 69 iv) and blaSHV-129, detected in an abscess specimen from a patient hospitalized in Italy in 2008 . blaSHV-46 was only described on a 70-kb conjugative plasmid also carrying blaTEM-1 and blaKPC-2 in a carbapenem-resistant strain of Klebsiella oxytoca from the urine of a hospitalized patient in New

61 York (USA) in 1998 . Finally, blaSHV-34 is an interesting example of extended-spectrum β-lactamase encoded by an epidemic plasmid circulating among Citrobacter koseri, E. coli, and K. pneumoniae in the same US hospital between 1998-2000 59. 4A

Majority of SHV ESBLs have been detected in K. pneumoniae or E. coli (Table 1). blaSHV-30 was the first variant to be detected in an E. cloacae isolate from a blood culture from a solid-organ transplant recipient in the USA in 2003 57. The gene, previously described in K. pneumoniae and Salmonella 60,70, was located on a 9.4 kb plasmid and contributed together with chromosomal ampC, blaSHV-7, and blaTEM-1 to the antibiotic resistance profile of theE. cloacae isolate, the first of its kind producing two different SHV enzymes. Three other novel ESBL variants have been solely identified as plasmid-encoded in clinicalE. cloacae: i) blaSHV-70, from a Chinese patient with history 63 71 of ceftazidime treatment and observed in other clinical Chinese settings ; ii) blaSHV-128, isolated in Tunisia in 2009, located on an IncFII conjugative plasmid, and conferring resistance to all β-lac-

68 tams except imipenem ; iii) and blaSHV-183, for which additional description is not available (http:// www.lahey.org/studies/).

SHV extended-spectrum β-lactamases: catalytic properties and resistance phenotype

Extended-spectrum SHV β-lactamases belong to functional group 2be, while very recently they were assigned to subclass A1 of serine β-lactamases, clustering with TEM and CTX-M enzymes among other clinically relevant β-lactamases 8,72. SHV ESBLs consist of two subdomains: an α/β that includes an antiparallel five-stranded β-sheet flanked by α-helices, and an all-α-helical sub-

A review of SHV extended-spectrum β- lactamases: neglected yet ubiquitous | 105 61 35 57 36 Reference# Reference# 37 62 62 63 64 34 34 65 65 65 66 67 11,12 23 24 25 26,48 28 47 49 50 51 21 21 52 http://www.lahey.org/studies/ 53 54 55 56 39 58 59 17 60 60 60 , ; OXY-2 TEM-1 OXA ; bla bla bla ; , ; SHV-7 KPC-2 TEM-1 AmpC CTX-M-2 SHV-27 TEM-1 bla bla bla Other Ab genes Other Ab genes bla ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND - ND ND ND ND ND - ND ND ND ND bla - ND - ND ND ND bla bla Plasmid size (Kb) Plasmid size (Kb) - 40-60 ND ND ND ND ND ND ND ND ND ND 50 ND 45 66 180 180 150 180 10 - ND 80 80 170 ND >100 80 ND 150 - 9.4 - >100 - ND ND ND 97-145 70 Conjugative plasmid Conjugative plasmid No Yes ND ND Yes Yes ND ND ND ND ND ND Yes Yes Yes Yes Yes Yes No Yes Yes - Yes Yes Yes Yes ND Yes Yes ND Yes - ND - Yes - ND ND ND ND Yes ¥ ¥ Genetic background Genetic background ND pMTY512 ND ND pEC04 P ND ND ND ND ND ND pML2011 Genetic Location p453 pBP60 pZMP1 pUD18 P pAFF1 pSLH06 P C pK318-1; pE77-1; pS24-1 P P P ND P P ND pCA- ZR001 C P Genetic Location C pOZ185 C ND ND ND IncA/C P

K. pneumoniae E. coli K. pneumoniae K. pneumoniae E. cloacae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae E. coli K. pneumoniae Bacterial Species E. coli K. ozaenae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae E. coli E. coli E. coli; K.pneumoniae; S. marcescens K. pneumoniae E. coli; K. pneumoniae K. pneumoniae E. coli K. pneumoniae K. pneumoniae K. pneumoniae E. coli K. pneumoniae E. cloacae Bacterial Species K. pneumoniae C. koseri; E. coli; K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. oxytoca NA 1998 2000-2002 2000-2002 2003-2004 2003 2003 2003 2005 2005 2005 2003-2004 2004 Year* 1972 1983 1987-1988 1986 1987 1987 1991 1993 1990 1995 1993-1995 1993-1995 1994 1998 1996 1994 1990 1996 1999 2003 Year* 2001 1998-2000 2001 1999-2000 1999-2000 1999-2000 NA 1998 -lactamases. β Isolation Isolation Portugal Taiwan China China China Colombia Portugal Portugal Algeria Algeria Algeria Spain Tunisia Location NA Germany Germany France France Chile France USA USA Greece Switzerland Switzerland Netherlands India France USA South Africa Japan Brazil USA Location Netherlands USA France Canada Canada Canada Brazil New York ND 8.3 ND ND 7.6 8.2 8.2 7.6 7.6 7.8 7.2 ND 7,3/8,6 pI 7.6 7.6 7.6 7.0 7.8 8.2 7.6 7.6 7.6 8.2 8.2 8.2 7.6 ND 7.6 7.8 ND 7.5 8.2 6.7 pI 7.8 ND 7.6 7.6 7.6 7.6 8.2 8.2 DQ054528 AY223863 DQ174304 DQ174306 DQ013287 DQ328802 NA NA AM941844 AM941845 AM941846 EU024485 EU274581 Accession Number AF148850 AF148851 X98102 KX092356 NA X55640 Y11069.1 U20270 U92041 S82452.1 X98101 JX268741 AF164577 AJ011428.2 AF072684.2 AF132290 AF117747 AB023477 AF293345.1 AY661885 Accession Number AY277255 AY036620 AY079099 AF535128 AF535129 AF535130 AF547625 AY210887 SHV-type extended-spectrum SHV-type Continued ** ** § § SHV-55 SHV-57 SHV-64 SHV-66 SHV-70 SHV-86 SHV-90 SHV-91 SHV-98 SHV-99 SHV-100 SHV-102 SHV-104 SHV-1 SHV-2 SHV-2a SHV-3 SHV-4 SHV-5 SHV-6 SHV-7 SHV-8 SHV-9 SHV-11 SHV-12 SHV-13 SHV-15 SHV-16 SHV-18 SHV-23 SHV-24 SHV-27 SHV-30 SHV-31 SHV-34 SHV-38 SHV-40 SHV-41 SHV-42 SHV-45 SHV-46 bla bla bla Table 1. Table Gene 1. Table Gene bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla bla

106 | Chapter 4A 61 35 57 36 Reference# Reference# 37 62 62 63 64 34 34 65 65 65 66 67 11,12 23 24 25 26,48 28 47 49 50 51 21 21 52 http://www.lahey.org/studies/ 53 54 55 56 39 58 59 17 60 60 60 , ; OXY-2 TEM-1 OXA ; bla bla bla ; , ; SHV-7 KPC-2 TEM-1 AmpC CTX-M-2 SHV-27 TEM-1 bla bla bla Other Ab genes Other Ab genes bla ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND - ND ND ND ND ND - ND ND ND ND bla - ND - ND ND ND bla bla Plasmid size (Kb) Plasmid size (Kb) - 40-60 ND ND ND ND ND ND ND ND ND ND 50 ND 45 66 180 180 150 180 10 - ND 80 80 170 ND >100 80 ND 150 - 9.4 - >100 - ND ND ND 97-145 70 Conjugative plasmid Conjugative plasmid No Yes ND ND Yes Yes ND ND ND ND ND ND Yes Yes Yes Yes Yes Yes No Yes Yes - Yes Yes Yes Yes ND Yes Yes ND Yes - ND - Yes - ND ND ND ND Yes ¥ ¥ 4A Genetic background Genetic background ND pMTY512 ND ND pEC04 P ND ND ND ND ND ND pML2011 Genetic Location p453 pBP60 pZMP1 pUD18 P pAFF1 pSLH06 P C pK318-1; pE77-1; pS24-1 P P P ND P P ND pCA- ZR001 C P Genetic Location C pOZ185 C ND ND ND IncA/C P

A review of SHV extended-spectrum β- lactamases: neglected yet ubiquitous | 107 46 33 Reference# 45 68 69 http://www.lahey.org/studies/ ; SHV-5 TEM-1 bla Ib; ; ; ; (6’)- VIM-1 SHV-1 TEM-1 CTX-M-32 aac dhfrII; aadA1; catB2; bla Other Ab genes bla aac(3’)-Ia bla bla ND ND bla ND Plasmid size (Kb) ND ND 100 ND 75 ND Conjugative plasmid ND ND Yes ND Yes ND ¥ ) ) 26 26 Genetic background Genetic Location ND ND IncFII (IS pEc6-66 IncFIIA (IS ND Bacterial Species K. pneumoniae K. pneumoniae E. cloacae E. coli K. pneumoniae E. cloacae Year* NA 1999 2009 2008 2009 NA Isolation Location USA Portugal Tunisia Italy Spain NA are provided as reference; provided are SHV-11 bla pI ND 7.6 8.6 ND ND ND and SHV-1 bla was not included in the table because no information is available (http://www.lahey.org/studies); Accession Number FJ194944 AM941847 GU932590 GU827715 HM559945 HG934764 SHV-115 Continued bla § SHV-105 SHV-106 SHV-128 SHV-129 SHV-134 SHV-183 Non ESBL genes Gene Isolation or first description; indicated; and Insertion Sequences are when known plasmid name or Inc group, P: plasmid; C: Chromosome; bla bla NA: not available and ND: determined. Table 1. Table Gene § * ** ¥ bla bla bla bla

108 | Chapter 4A domain 73. Similar to TEM β-lactamases 74, the active site is located within the cleft created by the subdomains and it contains the Ser70 residue that mediates the nucleophilic attack on the carbonyl group of the β-lactam ring. In the vicinity of this serine residue, several conserved structural and functional amino acid motifs have been identified. These include the Ser70XXLys (“SXXK” motif, with X representing variable amino acids), the Ser130AspAsn (“SDN” motif), the Glu166XXLysAsn (“EXXLN” motif), and the Lys234Thr/SerGly (“KTG” motif)72.

Each SHV ESBL has one (SHV-2, SHV-6, SHV-8, SHV-24, SHV-27, SHV-38, SHV-41, SHV-57, SHV- 98, SHV-99, SHV-102 and SHV-104) to six (SHV-128) amino acid substitutions when compared to SHV-1 (Table 2), indicating that even a single amino acid substitution is enough to convey an extended-spectrum phenotype. Therefore, we can speculate that other SHV ESBLs may still evolve from a parental SHV β-lactamase due to single spectrum-extending substitutions, although the majority of them have possibly emerged through a stepwise acquisition of several mutations (substitutions, deletions and/ or insertions) from pre-existing extended-spectrum SHV variants.

Among SHV ESBLs, amino acid substitutions are predominantly located at positions Leu35, Gly238 and Glu240, while other less frequent but critical substitutions for the extended-spectrum pheno- 4A type occur on several amino acids including Ile8, Arg43, Glu64, Gly156, Asp179 and Arg205 (Table 2). Although most of these residues are not involved directly in β-lactams hydrolysis, they result in the enhancement or relaxation of the active site, enabling it to accommodate and to efficiently react with oxyimino-β-lactams 16. Amino acid substitutions on some of these positions (Arg43, Asp179, Arg205, Gly238 and Glu240) have been also associated with the expansion towards an ESBL phenotype among TEM enzymes 75.

Residue Leu35 is located further away from the active site of class A β-lactamases and its substitution to Gln (e.g. SHV-2a, SHV-12) has been suggested to have an indirect role in enhancing the extended-spectrum capability of SHV β-lactamases 21. In contrast, Gly238 and Glu240 amino acids are part of the active site lying near the R1 side chain of the β-lactam 76. Substitutions in Gly238 either to Ser (e.g. SHV-2, SHV-2a) or Ala (e.g. SHV-13, SHV-18) displace the β3-strand from the reactive Ser70, resulting in a slightly expanded active site. This conformational change improves the binding to and the accommodation of newer cephalosporins with large C7 substituents, thereby expanding the substrate spectrum of these SHV ESBLs to include cefotaxime and to a lesser extent to ceftazidime 73,76,77. It has been suggested that Glu240 substitutions to Arg (SHV-86) or Lys (e.g. SHV-4, SHV-5) cause the ammonium group of the long side-chains of these residues to form an electrostatic bond with the carboxylic acid group on the oxyimino-substituents of ceftazidime and aztreonam 75. This interaction has a dual effect on the hydrolysis of ceftazidime by improving initial

A review of SHV extended-spectrum β- lactamases: neglected yet ubiquitous | 109 binding and facilitating proper positioning within the SHV β-lactamase, whereas the hydrolysis of other β-lactams is less affected 76. Gly238Ser and Glu240Lys amino acid substitutions characterize the majority of SHV ESBLs (Table 2) and mirror those seen in extended-spectrum TEM β-lactamases. Interestingly, a plethora of extended-spectrum SHV and TEM β-lactamases exhibit higher levels of hydrolytic activity against ceftazidime than against cefotaxime (ceftazidimases) (Table 3). This phenotype was attributed to the Glu240Lys substitution, in contrast with most CTX-M β-lactamases lacking this critical substitution and only showing a cefotaximase activity78.

Among the less frequent but critical substitutions, Ile8Phe in the signal sequence of the precursor of SHV ESBLs (e.g. SHV-7, SHV-18) has been associated with a more efficient β-lactamase transfer into the periplasm 79, a proof that, beside enzymatic structure and gene expression, also the rate of transfer plays a role in resistance phenotype. On the contrary, Arg43Ser (e.g. SHV-7, SHV-18) and Gly156Asp (SHV-27, SHV-45, SHV-105) substitutions affect the structural arrangement of the conserved residues 64 to 69 and 166 to 170, respectively. These changes, opposite to the active site cavity (Ser70) for the hydrolysis of the β-lactam molecules, expand the active site to accommodate bulkier cephalosporins 39,75. Asp179 amino acid is highly conserved among subclass A1 of serine β-lactamases and together with Arg164 form a salt bridge that links the two ends of the Ω loop. Substitutions Asp179Ala (SHV-6), Asp179Asn (SHV-8) and Asp179Gly (SHV-24) result in the elimination of the salt bridge with subsequent increase in ceftazidime resistance 80. Several other amino acid substitutions (Table 2) have been described as either responsible for or possibly contributing to the ESBL phenotype, the detailed description of which exceeds the scope of this review.

Apart from point mutations leading to amino acid substitutions, frame shift mutations have been observed with very low occurrence among SHV ESBLs resulting in amino acid insertions 53,65 or deletions 51. However, their role in the rising of the extended-spectrum phenotype remains unclear. SHV ESBL variants falling in this category are: i) SHV-9, with the deletion of Gly54 51; ii) SHV-16, with a 5-amino acid sequence duplication (Asp163aArgGluTrpGluThr-Asp163bArgGluTrpGluThr) of the amino acids between 163 to 167, including Glu166 in the omega loop 53; iii) SHV-100, with a 13-amino acid insertion (SerGluSerGlnLeuSerGlyArgValGlyMetIleGlu) between amino acids 35 and 36 65; and iv) SHV-183, with an Ala insertion between amino acids 186 and 187 (http://www. lahey.org/studies). Of note, the duplication observed in SHV-16 was shown to increase the conformational flexibility of the catalytic region facilitating the access of bulkier cephalosporins, such as ceftazidime, but resulted in enzymatic instability 53. This finding could explain the low incidence of frame shift mutations among extended-spectrum SHV β-lactamases, due to a deleterious effect on the enzymes.

110 | Chapter 4A Overall, the available SHV ESBL kinetic parameters show that most of the substitutions lead to more efficient hydrolysis of oxyimino-β-lactams than penicillins, as depicted by the low Kcat values for penicillins (Table 3). While they retain their ability to hydrolyze penicillins, they are not catalytically so efficient compared to SHV-181 and this is due to the decreased strength of the crucial hydro- gen-bonding network needed for penicillin catalysis (turnover). As a consequence, since β-lactam inhibitors (clavulanic acid, sulbactam and tazobactam) are structurally very similar to penicillin substrates, SHV ESBLs also exhibit increased susceptibility to β-lactam inhibitors compared to SHV-1

16 (Table 3) leading to less inhibitor required for inactivation (lower Ki and IC50s) .

Detection

There are at least 46 known SHV-ESBL genes together with more than 150 non-ESBL or unclassified alleles to date (http://www.lahey.org/studies/). Accurate identification of these variants is essential for surveillance and for epidemiological studies of transmission mode, particularly in clinical setting, where appropriate antimicrobial therapy is critical. 4A A panel of different phenotypic confirmatory tests is available to determine the presence of extended-spectrum β-lactamases, including SHV-variants: minimum inhibitory concentration (MIC) determination of β-lactam with and without clavulanic acid, double disk synergy test (DDST), inhibitor potentiated disk diffusion test (IPDDT), three-dimensional test (TDT) and commercially available methods (Etest for ESBLs, Vitek ESBL cards, MicroScan panels and BD Phoenix Automated Microbiology System 83,84. Standard microbiological procedures can take up to several days for culture, isolation and characterization and many comparative studies have shown that PCR-based methods have higher sensitivity 85-87, mostly due to variable levels of gene expression. Therefore, PCR and nucleotide sequence analysis 88, together with various PCR-based methods, remain the gold standard for extended-spectrum β-lactamase SHV-variants identification.

Chanawong and colleagues developed a PCR-restriction fragment length polymorphism (PCR-RFLP) method to allow the identification of new SHV β-lactamases variants through detection of known mutations that alter recognition sites of restriction endonucleases 89. PCR-RFLP complements pre-existing PCR-single strand conformational polymorphism (PCR-SSCP) limited by partial gene amplification, thus missing potential mutation sites90 . PCR-RFLP can also be used in combination with restriction site insertion-PCR (RSI-PCR), a method based on primers mismatches, allowing the unambiguous identification of up to 27 SHV variants by point mutation91 . Fluorescently labelled hybridization probes followed by melting curve analysis can also be used to discriminate between

A review of SHV extended-spectrum β- lactamases: neglected yet ubiquitous | 111 Table 2. Amino acid polymorphisms in SHV-type extended-spectrum β-lactamases. Table 2. Continued. Amino acid position Amino acid position 7 8 10 14 18 20 25 35 43 54 61 64 75 80 89 96 97 104 122 123 129 140 142 146 148 154 156 163 169 179 186 187 188 192 193 195 202 205 238 240 243 271 274 275 276 282 286

SHV-1 Y I L S T P A L R G R E V V E H Y D L C M A V A L Q G D L D M A A K L T R R G E A S E R N I L

SHV-2 ...... S ......

SHV-2A ...... Q ...... S ......

SHV-3 ...... L S ......

SHV-4 ...... L S K ......

SHV-5 ...... S K ......

SHV-6 ...... A ......

SHV-7 . F ...... S ...... S K ......

SHV-8 ...... N ......

SHV-9 ...... Del ...... R ...... N V . . . S K ......

SHV-11** ...... Q ......

SHV-12 ...... Q ...... S K ......

SHV-13 ...... Q ...... A ......

SHV-15 ...... Q . . . . M K ...... S K ......

SHV-16 ...... T H ...... Ins ......

SHV-18 . F ...... S ...... A K ......

SHV-23* ...... F ...... G . . . . . S K ......

SHV-24 ...... G ......

SHV-27 ...... D ......

SHV-30 . F ...... S ...... S ......

SHV-31 ...... Q ...... K ......

SHV-34 . F ...... S . . G ...... S ......

SHV-38 ...... V ......

SHV-40 ...... Q ...... G ......

SHV-41 ...... F ......

SHV-42 ...... S ...... V ......

SHV-45 ...... D ...... S K ......

SHV-46 ...... N . . S K ......

SHV-55 F ...... S K ......

SHV-57 ...... R ......

SHV-64 ...... Q . . . . L ...... S K ......

SHV-66 ...... Q . . . Q ...... S K ......

SHV-70 ...... Q ...... V ......

SHV-86 F ...... Q ...... S R ......

SHV-90 ...... T ...... S K ......

SHV-91 . . . . . S ...... K ......

SHV-98 ...... I . . . . .

SHV-99 ...... G ......

SHV-100 F ...... Ins ......

SHV-102 ...... A ......

SHV-104 ...... S ......

SHV-105 . F ...... S ...... D ...... S K ......

SHV-106* F ...... S ......

SHV-115 ...... H ...... K . . K . . . .

SHV-128 ...... Q ...... R ...... S K . . . . . T P

SHV-129 ...... Q ...... S K . . . L D . .

SHV-134 ...... Q ...... E ...... S K ......

SHV-183 ...... Q ...... Ins ...... S K ......

112 | Chapter 4A Table 2. Amino acid polymorphisms in SHV-type extended-spectrum β-lactamases. Table 2. Continued. Amino acid position Amino acid position 7 8 10 14 18 20 25 35 43 54 61 64 75 80 89 96 97 104 122 123 129 140 142 146 148 154 156 163 169 179 186 187 188 192 193 195 202 205 238 240 243 271 274 275 276 282 286

SHV-1 Y I L S T P A L R G R E V V E H Y D L C M A V A L Q G D L D M A A K L T R R G E A S E R N I L

SHV-2 ...... S ......

SHV-2A ...... Q ...... S ......

SHV-3 ...... L S ......

SHV-4 ...... L S K ......

SHV-5 ...... S K ......

SHV-6 ...... A ...... ) according to GenBank sequences: ) according SHV-7 . F ...... S ...... S K ......

SHV-8 ...... N ......

SHV-9 ...... Del ...... R ...... N V . . . S K ......

SHV-11** ...... Q ......

SHV-12 ...... Q ...... S K ......

SHV-13 ...... Q ...... A ......

SHV-15 ...... Q . . . . M K ...... S K ......

SHV-16 ...... T H ...... Ins ...... http://www.lahey.org/studies/ SHV-18 . F ...... S ...... A K ......

SHV-23* ...... F ...... G . . . . . S K ......

SHV-24 ...... G ......

SHV-27 ...... D ......

SHV-30 . F ...... S ...... S ......

SHV-31 ...... Q ...... K ......

SHV-34 . F ...... S . . G ...... S ...... , HG934764. 4A SHV-38 ...... V ...... SHV-183 SHV-40 ...... Q ...... G ...... bla SHV-41 ...... F ......

SHV-42 ...... S ...... V ......

SHV-45 ...... D ...... S K ......

SHV-46 ...... N . . S K ...... , AM941847;

SHV-55 F ...... S K ...... SHV-106 SHV-57 ...... R ...... bla SHV-64 ...... Q . . . . L ...... S K ......

