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.

Cerium oxide nanozymes confer a cytoprotective and bio-friendly surface micro-environment to methacrylate based oro-facial prostheses.

Poly-(methyl methacrylate) (PMMA) is the preferred biomaterial for orofacial prostheses used for the rehabilitation of naso-palatal defects. However, conventional PMMA has limitations determined by the complexity of the local microbiota and the friability of oral mucosa adjacent to these defects. Our purpose was to develop a new type of PMMA, i-PMMA, with good biocompatibility and better biological effects such as higher resistance to microbial adhesion of multiple species and enhanced antioxidant effect. The addition of cerium oxide nanoparticles to PMMA using a mesoporous nano-silica carrier and polybetaine conditioning, resulted in an increased release of cerium ions and enzyme mimetic activity, without tangible loss of mechanical properties. Ex vivo experiments confirmed these observations. In stressed human gingival fibroblasts, i-PMMA reduced the levels of reactive oxygen species and increased the expression of homeostasis-related proteins (PPARg, ATG5, LCI/III). Furthermore, i-PMMA increased the levels of expression of superoxide dismutase and mitogen-activated protein kinases (ERK and Akt), and cellular migration. Lastly, we demonstrated the biosafety of i-PMMA using two in vivo models: skin sensitization assay and oral mucosa irritation test, respectively. Therefore, i-PMMA offers a cytoprotective interface that prevents microbial adhesion and attenuates oxidative stress, thus supporting physiological recovery of the oral mucosa.

Effect of surface stiffness in initial adhesion of oral microorganisms under various environmental conditions.

Biofilms are three-dimensional structures formed as a result of microorganism's adhesion on a biotic or abiotic surface. Once a biofilm is established, it is onerous to eradicate it or kill the pathogens therein. Thus, targeting the microbial adhesion process, the initial stage of biofilm formation, is a reasonable approach to avoid challenges associated with subsequently formed biofilms. While many properties of interacting material that play significant roles in initial bacterial adhesion have been widely studied, the effect of surface stiffness on bacterial adhesion was relatively underexplored. In this study, we aimed to investigate the effect of surface stiffness on the adhesion of microbial species found in the oral cavity by employing representative oral bacteria, Streptococcus mutans and Streptococcus oralis, and the fungus, Candida albicans. We compared the adhesion behavior of these species alone or in combination toward various surface stiffness (0.06 - 3.01 MPa) by assessing the adhered and planktonic cell numbers at an early (4 h) adhesion stage under various carbon sources and the presence of conditioning film. Our data revealed that in general, a higher amount of microbial cells adhered to softer PDMS surfaces than stiffer ones, which indicates that surface stiffness plays a role in the adhesion of tested species (either single or co-cultured). This pattern was more obvious under sucrose conditions than glucose + fructose conditions. Interestingly, in monospecies, saliva coating did not alter the effect of surface stiffness on S. mutans adhesion while it diminished S. oralis and C. albicans adhesion. However, in the multispecies model, saliva coating rendered the percentage of all adhered microbes to varied PDMS not distinct. The data provide new insights into the role of surface stiffness on microbial mechanosensing and their initial adhesion behavior which may set a scientific foundation for future anti-adhesion strategies.

A dental implant-on-a-chip for 3D modeling of host-material-pathogen interactions and therapeutic testing platforms.

The precise spatiotemporal control and manipulation of fluid dynamics on a small scale granted by lab-on-a-chip devices provide a new biomedical research realm as a substitute for in vivo studies of host-pathogen interactions. While there has been a rise in the use of various medical devices/implants for human use, the applicability of microfluidic models that integrate such functional biomaterials is currently limited. Here, we introduced a novel dental implant-on-a-chip model to better understand host-material-pathogen interactions in the context of peri-implant diseases. The implant-on-a-chip integrates gingival cells with relevant biomaterials - keratinocytes with dental resin and fibroblasts with titanium while maintaining a spatially separated co-culture. To enable this co-culture, the implant-on-a-chip's core structure necessitates closely spaced, tall microtrenches. Thus, an SU-8 master mold with a high aspect-ratio pillar array was created by employing a unique backside UV exposure with a selective optical filter. With this model, we successfully replicated the morphology of keratinocytes and fibroblasts in the vicinity of dental implant biomaterials. Furthermore, we demonstrated how photobiomodulation therapy might be used to protect the epithelial layer from recurrent bacterial challenges (∼3.5-fold reduction in cellular damage vs. control). Overall, our dental implant-on-a-chip approach proposes a new microfluidic model for multiplexed host-material-pathogen investigations and the evaluation of novel treatment strategies for infectious diseases.

