This study aimed to develop strain-specific polymerase chain reaction (PCR) primers to detect Fusobacterium hwasookii KCOM 1249T, F. hwasookii KCOM 1253, F. hwasookii KCOM 1256, F. hwasookii KCOM 1258, and F. hwasookii KCOM 1268 on the basis of nucleotide sequences of a gene specific to each strain. The unique genes for each F. hwasookii strain were determined on the basis of their genome sequences using Roary. The strain-specific PCR primers based on each strain-specific gene were designed using PrimerSelect. The specificity of each PCR primer was determined using the genomic DNA of the 5 F. hwasookii strains and 25 strains of oral bacterial species. The detection limit and sensitivity of each strain-specific PCR primer pair were determined using the genomic DNA of each target strain. The results showed that the strain-specific PCR primers correspond to F. hwasookii KCOM 1249T, F. hwasookii KCOM 1253, F. hwasookii KCOM 1258, F. hwasookii KCOM 1256/F. nucleatum subsp. polymorphum KCOM 1260, or F. hwasookii KCOM 1268/Fusobacterium sp. oral taxon 203 were developed. The detection limits of these strain-specific PCR primers ranged from 0.2 to 2 ng of genomic DNA for each target strain. The results suggest that these strain-specific PCR primers are valuable in quality control for detecting specific F. hwasookii strains.

This study aimed to develop Lautropia mirabilis -specific quantitative real-time polymerase chain reaction (qPCR) primers based on the sequence of DNA-directed RNA polymerase subunit beta gene. The PrimerSelect program was used in designing of the qPCR primers, RTLam-F4 and RTLam-R3. The specificity of the qPCR primers were performed by conventional PCR with 37 strains of 37 oral bacterial species, including L. mirabilis . The sensitivity of the primers was determined by qPCR with the serial dilution of purified genomic DNA of L. mirabilis KCOM 3484, ranged from 4 ng to 4 fg. The data showed that the qPCR primers could detect only L. mirabilis strains and as little as 40 fg of genome DNA of L. mirabilis KCOM 3484. These results indicate that this qPCR primer pair (RTLam-F4/ RTLam-R3) may be useful for species-specific detection of L. mirabilis in epidemiological studies of oral bacterial infectious diseases such as periodontal disease.

The purpose of this study was to introduce a new in vitro method for evaluating the antimicrobial activity of toothpaste, reflecting the actual toothbrushing time and the dilution of toothpaste by salivation. We designed three experimental groups and one negative control group. The experimental groups were (1) 90 μL of toothpaste + 10 μL 1X phosphate-buffered saline (PBS, 9/10 dilution group), (2) 50 μL of toothpaste + 40 μL 1X PBS (1/2 dilution group), and (3) 25 μL of toothpaste + 65 μL 1X PBS (1/4 dilution group). During toothbrushing, saliva is continuously secreted into the oral cavity and the toothpaste concentration is diluted over time during toothbrushing. Therefore, the 1/2 and 1/4 dilution experimental groups were added. The negative control group was toothpaste diluted 20,000-fold with 1X PBS. Miracle Fresh Doctor toothpaste and Streptococcus mitis KCOM 1350, Prevotella intermedia KCOM 1107, Fusobacterium nucleatum subsp. polymorphum KCOM 1322, and Aggregatibacter actinomycetemcomitans KCOM 1306 were used as the toothpaste and target bacterial strains, respectively. The number of bacterial cells plated on agar plates in the negative control group was 1,000 CFU. If the number of colonies on the experimental group plate was less than one, the treatment was considered to have > 99.9% bactericidal activity. These results suggest that this new in vitro method for antimicrobial evaluation could be used as the standard method for testing the antimicrobial activity of toothpaste.

