## 3 Distribution of VBNC _L. monocytogenes_ in Food and Food Production Plants  
A few recent studies have investigated the presence of VBNC _L. monocytogenes_ in food considered ready-to-eat (RTE) salads and chicken meat [17, 36, 45]. RTE salads require extensive handling and are exposed to recontamination from processing surfaces and equipment after bactericidal treatments. The storage of these products in refrigerated conditions could allow the development of _L. monocytogenes_[17].  
[PERSON] et al. [17] reported that, based on hygiene indicators and _L. monocytogenes_ determination by culture-based methods, an RTE salad considered in the study could have a shelf-life of eight days when stored at 4 \({}^{\circ}\)C, according to Regulation (EC) No. 2073/2005 food safety criteria [27]. However, on day 8, the concentration of _L. monocytogenes_ obtained by vPCR with PMAxx was at least 3 Log CFU/g higher than that obtained by colony counts. No amplification was observed by vPCR from boiled salad extracts. Therefore, the vPCR results exceeded the food safety criteria of 2 Log CFU/g and showed that the standard culture-based methods might underestimate the number of viable _L. monocytogenes_ cells affected by processing treatments that hamper cultivability. However, the potential interference of dead cells was not evaluated [17].  
A study on poultry meat investigated the presence of VBNC cells of _L. monocytogenes_ in two slaughterhouses in 11 wings, three thighs, 12 drumsticks, and four breast samples. After homogenization of 25 g of sample in 225 mL of 0.1% peptone water, 1 mL was used to determine the total cell concentration and 1 mL was used for vPCR after treatment of cells with PMAxx 25 \(\upmu\)M. DNA extraction was performed on 750 \(\upmu\)L of the two aliquots, treated and not treated with PMAxx, using a commercial kit with proteinase K. The vPCR technique showed a sensitivity of 100%, a specificity of 66.7%, and an efficiency of 83.3%, with 0.67 kappa coefficient agreement when compared with culturing by enrichment and colony isolation on Oxoid Chromogenic Listeria agar (OCLA) followed by colony identification by species-specific PCR (OCLA-PCR). The rather low specificity and agreement were because five samples were positive on vPCR but negative on OCLA-PCR. These five samples most probably contained VBNC _L. monocytogenes_ cells, indicating the occurrence of these forms in poultry meat. Previous studies based on plate counts reported lower contamination levels for poultry, and this can be explained by the ability of vPCR to detect also VBNC cells. The high sensitivity score of vPCR (100%) against OCLA-PCR indicated the absence of false negative results for the method [36].  
A second investigation found _L. monocytogenes_ in 75.0% of 65 samples from two slaughterhouses by culturing methods and qPCR. The results were not fully concordant since some samples were positive only for one of the techniques. Viability PCR allowed detecting _L. monocytogenes_ in half of the carcass from one slaughterhouse, but the microorganism was not detected by culture-dependent methods. In another slaughterhouse, _L. monocytogenes_ was detected in 100% of samples by culture-dependent methods only (38.5%) or both by culturing and qPCR (61.5%). A comparison among culture-dependent detection, qPCR, and vPCR allowed the authors to determine that the contamination levels in the positive samples were 4.01 Log CFU/g for total cells, 3.21 Log CFU/g for viable cells, 1.00 Log CFU/g for culturable cells, 3.20 Log CFU/g for VBNC cells, and 3.93 Log CFU/g for dead cells [45].