The effects of sealing infected carious dentine below dental restorations on the phenotypic and genotypic diversity of the surviving microbiota was investigated. It was hypothesized that the microbiota would be subject to nutrient limitation or nutrient simplification, as it would no longer have access to dietary components or salivary secretion for growth. The available nutrients would be limited primarily to serum proteins passing from the pulp through the patent dentinal tubules to the infected dentine. Ten lesions were treated, and infected dentine was sealed below dental restorations for approximately 5 months. Duplicate standardized samples of infected dentine were taken at baseline and after the removal of the restorations. The baseline microbiota were composed primarily of Lactobacillus spp., Streptococcus mutans, Streptococcus parasanguinis, Actinomyces israelii, and Actinomyces gerencseriae. None of these taxa were isolated among the microbiota of the dentine samples taken after 5 months, which consisted of only Actinomyces naeslundii, Streptococcus oralis, Streptococcus intermedius, and Streptococcus mitis. The microbiota of the final sample exhibited a significantly (P < 0.001) increased ability to produce glycosidic enzymes (sialidase, ␤-N-acetylglucosaminidase, and ␤-galactosidase), which liberate sugars from glycoproteins. The genotypic diversity of S. oralis and A. naeslundii was significantly (P ‫؍‬ 0.002 and P ‫؍‬ 0.001, respectively) reduced in the final samples. There was significantly (P < 0.001) greater genotypic diversity within these taxa between the pairs of dentine samples taken at baseline than was found in the 5-month samples, indicating that the dentine was more homogenous than it was at baseline. We propose that during the interval between placement of the restorations and their removal, the available nutrient, primarily serum proteins, or the relative simplicity and homogeneity of the nutrient supply significantly affected the surviving microbiota. The surviving microbiota was less complex, based on compositional, phenotypic, and genotypic analyses, than that isolated from carious lesions which were also exposed to salivary secretions and pH perturbations.The survival of bacteria in the mouth and in the oral biofilm, dental plaque, in particular, depends on the ability of the adherent biota obtaining nutrients from their immediate environment and being resistant to fluctuating environmental acid and nutrient stresses (3, 31). Dental plaque rapidly ferments dietary carbohydrates to acids, reversibly demineralizing the underlying enamel, which may ultimately develop into a carious lesion. The fluctuating nature of these acid exposures has significant effects on the microbiota of the oral biofilm (25). Perhaps the best documented are the increased representation of yeasts, lactobacilli, and mutans streptococci in dental plaque and saliva, especially of individuals with high caries scores and xerostomia (2, 20, 21). Other investigators have studied the effects of acids on the oral flor...

The genotypic diversity of Actinomyces naeslundii genospecies 2 (424 isolates) and Streptococcus oralis (446 isolates) strains isolated from two sound approximal sites in all subjects who were either caries active (seven subjects) or caries free (seven subjects) was investigated by using the repetitive extragenic palindromic PCR. The plaque from the caries-active subjects harbored significantly greater proportions of mutans streptococci and lactobacilli and a smaller proportion of A. naeslundii organisms than the plaque sampled from the caries-free subjects. These data confirmed that the sites of the two groups of subjects were subjected to different environmental stresses, probably determined by the prevailing or fluctuating acidic pH values. We tested the hypothesis that the microfloras of the sites subjected to greater stresses (the plaque samples from the caries-active subjects) would exhibit reduced genotypic diversity since the sites would be less favorable. We found that the diversity of A. naeslundii strains did not change (2 ‫؍‬ 0.68; P ‫؍‬ 0.41) although the proportional representation of A. naeslundii was significantly reduced (P < 0.05). Conversely, the diversity of the S. oralis strains increased (2 ‫؍‬ 11.71; P ‫؍‬ 0.0006) and the proportional representation of S. oralis did not change. We propose that under these environmental conditions the diversity and number of niches within the oral biofilm that could be exploited by S. oralis increased, resulting in the increased genotypic diversity