SHV-66 ...... Q . . . Q ...... S K ......

SHV-70 ...... Q ...... V ......

SHV-86 F ...... Q ...... S R ...... , AM941846;

SHV-90 ...... T ...... S K ...... as reference. 2b) is provided (Subgroup SHV-11 ** SHV-100 SHV-91 . . . . . S ...... K ...... bla SHV-98 ...... I . . . . .

SHV-99 ...... G ......

SHV-100 F ...... Ins ......

SHV-102 ...... A ...... , DQ328802;

SHV-104 ...... S ...... SHV-86 SHV-105 . F ...... S ...... D ...... S K ...... bla

SHV-106* F ...... S ......

SHV-115 ...... H ...... K . . K . . . .

SHV-128 ...... Q ...... R ...... S K . . . . . T P

SHV-129 ...... Q ...... S K . . . L D . . , AF072684.2; SHV-134 ...... Q ...... E ...... S K ...... SHV-16

SHV-183 ...... Q ...... Ins ...... S K ...... 2be; not confirmed as belonging to subgroup Amino acid numbering is according to SHV-1 (Ambler numbering system, upper row). Dots indicate identical amino acids. Amino acid positions for SHV-16 (96, 97, 163), SHV-86 (7), (96, 97, 163), SHV-86 Dots indicate identical amino acids. Amino acid positions for SHV-16 (Ambler numbering system, upper row). to SHV-1 Amino acid numbering is according ( in the Lahey Clinic Website what reported (186) have been updated from (7, 8) and SHV-183 (7, 35), SHV-106 SHV-100 bla *

A review of SHV extended-spectrum β- lactamases: neglected yet ubiquitous | 113 0.057 0.04 0.036 0.150 0.049 0.022 TAZ 0.027 0.038 1.70 0.36 0.18 7.50 0.57 0.40 0.43 SUL 0.47 0.68 0.19 0.16 0.10 0.057 0.020 0.005 0.14 CLA 0.08 0.018 NH ND ND 1 5 0.5 3.3 ATM 13 3 10 0.02 >100 >3000 >35 FEP NA 0.008 NA NH ND ND 70 115 25 30 FOT 11 11 0.6 4 10 18 7 7 9 24 NH ND ND 6.5 52 60 13 CAZ 24 72 1 24 23 4 18 10 10 26 400 35 CEF 2.7 ND ND 5 53 1 ND 3 51 5 58 170 110 1500 CER 4 570 60 10000 PIP ND ND ND -lactamases. β 60 22 2700 TIC 900 90 10000 AMOX 206 17 13 100 12 11 100 12 215 AMP 455 20 23000 100 100 3.5 100 3.5 15 100 18 100 PEN m m m m K K K K / /

/ / 50 50 50 50 50 cat m cat max i i max m cat cat i max m i m max m max max m K K V IC IC IC V K K K K K V Parameter V K K IC K K K V K IC K V Kinetic parameters of available SHV-type extended-spectrum Kinetic parameters of available SHV-type ** SHV-5 SHV-9 Table 3. Table Enzyme SHV-2a SHV-2 SHV-7 SHV-1 SHV-4

114 | Chapter 4A 3 1.16 x 10 TAZ SUL 3 0.02 27 x 10 CLA 3 5500 0.5 5± 0.62 <0.1 ND 196± 0.60 ATM 0.735 500 0.00147 ND 77 0.86 ND 0.66 <1 3 1600 2 149± 2.61 30± 3.10 0.2± 0.02 FEP 1 800 1 21± 0.13 24± 0.34 1.1± 0.01 183± 0.72 FOT 11 11 24 12 26.9 -4 -5 110 3800 30 58± 7.40 9± 0.21 0.2± 0.02 136± 4.09 30.9 8.6 x 10 2.78 x 10 CAZ 0.043 30 0.000143 37 91 0.42 1.5 0.38 13.5 5 100 50 9± 0.68 38± 3.94 4.4± 0.78 102± 11.38 CEF 4A 40 150 270 CER 2.37 210 0.0113 ND 53 200 100 80 1300 8 ± 0.37 27± 1.53 3.7± 0.03 13± 1.43 PIP 18 76 136 10 14 700 6 ± 0.02 8± 0.00 1.5± 0.00 5± 0.93 TIC 1800 35 51000 10 ± 0.14 23± 0.17 2.5± 0.002 11± 0.26 AMOX 2 32 0.0625 57 28 64 178 AMP -3 -5 10 100 100 100 13 7700 5 ± 0.51 23± 0.76 5.3± 0.42 67 3.8 x 10 5.67 x 10 12± 0.11 100 100 PEN m m m m m m K K K K K K / / /

/ / / 50 m max max max max max m max i cat m cat m cat cat m cat cat i m K K K K IC K K K K K K K Parameter K V K V V V V V K Continued. $ * * SHV-55 SHV-57 Table 3. Table Enzyme SHV-99 SHV-38 SHV-18 SHV-24 SHV-13

A review of SHV extended-spectrum β- lactamases: neglected yet ubiquitous | 115 ; 67 0.03 0.04 TAZ ; SHV-104 ; SHV-104 0.4 SUL 65 ). Only antibiotics max V , 0.02 0.4 CLA m : SHV-99 : SHV-99 K 37 / cat K (

0.5± 0.5± 0.001 0.003 ATM ; SHV-57 ; SHV-57 35 ), and µM/min cat ; SHV-55 ; SHV-55 FEP 4.5± 0.5 52± 3.5 0.09± 0.01 K 17 (

-1 ), s m K , ; SHV-38 ; SHV-38 50 56 <0.1 <0.001 FOT 4.8± 3.4 26.7± 5.5 0.2± 0.5 >1.8 >600 0.003 , IC i K (

; SHV-24 ; SHV-24 54 <0.1 <0.001 CAZ 3.1± 1.5 24± 3 0.13± 0.5 ; SHV-18 ; SHV-18 52 37± 2 0.37± 0.04 CEF 26± 1 12.1± 3.7 2.2± 0.3 30 68 0.44 ; SHV-13 ; SHV-13 51 CER ; SHV-9 ; SHV-9 49 563± 13 43.5± 6.5 PIP 1688± 4 25± 9 7± 0.4 ; SHV-7 ; SHV-7 28 58± 2 13± 2.4 80 10 8 TIC ; SHV-5 ; SHV-5 48 563± 8 49.6± 1.8 AMOX ; SHV-4 ; SHV-4 24 22.8± 22.8± 11 46.8± 24 0.5± 0.7 AMP ; SHV-2a ; SHV-2a 28,49,82 778± 778± 616 62.3± 4.4 55 94 0.6 PEN ; SHV-2 ; SHV-2 ) represent mean ± standard deviation mean ± standard ) represent 17,28 m m m 50 K K K / / / 50 cat cat cat m cat cat m cat i K IC K K Parameter K K K K K K . 82 Continued. values are expressed as mM/sec expressed values are as µM/sec expressed values are * m m K K / / #* cat cat Non ESBL SHV-1 is provided as reference is provided Non ESBL SHV-1 K K (Except IC Values Table 3. Table Enzyme ** $ # * Cefepime (FOT); Cefotaxime (CAZ); Ceftazidime (CEF); Cephalothin (CER); Cephaloridine (PIP); Piperacillin Ticarcillin (TIC); (AMOX); Amoxicillin (AMP); Ampicillin (PEN); Penicillin Antibiotic: as µM expressed Parameters are (TAZ). Clavulanic Acid (CLA); Sulbactam (SUL); Tazobactam (ATM); (FEP); Aztreonam and ND: not determined. NH: not hydrolyzed and affinity; NA: not able to determine the rate of hydrolysis reported. available are SHV enzyme values were for which 3 or more SHV-1 References: SHV-129 SHV-104 SHV- 129 SHV-99

116 | Chapter 4A 92 ESBL and non-ESBL blaSHV genes . This method, termed the SHV melting curve mutation detection method, is also able to categorize SHV ESBL producers into phenotypically relevant subgroups: (i) weak ceftazidime resistance (SHV-6 and SHV-8); (ii) significant resistance to cefotaxime and ceftriaxone and moderate resistance to ceftazidime (SHV-2, SHV-2a, and SHV-3); and (iii), most effective against all expanded-spectrum cephalosporins (SHV-4, SHV-5, SHV-9, and SHV-12). Com- bined systems can also be developed ad hoc to rapidly screen local epidemiological settings 93. A modified SHV melting-curve mutation detection method able to distinguish between prevalent

Taiwanese blaSHV genes (SHV-1, SHV-2, SHV-2a, SHV-5, SHV-11, and SHV-12) was combined with a multiplex PCR to identify different β-lactamases families (blaSHV, blaCTX-M-3-like, and blaCTX-M-14). The design of this method can be easily adapted to other geographic areas where different ESBLs are prevalent. Multiplex real-time PCR assays for the fast detection of extended-spectrum β-lactamase and carbapenemase genes were developed with differential melting curves able to recognize up to 120 different SHV allelic variants 94.

New techniques for ESBL detection are employed alongside PCR-based methods these days. Loop-mediated isothermal amplification (LAMP) was applied to detect SHV- and other ESBL-producing bacteria in meat and proved to be more specific and sensitive than MacConkey agar or 4A cefpodoxime disc methods 95. Commercial DNA microarrays are also proving themselves to be accurate, with sensitivity and specificity values for ESBL detection being high. Up to 53 SHV-variants can be covered on a same array 96, but on the other hand some alleles may fail to be detected (i.e. SHV-12), as previously reported 97. Because arrays have major limitations to detect novel genes or variants, PCR and sequencing remains essential. Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is routinely used for bacteria identification and has been recently applied to detect ESBL-producing Enterobacteriaceae from positive blood cultures in clinical practice 98,99. Although this methodology has yet to be fully validated, preliminary results show 99% sensitivity and 100% specificity, and denote a novel approach to categorize bacteria as ESBL producers.

Pyrosequencing combines standard PCR and sequencing by synthesis to rapidly determine the sequence of a target DNA region; it has been extensively used for the detection of bacterial resistance genes and bacterial community composition 100,101. This technique has been used to perform mutation analysis of blaSHV to resolve heterogeneous sequences in clinical isolates of K. pneumoniae containing more than one SHV variant 102. An alternative protocol for pyrosequencing is the single-nucleotide polymorphism (SNP), ideal for the sequencing of mixed templates and determination of SNPs at the position of interest. This protocol has been applied to discriminate between eight blaSHV variants from clinical isolates of E. coli and K. pneumoniae, reporting great

A review of SHV extended-spectrum β- lactamases: neglected yet ubiquitous | 117 reproducibility and ability to discriminate between sequences 46. A multiplex pyrosequencing assay coupled with qPCR amplification has also been recently developed to enable rapid and accurate

103 detection of blaSHV and blaTEM -producing Enterobacteriaceae . Overall, pyrosequencing can be a useful epidemiological tool for the exact identification ofbla SHV as a prerequisite for analyzing the spread of certain SHV variants.

Finally, the advent of whole genome sequencing (WGS) has taken differentiation of bacterial strains and identification of the associated antibiotic resistance gene cargo to another level. Aside from the phylogenetic analysis that WGS provide, the complete resistome of a strain can be unraveled as well as its mobilome, i.e. the mobile genetic elements that are associated with antibiotic resistance diffusion. Only this information can provide us with full understanding of complex genomic structures as observed, for example, in clinical K. pneumoniae genomes carrying i) nineteen antibiotic resistance genes including blaOXA-1 and blaSHV-28 in the chromosome, blaNDM-1 in a plasmid, 104 and blaOXA-232 in a second plasmid ; ii) β-lactamase genes blaKPC-2, blaSHV-11, blaTEM-169, and blaOXA-9, together with aac(6’)-Ib, aadA2, and aph(3’)-Ia as aminoglycoside resistance encoding genes, mph(A) for macrolides, oqxA and oqxB for quinolone, catA1 for phenicol, sul1 for sulfonamide, and dfrA12 for trimethoprim 105; or iii) six different plasmids, adding up to 0.43 Mbp, coding for six beta-lactamases (blaSHV-12, blaOXA-9, blaTEM-1, blaCTX-M-2, and blaKPC-2), together with blaSHV-110 and adhesin-related gene clusters on the chromosome 106.

Expansion towards new ecological niches

Over the last years the presence of antibiotics as well as antibiotic resistant bacteria has been shown outside the clinical environment, including water, soil and, most notably, food producing animals. When looking at SHV-variants distribution it is evident that in recent years, as for most extended-spectrum β-lactamases 10, their presence has been confirmed in virtually all ecological niches (Supplementary Table S1), making it more challenging to restrain antibiotic resistance diffusion. The most representative cases and variants will be discussed.

Aquatic environment In an effort to control the release of antibiotics and antibiotic resistant bacteria in the environment, aquatic environments are being investigated worldwide, whether they be natural, drinking or wastewaters. The latter are particularly worrisome given the high prevalence of blaSHV alleles, as observed in untreated hospital wastewater in Australia 107, their possible association with determinants of quinolone and other β-lactamase resistance 108,109, and their relatively easy transmission to surface

118 | Chapter 4A water through waste water treatment plant discharges 110. Studies showed that SHV types, together with CTX-M and OXA genes can be significantly decreased by biological treatments such as activated sludge processing and anaerobic digestion, although not all can be effectively eliminated 111.

Urban waters are also exposed to relatively high population densities and therefore are often unprotected from biological contaminants, with people playing a crucial role in antibiotic resistance dissemination in the environment. Unusual finding of SHV-producing Stenotrophomonas maltophilia in a swimming recreational Serbian lake and its transient presence during summer months can be considered as a proof of its anthropogenic origin, given its nature of emerging nosocomial pathogen 112. Similar conclusions can be drawn for SHV-producing K. pneumoniae and E. cloacae isolated from a Bangladeshi lake, which receives waste water from surrounding residents, commercial buildings and clinics in Dhaka city 113, as well as for artificial water reservoirs in Poland 114, or urban surface waters in Malaysia 115. In recent surveillance studies of different rivers and lakes in Switzerland, blaSHV-12-producing Enterobacteriaceae were isolated only in 4% 116 21 of the cases , although this variant is predominant in clinical Swiss isolates . blaSHV-12 was also detected in Enterobacteriaceae from seawater, together with tet(A) and sul2 in Portugal 117, and 118 A plasmid-encoded together with blaTEM-1 and/or blaCTX-M-1 in Croatia . Finally, data on ESBL-produc- 4 ing Enterobacteriaceae isolated from drinking water is also increasing, reporting SHV alleles in rural water reservoirs in China 119, or drinking water sources for First Nation communities in Canada 120.

Food producing animals Food producing animals have become subject of increasing interest after several studies demonstrated that resistant strains of animal origin can be associated to human infections, possibly

121 through the food chain . Majority of SHV variants in this reservoir belong to blaSHV-2, blaSHV-2a, blaSHV-5 and blaSHV-12 (Supplementary Table S1) owing to their successful association with conjugative plasmids (see section 5).

Surveillance activities in healthy animals worldwide are generating a tremendous amount of data on ESBL distribution. Most SHV β-lactamase producers are E. coli from swine and broiler faecal

38 samples as observed in China ; in Spain, with blaSHV-2 associated with blaCTX-M-9 and blaSHV-12 with 122 blaCTX-M-1, in pigs and broilers respectively ; in layers, cattle, and broilers but not in swine in Japan 123,124 ; and in the Netherlands, where healthy broilers carried blaSHV-2 in combination with blaTEM-1 or 125 blaTEM-135 . Other Enterobacteriaceae like K. pneumoniae and Citrobacter freundii were positive 126 for blaSHV-2 or blaSHV-12 from poultry and swine, respectively .

Finding ESBL producers in food producing animals is also mirrored by positive food samples world-

A review of SHV extended-spectrum β- lactamases: neglected yet ubiquitous | 119 wide, mostly retail chicken meat, as reported in Tunisia, with E. coli carrying blaSHV-5 isolated from 127 different butcheries, supermarkets, and local markets , or Salmonella enterica carrying blaSHV-12 in Japan 128. The cross-contamination between food producing animals and retail meat has been internationally demonstrated due to the detection of plasmid-borne SHV variants, such as blaSHV-2 129 and blaSHV-2a from Canadian chicken meat and abattoir chicken cecum or blaSHV-2 and blaTEM-1 in Japan 130, presenting the potential for horizontal transfer between Enterobacteriaceae as a high public health concern.

SHV β-lactamase producing Enterobacteriaceae have been detected also in diseased animals, as reported for septicemic broilers due to avian pathogenic E. coli encoding a remarkable array of antibiotic resistance genes (dfrA17-aadA5, blaTEM-1, blaCTX-M-15, blaOXA-1, blaSHV-2, tet(A), tet(E), qnrB2, aac(6)-Ib-cr) 131; for K. pneumoniae isolated from bovine mastitis in the United Kingdom 132 and Egypt 133; and for multidrug resistant S. enterica serovars Enteritidis and Typhimurium isolated from diarrheic calves 134.

Finally, blaSHV-27 is the only other SHV variant frequently reported as chromosomally located in K. 135 pneumoniae from swine, in association with blaSHV-11 and blaCTX-M-1 in China ; in E. coli isolated 136 from farmed fish together with non ESBLs blaSHV-1, blaSHV-11, blaSHV-25, and blaSHV-26 ; and in opportunistic pathogens asymptomatically colonizing healthy milk cows 137.

Wildlife, companion animals and vegetables ESBL diffusion has been studied extensively in Enterobacteriaceae from humans and livestock, whereas information on antibiotic resistance in the environment is still limited. Yet, the dissemination success of blaSHV-12 is confirmed by its introduction into the wildlife, notably in birds, as reported in Spain 138, the Netherlands 139, Poland 140, and the Czech Republic 141. This success is likely associated to predominant avian clones and to efficient plasmids (Table 4, Figure 2) of the IncN incompatibility group, described to be more frequent in pathogenic than in commensal avian and

142 human E. coli strains . blaSHV-5 was also detected in E. coli from several birds of prey in Portugal, 143 alone or in associations with blaTEM-1b .

Emergence of Enterobacteriaceae producing β-lactamases in companion animals have been gradually reported, with CTX-M enzymes being prevalent as observed in the human scenario 144. Few studies, on both healthy and diagnostic clinical canine and feline samples, report finding other

29 145 146,147 ESBL variants including blaSHV-3 in the USA , blaSHV-2 in Mexico , blaSHV-12 in Italy and Poland 148 and blaSHV-12 in association with blaOXA-48, blaCMY-2, blaTEM-1, aac(6′)-Ib-cr, and qnrB2 in Germany . Lastly, SHV variants have been detected in imported vegetables in Switzerland together with

120 | Chapter 4A blaSHV-12 for the first time in the opportunistic foodborne pathogen Cronobacter sakazakii whose potential to cause bacteremia and meningitis is an actual concern 149. Similar results were observed in vegetables collected in South Korea 150, salads in the Netherlands 151, and Spain 152, displaying a new route of introduction for ESBLs and pathogenic Enterobacteriaceae.

Plasmid epidemiology of blaSHV-2, blaSHV-2a, blaSHV-5 and blaSHV-12

The role of plasmids in the successful spread of β–lactamase genes has been extensively described

153,154 and, among the SHV family, it finds its best examples inbla SHV-2, blaSHV-2a, blaSHV-5 and blaSHV-12. Combination of these alleles with different dissemination machineries has brought the enzymes to reach diverse niches worldwide (Figure 2). Plasmids belonging to seven replicon types (A/C, F, HI2, I1, L/M, N, X3) have been shown to drive the epidemiology of these four predominant SHV ESBLs, although their distribution varies on the plasmid families (Table 4). Other rep families that have been only incidentally associated with extended-spectrum SHV β–lactamases include the ColE, K, P and R (Table 4). 4A IncA/C blaSHV-12 has been identified on mostly conjugative broad-host range IncA/C plasmids in a variety of bacterial species, including E. coli, Proteus mirabilis and Aeromonas caviae, isolated from clinical samples in Tunisia, France, Korea and Italy 155-158. E. coli isolates recovered from clinical specimens

155,158 encoding blaSHV-2, blaSHV-2a, and blaSHV-5 have been also identified in Tunisia and France , whereas

Providencia stuartii isolates encoding blaSHV-5 on either IncA/C or multireplicon IncA/C-R plasmids have been reported from different outbreaks in Greece 159,160. Interestingly, these IncA/C plasmids (130-220 Kb) often carried multiple resistance genes, conferring multidrug resistant phenotypes 156,159,160, resulting in the proliferation of the SHV ESBLs by co-selection.

