Flagellar interference with plasmid uptake in biofilms: a joint experimental and modeling study.

Plasmid conjugation is a key facilitator of horizontal gene transfer (HGT), and plasmids encoding antibiotic resistance drive the increasing prevalence of antibiotic resistance. In natural, engineered, and clinical environments, bacteria often grow in protective biofilms. Therefore, a better understanding of plasmid transfer in biofilms is needed. Our aim was to investigate plasmid transfer in a biofilm-adapted wrinkly colony mutant of Xanthomonas retroflexus (XRw) with enhanced matrix production and reduced motility. We found that XRw biofilms had an increased uptake of the broad host-range IncP-1ϵ plasmid pKJK5 compared to the wild type (WT). Proteomics revealed fewer flagellar-associated proteins in XRw, suggesting that flagella were responsible for reducing plasmid uptake. This was confirmed by the higher plasmid uptake of non-flagellated fliM mutants of the X. retroflexus wrinkly mutant as well as the wild type. Moreover, testing several flagellar mutants of Pseudomonas putida suggested that the flagellar effect was more general. We identified seven mechanisms with the potential to explain the flagellar effect and simulated them in an individual-based model. Two mechanisms could thus be eliminated (increased distances between cells and increased lag times due to flagella). Another mechanism identified as viable in the modeling was eliminated by further experiments. The possibility of steric hindrance of pilus movement and binding by flagella, reducing the frequency of contact and thus plasmid uptake, proved viable, and the three other viable mechanisms had a reduced probability of plasmid transfer in common. Our findings highlight the important yet complex effects of flagella during bacterial conjugation in biofilms.IMPORTANCEBiofilms are the dominant form of microbial life and bacteria living in biofilms are markedly different from their planktonic counterparts, yet the impact of the biofilm lifestyle on horizontal gene transfer (HGT) is still poorly understood. Horizontal gene transfer by conjugative plasmids is a major driver in bacterial evolution and adaptation, as exemplified by the troubling spread of antibiotic resistance. To either limit or promote plasmid prevalence and dissemination, we need a better understanding of plasmid transfer between bacterial cells, especially in biofilms. Here, we identified a new factor impacting the transfer of plasmids, flagella, which are required for many types of bacterial motility. We show that their absence or altered activity can lead to enhanced plasmid uptake in two bacterial species, Xanthomonas retroflexus and Pseudomonas putida. Moreover, we demonstrate the utility of mathematical modeling to eliminate hypothetical mechanisms.

Genotypic variations and interspecific interactions modify gene expression and biofilm formation of Xanthomonas retroflexus.

Multispecies biofilms are important models for studying the evolution of microbial interactions. Co-cultivation of Xanthomonas retroflexus (XR) and Paenibacillus amylolyticus (PA) systemically leads to the appearance of an XR wrinkled mutant (XRW), increasing biofilm production. The nature of this new interaction and the role of each partner remain unclear. We tested the involvement of secreted molecular cues in this interaction by exposing XR and XRW to PA or its supernatant and analysing the response using RNA-seq, colony-forming unit (CFU) estimates, biofilm quantification, and microscopy. Compared to wild type, the mutations in XRW altered its gene expression and increased its CFU number. These changes matched the reported effects for one of the mutated genes: a response regulator part of a two-component system involved in environmental sensing. When XRW was co-cultured with PA or its supernatant, the mutations effects on XRW gene expression were masked, except for genes involved in sedentary lifestyle, being consistent with the higher biofilm production. It appears that the higher biofilm production was the result of the interaction between the genetic context (mutations) and the biotic environment (PA signals). Regulatory genes involved in environmental sensing need to be considered to shed further light on microbial interactions.

Associational Resistance to Predation by Protists in a Mixed Species Biofilm.