of this species. Apparently, A. naeslundii was not able to exploit the new niches since the prevailing conditions within the niches may have been deleterious and not supportive of its proliferation. These results suggest that environmental stress may modify a biofilm such that the diversity of the niches is increased and that these niches may be successfully exploited by some, but not necessarily all, members of the microbial community.Dental plaque bacteria colonize the hard tissues of the oral cavity by ecological succession (4,29,30). Early colonizers of the dentition, including Actinomyces naeslundii and Streptococcus oralis, facilitate further bacterial colonization (16, 29) and maintain biofilm integrity (23), protecting the host against colonization by extraoral pathogens (36). S. oralis is an oral commensal organism and is a member of the mitis group of viridans streptococci (41). A. naeslundii is a gram-positive pleomorphic rod which forms a significant component of commensal oral microfloras (7). A. naeslundii strains are assigned to two genospecies (1 and 2) on the basis of DNA homology (19) and may be identified by using genospecies-specific antisera (28).The ability of bacteria to survive and persist in a given environment will depend, in part, on their inherent genetic plasticity, which determines their ability to respond to fluctuating local environmental conditions or stresses (13). The presence of active carious lesions indicates that the oral cavity is subject to local stresses, including the in...

The predominant surface proteins of biofilm and planktonic Actinomyces naeslundii, a primary colonizer of the tooth surface, were examined. Seventy-nine proteins (the products of 52 genes) were identified in biofilm cells, and 30 of these, including adhesins, chaperones, and stress-response proteins, were significantly upregulated relative to planktonic cells.Actinomyces spp. are dominant dental plaque bacteria, and, with certain species of streptococci, they are early colonizers, attaching to the salivary pellicle coating the tooth surface and growing in biofilm (16,18). Actinomyces spp. therefore mediate establishment of the complex plaque community, via both their interactions with host salivary glycoproteins at the tooth surface and coaggregation with later-colonizing bacteria. Bacteria growing in biofilms are phenotypically and metabolically distinct from planktonic cells, and the transition to the biofilm phenotype is mediated following sensing of key environmental parameters (5, 6). Nutrient starvation and high cell density are two characteristics that are considered, at least in part, to define the physiological characteristics of biofilm cells, and in this respect they may be considered to resemble stationaryphase planktonic cultures (2, 9). Bacteria interact with their environment via surface-associated proteins (20), and these fulfill a range of functions, including adhesion, environmental sensing, and nutrient transport. We therefore studied the effects of biofilm growth on the proteome of surface-associated proteins of Actinomyces naeslundii genospecies 2, separating proteins by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and achieving protein identification using liquid chromatography/tandem mass spectrometry (LC-MS/MS).A. naeslundii genospecies 2, strain 4A7C, was isolated from the approximal plaque of a caries-free patient (21), cultured on fastidious anaerobe agar (LabM, Bury, Lancashire, United Kingdom) supplemented with 5% (vol/vol) horse blood, and incubated in an anaerobic atmosphere for 16 to 24 h. A clinical isolate was selected because of its biological relevance and because, unlike the type strain, it had not been subjected to long-term storage and repeated subculture. Saliva (unstimulated) was collected from healthy human volunteers, pooled, and sterilized as described by Söderling et al. (25). Sterile, polystyrene culture flasks with vented caps (175-cm 2 surface area; Sarstedt Inc., Newton, NC) were used for growth of A. naeslundii. Flasks were conditioned with saliva to coat surfaces with glycoproteins and provide attachment sites for bacteria. Excess saliva was decanted and replaced with 200 ml of brain heart infusion broth (BHI; Oxoid Ltd., Basingstoke, Hampshire, United Kingdom), which was inoculated with 5% (vol/ vol) of a mid-exponential-phase culture of A. naeslundii in BHI. Flasks were secured and incubated statically under anaerobic conditions at 37°C for 7 days to model mature plaque, by which time cells had entered stationary phase, as determined by followi...