Evolution of Listeria monocytogenes Strains in Long-Term Cultures

Listeria monocytogenes is a ubiquitous bacterium and the causative agent of listeriosis. Although rare, it is a severe foodborne illness with high hospitalization and mortality rates in humans. Thermal processing of food can inactivate the organism, but post-processing contamination remains a major threat in the industry. Unlike many other food-borne pathogens, L. monocytogenes tolerates high salt levels, grows at refrigeration temperatures, and forms persistent, viable-but-non-culturable (VBNC) cells that resist elimination even after rigorous cleaning and sanitation. Because VBNC cells evade routine microbiological tests, they create a false sense of security and increase the likelihood that contaminated foods reach consumers unnoticed. It is therefore crucial to thoroughly investigate the survival mechanisms adopted by L. monocytogenes to develop improved strategies for controlling contamination and ensuring food safety. This study investigates the long-term survival strategies and genetic mutations of a reference Listeria monocytogenes strain in a nutrient-poor environment. The strain was incubated in Minimal Bacterial Medium (MBM) for 180 days at 4 °C and 30 °C in five culture replicates. After 180 days of incubation, the results identified that the cultures survived at both temperatures under starvation. At 4 °C, the viable cell counts averaged 4.8 log CFU/mL with 0.6 log VBNC cells/mL; at 30 °C, the viable cell counts averaged 4.0 log CFU/mL with 1.3 log VBNC cells/mL. The replicates were then subjected to next-generation sequencing (NGS) to analyse the presence or absence of mutant genes and to gain deeper insight into the cellular processes they influence. The results revealed a higher frequency of mutations in cultures maintained at 30 °C than in those held at 4 °C. These findings advance our understanding of L. monocytogenes stress tolerance and will inform the development of targeted interventions to prevent contamination along the food-production chain.