REBEL-ing. against resistance. REsistance to BEta-Lactam antibiotics due to beta-lactamases. Elien Ascelijn Reuland

Transcription

1 REBEL-ing against resistance REsistance to BEta-Lactam antibiotics due to beta-lactamases Elien Ascelijn Reuland

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3 REBEL-ing against Resistance REsistance to BEta-Lactam antibiotics due to beta-lactamases

4 The studies in this thesis were funded by ZonMw, the Netherlands Organization for Health Research and Development (grant number ). Printing of this thesis was financially supported by the VU University Medical Center Amsterdam, Mediaproducts BV, Check-Points B.V., AlphaOmega Instruments - Diagnostics BV, the Netherlands Society of Medical Microbiology (NVMM) and the Royal Netherlands Society for Microbiology (KNVM). REBEL-ing against Resistance: REsistance to BEta-Lactam antibiotics due to beta-lactamases Thesis, VU University Amsterdam, the Netherlands. Copyright 2017 Elien Ascelijn Reuland, Amsterdam, the Netherlands. ISBN: Cover: Lay-out: Printing: Janno Heck Nikki Vermeulen Ridderprint BV Ridderprint BV

5 VRIJE UNIVERSITEIT REBEL-ing against Resistance REsistance to BEta-Lactam antibiotics due to beta-lactamases ACADEMISCH PROEFSCHRIFT ter verkrijging van de graad Doctor aan de Vrije Universiteit Amsterdam, op gezag van de rector magnificus prof.dr. V. Subramaniam, in het openbaar te verdedigen ten overstaan van de promotiecommissie van de Faculteit der Geneeskunde op vrijdag 3 februari 2017 om uur in de aula van de universiteit, De Boelelaan 1105 door Elien Ascelijn Reuland geboren te Groningen

6 promotoren: copromotor: prof.dr. C.M.J.E. Vandenbroucke-Grauls prof.dr. J.A.J.W. Kluytmans dr. N. al Naiemi

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8 leescommissie: paranimfen: prof.dr. H.E. van der Horst prof.dr. M.J.M. Bonten prof.dr. A.W. Friedrich prof.dr. C. Schultsz prof.dr. A. Voss dr. M.A. van Agtmael drs. Marre van den Brand dr. Oddeke van Ruler

9 Voor mijn ouders grote broer grote zus kleine broer

10 ...uiteindelijk krijgt iedereen dezelfde bonuskorting. - Nashwan

11 CONTENTS CHAPTER 1 Introduction and outline of the thesis 13 (adapted from ESBL in de kliniek: achtergrond, relevantie en epidemiologie) Tijdschrift voor infectieziekten 2011 vol 6; nr 4 CHAPTER 2 High prevalence of ESBL-producing Enterobacteriaceae 31 carriage in Dutch community patients with gastrointestinal complaints Clinical Microbiology and Infection 2013 Jun;19(6):542-9 CHAPTER 3 Prevalence and risk factors for carriage of ESBL-producing 47 Enterobacteriaceae in Amsterdam Journal of Antimicrobial Chemotherapy 2016 Apr;71(4): CHAPTER 4 Travel to Asia and traveller s diarrhoea with antibiotic treatment 75 are independent risk factors for acquiring ciprofloxacin-resistant and extended spectrum betalactamase-producing Enterobacteriaceae - a prospective cohort study Clinical Microbiology and Infection Aug;22(8):731 CHAPTER 5 Extended-Spectrum beta-lactamase- and Carbapenemase- 95 Producing Enterobacteriaceae Isolated from Egyptian Patients with Suspected Blood Stream Infection PLoS One 2015 May 22;10(5):e CHAPTER 6 Extended-Spectrum beta-lactamases and/or Carbapenemases- 107 Producing Enterobacteriaceae Isolated from Retail Chicken Meat in Zagazig, Egypt PLoS One 2015 Aug 18;10(8):e CHAPTER 7 Prevalence of ESBL-producing Enterobacteriaceae in raw vegetables 119 European Journal of Clinical Microbiology & Infectious Diseases 2014 Oct;33(10):1843-6

12 CHAPTER 8 A case of New Delhi metallo-beta-lactamase 1 (NDM-1)-producing 129 Klebsiella pneumoniae with putative secondary transmission from the Balkan region in the Netherlands Antimicrobial Agents and Chemotherapy 2012 May;56(5): CHAPTER 9 The cost-effectiveness of ESBL detection: towards molecular 135 detection methods? Clinical Microbiology and Infection 2013 Jul;19(7):662-5 CHAPTER 10 Detection and occurrence of plasmid-mediated AmpC in highly 147 resistant gram-negative rods PLoS One 2014 Mar 18;9(3):e91396 CHAPTER 11 Plasmid-mediated AmpC: prevalence in community-acquired 163 isolates in Amsterdam, the Netherlands, and risk factors for carriage PLoS One 2015 Jan 14;10(1):e CHAPTER 12 Summarizing discussion and future directions 177 Nederlandse samenvatting 189 List of publications 203 Curriculum Vitae 209 Dankwoord 215

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15 Introduction and outline of the thesis EA Reuland 1, CMJE Vandenbroucke-Grauls 1, N al Naiemi 1,2,3 1 Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam (adapted from ESBL in de kliniek: achtergrond, relevantie en epidemiologie) Tijdschrift voor infectieziekten 2011 vol 6; nr 4

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17 Introduction and outline of the thesis OUTLINE OF THE THESIS This thesis focuses on beta-lactamases, in particular on extended-spectrum betalactamases (ESBL) and plasmidal AmpC beta-lactamases. These enzymes produced by many bacterial species, cause resistance to beta-lactam antibiotics. The aim of the studies presented in this thesis was to investigate the epidemiology of carriage of beta-lactamaseproducing Enterobacteriaceae, and the determinants of such carriage in the Netherlands. A corollary to these studies is studies aimed at improving the detection of these enzymes in the clinical microbiology laboratory. In chapter 2 we determined how frequent carriage of ESBL-producing Enterobacteriaceae (ESBL-E) is in Dutch community patients with gastrointestinal complaints, as a pilot study for the larger study that is described in chapter 3. This study was performed to determine the prevalence of ESBL-E in the community in the Netherlands, and to analyze the most important risk factors for such carriage. One of the main risk factors for carriage of resistant strains in the community appeared to be travel. The role of travel was further elaborated in a study among travelers (chapter 4), and by studying the rate of resistant strains in suspected bloodstream infections (chapter 5) and contamination of chicken retail meat in Egypt (chapter 6). Carriage of resistant strains in the gut points to food as a possible source. Therefore we investigated whether resistant strains are also present in raw vegetables (chapter 7).The even more threatening development in resistance in Gram-negative bacteria is the production of carbapenemases, enzymes that cause resistance not only to beta-lactam antibiotics but also to carbapenems, the agents next in line for treatment of resistant infections. Also this type of resistance comes to the Netherlands through travelers, as illustrated by the case described in chapter 8. Halting the spread of resistance starts with its detection, but an important question is what type of detection method is most cost-effective? This question was addressed in chapter 9. In the course of this study, we realized the potential problem of plasmidal AmpC beta-lactamases, therefore we developed a screening method for detection of these enzymes (chapter 10), and searched for strains producing these enzymes in a subset of participants in our community study (chapter 11). Finally, in chapter 12, the main findings of this thesis and concerns arising from these findings are discussed, and placed in the context of possible directions for the future. 15

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19 Introduction and outline of the thesis INTRODUCTION Many types of infections like urinary tract infections, bloodstream infections, hospitalacquired pneumonias, and intra-abdominal infections are often caused by Gram-negative bacteria from the family of the Enterobacteriaceae. Increasing resistance of these bacteria to beta-lactam antibiotics, predominantly due to production of beta-lactamases, is a major problem worldwide. Extended-spectrum beta-lactamases (ESBLs) in particular are an increasing threat for public health. ESBLs are associated with outbreaks, multiresistance and therapeutic failure. In this thesis we will describe the background, relevance and the epidemiology of ESBL-producing Enterobacteriaceae (ESBL-E), especially focusing on the emerging problem in the community. Next to ESBLs, other resistance mechanisms are emerging, e.g. plasmidal AmpC (pampc) and carbapenemases. In order to describe the problem of ESBLs we could not ignore these other resistance threats and as a result these also will be included in the discussion. Background Beta-lactam antibiotics (penicillins, cephalosporins, carbapenems and monobactams) are used extensively to treat infections. The reason is because of their effectiveness: they have a broad spectrum of activity and are of low toxicity. 1,2 Oxyimino-cephalosporins -such as cefuroxim, cefotaxim, ceftriaxon and ceftazidim- constitute the main therapy for many infections in clinical healthcare settings worldwide. However, the frequent use of these antimicrobial agents has led to extensive resistance, a problem that is still increasing ( ecdc.europa.eu). 3 Resistance to beta-lactam antibiotics can develop in different ways. In Gram-negative bacteria, like Escherichia coli and Klebsiella species, beta-lactamases are the main cause of resistance. 4 6 Beta-lactamases are specific enzymes produced by bacteria that hydrolyze and thereby inactivate beta-lactam antibiotics. Beta-lactamases have undergone a major evolution since their first appearance, which has held parallel step, since the introduction of penicillin, with all later developed novel beta-lactam antibiotics. 7 Of these beta-lactamases especially the extended-spectrum beta-lactamases (ESBLs) play an important role. There is no uniform definition of ESBL. The definition is complicated by exceptions and the rise of new types of ESBL. 1,2,8 A much-used working definition is that these are beta-lactamases able to hydrolyze penicillins, first, second and third generation cephalosporins and aztreonam, but are inhibited by beta-lactamase inhibitors such as clavulanic acid. 1 On the other hand, ESBL-producing bacteria are sensitive to cephamycins (such as cefoxitin and cefotetan) and carbapenems (ertapenem, meropenem, imipenem and doripenem). 9 17

20 Chapter 1 The genes encoding for ESBL frequently lie on plasmids and are therefore easily transmissible between bacteria, both between bacteria of the same species and between bacteria of different species. 4 In this way these genes can strongly spread themselves worldwide. Characterization of these plasmids can be performed by PCR-based replicon typing. This method allows the examination of plasmids conferring drug resistance by typing them by incompatibility groups in a multiplex PCR setting. Next to the clonal dispersion of resistant bacteria themselves, this efficient spreading of plasmids is responsible not only for outbreaks of resistant bacteria but also for outbreaks of plasmids. The plasmids with ESBL genes frequently bear genes for resistance to other classes of antibiotics. For this reason ESBL-producing bacteria are frequently also resistant to aminoglycosides, co-trimoxazole and (fluoro) quinolones. Because of this the treatment options of infections with these ESBL-producing bacteria are particularly limited, thereby posing a significant challenge to antimicrobial therapy. 10,11 Carbapenems remain as only therapeutic option and are the first choice for serious infections. 1 Unfortunately, resistance is more and more observed also to carbapenems, and therefore bacteria, that are insensitive against the most available resources, are emerging and creating a widespread resistance phenomenon. Alternative resources, with just as good effectiveness and low toxicity, are hardly available. Also little to no really new antimicrobial resources is expected in the short term. 1,12 Several types of ESBL can be distinguished. These types have been classified in various ways; this classification is complicated because a number of classification systems exist. Therefore we will have to limit discussion, and just mention the most predominant types of ESBLs, which are frequently discussed in the literature. Most ESBLs are derivatives of ordinary betalactamases, i.e. beta-lactamases which cannot hydrolyze third generation cephalosporins and aztreonam. However, due to mutations varieties have arisen with the possibility to hydrolyze these. Several families of beta-lactamases can be distinguished like e.g., TEM, SHV and CTX-M. Within these families both beta-lactamases and ESBL appear which sometimes only differ by a single point mutation. To determine a genetic relationship between two ESBL-producing bacteria (e.g. in an outbreak situation) detection and identification at the gene level is necessary. The TEM family is formed by derivatives of TEM-1 and TEM-2, both ordinary beta-lactamases. TEM-1 has been described in 1965 as the first plasmid-mediated beta-lactamase. The name has been derived from a patient from Athens, Temoneira, where E. coli with these betalactamases were recognized for the first time. Since then already more than 200 TEMenzymes have been detected ( SHV (sulfhydryl reagent variable) is an abbreviation that refers to a biochemical characterization of this type of beta-lactamase just like for TEM also here applies that the SHV enzymes that were first described are ordinary beta-lactamases; later the ESBL varieties arose by mutations. The SHV-type ESBL 18

