Multispecies Biofilm Formation Research Articles

Difference in formation of a dental multi-species biofilm according to substratum direction

ObjectivesThe aim of this study was to investigate the difference in dental biofilm formation according to substratum direction, using an artificial biofilm model. MethodsA three-species biofilm, consisting of Streptococcus mutans, Streptococcus oralis, and Actinomyces naeslundii, was formed on saliva-coated hydroxyapatite (sHA) discs oriented in three directions: downward (the discs placed in the direction of gravity), vertical (the discs placed parallel to the direction of gravity), and upward (the discs placed in opposite direction of gravity). The biofilms at 22h and 46h of age were analyzed using microbiological and biochemical methods, fluorescence-based assays, and scanning electron microscopy to investigate difference in bacterial adhesion, early and mature biofilm formation. ResultsThe biofilms formed in the upward direction displayed the most complex structure, with the highest number and biovolume of bacteria, as well as the lowest pH conditions at both time points. The vertical and downward directions, however, had only scattered and small bacterial colonies. In the 22-h-old biofilms, the proportion of S. oralis was similar to, or slightly higher than, that of S. mutans in all directions of substratum surfaces. However, in the 46-h-old biofilms, S. mutans became the dominant bacteria in all directions, especially in the vertical and upward directions. ConclusionsThe direction of the substratum surface could impact the proportion of bacteria and cariogenic properties of the multi-species biofilm. Biofilms in an upward direction may exhibit a higher cariogenic potential, followed by those in the vertical and downward directions, which could be related to gravity.

Characterizing the formation and cross-contamination potential of multi-species biofilms in poultry processing environments: S. Enteritidis, C. jejuni and C. perfringens

This study investigated the cross-contamination dynamics of biofilms preformed on stainless steel (SS) coupons on chicken skin and chicken meat by modeling surface-to-food transfer by contact and surface-to-food transfer in fluids. Additionally, mono-, dual-, and multi-species biofilms were examined to understand the effects of environmental conditions on biofilm-forming abilities. The results of crystal violet (CV) assay revealed that a dual-species biofilm of Salmonella Enteritidis with Campylobacter jejuni and a multi-species biofilm of S. Enteritidis with C. jejuni and Clostridium perfringens cultivated for 72 h under aerobic conditions exhibited significantly higher biofilm-forming abilities than the other biofilm types. Consistent with the CV assay findings, the multi-species biofilm exhibited significantly higher extracellular DNA and protein levels than the other biofilm types. Furthermore, the results highlighted the significant increase in cultivable counts when S. Enteritidis formed dual- or multi-species biofilms with C. jejuni or C. perfringens, indicating a synergistic effect among the strains. Additionally, the analysis of cross-contamination models revealed that the pressing method induced greater cross-contamination across most biofilm types irrespective of the food sample or biofilm characteristics. This suggests that surface-to-food contact played a more influential role in cross-contamination than surface-to-food interactions in fluids. Overall, our findings provide insights into the intricate dynamics of preformed biofilms on SS coupons in contact with chicken skin and meat, underscoring the significance of the relationship between biofilm formation and cross-contamination.

Cold Atmospheric Helium Plasma in the Post-COVID-19 Era: A Promising Tool for the Disinfection of Silicone Endotracheal Prostheses.

Despite the excellent properties of silicone endotracheal prostheses, their main limitation is the formation of a polymicrobial biofilm on their surfaces. It can cause local inflammation, interfering with the local healing process and leading to further complications in the clinical scenario. The present study evaluated the inhibitory effect of cold atmospheric plasma (CAP) on multispecies biofilms grown on the silicone protheses' surfaces. In addition to silicone characterization before and after CAP exposure, CAP cytotoxicity on immortalized human bronchial epithelium cell line (BEAS-2B) was evaluated. The aging time test reported that CAP could temporarily change the silicone surface wetting characteristics from hydrophilic (80.5°) to highly hydrophilic (<5°). ATR-FTIR showed no significant alterations in the silicone surficial chemical composition after CAP exposure for 5 min. A significant log reduction in viable cells in monospecies biofilms (log CFU/mL) of C. albicans, S. aureus, and P. aeruginosa (0.636, 0.738, and 1.445, respectively) was detected after CAP exposure. Multispecies biofilms exposed to CAP showed significant viability reduction for C. albicans and S. aureus (1.385 and 0.831, respectively). The protocol was not cytotoxic to BEAS-2B. CAP can be a simple and effective method to delay multispecies biofilm formation inside the endotracheal prosthesis.

