- [Show abstract] [Hide abstract] ABSTRACT: International Journal of Paediatric Dentistry 2013; 23: 32–38 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 (χ2 = 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.
- [Show abstract] [Hide abstract] ABSTRACT: A collection of Streptococcus sanguinis strains from patients with endocarditis (n = 21) and from the oral cavity (n = 34) was subjected to a multi-locus sequence typing analysis using seven housekeeping genes, carbamoyl-phosphate synthetase (carB), Co/Zn/Cd efflux system component (czcD), d-alanyl-d-alanine ligase (ddl), DNA polymerase III (dnaX), glucose-6-phosphate dehydrogenase (gdh), DNA-directed RNA polymerase, beta subunit (rpoB) and superoxide dismutase (sodA). The scheme was expanded by the inclusion of two the putative virulence genes, bacitracin-resistance protein (bacA) and saliva-binding protein (ssaB), to increase strain discrimination. Extensive intra-species recombination was apparent in all genes but inter-species recombination was also apparent with strains apparently harbouring gdh and ddl from unidentified sources and one isolate harboured a sodA allele apparently derived from Streptococcus oralis. The recombination/mutation ratio for the concatenated housekeeping gene sequences was 1.67 (95% confidence limits 1.25-2.72) and for the two virulence genes the r/m ratio was 3.99 (95% confidence limits 1.61-8.72); recombination was the major driver for genetic variation. All isolates were distinct and the endocarditis strains did not form distinct sub-clusters when the data were analysed using ClonalFrame. These data support the widely held opinion that infecting S. sanguinis strains are opportunistic human pathogens.
Dataset: Table S1[Show abstract] [Hide abstract] ABSTRACT: Allelic profiles and sequence types of the A. oris and A. naeslundii strains. (DOC)
- [Show abstract] [Hide abstract] ABSTRACT: 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.
Conference Paper: Radiotherapy Effect on Saliva and Oral Microflora in Oncology Patients[Show abstract] [Hide abstract] ABSTRACT: Background: Radiotherapy, although curative, causes adverse effects upon salivary glands and changes in oral microflora in head and neck cancer patients. Knowledge of the implications of radiotherapy for saliva properties and underlying changes in microflora may offer a better understanding of the acute and chronic oral complications associated with these patients. Objective: To determine the effects of radiotherapy on saliva properties and selected aciduric microflora in head and neck cancer patients following radiotherapy. Material and Methods: With ethical approval (LREC Study Number:09/H0804/22), 14 head and neck cancer patients were examined at routine dental assessments at Guy's Hospital, London pre- and post-radiotherapy (3-24 weeks; median time 6 weeks). Stimulated and unstimulated saliva samples were obtained to determine the flow rate (5 min collection). The stimulated sample was used to enumerate mutans streptococci, Lactobacilli, Bifidobacteria, and yeasts as cfu/mL. Buffering capacity and saliva pH were analysed chairside (Saliva-Check Buffer, GC Europe N.V. Leuven, Belgium). The oral clearance time of glucose and consequent lactate formation were determined 10 min after glucose rinsing. Data were analysed using 2, Wilcoxon matched-pairs signed rank and Spearman's rank correlation coefficient tests. Results: Stimulated saliva flow rate reduced significantly from 2.601.20 mL/min to 0.690.76 mL/min (p<0.001). The unstimulated flow rates, pre- and post-treatment, were not significantly different. Significant inverse relationships were found, post treatment, only between saliva flow rate and yeasts (r=-0.6; p<0.05) and Bifidobacteria (r=-0.60; p<0.05). Salivary level of yeasts increased significantly post treatment (p=0.014). The saliva buffering capacity and saliva pH were reduced significantly (p=0.002 and p=0.03, respectively). Oral clearance time of glucose was increased significantly post treatment (p=0.03) but the salivary lactate concentrations were not significantly different. Conclusion: Irradiation caused a reduction in stimulated salivary flow rate, reflected in increased oral clearance time, and reduced buffering capacity resulting in a more acidic oral environment.
Article: Clonal structure of[Show abstract] [Hide abstract] ABSTRACT: SummaryA collection of Streptococcus sanguinis strains from patients with endocarditis (n = 21) and from the oral cavity (n = 34) was subjected to a multi‐locus sequence typing analysis using seven housekeeping genes, carbamoyl‐phosphate synthetase (carB), Co/Zn/Cd efflux system component (czcD), d‐alanyl‐d‐alanine ligase (ddl), DNA polymerase III (dnaX), glucose‐6‐phosphate dehydrogenase (gdh), DNA‐directed RNA polymerase, beta subunit (rpoB) and superoxide dismutase (sodA). The scheme was expanded by the inclusion of two the putative virulence genes, bacitracin‐resistance protein (bacA) and saliva‐binding protein (ssaB), to increase strain discrimination. Extensive intra‐species recombination was apparent in all genes but inter‐species recombination was also apparent with strains apparently harbouring gdh and ddl from unidentified sources and one isolate harboured a sodA allele apparently derived from Streptococcus oralis. The recombination/mutation ratio for the concatenated housekeeping gene sequences was 1.67 (95% confidence limits 1.25–2.72) and for the two virulence genes the r/m ratio was 3.99 (95% confidence limits 1.61–8.72); recombination was the major driver for genetic variation. All isolates were distinct and the endocarditis strains did not form distinct sub‐clusters when the data were analysed using ClonalFrame. These data support the widely held opinion that infecting S. sanguinis strains are opportunistic human pathogens.
