Michiyo Matsumoto-Nakano

Cross-cultural validity of a dietary questionnaire for studies of dental caries risk in Japanese.

BackgroundDiet is a major modifiable contributing factor in the etiology of dental caries. The purpose of this paper is to examine the reliability and cross-cultural validity of the Japanese version of the Food Frequency Questionnaire to assess dietary intake in relation to dental caries risk in Japanese.MethodsThe 38-item Food Frequency Questionnaire, in which Japanese food items were added to increase content validity, was translated into Japanese, and administered to two samples. The first sample comprised 355 pregnant women with mean age of 29.2 ± 4.2 years for the internal consistency and criterion validity analyses. Factor analysis (principal components with Varimax rotation) was used to determine dimensionality. The dietary cariogenicity score was calculated from the Food Frequency Questionnaire and used for the analyses. Salivary mutans streptococci level was used as a semi-quantitative assessment of dental caries risk and measured by Dentocult SM. Dentocult SM scores were compared with the dietary cariogenicity score computed from the Food Frequency Questionnaire to examine criterion validity, and assessed by Spearman’s correlation coefficient (rs) and Kruskal-Wallis test. Test-retest reliability of the Food Frequency Questionnaire was assessed with a second sample of 25 adults with mean age of 34.0 ± 3.0 years by using the intraclass correlation coefficient analysis.ResultsThe Japanese language version of the Food Frequency Questionnaire showed high test-retest reliability (ICC = 0.70) and good criterion validity assessed by relationship with salivary mutans streptococci levels (rs = 0.22; p < 0.001). Factor analysis revealed four subscales that construct the questionnaire (solid sugars, solid and starchy sugars, liquid and semisolid sugars, sticky and slowly dissolving sugars). Internal consistency were low to acceptable (Cronbach’s alpha = 0.67 for the total scale, 0.46-0.61 for each subscale). Mean dietary cariogenicity scores were 50.8 ± 19.5 in the first sample, 47.4 ± 14.1, and 40.6 ± 11.3 for the first and second administrations in the second sample. The distribution of Dentocult SM score was 6.8% (score = 0), 34.4% (score = 1), 39.4% (score = 2), and 19.4% (score = 3). Participants with higher scores were more likely to have higher dietary cariogenicity scores (p < 0.001; Kruskal-Wallis test).ConclusionsThese results provide the preliminary evidence for the reliability and validity of the Japanese language Food Frequency Questionnaire.

Contribution of the Interaction of Streptococcus mutans Serotype k Strains with Fibrinogen to the Pathogenicity of Infective Endocarditis

ABSTRACT Streptococcus mutans, a pathogen responsible for dental caries, is occasionally isolated from the blood of patients with bacteremia and infective endocarditis (IE). Our previous study demonstrated that serotype k-specific bacterial DNA is frequently detected in S. mutans-positive heart valve specimens extirpated from IE patients. However, the reason for this frequent detection remains unknown. In the present study, we analyzed the virulence of IE from S. mutans strains, focusing on the characterization of serotype k strains, most of which are positive for the 120-kDa cell surface collagen-binding protein Cbm and negative for the 190-kDa protein antigen (PA) known as SpaP, P1, antigen I/II, and other designations. Fibrinogen-binding assays were performed with 85 clinical strains classified by Cbm and PA expression levels. The Cbm+/PA− group strains had significantly higher fibrinogen-binding rates than the other groups. Analysis of platelet aggregation revealed that SA31, a Cbm+/PA− strain, induced an increased level of aggregation in the presence of fibrinogen, while negligible aggregation was induced by the Cbm-defective isogenic mutant SA31CBD. A rat IE model with an artificial impairment of the aortic valve created using a catheter showed that extirpated heart valves in the SA31 group displayed a prominent vegetation mass not seen in those in the SA31CBD group. These findings could explain why Cbm+/PA− strains are highly virulent and are related to the development of IE, and the findings could also explain the frequent detection of serotype k DNA in S. mutans-positive heart valve clinical specimens.

Distribution of periodontopathic bacterial species in dogs and their owners.

