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.

Clinical and microbiological parameters of naturally occurring periodontitis in the non-human primate Macaca mulatta

“…Supragingival plaque was removed with sterile gauze and discarded, and subgingival plaque was taken from the selected periodontal sites with Gracey curettes (Hu-Friedy), pooled and suspended in TE buffer (50 mM Tris HCL, 1 mM EDTA pH 7.6), placed on ice, and stored at −80°C until further analysis. Whole genomic bacterial DNA was extracted and purified from the pooled subgingival microbiome samples using the MasterPure™ DNA purification kit (Epicentre®, Madison, WI [17]. …”

“…Four libraries were constructed, two with broad range primers and two with Bacteroidetes-specific primers. 16S rRNA PCR amplification, cloning and sequencing were performed as previously described [17]. More than 80 clones were picked from each library, amplified, and sequenced, yielding 330 partial sequences.…”

Gene Regulation, Two Component Regulatory Systems, and Adaptive Responses in Treponema Denticola

“…However, this represents only a small subset of the total oral population (Paster et al 2001). The complexity and remarkable nature of the bacterial population in the oral cavity were noted over 340 years ago by Antonie van Leeuwenhoek, a "non-scientist" merchant in Holland who documented his observations in unpublished personal notes later recounted by others (Amako 2014).…”

“…The oral cavity of mammals hosts a remarkably diverse bacterial population consisting of 500-700 species (Paster et al 2001). Signature species differences in the composition of the bacterial population at specific niches have been demonstrated and shown to correlate with infections including caries, endodontic infections, gingivitis, subgingival plaque and periodontitis (Siqueira and Rocas 2004;Zijnge et al 2010).…”

“…The anaerobic environment of deep periodontal pockets (>3 mm) favors the proliferation of a subset of the oral bacterial community that consists primarily of Gram-negative anaerobes and anaerobic spirochetes (Dewhirst et al 2010(Dewhirst et al , 2012. All oral spirochetes belong exclusively to the genus Treponema (Paster et al 2001). Here we present an overview of the genetic regulatory mechanisms and adaptive responses that may contribute to the ability of T. denticola to compete and thrive in the competitive environment of subgingival plaque in periodontal pockets.…”