Re-evaluation of the taxonomy of the Mitis group of the genus Streptococcus based on whole genome phylogenetic analyses, and proposed reclassification of Streptococcus dentisani as Streptococcus oralis subsp. dentisani comb. nov., Streptococcus tigurinus as Streptococcus oralis subsp. tigurinus comb. nov., and Streptococcus oligofermentans as a later synonym of Streptococcus cristatus.

The Mitis group of the genus Streptococcus currently comprises 20 species with validly published names, including the pathogen S. pneumoniae. They have been the subject of much taxonomic confusion, due to phenotypic overlap and genetic heterogeneity, which has hampered a full appreciation of their clinical significance. The purpose of this study was to critically re-examine the taxonomy of the Mitis group using 195 publicly available genomes, including designated type strains for phylogenetic analyses based on core genomes, multilocus sequences and 16S rRNA gene sequences, combined with estimates of average nucleotide identity (ANI) and in silico and in vitro analyses of specific phenotypic characteristics. Our core genomic phylogenetic analyses revealed distinct clades that, to some extent, and from the clustering of type strains represent known species. However, many of the genomes have been incorrectly identified adding to the current confusion. Furthermore, our data show that 16S rRNA gene sequences and ANI are unsuitable for identifying and circumscribing new species of the Mitis group of the genus Streptococci. Based on the clustering patterns resulting from core genome phylogenetic analysis, we conclude that S. oligofermentans is a later synonym of S. cristatus. The recently described strains of the species Streptococcus dentisani includes one previously referred to as 'S. mitis biovar 2'. Together with S. oralis, S. dentisani and S. tigurinus form subclusters within a coherent phylogenetic clade. We propose that the species S. oralis consists of three subspecies: S. oralis subsp. oralis subsp. nov., S. oralis subsp. tigurinus comb. nov., and S. oralis subsp. dentisani comb. nov.

Arthritis-induced alveolar bone loss is associated with changes in the composition of oral microbiota.

Rheumatoid arthritis (RA) and periodontitis (PD) are chronic inflammatory disorders that cause bone loss. PD tends to be more prevalent and severe in RA patients. Previous experimental studies demonstrated that RA triggers alveolar bone loss similarly to PD. The aim of this study was to investigate if arthritis-induced alveolar bone loss is associated with modification in the oral microbiota. Checkerboard DNA-DNA hybridization was employed to analyze forty oral bacterial species in 3 groups of C57BL/6 mice: control (n = 12; without any challenge); Y4 (n = 8; received oral inoculation of Aggregatibacter Actinomycetemcomitans strain FDC Y4) and AIA group (n = 12; chronic antigen-induced arthritis). The results showed that AIA and Y4 group exhibited similar patterns of bone loss. The AIA group exhibited higher counts of most bacterial species analyzed with predominance of Gram-negative species similarly to infection-induced PD. Prevotella nigrescens and Treponema denticola were detected only in the Y4 group whereas Campylobacter showae, Streptococcus mitis and Streptococcus oralis were only found in the AIA group. Counts of Parvimonas micra, Selenomonas Noxia and Veillonella parvula were greater in the AIA group whereas Actinomyces viscosus and Neisseira mucosa were in large proportion in Y4 group. In conclusion, AIA is associated with changes in the composition of the oral microbiota, which might account for the alveolar bone loss observed in AIA mice.

Bacterial diversity and functional analysis of severe early childhood caries and recurrence in India.

Dental caries is the most prevalent oral disease affecting nearly 70% of children in India and elsewhere. Micro-ecological niche based acidification due to dysbiosis in oral microbiome are crucial for caries onset and progression. Here we report the tooth bacteriome diversity compared in Indian children with caries free (CF), severe early childhood caries (SC) and recurrent caries (RC). High quality V3-V4 amplicon sequencing revealed that SC exhibited high bacterial diversity with unique combination and interrelationship. Gracillibacteria_GN02 and TM7 were unique in CF and SC respectively, while Bacteroidetes, Fusobacteria were significantly high in RC. Interestingly, we found Streptococcus oralis subsp. tigurinus clade 071 in all groups with significant abundance in SC and RC. Positive correlation between low and high abundant bacteria as well as with TCS, PTS and ABC transporters were seen from co-occurrence network analysis. This could lead to persistence of SC niche resulting in RC. Comparative in vitro assessment of biofilm formation showed that the standard culture of S. oralis and its phylogenetically similar clinical isolates showed profound biofilm formation and augmented the growth and enhanced biofilm formation in S. mutans in both dual and multispecies cultures.

