Autoinducer 2 of Fusobacterium nucleatum as a target molecule to inhibit biofilm formation of periodontopathogens

Periodontitis is initiated by bacteria in subgingival biofilms, which are composed mostly of Gram-negative anaerobes. Autoinducer 2 (AI-2) is a universal quorum sensing (QS) molecule that mediates intergeneric signalling in multispecies bacterial communities and may induce biofilm formation. As Fusobacterium nucleatum is the major coaggregation bridge organism that links early colonising commensals and late pathogenic colonisers in dental biofilms via the accretion of periodontopathogens, we hypothesised that AI-2 of F. nucleatum contributes to this interspecies interaction, and interruption of this signalling could result in the inhibition of biofilm formation of periodontopathogens. To test this hypothesis, we evaluated the effect of partially purified F. nucleatum AI-2 on monospecies biofilm as well as mutualistic interactions between F. nucleatum and the so-called ‘red complex’ (Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia). Then we tested the effect of two QS inhibitors (QSIs), (5Z)-4-bromo-5-(bromomethylene)-2(5H)-furanone (furanone compound) and d-ribose, on AI-2-induced biofilm formation and coaggregation. F. nucleatum AI-2 remarkably induced biofilm growth of single and dual species and coaggregation between F. nucleatum and each species of the ‘red complex’, all of which were inhibited by the QSIs. F. nucleatum AI-2 induced the expression of the representative adhesion molecules of the periodontopathogens, which were inhibited by the QSIs. Our results demonstrate that F. nucleatum AI-2 plays an important role in inter- and intraspecies interactions between periodontopathogens via enhanced expression of adhesion molecules and may be a target for the inhibition of pathogenic dental biofilm formation.     

Role of an autoinducer-2-like molecule from Veillonella tobetsuensis in Streptococcus gordonii biofilm formation

Objectives

Dental plaque is a multispecies biofilm that causes dental caries and periodontal diseases. In particular, Streptococcus spp. and Veillonella spp. have been identified as early colonizers of oral biofilms. Coaggregation of bacterial species and/or autoinducers using the quorum sensing system has been suggested to plan an important role in biofilm formation. In our previous study, we showed that Veillonella tobetsuensis clearly promoted the development of Streptococcus gordonii biofilms. To clarify the mechanism, coaggregation between S. gordonii and V. tobetsuensis and the effect of an autoinducer-like molecule were investigated in this study.

Biocompatibility and antibiofilm activity of graphene-oxide functionalized titanium discs and collagen membranes

The aims of the study were: 1) to evaluate the effect on biofilm formation of barrier membranes and titanium surfaces coated with graphene-oxide (GO); 2) to analyze the connection between the superficial topography of the tested materials and the amount of bacterial accumulation on them and 3) to analyze the biocompatibility of GO functionalized discs using the zebrafish model.MethodsSingle species bacterial biofilms (Streptococcus oralis, Veilonella parvula, Fusobacterium nucleatum, Porphyomonas gingivalis) were grown on GO-free membranes, membranes coated with 2 and 10 μg/ml of GO, GO-free and GO-coated titanium discs. The biofilms were analyzed by determining the CFU count and by Scanning Electron Microscopy (SEM) and the materials’ topography by Atomic Force Microscopy (AFM). Zebrafish model was used to determine the materials’ toxicity and inflammatory effects.ResultsAFM showed similar roughness of control and GO-coated materials. CFU counts on GO-coated discs were significantly lower than on control discs for all species. CFU counts of S. oralis, V. parvula and P. gingivalis were lower on biofilms grown on both types of GO-coated membranes than on GO-free membrane. SEM analysis showed different formation of single species biofilm of S. oralis on control and GO-coated materials.GO-functionalized titanium discs do not induce toxic or inflammatory effects.SignificanceTitanium implant surfaces functionalized with GO have shown to be biocompatible and less susceptible to biofilm formation. These results encourage further in vivo investigation of the tested materials on infection prevention, specifically in prevention and reduction of peri-implant mucositis and periimplantitis incidence.     

