Exploring Shiga toxin's vector and its function in Escherichia coli and protozoan predator populations

In recent foodborne E. coli outbreaks, the key virulence factor Shiga toxins (Stxl and 2) are encoded by a lysogenic phage on Escherichia coli genome. In my research, I developed two PCR based techniques, inverse PCR (IPCR) and thermal asymmetric interlaced PCR (TAIL-PCR), to rapidly identify the flanking sequences of stx1 gene. Both methods could be used to detect possible genetic variation among stx gene carriers, which could help to study Stx phage variation and evolution, and trace the possible transmission route of Stx phages in STEC outbreaks.;The stx genes could possibly increase the fitness of their bacterial hosts against protozoal predators. A microcosm experiment was performed to study the impact of STEC on ciliate protozoa populations. The results indicated that E. coli strains with and without stx genes could affect the protozoal population differently in activated sludge environment. The 18S rDNA clone library analysis indicated that Epistylis wenrichi and Prorodon teres are the dominate ciliate species in the original activated sewage environment, while ciliates in the STEC treated sewage sample were identified as Prorodon teres, Dexitrichides pangi, Opisthonecta henneguyi and Vorticella fusca. The ciliate Epistylis wenrichi was less resistant to STEC than Prorodon teres, Dexitrichides pangi, Opisthonecta henneguyi and Vorticella fusca. This study could potentially provide useful information for identifying STEC sensitive and resistant ciliate protozoa as indicators in wastewater treatment facilities for preventing and tracking E. coli outbreaks.;The amoeboid protozoan Dictyostelium discoideum has been used as host model for microbial pathogenesis. A new bacterial virulence evaluation method was developed by using D. discoideum to evaluate the virulence of STEC strains in a simple liquid medium. E. coli strains that carry either stx1 or stx2 genes can significantly reduce the viability of Dictyostelium within 24 hours. This method could be used to test the virulence of different STEC strains on eukaryotic cells in a very short period of time.