A 3D-Printed Customizable Platform for Multiplex Dynamic Biofilm Studies.

Biofilms are communities of microbes that colonize surfaces. While several biofilm experimental models exist, they often have limited replications of spatiotemporal dynamics surrounding biofilms. For a better understanding dynamic and complex biofilm development, this manuscript presents a customizable platform compatible with off-the-shelf well plates that can monitor microbial adhesion, growth, and associated parameters under various relevant scenarios by taking advantage of 3D printing. The system i) holds any substrate in a stable, vertical position, ii) subjects samples to flow at different angles, iii) switches between static and dynamic modes during an experiment, and iv) allows multiplexing and real-time monitoring of biofilm parameters. Simulated fluid dynamics is employed to estimate flow patterns around discs and shear stresses at disc surfaces. A 3D printed peristaltic pump and a customized pH measurement system for real-time tracking of spent biofilm culture media are equipped with a graphical user interface that grants control over all experimental parameters. The system is tested under static and dynamic conditions with Streptococcus mutans using different carbon sources. By monitoring the effluent pH and characterizing biochemical, microbiological, and morphological properties of cultured biofilms, distinct properties are demonstrated. This novel platform liberates designing experimental strategies for investigations of biofilms under various conditions.

In it together: Candida-bacterial oral biofilms and therapeutic strategies.

Under natural environmental settings or in the human body, the majority of microorganisms exist in complex polymicrobial biofilms adhered to abiotic and biotic surfaces. These microorganisms exhibit symbiotic, mutualistic, synergistic, or antagonistic relationships with other species during biofilm colonization and development. These polymicrobial interactions are heterogeneous, complex and hard to control, thereby often yielding worse outcomes than monospecies infections. Concerning fungi, Candida spp., in particular, Candida albicans is often detected with various bacterial species in oral biofilms. These Candida-bacterial interactions may induce the transition of C. albicans from commensal to pathobiont or dysbiotic organism. Consequently, Candida-bacterial interactions are largely associated with various oral diseases, including dental caries, denture stomatitis, periodontitis, peri-implantitis, and oral cancer. Given the severity of oral diseases caused by cross-kingdom consortia that develop hard-to-remove and highly drug-resistant biofilms, fundamental research is warranted to strategically develop cost-effective and safe therapies to prevent and treat cross-kingdom interactions and subsequent biofilm development. While studies have shed some light, targeting fungal-involved polymicrobial biofilms has been limited. This mini-review outlines the key features of Candida-bacterial interactions and their impact on various oral diseases. In addition, current knowledge on therapeutic strategies to target Candida-bacterial polymicrobial biofilms is discussed.

Bimodal Nanocomposite Platform with Antibiofilm and Self-Powering Functionalities for Biomedical Applications.

Advances in microelectronics and nanofabrication have led to the development of various implantable biomaterials. However, biofilm-associated infection on medical devices still remains a major hurdle that substantially undermines the clinical applicability and advancement of biomaterial systems. Given their attractive piezoelectric behavior, barium titanate (BTO)-based materials have also been used in biological applications. Despite its versatility, the feasibility of BTO-embedded biomaterials as anti-infectious implantable medical devices in the human body has not been explored yet. Here, the first demonstration of clinically viable BTO-nanocomposites is presented. It demonstrates potent antibiofilm properties against Streptococcus mutans without bactericidal effect while retaining their piezoelectric and mechanical behaviors. This antiadhesive effect led to ∼10-fold reduction in colony-forming units in vitro. To elucidate the underlying mechanism for this effect, data depicting unfavorable interaction energy profiles between BTO-nanocomposites and S. mutans using the classical and extended Derjaguin, Landau, Verwey, and Overbeek theories is presented. Direct cell-to-surface binding force data using atomic force microscopy also corroborate reduced adhesion between BTO-nanocomposites and S. mutans. Interestingly, the poling process on BTO-nanocomposites resulted in asymmetrical surface charge density on each side, which may help tackle two major issues in prosthetics-bacterial contamination and tissue integration. Finally, BTO-nanocomposites exhibit superior biocompatibility toward human gingival fibroblasts and keratinocytes. Overall, BTO-embedded composites exhibit broad-scale potential to be used in biological settings as energy-harvestable antibiofilm surfaces.

