Hyposalivation but not Sjögren's syndrome associated with microbial dysbiosis in women.

Background: Saliva modulates the environment of the oral biofilm through pH buffer, microbial attachment to host surfaces, and nutritional source. The ecology of stress occurs when a physical factor adversely impacts an ecosystem or its biotic components. Therefore, reduced salivary flow can affect oral-host balance. The leading causes of hyposalivation include disease-associated Sjögren's syndrome (SS) and menopausal women as aging-associated. However, little is known about the oral microbiome integrated with sex hormones in hyposalivation. This study aimed to characterize the hyposalivation microbiome caused by aging or disease affecting the salivary glands in women. Methods: We included 50 women older than 40 years of age in any menopausal phase. We collected stimulated saliva from 25 women diagnosed with SS (SS) and 25 without SS (non-SS). The bacterial profile of the patients was obtained by 16S rRNA sequencing. Bioinformatics analysis used machine learning to analyze the cohort's signs, symptoms, and bacterial profile. Salivary estradiol as a sex hormone variation level was determined. Results: We obtained that 79% of the SS group, and 52% of the non-SS group had hyposalivation. We found a negatively correlated Prevotella-age and Rothia-estradiol in the SS group. Highlight, we found that the cause of the hyposalivation in the study did not explain differences in microbial diversity comparing non-SS and SS groups. Therefore, microbial communities found in hyposalivation but not related to systemic conditions suggest that changes in the oral environment might underpin host-microbial balance. Conclusion: The salivary microbiome was similar in women with and without SS. However, hyposalivation showed two distinctive clusters associated with the bacterial population profiles. Our study suggests that local ecological disturbances could drive the change in the microbiome.

Oral microbial biomap in the drought environment: Sjogren's syndrome.

Sjogren's syndrome (SS) is an autoimmune disease that affects primarily the salivary glands, making perturbations in the oral ecosystem and potential factors of salivary flow that influence the onset and development of the disease. The oral cavity contains diverse microorganisms that inhabit various niches such as the oral microbial "biomap." It does not seem specific enough to establish a characteristic microbiome, given the diversity of clinical manifestations, variable rates of salivary secretion, and influential risk factors in patients with SS. This review discusses the biogeography of the oral microbiome in patients with SS such as saliva, tongue, tooth, mucosa, and gum. The microorganisms that were more abundant in the different oral niches were Gram-positive species, suggesting a higher survival of cell wall bacteria in this arid oral environment. Reduced salivary flow appears not to be linked to the cause of dysbiosis alone but influences host-associated risk factors. However, much work remains to be done to establish the role of the microbiome in the etiopathogenesis of autoimmune diseases such as SS. Future studies of the microbiome in autoimmunity will shed light on the role of specific microorganisms that have never been linked before with SS.

Iron oxide nanozymes stabilize stannous fluoride for targeted biofilm killing and synergistic oral disease prevention.

Dental caries is the most common human disease caused by oral biofilms despite the widespread use of fluoride as the primary anticaries agent. Recently, an FDA-approved iron oxide nanoparticle (ferumoxytol, Fer) has shown to kill and degrade caries-causing biofilms through catalytic activation of hydrogen peroxide. However, Fer cannot interfere with enamel acid demineralization. Here, we show notable synergy when Fer is combined with stannous fluoride (SnF2), markedly inhibiting both biofilm accumulation and enamel damage more effectively than either alone. Unexpectedly, we discover that the stability of SnF2 is enhanced when mixed with Fer in aqueous solutions while increasing catalytic activity of Fer without any additives. Notably, Fer in combination with SnF2 is exceptionally effective in controlling dental caries in vivo, even at four times lower concentrations, without adverse effects on host tissues or oral microbiome. Our results reveal a potent therapeutic synergism using approved agents while providing facile SnF2 stabilization, to prevent a widespread oral disease with reduced fluoride exposure.

Iron oxide nanozymes stabilize stannous fluoride for targeted biofilm killing and synergistic oral disease prevention.

