Lantibiotics Produced by Oral Inhabitants as a Trigger for Dysbiosis of Human Intestinal Microbiota.

Lantibiotics are a type of bacteriocin produced by Gram-positive bacteria and have a wide spectrum of Gram-positive antimicrobial activity. In this study, we determined that Mutacin I/III and Smb (a dipeptide lantibiotic), which are mainly produced by the widespread cariogenic bacterium Streptococcus mutans, have strong antimicrobial activities against many of the Gram-positive bacteria which constitute the intestinal microbiota. These lantibiotics also demonstrate resistance to acid and temperature. Based on these features, we predicted that lantibiotics may be able to persist into the intestinal tract maintaining a strong antimicrobial activity, affecting the intestinal microbiota. Saliva and fecal samples from 69 subjects were collected to test this hypothesis and the presence of lantibiotics and the composition of the intestinal microbiota were examined. We demonstrate that subjects possessing lantibiotic-producing bacteria in their oral cavity exhibited a tendency of decreased species richness and have significantly reduced abundance of the phylum Firmicutes in their intestinal microbiota. Similar results were obtained in the fecal microbiota of mice fed with S. mutans culture supernatant containing the lantibiotic bacteriocin Mutacin I. These results showed that lantibiotic bacteriocins produced in the oral cavity perturb the intestinal microbiota and suggest that oral bacteria may be one of the causative factors of intestinal microbiota dysbiosis.

Differential expression of the Smb bacteriocin in Streptococcus mutans isolates.

The two-component lantibiotic Smb is produced by Streptococcus mutans GS5. In the present study, we identified seven strains of S. mutans containing the smb gene cluster. These strains could be classified into high- and low-level Smb producers relative to the levels of Smb production by indicator strains in vitro. This classification was dependent upon the transcription levels of the structural smbA and smbB genes. Sequence analysis upstream of smbA in the high- and low-level Smb-producing strains revealed differences at nucleotide position -46 relative to the smbA start codon. Interestingly, the transcription start site was present upstream of the point mutation, indicating that both groups of strains have the same promoter constructs and that the differential expression of smbA and smbB mRNA occurred subsequent to transcription initiation. In addition, smbA::lacZ fusion expression was higher when it was regulated by the sequences of strains with high-level Smb activity than when it was regulated by the comparable region from strains with low-level Smb activity. Taken together, we conclude that high- or low-level Smb expression is dependent on the presence of a G or a T nucleotide at position -46 relative to the smbA translational start site in S. mutans Smb producers.

[Assessment of gene associated with Streptococcus mutans biofilm formation].

Streptococcus mutans, the primary etiological agent of human dental caries and an obligate biofilm-forming bacterium, has developed a variety of mechanisms to colonize the tooth surface. Oral transmission of S. mutans through contact between mother and child is thought to be one of the risks of developing dental caries. In this study, I surveyed oral transmission frequency of S. mutans from mother to a 3-year-old child. In 19 (10.9%) of 174 mother-child pairs, S. mutans was isolated from both mother and chihld. The identities of genomic DNA from S. mutans in 5 (45.5%) of the 11 mother-child pairs were presented. Among those, the biofilms formed by S. mutans 3 c and 4 c, respectively, which had high and low ability of biofilm formation, were examined by confocal laser scanning microscopy (CLSM). Microscopic analysis revealed that the volume of biofilm formation in 3 c was higher than that in 4 c biofilm in the bottom. Furthermore, DNA microarrays were used to study the gene expression profiles of 3 c and 4 c biofilms. In this paper we describe that about 3.8% of genes showed differential expression; about 2.2% of genes were activated and about 1.6% were repressed in 3 c biofilm compared with 4 c biofilm. The present study suggests that biofilm gene expression is strongly associated with differential biofilm formation. Our identification of biofilm-involved genes points to mechanisms of the virulence of S. mutans and provides a first foothold for studying the natural history of S. mutans infections in mother and child.

Correlation between patient satisfaction and dental clinic credibility in regular dental check-ups in Japan.

The aim of this study was to investigate the relationship between dental clinic credibility and patient satisfaction with regular dental check-ups, to compare the level of satisfaction of patients who had regular check-ups with those who did not, and to identify factors associated with regular dental checkups. Thirty-nine private dental clinics in 17 prefectures throughout Japan participated in this study. A total of 9024 questionnaires were distributed to the patients at these clinics, and patients returned the questionnaires using a pre-paid envelope (response rate 56.8%). The questionnaires consisted of items related to patient demographics and 11 items concerning the level of patient satisfaction with the dental clinic. Using multiple logistic regression analysis, the strongest correlation was found between dental clinic credibility in regular checkups and the technical competence of the dentist. The responses to those items concerning making an appointment, and the cleanliness and neatness of the waiting room were different between patients who had regular check-ups and those who did not. To investigate the factors that encourage regular dental check-ups, correlation of factors with regular check up experience were analyzed. Multivariate-adjusted odds ratios indicated a strong association between regular dental check ups and both the technical competence of the dental hygienist and the cost of treatment.

