Four-species Biofilm Research Articles

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.

Interspecific interactions facilitate keystone species in a multispecies biofilm that promotes plant growth.

Microorganisms colonizing plant roots co-exist in complex, spatially structured multispecies biofilm communities. However, little is known about microbial interactions and the underlying spatial organization within biofilm communities established on plant roots. Here, a well-established four-species biofilm model (Stenotrophomonas rhizophila, Paenibacillus amylolyticus, Microbacterium oxydans, and Xanthomonas retroflexus, termed as SPMX) was applied to Arabidopsis roots to study the impact of multispecies biofilm on plant growth and the community spatial dynamics on the roots. SPMX co-culture notably promoted root development and plant biomass. Co-cultured SPMX increased root colonization and formed multispecies biofilms, structurally different from those formed by monocultures. By combining 16S rRNA gene amplicon sequencing and fluorescence in situ hybridization with confocal laser scanning microscopy, we found that the composition and spatial organization of the four-species biofilm significantly changed over time. Monoculture P. amylolyticus colonized plant roots poorly, but its population and root colonization were highly enhanced when residing in the four-species biofilm. Exclusion of P. amylolyticus from the community reduced overall biofilm production and root colonization of the three species, resulting in the loss of the plant growth-promoting effects. Combined with spatial analysis, this led to identification of P. amylolyticus as a keystone species. Our findings highlight that weak root colonizers may benefit from mutualistic interactions in complex communities and hereby become important keystone species impacting community spatial organization and function. This work expands the knowledge on spatial organization uncovering interspecific interactions in multispecies biofilm communities on plant roots, beneficial for harnessing microbial mutualism promoting plant growth.

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

Identifying interspecies interactions in mixed-species biofilms is a key challenge in microbial ecology and is of paramount importance given that interactions govern community functionality and stability. We previously reported a bacterial four-species biofilm model comprising Stenotrophomonas rhizophila, Bacillus licheniformis, Microbacterium lacticum, and Calidifontibacter indicus that were isolated from the surface of a dairy pasteuriser after cleaning and disinfection. These bacteria produced 3.13-fold more biofilm mass compared to the sum of biofilm masses in monoculture. The present study confirms that the observed community synergy results from dynamic social interactions, encompassing commensalism, exploitation, and amensalism. M. lacticum appears to be the keystone species as it increased the growth of all other species that led to the synergy in biofilm mass. Interactions among the other three species (in the absence of M. lacticum) also contributed towards the synergy in biofilm mass. Biofilm inducing effects of bacterial cell-free-supernatants were observed for some combinations, revealing the nature of the observed synergy, and addition of additional species to dual-species combinations confirmed the presence of higher-order interactions within the biofilm community. Our findings provide understanding of bacterial interactions in biofilms which can be used as an interaction–mediated approach for cultivating, engineering, and designing synthetic bacterial communities.

Dynamic interactions between Candida albicans and different streptococcal species in a multispecies oral biofilm.

The oral cavity is colonized by a plethora of bacteria, fungi, and archaea, including streptococci of the mitis group (MSG) and the yeast Candida albicans. This study aims to investigate the role of streptococcal species in the development of oral biofilm and the cross-kingdom interactions between some of the members of the commensal MSG and the pathogen yeast C. albicans using a multispecies supragingival biofilm model. A total of nine different in vitro biofilms were grown, quantified with culture analyses, and visually examined with confocal laser scanning microscopy (CLSM). A four-species biofilm without any streptococcal species was used as a basic biofilm. In each subsequent inoculum, one species of MSG was added and afterward combined with Streptococcus mutans. The eight-species biofilm contained all eight strains used in this study. Culture analyses showed that the presence of S. mutans in a four-species biofilm with Streptococcus oralis or S. oralis subsp. tigurinus did not differ significantly in C. albicans colony-forming unit (CFU) counts compared to biofilms without S. mutans. However, compared to other mitis species, Streptococcus gordonii combined with S. mutans resulted in the lowest CFUs of C. albicans. Visual observation by CLSM showed that biofilms containing both S. mutans and one species of MSG seemed to induce the formation of filamentous form of C. albicans. However, when several species of MSG were combined with S. mutans, C. albicans was again found in its yeast form.

