The influence of apical periodontitis on circulatory inflammatory mediators in peripheral blood: A prospective case-control study.

AIM: To explore the influence of apical periodontitis (AP) on inflammatory markers in blood of otherwise healthy individuals and to depict the inflammatory profile of the healing after dental extraction. METHODOLOGY: This is a prospective case-control intervention study, during which, individuals with a diagnosis of AP of one affected tooth were included, along with a control group matched for age and gender. A broad panel of blood inflammatory mediators was examined longitudinally in all subjects during six visits. In the case of the AP subjects, the tooth with AP was extracted at the third visit. Results were analysed by linear regression analyses and linear mixed-model analyses. RESULTS: A total of 53 subjects were included in the study, 27 with AP and 26 without. Fifteen females and 12 males were included in the AP group, and 14 females and 12 males in the control group. At baseline, granulocyte colony-stimulating factor (p < .001), interleukin (IL)-1ß (p = .03) and IL-4 (p = .01) were significantly lower in AP subjects than in controls. Comparison of the differences between baseline and the last visit, i.e. 3 months after the tooth extraction, showed a significant reduction in IL-10 (p = .03) and IL-12p70 (p = .01). CONCLUSIONS: The immunologic profile of chronic AP in one tooth and its healing profile reveals a systemic low-grade inflammation through compensatory immunosuppression. A larger lesion or multiple lesions could disrupt the balance that the system is trying to maintain, resulting in loss of homeostasis.

Host-microbiome interactions in apical periodontitis: The endodontic microbiome in relation to circulatory immunologic markers.

AIM: To explore microbial differences in the endodontic infection of teeth with primary or secondary apical periodontitis (AP), with or without symptomatology. Additionally, to investigate if these differences are depicted in immunologic markers in blood. METHODOLOGY: Twenty-nine teeth with primary or secondary AP were extracted and cryo-pulverized. Blood was drawn from the subjects at three different time-points before and three time-points after the extraction in a time period of four months. The V4 hypervariable region of the 16S rRNA gene was sequenced using Illumina MiSeq. The microbial profiles were ordinated using principal component analysis and tested for differences between groups with permutational multivariate analysis of variance using the Bray-Curtis distance. If significantly different, the microbial profiles were further analysed using the LDA effect size (LEfSe) biomarker discovery tool. A broad panel of inflammatory mediators in blood was examined longitudinally in all subjects during the six visits with mixed models. The Spearman correlation between these mediators and the zOTUs was calculated, and significant correlations (p < .05) were used as input for significant analysis of microarrays (SAM) using MeV. RESULTS: After subsampling, the 467 zOTUs were classified into 9 phyla and 99 genera or higher level taxa. The predominant genus in the entire sample set was Fusobacterium with a relative abundance of 12.3%, followed by Prevotella (9.9%), Actinomyces (7.7%) and Streptococcus (6.7%). The microbiomes of the endodontic infections were significantly associated with endodontic status (primary/secondary infection; p = .015) as well as with the presence or absence of pain (p = .011). There was also a difference in the concentration of inflammatory mediators, namely, C-reactive protein, Interleukin (IL)-8, IL-10, IL-12p70, RANKL and TNF-α, depending on the existence of pain. In addition, the presence of specific bacteria (zOTUs) was correlated, positively or negatively, with the expression of several circulating inflammatory markers. CONCLUSIONS: The microbial profiles and the concentration-time relationship of systemic inflammatory mediators of primary endodontic infection differed from those of secondary, and of symptomatic from those of asymptomatic cases. The fingerprint of associations between the immunological and microbiological profiles differed between asymptomatic and symptomatic patients.

Well-being, postoperative pain and outcome after clinical application of a novel root canal irrigation fluid-RISA-in teeth with apical periodontitis: A first-in-human study.

