High prevalence of multidrug resistant Enterobacteriaceae among residents of long term care facilities in Amsterdam, the Netherlands

High prevalence of multidrug resistant Enterobacteriaceae among residents of long term care... ONE 14(9): e0222200. https://doi.org/10.1371/ journal.pone.0222200 Editor: Vishnu Chaturvedi, Wadsworth Center, Introduction UNITED STATES The aim of this study was to determine the rate of asymptomatic carriage and spread of mul- Received: March 25, 2019 tidrug-resistant micro-organisms (MDRO) and to identify risk factors for extended spectrum Accepted: August 24, 2019 beta-lactamase-producing Enterobacteriaceae (ESBL-E) carriage in 12 long term care facili- ties (LTCFs) in Amsterdam, the Netherlands. Published: September 12, 2019 Copyright:© 2019 van Dulm et al. This is an open Materials and methods access article distributed under the terms of the Creative Commons Attribution License, which From November 2014 to august 2015, feces and nasal swabs from residents from LTCFs in permits unrestricted use, distribution, and Amsterdam, the Netherlands were collected and analyzed for presence of multidrug-resis- reproduction in any medium, provided the original tant Gram-negative bacteria (MDRGN), including ESBL-E, carbapenemase-producing author and source are credited. Enterobacteriaceae (CPE), colistin-resistant Enterobacteriaceae and methicillin-resistant Data Availability Statement: All relevant data are Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Logistic within the manuscript and its Supporting Information files. regression analysis was performed to assess associations between variables and ESBL- carriage. Funding: This study was funded by a research and development grant (R&D kp2157) of the Public Health Service Amsterdam. The funders had no Results role in study design, data collection and analysis, In total, 385 residents from 12 LTCFs (range 15–48 residents per LTCF) were enrolled. The decision to publish, or preparation of the manuscript. prevalence of carriage of MDRGN was 18.2% (range among LTCFs 0–47%) and the PLOS ONE | https://doi.org/10.1371/journal.pone.0222200 September 12, 2019 1 / 14 High prevalence of multidrug resistant Enterobacteriaceae among residents of Amsterdam LTCF, the Netherlands Competing interests: The authors have declared prevalence of ESBL-E alone was 14.5% (range among LTCFs: 0–34%). Of 63 MDRGN pos- that no competing interests exist. itive residents, 50 (79%) were ESBL-E positive of which 43 (86%) produced CTX-M. Among 44 residents with ESBL-E positive fecal samples of whom data on contact precautions were available at the time of sampling, only 9 (20%) were already known as ESBL-E carriers. The prevalence for carriage of MRSA was 0.8% (range per LTCF: 0–7%) and VRE 0%. One CPE colonized resident was found. All fecal samples tested negative for presence of plas- mid mediated resistance for colistin (MCR-1). Typing of isolates by Amplified Fragment Length Polymorphism (AFLP) showed five MDRGN clusters, of which one was found in mul- tiple LTCFs and four were found in single LTCFs, suggesting transmission within and between LTCFs. In multivariate analysis only the presence of MDRO in the preceding year remained a risk factor for ESBL-E carriage. Conclusions The ESBL-carriage rate of residents in LTCFs is nearly two times higher than in the general population but varies considerably among LTCFs in Amsterdam, whereas carriage of MRSA and VRE is low. The majority (80%) of ESBL-E positive residents had not been detected by routine culture of clinical specimens at time of sampling. Current infection con- trol practices in LTCFs in Amsterdam do not prevent transmission. Both improvement of basic hygiene, and funding for laboratory screening, should allow LTCFs in Amsterdam to develop standards of care to prevent transmission of ESBL-E. Introduction Antimicrobial resistance has been identified as a key public health challenge [1]. Amongst the multidrug-resistant micro-organisms (MDRO) are extended spectrum beta-lactamase-pro- ducing Enterobacteriaceae (ESBL-E), carbapenemase-producing Enterobacteriaceae (CPE), vancomycin-resistant enterococci (VRE), and methicillin-resistant Staphylococcus aureus (MRSA). The Netherlands is a country with low antibiotic use in humans and is among to the countries with the lowest antibiotic resistance rates in clinical isolates in Europe [2]. Dutch national guidelines for contact precautions for carriers of MDRO (other than MRSA) in Long Term Care Facilities (LTCFs) were published late 2014 [3]. In addition to European guidelines for the management of infection control precautions of multidrug-resis- tant Gram-negative bacteria (MDRGN) in hospitals [4], the Dutch national guidelines also define co-resistance to fluoroquinolones and aminoglycosides in Enterobacteriaceae as multi- drug resistance. Pseudomonas aeruginosa is considered MDRGN when resistance for three out of five of the following antibiotics is detected: carbapenems, aminoglycosides, fluorochino- lones, ceftazidim, piperacillin[3]. Previous studies in Amsterdam (2010–2011) showed a prevalence of ESBL-E carriage of 10.6% (95% CI: 9.7–11.5) and 8.6% (95% CI: 7.3–10.0) in patients attending their general prac- titioner with gastrointestinal symptoms and in the general population, respectively [5, 6]. A point prevalence study among 200 patients screened upon admission in a large general hospi- tal in Amsterdam in 2014 [7], showed a MDRGN prevalence of 10.5%, of which 76% was iden- tified as ESBL-E. Outbreaks of MDRGN are rarely detected and only incidentally reported in Dutch LTCFs [8]. Point prevalence studies in Dutch LTCFS have shown a large variation in MDRO carriage PLOS ONE | https://doi.org/10.1371/journal.pone.0222200 September 12, 2019 2 / 14 High prevalence of multidrug resistant Enterobacteriaceae among residents of Amsterdam LTCF, the Netherlands rates, ranging from 4% to 21% [8–12]. The role of LTCFs in the transmission of MDRO within the Dutch healthcare network and interventions needed to prevent transmission of MDRO in LTCFs are still under debate [13, 14]. Our aim was to study the prevalence, risk factors and molecular epidemiology of carriage of MDRO among residents of LTCFs in Amsterdam. Materials and methods Setting and data collection For this cross-sectional study we made a selection of LTCFs in Amsterdam that provided assis- ted-living and intensive nursing and harbored at least 50 residents. LTCFs with different types of nursing wards (psychogeriatric, somatic, rehabilitation, or a combination of these wards) were included to obtain an equal number of patients of each type of ward. Resident-related risk factors for carriage of MDRO were assessed by a questionnaire that was completed by LTCF nursing staff. Institutional risk factors were assessed through a questionnaire that was completed by the LTCF management staff and during a site visit at participating LTCF wards by an expert in infection control. Risk factors were scored, using scoring lists adapted from a previously validated infection risk scan (IRIS) [12]. Sample collection Nasal swabs (Copan eMRSA™, Brescia, Italy) and feces (COPAN FecalSwab™, Brescia, Italy) were collected from each participating resident by local nursing staff. MDRGN definition MDRGN were defined as used by the Dutch national guidelines. Enterobacteriaceae were con- sidered MDRGN when they were ESBL or carbapenemase-producing or if they harbored a co- resistance Laboratory detection After overnight incubation (37˚C), nasal swabs were cultured on chromID™ MRSA agar (bio- Merieux, Marcy l’Etoile, France). Feces was cultured on chromID™ MRSA agar after overnight incubation in nutrient broth no.2 + 6% NaCl (Media Products, Groningen, the Netherlands). Feces was additionally screened for 1) multidrug-resistant Gram-negative organisms using overnight incubation of an amoxicillin (16mg/L) containing BHI broth (Media Products) sub- cultured to MacConkey agar plates (Media Products) with cefotaxime (5ug) and ceftazidim (10 ug) neo-sensitabs (Rosco Diagnostica, Taastrup, Denmark) and MacConkey agar plates containing 16 ug/L gentamicin with a ciprofloxacin neo-sensitab (10ug) and 2) VRE using TM overnight incubation of an antibiotic free Enterococcosel enrichment broth (Becton Dick- inson, Utrecht, Netherlands) and chromID™ VRE (bioMerieux) agar plates. Identification and antimicrobial susceptibility testing (N200 card) of isolates was performed by standard methods and phenotypic confirmation of ESBL by E-test in accordance with EUCAST [15] and Dutch national guidelines [16]. Confirmation and genotyping of MRSA and CPE was performed by the Dutch reference laboratory at the National Institute for Public Health and the Environ- ment (RIVM). Amplified fragment length polymorphism (AFLP) was performed on all avail- able multidrug-resistant Enterobacteriaceae isolates as described in the S1 File. Isolates were considered indistinguishable (representing a cluster) when the band patterns were >90% iden- tical. For phylogenetic typing, a selection of Escherichia coli isolates were further analyzed by phylogroup-defining PCR [17]. Group B2 E. coli were further characterized by O25:ST131- specific PCR [18]. All phenotypically ESBL-positive isolates were tested for the presence of PLOS