ABSTRACT Streptococcus gordonii is a gram-positive oral bacterium capable of adhering to a variety of biotic and abiotic surfaces and forming biofilms. To characterize physiological changes associated with biofilm formation in S. gordonii , we investigated the roles of two putative GCN5-related N-acetyltransferases (GNATs), SGO_2030 and SGO_2031, in in vitro biofilm formation on saliva-coated surfaces using the laboratory strain DL1. Our results demonstrate that SGO_2031, but not SGO_2030, seems to contribute to biofilm formation by modulating the abundance of extracellular polysaccharides within the biofilm matrix. This defect in biofilm formation observed by the deletion of SGO_2031 resulted in a significant fitness disadvantage during colonization of the murine oral cavity compared to the wild-type parent strain. Consistent with the role of S. gordonii as an early colonizer of tooth surfaces that influences oral biofilm community structure, inoculation with either the wild-type or the SGO_2031 mutant strain led to distinct alterations in the murine oral microbiome composition. Deletion of SGO_2031 also resulted in changes in protein acetylation patterns, as assessed by Western immunoblot analysis, supporting the role of this enzyme as an acetyltransferase. Given that SGO_2031 is conserved and widely distributed among streptococci, we propose naming this enzyme Streptococcal Lysine Acetyltransferase A (SktA). IMPORTANCE Protein acetylation is a common posttranslational modification conserved across all domains of life. In bacteria, protein acetylation is carried out by homologs of the GCN5-related N-acetyltransferase (GNAT) family. GNATs catalyze the transfer of an acetyl group from acetyl-CoA to the ε-amino group of lysine residues on proteins. This process changes the charge and length of lysine residues, resulting in changes to protein function. Streptococcus gordonii is predicted to encode 17 GNAT homologs. Here, we report that one of them, SGO_2031 (SktA), plays an important role in S. gordonii biofilms.

Dental plaque is a complex oral biofilm responsible for periodontal diseases. Bacterial biofilms are often regulated by Quorum Sensing (QS) mediated by N-acyl homoserine lactones (AHLs). While their presence and roles in oral microbiota have been debated, emerging evidence suggests AHLs influence oral biofilm development. AHLs are detectable in a microbial community derived from human dental plaque cultured under 5% CO2 but not under anaerobic conditions. Manipulating QS in this community via AHL lactonases enriched commensals and pioneer colonizers under 5% CO₂, whereas in anaerobic conditions exogenous AHLs promoted late colonizers. QS disruption reduced biofilm formation, enhanced sucrose fermentation to lactate, and altered metabolic profiles of the community depending on the lactonase substrate specificity. Our findings highlight the importance of AHL-mediated QS in oral biofilm development and suggest its differential roles under aerobic versus anaerobic conditions. Targeting QS may offer a novel strategy for managing oral biofilms and preventing periodontal disease.

To evaluate the root canal microbiome composition and bacterial functional capability in cases of primary and secondary apical periodontitis utilizing whole-metagenome shotgun sequencing. Twenty-two samples from patients with primary root canal infections, and 18 samples obtained from previously treated teeth currently diagnosed with apical periodontitis were analysed with whole-metagenome shotgun sequencing at a depth of 20 M reads. Taxonomic and functional gene annotations were made using MetaPhlAn3 and HUMAnN3 software. The Shannon and Chao1 indices were utilized to measure alpha diversity. Differences in community composition were evaluated utilizing analysis of similarity (ANOSIM) using Bray-Curtis dissimilarities. The Wilcoxon rank sum test was used to compare differences in taxa and functional genes. Microbial community variations within a community were significantly lower in secondary relative to primary infections (alpha diversity p = .001). Community composition was significantly different in primary versus secondary infection ( R = .11, p = .005). The predominant taxa observed among samples (>2.5%) were Pseudopropionibacterium propionicum , Prevotella oris , Eubacterium infirmum , Tannerella forsythia , Atopobium rimae , Peptostreptococcus stomatis , Bacteroidetes bacterium oral taxon 272, Parvimonas micra , Olsenella profusa , Streptococcus anginosus , Lactobacillus rhamnosus , Porphyromonas endodontalis , Pseudoramibacter alactolyticus , Fusobacterium nucleatum , Eubacterium brachy and Solobacterium moorei . The Wilcoxon rank test revealed no significant differences in relative abundances of functional genes in both groups. Genes with greater relative abundances (top 25) were associated with genetic, signalling and cellular processes including the iron and peptide/nickel transport system. Numerous genes encoding toxins were identified: exfoliative toxin, haemolysins, thiol-activated cytolysin, phospholipase C, cAMP factor, sialidase, and hyaluronic glucosaminidase. Despite taxonomic differences between primary and secondary apical periodontitis, the functional capability of the microbiomes was similar.

