• Journal of Life Science
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• v.23 no.2
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• pp.290-298
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• 2013

The genus Mycobacterium includes crucial animal and human pathogens such as Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacterium bovis. Although it is important to understand the genetic basis for their virulence and persistence in host, genetic analysis in mycobacteria was hampered by a lack of sufficient genetic tools. Therefore, many functional vectors as molecular genetic tools have been designed for understanding mycobacterial biology, and the application of these tools to mycobacteria has accelerated the study of mechanisms involved in virulence and gene expression. To overcome the pre-existing problems in genetic manipulation of mycobacteria, this paper reports new vector systems as effective genetic tools in Mycobacterium smegmatis. Three vectors were developed; pKOTs is a suicide vector for mutagenesis containing a temperature-sensitive replication origin (TSRO) and the sacB gene encoding levansucrase as a counterselectable marker. pMV306lacZ is an integrative lacZ transcriptional fusion vector that can be inserted into chromosomal DNA by site-specific recombination. pTnMod-OKmTs is a minitransposon vector harboring the TSRO that can be used in random mutagenesis. It was demonstrated in this study that these vectors effectively worked in M. smegmatis. The vector systems reported here are expected to successfully applicable to future research of mycobacterial molecular genetics.

• Korean Journal of Clinical Laboratory Science
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• v.51 no.2
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• pp.171-176
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• 2019

A comparative study between commercially available mycobacteria growth indicator tubes (MGIT) in the BACTEC MGIT 960 System and the conventional Ogawa media was carried out to assess the effectiveness of the re-decontaminating process for the recovery of mycobacteria. Processed specimens with 5% sodium hydroxide and 0.5% N-acetyl-L-cysteine were inoculated into MGIT and Ogawa media. The acid fast bacilli (AFB) recovered from the cultures were identified using a mycobacterium tuberculosis (TB) antigen kit. If contaminants were observed in the MGIT tubes within five days, a decontaminating process was repeated. A total of 1,190 out of 4,790 (24.8%) specimens showed positive results using the BACTEC MGIT 960 system. Among them, 278 specimens were reprocessed. When the MGIT and Ogawa results were compared, it showed discordant results (weighted kappa value: 0.283). One TB and 10 nontuberculous mycobacteria (NTM) were newly detected in MGIT only. The likely benefit of the re-decontaminating process is the detection of additional mycobacteria that could not be detected without a re-decontaminating process despite being small in number. In addition to the combination of MGIT and Ogawa, the re-decontaminating process is recommended in the case of contaminations to recover mycobacteria.

• Korean Journal of Clinical Laboratory Science
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• v.51 no.2
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• pp.177-184
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• 2019

Mycobacteria grow slowly. Therefore, a solid medium should be used for eight weeks and a liquid medium for six weeks. The purpose of this study was to find the growth factors that can grow Mycobacterium rapidly and to help develop a solid medium for rapid identification. Three types of Mycobacterium growth factors were evaluated with 10 Mycobacteria by adding activated charcoal, defibrinated sheep blood, and L-ascorbic acid to $Difco^{TM}$ Mycobacteria 7H11 agar (Becton, Dickinson and Company, Sparks, MD, USA). The time to detection and the distinguishability of a colony were compared with that of the current method. In the rapidly growing Mycobacterium, the difference in detection time between the new media and conventional media confirmed that the new media was faster. M. kansasii and M. intracelluare grew faster in 7H11 C than in 7H11 medium. MTB grew faster than the other media in 7H11 C. This study confirmed that the two growth factors affect fast-growing Mycobacteria and slow-growing Mycobacteria. 7H11 C showed better distinguishability than the conventional media in all 10 Mycobacterium due to the color contrast. In particular, when the MTB was grown, the size of the colonies was larger than with other media, so visualization was easy.

• Molecules and Cells
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• v.38 no.7
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• pp.610-615
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• 2015

Alveolar epithelial cells have been functionally implicated in Mycobacterium tuberculosis infection. This study investigated the role of ursolic acid (UA)-a triterpenoid carboxylic acid with potent antioxidant, anti-tumor, anti-inflammatory, and anti-tuberculosis properties in mycobacterial infection of alveolar epithelial A549 cells. We observed that M. tuberculosis successfully entered A549 cells. Cytotoxicity was mediated by nitric oxide (NO). A549 toxicity peaked along with NO generation 72 h after infection. The NO generated by mycobacterial infection in A549 cells was insufficient to kill mycobacteria, as made evident by the mycobacteria growth indicator tube time to detect (MGIT TTD) and viable cell count assays. Treatment of mycobacteria-infected cells with UA reduced the expression of inducible nitric oxide synthase, NO generation, and eventually improved cell viability. Moreover, UA was found to quench the translocation of the transcription factor, nuclear factor kappa B (NF-${\kappa}B$), from the cytosol to the nucleus in mycobacteria-infected cells. This study is the first to demonstrate the cytotoxic role of NO in the eradication of mycobacteria and the role of UA in reducing this cytotoxicity in A549 cells.