21 Introduction and outline of the thesis is more common in hospitals than the TEM-type ESBL. SHV beta-lactamases seem to have their origin in Klebsiella species. 4 At this moment more than 175 SHV derivatives are known ( Another frequent group of ESBL is the CTX-M group. The name refers to the fact that these enzymes hydrolyze cefotaxim generally better than ceftazidim. These CTX-M beta-lactamases appear to be derived from chromosomally encoded betalactamases produced by Kluyvera spp., probably infrequent opportunistic pathogens of the Enterobacteriaceae found in the environment. 13 They were found in the late 1980s, and now more than 150 variants are detected. 6 The CTX-M beta-lactamases can be divided into five groups, i.e. CTX-M group 1, 2, 8, 9, and 25, based on their amino acid sequence similarities. CTX-M ESBLs are more often detected outside the hospital compared to SHV and TEM. Other classes of ESBLs are e.g. OXA, PER, VEB, CME, GES, IBC, BES, BEL, SFO and TLA. They are mainly found in Pseudomonas aeruginosa, where OXA is the predominantly present. The geographical distribution of these ESBLs has been restricted to a number of regions, like e.g. Mexico, Japan, Brazil and Turkey. 14 Resistance to broad-spectrum cephalosporins is considered to be mainly caused by extended-spectrum beta-lactamases (ESBLs). Another group of enzymes that can hydrolyze cephalosporins are the AmpC beta-lactamases. AmpC were originally described as chromosomally encoded beta-lactamases, particularly in Enterobacter spp., Citrobacter freundii, and Serratia spp. Plasmid-mediated AmpC (pampc) are AmpC beta-lactamases encoded on plasmids and hence transferable between species. These enzymes appeared in Enterobacteriaceae that lack chromosomal AmpC enzymes (Proteus mirabilis, Salmonella spp and Klebsiella spp) or only express low basal amounts of AmpC like Escherichia coli and Shigella spp. The frequency of pampc may be of larger concern than initially thought, especially if this resistance threat would mimic the trend that we have seen occurring over the past years for ESBL-E. 1,2 We consider it important therefore, to closely monitor the occurrence of this resistance threat. Carbapenem-resistant Enterobacteriaceae (CR-E), due to carbapenemases, have been reported all over the world. 15 The first was detected in 1993, thereafter several CR-E have been found. 16 Chromosome-encoded cephalosporinases (class C according to the Ambler classification) are able to hydrolyze carbapenems, however with no clear consequences yet. 8,17 However, KPC (class A) and e.g. the OXA-48 enzymes (oxacillinases known as class D) are of increasing clinically importance In addition, the metallo-beta-lactamases (MBLs), group B, are an alarming threat including the so called New-Delhi metallo-beta-lactamase-1 (NDM-1) originating from New Delhi and described in Detection of ESBLs, pampc and carbapenemases In the microbiological laboratory there are roughly two different methods for the detection of ESBLs. 19

22 Chapter 1 The phenotypical detection of ESBL is complex and far from uniform. Several factors (e.g.: subjective perception of inhibition or choice of the medium for the bacteria) hamper the detection of ESBL-producing bacteria. In the Netherlands, the Dutch Association for Medical Microbiology (NVMM) has developed a guideline for standardizing the detection of ESBL in daily routine. 23 However, even when this guideline is used, the phenotypical detection remains time-consuming and is not always easy to interpret. The genotypical detection of ESBL has become an important diagnostic tool. In addition to knowing whether an ESBL is really present when a less clear phenotype has been found, it is frequently also essential to determine exactly which type of ESBL it concerns, e.g. in cases of tracing outbreaks. When hospitalized patients prove to be carriers of the same type of bacteria, e.g. an ESBL-producing E. coli, transfer between patients is just probable and an epidemic likely as it has been shown that the genes responsible for the produced ESBL are identical. Genotypic detection is possible by means of PCR on the ESBL gene and thereafter sequencing of the PCR product. The identification of beta-lactamase genes is also possible by means of DNA-microarray; an advantage of this method is that it is accurate and fast The exact prevalence of pampc is still unknown because simple and valid detection methods are not available; hence pampc-producing organisms are often missed. While algorithms for the routine detection of resistance among Gram-negative bacteria, including detection of ESBL and carbapenemases, are widely available, such algorithms are still lacking for pampc. 23 Therefore evaluation of the current screening methods and confirmation tests for phenotypic plasmidal AmpC (pampc) detection are needed, and should be confirmed by PCR. Carbapenemases can be detected by testing susceptibility to carbapenems like imipenem, meropenem and ertapenem. Dutch guidelines on how to screen for these isolates were also developed; here to, phenotypic detection must be confirmed. A real-time multiplex PCR was developed to identify the specific genes found. Epidemiology and risk factors The prevalence of ESBLs has been underestimated for a long time probably because detection in microbiological laboratories has not always been adequate or because the importance was not recognized. Most of the ESBLs among clinical isolates belong to the CTX-M, SHV and TEM families. 1,27 Although ESBLs are found mainly in Klebsiella spp. and E. coli, they are found more and more also in other Enterobacteriaceae such as Enterobacter and Salmonella spp. and even from time to time in other Gram-negative bacteria such as Pseudomonas aeruginosa and Burkholderia cepacia. 28 In the meantime E. coli has replaced Klebsiella spp. as the main species of ESBL-producing Enterobacteriaceae in large parts of the world. 20

23 Introduction and outline of the thesis The prevalence of ESBL is higher in Europe than in the United States but lower compared to Asia and Southern America. 29 Within Europe there are considerable geographical differences. 1,29 Several studies show that the prevalence in Northern Europe is substantially lower (1-5%) than in Eastern Europe (39-47%), or for example Russia (almost 50% and Poland 40%). The precise size of the problem, the determinants of the increase in resistance, and the risk factors for the occurrence of ESBL-producing microorganisms in the Netherlands, however, were largely unknown. It has been shown that patients admitted to the hospital already carry ESBL in 4% of the cases. 30 Data of EARSS (European Antibiotic Resistance Surveillance Study) show that in the Netherlands the percentage of E. coli isolates from blood cultures resistant to third generation cephalosporins has increased from 0.6% in 2001, to 4.3% in 2009 and 5.8% in For K. pneumoniae this percentage has risen from 3.5% in 2005, to 5.5% in 2009, and to 7.5% in ( Pages/table_reports.aspx). A large Dutch research project, conducted in 2006, estimated the ESBL prevalence in nosocomial isolates close to 6%. 31 The difference between the research performed in 2006 and data of ISIS-AR are that the latter database data contained all E. coli and Klebsiella grown in a number of laboratories; it concerns therefore both hospital bacteria and bacteria found in nursing homes and in general practice. In Asia and Southern America, the number of ESBL-producing microorganisms is high compared to Europe. 32,33 Due to the high population density in India and China these two countries can be considered as the largest reservoirs of CTX-M ESBL genes in the world. A study performed in India shows a prevalence of 68% under E. coli and K. pneumoniae isolates. 32 In hospitals the intensive care frequently constitutes an epicenter of ESBL production. 4 Nursing homes and residential care can also be a focus of infections and thereby serve as reservoir for influx in hospitals. 34 A worrisome development is that ESBLs are found in E. coli isolates which cause infections outside the hospital; these ESBLs are often of the CTX-M type. 29 Next to clonal dispersion, the acquisition of multidrug-resistant plasmids plays a pivotal role in the dissemination of CTX-M-producing ESBLs. Various replicons, especially those widely distributed among E. coli strains, could be involved in part of this dissemination process. It has been proposed that most of the CTX-M-15 enzymes are encoded on IncF replicons (FIA, FIB and FII). 35 The cause of this sudden increase outside the hospital is not clear yet and probably multifactorial. Some risk factors are well known while other are not reported yet. Risk factors for colonization or infection with ESBL-producing bacteria are comparable with risk factors for other nosocomial infections. 36 Patients with a high risk for ESBL-producing 21

24 Chapter 1 bacteria are seriously ill patients with a long hospital stay and/or long stay on the intensive care unit where medical devices are frequently indicated (catheters, drains, central venous lines). 1 Also frequent use of antibiotics, especially cephalosporins, is a risk factor. 37 Different studies show a relation between the use of third generation cephalosporins and the acquisition of ESBL-producing bacteria by selection. 1,36,38 Use of broad-spectrum antibiotics selects ESBL-producing mutants. For pampc-producing Enterobacteriaceae risk factors are largely unknown, especially undefined in the community. In the community several other risk factors can be envisaged: the complex dynamics and dissemination of antibiotic resistance and its relation to different reservoirs has been depicted in other reports. 39,40 In addition to acquisition of resistance from high ESBL prevalence reservoirs (hospitals and long-term care facilities) the presence of resistant strains in the food chain, environment or water sources can be considered. 11 Especially, the food chain is of importance because a high prevalence of resistance genes in poultry was reported, related to the high rate of antimicrobial drug use in the livestock sector. 30,41 Research has shown that in the Netherlands a large part of the chickens in poultry farms carry ESBL-producing bacteria. 30,42 An important cause is the large quantities of antibiotics given to these animals. The main problem is that the antibiotics used belong to the same classes of antibiotics used in the human population. A Dutch study showed that one third of the genes found in human isolates of ESBL-producing E. coli correspond to genes found in poultry isolates. 43 As a result, the relation between use of antibiotics in livestock and the increase of resistance in the human population becomes more plausible. Furthermore, it has been shown that over the last seventy years resistance genes are being found in increasing amounts in soil (agriculture) in the Netherlands. 44 The influence of these resistance genes in soil on humans is still unclear. Further research is needed to ascertain whether these ESBLs, widespread in agriculture, can be transferred to human bacteria. 39,45 A French study observed that vegetables in France were often contaminated with resistance genes. 46 In 2011, a Shiga toxin-producing Escherichia coli (STEC) outbreak in Germany, caused by ESBL-producing E. coli, was traced to sprouts. 47 So, careful monitoring of resistance present in the food chain is needed. Traveling to foreign countries with a high prevalence of resistance genes is also a wellknown risk factor for the acquisition of these genes after return. 48 Even carbapenemase genes can be acquired by visiting e.g. Northern Africa due to the endemic presence of OXA To identify other (travel-related) risk factors, like traveler s diarrhea, is essential in order to restrain acquisition and spread. Outbreaks of pampc have been recognized in different settings worldwide. 49,50 Currently little information is available regarding the prevalence of this group of beta-lactamases in the Dutch community. Therefore, there is a need to determine the prevalence of pampc 22

25 Introduction and outline of the thesis beta-lactamases in community-acquired Gram-negative bacteria in the Netherlands, and to identify possible risk factors for carriage of these strains. Over the past fifteen years carbapenemase production in Enterobacteriaceae has increasingly been reported. 15,21 A marked endemicity for some of these enzymes has been seen in some parts of the world: e.g. United States and Greece for KPC, metallo-enzymes all over the world with a higher prevalence in Southern Europe and Asia. Oxacillinase-48 type (OXA-48 type) carbapenemases have been identified in Mediterranean and European countries and in India. A worrisome finding is that New Delhi metallo-beta-lactamase-1 producers (NDM-1), originally found in the United Kingdom, India, and Pakistan are now found worldwide. For these isolates identification and detection of carriers is of crucial importance to prevent further spread. Relevance for the clinic When a patient carries ESBL-producing bacteria it might be possible that the empirically indicated therapy to treat the infection, awaiting the laboratory results, has no effect. This might lead to an increased risk on therapeutic failure with subsequent increased risk of morbidity and mortality. In the absence of a favorable response to empirical therapy, or confirmation of an ESBL-producer, a switch to other antimicrobial agents is needed. As a consequence a more toxic or less effective antibiotic has to be used or only more expensive and intravenous therapy are options. 1 Due to the presence of frequently occurring co-resistance in ESBL-producing bacteria, carbapenems generally remain as the only therapeutic option. 1 Colistine, tigecycline, fosfomycine, and nitrofurantoin are also still possible alternatives if resistance to carbapenems has been encountered. These agents have their own disadvantages, like e.g. toxicity or unfavorable pharmacokinetics/ pharmacodynamics. A further increase of prevalence of ESBL-producing bacteria will lead eventually to adjustment of the empirical policy; aminoglycosides must be added to the current policy, or carbapenems will be part of the empirical therapy. Undoubtedly this will promote the strongly rise of carbapenem resistance, in many countries already noticed. Prevention of distribution of ESBL-producing bacteria in the hospital The principle for restraining multiresistant Gram-negative bacteria involves reducing the use of antimicrobials and therefore selection pressure. As a result the risk of therapeutic failure might be decreased. 51 Adequate use of antibiotics is essential, i.e. avoiding the use of broad-spectrum antibiotics (particularly extended-spectrum cephalosporins) as much as possible. 52 Guidelines to restrain antibiotic use have been developed by the foundation working group antibiotic policy (Stichting Werkgroep Antibiotica-beleid (SWAB)). A second important element to restrain multiresistance consists in infection prevention strategies, i.e. taking different measures to reduce the distribution of resistant 23

26 Chapter 1 microorganisms. In case of detection of ESBL-producing bacteria within a patient, contact isolation measures will have to be set up immediately. 53 Patients have to be separated from other patients by nursing in a single room, and during care and possible contact with contaminated patient material, gloves and apron have to be worn. According to WIPguidelines contact isolation measures can be stopped after two negative cultures, swabs taken with one day interval and at least two days after finishing antibiotic treatment. 53 It has been doubted by some, whether a single room is really necessary, beside the use of gloves and apron. For this reason a large randomized research has been started in the Netherlands, financed by ZonMw. 54 As long as the results of this research are not public yet, a single room is the option of preference. Conclusion The prevalence of ESBL-producing bacteria increases worldwide and has a large impact both clinically and economically. ESBLs have been associated with therapeutic failure and higher morbidity and mortality. Prevention by reducing unnecessary use of broadspectrum antibiotics in hospitals and reducing distribution by means of good infection prevention measures contribute to control ESBLs. An accurate and fast detection of ESBLs is therefore necessary. The uncontrolled use of antibiotics in livestock must also be reduced. Research to identify risk factors and possible sources of resistant bacteria remains important to improve policies for antibiotic use and improve infection prevention measures. 24