Diagnosis of Biofilm-Associated Peri-Implant Disease Using a Fluorescence-Based Approach.

Dental implants have become a routine component of daily dental practice and the demand for dental implants is expected to increase significantly in the future. Despite the high success rates of dental implants, failures do occur, resulting in discomfort, rampant destruction of the oral health, or painful and costly surgical replacement of a failed implant. Peri-implant diseases are inflammatory conditions affecting the soft/hard tissues surrounding a functional dental implant. Plenty of experimental evidence indicates that the accumulation of dental plaque at the soft tissue-implant interface and the subsequent local inflammatory response seems to be key in the pathogenesis of the peri-implant mucositis. Such peri-implant-soft tissue interface is less effective than natural teeth in resisting bacterial invasion, enhancing vulnerability to subsequent peri-implant disease. Furthermore, in certain individuals, it will progress to peri-implantitis, resulting in alveolar bone loss and implant failure. Although early diagnosis and accurate identification of risk factors are extremely important to effectively prevent peri-implant diseases, current systematic reviews revealed that a uniform classification and diagnostic methodology for peri-implantitis are lacking. Recent progress on fluorescence-based technology enabled rapid diagnosis of the disease and effective removal of plaques. Here, we briefly review biofilm-associated peri-implant diseases and propose a fluorescence-based approach for more accurate and objective diagnoses. A fluorescence-based diagnosis tool through headlights combined with special-filtered dental loupes may serve as a hands-free solution for both precise diagnosis and effective removal of plaque-biofilms.

Implication of Surface Properties, Bacterial Motility, and Hydrodynamic Conditions on Bacterial Surface Sensing and Their Initial Adhesion.

Biofilms are structured microbial communities attached to surfaces, which play a significant role in the persistence of biofoulings in both medical and industrial settings. Bacteria in biofilms are mostly embedded in a complex matrix comprised of extracellular polymeric substances that provide mechanical stability and protection against environmental adversities. Once the biofilm is matured, it becomes extremely difficult to kill bacteria or mechanically remove biofilms from solid surfaces. Therefore, interrupting the bacterial surface sensing mechanism and subsequent initial binding process of bacteria to surfaces is essential to effectively prevent biofilm-associated problems. Noting that the process of bacterial adhesion is influenced by many factors, including material surface properties, this review summarizes recent works dedicated to understanding the influences of surface charge, surface wettability, roughness, topography, stiffness, and combination of properties on bacterial adhesion. This review also highlights other factors that are often neglected in bacterial adhesion studies such as bacterial motility and the effect of hydrodynamic flow. Lastly, the present review features recent innovations in nanotechnology-based antifouling systems to engineer new concepts of antibiofilm surfaces.

Human Oral Motion-Powered Smart Dental Implant (SDI) for In Situ Ambulatory Photo-biomodulation Therapy.

Peri-implant disease is an inflammatory condition affecting the soft and hard tissues surrounding a dental implant. However, current preventative methods are insufficient due to the limited bioactivity on the dental implant and poor patient compliance. Recently, photo-biomodulation (PBM) therapy that can recover and regenerate peri-implant soft tissue has attracted considerable attention in dentistry. In this paper, a seamless human oral motion-powered dental implant system (called Smart Dental Implant or SDI) is presented as an ambulatory PBM therapy modality. SDI allows the in situ light delivery, which is enabled by the energy harvesting from dynamic human oral motions (chewing and brushing) via an engineered piezoelectric dental crown, an associated circuit, and micro light emitting diodes (LEDs). The SDI also offers adequate mechanical strength as the clinical standards. Using primary human gingival keratinocytes (HGKs) as a model host organism and Pseudomonas aeruginosa lipopolysaccharides (LPS) as a model inflammatory stimulus, effective SDI-mediated PBM therapy is demonstrated. A new class of dental implants could be an ambulatory PBM therapy platform for the prevention of peri-implant disease without patient dependency, warranting long-lasting dental implants.

Anti-biofilm activity of a novel pit and fissure self-adhesive sealant modified with metallic monomers.