- [Show abstract] [Hide abstract] ABSTRACT: 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.
Article: Oral Bifidobacteria[Show abstract] [Hide abstract] ABSTRACT: Bifidobacteria are aciduric bacteria that might play a role in the caries process. To test the hypothesis that Bifidobacteria behave as caries-associated organisms, as predicted by the ecological plaque hypothesis, we determined salivary levels of Bifidobacteria and caries-associated organisms for 156 older adults. Salivary levels of Bifidobacteria, mutans streptococci, lactobacilli, and yeasts were correlated with each other (p < 0.001), negatively correlated with salivary flow rate (p < 0.001), and positively correlated with plaque index (p < 0.05). Salivary Bifidobacteria levels were positively associated with the number of filled (p < 0.001) and decayed (p = 0.036) tooth surfaces and negatively associated with number of teeth (p < 0.001) and salivary flow rate (p = 0.049). In regression analyses, caries experience was significantly associated with only salivary Bifidobacteria (p < 0.001) and yeast (p < 0.001) levels and the individual's age (p = 0.021). Bifidobacteria should be regarded as caries-associated organisms whose role in the caries process and as markers of caries risk requires further investigation.
- [Show abstract] [Hide abstract] ABSTRACT: The microbiota of the denture plaque biofilm colonizing the fitting surface of dentures in edentulous subjects with healthy palates (n = 20) and in edentulous subjects with denture stomatitis (n = 20) was studied. The numbers of bacteria colonizing the dentures of healthy subjects was significantly less than the numbers colonizing the dentures of stomatitis subjects. The proportions and frequency of isolation of mutans streptococci, lactobacilli, bifidobacteria and yeasts were significantly (P < 0.05) greater in the subjects with denture stomatitis. The proportions of these organisms in the denture plaque biofilm of the subjects with denture stomatitis were similar to those found in carious lesions, indicating that the site is a low pH environment. The predominant bifidobacterial species in the mouths of dentate subjects is Bifidobacterium dentium but in the edentulous subjects wearing dentures B. dentium was isolated from only one of the 20 subjects with denture stomatitis and from none of the 20 subjects with healthy palates. Instead, Bifidobacterium breve, Bifidobacterium scardovii and Bifidobacterium longum subsp. longum were isolated. Only a single non-oral bifidobacterial species was isolated from each individual and repetitive extragenic palindromic- and BOX-polymerase chain reaction typing methods indicated that the same genotypes were shared between subjects. Using deferred antagonism spot plate assays, interspecies inhibition was demonstrated between oral isolates of B. dentium, B. breve, B. scardovii and B. longum subsp. longum. Here we have shown that bifidobacteria and caries-associated microbiota are present in denture plaque at levels similar to those of carious lesions and B. dentium cannot be maintained in an edentulous mouth.
- [Show abstract] [Hide abstract] ABSTRACT: 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.
Dataset: Figure S1[Show abstract] [Hide abstract] ABSTRACT: Splitstree analysis of the 8 loci. Only tyrS gave a significant PHI value. (0.11 MB TIF)
Dataset: Figure S2[Show abstract] [Hide abstract] ABSTRACT: Neighbour-joining tree constructed using MEGA v4.0, showing relationships between the concatenated sequences of all S. mutans STs (n = 122). Bootstrap values are indicated at corresponding nodes and STs at end of branches. Bar is 0.0005 substitutions per site. (0.06 MB TIF)
Dataset: Table S1[Show abstract] [Hide abstract] ABSTRACT: Sequence types (ST) and allelic profiles; with N, number of ST occurrences. (0.36 MB DOC)
Dataset: Figure S3[Show abstract] [Hide abstract] ABSTRACT: Radial diagram of the eBURST analysis of S. mutans STs. (0.07 MB TIF)
- [Show abstract] [Hide abstract] ABSTRACT: The aim of this study was to enumerate and identify bifidobacteria from occlusal carious lesions in permanent and deciduous teeth. Samples of infected dentine were obtained from 24 active occlusal lesions in deciduous teeth and from 15 occlusal lesions in permanent teeth. Plaque samples from sound occlusal surfaces of 12 caries-free adults and 12 children were also obtained. The bifidobacterial strains were isolated in mupirocin-containing selective media, Gram-stained and subcultured for identification. Total bacterial counts were determined using fastidious anaerobic agar, and isolates were identified using genus-specific PCR primers and were confirmed by 16S rRNA sequencing. Bifidobacteria were isolated from 13 of the 15 occlusal lesions in the adults and formed 5.09 +/- 2.11% of the total cultivable flora. In the children, bifidobacteria were isolated from 16 of the 24 occlusal lesions and formed 7.4 +/- 2.6% of the total flora. No bifidobacteria were isolated from the occlusal surfaces of caries-free adults or children. A total of 424 bifidobacteria were identified and these were Bifidobacteriumdentium, Parascardovia denticolens, Scardoviainopicata, Bifidobacterium longum, Scardovia genomosp. C1 and Bifidobacterium breve. B. dentium was present in 14 out of the 16 bifidobacteria-positive samples from the lesions on the deciduous teeth and in 7 out of the 13 positive lesions in adults (p = 0.04). The present data suggest that bifidobacteria may play a role in the progression of occlusal caries lesions in both children and adults.