Rapid and Accurate Species Identification of Mitis Group Streptococci Using the MinION Nanopore Sequencer.

Differentiation between mitis group streptococci (MGS) bacteria in routine laboratory tests has become important for obtaining accurate epidemiological information on the characteristics of MGS and understanding their clinical significance. The most reliable method of MGS species identification is multilocus sequence analysis (MLSA) with seven house-keeping genes; however, because this method is time-consuming, it is deemed unsuitable for use in most clinical laboratories. In this study, we established a scheme for identifying 12 species of MGS (S. pneumoniae, S. pseudopneumoniae, S. mitis, S. oralis, S. peroris, S. infantis, S. australis, S. parasanguinis, S. sinensis, S. sanguinis, S. gordonii, and S. cristatus) using the MinION nanopore sequencer (Oxford Nanopore Technologies, Oxford, UK) with the taxonomic aligner "What's in My Pot?" (WIMP; Oxford Nanopore's cloud-based analysis platform) and Kraken2 pipeline with the custom database adjusted for MGS species identification. The identities of the species in reference genomes (n = 514), clinical isolates (n = 31), and reference strains (n = 4) were confirmed via MLSA. The nanopore simulation reads were generated from reference genomes, and the optimal cut-off values for MGS species identification were determined. For 31 clinical isolates (S. pneumoniae = 8, S. mitis = 17 and S. oralis = 6) and 4 reference strains (S. pneumoniae = 1, S. mitis = 1, S. oralis = 1, and S. pseudopneumoniae = 1), a sequence library was constructed via a Rapid Barcoding Sequencing Kit for multiplex and real-time MinION sequencing. The optimal cut-off values for the identification of MGS species for analysis by WIMP and Kraken2 pipeline were determined. The workflow using Kraken2 pipeline with a custom database identified all 12 species of MGS, and WIMP identified 8 MGS bacteria except S. infantis, S. australis, S. peroris, and S. sinensis. The results obtained by MinION with WIMP and Kraken2 pipeline were consistent with the MGS species identified by MLSA analysis. The practical advantage of whole genome analysis using the MinION nanopore sequencer is that it can aid in MGS surveillance. We concluded that MinION sequencing with the taxonomic aligner enables accurate MGS species identification and could contribute to further epidemiological surveys.

Comparative structural and molecular characterization of ribitol-5-phosphate-containing Streptococcus oralis coaggregation receptor polysaccharides.

The antigenically related coaggregation receptor polysaccharides (RPS) of Streptococcus oralis strains C104 and SK144 mediate recognition of these bacteria by other members of the dental plaque biofilm community. In the present study, the structure of strain SK144 RPS was established by high resolution NMR spectroscopy as [6Galfbeta1-6GalNAcbeta1-3Galalpha1-2ribitol-5-PO(4)(-)-6Galfbeta1-3Galbeta1](n), thereby indicating that this polysaccharide and the previously characterized RPS of strain C104 are identical, except for the linkage between Gal and ribitol-5-phosphate, which is alpha1-2 in strain SK144 versus alpha1-1 in strain C104. Studies to define the molecular basis of RPS structure revealed comparable genes for six putative transferases and a polymerase in the rps loci of these streptococci. Cell surface RPS production was abolished by disrupting the gene for the first transferase of strain C104 with a nonpolar erm cassette. It was restored in the resulting mutant by plasmid-based expression of either wcjG, the corresponding gene of S. pneumoniae for serotype 10A capsular polysaccharide (CPS) biosynthesis or wbaP for the transferase of Salmonella enterica that initiates O-polysaccharide biosynthesis. Thus, WcjG, like WbaP, appears to initiate polysaccharide biosynthesis by transferring galactose-1-phosphate to a lipid carrier. In further studies, the structure of strain C104 RPS was converted to that of strain SK144 by replacing the gene (wefM) for the fourth transferase in the rps locus of strain C104 with the corresponding gene (wcrC) of strain SK144 or Streptococcus pneumoniae serotype 10A. These findings identify genetic markers for the different ribitol-5-phosphate-containing types of RPS present in S. oralis and establish a close relationship between these polysaccharides and serogroup 10 CPSs of S. pneumoniae.

