In the RNA-based study, it is important to extract high-quality RNA. However, RNA extraction from Mycobacterium tuberculosis is problematic due to its thick, waxy cell wall rich in mycolic acid, which renders the cells resistant to lysis. Using TRIzol reagent and several powerful bead-beating steps, a high quantity of RNA was obtained.
Cell Wall ; Mycobacterium tuberculosis* ; Mycobacterium* ; Mycolic Acids ; RNA*

Mycolic acids (MAs), i.e. 2-alkyl, 3-hydroxy long-chain fatty acids, are the hallmark of the cell envelope of Mycobacterium tuberculosis and are related with antibiotic resistance and host immune escape. Nowadays, they've become hot target of new anti-tuberculosis drugs. There are two main methods to detect MAs, ¹⁴C metabolic labeling thin-layer chromatography (TLC) and liquid chromatograph mass spectrometer (LC-MS). However, the user qualification of ¹⁴C or the lack of standards for LC-MS hampered the easy use of this method. TLC is a common way to analyze chemical substance and can be used to analyze MAs. In this study, we used tetrabutylammonium hydroxide and methyl iodide to hydrolyze and formylate MAs from mycobacterium cell wall. Subsequently, we used diethyl ether to extract methyl mycolate. By this method, we can easily extract and analyze MA in regular biological labs. The results demonstrated that this method could be used to compare MAs of different mycobacterium in different growth phases, MAs of mycobacteria treated by anti-tuberculosis drugs or MAs of mycobacterium mutants. Therefore, we can use this method as an initial validation for the changes of MAs in researches such as new drug screening without using radioisotope or when the standards are not available.
Mycolic Acids/metabolism* ; Chromatography, Thin Layer ; Mycobacterium tuberculosis ; Fatty Acids ; Antitubercular Agents/pharmacology*

BACKGROUND: Mycobacteria are traditionally identified with biochemical reactions. Since it takes 2 to 6 weeks, more rapid method is needed for timely treatment of mycobacterial infection. Mycolic acid analysis by high-performance liquid chromatography (HPLC) was recently introduced, which showed species-specificity with more than 95% sensitivity and 100% specificity for identifing Mycobacterium spp. within 2-4 hours. In this study, We performed mycolic acid analyses of standard strains of Mycobacterium spp. and two clinical isolates of known M. tuberculosis for demonstrating their species-specific nature and evaluated its reproduciblity. METHODS : 8 standard strains of Mycobacterium spp. (M. tuberculosis H37Rv, M. intracellurae, M. avium, M. fortuitum, M. chelonae subsp. chelonae, M. scrofulaceum, M. kansasii, M. gordonae) and 2 clinical isolates of known M. tuberculosis were analyzed. The extracted mycolic acids which were prepared by 3 steps were analyzed by HPLC with rC18 column. RESULTS: Mean retention time (MRT) of low and high molecular weight internal standards were 3.757min+/-0.017 (C.V. <0.455%) and 9.829min+/-0.015 (C.V. <0.015%), respectively (n=30). The C.V. of MRT for M. intracellurae for positive control showing double cluster pattern was less than 0.3% from 4 injection. The C.V. of MRT for M. tuberculosis H37Rv and 2 clinical isolates of M. tuberculosis with single cluster pattern were less than 0.4%, and 0.9%, respectively. The chromatographic patterns of M. kansasii and M. gordonae showed a single cluster pattern, and M. avium, M. fortuitum, M. chelonae subsp. chelonae, and M. scrofulaceum showed a double cluster pattern which were species-specific nature. CONCLUSIONS: We demonstrated HPLC method was rapid and highly reproducible.
Chromatography, High Pressure Liquid ; Chromatography, Liquid* ; Gordonia Bacterium ; Molecular Weight ; Mycobacterium* ; Mycolic Acids* ; Sensitivity and Specificity ; Tuberculosis

