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1.  As-yet-uncultivated oral bacteria: breadth and association with oral and extra-oral diseases 
Journal of Oral Microbiology  2013;5:10.3402/jom.v5i0.21077.
It has been shown that 40–60% of the bacteria found in different healthy and diseased oral sites still remain to be grown in vitro, phenotypically characterized, and formally named as species. The possibility exists that these as-yet-uncultivated bacteria play important ecological roles in oral bacterial communities and may participate in the pathogenesis of several oral infectious diseases. There is also a potential for these as-yet-uncultivated oral bacteria to take part in extra-oral infections. For a comprehensive characterization of physiological and pathogenic properties as well as antimicrobial susceptibility of individual bacterial species, strains need to be grown in pure culture. Advances in culturing techniques have allowed the cultivation of several oral bacterial taxa only previously known by a 16S rRNA gene sequence signature, and novel species have been proposed. There is a growing need for developing improved methods to cultivate and characterize the as-yet-uncultivated portion of the oral microbiome so as to unravel its role in health and disease.
doi:10.3402/jom.v5i0.21077
PMCID: PMC3664057  PMID: 23717756
uncultivated bacteria; oral microbiology; molecular biology methods; taxonomy
2.  The Human Oral Microbiome Database: a web accessible resource for investigating oral microbe taxonomic and genomic information 
The human oral microbiome is the most studied human microflora, but 53% of the species have not yet been validly named and 35% remain uncultivated. The uncultivated taxa are known primarily from 16S rRNA sequence information. Sequence information tied solely to obscure isolate or clone numbers, and usually lacking accurate phylogenetic placement, is a major impediment to working with human oral microbiome data. The goal of creating the Human Oral Microbiome Database (HOMD) is to provide the scientific community with a body site-specific comprehensive database for the more than 600 prokaryote species that are present in the human oral cavity based on a curated 16S rRNA gene-based provisional naming scheme. Currently, two primary types of information are provided in HOMD—taxonomic and genomic. Named oral species and taxa identified from 16S rRNA gene sequence analysis of oral isolates and cloning studies were placed into defined 16S rRNA phylotypes and each given unique Human Oral Taxon (HOT) number. The HOT interlinks phenotypic, phylogenetic, genomic, clinical and bibliographic information for each taxon. A BLAST search tool is provided to match user 16S rRNA gene sequences to a curated, full length, 16S rRNA gene reference data set. For genomic analysis, HOMD provides comprehensive set of analysis tools and maintains frequently updated annotations for all the human oral microbial genomes that have been sequenced and publicly released. Oral bacterial genome sequences, determined as part of the Human Microbiome Project, are being added to the HOMD as they become available. We provide HOMD as a conceptual model for the presentation of microbiome data for other human body sites.
Database URL: http://www.homd.org
doi:10.1093/database/baq013
PMCID: PMC2911848  PMID: 20624719
3.  High-throughput isotopic analysis of RNA microarrays to quantify microbial resource use 
The ISME Journal  2011;6(6):1210-1221.
Most microorganisms remain uncultivated, and typically their ecological roles must be inferred from diversity and genomic studies. To directly measure functional roles of uncultivated microbes, we developed Chip-stable isotope probing (SIP), a high-sensitivity, high-throughput SIP method performed on a phylogenetic microarray (chip). This approach consists of microbial community incubations with isotopically labeled substrates, hybridization of the extracted community rRNA to a microarray and measurement of isotope incorporation—and therefore substrate use—by secondary ion mass spectrometer imaging (NanoSIMS). Laboratory experiments demonstrated that Chip-SIP can detect isotopic enrichment of 0.5 atom % 13C and 0.1 atom % 15N, thus permitting experiments with short incubation times and low substrate concentrations. We applied Chip-SIP analysis to a natural estuarine community and quantified amino acid, nucleic acid or fatty acid incorporation by 81 distinct microbial taxa, thus demonstrating that resource partitioning occurs with relatively simple organic substrates. The Chip-SIP approach expands the repertoire of stable isotope-enabled methods available to microbial ecologists and provides a means to test genomics-generated hypotheses about biogeochemical function in any natural environment.