AIM: The aim of the study was to assess the tolerance to the new root canal irrigation fluid RISA after root canal treatment (RCT) by evaluating the subject's postoperative well-being, postoperative pain (PP) and treatment outcome. METHODOLOGY: A single-arm prospective study with 16 subjects (17 teeth) diagnosed with asymptomatic apical periodontitis. Endodontic treatment in one session performed using RISA for root canal irrigation. Well-being was assessed on the same day and after 24 h by telephone. For pain intensity, a visual analogue scale was used at 0-5 days. Clinical and radiographic evaluations were performed at ≥12 months. Well-being, occurrence of PP and outcome were qualitatively reported. Friedman test for paired samples and Spearman correlation coefficient were used. Significance was set at p < .05. RESULTS: At the same day and after 24 h, 14/16 subjects felt 'good'. 9/16 presented intra- or extra-oral swelling. The frequency of PP ≥36 (weak) was 82.4%. On the same day, 1 and 2 days postoperatively, there was more pain compared with preoperative pain p < .05. At Day 3, PP equalled preoperative pain (p > .05). 62.5% of subjects needed analgesics Day 0-2. The recall rate was 94.1%, and resolution of apical periodontitis was observed in 87.5%. CONCLUSIONS: The well-being of subjects was good, and the overall PP intensity was low. However, postoperative intra- and extra-oral swelling occurred often. At the recall visit, the effectiveness of the RCT with RISA appeared high (87.5%). The encouraging outcome results plus the fact that RISA has a broader action range than NaOCl in vitro, justify further work on the RISA solution. To reduce postoperative swelling, it is advised to further investigate the optimal way of application of RISA in the laboratory before clinical application is recommended.

The microbiome of dental and peri-implant subgingival plaque during peri-implant mucositis therapy: A randomized clinical trial.

AIM: To assess the microbial effects of mechanical debridement in conjunction with a mouthrinse on sites with peri-implant mucositis and gingivitis. MATERIALS AND METHODS: Eighty-nine patients with peri-implant mucositis were included in a double-blinded, randomized, placebo-controlled trial with mechanical debridement and 1-month use of either delmopinol, chlorhexidine (CHX), or a placebo mouthrinse. Submucosal and subgingival plaque samples of implants and teeth were collected at baseline and after 1 and 3 months, processed for 16S V4 rRNA gene amplicon sequencing, and analysed bioinformatically. RESULTS: The sites with peri-implant mucositis presented with a less diverse and less anaerobic microbiome. Exposure to delmopinol or CHX, but not to the placebo mouthrinse resulted in microbial changes after 1 month. The healthy sites around the teeth harboured a more diverse and more anaerobe-rich microbiome than the healthy sites around the implants. CONCLUSIONS: Peri-implant sites with mucositis harbour ecologically less complex and less anaerobic biofilms with lower biomass than patient-matched dental sites with gingivitis while eliciting an equal inflammatory response. Adjunctive antimicrobial therapy in addition to mechanical debridement does affect both dental and peri-implant biofilm composition in the short term, resulting in a less dysbiotic subgingival biofilm.

Comparability of microbiota of swabbed and spit saliva.

In general, saliva is used for microbiota analysis in longitudinal studies, and several collection methods are being used. Using a robust sample collection procedure is important, as it may influence salivary composition. This study explored the comparability of the microbiota of swabbed and spit saliva. Twenty-two females participated in this cross-sectional study. The bacterial composition of the three saliva samples (swab collected by the participant (SW-P), swab collected by the researcher (SW-R), and spit (SP) was assessed by 16S rRNA gene amplicon sequencing. The bacterial composition of the swabbed and the spit saliva was significantly different irrespective of the operator, and Shannon diversity was significantly higher in spit saliva than in SW-P and SW-R. The salivary microbiota of spit and swabbed adult saliva differs significantly. Research on microbial composition therefore requires collection of similar saliva sample types in all study participants.

Effect of high-fluoride dentifrice on root dentine and bacterial composition in a multispecies biofilm model.

