Isolation and characterization of the mutans streptococci from the dental plaques in Koreans

Mutans streptococci have been implicated as cariogenic bacteria in dental caries because they can produce high levels of dental caries-causing lactic acid and extracellular polysaccharide. The aim of this study was to isolate and characterize the mutans streptococci from the dental plaque obtained from Koreans. The dental plaque samples were collected from the anterior and molar teeth of both jaws in 155 subjects (aged 2 to 33.2 years, average age 13.7+/-4.7 years). The samples were diluted by 100-fold in 1x PBS and plated on mitis-salivarius bacitracin (MSB) agar plates. The mutans streptococci grown on MSB plates were screened by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) targeting dextranase gene (dex). The mutans streptococci were identified at the species level using a 16S rDNA sequencing comparison method. The biochemical tests were carried out to biotype the mutans streptococci. Ninety-five strains of the mutans streptococci out of 358 colonies, which were derived from 141 subjects, were isolated. Of them, 77 strains and 18 strains were Streptococcus mutans and Streptococcus sobrinus, respectively. The biotyping data showed that 62, 1, 20, 10, and 2 strains were biotypes I, II, IV, V and variant, respectively. Of the two strains of variant biotype, one strains was similar to biotype IV except that it was positive to the arginine hydrolysis test. We considered this one strain a new biotype, and classified it as biotype VII. In conclusion, S. mutans and its biotype I was most frequently isolated in Korean dental plaque. The mutans streptococci strains isolated in this study might be useful for the study of the pathogenesis and the prevention of dental caries.     

Characterization of a Streptococcus sp.-Veillonella sp. community micromanipulated from dental plaque

Streptococci and veillonellae occur in mixed-species colonies during formation of early dental plaque. One factor hypothesized to be important in assembly of these initial communities is coaggregation (cell-cell recognition by genetically distinct bacteria). Intrageneric coaggregation of streptococci occurs when a lectin-like adhesin on one streptococcal species recognizes a receptor polysaccharide (RPS) on the partner species. Veillonellae also coaggregate with streptococci. These genera interact metabolically; lactic acid produced by streptococci is a carbon source for veillonellae. To transpose these interactions from undisturbed dental plaque to an experimentally tractable in vitro biofilm model, a community consisting of RPS-bearing streptococci juxtaposed with veillonellae was targeted by quantum dot-based immunofluorescence and then micromanipulated off the enamel surface and cultured. Besides the expected antibody-reactive cell types, a non-antibody-reactive streptococcus invisible during micromanipulation was obtained. The streptococci were identified as Streptococcus oralis (RPS bearing) and Streptococcus gordonii (adhesin bearing). The veillonellae could not be cultivated; however, a veillonella 16S rRNA gene sequence was amplified from the original isolation mixture, and this sequence was identical to the sequence of the previously studied organism Veillonella sp. strain PK1910, an oral isolate in our culture collection. S. oralis coaggregated with S. gordonii by an RPS-dependent mechanism, and both streptococci coaggregated with PK1910, which was used as a surrogate during in vitro community reconstruction. The streptococci and strain PK1910 formed interdigitated three-species clusters when grown as a biofilm using saliva as the nutritional source. PK1910 grew only when streptococci were present. This study confirms that RPS-mediated intrageneric coaggregation occurs in the earliest stages of plaque formation by bringing bacteria together to create a functional community.     

A comparative study of the effect of probiotics on cariogenic biofilm model for preventing dental caries

Dental caries is induced by oral biofilm containing Streptococcus mutans. Probiotic bacteria were mainly studied for effect on the gastrointestinal tract and have been known to promote human health. However, the information of probiotics for oral health has been lack yet. In this study, we investigated influence of various probiotics on oral bacteria or cariogenic biofilm and evaluated candidate probiotics for dental caries among them. The antimicrobial activity of the spent culture medium of probiotics for oral streptococci was performed. Probiotics were added during the biofilm formation with salivary bacteria including S. mutans. The oral biofilms were stained with a fluorescent dye and observed using the confocal laser scanning microscope. To count bacteria in the biofilm, the bacteria were plated on MSB and BHI agar plates after disrupting the biofilm and cultivated. Glucosyltransferases (gtfs) expression of S. mutans and integration of lactobacilli into the biofilm were evaluated by real-time RT-PCR. Among probiotics, Lactobacillus species strongly inhibited growth of oral streptococci. Moreover, Lactobacillus species strongly inhibited formation of cariogenic biofilm model. The expression of gtfs was significantly reduced by Lactobacillus rhamnosus. The integration of L. rhamnosus into the biofilm model did not exhibit. However, L. acidophilus and L casei integrated into the biofilm model. These results suggest that L. rhamnosus may inhibit oral biofilm formation by decreasing glucan production of S. mutans and antibacterial activity and did not integrate into oral biofilm, which can be a candidate for caries prevention strategy.     

