REsistance to BEta-Lactam antibiotics · Beta-lactam antibiotics (penicillins, cephalosporins,...

REBEL-ing

against resistance

REsistance to BEta-Lactam antibiotics due to beta-lactamases

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again

st resistan

ce

Elien Ascelijn Reuland

Elie

n A

sce

lijn R

eu

lan

d

voor het bijwonen van de openbare verdediging

van het proefschrift

•

REBEL-ing against Resistance



•

Vrijdag 3 februari 2017om 13.45 uur in de aula van het hoofdgebouw

van de Vrije Universiteit De Boelelaan 1105

te Amsterdam

Aansluitend bent U van harte uitgenodigd voor de receptie

ter plaatse

Ascelijn ReulandKorte Prinsengracht 9 huis

1013 GN [email protected]

06 24 77 31 25

•

ParanimfenMarre van den Brand

[email protected] 42 26 12 98

Oddeke van [email protected]

06 17 45 05 70

UITNODIGING


REsistance to BEta-Lactam antibiotics due to beta-lactamasesREBEL-ing

against resistance


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BE

L-ing

again

st resistan

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Elie

n A

sce

lijn R

eu

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•




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te Amsterdam


ter plaatse



06 24 77 31 25

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06 17 45 05 70

UITNODIGING

The studies in this thesis were funded by ZonMw, the Netherlands Organization for Health Research and Development (grant number 125 020 011).

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-lactamasesThesis, VU University Amsterdam, the Netherlands.

Copyright © 2017 Elien Ascelijn Reuland, Amsterdam, the Netherlands.

ISBN: 978-94-6299-518-5

Cover: Janno Heck | www.jannoheck.comLay-out: Nikki Vermeulen | Ridderprint BV Printing: Ridderprint BV | www.ridderprint.nl

VRIJE UNIVERSITEIT



ACADEMISCH PROEFSCHRIFT

ter verkrijging van de graad Doctor aande Vrije Universiteit Amsterdam,

op gezag van de rector magnificusprof.dr. V. Subramaniam,

in het openbaar te verdedigenten overstaan van de promotiecommissie

van de Faculteit der Geneeskundeop vrijdag 3 februari 2017 om 13.45 uur

in de aula van de universiteit,De Boelelaan 1105

door


geboren te Groningen

promotoren: prof.dr. C.M.J.E. Vandenbroucke-Grauls prof.dr. J.A.J.W. Kluytmans

copromotor: dr. N. al Naiemi

leescommissie: 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

paranimfen: drs. Marre van den Brand dr. Oddeke van Ruler

Voor mijn ouders

grote broer

grote zus

‘ kleine’ broer

“...uiteindelijk krijgt iedereen dezelfde bonuskorting.”

- Nashwan

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):1076-82

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):e0128120

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):e0136052

CHAPTER 7 Prevalence of ESBL-producing Enterobacteriaceae in raw vegetables 119

European Journal of Clinical Microbiology & Infectious Diseases

2014 Oct;33(10):1843-6

http://www.ncbi.nlm.nih.gov/pubmed/22757622














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):2790-1

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):e0113033

CHAPTER 12 Summarizing discussion and future directions 177

Nederlandse samenvatting 189

List of publications 203

Curriculum Vitae 209

Dankwoord 215










Introduction and outline of the thesis

EA Reuland1, CMJE Vandenbroucke-Grauls1, N al Naiemi1,2,3

(adapted from ESBL in de kliniek: achtergrond, relevantie en epidemiologie)Tijdschrift voor infectieziekten 2011 vol 6; nr 4

1 Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam

IntroductIon and outlIne of the thesIs

15

OUTLINE OF THE THESIS

This thesis focuses on beta-lactamases, in particular on extended-spectrum beta-lactamases (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-lactamase-producing 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.


17

INTRODUCTION

Many types of infections like urinary tract infections, bloodstream infections, hospital-acquired 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.

BackgroundBeta-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 (http://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

http://ecdc.europa.eu/

http://ecdc.europa.eu/

Chapter 1

18

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 beta-lactamases, 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 beta-lactamases were recognized for the first time. Since then already more than 200 TEM-enzymes have been detected (http://lahey.org/studies/). 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

http://www.lahey.org/studies/


19

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 (http://lahey.org/studies/). 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 beta-lactamases 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.18–21 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 2009.22

Detection of ESBLs, pAmpC and carbapenemasesIn the microbiological laboratory there are roughly two different methods for the detection of ESBLs.

http://www.lahey.org/studies/

Chapter 1

20

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.24–26

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.


21

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 2013. For K. pneumoniae this percentage has risen from 3.5% in 2005, to 5.5% in 2009, and to 7.5% in 2013. (http://ecdc.europa.eu/en/healthtopics/antimicrobial_resistance/database/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

Chapter 1

22

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 well-known 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-48.21 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


23

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 clinicWhen 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 hospitalThe 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

Chapter 1

24

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 WIP-guidelines 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.

ConclusionThe 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 broad-spectrum 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.


25

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18 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: 1175–84.

19 Nordmann P, Cuzon G, Naas T. The real threat of Klebsiella pneumoniae carbapenemase-producing bacteria. Lancet Infect. Dis. 2009; 9: 228–36.

20 Walsh TR. Emerging carbapenemases: A global perspective. Int. J. Antimicrob. Agents. 2010; 36. DOI:10.1016/S0924-8579(10)70004-2.

21 Poirel L, Potron A, Nordmann P. OXA-48-like carbapenemases: The phantom menace. J Antimicrob Chemother 2012; 67: 1597–606.

22 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: 5046–54.

23 Cohen Stuart J, Leverstein van Hall M, Al Naiemi N. NVMM Guideline Laboratory detection of highly resistant microorganisms (HRMO), version 2.0. 2012; Available at: http://www.nvmm.nl/richtlijnen/hrmolaboratory-detection-highly-resistant-microorganisms.

24 Felmingham D, Brown DF. Instrumentation in antimicrobial susceptibility testing. J Antimicrob Chemother 2001; 48 Suppl 1: 81–5.

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25 Leinberger DM, Grimm V, Rubtsova M, et al. Integrated detection of extended-spectrum-beta-lactam resistance by DNA microarray-based genotyping of TEM, SHV, and CTX-M genes. J Clin Microbiol 2010. DOI:10.1128/JCM.00765-09.

26 Stuart JC, Dierikx C, Naiemi N Al, et al. Rapid detection of TEM, SHV and CTX-M extended-spectrum b-lactamases in Enterobacteriaceae using ligation-mediated amplification with microarray analysis. J Antimicrob Chemother 2010; 65: 1377–81.

27 Livermore DM, Canton R, Gniadkowski M, et al. CTX-M: Changing the face of ESBLs in Europe. J. Antimicrob. Chemother. 2007; 59: 165–74.

28 Bradford PA. Extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 2001; 14: 933–51, table of contents.

29 Canton R, Novais A, Valverde A, et al. Prevalence and spread of extended-spectrum beta-lactamase-producing Enterobacteriaceae in Europe. Clin Microbiol Infect 2008; 14 Suppl 1: 144–53.

30 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: 1216–22.

31 Mouton J, Voss A, Arends J, Bernards on behalf of the ONE study group. S. Prevalence of ESBL in the Netherlands: the ONE study. 2007.

32 Hawkey PM. Prevalence and clonality of extended-spectrum beta-lactamases in Asia. Clin Microbiol Infect 2008; 14 Suppl 1: 159–65.

33 Villegas M V, Kattan JN, Quinteros MG, Casellas JM. Prevalence of extended-spectrum beta-lactamases in South America. Clin Microbiol Infect 2008; 14 Suppl 1: 154–8.

34 Nicolas-Chanoine MH, Jarlier V. Extended-spectrum beta-lactamases in long-term-care facilities. Clin Microbiol Infect 2008; 14 Suppl 1: 111–6.

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: 67–71.

36 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: 834–44.

37 Lautenbach E, Patel JB, Bilker WB, Edelstein PH, Fishman NO. Extended-spectrum beta-lactamase-producing Escherichia coli and Klebsiella pneumoniae: risk factors for infection and impact of resistance on outcomes. Clin Infect Dis 2001; 32: 1162–71.

38 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: 26–32.

39 Davies J, Davies D. Origins and evolution of antibiotic resistance. Microbiol Mol Biol Rev 2010; 74: 417–33.

40 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: 155–65.

41 Mesa RJ, Blanc V, Blanch AR, et al. Extended-spectrum beta-lactamase-producing Enterobacteriaceae in different environments (humans, food, animal farms and sewage). J Antimicrob Chemother 2006; 58: 211–5.

42 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: 212–4.

43 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: 873–80.

44 Knapp CW, Dolfing J, Ehlert PA, Graham DW. Evidence of increasing antibiotic resistance gene abundances in archived soils since 1940. Env Sci Technol 2010; 44: 580–7.

45 Heuer H, Schmitt H, Smalla K. Antibiotic resistance gene spread due to manure application on agricultural fields. Curr Opin Microbiol 2011; 14: 236–43.

46 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: 608–15.

47 Buchholz U, Bernard H, Werber D, et al. German outbreak of Escherichia coli O104:H4 associated with sprouts. N Engl J Med 2011; 365: 1763–70.


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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 extended-spectrum beta-lactamases: a prospective study with Swedish volunteers. Antimicrob Agents Chemother 2010; 54: 3564–8.

49 Philippon A, Arlet G, Jacoby GA. Plasmid-determined AmpC-type beta-lactamases. Antimicrob Agents Chemother 2002; 46: 1–11.

50 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: 35–40.

51 Warren RE, Harvey G, Carr R, Ward D, Doroshenko A. Control of infections due to extended-spectrum beta-lactamase-producing organisms in hospitals and the community. Clin Microbiol Infect 2008; 14 Suppl 1: 124–33.

52 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: 353–8.

53 Maatregelen tegen overdracht van Bijzonder Resistente Micro-Organismen (BRMO). Available at: http://www.wip.nl/free_content/Richtlijnen/ BRMO.pdf.

54 Kluytmans - van den Bergh M. The prevention paradox of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E): species-specific risk and burden of transmission. Abstract #3418 O379 ECCMID Amsterdam, 2016.

High prevalence of ESBL-producing Enterobacteriaceae carriage in

Dutch community patients with gastrointestinal complaints

EA Reuland1, ITMA Overdevest2,3, N al Naiemi1,4, JS Kalpoe5, MC Rijnsburger1, SA Raadsen1, I Ligtenberg-Burgman5, KW van der Zwaluw6, M Heck6, PHM Savelkoul1,

JAJW Kluytmans1,2 and CMJE Vandenbroucke-Grauls1

Clinical Microbiology and Infection 2013 Jun;19(6):542-9

1 Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam2 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, Amsterdam6 Center for Infectious Disease Control, National Institute for Public Health and the Environment,

Bilthoven, the Netherlands

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ABSTRACT

ObjectivesThe 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.

MethodsFecal 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).

ResultsSeventy-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 ST131.

HigH prevalence of eSBl-producing enteroBacteriaceae carriage

33

INTRODUCTIONDue 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, http://www.ecdc.europa.eu/en/activities/surveillance/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. http://www.ecdc.europa.eu/en/activities/surveillance/ESAC-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.

http://www.ecdc.europa.eu/en/activities/surveillance/

http://www.ecdc.europa.eu/en/activities/surveillance/ESAC-Net/


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METHODS

Data collection/study designFecal 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 testingSpecies 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 typingThe 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 (http://www.ncbi.nlm.nih.gov/BLAST) and Lahey (http://www.lahey.org/studies/).


35

Characterization of plasmidsIdentification 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 typingSeventy ESBL-positive E. coli strains were analyzed for genetic relatedness by amplified-fragment 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 http://mlst.ucc.ie/mlst/dbs/Ecoli. Clonal complexes were determined by including whole E. coli MLST data using eBURST v3 (http://eburst.mlst.net).

Statistical analysesStatistical analyses were performed with SPSS, version 15.0. 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%, 9.7–11.5 95% 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%, 8.1–10.3 95% 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

http://eburst.mlst.net

http://eburst.mlst.net

Chapter 2

36

isolated in Amsterdam. This showed four blaCTX-M-1

, 24 blaCTX-M-15

, one blaCTX-M-14

, seven blaCTX-M-

14b, four bla

CTX-M-27, one bla

TEM-52, one bla

SHV-2a and one bla

SHV-12 genes. One gene belonging to

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

UrbanN (%)

RuralN (%)

Number of patients 471 249

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 40 20 16 16 14 14 14 5 1

CTX-M-2 group 2 1 0 1 0 0 1 0 0

CTX-M-9 group 18 10 14 9 8 4 7 1 0

SHV 10 6 6 3 4 7 2 1 0

TEM 2 0 0 0 1 2 0 1 0

Total 72* 37 36 29 27 27 24 8 1

* The genes belonging to the CTX-M-1 group were 6 blaCTX-M-1

and 34 blaCTX-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 51 70

ciprofloxacin 31 43

gentamicin 21 29

gentamicin/ciprofloxacin/cotrimoxazole 9 12


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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).

Chapter 2

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The ESBL-producing strains were genotyped by AFLP to assess their diversity. The AFLP-based 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).

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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.


39

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.

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Figure 3 - Multilocus sequencing typing showing several clusters in E. coli isolates (n = 70) obtained from fecal samples: Amsterdam vs. Brabant.

Chapter 2

40

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, http://www.ecdc.europa.eu/en/activities/surveillance/ESAC-Net/). No carbapenemase-producing 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, http://www.ecdc.europa.eu/en/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 2003.22 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 2005.23,24 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 in-hospital 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 ST131.26 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



41

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 2010. 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 blaCTX-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.30–32 A high prevalence of fecal carriage of ESBL-producing strains is observed in particular in patients with travelers’ diarrhea.30–32 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.

Chapter 2

42

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.


43

REFERENCES1 Medeiros AA. Evolution and dissemination of

beta-lactamases accelerated by generations of beta-lactam antibiotics. Clin Infect Dis 1997; 24 Suppl 1: S19-45.

2 Pitout JD, Laupland KB. Extended-spectrum beta-lactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect Dis 2008; 8: 159–66.

3 Rodriguez-Bano J, Navarro MD, Romero L, et al. Epidemiology and clinical features of infections caused by extended-spectrum beta-lactamase-producing Escherichia coli in nonhospitalized patients. J Clin Microbiol 2004; 42: 1089–94.


5 Rossolini GM, D'Andrea MM, Mugnaioli C. The spread of CTX-M-type extended-spectrum β-lactamases. Clin. Microbiol. Infect. 2008; 14: 33–41.

6 Livermore DM, Canton R, Gniadkowski M, et al. CTX-M: Changing the face of ESBLs in Europe. J. Antimicrob. Chemother. 2007; 59: 165–74.

7 Coque TM, Novais A, Carattoli A, et al. Dissemination of clonally related Escherichia coli strains expressing extended-spectrum beta-lactamase CTX-M-15. Emerg Infect Dis 2008; 14: 195–200.

8 Arpin C, Quentin C, Grobost F, et al. Nationwide survey of extended-spectrum {beta}-lactamase-producing Enterobacteriaceae in the French community setting. J Antimicrob Chemother 2009; 63: 1205–14.



11 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: 519–22.

12 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: 989–90.

13 CLSI. Clinical and Laboratory Standard Institute. Performance standards for antimicrobial susceptibility testing. CLSI M100-S18. Wayne, PA, USA. 2008.

14 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]. http://www.nvmm.nl/richtlijnen/esbl. 2008.


16 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: 4862–4.

17 Carattoli A, Bertini A, Villa L, Falbo V, Hopkins KL, Threlfall EJ. Identification of plasmids by PCR-based replicon typing. J Microbiol Methods 2005; 63: 219–28.

18 Savelkoul PH, Aarts HJ, de Haas J, et al. Amplified-fragment length polymorphism analysis: the state of an art. J Clin Microbiol 1999; 37: 3083–91.

19 Wirth T, Falush D, Lan R, et al. Sex and virulence in Escherichia coli: an evolutionary perspective. Mol Microbiol 2006; 60: 1136–51.

20 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: 268–81.

21 Hawkey PM. Prevalence and clonality of extended-spectrum beta-lactamases in Asia. Clin Microbiol Infect 2008; 14 Suppl 1: 159–65.

22 Valverde A, Coque TM, Sanchez-Moreno MP, Rollan A, Baquero F, Canton R. Dramatic increase in prevalence of fecal carriage of extended-spectrum

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beta-lactamase-producing Enterobacteriaceae during nonoutbreak situations in Spain. J Clin Microbiol 2004; 42: 4769–75.


24 al Naiemi N, Bart A, de Jong MD, et al. Widely distributed and predominant CTX-M extended-spectrum beta-lactamases in Amsterdam, The Netherlands. J Clin Microbiol 2006; 44: 3012–4.

25 Pitout JD. Infections with extended-spectrum beta-lactamase-producing enterobacteriaceae: changing epidemiology and drug treatment choices. Drugs 2010; 70: 313–33.

26 Peirano G, Pitout JD. Molecular epidemiology of Escherichia coli producing CTX-M beta-lactamases: the worldwide emergence of clone ST131 O25:H4. Int J Antimicrob Agents 2010; 35: 316–21.

27 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: 67–71.


29 NHG-Standaarden. Standards of the Dutch College of General Practitioners [in Dutch]. 2011.


31 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: 441–8.

32 Tham J, Odenholt I, Walder M, Brolund A, Ahl J, Melander E. Extended-spectrum beta-lactamase-producing Escherichia coli in patients with travellers’ diarrhoea. Scand J Infect Dis 2010; 42: 275–80.

Prevalence and risk factors for carriage of ESBL-producing

Enterobacteriaceae in Amsterdam

EA Reuland1, N al Naiemi1,2,3, AM Kaiser1, M Heck4, JAJW Kluytmans1 ,5,6, PHM Savelkoul1, PJM Elders7 and CMJE Vandenbroucke-Grauls1

Journal of Antimicrobial Chemotherapy 2016 Apr;71(4):1076-82

1 Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam2 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, Bilthoven5 Department of Medical Microbiology and Infection Control, Amphia Hospital, Breda

6 Department of Medical Microbiology, St Elisabeth Hospital, Tilburg7 EMGO Institute for Health and Care Research, VU University Medical Center, Amsterdam, The Netherlands

Chapter 3

48

ABSTRACT

ObjectivesThe objectives of this study were to determine the prevalence of carriage of ESBL-producing 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.

MethodsAdults 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.

ResultsESBL-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.

Prevalence and risk factors for esBl in amsterdam

49

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 collectionFor 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 2011.

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 NL29769.029.09) of the VU University Medical Center approved the study (NTR Trial ID NTR2453).

Chapter 3

50

ESBL detectionSamples 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).8–11 Sequences were analyzed with BioNumerics software (version 6.6; Applied Maths, Sint-Martens-Latem, Belgium) and compared with sequences in the NCBI (http://www.ncbi.nlm.nih.gov/BLAST) and Lahey database (http://www.lahey.org/studies/).

Molecular typingE. coli strains were typed by MLST (http://mlst.warwick.ac.uk/mlst/mlst/dbs/Ecoli). Clonal complexes were assigned using eBURST v3 (http://eburst.mlst.net).

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 methodsFor 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.


51

RESULTS

ParticipantsOf 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 characterizationESBL-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 blaCTX-M-14

. No difference was found in detection rate for fecal samples compared with perineal swabs (OR 1.0, 95% CI 0.7–1.4).

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 1393 48 (20 – 90) 50 (18 – 95) NA NA

Female, n (%); N=129 and 1393 75 (58.1) 852 (61.2) 0.9 0.6 – 1.3

Use of antibiotics, n (%); N=129 and 1393 33 (25.6) 195 (14.0) 2.1 1.4 – 3.2

PPIs or H2 blockers, n (%); N=129 and 1393 28 (21.7) 166 (11.9) 2.0 1.3 – 3.2

Travel to, n (%)a

Africa; N=111 and 1245 19 (17.1 88 (7.1) 2.7 1.6 – 4.7

Latin America/Caribbean; N=109 and 1264 12 (11.0) 112 (8.9) 1.3 0.7 – 2.4

Northern America; N=106 and 1255 23 (21.7) 133 (10.6) 2.3 1.4 – 3.8

Asia; N=118 and 1310 39 (33.1) 247 (18.9) 2.1 1.4 – 3.2

Australia/New Zealand; N=102 and 1222 0 (0) 18 (1.5) NA NA

NA, not applicable; PPIs, proton-pump inhibitors.aNumber 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.

Chapter 3

52

MLSTMLST 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 membersFifty 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 blaCTX-M-15

59

blaCTX-M-15

+ blaTEM-52

1

blaCTX-M-1

25

blaCTX-M-15

+ blaSHV-12

1

blaCTX-M-3

4

CTX-M-2 blaCTX-M-2

2

CTX-M-9 CTX-M 9 group 1

blaCTX-M-14

a 19

blaCTX-M-9

4

blaCTX-M-27

5

CTX-Mb blaCTX-M

2

TEM and SHV blaSHV-12

5

blaTEM-52

6

blaTEM-52

+ blaSHV-12

1

Other blaCTX-M-21

1

blaCTX-M-22

3

blaCTX-M-32

3

blaCTX-M-55

3

Total 145

aOne isolate also encoded OXA-48.bExact subtype of two CTX-M genes remained unresolved by sequencing.


53

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Chapter 3

54

Table 3 - Household members: strains and plasmids

Cluster Straina Gene(s) Plasmidb

Inc I1 FrepB ColE FIA Y B/O

A TY8857.1 blaCTX-M-1

+ - - - + -

TY8870.1 blaCTX-M-1

+ + - - - -

B TY8927 blaCTX-M-14/18

- + + - - -

TY8933 blaCTX-M-14/18

- + + - - -

C TY8892 blaCTX-M-15

- + - + + -

TY8896 blaCTX-M-15

- + - + + -

D TY8851 blaCTX-M-15

- + + + - -

TY8853 blaCTX-M-15

- + + + - -

E TY8879 blaCTX-M-3/TEM-1

+ + - + + +

TY8881 blaCTX-M-3/TEM-1

- - - - - -

aLaboratory strain numbers. bPlasmids R, ColEtp, FIIs, FIB, P, A/C, U, HI1, L/M, HI2, W, T, N, X, F/C and K were not detected

Risk factorsFor 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.

TravelIn 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 1.1–7.7) and Eastern Africa (OR 5.5, 95% CI 1.1–27.4) 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 1.7–5.7). The risk associated with travel to Asia was highest for South-Central Asia (OR 5.5, 95% CI 2.2–14.2), for India in particular (OR 4.7, 95% CI 1.4–16.0).


55

Antimicrobial and antacid useBoth 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) 1.0 1.0 – 1.0

Female 0.9 0.6 – 1.5

Use of antibiotics 2.2 1.4 – 3.7

PPIs or H2 blockers 1.9 1.1 – 3.3

Travel to

Africaa 2.2 1.1 – 4.6

Latin America/Caribbeana 0.7 0.3 – 1.9

Northern Americaa 2.7 1.6 – 4.8

Asiaa 2.1 1.3 – 3.6

Australia/New Zealanda NA NA

NA, not applicable; PPIs, proton-pump inhibitors.aCountries grouped according toWHO major area codes, reference. Europe (inclusive of persons who only travelled in the Netherlands or did not travel).

Other factorsWe 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 0.5 – 34.3) 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 0.3 – 27.1). 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 1.0 – 5.8), indicating that this risk was independent of the possible association with travel.

Chapter 3

56

Association of travel with specific ESBL genesThe association of travel with carriage of strains with specific ESBL genes is shown in Table 5. bla

CTX-M-1is typically found in poultry in the Netherlands, while bla

CTX-M-14 and bla

CTX-M-15 are

found in humans worldwide. blaCTX-M-15

and blaCTX-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 blaCTX-M-1 (N=26)

Europe (reference) 20 901

Africa 0 88 NA

Latin America/Caribbean 1 112 0.5 0.1– 3.7

Northern America 1 133 0.4 0.1 – 3.2

Asia 1 247 0.2 0.0 – 1.5

Australia/New Zealand 0 19 NA

ESBL blaCTX-M-14 and blaCTX-M-15 (N=79) a

Europe (reference) 19 901

Africa 18 88 5.5 3.0 – 9.9

Latin America/Caribbean 8 112 1.7 0.8 – 3.6

Northern America 15 133 3.1 1.7 – 5.7

Asia 25 247 3.0 1.8 – 5.1

Australia/New Zealand 0 18 NA

NA, not applicable.aCTX-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


57

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 2007.16 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.

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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


59

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).

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lactamase-producing Enterobacteriaceae: an emerging public-health concern. Lancet Infect Dis 2008; 8: 159–66.

2 EARS-Net. European Antimicrobial Resistance Surveillance Network. http://www.ecdc.europa.eu/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: 542–9.

4 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: 989–90.

5 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: 519–22.

6 Cohen Stuart J, Leverstein van Hall M, Al Naiemi N. NVMM Guideline Laboratory detection of highly resistant microorganisms (HRMO), version 2.0. 2012; Available at: http://www.nvmm.nl/richtlijnen/hrmolaboratory- detection-highly-resistant-microorganisms. .

7 EUCAST. European committee on antimicrobial susceptibility testing Breakpoint tables for interpretation of MICs and zone diameters Available at: http:// www.eucast.org/clinical_breakpoints/. .

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10 Poirel L, Walsh TR, Cuvillier V, Nordmann P. Multiplex PCR for detection of acquired carbapenemase genes. Diagn Microbiol Infect Dis 2011; 70: 119–23.

11 Voets GM, Fluit AC, Scharringa J, Cohen Stuart J, Leverstein-van Hall MA. A set of multiplex PCRs for genotypic detection of extended-spectrum beta-lactamases, carbapenemases, plasmid-mediated AmpC beta-lactamases and OXA beta-lactamases. Int J Antimicrob Agents 2011; 37: 356–9.


13 Boot M, Raadsen S, Savelkoul PH, Vandenbroucke-Grauls C. Rapid plasmid replicon typing by real time PCR melting curve analysis. BMC Microbiol 2013; 13: 83.


15 World Health Organization. United Nations, Department of Economic and Social Affairs. Available at: http://esa.un.org/unpd/wpp/General/Files/ Definition_of_Regions.pdf. .

16 Woerther PL, Burdet C, Chachaty E, Andremont A. Trends in human fecal carriage of extended-spectrum beta-lactamases in the community: Toward the globalization of CTX-M. Clin. Microbiol. Rev. 2013; 26: 744–58.

17 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: 1885–7.

18 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: 478–87.


20 Johnson JR, Johnston B, Clabots C, Kuskowski M a, Castanheira M. Escherichia coli sequence type ST131 as the major cause of serious multidrug-resistant E. coli infections in the United States. Clin Infect Dis 2010; 51: 286–94.


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21 Valenza G, Nickel S, Pfeifer Y, et al. Extended-spectrum-beta-lactamase-producing escherichia coli as intestinal colonizers in the German community. Antimicrob Agents Chemother 2014; 58: 1228–30.

22 Huijbers PMC, de Kraker M, Graat EAM, et al. Prevalence of extended-spectrum β-lactamase-producing Enterobacteriaceae in humans living in municipalities with high and low broiler density. Clin Microbiol Infect 2013; 19. DOI:10.1111/1469-0691.12150.


24 Hayakawa K, Gattu S, Marchaim D, et al. Epidemiology and risk factors for isolation of Escherichia coli producing CTX-M-type extended-spectrum β-lactamase in a large U.S. Medical Center. Antimicrob Agents Chemother 2013; 57: 4010–8.

25 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: 1181–91.

26 Giannella RA, Broitman SA, Zamcheck N. Salmonella enteritis - I. Role of reduced gastric secretion in pathogenesis. Am J Dig Dis 1971; 16: 1000–6.

27 Giannella RA, Broitman SA, Zamcheck N. Gastric acid barrier to ingested microorganisms in man: studies in vivo and in vitro. Gut 1972; 13: 251–6.


29 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: 212–4.

30 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: 1843–6.

31 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; : 1–6.

32 MARAN. Monitoring of Antimicrobial Resistance and Antibiotic Usage in Animals in the Netherlands in 2014. http://www.wageningenur. nl/upload_mm/2/2/2/0ab4b3f5-1cf0-42e7-a460-d67136870ae5_Nethmap Maran2015.pdf. .

33 Leistner R, Meyer E, Gastmeier P, et al. Risk factors associated with the community-acquired colonization of extended-spectrum beta-lactamase (ESBL) positive Escherichia Coli. an exploratory case-control study. PLoS One 2013; 8: e74323.

