Whole genome sequencing and protein structure analyses of target genes for the detection of Salmonella.

Rapid and sensitive detection of Salmonella is a critical step in routine food quality control, outbreak investigation, and food recalls. Although various genes have been the targets in the design of rapid molecular detection methods for Salmonella, there is limited information on the diversity of these target genes at the level of DNA sequence and the encoded protein structures. In this study, we investigated the diversity of ten target genes (invA, fimA, phoP, spvC, and agfA; ttrRSBCA operon including 5 genes) commonly used in the detection and identification of Salmonella. To this end, we performed whole genome sequencing of 143 isolates of Salmonella serotypes (Enteritidis, Typhimurium, and Heidelberg) obtained from poultry (eggs and chicken). Phylogenetic analysis showed that Salmonella ser. Typhimurium was more diverse than either Enteritidis or Heidelberg. Forty-five non-synonymous mutations were identified in the target genes from the 143 isolates, with the two most common mutations as T ↔ C (15 times) and A ↔ G (13 times). The gene spvC was primarily present in Salmonella ser. Enteritidis isolates and absent from Heidelberg isolates, whereas ttrR was more conserved (0 non-synonymous mutations) than ttrS, ttrB, ttrC, and ttrA (7, 2, 2, and 7 non-synonymous mutations, respectively). Notably, we found one non-synonymous mutation (fimA-Mut.6) across all Salmonella ser. Enteritidis and Salmonella ser. Heidelberg, C → T (496 nt postion), resulting in the change at AA 166 position, Glutamine (Q) → Stop condon (TAG), suggesting that the fimA gene has questionable sites as a target for detection. Using Phyre2 and SWISS-MODEL software, we predicted the structures of the proteins encoded by some of the target genes, illustrating the positions of these non-synonymous mutations that mainly located on the α-helix and ß-sheet which are key elements for maintaining the conformation of proteins. These results will facilitate the development of sensitive molecular detection methods for Salmonella.

Antimicrobial resistance and related gene analysis of Salmonella from egg and chicken sources by whole-genome sequencing.

Whole-genome sequencing (WGS) is a valuable tool in research on foodborne pathogens. In this study, a total of 143 isolates of Salmonella serotypes Enteritidis, Typhimurium, and Heidelberg sourced from eggs and chickens were analyzed for their antimicrobial resistance profiles using WGS data. The isolates carried high rate of genes resistant to aminoglycoside (70.63%), tetracycline (26.57%), fosfomycin (25.17%), sulfonamides (23.78%), and ß-lactamases (15.38%); and aadA was the most frequently observed antimicrobial resistance gene (ARG). Antimicrobial resistance varies by Salmonella serotypes, with Salmonella enterica serovar Enteritidis (Salmonella ser. Enteritidis) isolates being highly resistant to aminoglycoside (particularly streptomycin); Salmonella ser. Typhimurium more resistant to aminoglycoside, tetracycline, and sulfonamides; and Salmonella ser. Heidelberg more resistant to aminoglycoside and fosfomycin. Salmonella ser. Typhimurium isolates presented more varieties of ARG than Salmonella ser. Enteritidis and Salmonella ser. Heidelberg. Our data showed that 5 isolates of Salmonella ser. Typhimurium and Salmonella ser. Heidelberg contained ARG resistant to ≥ 5 antimicrobials. In addition, 23 Salmonella isolates carried ARG resistant to 4 antimicrobials.

Development of Models To Relate Microbiological and Headspace Volatile Parameters in Stored Atlantic Salmon to Acceptance and Willingness To Prepare the Product by Senior Consumers.

Microbial spoilage of salmon occurs during extended refrigerated storage and is often accompanied by unpleasant aromas. When spoilage is detected, it is assumed that consumers will reject the product for consumption. Because sensory panels of trained individuals or consumers are expensive and labor intensive, identification of microbiological or chemical indicators to characterize the extent to which fish has spoiled is needed when experimental process and storage treatments are being evaluated. A consumer panel of 53 senior citizens (60 to 85 years of age) evaluated in duplicate raw salmon subjected to 10 storage conditions, and the fish quality was targeted to range from fresh to very spoiled. This population group was chosen because they would be expected to have a greater prevalence of olfactory impairments and higher odor thresholds than the general population; in turn, a shorter safety margin or time period between product rejection due to spoilage and the generation of Clostridium botulinum toxins would be likely. Low hedonic scores for aroma and overall acceptance (2 or 3 of 9), corresponding to "dislike very much" to "dislike moderately," did not equate with unwillingness to prepare the sample for consumption by up to seven panelists (13%) when the product was presumed to have already been purchased. Despite these outliers, significant models (P = 0.0000) were developed for the willingness of consumers to prepare the sample for consumption and the sample's aerobic and anaerobic microbiological populations and two volatile peaks with Kovats indices of 640 and 753. However, these models revealed that the levels of microbiological and chemical markers must be very high before some consumers would reject the sample; hence, spoilage detection by smell would likely not be an adequate safeguard against consuming salmon in which C. botulinum toxin had been generated.

Clostridium botulinum Toxin Production in Relation to Spoilage of Atlantic Salmon (Salmo salar) Packaged in Films of Varying Oxygen Permeabilities and with Different Atmospheres.

Shelf life of fish packaged under modified atmosphere (MA) is extended, but within the United States, commercial application of MA with impermeable packaging films is restricted due to concerns that botulinum toxin production would precede spoilage when contaminated fish are held at abusive storage temperatures. Use of semipermeable packaging films has been advocated; however, previous studies are inconclusive in determining the oxygen transmission rate (OTR) of a film that is needed to achieve an acceptable margin of safety (i.e., toxin production occurs only after spoilage). This study was conducted to determine the influence of OTR (target OTRs of 3 to 15,000) on the development of spoilage volatiles and toxin in salmon inoculated with type E Clostridium botulinum and subjected to air, vacuum, or 75:25 CO2:N2 MA and storage temperatures of 4, 8, 12, or 16°C. The most dominant headspace volatile peak that was produced during spoilage of samples at 4, 8 or 12°C was a peak, having a Kovats retention index (KI) of 753, and at which external standards of 2- or 3-methyl 1-butanol also eluted. Under anaerobic conditions, both the aerobic microbial populations and the size of the KI 753 spoilage peak were less in inoculated samples compared with uninoculated samples. C. botulinum-inoculated samples that were stored at 12 or 16°C under conditions favorable for anaerobic growth were also characterized by a KI 688 peak. Using a previously developed model that related the percentage of elderly consumers who would prepare a sample having the KI 753 spoilage peak of a specific size, it was determined that for salmon packaged with 3 or 3,000 OTR films under any atmosphere and stored at 12 or 16°C, 2 to 61% of the consumers could potentially prepare toxin-contaminated samples. Hence, when abusive storage conditions are suspected, the fish should not be consumed.