ONE | https://doi.org/10.1371/journal.pone.0222200 September 12, 2019 3 / 14 High prevalence of multidrug resistant Enterobacteriaceae among residents of Amsterdam LTCF, the Netherlands CTX-M, SHV and TEM ESBL resistance genes by PCR as previously described [19, 20]. The CTX-M, SHV and TEM ESBL resistance genes were additionally typed by sequencing. Sequencing was performed as described in the S1 File. Primer sequences are listed in S1 Table. Consensus sequences were uploaded at The Comprehensive Antibiotic Resistance Database BLAST service for typing (Jia et al., at http://arpcard.mcmaster.ca) [21]. The MCR-1 PCR was performed by the Department of Medical Microbiology of Leiden University Medical Centre according to methods described previously by Nijhuis et al. [22] and Terveer et al. [23]. All lab- oratory detection methods are described in the S1 File. Statistical analysis Associations between variables and ESBL carriage were assessed by univariable logistic regres- sion analysis. All variables with an associated p<0.25 in univariable analyses were included in a multivariable model, with the exception of type of room and use of contact precautions at the time of sampling (since these factors might be a consequence of previously detected ESBL- carriage). A backwards-stepwise procedure was performed by sequentially removing any vari- able with a p-value >0.05 in order to obtain a final multivariable model. Results are presented as odds ratios (OR) with corresponding 95% confidence intervals (CI). Confidence intervals that did not contain 1 were considered statistically significant. All statistical analyses were per- formed with SPSS version 21.0 (SPSS Inc., Chicago, IL, USA). Ethical considerations This study was reviewed and approved by the Medical Ethical Committee of the VU Medical Center Amsterdam (protocol ID NL50241.018.14). The study was judged to be beyond the scope of the Medical Research Involving Human Subjects Act (in Dutch, Wet Medisch- wetenschappelijk Onderzoek met Mensen [WMO]), and a waiver of written informed consent was obtained. Patients who participated in the study provided verbal informed consent for use of demographic, clinical, and culture data. Results Participating LTCFs and residents Twenty-four Amsterdam LTCFs were approached of whom ten participated in this study. Because of a lower response rate than expected we additionally approached one LTCF in Zaan- dam (LTCF L, 15km from Amsterdam) and one LTCF post acute care ward located in a large teaching hospital (<50 residents, LTCF K). The main reason for non-participation was the expected workload of sampling. Characteristics of the participating LTCFs are listed in Table 1. From November 2014 to August 2015, 385 residents from 12 LTCFs (range 15–48 res- idents per LTCF, 1730 residents in total) were enrolled. For 30 residents sample collection was either not or inadequately performed and they were excluded from further analysis. For another 36 residents the questionnaire was missing. For 310 residents both a fecal swab and questionnaire were available for analysis for MDRO colonization. Key participant characteris- tics of residents with both a fecal swab and questionnaire available for analysis are summarized in Table 2. Prevalence of carriage of MDRO The prevalence of carriage of MDRGN was 18.2% (range among LTCFs 0–47%) and the preva- lence of ESBL-E alone was 14.5% (range among LTCFs: 0–34%) (Table 1). The prevalence of carriage of MRSA was 0.8% (range per LTCF: 0–7%) and of VRE 0%. The three carriers of PLOS ONE | https://doi.org/10.1371/journal.pone.0222200 September 12, 2019 4 / 14 High prevalence of multidrug resistant Enterobacteriaceae among residents of Amsterdam LTCF, the Netherlands Table 1. Characteristics and MDRGN prevalence of 12 participating LTCFs. LTCF No. of residents No. of samples ESBL+ residents Total MDRGN MDRGN cluster analysis by AFLP N % N % A 125 34 6 17.6% 7 20.6% Cluster 1 (5 residents), Cluster 5 B 130 34 6 17.6% 7 20.6% Cluster 1 (2 residents), Cluster 4 C 193 42 2 4.8% 5 11.9% D 189 17 0 0.0% 0 0.0% E 108 17 0 0.0% 0 0.0% F 144 32 11 34.4% 15 46.9% Cluster 1 (4 residents), Cluster 2, Cluster 3 G 110 33 6 18.2% 7 21.2% H 199 39 6 15.4% 8 20.5% Cluster 1 (1 resident) I 144 32 9 28.1% 9 28.1% J 96 18 1 5.6% 2 11.1% K 20 13 3 23.1% 3 23.1% L 272 35 0 0.0% 0 0.0% Total 1,730 346 50 14.5% 63 18.2% # Includes ESBL-E, Carbapenemase-producing Enterobacteriaceae, Multidrug-resistant P. aeruginosa and aminoglycoside-fluoroquinolones co-resistant Enterobacteriaceae Estimated number based on historical data Abbreviations: MDRGN = Multidrug-resistant Gram-negative bacteria; LTCF = Long term care facility; No. = number; ESBL-E = Extended Spectrum Beta Lactamase- producing Enterobacteriaceae; AFLP = Amplified Fragment Length Polymorphism https://doi.org/10.1371/journal.pone.0222200.t001 MRSA resided in three different LTCFs and did not carry MDRGN. In total, 71 unique MDRGN isolates were cultured from 63 residents; 53/71 (75%) isolates from 50 residents phe- notypically produced ESBL, of which 39 (74%) were identified as E. coli, 12 (23%) as Klebsiella pneumoniae and two as Enterobacter cloacae and Citrobacter freundii. Thirteen ESBL-produc- ing isolates were co-resistant to fluoroquinolones and aminoglycosides and one K. pneumoniae isolate also produced New Delhi Metallo-beta-lactamase-1 (NDM). The prevalence of ESBL-E carriage among LTCF residents was 14.5% (95% CI: 10.8–18.2). Of the remaining 18 non- ESBL isolates, 17 Enterobacteriaceae were resistant to the combination of aminoglycosides and fluoroquinolones and one isolate identified as P. aeruginosa additionally resistant to piperacil- lin. All 346 fecal samples were negative for presence of the MCR-1 gene. Molecular characterization and ESBL typing A total of 7/71 (10%) MDRGN isolates had not been stored and could not be retrieved from the small quantities of original sample kept by -80 degrees Celsius. The missing isolates have a similar distribution of species identification, resistance pattern and LTCF location as the selec- tion used for molecular analysis. The presence of genes encoding ESBL was confirmed in all phenotypically ESBL-producing isolates except for one isolate which had a TEM-1 gene only (no ESBL). However, another E. coli isolate of the same resident with a different AFLP-result was genotypically confirmed as ESBL. The ESBL-genes most frequently detected were CTX-M-15 (16/51, 31%) and CTX-M- 27 (12/51, 24%) (Table 3). In total, 5 clusters varying in size from 2 to 12 strains, were detected in 4 LTCFs by phylogenetic analysis of AFLP-results (Table 1). Fig 1 depicts AFLP-results of all E. coli isolates with one representative isolate per cluster. All E. coli isolates from clusters 1–3 are depicted in Fig 2. A total of 22/63 (35%) MDRGN carriers from our study could be clustered with at least one other MDRGN carrier. PLOS ONE | https://doi.org/10.1371/journal.pone.0222200 September 12, 2019 5 / 14 High prevalence of multidrug resistant Enterobacteriaceae among residents of Amsterdam LTCF, the Netherlands Table 2. Demographics and clinical characteristics of participating residents. Cases are ESBL-E carriers. Univariable associations of demographic and clinical charac- teristics with ESBL carriage of LTCF participants with both a fecal swab and questionnaire available for analysis (N = 310). Variable Cases /Total OR 95%CI p-value N % Sex .590 Female 30/199 15.1% Ref Male 14/109 12.8% 0.83 0.42–1.64 Age .168 <70 years 2/39 5.1% Ref 70–79 years 11/61 18.0% 4.07 0.85–19.47 80–89 years 22/129 17.1% 3.80 0.85–16.96 �90 years 9/73 12.3% 2.60 0.53–12.69 Nursing indication .689 Psychogeriatric 14/108 13.0% Ref Somatic 19/137 13.9% 1.08 0.51–2.27 Rehabilitation 11/62 17.7% 1.45 0.61–3.42 Antimicrobial use in previous 30 days .899 No 39/273 14.3% Ref Yes 5/37 13.5% 0.94 0.34–2.55 Current antimicrobial use .888 No 43/302 14.2% Ref Yes 1/8 12.5% 0.86 0.10–7.17 Hospitalization in previous 90 days .449 No 33/218 15.1% Ref Yes 9/77 11.7% 0.74 0.34–1.63 MDRO detected in previous year < .001 No 35/289 12.1% Ref Yes 9/15 60.0% 10.89 3.65–32.43 Type of room Single person 32/201 15.9% # # Multiple person 9/88 10.2% # # Contact precautions at time of sampling No 35/293 12.0% # # Yes 9/14 64.3% # # Length of stay .401 0–10 weeks 13/73 17.8% Ref 11–64 weeks 7/74 9.5% 0.48 0.18–1.29 65–161 weeks 10/73 13.7% 0.73 0.30–1.80 162–670 weeks 13/73 17.8% 1.00 0.43–2.33 Decubitus wounds .796 No 41/285 14.4% Ref Yes 3/24 12.5% 0.85 0.24–2.98 Other wounds .534 No 39/279 14.0% Ref Yes 5/27 18.5% 1.40 0.50–3.91 Pneumonia in medical history .508 No 34/251 13.6% Ref Yes 10/59 16.9% 1.30 0.60–2.81 Comorbidities (Continued ) PLOS ONE | https://doi.org/10.1371/journal.pone.0222200 September 12, 2019 6 / 14 High prevalence of multidrug resistant Enterobacteriaceae among residents of Amsterdam LTCF, the Netherlands Table 2. (Continued ) Variable Cases /Total OR 95%CI p-value N % Diabetes .097 No 29/236 12.3% Ref Yes 15/74 20.3% 1.81 0.92–3.61 COPD .050 No 34/270 12.6% Ref Yes 10/40 25.0% 2.31 1.04–5.15 Vascular disorder .819 No 21/143 14.7% Ref Yes 23/167 13.8% 0.93 0.49–1.76 Renal impairment .893 No 33/235 14.0% Ref Yes 11/75 14.7% 1.05 0.50–2.20 IBD No 44/305 14.4% - - Yes 0/5 0.0% - - Other No 43/307 14.0% Ref Yes 1/3 33.3% 3.07 