• Journal of Microbiology and Biotechnology
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• v.19 no.10
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• pp.1259-1264
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• 2009

We assessed the capacity of two liquid-medium culture methods with automated incubation and reading systems (MB/BacT ALERT 3D System and BACTEC MGIT 960 System) and one solid-medium culture method ($L\ddot{o}wenstein$-Jensen) to detect mycobacteria in different types of clinical samples. Out of 1,770 cultured clinical samples (1,519 of respiratory origin and 251 of non respiratory origin), mycobacteria were isolated in 156 samples (135 M. tuberculosis complex, 8 M. chelonae, 6 M. kansasii, 4 M. fortuitum, 2 M. gordonae, and 1 M. marinum) by at least one of the methods used. The BACTEC MGIT 960 System proved to be the most sensitive method (86.5%), especially in the detection of M. tuberculosis complex (89.1%). However, $L\ddot{o}wenstein$-Jensen culture was the most sensitive (76.2%) to detect nontuberculous mycobacteria. The BACTEC MGIT 960 System showed the lowest mean detection time for mycobacterial growth (15.3 days), significantly shorter than the other two methods. Highest sensitivity (95.5%) and specificity (99.6%) values were obtained using the BACTEC MGIT 960 System with the $L\ddot{o}wenstein$-Jensen culture method, which was also the only combination capable of detecting 100% of the nontuberculous mycobacteria.

• The Journal of Internal Korean Medicine
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• v.21 no.4
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• pp.555-563
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• 2000

Objective : In order to know the antibacterial effects of Kyungok-go against Mycobacteria tuberculosis, Methods : In this study, I inverstigated these effects in terms of combination of other antibiotics with and without Kyungok-go on several different media conditions. Results: On Ogawa medium, Kyungok-go of the $10{\mu}/ml$ concentration showed the anti-Mycobacteria tuberculosis activity against antibiotic drug-sensitive strain. On Middle-blue medium, Kyungok-go of the $10{\mu}/ml$ concentration showed the anti-Mycobacteria tuberculosis activity against antibiotic drug-sensitive strain. Kyungok-go showed the anti mycobacteria tuberculosis activity with the meaningful result above a certain concentration. The resistance against M, tuberculosis as the concentration of Kyungok-go was decreased significantlly on the high concentration($500{\mu}/ml$) When rifampicin and Kyungok-go were used together, the resistance was decreased with the statistical significance as to the persistant antibacterial effect against M. tuberculosis, When ciprofloxacin and Kyungok-go were used together, the resistance was decreased with the statistical significance as to the persistant antibacterial effect against M. tuberculosis, The combination of treatment, Kyungok-go with both rifampicin and ciprofloxacin, showed much better antibacterial effect against M, tuberculosis than antibiotics alone. Conclusions : This study shows that Kyungok-go has antibacterial effect against M. tuberculosis and in the combination of treatment, Kyungok-go with antibiotics, showed much better antibacterial effect against M. tuberculosis than antibiotics alone,.

• Journal of Microbiology and Biotechnology
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• v.29 no.9
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• pp.1401-1411
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• 2019

Mycobacterial cell walls comprise thick and diverse lipids and glycolipids that act as a permeability barrier to antibiotics or other chemical agents. The use of OH radicals from a non-thermal plasma jet (NTPJ) for the inactivation of mycobacteria in aqueous solution was adopted as a novel approach. Addition of water vapor in a nitrogen plasma jet generated OH radicals, which converted to hydrogen peroxide ($H_2O_2$) that inactivated non-pathogenic Mycobacterium smegmatis and pathogenic Mycobacterium tuberculosis H37Rv. A stable plasma plume was obtained from a nitrogen plasma jet with 1.91 W of power, killing Escherichia coli and mycobacteria effectively, whereas addition of catalase decreased the effects of the former. Mycobacteria were more resistant than E. coli to NTPJ treatment. Plasma treatment enhanced intracellular ROS production and upregulation of genes related to ROS stress responses (thiolrelated oxidoreductases, such as SseA and DoxX, and ferric uptake regulator furA). Morphological changes of M. smegmatis and M. tuberculosis H37Rv were observed after 5 min treatment with $N_2+H_2O$ plasma, but not of pre-incubated sample with catalase. This finding indicates that the bactericidal efficacy of NTPJ is related to the toxicity of OH and $H_2O_2$ radicals in cells. Therefore, our study suggests that NTPJ treatment may effectively control pulmonary infections caused by M. tuberculosis and nontuberculous mycobacteria (NTM) such as M. avium or M. abscessus in water.