Morphological plasticity of Streptococcus oralis isolates for biofilm production,invasiveness, and architectural patterns

Objective

Streptococcus oralis is an early coloniser of the oral cavity that contributes to dental plaque formation. Many different genotypes can coexist in the same individual and cause opportunistic infections such as bacterial endocarditis. However, little is known about virulence factors involved in those processes. The aim was to analyze the evolving growth of S. oralis colony/biofilm to find out potentially pathogenic features.

Design

Thirty-three S. oralis isolates were analyzed for: (1) biofilm production, by spectrophotometric microtiter plate assay; (2) colonial internal architecture, by histological methods and light and electron microscopy; (3) agar invasion, by a new colony-biofilm assay.

Results

S. oralis colonies showed two different growth patterns: (1) fast growth rate without invasion or minimally invasive; (2) slow growth rate, but high invasion ability. 12.1% of strains were biofilm non-producers and 24.2% not invasive, compared to 51.5% biofilm high-producers and 39.4% very invasive. Both phenotypic characteristics tended to be mutually exclusive. However, a limited number of strains (15%) co-expressed these features at the highest level.

Conclusions

Morphological plasticity of S. oralis highlighted in this study may have important ecological and clinical implications. Coexistence of strains with different growth patterns could produce a synergic effect in the formation and development of subgingival dental plaque. Moreover, invasiveness might regulate dissemination and colonisation mechanisms. Simultaneous co-expression of high-invasive and high-biofilm phenotypes gives a fitness advantage during colonisation and may confer higher pathogenic potential.     

Effect of arginine on the growth and biofilm formation of oral bacteria

BackgroundAlkali production via arginine deiminase system (ADS) of oral bacteria plays a significant role in oral ecology, pH homeostasis and inhibition of dental caries. ADS activity in dental plaque varies greatly between individuals, which may profoundly affect their susceptibility to caries.ObjectiveTo investigate the effect of arginine on the growth and biofilm formation of oral bacteria.Methods and resultsPolymicrobial dental biofilms derived from saliva were formed in a high-throughput active attachment biofilm model and l-arginine (Arg) was shown to reduce the colony forming units (CFU) counts of such biofilms grown for various periods or biofilms derived from saliva of subjects with different caries status. Arg hardly disturbed bacterial growth of Streptococcus mutans, Streptococcus sobrinus, Streptococcus sanguinis and Streptococcus gordonii in BHI medium, but only inhibited biofilm formation of S. mutans. Scanning electron microscope (SEM) showed S. mutans biofilms harboured fewer cells grown with Arg than that without Arg, even in the initial 2 h and 8 h phase. Confocal laser scanning microscope (CLSM) images of poly-microbial dental and S. mutans biofilms revealed the biofilms grown with Arg had lower exopolysaccharide (EPS)/bacteria ratios than those without Arg (P = 0.004, 0.002, respectively). Arg could significantly reduce the production of water-insoluble EPS in S. mutans biofilms (P < 0.001); however, quantitative real-time PCR (qRT-PCR) did not show significantly influence in gene expression of gtfB, gtfC or gtfD (P = 0.32, 0.06, 0.44 respectively).ConclusionsArg could reduce the biomass of poly-microbial dental biofilms and S. mutans biofilms, which may be due to the impact of Arg on water-insoluble EPS. Considering the contribution to pH homeostasis in dental biofilms, Arg may serve as an important agent keeping oral biofilms healthy thus prevent dental caries.     

Colonisation of gingival epithelia by subgingival biofilms in vitro: Role of “red complex” bacteria

Objectives

Biofilm formation on tooth surface results in colonisation and invasion of the juxtaposed gingival tissue, eliciting strong inflammatory responses that lead to periodontal disease. This in vitro study investigated the colonisation of human gingival multi-layered epithelium by multi-species subgingival biofilms, and evaluated the relative effects of the “red complex” species (Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola).

Methods

The grown biofilm consisted of Fusobacterium nucleatum, Campylobacter rectus, Veillonella dispar, P. gingivalis, Prevotella intermedia, T. forsythia, T. denticola, Actinomyces oris, Streptococcus anginosus and Streptococcus oralis, or its variant lacking the “red complex”. After 48 h in co-culture with the gingival epithelia, the bacterial species in the biofilm were quantified, whereas their localisation on the cell surface was investigated by combining confocal-laser scanning microscopy (CLSM) and fluorescence in situ hybridisation (FISH), as well as by scanning electron microscopy (SEM).

