Exploring the efficacy of in-vitro low-temperature plasma treatment on single and multispecies dental cariogenic biofilms.

The primary aim of this study was to investigate the impact of treatment with low-temperature plasma (LTP) for varying exposure durations on a multispecies cariogenic biofilm comprising C. albicans, L. casei, and S. mutans, as well as on single-species biofilms of L. casei and C. albicans, cultured on hydroxyapatite discs. Biofilms were treated with LTP-argon at a 10 mm distance for 30 s, 60 s, and 120 s. Chlorhexidine solution (0.12%) and NaCl (0.89%) were used as positive (PC) and negative controls (NC), respectively. Argon flow only was also used as gas flow control (F). Colony-forming units (CFU) recovery and confocal laser scanning microscopy (CLSM) were used to analyze biofilm viability. LTP starting at 30 s of application significantly reduced the viability of multispecies biofilms by more than 2 log10 in all treated samples (p < 0.0001). For single-species biofilms, L. casei showed a significant reduction compared to PC and NC of over 1 log10 at all exposure times (p < 0.0001). In the case of C. albicans biofilms, LTP treatment compared to PC and NC resulted in a significant decrease in bacterial counts when applied for 60 and 120 s (1.55 and 1.90 log10 CFU/mL, respectively) (p < 0.0001). A significant effect (p ≤ 0.05) of LTP in single-species biofilms was observed to start at 60 s of LTP application compared to F, suggesting a time-dependent effect of LTP for the single-species biofilms of C. albicans and L. casei. LTP is a potential mechanism in treating dental caries by being an effective anti-biofilm therapy of both single and multispecies cariogenic biofilms.

Targeted metabolomics analysis approach to unravel the biofilm formation pathways of Enterococcus faecalis clinical isolates.

AIM: (i) To characterize Enterococcus faecalis biofilm formation pathways by semi-targeted metabolomics and targeted nitrogen panel analysis of strong (Ef63) and weak (Ef 64) biofilm forming E. faecalis clinical isolates and (ii) to validate the identified metabolic markers using targeted inhibitors. METHODOLOGY: Previous proteomics profiling of E. faecalis clinical isolates with strong and weak biofilm formation revealed that differences in metabolic activity levels of small molecule, nucleotide and nitrogen compound metabolic processes and biosynthetic pathways, cofactor metabolic process, cellular amino acid and derivative metabolic process and lyase activity were associated with differences in biofilm formation. Hence, semi-targeted analysis of Ef 63, Ef 64 and ATC control strain Ef 29212 was performed by selecting metabolites that were part of both the previously identified pathways and a curated library with confirmed physical and chemical identity, followed by confirmatory targeted nitrogen panel analysis. Significantly regulated metabolites (p < .05) were selected based on fold change cut-offs of 1.2 and 0.8 for upregulation and downregulation, respectively, and subjected to pathway enrichment analysis. The identified metabolites and pathways were validated by minimum biofilm inhibitory concentration (MBIC) and colony forming unit (CFU) assays with targeted inhibitors. RESULTS: Metabolomics analysis showed upregulation of betaine, hypoxanthine, glycerophosphorylcholine, tyrosine, inosine, allantoin and citrulline in Ef 63 w.r.t Ef 64 and Ef 29212, and thesemetabolites mapped to purinemetabolism, urea cycle and aspartate metabolism pathways. MBIC and CFU assays using compounds against selected metabolites and metabolic pathways, namely glutathione against hypoxanthine and hydroxylamine against aspartate metabolism showed inhibitory effects against E. faecalis biofilm formation. CONCLUSIONS: The study demonstrated the importance of oxidative stress inducers such as hypoxanthine and aspartate metabolism pathway in E. faecalis biofilm formation. Targeted therapeutics against these metabolic markers can reduce the healthcare burden associated with E. faecalis infections.

Biochemical properties of a Flavobacterium johnsoniae dextranase and its biotechnological potential for Streptococcus mutans biofilm degradation.

