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.

Culture-based studies have shown that Streptococcus mutans and lactobacilli are associated with root caries (RC). The purpose of the present study was to assess the bacterial diversity of RC in elderly patients by use of culture-independent molecular techniques and to determine the associations of specific bacterial species or bacterial communities with healthy and carious roots. Plaque was collected from root surfaces of 10 control subjects with no RC and from 11 subjects with RC. The bacterial 16S rRNA genes from extracted DNA were PCR amplified, cloned, and sequenced to determine species identity. From a total of 3,544 clones, 245 predominant species or phylotypes were observed, representing eight bacterial phyla. The majority (54%) of the species detected have not yet been cultivated. Species of Selenomonas and Veillonella were common in all samples. The healthy microbiota included Fusobacterium nucleatum subsp. polymorphum, Leptotrichia spp., Selenomonas noxia, Streptococcus cristatus, and Kingella oralis. Lactobacilli were absent, S. mutans was present in one, and Actinomyces spp. were present in 50% of the controls. In contrast, the microbiota of the RC subjects was dominated by Actinomyces spp., lactobacilli, S. mutans, Enterococcus faecalis, Selenomonas sp. clone CS002, Atopobium and Olsenella spp., Prevotella multisaccharivorax, Pseudoramibacter alactolyticus, and Propionibacterium sp. strain FMA5. The bacterial profiles of RC showed considerable subject-to-subject variation, indicating that the microbial communities are more complex than previously presumed. The data suggest that putative etiological agents of RC include not only S. mutans, lactobacilli, and Actinomyces but also species of Atopobium, Olsenella, Pseudoramibacter, Propionibacterium, and Selenomonas.

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BackgroundDirect microscopy, anaerobic culture and DNA–DNA hybridization have previously demonstrated an association between microorganisms and osteoradionecrosis (ORN). The purpose of our study was to use culture independent molecular techniques to detect bacteria in necrotic bone lesions of the mandible after radiation therapy.DesignBacterial DNA was extracted from eight deep medullar specimens from resected mandibles (six cases), including one patient with relapse. 16S rRNA genes were PCR amplified, cloned, transformed into Escherichia coli and sequenced to determine species identity and closest relatives.ResultsFrom the analysis of 438 clones, 59 predominant species were detected, 27% of which have not been cultivated. The predominant species detected from radionecrotic mandibles were Campylobacter gracilis, Streptococcus intermedius, Peptostreptococcus sp. oral clone FG014, uncultured bacterium clone RL178, Fusobacterium nucleatum, and Prevotella spp. The study demonstrated intersubject variability of the bacteria present in ORN. In contrast to the diverse bacterial profile detected in primary infection, only a few members of the oral indigenous flora were identified from the relapse case.ConclusionsDiverse bacterial profiles in specimens of ORN in marrow spaces of the mandible were detected by culture independent molecular techniques. To better understand the pathogenesis and to improve the therapy of the infection, detection of all members of the complex bacterial flora associated with ORN is necessary.

This review gives an overview of the bacterial diversity of cultivated and not-yet-cultivated bacterial species in oral biofilms. Examples are given from the healthy oral cavity of youngsters, adults, and the elderly; caries in primary and permanent teeth; root caries in the elderly; subgingival plaque; aggressive periodontitis; chronic periodontitis; necrotizing ulcerative periodontitis; halitosis; noma; endodontic infections; and spreading odontogenic infections. Transfer of biofilm bacteria to the blood is also discussed. Techniques used for identifying these organisms are mainly PCR, cloning, and 16S rRNA gene sequencing, as well as microarrays. As much as 50% or more of the microbiota in oral biofilms cannot yet be cultured. This may have significant implications for our knowledge of the pathogens in major biofilm infections in the oral cavity such as caries, periodontitis, peri-implantitis, and mucositis. Furthermore, several bacterial species not traditionally believed to be oral pathogens have also been shown to be associated with disease.