Defining the normal bacterial flora of the oral cavity

Abstract

More than 700 bacterial species or phylotypes, of which over 50% have not been cultivated, have been detected in the oral cavity. Our purposes were (i) to utilize culture-independent molecular techniques to extend our knowledge on the breadth of bacterial diversity in the healthy human oral cavity, including not-yet-cultivated bacteria species, and (ii) to determine the site and subject specificity of bacterial colonization. Nine sites from five clinically healthy subjects were analyzed. Sites included tongue dorsum, lateral sides of tongue, buccal epithelium, hard palate, soft palate, supragingival plaque of tooth surfaces, subgingival plaque, maxillary anterior vestibule, and tonsils. 16S rRNA genes from sample DNA were amplified, cloned, and transformed into Escherichia coli. Sequences of 16S rRNA genes were used to determine species identity or closest relatives. In 2,589 clones, 141 predominant species were detected, of which over 60% have not been cultivated. Thirteen new phylotypes were identified. Species common to all sites belonged to the genera Gemella, Granulicatella, Streptococcus, and Veillonella. While some species were subject specific and detected in most sites, other species were site specific. Most sites possessed 20 to 30 different predominant species, and the number of predominant species from all nine sites per individual ranged from 34 to 72. Species typically associated with periodontitis and caries were not detected. There is a distinctive predominant bacterial flora of the healthy oral cavity that is highly diverse and site and subject specific. It is important to fully define the human microflora of the healthy oral cavity before we can understand the role of bacteria in oral disease.

FIG. 1.

Bacterial profiles of the buccal epithelium of healthy subjects. Distribution and levels of bacterial species/phylotypes among five subjects are shown by the columns of boxes to the right of the tree as either not detected (clear box), <15% of the total number of clones assayed (shaded box), or ≥15% of the total number of clones assayed (darkened box). The 15% was chosen arbitrarily. GenBank accession numbers are provided. Marker bar represents a 5% difference in nucleotide sequences.

FIG. 2.

Bacterial profiles of the maxillary anterior vestibule of healthy subjects. Distribution and levels of bacterial species/phylotypes among five subjects are as described for Fig. 1. Novel phylotypes identified in this study are indicated in bold. GenBank accession numbers are provided. Marker bar represents a 5% difference in nucleotide sequences.

FIG. 3.

Bacterial profiles of the tongue dorsum of healthy subjects. Distribution and levels of bacterial species/phylotypes among five subjects are as described for Fig. 1. Novel phylotypes identified in this study are indicated in bold. GenBank accession numbers are provided. Marker bar represents a 5% difference in nucleotide sequences.

FIG. 4.

Bacterial profiles of the lateral tongue surface of healthy subjects. Distribution and levels of bacterial species/phylotypes among five subjects are as described for Fig. 1. Novel phylotypes identified in this study are indicated in bold. GenBank accession numbers are provided. Marker bar represents a 5% difference in nucleotide sequences.

FIG. 5.

Bacterial profiles of the hard palate of healthy subjects. Distribution and levels of bacterial species/phylotypes among five subjects are as described for Fig. 1. Novel phylotypes identified in this study are indicated in bold. GenBank accession numbers are provided. Marker bar represents a 5% difference in nucleotide sequences.

FIG. 6.

Bacterial profiles of the soft palate of healthy subjects. Distribution and levels of bacterial species/phylotypes among five subjects are as described for Fig. 1. Novel phylotypes identified in this study are indicated in bold. GenBank accession numbers are provided. Marker bar represents a 5% difference in nucleotide sequences.

FIG. 7.

Bacterial profiles of the tonsils of healthy subjects. Distribution and levels of bacterial species/phylotypes among five subjects are as described for Fig. 1. Novel phylotypes identified in this study are indicated in bold. GenBank accession numbers are provided. Marker bar represents a 5% difference in nucleotide sequences.

FIG. 8.

Bacterial profiles of the tooth surfaces of healthy subjects. Distribution and levels of bacterial species/phylotypes among five subjects are as described for Fig. 1. Novel phylotypes identified in this study are indicated in bold. GenBank accession numbers are provided. Marker bar represents a 5% difference in nucleotide sequences.

FIG. 9.

Bacterial profiles of the subgingival plaque of healthy subjects. Distribution and levels of bacterial species/phylotypes among five subjects are as described for Fig. 1. Novel phylotypes identified in this study are indicated in bold. GenBank accession numbers are provided. Marker bar represents a 5% difference in nucleotide sequences.

FIG. 10.

Site specificity of predominant bacterial species in the oral cavity. In general, bacterial species or phylotypes were selected on the basis of their detection in multiple subjects for a given site. Distributions of bacterial species in oral sites among subjects are indicated by the columns of boxes to the right of the tree as follows: not detected in any subject (clear box), <15% of the total number of clones assayed (yellow box), ≥15% of the total number of clones assayed (green box). The 15% cutoff for low and high abundance was chosen arbitrarily. Marker bar represents a 10% difference in nucleotide sequences.