Determining antimicrobial susceptibility in Salmonella enterica serovar Typhimurium through whole genome sequencing: a comparison against multiple phenotypic susceptibility testing methods.

BACKGROUND: UK public health organisations perform routine antimicrobial susceptibility tests (ASTs) to characterise the potential for antimicrobial resistance in Salmonella enterica serovars. Genetic determinants of these resistance mechanisms are detectable by whole genome sequencing (WGS), however the viability of WGS-based genotyping as an alternative resistance screening tool remains uncertain. We compared WGS-based genotyping, disk diffusion and agar dilution to the broth microdilution reference AST for 102 Salmonella enterica serovar Typhimurium (S. Typhimurium) isolates across 11 antimicrobial compounds. RESULTS: Genotyping concordance, interpreted using epidemiological cut-offs (ECOFFs), was 89.8% (1007/1122) with 0.83 sensitivity and 0.96 specificity. For seven antimicrobials interpreted using Salmonella clinical breakpoints, genotyping produced 0.84 sensitivity and 0.88 specificity. Although less accurate than disk diffusion (0.94 sensitivity, 0.93 specificity) and agar dilution (0.83 sensitivity, 0.98 specificity), genotyping performance improved to 0.89 sensitivity and 0.97 specificity when two antimicrobials with relatively high very major error rates were excluded (streptomycin and sulfamethoxazole). CONCLUSIONS: An 89.8% concordance from WGS-based AST predictions using ECOFF interpretations suggest that WGS would serve as an effective screening tool for the tracking of antimicrobial resistance mechanisms in S. Typhimurium. For use as a standalone clinical diagnostic screen, further work is required to reduce the error rates for specific antimicrobials.

Study of Staphylococcus aureus collected at slaughter from dairy cows with chronic mastitis.

Staphylococcus aureus is one of the most important pathogens associated with bovine mastitis. Recent studies have shown that Staph. aureus strains may differ in virulence, and in their ability to disseminate across commercial dairy herds. The goal of this study was to determine whether Staph. aureus isolates differed in their ability to colonize mammary tissue, and whether such differences could be related to molecular characteristics. Quarter milk and mammary tissues of 22 cows from two dairy herds, were collected at slaughter and bacteriological analysis was performed. All Staph. aureus isolates were characterized by Pulsed Field Gel Electrophoresis (PFGE) and microarray. Overall 45 mammary quarters were infected and 20 Staph. aureus isolates were identified. The bacteria were mostly recovered from both milk and tissue of the same quarter in significantly higher numbers from herd A cows compared with herd B. Molecular characterization of the isolates showed distinct PFGE profiles for isolates from each herd. Differences in virulence factors between herds A and B isolates were evidenced The genes for enterotoxin D, J and R were present in herd A, those for G, I, N, M, O and U were shown in herd B, whilst both components of the leukocidin lukD/E genes were only carried by herd A isolates. Furthermore, all herd A isolates showed ß-haemolysin activity, which was absent in all but one isolate from herd B. Therefore our data indicate that Staph. aureus isolates showing differences in their ability to disseminate and colonize across quarters, also have significantly different virulence characteristics.

Comparative analysis of ESBL-positive Escherichia coli isolates from animals and humans from the UK, The Netherlands and Germany.

The putative virulence and antimicrobial resistance gene contents of extended spectrum ß-lactamase (ESBL)-positive E. coli (n=629) isolated between 2005 and 2009 from humans, animals and animal food products in Germany, The Netherlands and the UK were compared using a microarray approach to test the suitability of this approach with regard to determining their similarities. A selection of isolates (n=313) were also analysed by multilocus sequence typing (MLST). Isolates harbouring bla(CTX-M-group-1) dominated (66%, n=418) and originated from both animals and cases of human infections in all three countries; 23% (n=144) of all isolates contained both bla(CTX-M-group-1) and bla(OXA-1-like) genes, predominantly from humans (n=127) and UK cattle (n=15). The antimicrobial resistance and virulence gene profiles of this collection of isolates were highly diverse. A substantial number of human isolates (32%, n=87) did not share more than 40% similarity (based on the Jaccard coefficient) with animal isolates. A further 43% of human isolates from the three countries (n=117) were at least 40% similar to each other and to five isolates from UK cattle and one each from Dutch chicken meat and a German dog; the members of this group usually harboured genes such as mph(A), mrx, aac(6')-Ib, catB3, bla(OXA-1-like) and bla(CTX-M-group-1). forty-four per cent of the MLST-typed isolates in this group belonged to ST131 (n=18) and 22% to ST405 (n=9), all from humans. Among animal isolates subjected to MLST (n=258), only 1.2% (n=3) were more than 70% similar to human isolates in gene profiles and shared the same MLST clonal complex with the corresponding human isolates. The results suggest that minimising human-to-human transmission is essential to control the spread of ESBL-positive E. coli in humans.

Epidemic multidrug-resistant (MDR-AmpC) Salmonella enterica serovar Newport strains contain three phage regions and a MDR resistance plasmid.

Multidrug-resistant (MDR-AmpC) Salmonella enterica serovar Newport has caused serious disease in animals and humans in North America, whereas in the UK S. enterica serovar Newport is not associated with severe disease and usually sensitive to antibiotics; MDR S. Newport (not AmpC) strains have only been isolated from poultry. We found that UK poultry strains belonged to MLST type ST166 and were distinct from cattle isolates for being able to utilize D-tagotose and when compared by pulsed-field gel electrophoresis (PFGE), comparative genomic hybridization (CGH) and diversity arrays technology (DArT). Cattle strains belonged to the ST45 complex differing from ST166 at all seven loci. PFGE showed that 19 out of 27 cattle isolates were more than 85% similar to each other and some UK and US strains were indistinguishable. Both CGH and DArT identified genes (including phage-related ones) that were uniquely present in the US isolates and two such genes identified by DArT showed sequence similarities with the pertussis-like (artAB) toxin. This work demonstrates that MDR-AmpC S. Newport from the USA are genetically closely related to pan-susceptible strains from the UK, but contained three extra phage regions and a MDR plasmid.