Sortases are highly conserved enzymes with endopeptidase and transpeptidase activities in Gram-positive bacteria. Sortase A cleaves within an LPXTG-motif and covalently crosslinks cell wall proteins to become anchored to the peptidoglycan of the cell wall. We showed that a peptide cleaved by sortase A from the C-terminus (C-pep) of the LPXTG-adhesin SspA intercalates in the cell membrane. Nested in the membrane, this C-pep docks with the intramembrane sensor histidine kinase, SraS, to activate the response regulator, SraR. SraR signals that the C-pep has been cleaved as an indicator of the fidelity of sortase A processing. SraSR also signals that key LPXTG-proteins in concert with lipoteichoic acid engage the mucin, MUC5B, which elicits a different transcriptional response than the binding of other salivary constituents. To visualize the C-pep intercalating in the cell membrane in vivo, we used Structured Illumination Microscopy (SIM). And to show that the C-pep complexes with SraS, we used bimolecular fluorescence experiments. The C-pep and SraS were each expressed with one or the other half of yellow fluorescence protein (YFP). Reconstitution of the complete YFP signal indicated that the C-pep and SraS interacted at molecular distances within the cell membrane in vivo. Using these imaging protocols, we learned that the C-pep functions as a signaling molecule within the cell membrane of the streptococcal cell.

To analyze if the microbiome community composition in primary endodontic infections is associated with clinical or radiographic factors. Seventy-one patients with primary endodontic infections were evaluated for percussion tenderness, presence of a sinus tract, presence of caries, sex, probing depth > 4 mm, and age. Samples from the root canals were obtained and the microbiome was subsequently characterized by 16 S rRNA amplicon sequencing. For the radiographic analysis, a subset of 12 samples with a periapical index (PAI) ≤ 2 were compared with 19 samples with PAI of 5. The Shannon and Chao1 indices were used to measure alpha diversity. Differences in abundances of genera were evaluated using the Kruskal-Wallis test with Bonferroni’s correction. Differences in community composition were evaluated using analysis of similarity (ANOSIM) with Bray-Curtis dissimilarity matrices. No significant differences in microbiome composition relative to clinical factors were found using ANOSIM. Teeth within the two categories of periapical index showed a similar number of species richness, and alpha diversity values P > 0.05. Community composition was significantly affected by the periapical index (ANOSIM P = 0.039, R = 0.10). Larger radiographic lesions demonstrated significant increase in Prevotellaceae, Olsenella, and the motile bacteria Oribacterium, Selenomonadaceae spp., and Treponema. Clinical factors associated with apical periodontitis have a limited impact on the root canal microbiome composition. Community composition appears to be affected in teeth with large apical lesions.

