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Soil aggregates are integral parts of soil structure and play paramount roles in supporting microbial diversity, nutrient cycling and water retention. The formation of multispecies biofilms is a survival strategy for bacterial adaptation to the environment and help microorganisms access more complex nutrient sources via labor sharing, especially in soil aggregates. However, very little is known about the effect of species richness and composition on bacterial multispecies biofilms formation in different size soil aggregates. A random partition design strategy was used to identify the relative importance of bacterial richness and composition in driving multispecies biofilms. The strategy can separate the effects of species richness and composition from the soil aggregates occurring bacterial assemblage. Increasing species richness was found to be always positively correlated with multispecies biofilms productivity for bacteria from the same aggregate fractions. General linear model analysis revealed that species composition contributed more than species richness to forming multispecies biofilms, suggesting that "selection mechanism" plays a more important role than "complementarity mechanism". This "selection mechanism" relies mainly on culturable keystone species that can significantly enhance the formation of multispecies biofilms. The co-occurrence network was investigated to explore whether the culturable keystone species from the random partitions experiment are consistent with the keystone taxa. Four out of 10 culturable keystone species isolated from soil aggregates were matched the keystone taxa. It is concluded that the culturable keystone species determine the multispecies biofilms formation for bacteria residing in soil aggregates. This study provides insights into the role of culturable keystone species in multispecies biofilms. Understanding the formation of multispecies biofilms is fundamental to decipher how microbes interact with each other in soil aggregates. Meanwhile, it will enhance our knowledge of the quorum behavior of complex bacterial communities., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Biofilms are the predominant bacterial lifestyle and can protect microorganisms from environmental stresses. Multispecies biofilms can affect the survival of enteric pathogens that contaminate food products, and thus, investigating the underlying mechanisms of multispecies biofilms is essential for food safety and human health. In this study, we investigated the ability of the natural isolate Bacillus subtilis PS-216 to restrain Campylobacter jejuni biofilm formation and adhesion to abiotic surfaces as well as to disrupt preestablished C. jejuni biofilms. Using confocal laser scanning microscopy and colony counts, we demonstrate that the presence of B. subtilis PS-216 prevents C. jejuni biofilm formation, decreases growth of the pathogen by 4.2 log 10 , and disperses 26-h-old preestablished C. jejuni biofilms. Furthermore, the coinoculation of B. subtilis and C. jejuni interferes with the adhesion of C. jejuni to abiotic surfaces, reducing it by 2.4 log 10 . We also show that contact-independent mechanisms contribute to the inhibitory effect of B. subtilis PS-216 on C. jejuni biofilm. Using B. subtilis mutants in genes coding for nonribosomal peptides and polyketides revealed that bacillaene significantly contributes to the inhibitory effect of B. subtilis PS-216. In summary, we show a strong potential for the use of B. subtilis PS-216 against C. jejuni biofilm formation and adhesion to abiotic surfaces. Our research could bring forward novel applications of B. subtilis in animal production and thus contribute to food safety. IMPORTANCE Campylobacter jejuni is an intestinal commensal in animals (including broiler chickens) but also the most frequent cause of bacterial foodborne infection in humans. This pathogen forms biofilms which enhance survival of C. jejuni in food processing and thus threaten human health. Probiotic bacteria represent a potential alternative in the prevention and control of foodborne infections. The beneficial bacterium Bacillus subtilis has an excellent probiotic potential to reduce C. jejuni in the animal gastrointestinal tract. However, data on the effect of B. subtilis on C. jejuni biofilms are scarce. Our study shows that the B. subtilis natural isolate PS-216 prevents adhesion to the abiotic surfaces and the development of submerged C. jejuni biofilm during coculture and destroys the preestablished C. jejuni biofilm. These insights are important for development of novel applications of B. subtilis that will reduce the use of antibiotics in human and animal health and increase productivity in animal breeding.

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.