Streptococcus gordonii biofilm formation: identification of genes that code for biofilm phenotypes

Viridans streptococci, which include Streptococcus gordonii, are pioneer oral bacteria that initiate dental plaque formation. Sessile bacteria in a biofilm exhibit a mode of growth that is distinct from that of planktonic bacteria. Biofilm formation of S. gordonii Challis was characterized using an in vitro biofilm formation assay on polystyrene surfaces. The same assay was used as a nonbiased method to screen isogenic mutants generated by Tn916 transposon mutagenesis for defective biofilm formation. Biofilms formed optimally when bacteria were grown in a minimal medium under anaerobic conditions. Biofilm formation was affected by changes in pH, osmolarity, and carbohydrate content of the growth media. Eighteen biofilm-defective mutants of S. gordonii Challis were identified based on Southern hybridization with a Tn916-based probe and DNA sequences of the Tn916-flanking regions. Molecular analyses of these mutants showed that some of the genes required for biofilm formation are involved in signal transduction, peptidoglycan biosynthesis, and adhesion. These characteristics are associated with quorum sensing, osmoadaptation, and adhesion functions in oral streptococci. Only nine of the biofilm-defective mutants had defects in genes of known function, suggesting that novel aspects of bacterial physiology may play a part in biofilm formation. Further identification and characterization of biofilm-associated genes will provide insight into the molecular mechanisms of biofilm formation of oral streptococci.     

Differences in microbiological composition of saliva and dental plaque in subjects with different drinking habits

Several foods have been shown to contain natural components (especially polyphenols) which display anti-adhesive properties against Streptococcus mutans, the aetiological agent responsible for dental crown caries, as well as inhibition of glucosyltransferases, which are the S. mutans enzymes involved in the synthesis of an adherent, water-insoluble glucan from sucrose. Other studies have demonstrated an in vitro action on oral plaque biofilm formation and desorption. This study evaluated whether the activity displayed in vitro by food compounds could affect the microbiological composition of saliva and dental plaque of subjects with a diet rich in these foods, comparing the results with those obtained from subjects with a different diet. The foods considered were: coffee, barley coffee, tea and wine. A total of 93 subjects were recruited into the study. Six samples of both plaque and saliva were collected from each subject at roughly one-monthly intervals. Total bacteria, total streptococci, S. mutans and lactobacilli counts were determined by culture in both saliva and dental plaque. The highest bacterial titres were recorded for the control population, while each drinking habit subgroup showed counts roughly one log lower than the controls. These differences in bacterial counts proved statistically significant (P<0.05). As far as dental plaque was concerned, while total counts did not significantly vary per mg of plaque in the subjects belonging to the different drinking habit subgroups, a significant decrease (P<0.05) was observed in those subjects drinking coffee, tea, barley coffee and wine when mutans streptococci and lactobacilli were evaluated. In several cases a more than one log decrease was observed. Plaque indices were also determined, and a significant (P<0.05) reduction in values was recorded in the subjects belonging the specific drinking habit subgroups compared to the control group. This study indicates that there is a correlation between consumption of specific foods and oral health in terms of reduced plaque deposition and lower counts of odontopathogens.     

Carbohydrate uptake in the oral pathogen Streptococcus mutans: mechanisms and regulation by protein phosphorylation

Streptococcus mutans is the primary etiological agent of dental caries in man and other animals. This organism and other related oral streptococci use carbohydrates almost exclusively as carbon and energy sources, fermenting them primarily to lactic acid which initiates erosion of tooth surfaces. Investigations over the past decade have shown that the major uptake mechanism for most carbohydrates in S. mutans is the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS), although non-PTS systems have also been identified for glucose and sucrose. Regulation of sugar uptake occurs by induction/repression and inducer exclusion mechanisms in S. mutans, but apparently not by inducer expulsion as is found in some other streptococci. In addition, ATP-dependent protein kinases have also been identified in S. mutans and other oral streptococci, and a regulatory function for at least one of these has been postulated. Among a number of proteins that are phosphorylated by these enzymes, the predominant soluble protein substrate is the general phospho-carrier protein of the PTS, HPr, as had previously been observed in a variety of Gram-positive bacteria. Recent results have provided evidence for a role for ATP-dependent phosphorylation of HPr in the coordination of sugar uptake and its catabolism in S. mutans. In this review, these results are summarized, and directions for future research in this area are discussed.     