• The Journal of the Korean Society for Microbiology
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• v.21 no.3
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• pp.363-373
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• 1986

We developed a giant colony test system with rapidly growing mycobacteria by stab-culture with a loopful inoculum of cells into Middlebrook 7H10 agar medium containing soluble extracts of the fruits of Gardenia jasminoides(7H10-crocin agar medium) and assessed the significance of the giant colony test with 28 strains of 10 clusters of rapidly growing mycobacteria classified by the simple biological 5-test characters. Of the 10 clusters of mycobacteria tested, some of strains which belonged to cluster No. 1a, 5a and 11a did grow as gravis types, whereas most of other clusters gave mitis or intermedius types in their colonial sizes at 12 days culture. By this test, pathogenic strains of M. fortuitum-chelonei complex which belonged to cluster No. 5a, b, 7a and 8a, b could be divided into gravis, intermedius and mitis colony types and the gravis ones were characterized by bluish-white "mushroom-shaped" colonies with central complexes in the texture, whereas the intermedius gave grayish-white "flower-shaped" colonies with radiated folds, but without any central complexes. The mitis colonies were characterized by grayish-white smooth or smooth mucoid colonies and were common among the clusters in their shapes. The colony of M. chelonei was bluish-white mitis type and was characterized by its hilly rhizoid colony. The gravis colony of cluster No. 1a identified as M. phlei was characterized by yellow "round straw- mat-shaped" or "chrysanthemum-shaped" colony with whole complexes in the texture, and the gravis colonies of the cluster No. 11a gave grayish-white "flower-shaped" colonies with central stamens, radiating trough and fine cup-shaped strands in the texture. The four colony types of pathogenic species of M. fortuitum-chelonei complex on 7H10-crocin agar medium were distinctive from those of other clusters of rapidly growing mycobacteria and these results indicated that the giant colony test, in conjunction with the simple 5-test characters, would be of value in the differentiation of M. fortuitum complex from other clusters of rapidly growing mycobacteria.

• The Journal of the Korean Society for Microbiology
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• v.34 no.6
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• pp.561-571
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• 1999

Diagnosis of mycobacterial infection is dependent upon the isolation and identification of causative agents. The procedures involved are time consuming and technically demanding. To improve the laborious identification process mycobacterial systematics supported by gene analysis is feasible, being particularly useful for slowly growing or uncultivable mycobacteria. To complement genetic analysis for the differentiation and identification of mycobacterial species, an alternative marker gene, rpoB encoding the ${\beta}$ subunit of RNA polymerase, was investigated. rpoB DNAs (342 bp) were amplified from 52 reference strains of mycobacteria including Mycobacterium tuberculosis H37Rv (ATCC 27294) and clinical isolates by the PCR. The nucleotide sequences were directly determined (306 bp) and aligned using the multiple alignment algorithm in the MegAlign package (DNASTAR) and MEGA program. A phylogenetic tree was constructed with a neighborhood joining method. Comparative sequence analysis of rpoB DNA provided the basis for species differentiation. By being grouped into species-specific clusters with low sequence divergence among strains belonging to same species, all the clinical isolates could be easily identified. Furthermore RFLP analysis enabled rapid identification of clinical isolates.

• The Journal of the Korean Society for Microbiology
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• v.21 no.1
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• pp.53-62
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• 1986

An attempt was made to verify the antibacterial activities of the soluble extracts of Gardenia jasminoides(SEGJ) against mycobacteria other than tubercle bacilli (MOTT); M. kansasii, M. scrofulaceum, M. simiae, M. szulgai, M.xenopi, M. avium-intracellulare complex, M. gordonae and M. flarescens. In addition, the effect of the SEGJ on the yellow pigmentation and violet pigmentation (crocin reaction) of the colonies of the MOTT grown on7H10-crocin agar plates was observed. The SEGJ gave a growth stimulatory activity against most species of mycobacteria except for M. szulgai at low concentration of the SEGJ in terms of crocin pigment OD=0.02 or less, but exerted an inhibitory activity at high concentration of OD=0.04 or more. The growth-inhibitory activity of the SEGJ was dose-dependent but the yellow pigmentation of the scotochromogens was not affected by the dose of the SEGJ that was growth-inhibitory. Crocin positive reaction was observed only with M. kansasii.