IncF Plasmids belonging to the narrow-host range IncF group, including plasmids with fused replicons, have been reported to accommodate blaSHV-12 among clinical E. coli isolates from France (IncFII), Tunisia (IncFIA-FIB, IncFII-FIA, IncFII-FIA-FIB) and United Kingdom (IncFIB), but also among food-producing animals from Italy (IncFIB) 155,158,162,164. IncF plasmids account for the dissemination of blaSHV-2 gene among E. coli from both clinical specimens in France (IncFIB, IncFII, IncFII-FIB) and food-producing animals (avian and porcine sources) in Canada (IncFIB), as well as in clinical K. pneumoniae isolates belonging to ST654 and ST15 from China (IncFIB) and Portugal (IncFII), respectively 129,158,163,167. Finally, clinical E. coli and K. pneumoniae from Tunisia were found to encode

155,166 blaSHV-2a , clinical E. coli from Poland encoded blaSHV-5 on IncF plasmids, as well as clinical K. A review of SHV extended-spectrum β- lactamases: neglected yet ubiquitous | 121 Table 4. Plasmid epidemiology of SHV-type extended-spectrum β-lactamases. Plasmid Other Antibiotic Bacterial Inc Group bla allele§ Country Reference Size (Kb)* SHV Resistance Genes Species# IncA/C ND (C) SHV-12 or SHV-2a ND E. coli (H) Tunisia 155

156 150 (C) SHV-12 blaVIM-1, aac(6′)-Ib′, aadA1, A. caviae (H) Italy catB2, sul1, dfrA14

157 150 (NC) SHV-12 (IS26) blaCTX-M-14, blaDHA-1 P. mirabilis (H) Korea ND SHV-2, SHV-5 or ND E. coli (H) France 158 SHV-12

159 130 (C) SHV-5 (IS26) blaVEB-1, blaVIM-1, aacA7, P. stuartii (H) Greece

dfrA1, aadA1, blaOXA-1,

blaTEM-1, aadB, arr2, cmlA5

36 97-145 SHV-45 blaCTX-M-2; blaSHV-27 K. pneumoniae (H) Brazil

36 63.5-209 SHV-55 blaCTX-M-2; blaSHV-28 K. pneumoniae (H) Brazil

160 IncA/C-IncR 220 (C) SHV-5 blaVEB-1, blaVIM-1, rmtB, P. stuartii (H) Greece aacA7, dfrA1, aadA1 IncF 125 SHV-5 (IS26) ND E. coli (H) Poland 161 IncFIA-FIB ND (C) SHV-12 ND E. coli (H) Tunisia 155 IncFIB ND SHV-12 sul3 E. coli (A) Italy 162 95-200 (C) SHV-2 aadA1 E. coli (A) Canada 129 >23 SHV-2 ND K. pneumoniae (H) China 163 ND SHV-2 ND E. coli (H) France 158

ND (C) SHV-5 - E. coli (H) Uruguay

164 IncFIB10 ND SHV-12 (IS26) blaTEM-1 E. coli (H) UK IncFIC ND aac(6′)-Ib′, aadA1 S. marcescens (H) 165 IncF-N ND SHV-2 aac(6′)-Ib′ K. pneumoniae (H) Uruguay 165 IncFII ND SHV-2 or SHV-12 ND E. coli (H) France 158 70-80 (C) SHV-2a (IS26) ND K. pneumoniae (H) Tunisia 166 100 (C) SHV-128 (IS26) ND E. cloacae (H) Tunisia 68 IncFII-FIA ND (C) SHV-12 ND E. coli (H) Tunisia 155 IncFII-FIA-FIB ND (C) SHV-12 ND E. coli (H) Tunisia 155 IncFII-FIB ND SHV-2 ND E. coli (H) France 158 ND (C) SHV-2a ND E. coli (H) Tunisia 155 IncFIIk1 200-220 SHV-2, SHV-55 or ND K. pneumoniae (H) Portugal 167 SHV-106 IncFIIk5 220 SHV-55 ND K. pneumoniae (H) Portugal 167 IncHI2 ND (C) SHV-2a or SHV-12 ND E. coli (H) Tunisia 155 95 (C) SHV-12 (IS26) ND K. pneumoniae (H) Tunisia 166

168 310 (C) SHV-12 qnrB2, blaTEM-1, sul1, S. enterica Netherlands dfrA19, tet(D), strA, strB, Senftenberg (H) aac(6’)-Ib 200 (NC) SHV-12 tet(D) S. enterica Netherlands 168 Concord (H)

122 | Chapter 4A Table 4. Continued. Plasmid Other Antibiotic Bacterial Inc Group bla allele§ Country Reference Size (Kb)* SHV Resistance Genes Species#

168 290 (C) SHV-12 qnrB2, blaTEM-1, sul1, sul2, S. enterica Netherlands dfrA19, tet(D), strA, strB, Concord (H) 180, 350, SHV-12 ND K. pneumoniae (H) Portugal 167 380

400 SHV-12 ND E. cloacae (H) Portugal 167

320 (C) SHV-12 qnrB2, strA/B, tet(D), S. enterica Spain 169 clmA, sul1 Bredeney (H)

170 ND (C) SHV-12 (IS26) blaCTX-M-14 E. cloacae (H) Taiwan

170 ND (C) SHV-12 (IS26) blaCTX-M-3 E. cloacae(H) Taiwan IncHI2 (ST1) 300 (C) SHV-2 ND S. enterica Agona Senegal 171 or Keurmassar (H)

IncI1 ND (C) SHV-12 ND E. coli (H) Bulgaria 172

ND SHV-12 ND E. coli (H) France 158

ND SHV-12 sul3 E. coli (A) Italy 162

19 (C) SHV-12 - E. coli (A) Italy 173 340 (C) SHV-12 ND S. enterica Norway 174 Concord (H) (Ethiopia) 4A 95 (C) SHV-12 - E. coli (A) Poland 140 10 (NC) SHV-12 - S. enteritidis (H) Spain 175

176 60 (C) SHV-12 blaVIM-1-aacA4-dfrII- K. pneumoniae, Spain aadA1-catB2 E. coli (H)

170 ND (C) SHV-12 (IS26) blaCTX-M-3 E. cloacae (H) Taiwan 95-200 (C) SHV-2 aadA1 E. coli, S. enterica Canada 129 Heidelberg (A) 95-200 (C) SHV-2 - E. coli (A) Canada 129 95-200 (C) SHV-2a aadA1, dfrA1 E. coli, S. enterica Canada 129 Kiambu (A) 95-200 (C) SHV-2a aadA1 E. coli (A) Canada 129 IncI1 (ST26) ND SHV-12 ND E. coli (H) Italy 177 ND SHV-12 (IS26) ND E. coli (A) Portugal 178 IncI1 (ST27, 115 (C) SHV-2 aadA2 S. enterica Spain 175 CC26) Livingstone (H) IncI1 (ST29/ ND (C) SHV-12 (IS26) ND E. coli (E) Portugal 178 CC26) IncI1 (ST3) ND SHV-12 ND E. coli (A) Italy 177 104 (C) SHV-12 - E. coli (A) Italy 173 IncK ND (NC) SHV-12 ND K. pneumoniae (A) England 132 155 SHV-2 - E. coli (A) Netherlands 125 IncL/M ND (C) SHV-12 ND E. coli (H) Tunisia 155

A review of SHV extended-spectrum β- lactamases: neglected yet ubiquitous | 123 Table 4. Continued. Plasmid Other Antibiotic Bacterial Inc Group bla allele§ Country Reference Size (Kb)* SHV Resistance Genes Species#

179 65 SHV-12 blaKPC-2, rmtB K. pneumoniae (H) China 65 SHV-2 ND K. pneumoniae (H) Portugal 167 ND (C) SHV-2a ND E. coli (H) Tunisia 155 60-70 (C) SHV-2a (IS26) ND K. pneumoniae (H) Tunisia 166 ND (C) SHV-5 (IS26) aacA4, aacC1, aadA1, sul1 S. enterica Italy 180 Typhimurium (H) 90 (C) SHV-5 tet(A), aadA1, aacC1, K. oxytoca (H) USA 181 aacA4, dfrA1 IncN ND (C) SHV-12 ND E. coli (H) Tunisia 155 ND (C) SHV-12 ND K. pneumoniae (H) Bulgaria 172

182 50 SHV-12 blaVIM-1, qnrS K. pneumoniae, Norway E. coli (H)

183 50 (C) SHV-12 blaVIM-1, qnrS K. pneumoniae (H) Norway >23 SHV-2 (IS26) ND K. pneumoniae (H) China 163 ND (C) SHV-2a ND E. coli (H) Tunisia 155 IncN (ST1) ND SHV-12 aadA2 E. coli (H) Netherlands 184 IncN (ST16) 50 (C) SHV-2 ND S. enterica Miami Senegal 171 (U) IncP ND (C) SHV-12 (IS26) - E. cloacae (H) Taiwan 170 95-200 (C) SHV-2a aadA1, dfrA1 E. coli (A) Canada 129

185 IncX3 50 (C) SHV-12 blaKPC-2 K. pneumoniae (H) Australia

186 54 (C) SHV-12 blaNDM-1 K. pneumoniae (H) China

187 54 (C) SHV-12 (IS26) blaNDM-1 K. pneumoniae, China C. freundii, E. aerogenes, E. cloacae, E. coli (H)

188 60 (C) SHV-12 blaNDM-1, blaTEM-1 E. coli (H) China

188 60 (C) SHV-12 blaNDM-1 E. coli (H) China

189 54 (C) SHV-12 (IS26) blaNDM-1 E. coli (H) China

190 54 (C) SHV-12 (IS26) blaNDM-1 C. freundii (H) China 50 SHV-12 qnrB7 E. coli (A) Czech 191 Republic 40 SHV-12 qnrS1 E. coli (E) Czech 191 Republic

192 53 SHV-12 (IS26) blaKPC-2 K. pneumoniae (H) France

193 50 (C) SHV-12 blaNDM-1 E. cloacae (H) UAE

193 50 (C) SHV-12 blaNDM-1 E. coli (H) UAE

193 50 (C) SHV-12 blaNDM-1 C. freundii (H) UAE 43 (C) SHV-12 (IS26) - E. cloacae (H) USA 194

191 IncX3-N 80 SHV-12 blaTEM-1, qnrS1 E. coli (A) Germany

124 | Chapter 4A Table 4. Continued. Plasmid Other Antibiotic Bacterial Inc Group bla allele§ Country Reference Size (Kb)* SHV Resistance Genes Species# ColETp 10 (NC) SHV-12 qnrS1 S. enterica Spain 169 Typhimurium (H) R 70 SHV-12 ND K. pneumoniae (H) Portugal 167 R+IncFIIk1 300 SHV-2 ND K. pneumoniae (H) Portugal 167 Untypable 90-140 (C) SHV-12 (IS26) ND K. pneumoniae (H) Tunisia 166 ND SHV-12 (IS26) - E. coli (H) UK 164

164 ND SHV-12 (IS26) blaTEM-1 E. coli (H) UK

164 ND SHV-12 (IS26) blaTEM-1, blaOXA-1, qnrS1 E. coli (H) UK

193 50 (C) SHV-12 blaNDM-1 K. pneumoniae (H) UAE *C; conjugative; NC; non conjugative; when blank is because not determined §When present, IS26 is indicated in parenthesis #H: human; A: animal (mostly poultry, turkey and broilers; check reference for full description); E: environment ND: not determined pneumoniae and Serratia marcescens from Uruguay 161,165, whereas the same plasmids have been associated with less prevalent SHV ESBLs (blaSHV-55 and blaSHV-106) in clinical K. pneumoniae isolates from Portugal 167. 4A

IncHI2 In contrast with the IncA/C and IncF plasmids, the broad-host range IncHI2 group is responsible mainly for the dissemination of blaSHV-12, although this group has been found incidentally to also accommodate 155 blaSHV-2a . Plasmids of this group varying in sizes (95-400 Kb) have been reported to encode blaSHV-12 in various bacterial species, such as E. coli, K. pneumoniae, E. cloacae and at least three S. enterica serotypes (Bredeney, Concord, and Senftenberg) from human specimens with diverse geographical origin

155,166-170 (Netherlands, Portugal, Spain, Taiwan, Tunisia) . Apart from blaSHV-12, some of these conjugative plasmids have been reported to co-encode for other resistance genes, including additional SHV ESBLs

168,170 (blaCTX-M-3, blaCTX-M-14) .

IncI1α/γ The IncI1α/γ group, consisting of narrow-host range mostly conjugative plasmids, ranks amongst the top facilitators of blaSHV-2, blaSHV-2a and blaSHV-12 genes. The range of bacterial species they have encountered is limited to E. coli, K. pneumoniae, E. cloacae and the S. enterica serotypes Concord, Enteritidis, Heidel- berg and Kiambu. Nevertheless, IncI1α/γ plasmids (19-340 Kb) occur in very diverse settings: blaSHV-2- and 158,170,172,175-177 blaSHV-12-encoding isolates from human infections (Bulgaria, France, Italy, Spain, Taiwan) and 174 colonization (Ethiopia) ; blaSHV-2-, blaSHV-2a- and blaSHV-12-encoding isolates from poultry (Canada, Italy, 129,162,173,177,195 129 Portugal) , blaSHV-2- and blaSHV-2a-encoding isolates from pigs (Canada) ; blaSHV-12-encoding

A review of SHV extended-spectrum β- lactamases: neglected yet ubiquitous | 125 . Inc/rep types are represented in different colors; diverse symbols depict human, in different represented types are . Inc/rep SHV-12 bla , and SHV-5 bla , SHV-2a bla , SHV-2 bla Worldwide distribution of plasmid families encoding Worldwide Figure 2. Figure bovine, porcine, poultry, wildlife, soil, or wastewater sources. For a complete full reference list see Table 4. list see Table For a complete full reference wildlife, soil, or wastewater sources. poultry, bovine, porcine,

126 | Chapter 4A 140 isolates from aquatic birds (Poland) ; and blaSHV-12-encoding isolates from farming soil (Portugal) 178 . Remarkably, blaSHV-12 on IncI1α/γ plasmids belonging to pST26 have been identified among E. coli isolates of human and animal origin 177,195, indicating the potential transmission of these blaSHV-12-encoding vehicles from human to animals and/or vice versa.

IncL/M and IncN The broad-host range IncL/M and IncN plasmids contribute to a lesser extent to the epidemiology of blaSHV-2, blaSHV-2a, blaSHV-5 and blaSHV-12 than the above-mentioned families. IncL/M plasmids (60-90 Kb) carrying SHV ESBL genes have been reported only among E. coli, K. pneumoniae, K. oxytoca, and S. enterica serotype Typhimurium of human origin in Portugal (blaSHV-2), Tunisia (blaSHV-2a, blaSHV- 155,166,167,179-181 12), Italy (blaSHV-5), USA (blaSHV-5) and recently in China (blaSHV-12) . The same bacterial species mostly from human sources carry IncN plasmids (~50 Kb) encoding blaSHV-2 (China, Senegal), 155,163,171,172,182-184 blaSHV-2a (Tunisia) or blaSHV-12 (Bulgaria, Netherlands, Norway, Tunisia) . Interestingly, the presence of IncN (pST1) plasmids encoding blaSHV-12 has been reported among E. coli from human and animal sources 184, mirroring the situation for IncI1α/γ plasmids and underscoring the contribution of this plasmid family in the transmission of blaSHV-12 within or between these niches. 4A IncX3 The IncX3 plasmid subgroup consists of narrow-host range plasmids and plays an important role in the exclusive dissemination of blaSHV-12. Conjugative plasmids (40-60 Kb) of this subgroup have been identified in diverse bacterial species (E. coli, K. pneumoniae, C. freundii, E. aerogenes, E. cloacae), sources (human, animal, environment) and geographical areas (Australia, China, Czech Republic, France, United Arab Emirates, US) 185-194. Interestingly, the majority of these plasmids appear to co-harbor carbapenemase genes (blaKPC-2, blaNDM-1), whereas the co-localization of SHV

ESBL and carbapenemase genes was reported only on IncA/C or IncA/C-R (blaVIM-1), IncL/M (blaKPC- 159,160,182,183 2) and IncN (blaVIM-1) plasmids , enhancing the plasmid potential maintenance among bacterial populations and the subsequent preservation and dissemination of the SHV ESBL genes.

Miscellaneous plasmids blaSHV-12 has been incidentally found on: i) a ColE plasmid from S. enterica serotype Typhimurium DT104b in Spain 169; ii) an IncK plasmid from K. pneumoniae in the United Kingdom 132; iii) an IncP plasmid from E. cloacae in Taiwan 170; and iv) a plasmid assigned to the R replicon type from

167 K. pneumoniae in Portugal . E. coli and K. pneumoniae encoding blaSHV-2 on IncK plasmids were recovered from animal and human sources in the Netherlands and in Uruguay, respectively 125,165.

IncP plasmids encoding blaSHV-2a from animals in Canada and blaSHV-5 from human in Uruguay have 129,165 also been reported . Finally, a number of reports highlight the presence of blaSHV-12 on mostly

A review of SHV extended-spectrum β- lactamases: neglected yet ubiquitous | 127 conjugative non-typeable plasmids, according to the PCR-based replicon-typing scheme 196. These plasmids of human origin, varying between 50 to 140Kb in size, were mostly detected among E. coli from the United Kingdom 164 and K. pneumoniae from Tunisia 166 and United Arab Emirates 193, underscoring that their dissemination is wider than we know.

IS26 role in blaSHV mobilization

Analysis of the sequences bracketing several SHV ESBL genes (blaSHV-2, blaSHV-2a, blaSHV-5, blaSHV-12, blaSHV-106 and blaSHV-134) among Gram-negative bacteria, including Enterobacteriaceae and non-fer- menters, revealed that these β-lactamase genes are mostly associated with the IS26 element (Table 4, Supplementary Table S1). Beside SHV ESBLs, this member of the IS6 insertion sequence family 197, has been associated with a plethora of resistance genes 198-202 and has been found to contribute to their expression by supplying a promoter −35 box that can be coupled with a −10 box in the adjacent DNA 203,204. In contrast to other insertions sequences, it has been suggested that IS26 transposes preferentially within plasmids rather than into the chromosome 205, possibly explain- ing the linkage of IS26 and the four predominant SHV ESBL genes with IncA/C (blaSHV-5, blaSHV-12),

IncF (blaSHV-2a, blaSHV-5, blaSHV-12), IncHI2 (blaSHV-12), IncI1α/γ (blaSHV-12), IncL/M (blaSHV-2a, blaSHV-5), IncN

(blaSHV-2), IncP (blaSHV-12), IncX3 (blaSHV-12) and non-typeable (blaSHV-12) plasmids (Table 4). Similarly to most antibiotic resistance genes, IS26-mediated mobilization of SHV ESBL genes on conjugative plasmids facilitated their subsequent intra- and inter-species dissemination (Table 4). Available sequences of transposons flanked by copies of intact and/or truncated IS26 elements (Figure 3) and coding for SHV ESBL genes show the presence of other co-linear genes originating from the chromosome of K. pneumoniae (i.e. fucA, ygbI, ygbK, ygbJ, ygbM, deoR) 159,163,170,181,187,189,190,192, likely underscoring the involvement of IS26 in the mobilization of blaSHV from the chromosome of K. pneumoniae, as previously suggested 14.

Outside of the Enterobacteriaceae and a few peculiar SHV ESBLs

SHV β–lactamases have virtually invaded all human, environmental and animal sceneries, mostly associated to Enterobacteriaceae. In recent years, the first reports of alternative bacterial hosts have been described, notably in Aeromonads, ubiquitous in aquatic habitats and occasionally able to cause human infections. blaSHV-12 was detected, in association with blaFOX-2 and blaCTX-M-15, on the chromosome of the foodborne pathogens A. caviae and Aeromonas hydrophila from wild-growing mussels from Croatia 206. The first identification of plasmid-encoded SHV-12, together with

128 | Chapter 4A ; IncA/C 163

4A association. Common occurring features are color coded. Map is not to scale. References: IncN color coded. Map is not to scale. References: are association. Common occurring features 26 . genetic surroundings and IS genetic surroundings 187,189,190,192 SHV bla ; IncX3 170 ; IncHI2, IncI1, IncP 181 Schematic representation of Schematic representation ; IncL/M Figure 3. Figure 159

A review of SHV extended-spectrum β- lactamases: neglected yet ubiquitous | 129 VIM-1, occurred in clinical A. caviae accountable for a newborn bloodstream infection 156. The coproduction of these enzymes highlights the potential risks for public health and the role of Aer- omonads as reservoirs and dissemination tools of resistance determinants in both environmental and clinical settings.

Occasionally, SHV ESBL-producing Pseudomonas aeruginosa can be detected in nosocomial settings and can pose a serious threat as healthcare-associated infection in many regions of the world. blaSHV-2a was first identified on the chromosome of a 1995 clinicalP. aeruginosa strain, with high sequence homology to plasmid pMPA2a from K. pneumoniae indicating a likely enterobacterial gene origin

207 . Subsequent studies demonstrated the insertion of blaSHV alleles into P. aeruginosa chromosome: 208 209 210 211 212 blaSHV in China and Iran ; blaSHV-5 and blaSHV-12 in Greece; and blaSHV-2a in Tunisia and 213,214 France . The role of IS26 in the mobilization of blaSHV-12 was demonstrated by the chromosomal insertion of an IS26 composite transposon (>24 kb) thanks to the co-mobilization of antibiotic resistance aac(6’)-Ib, which confers amikacin resistance, likely occurred during the clinical course

215 of a burn infection, immediately after amikacin administration . blaSHV-5, blaSHV-11, blaSHV-12 were 216 also detected in different combinations, together with blaTEM-1b, on various plasmids in Thailand ,

Finally, one of the most effective associations outside of the Enterobacteriaceae is with Acineto- bacter baumannii, contributing to the worrisome spread of ESBL-producing strains especially in clinical outbreaks 217. Plasmid transfer from nosocomial SHV-encoding Enterobacteriaceae seems to be responsible for this phenomenon, as observed for SHV-12 in the Netherlands 218, or SHV-5 in the USA, a country where this variant is the most prevalent ESBL gene in Enterobacteriaceae 219. Among all ESBL SHV β-lactamases, few enzymes deserve special consideration because of their unique enzymatic features. SHV-38 is a unique allelic variant of the SHV family to have an expanded-spectrum to carbapenems. It was first described in K. pneumoniae from France 17 and it holds a point mutation (A146V) compared to the chromosome-encoded SHV-1. Among all 46 available SHV ESBL variants, only SHV-38 possess the A146V substitution (Table 2), likely inducing subtle structural conformational changes favoring imipenem but not meropenem hydrolysis 220.

SHV-129 is a novel clinically acquired variant identified in 2012 from an ItalianE. coli isolate (Table 1) 69 and it represents an interesting example of enzyme evolution due to antibiotic pressure. Alongside two well-known amino acid substitutions (G238S, E240K), SHV-129 contains new substitutions, R275L and N276D). The latter was recently demonstrated to be the first global sup- pressor substitution identified in the SHV β-lactamase family 82, likely helping in protein stabilization and functionality, as well as in the ability of the enzyme to acquire additional substitutions. It is also

130 | Chapter 4A proposed that due to the increasing clinical use of cefepime, SHV-129 might have evolved from SHV-2 or SHV-5 in an alternative conformation to expand its spectrum to hydrolyze cefepime, as mirrored by the kinetic parameters of the three enzymes (Table 3).

Finally, SHV-2 can be located on both chromosome and self-transmissible plasmids (Table 4 and

Supplementary Table S1). Association of blaSHV-2 with RCS47, a P1-like bacteriophage that infects 221 and lysogenizes E. coli and several other enteric bacteria, was recently reported . blaSHV-2 is flanked by two IS26 elements that likely drove the insertion in the phage backbone. The P1-like prophages were found with high prevalence in natural E. coli of both animal and human origin, including ESBL-producing isolates. This is kind of association was already reported for other

222 β-lactamases (blaTEM, blaCTX−M and mecA) from river and urban sewage water , suggesting that bacteriophages might play a wider role in favoring horizontal transfer of antibiotic resistance determinants than initially thought 223.

Concluding remarks 4A Tzouvelekis and Bonomo suggested than “it will not be surprising if (SHV) enzymes will continue to expand their substrate spectrum as long as the current antibiotics, or novel ones derived from the basic β-lactam structure, are used” 16. In the last two decades we observed the appearance of multiple SHV-type variants, with few ones significantly expanding their substrate. One exception is represented by SHV-38, the only known SHV allelic variant able to hydrolyze carbapenems 17, a feature that has not been associated with any TEM or CTX-M enzyme. In this image resides the fate of SHV extended β-lactamases, unable to undergo the dominant propagation observed, for instance, for the CTX-M family but yet contributing to β-lactam resistance in a not negligible way.

The persistence of SHV enzymes in the bacterial community might also be secured by co-selection with emerging resistance genes. Association of blaSHV-12 with IncX3 plasmids carrying carbapenemase genes blaKPC-2 and blaNDM has been observed in recent years (Table 4) and it seems to be a phenomenon occurring in clinical carbapenem-resistant Enterobacteriaceae worldwide 185,188,192,193.