Automated fluorescent in situ hybridization for the specific detection and quantification of oral streptococci in dental plaque

Our aim was to develop a rapid fluorescent in situ hybridization (FISH) assay for the identification of different oral groups of streptococci in dental plaque and to combine it with digital image analysis for the automated enumeration of target cells. Cy3-labeled oligonucleotide probes specific for 16S rRNA gene sequences of the anginosus, mitis, mutans, and salivarius groups of streptococci were hybridized under stringent conditions with bacterial cultures or supragingival plaque samples that had been permeabilized with lysozyme. Probe specificity was determined with strains from 30 different species, mainly of oral origin. Results showed that probes ANG541, MIT447, SSP001, and SAL090 with specificity for the anginosus, mitis, mutans, and salivarius groups, respectively, the pan-reactive streptococcal probe STR405, the S. mutans specific probe MUT590, and the S. sobrinus specific probe SOB174 were well-suited for the identification of cultured streptococci. Probes STR405, MIT447 and SSP001 were then successfully applied to enumerate automatically bacteria of the recognized taxa in 144 supragingival plaque samples. On the average, total streptococci accounted for 8.2%, streptococci of the mitis and mutans groups for 3.9 and 1.7%, respectively, of the plaques. The combined application of FISH and automated image analysis provides an objective time-saving alternative to culture or PCR for the enumeration of selected oral streptococci in dental plaque.     

Detection and cultivation of soil verrucomicrobia

Only one isolate each of the class "Spartobacteria" (subdivision 2 of the phylum Verrucomicrobia) and of subdivision 3 of Verrucomicrobia have previously been reported to grow in laboratory culture. Using media that had been used successfully in other studies to isolate members of diverse groups of soil bacteria, we generated a collection of over 1,200 isolates from soil from a pasture. An oligonucleotide probe that targets the 16S rRNA genes of verrucomicrobia was used to screen this collection, and 14 new verrucomicrobia were identified. Nine of these belonged to the class "Spartobacteria" and were related to "Chthoniobacter flavus." Five further isolates were members of subdivision 3 and were related to the only known isolate of this subdivision. The differences in the 16S rRNA gene sequences of the new isolates and previously described isolates, of up to 10%, indicated that the new isolates represent new species and genera. All but two of the verrucomicrobial isolates were from colonies that first became visible one or more months after inoculation of plates with soil, but subcultures grew more rapidly. Analysis of PCR-amplified 16S rRNA genes in the pasture soil showed that members of the class "Spartobacteria" were more numerous than members of subdivision 3. Isolates of subdivision 3 were only found on plates receiving an inoculum that yielded a mean of 29 colonies per plate, while members of the class "Spartobacteria" were only found on plates receiving a more dilute inoculum that resulted in a mean of five colonies per plate. This suggested that colony development by members of the class "Spartobacteria" was inhibited by other culturable bacteria.     

Effect of carbohydrate fatty acid esters on Streptococcus sobrinus and glucosyltransferase activity

Mutans streptococci are oral bacteria with a key role in the initiation of dental caries, because their glucosyltransferases synthesize polysaccharides from sucrose that allow them to colonize the tooth surface. Among the strategies to prevent dental caries that are being investigated are (1) the inhibition of bacterial growth of mutans streptococci or (2) the inhibition of glucosyltransferases involved in polysaccharide formation. Pure fatty acid esters of sucrose, maltose and maltotriose were synthesized by an enzyme-catalyzed process and tested as inhibitors of two glucosyltransferases of great homology, those from Streptococcus sobrinus and Leuconostoc mesenteroides NRRL B-512F. In spite of having their nonreducing end glucose blocked at 6-OH, they did not inhibit dextran synthesis. However, their effect on the growth of S. sobrinus in the solid and liquid phase was notable. 6-O-Lauroylsucrose, 6'-O-lauroylmaltose and 6"-O-lauroylmaltotriose at 100 microg/mL showed complete inhibition of S. sobrinus in agar plates. Consequently, these nontoxic derivatives are very promising for inclusion in oral-hygiene products aimed at disrupting plaque formation and preventing caries.     

