Interkingdom assemblages in human saliva display group-level surface mobility and disease-promoting emergent functions.

Fungi and bacteria often engage in complex interactions, such as the formation of multicellular biofilms within the human body. Knowledge about how interkingdom biofilms initiate and coalesce into higher-level communities and which functions the different species carry out during biofilm formation remain limited. We found native-state assemblages of Candida albicans (fungi) and Streptococcus mutans (bacteria) with highly structured arrangement in saliva from diseased patients with childhood tooth decay. Further analyses revealed that bacterial clusters are attached within a network of fungal yeasts, hyphae, and exopolysaccharides, which bind to surfaces as a preassembled cell group. The interkingdom assemblages exhibit emergent functions, including enhanced surface colonization and growth rate, stronger tolerance to antimicrobials, and improved shear resistance, compared to either species alone. Notably, we discovered that the interkingdom assemblages display a unique form of migratory spatial mobility that enables fast spreading of biofilms across surfaces and causes enhanced, more extensive tooth decay. Using mutants, selective inactivation of species, and selective matrix removal, we demonstrate that the enhanced stress resistance and surface mobility arise from the exopolymeric matrix and require the presence of both species in the assemblage. The mobility is directed by fungal filamentation as hyphae extend and contact the surface, lifting the assemblage with a "forward-leaping motion." Bacterial cell clusters can "hitchhike" on this mobile unit while continuously growing, to spread across the surface three-dimensionally and merge with other assemblages, promoting community expansion. Together, our results reveal an interkingdom assemblage in human saliva that behaves like a supraorganism, with disease-causing emergent functionalities that cannot be achieved without coassembly.

Microrobotics in endodontics: A perspective.

Microorganisms are the primary aetiological factor of apical periodontitis. The goal of endodontic treatment is to prevent and eliminate the infection by removing the microorganisms. However, microbial biofilms and the complex root canal anatomy impair the disinfection process. Effective and precise endodontic therapy could potentially be achieved using advanced multifunctional technologies that have the ability to access hard-to-reach surfaces and perform simultaneous biofilm killing, removal, and detection of microorganisms. Advances in microrobotics are providing novel therapeutic and diagnostic opportunities with high precision and efficacy to address current biofilm-related challenges in biomedicine. Concurrently, multifunctional magnetic microrobots have been developed to overcome the disinfection challenges of current approaches to disrupt, kill, and retrieve biofilms with the goal of enhancing the efficacy and precision of endodontic therapy. This article reviews the recent advances of microrobotics in healthcare and particularly advances to overcome disinfection challenges in endodontics, and provides perspectives for future research in the field.

Spatial mapping of polymicrobial communities reveals a precise biogeography associated with human dental caries.

Tooth decay (dental caries) is a widespread human disease caused by microbial biofilms. Streptococcus mutans, a biofilm-former, has been consistently associated with severe childhood caries; however, how this bacterium is spatially organized with other microorganisms in the oral cavity to promote disease remains unknown. Using intact biofilms formed on teeth of toddlers affected by caries, we discovered a unique 3D rotund-shaped architecture composed of multiple species precisely arranged in a corona-like structure with an inner core of S. mutans encompassed by outer layers of other bacteria. This architecture creates localized regions of acidic pH and acute enamel demineralization (caries) in a mixed-species biofilm model on human teeth, suggesting this highly ordered community as the causative agent. Notably, the construction of this architecture was found to be an active process initiated by production of an extracellular scaffold by S. mutans that assembles the corona cell arrangement, encapsulating the pathogen core. In addition, this spatial patterning creates a protective barrier against antimicrobials while increasing bacterial acid fitness associated with the disease-causing state. Our data reveal a precise biogeography in a polymicrobial community associated with human caries that can modulate the pathogen positioning and virulence potential in situ, indicating that micron-scale spatial structure of the microbiome may mediate the function and outcome of host-pathogen interactions.

The effect of Brazilian propolis type-3 against oral microbiota and volatile sulfur compounds in subjects with morning breath malodor.