Isolation and Bacteriocin-Related Typing of Streptococcus dentisani.

Streptococcus oralis subspecies dentisani is explored as an anti-cariogenic probiotic. Here, subjecting freshly stimulated saliva samples of 35 healthy volunteers, six epidemiologically unrelated and two related strains were isolated (prevalence around 20%) applying a newly developed three-step procedure. Furthermore, the probiotic strain S. dentisani 7746 (AB-Dentisanium®) was tested under a variety of environmental conditions for its inhibitory effect on six S. mutans, two S. sobrinus, 15 other oral or intestinal streptococci, 15 S. dentisani strains, and six representatives of other species including periodontopathogens. All except one of the S. mutans strains were inhibited by 7746 colonies or culture supernatant concentrate but only if either the test cell number was low or the producer or its bacteriocin concentration, respectively, was high. S. sanguinis OMI 332, S. salivarius OMI 315, S. parasanguinis OMI 335, S. vestibularis OMI 238, and the intestinal S. dysgalactiae OMI 339 were not inhibited, while the other 10 streptococcal strains (especially S. oralis OMI 334 and intestinal S. gallolyticus OMI 326) showed a certain degree of inhibition. From the panel of other bacterial species only Aggregatibacter actinomycetemcomitans was slightly inhibited. With the exception of OMI 285 and OMI 291 that possessed a 7746 bacteriocin-like gene cluster, all S. dentisani strains and especially type strain 7747T were strongly inhibited by 7746. In conclusion, probiotic strain 7746 might antagonize the initiation and progression of dental caries by reducing S. mutans if not too abundant. S. dentisani strains inhibit each other, but strains with similar bacteriocin-related gene clusters, including immunity genes, are able to co-exist due to cross-resistance. In addition, development of resistance and adaptation to 7746-bacteriocins was observed during our study and needs attention. Hence, mechanisms underlying such processes need to be further investigated using omics-approaches. On the manufacturing level, probiotic strains should be continuously tested for function. Further clinical studies investigating inhibition of S. mutans by AB-Dentisanium® are required that should also monitor the impact on the oral microbiome composition including resident S. dentisani strains.

Use of MALDI Biotyper plus ClinProTools mass spectra analysis for correct identification of Streptococcus pneumoniae and Streptococcus mitis/oralis.

BACKGROUND: Differentiation of Streptococcus pneumoniae from other viridans group streptococci is well known to be challenging in clinical laboratories. Matrix assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) had been reported to be a good alternative for Streptococcus species level identification. However, differentiation of S. pneumoniae from other Streptococcus mitis group organisms was found to be problematic using the Bruker MALDI Biotyper system. METHODS: This study used the Bruker MALDI Biotyper system in addition to a mass spectra model analysis generated by 10 reference strains of S. pneumoniae, 8 strains of S. mitis and 2 strains of S. oralis in the ClinProTools to identify 28 clinical isolates of S. pneumoniae and 47 isolates of S. mitis/oralis. The results were compared with those generated by the MALDI Biotyper system alone. RESULTS: The percentages of correct species level identification using the MALDI Biotyper system alone and the direct transfer and extraction method were 66.7% (50/75) and 70.7% (53/75), respectively. With the additional ClinProTools mass spectra analysis, the percentages of correct identification by the direct transfer and extraction method increased to 85.3% (64/75) and 100% (75/75), respectively. This new workflow significantly improved the accuracy of S. pneumoniae and S. mitis/oralis identification. CONCLUSIONS: The additional ClinProTools mass spectra analysis with extraction method after MALDI Biotyper identification significantly improved the accuracy of identification among S. pneumoniae, S. oralis and S. mitis. The extra 15 min processing time of spectra analysis should be affordable in most clinical laboratories. We suggest that the same approach could be further explored in handling other bacterial species with high similarities.

