Impact of mixed biofilm formation with environmental microorganisms on *E. coli* O157:H7 survival against sanitization

Many foodborne pathogens, including Escherichia coli O157:H7, Salmonella enterica, and Listeria monocytogenes, can form biofilms that threaten food safety because detached biofilm cells can cross-contaminate meat processing environments. Environmental biofilms in such facilities are typically multispecies communities shaped by local conditions, and mixed biofilms can enhance sanitizer tolerance of foodborne pathogens. Floor drains are recognized niches harboring diverse microorganisms and sometimes pathogens, influenced by drain location, processing activity, and cleaning/sanitization routines. Rinsates from carcasses and equipment collect in drains, making drain microbiota representative of the facility environment. Mixed biofilms could provide ecological niches that promote pathogen colonization and survival against routine sanitization, potentially increasing pathogen prevalence and contamination risk. Prior work shows environmental genera can drive synergistic or antagonistic interactions that affect pathogen biofilm development. Motivated by these findings, the authors hypothesized that interspecies interactions among resident microflora in meat processing facilities can either enhance or inhibit pathogen survival and persistence, affecting prevalence rates of pathogens such as E. coli O157:H7. Two beef plants with differing O157:H7 prevalence histories (Plant A higher, Plant B lower) were compared using floor drain samples to assess impacts on E. coli O157:H7 survival in mixed biofilms.

The introduction summarizes evidence that mixed-species biofilms can increase sanitizer tolerance in pathogens. Studies in household dishwashers showed synergistic interactions where Acinetobacter junii and Pseudomonas aeruginosa enhanced mixed biofilms facilitating Exophiala dermatitidis. Floor drain microbiota associated with Listeria colonization differed by genera: Prevotella and Janthinobacterium linked to Listeria-negative drains; Enterococcus and Rhodococcus enriched in Listeria-positive drains, with Janthinobacterium inhibiting and Enterococcus gallinarum enhancing Listeria biofilm formation. Additional cited work suggests rheotaxis can amplify drain contamination, and that in-house E. coli O157:H7 biofilms may contribute to high event period (HEP) contamination in meat plants. The literature thus supports the role of environmental community composition and interspecies interactions in shaping pathogen colonization and biofilm behavior.

