title: Quantitative analysis of viable, stressed and dead cells of Campylobacter jejuni strain 81-176.
abstract: Campylobacter jejuni is an important foodborne gastrointestinal pathogen and highly sensitive to environmental stresses. Research has shown that changes in culturability, cell morphology, and viability occur when C. jejuni cells are subjected to stresses. In this study, real-time PCR, ethidium monoazide (EMA) in combination with real-time PCR (EMA-PCR), BacLight bacterial viability staining, and agar plate counting methods were used to quantitatively analyze viable, stressed, and dead C. jejuni strain 81-176. The real-time PCR assay provides highly sensitive and specific quantification of total genome copies of C. jejuni culture in different growth phases. Our results also reveal that real-time PCR can be used for direct quantification of Campylobacter genome release into Phosphate Buffered Saline (PBS) as an indicator of cell lysis. Using EMA-PCR, we obtained a dynamic range of greater than 3 logs for differentiating viable vs. dead cells. The viability and morphological characteristics of the stressed cells after one-week incubation at 25 degrees C, in air, and under nutrient-poor conditions were investigated. Our results indicated that, over 99% of the stressed cells were converted from the spiral to the coccoid form and became non-culturable. However, more than 96% of the coccoid cells retained their membrane integrity as suggested by both the BacLight staining and EMA-PCR analyses. Thus, to detect C. jejuni under stress conditions, conventional culturing method in conjunction with EMA-PCR or BacLight staining might be a more appropriate approach.
authors.affiliation: Microbial Biophysics and Residue Chemistry Research Unit, U. S. Department of Agriculture, 600 E. Mermaid Lane, Wyndmoor, PA 19038, USA. yiping.he@ars.usda.gov
authors.forename: Yiping
authors.initials: Y
authors.lastname: He
authors.forename: Chin-Yi
authors.initials: CY
authors.lastname: Chen
mesh_terms: D000345:Affinity Labels / Q000494:pharmacology*; D001386:Azides / Q000494:pharmacology*; D016123:Campylobacter jejuni / Q000187:drug effects / Q000254:growth & development*; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000235:genetics*; D005506:Food Contamination / Q000032:analysis; D005519:Food Preservation / Q000379:methods; D050296:Microbial Viability / Q000187:drug effects / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D013997:Time Factors
chemical_list: D000345:Affinity Labels; D001386:Azides; D004269:DNA, Bacterial; C014535:8-azidoethidium