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-yetcultivated 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.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 abs...

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...

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347F/803R is the most suitable pair of primers for classification of foregut 16S rRNA genes but also possess universality suitable for analyses of other complex microbiomes.