Dental caries (tooth decay) is the most prevalent human disease caused by oral biofilms, affecting nearly half of the global population despite increased use of fluoride, the mainstay anticaries (tooth-enamel protective) agent. Recently, an FDA-approved iron oxide nanozyme formulation (ferumoxytol, Fer) has been shown to disrupt caries-causing biofilms with high specificity via catalytic activation of hydrogen peroxide, but it is incapable of interfering with enamel acid demineralization. Here, we find notable synergy when Fer is combined with stannous fluoride (SnF 2 ), markedly inhibiting both biofilm accumulation and enamel damage more effectively than either alone. Unexpectedly, our data show that SnF 2 enhances the catalytic activity of Fer, significantly increasing reactive oxygen species (ROS) generation and antibiofilm activity. We discover that the stability of SnF 2 (unstable in water) is markedly enhanced when mixed with Fer in aqueous solutions without any additives. Further analyses reveal that Sn 2+ is bound by carboxylate groups in the carboxymethyl-dextran coating of Fer, thus stabilizing SnF 2 and boosting the catalytic activity. Notably, Fer in combination with SnF 2 is exceptionally effective in controlling dental caries in vivo , preventing enamel demineralization and cavitation altogether without adverse effects on the host tissues or causing changes in the oral microbiome diversity. The efficacy of SnF 2 is also enhanced when combined with Fer, showing comparable therapeutic effects at four times lower fluoride concentration. Enamel ultrastructure examination shows that fluoride, iron, and tin are detected in the outer layers of the enamel forming a polyion-rich film, indicating co-delivery onto the tooth surface. Overall, our results reveal a unique therapeutic synergism using approved agents that target complementary biological and physicochemical traits, while providing facile SnF 2 stabilization, to prevent a widespread oral disease more effectively with reduced fluoride exposure.

Human Tooth as a Fungal Niche: Candida albicans Traits in Dental Plaque Isolates.

Candida albicans, a fungus typically found in the mucosal niche, is frequently detected in biofilms formed on teeth (dental plaque) of toddlers with severe childhood caries, a global public health problem that causes rampant tooth decay. However, knowledge about fungal traits on the tooth surface remains limited. Here, we assess the phylogeny, phenotype, and interkingdom interactions of C. albicans isolated from plaque of diseased toddlers and compare their properties to reference strains, including 529L (mucosal isolate). C. albicans isolates exhibit broad phenotypic variations, but all display cariogenic traits, including high proteinase activity, acidogenicity, and acid tolerance. Unexpectedly, we find distinctive variations in filamentous growth, ranging from hyphal defective to hyperfilamentous. We then investigate the ability of tooth isolates to form interkingdom biofilms with Streptococcus mutans (cariogenic partner) and Streptococcus gordonii (mucosal partner). The hyphal-defective isolate lacks cobinding with S. gordonii, but all C. albicans isolates develop robust biofilms with S. mutans irrespective of their filamentation state. Moreover, either type of C. albicans (hyphae defective or hyperfilamentous) enhances sucrose metabolism and biofilm acidogenicity, creating highly acidic environmental pH (<5.5). Notably, C. albicans isolates show altered transcriptomes associated with pH, adhesion, and cell wall composition (versus reference strains), further supporting niche-associated traits. Our data reveal that C. albicans displays distinctive adaptive mechanisms on the tooth surface and develops interactions with pathogenic bacteria while creating an acidogenic state regardless of fungal morphology, contrasting with interkingdom partnerships in mucosal infections. Human tooth may provide new insights into fungal colonization/adaptation, interkingdom biofilms, and contributions to disease pathogenesis. IMPORTANCE Severe early childhood caries is a widespread global public health problem causing extensive tooth decay and systemic complications. Candida albicans, a fungus typically found in mucosal surfaces, is frequently detected in dental plaque formed on teeth of diseased toddlers. However, the clinical traits of C. albicans isolated from tooth remain underexplored. Here, we find that C. albicans tooth isolates exhibit unique biological and transcriptomic traits. Notably, interkingdom biofilms with S. mutans can be formed irrespective of their filamentation state. Furthermore, tooth isolates commonly share dental caries-promoting functions, including acidogenesis, proteolytic activity, and enhanced sugar metabolism, while displaying increased expression of pH-responsive and adhesion genes. Our findings reveal that C. albicans colonizing human teeth displays distinctive adaptive mechanisms to mediate interkingdom interactions associated with a disease-causing state on a mineralized surface, providing new insights into Candida pathobiology and its role in a costly pediatric disease.

Dietary sugars modulate bacterial-fungal interactions in saliva and inter-kingdom biofilm formation on apatitic surface.