Mixed species biofilms exhibit increased tolerance to numerous stresses compared to single species biofilms. The aim of this study was to examine the effect of grazing by the heterotrophic protist, Tetrahymena pyriformis, on a mixed species biofilm consisting of Pseudomonas aeruginosa, Pseudomonas protegens, and Klebsiella pneumoniae. Protozoan grazing significantly reduced the single species K. pneumoniae biofilm, and the single species P. protegens biofilm was also sensitive to grazing. In contrast, P. aeruginosa biofilms were resistant to predation. This resistance protected the otherwise sensitive members of the mixed species biofilm consortium. Rhamnolipids produced by P. aeruginosa were shown to be the primary toxic factor for T. pyriformis. However, a rhamnolipid-deficient mutant of P. aeruginosa (P. aeruginosa ΔrhlAB) maintained grazing resistance in the biofilm, suggesting the presence of at least one additional protective mechanism. P. aeruginosa with a deleted gene encoding the type III secretion system also resisted grazing. A transposon library was generated in the ΔrhlAB mutant to identify the additional factor involved in community biofilm protection. Results indicated that the Pseudomonas Quinolone Signal (PQS), a quorum sensing signaling molecule, was likely responsible for this effect. We confirmed this observation by showing that double mutants of ΔrhlAB and genes in the PQS biosynthetic operon lost grazing protection. We also showed that PQS was directly toxic to T. pyriformis. This study demonstrates that residing in a mixed species biofilm can be an advantageous strategy for grazing sensitive bacterial species, as P. aeruginosa confers community protection from protozoan grazing through multiple mechanisms. IMPORTANCE Biofilms have been shown to protect bacterial cells from predation by protists. Biofilm studies have traditionally used single species systems, which have provided information on the mechanisms and regulation of biofilm formation and dispersal, and the effects of predation on these biofilms. However, biofilms in nature are comprised of multiple species. To better understand how multispecies biofilms are impacted by predation, a model mixed-species biofilm was here exposed to protozoan predation. We show that the grazing sensitive strains K. pneumonia and P. protogens gained associational resistance from the grazing resistant P. aeruginosa. Resistance was due to the secretion of rhamnolipids and quorum sensing molecule PQS. This work highlights the importance of using mixed species systems.

Community-wide changes reflecting bacterial interspecific interactions in multispecies biofilms.

Existence of most bacterial species, in natural, industrial, and clinical settings in the form of surface-adhered communities or biofilms has been well acknowledged for decades. Research predominantly focusses on single-species biofilms as these are relatively easy to study. However, microbiologists are now interested in studying multispecies biofilms and revealing interspecific interactions in these communities because of the existence of a plethora of different bacterial species together in almost all natural settings. Multispecies biofilms-led emergent properties are triggered by bacterial social interactions which have huge implication for research and practical knowledge useful for the control and manipulation of these microbial communities. Here, we discuss some important bacterial interactions that take place in multispecies biofilm communities and provide insights into community-wide changes that indicate bacterial interactions and elucidate underlying mechanisms.

Staphylococcus aureus coagulases are exploitable yet stable public goods in clinically relevant conditions.

Coagulation is an innate defense mechanism intended to limit blood loss and trap invading pathogens during infection. However, Staphylococcus aureus has the ability to hijack the coagulation cascade and generate clots via secretion of coagulases. Although many S. aureus have this characteristic, some do not. The population dynamics regarding this defining trait have yet to be explored. We report here that coagulases are public goods that confer protection against antimicrobials and immune factors within a local population or community, thus promoting growth and virulence. By utilizing variants of a methicillin-resistant S. aureus we infer that the secretion of coagulases is a cooperative trait, which is subject to exploitation by invading mutants that do not produce the public goods themselves. However, overexploitation, "tragedy of the commons," does not occur at clinically relevant conditions. Our micrographs indicate this is due to spatial segregation and population viscosity. These findings emphasize the critical role of coagulases in a social evolution context and provide a possible explanation as to why the secretion of these public goods is maintained in mixed S. aureus communities.

Unravelling interspecies interactions across heterogeneities in complex biofilm communities.