Synergistic interactions in multispecies biofilm combinations of bacterial isolates recovered from diverse food processing industries.

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.

Eradication of multiple-species biofilms from food industrial and domestic surfaces using essential oils.

Microbial biofilm formation represents a serious problem for both food industry and households. Natural biofilms are formed mostly by multiple species, and show resistance against most of the usual sanitizers. In this study, the effects of cinnamon (Cinnamomum zeylanicum), marjoram (Origanum majorana) and thyme (Thymus vulgaris) essential oils (EOs) and their main components (cinnamaldehyde, terpinene-4-ol, and thymol) were investigated on four-species biofilms of Escherichia coli, Listeria monocytogenes, Pseudomonas putida and Staphylococcus aureus. Minimum bactericide concentration (MBC) and killing time were determined by means of the microdilution method. MBC of the investigated EOs and components was between 0.5 mg/mL (cinnamaldehyde) to 25 mg/mL (terpinene-4-ol). Killing times for the four-species suspension were 5 or 10 min, time spans usable in the food industry. For eradication of the mixed-population biofilm from stainless steel (SS), polypropylene (PP), tile and wood surfaces, EO- or EO component-based disinfectant solutions were developed, and their effects were compared to a peracetic acid-based industrial sanitizer (HC-DPE). Total eradication of biofilms (99.9%) was achieved, with solutions containing cinnamon and thyme EO and EO components, from SS and PP, but not from tile or wood surfaces. Apparently, cinnamon EO, terpinene-4-ol and thymol have better disinfectant activity than HC-DPE.

Action of chitosan-based solutions against a model four-species biofilm formed on cobalt-chromium and acrylic resin surfaces.

To evaluate the anti-biofilm action of chitosan, nanoparticulate chitosan, and denture cleanser Nitradine™ against biofilms comprising Candida albicans, Candida glabrata, Staphylococcus aureus, and Streptococcus mutans. Biofilm removal from removable partial dentures (RPD) is important for success in prosthetic rehabilitation. The anti-biofilm action of the experimental chitosan-based solutions and Nitradine™ was evaluated on acrylic resin and cobalt-chromium alloy through assessing cell viability, cell metabolism, residual aggregated biofilm, and extracellular polymeric substance and biofilm morphology. Only chitosan reduced the viability of C. albicans on cobalt-chromium alloy surface, by 98% (a 1.7 log10 reduction in cfu). Chitosan-based solutions neither promoted substantial alteration of the metabolic activity of the four-species biofilm nor reduced the amount of the aggregated biofilm. After immersion in chitosan and nanoparticulate chitosan, viable microorganisms and extracellular polymeric substances distributed over the entire specimens' surfaces were observed. Nitradine™ reduced the viability and metabolic activity of biofilm grown on both surfaces, but it did not remove all aggregated biofilm and extracellular polymeric substances. After immersion in Nitradine™, approximately 35% of the specimens' surfaces remained covered by aggregated biofilm, mainly composed of dead cells. Although chitosan and Nitradine™ promoted changes in the viability of microorganisms, neither solution completely removed the four-species biofilm from the Co-Cr and acrylic resin surfaces. Thus, isolated use of hygiene solutions is not indicated for biofilm control on RPDs; this requires complementary mechanical removal.

In vitro bacterial vaginosis biofilm community manipulation using endolysin therapy