27 Introduction and outline of the thesis REFERENCES 1 Paterson D, Bonomo R. Extended-spectrum β-lactamases: a clinical update. Clin Microbiol Rev 2005; 18: Livermore DM. Defining an extended-spectrum beta-lactamase. Clin Microbiol Infect 2008; 14 Suppl 1: Paterson DL. Resistance in gram-negative bacteria: Enterobacteriaceae. Am J Infect Control 2006; 34: S Livermore DM. beta-lactamases in laboratory and clinical resistance. Clin Microbiol Rev 1995; 8: Jacoby GA, Munoz-Price LS. The new betalactamases. N Engl J Med 2005; 352: Bonnet R. Growing group of extended-spectrum beta-lactamases: the CTX-M enzymes. Antimicrob Agents Chemother 2004; 48: Medeiros AA. Evolution and dissemination of beta-lactamases accelerated by generations of beta-lactam antibiotics. Clin Infect Dis 1997; 24 Suppl 1: S Giske CG, Sundsfjord AS, Kahlmeter G, et al. Redefining extended-spectrum beta-lactamases: balancing science and clinical need. J Antimicrob Chemother 2009; 63: Pitout JD, Laupland KB. Extended-spectrum betalactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect Dis 2008; 8: Rodriguez-Bano J, Navarro MD, Romero L, et al. Epidemiology and clinical features of infections caused by extended-spectrum beta-lactamaseproducing Escherichia coli in nonhospitalized patients. J Clin Microbiol 2004; 42: Ben-Ami R, Schwaber MJ, Navon-Venezia S, et al. Influx of extended-spectrum beta-lactamaseproducing enterobacteriaceae into the hospital. Clin Infect Dis 2006; 42: Garau J. Other antimicrobials of interest in the era of extended-spectrum beta-lactamases: fosfomycin, nitrofurantoin and tigecycline. Clin Microbiol Infect 2008; 14 Suppl 1: Bush K, Jacoby GA, Medeiros AA. A functional classification scheme for beta-lactamases and its correlation with molecular structure. Antimicrob Agents Chemother 1995; 39: Naas T, Poirel L, Nordmann P. Minor extendedspectrum beta-lactamases. Clin Microbiol Infect 2008; 14 Suppl 1: Nordmann P, Naas T, Poirel L. Global spread of carbapenemase producing Enterobacteriaceae. Emerg Infect Dis 2011; 17: Naas T, Nordmann P. Analysis of a carbapenemhydrolyzing class A beta-lactamase from Enterobacter cloacae and of its LysR-type regulatory protein. Proc Natl Acad Sci U S A 1994; 91: Woodford N, Dallow JWT, Hill RLR, et al. Ertapenem resistance among Klebsiella and Enterobacter submitted in the UK to a reference laboratory. Int J Antimicrob Agents 2007; 29: Roberts RR, Hota B, Ahmad I, et al. Hospital and societal costs of antimicrobial-resistant infections in a Chicago teaching hospital: implications for antibiotic stewardship. Clin Infect Dis 2009; 49: Nordmann P, Cuzon G, Naas T. The real threat of Klebsiella pneumoniae carbapenemaseproducing bacteria. Lancet Infect. Dis. 2009; 9: Walsh TR. Emerging carbapenemases: A global perspective. Int. J. Antimicrob. Agents. 2010; 36. DOI: /S (10) Poirel L, Potron A, Nordmann P. OXA-48-like carbapenemases: The phantom menace. J Antimicrob Chemother 2012; 67: Yong D, Toleman MA, Giske CG, et al. Characterization of a new metallo-beta-lactamase gene, bla(ndm-1), and a novel erythromycin esterase gene carried on a unique genetic structure in Klebsiella pneumoniae sequence type 14 from India. Antimicrob Agents Chemother 2009; 53: Cohen Stuart J, Leverstein van Hall M, Al Naiemi N. NVMM Guideline Laboratory detection of highly resistant microorganisms (HRMO), version ; Available at: richtlijnen/hrmolaboratory-detection-highlyresistant-microorganisms. 24 Felmingham D, Brown DF. Instrumentation in antimicrobial susceptibility testing. J Antimicrob Chemother 2001; 48 Suppl 1:

28 Chapter 1 25 Leinberger DM, Grimm V, Rubtsova M, et al. Integrated detection of extended-spectrumbeta-lactam resistance by DNA microarray-based genotyping of TEM, SHV, and CTX-M genes. J Clin Microbiol DOI: /JCM Stuart JC, Dierikx C, Naiemi N Al, et al. Rapid detection of TEM, SHV and CTX-M extendedspectrum b-lactamases in Enterobacteriaceae using ligation-mediated amplification with microarray analysis. J Antimicrob Chemother 2010; 65: Livermore DM, Canton R, Gniadkowski M, et al. CTX-M: Changing the face of ESBLs in Europe. J. Antimicrob. Chemother. 2007; 59: Bradford PA. Extended-spectrum betalactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 2001; 14: , table of contents. 29 Canton R, Novais A, Valverde A, et al. Prevalence and spread of extended-spectrum beta-lactamaseproducing Enterobacteriaceae in Europe. Clin Microbiol Infect 2008; 14 Suppl 1: Overdevest I, Willemsen I, Rijnsburger M, et al. Extended-Spectrum B-Lactamase Genes of Escherichia coli in Chicken Meat and Humans, the Netherlands. Emerg Infect Dis 2011; 17: Mouton J, Voss A, Arends J, Bernards on behalf of the ONE study group. S. Prevalence of ESBL in the Netherlands: the ONE study Hawkey PM. Prevalence and clonality of extended-spectrum beta-lactamases in Asia. Clin Microbiol Infect 2008; 14 Suppl 1: Villegas M V, Kattan JN, Quinteros MG, Casellas JM. Prevalence of extended-spectrum betalactamases in South America. Clin Microbiol Infect 2008; 14 Suppl 1: Nicolas-Chanoine MH, Jarlier V. Extendedspectrum beta-lactamases in long-term-care facilities. Clin Microbiol Infect 2008; 14 Suppl 1: Marcade G, Deschamps C, Boyd A, et al. Replicon typing of plasmids in Escherichia coli producing extended-spectrum beta-lactamases. J Antimicrob Chemother 2009; 63: Safdar N, Maki DG. The commonality of risk factors for nosocomial colonization and infection with antimicrobial-resistant Staphylococcus aureus, enterococcus, gram-negative bacilli, Clostridium difficile, and Candida. Ann Intern Med 2002; 136: Lautenbach E, Patel JB, Bilker WB, Edelstein PH, Fishman NO. Extended-spectrum betalactamase-producing Escherichia coli and Klebsiella pneumoniae: risk factors for infection and impact of resistance on outcomes. Clin Infect Dis 2001; 32: Paterson DL, Ko WC, Von Gottberg A, et al. International prospective study of Klebsiella pneumoniae bacteremia: implications of extended-spectrum beta-lactamase production in nosocomial Infections. Ann Intern Med 2004; 140: Davies J, Davies D. Origins and evolution of antibiotic resistance. Microbiol Mol Biol Rev 2010; 74: Wellington EM, Boxall a B, Cross P, et al. The role of the natural environment in the emergence of antibiotic resistance in gram-negative bacteria. Lancet Infect Dis 2013; 13: Mesa RJ, Blanc V, Blanch AR, et al. Extendedspectrum beta-lactamase-producing Enterobacteriaceae in different environments (humans, food, animal farms and sewage). J Antimicrob Chemother 2006; 58: Cohen Stuart J, van den Munckhof T, Voets G, Scharringa J, Fluit A, Hall ML Van. Comparison of ESBL contamination in organic and conventional retail chicken meat. Int J Food Microbiol 2012; 154: Leverstein-van Hall MA, Dierikx CM, Cohen Stuart J, et al. Dutch patients, retail chicken meat and poultry share the same ESBL genes, plasmids and strains. Clin Microbiol Infect 2011; 17: Knapp CW, Dolfing J, Ehlert PA, Graham DW. Evidence of increasing antibiotic resistance gene abundances in archived soils since Env Sci Technol 2010; 44: Heuer H, Schmitt H, Smalla K. Antibiotic resistance gene spread due to manure application on agricultural fields. Curr Opin Microbiol 2011; 14: Ruimy R, Brisabois A, Bernede C, et al. Organic and conventional fruits and vegetables contain equivalent counts of Gram-negative bacteria expressing resistance to antibacterial agents. Environ Microbiol 2010; 12: Buchholz U, Bernard H, Werber D, et al. German outbreak of Escherichia coli O104:H4 associated with sprouts. N Engl J Med 2011; 365:

29 Introduction and outline of the thesis 48 Tangden T, Cars O, Melhus A, Lowdin E. Foreign travel is a major risk factor for colonization with Escherichia coli producing CTX-M-type extendedspectrum beta-lactamases: a prospective study with Swedish volunteers. Antimicrob Agents Chemother 2010; 54: Philippon A, Arlet G, Jacoby GA. Plasmiddetermined AmpC-type beta-lactamases. Antimicrob Agents Chemother 2002; 46: Nadjar D, Rouveau M, Verdet C, et al. Outbreak of Klebsiella pneumoniae producing transferable AmpC-type beta-lactamase (ACC-1) originating from Hafnia alvei. FEMS Microbiol Lett 2000; 187: Warren RE, Harvey G, Carr R, Ward D, Doroshenko A. Control of infections due to extendedspectrum beta-lactamase-producing organisms in hospitals and the community. Clin Microbiol Infect 2008; 14 Suppl 1: Meyer KS, Urban C, Eagan JA, Berger BJ, Rahal JJ. Nosocomial outbreak of Klebsiella infection resistant to late-generation cephalosporins. Ann Intern Med 1993; 119: Maatregelen tegen overdracht van Bijzonder Resistente Micro-Organismen (BRMO). Available at: BRMO.pdf. 54 Kluytmans - van den Bergh M. The prevention paradox of extended-spectrum beta-lactamaseproducing Enterobacteriaceae (ESBL-E): speciesspecific risk and burden of transmission. Abstract #3418 O379 ECCMID Amsterdam,

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33 High prevalence of ESBL-producing Enterobacteriaceae carriage in Dutch community patients with gastrointestinal complaints EA Reuland 1, ITMA Overdevest 2,3, N al Naiemi 1,4, JS Kalpoe 5, MC Rijnsburger 1, SA Raadsen 1, I Ligtenberg-Burgman 5, KW van der Zwaluw 6, M Heck 6, PHM Savelkoul 1, JAJW Kluytmans 1,2 and CMJE Vandenbroucke-Grauls 1 1 Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam 2 Department of Medical Microbiology and Infection Control, Amphia Hospital, Breda 3 Department of Medical Microbiology, St Elisabeth Hospital, Tilburg 4 Laboratory for Medical Microbiology and Public Health, Enschede 5 ATAL Medical Diagnostic Center, Amsterdam 6 Center for Infectious Disease Control, National Institute for Public Health and the Environment, Bilthoven, the Netherlands Clinical Microbiology and Infection 2013 Jun;19(6):542-9

34 Chapter 2 ABSTRACT Objectives The aim of this study was to determine the rate of carriage of ESBL-producing Enterobacteriaceae (ESBL-E) in the community in the Netherlands and to gain understanding of the epidemiology of these resistant strains. Methods Fecal samples from 720 consecutive patients presenting to their general practitioner, obtained in May 2010, and between December 2010 and January 2011, were analyzed for presence of ESBL-E. Species identification and antibiotic susceptibility testing were performed according to the Dutch national guidelines. PCR, sequencing and microarray were used to characterize the genes encoding for ESBL. Strain typing was performed with amplified-fragment length polymorphism (AFLP) and multilocus sequence typing (MLST). Results Seventy-three of 720 (10.1%) samples yielded ESBL-producing organisms, predominantly E. coli. No carbapenemases were detected. The most frequent ESBL was CTX-M-15 (34/73, 47%). Co-resistance to gentamicin, ciprofloxacin and cotrimoxazole was found in (9/73) 12% of the ESBL-E strains. AFLP did not show any clusters, and MLST revealed that CTX- M-15-producing E. coli belonged to various clonal complexes. Clonal complex ST10 was predominant. Conclusions This study showed a high prevalence of ESBL-producing Enterobacteriaceae in Dutch primary care patients with presumed gastrointestinal discomfort. Hence, also in the Netherlands, a country with a low rate of consumption of antibiotics in humans, resistance due to the expansion of CTX-M ESBLs, in particular CTX-M-15, is emerging. The majority of ESBL-producing strains do not appear to be related to the international clonal complex ST

