Identical plasmid AmpC beta-lactamase genes and plasmid types in E. coli isolates from patients and poultry meat in the Netherlands.

The increasing prevalence of third-generation cephalosporin-resistant Enterobacteriaceae is a worldwide problem. Recent studies showed that poultry meat and humans share identical Extended-Spectrum Beta-Lactamase genes, plasmid types, and Escherichia coli strain types, suggesting that transmission from poultry meat to humans may occur. The aim of this study was to compare plasmid-encoded Ambler class C beta-lactamase (pAmpC) genes, their plasmids, and bacterial strain types between E. coli isolates from retail chicken meat and clinical isolates in the Netherlands. In total, 98 Dutch retail chicken meat samples and 479 third-generation cephalosporin non-susceptible human clinical E. coli isolates from the same period were screened for pAmpC production. Plasmid typing was performed using PCR-based replicon typing (PBRT). E coli strains were compared using Multi-Locus-Sequence-Typing (MLST). In 12 of 98 chicken meat samples (12%), pAmpC producing E. coli were detected (all blaCMY-2). Of the 479 human E. coli, 25 (5.2%) harboured pAmpC genes (blaCMY-2 n = 22, blaACT n = 2, blaMIR n = 1). PBRT showed that 91% of poultry meat isolates harboured blaCMY-2 on an IncK plasmid, and 9% on an IncI1 plasmid. Of the human blaCMY-2 producing isolates, 42% also harboured blaCMY-2 on an IncK plasmid, and 47% on an IncI1 plasmid. Thus, 68% of human pAmpC producing E. coli have the same AmpC gene (blaCMY-2) and plasmid type (IncI1 or IncK) as found in poultry meat. MLST showed one cluster containing one human isolate and three meat isolates, with an IncK plasmid. These findings imply that a foodborne transmission route of blaCMY-2 harbouring plasmids cannot be excluded and that further evaluation is required.

International multicenter evaluation of the DiversiLab bacterial typing system for Escherichia coli and Klebsiella spp.

Successful multidrug-resistant clones are increasing in prevalence globally, which makes the ability to identify these clones urgent. However, adequate, easy-to-perform, and reproducible typing methods are lacking. We investigated whether DiversiLab (DL), an automated repetitive-sequence-based PCR bacterial typing system (bioMérieux), is suitable for comparing isolates analyzed at different geographic centers. A total of 39 Escherichia coli and 39 Klebsiella species isolates previously typed by the coordinating center were analyzed. Pulsed-field gel electrophoresis (PFGE) confirmed the presence of one cluster of 6 isolates, three clusters of 3 isolates, and three clusters of 2 isolates for each set of isolates. DL analysis was performed in 11 centers in six different countries using the same protocol. The DL profiles of 425 E. coli and 422 Klebsiella spp. were obtained. The DL system showed a lower discriminatory power for E. coli than did PFGE. The local DL data showed a low concordance, as indicated by the adjusted Rand and Wallace coefficients (0.132 to 0.740 and 0.070 to 1.0 [E. coli] and 0.091 to 0.864 and 0.056 to 1.0 [Klebsiella spp.], respectively). The central analysis showed a significantly improved concordance (0.473 to 1.0 and 0.290 to 1.0 [E. coli] and 0.513 to 0.965 and 0.425 to 1.0 [Klebsiella spp.], respectively). The misclassifications of profiles for individual isolates were mainly due to inconsistent amplification, which was most likely due to variations in the quality and amounts of the isolated DNA used for amplification. Despite local variations, the DL system has the potential to indicate the occurrence of clonal outbreaks in an international setting, provided there is strict adherence to standardized, reproducible DNA isolation methods and analysis protocols, all supported by a central database for profile comparisons.

Population distribution of Beta-lactamase conferring resistance to third-generation cephalosporins in human clinical Enterobacteriaceae in the Netherlands.

