Microbial transcriptional dynamics of beef-processing drain biofilm models revealed by enrichment-based...

Microbial biofilms in beef-processing facilities represent persistent reservoirs of foodborne pathogens and spoilage organisms, posing significant risks of cross-contamination of meat products. Floor-drains, as nutrient-rich convergence points within processing environments, are particularly conducive to multi-species biofilm formation. While previous studies have characterized the taxonomical composition and functional potential of drain microbial communities, their transcriptional activities remain largely unexplored. To address this gap, we developed a metatranscriptomic approach to study the transcriptional dynamics of drain-associated microbiomes in beef-processing facilities using enrichment-derived drain biofilm models (hereafter referred to as biofilm models). Floor drain swab samples were collected from hotbox and cooler areas of nine beef-processing plants in 2019 and 2021, and subjected to laboratory enrichment under processing-relevant ecological conditions prior to RNA extraction and sequencing. Metatranscriptomic analysis revealed a core set of highly transcriptionally active genera, including Pseudomonas, Carnobacterium, Acinetobacter, and Brochothrix, in the biofilm models. Functional profiling indicated high expression (Σlog₁₀TPM >3.5) of key biofilm-associated functions such as cell adhesion, exopolysaccharide biosynthesis, bacterial chemotaxis, and quorum sensing (QS), suggesting potential biofilm formation, migration and microbial communication. In comparing biofilm models developed from samples collected in 2019 and 2021, a significant upregulation of genes associated with biofilm formation were identified in 2021-derived communities, suggesting differences in transcriptional responses under identical enrichment conditions between microbial communities originating from the two sampling periods. Transcriptional activity of antimicrobial resistance (AMR) genes was also detected, particularly those associated with tolerance to quaternary ammonium compounds (QACs), the predominant sanitizers used in food-processing environments. The biofilm models employed in this study may introduce selection bias relative to the native drain community. However, by using drain-derived inocula incubated under processing-relevant conditions, this approach captures the transcriptional potential of the drain-associated microbial communities. This framework provides a reproducible and experimentally accessible platform for investigating gene expression dynamics in complex food-environment microbiomes, and establishes a foundation for future in situ and controlled in vitro studies. Collectively, these findings advance our understanding of drain microbiome ecology and may offer insights for designing intervention strategies to improve biofilm control in meat-processing environments.