doi:10.1038/ismej.2011.175
PMCID: PMC3358021  PMID: 22158395
biogeochemistry; marine; microarray; microbial function; NanoSIMS; stable isotope probing
4.  Resolving Genetic Functions within Microbial Populations: In Situ Analyses Using rRNA and mRNA Stable Isotope Probing Coupled with Single-Cell Raman-Fluorescence In Situ Hybridization ▿ †  
Prokaryotes represent one-half of the living biomass on Earth, with the vast majority remaining elusive to culture and study within the laboratory. As a result, we lack a basic understanding of the functions that many species perform in the natural world. To address this issue, we developed complementary population and single-cell stable isotope (13C)-linked analyses to determine microbial identity and function in situ. We demonstrated that the use of rRNA/mRNA stable isotope probing (SIP) recovered the key phylogenetic and functional RNAs. This was followed by single-cell physiological analyses of these populations to determine and quantify in situ functions within an aerobic naphthalene-degrading groundwater microbial community. Using these culture-independent approaches, we identified three prokaryote species capable of naphthalene biodegradation within the groundwater system: two taxa were isolated in the laboratory (Pseudomonas fluorescens and Pseudomonas putida), whereas the third eluded culture (an Acidovorax sp.). Using parallel population and single-cell stable isotope technologies, we were able to identify an unculturable Acidovorax sp. which played the key role in naphthalene biodegradation in situ, rather than the culturable naphthalene-biodegrading Pseudomonas sp. isolated from the same groundwater. The Pseudomonas isolates actively degraded naphthalene only at naphthalene concentrations higher than 30 μM. This study demonstrated that unculturable microorganisms could play important roles in biodegradation in the ecosystem. It also showed that the combined RNA SIP-Raman-fluorescence in situ hybridization approach may be a significant tool in resolving ecology, functionality, and niche specialization within the unculturable fraction of organisms residing in the natural environment.
doi:10.1128/AEM.01861-08
PMCID: PMC2612214  PMID: 18997025
5.  The oral metagenome in health and disease 
The ISME Journal  2011;6(1):46-56.
The oral cavity of humans is inhabited by hundreds of bacterial species and some of them have a key role in the development of oral diseases, mainly dental caries and periodontitis. We describe for the first time the metagenome of the human oral cavity under health and diseased conditions, with a focus on supragingival dental plaque and cavities. Direct pyrosequencing of eight samples with different oral-health status produced 1 Gbp of sequence without the biases imposed by PCR or cloning. These data show that cavities are not dominated by Streptococcus mutans (the species originally identified as the ethiological agent of dental caries) but are in fact a complex community formed by tens of bacterial species, in agreement with the view that caries is a polymicrobial disease. The analysis of the reads indicated that the oral cavity is functionally a different environment from the gut, with many functional categories enriched in one of the two environments and depleted in the other. Individuals who had never suffered from dental caries showed an over-representation of several functional categories, like genes for antimicrobial peptides and quorum sensing. In addition, they did not have mutans streptococci but displayed high recruitment of other species. Several isolates belonging to these dominant bacteria in healthy individuals were cultured and shown to inhibit the growth of cariogenic bacteria, suggesting the use of these commensal bacterial strains as probiotics to promote oral health and prevent dental caries.
doi:10.1038/ismej.2011.85
PMCID: PMC3246241  PMID: 21716308
metagenomics; human microbiome; dental caries; Streptococcus mutans; pyrosequencing; probiotics
6.  Saliva microbiomes distinguish caries-active from healthy human populations 
The ISME Journal  2011;6(1):1-10.
The etiology of dental caries remains elusive because of our limited understanding of the complex oral microbiomes. The current methodologies have been limited by insufficient depth and breadth of microbial sampling, paucity of data for diseased hosts particularly at the population level, inconsistency of sampled sites and the inability to distinguish the underlying microbial factors. By cross-validating 16S rRNA gene amplicon-based and whole-genome-based deep-sequencing technologies, we report the most in-depth, comprehensive and collaborated view to date of the adult saliva microbiomes in pilot populations of 19 caries-active and 26 healthy human hosts. We found that: first, saliva microbiomes in human population were featured by a vast phylogenetic diversity yet a minimal organismal core; second, caries microbiomes were significantly more variable in community structure whereas the healthy ones were relatively conserved; third, abundance changes of certain taxa such as overabundance of Prevotella Genus distinguished caries microbiota from healthy ones, and furthermore, caries-active and normal individuals carried different arrays of Prevotella species; and finally, no ‘caries-specific' operational taxonomic units (OTUs) were detected, yet 147 OTUs were ‘caries associated', that is, differentially distributed yet present in both healthy and caries-active populations. These findings underscored the necessity of species- and strain-level resolution for caries prognosis, and were consistent with the ecological hypothesis where the shifts in community structure, instead of the presence or absence of particular groups of microbes, underlie the cariogenesis.
doi:10.1038/ismej.2011.71
PMCID: PMC3246229  PMID: 21716312
caries; metagenomics; oral-microbiome; Prevotella; saliva
7.  Correlation Network Analysis Applied to Complex Biofilm Communities 
PLoS ONE  2011;6(12):e28438.
