Oral Biofilm Development Research Articles

The dynamics of bacterial proliferation, viability, and extracellular polymeric substances in oral biofilm development

ObjectivesThis study investigated the relationship between bacterial growth, viability, and extracellular polymeric substances (EPS) formation in biofilms, particularly regarding resistance development. It also examined the impact of chemical factors on the EPS matrix and bacterial proliferation in oral biofilms. MethodsThree multi-species oral biofilms were incubated in anaerobic conditions. Three strains of Enterococcus faecalis were incubated in aerobic conditions. The incubation periods ranged from 0 h to 7 days for short-term biofilms, and from 3 to 90 days for long-term biofilms. Fluorescent labeling with carboxyfluorescein diacetate succinimidyl ester (CFSE) and flow cytometry were used to track EPS and bacterial growth. Confocal laser scanning microscopy (CLSM) assessed bacterial viability and EPS structure. Biofilms aged 7, 14, and 21 days were treated with 2 % chlorhexidine (CHX) and 1 % sodium hypochlorite (NaOCl) to evaluate their effects on EPS and bacterial proliferation. ResultsShort-term biofilms showed rapid bacterial proliferation and a gradual increase in EPS, maintaining stable viability. In the first two weeks, a significant rise in CFSE indicated growing maturity. From 14 to 90 days, EPS and CFSE levels stabilized. Following treatment, CHX significantly reduced bacterial proliferation, while NaOCl decreased EPS volume. ConclusionsBiofilm development involves a balance between bacterial proliferation and EPS production. The complexity of this process poses challenges in treating biofilm-associated infections, requiring strategies tailored to the biofilm's developmental stage. Clinical significanceFor effective root canal treatment, it is imperative to focus on reducing bacterial proliferation during the early stages of oral infections. In contrast, strategies aimed at minimizing EPS production could be more beneficial for long-term management of these conditions.

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.

Glycerol metabolism supports oral commensal interactions.

During oral biofilm development, interspecies interactions drive species distribution and biofilm architecture. To understand what molecular mechanisms determine these interactions, we used information gained from recent biogeographical investigations demonstrating an association of corynebacteria with streptococci. We previously reported that Streptococcus sanguinis and Corynebacterium durum have a close relationship through the production of membrane vesicle and fatty acids leading to S. sanguinis chain elongation and overall increased fitness supporting their commensal state. Here we present the molecular mechanisms of this interspecies interaction. Coculture experiments for transcriptomic analysis identified several differentially expressed genes in S. sanguinis. Due to its connection to fatty acid synthesis, we focused on the glycerol-operon. We further explored the differentially expressed type IV pili genes due to their connection to motility and biofilm adhesion. Gene inactivation of the glycerol kinase glpK had a profound impact on the ability of S. sanguinis to metabolize C. durum secreted glycerol and impaired chain elongation important for their interaction. Investigations on the effect of type IV pili revealed a reduction of S. sanguinis twitching motility in the presence of C. durum, which was caused by a decrease in type IV pili abundance on the surface of S. sanguinis as determined by SEM. In conclusion, we identified that the ability to metabolize C. durum produced glycerol is crucial for the interaction of C. durum and S. sanguinis. Reduced twitching motility could lead to a closer interaction of both species, supporting niche development in the oral cavity and potentially shaping symbiotic health-associated biofilm communities.

Management of Chronic Atrophic Candidiasis (Denture Stomatitis)-A Narrative Review.

One of the most common oral diseases affecting people wearing dentures is chronic atrophic candidiasis or denture stomatitis (DS). The aim of the paper is to provide an update on the pathogenesis, presentation, and management of DS in general dental practice settings. A comprehensive review of the literature published in the last ten years was undertaken using multiple databases, including PubMed via MEDLINE, EMBASE, and Scopus. The eligible articles were analyzed to identify evidence-based strategies for the management of DS. Despite its multifactorial nature, the leading cause of DS is the development of oral Candida albicans biofilm, which is facilitated by poor oral and denture hygiene, long-term denture wear, ill-fitting dentures, and the porosity of the acrylic resin in the dentures. DS affects between 17 and 75% of the population wearing dentures, with a slight predominance in elderly females. The mucosal denture surfaces and posterior tongue are the common sites of DS, and the affected areas exhibit erythema, the swelling of the palatal mucosa and edema. Oral and denture hygiene protocols, adjusting or re-fabricating poorly adapting dentures, smoking cessation, avoiding nocturnal denture wear, and the administration of topical or systemic antifungals are the mainstay of management. Alternate treatments such as microwave disinfection, phytomedicine, photodynamic therapy, and incorporation of antifungals and nanoparticles into denture resins are being evaluated for the treatment of DS but require further evidence before routine use in clinical practice. In summary, DS is the most common oral inflammatory lesion experienced by denture wearers. Most patients with DS can be managed in general dental practice settings. Effective management by general dental practitioners may be supported by a thorough understanding of the pathogenesis, the recognition of the clinical presentation, and an awareness of contemporary treatment strategies.

