Biofilm capacity of the psychrophilic bacteria triggers their persistence in the equipment and their spread to beef products throughout processing

Microbial contamination in food processing remains a persistent and complex challenge. Understanding the sources, contributing factors, and control measures is essential for effective mitigation. In this study we employed a combination of metagenomic sequencing, targeted culturomics, and whole-genome sequencing of key isolates to gain a comprehensive view of bacterial dynamics and functional capabilities throughout a working shift in a beef slaughter and cutting facility. This allowed us to identify which bacteria are i) most prevalent in the clean facility before the start of the work, ii) able to establish themselves over time, and iii) detectable in the final product. We further generated a functional profile of the microbial community within the facility, with a particular focus on antimicrobial resistance and biofilm formation genes, and the presence of specific pathogens and spoilage organisms. Both culture-based and sequencing data showed that Psychrobacter and Pseudomonas strains present in the final product were also detected on the membrane skinner, a machine used to remove all the excess tissues from meat, and in the drains even after cleaning. We found a high number of genes involved in biofilm formation in Psychrobacter immobilis , a characteristic that may explain their biofilm capacity and the survival of this species during the cleaning process and persistence throughout the facility. Taken together, our findings suggest potential sources of contamination and highlight the advantages of integrating culture-dependent methods with high-throughput sequencing technologies to enhance microbial monitoring and control strategies in food production environments. • Pseudomonas fragi and Psychrobacter immobilis increased in abundance on most of the surfaces in the cutting and packaging facility of the abattoir. • Biofilm-associated genes were found throughout the facility, and the samples collected pre-shift, in the clean facility, contained the highest abundance. • P. bubulae 0015 and P. immobilis 1601 encoded for spoilage related genes, necessary for production of acetoin, ammonia and putrescine. • P. immobilis 1601 strain forms biofilms in nutrient poor environments.