Intracanal microbiome profiles of two apical periodontitis cases in one patient: A comparison with saliva and plaque profiles.

OBJECTIVES: To determine the characteristics of the endodontic microbiome. MATERIAL AND METHODS: Saliva, plaque, and infected root canal wall dentin of two teeth suffering from apical periodontitis were harvested from a 58-year-old man. Bacterial DNA was extracted from each sample, and 16S rRNA gene analysis targeting the V3-V4 region was conducted on the Illumina MiSeq platform using QIIME2. The functional potential of the microbiomes was inferred using PICRUSt2. RESULTS: The four microbiomes were different in structure and membership, yet the nine most abundant metabolic pathways were common among them. The two endodontic microbiomes were more anaerobic, rich in Firmicutes, and scarce in Actinobacteriota and Proteobacteria, compared with saliva and plaque microbiomes. Their profiles were dissimilar despite their clinical and radiographic similarities. CONCLUSIONS: The endodontic microbiomes were anaerobic, rich in Firmicutes, scarce in Actinobacteriota and Proteobacteria, and considerably varied within an individual.

Profiling of Microbiota at the Mouth of Bottles and in Remaining Tea after Drinking Directly from Plastic Bottles of Tea.

It has been speculated that oral bacteria can be transferred to tea in plastic bottles when it is drunk directly from the bottles, and that the bacteria can then multiply in the bottles. The transfer of oral bacteria to the mouth of bottles and bacterial survival in the remaining tea after drinking directly from bottles were examined immediately after drinking and after storage at 37 °C for 24 h. Twelve healthy subjects (19 to 23 years of age) were asked to drink approximately 50 mL of unsweetened tea from a plastic bottle. The mouths of the bottles were swabbed with sterile cotton, and the swabs and the remaining tea in the bottles were analyzed by anaerobic culture and 16S rRNA gene sequencing. Metagenomic analysis of the 16S rRNA gene was also performed. The mean amounts of bacteria were (1.8 ± 1.7) × 104 colony-forming units (CFU)/mL and (1.4 ± 1.5) × 104 CFU/mL at the mouth of the bottles immediately after and 24 h after drinking, respectively. In contrast, (0.8 ± 1.6) × 104 CFU/mL and (2.5 ± 2.6) × 106 CFU/mL were recovered from the remaining tea immediately after and 24 h after drinking, respectively. Streptococcus (59.9%) were predominant at the mouth of the bottles immediately after drinking, followed by Schaalia (5.5%), Gemella (5.5%), Actinomyces (4.9%), Cutibacterium (4.9%), and Veillonella (3.6%); the culture and metagenomic analyses showed similar findings for the major species of detected bacteria, including Streptococcus (59.9%, and 10.711%), Neisseria (1.6%, and 24.245%), Haemophilus (0.6%, and 15.658%), Gemella (5.5%, and 0.381%), Cutibacterium (4.9%, and 0.041%), Rothia (2.6%, and 4.170%), Veillonella (3.6%, and 1.130%), Actinomyces (4.9%, and 0.406%), Prevotella (1.6%, and 0.442%), Fusobacterium (1.0%, and 0.461%), Capnocytophaga (0.3%, and 0.028%), and Porphyromonas (1.0%, and 0.060%), respectively. Furthermore, Streptococcus were the most commonly detected bacteria 24 h after drinking. These findings demonstrated that oral bacteria were present at the mouth of the bottles and in the remaining tea after drinking.

Profiling of microbiota in liquid baby formula consumed with an artificial nipple.

