Biofilm formation of Streptococcus mutans, Streptococcus sanguinis, Staphylococcus epidermidis, Staphylococcus aureus, Lactobacillus casei, and Candida Albicans on 5 thermoform and 3D printed orthodontic clear aligner and retainer materials at 3 time points: an in vitro study.

INTRODUCTION: Orthodontic clear aligners and retainers have numerous advantages that is making them ever increasingly popular. However, they might, similar to any other oral appliance, contribute to biofilm formation and finally dental caries or white spot lesions or gingival inflammations. The literature on biofilm formation on orthodontic clear appliances is very scarce and limited to a few microorganisms and materials. Therefore, this experimental study evaluated the biofilm formation on 5 thermoformed and 3D printed CAD/CAM orthodontic retainers in 3 intervals. METHODS: In this in vitro study, 345 specimens (270 test discs and 45 negative controls) were created from fabricated retainers. Retainers included a 3D printed CAD/CAM material (Detax) and four thermoformed retainers [Erkodent (polyethylene terephthalate glycol [PETG]); EasyVac (polyethylene); DB (polyester based on terephthalic acid); and Clear Tech]. They were all 1 mm thick, and all completely fabricated, i.e., heated or printed. The discs were placed in 96-well plates. Microorganisms were cultured on 270 discs for 24 h (90 discs), 72 h (90 other discs), and 5 days or 120 h (90 other discs). Biofilm formation of the strains and negative controls was measured using the microtiter plate assay by ELISA reading. The microbes' ability to produce biofilm was categorized based on the comparison of average optical density (OD) of tests versus a cut-off point OD (ODc) calculated as the average of the OD of corresponding negative controls plus 3× its standard deviation: non-biofilm former [OD ≤ ODc], weak biofilm former [ODc < OD ≤ (2 × ODc)], moderate biofilm former [(2 × ODc) < OD ≤ (4 × ODc)], and strong biofilm former [(4 × ODc) < OD]. These were also converted to ranked scores between zero (no biofilm) and 3. The difference between ODs with control ODs were calculated. These were analyzed using 3-way ANOVA, 2-way ANOVA, and Tukey tests (α = 0.05, α = 0.008). RESULTS: The 3-way ANOVA showed that the overall difference among the ΔODs of 5 retainers (all microorganisms and all intervals combined, n = 270) was not significant (F = 1.860, P = 0.119). Nevertheless, the difference among 3 intervals (F = 31.607, P = 0.0000) and the difference among the 6 microorganisms (F = 24.044, P = 0.0000) were significant. According to the Tukey test, the differences between the 1st interval with either of the other two intervals was significant (both P values = 0.000). There were significant differences between Candida albicans with all other organisms (all 5 P values = 0.0000). All other pairwise comparisons were insignificant (all 10 P values ≥ 0.1). After taking the averages of the 3 intervals, the order of the biofilm generation for different materials were as follows: Detax (average score: 1.56), Easyvac (1.67), Erkodent (1.78), Clear Tech (1.83), BD (2.28). CONCLUSIONS: As far as these 6 microorganisms are of concern, there might not be a significant overall difference among the clear retainer materials tested in this study. A significant overall increase was observed between the first and third days, which later did not significantly increase more until day 5. The Candida albicans biofilm was more intense than the tested 5 bacteria, which themselves showed rather similar growth patterns to each other.

Caries-Prone Primary Teeth: A Hidden Reason and Prophylactic Treatment in the Viewpoint of Materials Science.

Dental caries, the most prevalent chronic disease across all age groups, has a high prevalence, particularly among children. However, there is no specific and effective treatment for the prevention of caries in primary teeth (Pr.T.), which stems from a lack of knowledge regarding the basic nature of the tooth surface. Herein, we observed that the adhesion energies of the caries-related bacteria Streptococcus mutans and Streptococcus sanguinis to Pr.T were approximately 10 and 5.5 times higher than those to permanent teeth (Pe.T). A lower degree of mineralization and more hydrophilic characteristics of the Pr.T enamel account for this discrepancy. Accordingly, we proposed that the on-target modification of both hydroxyapatite and organic components on Pr.T by dual modification would render a sufficient hydration layer. This resulted in an approximately 11-time decrease in bacterial adhesion energy after treatment. In contrast, a single hydroxyapatite modification on Pe.T and young permanent teeth (Y.Pe.T) was sufficient to achieve a similar effect. Theoretical simulation further verified the rationality of the approach. Our findings may help understand the reason for Pr.T being caries-prone and provide references for treatment using resin restorations. This strategy offers valuable insights into daily oral hygiene and dental prophylactic treatment in children.

