Comparison of hybrid-and mono-pathotype Escherichia coli isolates from South Korea based on whole genome analysis and cytotoxicity assay

BACKGROUND: Among intestinal pathogenic Escherichia coli, hybrid pathotypes carrying virulence determinants of multiple classical pathotypes have recently been identified. METHODS: A total of 292 E. coli isolates were collected from human, livestock, and environmental sources in South Korea, and their whole genome sequences were determined. They were classified into pathotypes based on virulence gene profiles. We constructed a phylogenetic tree based on cgSNPs. Synteny analysis on the LEE pathogenicity island was performed, and enriched accessory gene repertoires in particular hybrid-pathotypes were identified. Bacterial virulence was measured by lactate dehydrogenase (LDH) cytotoxicity assays. Genetic factors associated with increased cytotoxicity in the LDH assay were identified using SPLASH. RESULTS: Among 192 intestinal pathogenic E. coli isolates, 63 hybrid pathotype isolates (38 STEC/EPEC, 23 STEC/ETEC, and 2 STEC/EAEC) were identified. Two main groups of STEC/EPEC hybrid-pathotype isolates and one major STEC/ETEC group were identified. Both groups of STEC/EPEC hybrid isolates were closely associated with EPEC mono-pathotype isolates, and most STEC/ETEC hybrid groups clustered with STEC mono-pathotype isolates. Synteny analysis on the LEE pathogenicity island also showed that some STEC/EPEC hybrids might originate from EPEC genomic backbone. Based on the LDH assay, cytotoxicity was higher in STEC/ETEC, but lower in STEC/EPEC. Five gene modules (adhesion-pili, plasmid conjugation, flagella-motility, iron uptake, and T3SS) were identified to be genetic factors correlated with increased cytotoxicity in STEC/ETEC. CONCLUSION: High prevalence of hybrid-pathotype E coli isolates was identified. They might emerge repeatedly by independent incorporation of additional virulence factors, and genomic backbones and cytotoxicity differed according to the specific hybrid pathotype.