The present study evaluated the effect of high-fluoride dentifrice on dentine demineralization and bacterial composition in a multispecies biofilm model in vitro. A seven-organism bacterial consortium was grown on bovine dentine discs in a high-throughput active attachment model. The biofilms were submitted twice per day to the following dentifrices treatments: 5,000 ppm F, 1,100 ppm F, with placebo as a negative control. After 5 days of biofilm growth, dentine samples were assessed by transversal microradiography, the biofilm was collected for bacterial counts and the pH of the media was determined. Lower integrated mineral loss values were observed when 5,000 ppm F-treatment was used compared to the other treatments. Overall microbiological counts decreased with increasing F-concentration as well the pH of the media throughout the experiment. The 5,000 ppm F-treatment caused a shift in microbial composition and reduced dentine demineralization in the in-vitro experimental model.

Effects of Surface Prereacted Glass on Saliva-Derived Polymicrobial Biofilms in an Active Attachment Biofilm Model.

Bioactive restorative materials are being developed to either influence the de/remineralization balance of the dental hard tissues locally or to release components that interact with the oral microbiota. Surface prereacted glass (S-PRG, Shofu, Japan) is a material that may influence both processes. S-PRG releases fluoride, which can interact with the de/remineralization process, and a range of other compounds that may influence the oral microbiota. In the current study, several experiments were performed to investigate the potential of S-PRG to influence both the growth and lactic acid production of saliva-derived polymicrobial biofilms. Biofilm formation was studied using the Amsterdam Active Attachment model. An eluate of the S-PRG particles was tested by adding it to the growth medium or by exposing the biofilms to it for 1 h. The effect of S-PRG particles was tested by adding the particles to the growth medium. The current experiments showed that the presence of S-PRG eluate in the medium influenced biofilm growth and lactic acid production even at low concentrations. The composition of the biofilms changed in the presence of S-PRG eluate, even at concentrations of S-PRG eluate at which biofilm viability was not affected. Treatment of developing biofilms with S-PRG eluate did neither show an effect on biofilm viability nor on lactic acid production. The addition of S-PRG particles to the growth medium resulted in both a lower biofilm viability and lower lactic acid production, indicating that the release of ions from the particles was fast enough to influence biofilm formation. From the current experiments, it can be concluded that S-PRG has the potential to influence biofilm growth, but the presence of the released ions during biofilm formation is required to show an effect.

Oral health-related quality of life in patients with early rheumatoid arthritis is associated with periodontal inflammation and painful temporomandibular disorders: a cross-sectional study.

OBJECTIVES: To evaluate oral health-related quality of life (OHRQoL) in early rheumatoid arthritis (ERA) patients and individuals at risk of rheumatoid arthritis (RA) compared to healthy controls, and to explore possible associated factors. MATERIALS AND METHODS: Fifty ERA patients, 50 at-risk individuals, and 50 age and gender matched healthy controls were recruited. OHRQoL (Oral Health Impact Profile-14 (OHIP-14)); number of decayed, missing, and filled teeth (DMFT); denture use; periodontal inflamed surface area (PISA); xerostomia (xerostomia inventory (XI)); and possible TMD (-pain) diagnoses were recorded. The groups were compared on these variables. Subsequently, backward multiple regression analyses were performed for the ERA and at-risk groups, with OHRQoL as the dependent variable and gender, age, DMFT, denture use, PISA, XI, non-painful TMD, and TMD pain as independent variables. RESULTS: At-risk individuals had higher XI scores (U = 789.5, z = -3.181, p = 0.001, r = -0.32) and higher prevalence of TMD pain (p = 0.046, OR = 4.57; 95% CI 0.92-22.73) than healthy controls and higher OHIP-14 scores than the ERA group (U = 894.5, z = -2.418, p = 0.016, r = -0.24), while no difference in OHIP-14 was found between the control group and both other groups. For ERA patients, OHRQoL was associated with PISA and TMD pain (R2 = 0.498, p < 0.001). For at-risk individuals, OHRQoL was associated with XI score (R2 = 0.410, p < 0.001). CONCLUSIONS: Alertness of health professionals to TMD pain and periodontal inflammation in ERA patients and to xerostomia and TMD pain in at-risk individuals is recommended. CLINICAL RELEVANCE: The results of this study address orofacial aspects that require attention of health professionals in the timeframe around RA onset. TRIAL REGISTRATION: Dutch National Trial Register (NTR, NTR6362).