Glycerol metabolism contributes to competition by oral streptococci through production of hydrogen peroxide.

As a biological byproduct from both humans and microbes, glycerol's contribution to microbial homeostasis in the oral cavity remains understudied. In this study, we examined glycerol metabolism by Streptococcus sanguinis, a commensal associated with oral health. Genetic mutants of glucose-PTS enzyme II (manL), glycerol metabolism (glp and dha pathways), and transcriptional regulators were characterized with regard to glycerol catabolism, growth, production of hydrogen peroxide (H2O2), transcription, and competition with Streptococcus mutans. Biochemical assays identified the glp pathway as a novel source for H2O2 production by S. sanguinis that is independent of pyruvate oxidase (SpxB). Genetic analysis indicated that the glp pathway requires glycerol and a transcriptional regulator, GlpR, for expression and is negatively regulated by PTS, but not the catabolite control protein, CcpA. Conversely, deletion of either manL or ccpA increased the expression of spxB and a second, H2O2-non-producing glycerol metabolic pathway (dha), indicative of a mode of regulation consistent with conventional carbon catabolite repression (CCR). In a plate-based antagonism assay and competition assays performed with planktonic and biofilm-grown cells, glycerol greatly benefited the competitive fitness of S. sanguinis against S. mutans. The glp pathway appears to be conserved in several commensal streptococci and actively expressed in caries-free plaque samples. Our study suggests that glycerol metabolism plays a more significant role in the ecology of the oral cavity than previously understood. Commensal streptococci, though not able to use glycerol as a sole carbohydrate source for growth, benefit from the catabolism of glycerol through production of both ATP and H2O2. IMPORTANCE: Glycerol is an abundant carbohydrate in the oral cavity. However, little is understood regarding the metabolism of glycerol by commensal streptococci, some of the most abundant oral bacteria. This was in part because most streptococci cannot grow on glycerol as the sole carbon source. In this study, we show that Streptococcus sanguinis, a commensal associated with dental health, can degrade glycerol for persistence and competition through two pathways, one of which generates hydrogen peroxide at levels capable of inhibiting Streptococcus mutans. Preliminary studies suggest that several additional commensal streptococci are also able to catabolize glycerol, and glycerol-related genes are actively expressed in human dental plaque samples. Our findings reveal the potential of glycerol to significantly impact microbial homeostasis, which warrants further exploration.

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).

Dpr-mediated H2O2 resistance contributes to streptococcus survival in a cystic fibrosis airway model system.

The cystic fibrosis (CF) lung environment is conducive to the colonization of bacteria as polymicrobial biofilms, which are associated with poor clinical outcomes for persons with CF (pwCF). Streptococcus spp. are highly prevalent in the CF airway, but its role in the CF lung microbiome is poorly understood. Some studies have shown Streptococcus spp. to be associated with better clinical outcomes for pwCF, while others show that high abundance of Streptococcus spp. is correlated with exacerbations. Our lab previously reported a polymicrobial culture system consisting of four CF-relevant pathogens that can be used to study microbial behavior in a more clinically relevant setting. Here, we use this model system to identify genetic pathways that are important for Streptococcus sanguinis survival in the context of the polymicrobial community. We identified genes related to reactive oxygen species as differentially expressed in S. sanguinis monoculture versus growth of this microbe in the mixed community. Genetic studies identified Dpr as important for S. sanguinis survival in the community. We show that Dpr, a DNA-binding ferritin-like protein, and PerR, a peroxide-responsive transcriptional regulator of Dpr, are important for protecting S. sanguinis from phenazine-mediated toxicity in co-culture with Pseudomonas aeruginosa and when exposed to hydrogen peroxide, both of which mimic the CF lung environment. Characterizing such interactions in a clinically relevant model system contributes to our understanding of microbial behavior in the context of polymicrobial biofilm infections. IMPORTANCE: Streptococcus spp. are recognized as a highly prevalent pathogen in cystic fibrosis (CF) airway infections. However, the role of this microbe in clinical outcomes for persons with CF is poorly understood. Here, we leverage a polymicrobial community system previously developed by our group to model CF airway infections as a tool to investigate a Pseudomonas-Streptococcus interaction involving reactive oxygen species (ROS). We show that protection against ROS is required for Streptococcus sanguinis survival in a clinically relevant polymicrobial system. Using this model system to study interspecies interactions contributes to our broader understanding of the complex role of Streptococcus spp. in the CF lung.

