## 4 Factors Inducing the VBNC State in _L. monocytogenes_
### Effect of Physicochemical Stresses in Food Production on VBNC Induction in L. monocytogenes
The results showed that bacteria in a water microcosm lost their culturability after 13 days with 0% NaCl concentration and after 27 days of incubation in the presence of 30% NaCl. However, bacteria in rainbow trout fillets remained culturable with and without NaCl. RT-PCR targeted on the 16 S rRNA gene was positive for all treatments during the period of the study, indicating the entering of _L. monocytogenes_ into the VBNC state in water microcosm with 0% and 30% NaCl. Moreover, VBNC _L. monocytogenes_ expressed _hly_ and _inLA_ virulence genes, so the authors concluded that VBNC _L. monocytogenes_ retains pathogenicity [44]. Another study evaluated the ability of _L. monocytogenes_ to form VBNC cells in the presence of high NaCl concentrations at different temperatures and to resuscitate under different nutritional conditions. Viability PCR with PMA was used and compared with the Chinese national standard method for _L. monocytogenes_ enumeration. Four strains of _L. monocytogenes_ were examined including _L. monocytogenes_ ATCC 19115 (serotype 4b) and ATCC 19111 (serotype 1/2a). Single colonies were inoculated into TSBYE and incubated until reaching the exponential phase; then, an induction suspension containing around 10\({}^{6}\) CFU/mL of cells from the exponential phase was prepared with pH adjustedto 5.0. The suspension was aliquoted and stored at \(-20\)\({}^{\circ}\)C to ensure comparability among experiments [51]. At 4 \({}^{\circ}\)C, the number of culturable bacteria decreased significantly in the presence of 20% NaCl and 30% NaCl, and was below the LOD after 70 days in the 30% NaCl solution. Culturable _L. monocytogenes_ ATCC 19115 was below the limit of detection on day 70 in the presence of 20% NaCl, while _L. monocytogenes_ ATCC 19111 and MRL300007 decreased until day 21 or 49 and stabilized later. At 25 \({}^{\circ}\)C, a significant decrease in culturable bacteria was observed for all four strains. In particular, _L. monocytogenes_ ATCC 19115 at 20% and 30% NaCl and ATCC 19111 at 30% NaCl were below the LOD on day 14. In the presence of 20% NaCl, the loss of culturability occurred on day 21 for some strains, while _L. monocytogenes_ MRL300007 lost culturability in the presence of 30% NaCl on day 35 [51]. The VBNC counts of _L. monocytogenes_ ATCC 19115 were 4.31 Log CFU/mL at 4 \({}^{\circ}\)C and 3.23 Log CFU/mL at \(-20\)\({}^{\circ}\)C after 70 days at a high concentration of NaCl, and all cells were in VBNC state. _L. monocytogenes_ ATCC 19111 at 20% and 30% NaCl concentration showed a higher number of VBNC cells, 5.08 and 5.38 Log CFU/mL, respectively, compared to the other strains [51]. In the resuscitation experiments, only the strain ATCC 19111 was resuscitated without dilution of the VBNC cell suspension, while all strains recovered in BHI medium from 1000-fold diluted VBNC cell suspensions. Tryptic soy broth (TSB) plus 5% Tween 80 allowed a better resuscitation, except for _L. monocytogenes_ MRL300007. TSB added with 2 mg/mL sodium pyruvate also improved resuscitation. Recovery in pasteurized milk at 4 \({}^{\circ}\)C was observed only for _L. monocytogenes_ ATCC 19115 VBNC cells. Only this strain appeared to enter the VBNC state at 4 \({}^{\circ}\)C. When the NaCl stress was removed and pasteurized milk was added in the culture medium, only _L. monocytogenes_ MRL300007 did not recover, possibly for the too low number of cells in VBNC state formed during incubation in the stressful conditions [51]. The types of fresh produce implicated in listeriosis outbreaks in the US included cantaloupe, packaged lettuce, stone fruits, and celery [52]. A study regarding the survival of _L. monocytogenes_ on fresh-cut products during storage at the temperature values prevailing during commercialization found that for three strains from cantaloupe outbreaks--_L. monocytogenes_ FS 2025 1/2b, FS 2030 1/2a, and FS 2061 1/2b--the culturable cells significantly decreased on fresh-cut carrots, fresh-cut pineapple, and fresh-cut celery and broccoli at 4 \({}^{\circ}\)C. The populations of _L.