This study evaluated the antimicrobial activity of Endoseal TCS, an mineral trioxide aggregate-based root canal sealer, mixed with water-soluble mangostin derivatives (WsMD) of Garcinia mangostana L. (mangosteen) ethanol extract against Enterococcus faecalis and Staphylococcus aureus. The antibacterial activity of Endoseal TCS mixed with WsMD against three strains of E. faecalis and three strains of S. aureus was performed using agar diffusion test. The data showed that Endoseal TCS mixed with 0.115% WsMD had a zone of inhibition of 0.7 ± 0.2–2.4 ± 0.1 mm. The results suggest that Endoseal TCS mixed with WsMD of Garcinia mangostana L. ethanol extract is useful as a root canal sealer with antibacterial activity against E. faecalis and S. aureus .

The aim of this study was to identify strain KCOM 1265 isolated from subgingival plaque at the species level by comparing 16S ribosomal RNA gene (16S rDNA) and genome sequences. The whole genome of strain KCOM 1265 was extracted using the phenol–chloroform extraction method. 16S rDNA was amplified using polymerase chain reaction and sequenced using the dideoxy chain termination method. Pairwise genome comparison was performed using average nucleotide identity (ANI) and genome-to-genome distance (GGD) analyses. The data showed that the percent similarity of 16S rDNA sequence of strain KCOM 1265 was 99.6% as compared with those of Fusobacterium polymorphum ATCC 10953T and Fusobacterium hwasookii KCOM 1249T. The ANI values of strain KCOM 1265 with F. polymorphum ATCC 10953T and F. hwasookii KCOM 1249T were 95.8% and 93.0%, respectively. The GGD values of strain KCOM 1265 with F. polymorphum ATCC 10953T and F. hwasookii KCOM 1249T were 63.9% and 49.6%, respectively. These results indicate that strain KCOM 1265 belongs to F. polymorphum.

The purpose of this study was to investigate the antimicrobial effects of Australia propolis against cariogenic and periodontopathic bacteria. Antimicrobial activity was determined by evaluating the minimal bactericidal concentration (MBC). Cell cytotoxicity of propolis extract on normal human gingival fibroblast (HGF-1) cells was observed using the methylthiazolyldiphenyl-tetrazolium bromide assay. The data indicated that, with the exception of Aggregatibacter actinomycetemcomitans (KCOM 1306), the MBC values of the propolis strains were 0.25–1% without HGF-1 cell cytotoxicity. These results suggest that propolis can be used to develop oral hygiene products for the prevention of oral infectious disease.

The purpose of this study was to investigate the antimicrobial activity of the ethanol extract of Garcinia mangostana L. (mangosteen) against Cutibacterium acnes (6 strains) and Staphylococcus aureus (6 strains). The antimicrobial activity of the mangosteen extract was evaluated based on its minimal bactericidal concentration. Cytotoxicity of the mangosteen extract against human embryonic kidney 293 (HEK 293) cells was determined using the cell counting method. The data showed that the mangosteen extract was not toxic to HEK 293 cells at a concentration of up to 16 μg/mL and killed 87.0% and 99.9% of C. acnes and S. aureus after 10 minutes and 1 hour of treatment, respectively. These results suggest that ethanol extract of mangosteen can be used as an anti-acne agent.

The purpose of this study was to develop Peptoniphilus mikwangii -specific quantitative real-time polymerase chain reaction (qPCR) primers based on the 16S ribosomal RNA (16S rDNA) gene. The specificity of the primers was determined by conventional PCR using 29 strains of 27 oral bacterial species including P. mikwangii. The sensitivity of the primers was determined by qPCR using the purified genomic DNA of P. mikwangii KCOM 1628T (40 ng to 4 fg). The data showed that the qPCR primers (RTB134-F4/RTB134-R4) could detect P. mikwangii strains exclusively and as little as 40 fg of the genomic DNA of P. mikwangii KCOM 1628T. These results suggest that the developed qPCR primer pair can be useful for detecting P. mikwangii in epidemiological studies of oral bacterial infectious diseases.

