Location: Produce Safety and Microbiology ResearchTitle: Draft genome sequences of Shiga toxin-producing Escherichia coli O157:H7 strains recovered from a major production region for leafy greens in California
Submitted to: Microbiology Resource Announcements
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/7/2019
Publication Date: 7/3/2019
Citation: Quiñones, B., Yambao, J.C., Silva, C.J., Lee, B.G. 2019. Draft genome sequences of Shiga toxin-producing Escherichia coli O157:H7 strains recovered from a major production region for leafy greens in California. Microbiology Resource Announcements. 8(27):e00644-19. https://doi.org/10.1128/MRA.00644-19.
Interpretive Summary: Shiga toxin-producing Escherichia coli (STEC) are responsible for serious foodborne disease outbreaks. Of the many serotypes, O157:H7 is notorious for being commonly associated with the development of severe human illnesses that include hemorrhagic colitis as well as hemolytic uremic syndrome, a life-threatening condition. Serotype O157:H7 is responsible for 36% of the approximately 265,000 U.S. STEC infections each year with an economic burden of $405 million. A key virulence factor in STEC strains contributing to the development of severe human illness are Shiga toxins. Cattle are the primary reservoir for O157:H7, but over the past decade, the consumption of leafy vegetables has been significantly linked to foodborne illnesses caused by O157:H7 serotype. In the present study, whole genome sequencing was performed to obtain draft genome assemblies of nine O157:H7 cattle strains, recovered from a major production region for leafy greens in California’s Central Coast. The O157:H7 cattle strains were all collected during a short time period and a discrete sampling location. Preliminary routine screening revealed that all O157:H7 strains were positive for the Shiga toxin gene but further analysis of Shiga toxin activity demonstrated that some strains did not induce toxicity in human cells. The use of genome sequencing allowed us to differentiate between strains with a precision that other molecular technologies do not allow. High resolution genome sequencing enabled the identification of insertion sequences in the O157:H7 E. coli chromosome that inactivated the expression of the Shiga toxin gene. These findings provided an explanation for the observed variability in toxin activity, which can consequently lead to the attenuation of the severity of human infections caused by STEC.
Technical Abstract: Shiga toxin-producing Escherichia coli (STEC) is an enteric pathogen responsible for human gastroenteritis. In some cases, human infections progress to hemolytic uremic syndrome, a life-threatening disease due to the production of Shiga toxins (Stx). By using a robust isolation method, STEC O157:H7 strains were recovered from cattle in a major agricultural region for leafy greens and were identified based on a typical STEC colony morphology on selective chromogenic media and to be PCR-positive for the stx gene. Further proteomic analyses indicated that all O157:H7 cattle strains were found to express the toxin receptor binding B-subunit, but some were found not have a functional toxin. The genome sequencing of nine O157:H7 cattle strains with various levels of toxin activity is reported here. Genome sequence comparison showed all O157:H7 cattle strains were highly similar and had an average genome size of about 5,441,700 bp with either a 50.5% or 50.6% G+C content. Analysis of methylation patterns revealed DNA motifs GATC, GAAABCC, CACNNNNNNNCTGG and CCAGNNNNNNNGTG to be 98-99% methylated. The cattle strains harbored only one copy of the stx2c-encoding prophage, which was inserted in the chromosomal sbcB gene, an insertion site commonly used by stx2c prophages in virulent O157:H7 strains. Only strain RM10024 tested positive for cytotoxic activity, but all other O157:H7 cattle strains tested negative due to the presence of the insertion sequence variant, IS1203v, in the coding sequence of the catalytic A-subunit of Stx. The information provided by whole-genome sequencing of O157:H7 cattle strains, recovered during a short time period and discrete sampling location in a major agricultural region, have provided an explanation for the variability in Stx activity and have set the foundation for future studies on the persistence of strains with an attenuated pathogenic potential.