0.27–34.59 .401 Current infections Sepsis/bacteremia No 44/310 14.2% - - Yes 0/0 - - - Urinary tract infection .901 No 42/297 14.1% Ref Yes 2/13 15.4% 1.10 0.24–5.16 Upper respiratory tract infection .153 No 42/305 13.8% Ref Yes 2/5 40.0% 4.17 0.68–25.73 Lower respiratory tract infection .994 No 43/303 14.2% Ref Yes 1/7 14.3% 1.01 0.12–8.58 Gastro-intestinal tract infection No 44/307 14.3% - - Yes 0/3 0.0% - - Skin infection .994 No 43/303 14.2% Ref Yes 1/7 14.3% 1.01 0.12–8.58 Medical devices Urinary catheter .361 No 42/286 14.7% Ref Yes 2/24 8.3% 0.53 0.12–2.33 Suprabubic catheter No 44/303 14.5% - - Yes 0/7 0.0% - - PEG tube .721 (Continued ) PLOS ONE | https://doi.org/10.1371/journal.pone.0222200 September 12, 2019 7 / 14 High prevalence of multidrug resistant Enterobacteriaceae among residents of Amsterdam LTCF, the Netherlands Table 2. (Continued ) Variable Cases /Total OR 95%CI p-value N % No 43/305 14.1% Ref Yes 1/5 20.0% 1.52 0.17–13.95 Vacuum therapy No 43/294 14.6% - - Yes 0/0 - - - Intravascular catheter No 44/309 14.2% - - Yes 0/1 0.0% - - Incontinence Urine .672 No 19/143 13.3% Ref Yes 25/167 15.0% 1.14 0.60–2.19 Feces .926 No 22/157 14.0% Ref Yes 22/153 14.4% 1.03 0.54–1.95 Cases are defined as carriers of ESBL # Not estimated since contact measures at time of sampling and staying in a single vs. multiple person room might be a consequence of known ESBL-E carriage Missings: sex 2; age 8; nursing indication 3; decubitis wounds 1; other wounds 4; hospitalization in previous 90 days 15; MDRO detected in previous year 6; type of room 21; ICP at time of sampling 3; length of stay 17; vacuum therapy 16 Abbreviations: CI = Confidence interval; OR = Odds ratio; IQR = Inter quartile range; COPD = Chronic Obstructive Pulmonary Disease; IBD = Inflammatory Bowel Disease; PEG = Percutaneous Endogastric; MDRO = Multidrug-resistant micro-organisms; ESBL = Extended Spectrum Beta Lactamase https://doi.org/10.1371/journal.pone.0222200.t002 Phylogenetic typing was performed on a selection of 19 E. coli isolates, dividing the phylo- genetic tree in half with 22/45 (49%) non-ST131 E. coli isolates and 23/45 (51%) ST131 E. coli isolates (Fig 1). Isolates from clusters 1 and 3 belong to the ST131 genotype. Risk factors for carriage of ESBL For 310 of 385 residents both results from fecal sample cultures and from questionnaire were available; these were used for analysis of resident-related risk factors for carriage of ESBL-E. Among 44 residents with ESBL-E positive fecal samples of whom data on contact precautions were available at the time of sampling, only 9 (20%) were already known as ESBL-E carriers. In the univariable logistic regression analysis the following risk factors (p<0.25) were associated with ESBL-carriage: age, MDRO carriage in the preceding year, diabetes mellitus, COPD and having a current upper respiratory tract infection (Table 2). In the multivariable logistic regres- sion analyses only the presence of a MDRO in the preceding year remained a risk factor for ESBL-carriage (OR 10.9, 95%CI: 3.7–32.4). Discussion The present study showed that nearly one in five residents carried MDRGN in LTCFs in Amsterdam. Phylogenetic analysis showed five clusters of isolates in four LTCFs, suggesting transmission of ESBL-E within and between LTCFs. The large majority of MDRGN were ESBL-E, with a prevalence of carriage of nearly one in seven residents. The prevalence of MRSA was less than 1%, while no carriers of VRE were found. PLOS ONE | https://doi.org/10.1371/journal.pone.0222200 September 12, 2019 8 / 14 High prevalence of multidrug resistant Enterobacteriaceae among residents of Amsterdam LTCF, the Netherlands Table 3. ESBL-encoding genes (1 sequence per cluster, see Methods). ESBL family ESBL gene/type N CTX-M-1 family bla 16 CTX-M-15 bla 4 CTX-M-1 CTX-M-9 family bla 7 CTX-M-14 bla 1 CTX-M-14 /17$ bla 2 CTX-M-9 bla 12 CTX-M-27 CTX-M bla 1 CTX-M TEM and SHV bla 3 TEM-52 bla 1 TEM-20 bla 1 SHV-2 bla 2 SHV-12 Total 50 One isolate also encoded New Delhi Metallo-beta-lactamase-1 $ No discrimination between CTX-M-14 and CTX-M-17. One strain was phenotypically ESBL, but no ESBL gene could be detected # Exact subtype of one CTX-M gene remained unresolved by sequencing + Possibly, there were more TEM or SHV ESBL genes present. TEM or SHV was not sequenced from CTX-M- positive strains Abbreviations: N = number; ESBL = Extended Spectrum Beta Lactamase https://doi.org/10.1371/journal.pone.0222200.t003 Our study suggests a higher prevalence of ESBL-E carriage in nursing home residents than in the general population in the Amsterdam area. The majority of these carriers was only detected during the prevalence survey, hence, most carriers remain undetected. Verhoef et al [9] found an overall prevalence of ESBL-resistance genes of 4.2% in E. coli iso- lates isolated from urine samples in 107 Dutch LTCFs in 2012–2014. Only one LTCF from Amsterdam participated in that survey, and no cases of ESBL-E were detected among its resi- dents. This prevalence is likely to be an underestimation because Verhoef only focused on ESBL-producing E. coli in urine samples, and not on gastrointestinal carriage of ESBL-E. LTCFs in the Amsterdam area are underrepresented in national surveillance studies such as SNIV (surveillance network in LTCFs), hampering actual insight and control plans for MDRO. However, healthcare inspectorate reports suggest that quality and safety of care in LTCFs in Amsterdam are compromised more often compared to acute care facilities [9, 24]. Preliminary results of a recent national surveillance point-prevalence study for intestinal car- riage of resistant bacteria show an ESBL-E prevalence of 9.5% (range 0–22%) in eight nursing homes where feces samples were collected from 337/448 (75%) of residents [10]. In other Dutch studies, fecal ESBL-carriage was demonstrated in 70/643 (10.9%) nursing home residents [12] and in 50/579 (8.6%) residents of nursing homes screened upon hospital admission compared to 61/ 772 (7.9%) elderly who still lived in their own homes [11]. A study performed in region Leiden revealed fecal ESBL-carriage of 11% (E.M. Terveer and E.J. Kuijper, manuscript submitted). The ESBL-E prevalence in our study was significantly higher than that of nearly 9% found in the general population in Amsterdam in 2011 [6]. In that study, age was not associated with a higher risk of ESBL-E carriage. Although the prevalence of ESBL-E may have increased in the general population since 2011, our study indicates that LTCFs in Amsterdam may repre- sent a potential reservoir for MDRO in the healthcare network. The majority of ESBL-E carriers was not detected by routine culture of clinical specimens and were only detected during the prevalence survey. The high proportion of ESBL-E carriers PLOS ONE | https://doi.org/10.1371/journal.pone.0222200 September 12, 2019 9 / 14 High prevalence of multidrug resistant Enterobacteriaceae among residents of Amsterdam LTCF, the Netherlands Fig 1. AFLP-results of all E. Coli isolates with one representative isolate per cluster. Abbreviations: PIN = Patient Identification Number; EEG = ESBL Encoding Gene; P-PCR = Phylogroup defining Polymerase Chain Reaction. https://doi.org/10.1371/journal.pone.0222200.g001 that were additionally detected in our study, may be explained by the restrictive diagnostic pol- icy in LTCFs, and the absence of surveillance. Applying additional contact precautions only to the few known carriers of ESBL-E will very likely result in on-going transmission among resi- dents and to other healthcare institutions. The current infection control policy, which does not include surveillance or regular screening, is likely to be ineffective. The ESBL-E carriage prevalence ranged from 0% to 34% between participating LTCFs in our study. In a previous survey of a single LTCF in the South of the Netherlands, ESBL-E car- riage rates varied substantially between wards, between 0% and 47% [8]. This means that the outcome of a single survey is highly dependent on the selection of wards in the LTCF. This also indicates that good quality prognostic determinants of ESBL-E transmission in LTCFs are needed. The distribution of ESBL-encoding genes in our study is similar to that in the general popu- lation of Amsterdam [6] with the exception of CTX-M-27, which was more prevalent in nurs- ing homes. This, however may be related to the presence of a cluster of isolates with this gene (cluster 1). While nearly 16% of ESBL-E in the general population of Amsterdam belong to the PLOS ONE | https://doi.org/10.1371/journal.pone.0222200 September 12, 2019 10 / 14 High prevalence of multidrug resistant Enterobacteriaceae among residents of Amsterdam LTCF, the Netherlands Fig 2. E. Coli isolates from clusters 1–3. Abbreviations: PIN = Patient Identification Number; EEG = ESBL Encoding Gene; P-PCR = Phylogroup defining Polymerase Chain Reaction. https://doi.org/10.1371/journal.pone.0222200.g002 ST131 MLST genotype, in LTCFs, nearly 50% of ESBL-E belong to this easily expanding, more virulent and better persisting genotype [25, 26]. The only cluster of isolates that extended over more than one LTCF in our study belonged to ST131. The overrepresentation of ST131 in LTCFs could be due to clonal expansion since the study performed in the general population, or