Randomized Trial to Test a Chemo-Mechanical Antiplaque Regimen as Adjunct to Periodontal Therapy.

BACKGROUND: The control of dental biofilm regrowth after nonsurgical periodontal therapy is associated with better clinical outcomes. However, many patients have difficulty achieving optimal plaque control. Subjects with diabetes, in which immune and wound-healing responses are typically impaired, may benefit from intensive antiplaque control regimens after scaling and root planing (SRP). OBJECTIVES: This study aimed to evaluate the effects of an intensive, at-home, chemical, and mechanical antiplaque regimen as an adjunct to SRP for the treatment of moderate to severe periodontitis. A secondary objective was to compare responses in subjects with type 2 diabetes and nondiabetics. METHODS: This was a 6-mo, single-center, parallel-group, randomized trial. The test group received SRP and oral hygiene instructions, and subjects were instructed to use a 0.12% chlorhexidine gluconate mouthrinse twice a day for 3 mo and utilize rubber interproximal bristle cleaners twice a day for 6 mo. The control group received SRP and oral hygiene instructions. The main outcome was change in mean probing depth (PD) from baseline to 6 mo. Secondary outcomes included change in sites with deep PDs, mean clinical attachment level, bleeding on probing, plaque index, hemoglobin A1C, fasting blood glucose, C-reactive protein, and taste assessment. This study was registered at ClinicalTrials.gov as NCT04830969. RESULTS: In total, 114 subjects were randomized to either treatment. Eighty-six subjects completed the trial with no missing visits. Neither an intention-to-treat nor a per-protocol analysis showed statistically significant differences between treatment groups in mean PD at 6 mo. In a subgroup analysis, subjects with diabetes in the test group showed a statistically significant greater reduction in mean PD at 6 mo when compared to subjects with diabetes receiving the control treatment (Δ = 0.15, P = 0.04), while there were no differences within nondiabetics (Δ = 0.02, P = 0.75). CONCLUSION: Outcomes in subjects with diabetes may be improved by chemo-mechanical antiplaque measures after nonsurgical periodontal therapy. KNOWLEDGE TRANSFER STATEMENT: This study suggests diabetic subjects may benefit from an intensive, at-home, chemical, and mechanical antiplaque regimen to improve nonsurgical periodontal therapy outcomes.

Critically Appraising the Significance of the Oral Mycobiome.

Recent efforts to understand the oral microbiome have focused on its fungal component. Since fungi occupy a low proportion of the oral microbiome biomass, mycobiome studies rely on sequencing of internal transcribed spacer (ITS) amplicons. ITS-based studies usually detect hundreds of fungi in oral samples. Here, we review the oral mycobiome, critically appraising the significance of such large fungal diversity. When harsh lysis methods are used to extract DNA, 2 oral mycobiome community types (mycotypes) are evident, each dominated by only 1 genus, either Candida or Malassezia. The rest of the diversity in ITS surveys represents low-abundance fungi possibly acquired from the environment and ingested food. So far, Candida is the only genus demonstrated to reach a significant biomass in the oral cavity and clearly shown to be associated with a distinct oral ecology. Candida thrives in the presence of lower oral pH and is enriched in caries, with mechanistic studies in animal models suggesting it participates in the disease process by synergistically interacting with acidogenic bacteria. Candida serves as the main etiological agent of oral mucosal candidiasis, in which a Candida-bacteriome partnership plays a key role. The function of other potential oral colonizers, such as lipid-dependent Malassezia, is still unclear, with further studies needed to establish whether Malassezia are metabolically active oral commensals. Low-abundance oral mycobiome members acquired from the environment may be viable in the oral cavity, and although they may not play a significant role in microbiome communities, they could serve as opportunistic pathogens in immunocompromised hosts. We suggest that further work is needed to ascertain the significance of oral mycobiome members beyond Candida. ITS-based surveys should be complemented with other methods to determine the in situ biomass and metabolic state of fungi thought to play a role in the oral environment.

The Salivary Mycobiome Contains 2 Ecologically Distinct Mycotypes.