OBJECTIVES: To evaluate propolis type-3 mouthrinse effects on the concentration of volatile sulfur compounds (VSCs) and on tongue dorsum microbial profile. MATERIALS AND METHODS: A three-step double-blind, crossover, randomized study with 10 individuals divided into three groups: I-placebo (P); II-ethanolic extract of propolis type-3 3% (EEP); and III-chlorhexidine 0.12% (CHX) and instructed to rinse twice daily for 5 days. Each experimental period was followed by a 21-day washout interval. Morning mouth breath was assessed by VSC concentrations and microbiological samples were obtained from tongue dorsum at baseline and the end of period of rinses and analyzed using checkerboard DNA-DNA hybridization technique for 39 bacterial species. RESULTS: CHX and EEP presented the lowest VSC concentration when compared with placebo (p < 0.05). Even in the absence of mechanical plaque control, CHX and EEP treatments reduced VSC levels and there were no statistical differences for VSC measurement between CHX and EEP. There was a significant reduction in mean counts of 10 species including some VSC producers (Prevotella intermedia, Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia) by EEP. Total counts of organisms, gram-negative and gram-positive bacterial species showed a decrease for EEP and CHX (p < 0.05). In addition, no statistical difference was observed between EEP and CHX (p > 0.05). A positive correlation was observed between decrease of bacterial counts and decrease of VCSs concentration for the EEP and CHX. CONCLUSIONS: The use of a 3% propolis type-3 mouthrinse is an effective way to prevent morning bad breath. Thus, propolis may be a promising agent for the treatment of halitosis. CLINICAL RELEVANCE: Propolis type-3 may be used as adjuvant treatment for morning breath malodor.

Ferumoxytol Nanoparticles Target Biofilms Causing Tooth Decay in the Human Mouth.

Severe tooth decay has been associated with iron deficiency anemia that disproportionally burdens susceptible populations. Current modalities are insufficient in severe cases where pathogenic dental biofilms rapidly accumulate, requiring new antibiofilm approaches. Here, we show that ferumoxytol, a Food and Drug Administration-approved nanoparticle formulation for treating iron deficiency, exerts an alternative therapeutic activity via the catalytic activation of hydrogen peroxide, which targets bacterial pathogens in biofilms and suppresses tooth enamel decay in an intraoral human disease model. Data reveal the potent antimicrobial specificity of ferumoxytol iron oxide nanoparticles (FerIONP) against biofilms harboring Streptococcus mutans via preferential binding that promotes bacterial killing through in situ free-radical generation. Further analysis indicates that the targeting mechanism involves interactions of FerIONP with pathogen-specific glucan-binding proteins, which have a minimal effect on commensal streptococci. In addition, we demonstrate that FerIONP can detect pathogenic biofilms on natural teeth via a facile colorimetric reaction. Our findings provide clinical evidence and the theranostic potential of catalytic nanoparticles as a targeted anti-infective nanomedicine.

Antibacterial and Physical Properties of PVM/MA Copolymer- Incorporated Polymethyl Methacrylate as a Novel Antimicrobial Acrylic Resin Material.

Polymethyl methacrylate (PMMA), an acrylic resin used in orthodontic appliances and removable dentures for its biocompatibility and esthetics, may harbor bacteria on its surface. The present study investigated a new PMMA formula with Gantrez: an antibacterial copolymer of methyl vinyl ether and maleic acid (PVM/MA). Samples were tested for mechanical properties (surface hardness, flexural strength, water sorption, and water solubility) and effects against Streptococcus mutans. Six groups (0%-control, 5%, 10%, 15%, 20%, and 25% Gantrez) of n = 12 were fabricated for physical property tests and analyzed with one-way ANOVA and Prism 6. From these results, three groups (0%, 5%, and 10% Gantrez) were selected for antibacterial tests, and data were analyzed with one-way ANOVA and Tukey's multiple comparison test. Adding 5% and 10% Gantrez into PMMA significantly decreased S. mutans adhesion. There was no significant difference between the control vs. 5%, 10%, 15%, and 20% Gantrez (p > 0.05) for surface hardness, the control vs. 5% Gantrez (p > 0.05) for flexural strength, and the control vs. 5 and 10% Gantrez for water sorption and water solubility. Overall, incorporating 5% Gantrez into PMMA may be a promising solution to reduce bacterial adhesion without changing the acrylic resin's physical properties.