The quorum quenching enzyme Aii20J modifies in vitro periodontal biofilm formation.

Recent studies have revealed the presence of N-acyl-homoserine lactones (AHLs) quorum sensing (QS) signals in the oral environment. Yet, their role in oral biofilm development remains scarcely investigated. The use of quorum quenching (QQ) strategies targeting AHLs has been described as efficient for the control of pathogenic biofilms. Here, we evaluate the use of a highly active AHL-targeting QQ enzyme, Aii20J, to modulate oral biofilm formation in vitro. The effect of the QQ enzyme was studied in in vitro multispecies biofilms generated from oral samples taken from healthy donors and patients with periodontal disease. Subgingival samples were used as inocula, aiming to select members of the microbiota of the periodontal pocket niche in the in vitro biofilms. Biofilm formation abilities and microbial composition were studied upon treating the biofilms with the QQ enzyme Aii20J. The addition of the enzyme resulted in significant biofilm mass reductions in 30 - 60% of the subgingival-derived biofilms, although standard AHLs could not be found in the supernatants of the cultured biofilms. Changes in biofilm mass were not accompanied by significant alterations of bacterial relative abundance at the genus level. The investigation of 125 oral supragingival metagenomes and a synthetic subgingival metagenome revealed a surprisingly high abundance and broad distribution of homologous of the AHL synthase HdtS and several protein families of AHL receptors, as well as an enormous presence of QQ enzymes, pointing to the existence of an intricate signaling network in oral biofilms that has been so far unreported, and should be further investigated. Together, our findings support the use of Aii20J to modulate polymicrobial biofilm formation without changing the microbiome structure of the biofilm. Results in this study suggest that AHLs or AHL-like molecules affect oral biofilm formation, encouraging the application of QQ strategies for oral health improvement, and reinforcing the importance of personalized approaches to oral biofilm control.

Streptococcus mutans membrane vesicles inhibit the biofilm formation of Streptococcus gordonii and Streptococcus sanguinis

Streptococcus mutans, whose main virulence factor is glucosyltransferase (Gtf), has a substantial impact on the development of dental caries. S. mutans membrane vesicles (MVs), which are rich in Gtfs, have been shown to affect biofilm formation of other microorganisms. Streptococcus gordonii and Streptococcus sanguinis are initial colonizers of tooth surfaces, which provide attachment sites for subsequent microorganisms and are crucial in the development of oral biofilms. S. mutans and S. gordonii, as well as S. mutans and S. sanguinis, have a complex competitive and cooperative relationship, but it is unclear whether S. mutans MVs play a role in these interspecific interactions. Therefore, we co-cultured S. mutans MVs, having or lacking Gtfs, with S. gordonii and S. sanguinis. Our results showed that S. mutans MVs inhibited biofilm formation of S. gordonii and S. sanguinis but did not affect their planktonic growth; contrastingly, S. mutans ΔgtfBC mutant MVs had little effect on both their growth and biofilm formation. Additionally, there were fewer and more dispersed bacteria in the biofilms of the S. mutans MV-treated group than that in the control group. Furthermore, the expression levels of the biofilm-related virulence factors GtfG, GtfP, and SpxB in S. gordonii and S. sanguinis were significantly downregulated in response to S. mutans MVs. In conclusion, the results of our study showed that S. mutans MVs inhibited biofilm formation of S. gordonii and S. sanguinis, revealing an important role for MVs in interspecific interactions.

Adhesion Forces of Oral Bacteria to Titanium and the Correlation with Biophysical Cellular Characteristics.

