## 5 Ultrastructural, Molecular, and Transcriptomic Changes in _L. monocytogenes_ VBNC Cells  
A thorough description of the physiological characteristics of _L. monocytogenes_ VBNC cells is available for those induced by starvation in water. In the study of [PERSON] et al. [21], the _L. monocytogenes_ EGDe, serogroup 1/2a, was incubated for 28 days in commercial mineral water with low mineral content and quality-controlled composition at initial concentrations ranging from 10\({}^{9}\) to 10\({}^{6}\) cells/mL. Culturability decreased at a higher rate \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Stress Factor(s)** & **Intensity–Concentration** & **L. monocytogenes Strain** & **Reference** \\ \hline biofilm & 8 days at 15 \({}^{\circ}\)C and 33\% & 568 & [18] \\ desiccation & RH & & \\ starvation & Water microcosm & ATCC 19115 (serotype 4b) & [44] \\ NaCl & 30\% & ATCC 19115 (serotype 4b) & [44] \\ NaCl & 2–20\% & Lm1 (serogroup 1/2a-3a), EGD-e (serotype 1/2a) & [51] \\ NaCl & 20–30\% at 4 and \(-\)20 \({}^{\circ}\)C & Four strains including ATCC 19115 (serotype 4b), & [51] \\  & & ATCC 19111 (serotype 1/2a) & [51] \\ fresh-cut carrots & & FS 2025 serotype 1/2b, FS 2030 serotype 1/2a, FS 2061 & [53] \\  & & serotype 1/2b (cantaloupe outbreak) & [52] \\ Raw carrots & & FS2025 (serotype 1/2b cantaloupe outbreak) & [52] \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Physicochemical stress factors that induced the VBNC state in _L. monocytogenes_ during real or simulated food production conditions. and extent at a lower starting bacterial concentration of less than 1 CFU/mL at day 28, while with an initial concentration of 10\({}^{8}\) cells/mL, a reduction of 2 log culturable cells/mL occurred. FC carried out by staining the viable cell population with CFDA showed that the total and viable cell numbers remained nearly unchanged. Moreover, the ATP cell content, which is a marker of cell viability, declined after 7 days and increased later, reaching levels higher than those expected from the culturable cells from day 21, thus indicating a contribution from the VBNC forms [21]. The use of _L. monocytogenes_ cells expressing the Green fluorescent protein (GFP) allowed observing the formation of coccoid cells, sometimes near rod-shaped structures resembling empty cell walls, which increased in number from day 7 to day 28. The coccoid cells did not stain as Gram-positive because of the peptidoglycan loss that was complete on day 28, as shown by Ultra-High-Performance Liquid Chromatography (UHPLC) analysis [21]. Differential staining of the cell wall with a fluorescent lectin able to bind teichoic acids and of the cell membrane with an antibody specific for _L. monocytogenes_ of the 1/2 serogroup showed that rod-shaped cells, observed by cryogenic electron tomography (cryo-ET), presented cell wall gaps from which spherical protoplast egressed. This phenomenon was also observed for three other _L. monocytogenes_ strains and other _Listeria_ sensu stricto species, _L. ianovii, L. innocua, L. marthi, L. seeligeri, and _L. welsthimeri_, thus suggesting that this is a common mechanism in _Listeria_ species [21]. Known and putative autolysins, namely, _Imo0394, p60, aut, lmo1215, lmo1521, lmo2522, ami_, and _nannA_, were strongly downregulated after 7 days. Therefore, the cell wall degradation was probably carried out by a pre-existing pool of autolysins. However, 90% of NamA-deficient mutants of _L. monocytogenes_ EGDe still maintained the cell wall at day 14, compared to 44% of the wild-type. A similar behavior was observed in deletion mutants of the _secA2_ component of the Sec system ATPase that mediates the secretion of the NamA autolysin [21]. Gas chromatography coupled with mass spectrometry highlighted a decrease in the relative abundance of anteiso- branched-chain fatty acid species (a-BFA) a-C15:0 and a-C17:0, key regulators of membrane fluidity, and an increase in linear saturated (SFA) and unsaturated (UFA) fatty acids between day 0 and day 28.