The importance of microbial biofilms has been well-recognized for several decades, and focus is now shifting towards investigating multispecies biofilm communities rather than mono- or dual-species biofilms. Therefore, the demand for techniques that provide a sufficient amount of information at adequate resolution is increasing. One major challenge for multispecies studies is that diversity and spatial organization often lead to a high degree of spatial and chemical heterogeneity. Many current approaches do not account for such heterogeneity and therefore only provide average information (-omics techniques in particular), which could obscure important information about the community. Here, we bring attention to the issues of heterogeneity when analysing synthetic multi-species biofilms, in vitro, and the importance of multi-scale approaches. We provide an overview of current and newer approaches that can be applied to biofilm communities, in order to elucidate interactions at the appropriate scale.

Mixed-species biofilms in the food industry: Current knowledge and novel control strategies.

Attachment of microorganisms to food contact surfaces and the subsequent formation of biofilms may cause equipment damage, food spoilage and even diseases. Mixed-species biofilms are ubiquitous in the food industry and they generally exhibit higher resistance to disinfectants and antimicrobials compared to single-species biofilms. The physiology and metabolic activity of microorganisms in mixed-species biofilms are however rather complicated to study, and despite targeted research efforts, the potential role of mixed-species biofilms in food industry is still rather unexplored. In this review, we summarize recent studies in the context of bacterial social interactions in mixed-species biofilms, resistance to disinfectants, detection methods, and potential novel strategies to control the formation of mixed-species biofilms for enhanced food safety and food quality.

The T-shirt microbiome is distinct between individuals and shaped by washing and fabric type.

Activity of the microbial population in clothing causes unpleasant odor and textile deterioration. However, little is known about how the textile microbial community is shaped. In this study, we developed a method for extracting DNA from small amounts of detergent-washed clothing, and applied it to both worn and unworn, washed and unwashed cotton and polyester samples of the axillary region of T-shirts from 10 male subjects. The combined application of 16S rRNA gene amplicon sequencing and quantitative PCR allowed us to estimate the absolute abundances of bacteria in the samples. We found that the T-shirt microbiome was highly individual, both in composition, diversity and microbial biomass. Fabric type was influential where Acinetobacter was more abundant in cotton. Intriguingly, unworn cotton T-shirts had a native microbiome dominated by Acinetobacter, whereas unworn polyester had no detectable bacterial microbiome. The native textile microbiome did not seem to have any effect on the microbial composition emerging from wearing the garment. Surprisingly, washing in mild detergent had only minor effects on the composition and biomass of the microbial community, and only few Amplicon Sequence Variants (ASV)s were found to decrease in abundance after washing. Individual variations between test subjects shaped the microbial community more than the type of fabric or wash with detergent. The individuality of T-shirt microbiomes and specificity of the washing procedure suggests that personalized laundry regimes could be applied to increase efficient removal of undesired bacteria.

Antagonism correlates with metabolic similarity in diverse bacteria.

In the Origin of Species, Charles R. Darwin [Darwin C (1859) On the Origin of Species] proposed that the struggle for existence must be most intense among closely related species by means of their functional similarity. It has been hypothesized that this similarity, which results in resource competition, is the driver of the evolution of antagonism among bacteria. Consequently, antagonism should mostly be prevalent among phylogenetically and metabolically similar species. We tested the hypothesis by screening for antagonism among all possible pairwise interactions between 67 bacterial species from 8 different environments: 2,211 pairs of species and 4,422 interactions. We found a clear association between antagonism and phylogenetic distance, antagonism being most likely among closely related species. We determined two metabolic distances between our strains: one by scoring their growth on various natural carbon sources and the other by creating metabolic networks of predicted genomes. For both metabolic distances, we found that the probability of antagonism increased the more metabolically similar the strains were. Moreover, our results were not compounded by whether the antagonism was between sympatric or allopatric strains. Intriguingly, for each interaction the antagonizing strain was more likely to have a wider metabolic niche than the antagonized strain: that is, larger metabolic networks and growth on more carbon sources. This indicates an association between an antagonistic and a generalist strategy.