Bacterial adhesion to dental implants is the onset for the development of pathological biofilms. Reliable characterization of this initial process is the basis towards the development of anti-biofilm strategies. In the present study, single-cell force spectroscopy (SCFS), by means of an atomic force microscope connected to a microfluidic pressure control system (FluidFM), was used to comparably measure adhesion forces of different oral bacteria within a similar experimental setup to the common implant material titanium. The bacteria selected belong to different ecological niches in oral biofilms: the commensal pioneers Streptococcus oralis and Actinomyces naeslundii; secondary colonizer Veillonella dispar; and the late colonizing pathogens Porphyromonas gingivalis as well as fimbriated and non-fimbriated Aggregatibacter actinomycetemcomitans. The results showed highest values for early colonizing pioneer species, strengthening the link between adhesion forces and bacteria’s role in oral biofilm development. Additionally, the correlation between biophysical cellular characteristics and SCFS results across species was analyzed. Here, distinct correlations between electrostatically driven maximum adhesion force, bacterial surface elasticity and surface charge as well as single-molecule attachment points, stretching capability and metabolic activity, could be identified. Therefore, this study provides a step towards the detailed understanding of oral bacteria initial adhesion and could support the development of infection-resistant implant materials in future.

Genetic Determinants of Hydrogen Sulfide Biosynthesis in Fusobacterium nucleatum Are Required for Bacterial Fitness, Antibiotic Sensitivity, and Virulence.

ABSTRACTThe Gram-negative anaerobe Fusobacterium nucleatum is a major producer of hydrogen sulfide (H2S), a volatile sulfur compound that causes halitosis. Here, we dissected the genetic determinants of H2S production and its role in bacterial fitness and virulence in this important member of the oral microbiome. F. nucleatum possesses four enzymes, CysK1, CysK2, Hly, and MegL, that presumably metabolize l-cysteine to H2S, and CysK1 was previously shown to account for most H2S production in vitro, based on correlations of enzymatic activities with gene expression at mid-log phase. Our molecular studies showed that cysK1 and megL were highly expressed at the late exponential growth phase, concomitant with high-level H2S production, while the expression levels of the other genes remained substantially lower during all growth phases. Although the genetic deletion of cysK1 without supplementation with a CysK1-catalyzed product, lanthionine, caused cell death, the conditional ΔcysK1 mutant and a mutant lacking hly were highly proficient in H2S production. In contrast, a mutant devoid of megL showed drastically reduced H2S production, and a cysK2 mutant showed only minor deficiencies. Intriguingly, the exposure of these mutants to various antibiotics revealed that only the megL mutant displayed altered susceptibility compared to the parental strain: partial sensitivity to nalidixic acid and resistance to kanamycin. Most significantly, the megL mutant was attenuated in virulence in a mouse model of preterm birth, with considerable defects in the spread to amniotic fluid and the colonization of the placenta and fetus. Evidently, the l-methionine γ-lyase MegL is a major H2S-producing enzyme in fusobacterial cells that significantly contributes to fusobacterial virulence and antibiotic susceptibility.

Natural products from traditional medicine as promising agents targeting at different stages of oral biofilm development.

Oral cavity is an ideal habitat for more than 1,000 species of microorganisms. The diverse oral microbes form biofilms over the hard and soft tissues in the oral cavity, affecting the oral ecological balance and the development of oral diseases, such as caries, apical periodontitis, and periodontitis. Currently, antibiotics are the primary agents against infectious diseases; however, the emergence of drug resistance and the disruption of oral microecology have challenged their applications. The discovery of new antibiotic-independent agents is a promising strategy against biofilm-induced infections. Natural products from traditional medicine have shown potential antibiofilm activities in the oral cavity with high safety, cost-effectiveness, and minimal adverse drug reactions. Aiming to highlight the importance and functions of natural products from traditional medicine against oral biofilms, here we summarized and discussed the antibiofilm effects of natural products targeting at different stages of the biofilm formation process, including adhesion, proliferation, maturation, and dispersion, and their effects on multi-species biofilms. The perspective of antibiofilm agents for oral infectious diseases to restore the balance of oral microecology is also discussed.

New structural insights into the PI-2 pilus from Streptococcus oralis, an early dental plaque colonizer.

