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.     

Development of a Multispecies Oral Bacterial Community in a Saliva-Conditioned Flow Cell

Microbial communities within the human oral cavity are dynamic associations of more than 500 bacterial species that form biofilms on the soft and hard tissues of the mouth. Understanding the development and spatial organization of oral biofilms has been facilitated by the use of in vitro models. We used a saliva-conditioned flow cell, with saliva as the sole nutritional source, as a model to examine the development of multispecies biofilm communities from an inoculum containing the coaggregation partners Streptococcus gordonii, Actinomyces naeslundii, Veillonella atypica, and Fusobacterium nucleatum. Biofilms inoculated with individual species in a sequential order were compared with biofilms inoculated with coaggregates of the four species. Our results indicated that flow cells inoculated sequentially produced biofilms with larger biovolumes compared to those biofilms inoculated with coaggregates. Individual-species biovolumes within the four-species communities also differed between the two modes of inoculation. Fluorescence in situ hybridization with genus- and species-specific probes revealed that the majority of cells in both sequentially and coaggregate-inoculated biofilms were S. gordonii, regardless of the inoculation order. However, the representation of A. naeslundii and V. atypica was significantly higher in biofilms inoculated with coaggregates compared to sequentially inoculated biofilms. Thus, these results indicate that the development of multispecies biofilm communities is influenced by coaggregations preformed in planktonic phase. Coaggregating bacteria such as certain streptococci are especially adapted to primary colonization of saliva-conditioned surfaces independent of the mode of inoculation and order of addition in the multispecies inoculum. Preformed coaggregations favor other bacterial strains and may facilitate symbiotic relationships.     

Enhanced Biofilm Formation and Increased Resistance to Antimicrobial Agents and Bacterial Invasion Are Caused by Synergistic Interactions in Multispecies Biofilms

Most biofilms in their natural environments are likely to consist of consortia of species that influence each other in synergistic and antagonistic manners. However, few reports specifically address interactions within multispecies biofilms. In this study, 17 epiphytic bacterial strains, isolated from the surface of the marine alga Ulva australis, were screened for synergistic interactions within biofilms when present together in different combinations. Four isolates, Microbacterium phyllosphaerae, Shewanella japonica, Dokdonia donghaensis, and Acinetobacter lwoffii, were found to interact synergistically in biofilms formed in 96-well microtiter plates: biofilm biomass was observed to increase by >167% in biofilms formed by the four strains compared to biofilms composed of single strains. When exposed to the antibacterial agent hydrogen peroxide or tetracycline, the relative activity (exposed versus nonexposed biofilms) of the four-species biofilm was markedly higher than that in any of the single-species biofilms. Moreover, in biofilms established on glass surfaces in flow cells and subjected to invasion by the antibacterial protein-producing Pseudoalteromonas tunicata, the four-species biofilms resisted invasion to a greater extent than did the biofilms formed by the single species. Replacement of each strain by its cell-free culture supernatant suggested that synergy was dependent both on species-specific physical interactions between cells and on extracellular secreted factors or less specific interactions. In summary, our data strongly indicate that synergistic effects promote biofilm biomass and resistance of the biofilm to antimicrobial agents and bacterial invasion in multispecies biofilms.     

Constructing multispecies biofilms with defined compositions by sequential deposition of bacteria

Rationally-assembled multispecies biofilms could benefit applied processes including mixed waste biodegradation and drug biosynthesis by combining complementary metabolic pathways into single functional communities. We hypothesized that the cellular composition of mature multispecies biofilms could be manipulated by controlling the number of each cell type present on newly colonized surfaces. To test this idea, we developed a method for attaching specific numbers of bacteria to a flow cell by recirculating cell suspensions. Initial work revealed a nonlinear relationship between suspension cell density and areal density when two strains of Escherichia coli were simultaneously recirculated; in contrast, sequential recirculation resulted in a predictable deposition of cell numbers. Quantitative analysis of cell distributions in 48-h biofilms comprised of the E. coli strains demonstrated a strong relationship between their distribution at the substratum and their presence in mature biofilms. Sequentially depositing E. coli with either Pseudomonas aeruginosa or Bacillus subtilis determined small but reproducible differences in the areal density of the second microorganism recirculated relative to its areal density when recirculated alone. Overall, the presented method offers a simple and reproducible way to construct multispecies biofilms with defined compositions for biocatalytic processes.     

