ABSTRACT
The role of bacteria in the etiology of dental caries is long established, while the role of fungi has only recently gained more attention. The microbial invasion of dentin in advanced caries especially merits additional research. We evaluated the fungal and bacterial community composition and spatial distribution within carious dentin. Amplicon 16S rRNA gene sequencing together with quantitative PCR was used to profile bacterial and fungal species in caries-free children (n = 43) and 4 stages of caries progression from children with severe early childhood caries (n = 32). Additionally, healthy (n = 10) and carious (n = 10) primary teeth were decalcified, sectioned, and stained with Grocott's methenamine silver, periodic acid Schiff (PAS) and calcofluor white (CW) for fungi. Immunolocalization was also performed using antibodies against fungal ß-D-glucan, gram-positive bacterial lipoteichoic acid, gram-negative endotoxin, Streptococcus mutans, and Candida albicans. We also performed field emission scanning electron microscopy (FESEM) to visualize fungi and bacteria within carious dentinal tubules. Bacterial communities observed included a high abundance of S. mutans and the Veillonella parvula group, as expected. There was a higher ratio of fungi to bacteria in dentin-involved lesions compared to less severe lesions with frequent preponderance of C. albicans, C. dubliniensis, and in one case C. tropicalis. Grocott's silver, PAS, CW and immunohistochemistry (IHC) demonstrated the presence of fungi within carious dentinal tubules. Multiplex IHC revealed that fungi, gram-negative, and gram-positive bacteria primarily occupied separate dentinal tubules, with rare instances of colocalization. Similar findings were observed with multiplex immunofluorescence using anti-S. mutans and anti-C. albicans antibodies. Electron microscopy showed monomorphic bacterial and fungal biofilms within distinct dentin tubules. We demonstrate a previously unrecognized phenomenon in which fungi and bacteria occupy distinct spatial niches within carious dentin and seldom co-colonize. The potential significance of this phenomenon in caries progression warrants further exploration.
Subject(s)
Dental Caries , Dentin , Humans , Dental Caries/microbiology , Dental Caries/pathology , Dentin/microbiology , Male , Child , Female , Child, Preschool , Bacteria/genetics , Fungi , RNA, Ribosomal, 16SABSTRACT
OBJECTIVE: HIV disease is evolving with more HIV+ persons experiencing a high quality of life with well-controlled viremia. We recently enrolled a large cohort of HIV+ and clinically relevant HIV- persons for oral microbiome analyses that included a questionnaire related to oral hygiene and recreational behaviors. Here, the questionnaire responses were analyzed for behavioral trends within the cohort, together with trends over time by comparison to a previous geographically centered HIV+ cohort. METHODS: Data were collected by questionnaire at baseline visits as cross-sectional assessments. Multivariable analyses were conducted for associations of HIV status as well as age, race, and sex, on oral hygiene/recreational behaviors. RESULTS: HIV+ subjects had reduced brushing frequency, but increased incidence of past cleanings and frequency of dry mouth, compared to the HIV- subjects. Within the entire cohort, positive associations were identified between age and several oral hygiene practices, and between age, race, and sex for several recreational behaviors. In comparison to the historical cohort, the contemporary HIV+ cohort participated in fewer high-risk behaviors, but with similar trends for smoking and oral hygiene practices. CONCLUSION: HIV status had little association with oral hygiene and recreational behaviors despite several differences in age, race, and sex. Behavioral trends over time support a higher quality of life in people currently living with HIV.