Bacterial vaginosis (BV) affects approximately 26% of women of childbearing age globally, presenting with 3–5 times increased risk of miscarriage and two-fold risk of pre-term birth. Antibiotics (metronidazole and clindamycin) are typically employed to treat BV; however the success rate is low due to the formation of recalcitrant polymicrobial biofilms. As a novel therapeutic, promising results have been obtained in vitro using Gardnerella endolysins, although to date their efficacy has only been demonstrated against simple biofilm models.In this study, a four-species biofilm was developed consisting of Gardnerella vaginalis, Fannyhessea vaginae, Prevotella bivia and Mobiluncus curtisii. Biofilms were grown in NYC III broth and treated using antibiotics and an anti-Gardnerella endolysin (CCB7.1) for 24 h. Biofilm composition, viability and structure were assessed using colony counts, live/dead qPCR and scanning electron microscopy.All species colonised biofilms to varying degrees, with G. vaginalis being the most abundant. Biofilm composition remained largely unchanged when challenged with escalated concentrations of conventional antibiotics. A Gardnerella-targeted endolysin candidate (CCB7.1) showed efficacy against several Gardnerella species planktonically, and significantly reduced viable G. vaginalis within polymicrobial biofilms at 1 to 4X pMIC (p < 0.05 vs. vehicle control).Collectively, this study highlights the resilience of biofilm-embedded pathogens against the currently used antibiotics and provides a polymicrobial model that allows for more effective pre-clinical screening of BV therapies. The Gardnerella-specific endolysin CCB7.1 demonstrated significant activity against G. vaginalis within polymicrobial biofilms, altering the overall community dynamic and composition.

Assessing the interspecies relationship betweenPseudomonas aeruginosa and Staphylococcus aureus in mixed biofilms grown in alginate beads and collagen scaffolds

Polymicrobial biofilms in chronic infected wounds harbour different bacterial species that interact with each other, competing or co-operating to survive. The two most common pathogens co-isolated from chronic wound biofilms are Pseudomonas aeruginosa and Staphylococcus aureus. Evidence from in vitrobiofilms models have shown these two bacteria interactand data suggests that P. aeruginosa inhibits the growth of S. aureusin mixed biofilms. This study aimed to assess the growth of these two species in a complex polymicrobial biofilm in a 3D matrix comprised of either alginate (1.5% w/v) or a collagen scaffold. Using a five-species biofilm (S. aureus, P. aeruginosa, Citrobacter freundii, Enterococcus faecalisand Escherichia coli), with all bacteria inoculated at time zero, it was consistently observed that P. aeruginosa was not recoverable over a 72h period, with sampling every 24h. However, P. aeruginosagrew well if it was added to a pre-formed four-species biofilm. Further, P. aeruginosa was seen to inhibit the growth of S. aureus after 24h subsequent co-culture in the pre-formed biofilm, which resulted in the emergence of small colony variants of S. aureus. Interestingly when P. aeruginosawas co-inoculated in a four species biofilm that did not contain S. aureus,its growth was not inhibited, suggesting a competitive interaction between these two bacteria during establishment of the early biofilm. These data were consistent in alginate beads and collagen scaffolds. In a chronic wound P. aeruginosais regarded as a late coloniser and the phenomena observed in this study might be reflective of this.

Dynamic interspecies interactions and robustness in a four-species model biofilm.

Interspecific interactions within biofilms determine relative species abundance, growth dynamics, community resilience, and success or failure of invasion by an extraneous organism. However, deciphering interspecific interactions and assessing their contribution to biofilm properties and function remain a challenge. Here, we describe the constitution of a model biofilm composed of four bacterial species belonging to four different genera (Rhodocyclus sp., Pseudomonas fluorescens, Kocuria varians, and Bacillus cereus), derived from a biofilm isolated from an industrial milk pasteurization unit. We demonstrate that the growth dynamics and equilibrium composition of this biofilm are highly reproducible. Based on its equilibrium composition, we show that the establishment of this four‐species biofilm is highly robust against initial, transient perturbations but less so towards continuous perturbations. By comparing biofilms formed from different numbers and combinations of the constituent species and by fitting a growth model to the experimental data, we reveal a network of dynamic, positive, and negative interactions that determine the final composition of the biofilm. Furthermore, we reveal that the molecular determinant of one negative interaction is the thiocillin I synthesized by the B. cereus strain, and demonstrate its importance for species distribution and its impact on robustness by mutational analysis of the biofilm ecosystem.

Transcriptional Changes in Bifidobacterium bifidum Involved in Synergistic Multispecies Biofilms.

