Foodborne pathogens like *E. coli* O157:H7, *Salmonella enterica*, and *Listeria monocytogenes* form biofilms, posing a significant food safety risk in meat processing plants. Detached biofilms can lead to cross-contamination. Environmental biofilms are typically multispecies, their composition shaped by the environment. Mixed biofilms can enhance sanitizer tolerance of foodborne pathogens. Floor drains in meat processing plants are important niches harboring pathogens like *L. monocytogenes*, often containing diverse biofilm-forming microorganisms. Drain composition is dynamic, influenced by location, processing activity, and sanitation procedures. Bacteria can even move upstream via rheotaxis, amplifying contamination. Rinsing water accumulating in drains contains microorganisms from various sources, making drain communities representative of the processing environment. Mixed biofilm formation by environmental microorganisms and foodborne pathogens can create an ecological niche enhancing pathogen colonization and survival against sanitization, increasing pathogen prevalence and contamination risk. Interspecies interactions within the resident microflora can either promote or inhibit pathogen growth and colonization. Studies have shown synergistic interactions among bacteria (e.g., *Acinetobacter junii*, *Pseudomonas aeruginosa*) enhancing mixed biofilm formation and promoting fungal pathogen establishment. Other studies revealed variations in bacterial populations in floor drains with or without *Listeria* colonization, with genera like *Prevotella* and *Janthinobacterium* associated with *Listeria*-negative samples, while *Enterococcus* and *Rhodococcus* were more abundant in *Listeria*-positive samples. *Janthinobacterium* inhibited *L. monocytogenes* biofilm formation, while *Enterococcus gallinarum* enhanced it. This study hypothesized that interspecies interactions in meat processing facilities would either enhance or inhibit pathogen survival and persistence, impacting the prevalence of pathogens like *E. coli* O157:H7. Some plants experience higher *E. coli* O157:H7 prevalence than others without clear causes. Plant A in this study had a historically higher prevalence than Plant B. Plant A showed a 0.5–1.0% positive rate for *E. coli* O157:H7, with recurring “high event periods” (HEPs) reaching 3–10% positive rates, while Plant B had a lower positive rate. Biofilm formation by *E. coli* O157:H7 might contribute to HEP contamination. Therefore, the study collected floor drain samples from these two plants to compare the impact of environmental microorganisms on *E. coli* O157:H7 survival in mixed biofilms.

The literature review section of the paper focuses on previous research demonstrating the impact of mixed-species biofilms on the survival and persistence of foodborne pathogens, particularly *E. coli* O157:H7. Several studies highlighted the enhanced sanitizer tolerance of pathogens within these complex communities compared to single-species biofilms. The role of interspecies interactions, both synergistic and antagonistic, in shaping the composition and function of these biofilms was also discussed. Specific examples included research on synergistic interactions between bacterial species, such as *Acinetobacter junii* and *Pseudomonas aeruginosa*, in promoting the establishment of opportunistic fungal pathogens. Furthermore, the influence of bacterial genera on *Listeria* biofilm development was reviewed, illustrating the complex interplay between different bacterial species and their impact on pathogen survival. The previous work laid the groundwork for the current study by establishing the importance of mixed biofilm formation in the context of food safety and the need to understand the underlying mechanisms governing pathogen persistence in meat processing environments.

Floor drain samples were collected from two beef processing plants (Plant A with higher *E. coli* O157:H7 prevalence and Plant B with lower prevalence) using cellulose sponges moistened with buffered peptone water. Samples were taken from cooler and hotbox drain locations in each plant. Aerobic plate counts, psychrophilic bacteria counts, and *Enterobacteriaceae* counts were performed. Samples were checked for the absence of *E. coli* O157:H7 using O157 Chromagar plates. To simulate the plant environment, drain samples were inoculated into Lennox Broth (LB) without salt (LB-NS) and incubated at 7°C for 5 days. *E. coli* O157:H7 strain (isolated from contaminated ground beef) was cultured separately. Biofilms were developed on stainless steel chips immersed in the 5-day LB-NS cultures of drain samples, with and without the addition of *E. coli* O157:H7 (1:100 ratio). Single-strain *E. coli* O157:H7 biofilms served as controls. Biofilms were treated with 300 ppm of a quaternary ammonium chloride (QAC)-based sanitizer (Vanquish™) for one minute and then neutralized with Dey/Engley broth. Viable cells were quantified by colony enumeration on TSA and O157 Chromagar plates. Confocal laser scanning microscopy (CLSM) using FM 1-43 dye was used to visualize biofilm architecture. DNA was extracted from biofilm samples, and 16S rRNA gene amplicon sequencing (V4 region) was performed to analyze bacterial community composition using Illumina MiSeq. Data were analyzed using QIIME2.0, and statistical analyses (ANOVA, Tukey's or Dunnett's tests) were performed using GraphPad Prism. Bacterial diversity and community composition were assessed before and after QAC treatment. Samples were classified into *E. coli* O157:H7 “protectors” and “non-protectors” based on pathogen log reductions after sanitization to analyze the correlation between community composition and pathogen tolerance. Heatmaps and log2 fold changes were generated in R to visualize the relative abundance of bacterial families in each group.

