Experimental Inoculation of Aggregatibacter actinomycetemcomitans and Streptococcus gordonii and Its Impact on Alveolar Bone Loss and Oral and Gut Microbiomes.

Oral bacteria are implicated not only in oral diseases but also in gut dysbiosis and inflammatory conditions throughout the body. The periodontal pathogen Aggregatibacter actinomycetemcomitans (Aa) often occurs in complex oral biofilms with Streptococcus gordonii (Sg), and this interaction might influence the pathogenic potential of this pathogen. This study aims to assess the impact of oral inoculation with Aa, Sg, and their association (Aa+Sg) on alveolar bone loss, oral microbiome, and their potential effects on intestinal health in a murine model. Sg and/or Aa were orally administered to C57Bl/6 mice, three times per week, for 4 weeks. Aa was also injected into the gingiva three times during the initial experimental week. After 30 days, alveolar bone loss, expression of genes related to inflammation and mucosal permeability in the intestine, serum LPS levels, and the composition of oral and intestinal microbiomes were determined. Alveolar bone resorption was detected in Aa, Sg, and Aa+Sg groups, although Aa bone levels did not differ from that of the SHAM-inoculated group. Il-1ß expression was upregulated in the Aa group relative to the other infected groups, while Il-6 expression was downregulated in infected groups. Aa or Sg downregulated the expression of tight junction genes Cldn 1, Cldn 2, Ocdn, and Zo-1 whereas infection with Aa+Sg led to their upregulation, except for Cldn 1. Aa was detected in the oral biofilm of the Aa+Sg group but not in the gut. Infections altered oral and gut microbiomes. The oral biofilm of the Aa group showed increased abundance of Gammaproteobacteria, Enterobacterales, and Alloprevotella, while Sg administration enhanced the abundance of Alloprevotella and Rothia. The gut microbiome of infected groups showed reduced abundance of Erysipelotrichaceae. Infection with Aa or Sg disrupts both oral and gut microbiomes, impacting oral and gut homeostasis. While the combination of Aa with Sg promotes Aa survival in the oral cavity, it mitigates the adverse effects of Aa in the gut, suggesting a beneficial role of Sg associations in gut health.

Streptococcus gordonii Supragingival Bacterium Oral Infection-Induced Periodontitis and Robust miRNA Expression Kinetics.

Streptococcus gordonii (S. gordonii, Sg) is one of the early colonizing, supragingival commensal bacterium normally associated with oral health in human dental plaque. MicroRNAs (miRNAs) play an important role in the inflammation-mediated pathways and are involved in periodontal disease (PD) pathogenesis. PD is a polymicrobial dysbiotic immune-inflammatory disease initiated by microbes in the gingival sulcus/pockets. The objective of this study is to determine the global miRNA expression kinetics in S. gordonii DL1-infected C57BL/6J mice. All mice were randomly divided into four groups (n = 10 mice/group; 5 males and 5 females). Bacterial infection was performed in mice at 8 weeks and 16 weeks, mice were euthanized, and tissues harvested for analysis. We analyzed differentially expressed (DE) miRNAs in the mandibles of S. gordonii-infected mice. Gingival colonization/infection by S. gordonii and alveolar bone resorption (ABR) was confirmed. All the S. gordonii-infected mice at two specific time points showed bacterial colonization (100%) in the gingival surface, and a significant increase in mandible and maxilla ABR (p < 0.0001). miRNA profiling revealed 191 upregulated miRNAs (miR-375, miR-34b-5p) and 22 downregulated miRNAs (miR-133, miR-1224) in the mandibles of S. gordonii-infected mice at the 8-week mark. Conversely, at 16 weeks post-infection, 10 miRNAs (miR-1902, miR-203) were upregulated and 32 miRNAs (miR-1937c, miR-720) were downregulated. Two miRNAs, miR-210 and miR-423-5p, were commonly upregulated, and miR-2135 and miR-145 were commonly downregulated in both 8- and 16-week-infected mice mandibles. Furthermore, we employed five machine learning (ML) algorithms to assess how the number of miRNA copies correlates with S. gordonii infections in mice. In the ML analyses, miR-22 and miR-30c (8-week), miR-720 and miR-339-5p (16-week), and miR-720, miR-22, and miR-339-5p (combined 8- and 16-week) emerged as the most influential miRNAs.