Subject(s)
HIV Infections , Oral Hygiene , Humans , Quality of Life , Cross-Sectional Studies , Toothbrushing , HIV Infections/epidemiologyABSTRACT
The oral microbiome is an important predictor of health and disease. We recently reported significant yet modest effects of HIV under highly active antiretroviral therapy (ART) on the oral microbiome (bacterial and fungal) in a large cohort of HIV-positive (HIV+) and matched HIV-negative (HIV-) individuals. As it was unclear whether ART added to or masked further effects of HIV on the oral microbiome, the present study aimed to analyze the effects of HIV and ART independently, which also included HIV- subjects on preexposure prophylaxis (PrEP) therapy. Cross-sectional analyses of the effect of HIV devoid of ART (HIV+ ART- versus matched HIV- subjects) showed a significant effect on both the bacteriome and mycobiome (P < 0.024) after controlling for other clinical variables (permutational multivariate analysis of variance [PERMANOVA] of Bray-Curtis dissimilarity). Cross-sectional analyses evaluating the effects of ART (HIV+ ART+ versus HIV+ ART- subjects) revealed a significant effect on the mycobiome (P < 0.007) but not the bacteriome. In parallel longitudinal analyses, ART (before versus after the initiation of ART) had a significant effect on the bacteriome, but not the mycobiome, of HIV+ and HIV- PrEP subjects (P < 0.005 and P < 0.016, respectively). These analyses also revealed significant differences in the oral microbiome and several clinical variables between HIV- PrEP subjects (pre-PrEP) and the HIV-matched HIV- group (P < 0.001). At the species level, a small number of differences in both bacterial and fungal taxa were identified within the effects of HIV and/or ART. We conclude that the effects of HIV and ART on the oral microbiome are similar to those of the clinical variables but collectively are modest overall. IMPORTANCE The oral microbiome can be an important predictor of health and disease. For persons living with HIV (PLWH), HIV and highly active antiretroviral therapy (ART) may have a significant influence on their oral microbiome. We previously reported a significant effect of HIV with ART on both the bacteriome and mycobiome. It was unclear whether ART added to or masked further effects of HIV on the oral microbiome. Hence, it was important to evaluate the effects of HIV and ART independently. For this, multivariate cross-sectional and longitudinal oral microbiome analyses (bacteriome and mycobiome) were conducted within the cohort, including HIV+ ART+ subjects and HIV+ and HIV- (preexposure prophylaxis [PrEP]) subjects before and after the initiation of ART. While we report independent significant effects of HIV and ART on the oral microbiome, we conclude that their influence is similar to that of the clinical variables but collectively modest overall.
Subject(s)
HIV Infections , Microbiota , Mycobiome , Humans , Cross-Sectional Studies , Bacteria , HIV Infections/drug therapy , HIV Infections/microbiology , Multivariate AnalysisABSTRACT
Human microbiome composition is closely tied to health, but how the host manages its microbial inhabitants remains unclear. One important, but understudied, factor is the natural host environment: mucus, which contains gel-forming glycoproteins (mucins) that display hundreds of glycan structures with potential regulatory function. Leveraging a tractable culture-based system to study how mucins influence oral microbial communities, we found that mucin glycans enable the coexistence of diverse microbes, while resisting disease-associated compositional shifts. Mucins from tissues with unique glycosylation differentially tuned microbial composition, as did isolated mucin glycan libraries, uncovering the importance of specific glycan patterns in microbiome modulation. We found that mucins shape microbial communities in several ways: serving as nutrients to support metabolic diversity, organizing spatial structure through reduced aggregation, and possibly limiting antagonism between competing taxa. Overall, this work identifies mucin glycans as a natural host mechanism and potential therapeutic intervention to maintain healthy microbial communities.
Subject(s)
Microbiota , Mucins , Humans , Mucins/chemistry , Mucins/metabolism , Glycosylation , Mucus/metabolism , Polysaccharides/metabolismABSTRACT
OBJECTIVE: This randomized, prospective, blinded study compared pain in children following dental treatment under general anesthesia (GA) using 1 of 2 established analgesia methods. METHODS: Patients age 4 to 7 years were randomly assigned to a control group (intravenous [IV] analgesics) or experimental group (IV analgesics and intrapapillary local anesthetic infiltrations) between July 2017 and February 2018. During recovery from surgery, Faces, Legs, Activity, Cry, and Consolability (FLACC) scores were recorded upon regaining consciousness and reassessed every 15 minutes until discharge. Overall pain occurrence (FLACC ≥1) and moderate/severe pain occurrence (FLACC ≥4) were analyzed using mixed effects logistic regression (N = 88). RESULTS: The experimental group had a 17% lower overall pain occurrence than the control group (16 vs 33%; p = .02). Moderate/severe pain occurrence between the groups was not significant (9 vs 22%; p = .23). The dental treatment subjects received (number of completed stainless steel crowns, extractions, and/or pulpotomies) did not significantly affect pain occurrence. CONCLUSION: Local anesthesia intrapapillary infiltrations around stainless steel crowns decrease overall pain occurrence but not moderate/severe pain occurrence following dental treatment under GA in pediatric patients.
