Colonization of Native Dairy Facility Biofilms by Persistent Listeria monocytogenes Strains

Abstract Listeria (L.) monocytogenes , the causative agent of listeriosis, can persist in food processing environments for months to years and poses a significant food safety risk. Persistence is often linked to integration into multispecies biofilms, yet the influence of community composition and timing of L. monocytogenes integration remain poorly understood. This study investigates the integration of a long-term persistent L. monocytogenes strain into multispecies biofilms derived from two sites in an Austrian dairy facility: one where L. monocytogenes was detected, and one where it was absent. Biofilms were cultivated on stainless steel slides under laboratory conditions and L. monocytogenes was introduced at different stages of biofilm development. After 24 h and 48 h, biofilms were analysed using qPCR targeting the prfA gene and the bacterial 16S rRNA gene. Community composition was characterized using full-length 16S rRNA gene sequencing. Both communities formed reproducible multispecies biofilms and were successfully colonized by L. monocytogenes regardless of inoculation timing. Community composition differed markedly between the biofilms. Community background, sequential inoculation and timing influenced community development. Several taxa were associated with L. monocytogenes integration, with Serratia liquefaciens showing co-occurrence and Lactococcus lactis displaying patterns consistent with competitive exclusion. These findings demonstrate that L. monocytogenes can integrate into diverse multispecies biofilms and highlight specific microbial taxa that may influence pathogen persistence. The results provide potential targets for reducing L. monocytogenes persistence and contamination in dairy processing environments.