Precision long-read metagenomics sequencing for food safety by detection and assembly of Shiga toxin-producing Escherichia coli in irrigation water

• Published in: PloS one 2021-01, Vol.16 (1), p.e0245172-e0245172
• Main Authors: Maguire, Meghan, Kase, Julie A, Roberson, Dwayne, Muruvanda, Tim, Brown, Eric W, Allard, Marc, Musser, Steven M, González-Escalona, Narjol
• Format: Article
• Language: English
• Subjects: Antimicrobial agents; Bacterial toxins; Biology and Life Sciences; Cefixime; Computer programs; DIABLO protein; Drafting software; Drug resistance; E coli; Editing; Environmental aspects; Escherichia coli; Escherichia coli O157 - classification; Escherichia coli O157 - genetics; Food; Food Safety; Foodborne Diseases - genetics; Foodborne Diseases - microbiology; Foodborne pathogens; Funding; Genes; Genomes; Genomics; Health aspects; Hemolytic uremic syndrome; Humans; Identification and classification; Inspection; Irrigation; Irrigation water; Low concentrations; Medicine and Health Sciences; Metagenome; Metagenomics; Methodology; Methods; Microbiological research; Nutrition; Porosity; Regulatory agencies; Research and Analysis Methods; Reviews; Safety; Safety and security measures; Selective plating; Shiga toxin; Software; Sorbitol; Tellurite; Tellurium dioxide; Toxins; Virulence; Water; Water analysis; Water Microbiology; Water sampling; Yeasts
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0245172

Shiga toxin-producing Escherichia coli (STEC) contamination of agricultural water might be an important factor to recent foodborne illness and outbreaks involving leafy greens. Closed bacterial genomes from whole genome sequencing play an important role in source tracking. We aimed to determine the limits of detection and classification of STECs by qPCR and nanopore sequencing using 24 hour enriched irrigation water artificially contaminated with E. coli O157:H7 (EDL933). We determined the limit of STEC detection by qPCR to be 30 CFU/reaction, which is equivalent to 105 CFU/ml in the enrichment. By using Oxford Nanopore's EPI2ME WIMP workflow and de novo assembly with Flye followed by taxon classification with a k-mer analysis software (Kraken2), E. coli O157:H7 could be detected at 103 CFU/ml (68 reads) and a complete fragmented E. coli O157:H7 metagenome-assembled genome (MAG) was obtained at 105-108 CFU/ml. Using a custom script to extract the E. coli reads, a completely closed MAG was obtained at 107-108 CFU/ml and a complete, fragmented MAG was obtained at 105-106 CFU/ml. In silico virulence detection for E. coli MAGs for 105-108 CFU/ml showed that the virulotype was indistinguishable from the spiked E. coli O157:H7 strain. We further identified the bacterial species in the un-spiked enrichment, including antimicrobial resistance genes, which could have important implications to food safety. We propose this workflow provides proof of concept for faster detection and complete genomic characterization of STECs from a complex microbial sample compared to current reporting protocols and could be applied to determine the limit of detection and assembly of other foodborne bacterial pathogens.