Subject(s)
Anesthesia, Local , Anesthetics, Local , Anesthesia, General/adverse effects , Anesthesia, Local/adverse effects , Anesthesia, Local/methods , Anesthetics, Local/adverse effects , Child , Child, Preschool , Dental Care , Humans , Pain Measurement , Pain, Postoperative/etiology , Pain, Postoperative/prevention & control , Prospective StudiesABSTRACT
Purpose: The purpose of this study was to investigate an association between treatments on the primary second molars (PSMs) under general anesthesia (GA) and odds of repeat GA. Methods: This was a retrospective study of children who received dental treatment under GA between the ages of 24 to 48 months. Descriptive statistics and logistic regression models (P<0.05) were used to test the association between the treatment of PSMs at the first dental GA visit (GA1) and the odds of receiving GA a second time (GA2) within the next 55 months post-GA1. Results: A total of 819 children (53 percent male) with a mean (±SD) age of 36 (±seven SD) months and 3,276 PSMs were included. Only three percent of children with all PSMs covered at GA1 received GA2. The odds of GA2 significantly increased for children with any uncovered PSMs. Among children with four uncovered PSMs, 19 percent (odds ratio [OR] equals 13; 95 percent confidence interval [95% CI] equals 5.8 to 33.5; P<0.001) and among those with unerupted PSMs at GA1, 51 percent received GA2 (OR equals 62.9; 95% CI equals 23.5 to 189.2; P<0.001). In the group that received GA2, 79.1 percent of uncovered PSMs at GA1 eventually received a stainless steel crown at GA2. Conclusions: Restorative treatments other than stainless steel crowns were associated with higher odds of repeat general anesthesia. These findings support the preferential use of full-coverage restorations for the treatment of carious primary molars in young children undergoing GA to minimize the risk of the need for repeat GA.
Subject(s)
Crowns , Dental Caries , Anesthesia, General , Child , Child, Preschool , Dental Caries/therapy , Humans , Male , Molar/surgery , Retrospective Studies , Tooth, DeciduousABSTRACT
The oral microbiome is considered an important factor in health and disease. We recently reported significant effects of HIV and several other clinical variables on the oral bacterial communities in a large cohort of HIV-positive and -negative individuals. The purpose of the present study was to similarly analyze the oral mycobiome in the same cohort. To identify fungi, the internal transcribed spacer 2 (ITS2) of the fungal rRNA genes was sequenced using oral rinse samples from 149 HIV-positive and 88 HIV-negative subjects that had previously undergone bacterial amplicon sequencing. Quantitative PCR was performed for total fungal content and total bacterial content. Interestingly, samples often showed predominance of a single fungal species with four major clusters predominated by Candida albicans, Candida dubliniensis, Malassezia restricta, or Saccharomyces cerevisiae Quantitative PCR analysis showed the Candida-dominated sample clusters had significantly higher total fungal abundance than the Malassezia or Saccharomyces species. Of the 25 clinical variables evaluated for potential influences on the oral mycobiome, significant effects were associated with caries status, geographical site of sampling, sex, HIV under highly active antiretroviral therapy (HAART), and missing teeth, in rank order of statistical significance. Investigating specific interactions between fungi and bacteria in the samples often showed Candida species positively correlated with Firmicutes or Actinobacteria and negatively correlated with Fusobacteria, Proteobacteria, and Bacteroidetes Our data suggest that the oral mycobiome, while diverse, is often dominated by a limited number of species per individual; is affected by several clinical variables, including HIV positivity and HAART; and shows genera-specific associations with bacterial groups.IMPORTANCE The oral microbiome is likely a key element of homeostasis in the oral cavity. With >600 bacterial species and >160 fungal species comprising the oral microbiome, influences on its composition can have an impact on both local and systemic health. We recently reported significant effects of HIV and several other clinical variables on the oral bacterial community in a large cohort of HIV-positive and -negative subjects. We describe here a comprehensive analysis of the oral mycobiome in the same cohort. Similar to the bacterial community, HIV under highly active antiretroviral therapy (HAART) had a significant impact on the mycobiome composition, but with less impact compared to other clinical variables. Additionally, unlike the oral bacterial microbiome, the oral mycobiome is often dominated by a single species with 4 major clusters of fungal communities. Together, these results suggest the oral mycobiome has distinct properties compared with the oral bacterial community, although both are equally impacted by HIV.
