Abstract
Salmonella enterica subspecies enterica serovar Dublin (S. Dublin) figures among the most frequently isolated Salmonella strains in humans in France. This serovar may affect production and animal health mainly in cattle herds with corresponding high economic losses. Given that the current gold standard method, pulsed-field gel electrophoresis (PFGE), provides insufficient discrimination for epidemiological investigations, we propose a standard operating procedure in this study for multiple-locus variable number tandem repeat analysis (MLVA) of S. Dublin, suitable for inter-laboratory surveillance. An in silico analysis on the genome of S. Dublin strains CT_02021853 was performed to identify appropriate microsatellite regions. Of 21 VNTR loci screened, six were selected and 401 epidemiologically unrelated and related strains, isolated from humans, food and animals were analyzed to assess performance criteria such as typeability, discriminatory power and epidemiological concordance. The MLVA scheme developed was applied to an outbreak involving Saint-Nectaire cheese for which investigations were conducted in France in 2012, making it possible to discriminate between epidemiologically related strains and sporadic case strains, while PFGE assigned only a single profile. The six loci selected were sequenced on a large set of strains to determine the sequence of the repeated units and flanking regions, and their stability was evaluated in vivo through the analysis of the strains investigated from humans, food and the farm environment during the outbreak. The six VNTR selected were found to be stable and the discriminatory power of the MLVA method developed was calculated to be 0.954 compared with that for PFGE, which was only 0.625. Twenty-four reference strains were selected from the 401 examined strains in order to represent most of the allele diversity observed for each locus. This reference set can be used to harmonize MLVA results and allow data exchange between laboratories. This original MLVA protocol could be used easily and routinely for monitoring of serovar Dublin isolates and for conducting outbreak investigations.
Highlights
In order to assess the discriminatory power of the multiple-locus variable number tandem repeat analysis (MLVA) protocol developed here and the stability of tandem repeats (TRs), we used a set of 401 strains isolated between 1929 and 2015 from three different collections (Salmonella Network, National Reference Centre for Salmonella and Centre of Expertise for the Food Industry)
The strains isolated from animals and food sources were collected by the Salmonella Network of ANSES and the Centre of Expertise for the Food Industry (Actalia); the clinical strains came from the National Reference Centre for Salmonella at Institut Pasteur in Paris
Within the panel of 401 strains used for the development of the MLVA scheme, 51 Salmonella Dublin strains, isolated from 2002 to 2011, were pulsed-field gel electrophoresis (PFGE) subtyped according to a standardized protocol (PulseNet, 2013, EU) with some modifications in the composition of the cell lysis buffer and the concentration of the enzyme XbaI (Sigma–Aldrich, France)
Summary
In order to assess the discriminatory power of the MLVA protocol developed here and the stability of tandem repeats (TRs), we used a set of 401 strains isolated between 1929 and 2015 from three different collections (Salmonella Network, National Reference Centre for Salmonella and Centre of Expertise for the Food Industry). This panel of strains comprised human and food isolates recovered in the framework of a FBO investigation that occurred in 2012 in France. Dublin associated with raw milk cheeses were reported over the summer months of 2016
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