SepM mutation in Streptococcus mutans clinical isolates and related function analysis.

BACKGROUND: Streptococcus mutans (S. mutans) is an important pathogenic bacterium that causes dental caries, while Streptococcus gordonii (S. gordonii) is a non-cariogenic bacterium that inhibits the growth of S. mutans. The SepM protein can promote the inhibitory ability of S. mutans against S. gordonii by cleaving CSP-21 and activating the ComDE two-component system. This study was designed to explore sepM mutation in S. mutans clinical isolates and related function in the regulation of interactions with S. gordonii. METHODS: The S. mutans clinical strains that can inhibit the growth of S. gordonii constitute the inhibitory group. 286 C-serotype S. mutans strains were categorized into S. gordonii inhibitory (n = 114) and non-inhibitory bacteria (n = 172). We detected sanger sequencing of sepM gene, the expression levels of related genes and proteins in clinical isolates, obtained prokaryotic expression and purification of mutated proteins, and analyzed the effect of the target mutations on the binding between SepM and CSP-21. RESULTS: We found that C482T, G533A, and G661A missense mutations were presented at significantly higher frequency in the inhibitory group relative to the non-inhibitory group. There was no significant difference in the expression of the sepM gene between selected clinical isolates harboring the G533A mutation and the control group. The expression levels of SepM, phosphorylated ComD, and ComE in the mutation group were significantly higher than those in the control group. SepM_control and SepM_D221N (G661A at the gene level) were found to contain two residues close to the active center while SepM_G178D (G533A at the gene level) contained three residues close to the active center. At 25 °C and a pH of 5.5, SepM_D221N (G661A) exhibited higher affinity for CSP-21 (KD = 8.25 µM) than did the SepM control (KD = 33.1 µM), and at 25 °C and a pH of 7.5, SepM_G178D (G533A) exhibited higher affinity (KD = 3.02 µM) than the SepM control (KD = 15.9 µM). It means that it is pH dependent. CONCLUSIONS: Our data suggest that increased cleavage of CSP-21 by the the mutant SepM may be a reason for the higher inhibitory effect of S. mutans on S. gordonii .

Does dental material type influence bacterial adhesion under the same polishing conditions? Direct observation using a fluorescent staining technique: An in vitro study.

Highly polished 3, 4, and 5 mol% yttria-stabilized zirconia and CAD/CAM composite resin samples were prepared, and the influence of surface roughness (Ra and Sa, 21 areas/group), wettability (contact angle and surface energy, 3 samples/group), and surface chemical composition (2 samples/group) on single-strain bacterial adhesion models (Porphyromonas gingivalis, Streptococcus oralis, Streptococcus sanguinis, Streptococcus gordonii, and Streptococcus mutans) were compared via fluorescent staining with graphical analysis (21 areas/group). Statistical analysis was performed using the Shapiro-Wilk test followed by one-way analysis of variance with Tukey's test or the Kruskal-Wallis test with Dunn's test (α=0.05) and linear regression. For dental zirconia with the same surface roughness, the yttria content did not significantly influence the initial bacterial adhesion. However, higher bacterial adhesion was detected for the composite resin owing to its high C, O, and Si contents. There was no correlation between surface energy and bacterial adhesion for any bacterial strain (p<0.005).

Inverted amino acids reduce the adhesion and biofilm biomass of early oral colonizers.