Manipulation of Saliva-Derived Microcosm Biofilms To Resemble Dysbiotic Subgingival Microbiota.

Periodontitis is a highly prevalent oral inflammatory disease triggered by dysbiotic subgingival microbiota. For the development of microbiome modulators that can reverse the dysbiotic state and reestablish a health-associated microbiota, a high-throughput in vitro multispecies biofilm model is needed. Our aim is to establish a model that resembles a dysbiotic subgingival microbial biofilm by incorporating the major periodontal pathogen Porphyromonas gingivalis into microcosm biofilms cultured from pooled saliva of healthy volunteers. The biofilms were grown for 3, 7, and 10 days and analyzed for their microbial composition by 16S rRNA gene amplicon sequencing as well as measurement of dipeptidyl peptidase IV (DPP4) activity and butyric acid production. The addition of P. gingivalis increased its abundance in saliva-derived microcosm biofilms from 2.7% on day 3 to >50% on day 10, which significantly reduced the Shannon diversity but did not affect the total number of operational taxonomic units (OTUs). The P. gingivalis-enriched biofilms displayed altered microbial composition as revealed by principal-component analysis and reduced interactions among microbial species. Moreover, these biofilms exhibited enhanced DPP4 activity and butyric acid production. In conclusion, by adding P. gingivalis to saliva-derived microcosm biofilms, we established an in vitro pathogen-enriched dysbiotic microbiota which resembles periodontitis-associated subgingival microbiota in terms of increased P. gingivalis abundance and higher DPP4 activity and butyric acid production. This model may allow for investigating factors that accelerate or hinder a microbial shift from symbiosis to dysbiosis and for developing microbiome modulation strategies.IMPORTANCE In line with the new paradigm of the etiology of periodontitis, an inflammatory disorder initiated by dysbiotic subgingival microbiota, novel therapeutic strategies have been proposed targeting reversing dysbiosis and restoring host-compatible microbiota rather than eliminating the biofilms unselectively. Thus, appropriate laboratory models are required to evaluate the efficacy of potential microbiome modulators. In the present study, we used the easily obtainable saliva as an inoculum, spiked the microcosm biofilms with the periodontal pathogen Porphyromonas gingivalis, and obtained a P. gingivalis-enriched microbiota, which resembles the in vivo pathogen-enriched subgingival microbiota in severe periodontitis. This biofilm model circumvents the difficulties encountered when using subgingival plaque as the inoculum and achieves microbiota in a dysbiotic state in a controlled and reproducible manner, which is required for high-throughput and large-scale evaluation of strategies that can potentially modulate microbial ecology.

Submucosal microbiome of peri-implant sites: A cross-sectional study.

AIM: To study the peri-implant submucosal microbiome in relation to implant disease status, dentition status, smoking habit, gender, implant location, implant system, time of functional loading, probing pocket depth (PPD), and presence of bleeding on probing. MATERIALS AND METHODS: Biofilm samples were collected from the deepest peri-implant site of 41 patients with paper points, and analysed using 16S rRNA gene pyrosequencing. RESULTS: We observed differences in microbial profiles by PPD, implant disease status, and dentition status. Microbiota in deep pockets included higher proportions of the genera Fusobacterium, Prevotella, and Anaeroglobus compared with shallow pockets that harboured more Rothia, Neisseria, Haemophilus, and Streptococcus. Peri-implantitis (PI) sites were dominated by Fusobacterium and Treponema compared with healthy implants and peri-implant mucositis, which were mostly colonized by Rothia and Streptococcus. Partially edentulous (PE) individuals presented more Fusobacterium, Prevotella, and Rothia, whereas fully edentulous individuals presented more Veillonella and Streptococcus. CONCLUSIONS: PPD, implant disease status, and dentition status may affect the submucosal ecology leading to variation in composition of the microbiome. Deep pockets, PI, and PE individuals were dominated by Gram-negative anaerobic taxa.

