Viability Quantitative PCR Utilizing Propidium Monoazide, Spheroplast Formation, and Campylobacter coli as a Bacterial Model

Abstract

A viability quantitative PCR (qPCR) utilizing propidium monoazide (PMA) is presented for rapid quantification of viable cells using the foodborne pathogen Campylobacter 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 into dead cells, and exploitation of an 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 into viable cells since a strong linear relationship was detected between simple qPCR and PMA-qPCR quantification, and no differences were observed regardless of whether spheroplasting was utilized. The PMA-qPCR signal suppression of dead cells was elevated using spheroplast formation. With regard to the ISPC, cell inactivation by hydrogen peroxide resulted in higher signal suppression during qPCR than heat inactivation did. Viability quantification of C. coli cells by 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 to 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 PCR (qPCR) are negated by its inability to differentiate the DNAs from viable and dead cells. The combination of propidium monoazide (PMA), a DNA-intercalating dye, with qPCR assays is promising for detection of viable cells. Despite encouraging results, these assays still encounter various challenges, such as false-positive signals by dead cells and the lack of an internal control identifying these signals per sample. The significance of our research lies in enhancing the selective entrance of PMA into 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.

Method-Dependent Implications in Foodborne Pathogen Quantification: The Case of Campylobacter coli Survival on Meat as Comparatively Assessed by Colony Count and Viability PCR

“…This suspension was then homogenized and nine aliquots (18 μl each) were generated and transferred to equal number of transparent PCR tubes (0.2 ml; Kisker Biotech GmbH & Co). The treatments of an optimized PMA-qPCR utilizing spheroplast formation (induced by lysozyme and EDTA) and ISPC, as described by Lazou et al (2019) , were applied. In brief, the first set of thee aliquots was directly subjected to DNA extraction and qPCR in order to quantify the total C. coli cell equivalents (quantification controls).…”

“…The qPCR assay, as described by Lazou et al (2019) , targeting the serine hydroxymethyltransferase ( gly A) single-copy gene (one genome copy represents one cell equivalent) was applied. Briefly, the primers used were Cc-F (5'-TGTAAAACCAAAGCTTATCGTGTGC-3') and Cc-R (5'-AGTCCAGCAATGTGTGCAATG-3'), along with the Cc-FAM TaqMan probe (5'-6-FAM-AGCTCCAACTTCATCCGCAATCTCTCT-BHQ1-3').…”

“…The qPCR assay exhibited a linear range of 10 0 –10 7 copies per reaction ( R 2 > 0.99), with a PCR efficiency of 93.4% and an LOD of 7.046 copies per reaction (95% CI). The standard curve was described by the equation y = 10 [(41.763 ‐ Ct)/3.491)] , where y stands for the genome copies per qPCR reaction ( Lazou et al, 2019 ).…”

“…However, applications of v-qPCR protocols with PMA (PMA-qPCR) on meat samples have encountered various challenges and limitations attributed to a rather conditional and not absolute suppression of qPCR signals originating from dead Campylobacter bacteria (false-positive signals) due to a complex set of parameters including experimental, target and sample features ( Nocker et al, 2006 ; Pacholewicz et al, 2013 ; Krüger et al, 2014 ; Duarte et al, 2015 ; Vondrakova et al, 2018 ). The development of a PMA-qPCR assay utilizing spheroplast formation as a pretreatment for enhancing the selective entrance of PMA to dead cells and an internal sample process control (ISPC), unique for any given sample, that could address false-positive signals originating from dead cells, has only recently been achieved using C. coli as a bacterial model ( Lazou et al, 2019 ).…”

Transformation of Helicobacter pylori into Coccoid Forms as a Challenge for Research Determining Activity of Antimicrobial Substances

“…For example, the use of vPCR and MVT has been very helpful in distinguishing VBNC from dead cells of Pseudomonas aeruginosa treated with antibiotics [91]. The use of vPCR has also been successfully used in the detection of VBNC Campylobacter cells, bacteria closely related to Helicobacter [92]. Some hope in assessing the viability of coccoid H. pylori also gives the measurement of de novo synthesized ATP.…”