Functional Analysis of the Major Pilin Proteins of Type IV Pili in Streptococcus sanguinis CGMH010.

The pil gene cluster for Type IV pilus (Tfp) biosynthesis is commonly present and highly conserved in Streptococcus sanguinis. Nevertheless, Tfp-mediated twitching motility is less common among strains, and the factors determining twitching activity are not fully understood. Here, we analyzed the functions of three major pilin proteins (PilA1, PilA2, and PilA3) in the assembly and activity of Tfp in motile S. sanguinis CGMH010. Using various recombinant pilA deletion strains, we found that Tfp composed of different PilA proteins varied morphologically and functionally. Among the three PilA proteins, PilA1 was most critical in the assembly of twitching-active Tfp, and recombinant strains expressing motility generated more structured biofilms under constant shearing forces compared to the non-motile recombinant strains. Although PilA1 and PilA3 shared 94% identity, PilA3 could not compensate for the loss of PilA1, suggesting that the nature of PilA proteins plays an essential role in twitching activity. The single deletion of individual pilA genes had little effect on the invasion of host endothelia by S. sanguinis CGMH010. In contrast, the deletion of all three pilA genes or pilT, encoding the retraction ATPase, abolished Tfp-mediated invasion. Tfp- and PilT-dependent invasion were also detected in the non-motile S. sanguinis SK36, and thus, the retraction of Tfp, but not active twitching, was found to be essential for invasion.

Growing a Cystic Fibrosis-Relevant Polymicrobial Biofilm to Probe Community Phenotypes.

Most in vitro models lack the capacity to fully probe bacterial phenotypes emerging from the complex interactions observed in real-life environments. This is particularly true in the context of hard-to-treat, chronic, and polymicrobial biofilm-based infections detected in the airways of individuals living with cystic fibrosis (CF), a multiorgan genetic disease. While multiple microbiome studies have defined the microbial compositions detected in the airway of people with CF (pwCF), no in vitro models thus far have fully integrated critical CF-relevant lung features. Therefore, a significant knowledge gap exists in the capacity to investigate the mechanisms driving the pathogenesis of mixed species CF lung infections. Here, we describe a recently developed four-species microbial community model, including Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica grown in CF-like conditions. Through the utilization of this system, clinically relevant phenotypes such as antimicrobial recalcitrance of several pathogens were observed and explored at the molecular level. The usefulness of this in vitro model resides in its standardized workflow that can facilitate the study of interspecies interactions in the context of chronic CF lung infections.

Biological Activities of Virgin Coconut and Virgin Olive Oil Mixture against Oral Primary Colonizers: An In Vitro Study.

AIM AND BACKGROUND: This study aimed to explore the potential synergistic interaction of virgin coconut oil (VCO) and virgin olive oil (VOO) mixture against Streptococcus sanguinis, Streptococcus mutans, and Lactobacillus casei in a single and mixture species through the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), antiadherence, and antibiofilm activities. MATERIALS AND METHODS: The broth microdilution technique was used to individually determine the MIC of both oils and an oil mixture (in the ratio of 1:1) in a 96-well microtiter plate. As for the MBC, the subcultured method was used. The fractional inhibitory concentration index (ΣFIC) was determined to identify the interaction types between both oils. The oil mixture at its MIC was then tested on its antibiofilm and antiadherence effect. RESULTS: The MIC of the oil mixture against the tested microbiota was 50-100%. The oil mixture was bactericidal at 100% concentration for all the mentioned microbes except S. mutans. The ΣFIC value was 2 to 4, indicating that the VCO and VOO acted additively against the microbiota. Meanwhile, the oil mixture at MIC (50% for S. sanguinis and L. casei; 100% for S. mutans and mixture species) exhibited antiadherence and antibiofilm activity toward the microbiota in mixture species. CONCLUSION: The oil mixture possesses antibacterial, antibiofilm, and antiadherence properties toward the tested microbiota, mainly at 50-100% concentration of oil mixture. There was no synergistic interaction found between VCO and VOO. CLINICAL SIGNIFICANCE: Children and individuals with special care may benefit from using the oil mixture, primarily to regulate the biofilm formation and colonization of the bacteria. Furthermore, the oil mixture is natural and nontoxic compared to chemical-based oral healthcare products. How to cite this article: Ng YM, Sockalingam SNMP, Shafiei Z, et al. Biological Activities of Virgin Coconut and Virgin Olive Oil Mixture against Oral Primary Colonizers: An In Vitro Study. J Contemp Dent Pract 2024;25(3):260-266.

