Genomic Diversity among Actinomyces naeslundii Strains and Closely Related Species.

The aim of this study was to investigate and clarify the ambiguous taxonomy of Actinomyces naeslundii and its closely related species using state-of-the-art high-throughput sequencing techniques, and, furthermore, to determine whether sub-clusters identified within Actinomyces oris and Actinomyces naeslundii in a previous study by multi locus sequence typing (MLST) using concatenation of seven housekeeping genes should either be classified as subspecies or distinct species. The strains in this study were broadly classified under Actinomyces naeslundii group as A. naeslundii genospecies I and genospecies II. Based on MLST data analysis, these were further classified as A. oris and A. naeslundii. The whole genome sequencing of selected strains of A. oris (n = 17) and A. naeslundii (n = 19) was carried out using Illumina Genome Analyzer IIxe and Roche 454 allowing paired-end and single-reads sequencing, respectively. The sequences obtained were aligned using CLC Genomic workbench version 5.1 and annotated using RAST (Rapid Annotation using Subsystem Technology) release version 59 accessible online. Additionally, genomes of seven publicly available strains of Actinomyces (k20, MG1, c505, OT175, OT171, OT170, and A. johnsonii) were also included. Comparative genomic analysis (CGA) using Mauve, Progressive Mauve, gene-by-gene, Core, and Pan Genome, and finally Digital DNA-DNA homology (DDH) analysis was carried out. DDH values were obtained using in silico genome-genome comparison. Evolutionary analysis using ClonalFrame was also undertaken. The mutation and recombination events were compared using chi-square test among A. oris and A. naeslundii isolates (analysis methods are not included in the study). CGA results were consistent with previous traditional classification using MLST. It was found that strains of Actinomyces k20, MG1, c505, and OT175 clustered in A. oris group of isolates, while OT171, OT170, and A. johnsonii appeared as separate branches. Similar clustering to MLST was observed for other isolates. The mutation and recombination events were significantly higher in A. oris than A. naeslundii, highlighting the diversity of A. oris strains in the oral cavity. These findings suggest that A. oris forms six distinct groups, whereas A. naeslundii forms three. The correct designation of isolates will help in the identification of clinical Actinomyces isolates found in dental plaque. Easily accessible online genomic sequence data will also accelerate the investigation of the biochemical characterisation and pathogenesis of this important group of micro-organisms.

Role of microbial biofilms in the maintenance of oral health and in the development of dental caries and periodontal diseases. Consensus report of group 1 of the Joint EFP/ORCA workshop on the boundaries between caries and periodontal disease.

BACKGROUND AND AIMS: The scope of this working group was to review (1) ecological interactions at the dental biofilm in health and disease, (2) the role of microbial communities in the pathogenesis of periodontitis and caries, and (3) the innate host response in caries and periodontal diseases. RESULTS AND CONCLUSIONS: A health-associated biofilm includes genera such as Neisseria, Streptococcus, Actinomyces, Veillonella and Granulicatella. Microorganisms associated with both caries and periodontal diseases are metabolically highly specialized and organized as multispecies microbial biofilms. Progression of these diseases involves multiple microbial interactions driven by different stressors. In caries, the exposure of dental biofilms to dietary sugars and their fermentation to organic acids results in increasing proportions of acidogenic and aciduric species. In gingivitis, plaque accumulation at the gingival margin leads to inflammation and increasing proportions of proteolytic and often obligately anaerobic species. The natural mucosal barriers and saliva are the main innate defence mechanisms against soft tissue bacterial invasion. Similarly, enamel and dentin are important hard tissue barriers to the caries process. Given that the present state of knowledge suggests that the aetiologies of caries and periodontal diseases are mutually independent, the elements of innate immunity that appear to contribute to resistance to both are somewhat coincidental.

Acidogenicity of dual-species biofilms of bifidobacteria and Streptococcus mutans.

