Persistence and food safety: an in-vitro study of the persister state in Listeria monocytogens strains to simulate the persistence in the food environment

The persistence in the food environment (facilities and products) of Listeria monocytogenes (Lm) is a challenging problem in food safety management. Lm is still the first mortality cause in food-borne infections and the trend is always increasing. The objective of the project is to advance knowledge on persistence of Lm. The persistence and the formation of persister cells in five Lm strains will be investigated using an in-vitro system in presence of sublethal stress (starvation, refrigeration T°C, low pH, low Aw, thermal stress, antimicrobials), using two different approaches. The first approach involves the creation of long-term cultures that can simulate persistence in-vitro. A single stress (starvation) or two stress types (starvation and refrigeration) will be chronically imposed on long term cultures with the aim of simulating what could actually happen in a real food environment. The long-term culture populations (persister and not persister cells) will be analyzed as total population or separated by cytofluorimeter and analyzed separately. In addition, the long-term cultures will be analyzed for the ability to form biofilm, a characteristic of bacteria frequently associated with persistence. Finally, metagenomic and metatranscriptomic analysis will be carried out on total population and on the two separated populations and WGS and MALDI-TOF analysis will be directed to clones isolated from both populations. In the second approach, the formation of persister cells following different types of stress (Short-term cultures) will be evaluated, both classic stresses present in the food industry (temperature, acidity, detergents), or stress deriving from natural antimicrobials. The formation of the persister cells will be monitored using the MIC/MDK system or using the Scan-Lag. Persister isolates will be characterized both phenotypically and genotypically (WGS and MALDI-TOF analysis).