Cariogenic biofilms have a matrix rich in exopolysaccharides (EPS), mutans and dextrans, that contribute to caries development. Although several physical and chemical treatments can be employed to remove oral biofilms, those are only partly efficient and use of biofilm-degrading enzymes represents an exciting opportunity to improve the performance of oral hygiene products. In the present study, a member of a glycosyl hydrolase family 66 from Flavobacterium johnsoniae (FjGH66) was heterologously expressed and biochemically characterized. The recombinant FjGH66 showed a hydrolytic activity against an early EPS-containing S. mutans biofilm, and, when associated with a α-(1,3)-glucosyl hydrolase (mutanase) from GH87 family, displayed outstanding performance, removing more than 80% of the plate-adhered biofilm. The mixture containing FjGH66 and Prevotella melaninogenica GH87 α-1,3-mutanase was added to a commercial mouthwash liquid to synergistically remove the biofilm. Dental floss and polyethylene disks coated with biofilm-degrading enzymes also degraded plate-adhered biofilm with a high efficiency. The results presented in this study might be valuable for future development of novel oral hygiene products.

Revisiting gas-chromatography/mass-spectrometry molar response factors for quantitative analysis (FID or TIC) of glycosidic linkages in polysaccharides produced by oral bacterial biofilms.

Methylation analysis was performed on methylated alditol acetate standards and Streptococcus mutans extracellular polymeric substances (EPS) produced from wild-type and Gtf knockout strains (∆GtfB, ∆GtfB, and ∆GtfD). The methylated alditol acetate standards were representative of glycosidic linkages found in S. mutans EPS and were used to calibrate the GC-MS system for an FID detector and MS (TIC) and produce molar response factor, a necessary step in quantitative analysis. FID response factors were consistent with literature values (Sweet et al., 1975) and found to be the superior option for quantitative results, although the TIC response factors now give researchers without access to an FID detector a needed option for molar response factor correction. The GC-MS analysis is then used to deliver the ratio of the linkage types within a biofilm.

Antibiofilm and Immune-Modulatory Activity of Cannabidiol and Cannabigerol in Oral Environments-In Vitro Study.

OBJECTIVE: To evaluate the in vitro antimicrobial and antibiofilm properties and the immune modulatory activity of cannabidiol (CBD) and cannabigerol (CBG) on oral bacteria and periodontal ligament fibroblasts (PLF). METHODS: Cytotoxicity was assessed by propidium iodide flow cytometry on fibroblasts derived from the periodontal ligament. The minimum inhibitory concentration (MIC) of CBD and CBG for S. mutans and C. albicans and the metabolic activity of a subgingival 33-species biofilm under CBD and CBG treatments were determined. The Quantification of cytokines was performed using the LEGENDplex kit (BioLegend, Ref 740930, San Diego, CA, USA). RESULTS: CBD-treated cell viability was greater than 95%, and for CBG, it was higher than 88%. MIC for S. mutans with CBD was 20 µM, and 10 µM for CBG. For C. albicans, no inhibitory effect was observed. Multispecies biofilm metabolic activity was reduced by 50.38% with CBD at 125 µg/mL (p = 0.03) and 39.9% with CBG at 62 µg/mL (p = 0.023). CBD exposure at 500 µg/mL reduced the metabolic activity of the formed biofilm by 15.41%, but CBG did not have an effect. CBG at 10 µM caused considerable production of anti-inflammatory mediators such as TGF-ß and IL-4 at 12 h. CBD at 10 µM to 20 µM produced the highest amount of IFN-γ. CONCLUSION: Both CBG and CBD inhibit S. mutans; they also moderately lower the metabolic activity of multispecies biofilms that form; however, CBD had an effect on biofilms that had already developed. This, together with the production of anti-inflammatory mediators and the maintenance of the viability of mammalian cells from the oral cavity, make these substances promising for clinical use and should be taken into account for future studies.

Oral bacterial insights from a comparative study between healthy and comorbid diseased human individuals.

