Effects of short-term xylitol gum chewing on the oral microbiome.

OBJECTIVES: The aim of this study was to determine the effects of short-term xylitol gum chewing on the salivary microbiota of children. MATERIALS AND METHODS: The study was a randomised, controlled, double-blind trial. Healthy children used xylitol chewing gum (xylitol group, n = 35) or sorbitol chewing gum (control group, n = 38) for 5 weeks. The daily dose of xylitol/sorbitol was approximately 6 g/day. At baseline and at the end of the test period, unstimulated and paraffin-stimulated saliva were collected. The microbial composition of the saliva was assessed using human oral microbe identification microarray (HOMIM). Mutans streptococci (MS) were plate cultured. RESULTS: As judged by HOMIM results, no xylitol-induced changes in the salivary microbiota took place in the xylitol group. In the control group, Veillonella atypica showed a significant decrease (p = 0.0001). The xylitol gum chewing decreased viable counts of MS in both stimulated (p = 0.006) and unstimulated (p = 0.002) saliva, but similar effects were also seen in the control group. CONCLUSIONS: The use of xylitol gum decreased MS, in general, but did not change the salivary microbial composition. CLINICAL RELEVANCE: Short-term consumption of xylitol had no impact on the composition of the salivary microbiota, but resulted in a decrease in the levels of MS.

Impact of orally administered lozenges with Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis BB-12 on the number of salivary mutans streptococci, amount of plaque, gingival inflammation and the oral microbiome in healthy adults.

OBJECTIVES: The aim was to evaluate the effects of orally administered Lactobacillus rhamnosus GG (LGG) and Bifidobacterium animalis subsp. lactis BB-12 (BB-12) on the number of salivary mutans streptococci (MS), amount of plaque, gingival inflammation and the oral microbiota in healthy young adults. MATERIALS AND METHODS: The study was a randomised, controlled, double-blind trial. Healthy volunteers used lozenges containing a combination of LGG and BB-12 (test group, n = 29) or lozenges without added probiotics (control group, n = 31) for 4 weeks. At baseline and at the end of the test period, the plaque index (PI) and gingival index (GI) were determined, and stimulated saliva was collected. The microbial composition of saliva was assessed using human oral microbe identification microarray (n = 30). MS and lactobacilli (LB) were plate cultured. RESULTS: The probiotic lozenge decreased both PI and GI (p < 0.05) while no changes were observed in the control group. However, no probiotic-induced changes were found in the microbial compositions of saliva in either group. CONCLUSIONS: The probiotic lozenge improved the periodontal status without affecting the oral microbiota. CLINICAL RELEVANCE: Short-term consumption of LGG and BB-12 decreased the amount of plaque which was associated with a clinical impact: a decrease in gingival inflammation.

HIV infection and microbial diversity in saliva.

Limited information is available about the effects of HIV and subsequent antiretroviral treatment on host-microbe interactions. This study aimed to determine the salivary microbial composition for 10 HIV-seropositive subjects, before and 6 months after highly active antiretroviral therapy (HAART), compared with that for 10 HIV-seronegative subjects. A conventional culture and two culture-independent analyses were used and consistently demonstrated differences in microbial composition among the three sets of samples. HIV-positive subjects had higher levels of total cultivable microbes, including oral streptococci, lactobacilli, Streptococcus mutans, and Candida, in saliva than did HIV-negative subjects. The total cultivable microbial levels were significantly correlated with CD4+ T cell counts. Denaturing gradient gel electrophoresis (DGGE), which compared the overall microbial profiles, showed distinct fingerprinting profiles for each group. The human oral microbe identification microarray (HOMIM) assay, which compared the 16S rRNA genes, showed clear separation among the three sample groups. Veillonella, Synergistetes, and Streptococcus were present in all 30 saliva samples. Only minor changes or no changes in the prevalence of Neisseria, Haemophilus, Gemella, Leptotrichia, Solobacterium, Parvimonas, and Rothia were observed. Seven genera, Capnocytophaga, Slackia, Porphyromonas, Kingella, Peptostreptococcaceae, Lactobacillus, and Atopobium, were detected only in HIV-negative samples. The prevalences of Fusobacterium, Campylobacter, Prevotella, Capnocytophaga, Selenomonas, Actinomyces, Granulicatella, and Atopobium were increased after HAART. In contrast, the prevalence of Aggregatibacter was significantly decreased after HAART. The findings of this study suggest that HIV infection and HAART can have significant effects on salivary microbial colonization and composition.