Effect of hydroxyapatite nanoparticles coating of titanium surface on biofilm adhesion: An in vitro study.

AIM: To evaluate the adhesion of mono and duospecies biofilm on a commercially available dental implant surface coated with hydroxyapatite nanoparticles (nanoHA). MATERIAL AND METHODS: Titanium discs were divided into two groups: double acid-etched (AE) and AE coated with nanoHA (NanoHA). Surface characteristics evaluated were morphology, topography, and wettability. Mono and duospecies biofilms of Streptococcus sanguinis (S. sanguinis) and Candida albicans (C. albicans) were formed. Discs were exposed to fetal bovine serum (FBS) to form the pellicle. Biofilm was growth in RPMI1640 medium with 10% FBS and 10% BHI medium for 6 h. Microbial viability was evaluated using colony-forming unit and metabolic activity by a colorimetric assay of the tetrazolium salt XTT. Biofilm architecture and organization were evaluated by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). RESULTS: AE surface had more pores, while NanoHA had even nanoHA crystals distribution. Roughness was similar (AE: 0.59 ± 0.07 µm, NanoHA: 0.69 ± 0.18 µm), but wettability was different (AE: Θw= 81.79 ± 8.55°, NanoHA: Θw= 53.26 ± 11.86°; P = 0.01). NanoHA had lower S. sanguinis viability in monospecies biofilm (P = 0.007). Metabolic activity was similar among all biofilms. In SEM both surfaces on C. albicans biofilm show a similar distribution of hyphae in mono and duospecies biofilms. AE surface has more S. sanguinis than the NanoHA surface in the duospecies biofilm. CLSM showed a large proportion of live cells in all groups. CONCLUSIONS: The nanoHA surface reduced the adhesion of S. sanguinis biofilm but did not alter the adhesion of C. albicans or the biofilm formed by both species.

Mechanical and microbiological properties of orthodontic resin modified with nanostructured silver vanadate decorated with silver nanoparticles (ßAgVO3).

OBJECTIVE: To investigate the impact of incorporating the antimicrobial nanomaterial ß-AgVO3 into orthodontic resin, focusing on degree of conversion, surface characteristics, microhardness, adhesion properties, and antimicrobial activity. METHODS: The 3 M Transbond XT resin underwent modification, resulting in three groups (Control, 2.5% addition, 5% addition) with 20 specimens each. Fourier transform infrared spectroscopy assessed monomer conversion. Laser confocal microscopy examined surface roughness, and microhardness was evaluated using Knoop protocols. Shear strength was measured before and after artificial aging on 36 premolar teeth. Microbiological analysis against S. mutans and S. sanguinis was conducted using the agar diffusion method. RESULTS: Degree of conversion remained unaffected by time (P = 0.797), concentration (P = 0.438), or their interaction (P = 0.187). The 5% group exhibited the lowest surface roughness, differing significantly from the control group (P = 0.045). Microhardness showed no significant differences between concentrations (P = 0.740). Shear strength was highest in the control group (P < 0.001). No significant differences were observed in the samples with or without thermocycling (P = 0.759). Microbial analysis revealed concentration-dependent variations, with the 5% group exhibiting the largest inhibition halo (P < 0.001). CONCLUSIONS: Incorporating ß-AgVO3 at 2.5% and 5% concentrations led to significant differences in surface roughness, adhesion, and antimicrobial activity. Overall, resin modification positively impacted degree of conversion, surface characteristics, microhardness, and antimicrobial activity. Further research is warranted to determine clinically optimal concentrations that maximize antimicrobial benefits while minimizing adverse effects on adhesion properties. CLINICAL SIGNIFICANCE: Incorporating ß-AgVO3 into orthodontic resin could improve patient quality of life by prolonging intervention durability and reducing the impact of cariogenic microorganisms. The study's findings also hold promise for the industry, paving the way for the development of new materials with antimicrobial properties for potential applications in the health sector.