The human oral cavity is colonized by a diverse microbial community, which includes both native and transient colonizers. The microbial composition is crucial for maintaining oral homeostasis, but due to overgrowth or imbalances of these microbial communities, dysbiosis can occur. There is a lack of understanding of the research of native and transient colonizers in the oral cavity of the Indian subpopulation Therefore, in our present study, we explored the role and prevalence of transient and native colonizers between healthy and comorbid oral diseased human individuals. Culture-dependent techniques and culture-independent 16S r DNA metagenomic analyses were employed to isolate and study the interactions of native and transient colonizers from human oral samples. Among the 66 human individuals of both healthy and comorbid individuals, the most abundant isolate was found to be Bacillus amyloliquefaciens MCC 4424. In addition, the more prevalent culturable isolate from the healthy samples was Streptococcus salivarius MTCC 13009, whereas in comorbid samples Staphylococcus pasteuri MTCC 13076, Rothia dentocariosa MTCC 13010 and Pseudomonas aeruginosa MTCC 13077 were prevalent to a greater extent. 16S rDNA metagenomic analyses revealed the prevalence and abundance of genera such as Bacteroidetes and Proteobacteria in healthy individuals; consequently, Fusobacteria and Firmicutes were observed mostly in comorbid individuals. The significant differences in bacterial population density were observed in terms of the Shannon index (p = 0.5145) and Simpson index (p = 0.9061) between the healthy and comorbid groups. B. amyloliquefaciens MCC 4424 exhibits antagonistic behavior when grown as a dual-species with native and transient colonizers. This result is very consistent with the findings of antibiofilm studies using confocal laser scanning microscopy, which revealed a significant reduction in biofilm biovolume (73 %) and maximum thickness (80 %) and an increase in the rough coefficient of biofilms (30 %). Our data suggested that B. amyloliquefaciens MCC 4424 can be a native colonizer of Indian sub-populations. It may act as a novel candidate for oral healthcare applications and greatly aids in the regulation of transient species in the oral cavity.

Biofouling on titanium implants: a novel formulation of poloxamer and peroxide for in situ removal of pellicle and multi-species oral biofilm.

Eradicating biofouling from implant surfaces is essential in treating peri-implant infections, as it directly addresses the microbial source for infection and inflammation around dental implants. This controlled laboratory study examines the effectiveness of the four commercially available debridement solutions '(EDTA (Prefgel®), NaOCl (Perisolv®), H2O2 (Sigma-Aldrich) and Chlorhexidine (GUM® Paroex®))' in removing the acquired pellicle, preventing pellicle re-formation and removing of a multi-species oral biofilm growing on a titanium implant surface, and compare the results with the effect of a novel formulation of a peroxide-activated 'Poloxamer gel (Nubone® Clean)'. Evaluation of pellicle removal and re-formation was conducted using scanning electron microscope (SEM), energy-dispersive X-ray spectroscopy and X-ray photoelectron spectroscopy to assess the surface morphology, elemental composition and chemical surface composition. Hydrophilicity was assessed through contact angle measurements. The multi-species biofilm model included Streptococcus oralis, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans, reflecting the natural oral microbiome's complexity. Biofilm biomass was quantified using safranin staining, biofilm viability was evaluated using confocal laser scanning microscopy, and SEM was used for morphological analyses of the biofilm. Results indicated that while no single agent completely eradicated the biofilm, the 'Poloxamer gel' activated with 'H2O2' exhibited promising results. It minimized re-contamination of the pellicle by significantly lowering the contact angle, indicating enhanced hydrophilicity. This combination also showed a notable reduction in carbon contaminants, suggesting the effective removal of organic residues from the titanium surface, in addition to effectively reducing viable bacterial counts. In conclusion, the 'Poloxamer gel + H2O2' combination emerged as a promising chemical decontamination strategy for peri-implant diseases. It underlines the importance of tailoring treatment methods to the unique microbial challenges in peri-implant diseases and the necessity of combining chemical decontaminating strategies with established mechanical cleaning procedures for optimal management of peri-implant diseases.

Platelet Membrane-Enclosed Bioorthogonal Catalysis for Combating Dental Caries.