Affordable oral health care: dental biofilm disruption using chloroplast made enzymes with chewing gum delivery.

Current approaches for oral health care rely on procedures that are unaffordable to impoverished populations, whereas aerosolized droplets in the dental clinic and poor oral hygiene may contribute to spread of several infectious diseases including COVID-19, requiring new solutions for dental biofilm/plaque treatment at home. Plant cells have been used to produce monoclonal antibodies or antimicrobial peptides for topical applications to decrease colonization of pathogenic microbes on dental surface. Therefore, we investigated an affordable method for dental biofilm disruption by expressing lipase, dextranase or mutanase in plant cells via the chloroplast genome. Antibiotic resistance gene used to engineer foreign genes into the chloroplast genome were subsequently removed using direct repeats flanking the aadA gene and enzymes were successfully expressed in marker-free lettuce transplastomic lines. Equivalent enzyme units of plant-derived lipase performed better than purified commercial enzymes against biofilms, specifically targeting fungal hyphae formation. Combination of lipase with dextranase and mutanase suppressed biofilm development by degrading the biofilm matrix, with concomitant reduction of bacterial and fungal accumulation. In chewing gum tablets formulated with freeze-dried plant cells, expressed protein was stable up to 3 years at ambient temperature and was efficiently released in a time-dependent manner using a mechanical chewing simulator device. Development of edible plant cells expressing enzymes eliminates the need for purification and cold-chain transportation, providing a potential translatable therapeutic approach. Biofilm disruption through plant enzymes and chewing gum-based delivery offers an effective and affordable dental biofilm control at home particularly for populations with minimal oral care access.

Streptococcus mutans yidC1 and yidC2 Impact Cell Envelope Biogenesis, the Biofilm Matrix, and Biofilm Biophysical Properties.

Proper envelope biogenesis of Streptococcus mutans, a biofilm-forming and dental caries-causing oral pathogen, requires two paralogs (yidC1 and yidC2) of the universally conserved YidC/Oxa1/Alb3 family of membrane integral chaperones and insertases. The deletion of either paralog attenuates virulence in vivo, but the mechanisms of disruption remain unclear. Here, we determined whether the deletion of yidC affects cell surface properties, extracellular glucan production, and/or the structural organization of the exopolysaccharide (EPS) matrix and biophysical properties of S. mutans biofilm. Compared to the wild type, the ΔyidC2 mutant lacked staining with fluorescent vancomycin at the division septum, while the ΔyidC1 mutant resembled the wild type. Additionally, the deletion of either yidC1 or yidC2 resulted in less insoluble glucan synthesis but produced more soluble glucans, especially at early and mid-exponential-growth phases. Alteration of glucan synthesis by both mutants yielded biofilms with less dry weight and insoluble EPS. In particular, the deletion of yidC2 resulted in a significant reduction in biofilm biomass and pronounced defects in the spatial organization of the EPS matrix, thus modifying the three-dimensional (3D) biofilm architecture. The defective biofilm harbored smaller bacterial clusters with high cell density and less surrounding EPS than those of the wild type, which was stiffer in compression yet more susceptible to removal by shear. Together, our results indicate that the elimination of either yidC paralog results in changes to the cell envelope and glucan production that ultimately disrupts biofilm development and EPS matrix structure/composition, thereby altering the physical properties of the biofilms and facilitating their removal. YidC proteins, therefore, represent potential therapeutic targets for cariogenic biofilm control.IMPORTANCE YidC proteins are membrane-localized chaperone insertases that are universally conserved in all bacteria and are traditionally studied in the context of membrane protein insertion and assembly. Both YidC paralogs of the cariogenic pathogen Streptococcus mutans are required for proper envelope biogenesis and full virulence, indicating that these proteins may also contribute to optimal biofilm formation in streptococci. Here, we show that the deletion of either yidC results in changes to the structure and physical properties of the EPS matrix produced by S. mutans, ultimately impairing optimal biofilm development, diminishing its mechanical stability, and facilitating its removal. Importantly, the universal conservation of bacterial yidC orthologs, combined with our findings, provide a rationale for YidC as a possible drug target for antibiofilm therapies.