Bacteria and fungi can interact to form inter-kingdom biofilms in the oral cavity. Streptococcus mutans and Candida albicans are frequently detected in saliva and in dental biofilms associated with early childhood caries (tooth-decay), a prevalent oral disease induced by dietary sugars. However, how different sugars influence this bacterial-fungal interaction remains unclear. Here, we investigate whether specific sugars affect the inter-kingdom interaction in saliva and subsequent biofilm formation on tooth-mimetic surfaces. The microbes were incubated in saliva containing common dietary sugars (glucose and fructose, sucrose, starch, and combinations) and analyzed via fluorescence imaging and quantitative computational analyses. The bacterial and fungal cells in saliva were then transferred to hydroxyapatite discs (tooth mimic) to allow microbial binding and biofilm development. We found diverse bacterial-fungal aggregates which varied in size, structure, and spatial organization depending on the type of sugars. Sucrose and starch+sucrose induced the formation of large mixed-species aggregates characterized by bacterial clusters co-bound with fungal cells, whereas mostly single-cells were found in the absence of sugar or in the presence of glucose and fructose. Notably, both colonization and further growth on the apatitic surface were dependent on sugar-mediated aggregation, leading to biofilms with distinctive spatial organizations and 3D architectures. Starch+sucrose and sucrose-mediated aggregates developed into large and highly acidogenic biofilms with complex network of bacterial and fungal cells (yeast and hyphae) surrounded by an intricate matrix of extracellular glucans. In contrast, biofilms originated from glucose and fructose-mediated consortia (or without sugar) were sparsely distributed on the surface without structural integration, growing predominantly as individual species with reduced acidogenicity. These findings reveal the impact of dietary sugars on inter-kingdom interactions in saliva and how they mediate biofilm formation with distinctive structural organization and varying acidogenicity implicated with human tooth-decay.

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.

The Lung Allograft Microbiome Associates with Pepsin, Inflammation, and Primary Graft Dysfunction.

Rationale: Primary graft dysfunction (PGD) is the principal cause of early morbidity and mortality after lung transplantation. The lung microbiome has been implicated in later transplantation outcomes but has not been investigated in PGD. Objectives: To define the peritransplant bacterial lung microbiome and relationship to host response and PGD. Methods: This was a single-center prospective cohort study. Airway lavage samples from donor lungs before organ procurement and recipient allografts immediately after implantation underwent bacterial 16S ribosomal ribonucleic acid gene sequencing. Recipient allograft samples were analyzed for cytokines by multiplex array and pepsin by ELISA. Measurements and Main Results: We enrolled 139 transplant subjects and obtained donor lung (n = 109) and recipient allograft (n = 136) samples. Severe PGD (persistent grade 3) developed in 15 subjects over the first 72 hours, and 40 remained without PGD (persistent grade 0). The microbiome of donor lungs differed from healthy lungs, and recipient allograft microbiomes differed from donor lungs. Development of severe PGD was associated with enrichment in the immediate postimplantation lung of oropharyngeal anaerobic taxa, particularly Prevotella. Elevated pepsin, a gastric biomarker, and a hyperinflammatory cytokine profile were present in recipient allografts in severe PGD and strongly correlated with microbiome composition. Together, immediate postimplantation allograft Prevotella/Streptococcus ratio, pepsin, and indicator cytokines were associated with development of severe PGD during the 72-hour post-transplantation period (area under the curve = 0.81). Conclusions: Lung allografts that develop PGD have a microbiome enriched in anaerobic oropharyngeal taxa, elevated gastric pepsin, and hyperinflammatory phenotype. These findings suggest a possible role for peritransplant aspiration in PGD, a potentially actionable mechanism that warrants further investigation.

Polymicrobial Aggregates in Human Saliva Build the Oral Biofilm.

Biofilm community development has been established as a sequential process starting from the attachment of single cells on a surface. However, microorganisms are often found as aggregates in the environment and in biological fluids. Here, we conduct a comprehensive analysis of the native structure and composition of aggregated microbial assemblages in human saliva and investigate their spatiotemporal attachment and biofilm community development. Using multiscale imaging, cell sorting, and computational approaches combined with sequencing analysis, a diverse mixture of aggregates varying in size, structure, and microbial composition, including bacteria associated with host epithelial cells, can be found in saliva in addition to a few single-cell forms. Phylogenetic analysis reveals a mixture of complex consortia of aerobes and anaerobes in which bacteria traditionally considered early and late colonizers are found mixed together. When individually tracked during colonization and biofilm initiation, aggregates rapidly proliferate and expand tridimensionally, modulating population growth, spatial organization, and community scaffolding. In contrast, most single cells remain static or are incorporated by actively growing aggregates. These results suggest an alternative biofilm development process whereby aggregates containing different species or associated with human cells collectively adhere to the surface as "growth nuclei" to build the biofilm and shape polymicrobial communities at various spatial and taxonomic scales. IMPORTANCE Microbes in biological fluids can be found as aggregates. How these multicellular structures bind to surfaces and initiate the biofilm life cycle remains understudied. Here, we investigate the structural organization of microbial aggregates in human saliva and their role in biofilm formation. We found diverse mixtures of aggregates with different sizes, structures, and compositions in addition to free-living cells. When individually tracked during binding and growth on tooth-like surfaces, most aggregates developed into structured biofilm communities, whereas most single cells remained static or were engulfed by the growing aggregates. Our results reveal that preformed microbial consortia adhere as "buds of growth," governing biofilm initiation without specific taxonomic order or cell-by-cell succession, which provide new insights into spatial and population heterogeneity development in complex ecosystems.

