Dynamic social interactions and keystone species shape the diversity and stability of mixed-species biofilms – an example from dairy isolates

Biofilms are a concern across industries (food, water, health, industrial, marine) and typically comprise multiple interacting species forming associations from cooperation and commensalism to competition, amensalism, and exploitation. In dairy environments, multispecies biofilms can harbor pathogens and spoilers impacting product safety and quality due to elevated spoilage enzymes and toxins, and may persist after cleaning and disinfection. Despite recognition of their importance, interspecies interactions remain difficult to dissect. Pairwise interaction outcomes have been used to predict structure and function in simplified communities, but they miss higher-order interactions where additional species modulate pairwise effects. Keystone species—those exerting disproportionate effects irrespective of abundance—can shape community dynamics, stability, and function. Understanding variability and strength of both pairwise and higher-order interactions is essential to explain coexistence, stability, and functionality and to inform control strategies on food contact surfaces. Building on previous work identifying synergistic four-species biofilms on stainless steel from dairy pasteurizer isolates—with several communities sharing Stenotrophomonas rhizophila, Bacillus licheniformis, and Microbacterium lacticum and showing strong synergy when combined with Calidifontibacter indicus—this study asks: What interspecies social interactions underlie the observed biofilm synergy and stability? What is the role of keystone species and higher-order interactions in community assembly and function? Using a bottom-up approach, the study disentangles pairwise and higher-order interactions among these four species to reveal mechanisms driving synergy, stability, and coexistence in a dairy-relevant biofilm model.

- Multispecies biofilms commonly exhibit complex interactions affecting structure and function, with implications across sectors and particularly in food processing environments where persistence after sanitation is documented. - Prior studies have used pairwise interaction outcomes to predict community assembly, but higher-order interactions can modulate pair interactions, challenging predictive power based solely on pairs. - Keystone taxa can drive biofilm formation, antimicrobial tolerance, and community structure; removing certain strains can alter diversity and composition in synthetic communities. - Previous work by the authors characterized synergistic four-species biofilm communities on stainless steel from dairy pasteurizer surfaces; synergy frequently involved S. rhizophila, B. licheniformis, and M. lacticum, with strong synergy upon adding C. indicus. - Environmental modification (e.g., pH shifts) can mediate interactions and coexistence; pH stabilization has been shown to facilitate community synergy in soil isolates. - Literature debates whether pairwise interactions suffice to explain community assembly or whether higher-order interactions must be considered; multiple studies in host-associated microbiomes show higher-order effects dampen pairwise competition and affect species abundances.

Strains and culture conditions: - Four dairy pasteurizer surface isolates were used: Stenotrophomonas rhizophila (SR, B68), Bacillus licheniformis (BL, B65), Microbacterium lacticum (ML, B30), Calidifontibacter indicus (CI, 844). Strains grown in Brain Heart Infusion (BHI) at 30°C; freezer stocks at −70°C. Biofilm formation on polystyrene (96-well): - Overnight (~16 h) cultures in BHI at 30°C, static, diluted to OD595=0.05. - Inoculum volume per well: 160 µL. For mixed cultures (dual, tri, quad), equal volumes of each species combined to total 160 µL. - Incubation: 24 h, 30°C, static. Biofilms stained with 0.1% crystal violet; CV solubilized in 33% glacial acetic acid; absorbance measured at 595 nm (OD595) using a plate reader. Cell-free supernatant (CFS) assays: - CFS prepared by filtering overnight planktonic cultures (0.2 µm). In mixed-species setups, each species was individually replaced by its own CFS to test metabolite effects on partners’ biofilm mass. - Equal volumes of CFS substituted for viable cells in BHI. To control for nutrient dilution (“dilution effect”), in cases of decreased biofilm mass with CFS, equivalent volumes of sterile water were used as controls. - Note: CFS is more diluted in higher-order communities (three or four members) than in dual cultures, potentially limiting interpretability. Biofilm development on stainless steel (SS) coupons and selective counting: - Substrate: AISI 304 SS coupons (30×15 mm) in 6-well plates (5 mL medium/well), coupons placed horizontally. - Media: BHI and cow’s skim milk (SM) for four-species time-course; BHI for pairwise and three-species cell counts. - Inoculation: Overnight cultures diluted to ~1×10^5 CFU/mL for each species; single and mixed cultures prepared with equal proportions in mixtures. - Incubation: 30°C; time points at 4, 8, 12, 16, 20, and 24 h (for quad-species dynamics); otherwise 24 h. - Post-incubation: Coupons rinsed 3× in sterile water to remove loosely attached cells; cells detached by sonication (10 min) plus vortexing (2 min) into 9 mL saline. - Selective counting: Species-specific media and conditions (temp, media type, antibiotics) validated not to bias counts versus BHI agar (details in Supplementary File S1). Planktonic fractions were also enumerated at matched time points. pH of planktonic fractions measured at 24 h for monocultures and combinations. Scanning electron microscopy (SEM): - Biofilms on SS (24 h, BHI) fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate (>8 h), post-fixed in 1% osmium tetroxide (1–2 h), washed in cacodylate, dehydrated in graded ethanol (30–100%), critical point dried (CO2), sputter-coated (gold-palladium, 4–5 min), imaged with Zeiss Crossbeam 540 FIB-SEM. Statistics: - Each experiment repeated three times with three replicates per trial. For CV assays, OD measured from four wells per plate across three replicate plates per experiment. - ANOVA with Duncan’s Multiple Range Test (SPSS v23) for biofilm mass comparisons (P<0.05). Paired two-tailed t-tests (GraphPad Prism 9) for comparing cell counts between monocultures and co-cultures (P≤0.05 considered significant).