Candida albicans mannans mediate Streptococcus mutans exoenzyme GtfB binding to modulate cross-kingdom biofilm development in vivo.

Candida albicans is frequently detected with heavy infection by Streptococcus mutans in plaque-biofilms from children with early-childhood caries (ECC). This cross-kingdom biofilm contains an extensive matrix of extracellular α-glucans that is produced by an exoenzyme (GtfB) secreted by S. mutans. Here, we report that mannans located on the outer surface of C. albicans cell-wall mediates GtfB binding, enhancing glucan-matrix production and modulating bacterial-fungal association within biofilms formed in vivo. Using single-molecule atomic force microscopy, we determined that GtfB binds with remarkable affinity to mannans and to the C. albicans surface, forming a highly stable and strong bond (1-2 nN). However, GtfB binding properties to C. albicans was compromised in strains defective in O-mannan (pmt4ΔΔ) or N-mannan outer chain (och1ΔΔ). In particular, the binding strength of GtfB on och1ΔΔ strain was severely disrupted (>3-fold reduction vs. parental strain). In turn, the GtfB amount on the fungal surface was significantly reduced, and the ability of C. albicans mutant strains to develop mixed-species biofilms with S. mutans was impaired. This phenotype was independent of hyphae or established fungal-biofilm regulators (EFG1, BCR1). Notably, the mechanical stability of the defective biofilms was weakened, resulting in near complete biomass removal by shear forces. In addition, these in vitro findings were confirmed in vivo using a rodent biofilm model. Specifically, we observed that C. albicans och1ΔΔ was unable to form cross-kingdom biofilms on the tooth surface of rats co-infected with S. mutans. Likewise, co-infection with S. mutans defective in GtfB was also incapable of forming mixed-species biofilms. Taken together, the data support a mechanism whereby S. mutans-secreted GtfB binds to the mannan layer of C. albicans to promote extracellular matrix formation and their co-existence within biofilms. Enhanced understanding of GtfB-Candida interactions may provide new perspectives for devising effective therapies to disrupt this cross-kingdom relationship associated with an important childhood oral disease.

Candida-Bacterial Biofilms and Host-Microbe Interactions in Oral Diseases.

Oral cavity harbors a complex and highly diverse microbial community. Cross-kingdom interactions between Candida and oral bacteria are critical for their co-existence, which may also affect the course and the severity of biofilm-mediated bacterial-mediated diseases. C. albicans has been found in polymicrobial biofilms associated with denture stomatitis, oral mucositis, dental caries, periodontal diseases, peri-implantitis, and root canal infection. Thus, it is of utmost importance to unravel the mechanisms of Candida-bacterial interactions and their impact on the onset and severity of cross-kingdom biofilm-related diseases. Here, we highlight the potential role of Candida-bacterial biofilm interactions in the pathogenesis of oral diseases, especially mucosal infections and dental caries. The influence of Candida-bacterial biofilms on the mucosal host immune response is also discussed. Finally, we present some of the current and prospective therapeutic strategies for controlling these cross-kingdom interactions and their virulence properties associated with oral diseases.

Candida albicans and Early Childhood Caries: A Systematic Review and Meta-Analysis.

Oral Candida albicans has been detected in children with early childhood caries (ECC) and has demonstrated cariogenic traits in animal models of the disease. Conversely, other studies found no positive correlation between C. albicans and caries experience in children, while suggesting it may have protective effects as a commensal organism. Thus, this study aimed to examine whether oral C. albicans is associated with ECC. Seven electronic databases were searched. The data from eligible studies were extracted, and the risk of bias was evaluated. A fixed effects model (Mantel-Haenszel estimate) was used for meta-analysis, and the summary effect measure was calculated by odds ratio (OR) and 95% confidence interval (CI). Fifteen cross-sectional studies were included for the qualitative assessment and 9 studies for meta-analysis. Twelve studies revealed higher oral C. albicans prevalence in ECC children than in caries-free children, while 2 studies indicated an equivalent prevalence. A pooled estimate, with OR = 6.51 and 95% CI = 4.94-8.57, indicated a significantly higher ECC experience in children with oral C. albicans than those without C. albicans (p < 0.01). The odds of experiencing ECC in children with C. albicans versus children without C. albicans were 5.26 for salivary, 6.69 for plaque, and 6.3 for oral swab samples. This systematic review indicates that children with oral C. albicans have >5 times higher odds of having ECC compared to those without C. albicans. Further prospective cohort studies are needed to determine whether C. albicans could be a risk factor for ECC, and whether it is dependent on different sample sources (saliva/plaque).

l-Arginine Modifies the Exopolysaccharide Matrix and Thwarts Streptococcus mutans Outgrowth within Mixed-Species Oral Biofilms.

