Enumeration and Genomic Confirmation of Viable Shiga-Toxin-Producing Escherichia coli from Ground Beef by Integrating Serial Plating with Long-Read Sequencing

Consumption of food contaminated with Shiga toxin-producing Escherichia coli (STEC) causes thousands of illnesses in the United States annually. Long-read sequencing could 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 homogenized with a stomacher. 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 single colonies were subsequently sequenced. Initial experiments revealed that processing samples at room temperature led to a 1 log increase in STEC levels from the initial inoculated concentration, confounding accurate enumeration. In subsequent experiments, samples and reagents were kept 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. Quantification of STEC in food samples using this method would improve risk assessment and guide mitigation efforts in industry.