BACKGROUND: Mycobacterial disease is still greatly concerned in the developing and industrialized countries. Ogawa media has been used to diagnose mycobacterial disease in Korea in spite of a low sensitivity and long incubation time. Mycobacterium Growth Indicator Tube (MGIT) 960 system has been developed to overcome the pitfalls of Ogawa media. So, we investigated improvement in dectection rate and the detection time of mycobacteria using the MGIT 960 system along with 3% Ogawa media. METHODS: A total of 8,045 clinical specimens referred to the department of laboratory medicine in Ulsan University Hospital from January in 2001 to June in 2002 were cultured for mycobacteria. Specimens were processed with the NALC-NAOH (final concentration of NaOH: 1%) and inoculated into both MGIT and Ogawa media. Mycolic acid in the cultured products were analyzed by High performance liquid chromatography to discriminate between Mycobacterium tuberculosis and nontuberculous mycobacteria. RESULTS: Of 8,045 clinical specimens cultured, mycobacteria grew in 957 (11.9%) specimens, 840 (87.8%) M. tuberculosis and 117 (12.2%) nontuberculous mycobacteria. Mycobacteria were detected in 939 specimens (98.1%) by MGIT and 771 (80.6%) specimens by Ogawa media; 753 (78.7%) were detected by both media, 186 (19.4%) by MGIT only, and 18 (1.9%) by Ogawa media only. Mycobacteria were detected in 11.7 days by MGIT 960 and 28.4 days by Ogawa media. CONCLUSIONS: The detection rate and detection time of mycobacteri are improved considerably by the MGIT system; however a combined use of MGIT system and Ogawa media is the most ideally recommended for increasing the detection rate and shortening the detection time.
Chromatography, Liquid ; Developed Countries ; Korea ; Mycobacterium tuberculosis ; Mycobacterium* ; Mycolic Acids ; Nontuberculous Mycobacteria ; Tuberculosis ; Ulsan

BACKGROUND: Infections caused by nontuberculous mycobacteria (NTM) are significantly increasing over the last decade. Due to the uncertainty in the clinical significance of these organisms, their effective diagnosis and treatment has been challenging. The purpose of this study was to investigate the distribution and clinical significance of NTM in clinical specimens. METHODS: Acid-fast culture positive 3,107 clinical specimens were identified by mycolic acid analysis using high performance liquid chromatography (HPLC.) The HPLC patterns of 384 NTM strains were compared with those of standard mycobacterium species. Clinical significance of NTM was investigated by a retrospective study including acid-fast stain and culture, medical history, symptoms and signs, radiological and other laboratory findings, pathologic findings, response to treatment, and follow-up study, and was confirmed according to the guideline of American Thoracic Society. RESULTS: Among the 3,107 Mycobacterium-positive specimens, 384 (12.4%) were found to be positive for NTM. Of these, 367 (95.6%) were successfully identified by HPLC as 19 different species, each of which comprising 0.3% to 15.9% of the total NTM, Studies on the pathogenic role of NTM showed that 0~79.6% of each species or 0~100% of isolates from each specimen could be considered clinically significant. CONCLUSION: HPLC method is highly discriminative for the identification of NTM in clinical specimens. When NTM is isolated from clinical specimens in the Ulsan area, the findings from this study could serve as a database on which to determine its clinical significance depending on species type and also specimen type.
Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Follow-Up Studies ; Mycobacterium ; Mycolic Acids ; Nontuberculous Mycobacteria ; Retrospective Studies ; Uncertainty

BACKGROUND: Histoplasmosis (HP) is diagnosed by visualizing intracellular microorganisms in biopsy and/or culture. Periodic-acid Schiff (PAS) and Gomori methenamine silver (GMS) staining methods are routinely used for identification. The acid-fast property of Histoplasma was identified decades ago, but acid-fast staining has not been practiced in current surgical pathology. Awareness of the acid-fast property of Histoplasma, which is due to mycolic acid in the cell wall, is important in distinguishing Histoplasma from other infective microorganisms. Here, we examined acid-fastness in previously diagnosed cases of Histoplasma using the Ziehl-Neelsen (ZN) stain and correlated those findings with other known fungal stains. METHODS: All cases diagnosed as HP were retrieved and reviewed along with ZN staining and other fungal stains. We also stained cases diagnosed with Cryptococcus and Leishmania as controls for comparison. RESULTS: A total of 54 patients ranging in age from 11 to 69 years were examined. The most common sites of infection were the skin, adrenal tissue, and respiratory tract. Of the total 43 tissue samples, 20 (46.5%) stained positive with the ZN stain. In viable cases, a significant proportion of microorganisms were positive while necrotic cases showed only rare ZN-positive yeasts. In comparison to PAS and GMS stains, there was a low burden of ZN-positive yeasts. Cryptococcus showed characteristic ZN staining and all cases of Leishmania were negative. CONCLUSIONS: Although the morphology of fungal organisms is the foundation of identification, surgical pathologists should be aware of the acid-fast property of fungi, particularly when there is the potential for confusion with other infective organisms.
Biopsy ; Cell Wall ; Coloring Agents ; Cryptococcus ; Fungi ; Histoplasma* ; Histoplasmosis ; Humans ; Leishmania ; Methenamine ; Mycolic Acids ; Pathology, Surgical* ; Respiratory System ; Skin ; Yeasts