All drain samples formed biofilms on stainless steel at 7°C, with varying biofilm-forming ability. Total biofilm cells ranged from 5.3 to 6.4 log10 CFU/chip. Cooler drain samples generally had higher biofilm mass. *E. coli* O157:H7 cell numbers in mixed biofilms ranged from 2.9 to 4.0 log10 CFU/chip, significantly lower than in single-strain biofilms (~5.0 log10 CFU/chip). CLSM revealed biofilm thickness ranging from 20.19 µm to 157.55 µm, with increased thickness after adding *E. coli* O157:H7. QAC treatment reduced total bacterial cells by 1.3–3.0 log10 CFU/chip, with the greatest reduction in samples B-C2. *E. coli* O157:H7 survival varied significantly depending on the co-cultured drain sample. The lowest survival was observed with hotbox samples (0.6–0.8 log10 CFU/chip), while the highest survival (2.0–3.8 log10 CFU/chip) was seen with cooler samples from Plant A (A-C1, A-C2, A-H1). Only A-C1 showed significantly higher *E. coli* O157:H7 survival in the mixed biofilm than in the single-strain biofilm after QAC treatment. 16S rRNA gene sequencing revealed 28 bacterial families in the drain biofilms, with Pseudomonadaceae, Moraxellaceae, and Enterobacteriaceae present in all samples. Sample A-C1 (strongest *E. coli* O157:H7 protector) had the highest bacterial diversity. QAC treatment altered the relative abundance of different taxa. In A-C1, Flavobacteriaceae and Listeriaceae decreased after treatment and addition of *E. coli* O157:H7, while Carnobacteriaceae, Micrococcaceae, and Enterococcaceae increased. Samples grouped as *E. coli* O157:H7 “protectors” (A-C1, A-C2, A-H1) shared Weeksellaceae, Sphingibacteriaceae, and Brucellaceae. “Non-protectors” had Lactobacillaceae and Leuconostocaceae (LAB). After QAC treatment, Enterococcaceae and Oxalobacteraceae increased in protectors, while Listeriaceae and Flavobacteriaceae decreased. In non-protectors, Lactobacillaceae and Leuconostocaceae decreased. The strongest protector (A-C1) had the highest diversity and showed increased Enterococcaceae after sanitization.

The results support the hypothesis that environmental microorganisms influence *E. coli* O157:H7 colonization and sanitizer tolerance. *E. coli* O157:H7 survival was generally higher in mixed biofilms from Plant A (recurrent prevalence history) than Plant B. This protection wasn't solely related to overall biofilm tolerance or biovolume. Mixed biofilms develop through synergistic interactions, with species recruitment based on co-evolution. *E. coli* O157:H7 established itself in mixed biofilms, but its numbers were lower than in single-strain biofilms, likely due to competition for resources or inhibitory substances. Biofilm architecture acts as a physical barrier against sanitizer penetration. Low-abundance species like those in the *Microbacteriaceae* family in A-C1 might play crucial roles in spatial organization, creating a protective layered structure. Bacterial diversity, adhesion, competition, and biofilm formation on processing materials all impact pathogen tolerance. The high *E. coli* O157:H7 prevalence in Plant A might be due to adaptive responses and a “memory effect,” where the community is structured to protect *E. coli* O157:H7. Plant B's lower prevalence might be linked to its microbial community, including the presence of LAB, known to inhibit *E. coli* O157:H7. The study suggests using LAB as probiotic biofilm formers for pathogen control. Future studies are needed to identify specific species and mechanisms of pathogen protection or inhibition.

This study demonstrates that environmental microorganisms in meat processing plants significantly impact *E. coli* O157:H7 survival and sanitizer tolerance. Plant A's history of *E. coli* O157:H7 prevalence correlated with higher species diversity and better protection of the pathogen in mixed biofilms. Plant B's lower prevalence may be linked to its distinct microbial community, including LAB. This highlights the importance of understanding complex microbial interactions for developing targeted interventions in food processing facilities. Future research should focus on identifying specific species and mechanisms to improve sanitation strategies.

The study was conducted in only two meat processing plants, potentially limiting the generalizability of the findings. The chosen sanitizer (QAC) might not represent all sanitation practices in the industry. While the study analyzed bacterial community composition, the specific mechanisms of interaction between *E. coli* O157:H7 and other species remain to be elucidated. Further investigation is needed to confirm the role of low-abundance species in biofilm architecture and protection. Finally, the simulated biofilm conditions might not perfectly reflect the dynamic environment of actual processing plants.