OBJECTIVE: The aim of this study was to evaluate the acidogenicity of dual-species biofilms of bifidobacteria and Streptococcus mutans. MATERIALS AND METHODS: The following strains were tested: Bifidobacterium dentium DSM20436, Parascardovia denticolens DSM10105, and Scardovia inopinata DSM10107. Streptococcus mutans UA159 and Lactobacillus acidophilus ATCC4356 were used as control. Bifidobacteria were studied planktonically as they were not able to form monospecies biofilm, they were grown in biofilms associated with S. mutans. Endogenous polysaccharide reserves of cultures at log phase were depleted. Standardized suspensions of the microorganisms were incubated in growth media supplemented with 10 mM glucose, lactose, raffinose, glucose, or xylitol. S. mutans biofilms were grown on glass cover slips for 24 h to which bifidobacteria were added. After 24 h, the dual-species biofilms were exposed to the same carbon sources, and after 3 h, the pH of spent culture media and concentrations of organic acids were measured. Statistical analyses were carried out using ANOVA and Tukey's test (α = 0.05). RESULTS: A higher pH drop was observed when S. mutans was associated with P. denticolens or S. inopinata, in either planktonic or biofilm cultures, than with S. mutans alone. Bifidobacteria showed a higher pH drop in the presence of raffinose than S. mutans or L. acidophilus. CONCLUSIONS: Dual-species biofilms of bifidobacteria and S. mutans produced more acid and greater pH drops than biofilms of S. mutans alone. CLINICAL RELEVANCE: New insights on the complex process of caries pathogenicity contribute to the establishment of preventive and therapeutic measures, in particular in specific cases, such as in early childhood caries.

Bifidobacteria inhibit the growth of Porphyromonas gingivalis but not of Streptococcus mutans in an in vitro biofilm model.

There is growing interest in the use of probiotic bifidobacteria for enhancement of the therapy, and in the prevention, of oral microbial diseases. However, the results of clinical studies assessing the effects of bifidobacteria on the oral microbiota are controversial, and the mechanisms of actions of probiotics in the oral cavity remain largely unknown. In addition, very little is known about the role of commensal bifidobacteria in oral health. Our aim was to study the integration of the probiotic Bifidobacterium animalis subsp. lactis Bb12 and of oral Bifidobacterium dentium and Bifidobacterium longum isolates in supragingival and subgingival biofilm models and their effects on other bacteria in biofilms in vitro using two different in vitro biofilms and agar-overlay assays. All bifidobacteria integrated well into the subgingival biofilms composed of Porphyromonas gingivalis, Actinomyces naeslundii, and Fusobacterium nucleatum and decreased significantly only the number of P. gingivalis in the biofilms. The integration of bifidobacteria into the supragingival biofilms containing Streptococcus mutans and A. naeslundii was less efficient, and bifidobacteria did not affect the number of S. mutans in biofilms. Therefore, our results suggest that bifidobacteria may have a positive effect on subgingival biofilm and thereby potential in enhancing gingival health; however, their effect on supragingival biofilm may be limited.

Isolation of Propionibacterium acnes among the microbiota of primary endodontic infections with and without intraoral communication.

OBJECTIVES: The presence of opportunistic pathogens such as Propionibacterium acnes (P. acnes) may contribute to the endodontic pathology. The presence of P. acnes may be influenced by different endodontic conditions. The aims of the study were firstly, to identify P. acnes within the whole cultivable microbiota of primary endodontic infections, to investigate which P. acnes phylotypes predominate in such infections and secondly to determine if the presence of an "open" communication (e.g. a sinus) can be associated with the isolation of P. acnes from the root canal. MATERIAL AND METHODS: The predominant cultivable microbiota of 15 primary endodontic lesions (7 without communication with the oral environment and 8 with an open communication) were identified using partial 16S ribosomal RNA (rRNA) gene sequence analysis. The identification of the organism was determined by interrogating the Human Oral Microbiome Database. The P. acnes isolates were typed on the basis of the recA gene sequence comparison. A neighbor-joining tree was constructed using MEGA 4.1 with the inclusion of known recA sequences. RESULTS: There was no difference in the number of species identified from lesions without communication (5.86 ± 3.7) and those with communication (5.37 ± 3.6) (P > 0.05). PCR-based 16S rRNA gene sequencing revealed P. acnes as the most prevalent isolate recovered from lesions with communication. recA gene sequencing revealed two phylogenetic lineages present in lesion with communication, with mainly type I (further split into type IA and type IB) and type II. CONCLUSIONS: The presence of P. acnes as opportunistic pathogens has been confirmed and may sustain the traits observed in specific clinical presentations. CLINICAL RELEVANCE: Clinical management of open lesions may require further disinfection to eliminate opportunistic bacteria.

Influence of saliva on the oral microbiota.

