Living Together in a World of Extended-Spectrum Cephalosporin Resistance: Molecular Snapshots of a Complex Epidemiology
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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 cli- nical 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 mecha- nisms 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 iden- tified 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 con- stitute 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-produ- cing 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 cephalo- sporins (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-bin- ding 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 forβ -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 resis- tance 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 com- pounds, 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