Differentiation of Salmonella serotypes in meat using long-read sequencing

Authors: Counihan, Katrina; Tilman, Shannon

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/19/2025
Publication Date: N/A
Citation: N/A

Interpretive Summary:

Technical Abstract: Foodborne pathogens are a serious cause of illness in the United States that can result in life-threatening complications. In previous studies, we have shown that long-read, whole genome sequencing could significantly reduce the time needed to identify Escherichia coli O157:H7 in ground beef as compared to current methods. However, detection of Salmonella in food may be much more complex due to the existence of over 2600 serotypes with high genetic similarity. Therefore, the goal of this project was to evaluate the ability of various bioinformatic pipelines to accurately identify Salmonella serotypes when present in a mixture. Salmonella serotype Typhimurium and serotype Enteritidis were grown overnight in buffered peptone water for use in experiments. The DNA was extracted from each serotype individually and after being mixed 1:1 and sequenced using an Oxford Nanopore GridION for 24 hours. Ground beef was also inoculated with 100-103 cfu g-1 of each serotype or an equal mixture. The inoculated meat was diluted 1:4 with modified tryptone soy broth (mTSB), stomached, and incubated overnight at 42°C. The liquid was removed, filtered, centrifuged, and the DNA extracted from the pellet for sequencing on a GridION for 24 hours. The data was uploaded to Galaxy, underwent quality control, and then reads or Flye assemblies polished with Medaka were analyzed with SeqSero2, SISTR, or Masher to evaluate what Salmonella serotypes were identified in the samples. All of the tools were able to identify S. Typhimurium and S. Enteritidis when inoculated individually into meat at concentrations as low as 101 cfu g-1. However, only Masher was able to identify both serotypes when they were inoculated as a mixture into meat. Analyzing assemblies improved accuracy and reduced false identification of other serotypes. The ability to identify multiple Salmonella serotypes from an overnight enrichment could significantly reduce the time needed for testing. Additionally, Masher has the ability to identify multiple bacterial species, not just Salmonella, which would allow multiple pathogens to be targeted with one sequencing run.