The complexity of the human microbiome makes it difficult to reveal organizational principles of the community and even more challenging to generate testable hypotheses. It has been suggested that in the gut microbiome species such as Bacteroides thetaiotaomicron are keystone in maintaining the stability and functional adaptability of the microbial community. In this study, we investigate the interspecies associations in a complex microbial biofilm applying systems biology principles. Using correlation network analysis we identified bacterial modules that represent important microbial associations within the oral community. We used dental plaque as a model community because of its high diversity and the well known species-species interactions that are common in the oral biofilm. We analyzed samples from healthy individuals as well as from patients with periodontitis, a polymicrobial disease. Using results obtained by checkerboard hybridization on cultivable bacteria we identified modules that correlated well with microbial complexes previously described. Furthermore, we extended our analysis using the Human Oral Microbe Identification Microarray (HOMIM), which includes a large number of bacterial species, among them uncultivated organisms present in the mouth. Two distinct microbial communities appeared in healthy individuals while there was one major type in disease. Bacterial modules in all communities did not overlap, indicating that bacteria were able to effectively re-associate with new partners depending on the environmental conditions. We then identified hubs that could act as keystone species in the bacterial modules. Based on those results we then cultured a not-yet-cultivated microorganism, Tannerella sp. OT286 (clone BU063). After two rounds of enrichment by a selected helper (Prevotella oris OT311) we obtained colonies of Tannerella sp. OT286 growing on blood agar plates. This system-level approach would open the possibility of manipulating microbial communities in a targeted fashion as well as associating certain bacterial modules to clinical traits (e.g.: obesity, Crohn's disease, periodontal disease, etc).
doi:10.1371/journal.pone.0028438
PMCID: PMC3233593  PMID: 22163302
8.  CORE: A Phylogenetically-Curated 16S rDNA Database of the Core Oral Microbiome 
PLoS ONE  2011;6(4):e19051.
Comparing bacterial 16S rDNA sequences to GenBank and other large public databases via BLAST often provides results of little use for identification and taxonomic assignment of the organisms of interest. The human microbiome, and in particular the oral microbiome, includes many taxa, and accurate identification of sequence data is essential for studies of these communities. For this purpose, a phylogenetically curated 16S rDNA database of the core oral microbiome, CORE, was developed. The goal was to include a comprehensive and minimally redundant representation of the bacteria that regularly reside in the human oral cavity with computationally robust classification at the level of species and genus. Clades of cultivated and uncultivated taxa were formed based on sequence analyses using multiple criteria, including maximum-likelihood-based topology and bootstrap support, genetic distance, and previous naming. A number of classification inconsistencies for previously named species, especially at the level of genus, were resolved. The performance of the CORE database for identifying clinical sequences was compared to that of three publicly available databases, GenBank nr/nt, RDP and HOMD, using a set of sequencing reads that had not been used in creation of the database. CORE offered improved performance compared to other public databases for identification of human oral bacterial 16S sequences by a number of criteria. In addition, the CORE database and phylogenetic tree provide a framework for measures of community divergence, and the focused size of the database offers advantages of efficiency for BLAST searching of large datasets. The CORE database is available as a searchable interface and for download at http://microbiome.osu.edu.
doi:10.1371/journal.pone.0019051
PMCID: PMC3081323  PMID: 21544197
9.  Comparative Metagenomics Revealed Commonly Enriched Gene Sets in Human Gut Microbiomes 
Numerous microbes inhabit the human intestine, many of which are uncharacterized or uncultivable. They form a complex microbial community that deeply affects human physiology. To identify the genomic features common to all human gut microbiomes as well as those variable among them, we performed a large-scale comparative metagenomic analysis of fecal samples from 13 healthy individuals of various ages, including unweaned infants. We found that, while the gut microbiota from unweaned infants were simple and showed a high inter-individual variation in taxonomic and gene composition, those from adults and weaned children were more complex but showed a high functional uniformity regardless of age or sex. In searching for the genes over-represented in gut microbiomes, we identified 237 gene families commonly enriched in adult-type and 136 families in infant-type microbiomes, with a small overlap. An analysis of their predicted functions revealed various strategies employed by each type of microbiota to adapt to its intestinal environment, suggesting that these gene sets encode the core functions of adult and infant-type gut microbiota. By analysing the orphan genes, 647 new gene families were identified to be exclusively present in human intestinal microbiomes. In addition, we discovered a conjugative transposon family explosively amplified in human gut microbiomes, which strongly suggests that the intestine is a ‘hot spot’ for horizontal gene transfer between microbes.
doi:10.1093/dnares/dsm018
PMCID: PMC2533590  PMID: 17916580
metagenomics; human gut microbiota; gene family; conjugative transposon
10.  Deep Sequencing of the Oral Microbiome Reveals Signatures of Periodontal Disease 
PLoS ONE  2012;7(6):e37919.
