The oral cavity is comprised of many surfaces, each coated with a plethora of bacteria, the proverbial bacterial biofilm. Some of these bacteria have been implicated in oral diseases such as caries and periodontitis, which are among the most common bacterial infections in humans. For example, it has been estimated that at least 35% of dentate U.S. adults aged 30 to 90 years have periodontitis (
1). In addition, specific oral bacterial species have been implicated in several systemic diseases, such as bacterial endocarditis (
4), aspiration pneumonia (
26), osteomyelitis in children (
8), preterm low birth weight (
6,
20), and cardiovascular disease (
2,
34). Surprisingly, little is known about the microflora of the healthy oral cavity.
By using culture-independent molecular methods, we previously detected over 500 species or phylotypes in subgingival plaque of healthy subjects and subjects with periodontal diseases (
21), necrotizing ulcerative periodontitis in human immunodeficiency virus-positive subjects (
23), dental plaque in children with rampant caries (
3), noma (
22), and on the tongue dorsum of subjects with and without halitosis (
15). Other investigators have used similar techniques to determine the bacterial diversity of saliva (
25), subgingival plaque of a subject with gingivitis (
16), and dentoalveolar abscesses (
10,
29). Over half of the species detected have not yet been cultivated. Data from these studies have implicated specific species or phylotypes in a variety of diseases and oral infections, but still only limited information is available on species associated with health. Recently, Mager et al. (
19) demonstrated significant differences in the bacterial profiles of 40 oral cultivable species on soft and hard tissues in healthy subjects. They also found that the profiles of the soft tissues were more similar to each other than those of supragingival and subgingival plaques.
Our purposes were as follows: (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 phylotypes, and (ii) to determine the site and subject specificity of bacterial colonization.
RESULTS AND DISCUSSION
It has long been known that oral bacteria preferentially colonize different surfaces in the oral cavity as a result of specific adhesins on the bacterial surface binding to complementary specific receptors on a given oral surface (
11,
12). Indeed, the profiles of 40 cultivable bacterial species differed markedly on different oral soft tissue surfaces, saliva, and supragingival and subgingival plaques from healthy subjects (
19). The purpose of this study was to define the predominant bacterial flora of the healthy oral cavity by identifying and comparing the cultivable and the not-yet-cultivated bacterial species on different soft tissues and supra- and subgingival plaques.
Based on the analysis of 2,589 16S rRNA clones, the bacterial diversity of the microflora from nine different sites of five clinically healthy subjects was striking—a total of 141 different bacterial taxa representing six different bacterial phyla were detected. The six phyla included the
Firmicutes (previously referred to as the low-G+C gram positives, such as species of
Streptococcus,
Gemella,
Eubacterium,
Selenomonas,
Veillonella, and related ones), the
Actinobacteria (previously referred to as the high-G+C gram positives, such as species of
Actinomyces,
Atopobium,
Rothia, and related ones), the
Proteobacteria (e.g., species of
Neisseria,
Eikenella,
Campylobacter, and related ones), the
Bacteroidetes (e.g., species of
Porphyromonas,
Prevotella,
Capnocytophaga, and related ones), the
Fusobacteria (e.g., species of
Fusobacterium and
Leptotrichia), and the TM7 phylum, for which there are no cultivable representatives. As determined in our previous studies using culture-independent molecular techniques (
15,
21,
22), over 60% of the bacterial flora was represented by not-yet-cultivated phylotypes. Thirteen new phylotypes (see Fig.
2 through
9) were identified for the first time in this study. In a comparable ongoing study of caries in permanent teeth, using the same techniques, we detected 224 species in the analysis of 1,275 16S rRNA clones with about 60% as not-yet-cultivable phylotypes (J. A. Aas, S. R. Dardis, A. L. Griffen, L. N. Stokes, A. M. Lee, I. Olsen, F. E. Dewhirst, E. J. Leys, and B. J. Paster. J. Dent. Res.
84:abstract 2805, 2005). Consequently, it is important to identify both the cultivable and not-yet-cultivated bacterial flora in a given environment before we can ascribe association to health or disease status. There is no evidence to suggest that the not-yet-cultivated segment is any less important than the cultivable segment.
Bacterial profiles for each site tested for each subject are shown in nine phylogenetic trees (see Fig.
