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Characterization of Heat Resistant Escherichia coli Isolates Associated with Human Infection
- Author / Creator
- Ma, Angela
Shiga toxin-producing Escherichia coli (STEC) is a bacterial pathogen associated with foodborne diarrheal disease. Infection with STEC presents as a mild, watery diarrhea to hemorrhagic colitis and can progress to the life-threatening complication, hemolytic uremic syndrome. Cattle are recognized as a primary reservoir of STEC and beef products are a major vehicle for STEC infection. STEC is considered a heat sensitive pathogen and the food processing industry heavily relies on thermal inactivation processes to eliminate bacterial contaminants. However, non-pathogenic E. coli isolated from environmental sources have been found to survive heat exposure at temperatures of 60°C and above. These isolates possess the locus of heat resistance (LHR), which confers resistance to thermal, osmotic, and oxidative stress. It is unknown if heat resistant E. coli, specifically STEC, exist and if they have been involved in human disease. It is hypothesized that the LHR in clinical E. coli isolates facilitates the increased survival of pathogenic strains against heat inactivation measures in food processing and consumer cooking practices, and can be a contributing factor in human foodborne infection in Alberta, Canada.
This thesis presents the novel identification and characterization of 3 clinical E. coli isolates, two being STEC, which possess the LHR. Three real-time quantitative polymerase chain reaction assays were developed and validated for the LHR with heat resistant, environmental E. coli isolate AW1.7. These assays allow for rapid screening of E. coli for the LHR from foodborne outbreak investigations and food processing plants.
To investigate the threat of heat resistant E. coli in the food processing industry and human consumption, the isolates were characterized for their ability to survive heat exposure at 60°C and 71°C in liquid culture media for the calculation of decimal reduction times (D-values) and in ground beef. D60-values of heat resistant isolates all exceeded 10.20 minutes with one isolate's D60-values ranging from 20.46 to 72.47 minutes in the presence of increasing osmotic stress. Literature on D71-values of foodborne pathogens is limited and the level of heat resistance mediated by the LHR at 71°C is unknown. It was determined that heat resistant isolates possessed elevated D71-values compared to heat sensitive E. coli but to a far lesser extent than at 60°C. At temperatures of 71°C and above, it is suspected that the LHR is less effective at mediating turnover of misfolded and denatured proteins than at 60°C. Cell reductions of heat resistant isolates in ground beef patties grilled to 60°C and 71°C were 2.84 and 4.95 log colony forming units (CFU)/mL, respectively, compared to reductions of 6.08 log CFU/mL and greater in heat sensitive E. coli.
Despite the numerous thermal inactivation measures used in food processing plants to eliminate foodborne pathogens, E. coli biofilms remain a persistent source of contamination. Using an in-house, two-component apparatus, biofilm formation by the 3 clinical isolates was compared with 3 environmental isolates. All isolates harboured the LHR. Optimum conditions for biofilm formation in each of the isolates were determined by manipulating inoculum size, nutrient concentration, and temperature conditions. One out of the 3 clinical, heat resistant isolates was capable of forming biofilms whereas all 3 of the environmental isolates formed biofilms, suggesting that the LHR does not contribute to biofilm formation.
To elucidate the function of the components of the LHR in regards to their contributions to heat resistance, genetic and proteomic analyses of the LHR were conducted. Whole genome sequencing revealed that the LHR sequences in isolates AW1.7, 111, 128, and 8354 were 98.3% similar and that all of the clinical isolates encoded for a larger variant of the LHR compared to the LHR in E. coli AW1.7. Constitutive expression of novel Clp protease ClpK, encoded on open reading frame 3 of the LHR, was identified in all heat resistant isolates. However, transgenic strains that expressed ClpK without the entire LHR did not survive heat exposure at 60°C.
In conclusion, this research describes the emergence of multi-stress tolerant E. coli implicated in human gastrointestinal disease as a novel food safety risk. The heightened survivability of heat resistant E. coli facilitates their evasion from elimination along multiple stages of the farm-to-fork continuum and consequentially increases their potential to cause human foodborne infection.
- Graduation date
- Spring 2021
- Type of Item
- Doctor of Philosophy
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