Isolation of Serratia marcescens in the midgut of Rhodnius prolixus: impact on the establishment of the parasite Trypanosoma cruzi in the vector

The effects of resident bacteria in the stomach of 5th-instar larvae of Rhodnius prolixus on the erythrocyte lysis and Trypanosoma cruzi infection were studied. The bacteria population increased approximately 10,000-fold after feeding. Hemolysis rose to approximately 28% within 24h postfeeding and then linearly grew until day 4 attaining almost 100%. The number of surviving Y strain of T. cruzi in the stomach declined drastically, while the infection with Dm28c clone was maintained stable. Five days after feeding, few different types of bacterial colonies were obtained when stomach content suspensions were spread onto BHI agar plates. The hemolytic bacteria were isolated and identified as Serratia marcescens biotype A1a (referenced as RPH), which produces the pigment prodigiosin. In vitro experiments, comparing incubations of RPH with S. marcescens SM365, a prodigiosin pigment producer, and S. marcescens DB11, a nonpigment variant, as a control, with erythrocytes and T. cruzi demonstrated that: (i) at 30 degrees C, SM365 and RPH diminished the populations of Y strain, but not DM28c clone, and DB11 was unable to lyse both T. cruzi strains; (ii) at 0 degrees C, SM263 and RPH killed the flagellates, but DB11 did not; and (iii) all three strains of S. marcescens were able to lyse erythrocytes. These results suggest that S. marcescens trypanolytic activity from the SM365 and RPH strains is distinct from the hemolytic activity and that prodigiosin is an important factor for the trypanolytic action of the bacteria.     

Comparison of culture media and chairside assays for enumerating mutans streptococci

AIM: This study compared several traditional culture-based media and chairside cultural assays for ability to recover mutans streptococci (MS) from pure cultures and from saliva samples. METHODS AND RESULTS: When pure cultures were used with traditional culture-based media, mitis-salivarius bacitracin (MSB) agar demonstrated less support for bacterial recovery than trypticase-yeast extract-cysteine sucrose-bacitracin (TYCSB) agar and the modified medium of Ritz (HLR-S). One species of MS, Streptococcus ferus (c), was not recovered on MSB medium. Chairside cultural tests displayed considerable disparity between tests in recovering bacteria from pure cultures. On the glass adherence assay (Mucount), S. ferus was not detected and Streptococcus criceti was not detected on the dipslide assay (Cariescreen SM) or on the plastic adherence assay (Dentocult SM Strip mutans). The frequency of isolation of pure strains of bacteria other than MS was common. From saliva samples, the frequency of isolation of MS on HLR-S and TYCSB media and the glass adherence assay was 91-97%. The frequency of isolation on MSB medium and on the dip-slide and plastic adherence assays was significantly decreased (37, 47 and 69%, respectively). Recovery scores varied considerably among the culture methods studied and tended to be highest on the HLR-S medium and on the glass adherence assay. CONCLUSIONS: Growth and recovery profiles of pure bacterial cultures and of saliva samples for the MS varied according to different media. SIGNIFICANCE AND IMPACT OF THE STUDY: Caution should be exercised in comparing results between studies that employ different cultural methods for MS enumeration.     

Coaggregation-mediated interactions of streptococci and actinomyces detected in initial human dental plaque

Streptococci and actinomyces that initiate colonization of the tooth surface frequently coaggregate with each other as well as with other oral bacteria. These observations have led to the hypothesis that interbacterial adhesion influences spatiotemporal development of plaque. To assess the role of such interactions in oral biofilm formation in vivo, antibodies directed against bacterial surface components that mediate coaggregation interactions were used as direct immunofluorescent probes in conjunction with laser confocal microscopy to determine the distribution and spatial arrangement of bacteria within intact human plaque formed on retrievable enamel chips. In intrageneric coaggregation, streptococci such as Streptococcus gordonii DL1 recognize receptor polysaccharides (RPS) borne on other streptococci such as Streptococcus oralis 34. To define potentially interactive subsets of streptococci in the developing plaque, an antibody against RPS (anti-RPS) was used together with an antibody against S. gordonii DL1 (anti-DL1). These antibodies reacted primarily with single cells in 4-h-old plaque and with mixed-species microcolonies in 8-h-old plaque. Anti-RPS-reactive bacteria frequently formed microcolonies with anti-DL1-reactive bacteria and with other bacteria distinguished by general nucleic acid stains. In intergeneric coaggregation between streptococci and actinomyces, type 2 fimbriae of actinomyces recognize RPS on the streptococci. Cells reactive with antibody against type 2 fimbriae of Actinomyces naeslundii T14V (anti-type-2) were much less frequent than either subset of streptococci. However, bacteria reactive with anti-type-2 were seen in intimate association with anti-RPS-reactive cells. These results are the first direct demonstration of coaggregation-mediated interactions during initial plaque accumulation in vivo. Further, these results demonstrate the spatiotemporal development and prevalence of mixed-species communities in early dental plaque.     