The oral microbiome, the complex ecosystem of microbes inhabiting the human mouth, harbors several thousands of bacterial types. The proliferation of pathogenic bacteria within the mouth gives rise to periodontitis, an inflammatory disease known to also constitute a risk factor for cardiovascular disease. While much is known about individual species associated with pathogenesis, the system-level mechanisms underlying the transition from health to disease are still poorly understood. Through the sequencing of the 16S rRNA gene and of whole community DNA we provide a glimpse at the global genetic, metabolic, and ecological changes associated with periodontitis in 15 subgingival plaque samples, four from each of two periodontitis patients, and the remaining samples from three healthy individuals. We also demonstrate the power of whole-metagenome sequencing approaches in characterizing the genomes of key players in the oral microbiome, including an unculturable TM7 organism. We reveal the disease microbiome to be enriched in virulence factors, and adapted to a parasitic lifestyle that takes advantage of the disrupted host homeostasis. Furthermore, diseased samples share a common structure that was not found in completely healthy samples, suggesting that the disease state may occupy a narrow region within the space of possible configurations of the oral microbiome. Our pilot study demonstrates the power of high-throughput sequencing as a tool for understanding the role of the oral microbiome in periodontal disease. Despite a modest level of sequencing (∼2 lanes Illumina 76 bp PE) and high human DNA contamination (up to ∼90%) we were able to partially reconstruct several oral microbes and to preliminarily characterize some systems-level differences between the healthy and diseased oral microbiomes.
doi:10.1371/journal.pone.0037919
PMCID: PMC3366996  PMID: 22675498
11.  Identification of Bacterial Micropredators Distinctively Active in a Soil Microbial Food Web 
The understanding of microbial interactions and trophic networks is a prerequisite for the elucidation of the turnover and transformation of organic materials in soils. To elucidate the incorporation of biomass carbon into a soil microbial food web, we added 13C-labeled Escherichia coli biomass to an agricultural soil and identified those indigenous microbes that were specifically active in its mineralization and carbon sequestration. rRNA stable isotope probing (SIP) revealed that uncultivated relatives of distinct groups of gliding bacterial micropredators (Lysobacter spp., Myxococcales, and the Bacteroidetes) lead carbon sequestration and mineralization from the added biomass. In addition, fungal populations within the Microascaceae were shown to respond to the added biomass after only 1 h of incubation and were thus surprisingly reactive to degradable labile carbon. This RNA-SIP study identifies indigenous microbes specifically active in the transformation of a nondefined complex carbon source, bacterial biomass, directly in a soil ecosystem.
doi:10.1128/AEM.00400-06
PMCID: PMC1538704  PMID: 16885285
12.  Molecular Analysis of Bacterial Species Associated with Childhood Caries 
Journal of Clinical Microbiology  2002;40(3):1001-1009.
Although substantial epidemiologic evidence links Streptococcus mutans to caries, the pathobiology of caries may involve more complex communities of bacterial species. Molecular methods for bacterial identification and enumeration now make it possible to more precisely study the microbiota associated with dental caries. The purpose of this study was to compare the bacteria found in early childhood caries (ECC) to those found in caries-free children by using molecular identification methods. Cloning and sequencing of bacterial 16S ribosomal DNAs from a healthy subject and a subject with ECC were used for identification of novel species or uncultivated phylotypes and species not previously associated with dental caries. Ten novel phylotypes were identified. A number of species or phylotypes that may play a role in health or disease were identified and warrant further investigation. In addition, quantitative measurements for 23 previously known bacterial species or species groups were obtained by a reverse capture checkerboard assay for 30 subjects with caries and 30 healthy controls. Significant differences were observed for nine species: S. sanguinis was associated with health and, in order of decreasing cell numbers, Actinomyces gerencseriae, Bifidobacterium, S. mutans, Veillonella, S. salivarius, S. constellatus, S. parasanguinis, and Lactobacillus fermentum were associated with caries. These data suggest that A. gerencseriae and other Actinomyces species may play an important role in caries initiation and that a novel Bifidobacterium may be a major pathogen in deep caries. Further investigation could lead to the identification of targets for biological interventions in the caries process and thereby contribute to improved prevention of and treatment for this significant public health problem.
doi:10.1128/JCM.40.3.1001-1009.2002
PMCID: PMC120252  PMID: 11880430
13.  The Lung Microbiome in Moderate and Severe Chronic Obstructive Pulmonary Disease 
PLoS ONE  2012;7(10):e47305.
Chronic obstructive pulmonary disease (COPD) is an inflammatory disorder characterized by incompletely reversible airflow obstruction. Bacterial infection of the lower respiratory tract contributes to approximately 50% of COPD exacerbations. Even during periods of stable lung function, the lung harbors a community of bacteria, termed the microbiome. The role of the lung microbiome in the pathogenesis of COPD remains unknown. The COPD lung microbiome, like the healthy lung microbiome, appears to reflect microaspiration of oral microflora. Here we describe the COPD lung microbiome of 22 patients with Moderate or Severe COPD compared to 10 healthy control patients. The composition of the lung microbiomes was determined using 454 pyrosequencing of 16S rDNA found in bronchoalveolar lavage fluid. Sequences were analyzed using mothur, Ribosomal Database Project, Fast UniFrac, and Metastats. Our results showed a significant increase in microbial diversity with the development of COPD. The main phyla in all samples were Actinobacteria, Firmicutes, and Proteobacteria. Principal coordinate analyses demonstrated separation of control and COPD samples, but samples did not cluster based on disease severity. However, samples did cluster based on the use of inhaled corticosteroids and inhaled bronchodilators. Metastats analyses demonstrated an increased abundance of several oral bacteria in COPD samples.
doi:10.1371/journal.pone.0047305
PMCID: PMC3469539  PMID: 23071781
14.  Species-Level Analysis of DNA Sequence Data from the NIH Human Microbiome Project 
PLoS ONE  2012;7(10):e47075.