Streptococcus oralis is a member of the mitis group of oral streptococci and an early colonizer in dental plaque biofilm, a major cause of periodontal disease, dental caries, and other oral infections. S. oralis promotes biofilm growth by coaggregating in a mutualistic partnership with other early colonizers such as Actinomyces oris. For this cell-to-cell interaction, A. oris is known to use its sortase-dependent pilus (type 2), but whether S. oralis uses its PI-2 (pilus islet 2) pilus is still to be determined. The PI-2 pilus is predicted to have a heterodimeric structure consisting of two different protein subunits with their own location and function: the tip PitA pilin for adhesion and the backbone PitB pilin for length. Thus far, structural information remains incomplete about the role of PI-2 pili in the mutualistic mechanism between S. oralis and A. oris. We now report on the crystal structure analysis of PitA and PitB using X-ray crystallography, small-angle X-ray scattering, and molecular docking studies. Accordingly, we propose a structural model for the PI-2 pilus, wherein repeating PitB subunits are arranged head-to-tail to form the long backbone structure with PitA on the outer tip. By performing both in vitro and in vivo experiments, we examined the role played by PitA in mediating the mutualistic interaction between S. oralis and A. oris, which appears to involve the coaggregation factor CafA. We also reveal that the galactose monosaccharide is a conceivable ligand for PitA and thereby might be used to inhibit coaggregation and control oral biofilm development. DATABASE: Structural coordinates for the PitA fragment, PitA fragment TbXO4 derivative, full-length PitA, and PitB from S. oralis have been deposited at the Protein Data Bank as 7VCR, 7W7I, 7VCN, 7W6B, and 7W7I, respectively. Streptococcus pneumoniae PitB coordinates have been deposited as 7F7Y.

A cell wall-anchored glycoprotein confers resistance to cation stress in Actinomyces oris biofilms.

Actinomycesoris plays an important role in oral biofilm development. Like many gram-positive bacteria, A. oris produces a sizable number of surface proteins that are anchored to bacterial peptidoglycan by a conserved transpeptidase named the housekeeping sortase SrtA; however, the biological role of many A. oris surface proteins in biofilm formation is largely unknown. Here, we report that the glycoprotein GspA-a genetic suppressor of srtA deletion lethality-not only promotes biofilm formation but also maintains cell membrane integrity under cation stress. In comparison to wild-type cells, under elevated concentrations of mono- and divalent cations the formation of mono- and multi-species biofilms by mutant cells devoid of gspA was significantly diminished, although planktonic growth of both cell types in the presence of cations was indistinguishable. Because gspA overexpression is lethal to cells lacking gspA and srtA, we performed a genetic screen to identify GspA determinants involving cell viability. DNA sequencing and biochemical characterizations of viable clones revealed that mutations of two critical cysteine residues and a serine residue severely affected GspA glycosylation and biofilm formation. Furthermore, mutant cells lacking gspA were markedly sensitive to sodium dodecyl sulfate, a detergent that solubilizes the cytoplasmic membranes, suggesting the cell envelope of the gspA mutant was altered. Consistent with this observation, the gspA mutant exhibited increased membrane permeability, independent of GspA glycosylation, compared to the wild-type strain. Altogether, the results support the notion that the cell wall-anchored glycoprotein GspA provides a defense mechanism against cation stress in biofilm development promoted by A. oris.

Cleaning and Disinfection Protocols for Clear Orthodontic Aligners: A Systematic Review.

(1) Background: Clear orthodontic aligners support the development of oral biofilms, which could lead to interferences with the oral microbiota already existing and the deterioration of oral health, with the development of dental caries, periodontal disease and even systemic infections. Therefore, preventive oral health care requires a cleaning and disinfection procedure for aligners. (2) Methods: A systematic review of the literature was conducted across four databases following the PRISMA guidelines up to May 2021, combining an electronic and a manual search. Prospective studies, including randomized controlled trials (RCTs), crossover studies (COSs) and controlled clinical trials (CCTs), published in the English language without time restrictions, evaluating the efficacy of cleaning and disinfection protocols for clear orthodontic aligners by comparing them with a placebo or a negative control, were included. The article selection, data extraction and risk of bias assessment were performed by two independent blinded review authors. In case of disagreement, a third author was solicited throughout the selection process. (3) Results: Among the 221 articles screened in the search process, 4 studies were included in the review, all designed as crossover studies (single arm without randomization with the same sequence of different cleaning and disinfection protocols for each participant). Different cleaning and disinfection methods were studied such as mechanical methods (brushing with toothpaste or vibration), chemical methods/pharmaceutical products (chlorhexidine antibacterial substance, anionic or cationic detergents or effervescent tablets) or combinations of both. (4) Conclusion: Although the determination of the most remarkable method of cleaning and disinfection was impossible because no direct comparison was conducted between all these methods, a multi-step protocol, including the combination of a mechanical and a chemical method, seems to be the most effective approach. Further research is needed to define the most preventive oral health care protocol. Registration: PROSPERO CRD 42021278498.

