Conditional synergy: Impact of nutrient abundance on multispecies biofilm formation and sanitizer tolerance Krisha Pant * , Jon Palmer , Steve Flint School of Food Technology and Natural Sciences, Massey University, Private Bag, 11222, Palmerston North, New Zealand A R T I C L E I N F O Keywords: Listeria monocytogenes Exopolysaccharide Nutrient-deficient Compact biofilm A B S T R A C T Bacteria exist in varying nutrient conditions and complex microbial consortia. Pseudomonas fluorescens, Staph- ylococcus aureus, and Listeria monocytogenes are commonly occurring biofilm-formers, share a similar nutritional niche, and have been isolated from common surfaces in multispecies environments. Biofilm properties, including biomass (O.D590 nm), cell concentration (log CFU/cm2), exopolysaccharide content (μg/cm2), structure, and sanitizer tolerance (sodium hypochlorite), were observed under varying nutrient (full-strength TSB and 10 % TSB) conditions on polystyrene surfaces for single and multispecies biofilm. The synergistic interactions between the bacteria in multispecies biofilm were found to be nutrient-dependent, with significantly higher (p < 0.05) biofilm formation, exopolysaccharide content, and sanitizer tolerance in high nutrient conditions (TSB) compared with low nutrient conditions (10 % TSB). The cell concentrations in the biofilm (single and multi- species) were found to be comparable between TSB and 10 % TSB. All three bacteria involved showed increased tolerance against sanitizers in the multispecies arrangement compared to their single-species counterparts, with significantly higher survival for L. monocytogenes (5.3 log CFU/cm2) in a multispecies biofilm compared to its single-species counterpart (2.3 log CFU/cm2). A positive correlation was observed between exopolysaccharide concentration and sanitizer tolerance. This study highlights the importance of taking multiple bacteria and their growth environment into account when understanding sanitizer response, as it varies in multispecies biofilm setups and according to nutrient availability. 1. Introduction The presence of multispecies bacteria in the natural biofilms has been observed in a multitude of industrial settings and food processing areas, often leading to contamination and microbial corrosion (Bonneville et al., 2021; Di Pippo et al., 2018; Dula et al., 2021). Biofilm formers such as Pseudomonas fluorescens, Staphylococcus aureus, and Listeria monocytogenes have been isolated in combination from food surfaces (Cherif-Antar et al., 2016), such as those in the dairy industry (Oxaran et al., 2018). Staphylococcus and Pseudomonas are good biofilm formers with common nutritional niches such as meat, milk and dairy products, fresh fruits and vegetables, with Pseudomonas causing spoilage and Staphylococcus capable of causing food poisoning (Xu et al., 2019). L. monocytogenes can adhere and multiply on industrial surfaces in the presence of Pseudomonas (Agustín and Brugnoni, 2018) and has been isolated in multispecies communities from drains (Dzieciol et al., 2016). Pseudomonas can protect and harbor L. monocytogenes against sanitizers on various surfaces (Dos Santos et al., 2023; Fagerlund et al., 2017; Kocot and Olszewska, 2020; Thomassen et al., 2023). The multispecies biofilms show increased resistance to cleaning agents under synergistic interactions compared to single-species biofilms, which has been asso- ciated with the exchange of public goods, quorum sensing between the bacteria involved, and added resistance provided by the biofilm matrix (Alonso et al., 2020; Sanchez-Vizuete et al., 2015; Wicaksono et al., 2022; Yuan et al., 2020). The bacteria that survive these treatments on industrial processing surfaces can be released into the passing bulk fluid/substrate, partly due to shear stress, and contaminate the food (Rückerl et al., 2014), which has been previously noted for pathogens such as L. monocytogenes (Rodríguez-Campos et al., 2019). The cross-contamination that leads from failure to remove biofilm on food industry surfaces has so far accounted for 25 % of the world’s food safety outbreak cases (Yushina et al., 2024). The sanitizers commonly used in the food industry for cleaning and sanitation are quaternary ammonium compounds, peracetic acid, and chlorine compounds such as sodium hypochlorite (Chaves et al., 2024; Simões et al., 2010). Chlorine compounds are also strong oxidizers and * Corresponding author. E-mail address: kpant@massey.ac.nz (K. Pant). Contents lists available at ScienceDirect Food Microbiology journal homepage: www.elsevier.com/locate/fm https://doi.org/10.1016/j.fm.2025.104952 Received 12 August 2025; Received in revised form 8 October 2025; Accepted 13 October 2025 Food Microbiology 134 (2026) 104952 Available online 13 October 2025 0740-0020/© 2025 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license ( http://creativecommons.org/licenses/by/4.0/ ). https://orcid.org/0000-0003-2092-5679 https://orcid.org/0000-0003-2092-5679 mailto:kpant@massey.ac.nz www.sciencedirect.com/science/journal/07400020 https://www.elsevier.com/locate/fm https://doi.org/10.1016/j.fm.2025.104952 https://doi.org/10.1016/j.fm.2025.104952 http://crossmark.crossref.org/dialog/?doi=10.1016/j.fm.2025.104952&domain=pdf http://creativecommons.org/licenses/by/4.0/ can interact with proteins, enzymes, lipids, DNA, and