It is suspected that oral bacteria are transferred to the liquid baby formula through the artificial nipple and multiply in the bottle after feeding. In the present study, in order to understand the influence of bacteria on liquid baby formula after feeding, the transfer of oral bacteria through artificial nipples and their survival in liquid baby formula were examined immediately after drinking as well as after storage at 4°C for 3 h. Four healthy human subjects (20-23 years old) were asked to drink liquid baby formula (Aptamil®, ca. 50 mL) from baby bottles using artificial nipples. Samples of the liquid baby formula (immediately after drinking and 3 h later) were inoculated onto blood agar plates and incubated anaerobically at 37°C for 7 days. Salivary samples from each subject and 6 newborn infants were also cultured. Genomic DNA was extracted from individual colonies, and bacterial species were identified by 16S rRNA gene sequencing. The mean amounts of bacteria (CFU/mL) were (3.2 ± 3.0) ×104 and (3.4 ± 3.3) ×104 immediately after drinking and 3 h later, respectively. Streptococcus (41.6 and 40.5%), Actinomyces (24.3 and 21.5%) and Veillonella (16.2 and 11.0%) were recovered from the samples immediately after drinking and 3 h later, respectively. On the other hand, Streptococcus (38.9%), Actinomyces (17.1%), Neisseria (9.1%), Prevotella (6.9%), Rothia (6.9%) and Gemella (5.1%) were predominant in the saliva of adult subjects, and Streptococcus (65.2%), Staphylococcus (18.5%), Gemella (8.2%) and Rothia (5.4%) were predominant in the saliva of infant subjects. From these findings, oral bacteria, e.g., Streptococcus, Gemella and Rothia, were found to transfer into the liquid baby formula through artificial nipples, and the bacterial composition in the remaining liquid baby formula was found to resemble that of human saliva. The bacterial levels were similar between immediately after drinking and when stored at 4°C for 3 h, suggesting that the remaining liquid baby formula may be preserved in a refrigerator for a specified amount of time.

Improvement of periodontal condition by probiotics with Lactobacillus salivarius WB21: a randomized, double-blind, placebo-controlled study.

AIM: This randomized clinical study was designed to evaluate the effect of probiotic intervention using lactobacilli on the periodontal condition of volunteers without severe periodontitis. MATERIAL AND METHODS: Freeze-dried Lactobacillus salivarius WB21 (WB21)-containing tablets or a placebo were given to volunteers in a double-blind randomized study. A total of 66 volunteers were finally enrolled and randomly assigned to receive tablets containing WB21 (6.7 x 10(8) CFU) with xylitol or xylitol alone (placebo) three times a day for 8 weeks. Periodontal clinical parameters and whole saliva samples were obtained at baseline (BL), 4 weeks, and the end of the interventional period (8 weeks). Salivary lactoferrin (Lf) levels were measured by enzyme-linked immunosorbent assay. Lactobacilli in saliva and plaque samples was detected by semi-quantitative RT-PCR using 16S rRNA primers. RESULTS: Periodontal clinical parameters were improved in both groups after an 8-week intervention. Current smokers in the test group showed a significantly greater improvement of plaque index and probing pocket depth from BL when compared with those in the placebo group. Salivary Lf level was also significantly decreased in the test group smokers. CONCLUSION: Our results indicate that probiotics could be useful in the improvement/maintenance of oral health in subjects at a high risk of periodontal disease.

Prevalence and risk factors for peri-implant diseases in Japanese adult dental patients.

We investigated the prevalences and risk factors for peri-implant diseases in Japanese adult dental patients attending a follow-up visit at dental hospitals or clinics as part of their maintenance program. This cross-sectional multicenter study enrolled patients with dental implants who attended regular check-ups as part of a periodontal maintenance program during the period from October 2012 through September 2013. Patients with implants with at least 3 years of loading time were included in the study. The condition of peri-implant tissue was examined and classified into the following categories: healthy, peri-implant mucositis, and peri-implantitis. Patients were also evaluated for implant risk factors. A total of 267 patients (110 men, 157 women; mean age: 62.5 ± 10.7 years) were analyzed. The prevalence of patient-based peri-implant mucositis was 33.3% (n = 89), and the prevalence of peri-implantitis was 9.7% (n = 26). Poor oral hygiene and a history of periodontitis were strong risk factors for peri-implant disease. The present prevalences were lower than those previously reported. The quality of periodontal therapy before and after implant installation and patient compliance and motivation, as indicated by plaque control level, appear to be important in maintaining peri-implant tissue health.