A broad range of fungi has been detected in molecular surveys of the oral mycobiome. However, knowledge is still lacking on interindividual variability of these communities and the ecologic and clinical significance of oral fungal commensals. In this cross-sectional study, we use internal transcribed spacer 1 amplicon sequencing to evaluate the salivary mycobiome in 59 subjects, 36 of whom were scheduled to receive cancer chemotherapy. Analysis of the broad population structure of fungal communities in the whole cohort identified 2 well-demarcated genus-level community types (mycotypes), with Candida and Malassezia as the main taxa driving cluster partitioning. The Candida mycotype had lower diversity than the Malassezia mycotype and was positively correlated with cancer and steroid use in these subjects, smoking, caries, utilizing a removable prosthesis, and plaque index. Mycotypes were also associated with metabolically distinct bacteria indicative of divergent oral environments, with aciduric species enriched in the Candida mycotype and inflammophilic bacteria increased in the Malassezia mycotype. Similar to their fungal counterparts, coexisting bacterial communities associated with the Candida mycotype showed lower diversity than those associated with the Malassezia mycotype, suggesting that common environmental pressures affected bacteria and fungi. Mycotypes were also seen in an independent cohort of 24 subjects, in which cultivation revealed Malassezia as viable oral mycobiome members, although the low-abundance Malassezia sympodialis was the only Malassezia species recovered. There was a high degree of concordance between the molecular detection and cultivability of Candida, while cultivation showed low sensitivity for detection of the Malassezia mycotype. Overall, our work provides insights into the oral mycobiome landscape, revealing 2 community classes with apparently distinct ecologic constraints and specific associations with coexisting bacteria and clinical parameters. The utility of mycotypes as biomarkers for oral diseases warrants further study.

Microbial Interactions in Oral Communities Mediate Emergent Biofilm Properties.

Oral microbial communities are extraordinarily complex in taxonomic composition and comprise interdependent biological systems. The bacteria, archaea, fungi, and viruses that thrive within these communities engage in extensive cell-cell interactions, which are both beneficial and antagonistic. Direct physical interactions among individual cells mediate large-scale architectural biofilm arrangements and provide spatial proximity for chemical communication and metabolic cooperation. In this review, we summarize recent work in identifying specific molecular components that mediate cell-cell interactions and describe metabolic interactions, such as cross-feeding and exchange of electron acceptors and small molecules, that modify the growth and virulence of individual species. We argue, however, that although pairwise interaction models have provided useful information, complex community-like systems are needed to study the properties of oral communities. The networks of multiple synergistic and antagonistic interactions within oral biofilms give rise to the emergent properties of persistence, stability, and long-range spatial structure, with these properties mediating the dysbiotic transitions from health to oral diseases. A better understanding of the fundamental properties of interspecies networks will lead to the development of effective strategies to manipulate oral communities.

Chemotherapy-induced oral mucositis and associated infections in a novel organotypic model.

Oral mucositis is a common side effect of cancer chemotherapy, with significant adverse impact on the delivery of anti-neoplastic treatment. There is a lack of consensus regarding the role of oral commensal microorganisms in the initiation or progression of mucositis because relevant experimental models are non-existent. The goal of this study was to develop an in vitro mucosal injury model that mimics chemotherapy-induced mucositis, where the effect of oral commensals can be studied. A novel organotypic model of chemotherapy-induced mucositis was developed based on a human oral epithelial cell line and a fibroblast-embedded collagen matrix. Treatment of organotypic constructs with 5-fluorouracil (5-FU) reproduced major histopathologic characteristics of oral mucositis, such as DNA synthesis inhibition, apoptosis and cytoplasmic vacuolation, without compromising the three-dimensional structure of the multilayer organotypic mucosa. Although structural integrity of the model was preserved, 5-FU treatment resulted in a widening of epithelial intercellular spaces, characterized by E-cadherin dissolution from adherens junctions. In a neutrophil transmigration assay we discovered that this treatment facilitated transport of neutrophils through epithelial layers. Moreover, 5-FU treatment stimulated key proinflammatory cytokines that are associated with the pathogenesis of oral mucositis. 5-FU treatment of mucosal constructs did not significantly affect fungal or bacterial biofilm growth under the conditions tested in this study; however, it exacerbated the inflammatory response to certain bacterial and fungal commensals. These findings suggest that commensals may play a role in the pathogenesis of oral mucositis by amplifying the proinflammatory signals to mucosa that is injured by cytotoxic chemotherapy.