RNA (Barnes and Greive, 2013; Ran et al., 2019). The active compound in sodium hypo- chlorite is hypochlorous acid, which interacts with the enzyme’s sulf- hydryl group. Additionally, chlorine oxidizes the organic matter in the biofilm before diffusing into the layers for a bactericidal effect (Sarjit and Dykes, 2017). The recommended concentration of sanitizer, such as sodium hypochlorite (active chlorine 13 %), ranges from 0.05 to 2 % (500–20,000 ppm), depending on the intended application and treat- ment time (Aarnisalo et al., 2007). Environmental variables such as temperature (Yushina et al., 2024), nutrients, and substrate variables such as topography, material, and coating/conditioning impact the interspecies interaction in the biofilm properties, such as key species, chemical composition, and sanitizer resistance (Ramstedt and Burmølle, 2022). In the food industry, surfaces are cleaned and sanitized frequently, which results in a nutrient-depleted surface for bacterial biofilms. Hence, it is important to understand the responses of biofilm-forming bacteria under different nutrient conditions (Pang et al., 2019). The biofilm and its components, including exopolysaccharide components, proteins, and eDNA, are affected by the nutrition condi- tions and are reported to be different for nutrient-limiting conditions (Wang et al., 2022). The bacteria on the food contact surfaces rarely exist by themselves (Sadiq et al., 2017) hence the sanitizer response is defined by the complex interaction dynamics of the multiple bacteria present in the biofilm (Yuan et al., 2020). Although several researchers have studied the multispecies behavior of bacteria in the biofilm, few have focused on the relationship between nutrient conditions and interspecies in- teractions (Alonso and Kabuki, 2019; Halim et al., 2025; Pant et al., 2025). Nutrient concentration is a critical parameter for biofilm for- mation when the interspecies interactions of multiple species of bacteria are observed (Ibusquiza et al., 2012; Wang et al., 2022). This study aimed to understand the effect of nutrient variation on the single, dual, and triple-species biofilm formed by Pseudomonas fluorescens, S. aureus, and L. monocytogenes and their tolerance to sub-lethal concentrations of sodium hypochlorite (50 ppm/5 min). 2. Materials and methods 2.1. Bacterial strains and culture media used Stock cultures (− 80 ◦C in glycerol) of P. fluorescens (dairy), S. aureus ATCC 9144, and L. monocytogenes (environment) were inoculated into tryptone soy broth (TSB) (Difco™, Becton, Dickinson and Company, USA) and incubated at 30 ◦C for 18 h. All three bacteria are from a previously established culture collection of the Food Microbiology Lab, School of Natural Sciences and Food Technology, Massey University, Palmerston North, New Zealand. The cultures were then inoculated into TSB and incubated again in 15 mL centrifuge tubes (Falcon®, Corning, the USA) for another 18 h to prepare the working culture. After the secondary incubation, the cultures were centrifuged (Sigma® 6–16, John Morris Scientific Ltd., New Zealand) at 3000 g for 20 min at room temperature. The cell pellets after centrifugation were washed with sterile saline solution (0.85 % w/v) via vortex mixing (30 s, highest setting) (Scilogex, Germany) and centrifugation (3000 g for 10 min). The concentrated cells were collected by removing the saline solution and dissolving the final pellet into fresh sterile saline solution to a 6 log CFU/ mL cell concentration. The cell concentration of each bacterium was set to 6 log CFU/mL using a graph of optical density (O. D600nm) vs. bac- terial concentration (log CFU/mL) (R2 = 0.97). 2.2. Biofilm formation Polystyrene 96-well plates (Falcon, USA) (0.34 cm2) were used as substrates. For single-species biofilm formation, 20 μL of bacterial so- lution (in saline) (7 log CFU/mL) was added to 180 μL of 10 % tryptone soy broth (TSB) in each well for biofilm formation. For multispecies biofilm formation, P. fluorescens, S. aureus, and L. monocytogenes in 1:1 (10 μL each) and 1:1:1 (10 μL each) was added into the 180 μL media (TSB and 10 % TSB) in single, dual, and triple species in each well. The final cell concentration was fixed at 6 log CFU/mL. The wells were incubated for 24 h at 30 ◦C under static conditions. For blank samples, respective sterile media were added to the wells. The full-strength TSB and 10 % TSB were selected to create nutrient-abundant and nutrient- deficient conditions, respectively (Chen et al., 2020; Folsom et al., 2006). 2.3. Biofilm biomass The biofilm biomass was determined according to (Stepanović et al., 2004) with crystal violet dye (Acros Oranics, USA). Crystal violet stains all biofilm components, including cells and extracellular components. The 24 h incubated plates from section 2.2 were washed with saline solution ( × 3) (200 μL) and air-dried at room temperature for 20 min. Crystal violet dye (200 μL) (0.5 % w/v in water) was added to each well of a 96-well plate and allowed to rest for 20 min before washing with saline solution until the washed saline runs clear. The stained plate was allowed to air-dry at room temperature (30 min) before adding 200 μL of 95 % ethanol. After 20 min of ethanol leeching, the absorbance value was noted at an optical density of 590 nm using a spectrophotometer (Varioskan Lux 3020–1333, Thermo Fisher Scientific Ltd., USA). Depending on the crystal violet value for the single and multispecies biofilm, the interaction was classified into synergistic, neutral, and antagonistic. For synergy to be effective, the absorbance for the multi- species biofilm (OD590 MS) needed to be higher than the absorbance obtained from the highest biofilm producer in the single-species biofilm (OD590 HS). This can be written as (OD590 MS- S.D) > (O.D590 HS + S.D), where S.D is the standard deviation of respective values. Similarly for neutral interaction, (OD590 MS- S.D) = (O.D590 HS + S.D) and for competitive interactions, (OD590 MS- S.D) < (O.D590 HS + S.D) (Ren et al., 2015). 