Dental plaque is a biofilm composed of a complex oral microbial community. The accumulation of plaque in the pit and fissures of dental elements often leads to the development of tooth decay (dental caries). Here, potent anti-biofilm materials were developed by incorporating zinc methacrylates or di-n-butyl-dimethacrylate-tin into the light-curable sealant and their physical, mechanical, and biological properties were evaluated. The data revealed that 5% di-n-butyl-dimethacrylate-tin (SnM 5%) incorporated sealant showed strong anti-biofilm efficacy against various single-species (Streptococcus mutans or Streptococcus oralis or Candida albicans) and S. mutans-C. albicans cross-kingdom dual-species biofilms without either impairing the mechanical properties of the sealant or causing cytotoxicities against mouse fibroblasts. The findings indicate that the incorporation of SnM 5% in the experimental pit and fissure self-adhesive sealant may have the potential to be part of current chemotherapeutic strategies to prevent the formation of cariogenic oral biofilms that cause dental caries.

A Comprehensive Analysis of Near-Contact Photobiomodulation Therapy in the Host-Bacteria Interaction Model Using 3D-Printed Modular LED Platform.

One well-studied bacterial factor recognized by the host immune system is lipopolysaccharides (LPS) that stimulate host cells, resulting in cell inflammation. Although photobiomodulation (PBM) therapy demonstrates its potency on anti-inflammatory activity, the complete mechanism of action in the host-bacteria interaction model is still elusive. In addition, many studies were performed regarding a distance between the light source and biological sample (non-contact therapy) that may result in disparate reports on the efficacy of PBM therapy. Thus, it is critical to clearly understand the effect of this approach to maximize efficacy and minimize side effects. Here, a custom-built light-emitting diode (LED) platform that mimics near-contact therapy is developed. The effect and mechanism of PBM therapy on epithelial cells in response to LPS is systematically investigated under various conditions (wavelength, irradiation-time, pulse-frequency). The data show that the irradiation of near-infrared (NIR-LED) significantly improves the viability of inflamed cells. It reveals that NIR-LED inhibits the production of reactive oxygen species by regulating the Nox4-NF-κB pathway. Interestingly, however, high-pulse frequency stimulus causes the collapse of the mitochondrial membrane potential (ΔΨm) of cells, resulting in cell death. These results suggest that the optimized "PBM condition" is critical to assist the healthy immune system of the host against bacterial invasion.

Dynamics of bacterial population growth in biofilms resemble spatial and structural aspects of urbanization.

Biofilms develop from bacteria bound on surfaces that grow into structured communities (microcolonies). Although surface topography is known to affect bacterial colonization, how multiple individual settlers develop into microcolonies simultaneously remains underexplored. Here, we use multiscale population-growth and 3D-morphometric analyses to assess the spatiotemporal development of hundreds of bacterial colonizers towards submillimeter-scale microcolony communities. Using an oral bacterium (Streptococcus mutans), we find that microbial cells settle on the surface randomly under sucrose-rich conditions, regardless of surface topography. However, only a subset of colonizers display clustering behavior and growth following a power law. These active colonizers expand three-dimensionally by amalgamating neighboring bacteria into densely populated microcolonies. Clustering and microcolony assembly are dependent on exopolysaccharides, while population growth dynamics and spatial structure are affected by cooperative or antagonistic microbes. Our work suggests that biofilm assembly resembles certain spatial-structural features of urbanization, where population growth and expansion can be influenced by type of settlers, neighboring cells, and further community merging and scaffolding occurring at various scales.

Synergism of Streptococcus mutans and Candida albicans Reinforces Biofilm Maturation and Acidogenicity in Saliva: An In Vitro Study.

Early childhood caries, a virulent-form of dental caries, is painful, difficult, and costly to treat that has been associated with high levels of Streptococcus mutans (Sm) and Candida albicans (Ca) in plaque-biofilms on teeth. These microorganisms appear to develop a symbiotic cross-kingdom interaction that amplifies the virulence of plaque-biofilms. Although biofilm studies reveal synergistic bacterial-fungal association, how these organisms modulate cross-kingdom biofilm formation and enhance its virulence in the presence of saliva remain largely unknown. Here, we compared the properties of Sm and Sm-Ca biofilms cultured in saliva by examining the biofilm structural organization and capability to sustain an acidic pH environment conducive to enamel demineralization. Intriguingly, Sm-Ca biofilm is rapidly matured and maintained acidic pH-values (~4.3), while Sm biofilm development was retarded and failed to create an acidic environment when cultured in saliva. In turn, the human enamel slab surface was severely demineralized by Sm-Ca biofilms, while there was minimal damage to the enamel surface by Sm biofilm. Interestingly, Sm-Ca biofilms exhibited an acidic environment regardless of their hyphal formation ability. Our data reveal the critical role of symbiotic interaction between S. mutans and C. albicans in human saliva in the context of pathogenesis of dental caries, which may explain how the cross-kingdom interaction contributes to enhanced virulence of plaque-biofilm in the oral cavity.