AIM To analyse the effect of ultrasonic irrigant activation (UIA) and the GentleWave (GW) multisonic irrigation (GW) with minimal instrumentation on the root canal microbial diversity in an ex vivo model that used extracted molars with a history of pulp necrosis. METHODOLOGY Twenty-three mandibular molars were prepared ex vivo for collection of superficial (surface control), pre-treatment and post-treatment samples 24 h after extraction. Samples were divided into two groups: UIA using 6% NaOCl (n = 11) and GW group (n = 12). All samples were processed using quantitative real-time polymerase chain reaction (qPCR) and 16S rRNA next-generation sequencing to measure microbial diversity before and after the antimicrobial treatment. For qPCR, a t-test (α = .05) was used to compare the log10 reduction. The Chao1 and Shannon indices evaluated alpha diversity. Differences in community composition (beta diversity) were evaluated by analysis of similarity (ANOSIM). Kruskal-Wallis test with Bonferroni corrections was performed to evaluate the differences in abundances genera in the samples. RESULTS Quantitative real-time polymerase chain reaction revealed an estimated 1.6 and 2.6 log10 reduction for UIA and GW groups respectively (p = .048). An average of 5 ± 4 and 3 ± 5 operational taxonomic units (OTUs) were found in surface's samples in the UIA and GW group respectively. These values were significantly lower (p < .001) compared to the number of preoperative OTUs in those groups (155 ± 79 and 187 ± 121). In assessing beta diversity, there were no significant differences found in pre-treatment samples (R = .090, p = .070 ANOSIM with Bonferroni corrections). Also, no significant differences in community composition were observed in post-treatment samples (R = -.05, p = .829). After treatment, there was a significant reduction of Eubacterium using conventional treatment with UIA and a significant reduction of Prevotella using minimal instrumentation with GW irrigation (p = .007 and p = .002 respectively). CONCLUSION Quantitative PCR analysis revealed a significant reduction in microbial load for GW group. Overall, diversity changes were similar between UIA and GW irrigation in this ex vivo model that used extracted teeth with a history of pulp necrosis. OTUs obtained from the surface sample were negligible and did not affect the statistical outcome of the study.

Aim: To compare the effect of different sodium hypochlorite (NaOCl) agitation techniques on an ex vivo oral multispecies biofilm during passive disinfection of simulated immature roots. Methodology: Extracted human teeth were prepared to simulate immature roots. They were infected with a dental plaque-derived multispecies biofilm and cultured for 14 days. The roots were randomly designated into four groups: (1) negative control (PBS), (2) 1.5% NaOCl (CNI), (3) CNI + Ultrasonic activation (UA), (4) CNI + EasyClean agitation (ECA), (5) CNI + XP-endo finisher agitation (XPF), and (6) positive control (6% NaOCl). Biofilm samples were collected from the root canals and used to determine the number of viable cells (colony-forming units), scanning electron microscopy, and 16S rRNA gene sequencing. The mean colony-forming units per mL (CFU/mL) were analysed using One-way anova. 16S rRNA sequencing data were analysed for alpha (observed OTUs, Shannon index, and Chao1) and beta diversity (Bray-Curtis dissimilarities). The LEfSe analysis was used to determine the effect of treatment procedures on the abundance of root canal microbiota. The significance was set at .05. Results: PBS and CNI samples had significantly higher CFU/mL counts than UA, ECA, XPF, and 6% NaOCl samples (p < .05). The pre-treatment, PBS, and CNI groups had significantly greater alpha diversity than the UA, ECA, XPF, and 6% NaOCl groups (p < .05). NaOCl agitation groups and the 6% NaOCl group achieved a more pronounced reduction in bacteria from the genera Fusobacterium, Actinomyces, Porphyromonas, and Capnocytophaga. Conclusions: The effectiveness of passive disinfection protocols was enhanced by NaOCl agitation techniques, suggesting that this supplementary method can improve the outcome of revitalization procedures.