Background
Outbreaks of antibiotic-resistant bacterial infections emphasize the importance of surveillance of potentially pathogenic bacteria. Genomic sequencing of clinical microbiological specimens expands our capacity to study cultivable, fastidious and uncultivable members of the bacterial community. Herein, we compared the primary data collected by the NIH’s Human Microbiome Project (HMP) with published epidemiological surveillance data of Staphylococcus aureus.
Methods
The HMP’s initial dataset contained microbial survey data from five body regions (skin, nares, oral cavity, gut and vagina) of 242 healthy volunteers. A significant component of the HMP dataset was deep sequencing of the 16S ribosomal RNA gene, which contains variable regions enabling taxonomic classification. Since species-level identification is essential in clinical microbiology, we built a reference database and used phylogenetic placement followed by most recent common ancestor classification to look at the species distribution for Staphylococcus, Klebsiella and Enterococcus.
Main Results
We show that selecting the accurate region of the 16S rRNA gene to sequence is analogous to carefully selecting culture conditions to distinguish closely related bacterial species. Analysis of the HMP data showed that Staphylococcus aureus was present in the nares of 36% of healthy volunteers, consistent with culture-based epidemiological data. Klebsiella pneumoniae and Enterococcus faecalis were found less frequently, but across many habitats.
Conclusions
This work demonstrates that large 16S rRNA survey studies can be used to support epidemiological goals in the context of an increasing awareness that microbes flourish and compete within a larger bacterial community. This study demonstrates how genomic techniques and information could be critically important to trace microbial evolution and implement hospital infection control.
doi:10.1371/journal.pone.0047075
PMCID: PMC3468466  PMID: 23071716
15.  Defining the healthy "core microbiome" of oral microbial communities 
BMC Microbiology  2009;9:259.
Background
Most studies examining the commensal human oral microbiome are focused on disease or are limited in methodology. In order to diagnose and treat diseases at an early and reversible stage an in-depth definition of health is indispensible. The aim of this study therefore was to define the healthy oral microbiome using recent advances in sequencing technology (454 pyrosequencing).
Results
We sampled and sequenced microbiomes from several intraoral niches (dental surfaces, cheek, hard palate, tongue and saliva) in three healthy individuals. Within an individual oral cavity, we found over 3600 unique sequences, over 500 different OTUs or "species-level" phylotypes (sequences that clustered at 3% genetic difference) and 88 - 104 higher taxa (genus or more inclusive taxon). The predominant taxa belonged to Firmicutes (genus Streptococcus, family Veillonellaceae, genus Granulicatella), Proteobacteria (genus Neisseria, Haemophilus), Actinobacteria (genus Corynebacterium, Rothia, Actinomyces), Bacteroidetes (genus Prevotella, Capnocytophaga, Porphyromonas) and Fusobacteria (genus Fusobacterium).
Each individual sample harboured on average 266 "species-level" phylotypes (SD 67; range 123 - 326) with cheek samples being the least diverse and the dental samples from approximal surfaces showing the highest diversity. Principal component analysis discriminated the profiles of the samples originating from shedding surfaces (mucosa of tongue, cheek and palate) from the samples that were obtained from solid surfaces (teeth).
There was a large overlap in the higher taxa, "species-level" phylotypes and unique sequences among the three microbiomes: 84% of the higher taxa, 75% of the OTUs and 65% of the unique sequences were present in at least two of the three microbiomes. The three individuals shared 1660 of 6315 unique sequences. These 1660 sequences (the "core microbiome") contributed 66% of the reads. The overlapping OTUs contributed to 94% of the reads, while nearly all reads (99.8%) belonged to the shared higher taxa.
Conclusions
We obtained the first insight into the diversity and uniqueness of individual oral microbiomes at a resolution of next-generation sequencing. We showed that a major proportion of bacterial sequences of unrelated healthy individuals is identical, supporting the concept of a core microbiome at health.
doi:10.1186/1471-2180-9-259
PMCID: PMC2805672  PMID: 20003481
16.  The Canine Oral Microbiome 
PLoS ONE  2012;7(4):e36067.
