Shiga toxin encoding bacteriophages and horizontal gene transfer in Escherichia coli O157

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Shiga toxigenic Escherichia coli (STEC), most notably serotype O157, is a food borne pathogen of global public health concern. The progression to a severe disease state following an STEC infection is associated with the production of Shiga toxin (Stx). The ability to produce Stx is conferred upon STEC strains by Stx–encoding bacteriophages (Stx phages), which infect and integrate into the host bacterial genome. These phages carry either stx1 or stx2 genes which encode two immunologically distinct toxins with similar biological functions. However, not all STEC O157 strains are equally pathogenic as Australian STEC O157 strains, are associated with lower incidence of clinical disease than strains from other countries. This led to the aim of this thesis, which was to investigate how Stx phages differentially influence the virulence of STEC O157 strains The characterisation of two Stx1 phages, originating from clinical Australian STEC O157 strains, revealed that these phages are morphologically and genomically similar to Stx2a phages. It was also observed that the bacterial host genetic background could influence toxin production. Genomic analysis revealed that these phages can potentially induce a translational frameshift with two overlapping tail-coding sequences with different host recognition domains, which may account for the broad host range of Stx phages leading to the emergence of new Stx producing pathogens. In addition, in-silico analysis also revealed a possible mechanism on how the phage obtained the stx gene by means of a Miniature Inverted Transposable element. Stx prophages from three non-clinical Australian STEC O157 isolates were also characterised in this thesis. Two STEC O157 strains harboured both Stx1 and Stx2c prophages and it was observed in these strains that one Stx prophage was induced at a higher titre over the other. Genomic analysis predicted for the first time that the Stx2c phages package their DNA via cohesive ends. Further interrogation of the genomes also showed that two translational frameshift events, in different genes, are required for tail assembly and extension of host range respectively. In addition, each of the Stx2c prophages studied carried an anti-repression operon that may counteract the repression of the Stx1 prophage in-trans which could be a possible explaination for the increase in the production of Stx. This could be the mechanism as to why STEC strains that harbour a Stx2c prophage in conjunction with another Stx prophage, in particular the Stx2a prophage, appear to be more virulent than other combinations of Stx prophages. This thesis also reports the first evidence for Stx phage-mediated horizontal transfer of the locus of enterocyte effacement (LEE) pathogenicity island, as well as the characterisation of this Stx prophage. The mechanism of LEE mobilisation was determined to be likely due to a combination of both generalised and specialised transduction, and the incorporation of the LEE in recipient strains is likely due to homologous recombination. In addition to the discovery that an Stx phage can mediate the mobilisation of the LEE, genomic characterisation of this Stx prophage also revealed that it has a number of phage encoded genes that are predicted to enhance its ability to infect other susceptible bacterial strains by evading bacterial toxin-anti toxin defences and infecting a broad host range. Overall, the results presented in this thesis suggested that Stx phages contribute more to bacterial pathogenesis than just the conference of the stx genes and that there are other interactions between the host and the Stx phage, which have resulted in STEC O157 becoming a successful pathogen.
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