- Community-level synergy: - Three-species SR-BL-ML biofilm mass increased 2.65-fold relative to the sum of monocultures (polystyrene assay). Adding CI as a fourth species further increased biofilm mass by 21%. - Previously observed 3.13-fold increase in the specific four-species combination SR-BL-ML-CI was corroborated. - Pairwise interactions (24 h, SS cell counts and polystyrene biofilm mass): - ML stimulated growth of all other species. • SR–ML: Commensalism; SR growth increased by ~2 log CFU/cm²; biofilm mass showed synergy; SEM indicated ML as a basal layer with SR settling atop. • BL–ML: Exploitation; BL cell count increased by ~1.4 log CFU/cm²; SEM revealed extensive EPS associated with BL. • CI–ML: Exploitation; CI cell count increased by ~2 log CFU/cm². - SR–CI: Amensalism; SR reduced CI growth slightly without affecting its own growth. - BL–CI: Exploitation; BL benefited with negative effect on CI. - SR–BL: Neutral in cell counts but strong biofilm mass synergy (~4-fold increase vs sum of monocultures). CFS from SR increased BL biofilm mass 2.7-fold, and CFS from BL increased SR biofilm mass 3.1-fold relative to their respective monocultures, but viable SR+BL together produced >2-fold more biofilm mass than combinations where one partner was replaced by CFS. - ML’s biofilm mass increased by ~24% in presence of CI CFS (likely due to absence of competitive effects seen with viable CI). - Higher-order interactions (triads and tetrad): - SR-BL-ML (triad): SR attained highest abundance (8.31±0.18 log CFU/cm²), followed by BL (7.06±0.31) and ML (6.88±0.47). SEM confirmed SR abundance and BL matrix production. - CFS from BL increased biofilm mass of SR-ML; CFS from SR increased biofilm mass of BL-ML, consistent with SR–BL pairwise effects and indicating higher-order modulation by ML. - Adding CI to pairs altered absolute and relative abundances (e.g., SR increased from 7.86 in SR-ML to 8.31 log CFU/cm² upon addition of BL in presence of ML, indicating modulation by a third species). - CI CFS significantly increased biofilm mass of BL-ML and SR-BL combinations. - Four-species community and CFS substitutions: • Replacing SR with its CFS in BL-ML-CI increased biofilm mass by 36.3% vs BL-ML-CI without SR CFS. • Replacing BL with its CFS in SR-ML-CI increased biofilm mass by 21% vs SR-ML-CI without BL CFS. • Replacing ML with its CFS in SR-BL-CI increased biofilm mass by only 5.1%, whereas adding viable ML to SR-BL-CI increased biofilm mass by 71% (highlighting the necessity of viable ML for maximal synergy). - ML acted as a keystone species: involved in most synergistic pairs and trios (SR-ML, BL-ML, SR-BL-ML, BL-ML-CI), stimulating growth of all others. In the four-species biofilm, SR, BL, and CI achieved fitness advantages, while ML’s growth was reduced due to exploitation by BL and CI. - pH-mediated environmental effects (24 h, planktonic): - Monocultures: SR pH 8.26±0.06; BL 8.04±0.05; ML 6.00±0.03; CI 7.46±0.15. - Key combinations: SR-ML 7.62±0.13; BL-ML 7.70±0.18; SR-BL 8.08±0.09; SR-BL-ML 8.18±0.10; SR-CI 8.16±0.03; BL-CI 7.85±0.10; ML-CI 6.46±0.15; SR-ML-CI 7.45±0.09; BL-ML-CI 7.63±0.07; SR-BL-ML-CI 8.01±0.11. Elevated pH associated with SR/BL dominance; lower pH associated with ML activity. - Temporal dynamics in four-species community (SS biofilm and planktonic; BHI vs skim milk): - In BHI biofilms: SR and BL cell numbers increased up to ~20 h and ~16 h, respectively; CI and ML decreased after ~8 h and ~12 h. - Early dominance by ML: at 4 h, ML constituted ~89% of the biofilm community (SR ~8.9%). By 24 h, SR dominated (~85.7%) with BL ~11.4% and ML ~2%. - In skim milk (SM), ML achieved higher/stable cell numbers longer: ML increased or remained stable in biofilm up to 20 h (~1.5×10^8 cells/cm²) vs decrease after 12 h in BHI from ~3.5×10^9 cells/cm². At 24 h, SR proportion was 64.4% in SM vs 85.6% in BHI; ML stability in SM kept BL at lower proportions from 8–20 h. - Interaction types observed: commensalism (+/0), amensalism (0/−), exploitation (+/−). No mutualism (+/+) or altruism observed in pairwise tests. - Structural observations: SEM indicated ML forming an initial layer facilitating SR attachment; BL contributed substantial extracellular matrix (EPS), potentially linked to stress responses and exploited by partners.