Biofilms are subjected to many environmental pressures that can influence community structure and physiology. In the oral cavity, and many other environments, biofilms are exposed to forces generated by fluid flow; however, our understanding of how oral biofilms respond to these forces remains limited. In this study, we developed a linear rocker model of fluid flow to study the impact of shear forces on Streptococcus gordonii and dental plaque-derived multispecies biofilms. We observed that as shear forces increased, S. gordonii biofilm biomass decreased. Reduced biomass was largely independent of overall bacterial growth. Transcriptome analysis of S. gordonii biofilms exposed to moderate levels of shear stress uncovered numerous genes with differential expression under shear. We also evaluated an ex vivo plaque biofilm exposed to fluid shear forces. Like S. gordonii, the plaque biofilm displayed decreased biomass as shear forces increased. Examination of plaque community composition revealed decreased diversity and compositional changes in the plaque biofilm exposed to shear. These studies help to elucidate the impact of fluid shear on oral bacteria and may be extended to other bacterial biofilm systems.

To analyse the effect of ultrasonic irrigant activation (UIA) and the GentleWave (GW) multisonic irrigation (GW) with minimal instrumentation on the root canal microbial diversity in an ex vivo model that used extracted molars with a history of pulp necrosis. Twenty-three mandibular molars were prepared ex vivo for collection of superficial (surface control), pre-treatment and post-treatment samples 24 h after extraction. Samples were divided into two groups: UIA using 6% NaOCl ( n  = 11) and GW group ( n  = 12). All samples were processed using quantitative real-time polymerase chain reaction (qPCR) and 16S rRNA next-generation sequencing to measure microbial diversity before and after the antimicrobial treatment. For qPCR, a t -test ( α  = .05) was used to compare the log10 reduction. The Chao1 and Shannon indices evaluated alpha diversity. Differences in community composition (beta diversity) were evaluated by analysis of similarity (ANOSIM). Kruskal–Wallis test with Bonferroni corrections was performed to evaluate the differences in abundances genera in the samples. Quantitative real-time polymerase chain reaction revealed an estimated 1.6 and 2.6 log10 reduction for UIA and GW groups respectively ( p  = .048). An average of 5 ± 4 and 3 ± 5 operational taxonomic units (OTUs) were found in surface's samples in the UIA and GW group respectively. These values were significantly lower ( p  < .001) compared to the number of preoperative OTUs in those groups (155 ± 79 and 187 ± 121). In assessing beta diversity, there were no significant differences found in pre-treatment samples ( R  = .090, p  = .070 ANOSIM with Bonferroni corrections). Also, no significant differences in community composition were observed in post-treatment samples ( R  = −.05, p  = .829). After treatment, there was a significant reduction of Eubacterium using conventional treatment with UIA and a significant reduction of Prevotella using minimal instrumentation with GW irrigation ( p  = .007 and p  = .002 respectively). Quantitative PCR analysis revealed a significant reduction in microbial load for GW group. Overall, diversity changes were similar between UIA and GW irrigation in this ex vivo model that used extracted teeth with a history of pulp necrosis. OTUs obtained from the surface sample were negligible and did not affect the statistical outcome of the study.

IntroductionTo evaluate the antimicrobial activity of Triton irrigation versus 4% NaOCl utilizing a direct contact test and an extracted tooth model.MethodsIn the first experiment, a direct contact test was conducted to compare bacterial DNA removal and microbial diversity changes following irrigation with sodium hypochlorite (4% NaOCl) or Triton. Hydroxyapatite and dentin discs were inoculated with subgingival human-derived dental plaque for 2 weeks utilizing a CDC biofilm reactor and subsequently challenged with the root canal irrigants for 5 min. In the second experiment, teeth contaminated with a multispecies biofilm (n=24) were assigned into two treatment groups, NaOCl or Triton irrigation. Samples were obtained for qPCR and next-generation sequencing (NGS) analysis before and after instrumentation. The Shannon and Chao1 indices were used to measure alpha diversity. The Bray–Curtis dissimilarity and ANOSIM was used to measure beta diversity. Differences in abundances of genera were evaluated using Kruskal–Wallis test with Bonferroni corrections.ResultsThe direct contact test revealed no significant differences in the bacterial load based on 16S rRNA gene molecules/μL, reads, or differences in the Shannon index among groups. In the extracted tooth model, a bacterial load reduction of log10 3.08 ± 0.69 and 2.76 ± 0.91 were found for NaOCl and Triton respectively (P = 0.348). NGS showed fewer reads, lower Chao1 and beta diversity values when pre- and post-treatment samples were assessed in both experimental groups (P < 0.0001). The Kruskal Wallis analysis found that 17 genera of bacteria were overrepresented in minimal values in the Triton post-treatment group, 14 of these genera represented less than 1% of the microbial community.ConclusionsBoth irrigants had limited antimicrobial activity in the direct contact test. When used in conjunction with mechanical instrumentation both irrigants were able to reduce the bacterial DNA load and diversity in comparison with pretreatment communities. The NaOCl irrigation, followed by EDTA flush, was more effective in decreasing DNA counts from low-abundance organisms.