Saliva plays a major role in determining the composition and activity of the oral microbiota, via a variety of mechanisms. Molecules, mainly from saliva, form a conditioning film on oral surfaces, thus providing receptors for bacterial attachment. The attached cells use saliva components, such as glycoproteins, as their main source of nutrients for growth. Oral bacteria work sequentially and in a concerted manner to catabolize these structurally complex molecules. Saliva also buffers the pH in the biofilm to around neutrality, creating an environment which is conducive to the growth of many oral bacteria that provide important benefits to the host. Components of the adaptive and innate host defences are delivered by saliva, and these often function synergistically, and at sublethal concentrations, so a complex relationship develops between the host and the resident microbiota. Dysbiosis can occur rapidly if the flow of saliva is perturbed.

Transcriptomic analysis of three Veillonella spp. present in carious dentine and in the saliva of caries-free individuals.

Veillonella spp. are predominant bacteria found in all oral biofilms. In this study, a metatranscriptomic approach was used to investigate the gene expression levels of three oral Veillonella spp. (V. parvula, V. dispar and V. atypica) in whole stimulated saliva from caries-free volunteers and in carious lesions (n = 11 for each group). In the lesions the greatest proportion of reads were assigned to V. parvula and genes with the highest level of expression in carious samples were those coding for membrane transport systems. All three Veillonella spp. increased expression of genes involved in the catabolism of lactate and succinate, notably the alpha- and beta-subunits of L(+)-tartrate dehydratase (EC 4.2.1.32). There was also significantly increased expression of histidine biosynthesis pathway in V. parvula, suggesting higher intra-cellular levels of histidine that could provide intra-cellular buffering capacity and, therefore, assist survival in the acidic environment. Various other systems such as potassium uptake systems were also up regulated that may aid in the survival and proliferation of V. parvula in carious lesions.

Identification of the 'Streptococcus anginosus group' by matrix-assisted laser desorption ionization--time-of-flight mass spectrometry.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) provides rapid, accurate and cost-effective identification of a range of bacteria and is rapidly changing the face of routine diagnostic microbiology. However, certain groups of bacteria, for example streptococci (in particular viridans or non-haemolytic streptococci), are less reliably identified by this method. We studied the performance of MALDI-TOF MS for identification of the 'Streptococcus anginosus group' (SAG) to species level. In total, 116 stored bacteraemia isolates identified by conventional methods as belonging to the SAG were analysed by MALDI-TOF MS. Partial 16S rRNA gene sequencing, supplemented with sialidase activity testing, was performed on all isolates to provide 'gold standard' identification against which to compare MALDI-TOF MS performance. Overall, 100 % of isolates were correctly identified to the genus level and 93.1 % to the species level by MALDI-TOF MS. However, only 77.6 % were correctly identified to the genus level and 59.5 % to the species level by a MALDI-TOF MS direct transfer method alone. Use of a rapid in situ extraction method significantly improved identification rates when compared with the direct transfer method (P<0.001). We recommend routine use of this method to reduce the number of time-consuming full extractions required for identification of this group of bacteria by MALDI-TOF MS in the routine diagnostic laboratory. Only 22 % (1/9) of Streptococcus intermedius isolates were reliably identified by MALDI-TOF MS to the species level, even after full extraction. MALDI-TOF MS reliably identifies S. anginosus and Streptococcus constellatus to the species level but does not reliably identify S. intermedius.

Oral health patterns of independently living dentate older people: a cross-sectional survey of dental attendees in south-east London.

BACKGROUND: The oral health needs of older adults present increasing challenges to dental services. OBJECTIVES: To examine the clinical oral health status of dentate older people living in the community and attending dental services. METHODS: One hundred and eighty-six dentate adults, aged ≥60 years, underwent clinical examination (DMFS, Plaque and Gingival Indexes), salivary analysis and completed a questionnaire. RESULTS: Participants had an average of 21.4 (±6.2) teeth present and 1.2 (±3.0) decayed, 51.0 (±28.8) missing and 32.6 (±20.5) restored surfaces. Individuals living in the most deprived areas had significantly lower numbers of teeth than those in the least deprived areas (19.1 ± 7.5 cf 23.8 ± 4.1; p < 0.001). Whilst there were no significant differences in DMFS score, residents in the most deprived areas had significantly more missing and fewer filled surfaces than those in the least deprived areas (p = 0.001 and p < 0.001, respectively). Participants with ≥21 teeth (64%) had lower plaque scores, fewer decayed root surfaces, higher stimulated saliva flow rates and lower salivary lactobacilli and yeast counts than those with <21 teeth (p < 0.05 for all). CONCLUSIONS: The findings highlight differences in clinical oral health by age and deprivation status and underline the importance of saliva and retaining a functional dentition.