Toward organism identification through analysis of bacterial colonies.

The clinical problem of bacterial identification has been approached by applying pattern-recognition techniques to multi-wavelength surface-scattering and reflectance data derived from real-time scans of isolated colonies. Preliminary results, obtained from blood-agar plates inoculated with a mixture of staphylococci, streptococci and escherichieae, indicate that these organisms can be differentiated with better than 90% certainty, provided the colonies are physically separated and their growth conditions closely controlled. Data collection and classification characteristics of the experimental system are briefly described; it is felt that the technique, possibly expanded to include boundary characteristics of the colonies, may offer a viable means of identifying clinically important bacteria.     

Multiplex PCR screening of soil isolates for novel Bacillus-related lineages

A16S rDNA multiplex PCR-based high-throughput protocol is presented to screen bacterial isolates in large amounts for the appearance of novel lineages of bacteria, especially hitherto unknown Bacillus relatives. The 16S rDNAs of 4224 isolates from a comprehensive cultivation campaign were screened for similarity to predominant uncultured soil bacteria. Soil suspensions were plated in serial dilutions on various media. After 2, 4 and 6 weeks, colonies were collected with toothpicks and transferred to microtiterplates for cell lysis and storage plates for subculture. Cell lysis was a simple freeze-heating cycle in distilled water. The multiplex PCR was adapted to operate sufficiently for Gram positives under these conditions. Approximately 10.6% of all picked colonies reacted with a primer targeting a Bacillus fraction containing novel Bacillus benzoevorans-relatives previously detected as predominant soil bacteria by culture-independent studies. From these 446 colonies detected by multiplex PCR, 363 (81.4%) could be successfully used for continued subculture and 16S rDNA sequencing. All identification was done by 16S rDNA sequencing. This revealed that more than 60% of them represented a variety of candidates for potentially new species. Twelve colonies were identified as almost identical matches to 16S rDNA sequences of hitherto uncultured but apparently predominant soil bacteria cloned from directly extracted soil DNA. Also, novel lineages of unpredicted phylogenetic diversity like novel Paenibacillus, Sporosarcina and even Xanthomonads were represented.     

Glucanase-producing organisms in human dental plaques.

Selective media were used to isolate a wide range of bacteria from sixty human dental plaques. Glucanase activities of the isolates were determined on dextran- and starch-containing media. All sixty samples of dental plaque yielded some colonies showing amylolytic and dextranolytic activities. The glucanase-producing organisms comprised 20% of the isolates. Of these 38% were Gram-positive rods, 27% Gram-positive cocci, 28% Gram-negative rods and 7% were Gram-negative cocci. The cultural groups most commonly represented among the glucanase-producing isolates were Actinomycetaceae, streptococci, haemophili and Gram-negative anaerobes. Species prominent among these isolates included Streptococcus sanguis, Streptococcus mitior, Actinomyces naeslundii, Actinomyces viscosus, Bacterionema matruchotii, Bifidobacterium sp. and Bacteroides sp. No isolates capable of degrading starch or dextran were identified as Streptococcus milleri, Rothia dentocariosa or Fusobacterium sp. This study has shown that a wide range of bacterial species commonly isolated from human dental plaques exhibit both amylolytic and dextranolytic activities. In order to understand glucan metabolism in human dental plaques further investigation of these catabolic activities is necessary.     

Immunochemical study of nutritionally variant streptococci

Nutritionally variant streptococci (NVS) have been characterized by their growth as satellite colonies around colonies of staphylococci or several other gram-positive or gram-negative bacterial strains. The majority of the NVS strains were isolated from patients with subacute bacterial endocarditis. Organisms identified as NVS were subdivided into three serotypes by rocket-line electrophoresis and hemagglutination inhibition assays. Ninety-nine of 103 strains expressed one or more of the three serotype antigens; however, a group antigen was not demonstrated in the various extracts of these streptococci. Surface protein studies confirmed the NVS differentiation into serotypes. Serotype I organisms expressed surface protein(s) specific for the serotype, whereas the serotype II and III NVS demonstrated common protein(s) on their surface. Furthermore, SDS extraction released a greater amount of radioiodinatable surface protein from serotypes I and III bacteria than serotype II. Finally, there was no correlation between the serotype or the disease of the patients from which the NVS strains were isolated.     