Cell-cell communication enables bacteria to coordinate their behavior through the production, recognition, and response to chemical signals produced by their microbial neighbors. An important example of coordinated behavior in bacteria is biofilm formation, where individual cells organize into highly complex, matrix-encased communities that differentiate into distinct cell types and divide labor among individual cells. Bacteria rely on environmental cues to influence biofilm development, including chemical cues produced by other microbes. A multitude of recent studies have demonstrated that natural-product antibiotics at subinhibitory concentrations can impact biofilm formation in neighboring microbes, supporting the hypothesis that these compounds may have evolved as signaling molecules that mediate cell-cell interactions. In this review we discuss the role of antibiotics in modulating biofilm formation and interspecies communication in bacteria., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The high prevalence and significance of multispecies biofilms have now been demonstrated in various bacterial habitats with medical, industrial, and ecological relevance. It is highly evident that several species of bacteria coexist and interact in biofilms, which highlights the need for evaluating the approaches used to study these complex communities. This review focuses on the establishment of multispecies biofilms in vitro, interspecies interactions in microhabitats, and how to select communities for evaluation. Studies have used different experimental approaches; here we evaluate the benefits and drawbacks of varying the degree of complexity. This review aims to facilitate multispecies biofilm research in order to expand the current limited knowledge on interspecies interactions., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Attachment of potential spoilage and pathogenic bacteria to food contact surfaces and the subsequent biofilm formation represent serious challenges to the meat industry, since these may lead to cross-contamination of the products, resulting in lowered-shelf life and transmission of diseases. In meat processing environments, microorganisms are sometimes associated to surfaces in complex multispecies communities, while bacterial interactions have been shown to play a key role in cell attachment and detachment from biofilms, as well as in the resistance of biofilm community members against antimicrobial treatments. Disinfection of food contact surfaces in such environments is a challenging task, aggravated by the great antimicrobial resistance of biofilm associated bacteria. In recent years, several alternative novel methods, such as essential oils and bacteriophages, have been successfully tested as an alternative means for the disinfection of microbial-contaminated food contact surfaces. In this review, all these aspects of biofilm formation in meat processing environments are discussed from a microbial meat-quality and safety perspective., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Most biofilms within the food industry are formed by multiple bacterial species which co-exist on surfaces as a result of interspecies interactions. These ecological interactions often make these communities tolerant against antimicrobials. Our previous work led to the identification of a large number (327) of highly diverse bacterial species on food contact surfaces of the dairy, meat, and egg industries after routine cleaning and disinfection (C&D) regimes. In the current study, biofilm-forming ability of 92 bacterial strains belonging to 26 genera and 42 species was assessed and synergistic interactions in biofilm formation were investigated by coculturing species in all possible four-species combinations. Out of the total 455 four-species biofilm combinations, greater biofilm mass production, compared to the sum of biofilm masses of individual species in monoculture, was observed in 34 combinations. Around half of the combinations showed synergy in biofilm mass > 1.5-fold and most of the combinations belonged to dairy strains. The highest synergy (3.13-fold) was shown by a combination of dairy strains comprising Stenotrophomonas rhizophila, Bacillus licheniformis, Microbacterium lacticum, and Calidifontibacter indicus. The observed synergy in mixed biofilms turned out to be strain-specific rather than species-dependent. All biofilm combinations showing remarkable synergy appeared to have certain common species in all combinations which shows there are keystone industryspecific bacterial species which stimulate synergy or antagonism and this may have implication for biofilm control in the concerned food industries. [ABSTRACT FROM AUTHOR]

Most biofilms within the food industry are formed by multiple bacterial species which co-exist on surfaces as a result of interspecies interactions. These ecological interactions often make these communities tolerant against antimicrobials. Our previous work led to the identification of a large number (327) of highly diverse bacterial species on food contact surfaces of the dairy, meat, and egg industries after routine cleaning and disinfection (C&D) regimes. In the current study, biofilm-forming ability of 92 bacterial strains belonging to 26 genera and 42 species was assessed and synergistic interactions in biofilm formation were investigated by coculturing species in all possible four-species combinations. Out of the total 455 four-species biofilm combinations, greater biofilm mass production, compared to the sum of biofilm masses of individual species in monoculture, was observed in 34 combinations. Around half of the combinations showed synergy in biofilm mass > 1.5-fold and most of the combinations belonged to dairy strains. The highest synergy (3.13-fold) was shown by a combination of dairy strains comprising Stenotrophomonas rhizophila, Bacillus licheniformis, Microbacterium lacticum, and Calidifontibacter indicus. The observed synergy in mixed biofilms turned out to be strain-specific rather than species-dependent. All biofilm combinations showing remarkable synergy appeared to have certain common species in all combinations which shows there are keystone industry-specific bacterial species which stimulate synergy or antagonism and this may have implication for biofilm control in the concerned food industries.

Biofilms on the surface of food processing equipment act as potential reservoirs of microbial contamination. Bacterial interactions are believed to play key roles in both biofilm formation and antimicrobial tolerance. In this study, Aeromonas hydrophila, Chryseobacterium oncorhynchi, and Pseudomonas libanensis, which were previously isolated from Chinese raw milk samples, were selected to establish two dual-species biofilm models (P. libanensis plus A. hydrophila and P. libanensis plus C. oncorhynchi) on stainless steel at 78C. Subsequently, three disinfectants, hydrogen peroxide (100 ppm), peracetic acid (100 ppm), and sodium hypochlorite (100 ppm), were used to treat the developed sessile communities for 10 min. Structural changes after exposure to disinfectants were analyzed with confocal laser scanning microscopy. The cell numbers of both A. hydrophila and C. oncorhynchi recovered from surfaces increased when grown as dual species biofilms with P. libanensis. Dual-species biofilms were more tolerant of disinfectants than were each single-species biofilm. Peracetic acid was the most effective disinfectant for removing biofilms, followed by hydrogen peroxide and sodium hypochlorite. The results expand the knowledge of mixed-species biofilms formed by psychrotrophic bacteria and will be helpful for developing effective strategies to eliminate bacterial mixed-species biofilms. [ABSTRACT FROM AUTHOR]

