An investigation into the viability of coccoid cells of Campylobacter : detection of mRNA by reverse-transcriptase-PCR as an indicator of viability in Campylobacter jejuni.

In response to unfavourable environmental conditions, a number of bacterial species are shown to enter a viable but nonculturable (VNC) state. This phenomenon has led to the recognition of the inherent limitations of the standard microbiological cultural method and has resulted in the development of novel techniques to determine microbial viability. In this study, emphasis was placed on the analysis of viability by molecular amplification of the human and animal enteric bacterium, Campylobacter jejuni, which has been one of the paradigm organisms for the study of the VNC response. This gastrointestinal organism is of particular interest due to its high incidence in New Zealand and its ability to cause diarrhoeal infections in persons who consume contaminated poultry or untreated water. Due to the medical significance of the pathogen, it is essential to establish whether the VNC state is indeed living and, as such, may have a role in the contamination or infection cycle. In this thesis, the relationship between the detection of mRNA and cellular viability in C. jejuni was investigated in cultures aerobically incubated below the bacterium's optimum growth temperature. Reverse transcription-PCR (RT-PCR) methods were developed for detecting mRNA from the lpxA, dnaJ, hupB, and ciaB genes and rRNA from the 23S rDNA gene. Total RNA from C. jejuni was isolated and, following DNase treatment, the RNA was amplified by both PCR and RT-PCR with primers specific for each gene. The levels of expression differed markedly with respect to prolonged incubation time, but the results indicated that coccoid cell forms of Campylobacter retain structural integrity and a degree of protein synthesis for up to two weeks under the experimental conditions of this study. This research demonstrated that either the IpxA or dnaJ genes may provide suitable targets for development of a specific method for detection of viable C. jejuni based on RT -PCR amplification. The usefulness of the IpxA mRNA species as a viability determinant was validated in an artificially killed C. jejuni culture. Following a twenty minute heat treatment at 65°C, the IpxA-specific amplification product could be detected in the culture for 96 h postheating. In contrast, aerobic plate counts (once an indication of viability) demonstrated loss of culturability within five minutes of heat-shock induction. These results further demonstrate that nonculturability and non-viability are not synonymous. Thus, this study supports the usefulness of RT-PCR amplification of mRNA as a sensitive method for specific detection of viable bacteria and indicates this method may prove beneficial in the detection of C. jejuni in clinical and environmental samples