F E Dewhirst

Harvard University, Cambridge, Massachusetts, United States

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Publications (134)449.49 Total impact

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    ABSTRACT: Objective: Rhesus macaques are often used as animal models for periodontitis. The purpose of this study was to determine the presence of naturally-occurring Porphyromonas gingivalis (Pg) and other periodontal pathogens in an elder population of macaques. Method: Fifty-one elder animals (9 to 18 years) with clinical signs of mild-moderate periodontitis were studied. Bacterial DNA was isolated from subgingival plaque from the deepest sites and PCR-amplified using Pg-specific 16S rDNA bacterial primers. Amplicons were excised and sequenced. DNA was also PCR-amplified using Bacteroidetes-specific or broad-range 16S rDNA bacterial primers. Full-sized amplicons (about 1500 bp) were cloned and sequenced for species identification. Microarrays (HOMIM) were run on DNA samples from 7 animals. Result: Based on PCR and subsequent sequence analyses with 100% 250 bp match, all 51 macaques were colonized with Pg. Based on sequence analysis of 330 clones, 48 of 108 bacterial taxa (44%) were unique to the macaque. The remaining taxa were considered human species. Putative periodontal pathogens detected included Pg, Filifactor alocis, Parvimonas micra, several Treponema phylotypes, a Desulfobulbus phylotype, and, most notably, a monkey version of Aggregatibacter actinomycetemcomitans. Interestingly, caries-associated species, Streptococcus mutans and Scardovia wiggsiae were also detected. HOMIM was comparable to sequence analysis confirming the presence of Pg in all animals tested. Conclusion: The presence of naturally occurring Pg and other human periodontal pathogens in the subgingival plaque of elder macaques validates that they serve as an excellent animal model for human periodontitis.
    AADR Annual Meeting & Exhibition 2014; 03/2014
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    ABSTRACT: Objectives: Of the 688 taxa currently in the Human Oral Microbiome Database (HOMD), 65% have been cultivated and 244 taxa (35%) are as-yet-uncultivated phylotypes. The goal of this study was to determine the prevalence of uncultivated taxa in various oral niches. Methods: Ten adults who had not taken antibiotics within the past 3 months were sampled at 9 oral sites each: supragingival, subgingival (4 deepest sites), cheek, palate, tongue, tonsils (throat sampled for two subjects without tonsils). Soft tissue sites were sampled using nylon brushes, hard tissue sites with scalers. DNA was purified from the clinical samples, PCR amplified using 16S rRNA V3V4 primers, purified, and sequenced using an Illumina Miseq instrument. The 16S rRNA reads were parsed, and BLASTN analyzed using HOMD RefSeq Version 13.2. Results: A total of 5,586,237 sequences (V3-V4 assembled overlapped paired reads) matched HOMD reference sequences at >98.5% BLASTN identity. The reads were identified to 481 HOMD taxa: 317 cultivated and 164 uncultivated phylotypes. The number of uncultivated taxa per sample ranged from 15-68 with a mean of 40.6. The total number of oral taxa/subject-site ranged from 91-251 with a mean of 182. The mean number of uncultivated taxa/individual sample at oral sites was as follows: cheek 45.9, palate 38.4, tongue 29.0, tonsils 46.3, throat 45.0, supragingival 39.8 and subgingival 39.6. Uncultivated taxa identified included 22 from the rare phyla: Chloroflexi, 1; Synergistetes genus Fretibacterium, 4; GNO2, 3; SR1, 3; and TM7, 11 taxa. Conclusion: In screening just 10 subjects, we identified subject-sites with 164/244 (67%) of the uncultivated taxa currently in HOMD. The majority of these uncultivated taxa are thought to be uncultivable using standard cultivation methods; however, this study shows they are readily available for study and attempted cultivation.