Identification of proteins in Streptococcus pneumoniae by reverse vaccinology and genetic diversity of these proteins in clinical isolates.

Streptococcus pneumoniae is a major cause of morbidity and mortality worldwide. Virulence-associated proteins common and conserved among all capsular types now represent the best strategy to combat pneumococcal infections. Our aim was to identify conserved targets in pneumococci that showed positive prediction for lipoprotein and extracellular subcellular location using bioinformatics programs and verify the distribution and the degree of conservation of these targets in pneumococci. These targets can be considered potential vaccine candidate to be evaluated in the future. A set of 13 targets were analyzed and confirmed the presence in all pneumococci tested. These 13 genes were highly conserved showing around >96 % of amino acid and nucleotide identity, but they were also present and show high identity in the closely related species Streptococcus mitis, Streptococcus oralis, and Streptococcus pseudopneumoniae. S. oralis clusters away from S. pneumoniae, while S. pseudopneumoniae and S. mitis cluster closer. The divergence between the selected targets was too small to be observed consistently in phylogenetic groups between the analyzed genomes of S. pneumoniae. The proteins analyzed fulfill two of the initial criteria of a vaccine candidate: targets are present in a variety of different pneumococci strains including different serotypes and are conserved among the samples evaluated.

Origins of Staphylococcus epidermidis and Streptococcus oralis causing bacteraemia in a bone marrow transplant patient.

Coagulase-negative staphylococcal bacteraemia in immunocompromised patients is often associated with the use of central venous catheters, while the proposed origin of viridans streptococci causing bacteraemia in this patient group is the oral cavity. This report describes an episode of polymicrobial bacteraemia caused by Staphylococcus epidermidis and Streptococcus oralis followed by several further episodes of S. epidermidis bacteraemia in a 15-year-old boy after bone marrow transplantation. Pulsed-field gel electrophoresis (PFGE) of SmaI chromosomal DNA digests was used to compare blood culture and oral isolates of S. epidermidis and Str. oralis. The results indicated that the mouth was the source of both S. epidermidis and Str. oralis causing the first episode of bacteraemia. PFGE further demonstrated that the central venous catheter was the origin of a second strain of S. epidermidis responsible for subsequent episodes of staphylococcal bacteraemia. Both the oral mucosa and central venous lines should be considered as potential sources of organisms, including coagulase-negative staphylococci, associated with bacteraemia in immunocompromised patients.

[A new genus of oral bacteria in human].

A strain, No. 90-1, is isolated from the oral cavity of a patient with periodontophthy. This strain is a Gram-positive, non-endospore-forming, facultative anaerobe with spherical cells, 0.9-1.5 microns in diameter, occuring in pairs and seldom in short chains of four cells, and motile by one flagellum per cell. The optimum growing temperature is 35-37 degrees C; appreciable growth is not found below 10 degrees C, but growth at 53 degrees and tolerance to 60 degrees C for 30 min. This strain is microhalophilous and grows best, well and poorly in the medium containing 2%, 10%-15% and 25% NaCl respectively. Catalase and urease are positive and nitrate is reduced. Acid is produced from many carbohydrates, but no gas. Gelatin can be hydrolyzed, but starch, cellulose and dextrin do not. G + C content in DNA is 41.34 mol%(Tm). The strain(90-1) is considered to be a new species belonging to a new genus because its some characteristics are different from those of the known coccus genuera and designated as Stomatostreptococcus microhalophilus Ping, Zhou, Sun et Fan gen. nov. sp. nov. according to its source and microhalophilic trait.

Using high throughput sequencing to explore the biodiversity in oral bacterial communities.