2.4. Sanitizer treatment The response of single and multiple species bacteria in biofilm to sub- lethal concentrations of sodium hypochlorite sanitizer was analyzed according to (Lin et al., 2022). After 24 h incubation at 30 ◦C, the wells were washed with sterile saline (x2) to remove planktonic cells from the wells before treating with sodium hypochlorite (200 μL, 50 ppm, pH: 6.8–7) (Janola, Australia) for 5 min at room temperature immediately followed by 200 μL of 1 % (w/v) sodium thiosulphate neutralizer (AnalaR®, VWR International, England) for 2 min. The wells were then promptly saline solution washed and air dried. The wells were swabbed with cotton buds (Citoswab® Citotest Labware, China) in saline solution and then mixed by vortex (highest setting/1 min). The serial 10-fold dilutions for plate counting were carried out using saline solution (0.85 % w/v) and plated on selective agars. Pseudomonas Isolation agar (Difco™, USA) for Pseudomonas, Mannitol salt agar (Oxoid, UK) for Staphylococcus, and Modified Oxford agar (HiMedia, USA) for Listeria. 2.5. Exopolysaccharide concentration The biofilm matrix from the polystyrene surface was extracted using sonication and then quantified using phenol-sulphuric acid hydrolysis (Zhou et al., 2024). The 96-well plates with single and multispecies biofilms (3 days) were washed ( × 3) with sterile saline solutions to remove planktonic cells. Sterile distilled water (200 μL) was added into each well and sealed using microplate sealing tape (Nunc™, Thermo Scientific™, Denmark) before sonication for 20 min (40 kHz) (Ultra- sonic cleaner, DAIHAN Scientific Co., Ltd, Korea). The extraction pro- cess was completed by centrifugation (14,400 g/5 min) of the sample solution collected from the wells. The supernatant pooled from a plate (96-wells) was collected for filtration (0.20 μm), and the pellet was K. Pant et al. Food Microbiology 134 (2026) 104952 2 discarded. To 200 μL of filtrate, 100 μL of 6 % phenol and 500 μL of 98 % sulphuric acid were added and left to react for 20 min at room tem- perature. The optical density reading was taken at 490 nm and compared with the standard curve for dextran (Supplementary 1), and the EPS was determined in ug/cm2 of the polystyrene surface. To confirm the removal of biofilm from the surface through soni- cation, the wells incubated with the sample and blank were stained with crystal violet (0.5 % w/v) after sonication and compared. 2.6. Confocal microscopy and surface plots The co-localization of the bacteria and the structure of the biofilm formed under varying nutrient conditions was analyzed using two different stains: 4′,6-diamino-2-phenylindole (DAPI) (Invitrogen, Thermo Fisher Scientific, USA) for P. fluorescens and wheat germ agglutinin, Texas Red™- X conjugate (Invitrogen, Thermo-Fischer Sci- entific, Life Technologies corporation, USA) for L. monocytogenes (Zhou et al., 2024). Biofilm formed in 10 % TSB and full-strength TSB for 72 h at 30 ◦C on polystyrene surfaces was selected for staining and confocal microscopy (Nikon D-eclipse C1, Japan). The polystyrene coupons were washed with PBS (Oxoid, Oxoid Ltd, UK) and a staining solution con- taining 6 μL of DAPI (1 mg/mL) and 6 μL of wheat germ agglutinin- Texas red (1 mg/mL) in 6 mL of TSB. The staining was completed in the dark for 60 min at room temperature, after which the coupons were washed with PBS and allowed to air-dry before proceeding to the confocal imaging. The z-stack sequence was obtained using EZ-C1 (Gold version 3.80 build 860) and processing in ImageJ software (ImageJ 1.54g, National Institute of Health, USA) for surface plots. P. fluorescens and L. monocytogenes dual-species biofilm was chosen for imaging based on the lowest log reduction of each bacterium (higher sanitizer toler- ance) observed compared to other combinations and their single coun- terparts (Table 2). 2.7. Statistical analysis The significant differences between the samples were determined with one-way ANOVA (IBM SPSS version 29) with 95 % confidence (p < 0.05), together with Tukey analysis for post-hoc analysis. The normality of the data was verified using the Shapiro-wilk (p > 0.05) and Kolmogorov-Smirnov test (p > 0.05). The correlation between the properties of the biofilm was analyzed using Pearson’s Correlation Analysis (IBM SPSS version 29). All experiments had at least 2 biological replicates and 6 technical replicates, and results were presented as mean ± standard deviation. 