35 High prevalence of ESBL-producing Enterobacteriaceae carriage INTRODUCTION Due to the extensive use of beta-lactam antibiotics in human medicine, beta-lactamases have co-evolved with them. 1 Extended-spectrum beta-lactamases (ESBLs) are the main source of acquired antibiotic resistance in Gram-negative bacteria and are of particular concern. 2 These enzymes have a broad spectrum of activity against almost all beta-lactam antibiotics. The genes that encode ESBLs are transferred very efficiently due to their location on plasmids. Furthermore, these ESBL-encoding plasmids frequently bear resistance genes for additional antibiotic classes, thereby posing a significant challenge to antimicrobial therapy. 3,4 Recently, a major increase in the prevalence of ESBL has been observed, mainly due to an increase of CTX-M type ESBLs. 2 Today organisms producing these enzymes are the most common type of ESBL-producing bacteria found in most areas of the world. 5 The classic SHV and TEM enzymes, associated with nosocomial outbreaks, are substituted by CTX-M enzymes, principally in community-acquired infections caused by Escherichia coli. 6 This major shift in ESBL epidemiology is observed both in Europe and in other continents. 5,6 An increase in community-onset infections with ESBL-E due to CTX-M-producing E. coli is a large problem in many European countries, for example in Spain and France. 3,5 Especially, CTX-M-15 is predominant in community-acquired infections. 2,7,8 The Netherlands is well known for its low rate of resistance, and this also applies to resistance to third-generation cephalosporins, a surrogate marker for ESBL production (EARS-Net, Therefore it is interesting to gain insight into the prevalence of ESBLs in a country with a prudent use of antibiotics in human medicine (ESAC-Net. Net/). The presence of ESBL-producing Gram-negative bacteria in Dutch retail meat found in recent studies is quite worrying. 9,10 To the best of our knowledge, no data are available on the prevalence of carriage of ESBL-producing Enterobacteriaceae (ESBL-E) in the Dutch community. The aim of this study was to determine the prevalence of ESBL carriage in the primary care population in the region of Amsterdam (a densely populated urban area) and Brabant (a more rural area), to assess the susceptibility of these isolates to common antibiotics that are important for treating community-acquired infections, to characterize the ESBL genes and plasmids involved, and to type the ESBL-positive strains to gain understanding of the epidemiology of this emerging resistance in the Dutch outpatient population. 33

36 Chapter 2 METHODS Data collection/study design Fecal samples, obtained between 12 April and 19 May 2010, and between 21 November 2010 and 9 January 2011, from patients presenting to their general practitioner (GP) with mild gastrointestinal discomfort and/or diarrhea for more than 3 weeks were analyzed. Samples were collected at the ATAL Medical Diagnostic Centre, a laboratory servicing GPs in Amsterdam, and the Microbiological Laboratory of Sint Elisabeth Hospital in Tilburg, a laboratory servicing GPs in the region of Brabant. Fecal samples were inoculated in trypticase soy enrichment broth. Screening for ESBL-producing Enterobacteriaceae (ESBL-E) was performed by inoculation onto a selective screening agar, the EbSA ESBL screening agar (Cepheid Benelux, Apeldoorn, the Netherlands). 11,12 All broths and plates were incubated overnight at 37 C. Antimicrobial susceptibility testing Species identification and antibiotic susceptibility testing of colonies growing on the EbSA plates were performed with the Vitek 2 system (Vitek ID and Vitek AST; biomérieux, Marcy l Etoile, France). The MIC breakpoints used for interpreting the results were according to the criteria of the Clinical and Laboratory Standards Institute (CLSI). 13 ESBL production was confirmed with a combination disk diffusion test (Rosco, Taastrup, Denmark) and the E-test on Mueller-Hinton agar, interpreted according to the Dutch national guidelines. 14 Molecular characterization and ESBL typing The presence of ESBL genes was confirmed by molecular analysis of all phenotypically confirmed ESBL-positive strains. Bacterial DNA was isolated with the QIAamp DNA mini kit (Qiagen, Venlo, the Netherlands). Isolates obtained in Amsterdam were screened for ESBL resistance genes at the VUmc by Check-KPC ESBL microarray to identify CTX-M, TEM and SHV ESBL genes (Check-Points Health BV, Wageningen, the Netherlands). 15 Isolates obtained at Amphia Hospital were screened with Check-MDR CT103 (Check-Points Health BV), a newly developed microarray that enables the detection of two commonly encountered ESBLs: CTX-M-1 and CTX-M-15. In isolates obtained in Amsterdam ESBL-encoding genes were characterized by polymerase chain reaction (PCR) at the VUmc, followed by sequencing (BaseClear, Leiden, the Netherlands), as described by Naiemi et al. 16 Sequences were analyzed with BioNumerics software (version 6.5; Applied Maths, Sint-Martens-Latem, Belgium) and compared with sequences in the NCBI database ( gov/blast) and Lahey ( 34

37 High prevalence of ESBL-producing Enterobacteriaceae carriage Characterization of plasmids Identification of plasmids was performed by PCR-based replicon typing for the eight most prevalent plasmids. 17 This method allows the examination of plasmids conferring drug resistance by typing them by incompatibility groups in a multiplex PCR setting. Epidemiological typing Seventy ESBL-positive E. coli strains were analyzed for genetic relatedness by amplifiedfragment length polymorphism (AFLP). This DNA fingerprinting technique and the protocol used has been described by Savelkoul et al. 18 AFLP banding patterns were analyzed as described previously with BioNumerics software (Applied Maths). Multilocus sequencing typing (MLST) was performed on all the E. coli isolates by using seven conserved housekeeping genes (adka, fumc, gyrb, icd, mdh, pura and reca) as described by Wirth et al. 19 The MLST protocol is detailed at Clonal complexes were determined by including whole E. coli MLST data using eburst v3 ( eburst.mlst.net). Statistical analyses Statistical analyses were performed with SPSS, version Principal components analysis (PCA) was performed with BioNumerics version 6.5. RESULTS In total, 720 fecal samples were obtained from 720 consecutive patients presenting to their GP with complaints of gastrointestinal discomfort. Analysis of the samples for diagnosis was performed separately in a routine setting. These samples were considered to be community based because the specimens were obtained from a laboratory serving only general practitioners. Data regarding the patients history were not available. The median age of patients was 46 years (range, 2 87); 53% were female. Patients lived in different geographical areas and were not institutionalized. In the region of Amsterdam, 50 out of 471 (10.6%, % CI) samples yielded ESBL-E: 49 Escherichia coli isolates and one Shigella sonnei isolate. In the region of Brabant 23 out of 249 (9.2%, % CI) samples yielded ESBL-E (Table 1). These included 21 E. coli and two Klebsiella pneumoniae isolates. Hence the frequency of ESBL-producing isolates was the same in both regions. No strains with reduced sensitivity to imipenem or meropenem were detected. The microarray revealed that both in Amsterdam and in Brabant the isolates contained genes belonging to the CTX-M family; bla CTX-M-15 was predominant, found in 34/73 (47%) isolates (see also Table 2). We also performed PCR and sequencing on the 50 strains 35

38 Chapter 2 isolated in Amsterdam. This showed four bla CTX-M-1, 24 bla CTX-M-15, one bla CTX-M-14, seven bla CTX-M-, four bla, one bla, one bla and one bla genes. One gene belonging to 14b CTX-M-27 TEM-52 SHV-2a SHV-12 the CTX-M-1 family remained unidentified. No difference in the distribution of these genes in the two regions was seen. Table 1 - Number of patients and ESBL-producing bacterial isolates in the urban and rural communities Urban N (%) Rural N (%) Number of patients ESBL-positive bacterial isolates 50 (10.6) 23 (9.2) Table 2 - Distribution of ESBL genes and plasmids ESBL group * N Plasmids ColE FrepB FIB ColEtp IncI1 FIA R FIIs CTX-M-1 group CTX-M-2 group CTX-M-9 group SHV TEM Total 72* * The genes belonging to the CTX-M-1 group were 6 bla CTX-M-1 and 34 bla CTX-M-15 genes. One gene belonging to the CTX-M-1 group remained unidentified. TEM and SHV were ESBL (no wild-type). The majority of the isolates showed co-resistance to cotrimoxazole, followed by ciprofloxacin and gentamicin. A summary of the co-resistances is shown in Table 3. Twelve per cent (9/73) of the ESBL-producing isolates were multiresistant (i.e. resistant to at least one agent in three or more antimicrobial categories (aminoglycosides, quinolones and cotrimoxazole)). 20 Thirty per cent (22/73) of strains were intermediately susceptible to nitrofurantoin. All isolates were susceptible to meropenem and imipenem. Table 3 - Co-resistances in ESBL-producing isolates. Co-resistance ESBL N=73 % cotrimoxazole ciprofloxacin gentamicin gentamicin/ciprofloxacin/cotrimoxazole

39 High prevalence of ESBL-producing Enterobacteriaceae carriage LM-PCR LM-PCR CTX-M-15, TEM 1. CTX-M-15, TEM 1. CTX-M-15. CTX-M-15. CTX-M? (CTX-M-1 fam.. SHV 12. SHV 12, TEM 1. CTX-M-2 family. CTX-M-14b, TEM 1. CTX-M-14b, TEM 1. CTX-M-15. CTX-M-15. CTX-M-9 family. CTX-M-15. CTX-M-14b, TEM 1. CTX-M-15. CTX-M-14b, TEM 1. CTX-M-15. CTX-M-15, TEM 1. CTX-M-15. CTX-M-9 family. SHV 12, TEM 1. CTX-M-9 family. SHV 12, TEM 1. CTX-M-15. CTX-M-1. TEM 52. CTX-M-9 family. CTX-M-15, TEM 1. CTX-M-1, TEM 1. CTX-M-14b. CTX-M-15, TEM 1. CTX-M-1. SHV 12, TEM-1. CTX-M-15. SHV. SHV 12. CTX-M-15. CTX-M-1, TEM 1. SHV 12. CTX-M-1. CTX-M-27, TEM 1. SHV 2A, TEM 1. CTX-M-15. CTX-M-9 family. CTX-M-15. CTX-M-15. CTX-M-15. TEM. SHV. CTX-M-2 family. CTX-M-15, TEM 1. CTX-M-14b. CTX-M-15, TEM 1. CTX-M-15. CTX-M-15. CTX-M-15. CTX-M-1. CTX-M-15, TEM 1. CTX-M-15, TEM 1. CTX-M-27, TEM 1. CTX-M-15, TEM 1. CTX-M-15. CTX-M-14b, TEM 1. CTX-M-27. CTX-M-27. CTX-M-9 family. CTX-M-15. CTX-M-15. CTX-M-14, TEM 1 Amsterdam Amsterdam Amsterdam Amsterdam Amsterdam Amsterdam Amsterdam Brabant Amsterdam Amsterdam Brabant Brabant Brabant Amsterdam Amsterdam Amsterdam Amsterdam Amsterdam Amsterdam Amsterdam Brabant Amsterdam Brabant Amsterdam Brabant Amsterdam Amsterdam Brabant Amsterdam Amsterdam Amsterdam Amsterdam Brabant Amsterdam Amsterdam Brabant Amsterdam Brabant Amsterdam Amsterdam Amsterdam Amsterdam Amsterdam Brabant Brabant Amsterdam Amsterdam Amsterdam Brabant Brabant Brabant Amsterdam Amsterdam Amsterdam Brabant Amsterdam Amsterdam Brabant Amsterdam Amsterdam Amsterdam Amsterdam Brabant Amsterdam Amsterdam Amsterdam Brabant Amsterdam Brabant Amsterdam Figure 1 - Dendrogram showing the relatedness of AFLP patterns. Seventy ESBL-positive E. coli strains were analyzed for genetic relatedness by amplified fragment length polymorphism (AFLP). 37

40 Chapter 2 The ESBL-producing strains were genotyped by AFLP to assess their diversity. The AFLPbased dendogram showed a few pairs of isolates with identical AFLP patterns (Figure 1). All patients with strains with related AFLP patterns lived in different geographical areas. Further analysis by PCA did not reveal any clusters (Figure not shown) CTX-M-1 type CTX-M-2 family CTX-M-9 family CTX-M-15 type CTX-M-1 family (remained unidentified by sequencing) SHV TEM Figure 2 - Multilocus sequence typing of E. coli isolates (n = 70). The numbers indicate the different sequence types. Thick connecting lines indicate single-locus variants; thin connecting lines indicate variants with two or three loci difference; dashed connecting lines indicate variants with four loci difference; five loci differences are indicated by dotted connecting lines. Shadowing indicates that more than one sequence type belongs to the same complex. 38

41 High prevalence of ESBL-producing Enterobacteriaceae carriage The results of MLST are shown in Figure 2. Multilocus sequence typing revealed 43 different sequence types, and included nine new sequence types not present yet in the E. coli MLST database. Most isolates belonged to sequence types ST38 (seven isolates; 10%), ST131 (six isolates; 8.6%), ST648 (five isolates; 7.1%) and ST10 (four isolates; 5.7%). The main clonal complexes according to the MLST database (including sequence types with one locus difference) were ST10 (12 isolates; 17.1%) and ST38 (nine isolates; 12.8%). All but one cluster harbored different ESBL genes. CTX-M-15 was scattered over all the ST types. There was no difference in MLST types between Amsterdam and Brabant (Figure 3). The distribution of ESBL genes and plasmids is described in Table 2. ColE, FIB and FIA were the most prevalent plasmids Amsterdam Brabant Figure 3 - Multilocus sequencing typing showing several clusters in E. coli isolates (n = 70) obtained from fecal samples: Amsterdam vs. Brabant. 39