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SUPPLEMENTARY DATA

QuestionnaireStudy 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 databaseICPC: 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 code, explanation, starting date, mutation date

ICPC_J (journal): notes, ICPC code, explanation, contact date

ATC_J01 (antimicrobials): description, name of antimicrobial, description of group, prescription code, number, entry date, ICPC code, ICPC explanation, variable text

ATC_A02 (antacids): description, name of antacid, prescription code, number, entry date, variable text

ATC_H02 (corticosteroids): description, name of corticosteroid, prescription code, number, entry date, ICPC code, ICPC explanation


63

Table S1 - Co-resistance of ESBL-E isolates

Antibiotic Number of resistant strainsa % of resistant strains

gentamicin 37 25.5

ciprofloxacin 44 30.3

co-trimoxazole 88 60.7

nitrofurantoine 3 2.1

Multiresistance (to gentamicin, ciprofloxacin, co-trimoxazole)

48 33.1

aNumber of strains tested: 145

Table S2 - Univariate analysis of risk factors

Risk factor Cases (n=129)

Controls (n=1393) OR 95% CI

Median age, years (range) 48 (20 – 90) 50 (18 – 95)

Gender (female) 75 852 0.9 0.6 1.3

Underlying diseases/comorbidity

malignancies 13 117 1.3 0.7 2.3

benign neoplasma 1 64 0.2 0.0 1.2

bile/liver diseases 6 25 2.8 1.1 6.9

renal problems/diseases 5 41 1.4 0.5 3.6

cardiac problems/diseases 8 49 1.9 0.9 4.1

lung problems/diseases 15 140 1.2 0.7 2.2

gastric problems/diseases 0 1 NA NA NA

intestinal problems/diseases 8 48 1.9 0.9 4.2

urinary tract problems/diseases excl. cystitis 4 26 1.7 0.6 5.1

cystitis 9 93 1.1 0.5 2.2

diabetes mellitus 5 67 0.8 0.3 2.1

HIV 1 1 11.3 0.7 181.0

other 21 220 1.1 0.7 1.8

Antimicrobial treatment (previous 12 months)

all antimicrobial agents 33 195 2.1 1.4 3.2 narrow-spectrum beta-lactams 0 19 NA NA NA

broad-spectrum beta-lactams 10 48 2.4 1.2 4.8

tetracyclines 8 49 1.8 0.8 3.9

nitrofurans 12 65 2.1 1.1 4.0

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Table S2 - Univariate analysis of risk factors (Continued)



fluoroquinolones 3 18 1.8 0.5 6.3

macrolides 4 27 1.6 0.6 4.7

trimethoprim/sulfamethoxazole 2 17 1.3 0.3 5.6

other 1 4 2.7 0.3 24.5

Antacid therapy (previous 12 months)

antacids - proton pump inhibitors and H2 blockers 28 166 2.0 1.3 3.2 antacids - proton pump inhibitors 27 161 2.0 1.3 3.2

antacids - H2 blockers 1 6 1.8 0.2 15.1

antacids - other 1 7 1.5 0.2 12.7

Corticosteroid use (previous 12 months) 6 74 0.9 0.4 2.0

Country of birth

outside the Netherlands - participant 21 194 1.2 0.7 2.0

outside the Netherlands - mother 36 257 1.7 1.1 2.6

outside the Netherlands - father 35 242 1.8 1.2 2.7

outside the Netherlands - mother or father 42 310 1.7 1.1 2.5 participant

Africa 4 15 2.9 1.0 9.0

Latin America and the Caribbean 5 43 1.3 0.5 3.3

Northern America 0 5 NA NA NA

Europe 111 1257 0.7 0.4 1.1

Oceania 0 4 NA NA NA

Asia 8 61 1.4 0.7 3.1

mother

Africa 3 17 1.9 0.6 6.7


Northern America 1 7 1.5 0.2 12.7

Europe 102 1225 0.5 0.3 0.8


Asia 15 80 2.2 1.2 3.9

father

Africa 4 15 2.9 1.0 9.0


Northern America 0 5 NA NA NA

Europe 111 1252 0.7 0.4 1.1


65





Asia 8 61 1.4 0.7 3.1

Visit to a foreign country (previous 12 months) a

visit to a foreign country 112 1158 1.4 0.8 2.4

Europe reference

Africa 19 88 2.7 1.6 4.7

Eastern Africa 3 16 2.3 0.7 7.9

Middle Africa 1 6 2.0 0.2 16.8

Northern Africa 14 39 4.5 2.3 8.5

Egypt 12 16 9.4 4.3 20.5

Morocco 2 20 1.2 0.3 5.1

Tunisia 0 3 NA NA NA

Southern Africa 1 26 0.5 0.1 3.4

Western Africa 2 5 4.8 0.9 25.1

Asia 39 247 2.1 1.4 3.2

Eastern Asia 6 28 2.6 1.1 6.5

China 3 22 1.6 0.5 5.6

Japan 1 4 3.0 0.3 26.9

Mongolia 1 1 12.0 0.7 193.9

Republic of Korea 0 0 NA NA NA

Other non-specified areas 0 1 NA NA NA

South-Central Asia 11 26 5.4 2.6 11.3

Afghanistan 1 0 NA NA NA

Bangladesh 0 2 NA NA NA

India 6 18 4.1 1.6 10.6

Iran (Islamic Republic of ) 3 0 NA NA NA

Nepal 0 3 NA NA NA

Sri Lanka 1 5 2.4 0.3 20.8

South-Eastern Asia 11 77 1.7 0.9 3.3

Western Asia 20 133 1.9 1.1 3.2

Cyprus 0 7 NA NA NA

Georgia 0 1 NA NA NA

Israel 6 20 3.7 1.4 9.4

Jordan 0 6 NA NA NA

Lebanon 0 2 NA NA NA

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66




Syrian Arab Republic 0 3 NA NA NA

Turkey 12 85 1.7 0.9 3.3

United Arab Emirates 3 14 2.6 0.7 9.2


Caribbean 8 47 2.1 1.0 4.5

Central America 2 15 1.6 0.4 7.0

South America 3 59 0.6 0.2 1.9



Bermuda 0 1 NA NA NA

Canada 3 20 1.8 0.5 6.2

Greenland 0 1 NA NA NA

United States of America 22 118 2.5 1.5 4.2


Australia/New Zealand 0 18 NA NA NA

Profession

health care 17 136 1.4 0.8 2.4

cabin crew 4 11 4.0 1.3 12.9

education 8 68 1.3 0.6 2.8

catering 3 11 3.0 0.8 10.9

Admission to healthcare institution or long term care facility (previous 12 months)

hospital 18 155 1.3 0.8 2.2

long term care facility 2 14 1.6 0.4 7.0

hospital in a foreign country 5 8 7.0 2.3 21.7

NA, not applicable; PPIs, proton-pump inhibitors.aOnly countries visited are named


67

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) 0.9 0.6 1.5

Underlying diseases/comorbidity

malignancies 1.4 0.7 2.8

benign neoplasma 0.2 0.0 1.6

bile/liver diseases 2.7 0.8 8.7

renal problems/diseases 0.9 0.3 3.1

cardiac problems/diseases 2.2 0.8 5.8

lung problems/diseases 1.1 0.5 2.2

gastric problems/diseases NA NA NA

intestinal problems/diseases 2.6 1.1 5.9

urinary tract problems/diseases excl. cystitis 1.5 0.4 5.6

cystitis 0.6 0.2 1.6

diabetes mellitus 0.6 0.2 2.2

HIV NA NA NA

other 1.5 0.8 2.7

Antimicrobial treatment (previous 12 months)

all antimicrobial agents 2.2 1.4 3.7

narrow-spectrum beta-lactams NA NA NA

broad-spectrum beta-lactams 2.3 1.0 5.2

tetracyclines 2.0 0.8 4.6

nitrofurans 2.8 1.4 5.7

fluoroquinolones 1.8 0.5 6.6

macrolides 1.7 0.5 5.9

trimethoprim/sulfamethoxazole 1.5 0.3 6.8

other NA NA NA

Antacid therapy (previous 12 months)

antacids - proton pump inhibitors and H2 blockers 1.9 1.1 3.3

antacids - proton pump inhibitors 1.9 1.1 3.2

antacids - H2 blockers 1.7 0.2 15.5

antacids - other 1.7 0.2 14.6

Corticosteroid use (previous 12 months) 0.9 0.3 2.3

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Table S3 - Multivariate analysis of main risk factors (see table 4) with other risk factors included separately, i.e. one at a time (Continued)


Country of birth

outside the Netherlands - participant 1.3 0.7 2.3

outside the Netherlands - mother 1.7 1.1 2.8

outside the Netherlands - father 1.6 1.0 2.7

outside the Netherlands - mother or father 1.6 1.0 2.5

participant

Africa 4.4 1.2 15.8

Latin America/Caribbean 0.9 0.2 3.9

Northern America NA NA NA

Europe 0.7 0.3 1.3

Oceania NA NA NA

Asia 1.5 0.6 3.9

mother

Africa 2.7 0.7 10.5


Northern America 1.6 0.2 13.5

Europe 0.5 0.3 0.9

Oceania NA NA NA

Asia 2.0 0.9 4.4

father

Africa 4.4 1.2 15.7


Northern America NA NA NA

Europe 0.7 0.3 1.3

Oceania NA NA NA

Asia 1.5 0.6 3.9

Visit to a foreign country (previous 12 months) a

visit to a foreign country 1.5 0.9 2.7

Europe reference

Africa 2.2 1.1 4.6

Eastern Africa 5.5 1.1 27.4

Middle Africa 1.3 0.1 25.0

Northern Africa 2.9 1.1 7.7

Egypt 8.1 2.8 23.6


69



Morocco 0.0 0.0 NA

Tunisia 0.0 0.0 NA

Southern Africa NA NA NA

Western Africa 8.0 0.7 93.9

Asia 2.1 1.3 3.6

Eastern Asia 3.9 1.1 14.0

China 2.5 0.4 14.4

Japan 0.0 0.0 NA

Mongolia 21.7 1.3 356.7

Republic of Korea NA NA NA

Other non-specified areas 0.0 0.0 NA

South-Central Asia 5.5 2.2 14.2

Afghanistan NA NA NA

Bangladesh 0.3 0.0 NA

India 4.7 1.4 16.0

Iran (Islamic Republic of ) NA NA NA

Nepal 0.0 0.0 NA

Sri Lanka 2.7 0.3 27.0

South-Eastern Asia 1.3 0.5 3.7

Western Asia 1.9 1.0 3.8

Cyprus 0.0 0.0 NA

Georgia 0.0 0.0 NA

Israel 2.6 0.8 8.6

Jordan 0.0 0.0 NA

Lebanon 0.0 0.0 NA

Syrian Arab Republic 0.4 0.0 NA

Turkey 2.1 0.9 5.1

United Arab Emirates 2.7 0.6 12.7


Caribbean 1.7 0.6 4.9

Central America NA NA NA

South America NA NA NA



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Bermuda 0.0 0.0 NA

Canada 2.4 0.5 11.2

Greenland 0.0 0.0 NA

United States of America 3.1 1.7 5.7

Oceania NA NA NA

Australia/New Zealand NA NA NA

Profession

health care 1.4 0.8 2.7

cabin crew 4.5 0.6 35.8

education 1.9 0.9 4.3

catering 5.3 1.0 27.9

Admission to healthcare institution or long term care facility (previous 12 months)

hospital 1.1 0.6 2.2

long term care facility 2.3 0.5 11.0

hospital in a foreign country 2.9 0.3 26.0

NA, not applicable; PPIs, proton-pump inhibitors.aOnly countries visited are named


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398398398398398398398398398

730730730730730730730730730204120412041204120412041204120412041

360636063606360636063606360636063606

191191191191191191191191191184418441844184418441844184418441844

216521652165216521652165216521652165

360736073607360736073607360736073607

361236123612361236123612361236123612

360536053605360536053605360536053605

131131131131131131131131131

648648648648648648648648648

362136213621362136213621362136213621

405405405405405405405405405

383838383838383838

394394394394394394394394394

207620762076207620762076207620762076

696969696969696969

361136113611361136113611361136113611449449449449449449449449449

362036203620362036203620362036203620

362362362362362362362362362

279727972797279727972797279727972797

354354354354354354354354354

595959595959595959

550550550550550550550550550

127127127127127127127127127

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.

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.

Travel to Asia and traveler’s diarrhea with antibiotic treatment

are independent risk factors for acquiring ciprofloxacin-resistant and extended spectrum beta-lactamase-

producing Enterobacteriaceae - a prospective cohort study

EA Reuland1, GJB Sonder2,3, I Stolte2, N al Naiemi1,4,5, A Koek1, GB Linde6, TJW van de Laar1, CMJE Vandenbroucke-Grauls1, AP van Dam6,7

Clinical Microbiology and Infection 2016 Aug;22(8):731.e1-7

1 Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam2 Department of Infectious Diseases, Public Health Service (GGD) Amsterdam

3 Department of Internal Medicine, Division of Infectious Diseases, Tropical Medicine and AIDS, Academic Medical Center, Amsterdam

4 Laboratory for Medical Microbiology and Public Health, Enschede 5 Microbiology and Infection Control, Ziekenhuisgroep Twente, Hengelo

6 Public Health Laboratory, Division of Infectious Diseases, Amsterdam Health Service7 Medical Microbiology, Onze Lieve Vrouwe Gasthuis, Amsterdam, The Netherlands

Chapter 4

76

ABSTRACTObjectivesTravel to (sub) tropical countries is a well-known risk factor for acquiring resistant bacterial strains, which is especially of significance for travelers from countries with low resistance rates. In this study we investigated the rate of and risk factors for travel-related acquisition of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E), ciprofloxacin-resistant Enterobacteriaceae (CIPR-E) and carbapenem-resistant Enterobacteriaceae.

MethodsData before and after travel were collected from 445 participants. Swabs were cultured with an enrichment broth and sub-cultured on selective agar plates for ESBL detection, and on plates with a ciprofloxacin disc. ESBL production was confirmed with the double-disc synergy test. Species identification and susceptibility testing were performed with the Vitek-2 system. All isolates were subjected to ertapenem Etest. ESBL and carbapenemase genes were characterized by PCR and sequencing.

Results Twenty-seven out of 445 travelers (6.1%) already had ESBL-producing strains and 45 of 445 (10.1%) travelers had strains resistant to ciprofloxacin before travel. Ninety-eight out of 418 (23.4%) travelers acquired ESBL-E and 130 of 400 (32.5%) travelers acquired a ciprofloxacin-resistant strain. Of the 98 ESBL-E, predominantly Escherichia coli and predominantly bla

CTX-M-15,

56% (55/98) were resistant to gentamicin, ciprofloxacin and co-trimoxazole. Multivariate analysis showed that Asia was a high-risk area for ESBL-E as well as CIPR-E acquisition. Travelers with diarrhea combined with antimicrobial use were significantly at higher risk for acquisition of resistant strains. Only one carbapenemase-producing isolate was acquired, isolated from a participant after visiting Egypt.

Conclusions In conclusion, travelling to Asia and diarrhea combined with antimicrobial use are important risk factors for acquiring ESBL-E and CIPR-E.

Travel and risk facTors

77

INTRODUCTION

Resistance of Enterobacteriaceae to several classes of antimicrobials (AB) is increasing all over the world. Carriage of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) in healthy persons varies from 3% in Western European countries to 60% in India.1,2 Travelers to countries with a high prevalence of antimicrobial resistance might be at increased risk for the acquisition of antibiotic-resistant bacteria (ARB) and may facilitate the spread to countries with relatively low antimicrobial resistance rates. The identification of risk factors associated with the acquisition of resistant Enterobacteriaceae is crucial for developing strategies to prevent acquisition of ARB in travelers and to ensure best choice of empirical treatment if needed, either during travel or upon return to the country of origin, both in cases where carry-on empirical stand-by treatment is advised and when empirical treatment is started in returned travellers.3 In the present study we examined the acquisition rate and risk factors for travel-related acquisition of Enterobacteriaceae producing ESBL (ESBL-E), carbapenemases (CR-E), or resistant to ciprofloxacin (CIPR-E).

MATERIALS AND METHODS

Participants were individuals (age ≥18 years) attending the vaccination clinic of the Public Health Service, Amsterdam, between April 2012 and April 2013, who intended to travel to Africa, Asia or Latin America including the Caribbean. The Dutch LCR guidelines do not recommend that healthy travelers carry empirical antibiotic stand-by treatment. Stand-by treatment is only recommended for travelers who are at increased risk (such as immunocompromised persons and travelers to very remote areas).

Study populationThe study was an open, prospective, observational cohort study. Travelers were advised according to the Dutch National guidelines for traveler’s health advice (LCR guidelines).4 Within each continent, countries were categorized into distinct geographical regions following the definition by the United Nations Department of Economic and Social Affairs.5

Before travel, participants were asked for their age, gender, country of birth (participant and parents), country of destination, duration of travel, antimicrobial use in the previous 12 months, and whether they had been admitted to a (foreign) hospital in the last 12 months. People were not included in the analyses if their case record forms were not filled in completely and data were missing. Participants were given the option to take either a rectal swab or a stool sample before departure, which they had to send by mail to the infectious disease laboratory of the Public Health Service.

Chapter 4

78

After travel, participants were asked to fill in a questionnaire with questions about which countries they had actually visited, the amount of time they spent in each country, whether they had been hospitalized, whether they had traveler’s diarrhea (TD) and in which country, or had used AB. Together with the questionnaire, we included a transport medium to obtain a second rectal swab or stool sample. Participants were asked to collect this sample within 2 weeks after their return. A voucher of 25 € was given for participation.

Ethics statementWritten informed consent was obtained from all participants. The study was approved by the medical ethics committee (METc, NL29769.029.09) of the VU University Medical Center (NTR Trial ID NTR2453).

Phenotypic and genotypic detectionSwabs in Copan transport medium (Copan Italia, Brescia, Italy) were cultured in trypticase soy enrichment broth containing 50 mg/L ampicillin and thereafter sub-cultured. In the case of stool samples, feces was retrieved using a swab and thereafter treated in the same way as rectal swabs. Broths and plates were incubated overnight (37°C). Control plates were used to ensure proper growth.

Selective agar plates (EbSA ESBL screening agar; Cepheid Benelux, Apeldoorn, the Netherlands) were used for detection of cephalosporin-resistant strains, i.e. MIC >1 mg/L for cefotaxime and/or ceftazidime. ESBL production was confirmed with the double-disc synergy test with clavulanic acid (Rosco, Taastrup, Denmark) on Mueller-Hinton agar. A positive confirmation test was defined as a >5 mm zone difference for cephalosporins with clavulanic acid compared with cephalosporins without this inhibitor.6 Screening for ciprofloxacin-resistance was carried out by plating out enrichment broth on CLED agar (cystine lactose electrolyte-deficient medium) with a ciprofloxacin disc (concentration 5 mg), using zone diameter <21 mm as cut-off for further testing. Colonies growing within this zone that phenotypically resembled Enterobacteriaceae were subsequently further analyzed for CIPR-E as described below.

Species identification and susceptibility testing were performed with the Vitek-2 system (Vitek ID and Vitek AST; bioMérieux, Marcy l’Etoile, France). EUCAST criteria for resistance were used.7 All of these isolates were also subjected to ertapenem Etest on Mueller-Hinton agar to screen specifically for carbapenemases.

Molecular analysisMolecular analysis of ESBL genes was performed on all isolates with a positive confirmation test. Isolates with an increased MIC for ertapenem (≥0.25 mg/L), meropenem (MIC ≥0.5 mg/L) or imipenem (MIC ≥2 mg/L) were selected for molecular characterization of


79

carbapenemases.7 ESBL and carbapenemase genes were characterized using PCR followed by sequencing.8,9 Sequences were analyzed in BioNumerics (version 6.6; Applied Maths, Sint-Martens-Latem, Belgium) and compared with sequences in the NCBI (http://www.ncbi.nlm.nih.gov/BLAST) and Lahey (http://www.lahey.org/studies/) databases.

Statistical analysesPotential risk factors were based on preceding literature, e.g. antibiotic use, TD and travel to (sub) tropical areas. Risk factors for the acquisition of ESBL-E and/or CIPR-E before and during travel were identified by comparing travelers who did and who did not acquire ARB during travel using univariate and multivariate logistic regression models. Participants who already were colonized with ESBL-producing or ciprofloxacin-resistant Enterobacteriaceae before travel were excluded for risk factor analysis of ESBL-E and CIPR-E acquisition during travel, respectively. Also participants visiting more than one region were not present in the analyses. Two multivariable models were built following a backward procedure starting with only variables with a univariate p-value <0.05. Final multivariate models only included variables with a p-value <0.05. In addition, age was forced into the multivariate models to correct for potential confounding. In the final models interaction between all variables was investigated and interaction between two variables was considered present with a p-value <0.10. All statistical analyses were performed with Statistical Package for the Social Sciences (SPSS, version 20.0).

RESULTS

Study populationIn total, 622 travelers were asked to participate in the study, of whom 487 agreed and signed an informed consent (Figure 1). Of these, one was excluded because the first sample was not returned, and 41 were excluded because they did not return their second sample and questionnaire. Excluded participants (n = 42) were not significantly different from the final study population (see Supplementary material, Table S1). In total 445 participants were available for analysis to determine risk factors before travel. Almost all samples (>95%) were received within 1 month of return. Of all 932 specimens submitted either pre- or post-travel, 352 (38%) were fecal swabs, whereas the remainder were stool samples. The distribution of stool samples and fecal swabs in pre- and post-travel samples was similar among included participants. As there was no significant difference in the frequency of ESBL-E and CIPR-E between these two sample types, the term ‘samples’ is used throughout the manuscript. The median age of the participants was 33 years (IQR 27- 48) and 58% were female. Of these travelers, 27/445 participants (6.1%, 95% CI 4.2% - 8.7%) acquired ESBL-E before travel (15 only ESBL-E, and 12 ESBL-E in combination with ciprofloxacin-resistance) and 45 participants

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80

(10.1%, 95% CI 7.6% - 13.3%) acquired CIPR-E before travel (31 only with ciprofloxacin-resistance, 12 ESBL-E in combination with ciprofloxacin-resistance and two isolates with a wild-type beta-lactamase (TEM-1) in combination with ciprofloxacin-resistance). After exclusion of five travelers who did not provide information on previous travels, the only predictor for the presence of ESBL-E before travel was a previous visit to Northern Africa (OR 11.21, 95% CI 2.07- 60.84). No predictors for presence of CIPR-E before travel were found.

The study population for acquisition of resistant strains during travel was 418 for ESBL and 400 for ciprofloxacin-resistance. In line with LCR guidelines, none of these travelers was prescribed carry-on AB by the vaccination clinic of the Public Health Service.

Acquisition of resistant strainsESBLOf the 418 participants who did not have ESBL-E before travel, 98 acquired ESBL-E (23.44%, 95% CI 19.63% - 27.74%).

These 418 participants visited 79 different countries. The continent most frequently visited was Asia (n = 240), followed by Africa (n = 96) and Latin America/Caribbean (n = 79). The most visited countries were Thailand, Indonesia, India, Vietnam and China. More than one country was visited by 32% (133/418) of the travelers, more than one region by almost 9% (37/418) of the travelers and more than one continent by 1.2% (5/418). On average travelers stayed for 2 weeks, with a range from 1 to 105 days. Molecular analysis of the 98 ESBL-E (95 Escherichia coli, two Klebsiella pneumoniae and one Morganella morganii) yielded ESBL-encoding genes for all 98 isolates.

The ESBL-encoding genes that were most frequently acquired in our study were blaCTX-M-15

(n = 54), bla

CTX-M-14/18 (n = 16) and bla

CTX-M-27 (n = 9). Furthermore the isolates comprised one

blaCTX-M-3

, two blaCTX-M-8

, two blaCTX-M-9

, one blaCTX-M-24/130

, one blaCTX-M-32

, one blaCTX-M-55

, one bla

CTX-M-55/79 and one bla

CTX-M-104. Two isolates harbored bla

CTX-M-1, one isolate had two genes

(blaCTX-M-14

and blaCTX-M-15

). One gene belonging to the CTX-M family remained unidentified, also one gene per group included members of respectively the CTX-M-1, CTX-M-8 or CTX-M-9 family but could not be subdivided further by sequencing. In addition, two bla

SHV-12

were detected. Univariate analysis of association between the countries visited and the four most predominant gene types showed that bla

CTX-M-15 was mainly acquired in Western and

South-Central Asia, blaCTX-M-14

was acquired in Eastern Asia, and blaCTX-M-27

in South-East Asia and Western Asia. In travelers visiting India only bla

CTX-M-15 was found.

In addition, co-resistance was common: 50 isolates also comprised ciprofloxacin-resistance. Gentamicin resistance was detected in 39% (38/98) of the isolates, and resistance to cotrimoxazole in 70% (69/98); 56% (55/98) of strains were multidrug resistant as defined by Magiorakos et al.10


81

Figure 1 - Flowchart of the study population

Ciprofloxacin-resistanceIn total,130 out of 400 travelers (32.50%, 95% CI 28.09% - 37.24%) acquired CIPR-E. As the study group of 400 travelers largely overlapped with the study group for ESBL acquisition, the distribution of continents and countries visited was highly similar (data not shown). Of these ciprofloxacin-resistant strains 65.4% (85/130) were resistant to ciprofloxacin only and 34.6% (45/130) also produced ESBL. A total of 126 (97%) of these strains were Escherichia coli, two Morganella morganii, one Citrobacter freundii and one Shigella sonnei.

CarbapenemaseOnly one traveler returned with a carbapenemase (OXA-48) producing strain. This isolate, E. coli, harboring bla

CTX-M-9, had a reduced susceptibility to meropenem (MIC 1 mg/L), but

remained ertapenem- and imipenem-susceptible. Seven other isolates had reduced susceptibility to ertapenem but no carbapenemase-encoding gene could be detected using PCR.

Chapter 4

82

Risk factorsESBLUnivariate and multivariate results for the acquisition of ESBL-E were similar and are shown in Table 1. Participants who visited Asia were at significantly higher risk for acquisition of ESBL-producing strains (OR 7.53, 95% CI 3.12 - 18.18). Of all participants who visited Eastern Asia, 46% (10/22) acquired ESBL-E, for South-Central Asia this was 55% (27/49), for South-East Asia 23% (32/137) and for Western Asia 25% (3/12). The highest acquisition rate for ESBL-E was in travelers visiting India, 62.5% (20/32).Travelers with diarrhea who did not use AB were at slightly higher risk of acquisition of ESBL-E (OR 1.65, 95% CI 0.97 - 2.82) compared with those without diarrhea and without AB, although this association was not statistically significant. However, risk for ESBL-E acquisition was highly increased in those travelers that developed TD and used antimicrobial agents (OR 9.56, 95% CI 2.64 - 34.57).

Age, sex, country of birth, travel history before current travel and admission to a foreign hospital were not associated with acquisition of ESBL-E. One of the five participants who was admitted to a foreign hospital was colonized with an ESBL-E (CTXM-15) combined with ciprofloxacin-resistance upon return from Togo.

Ciprofloxacin-resistanceUnivariate and multivariate results for acquisition of isolates with ciprofloxacin-resistance are shown in Table 1. Like ESBL-E, the two independent risk factors were visits to Asia and the combination of TD with antibiotic use. No other risk factors were identified. One of the four participants was admitted to a foreign hospital in Togo and was colonized with an ESBL-E (CTX-M-15) combined with ciprofloxacin-resistance, and another participant was admitted to a foreign hospital in India and was colonized with a ciprofloxacin-resistant strain.

CarbapenemaseOnly one carbapenemase (OXA-48) producing E. coli was acquired by a participant after a 15-day visit to Egypt; however, the number of travelers visiting Northern Africa was low (n =12). This participant was a 50-year-old female, born in the Netherlands. No use of AB and no admission to a hospital in the previous episode was reported, but she did report an episode of TD.


83

Tabl

e 1 -

Uni

varia

te a

nd m

ultiv

aria

ble

risk

fact

ors

of a

cqui

sitio

n of

ESB

L-E

or c

ipro

floxa

cin-

resi

stan

ce C

IPR-

E du

ring

trav

el, a

mon

g 41

8 an

d 40

0 tr

avel

ers,

resp

ectiv

ely,

att

endi

ng th

e va

ccin

atio

n cl

inic

at t

he In

fect

ious

Dis

ease

s dep

artm

ent a

t the

Pub

lic H

ealth

Ser

vice

(GG

D) A

mst

erda

m (b

etw

een

Apr

il 20

12

and

Apr

il 20

13)

Risk

fact

orPr

eval

ence

Uni

vari

ate

Mul

tivar

iate

Prev

alen

ceU

niva

riat

eM

ultiv

aria

te

ESBL

n/N

(%)

OR

(95%

CI)

OR

(95%

CI)

CIPR

-E n

/N (%

)O

R (9

5% C

I)O

R (9

5% C

I)

Tota

l98

/418

(23,

4)(1

9,63

-27,

74)

130/

400

(32,

5)(2

8,09

-37,

24)

Age

(med

ian

(IQR)

) per

10

year

incr

ease

33 (2

7-49

)1,

06 (0

,89-

1,26

)1,

02 (1

,00-

1,04

)33

(27-

49)

0,97

(0,8

3-1,

14)

1,01

(0.9

9-1,

03)

Sex

Men

50/1

77 (2

8,2)

155

/172

(32,

0)1

Wom

en48

/240

(20,

0)0,

64 (0

,40-

1,00

)75

/227

( 33,

0)1,

05 (0

,69-

1,60

)

mis

sing

11

Coun

try

of b

irth

Non

-end

emic

cou

ntry

a85

/359

(23,

7)1

109/

340

(32,

1)1

Oth

erb

11/5

2 (2

1,2)

0,86

(0,4

3-1,

76)

18/5

2 (3

4,6)

1,12

(0,6

1-2,

08)

Mis

sing

78

Trav

el h

isto

ry (p

revi

ous

12 m

onth

s, b

efor

e cu

rren

t tra

vel)

Stay

in a

(sub

) tro

pica

l cou

ntry

- n

o8/

34 (2

3,5)

111

/32

(34,

4)1

Stay

in a

(sub

) tro

pica

l cou

ntry

- y

es85

/361

(23,

5)1,

00 (0

,44-

2,29

)11

0/34

4 (3

2,0)

0,90

(0,4

2-1,

93)

Mis

sing

2325

Med

ical

his

tory

(cur

rent

trav

el)

TD-,

AB-

38/2

13 (1

7,8)

11

53/2

03 (2

6,1)

11

TD +

, AB-

46/1

75 (2

6,3)

1,64

(1,0

1-2,

67)

1,65

(0,9

7-2,

82)

62/1

71 (3

6,3)

1,61

(1,0

4-2,

50)

1,51

(0,9

3-2,

45)

TD-,

AB+

2/8

(25,

0)1,

54 (0

,30-

7,90

)1,

49 (0

,24-

9,08

)3/

8 (3

7,5)

1,70

(0,3

9-7,

35)

1,35

(0,2

9-6,

22)

TD+

, AB+

9/14

(64,

3)8,

29 (2

,63-

26,1

3)9,

56 (2

,64-

34,5

7)7/

10 (7

0,0)

6,60

(1,6

5-26

,47)

5,69

(1,2

9-24

,99)

Mis

sing

98

Chapter 4

84

Tabl

e 1 -

Uni

varia

te a

nd m

ultiv

aria

ble

risk

fact

ors

of a

cqui

sitio

n of

ESB

L-E

or c

ipro

floxa

cin-

resi

stan

ce C

IPR-

E du

ring

trav

el, a

mon

g 41

8 an

d 40

0 tr

avel

ers,

resp

ectiv

ely,

att

endi

ng th

e va

ccin

atio

n cl

inic

at t

he In

fect

ious

Dis

ease

s dep

artm

ent a

t the

Pub

lic H

ealth

Ser

vice

(GG

D) A

mst

erda

m (b

etw

een

Apr

il 20

12

and

Apr

il 20

13) (

Cont

inue

d)

Risk

fact

orPr

eval

ence

Uni

vari

ate

Mul

tivar

iate

Prev

alen

ceU

niva

riat

eM

ultiv

aria

te

ESBL

n/N

(%)

OR

(95%

CI)

OR

(95%

CI)

CIPR

-E n

/N (%

)O

R (9

5% C

I)O

R (9

5% C

I)

Adm

issi

on to

a fo

reig

n ho

spita

l - n

o95

/407

(23,

3)1

125/

390

(32,

1)1

Adm

issi

on fo

reig

n ho

spita

l - y

es1/

5 (2

0,0)

0,82

(0,0

9-7,

44)

2/4

(50,

0)2,

12 (0

,30-

15,2

2)

Mis

sing

66

Trav

el d

estin

atio

ns

Onl

y A

frica

c7/

93 (7

,5)

11

18/8

8 (2

0,5)

11

Onl

y La

tin A

mer

ica/

Carib

bean

7/77

(9,1

)1,

23 (0

,41-

3,67

)1,

36 (0

,43-

4,31

)8/

70 (1

1,4)

0,50

(0,2

0-1,

24)

0,48

(0,1

8-1,

23)

Onl

y A

sia

81/2

35 (3

4,5)

6,46

(2,8

6-14

,61)

7,31

(3,0

3-17

,63)

102/

229

(44,

5)3,

12 (1

,75-

5,58

)3,

14 (1

,73-

5,71

)

Abb

revi

atio

ns: A

B, a

ntim

icro

bial

use

; CIP

R-E,

cip

roflo

xaci

n-re

sist

ant

Ente

roba

cter

iace

ae; E

SBL-

E, e

xten

ded-

spec

trum

bet

a-la

ctam

ase-

prod

ucin

g En

tero

bact

eria

ceae

; TD

, tr

avel

er’s

diar

rhea

.a Co

untr

y of

birt

h an

d co

untr

y of

birt

h of

bot

h pa

rent

s is

in a

non

-end

emic

are

a (E

urop

e, O

cean

ia, N

orth

ern

Am

eric

a).a

b Coun

try

of b

irth

or c

ount

ry o

f birt

h of

one

of b

oth

pare

nts

is in

an

ende

mic

are

a (A

frica

, Asi

a, L

atin

Am

eric

a/Ca

ribbe

an).

c Afri

ca h

ad lo

wes

t per

cent

age

of E

SBL

and

ther

efor

e ch

osen

as

refe

renc

e ca

tego

ry.