Antibacterial effects of <Emphasis Type="Italic">N</Emphasis>-acetylcysteine against endodontic pathogens

The success of endodontic treatment depends on the eradication of microorganisms from the root canal system and the prevention of reinfection. The purpose of this investigation was to evaluate the antibacterial and antibiofilm efficacy of N-acetylcysteine (NAC), an antioxidant mucolytic agent, as an intracanal medicament against selected endodontic pathogens. Minimum inhibitory concentrations (MICs) of NAC for Actinomyces naeslundii, Lactobacillus salivarius, Streptococcus mutans, and Enterococcus faecalis were determined using the broth microdilution method. NAC showed antibacterial activity, with MIC values of 0.78–1.56 mg/ml. The effect of NAC on biofilm formation of each bacterium and a multispecies culture consisting of the four bacterial species was assessed by crystal violet staining. NAC significantly inhibited biofilm formation by all the monospecies and multispecies bacteria at minimum concentrations of 0.78–3.13 mg/ml. The efficacy of NAC for biofilm disruption was evaluated by scanning electron microscopy and ATP-bioluminescence quantification using mature multispecies biofilms. Preformed mature multispecies biofilms on saliva-coated hydroxyapatite disks were disrupted within 10 min by treatment with NAC at concentrations of 25 mg/ml or higher. After 24 h of treatment, the viability of mature biofilms was reduced by > 99% compared with the control. Moreover, the biofilm disrupting activity of NAC was significantly higher than that of saturated calcium hydroxide or 2% chlorhexidine solution. Within the limitations of this in vitro study, we conclude that NAC has excellent antibacterial and antibiofilm efficacy against endodontic pathogens and may be used as an alternative intracanal medicament in root canal therapies.     

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.     

Ciliates as engineers of phototrophic biofilms

1. Phototrophic biofilms consist of a matrix of phototrophs, non‐photosynthetic bacteria and extracellular polymeric substances (EPS) which is spatially structured. Despite widespread exploitation of algae and bacteria within phototrophic biofilms, for example by protozoans, the ‘engineering’ effects of these ciliates on the spatial heterogeneity of phototrophic biofilms are poorly studied. 2. We studied the potential engineering effects of two ciliates, Urostyla sp. and Paramecium bursaria, on the spatial heterogeneity of synthetic multispecies biofilms. Biomass of phototrophic organisms, EPS and bacteria was analysed three dimensionally using confocal laser scanning microscopy. Spatial heterogeneity and cover of the phototrophs, bacteria and EPS were determined at several depths within the biofilm. 3. Ciliate species did not interfere with the overall development of phototrophic microorganisms, because the thickness of the biofilm was equal whether the ciliates were present or not, even though their abundance did affect spatial heterogeneity of biofilm components. When Urostyla was present, it reduced aggregation in EPS and bacteria and increased EPS biovolume. This implies a local facilitating effect of ciliates on photosynthetic activity. Biofilms to which Paramecium was added did not differ from controls in terms of phototrophs, EPS cover and biovolume. Nevertheless, ciliates affected the spatial heterogeneity of these components as phototrophs and EPS became more evenly distributed. 4. This study shows that ecosystem engineering by organisms does not only occur at large spatial scales, as in grasslands and estuaries, but also plays a role at the microscopic scale of biofilms. This effect on spatial heterogeneity was not driven by substantial exploitation of biofilm components, but via the subtle engineering effects of ciliates.     

Silver oxynitrate – an efficacious compound for the prevention and eradication of dual-species biofilms

Preventing and eradicating biofilms remains a challenge in clinical and industrial settings. Recently, the present authors demonstrated that silver oxynitrate (Ag₇NO₁₁) prevented and eradicated single-species planktonic and biofilm populations of numerous microbes at lower concentrations than other silver (Ag) compounds. Here, the antimicrobial and anti-biofilm efficacy of Ag₇NO₁₁ is elaborated by testing its in vitro activity against combinations of dual-species, planktonic and biofilm populations of Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. As further evidence emerges that multispecies bacterial communities are more common in the environment than their single-species counterparts, this study reinforces the diverse applicability of the minimal biofilm eradication concentration (MBEC?) assay for testing antimicrobial compounds against biofilms. Furthermore, this study demonstrated that Ag₇NO₁₁ had enhanced antimicrobial and anti-biofilm activity compared to copper sulfate (CuSO₄) and silver nitrate (AgNO₃) against the tested bacterial species.     