Antimicrobial Activity of Five Calcium Silicate Based Root Canal Sealers against a Multispecies Engineered Biofilm: An In Vitro Study.

The present study's objective was to compare the impact of CerasealR, total fill BC SealerR, Bio-C SealerR, AH Plus BioceramicR, and K-BiocerR on the elimination of a multispecies' endodontic biofilm at 3, 7 and 14 days. A total of 20 freshly extracted, caries-free premolars were prepared for the study to create dentinal disks. For the multispecies biofilm formation, Enterococcus faecalis, Proteus mirabilis, Pseudomonas aeruginosa, and Candida albicans were cultured and used to inoculate hydroxyapatite discs. After incubation, the biofilms were placed on blotting papers in petri dishes with an orthodontic bend. Different root canal sealers, including CeraSeal, total Fill BC Sealer, Bio-C Sealer, AH Plus Bioceramic, K-Biocer, and Sealite, were injected into the bend, facilitating contact with the biofilms. The samples were divided into seven groups, including a negative control. At specific intervals, 3, 7, and 14 days, 3 biofilm samples from each group were collected, diluted, and plated on Agar media for colony counting and analysis. In all tested groups, the total bacterial count significantly decreased between day 3 and 14 (p < 0.05) with no statistically significant differences among the different sealers' groups at all-time points for the total bacterial count, E. faecalis count, and P. mirabilis count. However, Sealite demonstrated the most consistent effectiveness in reducing bacterial counts across multiple categories. The sealite group was capable of decreasing the C. albicans count significantly between day 3 and day 14 (p < 0.05) in comparison with the bioceramic groups. All sealers had antibacterial activity against the multispecies biofilm between day 3 and day 14. The ascending order of sealers in terms of their effectiveness in killing bacteria, based on the provided results, is as follows: Sealite, Bio-C Sealer, AH Plus, CeraSeal, TotalFill, and K-Biocer. However, there were no statistically significant differences in the bacterial counts among the different sealer groups at any time point. The role of sealers in combating biofilm-associated infections highlights their potential clinical utility in preserving root canal health. Understanding the antimicrobial properties of these sealers is vital for informed decision-making in selecting the most effective materials for improved treatment outcomes and long-term success in endodontic procedures.

Stenotrophomonas maltophilia affects the gene expression profiles of the major pathogens Pseudomonas aeruginosa and Staphylococcus aureus in an in vitro multispecies biofilm model.

Stenotrophomonas maltophilia is an emerging lung pathogen. In its natural habitat, it lives together with Pseudomonas aeruginosa, Staphylococcus aureus, and other pathogens. Here, we provide the first evidence that S. maltophilia interferes with the metabolism and physiology of other species when co-cultivated in dual- and triple-species biofilms. CSLM analyses implied that S. maltophilia was in general the first to colonize the bottom layer in multispecies biofilms. Structural patterns and niche formation resulting in distinct layer formation within the biofilm were observed on a species- and strain-dependent level for S. maltophilia. Furthermore, gene expression profiles of S. aureus and P. aeruginosa were strongly affected by the presence of S. maltophilia. The S. maltophilia metabolism was mostly fermentative in multispecies biofilms with varying sets of cytochromes used for anaerobic respiration. One of the most striking observations was that S. maltophilia interfered with P. aeruginosa LasI-dependent expression of quorum sensing-regulated pathogenicity factors in multispecies biofilms. IMPORTANCE In the past, studies have focused on bacterial pathogenicity in mono-species infections, in part ignoring the clinical relevance of diseases caused by more than one pathogen (i.e., polymicrobial infections). However, it is now common knowledge that multiple bacteria species are often involved in the course of an infection. For treatment of such infections, it is absolutely important to understand the dynamics of species interactions at possible infection sites and the molecular mechanisms behind these interactions. Here, we studied the impact of Stenotrophomonas maltophilia on its commensals Pseudomonas aeruginosa and Staphylococcus aureus in multispecies biofilms. We analyzed the 3D structural architectures of dual- and triple-species biofilms, niche formation within the biofilms, and the interspecies interactions on a molecular level. RNAseq data identified key genes involved in multispecies biofilm formation and interaction as potential drug targets for the clinical combat of multispecies infection with these major pathogens.