The purpose of this study was to evaluate the effect of mangosteen extract complex (MEC; Garcinia mangostana L. and propolis extracts) on the inhibition of inflammation and prevention of alveolar bone loss using a ligature-induced periodontitis model. Rat molars were ligatured with silk, and 1 μg/mL of lipopolysaccharide of Porphyromonas gingivalis was injected into the buccal and palatal gingivae of the teeth with or without treatment with the MEC. Changes in the expression levels of prostaglandin E2 (PGE2), interleukin-8 (IL-8), inducible nitric oxide synthase (iNOS), matrix metalloproteinase-8 (MMP-8), cyclooxygenase (COX)-1, and COX-2 in gingival tissues were evaluated using enzyme-linked immunosorbent assays. Alveolar bone loss around the ligated molars was examined using micro-computed tomography. The expression levels of PGE2, IL-8, iNOS, MMP-8, COX-1, and COX-2 in gingival tissues were significantly reduced in the group treated with a mixture of 16 μg of mangosteen extract powder and 544 μg of propolis extract powder (ligation [Lig] + lipopolysaccharide extracted from P. gingivalis KCOM 2804 [L] + MEC 1:34). Additionally, alveolar bone loss was significantly reduced in the Lig + L + MEC 1:34 group compared with that in other groups. These results indicate that the MEC could be useful in preventing and treating periodontitis.

Enterococcus faecalis is a major causative agent of endodontic treatment failure. The purpose of this study was to investigate bactericidal effects of ethanol extract of Garcinia mangostana L. (mangosteen extract) on five strains of E. faecalis that were isolated from human oral cavities. The bactericidal effects of mangosteen extract were assessed by measurement of minimum bactericidal concentration (MBC) value. The cytotoxicity of mangosteen extract on immortalized human gingival fibroblasts, hTERT-hNOF, was determined based on cell counting method. The data revealed the MBC value of mangosteen extract against the E. faecalis strains was 4 ㎍/ml. Additionally, the cell viability of mangosteen extract on hTERT-hNOF was 83.7-89.1% at the 1 to 16 ㎍/ml. These findings indicated that mangosteen extract could be used as a root canal cleaner during management of endodontic treatment failure caused by E. faecalis.

Polyphenon 60 refers to the mixture of catechins present in green tea. The aim of this study was to investigate the antimicrobial activities of polyphenon 60 against 4 strains of Streptococcus mutans and 2 strains of Streptococcus sorbrinus, which are the major causative bacteria of dental caries. The minimum bactericidal concentration (MBC) values of polyphenon 60 for S. mutans and S. sobrinus were determined and the effect of biofilm formation inhibition of that was evaluated. The MBC value of polyphenon 60 against the bacterial strains was 2.5 mg/ml except for one particular strain, S. mutans KCOM 1128 for which the value was 1.25 mg/ml. The results of biofilm formation inhibition assay revealed that polyphenon 60 inhibited biofilm formation more than 90% at a concentration of 2.5 mg/ml. It was apparent that polyphenon exhibited biofilm formation inhibition activity along with bactericidal effect against S. mutans and S. sobrinus. Therefore, it is proposed that polyphenon 60 as one of the components of bactericidal agents could be useful in developing oral hygiene products, toothpaste or gargling solution.

In a previous study, Peptoniphilus mikwangii was isolated from the human oral cavity as a new species. The purpose of this study was to develop P. mikwangii-specific PCR primers. The PCR primers were designed, based on the nucleotide sequence of 16S ribosomal RNA (16S rDNA). The specificity of the primers was tested using genomic DNAs of 3 strains of P. mikwangii and 27 strains (27 species) of non-P. mikwangii bacteria. The sensitivity of primers sensitivity was determined using PCR, with serial dilutions of the purified genomic DNAs (4 ng to 4 fg) of P. mikwangii KCOM 1628T. The data showed that P. mikwangii-specific qPCR primers (B134-F11/B134-R1 & B134-F5/B134-R5) could detect only P. mikwangii strains, and 400 fg or 40 fg of P. mikwangii genome DNA. These results suggest that PCR primers are useful in detecting P. mikwangii from the oral cavity.

