David Beighton

Aim: To establish a nutrient‐stressed multispecies model biofilm and investigate the dynamics of biofilm killing and disruption by 1% trypsin and 1% proteinase K with or without ultrasonic activation.Methodology: Nutrient‐stressed biofilms (Propionibacterium acnes, Staphylococcus epidermidis, Actinomyces radicidentis, Streptococcus mitis and Enterococcus faecalis OMGS 3202) were grown on hydroxyapatite discs and in prepared root canals of single‐rooted teeth in modified fluid universal medium. The treatment groups included trypsin, proteinase K, 0.2% chlorhexidine gluconate and 1% sodium hypochlorite (NaOCl) (with and without ultrasonics). NaOCl and chlorhexidine were the positive controls and untreated group, and sterile saline was the negative control. The biofilms were investigated using confocal laser scanning microscopy (CLSM) with live/dead staining and quantitative microbial culture.Results: Nutrient stress in the multispecies biofilm was apparent as the medium pH became alkaline, glucose was absent, and serum proteins were degraded in the supernatant. The CLSM showed the percentage reduction in viable bacteria at the biofilm surface level due to nutrient starvation. On the disc model, trypsin and proteinase K were effective in killing bacteria; their aerobic viable counts were significantly lower (P < 0.01) than the negative control and chlorhexidine. NaOCl was the most effective agent (P < 0.001). In the tooth model, when compared to saline, trypsin with ultrasonics caused significant killing both aerobically and anaerobically (P < 0.05). Chlorhexidine (1.46 ± 0.42), trypsin (3.56 ± 1.18) and proteinase K (4.2 ± 1.01) with ultrasonics were significantly effective (P < 0.05) in reducing the substratum coverage as compared to saline with ultrasonics (12% ± 4.9).Conclusion: Trypsin with ultrasonic activation has a biofilm killing and disrupting potential.

Antimicrobial methods to augment fluoride-mediated caries inhibition are necessary. Several methods are described here, but none was considered likely to be as effective as fluoride usage. None had been tested in effective models to demonstrate their ability to act either additively or synergistically with fluoride-containing toothpastes. Dental caries is a biofilm-mediated disease: The composition of the biofilm associated with caries initiation and progression is diverse. Caries-associated taxa – including mutans streptococci, lactobacilli, bifidobacteria, and yeasts – may be useful surrogate markers for in vivo investigations. In vitro testing should progress from single-species planktonic cells to multi-species biofilms prior to essential testing in randomized control trials (RCTs). Modern high-throughput sequencing techniques need to be applied to the study of bacterial acquisition from birth and of the composition of the biofilm associated with the formation of white-spot lesions. The determination of the functions of the biofilm and the phenotype of the bacterial components may be determined by RNA-seq techniques, since they must be conserved between caries lesions and will include the ability to produce acids and survive and proliferate in acidic conditions. The application of such methods will significantly improve our understanding of the etiology and progression of dental caries.

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.

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.

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.

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.

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.

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.

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 π, 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.