Periodontitis has a polymicrobial etiology within the framework of a complex microbial ecosystem. With advances in sequencing technologies, comprehensive studies to elucidate bacterial community differences have recently become possible. We used 454 sequencing of 16S rRNA genes to compare subgingival bacterial communities from 29 periodontally healthy controls and 29 subjects with chronic periodontitis. Amplicons from both the V1-2 and V4 regions of the 16S gene were sequenced, yielding 1 393 579 sequences. They were identified by BLAST against a curated oral 16S database, and mapped to 16 phyla, 106 genera, and 596 species. 81% of sequences could be mapped to cultivated species. Differences between health-and periodontitis-associated bacterial communities were observed at all phylogenetic levels, and UniFrac and principal coordinates analysis showed distinct community profiles in health and disease. Community diversity was higher in disease, and 123 species were identified that were significantly more abundant in disease, and 53 in health. Spirochaetes, Synergistetes and Bacteroidetes were more abundant in disease, whereas the Proteobacteria were found at higher levels in healthy controls. Within the phylum Firmicutes, the class Bacilli was health-associated, whereas the Clostridia, Negativicutes and Erysipelotrichia were associated with disease. These results implicate a number of taxa that will be targets for future research. Some, such as Filifactor alocis and many Spirochetes were represented by a large fraction of sequences as compared with previously identified targets. Elucidation of these differences in community composition provides a basis for further understanding the pathogenesis of periodontitis.

Although Streptococcus mutans has been implicated as a major etiological agent of dental caries, our cross-sectional preliminary study indicated that 10% of subjects with rampant caries in permanent teeth do not have detectable levels of S. mutans. Our aims were to use molecular methods to detect all bacterial species associated with caries in primary and permanent teeth and to determine the bacterial profiles associated with different disease states. Plaque was collected from 39 healthy controls and from intact enamel and white-spot lesions, dentin lesions, and deep-dentin lesions in each of 51 subjects with severe caries. 16S rRNA genes were PCR amplified, cloned, and sequenced to determine species identities. In a reverse-capture checkerboard assay, 243 samples were analyzed for 110 prevalent bacterial species. A sequencing analysis of 1,285 16S rRNA clones detected 197 bacterial species/phylotypes, of which 50% were not cultivable. Twenty-two new phylotypes were identified. PROC MIXED tests revealed health-and disease-associated species. In subjects with S. mutans, additional species, e.g., species of the genera Atopobium, Propionibacterium, and Lactobacillus, were present at significantly higher levels than those of S. mutans. Lactobacillus spp., Bifidobacterium dentium, and low-pH non-S. mutans streptococci were predominant in subjects with no detectable S. mutans. Actinomyces spp. and non-S. mutans streptococci were predominant in white-spot lesions, while known acid producers were found at their highest levels later in disease. Bacterial profiles change with disease states and differ between primary and secondary dentitions. Bacterial species other than S. mutans, e.g., species of the genera Veillonella, Lactobacillus, Bifidobacterium, and Propionibacterium, low-pH non-S. mutans streptococci, Actinomyces spp., and Atopobium spp., likely play important roles in caries progression.Dental caries is one of the most common chronic infectious diseases in the world (2, 39). There are three major hypotheses for the etiology of dental caries: the specific plaque hypothesis, the nonspecific plaque hypothesis, and the ecological plaque hypothesis (24,26,37). The specific plaque hypothesis has proposed that only a few specific species, such as Streptococcus mutans and Streptococcus sobrinus, are actively involved in the disease. On the other hand, the nonspecific plaque hypothesis maintains that caries is the outcome of the overall activity of the total plaque microflora, which is comprised of many bacterial species (37). The ecological plaque hypothesis suggests that caries is a result of a shift in the balance of the resident microflora driven by changes in local environmental conditions (26).Caries-associated bacteria traditionally have been identified by using culture-based methods, which exclude not-yet-cultivated species. Molecular methods for bacterial identification and enumeration now are performed routinely to more precisely study bacterial species that are associated with dental caries, including those th...

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.

Most studies of the bacterial etiology of periodontitis have used either culture-based or targeted DNA approaches, and so it is likely that pathogens remain undiscovered. The purpose of this study was to use culture-independent, quantitative analysis of biofilms associated with chronic periodontitis and periodontal health to identify pathogens and beneficial species. Samples from subjects with periodontitis and controls were analyzed using ribosomal 16S cloning and sequencing. Several genera, many of them uncultivated, were associated with periodontitis, the most numerous of which were gram positive, including Peptostreptococcus and Filifactor. The genera Megasphaera and Desulfobulbus were elevated in periodontitis, and the levels of several species or phylotypes of Campylobacter, Selenomonas, Deferribacteres, Dialister, Catonella, Tannerella, Streptococcus, Atopobium, Eubacterium, and Treponema were elevated in disease. Streptococcus and Veillonella spp. were found in high numbers in all samples and accounted for a significantly greater fraction of the microbial community in healthy subjects than in those with periodontitis. The microbial profile of periodontal health also included the less-abundant genera Campylobacter, Abiotrophia, Gemella, Capnocytophaga, and Neisseria. These newly identified candidates outnumbered Porphyromonas gingivalis and other species previously implicated as periodontopathogens, and it is not clear if newly identified and more numerous species may play a more important role in pathogenesis. Finally, more differences were found in the bacterial profile between subjects with periodontitis and healthy subjects than between deep and shallow sites within the same subject. This suggests that chronic periodontitis is the result of a global perturbation of the oral bacterial ecology rather than a disease-site specific microbial shift.There is considerable evidence to show that bacterial plaque is the etiologic agent in chronic periodontitis. No single species has been implicated as a primary pathogen, and the available evidence is consistent with a polymicrobial disease etiology. Nearly all studies on the bacterial etiology of periodontitis have used either culture-based or directed DNA approaches, targeting known species. The prevailing paradigm that implicates minor constituents of the subgingival community, the gram-negative bacteria Porphyromonas gingivalis, Tanerella forsythensis, and Treponema denticola (24), as periodontopathogens is based on such approaches. However, culturing is not representative of the composition of a microbial community, since it is often too selective, especially for fastidious and as-yet-uncultivable species. Even culture-independent targeted approaches are limited to detecting the presence and levels of known species. Obviously cultivation will not detect uncultivated species, but the limitations of closed-ended molecular approaches such as PCR or hybridization assays such as checkerboard and microarrays are not as widely appreciated. Using these approaches it...

Recent investigations of the human subgingival oral flora based on ribosomal 16S cloning and sequencing have shown many of the bacterial species present to be novel species or phylotypes. The purpose of the present investigation was to identify potential periodontal pathogens among these newly identified species and phylotypes. Species-specific ribosomal 16S primers for PCR amplification were developed for detection of new species. Associations with chronic periodontitis were observed for several new species or phylotypes, including uncultivated clones D084 and BH017 from the Deferribacteres phylum, AU126 from the Bacteroidetes phylum, Megasphaera clone BB166, clone X112 from the OP11 phylum, and clone I025 from the TM7 phylum, and the named species Eubacterium saphenum, Porphyromonas endodontalis, Prevotella denticola, and Cryptobacterium curtum. Species or phylotypes more prevalent in periodontal health included two uncultivated phylotypes, clone W090 from the Deferribacteres phylum and clone BU063 from the Bacteroidetes, and named species Atopobium rimae and Atopobium parvulum.