UNLABELLED: l-Arginine, a ubiquitous amino acid in human saliva, serves as a substrate for alkali production by arginolytic bacteria. Recently, exogenous l-arginine has been shown to enhance the alkalinogenic potential of oral biofilm and destabilize its microbial community, which might help control dental caries. However, l-arginine exposure may inflict additional changes in the biofilm milieu when bacteria are growing under cariogenic conditions. Here, we investigated how exogenous l-arginine modulates biofilm development using a mixed-species model containing both cariogenic (Streptococcus mutans) and arginolytic (Streptococcus gordonii) bacteria in the presence of sucrose. We observed that 1.5% (wt/vol) l-arginine (also a clinically effective concentration) exposure suppressed the outgrowth of S. mutans, favored S. gordonii dominance, and maintained Actinomyces naeslundii growth within biofilms (versus vehicle control). In parallel, topical l-arginine treatments substantially reduced the amounts of insoluble exopolysaccharides (EPS) by >3-fold, which significantly altered the three-dimensional (3D) architecture of the biofilm. Intriguingly, l-arginine repressed S. mutans genes associated with insoluble EPS (gtfB) and bacteriocin (SMU.150) production, while spxB expression (H2O2 production) by S. gordonii increased sharply during biofilm development, which resulted in higher H2O2 levels in arginine-treated biofilms. These modifications resulted in a markedly defective EPS matrix and areas devoid of any bacterial clusters (microcolonies) on the apatitic surface, while the in situ pH values at the biofilm-apatite interface were nearly one unit higher in arginine-treated biofilms (versus the vehicle control). Our data reveal new biological properties of l-arginine that impact biofilm matrix assembly and the dynamic microbial interactions associated with pathogenic biofilm development, indicating the multiaction potency of this promising biofilm disruptor. IMPORTANCE: Dental caries is one of the most prevalent and costly infectious diseases worldwide, caused by a biofilm formed on tooth surfaces. Novel strategies that compromise the ability of virulent species to assemble and maintain pathogenic biofilms could be an effective alternative to conventional antimicrobials that indiscriminately kill other oral species, including commensal bacteria. l-Arginine at 1.5% has been shown to be clinically effective in modulating cariogenic biofilms via alkali production by arginolytic bacteria. Using a mixed-species ecological model, we show new mechanisms by which l-arginine disrupts the process of biofilm matrix assembly and the dynamic microbial interactions that are associated with cariogenic biofilm development, without impacting the bacterial viability. These results may aid in the development of enhanced methods to control biofilms using l-arginine.

The collagen binding protein Cnm contributes to oral colonization and cariogenicity of Streptococcus mutans OMZ175.

Streptococcus mutans is the etiological agent of dental caries and one of the many bacterial species implicated in infective endocarditis. The expression of the collagen-binding protein Cnm by S. mutans has been associated with extraoral infections, but its relevance for dental caries has only been theorized to date. Due to the collagenous composition of dentinal and root tissues, we hypothesized that Cnm may facilitate the colonization of these surfaces, thereby enhancing the pathogenic potential of S. mutans in advancing carious lesions. As shown for extraoral endothelial cell lines, Cnm mediates the invasion of oral keratinocytes and fibroblasts by S. mutans. In this study, we show that in the Cnm(+) native strain, OMZ175, Cnm mediates stringent adhesion to dentinal and root tissues as well as collagen-coated surfaces and promotes both cariogenicity and carriage in vivo. In vitro, ex vivo, and in vivo experiments revealed that while Cnm is not universally required for S. mutans cariogenicity, it contributes to (i) the invasion of the oral epithelium, (ii) enhanced binding on collagenous surfaces, (iii) implantation of oral biofilms, and (IV) the severity of caries due to a native Cnm(+) isolate. Taken together, our findings reveal that Cnm is a colonization factor that contributes to the pathogenicity of certain S. mutans strains in their native habitat, the oral cavity.