Subject(s)
Antiretroviral Therapy, Highly Active , HIV Infections/drug therapy , HIV/physiology , Mouth/microbiology , Mouth/virology , Multivariate Analysis , Mycobiome/genetics , Bacteria/classification , Bacteria/genetics , Bacteria/metabolism , Cohort Studies , DNA, Intergenic/genetics , Female , Fungi/classification , Fungi/genetics , Fungi/metabolism , HIV/genetics , HIV Infections/virology , Humans , Male , Mycobiome/physiologyABSTRACT
BACKGROUND: The oral microbiota is acquired very early, but the factors shaping its acquisition are not well understood. Previous studies comparing monozygotic (MZ) and dizygotic (DZ) twins have suggested that host genetics plays a role. However, all twins share an equal portion of their parent's genome, so this model is not informative for studying parent-to-child transmission. We used a novel study design that allowed us to directly examine the genetics of transmission by comparing the oral microbiota of biological versus adoptive mother-child dyads. RESULTS: No difference was observed in how closely oral bacterial community profiles matched for adoptive versus biological mother-child pairs, indicating little if any effect of host genetics on the fidelity of transmission. Both adopted and biologic children more closely resembled their own mother as compared to unrelated women, supporting the role of contact and environment. Mother-child strain similarity increased with the age of the child, ruling out early effects of host genetic influence that are lost over time. No effect on the fidelity of mother-child strain sharing from vaginal birth or breast feeding was seen. Analysis of extended families showed that fathers and mothers were equally similar to their children, and that cohabitating couples showed even greater strain similarity than mother-child pairs. These findings support the role of contact and shared environment, and age, but not genetics, as determinants of microbial transmission, and were consistent at both species and strain level resolutions, and across multiple oral habitats. In addition, analysis of individual species all showed similar results. CONCLUSIONS: The host is clearly active in shaping the composition of the oral microbiome, since only a few of the many bacterial species in the larger environment are capable of colonizing the human oral cavity. Our findings suggest that these host mechanisms are universally shared among humans, since no effect of genetic relatedness on fidelity of microbial transmission could be detected. Instead our findings point towards contact and shared environment being the driving factors of microbial transmission, with a unique combination of these factors ultimately shaping the highly personalized human oral microbiome. Video abstract.
Subject(s)
Adoption , Environment , Family Health , Microbiota , Mothers , Mouth/microbiology , Parent-Child Relations , Adult , Bacteria/genetics , Child , Child, Preschool , Disease Transmission, Infectious , Family Health/statistics & numerical data , Fathers , Female , Housing , Humans , Infant , Male , Microbiota/genetics , Pregnancy , Twin Studies as Topic , Twins/geneticsABSTRACT
BACKGROUND: The authors examined time to need new treatment of primary second molars in very young children treated under general anesthesia (GA). METHODS: During this retrospective chart review, the authors examined patients aged 2 through 4 years with severe early childhood caries (ECC) who received dental treatment under GA. Primary second molars were tracked in periodic recall visits after GA for 6 through 89 months. Using a random-effects Cox proportional hazards model, the authors compared hazards of teeth requiring new treatment based on treatments received at GA. RESULTS: Of 3,166 primary second molars included in the study, 367 (12%) were not erupted, 77 (2%) received topical fluoride only, 873 (28%) received a pit and fissure sealant, 242 (8%) received a composite restoration, and 1,607 (50%) received a stainless steel crown (SSC) at GA. SSCs had a survival probability of 98% by 84 months after GA, significantly higher than all other groups (P < .0001). The second molars that were not erupted at GA had the highest hazard, especially within the first 24 months after GA. Teeth that received sealant had longer time to need new treatment than nonsealed teeth; however, at 84 months after GA, only 33% of the sealed teeth did not require additional treatment. CONCLUSIONS: Preventive or restorative treatments other than SSCs resulted in need for new treatment in a substantial number of teeth. SSCs had the highest success in this population with severe ECC treated under GA and should be chosen over other restorative options to reduce risk of undergoing repeat dental treatment. PRACTICAL IMPLICATIONS: Aggressive treatment with SCC should be considered for young children with severe ECC especially those who are treated under GA at a young age.