In Vitro Community Synergy between Bacterial Soil Isolates Can Be Facilitated by pH Stabilization of the Environment.

The composition and development of naturally occurring microbial communities are defined by a complex interplay between the community and the surrounding environment and by interactions between community members. Intriguingly, these interactions can in some cases cause synergies, where the community is able to outperform its single-species constituents. However, the underlying mechanisms driving community interactions are often unknown and difficult to identify due to high community complexity. Here, we show how opposite pH drift induced by specific community members leads to pH stabilization of the microenvironment, acting as a positive interspecies interaction, driving in vitro community synergy in a model consortium of four coisolated soil bacteria, Microbacterium oxydans, Xanthomonas retroflexus, Stenotrophomonas rhizophila, and Paenibacillus amylolyticus We use microsensor pH measurements to show how individual species change the local pH microenvironment and how cocultivation leads to a stabilized pH regime over time. Specifically, in vitro acid production from P. amylolyticus and alkali production primarily from X. retroflexus led to an overall pH stabilization of the local environment over time, which in turn resulted in enhanced community growth. This specific type of interspecies interaction was found to be highly dependent on medium type and concentration; however, similar pH drift from the individual species could be observed across medium variants.IMPORTANCE Understanding interspecies interactions in bacterial communities is important for unraveling species dynamics in naturally occurring communities. These dynamics are fundamental for identifying evolutionary drivers and for the development of efficient biotechnological industry applications. Recently, pH interplay among community members has been identified as a factor affecting community development, and pH stabilization has been demonstrated to result in enhanced community growth. The use of model communities in which the effect of changing pH level can be attributed to specific species contributes to the investigation of community developmental drivers. This contributes to assessment of the extent of emergent behavior and members' contributions to community development. Here, we show that pH stabilization of the microenvironment in vitro in a synthetic coisolated model community results in synergistic growth. This observation adds to the growing diversity of community interactions leading to enhanced community growth and hints toward pH as a strong driver for community development in diverse environments.

Microbial Diversity and Putative Opportunistic Pathogens in Dishwasher Biofilm Communities.

Extreme habitats are not only limited to natural environments, but also exist in manmade systems, for instance, household appliances such as dishwashers. Limiting factors, such as high temperatures, high and low pHs, high NaCl concentrations, presence of detergents, and shear force from water during washing cycles, define microbial survival in this extreme system. Fungal and bacterial diversity in biofilms isolated from rubber seals of 24 different household dishwashers was investigated using next-generation sequencing. Bacterial genera such as Pseudomonas, Escherichia, and Acinetobacter, known to include opportunistic pathogens, were represented in most samples. The most frequently encountered fungal genera in these samples belonged to Candida, Cryptococcus, and Rhodotorula, also known to include opportunistic pathogenic representatives. This study showed how specific conditions of the dishwashers impact the abundance of microbial groups and investigated the interkingdom and intrakingdom interactions that shape these biofilms. The age, usage frequency, and hardness of incoming tap water of dishwashers had significant impact on bacterial and fungal community compositions. Representatives of Candida spp. were found at the highest prevalence (100%) in all dishwashers and are assumed to be one of the first colonizers in recently purchased dishwashers. Pairwise correlations in tested microbiomes showed that certain bacterial groups cooccur, as did the fungal groups. In mixed bacterial-fungal biofilms, early adhesion, contact, and interactions were vital in the process of biofilm formation, where mixed complexes of bacteria and fungi could provide a preliminary biogenic structure for the establishment of these biofilms.IMPORTANCE Worldwide demand for household appliances, such as dishwashers and washing machines, is increasing, as is the number of immunocompromised individuals. The harsh conditions in household dishwashers should prevent the growth of most microorganisms. However, our research shows that persisting polyextremotolerant groups of microorganisms in household appliances are well established under these unfavorable conditions and supported by the biofilm mode of growth. The significance of our research is in identifying the microbial composition of biofilms formed on dishwasher rubber seals, how diverse abiotic conditions affect microbiota, and which key microbial members were represented in early colonization and contamination of dishwashers, as these appliances can present a source of domestic cross-contamination that leads to broader medical impacts.