To compare the effect of different sodium hypochlorite (NaOCl) agitation techniques on an ex vivo oral multispecies biofilm during passive disinfection of simulated immature roots. Extracted human teeth were prepared to simulate immature roots. They were infected with a dental plaque-derived multispecies biofilm and cultured for 14 days. The roots were randomly designated into four groups: (1) negative control (PBS), (2) 1.5% NaOCl (CNI), (3) CNI + Ultrasonic activation (UA), (4) CNI + EasyClean agitation (ECA), (5) CNI + XP-endo finisher agitation (XPF), and (6) positive control (6% NaOCl). Biofilm samples were collected from the root canals and used to determine the number of viable cells (colony-forming units), scanning electron microscopy, and 16S rRNA gene sequencing. The mean colony-forming units per mL (CFU/mL) were analysed using One-way anova . 16S rRNA sequencing data were analysed for alpha (observed OTUs, Shannon index, and Chao1) and beta diversity (Bray-Curtis dissimilarities). The LEfSe analysis was used to determine the effect of treatment procedures on the abundance of root canal microbiota. The significance was set at .05. PBS and CNI samples had significantly higher CFU/mL counts than UA, ECA, XPF, and 6% NaOCl samples ( p  < .05). The pre-treatment, PBS, and CNI groups had significantly greater alpha diversity than the UA, ECA, XPF, and 6% NaOCl groups ( p  < .05). NaOCl agitation groups and the 6% NaOCl group achieved a more pronounced reduction in bacteria from the genera Fusobacterium , Actinomyces , Porphyromonas , and Capnocytophaga . The effectiveness of passive disinfection protocols was enhanced by NaOCl agitation techniques, suggesting that this supplementary method can improve the outcome of revitalization procedures.

AimTo evaluate the root canal microbiome composition and bacterial functional capability in cases of primary and secondary apical periodontitis utilizing whole-metagenome shotgun sequencing. MethodologyTwenty-two samples from patients with primary root canal infections, and 18 samples obtained from previously treated teeth currently diagnosed with apical periodontitis were analysed with whole-metagenome shotgun sequencing at a depth of 20 M reads. Taxonomic and functional gene annotations were made using MetaPhlAn3 and HUMAnN3 software. The Shannon and Chao1 indices were utilized to measure alpha diversity. Differences in community composition were evaluated utilizing analysis of similarity (ANOSIM) using Bray-Curtis dissimilarities. The Wilcoxon rank sum test was used to compare differences in taxa and functional genes. ResultsMicrobial community variations within a community were significantly lower in secondary relative to primary infections (alpha diversity p = .001). Community composition was significantly different in primary versus secondary infection (R = .11, p = .005). The predominant taxa observed among samples (>2.5%) were Pseudopropionibacterium propionicum, Prevotella oris, Eubacterium infirmum, Tannerella forsythia, Atopobium rimae, Peptostreptococcus stomatis, Bacteroidetes bacterium oral taxon 272, Parvimonas micra, Olsenella profusa, Streptococcus anginosus, Lactobacillus rhamnosus, Porphyromonas endodontalis, Pseudoramibacter alactolyticus, Fusobacterium nucleatum, Eubacterium brachy and Solobacterium moorei. The Wilcoxon rank test revealed no significant differences in relative abundances of functional genes in both groups. Genes with greater relative abundances (top 25) were associated with genetic, signalling and cellular processes including the iron and peptide/nickel transport system. Numerous genes encoding toxins were identified: exfoliative toxin, haemolysins, thiol-activated cytolysin, phospholipase C, cAMP factor, sialidase, and hyaluronic glucosaminidase. ConclusionsDespite taxonomic differences between primary and secondary apical periodontitis, the functional capability of the microbiomes was similar.