BACKGROUND: Early colonizers adhere to the dental surface and facilitate the initial adhesion of secondary colonizers to form oral biofilms, which may cause oral infections. OBJECTIVES: This study aimed to determine the antimicrobial, anti-adhesion and antibiofilm potency of inverted amino acids on early colonizer streptococci and their mixed species. MATERIAL AND METHODS: The following test strains were used: Streptococcus gordonii (American Type Culture Collection (ATCC) 35105); Streptococcus mitis (ATCC 49456); Streptococcus oralis (ATCC 10557); Streptococcus salivarius (ATCC 7073); and Streptococcus sanguinis (ATCC BAA-1455). The concentration-dependent antimicrobial potency of d-alanine (d-ala), d-arginine (d-arg), d-leucine (d-leu), d-methionine (d-met), and d-tryptophan (d-try) was determined using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method with AlamarBlue modification. The adhesion of primary colonizers in the presence of 25-mM d-amino acids (dAAs) was assessed using the colony forming unit (CFU) assay. The CFU assay was conducted on 24-h flow cell bacterial biofilm models after exposure to 25-mM inverted dAAs. RESULTS: No minimum inhibitory concentration (MIC) point was detected at any concentration tested. The minimum bactericidal concentration (MBC) point was not observed. The adhesion of S. mitis, S. oralis and mixed species was reduced by all tested dAAs. No adverse effects were observed on S. gordonii with any of the tested dAAs. The biofilm biomass of test strains under flow conditions was significantly reduced after a 5-min exposure to all tested dAAs at 25-mM concentration. CONCLUSIONS: D-amino acids did not inhibit bacterial growth and did not show bactericidal or bacteriostatic effects on test strains at any concentration tested (ranging from 6.25 mM to 100 mM). However, dAAs effectively inhibit the adhesion of early colonizers, thereby preventing the formation of oral biofilm.

Measurement and analysis of microbial fluoride resistance in dental biofilm models.

The interactions between communities of microorganisms inhabiting the dental biofilm is a major determinant of oral health. These biofilms are periodically exposed to high concentrations of fluoride, which is present in almost all oral healthcare products. The microbes resist fluoride through the action of membrane export proteins. This chapter describes the culture, growth and harvest conditions of model three-species dental biofilm comprised of cariogenic pathogens Streptococcus mutans and Candida albicans and the commensal bacterium Streptococcus gordonii. In order to examine the role of fluoride export by S. mutans in model biofilms, procedures for generating a strain of S. mutans with a genetic knockout of the fluoride exporter are described. We present a case study examining the effects of this mutant strain on the biofilm mass, acid production and mineral dissolution under exposure to low levels of fluoride. These general approaches can be applied to study the effects of any gene of interest in physiologically realistic multispecies oral biofilms.

Interpenetrating nanofibrillar membrane of self-assembled collagen and antimicrobial peptides for enhanced bone regeneration.

Bone regeneration remains a major clinical challenge, especially when infection necessitates prolonged antibiotic treatment. This study presents a membrane composed of self-assembled and interpenetrating GL13K, an antimicrobial peptide (AMP) derived from a salivary protein, in a collagen membrane for antimicrobial activity and enhanced bone regeneration. Commercially available collagen membranes were immersed in GL13K solution, and self-assembly was initiated by raising the solution pH to synthesize the multifunctional membrane called COL-GL. COL-GL was composed of interpenetrating large collagen fibers and short GL13K nanofibrils, which increased hydrophobicity, reduced biodegradation from collagenase, and stiffened the matrix compared to control collagen membranes. Incorporation of GL13K led to antimicrobial and anti-fouling activity against early oral surface colonizer Streptococcus gordonii while not affecting fibroblast cytocompatibility or pre-osteoblast osteogenic differentiation. GL13K in solution also reduced macrophage inflammatory cytokine expression and increased pro-healing cytokine expression. Bone formation in a rat calvarial model was accelerated at eight weeks with COL-GL compared to the gold-standard collagen membrane based on microcomputed tomography and histology. Interpenetration of GL13K within collagen sidesteps challenges with antimicrobial coatings on bone regeneration scaffolds while increasing bone regeneration. This strength makes COL-GL a promising approach to reduce post-surgical infections and aid bone regeneration in dental and orthopedic applications. STATEMENT OF SIGNIFICANCE: The COL-GL membrane, incorporating the antimicrobial peptide GL13K within a collagen membrane, signifies a noteworthy breakthrough in bone regeneration strategies for dental and orthopedic applications. By integrating self-assembled GL13K nanofibers into the membrane, this study successfully addresses the challenges associated with antimicrobial coatings, exhibiting improved antimicrobial and anti-fouling activity while preserving compatibility with fibroblasts and pre-osteoblasts. The accelerated bone formation observed in a rat calvarial model emphasizes the potential of this innovative approach to minimize post-surgical infections and enhance bone regeneration outcomes. As a promising alternative for future therapeutic interventions, this material tackles the clinical challenges of extended antibiotic treatments and antibiotic resistance in bone regeneration scenarios.

