Bacterial diversity in human subgingival plaque

Abstract

The purpose of this study was to determine the bacterial diversity in the human subgingival plaque by using culture-independent molecular methods as part of an ongoing effort to obtain full 16S rRNA sequences for all cultivable and not-yet-cultivated species of human oral bacteria. Subgingival plaque was analyzed from healthy subjects and subjects with refractory periodontitis, adult periodontitis, human immunodeficiency virus periodontitis, and acute necrotizing ulcerative gingivitis. 16S ribosomal DNA (rDNA) bacterial genes from DNA isolated from subgingival plaque samples were PCR amplified with all-bacterial or selective primers and cloned into Escherichia coli. The sequences of cloned 16S rDNA inserts were used to determine species identity or closest relatives by comparison with sequences of known species. A total of 2,522 clones were analyzed. Nearly complete sequences of approximately 1,500 bases were obtained for putative new species. About 60% of the clones fell into 132 known species, 70 of which were identified from multiple subjects. About 40% of the clones were novel phylotypes. Of the 215 novel phylotypes, 75 were identified from multiple subjects. Known putative periodontal pathogens such as Porphyromonas gingivalis, Bacteroides forsythus, and Treponema denticola were identified from multiple subjects, but typically as a minor component of the plaque as seen in cultivable studies. Several phylotypes fell into two recently described phyla previously associated with extreme natural environments, for which there are no cultivable species. A number of species or phylotypes were found only in subjects with disease, and a few were found only in healthy subjects. The organisms identified only from diseased sites deserve further study as potential pathogens. Based on the sequence data in this study, the predominant subgingival microbial community consisted of 347 species or phylotypes that fall into 9 bacterial phyla. Based on the 347 species seen in our sample of 2,522 clones, we estimate that there are 68 additional unseen species, for a total estimate of 415 species in the subgingival plaque. When organisms found on other oral surfaces such as the cheek, tongue, and teeth are added to this number, the best estimate of the total species diversity in the oral cavity is approximately 500 species, as previously proposed.

FIG. 1

Bacterial phyla identified from subgingival plaque. Nine bacterial phyla were represented from clonal analysis. As shown, the first two phyla, Obsidian Pool OP11 and TM7, have no cultivable representatives. Oral members of the phylum Deferribacteres form a cluster consisting of only not-yet-cultivated phylotypes. The information presented in Fig. 1 to 7 includes bacterial species or phylotype, strain or clone identification, sequence accession number, total number of retrieved clones, and number of subjects in each health status category in which each species was identified (as indicated by color-coordinated symbols). Novel phylotypes are defined as those taxa that are <98.5 to 99% similar in sequence comparisons to the phylotype's closest relative. Prevalent species are defined as those phylotypes or species identified in four or more subjects and are noted in boldface and underlined in each dendrogram. Two hundred bootstrap trees were generated, and bootstrap confidence levels as percentages (only values over 40%) are shown at tree nodes.

FIG. 2

Phylogenetic tree of oral treponemes identified from clone libraries. All species identified thus far fall in the genus Treponema, and presently there are about 60 oral treponemal species or phylotypes (6). The marker bar represents a 10% difference in nucleotide sequences.

FIG. 3

Phylogenetic trees of the phyla Fusobacteria and Actinobacteria identified from clone libraries. The members of the oral Fusobacteria identified include species of the genera Fusobacterium and Leptotrichia. The marker bar represents a 5% difference in nucleotide sequences.

FIG. 4

Phylogenetic tree of the phylum Firmicutes identified from clone libraries. The phylum Firmicutes is presently divided into three classes: “Bacilli,” Mollicutes, and “Clostridia.” This phylum represented the largest group detected; 115 species or novel phylotypes were identified (Table 4). Streptococcus oralis and S. mitis could not be differentiated based on sequence comparisons. Consequently, the two species are grouped together. The marker bar represents a 5% difference in nucleotide sequences.

FIG. 5

Phylogenetic tree of the class “Clostridia” of the phylum Firmicutes identified from clone libraries. The marker bar represents a 5% difference in nucleotide sequences.

FIG. 6

Phylogenetic tree of the phylum Proteobacteria identified from clone libraries. The marker bar represents a 5% difference in nucleotide sequences.

FIG. 7

Phylogenetic tree of the phylum Bacteroidetes identified from clone libraries. Oral species identified generally fall within the genera Porphyromonas, Prevotella, and Capnocytophaga. An asterisk denotes sequence of only about 1,200 bp. The marker bar represents a 5% difference in nucleotide sequences.