To evaluate the root canal microbiome composition in cases of primary and secondary apical periodontitis. Thirty-nine samples from patients with primary root canal infections obtained before root canal treatment, and 40 samples obtained during root-end resection procedures from previously filled cases with apical periodontitis were evaluated using 16S rRNA next-generation sequencing analysis (NGS). Demographic and clinical factors included age, sex, infection type, percussion sensitivity, and presence of pain. Differences in abundances of genera were evaluated using Kruskal-Wallis test. Alpha and beta diversity indices were calculated using mothur. The Shannon and Chao1 indices were used to measure alpha diversity. The Bray–Curtis dissimilarity was used to measure beta diversity. Differences in community composition were evaluated using analysis of similarity (ANOSIM) with Bonferroni correction for multiple comparisons. Significantly fewer operational taxonomic units values were observed from samples from secondary infections ( p < .0001). While no significant differences were observed in the Chao 1 index between primary and secondary infections, the Shannon alpha diversity was significantly lower in secondary relative to primary infections ( p = .008). Among samples, sex, age (adult vs. older adult), percussion sensitivity, and presence of pain all showed no significant effects on community composition via an analysis of similarity (ANOSIM). However, community composition was significantly different depending on whether the sample was from a primary or secondary infection ( R = .051, p = .03). Nine microbial genera comprised the predominant taxa observed among samples (>3.3%) and included Parvimonas , Fusobacterium , Campylobacter , Arachnia, Eubacterium , Prevotella , Peptostreptococcus , Fretibacterirum , and Pseudoramibacter. Significantly greater relative abundances of Prevotella , Peptostreptococcus , Veillonella , Lactucaseibacillus , and Dialister were observed in primary infections. Primary endodontic infections are more diverse than secondary infections. The microbial composition is not associated with the clinical manifestations of apical periodontitis.

Understanding the axis of the human microbiome and physiological homeostasis is an essential task in managing deep-space-travel-associated health risks. The NASA-led Rodent Research 5 mission enabled an ancillary investigation of the gut microbiome, varying exposure to microgravity (flight) relative to ground controls in the context of previously shown bone mineral density (BMD) loss that was observed in these flight groups. We demonstrate elevated abundance of Lactobacillus murinus and Dorea sp. during micro -gravity exposure relative to ground control through whole-genome sequencing and 16S rRNA analyses. Specific functionally assigned gene clusters of L. murinus and Dorea sp. capable of producing metabolites, lactic acid, leucine/isoleucine, and glutathione are enriched. These metabolites are elevated in the micro -gravity-exposed host serum as shown by liquid chromatography-tandem mass spectrometry (LC-MS/ MS) metabolomic analysis. Along with BMD loss, ELISA reveals increases in osteocalcin and reductions in tartrate-resistant acid phosphatase 5b signifying additional loss of bone homeostasis in flight.

All organisms throughout the tree of life sense and respond to their surface environments. To discriminate among mucosal surface environmental cues, we report that Streptococcus gordonii recognizes a high-molecular-weight mucin glycoprotein, MUC5B, using the paired adhesins SspAB and lipoteichoic acid; the latter bridges the outside signal to an intramembrane two-component system to transcriptionally regulate a MUC5B-specific adhesin and genes that may facilitate glycan catabolism.

