Developing species-specific quantitative real-time polymerase chain reaction primers for detecting Lautropia mirabilis

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.

Developing species-specific quantitative real-time polymerase chain reaction primers for detecting Lautropia mirabilis

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.

Genome-based identification of strain KCOM 1265 isolated from subgingival plaque at the species level

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.

Peptoniphilus mikwangii-specific quantitative real-time polymerase chain reaction primers

The purpose of this study was to develop Peptoniphilus mikwangii

Antimicrobial activity of Garcinia mangostana L. ethanol extract against Cutibacterium acnes and Staphylococcus aureus

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.

Development of Species-specific PCR Primers for Detecting Peptoniphilus mikwangii

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 1628(T). 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.

Identification of Non-Aggregatibacter actinomycetemcomitans Bacteria Grown on the Tryptic soy-Serum-Bacitracin-Vancomycin Medium

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.

Development of Quantitative Real-Time PCR Primers for Detection of Streptococcus sobrinus

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 33478(T). 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.

Identification of the Bacteria Isolated from Oral Cavities in Korea

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. Gram-negative 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.

Antimicrobial Effect of Coptidis rhizome Extract against Mutans Streptococci and Periodontopathogens

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 microg/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.

Development of Quantitative Real-Time PCR Primers for Detection of Prevotella intermedia

Prevotella intermedia-specific quantitative real-time PCR (qPCR) primers were previously designed based on the nucleotide sequences of RNA polymerase beta-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.

Antimicrobial Effects of Oleanolic Acid, Ursolic Acid, and Sophoraflavanone G against Enterococcus faecalis and Propionibacterium acnes

The aim of this study was to investigate antimicrobial effect of oleanolic acid (OA), ursolic acid (UA), and sophoraflavanone G against Enterococcus faecalis and Propionibacterium acnes, which are the major causative bacteria of endodontic infections. The antimicrobial activity was evaluated by the minimal inhibitory concentration (MIC). The data showed that the OA, UA, and sophoraflavanone G had antimicrobial effect on all the strains use in the study with 16-64 microg/ml, 8-64 microg/ml, and 1-8 microg/ml of MIC values, respectively. These results indicate that OA, UA, and sophoraflavanone G could be useful in the development of antiseptic solution for washing the root canal in endodontic treatments.

Identification and Antibiotic Susceptibility of the Bacteria from Non-odontogenic Infectious Lesions

The purpose of this study was to isolate and identify bacteria from the 4 patients with non-odontogenic infectious lesions (mucormycosis, chronic inflammation from wound infection, and two actinomycosis) and determine their antimicrobial susceptibility against eight antibiotics. Bacterial culture was performed under three culture conditions (anaerobic, CO2, and aerobic incubator). The bacterial strains were identified by 16S rRNA gene (16S rDNA) sequence comparison analysis method. For investigating the antimicrobial susceptibility of the bacteria against eight antibiotics, penicillin G, amoxicillin, tetracycline, cefuroxime, erythromycin, clindamycin, vancomycin, and Augmentin(R) (amoxicillin + clavulanic acid), minimum inhibitory concentration (MIC) measurement was performed using broth microdilution assay. Nosocomial pathogens such as Enterococcus faecalis, Klebsiella pneumoniae, Bacillus subtilis, and Neisseria flavescens were isolated from mucormycosis. Veillonella parvula, Enterobacter hormaechei, and Acinetobacter calcoaceticus were isolated from chronic inflammatory lesion. Actinomyces massiliensis was isolated from actinomycosis in parotid gland. Capnocytophaga ochracea was isolated from actinomycosis in buccal region in anaerobic condition. There was no susceptible antibiotic to all bacteria in mucormycosis. Tetracycline was susceptible to all bacteria in chronic inflammation. C. ochracea was resistant to vancomycin and penicillin G; and other antibiotics showed susceptibility to all bacteria in actinomycosis. The results indicated that the combined treatment of two or more antibiotics is better than single antibiotic treatment in mucormycosis, and penicillin is the first recommended antibiotic to treat actinomycosis.

Antimicrobial Activity of Mulberry Leaf against Mutans Streptococci and Periodontopathogens

This study investigated the antimicrobial activity of methanol extract of mulberry leaf against 16 strains of mutans streptococci and four species of periodontopathogens: Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Aggregatibacter actinomycetemcomitans. The antimicrobial activities of the crude extracts or silica gel chromatography fractions of methanol-extracted mulberry leaf were evaluated by determining minimal inhibitory concentrations using an established microdilution method. The cytotoxicity of the extracts of mulberry leaf on KB cells was tested by the methyl thiazolyl tetrazolium assay. Chromatography fraction 12 displayed the most potent antimicrobial activity against all 16 strains of mutans streptococci, P. gingivalis, and P. intermedia. No KB cell cytotoxicity was evident up to 128 microg/ml of fraction 12. The methanol extract had no antimicrobial activity against F. nucleatum and A. actinomycetemcomitans. These results suggest chromatography fraction 12 methanol extract of mulberry leaf could be useful in the development of oral hygiene products, such as dentifrice and oral hygiene solution, for the prevention of dental caries.