Unsweetened and sucrose-sweetened black and green tea modifies the architecture of in vitro oral biofilms

ObjectiveIt is unclear whether tea infusions with or without sucrose supplementation alter oral biofilm development, so we evaluated the effect of unsweetened and sucrose-sweetened black and green tea infusions on in vitro saliva-derived biofilms. DesignBiofilms were developed from human saliva for 20 h in cell-free 25% human saliva within static glass-bottom microplates. During biofilm development, biofilms were treated with either (i) unsweetened black tea, (ii) unsweetened green tea, (iii) 10% sucrose-sweetened black tea, (iv) 10% sucrose-sweetened green tea (v) deionized water (negative control), or (vi) 10% sucrose (positive control). Biofilms were incubated at 37 °C in 5% CO2. After 20 h of development, biofilms were imaged using a CLSM, and biofilm architecture and viability were evaluated. ResultsAll the tea infusions reduced biofilm biomass and altered some other biofilm architectural outcomes (e.g., biofilm surface area) compared to the control groups. Statistically significant differences in biofilm biomass, number of objects, surface area, and convex-hull porosity were observed between biofilms treated with green and black tea. The addition of sugar to tea did not significantly modify the ability of tea to alter biofilm architecture. Only the treatment of biofilms with unsweetened black tea significantly reduced bacterial viability. ConclusionsWhile both teas reduced biofilm biomass and altered biofilm architecture, black tea had an enhanced effect that may relate to this tea's observed antimicrobial activity. The addition of sucrose to tea infusions did not appear to reduce the impact of either tea in modifying oral biofilm architecture

Veillonellae: Beyond Bridging Species in Oral Biofilm Ecology.

The genus Veillonella comprises 16 characterized species, among which eight are commonly found in the human oral cavity. The high abundance of Veillonella species in the microbiome of both supra- and sub-gingival biofilms, and their interdependent relationship with a multitude of other bacterial species, suggest veillonellae to play an important role in oral biofilm ecology. Development of oral biofilms relies on an incremental coaggregation process between early, bridging and later bacterial colonizers, ultimately forming multispecies communities. As early colonizer and bridging species, veillonellae are critical in guiding the development of multispecies communities in the human oral microenvironment. Their ability to establish mutualistic relationships with other members of the oral microbiome has emerged as a crucial factor that may contribute to health equilibrium. Here, we review the general characteristics, taxonomy, physiology, genomic and genetics of veillonellae, as well as their bridging role in the development of oral biofilms. We further discuss the role of Veillonella spp. as potential “accessory pathogens” in the human oral cavity, capable of supporting colonization by other, more pathogenic species. The relationship between Veillonella spp. and dental caries, periodontitis, and peri-implantitis is also recapitulated in this review. We finally highlight areas of future research required to better understand the intergeneric signaling employed by veillonellae during their bridging activities and interspecies mutualism. With the recent discoveries of large species and strain-specific variation within the genus in biological and virulence characteristics, the study of Veillonella as an example of highly adaptive microorganisms that indirectly participates in dysbiosis holds great promise for broadening our understanding of polymicrobial disease pathogenesis.

In vitro model systems for exploring oral biofilms: From single-species populations to complex multi-species communities.

