Multicentre evaluation of a selective isolation protocol for detection of mcr-positive E. coli and Salmonella spp. in food-producing animals and meat.

This study was conducted to evaluate the performance of a screening protocol to detect and isolate mcr-positive Escherichia coli and Salmonella spp. from animal caecal content and meat samples. We used a multicentre approach involving 12 laboratories from nine European countries. All participants applied the same methodology combining a multiplex PCR performed on DNA extracted from a pre-enrichment step, followed by a selective culture step on three commercially available chromogenic agar plates. The test panel was composed of two negative samples and four samples artificially contaminated with E. coli and Salmonella spp. respectively harbouring mcr-1 or mcr-3 and mcr-4 or mcr-5 genes. PCR screening resulted in a specificity of 100% and a sensitivity of 83%. Sensitivity of each agar medium to detect mcr-positive colistin-resistant E. coli or Salmonella spp. strains was 86% for CHROMID® Colistin R, 75% for CHROMagarTM COL-APSE and 70% for COLISTIGRAM. This combined method was effective to detect and isolate most of the E. coli or Salmonella spp. strains harbouring different mcr genes from food-producing animals and food products and might thus be used as a harmonized protocol for the screening of mcr genes in food-producing animals and food products in Europe.

Novel Carbapenemases FLC-1 and IMI-2 Encoded by an Enterobacter cloacae Complex Isolated from Food Products.

Food for human consumption is screened widely for the presence of antibiotic-resistant bacteria to assess the potential for transfer of resistant bacteria to the general population. Here, we describe an Enterobacter cloacae complex isolated from imported seafood that encodes two carbapenemases on two distinct plasmids. Both enzymes belong to Ambler class A ß-lactamases, the previously described IMI-2 and a novel family designated FLC-1. The hydrolytic activity of the novel enzyme against aminopenicillins, cephalosporins, and carbapenems was determined.

High Prevalence of Intra-Familial Co-colonization by Extended-Spectrum Cephalosporin Resistant Enterobacteriaceae in Preschool Children and Their Parents in Dutch Households.

Extended-spectrum cephalosporin-resistant (ESCR) Enterobacteriaceae pose a serious infection control challenge for public health. The emergence of the ESCR phenotype is mostly facilitated by plasmid-mediated horizontal extended-spectrum ß-lactamases (ESBLs) and AmpC gene transfer within Enterobacteriaceae. Current data regarding the plasmid contribution to this emergence within the Dutch human population is limited. Hence, the aim of this study was to gain insight into the role of plasmids in the dissemination of ESBL/AmpC genes inside Dutch households with preschool children and precisely delineate co-colonization. In 87 ESCREnterobacteriaceae from fecal samples of parents and preschool children within 66 Dutch households, genomic localization, plasmid type and insertion sequences linked to ESBL/AmpC genes were determined. Chromosomal location of ESBL/AmpC genes was confirmed when needed. An epidemiologically relevant subset of the isolates based on household co-carriage was assessed by Multilocus Sequence Typing and Pulsed-Field Gel Electrophoresis for genetic relatedness. The narrow-host range I1α and F plasmids were the major facilitators of ESBL/AmpC-gene dissemination. Interestingly, we documented a relatively high occurrence of chromosomal integration of typically plasmid-encoded ESBL/AmpC-genes. A high diversity of non-epidemic Escherichia coli sequence types (STs) was revealed; the predominant STs belonged to the pandemic lineages of extraintestinal pathogenic E. coli ST131 and ST69. Intra-familiar co-carriage by identical ESCREnterobacteriaceae was documented in 7 households compared to 14 based on sole gene typing, as previously reported. Co-carriage was more frequent than expected based on pure chance, suggesting clonal transmission between children and parents within the household.

Molecular Characterization of Extended-Spectrum-Cephalosporin-Resistant Enterobacteriaceae from Wild Kelp Gulls in South America.

Extended-spectrum-cephalosporin-resistant Enterobacteriaceae are a public health concern due to limited treatment options. Here, we report on the occurrence and the molecular characteristics of extended-spectrum-cephalosporin-resistant Enterobacteriaceae recovered from wild birds (kelp gulls). Our results revealed kelp gulls as a reservoir of various extended-spectrum cephalosporinase genes associated with different genetic platforms. In addition, we report for the first time the presence of a known epidemic clone of Salmonella enterica serotype Heidelberg (JF6X01.0326/XbaI.1966) among wild birds.

Extended-Spectrum Cephalosporin-Resistant Salmonella enterica serovar Heidelberg Strains, the Netherlands(1).

Extended-spectrum cephalosporin-resistant Salmonella enterica serovar Heidelberg strains (JF6X01.0022/XbaI.0251, JF6X01.0326/XbaI.1966, JF6X01.0258/XbaI.1968, and JF6X01.0045/XbaI.1970) have been identified in the United States with pulsed-field gel electrophoresis. Our examination of isolates showed introduction of these strains in the Netherlands and highlight the need for active surveillance and intervention strategies by public health organizations.

Bluetongue virus serotype 6 in Europe in 2008-Emergence and disappearance of an unexpected non-virulent BTV.

Bluetongue viruses (BTVs) could invade N-W Europe similar to BTV serotype 8 (BTV8/net06), since the source and route of introduction of this virus has not been solved. Therefore, the Dutch survey for Bluetongue by PCR testing was extended by further analysis of PCR positives to identify the involved BTV. In late August 2008, BTV was reported with 12 nucleotide differences in the S10 amplicon (S10 genotyping). This virus was identified as serotype 6, here named BTV6/net08. Promptly, serotype specific real-time PCR tests were developed for serotypes 1, 6, and 8 (S2 genotyping). Agreement was found between results by S10- and S2 genotyping. Further, BTV1 was identified by both S10- and S2 genotyping in one imported animal. After initial discovery of BTV6 in the Netherlands, animals from 18 holdings tested PCR positive for BTV6/net08 in 2008. Remarkably only one or two PCR positive animals per holding were found. Serum neutralization tests did not result in the discovery of more BTV6 infected animals. Retrospective studies indicated no evidence for infections by BTV6/net08 prior to the first discovery. Experimental infections with BTV6/net08 did not cause clinical disease in sheep, calves and cattle, except for a very short fever in some animals. This clearly showed that the vaccine-related BTV6/net08 is not virulent. BTV6/net08 was not found by passive and active surveys in the years after its discovery. Apparently, BTV6/net08 was not efficiently transmitted by endemic species of Culicoides in N-W Europe, and disappeared without the need of any control measure.