Effect of tranilast on matrix metalloproteinase-1 secretion from human gingival fibroblasts in vitro.

BACKGROUND: Some drugs such as phenytoin, calcium blockers, or cyclosporins are known to cause gingival fibrous hyperplasia, an unwanted side effect. Decreased collagen catabolism in overgrown gingival tissue has been proposed as one of the reasons causing the disease. The effect of tranilast, which suppresses collagen synthesis and cell proliferation, on matrix metalloproteinase (MMP-1) secretion from human gingival fibroblast, was studied in vitro. METHODS: Human gingival fibroblasts were cultured from specimens taken from healthy, periodontal, and overgrown gingival tissues. The effects of tranilast on cell proliferation and MMP-1 secretion from gingival fibroblast were assessed. Inhibitory effect of transforming growth factor (TGF)-beta secretion from gingival fibroblast by tranilast was also evaluated. RESULTS: Tranilast did not interfere with cell proliferation at the low concentrations. MMP-1 concentration significantly increased at the lower doses of tranilast up to about 2-fold compared to controls (P < 0.05). In contrast, higher doses of tranilast significantly decreased activity to 30% and 20%, respectively. MMP-1 secretion was inhibited significantly by phenytoin, nifedipine, and cyclosporin A and the depressed MMP-1 recovered to the control level with tranilast. The amount of secretion from normal and periodontitis gingival fibroblast specimens did not differ, but that from the overgrown gingiva was significantly less than the other types. Moreover, TGF-beta secretion was significantly inhibited by 300 microM of tranilast. CONCLUSIONS: Tranilast upregulates the expression of type 1 collagenase suppressed by gingival overgrowth-inducing drugs, and inhibits TGF-beta secretion from gingival fibroblasts. Therefore, tranilast could be considered as an agent for controlling gingival over-growth.

Microflora profiling of infected root canal before and after treatment using culture-independent methods.

This study aimed to profile the microflora in infected root canals before and after root canal treatment using culture-independent methods. Six infected root canals in single-rooted teeth with periapical lesions from five subjects were included. Quantification of total bacteria was performed by real-time PCR with primers targeting 16S rRNA genes. PCR products with universal 16S rRNA gene primers were cloned and partially sequenced, and bacterial identification at the species level was performed by comparative analysis with the GenBank database. The concentration of extracted DNA before treatment was higher than that after root canal treatment, although the difference was not statistically significant. Sequence analysis revealed that oral bacteria such as Fusobacterium, Streptococcus, Olsenella, and Pseudoramibacter detected in cases before root canal treatment disappeared after treatment. These results suggest that the root canal microflora are distinct before and after root canal treatment, and that treatment changes the microflora in both quantity and quality.

Rapid quantification of bacteria in infected root canals using fluorescence reagents and a membrane filter: a pilot study on its clinical application to the evaluation of the outcomes of endodontic treatment.

Objective. The bacterial examination has been performed during the course of the root canal treatment. In the present pilot study, the new developed method, using fluorescence reagents and a membrane filter, was applied to the detection and quantification of bacteria in infected root canals, in order to evaluate the outcomes of the treatment. Methods. Six infected root canals with periapical lesions from 5 subjects were included. Informed consent was obtained from all subjects (age ranges, 23-79 years). Samples from infected root canals were collected at the beginning of the treatment (termed #25 First), the end of the first day of treatment (termed #55 First), and the next appointment day (termed #55 Second). Then, the bacterial count (CFU) was measured using fluorescence reagents (4',6'-diamidino-2-phenylindole and propidium iodide) and the polycarbonate membrane filter by Bioplorer. Results. The mean ± SD of CFU in the sample of "#25 First" was (1.0 ± 1.4) × 10(5). As the root canal treatment progressed, the CFU decreased as 7.9 × 10(3) (#55 First) and 4.3 × 10(2) (#55 Second). Conclusion. In the present pilot study, rapid detection and quantification of bacteria in infected root canals were found to be successfully performed using fluorescence reagents and a membrane filter (Bioplorer analysis).