## 4 Factors Inducing the VBNC State in _L. monocytogenes_
### Role of Disinfecting Agents
After 24 h of recovery treatment, the cells with a viability phenotype (BOX\({}^{-}\) PI\({}^{-}\)), especially for cells exposed to ROEO, increased, reaching 69.1-83.5% in PBS and 44.9-75.5% in meat broth [31]. The subpopulations FDA\({}^{+}\) PI\({}^{+}\), i.e., with damaged cell membrane but with enzymatic activity, ranged from 33.5 to 55.8% and 19.7 to 44.8% for cells exposed to OVEO and ROEO in PBS and meat broth, respectively. The number of dead cells (FDA\({}^{-}\)PI\({}^{+}\)) ranged from 10.8 to 22.7% and 8.6 to 29.5% for cells exposed to OVEO and ROEO in PBS and meat broth, respectively. However, the subpopulations with undamaged cell membranes increased after 6 h of recovery treatment and, for cells exposed to ROEO, reached percentages of 72.6 and 69.7% after exposure to PBS and 99.9 and 86.6% after exposure to meat broth. After 24 h of recovery, cells exposed to ROEO in PBS showed 79.5 and 91% subpopulations with undamaged cell membranes. Subpopulations with enzymatic activity after exposure to OVEO and ROEO in meat broth increased from 58.5 to 71.7% after 24 h of recovery [31]. Cell efflux and respiratory functions, beyond membrane damage, might influence culturability. Therefore, ethidium bromide (EB) staining was performed to investigate efflux activity. This dye can cross intact cell membranes and is pumped out by cells with active efflux systems, so cells with impaired efflux activity are EB+. Cells exposed to OVEO and ROEO originated similar percentages of EB+ subpopulations, namely, 57.2-84.1% and 55.6-81% in PBS and meat broth. Gradual increases in EB\({}^{-}\) subpopulations were observed after 24 h of recovery from exposure to ROEO in PBS (34.1 and 61.4% for the concentrations 10 and 5 \(\mu\)L/mL). After 24 h of recovery treatment, cells exposed to OVEO increased the efflux activity, whereas those exposed to ROEO increased the EB+ subpopulation. The results of viable counts after recovery diverged for the two essential oils but cells in which the culturability was not restored had improved physiological functions and appeared to be viable. Further investigations should elucidate whether OVEO- and ROEO-induced VBNC cells can restore the ability to multiply in food matrices [31]. Lavender essential oil (LEO) has an attractive aroma and is widely used in the food industry because of its broad antibacterial, antifungal, antiviral, nematocidal, and anti-mite properties. It is also largely used in pharmaceutical products and cosmetics for its multiple health-promoting properties. The effects of LEO against _L. monocytogenes_ ATCC19115 were defined toward biofilms of the pathogen by using plate counts and vPCR with PMA. Biofilm formation of _L. monocytogenes_ on sterile coverslips in polystyrene microtiter plates was evaluated by the crystal violet staining method [49]. The effect of LEO on _L. monocytogenes_ was compared with that of sodium hypochlorite. The minimum inhibitory concentration (MIC) for LEO and sodium hypochlorite against _L. monocytogenes_ was 1.6 \(v/v\) and 0.219 mg/mL, respectively. In the treatment with 4 MIC of LEO, viable and culturable cells in biofilm formed at 32 \({}^{\circ}\)C (warm biofilm) decreased by 6.35 and 9.01 Log CFU/mL, and those in the biofilm formed at 10 \({}^{\circ}\)C (cold biofilm) decreased by 3.16 and 1.93 Log CFU/mL, respectively. In the treatment with 4 MIC of sodium hypochlorite, the proportion of viable and culturable cells decreased by 3.68 and 4.76 Log CFU/mL in the warm biofilm, and 2.88 and 2.91 Log CFU/mL in the cold biofilm, respectively. Consequently, LEO reduced the number of viable cells more effectively than sodium hypochlorite. However, both LEO and sodium hypochlorite-induced VBNC populations that ranged in number from 1.74 to 3.58 and from 2.10 to 3.26 Log CFU/mL in the warm biofilm and cold biofilm, respectively, in the treatment with LEO [49].