Targeted Next-Generation Sequencing Assay for Direct Detection and Serotyping of Salmonella from Enrichment.

In this study, an automated, targeted next-generation sequencing (tNGS) assay to detect and serotype Salmonella from sample enrichments was evaluated. The assay generates millions of reads to detect multiple Salmonella-specific genes and serotype-specific alleles, detecting all Salmonella spp. tested to date, and serotyping 62 common Salmonella serotypes. Accuracy was tested on 291 pure reference cultures (251 Salmonella, 40 non-Salmonella), 21 artificially contaminated poultry carcass rinse samples, and 363 naturally contaminated poultry environmental samples. Among the 291 pure reference cultures, the automated tNGS assay resulted in 100% detection accuracy, 100% serotyping accuracy for the claimed serotypes, and 0% false positives. The limit of detection was estimated at 5 × 104 CFU/mL by testing enumerated cultures of strains representative of six serotypes. In cocontamination studies with mixtures of two serotypes (Enteritidis, Typhimurium, Kentucky, Infantis, and Newport) at a 1:1 ratio, tNGS detected both serotypes with 100% accuracy. The assay demonstrated 100% accuracy in artificially contaminated poultry carcass rinse sample enrichments. Targeted NGS was highly effective in detecting Salmonella in samples collected from poultry production facilities. Results demonstrated that tNGS could detect Salmonella and provide accurate serotyping information consistent with conventional serology. These findings highlight the reliable and efficient performance of a fully automated tNGS Salmonella assay in detecting and identifying Salmonella strains in complex matrices, reducing the time to results from 4 to 5 days required by the traditional isolation and serotyping to 10-12 h for tNGS after primary enrichment.

Regional Salmonella Differences in United States Broiler Production from 2016 to 2020 and the Contribution of Multiserovar Populations to Salmonella Surveillance.

Poultry remains a considerable source of foodborne salmonellosis despite significant reduction of Salmonella incidence during processing. There are multiple entry points for Salmonella during production that can lead to contamination during slaughter, and it is important to distinguish the serovars present between the different stages to enact appropriate controls. National Salmonella data from the U.S. Department of Agriculture-Food Safety Inspection Service (USDA-FSIS) monitoring of poultry processing was analyzed from 2016 to 2020. The overall Salmonella incidence at processing in broiler carcasses and intact parts (parts) decreased from 9.00 to 6.57% over this period. The incidence in parts was higher (11.15%) than in carcasses (4.78%). Regional differences include higher proportions of serovars Infantis and Typhimurium in the Atlantic and higher proportion of serovar Schwarzengrund in the Southeast. For Georgia, the largest broiler-producing state, USDA-FSIS data were compared to Salmonella monitoring data from breeder flocks over the same period, revealing serovar Kentucky as the major serovar in breeders (67.91%) during production but not at processing, suggesting that it is more effectively removed during antimicrobial interventions. CRISPR-SeroSeq was performed on breeder samples collected between 2020 and 2021 to explain the incongruence between pre- and postharvest and showed that 32% of samples contain multiple serovars, with up to 11 serovars found in a single flock. High-resolution sequencing identifies serovar patterns at the population level and can provide insight to develop targeted controls. The work presented may apply to other food production systems where Salmonella is a concern, since it overcomes limitations associated with conventional culture. IMPORTANCE Salmonella is a leading cause of bacterial foodborne illness in the United States, with poultry as a significant Salmonella reservoir. We show the relative decrease in Salmonella over a 5-year period from 2016 to 2020 in processed chicken parts and highlight regional differences with respect to the prevalence of clinically important Salmonella serovars. Our results show that the discrepancy between Salmonella serovars found in pre- and postharvest poultry during surveillance are due in part by the limited detection depth offered by traditional culture techniques. Despite the reduction of Salmonella at processing, the number of human salmonellosis cases has remained stable, which may be attributed to differences in virulence among serovars and their associated risk. When monitoring for Salmonella, it is imperative to identify all serovars present to appropriately assess public health risk and to implement the most effective Salmonella controls.