Short-Chain N-Acylhomoserine Lactone Quorum-Sensing Molecules Promote Periodontal Pathogens in In Vitro Oral Biofilms.

Acylhomoserine lactones (AHLs), the quorum-sensing (QS) signals produced by a range of Gram-negative bacteria, are involved in biofilm formation in many pathogenic and environmental bacteria. Nevertheless, the current paradigm excludes a role of AHLs in dental plaque formation, while other QS signals, such as AI-2 and autoinducer peptides, have been demonstrated to play an important role in biofilm formation and virulence-related gene expression in oral pathogens. In the present work, we have explored the effect of externally added AHLs on in vitro oral biofilm models for commensal, cariogenic, and periodontal dental plaque. While little effect on bacterial growth was observed, some AHLs specifically affected the lactic acid production and protease activity of the biofilms. Most importantly, the analysis of bacterial diversity in the biofilms showed that the addition of C6-homoserine lactone (C6-HSL) results in a shift toward a periodontal bacterial composition profile by increasing the relative presence of the orange-complex bacteria Peptostreptococcus and Prevotella These results point to a relevant role of AHL-mediated QS in dental plaque formation and might be involved in the development of dysbiosis, the mechanism of which should be further investigated. This finding potentially opens new opportunities for the prevention or treatment of the periodontal disease.IMPORTANCE Dental plaque is omnipresent in healthy oral cavities and part of our commensal microbial colonization. At the same time, dental plaque is the cause of the most common human diseases, caries and gum disease. Dental plaque consists of billions of microbes attached to the surface of your teeth. Communication among these microbes is pivotal for development of these complex communities yet poorly studied in dental plaque. In the present study, we show that a specific communication molecule induces changes within the community related to the development of gum disease. This finding suggests that interfering with microbial communication may represent an interesting novel strategy to prevent gum disease that should be further investigated.

Qualitative and quantitative differences in the subgingival microbiome of the restored and unrestored teeth.

BACKGROUND AND OBJECTIVE: Metal-based dental restorations with a subgingival outline may enhance plaque accumulation and bacterial colonization. This study aimed to investigate whether metal-based restorations influence the composition of subgingival microbiome. MATERIAL AND METHODS: Per subject one site with a metal-based restoration and one contra-lateral site without a restoration were selected on basis of radiographic bone loss ≤2 mm, restoration outline at sulcus level/subgingivally, pocket depth ≤4 mm, and no root canal treatments. Subgingival samples were collected with sterile paper-points, and microbial profiles were obtained by 16S rRNA gene amplicon sequencing. Restorations were sampled with an Arkansas-stone and the metal composition was determined using energy-dispersive X-ray spectroscopy. RESULTS: A total of 22 sites from 11 subjects were included. No significant differences for the clinical parameters were found between the restored and unrestored sites. The average age of the restorations was 14.9 ± 7.1 years. Firmicutes was the most prevalent phylum at the restored sites (32% vs 20% of the reads of the unrestored sites, P = 0.016), and Actinobacteria at the unrestored sites (33% vs 18% of the reads of the restored sites, P = 0.01). Overall, sequences clustered into 573 operational taxonomic units (OTUs). Species richness of the restored sites was significantly higher than species richness of the unrestored sites (117 ± 32 and 96 ± 20 OTUs, respectively, P = 0.013). No associations between the metal composition and bacterial profiles were found. CONCLUSION: This study shows that metal-based restorations may enhance colonization of Firmicutes and the neighboring pocket may harbor more diverse microbial communities.