    AADR Annual Meeting & Exhibition 2014; 03/2014
  • T. CHEN, J. MCCAFFERTY, F.E. DEWHIRST
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    ABSTRACT: Objective: Next generation sequencing platforms such as those based on the Illumina or Roche 454, have become cost-effective and commonly applied to study human oral microbiome by deep 16S rRNA gene sequencing of microbial samples. New and existing bioinformatics software pipelines are being developed and modified for analyzing the sequence reads and interpreting the microbial richness and diversity of the samples. Different software and analyzing procedures may result in different conclusions even on the same dataset. In this report, we evaluated different software pipelines and proposed a comprehensive approach that is optimized specifically for analyzing the 16S rRNA short read sequences derived from human oral samples. Method: Bioinformatics programs were evaluated for the following stages of data analysis: 1) sequence quality filtering; 2) pair-ending sequence merging; 3) operational taxonomic unit (OTU) calling; 4) classification; and 5) diversity and richness estimation. Result: The human oral microbiome has been well-characterized and full length 16S rRNA gene sequences of most abundant species are available as references. Based on the evaluation of the current available software and pipeline, we propose the use of two-stage, open-ended reference-base OTU calling pipeline: 1) reference-based OTU calling using HOMD 16S rRNA references and taxonomy inferred from the HOMD taxonomy; 2) de-novo OTU calling of the reads not mapped in stage 1 and taxonomy inferred from non-HOMD references, such as GreenGene or Silva databases. Conclusion: The proposed two-stage approach for NGS 16S rRNA data is comprehensive in mapping the reads to known human oral taxa as well as in discovering novel taxa in the oral microbial samples.
    AADR Annual Meeting & Exhibition 2014; 03/2014
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    ABSTRACT: Objective: The Human Oral Microbiome Database (HOMD) is a public database providing comprehensive information on the prokaryotic species present in the human oral cavity. Our team maintains and curates the HOMD with up-to-date information on genomics, phylogeny, and taxonomy for prevalent human oral microbial species. We have also been developing bioinformatics tools that are integrated into the database for use by the research community. This report summarizes the latest content updates as well as newly available tools and features. Method: The HOMD was created with PHP, Perl, MySQL and Apache and is hosted on a cluster of computers and housed in the Forsyth Institute Data Center. The HOMD Genomic BLAST was constructed based on the NCBI BLAST+. The new HOMD Genome Browser was implemented based on the “JBrowse” system. Novel human oral taxa were created based on 16S rRNA reference sequences of recently described oral species or recognized phylotypes. New genomic sequences were obtained through the collaboration with the Human Microbiome Project (HMP) sequencing centers and from repositories world-wide. Result: Currently HOMD contains a total of 688 human oral taxa. Of these, 342 (49.7%) are validly described, 102 (14.8%) unnamed (but cultivated) and 244 (35.5%) known only as uncultivated phylotypes. The current version (13.2) of the 16S rRNA reference sequences contains 831 full length sequences representing prevalent human oral taxa. The HOMD Genomics database currently contains 1292 genomes, representing 325 oral taxa, with either static or dynamic annotations. Additional genomic sequences are being added frequently as they appear at NCBI and other sequence repositories. The HOMD Genomic BLAST and the HOMD JBrowse Genome Browser have also been implemented for searching and navigating among all the human oral microbes with available sequences. Conclusion: HOMD is a comprehensive bioinformatics resource for the scientific community. HOMD is available at http://www.homd.org.
    AADR Annual Meeting & Exhibition 2014; 03/2014
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    ABSTRACT: Objective: We examined the metatranscriptome of the bacterial community associated with early childhood caries (ECC) compared with caries-free-associated plaques to evaluate which gene activities and species were contributing to caries pathogenesis. Method: Incisor and molar plaque samples were taken from 2-6 year old children attending a community dental center serving mainly low-income families. RNA was purified from plaque samples of 10 children (ECC n=5, caries-free n=5), and sequenced on the Illumina MiSeq platform. Gene expression analysis was conducted by aligning sequences to the genome sequences in the Human Oral Microbiome Database (HOMD) and counting total hits to genes. The edgeR package was used identify and visualize gene expression levels between the two groups. Result: Multidimensional scaling gene counts grouped four of five children in each group by caries or caries-free disease status. Over and under expressed genes were detected, however, no expressed genes differed significantly between ECC or caries-free groups (FDR < 0.1). Species with over expressed genes in ECC compared with caries-free included for Streptococcus mutans, (p=0.008) with higher gene expression for Oribacterium sp HOT 078, Actinomyces HOT 176, Scardovia wiggsiae Selenomonas sputigena and Prevotella sp. HOT 317, but not significantly. Over expressed genes included those for L-lactate dehydrogenase, glucosyltransferase GtfG, surface antigen-related protein, glycogen phosphorylase and lantibiotic streptococcin A-FF22 precursor all in S. mutans. There were only modest gene expression increases in taxa detected more frequently in caries-free children, including Streptococcus oralis, Neisseria sicca, Catonell morbi, Cardiobacterium hominis Cardiobacterium valvarum, and Actinomyces sp HOT 848. Conclusion: We detected species with increased gene expression activity in the caries-associated microbiome, and linked activity to individual species. This is a promising approach to determine species and mechanisms active in dental caries progression.