Numerous in vitro biofilm model systems are available to study oral biofilms. Over the past several decades, increased understanding of oral biology and advances in technology have facilitated more accurate simulation of intraoral conditions and have allowed for the increased generalizability of in vitro oral biofilm studies. The integration of contemporary systems with confocal microscopy and 16S rRNA community profiling has enhanced the capabilities of in vitro biofilm model systems to quantify biofilm architecture and analyse microbial community composition. In this review, we describe several model systems relevant to modern in vitro oral biofilm studies: the constant depth film fermenter, Sorbarod perfusion system, drip-flow reactor, modified Robbins device, flowcells and microfluidic systems. We highlight how combining these systems with confocal microscopy and community composition analysis tools aids exploration of oral biofilm development under different conditions and in response to antimicrobial/anti-biofilm agents. The review closes with a discussion of future directions for the field of in vitro oral biofilm imaging and analysis.

Plant-based oral care product exhibits antibacterial effects on different stages of oral multispecies biofilm development in vitro

BackgroundExcessive biofilm formation on surfaces in the oral cavity is amongst the main reasons for severe infection development like periodontitis and peri-implantitis. Mechanical biofilm removal as well as the use of adjuvant antiseptics supports the prevention of pathogenic biofilm formation. Recently, the antibacterial effect of the oral care product REPHA-OS®, based on medicinal plant extracts and essential oils, has been demonstrated on oral pathogens grown on agar plates. In the present study, the effectiveness of the product on medical relevant oral biofilm development should be demonstrated for the first time.MethodsAn established in vitro oral multispecies biofilm, composed of Streptococcus oralis, Actinomyces naeslundii, Veillonella dispar and Porphyromonas gingivalis, was used to analyze the antibacterial effect of different REPHA-OS® concentrations on planktonic bacteria, biofilm formation and mature biofilms. It was quantified using metabolic activity assays and live/dead fluorescence staining combined with three-dimensional confocal laser-scanning microscopy. Additionally, effects on species distribution inside the biofilm were assessed by means of quantitative real-time PCR.ResultsREPHA-OS® showed statistically significant antimicrobial effects on all stages of biofilm development: a minimal inhibitory concentration of 5% could be detected for both, for planktonic bacteria and for biofilm formation. Interestingly, only a slightly higher concentration of 10% was necessary to completely kill all bacteria in mature biofilms also. In contrast, an influence on the biofilm matrix or the species distribution could not be observed. The effect could be attributed to the herbal ingredients, not to the contained ethanol.ConclusionThe strong antibacterial effect of REPHA-OS® on different stages of oral biofilm development strengthens its application as an alternative adjuvant in oral care therapies.

EpsR Negatively Regulates Streptococcus mutans Exopolysaccharide Synthesis

Streptococcus mutans is considered the primary etiological agent of human dental caries. Glucosyltransferases (Gtfs) from S. mutans play important roles in the formation of biofilm matrix and the development of cariogenic oral biofilm. Therefore, Gtfs are considered an important target to prevent the development of dental caries. However, the role of transcription factors in regulating gtf expression is not yet clear. Here, we identify a MarR (multiple antibiotic resistance regulator) family transcription factor named EpsR (exopolysaccharide synthesis regulator), which negatively regulates gtfB expression and exopolysaccharide (EPS) production in S. mutans. The epsR in-frame deletion strain grew slowly, aggregated more easily in the presence of dextran, and displayed different colony morphology and biofilm structure. Notably, epsR deletion resulted in altered 3-dimensional biofilm architecture, increased water-insoluble EPS production, and upregulated GtfB protein content and activity. In addition, global gene expression profiling revealed differences in the expression levels of 69 genes in which gtfB was markedly upregulated. The conserved DNA motif for EpsR binding was determined by electrophoretic mobility shift assay and DNase I footprinting assays. Moreover, analysis of β-galactosidase activity suggested that EpsR acted as a repressor and inhibited gtfB expression. Taken together, our findings indicate that EpsR is an important transcription factor that regulates gtfB expression and EPS production in S. mutans. These results add new aspects to the complexity of regulating the expression of genes involved in the cariogenicity of S. mutans, which might lead to novel strategies to prevent the formation of cariogenic biofilm that may favor diseases.