3. Results 3.1. Biofilm formation All three bacteria formed biofilm on the polystyrene surface under both nutrient conditions (TSB and 10 % TSB) as observed through the O. D590 nm value after crystal violet staining. Significantly higher biofilm (p < 0.001) was observed in full-strength TSB compared to 10 % TSB for all three bacteria in single and multispecies (Fig. 1). In 10 % TSB, P. fluorescens (0.09) and S. aureus (0.06) formed significantly higher biofilm (p < 0.001) compared to L. monocytogenes (0.02) in single spe- cies. A similar pattern was observed for single-species biofilm formed in TSB, with L. monocytogenes being the poor biofilm former (0.11) compared to P. fluorescens (0.21) and S. aureus (0.37) (Fig. 1). The relationship equation by (Ren et al., 2015) provided insight into the interaction between the bacteria in both nutrient-sufficient (TSB) and nutrient-deficient (10 % TSB) conditions. In TSB, the combination of P. fluorescens, S. aureus, and L. monocytogenes in dual and triple species biofilm resulted in synergistic interactions except for P. fluorescens and L. monocytogenes dual species biofilm, which resulted in neutral inter- action (0.2) (Table 1). Synergistic interaction: MS (O.D590 - S. D) > SS (O. D590 + S. D); Neutral interaction: MS (O.D590 - S. D) = SS (O. D590 + S. D); Fig. 1. Crystal violet values (O.D590 nm) of single, dual, and triple species biofilm (24h) formed in 10 % TSB and full-strength TSB. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) Table 1 Interaction between P. fluorescens (P1), S. aureus (S2), and L. monocytogenes (LM1) in dual and triple species biofilm formed in TSB and 10 % TSB based on the relationship equation by (Ren et al., 2015). Media Sample Multispecies biofilm MS (O. D590 - S. D) Highest biofilm former- single species SS (O. D590 + S. D) Interaction 10 % TSB P1+S2 0.09 0.16 Antagonistic P1+LM1 0.07 0.11 Antagonistic S2+LM1 0.11 0.07 Synergistic P1+S2+LM1 0.06 0.11 Antagonistic TSB P1+S2 0.75 0.37 Synergistic P1+LM1 0.22 0.23 Neutral S2+LM1 0.78 0.37 Synergistic P1+S2+LM1 0.81 0.37 Synergistic K. Pant et al. Food Microbiology 134 (2026) 104952 3 Antagonistic interaction MS (O.D590 - S. D) < SS (O. D590 + S. D) Biofilm formed under 10 % TSB resulted in antagonistic interactions between all combinations of dual and triple species, except S. aureus and L. monocytogenes dual species, indicating the nutrient content-based synergy of the bacteria involved (Table 1). 3.2. Biofilm formation: cell concentrations Under nutrient-limiting concentrations (10 % TSB), there was no significant difference (p > 0.05) in the cell concentration between single and multispecies biofilm for all three bacteria involved (P. fluorescens, S. aureus, and L. monocytogenes). P. fluorescens, S. aureus, and L. monocytogenes reached the cell concentration of 6.1–6.3 log CFU/cm2, 7.4–7.7 log CFU/cm2, and 6.1–7.0 log CFU/cm2, respectively, indicating strong biofilm formation in both single and multispecies biofilms (Fig. 2). The co-inoculation of the second bacterium did not impact the cell concentrations of any bacteria involved in the dual and triple species biofilm formed in 10 % TSB (Fig. 2). In full-strength TSB, the cell concentration of all three bacteria involved, P. fluorescens, S. aureus, and L. monocytogenes, were compa- rable in single and dual species, reaching 7.0–7.7 log CFU/cm2, 6.5–7.6 log CFU/cm2, and 6.1–6.6 log CFU/cm2, respectively, for each bacte- rium. Significantly higher cell concentration (p < 0.001) was observed for L. monocytogenes in the three-species biofilm (7.9 log CFU/cm2) compared to its single (6.6 log CFU/cm2) and dual species (6.1–6.4 log CFU/cm2) counterparts (Fig. 2). There was no significant difference (p > 0.05) between the cell concentrations of biofilm in TSB and 10 % TSB. In the three-species biofilm, the predominance of the bacteria varied depending on the media used. In the three-species biofilm formed in 10 % TSB, the cell concentration of S. aureus was higher (7.7 log CFU/cm2) compared to L. monocytogenes (6.18 log CFU/cm2) and P. fluorescens (6.76 log CFU/cm2), indicating the predominance of S. aureus. In contrast, observations showed that the three species biofilm formed in full-strength TSB had no significant differences (p > 0.05) in the cell concentration among P. fluorescens (7.7 log CFU/cm2), S. aureus (8.3 log CFU/cm2), and L. monocytogenes (7.9 log CFU/cm2) (Fig. 2). 3.3. Exopolysaccharide content The exopolysaccharide (EPS) content of the biofilm formed under nutrient-abundant conditions (TSB) was significantly higher (p < 0.001) than that under nutrient-limiting conditions (10 % TSB) (Fig. 3). Under both nutrient conditions, L. monocytogenes produced significantly lower exopolysaccharides (2.5–11.8 μg/cm2) compared to S. aureus and P. fluorescens. In nutrient-limited conditions, the triple species combi- nation resulted in significantly higher (p < 0.001) EPS content (16.9 μg/ cm2) compared to their single species counterparts (1.3–8.0 μg/cm2) but was comparable to their dual species combinations (13.0–15.0 μg/cm2). A similar observation was made for a multispecies biofilm formed in full- strength TSB, where the three species biofilm showed significantly higher (p < 0.001) EPS concentration (60.9 μg/cm2) compared to their single species (11.8–33.5 μg/cm2) and dual species counterpart (36.7–42.3 μg/cm2). The EPS concentration (Fig. 3) reflected the find- ings of biofilm formation (crystal violet staining) (Fig. 1). 