Specific host metabolite and gut microbiome alterations are associated with bone loss during spaceflight.

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 microgravity 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 microgravity-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.

Construction of habitat-specific training sets to achieve species-level assignment in 16S rRNA gene datasets.

BACKGROUND: The low cost of 16S rRNA gene sequencing facilitates population-scale molecular epidemiological studies. Existing computational algorithms can resolve 16S rRNA gene sequences into high-resolution amplicon sequence variants (ASVs), which represent consistent labels comparable across studies. Assigning these ASVs to species-level taxonomy strengthens the ecological and/or clinical relevance of 16S rRNA gene-based microbiota studies and further facilitates data comparison across studies. RESULTS: To achieve this, we developed a broadly applicable method for constructing high-resolution training sets based on the phylogenic relationships among microbes found in a habitat of interest. When used with the naïve Bayesian Ribosomal Database Project (RDP) Classifier, this training set achieved species/supraspecies-level taxonomic assignment of 16S rRNA gene-derived ASVs. The key steps for generating such a training set are (1) constructing an accurate and comprehensive phylogenetic-based, habitat-specific database; (2) compiling multiple 16S rRNA gene sequences to represent the natural sequence variability of each taxon in the database; (3) trimming the training set to match the sequenced regions, if necessary; and (4) placing species sharing closely related sequences into a training-set-specific supraspecies taxonomic level to preserve subgenus-level resolution. As proof of principle, we developed a V1-V3 region training set for the bacterial microbiota of the human aerodigestive tract using the full-length 16S rRNA gene reference sequences compiled in our expanded Human Oral Microbiome Database (eHOMD). We also overcame technical limitations to successfully use Illumina sequences for the 16S rRNA gene V1-V3 region, the most informative segment for classifying bacteria native to the human aerodigestive tract. Finally, we generated a full-length eHOMD 16S rRNA gene training set, which we used in conjunction with an independent PacBio single molecule, real-time (SMRT)-sequenced sinonasal dataset to validate the representation of species in our training set. This also established the effectiveness of a full-length training set for assigning taxonomy of long-read 16S rRNA gene datasets. CONCLUSION: Here, we present a systematic approach for constructing a phylogeny-based, high-resolution, habitat-specific training set that permits species/supraspecies-level taxonomic assignment to short- and long-read 16S rRNA gene-derived ASVs. This advancement enhances the ecological and/or clinical relevance of 16S rRNA gene-based microbiota studies. Video Abstract.

Subgingival microbiome and clinical periodontal status in an elderly cohort: The WHICAP ancillary study of oral health.

BACKGROUND: There is a sparsity of data describing the periodontal microbiome in elderly individuals. We analyzed the association of subgingival bacterial profiles and clinical periodontal status in a cohort of participants in the Washington Heights-Inwood Columbia Aging Project (WHICAP). METHODS: Dentate individuals underwent a full-mouth periodontal examination at six sites/tooth. Up to four subgingival plaque samples per person, each obtained from the mesio-lingual site of the most posterior tooth in each quadrant, were harvested and pooled. Periodontal status was classified according to the Centers for Disease Control/American Academy of Periodontology (CDC/AAP) criteria as well as based on the percentage of teeth/person with pockets ≥4 mm deep. Bacterial DNA was isolated and was processed and analyzed using Human Oral Microbe Identification using Next Generation Sequencing (HOMINGS). Differential abundance across the periodontal phenotypes was calculated using the R package DESeq2. α- and ß-diversity metrics were calculated using DADA2-based clustering. RESULTS: The mean age of the 739 participants was 74.5 years, and 32% were male. Several taxa including Sneathia amnii-like sp., Peptoniphilaceae [G-1] bacterium HMT 113, Porphyromonas gingivalis, Fretibacterium fastidiosum, Filifactor alocis, and Saccharibacteria (TM7) [G-1] bacterium HMT 346 were more abundant with increasing severity of periodontitis. In contrast, species such as Veillonella parvula, Veillonella dispar, Rothia dentocariosa, and Lautropia mirabilis were more abundant in health. Microbial diversity increased in parallel with the severity and extent of periodontitis. CONCLUSIONS: The observed subgingival bacterial patterns in these elderly individuals corroborated corresponding findings in younger cohorts and were consistent with the concept that periodontitis is associated with perturbations in the resident microbiome.

