Steven C. Gilbert

Gordonia amicalis sp. nov., a novel dibenzothiophene-desulphurizing actinomycete

The taxonomic position of a dibenzothiophene-desulphurizing soil actinomycete was established using a polyphasic taxonomic approach. The organism, strain IEGMT, was shown to have chemical and morphological properties typical of members of the genus Gordonia. The tested strain formed a distinct phyletic line within the evolutionary radiation occupied by the genus Gordonia, with Gordonia alkanivorans DSM 44369T, Gordonia desulfuricans NCIMB 40816T and Gordonia rubropertincta DSM 43197T as the most closely related organisms. Strain IEGMT has a range of phenotypic properties that distinguish it from representatives of all of the validly described species of Gordonia. It was also sharply distinguished from the type strains of Gordonia desulfuricans and Gordonia rubropertincta on the basis of DNA-DNA relatedness data. The combined genotypic and phenotypic data show that strain IEGMT merits recognition as a new species of Gordonia. The name proposed for the new species is Gordonia amicalis; the type strain is IEGMT (= DSM 44461T = KCTC 9899T).

The Isolation of Bifidobacteria from Occlusal Carious Lesions in Children and Adults

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.

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.

Propionibacterium acnes and Staphylococcus epidermidis Isolated from Refractory Endodontic Lesions Are Opportunistic Pathogens

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.

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/).

Gordonia desulfuricans sp. nov., a benzothiophene-desulphurizing actinomycete

The taxonomic position of two actinomycetes isolated from soil was established using a polyphasic approach. The organisms, designated 213ET and 213F, were found to have chemical and morphological properties consistent with their assignment to the genus Gordonia. Nearly complete sequences of the 16S rDNA genes of the two strains were determined following the isolation and direct sequencing of the amplified genes. The tested strains were found to have identical 16S rDNA sequences and formed a phylogenetic line within the evolutionary radiation occupied by the genus Gordonia that was most closely related to Gordonia rubropertincta DSM 43197T. However, DNA-DNA relatedness data showed that strain 213ET and Gordonia rubropertincta DSM 43197T belonged to distinct genomic species. Strains 213ET and 213F also shared an identical phenotypic profile which distinguished them from representatives of validly described Gordonia species. The combined genotypic and phenotypic data show that strains 213ET and 213F merit recognition as a new species of Gordonia. The name proposed for the new species is Gordonia desulfuricans, for which the type strain is 213ET (= NCIMB 40816T).

Isolation and Identification of Bifidobacteriaceae from Human Saliva

ABSTRACT Bifidobacteriaceae were isolated from saliva and infected dentine by using a mupirocin-based selective medium. Of the saliva samples, 94% harbored bifids. The mean concentration (± the standard error) was 4.46 (±0.12) log10(CFU per ml + 1), and the predominant isolates were Bifidobacterium dentium, B. longum, Scardovia inopinata, Parascardovia denticolens, and Alloscardovia omnicolens.

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.

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.

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).