Evolutionary and population genomics of the cavity causing bacteria Streptococcus mutans.

Streptococcus mutans is widely recognized as one of the key etiological agents of human dental caries. Despite its role in this important disease, our present knowledge of gene content variability across the species and its relationship to adaptation is minimal. Estimates of its demographic history are not available. In this study, we generated genome sequences of 57 S. mutans isolates, as well as representative strains of the most closely related species to S. mutans (S. ratti, S. macaccae, and S. criceti), to identify the overall structure and potential adaptive features of the dispensable and core components of the genome. We also performed population genetic analyses on the core genome of the species aimed at understanding the demographic history, and impact of selection shaping its genetic variation. The maximum gene content divergence among strains was approximately 23%, with the majority of strains diverging by 5-15%. The core genome consisted of 1,490 genes and the pan-genome approximately 3,296. Maximum likelihood analysis of the synonymous site frequency spectrum (SFS) suggested that the S. mutans population started expanding exponentially approximately 10,000 years ago (95% confidence interval [CI]: 3,268-14,344 years ago), coincidental with the onset of human agriculture. Analysis of the replacement SFS indicated that a majority of these substitutions are under strong negative selection, and the remainder evolved neutrally. A set of 14 genes was identified as being under positive selection, most of which were involved in either sugar metabolism or acid tolerance. Analysis of the core genome suggested that among 73 genes present in all isolates of S. mutans but absent in other species of the mutans taxonomic group, the majority can be associated with metabolic processes that could have contributed to the successful adaptation of S. mutans to its new niche, the human mouth, and with the dietary changes that accompanied the origin of agriculture.

Salivary levels of Bifidobacteria in caries-free and caries-active children.

BACKGROUND: Salivary levels of Bifidobacteria have been shown to be significantly correlated with caries experience in adults but not as yet in children. HYPOTHESIS: Salivary levels of Bifidobacteria are positively associated with caries experience in children. AIM: To compare the salivary concentrations of Bifidobacteria of caries-free and caries-active children. DESIGN: Saliva was collected using the tongue-loop method from 38 caries-active children and from 22 clinically caries-free children, and the numbers of Bifidobacteria, mutans streptococci, lactobacilli and yeasts were determined. Additionally, the age and gender of the children, a plaque index, sugar amount in diet, sugar frequency in diet, hygiene practice and fluoride toothpaste usage were recorded. RESULTS: Bifidobacteria were isolated from 95% of the caries-active children and from only 9% of the caries-free children (P < 0.001). Salivary levels of Bifidobacteria were significantly correlated with amount of sugar in the diet, frequency of sugar consumption and oral hygiene practice. The significant variables that discriminated between the caries-free and caries-active subjects were salivary levels of Bifidobacteria, salivary levels of mutans streptococci and oral hygiene practice (χ(2) = 72.57, P < 0.001) and overall 90.0% of cases were correctly classified. CONCLUSIONS: Salivary levels of Bifidobacteria are significantly associated with caries experience in children. The salivary levels of this genus may be a useful marker of caries risk.

Application of MLST and pilus gene sequence comparisons to investigate the population structures of Actinomyces naeslundii and Actinomyces oris.

Actinomyces naeslundii and Actinomyces oris are members of the oral biofilm. Their identification using 16S rRNA sequencing is problematic and better achieved by comparison of metG partial sequences. A. oris is more abundant and more frequently isolated than A. naeslundii. We used a multi-locus sequence typing approach to investigate the genotypic diversity of these species and assigned A. naeslundii (n = 37) and A. oris (n = 68) isolates to 32 and 68 sequence types (ST), respectively. Neighbor-joining and ClonalFrame dendrograms derived from the concatenated partial sequences of 7 house-keeping genes identified at least 4 significant subclusters within A. oris and 3 within A. naeslundii. The strain collection we had investigated was an under-representation of the total population since at least 3 STs composed of single strains may represent discrete clusters of strains not well represented in the collection. The integrity of these sub-clusters was supported by the sequence analysis of fimP and fimA, genes coding for the type 1 and 2 fimbriae, respectively. An A. naeslundii subcluster was identified with both fimA and fimP genes and these strains were able to bind to MUC7 and statherin while all other A. naeslundii strains possessed only fimA and did not bind to statherin. An A. oris subcluster harboured a fimA gene similar to that of Actinomyces odontolyticus but no detectable fimP failed to bind significantly to either MUC7 or statherin. These data are evidence of extensive genotypic and phenotypic diversity within the species A. oris and A. naeslundii but the status of the subclusters identified here will require genome comparisons before their phylogenic position can be unequivocally established.