Task Force on Design and Analysis in Oral Health Research: Host-Microbiome Interactions in Dysbiosis.

Knowledge Transfer Statement: This article discusses the proceedings of the conference organized by the Task Force on Design and Analysis in Oral Health Research on the new advances in host-microbiome interactions, analytical methods, and their implication in inflammatory periodontal disease management.

Clinical, Immune, and Microbiome Traits of Gingivitis and Peri-implant Mucositis.

Tissues surrounding dental implants and teeth develop clinical inflammation in response to microbial stimuli. However, the literature suggests that differences exist in the microbial insult and inflammatory responses leading to gingivitis and peri-implant mucositis. In this pilot study, the authors use for the first time a systems biology approach to comprehensively evaluate clinical parameters, selected inflammatory markers, and the microbiome of subject-matched tooth and implant sites during native inflammation and in response to experimental plaque accumulation. Fifteen subjects with 2 posterior implants and corresponding contralateral teeth were examined at enrollment; at day 0, after reinstitution of gingival/mucosal health; at days 7, 14, and 21, during stent-mediated oral hygiene (OH) abstention; and at day 42, after resumption of OH. The subgingival microbiome was evaluated via 16S rRNA gene sequencing and 8 selected inflammatory markers measured in crevicular fluid. Comparison of teeth and implants via general linear models based on orthogonal polynomials showed similar responses in clinical parameters, inflammatory mediators, and proportions of individual microbial taxa during OH abstention. Implants, however, accumulated less plaque and underwent more heterogeneous shifts in microbiome structure. A multilevel, within-group, sparse partial least squares analysis of covariation of microbial, inflammatory, and clinical parameters throughout all study visits found inflammation around teeth and implants positively correlated with IL-1 alpha and IL-1 beta and with the proportions of Selenomonas, Prevotella, and 5 species-level phylotypes. Gingivitis, however, showed a stronger positive correlation with lactoferrin and IL-1ra and a stronger negative correlation with Rothia. Peri-implant mucositis, on the contrary, correlated positively with certain microbial taxa not associated with gingivitis by a previous study or the current one. In summary, differences existed between implants and tooth sites in microbiome evolution during OH abstention and in the correlation of specific inflammatory mediators and microbial taxa with clinical inflammation. Common biological features, however, were also identified for gingivitis and mucositis.

Streptococcal co-infection augments Candida pathogenicity by amplifying the mucosal inflammatory response.

Mitis-group streptococci are ubiquitous oral commensals that can promote polybacterial biofilm virulence. Using a novel murine oral mucosal co-infection model we sought to determine for the first time whether these organisms promote the virulence of C. albicans mucosal biofilms in oropharyngeal infection and explored mechanisms of pathogenic synergy. We found that Streptococcus oralis colonization of the oral and gastrointestinal tract was augmented in the presence of C. albicans. S. oralis and C. albicans co-infection significantly augmented the frequency and size of oral thrush lesions. Importantly, S. oralis promoted deep organ dissemination of C. albicans. Whole mouse genome tongue microarray analysis showed that when compared with animals infected with one organism, the doubly infected animals had genes in the major categories of neutrophilic response/chemotaxis/inflammation significantly upregulated, indicative of an exaggerated inflammatory response. This response was dependent on TLR2 signalling since oral lesions, transcription of pro-inflammatory genes and neutrophil infiltration, were attenuated in TLR2(-/-) animals. Furthermore, S. oralis activated neutrophils in a TLR2-dependent manner in vitro. In summary, this study identifies a previously unrecognized pathogenic synergy between oral commensal bacteriaand C. albicans. This is the first report of the ability of mucosal commensal bacteria to modify the virulence of an opportunistic fungal pathogen.