The Oral Mouse Microbiome Promotes Tumorigenesis in Oral Squamous Cell Carcinoma.

Oral squamous cell carcinoma (OSCC) is the most common malignancy of the head and neck worldwide. Dysbiosis of the microbiome has increasingly been linked to the development of different kinds of cancer. Applying 16S rRNA gene sequence analysis and metatranscriptomic analyses, we characterized the longitudinal changes in the profiles and the function of the oral microbiome in a 4-nitroquinoline-1-oxide (4-NQO)-induced model of OSCC in gnotobiotic mice. We characterized the dynamics of the oral microbiome in this model using two different microbiome inocula: one from healthy mice and the other from mice bearing a 4-NQO-induced tumor. Mice colonized with different oral microbiomes and exposed to 4-NQO had increased tumor numbers and sizes compared to controls exposed to 4-NQO but lacking a microbiome. We observed an overall increase in diversity in the tumorigenic samples compared to that in the nontumor group not exposed to 4-NQO. Despite the variability in community dynamics, specific patterns emerged during the progression of the disease. In the two groups that were inoculated with the OSCC-associated microbiome, we observed opposite profiles of abundance in Parabacteroides and Corynebacterium While the percentage of Parabacteroides bacteria decreased in the control group, it increased in the OSCC group, and the opposite was observed for Corynebacterium The metatranscriptomic analysis revealed overexpression of the same metabolic signatures associated with OSCC regardless of the community profile. These included nitrogen transport, response to stress, interspecies interactions, Wnt pathway modulation, and amino acid and lipid biosynthesis. Thus, these results seem to suggest that certain collective physiological activities are critical for microbiome-mediated OSCC progression.IMPORTANCE There is growing evidence that changes in the microbiome are associated with carcinogenesis. To date, no consistent oral microbiome composition associated with OSCC has been identified. Longitudinal and functional studies like the study presented here should yield a better understanding of the role that the oral microbiome plays in OSCC. Our findings, obtained using a germ-free mouse model, indicate that the presence of different oral microbiomes enhances tumorigenesis and increases the final number of tumors in mice. By studying community-wide expression profiles, we found that regardless of the phylogenetic composition of the microbiome, the same metabolic activities were consistently associated with OSCC. Therefore, due to the functional redundancy of the microbiome, the critical element in explaining the contribution of the microbiota in OSCC is the collective physiological activity of the community, thus accounting for the previous inability to identify a consensus community profile or etiologic agents for OSCC.

Analysis of oral bacterial communities: comparison of HOMINGS with a tree-based approach implemented in QIIME.

Background: Molecular taxonomic assignments in oral microbial communities have been made using probe-matching approaches, but never compared to those obtained by more readily accepted tree-based approaches. Objective:  To compare community composition profiles obtained from a probe-matching approach (HOMINGS) to those from a closed-ended tree-based approach (QIIME using the eHOMD database). Design:  HOMINGS and QIIME were used for parallel analysis of ten mock community samples, and of 119 supragingival plaque samples from ecologically unique sites (sound tooth surfaces in healthy subjects, sound tooth surfaces in patients with primary Sjögren's Syndrome, and carious lesions in Sjögren's Syndrome patients). Linear discriminant analysis Effective Size (LEfSe) was used to identify discriminating taxa among the natural plaque samples. Results: Community composition profiles of all samples were congruent between the two analysis aproaches. Alpha and beta diversity of the natural plaque communities were likewise similar. Communities from pSS patients and those from individuals with normal salivary flow differed in alpha and beta diversity. Both classification approaches yielded differences in composition predicted for samples from these subject cohorts, and discriminating taxa were similar between approaches. Conclusions: A direct comparison demonstrates that HOMINGS is largely equivalent to the tree-based approach as implemented here.

Salivary microbiota in individuals with different levels of caries experience.

