Viability qPCR utilizing propidium monoazide, spheroplast formation and Campylobacter coli as a bacterial model

A viability quantitative PCR (qPCR) utilizing propidium monoazide (PMA) is presented, for rapid quantification of viable cells using the foodborne pathogen Campylobacter coli (C. coli) as a bacterial model. It includes optimized spheroplast formation via lysozyme and EDTA, induction of a mild osmotic shock for enhancing the selective penetration of PMA to dead cells, and exploitation of internal sample process control (ISPC) involving cell inactivation to assess residual false-positive signals within each sample. Spheroplasting of bacteria in exponential phase did not permit PMA entrance to viable cells since a strong linear relationship was detected between simple qPCR and PMA-qPCR quantification, and no differences were observed regardless of utilizing spheroplasting. The PMA-qPCR signal suppression of dead cells was elevated using spheroplast formation. Regarding the ISPC, cell inactivation by hydrogen peroxide resulted in higher signal suppression during qPCR compared to heat-inactivation. Viability quantification of C. coli by the optimized spheroplasting-PMA-qPCR with ISPC was successfully applied in an aging pure culture under aerobic conditions and artificially inoculated meat. The same method exhibited a high linear range of quantification (1.5 – 8.5 log10 viable cells mL-1) and results were highly correlated with culture-based enumeration. PMA-qPCR quantification of viable cells can be affected by their rigidity, age, culture media and niches, but spheroplast formation along with osmotic shock and the use of a proper ISPC can address such variations. The developed methodology could detect cells in a viable but nonculturable state and might be utilized for the quantification of other Gram-negative bacteria. IMPORTANCE There is need for rapid and accurate methods to detect viable bacterial cells of foodborne pathogens. Conventional culture-based methods are time-consuming and unable to detect bacteria in a viable but nonculturable state. The high sensitivity and specificity of the quantitative polymerase chain reaction (qPCR) are negated by its inability to differentiate the DNA from viable and dead cells. The combination of propidium monoazide (PMA), a DNA intercalating dye, with qPCR assays is promising for viability detection. Despite encouraging results, these assays still encounter various challenges such as false-positive signals by dead cells and lack of an internal control identifying these signals per sample. The significance of our research relies in enhancing the selective entrance of PMA to dead Campylobacter coli cells via spheroplasting and in developing an internal sample process control thus delivering reliable results in pure cultures and meat samples, approaches that can be applicable to other Gram-negative pathogens.