All together now: experimental multispecies biofilm model systems.

Studies of microorganisms have traditionally focused on single species populations, which have greatly facilitated our understanding of the genetics and physiology that underpin microbial growth, adaptation and biofilm development. However, given that most microorganisms exist as multispecies consortia, the field is increasingly exploring microbial communities using a range of technologies traditionally limited to populations, including meta-omics based approaches and high resolution imaging. The experimental communities currently being explored range from relatively low diversity, for example, two to four species, to significantly more complex systems, comprised of several hundred species. Results from both defined and undefined communities have revealed a number of emergent properties, including improved stress tolerance, increased biomass production, community level signalling and metabolic cooperation. Based on results published to date, we submit that community-based studies are timely and increasingly reveal new properties associated with multispecies consortia that could not be predicted by studies of the individual component species. Here, we review a range of defined and undefined experimental systems used to study microbial community interactions.

Low-abundant species facilitates specific spatial organization that promotes multispecies biofilm formation.

Microorganisms frequently co-exist in matrix-embedded multispecies biofilms. Within biofilms, interspecies interactions influence the spatial organization of member species, which likely play an important role in shaping the development, structure and function of these communities. Here, a reproducible four-species biofilm, composed of Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus, was established to study the importance of individual species spatial organization during multispecies biofilm development. We found that the growth of species that are poor biofilm formers, M. oxydans and P. amylolyticus, were highly enhanced when residing in the four-species biofilm. Interestingly, the presence of the low-abundant M. oxydans (0.5% of biomass volume) was observed to trigger changes in the composition of the four-species community. The other three species were crucially needed for the successful inclusion of M. oxydans in the four-species biofilm, where X. retroflexus was consistently positioned in the top layer of the mature four-species biofilm. These findings suggest that low abundance key species can significantly impact the spatial organization and hereby stabilize the function and composition of complex microbiomes.

A post-planktonic era of in vitro infectious models: issues and changes addressed by a clinically relevant wound like media.

Medical science is pitted against an ever-increasing rise in antibiotic tolerant microorganisms. Concurrently, during the past decade, biofilms have garnered much attention within research and clinical practice. Although the significance of clinical biofilms is becoming very apparent, current methods for diagnostics and direction of therapy plans in many hospitals do not reflect this knowledge; with many of the present tools proving to be inadequate for accurately mimicking the biofilm phenomenon. Based on current findings, we address some of the fundamental issues overlooked by clinical labs: the paradigm shifts that need to occur in assessing chronic wounds; better simulation of physiological conditions in vitro; and the importance of incorporating polymicrobial populations into biofilm models. In addition, this review considers using a biofilm relevant in vitro model for cultivating and determining the antibiotic tolerance and susceptibility of microorganisms associated with chronic wounds. This model presents itself as a highly rapid and functional tool that can be utilized by hospitals in an aim to improve bedside treatments.

Coexistence facilitates interspecific biofilm formation in complex microbial communities.

Social interactions in which bacteria respond to one another by modifying their phenotype are central determinants of microbial communities. It is known that interspecific interactions influence the biofilm phenotype of bacteria; a phenotype that is central to the fitness of bacteria. However, the underlying role of fundamental ecological factors, specifically coexistence and phylogenetic history, in biofilm formation remains unclear. This study examines how social interactions affect biofilm formation in multi-species co-cultures from five diverse environments. We found prevalence of increased biofilm formation among co-cultured bacteria that have coexisted in their original environment. Conversely, when randomly co-culturing bacteria across these five consortia, we found less biofilm induction and a prevalence of biofilm reduction. Reduction in biofilm formation was even more predominant when co-culturing bacteria from environments where long-term coexistence was unlikely to have occurred. Phylogenetic diversity was not found to be a strong underlying factor but a relation between biofilm induction and phylogenetic history was found. The data indicates that biofilm reduction is typically correlated with an increase in planktonic cell numbers, thus implying a behavioral response rather than mere growth competition. Our findings suggest that an increase in biofilm formation is a common adaptive response to long-term coexistence.

