VBNC induction and persistence of Listeria monocytogenes Scott A as a defence mechanism against free chlorine stress

Sodium hypochlorite (SH) belongs to the chlorine-releasing agents (CRAs) and is widely used as a disinfectant or a bleaching agent for sanitizing in the food processing environment and fresh-cut industry. In the present study, the potential induction of dormancy states, i.e. the VBNC state and persistence, in Listeria monocytogenes, Scott A strain, was evaluated after exposure to SH for 3 h at 20 °C. Our results showed that the concentration of free chlorine after cells (10^9.5 CFU/mL) resuspension into the working solution decreased down to 3.7 ppm (SD ± 0.4 ppm; pH 6.64 ± 0.1). To detect VBNC fractions we evaluated comparatively the results of plate counting with fluorescence microscopy, using 5(6)-carboxy-fluorescein diacetate (CFDA; metabolic activity) and propidium iodide (PI; death) staining. The resuscitation capacity of L. monocytogenes stressed single cells was monitored real-time on TSAYE at 37°C, using time-lapse microscopy. Thus, colony outgrowth kinetics were estimated and non-diving fractions were detected. Furthermore, variability in the division time per generation was examined. Our analyses showed that SH induces the VBNC state and persistence in L. monocytogenes. Phenotypic variants of “high” fitness, i.e. size colony variations (SCVs) were also detected in response to SH stress. L. monocytogenes cells presented a prolonged lag time after exposure to SH. This phenomenon is a defence mechanism that allows cells to tolerate stress and maximize population fitness. The investigation of the VBNC state is of high importance for the food industry, as the impacts of VBNC induction and single cell outgrowth heterogeneity can contribute to false-negative detection outcomes.