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Epidemiology of Extended-Spectrum β-Lactamase-Producing E. coli and Vancomycin-Resistant Enterococci in the Northern Dutch–German Cross-Border Region

Epidemiology of Extended-Spectrum β-Lactamase-Producing E. coli and Vancomycin-Resistant... fmicb-08-01914 October 4, 2017 Time: 12:53 # 1 ORIGINAL RESEARCH published: 05 October 2017 doi: 10.3389/fmicb.2017.01914 Epidemiology of Extended-Spectrum b-Lactamase-Producing E. coli and Vancomycin-Resistant Enterococci in the Northern Dutch–German Cross-Border Region Edited by: John R. Battista, Louisiana State University, 1 † 1 † 2 1 Xuewei Zhou * , Silvia García-Cobos * , Gijs J. H. M. Ruijs , Greetje A. Kampinga , United States 1 1 3 4 5 Jan P. Arends , Dirk M. Borst , Lieke V. Möller , Nicole D. Holman , Theo A. Schuurs , 2 5 5 6,7 Reviewed by: Lesla E. Bruijnesteijn van Coppenraet , Jan F. Weel , Jan H. van Zeijl , Robin Köck , 1† 1† Zhuofei Xu, John W. A. Rossen and Alexander W. Friedrich Huazhong Agricultural University, University of Groningen, University Medical Center Groningen, Department of Medical Microbiology, Groningen, China 2 3 Netherlands, Laboratory of Medical Microbiology and Infectious Diseases, Isala Clinics, Zwolle, Netherlands, Department Jose M. Munita, of Microbiology, CERTE Medische Diagnostiek en Advies, Groningen, Netherlands, Department of Intensive Care Medicine, University of Texas Health Science Martini Hospital, Groningen, Netherlands, Centre for Infectious Diseases Friesland, Izore, Leeuwarden, Netherlands, Center at Houston, United States 6 7 Institute of Medical Microbiology, University Hospital Münster, Münster, Germany, Institute of Hospital Hygiene, Klinikum *Correspondence: Oldenburg, Oldenburg, Germany Xuewei Zhou x.w.zhou@umcg.nl Silvia García-Cobos Objectives: To reveal the prevalence and epidemiology of extended-spectrum s.garcia.cobos@umcg.nl b-lactamase (ESBL)- and/or plasmid AmpC (pAmpC)- and carbapenemase (CP) These authors have contributed producing Enterobacteriaceae and vancomycin-resistant enterococci (VRE) across the equally to this work. Northern Dutch–German border region. Specialty section: Methods: A point-prevalence study on ESBL/pAmpC/CP producing This article was submitted to Evolutionary and Genomic Enterobacteriaceae and VRE was carried out in hospitalized patients in the Northern Microbiology, Netherlands (n D 445, 2012–2013) and Germany (n D 242, 2012). Healthy individuals a section of the journal from the Dutch community (n D 400, 2010–2012) were also screened. In addition, Frontiers in Microbiology a genome-wide gene-by-gene approach was applied to study the epidemiology of Received: 24 April 2017 Accepted: 20 September 2017 ESBL-Escherichia coli and VRE. Published: 05 October 2017 Results: A total of 34 isolates from 27 patients (6.1%) admitted to Dutch hospitals Citation: Zhou X, García-Cobos S, were ESBL/pAmpC positive and 29 ESBL-E. coli, three pAmpC-E. coli, one Ruijs GJHM, Kampinga GA, ESBL-Enterobacter cloacae, and one pAmpC-Proteus mirabilis were found. In the Arends JP, Borst DM, Möller LV, German hospital, 18 isolates (16 E. coli and 2 Klebsiella pneumoniae) from 17 patients Holman ND, Schuurs TA, Bruijnesteijn van Coppenraet LE, (7.7%) were ESBL positive. In isolates from the hospitalized patients CTX-M-15 was the Weel JF, van Zeijl JH, Köck R, most frequently detected ESBL-gene. In the Dutch community, 11 individuals (2.75%) Rossen JWA and Friedrich AW (2017) Epidemiology were ESBL/pAmpC positive: 10 ESBL-E. coli (CTX-M-1 being the most prevalent gene) of Extended-Spectrum and one pAmpC E. coli. Six Dutch (1.3%) and four German (3.9%) hospitalized patients -Lactamase-Producing E. coli were colonized with VRE. Genetic relatedness by core genome multi-locus sequence and Vancomycin-Resistant Enterococci in the Northern typing (cgMLST) was found between two ESBL-E. coli isolates from Dutch and German Dutch–German Cross-Border Region. cross-border hospitals and between VRE isolates from different hospitals within the Front. Microbiol. 8:1914. doi: 10.3389/fmicb.2017.01914 same region. Frontiers in Microbiology | www.frontiersin.org 1 October 2017 | Volume 8 | Article 1914 fmicb-08-01914 October 4, 2017 Time: 12:53 # 2 Zhou et al. Epidemiology of ESBL and VRE in Hospitals and the Community Conclusion: The prevalence of ESBL/pAmpC-Enterobacteriaceae was similar in hospitalized patients across the Dutch–German border region, whereas VRE prevalence was slightly higher on the German side. The overall prevalence of the studied pathogens was lower in the community than in hospitals in the Northern Netherlands. Cross- border transmission of ESBL-E. coli and VRE seems unlikely based on cgMLST analysis, however continuous monitoring is necessary to control their spread and stay informed about their epidemiology. Keywords: WGS, cgMLST, VRE, ESBL, hospital, community, prevalence, cross-border research to study hospital and cross-border dissemination of ESBL-E. coli INTRODUCTION and VRE. International travel and patient care are risk factors for dissemination of bacteria including multidrug-resistant MATERIALS AND METHODS microorganisms (MDRO), such as extended-spectrum b-lactamase (ESBL) and carbapenemase (CP)-producing Study Design Enterobacteriaceae (Woerther et al., 2013; Muller et al., A prospective point prevalence study was conducted in four 2015), and vancomycin-resistant enterococci (VRE). The of the largest hospitals (in total 3550 beds) in the Northern prevalence of the latter has increased in the last years due to Netherlands between November 2012 and February 2013, successful polyclonal subpopulations of hospital-associated (HA) covering a total population of approximately 2.85 million people. Enterococcus faecium [previously designated clonal complex The Hospital Ethical Committee of the University Medical (CC) CC17] and which are also associated with amoxicillin Center Groningen (UMCG) was informed and patients were resistance (ARE) (Arias and Murray, 2012). These populations approached to voluntarily participate in the study. Patients are distinct from E. faecium isolates in the community and included in this study provided their written informed consent isolates from non-human sources (Galloway-Pena et al., 2012; and a questionnaire concerning epidemiological and clinical Lebreton et al., 2013). data. The following high-risk wards for antibiotic-resistant Netherlands and Germany as bordering countries with microorganisms were selected: intensive care units (ICU), possible transfer of patients between them, created a vascular surgery, internal medicine hematology/oncology, and cooperative network to prevent the spread of MDRO and dialysis wards (both for in- and outpatients). Gynecology and to harmonize guidelines in healthcare settings (Muller neurology (low-risk wards) were also included for comparison. et al., 2015). Surveillance studies to monitor the prevalence, From the largest German university hospital in the same resistance patterns, and molecular background of MDRO (border) region, patients from four ICUs, a surgical ward, and in hospitals and the community are essential to get insights a hematology/oncology ward were screened during October into their epidemiology to implement infection prevention and November 2012 and included in the study. After consent measures. Bacterial whole-genome sequencing (WGS) has agreement, all admitted patients from the studied wards were been demonstrated to be very useful for epidemiological screened until completing a minimum of 100 samples per surveillance and detection of antimicrobial resistance (Kwong hospital. et al., 2015). The gene-by-gene approach uses a defined set The study in healthy people living in the Northern of genes to extract an allele-based profile which makes it Netherlands was conducted retrospectively, using control scalable and portable between laboratories (Maiden et al., 2013; patients included in a previous case–control study on Leopold et al., 2014). A core genome multi-locus sequence microorganisms causing gastroenteritis. Control subjects typing (cgMLST) scheme has been developed for E. faecium were patients attending their general practitioner for a variety to distinguish between epidemiologically related and unrelated of medical questions, but no gastrointestinal problems, in the isolates (de Been et al., 2015). Although there is no cgMLST period between August 2010 and December 2012 (Bruijnesteijn scheme nor threshold publicly approved yet for Escherichia coli, van Coppenraet et al., 2015). No prevalence study was performed there are several tools available that allow to define an ad hoc in the community in Germany. cgMLST. The aim of this study was to perform a point-prevalence study Sample Collection on ESBL/plasmid-mediated AmpC (pAmpC)/CP-producing TM A total of 445 rectal swabs (Copan ESwab ) were taken from Enterobacteriaceae and HA E. faecium (VRE and ARE) in hospitalized patients (median ageD 66 years, range 18–99 years) hospitals in the Northern Dutch–German border region and in the Northern Netherlands, 51.7% (n D 230) from men and to determine the predominant resistance genes. In addition, 48.3% (n D 215) from women. A total of 328 (73.7%) patients stool community samples from the Northern Netherlands were were screened at high-risk wards and 117 (26.3%) patients were screened for the same resistant pathogens. A cgMLST was used screened at low-risk wards (Table 1). In the German university https://www.Deutschland- Nederland.Eu hospital, 242 patients (median ageD 64 years, range 0–94 years) Frontiers in Microbiology | www.frontiersin.org 2 October 2017 | Volume 8 | Article 1914 fmicb-08-01914 October 4, 2017 Time: 12:53 # 3 Zhou et al. Epidemiology of ESBL and VRE in Hospitals and the Community were included, 64.5% (nD 156) men and 35.5% (nD 86) women. German Hospital Of these 242 patients, 140 were screened only for ESBL, 22 only Rectal swabs were directly plated on chromID ESBL agar TM for VRE, and 80 for both. From the Dutch community study, 400 (bioMérieux) for ESBL screening and enriched Enterococcosel frozen faeces samples were included; 41% (n D 164) from men Broth (Bile Esculin Azide Broth) (BD; Becton, Dickinson and and 59% (nD 236) from women, 12% of the samples were from Company) was used for VRE screening and subsequently children. The median age of the healthy individuals was 47.5 years cultured on chromID VRE agar (bioMérieux). (range 0–84 years). Species identification and antibiotic susceptibility testing was done by MALDI-TOF MS (Bruker Daltonik GmbH, Bremen) and VITEK 2 (bioMérieux), respectively, following EUCAST criteria. Microbiological Detection, Identification, Confirmation of ESBL was performed using disk diffusion and Susceptibility Testing (cefotaxime 30 mg, cefotaxime 30 mg plus clavulanic acid 10 mg, Dutch Hospitals and Retrospective Dutch Community ceftazidime 30 mg, ceftazidime plus clavulanic acid 10 mg, Study cefepime 30 mg, cefepime 30 mg plus clavulanic acid 10 mg, and Rectal swabs (Dutch hospitalized patients) and approximately cefoxitin 30 mg) (Mast Diagnostics, Bootle, UK). 50 mg of feces per sample (Dutch community patients) were enriched in selective broths: VRA broth containing BHI PCRs and Microarray (brain heart infusion) with 20 mg/L amphoterin-B, 20 mg/L Enterococci isolates from Netherlands were screened by in-house aztreonam, 20 mg/L colistin, and 16 mg/L amoxicillin and PCR for IS16 (a marker for specific hospital-associated strains), TSB-VC broth containing tryptic soy broth with 8 mg/L vanA and vanB genes as described previously (Clark et al., vancomycin and 0.25 mg/L cefotaxime. Both broths were 1993; Werner et al., 2011). The GenoType Enterococcus (Hain incubated for 24 h at 35  1 C. Subsequently, 10 mL Lifescience GmbH) was used in enterococci isolates from of VRA broth was subcultured on VRE Brilliance agar R Germany, which detects species and genotypes vanA, vanB, (Oxoid ) and BMEG-2 agar (blood agar containing 64 mg/L vanC1, and vanC2. ESBL and VRE positive isolates were sent to meropenem, 2 mg/L gentamicin, 10 mg/L oxacillin, and our hospital for further characterization. 20 mg/L amphotericin-B) for identification of VRE and all ARE, Enterobacteriaceae isolates resistant to third generation respectively. Ten microliters of TSB-VC broth was subcultured cephalosporins and natural chromosomal AmpC producers onto ME/CF/CX comparted plates, containing iso-sensitest agar intermediate or resistant to cefepime were selected for DNA with 1 mg/L meropenem, 1 mg/L ceftazidime, or 1 mg/L extraction using the UltraClean Microbial DNA Isolation Kit (Mo cefotaxime, respectively, plus 20 mg/L vancomycin and 20 mg/L Bio Laboratories, Inc.) and further characterized for the presence amphotericin-B (Mediaproducts, Groningen), for selection of of ESBL/AmpC genes using a DNA-array (Check-MDR CT103, ESBL/pAmpC/CP-producing bacteria. Plates were incubated for Check-points, Wageningen, Netherlands) (Garcia-Cobos et al., 24 h at 35 1 C, except for VRE Brilliance agar plates that were 2015). incubated for 48 h. Suspected colonies on VRE Brilliance, BMEG-2 and Whole-Genome Sequencing of VRE and ME/CF/CX agar plates were streaked on blood agar ESBL-E. coli (one isolate per morphotype). Species identification was done by matrix-assisted laser desorption/ionization-time WGS was performed for all ESBL-E. coli and VRE isolates. of flight mass spectrometry (MALDI-TOF MS) (Bruker For each isolate, several colonies (about 5 mL) of the culture Daltonik GmbH, Bremen). Confirmed Enterococcus spp. and were suspended in 300 mL microbead solution, which was Enterobacteriaceae spp., were tested for antibiotic susceptibility subjected to DNA extraction with the Ultraclean Microbial DNA using VITEK 2 (bioMérieux) automatic system and EUCAST isolation kit (Mo Bio Laboratories, Carlsbad, CA, United States). clinical breakpoints. The DNA concentration and purity were measured using a TABLE 1 | Distribution of ESBL/pAmpC producing Enterobacteriaceae, and amoxicillin- and vancomycin-resistant E. faecium among the different wards in Dutch hospitals. Ward ESBL/pAmpC producing Amoxicillin-resistant Vancomycin-resistant Enterobacteriaceae E. faecium E. faecium High-risk (n D 328) 19 .5:8%/ 99 .30:2%/ 6 .1:8%/ Intensive care unit (n D 102) 6 .5:9%/ 31 .30:4%/ 1 .1%/ Vascular surgery (n D 54) 6 .11:1%/ 15 .27:8%/ 1 .1%/ Internal medicine hematology/oncology (n D 81) 1 .1:2%/ 36 .44:4%/ 2 .2:5%/ Dialysis (n D 91) 6 .6:6%/ 17 .18:7%/ 2 .2:2%/ Low-risk (n D 117) 8 .6:8%/ 6 .5:1%/ 0 .0%/ Gynecology (n D 55) 3 .5:5%/ 1 .1:8%/ 0 .0%/ Neurology (n D 62) 5 .8:1%/ 5 .8:1%/ 0 .0%/ Total (n D 445) 27 .6:1%/ 105 .23:6%/ 6 .1:3%/ Frontiers in Microbiology | www.frontiersin.org 3 October 2017 | Volume 8 | Article 1914 fmicb-08-01914 October 4, 2017 Time: 12:53 # 4 Zhou et al. Epidemiology of ESBL and VRE in Hospitals and the Community NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, (Supplementary Table S2b) were added to exclude such genes Waltham, MA, United States) and the Qubit double-stranded are part of the cgMLST typing scheme. The final cgMLST scheme DNA (dsDNA) HS and BR assay kits (Life Technologies, consisted of 1.771 targets/genes, and 2329 accessory genes were Carlsbad, CA, United States). One nanogram of bacterial DNA additionally included for the wgMLST scheme (Supplementary was used for library preparation. The DNA library was prepared Tables S3, S4). The minimum coverage of the genome assemblies using the Nextera XT library preparation kit with the Nextera was 20 times (Supplementary Table S1) and the percentage of XT v2 index kit (Illumina, San Diego, CA, United States). The good targets/genes included in the cgMLST were 97.6% for E. coli library fragment length was aimed at fragments with a median and 98.6% for E. faecium (Supplementary Tables S5, S6). size of 575 bases and was assessed with the Genomic DNA Furthermore, to determine the genetic relatedness, the genetic ScreenTape assay with the 2200 TapeStation system (Agilent distance for the E. coli isolates was calculated as the proportion Technologies, Waldbronn, Germany). Subsequently, the library of allele differences: dividing the number of allele differences was sequenced on a MiSeq sequencer, using the MiSeq reagent between two genomes by the total number of genes commonly kit v2 generating 250-bp paired-end reads. Sequencing was aimed shared by those two genomes (Kluytmans-van den Bergh et al., at a coverage of at least 60-fold. MiSeq data were processed 2016). In this study, thresholds for genetic distance were with MiSeq control software v2.4.0.4 and MiSeq Reporter v2.4 described to discriminate between epidemiologically related and (Illumina, San Diego, CA, United States). Reads were quality- unrelated E. coli isolates as 0.0095 when using wgMLST and trimmed using the CLC Genomics Workbench software version 0.0105 for cgMLST. 