Enhanced plasmid loss in bacterial populations exposed to the antimicrobial compound irgasan delivered from interpenetrating polymer network silicone hydrogels.

The spread of antimicrobial resistance, usually mediated by horizontal transfer of plasmids, limits the options of treating bacterial infections and thereby poses a crucial human health problem. The disturbance of plasmid stability within bacterial species in clinical environments serves as a novel strategy to reduce the development and dissemination of antibiotic resistance. We tested the ability of irgasan to destabilize plasmids from Escherichia coli K-12 cells when added directly into liquid growth medium at concentrations below levels of marked bacterial growth inhibition, or when released into liquid growth medium from irgasan-impregnated Interpenetrating Polymer Network (IPN) silicone hydrogel objects, a novel technology developed as drug-delivery platform. IPN-mediated irgasan-release was indirectly monitored as the extent of plasmid loss from bacterial cells during a 24-hour period or during repeated exposure to new irgasan-loaded IPN devices every 24h for a total of 10days. The cells were genetically modified so that plasmid loss could be quantified by applying a combination of fluorescence-based reporter gene technology and flow cytometry. When exposing bacterial cells to the irgasan-impregnated IPNs for 24h, we observed a modest (2.8-4.7%), but significant (P<0.05), plasmid loss as well as an inhibition of bacterial growth, both gradually increasing with increasing impregnation concentration. Repeated exposure to irgasan-impregnated IPNs drastically increased the plasmid loss of up to 83%, but cells adapted over time, which indicated the limitations of this specific drug for future medical applications. This study, however, illustrates the ability of IPNs to release an impregnated compound into a liquid suspension to induce a significant biological impact on growing bacterial cells.

Interspecies interactions result in enhanced biofilm formation by co-cultures of bacteria isolated from a food processing environment.

Bacterial attachment and biofilm formation can lead to poor hygienic conditions in food processing environments. Furthermore, interactions between different bacteria may induce or promote biofilm formation. In this study, we isolated and identified a total of 687 bacterial strains from seven different locations in a meat processing environment and evaluated their biofilm formation capability. A diverse group of bacteria was isolated and most were classified as poor biofilm producers in a Calgary biofilm device assay. Isolates from two sampling sites, the wall and the meat chopper, were further examined for multispecies biofilm formation. Eight strains from each sampling site were chosen and all possible combinations of four member co-cultures were tested for enhanced biofilm formation at 15 °C and 24 °C. In approximately 20% of the multispecies consortia grown at 15 °C, the biofilm formation was enhanced when comparing to monospecies biofilms. Two specific isolates (one from each location) were found to be present in synergistic combinations with higher frequencies than the remaining isolates tested. This data provides insights into the ability of co-localized isolates to influence co-culture biofilm production with high relevance for food safety and food production facilities.

High-throughput screening of multispecies biofilm formation and quantitative PCR-based assessment of individual species proportions, useful for exploring interspecific bacterial interactions.

Multispecies biofilms are predominant in almost all natural environments, where myriads of resident microorganisms interact with each other in both synergistic and antagonistic manners. The interspecies interactions among different bacteria are, despite the ubiquity of these communities, still poorly understood. Here, we report a rapid, reproducible and sensitive approach for quantitative screening of biofilm formation by bacteria when cultivated as mono- and multispecies biofilms, based on the Nunc-TSP lid system and crystal violet staining. The relative proportion of the individual species in a four-species biofilm was assessed using quantitative PCR based on SYBR Green I fluorescence with specific primers. The results indicated strong synergistic interactions in a four-species biofilm model community with a more than 3-fold increase in biofilm formation and demonstrated the strong dominance of two strains, Xanthomonas retroflexus and Paenibacillus amylolyticus. The developed approach can be used as a standard procedure for evaluating interspecies interactions in defined microbial communities. This will be of significant value in the quantitative study of the microbial composition of multispecies biofilms both in natural environments and infectious diseases to increase our understanding of the mechanisms that underlie cooperation, competition and fitness of individual species in mixed-species biofilms.

