PFGE diversity within the methicillin-resistant Staphylococcus aureus clonal lineage ST398

<p>Abstract</p> <p>Background</p> <p>Livestock has recently been identified as a new reservoir of methicillin-resistant <it>Staphylococcus aureus </it>(MRSA). Most isolates belong to ST398 and are non-typeable with PFGE using <it>Sma</it>I, making it difficult to study transmission and outbreaks. Therefore, a new PFGE using <it>Cfr</it>9I, a neoschizomer of <it>Sma</it>I was optimized and evaluated to investigate ST398 isolates.</p> <p>Results</p> <p>After optimizing and evaluating the <it>Cfr</it>9I PFGE, clear and reproducible banding patterns were obtained from all previously non-typeable MRSA (NT_{<it>Sma</it>I}-MRSA) isolates. The PFGE patterns of ST398 isolates showed more diversity than with <it>spa</it>-typing and/or MLST. The PFGE results showed diversity within and between the two most prevalent <it>spa</it>-types of NT_{<it>Sma</it>I}-MRSA (t011 and t108). No match was found, when comparing banding patterns of the NT_{<it>Sma</it>I}-MRSA with 700 different PFGE types, obtained with <it>Sma</it>I digestion, in our database of more than 4000 strains. Furthermore, possible transmission among veterinarians and their family members was investigated and an outbreak of ST398 MRSA in a residential care facility was confirmed with the <it>Cfr</it>9I PFGE.</p> <p>Conclusions</p> <p>The adjusted PFGE can be used as a method for selecting important and distinct ST398 isolates for further research. The adjustments in the PFGE protocol using <it>Cfr</it>9I are easy to implement to study the ST398 clonal lineage in laboratories which already have a PFGE facility.</p

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

Evaluation of 5 '-nuclease and hybridization probe assays for the detection of shiga toxin-producing Escherichia coli in human stools

5'-Nuclease and a hybridization probe assays for the detection of shiga toxin-producing Escherichia coli were validated with regard to selectivity, analytical sensitivity, reproducibility and clinical performance. Both assays were capable of detecting the classical stx(1) and stx(2) genes when challenged with reference strains of E. coli (n=40), although 1 to 4 minority sequence variants, whose clinical relevance is limited (stx(1c), stx(1d), and stx(2f)), were detected less efficiently or not at all by one or both assays. No cross reaction was observed for both assays with 37 strains representing other gastrointestinal pathogens, or normal gastrointestinal flora. Analytical sensitivity ranged from 3.07 to 3.52 log(10) and 3.42 to 4.63 log(10) CFU/g of stool for 5'-nuclease and hybridization probe assay, respectively. Reproducibility was high with coefficients of variation o