9.0.1 (CLC bio, Aarhus, Denmark) using default settings except Escherichia coli STs were determined uploading genome for the following modifications: “trim using quality scores was assemblies to SeqSphereC software following the scheme of set to 0.02” and “discard reads below length was set to 15.” Wirth et al. (2006). Sequence genomes with no conclusive results Subsequently, trimmed-reads were de novo assembled with an for the 7-gene MLST were uploaded to the Enterobase database . optimal word size of 29 and a minimum contig length of Additionally, E. coli major phylogenetic groups (A, B1, B2, and D) 500. Metrics on raw read and assembly level are provided in were analyzed in silico by using MLST Target Definer function Supplementary Table S1. of SeqSphere , including the chuA, yjaA, and TSPE4.C2 loci (Clermont et al., 2000). Genome assemblies were also uploaded to the Center for cgMLST of VRE and ESBL-E. coli Genomic Epidemiology to extract information on resistance A genome-wide gene-by-gene comparison approach was used to genes (ResFinder) and virulence factors (VirulenceFinder), and determine the genetic relatedness using SeqSphere version 3.4.0 species confirmation for VRE and ESBL-E. coli (KmerFinder), (Ridom GmbH, Münster, Germany) (Leopold et al., 2014). and serotype (SerotypeFinder) and plasmid replicons Genome assemblies from the VRE isolates were analyzed using (PlasmidFinder) for ESBL-E. coli (Zankari et al., 2012; Carattoli the E. faecium cgMLST scheme previously published, considering et al., 2014; Hasman et al., 2014; Joensen et al., 2014, 2015; Larsen a cluster alert distance of 20 different alleles (de Been et al., 2015). et al., 2014). An ad hoc cgMLST and whole-genome MLST (wgMLST) scheme was determined for E. coli isolates using the MLST Statistical Analysis target definer function with default parameters and E. coli K-12 In the Dutch hospital prevalence study, associations between as a reference (GenBank accession no. NC_010473.1). The filters ESBL and ARE carriage and the following variables were applied to reference genome were: “minimum length filter” that analyzed: length of hospital stay, antibiotic use, and (low- or discards genes shorter than 50 bases; “start codon filter” that high-risk) ward. Information was gathered by the questionnaires. discards all genes that contain no start codon at the beginning Statistical analyses were performed using SPSS for Windows, v. of the gene: “stop codon filter” that discards all genes that contain 20.0. Univariate analyses were performed using the Fisher’s exact no stop codon, more than one stop codon or if the stop codon or Chi-square methods for categorical variables. The Mann– is not at the end of the gene: “homologous gene filter” that Whitney U -test was used as a non-parametric tests in variables discards all genes that have fragments that occur in multiple with no normal distribution. Results with a p-value of 0.05 copies in a genome (with identity 90% and more than 100 were considered to be statistically significant. All p-values are bases overlap); “gene overlap filter” that discards the shorter of two-tailed. two overlapping flanking genes if these genes overlap >4 bp. The remaining genes were then used in a pairwise comparison using BLAST (Leopold et al., 2014) with 45 query genomes RESULTS (Supplementary Table S2a). All genes of the reference genome that were common in all query genomes with a sequence identity ESBL/pAmpC-Producing of 90 and 100 overlap, and with the default parameter stop codon percentage filter turned on, formed the final cgMLST Enterobacteriaceae scheme; this discards all genes that have internal stop codons in Thirty-four isolates from 27 of the 445 included patients admitted >20% of the query genomes. Additionally, 26 plasmid sequences to hospitals in the Northern Netherlands (6.1%) were confirmed ESBL and/or pAmpC positive. A total of 85.2% (23/27), 14.8% http://www.ridom.de/seqsphere/ug/v20/Tutorial_for_MLST+_Target_Definer. html https://enterobase.Warwick.Ac.Uk Frontiers in Microbiology | www.frontiersin.org 4 October 2017 | Volume 8 | Article 1914 fmicb-08-01914 October 4, 2017 Time: 12:53 # 5 Zhou et al. Epidemiology of ESBL and VRE in Hospitals and the Community (4/27), and 3.7% (1/27) of these patients were positive for ESBL, (n D 12)] of this study were analyzed by a gene-by-gene pAmpC, and both, respectively. Among the 34 isolates, 32 were approach and the allelic distance from the cgMLST and wgMLST E. coli, of which 29 were ESBL positive and three were pAmpC were visualized in a minimum spanning tree (Figure 1 and producers. Resistance genes detected in the E. coli isolates are Supplementary Figure S1, respectively). shown in Table 2. CTX-M-15 (n D 8) and CTX-M-14 (n D 8) Six groups of isolates with a lower number of different were the most prevalent ones. The other two isolates were alleles (35) by cgMLST were further analyzed. Supplementary an Enterobacter cloacae, containing a CTX-M-1-like gene and Table S7 summarizes the origin of the isolates in every group a pAmpC CMY-II producing Proteus mirabilis. At high-risk and the core and whole-genome genetic distance. Those groups wards, 19 patients (5.8%) were found with ESBL/pAmpC isolates formed by isolates with an epidemiological link (isolated from compared to eight patients (6.8%) at low-risk wards (p D 0.68; the same patient; group 1, 4, 5a, 6a, and 7), showed a core and NS). No association was found between ESBL/pAmpC carriage whole-genome genetic distance lower than 0.0030 and 0.0046, and antibiotic use, length of hospital stay or ward (Table 1). respectively. In addition, isolates of group 5b, although with In the German hospital, a total of 18 isolates from 17 patients unknown epidemiological link, had a core genetic distance of (17/220; 7.7%) were ESBL positive. Sixteen isolates were E. coli 0.0063 and a whole-genome genetic distance of 0.0076. Both and two were Klebsiella pneumoniae. Of these, 12 E. coli and one isolates were positive for CTX-M-14, however, no plasmid K. pneumoniae isolates were available for molecular testing. Six replicons were found in one of them (isolate 38_Esco_HA-DE) out of 12 (50%) E. coli isolates and the K. pneumoniae isolate had (Supplementary Table S7). a CTX-M-15 gene (Table 2). Among those groups including isolates with non In the retrospective Dutch community study, 11 patients (or unknown) epidemiological link, the core genome genetic (11/400; 2.75%) were ESBL/pAmpC positive: 10 ESBL-E. coli distance was between 0.0122 and 0.0199 and the whole-genome (CTX-M-1 being the most prevalent gene) and one pAmpC genetic distance was between 0.0104 and 0.0208 (groups 2, 3, 6b, E. coli (Table 2). Overall, no carbapenem resistance was observed and 6c; Figure 1). Resistance and virulence profiles of the isolates neither in the community nor in the hospitals. are shown in Supplementary Table S8. Escherichia coli MLST and Phylogenetic cgMLST Comparison of VRE Isolates Groups from the Community and Hospitals Among ESBL/pAmpC-E. coli isolates from Dutch hospitals, the A minimum spanning tree was created for the 11 VRE isolates most prevalent ST was ST131 (CC, ST131; n D 5, 15.6%), all [Dutch community (nD 1), Dutch hospitals (nD 6), and German of them belonging to phylogroup B2 (Table 2). In the Dutch hospital (nD 4)]. Two clusters of isolates from different patients community isolates, 10 different STs were found, most of them were observed (Figure 2). One cluster of four vanB-VRE isolates belonging to CC ST10 (n D 3, 27.3%) and one isolate to ST131 from the Dutch hospital belonged to cluster type (CT) 110 (ST17); (phylogroup B2). In the German hospital, the most prevalent STs two isolates were from the same ward in hospital A and the other were ST38 (33.3%) and ST10 (33.3%) (Table 2). two isolates were isolated from different wards in hospital B. The other cluster of two vanA-VRE isolates were isolated from Amoxicillin- and Vancomycin-Resistant different wards from the German hospital (CT 20, ST203). The resistance and virulence genotypes of VRE isolates are shown in E. faecium (ARE and VRE) Supplementary Table S8. In the Dutch hospitals, 105 patients (105/445; 23.6%) were colonized with ARE, including six patients (6/445; 1.3%) with Nucleotide Sequence Accession Number VRE. All ARE were positive for IS16 and all VRE were Sequence data obtained in this study has been deposited at the vanB positive. Colonization of ARE (and VRE) was associated National Center for Biotechnology Information under BioProject with high-risk wards (p < 0.001), prolonged hospitalization no. PRJNA352198. (p < 0.001), and use of antibiotics (pD 0.05), especially penicillins and fluoroquinolones (p < 0.001) (Table 3). In the border German university hospital four (4/102; 3.9%) DISCUSSION VRE isolates were isolated. Three of them were vanA positive and one was vanB positive. This study shows the molecular epidemiology of ESBL/pAmpC- In the retrospective Dutch community study, six ARE (6/400; E. coli and HA E. faecium in hospitals in the Northern 1.5%) were found, three of them were IS16 positive. Only one Dutch–German border region and the community in the vanA-VRE (1/400; 0.25%) was found, this strain was amoxicillin Northern Netherlands. Dutch hospitals showed a prevalence susceptible and IS16 negative. for ESBL/pAmpC – Enterobacteriaceae, VRE, and ARE of 6.1, 1.3, and 23.6%, respectively, whereas the prevalence in the cgMLST and wgMLST Comparison of community was 2.75, 0.25, and 1.5%, respectively. The German ESBL-E. coli Isolates from the hospital had an ESBL/pAmpC prevalence of 7.7 and 3.9% for Community and Hospitals VRE. Genome assemblies of 55 ESBL-E. coli [Dutch community A previous study reported a prevalence of ESBL-producing (n D 11), Dutch hospitals (n D 32), and German hospital bacteria of 4.9% in the Netherlands (Overdevest et al., 2014), Frontiers in Microbiology | www.frontiersin.org 5 October 2017 | Volume 8 | Article 1914 fmicb-08-01914 October 4, 2017 Time: 12:53 # 6 Zhou et al. Epidemiology of ESBL and VRE in Hospitals and the Community TABLE 2 | Molecular characterization of the E. coli isolates from the community and hospital patients in the Netherlands and Germany. Sample Hospital/ward b-Lactamase genes Phylogroup ST CC Community 1_Esco_CA-NL blaCTX-M-1, blaTEM-1B B2 131 ST131 2_Esco_CA-NL blaSHV-12 B2 117 None 3_Esco_CA-NL blaCMY-2 D 2309 None 4_Esco_CA-NL blaCTX-M-1 D 57 ST350 5_Esco_CA-NL blaCTX-M-1, blaTEM-1B A 10 ST10 6_Esco_CA-NL blaCTX-M-1, blaTEM-1B B1 1079 None 7_Esco_CA-NL blaCTX-M-1, blaTEM-1B A 10 ST10 8_Esco_CA-NL blaCTX-M-15 D 648 ST648 9_Esco_CA-NL blaCTX-M-15 A 617 ST10 10_Esco_CA-NL blaCTX-M-15 A 1312 None 11_Esco_CA-NL blaCTX-M-14b, blaTEM-1B D 38 ST38 Hospital 12_Esco_HA-NL A/Gynecology blaCTX-M-15, blaTEM-1B D 5463 None 12b_Esco_HA-NL A/Gynecology blaCTX-M-15, blaTEM-1B D 5463 None 13_Esco_HA-NL A/Neurology blaCTX-M-27 B2 131 ST131 14_Esco_HA-NL A/Dialysis outpatient blaCTX-M-15, blaTEM-1B A 93 ST168 15_Esco_HA-NL A/ICU blaCMY-2, blaTEM-1B D 354 ST354 16_Esco_HA-NL A/ICU blaCTX-M-15, blaTEM-1B, blaOXA-1 B1 58 ST155 17_Esco_HA-NL A/ICU blaCTX-M-15, blaTEM-1B B1 38 ST38 18_Esco_HA-NL A/ICU blaTEM-52C B1 453 ST86 19_Esco_HA-NL A/ICU blaCTX-M-1 B1 641 ST86 20_Esco_HA-NL A/ICU blaSHV-12 A 5888 None 20b_Esco_HA-NL A/ICU blaCTX-M-1 B1 58 ST155 21_Esco_HA-NL B/Gynecology blaCTX-M-14 B1 101 ST101 22_Esco_HA-NL B/Dialysis outpatient blaCTX-M-14 B1 38 ST38 22c_Esco_HA-NL B/Dialysis outpatient blaCTX-M-14 D 38 ST38 23_Esco_HA-NL B/Vascular surgery blaCMY-2, blaTEM-1B D 1508 None 24_Esco_HA-NL B/Neurology blaTEM-52C D 2064 None 25_Esco_HA-NL B/Neurology blaCTX-M-3, blaTEM-1B B2 95 ST95 25b_Esco_HA-NL B/Neurology blaCTX-M-3, blaTEM-1B D 95 ST95 26_Esco_HA-NL C/Gynecology blaCTX-M-15, blaOXA-1 B2 131 ST131 27_Esco_HA-NL C/Dialysis outpatient blaCTX-M-1, blaTEM-33 A 3478 None 28_Esco_HA-NL C/Dialysis outpatient blaCTX-M-14 A 10 ST10 29_Esco_HA-NL C/Neurology blaCTX-M-1 B1 603 None 30_Esco_HA-NL C/Vascular surgery blaCTX-M-14 A 410 ST23 31_Esco_HA-NL D/Vascular surgery blaCTX-M-14, blaTEM-1B, blaOXA-1 B1 58 ST155 32_Esco_HA-NL D/Vascular surgery blaCTX-M-1 D 117 None 32b_Esco_HA-NL D/Vascular surgery blaDHA-1, blaTEM-1B B2 131 ST131 33_Esco_HA-NL D/Vascular surgery blaCTX-M-14 D 69 ST69 33b_Esco_HA-NL D/Vascular surgery blaCTX-M-14 D 69 ST69 34_Esco_HA-NL D/Internal medicine blaCTX-M-55, blaOXA-1 B1 4385 None 35_Esco_HA-NL D/Dialysis outpatient blaCTX-M-15, blaTEM-1B, blaOXA-1 B2 131 ST131 35b_Esco_HA-NL D/Dialysis outpatient blaCTX-M-15, blaOXA-1 B2 131 ST13 36_Esco_HA-NL D/Dialysis outpatient blaCTX-M-1, blaTEM-1B B1 58 ST155 37_Esco_HA-DE ICU 1 blaCTX-M-15 D 38 ST38 38_Esco_HA-DE ICU 6 blaCTX-M-14 D 38 ST38 39_Esco_HA-DE ICU 2 blaCTX-M-14 