Combination of serial plating and long-read sequencing to detect and quantify live Shiga-toxin producing Escherichia coli

Authors: Counihan, Katrina; TILMAN, SHANNON - Former ARS Employee

Submitted to: Pathogens
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/21/2026
Publication Date: 5/27/2026
Citation: Counihan, K.L., Tilman, S. 2026. Combination of serial plating and long-read sequencing to detect and quantify live Shiga-toxin producing Escherichia coli. Pathogens. https://doi.org/10.3390/pathogens15060573.
DOI: https://doi.org/10.3390/pathogens15060573

Interpretive Summary: Eating food contaminated with Shiga toxin-producing Escherichia coli (STEC) causes millions of illnesses in the United States every year. Long-read sequencing could be used to reduce the time needed to test food for STEC, but sequencing cannot determine how many STEC are in a sample or whether they are alive or dead. Therefore, this study combined serial plating with long-read sequencing to quantify only live STEC in a sample. Ground beef was mixed with STEC and then the bacteria in the sample were concentrated. The concentrated bacteria were diluted in standard amounts of liquid and plated on a medium that selects for STEC. It was determined that the samples had to be kept cold during processing to prevent the STEC from growing. The amount of STEC in a sample could be successfully determined, and very low amounts could be recovered. Sequencing also identified the correct type of E. coli in the samples. This method could be used by industry to improve the safety of food for consumers.

Technical Abstract: 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.