Influence Of Surface Treatment On The Physical Properties And Biofilm Formation Of Zirconia-Reinforced Lithium Silicate Ceramics: In Vitro Trial.

To evaluate the performance of fully (Celtra Duo, Dentsply Sirona) and partially (VITA Suprinity, VITA) crystallized zirconia-reinforced lithium silicate and partially sintered lithium disilicate (IPS e.max CAD, Ivoclar Vivadent) glass-ceramics submitted to polishing, glazing, or no surface treatment after aging. Specimens of each glass-ceramic were subjected to polishing with rubber cups (POL), glazing (GL), or control (C; no treatment) and afterwards aged with 18,000 thermal cycles (5°C to 55°C). The average roughness (Ra), 2D and 3D morphology, contact angle, multispecies biofilm formation (Streptococcus mutans and Candida albicans), and mechanical strength were evaluated by atomic force microscopy (AFM, n = 5), sessile-drop goniometry (n = 5), spectrophotometry (n = 5), and flexural strength test (n = 10), respectively. Data were analyzed using two-way analysis of variance and Tukey test (α = 5%). POL promoted lower Ra than glazing, and Celtra Duo presented higher Ra than IPS e.max CAD (P < .05). Surfaces without polishing promoted higher Ra than the POL group (P < .001), greater contact angle (P < .001), and significant morphologic changes, regardless of the glass-ceramic. Irrespective of the treatment, the contact angle was higher in Celtra Duo, and regardless of the material, there was higher biofilm formation and lower flexural strength of unpolished compared to POL or GL ceramics. Polishing with rubber cups promoted lower roughness and minor morphologic surface alterations, but biofilm formation and flexural strength were similar to the glazed surface. In general, Celtra Duo and VITA Suprinity showed similar behavior to IPS e.max CAD, which makes zirconia-reinforced lithium silicate glass-ceramics a good option for indirect restorations.

Amyloid hexapeptide prevent dental caries by antibiofilm formation

ObjectivesBiofilm formed by cariogenic microbes is the direct cause of dental caries, therefore, prevention of dental caries should be anti-biofilm-based. Previously, we found the amyloid hexapeptides efficiently inhibited biofilm formation by aggregating into amyloid fibrils agglutinating microbes. This study aimed to select the most stable amyloid hexapeptide GIDLKI (GI6) and study its anti-caries effect. MethodsBiofilms of multi-species bacteria, derived from mixed saliva, were cultured to evaluate the anti-biofilm formation effect of GI6. And then, the primary cariogenic bacterium Streptococcus mutans (S.mutans) was cultured in BHI with various pH, gradient concentrations of sucrose, glucose, and calcium ions to evaluate the anti-biofilm formation effects of GI6. Then models of human enamel block caries and twenty male SPF-SD rat caries induced by S. mutans biofilm were constructed, and confocal laser scanning microscopy, scanning electron microscopy, and micro-computed tomography were applied to investigate the anti-biofilm formation, anti-caries effects and use safety of GI6. ResultsGI6 could inhibit the multi-species bacteria biofilm formation and remained effective in anti-biofilm activity against S. mutans in environments closely related to caries. GI6 suppressed S. mutans biofilm formation and thus prevented or alleviated the development of caries in human tooth blocks and rat teeth. GI6 did not affect the intestinal flora, serum biochemical parameters, and the pathological changes of various organs. ConclusionsAmyloid hexapeptides, including but not limited to GI6, are novel effective anti-caries agents that can be used to prevent dental caries safely. Clinical significanceThis study explored the anti-biofilm formation and anti-caries effect of GI6 in vitro, highlighting the anti-biofilm formation therapy for dental caries and setting a foundation for the practical application of GI6 for the treatment of dental caries.