3.4. Sanitizer tolerance The sanitizer treatment of biofilms (single and multispecies) formed in nutrient-limiting conditions (10 % TSB) reduced the cell concentra- tion of all three bacteria, P. fluorescens, S. aureus, and L. monocytogenes below the detection limit (<2.1 log CFU/cm2) (Fig. 4). In full-strength TSB, the highest reduction post-sanitizer treatment was observed for L. monocytogenes in single species (4.3 log reduction), which improved in dual species, in the presence of P. fluorescens (1.1 log reduction) and S. aureus (2.2 log reduction). Similar observations were made for P. fluorescens with 2.8 log reduction in single species, 2.0 log reduction with S. aureus in dual species, and 1.4 log reduction with L. monocytogenes in dual species. The lowest log reduction was observed for S. aureus in single species (2.4 log reduction), which further improved in dual species (1.9 log reduction with P. fluorescens, and 2.2 log reduction with L. monocytogenes) (Table 2). In the three-species biofilm formed in full-strength TSB, the survival of all three bacteria increased with the log reduction ranging 1.3 log CFU/cm2, 1.8 log CFU/cm2, and 3.3 log CFU/cm2 for P. fluorescens, S. aureus, and L. monocytogenes, respectively. This indicated that all three bacteria in this study benefit from the synergy in dual and triple- species biofilm (Fig. 4). Fig. 2. Cell concentrations of P. fluorescens (P1), S. aureus (S2), and L. monocytogenes (LM1) in single, dual, and triple species biofilm formed in nutrient-limited conditions (10 % TSB) and nutrient-abundant conditions (TSB). K. Pant et al. Food Microbiology 134 (2026) 104952 4 3.5. Correlation between biofilm formation and sanitizer resistance A significant positive correlation (0.6) was observed between exo- polysaccharide and sanitizer tolerance (Table 3), indicating that the higher exopolysaccharide concentration in combinations could be one of the reasons for increased tolerance in multispecies biofilm (Fig. 3). The cell concentration in the biofilm and the sanitizer tolerance did not have a significant correlation (0.1) (Table 3), clarifying the variation between sanitizer tolerance for biofilm formed in 10 % TSB and TSB despite having comparable cell concentrations (Fig. 4). 3.6. Structural variation as the result of nutrient conditions The biofilm formed in 10 % TSB was observed to consist of mound- like protrusions compared to the compact layers formed in full-strength TSB. (Fig. 5A and B). The compact biofilm formed in full-strength TSB consisted of higher EPS concentration (Fig. 3) and higher tolerance to sanitizer (Table 2) compared to the biofilm with uneven surfaces formed in 10 % TSB. 4. Discussion Significantly higher (p < 0.05) biofilm formation was observed for single and multispecies biofilms formed in TSB, compared to 10 % TSB Fig. 3. Exopolysaccharide concentration (μg/cm2) of single, dual, and triple species biofilm consisting of P. fluorescens (P1), S. aureus (S2), and L. monocytogenes (LM1). Fig. 4. Surviving cell concentrations (log CFU/cm2) of P. fluorescens (P1), S. aureus (S2), and L. monocytogenes (LM1) in single, dual, and triple species biofilm formed in 10 % TSB and full-strength TSB after treatment with sodium hypochlorite (50 ppm/5 min). The dotted line represents the detection limit (2.1 log CFU/cm2). K. Pant et al. Food Microbiology 134 (2026) 104952 5 for all three bacteria involved: P. fluorescens, S. aureus, and L. monocytogenes, as observed through crystal violet staining (Fig. 1) and confirmed by exopolysaccharide concentrations (Fig. 3). Similar varia- tions in the biofilm as a result of nutrient conditions have been mentioned before (S. Yuan et al., 2020). observed that the increased access to nutrients resulted in biofilms with higher thickness, bio- volume, and roughness. Interestingly, the variation in nutrient content had no significant impact (p > 0.05) on the cell concentrations in single and multispecies biofilms (Fig. 2). Pseudomonas species have been pre- viously reported as one of the major microbial contamination risks in the food industry because of their ability to grow and form biofilm even under low nutrient conditions and at low temperatures (frequently iso- lated from water - Centre for Disease Control and Prevention) (Xu et al., 2019). The supplementation of media with glucose, sucrose, and sodium chloride yielded higher biofilm (biomass) measured by crystal violet staining for Staphylococcus (Liu et al., 2020; Singh et al., 2017). A similar observation was made by (Karatan and Watnick, 2009) for Staphylo- coccus aureus and Staphylococcus epidermis where the presence of glucose and related sugars resulted in multilayer biofilm formation. Another study shows that the growth and attachment of Pseudomonas and Staphylococcus in pure and mixed (1:1) cultures in the presence of four different media (BHI, NB, LB, and RPMI 1640) resulted in higher growth and attachment in BHI broth (Wijesinghe et al., 2019). Enhanced planktonic growth and biofilm attachment have been positively associ- ated with high nutrient concentrations. In a study of biofilm formation in different media, such as chicken juice and TSB, L. monocytogenes showed higher biofilm formation in TSB (Dong et al., 2022). In contrast, Wang et al. (2022) reported the increased production of exopoly- saccharides for L. monocytogenes strains under nutrient-limiting condi- tions (dTSB-YE) compared with nutrient-rich conditions (TSB-YE), indicating the response is strain dependent. The order of predominance of bacteria in a three-species biofilm was found to be dependent on the media as well. In 10 % TSB media, S. aureus predominated the biofilm, followed by P. fluorescens and L. monocytogenes (Fig. 2). Previous reports have mentioned the predominance of P. fragi and S. xylosus in the steady-state multispecies setup containing P. fragi, S. xylosus, and L. monocytogenes in dilute tryptone soy broth (2 g/L) formed in a constant-depth film fermenter (Norwood and Gilmour, 2000). The order of dominance between Acinetobacter and Enterobacter in dual-species biofilm was also found to be dependent on the nutrient concentration in an 8-day study (Yuan et al., 2020). The nutrient abundance was associated with higher biofilm formation (Fig. 1) and synergistic in- teractions (Table 1) between the bacteria in multispecies biofilm. In this study, synergistic interaction in multispecies biofilm was observed to be limited to high nutrient conditions (full-strength TSB). Under nutrient-deficient conditions, the relationship equations showed higher antagonistic combinations than nutrient-sufficient conditions (Table 1). While the biofilm formation of S. aureus and L. monocytogenes was found to be lowered in 10 % TSB, the dual species combination of these resulted in synergistic interactions under both nutrient conditions, indicating the species-dependent interactions despite the nutrient un- availability. These observations were based solely on biofilm biomass and are indicative of interactions between bacteria in multispecies biofilm (Røder et al., 2020). Depending on the nature of the bacteria, a similar shift in interaction is noted for a combination of oligotrophic bacteria (stress-preferring) and a fungus, which shifts their interaction from competition to cooperation when evaluated in a low-nutrient environment (Velez et al., 2018). While the ¼ TSB did not have a sig- nificant effect on the growth of the strains, lowered synergy was observed in the multispecies biofilm formed by Stenotrophomonas rhi- zophila, Xanthomonas retroflexus, Microbacterium oxydans, and Paeniba- cillus amylolyticus compared to the multispecies biofilm formed in full TSB (Olsen et al., 2019). Competitive interactions are reported to occur when there is competition for nutritional niches and space (Parijs and Steenackers, 2018). Hence, under nutrient-limiting conditions, the interaction between the bacteria has been reported to evolve into competition (Xu et al., 2019). Under stressful conditions, bacteria can shut down their regulatory mechanisms to focus on survival, giving rise to cells that are resistant to other stresses, such as antibiotics (Mok and Brynildsen, 2019). In biofilm, starvation causes changes in the compo- sition of EPS, and also modifies the cell envelope for cell maintenance (Myszka and Czaczyk, 2009). The synergistic interaction occurs in many ways: physical interaction (co-aggregation) and metabolite interaction (secretion of enzymes, metabolic cross-feeding), but does not necessarily assure higher fitness of the biofilm (Burmølle et al., 2006). The inter- action between the dual-species biofilm formed by P. aeruginosa and K. pneumoniae changed from neutral to competition when the nutrient feeding and waste removal rates were decreased (Tan et al., 2017). These observations collectively indicate that the interaction between the bacteria is conditional and dynamic. In this study, the synergy between the bacteria in the multispecies biofilm provided the increased fitness of the mixed biofilm, showing increased survival of all three bacteria in the multispecies biofilm formed in full-strength TSB (Fig. 4). The sanitizer tolerance of the pathogen L. monocytogenes increased significantly (p < 0.05) in dual and triple species combinations (Fig. 4). In a similar disinfection study, eighteen L. monocytogenes cultures in single and dual species (with Table 2 Log reduction of P. fluorescens (P1), S. aureus (S2), and L. monocytogenes (LM1) in single, dual, and triple species biofilm formed in 10 % TSB and full-TSB. Biofilm Bacteria Log reduction (log CFU/ cm2) 10 % TSB TSB P. fluorescens ​ 5.21 ± 0.39y1 2.86 ± 0.23cd2 S. aureus ​ 5.38 ± 0.21y1 2.49 ± 0.41bcd2 L. monocytogenes ​ 4.99 ± 0.37xy1 4.32 ± 0.09e2 P. fluorescens + S. aureus P. fluorescens 5.01 ± 0.20xy1 2.08 ± 0.42abcd2 ​ S. aureus 5.32 ± 0.17y1 1.96 ± 0.06abc2 P. fluorescens + L. monocytogenes P. fluorescens 4.90 ± 0.43xy1 1.46 ± 0.76ab2 ​ L. monocytogenes 4.76 ± 0.36xy1 1.12 ± 0.36a2 S. aureus + L. monocytogenes S. aureus 5.64 ± 0.40y1 2.25 ± 0.40abcd2 ​ L. monocytogenes 4.74 ± 0.25xy1 2.23 ± 0.10abcd2 P. fluorescens + S. aureus + L. monocytogenes P. fluorescens 4.67 ± 0.02xy1 1.31 ± 0.51ab2 ​ S. aureus 5.64 ± 0.22y1 1.88 ± 0.02abc2 ​ L. monocytogenes 4.08 ± 0.10x1 3.34 ± 0.09de2 Note: letters (x,y) indicate the significant differences between the log reduction of bacteria for biofilm formed in 10 % TSB (between rows). Letters (a,b,c,d,e) indicate the significant differences between the log reduction of bacteria formed in TSB (between rows). Numbers (1,2) indicate the significant differences be- tween the log reduction of bacteria for biofilm formed in 10 % TSB and TSB (between columns). Table 3 Pearson’s coefficient analysis between biofilm biomass (CV), biofilm cells, sanitizer