This study compared salivary bacterial profiles in two groups having a 10-fold difference in levels of caries experience, as it was hypothesized that the composition of the salivary microbiota might associate with the levels of caries experience. Bacterial profiles in stimulated saliva samples from 85 individuals with low levels of caries experience (healthy group) and 79 individuals with high levels of caries experience (caries group) were analyzed by means of the Human Oral Microbiome Identification Next Generation Sequencing (HOMINGS) technique. Subsequently, saliva samples from caries-free individuals in the healthy group (n = 57) and the caries group (n = 31) were compared. A significantly higher α-diversity (p < 0.0001) and a twofold higher relative abundance of Neisseria, Haemophilus, and Fusobacterium were recorded in saliva samples from the healthy group compared with the caries group. Differences observed were more pronounced when limiting the analyses to caries-free individuals in each group. Data from this cross-sectional analysis suggest that low levels of caries experience might associate with a characteristic salivary bacterial composition different from that in individuals with high caries experience. Consequently, longitudinal studies are required to determine if the composition of the salivary microbiota might be a predictive factor of caries risk at the individual level.

Bacterial Composition at the Implant-Abutment Connection under Loading in vivo.

PURPOSE: Platform-switched implants have been demonstrated to prevent bone loss after loading. The present study evaluated bacterial composition of sites from implant-abutment connections of immediately loaded implants, which were placed in the anterior mandible. Ten patients participated in this study. MATERIALS AND METHODS: A and B implant systems with two different prosthetic connections (Morse tapered vs internal polygonal butt-joint connections, respectively) were placed and loaded for 2 years. The abutments were removed (AB sample) after careful decontamination. Bacterial sampling of the abutments, inner part of the implants (before/visit 1 and after rinsing with chlorhexidine [CHX]/visit 2), and after new abutment connection and loading for 1 additional month, a new sampling (visit 3) was taken to compare the bacteria composition in association with the two connections. Bacterial profiles of samples were determined by using the human oral microbe identification microarray. RESULTS: A total of 240 samples were analyzed taken at different time intervals. Nonparametric statistical analysis (Wilcoxon Rank sum) with uncorrected alpha (p < .05) and after corrections (Benjamini-Hochberg) found no statistical significance between the two connections. No significant changes in the overall microbial profiles were detected at the different time intervals. However, there were trends toward presence of periodontitis-associated species at the B implants in all samples (AB, visit 1, even after CHX irrigation) and after decontamination, abutment replacement, and 1-month loading period. CONCLUSIONS: CHX irrigation does not seem to have any effect on decontamination of connections. As shown previously, there is significantly more bone loss around B implants compared with A implants. Although there was no statistical difference in the microbial profiles, there was indeed a trend for the presence of typical periodontal pathogens associated with the internal polygonal butt-joint connection. A possible scenario is that this connection tends to harbor the pathogens that may be involved in subsequent bone loss.

Comparative analysis of bacterial profiles in unstimulated and stimulated saliva samples.

BACKGROUND AND OBJECTIVE: The microbial profiles of stimulated saliva samples have been shown to differentiate between patients with periodontitis, patients with dental caries, and orally healthy individuals. Saliva was stimulated to allow for easy and rapid collection; however, microbial composition may not reflect the more natural, unstimulated state. The purpose of this study was to validate whether stimulated saliva is an adequate surrogate for unstimulated saliva in determining salivary microbiomes. DESIGN: Unstimulated (n=20) and stimulated (n=20) saliva samples were collected from 20 orally and systemically healthy, non-smoking participants. Salivary bacterial profiles were analyzed by means of the Human Oral Microbe Identification using Next Generation Sequencing (HOMINGS), and statistical analysis was performed using Mann-Whitney test with Benjamini-Hochberg's correction for multiple comparison, cluster analysis, principal component analysis, and correspondence analysis. RESULTS: From a total of 40 saliva samples, 496 probe targets were identified with a mean number of targets per sample of 203 (range: 146-303), and a mean number of probe targets of 206 and 200 in unstimulated and stimulated saliva samples, respectively (p=0.62). Based on all statistical methods used for this study, the microbial profiles of unstimulated and stimulated saliva samples collected from the same person were not statistically significantly different. CONCLUSIONS: Analysis of bacterial salivary profiles in unstimulated and stimulated saliva samples collected from the same individual showed comparable results. Thus, the results verify that stimulated saliva is an adequate surrogate of unstimulated saliva for microbiome-related studies.

Temporal Stability of the Salivary Microbiota in Oral Health.