The effect of bovine trypsin on dental biofilm dispersion: an in vitro study.

To investigate the degradation effect of bovine trypsin on multispecies biofilm of caries-related bacteria and provide an experimental foundation for the prevention of dental caries. Standard strains of S. mutans, S. sanguis, S. gordonii, and L. acidophilus were co-cultured to form 24 h, 48 h, and 72 h biofilms. The experimental groups were treated with bovine trypsin for 30 s, 1 min, and 3 min. Morphological observation and quantitative analysis of extracellular polymeric substances (EPS), live bacteria, and dead bacteria were conducted using the confocal laser scanning microscope (CLSM). The morphological changes of EPS and bacteria were also observed using a scanning electron microscope (SEM). When biofilm was treated for 1 min, the minimal inhibitory concentration (MIC) of bovine trypsin to reduce EPS was 0.5 mg/mL in 24 h and 48 h biofilms, and the MIC of bovine trypsin was 2.5 mg/mL in 72 h biofilms (P < 0.05). When biofilm was treated for 3 min, the MIC of bovine trypsin to reduce EPS was 0.25 mg/mL in 24 h and 48 h biofilms, the MIC of bovine trypsin was 1 mg/mL in 72 h biofilm (P < 0.05). The ratio of live-to-dead bacteria in the treatment group was significantly lower than blank group in 24 h, 48 h, and 72 h multispecies biofilms (P < 0.05). Bovine trypsin can destroy multispecies biofilm structure, disperse biofilm and bacteria flora, and reduce the EPS and bacterial biomass in vitro, which are positively correlated with the application time and concentration.

Noncanonical Streptococcus sanguinis ComCDE circuitry integrates environmental cues in transformation outcome decision.

Natural competence is the principal driver of streptococcal evolution. While acquisition of new traits could facilitate rapid fitness improvement for bacteria, entry into the competent state is a highly orchestrated event, involving an interplay between various pathways. We present a new type of competence-predation coordination mechanism in Streptococcus sanguinis. Unlike other streptococci that mediate competence through the ComABCDE regulon, several key components are missing in the S. sanguinis ComCDE circuitry. We assembled two synthetic biology devices linking competence-stimulating peptide (CSP) cleavage and export with a quantifiable readout to unravel the unique features of the S. sanguinis circuitry. Our results revealed the ComC precursor cleavage pattern and the two host ABC transporters implicated in the export of the S. sanguinis CSP. Moreover, we discovered a ComCDE-dependent bacteriocin locus. Overall, this study presents a mechanism for commensal streptococci to maximize transformation outcome in a fluid environment through extensive circuitry rewiring.

Systematic functional analysis of the Com pilus in Streptococcus sanguinis: a minimalistic type 4 filament dedicated to DNA uptake in monoderm bacteria.

IMPORTANCE: Type 4 filaments (T4F) are nanomachines ubiquitous in prokaryotes, centered on filamentous polymers of type 4 pilins. T4F are exceptionally versatile and widespread virulence factors in bacterial pathogens. The mechanisms of filament assembly and the many functions they facilitate remain poorly understood because of the complexity of T4F machineries. This hinders the development of anti-T4F drugs. The significance of our research lies in characterizing the simplest known T4F-the Com pilus that mediates DNA uptake in competent monoderm bacteria-and showing that four protein components universally conserved in T4F are sufficient for filament assembly. The Com pilus becomes a model for elucidating the mechanisms of T4F assembly.

Infective Endocarditis Caused by Streptococcus sanguinis Resulting in Stroke, Ruptured Infected Pseudoaneurysm of Superior Mesenteric Artery, and Rapidly Progressive Glomerulonephritis.

A 71-year-old-man was admitted to our hospital with a cerebral embolism and diagnosed with infective endocarditis (IE) caused by Streptococcus sanguinis. Mitral valve replacement was performed. About one month later, he experienced sudden abdominal pain and shock due to a ruptured infected mesenteric artery pseudoaneurysm. Forty-four days after abdominal surgery, he presented with rapidly progressive glomerulonephritis with anti-glomerular basement membrane antibodies. He was treated with plasma exchange and prednisolone, and his renal function gradually improved. Since postoperative complications often occur within a few years after surgery for IE, careful follow-up is important, even after antimicrobial therapy and valve surgery.