    AADR Annual Meeting & Exhibition 2014; 03/2014
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    ABSTRACT: Routine necropsies of 27 asymptomatic juvenile chinchillas revealed a high prevalence of gastric ulcers with microscopic lymphoplasmacytic gastroenteritis and typhlocolitis. Polymerase chain reaction (PCR) analysis using Campylobacter genus-specific partial 16S rRNA primers revealed the presence of Campylobacter spp. DNA in the faeces of 12 of 27 animals (44.4%). Species-specific partial 16S rRNA PCR and sequencing confirmed that these animals were colonized with Campylobacter lanienae, a gram-negative, microaerophilic bacterium that was first identified on routine faecal screening of slaughterhouse employees and subsequently isolated from faeces of livestock. Campylobacter lanienae was isolated from the faeces of six PCR-positive animals and identified with species-specific PCR and full 16S rRNA sequencing. Phylogenetic analysis showed that these isolates clustered with C. lanienae strain NCTC 13004. PCR analysis of DNA extracted from gastrointestinal tissues revealed the presence of C. lanienae DNA in the caecum and colon of these chinchillas. Gastrointestinal lesions were scored and compared between C. lanienae-positive and C. lanienae-negative animals. There was no correlation between colonization status and lesion severity in the stomach, liver, duodenum, or colon. Possible routes of C. lanienae infection in chinchillas could include waterborne transmission and faecal–oral transmission from wild mice and rats or livestock. Based on these findings, the authors conclude that C. lanienae colonizes the lower bowel of chinchillas in the absence of clinical disease. This is the first report of C. lanienae in any rodent species. Campylobacter lanienae isolates from different mammalian species demonstrate heterogeneity by 16S rRNA sequence comparison. Analysis using rpoB suggests that isolates and clones currently identified as C. lanienae may represent multiple species or subspecies.
    Zoonoses and Public Health 03/2014; 61(8). DOI:10.1111/zph.12107 · 2.07 Impact Factor
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    ABSTRACT: Limited information is available about the effect of human immunodeficiency virus (HIV) and subsequent antiretroviral treatment on host-microbe interaction. This study aimed to determine the salivary microbial composition in 10 HIV-seropositive subjects, before and 6 months after highly active antiretroviral therapy (HAART), compared with that of 10 HIV-seronegative subjects. Both a conventional culture and two culture-independent analyses were used and consistently demonstrated differences in microbial composition among the three sets of samples. HIV(+) subjects had higher levels of total cultivable microbes, including oral streptococci, lactobacilli, S. mutans, and Candida, in saliva as compared to HIV(-) subjects. The total cultivable microbial level was 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. Human oral microbe identification microarray (HOMIM), which compared the 16S rRNA genes, showed a clear separation among the three sample groups. Veillonella, Synergistetes, and Streptococcus, were present in all 30 saliva samples. Only minor changes or no changes were observed in the prevalence of Neisseria, Haemophilus, Gemella, Leptotrichia, Solobacterium, Parvimonas and RothiaI. Severn genera were detected only in HIV(-) samples, including Capnocytophaga, Slackia, Porphyromonas, Kingella, Peptostreptococcaceae, Lactobacillus, and Atopobium. The prevalence of Fusobacterium, Campylobacter, Prevotella, Capnocytophaga, Selenomonas, Actinomyces, and Atopobium was increased after therapy with HAART. In contrast, the prevalence of Aggregatibacter was significantly decreased after HAART. Findings of this study suggest that HIV infection and therapy with HAART could have a significant effect on salivary microbial colonization and composition.
