Growth phase-specific induction of a viable but nonculturable (VBNC) state in <i>Listeria innocua</i> in response to a green food sanitizer

Sustainable food sanitation and preservation technologies are highly needed to meet the increasing demand for healthy and safe food. In this work, we investigated the effects of a recently developed antimicrobial triple formulation (TF) comprised of a natural polyphenol gallic acid amended with the generally recognized as safe materials hydrogen peroxide and DL-lactic acid, on a gram-positive bacterium <i>Listeria innocua</i> during its exponential and stationary growth phases. Our findings revealed the physiological changes associated with the application of this material, as observed using single-cell flow cytometry, photometric assays, as well as confocal and scanning electron microscopy. Both exponential- and stationary-phase <i>L. innocua</i> cells lost culturability after incubation with TF, but their cellular responses were different. After 30 min of contact with the TF, the nonculturable, stationary-phase cells maintained membrane integrity, membrane potential, stable respiratory electron-transport chains, and high levels of intracellular ATP. These phenotypic features, characteristic of the viable but nonculturable (VBNC) adaptation strategy, appear to be associated with upregulation of the stress-response factor <i>sigB</i>. In contrast, in TF-treated exponential-phase cells, membrane integrity was severely compromised and membrane potential showed hyperpolarization, suggesting sublethal injury accompanied by downregulation of <i>sigB</i>. Furthermore, the TF treatment enhanced the formation of extracellular vesicles in <i>L. innocua</i> and the deposition of extracellular polymeric substances in stationary-phase cells. The overall findings of this work demonstrate that sensitivity of <i>Listeria</i> cells to nature-based antimicrobials depends on their growth phase and, therefore, modulating their growth-phase-associated physiological status may counteract their VBNC defense strategy and improve biocidal efficacy.IMPORTANCEIn this work, we examined the efficacy of our novel, nature-based green sanitizer as a means to improve microbiological food safety without health and environmental risks, using gram-positive <i>Listeria innocua</i> as a model organism. <i>L. innocua</i> serves as a surrogate of the foodborne pathogen <i>L. monocytogenes</i> for evaluating sanitation efficacy and may also be involved in virulence and multidrug resistance transfer between the <i>Listeria</i> species. We demonstrate that the sanitizer effect depends on the physiological state of bacterial cells, which is affected by their growth phase. The treatment caused membrane damage to <i>L. innocua</i> in its exponential phase but induced the viable but nonculturable state during its stationary phase. Thus, this study demonstrates a way to overcome bacterial defense mechanisms and improve sanitizer efficacy by modulating the growth-phase-associated physiological status of targeted bacteria.