Scardovia wiggsiae sp. nov., isolated from the human oral cavity and clinical material, and emended descriptions of the genus Scardovia and Scardovia inopinata.

Six strains of anaerobic, pleomorphic Gram-positive bacilli, isolated from the human oral cavity and an infected arm wound, were subjected to a comprehensive range of phenotypic and genotypic tests and were found to comprise a homogeneous group. 16S rRNA gene sequence analysis revealed that the isolates were most closely related to Scardovia inopinata CCUG 35729(T) (94.8-94.9 % 16S rRNA gene sequence similarity). The isolates were saccharolytic and produced acetic and lactic acids as end products of fermentation. The major fatty acids were C(16 : 0) (49.8 %) and C(18 : 1)ω9c (35.8 %). Polar lipid analysis revealed a variety of glycolipids, diphosphatidylglycerol, an unidentified phospholipid and an unidentified phosphoglycolipid. No respiratory quinones were detected. The peptidoglycan was of the type A4α L-Lys-Thr-Glu, with L-lysine partially replaced by L-ornithine. The DNA G+C content of one of the strains, C1A_55(T)(,) was 55 mol%. A novel species, Scardovia wiggsiae sp. nov., is proposed to accommodate the six isolates, with the type strain C1A_55(T) (=DSM 22547(T)=CCUG 58090(T)).

Propionibacterium acnes and Staphylococcus epidermidis isolated from refractory endodontic lesions are opportunistic pathogens.

The predominant cultivable microbiota from 20 refractory endodontic lesions (9 with abscesses and 11 without abscesses) were determined, and Propionibacterium acnes and Staphylococcus epidermidis were among the most predominant organisms. The number of species identified from lesions with abscesses (14.1 ± 2.6) was significantly greater (P < 0.001) than the number from lesions without abscesses (7.4 ± 5.9). Comparison of perioral isolates using repetitive extragenic palindromic PCR of the same species from the same subjects demonstrated that the endodontic and skin populations were significantly different. The P. acnes isolates were typed on the basis of recA gene sequence comparison, and only three types (types I, II, and III) were identified among 125 isolates examined. However, we found that type I (type IA and IB) isolates were primarily isolated from the skin, while types II and III were significantly more likely to be isolated from the endodontic lesions (P < 10(-10)). We found that the robustness of the recA phylotypes was not strong by comparing the partial gene sequences of six putative virulence determinants, PAmce, PAp60, PA-25957, PA-5541, PA-21293, and PA-4687. The resulting neighbor-joining trees were incongruent, and significant (phi test; P = 2.2 × 10(-7)) evidence of recombination was demonstrated, with significant phylogenetic heterogeneity being apparent within the clusters. P. acnes and S. epidermidis isolated from refractory endodontic infections, with or without periapical abscesses, are likely to be nosocomial infections.

Generation of diversity in Streptococcus mutans genes demonstrated by MLST.

Streptococcus mutans, consisting of serotypes c, e, f and k, is an oral aciduric organism associated with the initiation and progression of dental caries. A total of 135 independent Streptococcus mutans strains from caries-free and caries-active subjects isolated from various geographical locations were examined in two versions of an MLST scheme consisting of either 6 housekeeping genes [accC (acetyl-CoA carboxylase biotin carboxylase subunit), gki (glucokinase), lepA (GTP-binding protein), recP (transketolase), sodA (superoxide dismutase), and tyrS (tyrosyl-tRNA synthetase)] or the housekeeping genes supplemented with 2 extracellular putative virulence genes [gtfB (glucosyltransferase B) and spaP (surface protein antigen I/II)] to increase sequence type diversity. The number of alleles found varied between 20 (lepA) and 37 (spaP). Overall, 121 sequence types (STs) were defined using the housekeeping genes alone and 122 with all genes. However pi, nucleotide diversity per site, was low for all loci being in the range 0.019-0.007. The virulence genes exhibited the greatest nucleotide diversity and the recombination/mutation ratio was 0.67 [95% confidence interval 0.3-1.15] compared to 8.3 [95% confidence interval 5.0-14.5] for the 6 concatenated housekeeping genes alone. The ML trees generated for individual MLST loci were significantly incongruent and not significantly different from random trees. Analysis using ClonalFrame indicated that the majority of isolates were singletons and no evidence for a clonal structure or evidence to support serotype c strains as the ancestral S. mutans strain was apparent. There was also no evidence of a geographical distribution of individual isolates or that particular isolate clusters were associated with caries. The overall low sequence diversity suggests that S. mutans is a newly emerged species which has not accumulated large numbers of mutations but those that have occurred have been shuffled as a consequence of intra-species recombination generating genotypes which can be readily distinguished by sequence analysis.