    Journal of clinical microbiology 02/2014; 52(5). DOI:10.1128/JCM.02954-13 · 4.23 Impact Factor
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    ABSTRACT: Objective: Around half of oral bacteria have yet to be cultured. The family Lachnospiraceae includes a major lineage of uncultivated bacteria, some of which are disease-associated. The aim of this study was to use colony hybridisation directed enrichment co-culture to isolate a representative of LachnospiraceaeHuman Oral Taxon (HOT) 500. Methods: Samples of subgingival plaque from pockets > 8 mm in depth were collected from subjects with periodontitis patients and used to inoculate hydroxyapatite-coated pegs immersed in Periodontitis Peg Medium broth (PPB) in the Calgary Biofilm Device (CBD). The CBD was incubated anaerobically and the medium changed every 3.5 days. PCR primers and oligonucleotide probes specific for LachnospiraceaeHOT 500 were designed and validated. Colony hybridisation with DIG-labelled probes was performed on Periodontitis Peg Medium agar plates (PPA) inoculated with biofilms harvested from CBD biofilms incubated anaerobically for 10 days. Hybridisation-positive areas of the plates were subcultured onto fresh media to enrich for the target. Results: Lachnospiraceae HOT 500-positive regions were seen on colony hybridisation blots from CBD culture plates and, after enrichment, a simple community was seen consisting of Veillonella parvula, Parvimonas micra and tiny colonies growing in close proximity to the Veillonella parvula colonies. 16S rRNA gene sequence analysis identified the tiny colonies, designated strain SP1_1, as be Lachnospiraceae HOT 500. SP1_1 was found to be entirely dependent on V. parvula for growth. Growth stimulation was also seen with Fusobacterium nucleatum and Propionibacterium acnes. The addition of V. parvulaculture sonicates allowed SP1_1 to be cultured in PPB. Conclusion: A strain representative of the previously uncultivated human oral taxon Lachnospiraceae 500 has been successfully cultured using a colony hybridisation directed enrichment approach. This abstract is based on research that was funded entirely or partially by an outside source: NIH-NIDCR DE016937 Keywords: Ecology, Microbiology and Periodontal organisms Presenting author's disclosure statement: ** MISSING DISCLOSURE **
    09/2013
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    ABSTRACT: Coeliac disease is characterized by intestinal inflammation caused by gluten, proteins which are widely contained in the Western diet. Mammalian digestive enzymes are only partly capable of cleaving gluten, and fragments remain that induce toxic responses in patients with coeliac disease. We found that the oral microbiome is a novel and rich source of gluten-degrading organisms. Here we report on the isolation and characterization of the cultivable resident oral microbes that are capable of cleaving gluten, with special emphasis on the immunogenic domains. Bacteria were obtained by a selective culturing approach and enzyme activities were characterized by: (i) hydrolysis of paranitroanilide-derivatized gliadin-derived tripeptide substrates; (ii) gliadin degradation in-gel (gliadin zymography); (iii) gliadin degradation in solution; (iv) proteolysis of the highly immunogenic α-gliadin-derived 33-mer peptide. For selected strains pH activity profiles were determined. The culturing strategy yielded 87 aerobic and 63 anaerobic strains. Species with activity in at least two of the four assays were typed as: Rothia mucilaginosa HOT-681, Rothia aeria HOT-188, Actinomyces odontolyticus HOT-701, Streptococcus mitis HOT-677, Streptococcus sp. HOT-071, Neisseria mucosa HOT-682 and Capnocytophaga sputigena HOT-775, with Rothia species being active in all four assays. Cleavage specificities and substrate preferences differed among the strains identified. The approximate molecular weights of the enzymes were ~75 kD (Rothia spp.), ~60 kD (A. odontolyticus) and ~150 kD (Streptococcus spp.). In conclusion, this study identified new gluten-degrading microorganisms in the upper gastrointestinal tract. A cocktail of the most active oral bacteria, or their isolated enzymes, may offer promising new treatment modalities for coeliac disease.