High throughput sequencing of 16S ribosomal RNA gene amplicons is a cost-effective method for characterization of oral bacterial communities. However, before undertaking large-scale studies, it is necessary to understand the technique-associated limitations and intrinsic variability of the oral ecosystem. In this work we evaluated bias in species representation using an in vitro-assembled mock community of oral bacteria. We then characterized the bacterial communities in saliva and buccal mucosa of five healthy subjects to investigate the power of high throughput sequencing in revealing their diversity and biogeography patterns. Mock community analysis showed primer and DNA isolation biases and an overestimation of diversity that was reduced after eliminating singleton operational taxonomic units (OTUs). Sequencing of salivary and mucosal communities found a total of 455 OTUs (0.3% dissimilarity) with only 78 of these present in all subjects. We demonstrate that this variability was partly the result of incomplete richness coverage even at great sequencing depths, and so comparing communities by their structure was more effective than comparisons based solely on membership. With respect to oral biogeography, we found inter-subject variability in community structure was lower than site differences between salivary and mucosal communities within subjects. These differences were evident at very low sequencing depths and were mostly caused by the abundance of Streptococcus mitis and Gemella haemolysans in mucosa. In summary, we present an experimental and data analysis framework that will facilitate design and interpretation of pyrosequencing-based studies. Despite challenges associated with this technique, we demonstrate its power for evaluation of oral diversity and biogeography patterns.

The rgg gene is a specific marker for Streptococcus oralis.

Although the pathogenesis of Streptococcus oralis may be different from that of other viridans group streptococci, S. oralis shares a high degree of DNA sequence similarity with these streptococci. As a result, discrimination of S. oralis from its close relatives has long been considered difficult. This study was conducted to find specific genes that allow for the in vitro identification of S. oralis, but not other oral commensals. Four hundred ninety S. oralis clones obtained by suppressive subtractive hybridization were used for Southern hybridization, and positive clones were sequenced. Of 5 S. oralis-specific clones, newly designed primer sets based on the glucosyltransferase regulatory gene amplified genomic DNA only from S. oralis strains, but not from any of the other 125 strains tested. Our findings may be useful for the future development of efficient diagnostic tools for the rapid identification and differentiation of S. oralis from other oral streptococci strains.

Molecular and antigenic characterization of a Streptococcus oralis coaggregation receptor polysaccharide by carbohydrate engineering in Streptococcus gordonii.

The coaggregation receptor polysaccharides (RPS) of Streptococcus oralis and related species are recognized by lectin-like adhesins on other members of the oral biofilm community and by RPS-specific antibodies. The former interactions involve beta-GalNAc or beta-Gal containing host-like motifs in the oligosaccharide repeating units of these polysaccharides, whereas the latter involves features of these molecules that are immunogenic. In the present investigation, the molecular and corresponding structural basis for the serotype specificity of S. oralis ATCC 10557 RPS was determined by engineering the production of this polysaccharide in transformable Streptococcus gordonii 38. This involved the systematic replacement of genes in the rps cluster of strain 38 with different but related genes from S. oralis 10557 and structural characterization of the resulting polysaccharides. The results identify four unique genes in the rps cluster of strain 10557. These include wefI for an alpha-Gal transferase, wefJ for a GalNAc-1-phosphotransferase that has a unique acceptor specificity, wefK for an acetyl transferase that acts at two positions in the hexasaccharide repeating unit, and a novel wzy associated with the beta1-3 linkage between these units. The serotype specificity of engineered polysaccharides correlated with the wefI-dependent presence of alpha-Gal in these molecules rather than with partial O-acetylation or with the linkage between repeating units. The findings illustrate a direct approach for defining the molecular basis of polysaccharide structure and antigenicity.

Fluoroquinolone resistance in atypical pneumococci and oral streptococci: evidence of horizontal gene transfer of fluoroquinolone resistance determinants from Streptococcus pneumoniae.

