Nationwide outbreak of STEC O157 infection in the Netherlands, December 2008-January 2009: continuous risk of consuming raw beef products.

The Netherlands experienced a nationwide outbreak of Shiga toxin-producing Escherichia coli (STEC) O157 with onset of symptoms from the end of December 2008 until the end of January 2009. A total of 20 laboratory-confirmed cases were linked to the outbreak strain, serotype O157: H-, stx1, stx2, eae and e-hly positive. The investigation into the source of this outbreak is still ongoing, but evidence so far suggests that infection occurred as a result of consuming contaminated raw meat (steak tartare)

Genetic Features Differentiating Bovine, Food, and Human Isolates of Shiga Toxin-Producing Escherichia coli O157 in The Netherlands

The frequency of Escherichia coli O157 genotypes among bovine, food, and human clinical isolates from The Netherlands was studied. Genotyping included the lineage-specific polymorphism assay (LSPA6), the Shiga-toxin-encoding bacteriophage insertion site assay (SBI), and PCR detection and/or subtyping of virulence factors and markers [stx1, stx2a/stx2c, q21/Q933, tir(A255T), and rhsA(C3468G)]. LSPA6 lineage II dominated among bovine isolates (63%), followed by lineage I/II (35.6%) and lineage I (1.4%). In contrast, the majority of the human isolates were typed as lineage I/II (77.6%), followed by lineage I (14.1%) and lineage II (8.2%). Multivariate analysis revealed that the tir(A255T) SNP and the stx2a/stx2c gene variants were the genetic features most differentiating human from bovine isolates. Bovine and food isolates were dominated by stx2c (86.4% and 65.5%, respectively). Among human isolates, the frequency of stx2c was 36.5%, while the frequencies of stx2a and stx2a plus stx2c were 41.2% and 22.4%, respectively. Bovine isolates showed equal distribution of tir(255A) (54.8%) and tir(255T) (45.2%), while human isolates were dominated by the tir(255T) genotype (92.9%). LSPA6 lineage I isolates were all genotype stx2c and tir(255T), while LSPA6 lineage II was dominated by tir(255A) (86.4%) and stx2c (90.9%). LSPA6 lineage I/II isolates were all genotype tir(255T) but showed more variation in stx2 types. The results support the hypothesis that in The Netherlands, the genotypes primarily associated with human disease form a minor subpopulation in the bovine reservoir. Comparison with published data revealed that the distribution of LSPA6 lineages among bovine and human clinical isolates differs considerably between The Netherlands and North Americ

Molecular characteristics of carbapenemase-producing Enterobacterales in the Netherlands; results of the 2014–2018 national laboratory surveillance

Objectives: Carbapenem resistance mediated by mobile genetic elements has emerged worldwide and has become a major public health threat. To gain insight into the molecular epidemiology of carbapenem resistance in The Netherlands, Dutch medical microbiology laboratories are requested to submit suspected carbapenemase-producing Enterobacterales (CPE) to the National Institute for Public Health and the Environment as part of a national surveillance system. Methods: Meropenem MICs and species identification were confirmed by E-test and MALDI-TOF and carbapenemase production was assessed by the Carbapenem Inactivation Method. Of all submitted CPE, one species/carbapenemase gene combination per person per year was subjected to next-generation sequencing (NGS). Results: In total, 1838 unique isolates were received between 2014 and 2018, of which 892 were unique CPE isolates with NGS data available. The predominant CPE species were Klebsiella pneumoniae (n = 388, 43%), Escherichia coli (n = 264, 30%) and Enterobacter cloacae complex (n = 116, 13%). Various carbapenemase alleles of the same carbapenemase gene resulted in different susceptibilities to meropenem and this effect varied between species. Analyses of NGS data showed variation of prevalence of carbapenemase alleles over time with blaOXA-48 being predominant (38%, 336/892), followed by blaNDM-1 (16%, 145/892). For the first time in the Netherlands, blaOXA-181, blaOXA-232 and blaVIM-4 were detected. The genetic background of K. pneumoniae and E. coli isolates was highly diverse. Conclusions: The CPE population in the Netherlands is diverse, suggesting multiple introductions. The predominant carbapenemase alleles are blaOXA-48 and blaNDM-1. There was a clear association between species, carbapenemase allele and susceptibility to meropenem

The Carbapenem Inactivation Method (CIM), a Simple and Low-Cost Alternative for the Carba NP Test to Assess Phenotypic Carbapenemase Activity in Gram-Negative Rods

Contains fulltext : 154105.PDF (publisher's version ) (Open Access)A new phenotypic test, called the Carbapenem Inactivation Method (CIM), was developed to detect carbapenemase activity in Gram-negative rods within eight hours. This method showed high concordance with results obtained by PCR to detect genes coding for the carbapenemases KPC, NDM, OXA-48, VIM, IMP and OXA-23. It allows reliable detection of carbapenemase activity encoded by various genes in species of Enterobacteriaceae (e.g., Klebsiella pneumoniae, Escherichia coli and Enterobacter cloacae), but also in non-fermenters Pseudomonas aeruginosa and Acinetobacter baumannii. The CIM was shown to be a cost-effective and highly robust phenotypic screening method that can reliably detect carbapenemase activity

A longitudinal study of Escherichia coli O157 in cattle of a Dutch dairy farm and in the farm environment