Bifidobacterium bifidum is part of the core microbiota of healthy infant guts where it may form biofilms on epithelial cells, mucosa, and food particles in the gut lumen. Little is known about transcriptional changes in B. bifidum engaged in synergistic multispecies biofilms with ecologically relevant species of the human gut. Recently, we reported prevalence of synergism in mixed-species biofilms formed by the human gut microbiota. This study represents a comparative gene expression analysis of B. bifidum when grown in a single-species biofilm and in two multispecies biofilm consortia withBifidobacterium longum subsp. infantis, Bacteroides ovatus, and Parabacteroides distasonis in order to identify genes involved in this adaptive process in mixed biofilms and the influence on its metabolic and functional traits. Changes up to 58% and 43% in its genome were found when it grew in three- and four-species biofilm consortia, respectively. Upregulation of genes of B. bifidum involved in carbohydrate metabolism (particularly the galE gene), quorum sensing (luxS and pfs), and amino acid metabolism (especially branched chain amino acids) in both multispecies biofilms, compared to single-species biofilms, suggest that they may be contributing factors for the observed synergistic biofilm production when B. bifidum coexists with other species in a biofilm.

Synergistic interactions prevail in multispecies biofilms formed by the human gut microbiota on mucin.

Bacterial species in the human gut predominantly exist in the form of mixed-species biofilms on mucosal surfaces. In this study, the biofilm-forming ability of many human gut bacterial strains (133 strains recovered from human faeces) on mucin-coated and non-coated polystyrene surfaces was determined. A significant variation (P< 0.05) in the biofilm-forming ability of many bacterial species on both surfaces was noticed. Based on some preliminary trials, four bacterial species were selected (Bifidobacterium bifidum, Bifidobacterium longum subsp. infantis, Parabacteroides distasonis and Bacteroides ovatus), which could not form any abundant biofilm individually under the invitro conditions investigated, but produced abundant biofilms when co-cultured in different combinations of two, three and four species, giving an evidence of synergistic interactions in multispecies biofilm formation. There was a 4.74-fold increase in the biofilm mass when all strains developed a biofilm together. Strain-specific qPCR analysis showed that B.bifidum was the most dominant species (56%) in the four-species biofilm after 24 h, followed by B.longum subsp. infantis (36.2%). Study involving cell free supernatant of the cooperating strains showed that cell viability as well as physical presence of cooperating cells were prerequisites for the observed synergy in biofilms. The molecular mechanism behind these interactions and subsequent effects on the functionality of the strains involved were not determined in our study but merit further work.

Interspecies interactions reduce selection for a biofilm-optimized variant in a four-species biofilm model.

Multispecies biofilms are structured and spatially defined communities, where interspecies interactions impact assembly and functionality. Here, we compared the spatial organization and growth of bacterial cells in differently composed biofilm communities over time to determine links between interspecies interactions and selection for biofilm phenotypes of individual species. An established model community consisting of Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus was used. It was found that interspecies interactions led to varying levels of selection for a new colony phenotype of X. retroflexus, depending on the presence/absence of other species. When M. oxydans was absent, X. retroflexus was not able to establish in the top layers of the biofilm, which led to selection for a hyper-matrix forming phenotype of X. retroflexus that successfully established in the biofilm top layers. No such phenotypic X. retroflexus variants were identified in the presence of M. oxydans. These findings indicate that interspecies interactions may lead to favourable localization of individual species in a multispecies biofilm and thereby reduce selection for competitive phenotypes.

Deciphering links between bacterial interactions and spatial organization in multispecies biofilms.