Amyloid Fibrils Produced by Streptococcus sanguinis Contribute to Biofilm Formation and Immune Evasion.

Bacterial surface proteins assembled into amyloids contribute to biofilm formation and host immune evasion. Streptococcus sanguinis, a pioneer colonizer of teeth commonly involved in cardiovascular infections, expresses about thirty-three proteins anchored to the cell wall by sortase A. Here, we characterized the production of amyloid in S. sanguinis strains differing in biofilm and immune evasion phenotypes and investigated the role of sortase A in amyloidogenesis. Amyloid was identified in biofilms formed by nine strains, using Congo red (CR) staining and cross-polarized light microscopy. Additionally, EGCG, an amyloid inhibitor, impaired biofilm maturation in a strain-specific fashion. The amounts of amyloid-like components quantified in culture fluids of nine strains using thioflavin T and fluorimetry negatively correlated with bacterial binding to complement-activating proteins (SAP, C1q), C3b deposition and rates of opsonophagocytosis in PMNs, implying amyloid production in immune evasion. The deletion of the sortase A gene (srtA) in strain SK36 compromised amyloid production and sucrose-independent biofilm maturation. The srtA mutant further showed increased susceptibility to C3b deposition and altered interactions with PMNs as well as reduced persistence in human blood. These findings highlight the contribution of amyloids to biofilm formation and host immune evasion in S. sanguinis strains, further indicating the participation of sortase A substrates in amyloidogenesis.

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.

Antibacterial Activity of a Lysin LysP53 against Streptococcus mutans.

Dental caries is a common disease affecting quality of life globally. In the present study, we found that a bacteriophage lysin LysP53 against Acinetobacter baumannii possesses selective activity on Streptococcus mutans, the main etiological agent of dental caries, even in low pH caries microenvironments, whereas only minor LysP53 activity was detected against Streptococcus sanguinis, Streptococcus oralis, and Streptococcus mitis. Testing activity against S. mutans planktonic cells showed that 4 µM LysP53 could kill more than 84% of S. mutans within 1 min in buffer with optimal pHs ranging from 4.0 to 6.5. Daily application of LysP53 on biofilms formed in BHI medium supplemented or not with sucrose could reduce exopolysaccharides, expression of genes related to acid resistance and adhesion, and the number of live bacteria in the biofilms. LysP53 treatment also showed similar effects as 0.12% chlorhexidine in preventing enamel demineralization due to S. mutans biofilms, as well as effective removal of S. mutans colonization of tooth surfaces in mice without observed toxic effects. Because of its selective activity against main cariogenic bacteria and good activity in low pH caries microenvironments, it is advantageous to use LysP53 as an active agent for preventing caries.

PepO and CppA modulate Streptococcus sanguinis susceptibility to complement immunity and virulence.

Streptococcus sanguinis is a ubiquitous commensal species of the oral cavity commonly involved as an opportunistic pathogen in cardiovascular infections. In this study, we investigated the functions of endopeptidase O (PepO) and a C3-degrading protease (CppA) in the systemic virulence of S. sanguinis. Isogenic mutants of pepO and cppA obtained in strain SK36 showed increased susceptibility to C3b deposition and to opsonophagocytosis by human polymorphonuclear neutrophils (PMN). These mutants differ, however, in their profiles of binding to serum amyloid P component (SAP) and C1q, whereas both showed reduced interaction with C4b-binding protein (C4BP) and/or factor H (FH) regulators as compared to SK36. The two mutants showed defects in ex vivo persistence in human blood, serum-mediated invasion of HCAEC endothelial cells, and virulence in a Galleria mellonella infection model. The transcriptional activities of pepO and cppA, assessed by RT-qPCR in nine wild-type strains, further indicated strain-specific profiles of pepO/cppA expression. Moreover, non-conserved amino acid substitutions were detected among the strains, mostly in CppA. Phylogenetic comparisons with homologues of streptococcal species of the oral and oropharyngeal sites suggested that S. sanguinis PepO and CppA have independent ancestralities. Thus, this study showed that PepO and CppA are complement evasion proteins expressed by S. sanguinis in a strain-specific manner, which are required for multiple functions associated with cardiovascular virulence.