Interspecies interactions in dairy biofilms drive community structure and response against cleaning and disinfection.

Interspecies interactions within a biofilm community influence population dynamics and community structure, which in turn may affect the bacterial stress response to antimicrobials. This study was conducted to assess the impact of interactions between Kocuria salsicia and a three-species biofilm community (comprising Stenotrophomonas rhizophila, Bacillus licheniformis, and Microbacterium lacticum) on biofilm mass, the abundance of individual species, and their survival under a laboratory-scale cleaning and disinfection (C&D) regime. The presence of K. salsicia enhanced the cell numbers of all three species in pairwise interactions. The outcomes derived from summing up pairwise interactions did not accurately predict the bacterial population dynamics within communities of more than two species. In four-species biofilms, we observed the dominance of S. rhizophila and B. licheniformis, alongside a concurrent reduction in the cell counts of K. salsicia and M. lacticum. This pattern suggests that the underlying interactions are not purely non-transitive; instead, a more complex interplay results in the dominance of specific species. We observed that bacterial spatial organization and matrix production in different mixed-species combinations affected survival in response to C&D. Confocal microscopy analysis of spatial organization showed that S. rhizophila localized on the biofilm formed by B. licheniformis and M. lacticum, and S. rhizophila was more susceptible to C&D. Matrix production in B. licheniformis, evidenced by alterations in biofilm mass and by scanning electron microscopy, demonstrated its protective role against C&D, not only for this species itself, but also for neighbouring species. Our findings emphasise that various social interactions within a biofilm community not only affect bacterial population dynamics but also influence the biofilm community's response to C&D stress.

The use of biomimetic surfaces to reduce single- and dual-species biofilms of Escherichia coli and Pseudomonas putida.

The ability of bacteria to adhere to and form biofilms on food contact surfaces poses serious challenges, as these may lead to the cross-contamination of food products. Biomimetic topographic surface modifications have been explored to enhance the antifouling performance of materials. In this study, the topography of two plant leaves, Brassica oleracea var. botrytis (cauliflower, CF) and Brassica oleracea capitate (white cabbage, WC), was replicated through wax moulding, and their antibiofilm potential was tested against single- and dual-species biofilms of Escherichia coli and Pseudomonas putida. Biomimetic surfaces exhibited higher roughness values (SaWC = 4.0 ± 1.0 µm and SaCF = 3.3 ± 1.0 µm) than the flat control (SaF = 0.6 ± 0.2 µm), whilst the CF surface demonstrated a lower interfacial free energy (ΔGiwi) than the WC surface (-100.08 mJ m-2 and -71.98 mJ m-2, respectively). The CF and WC surfaces had similar antibiofilm effects against single-species biofilms, achieving cell reductions of approximately 50% and 60% for E. coli and P. putida, respectively, compared to the control. Additionally, the biomimetic surfaces led to reductions of up to 60% in biovolume, 45% in thickness, and 60% in the surface coverage of single-species biofilms. For dual-species biofilms, only the E. coli strain growing on the WC surface exhibited a significant decrease in the cell count. However, confocal microscopy analysis revealed a 60% reduction in the total biovolume and surface coverage of mixed biofilms developed on both biomimetic surfaces. Furthermore, dual-species biofilms were mainly composed of P. putida, which reduced E. coli growth. Altogether, these results demonstrate that the surface properties of CF and WC biomimetic surfaces have the potential for reducing biofilm formation.