Results

Exclusion of the “red complex” quantitatively affected S. oralis, but not other species. The “red-complex” species were all able to colonise the gingival epithelial cells. A co-localisation trend was observed between P. gingivalis and T. denticola, as determined by FISH. However, in the absence of all three “red complex” bacteria from the biofilm, an immense colonisation of streptococci (potentially S. oralis) was observed on the gingival epithelia, as confirmed by both CLSM and SEM.

Conclusions

While the “red complex” species synergise in colonizing gingival epithelia, their absence from the biofilm enhances streptococcal colonisation. This antagonism with streptococci reveals that the “red complex” may regulate biofilm virulence, with potential implications in periodontal pathogenesis.     

Antimicrobial and antibiofilm action of Casbane Diterpene from Croton nepetaefolius against oral bacteria

ObjectiveThe antibacterial activity of Casbane Diterpene (CD) was evaluated in vitro against Streptococcus oralis, S. mutans, S. salivarius, S. sobrinus, S. mitis and S. sanguinis. The viability of planktonic cells was analysed by susceptibility tests (MIC and MBC) and antibiofilm action was assayed.MethodsThe minimal inhibitory and bactericidal concentrations (MIC and MBC) of oral Streptococcus were evaluated through microdilution tests. To assay antibiofilm activity, biofilms were generated on 96-wells polystyrene plates under the presence of CD and quantified by a crystal violet technique and colonies forming units counting.ResultsThe CD isolated from Croton nepetaefolius showed antimicrobial effect on planktonic forms and biofilms of oral pathogens, with MIC values of 62.5 μg/mL against Streptococcus oralis and values between 125 and 500 μg/mL against S. mutans, S. salivarius, S. sobrinus, S. mitis and S. sanguinis. CD showed an inhibitory effect on S. mutans biofilm formation at 250 μg/mL, and a decrease on viable cell of 94.28% compared to the normal biofilm growth.ConclusionsThe compound CD can be considered as a promising molecule for the treatment against oral pathogens responsible for dental biofilm.     

Salivary pellicle composition and multispecies biofilm developed on titanium nitrided by cold plasma

Objective

The aim of this study was to evaluate the composition of the salivary pellicle (SP) and multispecies biofilm developed on titanium nitrided by cold plasma.

Methods

Titanium discs were allocated into a control group (Ti) and an experimental group (TiN – titanium-nitrided by cold plasma). The disc surface topography was characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). The chemical composition of the disc surface was determined by X-ray photoelectron spectroscopy (XPS). Stimulated, clarified, and filtered saliva was used to form pellicles on the discs. Proteome analysis of the adsorbed SP proteins was performed by liquid chromatography–mass spectrometry (LC–MS). The surface free energy (SFE) was evaluated before and after SP formation. A multispecies biofilm composed of Actinomyces naeslundii, Streptococcus oralis, Streptococcus mutans, Fusobacterium nucleatum, Veillonella dispar, and Candida albicans was developed on the SP-coated discs. Viable microorganism counts were determined. The biomass and average thickness of biofilms were analyzed by confocal laser-scanning microscopy (CLSM) with COMSTAT software. The biofilm organization was visualized by SEM.

Results

The surface topography was similar in both groups. The SFE of the TiN group did not differ from that of the Ti group (p > 0.05), although the adsorption of pellicle proteins increased the SFE in both pellicle-coated groups (p < 0.001). Different proteins were identified on the Ti and TiN surfaces. The amount of biofilm was similar for both groups (p = 0.416), but the counts of F. nucleatum and S. oralis were higher in the TiN group (p < 0.001). Similar biofilms were characterized by the COMSTAT data, CLSM images, and SEM images.

Conclusion

The titanium nitrided by cold plasma exhibited differences in SP composition and multispecies microbial biofilm population compared to the control titanium surface.     

