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Consumption of food contaminated with Shiga toxin-producing Escherichia coli (STEC) causes millions of illnesses in the United States annually. Long-read sequencing could be used to reduce the time needed to test food for STEC, but sequencing is not quantitative and cannot differentiate between live and dead bacteria. Therefore, this study investigated combining serial plating with long-read sequencing to quantify only live STEC in a sample. Ground beef was inoculated with STEC and stomached. The liquid was filtered to remove eukaryotic cells and then centrifuged to pellet the bacteria. Serial dilutions of the pellet were plated on selective agar and then single colonies were selected for sequencing. During initial experiments where sample processing was conducted at room temperature, a 1 log increase in STEC concentration from the initial inoculated amount was observed. Subsequent experiments kept samples and reagents cold, and the amount of STEC recovered did not significantly differ from the amount inoculated. The DNA extracted from a single colony provided enough data to identify all virulence genes of interest multiple times. The amount of STEC in a sample could be quantified down to 1 cfu g-1. The ability of this method to quantify STEC in samples would improve risk assessment and guide mitigation in industry.

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