As highlighted in this review, the association of successful variants blaSHV-2, blaSHV-2a, blaSHV-5 and blaSHV-12 with different families of conjugative plasmids (IncA/C, IncF, IncHI2) might also underlie the colonization of virtually all ecological niches encompassing food producing animals, aquatic environment, wildlife, companion animals and vegetables. Plasmid mediated transfer from nosocomial Enterobacteriaceae enabled SHV dispersion towards alternative bacterial hosts such as the emerging nosocomial pathogens of aquatic origin S. maltophilia and A. caviae, or contributed to

A review of SHV extended-spectrum β- lactamases: neglected yet ubiquitous | 131 the worrisome spread of ESBL-producing strains of A. baumannii and P. aeruginosa. Most interestingly, the ubiquitous presence of SHV ESBL genes and plasmids is suggestive for transmission in human, animals and the environment, most likely through the food chain, highlighting the potential risks for public health and endorsing a one health research approach.

Overall, SHV ESBL enzymes have kept a stable role in antibiotic resistance over the years. Allele diversification is still occurring, the latest variant being identified in E. cloacae in 2014 (blaSHV-183), and effective associations with new genetic platforms are taking place helping expansion towards novel bacterial hosts and reservoirs.

Acknowledgements

The authors would like to thank Mr. Bo Derks for valuable technical help in Figure 2 production.

Funding

AL was partially supported by the ESBLAT project (BO-22.04-008-001). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

132 | Chapter 4A Supporting material

blaSHV-57

blaSHV-24 blaSHV-100

blaSHV-43

blaSHV-106 blaSHV-8 blaSHV-55 blaSHV-99 blaSHV-16 blaSHV-28

blaSHV-1 blaSHV-51 blaSHV-75 blaSHV-137 blaSHV-49 blaSHV-33

blaSHV-48 blaSHV-102 blaSHV-190 blaSHV-108

blaSHV-52 blaSHV-143 blaSHV-92blaSHV-62 blaSHV-120 blaSHV-46 blaSHV-157

blaSHV-94 blaSHV-2 blaSHV-96

blaSHV-153 blaSHV-35 blaSHV-40 blaSHV-165 blaSHV-89 blaSHV-9 blaSHV-103 blaSHV-5 blaSHV-56

blaSHV-161 blaSHV-122 blaSHV-172 blaSHV-79 blaSHV-70 blaSHV-85

blaSHV-13 blaSHV-29

blaSHV-2a blaSHV-69 blaSHV-105 blaSHV-15 blaSHV-7 blaSHV-64 blaSHV-18 blaSHV-129 blaSHV-189 blaSHV-128 blaSHV-30

blaSHV-12 blaSHV-34

blaSHV-134 blaSHV-14

blaSHV-160 blaSHV-154 blaSHV-183 blaSHV-163 blaSHV-141 blaSHV-86 blaSHV-162 blaSHV-66 blaSHV-188 blaSHV-11 blaSHV-83 blaSHV-67 blaSHV-187 blaSHV-152 blaSHV-140 blaSHV-81 blaSHV-132 4A blaSHV-77 blaSHV-144

blaSHV-93 blaSHV-38

blaSHV-110 blaSHV-71

bla-SHV-32 blaSHV-73 blaSHV-168 blaSHV-27 blaSHV-72 blaSHV-45 blaSHV-121/136 blaSHV-191 blaSHV-25 blaSHV-74 blaSHV-37 blaSHV-173 blaSHV-76 blaSHV-119

blaSHV-107 blaSHV-156

blaSHV-82 blaSHV-155 blaSHV-36 blaSHV-158

blaSHV-193 blaSHV-159

blaSHV-98 blaSHV-182 blaSHV-80 blaSHV-41

blaSHV-95

blaSHV-178 blaSHV-78

blaSHV-179 blaSHV-97

blaSHV-147 blaSHV-44

blaSHV-145

blaSHV-104 blaSHV-42 blaSHV-148 blaSHV-59

blaSHV-164

blaSHV-63

blaSHV-150

blaSHV-151 blaSHV-31

blaSHV-149 blaSHV-101 blaSHV-109

blaSHV-65 blaSHV-133 blaSHV-146 blaSHV-61

blaSHV-142

blaSHV-135 blaSHV-50

blaSHV-180/181 blaSHV-186 blaSHV-60 blaSHV-26

Figure S1. Maximum likelihood nucleotide tree of 142 SHV-type β-lactamases. Variants whose sequence was not released in GenBank as of June 2016, that showed partial sequence or were identical to others (http://www.lahey.org/

studies/) were not included in the analysis. blaSHV-180 and blaSHV-181 share the same sequence, as well as blaSHV-121 and blaSHV- 22 136. The tree was implemented in Mega version 6.06 . Solid circles represent: red, extended-spectrum β-lactamases (2be; n=39); green, broad-spectrum β-lactamases (2br, n=5); and blue, penicillinases (2b, n=30). Unclassified alleles are reported in black (n=68).

A review of SHV extended-spectrum β- lactamases: neglected yet ubiquitous | 133 # # 225 228 43 229 122 23 66 224 34 60 221 171 165 226 227 230 231 233 234 235 129 149 131 145 236 141 125 126 Reference Reference 232 38 - ;

TEM-1 CMY-2 ; or strA/B TEM-1 TEM-135 bla bla CMY-2 OXY-1 CTX-M-15 bla bla ; or or tet(A), tet(E), bla bla bla ; ; ; ; or and/or CTX-M-14 TEM-1 TEM-1 CTX-M-9 TEM-1 TEM-1 CTX-M-9 TEM-1 OXA-1 TEM-1 qnrB2, aac(6)-Ib-cr; qnrA1, tet(E); tet(B) bla Other Antibiotic Resistance Genes Other Antibiotic Resistance Genes ND ND ND ND bla - - - bla ND - bla bla aadA1 ND bla aacC2; strA; strB; catA2; sul2; tetD; dfrA14; qnrS - ND bla tetA/B; sul1; dfrA1; aadA; - bla dfrA17-aadA5 bla - - dhfr17-aadA5; or dhfr1-aadA1 bla dfrA12-orfF-aadA2 ND Plasmid Size (Kb) Plasmid Size (Kb) ND ND ND Variable ND - 50 ND ND ND ND ND ND 125 Variable - 98 ND ND ND - ND - - ND ND ND ND ND 45 Conjugative Plasmid Conjugative Plasmid ND ND ND Yes Yes - Yes No ND Yes ND Yes ND Yes Yes - Yes Yes Yes ND - ND - No ND ND ND Yes Yes Yes ¥ ¥ ) 5 , IS 26 Genetic Location Genetic Location ND ND ND P P Phage (IS IncN IncN; IncFIC; IncF ND P P P ND IncI1 P C pK245 P P ND IncI1; IncFIB ND C C ND ND ND IncK P pBP60 (A) E. coli

§ § (E) (A) Miami; Isangi and Saint-paul

Corvallis Livingstone ; S. enterica K. pneumoniae (A) (E) (A) (A) (E) (A) (A) ; (A) E. coli E. coli K. pneumoniae K. pneumoniae K. pneumoniae E. coli Salmonella E. coli Salmonella K. pneumoniae; S. marcescens; S. enterica E. cloacae K. oxytoca E. coli; K. pneumoniae S. enterica K. pneumoniae; variicola E. coli K. pneumoniae E. coli; K. pneumoniae S. flexneri E. coli E. coli K. pneumoniae E. coli E. coli E. coli E. coli E. coli E. coli K. pneumoniae K. ozaenae Bacterial Species Bacterial Species 2003- 2004 2000- 2001 2003 1999- 2000 2004- 2005 2002 1990 2009 2001 1999- 2000 2003- 2004 2001- 2003 2001- 2002 2001- 2005 1990- 1996 2005 NA NA 1998 2006- 2009 2006- 2007 2014 NA 2011 2001 2010 2005 2006 2003 1983 Year* Year* -lactamase producing bacteria of human, animal or environmental origin, their geographical distribution, year of isolation and genetic background. bacteria of human, animal or environmental -lactamase producing β Spain Vietnam Portugal Canada Thailand France Senegal Uruguay South Africa Bulgaria USA France Indonesia Spain Mexico Bolivia, Peru Taiwan Turkey Argentina China Canada Dominican Republic, Vietnam, Thailand Spain Egypt Mexico Netherlands Czech Re- public Netherlands Portugal Germany Location Location 7.6 pI pI AF148851 Accession Number Accession Number SHV-type extended-spectrum SHV-type Continued. SHV-2 Table S1. Table Gene bla S1. Table Gene

134 | Chapter 4A # # 225 228 43 229 122 23 66 224 34 60 221 171 165 226 227 230 231 233 234 235 129 149 131 145 236 141 125 126 Reference Reference 232 38 - ;

§ § (E) (A) Miami; Isangi and Saint-paul

A review of SHV extended-spectrum β- lactamases: neglected yet ubiquitous | 135 # # 249 251 246 253 24 237 238 241 242 243 244 245 247 149 129 123 25 29 48 30 31 248 250 249 28,252 71 Reference Reference 130 239 240 32 32 ; strA/B OXA CTX-M-24 CTX-M-15 SHV-1 SHV-5 bla bla bla bla ;

bla aacA7; aacA or ; and/or or (on various ; variable or ; ; ; and/or TEM-1 DHA-1 TEM-15 VIM-4 VIM-4 TEM-1 AmpC CTX-M-15 CMY-2 TEM-1 AmpC SHV-3 TEM-4 ESBL-like CTX-M TEM-1 dhfrA1; aadA1; sul1 bla bla Other Antibiotic Resistance Genes Other Antibiotic Resistance Genes plasmids) bla bla ND ND bla bla bla - aac(6’)-Ib bla ND bla - - tetA/B; sul1; dfrA1; aadA; - bla bla ND ND bla ND bla bla bla ND ND bla ND ND ND ND Plasmid Size (Kb) Plasmid Size (Kb) ND ND ND ND 120-150 40 Variable 60-100 - ND ND - ND - - 10-50 - ND ND Variable ND Variable ND ND ND ND - 66 180 180 150 Conjugative Plasmid Conjugative Plasmid Yes ND ND Yes Yes Yes Yes Yes - ND ND - ND - - ND - ND Yes ND ND ND ND ND ND Yes - Yes Yes Yes No ¥ ¥ ) ) 26 26 Genetic Location Genetic Location P ND ND P C and P P IncR P C (IS ND P C (IS ND IncI1; IncP C C and/or P C P (different profiles) P (different profiles) P ND P P ND P P C pZMP1 pUD18 P pAFF1

(E) 4,5 (A)

§ § ; S. enterica (A) (E) (A) (A) E. coli K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae; E. coli; E. cloacae; C. freundii E. cloacae K. pneumoniae; oxytoca K. pneumoniae aeruginosa P. S. typhimurium; enterica K. pneumoniae aeruginosa P. C. sakazakii; K. pneumoniae E. coli E. coli E. aerogenes E. coli E. aerogenes C. diversus E. aerogenes E. coli; K. pneumoniae E. cloacae E. coli K. pneumoniae E. cloacae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae Bacterial Species Bacterial Species 2004- 2006 1989- 2015 1994- 1995 2004 1998- 2002 2006 1998- 2002 2006 2011 2007- 2011 2002- 2003 2005- 2007 2014 2006- 2007 2004 1993- 1995 2008- 2009 1993 1988- 1989 1993- 1995 1997 1994 2002- 2005 2008- 2010 1994 2003- 2004 1997- 2001 1987- 1988 1986 1987 1987 Year* Year* Japan Australia Croatia Korea Slovenia Greece Hungary Mexico France DR Congo Egypt Tunisia Thailand Canada Japan USA USA France France USA Turkey Turkey France Iraq Belgium China Taiwan Germany France France Chile Location Location 7.6 7 7.8 8.2 pI pI X98102 KX092356 NA X55640 Accession Number Accession Number Continued. Continued. SHV-2a SHV-3 SHV-4 SHV-5 Table S1. Table Gene bla S1. Table Gene bla bla bla

136 | Chapter 4A # # 249 251 246 253 24 237 238 241 242 243 244 245 247 149 129 123 25 29 48 30 31 248 250 249 28,252 71 Reference Reference 130 239 240 32 32 ; strA/B OXA CTX-M-24 CTX-M-15 SHV-1 SHV-5 bla bla bla bla ;

(E) 4 4,5 (A)

A review of SHV extended-spectrum β- lactamases: neglected yet ubiquitous | 137 # # 263 266 122 60 249 255 256 219 257 165 123 258 259 47 49 261 50 259 51 262 264 21 156 265 71 Reference Reference 32 159 254 124 143 260 33 - D ; ; bla ; CMY-2 ; OXA-1 TEM-1 )-Ib ′ CMY-7 bla KPC-3 VIM-1 OXA-10 bla bla ; ; bla or bla qnr bla dfrA14 bla ; ; ; ; ; ; and/or ; aac(6”)-Ib-cr ; AmpC SHV-5 VEB-1 TEM-1 CTX-M-15 CTX-M-14 ACT-1 CMY-2 CTX-M-9 TEM-1b TEM-1 CTX-M-14 OXA-30 VIM-1 TEM-1 TEM-1 CTX-M-59 bla HA-1 bla Other Antibiotic Resistance Genes Other Antibiotic Resistance Genes bla bla ND bla ND bla bla - bla - - aadA1/aac(6 - ND bla bla bla ND bla qnr ND ND bla ND bla bla ND bla bla ND ND - ND ND Plasmid Size (Kb) Plasmid Size (Kb) Variable Variable - ND 130 Variable 120 ND - - ND - ND ND - ND ND - ND ND ND ND ND 150 ND ND ND ND 180 10 - ND 80 Conjugative Plasmid Conjugative Plasmid Yes - ND Yes ND Y ND - - Yes - ND ND - ND ND - Yes ND ND ND ND Yes Yes Yes ND ND Yes Yes - Yes Yes ¥ ¥ ) 26 Genetic Location Genetic Location P C P IncA/C P P P C (IS C IncP; IncFIB; IncFIC; IncA/C C IncI1, IncFIB, IncFII P C ND ND C P ND P ND ND pAOUC-AA14 P P ND ND pSLH06 P C pK318-1; pE77-1; pS24-1 P

§ § Give Brandenburg Paratyphi B Typhimurium Typhimurium (A) (A) (A) (A) (A) K. pneumoniae E. aerogenes K. pneumoniae stuartii P. K. pneumoniae; E. coli S. enterica S. marcescens A. baumannii aeruginosa P. S. marcescens E. coli E. coli E. coli E. coli E. coli K. pneumoniae E. coli; C. freundii K. pneumoniae K. pneumoniae K. pneumoniae S. enterica S. enterica A. caviae Salmonella E. cloacae K. pneumoniae K. pneumoniae K. pneumoniae E. coli E. coli E. coli; K. pneumoniae; S. marcescens E. coli; K. pneumoniae Bacterial Species Bacterial Species 1999- 2000 1993- 1995 1994 2012 2010- 2011 2000 2005- 2011 2004 1998- 2002 2009 2004 2006- 2007 2011 NA 2008 1991- 1995 NA 1999- 2002 1991- 1995 1998- 2002 2000 NA 2014 2011 2003- 2004 2006 2005- 2007 1991 1993 1990 1995 1993- 1995 Year* Year* Canada USA Belgium Greece Mexico Greece China USA Greece Uruguay Japan Tunisia Japan Spain Portugal Australia China USA Australia China Australia Turkey Italy Venezuela China Portugal Brazil France USA USA Greece Switzerland Location Location 7.6 7.6 7.6 8.2 8.2 pI pI Y11069.1 U20270 U92041 S82452.1 JX268741 Accession Number Accession Number Continued. Continued. SHV-6 SHV-7 SHV-8 SHV-9 SHV-12 Table S1. Table Gene S1. Table Gene bla bla bla bla bla

138 | Chapter 4A # # 263 266 122 60 249 255 256 219 257 165 123 258 259 47 49 261 50 259 51 262 264 21 156 265 71 Reference Reference 32 159 254 124 143 260 33 - D ; ; bla ; CMY-2 ; OXA-1 TEM-1 )-Ib ′ CMY-7 bla KPC-3 VIM-1 OXA-10 bla bla ; ; bla or bla qnr bla dfrA14 bla ; ; ; ; ; ; and/or ; aac(6”)-Ib-cr ; AmpC SHV-5 VEB-1 TEM-1 CTX-M-15 CTX-M-14 ACT-1 CMY-2 CTX-M-9 TEM-1b TEM-1 CTX-M-14 OXA-30 VIM-1 TEM-1 TEM-1 CTX-M-59 bla HA-1 bla Other Antibiotic Resistance Genes Other Antibiotic Resistance Genes bla bla ND bla ND bla bla - bla - - aadA1/aac(6 - ND bla bla bla ND bla qnr ND ND bla ND bla bla ND bla bla ND ND - ND ND Plasmid Size (Kb) Plasmid Size (Kb) Variable Variable - ND 130 Variable 120 ND - - ND - ND ND - ND ND - ND ND ND ND ND 150 ND ND ND ND 180 10 - ND 80 Conjugative Plasmid Conjugative Plasmid Yes - ND Yes ND Y ND - - Yes - ND ND - ND ND - Yes ND ND ND ND Yes Yes Yes ND ND Yes Yes - Yes Yes ¥ ¥ ) 26 Genetic Location Genetic Location P C P IncA/C P P P C (IS C IncP; IncFIB; IncFIC; IncA/C C IncI1, IncFIB, IncFII P C ND ND C P ND P ND ND pAOUC-AA14 P P ND ND pSLH06 P C pK318-1; pE77-1; pS24-1 P 4A

A review of SHV extended-spectrum β- lactamases: neglected yet ubiquitous | 139 # # 157 43 34 60 70 267 268 250 269 270 271 272 171 149 126 123 195 128 148 184 134 52 53 Reference Reference 41 206 132 116 118 273 http://www. lahey.org/ studies/ ; or

; , ; )-Ib-cr, )-Ib-cr, ′ TEM-1 DHA-1 )-Ib-cr CTX-M-15 ′ CTX-M-9 OXA-10 ESBL-like TEM-1 SHV-12 bla bla bla ; ; aac(6 bla bla bla bla qnrB2 qnrB bla ; ; ; ; ; ; and/or ; or )-Ib ′ CTX-M-14 SHV-12 VEB-1 CTX-M-15 TEM-24 TEM-1 CTX-M-14 VIM-1 CTX-M-15 TEM-1 CMY-2 OXA-48 TEM-1 CTX-M TEM-1 CTX-M-15 TEM-1 bla qnrB2 bla qnrS1; aacA4; dfrA14 bla variable Other Antibiotic Resistance Genes Other Antibiotic Resistance Genes bla - ND ND ND ND bla bla bla ND bla bla bla aac(6 qnrB2, aac(6 - - - bla - ND bla bla aadA2 bla bla bla bla ND ND - Plasmid Size (Kb) Plasmid Size (Kb) Variable Variable Variable ND ND ND ND ND ND 123 315 - 55 - Variable ND ND - - ND Variable ND - ND ND ND ND ND 170 ND >100 Conjugative Plasmid Conjugative Plasmid ND Yes ND ND ND Yes ND ND Yes Yes - Yes - Yes ND Yes - - ND ND No - Yes ND ND ND ND Yes ND Yes ¥ ¥ ) ) 26 903 ; IS 26 ) 26 Genetic Location Genetic Location P P ND ND ND P ND P pK7746-C1 (IS pB1004 (IncHI) C IncN C (IS IncHI2, IncF ND P C C IncI1 (IS P IncK/B C IncFIB, IncK, IncB/O, ColE ND ND P P P ND P (E)

;

(E)

§ § Agona and (E) (E)

E. coli;

(A) Senftenberg Typhimurium Bredeney Manhattan (E) (A) Typhimurium (E) (A) (A) (A) (A) K. oxytoca K. pneumoniae S. enterica S. enterica K. pneumoniae K. oxytoca; E. cloacae; K. pneumoniae E. coli; K. pneumoniae E. coli; K. pneumoniae; E. cloacae E. cloacae K. pneumoniae S. enterica mirabilis P. Klebsiella oxytoca aeruginosa P. E. coli; S. enterica Kentucky C. sakazakii; K. pneumoniae C. freundii E. coli Aeromonas spp E. coli S. enterica K. pneumoniae E. coli E. coli S. enterica Enterobacteriaceae K. pneumoniae; E. coli; E. cloacae; aerogenes; K. oxytoca; R. terrigena E. coli K. pneumoniae E. coli K. pneumoniae Bacterial Species Bacterial Species 2003- 2004 1999- 2000 1999- 2004 2011 2004- 2005 2004- 2005 2004- 2006 2002- 2005 1997 NA 2008 2005 NA 2000 2014 2004- 2005 2004 2009- 2010 NA 2002- 2003 2010 2011- 2012 2009 2006- 2007 2012 2009- 2013 2013- 2014 1994 1998 1996 Year* Year* Portugal Canada USA Saudi Arabia Malawi Thailand Mali France Korea Spain Korea Italy Japan Senegal Dominican Republic, Thailand Portugal Japan Croatia Portugal Japan UK Germany Netherlands Egypt Switzerland Croatia Spain Netherlands India France Location Location 7.6 ND 7.6 pI pI AF164577 AJ011428.2 AF072684.2 Accession Number Accession Number Continued. Continued. SHV-13 SHV-15 SHV-16 Table S1. Table Gene S1. Table Gene bla bla bla

140 | Chapter 4A # # 157 43 34 60 70 267 268 250 269 270 271 272 171 149 126 123 195 128 148 184 134 52 53 Reference Reference 41 206 132 116 118 273 http://www. lahey.org/ studies/ ; or

;

(E)

§ § Agona and (E) (E)

E. coli;

A review of SHV extended-spectrum β- lactamases: neglected yet ubiquitous | 141 # # 275 33 274 266 36 44 54 55 56 39 33 268 40 135 136 137 60 70 58 266 276 59 17 60 60 60 278 Reference Reference 253 43 41 57 277 253 A2 ; or ere ; , IMP TEM-1b SHV-12 TEM-1 SHV-27 DHA-1 TEM-1 A5; CTX-M-14 TEM-1 TEM-24 bla bla bla bla bla ; , bla ; ; ; bla dfr bla bla bla or ; ; , ; , ; TEM-116 DHA-1 CMY-2 TEM-1 CTX-M-15 CTX-M-15 OXA-1 CTX-M-15 CTX-M-15 AmpC SHV-7 CMY-2 SHV-1 CTX-M-2 TEM-116 GES-7 TEM-116 CTX-M-2 bla bla bla bla bla Other Antibiotic Resistance Genes Other Antibiotic Resistance Genes - bla ND bla bla bla bla bla ND ND ND ND ND bla - ND bla bla bla bla ND ND ND ND ND bla - ND - ND ND ND bla Plasmid Size (Kb) Plasmid Size (Kb) ND - ND ND ND 70 ND ND ND ND ND Variable ND ND ND ND ND ND ND - ND 80 ND 150 - 9.4 - >100 - ND ND ND 97-145 Conjugative Plasmid Conjugative Plasmid ND - Yes ND Yes Yes ND ND ND ND ND Yes ND Yes ND ND ND ND ND - ND Yes ND Yes - ND - Yes - ND ND ND ND ¥ ¥ 1) Ecp Genetic Location Genetic Location ND C P P P P (IS ND ND ND ND ND P ND P ND ND C ND ND C P P ND pCAZR001 C P C pOZ185 C ND ND ND IncA/C

