How bacteria survive in food-related environments: L. monocytogenes and P. fluorescens dual-species biofilms in the dairy industry

In nature, bacterial biofilms are typically composed of two or more microbial species. For instance, in the dairy industry Listeria monocytogenes and Pseudomonas fluorescens are often associated in mixed-species biofilms that increase bacterial resistance to disinfectants. Our study evaluated the behaviour of L. monocytogenes and P. fluorescens alone and their interactions in a dual-species biofilm, mimicking the conditions of dairy processing environments. Using a Ricotta-based model system as growth medium, biofilm formation was assessed on stainless steel (SS) surfaces at 12 °C for 7 days. Hence, the biofilm biomass was determined through crystal violet staining, also measuring the sessile cells loads. Moreover, the carbohydrates produced within the extracellular polymeric substances (EPS) were quantified by the anthrone method, and Confocal Laser Scanning Microscopy (CLSM) was used to analyse the biofilm structure. The bacterial species, both alone and in combination, were able to produce biofilm on SS surface. It is noteworthy that P. fluorescens increased L. monocytogenes sessile load from 1.40 ± 0.20 to 3.58 ± 0.16 Log CFU/cm2, as well as the total EPS carbohydrates, which reached 2.91 µg/cm² after 72 h. CLSM evidenced the presence of green agglomerates, probably due to the formation of P. fluorescens blue pigment. This study highlights the different behaviour of the two bacteria when considered alone and suggests that the interactions between the two species can influence biofilm formation, but not the capability of P. fluorescens to produce blue pigment. Given the potential risk for consumers, the stimulating effect of P. fluorescens on L. monocytogenes adhesion on food contact surfaces should be carefully considered. The results also underline the need to investigate biofilms made of bacterial consortia, which represent the real situation observed in food environments.