Erratum: Antimicrobial Effects of Oleanolic Acid, Ursolic Acid, and Sophoraflavanone G against Enterococcus faecalis and Propionibacterium acnes

We would like to correct the article.

Molecular Identification of Anginosus Group Streptococci Isolated from Korean Oral Cavities

Anginosus group streptococci (AGS) were classified based on the nucleotide sequences of the 16S rRNA gene (16S rDNA) and comprised Streptococcus anginosus, Streptococcus intermedius, and Streptococcus constellatus. It is known that AGS is a causative factor of oral and systematic diseases. The purpose of this study was to discriminate the 56 clinical strains of AGS isolated from Korean oral cavities using phylogenetic analysis of 16S rDNA and species-specific PCR at the species-level. The 16S rDNA of clinical strains of AGS was sequenced using the dideoxy chain termination method and analyzed using MEGA version 5 software. PCR was performed to identify the clinical strains using species-specific primers described in previous studies and S. intermedius-specific PCR primers developed in our laboratory. The resulting phylogenetic data showed that the 16S rDNA sequences can delineate the S. anginosus, S. intermedius, and S. constellatus strains even though the 16S rDNA sequence similarity between S. intermedius and S. constellatus is above 98%. The PCR data showed that each species-specific PCR primer pair could discriminate between clinical strains at the species-level through phylogenetic analysis of 16S rDNA nucleotide sequences. These results suggest that phylogenetic analysis of 16S rDNA and PCR are useful tools for discriminating between AGS strains at the species-level.

Molecular Discrimination of Mitis Group Streptococci Isolated from Koreans using RpoB Nucleotide Sequences

Mitis group streptococci (MGS) were classified based on the nucleotide sequences 16S rRNA gene (16S rDNA) and comprised 13 Streptococcus species. However, 16S rDNA homogeneity among MGS was too high to discriminate between clinical strains at the species level, notably between Streptococcus mitis, Streptococcus oralis, Streptococcus pneumoniae, and Streptococcus pseudopneumoniae. The purpose of this study was to discriminate between 37 strains of MGS isolated from Korean oral cavities using phylogenetic analysis of the DNA-dependant RNA polymerase beta-subunit gene (rpoB). 16S rDNA and rpoB from clinical strains of MGS were sequenced using the dideoxy chain termination method and analyzed using MEGA version 5 software. The resulting phylogenetic data showed that the rpoB sequences could delineate clinical strains of MGS at the species level. Phylogenetic analysis of rpoB is therefore a useful approach for identifying MGS at the species level.

Antimicrobial Activity of Berberine against Oral Bacteria Related to Endodontic Infections

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 microg/ml and 25 microg/ml, respectively. In the cytotoxicity studies, cell viability was maintained at 66.1% following exposure to 31.3 microg/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.

Antimicrobial Activity of Oleanolic Acid, Ursolic Acid, and Sophoraflavanone G against Periodontopathogens

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 microg/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.

Development of Streptococcus sanguinis-, Streptococcus parasanguinis-, and Streptococcus gordonii-PCR Primers Based on the Nucleotide Sequences of Species-specific DNA Probes Screened by Inverted Dot Blot Hybridization

The objective of this study was to develop PCR primers that are specific for Streptococcus sanguinis, Streptococcus parasanguinis, and Streptococcus gordonii. We designed the S. sanguinis-, S. parasanguinis-, and S. gordonii-specific primers, Ssa21-F3/Ssa21-R2, Spa17-F/Spa17-R, and Sgo41-F1/Sgo41-R1 respectively, based on the nucleotide sequences of the Ssa21, Spa17, and Sgo41 DNA probes that were screened using inverted dot blot hybridization (IDBH). The species-specificity of these primers was assessed against 43 strains of mitis group streptococci, including clinical strains of S. sanguinis, S. parasanguinis, and S. gordonii. The resulting PCR data revealed that species-specific amplicons had been obtained from all strains of the target species tested, and that none of these amplicons occurred in any other strains from other species. These results suggest that the Ssa21-F3/Ssa21-R2, Spa17-F/Spa17-R, and Sgo41-F1/Sgo41-R1 primers may be useful in detecting S. sanguinis, S. parasanguinis, and S. gordonii at the species level, respectively.