A 10 ST10 40_Esco_HA-DE ICU 6 blaCTX-M-15, blaTEM-1B, blaOXA-1 B1 448 ST448 41_Esco_HA-DE Surgical ward blaCTX-M-1, blaTEM-1B A 10 ST10 42_Esco_HA-DE Hemato-oncology ward blaCTX-M-15, blaTEM-1B, blaOXA-1 A 90 ST23 (Continued) Frontiers in Microbiology | www.frontiersin.org 6 October 2017 | Volume 8 | Article 1914 fmicb-08-01914 October 4, 2017 Time: 12:53 # 7 Zhou et al. Epidemiology of ESBL and VRE in Hospitals and the Community TABLE 2 | Continued Sample Hospital/ward b-Lactamase genes Phylogroup ST CC 43_Esco_HA-DE ICU 4 blaCTX-M-15, blaOXA-1 A 34 ST10 44_Esco_HA-DE ICU 3 blaTEM-187 A 10 ST10 45_Esco_HA-DE ICU 3 blaCTX-M-15, blaOXA-1 D 38 ST38 46_Esco_HA-DE ICU 3 blaCTX-M-1, blaTEM-1B A 10 ST10 47_Esco_HA-DE ICU 1 blaCTX-M-15 D 38 ST38 48_Esco_HA-DE ICU 1 blaCTX-M-14, blaTEM-1B D 1177 – CA, community acquired; HA, hospital acquired; NL, Netherlands; DE, Germany; numbers refer to individual patients and a letter behind a number indicates that more than one isolate was obtained from the patient. comparable to the 6.1% prevalence observed in Dutch hospitals The pAmpC prevalence in E. coli in our study was 0.3%, in this study. A prevalence of 5.6% ESBL-producing E. coli comparable to the prevalence of 0.6% what was reported in the isolates in hospitalized and ambulatory patients in Germany study of van Hoek et al. (2015) (0.6% pAmpC Enterobacteriaceae) has been reported recently (Pietsch et al., 2015), which and somewhat lower to findings of Reuland et al. (2015) is slightly lower than the 7.7% observed in the present (1.3% pAmpC-E. coli). The most common pAmpC gene found in study. hospital and community isolates were CMY-II, which is together Furthermore, we observed an ESBL-E. coli prevalence of with DHA frequently found in human isolates (Reuland et al., 2.5% in the Northern Netherlands community, which is low 2015). compared to previous studies in other regions, in which the ESBL-producing E. coli belonging to CC ST131-phylogroup prevalence in the community ranged from 4.7% (2009) to 10.1% B2 are usually associated with more virulent strains (Overdevest (2011) (Reuland et al., 2013; van Hoek et al., 2015). This et al., 2015). These were frequently found in the Dutch hospitals difference may have several reasons. First, ESBL prevalence may included in the present study but only sporadically in the vary between regions and over time, and natural eradication community samples. This CC ST131-phylogroup B2 was also of resistant Enterobacteriaceae might occur over time in the prevalent in a study carried out in hospitals in the Rotterdam community (Bar-Yoseph et al., 2016). Additionally, samples region (van der Bij et al., 2011). CC ST10 was predominant included in this study were only chosen from patients without any among the ESBL-producing E. coli in the community, the same gastrointestinal complaints, a factor which otherwise has been CC was also described to be prevalent in another Dutch study in described to be associated with high ESBL prevalence (Reuland community patients (Reuland et al., 2013). et al., 2013). We observed an overall ARE and VRE prevalence in The majority of the resistance genes found in our community hospitalized patients of 23.6 and 1.3%, respectively. Similar isolates were CTX-M-1 which is broadly disseminated among observations were made in a study performed in Dutch animals in Europe, especially in cattle and pigs, followed by hospitals in 2008 reporting ARE carriage rates of 10–16% the CTX-M-15 gene, commonly associated with human origin upon admission and 15–39% on acquisition in hematology (Reuland et al., 2013; Pietsch et al., 2015). The latter was the most and gastroenterology/nephrology wards (de Regt et al., 2008). frequent gene among the Dutch and German hospital isolates, in The clinical significance of enterococcal infections and active concordance with previous studies (Ewers et al., 2012; Reuland VRE screening has been a matter of discussion. However, in et al., 2013; Pietsch et al., 2015). immunocompromised patients, high morbidity and mortality TABLE 3 | Variables associated with carriage of amoxicillin-resistant E. faecium (ARE) and extended-spectrum b-lactamase (ESBL)- and/or plasmid AmpC (pAmpC)- producing Enterobacteriaecae. Variables ARE (n D 105) No ARE (n D 340) p-Value ESBL/pAmpC (n D 27) No p-Value ESBL/pAmpC (n D 418) Hospitalization days median (range) 12 .1 127/ 3 .1 107/ p < 0:001 4 .1 127/ 4 .1 36/ p D 0.886 Ward p < 0:001 p = 0.657 High-risk (n = 328) 99 .94:3%/ 229 .67:4%/ 19 .70:4%/ 309 .73:9%/ Low-risk (n = 117) 6 .5:7%/ 111 .32:6%/ 8 .29:6%/ 109 .26:1%/ Antibiotic use (n = 145) 62 .59%/ 83 .24:4%/ p < 0:001 7 .25:9%/ 138 .33%/ p = 0.529 Penicillins 26 .24:8%/ 29 .8:5%/ p < 0:001 3 .11:1%/ 35 .8:4%/ p = 0.494 Fluoroquinolones 28 .26:7%/ 15 .4:4%/ p < 0:001 1 .3:7%/ 42 .10%/ p = 0.499 Third generation cephalosporins 11 .10:5%/ 19 .5:6%/ p D 0:081 1 .3:7%/ 29 .6:9%/ p = 1.00 Results with a p-value of 0.05 were considered to be statistically significant. All p-values are two-tailed. Used penicillins: benzylpenicillin, flucloxacillin, amoxicillin– clavulanic acid, and piperacillin–tazobactam. Frontiers in Microbiology | www.frontiersin.org 7 October 2017 | Volume 8 | Article 1914 fmicb-08-01914 October 4, 2017 Time: 12:53 # 8 Zhou et al. Epidemiology of ESBL and VRE in Hospitals and the Community FIGURE 1 | Minimum spanning tree of ESBL-E. coli isolates from hospitals and the community. Distance based on a cgMLST of 1771 genes using the parameters “pairwise ignoring missing values” during calculation. Each circle represents a genotype, colors indicate geographical origin and community or hospital. Orange: hospital—Netherlands; blue: hospital—Germany; green: community—Netherlands. Number of different alleles are indicated on the edges between connected isolates (nodes). The cut-off values for defining a group was 35 alleles. Isolates are presented by their ID and ST. rates have been reported in infections caused by enterococci 2013). Endtz et al. (1997) reported a higher number of VRE (Zhou et al., 2013). In this study, ARE/VRE carriage was in the community (2%), however, this study did not include associated with prolonged hospitalization and antibiotic use, information about amoxicillin resistance nor IS16 which makes it which is in line with previous literature (Torell et al., 1999). We difficult to determine if they had a hospital or non-hospital origin found a high carriage rate of ARE in high-risk wards (30.2%). (Galloway-Pena et al., 2012; Lebreton et al., 2013). Notably, these patients may be at risk for a subsequent infection. The cgMLST analysis in our study showed heterogeneity Since 2011, VRE started to become a problem in multiple among E. coli species, and isolates were genetically distributed hospitals in the Netherlands: a total of 14 hospitals were affected independently of their origin. The hospital and community with outbreaks of VRE in October 2012 (van den Brink, 2013). ESBL-E. coli isolates included in this study did not show any However, in this study a prevalence of VRE (vanB) carriage of genetic relatedness except for the ones isolated from the same only 1.3% was found. This is probably due to extensive infection patient and for two isolates (group 5b) from patients in different prevention measures and successful outbreak management hospitals across the Dutch–German border, in a distance of approximately 200 km and with no known epidemiological link. control. The prevalence of 1.3% is similar to what has been The patient from the Dutch hospital was a dialyses outpatient previously published in the Netherlands, with prevalence rates (isolation date December 2012) whereas the patient from the ranging from 1.4 to 2% in the 1990s (Endtz et al., 1997; van den German hospital was admitted to ICU (isolation date November Braak et al., 2000). The VRE prevalence in the German hospital 2012). Interestingly, both isolates harbored the same ESBL gene was slightly higher (3.9%), though it is known that Germany has and virulence factors. a higher VRE prevalence compared to the Netherlands . Genetic relatedness was found between four VRE isolates In our Dutch community one vanA-VRE was found, that (CT110) from patients from two different Dutch hospitals was amoxicillin susceptible and IS16 negative, indicative for a (Figure 2), which indicates transmission between wards, but non-hospital origin (Galloway-Pena et al., 2012; Lebreton et al., also between hospitals in a close geographical region similar 4 to findings of a previous population-based study of VRE using Antimicrobial Resistance Interactive Database (EARS-Net) (http://ecdc.Europa. WGS that also showed intra- and inter-regional spread of closely eu/en/) Frontiers in Microbiology | www.frontiersin.org 8 October 2017 | Volume 8 | Article 1914 fmicb-08-01914 October 4, 2017 Time: 12:53 # 9 Zhou et al. Epidemiology of ESBL and VRE in Hospitals and the Community FIGURE 2 | Minimum spanning tree of VREfcm, cgMLST based on 1423 genes using the parameters “pairwise ignoring missing values” during distance calculation. Each circle represents a genotype and colors indicate cluster types (CT). Number of different alleles are indicated on the edges between connected isolates (nodes). Isolates are presented by their ID, ST, and CT. related VRE isolates (Pinholt et al., 2015). Although no genetic analyzing more than 2000 ESBL-Enterobacteriaceae isolates from relatedness was found between VRE isolates of Dutch and Dutch hospitals (Kluytmans-van den Bergh et al., 2016). German hospitals, the numbers of VRE isolates were too low In another study, a cgMLST approach for several MDR to draw definite conclusions. It is known that several VRE CT bacteria was prospectively used for taking relevant infection co-circulate in Germany and the Netherlands (data not shown). control decisions in a hospital setting (Mellmann et al., 2016). However, only some laboratories have implemented the use of A threshold of >10 differing alleles was defined to exclude cgMLST in their routine to analyze VRE outbreaks and more nosocomial transmission of MDR E. coli (Mellmann et al., 2016). epidemiological studies are needed to investigate cross-border If we would have applied this threshold we would have missed transmission of VRE. the genetic relatedness between isolates belonging to group 5b, To our knowledge, there are no similar studies that compare presenting 11 different alleles (Figure 1 and Supplementary and investigate the molecular epidemiology of ESBL-E. coli and Table S7). This highlights that thresholds based on number of VRE in hospitals and the community by WGS. Recently, the same allele differences are only applicable to specific collections within approach has been used to study the clonality of ESBL-producing a study, whereas the genetic distance calculation seems to give a Enterobacteriaceae from environmental and stool samples from more objective result, independently of the analyzed population. farmers suggesting possible cross-transmission between the We acknowledge this study has some limitations. No farmers and the environment. This was only based on number of community study in the German cross-border region, neither allele differences (Fischer et al., 2016) which makes it difficult to ARE monitoring in the German hospital were performed. interpret results without considering the total number of genes Laboratory methods for isolation of ESBL Enterobacteriaceae and included in the cgMLST scheme. In our study, we determined VRE differed between Dutch and German hospitals since no the genetic relatedness between ESBL-E. coli using cgMLST or enrichment broth was used in Germany, however, selective media wgMLST comparison and genetic distance calculation. These agar was used in both regions. Since this study was anonymous, results were in concordance with the genetic distance thresholds some epidemiological data were not available which makes it of 0.0095 (wgMLST) and 0.0105 (cgMLST) previously established more difficult to draw conclusions regarding genetic relatedness for E. coli based on known existing epidemiological links by among isolates between patients. Frontiers in Microbiology | www.frontiersin.org 9 October 2017 | Volume 8 | Article 1914 fmicb-08-01914 October 4, 2017 Time: 12:53 # 10 Zhou et al. Epidemiology of ESBL and VRE in Hospitals and the Community (III-2-03D025), part of a Dutch–German cross-border network CONCLUSION supported by the European Commission, the German In conclusion, the results of this study suggest that Federal States of Nordrhein-Westfalen and Niedersachsen, ESBL/pAmpC-E. coli circulate in the hospital and the and the Dutch Provinces of Overijssel, Gelderland, and community, although a higher prevalence of ESBL/pAmpC- Limburg. E. coli was observed in hospitals compared to the community in the Northern Netherlands. Hospitals in the Northern Dutch–German region showed a similar prevalence of ESBL/ ACKNOWLEDGMENT pAmpC-Enterobacteriaceae. VRE prevalence was still low in the hospital as well as in the community in the Northern We would like to thank Dr. C. R. C. Doorenbos, M. Zigterman, Netherlands. The German hospital showed a slightly higher VRE W. Postma, P. Rurenga, N. Welles, A. Woudstra, S. de Vries, and prevalence compared to hospitals in the Northern Netherlands. A. J. Stellingwerf for their participation in this study. Nosocomial but no cross-border transmission of VRE was observed in this study. Epidemiologically related ESBL-E. coli and VRE were uncommon across the Northern Dutch–German SUPPLEMENTARY MATERIAL border in the studied population, as only two ESBL-E. coli isolates from a Dutch and a German hospital were genetically similar. The Supplementary Material for this article can be found Cooperation between bordering countries and continuous online at: https://www.frontiersin.org/articles/10.3389/fmicb. monitoring using high discriminatory typing methods are still 2017.01914/full#supplementary-material necessary to keep the epidemiology of resistant pathogens updated thereby helping to control their spread. FIGURE S1 | Minimum spanning tree of ESBL-E. coli isolates from hospitals and the community. Distance based on a wgMLST of 4100 genes (cgMLST of 1771 These results were partially presented at the ECCMID genes and 2329 accessory genes) using the parameters “pairwise ignoring conference 2016, Amsterdam. missing values” during calculation. Each circle represents a genotype, colors indicate geographical origin and community or hospital. Orange: hospital— Netherlands; blue: hospital—Germany; green: community—Netherlands. Number ETHICS STATEMENT of different alleles are indicated on the edges between connected isolates (nodes). The same groups considered by cgMLST analysis are highlighted. Isolates are The “METc (medical ethical committee) UMCG” believes that presented by their ID and ST. this research is not a research involving humans as is ment in TABLE S1 | Metrics on raw read data and assemblies. the Law on Medical Scientific Research involving Human Beings TABLE S2a | Finished E. coli query genomes used in this study to develop and (WMO). Therefore, the METc UMCG has decided that no WMO ad hoc cgMLST scheme (n D 45). One representative isolate of every ST from approval is needed. every collection community NL (n D 10), Dutch hospitals (n D 20) and German hospital (n D 6) of the present study and 9 E. coli genomes from Dutch patients and farmers previously published (de Been et al., 2014). AUTHOR CONTRIBUTIONS TABLE S2b | Finished plasmid genomes for exclusion of genes with BLAST matches >90% and >100 bp length found within the query sequences used in GR, DB, LM, NH, TS, LB, JW, JvZ, and RK have all contributed this study to develop a cgMLST scheme. to the collection of all samples, they have all reviewed the article TABLE S3 | Genes included in the cgMLST scheme of E. coli. carefully. XZ, SG-C, GK, JA, DB, JR, and AF have contributed to the study design and reviewed the article. XZ and SG-C wrote the TABLE S4 | Accessory genes included in the wgMLST scheme of E. coli. manuscript together and analyzed the data. JR and AF reviewed TABLE S5 | E. coli cgMLST allele types for distance calculation and percentage of the manuscript several times, before sending to the other authors good targets/genes. for review. TABLE S6 | E. faecium cgMLST allele types for distance calculation and percentage of good targets/genes. TABLE S7 | Genetic distance for pairwise comparisons of grouped ESBL- E. coli FUNDING isolates. This study was supported by the Interreg IVa-funded TABLE S8 | Results of ResFinder, VirulenceFinder, PlasmidFinder, and projects EurSafety Health-net (III-1-02D73) and SafeGuard SerotypeFinder for E. coli and E. faecium isolates. carriage: systematic review and meta-analysis. J. Antimicrob. Chemother. 