## 4 Factors Inducing the VBNC State in _L. monocytogenes_
### Role of Disinfecting Agents
monocytogenes_ Lm1 (serogroup 1/2a-3a), _Carnobacterium maltaromatic_ P1 AV5, and _C. divergens_ P9 AV5, all isolated from food contact surfaces of seafood processing plants, were used to produce biofilms on stainless steel and polyvinyl chloride (PVC) coupons. The biofilms were treated for 20 min at room temperature with 2% (\(v/v\)) solutions of the disinfecting agents according to the recommendations for use [48]. The difference between the number of cells determined by qPCR and vPCR with PMA, expressed as genome equivalents (GE), represented the number of dead cells while the difference between the number of cells determined by vPCR and counts on agar plates corresponded to the number of VBNC cells. _Listeria_ cells remained viable and culturable in the biofilm not treated with disinfectants. The difference between qPCR and vPCR measurements in biofilms of stainless steel were 2.06 Log GE/cm\({}^{2}\) and 1.70 Log GE/cm\({}^{2}\) after HP and QA treatment, respectively. However, the decreases in _L. monocytogenes_ culturable cell numbers were 5.22 Log CFU/cm\({}^{2}\) and 4.49 Log CFU/cm\({}^{2}\), respectively, indicating the presence of VBNC cells in the biofilms with differences of 2.95 Log GE/cm\({}^{2}\) and 2.12 Log GE/cm\({}^{2}\) compared to the culturable cells. Similar results were obtained for the biofilms on PVC [48]. Sampling of food contact surfaces in four commercial smoked salmon processing plants was also carried out before and after cleaning and disinfection for 8 months to examine the prevalence of culturable and VBNC _L. monocytogenes_. Before cleaning and disinfection, the prevalence of _L. monocytogenes_ cells was 3.17% for culturable cells, 10.32% for viable cells, and 15.87% for total cells, while after cleaning and disinfection, the prevalence of _L. monocytogenes_ was 1.59% for culturable cells, 9.52% for viable cells, and 14.29% for total cells, thus indicating a high rate of VBNC formation though with differences in prevalence among single plants [48]. Essential oils are natural antimicrobials particularly attractive for use in food production. Those extracted from oregano (_Origanum vulgare_ essential oil, OVEO) and rosemary (_Rosmarinus officinalis_ essential oil, ROEO), alone or in combination, inhibited _L. monocytogenes_ in food matrices. Therefore, their capacity to induce _L. monocytogenes_ VBNC formation in culture media and conditions mimicking food matrices was analyzed for a mixture of _L. monocytogenes_ strains ATCC 7644 and ATCC 19112 (serotype 1/2c of human origin), and ATCC 19117 (serotype 4d of sheep origin). Exposure to 5 and 2.5 uL/mL of OVEO determined culturability loss after 60 min in PBS and after 180 min in meat broth. Exposure to 10 and 5 uL/mL of ROEO in PBS determined culturability loss after 60 and 180 min, respectively, while in meat broth, culturability loss occurred after 180 min at both concentrations, thus appearing delayed in the presence of nutrients [31]. _L. monocytogenes_ cells that lost culturability did not recover after 6 h, in agreement with previous observations with _L. monocytogenes_ [PERSON] With a recovery time of 24 h, _L. monocytogenes_ cells exposed to OVEO did not regain culturability, while those exposed to 5 uL/mL of ROEO in PBS and 10 uL/mL in meat broth restored culturability, with average viable counts in the range of 3.0-4.8 Log CFU/mL from initial viable counts of about 7 Log CFU/mL. These results indicated that _L. monocytogenes_ cells exposed to ROEO in PBS and meat broth entered the VBNC state [31]. The presence of VBNC cells was analyzed by FC and double staining with bis-1,3-dibutyl barbituric acid (BOX) and PI as indicators of membrane potential and membrane integrity, respectively. Exposure to OVEO and ROEO caused the formation of 76.4 to 97.2% of cells with depolarized and damaged cell membranes (BOX\({}^{+}\) PI\({}^{+}\)) in PBS and 97 to 98.5% in meat broth.