Fluoride export is required for the competitive fitness of pathogenic microorganisms in dental biofilm models.

Microorganisms resist fluoride toxicity using fluoride export proteins from one of several different molecular families. Cariogenic species Streptococcus mutans and Candida albicans extrude intracellular fluoride using a CLCF F-/H+ antiporter and FEX fluoride channel, respectively, whereas oral commensal eubacteria, such as Streptococcus gordonii, export fluoride using a Fluc fluoride channel. In this work, we examine how genetic knockout of fluoride export impacts pathogen fitness in single-species and three-species dental biofilm models. For biofilms generated using S. mutans with the genetic knockout of the CLCF transporter, exposure to low fluoride concentrations decreased S. mutans counts, synergistically reduced the populations of C. albicans, increased the relative proportion of oral commensal S. gordonii, and reduced properties associated with biofilm pathogenicity, including acid production and hydroxyapatite dissolution. Biofilms prepared with C. albicans with genetic knockout of the FEX channel also exhibited reduced fitness in the presence of fluoride but to a lesser degree. Imaging studies indicate that S. mutans is highly sensitive to fluoride, with the knockout strain undergoing complete lysis when exposed to low fluoride for a moderate amount of time. Biochemical purification of the S. mutans CLCF transporter and functional reconstitution establishes that the functional protein is a dimer encoded by a single gene. Together, these findings suggest that fluoride export by oral pathogens can be targeted by specific inhibitors to restore biofilm symbiosis in dental biofilms and that S. mutans is especially susceptible to fluoride toxicity. IMPORTANCE: Dental caries is a globally prevalent condition that occurs when pathogenic species, including Streptococcus mutans and Candida albicans, outcompete beneficial species, such as Streptococcus gordonii, in the dental biofilm. Fluoride is routinely used in oral hygiene to prevent dental caries. Fluoride also has antimicrobial properties, although most microbes possess fluoride exporters to resist its toxicity. This work shows that sensitization of cariogenic species S. mutans and C. albicans to fluoride by genetic knockout of fluoride exporters alters the microbial composition and pathogenic properties of dental biofilms. These results suggest that the development of drugs that inhibit fluoride exporters could potentiate the anticaries effect of fluoride in over-the-counter products like toothpaste and mouth rinses. This is a novel strategy to treat dental caries.

In vitro bacterial penetration and dissemination through dentinal tubules in roots filled with calcium silicate-based cements.

The aim of this study was to assess whether calcium silicate root fillings prevent bacterial penetration and to determine how bacteria penetrate roots. Extracted single-rooted, single-canal human teeth were decoronated, prepared and filled with ProRootMTA or Biodentine (n = 12 each). Positive and negative (n = 2 each) controls were not filled. A two-chamber model was used with Streptococcus gordonii. The lower compartment was evaluated for turbidity over 150 days. Roots were split and examined for bacteria via SEM. The chi-squared test was used for comparisons (α = 0.05). Experimental groups had bacteria in their coronal thirds. Tubules contained bacteria in 90.9% and 91.7% of areas examined in the Biodentine and ProRootMTA groups, respectively, with no significant difference (p = 0.914). Experimental and negative roots had no turbidity with no significant difference between Biodentine and ProRootMTA (p = 1.000). Positive controls had turbidity. Bacteria penetrate roots via dentine tubules of root-filled teeth. Biodentine was comparable to ProRoot MTA.