§ § (A) (E) Mbandaka (A) K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae E. coli E. cloacae K. pneumoniae E. coli K. pneumoniae K. pneumoniae S. enterica K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae E. coli K. pneumoniae E. cloacae K. pneumoniae C. koseri; E. coli; K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae Bacterial Species Bacterial Species 1999 1997- 2001 2004- 2005 2000- 2002 2004- 2005 2006 2004- 2006 2009 2007- 2009 2010 2007- 2009 1999- 2000 2002 ND 2005- 2007 2006- 2007 1999 2005- 2007 2004 1997- 2001 2001- 2002 1994 1990 1996 1999 2003 2001 1998- 2000 2001 1999- 2000 1999- 2000 1999- 2000 NA Year* Year* Portugal Taiwan Malawi Japan Thailand Tunisia Mali Tunisia China China Japan Canada USA Taiwan Brazil Iran Portugal Brazil Brazil Taiwan India USA South Africa Japan Brazil USA Netherlands USA France Canada Canada Canada Brazil Location Location 7.8 ND 7.5 8.2 6.7 7.8 ND 7.6 7.6 7.6 7.6 8.2 pI pI AF132290 AF117747 AB023477 AF293345.1 AY661885 AY277255 AY036620 AY079099 AF535128 AF535129 AF535130 AF547625 Accession Number Accession Number Continued. Continued. SHV-18 SHV-23 SHV-24 SHV-27 SHV-30 SHV-31 SHV-34 SHV-38 SHV-40 SHV-41 SHV-42 SHV-45 Table S1. Table Gene bla bla bla bla bla S1. Table Gene bla bla bla bla bla bla bla

142 | Chapter 4A # # 275 33 274 266 36 44 54 55 56 39 33 268 40 135 136 137 60 70 58 266 276 59 17 60 60 60 278 Reference Reference 253 43 41 57 277 253 A2 ; or ere ; , IMP TEM-1b SHV-12 TEM-1 SHV-27 DHA-1 TEM-1 A5; CTX-M-14 TEM-1 TEM-24 bla bla bla bla bla ; , bla ; ; ; bla dfr bla bla bla or ; ; , ; , ; TEM-116 DHA-1 CMY-2 TEM-1 CTX-M-15 CTX-M-15 OXA-1 CTX-M-15 CTX-M-15 AmpC SHV-7 CMY-2 SHV-1 CTX-M-2 TEM-116 GES-7 TEM-116 CTX-M-2 bla bla bla bla bla Other Antibiotic Resistance Genes Other Antibiotic Resistance Genes - bla ND bla bla bla bla bla ND ND ND ND ND bla - ND bla bla bla bla ND ND ND ND ND bla - ND - ND ND ND bla Plasmid Size (Kb) Plasmid Size (Kb) ND - ND ND ND 70 ND ND ND ND ND Variable ND ND ND ND ND ND ND - ND 80 ND 150 - 9.4 - >100 - ND ND ND 97-145 Conjugative Plasmid Conjugative Plasmid ND - Yes ND Yes Yes ND ND ND ND ND Yes ND Yes ND ND ND ND ND - ND Yes ND Yes - ND - Yes - ND ND ND ND ¥ ¥ 1) Ecp Genetic Location Genetic Location ND C P P P P (IS ND ND ND ND ND P ND P ND ND C ND ND C P P ND pCAZR001 C P C pOZ185 C ND ND ND IncA/C 4A

A review of SHV extended-spectrum β- lactamases: neglected yet ubiquitous | 143 # # 33 61 279 36 33 34 37 38 62 62 63 71 64 34 34 65 65 65 66 67 68 69 45 Reference Reference 35 http://www. lahey.org/ studies/ TEM-1 Ib; ; SHV-28 bla (6’)- OXY-2 CTX-M-32 OXA SHV-5 bla or ; bla bla bla bla aac ; ; ; aac(3’)-IIa ; ; ; TEM-1 KPC-2 TEM-1 CTX-M-2 SHV-1 TEM-1 CTX-M-32 VIM-1 TEM-1 bla dhfrII; aadA1; catB2; bla Other Antibiotic Resistance Genes Other Antibiotic Resistance Genes - bla ND ND bla bla bla ND ND ND ND ND ND ND ND ND ND ND ND bla bla ND ND bla ND Plasmid Size (Kb) Plasmid Size (Kb) - 63.5/112/209 ND ND ND 70 - 40-60 ND ND ND ND ND ND ND ND ND ND 50 ND ND 100 ND 75 ND Conjugative Plasmid Conjugative Plasmid No ND ND Yes ND Yes No Yes ND ND Yes Yes ND ND ND ND ND ND Yes ND ND Yes ND Yes ND ¥ ¥ ) ) 26 26 Genetic Location Genetic Location P IncA/C ND P ND P ND pMTY512 ND ND pEC04 P ND ND ND ND ND ND pML2011 ND ND IncFII (IS pEc6-66 IncFIIA (IS ND

§ § K. pneumoniae K. pneumoniae E. coli E. cloacae K. pneumoniae K. oxytoca K. pneumoniae E. coli K. pneumoniae K. pneumoniae E. cloacae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae K. pneumoniae E. coli K. pneumoniae K. pneumoniae K. pneumoniae E. cloacae E. coli K. pneumoniae E. cloacae Bacterial Species Bacterial Species 2003 NA 2006- 2007 2003- 2004 2006 1998 NA 1998 2000- 2002 2000- 2002 2003- 2004 2003 2003 2003 2005 2005 2005 2003- 2004 2004 NA 1999 2009 2008 2009 NA Year* Year* Portugal Brazil China China Portugal New York Portugal Taiwan China China China Colombia Portugal Portugal Algeria Algeria Algeria Spain Tunisia USA Portugal Tunisia Italy Spain NA Location Location 8.2 ND 8.3 ND ND 7.6 8.2 8.2 7.6 7.6 7.8 7.2 ND 7,3/8,6 ND 7.6 8.6 ND ND ND pI pI AY210887 DQ054528 AY223863 DQ174304 DQ174306 DQ013287 DQ328802 NA NA AM941844 AM941845 AM941846 EU024485 EU274581 FJ194944 AM941847 GU932590 GU827715 HM559945 HG934764 Accession Number Accession Number Continued. Continued. SHV-46 SHV-55 SHV-57 SHV-64 SHV-66 SHV-70 SHV-86 SHV-90 SHV-91 SHV-98 SHV-99 SHV-100 SHV-102 SHV-104 SHV-105 SHV-106 SHV-128 SHV-129 SHV-134 SHV-183 Table S1. Table Gene bla bla bla bla bla bla bla bla bla bla bla bla bla bla S1. Table Gene bla bla bla bla bla bla *Isolation or first description; §A: animal sample; E: environmental sample *Isolation or first description; §A: animal sample; E: environmental and Insertion Sequences indicated; provided are when known plasmid name or Inc group ¥P: plasmid; C: Chromosome; reported; are references only representative SHV-12) SHV4, SHV-5, #For some enzymes (SHV2, SHV2a, SHV-3, NA: not available; ND: determined.

144 | Chapter 4A # # 33 61 279 36 33 34 37 38 62 62 63 71 64 34 34 65 65 65 66 67 68 69 45 Reference Reference 35 http://www. lahey.org/ studies/ TEM-1 Ib; ; SHV-28 bla (6’)- OXY-2 CTX-M-32 OXA SHV-5 bla or ; bla bla bla bla aac ; ; ; aac(3’)-IIa ; ; ; TEM-1 KPC-2 TEM-1 CTX-M-2 SHV-1 TEM-1 CTX-M-32 VIM-1 TEM-1 bla dhfrII; aadA1; catB2; bla Other Antibiotic Resistance Genes Other Antibiotic Resistance Genes - bla ND ND bla bla bla ND ND ND ND ND ND ND ND ND ND ND ND bla bla ND ND bla ND Plasmid Size (Kb) Plasmid Size (Kb) - 63.5/112/209 ND ND ND 70 - 40-60 ND ND ND ND ND ND ND ND ND ND 50 ND ND 100 ND 75 ND Conjugative Plasmid Conjugative Plasmid No ND ND Yes ND Yes No Yes ND ND Yes Yes ND ND ND ND ND ND Yes ND ND Yes ND Yes ND ¥ ¥ ) ) 26 26 Genetic Location Genetic Location P IncA/C ND P ND P ND pMTY512 ND ND pEC04 P ND ND ND ND ND ND pML2011 ND ND IncFII (IS pEc6-66 IncFIIA (IS ND 4A

A review of SHV extended-spectrum β- lactamases: neglected yet ubiquitous | 145 References

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A review of SHV extended-spectrum β- lactamases: neglected yet ubiquitous | 159 271 Carattoli, A. et al. Complete nucleotide sequence CMY-2, and DHA-1. Biomed Res. Int. 2015, 5, of the IncN plasmid pKOX105 encoding VIM-1, doi:10.1155/2015/568079 (2015). QnrS1 and SHV-12 proteins in Enterobacteria- 276 Feizabadi, M. M. et al. Genetic characterization ceae from Bolzano, Italy compared with IncN of ESBL producing strains of Klebsiella pneumo- plasmids encoding KPC enzymes in the USA. niae from Tehran hospitals. J. Infect. Dev. Ctries J. Antimicrob. Chemother. 65, 2070-2075, 4 (2010). doi:10.1093/jac/dkq269 (2010). 277 Dropa, M. et al. Emergence of Klebsiella pneu- 272 Uemura, S. et al. Acquisition of a transposon moniae carrying the novel extended-spectrum encoding extended-spectrum β-lactamase β-lactamase gene variants blaSHV-40, blaTEM- SHV-12 by Pseudomonas aeruginosa isolates 116 and the class 1 integron associated during the clinical course of a burn patient. blaGES-7 in Brazil. Clin. Microbiol. Infect. 16, Antimicrob. Agents Chemother. 54, 3956-3959, 630-632, doi:http://dx.doi.org/10.1111/j.1469- doi:10.1128/aac.00110-10 (2010). 0691.2009.02944.x (2010). 273 Alcala, L. et al. Wild Birds, Frequent Carriers of 278 Dhara, L. & Tripathi, A. Genetic and structural Extended-Spectrum beta-Lactamase (ESBL) Pro- insights into plasmid-mediated extended-spec- ducing Escherichia coli of CTX-M and SHV-12 trum β-lactamase activity of CTX-M and SHV Types. Microbial ecology, doi:10.1007/s00248- variants among pathogenic Enterobacteriaceae 015-0718-0 (2015). infecting Indian patients. Int. J. Antimi- 274 Hammami, S. et al. Characterization and crob. Agents 43, 518-526, doi:http://dx.doi. molecular epidemiology of extended spectrum org/10.1016/j.ijantimicag.2014.03.002 (2014). beta-lactamase producing Enterobacter cloacae 279 Jones, C. H. et al. Pyrosequencing using the sin- isolated from a Tunisian hospital. Microb. Drug gle-Nucleotide polymorphism protocol for rapid Res. 18, 59-65, doi:10.1089/mdr.2011.0074 determination of TEM- and SHV-type extend- (2011). ed-spectrum β-lactamases in clinical isolates and 275 Tang, H.-J., Ku, Y.-H., Lee, M.-F., Chuang, Y.-C. identification of the novelβ -lactamase genes & Yu, W.-L. In vitro activity of imipenem and blaSHV-48, blaSHV-105, and blaTEM-155. colistin against a carbapenem-resistant Klebsi- Antimicrob. Agents Chemother. 53, 977-986, ella pneumoniae isolate coproducing SHV-31, doi:10.1128/aac.01155-08 (2009).

160 | Chapter 4A

CHAPTER 4 B

Plasmid epidemiology of SHV-12- producing Escherichia coli from human and animal origin: X factor(s) of an emerging plasmid family

Apostolos Liakopoulos1, Jeanet van der Goot1, Alex Bossers2, Jonathan Betts3, Michael S. M. Brouwer1, Arie Kant1, Hilde Smith2, Daniela Ceccarelli1, Dik J. Mevius1, 4

1Department of Bacteriology and Epidemiology, Wageningen Bioveterinary Research, Lelystad, the Netherlands; 2Department of Infection Biology, Wageningen Bioveterinary Research, Lelystad, the Netherlands; 3Department of Bacteriology, School of Veterinary Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, United Kingdom; 4Department of Infectious Diseases and Immunology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, the Netherlands.

Submitted Abstract

The blaSHV-12 β-lactamase gene ranks amongst the most predominant extended-spectrum β-lactamases in Enterobacteriaceae disseminating within and between reservoirs, mostly via plasmid-mediated horizontal gene transfer. Yet, studies regarding the biology of plasmids encoding blaSHV-12 are very limited. In this study we revealed a shift in blaSHV-12 plasmid epidemiology from

IncI1α/γ to IncX3 in animal-related Escherichia coli isolates. Four representative blaSHV-12-encoding IncX3 plasmids were selected for genome sequencing and further genetic and functional characterization. We report here the first complete sequences of IncX3 plasmids of animal origin and show that IncX3 plasmids exhibit remarkable synteny in their backbone, while the major differences lie in their blaSHV-12-flanking region. Our findings indicate that plasmids of this subgroup are conjugative and highly stable, while they exert no fitness cost on their bacterial host. These favourable features might have contributed to the shift in plasmid prevalence amongst SHV-12-producing E. coli in the Netherlands, highlighting the epidemic potential of IncX3 plasmids.

164 | Chapter 4B Introduction

The blaSHV-12 gene ranks amongst the most predominant extended-spectrum β-lactamases within Enterobacteriaceae of diverse origins1. Plasmid-mediated horizontal gene transfer constitutes a key mechanism by which this gene disseminates among bacterial populations, therefore monitoring the spread of plasmids is essential to track the transmission of the blaSHV-12 gene between different reservoirs1. Several plasmid replicon types have been associated with the worldwide dissemination

1 of blaSHV-12, including A/C, colE, F, HI2, I1α/γ, K, L/M, N, P, R, as well as the recently emerging X3 .

The few available data on the prevalence of blaSHV-12-encoding plasmids in the Netherlands, report IncHI2 plasmids in human Salmonella enterica isolates, IncN and IncF plasmids in human Escherichia coli1,2, as well as IncK plasmids in E. coli from poultry1.

The emerging IncX plasmid family consists of narrow host-range, self-transferable, iteron-containing plasmids with class A theta replication and sizes ranging approximately between 30 and 50 kb3,4. IncX plasmids have a highly syntenic backbone; yet based on phylogenetic analysis they can be assigned to six distinct subgroups, namely IncX1 to IncX64-6. Although it has been demonstrated that IncX plasmids occur only infrequently among commensal and pathogenic E. coli isolates7, plasmids of this family encoding various resistance genes were recently described in Enterobacteriaceae originating from diverse sources and geographical areas4,5,8-12. Among this plasmid family, the IncX3 subgroup mediates the spread of genes encoding resistance for clini- 4B cally relevant first-line (fluoroquinolones and extended-spectrum cephalosporins) and last-resort (carbapenems) antibiotics. IncX3 plasmids have been reported to encode qnrB710, qnrS14,10,13-16,

12 10,17-20 21,22 23 19,20,24 25 26-29 16,27,30-34 blaCTX-M-3 , blaSHV-12 , blaKPC-2 , blaKPC-3 , blaNDM-1 , blaNDM-4 , blaNDM-5 , blaNDM-7 , 17 35 13-15,36,37 blaNDM-13 , blaNDM-17 and blaOXA-181 . Overall, these reports highlight the importance of this plasmid subgroup for the dissemination of antibiotic resistance genes within Enterobacteriaceae.

In an attempt to better understand the dynamics of blaSHV-12 diffusion in the Netherlands, also in light of the emerging role of IncX3 plasmids in their epidemiology, we investigated a collection of previously uncharacterized SHV-12-encoding E. coli isolates. We report here the plasmid epidemiology of SHV-12-producing E. coli from different reservoirs in the Netherlands, the first fully assembled and annotated sequence of three blaSHV-12 encoding-IncX3 plasmids of animal origin, and the genetic and functional characteristics of IncX3 plasmids from both human and animal origin.

Plasmid epidemiology of SHV-12-producing E. coli from human and animal origin: X factor(s) of an emerging plasmid family | 165 Results

Emergence of IncX3 plasmid encoding blaSHV-12

Among the 129 blaSHV-12 encoding E. coli isolates included in this study, 49.6% (n=64) was isolated from food-producing animals, 41.1% (n=53) from retail meat and 9.3% (n=12) from humans

(Table S1). Plasmid typing revealed that blaSHV-12 was encoded by nine different plasmid families: I1α/γ (n=86; 66.7%), X3 (n=21; 16.3%), X1 (n=6; 4.7%), F (n=5; 3.9%), B/O (n=4; 3.1%), K (n=4; 3.1%), N (n=1; 0.8%), colE (n=1; 0.8%) and multi-replicon F-X1 (n=1; 0.8%). A significant increase

(p = 0.041) in the prevalence of blaSHV-12-IncX3-harbouring isolates was documented between 2011 and 2014 among food-producing animals and retail meat. As a result blaSHV-12-IncX3-harbouring plasmids were among the predominant rep-types encoding blaSHV-12 in the Netherlands from 2012 onwards (Figure 1).

Four of these IncX3 plasmids (pEC-NRS18, pEC-393, pEC-125 and pEC-243) recovered from epidemiologically unrelated E. coli isolates of human (n=1; ST69) and animal origin (n=3; ST117, ST315 and ST410) were fully sequenced and functionally characterized in this study (Table 1).

Figure 1. Prevalence of IncI1α/γ and IncX3 plasmids encoding blaSHV-12 among E. coli between 2009 and 2014 in the Netherlands. Isolates were recovered from food-producing animals, retail meat and humans during national antimicrobial resistance monitoring programmes or national projects.

166 | Chapter 4B Table 1. IncX3 plasmids included in this study and their characteristics. Plasmid ID Year Host Host source Resistance gene(s) Size GC Open (bp) Content reading % frames

pEC-NRS18 2009 E. coli ST69/CC69 Human UTI blaSHV-12, blaTEM-1, qnrS1 48,250 46.4 74

pEC-393 2013 E. coli ST410/CC23 Turkey meat blaSHV-12 43,506 46.8 65

pEC-125 2014 E. coli ST117 Chicken faeces blaSHV-12, qnrS1 46,338 46.4 73

pEC-243 2014 E. coli ST315/CC38 Chicken faeces blaSHV-12, qnrS1 46,338 46.4 73

IncX3 plasmid backbone is highly syntenic and conserved Comparison between whole sequences of the four IncX3 plasmids from this study and twenty IncX3 plasmids available in GenBank (last accessed 10.05.2017) revealed a highly conserved plasmid backbone and their organization into a number of distinct clades (Figure 2). Plasmids of animal origin pEC-125 and pEC-243 were closely clustered together (MUMi distance 0.018) and grouped with them was the human-derived plasmid pEC-NRS18 that showed distance from 0.085 (pEC-125) to 0.093 (pEC-243). The animal-derived plasmid pEC-393 (turkey meat) clustered with a Klebsiella pneumoniae-encoded pIncX-SHV from human source in Italy (MUMi distance 0.002). IncX3 plasmids recovered in the Netherlands clustered closely with pOXA181 (China) and pKS22

(Switzerland) encoding blaOXA-181 and qnrS1, respectively, with MUMi distances varying from 0.140 (pOXA181 with pEC-125) to 0.179 (pKS22with pEC-393). 4B The four plasmids sequenced, assembled and fully annotated in this study had sizes varying from 43,506 (pEC-393) to 48,250 (pEC-NRS18) bp with an average GC content of 46.5% (Table 1). Similar to other IncX3 plasmids, they carried three putative origins of replication (oriV-α, oriV-β and oriV-γ), two origins of conjugal transfer (oriT-α and oriT-β), and approximately 6 iteron sequences. Nucleotide sequence analysis revealed 65 to 74 predicted open reading frames (Figure 3). All four plasmids encoded genes for replication (pir: replication initiation protein and bis: replication accessory protein), partitioning (parAB), entry exclusion (eex), maintenance (topB and hns), tran- scriptional activation (actX), and conjugal transfer (pilX1-11 and taxA-C). In addition, a mosaic and variable region containing resistance genes as well as intact and/or defective insertion sequences was identified in all four plasmids upstream of the partitioning gene parA (Figure 3). The variable region of pEC-NRS18 contained blaTEM-1 embedded in a Tn3 transposon, as well as genes blaSHV-12 and qnrS1 associated with the upstream presence of IS26 in the opposite and same orientation, respectively. Similarly, pEC-125 and pEC-243 contained both blaSHV-12 and qnrS1 genes, whereas pEC-393 encoded only blaSHV-12 associated to IS26 (Figure 3).

Plasmid epidemiology of SHV-12-producing E. coli from human and animal origin: X factor(s) of an emerging plasmid family | 167 Figure 2. BioNJ MUMi distances phylogram of IncX3 plasmids. The plasmid sequences obtained here and the ones available in GenBank database (last accessed 10.05.2017) were compared pair-wise and maximum unique matches converted to MUMi distances were hierarchically clustered and displayed as a phylogram using the BioNJ algorithm. GenBank accession number, country and source of isolation, as well as their antibiotic resistant gene content are indicated. NL: Netherlands, CN: China, CH: Switzerland, IT: Italy, US: United States, BR: Brazil, NG: Nigeria, FR: France, CA: Canada, IN: India and AMR: antimicrobial resistance.

168 | Chapter 4B 4B

Figure 3. Linear comparison in scale of IncX3 plasmids. The open reading frames identified in each sequence are represented with arrows, with the arrowhead indicating the direction of transcription. Their involvement in replication, partitioning, transfer, or antibiotic resistance, their association to mobile genetic elements, as well as other known or unknown function and pseudogenes are colour-coded. The areas shaded in grey indicate nucleotide identity in plasmid sequences.