Inhibitory effects of piperine and black pepper essential oil on multispecies biofilm formation by Listeria monocytogenes, Salmonella Typhimurium, and Pseudomonasaeruginosa

This study aimed to evaluate the antimicrobial and antibiofilm activities of black pepper essential oil (BPEO) and piperine and the effect of piperine on gene expression in a multispecies biofilm composed of Listeria monocytogenes, Salmonella Typhimurium, and Pseudomonas aeruginosa on a polypropylene surface. The minimal inhibitory concentrations of BPEO and piperine were 100 and 25 mg/mL, respectively, against this consortium of microorganisms. Sessile cell counts were 5.35–7.35 log CFU/cm2 and varied over time. The total population eradicated in the biofilm ranged from 78.9% (5.88 log CFU/cm2) to 99.8% (4.16 log CFU/cm2). Evaluation of biofilm-related gene expression showed upregulation of the L. monocytogenes genes agrC (24 and 72 h), agrD (72 h), and prfA (72 h) and downregulation of all evaluated S. Typhimurium and P. aeruginosa genes (24 and 72 h) in the untreated control biofilm. The addition of piperine resulted in upregulation of the L. monocytogenes genes agrB (24 and 72 h), agrC (72 h), agrD (24 and 72 h), and prfA (24 h); the S. Typhimurium genes agfA (24 and 72 h), adrA (24 and 72 h), and csgD (72 h); and all evaluated P. aeruginosa genes (24 and 72 h). Piperine more effectively controlled the multispecies biofilm.

Effect of outdoor pipe materials and community-intrinsic properties on biofilm formation and chlorine resistance: Black sheep or team leader

Community-intrinsic properties affect the function and composition of microbial community, and understanding of the microbial community-intrinsic properties in different pipe materials of drinking water distribution systems can help to prevent microbial pollution more reasonably and efficiently. This study investigated the biofilm formation and chlorine resistance of multi-species biofilms in two representative outdoor pipe materials (polyethylene (PE) and cast iron pipe). The results showed that cast iron pipes have higher biofilm biomass but lower biofilm activity than PE pipes. The different growth environments of the two pipe materials resulted in diverse microbial community-intrinsic properties and affected multi-species biofilm formation. The microbial communities without Sphingomonas or Acidovorax in cast iron pipe exhibited higher biofilm biomass, when compared with those in PE pipe. In contrast, the microbial community without Bacillus in PE pipe presented higher biofilm biomass, when compared with those in cast iron pipe. Extracellular polymeric substance (EPS) and corrosion products protected bacteria from chlorination. Although community-intrinsic properties helped bacteria resist chlorination, the same microbial community exhibited diverse chlorine resistance in different pipe materials owing to different community-intrinsic properties. While the chlorine resistance of microbial community without Bacillus was low in PE pipe, that in cast iron pipe was better owing to high corrosion degree. Multi-species biofilm without Sphingomonas or Acidovorax presented higher biofilm biomass, but lower EPS content and corrosion products in cast iron pipe, resulting in low chlorine resistance. In summary, this study revealed that outdoor pipe materials could affect microbial community-intrinsic properties, which in turn could influence biofilm characteristics, and provided unique insights for ensuring drinking water safety.