71, REFERENCES 2729–2739. doi: 10.1093/jac/dkw221 Arias, C. A., and Murray, B. E. (2012). The rise of the Enterococcus: Bruijnesteijn van Coppenraet, L. E., Dullaert-de Boer, M., Ruijs, G. J., van der beyond vancomycin resistance. Nat. Rev.Microbiol. 10, 266–278. doi: 10.1038/ Reijden, W. A., van der Zanden, A. G., Weel, J. F., et al. (2015). Case- nrmicro2761 control comparison of bacterial and protozoan microorganisms associated with Bar-Yoseph, H., Hussein, K., Braun, E., and Paul, M. (2016). Natural history and gastroenteritis: application of molecular detection. Clin. Microbiol. Infect. 21, decolonization strategies for ESBL/carbapenem-resistant Enterobacteriaceae 592.e9–e19. doi: 10.1016/j.cmi.2015.02.007 Frontiers in Microbiology | www.frontiersin.org 10 October 2017 | Volume 8 | Article 1914 fmicb-08-01914 October 4, 2017 Time: 12:53 # 11 Zhou et al. Epidemiology of ESBL and VRE in Hospitals and the Community Carattoli, A., Zankari, E., Garcia-Fernandez, A., Voldby Larsen, M., Lund, O., Larsen, M. V., Cosentino, S., Lukjancenko, O., Saputra, D., Rasmussen, S., Villa, L., et al. (2014). In silico detection and typing of plasmids using Hasman, H., et al. (2014). Benchmarking of methods for genomic taxonomy. PlasmidFinder and plasmid multilocus sequence typing. Antimicrob. Agents J. Clin. Microbiol. 52, 1529–1539. doi: 10.1128/JCM.02981- 13 Chemother. 58, 3895–3903. doi: 10.1128/AAC.02412- 14 Lebreton, F., van Schaik, W., McGuire, A. M., Godfrey, P., Griggs, A., Clark, N. C., Cooksey, R. C., Hill, B. C., Swenson, J. M., and Tenover, F. C. (1993). Mazumdar, V., et al. (2013). Emergence of epidemic multidrug-resistant Characterization of glycopeptide-resistant enterococci from U.S. hospitals. Enterococcus faecium from animal and commensal strains. mBio 4:e00534-13. Antimicrob. Agents Chemother. 37, 2311–2317. doi: 10.1128/AAC.37.11. doi: 10.1128/mBio.00534- 13 2311 Leopold, S. R., Goering, R. V., Witten, A., Harmsen, D., and Mellmann, A. Clermont, O., Bonacorsi, S., and Bingen, E. (2000). Rapid and simple (2014). Bacterial whole-genome sequencing revisited: portable, scalable, and determination of the Escherichia coli phylogenetic group. Appl. Environ. standardized analysis for typing and detection of virulence and antibiotic Microbiol. 66, 4555–4558. doi: 10.1128/AEM.66.10.4555- 4558.2000 resistance genes. J. Clin. Microbiol. 52, 2365–2370. doi: 10.1128/JCM. de Been, M., Lanza, V. F., de Toro, M., Scharringa, J., Dohmen, W., Du, Y., et al. 00262-14 (2014). Dissemination of cephalosporin resistance genes between Escherichia Maiden, M. C., Jansen van Rensburg, M. J., Bray, J. E., Earle, S. G., Ford, S. A., Jolley, coli strains from farm animals and humans by specific plasmid lineages. PLOS K. A., et al. (2013). MLST revisited: the gene-by-gene approach to bacterial Genet. 10:e1004776. doi: 10.1371/journal.pgen.1004776 genomics. Nat. Rev. Microbiol. 11, 728–736. doi: 10.1038/nrmicro3093 de Been, M., Pinholt, M., Top, J., Bletz, S., Mellmann, A., van Schaik, W., Mellmann, A., Bletz, S., Boking, T., Kipp, F., Becker, K., Schultes, A., et al. et al. (2015). A core genome MLST scheme for high-resolution typing of (2016). Real-time genome sequencing of resistant bacteria provides precision Enterococcus faecium. J. Clin. Microbiol. 53, 3788–3797. doi: 10.1128/JCM. infection control in an institutional setting. J. Clin. Microbiol. 54, 2874–2881. 01946- 15 doi: 10.1128/JCM.00790- 16 de Regt, M. J., van der Wagen, L. E., Top, J., Blok, H. E., Hopmans, Muller, J., Voss, A., Kock, R., Sinha, B., Rossen, J. W., Kaase, M., et al. (2015). T. E., Dekker, A. W., et al. (2008). High acquisition and environmental Cross-border comparison of the dutch and German guidelines on multidrug- contamination rates of CC17 ampicillin-resistant Enterococcus faecium in a resistant gram-negative microorganisms. Antimicrob. Res. Infect. Control 4:7. dutch hospital. J. Antimicrob. Chemother. 62, 1401–1406. doi: 10.1093/jac/ doi: 10.1186/s13756- 015- 0047- 6 dkn390 Overdevest, I. T., Bergmans, A. M., Verweij, J. J., Vissers, J., Bax, N., Endtz, H. P., van den Braak, N., van Belkum, A., Kluytmans, J. A., Koeleman, J. G., Snelders, E., et al. (2015). Prevalence of phylogroups and O25/ST131 Spanjaard, L., et al. (1997). Fecal carriage of vancomycin-resistant enterococci in susceptible and extended-spectrum beta-lactamase-producing Escherichia in hospitalized patients and those living in the community in the Netherlands. coli isolates, the Netherlands. Clin. Microbiol. Infec. 21, 570.e1–e4. doi: J. Clin. Microbiol. 35, 3026–3031. 10.1016/j.cmi.2015.02.020 Ewers, C., Bethe, A., Semmler, T., Guenther, S., and Wieler, L. H. (2012). Extended- Overdevest, I. T., Heck, M., van der Zwaluw, K., Huijsdens, X., van Santen, M., spectrum beta-lactamase-producing and AmpC-producing Escherichia coli Rijnsburger, M., et al. (2014). Extended-spectrum beta-lactamase producing from livestock and companion animals, and their putative impact on public Klebsiella spp. in chicken meat and humans: a comparison of typing methods. health: a global perspective. Clin. Microbiol. Infect. 18, 646–655. doi: 10.1111/j. Clin. Microbiol. Infec. 20, 251–255. doi: 10.1111/1469-0691.12277 1469- 0691.2012.03850.x Pietsch, M., Eller, C., Wendt, C., Holfelder, M., Falgenhauer, L., Fruth, A., Fischer, J., Hille, K., Ruddat, I., Mellmann, A., Kock, R., and Kreienbrock, L. et al. (2015). Molecular characterisation of extended-spectrum beta-lactamase (2016). Simultaneous occurrence of MRSA and ESBL-producing (ESBL)-producing Escherichia coli isolates from hospital and ambulatory Enterobacteriaceae on pig farms and in nasal and stool samples from patients in Germany. Vet. Microbiol. 200, 130–137. doi: 10.1016/j.vetmic.2015. farmers. Vet. Microbiol. 200, 107–113. doi: 10.1016/j.vetmic.2016. 11.028 05.021 Pinholt, M., Larner-Svensson, H., Littauer, P., Moser, C. E., Pedersen, M., Galloway-Pena, J., Roh, J. H., Latorre, M., Qin, X., and Murray, B. E. (2012). Lemming, L. E., et al. (2015). Multiple hospital outbreaks of vanA Enterococcus Genomic and SNP analyses demonstrate a distant separation of the hospital and faecium in Denmark, 2012-13, investigated by WGS, MLST and PFGE. community-associated clades of Enterococcus faecium. PLOS ONE 7:e30187. J. Antimicrob. Chemother. 70, 2474–2482. doi: 10.1093/jac/dkv142 doi: 10.1371/journal.pone.0030187 Reuland, E. A., Halaby, T., Hays, J. P., de Jongh, D. M., Snetselaar, H. D., van Garcia-Cobos, S., Kock, R., Mellmann, A., Frenzel, J., Friedrich, A. W., and Keulen, M., et al. (2015). Plasmid-mediated AmpC: prevalence in community- Rossen, J. W. (2015). Molecular typing of Enterobacteriaceae from pig acquired isolates in Amsterdam, the Netherlands, and risk factors for carriage. holdings in north-western Germany reveals extended- spectrum and AmpC PLOS ONE 10:e0113033. doi: 10.1371/journal.pone.0113033 beta-lactamases producing but no carbapenem resistant ones. PLOS ONE Reuland, E. A., Overdevest, I. T., Al Naiemi, N., Kalpoe, J. S., Rijnsburger, 10:e0134533. doi: 10.1371/journal.pone.0134533 M. C., Raadsen, S. A., et al. (2013). High prevalence of ESBL-producing Hasman, H., Saputra, D., Sicheritz-Ponten, T., Lund, O., Svendsen, C. A., Frimodt- Enterobacteriaceae carriage in dutch community patients with gastrointestinal Moller, N., et al. (2014). Rapid whole-genome sequencing for detection and complaints. Clin. Microbiol. Infect. 19, 542–549. doi: 10.1111/j.1469- 0691.2012. characterization of microorganisms directly from clinical samples. J. Clin. 03947.x Microbiol. 52, 139–146. doi: 10.1128/JCM.02452- 13 Torell, E., Cars, O., Olsson-Liljequist, B., Hoffman, B. M., Lindback, J., and Joensen, K. G., Scheutz, F., Lund, O., Hasman, H., Kaas, R. S., Nielsen, E. M., et al. Burman, L. G. (1999). Near absence of vancomycin-resistant enterococci (2014). Real-time whole-genome sequencing for routine typing, surveillance, but high carriage rates of quinolone-resistant ampicillin-resistant enterococci and outbreak detection of verotoxigenic Escherichia coli. J. Clin. Microbiol. 52, among hospitalized patients and nonhospitalized individuals in Sweden. J. Clin. 1501–1510. doi: 10.1128/JCM.03617- 13 Microbiol. 37, 3509–3513. Joensen, K. G., Tetzschner, A. M., Iguchi, A., Aarestrup, F. M., and Scheutz, F. van den Braak, N., Ott, A., van Belkum, A., Kluytmans, J. A., Koeleman, (2015). Rapid and easy in silico serotyping of Escherichia coli isolates by J. G., Spanjaard, L., et al. (2000). Prevalence and determinants of fecal use of whole-genome sequencing data. J. Clin. Microbiol. 53, 2410–2426. colonization with vancomycin-resistant Enterococcus in hospitalized patients doi: 10.1128/JCM.00008- 15 in the Netherlands. Infect. Control Hosp. Epidemiol. 21, 520–524. doi: 10.1086/ Kluytmans-van den Bergh, M. F., Rossen, J. W., Bruijning-Verhagen, P. C., Bonten, 501797 M. J., Friedrich, A. W., Vandenbroucke-Grauls, C. M., et al. (2016). Whole van den Brink, R. (2013). Het Einde van de Antibiotica. Hoe Bacteriën Winnen van genome multilocus sequence typing of extended-spectrum beta-lactamase- een Wondermiddel. Breda: Uitgeverij De Geus B.V. producing Enterobacteriaceae. J. Clin. Microbiol. 54, 2919–2927. doi: 10.1128/ van der Bij, A. K., Peirano, G., Goessens, W. H., van der Vorm, E. R., JCM.01648- 16 van Westreenen, M., and Pitout, J. D. (2011). Clinical and molecular Kwong, J. C., McCallum, N., Sintchenko, V., and Howden, B. P. (2015). Whole characteristics of extended-spectrum-beta-lactamase-producing Escherichia genome sequencing in clinical and public health microbiology. Pathology 47, coli causing bacteremia in the rotterdam area, Netherlands. Antimicrob. Agents 199–210. doi: 10.1097/PAT.0000000000000235 Chemother. 55, 3576–3578. doi: 10.1128/AAC.00074- 11 Frontiers in Microbiology | www.frontiersin.org 11 October 2017 | Volume 8 | Article 1914 fmicb-08-01914 October 4, 2017 Time: 12:53 # 12 Zhou et al. Epidemiology of ESBL and VRE in Hospitals and the Community van Hoek, A. H., Schouls, L., van Santen, M. G., Florijn, A., de Greeff, S. C., Zhou, X., Arends, J. P., Span, L. F., and Friedrich, A. W. (2013). and van Duijkeren, E. (2015). Molecular characteristics of extended-spectrum Algorithm for pre-emptive glycopeptide treatment in patients with cephalosporin-resistant Enterobacteriaceae from humans in the community. haematologic malignancies and an Enterococcus faecium bloodstream PLOS ONE 10:e0129085. doi: 10.1371/journal.pone.0129085 infection. Antimicrob. Resist. Infect. Control 2:24. doi: 10.1186/2047-2994- Werner, G., Fleige, C., Geringer, U., van Schaik, W., Klare, I., and Witte, W. (2011). 2-24 IS element IS16 as a molecular screening tool to identify hospital-associated strains of Enterococcus faecium. BMC Infect. Dis. 11:80. doi: 10.1186/1471- Conflict of Interest Statement: The authors declare that the research was 2334- 11- 80 conducted in the absence of any commercial or financial relationships that could Wirth, T., Falush, D., Lan, R., Colles, F., Mensa, P., Wieler, L. H., et al. (2006). Sex be construed as a potential conflict of interest. and virulence in Escherichia coli: an evolutionary perspective. Mol. Microbiol. 60, 1136–1151.. doi: 10.1111/j.1365- 2958.2006.05172.x Copyright © 2017 Zhou, García-Cobos, Ruijs, Kampinga, Arends, Borst, Möller, Woerther, P. L., Burdet, C., Chachaty, E., and Andremont, A. (2013). Trends in Holman, Schuurs, Bruijnesteijn van Coppenraet, Weel, van Zeijl, Köck, Rossen and human fecal carriage of extended-spectrum beta-lactamases in the community: Friedrich. This is an open-access article distributed under the terms of the Creative toward the globalization of CTX-M. Clin. Microbiol. Rev. 26, 744–758. Commons Attribution License (CC BY). The use, distribution or reproduction in doi: 10.1128/CMR.00023- 13 other forums is permitted, provided the original author(s) or licensor are credited Zankari, E., Hasman, H., Cosentino, S., Vestergaard, M., Rasmussen, S., Lund, O., and that the original publication in this journal is cited, in accordance with accepted et al. (2012). Identification of acquired antimicrobial resistance genes. academic practice. No use, distribution or reproduction is permitted which does not J. Antimicrob. Chemother. 67, 2640–2644. doi: 10.1093/jac/dks261 comply with these terms. Frontiers in Microbiology | www.frontiersin.org 12 October 2017 | Volume 8 | Article 1914 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Frontiers in Microbiology Pubmed Central

Epidemiology of Extended-Spectrum β-Lactamase-Producing E. coli and Vancomycin-Resistant Enterococci in the Northern Dutch–German Cross-Border Region

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Copyright © 2017 Zhou, García-Cobos, Ruijs, Kampinga, Arends, Borst, Möller, Holman, Schuurs, Bruijnesteijn van Coppenraet, Weel, van Zeijl, Köck, Rossen and Friedrich.