85

Use of antibioticsTwenty-two participants reported antibiotic use during travel. Details about the type of antibiotics, the presence of TD and the acquisition of ESBL-E and CIPR-E have been provided in Table 2. Although the numbers of patients having received different groups of antibiotics are low, it is remarkable that all six persons who used quinolones during travel, who all had TD, acquired both ESBL-E and CIPR-E. Five of these participants had visited Asia (Indonesia, India and Nepal), one participant travelled to Africa (Malawi). In contrast, the two persons who used doxycycline only, probably for malaria prophylaxis, did not acquire CIPR-E or ESBL-E.

Table 2 - Baseline participant characteristics with the antimicrobials used

Antimicrobial n TD- TD+ Resistance mechanism

amoxicillin 3 2 none

amoxicillin 1 ESBL

augmentin 1 1 ESBL

azitromycin 1 1 ciprofloxacin-resistance only

ciprofloxacin 4 4 ESBL and ciprofloxacin-resistance

ciprofloxacin + doxycycline 1 1 ESBL and ciprofloxacin-resistance

ofloxacin, aciloc/ranitidin, tinidazol 1 1 ESBL and ciprofloxacin-resistance

cotrimoxazole 1 1 ESBL and ciprofloxacin-resistance

nitrofurantoin 1 1 ciprofloxacin-resistance only

doxycycline 2 2 none

unspecified 2 1 ciprofloxacin-resistance only

unspecified 1 ESBL and ciprofloxacin-resistance

unspecified: bacterial infection 1 1 ciprofloxacin-resistance only

unspecified: stitches after small accident 1 1 none

unspecified: salmonella infection 1 1 none

unspecified: cystitis 1 1 ESBL and ciprofloxacin-resistance

unspecified: salmonella infection and parasite 1 1 ciprofloxacin-resistance only

Abbreviations ESBL, extended spectrum beta-lactamase; TD, traveler’s diarrhea.

DISCUSSION

Colonization with resistant strains after travel to (sub)tropical areas was detected in a large proportion of Dutch travelers in the present study. This included ESBL-E, CIPR-E and even one CR-E was detected. Independent risk factors associated with the acquisition of antimicrobial resistance were travel to Asia, and TD in combination with the use of AB. People with TD without AB use was slightly at higher risk of acquisition of ARB. This confirms

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the recent finding that TD and the use of AB during travel contribute to the acquisition of ESBL-E.11 Although a prospective study has found that travel is a risk for the introduction of quinolone-resistance genes, as far as we know, this is the first study that finds that antibiotic use during travel contributes to the acquisition of CIPR-E.12

The percentage of carriage of ESBL-E before travel (about 6.0%) was comparable with 8.6% found in a study in the Dutch community and the high pre-travel ESBL-E carriage rate in another prospective cohort study among healthy travelers in the Netherlands.13,14 We found an even higher prevalence of colonization with CIPR-E (10.1%) before departure; comparable with the prevalence of quinolone resistance encoding genes found pre-travel by von Wintersdorff et al.12

The percentage of ESBL acquisition during travel was similar to that found in other studies from Sweden, Finland and the Netherlands, where approximately one in five to one in three travelers was colonized upon return.11,14,15 Indeed, other retrospective16–18 and prospective11,19

studies have confirmed this issue. Import of quinolone-resistance genes by travel was seen in one-third to one-half of travelers and recently also import of CRE has been reported in asymptomatic travelers, although only in 0.5%.12,20

Overall, predominantly CTX-M-producing E. coli, mainly CTX-M-15, were found, which is comparable with what has been described in other studies.11,15,21,22 Multidrug resistance was seen in 56% of the ESBL-E.21,22 The present study, where we also determined CIPR-E besides the association of quinolone resistance with ESBL-E, showed even more clearly that acquisition of multidrug-resistant strains in general is a major threat.

The risk of acquiring CIPR-E during travel was even higher than the risk of acquiring ESBL-E, comparable with other data concerning quinolone-resistance-encoding genes after travel.12 As with ESBL, Asia was the highest risk area for acquiring such strains.

When focusing on CIPR-E, all six participants in our study who had taken quinolones during travel also reported TD. All six travelers treated with quinolones acquired ESBL-E as well as CIPR-E. Also Tangden et al. reported acquisition of ESBL-E in three patients treated with ciprofloxacin.15 We assume that antimicrobial use is a trigger for inducing ciprofloxacin-resistance in plasmid-mediated ESBL-producing strains or for selection of co-resistant strains. However, ciprofloxacin use could also lead to horizontal dissemination of antibiotic resistance genes between species by inducing an SOS response.23

In clinical studies as well as in studies among travelers, use of AB and gastrointestinal symptoms has been reported as a risk factor for the acquisition of ARB.11,15,24,25 Findings from ours and other mentioned studies suggest that routinely prescribing (stand-by) AB for TD should be strongly reconsidered, also because TD is usually self-limiting.3


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In this study we only found one CR-E in a traveler to Egypt, therefore the risk for acquisition of CR-E might seem low. However, the number of travelers acquiring OXA-48 may be underestimated because in our study only a few travelers to Northern African countries were included, known for their high endemic levels of OXA-48-producing strains.26–28 Several other studies also described the introduction of CR-E by returning (asymptomatic) travellers.13,29–31

One limitation of our study is that data on AB use during travel were self-reported, and that we did not ask whether the AB were used specifically for TD. In addition, we did not provide a standardized definition of TD to travelers responding to the questionnaire. Another possible limitation of the study is that we did not follow up participants to assess the duration of carriage. However, other studies have already demonstrated that colonization persists at least 6 months after return.14,32 Also, travelers to countries were no vaccination was needed were not included. The number of participants who took AB during travel was small, so we cannot draw conclusions on risk based on specific AB used. Also, because of the enrichment broth used, quinolone-resistant but ampicillin susceptible strains were not detected, so our screening method might not detect all CIPR-E.

In conclusion, international travel to (sub) tropical areas, especially travel to Asia, is an important risk factor for acquiring and importing ESBL-E and CIPR-E. A high percentage of multidrug resistance was found among these isolates. The contribution of AB to the high risk of acquisition of ARB is important in this era of increasing antimicrobial resistance. It may be useful to advise returning travelers regarding personal hygiene to decrease the risk of transmission of acquired ARB to household members. A careful patient’s travel history asked on admission to hospital might prevent unnecessary delay of appropriate therapy and allow adequate infection control policies.

ACKNOWLEDGEMENTS

We thank Joan Kint for enrolling participants at the vaccination clinic and Kaoutar el Faouzi for organizing/assisting the laboratory work at the Department of Infectious Diseases, GGD, Amsterdam, the Netherlands.

FUNDING

This work was supported by Research & Development fund GGD Amsterdam and ZonMw, the Netherlands Organisation for Health Research and Development [grant number 125020011].

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REFERENCES1 Mathai D, Rhomberg PR, Biedenbach DJ, Jones RN,

India Antimicrobial Resistance Study G. Evaluation of the in vitro activity of six broad-spectrum beta-lactam antimicrobial agents tested against recent clinical isolates from India: a survey of ten medical center laboratories. Diagnostic Microbiol Infect Dis 2002; 44: 367–77.

2 Struwe J O-LB. Short summary of swedres 2008, a report on antimicrobial utilisation and resistance in humans in sweden. Euro Surveill 2009;14. .

3 Belderok SM, van den Hoek A, Kint JA, Schim van der Loeff MF, Sonder GJ. Incidence, risk factors and treatment of diarrhoea among Dutch travellers: reasons not to routinely prescribe antibiotics. BMC Infect Dis 2011; 11: 295.

4 LCR. National Coordination Center for Travelers Health Available at: https:// www.lcr.nl/Landen. .

5 World Health Organization. United Nations, Department of Economic and Social Affairs. Available at: http://esa.un.org/unpd/wpp/General/Files/ Definition_of_Regions.pdf. .


7 EUCAST. European committee on antimicrobial susceptibility testing Breakpoint tables for interpretation of MICs and zone diameters Available at: http:// www.eucast.org/clinical_breakpoints/. .




11 Kantele A, Lääveri T, Mero S, et al. Antimicrobials increase travelers’ risk of colonization by extended-spectrum betalactamase-producing enterobacteriaceae. Clin Infect Dis 2015; 60: 837–46.

12 von Wintersdorff CJH, Penders J, Stobberingh EE, et al. High rates of antimicrobial drug resistance gene acquisition after international travel, The Netherlands. Emerg Infect Dis 2014; 20: 649–57.

13 Reuland EA, Al Naiemi N, Kaiser AM, et al. Prevalence and risk factors for carriage of ESBL-producing Enterobacteriaceae in Amsterdam. J Antimicrob Chemother 2016; 71: 1076–82.

14 Paltansing S, Vlot JA, Kraakman MEM, et al. Extended-spectrum beta-lactamase-producing enterobacteriaceae among travelers from the Netherlands. Emerg Infect Dis 2013; 19: 1206–13.


16 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: 441–8.


18 Peirano G, Laupland KB, Gregson DB, Pitout JDD. Colonization of returning travelers with CTX-M-producing Escherichia coli. J Travel Med 2011; 18: 299–303.

19 Lübbert C, Straube L, Stein C, et al. Colonization with extended-spectrum beta-lactamase-producing and carbapenemase-producing Enterobacteriaceae in international travelers returning to Germany. Int J Med Microbiol 2015; 305: 148–56.

20 Ruppé E, Armand-Lefèvre L, Estellat C, et al. High Rate of Acquisition but Short Duration of Carriage of Multidrug-Resistant Enterobacteriaceae after Travel to the Tropics. Clin Infect Dis 2015; 61: 593–600.


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21 Hawkey PM, Jones AM. The changing epidemiology of resistance. J Antimicrob Chemother 2009; 64. DOI:10.1093/jac/dkp256.

22 Rossolini GM, D’Andrea MM, Mugnaioli C. The spread of CTX-M-type extended-spectrum beta-lactamases. Clin Microbiol Infect 2008; 14 Suppl 1: 33–41.

23 Beaber JW, Hochhut B, Waldor MK. SOS response promotes horizontal dissemination of antibiotic resistance genes. Nature 2004; 427: 72–4.

24 Woerther PL, Burdet C, Chachaty E, Andremont A. Trends in human fecal carriage of extended-spectrum β-lactamases in the community: Toward the globalization of CTX-M. Clin. Microbiol. Rev. 2013; 26: 744–58.

25 Kennedy K, Collignon P. Colonisation with Escherichia coli resistant to ‘critically important’ antibiotics: A high risk for international travellers. Eur J Clin Microbiol Infect Dis 2010; 29: 1501–6.

26 Poirel L, Potron A, Nordmann P. OXA-48-like carbapenemases: the phantom menace. J Antimicrob Chemother 2012; 67: 1597–606.

27 Van der Bij AK, Pitout JDD. The role of international travel in the worldwide spread of multiresistant enterobacteriaceae. J Antimicrob Chemother 2012; 67: 2090–100.

28 Naas T, Nordmann P. OXA-type beta-lactamases. Curr Pharm Des 1999; 5: 865–79.

29 Leverstein-Van Hall MA, Stuart JC, Voets GM, Versteeg D, Tersmette T FA. Global spread of New Delhi metallo-beta-lactamase 1. Lancet Infect Dis 2010;10:830e1. .

30 Kalpoe JS, Al Naiemi N, Poirel L, Nordmann P. Detection of an ambler class d oxa-48-type β-lactamase in a klebsiella pneumoniae strain in the netherlands. J Med Microbiol 2011; 60: 677–8.

31 Hashimoto A, Nagamatsu M, Ohmagari N, Hayakawa K, Kato Y, Kirikae T. Isolation of OXA-48 carbapenemase-producing Klebsiella pneumonia ST101 from an overseas traveler returning to Japan. Jpn J Infect Dis 2014; 67: 120–1.

32 Platteel TN, Leverstein-van Hall MA, Cohen Stuart JW, et al. Predicting carriage with extended-spectrum beta-lactamase-producing bacteria at hospital admission: A cross-sectional study. Clin Microbiol Infect 2015; 21: 141–6.

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SUPPLEMENTARY DATASupplemenTary Table S1 - Comparison for known data of the 42 participants that did not return the second sample versus the participants that did return their sample to the Public Health Service (GGD) Amsterdam

Risk factorFrequencies Frequencies

n/N (%) n/N (%)

Total 42 445

Age (median (IQR)) 28 (23-40) 33 (27-49)

Sex

Men 9/24 (37,5) 185/444 (41,7)

Women 15/24 (62,5) 259/444(58,3)

missing 18 1

Country of birth

Non-endemic country a 38/42 (90,5) 344/437 (78,7)

Other b 4/42 (9,5) 93/437 (21,3)

Missing 0 8

Travel history (previous 12 months, before current travel)

Stay in a (sub) tropical country - no 4/40 (10,0) 38/418 (9,1)

Stay in a (sub) tropical country - yes 36/40 (90,0) 380/418 (90,9)

Missing 2 27

Duration of travel

Medical history (current travel)

Antimicrobial use - no 30/35 (85,7) 359/409 (87,8)

Antimicrobial use - yes 5/35 (14,3) 50/409 (12,2)

Missing 7 36

Admission to a foreign hospital - no 40/40 (100,0) 423/424 (99,8)

Admission foreign hospital - yes 0/40 (0,0) 1/424 (0,0)

Missing 2 21

Admission to a Dutch hospital - no 39/40 (97,5) 416/430 (96,7)

Admission to a Dutch hospital - yes 1/40 (2,5) 14/430 (3,3)

Missing 2 15

Travel destinations

Only Africa c 4/16 (25,0) 37/428 (8,6)

Only Latin America/Caribbean 4/16 (25,0) 42/428 (9,8)

Only Asia 5/16 (31,3) 71/428 (16,6)


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SupplemenTary Table S1 - Comparison for known data of the 42 participants that did not return the second sample versus the participants that did return their sample to the Public Health Service (GGD) Amsterdam (Continued)

Risk factorFrequencies Frequencies

n/N (%) n/N (%)

Sample

Rectal swab 24/39 (61,5) 167/418 (40,0)

Stool sample 15/39 (38,5) 251/418 (60,0)

Missing 3 27

aCountry of birth and country of birth of both parents is in a non-endemic area (Europe, Oceania, Northern America).

bCountry of birth or country of birth of one of both parents is in an endemic area (Africa, Asia, Latin America/Caribbean).

cAfrica had lowest percentage of ESBL and therefore chosen as reference category.

Extended-Spectrum Beta-Lactamase- and Carbapenemase-Producing

Enterobacteriaceae Isolated from Egyptian Patients with Suspected

Blood Stream Infection

HM Abdallah1,2, BB Wintermans1, EA Reuland1, A Koek1, N al Naiemi1,3,4, AM Ammar2, AA Mohamed2, CMJE Vandenbroucke-Grauls1

PLoS One 2015 May 22;10(5):e0128120

1 Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam the Netherlands2 Department of Bacteriology, Mycology and Immunology, Faculty of Veterinary Medicine,

Zagazig University, Zagazig, Egypt3 Laboratory for Medical Microbiology and Public Health, Hengelo, the Netherlands

4 Microbiology and Infection Control, Ziekenhuisgroep Twente, Almelo, Amsterdam, the Netherlands

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ABSTRACT

ObjectivesThe aim of the study was to investigate the prevalence of extended-spectrum beta-lactamase and carbapenemase production among Enterobacteriaceae isolated from Egyptian patients with suspected blood stream infection.

MethodsNinety-four Enterobacteriaceae blood culture isolates from Egyptian patients with suspected blood stream infection were collected, one isolate per patient. Identification of bacterial isolates was performed with MALDI-TOF (MS-based Vitek MS system, bioMérieux). Screening for ESBLs and carbapenemase production was done with the Vitek 2 system (bioMérieux). ESBL production was confirmed using the combined disk diffusion method for cefotaxime, ceftazidime, and cefepime, all with and without clavulanic acid (Rosco). Real-time PCR and sequencing were used to characterize the resistance genes. The phylogenetic groups of E. coli were identified by a PCR-based method.

ResultsOf the 94 Enterobacteriaceae isolates 46 (48.93%) showed an ESBL phenotype. One Enterobacter spp isolate was ESBL-producer and meropenem-resistant. The genetic analysis showed that CTX-M was present in 89.13% (41/46) of the ESBL-producing Enterobacteriaceae, whereas TEM and SHV were detected in 56.52% (26/46) and 21.74% (10/46) respectively (47.83%) of the ESBL-producing isolates were multidrug resistant (MDR). Eleven out of 30 ESBL-producing E. coli isolates were assigned to phylogroup B2, followed by groups B1 (8 isolates), A (6 isolates) and D (5 isolates).

ConclusionsThe high ESBL-E rates (48.93%) found in this study together with the identification of one carbapenem-resistant Enterobacter spp isolate is worrisome. Our results indicate that systems for monitoring and detection of ESBL-producing bacteria in Egyptian hospitals have to be established. Also strict hospital infection control policies with the restriction of the consumption of extended-spectrum cephalosporins are necessary.

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INTRODUCTION

The beta-lactam antibiotics are the most commonly used therapeutic class of antimicrobials for treatment of bacterial infection because of their broad antibacterial spectrum and excellent safety profile.1 Bacterial resistance to antibiotics is increasing worldwide in healthcare settings and in the community.2 Resistance to beta-lactam antibiotics can be caused by either the production of beta-lactamase enzymes, the presence of beta-lactam-insensitive cell wall transpeptidases, or the active expulsion of beta-lactam molecules from Gram-negative cells by efflux pumps.3 In Enterobacteriaceae, beta-lactamase production remains the most important mediator of beta-lactam resistance.4 Extended-spectrum beta-lactamases (ESBLs) are a rapidly evolving group of beta-lactamases which hydrolyze the extended-spectrum cephalosporins, the penicillins, as well as aztreonam, but not carbapenems.5,6 The spread of extended-spectrum beta-lactamase-producing Enterobacteriaceae (ESBL-E) is increasing worldwide.7 They cause community- and hospital- associated infections in both humans and animals.8 Carbapenems have served as the first-choice drugs for the treatment of ESBL-E especially with the increasing reports of ESBL-producing clinical isolates expressing multidrug resistance (MDR).9 The emergence of carbapenemases-producing Enterobacteriaceae (CPE), is causing an unprecedented public health threat leaving few treatment options. CPE not only infect hospitalized patients, but have also spread in long-term care facilities.10 In Egypt, information about the prevalence of ESBL-E is limited. According to the Pan European Antimicrobial Resistance Local Surveillance (PEARLS) study (2001–2002), performed in 13 European, three Middle Eastern countries and South Africa, Egypt was one of the countries with the highest rate of ESBL among Enterobacteriaceae (38.5%).11A more recent surveillance program for nosocomial bloodstream infections revealed that ESBL production was found to be 80.6% among K. pneumoniae and 40.9% among E. coli.12 Moreover, the majority of CTX-M-15-producing E. coli was related to the international clonal complex ST131.13 CPE was also recently reported in Egypt.14 The aim of this study was to investigate the prevalence of extended-spectrum beta-lactamase and carbapenemase production among Enterobacteriaceae isolated from Egyptian patients with suspected blood stream infection.

MATERIALS AND METHODS

Bacterial isolatesThis is study was conducted in the period between January 2013 and May 2013, at El-Ahrar General Hospital, Zagazig, Egypt, a 608-bed hospital affiliated to the Egyptian health ministry. 10 ml of blood was collected aseptically by peripheral venipuncture from every patient with suspected blood stream infection. Blood culture was done by conventional method described elsewhere.15 A total of 94 enterobacterial isolates, one isolate per patient were identified by the automated VitekMS system (BioMérieux, Marcy l’Étoile, France).

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Phenotypic screening and confirmation of ESBL-E and CPEThe antimicrobial susceptibility testing was performed with the Vitek 2 system with AST N198 (BioMérieux, Marcy l’Étoile, France). The interpretation of antimicrobial susceptibility test results followed recommendations of European Committee on Antimicrobial Susceptibility Testing (EUCAST).16 Phenotypic ESBL production was confirmed with the combination disc diffusion test with clavulanic acid (Rosco, Taastrup, Denmark).17 The inhibition zone around the cephalosporin (cefotaxime, ceftazidime and cefepime) tablet combined with clavulanic acid is compared to the zone around the tablet with the cephalosporin alone. The test is positive if the inhibition zone is > 5 mm larger with clavulanic acid than without.17

Carbapenemases production was confirmed by carbapenemases double disk synergy test.18 Enhancement of the inhibition zone in the area between the carbapenems (meropenem and/or imipenem) and the inhibitor-containing disk (boronic acid and/or dipicolinic acid) was considered to be a positive result.17,19

Real-time PCR for characterization of beta-lactamase-encoding genesAll phenotypic ESBL producers were screened by real-time PCR to identify their ESBL-carrying genes with specific primers for TEM, SHV, and CTX-M.20–22 Phenotypic carbapenemases were analyzed for the presence of genes encoding KPC, NDM, OXA-48, IMP, and VIM by multiplex PCRs using primers described before.23 DNA was extracted by a boiling lysis method as described.24 All real-time PCR amplifications and melting curve analysis were carried out on the LightCycler 480 II system with software version 3.5 (Roche) in a total volume of 20 μl. Amplification conditions were described elsewhere.25,26

DNA sequencing analysisThe amplicons of ESBLs producers were sequenced with the Sanger ABI 3730 XL automated DNA sequencer (BaseClear, Leiden, The Netherlands). The nucleotide sequences were analyzed using the Codon Code Aligner software (Version 5.0.2) and were compared to sequences available at the National Center for Biotechnology Information website (http:// www.ncbi.nlm.nih.gov).

Determination of E. coli PhylogroupsThe assignation of the E. coli isolates to one of the four main phylogenetic groups (A, B1, B2, or D) was performed by a PCR-based method targeted to the chuA, yjaA genes and the TspE4.C2 DNA fragment, as developed by Clermont et al.27


99

Ethics StatementFormal permission was obtained from the managers of Al Ahrar hospitals. Informed written consent was obtained from all participants in this study after explanation of the procedure and the purpose of the study. The study was approved by the review boards of the Research Ethics Committee, Faculty of Medicine, Zagazig University.

RESULTS

Of the 94 tested clinical strains of Enterobacteriaceae isolated from blood of Egyptian patients with suspected blood stream infection, 46 (48.93%) were ESBL positive.

The frequency of ESBL production per species among the tested isolates was the following: 54.5% (30/55) E. coli, 66.66% (10/15) Klebsiella pneumoniae, 35.71% (5/14) Enterobacter spp, and one out of 2 isolates of Morganella morganii. One Enterobacter aerogenes isolate was ESBL-producer and resistant to both imipenem and meropenem.

The genetic analysis showed that CTX-M was present in 89.13% (41/46) of the ESBL-producing Enterobacteriaceae, whereas TEM and SHV were detected in 56.52% (26/46) and 21.74% (10/46) respectively. A summary of the different types of beta-lactamase-encoding genes among different Enterobacteriaceae is provided in Table 1.

Table 1 - Prevalence of the different types of beta-lactamases among different Enterobacteriaceae species.

E.coli Klebsiella pneumoniae

Enterobacter spp

Morganella morganii

Other species Total

CTX-M-15 alone 15 0 0 0 1 5

CTX-M-15 + TEM1 12 0 1 0 0 13

CTX-M-14 + TEM1 2 0 0 0 0 2

CTX-M-15 + CTX-M-14 + TEM1 0 0 1 0 0 1

CTX-M-14 + SHV1 0 2 0 0 2

CTX-M-15 + SHV11 0 1 0 0 0 1

CTX-M-15 + SHV12 0 1 0 0 0 1

TEM1 alone 1 0 3 0 0 4

CTX-M-15 + TEM1 + SHV1 0 2 0 0 0 2

CTX-M-15 + TEM1 + SHV11 0 3 0 0 0 3

CTX-M-15 + TEM1 + SHV12 0 1 0 0 0 1

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Sequence analysis revealed that CTX-M comprised mainly CTX-M-15 (37/41, 90.24%) and CTX-M-14 (5/41, 12.20%). One Enterobacter spp isolate harbored both alleles. All TEM positive isolates were found to harbor the prototype TEM-1 enzyme, while SHV belonged to SHV- 1 (n = 4), SHV-11 (n = 4), and SHV-12 (n = 2).One Morganella morganii isolate expressed ESBL phenotype but no TEM, SHV or CTX-M was detected by PCR. The carbapenem resistant Enterobacter aerogenes isolate was found to be positive for NDM.

Of the 46 ESBL-producing isolates, 37 (80.43%) showed combined resistance to quinolones (ciprofloxacin and/or norfloxacin), 36 (78.26%) to trimethoprim/sulfamethoxazole, 30 (56.22%) to aminoglycosides (gentamicin and/or tobramycin) and 9 (19.57%) to nitrofurantoin. Twenty-two (47.83%) of the ESBL-producing isolates were multidrug resistant (MDR) (i.e. not susceptible to at least one agent in three or more classes of antimicrobials (aminoglycosides, quinolones and cotrimoxazole).28

Phylogenic analysis revealed that 11 out of 30 ESBL-producing E. coli isolates belonged to phylogroup B2, 8 to group B1, 6 to group A, and 5 to group D. The distribution of the different ESBL types over the phylogroups is shown in Table 2.

Table 2 - Phylogenetic groups of ESBL producing E. coli isolates.

Type of ESBL Number of isolates belonging to phylogenetic groups

A B1 B2 D

CTX-M alone (n = 15) 2 5 7 1

CTX-M +TEM (n = 14) 4 3 4 3

TEM alone(n = 1) 0 0 0 1

Total(n = 30) 6 8 11 5

DISCUSSION

This study was carried out to determine the prevalence of extended-spectrum beta-lactamase and carbapenemase production among Enterobacteriaceae isolated from Egyptian hospitalized patients with suspected blood stream infection. Our findings showed that 48.93% of enterobacterial strains isolated from blood specimens were ESBL producers. This is a high prevalence compared to many European countries (http://www.ecdc.europa.eu/en/healthtopics/antimicrobial_resistance/database/Pages/database.aspx). Possibly, this high prevalence is related to the less controlled use of antibiotics in Egypt, where many drugs are still available over the counter. One carbapenem-resistant strain was detected.

Few studies have investigated the prevalence of ESBL-E in Egyptian hospitals. Bouchillon et

al. conducted the PEARLS study in 2001–2002, and found that 38.5% of Enterobacteriaceae

http://www.ecdc.europa.eu/en/healthtopics/

http://www.ecdc.europa.eu/en/healthtopics/


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isolates did produce an ESBL.11 A lower ESBL prevalence rate (16%) was found among 120 isolates collected between May 2007 and August 2008 at the Theodor Bilharz Research Institute, Cairo, Egypt. In 2009, Ahmed et al. detected 64.7% ESBL-producing Enterobacteriaceae among strains isolated from patients in the intensive care unit of a university hospital.29 Considering that we included bloodstream infections from all departments in El-Ahrar General Hospital, Zagazig, Egypt, the rate that we observed is in line with the findings of Ahmed et al.

The results obtained in this study showed that blaCTX-M

type was the most prevalent beta-lactamase-encoding gene. It was detected in almost 90% of the ESBL-producing Enterobacteriaceae, whereas bla

TEM and bla

SHV were present in about half and one fifth of

isolates, respectively. These findings agree with other contemporary studies from around the world that show that ESBL genes of the CTX-M are dominant.30,31 In contrast to our findings, Ahmed et al. reported that bla

TEM was the most frequent beta-lactamase-encoding

gene.29

One NDM-producing Enterobacter aerogenes isolate was found. This goes along with the recent detection of NDM-producing enterobacterial strains in Egypt, as well as other countries in the Middle East and North Africa.14,32–34

Our data showed that the CTX-M-15 allele was the dominant CTX-M type ESBL with a frequency of over 90% followed by the CTX-M-14 allele with a frequency rate of over 12%. One isolate expressed the two alleles. These findings were concordant with the results of other studies carried out in Egypt and Europe.13,35,36

Although TEM-1 and SHV-1 are not regarded as extended-spectrum beta-lactamases, presence of these enzymes, combined with changes in the outer membrane proteins lead to reduced susceptibility to third-generation cephalosporins, that phenotypically suggest ESBL production.37 Both SHV-11 and SHV-12 identified in this study are SHV-type ESBL.38 SHV-12-producing Klebsiella pneumoniae has been recently reported in Egypt.39

As expected, the ESBL-producing blood isolates showed a high frequency of co-resistance to quinolones, trimethoprim/sulfamethoxazole, aminoglycosides, and nitrofurantoin. Co-resistance was multiple in many cases, which means that nearly half of the isolates were multidrug resistant. This high level of multidrug resistance poses even more treatment problems than just the production of beta-lactamases.

