Phenotypic and Genotypic Characterisation of Escherichia coli O26 Strains Isolated from Cattle and Clinical sources in Australia

Abstract

Escherichia coli (E. coli) O26 is the second most common serogroup of enterohaemorrhagic E. coli (EHEC) implicated in foodborne illness globally with outbreaks occasionally linked to the consumption of cattle related food products. This study was performed to gain insights into the phenotypic and genotypic characteristics of E. coli O26 circulating in Australian cattle and those strains from clinical cases. Initial characterisation determined that E. coli O26 are a genetically diverse group that can belong to a range of pathotypes including: EHEC, atypical enteropathogenic E. coli (aEPEC), potential EHEC (pEHEC) and non-toxigenic E. coli (NTEC) and split into two main clusters by pulsed field gel electrophoresis. When the preliminary characterisation data were combined with spatial and temporal data a subset of 40 out of 88 isolates comprising ten human and 30 cattle E. coli O26 isolates were selected for subsequent analysis. The response of E. coli O26 strains in planktonic or biofilm forms to a range of antimicrobials and sanitisers and the possible influence of pathotype or strain source was assessed. The survival of E. coli O26 to disinfectants in a planktonic state varies depending on which stressor is applied. Disinfectants containing quaternary ammonium compounds or peracetic acid inhibited the growth of E. coli O26 whereas hypochlorite and acid anionic based disinfectants were not effective. It was also demonstrated that O26 strains of all pathotypes examined here had elevated MICs (1,024 to 4,096 μg/ml) to acetic, propionic, lactic and citric acids. E. coli O26 in planktonic state did not demonstrate variability in resistance when source or pathotype was considered. To evaluate the survival capability of strains in biofilm state, biofilm formation on different surfaces was investigated. EHEC strains were more likely to form biofilms after 48 hr than aEPEC strains on polystyrene plates. However, biofilm formation was generally enhanced with increasing incubation time and was further enhanced for all pathotypes when stainless steel and glass slides were used as a contact surface. Interestingly, investigating pellicle formation at the air-liquid interface indicated that pellicle formation was primarily produced by EHEC strains and NTEC while it was absent from aEPEC. The study also demonstrated that prophage insertion in mlrA (yehV), mutations in RpoS, lack of motility and failure to produce cellulose and/or curli was associated with reduced biofilm forming capacity. It was also demonstrated that biofilms provided a protective effect to E. coli O26 strains against the three sanitisers, previously shown to successfully control the growth of their planktonic counterparts, regardless of pathotype, source and the amount of biofilm formed. The carriage of stx is a key factor in the ability of E. coli O26 to cause human disease. Stx bacteriophage insertion sites and the ability for stx to be gained or lost were investigated. It was found that Stx phages integrated into wrbA, yehV or near the torS gene, a novel not previously described insertion site. Although these loci were shown to be intact in stx-negative strains, this study was unable to detail the ability or frequency at which Stx phage are acquired by stx-negative O26 strains. The carriage of non Stx phage integrases was also assessed with pathotype specificity observed which likely promotes the differing genetic profiles observed between pathotypes. Comparative analysis of Stx phage induction, toxin production and stx expression (copy number) identified that lysogens were found to produce greater quantities of Stx than their parent counterparts. Overall, stx expression and Stx production were comparable between EHEC regardless of source thereby confirming the pathogenic potential of EHEC cattle strains. Finally, the use of whole genome sequencing (WGS) provides evidence of the continuous evolution and plasticity of the genome of this organism and its ability to undergo genetic rearrangements and further confirmed the relationship between human and cattle strains. This study determined that while pathotypes of E. coli O26 appear to be highly clonal regardless of source, certain pEHEC and aEPEC strains share similar genetic profiles to EHEC strains and could rapidly transition to EHEC via the acquisition of stx. E. coli O26 remains an organism of significance to global public health and cattle processing systems. This study has confirmed that cattle are a potential reservoir of EHEC strains capable of causing human disease and has demonstrated the ability of EHEC strains to survive in the presence of anti-bactericidal substances. Continued implementation of regular and effective sanitisation will assist in controlling the hazard and associated risk caused by this global public health and economically important pathogen to the food industry.