Evaluation of possible biomarkers for caries risk in children 6 to 12 years of age.

Background: Electrolytes, proteins, and other salivary molecules play an important role in tooth integrity and can serve as biomarkers associated with caries. Objective: To determine the concentration of potential biomarkers in children without caries (CF) and children with caries (CA). Methods: Unstimulated saliva was collected, and the biomarkers quantified in duplicate, using commercial Enzyme Linked Immunosorbent Assay (ELISA) kits to determine IgA, fibronectin, cathelicidin LL-37, and statherin levels, as well as colorimetric tests to detect formate and phosphate. Results: Significantly higher concentrations of statherin was detected in the CF group (Median: 94,734.6; IQR: 92,934.6-95,113.7) compared to the CA2 group (90,875.0; IQR: 83,580.2-94,633.4) (p = 0.03). Slightly higher median IgA (48,250.0; IQR: 31,461.9-67,418.8) and LL-37 levels (56.1; IQR 43.6-116.2) and a lower concentration of formate were detected in the CF group (0.02; IQR 0.0034-0.15) compared to the group with caries (IgA: 37,776.42; IQR: 33,383.9-44,128.5; LL-37: 46.3; IQR: 40.1011-67.7; formate: 0.10; IQR: 0.01-0.18), but these differences were not statistically significant. Conclusion: The fact that these compounds have been identified as good markers for caries among European adults highlights the difficulty of identifying universal biomarkers that are applicable to all ages or to different populations.

The lung microbiome in lung transplantation.

Culture-independent study of the lower respiratory tract after lung transplantation has enabled an understanding of the microbiome - that is, the collection of bacteria, fungi, and viruses, and their respective gene complement - in this niche. The lung has unique features as a microbial environment, with balanced entry from the upper respiratory tract, clearance, and local replication. There are many pressures impacting the microbiome after transplantation, including donor allograft factors, recipient host factors such as underlying disease and ongoing exposure to the microbe-rich upper respiratory tract, and transplantation-related immunosuppression, antimicrobials, and postsurgical changes. To date, we understand that the lung microbiome after transplant is dysbiotic; that is, it has higher biomass and altered composition compared to a healthy lung. Emerging data suggest that specific microbiome features may be linked to host responses, both immune and non-immune, and clinical outcomes such as chronic lung allograft dysfunction (CLAD), but many questions remain. The goal of this review is to put into context our burgeoning understanding of the lung microbiome in the postlung transplant patient, the interactions between microbiome and host, the role the microbiome may play in post-transplant complications, and critical outstanding research questions.

Impact of the repurposed drug thonzonium bromide on host oral-gut microbiomes.

Drug repurposing is a feasible strategy for the development of novel therapeutic applications. However, its potential use for oral treatments and impact on host microbiota remain underexplored. Here, we assessed the influences of topical oral applications of a repurposed FDA-approved drug, thonzonium bromide, on gastrointestinal microbiomes and host tissues in a rat model of dental caries designed to reduce cross-contamination associated with coprophagy. Using this model, we recapitulated the body site microbiota that mirrored the human microbiome profile. Oral microbiota was perturbed by the treatments with specific disruption of Rothia and Veillonella without affecting the global composition of the fecal microbiome. However, disturbances in the oral-gut microbial interactions were identified using nestedness and machine learning, showing increased sharing of oral taxon Sutterella in the gut microbiota. Host-tissue analyses revealed caries reduction on teeth by thonzonium bromide without cytotoxic effects, indicating bioactivity and biocompatibility when used orally. Altogether, we demonstrate how an oral treatment using a repurposed drug causes localized microbial disturbances and therapeutic effects while promoting turnover of specific oral species in the lower gut in vivo.