Atypical strains, presumed to be pneumococcus, with ciprofloxacin MICs of > or =4.0 microg/ml and unique sequence variations within the quinolone resistance-determining regions (QRDRs) of the gyrase and topoisomerase genes in comparison with the Streptococcus pneumoniae R6 strain, were examined. These strains were reidentified using phenotypic methods, including detection of optochin susceptibility, bile solubility, and agglutination by serotype-specific antisera, and genotypic methods, including detection of pneumolysin and autolysin genes by PCR, 16S rRNA sequencing, and multilocus sequence typing (MLST). The analysis based on concatenated sequences of the six MLST loci distinguished the "atypical" strains from pneumococci, and these strains clustered closely with S. mitis. However, all these strains and five of nine strains from the viridans streptococcal group possessed one to three gyrA, gyrB, parC, and parE genes whose QRDR sequences clustered with those of S. pneumoniae, providing evidence of horizontal transfer of the QRDRs of the gyrase and topoisomerase genes from pneumococci into viridans streptococci. These genes also conferred fluoroquinolone resistance to viridans streptococci. In addition, the fluoroquinolone resistance determinants of 32 well-characterized Streptococcus mitis and Streptococcus oralis strains from bacteremic patients were also compared. These strains have unique amino acid substitutions in GyrA and ParC that were distinguishable from those in fluoroquinolone-resistant pneumococci and the "atypical" isolates. Both recombinational events and de novo mutations play an important role in the development of fluoroquinolone resistance.

Differentiation of mutans streptococci by intact cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

It is difficult to distinguish mutans streptococci on the species level, and even more so on the subspecies level. Intact cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) (ICM) was applied to reference strains of five of the species of the mutans group (Streptococcus criceti, Streptococcus downei, Streptococcus mutans, Streptococcus ratti, Streptococcus sobrinus), nonmutans streptococci (Streptococcus oralis, Streptococcus mitis, Streptococcus salivarius, and Streptococcus sanguinis), and 177 mutans streptococci isolated from saliva of 10 children. From the analysis of the reference strains, readily distinguishable ICM mass spectra were obtained for the different species. Based on multivariate statistical analysis, a correct and unambiguous assignment was made of the spectra of 159 isolated mutans streptococci to S. mutans and 16 isolates to S. sobrinus. Two isolates were sorted out and were identified by sequencing of their 16S rRNA genes as Streptococcus anginosus. In addition, ICM indicated a misclassification for some reference strains (AHT, V 100 and E 49) and re-classified AHT and E 49 as S. ratti and V 100 as S. sobrinus. This was confirmed by 16S rDNA sequencing. Based on a statistical similarity analysis of the spectra of reference strains and a quantitative assessment of the reproducibility of ICM, the isolates identified as either S. mutans or S. sobrinus were phenotyped on the subspecies level. In the population of the clinical isolates, 14 unambiguously different S. mutans and three different S. sobrinus phenotypes were detected. ICM proved to be a powerful tool for a differentiation of mutans streptococci down to the subspecies level.

Discrimination of Streptococcus pneumoniae from viridans group streptococci by genomic subtractive hybridization.

Two oligonucleotide primer sets for the discrimination of Streptococcus pneumoniae from "pneumococcus-like" oral streptococcal isolates by PCR were developed. Genomic subtractive hybridization was performed to search for differences between Streptococcus pneumoniae strain WU2 and the most closely related oral streptococcus, Streptococcus mitis strain 903. We identified 19 clones that contained S. pneumoniae-specific nucleotide fragments that were absent from the chromosomal DNA of typical laboratory strains of S. mitis and other oral bacteria. Subsequently, oligonucleotide PCR primers for the detection of S. pneumoniae were designed from the sequences of the subtracted DNA fragments, and the specificities of the 19 primer sets were evaluated by PCR using chromosomal DNAs extracted from four S. pneumoniae clinical isolates and from 20 atypical organisms classified as S. mitis or S. oralis, which harbored genes encoding the pneumococcal virulence factors autolysin (lytA) or pneumolysin (ply), as templates. Of the 19 primer sets, two (Spn9802 and Spn9828) did not amplify PCR products from any of the pneumococcus-like streptococcal strains that we examined. The genes containing the Spn9802 and Spn9828 sequences encoded proteins of unknown function that did not correspond to any previously described proteins in other bacteria. These new oligonucleotide primers may be very useful for early and correct diagnosis of S. pneumoniae infections.

Genotypic heterogeneity of Streptococcus oralis and distinct aciduric subpopulations in human dental plaque.