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Abstract

fmicb-08-01914 October 4, 2017 Time: 12:53 # 1 ORIGINAL RESEARCH published: 05 October 2017 doi: 10.3389/fmicb.2017.01914 Epidemiology of Extended-Spectrum b-Lactamase-Producing E. coli and Vancomycin-Resistant Enterococci in the Northern Dutch–German Cross-Border Region Edited by: John R. Battista, Louisiana State University, 1 † 1 † 2 1 Xuewei Zhou * , Silvia García-Cobos * , Gijs J. H. M. Ruijs , Greetje A. Kampinga , United States 1 1 3 4 5 Jan P. Arends , Dirk M. Borst , Lieke V. Möller , Nicole D. Holman , Theo A. Schuurs , 2 5 5 6,7 Reviewed by: Lesla E. Bruijnesteijn van Coppenraet , Jan F. Weel , Jan H. van Zeijl , Robin Köck , 1† 1† Zhuofei Xu, John W. A. Rossen and Alexander W. Friedrich Huazhong Agricultural University, University of Groningen, University Medical Center Groningen, Department of Medical Microbiology, Groningen, China 2 3 Netherlands, Laboratory of Medical Microbiology and Infectious Diseases, Isala Clinics, Zwolle, Netherlands, Department Jose M. Munita, of Microbiology, CERTE Medische Diagnostiek en Advies, Groningen, Netherlands, Department of Intensive Care Medicine, University of Texas Health Science Martini Hospital, Groningen, Netherlands, Centre for Infectious Diseases Friesland, Izore, Leeuwarden, Netherlands, Center at Houston, United States 6 7 Institute of Medical Microbiology, University Hospital Münster, Münster, Germany, Institute of Hospital Hygiene, Klinikum *Correspondence: Oldenburg, Oldenburg, Germany Xuewei Zhou x.w.zhou@umcg.nl Silvia García-Cobos Objectives: To reveal the prevalence and epidemiology of extended-spectrum s.garcia.cobos@umcg.nl b-lactamase (ESBL)- and/or plasmid AmpC (pAmpC)- and carbapenemase (CP) These authors have contributed producing Enterobacteriaceae and vancomycin-resistant enterococci (VRE) across the equally to this work. Northern Dutch–German border region. Specialty section: Methods: A point-prevalence study on ESBL/pAmpC/CP producing This article was submitted to Evolutionary and Genomic Enterobacteriaceae and VRE was carried out in hospitalized patients in the Northern Microbiology, Netherlands (n D 445, 2012–2013) and Germany (n D 242, 2012). Healthy individuals a section of the journal from the Dutch community (n D 400, 2010–2012) were also screened. In addition, Frontiers in Microbiology a genome-wide gene-by-gene approach was applied to study the epidemiology of Received: 24 April 2017 Accepted: 20 September 2017 ESBL-Escherichia coli and VRE. Published: 05 October 2017 Results: A total of 34 isolates from 27 patients (6.1%) admitted to Dutch hospitals Citation: Zhou X, García-Cobos S, were ESBL/pAmpC positive and 29 ESBL-E. coli, three pAmpC-E. coli, one Ruijs GJHM, Kampinga GA, ESBL-Enterobacter cloacae, and one pAmpC-Proteus mirabilis were found. In the Arends JP, Borst DM, Möller LV, German hospital, 18 isolates (16 E. coli and 2 Klebsiella pneumoniae) from 17 patients Holman ND, Schuurs TA, Bruijnesteijn van Coppenraet LE, (7.7%) were ESBL positive. In isolates from the hospitalized patients CTX-M-15 was the Weel JF, van Zeijl JH, Köck R, most frequently detected ESBL-gene. In the Dutch community, 11 individuals (2.75%) Rossen JWA and Friedrich AW (2017) Epidemiology were ESBL/pAmpC positive: 10 ESBL-E. coli (CTX-M-1 being the most prevalent gene) of Extended-Spectrum and one pAmpC E. coli. Six Dutch (1.3%) and four German (3.9%) hospitalized patients -Lactamase-Producing E. coli were colonized with VRE. Genetic relatedness by core genome multi-locus sequence and Vancomycin-Resistant Enterococci in the Northern typing (cgMLST) was found between two ESBL-E. coli isolates from Dutch and German Dutch–German Cross-Border Region. cross-border hospitals and between VRE isolates from different hospitals within the Front. Microbiol. 8:1914. doi: 10.3389/fmicb.2017.01914 same region. Frontiers in Microbiology | www.frontiersin.org 1 October 2017 | Volume 8 | Article 1914 fmicb-08-01914 October 4, 2017 Time: 12:53 # 2 Zhou et al. Epidemiology of ESBL and VRE in Hospitals and the Community Conclusion: The prevalence of ESBL/pAmpC-Enterobacteriaceae was similar in hospitalized patients across the Dutch–German border region, whereas VRE prevalence was slightly higher on the German side. The overall prevalence of the studied pathogens was lower in the community than in hospitals in the Northern Netherlands. Cross- border transmission of ESBL-E. coli and VRE seems unlikely based on cgMLST analysis, however continuous monitoring is necessary to control their spread and stay informed about their epidemiology. Keywords: WGS, cgMLST, VRE, ESBL, hospital, community, prevalence, cross-border research to study hospital and cross-border dissemination of ESBL-E. coli INTRODUCTION and VRE. International travel and patient care are risk factors for dissemination of bacteria including multidrug-resistant MATERIALS AND METHODS microorganisms (MDRO), such as extended-spectrum b-lactamase (ESBL) and carbapenemase (CP)-producing Study Design Enterobacteriaceae (Woerther et al., 2013; Muller et al., A prospective point prevalence study was conducted in four 2015), and vancomycin-resistant enterococci (VRE). The of the largest hospitals (in total 3550 beds) in the Northern prevalence of the latter has increased in the last years due to Netherlands between November 2012 and February 2013, successful polyclonal subpopulations of hospital-associated (HA) covering a total population of approximately 2.85 million people. Enterococcus faecium [previously designated clonal complex The Hospital Ethical Committee of the University Medical (CC) CC17] and which are also associated with amoxicillin Center Groningen (UMCG) was informed and patients were resistance (ARE) (Arias and Murray, 2012). These populations approached to voluntarily participate in the study. Patients are distinct from E. faecium isolates in the community and included in this study provided their written informed consent isolates from non-human sources (Galloway-Pena et al., 2012; and a questionnaire concerning epidemiological and clinical Lebreton et al., 2013). data. The following high-risk wards for antibiotic-resistant Netherlands and Germany as bordering countries with microorganisms were selected: intensive care units (ICU), possible transfer of patients between them, created a vascular surgery, internal medicine hematology/oncology, and cooperative network to prevent the spread of MDRO and dialysis wards (both for in- and outpatients). Gynecology and to harmonize guidelines in healthcare settings (Muller neurology (low-risk wards) were also included for comparison. et al., 2015). Surveillance studies to monitor the prevalence, From the largest German university hospital in the same resistance patterns, and molecular background of MDRO (border) region, patients from four ICUs, a surgical ward, and in hospitals and the community are essential to get insights a hematology/oncology ward were screened during October into their epidemiology to implement infection prevention and November 2012 and included in the study. After consent measures. Bacterial whole-genome sequencing (WGS) has agreement, all admitted patients from the studied wards were been demonstrated to be very useful for epidemiological screened until completing a minimum of 100 samples per surveillance and detection of antimicrobial resistance (Kwong hospital. et al., 2015). The gene-by-gene approach uses a defined set The study in healthy people living in the Northern of genes to extract an allele-based profile which makes it Netherlands was conducted retrospectively, using control scalable and portable between laboratories (Maiden et al., 2013; patients included in a previous case–control study on Leopold et al., 2014). A core genome multi-locus sequence microorganisms causing gastroenteritis. Control subjects typing (cgMLST) scheme has been developed for E. faecium were patients attending their general practitioner for a variety to distinguish between epidemiologically related and unrelated of medical questions, but no gastrointestinal problems, in the isolates (de Been et al., 2015). Although there is no cgMLST period between August 2010 and December 2012 (Bruijnesteijn scheme nor threshold publicly approved yet for Escherichia coli, van Coppenraet et al., 2015). No prevalence study was performed there are several tools available that allow to define an ad hoc in the community in Germany. cgMLST. The aim of this study was to perform a point-prevalence study Sample Collection on ESBL/plasmid-mediated AmpC (pAmpC)/CP-producing TM A total of 445 rectal swabs (Copan ESwab ) were taken from Enterobacteriaceae and HA E. faecium (VRE and ARE) in hospitalized patients (median ageD 66 years, range 18–99 years) hospitals in the Northern Dutch–German border region and in the Northern Netherlands, 51.7% (n D 230) from men and to determine the predominant resistance genes. In addition, 48.3% (n D 215) from women. A total of 328 (73.7%) patients stool community samples from the Northern Netherlands were were screened at high-risk wards and 117 (26.3%) patients were screened for the same resistant pathogens. A cgMLST was used screened at low-risk wards (Table 1). In the German university https://www.Deutschland- Nederland.Eu hospital, 242 patients (median ageD 64 years, range 0–94 years) Frontiers in Microbiology | www.frontiersin.org 2 October 2017 | Volume 8 | Article 1914 fmicb-08-01914 October 4, 2017 Time: 12:53 # 3 Zhou et al. Epidemiology of ESBL and VRE in Hospitals and the Community were included, 64.5% (nD 156) men and 35.5% (nD 86) women. German Hospital Of these 242 patients, 140 were screened only for ESBL, 22 only Rectal swabs were directly plated on chromID ESBL agar TM for VRE, and 80 for both. From the Dutch community study, 400 (bioMérieux) for ESBL screening and enriched Enterococcosel frozen faeces samples were included; 41% (n D 164) from men Broth (Bile Esculin Azide Broth) (BD; Becton, Dickinson and and 59% (nD 236) from women, 12% of the samples were from Company) was used for VRE screening and subsequently children. The median age of the healthy individuals was 47.5 years cultured on chromID VRE agar (bioMérieux). (range 0–84 years). Species identification and antibiotic susceptibility testing was done by MALDI-TOF MS (Bruker Daltonik GmbH, Bremen) and VITEK 2 (bioMérieux), respectively, following EUCAST criteria. Microbiological Detection, Identification, Confirmation of ESBL was performed using disk diffusion and Susceptibility Testing (cefotaxime 30 mg, cefotaxime 30 mg plus clavulanic acid 10 mg, Dutch Hospitals and Retrospective Dutch Community ceftazidime 30 mg, ceftazidime plus clavulanic acid 10 mg, Study cefepime 30 mg, cefepime 30 mg plus clavulanic acid 10 mg, and Rectal swabs (Dutch hospitalized patients) and approximately cefoxitin 30 mg) (Mast Diagnostics, Bootle, UK). 50 mg of feces per sample (Dutch community patients) were enriched in selective broths: VRA broth containing BHI PCRs and Microarray (brain heart infusion) with 20 mg/L amphoterin-B, 20 mg/L Enterococci isolates from Netherlands were screened by in-house aztreonam, 20 mg/L colistin, and 16 mg/L amoxicillin and PCR for IS16 (a marker for specific hospital-associated strains), TSB-VC broth containing tryptic soy broth with 8 mg/L vanA and vanB genes as described previously (Clark et al., vancomycin and 0.25 mg/L cefotaxime. Both broths were 1993; Werner et al., 2011). The GenoType Enterococcus (Hain incubated for 24 h at 35  1 C. Subsequently, 10 mL Lifescience GmbH) was used in enterococci isolates from of VRA broth was subcultured on VRE Brilliance agar R Germany, which detects species and genotypes vanA, vanB, (Oxoid ) and BMEG-2 agar (blood agar containing 64 mg/L vanC1, and vanC2. ESBL and VRE positive isolates were sent to meropenem, 2 mg/L gentamicin, 10 mg/L oxacillin, and our hospital for further characterization. 20 mg/L amphotericin-B) for identification of VRE and all ARE, Enterobacteriaceae isolates resistant to third generation respectively. Ten microliters of TSB-VC broth was subcultured cephalosporins and natural chromosomal AmpC producers onto ME/CF/CX comparted plates, containing iso-sensitest agar intermediate or resistant to cefepime were selected for DNA with 1 mg/L meropenem, 1 mg/L ceftazidime, or 1 mg/L extraction using the UltraClean Microbial DNA Isolation Kit (Mo cefotaxime, respectively, plus 20 mg/L vancomycin and 20 mg/L Bio Laboratories, Inc.) and further characterized for the presence amphotericin-B (Mediaproducts, Groningen), for selection of of ESBL/AmpC genes using a DNA-array (Check-MDR CT103, ESBL/pAmpC/CP-producing bacteria. Plates were incubated for Check-points, Wageningen, Netherlands) (Garcia-Cobos et al., 24 h at 35 1 C, except for VRE Brilliance agar plates that were 2015). incubated for 48 h. Suspected colonies on VRE Brilliance, BMEG-2 and Whole-Genome Sequencing of VRE and ME/CF/CX agar plates were streaked on blood agar ESBL-E. coli (one isolate per morphotype). Species identification was done by matrix-assisted laser desorption/ionization-time WGS was performed for all ESBL-E. coli and VRE isolates. of flight mass spectrometry (MALDI-TOF MS) (Bruker For each isolate, several colonies (about 5 mL) of the culture Daltonik GmbH, Bremen). Confirmed Enterococcus spp. and were suspended in 300 mL microbead solution, which was Enterobacteriaceae spp., were tested for antibiotic susceptibility subjected to DNA extraction with the Ultraclean Microbial DNA using VITEK 2 (bioMérieux) automatic system and EUCAST isolation kit (Mo Bio Laboratories, Carlsbad, CA, United States). clinical breakpoints. The DNA concentration and purity were measured using a TABLE 1 | Distribution of ESBL/pAmpC producing Enterobacteriaceae, and amoxicillin- and vancomycin-resistant E. faecium among the different wards in Dutch hospitals. Ward ESBL/pAmpC producing Amoxicillin-resistant Vancomycin-resistant Enterobacteriaceae E. faecium E. faecium High-risk (n D 328) 19 .5:8%/ 99 .30:2%/ 6 .1:8%/ Intensive care unit (n D 102) 6 .5:9%/ 31 .30:4%/ 1 .1%/ Vascular surgery (n D 54) 6 .11:1%/ 15 .27:8%/ 1 .1%/ Internal medicine hematology/oncology (n D 81) 1 .1:2%/ 36 .44:4%/ 2 .2:5%/ Dialysis (n D 91) 6 .6:6%/ 17 .18:7%/ 2 .2:2%/ Low-risk (n D 117) 8 .6:8%/ 6 .5:1%/ 0 .0%/ Gynecology (n D 55) 3 .5:5%/ 1 .1:8%/ 0 .0%/ Neurology (n D 62) 5 .8:1%/ 5 .8:1%/ 0 .0%/ Total (n D 445) 27 .6:1%/ 105 .23:6%/ 6 .1:3%/ Frontiers in Microbiology | www.frontiersin.org 3 October 2017 | Volume 8 | Article 1914 fmicb-08-01914 October 4, 2017 Time: 12:53 # 4 Zhou et al. Epidemiology of ESBL and VRE in Hospitals and the Community NanoDrop 2000c spectrophotometer (Thermo Fisher Scientific, (Supplementary Table S2b) were added to exclude such genes Waltham, MA, United States) and the Qubit double-stranded are part of the cgMLST typing scheme. The final cgMLST scheme DNA (dsDNA) HS and BR assay kits (Life Technologies, consisted of 1.771 targets/genes, and 2329 accessory genes were Carlsbad, CA, United States). One nanogram of bacterial DNA additionally included for the wgMLST scheme (Supplementary was used for library preparation. The DNA library was prepared Tables S3, S4). The minimum coverage of the genome assemblies using the Nextera XT library preparation kit with the Nextera was 20 times (Supplementary Table S1) and the percentage of XT v2 index kit (Illumina, San Diego, CA, United States). The good targets/genes included in the cgMLST were 97.6% for E. coli library fragment length was aimed at fragments with a median and 98.6% for E. faecium (Supplementary Tables S5, S6). size of 575 bases and was assessed with the Genomic DNA Furthermore, to determine the genetic relatedness, the genetic ScreenTape assay with the 2200 TapeStation system (Agilent distance for the E. coli isolates was calculated as the proportion Technologies, Waldbronn, Germany). Subsequently, the library of allele differences: dividing the number of allele differences was sequenced on a MiSeq sequencer, using the MiSeq reagent between two genomes by the total number of genes commonly kit v2 generating 250-bp paired-end reads. Sequencing was aimed shared by those two genomes (Kluytmans-van den Bergh et al., at a coverage of at least 60-fold. MiSeq data were processed 2016). In this study, thresholds for genetic distance were with MiSeq control software v2.4.0.4 and MiSeq Reporter v2.4 described to discriminate between epidemiologically related and (Illumina, San Diego, CA, United States). Reads were quality- unrelated E. coli isolates as 0.0095 when using wgMLST and trimmed using the CLC Genomics Workbench software version 0.0105 for cgMLST. 