may be due to a higher transmission rate of ESBL-E (or exposure to a common source) in LTCFs. ST131 clone is associated with community-acquired infections and older age and is frequently observed in nursing homes throughout Europe [27]. In our study, one third of MDRGN isolates could be clustered with at least one other MDRGN isolate, suggesting a high transmission rate of MDRGN. A similar high rate (54%) was found in two geriatric rehabilitation wards in Israel [28]. In a recent study, Kluytmans-van den Berg et al. analyzed 2005 ESBL-E isolates from 690 ward-based prevalence surveys per- formed in 14 Dutch hospitals over a period of three years. With core genome Multilocus Sequence Typing (cgMLST) they showed a clonal relation between 2.3% of the isolates at ward level, 1.0% at institution level and 0.5% between institutions [29]. This finding suggests that in Dutch hospitals the transmission rate of ESBL-E between patients is low, which was also found in Swiss hospitals [30, 31]. Our findings, however, indicate that ESBL-E transmission within LTFCs might be higher. Our study has some limitations. More than half of the initially selected LTCFs refused to participate, mainly because of time constraints. The LTCFs that did participate endorsed the importance of a point prevalence survey, and of infection control. This selection bias may have resulted in an underestimation of the MDRO prevalence. Due to the low participation rate of residents within participating LTCFs (<20% in some LTCFs), it is not possible to make robust statements concerning transmission. Furthermore, in our study we could not associate current carriage of ESBL with known risk factors described in PLOS ONE | https://doi.org/10.1371/journal.pone.0222200 September 12, 2019 11 / 14 High prevalence of multidrug resistant Enterobacteriaceae among residents of Amsterdam LTCF, the Netherlands literature [32], except for being diagnosed with a MDRO in the preceding year. This could be due to the relative small sample size. In conclusion, our data show that the carriage rate of ESBL-E in Amsterdam is significantly higher in LTCFs than in the general population, and varies considerably between LTCFs. The prevalence of MRSA and VRE, on the contrary, is low. No MCR-1 colistin-resistance was detected in the MDRGN isolates. Resistance due to the expansion of CTX-M ESBLs, in partic- ular CTX-M-15, is emerging in LTCFs in Amsterdam. About half of multidrug-resistant E. coli appear to be related to the international clonal complex ST131. The majority of ESBL-E carri- ers are undetected in LTCFs in Amsterdam and current infection control practices do not pre- vent transmission. Both improvement of basic hygiene, and funding for laboratory screening, should allow LTCFs in Amsterdam to develop standards of care to prevent transmission of ESBL-E. Supporting information S1 Table. Primer sequences. (DOCX) S1 File. Supplementary methods. (DOCX) Acknowledgments Part of the results of this study were presented at the twenty-sixth European Congress of Clini- cal Microbiology and Infectious Diseases, Amsterdam, The Netherlands, 2016 (eposter presen- tation EV0332). We thank Tineke Roest for providing medication data, Daan Uitenbroek for help with cal- culating the sample size, Ellen Stobberingh for advice on study procedures, Kirsten Smoren- berg for sample collection, Bianca Blok for technical assistance for AFLP typing and sequencing and Ingrid Bos-Sanders for performing PCRs for colistin-resistance. We kindly thank COPAN ITALIA SPA (Italy), bioMe ´rieux Benelux bv (the Netherlands) and 3M Neder- land B.V. (the Netherlands), for providing study materials for free or with discount. Author Contributions Conceptualization: Aletta T. R. Tholen, Ina Willemsen, Peter Molenaar, Marjolein Damen, Paul Gruteke, Cees M. P. M. Hertogh, Christina M. J. E. Vandenbroucke-Grauls, Maarten Scholing. Data curation: Martijn S. van Rooijen. Formal analysis: Eline van Dulm, Martijn S. van Rooijen, Maarten Scholing. Funding acquisition: Marjolein Damen, Maarten Scholing. Investigation: Aletta T. R. Tholen, Annika Pettersson, Peter Molenaar, Ed J. Kuijper, Maarten Scholing. Methodology: Aletta T. R. Tholen, Annika Pettersson, Ina Willemsen, Peter Molenaar, Marjo- lein Damen, Paul Gruteke, Paul Oostvogel, Cees M. P. M. Hertogh, Christina M. J. E. Van- denbroucke-Grauls, Maarten Scholing. Project administration: Paul Oostvogel, Maarten Scholing. Resources: Ed J. Kuijper, Maarten Scholing. PLOS ONE | https://doi.org/10.1371/journal.pone.0222200 September 12, 2019 12 / 14 High prevalence of multidrug resistant Enterobacteriaceae among residents of Amsterdam LTCF, the Netherlands Supervision: Christina M. J. E. Vandenbroucke-Grauls, Maarten Scholing. Validation: Eline van Dulm, Aletta T. R. Tholen, Martijn S. van Rooijen, Maarten Scholing. Visualization: Eline van Dulm. Writing – original draft: Eline van Dulm. Writing – review & editing: Eline van Dulm, Aletta T. R. Tholen, Annika Pettersson, Martijn S. van Rooijen, Ina Willemsen, Peter Molenaar, Paul Gruteke, Paul Oostvogel, Ed J. Kuijper, Cees M. P. M. Hertogh, Christina M. J. E. Vandenbroucke-Grauls, Maarten Scholing. References 1. Spellberg B, Guidos R, Gilbert D, Bradley J, Boucher HW, Scheld WM, et al. The epidemic of antibiotic- resistant infections: a call to action for the medical community from the Infectious Diseases Society of America. Clin Infect Dis. 2008; 46(2):155–64. https://doi.org/10.1086/524891 PMID: 18171244 2. European Antimicrobial Resistance Surveillance Network (EARS-Net). Data from the ECDC Surveil- lance Atlas—Antimicrobial resistance [Internet]. Available from: https://ecdc.europa.eu/en/ antimicrobial-resistance/surveillance-and-disease-data/data-ecdc. 3. WIP richtlijn Bijzonder resistente micro-organismen. 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Improved multiplex PCR strategy for rapid assign- ment of the four major Escherichia coli phylogenetic groups. J Clin Microbiol. 2012; 50(9):3108–10. https://doi.org/10.1128/JCM.01468-12 PMID: 22785193 18. Dhanji H, Doumith M, Clermont O, Denamur E, Hope R, Livermore DM, et al. Real-time PCR for detec- tion of the O25b-ST131 clone of Escherichia coli and its CTX-M-15-like extended-spectrum beta-lacta- mases. Int J Antimicrob Agents. 2010; 36(4):355–8. https://doi.org/10.1016/j.ijantimicag.2010.06.007 PMID: 20691573 19. Mulvey MR, Soule G, Boyd D, Demczuk W, Ahmed R, Multi-provincial Salmonella Typhimurium Case Control Study G. Characterization of the first extended-spectrum beta-lactamase-producing Salmonella isolate identified in Canada. J Clin Microbiol. 2003; 41(1):460–2. https://doi.org/10.1128/JCM.41.1.460- 462.2003 PMID: 12517894 20. Agerso Y, Aarestrup FM, Pedersen K, Seyfarth AM, Struve T, Hasman H. 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Terveer EM, Nijhuis RHT, Crobach MJT, Knetsch CW, Veldkamp KE, Gooskens J, et al. Prevalence of colistin resistance gene (mcr-1) containing Enterobacteriaceae in feces of patients attending a tertiary care hospital and detection of a mcr-1 containing, colistin susceptible E. coli. PLoS One. 2017; 12(6): e0178598. https://doi.org/10.1371/journal.pone.0178598 PMID: 28575076 24. IGZ, Eindrapportage toezicht IGZ op 150 verpleegzorginstellingen, Utrecht, July 2016. http://www.igz. nl/zoeken/download.aspx?download=Eindrapportage+toezicht+IGZ+op+150+verpleegzorg instellingen.pdf. 25. Dautzenberg MJ, Haverkate MR, Bonten MJ, Bootsma MC. Epidemic potential of Escherichia coli ST131 and Klebsiella pneumoniae ST258: a systematic review and meta-analysis. BMJ Open. 2016; 6 (3):e009971. https://doi.org/10.1136/bmjopen-2015-009971 PMID: 26988349 26. Overdevest I, Haverkate M, Veenemans J, Hendriks Y, Verhulst C, Mulders A, et al. Prolonged coloni- sation with Escherichia coli O25:ST131 versus other extended-spectrum beta-lactamase-producing E. coli in a long-term care facility with high endemic level of rectal colonisation, the Netherlands, 2013 to 2014. Euro Surveill. 2016; 21(42). 27. Price LB, Johnson JR, Aziz M, Clabots C, Johnston B, Tchesnokova V, et al. The epidemic of extended-spectrum-beta-lactamase-producing Escherichia coli ST131 is driven by a single highly path- ogenic subclone, H30-Rx. MBio. 2013; 4(6):e00377–13. https://doi.org/10.1128/mBio.00377-13 PMID: 28. Adler A, Gniadkowski M, Baraniak A, Izdebski R, Fiett J, Hryniewicz W, et al. Transmission dynamics of ESBL-producing Escherichia coli clones in rehabilitation wards at a tertiary care centre. Clin Microbiol Infect. 2012; 18(12):E497–505. https://doi.org/10.1111/j.1469-0691.2012.03999.x PMID: 22963432 29. Kluytmans-van den Bergh MF, Rossen JW, Bruijning-Verhagen PC, Bonten MJ, Friedrich AW, Vanden- broucke-Grauls CM, et al. 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High prevalence of multidrug resistant Enterobacteriaceae among residents of long term care facilities in Amsterdam, the Netherlands

PLoS ONE, Volume 14 (9) – Sep 12, 2019

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Copyright: © 2019 van Dulm et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the manuscript and its Supporting Information files. Funding: This study was funded by a research and development grant (R&D kp2157) of the Public Health Service Amsterdam. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.