Precision targeting of bacterial pathogen via bi-functional nanozyme activated by biofilm microenvironment.

Human dental caries is an intractable biofilm-associated disease caused by microbial interactions and dietary sugars on the host's teeth. Commensal bacteria help control opportunistic pathogens via bioactive products such as hydrogen peroxide (H2O2). However, high-sugar consumption disrupts homeostasis and promotes pathogen accumulation in acidic biofilms that cause tooth-decay. Here, we exploit the pathological (sugar-rich/acidic) conditions using a nanohybrid system to increase intrinsic H2O2 production and trigger pH-dependent reactive oxygen species (ROS) generation for efficient biofilm virulence targeting. The nanohybrid contains glucose-oxidase that catalyzes glucose present in biofilms to increase intrinsic H2O2, which is converted by iron oxide nanoparticles with peroxidase-like activity into ROS in acidic pH. Notably, it selectively kills Streptococcus mutans (pathogen) without affecting Streptococcus oralis (commensal) via preferential pathogen-binding and in situ ROS generation. Furthermore, nanohybrid treatments potently reduced dental caries in a rodent model. Compared to chlorhexidine (positive-control), which disrupted oral microbiota diversity, the nanohybrid had significant higher efficacy without affecting soft-tissues and the oral-gastrointestinal microbiomes, while modulating dental health-associated microbial activity in vivo. The data reveal therapeutic precision of a bi-functional hybrid nanozyme against a biofilm-related disease in a controlled-manner activated by pathological conditions.

Potential implications of SARS-CoV-2 oral infection in the host microbiota.

The oral cavity, as the entry point to the body, may play a critical role in the pathogenesis of SARS-CoV-2 infection that has caused a global outbreak of the coronavirus disease 2019 (COVID-19). Available data indicate that the oral cavity may be an active site of infection and an important reservoir of SARS-CoV-2. Considering that the oral surfaces are colonized by a diverse microbial community, it is likely that viruses have interactions with the host microbiota. Patients infected by SARS-CoV-2 may have alterations in the oral and gut microbiota, while oral species have been found in the lung of COVID-19 patients. Furthermore, interactions between the oral, lung, and gut microbiomes appear to occur dynamically whereby a dysbiotic oral microbial community could influence respiratory and gastrointestinal diseases. However, it is unclear whether SARS-CoV-2 infection can alter the local homeostasis of the resident microbiota, actively cause dysbiosis, or influence cross-body sites interactions. Here, we provide a conceptual framework on the potential impact of SARS-CoV-2 oral infection on the local and distant microbiomes across the respiratory and gastrointestinal tracts ('oral-tract axes'), which remains largely unexplored. Studies in this area could further elucidate the pathogenic mechanism of SARS-CoV-2 and the course of infection as well as the clinical symptoms of COVID-19 across different sites in the human host.

Upper Respiratory Dysbiosis with a Facultative-dominated Ecotype in Advanced Lung Disease and Dynamic Change after Lung Transplant.

Rationale: The oropharyngeal microbiome is a primary source of lung microbiota, contributes to lower respiratory infection, and is also a driver of oral health.Objectives: We sought to understand oropharyngeal microbial communities in advanced lung disease, community dynamics after lung transplantation, and ecological features of dysbiosis.Methods: Oropharyngeal wash samples were obtained from individuals with end-stage disease awaiting transplantation (n = 22) and longitudinally from individuals at 6 weeks, 3 months, and 6 months after transplantation (n = 33), along with healthy control subjects (n = 14). Bacterial 16S and fungal internal transcribed spacer rRNA regions were deep-sequenced, and bacterial community respiratory patterns were imputed from taxonomic composition.Results: Healthy subjects' oropharyngeal microbiomes showed a gradient of community types reflecting relative enrichment of strictly anaerobic, aerobic, or facultative anaerobic bacteria. Patients with end-stage lung disease showed severe dysbiosis by both taxonomic composition and respiration phenotypes, with reduced richness and diversity, increased facultative and decreased aerobic bacteria, and absence of communities characterized by obligate aerobes. In patients at 6 weeks and 3 months post-transplant, richness and diversity were intermediate between healthy and pretransplant subjects, with near-normal distribution of community types. However, by 6 months post-transplant, oropharyngeal wash resembled the low-diversity facultative-dominated profile of pretransplant subjects. Community ecotype correlated with Candida abundance.Conclusions: End-stage lung disease is associated with marked upper respiratory tract dysbiosis involving both community structure and respiratory metabolism profiles of constituent bacteria. Dynamic changes occur after lung transplantation, with partial normalization early but later appearance of severe dysbiosis similar to pretransplant patients. Aberrant oropharyngeal communities may predispose to abnormal lung microbiota and infection risk both in advanced lung disease and after transplantation.