    Clinical Microbiology and Infection 04/2013; 9. DOI:10.1111/1469-0691.12249 · 5.20 Impact Factor
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    ABSTRACT: Objective: 2,058 children ages 11-17 years were screened for the presence of Aggregatibacter actinomycetemcomitans (Aa) and localized aggressive periodontitis (LAP). Goals were to determine; 1) the relationship of Aa to BL, and 2) the relationship of Aa to other subgingival bacteria. Methods: 134 periodontally healthy children (73 Aa-negative and 61 Aa-positives) were enrolled in a longitudinal study. Subjects were sampled, examined, and x-rayed every 6 months for 3 years. LAP was diagnosed by radiographic evidence of bone loss (BL). Results: 227 of 2,058 subjects had Aa at screening. 64 of 2058 had LAP by probe. 54 of 64 with LAP had Aa at screening (84.3%). 54 of 227 Aa carriers had LAP (23.7%). In the longitudinal study 16 of 61 Aa-positives developed LAP (26.2%). 39 of 134 enrolled had pockets. 33 of 39 with pockets had Aa (84.6%)}. 16 subjects who developed BL were Aa-positive. Cross-sectional and longitudinal data in relation to Aa and disease were similar. Assessment of over 200 organisms by HOMIM showed pooled samples from subjects with pockets had elevated levels of Parvimonas micra, Filofactor alocis, Aa and Peptostreptococcus DA014 compared to pooled samples from healthy subjects. Streptococcus mitis, S. sanguis, S. salivarius, Veillonella parvula, and others were lower when these two groups were compared. Aa, F. alocis and S. parasanguis I and II were elevated in sites 6 months prior to BL. By combining the prevalence and levels of Aa, F. alocis and S. parasanguis the specificity to detect BL was elevated to 99% while the specificity was 89%. Conclusions: 1) Detection of Aa improves the chances of detecting patients susceptible to LAP from 2% (ethnicity alone) to over 20% (ethnicity and Aa). 2) Aa is necessary but not sufficient to initiate LAP. 2) LAP requires a consortium of organisms including F. alocis and S. parasanguis.
    IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
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    ABSTRACT: Previous studies have demonstrated that HIV-infected individuals are at greater risk for oral microbial infections compared with non-HIV-infected controls. Objective: To quantitatively and qualitatively compare salivary microbiota profiles using two culture-independent survey methods, denaturing gradient gel electrophoresis (DGGE) and the human oral microbe identification microarray (HOMIM). Methods: Stimulated whole saliva samples were collected from 10 HIV-infected individuals before and 6 month after highly active antiretroviral therapy (HAART), and from 10 non-HIV-infected individuals. Total bacterial genomic DNA was isolated from each salivary sample and targeted 16S rRNA genes were PCR amplified. DGGE profiles of the 16S rRNA genes were analyzed using BioNumerics (Applied Maths, NV). Meanwhile, the same DNA samples were processed by HOMIM hybridization and were analyzed using MeV program (Dana-Farber Cancer Ins. MA). Results: The DGGE profile showed differences in the microbial distribution among the three groups; HAART reduced the microbial diversity. HOMIM data analysis showed that, of the 425 target probes, 121 were detected in the saliva samples. HIV-infected samples clustered separately from the non-HIV-infected samples. Species and phylotypes identified as different among the three groups were: Actinomyces gerencseriae (p<0.001), Aggregatibacter segnis (p<0.01), Atopobium sp (p<0.01), Capnocytophaga granulosa sp (p<0.05), Fusobacterium nucleatum (p<0.05), Fusobacterium periodontiumI (p<0.01), Haemophilus parainfluenzae (p<0.05), Neisseria Cluster II (p<0.05), Prevotella sp (p<0.05), and Streptococcus australis (p<0.05). Conclusion: HIV infection and subsequent HAART can significantly affect the oral microbial colonization and the microbial diversity in the saliva. Supported by research grant U19 DE018385 from the NIDCR/NIH.
    IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
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    ABSTRACT: Objectives: The search for therapies for celiac disease includes investigations into luminal enzymes capable of cleaving gluten into fragments that are unable to elicit inflammatory immune responses. We recently provided evidence that the oral cavity, representing the port of entry to the gastro-intestinal tract, harbors gluten-degrading microorganisms. The goal of this study was to conduct a comprehensive screening of human dental plaque and saliva samples to isolate and identify novel resident gluten/gliadin-degrading bacteria. Methods: Bacteria were obtained by a selective culturing approach and enzyme activities were characterized by: 1) Hydrolysis of paranitroanilide-derivatized gliadin-derived tripeptide substrates; 2) Gliadin degradation in gel (gliadin zymography); 3) Gliadin degradation in solution; 4) Proteolysis of the highly immunogenic α-gliadin-derived 33-mer. For the selected strains pH activity profiles were determined. Results: The culturing strategy yielded 87 aerobic and 63 anaerobic strains. Twenty one aerobic strains representing seven oral species showed activity in at least two of the four assays with two species being active in all four assays. Tripeptide substrates cleaved most readiliy exhibited the sequence of YPQ and LPY and occur frequently in immunogenic gliadin domains. Conclusions: New gluten-degrading microorganisms were identified that naturally colonize the upper gastro-intestinal tract. A cocktail of the most active oral bacteria, or their isolated enzymes, may offer promising new treatment modalities for celiac disease. Supported by NIH grants AI078385 (DS), DE07652 (FO), AI087803 (EJH).
    IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
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    ABSTRACT: Objective: Anaerobic culture of plaque samples from children with severe-early childhood caries (S-ECC) detected a diverse microbiota (Tanner et al. 2011) comparable to that from molecular clonal analyses. This study sought microbial complexes from samples cultured on acidic and blood agars from caries-free and S-ECC children. Method: The study population comprised 42 S-ECC and 40 caries-free children (2-6 years old). Over 5,000 isolates from plaque cultured anaerobically on blood (pH 7), and acid (pH 5) agars were identified by comparing partial 16S rRNA sequences with taxa/species in the Forsyth Human Oral Microbiome Database. The microbiotas in samples were clustered by disease category and isolation medium using principal component analysis and two-way cluster analysis. Result: The microbiotas of blood and acidic agars grouped separately by cluster analysis. There was some overlap in the microbiotas of S-ECC and caries-free samples isolated on blood. In contrast, samples cultured on the acidic agar clustered into two major groups, one of S-ECC and another of caries-free children. When analyzed separately, three clusters were formed in both S-ECC and caries-free children. Species detected in the two major clusters were similar from both S-ECC and caries-free children. From the S-ECC children the third, loosely-grouped, cluster comprised of Streptococcus mutans, Scardovia wiggsiae, Streptococcus intermedius and Veillonella atypica. From caries-free children, the third cluster included S. intermedius and V. atypica with 3 unnamed Actinomyces species, Streptococcus gordonii, Streptococcus mitis, Streptococcus thermophilus, Veillonella parvula and Veillonella dispar. Conclusion: Microbial complexes varied between S-ECC and caries-free children although the majority of taxa detected grouped similarly in both disease categories. A microbial complex containing S. mutans and Scardovia wiggsiae was detected in the S-ECC children but not observed in the caries-free children.
