## 4 Factors Inducing the VBNC State in _L. monocytogenes_
### Role of Disinfecting Agents  
Benzalkonium chloride (BC) is a widely used quaternary ammonium compound (QAC) in food production plants and damages the cell membrane. BC was tested on non-adapted and pre-adapted cells of _L. monocytogenes_ SLCC2540 serotype 3b and was found to induce the VBNC state, expressed as metabolically active cells, more efficiently in pre-adapted cells. Pre-adaptation was carried out by exposure to BC concentrations from 2 \(\mu\)g/mL to 13 \(\mu\)g/mL, gradually increasing by 1 \(\mu\)g/mL in BHI broth. Pre-adapted cells were also less susceptible than parent cells to 13 antibiotics of different classes except vancomycin and erythromycincin. However, when looking only at membrane integrity by LIVE/DEAD BacLight kit staining and FC, non-adapted cultures also showed high numbers of apparently viable cells, about 10\({}^{8}\) cells/mL, at BC concentrations between 4 \(\mu\)g/mL and 13 \(\mu\)g/mL. However, when the metabolically active cells were identified by measuring the uptake of 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG), their number was much higher than for non-adapted cells at concentrations of BC between 5 \(\mu\)g/mL and 11 \(\mu\)g/mL. With BC above 10 \(\mu\)g/mL, no colonies were obtained by plate count. It must be underlined that also for non-adapted cells, low percentages of metabolically active cells were detected at BC concentrations between 5 \(\mu\)g/mL and 13 \(\mu\)g/mL [46]. One of the concerns dealt with in studies regarding VBNC _L. monocytogenes_ was the effect of RTE fresh produce process wash water (PWW) in the induction of VBNC cells. PWW is indeed a relevant vector of microbial cross-contamination of clean products from contaminated products that is prevented by adding sanitizers, among which chlorine is the most commonly used. However, it was reported that chlorine induces the VBNC state in _L. monocytogenes_ and _S. enterica_, so suitable quantification methods should be applied to detect and control VBNC cells in PWW [29]. An optimized detection/quantification method of VBNC cells in PWW was developed after comparing different combinations of dyes in FC, and of PMAxx, or a combination of EMA and PMAxx in vPCR. The method was validated in industrial settings by using six _L. monocytogenes_ isolates from leafy vegetables in a mixture. A PMAxx concentration of 75 \(\mu\)M was tested since a 50 \(\mu\)M concentration added to PWW did not completely inhibit amplification from dead cells, possibly due to the presence of interfering organic matter, while 100 \(\mu\)M appeared toxic for culturable cells. Amplification from heat-treated cells was abolished after 30 min of incubation with the dye at 40 \({}^{\circ}\)C but the PCR signal from cells treated with chlorine was not completely inactivated. PMAxx combined with EMAat concentrations of 75 \(\upmu\)M and 10 \(\upmu\)M, respectively, and incubation at 40 \({}^{\circ}\)C for 40 min followed by 15 min exposure to light, reduced the amplification from dead cells after chlorine treatment [29]. In the shredded lettuce PWW, no significant changes in the concentration of free chlorine (10 mg/L) and pH were observed, and the culturable bacteria maintained a total count below 3 Log CFU/mL. However, a higher number of viable cells was detected by using EMA-PMAxx-vPCR, thus showing the induction of VBNC _L. monocytogenes_. FC with double-staining indicated a higher number of viable cells compared to vPCR, possibly for an overestimation caused by the presence of cells with intact membranes but damaged DNA, proteins, and lipids. Therefore, fluorescent markers able to highlight damages to these molecules should be used in FC for improved viable cell discrimination. The VBNC _L. monocytogenes_ cells induced by 10 mg/L chlorine could be detected after 1 h and remained stable after 3 h [29].