Interspecies metabolite transfer fuels the methionine metabolism of Fusobacterium nucleatum to stimulate volatile methyl mercaptan production.

The major oral odor compound methyl mercaptan (CH3SH) is strongly associated with halitosis and periodontitis. CH3SH production stems from the metabolism of polymicrobial communities in periodontal pockets and on the tongue dorsum. However, understanding of CH3SH-producing oral bacteria and their interactions is limited. This study aimed to investigate CH3SH production by major oral bacteria and the impact of interspecies interactions on its generation. Using a newly constructed large-volume anaerobic noncontact coculture system, Fusobacterium nucleatum was found to be a potent producer of CH3SH, with that production stimulated by metabolic interactions with Streptococcus gordonii, an early dental plaque colonizer. Furthermore, analysis of extracellular amino acids using an S. gordonii arginine-ornithine antiporter (ArcD) mutant demonstrated that ornithine excreted from S. gordonii is a key contributor to increased CH3SH production by F. nucleatum. Further study with 13C, 15N-methionine, as well as gene expression analysis, revealed that ornithine secreted by S. gordonii increased the demand for methionine through accelerated polyamine synthesis by F. nucleatum, leading to elevated methionine pathway activity and CH3SH production. Collectively, these findings suggest that interaction between S. gordonii and F. nucleatum plays a key role in CH3SH production, providing a new insight into the mechanism of CH3SH generation in oral microbial communities. A better understanding of the underlying interactions among oral bacteria involved in CH3SH generation can lead to the development of more appropriate prophylactic approaches to treat halitosis and periodontitis. An intervention approach like selectively disrupting this interspecies network could also offer a powerful therapeutic strategy.IMPORTANCEHalitosis can have a significant impact on the social life of affected individuals. Among oral odor compounds, CH3SH has a low olfactory threshold and halitosis is a result of its production. Recently, there has been a growing interest in the collective properties of oral polymicrobial communities, regarded as important for the development of oral diseases, which are shaped by physical and metabolic interactions among community participants. However, it has yet to be investigated whether interspecies interactions have an impact on the production of volatile compounds, leading to the development of halitosis. The present findings provide mechanistic insights indicating that ornithine, a metabolite excreted by Streptococcus gordonii, promotes polyamine synthesis by Fusobacterium nucleatum, resulting in a compensatory increase in demand for methionine, which results in elevated methionine pathway activity and CH3SH production. Elucidation of the mechanisms related to CH3SH production is expected to lead to the development of new strategies for managing halitosis.

Identification of a glutathione transporter in A. actinomycetemcomitans.

IMPORTANCE: Microbes produce a large array of extracellular molecules, which serve as signals and cues to promote polymicrobial interactions and alter the function of microbial communities. This has been particularly well studied in the human oral microbiome, where key metabolites have been shown to impact both health and disease. Here, we used an untargeted mass spectrometry approach to comprehensively assess the extracellular metabolome of the pathogen Aggregatibacter actinomycetemcomitans and the commensal Streptococcus gordonii during mono- and co-culture. We generated and made publicly available a metabolomic data set that includes hundreds of potential metabolites and leveraged this data set to identify an operon important for glutathione secretion in A. actinomycetemcomitans.

Sarcoidosis Associated with Enlarged Mediastinal Lymph Nodes with the Detection of Streptococcus gordonii and Cutibacterium acnes Using a Clone Library Method.

A 77-year-old Japanese woman with mediastinal lymphadenopathy and uveitis was diagnosed with sarcoidosis. The bacterial flora in biopsied samples from mediastinal lymph nodes was analyzed using a clone library method with Sanger sequencing of the 16S rRNA gene, and Streptococcus gordonii (52 of 71 clones) and Cutibacterium acnes (19 of 71 clones) were detected. No previous study has conducted a bacterial floral analysis using the Sanger method for the mediastinal lymph node in sarcoidosis, making this case report the first to document the presence of S. gordonii and C. acnes in the mediastinal lymph node of a patient with sarcoidosis.