OBJECTIVE Presently, a large number of individuals consider their companion animals as family members and have close contact with them in daily life. The purpose of the present study was to analyze the distribution of periodontopathic bacterial species in oral specimens taken from dogs and their owners. DESIGN Dental plaque specimens were collected from 66 dogs and 81 members of 64 families who came to an animal clinic or dog training school in Okayama, Japan, in 2011. Bacterial DNA was extracted from each specimen and PCR analyses using primers specific for 11 periodontopathic species, Porphyromonas gingivalis, Porphyromonas gulae, Treponema denticola, Tannerella forsythia, Capnocytophaga ochracea, Capnocytophaga sputigena, Prevotella intermedia, Prevotella nigrescens, Aggregatibacter actinomycetemcomitans, Campylobacter rectus, and Eikenella corrodens were performed. RESULTS P. gulae (71.2%), T. forsythia (77.3%), and C. rectus (66.7%) were frequently found in the dogs, whereas the detection rates of those species in humans were less frequent at 16.0%, 30.9%, and 21.0%, respectively. P. gulae was identified in 13 human subjects and each of their dogs was also positive for the species. Furthermore, E. corrodens and T. denticola in specimens obtained from dogs were correlated with their presence in specimens from owners who had close contact with them. CONCLUSIONS These results suggest that several periodontopathic species could be transmitted between humans and their companion dogs, though the distribution of periodontopathic species in both is generally different.

Contribution of glucan-binding protein A to firm and stable biofilm formation by Streptococcus mutans

Glucan-binding proteins (Gbps) of Streptococcus mutans, a major pathogen of dental caries, mediate the binding of glucans synthesized from sucrose by the action of glucosyltransferases (GTFs) encoded by gtfB, gtfC, and gtfD. Several stress proteins, including DnaK and GroEL encoded by dnaK and groEL, are related to environmental stress tolerance. The contribution of Gbp expression to biofilm formation was analyzed by focusing on the expression levels of genes encoding GTFs and stress proteins. Biofilm-forming assays were performed using GbpA-, GbpB-, and GbpC-deficient mutant strains and the parental strain MT8148. The expression levels of gtfB, gtfC, gtfD, dnaK, and groEL were evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Furthermore, the structure of biofilms formed by these Gbp-deficient mutant strains was observed using confocal laser scanning microscopy (CLSM). Biofilm-forming assay findings demonstrated that the amount formed by the GbpA-deficient mutant strain (AD1) was nearly the same as that by the parental strain, while the GbpB- and GbpC-deficient mutant strains produced lower amounts than MT8148. Furthermore, RT-qPCR assay results showed that the expressions of gtfB, dnaK, and groEL in AD1 were elevated compared with MT8148. CLSM also revealed that the structure of biofilm formed by AD1 was prominently different compared with that formed by the parental strain. These results suggest that a defect in GbpA influences the expression of genes controlling biofilm formation, indicating its importance as a protein for firm and stable biofilm formation.

Identification and Functional Analysis of an Ammonium Transporter in Streptococcus mutans

Streptococcus mutans, a Gram-positive bacterium, is considered to be a major etiologic agent of human dental caries and reported to form biofilms known as dental plaque on tooth surfaces. This organism is also known to possess a large number of transport proteins in the cell membrane for export and import of molecules. Nitrogen is an essential nutrient for Gram-positive bacteria, though alternative sources such as ammonium can also be utilized. In order to obtain nitrogen for macromolecular synthesis, nitrogen-containing compounds must be transported into the cell. However, the ammonium transporter in S. mutans remains to be characterized. The present study focused on characterizing the ammonium transporter gene of S. mutans and its operon, while related regulatory genes were also analyzed. The SMU.1658 gene corresponding to nrgA in S. mutans is homologous to the ammonium transporter gene in Bacillus subtilis and SMU.1657, located upstream of the nrgA gene and predicted to be glnB, is a member of the PII protein family. Using a nrgA-deficient mutant strain (NRGD), we examined bacterial growth in the presence of ammonium, calcium chloride, and manganese sulfate. Fluorescent efflux assays were also performed to reveal export molecules associated with the ammonium transporter. The growth rate of NRGD was lower, while its fluorescent intensity was much higher as compared to the parental strain. In addition, confocal laser scanning microscopy revealed that the structure of biofilms formed by NRGD was drastically different than that of the parental strain. Furthermore, transcriptional analysis showed that the nrgA gene was co-transcribed with the glnB gene. These results suggest that the nrgA gene in S. mutans is essential for export of molecules and biofilm formation.

Contributions of Streptococcus mutans Cnm and PA antigens to aggravation of non-alcoholic steatohepatitis in mice.

Streptococcus mutans, a major pathogen of dental caries, can cause infective endocarditis after invading the bloodstream. Recently, intravenous administration of specific S. mutans strains was shown to aggravate non-alcoholic steatohepatitis (NASH) in a mouse model fed a high-fat diet. Here, we investigated the mechanism of this aggravation in a NASH mouse model by focusing on the S. mutans cell surface collagen-binding protein (Cnm) and the 190-kDa protein antigen (PA). Mice that were intravenously administered a S. mutans strain with a defect in Cnm (TW871CND) or PA (TW871PD) did not show clinical or histopathological signs of NASH aggravation, in contrast to those administered the parent strain TW871. The immunochemical analyses demonstrated higher levels of interferon-γ and metallothionein expression in the TW871 group than in the TW871CND and TW871PD groups. Analysis of bacterial affinity to cultured hepatic cells in the presence of unsaturated fatty acids revealed that the incorporation rate of TW871 was significantly higher than those of TW871CND and TW871PD. Together, our results suggest that Cnm and PA are important cell surface proteins for the NASH aggravation caused by S. mutans adhesion and affinity for hepatic cells.

