Investigation of expression of cytotoxic genes of Escherichia coli 0157:H7 in foodstuffs of animal origin. [Doctoral dissertation] | Ispitivanje ekspresije gena za citotoksin Escherichia coli O 157: H 7 u namirnicama animalnog porekla. Doktorska disertacija
2010
Velebit, B.
Expression of cytotoxic genes of E. coli 0157:H7 (stx1, stx2 and eaeA) in different foodstuffs of animal origin has been investigated. Experiment was set up in a way it simulated the most common storage conditions. Minced beef, UHT milk and soft cheese were inoculated by an overnight broth culture of reference strain ATCC 35150 E. coli 0157:H7. Samples were incubated at the temperatures of 4 deg C and 12 deg C during 24 hours and 48 hours, respectively. Genetic expression profile was frozen at determined time points. In order to measure relative expression of cytotoxic genes, prokaryotic mRNA has been purified and converted to cDNA to be used in a RealTime PCR. Simultaneously, changes in growth kinetics were recorded. Results indicated that growth of E. coli 0157:H7 in experimentally contaminated minced beef kept at the temperature of 12 deg C during 48 hours and expression of stx1 and stx2 genes were considerably higher rather than in foodstuffs subjected to other experimental conditions. Regarding UHT milk growth kinetics and gene expression remained as similar as in minced beef, however at a somewhat lower intensity. Growth kinetics in soft cheese appeared to be not affected. Interestingly, stx1 gene was down-regulated during initial phase of incubation; while later up-regulation to a base level occurred by the end of experiment. Expression of eaeA gene in case of experimentally contaminated beef, milk and cheese and expression of stx2 and eaeA genes in milk and cheese was negligible i.e. experimental conditions did not provoked up-regulation of respective genes. It seemed that foodstuffs with higher fat content down-regulated stx2 genes. A strong correlation has been established between growth kinetics and gene expression in minced beef and partially UHT milk while no correlation has been established between growth kinetics and gene expression in soft cheese.
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