1 through
9). In these dendrograms, the distribution of bacterial species or phylotypes in each subject can be observed at a glance. For example, it is clear that
Streptococcus mitis,
S. mitis bv. 2, and
Gemella hemolysans are the predominant species of the buccal epithelium (Fig.
1). Each of these species was detected in most or all of the subjects and represented ≥15% of the total number of assayed clones (Fig.
1). Similarly, the predominant bacterial flora for the other eight oral sites was examined. In the maxillary anterior vestibule,
S. mitis,
Granulicatella spp., and
Gemella spp. predominated (Fig.
2). On the tongue dorsum, several species of
Streptococcus, such as
S. mitis,
Streptococcus australis,
Streptococcus parasanguinis,
Streptococcus salivarius,
Streptococcus sp. clone FP015, and
Streptococcus sp. clone FN051,
Granulicatella adiacens, and
Veillonella spp. were the predominant species (Fig.
3). On the lateral tongue surface (Fig.
4),
S. mitis,
S. mitis bv. 2,
Streptococcus sp. clone DP009,
Streptococcus sp. clone FN051,
S. australis,
G. adiacens,
G. hemolysans, and
Veillonella spp. predominated. It is interesting that there were considerable differences in the bacterial profiles of the tongue dorsum and the lateral tongue surface. For example,
S. mitis bv. 2 was well represented at the lateral side of the tongue but not detected on the tongue dorsum. Conversely,
S.parasanguinis strain 85-81 was present on the tongue dorsum, but was absent on the lateral side. This was not surprising, because these surfaces are known to be different in ultrastructure and function. For instance, the lateral side of the tongue has a smooth nonkeratinized surface, in contrast to the dorsum of the tongue, which is a keratinized, highly papillated surface with a large surface area and underlying serous glands. These anatomic differences likely influence the ecology of these habitats and create microbial environmental differences.
On the hard palate, the predominant bacterial species included
S. mitis,
S. mitis biovar 2,
Streptococcus sp. clone FN051,
Streptococcus infantis,
Granulicatella elegans,
G. hemolysans, and
Neisseria subflava (Fig.
5). On the soft palate,
S. mitis, other cultivable and not-yet-cultivable species of
Streptococcus,
G. adiacens and
G. hemolysans were predominant (Fig.
6). The tonsil bacterial flora (Fig.
7) was rather diverse—some subjects had
Prevotella and
Porphyromonas spp. (subjects 1 and 5), and others had
Firmicutes species (subjects 2, 3, and 4). On the tooth surface, several species of
Streptococcus, including
Streptococcus sp. clone EK048,
S. sanguinis, and
S. gordonii, and
Rothia dentocariosa,
G. hemolysans,
G. adiacens,
Actinomyces sp. clone BL008, and
Abiotrophia defectiva were often detected (Fig.
8). Finally, in subgingival plaque, several species of
Streptococcus and
Gemella were often found (Fig.
9).
The number of cultivable and not-yet-cultivable species detected in each subject for each site is also shown in Fig.
1 through
9. For example, in Fig.
1, only four species (
S. mitis,
S.mitis bv. 2,
A. defectiva, and
G. hemolysans) were detected on the buccal epithelium in subject 5. For convenience, the number of bacterial species detected in each oral site for each subject is summarized in Table
1. Surprisingly, the highest number of different species was found on the tonsils in subject 1, with 28 predominant species; collectively, 57 species were detected on the tonsils (Table
1; Fig.
7). In contrast, the maxillary anterior vestibule had the lowest diversity of the bacterial flora compared with the other sites, with as few as three to nine species detected among the subjects (Table
1; Fig.
2). A common question asked is how many bacterial species are present in the oral cavity of a single individual. The last column of Table
1 indicates the number of predominant species in each healthy individual from all nine different sites; thus, the numbers ranged from a low of 34 in subject 4 to a high of 72 in subject 3.
Overall, cultivable and not-yet-cultivable species of
Gemella,
Granulicatella,
Streptococcus, and
Veillonella were commonly detected in most sites.