9.0.1 (CLC bio, Aarhus, Denmark) using default settings except Escherichia coli STs were determined uploading genome for the following modifications: “trim using quality scores was assemblies to SeqSphereC software following the scheme of set to 0.02” and “discard reads below length was set to 15.” Wirth et al. (2006). Sequence genomes with no conclusive results Subsequently, trimmed-reads were de novo assembled with an for the 7-gene MLST were uploaded to the Enterobase database . optimal word size of 29 and a minimum contig length of Additionally, E. coli major phylogenetic groups (A, B1, B2, and D) 500. Metrics on raw read and assembly level are provided in were analyzed in silico by using MLST Target Definer function Supplementary Table S1. of SeqSphere , including the chuA, yjaA, and TSPE4.C2 loci (Clermont et al., 2000). Genome assemblies were also uploaded to the Center for cgMLST of VRE and ESBL-E. coli Genomic Epidemiology to extract information on resistance A genome-wide gene-by-gene comparison approach was used to genes (ResFinder) and virulence factors (VirulenceFinder), and determine the genetic relatedness using SeqSphere version 3.4.0 species confirmation for VRE and ESBL-E. coli (KmerFinder), (Ridom GmbH, Münster, Germany) (Leopold et al., 2014). and serotype (SerotypeFinder) and plasmid replicons Genome assemblies from the VRE isolates were analyzed using (PlasmidFinder) for ESBL-E. coli (Zankari et al., 2012; Carattoli the E. faecium cgMLST scheme previously published, considering et al., 2014; Hasman et al., 2014; Joensen et al., 2014, 2015; Larsen a cluster alert distance of 20 different alleles (de Been et al., 2015). et al., 2014). An ad hoc cgMLST and whole-genome MLST (wgMLST) scheme was determined for E. coli isolates using the MLST Statistical Analysis target definer function with default parameters and E. coli K-12 In the Dutch hospital prevalence study, associations between as a reference (GenBank accession no. NC_010473.1). The filters ESBL and ARE carriage and the following variables were applied to reference genome were: “minimum length filter” that analyzed: length of hospital stay, antibiotic use, and (low- or discards genes shorter than 50 bases; “start codon filter” that high-risk) ward. Information was gathered by the questionnaires. discards all genes that contain no start codon at the beginning Statistical analyses were performed using SPSS for Windows, v. of the gene: “stop codon filter” that discards all genes that contain 20.0. Univariate analyses were performed using the Fisher’s exact no stop codon, more than one stop codon or if the stop codon or Chi-square methods for categorical variables. The Mann– is not at the end of the gene: “homologous gene filter” that Whitney U -test was used as a non-parametric tests in variables discards all genes that have fragments that occur in multiple with no normal distribution. Results with a p-value of 0.05 copies in a genome (with identity 90% and more than 100 were considered to be statistically significant. All p-values are bases overlap); “gene overlap filter” that discards the shorter of two-tailed. two overlapping flanking genes if these genes overlap >4 bp. The remaining genes were then used in a pairwise comparison using BLAST (Leopold et al., 2014) with 45 query genomes RESULTS (Supplementary Table S2a). All genes of the reference genome that were common in all query genomes with a sequence identity ESBL/pAmpC-Producing of 90 and 100 overlap, and with the default parameter stop codon percentage filter turned on, formed the final cgMLST Enterobacteriaceae scheme; this discards all genes that have internal stop codons in Thirty-four isolates from 27 of the 445 included patients admitted >20% of the query genomes. Additionally, 26 plasmid sequences to hospitals in the Northern Netherlands (6.1%) were confirmed ESBL and/or pAmpC positive. A total of 85.2% (23/27), 14.8% http://www.ridom.de/seqsphere/ug/v20/Tutorial_for_MLST+_Target_Definer. html https://enterobase.Warwick.Ac.Uk Frontiers in Microbiology | www.frontiersin.org 4 October 2017 | Volume 8 | Article 1914 fmicb-08-01914 October 4, 2017 Time: 12:53 # 5 Zhou et al. Epidemiology of ESBL and VRE in Hospitals and the Community (4/27), and 3.7% (1/27) of these patients were positive for ESBL, (n D 12)] of this study were analyzed by a gene-by-gene pAmpC, and both, respectively. Among the 34 isolates, 32 were approach and the allelic distance from the cgMLST and wgMLST E. coli, of which 29 were ESBL positive and three were pAmpC were visualized in a minimum spanning tree (Figure 1 and producers. Resistance genes detected in the E. coli isolates are Supplementary Figure S1, respectively). shown in Table 2. CTX-M-15 (n D 8) and CTX-M-14 (n D 8) Six groups of isolates with a lower number of different were the most prevalent ones. The other two isolates were alleles (35) by cgMLST were further analyzed. Supplementary an Enterobacter cloacae, containing a CTX-M-1-like gene and Table S7 summarizes the origin of the isolates in every group a pAmpC CMY-II producing Proteus mirabilis. At high-risk and the core and whole-genome genetic distance. Those groups wards, 19 patients (5.8%) were found with ESBL/pAmpC isolates formed by isolates with an epidemiological link (isolated from compared to eight patients (6.8%) at low-risk wards (p D 0.68; the same patient; group 1, 4, 5a, 6a, and 7), showed a core and NS). No association was found between ESBL/pAmpC carriage whole-genome genetic distance lower than 0.0030 and 0.0046, and antibiotic use, length of hospital stay or ward (Table 1). respectively. In addition, isolates of group 5b, although with In the German hospital, a total of 18 isolates from 17 patients unknown epidemiological link, had a core genetic distance of (17/220; 7.7%) were ESBL positive. Sixteen isolates were E. coli 0.0063 and a whole-genome genetic distance of 0.0076. Both and two were Klebsiella pneumoniae. Of these, 12 E. coli and one isolates were positive for CTX-M-14, however, no plasmid K. pneumoniae isolates were available for molecular testing. Six replicons were found in one of them (isolate 38_Esco_HA-DE) out of 12 (50%) E. coli isolates and the K. pneumoniae isolate had (Supplementary Table S7). a CTX-M-15 gene (Table 2). Among those groups including isolates with non In the retrospective Dutch community study, 11 patients (or unknown) epidemiological link, the core genome genetic (11/400; 2.75%) were ESBL/pAmpC positive: 10 ESBL-E. coli distance was between 0.0122 and 0.0199 and the whole-genome (CTX-M-1 being the most prevalent gene) and one pAmpC genetic distance was between 0.0104 and 0.0208 (groups 2, 3, 6b, E. coli (Table 2). Overall, no carbapenem resistance was observed and 6c; Figure 1). Resistance and virulence profiles of the isolates neither in the community nor in the hospitals. are shown in Supplementary Table S8. Escherichia coli MLST and Phylogenetic cgMLST Comparison of VRE Isolates Groups from the Community and Hospitals Among ESBL/pAmpC-E. coli isolates from Dutch hospitals, the A minimum spanning tree was created for the 11 VRE isolates most prevalent ST was ST131 (CC, ST131; n D 5, 15.6%), all [Dutch community (nD 1), Dutch hospitals (nD 6), and German of them belonging to phylogroup B2 (Table 2). In the Dutch hospital (nD 4)]. Two clusters of isolates from different patients community isolates, 10 different STs were found, most of them were observed (Figure 2). One cluster of four vanB-VRE isolates belonging to CC ST10 (n D 3, 27.3%) and one isolate to ST131 from the Dutch hospital belonged to cluster type (CT) 110 (ST17); (phylogroup B2). In the German hospital, the most prevalent STs two isolates were from the same ward in hospital A and the other were ST38 (33.3%) and ST10 (33.3%) (Table 2). two isolates were isolated from different wards in hospital B. The other cluster of two vanA-VRE isolates were isolated from Amoxicillin- and Vancomycin-Resistant different wards from the German hospital (CT 20, ST203). The resistance and virulence genotypes of VRE isolates are shown in E. faecium (ARE and VRE) Supplementary Table S8. In the Dutch hospitals, 105 patients (105/445; 23.6%) were colonized with ARE, including six patients (6/445; 1.3%) with Nucleotide Sequence Accession Number VRE. All ARE were positive for IS16 and all VRE were Sequence data obtained in this study has been deposited at the vanB positive. Colonization of ARE (and VRE) was associated National Center for Biotechnology Information under BioProject with high-risk wards (p < 0.001), prolonged hospitalization no. PRJNA352198. (p < 0.001), and use of antibiotics (pD 0.05), especially penicillins and fluoroquinolones (p < 0.001) (Table 3). In the border German university hospital four (4/102; 3.9%) DISCUSSION VRE isolates were isolated. Three of them were vanA positive and one was vanB positive. This study shows the molecular epidemiology of ESBL/pAmpC- In the retrospective Dutch community study, six ARE (6/400; E. coli and HA E. faecium in hospitals in the Northern 1.5%) were found, three of them were IS16 positive. Only one Dutch–German border region and the community in the vanA-VRE (1/400; 0.25%) was found, this strain was amoxicillin Northern Netherlands. Dutch hospitals showed a prevalence susceptible and IS16 negative. for ESBL/pAmpC – Enterobacteriaceae, VRE, and ARE of 6.1, 1.3, and 23.6%, respectively, whereas the prevalence in the cgMLST and wgMLST Comparison of community was 2.75, 0.25, and 1.5%, respectively. The German ESBL-E. coli Isolates from the hospital had an ESBL/pAmpC prevalence of 7.7 and 3.9% for Community and Hospitals VRE. Genome assemblies of 55 ESBL-E. coli [Dutch community A previous study reported a prevalence of ESBL-producing (n D 11), Dutch hospitals (n D 32), and German hospital bacteria of 4.9% in the Netherlands (Overdevest et al., 2014), Frontiers in Microbiology | www.frontiersin.org 5 October 2017 | Volume 8 | Article 1914 fmicb-08-01914 October 4, 2017 Time: 12:53 # 6 Zhou et al. Epidemiology of ESBL and VRE in Hospitals and the Community TABLE 2 | Molecular characterization of the E. coli isolates from the community and hospital patients in the Netherlands and Germany. Sample Hospital/ward b-Lactamase genes Phylogroup ST CC Community 1_Esco_CA-NL blaCTX-M-1, blaTEM-1B B2 131 ST131 2_Esco_CA-NL blaSHV-12 B2 117 None 3_Esco_CA-NL blaCMY-2 D 2309 None 4_Esco_CA-NL blaCTX-M-1 D 57 ST350 5_Esco_CA-NL blaCTX-M-1, blaTEM-1B A 10 ST10 6_Esco_CA-NL blaCTX-M-1, blaTEM-1B B1 1079 None 7_Esco_CA-NL blaCTX-M-1, blaTEM-1B A 10 ST10 8_Esco_CA-NL blaCTX-M-15 D 648 ST648 9_Esco_CA-NL blaCTX-M-15 A 617 ST10 10_Esco_CA-NL blaCTX-M-15 A 1312 None 11_Esco_CA-NL blaCTX-M-14b, blaTEM-1B D 38 ST38 Hospital 12_Esco_HA-NL A/Gynecology blaCTX-M-15, blaTEM-1B D 5463 None 12b_Esco_HA-NL A/Gynecology blaCTX-M-15, blaTEM-1B D 5463 None 13_Esco_HA-NL A/Neurology blaCTX-M-27 B2 131 ST131 14_Esco_HA-NL A/Dialysis outpatient blaCTX-M-15, blaTEM-1B A 93 ST168 15_Esco_HA-NL A/ICU blaCMY-2, blaTEM-1B D 354 ST354 16_Esco_HA-NL A/ICU blaCTX-M-15, blaTEM-1B, blaOXA-1 B1 58 ST155 17_Esco_HA-NL A/ICU blaCTX-M-15, blaTEM-1B B1 38 ST38 18_Esco_HA-NL A/ICU blaTEM-52C B1 453 ST86 19_Esco_HA-NL A/ICU blaCTX-M-1 B1 641 ST86 20_Esco_HA-NL A/ICU blaSHV-12 A 5888 None 20b_Esco_HA-NL A/ICU blaCTX-M-1 B1 58 ST155 21_Esco_HA-NL B/Gynecology blaCTX-M-14 B1 101 ST101 22_Esco_HA-NL B/Dialysis outpatient blaCTX-M-14 B1 38 ST38 22c_Esco_HA-NL B/Dialysis outpatient blaCTX-M-14 D 38 ST38 23_Esco_HA-NL B/Vascular surgery blaCMY-2, blaTEM-1B D 1508 None 24_Esco_HA-NL B/Neurology blaTEM-52C D 2064 None 25_Esco_HA-NL B/Neurology blaCTX-M-3, blaTEM-1B B2 95 ST95 25b_Esco_HA-NL B/Neurology blaCTX-M-3, blaTEM-1B D 95 ST95 26_Esco_HA-NL C/Gynecology blaCTX-M-15, blaOXA-1 B2 131 ST131 27_Esco_HA-NL C/Dialysis outpatient blaCTX-M-1, blaTEM-33 A 3478 None 28_Esco_HA-NL C/Dialysis outpatient blaCTX-M-14 A 10 ST10 29_Esco_HA-NL C/Neurology blaCTX-M-1 B1 603 None 30_Esco_HA-NL C/Vascular surgery blaCTX-M-14 A 410 ST23 31_Esco_HA-NL D/Vascular surgery blaCTX-M-14, blaTEM-1B, blaOXA-1 B1 58 ST155 32_Esco_HA-NL D/Vascular surgery blaCTX-M-1 D 117 None 32b_Esco_HA-NL D/Vascular surgery blaDHA-1, blaTEM-1B B2 131 ST131 33_Esco_HA-NL D/Vascular surgery blaCTX-M-14 D 69 ST69 33b_Esco_HA-NL D/Vascular surgery blaCTX-M-14 D 69 ST69 34_Esco_HA-NL D/Internal medicine blaCTX-M-55, blaOXA-1 B1 4385 None 35_Esco_HA-NL D/Dialysis outpatient blaCTX-M-15, blaTEM-1B, blaOXA-1 B2 131 ST131 35b_Esco_HA-NL D/Dialysis outpatient blaCTX-M-15, blaOXA-1 B2 131 ST13 36_Esco_HA-NL D/Dialysis outpatient blaCTX-M-1, blaTEM-1B B1 58 ST155 37_Esco_HA-DE ICU 1 