OBJECTIVES: Saliva is a biological fluid suitable for biomarker analysis, and differences in the salivary microbiota in oral health and disease have been reported. For such comparative analyses, time of sampling is critical since the bacterial composition may vary throughout the day, i.e., diurnal variation. The purpose of this study is to compare the salivary microbiome over time to determine the optimal time for sampling. DESIGN: Stimulated saliva samples were collected from 5 orally healthy individuals in 4 h intervals for 24 h, and collection was repeated 7 days later (number of samples per person, n = 12, total number of samples, n = 60). Salivary microbiota was analyzed using the Human Oral Microbe Identification using Next Generation Sequencing (HOMINGS), and statistical analysis was performed using the Kruskal-Wallis test with Benjamini-Hochberg's correction for multiple comparisons, cluster analysis, principal component analysis and correspondence analysis. RESULTS: From a total of 60 saliva samples, 477 probe targets were collectively identified with a mean number of probes per sample of 207 (range: 153-307). Little or no variation in microbial profiles within subjects was observed over time. CONCLUSIONS: Although there was considerable variation between subjects, microbial profiles within subjects were stable throughout a 24 hour period and after 1 week. Since there is little or no evidence of diurnal variation of the salivary microbiome, time of sampling of saliva is not critical for perturbation or other microbial studies.

Microbiomes of Endodontic-Periodontal Lesions before and after Chemomechanical Preparation.

INTRODUCTION: This study was conducted to evaluate the microbiomes of endodontic-periodontal lesions before and after chemomechanical preparation (CMP). METHODS: Clinical samples were taken from 15 root canals (RCs) with necrotic pulp tissues and from their associated periodontal pockets (PPs) (n = 15) of teeth with endodontic-periodontal lesions before and after CMP. The Human Oral Microbe Identification using Next Generation Sequencing (NGS) protocol and viable culture were used to analyze samples from RCs and PPs. The Mann-Whitney U test and Benjamini-Hochberg corrections were performed to correlate the clinical and radiographic findings with microbial findings (P < .05). RESULTS: Bacteria were detected in 100% of the samples in both sites (15/15) using NGS. Firmicutes was the most predominant phylum in both sites using both methods. The most frequently detected species in the RCs before and after CMP using NGS were Enterococcus faecalis, Parvimonas micra, Mogibacterium timidum, Filifactor alocis, and Fretibacterium fastidiosum. The species most frequently detected in the PPs before and after CMP using NGS were P. micra, E. faecalis, Streptococcus constellatus, Eubacterium brachy, Tannerella forsythia, and F. alocis. Associations were found between periapical lesions ≤ 2 mm and Desulfobulbus sp oral taxon 041 and with periodontal pockets ≥ 6 mm and Dialister invisius and Peptostreptococcus stomatis (all P < .05, found in the RCs before CMP). CONCLUSIONS: It is concluded that the microbial community present in combined endodontic-periodontal lesions is complex and more diverse than previously reported. It is important to note that bacteria do survive in some root canals after CMP. Finally, the similarity between the microbiota of both sites, before and after CMP, suggests there may be a pathway of infection between the pulp and periodontium.

Alterations in diversity of the oral microbiome in pediatric inflammatory bowel disease.

BACKGROUND: Oral pathology is a commonly reported extraintestinal manifestation of Crohn's disease (CD). The host-microbe interaction has been implicated in the pathogenesis of inflammatory bowel disease (IBD) in genetically susceptible hosts, yet limited information exists about oral microbes in IBD. We hypothesize that the microbiology of the oral cavity may differ in patients with IBD. Our laboratory has developed a 16S rRNA-based technique known as the Human Oral Microbe Identification Microarray (HOMIM) to study the oral microbiome of children and young adults with IBD. METHODS: Tongue and buccal mucosal brushings from healthy controls, CD, and ulcerative colitis (UC) patients were analyzed using HOMIM. Shannon Diversity Index (SDI) and Principal Component Analysis (PCA) were employed to compare population and phylum-level changes among our study groups. RESULTS: In all, 114 unique subjects from the Children's Hospital Boston were enrolled. Tongue samples from patients with CD showed a significant decrease in overall microbial diversity as compared with the same location in healthy controls (P = 0.015) with significant changes seen in Fusobacteria (P < 0.0002) and Firmicutes (P = 0.022). Tongue samples from patients with UC did not show a significant change in overall microbial diversity as compared with healthy controls (P = 0.418). CONCLUSIONS: As detected by HOMIM, we found a significant decrease in overall diversity in the oral microbiome of pediatric CD. Considering the proposed microbe-host interaction in IBD, the ease of visualization and direct oral mucosal sampling of the oral cavity, further study of the oral microbiome in IBD is of potential diagnostic and prognostic value.