From July 1999 till November 2000, a longitudinal study was conducted on a dairy farm in The Netherlands to study within herd prevalence and types of verocytotoxin producing Escherichia coli (VTEC) of serogroup O157 over time, and determine environmental reservoirs and possible transmission routes. Faeces, blood, milk and environmental samples were collected 14 times with intervals varying from 4 to 10 weeks during the study period. Faecal samples were selectively cultured for Escherichia coli O157. Isolates were tested by PCR for the most common virulence genes, VTI, VTII and eae, and typed by pulsed field gel electrophoresis. In total, 71 isolates were obtained, of which 49 from dairy cows, 8 from young stock, 5 from other animals and 9 from the environment. Positive samples were all detected in summer and early fall. VT- and eae-genes were found in all tested isolates, except in one. DNA typing showed that three clusters of O157 isolates could be identified. One of these clusters contained samples of two shedding seasons, indicating persistence on the farm during winter and spring. Repeated measures analysis of variance showed that cows with O157 VTEC infection had higher daily milk production in the period preceding sampling (p = 0.0055). There was no significant association between the results of the LPS-ELISA on serum samples from dairy cows and their O157 statu

Emergence of Escherichia coli encoding Shiga toxin 2f in human Shiga toxin-producing E-coli (STEC) infections in the Netherlands, January 2008 to December 2011

The Shiga toxins of Shiga toxin-producing Escherichia coli (STEC) can be divided into Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2) with several sub-variants. Variant Stx(2f) is one of the latest described, but has been rarely associated with symptomatic human infections. In the enhanced STEC surveillance in the Netherlands, 198 STEC O-157 cases and 351 STEC non-O157 cases, including 87 stx(2f) STEC isolates, were reported between 2008 and 2011. Most stx2f strains belonged to the serogroups O63:H6 (n=47, 54%), O113:H6 (n=12, 14%) and O125:H6 (n=12, 14%). Of the 87 stx2f isolates, 84 (97%) harboured the E. coli attaching and effacing (eae) gene, but not the enterohaemorrhagic E. coli haemolysin (hly) gene. stx2f STEC infections show milder symptoms and a less severe clinical course than STEC O157 infections. Almost all infections with stx2f (n=83, 95%) occurred between June and December, compared to 170/198 (86%) of STEC O157 and 173/264 (66%) of other STEC non-O157. stx(2f) STEC infections in the Netherlands are more common than anticipated, and form a distinct group within STEC with regard to virulence genes and the relatively mild disease

A longitudinal study of Escherichia coli O157 in cattle of a Dutch dairy farm and in the farm environment

From July 1999 till November 2000, a longitudinal study was conducted on a dairy farm in The Netherlands to study within herd prevalence and types of verocytotoxin producing Escherichia coli (VTEC) of serogroup O157 over time, and determine environmental reservoirs and possible transmission routes. Faeces, blood, milk and environmental samples were collected 14 times with intervals varying from 4 to 10 weeks during the study period. Faecal samples were selectively cultured for Escherichia coli O157. Isolates were tested by PCR for the most common virulence genes, VTI, VTII and eae, and typed by pulsed field gel electrophoresis. In total, 71 isolates were obtained, of which 49 from dairy cows, 8 from young stock, 5 from other animals and 9 from the environment. Positive samples were all detected in summer and early fall. VT- and eae-genes were found in all tested isolates, except in one. DNA typing showed that three clusters of O157 isolates could be identified. One of these clusters contained samples of two shedding seasons, indicating persistence on the farm during winter and spring. Repeated measures analysis of variance showed that cows with O157 VTEC infection had higher daily milk production in the period preceding sampling (p = 0.0055). There was no significant association between the results of the LPS-ELISA on serum samples from dairy cows and their O157 statu

Occurrence and characteristics of extended-spectrum-b-lactamase and AmpC-producing clinical isolates derived from companion animals and horses

Objectives To investigate the occurrence and characteristics of extended-spectrum β-lactamase (ESBL)- and AmpC-producing Enterobacteriaceae isolates in clinical samples of companion animals and horses and compare the results with ESBL/AmpC-producing isolates described in humans. Methods Between October 2007 and August 2009, 2700 Enterobacteriaceae derived from clinical infections in companion animals and horses were collected. Isolates displaying inhibition zones of ≤25 mm for ceftiofur and/or cefquinome by disc diffusion were included. ESBL/AmpC production was confirmed by combination disc tests. The presence of resistance genes was identified by microarray, PCR and sequencing, Escherichia coli genotypes by multilocus sequence typing and antimicrobial susceptibility by broth microdilution. Results Sixty-five isolates from dogs (n = 38), cats (n = 14), horses (n = 12) and a turtle were included. Six Enterobacteriaceae species were observed, mostly derived from urinary tract infections (n = 32). All except 10 isolates tested resistant to cefotaxime and ceftazidime by broth microdilution using clinical breakpoints. ESBL/AmpC genes observed were blaCTX-M-1, -2, -9, -14, -15,blaTEM-52, blaCMY-2 and blaCMY-39. blaCTX-M-1 was predominant (n = 17). blaCTX-M-9 occurred in combination with qnrA1 in 3 of the 11 Enterobacter cloacae isolates. Twenty-eight different E. coli sequence types (STs) were found. E. coli carrying blaCTX-M-1 belonged to 13 STs of which 3 were previously described in Dutch poultry and patients. Conclusions This is the first study among a large collection of Dutch companion animals and horses characterizing ESBL/AmpC-producing isolates. A similarity in resistance genes and E. coli STs among these isolates and isolates from Dutch poultry and humans may suggest exchange of resistance between different reservoirs