Transcriptome and metabolome analyses of Streptococcus gordonii DL1 under acidic conditions.

OBJECTIVES: Streptococcus gordonii is associated with the formation of biofilms, especially those that comprise dental plaque. Notably, S. gordonii DL1 causes infective endocarditis (IE). Colonization of this bacterium requires a mechanism that can tolerate a drop in environmental pH by producing acid via its own sugar metabolism. The ability to survive acidic environmental conditions might allow the bacterium to establish vegetative colonization even in the endocardium due to inflammation-induced lowering of pH, increasing the risk of IE. At present, the mechanism by which S. gordonii DL1 survives under acidic conditions is not thoroughly elucidated. The present study was thus conducted to elucidate the mechanism(s) by which S. gordonii DL1 survives under acidic conditions. METHODS: We analyzed dynamic changes in gene transcription and intracellular metabolites in S. gordonii DL1 exposed to acidic conditions, using transcriptome and metabolome analyses. RESULTS: Transcriptome analysis revealed upregulation of genes involved in heat shock response and glycolysis, and down regulation of genes involved in phosphotransferase systems and biosynthesis of amino acids. The most upregulated genes were a beta-strand repeat protein of unknown function (SGO_RS06325), followed by copper-translocating P-type ATPase (SGO_RS09470) and malic enzyme (SGO_RS01850). The latter two of these contribute to cytoplasmic alkalinization. S. gordonii mutant strains lacking each of these genes showed significantly reduced survival under acidic conditions. Metabolome analysis revealed that cytoplasmic levels of several amino acids were reduced. CONCLUSIONS: S. gordonii survives the acidic conditions by recovering the acidic cytoplasm using the various activities, which are regulated at the transcriptional level.

The Effect of Ficin Immobilized on Carboxymethyl Chitosan on Biofilms of Oral Pathogens.

In the last decade, Ficin, a proteolytic enzyme extracted from the latex sap of the wild fig tree, has been widely investigated as a promising tool for the treatment of microbial biofilms, wound healing, and oral care. Here we report the antibiofilm properties of the enzyme immobilized on soluble carboxymethyl chitosan (CMCh) and CMCh itself. Ficin was immobilized on CMCh with molecular weights of either 200, 350 or 600 kDa. Among them, the carrier with a molecular weight of 200 kDa bound the maximum amount of enzyme, binding up to 49% of the total protein compared to 19-32% of the total protein bound to other CMChs. Treatment with pure CMCh led to the destruction of biofilms formed by Streptococcus salivarius, Streptococcus gordonii, Streptococcus mutans, and Candida albicans, while no apparent effect on Staphylococcus aureus was observed. A soluble Ficin was less efficient in the destruction of the biofilms formed by Streptococcus sobrinus and S. gordonii. By contrast, treatment with CMCh200-immobilized Ficin led to a significant reduction of the biofilms of the primary colonizers S. gordonii and S. mutans. In model biofilms obtained by the inoculation of swabs from teeth of healthy volunteers, the destruction of the biofilm by both soluble and immobilized Ficin was observed, although the degree of the destruction varied between artificial plaque samples. Nevertheless, combined treatment of oral Streptococci biofilm by enzyme and chlorhexidine for 3 h led to a significant decrease in the viability of biofilm-embedded cells, compared to solely chlorhexidine application. This suggests that the use of either soluble or immobilized Ficin would allow decreasing the amount and/or concentration of the antiseptics required for oral care or improving the efficiency of oral cavity sanitization.

Structural and dynamical changes of the Streptococcus gordonii metalloregulatory ScaR protein induced by Mn2+ ion binding.

