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Fusobacterium nucleatum is classified as four subspecies, subsp. nucleatum, polymorphum, vincentii, and animalis, based on DNA-DNA hybridization (DDH) patterns, phenotypic characteristics, and/or multilocus sequence analysis (MLSA). The gold standards for classification of bacterial species are DDH and 16S ribosomal RNA gene (16S rDNA) sequence homology. The thresholds of DDH and 16S rDNA similarity for delineation of bacterial species have been suggested to be >70 and 98.65%, respectively. Average nucleotide identity (ANI) and genome-to-genome distance (GGD) analysis based on genome sequences were recently introduced as a replacement for DDH to delineate bacterial species with ANI (95-96%) and GGD (70%) threshold values. In a previous study, F. hwasookii was classified as a new species based on MLSA and DDH results. 16S rDNA similarity between F. hwasookii type strain and F. nucleatum subspecies type strains was higher than that between F. nucleatum subspecies type strains. Therefore, it is possible that the four F. nucleatum subspecies can be classified as Fusobacterium species. In this study, we performed ANI and GGD analyses using the genome sequences of 36 F. nucleatum, five F. hwasookii, and one Fusobacterium periodonticum strain to determine whether the four F. nucleatum subspecies could be classified as species using OrthoANI and ANI web-based softwares provided by ChunLab and Kostas lab, respectively, and GGD calculator offered by German Collection of Microorganisms and Cell Cultures. ANI values calculated from OrthoANI and ANI calculators between the type strains of F. nucleatum subspecies ranged from 89.80 to 92.97 and from 90.40 to 91.90%, respectively. GGD values between the type strains of F. nucleatum subspecies ranged from 42.3 to 46.0%. ANI and GGD values among strains belonging to the same F. nucleatum subspecies, subsp. nucleatum, subsp. polymorphum, subsp. vincentii, and subsp. animalis were >96 and >68.2%, respectively. These results strongly suggest that F. nucleatum subsp. nucleatum, subsp. polymorphum, subsp. vincentii, and subsp. animalis should be classified as F. nucleatum, F. polymorphum, F. vincentii, and F. animalis, respectively.

The aim of this study was to determine the optimal concentration of Korean propolis against clinical isolates of mutans streptococci (MS) from Koreans. The antimicrobial activity was evaluated using the minimum inhibitory concentration (MIC) and time-kill curves against mutans streptococci. The MIC(90) values of propolis for MS were 35 μg/ml. Propolis had a bacteriostatic effect on Streptococcus mutans ATCC 25175(T) and bactericidal effects on Streptococcus sobrinus ATCC 33478(T) at > 2 × MIC (70 μg/ml). These results suggest that the propolis can be used in the development of oral hygiene products for the prevention of dental caries.

Periodontal diseases are infectious polymicrobial inflammatory diseases that lead to destruction of the periodontal ligament, gingiva, and alveolar bone. Sequential colonization of a broad range of bacteria, including Fusobacterium nucleatum and Porphyromonas gingivalis, is an important phenomenon in this disease model. F. nucleatum is a facultative anaerobic species thought to be a key mediator of dental plaque maturation due to its extensive coaggregation with other oral bacteria, while P. gingivalis is an obligate anaerobic species that induces gingival inflammation by secreting various virulence factors. The formation of a bacterial complex by these two species is central to the pathogenesis of periodontal disease. Reactive oxygen species (ROS) are produced during bacterial infections and are involved in intracellular signaling. However, the impact of oral bacteria-induced ROS on the ecology of F. nucleatum and P. gingivalis has yet to be clarified. In the present study, we investigated ROS production induced in primary human oral cells by F. nucleatum and P. gingivalis and its effect on the formation of their bacterial complexes and further host cell apoptosis. We found that in primary human gingival fibroblasts (GFs), two NADPH oxidase isoforms, NOX1 and NOX2, were activated in response to F. nucleatum infection but not P. gingivalis infection. Accordingly, increased NADPH oxidase activity and production of superoxide anion were observed in GFs after F. nucleatum infection, but not after P. gingivalis infection. Interestingly, in NOX1, NOX2, or NOX1/NOX2 knockdown cells, the number of P. gingivalis decreased when the cells were coinfected with F. nucleatum. A similar pattern of host cell apoptosis was observed. This implies that F. nucleatum contributes to attachment of P. gingivalis by triggering activation of NADPH oxidase in host cells, which may provide an environment more favorable to strict anaerobic bacteria and have a subsequent effect on apoptosis of host cells.

