• Journal of Microbiology and Biotechnology
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• 제33권8호
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• pp.998-1005
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• 2023

Streptococcus salivarius is a beneficial bacterium in oral cavity, and some strains of this bacterium are known to be probiotics. The purpose of this study was to investigate the anti-inflammatory effect and mechanism of S. salivarius G7 lipoteichoic acid (LTA) on lipopolysaccharide (LPS) and LTA of periodontopathogens. The surface molecules of S. salivarius G7 was extracted, and single- or co-treated on human monocytic cells with LPS and LTA of periodontopathogens. The induction of cytokine expression was evaluated by real-time PCR and ELISA. After labeling fluorescence on LPS and LTA of periodontopathogens, it was co-treated with S. salivarius LTA to the cell. The bound LPS and LTA were measured by a flow cytometer. Also, the biding assay of the LPS and LTA to CD14 and LPS binding protein (LBP) was performed. The surface molecules of S. salivarius G7 did not induce the expression of inflammatory cytokines, and S. salivarius G7 LTA inhibited the inflammatory cytokines induced by LPS and LTA of periodontopathogens. S. salivarius G7 LTA inhibited the binding of its LPS and LTA to cells. Also, S. salivarius G7 LTA blocked the binding of its LPS and LTA to CD14 and LBP. S. salivarius G7 has an inhibitory effect on inflammation induced by LPS or LTA of periodontopathogens, and may be a candidate probiotics for prevention of periodontitis.

• Journal of Microbiology and Biotechnology
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• 제17권12호
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• pp.2061-2065
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• 2007

The chemical components of the essential oil from Artemisia feddei LEV et VNT. were analyzed using GC-MS. Ninety-nine compounds, accounting for 96.23% of the extracted essential oil, were identified. The main oil compounds were 1,8-cineole (16.86%), chamazulene (9.04%), ${\alpha}$-terpineol (8.18%), ${\alpha}$-phellandrene (5.78%), ${\alpha}$-thujone (5.51%), ${\alpha}$-terpinyl acetate (5.07%), borneol (5.08%), ${\beta}$-caryophyllene (4.71%), camphor (4.04%), and terpinen-4-ol (3.04%). The antimicrobial activity of the essential oil and some of its compounds was tested against 15 different genera of oral bacteria. The essential oil from A. feddei had a considerable inhibitory effect on all the obligate anaerobic bacteria tested (MICs, 0.025 to 0.05 mg/ml; MBCs, 0.025 to 0.1 mg/ml), whereas the major compounds demonstrated different degrees of growth inhibition.

• International Journal of Oral Biology
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• 제48권2호
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• pp.9-18
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• 2023

The rapid detection of bacteria in the oral cavity, its species identification, and bacterial count determination are important to diagnose oral diseases caused by pathogenic bacteria. The existing clinical microbial diagnosis methods are time-consuming as they involve observing patients' samples under a microscope or culturing and confirming bacteria using polymerase chain reaction (PCR) kits, making the process complex. Therefore, it is required to analyze the development status of substances and systems that can rapidly detect and analyze pathogenic microorganisms in the oral cavity. With research advancements, a close relationship between oral and systemic diseases has been identified, making it crucial to identify the changes in the oral cavity bacterial composition. Additionally, an early and accurate diagnosis is essential for better prognosis in periodontal disease. However, most periodontal disease-causing pathogens are anaerobic bacteria, which are difficult to identify using conventional bacterial culture methods. Further, the existing PCR method takes a long time to detect and involves complicated stages. Therefore, to address these challenges, the concept of point-of-care (PoC) has emerged, leading to the study and implementation of various chair-side test methods. This study aims to investigate the different PoC diagnostic methods introduced thus far for identifying pathogenic microorganisms in the oral cavity. These are classified into three categories: 1) microbiological tests, 2) microchemical tests, and 3) genetic tests. The microbiological tests are used to determine the presence or absence of representative causative bacteria of periodontal diseases, such as A. actinomycetemcomitans, P. gingivalis, P. intermedia, and T. denticola. However, the quantitative analysis remains impossible, and detecting pathogens other than the specific ones is challenging. The microchemical tests determine the activity of inflammation or disease by measuring the levels of biomarkers present in the oral cavity. Although this diagnostic method is based on increase in the specific biomarkers proportional to inflammation or disease progression in the oral cavity, its commercialization is limited due to low sensitivity and specificity. The genetic tests are based on the concept that differences in disease vulnerability and treatment response are caused by the patient's DNA predisposition. Specifically, the IL-1 gene is used in such tests. PoC diagnostic methods developed to date serve as supplementary diagnostic methods and tools for patient education, in addition to existing diagnostic methods, although they have limitations in diagnosing oral diseases alone. Research on various PoC test methods that can analyze and manage the oral cavity bacterial composition is expected to become more active, aligning with the shift from treatment-oriented to prevention-oriented approaches in healthcare.