Divalent metal ions are essential micronutrients for many intercellular reactions. Maintaining their homeostasis is necessary for the survival of bacteria. In Streptococcus gordonii, one of the primary colonizers of the tooth surface, the cellular concentration of manganese ions (Mn2+) is regulated by the manganese-sensing transcriptional factor ScaR which controls the expression of proteins involved in manganese homeostasis. To resolve the molecular mechanism through which the binding of Mn2+ ions increases the binding affinity of ScaR to DNA, a variety of computational (QM and MD) and experimental (ITC, DSC, EMSA, EPR, and CD) methods were applied. The computational results showed that Mn2+ binding induces a conformational change in ScaR that primarily affects the position of the DNA binding domains and, consequently, the DNA binding affinity of the protein. In addition, experimental results revealed a 1:4 binding stoichiometry between ScaR dimer and Mn2+ ions, while the computational results showed that the binding of Mn2+ ions in the primary binding sites is sufficient to induce the observed conformational change of ScaR.

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

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

Spatiotemporal monitoring of a periodontal multispecies biofilm model: demonstration of prebiotic treatment responses.

Biofilms are complex polymicrobial communities which are often associated with human infections such as the oral disease periodontitis. Studying these complex communities under controlled conditions requires in vitro biofilm model systems that mimic the natural environment as close as possible. This study established a multispecies periodontal model in the drip flow biofilm reactor in order to mimic the continuous flow of nutrients at the air-liquid interface in the oral cavity. The design is engineered to enable real-time characterization. A community of five bacteria, Streptococcus gordonii-GFPmut3*, Streptococcus oralis-GFPmut3*, Streptococcus sanguinis-pVMCherry, Fusobacterium nucleatum, and Porphyromonas gingivalis-SNAP26 is visualized using two distinct fluorescent proteins and the SNAP-tag. The biofilm in the reactor develops into a heterogeneous, spatially uniform, dense, and metabolically active biofilm with relative cell abundances similar to those in a healthy individual. Metabolic activity, structural features, and bacterial composition of the biofilm remain stable from 3 to 6 days. As a proof of concept for our periodontal model, the 3 days developed biofilm is exposed to a prebiotic treatment with L-arginine. Multifaceted effects of L-arginine on the oral biofilm were validated by this model setup. L-arginine showed to inhibit growth and incorporation of the pathogenic species and to reduce biofilm thickness and volume. Additionally, L-arginine is metabolized by Streptococcus gordonii-GFPmut3* and Streptococcus sanguinis-pVMCherry, producing high levels of ornithine and ammonium in the biofilm. In conclusion, our drip flow reactor setup is promising in studying spatiotemporal behavior of a multispecies periodontal community.ImportancePeriodontitis is a multifactorial chronic inflammatory disease in the oral cavity associated with the accumulation of microorganisms in a biofilm. Not the presence of the biofilm as such, but changes in the microbiota (i.e., dysbiosis) drive the development of periodontitis, resulting in the destruction of tooth-supporting tissues. In this respect, novel treatment approaches focus on maintaining the health-associated homeostasis of the resident oral microbiota. To get insight in dynamic biofilm responses, our research presents the establishment of a periodontal biofilm model including Streptococcus gordonii, Streptococcus oralis, Streptococcus sanguinis, Fusobacterium nucleatum, and Porphyromonas gingivalis. The added value of the model setup is the combination of simulating continuously changing natural mouth conditions with spatiotemporal biofilm profiling using non-destructive characterization tools. These applications are limited for periodontal biofilm research and would contribute in understanding treatment mechanisms, short- or long-term exposure effects, the adaptation potential of the biofilm and thus treatment strategies.

Salivary pellicle modulates biofilm formation on titanium surfaces.