    IADR/AADR/CADR General Session and Exhibition 2013; 03/2013
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    Nature 06/2012; 486(7402):215–221. DOI:10.1038/nature11209 · 42.35 Impact Factor
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    Nature 06/2012; 486(7402):207–214. DOI:10.1038/nature11234 · 42.35 Impact Factor
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    ABSTRACT: Gluten proteins are prominent constituents of barley, wheat and rye. They have an unusual amino acid composition and are difficult to digest by mammalian proteolytic enzymes. The protease-resistant domains contain multiple immunogenic epitopes causing celiac disease in genetically pre-disposed individuals. Objective: To identify novel sources of gluten-digesting microbial enzymes from the oral cavity, representing the upper gastro-intestinal tract, with the potential to neutralize gluten epitopes. Method: Oral microorganisms with gluten-degrading capacity were obtained by a selective plating strategy using gluten agar. Microbial speciations were carried out by 16S rDNA gene sequencing. Enzyme activities were assessed using gliadin-derived enzymatic substrates, gliadins in solution, gliadin zymography, and 33-mer α-gliadin and 26-mer γ-gliadin immunogenic peptides. Fragments of the gliadin peptides were separated by RP-HPLC and structurally characterized by mass spectrometry. Result: Rothia mucilaginosa and Rothia aeria were identified as gluten-degrading strains in the oral cavity. Gliadins as well as immunogenic 33-mer and 26-mer peptides (250 µg/ml) added to Rothia cell suspensions (OD620 1.2) were degraded. Cleavage occured primarily C-terminal to Xaa-Pro-Gln (XPQ) and Xaa-Pro-Tyr (XPY). The major gliadin-degrading enzymes produced by the Rothia strains were ~70–75 kDa in size and the enzyme expressed by Rothia aeria was active over a wide pH range (pH 3–10). The enzymes also demonstrated activity in the presence of EDTA, which suggest that the enzymes appears to be non-metalloproteases. Conclusion: While the human digestive enzyme system lacks the capacity to cleave immunogenic gluten, such activities are naturally present in the oral microbial enzyme repertoire. The identified bacteria may be exploited for physiologic degradation of harmful gluten peptides and lead to novel and effective strategies to combat celiac disease.
    AADR Annual Meeting 2012; 03/2012
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    ABSTRACT: Objectives: For the past six years our group has been supported by NIDCR grant DE016937 to fulfil the following major goals: 1) To create a publically available database with access to a curated taxonomic scheme for provisional naming of the more than 600 oral taxa, and to allow viewing of annotated genomes for all sequenced oral bacteria; 2) To prepare DNA from phylogenetically diverse oral bacteria for genome sequencing by the Human Microbiome Project (HMP) sequencing centers; and 3) To identify isolates of previously uncultivated oral taxa and deposit them with a major culture collection. Methods: Our approach to this this project has been described extensively in the publication (Dewhirst et al. 2010. J. Bacteriol 192:5002-17). Results: The HOMD database currently contains 574 annotated genomes of oral bacteria from 184 taxa (multiple genomes being available for a few taxa). We have sent DNA from 173 oral strains representing 154 taxa to the HMP sequencing centers. Of the 173 strains, 120 are from named taxa and 53 from unnamed taxa. Examination of the Wade, Tanner and Moore’s culture collections by 16S rRNA sequencing has allowed us to identify strains for over 200 previously uncultivated oral taxa. The majority of these isolates represent Human Oral Taxa (HOT) known previously as phylotypes, but a few are totally novel and are assigned new HOT numbers. These isolates will have their genomes sequenced and be deposited with the BEI/ATCC resource. Conclusions: Our group has created the Human Oral Microbiome Database as a bioinformatics resource for the scientific community. We have been the major contributor of highly purified DNA for generating reference genomes for the oral microbiome as part of the HMP. We have successfully identified strains from a large number of previously uncultivated oral taxa and are making them available through a permanent culture collection.
    AADR Annual Meeting 2012; 03/2012
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    ABSTRACT: The majority of taxa in the oral microbiome are members of six well known phyla: Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Spirochaetes, and Fusobacteria. Less commonly encountered phyla include the Tenericutes, Chlamydiae and Synergistetes, and the uncultivated candidate division, TM7. Objectives: The primary objective of this study was to develop primers for selective PCR amplification and cloning of the 16S rRNA genes from five truly rare phyla/candidate divisions: Chloroflexi, Chlorobi, GN02, SR1, and WPS-2. Methods: 16S rRNA clone sequences demonstrating the existence of these five phyla/divisions are known from our previous cloning studies (Dewhirst et al. 2010. J. Bacteriol 192:5002-17), the Human Microbiome Project, and unpublished studies of the oral microbiomes of dogs and cats. 16S rRNA sequences from these studies were aligned and used to retrieve additional related sequences from GenBank and Greengenes. Based on aligned sequences from these phyla/divisions with reference sequences from the Human Oral Microbiome Database (HOMD), taxa selective forward and reverse primers were designed heuristically. DNA pools were created from previous oral cloning studies. Probe pairs, including “universal” 9-27F and 1492-1509R and 1525-1541R primers, were tested for amplification of DNA pools. Primer combinations producing amplicons were cloned using TA cloning kits. Results: Nineteen primers were designed and synthesized. Primer pairs for each of the five phyla/divisions produced an amplicon with at least one oral DNA pool. Twenty-one 16S rRNA libraries were constructed and approximately 50 clones sequenced from each library. Library analysis validated highly specific primer pairs for Chloroflexi, GN02 and SR1. Primer pairs for WPS-2 and Chlorobi produced amplicons for Canine DNA pools but not the two tested human DNA pools. Conclusions: We have designed highly selective primers for use in analyzing the presence and diversity of five rare oral phyla/divisions, and have identified six previously unrecognized oral taxa. Supported by NIDRC grant DE016937.