Platelets have shown promise as a means to combat bacterial infections, fostering the development of innovative therapeutic approaches. However, several challenges persist, including cargo loading issues, limited efficacy against biofilms, and concerns regarding the impact of payloads on the platelet carriers. Here, human platelet membrane vesicles (h-PMVs) encapsulating supramolecular metal catalysts (SMCs) as "nanofactories" to convert prodrugs into antimicrobial compounds within close proximity to bacteria are introduced. Having established the feasibility and effectiveness of the SMCs within h-PMVs, referred to as the PLT-reactor, to activate pro-antibiotic drugs (pro-ciprofloxacin and pro-moxifloxacin) using model organisms (Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923), the investigation is subsequently extended to oral biofilms, with a particular emphasis on Streptococcus mutans 3065. This "bind and kill" strategy demonstrates the potent antimicrobial specificity of the PLT-reactor through localized antibiotic production. h-PMVs play a pivotal role by enabling precise targeting of pathogenic biofilms on natural teeth while minimizing potential hemolytic effects. The finding indicates that platelet membrane-cloaked surfaces exhibit robust, multifaceted, and pathogen-specific binding affinity with excellent biocompatibility, making them a promising alternative to antibody-based therapies for infectious diseases.

Revisiting Oral Antiseptics, Microorganism Targets and Effectiveness.

A good oral health status is mostly dependent on good oral hygiene habits, which knowingly impacts systemic health. Although controversial, chemical oral antiseptics can be useful in adjunct use to mechanical dental plaque control techniques in the prevention and management of local and overall health and well-being. This review aims to revisit, gather and update evidence-based clinical indications for the use of the most popular oral antiseptics, considering different types, microorganism targets and effectiveness in order to establish updated clinical recommendations.

Novel Bioactive Nanocomposites Containing Calcium Fluoride and Calcium Phosphate with Antibacterial and Low-Shrinkage-Stress Capabilities to Inhibit Dental Caries.

OBJECTIVES: Composites are commonly used for tooth restorations, but recurrent caries often lead to restoration failures due to polymerization shrinkage-stress-induced marginal leakage. The aims of this research were to: (1) develop novel low-shrinkage-stress (L.S.S.) nanocomposites containing dimethylaminododecyl methacrylate (DMADDM) with nanoparticles of calcium fluoride (nCaF2) or amorphous calcium phosphate (NACP) for remineralization; (2) investigate antibacterial and cytocompatibility properties. METHODS: Nanocomposites were made by mixing triethylene glycol divinylbenzyl ether with urethane dimethacrylate containing 3% DMADDM, 20% nCaF2, and 20% NACP. Flexural strength, elastic modulus, antibacterial properties against Streptococcus mutans biofilms, and cytotoxicity against human gingival fibroblasts and dental pulp stem cells were tested. RESULTS: Nanocomposites with DMADDM and nCaF2 or NACP had flexural strengths matching commercial composite control without bioactivity. The new nanocomposite provided potent antibacterial properties, reducing biofilm CFU by 6 logs, and reducing lactic acid synthesis and metabolic function of biofilms by 90%, compared to controls (p < 0.05). The new nanocomposites produced excellent cell viability matching commercial control (p > 0.05). CONCLUSIONS: Bioactive L.S.S. antibacterial nanocomposites with nCaF2 and NACP had excellent bioactivity without compromising mechanical and cytocompatible properties. The new nanocomposites are promising for a wide range of dental restorations by improving marginal integrity by reducing shrinkage stress, defending tooth structures, and minimizing cariogenic biofilms.

Effects of DJK-5 and chlorhexidine on exopolysaccharide volume and pH in oral biofilms.

