High-pressure processing of Listeria monocytogenes: flow-cytometric quantification of viability beyond culturability

High-pressure processing (HPP) is widely applied as a non-thermal preservation technology to improve the microbiological safety of foods, including products at risk of contamination with Listeria monocytogenes . However, the efficacy of HPP is commonly assessed using culture-based methods, which detect only cells capable of forming colonies under the applied recovery conditions. This may lead to underestimation of pressure-stressed, sublethally injured, or non-culturable but physiologically active cells. In this study, the survival and physiological status of L. monocytogenes strains representing clonal complexes CC31 and CC101 were evaluated after HPP treatment at 200, 400, and 600 MPa for 5 min. Culture-based enumeration was performed on nutrient-rich and selective media, while flow cytometry was used to assess membrane integrity and metabolic activity. Plate counts showed pressure-dependent reductions, with 600 MPa reducing culturable cells below the detection limit. Recovery was lower on selective agar than on nutrient-rich medium, particularly after treatment at 200 and 400 MPa, indicating sublethal injury. In contrast, flow-cytometric analyses revealed that a proportion of cells retained membrane integrity and residual metabolic activity even when no culturable cells were detected. These results demonstrate that plate counting alone may not fully reflect the survival potential of L. monocytogenes after HPP. The findings support the use of multiparametric approaches combining culture-based methods with flow cytometry to obtain a more accurate assessment of HPP efficacy and microbiological safety.