Plasmid epidemiology of SHV-12-producing E. coli from human and animal origin: X factor(s) of an emerging plasmid family | 169 Figure 4. Conjugation frequencies of IncX3 plasmids. The reported values represent the average of three independent solid mating experiments (30 ºC and 37 ºC) and the error bars the 95% confidence interval for the ratio.

IncX3 plasmids transfer at high frequency The conjugation frequency of the four IncX3 plasmids was determined and relevant results are shown in Figure 4. Transfer rates differed between solid mating assays at different temperatures. Frequencies ranged from 6.36 X 10-6 (pEC-393) to 7.16 X 10-5 (pEC-243) at 30 ºC, and from 1.33 X 10-6 (pEC-393) to 1.46 X 10-4 (pEC-NRS18) at 37 ºC. Pairwise comparison of single plasmids at 30 ºC and 37 ºC indicated lower conjugation frequencies at 37 ºC for animal-originated plasmids (pEC-125, pEC-243 and pEC-393), with a significantly lower frequency documented for pEC-393 (p = 0.009). The human-originated plasmid pEC-NRS18 showed a slightly higher but not significant frequency of transfer at 37 ºC.

IncX3 plasmids exert no fitness cost and are highly stable The cost of IncX3 plasmid presence on the host cell fitness was assessed in the absence and in the presence of cefotaxime by comparing the exponential growth rate of E. coli DH10b with and without plasmid (Figure 5). The exponential growth rates of E. coli DH10b harbouring each of the IncX3 plasmids varied from 0.91 (95% CI 0.84-0.99; DH10b::pEC-125) to 1.22 (95% CI 1.13- 1.32; DH10b::pEC-NRS18) in the absence of antibiotic selective pressure, and from 1.48 (95% CI 1.14-1.83; DH10b::pEC-NRS18) to 1.94 (95% CI 1.77-2.12; DH10b::pEC-243) in the presence of cefotaxime. In the absence of cefotaxime, DH10b::pEC-125 showed significantly lower p( = 0.04) and DH10b::pEC-NRS18 significantly higher (p < 0.001) exponential growth rates compared

170 | Chapter 4B to the E. coli DH10b control strain. Pairwise comparison of the relative growth rates for E. coli DH10b harbouring each of the IncX3 plasmids in the absence and presence cefotaxime indicated significantly higher rates for every plasmid-harbouring strain in the presence of selective pressure (p<0.001) except for DH10b::pEC-NRS18.

E. coli DH10b transformed strains were propagated without positive antibiotic selective pressure. After approximately 180 generations of growth, the percentage of plasmid-harbouring cells in each population (for all four plasmids singularly) was determined as a measure of plasmid stability. All plasmids exhibited very high stability, ranging from 99.9% (95% CI 99.98-99.99) (pEC-NRS18) to 100% (95% CI 99.9-100) (pEC-393, pEC-125 and pEC-243) plasmid-harbouring cells per generation.

Figure 5. Relative exponential growth rates of IncX3-harbouring plasmid E. coli DH10b strains. All growth rates are set relative to plasmid-free E. coli DH10b. The reported values represent the average of three independent experiments and the error bars represent the 95% confidence interval for the ratio.

IncX3 plasmids do not contribute to bacterial pathogenicity The Galleria mellonella in vivo infection model was employed to evaluate the impact of harbouring

7 an IncX3 plasmid on bacterial pathogenicity. The LD50 value after 24 h was determined to be 10 CFU/larvae and survival curves were compared between the isogenic control E. coli DH10b strain and DH10b transformed strains harbouring each of the IncX3 plasmids (Figure 6). All four transformed strains displayed comparable virulence to the control strain (mortality = 40-86 %), with no significant difference in the 96 h survival curves (Figure 6). In both control groups all larvae survived.

Plasmid epidemiology of SHV-12-producing E. coli from human and animal origin: X factor(s) of an emerging plasmid family | 171 Figure 6. Impact of harbouring an IncX3 plasmid on E. coli DH10b strain pathogenicity. Kaplan-Meier plot of G. mellonella survival after injection with 107 CFU/larva of plasmid-free and IncX3-harbouring plasmid (pEC-NRS18, pEC-393, pEC-125 and pEC-243) E. coli DH10b strain is shown. Experiments were performed in triplicate and the plot represents the combined (additive) data from all experiments. No larval death was observed in control larvae injected with an equivalent volume of PBS.

Discussion

Our results confirmed that IncI1α/γ plasmids are the major facilitators of the blaSHV-12 diffusion in E. coli 1,38 of human and animal origin and mirrored the global plasmid repertoire associated with blaSHV-12 . However, a gradual decrease in the prevalence of IncI1α/γ and a parallel increase in IncX3 plasmids encoding blaSHV-12 was documented in animal-related E. coli, suggesting a shift in plasmid 10,17-20 epidemiology. The IncX3 plasmid subgroup was only incidentally associated with blaSHV-12 and/ or qnrS14,10,13,15,16,39 among clinically recovered E. coli isolates, and very recently it was identified among poultry isolates in Germany40. IncX3 plasmids have been documented in other Enterobac- teriaceae worldwide in association with multi-resistance, including to carbapenems19,21,23,25,27,35,37. Nevertheless, no association between IncX3 plasmids and other resistance genes (apart from blaSHV-12, blaTEM-1 and qnrS1) in Enterobacteriaceae was found in the Netherlands (data not shown).

The high degree of synteny and conservation in the backbone of IncX3 plasmids among E.coli isolates of both human and animal origin reflects the ecological success of this plasmid subgroup4. In addition to encoding genes essential for their maintenance and dissemination, IncX3 plasmids contained a variable region encoding resistance to clinically important antimicrobial agents (fluoroquinolones and/ or extended spectrum cephalosporins). Our findings confirm the potential of this subgroup for accu-

172 | Chapter 4B mulation of resistance genes via IS-mediated transposition, with the likely consequence of limiting effective treatment options for potential human infections10,16,17,19,22,39. As previously described41, the presence of IS26 linked to blaSHV-12 and other co-linear genes originating from the chromosome of K. pneumoniae was documented within the IncX3 variable region, confirming the hypothesis of the

IS26-mediated mobilization of the blaSHV ancestor gene from the chromosome of K. pneumoniae.

IncX3 plasmids, as well as the archetypal R6K plasmid of the IncX family, have been investigated for their ability of conjugative transfer16,17,19,20,39,42. We documented the interesting temperature- dependent conjugal transfer of animal-related IncX3 plasmids with conjugation frequencies higher at low temperatures, as already observed for plasmids belonging to IncHI1 family43. This finding suggests that IncX3 plasmids might transfer more efficiently in the environment than in the animal gut. Similarly to other conjugative plasmids, analysis of IncX3 plasmids revealed the presence of a gene encoding a H-NS-like protein4,43-47. Several studies demonstrated the inhibitory role of H-NS-like proteins on gene thermoregulation owing to their ability to polymerize along and bridge adjacent DNA regions at 37 ºC and the derepression of H-NS-regulated genes at lower tempe- ratures43,44,48. The involvement of these proteins in the temperature-dependent conjugal transfer of IncHI1 plasmids suggests a similar role in conjugal transfer of IncX3 plasmids, that can only be speculated here. The reported ability of IncX3 plasmids to replicate and be stably maintained in α-, β- and γ-Proteobacteria49, in combination with the higher conjugation frequency at low temperature, underscore a potential environmental reservoir of this plasmid subgroup. 4B

In contrast with studies describing that plasmids impose fitness cost on their bacterial hosts50, growth kinetics obtained over 24 h showed no evidence of fitness cost exercised by the majority of IncX3 plasmids tested here on the bacterial E. coli host. It has been demonstrated that H-NS- like proteins are able to silence newly introduced foreign sequences (including plasmids), based on increased adenine and thymine (AT) content in comparison with the chromosome47,51,52. Taking into consideration the high AT content of the IncX3 plasmids, we postulate that the presence of the H-NS gene on these plasmids allows them to invade bacterial hosts with a minimal impact on their fitness, ensuring the future competitiveness of the new plasmid-host combination even without the presence of antibiotic selective pressure. The significant IncX3 plasmid-mediated fitness enhancement of E. coli under antibiotic selective pressure, highlights the ecological advantage and subsequent successful proliferation of these plasmids in antibiotic-rich reservoirs.

All IncX3 plasmids encoded the widespread partitioning system ParAB ensuring the correct inheritance of these plasmids to the daughter cells53. The observed high stability of IncX3 plasmids is potentially due to their high conjugation frequency and absence of a fitness burden, as well as to

Plasmid epidemiology of SHV-12-producing E. coli from human and animal origin: X factor(s) of an emerging plasmid family | 173 a low rate of segregational loss.

Our data show that IncX3 plasmids encode a type IV secretion system (T4SS), typically used for the exchange of genetic material within bacteria and the translocation of virulent effector proteins into eukaryotic host cells54. Yet, we did not observe the alteration of the virulence potential of IncX3-carrying DH10b E. coli in the in vivo G. mellonella infection model, suggesting that T4SS does not play a role in the virulence of E. coli, at least in this invertebrate model, conversely from other Gram-negative pathogens54.

In conclusion, we report the first genetic characterisation of IncX3 plasmids of animal origin, as well as the first functional analysis of human- and animal-related plasmids of this subgroup, including their conjugation frequencies, stability, fitness cost and virulence potential. IncX3 plasmids are highly conserved, syntenic, conjugative and highly stable, while they exert no fitness cost on their bacterial host, independent of their origin. Although clonal expansion of E. coli strains could also play a role as suggested by the finding ofE. coli from the same clonal complex and carrying the same IncX3 plasmid (data not shown), the favourable plasmid functional features potentially contributed to the shifting plasmid epidemiology amongst SHV-12-producing E. coli in the Nether- lands, highlighting the epidemic potential of this plasmid subgroup.

Materials and Methods

Bacterial strains, transformants and plasmids

A total of 129 non duplicate blaSHV-12 encoding E. coli, consecutively recovered during national antimicrobial resistance monitoring programmes or national projects between 2009 and 2014 were included in the study. Identification of the isolates was performed by MALDI-TOF Mass spectrometry (Brucker, Coventry, UK). Genes conferring an ESC-resistant phenotype were sought by

55 microarray analysis followed by PCR amplification and sequencing . Plasmid location of blaSHV-12 was determined using a transformation-based approach. Briefly, plasmids encoding blaSHV-12 were isolated from the parental strain using a miniprep method and transformed into E. coli DH10b cells (Invitrogen, Van Allen Way, CA USA) by electroporation under the following conditions: 1.25 kV/ cm, 200 Ω, 25 μFar, as previously described55. Transformants were selected on Luria-Bertani (LB) agar plates supplemented with cefotaxime (1 mg/L) and confirmed for the presence of blaSHV-12 gene. Plasmid typing was confirmed by PCR-based replicon typing (PBRT KIT, DIATHEVA, Fano, Italy). Host E. coli sequence type were assigned by MLST based on the allelic profiles of seven housekeeping genes (adk, fumC, gyrB, icd, mdh, purA and recA)56.

174 | Chapter 4B Plasmid sequencing, assembly and analysis

Four IncX3 plasmids encoding blaSHV-12 were selected for further analysis based on their source of isolation and their relevant characteristics, as specified in Table 1. Plasmid DNA from transformants was isolated using the QIAfilter Plasmid Midi Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s recommendations. Deep sequencing of the plasmid genomes was performed using 300-bp paired-end sequencing libraries (Nextera TAG-mentation sequencing kits [Epicen- tre]) on an Illumina MiSeq sequencer. High-quality filtered reads were subsequently assembledde novo using SPAdes algorithm (SPAdes version 3.7.1) for Illumina-derived reads and then manually curated to close the gaps. Putative open reading frames (ORFs) were identified by RAST version 2.0 and manually curated when necessary57. BLASTP analyses of the putative ORFs against the NCBI nonredundant proteins (NR) database, Pfam, and Interpro scan were used to assess their putative functions by identification of structural features and motifs58,59. ResFinder (version 2.1), PlasmidFinder (version 1.3) and ISfinder were used to determine the presence of resistance genes, replicon types and insertion sequences, respectively60-62. Plasmid sequences were hierarchically clustered and displayed as a phenogram using the BioNJ algorithm, where the underlying distance matrix was calculated from the pairwise non-overlapping maximal unique matches (MUMs) using Nucmer version 3.0763,64. Relative pairwise distances were obtained by dividing the pairwise MUMs’ sum by the average genome size of the two paired genomes (MUMi genomic distance)65. BioNJ trees were generated from the MUMi distance matrix using SplitsTree466. 4B Mating assays Plasmid conjugation was assessed in solid mating assays at 30 ºC and 37 ºC conducted in triplicate. Chloramphenicol resistant (chlorR) E. coli MG1655::yfp was used as a recipient strain in 1:1 ratio with donor DH10b transformed strains carrying the different IncX3 plasmids, as previously described67. Overnight cultures of recipient and donor strains in mid-exponential phase were co-incubated (100 μl each) onto sterile nitrocellulose filters of 0.45 μm pore size (Schleicher and Schuell GmbH, Dassel, Germany) for 4h at 30 ºC and 37 ºC. Transconjugants were selected on LB agar supplemented with chloramphenicol (32 mg/L) and cefotaxime (1 mg/L). Positive transconjugants were confirmed by PCR amplification for the resistance and yfp genes. Conjugation frequency was calculated as the number of transconjugants per donor cell. For statistical analysis, conjugation frequencies were transformed to log10 values, and a p value <0.05 was considered to be statistically significant.

Plasmid epidemiology of SHV-12-producing E. coli from human and animal origin: X factor(s) of an emerging plasmid family | 175 Fitness cost assays Liquid cultures of DH10b transformed strains carrying different IncX3 plasmids were incubated overnight in 3 mL LB medium at 37ºC with 180 rpm shaking. Cultures were then diluted 100-fold into 3 mL of fresh pre-warmed LB medium with and without antibiotic (1 mg/L of cefotaxime) and incubated under the same conditions until mid-exponential phase (OD600 of ≈ 0.5). 200 µL of each culture were loaded in triplicate in wells of a 100-well honeycomb plate and incubated at 37°C with shaking for 24 h . Growth rates were obtained by measuring optical density at 600 nm every 30 min by using a Bioscreen C Reader (Oy Growth Curves, Helsinki, Finland). Assays were performed in triplicate. Relative growth rates were calculated by dividing the generation time of each DH10b transformed strain by the generation time of the wild-type DH10b strain which was included in each individual assay68. Growth rates between strains were compared using the Wil- coxon rank sum test with a Bonferroni adjustment for multiple comparisons. All statistical analysis were performed in R studio (version 1.0.143)69.

Stability assays E. coli DH10b transformants carrying different IncX3 plasmids were propagated in antibiotic-free LB medium at 37 ºC with 180 rpm shaking for 10 days (~180 generations). Cultures of each strain were daily diluted 1000-fold into 3 mL of fresh pre-warmed LB medium without antibiotics. On day 10, cultures were plated onto antibiotic-free LB agar and 100 randomly chosen colonies of each evolved line were replica-plated onto antibiotic-free and antibiotic-containing (1 mg/L of cefotaxime) LB agar plates. Plasmid presence was confirmed by colony PCR targeting the taxC gene of the IncX3 plasmids4. Colony growth on antibiotic-free but not on antibiotic-containing plates indicated the proportion of bacteria that lost the plasmid. Assays were performed in triplicate. The chance of E. coli DH10b keeping the plasmid was estimated for every plasmid using @Risk 6.3.1 (Palisade Corporation, Newfield, NY, USA), and the proportions of plasmid-harbouring colonies for each plasmid were compared.

Galleria mellonella survival assays G. mellonella caterpillars in the final-instar larval stage were obtained in bulk from Livefood UK Limited (Rooks Bridge, Somerset, United Kingdom) and stored at 15°C in the dark on wood shavings prior to use. Ten randomly chosen larvae weighing between 250 mg and 350 mg were employed for each group of an experiment. Strains included in the assay were grown overnight in LB broth and washed twice in sterile phosphate-buffered saline (PBS). The optimal bacterial inoculum was determined by injecting 10 larvae with 10 μl of bacterial suspensions containing 104 to 107 CFU/larva of organism in PBS. Bacterial inoculum concentration was determined by viable bacterial count on LB agar identifying the inoculum which killed 50% of larvae after 24 hours incubation

176 | Chapter 4B at 37°C (LD50). The optimal inoculum was then injected into the hemocoels of the caterpillars via a left proleg using 25-μl Hamilton syringes (Cole-Parmer, London, United Kingdom). Following injection, larvae were incubated in petri dishes lined with filter paper at 37°C for 96 h and scored for survival by 2 independent observers daily. Larvae were considered dead when they displayed no movement in response to touch. Two control groups were used per experiment, including larvae that were inoculated with PBS to control for any lethal effects of the injection process and larvae that received no injection. All G. mellonella survival assays were performed in triplicate using different batches of larvae. Survival curves were plotted using the Kaplan-Meier method and differences in survival were calculated by the log-rank test using R studio (version 1.0.143)69.

Accession codes: The reported plasmid sequences are deposited in GenBank under the following accession numbers: KX618696 (pEC-NRS18), KX618697 (pEC-393), KX618698 (pEC-243) and KX618703 (pEC-125).

Acknowledgements

We thank Nadine Handel and Benno ter Kuile for donation of the recipient strain E. coli MG1655::yfp and Frank Harders for technical assistance in plasmid genome sequencing. 4B Funding

This work was supported by the Dutch Ministry of Economic Affairs (BO-22.04-008-001).

Plasmid epidemiology of SHV-12-producing E. coli from human and animal origin: X factor(s) of an emerging plasmid family | 177 Supporting material

Table S1. blaSHV-12-encoding E. coli isolates analysed in this study. Inc/rep-type Date of Animal Other Strain ID Origin ESBL gene(s)* of bla - en- isolation Species genes SHV-12 coding plasmid

1954014 2009 Livestock pig blaSHV-12 blaTEM-1b IncF

35474 2009 Livestock poultry blaSHV-12 IncF

36289 2009 Livestock poultry blaSHV-12 IncI1α/γ

36278 2009 Livestock poultry blaSHV-12 IncI1α/γ

513768 2009 Livestock poultry blaSHV-12 IncI1α/γ

37318 2009 Livestock poultry blaSHV-12 IncI1α/γ

35078 2009 Livestock poultry blaSHV-12 IncI1α/γ

37881 2009 Livestock poultry blaCTX-M-1 (IncI1α/γ), blaCMY-2 (IncK), IncX3

blaSHV-12

35659 2009 Livestock poultry blaCTX-M-1 (IncN), blaSHV-12 IncN, IncI1α/γ

36498 2010 Livestock poultry blaSHV-12 blaTEM-1b IncI1α/γ

36700 2010 Livestock poultry blaSHV-12 IncI1α/γ

36809 2010 Livestock poultry blaSHV-12 IncI1α/γ

66191451 2010 Livestock poultry blaSHV-12 IncI1α/γ

35568 2010 Livestock poultry blaSHV-12 IncI1α/γ

636942 2010 Livestock poultry blaSHV-12 IncI1α/γ

1026302 2010 Livestock poultry blaSHV-12 IncI1α/γ

1025601 2010 Livestock poultry blaSHV-12 IncI1α/γ

55833907 2011 Livestock pig blaSHV-12 IncF

55927588 2011 Livestock pig blaSHV-12 IncI1α/γ

55927758 2011 Livestock pig blaSHV-12 IncI1α/γ

37156 2011 Livestock poultry blaSHV-12 IncI1α/γ

36239 2011 Livestock poultry blaSHV-12 IncI1α/γ

55727422 2011 Livestock cattle blaSHV-12 IncI1α/γ

884 2011 Livestock poultry blaSHV-12 IncI1α/γ

1105 2011 Livestock poultry blaSHV-12 IncI1α/γ

1109 2011 Livestock poultry blaSHV-12 IncI1α/γ

984 2011 Livestock poultry blaSHV-12 IncI1α/γ

65268442 2012 Livestock pig blaSHV-12 IncI1α/γ

36788 2012 Livestock poultry blaSHV-12 IncI1α/γ

29062012-02 2012 Livestock poultry blaSHV-12 IncI1α/γ

55580200139 2012 Livestock poultry blaSHV-12, blaTEM-52c IncK

65094754 2012 Livestock cattle blaSHV-12 IncI1α/γ

36458 2013 Livestock poultry blaSHV-12 IncI1α/γ

178 | Chapter 4B Table S1. Continued. Inc/rep-type Date of Animal Other Strain ID Origin ESBL gene(s)* of bla - en- isolation Species genes SHV-12 coding plasmid

35658 2013 Livestock poultry blaSHV-12 IncI1α/γ

1399001 2013 Livestock poultry blaSHV-12 IncX3

859 2014 Livestock poultry blaSHV-12 IncX3

219 2014 Livestock poultry blaSHV-12 IncX3

1041 2014 Livestock poultry blaSHV-12 IncI1α/γ

900 2014 Livestock poultry blaSHV-12 blaTEM-1b IncI1α/γ

570 2014 Livestock poultry blaSHV-12 IncX3

500 2014 Livestock poultry blaSHV-12 blaTEM-1 IncB/O

374 2014 Livestock poultry blaSHV-12 IncI1α/γ

287 2014 Livestock poultry blaSHV-12 IncX3

73 2014 Livestock poultry blaSHV-12 IncX3

1424 2014 Livestock poultry blaSHV-12 IncX3

71 2014 Livestock poultry blaSHV-12 IncI1α/γ

828 2014 Livestock poultry blaSHV-12 IncX1

1003 2014 Livestock poultry blaSHV-12 IncI1α/γ

11 2014 Livestock poultry blaSHV-12 IncI1α/γ

240 2014 Livestock poultry blaSHV-12 IncF-X1

386 2014 Livestock poultry blaSHV-12 IncX3 990 2014 Livestock poultry bla IncI1 / SHV-12 α γ 4B

1341 2014 Livestock poultry blaSHV-12 IncI1α/γ

1420 2014 Livestock poultry blaSHV-12 IncX3

118 2014 Livestock poultry blaSHV-12 IncI1α/γ

139 2014 Livestock poultry blaSHV-12 IncX3

864 2014 Livestock poultry blaSHV-12 IncX1

1206 2014 Livestock poultry blaSHV-12 IncI1α/γ

876 2014 Livestock poultry blaSHV-12 IncI1α/γ

20 2014 Livestock poultry blaSHV-12 IncX1

229 2014 Livestock poultry blaSHV-12 IncX3

1096 2014 Livestock poultry blaSHV-12 blaTEM-1b IncF

1433 2014 Livestock pig blaSHV-12 IncX3

1116 2014 Livestock cattle blaSHV-12 IncI1α/γ

69438407 2012 Meat poultry blaCTX-M-1 (IncI1α/γ), blaSHV-12 IncX3

69606962 2012 Meat poultry blaSHV-12 IncX3

76495084 2012 Meat cattle blaSHV-12 IncI1α/γ

76495084 02 2012 Meat cattle blaSHV-12 IncI1α/γ

69843204 2012 Meat poultry blaSHV-12 IncI1α/γ

69210023 2012 Meat poultry blaSHV-12 IncK

Plasmid epidemiology of SHV-12-producing E. coli from human and animal origin: X factor(s) of an emerging plasmid family | 179 Table S1. Continued. Inc/rep-type Date of Animal Other Strain ID Origin ESBL gene(s)* of bla - en- isolation Species genes SHV-12 coding plasmid