tolerance, and the exopolysaccharide concentration. CV Biofilm cells Tolerance EPS CV 1 0.247 0.555** 0.807** Biofilm cells 0.247 1 0.146 0.284 Tolerance 0.555** 0.146 1 0.618** EPS 0.807** 0.284 0.618** 1 ** Correlation is significant at the 0.01 level (2-tailed). K. Pant et al. Food Microbiology 134 (2026) 104952 6 Pseudomonas) were treated with peracetic acid and antibiotics. After the disinfection, survival of ten cultures of Listeria was noted in dual species, whereas in single species, all eighteen cultures of Listeria were reduced to undetectable levels (Thomassen et al., 2023). The dual species of Pseudomonas and S. enteritidis showed increased tolerance against qua- ternary ammonium compounds in a dual species combination (Pang et al., 2020). The protective effect of the binary biofilm of E. coli and P. aeruginosa against benzalkonium chloride has been noted before (Machado et al., 2012). In addition to the binary biofilm, the persistence of L. monocytogenes was also found to be elevated in the presence of S. Typhimurium and P. aeruginosa against black pepper essential oil in a three-species biofilm (Dos Santos et al., 2023). The results show enhanced resistance of the Listeria in binary biofilm with Pseudomonas, which can be added to the fact that Pseudomonas produces higher EPS, which acts as a barrier for the sanitizer before it reaches the Listeria or Staphylococcus (Kocot and Olszewska, 2020). The formation of denser biofilm (higher EPS) in multiple species and its correlation with increased sanitizer resistance has been discussed before (Table 2). In resistance studies, the survival of susceptible bacteria was found to be largely dependent on the bacteria present alongside them, owing to the thickness and distribution of EPS around the accompanying bacteria (Dos Santos et al., 2023). P. aeruginosa cells are good producers of exopolysaccharides, which can delay/inhibit the penetration of sanitizer in the biofilm (Bridier et al., 2015; Thomassen et al., 2023). In a multispecies biofilm, a ‘sheltering effect’ can be observed, where a low EPS producer, such as Listeria, benefits from higher EPS-producing bacteria by association (Sanchez-Vizuete et al., 2015; Wagner et al., 2020). In this study, despite the concentration of exopolysaccharide increasing significantly in three species biofilm (Fig. 3), the surviving bacteria in the biofilm after sanitizer treatment were comparable to dual species (Fig. 4), which might suggest that in addition to exopoly- saccharides, the higher survival of Listeria in multispecies biofilm could be the result of quorum sensing (horizontal gene transfer) and/or spatial arrangement (Wagner et al., 2020) with one specific bacterium present in the combination (Guillonneau et al., 2018). observed that irrespective of the number of bacteria (dual and triple) in the biofilm, P. mediterranea was able to benefit from only one bacterium (Shewanella sp.) in the combination. Previous reports have indicated the importance of spatial arrangement on the composition and behavior of multispecies biofilm (Dong et al., 2023; Ibusquiza et al., 2012). In the dual-species biofilm consisting of P. aeruginosa and S. aureus, S. aureus was located at the bottom of the biofilm (facultative anaerobic), whereas P. aeruginosa was found on the top layers owing to its aerobic metabolism. The poly- saccharide Psl produced by P. aeruginosa was found to form a protective barrier between S. aureus and P. aeruginosa, protecting the former bac- teria from the lytic effect of the latter (Xu et al., 2019). In addition to sanitizer tolerance, the synergistic interaction in multispecies biofilm has been noted to provide the bacteria with higher resistance towards invasive bacterial species (Burmølle et al., 2006) and surfactant and antibiotics (Lee et al., 2014). In this study, major structural differences were observed for biofilm formed under different nutrient conditions (Fig. 5). Heydorn et al. (2000) concluded that the Pseudomonas biofilm formed under nutrient-abundant conditions had a thick, compact, and uniform struc- tural appearance compared to the biofilm formed under nutrient-deficient conditions, where a porous biofilm containing inter- stitial channels and voids can be observed. The observations made in Fig. 5 have also been previously recognized as rugose morphotype (uneven) vs smooth morphotype in S. Typhimurium, observed as the Fig. 5. Confocal microscopy images showing dual-species biofilm formed with P. fluorescens (blue) and L. monocytogenes (red) in A) 10 % TSB and B) full-strength TSB. A.1 and B.1 are 2-D images obtained from the microscope after dual staining. A.2 and B.2 are the surface plots showing the structural differences between the biofilm formed under varying nutrient conditions for 10 % TSB and full-TSB, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.) K. Pant et al. Food Microbiology 134 (2026) 104952 7 result of continuous exposure to sodium hypochlorite (Bansal et al., 2019). Nutrient stress can cause a nutrient gradient across the biofilm (rapid proliferation of bacteria on the surface compared to deeper layers), resulting in wrinkled, mound-like, or towered structures, and overall mechanical instability (Flemming et al., 2016). The relationship between the nutrient content and structure of the biofilm has direct implications for the sanitizer