## 4 Factors Inducing the VBNC State in _L. monocytogenes_
### Effect of Physicochemical Stresses in Food Production on VBNC Induction in L. monocytogenes
monocytogenes_ slightly increased on fresh-cut cantaloupe and mango and did not change significantly on fresh-cut apple, cauliflower, romaine lettuce, pepper, and onion [53]. Temperature abuse at 15 \({}^{\circ}\)C above the normal storage temperature did not change the overall trends of _L. monocytogenes_ survival except for blueberry, on which _L. monocytogenes_ showed a lower decline. _L. monocytogenes_ population declined at higher rates on fresh-cut carrots, apples, tomatoes, grapes, and avocados while the growth rate on cantaloupe and mango increased. For cauliflower, celery, onion, and romaine lettuce, _L. monocytogenes_ growth was observed in temperature abuse conditions, while for fresh-cut broccoli and green peppers, the _L. monocytogenes_ populations did not change significantly [53]. Live/dead staining of _L. monocytogenes_ cells recovered from carrot showed a ratio of cells with intact membranes comparable to the initial inoculum, indicating that the loss of culturability of _L. monocytogenes_ did not reflect viability. When _L. monocytogenes_ recovered from fresh-cut carrot was examined by vPCR with PMAxx, a viable population of 3.84 Log higher than that quantified by selective plating on Day 0 was detected. Moreover, the population determined by plating decreased below the LOD after storage at 4, 10, and 15 \({}^{\circ}\)C for 6 and 14 days, whereas vPCR showed a reduction of only 1 Log, indicating the presence of VBNC _L. monocytogenes_ remaining after 2 weeks [53]. Ready-to-eat (RTE) foods with a pH lower than 4.4 and water activity lower than 0.92 are considered not permissive for the growth of _L. monocytogenes_, but those lacking the above characteristics can support the growth of _L. monocytogenes_ and must be subjected to time/temperature control measures to prevent _L. monocytogenes_ development during storage [26]. Raw carrots could be considered a food that supports the growth of _L. monocytogenes_ based on pH (6.43-6.7) and water activity (a\({}_{\text{w}}\) > 0.98) values, but several studies have shown that this food matrix does not support the growth of _L. monocytogenes_ and can even suppress its survival under various storage conditions, thus showing intrinsic antilisterial activity by as yet unidentified substances [52]. Strains of the _Listeria_ species--_L. grayi, L. innocua, L. monocytogenes, L. seeligeri, and L. welsthimeri_--exposed to fresh-cut carrots for 15 min in 10% buffered peptone water (BPW) from initial concentrations of about 9 Log CFU/mL showed a recovery range of 6.6-7.3 Log CFU/mL. In addition, _L. monocytogenes_ FS2025, a strain associated with an outbreak from cantaloupe, showed a recovery from boiled carrots ranging from 7.1 to 7.6 Log CFU/g against an average 4.7 \(\pm\) 0.3 Log CFU/g recovery from fresh-cut carrot samples, indicating the inhibition of _Listeria_ spp. in fresh carrots [52]. In qPCR without PMAxx pre-treatment rinsates from fresh-cut and boiled carrots contained comparable levels of _L. monocytogenes_ cells. Moreover, in the rinsate from boiled carrot, _L. monocytogenes_ showed comparable numbers in culture-dependent or culture-independent analysis, while in the fresh-cut carrot rinsate, more than 99% of _L. monocytogenes_ cells were non-culturable. On the other hand, vPCR showed similar numbers of _L. monocytogenes_ in fresh-cut carrots and boiled carrot rinsates, implying that exposure to fresh-cut carrots could induce the VBNC state in _L. monocytogenes_. Transmission electron microscopy (TEM) showed the formation of mesosome-like structures in cells exposed to the rinsate from fresh-cut carrots, whose role in the formation of VBNC cells should be elucidated [52]. The physicochemical stress factors other than antimicrobials that favored the formation of VBNC cells of _L. monocytogenes_ in food or mimicking food stressors are synthesized in Table 2.