Environmental microbes frequently live in multispecies biofilms where mutualistic relationships and co-evolution may occur, defining spatial organization for member species and overall community functions. In this context, intrinsic properties emerging from microbial interactions, such as efficient organization optimizing growth and activities in multispecies biofilms, may become the object of fitness selection. However, little is known on the nature of underlying interspecies interactions during establishment of a predictable spatial organization within multispecies biofilms. We present a comparative metatranscriptomic analysis of bacterial strains residing in triple-species and four-species biofilms, aiming at deciphering molecular mechanisms underpinning bacterial interactions responsible of the remarkably enhanced biomass production and associated typical spatial organization they display. Metatranscriptomic profiles concurred with changes in micro-site occupation in response to the addition/removal of a single species, being driven by both cooperation, competition, and facilitation processes. We conclude that the enhanced biomass production of the four-species biofilm is an intrinsic community property emerging from finely tuned space optimization achieved through concerted antagonistic and mutualistic interactions, where each species occupies a defined micro-site favoring its own growth. Our results further illustrate how molecular mechanisms can be better interpreted when supported by visual imaging of actual microscopic spatial organization, and we propose phenotypic adaptation selected by social interactions as molecular mechanisms stabilizing microbial communities.

Multispecies Biofilm Development of Marine Bacteria Implies Complex Relationships Through Competition and Synergy and Modification of Matrix Components.

Microbial communities composition is largely shaped by interspecies competition or cooperation in most environments. Ecosystems are made of various dynamic microhabitats where microbial communities interact with each other establishing metabolically interdependent relationships. Very limited information is available on multispecies biofilms and their microhabitats related to natural environments. The objective of this study is to understand how marine bacteria isolated from biofilms in the Mediterranean Sea interact and compete with each other when cultivated in multispecies biofilms. Four strains (Persicivirga mediterranea TC4, Polaribacter sp. TC5, Shewanella sp. TC10 and TC11) with different phenotypical traits and abilities to form a biofilm have been selected from a previous study. Here, the results show that these strains displayed a different capacity to form a biofilm in static versus dynamic conditions where one strain, TC11, was highly susceptible to the flux. These bacteria appeared to be specialized in the secretion of one or two exopolymers. Only TC5 seemed to secrete inhibitory molecule(s) in its supernatant, with a significant effect on TC10. Most of the strains negatively impacted each other, except TC4 and TC10, which presented a synergetic effect in the two and three species biofilms. Interestingly, these two strains produced a newly secreted compound when grown in dual-species versus mono-species biofilms. TC5, which induced a strong inhibition on two of its partners in dual-species biofilms, outfitted the other bacteria in a four-species biofilm. Therefore, understanding how bacteria respond to interspecific interactions should help comprehending the dynamics of bacterial populations in their ecological niches.

INFLUENCE OF BACTERIAL DIVERSITY AND INTERSPECIES INTERACTIONS ON DRINKING WATER-ASSOCIATED BIOFILMS OF YЕRSINIA ЕNTEROCOLITICA

The capability of the enteropathogenic Yersinia enterocolitica 8081 bio/serotype 1B/O:8 to form binary and multi-species biofilms with defined bacterial strains was studied. The interspecies interactions in the binary biofilms of the enteropathogen with three bacterial isolates from drinking water and water-associated biofilms were assessed. The effect of each individual partner strain for the Y. enterocolitica involvement in the four-species bacterial biofilm was evaluated by excluding one by one the isolates from the sessile community. It was found out that Y. enterocolitica and the tested bacterial strains interact each other in the binary biofilm formation. Moreover, the Y. enterocolitica involvement in the biofilms depends on the partner strain. In the multi-species biofilms, a synergetic effect of one of the bacterial partner strains on the Y. enterocolitica attachment was detected in contrast to the weak inhibitory effect of another one.

Micro-scale intermixing: a requisite for stable and synergistic co-establishment in a four-species biofilm

Microorganisms frequently coexist in complex multispecies communities, where they distribute non-randomly, reflective of the social interactions that occur. It is therefore important to understand how social interactions and local spatial organization influences multispecies biofilm succession. Here the localization of species pairs was analyzed in three dimensions in a reproducible four-species biofilm model, to study the impact of spatial positioning of individual species on the temporal development of the community. We found, that as the biofilms developed, species pairs exhibited distinct intermixing patterns unique to the four-member biofilms. Higher biomass and more intermixing were found in four-species biofilms compared to biofilms with fewer species. Intriguingly, in local regions within the four member biofilms where Microbacterium oxydans was scant, both biomass and intermixing of all species were lowered, compared to regions where M. oxydans was present at typical densities. Our data suggest that Xanthomonas retroflexus and M. oxydans, both low abundant biofilm-members, intermixed continuously during the development of the four-species biofilm, hereby facilitating their own establishment. In turn, this seems to have promoted distinct spatial organization of Stenotrophomonas rhizophila and Paenibacillus amylolyticus enabling enhanced growth of all four species. Here local intermixing of bacteria advanced the temporal development of a multi-species biofilm.