In vitro biofilm formation on different ceramic biomaterial surfaces: Coating with two bactericidal glasses

ObjectivesTo compare biofilm formation on the surface of different ceramic biomaterials to be used in implant dentistry.MethodsIn vitro biofilm formation was investigated from mixtures of standard reference strains of Streptococcus oralis, Veillonella parvula, Actinomyces naeslundii, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans and Porphyromonas gingivalis. Sterile ceramic calcium hydroxyapatite discs (HA) as control, sterile Al₂O₃/Ce-TZP nanocomposite sandblasted discs (material A1) and sterile Al₂O₃/Ce-TZP nanocomposite sandblasted discs and coated with two types of antimicrobial glasses (materials A2 and A3) were used. Biofilms were grown on the four surfaces and evaluated after 12, 24, 48 and 72 h of incubation. Biofilms were examined by confocal laser scanning microscopy (CLSM). In addition, counts of live bacterial cells of the target species A. actinomycetemcomitans, F. nucleatum and P. gingivalis were calculated by quantitative polymerase chain reaction (qPCR) combined with propidium monoazide (PMA). For data analysis, bacterial counts were compared with a multivariate general lineal model.ResultsUsing CLSM, cell vitality decreased in A2 and A3. With qPCR-PMA, significant differences in vitality were observed forA. actinomycetemcomitans in A3 after 48 and 72 h of incubation. With respect to the development of the biofilms, a significant increase in counts on HA and materials A1 and A2 was observed for A. actinomycetemcomitans and F. nucleatum. Conversely, for P. gingivalis, no differences were found for HA and materials A1 and A2.SignificanceDifferences in biofilm formation were detected among the different tested materials. The ceramic material A3 has an effect on the vitality of A. actinomycetemcomitans growing in an in vitro biofilm model.     

Interactions between Streptococcus oralis,Actinomyces oris,and Candida albicans in the development of multispecies oral microbial biofilms on salivary pellicle

The fungus Candida albicans is carried orally and causes a range of superficial infections that may become systemic. Oral bacteria Actinomyces oris and Streptococcus oralis are abundant in early dental plaque and on oral mucosa. The aims of this study were to determine the mechanisms by which S. oralis and A. oris interact with each other and with C. albicans in biofilm development. Spatial distribution of microorganisms was visualized by confocal laser scanning microscopy of biofilms labeled by differential fluorescence or by fluorescence in situ hybridization (FISH). Actinomyces oris and S. oralis formed robust dual‐species biofilms, or three‐species biofilms with C. albicans. The bacterial components tended to dominate the lower levels of the biofilms while C. albicans occupied the upper levels. Non‐fimbriated A. oris was compromised in biofilm formation in the absence or presence of streptococci, but was incorporated into upper biofilm layers through binding to C. albicans. Biofilm growth and hyphal filament production by C. albicans was enhanced by S. oralis. It is suggested that the interkingdom biofilms are metabolically coordinated to house all three components, and this study demonstrates that adhesive interactions between them determine spatial distribution and biofilm architecture. The physical and chemical communication processes occurring in these communities potentially augment C. albicans persistence at multiple oral cavity sites.     

Genetic basis of coaggregation receptor polysaccharide biosynthesis in Streptococcus sanguinis and related species

Interbacterial adhesion between streptococci and actinomyces promotes early dental plaque biofilm development. Recognition of coaggregation receptor polysaccharides (RPS) on strains of Streptococcus sanguinis, Streptococcus gordonii and Streptococcus oralis by Actinomyces spp. type 2 fimbriae is the principal mechanism of these interactions. Previous studies of genetic loci for synthesis of RPS (rps) and RPS precursors (rml, galE1 and galE2) in S. gordonii 38 and S. oralis 34 revealed differences between these strains. To determine whether these differences are strain‐specific or species‐specific, we identified and compared loci for polysaccharide biosynthesis in additional strains of these species and in several strains of the previously unstudied species, S. sanguinis. Genes for synthesis of RPS precursors distinguished the rps loci of different streptococci. Hence, rml genes for synthesis of TDP‐L‐Rha were in rps loci of S. oralis strains but at other loci in S. gordonii and S. sanguinis. Genes for two distinct galactose epimerases were also distributed differently. Hence, galE1 for epimerization of UDP‐Glc and UDP‐Gal was in galactose operons of S. gordonii and S. sanguinis strains but surprisingly, this gene was not present in S. oralis. Moreover, galE2 for epimerization of both UDP‐Glc and UDP‐Gal and UDP‐GlcNAc and UDP‐GalNAc was at a different locus in each species, including rps operons of S. sanguinis. The findings provide insight into cell surface properties that distinguish different RPS‐producing streptococci and open an approach for identifying these bacteria based on the arrangement of genes for synthesis of polysaccharide precursors.     