Catalytic antimicrobial robots for biofilm eradication.

Magnetically driven robots can perform complex functions in biological settings with minimal destruction. However, robots designed to damage deleterious biostructures could also have important impact. In particular, there is an urgent need for new strategies to eradicate bacterial biofilms as we approach a post-antibiotic era. Biofilms are intractable and firmly attached structures ubiquitously associated with drug-resistant infections and destruction of surfaces. Existing treatments are inadequate to both kill and remove bacteria leading to reinfection. Here we design catalytic antimicrobial robots (CARs) that precisely and controllably kill, degrade and remove biofilms with remarkable efficiency. CARs exploit iron oxide nanoparticles (NPs) with dual catalytic-magnetic functionality that (i) generate bactericidal free radicals, (ii) breakdown the biofilm exopolysaccharide (EPS) matrix, and (iii) remove the fragmented biofilm debris via magnetic field driven robotic assemblies. We develop two distinct CAR platforms. The first platform, the biohybrid CAR, is formed from NPs and biofilm degradation products. After catalytic bacterial killing and EPS disruption, magnetic field gradients assemble NPs and the biodegraded products into a plow-like superstructure. When driven with an external magnetic field, the biohybrid CAR completely removes biomass in a controlled manner, preventing biofilm regrowth. Biohybrid CARs can be swept over broad swathes of surface or can be moved over well-defined paths for localized removal with microscale precision. The second platform, the 3D molded CAR, is a polymeric soft robot with embedded catalytic-magnetic NPs, formed in a customized 3D printed mold to perform specific tasks in enclosed domains. Vane-shaped CARs remove biofilms from curved walls of cylindrical tubes, and helicoid-shaped CARs drill through biofilm clogs, while simultaneously killing bacteria. In addition, we demonstrate applications of CARs to target highly confined anatomical surfaces in the interior of human teeth. These 'kill-degrade-and-remove' CARs systems could have significant impact in fighting persistent biofilm-infections and in mitigating biofouling of medical devices and diverse surfaces.

Dextran-Coated Iron Oxide Nanoparticles as Biomimetic Catalysts for Localized and pH-Activated Biofilm Disruption.

Biofilms are surface-attached bacterial communities embedded within an extracellular matrix that create localized and protected microenvironments. Acidogenic oral biofilms can demineralize the enamel-apatite on teeth, causing dental caries (tooth decay). Current antimicrobials have low efficacy and do not target the protective matrix and acidic pH within the biofilm. Recently, catalytic nanoparticles were shown to disrupt biofilms but lacked a stabilizing coating required for clinical applications. Here, we report dextran-coated iron oxide nanoparticles termed nanozymes (Dex-NZM) that display strong catalytic (peroxidase-like) activity at acidic pH values, target biofilms with high specificity, and prevent severe caries without impacting surrounding oral tissues in vivo. Nanoparticle formulations were synthesized with dextran coatings (molecular weights from 1.5 to 40 kDa were used), and their catalytic performance and bioactivity were assessed. We found that 10 kDa dextran coating provided maximal catalytic activity, biofilm uptake, and antibiofilm properties. Mechanistic studies indicated that iron oxide cores are the source of catalytic activity, whereas dextran on the nanoparticle surface provided stability without blocking catalysis. Dextran-coating facilitated NZM incorporation into exopolysaccharides (EPS) structure and binding within biofilms, which activated hydrogen peroxide (H2O2) for localized bacterial killing and EPS-matrix breakdown. Surprisingly, dextran coating enhanced selectivity toward biofilms while avoiding binding to gingival cells. Furthermore, Dex-NZM/H2O2 treatment significantly reduced the onset and severity of caries lesions (vs control or either Dex-NZM or H2O2 alone) without adverse effects on gingival tissues or oral microbiota diversity in vivo. Therefore, dextran-coated nanozymes have potential as an alternative treatment to control tooth decay and possibly other biofilm-associated diseases.

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.

Enhanced design and formulation of nanoparticles for anti-biofilm drug delivery.

