Molecular Prediction of the O157:H-Negative Phenotype Prevalent in Australian Shiga Toxin-Producing Escherichia coli Cases Improves Concordance of In Silico Serotyping with Phenotypic Motility

Abstract

Shiga toxin-producing (STEC) is a foodborne pathogen, and serotype O157:H7 is typically associated with severe disease. Australia is unique in its STEC epidemiology, as severe cases are typically associated with non-O157 serogroups, and locally acquired O157 isolates are H-negative/nonmotile. TheH-negative phenotype and reduced severity of disease compared to that associated with H7/motile strains are distinct features of Australian O157 strains, but the molecular mechanism behind this phenotype has not been reported. Accurate characterization of the H-negative phenotype is important in epidemiological surveillance of STEC. Serotyping is moving away from phenotype-based methods, as next generation sequencing allows rapid extrapolation of serotype through detection of the O-antigen processing genes,, ,, and , and the H-antigen gene, The detection and genotyping of alone is unable to determine the motility of the strain. Typically, most Australian O157:H-negative strains carry an H7 genotype yet phenotypically are nonmotile; thus, many are mischaracterized as H7 strains by serotyping tools. Comparative genomic analysis of flagellar genes between Australian and international isolates was performed and an insertion at nucleotide (nt) 125 in the gene was identified in H-negative isolates. Chi-square results showed that this insertion was significantly associated with the H-negative phenotype ( < 0.0001). Phylogenetic analysis was also completed and showed that the Australian H-negative isolates with the insertion in represent a clade within the O157 serogroup, distinct from O157:H7 serotypes. This study provides a genetic target for inferring the nonmotile phenotype of Australian O157 STEC, which increases the predictive value of serotyping.

Core and Accessory Genome Comparison of Australian and International Strains of O157 Shiga Toxin-Producing Escherichia coli

“…Surprisingly, the United Kingdom 2015 isolates appear to be most closely related to the Australian clinical isolates, rather than to any of the international isolates. All United Kingdom isolates also carry the single cytosine base insertion in flgF at 125nt, a feature described previously for Australian O157 STEC (Pintara et al, 2018;Ingle et al, 2019). The apparent phylogenetic similarity between the Australian clinical and United Kingdom isolates as shown in Figure 1, despite their geographical difference, may be likely due to the lineage shared by the strains.…”

“…Whole genome sequencing of all the study isolates was done using previously described methods (Pintara et al, 2018).…”

“…We have shown that an O157 clone prevalent in Australia clinical cases contains a unique single base insertion potentially responsible for its non-motile phenotype (Pintara et al, 2018), and in this study, we seek to improve our understanding of genotypes associated with different incidences and severity of disease by comparing this Australian O157 clone with strains related to the United Kingdom 2015 outbreak and additional international O157 STEC strains. Multiple genetic analyses were performed to determine differences between the core and accessory genomes of Australian and international STEC strains.…”

“…Australian STEC epidemiology differs from many other countries, as severe outbreaks and HUS cases appear to be more often associated with non-O157 serogroups ( Paton et al, 1996 , 1999 ; McPherson et al, 2009 ; Vally et al, 2012 ). Australian O157 STEC strains are also predominantly non-motile, therefore having an O157:H- serotype, and this has been associated with a single insertion in the flgF gene ( Pintara et al, 2018 ). This is exemplified by two of Australia’s largest outbreaks; an outbreak that occurred in 1995 in South Australia, and a 2013 outbreak that was associated with an annual agricultural show in Queensland ( Centers for Disease Control and Prevention, 1995 ; Vasant et al, 2017 ).…”

Emergence and divergence of major lineages of Shiga-toxin-producing Escherichia coli in Australia

“…The binary data was processed in R using tidyverse (v1.2.1) (https://CRAN.R-project.org/package=tidyverse). The insertion of a cytosine base in position 125 in flgF associated with a H-non-motile phenotype [49] in Australian CG11 isolates from human cases between 2007–2016 was investigated from the Snippy output by manually identifying insertions at this site.…”