BACKGROUND: Exopolysaccharides (EPS) are essential constituents of the extracellular matrix within oral biofilms and are significantly influenced by the local microenvironment. This study aimed to investigate the impact of two distinct antimicrobial agents, DJK-5 and chlorhexidine (CHX), on the EPS volume and pH levels in oral biofilms. METHODS: Oral biofilms obtained from two donors were cultured on hydroxyapatite discs for durations of 3 days, 1 week, 2 weeks, 3 weeks, and 4 weeks. Subsequently, these biofilms were subjected to treatment with 10 µg/mL DJK-5 or 2% CHX for 3 min. The impact of these antimicrobial treatments on factors such as the proportion of dead bacterial, in situ pH, and EPS volume within the biofilms was assessed using corresponding fluorescent probes. The examination was carried out utilizing confocal laser scanning microscopy, and the resulting images were analyzed with a focus on the upper and lower layers of the biofilm, respectively. RESULTS: DJK-5 exhibited a more potent bactericidal effect compared to CHX across the 3-day to 4-week duration of the biofilm (P < 0.05). The biofilms were acidic, with the upper layer being less acidic than the lower layer (P < 0.05). Both antimicrobial agents increased the pH, but DJK-5 had a greater effect than CHX (P < 0.05). The volume of EPS was significantly lower in DJK-5 treated biofilms compared to that of CHX, regardless of age or layer (P < 0.05). CONCLUSION: DJK-5 exhibited superior effectiveness in reducing viable bacteria and EPS volume, as well as in raising extracellular pH, as compared to chlorhexidine.

Rational Designs of Biomaterials for Combating Oral Biofilm Infections.

Oral biofilms, which are also known as dental plaque, are the culprit of a wide range of oral diseases and systemic diseases, thus contributing to serious health risks. The manner of how to achieve good control of oral biofilms has been an increasing public concern. Novel antimicrobial biomaterials with highly controllable fabrication and functionalization have been proven to be promising candidates. However, previous reviews have generally emphasized the physicochemical properties, action mode, and application effectiveness of those biomaterials, whereas insufficient attention has been given to the design rationales tailored to different infection types and application scenarios. To offer guidance for better diversification and functionalization of anti-oral-biofilm biomaterials, this review details the up-to-date design rationales in three aspects: the core strategies in combating oral biofilm, as well as the biomaterials with advanced antibiofilm capacity and multiple functions based on the improvement or combination of the abovementioned antimicrobial strategies. Thereafter, insights on the existing challenges and future improvement of biomaterial-assisted oral biofilm treatments are proposed, hoping to provide a theoretical basis and reference for the subsequent design and application of antibiofilm biomaterials.

Cyclic strain of poly (methyl methacrylate) surfaces triggered the pathogenicity of Candida albicans.

Candida albicans is an opportunistic yeast and the primary etiological factor in oral candidiasis and denture stomatitis. The pathogenesis of C. albicans could be triggered by several variables, including environmental, nutritional, and biomaterial surface cues. Specifically, biomaterial interactions are driven by different surface properties, including wettability, stiffness, and roughness. Dental biomaterials experience repetitive (cyclic) stresses from chewing and biomechanical movements. Pathogenic biofilms are formed over these biomaterial surfaces under cyclic strain. This study investigated the effect of the cyclic strain (deformation) of biomaterial surfaces on the virulence of Candida albicans. Candida biofilms were grown over Poly (methyl methacrylate) (PMMA) surfaces subjected to static (no strain) and cyclic strain with different levels (ε˜x=0.1 and 0.2%). To evaluate the biomaterial-biofilm interactions, the biofilm characteristics, yeast-to-hyphae transition, and the expression of virulent genes were measured. Results showed the biofilm biomass and metabolic activity to be significantly higher when Candida adhered to surfaces subjected to cyclic strain compared to static surfaces. Examination of the yeast-to-hyphae transition showed pseudo-hyphae cells (pathogenic) in cyclically strained biomaterial surfaces, whereas static surfaces showed spherical yeast cells (commensal). RNA sequencing was used to determine and compare the transcriptome profiles of cyclically strained and static surfaces. Genes and transcription factors associated with cell adhesion (CSH1, PGA10, and RBT5), biofilm formation (EFG1), and secretion of extracellular matrix (ECM) (CRH1, ADH5, GCA1, and GCA2) were significantly upregulated in the cyclically strained biomaterial surfaces compared to static ones. Genes and transcription factors associated with virulence (UME6 and HGC1) and the secretion of extracellular enzymes (LIP, PLB, and SAP families) were also significantly upregulated in the cyclically strained biomaterial surfaces compared to static. For the first time, this study reveals a biomaterial surface factor triggering the pathogenesis of Candida albicans, which is essential for understanding, controlling, and preventing oral infections. STATEMENT OF SIGNIFICANCE: Fungal infections produced by Candida albicans are a significant contributor to various health conditions. Candida becomes pathogenic when certain environmental conditions change, including temperature, pH, nutrients, and CO2 levels. In addition, surface properties, including wettability, stiffness, and roughness, drive the interactions between Candida and biomaterials. Clinically, Candida adheres to biomaterials that are under repetitive deformation due to body movements. In this work, we revealed that when Candida adhered to biomaterial surfaces subjected to repetitive deformation, the microorganism becomes pathogenic by increasing the formation of biofilms and the expression of virulent factors related to hyphae formation and secretion of enzymes. Findings from this work could aid the development of new strategies for treating fungal infections in medical devices or implanted biomaterials.