blaCTX-M-15 D 38 ST38 38_Esco_HA-DE ICU 6 blaCTX-M-14 D 38 ST38 39_Esco_HA-DE ICU 2 blaCTX-M-14 A 10 ST10 40_Esco_HA-DE ICU 6 blaCTX-M-15, blaTEM-1B, blaOXA-1 B1 448 ST448 41_Esco_HA-DE Surgical ward blaCTX-M-1, blaTEM-1B A 10 ST10 42_Esco_HA-DE Hemato-oncology ward blaCTX-M-15, blaTEM-1B, blaOXA-1 A 90 ST23 (Continued) Frontiers in Microbiology | www.frontiersin.org 6 October 2017 | Volume 8 | Article 1914 fmicb-08-01914 October 4, 2017 Time: 12:53 # 7 Zhou et al. Epidemiology of ESBL and VRE in Hospitals and the Community TABLE 2 | Continued Sample Hospital/ward b-Lactamase genes Phylogroup ST CC 43_Esco_HA-DE ICU 4 blaCTX-M-15, blaOXA-1 A 34 ST10 44_Esco_HA-DE ICU 3 blaTEM-187 A 10 ST10 45_Esco_HA-DE ICU 3 blaCTX-M-15, blaOXA-1 D 38 ST38 46_Esco_HA-DE ICU 3 blaCTX-M-1, blaTEM-1B A 10 ST10 47_Esco_HA-DE ICU 1 blaCTX-M-15 D 38 ST38 48_Esco_HA-DE ICU 1 blaCTX-M-14, blaTEM-1B D 1177 – CA, community acquired; HA, hospital acquired; NL, Netherlands; DE, Germany; numbers refer to individual patients and a letter behind a number indicates that more than one isolate was obtained from the patient. comparable to the 6.1% prevalence observed in Dutch hospitals The pAmpC prevalence in E. coli in our study was 0.3%, in this study. A prevalence of 5.6% ESBL-producing E. coli comparable to the prevalence of 0.6% what was reported in the isolates in hospitalized and ambulatory patients in Germany study of van Hoek et al. (2015) (0.6% pAmpC Enterobacteriaceae) has been reported recently (Pietsch et al., 2015), which and somewhat lower to findings of Reuland et al. (2015) is slightly lower than the 7.7% observed in the present (1.3% pAmpC-E. coli). The most common pAmpC gene found in study. hospital and community isolates were CMY-II, which is together Furthermore, we observed an ESBL-E. coli prevalence of with DHA frequently found in human isolates (Reuland et al., 2.5% in the Northern Netherlands community, which is low 2015). compared to previous studies in other regions, in which the ESBL-producing E. coli belonging to CC ST131-phylogroup prevalence in the community ranged from 4.7% (2009) to 10.1% B2 are usually associated with more virulent strains (Overdevest (2011) (Reuland et al., 2013; van Hoek et al., 2015). This et al., 2015). These were frequently found in the Dutch hospitals difference may have several reasons. First, ESBL prevalence may included in the present study but only sporadically in the vary between regions and over time, and natural eradication community samples. This CC ST131-phylogroup B2 was also of resistant Enterobacteriaceae might occur over time in the prevalent in a study carried out in hospitals in the Rotterdam community (Bar-Yoseph et al., 2016). Additionally, samples region (van der Bij et al., 2011). CC ST10 was predominant included in this study were only chosen from patients without any among the ESBL-producing E. coli in the community, the same gastrointestinal complaints, a factor which otherwise has been CC was also described to be prevalent in another Dutch study in described to be associated with high ESBL prevalence (Reuland community patients (Reuland et al., 2013). et al., 2013). We observed an overall ARE and VRE prevalence in The majority of the resistance genes found in our community hospitalized patients of 23.6 and 1.3%, respectively. Similar isolates were CTX-M-1 which is broadly disseminated among observations were made in a study performed in Dutch animals in Europe, especially in cattle and pigs, followed by hospitals in 2008 reporting ARE carriage rates of 10–16% the CTX-M-15 gene, commonly associated with human origin upon admission and 15–39% on acquisition in hematology (Reuland et al., 2013; Pietsch et al., 2015). The latter was the most and gastroenterology/nephrology wards (de Regt et al., 2008). frequent gene among the Dutch and German hospital isolates, in The clinical significance of enterococcal infections and active concordance with previous studies (Ewers et al., 2012; Reuland VRE screening has been a matter of discussion. However, in et al., 2013; Pietsch et al., 2015). immunocompromised patients, high morbidity and mortality TABLE 3 | Variables associated with carriage of amoxicillin-resistant E. faecium (ARE) and extended-spectrum b-lactamase (ESBL)- and/or plasmid AmpC (pAmpC)- producing Enterobacteriaecae. Variables ARE (n D 105) No ARE (n D 340) p-Value ESBL/pAmpC (n D 27) No p-Value ESBL/pAmpC (n D 418) Hospitalization days median (range) 12 .1 127/ 3 .1 107/ p < 0:001 4 .1 127/ 4 .1 36/ p D 0.886 Ward p < 0:001 p = 0.657 High-risk (n = 328) 99 .94:3%/ 229 .67:4%/ 19 .70:4%/ 309 .73:9%/ Low-risk (n = 117) 6 .5:7%/ 111 .32:6%/ 8 .29:6%/ 109 .26:1%/ Antibiotic use (n = 145) 62 .59%/ 83 .24:4%/ p < 0:001 7 .25:9%/ 138 .33%/ p = 0.529 Penicillins 26 .24:8%/ 29 .8:5%/ p < 0:001 3 .11:1%/ 35 .8:4%/ p = 0.494 Fluoroquinolones 28 .26:7%/ 15 .4:4%/ p < 0:001 1 .3:7%/ 42 .10%/ p = 0.499 Third generation cephalosporins 11 .10:5%/ 19 .5:6%/ p D 0:081 1 .3:7%/ 29 .6:9%/ p = 1.00 Results with a p-value of 0.05 were considered to be statistically significant. All p-values are two-tailed. Used penicillins: benzylpenicillin, flucloxacillin, amoxicillin– clavulanic acid, and piperacillin–tazobactam. Frontiers in Microbiology | www.frontiersin.org 7 October 2017 | Volume 8 | Article 1914 fmicb-08-01914 October 4, 2017 Time: 12:53 # 8 Zhou et al. Epidemiology of ESBL and VRE in Hospitals and the Community FIGURE 1 | Minimum spanning tree of ESBL-E. coli isolates from hospitals and the community. Distance based on a cgMLST of 1771 genes using the parameters “pairwise ignoring missing values” during calculation. Each circle represents a genotype, colors indicate geographical origin and community or hospital. Orange: hospital—Netherlands; blue: hospital—Germany; green: community—Netherlands. Number of different alleles are indicated on the edges between connected isolates (nodes). The cut-off values for defining a group was 35 alleles. Isolates are presented by their ID and ST. rates have been reported in infections caused by enterococci 2013). Endtz et al. (1997) reported a higher number of VRE (Zhou et al., 2013). In this study, ARE/VRE carriage was in the community (2%), however, this study did not include associated with prolonged hospitalization and antibiotic use, information about amoxicillin resistance nor IS16 which makes it which is in line with previous literature (Torell et al., 1999). We difficult to determine if they had a hospital or non-hospital origin found a high carriage rate of ARE in high-risk wards (30.2%). (Galloway-Pena et al., 2012; Lebreton et al., 2013). Notably, these patients may be at risk for a subsequent infection. The cgMLST analysis in our study showed heterogeneity Since 2011, VRE started to become a problem in multiple among E. coli species, and isolates were genetically distributed hospitals in the Netherlands: a total of 14 hospitals were affected independently of their origin. The hospital and community with outbreaks of VRE in October 2012 (van den Brink, 2013). ESBL-E. coli isolates included in this study did not show any However, in this study a prevalence of VRE (vanB) carriage of genetic relatedness except for the ones isolated from the same only 1.3% was found. This is probably due to extensive infection patient and for two isolates (group 5b) from patients in different prevention measures and successful outbreak management hospitals across the Dutch–German border, in a distance of approximately 200 km and with no known epidemiological link. control. The prevalence of 1.3% is similar to what has been The patient from the Dutch hospital was a dialyses outpatient previously published in the Netherlands, with prevalence rates (isolation date December 2012) whereas the patient from the ranging from 1.4 to 2% in the 1990s (Endtz et al., 1997; van den German hospital was admitted to ICU (isolation date November Braak et al., 2000). The VRE prevalence in the German hospital 2012). Interestingly, both isolates harbored the same ESBL gene was slightly higher (3.9%), though it is known that Germany has and virulence factors. a higher VRE prevalence compared to the Netherlands . Genetic relatedness was found between four VRE isolates In our Dutch community one vanA-VRE was found, that (CT110) from patients from two different Dutch hospitals was amoxicillin susceptible and IS16 negative, indicative for a (Figure 2), which indicates transmission between wards, but non-hospital origin (Galloway-Pena et al., 2012; Lebreton et al., also between hospitals in a close geographical region similar 4 to findings of a previous population-based study of VRE using Antimicrobial Resistance Interactive Database (EARS-Net) (http://ecdc.Europa. WGS that also showed intra- and inter-regional spread of closely eu/en/) Frontiers in Microbiology | www.frontiersin.org 8 October 2017 | Volume 8 | Article 1914 fmicb-08-01914 October 4, 2017 Time: 12:53 # 9 Zhou et al. Epidemiology of ESBL and VRE in Hospitals and the Community FIGURE 2 | Minimum spanning tree of VREfcm, cgMLST based on 1423 genes using the parameters “pairwise ignoring missing values” during distance calculation. Each circle represents a genotype and colors indicate cluster types (CT). Number of different alleles are indicated on the edges between connected isolates (nodes). Isolates are presented by their ID, ST, and CT. related VRE isolates (Pinholt et al., 2015). Although no genetic analyzing more than 2000 ESBL-Enterobacteriaceae isolates from relatedness was found between VRE isolates of Dutch and Dutch hospitals (Kluytmans-van den Bergh et al., 2016). German hospitals, the numbers of VRE isolates were too low In another study, a cgMLST approach for several MDR to draw definite conclusions. It is known that several VRE CT bacteria was prospectively used for taking relevant infection co-circulate in Germany and the Netherlands (data not shown). control decisions in a hospital setting (Mellmann et al., 2016). However, only some laboratories have implemented the use of A threshold of >10 differing alleles was defined to exclude cgMLST in their routine to analyze VRE outbreaks and more nosocomial transmission of MDR E. coli (Mellmann et al., 2016). epidemiological studies are needed to investigate cross-border If we would have applied this threshold we would have missed transmission of VRE. the genetic relatedness between isolates belonging to group 5b, To our knowledge, there are no similar studies that compare presenting 11 different alleles (Figure 1 and Supplementary and investigate the molecular epidemiology of ESBL-E. coli and Table S7). This highlights that thresholds based on number of VRE in hospitals and the community by WGS. Recently, the same allele differences are only applicable to specific collections within approach has been used to study the clonality of ESBL-producing a study, whereas the genetic distance calculation seems to give a Enterobacteriaceae from environmental and stool samples from more objective result, independently of the analyzed population. farmers suggesting possible cross-transmission between the We acknowledge this study has some limitations. No farmers and the environment. This was only based on number of community study in the German cross-border region, neither allele differences (Fischer et al., 2016) which makes it difficult to ARE monitoring in the German hospital were performed. interpret results without considering the total number of genes Laboratory methods for isolation of ESBL Enterobacteriaceae and included in the cgMLST scheme. In our study, we determined VRE differed between Dutch and German hospitals since no the genetic relatedness between ESBL-E. coli using cgMLST or enrichment broth was used in Germany, however, selective media wgMLST comparison and genetic distance calculation. These agar was used in both regions. Since this study was anonymous, results were in concordance with the genetic distance thresholds some epidemiological data were not available which makes it of 0.0095 (wgMLST) and 0.0105 (cgMLST) previously established more difficult to draw conclusions regarding genetic relatedness for E. coli based on known existing epidemiological links by among isolates between patients. Frontiers in Microbiology | www.frontiersin.org 9 October 2017 | Volume 8 | Article 1914 fmicb-08-01914 October 4, 2017 Time: 12:53 # 10 Zhou et al. Epidemiology of ESBL and VRE in Hospitals and the Community (III-2-03D025), part of a Dutch–German cross-border network CONCLUSION supported by the European Commission, the German In conclusion, the results of this study suggest that Federal States of Nordrhein-Westfalen and Niedersachsen, ESBL/pAmpC-E. coli circulate in the hospital and the and the Dutch Provinces of Overijssel, Gelderland, and community, although a higher prevalence of ESBL/pAmpC- Limburg. E. coli was observed in hospitals compared to the community in the Northern Netherlands. Hospitals in the Northern Dutch–German region showed a similar prevalence of ESBL/ ACKNOWLEDGMENT pAmpC-Enterobacteriaceae. VRE prevalence was still low in the hospital as well as in the community in the Northern We would like to thank Dr. C. R. C. Doorenbos, M. Zigterman, Netherlands. The German hospital showed a slightly higher VRE W. Postma, P. Rurenga, N. Welles, A. Woudstra, S. de Vries, and prevalence compared to hospitals in the Northern Netherlands. A. J. Stellingwerf for their participation in this study. Nosocomial but no cross-border transmission of VRE was observed in this study. Epidemiologically related ESBL-E. coli and VRE were uncommon across the Northern Dutch–German SUPPLEMENTARY MATERIAL border in the studied population, as only two ESBL-E. coli isolates from a Dutch and a German hospital were genetically similar. The Supplementary Material for this article can be found Cooperation between bordering countries and continuous online at: https://www.frontiersin.org/articles/10.3389/fmicb. monitoring using high discriminatory typing methods are still 2017.01914/full#supplementary-material necessary to keep the epidemiology of resistant pathogens updated thereby helping to control their spread. FIGURE S1 | Minimum spanning tree of ESBL-E. coli isolates from hospitals and the community. Distance based on a wgMLST of 4100 genes (cgMLST of 1771 These results were partially presented at the ECCMID genes and 2329 accessory genes) using the parameters “pairwise ignoring conference 2016, Amsterdam. missing values” during calculation. Each circle represents a genotype, colors indicate geographical origin and community or hospital. Orange: hospital— Netherlands; blue: hospital—Germany; green: community—Netherlands. Number ETHICS STATEMENT of different alleles are indicated on the edges between connected isolates (nodes). The same groups considered by cgMLST analysis are highlighted. Isolates are The “METc (medical ethical committee) UMCG” believes that presented by their ID and ST. this research is not a research involving humans as is ment in TABLE S1 | Metrics on raw read data and assemblies. the Law on Medical Scientific Research involving Human Beings TABLE S2a | Finished E. coli query genomes used in this study to develop and (WMO). Therefore, the METc UMCG has decided that no WMO ad hoc cgMLST scheme (n D 45). One representative isolate of every ST from approval is needed. every collection community NL (n D 10), Dutch hospitals (n D 20) and German hospital (n D 6) of the present study and 9 E. coli genomes from Dutch patients and farmers previously published (de Been et al., 2014). AUTHOR CONTRIBUTIONS TABLE S2b | Finished plasmid genomes for exclusion of genes with BLAST matches >90% and >100 bp length found within the query sequences used in GR, DB, LM, NH, TS, LB, JW, JvZ, and RK have all contributed this study to develop a cgMLST scheme. to the collection of all samples, they have all reviewed the article TABLE S3 | Genes included in the cgMLST scheme of E. coli. carefully. XZ, SG-C, GK, JA, DB, JR, and AF have contributed to the study design and reviewed the article. XZ and SG-C wrote the TABLE S4 | Accessory genes included in the wgMLST scheme of E. coli. manuscript together and analyzed the data. JR and AF reviewed TABLE S5 | E. coli cgMLST allele types for distance calculation and percentage of the manuscript several times, before sending to the other authors good targets/genes. for review. TABLE S6 | E. faecium cgMLST allele types for distance calculation and percentage of good targets/genes. TABLE S7 | Genetic distance for pairwise comparisons of grouped ESBL- E. coli FUNDING isolates. This study was supported by the Interreg IVa-funded TABLE S8 | Results of ResFinder, VirulenceFinder, PlasmidFinder, and projects EurSafety Health-net (III-1-02D73) and SafeGuard SerotypeFinder for E. coli and E. faecium isolates. carriage: systematic review and meta-analysis. J. Antimicrob. Chemother. 71, REFERENCES 2729–2739. doi: 10.1093/jac/dkw221 Arias, C. A., and Murray, B. E. (2012). The rise of the Enterococcus: Bruijnesteijn van Coppenraet, L. E., Dullaert-de Boer, M., Ruijs, G. J., van der beyond vancomycin resistance. Nat. Rev.Microbiol. 10, 266–278. doi: 10.1038/ Reijden, W. A., van der Zanden, A. G., Weel, J. F., et al. (2015). Case- nrmicro2761 control comparison of bacterial and protozoan microorganisms associated with Bar-Yoseph, H., Hussein, K., Braun, E., and Paul, M. (2016). Natural history and gastroenteritis: application of molecular detection. Clin. Microbiol. Infect. 21, decolonization strategies for ESBL/carbapenem-resistant Enterobacteriaceae 592.e9–e19. doi: 10.1016/j.cmi.2015.02.007 Frontiers in Microbiology | www.frontiersin.org 10 October 2017 | Volume 8 | Article 1914 fmicb-08-01914 October 4, 2017 Time: 12:53 # 11 Zhou et al. Epidemiology of ESBL and VRE in Hospitals and the Community Carattoli, A., Zankari, E., Garcia-Fernandez, A., Voldby Larsen, M., Lund, O., Larsen, M. V., Cosentino, S., Lukjancenko, O., Saputra, D., Rasmussen, S., Villa, L., et al. (2014). In silico detection and typing of plasmids using Hasman, H., et al. (2014). Benchmarking of methods for genomic taxonomy. PlasmidFinder and plasmid multilocus sequence typing. Antimicrob. Agents J. Clin. Microbiol. 52, 1529–1539. doi: 10.1128/JCM.02981- 13 Chemother. 58, 3895–3903. doi: 10.1128/AAC.02412- 14 Lebreton, F., van Schaik, W., McGuire, A. M., Godfrey, P., Griggs, A., Clark, N. C., Cooksey, R. C., Hill, B. C., Swenson, J. M., and Tenover, F. C. (1993). Mazumdar, V., et al. (2013). Emergence of epidemic multidrug-resistant Characterization of glycopeptide-resistant enterococci from U.S. hospitals. Enterococcus faecium from animal and commensal strains. mBio 4:e00534-13. Antimicrob. Agents Chemother. 37, 2311–2317. doi: 10.1128/AAC.37.11. doi: 10.1128/mBio.00534- 13 2311 Leopold, S. R., Goering, R. V., Witten, A., Harmsen, D., and Mellmann, A. Clermont, O., Bonacorsi, S., and Bingen, E. (2000). Rapid and simple (2014). Bacterial whole-genome sequencing revisited: portable, scalable, and determination of the Escherichia coli phylogenetic group. Appl. Environ. standardized analysis for typing and detection of virulence and antibiotic Microbiol. 66, 4555–4558. doi: 10.1128/AEM.66.10.4555- 4558.2000 resistance genes. J. Clin. Microbiol. 52, 2365–2370. doi: 10.1128/JCM. de Been, M., Lanza, V. F., de Toro, M., Scharringa, J., Dohmen, W., Du, Y., et al. 00262-14 (2014). Dissemination of cephalosporin resistance genes between Escherichia Maiden, M. C., Jansen van Rensburg, M. J., Bray, J. E., Earle, S. G., Ford, S. A., Jolley, coli strains from farm animals and humans by specific plasmid lineages. PLOS K. A., et al. (2013). MLST revisited: the gene-by-gene approach to bacterial Genet. 10:e1004776. doi: 10.1371/journal.pgen.1004776 genomics. Nat. Rev. Microbiol. 11, 728–736. doi: 10.1038/nrmicro3093 de Been, M., Pinholt, M., Top, J., Bletz, S., Mellmann, A., van Schaik, W., Mellmann, A., Bletz, S., Boking, T., Kipp, F., Becker, K., Schultes, A., et al. et al. (2015). A core genome MLST scheme for high-resolution typing of (2016). Real-time genome sequencing of resistant bacteria provides precision Enterococcus faecium. J. Clin. Microbiol. 53, 3788–3797. doi: 10.1128/JCM. infection control in an institutional setting. J. Clin. Microbiol. 54, 2874–2881. 01946- 15 doi: 10.1128/JCM.00790- 16 de Regt, M. J., van der Wagen, L. E., Top, J., Blok, H. E., Hopmans, Muller, J., Voss, A., Kock, R., Sinha, B., Rossen, J. W., Kaase, M., et al. (2015). T. E., Dekker, A. W., et al. (2008). High acquisition and environmental Cross-border comparison of the dutch and German guidelines on multidrug- contamination rates of CC17 ampicillin-resistant Enterococcus faecium in a resistant gram-negative microorganisms. Antimicrob. Res. Infect. Control 4:7. dutch hospital. J. Antimicrob. Chemother. 62, 1401–1406. doi: 10.1093/jac/ doi: 10.1186/s13756- 015- 0047- 6 dkn390 Overdevest, I. T., Bergmans, A. M., Verweij, J. J., Vissers, J., Bax, N., Endtz, H. P., van den Braak, N., van Belkum, A., Kluytmans, J. A., Koeleman, J. G., Snelders, E., et al. (2015). Prevalence of phylogroups and O25/ST131 Spanjaard, L., et al. (1997). Fecal carriage of vancomycin-resistant enterococci in susceptible and extended-spectrum beta-lactamase-producing Escherichia in hospitalized patients and those living in the community in the Netherlands. coli isolates, the Netherlands. Clin. Microbiol. Infec. 21, 570.e1–e4. doi: J. Clin. Microbiol. 35, 3026–3031. 10.1016/j.cmi.2015.02.020 Ewers, C., Bethe, A., Semmler, T., Guenther, S., and Wieler, L. H. (2012). Extended- Overdevest, I. T., Heck, M., van der Zwaluw, K., Huijsdens, X., van Santen, M., spectrum beta-lactamase-producing and AmpC-producing Escherichia coli Rijnsburger, M., et al. (2014). Extended-spectrum beta-lactamase producing from livestock and companion animals, and their putative impact on public Klebsiella spp. in chicken meat and humans: a comparison of typing methods. health: a global perspective. Clin. Microbiol. Infect. 18, 646–655. doi: 10.1111/j. Clin. Microbiol. Infec. 20, 251–255. doi: 10.1111/1469-0691.12277 1469- 0691.2012.03850.x Pietsch, M., Eller, C., Wendt, C., Holfelder, M., Falgenhauer, L., Fruth, A., Fischer, J., Hille, K., Ruddat, I., Mellmann, A., Kock, R., and Kreienbrock, L. et al. (2015). Molecular characterisation of extended-spectrum beta-lactamase (2016). Simultaneous occurrence of MRSA and ESBL-producing (ESBL)-producing Escherichia coli isolates from hospital and ambulatory Enterobacteriaceae on pig farms and in nasal and stool samples from patients in Germany. Vet. Microbiol. 200, 130–137. doi: 10.1016/j.vetmic.2015. farmers. Vet. Microbiol. 200, 107–113. doi: 10.1016/j.vetmic.2016. 11.028 05.021 Pinholt, M., Larner-Svensson, H., Littauer, P., Moser, C. E., Pedersen, M., Galloway-Pena, J., Roh, J. H., Latorre, M., Qin, X., and Murray, B. E. (2012). Lemming, L. E., et al. (2015). Multiple hospital outbreaks of vanA Enterococcus Genomic and SNP analyses demonstrate a distant separation of the hospital and faecium in Denmark, 2012-13, investigated by WGS, MLST and PFGE. community-associated clades of Enterococcus faecium. PLOS ONE 7:e30187. J. Antimicrob. Chemother. 70, 2474–2482. doi: 10.1093/jac/dkv142 doi: 10.1371/journal.pone.0030187 Reuland, E. A., Halaby, T., Hays, J. P., de Jongh, D. M., Snetselaar, H. D., van Garcia-Cobos, S., Kock, R., Mellmann, A., Frenzel, J., Friedrich, A. W., and Keulen, M., et al. (2015). Plasmid-mediated AmpC: prevalence in community- Rossen, J. W. (2015). Molecular typing of Enterobacteriaceae from pig acquired isolates in Amsterdam, the Netherlands, and risk factors for carriage. holdings in north-western Germany reveals extended- spectrum and AmpC PLOS ONE 10:e0113033. doi: 10.1371/journal.pone.0113033 beta-lactamases producing but no carbapenem resistant ones. PLOS ONE Reuland, E. A., Overdevest, I. T., Al Naiemi, N., Kalpoe, J. S., Rijnsburger, 10:e0134533. doi: 10.1371/journal.pone.0134533 M. C., Raadsen, S. A., et al. (2013). High prevalence of ESBL-producing Hasman, H., Saputra, D., Sicheritz-Ponten, T., Lund, O., Svendsen, C. A., Frimodt- Enterobacteriaceae carriage in dutch community patients with gastrointestinal Moller, N., et al. (2014). Rapid whole-genome sequencing for detection and complaints. Clin. Microbiol. Infect. 19, 542–549. doi: 10.1111/j.1469- 0691.2012. characterization of microorganisms directly from clinical samples. J. Clin. 03947.x Microbiol. 52, 139–146. doi: 10.1128/JCM.02452- 13 Torell, E., Cars, O., Olsson-Liljequist, B., Hoffman, B. M., Lindback, J., and Joensen, K. G., Scheutz, F., Lund, O., Hasman, H., Kaas, R. S., Nielsen, E. M., et al. Burman, L. G. (1999). Near absence of vancomycin-resistant enterococci (2014). Real-time whole-genome sequencing for routine typing, surveillance, but high carriage rates of quinolone-resistant ampicillin-resistant enterococci and outbreak detection of verotoxigenic Escherichia coli. J. Clin. Microbiol. 52, among hospitalized patients and nonhospitalized individuals in Sweden. J. Clin. 1501–1510. doi: 10.1128/JCM.03617- 13 Microbiol. 37, 3509–3513. Joensen, K. G., Tetzschner, A. M., Iguchi, A., Aarestrup, F. M., and Scheutz, F. van den Braak, N., Ott, A., van Belkum, A., Kluytmans, J. A., Koeleman, (2015). Rapid and easy in silico serotyping of Escherichia coli isolates by J. G., Spanjaard, L., et al. (2000). Prevalence and determinants of fecal use of whole-genome sequencing data. J. Clin. Microbiol. 53, 2410–2426. colonization with vancomycin-resistant Enterococcus in hospitalized patients doi: 10.1128/JCM.00008- 15 in the Netherlands. Infect. Control Hosp. Epidemiol. 21, 520–524. doi: 10.1086/ Kluytmans-van den Bergh, M. F., Rossen, J. W., Bruijning-Verhagen, P. C., Bonten, 501797 M. J., Friedrich, A. W., Vandenbroucke-Grauls, C. M., et al. (2016). Whole van den Brink, R. (2013). Het Einde van de Antibiotica. Hoe Bacteriën Winnen van genome multilocus sequence typing of extended-spectrum beta-lactamase- een Wondermiddel. Breda: Uitgeverij De Geus B.V. producing Enterobacteriaceae. J. Clin. Microbiol. 54, 2919–2927. doi: 10.1128/ van der Bij, A. K., Peirano, G., Goessens, W. H., van der Vorm, E. R., JCM.01648- 16 van Westreenen, M., and Pitout, J. D. (2011). Clinical and molecular Kwong, J. C., McCallum, N., Sintchenko, V., and Howden, B. P. (2015). Whole characteristics of extended-spectrum-beta-lactamase-producing Escherichia genome sequencing in clinical and public health microbiology. Pathology 47, coli causing bacteremia in the rotterdam area, Netherlands. Antimicrob. Agents 199–210. doi: 10.1097/PAT.0000000000000235 Chemother. 55, 3576–3578. doi: 10.1128/AAC.00074- 11 Frontiers in Microbiology | www.frontiersin.org 11 October 2017 | Volume 8 | Article 1914 fmicb-08-01914 October 4, 2017 Time: 12:53 # 12 Zhou et al. Epidemiology of ESBL and VRE in Hospitals and the Community van Hoek, A. H., Schouls, L., van Santen, M. G., Florijn, A., de Greeff, S. C., Zhou, X., Arends, J. P., Span, L. F., and Friedrich, A. W. (2013). and van Duijkeren, E. (2015). Molecular characteristics of extended-spectrum Algorithm for pre-emptive glycopeptide treatment in patients with cephalosporin-resistant Enterobacteriaceae from humans in the community. haematologic malignancies and an Enterococcus faecium bloodstream PLOS ONE 10:e0129085. doi: 10.1371/journal.pone.0129085 infection. Antimicrob. Resist. Infect. Control 2:24. doi: 10.1186/2047-2994- Werner, G., Fleige, C., Geringer, U., van Schaik, W., Klare, I., and Witte, W. (2011). 2-24 IS element IS16 as a molecular screening tool to identify hospital-associated strains of Enterococcus faecium. BMC Infect. Dis. 11:80. doi: 10.1186/1471- Conflict of Interest Statement: The authors declare that the research was 2334- 11- 80 conducted in the absence of any commercial or financial relationships that could Wirth, T., Falush, D., Lan, R., Colles, F., Mensa, P., Wieler, L. H., et al. (2006). Sex be construed as a potential conflict of interest. and virulence in Escherichia coli: an evolutionary perspective. Mol. Microbiol. 60, 1136–1151.. doi: 10.1111/j.1365- 2958.2006.05172.x Copyright © 2017 Zhou, García-Cobos, Ruijs, Kampinga, Arends, Borst, Möller, Woerther, P. L., Burdet, C., Chachaty, E., and Andremont, A. (2013). Trends in Holman, Schuurs, Bruijnesteijn van Coppenraet, Weel, van Zeijl, Köck, Rossen and human fecal carriage of extended-spectrum beta-lactamases in the community: Friedrich. This is an open-access article distributed under the terms of the Creative toward the globalization of CTX-M. Clin. Microbiol. Rev. 26, 744–758. Commons Attribution License (CC BY). The use, distribution or reproduction in doi: 10.1128/CMR.00023- 13 other forums is permitted, provided the original author(s) or licensor are credited Zankari, E., Hasman, H., Cosentino, S., Vestergaard, M., Rasmussen, S., Lund, O., and that the original publication in this journal is cited, in accordance with accepted et al. (2012). Identification of acquired antimicrobial resistance genes. academic practice. No use, distribution or reproduction is permitted which does not J. Antimicrob. Chemother. 67, 2640–2644. doi: 10.1093/jac/dks261 comply with these terms. Frontiers in Microbiology | www.frontiersin.org 12 October 2017 | Volume 8 | Article 1914

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