Veillonella nakazawae sp. nov., an anaerobic Gram-negative coccus isolated from the oral cavity of Japanese children.

Two strains of previously unknown Gram-negative cocci, T1-7T and S6-16, were isolated from the oral cavity of healthy Japanese children. The two strains showed atypical phenotypic characteristics of members of the genus Veillonella, including catalase production. Sequencing of their 16S rRNA genes confirmed that they belong to genus Veillonella. Under anaerobic conditions, the two strains produced acetic acid and propionic acid as metabolic end-products in a trypticase-yeast extract-haemin medium containing 1 % (w/v) glucose, 1 % (w/v) fructose and 1 % (v/v) sodium lactate. Comparative analysis of the 16S rRNA, dnaK, rpoB and gltA gene sequences revealed that the two strains are phylogenetically homogeneous and comprise a distinct, novel lineage within the genus Veillonella. The sequences from the two strains shared the highest similarity, at 99.9, 95.8, 96.9 and 96.7 %, using the partial 16S rRNA, dnaK, rpoB and gltA gene sequences, respectively, with the type strains of the two most closely related species, Veillonella dispar ATCC 17748T and Veillonella infantium JCM 31738T. Furthermore, strain T1-7T shared the highest average nucleotide identity (ANI) value (94.06 %) with type strain of the most closely related species, V. infantium. At the same time, strain T1-7T showed the highest digital DNA-DNA hybridization (dDDH) value (55.5 %) with the type strain of V. infantium. The two strains reported in this study were distinguished from the previously reported species from the genus Veillonella based on catalase production, partial dnaK, rpoB and gltA sequences, average ANI and dDDH values. Based on these observations, the two strains represent a novel species, for which the name Veillonella nakazawae sp. nov. is proposed. The type strain is T1-7T (JCM 33966T=CCUG 74597T).

Description of Streptococcus mutans, Streptococcus sanguinis, and Candida albicans biofilms after exposure to propolis dentifrice by using OpenCFU method.

CONTEXT: Dental caries is a major and chronic dental public health problem, which can usually be prevented by regular oral hygiene. The most common oral hygiene practice is brushing teeth with a dentifrice. Propolis has emerged as a promising anti-cariogenic agent, which is considered to be a good oral antiseptic for prevention of caries. Several studies have shown that the use of C has an influence in the growth of oral biofilms. There are several standard methods used to count bacterial colonies, such as crystal violet and CFU Count assays. OpenCFU method is a technique that can be used to calculate biofilm colonies more faster, precisely, and accurately. AIM: To compare several methods for evaluating the number of biofilm colonies formed with exposure to a standard dentifrice and propolis. METHODS AND MATERIALS: Biofilm assays were carried out on 96-well microplates. Reference strains of oral Streptococcus species (S. mutans ATCC 25175T and S. sanguinis ATCC 10566T) and yeast (Candida albicans ATCC 10231T) were inoculated into wells, and 200 µL of standard and propolis dentifrice solution were added to each well and incubated for 18 h at 37 °C. Bacteria and yeast were then sub-cultured on respective media and the colony-forming units (CFU) were counted manually. The other wells were stained by crystal violet and incubated for 15 min, followed by observation using an inverted microscope and evaluated using crystal violet analysis and the OpenCFU automated method. RESULTS: The numbers of CFUs determined for all strains were similar in the standard-dentifrice group and propolis-dentifrice group, and were similar among the three methods: crystal violet staining, manual CFU count, and OpenCFU analysis. CONCLUSION: OpenCFU analysis can be reliably used as a rapid and a more practical method to analyse the growth of oral microorganism biofilms. However, high digital image quality is required to provide an accurate analysis for colony counting.

Identification and phylogenetic analysis of oral Veillonella species isolated from the saliva of Japanese children.

Background: As the most frequent infectious disease among children worldwide, dental caries have a strong relationship with oral hygiene status, specifically in the development of infection. However, the study regarding the identification and distribution of oral Veillonella are limited. The oral Veillonella community may affected by the differences in geographical location, age, diet, lifestyle, socio-economic status and oral hygiene status. Here, we studied the oral hygiene status by examining the composition and proportion of oral Veillonella species in saliva of Japanese children. Methods: Microbial samples collected from 15 Japanese children divided into three oral hygiene groups were cultured under anaerobic conditions after homogenization and dilution, and inoculated onto brain heart infusion and selective medium Veillonella agar. Genomic DNA was extracted from each isolate. Veillonella species were detected by one-step PCR using rpoB species-specific primers. To analyse the phylogenetic properties of the unknown Veillonella strains, PCR amplification and sequence analysis of rpoB were conducted for 10 representative strains. Results: Although V. rogosae was found as the predominant species among all groups, its prevalence was significantly lower in the children with poor oral hygiene than in those with good oral hygiene. V. parvula was the prevalent species in the poor oral hygiene group. Approximately 10% of the isolated Veillonella strains were not classified to any established species; the phylogenetic analysis showed that they were most closely related to V.infantiumConclusions: This study demonstrates that the composition and proportion of oral Veillonella species in the saliva of Japanese children is correlated with different oral hygiene status. Changes in detection ratios of V. parvula and V. rogosae can be useful indicators of oral hygiene status. Furthermore, new strains closely related to V. infantium were isolated from the saliva of Japanese children.

Establishment of a species-specific primer pair for detecting Veillonella infantium based on the 70 kDa heat shock protein gene dnaK.

Recently, Veillonella infantium was isolated from tongue biofilm of a Thai child and established as a novel Veillonella species. In this study, a species-specific primer was designed to identify V. infantium on the basis of the sequence of the 70 kDa heat shock protein (dnaK) gene of Veillonella infantium JCM 31738T (= TSD-88T). The primer pair generated a specific PCR (Polymerase Chain Reaction) product specific for V. infantium, but not for other oral Veillonella species. This specific primer pair could detect dnaK even from 1 pg of genomic DNA extracted from the V. infantium type strain. To validate the primer pair, a number of strains of Veillonella species were isolated from tongue biofilm of 3 Japanese children, DNA was isolated from each strain, and PCR was performed using species-specific primers. All oral Veillonella species except V. infantium were identified by one-step PCR method reported previously. Four kinds of Veillonella species were detected in these subjects. V. rogosae was detected in all subjects and the most predominant species with an average prevalence of 82%. However, V. infantium was detected in 2 of 3 subjects and it was the second most predominant species of oral Veillonella detected in these subjects with an average prevalence of 9.4%. V. infantium appears to coexist with other oral Veillonella species in tongue biofilm. This species-specific primer pair established in this study could be useful to detect V. infantium and support the study of Veillonella for oral health in the future.