Oral biofilm models for mechanical plaque removal

In vitro plaque removal studies require biofilm models that resemble in vivo dental plaque. Here, we compare contact and non-contact removal of single and dual-species biofilms as well as of biofilms grown from human whole saliva in vitro using different biofilm models. Bacteria were adhered to a salivary pellicle for 2 h or grown after adhesion for 16 h, after which, their removal was evaluated. In a contact mode, no differences were observed between the manual, rotating, or sonic brushing; and removal was on average 39%, 84%, and 95% for Streptococcus mutans, Streptococcus oralis, and Actinomyces naeslundii, respectively, and 90% and 54% for the dual- and multi-species biofilms, respectively. However, in a non-contact mode, rotating and sonic brushes still removed considerable numbers of bacteria (24–40%), while the manual brush as a control (5–11%) did not. Single A. naeslundii and dual-species (A. naeslundii and S. oralis) biofilms were more difficult to remove after 16 h growth than after 2 h adhesion (on average, 62% and 93% for 16- and 2-h-old biofilms, respectively), while in contrast, biofilms grown from whole saliva were easier to remove (97% after 16 h and 54% after 2 h of growth). Considering the strong adhesion of dual-species biofilms and their easier more reproducible growth compared with biofilms grown from whole saliva, dual-species biofilms of A. naeslundii and S. oralis are suggested to be preferred for use in mechanical plaque removal studies in vitro.     

Succession of oral bacterial colonizers on dental implant materials: An in vitro biofilm model

ObjectivesOral bacterial adhesion on dental implant materials has been extensively studied using in vitro systems but has yielded results restricted to in vitro growth patterns due to limitations in species selection, sustained fastidious anaerobe growth, and mixed culture longevity. The aim of this study was to develop an oral bacterial biofilm model consisting of colonizers representative of the oral microbiome exhibiting temporal shifts characteristic of plaque development and maturation in vivo.MethodsStreptococcus oralis, Actinomyces naeslundii, Aggregatibacter actinomycetemcomitans, Veillonella parvula, Fusobacterium nucleatum, and Porphyromonas gingivalis were grown in monoculture prior to combination in mixed culture. Commercially pure titanium (cpTi) and yttria-stabilized zirconia (ZrO₂) disks with polished, acid-etched, or sandblasted surfaces were prepared to evaluate oral bacterial adhesion. After 6 h, 1, 3, 7, 14 and 21 days, genomic DNA from planktonic and adherent bacteria was isolated. Quantitative polymerase chain reaction (qPCR) was used to enumerate the amount and proportion of each species.ResultsEarly-colonizing S. oralis and A. actinomycetemcomitans, dominated after 6 h prior to secondary colonization by F. nucleatum and V. parvula in planktonic (1 day) and sessile (3 days) form. A. naeslundii maintained relatively low but stable bacterial counts throughout testing. After 14 days, late-colonizing P. gingivalis became established in mixed culture and persisted, becoming the dominant species after 21 days. The composition of adherent bacteria across all substrates was statistically similar at all timepoints with notable exceptions including lower S. oralis bacterial counts on polished cpTi (3 days).SignificanceWithin the present model’s limitations, multispecies oral bacterial attachment is similar on surface-treated cpTi and ZrO₂.     

