Physiological adaptations contributing to stress survival in the foodborne pathogen Campylobacter jejuni
SubjectCampylobacter in food and water
Viable but non-culturable (VBNC) state
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n spite of being considered fragile and fastidious, the zoonotic pathogen Campylobacter jejuni remains the leading cause of foodborne bacterial gastroenteritis in the developed world. Lacking many of the stress responses common to other enteric pathogens, C. jejuni employs the survival strategies, biofilm formation and entry into the viable but non-culturable (VBNC) state, which have not been well characterized. Recent studies have indicated that these strategies are likely related at the molecular level. The purpose of this thesis was threefold: 1) to characterize entry into the VBNC state for planktonic and biofilm cells of C. jejuni with starvation at 4°C; 2) to evaluate a novel PMAqPCR method to quantify viable cells (both culturable and viable but non-culturable) in planktonic and biofilm cells of C. jejuni during starvation at 4°C; and 3) to investigate changes in gene expression of selected genes involved in biofilm formation and entry into the VBNC state. The three strains C. jejuni NCTC 11168 V1, C. jejuni NCTC V26 and C. jejuni 16-2R were included in all studies to compare variation based on strains. Cells were considered VBNC when there was no growth with enrichment, but cells scored as viable based on membrane integrity. Biofilm cells which became VBNC in some cases after 10 days of stress were found to enter the VBNC state earlier than planktonic cells by 10 to 50 days. Additionally, no significant reductions occurred in viable cell counts over the course of the experiments, confirming that the loss of culturability was not due to cell death (p<0.05). To date, no methods have been used to quantify viable but non-culturable biofilm cells of C. jejuni. The novel method PMAqPCR which has been successful for the enumeration of planktonic C. jejuni as well as for biofilm cells of other species was validated for quantifying C. jejuni biofilm cells in late log phase (20 h) and once cells had entered the VBNC state. The genes that affect both entry into a VBNC state and the ability to form biofilm in C. jejuni were upregulated during biofilm formation. Gene expression prior to stress treatment was 5 to 37 fold higher in biofilm cells than in their planktonic counterparts for all three strains (p<0.001). For the planktonic samples, only one of the 3 strains showed significant changes in gene expression during the transition to the VBNC state. In this case, all 4 target genes were significantly upregulated 4-6 fold just prior to cells becoming VBNC (p<0.05). At present food and drinking water safety in Canada continues to be assessed primarily using culture-based methodology. As validated in this thesis, the ability to quantify both culturable and viable but non-culturable C. jejuni cells in both planktonic and biofilm forms will allow for improved evaluation of quality control methods in both research and industries where these pathogens are a concern. Also, the understanding of the interaction between biofilm formation and entry into the VBNC state at the molecular level described herein provides information which can be used to develop appropriate interventions and reduce the incidence of campylobacteriosis.