Listeria monocytogenes colonises established multispecies biofilms and resides within them without altering biofilm composition or gene expression

Listeria (L.) monocytogenes can survive for extended periods in the food producing environment. Here, biofilms possibly provide a niche for long-term survival due to their protective nature against environmental fluctuations and disinfectants. This study examined the behaviour of a L. monocytogenes ST121 isolate in a multispecies biofilm composed of Pseudomonas (P.) fragi, Brochothrix (B.) thermosphacta, and Carnobacterium (C.) maltaromaticum, previously isolated from a meat processing facility. The composition of the biofilm community and matrix, and transcriptional activity were analysed. L. monocytogenes colonised the multispecies biofilm, accounting for 6.4 % of all total biofilm cells after six hours. Transcriptomic analysis revealed 127 significantly up-regulated L. monocytogenes genes compared to the inoculum, including motility, chemotaxis, iron, and protein transport related genes. When comparing the differentially expressed transcripts within the multispecies biofilm with and without L. monocytogenes, only a cadmium/zinc exporting ATPase gene in C. maltaromaticum was significantly upregulated, while the other 9313 genes in the biofilm community showed no significant differential expression. We further monitored biofilm development over time (6, 24 hours and 7 days). P. fragi remained the dominant species, while L. monocytogenes was able to survive in the multispecies biofilm accounting for 2.4 % of total biofilm cells after 7 days, without any significant changes in its abundance. The presence of L. monocytogenes did neither alter the biofilm community nor its matrix composition (amount of extracellular DNA, carbohydrates, and protein). Our data indicate that L. monocytogenes resides in multispecies biofilms, potentially increasing survival against cleaning and disinfection in food processing environments, supporting persistence.