Phylogenetic analysis of CTX-M-producing E. coli isolates revealed that most isolates belonged to the extraintestinal pathogenic group B2.This finding is consistent with previous studies that showed the dominance of phylogroup B2 among CTX-M-producers, and further confirms that this phylogroup contains resistant and virulent clones.40,41 On the other hand, a study carried out by Branger et al. demonstrated that CTX-M is present mainly in strains belonging to group D, while TEM was mainly found in E. coli with genetic background B2.42

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In our strain collection, TEM-producing E. coli isolates were nearly equally divided over phylogroups A, B2 and D, while group B1 was less represented. The differences between Branger’s and our findings are probably related to the different geographical background of the patients from whom the strains were collected. They show that interpretation of genetic background in relation to plasmid-borne resistance genes is influenced by where and when a strain collection is assembled.

In conclusion, nearly half of Enterobacteriaceae isolated from blood in patients suspected of blood stream infection were ESBL producers. This high frequency of ESBL-E is worrisome. Our results indicate that systems for detection and monitoring of ESBL-producing bacteria in Egyptian hospitals have to be established. Also strict hospital infection control policies with restriction of the consumption of expanded-spectrum cephalosporins are necessary.

ACKNOWLEDGEMENTS

We would like to express our gratitude to the staff of the microbiology department, El-Ahrar General Hospital, Zagazig, Egypt, for collecting the blood samples.


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7 Biehl LM, Schmidt-Hieber M, Liss B, Cornely O a, Vehreschild MJGT. Colonization and infection with extended spectrum beta-lactamase producing Enterobacteriaceae in high-risk patients - Review of the literature from a clinical perspective. Crit Rev Microbiol 2014; 7828: 1–16.

8 Rubin JE, Pitout JDD. Extended-spectrum β-lactamase, carbapenemase and AmpC producing Enterobacteriaceae in companion animals. Vet. Microbiol. 2014; 170: 10–8.

9 Morosini MI, García-Castillo M, Coque TM, et al. Antibiotic coresistance in extended-spectrum-β-lactamase-producing Enterobacteriaceae and in vitro activity of tigecycline. Antimicrob Agents Chemother 2006; 50: 2695–9.

10 Tzouvelekis LS, Markogiannakis A, Psichogiou M, Tassios PT, Daikos GL. Carbapenemases in Klebsiella pneumoniae and other Enterobacteriaceae: An evolving crisis of global dimensions. Clin Microbiol Rev 2012; 25: 682–707.

11 Bouchillon SK, Johnson BM, Hoban DJ, et al. Determining incidence of extended spectrum beta-lactamase producing Enterobacteriaceae, vancomycin-resistant Enterococcus faecium and methicillin-resistant Staphylococcus aureus in 38 centres from 17 countries: the PEARLS study 2001-2002. Int J Antimicrob Agents 2004; 24: 119–24.

12 Saied T, Elkholy A, Hafez SF, et al. Antimicrobial resistance in pathogens causing nosocomial bloodstream infections in university hospitals in Egypt. Am J Infect Control 2011; 39. DOI:10.1016/j.ajic.2011.04.009.

13 Fam N, Leflon-Guibout V, Fouad S, et al. CTX-M-15-producing Escherichia coli clinical isolates in Cairo (Egypt), including isolates of clonal complex ST10 and clones ST131, ST73, and ST405 in both community and hospital settings. Microb Drug Resist 2011; 17: 67–73.

14 Abdelaziz MO, Bonura C, Aleo A, Fasciana T, Mammina C. NDM-1- and OXA-163-producing Klebsiella pneumoniae isolates in Cairo, Egypt, 2012. J Glob Antimicrob Resist 2013; 1: 213–5.

15 Nagoba BS NB. Microbiology for Dental Students [Internet]. BI Publications Pvt Ltd; 2007. p. 342. Available: https://books.google.com/books?id = w1rbVel7Q6YC&pgis=1. .

16 EUCAST. European Committee on Antimicrobial Susceptibility Testing Breakpoint tables for interpretation of MICs and zone diameters European Committee on Antimicrobial Susceptibility Testing Breakpoint tables for interpretation of MICs and zone diameters. 2014; : 0–79.


18 Pasteran F, Mendez T, Guerriero L, Rapoport M, Corso A. Sensitive screening tests for suspected class A carbapenemase production in species of Enterobacteriaceae. J Clin Microbiol 2009; 47: 1631–9.

19 Tsakris A, Poulou A, Themeli-Digalaki K, et al. Use of boronic acid disk tests to detect extended-spectrum beta-lactamases in clinical isolates of KPC carbapenemase-possessing Enterobacteriaceae. J Clin Microbiol 2009; 47: 3420–6.

20 Olesen I, Hasman H, Aarestrup FM. Prevalence of beta-lactamases among ampicillin-resistant Escherichia coli and Salmonella isolated from food animals in Denmark. Microb drug Resist 2004; 10: 334–40.

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21 Weill FX, Demartin M, Tandé D, Espié E, Rakotoarivony I, Grimont PAD. SHV-12-like extended-spectrum-β-lactamase-producing strains of Salmonella enterica serotypes Babelsberg and Enteritidis isolated in France among infants adopted from Mali. J Clin Microbiol 2004; 42: 2432–7.

22 Mulvey MR, Soule G, Boyd D, Demczuk W, Ahmed R. Characterization of the first extended-spectrum beta-lactamase-producing Salmonella isolate identified in Canada. J Clin Microbiol 2003; 41: 460–2.


24 De Medici D, Croci L, Delibato E, Di Pasquale S, Filetici E, Toti L. Evaluation of DNA extraction methods for use in combination with SYBR green I real-time PCR to detect Salmonella enterica serotype Enteritidis in poultry. Appl Environ Microbiol 2003; 69: 3456–61.

25 Naas T, Oxacelay C, Nordmann P. Identification of CTX-M-Type Extended-Spectrum- -Lactamase Genes Using Real-Time PCR and Pyrosequencing. Antimicrob Agents Chemother 2007; 51: 223–30.

26 Wang L, Gu H, Lu X. A rapid low-cost real-time PCR for the detection of Klebsiella pneumonia carbapenemase genes. Ann Clin Microbiol Antimicrob 2012; 11: 9.

27 Clermont O, Bonacorsi S, Bingen E. Rapid and simple determination of the Escherichia coli phylogenetic group. Appl Environ Microbiol 2000; 66: 4555–8.

28 Epstein S. Small Animal Critical Care Medicine [Internet]. Small Animal Critical Care Medicine. Elsevier; 2015. pp. 537–540. doi: 10.1016/B978-1-4557-0306-7.00103–3. .

29 Ahmed SH, Daef EA, Badary MS, Mahmoud MA, Abd-Elsayed AA. Nosocomial blood stream infection in intensive care units at Assiut University Hospitals (Upper Egypt) with special reference to extended spectrum beta-lactamase producing organisms. BMC Res Notes 2009; 2: 76.

30 Livermore DM, Canton R, Gniadkowski M, et al. CTX-M: changing the face of ESBLs in Europe. J Antimicrob Chemother 2007; 59: 165–74.

31 Cantón R, González-Alba JM, Galán JC. CTX-M enzymes: Origin and diffusion. Front. Microbiol. 2012; 3. DOI:10.3389/fmicb.2012.00110.

32 Poirel L, Benouda A, Hays C, Nordmann P. Emergence of NDM-1-producing Klebsiella pneumoniae in Morocco. J Antimicrob Chemother 2011; 66: 2781–3.

33 Sonnevend A, Al Baloushi A, Ghazawi A, et al. Emergence and spread of NDM-1 producer Enterobacteriaceae with contribution of IncX3 plasmids in the United Arab Emirates. J Med Microbiol 2013; 62: 1044–50.

34 Shahcheraghi F, Nobari S, Rahmati Ghezelgeh F, et al. First report of New Delhi metallo-beta-lactamase-1-producing Klebsiella pneumoniae in Iran. Microb Drug Resist 2013; 19: 30–6.


36 Reuland EA, Overdevest IT, Al Naiemi N, et al. High prevalence of ESBL-producing Enterobacteriaceae carriage in Dutch community patients with gastrointestinal complaints. Clin Microbiol Infect 2012. DOI:10.1111/j.1469-0691.2012.03947.x.

37 Wu T-L. Outer membrane protein change combined with co-existing TEM-1 and SHV-1 beta-lactamases lead to false identification of ESBL-producing Klebsiella pneumoniae. J Antimicrob Chemother. 2001; 47: 755–761. doi: 10.1093/jac/47.6.755. .

38 Bradford PA. Extended-spectrum beta-lactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. Clin Microbiol Rev 2001; 14: 933–51, table of contents.

39 Newire EA, Ahmed SF, House B, Valiente E, Pimentel G. Detection of new SHV-12, SHV-5 and SHV-2a variants of extended spectrum Beta-lactamase in Klebsiella pneumoniae in Egypt. Ann Clin Microbiol Antimicrob 2013; 12: 1.

40 Pitout JDD, Laupland KB, Church DL, Menard ML, Johnson JR. Virulence factors of Escherichia coli isolates that produce CTX-M-type extended-spectrum beta-lactamases. Antimicrob Agents Chemother 2005; 49: 4667–70.

41 Lee S, Yu JK, Park K, Oh EJ, Kim SY, Park YJ. Phylogenetic groups and virulence factors in pathogenic and commensal strains of escherichia coli and their association with blaCTX-M. Ann Clin Lab Sci 2010; 40: 361–7.

42 Branger C, Zamfir O, Geoffroy S, et al. Genetic background of Escherichia coli and extended-spectrum beta-lactamase type. Emerg Infect Dis 2005; 11: 54–61.

Extended-Spectrum Beta-Lactamases and/or Carbapenemases-Producing

Enterobacteriaceae Isolated from Retail Chicken Meat in Zagazig, Egypt

HM Abdallah1, EA Reuland1, BB Wintermans1, N al Naiemi1,2,3, A Koek1, AM Abdelwahab4, AM Ammar4, AA Mohamed4, CMJE Vandenbroucke-Grauls1

PLoS One 2015 Aug 18;10(8):e0136052

1 Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, the Netherlands2 Laboratory for Medical Microbiology and Public Health, Hengelo, the Netherlands

3 Microbiology and Infection Control, Ziekenhuisgroep Twente, Almelo, Amsterdam, the Netherlands4 Department of Bacteriology, Mycology and Immunology, Faculty of Veterinary Medicine,

Zagazig University, Zagazig, Egypt

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ABSTRACT

ObjectivesThe aim of the present study was to determine the prevalence and to characterize extended-spectrum beta-lactamases- and/or carbapenemase-producing Enterobacteriaceae among Enterobacteriaceae isolated from retail chicken meat in Zagazig, Egypt.

MethodsOne hundred and six Enterobacteriaceae isolates were collected from retail chicken meat samples purchased in Zagazig, Egypt in 2013. Species identification was done by MALDI-TOF MS. Screening for ESBL-E was performed by inoculation of isolates recovered from meat samples onto the EbSA (Cepheid Benelux, Apeldoorn, the Netherlands) selective screening agar. ESBL production was confirmed by combination disc diffusion test with clavulanic acid (Rosco, Taastrup, Denmark). Carbapenemase production was confirmed with double disk synergy tests. Resistance genes were characterized by PCR with specific primers for TEM, SHV, and CTX-M and carbapenemases (KPC, NDM, OXA-48, IMP and VIM). PCR products of CTX-M genes were purified and sequenced. Phylogenetic grouping of E. coli was performed by a PCR-based method.

ResultsOf these 106 isolates 69 (65.09%) were ESBL producers. Twelve (11.32%) of these isolates were also phenotypically class B carbapenemase producer. TEM genes were detected in 61 (57.55%) isolates. 49 (46.23%) isolates harbored CTX-M genes, and 25 (23.58%) carried genes of the SHV family. All CPE belonged to the NDM group. The predominant CTXM sequence type was CTX-M-15 (89.80%). The majority (80%) of the ESBL-EC belonged to low virulence phylogroups A and B1.

ConclusionsThis is the first study from Egypt reporting high rates of ESBLs and carbapenemases (65.09% and 11.32%, respectively) in Enterobacteriaceae isolated from retail chicken meat. These results raise serious concerns about public health and food safety as retail meat could serve as a reservoir for these resistant bacteria which could be transferred to humans through the food chain.

ChiCken Meat ContaMination with eSBL-e and CPe

109

INTRODUCTION

The beta-lactam antibiotics have been amongst the most successful drugs for the treatment of bacterial infections for the past 60 years.1 They are arguably the most important and widely used antimicrobial class for treating bacterial infections in both human and veterinary medicine, because of their excellent safety profile, broad antimicrobial spectrum, availability of orally bioavailable formulations, and the low cost of many products.2 More than half of all currently used antibiotics belong to the beta-lactam group, but their clinical effectiveness is severely limited by the emergence of beta-lactam resistant bacteria.3 The resistance to beta-lactam antibiotics occurs as a result of drug inactivation by beta-lactamases, target site (penicillin-binding proteins) alterations, diminished permeability and efflux.4 In Gram-negative pathogens, beta-lactamases are the major determinant of this resistance.5 Extended-spectrum beta-lactamases (ESBLs) are a rapidly evolving group of beta-lactamases which hydrolyze third-generation cephalosporins and aztreonam but not carbapenems.6 Extended-spectrum beta-lactamase producing Enterobacteriaceae (ESBL-E) are prevalent worldwide.7 Chicken meat has been proposed to constitute a source for ESBL-E that colonize and infect humans.8 Close genetic similarities among extended-spectrum beta-lactamase-producing Escherichia coli (ESBL-EC) isolated from chicken meat and humans together with the concurrent presence of CTX-M-1 and TEM-52 genes on similar plasmids of Escherichia coli isolated from both sources support the occurrence of food-borne transmission of ESBL genes.9,10 Furthermore, ESBL-EC isolated from chicken meat was documented as a source of ESBL-EC in humans.11 Previous studies reported high ESBL contamination rates of chicken meat in the Netherlands, Sweden and recently in Germany.8,10,12–14 A recent study demonstrated the presence of carbapenemase-producing Enterobacteriaceae (CPE) in Broiler Chicken Fattening Farms but there are no reports on acquired carbapenemase producers from retail chicken meat.15,16

In Egypt, ESBL and/or CPE have been reported in hospitalized patients.17,18 It is not known, however, whether Egyptian chicken meat is contaminated with ESBL-E and/or CPE. Therefore, we carried out this study to determine the prevalence and to characterize ESBL-E and/or CPE isolated from retail chicken meat in Zagazig, Egypt.

METHODS

Bacterial isolatesOver a period of eight weeks between January and March, 2013, seven butcher shops, located in different districts of Zagazig City, Egypt (latitude 30°35015@ N; longitude 31°30007@ E and altitude 16 meter above sea level), were visited once a week. At each visit, two random fresh chicken carcasses were bought at each shop, and immediately transported to the

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laboratory for culture. Sampling was done by whole carcass rinse method.19 The rinse fluid was collected, plated in parallel on selective EbSA-ESBL Screening Agar for the isolation of bacteria resistant to broad-spectrum cephalosporins and on MacConkey agar for the characterization of the dominant flora.20 The two plates were incubated aerobically at 37°C for 24 h. A pure colony was picked up from both plates for further identification by Vitek MS system (BioMérieux, Marcy l’Étoile, France).

Phenotypic screening and confirmation of ESBL-E and CPEESBL and carbapenemase production were screened by disk diffusion method on Mueller- Hinton agar using ceftazidime (30 μg), cefotaxime (30 μg), meropenem (10 μg), imipenem (10 μg) and ertapenem (10 μg), and interpreted according to the clinical breakpoints recommended by CLSI and NVMM.21,22 Confirmation of ESBL production was carried out by the combination disc diffusion test with clavulanic acid (Rosco, Taastrup, Denmark). The inhibition zone around the cephalosporin (cefotaxime, ceftazidime and cefepime) discs combined with clavulanic acid (CA) is compared to the zone around the discs with the cephalosporin alone. A positive test is defined as ≥5 mm increase in zone diameter around the cephalosporin disc with CA in comparison to a disc without.21,22

Carbapenemase production was confirmed by carbapenemase double disk synergy test.23 Enhancement of the inhibition zone in the area between the carbapenems (meropenem and/or Imipenem) and the inhibitor-containing disk (3-aminophenylboronic acid (APBA), or dipicolinic acid (DPA)) was considered to be a positive result.24

Real-time PCR for characterization of beta-lactamase-encoding genesDNAs of all phenotypic ESBL- and carbapenemase positive isolates were extracted by boiling lysis method as described previously.25,26 The phenotypic ESBL-positive isolates were analyzed for the presence of genes encoding TEM, SHV and CTX-M by real-time PCR using primers described before.27–29 Carbapenemase positive isolates were screened for KPC, NDM, OXA-48, IMP and VIM by multiplex PCRs using primers described before.30 All real-time PCR amplifications and melting curve analysis were carried out on the LightCycler 480 II system with software version 3.5 (Roche, Mannheim, Germany) in a total volume of 20 μl. Amplification conditions were as described elsewhere.31,32

DNA sequencing analysisPurified PCR products of ESBL-E were sequenced with Sanger ABI 3730 XL automated DNA sequencer (BaseClear, Leiden, The Netherlands). The nucleotide sequences were analyzed using the CodonCode Aligner software (Version 5.0.2), compared, and aligned with reference sequences available at the National Center for Biotechnology Information website (http:// www.ncbi.nlm.nih.gov).

http://www.ncbi.nlm.nih.gov


111

Phylogenetic grouping of E.coliE. coli isolates were allotted to one of the four main phylogenetic groups (A, B1, B2, or D) using a PCR-based method targeted to the chuA and yjaA genes and the TspE4.C2 DNA fragment, developed by Clermont et al.33

RESULTS

Of the total 112 carcasses collected, cultures of 12 carcasses had to be discarded because of the growth of Pseudomonas spp. (n = 7) or Gram-positive cocci (n = 5). ESBL-E was found in 63% (63/100) of the carcasses contaminated with Enterobacteriaceae, whereas 12 carcasses harbored CPE. Some carcasses showed growth of more than one species of Enterobacteriaceae, resulting in 106 isolates available for analysis, 69 (65.1%) isolates were ESBL producers (Table 1). The distribution of ESBL-producing species among different Enterobacteriaceae was: 44(63.77%) Klebsiella pneumoniae, 10 (14.49%) E. coli, 13 (18.84%) Enterobacter cloacae, and 2 (2.90%) Klebsiella oxytoca. Twelve (11.32%) of these isolates were also phenotypically class B carbapenemase-producers. CPE were Klebsiella pneumoniae (n = 11), and Klebsiella oxytoca (n = 1).

Table 1 - Prevalence of the different types of beta-lactamase-encoding genes among different Enterobacteriaceae.

species No. ofisolates

No. of ESBL

positive

TEMalone

TEM+ CTX-M

TEM+ SHV

TEM + CTX-M+ SHV

CTX-Malone

CTX-M+ SHV

Klebsiella pneumoniae 44 44 11 7 3 20 1 2

E.coli 38 10 2 4 0 0 4 0

Enterobacter spp 21 13 4 9 0 0 0 0

Klebsiella oxytoca 2 2 0 1 0 0 1 0

Citrobacter spp 1 0 0 0 3 0 0 0

Total 106 69 17 21 3 20 6 2

The TEM gene was detected in 61 (57.55%) isolates. 49 (46.23%) isolates contained CTX-M genes; of these, 47 (95.92%) belonged to CTX-M-1 group (44 CTX-M-15 and 3 unidentified) and 2 (4.08%) belonged to CTX-M-9 group (all were CTX-M-14) and 25 (23.58%) belonged to the SHV family.

20 isolates coproduced TEM, SHV, and CTX-M genes, 21 harbored CTX-M and TEM genes, 2 contained CTX-M and SHV genes, 3 expressed TEM and SHV genes, 6 possessed CTX-M genes alone and 17 produced TEM genes only (see also Table 1). All CPE belonged to the NDM group. The numbers of carcasses contaminated with ESBL-E and/or CPE per each shop during the different sampling period is shown in Table 2.

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Tabl

e 2 -

Num

bers

of c

arca

sses

con

tam

inat

ed w

ith E

SBL-

E an

d/or

CPE

per

eac

h sh

op d

urin

g th

e di

ffere

nt s

ampl

ing

perio

d.

Sam

plin

gPe

riod

sSh

op 1

Shop

2Sh

op 3

Shop

4Sh

op 5

Shop

6Sh

op 7

Tota

l

ESBL

-ECP

EES

BL-E

CPE

ESBL

-ECP

EES

BL-E

CPE

ESBL

-ECP

EES

BL-E

CPE

ESBL

-ECP

EES

BL-E

CPE

11

02

01

01

01

12

01

19

2

22

01

10

01

01

01

02

28

3

31

01

01

00

01

01

01

06

0

41

11

02

11

02

10

01

08

3

51

02

01

02

11

00

01

08

1

61

01

02

11

01

01

11

08

2

72

00

00

02

01

01

02

18

1

81

01

01

01

02

00

02

08

0

Tota

l10

19

18

29

110

26

111

463

12


113

Phylogenetic grouping revealed, of the 10 ESBL-EC isolates, 4 belonged to group A, 4 to group B1, 2 to group D and none to group B2. In the ESBL-negative E. coli, 6 of the isolates belonged to group A, 11 to group B1, 7 to group B2 and 4 to group D.

DISCUSSION

Our data showed that two thirds of Enterobacteriaceae isolates recovered from chicken meat samples were ESBL positive; more than one in ten isolates were also resistant to carbapenems. To the best of our knowledge, this is the first study conducted to determine the prevalence and to characterize ESBL-E and/or CPE isolated from retail chicken meat in Egypt. Nearly similar results were found in Spain, where 67% of the chicken meat was reported to be contaminated with ESBL or ESBL-like resistance genes.34 However, higher rates of ESBL-E in chicken meat were reported in Switzerland and the Netherlands.35–37 On the other hand, lower rates of ESBL-E were found in chicken meat in Gabon and Germany.14,38

The detection of high numbers of carbapenem-resistant isolates harboring NDM raises serious concerns about public health since carbapenems are considered the first-line drugs for the treatment of serious infections due to ESBL-producing bacteria.39 Carbapenemase-producing isolates have been detected in poultry farms but there are no reports on acquired carbapenemase producers from retail chicken meat.15,16 NDM-producing Enterobacteriaceae isolated from human clinical setting were recently reported in Egypt, Morocco, Oman, United Arab Emirates, and Iran.18,40–42

The predominant CTX-M sequence type was CTX-M-15, amounting to nearly 90%, while CTX-M-14 accounted for less than 5% of the CTX-M-producing isolates. A study on Dutch retail chicken meat revealed that CTX-M-15 was not detected and CTX-M-1 was the most prevalent CTX-M ESBL type.9 Another study of broiler chickens in Great Britain found that CTX-M-1 was the most common CTX-M sequence type followed by CTX-M-15.43

Phylogenetic analysis of E. coli isolates revealed that the vast majority (80%) of the ESBL-EC belonged to phylogroups A and B1, which include E. coli isolates of low virulence and commensal origin. This finding elucidates the pivotal silent role played by these commensal isolates in the spread of ESBL resistance genes. On the other hand, non-ESBL-producing isolates belonged mainly to the commensal phylogroup B1 and, to lesser extents, to phylogroups B2 and A, while the minority of isolates were phylogroup D.

In conclusion, this is the first study from Egypt showing high rates of ESBLs and carbapenemases (65.09% and 11.32%, respectively) in Enterobacteriaceae isolated from retail chicken meat. These results raise serious concerns about public health and food safety as retail meat could serve as a reservoir for these resistant bacteria which could be potentially transferred to humans through the food chain.

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39 Poirel L, Benouda A, Hays C, Nordmann P. Emergence of NDM-1-producing Klebsiella pneumoniae in Morocco. J Antimicrob Chemother 2011; 66: 2781–3.

40 Sonnevend A, Al Baloushi A, Ghazawi A, et al. Emergence and spread of NDM-1 producer Enterobacteriaceae with contribution of IncX3 plasmids in the United Arab Emirates. J Med Microbiol 2013; 62: 1044–50.

41 Shahcheraghi F, Nobari S, Rahmati Ghezelgeh F, et al. First report of New Delhi metallo-beta-lactamase-1-producing Klebsiella pneumoniae in Iran. Microb Drug Resist 2013; 19: 30–6.

42 Randall LP, Clouting C, Horton RA, et al. Prevalence of Escherichia coli carrying extended-spectrum β-lactamases (CTX-M and TEM-52) from broiler chickens and turkeys in Great Britain between 2006 and 2009. J Antimicrob Chemother 2011; 66: 86–95.

43 Livermore DM, Hawkey PM. CTX-M: Changing the face of ESBLs in the UK. J. Antimicrob. Chemother. 2005; 56: 451–4.

Prevalence of ESBL-producing Enterobacteriaceae in raw vegetables

EA Reuland1, N al Naiemi1 ,2,3, SA Raadsen1, PHM Savelkoul1, JAJW Kluytmans1,4, CMJE Vandenbroucke-Grauls1

European Journal of Clinical Microbiology & Infectious Diseases 2014 Oct;33(10):1843-6


3 Medical Microbiology and Infection Control, Ziekenhuisgroep Twente, Almelo4 Department of Medical Microbiology and Infection Control, Amphia Hospital, Breda, The Netherlands

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ABSTRACT

ObjectivesTo determine whether extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E) are present in retail raw vegetables in Amsterdam, the Netherlands.

MethodsWe collected 119 samples of 15 different types of vegetables from various sources. After culture, strain identification and susceptibility testing, ESBL-encoding genes were characterized by a microarray.

ResultsFour of the 15 vegetable types were contaminated with ESBL-E. Seven samples (6%) yielded ESBL-E. Three bla

CTX-M-15, one bla

CTX-M-1, two genes of the CTX-M-9 group and one SHV ESBL-

encoding gene were found.

Conclusions The ESBL genes were similar to what is found in enterobacterial strains from human origin. Therefore, raw vegetables might be a source of resistance genes for the enterobacterial strains found in humans.

Prevalence of eSBl-e in raw vegetaBleS

121

INTRODUCTION

Extended-spectrum beta-lactamases (ESBLs) are emerging rapidly worldwide. ESBLs have been detected in patients without prior healthcare contact, even in countries with low consumption of antibiotics (ESAC-Net; http://www.ecdc.europa.eu/en/activities/surveillance/esac-net/pages/index. aspx). Major sources of ESBLs in the community can be envisaged: resistant strains from high ESBL prevalence reservoirs (hospitals and long-term care facilities) or resistant strains present in the food chain, environment or water sources.1 The complex dynamics and dissemination of antibiotic resistance and its relation to different reservoirs has been depicted by Davies and Davies and by Wellington et al.2,3 Especially, the food chain has recently attracted attention because a high prevalence of resistance genes in foodproducing animals like poultry was reported; this is related to the high rate of antimicrobial drug use in the livestock sector.4,5 In addition, resistance genes are also widespread in agriculture.2,6 Ruimy et al. observed that vegetables in France were often contaminated with resistance genes.7 In 2011, a Shiga toxin-producing Escherichia coli (STEC) outbreak in Germany, caused by ESBL-producing E. coli, was traced to sprouts.8

The aim of this study was to evaluate the presence of ESBL-producing Enterobacteriaceae (ESBL-E) in raw vegetables in the region of Amsterdam, the Netherlands.

METHODS

Study designIn October, 2010, and March, 2011, 119 samples from 15 different types of retail vegetables were purchased from an organic store, the market, a store of a large Dutch supermarket chain and a local supermarket. We obtained two samples from each vegetable type and from each different source. We focused on vegetables grown on and in the ground/soil, and on (mung) bean sprouts. The 15 vegetables included: beet root, Brussels sprouts, carrots, cauliflower, celery, chicory, cucumber, lettuce, mushrooms, parsnip, potatoes, radish, spinach, spring onion and (mung) bean sprouts. Of each unwashed sample, 1 g was ground and inoculated in 10 ml of trypticase soy broth (Becton Dickinson, Breda, the Netherlands) supplemented with ceftazidime 0.5 mg/ L (incubation overnight, at 37 °C). Thereafter, the broth was subcultured on a selective screening agar (EbSA, Cepheid Benelux, Apeldoorn, the Netherlands) (incubation overnight, 37 °C).9 We only analyzed enterobacterial species.

Phenotypic and genotypic testingIdentification and antibiotic susceptibility testing were performed with the Vitek 2 system (bioMérieux, Marcy l’Etoile, France). The minimum inhibitory concentration (MIC) breakpoints according to the Clinical and Laboratory Standards Institute (CLSI) were used.10

http://www.ecdc.europa.eu/en/activities/surveillance/esac-net/pages/index

http://www.ecdc.europa.eu/en/activities/surveillance/esac-net/pages/index

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Phenotypic ESBL production was confirmed with the combination disc diffusion test with clavulanic acid (Rosco, Taastrup, Denmark).11

After DNA isolation (QIAamp DNA mini kit, Qiagen, Venlo, the Netherlands), isolates were screened for ESBL resistance genes by a microarray, which enables the distinction between the most prevalent ESBLs, including CTX-M-1 and CTX-M-15 (Check-MDR CT103, Check-Points, Wageningen, the Netherlands).12 The presence of carbapenemase genes and plasmid-mediated AmpCs was also tested with the microarray. Plasmids were identified by polymerase chain reaction (PCR)-based replicon typing.13 The strains were tested with 22 primer sets for Inc groups (ColE, FIA, ColEtp, HI1, HI2, T, Inc I1, FrepB, R, FIIs, FIB, P, B/O, A/C, K, U, L/M, W, N, X, FIC, Y).

RESULTS

Seven of the 119 samples (6%) yielded ESBL-E (Table 1). The type of contaminated vegetables, ESBL-producing strains, genes and plasmids found are described in Table 1. The contaminated samples were from four of the 15 vegetable types (27%). The majority of ESBL-positive samples (5/7) were derived from an organic store, the prevalence of ESBL-E in organically grown vegetables was 15.6% (5/32) [95% confidence interval (CI): 6.4–32.2] and in conventionally grown vegetables it was 2.3% (2/87) (95% CI: 0.14–8.5). The risk difference (RD) between the two types of vegetables was significant (RD 13.3%, 95% CI: 0.35–26.31). The variation in plasmids and genes found is in accordance with the findings of Carattoli et

al.13

A high rate of co-resistance to gentamicin, co-trimoxazole and nitrofurantoin was observed. Two out of these seven ESBL-producing strains were multi-resistant, but all were susceptible to meropenem and imipenem.14

TABLE 1 - Overview vegetable types in relation to extended-spectrum beta-lactamase (ESBL)-encoding genes and plasmids.