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ONE 14(9): e0222200. https://doi.org/10.1371/ journal.pone.0222200 Editor: Vishnu Chaturvedi, Wadsworth Center, Introduction UNITED STATES The aim of this study was to determine the rate of asymptomatic carriage and spread of mul- Received: March 25, 2019 tidrug-resistant micro-organisms (MDRO) and to identify risk factors for extended spectrum Accepted: August 24, 2019 beta-lactamase-producing Enterobacteriaceae (ESBL-E) carriage in 12 long term care facili- ties (LTCFs) in Amsterdam, the Netherlands. Published: September 12, 2019 Copyright:© 2019 van Dulm et al. This is an open Materials and methods access article distributed under the terms of the Creative Commons Attribution License, which From November 2014 to august 2015, feces and nasal swabs from residents from LTCFs in permits unrestricted use, distribution, and Amsterdam, the Netherlands were collected and analyzed for presence of multidrug-resis- reproduction in any medium, provided the original tant Gram-negative bacteria (MDRGN), including ESBL-E, carbapenemase-producing author and source are credited. Enterobacteriaceae (CPE), colistin-resistant Enterobacteriaceae and methicillin-resistant Data Availability Statement: All relevant data are Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE). Logistic within the manuscript and its Supporting Information files. regression analysis was performed to assess associations between variables and ESBL- carriage. Funding: This study was funded by a research and development grant (R&D kp2157) of the Public Health Service Amsterdam. The funders had no Results role in study design, data collection and analysis, In total, 385 residents from 12 LTCFs (range 15–48 residents per LTCF) were enrolled. The decision to publish, or preparation of the manuscript. prevalence of carriage of MDRGN was 18.2% (range among LTCFs 0–47%) and the PLOS ONE | https://doi.org/10.1371/journal.pone.0222200 September 12, 2019 1 / 14 High prevalence of multidrug resistant Enterobacteriaceae among residents of Amsterdam LTCF, the Netherlands Competing interests: The authors have declared prevalence of ESBL-E alone was 14.5% (range among LTCFs: 0–34%). Of 63 MDRGN pos- that no competing interests exist. itive residents, 50 (79%) were ESBL-E positive of which 43 (86%) produced CTX-M. Among 44 residents with ESBL-E positive fecal samples of whom data on contact precautions were available at the time of sampling, only 9 (20%) were already known as ESBL-E carriers. The prevalence for carriage of MRSA was 0.8% (range per LTCF: 0–7%) and VRE 0%. One CPE colonized resident was found. All fecal samples tested negative for presence of plas- mid mediated resistance for colistin (MCR-1). Typing of isolates by Amplified Fragment Length Polymorphism (AFLP) showed five MDRGN clusters, of which one was found in mul- tiple LTCFs and four were found in single LTCFs, suggesting transmission within and between LTCFs. In multivariate analysis only the presence of MDRO in the preceding year remained a risk factor for ESBL-E carriage. Conclusions The ESBL-carriage rate of residents in LTCFs is nearly two times higher than in the general population but varies considerably among LTCFs in Amsterdam, whereas carriage of MRSA and VRE is low. The majority (80%) of ESBL-E positive residents had not been detected by routine culture of clinical specimens at time of sampling. Current infection con- trol practices in LTCFs in Amsterdam do not prevent transmission. Both improvement of basic hygiene, and funding for laboratory screening, should allow LTCFs in Amsterdam to develop standards of care to prevent transmission of ESBL-E. Introduction Antimicrobial resistance has been identified as a key public health challenge [1]. Amongst the multidrug-resistant micro-organisms (MDRO) are extended spectrum beta-lactamase-pro- ducing Enterobacteriaceae (ESBL-E), carbapenemase-producing Enterobacteriaceae (CPE), vancomycin-resistant enterococci (VRE), and methicillin-resistant Staphylococcus aureus (MRSA). The Netherlands is a country with low antibiotic use in humans and is among to the countries with the lowest antibiotic resistance rates in clinical isolates in Europe [2]. Dutch national guidelines for contact precautions for carriers of MDRO (other than MRSA) in Long Term Care Facilities (LTCFs) were published late 2014 [3]. In addition to European guidelines for the management of infection control precautions of multidrug-resis- tant Gram-negative bacteria (MDRGN) in hospitals [4], the Dutch national guidelines also define co-resistance to fluoroquinolones and aminoglycosides in Enterobacteriaceae as multi- drug resistance. Pseudomonas aeruginosa is considered MDRGN when resistance for three out of five of the following antibiotics is detected: carbapenems, aminoglycosides, fluorochino- lones, ceftazidim, piperacillin[3]. Previous studies in Amsterdam (2010–2011) showed a prevalence of ESBL-E carriage of 10.6% (95% CI: 9.7–11.5) and 8.6% (95% CI: 7.3–10.0) in patients attending their general prac- titioner with gastrointestinal symptoms and in the general population, respectively [5, 6]. A point prevalence study among 200 patients screened upon admission in a large general hospi- tal in Amsterdam in 2014 [7], showed a MDRGN prevalence of 10.5%, of which 76% was iden- tified as ESBL-E. Outbreaks of MDRGN are rarely detected and only incidentally reported in Dutch LTCFs [8]. Point prevalence studies in Dutch LTCFS have shown a large variation in MDRO carriage PLOS ONE | https://doi.org/10.1371/journal.pone.0222200 September 12, 2019 2 / 14 High prevalence of multidrug resistant Enterobacteriaceae among residents of Amsterdam LTCF, the Netherlands rates, ranging from 4% to 21% [8–12]. The role of LTCFs in the transmission of MDRO within the Dutch healthcare network and interventions needed to prevent transmission of MDRO in LTCFs are still under debate [13, 14]. Our aim was to study the prevalence, risk factors and molecular epidemiology of carriage of MDRO among residents of LTCFs in Amsterdam. Materials and methods Setting and data collection For this cross-sectional study we made a selection of LTCFs in Amsterdam that provided assis- ted-living and intensive nursing and harbored at least 50 residents. LTCFs with different types of nursing wards (psychogeriatric, somatic, rehabilitation, or a combination of these wards) were included to obtain an equal number of patients of each type of ward. Resident-related risk factors for carriage of MDRO were assessed by a questionnaire that was completed by LTCF nursing staff. Institutional risk factors were assessed through a questionnaire that was completed by the LTCF management staff and during a site visit at participating LTCF wards by an expert in infection control. Risk factors were scored, using scoring lists adapted from a previously validated infection risk scan (IRIS) [12]. Sample collection Nasal swabs (Copan eMRSA™, Brescia, Italy) and feces (COPAN FecalSwab™, Brescia, Italy) were collected from each participating resident by local nursing staff. MDRGN definition MDRGN were defined as used by the Dutch national guidelines. Enterobacteriaceae were con- sidered MDRGN when they were ESBL or carbapenemase-producing or if they harbored a co- resistance Laboratory detection After overnight incubation (37˚C), nasal swabs were cultured on chromID™ MRSA agar (bio- Merieux, Marcy l’Etoile, France). Feces was cultured on chromID™ MRSA agar after overnight incubation in nutrient broth no.2 + 6% NaCl (Media Products, Groningen, the Netherlands). Feces was additionally screened for 1) multidrug-resistant Gram-negative organisms using overnight incubation of an amoxicillin (16mg/L) containing BHI broth (Media Products) sub- cultured to MacConkey agar plates (Media Products) with cefotaxime (5ug) and ceftazidim (10 ug) neo-sensitabs (Rosco Diagnostica, Taastrup, Denmark) and MacConkey agar plates containing 16 ug/L gentamicin with a ciprofloxacin neo-sensitab (10ug) and 2) VRE using TM overnight incubation of an antibiotic free Enterococcosel enrichment broth (Becton Dick- inson, Utrecht, Netherlands) and chromID™ VRE (bioMerieux) agar plates. Identification