An investigation into the effects of a simulated human gastro-intestinal tract has on Bacillus cereus and Bacillus weihenstephanensis viability and pathogenicity
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Bacillus cereus is one of the known causes of diarrhoeal food poisoning. In their natural environment of soil surviving as spores facilitates their colonisation of raw food ingredients enabling their access to the food chain. Recently psychrotrophic strains of B. cereus have been reclassified based on divergent cold shock gene (cspA) sequences and renamed B. weihenstephanensis. It is the modified cspA gene that is thought to confer the psychrotolerant phenotype witnessed by these strains. Aside from cspA, B. cereus and B. weihenstephanensis are closely related, leading to questions about its pathogenicity and ability to mediate diarrhoeal food poisoning outbreaks. Food producers use a variety of processes to limit microbial contamination within food products. Although effective against vegetative cells, spores are often resistant and as such can persist within this environment. Chilled temperatures (4°C) are often used to limit the growth of any contaminating microbes. Under such conditions B. cereus spores would remain dormant however B. weihenstephanensis spores have been shown to germinate and outgrow under refrigerated conditions. This could result in the consumption of both B. cereus and B. weihenstephanensis spores and vegetative cells. The effect that the human gastro-intestinal tract (GI) has on B. cereus and B. weihenstephanensis vegetative cells and spores is unclear. This study showed no difference in the viability of B. cereus or B. weihenstephanensis strains to survive and grow within a simulated human GI tract. Vegetative cells were revealed to die quickly in the stomach. Spore viability was shown to reduce in the stomach environment by approximately 10⁴-fold. With a larger initial inoculum, 10⁷ spore/ml, viable spores were still recorded after 4 hours. These spores subsequently germinated within the small intestinal simulation and the resulting vegetative cells rapidly proliferated. Mass spectrometry illustrated the ability of vegetative cells from both B. cereus and B. weihenstephanensis to produce an array of secreted proteins whose function were predominately related to virulence and pathogenesis. B.weihenstephanensis strain 10202 was shown to produce the potent cytotoxin, CytK-1, while other B. weihenstephanensis and B. cereus tested strains possessed either or both Nhe and Hbl toxins. The primary diarrhoeal virulence factor/haemolysin BL was shown to be present in the supernatant of each strain through western blotting. Significantly smaller concentrations of each protein were detected, however, under simulated human GI tract conditions when compared to optimal conditions. The effects of the simulated human GI tract on virulence gene expression were monitored through real time PCR. No pattern between B. cereus and B. weihenstephanensis strains was found confirming that virulence gene expression is strain specific. Some genes were shown to be significantly upregulated such as fur, (the ferric iron uptake regulator and groEL, encoding a molecular chaperone. The expression of others however was reduced such as haemolysin BL components, hblA and hblC. Overall there were no significant differences detected between B. cereus and B. weihenstephanensis strains in their ability to survive the human GI tract and express virulence factors associated with diarrhoeal food poisoning.
Thesis, PhD Doctor of Philosophy
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