Surface-induced changes in the conformation and glucan production of glucosyltransferase adsorbed on saliva-coated hydroxyapatite.

Glucosyltransferases (Gtfs) from S. mutans play critical roles in the development of virulent oral biofilms associated with dental caries disease. Gtfs adsorbed to the tooth surface produce glucans that promote local microbial colonization and provide an insoluble exopolysaccharides (EPS) matrix that facilitates biofilm initiation. Moreover, agents that inhibit the enzymatic activity of Gtfs in solution often have reduced or no effects on surface-adsorbed Gtfs. This study elucidated the mechanisms responsible for the differences in functionality that GtfB exhibits in solution vs surface-adsorbed. Upon adsorption to planar fused-quartz substrates, GtfB displayed a 37% loss of helices and 36% increase of ß-sheets, as determined by circular dichroism (CD) spectroscopy, and surface-induced conformational changes were more severe on substrates modified with CH3- and NH2-terminated self-assembled monolayers. GtfB also underwent substantial conformation changes when adsorbing to hydroxyapatite (HA) microspheres, likely due to electrostatic interactions between negatively charged GtfB and positively charged HA crystal faces. Conformational changes were lessened when HA surfaces were coated with saliva (sHA) prior to GtfB adsorption. Furthermore, GtfB remained highly active on sHA, as determined by in situ attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy, producing glucans that were structurally different than GtfB in solution and known to increase the accumulation and virulence of biofilms. Our data provide the first insight into the structural underpinnings governing Gtf conformation and enzymatic function that occur on tooth surfaces in vivo, which may lead to designing potent new inhibitors and improved strategies to combat the formation of pathogenic oral biofilms.

Clinical outcome of transcatheter arterial embolization with N-butyl-2-cyanoacrylate for control of acute gastrointestinal tract bleeding.

OBJECTIVE. The purpose of this article is to evaluate the clinical effectiveness of trans-catheter arterial embolization (TAE) with N-butyl-2-cyanoacrylate (NBCA), with or without other embolic materials for acute nonvariceal gastrointestinal tract bleeding, and to determine the factors associated with clinical outcomes. MATERIALS AND METHODS. TAE using NBCA only or in conjunction with other materials was performed for 102 patients (80 male and 22 female patients; mean age, 61.3 years) with acute nonvariceal gastrointestinal tract bleeding. Technical success, clinical success, and clinical factors, including age, sex, bleeding tendency, endoscopic attempts at hemostasis, number of transfusions, and bleeding causes (i.e., cancer vs noncancer), were retrospectively evaluated. Univariate and multivariable logistic regression analyses were performed to evaluate clinical factors and their ability to predict patient outcomes. Survival curves were obtained using Kaplan-Meier analyses and log-rank tests. RESULTS. There were 36 patients with cancer-related bleeding and 66 with non-cancer-related bleeding. Overall technical and clinical success rates were 100% (102/102) and 76.5% (78/102), respectively. Procedure-related complications included bowel infarction, which was noted in two patients. Recurrent bleeding and bleeding-related 30-day mortality rates were 15.7% (16/102) and 8.8% (9/102), respectively. Cancer-related bleeding increased clinical failure significantly (p = 0.003) and bleeding-related 30-day mortality with marginal significance (p = 0.05). Overall survival was poorer in patients with cancer-related bleeding. CONCLUSION. TAE with NBCA with or without other embolic agents showed high technical and clinical effectiveness in the management of acute nonvariceal gastrointestinal tract bleeding. Cancer-related bleeding was the only factor related to clinical failure, and possibly related to bleeding-related 30-day mortality.

Streptococcus mutans extracellular DNA is upregulated during growth in biofilms, actively released via membrane vesicles, and influenced by components of the protein secretion machinery.