and antimicrobial susceptibility testing (N200 card) of isolates was performed by standard methods and phenotypic confirmation of ESBL by E-test in accordance with EUCAST [15] and Dutch national guidelines [16]. Confirmation and genotyping of MRSA and CPE was performed by the Dutch reference laboratory at the National Institute for Public Health and the Environ- ment (RIVM). Amplified fragment length polymorphism (AFLP) was performed on all avail- able multidrug-resistant Enterobacteriaceae isolates as described in the S1 File. Isolates were considered indistinguishable (representing a cluster) when the band patterns were >90% iden- tical. For phylogenetic typing, a selection of Escherichia coli isolates were further analyzed by phylogroup-defining PCR [17]. Group B2 E. coli were further characterized by O25:ST131- specific PCR [18]. All phenotypically ESBL-positive isolates were tested for the presence of PLOS ONE | https://doi.org/10.1371/journal.pone.0222200 September 12, 2019 3 / 14 High prevalence of multidrug resistant Enterobacteriaceae among residents of Amsterdam LTCF, the Netherlands CTX-M, SHV and TEM ESBL resistance genes by PCR as previously described [19, 20]. The CTX-M, SHV and TEM ESBL resistance genes were additionally typed by sequencing. Sequencing was performed as described in the S1 File. Primer sequences are listed in S1 Table. Consensus sequences were uploaded at The Comprehensive Antibiotic Resistance Database BLAST service for typing (Jia et al., at http://arpcard.mcmaster.ca) [21]. The MCR-1 PCR was performed by the Department of Medical Microbiology of Leiden University Medical Centre according to methods described previously by Nijhuis et al. [22] and Terveer et al. [23]. All lab- oratory detection methods are described in the S1 File. Statistical analysis Associations between variables and ESBL carriage were assessed by univariable logistic regres- sion analysis. All variables with an associated p<0.25 in univariable analyses were included in a multivariable model, with the exception of type of room and use of contact precautions at the time of sampling (since these factors might be a consequence of previously detected ESBL- carriage). A backwards-stepwise procedure was performed by sequentially removing any vari- able with a p-value >0.05 in order to obtain a final multivariable model. Results are presented as odds ratios (OR) with corresponding 95% confidence intervals (CI). Confidence intervals that did not contain 1 were considered statistically significant. All statistical analyses were per- formed with SPSS version 21.0 (SPSS Inc., Chicago, IL, USA). Ethical considerations This study was reviewed and approved by the Medical Ethical Committee of the VU Medical Center Amsterdam (protocol ID NL50241.018.14). The study was judged to be beyond the scope of the Medical Research Involving Human Subjects Act (in Dutch, Wet Medisch- wetenschappelijk Onderzoek met Mensen [WMO]), and a waiver of written informed consent was obtained. Patients who participated in the study provided verbal informed consent for use of demographic, clinical, and culture data. Results Participating LTCFs and residents Twenty-four Amsterdam LTCFs were approached of whom ten participated in this study. Because of a lower response rate than expected we additionally approached one LTCF in Zaan- dam (LTCF L, 15km from Amsterdam) and one LTCF post acute care ward located in a large teaching hospital (<50 residents, LTCF K). The main reason for non-participation was the expected workload of sampling. Characteristics of the participating LTCFs are listed in Table 1. From November 2014 to August 2015, 385 residents from 12 LTCFs (range 15–48 res- idents per LTCF, 1730 residents in total) were enrolled. For 30 residents sample collection was either not or inadequately performed and they were excluded from further analysis. For another 36 residents the questionnaire was missing. For 310 residents both a fecal swab and questionnaire were available for analysis for MDRO colonization. Key participant characteris- tics of residents with both a fecal swab and questionnaire available for analysis are summarized in Table 2. Prevalence of carriage of MDRO The prevalence of carriage of MDRGN was 18.2% (range among LTCFs 0–47%) and the preva- lence of ESBL-E alone was 14.5% (range among LTCFs: 0–34%) (Table 1). The prevalence of carriage of MRSA was 0.8% (range per LTCF: 0–7%) and of VRE 0%. The three carriers of PLOS ONE | https://doi.org/10.1371/journal.pone.0222200 September 12, 2019 4 / 14 High prevalence of multidrug resistant Enterobacteriaceae among residents of Amsterdam LTCF, the Netherlands Table 1. Characteristics and MDRGN prevalence of 12 participating LTCFs. LTCF No. of residents No. of samples ESBL+ residents Total MDRGN MDRGN cluster analysis by AFLP N % N % A 125 34 6 17.6% 7 20.6% Cluster 1 (5 residents), Cluster 5 B 130 34 6 17.6% 7 20.6% Cluster 1 (2 residents), Cluster 4 C 193 42 2 4.8% 5 11.9% D 189 17 0 0.0% 0 0.0% E 108 17 0 0.0% 0 0.0% F 144 32 11 34.4% 15 46.9% Cluster 1 (4 residents), Cluster 2, Cluster 3 G 110 33 6 18.2% 7 21.2% H 199 39 6 15.4% 8 20.5% Cluster 1 (1 resident) I 144 32 9 28.1% 9 28.1% J 96 18 1 5.6% 2 11.1% K 20 13 3 23.1% 3 23.1% L 272 35 0 0.0% 0 0.0% Total 1,730 346 50 14.5% 63 18.2% # Includes ESBL-E, Carbapenemase-producing Enterobacteriaceae, Multidrug-resistant P. aeruginosa and aminoglycoside-fluoroquinolones co-resistant Enterobacteriaceae Estimated number based on historical data Abbreviations: MDRGN = Multidrug-resistant Gram-negative bacteria; LTCF = Long term care facility; No. = number; ESBL-E = Extended Spectrum Beta Lactamase- producing Enterobacteriaceae; AFLP = Amplified Fragment Length Polymorphism https://doi.org/10.1371/journal.pone.0222200.t001 MRSA resided in three different LTCFs and did not carry MDRGN. In total, 71 unique MDRGN isolates were cultured from 63 residents; 53/71 (75%) isolates from 50 residents phe- notypically produced ESBL, of which 39 (74%) were identified as E. coli, 12 (23%) as Klebsiella pneumoniae and two as Enterobacter cloacae and Citrobacter freundii. Thirteen ESBL-produc- ing isolates were co-resistant to fluoroquinolones and aminoglycosides and one K. pneumoniae isolate also produced New Delhi Metallo-beta-lactamase-1 (NDM). The prevalence of ESBL-E carriage among LTCF residents was 14.5% (95% CI: 10.8–18.2). Of the remaining 18 non- ESBL isolates, 17 Enterobacteriaceae were resistant to the combination of aminoglycosides and fluoroquinolones and one isolate identified as P. aeruginosa additionally resistant to piperacil- lin. All 346 fecal samples were negative for presence of the MCR-1 gene. Molecular characterization and ESBL typing A total of 7/71 (10%) MDRGN isolates had not been stored and could not be retrieved from the small quantities of original sample kept by -80 degrees Celsius. The missing isolates have a similar distribution of species identification, resistance pattern and LTCF location as the selec- tion used for molecular analysis. The presence of genes encoding ESBL was confirmed in all phenotypically ESBL-producing isolates except for one isolate which had a TEM-1 gene only (no ESBL). However, another E. coli isolate of the same resident with a different AFLP-result was genotypically confirmed as ESBL. The ESBL-genes most frequently detected were CTX-M-15 (16/51, 31%) and CTX-M- 27 (12/51, 24%) (Table 3). In total, 5 clusters varying in size from 2 to 12 strains, were detected in 4 LTCFs by phylogenetic analysis of AFLP-results (Table 1). Fig 1 depicts AFLP-results of all E. coli isolates with one representative isolate per cluster. All E. coli isolates from clusters 1–3 are depicted in Fig 2. A total of 22/63 (35%) MDRGN carriers from our study could be clustered with at least one other MDRGN carrier. PLOS ONE | https://doi.org/10.1371/journal.pone.0222200 September 12, 2019 5 / 14 High prevalence of multidrug resistant Enterobacteriaceae among residents of Amsterdam LTCF, the Netherlands Table 2. Demographics and clinical characteristics of participating residents. Cases are ESBL-E carriers. Univariable associations of demographic and clinical charac- teristics with ESBL carriage of