The study addresses how interspecies interactions and specific keystone taxa shape diversity, synergy, and stability in a dairy-relevant multispecies biofilm. By systematically analyzing all pairwise, three-species, and four-species combinations, the work demonstrates that community synergy cannot be fully explained by pairwise outcomes alone; higher-order interactions modulate pairwise relationships. Microbacterium lacticum acts as a keystone species, disproportionately promoting the growth of Stenotrophomonas rhizophila, Bacillus licheniformis, and Calidifontibacter indicus, thereby driving overall biofilm mass synergy. However, in the full community ML is exploited by BL and CI, resulting in reduced ML growth while conferring fitness advantages to others. Environmental mediation, particularly via pH shifts, likely contributes to interaction outcomes: SR and BL elevate pH, favoring their growth and potentially suppressing ML, whereas ML lowers pH. CFS experiments indicate that metabolites from SR and BL can enhance biofilm formation in partner combinations, especially in the presence of ML, highlighting higher-order effects and potential metabolite-driven niche construction. Temporal analyses reveal that early advantages provided by ML (rapid initial adherence and growth) set the stage for subsequent dominance by SR and matrix production by BL, reshaping community structure over 24 h. The findings underscore that predicting community assembly and function requires accounting for keystone species and higher-order interactions, informing strategies to manage biofilms on food contact surfaces and design synthetic communities.

- Viable keystone species (Microbacterium lacticum) in close association with partners was essential for biofilm synergy; substituting ML with its CFS did not recapitulate the full synergistic effect. - Community dynamics arose from both pairwise and higher-order interactions; third species frequently modulated pairwise outcomes. - Understanding these interaction networks enables better prediction and manipulation of biofilm behavior in environmental, industrial, and clinical contexts. - Future directions include transcriptional and mechanistic studies to resolve metabolite exchanges, EPS regulation, and environmental mediation (e.g., pH), and testing control strategies targeting keystone-driven interactions to disrupt persistence on food contact surfaces.

- CFS assays are confounded by a dilution effect (replacement of viable cells with supernatant reduces available nutrients), particularly in higher-order communities; thus, some decreases in biofilm mass may reflect nutrient dilution rather than absence/presence of interaction-mediating metabolites. - CFS was derived from monocultures; metabolite profiles and stability may differ from those in co-culture biofilms, potentially underestimating interaction effects. - Skim milk (SM) could not be used for all combinations (e.g., pairwise, triads) for biofilm mass quantification due to protein coagulation, limiting direct comparability across media; SM and BHI were used primarily for four-species population dynamics. - Results are environment- and nutrient-dependent (e.g., pH shifts, medium type), so generalization should be cautious. - The study focuses on 24 h dynamics; longer-term stability and succession were not assessed. - Mechanistic underpinnings (gene expression, metabolite identities) were not resolved and require transcriptomic/metabolomic follow-up.