Determining the bacterial composition of the canine oral microbiome is of interest for two primary reasons. First, while the human oral microbiome has been well studied using molecular techniques, the oral microbiomes of other mammals have not been studied in equal depth using culture independent methods. This study allows a comparison of the number of bacterial taxa, based on 16S rRNA-gene sequence comparison, shared between humans and dogs, two divergent mammalian species. Second, canine oral bacteria are of interest to veterinary and human medical communities for understanding their roles in health and infectious diseases. The bacteria involved are mostly unnamed and not linked by 16S rRNA-gene sequence identity to a taxonomic scheme. This manuscript describes the analysis of 5,958 16S rRNA-gene sequences from 65 clone libraries. Full length 16S rRNA reference sequences have been obtained for 353 canine bacterial taxa, which were placed in 14 bacterial phyla, 23 classes, 37 orders, 66 families, and 148 genera. Eighty percent of the taxa are currently unnamed. The bacterial taxa identified in dogs are markedly different from those of humans with only 16.4% of oral taxa are shared between dogs and humans based on a 98.5% 16S rRNA sequence similarity cutoff. This indicates that there is a large divergence in the bacteria comprising the oral microbiomes of divergent mammalian species. The historic practice of identifying animal associated bacteria based on phenotypic similarities to human bacteria is generally invalid. This report describes the diversity of the canine oral microbiome and provides a provisional 16S rRNA based taxonomic scheme for naming and identifying unnamed canine bacterial taxa.
doi:10.1371/journal.pone.0036067
PMCID: PMC3338629  PMID: 22558330
17.  The Personal Human Oral Microbiome Obscures the Effects of Treatment on Periodontal Disease 
PLoS ONE  2014;9(1):e86708.
Periodontitis is a progressive disease of the periodontium with a complex, polymicrobial etiology. Recent Next-Generation Sequencing (NGS) studies of the microbial diversity associated with periodontitis have revealed strong, community-level differences in bacterial assemblages associated with healthy or diseased periodontal sites. In this study, we used NGS approaches to characterize changes in periodontal pocket bacterial diversity after standard periodontal treatment. Despite consistent changes in the abundance of certain taxa in individuals whose condition improved with treatment, post-treatment samples retained the highest similarity to pre-treatment samples from the same individual. Deeper phylogenetic analysis of periodontal pathogen-containing genera Prevotella and Fusobacterium found both unexpected diversity and differential treatment response among species. Our results highlight how understanding interpersonal variability among microbiomes is necessary for determining how polymicrobial diseases respond to treatment and disturbance.
doi:10.1371/journal.pone.0086708
PMCID: PMC3906071  PMID: 24489772
18.  Uncultivated Phylotypes and Newly Named Species Associated with Primary and Persistent Endodontic Infections 
Journal of Clinical Microbiology  2005;43(7):3314-3319.
Endodontic infections have been traditionally studied by culture methods, but recent reports showing that over 50% of the oral microbiota is still uncultivable (B. J. Paster et al., J. Bacteriol. 183:3770-3783, 2001) raise the possibility that many endodontic pathogens remain unknown. This study intended to investigate the prevalence of several uncultivated oral phylotypes, as well as newly named species in primary or persistent endodontic infections associated with chronic periradicular diseases. Samples were taken from the root canals of 21 untreated teeth and 22 root-filled teeth, all of them with radiographic evidence of periradicular bone destruction. Genomic DNA was isolated directly from each sample, and 16S rRNA gene-based nested or heminested PCR assays were used to determine the presence of 13 species or phylotypes of bacteria. Species-specific primers had already been validated in the literature or were developed by aligning closely related 16S rRNA gene sequences. Species specificity for each primer pair was confirmed by running PCRs against a panel of several oral bacteria and by sequencing DNA from representative positive samples. All species or phylotypes were detected in at least one case of primary infections. The most prevalent species or phylotypes found in primary infections were Dialister invisus (81%), Synergistes oral clone BA121 (33%), and Olsenella uli (33%). Of the target bacteria, only these three species were detected in persistent infections. Detection of uncultivated phylotypes and newly named species in infected root canals suggests that there are previously unrecognized bacteria that may play a role in the pathogenesis of periradicular diseases.
doi:10.1128/JCM.43.7.3314-3319.2005
PMCID: PMC1169097  PMID: 16000454
19.  Oral Microbiome of Deep and Shallow Dental Pockets In Chronic Periodontitis 
PLoS ONE  2013;8(6):e65520.