The aim of this study was to identify the non-Aggregatibacter actinomycetemcomitans bacteria grown on the tryptic soy-serum-bacitracin-vancomycin (TSBV) medium, an A. actinomycetemcomitans selective medium. A total of 82 unidentified bacterial isolates from the oral cavities of a Korean population were kindly provide by the Korean Collection for Oral Microbiology. All the clinical isolates were grown on TSBV medium and bacterial DNA purified from each isolate was subjected to PCR with universal primers specific for bacterial 16S rRNA genes (16S rDNAs) sequence. The each bacterial 16S rDNA was amplified by PCR and the nucleotide sequences of it was determined by the dideoxynucleotide chain termination method. They were identified by 16S rDNA sequence comparison method at the specie-level. The data showed that Neisseria spp. (42 strains), Fusobacterium spp. (10 strains), Capnocytophaga spp. (8 strains), Propionibacterium acnes (5 strains), Aggregatibacter aprophilus (4 strains), Campylobacter spp. (5 strains), Veillonella dispar (3 strains), Streptococcus sp. (1 strain), Haemophilus parainfluenzae (1 strain), Leptotrichia wadei (1 strain), Morococcus sp./Neisseria sp. (1 strain), and Staphylococcus sp. (1 strain) were identified. These results could be used to develop a new A. actinomycetemcomitans-selective medium which is more effective than the TSBV medium in future studies.

The purpose of this study was to develop Streptococcus sobrinus-specific qPCR primers based on the nucleotide sequence of the RNA polymerase β-subunit gene (rpoB). The specificity of the primers was determined by conventional polymerase chain reaction (PCR) with 12 strains of S. sobrinus and 50 strains (50 species) of non-S. sobrinus bacteria. The sensitivity of the primers was determined by quantitative real-time PCR (qPCR) with serial dilutions of the purified genomic DNAs (40 ng to 4 fg) of S. sobrinus ATCC 33478T. The specificity data showed that the S. sobrinus-specific qPCR primers (RTSsob-F4/RTSsob-R4) detected only the genomic DNAs of S. sobrinus strains with a detection limit of up to 4 fg of S. sobrinus genomic DNA. Our results suggest that the RTSsob-F4/RTSsob-R4 primers are useful in detecting S. sobrinus with high sensitivity and specificity for epidemiological studies of dental caries..

Prevotella intermedia-specific quantitative real-time PCR (qPCR) primers were previously designed based on the nucleotide sequences of RNA polymerase β-subunit gene (rpoB). However, the several clinical strains isolated from Korean populations are not detectable by the qPCR primers. The purpose of this study was to develop new P. intermedia-specific qPCR primers based on the rpoB. The specificity of the primers was determined by conventional PCR with 12 strains of P. intermedia and 52 strains (52 species) of non-P. intermedia bacteria. The sensitivity of primers was determined by qPCR with serial dilutions of the purified genomic DNAs (40 ng to 4 fg) of P. intermedia ATCC 25611T. The data indicated that only P. intermedia strains were detected by the P intermedia-specific qPCR primers (RTPiF2/RTPiR2); in addition, as little as 40 fg of P. intermedia genomic DNA could be detected. These results suggest that these qPCR primers are useful in detecting P. intermedia from the bacterial infectious lesions including dental plaque and oral tissue lesions.

The purpose of the study was to investigate the antimicrobial activity of the methanol extract of Coptidis rhizome against the type strains of cariogenic bacteria, Streptococcus mutans and Streptococcus sobrinus, and the periodontopathogens, Porphyromonas gingivalis, Prevotella intermedia, Treponema denticola and Aggregatibacter actinomycetemcomitans. The antimicrobial activities of the crude extract and the methanol extract fractions of Coptidis rhizome separated by silica gel chromatography were evaluated by determining the minimal bactericidal concentration (MBC) values, using the microdilution method. The cell viability test of the extracts of Coptidis rhizome on the KB cells was also studied by methyl thiazolyl tetrazolium (MTT) assay. Our results showed that the 11th fraction (F11) of the methanol extract had the greatest antimicrobial activity against the tested bacteria, with no associated cytotoxicity on the KB cells, upto a concentration of 50 μg/ml. These results suggest that the silica gel chromatography fraction F11 of the methanol extract of Coptidis rhizome, could be useful in the development of oral hygiene products as an antimicrobial agent for the prevention of dental caries and periodontal diseases.