LTCF participants with both a fecal swab and questionnaire available for analysis (N = 310). Variable Cases /Total OR 95%CI p-value N % Sex .590 Female 30/199 15.1% Ref Male 14/109 12.8% 0.83 0.42–1.64 Age .168 <70 years 2/39 5.1% Ref 70–79 years 11/61 18.0% 4.07 0.85–19.47 80–89 years 22/129 17.1% 3.80 0.85–16.96 �90 years 9/73 12.3% 2.60 0.53–12.69 Nursing indication .689 Psychogeriatric 14/108 13.0% Ref Somatic 19/137 13.9% 1.08 0.51–2.27 Rehabilitation 11/62 17.7% 1.45 0.61–3.42 Antimicrobial use in previous 30 days .899 No 39/273 14.3% Ref Yes 5/37 13.5% 0.94 0.34–2.55 Current antimicrobial use .888 No 43/302 14.2% Ref Yes 1/8 12.5% 0.86 0.10–7.17 Hospitalization in previous 90 days .449 No 33/218 15.1% Ref Yes 9/77 11.7% 0.74 0.34–1.63 MDRO detected in previous year < .001 No 35/289 12.1% Ref Yes 9/15 60.0% 10.89 3.65–32.43 Type of room Single person 32/201 15.9% # # Multiple person 9/88 10.2% # # Contact precautions at time of sampling No 35/293 12.0% # # Yes 9/14 64.3% # # Length of stay .401 0–10 weeks 13/73 17.8% Ref 11–64 weeks 7/74 9.5% 0.48 0.18–1.29 65–161 weeks 10/73 13.7% 0.73 0.30–1.80 162–670 weeks 13/73 17.8% 1.00 0.43–2.33 Decubitus wounds .796 No 41/285 14.4% Ref Yes 3/24 12.5% 0.85 0.24–2.98 Other wounds .534 No 39/279 14.0% Ref Yes 5/27 18.5% 1.40 0.50–3.91 Pneumonia in medical history .508 No 34/251 13.6% Ref Yes 10/59 16.9% 1.30 0.60–2.81 Comorbidities (Continued ) PLOS ONE | https://doi.org/10.1371/journal.pone.0222200 September 12, 2019 6 / 14 High prevalence of multidrug resistant Enterobacteriaceae among residents of Amsterdam LTCF, the Netherlands Table 2. (Continued ) Variable Cases /Total OR 95%CI p-value N % Diabetes .097 No 29/236 12.3% Ref Yes 15/74 20.3% 1.81 0.92–3.61 COPD .050 No 34/270 12.6% Ref Yes 10/40 25.0% 2.31 1.04–5.15 Vascular disorder .819 No 21/143 14.7% Ref Yes 23/167 13.8% 0.93 0.49–1.76 Renal impairment .893 No 33/235 14.0% Ref Yes 11/75 14.7% 1.05 0.50–2.20 IBD No 44/305 14.4% - - Yes 0/5 0.0% - - Other No 43/307 14.0% Ref Yes 1/3 33.3% 3.07 0.27–34.59 .401 Current infections Sepsis/bacteremia No 44/310 14.2% - - Yes 0/0 - - - Urinary tract infection .901 No 42/297 14.1% Ref Yes 2/13 15.4% 1.10 0.24–5.16 Upper respiratory tract infection .153 No 42/305 13.8% Ref Yes 2/5 40.0% 4.17 0.68–25.73 Lower respiratory tract infection .994 No 43/303 14.2% Ref Yes 1/7 14.3% 1.01 0.12–8.58 Gastro-intestinal tract infection No 44/307 14.3% - - Yes 0/3 0.0% - - Skin infection .994 No 43/303 14.2% Ref Yes 1/7 14.3% 1.01 0.12–8.58 Medical devices Urinary catheter .361 No 42/286 14.7% Ref Yes 2/24 8.3% 0.53 0.12–2.33 Suprabubic catheter No 44/303 14.5% - - Yes 0/7 0.0% - - PEG tube .721 (Continued ) PLOS ONE | https://doi.org/10.1371/journal.pone.0222200 September 12, 2019 7 / 14 High prevalence of multidrug resistant Enterobacteriaceae among residents of Amsterdam LTCF, the Netherlands Table 2. (Continued ) Variable Cases /Total OR 95%CI p-value N % No 43/305 14.1% Ref Yes 1/5 20.0% 1.52 0.17–13.95 Vacuum therapy No 43/294 14.6% - - Yes 0/0 - - - Intravascular catheter No 44/309 14.2% - - Yes 0/1 0.0% - - Incontinence Urine .672 No 19/143 13.3% Ref Yes 25/167 15.0% 1.14 0.60–2.19 Feces .926 No 22/157 14.0% Ref Yes 22/153 14.4% 1.03 0.54–1.95 Cases are defined as carriers of ESBL # Not estimated since contact measures at time of sampling and staying in a single vs. multiple person room might be a consequence of known ESBL-E carriage Missings: sex 2; age 8; nursing indication 3; decubitis wounds 1; other wounds 4; hospitalization in previous 90 days 15; MDRO detected in previous year 6; type of room 21; ICP at time of sampling 3; length of stay 17; vacuum therapy 16 Abbreviations: CI = Confidence interval; OR = Odds ratio; IQR = Inter quartile range; COPD = Chronic Obstructive Pulmonary Disease; IBD = Inflammatory Bowel Disease; PEG = Percutaneous Endogastric; MDRO = Multidrug-resistant micro-organisms; ESBL = Extended Spectrum Beta Lactamase https://doi.org/10.1371/journal.pone.0222200.t002 Phylogenetic typing was performed on a selection of 19 E. coli isolates, dividing the phylo- genetic tree in half with 22/45 (49%) non-ST131 E. coli isolates and 23/45 (51%) ST131 E. coli isolates (Fig 1). Isolates from clusters 1 and 3 belong to the ST131 genotype. Risk factors for carriage of ESBL For 310 of 385 residents both results from fecal sample cultures and from questionnaire were available; these were used for analysis of resident-related risk factors for carriage of ESBL-E. Among 44 residents with ESBL-E positive fecal samples of whom data on contact precautions were available at the time of sampling, only 9 (20%) were already known as ESBL-E carriers. In the univariable logistic regression analysis the following risk factors (p<0.25) were associated with ESBL-carriage: age, MDRO carriage in the preceding year, diabetes mellitus, COPD and having a current upper respiratory tract infection (Table 2). In the multivariable logistic regres- sion analyses only the presence of a MDRO in the preceding year remained a risk factor for ESBL-carriage (OR 10.9, 95%CI: 3.7–32.4). Discussion The present study showed that nearly one in five residents carried MDRGN in LTCFs in Amsterdam. Phylogenetic analysis showed five clusters of isolates in four LTCFs, suggesting transmission of ESBL-E within and between LTCFs. The large majority of MDRGN were ESBL-E, with a prevalence of carriage of nearly one in seven residents. The prevalence of MRSA was less than 1%, while no carriers of VRE were found. PLOS ONE | https://doi.org/10.1371/journal.pone.0222200 September 12, 2019 8 / 14 High prevalence of multidrug resistant Enterobacteriaceae among residents of Amsterdam LTCF, the Netherlands Table 3. ESBL-encoding genes (1 sequence per cluster, see Methods). ESBL family ESBL gene/type N CTX-M-1 family bla 16 CTX-M-15 bla 4 CTX-M-1 CTX-M-9 family bla 7 CTX-M-14 bla 1 CTX-M-14 /17$ bla 2 CTX-M-9 bla 12 CTX-M-27 CTX-M bla 1 CTX-M TEM and SHV bla 3 TEM-52 bla 1 TEM-20 bla 1 SHV-2 bla 2 SHV-12 Total 50 One isolate also encoded New Delhi Metallo-beta-lactamase-1 $ No discrimination between CTX-M-14 and CTX-M-17. One strain was phenotypically ESBL, but no ESBL gene could be detected # Exact subtype of one CTX-M gene remained unresolved by sequencing + Possibly, there were more TEM or SHV ESBL genes present. TEM or SHV was not sequenced from CTX-M- positive strains Abbreviations: N = number; ESBL = Extended Spectrum Beta Lactamase https://doi.org/10.1371/journal.pone.0222200.t003 Our study suggests a higher prevalence of ESBL-E carriage in nursing home residents than in the general population in the Amsterdam area. The majority of these carriers was only detected during the prevalence survey, hence, most carriers remain undetected. Verhoef et al [9] found an overall prevalence of ESBL-resistance genes of 4.2% in E. coli iso- lates isolated from urine samples in 107 Dutch LTCFs in 2012–2014. Only one LTCF from Amsterdam participated in that survey, and no cases of ESBL-E were detected among its resi- dents. This prevalence is likely to be an underestimation because Verhoef only focused on ESBL-producing E. coli in urine samples, and not on gastrointestinal carriage of ESBL-E. LTCFs in the Amsterdam area are underrepresented in national surveillance studies such as SNIV (surveillance network in LTCFs), hampering actual insight and control plans for MDRO. However, healthcare inspectorate reports suggest that quality and safety of care in LTCFs in Amsterdam are compromised more often compared to acute care facilities [9, 24]. Preliminary results of a recent national surveillance point-prevalence study for intestinal car- riage of resistant bacteria show an ESBL-E prevalence of 9.5% (range 0–22%) in eight nursing homes where feces samples were collected from 337/448 (75%) of residents [10]. In other Dutch studies, fecal ESBL-carriage was demonstrated in 70/643 (10.9%) nursing home residents [12] and in 50/579 (8.6%) residents of nursing homes screened upon hospital admission compared to 61/ 772 (7.9%) elderly who still lived in their own homes [11]. A study performed in region Leiden revealed fecal ESBL-carriage of 11% (E.M. Terveer and E.J. Kuijper, manuscript submitted). The ESBL-E prevalence