Streptococcus mutans, a major etiological agent of human dental caries, lives primarily on the tooth surface in biofilms. Limited information is available concerning the extracellular DNA (eDNA) as a scaffolding matrix in S. mutans biofilms. This study demonstrates that S. mutans produces eDNA by multiple avenues, including lysis-independent membrane vesicles. Unlike eDNAs from cell lysis that were abundant and mainly concentrated around broken cells or cell debris with floating open ends, eDNAs produced via the lysis-independent pathway appeared scattered but in a structured network under scanning electron microscopy. Compared to eDNA production of planktonic cultures, eDNA production in 5- and 24-h biofilms was increased by >3- and >1.6-fold, respectively. The addition of DNase I to growth medium significantly reduced biofilm formation. In an in vitro adherence assay, added chromosomal DNA alone had a limited effect on S. mutans adherence to saliva-coated hydroxylapatite beads, but in conjunction with glucans synthesized using purified glucosyltransferase B, the adherence was significantly enhanced. Deletion of sortase A, the transpeptidase that covalently couples multiple surface-associated proteins to the cell wall peptidoglycan, significantly reduced eDNA in both planktonic and biofilm cultures. Sortase A deficiency did not have a significant effect on membrane vesicle production; however, the protein profile of the mutant membrane vesicles was significantly altered, including reduction of adhesin P1 and glucan-binding proteins B and C. Relative to the wild type, deficiency of protein secretion and membrane protein insertion machinery components, including Ffh, YidC1, and YidC2, also caused significant reductions in eDNA.

Symbiotic relationship between Streptococcus mutans and Candida albicans synergizes virulence of plaque biofilms in vivo.

Streptococcus mutans is often cited as the main bacterial pathogen in dental caries, particularly in early-childhood caries (ECC). S. mutans may not act alone; Candida albicans cells are frequently detected along with heavy infection by S. mutans in plaque biofilms from ECC-affected children. It remains to be elucidated whether this association is involved in the enhancement of biofilm virulence. We showed that the ability of these organisms together to form biofilms is enhanced in vitro and in vivo. The presence of C. albicans augments the production of exopolysaccharides (EPS), such that cospecies biofilms accrue more biomass and harbor more viable S. mutans cells than single-species biofilms. The resulting 3-dimensional biofilm architecture displays sizeable S. mutans microcolonies surrounded by fungal cells, which are enmeshed in a dense EPS-rich matrix. Using a rodent model, we explored the implications of this cross-kingdom interaction for the pathogenesis of dental caries. Coinfected animals displayed higher levels of infection and microbial carriage within plaque biofilms than animals infected with either species alone. Furthermore, coinfection synergistically enhanced biofilm virulence, leading to aggressive onset of the disease with rampant carious lesions. Our in vitro data also revealed that glucosyltransferase-derived EPS is a key mediator of cospecies biofilm development and that coexistence with C. albicans induces the expression of virulence genes in S. mutans (e.g., gtfB, fabM). We also found that Candida-derived ß1,3-glucans contribute to the EPS matrix structure, while fungal mannan and ß-glucan provide sites for GtfB binding and activity. Altogether, we demonstrate a novel mutualistic bacterium-fungus relationship that occurs at a clinically relevant site to amplify the severity of a ubiquitous infectious disease.

The exopolysaccharide matrix modulates the interaction between 3D architecture and virulence of a mixed-species oral biofilm.

