## 4 Factors Inducing the VBNC State in _L. monocytogenes_
### Role of Disinfecting Agents
monocytogenes_ at an initial level of 7.88 Log CFU/mL became undetectable by selective plating, non-selective enrichment, and vPCR with PMAxx when exposed to 50 mg/L free chlorine for 30 s; however, it was detected by vPCR, but not by selective plating, following treatment with 100 mg/L PAA. Cells treated with free chlorine stained red or brown with the LIVE/DEAD BacLight Bacterial viability kit, while in the PAA-treated samples, most cells stained green, suggesting uncompromised membrane integrity and viability [47]. Negative stain transmission electron microscopy (TEM) showed that most cells were damaged with electron-dense internal aggregations, large electron-lucent areas, and shrunk cytoplasm separated from the cell wall. However, a small proportion of cells resembled untreated cells and were probably VBNC. These cells could not be resuscitated, so the authors hypothesized that they could be damaged though the cell membrane was intact. Nevertheless, since chlorine-induced VBNC _L. monocytogenes_ had been previously shown to remain infectious for _Caenorhabditis elegans_, the possibility that these cells pose a food safety risk was not excluded [47]. CFDA/PI differential staining coupled with FC revealed three different subpopulations of _L. monocytogenes_ Scott A outbreak strain, serogroup 4b, when exposed to PAA or low pH adjusted with HCl or acetic acid (AA), namely, viable and metabolically active cells with an intact membrane (CFDA+PI-), dead cells with a damaged membrane (CFDA-PI+), and metabolically active cells with a damaged membrane classified as sub-lethally injured (CFDA+PI+). The CFDA+PI- cells were considered to be in a VBNC state if they were unable to restart growth in tryptic soy yeast extract broth or agar (TSBYE or TSAYE) [22]. After exposure to 20, 30, and 40 ppm of PAA at 20 \({}^{\circ}\)C for 1, 2, and 3 h, CFDA+ metabolically active cells ranged between 40.00% and 47.27%. The percentages of sub-lethally injured cells increased after 2 h of incubation. After exposure for 3 h to 40 ppm PAA at 20 \({}^{\circ}\)C, the total culturable and non-injured populations significantly decreased, and the whole population was nonculturable and CFDA+, thus indicating the induction of the VBNC state [22]. Exposure to 20 ppm PAA at 20 \({}^{\circ}\)C for 90 min and 180 min resulted in high percentages of CFDA+PI- cells, 16.17- and 13.33-fold compared to the control, while exposure to 30 and 40 ppm of PAA for 90 and 180 min increased the proportion of dead cells. However, exposure to higher PAA concentrations for 30 min led to the formation of numbers of 5.8- to 8.5-fold higher injured cells than the control [14]. Exposure to high acidity at pH 3.0 and 2.7 adjusted with HCl determined a small decline in metabolically active cells after 150 and 300 min. The decline was higher when AA was used as an acidifying agent, with a reduced formation of CFDA+PI+ cells and an increase in dead cells. The number of cells recovering on TSAYE compared to the non-agarized medium was significantly lower in all conditions, except for treatment with 20 ppm PAA for 180 min and 30 ppm PAA for 90 min, interpreted as the ability of PAA to induce the VBNC state in _L. monocytogenes_. After exposure to pH 3.0 adjusted with HCl, a small percentage of cells became non-culturable and the percentage increased to 33% at pH 2.7 after 300 min but only a small fraction of cells seemed to be in the VBNC state. When acidification to pH 2.7 was performed with AA, a significant percentage of CFDA+PI- cells- lost culturability, thus indicating VBNC induction. The lag phases for cells able to recover increased to a maximum of 120 h for cells exposed to PAA and to 21-24 h for exposure to pH 2.7 for 150-300 min [14]. The effects of two disinfectants commonly used in the seafood industry, one containing a quaternary ammonium compound and glutaraldehyde (QA) and one containing hydrogen peroxide (HP), were evaluated for the ability to induce VBNC cells of _L. monocytogenes_ in single-species and mixed-species biofilms. _L.