## 4 Factors Inducing the VBNC State in _L. monocytogenes_
### Effect of Physicochemical Stresses in Food Production on VBNC Induction in L. monocytogenes  
Some stress factors such as the presence of particular ingredients, sub-optimal temperature, and the presence of inhibitors are inherent to food and food production processes. In a study regarding the use of Raman-deuterium isotope probing (Raman-DIP) microspectroscopy to characterize VBNC cells of _L. monocytogenes_, this technique was evaluated for the analysis of the effects of different food-related stressors, namely, storage temperature, presence of salts, and exposure to disinfectants using strains _L. monocytogenes_ Lm1 and EGD-e. Viability was also analyzed by using qPCR, vPCR with PMA, and culturing. An incubation temperature effect was observed with the presence of a VBNC population at 37 \({}^{\circ}\)C and at 4 \({}^{\circ}\)C. For cells exposed to 2% and 20% NaCl, differences of 1.5 Log UFC/mL and 0.72 Log UFC/mL between viable and culturable cells were observed for _L. monocytogenes_, which were observed for _L. togenes_ Lm1, thus showing the presence of a VBNC population. Results obtained for _L. monocytogenes_ EGD-e were similar [50]. The persistence of _L. monocytogenes_ in food processing environments is the most important source of contamination of RTE foods. Therefore, a study was conducted to devise the rapid enumeration of viable _L. monocytogenes_ cells by vPCR in a food processing environment after heat treatment and desiccation on stainless steel with and without biofilm formation. PMA concentration, incubation time and temperature, light source for photoactivation and exposure time, target genes sequence, and amplicon length were optimized to suppress the signal from dead cells. A 50 uM concentration of PMA was used since it appeared non-toxic to cells. A linear correlation was observed between vPCR and plate count enumeration of _L. monocytogenes_ when the difference between sanitizer-killed cells and viable cells did not exceed 4 Log CFU. However, vPCR with a short amplicon gave false positive results from cells killed by heat or isopropanol [18]. A long-amplicon vPCR assay with an estimated LOQ of 1.32 Log CFU/reaction correctly indicated that heat treatment at 90 \({}^{\circ}\)C for 15 min reduced the _L. monocytogenes_ cell number from 8.02 Log CFU/mL to below the detection limit of 1.22 log CFU/mL, whereas a short-amplicon PMA-qPCR assay used for comparison provided false positive results. Viability PCR slightly but not significantly underestimated the viable cell counts, and the membrane destabilization treatment with SDS before PMA treatment did not significantly improve false positive results, while it was toxic for viable _L. monocytogenes_ cells. Similar vPCR results were obtained for two strains, _L. monocytogenes_ 568 and 08-5578, throughout an experiment of biofilm desiccation, except for the overestimation of the viable cell concentration at day 10 for _L. monocytogenes_ 568. At day 10, both strains exhibited a drastic decrease in culturable cells, which was also observed with vPCR but only for _L. monocytogenes_ 08-5578 [18]. In an experiment of biofilm desiccation for 8 days at 15 \({}^{\circ}\)C and 33% RH, the plate count quantified a reduction in culturable cells of 1.38 Log CFU/cm\({}^{2}\) for the two _L. monocytogenes_ strains. The reduction in viable cells measured by long-amplicon vPCR was 1.40 \(\pm\) 0.09 log CFU/cm\({}^{2}\) for _L. monocytogenes_ 08-5578 and 0.71 \(\pm\) 0.06 log CFU/cm\({}^{2}\) for _L. monocytogenes_ 568. Therefore, the survival to desiccation of _L. monocytogenes_ 568 was higher for biofilm cells than non-biofilm cells. The DNA extraction procedure was optimized to obtain a reliable cell number determination by the long-amplicon PMA-qPCR [18]. [PERSON] et al. [42] evaluated the fate of _L. monocytogenes_ ATCC 19115 (serotype 4b) in water microcosm and rainbow trout fillets with 0% or 30% NaCl at refrigeration temperature (4 \(\pm\) 2 \({}^{\circ}\)C).