Resistance and resilience to experimental gingivitis: a systematic scoping review.

BACKGROUND: This systematic scoping review aimed to identify changes in biomarkers of microbiological, immunological and biochemical origin during experimental gingivitis (EG) studies that might indicate resistance and resilience. METHODS: The term 'experimental gingivitis' was run in PubMed from inception to April 11th, 2018. From the 411 studies retrieved, 22 studies were included for this review. RESULTS: Studies reporting data on biomarker changes during and after full mouth EG trial were included. Two studies reported findings on changes in biomarkers of microbiological, 12 on immunological and eight on biochemical origin. Changes were reported in the induction phase, and occasionally in the resolution phase. The microbiological composition of both supragingival and subgingival dental plaque changed over the course of EG to a more pathogenic direction, but showed a shift back to a more normal composition. This indicates resilience of the oral microbiome. For immunological biomarkers, it was challenging to retrieve a robust pattern of changes across multiple studies. IL-1ß and IL-6 in saliva and in gingival crevicular fluid increased during induction phase and returned in the resolution phase below baseline values. The biochemical parameters cystatin-SN, cystatin-S and lactoferrin in saliva were increased at the end of induction phase, however also here no clear pattern emerged based on all available studies. CONCLUSIONS: More research is needed to investigate which microbiological, immunological, and biochemical biomarkers can be useful for future investigations into the resistance and resilience of the oral cavity to experimental gingivitis.

metaModules identifies key functional subnetworks in microbiome-related disease.

MOTIVATION: The human microbiome plays a key role in health and disease. Thanks to comparative metatranscriptomics, the cellular functions that are deregulated by the microbiome in disease can now be computationally explored. Unlike gene-centric approaches, pathway-based methods provide a systemic view of such functions; however, they typically consider each pathway in isolation and in its entirety. They can therefore overlook the key differences that (i) span multiple pathways, (ii) contain bidirectionally deregulated components, (iii) are confined to a pathway region. To capture these properties, computational methods that reach beyond the scope of predefined pathways are needed. RESULTS: By integrating an existing module discovery algorithm into comparative metatranscriptomic analysis, we developed metaModules, a novel computational framework for automated identification of the key functional differences between health- and disease-associated communities. Using this framework, we recovered significantly deregulated subnetworks that were indeed recognized to be involved in two well-studied, microbiome-mediated oral diseases, such as butanoate production in periodontal disease and metabolism of sugar alcohols in dental caries. More importantly, our results indicate that our method can be used for hypothesis generation based on automated discovery of novel, disease-related functional subnetworks, which would otherwise require extensive and laborious manual assessment. AVAILABILITY AND IMPLEMENTATION: metaModules is available at https://bitbucket.org/alimay/metamodules/ CONTACT: a.may@vu.nl or s.abeln@vu.nl SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Effect of mouthwashes on the composition and metabolic activity of oral biofilms grown in vitro.

OBJECTIVE: The aim of this study was to determine the effect of an oxygenating mouthwash compared to two other established mouthwash products on bacterial composition and metabolic activity of oral biofilms in vitro. MATERIAL AND METHODS: Twelve healthy subjects participated as donors. Plaque-saliva mixture inoculated biofilms were grown and treated with 3 different chemotherapeutic mouthwashes [amine fluoride/stannous fluoride (MD), oxygenating agent (AX), chlorhexidine 0.12 % (PA), and water (W)]. Effects of treatments were assessed on biofilm composition (16S rRNA gene amplicon sequencing), production of organic acids (formate, acetate, lactate, propionate, butyrate using capillary electrophoresis), and viability of the remaining biofilm (CFUs). RESULTS: Microbial profiles of biofilms clustered per inoculum donor and were dominated by the genera Veillonella, Streptococcus, and Prevotella. Microbial diversity was only reduced after PA treatment. Significant changes in composition occurred after treatment with AX, resulting in lower proportions of Veillonella and higher proportions of non-mutans streptococci. Production of all organic acids after PA and lactate after MD was significantly lower as compared to W. AX resulted in reduction of acetate, butyrate, and propionate and increase in lactate production (p < 0.05). Viable counts were significantly lower after PA and AX treatments compared to W, while no significant reduction was observed after MD. CONCLUSIONS: All studied mouthwashes affected the in vitro biofilms differently. The effects of the AX treatment were the most prominent which resulted in changes of the bacterial composition and metabolism. CLINICAL IMPLICATIONS: Awareness by the dental team that mouthwashes can change the bacterial composition and metabolism is important when advising its use.