Study design: Floor drain samples were collected from two beef processing plants (Plant A with historically higher E. coli O157:H7 prevalence; Plant B with lower prevalence). Drains from hotbox and cooler areas on opposing sides (≥30 m apart; separate drainage lines) were sampled using BPW-wetted cellulose sponges over ~500 cm² of grates, baskets, and entry vents. Samples were transported on ice. Microbiological characterization included Aerobic Plate Count, psychrophiles, and Enterobacteriaceae on Petrifilm; absence of E. coli O157:H7 was confirmed on O157 Chromagar. Culture and storage: Each drain sample was 50-fold inoculated into Lennox Broth without salt (LB-NS) and incubated at 7 °C for 5 days (200 rpm), then aliquoted with 15% glycerol and stored at −20 °C. The E. coli O157:H7 strain (from naturally contaminated beef trim) was maintained at −70 °C in LB-NS with 15% glycerol, streaked to TSA, and grown to stationary phase in LB-NS at 37 °C (200 rpm) to ~5×10^8 CFU/mL before dilution for experiments. Sanitizer: A QAC-based sanitizer (Vanquish™, alkylbenzyldimethyl-ammonium chloride mixture) was used at 300 ppm per manufacturer instructions. Biofilm formation: Thawed drain sample stocks were diluted 1:1000 into sterile LB-NS and incubated 5 days at 7 °C (200 rpm). Aliquots were plated on TSA and O157 Chromagar to confirm absence of O157 before adding the pathogen. For biofilm development, 15 mL cultures were placed in 50 mL tubes with a sterile stainless steel (SS) chip (18×18×2 mm) and incubated statically at 7 °C for 5 days. Mixed biofilms were formed by adding overnight E. coli O157:H7 culture (~5×10^8 CFU/mL) to 5-day drain cultures at 1:100 and incubating with SS chips for 5 days. Single-strain O157 biofilms were formed by incubating SS chips in 100-fold diluted O157 overnight culture for 5 days at 7 °C. Sanitization and recovery: Chips were rinsed with 10 mL sterile water (5 mL per side) and exposed to sterile water (control) or 300 ppm QAC for 1 min (minimum recommended exposure). Chips were then transferred to Dey/Engley broth (10 mL; with 0.3% soytone and 0.25% NaCl) with sterile glass beads (425–600 µm), followed by 1 min sonication and 2 min vortexing to detach biofilm cells. Serial dilutions were plated on TSA (total biofilm bacteria) or O157 Chromagar (E. coli O157:H7 in mixed biofilms) and incubated overnight at 37 °C. Log10 CFU per chip were calculated. Confocal laser scanning microscopy (CLSM): Biofilms on SS chips were stained with FM 1–43 (1:1000) for 15 min, washed, and imaged the same day using Nikon CLSM (excitation 488 nm, emission 500–550 nm) with 60× WI objective. Z-stacks were acquired. Biofilm structure was analyzed with COMSTAT2; 3D renderings were generated in FIJI 3D viewer. 16S rRNA gene amplicon sequencing: Biofilm cells (pre-treatment; post-QAC with and without O157 addition) were harvested, washed in PBS, and DNA extracted using the PowerSoil kit. DNA was quantified by Qubit HS assay. The V4 region (primers 515F/806R) was sequenced (Illumina MiSeq 2×250 bp; NeoSeek). QIIME2 was used for demultiplexing, quality control (truncation: F 240 bp, R 200 bp), ASV generation, and taxonomy assignment (Greengenes 99% classifier). In total, 24 samples yielded 3,572,808 sequences and 251 features. Heatmaps were created from CSS-normalized OTU tables (metagenomeSeq), visualized in ggplot2; log2 fold changes were computed on log2-transformed CSS counts (gtools). Only families >1% of total microbiome were considered for differential analyses. Statistics: Biofilm counts (log10 CFU/chip) were analyzed via one-way ANOVA with post hoc Tukey’s or Dunnett’s tests (GraphPad Prism). Standard deviations and 95% CIs were calculated; P<0.05 considered significant. Log reductions’ SDs were computed as SD = sqrt(SD_pre^2/n_pre + SD_post^2/n_post).

- Environmental drain samples from both plants formed substantial biofilms on stainless steel at 7 °C; total biofilm cells ranged from 5.3 to 6.4 log10 CFU/chip. Cooler drains from both plants produced >6.0 log10 CFU/chip biofilms; sample A-C1 (Plant A cooler) had the highest biomass. - In mixed biofilms, E. coli O157:H7 recovery ranged from 2.9 to 4.0 log10 CFU/chip; single-strain O157 biofilms reached ~5.0 log10 CFU/chip, significantly higher than in mixed biofilms. - CLSM showed biofilm thicknesses from ~20 to 158 µm; adding O157:H7 increased biofilm thickness. A-C1 formed thick, dense, sanitizer-tolerant biofilms that protected O157:H7; B-H2 formed thin biofilms, was sanitizer-susceptible, and provided minimal protection. - QAC (300 ppm) reduced total viable biofilm bacteria by 1.3–3.0 log10 CFU/chip. Largest reductions: A-H2 (Plant A hotbox, 2.0 log10) and B-C2 (Plant B cooler, 3.0 log10). Others showed 1.3–1.8 log10 reductions. - O157:H7 survival after QAC in mixed biofilms depended on the co-cultured drain community. Highest survivors: with A-C1 (3.8 log10 CFU/chip), A-C2 (2.5), A-H1 (2.0) — all Plant A. Lowest survivors: with A-H2 (0.8), B-H1 (0.7), B-H2 (0.6). O157 survival in mixed biofilm with A-C1 was significantly higher than most others and higher than O157 in single-strain biofilm; in other mixes, survival was not significantly higher than single-strain. - Microbiome profiling (pre-QAC) showed Pseudomonadaceae, Moraxellaceae, and Enterobacteriaceae in all samples. A-C1 (strongest protector) had the highest diversity and included dominant Flavobacteriaceae, Moraxellaceae, Listeriaceae, Pseudomonadaceae, Enterobacteriaceae, Weeksellaceae, Sphingobacteriaceae, Aeromonadaceae, plus 7 unique families. - Protector group (A-C1, A-C2, A-H1) shared Weeksellaceae, Sphingibacteriaceae, and Brucellaceae. Non-protectors (A-H2, B-C1, B-C2, B-H1, B-H2) commonly included lactic acid bacteria families Lactobacillaceae and Leuconostocaceae. - After QAC (with O157), protectors showed increased Enterococcaceae and Oxalobacteraceae; decreases in Listeriaceae and Flavobacteriaceae. In non-protectors, LAB families decreased after treatment. Pseudomonadaceae and Oxalobacteraceae, initially higher in non-protectors, were reduced by QAC in non-protector samples. - Protective effects on O157 survival were not directly predicted by overall mixed-biofilm sanitizer tolerance or total biovolume alone, implicating community composition and spatial organization as key factors.