69843409 2012 Meat poultry blaSHV-12 IncI1α/γ

69438105 2012 Meat poultry blaSHV-12 IncX3

69585604 2012 Meat poultry blaSHV-12 IncI1α/γ

69064655 2012 Meat poultry blaSHV-12 IncI1α/γ

69770576 2012 Meat poultry blaSHV-12, blaTEM-52c IncI1α/γ

69477895 2012 Meat poultry blaSHV-12, blaTEM-52c IncI1α/γ

69927807 2013 Meat poultry blaCMY-2 (IncK), blaSHV-12 IncB/O

698975250004 2013 Meat poultry blaSHV-12 colE

699561810004 2013 Meat poultry blaSHV-12 IncI1α/γ

699819760004 2013 Meat poultry blaSHV-12 IncI1α/γ

69986056 2013 Meat poultry blaSHV-12 IncI1α/γ

693784120004 2013 Meat poultry blaSHV-12 IncI1α/γ

693785440004 2013 Meat poultry blaSHV-12 IncI1α/γ

693562060004 2013 Meat poultry blaSHV-12 IncI1α/γ

699898610004 2013 Meat poultry blaSHV-12 IncI1α/γ

699229530004 2013 Meat poultry blaSHV-12 IncI1α/γ

698980410004 2013 Meat poultry blaSHV-12 IncI1α/γ

69960219 2013 Meat poultry blaSHV-12 IncI1α/γ

69960316 2013 Meat poultry blaSHV-12 IncI1α/γ

69345093 2013 Meat pig blaSHV-12 blaTEM-1b IncI1α/γ

69799639 2013 Meat cattle blaSHV-12 blaTEM-1b IncI1α/γ

699799710004 2013 Meat cattle blaSHV-12 IncI1α/γ

694658380004 2013 Meat poultry blaSHV-12, blaSHV-2A, blaTEM-52c IncI1α/γ (IncI1α/γ)

699813050004 2013 Meat poultry blaSHV-12, blaTEM-52c (IncI1α/γ) IncI1α/γ

699900020004 2013 Meat poultry blaSHV-12 IncK

693503480004 2013 Meat poultry blaSHV-12 IncX1

699953490004 2013 Meat poultry blaSHV-12 IncX1

699081950004 2013 Meat poultry blaSHV-12 blaTEM-1b IncX1

699952170004 2013 Meat poultry blaSHV-12 IncX3

69960189 2013 Meat poultry blaSHV-12 IncX3

79158224 2014 Meat cattle blaSHV-12 IncF

M14P0112 2014 Meat poultry blaSHV-12 IncI1α/γ

79197637 2014 Meat poultry blaSHV-12 IncI1α/γ

79059943 2014 Meat poultry blaSHV-12 IncI1α/γ

79230383 2014 Meat poultry blaSHV-12 IncB/O

180 | Chapter 4B Table S1. Continued. Inc/rep-type Date of Animal Other Strain ID Origin ESBL gene(s)* of bla - en- isolation Species genes SHV-12 coding plasmid

79696536 2014 Meat poultry blaSHV-12 IncI1α/γ

79194778 2014 Meat pig blaSHV-12 IncI1α/γ

79195006 2014 Meat poultry blaSHV-12 IncI1α/γ

79156655 2014 Meat poultry blaSHV-12 IncI1α/γ

79207004 2014 Meat poultry blaSHV-12 IncI1α/γ

79626295 2014 Meat poultry blaSHV-12 IncB/O

79207101 2014 Meat poultry blaSHV-12 IncI1α/γ

79352292 2014 Meat poultry blaSHV-12 IncK

79771872 2014 Meat poultry blaSHV-12 IncX3

79445126 2014 Meat poultry blaSHV-12 IncI1α/γ

79042587 2014 Meat poultry blaSHV-12 IncX3

1190900169 2009 Human urine blaSHV-12 IncX3

1190900881 2009 Human urine blaSHV-12 IncI1α/γ

1190900890 2009 Human urine blaSHV-12 IncI1α/γ

306 2014 Human faeces blaSHV-12 IncI1α/γ

1.1 2014 Human faeces blaSHV-12 IncI1α/γ

1.58 2014 Human faeces blaSHV-12 IncI1α/γ

2.12 2014 Human faeces blaSHV-12 IncI1α/γ 2.25 2014 Human faeces bla IncI1α/γ SHV-12 4B 2.48 2014 Human faeces blaSHV-12 IncI1α/γ

2.49 2014 Human faeces blaSHV-12 IncI1α/γ

2.52 2014 Human faeces blaSHV-12 IncI1α/γ

1.71 2014 Human faeces blaSHV-12 IncN

*When known the inc/rep-type of the plasmid encoding ESBL genes (exculing the blaSHV-12) is given in parenthesis. Highlight in grey are indicated the 4 plasmids that were sequenced and functionally characterized.

Plasmid epidemiology of SHV-12-producing E. coli from human and animal origin: X factor(s) of an emerging plasmid family | 181 References

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Plasmid epidemiology of SHV-12-producing E. coli from human and animal origin: X factor(s) of an emerging plasmid family | 185

CHAPTER 5

General discussion

Enterobacteriaceae rank amongst the most significant causes of nosocomial and community acquired infections, the therapeutic treatment of which depends on β-lactams [predominantly extended spectrum cephalosporins (ESCs) and carbapenems] and fluoroquinolones1. However, rapidly emerging resistance to these compounds, especially to ESCs, is being reported worldwide as a natural evolutionary bacterial response to antimicrobial exposure2. According to the CDC assessment based on clinical impact, economic impact, incidence, 10-year projection of incidence, transmissibility, availability of effective antibiotics and barriers to prevention, ESC-resistant Ente- robacteriaceae have been identified as a serious health threat and subsequently as a substantial clinical and financial burden on health care system, patients and their families3.

Among the many risk factors associated with colonization or infection with ESC–resistant Entero- bacteriaceae4,5, previous antibiotic use has been consistently reported, especially with β-lactams6,7, combinations of β-lactams and β-lactamase inhibitors8, and fluoroquinolones8. In addition to their transmission from person-to-person in hospital, community, and household settings via human intestinal colonization9-11, the zoonotic and foodborne transmission of ESC–resistant Enterobacte- riaceae has been strongly suggested12. As a consequence, collection of data to inform decisions, efforts to reduce antibiotic abuse in people and animals, antibiotic stewardship, optimal use of newer diagnostics, better support for clinical and basic resistance-related research, as well as novel methods to foster new antibiotic development are interventions proposed to resolve this well-acknowledged health threat13. 5 The studies presented in this thesis used the available molecular microbiology techniques and the incorporation of human and animal components in a “One Health” approach, providing insights on emerging trends among ESC-resistant Enterobacteriaceae of human and animal origin, their cross-transmission within and between reservoirs, as well as the complexity of their “One Health” epidemiology in order to assess the role of animals as a relevant source of such resistant bacteria for humans.

Emerging trends in extended-spectrum cephalosporin resistance

In this thesis, the emergence of ESC-resistant S. Heidelberg isolates for the first time in a European country via the importation of poultry products was documented (Chapter 2a). Salmonellosis is a significant public health concern, accountable for 82,694 confirmed cases, 7,841 hospitalizations

General discussion | 189 and 59 reported deaths within European countries in 201314. While S. Heidelberg has been infrequently reported in European countries, including the Netherlands, this serotype is recognised to be among the most commonly identified serotypes from poultry meat and human salmonellosis in the United States (US) and Canada14-17. In contrast with other non-typhoidal Salmonella serotypes, which are usually associated with mild to moderate self-limiting infections, S. Heidelberg is associated with systemic infections and hospitalizations that require antimicrobial drug therapy18. For the treatment of these infections, ESC are the drugs of choice for children or for adults with fluoroquinolone contraindications19.

In addition to the S. Heidelberg variant with PFGE-type XbaI.0251 (JF6X01.0022) that is the most common S. Heidelberg reported nationally in the US, at least three more PFGE-types (XbaI.1966/ JF6X01.0326, XbaI.1968/JF6X01.0258 and XbaI.1970/JF6X01.0045) associated with a multistate outbreak in the US were identified among the newly introducedS. Heidelberg isolates in the Netherlands20. This outbreak of multidrug resistant S. Heidelberg infections was linked to a single poultry company and involved 634 case-patients of whom 200 were hospitalized, while no deaths were reported20, underscoring the epidemic potential of isolates belonging to these PFGE-types. Although no human infections linked to ESC-resistant S. Heidelberg contaminated imported products have been documented in the Netherlands until now, the majority of these isolates exhibited additional non-susceptibility to fluoroquinolones. This finding highlights the limited effective treatment options for potential human infections by these isolates. Hence, the emergence of extended spectrum cephalosporin- and/ or fluoroquinolone-resistant S. Heidelberg in the food chain requires for public heath vigilance.

Of concern is the occurrence in the Netherlands of the ESC-resistant S. Heidelberg XbaI.0251 that has been previously documented to be highly virulent in the Caenorhabditis elegans infection model21. Isolates assigned to this PFGE-type were found to carry an extended repertoire of virulence-associated determinants21. It has been shown that they carry VirB/D4 type IV secretion system plasmids, enhancing their potency for entrance and survival in both epithelial cells and macrophages, as well as determinants contributing to Salmonella pathogenicity, such as the lambda-like prophage Gifsy-2, the msgA gene and genes encoding adhesins and type III secretion system proteins21. However, since the presence of these determinants was not investigated here, no conclusive assumptions regarding the virulence potential of these S. Heidelberg strains circulating in the Netherlands can be made.

The chromosomal integration of extended-spectrum β-lactamase genes typically plasmid-encoded is regarded as a relatively uncommon event22,23. Nevertheless, we described the emergence of

190 | Chapter 5 Enterobacteriaceae with chromosomal integration of blaCTX-M genes among wild birds (Chapter 2b) and Dutch individuals (Chapter 3a). Our findings add to previous reports on incidental integration of these genes on the chromosome of several Enterobacteriaceae22,24-28 and underscore that this event is apparently not so uncommon. IncF plasmids have been mostly associated with the world-

29 wide dissemination of blaCTX-M genes among Enterobacteriaceae from human and animal sources . These plasmids have been also shown to participate frequently in extensive recombination events30. Such recombination events between IncF plasmids and the chromosome may have contributed to integration of the blaCTX-M gene region on the latter.

Given that plasmids must carry genes that are beneficial with a sufficient frequency to outweigh

31 the cost of their carriage , the chromosomal integration of blaCTX-M gene seems to be an effective evolutionary strategy amongst Enterobacteriaceae recovered from wild birds and Dutch individuals. The positive selective pressure for blaCTX-M genes is limited among these reservoirs due to the fact that wild birds do not naturally come into contact with antibiotics and the low antibiotic consumption in the Dutch general population32, respectively. Under the limited selective pressure, the more stable inheritance of the chromosome, and the higher plasmid-associated cost suggest that chromosomal location of blaCTX-M genes is favorable and could lead to the maintenance of ESC-resistant phenotype without further selective pressure33.

The emergence of IncX3 plasmids encoding blaSHV-12 was documented worldwide (Chapter 4a), including animal-related E. coli in the Netherlands (Chapter 4b). Apart from the blaSHV-12 gene, this recently emerging plasmid subgroup has been reported to encode a great repertoire of resis-

34 34-39 40 41,42 43 44-46 47 tance genes, namely qnrB7 , qnrS1 , blaCTX-M-3 , blaKPC-2 , blaKPC-3 , blaNDM-1 , blaNDM-4 , 5 48-51 38,49,52-56 57 58 35-37,59,60 blaNDM-5 , blaNDM-7 , blaNDM-13 , blaNDM-17 and blaOXA-181 , and to confer resistance to clinically relevant first-line (fluoroquinolones and extended-spectrum cephalosporins) and last- resort (carbapenems) antibiotics.

The genetic and functional characterisation of IncX3 plasmids from human and animal origin in this thesis indicates that these plasmids are conjugative, highly stable and they exert no apparent fitness cost on their bacterial host, as a result of encoding all the necessary genetic machinery for their replication, stability and conjugative transfer. These favourable features potentially contributed to IncX3 plasmids emergence in the Netherlands and worldwide, highlighting the epidemic potential of this plasmid subgroup. Based on the high-shared synteny among the backbones of IncX plasmids39, it is possible that all subgroups of the IncX family (IncX1-IncX6) share these favourable features. This could explain the recent emergence of plasmids of the IncX4 subgroup encoding colistin resistance genes, mcr-161 and mcr-262, conferring resistance to one of the last-resort anti-

General discussion | 191 biotics for the treatment of severe clinical infections caused by multidrug-resistant Gram-negative bacteria.

The aforementioned epidemiological shifts in ESC-resistance among Enterobacteriaceae of human and animal origin described in this thesis highlight the requirement for constant surveillance adopting a “One Health” approach.

Complex “One Health” epidemiology and potential transmission between reservoirs

Several factors have been shown to contribute to the diffusion of the ESC-resistant phenotype, namely the diversity of acquired extended-spectrum cephalosporinase genes63,64, their mobility among Enterobacteriaceae predominantly due to plasmid horizontal transfer65, epidemic bacterial clones66 and their transmission between and within different reservoirs67. The studies included in this thesis indicate clearly the attribution of these interlinked factors to the complexity of the epidemiology of ESC-resistant Enterobacteriaceae.

Among them, the diversity of acquired genes conferring ESC-resistance within each reservoir as seen especially in wild birds (Chapter 2b) and Dutch households with pre-school children (Chapter 3a). In addition, the association of an ESC-resistance gene within the same reservoir with either the chromosome and/or plasmids belonging to different (sub)types (Chapters 2a, 2b, 3a, 3b, 4a and 4b). Similar plasmids encoding the same ESC-resistance gene were found among isolates with different genotypes (Chapters 2a, 2b, 3b and 4b). Conversely, similar genotypes carried different plasmids and ESC-resistance gene combinations (Chapters 2a, 2b, 3a and 3b). Finally, the contribution of trade to the epidemiology of ESC-resistant Enterobacteriaceae was confirmed by the importation of ESC-resistant S. Heidelberg isolates in the Netherlands via Brazilian poultry meat (Chapters 2a), as well as the potential contribution of wild life (Chapters 2b). Even though clonal and/or plasmid transmission was documented within epidemiologically linked settings such as households with pre-school children and pig farms (Chapters 3a and 3b), the great diversity at gene, plasmid and strain level observed within these reservoirs confirms the dynamic and complex epidemiology of ESC-resistant Enterobacteriaceae.

Overall, our findings suggest that apart from establishing a harmonized and broad surveillance system based on the “One Health” approach, the integration of state-of-the-art molecular methods is essential to increase our understanding of the genetic diversity of ESC-resistant Ente- robacteriaceae between and within reservoirs and ultimately determine the attribution route of

192 | Chapter 5 these microorganisms in human infections.

Plasmid contribution to the diffusion of extended-spectrum cephalosporin resistance

Various epidemic clones (e.g., E. coli ST131) were infrequently documented in the studies included in this thesis, whereas a great genetic diversity among ESC-resistant Enterobacteriaceae not belonging to known epidemic clones was observed. This suggests that commensal Enterobacteriaceae act as the main reservoir of the ESC-resistant phenotype. On the contrary, a limited number of known epidemic plasmid types and/or subtypes (e.g., IncIα/γ and IncF) were found to facilitate the diffusion of the ESC-resistant phenotype, highlighting the importance of horizontal transfer of plasmid-encoded ESC-resistance genes as important at the origin of acquisition of resistance in Enterobacteriaceae.

Plasmids have been shown to encode the genetic machinery for their own efficient autonomous replication, maintenance and stable inheritance during bacterial cell division29,68,69. In addition, they promote the lateral transfer of the ESC-resistant phenotype among bacterial hosts through their conjugation in human70-72 and animal73 gut, as well as in the environment74,75. Recent studies pinpoint that plasmids may transfer frequently between strains, even over short timescales76,77. This recurrent plasmid transfer among bacterial strains with diverse genetic backgrounds indicates that the chain of gene transmission no longer simply depends on strain transmission and that plasmids play a prominent dissemination role. These findings undermine the value of strain typing and 5 impose the inclusion of plasmid typing (and subtyping) in the routine surveillance of ESC-resistant Enterobacteriaceae and other resistant phenotypes.

On the basis of the inclusion of plasmid typing (and subtyping) in epidemiological studies, guide- lines and interpretive criteria aiming at the analysis of epidemiologically linked isolates obtained within relatively short periods are suggested here: • Dissimilarity in ESC-resistance genes and linked insertion sequences (IS) encoded by the isolates, their genetic location [plasmid types (and subtypes) or chromosome] and the isolates (ST or PFGE-type) should be considered as indication of no host-to-host or source-to-host transmission of the ESC-resistant phenotype. • Similarity only in ESC-resistance genes (and IS) encoded by the isolates indicates possible host-to-host or source-to-host transmission of the ESC-resistant phenotype. • Similarity in ESC-resistance genes (and IS) encoded by the isolates and their genetic location

General discussion | 193 [plasmid types (and subtypes)] indicates host-to-host or source-to-host transmission of ESC- resistant phenotype through plasmid transfer. • imilarity in ESC-resistance genes (and IS) encoded by the isolates, their genetic location [plasmid types (and subtypes) or chromosome] and the isolates (ST or PFGE-type) indicates of host-to-host or source-to-host transmission of the ESC-resistant phenotype through bacterial dissemination. • To confirm an epidemiological link of either the isolated plasmids or bacterial STs or PFGE- types, whole genome sequencing is required.

Genomic epidemiology: the spearhead in the fight of ESC resistant Enterobacte- riaceae

Whole genome sequencing (WGS) provides maximal details for strain or plasmid identification and detects clinically and epidemiologically relevant phenotypes78, thus it is widely seen as the ultimate tool for epidemiological typing. Although WGS has already been proven to be highly able to trace, control and characterize the diffusion of ESC-resistant Enterobacteriaceae79-82, this technology currently faces three major challenges: speed, data analysis and interpretation, and cost78.

Given the mainly plasmid-driven epidemiology of ESC-resistant Enterobacteriaceae and that bacterial cells may contain multiple plasmids and/or a single plasmid may contain multiple replicons, there are limitations to the usage of the popular high-throughput sequencing technologies (e.g., Illumina) producing short (~100–300 bp) reads. Although strategies for plasmid reconstruction from short-read WGS data have been proposed83-85, complete plasmid assembly is infrequently possible86. Repetitive mobile elements often flanking ESC-resistance genes introduce assembly ambiguities and can subsequently hinder the complete assembly of plasmids and their association with these genes87.

On the contrary, long-read sequencing technologies (e.g., Pacific Biosciences, Oxford Nanopore) seems promising to generate accurate and complete plasmid assemblies76,88, allowing the deci- phering of plasmid epidemiology. However, these technologies are not accessible to everyone because of financial constraints89, highlighting that the isolation of individual plasmids of interest prior to plasmid (sub)typing and/or targeted sequencing is more feasible at the moment for routine epidemiological studies. Overall, the future increased usage of long-read sequencing will render the currently popular replicon90 and MOB91 plasmid-typing schemes obsolete, revolutionizing epidemiology of plasmid-encoded resistant phenotypes.

194 | Chapter 5 Conclusion

The omnipresence of ESC-resistant Enterobacteriaceae confirms that ‘‘resistance anywhere is resistance everywhere’’ and reinforces the ecological character of antibiotic resistance92. In addition, there are genetic, spatial and temporal evidences underscoring the link between ESC-resistant Enterobacteriaceae of human and non-human origin93,94. As a result, the implementation of a “One Health” approach in global and harmonized surveillance and reporting system is required in order to tackle antibiotic resistance threat efficiently. In such a surveillance framework, the case of ESC- resistant Enterobacteriaceae could constitute an index of diffusion of other resistant phenotypes to last-resort antibiotic, namely carbapenems and colistin, that have shown potential for spread within and between human and non-human reservoirs95,96.

Based on the reported prevalence of antibiotic resistance by this system, the enforcement of financial fines upon the countries or regions with high prevalence might result in the implementation of strategies (e.g., prophylactic vaccines and antibiotic stewardship) for the reduction of antibiotic- resistance prevalence. At the same time, funding relevant, strategic and targeted research for decreasing the morbidity and mortality of antibiotic-resistant bacterial infection is essential. In principal, the countries facing the antibiotic-resistant threat would be the main contributors of the financial support of the relative research as they would also be the main beneficiaries of the results. A sound surveillance system should integrate high-resolution sequence-based methods such as WGS, resulting in both tracing of host-to-host or source-to-host transmission of antibiotic-resistant bacteria and better understanding their metabolism and physiology. This would allow the development of novel antibacterial compounds against novel gene targets and critical cell functions 5 alternative to those currently targeted by the well-established antibiotics, and the design of more targeted, narrow-spectrum97,98, pathogen-specific antibiotics, as well as to the improvement of frontline diagnostics99,100.

General discussion | 195 References

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General discussion | 201

APPENDIX

Summary in Dutch Summary in English Acknowledgments About the author List of publications

Summary in Dutch

Enterobacteriaceae die breed-spectrum cefalosporinase produceren zijn bij mensen en dieren wereldwijd al tientallen jaren in opkomst.

Hoewel onderzoek heeft aangetoond dat de directe verspreiding tussen mensen en dieren zeldzaam is bestaan er wel gemeenschappelijke reservoirs voor cefalosporinase genen, plasmiden en sequentie-typen die suggereren dat verspreiding plaats vindt en die zorg wekken voor een mogelijke zoönotische bron van ESBL/AmpC producerende bacteriën voor mensen.

Dit proefschrift heeft als doel om de moleculaire relaties tussen breed-spectrum cefalosporine resistente Enterobacteriaceae van humane en dierlijke monsters te onderzoeken en de verspreiding en epidemiologie vanuit een “One Health” perspectief te analyseren.

Hoofdstuk 1 dient als een introductie en geeft achtergrond informatie over breed-spectrum cefalosporine resistente Enterobacteriaceae en hun complexe epidemiologie, en helpt om de studies die in dit proefschrift zijn gepresenteerd te begrijpen.