To evaluate the physicochemical properties of five root canal sealers and assess their effect on an ex vivo dental plaque-derived polymicrobial community. Dental plaque-derived microbial communities were exposed to the sealers (AH Plus [AHP], GuttaFlow Bioseal [GFB], Endoseal MTA [ESM], Bio-C sealer [BCS] and BioRoot RCS [BRR]) for 3, 6 and 18 h. The sealers' effect on the biofilm biomass and metabolic activity was quantified using crystal violet (CV) staining and MTT assay, respectively. Biofilm community composition and morphology were assessed by denaturing gradient gel electrophoresis (DGGE), 16S rRNA sequencing and scanning electron microscopy. The ISO6876:2012 specifications were followed to determine the setting time, radiopacity, flowability and solubility. Obturated acrylic teeth were used to assess the sealers' effect on pH. Surface chemical characterization was performed using SEM with coupled energy-dispersive spectroscopy. Data normality was assessed using the Shapiro–Wilk test. One-way anova and Tukey's tests were used to analyze data from setting time, radiopacity, flowability and solubility. Two-way anova and Dunnett's tests were used for the data analysis from CV, MTT and pH. 16S rRNA sequencing data were analyzed for alpha (Shannon index and Chao analysis) and beta diversity (Bray–Curtis dissimilarities). Differences in community composition were evaluated by analysis of similarity ( p  < .05). The sealers significantly influenced microbial community composition and morphology. All sealers complied with ISO6876:2012 requirements for setting time, radiopacity and flowability. Although only AHP effectively reduced the biofilm biomass, all sealers, except BRR, reduced biofilm metabolic activity. Despite adequate physical properties, none of the sealers tested prevented biofilm growth. Significant changes in community composition were observed. If observed in vivo , these changes could affect intracanal microbial survival, pathogenicity and treatment outcomes.

AIM:To evaluate the root canal microbiome composition in cases of primary and secondary apical periodontitis. METHODOLOGY:Thirty-nine samples from patients with primary root canal infections obtained before root canal treatment, and 40 samples obtained during root-end resection procedures from previously filled cases with apical periodontitis were evaluated using 16S rRNA next-generation sequencing analysis (NGS). Demographic and clinical factors included age, sex, infection type, percussion sensitivity, and presence of pain. Differences in abundances of genera were evaluated using Kruskal-Wallis test. Alpha and beta diversity indices were calculated using mothur. The Shannon and Chao1 indices were used to measure alpha diversity. The Bray-Curtis dissimilarity was used to measure beta diversity. Differences in community composition were evaluated using analysis of similarity (ANOSIM) with Bonferroni correction for multiple comparisons. RESULTS:Significantly fewer operational taxonomic units values were observed from samples from secondary infections (p < .0001). While no significant differences were observed in the Chao 1 index between primary and secondary infections, the Shannon alpha diversity was significantly lower in secondary relative to primary infections (p = .008). Among samples, sex, age (adult vs. older adult), percussion sensitivity, and presence of pain all showed no significant effects on community composition via an analysis of similarity (ANOSIM). However, community composition was significantly different depending on whether the sample was from a primary or secondary infection (R = .051, p = .03). Nine microbial genera comprised the predominant taxa observed among samples (>3.3%) and included Parvimonas, Fusobacterium, Campylobacter, Arachnia, Eubacterium, Prevotella, Peptostreptococcus, Fretibacterirum, and Pseudoramibacter. Significantly greater relative abundances of Prevotella, Peptostreptococcus, Veillonella, Lactucaseibacillus, and Dialister were observed in primary infections. CONCLUSIONS:Primary endodontic infections are more diverse than secondary infections. The microbial composition is not associated with the clinical manifestations of apical periodontitis.