The genotypic heterogeneity of Streptococcus oralis isolated from the oral cavity was investigated using repetitive extragenic palindromic PCR. Unrelated subjects harbored unique genotypes, with numerous genotypes being isolated from an individual. S. oralis is the predominant aciduric bacterium isolated from noncarious tooth sites. Genotypic comparison of the aciduric populations isolated at pH 5.2 with those isolated from mitis-salivarius agar (MSA) (pH 7.0) indicated that the aciduric populations were genotypically distinct in the majority of subjects (chi(2) = 13.09; P = 0.0031). Neither the aciduric nor the MSA-isolated strains were stable, with no strains isolated at baseline being isolated 4 or 12 weeks later in the majority of subjects. The basis of this instability is unknown but is similar to that reported for Streptococcus mitis. Examination of S. oralis strains isolated from cohabiting couples demonstrated that in three of five couples, genotypically identical strains were isolated from both partners and this was confirmed by using Salmonella enteritidis repetitive element PCR and enterobacterial PCR typing. These data provide further evidence of the physiological and genotypic heterogeneity of non-mutans streptococci. The demonstration of distinct aciduric populations of S. oralis implies that the role of these and other non-mutans streptococci in the caries process requires reevaluation.

Identification of oral mitis group streptococci by arbitrarily primed polymerase chain reaction.

"Mitis group" streptococci are commensal but may play some role in dental caries, septicemia or endocarditis. Rapid genotypic identification would aid studies of dental plaque ecology, or diagnostic use. AP-PCR with 58 unpaired arbitrary primers was used to characterize 7 Streptococcus gordonii, 11 Streptococcus sanguis, 2 Streptococcus crista, 5 Streptococcus parasanguis, 18 Streptococcus oralis, and 36 Streptococcus mitis (22 biovar 1 and 14 biovar 2). S. parasanguis 16S rRNA variable region primer RR2 produced species-specific bands with all S. gordonii and S. sanguis. Human V beta 1 T-cell receptor primer 434 yielded concordant genotypic identification of all phenotypically defined S. crista and S. parasanguis, 83% of S. oralis, and 74% of S. mitis biovar 1. Amplicon patterns for S. mitis biovar 2 were heterogeneous. Findings suggest that primers RR2 and 434 in succession will allow rapid identification of genotypic groups corresponding closely to mitis group species established by phenotype.

Similarity of bacteriocin activity profiles of mutans streptococci within the family when the children acquire the strains after the age of 5.

It has been shown that there is a window of infectivity for mutans streptococci between the ages of 19 and 31 months, when many children acquire mutans streptococci transmitted from their mothers. Part of the children that escape this window acquire mutans streptococci at a later age. In this group, maternal transmission is expected to be less prevalent. The present study compared the bacteriocin activity profiles of mutans streptococci isolated from mothers, fathers and children when the children acquired the mutans streptococci between the ages of 5 and 11. Twelve families were randomly selected from a group of 11-year-old children who were known to have acquired mutans streptococci during this age period. From the saliva of the mothers (n = 12), fathers (n = 8) and children (n = 12) approximately 30 mutans streptococci strains were isolated. All isolates were tested twice for bacteriocin activity against 21 indicator strains with a double-layer technique. Bacteriocin activity of strains was considered to be different when the number of strains against which bacteriocin was produced differed >1 or when the width of one or more inhibition zones differed > or =4 mm. In 7/12 mother-child pairs similar profiles were found. In the 8 father-child pairs similar profiles were only found on 2 occasions. In these 2 families, all 3 ( mother, father and child) harboured strains with a similar profile. In 4/8 father-mother pairs similar profiles were found. There was no correlation between the prevalence of mutans streptococci strains, the number of indicator strains against which the strains made bacteriocin, nor the mean size of the inhibition zones and the presence of similarity of bacteriocin activity profiles of mutans streptococci within the family members. The results show that even when a child acquires mutans streptococci after the age of 5, there may be similarity between mutans streptococci in mother, father and child, indicating that transmission between the family members occurs.

Effect of the environment on genotypic diversity of Actinomyces naeslundii and Streptococcus oralis in the oral biofilm.

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 (chi2 = 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 (chi2 = 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.