The objective of the study was to investigate the antimicrobial effects of purified single compounds from ethanol-extracted licorice root on Streptococcus mutans. The crude licorice root extract (CLE) was obtained from Glycyrrhiza uralensis, which was subjected to column chromatography to separate compounds. Purified compounds were identified by mass spectrometry and nuclear magnetic resonance. Antimicrobial activities of purified compounds from CLE were evaluated by determining the minimum inhibitory concentration and by performing time-kill kinetics. The inhibitory effects of the compounds on biofilm development were evaluated using crystal violet assay and confocal microscopy. Cell toxicity of substances to normal human gingival fibroblast (NHGF) cells was tested using a methyl thiazolyl tetrazolium assay. Chlorhexidine digluconate (CHX) was used in the control group. Three antimicrobial flavonoids, 1-methoxyficifolinol, licorisoflavan A, and 6,8-diprenylgenistein, were isolated from the CLE. We found that the three flavonoids and CHX had bactericidal effects on S. mutans UA159 at the concentration of ≥4 and ≥1 µg/ml, respectively. The purified compounds completely inhibited biofilm development of S. mutans UA159 at concentrations over 4 μg/ml, which was equivalent to 2 μg/ml of CHX. Confocal analysis showed that biofilms were sparsely scattered in the presence of over 4 μg/ml of the purified compounds. However, the three compounds purified from CLE showed less cytotoxic effects on NHGF cells than CHX at these biofilm-inhibitory concentrations. Our results suggest that purified flavonoids from CLE can be useful in developing oral hygiene products, such as gargling solutions and dentifrices for preventing dental caries.

On the basis of the DNA-DNA hybridization patterns and phenotypic characteristics, Fusobacterium nucleatum was classified into five subspecies. Previous studies have suggested that F. nucleatum subsp. vincentii is genetically similar to F. nucleatum subsp. fusiforme. The aim of this study was to investigate the possibility of classifying these two subspecies into a single subspecies by phylogenetic analysis using a single sequence (24,715 bp) concatenated 22 housekeeping genes of eight F. nucleatum strains including type strains of five F. nucleatum subspecies. The phylogenetic analysis indicated that F. nucleatum subsp. vincentii and F. nucleatum subsp. fusiforme were clustered in the same group and each strain of other F. nucleatum subspecies were also separated into the same cluster. These results suggested that F. nucleatum subsp. fusiforme and F. nucleatum subsp. vincentii can be classified into a single subspecies. F. nucleatum subsp. vincentii was early published name; therefore, F. nucleatum subsp. fusiforme Gharbia and Shah 1992 can be regarded as a later synonym of F. nucleatum subsp. vincentii Dzink et al. 1990.

In this study, we introduced species-specific quantitative real-time PCR (qPCR) primers designed based on a DNA-dependent RNA polymerase beta-subunit gene (rpoB) for detecting 42 oral bacterial species. The specificity of the qPCR primers was confirmed by conventional PCR with the genomic DNAs of 73-79 strains regarding 73-75 bacterial species including the type strain for the target species. The standard curves revealed the lower detection limits of 42 bacterial species-specific qPCR primers ranged from 4 to 40 fg below a cycle threshold (C T) value of 35, except Atopobium rimae, Fusobacterium nucleatum, Neisseria meningitidis, and Porphyromonas asaccharolytica which were 400 fg. These results suggest that 42 bacterial species-specific qPCR primers are suitable for applications in epidemiological studies related to oral infectious diseases such as periodontal diseases, endodontic infection, and dental caries.

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