Screening and mode of action of natural antimicrobials to control Listeria monocytogenes in foods

Listeria monocytogenes is an important foodborne pathogen for which adequate control measures need to be implemented along the food production chain. The organism is widespread in the natural environment and can thus contaminate raw materials of both plant and animal origin. It can also be found and persist in food production facilities and from there contaminate food products during manufacturing. Despite its wide distribution and frequent presence in foods, the disease caused by the pathogen – listeriosis- is relatively rare, but it has a high mortality of around 20%. Listeriosis normally occurs only upon ingestion of large numbers of the pathogen, and the highest listeriosis risk is represented by foods in which L. monocytogenes can grow and which are not heated before consumption, such as cooked ham, ready-to-eat sandwich spreads etc. Prevention of listeriosis is therefore aimed at controlling pathogen outgrowth, and this is often achieved by a multi-hurdle approach that incorporates food preservatives. However, there is an increasing pressure to omit artificial food preservatives or replace them by natural alternatives because of safety concerns and because of the trend towards more natural foods. Despite extensive screening, natural preservatives are generally less effective and often cause unacceptable sensory deviations at effective concentrations. In the current project, we propose two basic research tracks that could lead to the development of more effective antilisterial preservatives. First, we will adopt an alternative screening method to detect more effective compounds. Screening for natural food preservatives has hitherto been conducted under standardized optimal growth conditions in the laboratory, and we hypothesize that compounds resulting from such screens are not necessarily the most effective under conditions where L. monocytogenes is a hazard in foods, i.e. at low temperature and/or elevated salt concentration. L. monocytogenes has a unique capability to cope with these conditions by activating specific stress adaptation pathways. By conducting a screen of a wide range of plant extracts and their pure constituents under these conditions, we expect to identify highly effective antimicrobials that specifically target the capability of L. monocytogenes to grow at low temperature or at elevated salt concentration. Second, once we have provided evidence for our hypothesis, at least one of the identified compounds will be subjected to a detailed mode of action study consisting of different approaches. A powerful approach, not requiring an a priori hypothesis on the mode of action, is a genetic approach, in which bacterial mutants with altered sensitivity to the compound will be isolated and wherein hypotheses on the mode of action will be deduced from the function of the mutated genes. Mutants will be generated by transposon mutagenesis (mainly loss of function) and by adaptive laboratory evolution (also gain of function). Another -more targeted- approach will be the use of in silico molecular docking, to study whether the selected compound effectively targets a key enzyme in the cold or salt adaptation pathways of the pathogen. An attempt will also be made to use molecular docking for de novo identification of compounds that target one or more of these key enzymes. In the long run, insight in the mode of action of the antilisterial compounds will allow to design blends of antimicrobials which have a synergistic effect, in order to enhance their effectiveness and reduce undesired side-effects.