Core Species Derived from Multispecies Interactions Facilitate the Immobilization of Cadmium

Microbial consortia have opened new avenues for heavy-metal remediation. However, the limited understanding of the overall effect of interspecific interactions on remediation efficacy hinders its application. Here, the effects of multispecies growth and biofilm formation on Cd immobilization were explored from direct and multiple interactions through random combinations of two or three rhizosphere bacteria. In monocultures, Cd stress resulted in an average decrease in planktonic biomass of 26%, but through cooperation, the decrease was attenuated in dual (21%) and triple cultures (13%), possibly involving an increase in surface polysaccharides. More than 65% of the co-cultures exhibited induction of biofilm formation under Cd stress, which further enhanced the role of biofilms in Cd immobilization. Notably, excellent biofilm-forming ability or extensive social induction makes Pseudomonas putida and Brevundimonas diminuta stand out in multispecies biofilm formation and Cd immobilization. These two core species significantly increase the colonization of soil microorganisms on rice roots compared to the control, resulting in a 40% decrease in Cd uptake by rice. Our study enhances the understanding of bacterial interactions under Cd stress and provides a novel strategy for adjusting beneficial soil consortia for heavy-metal remediation.

Inhibition of multi-species biofilm formation using chitosan-based film supplemented with essential oils

The biofilm development following microbial attachment on food-grade surfaces represents a main problem in the food sector, leading to significant economic losses. To overcome that, the present investigation aimed to develop biodegradable films that protect against heterogeneous biofilms using chitosan and essential oils (EOs). First, the cross-streak method revealed the absence of antagonism between Staphylococcus aureus, Enterococcus hirae, Escherichia coli, and Pseudomonas aeruginosa strains. The contact angle findings revealed a drop in the electron acceptor (γ+) and qualitative hydrophobicity (θw) associated with an increase in the Lifshitz-van Der Waals component (γLW) and electron donor (γ−) regarding the treated chitosan-based films (CH-films). The environmental scanning electron microscopy (ESEM) images showed that among all the treatments, the CH-film treated with the Pelargonium asperum EO exhibited the weakest antiadhesion activity (73.89 ± 1.42% of the covered surface) while the CH-film treated with the Pelargonium asperum, Thymus satureioides, Cinnamomum zeylanicum, and Eugenia caryophyllus EOs mixture had the strongest activity (9.23 ± 1.66% of the covered surface). From these results, the CH-film containing the EOs mixture presents a high potential for utilization in the food industry as an active coating and packaging that prevent the microorganisms' attachment and the eventual biofilm development.

Silver Nanoparticles Produced by Laser Ablation and Re-Irradiation Are Effective Preventing Peri-Implantitis Multispecies Biofilm Formation

Implant-associated infection due to biofilm formation is a growing problem. Given that silver nanoparticles (Ag-NPs) have shown antibacterial effects, our goal is to study their effect against multispecies biofilm involved in the development of peri-implantitis. To this purpose, Ag-NPs were synthesized by laser ablation in de-ionized water using two different lasers, leading to the production of colloidal suspensions. Subsequently, part of each suspension was subjected to irradiation one and three times with the same laser source with which it was obtained. Ag-NPs were immobilized on the surface of titanium discs and the resultant materials were compared with unmodified titanium coupons. Nanoparticles were physico-chemically analysed to determine their shape, crystallinity, chemical composition, and mean diameter. The materials were incubated for 90 min or 48 h, to evaluate bacterial adhesion or biofilm formation respectively with Staphylococcus aureus or oral mixed bacterial flora composed of Streptococcus oralis, Actinomyces naeslundii, Veionella dispar, and Porphyromonas gingivalis. Ag-NPs help prevent the formation of biofilms both by S. aureus and by mixed oral bacterial flora. Nanoparticles re-irradiated three times showed the biggest antimicrobial effects. Modifying dental implants in this way could prevent the development of peri-implantitis.

Soybean peptide inhibits the biofilm of periodontopathic bacteria via bactericidal activity