Synergistic Interactions within a Multispecies Biofilm Enhance Individual Species Protection against Grazing by a Pelagic Protozoan.

Biofilm formation has been shown to confer protection against grazing, but little information is available on the effect of grazing on biofilm formation and protection in multispecies consortia. With most biofilms in nature being composed of multiple bacterial species, the interactions and dynamics of a multispecies bacterial biofilm subject to grazing by a pelagic protozoan predator were investigated. To this end, a mono and multispecies biofilms of four bacterial soil isolates, namely Xanthomonas retroflexus, Stenotrophomonas rhizophila, Microbacterium oxydans and Paenibacillus amylolyticus, were constructed and subjected to grazing by the ciliate Tetrahymena pyriformis. In monocultures, grazing strongly reduced planktonic cell numbers in P. amylolyticus and S. rhizophila and also X. retroflexus. At the same time, cell numbers in the underlying biofilms increased in S. rhizophila and X. retroflexus, but not in P. amylolyticus. This may be due to the fact that while grazing enhanced biofilm formation in the former two species, no biofilm was formed by P. amylolyticus in monoculture, either with or without grazing. In four-species biofilms, biofilm formation was higher than in the best monoculture, a strong biodiversity effect that was even more pronounced in the presence of grazing. While cell numbers of X. retroflexus, S. rhizophila, and P. amylolyticus in the planktonic fraction were greatly reduced in the presence of grazers, cell numbers of all three species strongly increased in the biofilm. Our results show that synergistic interactions between the four-species were important to induce biofilm formation, and suggest that bacterial members that produce more biofilm when exposed to the grazer not only protect themselves but also supported other members which are sensitive to grazing, thereby providing a “shared grazing protection” within the four-species biofilm model. Hence, complex interactions shape the dynamics of the biofilm and enhance overall community fitness under stressful conditions such as grazing. These emerging inter- and intra-species interactions could play a vital role in biofilm dynamics in natural environments like soil or aquatic systems.

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.

An oral multispecies biofilm model for high content screening applications.

Peri-implantitis caused by multispecies biofilms is a major complication in dental implant treatment. The bacterial infection surrounding dental implants can lead to bone loss and, in turn, to implant failure. A promising strategy to prevent these common complications is the development of implant surfaces that inhibit biofilm development. A reproducible and easy-to-use biofilm model as a test system for large scale screening of new implant surfaces with putative antibacterial potency is therefore of major importance. In the present study, we developed a highly reproducible in vitro four-species biofilm model consisting of the highly relevant oral bacterial species Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar and Porphyromonas gingivalis. The application of live/dead staining, quantitative real time PCR (qRT-PCR), scanning electron microscopy (SEM) and urea-NaCl fluorescence in situ hybridization (urea-NaCl-FISH) revealed that the four-species biofilm community is robust in terms of biovolume, live/dead distribution and individual species distribution over time. The biofilm community is dominated by S. oralis, followed by V. dispar, A. naeslundii and P. gingivalis. The percentage distribution in this model closely reflects the situation in early native plaques and is therefore well suited as an in vitro model test system. Furthermore, despite its nearly native composition, the multispecies model does not depend on nutrient additives, such as native human saliva or serum, and is an inexpensive, easy to handle and highly reproducible alternative to the available model systems. The 96-well plate format enables high content screening for optimized implant surfaces impeding biofilm formation or the testing of multiple antimicrobial treatment strategies to fight multispecies biofilm infections, both exemplary proven in the manuscript.