Tea extracts modulate oral biofilm development by altering bacterial hydrophobicity and aggregation

ObjectivesThis study aims to investigate the effects of tea extracts on biofilm formation by oral streptococci and the potential mechanisms behind the effects. DesignWe examined the effects of five types of tea extracts (green, oolong, black, pu-erh and chrysanthemum tea) on cell surface hydrophobicity and auto-aggregation of three different streptococcal species (Streptococcus mutans, Streptococcus salivarius and Streptococcus mitis) and evaluated their biofilm formation on four disparate hard surfaces (glass, stainless steel, hydroxyapatite and titanium). The correlation between biofilm formation and the cellular properties were investigated in order to study the mechanisms by which the tea extracts affect biofilm formation. ResultsResults show that the tea extracts reduced cell surface hydrophobicity (by up to 57.9 %) and, in some cases, altered cellular auto-aggregation (by up to 12 %) and biofilm formation (by up to 2.61 log CFU cm−2). Specifically, oolong tea extract was found to enhance biofilm formation by increasing cellular auto-aggregation and pu-erh tea extract retarded biofilm formation by increasing auto-aggregation. Biofilm formation correlated well to cell surface hydrophobicity and auto-aggregation in combination, but not to either one alone as determined by multiple linear regression analysis. ConclusionsTea extracts have the ability to modulate streptococcal biofilm formation by altering cell surface hydrophobicity and cellular aggregation.

Isolation and Characterization of Nitrate-Reducing Bacteria as Potential Probiotics for Oral and Systemic Health.

Recent evidence indicates that the reduction of salivary nitrate by oral bacteria can contribute to prevent oral diseases, as well as increase systemic nitric oxide levels that can improve conditions such as hypertension and diabetes. The objective of the current manuscript was to isolate nitrate-reducing bacteria from the oral cavity of healthy donors and test their in vitro probiotic potential to increase the nitrate-reduction capacity (NRC) of oral communities. Sixty-two isolates were obtained from five different donors of which 53 were confirmed to be nitrate-reducers. Ten isolates were selected based on high NRC as well as high growth rates and low acidogenicity, all being Rothia species. The genomes of these ten isolates confirmed the presence of nitrate- and nitrite reductase genes, as well as lactate utilization genes, and the absence of antimicrobial resistance, mobile genetic elements and virulence genes. The pH at which most nitrate was reduced differed between strains. However, acidic pH 6 always stimulated the reduction of nitrite compared to neutral pH 7 or slightly alkaline pH 7.5 (p < 0.01). We tested the effect of six out of 10 isolates on in vitro oral biofilm development in the presence or absence of 6.5 mM nitrate. The integration of the isolates into in vitro communities was confirmed by Illumina sequencing. The NRC of the bacterial communities increased when adding the isolates compared to controls without isolates (p < 0.05). When adding nitrate (prebiotic treatment) or isolates in combination with nitrate (symbiotic treatment), a smaller decrease in pH derived from sugar metabolism was observed (p < 0.05), which for some symbiotic combinations appeared to be due to lactate consumption. Interestingly, there was a strong correlation between the NRC of oral communities and ammonia production even in the absence of nitrate (R = 0.814, p < 0.01), which indicates that bacteria involved in these processes are related. As observed in our study, individuals differ in their NRC. Thus, some may have direct benefits from nitrate as a prebiotic as their microbiota naturally reduces significant amounts, while others may benefit more from a symbiotic combination (nitrate + nitrate-reducing probiotic). Future clinical studies should test the effects of these treatments on oral and systemic health.

Acyl homoserine lactone-mediated quorum sensing in the oral cavity: a paradigm revisited

Acyl homoserine lactones (AHLs), the quorum sensing (QS) signals produced by Gram-negative bacteria, are currently considered to play a minor role in the development of oral biofilm since their production by oral pathogens has not been ascertained thus far. However, we report the presence of AHLs in different oral samples and their production by the oral pathogen Porphyromonas gingivalis. The importance of AHLs is further supported by a very high prevalence of AHL-degradation capability, up to 60%, among bacteria isolated from dental plaque and saliva samples. Furthermore, the wide-spectrum AHL-lactonase Aii20J significantly inhibited oral biofilm formation in different in vitro biofilm models and caused important changes in bacterial composition. Besides, the inhibitory effect of Aii20J on a mixed biofilm of 6 oral pathogens was verified using confocal microscopy. Much more research is needed in order to be able to associate specific AHLs with oral pathologies and to individuate the key actors in AHL-mediated QS processes in dental plaque formation. However, these results indicate a higher relevance of the AHLs in the oral cavity than generally accepted thus far and suggest the potential use of inhibitory strategies against these signals for the prevention and treatment of oral diseases.