Subject(s)
Dental Caries , Tooth, Deciduous , Anesthesia, General , Child , Child, Preschool , Humans , Molar , Pit and Fissure Sealants , Retrospective Studies , Survival AnalysisABSTRACT
Persons infected with HIV are particularly vulnerable to a variety of oral microbial diseases. Although various study designs and detection approaches have been used to compare the oral microbiota of HIV-negative and HIV-positive persons, both with and without highly active antiretroviral therapy (HAART), methods have varied, and results have not been consistent or conclusive. The purpose of the present study was to compare the oral bacterial community composition in HIV-positive persons under HAART to an HIV-negative group using 16S rRNA gene sequence analysis. Extensive clinical data was collected, and efforts were made to balance the groups on clinical variables to minimize confounding. Multivariate analysis was used to assess the independent contribution of HIV status. Eighty-nine HIV-negative participants and 252 HIV-positive participants under HAART were sampled. The independent effect of HIV under HAART on the oral microbiome was statistically significant, but smaller than the effect of gingivitis, periodontal disease, smoking, caries, and other clinical variables. In conclusion, a multivariate comparison of a large sample of persons with HIV under HAART to an HIV-negative control group showed a complex set of clinical features that influenced oral bacterial community composition, including the presence of HIV under HAART.
Subject(s)
Dental Caries/microbiology , HIV Infections/microbiology , Microbiota/drug effects , Adult , Anti-Retroviral Agents/pharmacology , Antiretroviral Therapy, Highly Active/methods , CD4 Lymphocyte Count/methods , Female , HIV Infections/drug therapy , HIV-1/drug effects , HIV-1/pathogenicity , Humans , Male , Metagenomics/methods , Multivariate Analysis , RNA, Ribosomal, 16S/geneticsABSTRACT
BACKGROUND: Localized aggressive periodontitis (LAgP) occurs in 2% of African-American adolescents but only 0.15% of white adolescents. First molars and incisors are affected by rapid onset and progression. METHODS: This nonsystematic critical review evaluated published data for LAgP and chronic periodontitis (CP), focusing on potential differences in epidemiology, microbiology, immunology, genetics, and response to therapy. RESULTS: LAgP differs from CP by localization to incisors and first molars, early onset and rapid progression in adolescents and young adults, and a 10-fold higher prevalence in populations of African or Middle Eastern origin, often with strong familial aggregation. The bacterium Aggregatibacter actinomycetemcomitans and hyperresponsive neutrophils are frequently observed. Antibiotic and nonsurgical therapies are highly effective. CONCLUSIONS: LAgP differs in many ways from the far more common CP that affects older adults. The substantial evidence of dissimilarities summarized in this review strongly supports the classification of LAgP as a distinct form of periodontitis. PRACTICAL IMPLICATIONS: Classifying LAgP as a distinct subcategory of periodontitis will encourage future research and does not conflict with the newly proposed "staging and grading" system. The silent onset and rapid progression of LAgP make early diagnosis and frequent follow-up with patients essential for effective treatment.
Subject(s)
Aggressive Periodontitis , Chronic Periodontitis , Adolescent , Aged , Aggregatibacter actinomycetemcomitans , Demography , Humans , Molar , Young AdultABSTRACT
Most microorganisms from all taxonomic levels are uncultured. Single-cell genomes and metagenomes continue to increase the known diversity of Bacteria and Archaea; however, while 'omics can be used to infer physiological or ecological roles for species in a community, most of these hypothetical roles remain unvalidated. Here, we report an approach to capture specific microorganisms from complex communities into pure cultures using genome-informed antibody engineering. We apply our reverse genomics approach to isolate and sequence single cells and to cultivate three different species-level lineages of human oral Saccharibacteria (TM7). Using our pure cultures, we show that all three Saccharibacteria species are epibionts of diverse Actinobacteria. We also isolate and cultivate human oral SR1 bacteria, which are members of a lineage of previously uncultured bacteria. Reverse-genomics-enabled cultivation of microorganisms can be applied to any species from any environment and has the potential to unlock the isolation, cultivation and characterization of species from as-yet-uncultured branches of the microbial tree of life.