tolerance (Bas et al., 2017). Compact biofilm with higher EPS density has been noted to increase resistance against external factors (hydraulic flow, antimicrobials). Mathematical modelling of biofilm showed that the structure of the biofilm is depen- dent on the nutrient uptake and diffusion of the nutrient throughout the biofilm. Once the microcolonies are formed, the proliferation of the bacteria is dependent on the nutrient uptake by the bacteria on the outer layer of the structures (Lobo-Cabrera et al., 2024). A porous and het- erogeneous structure can be observed when diffusion of the nutrients is limited in the biofilm, compared to a homogenous and compact biofilm when the diffusion limitations are absent (Van Loosdrecht et al., 2002). These structural observations are significant because the efficiency of antimicrobials (hydrogen peroxide, chlorine dioxide, and quaternary ammonium compounds) was found to be dependent on the proportion of biofilm coverage on the surface (Bas et al., 2017). Under different nutrient conditions, different genes may be upregulated, leading to differences in the EPS production and hence structural properties (Campanac et al., 2002). While the mechanisms are yet to be under- stood, the mound-like protrusions relative to mushrooms or towers have been observed for biofilm formed under stress, in contrast to non-stressed ones showing flat biofilm (Stoodley et al., 2002). This higher EPS production and compact biofilm result in higher proximity of the cells to each other and allow cell-cell interactions and the formation of a synergistic biofilm (Flemming and Wingender, 2010). While this study explores the nutrient variation on polystyrene sur- faces under static conditions, providing fundamental information on multispecies arrangements, the interactions on industrial surfaces (e.g, stainless steel) under dynamic conditions (continuous supply of media/ shear stress) might vary. Further studies are required to understand the variations in the interactions for practical applications such as industry surfaces. 5. Conclusion While the O.D595 nm was an accurate indicator of the biofilm biomass, the relationship equation based on these values did not correlate with the synergistic interactions in all the cases studied. Be- tween the three bacteria analyzed in this study, L. monocytogenes was the weakest biofilm former with the highest susceptibility to sanitizers in single-species and benefited most in the multispecies arrangement. While it’s clear that higher exopolysaccharide concentration results in higher tolerance, the uneven log reduction of three bacteria (compared to dual species) indicates that the role of spatial arrangement might be significant in defining which bacteria will be sheltered more in multi- species biofilms. Additionally, the difference in the sanitizer tolerance between biofilms formed under different nutrient conditions raises the question for future study: Is there a threshold concentration of extra- cellular matrix that the sanitizer would be able to penetrate? Further studies relating to the spatial arrangement and thickness of the biofilms could answer this question. In addition to spatial arrangement, gene expression related to resistant genes and compositional changes (pro- tein, eDNA) are also vital to understand in multispecies arrangements. In conclusion, by limiting the nutrient condition, more impactful removal methodologies can be developed without having to increase the con- centration of the chemical sanitizers. Overall, understanding the in- teractions between bacteria in a multispecies arrangement and their response to sanitizers under different environmental variables can help to develop a targeted, efficient, sustainable cleaning regime. CRediT authorship contribution statement Krisha Pant: Writing – original draft, Investigation, Formal analysis, Conceptualization. Jon Palmer: Writing – review & editing, Supervi- sion, Methodology, Conceptualization. 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Food Microbiology 134 (2026) 104952 10 https://doi.org/10.1016/j.foodres.2022.111143 https://doi.org/10.1016/j.foodres.2022.111143 https://doi.org/10.1016/j.jhazmat.2021.126836 https://doi.org/10.1159/000494757 https://doi.org/10.1016/j.cofs.2019.03.006 https://doi.org/10.1080/10408398.2019.1632790 https://doi.org/10.1080/10408398.2019.1632790 https://doi.org/10.4315/0362-028X.JFP-19-396 https://doi.org/10.4315/0362-028X.JFP-19-396 https://doi.org/10.1016/j.scitotenv.2020.139961 https://doi.org/10.1134/S0026261724604792 https://doi.org/10.1016/j.fm.2023.104394 Conditional synergy: Impact of nutrient abundance on multispecies biofilm formation and sanitizer tolerance 1 Introduction 2 Materials and methods 2.1 Bacterial strains and culture media used 2.2 Biofilm formation 2.3 Biofilm biomass 2.4 Sanitizer treatment 2.5 Exopolysaccharide concentration 2.6 Confocal microscopy and surface plots 2.7 Statistical analysis 3 Results 3.1 Biofilm formation 3.2 Biofilm formation: cell concentrations 3.3 Exopolysaccharide content 3.4 Sanitizer tolerance 3.5 Correlation between biofilm formation and sanitizer resistance 3.6 Structural variation as the result of nutrient conditions 4 Discussion 5 Conclusion CRediT authorship contribution statement Declaration of competing interest Appendix A Supplementary data Data availability References