42 Chapter 2 DISCUSSION This study showed that one out of ten Dutch outpatients with gastrointestinal complaints carried ESBL-producing Enterobacteriaceae in their feces. This was an unexpected high prevalence because the Netherlands is a country well known for its prudent antimicrobial use in human clinical practice, both in the outpatient setting and in the hospital (ESAC-Net, No carbapenemaseproducing strains were detected. It is well-known that the prevalence of ESBL-E differs markedly between countries. To date a high prevalence is found in clinical isolates in southern European countries, in Turkey and in India while the prevalence is low in northern European countries, including the Netherlands (EARS-Net, activities/surveillance/ears-net/). 6,21 Few studies have investigated the fecal carriage rate of ESBL-E in non-hospitalized patients. Valverde et al. showed that the fecal carriage rate of ESBL-E in a Spanish community was 5.5% in In the same study a prevalence of 3.7% was seen in healthy volunteers. 22 Hence the prevalence we measured is high compared with surveys performed previously in surrounding European countries. Possibly, the high rate we measured in our more recent study is due to the steep increase in ESBL-producing strains that is being observed over the last few years all over the world. The prevalence of rectal carriage among hospitalized patients in the Netherlands in previous years was lower. Before 2000 a prevalence of <1% was recorded in Dutch hospitals. This increased to 4 8% after ,24 Recently, Overdevest et al. found a percentage of 6% in hospitalized patients and 4% in patients at time of admission to the hospital. 10 This high percentage of carriage of ESBL-producing bacteria on admission already pointed towards a community reservoir. Initially, ESBL-producing Enterobacteriaceae were considered an inhospital problem, but now this study also reveals an unexpected increase in the Dutch community. Therefore, our results confirm the worrisome element that a continuous influx from the community into the hospital might be possible. 3,4 In our study, the most prevalent ESBLs were CTX-M. This is consistent with the worldwide dissemination of this type of ESBL and is comparable with the CTX-M pandemic in the community in other European countries. 3,6,8 The most prevalent CTX-M ESBL in our survey was CTX-M-15, again as noticed elsewhere. 6,7,21,25 In several countries the expansion of CTX- M-15-producing E. coli is due to the worldwide pandemic clone ST In contrast, the E. coli strains that we identified belonged to multiple sequence type clonal complexes and the presence of CTX-M-15 in these community-acquired isolates was scattered over different clusters. AFLP and PCA confirmed the data obtained with MLST, and showed that there was no epidemiological relationship between the strains. Next to clonal dispersion, the acquisition of multidrug-resistant plasmids plays a pivotal role in the dissemination of CTX-M-15-producing ESBLs. Various replicons, especially those 40

43 High prevalence of ESBL-producing Enterobacteriaceae carriage widely distributed among E. coli strains, could be involved in part of this dissemination process. It has been proposed that most of the CTX-M-15 enzymes are encoded on IncF replicons (FIA, FIB and FII). 27 Indeed, also in our study IncF replicons, FIB and FIA-type plasmids, were associated with the presence of bla CTX-M-15. Taken together, MLST and plasmid replicon typing point to both dispersion of several different clones and to spread of mobile genetic elements as drivers of the dissemination of ESBL genes in the Dutch community. The distribution of ESBL genes and plasmids in carriers of ESBL-positive E. coli isolates in this outpatient population differs from the distribution described recently in E. coli strains recovered from patients from hospitals and long-term care facilities in the Netherlands in 2009 and In these studies bla CTX-M-1 and IncI1 were the most frequent genes and plasmids. 9,10 It has been postulated that these are acquired through contaminated poultry, because Dutch chicken meat has been shown to be heavily colonized with E. coli strains containing bla CTX-M-1 and IncI1: 94% of chickens are colonized with these strains. 9,10,28 In human isolates from other countries bla CTX-M-15 is the most frequent gene. 7 In our patient population, strains producing CTX-M-15 were predominant. Possibly, the difference between our study and the previous Dutch studies is due to the difference in patient populations; we analyzed fecal samples from outpatients presenting to their GP with complaints of gastrointestinal discomfort. In Dutch general practice feces cultures are only requested for patients with gastrointestinal complaints that last for more than 10 days or gastrointestinal complaints after travel to foreign countries, especially to the (sub) tropics. 29 Various studies show that foreign travel, especially to countries with a high prevalence of ESBL-E, is a risk factor for colonization with ESBL-E A high prevalence of fecal carriage of ESBL-producing strains is observed in particular in patients with travelers diarrhea We have no data on travel history, previous use of antibiotics or recent hospitalization for the individual patients in our study, but Dutch general practitioners very seldom prescribe antibiotics for treatment of gastrointestinal complaints, according to the algorithms laid down in their own professional standards. 29 Thus, knowing that diagnostics for diarrhea is mainly performed after foreign travel, and that use of antibiotics in the treatment of diarrhea is very unusual in Dutch general practice, it seems likely that foreign travel might be responsible for at least part of the prevalence of ESBL-E in Dutch outpatients. Whatever the source of the resistance, however, the prevalence of ESBL-E in this specific patient population was worryingly high. At the same time, it is reassuring that carbapenemase-producing strains were still absent in the community. The association of ESBL production with multidrug resistance adds to the magnitude of the problem. 5,22 In this study we noted that nearly half of the ESBL-producing strains were resistant to ciprofloxacin, and nearly three-quarters were resistant to cotrimoxazole. None of the isolates were resistant to nitrofurantoin, the drug currently recommended for uncomplicated urinary tract infections in the Netherlands. 41

44 Chapter 2 In conclusion, this study showed an unexpected high prevalence of ESBL-E in Dutch outpatients presenting to their GP with gastrointestinal complaints. The present study emphasizes that multidrug-resistant CTX-M-producing (in particular CTX-M-15) E. coli are present in the community even in the Netherlands, a country well known for its prudent antimicrobial use in human medicine. Therefore it is important to monitor systematically the epidemiology of ESBL-E in hospitals, in the community and in other reservoirs such as food and the environment. ACKNOWLEDGEMENTS We would like to thank Martijn van Luit (RIVM, Bilthoven, the Netherlands) for the MLST experiments. 42

45 High prevalence of ESBL-producing Enterobacteriaceae carriage REFERENCES 1 Medeiros AA. Evolution and dissemination of beta-lactamases accelerated by generations of beta-lactam antibiotics. Clin Infect Dis 1997; 24 Suppl 1: S Pitout JD, Laupland KB. Extended-spectrum betalactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect Dis 2008; 8: Rodriguez-Bano J, Navarro MD, Romero L, et al. Epidemiology and clinical features of infections caused by extended-spectrum beta-lactamaseproducing Escherichia coli in nonhospitalized patients. J Clin Microbiol 2004; 42: Ben-Ami R, Schwaber MJ, Navon-Venezia S, et al. Influx of extended-spectrum beta-lactamaseproducing enterobacteriaceae into the hospital. Clin Infect Dis 2006; 42: Rossolini GM, D&apos;Andrea MM, Mugnaioli C. The spread of CTX-M-type extendedspectrum β-lactamases. Clin. Microbiol. Infect. 2008; 14: Livermore DM, Canton R, Gniadkowski M, et al. CTX-M: Changing the face of ESBLs in Europe. J. Antimicrob. Chemother. 2007; 59: Coque TM, Novais A, Carattoli A, et al. Dissemination of clonally related Escherichia coli strains expressing extended-spectrum betalactamase CTX-M-15. Emerg Infect Dis 2008; 14: Arpin C, Quentin C, Grobost F, et al. Nationwide survey of extended-spectrum {beta}-lactamaseproducing Enterobacteriaceae in the French community setting. J Antimicrob Chemother 2009; 63: Leverstein-van Hall MA, Dierikx CM, Cohen Stuart J, et al. Dutch patients, retail chicken meat and poultry share the same ESBL genes, plasmids and strains. Clin Microbiol Infect 2011; 17: Overdevest I, Willemsen I, Rijnsburger M, et al. Extended-Spectrum B-Lactamase Genes of Escherichia coli in Chicken Meat and Humans, the Netherlands. Emerg Infect Dis 2011; 17: Overdevest IT, Willemsen I, Elberts S, Verhulst C, Kluytmans JA. Laboratory detection of extended-spectrum-beta-lactamase-producing Enterobacteriaceae: evaluation of two screening agar plates and two confirmation techniques. J Clin Microbiol 2011; 49: Al Naiemi N, Murk JL, Savelkoul PHM, Vandenbroucke-Grauls CMJ, Debets-Ossenkopp YJ. Extended-spectrum beta-lactamases screening agar with AmpC inhibition. Eur J Clin Microbiol Infect Dis 2009; 28: CLSI. Clinical and Laboratory Standard Institute. Performance standards for antimicrobial susceptibility testing. CLSI M100-S18. Wayne, PA, USA al Naiemi N, Cohen Stuart J, Leverstein van Hall M. NVMM guideline of the Dutch Society for Medical Microbiology for screening and confirmation of extended-spectrum beta-lactamases (ESBLs) in Enterobacteriaceae [in Dutch]. nvmm.nl/richtlijnen/esbl Stuart JC, Dierikx C, Naiemi N Al, et al. Rapid detection of TEM, SHV and CTX-M extendedspectrum b-lactamases in Enterobacteriaceae using ligation-mediated amplification with microarray analysis. J Antimicrob Chemother 2010; 65: Al Naiemi N, Duim B, Savelkoul PHM, et al. Widespread transfer of resistance genes between bacterial species in an intensive care unit: Implications for hospital epidemiology. J Clin Microbiol 2005; 43: Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. Identification of plasmids by PCRbased replicon typing. J Microbiol Methods 2005; 63: Savelkoul PH, Aarts HJ, de Haas J, et al. Amplifiedfragment length polymorphism analysis: the state of an art. J Clin Microbiol 1999; 37: Wirth T, Falush D, Lan R, et al. Sex and virulence in Escherichia coli: an evolutionary perspective. Mol Microbiol 2006; 60: Magiorakos AP, Srinivasan A, Carey RB, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 2012; 18: Hawkey PM. Prevalence and clonality of extended-spectrum beta-lactamases in Asia. Clin Microbiol Infect 2008; 14 Suppl 1: Valverde A, Coque TM, Sanchez-Moreno MP, Rollan A, Baquero F, Canton R. Dramatic increase in prevalence of fecal carriage of extended-spectrum 43

46 Chapter 2 beta-lactamase-producing Enterobacteriaceae during nonoutbreak situations in Spain. J Clin Microbiol 2004; 42: Mouton J, Voss A, Arends J, Bernards on behalf of the ONE study group. S. Prevalence of ESBL in the Netherlands: the ONE study al Naiemi N, Bart A, de Jong MD, et al. Widely distributed and predominant CTX-M extendedspectrum beta-lactamases in Amsterdam, The Netherlands. J Clin Microbiol 2006; 44: Pitout JD. Infections with extended-spectrum beta-lactamase-producing enterobacteriaceae: changing epidemiology and drug treatment choices. Drugs 2010; 70: Peirano G, Pitout JD. Molecular epidemiology of Escherichia coli producing CTX-M betalactamases: the worldwide emergence of clone ST131 O25:H4. Int J Antimicrob Agents 2010; 35: Marcade G, Deschamps C, Boyd A, et al. Replicon typing of plasmids in Escherichia coli producing extended-spectrum beta-lactamases. J Antimicrob Chemother 2009; 63: Overdevest I, Willemsen I, Rijnsburger M, et al. Extended-Spectrum B-Lactamase Genes of Escherichia coli in Chicken Meat and Humans, the Netherlands. Emerg Infect Dis 2011; 17: NHG-Standaarden. Standards of the Dutch College of General Practitioners [in Dutch] Tangden T, Cars O, Melhus A, Lowdin E. Foreign travel is a major risk factor for colonization with Escherichia coli producing CTX-M-type extendedspectrum beta-lactamases: a prospective study with Swedish volunteers. Antimicrob Agents Chemother 2010; 54: Laupland KB, Church DL, Vidakovich J, Mucenski M, Pitout JD. Community-onset extended-spectrum beta-lactamase (ESBL) producing Escherichia coli: importance of international travel. J Infect 2008; 57: Tham J, Odenholt I, Walder M, Brolund A, Ahl J, Melander E. Extended-spectrum beta-lactamaseproducing Escherichia coli in patients with travellers diarrhoea. Scand J Infect Dis 2010; 42:

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49 Prevalence and risk factors for carriage of ESBL-producing Enterobacteriaceae in Amsterdam EA Reuland 1, N al Naiemi 1,2,3, AM Kaiser 1, M Heck 4, JAJW Kluytmans 1,5,6, PHM Savelkoul 1, PJM Elders 7 and CMJE Vandenbroucke-Grauls 1 1 Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam 2 Laboratory for Medical Microbiology and Public Health, Hengelo 3 Medical Microbiology and Infection Control, Ziekenhuisgroep Twente, Almelo 4 Center for Infectious Disease Control, National Institute for Public Health and the Environment, Bilthoven 5 Department of Medical Microbiology and Infection Control, Amphia Hospital, Breda 6 Department of Medical Microbiology, St Elisabeth Hospital, Tilburg 7 EMGO Institute for Health and Care Research, VU University Medical Center, Amsterdam, The Netherlands Journal of Antimicrobial Chemotherapy 2016 Apr;71(4):

50 Chapter 3 ABSTRACT Objectives The objectives of this study were to determine the prevalence of carriage of ESBLproducing Enterobacteriaceae (ESBL-E) in a representative sample of the general adult Dutch community, to identify risk factors and to gain understanding of the epidemiology of these resistant strains. Methods Adults enrolled in five general practices in Amsterdam were approached by postal mail and asked to fill in a questionnaire and to collect a fecal sample. Samples were analyzed for the presence of ESBL-E. ESBL genes were characterized by PCR and sequencing. Strains were typed using MLST and amplified fragment length polymorphism (AFLP) and plasmids were identified by PCR-based replicon typing. Risk factors for carriage were investigated by multivariate analysis. Results ESBL-E were found in 145/1695 (8.6%) samples; 91% were Escherichia coli. Most ESBL genes were of the CTX-M group (bla CTX-M-1 and bla CTX-M-15 ). MLST ST131 was predominant and mainly associated with CTX-M-15-producing E. coli. One isolate with reduced susceptibility to ertapenem produced OXA-48. In multivariate analyses, use of antimicrobial agents, use of antacids and travel to Africa, Asia and Northern America were associated with carriage of ESBL-E, in particular strains with bla CTX-M-14/15. Conclusions This study showed a high prevalence of ESBL-E carriage in the general Dutch community. Also, outside hospitals, the use of antibiotics was a risk factor; interestingly, use of antacids increased the risk of carriage. A major risk factor in the general population was travel to countries outside Europe, in particular to Asia, Africa and Northern America. 48

51 Prevalence and risk factors for ESBL in Amsterdam INTRODUCTION Resistance to beta-lactam antibiotics due to ESBLs has become a common problem worldwide. 1 The prevalence of this resistance mechanism has increased rapidly, even in countries known for prudent antibiotic use. 2 In a previous study we showed that over 10% of Dutch community patients with gastrointestinal complaints carry ESBL-producing Enterobacteriaceae (ESBL-E) in their gastrointestinal tract. 3 This is remarkable, because the Netherlands is a country with low antibiotic use in humans and has among the lowest resistance rates in clinical isolates in Europe. 2 This triggered us to perform the present study, which focused on the prevalence and molecular epidemiology of carriage of ESBL-E in the general population and on risk factors for carriage. METHODS Study design and data collection For this cross-sectional study we approached all adult persons (individuals aged 18 years), excepting those who were terminally ill, present in the databases of five general practices (~10000 persons), affiliated to the Academic General Practice Network (AGPN), VU University Medical Center, Amsterdam. In the Netherlands, citizens are registered with a general practitioner, regardless of health status. The database therefore is a representative sample of the general population. Individuals were approached by postal mail with a questionnaire, an informed consent form and a container for a fecal sample or perineal swab (according to their preference). Samples were returned in transport medium (Copan Italia, Brescia, Italy) between June 2011 and November The questionnaire asked about sampling date, sample type (perineal swab or fecal sample), age, gender, profession, country of birth of the participant and his/her parents, years living in the Netherlands, admission to a (foreign) hospital, healthcare institution or long-term care facility and travel to foreign countries, all in the previous 12 months. Data on antimicrobial, antacid and corticosteroid use and comorbid conditions in the past 12 months were extracted from the database of the AGPN. Please see the Supplementary data (available at JAC Online) for items included in the questionnaire and data extracted from the AGPN database. Fifty ESBL-positive participants were asked for participation of their household members, with the same questionnaires and request for samples. The medical ethics committee (METc ID NL ) of the VU University Medical Center approved the study (NTR Trial ID NTR2453). 49

52 Chapter 3 ESBL detection Samples were inoculated in selective enrichment broth (trypticase soy broth with ampicillin). After overnight incubation (37 C) an aliquot was inoculated on EbSA-ESBL screening agar (Cepheid Benelux, Apeldoorn, The Netherlands) and on blood agar. 4,5 Growth on the blood agar plate indicated the sample was suitable for analysis. Three colonies of each distinct morphotype on the EbSA-ESBL agar were characterized. ESBL production was confirmed by combination disc diffusion test with both cefotaxime and ceftazidime, with and without clavulanic acid (Rosco, Taastrup, Denmark), interpreted according to the Dutch national guideline. 6 Species identification and antibiotic susceptibility testing were performed with Vitek 2 (biomérieux, Marcy-l Etoile, France). MIC breakpoints were according to EUCAST. 7 Reduced susceptibility (MIC 0.25 mg/l) to ertapenem (Etest, biomérieux) indicated the possible presence of a carbapenemase. ESBL- and carbapenemase-encoding genes were characterized by PCR and sequencing (BaseClear, Leiden, The Netherlands) Sequences were analyzed with BioNumerics software (version 6.6; Applied Maths, Sint-Martens-Latem, Belgium) and compared with sequences in the NCBI ( and Lahey database ( Molecular typing E. coli strains were typed by MLST ( Clonal complexes were assigned using eburst v3 ( A subset of E. coli strains was typed by amplified fragment length polymorphism (AFLP). 12 Plasmids were identified by PCR-based replicon typing, as described by Carattoli and adapted by Boot et al. 13 Analysis of risk factors and statistical methods For a case control analysis of risk factors, cases were carriers of ESBL-E and controls were persons free of ESBL-E. Statistical analyses were performed with Statistical Package for the Social Sciences, version 20.0 (SPSS, Chicago, IL, USA). Possible risk factors were analyzed by univariate and multivariate logistic regression. ORs and 95% CIs were calculated. 50

53 Prevalence and risk factors for ESBL in Amsterdam RESULTS Participants Of 7000 persons approached, 1695 (24.2%) returned the questionnaire with a completed consent form and a specimen. Participants lived in the region of Amsterdam. Age and gender characteristics are given in Table 1. Prevalence of carriage of ESBL-E and ESBL gene characterization ESBL-E were detected in 145 of 1695 samples (8.6%, 95% CI 7.3% 10.0%): 132 (91.0%) Escherichia coli, 11 (7.6%) Klebsiella pneumoniae, 1 Enterobacter cloacae (0.7%) and 1 Serratia plymuthica (0.7%). The presence of genes encoding ESBL was confirmed in all phenotypically ESBL-producing strains (Table 2); these genes comprised mainly bla CTX-M-15 and bla CTX-M-1. Co-resistance to other antibiotics was common: 33% of strains were multiresistant as defined by Magiorakos et al. 14 (Table S1, available as Supplementary data at JAC Online). No strains with reduced susceptibility to imipenem or meropenem were found; one E. coli strain had reduced susceptibility to ertapenem (MIC 0.75 mg/l); this strain carried bla OXA-48 and bla CTX-M-14. No difference was found in detection rate for fecal samples compared with perineal swabs (OR 1.0, 95% CI ). The prevalence of carriage of ESBL-E in participants not using antibiotics (N.1294) was 7.4% (95% CI 6.0% 8.9%). Table 1 - Participant characteristics and main risk factors for ESBL-E carriage in univariate analysis Risk factor Cases Controls OR 95% CI Age, median (range); N=129 and (20 90) 50 (18 95) NA NA Female, n (%); N=129 and (58.1) 852 (61.2) Use of antibiotics, n (%); N=129 and (25.6) 195 (14.0) PPIs or H2 blockers, n (%); N=129 and (21.7) 166 (11.9) Travel to, n (%) a Africa; N=111 and ( (7.1) Latin America/Caribbean; N=109 and (11.0) 112 (8.9) Northern America; N=106 and (21.7) 133 (10.6) Asia; N=118 and (33.1) 247 (18.9) Australia/New Zealand; N=102 and (0) 18 (1.5) NA NA NA, not applicable; PPIs, proton-pump inhibitors. a Number of patients who travelled to WHO region/total number of patients with exclusion of those patients that also travelled to one of the other WHO regions or did not travel outside the Netherlands. 51

54 Chapter 3 MLST MLST showed 47 different STs, 6 of which represented new types. Types ST131 (21 isolates; 15.9%), ST10 (18 isolates; 13.6%) and ST38 (9 isolates; 6.8%) were most frequent. ST10 was the main clonal complex, including 26 isolates (19.7%) (Figure S1). MLST ST131 was mainly associated with CTX-M-15-producing E. coli. Household members Fifty carriers volunteered 41 household members, of which five (12.2%, 95% CI 4.9% 26.0%) were carriers of ESBL-E. Figure 1 and Table 3 present the distribution of ESBL genes and plasmids within households. Three of five clusters of isolates from single households had identical AFLP patterns and shared the same ESBL genes and plasmids. In cluster A, two E. coli strains shared an ESBL gene and an Inc1 plasmid, but each contained an additional plasmid, resulting in one band difference in the AFLP pattern. E. coli strains in cluster E also belonged to the same CTX-M-1 family, however had different ESBL genes, did not share plasmids, and the AFLP pattern was different. Table 2 - ESBL-encoding genes ESBL family ESBL gene/type n CTX-M-1 bla CTX-M bla CTX-M-15 + bla TEM-52 1 bla CTX-M-1 25 bla CTX-M-15 + bla SHV-12 1 bla CTX-M-3 4 CTX-M-2 bla CTX-M-2 2 CTX-M-9 CTX-M 9 group 1 a bla CTX-M bla CTX-M-9 4 bla CTX-M-27 5 CTX-M b bla CTX-M 2 TEM and SHV bla SHV-12 5 bla TEM-52 6 bla TEM-52 + bla SHV-12 1 Other bla CTX-M-21 1 bla CTX-M-22 3 bla CTX-M-32 3 bla CTX-M-55 3 Total 145 a One isolate also encoded OXA-48. b Exact subtype of two CTX-M genes remained unresolved by sequencing. 52

55 Prevalence and risk factors for ESBL in Amsterdam LM-PCR LM-PCR TY TY TY TY 8927 TY 8933 TY 8892 TY 8896 TY 8851 TY 8853 TY 8879 TY 8881 TY 4970 E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli E. coli A B C D E E. vulneris Figure 1 - AFLP household members. TY numbers are used for numbering laboratory strains; Escherichia vulneris (DSM 4564) and TY (ATCC 35218) are reference strains used for AFLP. LM-PCR, ligation-mediated PCR

56 Chapter 3 Table 3 - Household members: strains and plasmids Cluster Strain a Gene(s) Plasmid b Inc I1 FrepB ColE FIA Y B/O A TY bla CTX-M TY bla CTX-M B TY8927 bla CTX-M-14/ TY8933 bla CTX-M-14/ C TY8892 bla CTX-M TY8896 bla CTX-M D TY8851 bla CTX-M TY8853 bla CTX-M E TY8879 bla CTX-M-3/TEM TY8881 bla CTX-M-3/TEM a Laboratory strain numbers. b Plasmids R, ColEtp, FIIs, FIB, P, A/C, U, HI1, L/M, HI2, W, T, N, X, F/C and K were not detected Risk factors For the case control analysis, we included 1522 (129 ESBL-E carriers and 1393 non-carriers) of the 1695 participants who sent in a sample with the questionnaire and for whom data from the electronic database of the AGPN were available. Table 1 shows the main risk factors, with their univariate ORs and 95% CIs. Table S2 shows the full list of potential risk factors with univariate ORs. Countries were classified according to the format of the United Nations Department of Economic and Social Affairs into regions and major areas. 15 Europe was chosen as the reference category. Table 4 shows the multivariate analysis of the main potential risk factors. Travel to the different continents, antimicrobial use and antacid use (use of proton-pump inhibitors or H2 blockers) were identified as relevant factors and were therefore included in the multivariate analysis. The full list of factors with multivariate ORs and 95% CIs can be found in Table S3. Travel In the multivariate analysis, travel to Northern America, Africa and Asia remained associated with an increased risk of acquisition of ESBL-E relative to travel in Europe (Table 4). Detailed analysis by region, sub-region and country is given in Table S3. These analyses showed a statistically robust increase in the risk for Northern Africa (OR 2.9, 95% CI ) and Eastern Africa (OR 5.5, 95% CI ) and that the risk of travelling to Northern America was increased more than 3-fold and limited to the USA (OR 3.1, 95% CI ). The risk associated with travel to Asia was highest for South-Central Asia (OR 5.5, 95% CI ), for India in particular (OR 4.7, 95% CI ). 54