Streptococcus mutans and oral streptococci in dental plaque

The human oral microbial biota represents a highly diverse biofilm. Twenty-five species of oral streptococci inhabit the human oral cavity and represent about 20 % of the total oral bacteria. Taxonomy of these bacteria is complex and remains provisional. Oral streptococci encompass friends and foes bacteria. Each species has developed specific properties for colonizing the different oral sites subjected to constantly changing conditions, for competing against competitors, and for resisting external agressions (host immune system, physico-chemical shocks, and mechanical frictions). Imbalance in the indigenous microbial biota generates oral diseases, and under proper conditions, commensal streptococci can switch to opportunistic pathogens that initiate disease in and damage to the host. The group of "mutans streptococci" was described as the most important bacteria related to the formation of dental caries. Streptococcus mutans, although naturally present among the human oral microbiota, is the microbial species most strongly associated with carious lesions. This minireview describes the oral streptococci ecology and their biofilm life style by focusing on the mutans group, mainly S. mutans. Virulence traits, interactions in the biofilm, and influence of S. mutans in dental caries etiology are discussed.     

Acidobacteria, Rubrobacteridae and Chloroflexi are abundant among very slow-growing and mini-colony-forming soil bacteria

Easily visible colonies of bacteria continued to form on plates inoculated with soil and incubated for 24 weeks. Using two different media, 13% and 29% of easily visible colonies appeared after more than 12 weeks. In addition, 10% and 18% of all colonies had diameters of 25-200 μm (mini-colonies), which could not be readily seen with the unaided eye. Members of soil bacterial groups that are only rarely cultured, such as members of the subclass Rubrobacteridae of the phylum Actinobacteria, members of subdivisions 1 and 2 of the phylum Acidobacteria and members of three subphyla of the phylum Chloroflexi, were more abundant among the easily visible colonies and mini-colonies that developed after > 12 weeks of incubation. Our results indicate that there is a hidden culturable diversity of soil bacteria that may require laboratory study at colony sizes and incubation periods outside those commonly anticipated by most microbiologists. Working at these scales increases the likelihood of obtaining cultures from groups of soil bacteria that have generally eluded laboratory study by cultivation methods.     

Cultivation of a Synergistetes strain representing a previously uncultivated lineage

Subgingival plaque samples obtained from human subjects with periodontitis, shown to include previously uncultivable members of the phylum Synergistetes, were used to inoculate Cooked Meat Medium (CMM). The presence of Cluster A (uncultivable) Synergistetes was monitored by fluorescent in situ hybridization (FISH) and quantitative PCR (Q‐PCR). Cluster A Synergistetes were found to grow in CMM in co‐culture with other plaque bacteria and growth was stimulated by the addition of mucin and serum. Plaque samples were also used to inoculate Blood Agar (BA) plates and growth of Cluster A Synergistetes was revealed after anaerobic incubation, by colony hybridization with specific probes. Surface growth on the plates in regions identified by colony hybridization was harvested and used to inoculate fresh plates, thus enriching for Cluster A Synergistetes. Cross‐streaks of other plaque bacteria were also used to stimulate Synergistetes growth. In the early passages, no discrete Synergistetes colonies were seen, but after eight passages and the use of cross‐streaks of other bacteria present in the enriched community, colonies arose, which consisted solely of Cluster A Synergistetes cells, as determined by 16S rRNA gene PCR and cloning. This is the first report of the successful culture of a member of the uncultivable branch of this phylum.     

First isolation of Streptococcus downei from human dental plaques

In this study, we isolated four bacterial strains grown on mitis-salivarius sucrose bacitracin agar. The strains had similar biochemical characteristics to biotypes I or II of mutans streptococci. The four isolates were identified as Streptococcus downei by 16S rDNA and dextranase gene (dex) sequencing as well as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) targeting dex. To our knowledge, this is the first report of the isolation and identification of S. downei from dental plaque in humans. The results suggest that S. downei can inhabit the human oral cavity.