Using high throughput sequencing to explore the biodiversity in oral bacterial communities.

High throughput sequencing of 16S ribosomal RNA gene amplicons is a cost-effective method for characterization of oral bacterial communities. However, before undertaking large-scale studies, it is necessary to understand the technique-associated limitations and intrinsic variability of the oral ecosystem. In this work we evaluated bias in species representation using an in vitro-assembled mock community of oral bacteria. We then characterized the bacterial communities in saliva and buccal mucosa of five healthy subjects to investigate the power of high throughput sequencing in revealing their diversity and biogeography patterns. Mock community analysis showed primer and DNA isolation biases and an overestimation of diversity that was reduced after eliminating singleton operational taxonomic units (OTUs). Sequencing of salivary and mucosal communities found a total of 455 OTUs (0.3% dissimilarity) with only 78 of these present in all subjects. We demonstrate that this variability was partly the result of incomplete richness coverage even at great sequencing depths, and so comparing communities by their structure was more effective than comparisons based solely on membership. With respect to oral biogeography, we found inter-subject variability in community structure was lower than site differences between salivary and mucosal communities within subjects. These differences were evident at very low sequencing depths and were mostly caused by the abundance of Streptococcus mitis and Gemella haemolysans in mucosa. In summary, we present an experimental and data analysis framework that will facilitate design and interpretation of pyrosequencing-based studies. Despite challenges associated with this technique, we demonstrate its power for evaluation of oral diversity and biogeography patterns.

Characterization of the invasive and inflammatory traits of oral Campylobacter rectus in a murine model of fetoplacental growth restriction and in trophoblast cultures.

Campylobacter species (C. jejuni, C. fetus) are enteric abortifacient bacteria in humans and ungulates. Campylobacter rectus is a periodontal pathogen associated with human fetal exposure and adverse pregnancy outcomes including preterm delivery. Experiments in pregnant mice have demonstrated that C. rectus can translocate from a distant site of infection to the placenta to induce fetal growth restriction and impair placental development. However, placental tissues from human, small-for-gestational age deliveries have not been reported to harbor C. rectus despite evidence of maternal infection and fetal exposure by fetal IgM response. This investigation examined the temporal relationship between the placental translocation of C. rectus and the effects on fetal growth in mice. BALB/c mice were infected at gestational day E7.5 to examine placental translocation of C. rectus by immunohistology. C. rectus significantly decreased fetoplacental weight at E14.5 and at E16.5. C. rectus was detected in 63% of placentas at E14.5, but not at E16.5. In in vitro trophoblast invasion assays, C. rectus was able to effectively invade human trophoblasts (BeWo) but not murine trophoblasts (SM9-1), and showed a trend for more invasiveness than C. jejuni. C. rectus challenge significantly upregulated both mRNA and protein levels of IL-6 and TNFalpha in a dose-dependent manner in human trophoblasts, but did not increase cytokine expression in murine cells, suggesting a correlation between invasion and cytokine activation. In conclusion, the trophoblast-invasive trait of C. rectus that appears limited to human trophoblasts may play a role in facilitating bacterial translocation and placental inflammation during early gestation.

Studies on NADH oxidase and alkyl hydroperoxide reductase produced by Porphyromonas gingivalis.

Enzymes that detoxify oxygen or oxygen radicals are important to anaerobic microorganisms that inhabit oxygenated environments. In previous studies we have determined that Porphyromonas gingivalis W50 cell extracts possess NADH oxidase-like activity, which increases slightly under oxygenated conditions. The aim of this study was to characterize the protein responsible for this activity in order to establish whether it protects the microorganism from oxidative stress. Protein purification based on NADH oxidase activity did not isolate a conventional NADH oxidase. Instead, the NADH oxidase activity was found to be associated with a FAD-dependent enzyme identified as 4-hydroxybutyryl-CoA dehydratase (AbfD). The biological significance of this activity with respect to protection against oxidative stress is not clear; hydrogen peroxide (H2O2) was present after completion of the NADH oxidase assay with the purified protein. Northern blot analysis, examining the expression of other proteins likely to function as NADH oxidases/peroxidases in P. gingivalis, revealed the transcription of a protein similar to alkyl-hydroperoxide reductase (AhpF-C), which could serve as an NADH oxidase and H2O2-detoxification system. AhpF is transcribed in a polycystronic way with its neighboring gene, which encodes for the coupling protein AhpC. No transcript could be detected for the closest match to an NADH oxidase identified in the P. gingivalis genome sequence. In conclusion, P. gingivalis seems to lack a protective NADH oxidase but AhpF-C could contribute to its moderate tolerance to reactive oxygen species by metabolizing H2O2.

