Improved Genomic Identification, Clustering, and Serotyping of Shiga Toxin-Producing Escherichia coli Using Cluster/Serotype-Specific Gene Markers

Shiga toxin-producing Escherichia coli (STEC) have more than 470 serotypes. The well-known STEC O157:H7 serotype is a leading cause of STEC infections in humans. However, the incidence of non-O157:H7 STEC serotypes associated with foodborne outbreaks and human infections has increased in recent years. Current detection and serotyping assays are focusing on O157 and top six (“Big six”) non-O157 STEC serogroups. In this study, we performed phylogenetic analysis of nearly 41,000 publicly available STEC genomes representing 460 different STEC serotypes and identified 19 major and 229 minor STEC clusters. STEC cluster-specific gene markers were then identified through comparative genomic analysis. We further identified serotype-specific gene markers for the top 10 most frequent non-O157:H7 STEC serotypes. The cluster or serotype specific gene markers had 99.54% accuracy and more than 97.25% specificity when tested using 38,534 STEC and 14,216 non-STEC E. coli genomes, respectively. In addition, we developed a freely available in silico serotyping pipeline named STECFinder that combined these robust gene markers with established E. coli serotype specific O and H antigen genes and stx genes for accurate identification, cluster determination and serotyping of STEC. STECFinder can assign 99.85% and 99.83% of 38,534 STEC isolates to STEC clusters using assembled genomes and Illumina reads respectively and can simultaneously predict stx subtypes and STEC serotypes. Using shotgun metagenomic sequencing reads of STEC spiked food samples from a published study, we demonstrated that STECFinder can detect the spiked STEC serotypes, accurately. The cluster/serotype-specific gene markers could also be adapted for culture independent typing, facilitating rapid STEC typing. STECFinder is available as an installable package (https://github.com/LanLab/STECFinder) and will be useful for in silico STEC cluster identification and serotyping using genome data.

Nitric Oxide Induced stx2 Expression Is Inhibited by the Nitric Oxide Reductase, NorV, in a Clade 8 Escherichia coli O157:H7 Outbreak Strain

Escherichia coli O157:H7 pathogenesis is due to Shiga toxin (Stx) production, though variation in virulence has been observed. Clade 8 strains, for instance, were shown to overproduce Stx and were more common among hemolytic uremic syndrome cases. One candidate gene, norV, which encodes a nitric oxide (NO) reductase found in a clade 8 O157:H7 outbreak strain (TW14359), was thought to impact virulence. Hence, we screened for norV in 303 O157 isolates representing multiple clades, examined stx2 expression following NO exposure in TW14359 for comparison to an isogenic mutant (ΔnorV), and evaluated survival in THP-1 derived macrophages. norV was intact in strains representing clades 6–9, whereas a 204 bp deletion was found in clades 2 and 3. During anaerobic growth, NO induced stx2 expression in TW14359. A similar increase in stx2 expression was observed for the ΔnorV mutant in anaerobiosis, though it was not impaired in its ability to survive within macrophages relative to TW14359. Altogether, these data suggest that NO enhances virulence by inducing Stx2 production in TW14359, and that toxin production is inhibited by NorV encoded by a gene found in most clade 8 strains. The mechanism linked to these responses, however, remains unclear and likely varies across genotypes.

Prevalence of non-O157 Shiga Toxin-producing E. Coli in Children and Calves in Al- Muthanna Province, Iraq

The present study focus on non-O157 Shiga toxin-producing E. Coli (STEC), included a bacteriological study was subjected to provide additional information for non-O157 STEC prevalence in children and calves. Isolation by using selective culturing media (CHROMagar STEC and CHROMagar O157) from 127 children suffering from diarrhea and 133 calves in Al- Muthanna province. Characterization depends on culturing positive colony on MacConkey agar and Levin’s Eosin Methylene blue agar, staining single colony from the growth by gram stain, biochemical tests; Indole, the Methyl Red, Voges-Proskauer, Citrate test, Oxidase, Catalase, Urease, Motility, Kligler Iron and Api-20E, were done to confirm a diagnosis of non-O157 STEC, The reliable isolation as non-O157 STEC serotyping by specific latex agglutination test for the target non-O157 STEC (big six) serogroup (O26, O45, O103, O111, O121 and O145). The current study showed the prevalence of non-O157 STEC was 20 of out 127 (15.73%) in samples collected from children and 27 / 133 (20.30%) in calves samples in conclusion the Non-O157 STEC is an important cause of diarrhea in children, and calves; finally, the calves play an important reservoir for Non-O157 STEC.