Abstract Aim To evaluate the physicochemical properties of five root canal sealers and assess their effect on an ex vivo dental plaque‐derived polymicrobial community. Methodology Dental plaque‐derived microbial communities were exposed to the sealers (AH Plus [AHP], GuttaFlow Bioseal [GFB], Endoseal MTA [ESM], Bio‐C sealer [BCS] and BioRoot RCS [BRR]) for 3, 6 and 18 h. The sealers' effect on the biofilm biomass and metabolic activity was quantified using crystal violet (CV) staining and MTT assay, respectively. Biofilm community composition and morphology were assessed by denaturing gradient gel electrophoresis (DGGE), 16S rRNA sequencing and scanning electron microscopy. The ISO6876:2012 specifications were followed to determine the setting time, radiopacity, flowability and solubility. Obturated acrylic teeth were used to assess the sealers' effect on pH. Surface chemical characterization was performed using SEM with coupled energy‐dispersive spectroscopy. Data normality was assessed using the Shapiro–Wilk test. One‐way anova and Tukey's tests were used to analyze data from setting time, radiopacity, flowability and solubility. Two‐way anova and Dunnett's tests were used for the data analysis from CV, MTT and pH. 16S rRNA sequencing data were analyzed for alpha (Shannon index and Chao analysis) and beta diversity (Bray–Curtis dissimilarities). Differences in community composition were evaluated by analysis of similarity (p < .05). Results The sealers significantly influenced microbial community composition and morphology. All sealers complied with ISO6876:2012 requirements for setting time, radiopacity and flowability. Although only AHP effectively reduced the biofilm biomass, all sealers, except BRR, reduced biofilm metabolic activity. Conclusion Despite adequate physical properties, none of the sealers tested prevented biofilm growth. Significant changes in community composition were observed. If observed in vivo, these changes could affect intracanal microbial survival, pathogenicity and treatment outcomes.

Streptococcus gordonii is a commensal oral organism. Harmless in the oral cavity, S. gordonii is an opportunistic pathogen. S. gordonii adheres to body surfaces using surface adhesive proteins (adhesins), which are critical to subsequent formation of biofilm communities. As in most Gram-positive bacteria, S. gordonii surface proteins containing the C-terminal LPXTG motif cleavage sequence are processed by sortase A (SrtA) to become covalently attached to the cell wall. To characterize the functional diversity and redundancy in the family of SrtA-processed proteins, an S. gordonii DL1 markerless deletion mutant library was constructed of each of the 26 putative SrtA-processed proteins. Each library member was evaluated for growth in rich medium, biofilm formation on plastic, saliva and salivary fractions, cell surface hydrophobicity (CSH), hemagglutination, and integration into an ex vivo plaque biofilm community. Library members were compared to the non-SrtA-processed adhesins AbpA and AbpB. While no major growth differences in rich medium were observed, many S. gordonii LPXTG/A proteins impacted biofilm formation on one or more of the substrates. Several mutants showed significant differences in hemagglutination, hydrophobicity, or fitness in the ex vivo plaque model. From the identification of redundant and unique functions in these in vitro and ex vivo systems, functional stratification among the LPXTG/A proteins is apparent. IMPORTANCE S. gordonii interactions with its environment depend on the complement of cell wall proteins. A subset of these cell wall proteins requires processing by the enzyme sortase A (SrtA). The identification of SrtA-processed proteins and their functional characterization will help the community to better understand how S. gordonii engages with its surroundings, including other microbes, integrates into the plaque community, adheres to the tooth surface, and hematogenously disseminates to cause blood-borne infections. This study identified 26 putative SrtA-processed proteins through creation of a markerless deletion mutant library. The library was subject to functional screens that were chosen to better understand key aspects of S. gordonii physiology and pathogenesis.

In this study, a mutant library was screened to identify the Streptococcus sanguinis proteins involved in binding to Fusobacterium nucleatum during biofilm formation. Seven mutants showed a significant defect in the interaction with F. nucleatum, and the affected genes encoded the adhesins SspD and CrpA, three proteins with pleiotropic function (YidC, SrtB and DltB), a predicted stomatin/prohibitin-like membrane protease subunit and a hypothetical protein. These proteins could serve as therapeutic targets to prevent biofilm-related oral diseases.