Store Vegetable Species ESBL gene Plasmids

Organic store Bean sprouts Klebsiella pneumoniae blaSHV-12

ColEtp, T

Bean sprouts Klebsiella pneumoniae blaCTX-M-14

ColE, FIA

Radish Enterobacter cloacae blaCTX-M-15

ColEtp, HI2

Spring onion Enterobacter amnigenus blaCTX-M-15

HI2

Parsnip Citrobacter braakii blaCTX-M-1

HI1

Market Bean sprouts Klebsiella pneumoniae blaCTX-M-14

ColEtp, FIA, T

Supermarket (local) Bean sprouts Enterobacter cloacae blaCTX-M-15

ColEtp, HI2


123

DISCUSSION

Our results document the presence of ESBL-E in retail raw vegetables obtained in Amsterdam, the Netherlands, which implies that raw vegetables may be a source of resistance genes. These results correlate with other studies pointing to vegetables as a possible route for the dissemination of resistance genes in the community.4,7,15,16

Different pathways might be relevant to explain why these resistance genes were found on raw vegetables.2,6 Knapp et al. showed that the levels of antibiotic resistance genes in soil have increased considerably over the past 70 years in the Netherlands.17 Other reservoirs of antibiotic resistance genes are the aquatic system and sewage, created by antibiotic use and waste disposal.2,4,18,19 Fresh produce can also become contaminated during processing.15 We bought the vegetables at the market or at the shop itself, thereby providing information on the vegetables actually bought by the consumer, but this implies that contamination could have occurred during human handling.

A remarkable finding of the present study is that organic vegetables were more often contaminated with ESBL-E than conventionally produced vegetables. Several studies examined the potential differences between organic and conventional products .18 It has been shown that different microorganisms and contaminants are found on organic produce compared to conventionally grown vegetables due to the use of manure, no pesticides and a different processing.18

The resistance genes present in our samples belonged mainly to the CTX-M family, and especially CTX-M-15 was found. CTX-M-15 is the most common type of ESBL in Europe and has been increasingly described in community isolates. Also, a study in Amsterdam, among Dutch outpatients with gastrointestinal complaints, showed that the most prevalent ESBL-E is CTX-M-15-producing E. coli.20 A study performed in the region of Rotterdam describes the clinical and molecular characteristics of bacteraemia caused by ESBLs, also showing that these strains are the most prevalent.21 Therefore, although the species containing these genes were not E. coli but other enterobacterial species, these may act as a reservoir for mobile drug resistance genes and transfer these genes to the non-pathogenic E. coli present in the gut.22,23 The high rate of co-resistance that we noted may add to the acquisition of multi-drug resistance from the food chain.

Sprouts were found to be most often contaminated, but with other ESBL-producing species than E. coli. These vegetables have often been implicated in STEC outbreaks and it has been postulated that they may constitute a common gene pool for antibiotic resistance.8

In a previous study, we found that ESBL-E carried several different plasmids, some of which we also detected in the enterobacterial strains found on vegetables in the present study.20 The most common plasmids in humans were ColE and FrepB, but ColEtp and HI2 were also found in a substantial number of strains. Hence, plasmid transfer among Enterobacteriaceae

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found on vegetables and those of the human gut seems possible. It is, of course, not possible, from the present study, to infer how frequent such transfer could be.

In conclusion, we found classic ESBL genes in raw vegetables, namely CTX-M-1 and CTX-M-15, and several plasmids that are commonly associated with these genes. The possible impact of our findings on human health highlights the need to further evaluate the presence of ESBL-E in raw vegetables and to explore whether the exchange of resistance genes between these ESBL-E and other enterobacterial species in the human gut does indeed occur.


125

REFERENCES1 Ben-Ami R, Schwaber MJ, Navon-Venezia S, et al.

Influx of extended-spectrum beta-lactamase-producing enterobacteriaceae into the hospital. Clin Infect Dis 2006; 42: 925–34.

2 Davies J, Davies D. Origins and evolution of antibiotic resistance. Microbiol Mol Biol Rev 2010; 74: 417–33.

3 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: 155–65.

4 Mesa RJ, Blanc V, Blanch AR, et al. Extended-spectrum beta-lactamase-producing Enterobacteriaceae in different environments (humans, food, animal farms and sewage). J Antimicrob Chemother 2006; 58: 211–5.


6 Heuer H, Schmitt H, Smalla K. Antibiotic resistance gene spread due to manure application on agricultural fields. Curr Opin Microbiol 2011; 14: 236–43.

7 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. Env Microbiol 2009. DOI:EMI2100 [pii]10.1111/j.1462-2920.2009.02100.x.

8 Buchholz U, Bernard H, Werber D, et al. German outbreak of Escherichia coli O104:H4 associated with sprouts. N Engl J Med 2011; 365: 1763–70.




12 Cuzon G, Naas T, Bogaerts P, Glupczynski Y, Nordmann P. Evaluation of a DNA microarray for the rapid detection of extended-spectrum beta-lactamases (TEM, SHV and CTX-M), plasmid-mediated cephalosporinases (CMY-2-like, DHA, FOX, ACC-1, ACT/MIR and CMY-1-like/MOX) and carbapenemases (KPC, OXA-48, VIM, IMP and ND. J Antimicrob Chemother 2012; 67: 1865–9.



15 Schwaiger K, Helmke K, Hölzel CS, Bauer J. Antibiotic resistance in bacteria isolated from vegetables with regards to the marketing stage (farm vs. supermarket). Int J Food Microbiol 2011; 148: 191–6.

16 Hassan, Sabry A.; Altalhi, Abdullah D.; Gherbawy, Youssuf A.; El-Deeb BA. Bacterial Load of Fresh Vegetables and Their Resistance to the Currently Used Antibiotics in Saudi Arabia. Foodborne Pathog Dis 2011; 8: 1011–8.

17 Knapp CW, Dolfing J, Ehlert PA, Graham DW. Evidence of increasing antibiotic resistance gene abundances in archived soils since 1940. Env Sci Technol 2010; 44: 580–7.

18 Hoogenboom LA, Bokhorst JG, Northolt MD, et al. Contaminants and microorganisms in Dutch organic food products: a comparison with conventional products. Food Addit Contam Part A Chem Anal Control Expo Risk Assess 2008; 25: 1195–207.

19 Blaak H, Schets FM, Italiaander R, Schmitt H, de Roda Husman AM. Antibiotic resistant bacteria in surface water in an area with a high density of animal farms in the Netherlands. RIVM Rapp 703719031 2010.


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21 Van Der Bij AK, Peirano G, Goessens WHF, Van Der Vorm ER, Van Westreenen M, Pitout JDD. Clinical and molecular characteristics of extended-spectrum-β- lactamase-producing Escherichia coli causing bacteremia in the Rotterdam Area, Netherlands. Antimicrob Agents Chemother 2011; 55: 3576–8.

22 Canton R, Coque TM. The CTX-M beta-lactamase pandemic. Curr Opin Microbiol 2006; 9: 466–75.

23 Raphael E, Wong LK, Riley LW. Extended-spectrum Beta-lactamase gene sequences in gram-negative saprophytes on retail organic and nonorganic spinach. Appl Env Microbiol 2011; 77: 1601–7.

A case of New Delhimetallo-beta-lactamase 1 (NDM-1)-producing

Klebsiella pneumoniae with putative secondary transmission from the Balkan region in the Netherlands

T Halaby1, EA Reuland2, N al Naiemi2, A Potron3, PHM Savelkoul2, CMJE Vandenbroucke-Grauls2, P Nordmann3

Antimicrobial Agents and Chemotherapy 2012 May;56(5):2790-1

1 Laboratory for Medical Microbiology and Public Health, Hengelo, The Netherlands2 Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam, The Netherlands

3 Service de Bactériologie-Virologie, Hôpital de Bicêtre et Unité INSERM U914, Le Kremlin Bicêtre, France

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New Delhi metallo-beta-lactamase 1 (NDM-1) was first identified in 2008 in a Klebsiella

pneumoniae strain from a Swedish patient of Indian origin with a history of medical treatment in a New Delhi hospital for a urinary tract infection.1 Since then, more such cases in different parts of the world have been reported.2 The majority of these patients were found to have traveled to or been admitted to hospitals in the Indian subcontinent. A few patients had a travel history in the Balkan area.3 Here we describe a Dutch patient proven to be carrying an NDM-1-producing K. pneumoniae strain that was imported from the Balkan area. Furthermore, a second patient apparently acquired this strain during her stay in the same hospital as the index case.

Patient A, a 66-year-old female with a cerebrovascular accident, was transferred from a hospital in Belgrade, Serbia, to the neurology department of a hospital in the east of the Netherlands on 27 August 2008. Since the patient was known to carry methicillin-resistant Staphylococcus aureus (MRSA), she was directly placed in a separate room in isolation. During admission, extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae, as determined according to the 2008 CLSI guidelines, was isolated from different sites, including throat, rectum, and urinary tract.4 The patient was treated for the urinary tract infection with nitrofurantoin, to which the isolate was susceptible, and with removal of the urinary catheter. She was discharged from the hospital on 15 October to a nursing home, where contact isolation measures were maintained. Follow-up screening cultures remained positive for ESBL-producing K. pneumoniae up to March 2009, but subsequent cultures obtained on several occasions between April and September 2009 were negative.

Patient B, a 73-year-old female with no travel history outside the Netherlands, was admitted with exacerbation of pulmonary symptoms to the department of pulmonary disease (DPD) in the same hospital as the first patient between 10 October and 7 November 2008. At the end of October, ESBL-producing K. pneumonia was cultured from a urine specimen. The patient was treated with oral amoxicillin-clavulanate in addition to removal of the urinary catheter and was discharged from the hospital in good condition. A urinary culture obtained during readmission to the DPD on 25 June 2010 with pulmonary symptoms and urinary tract infection yielded only Escherichia coli. For the urinary tract infection, the patient was treated with ciprofloxacin, to which the E. coli strain was susceptible. The patient died on 18 July 2010.

Two ESBL-producing K. pneumoniae isolates, one from each of these two patients, were selected for further analysis aimed at the presence of carbapenemase because of their elevated MICs to meropenem according to the 2008 CLSI guidelines.4 Antimicrobial susceptibility testing using the EUCAST breakpoints (http://www.eucast.org) was performed with the Vitek 2 automated system (bioMérieux). Ertapenem, imipenem, meropenem, tigecycline, and colistin MIC values were determined by the Etest. The results of retrospective

Secondary tranSmiSSion of ndm-1

131

antimicrobial susceptibility testing and phenotypic confirmations are shown in Table 1. PCR and sequencing confirmed the presence of NDM-1, CTX-M-15, SHV-12, and OXA-1.5 Molecular plasmid analysis revealed the presence of a 70-kb IncII plasmid containing bla

NDM-1and a 140-kb plasmid.5 By amplified fragment length polymorphism (AFLP) typing,

the strains were shown to be identical (data not shown), a result which strongly suggests that the strain was transmitted from the index patient to the secondary case, although the transmission pathway remains unknown.6

By multilocus sequence typing (MLST), the strain of the index patient was found to belong to sequence type 15 (ST15).7 This MLST type was recently described in Belgium for an NDM- 1-producing K. pneumoniae isolate from a patient who had been previously hospitalized in Podgorica, Montenegro.8 The strain from the secondary case was ST431, an MLST type with only one base pair change from ST15.

Table 1 - Results of antimicrobial susceptibility testing and phenotypic confirmations for two ESBL-producing K. pneumoniae isolates, one from each of the two cases

MIC (µg/ml) Phenotypic confirmationc

MHT fore:

Patie

nt

Spec

imen

Ceft

azid

ime

Cefe

pim

e

Gen

tam

icin

Tobr

amyc

in

Cipr

oflox

acin

Imip

enem

Mer

open

em

Erta

pene

m

Colis

tin

Tige

cycl

ine

ESBL

d

Imip

enem

Mer

open

em

CDf im

ipen

em-E

DTA

Erta

pene

m-b

oron

ic a

cidg

1a Urine >64 32 >16 >16 >4 8 3 6 0.25 1.5 pos pos pos pos neg

2b Urine >64 32 >16 >16 >4 6 3 6 0.19 1.5 pos pos pos pos neg

aIndex patient. bSecondary case.cPos, positive; neg, negative.dESBL, extended-spectrum beta-lactamase (laboratory detection of highly resistant microorganisms [HRMO];

http://www.nvmm.nl).eMHT, modified Hodge test.5fCD, combined disc.6gSee reference.7

In conclusion, our study describes one of the first cases of NDM-1-producing K. pneumoniae with apparent secondary transmission, as evidenced by the findings from AFLP typing. Furthermore, the strain belongs to ST15, which has recently been isolated from a patient returning from the Balkan area, supporting the hypothesis of the Balkan region as a reservoir of NDM-1-producing Gram-negative bacteria.9

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REFERENCES1 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: 5046–54.

2 Grundmann H, Livermore DM, Giske CG, et al. Carbapenem-non-susceptible Enterobacteriaceae in Europe: Conclusions from a meeting of national experts. Eurosurveillance 2010; 15. DOI:19711 [pii].

3 Struelens MJ, Monnet DL, Magiorakos AP, et al. New Delhi metallo-beta-lactamase 1-producing Enterobacteriaceae: Emergence and response in Europe. Eurosurveillance 2010; 15. DOI:19716 [pii].

4 CLSI. Clinical and Laboratory Standards Institute. 2008. Performance standards for antimicrobial susceptibility testing; 18th informational supplement. CLSI M100-S18. Clinical and Laboratory Standards Institute, Wayne, PA. .

5 Poirel L, et al. Poirel L, et al. 2011. Cross-border transmission of OXA-48-producing Enterobacter cloacae from Morocco to France. J. Antimicrob. Chemother. 66:1181–1182. .


7 Diancourt L, Passet V, Verhoef J, Grimont PAD, Brisse S. Multilocus Sequence Typing of Klebsiella pneumoniae Nosocomial Isolates Multilocus Sequence Typing of Klebsiella pneumoniae Nosocomial Isolates. J Clin Microbiol 2005; 43: 4178–82.

8 Bogaerts P, Bouchahrouf W, De Castro RR, et al. Emergence of NDM-1-producing enterobacteriaceae in Belgium. Antimicrob Agents Chemother 2011; 55: 3036–8.

9 Livermore D, Walsh T, Toleman M, Woodford N. Escape or transplant? Lancet Infect. Dis. 11:164. 2011.

The cost-effectiveness of ESBL detection: towards molecular detection methods?

BB Wintermans1, EA Reuland1, RGF Wintermans2, AMC Bergmans2,3, JAJW Kluytmans1,3

Clinical Microbiology and Infection 2013 Jul;19(7):662-5

1 Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam2 Medical Microbiology and Infection Control, Franciscus Medical Center, Roosendaal

3 Medical Microbiology and Infection Control, Amphia Medical Center, Breda, the Netherlands

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ABSTRACT

ObjectivesCorrect detection of extended-spectrum beta-lactamases (ESBLs) is crucial for infection control and antibiotic choice. We performed a study to determine the cost-effectiveness of phenotypical testing, which can be inaccurate, and genotypical tests, which are considered to be more reliable but also more expensive.

MethodsAll patients that had been in isolation in the Amphia hospital because of the detection of ESBL according to the ESBL Etest were included in the survey. All strains were retested using the double disk confirmation test (DDCT) and a genotypical method. This was a commercially available microarray (Check-Points). Discordant results were confirmed by PCR and sequencing.

Results In total 174 patients were included. In 24 of 174 (14%) patients, ESBL carriage could not be confirmed with the microarray. This was verified with PCR and sequencing. The mean duration of isolation was 15 days, adding up to a total number of isolation days of 2571. False-positive results according to the microarray resulted in a total of 279 days of unnecessary isolation for the Etest and 151 days for the DDCT.

Conclusions Using Etest to detect the presence of ESBL results in a false-positive outcome in 14% of the cases. This results in unnecessary isolation of patients, which can be omitted by using a genotypic method.

Cost-effeCtiveness of esBL deteCtion

137

INTRODUCTION

The prevalence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae is increasing rapidly.1–3 Infections with ESBL are associated with increased costs and mortality.4 The control of ESBL is difficult as the resistance genes of these microorganisms are located on plasmids and may be transferred between different bacterial species and even different genera of Enterobacteriaceae.5 According to the Dutch guidelines for infection control all hospitalized patients colonized with ESBL have to be placed in isolation in a separate room.6 The current national guideline for microbiological detection of ESBL in the Netherlands recommends the use of phenotypical tests for confirmation of ESBL. These tests are sometimes difficult to interpret and may lead to false-positive tests, resulting in unnecessary isolation of patients and possibly inappropriate treatment, leading to higher costs. Phenotypical testing takes at least 1 day to provide results.

Genotypical methods are considered to be more accurate, can provide characterization of the ESBL genes and have the potential to provide results on the same day. Recently, a new diagnostic microarray for detection and identification of ESBL in Enterobacteriaceae has become available. Several studies have concluded that the diagnostic microarray is superior to phenotypical methods. Sensitivity for ESBL was comparable to or higher than phenotypic methods, whereas specificity was consistently higher.7–10 The microarray had almost the same sensitivity and specificity as PCR with additional sequencing and was therefore used as the standard for testing.11

We wanted to determine the effect of false-positive phenotypic testing on infection control measures and also evaluate the cost-effectiveness of ESBL testing. This was done to determine if implementation of a genotypical test is warranted.

METHODS

Patients who had been isolated because of ESBL from November 2006 until August 2010 in Amphia Hospital in Breda were included. The Amphia Hospital is a large teaching hospital located in the south of the Netherlands. The presumed ESBL-positive strains had been stored at -70°C. Information about type of organism, interpretation of phenotypical test and duration of isolation was obtained from the laboratory information system.

Primary phenotypic detection of ESBLFor species identification and susceptibility testing the Vitek 2 system (bioMérieux, Marcy l’Etoile, France) was used. According to the Dutch national guideline for ESBL detection, classification of Enterobacteriaceae was done to the presence of chromosomal AmpC beta-lactamases in two groups. Presence of ESBL production was determined using the ESBL

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Etest (bioMérieux).12 The hands-on time and the turn-around time were, respectively, 15 min and 16 h.

Genotypic detection of ESBLThe strains that had been stored at -70°C were recollected and DNA isolation was performed using the QIAamp DNA Mini Kit system (QIAGEN, Hilden, Germany). The ESBL Array (Check-Points, Wageningen, the Netherlands) is supplied as a kit. It is designed to detect single nucleotide polymorphisms (SNPs) of essential bla

TEM and bla

SHV variants and the following

blaCTX-M

groups: blaCTX-M-1

, blaCTX-M-2

, blaCTX-M-9

and blaCTX-M-8/25

, as described by Cohen Stuart et

al.7

Microarray images are read using a microarray reader (ArrayTube Reader; ClonDiag Chip Technologies, Jena, Germany) connected to a computer running dedicated software for analysis of the images. The software indicates whether bla

TEM, bla

SHV, bla

CTX-M or a combination

of these genes is present. The hands-on time and the turn-around time were, respectively, 2 and 8 h.

Secondary phenotypic detection of ESBLAll strains were retested using the DDCT (Rosco diagnostica, Taastrup, Denmark). Interpretation of the test was carried out according to the Dutch national guideline for the detection of ESBL in Enterobacteriaceae.12 The hands-on time and the turn-around time were, respectively, 15 min and 16 h.

Retesting of primary phenotypical test (Etest)After testing the strains with the microarray, the false-positive results obtained using the primary phenotypical test (Etest) were retested using the same test and the same algorithm.

Verifying the reference test (microarray)The beta-lactamase genes were characterized by PCR at the VUmc, followed by sequencing (BaseClear, Leiden, the Netherlands), as described by Naiemi et al.13 Sequences were analyzed with BioNumerics software (version 6.5; Applied Maths, Sint-Martens-Latem, Belgium) and compared with sequences in the NCBI database (http://www.ncbi.nlm.nih.gov/BLAST) and Lahey (http://www.lahey.org/studies/).


139

RESULTS

Primary phenotypical test (Etest)In total, 174 patients were included based on the initial Etest results. Among these strains, 97% were group I Enterobacteriaceae. The organisms are shown in (Table 1). The total number of days in isolation was 2571. The average duration of isolation was 15 days (range, 1–93; median, 10).

Genotypical testAll 174 phenotypical ESBL-positive strains were retested using the microarray. The microarray detected at least one ESBL-gene in 149 strains. Among the ESBLs there were 90 bla

CTX-M-1, 33

blaCTX-M-9

, 5 blaCTX-M-2/8/25

and 25 blaTEM/SHV

. There were four strains that contained multiple ESBL genes.

According to the microarray there were 25 false-positive phenotypical tests, resulting in a positive predictive value of 86% for the Etest. In group I Enterobacteriaceae 12% (n = 21) were discordant and showed a phenotypical positive test, whereas the genotypical test was negative. In group II Enterobacteriaceae 67% (n = 4) were discordant (Table 1).

Among the 25 false-positive results there were three patients that were tested with the same bacterial species twice. This was in different episodes of isolation. The total number of isolation days of these false-positive tests was 279 days (Table 2).

Table 1 - Microorganisms found among positive primary phenotypical test (Etest). Discordant results in the phenotypical test compared with the microarray. Classification according to Group I and Group II Enterobacteriaceae

Discordant results

Etest 14% DDCT 7%

Group I (n = 168) Escherichia coli 145 7 12% 3 5%

Klebsiella (pneumoniae 14, oxytoca 7) 21 11 4

Proteus mirabilis 2 2 1

Group II (n = 6) Enterobacter cloacae 4 2 67% 2 67%

Citrobacter freundii 2 2 2

Secondary phenotypical test (DDCT)At retesting of the 174 strains, 161 tested ESBL-positive using the DDCT. Among these 161 positive phenotypical tests are 13 false-positive tests according to the microarray. Retesting of these strains with the DDCT led to the same results.

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Among these false-positive tests, eight out of 13 tests were just over the cut-off. The DDCT had no false-negative tests. The total number of isolation days of these false-positive tests was 151 days (Table 2).

Table 2 - Performance and costs (material only) of different diagnostic tools compared with the microarray, which was chosen as the reference test

Costper test

Total costfor testing (n = 174)

Total daysof isolation

Extraisolation days

Etest € 25 € 4.350 2.571 +279

DDCT € 4 € 696 2.443 +151

Microarray € 33 € 5.742 2.292 –

Retesting of primary phenotypical test (Etest)At retesting of the 25 strains that were false-positive with the Etest, 20 tested ESBL-positive using the Etest and one could not be interpreted because of overgrowth.

Verifying the reference test (microarray)The results of PCR and sequencing did not show ESBL in bla

TEM/SHV in the 25 strains where

the microarray could not find any ESBL genes. The PCR for blaCTX-M

showed PCR product in seven out of 25 strains. Then sequence analysis of the PCR product was done. In six strains, all Klebsiella oxytoca, the PCR product was non-specific; these were bla

OXY genes and no ESBL

genes were found. In one E. coli we found blaCTX-M-8

, which is an ESBL gene.

DISCUSSION

Comparing the results of Etest with those of the microarray, 14% (n = 24) were discordant. In group I Enterobacteriaceae 12% (n = 20) of the strains were discordant and in group II Enterobacteriaceae 67% (n = 4). This indicates that a larger amount of group II organisms could increase the percentage of discordant results.

As reported by others, the microarray is more accurate than phenotypical tests. However, the microarray is more expensive to perform. We quantified the number of false-positive findings in clinical practice, which enabled us to estimate the savings that can be achieved. This is based on the reduction of the number of isolation days on the wards in relation to the costs of the microarray.

In this study the total cost of retesting the 174 samples (cost materials for DNA isolation and microarray) was c. € 6000. The total number of days of isolation that could have been avoided was 279 days. This means that the additional costs of an isolation day should be at


141

least 22 euro to result in a net savings. The costs of isolation may vary between hospitals or countries but are likely to be substantially higher than 22 euro in most settings. Changing to a cheaper ESBL confirmation test or not performing one at all can make further reductions in costs. This study does not provide enough data for discussion of this subject.

We decided not to include the cost of laboratory labor because this is highly dependent on throughput of tests and differences in the salaries of technicians. To do so, one could use the hands-on time of the phenotypical vs. the genotypical test, which is respectively 15 vs. 120 min.

Because of the possible limitations of the microarray compared with the gold standard, PCR and gene sequencing were carried out. We retested the 25 strains in which the microarray could not find any ESBL genes. In 24 strains, results were consistent with PCR and sequencing. In one strain the microarray showed a lack of sensitivity and did not pick-up bla

CTX-M-8. This

gene is included in the kit and after retesting the microarray did pick-up the blaCTX-M-8

. This lack of sensitivity did not have a major impact on this study because both tests, DDCT and Etest tested positive for this strain and the duration of this isolation episode was only 5 days.

We also showed that the PCR has a lack of specificity for blaCTX-M

in K. oxytoca; therefore all results should be verified by gene sequencing. The false-positive results in the phenotypic assays are likely to be caused by hyperproduction of the chromosomal K1 beta-lactamase or by effects of other resistance mechanisms that are also inhibited by clavulanic acid, for example Enterobacteriaceae carrying wild-type betalactamase genes. Retrospectively, we could also determine two misinterpretations that caused a false-positive result and one strain with overgrowth that made the test uninterpretable.

It is remarkable that the DDCT did not lead to false-positive results when testing strains of K. oxytoca with bla

OXY, whereas the Etest did. In other strains the DDCT also had fewer false-

positive results than the Etest.

This indicates that the performance of the DDCT is better than the Etest but this could be biased. When the DDCT was used as the initial screening and the Etest as a confirmatory method it is possible that the Etest would have found false-positive DDCT results. The low cost and more objective reading of the DDCT warrants a further prospective evaluation of the DDCT. After retrospective correction for the false-negative microarray result, among 12 false-positive results in the DDCT there were eight tests in which the result was just above the cut-off. Changing this cut-off slightly for a positive test could result in a better performance but this should be evaluated in a prospective clinical study.

A major drawback of the microarray is that it is not capable of picking up new and unknown ESBL genes that have not been incorporated in the microarray. When genotypic assays are to be implemented on a larger scale this should be accompanied by prospective surveillance for the emergence of new resistance mechanisms.

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In conclusion, the currently used phenotypical tests to detect ESBL have a relatively high rate of false-positive results. The microarray provides more reliable results and thereby avoids unnecessary isolation days. In our study the additional cost of 1 day of isolation had to be at least € 22 for the microarray to be cost-effective and costs can be further reduced by changing phenotypical confirmation. Also the microarray can provide results more rapidly than phenotypic tests. Therefore, the microarray should be considered for implementation in the routine diagnostic laboratory.


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REFERENCES1 Bush K. Extended-spectrum beta-lactamases in

North America, 1987-2006. Clin Microbiol Infect 2008; 14 Suppl 1: 134–43.

2 Cantón R, Novais A, Valverde A, et al. Prevalence and spread of extended-spectrum beta-lactamase-producing Enterobacteriaceae in Europe. Clin Microbiol Infect Off Publ Eur Soc Clin Microbiol Infect Dis 2008; 14 Suppl 1: 144–53.

3 Antimicrobial resistance surveillance in Europe Annual report of the European Antimicrobial Resistance Surveillance Network (EARS-Net); 2009. Available at: http://www.ecdc.europa.eu/en/publications/ Publications/Forms/ECDC_DispForm.aspx?ID=580.

4 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: 1175–84.

5 Paterson DL, Bonomo RA. Extended-spectrum beta-lactamases: a clinical update. Clin Microbiol Rev 2005; 18: 657–86.

6 W.I.P. Werkgroep Infectie Preventie. Maatregelen tegen overdracht van bijzonder-resistente micro-organismen (BRMO). .


8 Endimiani A, Hujer AM, Hujer KM, et al. Evaluation of a commercial microarray system for detection of SHV-, TEM-, CTX-M-, and KPC-type β-lactamase genes in gram-negative isolates. J Clin Microbiol 2010; 48: 2618–22.


10 Willemsen I, Overdevest I, Al Naiemi N, et al. New Diagnostic microarray (check-KPC ESBL) for detection and identification of extended-spectrum beta-lactamases in highly resistant Enterobacteriaceae. J Clin Microbiol 2011; 49: 2985–7.

11 Platteel TN, Stuart JWC, Voets GM, et al. Evaluation of a commercial microarray as a confirmation test for the presence of extended-spectrum β-lactamases in isolates from the routine clinical setting. Clin Microbiol Infect 2011; 17: 1435–8.

12 Bernards AT, Bonten MJ, Cohen J et al. NVMM-guideline Laboratory detection of highly resistant microorganisms (HRMO). Available at: http://www.nvmm.nl/richtlijnen/esbl-screening-en-confirmatie.


Detection and occurrence of plasmid-mediated AmpC in highly

resistant gram-negative rods

EA Reuland1, JP Hays2, DMC de Jongh2, E Abdelrehim1, I Willemsen3, JAJW Kluytmans1,3, PHM Savelkoul1, CMJE Vandenbroucke-Grauls1, N al Naiemi1,4,5

PLoS One 2014 Mar 18;9(3):e91396

1 Medical Microbiology and Infection Control, VU University Medical Center, Amsterdam2 Department of Medical Microbiology and Infectious Diseases, Erasmus MC, Rotterdam

3 Department of Medical Microbiology and Infection Control, Amphia Hospital, Breda4 Laboratory for Medical Microbiology and Public Health, Hengelo

5 Medical Microbiology and Infection Control, Ziekenhuisgroep Twente, Almelo, The Netherlands

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ABSTRACT

Objectives The aim of this study was to compare the current screening methods and to evaluate confirmation tests for phenotypic plasmidal AmpC (pAmpC) detection.

MethodsFor this evaluation we used 503 Enterobacteriaceae from 18 Dutch hospitals and 21 isolates previously confirmed to be pAmpC positive. All isolates were divided into three groups: isolates with 1) reduced susceptibility to ceftazidime and/or cefotaxime; 2) reduced susceptibility to cefoxitin; 3) reduced susceptibility to ceftazidime and/or cefotaxime combined with reduced susceptibility to cefoxitin. Two disk-based tests, with cloxacillin or boronic acid as inhibitor, and Etest with cefotetan-cefotetan/cloxacillin were used for phenotypic AmpC confirmation. Finally, presence of pAmpC genes was tested by multiplex and singleplex PCR.

ResultsWe identified 13 pAmpC producing Enterobacteriaceae isolates among the 503 isolates (2.6%): 9 CMY-2, 3 DHA-1 and 1 ACC-1 type in E. coli isolates. The sensitivity and specificity of reduced susceptibility to ceftazidime and/or cefotaxime in combination with cefoxitin was 97% (33/34) and 90% (289/322) respectively. The disk-based test with cloxacillin showed the best performance as phenotypic confirmation method for AmpC production.

Conclusions For routine phenotypic detection of pAmpC the screening for reduced susceptibility to third generation cephalosporins combined with reduced susceptibility to cefoxitin is recommended. Confirmation via a combination disk diffusion test using cloxacillin is the best phenotypic option. The prevalence found is worrisome, since, due to their plasmidal location, pAmpC genes may spread further and increase in prevalence.