Virulent biofilms are responsible for a range of infections, including oral diseases. All biofilms harbor a microbial-derived extracellular-matrix. The exopolysaccharides (EPS) formed on tooth-pellicle and bacterial surfaces provide binding sites for microorganisms; eventually the accumulated EPS enmeshes microbial cells. The metabolic activity of the bacteria within this matrix leads to acidification of the milieu. We explored the mechanisms through which the Streptococcus mutans-produced EPS-matrix modulates the three-dimensional (3D) architecture and the population shifts during morphogenesis of biofilms on a saliva-coated-apatitic surface using a mixed-bacterial species system. Concomitantly, we examined whether the matrix influences the development of pH-microenvironments within intact-biofilms using a novel 3D in situ pH-mapping technique. Data reveal that the production of the EPS-matrix helps to create spatial heterogeneities by forming an intricate network of exopolysaccharide-enmeshed bacterial-islets (microcolonies) through localized cell-to-matrix interactions. This complex 3D architecture creates compartmentalized acidic and EPS-rich microenvironments throughout the biofilm, which triggers the dominance of pathogenic S. mutans within a mixed-species system. The establishment of a 3D-matrix and EPS-enmeshed microcolonies were largely mediated by the S. mutans gtfB/gtfC genes, expression of which was enhanced in the presence of Actinomyces naeslundii and Streptococcus oralis. Acidic pockets were found only in the interiors of bacterial-islets that are protected by EPS, which impedes rapid neutralization by buffer (pH 7.0). As a result, regions of low pH (<5.5) were detected at specific locations along the surface of attachment. Resistance to chlorhexidine was enhanced in cells within EPS-microcolony complexes compared to those outside such structures within the biofilm. Our results illustrate the critical interaction between matrix architecture and pH heterogeneity in the 3D environment. The formation of structured acidic-microenvironments in close proximity to the apatite-surface is an essential factor associated with virulence in cariogenic-biofilms. These observations may have relevance beyond the mouth, as matrix is inherent to all biofilms.

Clinical values of (18) F-FDG PET/CT in oral cavity cancer with dental artifacts on CT or MRI.

LEVEL OF EVIDENCE: 2a BACKGROUND AND OBJECTIVES: To investigate the role of (18) F-FDG PET/CT in tumor staging, extent, and volume measurements in oral cavity squamous cell carcinoma (OSCC) patients with/without dental artifacts on CT or MRI. METHODS: This study was conducted in 63 consecutive patients with OSCC who received initial workups including (18) F-FDG PET/CT and MRI. The results of the imaging modalities were compared to those of pathology, using McNemar's test and the paired t-test. RESULTS: Thirty-seven patients (59%) had dental or metallic artifacts obscuring primary tumors. (18) F-FDG PET/CT scanning was superior to MRI in tumor staging (weighted κ = 0.870 vs. 0.518, P = 0.004) in patients with dental artifacts. In addition, (18) F-FDG PET/CT scans were more specific than MRI in detecting sublingual gland (P = 0.014) and mouth floor (P = 0.011) involvement. In patients with dental artifacts, there was a significant discrepancy between primary tumor volume (PTV) measured by pathology and MRI (P = 0.018), but not between PTV measured from pathology and (18) F-FDG PET/CT at SUV2.5 (P = 0.245), which showed the highest intraclass correlation coefficient value (0.860). CONCLUSION: (18) F-FDG PET/CT scans provide accurate tumor staging and volume measurements in OSCC patients with CR/MRI dental artifacts, leading to improved preoperative planning. LEVEL OF EVIDENCE: 2b CONDENSED ABSTRACT This study evaluated the clinical value of (18) F-FDG PET/CT in 63 patients with oral cavity cancers. In 37 (59%) patients with dental artifacts on CT/MRI, (18) F-FDG PET/CT showed superior results compared to MRI in tumor staging and represented the highest intraclass correlation coefficient value to tumor volume determined by pathology.

Analysis of the mechanical stability and surface detachment of mature Streptococcus mutans biofilms by applying a range of external shear forces.

Well-established biofilms formed by Streptococcus mutans via exopolysaccharide matrix synthesis are firmly attached to tooth surfaces. Enhanced understanding of the physical properties of mature biofilms may lead to improved approaches to detaching or disassembling these highly organized and adhesive structures. Here, the mechanical stability of S. mutans biofilms was investigated by determining their ability to withstand measured applications of shear stress using a custom-built device. The data show that the initial biofilm bulk (~ 50% biomass) was removed after exposure to 0.184 and 0.449 N m(-2) for 67 and 115 h old biofilms. However, removal of the remaining biofilm close to the surface was significantly reduced (vs initial bulk removal) even when shear forces were increased 10-fold. Treatment of biofilms with exopolysaccharide-digesting dextranase substantially compromised their mechanical stability and rigidity, resulting in bulk removal at a shear stress as low as 0.027 N m(-2) and > a two-fold reduction in the storage modulus (G'). The data reveal how incremental increases in shear stress cause distinctive patterns of biofilm detachment, while demonstrating that the exopolysaccharide matrix modulates the resistance of biofilms to mechanical clearance.