The mycobiome of root canal infections is correlated to the bacteriome.

OBJECTIVES: Bacterial infection of the root canal system causes apical periodontitis. Less is known about the role of fungi in these infections. This study aimed to assess the fungal prevalence, abundance, and diversity of root canal infections, as well as the relation between fungi and bacteria present in different parts of the root canal. MATERIALS AND METHODS: Twenty-six teeth with primary apical periodontitis were extracted, split in apical and coronal root segments, and cryo-pulverized. Bacteriome profiles of 23 teeth were analyzed based on the V3-V4 hypervariable region of the 16S ribosomal RNA gene. Mycobiome profiles of six teeth were analyzed based on the internal transcribed spacer (ITS) 1 or ITS2 region. Samples were sequenced on the Illumina MiSeq platform. RESULTS: A total of 338 bacterial operational taxonomic units (OTUs), 28 ITS1 OTUs, and 24 ITS2 OTUs were identified. Candida and Malassezia were the most frequently identified fungi. No differences could be found between the bacteriome and mycobiome profiles of the apical and coronal root segments. The bacteriome of fungi-positive root segments contained more Actinomyces, Bifidobacterium, four different Lactobacillus OTUs, Propionibacterium, and Streptococcus. A Spearman correlation matrix between bacteriomes and mycobiomes identified no correlations, but separate clusters could be observed. CONCLUSIONS: A considerable proportion of the root canal infections contain fungi, although fungal diversity is limited. However, when fungi are present, the composition of the bacteriome is clearly different. CLINICAL RELEVANCE: Interaction between bacteria and fungi in root canal infections may complicate the infection and require alternative treatment strategies.

The microbiome associated with equine periodontitis and oral health.

Equine periodontal disease is a common and painful condition and its severe form, periodontitis, can lead to tooth loss. Its aetiopathogenesis remains poorly understood despite recent increased awareness of this disorder amongst the veterinary profession. Bacteria have been found to be causative agents of the disease in other species, but current understanding of their role in equine periodontitis is extremely limited. The aim of this study was to use high-throughput sequencing to identify the microbiome associated with equine periodontitis and oral health. Subgingival plaque samples from 24 horses with periodontitis and gingival swabs from 24 orally healthy horses were collected. DNA was extracted from samples, the V3-V4 region of the bacterial 16S rRNA gene amplified by PCR and amplicons sequenced using Illumina MiSeq. Data processing was conducted using USEARCH and QIIME. Diversity analyses were performed with PAST v3.02. Linear discriminant analysis effect size (LEfSe) was used to determine differences between the groups. In total, 1308 OTUs were identified and classified into 356 genera or higher taxa. Microbial profiles at health differed significantly from periodontitis, both in their composition (p < 0.0001, F = 12.24; PERMANOVA) and in microbial diversity (p < 0.001; Mann-Whitney test). Samples from healthy horses were less diverse (1.78, SD 0.74; Shannon diversity index) and were dominated by the genera Gemella and Actinobacillus, while the periodontitis group samples showed higher diversity (3.16, SD 0.98) and were dominated by the genera Prevotella and Veillonella. It is concluded that the microbiomes associated with equine oral health and periodontitis are distinct, with the latter displaying greater microbial diversity.

Nitrate and the Origin of Saliva Influence Composition and Short Chain Fatty Acid Production of Oral Microcosms.