Detection anD occurrence of pampc in Hr-Gnr

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INTRODUCTION

The frequency of highly resistant gram-negative rods (HRGNRs) is still increasing worldwide.1 Gram-negative rods with resistance to carbapenems or to third generation cephalosporins only due to ESBL-production were defined as highly resistant isolates. Furthermore, strains resistant to two agents of the antimicrobial groups quinolones and aminoglycosides were also defined as highly resistant (adapted from the Dutch guideline for preventing nosocomial transmission of highly resistant microorganisms (HRMO)).2

Apart from ESBLs, one class of these enzymes has received relatively little attention, namely the AmpC-type beta-lactamases. Although these ‘‘Class C’’ beta-lactamases are often found to be associated with the bacterial chromosome, an increasing prevalence of plasmid-encoded AmpC enzymes (pAmpC) has been reported.3–5 Traditionally, chromosomally encoded AmpC is mainly present in group II Enterobacteriaceae (Enterobacter spp., Citrobacter freundii, Hafnia alvei, Providencia spp., Serratia spp., Morganella morganii), but pAmpC is gaining more and more importance in group I Enterobacteriaceae (Proteus mirabilis, Klebsiella spp., Salmonella spp., Escherichia coli, and Shigella spp.).3 Furthermore, carriage of plasmid-mediated AmpC is often associated with multidrug resistance (e.g. resistance to aminoglycosides, quinolones and cotrimoxazole), and worryingly, isolates with porin loss that carry pAmpC may also be resistant to carbapenems.4,6,7 The occurrence of pAmpC has been investigated in several studies.6,8–10 In a selection of clinical Enterobacteriaceae from a national survey a high prevalence of ampC genes among Enterobacteriaceae was found; 32 out of 181 isolates with reduced susceptibility to cefoxitin concerned pAmpC.11 Another study showed a high prevalence of ESBL/AmpC-producing E. coli in birds and farmers at Dutch broiler farms.12

The prevalence of pAmpC carriage reported in these studies is still low, though this is most likely an underestimation due to the difficulties associated with routine phenotypic screening for pAmpC. This means that molecular detection techniques are the current ‘gold standard’ for the detection of pAmpC, although these are more expensive and difficult to implement for routine use.3,13 For this reason, several previous studies have attempted to compare and evaluate current phenotypic tests for the detection of pAmpC.14–16 However, most of these reports did not analyze different screening methodologies. Therefore, the objective of this study was to compare the current pAmpC phenotypic screening methodologies used in the literature and to evaluate the different confirmation methods. The methodology was further used to assess the prevalence of pAmpC among 502 group I HR-GNRs collected from 18 Dutch hospitals in 2007.

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METHODS

Bacterial isolatesBacterial isolates were retrospectively screened using a collection of group I HR-GNR Enterobacteriaceae previously collected during a prospective observational multicenter study in 18 hospitals in the Netherlands.17 Gram-negative rods were defined as highly resistant (HR-GNR), according to the criteria of the Dutch Working Party on Infection Prevention.2 Isolates were obtained from patients hospitalized between January 1 and October 1, 2007 and comprised strains isolated from clinical and screening specimens. In total 892 different HR-GNR isolates were recovered from 786 patients.

Identification of strains, susceptibility testing and ESBL detection was performed according to Dutch guidelines.17,18 ESBL-encoding genes (bla

CTX-M, bla

SHV and bla

TEM), bla

OXA and

carbapenemase-encoding genes (blaKPC

, blaNDM

, blaOXA-48

, blaIMP

and blaVIM

) were detected by microarray and if necessary confirmed by PCR and sequencing (BaseClear) at the VU University Medical Center (VUmc).19,20 The authors specifically focused on enterobacterial species that are known to lack a chromosomal AmpC gene (P. mirabilis, Klebsiella spp., Salmonella spp.), or that are known to carry a chromosomal AmpC gene, but produce only low levels of AmpC enzyme (E. coli and Shigella spp.). Therefore, 503 of the 892 HR-GNR isolates from the original study were included in the present study. The 503 highly resistant isolates comprised E. coli (333), Klebsiella spp. (123), Proteus spp. (42), Salmonella spp. (3) and Shigella spp. (2). Duplicate isolates from the same patient were excluded; isolates were obtained from screening samples (158), and from clinical samples (345); in 61 samples the HRMO was also found in blood cultures during hospitalization. The samples were obtained from 18 different hospitals. Finally, a further 21 pAmpC-producing isolates, previously characterized by PCR, were included (as positive controls) in the study collection. Fifteen of the pAmpC control strains were obtained from the isolate biobank available at Erasmus Medical Center, having been collected from various non-Dutch sources over different years. The isolates were identified as E. coli by classical biochemical methods and confirmed to be pAmpC positive by PCR. Six isolates (confirmed by PCR at the Erasmus MC) were isolated during a study on community-acquired ESBL-producing Enterobacteriaceae at the VU University Medical Center, between August 12 and December 13, 2011.

Screening for AmpCThree screening strategies were evaluated: reduced susceptibility to third generation cephalosporins, reduced susceptibility to cefoxitin, and a combination of reduced susceptibility to third generation cephalosporins and cefoxitin.21 Reduced susceptibility to third generation cephalosporins was defined as a MIC for cefotaxime and/or ceftazidime that was >1 mg/L, corresponding to inhibition zone diameters for cefotaxime of ≤27 mm


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and for ceftazidime of ≤22 mm (following ESBL screening protocols defined by Dutch national guidelines).18 Reduced susceptibility to cefoxitin was determined using Vitek 2 (bioMérieux, Marcyl’Etoile, France), and was defined as a MIC.8 mg/L according to EUCAST guidelines.21 If isolates were positive for pAmpC by PCR but susceptible to cefoxitin (MIC≤8 mg/L; inhibition zone >18 mm), Vitek testing was repeated and phenotypic testing using cefoxitin Etest (bioMérieux, Solna, Sweden) on Mueller Hinton agar was performed to ensure cefoxitin sensitivity.

Confirmation of AmpCAmpC production was confirmed phenotypically using a two disk-based test and an Etest with boronic acid or cloxacillin as inhibitors. The combination disk diffusion tests consisted of cefotaxime and ceftazidime combined with boronic acid or cloxacillin as inhibitor (Rosco, Taastrup, Denmark). A positive test was considered when the zone of inhibition was ≥5 mm larger than the zone generated without inhibitor. The Etest cefotetan/cefotetan-cloxacillin (CN/CNI, bioMérieux, Marcy-l’Etoile, France) methodology was also used to confirm AmpC production, where either a ratio of cefotetan/cefotetan-cloxacillin ≥8, deformation of the ellipse, or the presence of a phantom zone were interpreted as positive for an AmpC producer.

Molecular pAmpC gene screeningIsolates that were suspected to be pAmpC producers by one or more of the screening methods were further tested by multiplex PCR. Thus, all 335 isolates with reduced susceptibility to third generation cephalosporins and/or reduced susceptibility to cefoxitin, were analyzed by PCR. DNA was isolated using the easyMag system (bioMérieux, Marcy-l’Etoile, France). Plasmid-mediated AmpC types were characterized using a variation of a standard multiplex PCR (Erasmus MC, Rotterdam) that can identify six family-specific pAmpC genes: bla

CMY-II, bla

MOX, bla

FOX, bla

DHA, bla

ACT/MIR and bla

ACC genes.13 In this variation,

the annealing temperature was increased to 70°C and multiplex PCR positive isolates were further tested using specific singleplex AmpC PCRs under the same reaction conditions, to ensure that the PCR-products found in the multiplex PCR positive isolates were correct. This multiplex AmpC PCR methodology was used as the gold standard AmpC detection methodology.

Analysis of genetic relatedness among the tested isolates in this study was performed using amplified-fragment length polymorphism (AFLP) as described by Savelkoul et al.22 Clustering and interpretation of AFLP banding patterns were performed using BioNumerics software, version 6.6 (Applied Maths, Sint-Martens-Latem, Belgium).

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Data analysisStatistical analyses were performed using SPSS, version 20.0. Sensitivity and specificity of the screening and confirmation methods were calculated using multiplex AmpC PCR results as the gold standard.

RESULTS

Phenotypic detection methodologies for (plasmid-mediated) AmpCOf the 503 HR-GNR isolates from Dutch hospitals 335 isolates (67%) showed reduced susceptibility to third generation cephalosporins and/or reduced susceptibility to cefoxitin. In addition three isolates had increased MICs (0.25 mg/L) for meropenem (2 mg/L, 4 mg/L and 8 mg/L) and imipenem (4 mg/L, 2 mg/L and 4 mg/L, respectively).23 The number of isolates for each species included E. coli (224), Klebsiella spp. (106), Proteus spp. (4) and Salmonella spp. (1). In total 101 screenings samples were isolated, the remaining 234 samples were from clinical samples. Of these, 12.9% (43/335) isolates were detected in blood cultures at a later stage. Nearly half of the samples (42.4%, 142/335) were obtained on the Intensive Care Unit.

Thirteen out of these 335 (3.9%) isolates were found to be pAmpC positive using a multiplex pAmpC PCR, i.e. CMY-2 (9), DHA-1 (3) and ACC-1 (1). Also included in the phenotypic screening was a collection of 21 previously characterized pAmpC positive E. coli isolates, 20 CMY-2 and one isolate with DHA-1(data not published), generating a total of 356 isolates for phenotypic comparison and evaluation (Table 1). Using both screening and confirmatory phenotypic methodologies on these 356 isolates revealed three major phenotypic groups. Phenotypic group I comprised 327 isolates that were found to be reduced susceptible to third generation cephalosporins (regardless of resistance to cefoxitin), with 34 (10.4%) of these isolates being pAmpC positive by PCR. This group included all pAmpC PCR-positive isolates. This results in a sensitivity of 100% (34/34) and a specificity of 9% (29/322).

Phenotypic group II comprised 122 isolates with reduced susceptibility to cefoxitin (regardless of reduced susceptibility to third generation cephalosporins). Thirty three of these 122 (27%) isolates were found to be pAmpC positive by PCR. An ACC-1gene positive isolate remained undetected due to a lack of cefoxitin resistance. This results in a sensitivity of 97% (33/34) and a specificity of 72% (233/322).

Phenotypic group III comprised 66 isolates with reduced susceptibility to cefoxitin combined with reduced susceptibility to third generation cephalosporins. Thirty three of these 66 (50%) isolates were found to be pAmpC positive by PCR, but again the ACC-1-positive isolate remained undetected. These results generated a sensitivity of 97% (33/34), but a higher specificity of 90% (289/322).


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The performance of different AmpC confirmatory tests in combination with different antibiotic and inhibitor combinations is shown in Table 2. A maximum sensitivity of 94% (range 91%–94%) was obtained for all the three screening strategies in combination with the combination disk diffusion tests with cloxacillin. By combining reduced susceptibility to third generation cephalosporins with reduced susceptibility to cefoxitin and in combination with the inhibitor-based combination disk diffusion test using cloxacillin yielded a sensitivity of 91% (31/34) with a specificity of 96% (309/322).

The DDCT with boronic acid missed two E. coli with CMY-2. The CN/CNI Etest did not detect three E. coli with CMY-2, one E. coli with DHA-1 and one Klebsiella oxytoca with ACC-1. Importantly, two E. coli isolates possessing CMY-2 type enzymes, one coproducing CTX-M-1 and one coproducing OXA-1, were not detected with any of the confirmation methodologies shown in Table 2. No other isolates additionally producing ESBL were negative in the phenotypic confirmation. All isolates with partly negative confirmation results were fully resistant to cefotaxime, ceftazidime and/or cefoxitin.

Table 1 - AmpC production in 356 highly resistant enterobacterial isolates

Species Total collection pAmpC positive pAmpC type

Total 356 34

Escherichia coli 245 (68.8%) 28 (82.4%) 27 CMY-2, 1 DHA-1

Klebsiella pneumoniae 82 (23.0%) 4 (11.8%) 3 DHA-1, 1 CMY-2

Klebsiella oxytoca 24 (6.8%) 1 (2.9%) ACC-1

Proteus mirabilis 4 (1.1%) 1 (2.9%) CMY-2

Salmonella species 1 (0.3%) 0

The 356 isolates were selected based on resistance to third generation cephalosporins (cefotaxime and/or ceftazidime) and/or cefoxitin. Reduced susceptibility was defined as a MIC > 1 mg/L corresponding to inhibition zone diameter of ≤ 27 mm for cefotaxim, MIC > 1 mg/L corresponding to inhibition zone diameter of ≤ 22 mm for ceftazidim, MIC > 8 mg/L for cefoxitin.

Molecular epidemiologyMultiplex pAmpC PCR screening revealed a prevalence of 2.6% (13/503) for pAmpC carriage among the group I Enterobacteriaceae tested in this study. Further molecular analysis revealed that nine of the 13 pAmpC multiplex PCR-positive isolates obtained in the multicenter study and isolated in five of the 18 different hospitals between between January 1 and October 1, 2007, contained the CMY-2 gene (predominantly E. coli except for one P. mirabilis and one Klebsiella pneumoniae). Three isolates contained DHA-1 (all K.

pneumoniae) and one isolate ACC-1 (K. oxytoca). Two of these isolates, one E. coli with CMY-2 and one K. pneumoniae with DHA-1, were obtained out of screening material and the rest were clinical samples.

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In three of these 13 pAmpC isolates an ESBL-encoding gene was also detected, these ESBL genes were determined as CTX-M-1 group (2/13) and CTX-M-9 group (1/13).

The 15 pAmpC-producers from the isolate biobank available at Erasmus Medical Center (Erasmus MC) were obtained from various non-Dutch sources over different years, therefore no identical strains were expected. However, AFLP was performed on the 6 pAmpC isolates derived from the community-acquired ESBL-producing strains to assure that these isolates were not identical. Furthermore, AFLP analysis of the 13 pAmpC producers from the multicenter study revealed no epidemiological relationship.

Table 2 - Comparison of phenotypic pAmpC confirmation tests

Phenotypic detection methods*Total Number of

isolates positive by pAmpC PCR

Total Number of isolates pAmpC positive using

phenotypic methods

Sensitivity (%)

Specificity (%)

Phenotypic Group I:

Analysis after screening for reduced susceptibility to third generation cephalosporins (n=327) 34 34 100 9

DDCT with cloxacillin 32 94 56

DDCT with boronic acid 30 88 65

Etest CN/CNI 27 79 98

Phenotypic Group II:

Analysis after screening for reduced susceptibility to cefoxitin (n=122)** 34 33** 97 72




Phenotypic Group III:

Analysis after screening for reduced susceptibility to all these cephalosporins together (n=66)** 34 33** 97 90




The antibiotics used in the DDCT tests were cefotaxime and ceftazidime combined with cloxacilllin or boronic acid. Etest CN/CNI consisted of cefotetan (CN) with cefotetan/cloxacillin (CNI).

*DDCT with cloxacillin/boronic acid and CN/CNI Etest.**Sensitivity and specificity of these confirmation tests is performed without ACC-1 in the analysis due to susceptibility for cefoxitin.


155

DISCUSSION

Our results show that among HR-GNR in Dutch hospitals, 2.6% (13/505) pAmpC-producing isolates were found retrospectively in a selected subgroup of group I Enterobacteriaceae. That the majority of isolates possessed CMY-2 type pAmpC is in line with molecular epidemiological results published elsewhere.6,11,24,25

Several reports reveal that pAmpC-producing nosocomial isolates have become endemic in some hospitals that they can cause outbreaks, and that they affect therapeutic choices.26–28 Reports from Spain suggest that compared to ESBL-producing organisms the acquisition of pAmpC-producing Enterobacteriaceae is still mainly hospital- or healthcare-associated.6 The isolates in our study were clinical isolates, but we cannot differentiate between healthcare-associated or community-based sources of nosocomial pAmpC infections. A rise in pAmpC carriage and infections could in theory mirror the rapid increase in global enterobacterial ESBL isolates observed over the last 10 years, not least because pAmpC carriage is reportedly becoming a serious global infectious disease health concern.5

Of great concern, treatment of infections caused by pAmpC-producing strains with cephalosporins is associated with adverse clinical outcomes.29,30

Recently Gude et al. evaluated different AmpC confirmatory tests.14 In contrast to these authors, we found not the Etest but DDCT cloxacillin as the best test, with the best sensitivity and specificity after the combination of screening criteria. In general the same genes were identified, except that we detected also bla

ACC-1. This difference may be due to differences

in the selection of strains. We included not only cefoxitin-resistant strains, but also strains of group I Enterobacteriaceae that were resistant to third generation cephalosporins alone. Therefore, cefoxitin susceptible isolates producing pAmpC (ACC-1) could be detected. In addition a MIC>8 mg/L was used as breakpoint for cefoxitin, as to eliminate less resistant isolates. These more stringent MICs were used to detect more isolates that fulfilled the screening criteria. We used the same cefotetan/cefotetan-cloxacillin Etest, however the other phenotypic tests were DDCT with cefotaxime/ceftazidime combined with boronic acid or cloxacillin as inhibitor. The latter were selected because these are commercially available, cheap and less prone to interobserver variability (like for example a three dimensional (3D) test or double disk approximation test).

The use of molecular testing strategies such as multiplex AmpC PCRs are currently the gold standard for pAmpc detection. A more convenient strategy for many institutions would be to optimize the phenotypic screening and confirmatory methodologies that are currently available in order to maximize the sensitivity and specificity of pAmpC detection. Results using pAmpC phenotypic screening assays on our set of isolates showed that a reduced susceptibility to cefotaxime and/or ceftazidime alone generated the best sensitivity (100%, i.e. 34/34). However, a major disadvantage of this methodology was found to be a low

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specificity (9%, i.e. 29/322) of detection. This means that no false negative results were generated using this methodology, but that there was a relatively high frequency of false positives. The end result is that many unnecessary confirmatory tests would have to be performed using this methodology.

In general resistance to cefoxitin is often used as indicator for the production of class C beta-lactamases (which include pAmpC beta-lactamases), with most reports only investigating isolates resistant to cephamycins.24,31 Though cefoxitin resistance is a sensitive test, it is not specific, mainly because a reduced permeability of the bacterial outer membrane, as well as the expression of some carbapenemase enzymes, may also lead to cefoxitin resistance.32,33 Further, hyperproduction of chromosomal AmpC, may lead to cephamycin resistance.3,14,34 Another disadvantage of using cefoxitin resistance as a phenotypic screening methodology is that ACC-1-type enzymes are susceptible to cefoxitin, which means that isolates possessing these genes will be regarded as pAmpC negative. This is an important point, because ACC-type enzymes have been detected in several different countries in Europe, including a large outbreak in a teaching hospital in Garches, France.25,27,35

From our results, we conclude that a combination of reduced sensitivity to the third generation cephalosporins (cefotaxime and/or ceftazidime) and reduced susceptibility to cefoxitin may generate the best specificity (90%) for phenotypic pAmpC screening (Table 2). A limitation however is that ACC-like enzymes will not be detected. In combination with this screening strategy our results suggest that the combination disk diffusion test with cloxacillin is the best phenotypic confirmation method.With respect to the disk-based phenotypic confirmatory AmpC methodologies used, it is well known that boronic acid and cloxacillin are well-known inhibitors of AmpC.14,16,36–39 Boronic acid is an AmpC inhibitor (both plasmidal and chromosomal) and also an inhibitor of KPC beta-lactamases. We found one isolate with a positive AmpC confirmation test with boronic acid but negative results using the test with cloxacillin. This K. pneumoniae isolate showed an increased MIC for meropenem (2 mg/L) and was KPC positive. Of the three isolates with MIC meropenem >0.25 mg/L one isolate (K. pneumoniae) was KPC positive. The two other isolates (one K. pneumoniae and one E. coli) were resistant to third generation cephalosporins and cefoxitin, showed decreased susceptibility to ertapenem, had negative AmpC confirmation test results and negative PCR result for KPC. Therefore, other mechanisms could be responsible for the resistance, e.g. porin loss and ESBLs (both isolates harbored SHV-type ESBL and CTX-M-1, respectively) or other carbapenemases.

In conclusion, our data suggest that phenotypic AmpC detection methods can be improved by combining the screening results of susceptibility testing to third generation cephalosporins and the susceptibility results to cefoxitin. Reduced susceptibility to both being a good indicator for the presence of pAmpC gene expression. However, it should


157

be noted that the presence of ACC-1 type AmpC will still be missed using these combined methodologies. The presence of pAmpC can be confirmed with the combination disk diffusion test; cefotaxime and ceftazidime with cloxacillin showed the best results. For the future, it is desirable to evaluate a larger collection of different enterobacterial species with pAmpC and to perform more studies to define the frequency of occurrence of pAmpC in comparison to 2007.

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5 Pitout JDD. Enterobacteriaceae that produce extended-spectrum beta-lactamases and AmpC beta-lactamases in the community: the tip of the iceberg? Curr Pharm Des 2013; 19: 257–63.

6 Rodriguez-Bano J, Miro E, Villar M, et al. Colonisation and infection due to Enterobacteriaceae producing plasmid-mediated AmpC beta-lactamases. J Infect 2012; 64: 176–83.

7 Dahmen S, Mansour W, Charfi K, Boujaafar N, Arlet G et al. Imipenem resistance in Klebsiella pneumoniae is associated to the combination of plasmid-mediated CMY-4 AmpC beta-lactamase and loss of an outer membrane protein. Microb Drug Resist 2012; 18: 479–83.

8 Gude MJ, Seral C, Sáenz Y, et al. Molecular epidemiology, resistance profiles and clinical features in clinical plasmid-mediated AmpC-producing Enterobacteriaceae. Int J Med Microbiol 2013; 303: 553–7.

9 Seiffert SN, Hilty M, Kronenberg A, Droz S, Perreten V, Endimiani A. Extended-spectrum cephalosporin-resistant escherichia coli in community, specialized outpatient clinic and hospital settings in Switzerland. J Antimicrob Chemother 2013; 68: 2249–54.

10 Miró E, Agüero J, Larrosa MN, et al. Prevalence and molecular epidemiology of acquired AmpC β-lactamases and carbapenemases in Enterobacteriaceae isolates from 35 hospitals in Spain. Eur J Clin Microbiol Infect Dis 2013; 32: 253–9.

11 Voets GM, Platteel TN, Fluit AC, et al. Population Distribution of Beta-Lactamase Conferring Resistance to Third-Generation Cephalosporins in Human Clinical Enterobacteriaceae in The Netherlands. PLoS One 2012; 7. DOI:10.1371/journal.pone.0052102.

12 Dierikx C, van der Goot J, Fabri T, van Essen-Zandbergen A, Smith H, Mevius D. Extended-spectrum-β-lactamase- and AmpC-β-lactamase-producing Escherichia coli in Dutch broilers and broiler farmers. J Antimicrob Chemother 2013; 68: 60–7.

13 Perez-Perez FJ, Hanson ND. Detection of plasmid-mediated AmpC beta-lactamase genes in clinical isolates by using multiplex PCR. J Clin Microbiol 2002; 40: 2153–62.

14 Gude MJ, Seral C, Saenz Y, Gonzalez-Dominguez M, Torres C, Castillo FJ. Evaluation of four phenotypic methods to detect plasmid-mediated AmpC beta-lactamases in clinical isolates. Eur J Clin Microbiol Infect Dis 2012; 31: 2037–43.

15 Black JA, Moland ES, Thomson KS. AmpC disk test for detection of plasmid-mediated AmpC beta-lactamases in Enterobacteriaceae lacking chromosomal AmpC beta-lactamases. J Clin Microbiol 2005; 43: 3110–3.

16 Doi Y, Paterson DL. Detection of plasmid-mediated class C beta-lactamases. Int J Infect Dis 2007; 11: 191–7.

17 Willemsen I, Elberts S, Verhulst C, et al. Highly resistant gram-negative microorganisms: incidence density and occurrence of nosocomial transmission (TRIANGLe Study). Infect Control Hosp Epidemiol 2011; 32: 333–41.

18 al Naiemi N, Cohen Stuart J, leverstein-van Hall namens de leden van werkgroep ESBL van de NVMM MA. NVMM-Richtlijn voor screening en confirmatie van extended-spectrum beta-lactamases (ESBL’s) in Enterobacteriaceae. 2008.

19 Willemsen I, Overdevest I, Al Naiemi N, et al. New Diagnostic microarray (check-KPC ESBL) for detection and identification of extended-spectrum beta-lactamases in highly resistant Enterobacteriaceae. J Clin Microbiol 2011; 49: 2985–7.


21 EUCAST. European Committee on Antimicrobial Susceptibility Testing Breakpoint tables for interpretation of MICs and zone diameters Version 20, valid from 2012-01-01 [Accessed: November, 2012] Växjö : EUCAST Available: http://www.eucast.org/fileadmin/src/me. .


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23 Cohen Stuart J, Leverstein van Hall M, Al Naiemi N. NVMM Guideline Laboratory detection of highly resistant microorganisms (HRMO), version 2.0. 2012; Available at: http://www.nvmm.nl/richtlijnen/hrmolaboratory-detection-highly-resistant-microorganisms. .

24 Pitout JD, Gregson DB, Church DL, Laupland KB. Population-based laboratory surveillance for AmpC beta-lactamase-producing Escherichia coli, Calgary. Emerg Infect Dis 2007; 13: 443–8.

25 Woodford N, Reddy S, Fagan EJ, et al. Wide geographic spread of diverse acquired AmpC beta-lactamases among Escherichia coli and Klebsiella spp. in the UK and Ireland. J Antimicrob Chemother 2007; 59: 102–5.


27 Ohana S, Leflon V, Ronco E, et al. Spread of a Klebsiella pneumoniae strain producing a plasmid-mediated ACC-1 AmpC beta-lactamase in a teaching hospital admitting disabled patients. Antimicrob Agents Chemother 2005; 49: 2095–7.

28 Huang IF, Chiu CH, Wang MH, Wu CY, Hsieh KS, Chiou CC. Outbreak of dysentery associated with ceftriaxone-resistant Shigella sonnei: First report of plasmid-mediated CMY-2-type AmpC β-lactamase resistance in S. sonnei. J Clin Microbiol 2005; 43: 2608–12.

29 Pai H, Kang CI, Byeon JH, et al. Epidemiology and clinical features of bloodstream infections caused by AmpC-type-beta-lactamase-producing Klebsiella pneumoniae. Antimicrob Agents Chemother 2004; 48: 3720–8.

30 Naseer U, Haldorsen B, Simonsen GS, Sundsfjord a. Sporadic occurrence of CMY-2-producing multidrug-resistant Escherichia coli of ST-complexes 38 and 448, and ST131 in Norway. Clin Microbiol Infect 2010; 16: 171–8.

31 Tan TY, Ng SY, Teo L, Koh Y, Teok CH. Detection of plasmid-mediated AmpC in Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. J Clin Pathol 2008; 61: 642–4.

32 Hernandez-Alles S, Conejo M, Pascual A, Tomas JM, Benedi VJ, Martinez-Martinez L. Relationship between outer membrane alterations and susceptibility to antimicrobial agents in isogenic strains of Klebsiella pneumoniae. J Antimicrob Chemother 2000; 46: 273–7.

33 Poirel L, Naas T, Nicolas D, et al. Characterization of VIM-2, a carbapenem-hydrolyzing metallo-beta-lactamase and its plasmid- and integron-borne gene from a Pseudomonas aeruginosa clinical isolate in France. Antimicrob Agents Chemother 2000; 44: 891–7.


35 Mata C, Miró E, Rivera a, Mirelis B, Coll P, Navarro F. Prevalence of acquired AmpC beta-lactamases in Enterobacteriaceae lacking inducible chromosomal ampC genes at a Spanish hospital from 1999 to 2007. Clin Microbiol Infect 2010; 16: 472–6.


37 Beesley T, Gascoyne N, Knott-Hunziker V, et al. The inhibition of class C beta-lactamases by boronic acids. Biochem J 1983; 209: 229–33.

38 Yagi T, Wachino J, Kurokawa H, et al. Practical methods using boronic acid compounds for identification of class C beta-lactamase-producing Klebsiella pneumoniae and Escherichia coli. J Clin Microbiol 2005; 43: 2551–8.

39 Tan TY, Ng LS, He J, Koh TH, Hsu LY. Evaluation of screening methods to detect plasmid-mediated AmpC in Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis. Antimicrob Agents Chemother 2009; 53: 146–9.

Plasmid-mediated AmpC: prevalence in community-acquired

isolates in Amsterdam, the Netherlands, and risk factors for carriage

EA Reuland1, T Halaby2, JP Hays3, DMC de Jongh3, HDR Snetselaar1, M van Keulen1, PJM Elders4, PHM Savelkoul1, CMJE Vandenbroucke-Grauls1, N al Naiemi1,2,5

PLoS One 2015 Jan 14;10(1):e0113033


3 Department of Medical Microbiology and Infectious Diseases, Erasmus MC, Rotterdam4 EMGO Institute for Health and Care Research, VU University Medical Center, Amsterdam

5 Medical Microbiology and Infection Control, Ziekenhuisgroep Twente, Almelo, The Netherlands

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ABSTRACT

ObjectivesThe objective of this study was to determine the prevalence of pAmpC beta-lactamases in community-acquired Gram-negative bacteria in the Netherlands, and to identify possible risk factors for carriage of these strains.

MethodsFecal samples were obtained from community-dwelling volunteers. Participants also returned a questionnaire for analysis of risk factors. Screening for pAmpC was performed with selective enrichment broth and a selective screening agar. Confirmation of AmpC-production was performed with two double disc combination tests: cefotaxime and ceftazidime with either boronic acid or cloxacillin as inhibitor. Multiplex PCR was used as gold standard for detection of pAmpC. 16S rRNA PCR and AFLP were performed as required, plasmids were identified by PCR-based replicon typing. Questionnaire results were analyzed with SPSS, version 20.0.

ResultsFecal samples were obtained from 550 volunteers; mean age 51 years (range: 18–91), 61% were females. pAmpC was present in seven E. coli isolates (7/550, 1.3%, 0.6–2.7 95% CI): six CMY-2-like pAmpC and one DHA. ESBL-encoding genes were found in 52/550 (9.5%, 7.3–12.2 95% CI) isolates; these were predominantly bla

CTX-M genes. Two isolates had both

ESBL and pAmpC. Admission to a hospital in the previous year was the only risk factor we identified.

ConclusionsOur data indicate that the prevalence of pAmpC in the community seems still low. However, since pAmpC-producing isolates were not identified as ESBL producers by routine algorithms, there is consistent risk that further increase of their prevalence might go undetected.