ObjectiveThis study aimed to clarify the antibacterial mechanism and antibiofilm effect of soybean-derived peptide BCBS-11 against periodontopathic bacteria. DesignThe minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of BCBS-11 against Porphyromonas gingivalis (P. gingivalis), Fusobacterium nucleatum (F. nucleatum), and Streptococcus mitis (S. mitis) were determined for the antibacterial mechanism. The effect of BCBS-11 on membrane permeability and depolarization activity were investigated using propidium iodide (PI) staining and 3, 3′-dipropylthiadicarbocyanine iodide (DiSC3-(5)) analysis. Monospecies and multispecies biofilms were cultured on 96-well plates. The amount of biofilm was determined using crystal violet staining to determine the inhibition of biofilm formation and the eradication of established biofilm using BCBS-11. The cytotoxicity of BCBS-11 was evaluated using 3-(4, 5-Dimethylthiazol-2-yl)− 2, 5-diphenyltetrazolium bromide (MTT) assay. ResultsThe MIC and MBC indicated the bactericidal activity of BCBS-11 against P. gingivalis and F. nucleatum. The PI staining revealed that BCBS-11 disrupted the bacterial membrane integrity. The DiSC3-(5) analysis indicated that BCBS-11 depolarized the bacterial cytoplasmic membrane. These results indicate the antimicrobial action of BCBS-11 through membrane disruption and the collapse of membrane electrochemical gradient. BCBS-11 significantly inhibited the monospecies biofilm formation of P. gingivalis and F. nucleatum and also inhibited dual-species biofilm. BCBS-11 was not cytotoxic toward human oral epithelial cells. ConclusionsBCBS-11 inhibits the monospecies and multispecies biofilm formation of P. gingivalis and F. nucleatum, and their bactericidal activity results from membrane disruption.

Multispecies biofilms in fermentation: Biofilm formation, microbial interactions, and communication

Food fermentation is driven by microorganisms, which usually coexist as multispecies biofilms. The activities and interactions of functional microorganisms and pathogenic bacteria in biofilms have important implications for the quality and safety of fermented foods. It was verified that the biofilm lifestyle benefited the fitness of microorganisms in harsh environments and intensified the cooperation and competition between biofilm members. This review focuses on multispecies biofilm formation, microbial interactions and communication in biofilms, and the application of multispecies biofilms in food fermentation. Microbial aggregation and adhesion are important steps in the early stage of multispecies biofilm formation. Different biofilm-forming abilities and strategies among microorganisms lead to several types of multispecies biofilm formation. Thespatial distribution of multispecies biofilms reflects microbial interactions and biofilm function. Then, we discuss the intrinsic factors and external manifestations of multispecies biofilm system succession. Several typical interspecies cooperation and competition modes and mechanisms of microbial communication were reviewed in this review. The main limitations of the studies included in this review are the relatively small number of studies of biofilms formed by functional microorganisms during fermentation and the lack of directevidenceforthe formation process of multispecies biofilms and microbial interactions and communication within biofilms. This review aims to provide the food industry with a sufficient understanding of multispecies biofilms in food fermentation. Practical Application: Meanwhile, it offers a reference value for better controlling and utilizing biofilms during food fermentation process, and the improvement of the yield, quality, and safety of fermented products including Chinese Baijiu, cheeese,kefir, soy sauce, kombucha, and fermented olive.

Impact of a human gut microbe on Vibrio cholerae host colonization through biofilm enhancement.

Recent studies indicate that the human intestinal microbiota could impact the outcome of infection by Vibrio cholerae, the etiological agent of the diarrheal disease cholera. A commensal bacterium, Paracoccus aminovorans, was previously identified in high abundance in stool collected from individuals infected with V. cholerae when compared to stool from uninfected persons. However, if and how P. aminovorans interacts with V. cholerae has not been experimentally determined; moreover, whether any association between this bacterium alters the behaviors of V. cholerae to affect the disease outcome is unclear. Here, we show that P. aminovorans and V. cholerae together form dual-species biofilm structure at the air-liquid interface, with previously uncharacterized novel features. Importantly, the presence of P. aminovorans within the murine small intestine enhances V. cholerae colonization in the same niche that is dependent on the Vibrio exopolysaccharide and other major components of mature V. cholerae biofilm. These studies illustrate that multispecies biofilm formation is a plausible mechanism used by a gut microbe to increase the virulence of the pathogen, and this interaction may alter outcomes in enteric infections.

Fusobacterium nucleatum Subspecies Differ in Biofilm Forming Ability in vitro.

