Serotype screening of salmonella enterica subspecies I by intergenic sequence ribotyping (ISR): Critical updates

Authors: Guard, Jean; Jones, Deana; Gast, Richard; Garcia, Javier; Rothrock, Michael

Submitted to: Microorganisms
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/21/2022
Publication Date: 12/30/2022
Citation: Guard, J.Y., Jones, D.R., Gast, R.K., Garcia, J.S., Rothrock Jr, M.J. 2022. Serotype screening of salmonella enterica subspecies I by intergenic sequence ribotyping (ISR): Critical updates. Microorganisms. 11(1):97. https://doi.org/10.3390/microorganisms11010097.
DOI: https://doi.org/10.3390/microorganisms11010097

Interpretive Summary: Domestic and International industries producing food for consumption by people and animals have great responsibility to produce safe products free of contamination by infectious agents. Salmonella is a persistent threat to the safety of food and regulations are in place to assure regular submission of products and oversight of agricultural commodities for the worst offending strains such as Salmonella serotypes Enteritidis, Typhimurium, Newport, Heidelberg, and Javiana. Regulatory agencies such as the Food Safety Inspection Service and the Food and Drug Administration are now employing whole genome sequencing (WGS) and closely related DNA-based technologies such as multilocus sequence targeting (MLST) to track dangerous serotypes and to trace sources of contamination. However, agricultural industries need a simpler Salmonella serotype screening method to facilitate in-house testing for the worst offending serotypes so that interventions can be applied quickly and consistently across time. The ideal method should be relatively inexpensive as compared to WGS, able to be conducted as quality control measures within in-house laboratories with a minimal need for equipment and bioinformatics software, confidential, and secure. Intergenic Sequence Ribotyping (ISR) was released in 2012 for application as a screening method for Salmonella serotype that is free, available, and requires no formal interaction with government agencies. The WGS database at the National Center for Biotechnology (NCBI) increased its number of whole genomes exponentially since 2012. Therefore, the ISR database was updated in this study to include new information at NCBI. The efficacy of primers for producing de novo sequence was tested, issues in data interpretation were addressed, and guidance about further processing for 7% of sequences requiring additional processing was provided. The 2022 ISR database is included in FASTA format and now has 268 separate sequences that can be used to screen for Salmonella serotypes that threaten the safety of the food supply.

Technical Abstract: Abstract: (1) Background: Foodborne illness from Salmonella enterica subspecies I is most associated with approximately 32 out of 1,600 serotypes. While whole genome sequencing and other nucleic acid-based methods are preferred for serotyping, they require expertise in bioinformatics. Intergenic Sequence Ribotyping (ISR) assigns serotype to Salmonella in coordination with the National Center for Biotechnology Information. ISR requires updating because it was developed from 26 genomes but there are currently 1,804 genomes and 1,685 plasmids. (2) Methods: Serotypes available for sequencing were analyzed by ISR to confirm primers efficacy and to identify any issues in application. Differences between the 2012 and 2022 ISR database were tabulated, nomenclature edited, and instances of multiple serotypes aligning to a single ISR were examined. (3) Results: The 2022 ISR database has 268 sequences, 40 ISR sequences were assigned new NCBI accession numbers, and 100% of the 2012 database was retained with some modifications. Extending boundaries of sequences resolved hdfR cross-alignment and reduced multiplicity of alignment with a single ISR. Comparison of gene cyaA sequences and some cell surface epitopes provided evidence that homologous recombination was potentially impacting results. (4) The 2022 ISR database is available for use as a serotype screening method for Salmonella enterica subspecies I.