The Bifidobacterium dentium Bd1 genome sequence reflects its genetic adaptation to the human oral cavity.

Bifidobacteria, one of the relatively dominant components of the human intestinal microbiota, are considered one of the key groups of beneficial intestinal bacteria (probiotic bacteria). However, in addition to health-promoting taxa, the genus Bifidobacterium also includes Bifidobacterium dentium, an opportunistic cariogenic pathogen. The genetic basis for the ability of B. dentium to survive in the oral cavity and contribute to caries development is not understood. The genome of B. dentium Bd1, a strain isolated from dental caries, was sequenced to completion to uncover a single circular 2,636,368 base pair chromosome with 2,143 predicted open reading frames. Annotation of the genome sequence revealed multiple ways in which B. dentium has adapted to the oral environment through specialized nutrient acquisition, defences against antimicrobials, and gene products that increase fitness and competitiveness within the oral niche. B. dentium Bd1 was shown to metabolize a wide variety of carbohydrates, consistent with genome-based predictions, while colonization and persistence factors implicated in tissue adhesion, acid tolerance, and the metabolism of human saliva-derived compounds were also identified. Global transcriptome analysis demonstrated that many of the genes encoding these predicted traits are highly expressed under relevant physiological conditions. This is the first report to identify, through various genomic approaches, specific genetic adaptations of a Bifidobacterium taxon, Bifidobacterium dentium Bd1, to a lifestyle as a cariogenic microorganism in the oral cavity. In silico analysis and comparative genomic hybridization experiments clearly reveal a high level of genome conservation among various B. dentium strains. The data indicate that the genome of this opportunistic cariogen has evolved through a very limited number of horizontal gene acquisition events, highlighting the narrow boundaries that separate commensals from opportunistic pathogens.

Proteolytic degradation of human salivary MUC5B by dental biofilms.

The degradation of complex substrates, like salivary mucins, requires an arsenal of glycosidases and proteases to sequentially degrade the oligosaccharides and polypeptide backbone. The mucin MUC5B is a complex oligomeric glycoprotein, heterogeneous in molecular mass (14-40 x 10(6) Da), with a diverse repertoire of oligosaccharides, differing in composition and charge. The aim of this study was to investigate whether proteolytic degradation of the mucin polypeptide backbone could be identified and if cooperation of dental biofilm bacteria was required. Cooperative bacteria-mediated proteolysis of MUC5B was determined by comparing individual species and mixed consortia of strains isolated from supragingival plaque, and freshly harvested supragingival plaque. Proteolytic activity was analysed using fluorescent labelled substrate and by visualizing mucin degradation by SDS-PAGE. Dental plaque degraded the polypeptide backbone of the salivary MUC5B mucin. The mucin was also degraded by a specific consortium of isolated species from supragingival plaque, although individual species and other consortia did not. Certain bacteria in supragingival dental plaque therefore cooperate as a consortium to proteolyse human salivary MUC5B and hydrolyse glycosides.

Population structure of Streptococcus oralis.

Streptococcus oralis is a member of the normal human oral microbiota, capable of opportunistic pathogenicity; like related oral streptococci, it exhibits appreciable phenotypic and genetic variation. A multilocus sequence typing (MLST) scheme for S. oralis was developed and the resultant data analysed to examine the population structure of the species. Analysis of 113 isolates, confirmed as belonging to the S. oralis/mitis group by 16S rRNA gene sequencing, characterized the population as highly diverse and undergoing inter- and intra-species recombination with a probable clonal complex structure. ClonalFrame analysis of these S. oralis isolates along with examples of Streptococcus pneumoniae, Streptococcus mitis and Streptococcus pseudopneumoniae grouped the named species into distinct, coherent populations and did not support the clustering of S. pseudopneumoniae with S. mitis as reported previously using distance-based methods. Analysis of the individual loci suggested that this discrepancy was due to the possible hybrid nature of S. pseudopneumoniae. The data are available on the public MLST website (http://pubmlst.org/soralis/).