Development of dysbiosis in complex multispecies bacterial biofilms forming on teeth, known as dental plaque, is one of the factors causing periodontitis. Fusobacterium nucleatum (F. nucleatum) is recognised as a key microorganism in subgingival dental plaque, and is linked to periodontitis as well as colorectal cancer and systemic diseases. Five subspecies of F. nucleatum have been identified: animalis, fusiforme, nucleatum, polymorphum, and vincentii. Differential integration of subspecies into multispecies biofilm models has been reported, however, biofilm forming ability of individual F. nucleatum subspecies is largely unknown. The aim of this study was to determine the single-subspecies biofilm forming abilities of F. nucleatum ATCC type strains. Static single subspecies F. nucleatum biofilms were grown anaerobically for 3 days on untreated or surface-modified (sandblasting, artificial saliva, fibronectin, gelatin, or poly-L-lysine coating) plastic and glass coverslips. Biofilm mass was quantified using crystal violet (CV) staining. Biofilm architecture and thickness were analysed by scanning electron microscopy and confocal laser scanning microscopy. Bioinformatic analysis was performed to identify orthologues of known adhesion proteins in F. nucleatum subspecies. Surface type and treatment significantly influenced single-subspecies biofilm formation. Biofilm formation was overall highest on poly-L-lysine coated surfaces and sandblasted glass surfaces. Biofilm thickness and stability, as well as architecture, varied amongst the subspecies. Interestingly, F. nucleatum ssp. polymorphum did not form a detectable, continuous layer of biofilm on any of the tested substrates. Consistent with limited biofilm forming ability in vitro, F. nucleatum ssp. polymorphum showed the least conservation of the adhesion proteins CmpA and Fap2 in silico. Here, we show that biofilm formation by F. nucleatum in vitro is subspecies- and substrate-specific. Additionally, F. nucleatum ssp. polymorphum does not appear to form stable single-subspecies continuous layers of biofilm in vitro. Understanding the differences in F. nucleatum single-subspecies biofilm formation may shed light on multi-species biofilm formation mechanisms and may reveal new virulence factors as novel therapeutic targets for prevention and treatment of F. nucleatum-mediated infections and diseases.

Combined utilization of metabolic inhibitors to prevent synergistic multi-species biofilm formation

Biofilm is ubiquitous in industrial water systems, causing biofouling and leading to heat transfer efficiency decreases. In particular, multi-species living in biofilms could boost biomass production and enhance treatment resistance. In this study, a total of 37 bacterial strains were isolated from a cooling tower biofilm where acetic acid and propionic acid were detected as the main carbon sources. These isolates mainly belonged to Proteobacteria and Firmicutes, which occupied more than 80% of the total strains according to the 16S rRNA gene amplicon sequencing. Four species (Acinetobacter sp. CTS3, Corynebacterium sp. CTS5, Providencia sp. CTS12, and Pseudomonas sp. CTS17) were observed co-existing in the synthetic medium. Quantitative comparison of biofilm biomass from mono- and multi-species showed a synergistic effect towards biofilm formation among these four species. Three metabolic inhibitors (sulfathiazole, 3-bromopyruvic acid, and 3-nitropropionic acid) were employed to prevent biofilm formation based on their inhibitory effect on corresponding metabolic pathways. All of them displayed evident inhibition profiles to biofilm formation. Notably, combining these three inhibitors possessed a remarkable ability to block the multi-species biofilm development with lower concentrations, suggesting an enhanced effect appeared in simultaneous use. This study demonstrates that combined utilization of metabolic inhibitors is an alternative strategy to prevent multi-species biofilm formation.

The Role of Candida albicans Virulence Factors in the Formation of Multispecies Biofilms With Bacterial Periodontal Pathogens.

