Molecular detection and gene expression of campylobacter during stress conditions.

The magnetic immuno-polymerase chain reaction assay (MIPA), was developed for the detection of Campylobacter jejuni and Campylobacter coli initially in milk and chicken products. After 18 hours pre-enrichment the MIPA could detect 420 cfu g-1 of chicken and 63 cfu ml-1 of artificially contaminated milk. MIPA was then applied for the detection of C. jejuni / C. coli in cloacal swabs and in retail poultry, where the sensitivity was found to be comparable to cultural methods. However, results were available significantly faster, within 24 hours compared to the 4-5 days of cultural methods. MIPA was also evaluated as a technique for the detection of "viable but non-culturable" (VBNC) forms in the environment. It was found that there was a reduction in sensitivity for the detection of these non-culturable forms (NCFs) and it was concluded that the MIPA had limited use in their detection. The lack of sensitivity of MIPA for detecting VBNC campylobacters in the environment may indicate that they are either antigenically or genetically distinct from the culturable forms. This result highlights the question of whether these VBNC forms actually represent a viable potentially infectious form of Campylobacter. This question can be answered at a molecular level; promoter activity can be monitored as a representation of viability of the bacterial cell during stress conditions. The promoter activity could be measured using a promoter probe vector. Initially we decided to construct a promoter probe vector which based on the recombinase system of the PI bacteriophage. However, the final construct was unstable and due to the constraints of time we decided to use a pre-existing promoter probe vector pSP73 constructed by Purdy and Park, (1993). This promoter probe vector contains promoterless luxAB genes which induce light emission as a reporter of gene expression. In order to investigate the genetic regulation during the conversion of C. jejuni to non-culturable forms (NCFs), we monitored gene expression during the transformation of C. jejuni into the non-culturable state. The promoterless copy of the luxAB genes was placed under the control of promoter regions of the C. jejuni flaA gene. Transformed C. jejuni was incubated under stress conditions that induce transition to NCFs (Non-culturable forms). Promoter activity, plate counts and direct microscope counts were simultaneously monitored. It was found that during the initial conversion of C. jejuni to NCFs there is an up-regulation of the flaA promoter. This regulation occurs in response to agitation and did not occur in NCFs and indicates that they are functionally non-viable