Akihiro Yoshida

β-glycyrrhetinic acid (BGA) possesses antibacterial effects against human supragingival plaque bacteria and inhibits biofilm formation. However, the effect of BGA on already formed dental plaque has not been investigated. We analyzed and compared the effects of BGA on preformed supragingival plaque biofilms with those of cetylpyridinium chloride (CPC). All experiments were performed in vitro using biofilms formed by incubating supragingival plaque bacteria for 24 h. First, we analyzed the number of viable and dead bacteria in the biofilms following BGA and CPC application. The number of viable bacteria was significantly reduced by BGA treatment compared with by the solvent control. However, the viable/dead bacterial ratios did not significantly vary. Conversely, the turbidity in the supernatants (optical density at 600 nm [OD ₆₀₀ ]) was 0.237 ± 0.003, 0.136 ± 0.002, and 0.096 ± 0.002 for the BGA, CPC, and solvent control groups, respectively, indicating superior ability of BGA in biofilm exfoliation. This study suggests that BGA may inhibit dental plaque adhesion and eliminate and disinfect dental plaque through a mechanism different from CPC, contributing to the prevention of periodontal disease.

The oral microbiome plays an important role in the human microbial community and in maintaining the health of an individual. Imbalances in the oral microbiome may contribute to oral and systemic diseases. The progression of periodontal disease is closely related to the growth of bacteria, such as Porphyromonas gingivalis, in the oral cavity. However, the pathogen growth mechanism specific to periodontal disease remains unknown. This study aimed to identify bacteria associated with periodontal health by focusing on hemolytic bacteria. Unstimulated saliva samples were collected from ten periodontitis patients and five healthy subjects to detect and identify the presence of hemolytic bacteria. The saliva of healthy subjects contained a higher proportion of G. haemolysans than saliva samples from patients with periodontitis. Growth inhibition assays indicated that the protein components contained in the culture supernatant of G. haemolysans directly suppressed the growth of P. gingivalis. This study shows that the presence of G. haemolysans in saliva is associated with periodontal health and that it inhibits the growth of P. gingivalis in vitro.

β‐Glycyrrhetinic acid (BGA) is a natural antibacterial agent. Previous studies reported that BGA has antibacterial effects against several bacteria. This study evaluated the effects of BGA on the regulation of supragingival plaque bacteria. First, the minimum inhibitory concentrations (MICs) of BGA against oral bacteria were measured. Next, the minimum concentrations for inhibition of biofilm formation were evaluated against Streptococcus mutans and Streptococcus sobrinus, possessing insoluble glucan synthesis abilities. The MICs of biofilm formation by these bacteria ranged from 1/8 to 2× MIC. Furthermore, the inhibition effects of BGA against the coaggregation of Porphyromonas gingivalis and Streptococcus gordonii were evaluated. BGA at 32 or 64 μg/mL inhibited the coaggregation of these bacteria after a 30 min incubation. Lastly, the inhibition effects of BGA against human supragingival plaque bacteria were evaluated. Human supragingival plaque samples were obtained from 12 healthy donors. The inhibition effects of BGA against biofilm formation by these plaque bacteria were evaluated. Of 12 samples, the biofilm formation by 11 was significantly attenuated by 128–256 μg/mL of BGA. The number of colony forming units in these biofilms was also significantly attenuated. In conclusion, it was revealed that BGA inhibits the growth and biofilm formation of bacteria, furthermore, the same effect was confirmed with supragingival plaque bacteria. BGA is a good candidate for a natural agent that prevents the outbreak and progression of periodontal disease because it suppresses not only the growth and biofilm formation of bacteria, but also the coaggregation of P. gingivalis with plaque bacteria.