The effect of oxygen on the growth and physiology of Porphyromonas gingivalis.

Oxygen constitutes a constant challenge for the survival of strict anaerobes in the oral environment. The aim of this study was to investigate the effect of oxygen on the physiology and growth of Porphyromonas gingivalis in a continuous culture system when grown under conditions of hemin limitation and excess. Results showed that, when grown in the presence of hemin at 0.5 mg/l, P. gingivalis could tolerate low levels of oxygen, being able to reach steady-state when 6% oxygen was present in the incoming gas mixture. When the hemin concentration was increased to 5 mg/l, the culture tolerated 10% oxygen. Anaerobically-grown cells were coccoid in shape, whereas those grown in the presence of oxygen were bacillary. Acetate was the predominant end-product in cultures grown in the presence of oxygen or in cultures hemin-limited. Despite some changes in the activity of Arg- and Lys-gingipain, most of the proteolytic activity was retained in the presence of oxygen. Activity of each of the three anti-oxidant enzymes tested (NADH oxidase, NADH peroxidase and SOD) was detected under all conditions and usually increased under oxygenated environments. Higher activities were also seen in the hemin-limited cultures. These results show some of the changes that occur in the physiology of P. gingivalis as a result of oxidative stress and confirm that hemin has a protective effect on the growth of the microorganism in the presence of oxygen.

Fusobacterium nucleatum supports the growth of Porphyromonas gingivalis in oxygenated and carbon-dioxide-depleted environments.

The authors compared the differences in tolerance to oxygen of the anaerobic periodontopathic bacteria Fusobacterium nucleatum and Porphyromonas gingivalis, and explored the possibility that F. nucleatum might be able to support the growth of P. gingivalis in aerated and CO2-depleted environments. Both micro-organisms were grown as monocultures and in co-culture in the presence and absence of CO2 and under different aerated conditions using a continuous culture system. At steady state, viable counts were performed and the activities of the enzymes superoxide dismutase and NADH oxidase/peroxidase were assayed in P. gingivalis. In co-culture, F. nucleatum was able to support the growth of P. gingivalis in aerated and CO2-depleted environments in which P. gingivalis, as a monoculture, was not able to survive. F. nucleatum not only appeared to have a much higher tolerance to oxygen than P. gingivalis, but a significant increase in its numbers occurred under moderately oxygenated conditions. F. nucleatum might have an additional indirect role in dental plaque maturation, contributing to the reducing conditions necessary for the survival of P. gingivalis and possibly other anaerobes less tolerant to oxygen. Additionally, F. nucleatum is able to generate a capnophilic environment essential for the growth of P. gingivalis.

The response to oxidative stress of Fusobacterium nucleatum grown in continuous culture.

Fusobacterium nucleatum ATCC 10953 was grown in continuous culture and the atmosphere changed stepwise from nitrogen (anaerobiosis) to a mixture of air: oxygen (40:60). No significant differences in biomass were observed and the baseline low level of superoxide dismutase increased only slightly; catalase and peroxidase activities were never detected but the level of NADH oxidase increased more than three-fold when oxygen was introduced into the system. In relation to acidic end-products, the relative proportion of acetate increased while that of butyrate decreased. Due mainly, it would seem, to NADH oxidase activity, the culture maintained a low redox potential (E(h)=-274 mV) even under an atmosphere of 40% oxygen in air and dissolved oxygen was not detected. This may, in part, explain the protective role of F. nucleatum for anaerobes in both biofilm and planktonic phases of aerated, mixed cultures of oral bacteria.