Community-ACquired pAmpC: prevAlenCe And risk FACtors

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INTRODUCTION

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 mimick the trend that we have seen occurring over the past years for ESBLs.1,2 We consider it important therefore, to closely monitor the occurrence of this resistance threat.

Outbreaks of pAmpC have been recognized in different settings worldwide.3–8 Currently little information is available regarding the prevalence of this group of beta-lactamases in the Dutch community. 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.9,10

The objective of the present study was to determine the prevalence of pAmpC beta-lactamases in community-acquired Gram-negative bacteria in the Netherlands, and to identify possible risk factors for carriage of these strains.

METHODS

Study populationIn the context of a larger study aimed at determining the prevalence of carriage of ESBL-positive isolates in the community in the Netherlands, volunteers for this study were approached through five general practices, affiliated to the Academic General Practice Network, VU University Medical Center, in the region of Amsterdam. In the Netherlands, health insurance is obligatory and all inhabitants have to be registered with a general practitioner, regardless of their health status. We took advantage of this registration system, and used it to approach the study subjects. All persons older than 18 years, registered in the above mentioned five general practices were approached by postal mail, except for a small group of terminally ill patients registered with a single practitioner who preferred that these patients were not asked to participate. This means that the persons who participated

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in the study were not hospitalized, nor visiting their physician at that moment, hence truly recruited from the community. Volunteers were asked to send in a fecal sample and to fill in a questionnaire.

Ethics StatementWritten informed consent was obtained from all participants, and the study was approved by the medical ethics committee (METc, NL29769.029.09) of the VU University Medical Center (NTR Trial ID NTR2453).

Antimicrobial susceptibility testing and phenotypic confirmation of ESBL and pAmpCFecal samples were inoculated into Trypticase Soy enrichment Broth containing 50 mg/L ampicillin (TSB-amp) and incubated overnight at 37°C. For ESBL and AmpC screening, an aliquot of the overnight culture was subcultured on a selective screening agar which is routinely used for ESBL screening (EbSA ESBL agar, Cepheid Benelux, Apeldoorn, the Netherlands). This agar consists of a double MacConkey agar plate supplemented with vancomycin to inhibit gram-positive enterococci (64 mg/L) and cloxacillin to inhibit AmpC producers (400 mg/L) on both sides. Additionally, cefotaxime (1mg/L) was added to one of the sides, and ceftazidime (1 mg/L) to the other side to screen for isolates resistant to third generation cephalosporins. In order to detect also AmpC producers (both chromosomally and plasmid encoded AmpC), an adapted agar without cloxacillin was used in this study. Colonies growing on either side of the adapted screening agar were regarded as suspect for ESBL- and/or AmpC production and were further analyzed.

Species identification and antibiotic susceptibility testing were performed with the Vitek 2 system (bioMérieux, Marcy l’Etoile, France). The MIC breakpoints used for interpreting the results were set according to EUCAST criteria.11

For species identification, amplification and sequencing of the 16s rRNA gene was performed on isolates for which Vitek 2 did not provide conclusive results. Phenotypic confirmation of ESBL production was performed according to the Dutch national guidelines for ESBL detection with the double disk combination test, i.e. synergy with clavulanic acid was tested for cefotaxime, ceftazidime and cefepime on Mueller-Hinton agar (Rosco, Taastrup, Denmark).9 In the Netherlands we assume that ESBL detection algorithms will detect all plasmid-mediated resistance to extended-spectrum cephalosporins.

According to this guideline for detection of ESBL, enterobacterial species can be divided, with regard to the presence of chromosomal AmpC which may interfere with ESBL detection, in two groups: group I comprises Escherichia coli, Klebsiella spp., Proteus mirabilis, Salmonella spp., and Shigella spp., in which inducible or derepressed chromosomal AmpC


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enzyme are uncommon or absent, and group II including Citrobacter freundii, Enterobacter spp., Hafnia alvei, Morganella morganii, Providencia spp. and Serratia spp. in which the presence of inducible chromosomal AmpC beta-lactamase is more rule than exception.9 Reduced susceptibility to cefoxitin was determined with Vitek 2 (bioMérieux, Marcy-l’Etoile, France), and was defined as an MIC > 8 mg/L according to EUCAST guidelines.11 In the present study we included all isolates of group I and II Enterobacteriaceae, regardless of their susceptibility to cefoxitin.11 A MIC≤ 8 mg/L for cefoxitin was repeated with Vitek 2 and confirmed with Etest cefoxitin on Mueller-Hinton agar (bioMérieux, Solna, Sweden).

Two disk-based tests have been proposed to detect AmpC activity, namely cefotaxime and ceftazidime combined with well-known inhibitors of pAmpC: boronic acid (PBA) or cloxacillin (Rosco, Taastrup, Denmark).12,13 An increase in zone diameter of ≥ 5 mm in the presence of the inhibitor indicated a positive AmpC test.

Molecular analysesAll phenotypically confirmed AmpC and ESBL positive isolates were analyzed by PCR for molecular detection of pAmpC genes. Bacterial DNA was isolated using the QIAamp DNA mini kit (QIAGEN, Venlo, the Netherlands), and an initial multiplex screening PCR was performed, followed by a confirmatory singleplex PCR for multiplex PCR positive DNA. The primers used were specific for MOX-type, CMY-type, DHA-type, ACC-type, MIR-/ACT-type and FOXtype, with a slightly increased primer annealing step of 70°C for 30 seconds.14 Detection by PCR was considered the gold standard for pAmpC detection. Group I isolates were also screened for AmpC-encoding genes by Check-MDR CT103 microarray to identify CMY I/MOX, CMY II, FOX, DHA, ACT/MIR and ACC (Check-Points Health BV,Wageningen, the Netherlands).15 In isolates positive for ESBL in the phenotypic tests, ESBL genes were characterized by PCR (VUmc) and sequencing (BaseClear, Leiden, the Netherlands).16,17

Identification of plasmids and epidemiological typingCharacterization of enterobacterial plasmids was performed with PCR-based replicon typing for the detection of the eight most prevalent replicon types.18 Seven AmpC-positive E. coli isolates were analyzed for genetic relatedness by amplified-fragment length polymorphism (AFLP), as described.19 BioNumerics software, version 6.6, (Applied Maths, Sint-Martens- Latem, Belgium) was used to analyze AFLP banding patterns.

Statistical analysesStatistical analyses were performed with SPSS, version 20.0.

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RESULTS

Between August 12 and December 13, 2011, fecal samples and questionnaires were obtained from 550 volunteers. The mean age of participants was 51 years (range: 18–91), and 61% of participants were female.

With the phenotypic AmpC confirmation test, (AmpC disk diffusion combination test with boronic acid and cloxacillin), we detected 176/550 (32%) AmpC positive isolates; these included Enterobacteriaceae from both Group I and Group II. Among group I species, 45/176 isolates (42 Escherichia coli, 2 Klebsiella pneumoniae, 1 Salmonella spp) were positive in the phenotypic confirmation test. Only 7/42 of the E. coli isolates, however, were confirmed to be positive for pAmpC by multiplex AmpC PCR. Among Group II species, which consisted of 60 Enterobacter spp, 47 Citrobacter spp, 16 Hafnia alvei, 5 Morganella morganii, 1 Raoultella ornithinolytica, 1 Aeromonas hydrophila/caviae, 1 Serratia plymuthica, 131 isolates were found to be positive using the phenotypic confirmation test, whereas only 52 were found to be positive using the multiplex AmpC screening PCR.

If an AmpC gene was detected by PCR that is specific for the species concerned, it was considered as chromosomal. The AmpC genes detected in group II species were all species-related chromosomal genes: CMY-2-like pAmpC was found in Citrobacter freundii, DHA in Morganella morganii spp., ACC in Hafnia alvei, and ACT/MIR-1 in Enterobacter species. A non-species related pAmpC was detected by PCR in 7 strains, all E. coli (7/550, 1.3%, 0.6–2.7 95% CI). The genes were 6 bla

CMY-2-like and 1 bla

DHA. Microarray results confirmed the results

obtained by PCR in group I Enterobacteriaceae.20

ESBL-encoding genes were detected in 52/550 samples (9.5%, 7.3–12.2 95% CI), these were predominantly bla

CTX-M genes. Interestingly, two pAmpC-producing isolates also

produced an ESBL: one CTX-M-1 and one CTX-M-15. Two of the seven pAmpC positive E. coli isolates were also resistant to cotrimoxazole, one to ciprofloxacin and one to gentamicin. Multiresistance, i.e. resistance to at least one antimicrobial agent from three or more antimicrobial categories (aminoglycosides, quinolones and cotrimoxazole), was detected in a single isolate.21 One isolate was also resistant to nitrofurantoin. All pAmpC-producing strains were susceptible to meropenem and imipenem. No cefoxitin susceptible (as determined by E-test) pAmpC-positive isolates were found.

AFLP was performed on the pAmpC-producing E. coli strains, which showed that the seven isolates were not genetically related. In these seven pAmpC-producing isolates, the plasmid replicon types IncI1 (5/7), ColE (5/7), ColEtp (3/7), FIB (3/7), Frep (2/7), R (1/7) and FIA (1/7) were identified. We did not perform transformation experiments to precisely identify the pAmpC-carrying plasmid types. The types of plasmids we detected, however, are in accordance with previous publications that show that a wide range of plasmid replicon types may be associated with pAmpC-positive bacterial isolates, including A/C, I1, Y, F, K, FII, L/M and B/O.22–24


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After analysis of the 544 questionnaires (six participants did not complete the questionnaire), our data showed that all the seven cases were women, with four out of six aged between 18 and 30 years (Table 1). No comorbidities were present. Only two out of seven cases had used antibiotics in the previous year (tetracyclines seven months and quinolones 12 months earlier), compared to 80 out of 495 persons who did not carry pAmpC (OR 2.6, CI 0.5–14.4).Three out of seven cases were admitted to a hospital in the Netherlands (OR 7.2; CI 1.6–33.2), one had an admission to a foreign hospital and one participant was admitted to a rehabilitation center. Healthcare-associated pAmpC carriage overall turned out to be significant (OR 6.9; CI 1.5–31.5). Four carriers visited countries inside Europe, only one travelled to Asia (5 months ago) and one to Africa (more than one month ago).

Table 1 - Characteristics of the participants (n=544) included.

pAmpC carrier (7) pAmpC non-carrier (537) total OR CI

18-30 years 4/6 148/537 544 5.4 1.0-9.5

Previous use of antibiotics 2/7 80/495 501 2.6 0.5-14.4

Healthcare-associated* 3/7 52/528 535 6.9 1.5-1.5

Travel outside Europe 2/7 192/533 540 0.7 0.1-3.7

*Healthcare-associated acquisition included hospital admission in the Netherlands or in a foreign country, or admission to a rehabilitation center.

DISCUSSION

In this study we detected pAmpC-positive enterobacterial isolates in seven out of 550 (1.3%) fecal samples obtained from community-dwelling individuals in the region of Amsterdam. The participants in the study were approached by postal mail, hence they were not hospitalized, nor attending their physician at the moment of recruitment. Because of the unbiased way we approached the study population, participants represent a cross-section of the general population (older than 18 years) and therefore include healthy persons, and persons that may have been in hospital before or may have recently visited their general practitioner for any reason. We consider therefore the prevalence that we measured to reflect the actual prevalence of carriage of pAmpC in the general population.

To date, pAmpC has been found mainly in clinical isolates obtained from hospitalized patients.25,26 Only very limited data are available regarding the prevalence of pAmpC circulating in the community.2 Data vary from 0.59% in outpatients in Spain to 6.7% in Libya and 16% in another study performed in Spain.2,27–29 More than one percent in the present study seems however to be high in strains isolated from community patients in a country with prudent antibiotic use. The possible relevance of community circulating pAmpC has been previously indicated by Pitout et al., who showed that pAmpC-producing

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Enterobacteriaceae are relevant community pathogens with important implications for public health, especially as a cause of urinary tract infections in older women.30 In addition, studies from different countries show that the community serves as a reservoir for the introduction of pAmpC in the hospital setting.31,32

A few studies analyzed possible risk factors for infections due to pAmpC and ESBL-producers. Our results should be interpreted with caution, because of the very small number of cases, but the possible risk factors that we found are similar to those described previously, i.e. contact with healthcare.1,2 It is interesting that, despite the small number of carriers we found, the association with female gender is apparent and comparable to that found by Pitout (although there is a difference in age).30

All seven pAmpC-producing Enterobacteriaceae we found were E. coli isolates, a species which is also the predominant ESBL-producing species among community-acquired ESBLproducers.33,34 The most frequent pAmpC gene belonged to CMY group II; this is comparable to what has been found in several previous studies, where bla

CMY-2 was found

to be the most widely distributed pAmpC gene geographically.30,31,35 pAmpC-encoding genes are often located on large plasmids, which are associated with multidrug resistance.36 Two of the seven pAmpC-positive E. coli strains that we identified in the present study also harbored an ESBL gene. These strains were therefore resistant to cephalosporin antibiotics by multiple mechanisms and could easily be detected by routine diagnostic methods. Five of the seven pAmpC-producing isolates, however, were phenotypically ESBL negative, meaning that these strains would be missed as strains possessing a plasmid-mediated resistance to extended-spectrum cephalosporins. Hence, in the routine setting, the prevalence of pAmpC-producing strains is probably largely underestimated. Phenotypic detection of pAmpC has such poor specificity that it cannot be used for routine pAmpC detection. The only reliable pAmpC detection methods are multiplex PCR and specific microarrays such as the Check-MDR CT103.20 These molecular methods however, are only applicable to species of Group I Enterobacteriaceae, since in Group II Enterobacteriaceae isolates, a positive pAmpC PCR result is most likely due to the presence of chromosomal AmpC genes. Indeed, plasmid-encoded genes can be identical to chromosomally located AmpC beta-lactamase genes in this group of Enterobacteriaceae.1

Several of the pAmpC-producing strains were also resistant to aminoglycosides, quinolones, cotrimoxazol and nitrofurantoin. High rates of co-resistance have also been reported in other studies, which means that also for infections caused by pAmpC-producing strains there may be few therapeutic options. This could increase morbidity and mortality in affected patients.2,37,38


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In conclusion, a pAmpC prevalence of 1.3% (all E. coli) was observed in a Dutch community setting. Importantly, the majority of the pAmpC-producing isolates were not detected by routine phenotypic screening algorithms for ESBLs. Although plasmid-mediated ESBL production seems to spread much more easily than plasmid-mediated AmpC production, careful monitoring of this plasmid-mediated resistance mechanism seems appropriate.

ACKNOWLEDGEMENTS

The European Community Seventh Framework Program FP7/2007-2013, TEMPOtest-QC, under grant agreement number 241742, supported this study by enabling the multiplex pAmpC PCR investigations.

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31 Hanson ND, Moland ES, Hong SG, Propst K, Novak DJ, Cavalieri SJ. Surveillance of community-based reservoirs reveals the presence of CTX-M, imported AmpC, and OXA-30 beta-lactamases in urine isolates of Klebsiella pneumoniae and Escherichia coli in a U.S. community. Antimicrob Agents Chemother 2008; 52: 3814–6.

32 Wiener J, Quinn JP, Bradford PA, et al. Multiple antibiotic-resistant Klebsiella and Escherichia coli in nursing homes. JAMA 1999; 281: 517–23.


34 Pitout JD, Nordmann P, Laupland KB, Poirel L. Emergence of Enterobacteriaceae producing extended-spectrum beta-lactamases (ESBLs) in the community. J Antimicrob Chemother 2005; 56: 52–9.

35 Woodford N, Reddy S, Fagan EJ, et al. Wide geographic spread of diverse acquired AmpC beta-lactamases among Escherichia coli and Klebsiella spp. in the UK and Ireland. J Antimicrob Chemother 2007; 59: 102–5.

36 Jacoby GA, Munoz-Price LS. The new beta-lactamases. N Engl J Med 2005; 352: 380–91.

37 Tumbarello M, Sanguinetti M, Montuori E, et al. Predictors of mortality in patients with bloodstream infections caused by extended-spectrum-beta-lactamase-producing Enterobacteriaceae: importance of inadequate initial antimicrobial treatment. Antimicrob Agents Chemother 2007; 51: 1987–94.

38 Naseer U, Haldorsen B, Simonsen GS, Sundsfjord A. Sporadic occurrence of CMY-2-producing multidrug-resistant Escherichia coli of ST-complexes 38 and 448, and ST131 in Norway. Clin Microbiol Infect 2010; 16: 171–8.

Summarizing discussion and future directions

Summarizing diScuSSion and future directionS

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SUMMARIZING DISCUSSION

Resistance to beta-lactam antibiotics due to carriage of ESBL-producing Enterobacteriaceae (ESBL-E) is increasing at an alarming rate, worldwide, not only in hospitalized patients but also in the community. Percentages of carriage of ESBL range from about 7% in Europe up to nearly 70% in some other parts of the world.1 Not only ESBLs are found but also carbapenemases are being detected with increasing frequency.

With respect to antibiotic resistance in general, and to ESBL-E in particular, the Netherlands appears as an island of low prevalence compared to other European regions. It is a country with low and prudent use of antimicrobials in the human population. However, due to several risk factors the prevalence may well be on the verge of a substantial increase. International travel for example may contribute to the acquisition and therefore introduction of resistant strains. Also use of antimicrobials -one of the main problems, due to its huge impact on emerging resistance- increases in the Netherlands due to the rise in immunocompromised patients, in use of invasive therapies and foreign bodies. Obtaining an estimate of the prevalence of ESBL-producing strains is important as a starting point for infection control policies and to establish empirical regimens for antimicrobial therapy. An increase in prevalence may necessitate adjustments in antimicrobial strategies.

For a proper assessment of the situation, it is, first of all, important to determine the prevalence of ESBL-E in the community in the Netherlands. Before 2000, ESBL-E was recorded in <1% among hospitalized patients in Dutch hospitals. After 2005 two studies reported an increase of this prevalence to 6 - 8%.2,3 These studies were performed more than ten years ago in hospitalized patients. Later on, in 2011, a high percentage of carriage of ESBL-producing bacteria on admission, nearly 5%, pointed towards the possible existence of a community reservoir.4

Before the start of a large study in the Dutch open community to determine the prevalence of carriage of ESBL-E, a pilot study was conducted to obtain an estimate of this percentage (Chapter 2). Therefore, we focused on Dutch primary care patients with presumed gastrointestinal discomfort. Unexpectedly, a high percentage (10.1%) of carriage was found. The participants included in this study were from two regions in the Netherlands: a primary care population in the region of Amsterdam (a densely populated urban area) and Brabant (a more rural area). Although two completely different regions in the Netherlands were taken, the study still involved a select population because these patients all had gastrointestinal complaints. According to the guidelines for the Dutch general practice, diagnostics by fecal culture is only requested for patients with gastrointestinal complaints that last for more than 10 days or gastrointestinal complaints after visiting foreign countries, especially the (sub) tropics.5 One of the major aims of the research carried out was to identify risk factors for the acquisition of resistant strains. In this pilot study no risk factors could be analyzed

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because no such data were available. There was for example no information on travel for these patients. However, it seems likely that visiting foreign countries is responsible for at least part of the prevalence of ESBL-E in Dutch outpatients, in view of the algorithms laid down in the professional standards for general practitioners mentioned above.5 Of the resistant strains found the ESBL-encoding genes were characterized, and strain typing was performed to gain insight in the epidemiology and to identify any genetic relatedness. We detected predominantly ESBL-producing E. coli, with CTX-M-15 as the most frequent ESBL type. This is comparable to the epidemiology in the community worldwide.1 Assessing our results from the latter perspective, note that in several countries the expansion of CTX-M-15-producing E. coli is due to the worldwide pandemic clone ST131, a more virulent clone that is associated with more severe infections.6,7 In contrast, the E. coli strains that we identified belonged to multiple sequence type clonal complexes and the presence of CTX-M-15 was scattered over different clusters; so, we could not confirm these data with our research. An important feature of ESBL-producing Enterobacteriaceae is co-resistance to other antibiotics. Co-resistance is associated with carriage of ESBL-E due to genes encoding for other plasmid-mediated resistance to other classes of antibiotics. In this study the problem of multiresistance was a significant problem as well: 45% was resistant to at least one agent in three or more antimicrobial categories and therefore likely to cause therapeutic failure.8 Twelve percent of the ESBL-E was resistant to gentamicin, ciprofloxacin and cotrimoxazole.

Because this first study showed emerging resistance by ESBL-E in just a subset of the Dutch outpatient population, it was interesting to find out the prevalence in the overall Dutch community.

To this end, a cross-sectional study was performed to determine prevalence and risk factors (Chapter 3). We initially assumed that the prevalence of carriage in the general adult population would be lower than in a specific subset of patients with gastrointestinal symptoms. With a percentage of 8.6% of ESBL-E carriage, the data from the general population confirmed the pilot study. In conclusion, the rate of ESBL carriage was still higher than expected and therefore identifying risk factors even more valuable. Several studies report risk factors in hospitalized patients such as diabetes, antimicrobial use and comorbidity.9 However, these risk factors cannot be applied to the more healthy community. Unfortunately, fewer studies have been conducted where risk factors were identified in the community; only a few European studies are available.1,10,11 In general, one of the main problems in studies focusing on antibiotic resistance in the community, is how to obtain a true representation of the community together with a reasonable sample size, as both are necessary to obtain a reliable answer. In this study we did obtain true community-derived isolates because participants were randomly selected from databases of general practices affiliated at the Academic General Practice Network (AGPN), VU University Medical Center, Amsterdam. In the Netherlands, health insurance is obligatory and all inhabitants have to


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be registered with a general practitioner, regardless of their health status. The database therefore is a representative sample of the general population.

We found that in this community setting the 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 a cabin crew member for an airline. The effects of antibiotic use and travel to Asia and Africa are not unexpected, and also frequently reported in other studies. Two other risk factors we found, however, stood out: the effects of antacid use and the more than threefold risk associated with travel to the USA have not been clearly shown before.1 The role of acid suppression has been noticed before, but received little attention.12,13 An association between antacid use and colonization with ESBL-E seems plausible and might be due to the mechanism of a disrupted barrier due to an increased gastric pH, and therefore diminished defense system.14–16 The risk of antacid use is particularly important, because antacids are used widely, and because it also points to ingestion as a route of acquisition of ESBL-E. This is in line with other reports suggesting the food chain as a possible source of resistance genes.

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. Referring to travel again, an advantage of our approach (i.e. using the general practitioner’s databases to draw a sample from the general population) is that we did not select for persons attending a travel clinic, which introduces strong bias towards countries that require vaccination or malaria prophylaxis. Our study comprised a cross-sectional study design with all WHO regions included in the analysis, and so we could identify Northern America or Northern Africa as high-risk areas. In addition to ESBL also one case of carbapenemase, OXA-48, was detected in a participant that had recently visited the USA and Egypt. Egypt is well-known for the presence of OXA-48.17

Since travel appeared as a major risk factor for ESBL-E acquisition, we also performed a study in travelers (Chapter 4). In this study we investigated the rate of and risk factors for travel-related acquisition of ESBL-producing Enterobacteriaceae (ESBL-E), ciprofloxacin-resistant Enterobacteriaceae (CIPR-E) and carbapenem-resistant Enterobacteriaceae (CR-E). For ESBL-E this rate increased from 6.1% pre-travel to 23.4% post-travel, comparable with data published in other studies.18–20 For CIPR-E the rate increased from 10.1% to 32.5% respectively. Import of quinolone resistance genes by travel was seen in one third to half of travelers in another study.21 In our prospective cohort study one carbapenemase-producing isolate, OXA-48, was acquired, also after a visit to Egypt and in line with more studies showing the acquisition of CR-E in asymptomatic travelers.22 The presence of traveler’s diarrhea and use of antimicrobials are found as risk factors for acquisition of resistance in other studies. The present study shows an increased risk of acquisition of ESBL-E as well as CIPR-E in travelers with diarrhea and a highly increased risk in those travelers that developed traveler’s diarrhea

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and used antimicrobial agents. In conclusion, international travel to (sub) tropical areas, especially travel to Asia, and diarrhea combined with antimicrobial use are important risk factors for acquiring ESBL-E and CIPR-E. Findings from ours and other studies we mentioned suggest that routinely prescribing (stand-by) antibiotics for traveler’s diarrhea should be strongly reconsidered, also because traveler’s diarrhea is usually self-limiting.23

Our study in the community pointed to Northern Africa as a region that brings a risk for ESBL-E acquisition. We had the opportunity to study the prevalence of ESBL-E, both in Egyptian patients and in Egyptian meat. These studies showed that the problem in Egypt is quite substantial. ESBL production was detected in nearly half of the Enterobacteriaceae causing bloodstream infections (Chapter 5). With respect to food in Egypt, nearly two thirds of chicken meat samples were contaminated with ESBL-E, and, even worse, around 10% of meat samples were also contaminated with carbapenemase producers (Chapter 6).

Also a very interesting issue is the possible transmission and acquisition of resistant strains via the food chain. In our study in the community, we found that CTX-M-1-encoding genes, associated with poultry, were not associated with travel while CTX-M-14 and CTX-M-15 were. This suggests that the CTX-M-1-encoding genes were acquired in the Netherlands, with food as a possible source. Several Dutch studies pose an association between ESBL-encoding genes in poultry and those found in humans.24–26 We detected ESBL genes also in Enterobacteriaceae recovered from vegetables (Chapter 7). Six percent of the samples we tested were contaminated by ESBL-E, and the genes found were comparable to what is found in enterobacterial strains from human origin. This finding, also points to the food chain as a possible source of resistance genes. However, a study design to prove the association of acquisition of resistant strains and the food chain is far from easy. Because ESBL-E are found in poultry and meat, one could envisage a study in vegetarians, but, since we detected ESBL-E also in vegetables such a study would not provide a good estimate of the risk associated with eating meat.

An important question is whether the very large amount of antibiotics used in livestock in the Netherlands does play a role in the high prevalence of ESBL-E carriage found in the Dutch community. In the past years, the amount of antibiotics used in poultry decreased substantially, due to a new policy introduced by the Dutch government in 2010. At that time, the Ministry of Agriculture, Nature and Food Quality decreed that antibiotic prescriptions had to be reduced by 50% within five years. In 2011 a further reduction to only 20% was imposed by what is now the Ministry of Economic Affairs, Agriculture and Innovation. By 2015 total sales of antimicrobial veterinary medicinal products have dropped to 207.000 kg/year, indeed a reduction of approximately 50% compared to total sales in 2010 (MARAN report 2015).27 A study by Kluytmans et al. suggests that the percentage of CTX-M-1 decreased the last few years, possibly as a consequence of reduced antibiotic use in food animals.28


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Influx from hospitals to the community is also an important source of resistant strains.12,29 Transmission in the community can occur by e.g. persistent carriers of resistant strains to household members. Several studies describe person-to-person transmission of resistant strains. In the study on ESBL-E carriage in the community, we too described three households where two members were carriers. The presence of different strains and plasmids in two households suggests that acquisition of ESBL-E within households is not only due to strain transmission.

Travel appeared as a source of carbapenemase-producing strains, however only very few were detected in the community or after travel. Influx, however, can occur with patients that have been treated in foreign hospitals (Chapter 8). We described an NDM-1-producing Klebsiella pneumoniae that was imported from the Balkan region, and found in a patient hospitalized in the east of the Netherlands. The same strain was detected in another patient during her stay in the same hospital as the index case. The strains were shown to be identical by amplified-fragment length polymorphism (AFLP). Halting the spread of resistance starts with its detection, but an important question is what type of detection method is most cost-effective. Our study favors the use of molecular methods (Chapter 9).

One resistance mechanism has been quite neglected so far. Plasmidal AmpC (pAmpC) has not been registered yet as a public health problem with consequences. Since pAmpC-producing isolates were not identified as ESBL producers by routine algorithms, a consistent risk is present that due to their plasmidal location, further increase of their prevalence might go undetected. The crucial point for determining prevalence and risk factors, and therefore impact on public health, is accurate detection in the laboratory. The Dutch national guidelines report screening and confirmation of ESBLs and carbapenemases. However, the detection of pAmpC remains difficult. In our study several phenotypic tests were evaluated and we found that the best method is to screen for reduced susceptibility to third generation cephalosporins combined with reduced susceptibility to cefoxitin. Subsequent confirmation via a combination disk diffusion test using cloxacillin was shown to have the best sensitivity and specificity in relation to costs (Chapter 10). We then measured the prevalence of carriage of Enterobacteriaceae producing pAmpC in the community, and this proved to be low, at a rate of 1.3%. This is difficult to compare with other studies because, to the best of our knowledge, no studies on pAmpC in the community have been performed so far. Furthermore, risk factors were difficult to analyze due to the small study population. However, within this small sample size we found admission to a hospital in the previous year as the only risk factor (Chapter 11).

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CONCLUSION AND FUTURE DIRECTIONS

In summary, resistant genes are present in humans, animals, food and the environment. Resistance due to beta-lactamases has been linked to several known and unknown pathways and reservoirs. What was first mainly a hospital problem is now emerging in the community and several risk factors have been identified. The food chain may play an increasing role, in addition to e.g. the increase in international travel and medical tourism. Hence, also in the Netherlands, a country with a low consumption rate of antibiotics in humans, resistant strains are more and more frequently detected. Not only ESBLs are a concern, but also carbapenemases and plasmidal AmpC. Reliable detection methods are crucial to detect carriers of resistant strains, and to be able to take adequate measures for infection control in order to restrain further spread. Identification of risk factors can contribute to improving empiric therapy in an essential way, minimizing therapeutic failure and thereby reducing morbidity and mortality.

On the basis of the results presented in this thesis we feel that important questions to ask when a patient is admitted to a hospital with severe infection and suspicion of Gram-negative bacterial cause are:

Over the last year:- Did you travel?- Did you use antibiotics?- Did you use antacids?


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REFERENCES1 Woerther PL, Burdet C, Chachaty E, Andremont

A. Trends in human fecal carriage of extended-spectrum β-lactamases in the community: Toward the globalization of CTX-M. Clin. Microbiol. Rev. 2013; 26: 744–58.

2 Al Naiemi N, Bart A, De Jong MD, et al. Widely distributed and predominant CTX-M extended-spectrum beta-lactamases in Amsterdam, the Netherlands. J Clin Microbiol 2006; 44: 3012–4.








10 Valenza G, Nickel S, Pfeifer Y, et al. Extended-spectrum beta-lactamase-producing escherichia coli as intestinal colonizers in the German community. Antimicrob Agents Chemother 2014; 58: 1228–30.













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23 Belderok S-M, van den Hoek A, Kint J a, Schim van der Loeff MF, Sonder GJ. Incidence, risk factors and treatment of diarrhoea among Dutch travellers: reasons not to routinely prescribe antibiotics. BMC Infect Dis 2011; 11: 295.