Inhibition of Streptococcus mutans adherence and biofilm formation using analogues of the SspB peptide

Objective

Streptococcus gordonii is a pioneer colonizer of the enamel salivary pellicle that forms biofilm on the tooth surfaces. Recent reports show the surface protein analogue peptide {400 (T) of SspB 390-402 is substituted to K forming SspB (390-T400K-402)} from S. gordonii interacts strongly with salivary receptors to cariogenic bacteria, Streptococcus mutans. To characterize the analogue peptide biological activities, we investigated its binding and inhibiting effects, and the role of its amino acid moities.

Methods

We measured binding activity of analogue peptides to salivary components using the BIAcore assay; assayed inhibition activities of peptides for bacterial binding and growth on saliva-coated hydroxyapatite beads (s-HA); and describe the peptides interfering with biofilm formation of S. mutans on polystyrene surfaces.

Results

The SspB (390-T400K-402 and -401) peptides significantly bound with salivary components and inhibited the binding of S. mutans and S. gordonii to s-HA without bactericidal activity; but did not inhibit binding of Streptococcus mitis, a beneficial commensal. Further, the lack of D and E-L at position 390 and 401-402 in the peptide, and substituted peptide SspB (D390H- or D390K-T400K-402) did not bind to salivary components or inhibit binding of S. mutans. The SspB (390-T400K-402) peptide inhibited biofilm formation on salivary components-coated polystyrene surfaces in absence of conditioned planktonic cells.

Conclusions

We found constructing the peptide to include positions 390(D), 400(K) and 401(E), two surface positive and negative connective charges, and at least 12 amino acids are required to bind salivary components and inhibit the binding of S. mutans and S. gordonii.     

Identification and characterization of a salivary-pellicle-binding peptide by phage display

Objective

Dental biofilms are associated with oral diseases, making their control necessary. One way to control them is to prevent initial bacterial adherence to the salivary pellicle and thereby eventually decrease binding of late colonizing potential pathogens. The goal of this study was to generate a salivary-pellicle-binding peptide (SPBP) with antifouling activity towards primary colonizing bacteria. In order to achieve this goal we aimed to: (i) identify novel SPBPs by phage display; (ii) characterize the binding and antifouling properties of the selected SPBPs.

Methods

A library of 2 × 10⁹ phages displaying a random sequence of 12-mer peptides was used to identify peptides that bound selectively to the in vitro salivary pellicle. Three rounds of panning resulted in the selection of 10 pellicle-binding phages, each displaying a novel peptide sequence. The peptides were synthesized and their binding to the in vitro salivary pellicle was characterized in the presence and absence of calcium ions and Tween-20. The antifouling property of hydroxyapatite (HA) and saliva-coated HA discs treated with and without SPBPs were evaluated against Streptococcus gordonii.

Results

Ten unique SPBPs were identified using the phage display. One of these peptides, SPBP 10 (NSAAVRAYSPPS), exhibited significant binding to the in vitro salivary pellicle which was neither influenced by calcium ions, nor affected by up to 0.5% Tween-20. Its antifouling property against S. gordonii was significantly higher on the treated surfaces than on untreated surfaces.

Conclusions

Use of the phage display library enabled us to find a specific SPBP with antifouling property towards S. gordonii.     

Synthetic bromated furanone inhibits autoinducer-2-mediated communication and biofilm formation in oral streptococci

INTRODUCTION: Autoinducer-2 (AI-2) is a widespread communication-signal molecule that allows bacteria to sense and react to environmental factors. In some streptococci AI-2 is reported to be involved in virulence expression and biofilm formation. It has earlier been shown that the alga Delisea pulchra produces bromated furanones, which prevent bacterial colonization of the algae. METHODS AND RESULTS: We have previously published a novel and simple synthesis of (Z)-5-bromomethylene-2(5H)-furanone. In this study we showed that our synthesized furanone inhibited biofilm formation and bioluminescence induction by Streptococcus anginosus, Streptococcus intermedius, and Streptococcus mutans, as well as bioluminescence induction by Vibrio harveyi BB152. CONCLUSION: We suggest that the effect is linked to interference with the AI-2 signaling pathway because adding furanone to the medium had no effect on the ability of the AI-2-defective S. anginosus luxS and S. intermedius luxS mutants to form biofilms.