Biofilms are surface-bound, structured microbial communities underpinning persistent bacterial infections. Biofilms often create acidic pH microenvironments, providing opportunities to leverage responsive drug delivery systems to improve antibacterial efficacy. Here, the antibacterial efficacy of novel formulations containing pH-responsive polymer nanoparticle carriers (NPCs) and farnesol, a hydrophobic antibacterial drug, were investigated. Multiple farnesol-loaded NPCs, which varied in overall molecular weight and corona-to-core molecular weight ratios (CCRs), were tested using standard and saturated drug loading conditions. NPCs loaded at saturated conditions exhibited ∼300% greater drug loading capacity over standard conditions. Furthermore, saturated loading conditions sustained zero-ordered drug release over 48 hours, which was 3-fold longer than using standard farnesol loading. Anti-biofilm activity of saturated NPC loading was markedly amplified using Streptococcus mutans as a biofilm-forming model organism. Specifically, reductions of ∼2-4 log colony forming unit (CFU) were obtained using microplate and saliva-coated hydroxyapatite biofilm assays. Mechanistically, the new formulation reduced total biomass by disrupting insoluble glucan formation and increased NPC-cell membrane localization. Finally, thonzonium bromide, a highly potent, FDA-approved antibacterial drug with similar alkyl chain structure to farnesol, was also loaded into NPCs and used to treat S. mutans biofilms. Similar to farnesol-loaded NPCs, thonzonium bromide-loaded NPCs increased drug loading capacity ≥2.5-fold, demonstrated nearly zero-order release kinetics over 96 hours, and reduced biofilm cell viability by ∼6 log CFU. This work provides foundational insights that may lead to clinical translation of novel topical biofilm-targeting therapies, such as those for oral diseases.

Topical ferumoxytol nanoparticles disrupt biofilms and prevent tooth decay in vivo via intrinsic catalytic activity.

Ferumoxytol is a nanoparticle formulation approved by the U.S. Food and Drug Administration for systemic use to treat iron deficiency. Here, we show that, in addition, ferumoxytol disrupts intractable oral biofilms and prevents tooth decay (dental caries) via intrinsic peroxidase-like activity. Ferumoxytol binds within the biofilm ultrastructure and generates free radicals from hydrogen peroxide (H2O2), causing in situ bacterial death via cell membrane disruption and extracellular polymeric substances matrix degradation. In combination with low concentrations of H2O2, ferumoxytol inhibits biofilm accumulation on natural teeth in a human-derived ex vivo biofilm model, and prevents acid damage of the mineralized tissue. Topical oral treatment with ferumoxytol and H2O2 suppresses the development of dental caries in vivo, preventing the onset of severe tooth decay (cavities) in a rodent model of the disease. Microbiome and histological analyses show no adverse effects on oral microbiota diversity, and gingival and mucosal tissues. Our results reveal a new biomedical application for ferumoxytol as topical treatment of a prevalent and costly biofilm-induced oral disease.

Bacterial-derived exopolysaccharides enhance antifungal drug tolerance in a cross-kingdom oral biofilm.

Fungal-bacterial interactions generate unique biofilms that cause many infections in humans. Candida albicans interact with Streptococcus mutans in dental biofilms associated with severe childhood tooth-decay, a prevalent pediatric oral disease. Current modalities are ineffective and primarily based on antimicrobial monotherapies despite the polymicrobial nature of the infection. Here, we show that the combination of clinically used topical antifungal fluconazole with povidone iodine (PI) can completely suppress C. albicans carriage and mixed-biofilm formation without increasing bacterial killing activity in vivo. We unexpectedly found that the inclusion of PI enhanced fluconazole efficacy by potently disrupting the assembly of a protective bacterial exopolysaccharide (EPS) matrix through inhibition of α-glucan synthesis by S. mutans exoenzyme (GtfB) bound on the fungal surface. Further analyses revealed that the EPS produced in situ directly bind and sequester fluconazole, reducing uptake and intracellular transportation of the drug. Conversely, inhibition of GtfB activity by PI, enzymatic degradation of the α-glucan matrix or co-culturing with gtfB-defective S. mutans re-established antifungal susceptibility. Hence, topical antifungal has limitations in mixed oral biofilms due to enhanced C. albicans tolerance to fluconazole afforded by the shielding effect of bacterial-derived EPS. The data provide new insights for treatment of C. albicans in cross-kingdom biofilms, indicating that EPS inhibitors may be required for enhanced killing efficacy and optimal anti-biofilm activity.