27 MARAN. Monitoring of Antimicrobial Resistance and Antibiotic Usage in Animals in the Netherlands in 2014. http://www.wageningenur. nl/upload_mm/2/2/2/0ab4b3f5-1cf0-42e7-a460-d67136870ae5_Nethmap Maran2015.pdf. .

28 Willemsen I, Oome S, Verhulst C, Pettersson A, Verduin K, Kluytmans J. Trends in Extended Spectrum Beta-Lactamase (ESBL) producing enterobacteriaceae and ESBL genes in a Dutch teaching hospital, measured in 5 yearly point prevalence surveys (2010-2014). PLoS One 2015; 10. DOI:10.1371/journal.pone.0141765.

29 Rodríguez-baño J, Navarro MD, Martínez-martínez L, et al. Epidemiology and Clinical Features of Infections Caused by Extended-Spectrum Beta-Lactamase-Producing Escherichia coli in Nonhospitalized Patients Epidemiology and Clinical Features of Infections Caused by Extended-Spectrum Beta-Lactamase-Producing Escher. J Clin Microbiol 2004; 42: 1089–94.

Nederlandse samenvatting

NederlaNdse sameNvattiNg

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SAMENVATTING EN DISCUSSIE

Het veelvuldig gebruik van beta-lactam antibiotica heeft resistentie tegen deze middelen

in de hand gewerkt. Voornamelijk de extended-spectrum beta-lactamasen (ESBL) en de

carbapenemasen die door resistente bacteriën geproduceerd worden, zijn een bron van zorg

omdat deze enzymen antibiotica met een breed activiteitsspectrum inactiveren en zich makkelijk

verspreiden van de ene bacteriesoort naar de andere. Een extra probleem is dat resistentie door

ESBL productie vaak gekoppeld is aan andere resistentiemechanismen waardoor multiresistentie

ontstaat. De stijgende prevalentie van ESBL beperkt de therapeutische mogelijkheden aanzienlijk

en doet de kans op therapiefalen toenemen. ESBL producerende bacteriën zijn geregeld

verantwoordelijk voor uitbraken en infecties in ziekenhuizen en andere zorginstellingen. Tevens

worden deze bacteriën in toenemende mate ook aangetroffen bij personen buiten het ziekenhuis.

Zij vormen inmiddels een wereldwijd probleem dat de hele gezondheidszorg aangaat. Snelle

en nauwkeurige detectie van ESBL is essentieel aangezien infectiepreventiemaatregelen en

adequaat antibioticagebruik de enige middelen zijn om verdere toename van ESBL tegen te gaan.

Resistentie tegen beta-lactam antibiotica als gevolg van dragerschap met ESBL-producerende Enterobacteriaceae (ESBL-E) neemt wereldwijd alarmerend snel toe, niet alleen bij patiënten die opgenomen zijn in het ziekenhuis maar ook in de open bevolking. ESBL-dragerschap varieert van ongeveer 7% in Europa tot ongeveer 70% in andere delen van de wereld.1 Niet alleen ESBLs maar ook carbapenemasen worden in toenemende mate gevonden.

Met betrekking tot resistentie tegen antibiotica in het algemeen, en ESBL-E in het bijzonder, is Nederland te beschouwen als een eiland met een lage prevalentie in vergelijking met andere Europese regio’s. Nederland is een land met een laag en terughoudend gebruik van antibiotica bij mensen. Echter, als gevolg van een aantal risicofactoren zou de prevalentie in de toekomst wellicht verder kunnen stijgen. Het reizen naar internationale bestemmingen bijvoorbeeld kan bijdragen aan het verwerven - en dus de invoer - van resistente stammen. Ook het gebruik van antimicrobiële middelen - een van de hoofdproblemen vanwege de enorme bijdrage aan de toenemende resistentie - stijgt in Nederland als gevolg van de stijging van het aantal immuungecompromitteerde patiënten, van het gebruik van invasieve therapieën en het gebruik van lichaamsvreemd materiaal. Het verkrijgen van een schatting van de prevalentie van ESBL-producerende stammen is belangrijk als uitgangspunt voor infectiepreventie maatregelen en het vaststellen van het empirisch antibioticabeleid. Een toename in de prevalentie kan een aanpassing van antimicrobiële strategieën noodzakelijk maken.

Voor een adequate beoordeling van de situatie, is het allereerst van belang om de prevalentie van ESBL-E in de open bevolking in Nederland te bepalen. Vóór 2000 werd bij minder dan


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1% van de patiënten opgenomen in Nederlandse ziekenhuizen ESBL-E gerapporteerd. Twee studies die na 2005 werden uitgevoerd lieten een stijging van de prevalentie zien tot 6 - 8%.2,3 Deze studies zijn meer dan tien jaar geleden uitgevoerd bij ziekenhuispatiënten. Later, in 2011, wees een hoog percentage dragerschap van ESBL-producerende bacteriën bij patiënten bij opname in het ziekenhuis, bijna 5%, in de richting van het mogelijke bestaan van een reservoir in de algemene bevolking.4

Voordat gestart werd met een grote studie in de Nederlandse open bevolking om de prevalentie en risicofactoren voor ESBL-dragerschap in kaart te brengen werd er eerst een pilot studie uitgevoerd om een globale schatting te kunnen maken (Hoofdstuk 2). We hebben ons hierbij gericht op patiënten uit de Nederlandse huisartspopulatie die maagdarmklachten hadden. In deze studie werd een hoog percentage (10.1%) ESBL-dragerschap gevonden, veel hoger dan aanvankelijk werd verwacht.

De deelnemers die in deze studie waren geïncludeerd kwamen uit twee regio’s in Nederland, namelijk uit een huisartspopulatie in de regio Amsterdam (een dichtbevolkt stedelijk gebied) en een in de regio Brabant (een meer landelijke regio). Alhoewel er dus twee compleet verschillende regio’s in Nederland werden onderzocht, betrof het nog steeds een selecte populatie omdat de patiënten zich gemeld hadden bij de huisarts met gastrointestinale klachten. Volgens de richtlijnen van het Nederlands Huisartsen Genootschap wordt alleen feces diagnostiek verricht bij patiënten met gastrointestinale klachten die langer dan tien dagen duren of wanneer deze klachten zijn ontstaan na een buitenlandse reis, waarbij met name aan een bezoek aan de (sub)tropen gedacht moet worden.5 Een van de voornaamste redenen om dit onderzoek uit te voeren was om de risicofactoren voor het verwerven van resistente stammen in kaart te brengen. In deze pilot studie kon hier echter geen onderzoek naar gedaan worden aangezien deze data niet beschikbaar waren. Er was bijvoorbeeld geen informatie met betrekking tot reishistorie bij deze patiëntenpopulatie. Echter, als we de richtlijnen -zoals die vastgelegd zijn in de professionele NHG-standaarden- ter hand nemen, lijkt het erop dat bezoek aan het buitenland tenminste voor een deel de prevalentie van ESBL-E in deze Nederlandse huisartsenpopulatie kan verklaren.5

Van de gevonden resistente stammen zijn de genen die coderen voor ESBL geïdentificeerd en vervolgens getypeerd om zo inzicht te krijgen in de epidemiologie en ook hun onderlinge genetische verwantschap te achterhalen. We hebben voornamelijk ESBL-producerende E. coli gevonden, met CTX-M-15 als het meest frequente ESBL type. Dit is vergelijkbaar met de epidemiologie van resistentiegenen die wereldwijd in de open bevolking wordt gezien.1 Als we onze resultaten hiertegen afzetten, moeten we in ogenschouw nemen dat in diverse landen de toename van CTX-M-15-producerende E. coli het gevolg is van de wereldwijd aanwezige pandemische kloon ST131, een meer virulente kloon die geassocieerd is met ernstigere infecties.6,7 In onze studie daarentegen vonden wij E. coli stammen die tot meerdere klonale complexen behoorden en waarbij de CTX-M-15 over


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verschillende clusters was verdeeld; onze Nederlandse data sloten dus minder goed aan bij de internationale epidemiologie. Een belangrijke eigenschap van ESBL-producerende Enterobacteriaceae is dat zij vaak ook resistent zijn tegen andere antibiotica. Reden hiervoor is dat ESBL-genen gelocaliseerd zijn op plasmiden waarop ook genen aanwezig kunnen zijn die coderen voor resistentie tegen andere klassen van antibiotica. In deze studie werd het probleem van multiresistentie eveneens goed zichtbaar aangezien 45% van de gevonden stammen resistent was tegen tenminste één middel uit drie of meer antibiotica klassen en derhalve een aannemelijke oorzaak voor therapeutisch falen.8 Twaalf procent van de gevonden ESBL-E was resistent tegen gentamicine, ciprofloxacine en cotrimoxazol.

Aangezien deze eerste studie in een selecte groep patiënten duidelijk liet zien dat de resistentie problematiek aan het toenemen is, was het interessant om te kijken naar de prevalentie in de gehele open bevolking in Nederland. Daarom werd er een cross-sectioneel onderzoek opgezet om zodoende de prevalentie en risicofactoren in kaart te brengen (Hoofdstuk 3). Aanvankelijk gingen we ervan uit dat de prevalentie in de volwassen bevolking lager zou zijn dan in deze specifieke subgroep van patiënten met gastrointestinale klachten. Echter, het gevonden percentage van 8.6% ESBL-dragerschap in de open bevolking bevestigde dus de resultaten uit de pilot studie.

Concluderend blijkt de prevalentie van ESBL-dragerschap hoger dan verwacht en is het belang om risicofactoren te identificeren alleen maar groter geworden. Verscheidene studies rapporteren risicofactoren in gehospitaliseerde patiënten zoals diabetes, antibiotica gebruik en comorbiditeit.9 Echter, deze risicofactoren kunnen niet worden geëxtrapoleerd naar de in principe gezondere open bevolking. Helaas zijn er minder studies uitgevoerd waarbij gekeken is naar het risicoprofiel in deze laatste groep; er zijn in feite maar een paar studies beschikbaar.1,10,11

Over het algemeen is een van de grootste problemen bij het verrichten van onderzoek in de open bevolking het verkrijgen van een ware afspiegeling van deze populatie door een redelijke steekproefgrootte. Beide zijn noodzakelijk om een betrouwbaar antwoord te verkrijgen. De deelnemers aan ons onderzoek werden willekeurig geselecteerd via het gegevensbestand van huisartspraktijken aangesloten bij het Academisch Netwerk Huisartsgeneeskunde (ANH), VU medisch centrum, Amsterdam. In Nederland is de ziektekostenverzekering verplicht en moeten alle inwoners geregistreerd zijn bij een huisarts, ongeacht hun gezondheidsstatus. Het gebruikte gegevensbestand is dus een getrouwe afspiegeling van de open bevolking.

Het onderzoek wees als risicofactoren aan: antibioticagebruik, gebruik van maagzuurremmers, en reizen naar Afrika, Azië of de Verenigde Staten. Bijkomende risicofactoren waren het hebben van een moeder geboren in Azië en mogelijk het werken als lid van het cabinepersoneel bij een luchtvaartmaatschappij. Dat antibiotica gebruik en reizen naar Azië en Afrika risicofactoren zijn, kwam niet onverwacht aangezien deze factoren


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ook in andere studies vaak gerapporteerd zijn. De twee andere gevonden risicofactoren zijn echter wel opvallend: het gebruik van zuurremmers en het meer dan drievoudig verhoogd risico geassocieerd met reizen naar de Verenigde Staten is niet eerder aangetoond.1

De mogelijke rol van maagzuurremmers is eerder beschreven, maar heeft vooralsnog weinig aandacht gehad.12,13 Een verband tussen het gebruik van zuurremmers en kolonisatie met ESBL-E lijkt plausibel: door het remmen van de zure pH in de maag wordt deze natuurlijke barrière verstoord.14–16 Aangezien zuurremmers op grote schaal gebruikt worden, zou deze risiscofactor heel belangrijk kunnen zijn. Het is ook een indirecte aanwijzing dat ingestie een belangrijke route voor het verwerven van ESBL-E is. Dit komt vervolgens weer overeen met ander onderzoek waarbij gesuggereerd wordt dat de voedselketen een mogelijke bron van resistentiegenen is.

Onze bevindingen, in combinatie met eerdere studies waarbij ESBL-E in overvloed wordt gevonden in de voedselketen, rechtvaardigen dat meer aandacht besteed moet worden aan de mogelijke gezondheidszorgrisico’s van resistente microorganismen in water en voedsel. Terugkomend op reizen als risicofactor kunnen we constateren dat het een voordeel is van onze benadering (i.e. het gebruiken van de gegevensbestanden via het ANH om een representatief beeld te krijgen van de open bevolking) dat er geen selectie heeft plaatsgevonden op basis van personen die een bezoek brengen aan een vaccinatiekliniek, hetgeen een sterke bias introduceert ten opzichte van landen waarbij vaccinatie of malaria profylaxe noodzakelijk is. Onze studie behelst een cross-sectionele onderzoeksopzet waarbij reizen naar alle WHO regio’s geïncludeerd zijn in de analyse, en waardoor zowel Noord-Amerika als Noord-Afrika geïdentificeerd konden worden als hoog-risico gebieden. Naast ESBL is ook een geval van carbapenemase, OXA-48, gedetecteerd bij een studiedeelnemer die recent een bezoek had gebracht aan de Verenigde Staten en Egypte. Egypte staat bekend om de endemische aanwezigheid van OXA-48.17

Aangezien reizen een belangrijke risicofactor blijkt voor het verkrijgen van ESBL-E, hebben we ook een reizigersstudie opgezet (Hoofdstuk 4). In deze studie hebben we gekeken naar de mate van en de risicofactoren voor reisgerelateerde verwerving van ESBL-producerende Enterobacteriaceae (ESBL-E), ciprofloxacine-resistente Enterobacteriaceae (CIPR-E) and carbapenem-resistente Enterobacteriaceae (CR-E). Wat betreft ESBL-E nam het percentage dragerschap onder de deelnemers toe van 6.1% voor het reizen naar 23.4% na het reizen, hetgeen overeenkomt met resultaten gepubliceerd in andere studies.18–20 Wat betreft CIPR-E nam dit toe van 10.1% naar 32.5% respectievelijk. De import van genen die coderen voor quinolonenresistentie als gevolg van reizen werd in een andere studie bij een derde tot de helft van alle reizigers gezien.21 In onze prospectieve cohortstudie vonden wij één reziger die, na een reis naar Egypte, een OXA-48 carbapenemase-producerende bacterie bij zich had.22


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In een aantal studies zijn reizigersdiarree en het gebruik van antibiotica gevonden als risicofactoren voor het verkrijgen van resistente bacteriën. Dit konden wij in onze studie bevestigen: zowel voor het verkrijgen van ESBL-E als wel CIPR-E brachten diarree en antibioticagebruik een sterk verhoogd risico met zich mee. Concluderend kunnen we zeggen dat internationale reizen naar (sub)tropische bestemmingen, voornamelijk Azië, en diarree in combinatie met antibiotica gebruik belangrijke risicofactoren zijn voor het verkrijgen van ESBL-E en CIPR-E. Onze bevindingen alsmede de andere genoemde studies suggereren dat het routinematig voorschrijven van (stand-by) antibiotica voor reizigersdiarree sterk heroverwogen moet worden, ook omdat reizigersdiarree over het algemeen vanzelf over gaat.23

Onze studie in de open bevolking liet zien dat Noord-Afrika een hoog-risico regio is voor het oplopen van ESBL-E. We hadden de mogelijkheid om de prevalentie van ESBL-E te bestuderen, zowel in patiënten uit Egypte als in vlees afkomstig uit Egypte. Deze studies toonden aan dat het probleem in Egypte vrij aanzienlijk is. ESBL-productie werd in bijna de helft van de Enterobacteriaceae gezien die bloedbaan infecties veroorzaakten (Hoofdstuk 5). Met betrekking tot het voedsel in Egypte bleek bijna twee derde van de kippenvleesmonsters besmet met ESBL-E, en zelfs rond de 10% van de vleesmonsters waren ook besmet met carbapenemase-producerende Enterobacteriaceae (Hoofdstuk 6).

Ook een zeer interessante kwestie is de mogelijke overdracht en het verkrijgen van resistente stammen via de voedselketen. In onze studie in de open bevolking vonden we dat reizen naar het buitenland het risico op het oplopen van stammen met ESBL van het type CTX-M-1 niet verhoogde, maar wel het risico op stammen met andere ESBL typen. Deze bevinding is interessant, omdat ESBL CTX-M-1 in Nederland vooral gevonden wordt in gevogelte. Dit zou erop kunnen duiden dat bacteriën met genen die coderen voor CTX-M-1 verworven zijn in Nederland. Diverse Nederlandse studies laten een associatie zien tussen de ESBL coderende genen die gevonden worden in gevogelte en die in de mens.24–26

We hebben ook ESBL-genen in Enterobacteriaceae aangetoond die we aantroffen op groenten (Hoofdstuk 7). Zes procent van de monsters die we hebben getest waren besmet met ESBL-E, en de gedetecteerde genen waren vergelijkbaar met de genen die gevonden worden in enterobacteriën van humane oorsprong. Ook deze bevinding wijst in de richting van de voedselketen als mogelijke bron van resistentiegenen. Echter, een studie naar de associatie tussen het verwerven van resistente genen en de voedselketen is verre van eenvoudig. Omdat ESBL-E zowel in gevogelte als vlees wordt gevonden, zou men een studie onder vegetariërs kunnen overwegen, echter omdat we ESBL-E ook gedetecteerd hebben in groenten zou een dergelijke studie niet bijdragen aan een goede inschatting van de risico’s geassocieerd met het eten van vlees.

Een belangrijke vraag is in hoeverre de enorme hoeveelheid antibiotica die gebruikt wordt in de veehouderij in Nederland een rol speelt in de hoge prevalentie van ESBL-E dragerschap


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die in de Nederlandse bevolking gevonden wordt. In de afgelopen jaren is de hoeveelheid gebruikte antibiotica bij pluimvee aanzienlijk gedaald, dit als gevolg van een nieuw beleid ingevoerd door de Nederlandse regering in 2010. Destijds werd door het ministerie van Landbouw, Natuur en Voedselkwaliteit overeengekomen dat antibioticavoorschriften met 50% verminderd moesten worden binnen vijf jaar. In 2011 werd een verdere verlaging tot slechts 20% opgelegd door wat inmiddels het Ministerie van Economische Zaken, Landbouw en Innovatie heet. In 2015 is de totale omzet van antimicrobiële diergeneesmiddelen gedaald tot 207.000 kg/jaar, inderdaad een vermindering van ongeveer 50% ten opzichte van de totale omzet in 2010 (MARAN rapport 2015).27 Een studie van Kluytmans et al. suggereert dat het percentage CTX-M-1 de laatste paar jaar is afgenomen, mogelijk als gevolg van de afname van antibiotica gebruik in de voedselindustrie.28

Instroom vanuit ziekenhuizen naar de gemeenschap is ook een belangrijke bron van resistente stammen.12,29 In de open bevolking kan transmissie optreden door bijvoorbeeld overdracht van resistente stammen van persisterende dragers naar huisgenoten. Verscheidene studies beschrijven overdracht van resistente stammen van persoon naar persoon. In de studie naar dragerschap onder de open bevolking hebben we dit fenomeen ook beschreven waarbij in drie huishoudens twee gezinsleden drager bleken. De aanwezigheid van verschillende stammen en plasmiden in twee andere huishoudens suggereert dat het verkrijgen van ESBL-E binnen huishoudens niet alleen het gevolg is van onderlinge transmissie van stammen.

Reizen bleek een bron van carbapenemase-producerende stammen te zijn, hoewel er slechts maar een aantal in de open bevolking of na het reizen zijn gedetecteerd. Echter, instroom hiervan kan optreden bij patiënten die eerder behandeld zijn in ziekenhuizen in het buitenland (Hoofdstuk 8). We hebben een NDM-1-producerende Klebsiella pneumoniae

beschreven die is geïmporteerd vanuit de Balkan, en gevonden is in een patiënt die in een ziekenhuis in het oosten van Nederland heeft gelegen. Dezelfde stam is vervolgens ook gedetecteerd in een andere patiënt gedurende haar verblijf in hetzelfde ziekenhuis als de index patiënt. De stammen bleken identiek aan elkaar, aangetoond door amplified-fragment length polymorphism (AFLP). Het tegengaan van verspreiding van resistentie begint met een goede detectie, maar wel een belangrijke vraag is wat voor soort detectiemethode het meest kosteneffectief is. Onze studie toont aan dat gebruik van moleculaire methoden meer kosteneffectief is (Hoofdstuk 9).

Één soort van resistentiemechanisme is tot nu toe vrijwel geheel genegeerd. Plasmidaal AmpC (pAmpC) wordt tot nu toe nog niet gezien als een probleem met duidelijke gevolgen voor de volksgezondheid. Zolang pAmpC-producerende isolaten in de microbiologische laboratoria niet door routinematige diagnostische algoritmes herkend worden als ESBL-producerende stammen, is er een continu risico aanwezig dat toename van hun prevalentie onopgemerkt blijft. Omdat het gen voor pAmpC op een plasmide gelokaliseerd


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is, is gemakkelijke verspreiding hiervan onder Gram-negatieve bacteriën niet ondenkbaar. Cruciaal voor het bepalen van de prevalentie en risicofactoren - en daarbij het bepalen van de gevolgen voor de volksgezondheid - is een nauwkeurige detectie van pAmpC in het laboratorium. De Nederlandse nationale richtlijnen zijn gericht op detectie van van ESBL en carbapenemases. Echter de detectie van pAmpC blijft lastig. In ons onderzoek werden verschillende fenotypische testen geëvalueerd en we vonden dat de beste methode is om te screenen op verminderde gevoeligheid voor derde generatie cefalosporinen in combinatie met verminderde gevoeligheid voor cefoxitin. Confirmatie door middel van een combinatie disk diffusie test met cloxacillin bleek vervolgens de beste sensitiviteit en specificiteit in relatie tot kosten te hebben (Hoofdstuk 10).

Vervolgens hebben we de prevalentie van dragerschap van Enterobacteriaceae die pAmpC produceren in de open bevolking gemeten. Deze bleek laag te zijn: 1.3%. Dit kunnen wij niet vergelijken met gegevens van anderen, omdat voor zover wij weten, tot nu toe geen studies zijn uitgevoerd waarbij is gekeken naar pAmpC in de open bevolking. Verder was onderzoek naar de mogelijke risicofactoren moeilijk vanwege de kleine studiepopulatie. Echter, binnen deze kleine steekproef vonden we opname in een ziekenhuis in het voorgaande jaar als enige risicofactor (Hoofdstuk 11).


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CONCLUSIE EN TOEKOMSTPERSPECTIEF

Kort samengevat, zijn resistentiegenen aanwezig in mensen, dieren, voeding en het milieu. De verspreiding van resistentie als gevolg van beta-lactamasen is in verband gebracht met verscheidene bekende maar ook minder bekende routes en reservoirs. Wat eerst vooral een ziekenhuisprobleem was, is nu in opkomst in de open bevolking waarbij inmiddels verscheidene mogelijke risicofactoren zijn geïdentificeerd. De voedselketen zou een belangrijke rol kunnen spelen, naast bijvoorbeeld de toename van het internationale reizigersverkeer en het medisch toerisme. Kortom, ook in Nederland, een land met een laag gebruik van antibiotica bij mensen, worden resistente stammen steeds vaker waargenomen. Niet alleen ESBL’s zijn een punt van zorg, maar ook carbapenemasen en plasmidaal AmpC. Betrouwbare detectiemethoden zijn hierbij van cruciaal belang om dragers van resistente stammen op te sporen, en zo adequate maatregelen voor infectiebestrijding te kunnen nemen en verdere verspreiding in te dammen. Identificatie van risicofactoren kan een wezenlijke bijdrage leveren aan de verbetering van empirische therapie, het minimaliseren van therapeutisch falen en daardoor het terugdringen van morbiditeit en mortaliteit.

Op basis van de resultaten gepresenteerd in dit proefschrift, zijn wij van mening dat het belangrijk is om, wanneer een patiënt wordt opgenomen in een ziekenhuis met een ernstige infectie met mogelijk Gram-negatieve bacteriën als oorzaak, de volgende vragen te stellen:

Heeft u het afgelopen jaar:- gereisd buiten Europa?- antibiotica gebruikt?- maagzuurremmers gebruikt?


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Reuland EA, Sonder GJ, Stolte I, Al Naiemi N, Koek A, Linde GB, van de Laar TJ, Vandenbroucke-Grauls CMJE, van Dam AP. Travel to Asia and traveller’s diarrhoea with antibiotic treatment are independent risk factors for acquiring ciprofloxacin-resistant and extended spectrum beta-lactamase-producing Enterobacteriaceae-a prospective cohort study. Clin Microbiol Infect 2016. DOI:10.1016/j.cmi.2016.05.003.

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Abdallah HM, Wintermans BB, Reuland EA, Koek A, al Naiemi N, Ammar AM, Mohamed AA, Vandenbroucke-Grauls CMJE. Extended-spectrum beta-lactamase- and carbapenemase-producing enterobacteriaceae isolated from Egyptian patients with suspected blood stream infection. PLoS One 2015; 10. DOI:10.1371/journal.pone.0128120.

Reuland EA, Halaby T, Hays JP, de Jongh DM, Snetselaar HD, van Keulen M, Elders PJ, Savelkoul PHM, Vandenbroucke-Grauls CMJE, al Naiemi N. Plasmid-mediated AmpC: Prevalence in community-acquired isolates in Amsterdam, the Netherlands, and risk factors for carriage. PLoS One 2015; 10. DOI:10.1371/journal.pone.0113033.

Hendriksen RS, Leekitcharoenphon P, Mikoleit M, Jensen JD, Kaas RS, Roer L, Joshi HB, Pornruangmong S, Pulsrikarn C, Gonzalez-Aviles GD, Reuland EA, Al Naiemi N, Wester AL, Aarestrup FM, Hasman H. Genomic dissection of travel-associated extended-spectrum-beta-lactamase-producing salmonella enterica serovar typhi isolates originating from the philippines: A one-off occurrence or a threat to effective treatment of typhoid fever? J Clin Microbiol 2015; 53: 677–80.

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: 1843–6.

Reuland EA, Hays JP, de Jongh DM, Abdelrehim E, Willemsen I, Kluytmans JAJW, Savelkoul PHM, Vandenbroucke-Grauls CMJE, al Naiemi N. Detection and occurrence of plasmid-mediated AmpC in highly resistant gram-negative Rods. PLoS One 2014; 9. DOI:10.1371/journal.pone.0091396.

Stewardson AJ, Renzi G, Maury N, Vaudaux C, Brossier C, Fritsch E, Pittet D, Heck M, van der Zwaluw K, Reuland EA, van de Laar T, Snelders E, Vandenbroucke-Grauls CMJE, Kluytmans JAJW, Edder P, Schrenzel J, Harbarth S. Extended-spectrum beta-lactamase-producing Enterobacteriaceae in hospital food: a risk assessment. Infect Control Hosp Epidemiol 2014; 35: 375–83.

Wintermans BB, Reuland EA, Wintermans RGF, Bergmans AMC, Kluytmans JAJW. The cost-effectiveness of ESBL detection: towards molecular detection methods? Clin Microbiol Infect 2013; 19: 662–5.

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Halaby T, Reuland EA, al Naiemi N, Potron A, Savelkoul PHM, Vandenbroucke-Grauls CMJE, Nordmann P. A case of New Delhi metallo-beta-lactamase 1 (NDM-1)-producing Klebsiella pneumoniae from the Balkan in the Netherlands with putative secondary transmission. Antimicrob Agents Chemother 2012. DOI:10.1128/AAC.00111-12.

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van Ruler O, Mahler CW, Boer KR, Reuland EA, Gooszen HG, Opmeer BC, de Graaf PW, Lamme B, Gerhards MF, Steller EP, van Till JW, de Borgie CJ, Gouma DJ, Reitsma JB, Boermeester MA; Dutch Peritonitis Study Group. Comparison of on-demand vs planned relaparotomy strategy in patients with severe peritonitis: a randomized trial. JAMA 2007; 298: 865–72.

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Curriculum Vitae

CurriCulum Vitae

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Op 27 mei 1975 werd Ascelijn in Groningen geboren als derde telg van vier in het gezin Reuland. Haar jeugd heeft ze doorgebracht in Roden en in 1993 behaalde zij haar eindexamen aan het Willem Lodewijk Gymnasium te Groningen. Nadat zij aanvankelijk was uitgeloot voor de studie geneeskunde in Nederland heeft zij onder meer een jaar geneeskunde gedaan aan de Universiteit Antwerpen, België. Toen zij uiteindelijk werd ingeloot startte zij haar studie Geneeskunde aan de Universiteit van Amsterdam. Nu woont zij met veel plezier aan de grachten van diezelfde stad. In het kader van haar wetenschappelijke stage deed zij een half jaar onderzoek aan het Aphasia Research Center, V.A. Medical Center at Boston University, Boston, USA. De hier ontwikkelde affiniteit met de wetenschap resulteerde in een aanstelling aan de afdeling Heelkunde van het AMC tijdens de afronding van haar studie geneeskunde. Hier participeerde zij in onderzoek naar abdominale sepsis onder leiding van prof. dr. M.A. Boermeester. Tijdens dit onderzoek groeide de fascinatie voor de microbiologie en werd Ascelijn ANIOS medische microbiologie bij Tergooiziekenhuizen. In 2009 startte zij haar promotieonderzoek in het VU medisch centrum naar antibioticaresistentie onder leiding van prof. dr. C.M.J.E Vandenbroucke-Grauls, prof. dr. J.A.J.W. Kluytmans en dr. N. al Naiemi. Na twee jaar combineerde zij dit met haar specialisatie tot arts-microbioloog. Inmiddels heeft zij haar specialisatie afgerond en is zij werkzaam als arts-microbioloog aan het Universitair Medisch Centrum Utrecht gedetacheerd bij Saltro met antibioticaresistentie en infectiepreventie als aandachtsgebied. Het promotie onderzoek heeft geresulteerd in dit proefschrift ‘REBEL-ing against resistance, REsistance to BEta-Lactam antibiotics due to beta-lactamases’ dat zij op 3 februari 2017 aan de Vrije Universiteit verdedigt.

M.v.d.B., O.v.R.

Dankwoord

DankwoorD

217

Dank (in weinig woorden, maar wel voor iedereen)

Dank is als een tere vlinder

die je onbedoeld verrast

en een vleugje geeft aan ieder

die die dank ook past

REBEL-ing

against resistance


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again

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sce

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