Distribution and molecular characterization of Porphyromonas gulae carrying a new fimA genotype

Porphyromonas gulae is a gram-negative black-pigmented anaerobe which is known to be a pathogen for periodontitis in dogs. Approximately 41kDa filamentous appendages on the cell surface (FimA) encoded by the fimA gene are regarded as important factors associated with periodontitis. The fimA genotype was classified into two major types and strains in type B were shown to be more virulent than those in type A. In the present study, we characterized a strain with a novel fimA genotype and designated it as type C. The putative amino acid sequence was shown to be similar to the genotype IV fimA of Porphyromonas gingivalis, a major pathogen of human periodontitis. Analyses using an oral squamous cell carcinoma cell line derived from tongue primary lesions revealed that the type C strain inhibited proliferation and scratch closure more than genotype A and B strains. In addition, experiments using a mouse abscess model demonstrated that the type C strain could induce much higher systemic inflammation when compared with strains of the other genotypes. Furthermore, molecular analyses of oral swab specimens collected from dogs demonstrated that the detection frequencies of P. gulae and the genotype C in the periodontitis group were significantly higher than those in the periodontally healthy group. These results suggest that FimA of P. gulae is diverse with the virulence of genotype C strains the highest and that molecular identification of genotype C P. gulae could be a possible useful marker for identifying dogs at high risk of developing periodontitis.

Characterization of the dextran‐binding domain in the glucan‐binding protein C of Streptococcus mutans

Streptococcus mutans produces multiple glucan‐binding proteins (Gbps), among which GbpC encoded by the gbpC gene is known to be a cell‐surface‐associated protein involved in dextran‐induced aggregation. The purpose of the present study was to characterize the dextran‐binding domain of GbpC using bioinformatics analysis and molecular techniques.

Regulation of Recombination between gtfB/gtfC Genes in Streptococcus mutans by Recombinase A

Streptococcus mutans produces 3 types of glucosyltransferases (GTFs), whose cooperative action is essential for cellular adhesion. The recombinase A (RecA) protein is required for homologous recombination. In our previous study, we isolated several strains with a smooth colony morphology and low GTF activity, characteristics speculated to be derived from the GTF fusions. The purpose of the present study was to investigate the mechanism of those fusions. S. mutans strain MT8148 was grown in the presence of recombinant RecA (rRecA) protein, after which smooth colonies were isolated. The biological functions and sequences of the gtfB and gtfC genes of this as well as other clinical strains were determined. The sucrose-dependent adherence rates of those strains were reduced as compared to that of MT8148. Determination of the sequences of the gtfB and gtfC genes showed that an approximately 3500 bp region was deleted from the area between them. Furthermore, expression of the recA gene was elevated in those strains as compared to MT8148. These results suggest that RecA has an important role in fusions of gtfB and gtfC genes, leading to alteration of colony morphology and reduction in sucrose-dependent adhesion.

Variation of expression defects in cell surface 190-kDa protein antigen of Streptococcus mutans.

Streptococcus mutans, which consists of four serotypes, c, e, f, and k, possesses a 190-kDa cell surface protein antigen (PA) for initial tooth adhesion. We used Western blot analysis to determine PA expression in 750 S. mutans isolates from 150 subjects and found a significantly higher prevalence of the isolates with PA expression defects in serotypes f and k compared to serotypes c and e. Moreover, the defect patterns could be classified into three types; no PA expression on whole bacterial cells and in their supernatant samples (Type N1), PA expression mainly seen in supernatant samples (Type N2), and only low expression of PA in the samples of whole bacterial cells (Type W). The underlying reasons for the defects were mutations in the gene encoding PA as well as in the transcriptional processing of this gene for Type N1, defects in the sortase gene for Type N2, and low mRNA expression of PA for Type W. Since cellular hydrophobicity and phagocytosis susceptibility of the PA-defective isolates were significantly lower than those of the normal expression isolates, the potential implication of such defective isolates in systemic diseases involving bacteremia other than dental caries was suggested. Additionally, multilocus sequence typing was utilized to characterize S. mutans clones that represented a proportion of isolates with PA defects of 65-100%. Therefore, we described the molecular basis for variation defects in PA expression of S. mutans. Furthermore, we also emphasized the strong association between PA expression defects and serotypes f and k as well as the clonal relationships among these isolates.