In Vitro Survival of Campylobacter jejuni And Campylobacter coli at Low pH
Type of DegreeThesis
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A series of experiments were performed to investigate the effect of pre-exposure to acidic conditions of early exponential and stationary-phase cells of Campylobacter jejuni and C. coli. The sublethal acid exposure to protect C. jejuni and C. coli against low pH treatment was also studied. Twelve C. jejuni strains, including ten fluoroquinolone-resistant C. jejuni isolated from humans, and nine C. coli strains, were grown at 42oC under microaerophilic conditionss (85% N2, 10% CO2, 5% and O2) in modified Campy Cefex (mCC) agar to create stationary-phase cells. The colonies were then suspended in Tryptic Soya broth (TSB, at final pH 7), and grown to the appropriate early exponential-phase under microaerophilic conditions. The cells in both phases were exposed to TSB at pH 5 for 4 h before the final challenge (TSB at final pH 4). The same procedures were performed utilizing Brucella broth. Survival was determined after 2-3 h of the final challenge by using viable cell counts and utilizing Campylobacter blood free selective media (mCCDA). Pre-exposure of the early exponential-phase and the late stationary-phase cells to moderate pH 5 shows no induction of resistance to the pH 4, where the control cells show better survival. Moreover, the survival time increased from 2 to 3 h when using Brucella broth instead of TSB. Campylobacter coli were very sensitive to the lethal acidic treatments. The results showed that Campylobacter jejuni and C. coli failed to produce an acid adaptation response in early-exponential or stationary-phase in Tryptic Soya broth or Brucella broth (pH 5). The images obtained from scanning electron microscopy also indicated that cells displayed different morphological changes at different pH values. The traditionally used liquid broth and chemically defined medium M 199 was also evaluated for Campylobacter jejuni in vitro. The liquid media compared were M 199, Brucella broth (BB), Brain Heart Infusion broth supplemented with yeast extract (BHIYE), and Tryptone Soya broth supplemented with yeast extract (TSBYE). An initial inoculum of 103 to 104 CFU/ml resulted in greater than or equal to 106 CFU/ml in BB and M 199 after incubation for up to 72 h, while lower growth rate was observed in both TSBYE and BHIYE. Among all liquid media compared, M 199 exhibited the least percent of coccoid cell formation. On M 199, C. jejuni grew over a pH range of 5 to 8.