Novel Dental Low-Shrinkage-Stress Composite with Antibacterial Dimethylaminododecyl Methacrylate Monomer.

OBJECTIVES: Current dental resins exhibit polymerization shrinkage causing microleakage, which has the potential to cause recurrent caries. Our objectives were to create and characterize low-shrinkage-stress (LSS) composites with dimethylaminododecyl methacrylate (DMADDM) as an antibacterial agent to combat recurrent caries. METHODS: Triethylene glycol divinylbenzyl ether and urethane dimethacrylate were used to reduce shrinkage stress. DMADDM was incorporated at different mass fractions (0%, 1.5%, 3%, and 5%). Flexural strength, elastic modulus, degree of conversion, polymerization stress, and antimicrobial activity were assessed. RESULTS: The composite with 5% DMADDM demonstrated higher flexural strength than the commercial group (p < 0.05). The addition of DMADDM in BisGMA-TEGDMA resin and LSS resin achieved clinically acceptable degrees of conversion. However, LSS composites exhibited much lower polymerization shrinkage stress than BisGMA-TEGDMA composite groups (p < 0.05). The addition of 3% and 5% DMADDM showed a 6-log reduction in Streptococcus mutans (S. mutans) biofilm CFUs compared to commercial control (p < 0.001). Biofilm biomass and lactic acid were also substantially decreased via DMADDM (p < 0.05). CONCLUSIONS: The novel LSS dental composite containing 3% DMADDM demonstrated potent antibacterial action against S. mutans biofilms and much lower polymerization shrinkage-stress, while maintaining excellent mechanical characteristics. The new composite is promising for dental applications to prevent secondary caries and increase restoration longevity.

Limosilactobacillus reuteri inhibits the acid tolerance response in oral bacteria.

Probiotic bacteria show promising results in prevention of the biofilm-mediated disease caries, but the mechanisms are not fully understood. The acid tolerance response (ATR) allows biofilm bacteria to survive and metabolize at low pH resulting from microbial carbohydrate fermentation. We have studied the effect of probiotic strains: Limosilactobacillus reuteri and Lacticaseibacillus rhamnosus on ATR induction in common oral bacteria. Communities of L. reuteri ATCC PTA5289 and Streptoccus gordonii, Streptococcus oralis, Streptococcus mutans or Actinomyces naeslundii in the initial stages of biofilm formation were exposed to pH 5.5 to allow ATR induction, followed by a low pH challenge. Acid tolerance was evaluated as viable cells after staining with LIVE/DEAD®BacLight™. The presence of L. reuteri ATCC PTA5289 caused a significant reduction in acid tolerance in all strains except S. oralis. When S. mutans was used as a model organism to study the effects of additional probiotic strains (L. reuteri SD2112, L. reuteri DSM17938 or L. rhamnosus GG) as well as L. reuteri ATCC PTA5289 supernatant on ATR development, neither the other probiotic strains nor supernatants showed any effect. The presence of L. reuteri ATCC PTA5289 during ATR induction led to down-regulation of three key genes involved in tolerance of acid stress (luxS, brpA and ldh) in Streptococci. These data suggest that live cells of probiotic L. reuteri ATCC PTA5289 can interfere with ATR development in common oral bacteria and specific strains of L. reuteri may thus have a role in caries prevention by inhibiting development of an acid-tolerant biofilm microbiota.