There is a global increase in infections caused by Enterobacteriaceae with plasmid-borne ß-lactamases that confer resistance to third-generation cephalosporins. The epidemiology of these bacteria is not well understood, and was, therefore, investigated in a selection of 636 clinical Enterobacteriaceae with a minimal inhibitory concentration >1 mg/L for ceftazidime/ceftriaxone from a national survey (75% E. coli, 11% E. cloacae, 11% K. pneumoniae, 2% K. oxytoca, 2% P. mirabilis). Isolates were investigated for extended-spectrum ß-lactamases (ESBLs) and ampC genes using microarray, PCR, gene sequencing and molecular straintyping (Diversilab and multi-locus sequence typing (MLST)). ESBL genes were demonstrated in 512 isolates (81%); of which 446 (87%) belonged to the CTX-M family. Among 314 randomly selected and sequenced isolates, bla(CTX-M-15) was most prevalent (n = 124, 39%), followed by bla(CTX-M-1) (n = 47, 15%), bla(CTX-M-14) (n = 15, 5%), bla(SHV-12) (n = 24, 8%) and bla(TEM-52) (n = 13, 4%). Among 181 isolates with MIC ≥16 mg/L for cefoxitin plasmid encoded AmpCs were detected in 32 and 27 were of the CMY-2 group. Among 102 E. coli isolates with MIC ≥16 mg/L for cefoxitin ampC promoter mutations were identified in 29 (28%). Based on Diversilab genotyping of 608 isolates (similarity cut-off >98%) discriminatory indices of bacteria with ESBL and/or ampC genes were 0.994, 0.985 and 0.994 for E. coli, K. pneumoniae and E. cloacae, respectively. Based on similarity cut-off >95% two large clusters of E. coli were apparent (of 43 and 30 isolates) and 21 of 21 that were typed by belonged to ST131 of which 13 contained bla(CTX-M-15). Our findings demonstrate that bla(CTX-M-15) is the most prevalent ESBL and we report a larger than previously reported prevalence of ampC genes among Enterobacteriaceae responsible for resistance to third-generation cephalosporins.

A set of multiplex PCRs for genotypic detection of extended-spectrum ß-lactamases, carbapenemases, plasmid-mediated AmpC ß-lactamases and OXA ß-lactamases.

Worldwide, resistance of Gram-negative micro-organisms to third-generation cephalosporins and carbapenems owing to ß-lactamases is an increasing problem. Although the CTX-M, TEM and SHV extended-spectrum ß-lactamases (ESBLs) are most widely disseminated, other ß-lactamase families have also recently emerged, such as plasmid-mediated AmpC ß-lactamases and carbapenemases. Here we describe a new set of multiplex polymerase chain reactions (PCRs) with one amplification protocol enabling detection of 25 prevalent ß-lactamase families, including ESBLs, carbapenemases, plasmid-mediated AmpC ß-lactamases and OXA ß-lactamases.

[Carbapenem-resistant Klebsiella pneumoniae following foreign travel]. / Carbapenem-resistente Klebsiella pneumoniae na verblijf in het buitenland.

This is the first report of 3 patients in whom carbapenemase-producing Klebsiella pneumoniae was identified in the Netherlands following foreign travel. They were a 55-year-old man who had undergone chemotherapy for lung cancer metastases, a 66-year-old woman and a 30-year-old man. The first patient was transferred from a Greek hospital; his isolate belonged to an epidemic clone (multilocus sequence type 258) with a KPC-2 carbapenemase gene. The patient died from pneumonia. The other two patients, who had been travelling around in India, were found to be colonised in the gasto-intestinal tract with different multiresistant K. pneumoniae isolates containing a New Delhi metallo-carbapenemase gene (NDM-1). The rapid emergence and dissemination of Enterobacteriaceae resistant to carbapenems such as imipenem and meropenem poses a considerable threat to clinical patient care and public health. Carbapenemase-producing strains are characterized by resistance to nearly all available beta-lactam antibiotics including cephalosporins and carbapenems. These strains are often also resistant to other classes of antibiotics. Invasive infections by these strains are associated with high morbidity and mortality rates. Adequate microbiological laboratory detection and infection control measures in hospital are pivotal to preventing dissemination in the Dutch healthcare setting.