    AADR Annual Meeting 2012; 03/2012
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    ABSTRACT: Gluten proteins, prominent constituents of barley, wheat and rye, cause celiac disease in genetically predisposed subjects. Gluten is notoriously difficult to digest by mammalian proteolytic enzymes and the protease-resistant domains contain multiple immunogenic epitopes. The aim of this study was to identify novel sources of gluten-digesting microbial enzymes from the upper gastro-intestinal tract with the potential to neutralize gluten epitopes. Oral microorganisms with gluten-degrading capacity were obtained by a selective plating strategy using gluten agar. Microbial speciations were carried out by 16S rDNA gene sequencing. Enzyme activities were assessed using gliadin-derived enzymatic substrates, gliadins in solution, gliadin zymography, and 33-mer α-gliadin and 26-mer γ-gliadin immunogenic peptides. Fragments of the gliadin peptides were separated by RP-HPLC and structurally characterized by mass spectrometry. Strains with high activity towards gluten were typed as Rothia mucilaginosa and Rothia aeria. Gliadins (250 µg/ml) added to Rothia cell suspensions (OD(620) 1.2) were degraded by 50% after ∼30 min of incubation. Importantly, the 33-mer and 26-mer immunogenic peptides were also cleaved, primarily C-terminal to Xaa-Pro-Gln (XPQ) and Xaa-Pro-Tyr (XPY). The major gliadin-degrading enzymes produced by the Rothia strains were ∼70-75 kDa in size, and the enzyme expressed by Rothia aeria was active over a wide pH range (pH 3-10). While the human digestive enzyme system lacks the capacity to cleave immunogenic gluten, such activities are naturally present in the oral microbial enzyme repertoire. The identified bacteria may be exploited for physiologic degradation of harmful gluten peptides.
    PLoS ONE 09/2011; 6(9):e24455. DOI:10.1371/journal.pone.0024455 · 3.53 Impact Factor

Publication Stats

8k Citations
449.49 Total Impact Points

Institutions

  • 2002–2014
    • Harvard University
      • Department of Oral Medicine, Infection, and Immunity
      Cambridge, Massachusetts, United States
  • 1991–2014
    • The Forsyth Institute
      • Department of Cytokine Biology
      Cambridge, Massachusetts, United States
  • 1994–2003
    • University of Massachusetts Boston
      Boston, Massachusetts, United States
  • 1993–2003
    • Massachusetts Institute of Technology
      • Division of Comparative Medicine
      Cambridge, MA, United States
  • 2001
    • University of Missouri
      • Department of Veterinary Pathobiology
      Columbia, MO, United States
    • University of Zurich
      • Institut für Orale Biologie
      Zürich, ZH, Switzerland
    • Harvard Medical School
      Boston, Massachusetts, United States
  • 2000
    • Virginia Commonwealth University
      • Department of Periodontics
      Richmond, VA, United States
  • 1999
    • Biomedical Research Institute, Rockville
      Maryland, United States
  • 1998
    • Beverly Hospital, Boston MA
      BVY, Massachusetts, United States
  • 1996
    • University of Glasgow
      Glasgow, Scotland, United Kingdom
    • Ghent University
      • Laboratory of Microbiology
      Gent, VLG, Belgium
  • 1995
    • University of Minnesota Duluth
      Duluth, Minnesota, United States
    • Melbourne Institute of Technology
      Melbourne, Victoria, Australia