Abstract Dental biofilms are complex ecosystems containing many bacterial species that live in mutualistic relationships. These interactions can profoundly affect the virulence properties of the community. In this study we investigated how the production of gingipains, virulence factors from Porphyromonas gingivalis important in periodontal disease, was affected by other commonly found members of the sub-gingival microbiome. To mimic the subgingival microbiome, multispecies consortia (P. gingivalis, Fusobacterium nucleatum, Actinomyces naeslundii, Streptococus oralis, Streptococcus mitis, Streptococcus gordonii and Streptococcus cristatus , with or without Parvimonas micra) as well as dual species consortia (P. gingivalis with P. micra , S. oralis or F. nucleatum) were constructed and maintained anaerobically in 10% serum for up to seven days. The number of P. gingivalis was determined by plating on Brucella agar and the gingipain specific fluorogenic substrate BikKam-10 was used to investigate gingipain activity. The effect of secreted products from P. micra on gingipain activity was investigated by adding supernatants from P. micra to P. gingivalis cultures. The most prominent secreted proteins in the supernatant were identified using mass spectrometry. P. gingivalis was unable to grow in serum, either alone or in the presence of S. oralis or F. nucleatum. In contrast, with P. micra growth was significantly enhanced and this was associated with an increase in gingipain activity. In the multi-species consortia, the presence of P. micra caused a 13-fold increase in gingipain activity. Exposure of P. gingivalis to supernatants from P. micra for 24 h caused a 3-fold increase in gingipain activity. This effect was reduced by 43% after heat-treatment of the supernatant. Two dimensional gel electrophoresis revealed that several of the most prominent proteins in the P. micra supernatant were glycolytic enzymes. The results from this study suggests that gingipains are produced in response to a P. micra derived signalling molecule that is most likely a protein. This is the first time it has been shown that P. micra can affect P. gingivalis virulence properties. This is likely to be of significance for the development of be of periodontitis since these two microorganisms are often found together in the subgingival biofilm. Highlights • Parvimonas micra enhance growth of Porphyromonas gingivalis in 10% serum. • P. micra significantly enhance gingipain activity in multispecies consortia. • P. micra secretes glycolytic enzymes into the external environment. • Gingipains are upregulated by soluble factors released from P. micra. [ABSTRACT FROM AUTHOR]

Neisseria meningitidis (Nm) and Neisseria lactamica (Nl) are commensal bacteria that live in the human nasopharynx, where they form microcolonies. In contrast to Nl, Nm occasionally causes blood and/or meningitis infection with often fatal consequences. Here, we studied interactions between neisserial strains during biofilm formation. Fluorescent strains were engineered and analyzed for growth in single- and dual-strain biofilms with confocal laser-scanning microscopy. Different strains of diverse Neisseria species formed microcolonies of different sizes and morphologies. Pair-wise combinations of two invasive Nm strains and one Nm carrier isolate showed that these strains can coexist in spite of the fact that they produce toxins to combat congeners. This lack of competition was even observed when the biofilms were formed under nutrient limitation and can be explained by the observation that the separate microcolonies within mixed biofilms are mostly lineage specific. However, these microcolonies showed different levels of interaction. The coexistence of two strains was also observed in mixed biofilms of Nm and Nl strains. Inactivation of the autotransporter NalP, which prevents the release of the heparin-binding antigen NHBA and the α-peptide of IgA protease from the cell surface, and/or the production of autotransporter AutA increased interactions between microcolonies, as evidenced by close contacts between microcolonies on the substratum. Qualitative and quantitative analysis revealed an altered spatial distribution of each strain in mixed biofilms with consequences for the biomass, biofilm architecture and bacterial viability depending on the synthesis of NalP and AutA, the expression of which is prone to phase variation. Being in a consortium resulted in some cases in commensalism and cooperative behavior, which promoted attachment to the substratum or increased survival, possibly as result of the shared use of the biofilm matrix. We hypothesize that Nm strains can cooperate during host colonization, but, possibly, the different capacities of the microcolonies of each strain to resist the host's defenses limits the long-term coexistence of strains in the host.