The effects of metabolite molecules produced by drinking water-isolated bacteria on their single and multispecies biofilms

The elucidation of the mechanisms by which diverse species survive and interact in drinking water (DW) biofilm communities may allow the identification of new biofilm control strategies. The purpose of the present study was to investigate the effects of metabolite molecules produced by bacteria isolated from DW on biofilm formation. Six opportunistic bacteria, viz. Acinetobacter calcoaceticus, Burkholderia cepacia, Methylobacterium sp., Mycobacterium mucogenicum, Sphingomonas capsulata and Staphylococcus sp. isolated from a drinking water distribution systems (DWDS) were used to form single and multispecies biofilms in the presence and absence of crude cell-free supernatants produced by the partner bacteria. Biofilms were characterized in terms of mass and metabolic activity. Additionally, several physiological aspects regulating interspecies interactions (sessile growth rates, antimicrobial activity of cell-free supernatants, and production of iron chelators) were studied to identify bacterial species with biocontrol potential in DWDS. Biofilms of Methylobacterium sp. had the highest growth rate and M. mucogenicum biofilms the lowest. Only B. cepacia was able to produce extracellular iron-chelating molecules. A. calcoaceticus, B. cepacia, Methylobacterium sp. and M. mucogenicum biofilms were strongly inhibited by crude cell-free supernatants from the other bacteria. The crude cell-free supernatants of M. mucogenicum and S. capsulata demonstrated a high potential for inhibiting the growth of counterpart biofilms. Multispecies biofilm formation was strongly inhibited in the absence of A. calcoaceticus. Only crude cell-free supernatants produced by B. cepacia and A. calcoaceticus had no inhibitory effects on multispecies biofilm formation, while metabolite molecules of M. mucogenicum showed the most significant biocontrol potential.     

Identification of ypqP as a New Bacillus subtilis Biofilm Determinant That Mediates the Protection of Staphylococcus aureus against Antimicrobial Agents in Mixed-Species Communities

In most habitats, microbial life is organized in biofilms, three-dimensional edifices sustained by extracellular polymeric substances that enable bacteria to resist harsh and changing environments. Under multispecies conditions, bacteria can benefit from the polymers produced by other species (“public goods”), thus improving their survival under toxic conditions. A recent study showed that a Bacillus subtilis hospital isolate (NDmed) was able to protect Staphylococcus aureus from biocide action in multispecies biofilms. In this work, we identified ypqP, a gene whose product is required in NDmed for thick-biofilm formation on submerged surfaces and for resistance to two biocides widely used in hospitals. NDmed and S. aureus formed mixed biofilms, and both their spatial arrangement and pathogen protection were mediated by YpqP. Functional ypqP is present in other natural B. subtilis biofilm-forming isolates. However, the gene is disrupted by the SPβ prophage in the weak submerged-biofilm-forming strains NCIB3610 and 168, which are both less resistant than NDmed to the biocides tested. Furthermore, in a 168 laboratory strain cured of the SPβ prophage, the reestablishment of a functional ypqP gene led to increased thickness and resistance to biocides of the associated biofilms. We therefore propose that YpqP is a new and important determinant of B. subtilis surface biofilm architecture, protection against exposure to toxic compounds, and social behavior in bacterial communities.     

Integration and Proliferation of Pseudomonas aeruginosa PA01 in Multispecies Biofilms

Despite an increased awareness of biofilm formation by pathogens and the role of biofilms in human infections, the potential role of environmental biofilms as an intermediate stage in the host-to-host cycle is poorly described. To initiate infection, pathogens in biofilms on inanimate environmental surfaces must detach from the biofilm and be transmitted to a susceptible individual in numbers large enough to constitute an infectious dose. Additionally, while detachment has been recognized as a discrete event in the biofilm lifestyle, it has not been studied to the same extent as biofilm development or biofilm physiology. Successful integration of Pseudomonas aeruginosa strain PA01 expressing green fluorescent protein (PA01GFP), employed here as a surrogate pathogen, into multispecies biofilm communities isolated and enriched from sink drains in public washrooms and a hospital intensive care unit is described. Confocal laser scanning microscopy indicated that PA01GFP cells were most frequently located in the deeper layers of the biofilm, near the attachment surface, when introduced into continuous flow cells before or at the same time as the multispecies drain communities. A more random integration pattern was observed when PA01GFP was introduced into established multispecies biofilms. Significant numbers of single PA01GFP cells were continuously released from the biofilms to the bulk liquid environment, regardless of the order of introduction into the flow cell. Challenging the multispecies biofilms containing PA01GFP with sub-lethal concentrations of an antibiotic, chelating agent and shear forces that typically prevail at distances away from the point of treatment showed that environmental biofilms provide a suitable habitat where pathogens are maintained and protected, and from where they are continuously released.     