The aim of this study was to identify bacteria isolated from the oral cavities and to determine their antimicrobial susceptibility against eight antibiotics. The bacterial strains were obtained from the Korean Collection for Oral Microbiology (KCOM). The bacteria were identified by comparing 16S rDNA sequences at the species level. The data showed that 77 bacterial strains were predominantly identified as streptococci (49.4%) and staphylococci (14.3%). Minimum inhibitory concentrations (MIC) were determined using a broth dilution assay to test the sensitivity of the bacterial strains. The MIC values of the oral bacterial strains against antibiotics were different. Streptococci were sensitive to clindamycin, cefuroxime axetil, and vancomycin, and they were resistant to tetracycline. Staphylococci also were sensitive to clindamycin, cefuroxime axetil, and vancomycin, and they were resistant to penicillin antibiotics. Gramnegative bacterial strains were sensitive to tetracycline and were resistant to clindamycin. These results suggest that the antimicrobial susceptibility test is necessary in deciding the prescription for antibiotics, to prevent the misuse or abuse of antibiotics.

In general, oleanolic acid (OA) and ursolic acid (UA) have antimicrobial effect against Gram-positive bacteria but not Gram-negative bacteria whereas sophoraflavanone G has antimicrobial activity against both bacterial types. However, the antimicrobial effects of OA, UA, and sophoraflavanone G against periodontopathogens have not been studied to any great extent. The aim of this study was to investigate antimicrobial effect of OA, UA, and sophoraflavanone G against 15 strains (5 species) of oral Gram-negative bacteria, which are the major causative bacteria of periodontal disease. The antimicrobial activity was evaluated by minimal inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determinations. OA and UA showed antimicrobial effects against all of the Porphyromonas gingivalis strains tested and also Prevotella intermedia ATCC 25611T. Interestingly, P. intermedia ATCC 49046 showed greater resistance to OA and UA than P. intermedia ATCC 25611T. In contrast, sophoraflavanone G had antimicrobial activity against all strains, with MIC and MBC values below 32 μg/ml, except Aggregatibacter actinomycetemcomitans. These results indicate that sophoraflavanone G may have potential for use in future oral hygiene products such as dentifrices and gargling solution to prevent periodontitis.

It has been established that berberine has strong antimicrobial effects. Little is known however regarding the antimicrobial activity of berberine against endodontic pathogenic bacteria or its cytotoxicity in human oral tissue cells. The antibacterial properties of berberine were tested against 5 strains of Enterococcus faecalis and type strains of Aggregatibacter actinomycetemcomitans, Prevotella nigrescens, Prevotella intermedia, and Tannerella forsythia, which are involved in endodontic infections. Antimicrobial activity was evaluated through minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) measurements. The viability of normal human gingival fibroblast (NHGF) cells after exposure to berberine was measured using a methyl thiazolyl tetrazolium (MTT) assay. The data showed that berberine has antimicrobial effects against A. actinomycetemcomitans with an MIC and MBC of 12.5 μg/ml and 25 μg/ml, respectively. In the cytotoxicity studies, cell viability was maintained at 66.1% following exposure to 31.3 μg/ml berberine. Overall, these findings suggest that berberine has antimicrobial activity against the tested bacteria. Nevertheless, lower concentrations in combination with other reagents will need to be tested before these in vitro results can be translated to clinical use.

Methicillin-resistant Staphylococcus aureus (MRSA) is one of the important causative microbes for nosocomial infe- ction and has been isolated from the dental environment. The purpose of this study was to investigate the antimi- crobial activity of linalool and α‐terpineol against MRSA isolates from a Korean population. In the experiments, we determined the minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of these two compounds against 18 strains of MRSA. The data re- vealed that the MIC90/MBC90 values of linalool and α‐ terpineol against MRSA were >12.8 mg/ml and 6.4 mg/ml, respectively. These results indicate that α-terpineol has more potent antimicrobial activity against MRSA than lina- lool and may have utility as an anti‐MRSA cleansing agent for dental instruments and dental unit chairs.