Nitrate is emerging as a possible health benefactor. Especially the microbial conversion of nitrate to nitrite in the oral cavity and the subsequent conversion to nitric oxide in the stomach are of interest in this regard. Yet, how nitrate influences the composition and biochemistry of the oral ecosystem is not fully understood. To investigate the effect of nitrate on oral ecology, we performed a 4-week experiment using the multiplaque artificial mouth (MAM) biofilm model. This model was inoculated with stimulated saliva of two healthy donors. Half of the microcosms (n = 4) received a constant supply of nitrate, while the other half functioned as control (n = 4). Additionally, all microcosms received a nitrate and sucrose pulse, each week, on separate days to measure nitrate reduction and acid formation. The bacterial composition of the microcosms was determined by 16S rDNA sequencing. The origin of the saliva (i.e., donor) showed to be the strongest determinant for the development of the microcosms. The supplementation of nitrate was related to a relatively high abundance of Neisseria in the microcosms of both donors, while Veillonella was highly abundant in the nitrate-supplemented microcosms of only one of the donors. The lactate concentration after sucrose addition was similarly high in all microcosms, irrespective of treatment or donor, while the concentration of butyrate was lower after nitrate addition in the nitrate-receiving microcosms. In conclusion, nitrate influences the composition and biochemistry of oral microcosms, although the result is strongly dependent on the inoculum.

Epithelial cell detachment by Porphyromonas gingivalis biofilm and planktonic cultures.

Porphyromonas gingivalis is present as a biofilm at the sites of periodontal infections. The detachment of gingival epithelial cells induced by P. gingivalis biofilms was examined using planktonic cultures as a comparison. Exponentially grown planktonic cultures or 40-h biofilms were co-incubated with epithelial cells in a 24-well plate for 4 h. Epithelial cell detachment was assessed using imaging. The activity of arginine-gingipain (Rgp) and gene expression profiles of P. gingivalis cultures were examined using a gingipain assay and quantitative PCR, respectively. P. gingivalis biofilms induced significantly higher cell detachment and displayed higher Rgp activity compared to the planktonic cultures. The genes involved in gingipain post-translational modification, but not rgp genes, were significantly up-regulated in P. gingivalis biofilms. The results underline the importance of including biofilms in the study of bacterial and host cell interactions.

Stability and resilience of oral microcosms toward acidification and Candida outgrowth by arginine supplementation.

Dysbiosis induced by low pH in the oral ecosystem can lead to caries, a prevalent bacterial disease in humans. The amino acid arginine is one of the pH-elevating agents in the oral cavity. To obtain insights into the effect of arginine on oral microbial ecology, a multi-plaque "artificial mouth" (MAM) biofilm model was inoculated with saliva from a healthy volunteer and microcosms were grown for 4 weeks with 1.6 % (w/v) arginine supplement (Arginine) or without (Control), samples were taken at several time-points. A cariogenic environment was mimicked by sucrose pulsing. The bacterial composition was determined by 16S rRNA gene amplicon sequencing, the presence and amount of Candida and arginine deiminase system genes arcA and sagP by qPCR. Additionally, ammonium and short-chain fatty acid concentrations were determined. The Arginine microcosms were dominated by Streptococcus, Veillonella, and Neisseria and remained stable in time, while the composition of the Control microcosms diverged significantly in time, partially due to the presence of Megasphaera. The percentage of Candida increased 100-fold in the Control microcosms compared to the Arginine microcosms. The pH-raising effect of arginine was confirmed by the pH and ammonium results. The abundances of sagP and arcA were highest in the Arginine microcosms, while the concentration of butyrate was higher in the Control microcosms. We demonstrate that supplementation with arginine serves a health-promoting function; it enhances microcosm resilience toward acidification and suppresses outgrowth of the opportunistic pathogen Candida. Arginine facilitates stability of oral microbial communities and prevents them from becoming cariogenic.