Objective: To evaluate the association between alveolar bone loss (ABL) detected on panoramic radiographs and carotid artery calcification (CAC) detected on computed tomography (CT). Methods: The study subjects included 295 patients (mean age ± SD: 64.6 ± 11.8 years) who visited the Matsumoto Dental University Hospital. The rate of ABL and the number of present teeth were measured on panoramic radiographs. Univariate analyses with t-tests and chi-squared tests were performed to evaluate the differences in age, gender, history of diseases, number of present teeth, and the ABL between subjects, with and without CAC. Moreover, multivariate logistic regression analysis, with forward selection and receiver operating characteristic curve (ROC) analysis, was performed. Results: The number of subjects without and with CAC was 174 and 121, respectively. Univariate analyses revealed that CAC was significantly associated with age, hypertension, osteoporosis, number of present teeth, and ABL. Multivariate logistic regression analysis adjusted for covariates revealed that the presence of CAC was significantly associated with ABL (OR = 1.233, 95% CI = 1.167–1.303). In the ROC analysis for predicting the presence of CAC, the the area under the ROC curve was the highest at 0.932 (95% CI = 0.904–0.960) for ABL, which was significant. Conclusions: Our results suggest that the measurement of ABL on panoramic radiographs may be an effective approach to identifying patients with an increased risk of CAC.

Fusobacterium nucleatum is a human periodontal pathogen that causes opportunistic infections. It has been implicated in preterm birth and has as a pathogen of colorectal cancer. However, it is a common member of the oral microbiota and can have a symbiotic relationship with its hosts. To date, studies of F. nucleatum have been hindered by a lack of effective genetic tools, and the transformation of F. nucleatum has not been investigated. In this chapter, protocols for the transformation of F. nucleatum strain 12230 using sonoporation are presented. We also include a genetic complementation protocol for a F. nucleatum knockout mutant.

This study investigated whether oral malodour with plaque-related periodontal disease was associated with the prevalence of periodontopathic bacteria in saliva and tongue debris. The subjects were 30 patients who visited the Breath Odour Clinic. An oral examination was performed at baseline and two months after periodontal treatment, and included measuring the volatile sulphur compounds (VSCs) in mouth air. The proportions of five periodontopathic bacteria versus all bacteria in saliva and tongue debris were determined using real-time polymerase chain reaction. The subjects were classified into four groups based on the existence of VSCs above the organoleptic threshold level (TVSC) and a periodontal pocket ⩾4 mm (PD4). At baseline, the group with TVSC/PD4 had higher proportions of periodontopathic bacteria in saliva compared to those with TVSC/no PD4 and no TVSC/no PD4, but there were no differences in the proportions in tongue debris among the four groups. At follow-up, the proportion of periodontopathic bacteria in the saliva of the group with TVSC/PD4 was significantly reduced, as compared to that at baseline, and the VSCs and periodontal health improved. The results suggested that the reduction in VSCs in mouth air of subjects with periodontal disease might be associated with the prevalence of periodontopathic bacteria in saliva.

Introduction:  Enterococcus faecalis is a major pathogen in the etiology of apical periodontitis after root canal treatment. A loop‐mediated isothermal amplification method, which amplifies DNA with high specificity, efficiency and rapidity under isothermal conditions using a set of four specially designed primers and a DNA polymerase with strand‐displacement activity, was developed for the rapid detection of E. faecalis in clinical specimens from root canals.Methods:  Primers for detecting E. faecalis from the azoA gene were designed. The specificity of this assay was evaluated using various oral bacteria and the sensitivity was evaluated using serially diluted E. faecalis chromosomal DNA. In addition, loop‐mediated isothermal amplification assays were applied to the rapid detection of E. faecalis from endodontic samples.Results:  The loop‐mediated isothermal amplification products amplified with the primer set were specific for E. faecalis. To confirm the specificity of the amplicon, the amplified products were digested with the restriction endonuclease Sau3AI. The lower detection limit of the E. faecalis primer set without the loop primer set was 10 μg/tube for a 50‐min loop‐mediated isothermal amplification reaction. Using loop primers increased the detection sensitivity by several orders of magnitude. Furthermore, E. faecalis was detected with the loop‐mediated isothermal amplification assay in four root canals from 18 individuals and the detection results were consistent with those of conventional polymerase chain reactions.Conclusion:  These results indicate that the loop‐mediated isothermal amplification assay is very useful for rapid detection of E. faecalis and diagnosis of endodontic infection.