in our study was significantly higher than that of nearly 9% found in the general population in Amsterdam in 2011 [6]. In that study, age was not associated with a higher risk of ESBL-E carriage. Although the prevalence of ESBL-E may have increased in the general population since 2011, our study indicates that LTCFs in Amsterdam may repre- sent a potential reservoir for MDRO in the healthcare network. The majority of ESBL-E carriers was not detected by routine culture of clinical specimens and were only detected during the prevalence survey. The high proportion of ESBL-E carriers PLOS ONE | https://doi.org/10.1371/journal.pone.0222200 September 12, 2019 9 / 14 High prevalence of multidrug resistant Enterobacteriaceae among residents of Amsterdam LTCF, the Netherlands Fig 1. AFLP-results of all E. Coli isolates with one representative isolate per cluster. Abbreviations: PIN = Patient Identification Number; EEG = ESBL Encoding Gene; P-PCR = Phylogroup defining Polymerase Chain Reaction. https://doi.org/10.1371/journal.pone.0222200.g001 that were additionally detected in our study, may be explained by the restrictive diagnostic pol- icy in LTCFs, and the absence of surveillance. Applying additional contact precautions only to the few known carriers of ESBL-E will very likely result in on-going transmission among resi- dents and to other healthcare institutions. The current infection control policy, which does not include surveillance or regular screening, is likely to be ineffective. The ESBL-E carriage prevalence ranged from 0% to 34% between participating LTCFs in our study. In a previous survey of a single LTCF in the South of the Netherlands, ESBL-E car- riage rates varied substantially between wards, between 0% and 47% [8]. This means that the outcome of a single survey is highly dependent on the selection of wards in the LTCF. This also indicates that good quality prognostic determinants of ESBL-E transmission in LTCFs are needed. The distribution of ESBL-encoding genes in our study is similar to that in the general popu- lation of Amsterdam [6] with the exception of CTX-M-27, which was more prevalent in nurs- ing homes. This, however may be related to the presence of a cluster of isolates with this gene (cluster 1). While nearly 16% of ESBL-E in the general population of Amsterdam belong to the PLOS ONE | https://doi.org/10.1371/journal.pone.0222200 September 12, 2019 10 / 14 High prevalence of multidrug resistant Enterobacteriaceae among residents of Amsterdam LTCF, the Netherlands Fig 2. E. Coli isolates from clusters 1–3. Abbreviations: PIN = Patient Identification Number; EEG = ESBL Encoding Gene; P-PCR = Phylogroup defining Polymerase Chain Reaction. https://doi.org/10.1371/journal.pone.0222200.g002 ST131 MLST genotype, in LTCFs, nearly 50% of ESBL-E belong to this easily expanding, more virulent and better persisting genotype [25, 26]. The only cluster of isolates that extended over more than one LTCF in our study belonged to ST131. The overrepresentation of ST131 in LTCFs could be due to clonal expansion since the study performed in the general population, or may be due to a higher transmission rate of ESBL-E (or exposure to a common source) in LTCFs. ST131 clone is associated with community-acquired infections and older age and is frequently observed in nursing homes throughout Europe [27]. In our study, one third of MDRGN isolates could be clustered with at least one other MDRGN isolate, suggesting a high transmission rate of MDRGN. A similar high rate (54%) was found in two geriatric rehabilitation wards in Israel [28]. In a recent study, Kluytmans-van den Berg et al. analyzed 2005 ESBL-E isolates from 690 ward-based prevalence surveys per- formed in 14 Dutch hospitals over a period of three years. With core genome Multilocus Sequence Typing (cgMLST) they showed a clonal relation between 2.3% of the isolates at ward level, 1.0% at institution level and 0.5% between institutions [29]. This finding suggests that in Dutch hospitals the transmission rate of ESBL-E between patients is low, which was also found in Swiss hospitals [30, 31]. Our findings, however, indicate that ESBL-E transmission within LTFCs might be higher. Our study has some limitations. More than half of the initially selected LTCFs refused to participate, mainly because of time constraints. The LTCFs that did participate endorsed the importance of a point prevalence survey, and of infection control. This selection bias may have resulted in an underestimation of the MDRO prevalence. Due to the low participation rate of residents within participating LTCFs (<20% in some LTCFs), it is not possible to make robust statements concerning transmission. Furthermore, in our study we could not associate current carriage of ESBL with known risk factors described in PLOS ONE | https://doi.org/10.1371/journal.pone.0222200 September 12, 2019 11 / 14 High prevalence of multidrug resistant Enterobacteriaceae among residents of Amsterdam LTCF, the Netherlands literature [32], except for being diagnosed with a MDRO in the preceding year. This could be due to the relative small sample size. In conclusion, our data show that the carriage rate of ESBL-E in Amsterdam is significantly higher in LTCFs than in the general population, and varies considerably between LTCFs. The prevalence of MRSA and VRE, on the contrary, is low. No MCR-1 colistin-resistance was detected in the MDRGN isolates. Resistance due to the expansion of CTX-M ESBLs, in partic- ular CTX-M-15, is emerging in LTCFs in Amsterdam. About half of multidrug-resistant E. coli appear to be related to the international clonal complex ST131. The majority of ESBL-E carri- ers are undetected in LTCFs in Amsterdam and current infection control practices do not pre- vent transmission. Both improvement of basic hygiene, and funding for laboratory screening, should allow LTCFs in Amsterdam to develop standards of care to prevent transmission of ESBL-E. Supporting information S1 Table. Primer sequences. (DOCX) S1 File. Supplementary methods. (DOCX) Acknowledgments Part of the results of this study were presented at the twenty-sixth European Congress of Clini- cal Microbiology and Infectious Diseases, Amsterdam, The Netherlands, 2016 (eposter presen- tation EV0332). We thank Tineke Roest for providing medication data, Daan Uitenbroek for help with cal- culating the sample size, Ellen Stobberingh for advice on study procedures, Kirsten Smoren- berg for sample collection, Bianca Blok for technical assistance for AFLP typing and sequencing and Ingrid Bos-Sanders for performing PCRs for colistin-resistance. We kindly thank COPAN ITALIA SPA (Italy), bioMe ´rieux Benelux bv (the Netherlands) and 3M Neder- land B.V. (the Netherlands), for providing study materials for free or with discount. Author Contributions Conceptualization: Aletta T. R. Tholen, Ina Willemsen, Peter Molenaar, Marjolein Damen, Paul Gruteke, Cees M. P. M. Hertogh, Christina M. J. E. Vandenbroucke-Grauls, Maarten Scholing. Data curation: Martijn S. van Rooijen. Formal analysis: Eline van Dulm, Martijn S. van Rooijen, Maarten Scholing. Funding acquisition: Marjolein Damen, Maarten Scholing. Investigation: Aletta T. R. Tholen, Annika Pettersson, Peter Molenaar, Ed J. Kuijper, Maarten Scholing. Methodology: Aletta T. R. Tholen, Annika Pettersson, Ina Willemsen, Peter Molenaar, Marjo- lein Damen, Paul Gruteke, Paul Oostvogel, Cees M. P. M. Hertogh, Christina M. J. E. Van- denbroucke-Grauls, Maarten Scholing. Project administration: Paul Oostvogel, Maarten Scholing. Resources: Ed J. Kuijper, Maarten Scholing. PLOS ONE | https://doi.org/10.1371/journal.pone.0222200 September 12, 2019 12 / 14 High prevalence of multidrug resistant Enterobacteriaceae among residents of Amsterdam LTCF, the Netherlands Supervision: Christina M. J. E. Vandenbroucke-Grauls, Maarten Scholing. Validation: Eline van Dulm, Aletta T. R. Tholen, Martijn S. van Rooijen, Maarten Scholing. Visualization: Eline van Dulm. Writing – original draft: Eline van Dulm. 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PLOS ONE | https://doi.org/10.1371/journal.pone.0222200 September 12, 2019 14 / 14

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