• International Journal of Oral Biology
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• 제43권3호
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• pp.123-127
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• 2018

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.

• International Journal of Oral Biology
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• 제46권2호
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• pp.94-97
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• 2021

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.

• Journal of Microbiology and Biotechnology
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• 제25권2호
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• pp.234-237
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• 2015

In this study, we developed lysosome-alginate beads for application as an oral drug delivery system (ODDS). The beads harboring lysosomes, which have antimicrobial activity, and various concentrations of alginate were characterized and optimized. For application as an ODDS, pH-dependent lysosome-alginate beads were generated, and the level of lysosome release was investigated by using antimicrobial tests. At low pH, lysosomes were not released from the lysosome-alginate beads; however, at neutral pH, similar to the pH in the intestine, lysosome release was confirmed, as determined by a high antimicrobial activity. This study shows the potential of such an ODDS for the in vivo treatment of infection with pathogens.

• International Journal of Oral Biology
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• 제41권3호
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• pp.149-154
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• 2016

The purpose of this study was to develop Streptococcus sobrinus-specific qPCR primers based on the nucleotide sequence of the RNA polymerase ${\beta}-subunit$ 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..

• International Journal of Oral Biology
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• 제39권2호
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• pp.115-120
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• 2014

Minimal inhibitory concentration (MIC) is the lowest concentration of antibiotics that inhibits the visible growth of a microorganism. It has been reported that sub-MIC of antibiotics may result in morphological alterations along with biochemical and physiological changes in bacteria. The purpose of this study was to examine morphological changes of periodontal pathogens after treatment with sub-MIC antibiotics. Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, and Porphyromonas gingivalis were used in this study. The MIC for amoxicillin, doxycycline, metronidazole, penicillin, and tetracycline were determined by broth dilution method. The bacterial morphology was observed with bright field microscope after incubating with sub-MIC antibiotics. The length of A. actinomycetemcomitans and F. nucleatum were increased after incubation with metronidazole; penicillin and amoxicillin. P. gingivalis were increased after incubating with metronidazole and penicillin. However, F. nucleatum showed decreased length after incubation with doxycycline and tetracycline. In this study, we observed that sub-MIC antibiotics can affect the morphology of periodontal pathogens.

• Journal of Microbiology and Biotechnology
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• 제14권1호
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• pp.110-115
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• 2004

This study was undertaken to develop PCR primers for the simultaneous detection of Fusobacterium nucleatum and Actinobacillus actinomycetemcomitans, using two species-specific reverse primers in combination with a single conserved forward primer. These primers target the variable and conserved regions of the 16S rDNA. The primer specificity was tested against (i) four F. nucleatum and three A. actinomycetemcomitans strains and (ii) seven representatives of the different species of oral bacteria. The primer sensitivity was determined by testing serial dilutions of the purified genomic DNA of F. nucleatum and A. actinomycetemcomitans. The data indicate that species-specific amplicons could be obtained for all the F. nucleatum and A. actinomycetemcomitans strains tested, which were not found in the seven other species. The multiplex PCR could detect as little as 4 fg of chromosomal DNA of F. nucleatum and A. actinomycetemcomitans simultaneously. These findings suggest that these PCR primers are highly sensitive and are suitable for applications in epidemiological studies, diagnosis, and monitoring F. nucleatum and A. actinomycetemcomitans after the treatment of periodontitis.

• International Journal of Oral Biology
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• 제42권2호
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• pp.63-70
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• 2017

Selecting an appropriate antigen with optimal immunogenicity and physicochemical properties is a pivotal factor to develop a protein based subunit vaccine. Despite rapid progress in modern molecular cloning and recombinant protein technology, there remains a huge challenge for purifying and using protein antigens rich in hydrophobic domains, such as membrane associated proteins. To overcome current limitations using hydrophobic proteins as vaccine antigens, we adopted in silico analyses which included bioinformatic prediction and sequence-based protein 3D structure modeling, to develop a novel periodontitis subunit vaccine against the outer membrane protein FomA of Fusobacterium nucleatum. To generate an optimal antigen candidate, we predicted hydrophilicity and B cell epitope parameter by querying to web-based databases, and designed a truncated FomA (tFomA) candidate with better solubility and preserved B cell epitopes. The truncated recombinant protein was engineered to expose epitopes on the surface through simulating amino acid sequence-based 3D folding in aqueous environment. The recombinant tFomA was further expressed and purified, and its immunological properties were evaluated. In the mice intranasal vaccination study, tFomA significantly induced antigen-specific IgG and sIgA responses in both systemic and oral-mucosal compartments, respectively. Our results testify that intelligent in silico designing of antigens provide amenable vaccine epitopes from hard-to-manufacture hydrophobic domain rich microbial antigens.