57 Prevalence and risk factors for ESBL in Amsterdam Antimicrobial and antacid use Both in univariate (Table 1) and multivariate (Table 4) analysis the use of antibiotics or antacids increased the risk of carriage of ESBL-E ~2-fold. Table 4 - Main risk factors included in multivariate analysis Risk factor Multivariate OR 95% CI Age (continuous variable) Female Use of antibiotics PPIs or H2 blockers Travel to Africa a Latin America/Caribbean a Northern America a Asia a Australia/New Zealand a NA NA NA, not applicable; PPIs, proton-pump inhibitors. a Countries grouped according towho major area codes, reference. Europe (inclusive of persons who only travelled in the Netherlands or did not travel). Other factors We explored other potential risk factors (Table S2) by adding them separately, i.e. one at a time, into the multivariate analysis shown in Table 4. Factors that stood out in the multivariate analysis were all travel related: working as airline cabin crew, admission to a foreign hospital, being born in Africa or having a father or mother born in Africa or Asia. We also performed an analysis for the risk associated with these travel-related factors, restricted to those participants who did travel outside the Netherlands. These were 1270 persons: 112 cases and 1158 controls. This restricted analysis suggested that working for an airline (multivariate OR 4.3, 95% CI ) may pose an extra risk, since the OR in the restricted analysis did not change substantially. The OR associated with admission to a foreign hospital was halved in the restricted analysis, with a wide CI (multivariate OR 3.0, 95% CI ). The OR associated with being born outside the Netherlands or having a father or a mother born outside the Netherlands was slightly different in the restricted analysis; the highest risk was having a mother born in Asia (multivariate OR 2.4, 95% CI ), indicating that this risk was independent of the possible association with travel. 55

58 Chapter 3 Association of travel with specific ESBL genes The association of travel with carriage of strains with specific ESBL genes is shown in Table 5. bla CTX-M-1 is typically found in poultry in the Netherlands, while bla CTX-M-14 and bla CTX-M-15 are found in humans worldwide. bla CTX-M-15 and bla CTX-M-14 were associated with travel (Africa, Asia and Northern America); bla CTX-M-1 was not. The carbapenemase OXA-48 was found in an E. coli strain from a participant who visited Egypt and the USA; he was born in the Netherlands and the country of origin of both parents was Southern Asia. He had no other risk factors. Table 5 - Association of genes with travel to different regions (univariate) ESBL gene(s) No ESBL OR 95% CI ESBL bla CTX-M-1 (N=26) Europe (reference) Africa 0 88 NA Latin America/Caribbean Northern America Asia Australia/New Zealand 0 19 NA ESBL bla CTX-M-14 and bla CTX-M-15 (N=79) a Europe (reference) Africa Latin America/Caribbean Northern America Asia Australia/New Zealand 0 18 NA NA, not applicable. a CTX-M-14 and -15 are grouped together because of their similar epidemiological distribution. DISCUSSION We showed a fecal carriage rate of ESBL-E of >8% in the general adult population in Amsterdam. This confirms and extends our previous finding of a 10% carriage rate in patients who visit their general practitioner with gastrointestinal complaints. 3 Main risk factors were antibiotic use, use of gastric acid-suppressing medication and travel to Africa, Asia or the USA. Additional risk factors were having a mother born in Asia and possibly working as cabin crew for an airline. While the findings that antibiotic use and travel to Asia and Africa increase the risk of carriage of ESBL-E are not unexpected, the association with antacid use and the >3-fold increased risk associated with travel to the USA have, to the best of our 56

59 Prevalence and risk factors for ESBL in Amsterdam knowledge, not been clearly shown before. 16 An interesting finding was that carriage of Enterobacteriaceae producing CTX-M-14 or CTX-M-15 was associated with travel to Africa, Asia or Northern America/the USA, while carriage of strains producing CTX-M-1 was not. A strength of our study is that it aimed at the carriage rate in the general adult population, because we did not select patients upon a visit to their general practitioner or on admission to hospital, but used the general practitioner s databases to draw a sample from the general population. This was possible because in the Netherlands health insurance is obligatory and inhabitants are registered with a general practitioner. A second advantage of our approach is that we did not select for persons attending a travel clinic, which introduces strong bias towards countries that require vaccination or malaria prophylaxis. The weakness of our study was the participation rate of ~25%. Participants had been informed that we were screening for resistant strains and of a possible relation with antibiotic use. This could have introduced self-selection bias for those participants that were concerned, because of previous antibiotic use, and could have affected the prevalence rate, rendering it higher. Therefore, we also determined the prevalence of carriage of resistant strains in participants who had not used antibiotics. This was also high, nearly 7.5%, and confirmed the high prevalence of ESBL-E in the Dutch population. Participants were unaware of other interests, such as types of ESBL, travel, ethnicity or acid-suppressing medication. Finally, our study was restricted to Amsterdam, a large, cosmopolitan city with inhabitants from many different origins and possibly a high propensity for international travel. Such selection does not invalidate the analysis of risk factors, because this selection is likely to be the same in all participants; numerically, it may have the effect of making the ORs closer to unity. Several reports describe increasing rates of fecal carriage of ESBL-E in the community (reviewed by Woerther et al. 16 ). The review by Woerther et al. shows that in Europe percentages of carriage of ESBL increased between 2002 and 2011, with the highest figures in Spain, where carriage rates of >7% were already noted in Overall, rates are quite similar to those we measured, albeit that our carriage rate of 8.6% is the highest determined so far in Europe. Possibly, this is due to our sensitive detection method, with an enrichment step. 17 In other regions of the world, especially in South-East Asia and China, ESBL-E carriage rates can be as high as nearly 70%. 16 The majority of ESBL-positive isolates in our study were E. coli and the predominant CTX-M allele was bla CTX-M-15, although a substantial proportion, almost one-fifth, of strains produced CTX-M-1. The predominance of CTX-M-15 is in concordance with the epidemiology in the community worldwide and comparable to what we found in our study in patients with gastrointestinal complaints. 3,16 In the present study, carriage of CTX-M-15- and 14-producing ESBL-E was associated with visiting a foreign country, while carriage of CTX-M-1-producing strains was not. Possibly, Enterobacteriaceae producing ESBLs of this allele are acquired in the Netherlands, since CTX-M-1 is the main ESBL type found in E. coli on chicken meat. 18,19 A large proportion of the ESBL-producing E. 57

60 Chapter 3 coli appeared to be related to ST131. This more virulent clone could lead to more adverse outcomes in case of infection. 20 Risk factors for fecal carriage of ESBL-E in Europe have been investigated especially in healthcare settings. Because of our focus on the community we will limit our discussion to studies in community settings. Only a few European studies are available. 16,21,22 With the exception of a study in Germany and one in the Netherlands, no risk factors for carriage were identified, possibly due to the limited size of the studies. The German study showed an association of ESBL-E carriage with travel to Greece and Africa and with ownership of a pet, while antibiotic use was not a risk factor. 21 The Dutch study found ownership of a horse to be the only risk factor. 22 In the present study, previous antimicrobial use increased the risk of ESBL-E carriage ~2-fold. This finding is interesting because it is biologically plausible, but has not been shown in other European community studies. Possibly, the low level of antibiotic use, compared with other countries, makes this risk factor stand out in our country. Noteworthy is the relationship between use of acid-suppressing medication and ESBL-E carriage. Two clinical studies showed an association between antacid use and colonization with ESBL-E, one conducted among hospitalized patients in Israel and the other in the USA. 23,24 The authors of these studies noticed the role of acid suppression, but did not discuss it. Our study indicates that antacids play a role as risk factors for acquisition of ESBL-E in the community too. Gastric acid suppression by bicarbonate has been shown to lower the infective dose of Vibrio cholerae in seminal studies conducted in the 1960s on inmates in correctional facilities that received oral doses of V. cholera. 25 An association has been shown between gastric ph and non-typhoidal salmonellosis. 26,27 The risk of antacid use points to ingestion as a main route of acquisition of ESBL-E. Possibly, the use of antacids or antibiotics while abroad may pose an additive risk for acquisition of ESBL. Our study, however, did not have the statistical power to test this hypothesis. Several studies have shown that travelers to foreign countries can be colonized with ESBL-E upon return. 16 Travel, especially to Asia, but also to Africa, may be the most important risk factor also in the general population. An intriguing finding is the risk associated with travel to the USA, which was not identified before. Studies on travel so far, however, have used travel clinics as sources of participants. This means that participants mainly travelled to countries for which vaccinations or malaria prophylaxis is needed and not to countries in Northern America. Such studies, therefore, cannot detect risk associated with travel to the USA. It would be interesting to investigate the prevalence of ESBL-E in the community in the USA. Like in the Netherlands, ESBL-E might be present in the food chain. Different studies reported that >90% of chicken meat in our country is contaminated with ESBLs and we showed that raw vegetables may be contaminated as well. 19,28 30 Also in the USA the use of antimicrobials is high in the food industry. 31 Our finding that carriage of strains producing CTX-M-1 was not associated with travel, and therefore was probably acquired 58

61 Prevalence and risk factors for ESBL in Amsterdam in the Netherlands, further points to the possibility of contaminated food as a source of ESBL. It would be interesting to investigate whether the decrease in antibiotic use in animals that was noted recently will reflect in a decrease in CTX-M-1 carriage in humans in the future. 32 Like Leistner et al., we found that having an Asian mother is a risk factor for ESBL-E. 33 Admission to a foreign hospital did not stand out as a risk for ESBL-E carriage after adjustment for travel. Hence, in the general population travel seems to be the major risk factor, irrespective of hospitalization. Several studies describe person-to-person transmission of resistant strains. In three households in our study, the ESBL carrying E. coli strains were genetically identical and carried the same plasmids and ESBL genes, pointing to person-to-person transmission. The presence of different strains and plasmids in two households suggests that acquisition of ESBL-E within households is not due only to strain transmission. In summary, this study shows that ESBL-E carriage is prevalent in the Dutch community, a worrying finding in a country with low resistance rates in healthcare facilities. Risk factors included use of antimicrobial agents, use of antacids and visits to foreign countries, in particular Asia, Africa and, surprisingly, the USA. That the use of antacids posed a risk points to ingestion as a mode of acquisition of ESBL-E. Our findings, combined with previous studies that show an abundant presence of ESBL-E in the food chain, warrant more attention to the potential risk to public health of resistant microorganisms in food and water. ACKNOWLEDGEMENTS This research could only be performed thanks to the collaboration of the general practices affiliated to the Academic General Practice Network (AGPN), VU University Medical Center. We are grateful for the help and expertise of Alex Koek and Martine Rijnsburger, and the assistance of Eman Abdelrehim, Czikjain Heijblom, Marte van Keulen and Henrieke Snetselaar with the laboratory work (VU University Medical Center). We thank Kim van der Zwaluw and Martijn van Luit for the MLST experiments and analysis (Center for Infectious Disease Control, National Institute for Public Health and the Environment, Bilthoven, The Netherlands). 59

62 Chapter 3 REFERENCES 1 Pitout JD, Laupland KB. Extended-spectrum betalactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect Dis 2008; 8: EARS-Net. European Antimicrobial Resistance Surveillance Network. en/activities/surveillance/ears-net/. 3 Reuland EA, Overdevest ITMA, al Naiemi N, et al. High prevalence of ESBL-producing Enterobacteriaceae carriage in Dutch community patients with gastrointestinal complaints. Clin Microbiol Infect 2013; 19: Al Naiemi N, Murk JL, Savelkoul PHM, Vandenbroucke-Grauls CMJ, Debets-Ossenkopp YJ. Extended-spectrum beta-lactamases screening agar with AmpC inhibition. Eur J Clin Microbiol Infect Dis 2009; 28: Overdevest IT, Willemsen I, Elberts S, Verhulst C, Kluytmans JA. Laboratory detection of extended-spectrum-beta-lactamase-producing Enterobacteriaceae: evaluation of two screening agar plates and two confirmation techniques. J Clin Microbiol 2011; 49: Cohen Stuart J, Leverstein van Hall M, Al Naiemi N. NVMM Guideline Laboratory detection of highly resistant microorganisms (HRMO), version ; Available at: richtlijnen/hrmolaboratory- detection-highlyresistant-microorganisms.. 7 EUCAST. European committee on antimicrobial susceptibility testing Breakpoint tables for interpretation of MICs and zone diameters Available at: breakpoints/.. 8 Mulvey MR, Bryce E, Boyd DA, et al. Molecular characterization of cefoxitin-resistant Escherichia coli from Canadian hospitals. Antimicrob Agents Chemother 2005; 49: Al Naiemi N, Duim B, Savelkoul PHM, et al. Widespread transfer of resistance genes between bacterial species in an intensive care unit: Implications for hospital epidemiology. J Clin Microbiol 2005; 43: Poirel L, Walsh TR, Cuvillier V, Nordmann P. Multiplex PCR for detection of acquired carbapenemase genes. Diagn Microbiol Infect Dis 2011; 70: Voets GM, Fluit AC, Scharringa J, Cohen Stuart J, Leverstein-van Hall MA. A set of multiplex PCRs for genotypic detection of extended-spectrum betalactamases, carbapenemases, plasmid-mediated AmpC beta-lactamases and OXA beta-lactamases. Int J Antimicrob Agents 2011; 37: Savelkoul PH, Aarts HJ, de Haas J, et al. Amplifiedfragment length polymorphism analysis: the state of an art. J Clin Microbiol 1999; 37: Boot M, Raadsen S, Savelkoul PH, Vandenbroucke- Grauls C. Rapid plasmid replicon typing by real time PCR melting curve analysis. BMC Microbiol 2013; 13: Magiorakos AP, Srinivasan A, Carey RB, et al. Multidrug-resistant, extensively drug-resistant and pandrug-resistant bacteria: an international expert proposal for interim standard definitions for acquired resistance. Clin Microbiol Infect 2012; 18: World Health Organization. United Nations, Department of Economic and Social Affairs. Available at: General/Files/ Definition_of_Regions.pdf.. 16 Woerther PL, Burdet C, Chachaty E, Andremont A. Trends in human fecal carriage of extendedspectrum beta-lactamases in the community: Toward the globalization of CTX-M. Clin. Microbiol. Rev. 2013; 26: Murk JLAN, Heddema ER, Hess DLJ, Bogaards JA, Vandenbroucke-Grauls CMJE, Debets-Ossenkopp YJ. Enrichment broth improved detection of extended-spectrum-beta-lactamase- producing bacteria in throat and rectal surveillance cultures of samples from patients in intensive care units. J Clin Microbiol 2009; 47: Kluytmans JAJW, Overdevest ITMA, Willemsen I, et al. Extended-spectrum β-lactamase-producing Escherichia coli from retail chicken meat and humans: comparison of strains, plasmids, resistance genes, and virulence factors. Clin Infect Dis 2013; 56: Leverstein-van Hall MA, Dierikx CM, Cohen Stuart J, et al. Dutch patients, retail chicken meat and poultry share the same ESBL genes, plasmids and strains. Clin Microbiol Infect 2011; 17: Johnson JR, Johnston B, Clabots C, Kuskowski M a, Castanheira M. Escherichia coli sequence type ST131 as the major cause of serious multidrugresistant E. coli infections in the United States. Clin Infect Dis 2010; 51:

63 Prevalence and risk factors for ESBL in Amsterdam 21 Valenza G, Nickel S, Pfeifer Y, et al. Extendedspectrum-beta-lactamase-producing escherichia coli as intestinal colonizers in the German community. Antimicrob Agents Chemother 2014; 58: Huijbers PMC, de Kraker M, Graat EAM, et al. Prevalence of extended-spectrum β-lactamaseproducing Enterobacteriaceae in humans living in municipalities with high and low broiler density. Clin Microbiol Infect 2013; 19. DOI: / Ben-Ami R, Schwaber MJ, Navon-Venezia S, et al. Influx of extended-spectrum beta-lactamaseproducing enterobacteriaceae into the hospital. Clin Infect Dis 2006; 42: Hayakawa K, Gattu S, Marchaim D, et al. Epidemiology and risk factors for isolation of Escherichia coli producing CTX-M-type extendedspectrum β-lactamase in a large U.S. Medical Center. Antimicrob Agents Chemother 2013; 57: Hornick R, Music S, Wenzel R, Al. E. The Broad Street pump revisited: response of volunteers to ingested cholera vibrios. Bull N Y Acad Med 1971; 47: Giannella RA, Broitman SA, Zamcheck N. Salmonella enteritis - I. Role of reduced gastric secretion in pathogenesis. Am J Dig Dis 1971; 16: Giannella RA, Broitman SA, Zamcheck N. Gastric acid barrier to ingested microorganisms in man: studies in vivo and in vitro. Gut 1972; 13: Overdevest I, Willemsen I, Rijnsburger M, et al. Extended-Spectrum B-Lactamase Genes of Escherichia coli in Chicken Meat and Humans, the Netherlands. Emerg Infect Dis 2011; 17: Cohen Stuart J, van den Munckhof T, Voets G, Scharringa J, Fluit A, Hall ML. Comparison of ESBL contamination in organic and conventional retail chicken meat. Int J Food Microbiol 2012; 154: Reuland EA, al Naiemi N, Raadsen SA, Savelkoul PHM, Kluytmans JAJW, Vandenbroucke- Grauls CMJE. Prevalence of ESBL-producing Enterobacteriaceae in raw vegetables. Eur J Clin Microbiol Infect Dis 2014; 33: Van Boeckel TP, Brower C, Gilbert M, et al. Global trends in antimicrobial use in food animals. Proc Natl Acad Sci U S A 2015; : MARAN. Monitoring of Antimicrobial Resistance and Antibiotic Usage in Animals in the Netherlands in nl/upload_mm/2/2/2/0ab4b3f5-1cf0-42e7-a460- d ae5_nethmap Maran2015.pdf.. 33 Leistner R, Meyer E, Gastmeier P, et al. Risk factors associated with the community-acquired colonization of extended-spectrum betalactamase (ESBL) positive Escherichia Coli. an exploratory case-control study. PLoS One 2013; 8: e

64 Chapter 3 SUPPLEMENTARY DATA Questionnaire Study number Date of sample Date of birth Gender Profession Country of birth (participant) Country of birth (mother) Country of birth (father) Since how many years are you living in the Netherlands? Did you use antimicrobials in the previous 12 months? Have you been admitted to a Dutch hospital in the previous 12 months? Have you been admitted to a foreign hospital in the previous 12 months? Have you been admitted (in the previous 12 months) to a nursing home, long-term care facility and/or rehabilitation center? Did you visit a foreign country in the previous 12 months? If yes, which country? When? For how long? In which way has the sample been taken, rectal swab or fecal sample? Data extracted from the AGPN database ICPC: International Classification of Primary Care ATC/DDD-system: Anatomical Therapeutic Chemical (ATC) Classification System with defined daily dose Patient data used to extract results from the AGPN database were date of birth, gender and general practitioner/general practice. ICPC_PL (problem list): ICPC_J (journal): ATC_J01 (antimicrobials): ATC_A02 (antacids): ATC_H02 (corticosteroids): ICPC code, explanation, starting date, mutation date notes, ICPC code, explanation, contact date description, name of antimicrobial, description of group, prescription code, number, entry date, ICPC code, ICPC explanation, variable text description, name of antacid, prescription code, number, entry date, variable text description, name of corticosteroid, prescription code, number, entry date, ICPC code, ICPC explanation 62

65 Prevalence and risk factors for ESBL in Amsterdam Table S1 - Co-resistance of ESBL-E isolates Antibiotic Number of resistant strains a % of resistant strains gentamicin ciprofloxacin co-trimoxazole nitrofurantoine Multiresistance (to gentamicin, ciprofloxacin, co-trimoxazole) a Number of strains tested: 145 Table S2 - Univariate analysis of risk factors Risk factor Cases (n=129) Controls (n=1393) Median age, years (range) 48 (20 90) 50 (18 95) OR 95% CI Gender (female) Underlying diseases/comorbidity malignancies benign neoplasma bile/liver diseases renal problems/diseases cardiac problems/diseases lung problems/diseases gastric problems/diseases 0 1 NA NA NA intestinal problems/diseases urinary tract problems/diseases excl. cystitis cystitis diabetes mellitus HIV other Antimicrobial treatment (previous 12 months) all antimicrobial agents narrow-spectrum beta-lactams 0 19 NA NA NA broad-spectrum beta-lactams tetracyclines nitrofurans

66 Chapter 3 Table S2 - Univariate analysis of risk factors (Continued) Risk factor Cases (n=129) Controls (n=1393) OR 95% CI fluoroquinolones macrolides trimethoprim/sulfamethoxazole other Antacid therapy (previous 12 months) antacids - proton pump inhibitors and H2 blockers antacids - proton pump inhibitors antacids - H2 blockers antacids - other Corticosteroid use (previous 12 months) Country of birth outside the Netherlands - participant outside the Netherlands - mother outside the Netherlands - father outside the Netherlands - mother or father participant Africa Latin America and the Caribbean Northern America 0 5 NA NA NA Europe Oceania 0 4 NA NA NA Asia mother Africa Latin America and the Caribbean Northern America Europe Oceania 0 2 NA NA NA Asia father Africa Latin America and the Caribbean Northern America 0 5 NA NA NA Europe

67 Prevalence and risk factors for ESBL in Amsterdam Table S2 - Univariate analysis of risk factors (Continued) Risk factor Cases (n=129) Controls (n=1393) OR 95% CI Oceania 0 4 NA NA NA Asia Visit to a foreign country (previous 12 months) a visit to a foreign country Europe reference Africa Eastern Africa Middle Africa Northern Africa Egypt Morocco Tunisia 0 3 NA NA NA Southern Africa Western Africa Asia Eastern Asia China Japan Mongolia Republic of Korea 0 0 NA NA NA Other non-specified areas 0 1 NA NA NA South-Central Asia Afghanistan 1 0 NA NA NA Bangladesh 0 2 NA NA NA India Iran (Islamic Republic of ) 3 0 NA NA NA Nepal 0 3 NA NA NA Sri Lanka South-Eastern Asia Western Asia Cyprus 0 7 NA NA NA Georgia 0 1 NA NA NA Israel Jordan 0 6 NA NA NA Lebanon 0 2 NA NA NA 65

68 Chapter 3 Table S2 - Univariate analysis of risk factors (Continued) Risk factor Cases (n=129) Controls (n=1393) OR 95% CI Syrian Arab Republic 0 3 NA NA NA Turkey United Arab Emirates Latin America and the Caribbean Caribbean Central America South America Northern America Northern America Bermuda 0 1 NA NA NA Canada Greenland 0 1 NA NA NA United States of America Oceania 0 19 NA NA NA Australia/New Zealand 0 18 NA NA NA Profession health care cabin crew education catering Admission to healthcare institution or long term care facility (previous 12 months) hospital long term care facility hospital in a foreign country NA, not applicable; PPIs, proton-pump inhibitors. a Only countries visited are named 66

69 Prevalence and risk factors for ESBL in Amsterdam Table S3 - Multivariate analysis of main risk factors (see table 4) with other risk factors included separately, i.e. one at a time Risk factor OR 95% CI Median age, years (range) continous variable Gender (female) Underlying diseases/comorbidity malignancies benign neoplasma bile/liver diseases renal problems/diseases cardiac problems/diseases lung problems/diseases gastric problems/diseases NA NA NA intestinal problems/diseases urinary tract problems/diseases excl. cystitis cystitis diabetes mellitus HIV NA NA NA other Antimicrobial treatment (previous 12 months) all antimicrobial agents narrow-spectrum beta-lactams NA NA NA broad-spectrum beta-lactams tetracyclines nitrofurans fluoroquinolones macrolides trimethoprim/sulfamethoxazole other NA NA NA Antacid therapy (previous 12 months) antacids - proton pump inhibitors and H2 blockers antacids - proton pump inhibitors antacids - H2 blockers antacids - other Corticosteroid use (previous 12 months)

70 Chapter 3 Table S3 - Multivariate analysis of main risk factors (see table 4) with other risk factors included separately, i.e. one at a time (Continued) Risk factor OR 95% CI Country of birth outside the Netherlands - participant outside the Netherlands - mother outside the Netherlands - father outside the Netherlands - mother or father participant Africa Latin America/Caribbean Northern America NA NA NA Europe Oceania NA NA NA Asia mother Africa Latin America/Caribbean Northern America Europe Oceania NA NA NA Asia father Africa Latin America/Caribbean Northern America NA NA NA Europe Oceania NA NA NA Asia Visit to a foreign country (previous 12 months) a visit to a foreign country Europe reference Africa Eastern Africa Middle Africa Northern Africa Egypt

71 Prevalence and risk factors for ESBL in Amsterdam Table S3 - Multivariate analysis of main risk factors (see table 4) with other risk factors included separately, i.e. one at a time (Continued) Risk factor OR 95% CI Morocco NA Tunisia NA Southern Africa NA NA NA Western Africa Asia Eastern Asia China Japan NA Mongolia Republic of Korea NA NA NA Other non-specified areas NA South-Central Asia Afghanistan NA NA NA Bangladesh NA India Iran (Islamic Republic of ) NA NA NA Nepal NA Sri Lanka South-Eastern Asia Western Asia Cyprus NA Georgia NA Israel Jordan NA Lebanon NA Syrian Arab Republic NA Turkey United Arab Emirates Latin America/Caribbean Caribbean Central America NA NA NA South America NA NA NA Northern America Northern America

72 Chapter 3 Table S3 - Multivariate analysis of main risk factors (see table 4) with other risk factors included separately, i.e. one at a time (Continued) Risk factor OR 95% CI Bermuda NA Canada Greenland NA United States of America Oceania NA NA NA Australia/New Zealand NA NA NA Profession health care cabin crew education catering Admission to healthcare institution or long term care facility (previous 12 months) hospital long term care facility hospital in a foreign country NA, not applicable; PPIs, proton-pump inhibitors. a Only countries visited are named 70

73 Prevalence and risk factors for ESBL in Amsterdam Figure S1. Multilocus sequence typing (MLST) of E. coli isolates (n =132). The numbers indicate the different sequence types. Thick connecting lines indicate single-locus variants; thin connecting lines indicate variants with two or three loci difference; dashed connecting lines indicate variants with four loci difference; five loci differences are indicated by dotted connecting lines. Shadowing indicates that more than one sequence type belongs to the same complex. 71