Molecular analysis of the endobronchial stent microbial biofilm reveals bacterial communities that associate with stent material and frequent fungal constituents.

Endobronchial stents are increasingly used to treat airway complications in multiple conditions including lung transplantation but little is known about the biofilms that form on these devices. We applied deep sequencing to profile luminal biofilms of 46 endobronchial stents removed from 20 subjects primarily with lung transplantation-associated airway compromise. Microbial communities were analyzed by bacterial 16S rRNA and fungal ITS marker gene sequencing. Corynebacterium was the most common bacterial taxa across biofilm communities. Clustering analysis revealed three bacterial biofilm types: one low diversity and dominated by Corynebacterium; another was polymicrobial and characterized by Staphylococcus; and the third was polymicrobial and associated with Pseudomonas, Streptococcus, and Prevotella. Biofilm type was significantly correlated with stent material: covered metal with the Staphylococcus-type biofilm, silicone with the Corynebacterium-dominated biofilm, and uncovered metal with the polymicrobial biofilm. Subjects with sequential stents had frequent transitions between community types. Fungal analysis found Candida was most prevalent, Aspergillus was common and highly enriched in two of three stents associated with airway anastomotic dehiscence, and fungal taxa not typically considered pathogens were highly enriched in some stents. Thus, molecular analysis revealed a complex and dynamic endobronchial stent biofilm with three bacterial types that associate with stent material, a central role for Corynebacterium, and that both expected and unexpected fungi inhabit this unique niche. The current work provides a foundation for studies to investigate the relationship between stent biofilm composition and clinical outcomes, mechanisms of biofilm establishment, and strategies for improved stent technology and use in airway compromise.

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.

In situ substrate-formed biofilms using IDODS mimic supragingival tooth-formed biofilms.

This study aimed to compare the bacterial viability and diversity of a substrate-formed biofilm (SF-biofilm) in situ to a supragingival tooth-formed biofilm (TF-biofilm) in the same group of individuals. The impact of the device/disc position and toothbrushing during the formation of SF-biofilm was also assessed. Two tests were run. In test 1, 15 volunteers wore two hemi-splints carrying six discs of human enamel, glass, and hydroxyapatite for 2 days, and were instructed to not perform any oral hygiene measure. Biofilm samples were collected from the substrates and the contralateral tooth and were analysed using CLSM. In five volunteers, half of the biofilm present on the discs and their contralateral teeth were scraped and analysed using 16S  pyrosequencing. In test 2, the microscopic analysis was repeated only on the SF-biofilm samples, and the volunteers were allowed to brush their teeth. Multivariate analyses revealed that the donors had a significant effect on the composition of the biofilm, confirming its subject-dependent character. The bacterial composition of the SF-biofilm was similar to the TF-biofilm, with significant differential abundance detected in very few taxa of low abundance. The toothbrushing during the formation of SF-biofilm was the only factor that conditioned the thickness or bacterial viability.

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.

Stimulated and unstimulated saliva samples have significantly different bacterial profiles.

Epidemiological studies use saliva on a regular basis as a non-invasive and easy-to-take sample, which is assumed to be a microbial representative of the oral cavity ecosystem. However, comparative studies between different kinds of saliva samples normally used in microbial studies are scarce. The aim of the current study was to compare oral microbiota composition between two different saliva samples collected simultaneously: non-stimulated saliva with paper points and stimulated saliva collected after chewing paraffin gum. DNA was extracted from saliva samples of ten individuals, then analyzed by 16S rRNA pyrosequencing to describe bacterial diversity. The results demonstrate significant differences between the microbiota of these two kinds of saliva. Stimulated saliva was found to contain an estimated number of species over three times higher than unstimulated saliva. In addition, bacterial composition at the class and genus level was radically different between both types of samples. When compared to other oral niches, both types of saliva showed some similarity to tongue and buccal mucosa, but they do not correlate at all with the bacterial composition described in supra- or sub-gingival dental plaque, questioning their use in etiological and epidemiological studies of oral diseases of microbial origin.