The study demonstrates that mixed microbial communities from meat plant floor drains can modulate E. coli O157:H7 colonization and sanitizer tolerance. Consistent with prior work, O157:H7 in mixed biofilms generally survived QAC better than in single-strain biofilms, but survival varied markedly by environmental community. Plant A communities, from a facility with recurrent O157:H7 prevalence, tended to protect O157:H7 more than Plant B communities. This protection did not strictly correlate with total biofilm sanitizer tolerance or biomass, indicating that specific taxa, diversity, and spatial organization within the 3D biofilm structure contribute to sanitizer protection (e.g., layered architectures that impede sanitizer penetration). Microbial diversity and adherence traits affect tolerance; Enterococcaceae increased in protector communities post-sanitization, while Flavobacteriaceae decreased. A “memory effect” from prior co-habitation may favor recruitment and protection of recurrent pathogens like O157:H7 in Plant A. Conversely, Plant B communities, enriched in lactic acid bacteria, may antagonize O157:H7 via competition and antimicrobial metabolite production, contributing to lower pathogen prevalence. These findings suggest that local microecology, history of contamination, and sanitation practices shape communities that either protect or inhibit pathogen persistence, and that targeting community composition and structure could improve control strategies.

Environmental microorganisms in meat processing plants influence E. coli O157:H7 colonization and sanitizer tolerance through mixed biofilm formation. Communities from the higher-prevalence Plant A, especially the diverse A-C1 community, provided strong protection to O157:H7 after QAC treatment, whereas certain Plant B communities were associated with poor O157:H7 survival, likely due to competitive and inhibitory interactions (e.g., lactic acid bacteria). Protection was associated with specific community compositions and not solely with overall biofilm mass or bulk sanitizer tolerance. Future research should identify critical species and mechanisms (including spatial organization) that mediate protection or inhibition, enabling design of niche-specific antimicrobial interventions and potentially leveraging protective probiotics (e.g., LAB) to prevent pathogen biofilms in processing environments.

- The study examined floor drain communities from only two processing plants and four drain locations per plant, which may limit generalizability across facilities. - Biofilms were modeled in vitro on stainless steel at 7 °C in LB-NS, which may not fully replicate in-plant conditions and nutrient environments. - 16S rRNA amplicon sequencing provided family-level resolution for many taxa; species/strain-level contributors and mechanisms were not resolved. - Spatial organization within mixed biofilms and precise locations of E. coli O157:H7 cells were not directly determined; authors note the need for fluorescent tagging and time-lapse imaging. - The study focused on a single O157:H7 strain; results may vary with other strains or pathogens.