Hoofdstuk 2 behandelt het verschijnen van breed-spectrum cefalosporine resistente Salmonella enterica serotype Heidelberg isolaten en hun moleculaire eigenschappen. Deze recente verschijning wordt waarschijnlijk veroorzaakt door de import van dieren en pluimvee producten vanuit Brazilië. Momenteel zijn er in Nederland nog geen gevallen bekend van humane infecties door verspreiding vanuit deze dieren of producten (Hoofdstuk 2a). Verder wordt de rol beschreven die kelpmeeuwen op het zuidelijk halfrond mogelijk spelen in de verspreiding van breed-spectrum cefalosporine resistente S. Heidelberg en andere Enterobacteriaceae (Hoofdstuk 2b). A In hoofdstuk 3 wordt de verspreiding van Enterobacteriaceae onder mensen en tussen mens en dier bediscussieerd. Een cross-sectioneel onderzoek onder kleuters en hun ouders wordt gepresenteerd waarin de moleculaire eigenschappen van de gevonden breed-spectrum cefalosporine resistente Enterobacteriaceae is onderzocht en de frequentie wordt beschreven waarin identieke isolaten binnen een gezin voorkomen (Hoofdstuk 3a). In een longitudinale studie is de aanwezigheid en moleculaire diversiteit van breed-spectrum cefalosporine resistente E. coli beschreven van boeren en varkens binnen varkensboerderijen waarin ook verspreiding tussen boeren en hun varkens is vast gesteld (Hoofdstuk 3b).

Summary in Dutch | 205 In hoofdstuk 4 worden de SHV breed-spectrum β-lactamasen bediscussieerd. De mondiale epidemiologie van de Enterobacteriaceae die SHV produceren is in een literatuurstudie in hoofdstuk 4a beschreven waaruit de structurele aanwezigheid van deze breed-spectrum cefalosporinasen blijkt. Verder wordt de recente associatie tussen blashv-12 en IncX3 plasmiden beschreven uit E. coli isolaten uit Nederland samen met de genetisch en functionele eigenschappen van deze plasmiden die bijdragen aan de toenemende frequentie waarin blaSHV-12 wordt gevonden (Hoofstuk 4b).

In de algemene discussie (Hoofdstuk 5) worden de resultaten uit dit proefschrift bediscussieerd in relatie tot de epidemiologie van breed-spectrum cefalosporine resistente Enterobacteriaceae en de beschikbare internationale literatuur. De belangrijkste resultaten uit dit proefschrift geven, door gebruik te maken van moleculaire microbiologische technieken voor de analyse van humane en veterinaire monsters in een “One health” aanpak, een inzicht in opkomende trends in breed- spectrum cefalosporine resistente Enterobacteriaceae van humane en dierlijke oorsprong, de verspreiding in en tussen reservoirs en de complexiteit van hun “One Health” epidemiologie om te bepalen hoe belangrijk de bijdrage is van dieren in de verspreiding van resistente bacteriën naar de mens.

206 | Appendix

Summary in English

Extended-spectrum cephalosporinase (ESCase)-producing Enterobacteriaceae from human and animal origin have emerged worldwide during the last decades. Although studies documenting direct transmission between humans and animals are rare, the existence of shared reservoirs of extended-spectrum cephalosporinase genes, plasmids and/or STs suggests cross-transmissions and raises the concern of a possible zoonotic source of ESBL/AmpC-producers for humans. The aim of this thesis is to explore the molecular relatedness of extended-spectrum cephalosporin (ESC)-resistant Enterobacteriaceae of human and animal origin and assess their cross-transmission and epidemiology from a “One health” perspective.

Chapter 1, as an introduction provides the necessary background information of the extended- spectrum cephalosporinase-producing Enterobacteriaceae and their complex epidemiology, to offer a better understanding of the studies presented in this thesis.

Chapter 2 focuses on the emergence and the molecular characteristics of ESC-resistant Salmonella enterica serotype Heidelberg isolates in the Netherlands. Their recent emergence was attributed to food-producing animals and poultry products imported from Brazil, while no human infections linked to these contaminated animals and products have been yet documented in the Netherlands (Chapter 2a). In addition, the potential contribution of Kelp gulls of the southern hemisphere in the dissemination of ESC-resistant S. Heidelberg and other Enterobacteriaceae is presented (Chapter 2b).

In Chapter 4, SHV extended-spectrum β-lactamases are discussed. The global epidemiology of Enterobacteriaceae encoding SHV ESCases is reviewed in Chapter 4a, highlighting the ubiquity of these extended-spectrum cephalosporinases. Finally, the recent association of blaSHV-12 with IncX3 plasmids among E. coli isolates in the Netherlands, as well as the genetic and functional characteristics of these plasmids contributing to blaSHV-12 emergence are reported (Chapter 4b).

Summary in English | 209 In the general discussion (Chapter 5), the results of the thesis are presented and discussed in relation to the epidemiology of ESC-resistant Enterobacteriaceae and the available international literature. Major outcomes of the studies presented in this thesis, using the available molecular microbiology techniques and the incorporation of human and animal components in a “One Health” approach, provide insights on emerging trends among ESC-resistant Enterobacteriaceae of human and animal origin, their cross-transmission within and between reservoirs, as well as the complexity of their “One Health” epidemiology in order to assess the role of animals as a relevant source of such resistant bacteria for humans.

210 | Appendix

Acknowledgments

My adventurous PhD journey has come to its end and throughout this journey I have been blessed with the help and support from my family, friends and colleagues. Therefore, I would like to express my genuine gratefulness to everyone without whom the completion of this thesis would not have been feasible.

First of all, I would like to express my sincere thankfulness to my promoter. Dear Dik thank you for giving me the opportunity to fulfill my dream to obtain a PhD degree. Thank you for guiding me from my previous hospital-oriented to the more holistic “One health”-oriented aspect of the antibiotic resistance problem. I am also thankful for your sharp critical view and for teaching me to see the bigger picture instead of sticking exclusively to the details. I thank you so much for your contribution in my scientific maturity and your faith in me! I extend my gratitude to my co- promoters Hilde and Mike for accepting this role, their continuous support and valuable scientific suggestions.

I would like to express my gratitude to all the past and present members of the antibiotic resistance group, Alieda, Arie, Ayla, Cindy, Daniela, Joop, Kees, Marga and Yvon, for the friendly atmosphere within the group and the times we spent together. I am especially thankful to Arie and Yvon for their help and support with my sometimes-overwhelming experimental burden, as well as to Daniela for her friendly mentorship, constant motivation and encouragement, and for being a shoulder to cry on more often than someone could withstand! In addition, I am grateful to all the personnel of the Wageningen Bioveterinary Research for their warm hospitality and particularly to Hendrik-Jan for our discussions and his guidance, Annemieke for our giggles and laughter, Alex and Jeanet for introducing me to bioinformatics and biostatistics respectively. A I would, also, like to express my sincere appreciation to my fellow PhD candidates, Alejandro, Geritta, Ewa, Runa and Wietske, not only for our close and very productive collaborations, but also their support, sympathy and words of comfort when things seemed to go wrong in my PhD life. Dear Runa apart from fellow PhD candidates, we were housemates and friends. Thank you for the lovely times we had at home and at the institute, and for making my life easier when I firstly arrived in the Netherlands.

I would like to express my gratitude to all my co-authors for contributing to the work descripted in the present thesis, and to all the members of my assessment committee for reviewing this thesis and accepting to take part as opponents in my defense.

Acknowledgments | 213 Special thanks to my chosen family and partners in crime Bo, Ioanna, Jeroen, Klaas, Linda, Melvin, Patrick and Steve for filling my life with adventures, love, joy, hope, laughter, craziness and lots of nice memories. My dear Bo thank you for sharing with me some of my brightest and definitely one of my darkest moments in life…without your love, patience and support I may have never seen the light at the end of the tunnel!!! Dear Ioanna thank you for our Greek moments full of passion and emotions that soothed the nostalgia for our home country! Dear Jeroen thank you so much for the sense of security in your presence and for our non-stop techno dancing! Dear Klaas thank you for your contagious placid and carefree attitude in life! Dear Linda thank you for holding my hand and showing me another world! Dear Melvin thank you for seeing something more in me than a shy guy and for helping me to find out pieces of myself! Dear Patrick thank you for being an example of unconditional love! Dear Steve thank you so much (among many other things) for advising me, encouraging me, being proud of me and always making me laugh!

To my oldest friend Vaso, although I will never forget your full of aversion look on me when we first met, thank you for your long lasting friendship!!! Together we grew up, dreamed, laughed out loud, cried, fought and tried! Our friendship means a lot to me…thank you for everything!!!

Last but not least to my beloved sister Eleni, who until very recently was my only family, I owe an enormous “Thank you” for all her love and support, as well as the best present I have ever received in my life, my niece Sotiria and my nephew Dimitrio, to whom I dedicate this thesis!!! Little sister, life was not easy for us but having you by my side lessened the pain and the despair!!! You should know that living far from you is the most intense suffering I have ever had to endure!!! Although far from each other I am always here for you!!! Take care of yourself and the kids!!! I love you and I miss you immensely!!!

214 | Appendix

About the author

Apostolos Liakopoulos was born on the 29th of December 1984 in Athens, Greece. After finishing his internship in the Department of Microbiology, ‘‘The Evaggelismos’’ General Hospital of Athens and his bachelor thesis in the Department of Microbiology, University Hospital of Larissa, he grad- uated in 2010 from the School of Health Sciences of University of Thessaly, Greece. He continued his postgraduate studies in the National and Kapodistrian University of Athens, where he obtained his M.Sc in Microbial Biotechnology (cum laude) in 2013. His master thesis took place in collaboration with the Department of Microbiology, University Hospital of Larissa and the Laboratory of Bacteriology, Hellenic Pasteur Institute (Athens, Greece) and was focused on carbapenem-resistant non-fermentative Gram-negative bacteria. In October 2013 Apostolos moved to the Netherlands and joined as a junior scientist the Department of Bacteriology and Epidemiology, Wageningen Bioveterinary Research, while he started his doctoral studies in “Infection and immunity” in the graduate School of Life Sciences, University of Utrecht. His doctoral research was focused on the molecular epidemiology of extended-spectrum cephalosporin-resistant Enterobacteriaceae and his results are presented in this thesis. Since 1st of July, Apostolos continues his career as postdoctoral researcher in microbial evolution, at the Institute of Biology, Leiden University (The Netherlands). His work has been published in peer-reviewed journals (30 publications) and presented in Inter- national, European (28 abstracts) and National (19 abstracts) conferences.

About the author | 217

List of publications

Liakopoulos A, van der Goot J, Bossers A, Betts J, Brouwer MSM, Kant A, Smith H, Ceccarelli D and Mevius DJ (2017). Plasmid epidemiology of SHV-12-producing Escherichia coli from human and animal origin: X factor(s) of an emerging plasmid family. Scientific Reports [Under revision].

Liakopoulos A, van den Bunt G, Geurts Y, Toleman M, van Pelt W and Mevius DJ (2017). Molecu- lar characterization of extended-spectrum cephalosporin resistant Enterobacteriaceae from Dutch preschool children and their parents: insight on intra-familial transmission. Frontiers in Microbiology [Under revision]. de Vries D, Pacholewicz E, Geurts Y, Liakopoulos A, Smid J, Mevius DJ, Heederik D and Schmitt H (2017). Risk factors for ESBL/AmpC producing Escherichia coli in recreational waters and potential human exposure. Water Research [Under revision].

Dorado-Garcia A*, Smid JH*, van Pelt W, Bonten MJM, Fluit AC, van den Bunt G, Wagenaar JA, Hordijk J, Dierikx CM, Veldman KT, de Koeijer A, Dohmen W, Schmitt H, Liakopoulos A, Pacholewicz E, Lam TJGM, Velthuis AG, Heuvelink A, Gonggrijp MA, van Duijkeren E, van Hoek AHAM, de Roda Husman AM, Blaak H, Havelaar AH, Mevius DJ and Heederik DJJ (2017). Molec- ular relatedness of ESBL/AmpC-producing Escherichia coli from humans, animals, food and the environment: a pooled analysis. J. Antimicrobial Chemotherapy [Under revision].

Liakopoulos A, Betts J, La Ragione RM, van Essen-Zandbergen A, Ceccarelli D, Petinaki E, Koutinas CK, La Ragione RM and Mevius DJ (2017). First report of extended-spectrum cephalosporin-resistant Enterobacteriaceae from household dogs in Greece. Plos One [Under revision]. A Abdul Momin MH*, Liakopoulos A*and Wareham DW (2017). Draft genome sequence of an outbreak-associated Klebsiella pneumoniae strain producing OXA-232 carbapenemase. Genome Announcements 6;5(27). pii: e00604-17.

Dohmen W, van Gompel L, Schmitt H, Liakopoulos A, Heres L, Urlings BA, Mevius DJ, Bonten MJM and Heederik DJJ (2017). ESBL carriage in pig slaughterhouse workers is associated with occupational exposure. Epidemiology and Infection 2:1-8.

List of publications | 219 Abdul Momin MH, Liakopoulos A, Phee LM and Wareham DW (2017). Emergence and nosocomial spread of carbapenem-resistant OXA-232 producing Klebsiella pneumoniae in Brunei Darussalam. Journal of Global Antimicrobial Resistance pii: S2213-7165(17)30051-6.

Liakopoulos A, Oikonomou O and Wareham DW (2017). Draft genome sequence of Providencia stuartii PS71, a multidrug-resistant strain associated with nosocomial infections in Greece. Genome Announcements 23;5(12). pii: e00056-17. van den Bunt G, Liakopoulos A, Mevius DJ, Geurts Y, Fluit AC, Bonten MJ, Mughini-Gras L, van Pelt W (2016). ESBL/AmpC-producing Enterobacteriaceae in households with children of preschool age: prevalence, risk factors and co-carriage. J. Antimicrobial Chemotherapy 72(2):589-595.

Liakopoulos A, Mevius D and Ceccarelli D (2016) A Review of SHV Extended-Spectrum β-Lacta- mases: Neglected Yet Ubiquitous. Frontiers in Microbiology 7:1374-1400.

Liakopoulos A, Olsen B, Geurts Y, Artursson K, Berg Charlotte, Mevius D and Bonnedahl J (2016). Molecular Characterization of Extended-Spectrum Cephalosporin-Resistant Enterobacte- riaceae from Wild Birds (Kelp Gulls) in South America. Antimicrobial Agents and Chemotherapy 60(11):6924-6927.

Liakopoulos A, Mevius D, Olsen B and Bonnedahl J (2016). The colistin resistance mcr-1 gene is going wild. J. Antimicrobial Chemotherapy 71(8):2335-2336.

Liakopoulos A, Geurts Y, Dierikx C, Brouwer MSM, Kant A, Wit B, Heymans R, van Pelt W and Mevius D (2016). Introduction of extended-spectrum cephalosporin-resistant Salmonella enterica serotype Heidelberg outbreak strains in the Netherlands. Emerging Infectious Diseases 22(7):1257- 1261.

Oikonomou O*, Liakopoulos A*, Phee LM, Betts J, Mevius D and Wareham DW (2016). Provi- dencia stuartii isolates from Greece: Co-carriage of cephalosporin (blaSHV-5, blaVEB-1), carbapenem

(blaVIM-1) and aminoglycoside (RmtB) resistance determinants by a multi-drug resistant outbreak clone. Microbial Drug Resistance 22(5):379-386.

Sarrou S, Liakopoulos A, Tsoumani K, Sagri E, Mathiopoulos KD, Tzouvelekis LS, Miriagou V and Petinaki E (2015). Characterization of a novel lsa(E)- and lnu(B)-carrying structure located in the chromosome of Staphylococcus aureus ST398. Antimicrobial Agents and Chemotherapy 60:1164–1166.

220 | Appendix Pacholewicz E, Liakopoulos A, Swart, A, Gortemaker B, Dierikx C, Havelaar A and Schmitt H (2015). Reduction of extended-spectrum-β-lactamase- and AmpC-β-lactamase-producing Escher- ichia coli through processing in two broiler chicken slaughterhouses. International Journal of Food Microbiology 215:57-63.

Sarrou S, Liakopoulos A, Chasioti M, Foka A, Fthenakis G, Billinis C, Spyrou V, Pantelidi K, Rous- saki-Schulze A, Lachanas V, Makaritsis K, Skoulakis C, Daikos GL, Dalekos G, Spiliopoulou I and Petinaki E (2015). Dissemination of methicillin-susceptible CC398 Staphylococcus aureus strains in a rural Greek area. PloS One 10(4):e0122761.

Mavroidi A, Liakopoulos A, Sarrou S, Miriagou V and Petinaki E (2015). Identification and characterization of genetic structures coding for carbapenemases in Enterobacteria from Central Greece. Diagnostic Microbiology and Infectious Disease 81(1):47-49.

Papadimitriou-Olivgeris M, Drougka E, Fligou F, Kolonitsiou F, Liakopoulos A, Dodou V, Anastas- siou ED, Petinaki E, Marangos M, Filos KS and Spiliopoulou I (2014). Risk factors for enterococcal infection and colonization by vancomycin-resistant enterococci in critically ill patients. Infection 42(6):1013-1022.

Giormezis N, Kolonitsiou F, Foka A, Drougka E, Liakopoulos A, Makri A, Papanastasiou AD, Vogiatzi A, Dimitriou G, Marangos MN, Christofidou M, Anastassiou ED, Petinaki E & Spiliopou- lou I (2014). Coagulase-negative staphylococcal bloodstream and prosthetic-device-associated infections: the role of biofilm formation and distribution of adhesin and toxin genes. Journal of Medical Microbiology 63:1500-1508.

Mavroidi A, Liakopoulos A, Gounaris A, Goudesidou M, Miriagou V and Petinaki E (2014). Suc- A cessful control of a neonatal outbreak caused mainly by ST20 multidrug-resistant SHV-5-producing Klebsiella pneumoniae, Greece.. BMC Pediatrics 14(1):105-112.

Drougka E, Foka A, Liakopoulos A, Doudoulakis A, Jelastopulu E, Chini V, Spiliopoulou A, Levid- iotou S, Panagea T, Vogiatzi A, Lebessi E, Petinaki E & Spiliopoulou I (2014). A twelve-year survey of methicillin-resistant Staphylococcus aureus infections in Greece: ST80-IV epidemic? Clinical Microbiology & Infection 20(11):O796-803.

List of publications | 221 Neocleous C, Gerogianni I, Liakopoulos A, Gourgoulianis K and Petinaki E (2014). Bacterial aetiology of community-acquired pneumonia in hospitalised patients with chronic obstructive pulmonary disease in central Greece. British Journal of Biomedical Science 71(1):46-47.

Liakopoulos A, Mavroidi A, Vourli S, Panopoulou M, Zachariadou L, Chatzipanagiotou S, Spili- opoulou I, Zerva L and Petinaki E (2014). Molecular characterization of Streptococcus agalactiae from vaginal colonization and neonatal infections: a four year multicenter study in Greece. Diag- nostic Microbiology and Infectious Disease 78(4):487-490.

Papadimitriou-Olivgeris M, Giormezis N, Fligou F, Liakopoulos A, Marangos M, Anastassiou ED, Petinaki E, Filos KS and Spiliopoulou I (2013). Factors influencing linezolid-nonsusceptible coagulase-negative staphylococci dissemination among patients in the intensive care unit: a retrospective cohort study. Chemotherapy 59(6):420-426.

Liakopoulos A, Mavroidi A, Katsifas EA, Theodosiou A, Karagouni AD, Miriagou V and Petinaki E (2013). Carbapenemase-producing Pseudomonas aeruginosa from Central Greece: molecular epidemiology and genetic analysis of class I integrons. BMC Infectious Diseases 13(1):505-511.

Koutsogiannou M, Drougka E, Liakopoulos A, Jelastopulu E, Petinaki E, Anastassiou E.D, Spili- opoulou I and Christofidou M (2013). Spread of Multi Drug Resistant Pseudomonas aeruginosa clones in a University Hospital. Journal of Clinical Microbiology 51(2):665-668.

Mavroidi A, Miriagou V, Liakopoulos A, Tzelepi E, Stefos A, Dalekos GN and Petinaki E (2012). Ciprofloxacin-resistantEscherichia coli in Central Greece: mechanism of resistance and molecular identification. BMC Infectious Diseases 12(1):371-377.

Drougka E, Foka A, Marangos MN, Liakopoulos A, Makatsoris T, Anastassiou ED, Petinaki E and Spiliopoulou I (2012). The first case of Staphylococcus aureus ST398 causing bacteremia in an immunocompromised patient in Greece. Indian Journal of Medical Microbiology 30(2):232-236.

Liakopoulos A, Miriagou V, Katsifas EA, Karagouni AD, Daikos GL, Tzouvelekis LS and Petinaki E (2012). Identification of OXA-23-producing Acinetobacter baumannii in Greece, 2010 to 2011. Eurosurveillance 17(11):pii=20117.

222 | Appendix Kotsakis S.D, Tzouvelekis L.S, Zerva L, Liakopoulos A and Petinaki E (2012). Staphylococcus lug- dunensis strain with a modified PBP1A/1B expressing resistance to β-lactams. European Journal of Clinical Microbiology & Infectious Diseases 31(2):169-172.

Mavroidi A, Neonakis I, Liakopoulos A, Papaioannou A, Ntala M, Tryposkiadis F, Miriagou V and Petinaki E (2011). Detection of Citrobacter koseri carrying β-lactamase KPC-2 in a hospitalized patient, Greece, July 2011. Eurosurveillance 16(41):pii=19990.

Liakopoulos A, Foka A, Vourli S, Zerva L, Tsiapara F, Protonotariou E, Dailiana Z, Economou M, Papoutsidou E, Koutsia-Carouzou C, Anastassiou E.D, Diza E, Zintzaras E, Spiliopoulou I and Petinaki E (2011). Aminoglycoside-resistant staphylococci in Greece: prevalence and resistance mechanisms. European Journal of Clinical Microbiology & Infectious Diseases 30(5):701–705.

Liakopoulos A, Spiliopoulou I, Damani A, Kanellopoulou M, Schoina S, Papafrangas E, Marangos M, Fligou F, Zakynthinos E, Makris D, Protonotariou E, Tsiapara F, Filos K, Diza E, Anastassiou E.D and Petinaki E (2010). Dissemination of two international linezolid-resistant Staphylococcus epidermidis clones in Greek hospitals. J. Antimicrobial Chemotherapy 65(5): 1070-1071.

Liakopoulos A, Neocleous C, Klapsa D, Kanellopoulou M, Spiliopoulou I, Mathiopoulos KD, Papafrangas E and Petinaki E (2009). A T2504A mutation in the 23S rRNA gene responsible for high-level resistance to linezolid of Staphylococcus epidermidis. J. Antimicrobial Chemotherapy 64(1): 206-207.

*These authors contributed equally. A

List of publications | 223