## 4 Factors Inducing the VBNC State in _L. monocytogenes_
### Role of Disinfecting Agents
After 40 days of exposure to 1/2 MIC of LEO, VBNC cells for planktonic cells and biofilm were 2.51 and 0.50 Log CFU/mL, respectively, while in the 1/2 MIC, the sodium hypochlorite treatment was 4.93 Log CFU/mL for planktonic cells and 1.08 Log CFU/mL for the biofilm, respectively. In the treatments with LEO or sodium hypochlorite at the MIC for 40 days, the numbers of VBNC cells for planktonic cells and biofilm were 2.61 and 0.64 Log CFU/mL and 4.93 and 1.08 Log CFU/mL, respectively. Therefore, a low concentration of LEO induced fewer VBNC cells than sodium hypochlorite [49]. Electrolyzed water (EW) is obtained by electrolysis of diluted NaCl or HCl solutions in a specialized chamber. One type of EW is slightly acidic electrolyzed water (SAEW), with disinfecting properties. SAEW has a pH of 5-6.5 and is gentle on surfaces, so it is suitable for use in the food industry. SAEW contains chlorine, which has a strong oxidizing capacity and covalently binds to proteins, modifying their structure in the chlorination process. To determine the concentration of chlorine required for VBNC induction, _L. monocytogenes_ cells were treated with SAEW for 5 min at different concentrations. With SAEW containing 4 and 6 mg/L of available chlorine (ACC), only 0.31 and 0.72 Log CFU/mL reductions were achieved, respectively, while 8 mg/L chlorine completely abolished culturability. Staining with the LIVE/DEADTMBacLightTM kit showed that 23 to 34% of the cells were viable after treatment with 8 and 10 mg/L of ACC, while standard plating provided negative results, indicating induction of the VBNC state. Most cells appeared to be intact by optical microscopy, but scanning electron microscopy (SEM) showed a shrinkage of the cell membrane and the formation of aggregates in the VBNC cells [32]. Iron is essential for different cell functions, including energy metabolism, cell signaling, gene expression, growth regulation, and cell differentiation, but when the redox reactions catalyzed by iron are poorly controlled, the process of ferreptosis, an iron-dependent nonapoptotic cell death, may occur. This process is also exploited for cancer therapy, in which the Fenton reaction causes lipid peroxidation by hydroxyl radicals production catalyzed by iron in excess and H\({}_{2}\)O\({}_{2}\)[19]. _L. monocytogenes_ ATCC51914 was susceptible to inorganic ferrous and ferric iron at a final concentration of 200 \(\upmu\)M for 3 h. Cells exposed to FeSO\({}_{4}\) lost culturability, as well as 90% of those exposed to ferric citrate (Cit-Fe (III)). _L. monocytogenes_ ATCC519114 cells exposed to FeSO\({}_{4}\) did not stain with PI nor with SYTOX in FC analysis; so, it was deduced that FeSO\({}_{4}\) did not cause cell death. To elucidate if FeSO\({}_{4}\) induced DNA degradation, the cells exposed to FeSO\({}_{4}\) for 3 h were incubated in a saline solution containing 400 \(\upmu\)g/mL thymol for 30 min, and it was observed that thymol led to PI-stained cells, indicating that cells were viable after FeSO\({}_{4}\) exposure [19]. The induction of VBNC cells by FeSO\({}_{4}\) occurred in a dose-dependent manner, but even at a low concentration of 25 \(\upmu\)M, FeSO\({}_{4}\) induced the VBNC state in approximately 90% of cells. The process occurred in less than 15 min at temperatures between 5 and 42 \({}^{\circ}\)C and for strains belonging to different serotypes. Morphological changes from rod-shaped to spheroids, size reduction, and shrinkage were observed in the FeSO\({}_{4}\)-induced VBNC cells. These were able to resuscitate in fresh BHI broth after a prolonged lag phase and were virulent for _C. elegans_ with survival rates similar to normal _L. monocytogenes_ cells after 10 days from infection [19]. Notably, while normal _L. monocytogenes_ cells were killed by 240 \(\upmu\)g/mL ampicillin, 60 \(\upmu\)g/mL tetracycline, and 60 \(\upmu\)g/mL chloramphenicol, in BHI broth, VBNC cells developed resistance, except to tetracycline. When resuscitated, the population of VBNC cells was, again, sensitive to ampicillin [19].