## 4 Factors Inducing the VBNC State in _L. monocytogenes_
### Role of Disinfecting Agents
Residual concentrations in PWW of 20 mg/L for chlorine, 80 mg/L for peroxyacetic acid (PAA), and 3 mg/L for chlorine dioxide (ClO\({}_{2}\)), the most commonly used sanitizers in vegetable washing, are recommended by scientific studies, guidelines, and regulations. The operational limits for these sanitizers must be set above these minimum residual levels, and their optimization for different types of fresh-cut produce was the object of many studies [37]. It has been reported that in industrial settings with free chlorine concentrations below the operational limit of 10 mg/L, there was an induction of VBNC bacteria; further, in one study, 100% of 10\({}^{6}\) CFU/mL _L. monocytogenes_ cells exposed to 12 mg/L free chlorine entered the VBNC state. The induction of the VBNC state was higher at higher concentrations of organic matter and using the recommended operational limits for chlorine of 20-25 mg/L effectively inactivated _L. monocytogenes_ in PWW without the induction of VBNC cells. Therefore, the efficacy of the recommended washing practices was evaluated in a pilot plant [37]. Culturable and VBNC _L. monocytogenes_ cells were undetectable in PWW with chlorine after 1 min of contact for all types of washed produce. Instead, PAA at 80 mg/L concentration did not completely inactivate the pathogen in any type of PWW [37]. Significant differences were observed among different types of PWW for ClO\({}_{2}\) used at the 3 mg/L recommended concentration. In particular, the presence of VBNC cells was detected in shredded lettuce PWW. Since the recommended concentrations of PAA and ClO\({}_{2}\) induced the VBNC state, the necessity to revise the current operational limits of these disinfecting agents by increasing the concentration or the time of contact was suggested [37]. The optimized vPCR PMAxx method developed by [PERSON] et al. [29] was used to evaluate the ability of VBNC _L. monocytogenes_ and _E. coli_ O157:H7 cells to tolerate sanitizers, cause cross-contamination, and resuscitate in leafy greens during storage [20]. To determine the free chlorine concentration needed in PWW to induce the VBNC state, the concentrations 2.5 and 5 mg/L were tested on a mixture of six _L. monocytogenes_ serotype 1/2a isolates from leafy vegetables. With 2.5 mg/L chlorine, VBNC and culturable _L. monocytogenes_ cells were detected, while with 5 mg/L chlorine, all cells were inactivated. The VBNC cells in the PWW cross-contaminated the product after 1 min wash with 3 mg/L of ClO\({}_{2}\), and after 5, 10, and 15 days of storage at 7 \({}^{\circ}\)C, at least one replicate originated colonies indicating the resuscitation of VBNC cells. On iceberg lettuce and baby spinach during the 15-day shelf life, the levels of inoculated VBNC cells did not significantly change, though these were not culturable [20]. Sterile distilled water (SDW), baby spinach rinse water (SRW), and romaine lettuce rinse water (LRW) were used as media to carry out sanitizer treatments against 8 Log CFU/mL of two foodborne strains of _L. monocytogenes_ serogroup 1/2a. In SDW, _L. monocytogenes_ became undetectable by selective plating after exposure to chlorine at a concentration of 0.5 mg/L for 30 s, while in spinach or lettuce rinse water, due to the presence of organic matter, inhibition of growth in the plate was obtained with 10 mg/L of free chlorine. _L. monocytogenes_ was undetectable in the plate after treatment with 30 mg/L PAA, except for one colony in one of the four replicates for LRW. For PAA treatment, the effect of organic matter on pathogen inactivation was much less pronounced than for chlorine. After exposure to PAA 30 and 50 mg/L in different media, the pathogens were detected only by vPCR with a decrease of only 1-2 Log from the initial numbers [47]. For treatments with free chlorine in SRW and LRW, selective plating and vPCR were significantly correlated. In SDW, _L.