- [Show abstract] [Hide abstract] ABSTRACT: 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/).
- [Show abstract] [Hide abstract] ABSTRACT: To determine the antimicrobial properties of a selection of dentine bonding agents [DBAs] using the disc diffusion and direct contact methods and an ex vivo method using extracted carious permanent molar teeth. DBAs (n=15) were tested using Streptococcus mutans UA159 in disc diffusion and direct contact methods. In the ex vivo study 6 DBAs were selected and pre- and post-treatment samples of carious dentine (n< or =12) were taken. Samples were also taken post-acid-etching. The number of microorganisms in dentine sample was determined and compared. The inhibition zones and percent growth inhibition were related to the pH of the culture medium containing the DBA (p<0.01). Clearfill Protect Bond exhibited the greatest bacterial killing followed by ibond (99.8%+/-0.08 and 98.2+/-1.4, respectively). The phosphoric acid etchant alone resulted in an 83% killing. The in vitro tests results did not correlate. The ex vivo killing reflected the percent growth inhibition observed in the direct contact method. A guide to the potential antimicrobial activity of a DBA may be gained from an assessment of its pH when added to bacteriological culture medium. The direct contact method gives a better reflection of the killing of bacteria in infected dentine than the disk diffusion method. Killing in the ex vivo model gives a more realistic and more reliable method for determining the antibacterial activity of a given DBA and that comparisons of the relative inhibitory activity of DBAs should be tested using this ex vivo model.
- [Show abstract] [Hide abstract] ABSTRACT: Actinomyces naeslundii is an important early colonizer in the oral biofilm and consists of three genospecies (1, 2 and WVA 963) which cannot be readily differentiated using conventional phenotypic testing or on the basis of 16S rRNA gene sequencing. We have investigated a representative collection of type and reference strains and clinical and oral isolates (n=115) and determined the partial gene sequences of six housekeeping genes (atpA, rpoB, pgi, metG, gltA and gyrA). These sequences identified the three genospecies and differentiated them from Actinomyces viscosus isolated from rodents. The partial sequences of atpA and metG gave best separation of the three genospecies. A. naeslundii genospecies 1 and 2 formed two distinct clusters, well separated from both genospecies WVA 963 and A. viscosus. Analysis of the same genes in other oral Actinomyces species (Actinomyces gerencseriae, A. israelii, A. meyeri, A. odontolyticus and A. georgiae) indicated that, when sequence data were obtained, these species each exhibited <90 % similarity with the A. naeslundii genospecies. Based on these data, we propose the name Actinomyces oris sp. nov. (type strain ATCC 27044(T) =CCUG 34288(T)) for A. naeslundii genospecies 2 and Actinomyces johnsonii sp. nov. (type strain ATCC 49338(T) =CCUG 34287(T)) for A. naeslundii genospecies WVA 963. A. naeslundii genospecies 1 should remain as A. naeslundii sensu stricto, with the type strain ATCC 12104(T) =NCTC 10301(T) =CCUG 2238(T).
- [Show abstract] [Hide abstract] ABSTRACT: Actinomyces spp., predominant members of human oral biofilms, may use extracellular sialidase to promote adhesion, deglycosylate immunoglobulins and liberation of nutrients. Partial nanH gene sequences (1,077 bp) from Actinomyces oris (n=74), Actinomyces naeslundii (n=30), Actinomyces viscosus (n=1) and Actinomyces johnsonii (n=2) which included the active-site region and the bacterial neuraminidase repeats (BNRs) were compared. The sequences were aligned and each species formed a distinct cluster with five isolates having intermediate positions. These five isolates (two A. oris and three A. naeslundii) exhibited interspecies recombination. The nonsynonymous/synonymous ratio was <1 for both A. oris and A. naeslundii indicating that nanH in both species is under stabilizing selective pressure; nonsynonymous mutations are not selected. However, for A. oris significant negative values in tests for neutral selection suggested the rate of mutation in A. oris was greater than in A. naeslundii but with selection against nonsynonymous mutations. This was supported by the observation that the frequency of polymorphic sites in A. oris, which were monomorphic in A. naeslundii was significantly greater than the frequency of polymorphic sites in A. naeslundii which were monomorphic in A. oris (chi(2)=7.011; P=0.00081). The higher proportions of A. oris in the oral biofilm might be explained by the higher mutation rate facilitating an increased ability to respond successfully to environmental stress.
Dataset: Supplementary Material