Physical and metabolic causes of sub-lethal damage in Listeria monocytogenes after long-term chilled storage at 4°C

Cells of Listeria monocytogenes display sub-lethal injury when subjected to long-term chill-storage in a nutrient-poor environment. The physical and metabolic causes or sub-lethal injury to two meat (L61 and L62) and two clinical (L98 and L99) L. monocytogenes strains chill-stored (4 degrees C) for 4 weeks in phosphate-buffered saline at pH 7.0 and pH 5.5, and pH 5.5 in the presence of 0.3% potassium sorbate, were characterized. Causes of sub-lethal injury were determined by examining changes in the cell structure, leakage of nucleic acids and proteins from the cells, and cell recovery from injury in the presence of the metabolic inhibitors rifampicin, D-cycloserine, carbonyl cyanide m-chlorophenylhydrazone and chloramphenicol. Visible shrinkage of the cytoplasm and slight cell wall damage were apparent over the 4 week storage period by electron microscopy for all four strains and three storage conditions. By contrast, over the same storage period, only three of the strains (L62, L98 and L99) displayed slight leakage of cellular content in all three storage media, while one strain (L61) displayed greater leakage. The three strains also displayed similar storage media-dependent metabolic damage. For these strains, phosphate-buffered saline pH 5.5 caused the least damage and potassium sorbate, the most. Recovery experiments also indicated that at pH 5.5, the energy transduction system of these three strains remained undamaged, and that injury to the cell transcription machinery was greatest at pH 7.0. The fourth strain, L61, displayed less damage than the others but this was attributed to the death of the injured cell sub-population in this strain. In this study, damage to sub-lethally injured chill-stored L. monocytogenes was different from that caused by other agents, such as heat. Therefore, cells injured by chill-storage under starvation conditions may require novel protocols to assure their effective recovery.