Detection and Quantification Methods for Viable but Non-culturable (VBNC) Cells in Process Wash Water of Fresh-Cut Produce: Industrial Validation

Abstract

The significance of viable but non-culturable (VBNC) cells in the food industry is not well known, mainly because of the lack of suitable detection methodologies to distinguish them from dead cells. The study aimed at the selection of the method to differentiate dead and VBNC cells of Listeria monocytogenes in process wash water (PWW) from the fruit and vegetable industry. Different methodologies were examined including (i) flow cytometry, (ii) viability quantitative polymerase chain reaction (v-qPCR) using an improved version of the propidium monoazide (PMAxx) dye as DNA amplificatory inhibitor, and (iii) v-qPCR combining ethidium monoazide (EMA) and PMAxx. The results showed that the flow cytometry, although previously recommended, was not a suitable methodology to differentiate between dead and VBNC cells in PWW, probably because of the complex composition of the water, causing interferences and leading to an overestimation of the dead cells. Based on results obtained, the v-qPCR combined with EMA and PMAxx was the most suitable technique for the detection and quantification of VBNC cells in PWW. Concentrations of 10 µM EMA and 75 µM PMAxx incubated at 40 • C for 40 min followed by a 15-min light exposure inhibited most of the qPCR amplification from dead cells. For the first time, this methodology was validated in an industrial processing line for shredded lettuce washed with chlorine (10 mg/L). The analysis of PWW samples allowed the differentiation of dead and VBNC cells. Therefore, this method can be considered as a rapid and reliable one recommended for the detection of VBNC cells in complex water matrixes such as those of the food industry. However, the complete discrimination of dead and VBNC cells was not achieved, which led to a slight overestimation of the percentage of VBNC cells in PWW, mostly, due to the complex composition of this type of water. More studies are needed to determine the significance of VBNC cells in case of potential cross-contamination of fresh produce during washing.

Listeria monocytogenes Assessment in a Ready-to-Eat Salad Shelf-Life Study Using Conventional Culture-Based Methods, Genetic Profiling, and Propidium Monoazide Quantitative PCR

“…At least a 3 log cfu/g difference was obtained, considering the lower limit of quantification of the conventional method (ISO 11290-2:2017). Similar results were reported by other authors when comparing both methods for bacterial quantification in food and food-processing environments [22,64,65]. No amplification was detected in salad extracts controls that were submitted to boiling prior to PMAxx-qPCR.…”

“…In food, L. monocytogenes is often affected by processing treatments that may hamper bacterial cultivability, while viability remains unaltered [11,12]. PMAxx has the ability to penetrate cells with compromised membrane integrity, covalently binding to DNA and free DNA, preventing PCR amplification and presenting a higher discriminative effect when compared to PMA in complex matrixes [65]. However, Nocker and Camper (2009) [67] highlighted the potential risk of overestimating VBNC cells because dead cells may present an intact membrane.…”

Environmental Management of Legionella in Domestic Water Systems: Consolidated and Innovative Approaches for Disinfection Methods and Risk Assessment

“…In this way, the amplification occurs only for viable cells, reducing the risk of VBNCs discarding and dead cells overestimation, which is typical of conventional qPCR and culture methods [ 148 , 151 ]. Moreover, some evidences of combined v-PCR approaches, such as that including a preliminary immunomagnetic separation step before the PMA-PCR reaction to increase the level of cells purification [ 152 ], suggest the possibility to improve these amplification methods, providing sensitive, selective, simple, and rapid bacteria detections in water towards innovative environmental monitoring applications.…”

Battling microbes: disinfecting water during fresh-cut lettuce processing

“…Research shows that techniques like flow cytometry have been recommended to quantify viable but non-culturable (VBNC) cells of E. coli when water was treated with sodium hypochlorite and PAA (Teixeira, Fernandes, Silva, Dias and Azeredo, 2020); however, other research has shown that flow cytometry can lead to an overestimation of dead cells (Truchado, Gil, Larrosa and Allende, 2020). Instead, a viability quantitative polymerase chain reaction (v-qPCR) with ethidium monoazide and propidium monoazide dyes as DNA amplificatory inhibitors were promising when trying to distinguish VBNC cells in chlorine-treated PWW (Truchado et al, 2020). If the same technique holds for other (chemical) wash water disinfectants, and other pathogens (besides that researched of Listeria monocytogenes) has yet to be validated in practice and remains a data gap.…”

“…If the same technique holds for other (chemical) wash water disinfectants, and other pathogens (besides that researched of Listeria monocytogenes) has yet to be validated in practice and remains a data gap. Overall, understanding how much VBNC cells contribute to cross-contamination during fresh(-cut) lettuce washing, and eventually, the impact on public health is warranted (Truchado et al, 2020).…”