We examined the subgingival bacterial biodiversity in untreated chronic periodontitis patients by sequencing 16S rRNA genes. The primary purpose of the study was to compare the oral microbiome in deep (diseased) and shallow (healthy) sites. A secondary purpose was to evaluate the influences of smoking, race and dental caries on this relationship. A total of 88 subjects from two clinics were recruited. Paired subgingival plaque samples were taken from each subject, one from a probing site depth >5 mm (deep site) and the other from a probing site depth ≤3mm (shallow site). A universal primer set was designed to amplify the V4–V6 region for oral microbial 16S rRNA sequences. Differences in genera and species attributable to deep and shallow sites were determined by statistical analysis using a two-part model and false discovery rate. Fifty-one of 170 genera and 200 of 746 species were found significantly different in abundances between shallow and deep sites. Besides previously identified periodontal disease-associated bacterial species, additional species were found markedly changed in diseased sites. Cluster analysis revealed that the microbiome difference between deep and shallow sites was influenced by patient-level effects such as clinic location, race and smoking. The differences between clinic locations may be influenced by racial distribution, in that all of the African Americans subjects were seen at the same clinic. Our results suggested that there were influences from the microbiome for caries and periodontal disease and these influences are independent.
doi:10.1371/journal.pone.0065520
PMCID: PMC3675156  PMID: 23762384
20.  Dental caries pathogenicity: a genomic and metagenomic perspective 
International dental journal  2011;61(0 1):11-22.
In this review we address the subject of dental caries pathogenicity from a genomic and metagenomic perspective. The application of genomic technologies is certain to yield novel insights into the relationship between the bacterial flora, dental health and disease. Three primary attributes of bacterial species are thought to have direct impact on caries development, these include: adherence on tooth surfaces (biofilm formation), acid production and acid tolerance. Attempts to define the specific aetiological agents of dental caries have proven to be elusive, supporting the notion that caries aetiology is perhaps complex and multi-faceted. The recently introduced Human Microbiome Project (HMP) that endeavors to characterise the micro-organisms living in and on the human body is likely to shed new light on these questions and improve our understanding of polymicrobial disease, microbial ecology in the oral cavity and provide new avenues for therapeutic and molecular diagnostics developments.
doi:10.1111/j.1875-595X.2011.00025.x
PMCID: PMC3699854  PMID: 21726221
Caries; biofilm; bacterial species; genomic; metagenomic; Human Microbiome Project
21.  Levels of Selenomonas species in generalized aggressive periodontitis 
Journal of periodontal research  2012;47(6):711-718.
Aim
To compare the levels of Selenomonas sputigena and uncultivated/unrecognized Selenomonas species in subgingival biofilms from generalized aggressive periodontitis (GAgP) and periodontaly healthy (PH) subjects.
Material and Methods
GAgP (n=15) and PH (n=15) subjects were recruited and their clinical periodontal parameters were evaluated. Subgingival plaque samples were collected (9 samples/subject) and analyzed for the levels of 10 bacterial taxa, including cultivated and uncultivated/unrecognized microorganisms using the RNA-oligonucleotide quantification technique (ROQT). Differences in the levels of the test taxa between groups were sought using the Mann-Whitney test.
Results
GAgP subjects showed significantly higher mean counts of Porphyromonas gingivalis, Selenomonas sputigena and Selenomonas oral clone CS002 (Human Oral Microbial Database (HOMD) Oral Taxon 131), while Actinomyces gerencseriae and Streptococcus sanguinis were found in higher mean counts in PH subjects (p<0.01). Selenomonas EW084 (HOMD OT 146) was only detected in the GAgP group. In the GAgP group, levels of P. gingivalis and S. sputigena were higher in sites with probing depth (PD) ≥5mm than in shallow sites (PD ≤3mm) (p<0.01). Furthermore, sites with PD≤3mm in GAgP subjects harbored higher levels of these two species than sites in PH subjects. There were positive correlations between PD and levels of P. gingivalis (r=0.77; p<0.01), S. sputigena (r=0.60; p<0.01) and Selenomonas sp. EW076 (OT 139) (r=042, p<0.05).
Conclusion
S. sputigena, Selenomonas sp. oral CS002 (OT 131) and Selenomonas sp. oral clone EW084 (OT 146) may be associated with the pathogenesis of GAgP, and their role in the onset and progression of this infection should be further investigated.
doi:10.1111/j.1600-0765.2012.01485.x
PMCID: PMC3678906  PMID: 22612405
Selenomonas sputigena; molecular biology; 16S rRNA; generalized aggressive periodontitis; not-yet-cultivated species
22.  Stable Isotope Probing of an Algal Bloom To Identify Uncultivated Members of the Rhodobacteraceae Associated with Low-Molecular-Weight Polycyclic Aromatic Hydrocarbon Degradation▿ 
Applied and Environmental Microbiology  2011;77(21):7856-7860.
