D Clark

Emended description of Actinomyces naeslundii and descriptions of Actinomyces oris sp. nov. and Actinomyces johnsonii sp. nov., previously identified as Actinomyces naeslundii genospecies 1, 2 and WVA 963

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 27044T =CCUG 34288T) for A. naeslundii genospecies 2 and Actinomyces johnsonii sp. nov. (type strain ATCC 49338T =CCUG 34287T) for A. naeslundii genospecies WVA 963. A. naeslundii genospecies 1 should remain as A. naeslundii sensu stricto, with the type strain ATCC 12104T =NCTC 10301T =CCUG 2238T.

Dental Caries, Oral Hygiene, and Oral Clearance in Children with Craniofacial Disorders

The reason that children with cleft palates tend to have a greater prevalence of tooth decay than normal children is unclear. We hypothesized that children with cleft palates would have increased oral clearance times for foods and, consequently, higher levels of caries and caries-associated micro-organisms than control children. Children aged 6–16 yrs, with (n = 81) or without (n = 61) cleft palates, were studied. Children with cleft palates had DMFT and dmft scores greater (p < 0.01) than those of the control group. The number of caries-associated organisms was greater in the saliva of the cleft palate children (all p < 0.001). The oral hygiene, plaque and gingival index scores were greater (p < 0.0001), oral clearance was longer (p < 0.01), and levels of sucrose and starch-derived saccharides higher (p < 0.01) in the cleft palate group. However, salivary concentrations of organic acids were lower in the children with craniofacial disorders, probably reflecting the altered physiology of the more mature dental biofilm. The longer oral clearance times of foods and the consequent generation of fermentable sugars from starches may contribute to the higher caries prevalence observed in children with cleft palates.

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/).

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 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.

Veillonella rogosae sp. nov., an anaerobic, Gram- negative coccus isolated from dental plaque

Strains of a novel anaerobic, Gram-negative coccus were isolated from the supra-gingival plaque of children. Independent strains from each of six subjects were shown, at a phenotypic level and based on 16S rRNA gene sequencing, to be members of the genus Veillonella. Analysis revealed that the six strains shared 99.7 % similarity in their 16S rRNA gene sequences and 99.0 % similarity in their rpoB gene sequences. The six novel strains formed a distinct group and could be clearly separated from recognized species of the genus Veillonella of human or animal origin. The novel strains exhibited 98 and 91 % similarity to partial 16S rRNA and rpoB gene sequences of Veillonella parvula ATCC 10790T, the most closely related member of the genus. The six novel strains could be differentiated from recognized species of the genus Veillonella based on partial 16S rRNA and rpoB gene sequencing. The six novel strains are thus considered to represent a single novel species of the genus Veillonella, for which the name Veillonella rogosae sp. nov. is proposed. The type strain is CF100T (=CCUG 54233T=DSM 18960T).

The predominant cultivable Veillonella spp. of the tongue of healthy adults identified using rpoB sequencing

The predominant Veillonella spp. were isolated from the dorsum surface of the tongues of 11 healthy adults and identified to species level using rpoB sequencing because 16S ribosomal RNA sequence analysis does not reliably differentiate between all members of this genus. In all, 253 isolates were identified and the mean proportion (+/- SE) of Veillonella spp. per sample was 16.2 (+/- 3.6) with a range of 3.0% to 36.3% of the total anaerobic colony count. The predominant species were Veillonella atypica (10/11), Veillonella dispar (9/11) and Veillonella rogosae (8/11) because they were isolated from the majority of subjects. Veillonella parvula was isolated from only one subject while Veillonella dentocariosi and Veillonella montpelleriensis were not isolated from any subject.

The microflora of the erupting first permanent molar

The relationship between microflora, eruption status and caries status in the first permanent molar of young children was investigated in 177 children aged 6–7 years. A significantly greater proportion of fully erupted teeth were classified as sound and plaque-free compared to partially erupted teeth. Fully erupted teeth yielded greater numbers and proportions of mutans streptococci compared with significantly greater numbers and proportions of Actinomyces israelii in partially erupted teeth. Logistical regression analysis showed significant associations between white spot lesions in partially erupted teeth and increased numbers of Streptococcus oralis, mutans streptococci and Streptococcus salivarius whereas the presence of Actinomyces naeslundii was associated with health. Significantly greater numbers and proportions of S. oralis and S. salivarius were isolated from partially erupted teeth with white spot lesions whereas Streptococcus mutans was isolated in significantly greater numbers and proportions from fully erupted molars with white spots. This study suggests that organisms other than mutans streptococci are associated with caries development in erupting permanent molar teeth.

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

ABSTRACT 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 (χ2 = 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 (χ2 = 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.

The metabolism of cholesterol and certain hormonal steroids by Treponema denticola.

The aim was to investigate whether reference cultures and fresh isolates of Treponema denticola are able to 5alpha-reduce and further metabolise testosterone, 4-androstenedione, progesterone, corticosterone, cortisol or cholesterol. Two reference and five freshly isolated cultures of T. denticola were incubated with either radiolabeled or unlabeled steroid substrates; in the first case products were identified by thin layer chromatography and in the latter by gas chromatography-mass spectroscopy. All the substrates were 5alpha-reduced. Both reference cultures and fresh isolates of T. denticola presented 3beta- and 17beta-hydroxy steroid dehydrogenase activity. It was concluded that T. denticola was capable of steroid metabolism and hypotheses are discussed regarding the in vivo function of this metabolism including, T. denticola utilising host supplied steroids as growth factors and T. denticola steroid metabolism acting as a virulence factor.

The Influence of Specific Foods and Oral Hygiene on the Microflora of Fissures and Smooth Surfaces of Molar Teeth: A 5–Day Study

A group of 20 students, harbouring >104 mutans streptococci per millilitre of saliva, was enrolled into the study. Models for sampling, reproducibly, the dental plaque present in specific sites (fissure and smooth surface) on the dentition were developed and validated. Withdrawal of normal oral hygiene procedures for only 1 day resulted in approximately 10–fold increases in the number of micro–organisms recovered from both sites. The effect of supplementing the subjects’ diets with particular food items given 5 times per day [lemonade (5.8% w/v sugars, 250 ml), biscuits (digestive biscuits, 67.6% w/w carbohydrate of which 22% w/w was sugars and 45.6% w/w was starch), caramel toffees and sugar lumps] on the number of micro–organisms recovered and on the composition of the flora at both sites was determined. Dental plaque samples were taken after 5 days and it was found that supplementation of the diet with toffee and sugar lumps resulted in significantly more micro–organisms at both sampling sites. The supplementation of the diets with lemonade or biscuits did not significantly alter the numbers of micro–organisms recovered from either site. The percentage composition of the plaque samples from both dental sites remained relatively unaffected by oral hygiene although there were lower levels of mutans streptococci which might be related to the use of an antimicrobial toothpaste containing fluoride, triclosan and zinc citrate. This study suggests that the cariogenicity of certain sucrose–containing foods may, in part, be due to the enhancement of plaque accumulation in addition to other effects on the percentage composition of the plaque which may become manifest on prolonged usage of these dietary foodstuffs. These observations are consistent with dietary survey findings which often find consumption of confectionery related to caries experience or incidence.