S. mitis was the most commonly found species in essentially all sites and subjects (Fig.
1 through
9). In one subject, 79% of the clones identified were
S. mitis (data not shown in figures). Note that in this study,
S. mitis and
Streptococcus pneumoniae have been grouped together. Some members of the mitis group share more than 99% 16S rRNA sequence similarity, although DNA-DNA similarity values for the entire chromosomes are estimated to be less than 60% (
14). Both
S. mitis and
Streptococcus oralis have been associated with bacterial endocarditis, especially in patients with prosthetic valves (
9). In addition, they are now recognized as frequent causes of infection in immunocompromised patients, particularly immediately after tissue transplants and in neutropenic cancer patients (
30).
G. adiacens, often considered an opportunistic pathogen, was also detected at all sites in the healthy oral cavity.
G. adiacens isolates have been reported from bacteremia/septicemia in patients with infective endocarditis/atheroma (
33) and in primary bacteremia in patients with neutropenic fever (
33). A high mortality rate for endocarditis by
G. adiacens has been reported (
5).
Figure
10 represents the overall summary of this study—namely, that there are emerging bacterial profiles that help define the healthy oral cavity. As already discussed, several species, such as
S. mitis and
G. adiacens, were detected in most or all oral sites, whereas several species were quite site specific. For example,
R. dentocariosa,
Actinomyces spp.,
S. sanguinis,
S.gordonii, and
A. defectiva appeared to preferentially colonize the teeth, while
S. salivarius was found mostly on the tongue dorsum. Some species appeared to have a predilection for soft tissue, e.g.,
S. sanguinis and
S. australis did not colonize the teeth or subgingival crevice.
S. intermedius preferentially colonized the subgingival plaque in most of the subjects but was not detected in most other sites. On the other hand,
Neisseria spp. were not found in subgingival plaque but were present in most other sites.
Simonsiella muelleri colonized only the hard palate. Indeed,
S. muelleri was initially isolated from the human hard palate (
32), although it has been isolated from a neonate with dental cyst and early eruption of teeth (
31). Several
Prevotella species were detected in most sites, but only in one or two subjects. For example,
P. melaninogenica and
Prevotella sp. clone BE073 were abundant in seven out of nine sites of one subject and were detected sporadically in other subjects (Fig.
10).
Prevotella sp. clone HF050 was found in the maxillary anterior vestibule of one subject, dominating the bacterial flora as 44% of the clones (Fig.
2 and
10). This clone was also found in lower proportions on the soft palate and tonsils of another subject (Fig.
6,
7, and
10).
In conclusion, there is a distinctive bacterial flora in the healthy oral cavity which is different from that of oral disease. For example, many species specifically associated with periodontal disease, such as
Porphyromonas gingivalis,
Tannerella forsythia, and
Treponema denticola, were not detected in any sites tested. In addition, the bacterial flora commonly thought to be involved in dental caries and deep dentin cavities, represented by
Streptococcus mutans,
Lactobacillus spp.,
Bifidobacterium spp., and
Atopobium spp., were not detected in supra- and subgingival plaques from clinically healthy teeth. A more detailed description of bacterial species associated with oral disease is discussed elsewhere (
17). As noted in this study on healthy subjects, some species are site specific at one or multiple sites, while other species are subject specific. As much as 60% of the species detected are not presently cultivable. Overall, there are still more species to be discovered, although the number of new species is beginning to reach saturation.
As we have previously asserted (
21), to rigorously assess the association of specific species or phylotypes with oral health or disease, it is necessary to analyze larger numbers of clinical samples for the levels of essentially all oral bacteria in well-controlled clinical studies. The bacterial complexes involved in periodontal disease as defined by Socransky et al. (
27) were based on the microbial analyses of 185 subjects representing about 13,000 plaque samples using DNA probes to 40 bacterial cultivable species in checkerboard hybridization assays. We are currently developing DNA probes for approximately 500 known bacterial species and novel phylotypes for use in similar studies. These DNA probes are being developed for use in the checkerboard hybridization assay (
3,
22,
23) and DNA microarray formats (S. K. Boches, A. M. Lee, B. J. Paster, and F. E. Dewhirst, J. Dent. Res.
83:abstract 2263, 2004). Our intent was to first establish the full bacterial diversity of the oral cavity and then to determine variation and reproducibility using the microarrays. Consequently, it will be relatively easy to compare the bacterial composition of a statistically significant number of samples to more precisely identify those species that are associated with health and with oral disease from different anatomic sites. It is necessary to first define the bacterial flora of the healthy oral cavity before we can determine the role of oral bacteria in disease.