Antimicrobial photodynamic therapy against oral biofilm: influencing factors, mechanisms, and combined actions with other strategies.

Oral biofilms are a prominent cause of a wide variety of oral infectious diseases which are still considered as growing public health problems worldwide. Oral biofilms harbor specific virulence factors that would aggravate the infectious process and present resistance to some traditional therapies. Antimicrobial photodynamic therapy (aPDT) has been proposed as a potential approach to eliminate oral biofilms via in situ-generated reactive oxygen species. Although numerous types of research have investigated the effectiveness of aPDT, few review articles have listed the antimicrobial mechanisms of aPDT on oral biofilms and new methods to improve the efficiency of aPDT. The review aims to summarize the virulence factors of oral biofilms, the progress of aPDT in various oral biofilm elimination, the mechanism mediated by aPDT, and combinatorial approaches of aPDT with other traditional agents.

Addition of cariogenic pathogens to complex oral microflora drives significant changes in biofilm compositions and functionalities.

BACKGROUND: Dental caries is a microbe and sugar-mediated biofilm-dependent oral disease. Of particular significance, a virulent type of dental caries, known as severe early childhood caries (S-ECC), is characterized by the synergistic polymicrobial interaction between the cariogenic bacterium, Streptococcus mutans, and an opportunistic fungal pathogen, Candida albicans. Although cross-sectional studies reveal their important roles in caries development, these exhibit limitations in determining the significance of these microbial interactions in the pathogenesis of the disease. Thus, it remains unclear the mechanism(s) through which the cross-kingdom interaction modulates the composition of the plaque microbiome. Here, we employed a novel ex vivo saliva-derived microcosm biofilm model to assess how exogenous pathogens could impact the structural and functional characteristics of the indigenous native oral microbiota. RESULTS: Through shotgun whole metagenome sequencing, we observed that saliva-derived biofilm has decreased richness and diversity but increased sugar-related metabolism relative to the planktonic phase. Addition of S. mutans and/or C. albicans to the native microbiome drove significant changes in its bacterial composition. In addition, the effect of the exogenous pathogens on microbiome diversity and taxonomic abundances varied depending on the sugar type. While the addition of S. mutans induced a broader effect on Kyoto Encyclopedia of Genes and Genomes (KEGG) ortholog abundances with glucose/fructose, S. mutans-C. albicans combination under sucrose conditions triggered unique and specific changes in microbiota composition/diversity as well as specific effects on KEGG pathways. Finally, we observed the presence of human epithelial cells within the biofilms via confocal microscopy imaging. CONCLUSIONS: Our data revealed that the presence of S. mutans and C. albicans, alone or in combination, as well as the addition of different sugars, induced unique alterations in both the composition and functional attributes of the biofilms. In particular, the combination of S. mutans and C. albicans seemed to drive the development (and perhaps the severity) of a dysbiotic/cariogenic oral microbiome. Our work provides a unique and pragmatic biofilm model for investigating the functional microbiome in health and disease as well as developing strategies to modulate the microbiome. Video Abstract.

Osteosynthesis-associated infection in maxillofacial surgery by bacterial biofilms: a retrospective cohort study of 11 years.