Subject(s)
Actinobacteria/metabolism , Antibodies/metabolism , Membrane Proteins/immunology , Mouth/microbiology , Single-Cell Analysis/methods , Actinobacteria/classification , Actinobacteria/genetics , Actinobacteria/isolation & purification , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Genomics , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Models, Molecular , Phylogeny , Protein Conformation , Reverse Genetics , Sequence Analysis, DNAABSTRACT
The human oral cavity is sterile prior to birth, and we have limited knowledge of how complex oral communities are assembled. To examine bacterial acquisition and community assembly over the first year of life, oral samples from a cohort of nine infants and their mothers were collected, and bacterial community composition was studied by 16S rRNA gene sequencing. Exogenous species including skin and environmental bacteria were present initially, but were quickly replaced by a small, shared microbial community of species common to all infants and adults. Subsequent ordered microbial succession and the formation of increasingly complex communities was observed. By one year of age oral microbial community composition converged to a profile that was remarkably similar among children. The introduction of new nutrient sources, but not tooth eruption, was associated with increasing complexity. Infants had fewer species than mothers, mostly accounted for by the lack of certain anaerobes, and showing that the acquisition and assembly of oral microbial communities continues past infancy. When relative abundance was considered, a shared set of species accounted for the majority of the microbial community at all ages, indicating that the dominant structure of the oral microbiome establishes early, and suggesting that it persists throughout life.
Subject(s)
Microbiota , Mouth/microbiology , Adult , Age Factors , Child Development , Cohort Studies , Female , Humans , Infant , Infant Nutritional Physiological Phenomena , Infant, Newborn , Longitudinal Studies , Microbiota/genetics , Mothers , RNA, Ribosomal, 16S/genetics , Saliva/microbiology , Species SpecificityABSTRACT
Following publication of the original article, the authors recognized that the left and right panels in Fig. 6b had been inadvertently switched during reformatting.
ABSTRACT
BACKGROUND: Sequencing of the 16S rRNA gene has been the standard for studying the composition of microbial communities. While it allows identification of bacteria at the level of species, this method does not usually provide sufficient information to resolve communities at the sub-species level. Species-level resolution is not adequate for studies of transmission or stability or for exploring subspecies variation in disease association. Strain level analysis using whole metagenome shotgun sequencing has significant limitations that can make it unsuitable for large-scale studies. Achieving sufficient depth of sequencing can be cost-prohibitive, and even with adequate coverage, deconvoluting complex communities such as the oral microbiota is computationally very challenging. Thus, there is a need for high-resolution, yet cost-effective, high-throughput methods for characterizing microbial communities. RESULTS: Significant improvement in resolution for amplicon-based bacterial community analysis was achieved by combining amplicon sequencing of a high-diversity marker gene, the ribosomal 16-23S intergenic spacer region (ISR), with a probabilistic error modeling based denoising algorithm, DADA2. The resolving power of this new approach was compared to that of both standard and high-resolution 16S-based approaches using a set of longitudinal subgingival plaque samples. The ISR strategy resulted in a 5.2-fold increase in community resolution compared to reference-based 16S rRNA gene analysis and showed 100% accuracy in predicting the correct source of a clinical sample. Individuals' microbial communities were highly personalized, and although they exhibited some drift in membership and levels over time, that difference was always smaller than the differences between any two subjects, even after 1 year. The construction of an ISR database from publicly available genomic sequences allowed us to explore genomic variation within species, resulting in the identification of multiple variants of the ISR for most species. CONCLUSIONS: The ISR approach resulted in significantly improved resolution of communities and revealed a highly personalized human oral microbiota that was stable over 1 year. Multiple ISR types were observed for all species examined, demonstrating a high level of subspecies variation in the oral microbiota. The approach is high-throughput, high-resolution yet cost-effective, allowing subspecies-level community fingerprinting at a cost comparable to that of 16S rRNA gene amplicon sequencing. It will be useful for a range of applications that require high-resolution identification of organisms, including microbial tracking, community fingerprinting, and potentially for identification of virulence-associated strains.
