Characterization of Listeria monocytogenes responses to food-related stress and population dynamics in soft cheese

Abstract

The success of Listeria monocytogenes as a pathogen depends on its ability to survive the many environmental stresses that it encounters along the food chain. However, response heterogeneity among strains and individual cells within populations affects the physiological states of the cells which subsequently influences the behavior and fate of the pathogen in contaminated foods. This study sought to investigate the influence of stress on the physiological states of L. monocytogenes strains and the potential influence on the persistence and survival of the pathogen in a ready-to-eat (RTE) food. Furthermore, the study also sought to elucidate the bimolecular changes related to the acid stress response of the pathogen. Flow cytometry coupled with cell membrane integrity indicators showed that the degree of cell injury in L. monocytogenes strains (69, 159/10, 243 and ATCC19115) subjected to acid, osmotic and heat stress treatments was influenced by individual strain susceptibilities and the extent to which the stress exposure affects cell membrane integrity. Regardless of strain susceptibilities, acid stress induced the highest level of cell damage with osmotic stress causing the least. Following sorting of injured cells, the lag phase duration was the main difference in the resuscitation behavior of the stress-injured L. monocytogenes strains an indication that repair of cell injury was influenced by strain heterogeneity and extent of cell membrane damage. Importantly, once the injury was repaired, the resuscitated cells possessed a growth potential similar to non-injured cells regardless of strain or stress treatment differences. Despite having a lower level of cell membrane injury, heat-injured cells were incapable of resuscitation in the majority of strains, an indication that the cellular targets of heat-induced injury are not necessarily limited to the cell membrane. Thus on its own, membrane integrity may not be a sufficient indicator of cell injury. Stress pre-exposure and individual strain susceptibilities also influenced the survival responses and population dynamics of the pathogen in a lactic soft cheese. Kinetic model analysis revealed that while acid and osmotic stress pre-exposures resulted in sensitization of the susceptible strain (69), the same exposures resulted in induction of tolerance responses that protected tolerant strains against the acidity of the cheese. Although the osmotolerance response conferred cross-protection to food stress, it was not as high as the acid tolerance response. Genetic diversity analysis of surviving populations from mixed strain inoculations of the soft cheese revealed that after 15 days of storage, one persistent strain (159/10) remained as the dominant survivor. As revealed by Fourier Transform Infrared (FT-IR) spectroscopy analysis, the effects of acid stress on both stress-susceptible (strain 69) and tolerant (159/10) strains involved disruptions in protein secondary structure, conformational changes in nucleic acids, and disruptions in cellular lipids and polysaccharides. However, changes in cell membrane lipid acyl chains related to membrane fluidity appeared to be an important factor in the acid stress response of the susceptible strain. Scanning electron microscopy showed that biomolecular changes were accompanied by a physical damage to the cell surface structures. When subjected to lethal acid stress, a highly tolerant and persistent cell subpopulation that survived, owed its persistence to a phenotypic differentiation into a metabolically inactive state characteristic of persister cells. When the persister survivors were re-grown under mildly acidic and cold conditions, the stress response gene expression profiles of the regrown cell populations were not different from control cells indicating a general lack of heritable stress resistance. However, there was an exception with respect to the reduced expression levels of the phosphotransferase system (PTS) coding gene lmo1038. A downregulation of PTS systems potentially infers a suppressed role for sugar uptake systems in persister survivors with a subsequent carry-over of such expression patterns in re-grown cells through epigenetic means. The findings of this study indicated that stress-injured, and stress hardened tolerant cells can be a food safety risk if conditions in contaminated foods allow for their growth or survival. The stress-induced formation of persister cells provides a potential explanation for the challenge of L. monocytogenes persistence in food processing environments.