Neisseria meningitidis (Nm) and Neisseria lactamica (Nl) are commensal bacteria that live in the human nasopharynx, where they form microcolonies. In contrast to Nl, Nm occasionally causes blood and/or meningitis infection with often fatal consequences. Here, we studied interactions between neisserial strains during biofilm formation. Fluorescent strains were engineered and analyzed for growth in single- and dual-strain biofilms with confocal laser-scanning microscopy. Different strains of diverse Neisseria species formed microcolonies of different sizes and morphologies. Pair-wise combinations of two invasive Nm strains and one Nm carrier isolate showed that these strains can coexist in spite of the fact that they produce toxins to combat congeners. This lack of competition was even observed when the biofilms were formed under nutrient limitation and can be explained by the observation that the separate microcolonies within mixed biofilms are mostly lineage specific. However, these microcolonies showed different levels of interaction. The coexistence of two strains was also observed in mixed biofilms of Nm and Nl strains. Inactivation of the autotransporter NalP, which prevents the release of the heparin-binding antigen NHBA and the a-peptide of IgA protease from the cell surface, and/or the production of autotransporter AutA increased interactions between microcolonies, as evidenced by close contacts between microcolonies on the substratum. Qualitative and quantitative analysis revealed an altered spatial distribution of each strain in mixed biofilms with consequences for the biomass, biofilm architecture and bacterial viability depending on the synthesis of NalP and AutA, the expression of which is prone to phase variation. Being in a consortium resulted in some cases in commensalism and cooperative behavior, which promoted attachment to the substratum or increased survival, possibly as result of the shared use of the biofilm matrix. We hypothesize that Nm strains can cooperate during host colonization, but, possibly, the different capacities of the microcolonies of each strain to resist the host's defenses limits the long-term coexistence of strains in the host. [ABSTRACT FROM AUTHOR]

Intercellular interactions encountered within and between different bacterial species are believed to play key roles in both biofilm formation and antimicrobial resistance. In this study, Salmonella Typhimurium and Staphylococcus aureus (3 strains per species) were left to form biofilms on stainless steel coupons incubated at 20 °C for 144 h (i.e. 6 days), in periodically renewable growth medium, under either mono- or dual-species conditions. Subsequently, the developed sessile communities were exposed for 6 min to sub-lethal concentrations of: (i) benzalkonium chloride (BC, 50 ppm), (ii) sodium hypochlorite (NaClO, 10 ppm), or (iii) peroxyacetic acid (PAA, 10 ppm). The dominance of each strain in the mono- and dual-species biofilm communities, both before and after disinfection, was monitored by pulsed field gel electrophoresis (PFGE). Results revealed that dual-species conditions led to a significant ( ca . 10-fold) reduction in the number of sessile cells for both species, compared to mono-species ones, with interspecies interactions however found to not exert any significant effect on the disinfection resistance of each species as a whole. However, PFGE analysis revealed that the different strains here employed behaved differently with regard to biofilm formation and disinfection resistance, with this effect to be also strongly dependent on the culture conditions (mono-/dual-species) and the disinfectant applied. Such results expand our knowledge on multi-species biofilms formed by foodborne pathogenic bacteria and could hopefully be helpful in our efforts to develop effective elimination strategies and thus improve food safety. [ABSTRACT FROM AUTHOR]

Abstract: Three selected Pseudomonas fluorescens strains (the type strain and two strains originally isolated from a dairy processing plant — D3-348 and D3-350) were used to form turbulent and laminar flow-generated biofilms under laboratorial conditions using flow cell reactors with stainless steel substrata. The D3-348 and D3-350 strains were also used to form dual biofilms. Biofilm phenotypic characteristics, such as respiratory activity, total and culturable cells, biomass, total and matrix proteins and polysaccharides were compared. Biofilm mechanical stability, as a major feature involved in biofilm persistence, was also assessed using a rotating device system. The results indicate that hydrodynamic conditions have a remarkable impact on biofilm phenotype. Turbulent biofilms were more active, had more mass per adhesion surface area, a higher number of total and culturable cells, a higher amount of total proteins per gram of biofilm, similar matrix proteins and identical (D3-348 and D3-350 single and dual biofilms) or smaller (type strain) total and matrix polysaccharides content than their laminar counterparts. Biofilms formed by the type strain revealed a considerable higher amount of total and culturable cells and a higher amount of total proteins (turbulent biofilms) and total and matrix polysaccharides per gram of biofilm than single and dual biofilms formed by the other strains. Mechanical stability assays disclosed that biofilms formed by both type and D3-348 strains had the highest resistance to removal when exposed to mechanical stress. Dual strain biofilms population analysis revealed an apparent co-existence, evidencing neutral interactions. The overall results provided useful information regarding a broad spectrum of P. fluorescens biofilm phenotypic parameters, which can contribute to control and model biofilm processes in food industry. [Copyright &y& Elsevier]