Brazilian red propolis reduces orange-complex periodontopathogens growing in multispecies biofilms

This study investigated the antimicrobial effects of the ethanolic extract of Brazilian red propolis (BRP) on multispecies biofilms. A seven-day-old subgingival biofilm with 32 species was grown in a Calgary device. Biofilms were treated with BRP (1,600, 800, 400 and 200?μg ml^?1) twice a day for 1?min, starting from day 3. Chlorhexidine (0.12%) and dilution-vehicle were used as positive and negative controls, respectively. On day 7, metabolic activity and the microbial composition of the biofilms by DNA-DNA hybridization were determined. The viability data were analyzed by one-way ANOVA followed by Tukey’s post hoc, whereas the microbial composition data were transformed via BOX-COX and analyzed using Dunnett’s post hoc. BRP (1,600?μg ml^?1) decreased biofilm metabolic activity by 45%, with no significant difference from chlorhexidine-treated samples. BRP (1,600?μg ml^?1) and chlorhexidine significantly reduced levels of 14 bacterial species compared to the vehicle control. Taken together, BRP showed promising antimicrobial properties which may be useful in periodontal disease control.     

Mini-review: efficacy of lytic bacteriophages on multispecies biofilms

There is potential for phages to prevent and control bacterial biofilms, but few studies have examined the effect of phages on the multispecies biofilms that characterize most bacterial infections. This paper reviews the mechanism of action of phages, the evidence supporting the view that phage therapy will be effective against bacterial targets and the opposite viewpoint, phage application approaches, and the comparative advantage of phage therapy in multispecies biofilms. The few reports measuring the actions of lytic phages against multispecies biofilms are also reviewed. The authors are cautiously optimistic about the application of phages against their targets when in multispecies biofilms because some lysis mechanisms do not require species specificity.     

New Methods for Analysis of Spatial Distribution and Coaggregation of Microbial Populations in Complex Biofilms

In biofilms, microbial activities form gradients of substrates and electron acceptors, creating a complex landscape of microhabitats, often resulting in structured localization of the microbial populations present. To understand the dynamic interplay between and within these populations, quantitative measurements and statistical analysis of their localization patterns within the biofilms are necessary, and adequate automated tools for such analyses are needed. We have designed and applied new methods for fluorescence in situ hybridization (FISH) and digital image analysis of directionally dependent (anisotropic) multispecies biofilms. A sequential-FISH approach allowed multiple populations to be detected in a biofilm sample. This was combined with an automated tool for vertical-distribution analysis by generating in silico biofilm slices and the recently developed Inflate algorithm for coaggregation analysis of microbial populations in anisotropic biofilms. As a proof of principle, we show distinct stratification patterns of the ammonia oxidizers Nitrosomonas oligotropha subclusters I and II and the nitrite oxidizer Nitrospira sublineage I in three different types of wastewater biofilms, suggesting niche differentiation between the N. oligotropha subclusters, which could explain their coexistence in the same biofilms. Coaggregation analysis showed that N. oligotropha subcluster II aggregated closer to Nitrospira than did N. oligotropha subcluster I in a pilot plant nitrifying trickling filter (NTF) and a moving-bed biofilm reactor (MBBR), but not in a full-scale NTF, indicating important ecophysiological differences between these phylogenetically closely related subclusters. By using high-resolution quantitative methods applicable to any multispecies biofilm in general, the ecological interactions of these complex ecosystems can be understood in more detail.     

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.     