OBJECTIVES: The present study aimed to evaluate the potential of the salivary pellicle (SP) formed on titanium (Ti) surfaces to modulate the formation of a biofilm composed of Streptococcus gordonii, Actinomyces naeslundii, Fusobacterium nucleatum, and Porphyromonas gingivalis. MATERIALS AND METHODS: Ti substrates were incubated for 2 h with a pool of saliva samples obtained from 10 systemically and periodontally healthy subjects. Enamel substrates were included as a biological reference. Scanning electron microscopy (SEM) and Raman spectroscopy analysis were used to analyze the formation of the salivary pellicle. After the SP formation, the surfaces were incubated for 12 h with a mix of Streptococcus gordonii, Actinomyces naeslundii, Fusobacterium nucleatum, and Porphyromonas gingivalis. The number of bacterial cells attached to each surface was determined by the XTT assay while bacterial viability was analyzed by fluorescence microscopy using the LIVE/DEAD® BacLightTM kit. RESULTS: The SEM and Raman spectroscopy analysis confirmed the presence of a salivary pellicle formed on the tested surfaces. Regarding the biofilm formation, the presence of the SP decreases the number of the bacterial cells detected in the test surfaces, compared with the uncover substrates. Even more, the SP-covered substrates showed similar bacterial counts in both Ti and enamel surfaces, meaning that the physicochemical differences of the substrates were less determinant than the presence of the SP. While on the SP-uncover substrates, differences in the bacterial adhesion patterns were directly related to the physicochemical nature of the substrates. CONCLUSIONS: The salivary pellicle was the main modulator in the development of the biofilm consisting of representative oral bacteria on the Ti substrates. CLINICAL RELEVANCE: The results of this study provide valuable information on the modulatory effect of the salivary pellicle on biofilm formation; such information allows us to understand better the events involved in the formation of oral biofilms on Ti dental implants.

Efficient clearance of periodontitis pathogens by S. gordonii membrane-coated H2O2 self-supplied nanocomposites in a "Jenga" style.

As a key pathogen of periodontitis, P. gingivalis requires support of the initial colonizing bacterium (S. gordonii preferably) to form symbiotic biofilms on gingival tissues with enhanced antibiotic resistance. Here, we report a new strategy to treat periodontitis biofilms with S. gordonii membrane-coated H2O2 self-supplied nanocomposites (ZnO2/Fe3O4@MV NPs) in a "Jenga" style. Integration of our special MV coatings enables selectively enhanced internalization of the cargos in S. gordonii, thus inducing severe damage to the foundational bacterial layer and collapse/clearance of symbiotic biofilms consequently. This strategy allows us to clear the symbiotic biofilms of S. gordonii and P. gingivalis with active hydroxyl radicals (˙OH) derived from ZnO2-Fe3O4@MV NPs in a H2O2 self-supplied, nanocatalyst-assisted manner. This "Jenga-style" treatment provides a cutting-edge proof of concept for the removal of otherwise robust symbiotic biofilms of periodontitis where the critical pathogens are difficult to target and have antibiotic resistance.

Inflammatory macrophages exploited by oral streptococcus increase IL-1B release via NLRP6 inflammasome.

Chronic inflammatory periodontal disease develops in part from the infiltration of a large number of classically activated inflammatory macrophages that release inflammatory cytokines important for disease progression, including inflammasome-dependent interleukin (IL)-1ß. Streptococcus gordonii is a normally commensal oral microorganism; while not causative, recent evidence indicates that commensal oral microbes are required for the full development of periodontal disease. We have recently reported that inflammatory macrophages counterintuitively allow for the increased survival of phagocytosed S. gordonii over nonactivated or alternatively activated macrophages. This survival is dependent on increased reactive oxygen species production within the phagosome of the inflammatory macrophages, and resistance by the bacterium and can result in S. gordonii damaging the phagolysosomes. Here, we show that activated macrophages infected with live S. gordonii release more IL-1ß than non-activated macrophages infected with either live or dead S. gordonii, and that the survival of oral Streptococci are more dependent on macrophage activation than other Gram positive microbes, both classical pathogens and commensals. We also find that S. gordonii-dependent inflammatory macrophage inflammasome activation requires the cytoplasmic NLRP6. Overall, our results suggest S. gordonii is capable of evading immune destruction, increasing inflammatory mediators, and increasing inflammatory macrophage response, and that this ability is increased under conditions of inflammation. This work reveals additional mechanisms by which normally commensal oral streptococci-macrophage interactions can change, resulting in increased release of mature IL-1ß, potentially contributing to an environment that perpetuates inflammation.