Detection and Occurrence of Plasmid-Mediated AmpC in Highly Resistant Gram-Negative Rods

et al. (2014) Detection and Occurrence of Plasmid-Mediated AmpC in Highly Resistant Gram-Negative Rods. PLoS ONE 9(3): e91396. doi:10.1371/journal.pone.0091396 Detection and Occurrence of Plasmid-Mediated AmpC in Highly Resistant Gram-Negative Rods E. Ascelijn Reuland 0 John P. Hays 0 Denise M. C. de Jongh 0 Eman Abdelrehim 0 Ina Willemsen 0 Jan A. J. W. Kluytmans 0 Paul H. M. Savelkoul 0 Christina M. J. E. Vandenbroucke-Grauls 0 Nashwan al 0 Asad U. Khan, Aligarh Muslim University, India 0 1 Medical Microbiology and Infection Control, VU University Medical Center , Amsterdam , The Netherlands , 2 Department of Medical Microbiology and Infectious Diseases, Erasmus MC , Rotterdam , The Netherlands , 3 Department of Medical Microbiology and Infection Control, Amphia Hospital , Breda , The Netherlands , 4 Laboratory for Medical Microbiology and Public Health , Hengelo , The Netherlands, 5 Medical Microbiology and Infection Control, Ziekenhuisgroep Twente, Almelo , The Netherlands Objectives: The aim of this study was to compare the current screening methods and to evaluate confirmation tests for phenotypic plasmidal AmpC (pAmpC) detection. Methods: For this evaluation we used 503 Enterobacteriaceae from 18 Dutch hospitals and 21 isolates previously confirmed to be pAmpC positive. All isolates were divided into three groups: isolates with 1) reduced susceptibility to ceftazidime and/ or cefotaxime; 2) reduced susceptibility to cefoxitin; 3) reduced susceptibility to ceftazidime and/or cefotaxime combined with reduced susceptibility to cefoxitin. Two disk-based tests, with cloxacillin or boronic acid as inhibitor, and Etest with cefotetan-cefotetan/cloxacillin were used for phenotypic AmpC confirmation. Finally, presence of pAmpC genes was tested by multiplex and singleplex PCR. Results: We identified 13 pAmpC producing Enterobacteriaceae isolates among the 503 isolates (2.6%): 9 CMY-2, 3 DHA-1 and 1 ACC-1 type in E. coli isolates. The sensitivity and specificity of reduced susceptibility to ceftazidime and/or cefotaxime in combination with cefoxitin was 97% (33/34) and 90% (289/322) respectively. The disk-based test with cloxacillin showed the best performance as phenotypic confirmation method for AmpC production. Conclusions: For routine phenotypic detection of pAmpC the screening for reduced susceptibility to third generation cephalosporins combined with reduced susceptibility to cefoxitin is recommended. Confirmation via a combination disk diffusion test using cloxacillin is the best phenotypic option. The prevalence found is worrisome, since, due to their plasmidal location, pAmpC genes may spread further and increase in prevalence. - Competing Interests: Co-author Jan Kluytmans is a PLOS ONE Editorial Board member. This does not alter the authors adherence to all the PLOS ONE policies on sharing data and materials. The frequency of highly resistant gram-negative rods (HRGNRs) is still increasing worldwide [1]. Gram-negative rods with resistance to carbapenems or to third generation cephalosporins only due to ESBL-production were defined as highly resistant isolates. Furthermore, strains resistant to two agents of the antimicrobial groups quinolones and aminoglycosides were also defined as highly resistant (adapted from the Dutch guideline for preventing nosocomial transmission of highly resistant microorganisms (HRMO)) [2]. Apart from ESBLs, one class of these enzymes has received relatively little attention, namely the AmpC-type beta-lactamases. Although these Class C beta-lactamases are often found to be associated with the bacterial chromosome, an increasing prevalence of plasmid-encoded AmpC enzymes (pAmpC) has been reported [35]. Traditionally, chromosomally encoded AmpC is mainly present in group II Enterobacteriaceae (Enterobacter spp., Citrobacter freundii, Hafnia alvei, Providencia spp., Serratia spp., Morganella morganii), but pAmpC is gaining more and more importance in group I Enterobacteriaceae (Proteus mirabilis, Klebsiella spp., Salmonella spp., Escherichia coli, and Shigella spp.) [3]. Furthermore, carriage of plasmid-mediated AmpC is often associated with multidrug resistance (e.g. resistance to aminoglycosides, quinolones and cotrimoxazole), and worryingly, isolates with porin loss that carry pAmpC may also be resistant to carbapenems [4,6,7]. The occurrence of pAmpC has been investigated in several studies [6,810]. In a selection of clinical Enterobacteriaceae from a national survey a high prevalence of ampC genes among Enterobacteriaceae was found; 32 out of 181 isolates with reduced susceptibility to cefoxitin concerned pAmpC [11]. Another study showed a high prevalence of ESBL/AmpCproducing E. coli in birds and farmers at Dutch broiler farms [12]. The prevalence of pAmpC carriage reported in these studies is still low, though this is most likely an underestimation due to the difficulties associated with routine phenotypic screening for pAmpC. This means that molecular detection techniques are the current gold standard for the detection of pAmpC, although these are more expensive and difficult to implement for routine use [3,13]. For this reason, several previous studies have attempted to compare and evaluate current phenotypic tests for the detection of pAmpC [1416]. However, most of these reports did not analyze different screening methodologies. Therefore, the objective of this study was to compare the current pAmpC phenotypic screening methodologies used in the literature and to evaluate the different confirmation methods. The methodology was further used to assess the prevalence of pAmpC among 502 group I HR-GNRs collected from 18 Dutch hospitals in 2007. Materials and Methods Bacterial isolates Bacterial isolates were retrospectively screened using a collection of group I HR-GNR Enterobacteriaceae previously collected during a prospective observational multicenter study in 18 hospitals in the Netherlands [17]. Gram negative rods were defined as highly resistant (HR-GNR), according to the criteria of the Dutch Working Party on Infection Prevention [2]. Isolates were obtained from patients hospitalized between January 1 and October 1, 2007 and comprised strains isolated from clinical and screening specimens. In total 892 different HR-GNR isolates were recovered from 786 patients. Identification of strains, susceptibility testing and ESBL detection was performed according to Dutch guidelines [17,18]. ESBL-encoding genes (blaCTX-M, blaSHV and blaTEM), blaOXA and carbapenemase-encoding genes (blaKPC, blaNDM, blaOXA-48, blaIMP and blaVIM) were detected by microarray and if necessary confirmed by PCR and sequencing (BaseClear) at the VU University Medical Center (VUmc) [19,20]. The authors specifically focused on Enterobacterial species that are known to lack a chromosomal AmpC gene (P. mirabilis, Klebsiella spp., Salmonella spp.), or that are known to carry a chromosomal AmpC gene, but produce only low levels of AmpC enzyme (E. coli (...truncated)