High prevalence of biofilm synergy among bacterial soil isolates in cocultures indicates bacterial interspecific cooperation

Biofilms that form on roots, litter and soil particles typically contain multiple bacterial species. Currently, little is known about multispecies biofilm interactions and few studies have been based on environmental isolates. Here, the prevalence of synergistic effects in biofilm formation among seven different soil isolates, cocultured in combinations of four species, was investigated. We observed greater biofilm biomass production in 63% of the four-species culture combinations tested than in biofilm formed by single-species cultures, demonstrating a high prevalence of synergism in multispecies biofilm formation. One four-species consortium, composed of Stenotrophomonas rhizophila, Xanthomonas retroflexus, Microbacterium oxydans and Paenibacillus amylolyticus, exhibited strong synergy in biofilm formation and was selected for further study. Of the four strains, X. retroflexus was the only one capable of forming abundant biofilm in isolation, under the in vitro conditions investigated. In accordance, strain-specific quantitative PCR revealed that X. retroflexus was predominant within the four-species consortium (>97% of total biofilm cell number). Despite low relative abundance of all the remaining strains, all were indispensable for the strong synergistic effect to occur within the four-species biofilm. Moreover, absolute individual strain cell numbers were significantly enhanced when compared with those of single-species biofilms, indicating that all the individual strains benefit from inclusion in the multispecies community. Our results show a high prevalence of synergy in biofilm formation in multispecies consortia isolated from a natural bacterial habitat and suggest that interspecific cooperation occurs.     

Growth of Escherichia coli in Model Distribution System Biofilms Exposed to Hypochlorous Acid or Monochloramine

Bacteria indigenous to water distribution systems were used to grow multispecies biofilms within continuous-flow slide chambers. Six flow chambers were also inoculated with an Escherichia coli isolate obtained from potable water. The effect of disinfectants on bacterial populations was determined after exposure of established biofilms to 1 ppm of hypochlorous acid (ClOH) for 67 min or 4 ppm of monochloramine (NH₂Cl) for 155 min. To test the ability of bacterial populations to initiate biofilm formation in the presence of disinfectants, we assessed the biofilms after 2 weeks of exposure to residual concentrations of 0.2 ppm of ClOH or 4 ppm of NH₂Cl. Lastly, to determine the effect of recommended residual concentrations on newly established biofilms, we treated systems with 0.2 ppm of ClOH after 5 days of growth in the absence of disinfectant. Whole-cell in situ hybridizations using fluorescently tagged, 16S rRNA-targeted oligonucleotide probes performed on cryosectioned biofilms permitted the direct observation of metabolically active bacterial populations, including certain phylogenetic groups and species. The results of these studies confirmed the resistance of established bacterial biofilms to treatment with recommended levels of disinfectants. Specifically, Legionella pneumophila, E. coli, and β and δ proteobacteria were identified within biofilms both before and after treatment. Furthermore, although it was undetected using routine monitoring techniques, the observation of rRNA-containing E. coli within biofilms demonstrated not only survival but also metabolic activity of this organism within the model distribution systems. The persistence of diverse bacterial species within disinfectant-treated biofilms suggests that current testing practices underestimate the risk to immunocompromised individuals of contracting waterborne disease.     

Survival Response of <Emphasis Type="Italic">Bacteriovorax</Emphasis> in Surface Biofilm Versus Suspension when Stressed by Extremes in Environmental Conditions

The Bacteriovorax, previously in the genus Bdellovibrio, are prokaryotes that prey upon many Gram-negative bacteria. They are ubiquitous in salt-water environments and have been reported to have a strong association with biofilms. The purpose of this study was to test the hypothesis that this association affords protection for the Bacteriovorax and enhances their survival in nature when exposed to extreme environmental conditions. Experiments were designed to compare their survival in biofilms versus in suspension when exposed to extremes in salinity and temperature. Natural mixed-population biofilms generated in moderate-salinity (16‰) Patuxent River water and containing Bacteriovorax were exposed to drastic changes in salinity by placing in low-salinity (1‰) river water and salt-free (no measurable salinity) distilled water for up to 14 days. In a separate trial, the biofilm was exposed to extremes in temperature, 5°C and 35°C, for up to 12 weeks in aquarium mesocosms. Simultaneously, suspensions of the Bacteriovorax were exposed to the same extremes in salinity and temperature as biofilms. The results revealed that the Bacteriovorax typically were able to survive for a week or longer while in association with biofilms than when in suspension. These results are consistent with observations from nature and establish that biofilms are important in the survival and ecology of the Bacteriovorax.