Periodontal disease depends on the presence of different microorganisms in the oral cavity that during the colonization of periodontal tissues form a multispecies biofilm community, thus allowing them to survive under adverse conditions or facilitate further colonization of host tissues. Not only numerous bacterial species participate in the development of biofilm complex structure but also fungi, especially Candida albicans, that often commensally inhabits the oral cavity. C. albicans employs an extensive armory of various virulence factors supporting its coexistence with bacteria resulting in successful host colonization and propagation of infection. In this article, we highlight various aspects of individual fungal virulence factors that may facilitate the collaboration with the associated bacterial representatives of the early colonizers of the oral cavity, the bridging species, and the late colonizers directly involved in the development of periodontitis, including the “red complex” species. In particular, we discuss the involvement of candidal cell surface proteins—typical fungal adhesins as well as originally cytosolic “moonlighting” proteins that perform a new function on the cell surface and are also present within the biofilm structures. Another group of virulence factors considered includes secreted aspartic proteases (Sap) and other secreted hydrolytic enzymes. The specific structure of the candidal cell wall, dynamically changing during morphological transitions of the fungus that favor the biofilm formation, is equally important and discussed. The non-protein biofilm-composing factors also show dynamic variability upon the contact with bacteria, and their biosynthesis processes could be involved in the stability of mixed biofilms. Biofilm-associated changes in the microbe communication system using different quorum sensing molecules of both fungal and bacterial cells are also emphasized in this review. All discussed virulence factors involved in the formation of mixed biofilm pose new challenges and influence the successful design of new diagnostic methods and the application of appropriate therapies in periodontal diseases.

Bioresponsive nanotherapy for preventing dental caries by inhibiting multispecies cariogenic biofilms

Early childhood caries (ECC) is a public healthcare concern that greatly reduces the quality of life of young children. As a leading factor of ECC, cariogenic biofilms are composed of acidogenic/aciduric pathogens and extracellular polysaccharides (EPSs), creating an acidic and protected microenvironment. Antimicrobial photodynamic therapy (aPDT) is a noninvasive, painless, and efficient therapeutic approach that is suitable for treating ECC. However, due to the hyperfine structure of cariogenic biofilms, most photosensitizers (PSs) could not access and penetrate deeply in biofilms, which dramatically hamper their efficiency in the clinic. Herein, bioresponsive nanoparticle loaded with chlorin e6 (MPP-Ce6) is developed, which largely increases the penetration depth (by over 75%) and retention (by over 100%) of PS in the biofilm compared with free Ce6. Furthermore, MPP-Ce6-mediated aPDT not only kills the bacteria in preformed biofilms but also inhibits multispecies biofilm formation. A rampant caries model is established to mimic ECC in vivo, where the population of cariogenic bacteria is decreased to 10% after MPP-Ce6-mediated aPDT. Importantly, the number and severity of carious lesions are efficiently reduced via Keyes’ scoring and micro-CT analysis. This simple but effective strategy can serve as a promising approach for daily oral hygiene in preventing ECC.

CO2 accelerates Candida albicans biofilm formation and reveals novel approaches to their inhibition on airway management devices

C. albicans is the predominant human fungal pathogen worldwide and frequently colonises medical devices, such as voice prosthesis, as a biofilm. It is a dimorphic yeast that can switch between yeast and hyphal forms in response to environmental cues, a property that is essential during biofilm establishment and maturation. One such cue is elevation of CO2 levels, as observed in exhaled breath for example. However, despite the clear medical relevance the effects of high CO2 levels on C. albicans biofilm growth has not been investigated to date. Here, we show that 5% CO2 significantly enhances each stage of the C. albicans biofilm forming process; from attachment through maturation to dispersion, via stimulation of the Ras/cAMP/PKA signalling pathway. Transcriptome analysis of biofilm formation under elevated CO2 conditions revealed the activation of key biofilm formation pathways governed by the central biofilm regulators Efg1, Brg1, Bcr1 and Ndt80. Biofilms grown in under elevated CO2 conditions also exhibit increases in azole resistance, tolerance to nutritional immunity and enhanced glucose uptake capabilities. We thus characterise the mechanisms by which elevated CO2 promote C. albicans biofilm formation. We also investigate the possibility of re-purposing drugs that can target the CO2 activated metabolic enhancements observed in C. albicans biofilms. Using this approach we can significantly reduce multi-species biofilm formation in a high CO2 environment and demonstrate a significant extension of the lifespan of voice prostheses in a patient trial. Our research demonstrates a bench to bedside approach to tackle Candida albicans biofilm formation.