Subject(s)
Bacteria/isolation & purification , DNA, Intergenic/genetics , High-Throughput Nucleotide Sequencing/methods , Microbiota , Mouth/microbiology , Adult , Bacteria/classification , Bacteria/genetics , DNA, Bacterial/genetics , Female , Genetic Variation , Humans , Male , Middle Aged , Phylogeny , RNA, Ribosomal, 16S/genetics , Young AdultABSTRACT
Despite decades of research into the human oral microbiome, many species remain uncultivated. The technique of single-cell whole-genome amplification and sequencing provides a means of deriving genome sequences for species that can be informative on biological function and suggest pathways to cultivation. Tannerella forsythia has long been known to be highly associated with chronic periodontitis and to cause periodontitis-like symptoms in experimental animals, and Tannerella sp. BU045 (human oral taxon 808) is an uncultivated relative of this organism. In this work, we extend our previous sequencing of the Tannerella sp. BU063 (human oral taxon 286) genome by sequencing amplified genomes from 11 cells of Tannerella sp. BU045, including 3 genomes that are at least 90% complete. Tannerella sp. BU045 is more closely related to Tannerella sp. BU063 than to T. forsythia by gene content and average nucleotide identity. However, two independent data sets of association with periodontitis, one based on 16S rRNA gene abundance and the other based on gene expression in a metatranscriptomic data set, show that Tannerella sp. BU045 is more highly associated with disease than Tannerella sp. BU063. Comparative genomics shows genes and functions that are shared or unique to the different species, which may direct further research of the pathogenesis of chronic periodontitis. IMPORTANCE Periodontitis (gum disease) affects 47% of adults over 30 in the United States (P. I. Eke, B. A. Dye, L. Wei, G. O. Thornton-Evans, R. J. Genco, et al., J Dent Res 91:914-920, 2012), and it cost between $39 and $396 billion worldwide in 2015 (A. J. Righolt, M. Jevdjevic, W. Marcenes, and S. Listl, J Dent Res, 17 January 2018, https://doi.org/10.1177/0022034517750572). Many bacteria associated with the disease are known only by the DNA sequence of their 16S rRNA gene. In this publication, amplification and sequencing of DNA from single bacterial cells are used to obtain nearly complete genomes of Tannerella sp. BU045, a species of bacteria that is more prevalent in patients with periodontitis than in healthy patients. Comparing the complete genome of this bacterium to genomes of related bacterial species will help to better understand periodontitis and may help to grow this organism in pure culture, which would allow a better understanding of its role in the mouth.
ABSTRACT
The human oral microbiota encompasses representatives of many bacterial lineages that have not yet been cultured. Here we describe the isolation and characterization of previously uncultured Desulfobulbus oralis, the first human-associated representative of its genus. As mammalian-associated microbes rarely have free-living close relatives, D. oralis provides opportunities to study how bacteria adapt and evolve within a host. This sulfate-reducing deltaproteobacterium has adapted to the human oral subgingival niche by curtailing its physiological repertoire, losing some biosynthetic abilities and metabolic independence, and by dramatically reducing environmental sensing and signaling capabilities. The genes that enable free-living Desulfobulbus to synthesize the potent neurotoxin methylmercury were also lost by D. oralis, a notably positive outcome of host association. However, horizontal gene acquisitions from other members of the microbiota provided novel mechanisms of interaction with the human host, including toxins like leukotoxin and hemolysins. Proteomic and transcriptomic analysis revealed that most of those factors are actively expressed, including in the subgingival environment, and some are secreted. Similar to other known oral pathobionts, D. oralis can trigger a proinflammatory response in oral epithelial cells, suggesting a direct role in the development of periodontal disease.IMPORTANCE Animal-associated microbiota likely assembled as a result of numerous independent colonization events by free-living microbes followed by coevolution with their host and other microbes. Through specific adaptation to various body sites and physiological niches, microbes have a wide range of contributions, from beneficial to disease causing. Desulfobulbus oralis provides insights into genomic and physiological transformations associated with transition from an open environment to a host-dependent lifestyle and the emergence of pathogenicity. Through a multifaceted mechanism triggering a proinflammatory response, D. oralis is a novel periodontal pathobiont. Even though culture-independent approaches can provide insights into the potential role of the human microbiome "dark matter," cultivation and experimental characterization remain important to studying the roles of individual organisms in health and disease.