Polycyclic aromatic hydrocarbon (PAH)-degrading bacteria associated with an algal bloom in Tampa Bay, FL, were investigated by stable isotope probing (SIP) with uniformly labeled [13C]naphthalene. The dominant sequences in clone libraries constructed from 13C-enriched bacterial DNA (from naphthalene enrichments) were identified as uncharacterized members of the family Rhodobacteraceae. Quantitative PCR primers targeting the 16S rRNA gene of these uncultivated organisms were used to determine their abundance in incubations amended with unlabeled naphthalene and phenanthrene, both of which showed substantial increases in gene copy numbers during the experiments. As demonstrated by this work, the application of uniformly 13C-labeled PAHs in SIP experiments can successfully be used to identify novel PAH-degrading bacteria in marine waters.
doi:10.1128/AEM.06200-11
PMCID: PMC3209190  PMID: 21926219
23.  Microbial Eukaryotes in the Human Microbiome: Ecology, Evolution, and Future Directions 
High throughput sequencing technology has opened a window into the vast communities of bacteria that live on and in humans, demonstrating tremendous variability, and that they play a large role in health and disease. The eukaryotic component of the human gut microbiome remains relatively unexplored with these methods, but turning these tools toward microbial eukaryotes in the gut will likely yield myriad insights into disease as well as the ecological and evolutionary principles that govern the gut microbiota. Microbial eukaryotes are common inhabitants of the human gut worldwide and parasitic taxa are a major source of morbidity and mortality, especially in developing countries, though there are also taxa that cause no harm or are beneficial. While the role microbial eukaryotes play in healthy individuals is much less clear, there are likely many complex interactions between the bacterial, archaeal, and eukaryotic microbiota that influence human health. Integrating eukaryotic microbes into a broad view of microbiome function requires an integrated ecological approach rather than one focused on specific, disease-causing taxa. Moving forward, we expect broad surveys of the eukaryotic microbiota and associated bacteria from geographically and socioeconomically diverse populations to paint a more complete picture of the human gut microbiome in health and disease.
doi:10.3389/fmicb.2011.00153
PMCID: PMC3135866  PMID: 21808637
intestinal protozoa; host-associated communities; eukaryotic diversity
24.  Analyses of the Microbial Diversity across the Human Microbiome 
PLoS ONE  2012;7(6):e32118.
Analysis of human body microbial diversity is fundamental to understanding community structure, biology and ecology. The National Institutes of Health Human Microbiome Project (HMP) has provided an unprecedented opportunity to examine microbial diversity within and across body habitats and individuals through pyrosequencing-based profiling of 16 S rRNA gene sequences (16 S) from habits of the oral, skin, distal gut, and vaginal body regions from over 200 healthy individuals enabling the application of statistical techniques. In this study, two approaches were applied to elucidate the nature and extent of human microbiome diversity. First, bootstrap and parametric curve fitting techniques were evaluated to estimate the maximum number of unique taxa, Smax, and taxa discovery rate for habitats across individuals. Next, our results demonstrated that the variation of diversity within low abundant taxa across habitats and individuals was not sufficiently quantified with standard ecological diversity indices. This impact from low abundant taxa motivated us to introduce a novel rank-based diversity measure, the Tail statistic, (“τ”), based on the standard deviation of the rank abundance curve if made symmetric by reflection around the most abundant taxon. Due to τ’s greater sensitivity to low abundant taxa, its application to diversity estimation of taxonomic units using taxonomic dependent and independent methods revealed a greater range of values recovered between individuals versus body habitats, and different patterns of diversity within habitats. The greatest range of τ values within and across individuals was found in stool, which also exhibited the most undiscovered taxa. Oral and skin habitats revealed variable diversity patterns, while vaginal habitats were consistently the least diverse. Collectively, these results demonstrate the importance, and motivate the introduction, of several visualization and analysis methods tuned specifically for next-generation sequence data, further revealing that low abundant taxa serve as an important reservoir of genetic diversity in the human microbiome.
doi:10.1371/journal.pone.0032118
PMCID: PMC3374608  PMID: 22719823
25.  Lignocellulose-responsive bacteria in a southern California salt marsh identified by stable isotope probing 
Carbon cycling by microbes has been recognized as the main mechanism of organic matter decomposition and export in coastal wetlands, yet very little is known about the functional diversity of specific groups of decomposers (e.g., bacteria) in salt marsh benthic trophic structure. Indeed, salt marsh sediment bacteria remain largely in a black box in terms of their diversity and functional roles within salt marsh benthic food web pathways. We used DNA stable isotope probing (SIP) utilizing 13C-labeled lignocellulose as a proxy to evaluate the fate of macrophyte-derived carbon in benthic salt marsh bacterial communities. Overall, 146 bacterial species were detected using SIP, of which only 12 lineages were shared between enriched and non-enriched communities. Abundant groups from the 13C-labeled community included Desulfosarcina, Spirochaeta, and Kangiella. This study is the first to use heavy-labeled lignocellulose to identify bacteria responsible for macrophyte carbon utilization in salt marsh sediments and will allow future studies to target specific lineages to elucidate their role in salt marsh carbon cycling and ultimately aid our understanding of the potential of salt marshes to store carbon.
doi:10.3389/fmicb.2014.00263
PMCID: PMC4040508  PMID: 24917856
salt marsh; bacteria; lignocellulose; stable isotope probing (SIP); food web; macrophytes; sediments

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