PURPOSE: Recognition of microbes is important to trigger the innate immune system. Mycolic acid (MA) is a component of the cell walls of mycobacteria such as Mycobacterium bovis Bacillus Calmette-Guerin. MA has immunogenic properties, which may modulate the innate and adaptive immune response. This study aimed to investigate whether a novel synthetic MA (sMA) inhibits allergic inflammatory responses in a mouse model of asthma. METHODS: BALB/c mice were injected intraperitoneally with sMA followed by sensitization and challenge with ovalbumin (OVA). Mice were examined for bronchial hyperresponsiveness (BHR), the influx of inflammatory cells into the lung tissues, histopathological changes in the lungs and CD4+CD25+Foxp3+ T cells in the spleen, and examined the response after the depleting regulatory T cells (Tregs) with an anti-CD25mAb. RESULTS: Treatment of mice with sMA suppressed the asthmatic response, including BHR, bronchoalveolar inflammation, and pulmonary eosinophilic inflammation. Anti-CD25mAb treatment abrogated the suppressive effects of sMA in this mouse model of asthma and totally depleted CD4+CD25+Foxp3+ T cells in the spleen. CONCLUSIONS: sMA attenuated allergic inflammation in a mouse model of asthma, which might be related with CD4+CD25+Foxp3+ T cell.
Adaptive Immunity ; Animals ; Asthma* ; Bacillus ; Cell Wall ; Eosinophils ; Immune System ; Inflammation* ; Lung ; Mice* ; Mycobacterium bovis ; Mycolic Acids* ; Ovalbumin ; Spleen ; T-Lymphocytes ; T-Lymphocytes, Regulatory

BACKGROUND: Infections caused by mycobacteria have been significantly increasing. Due to the difficulty of making a decision about the pathogenicity of mycobacteria, species-level identification is very important for patients' diagnosis and treatment. The purpose of this study was to identify mycobacteria species using a high performance liquid chromatography (HPLC) method and to provide an initial database for the distribution of mycobacteria in Korea. METHODS: Acid fast bacteria isolated from 3,107 clinical specimens were identified by mycolic acid analysis using HPLC. The HPLC patterns were compared with those of standard mycobacteria species. RESULTS: The HPLC patterns were divided into single, double, and triple cluster groups, each group comprising 9, 20, and 4 species, respectively. Mycobacteria and non-tuberculous mycobacteria (NTM) were identifies by HPLC at the rates of 99.5% and 95.6%, respectively. NTM was isolated in 12.4% of the mycobacteria positive specimens. This study also found that there were 20 different NTM species with the distribution of each species ranging from 0.3% to 15.9% of the total NTM. While the rate of NTM has been increasing in Korea, M. avium-intracellulare, M. fortuitum, and M. chelonae are relatively decreasing, and M. kansasii and M. gordonae are relatively increasing. CONCLUSIONS: HPLC method was highly discriminative for the identification of NTM in clinical specimens.
Bacterial Typing Techniques ; Chromatography, High Pressure Liquid/*methods ; Hospitals, University ; Humans ; Korea ; Mycobacteria, Atypical/chemistry/*isolation & purification ; Mycobacterium Infections, Atypical/drug therapy/microbiology ; Mycolic Acids/analysis

A slowly growing, non-chromogenic mycobacterial strain was isolated from sputum and bronchial lavage fluid samples of a patient presenting with productive cough, blood-tinged sputum, low-grade fever, and weakness. A positive acid-fast bacilli sputum smear result prompted the initiation of an anti-tuberculosis regimen. Multiplex real-time PCR showed a negative result for Mycobacterium tuberculosis complex and a positive result for nontuberculous mycobacteria. The DNA chip test confirmed this organism as a member of the genus Mycobacterium, but could not specify the species. Interestingly, the mycolic acid patterns obtained by HPLC nearly overlapped with those of M. simulans. The sequences of the Mycobacterium 16S rRNA gene and 16S-23S internal transcribed spacer region were unique and were found to have 100% similarity with those of M. riyadhense. After a review of the literature, we report this case as the first Korean case of M. riyadhense lung infection.
Adult ; Antitubercular Agents/pharmacology ; Chromatography, High Pressure Liquid ; Female ; Humans ; Lung Diseases/*microbiology ; Microbial Sensitivity Tests ; Mycobacterium/classification/drug effects/*isolation & purification ; Mycobacterium Infections/microbiology ; Mycobacterium tuberculosis/genetics/isolation & purification ; Mycolic Acids/analysis ; Oligonucleotide Array Sequence Analysis ; Phylogeny ; RNA, Ribosomal, 16S/chemistry/genetics ; RNA, Ribosomal, 23S/chemistry/genetics ; Republic of Korea ; Sequence Analysis, DNA