Pathogenomes of Atypical Non-shigatoxigenic Escherichia coli NSF/SF O157:H7/NM: Comprehensive Phylogenomic Analysis Using Closed Genomes

The toxigenic conversion of Escherichia coli strains by Shiga toxin-converting (Stx) bacteriophages were prominent and recurring events in the stepwise evolution of enterohemorrhagic E. coli (EHEC) O157:H7 from an enteropathogenic (EPEC) O55:H7 ancestor. Atypical, attenuated isolates have been described for both non-sorbitol fermenting (NSF) O157:H7 and SF O157:NM serotypes, which are distinguished by the absence of Stx, the characteristic virulence hallmark of Stx-producing E. coli (STEC). Such atypical isolates either never acquired Stx-phages or may have secondarily lost stx during the course of infection, isolation, or routine subculture; the latter are commonly referred to as LST (Lost Shiga Toxin)-isolates. In this study we analyzed the genomes of 15 NSF O157:H7 and SF O157:NM strains from North America, Europe, and Asia that are characterized by the absence of stx, the virulence hallmark of STEC. The individual genomic basis of the Stx (−) phenotype has remained largely undetermined as the majority of STEC genomes in public genome repositories were generated using short read technology and are in draft stage, posing a major obstacle for the high-resolution whole genome sequence typing (WGST). The application of LRT (long-read technology) sequencing provided us with closed genomes, which proved critical to put the atypical non-shigatoxigenic NSF O157:H7 and SF O157:NM strains into the phylogenomic context of the stepwise evolutionary model. Availability of closed chromosomes for representative Stx (−) NSF O157:H7 and SF O157:NM strains allowed to describe the genomic basis and individual evolutionary trajectories underlying the absence of Stx at high accuracy and resolution. The ability of LRT to recover and accurately assemble plasmids revealed a strong correlation between the strains’ featured plasmid genotype and chromosomally inferred clade, which suggests the coevolution of the chromosome and accessory plasmids. The identified ancestral traits in the pSFO157 plasmid of NSF O157:H7 strain LSU-61 provided additional evidence for its intermediate status. Taken together, these observations highlight the utility of LRTs for advancing our understanding of EHEC O157:H7/NM pathogenome evolution. Insights into the genomic and phenotypic plasticity of STEC on a lineage- and genome-wide scale are foundational to improve and inform risk assessment, biosurveillance, and prevention strategies.