Characteristics and willingness of patients to pay for regular dental check-ups in Japan.

The purpose of this survey was to investigate the relationship between demographic characteristics and willingness of patients to pay for regular dental check-ups in Japan. Questionnaires were distributed at private dental offices and collected via postage-paid return envelopes addressed to the center of the study groups. Questions focused on demographics and willingness to pay for regular check-ups. Five thousand one hundred thirty-two questionnaires were collected (response rate 56.8%). The 3 groups most likely to have regular dental check-ups were found to be the under 20s, 50 to 59 year olds and civil servants. Of these groups, civil servants were found to be the most likely of all to have regular check-ups. More females than males were represented in the sample. More than 60% of the patients responded that they would be willing to pay for regular check-ups if the cost were less than 2,000 yen (about 20 dollars). However, no statistically significant differences were observed in relation to household income. The results suggested that participation in regular dental check-ups might be related to gender and age, but not to household income.

Inhibiting effects of Enterococcus faecium non-biofilm strain on Streptococcus mutans biofilm formation.

BACKGROUND AND PURPOSE: Enterococcus faecium is a normal bowel commensal and lactic acid bacterium that is rarely found in the oral cavity. This study investigated whether a non-pathogenic and non-biofilm strain of E. faecium functioned as a probiotic strain toward biofilm formation by Streptococcus mutans, which is an etiological agent for dental caries. METHODS: The effects of E. faecium on streptococcal biofilm was evaluated by absorbance of safranin-stained biofilm at 492 nm in a 96-well microtiter plate. RESULTS: The E. faecium strain demonstrated cell-number-dependent inhibition of biofilm in dual cultures with 4 laboratory and 16 clinical S. mutans strains, as well as laboratory strains of Streptococcus sobrinus and Streptococcus sanguinis, in 96-well microtiter plates. The inhibiting effects of E. faecium were not dependent on the production of bacteriocin from streptococci and E. faecium, low pH after mix culture, or biofilm formation levels of S. mutans. A culture supernatant sample of more than 10 kDa from E. faecium showed direct inhibiting effects toward S. mutans biofilm formation. Treatment of heat, butanol, and phenol to a supernatant sample restored biofilm formation in culture of S. mutans with the sample. Moreover, the tendencies of inhibition levels by the supernatant sample were associated with those by bacterial cells of E. faecium to S. mutans strains. CONCLUSIONS: The E. faecium non-biofilm strain produced an inhibiting protein to streptococci biofilm formation, showed various susceptibilities to inhibit streptococcal biofilm, and acted as a probiotic bacterial inhibitor of streptococcal biofilm formation.

Assessment of genes associated with Streptococcus mutans biofilm morphology.

Streptococcus mutans, the major pathogen responsible for dental caries in humans, is a biofilm-forming bacterium. In the present study, 17 different pulsed-field gel electrophoresis patterns of genomic DNA were identified in S. mutans organisms isolated clinically from whole saliva. The S. mutans isolates showed different abilities to form biofilms on polystyrene surfaces in semidefined minimal medium cultures. Following cultivation in a flow cell system in tryptic soy broth with 0.25% sucrose and staining using a BacLight LIVE/DEAD system, two strains, designated FSC-3 and FSC-4, showed the greatest and least, respectively, levels of biofilm formation when examined with confocal laser scanning microscopy. Further, image analyses of spatial distribution and architecture were performed to quantify the merged green (live cells) and red (dead cells) light. The light intensity of the FSC-3 biofilm was greater than that of the FSC-4 biofilm in the bottom area but not in the top area. S. mutans whole-genome array results showed that approximately 3.8% of the genes were differentially expressed in the two strains, of which approximately 2.2%, including bacitracin transport ATP-binding protein gene glrA and a BLpL-like putative immunity protein gene, were activated in FSC-3. In addition, about 1.6% of the genes, including those associated with phosphotransferase system genes, were repressed. Analyses of the glrA-deficient strains and reverse transcription-PCR confirmed the role of the gene in biofilm formation. Differential assessment of biofilm-associated genes in clinical strains may provide useful information for understanding the morphological development of streptococcal biofilm, as well as for colonization of S. mutans.