Subject(s)
Adaptation, Biological , Deltaproteobacteria/genetics , Deltaproteobacteria/isolation & purification , Evolution, Molecular , Genome, Bacterial , Gingiva/microbiology , Gene Expression Profiling , Gene Transfer, Horizontal , Humans , Ohio , Periodontitis/microbiology , Phylogeny , Proteome/analysisABSTRACT
BACKGROUND: Glaucoma is a progressive optic nerve degenerative disease that often leads to blindness. Local inflammatory responses are implicated in the pathology of glaucoma. Although inflammatory episodes outside the CNS, such as those due to acute systemic infections, have been linked to central neurodegeneration, they do not appear to be relevant to glaucoma. Based on clinical observations, we hypothesized that chronic subclinical peripheral inflammation contributes to neurodegeneration in glaucoma. METHODS: Mouthwash specimens from patients with glaucoma and control subjects were analyzed for the amount of bacteria. To determine a possible pathogenic mechanism, low-dose subcutaneous lipopolysaccharide (LPS) was administered in two separate animal models of glaucoma. Glaucomatous neurodegeneration was assessed in the retina and optic nerve two months later. Changes in gene expression of toll-like receptor 4 (TLR4) signaling pathway and complement as well as changes in microglial numbers and morphology were analyzed in the retina and optic nerve. The effect of pharmacologic blockade of TLR4 with naloxone was determined. FINDINGS: Patients with glaucoma had higher bacterial oral counts compared to control subjects (p<0.017). Low-dose LPS administration in glaucoma animal models resulted in enhancement of axonal degeneration and neuronal loss. Microglial activation in the optic nerve and retina as well as upregulation of TLR4 signaling and complement system were observed. Pharmacologic blockade of TLR4 partially ameliorated the enhanced damage. CONCLUSIONS: The above findings suggest that the oral microbiome contributes to glaucoma pathophysiology. A plausible mechanism by which increased bacterial loads can lead to neurodegeneration is provided by experiments in animal models of the disease and involves activation of microglia in the retina and optic nerve, mediated through TLR4 signaling and complement upregulation. The finding that commensal bacteria may play a role in the development and/or progression of glaucomatous pathology may also be relevant to other chronic neurodegenerative disorders.
Subject(s)
Glaucoma/complications , Glaucoma/microbiology , Microbiota , Mouth/microbiology , Nerve Degeneration/complications , Nerve Degeneration/microbiology , Black or African American , Animals , Brain/drug effects , Brain/metabolism , Complement System Proteins/genetics , Complement System Proteins/metabolism , Female , Glaucoma/pathology , Glaucoma/physiopathology , Humans , Intraocular Pressure/drug effects , Lipopolysaccharides/administration & dosage , Lipopolysaccharides/pharmacology , Male , Mice, Inbred C57BL , Mice, Inbred DBA , Microbiota/drug effects , Microglia/drug effects , Microglia/metabolism , Microglia/pathology , Mouth/drug effects , Mouth/pathology , Nerve Degeneration/pathology , Nerve Degeneration/physiopathology , Optic Disk/drug effects , Optic Disk/pathology , Optic Disk/physiopathology , Toll-Like Receptor 4/metabolism , Up-Regulation/drug effectsABSTRACT
Many microbial phyla that are widely distributed in open environments have few or no representatives within animal-associated microbiota. Among them, the Chloroflexi comprises taxonomically and physiologically diverse lineages adapted to a wide range of aquatic and terrestrial habitats. A distinct group of uncultured chloroflexi related to free-living anaerobic Anaerolineae inhabits the mammalian gastrointestinal tract and includes low-abundance human oral bacteria that appear to proliferate in periodontitis. Using a single-cell genomics approach, we obtained the first draft genomic reconstruction for these organisms and compared their inferred metabolic potential with free-living chloroflexi. Genomic data suggest that oral chloroflexi are anaerobic heterotrophs, encoding abundant carbohydrate transport and metabolism functionalities, similar to those seen in environmental Anaerolineae isolates. The presence of genes for a unique phosphotransferase system and N-acetylglucosamine metabolism suggests an important ecological niche for oral chloroflexi in scavenging material from lysed bacterial cells and the human tissue. The inferred ability to produce sialic acid for cell membrane decoration may enable them to evade the host defence system and colonize the subgingival space. As with other low abundance but persistent members of the microbiota, discerning community and host factors that influence the proliferation of oral chloroflexi may help understand the emergence of oral pathogens and the microbiota dynamics in health and disease states.