OBJECTIVES: The aim of this retrospective cohort study was to determine risk factors for osteosynthesis-associated infections (OAI) with subsequent necessity of implant removal in oral and maxillofacial surgery. MATERIALS AND METHODS: A total of 3937 records of patients who received either orthognathic, trauma, or reconstructive jaw surgery from 2009 to 2021 were screened for osteosynthetic material removal due to infection. Treatment-intervals, volume of applied osteosynthetic material, and respective surgical procedures were also assessed. Moreover, intraoperatively harvested microbial flora was cultured and subsequently identified by MALDI TOF. Bacteria were then screened for antibiotic resistance via VITEK system or, if necessary, via agar diffusion or epsilometer test. Data was analyzed utilizing SPSS statistical software. For statistical analysis of categorical variables, chi-square tests or Fisher exact tests were used. Continuous variables were compared via non-parametric tests. The level of significance for p-values was set at < 0.05. Descriptive analysis was also performed. RESULTS: The lower jaw was more prone to OAI than the mid face region. Larger volumes of osteosynthetic material led to significantly more OAI, resulting in reconstruction plates bearing the highest risk for OAI especially when compared to small-volume mini-plates frequently applied in trauma surgery. Among OAI associated with implant volumes smaller than 1500 mm3, the detection of Streptococcus spp., Prevotella spp., Staphylococcus spp., and Veillonella spp. was significantly elevated, whereas implant volumes larger than 1500 mm3 showed a significant increase of Enterococcus faecalis, Proteus mirabilis and Pseudomonas aeruginosa. High susceptibility rates (87.7-95.7%) were documented for 2nd- and 3rd-generation cephalosporines and piperacillin/tazobactam. CONCLUSION: High material load and lower jaw reconstruction bear the greatest risks for OAI. When working with large volume osteosynthetic implants, gram-negative pathogens must be considered when choosing an appropriate antibiotic regime. Suitable antibiotics include, e.g., piperacillin/tazobactam and 3rd-generation cephalosporines. CLINICAL RELEVANCE: Osteosynthetic material utilized in reconstructive procedures of the lower jaw may be colonized with drug-resistant biofilms.

Novel bioactive dental restorations to inhibit secondary caries in enamel and dentin under oral biofilms.

OBJECTIVE: To provide the first review on cutting-edge research on the development of new bioactive restorations to inhibit secondary caries in enamel and dentin under biofilms. State-of-the-art bioactive and therapeutic materials design, structure-property relationships, performance and efficacies in oral biofilm models. DATA, SOURCES AND STUDY SELECTION: Researches on development and assessment new secondary caries inhibition restorations via in vitro and in vivo biofilm-based secondary caries models were included. The search of articles was carried out in Web of Science, PubMed, Medline and Scopus. CONCLUSIONS: Based on the found articles, novel bioactive materials are divided into different categories according to their remineralization and antibacterial biofunctions. In vitro and in vivo biofilm-based secondary caries models are effective way of evaluating the materials efficacies. However, new intelligent and pH-responsive materials were still urgent need. And the materials evaluation should be performed via more clinical relevant biofilm-based secondary caries models. CLINICAL SIGNIFICANCE: Secondary caries is a primary reason for dental restoration failures. Biofilms produce acids, causing demineralization and secondary caries. To inhibit dental caries and improve the health and quality of life for millions of people, it is necessary to summarize the present state of technologies and new advances in dental biomaterials for preventing secondary caries and protecting tooth structures against oral biofilm attacks. In addition, suggestions for future studies are provided.

The Antimicrobial Activity of Curcumin and Xanthohumol on Bacterial Biofilms Developed over Dental Implant Surfaces.

In search for natural products with antimicrobial properties for use in the prevention and treatment of peri-implantitis, the purpose of this investigation was to evaluate the antimicrobial activity of curcumin and xanthohumol, using an in vitro multi-species dynamic biofilm model including Streptococcus oralis, Actinomyces naeslundii, Veillonella parvula, Fusobacterium nucleatum, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. The antimicrobial activities of curcumin (5 mM) and xanthohumol (100 µM) extracts, and the respective controls, were evaluated with 72-h biofilms formed over dental implants by their submersion for 60 seconds. The evaluation was assessed by quantitative polymerase chain reaction (qPCR), confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). For the data analysis, comparisons were tested applying ANOVA tests with post-hoc Bonferroni corrections to evaluate the antimicrobial activity of both extracts. With qPCR, statistically significant reductions in bacterial counts were observed for curcumin and xanthohumol, when compared to the negative control. The results with CLSM and SEM were consistent with those reported with qPCR. It was concluded that both curcumin and xanthohumol have demonstrated antimicrobial activity against the six bacterial species included in the dynamic in vitro biofilm model used.