Plasmid-Mediated AmpC: Prevalence in Community-Acquired Isolates in Amsterdam, the Netherlands, and Risk Factors for Carriage

January Plasmid-Mediated AmpC: Prevalence in Community-Acquired Isolates in Amsterdam, the Netherlands, and Risk Factors for Carriage E. Ascelijn Reuland 0 1 Teysir Halaby 0 1 John P. Hays 0 1 Denise M. C. de Jongh 0 1 Henrieke D. R. Snetselaar 0 1 Marte van Keulen 0 1 Petra J. M. Elders 0 1 Paul H. M. Savelkoul 0 1 Christina M. J. E. Vandenbroucke-Grauls 0 1 Nashwan al Naiemi 0 1 0 1 Medical Microbiology and Infection Control, VU University Medical Center , Amsterdam , The Netherlands , 2 Laboratory for Medical Microbiology and Public Health , Hengelo , The Netherlands , 3 Department of Medical Microbiology and Infectious Diseases, Erasmus MC , Rotterdam , The Netherlands , 4 EMGO Institute for Health and Care Research, VU University Medical Center , Amsterdam , The Netherlands, 5 Medical Microbiology and Infection Control, Ziekenhuisgroep Twente, Almelo , The Netherlands 1 Academic Editor: Herman Tse, The University of Hong Kong , HONG KONG - Funding: The European Community Seventh Framework Program FP7/20072013, TEMPOtest-QC, under grant agreement number 241742, supported this study by enabling the multiplex pAmpC PCR investigations. This work was supported by ZonMw, the Netherlands Organisation for Health Research and The objective of this study was to determine the prevalence of pAmpC beta-lactamases in community-acquired Gram negative bacteria in the Netherlands, and to identify possible risk factors for carriage of these strains. Fecal samples were obtained from community-dwelling volunteers. Participants also returned a questionnaire for analysis of risk factors. Screening for pAmpC was performed with selective enrichment broth and a selective screening agar. Confirmation of AmpC-production was performed with two double disc combination tests: cefotaxime and ceftazidime with either boronic acid or cloxacillin as inhibitor. Multiplex PCR was used as gold standard for detection of pAmpC. 16S rRNA PCR and AFLP were performed as required, plasmids were identified by PCR-based replicon typing. Questionnaire results were analyzed with SPSS, version 20.0. Fecal samples were obtained from 550 volunteers; mean age 51 years (range: 1891), 61% were females. pAmpC was present in seven E. coli isolates (7/550, 1.3%, 0.62.7 95% CI): six CMY-2-like pAmpC and one DHA. ESBL-encoding genes were found in 52/550 (9.5%, 7.312.2 95% CI) isolates; these were predominantly blaCTX-M genes. Two isolates had both ESBL and pAmpC. Admission to a hospital in the previous year was the only risk factor we identified. Development (grant number 125020011 to CMJEV-G and NaN). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Our data indicate that the prevalence of pAmpC in the community seems still low. However, since pAmpC-producing isolates were not identified as ESBL producers by routine algorithms, there is consistent risk that further increase of their prevalence might go undetected. Resistance to broad-spectrum cephalosporins is considered to be mainly caused by extendedspectrum beta-lactamases (ESBLs). Another group of enzymes that can hydrolyze cephalosporins are the AmpC beta-lactamases. AmpC were originally described as chromosomally encoded beta-lactamases, particularly in Enterobacter spp., Citrobacter freundii, and Serratia spp. Plasmid-mediated AmpC (pAmpC) are AmpC beta-lactamases encoded on plasmids and hence transferable between species. These enzymes appeared in Enterobacteriaceae that lack chromosomal AmpC enzymes (Proteus mirabilis, Salmonella spp and Klebsiella spp) or only express low basal amounts of AmpC like Escherichia coli and Shigella spp. The frequency of pAmpC may be of larger concern than initially thought, especially if this resistance threat would mimick the trend that we have seen occurring over the past years for ESBLs [1, 2]. We consider it important therefore, to closely monitor the occurrence of this resistance trait. Outbreaks of pAmpC have been recognized in different settings worldwide [38]. Currently little information is available regarding the prevalence of this group of beta-lactamases in the Dutch community. The exact prevalence of pAmpC is still unknown because simple and valid detection methods are not available, hence pAmpC-producing organisms are often missed. While algorithms for the routine detection of resistance among Gram-negative bacteria, including detection of ESBL and carbapenemases, are widely available, such algorithms are still lacking for pAmpC [9, 10]. The objective of the present study was to determine the prevalence of pAmpC beta-lactamases in community-acquired Gram negative bacteria in the Netherlands, and to identify possible risk factors for carriage of these strains. Materials and Methods Study population In the context of a larger study aimed at determining the prevalence of carriage of ESBLpositive isolates in the community in The Netherlands, volunteers for this study were approached through five general practices, affiliated to the Academic General Practice Network, VU University Medical Center, in the region of Amsterdam. In the Netherlands, health insurance is obligatory and all inhabitants have to be registered with a general practitioner, regardless of their health status. We took advantage of this registration system, and used it to approach the study subjects. All persons older than 18 years, registered in the above mentioned five general practices were approached by postal mail, except for a small group of terminally ill patients registered with a single practitioner who preferred that these patients were not asked to participate. This means that the persons who participated in the study were not hospitalized, nor visiting their physician at that moment, hence truly recruited from the community. Volunteers were asked to send in a fecal sample and to fill in a questionnaire. Written informed consent was obtained from all participants, and the study was approved by the medical ethics committee (METc, NL29769.029.09) of the VU University Medical Center (NTR Trial ID NTR2453). Fecal samples were inoculated into Trypticase Soy enrichment Broth containing 50 mg/L ampicillin (TSB-amp) and incubated overnight at 37C. For ESBL and AmpC screening, an aliquot of the overnight culture was subcultured on a selective screening agar which is routinely used for ESBL screening (EbSA ESBL agar, Cepheid Benelux, Apeldoorn, the Netherlands). This agar consists of a double MacConkey agar plate supplemented with vancomycin to inhibit gram-positive enterococci (64 mg/L) and cloxacillin to inhibit AmpC producers (400 mg/L) on both sides. Additionally, cefotaxime (1mg/L) was added to one of the sides, and ceftazidime (1 mg/L) to the other side to screen for isolates resistant to third generation cephalosporins. In order to detect also AmpC producers (both chromosomally and plasmid encoded AmpC), an adapted agar without cloxacillin was used in this st (...truncated)