OBJECTIVETo explore the correlation between Beijing genotype (Beijing family) strains of Mycobacterium tuberculosis (MTB) and drug resistance.

METHODSA computer retrieval of Medline, Embase, SCI, EBSCO, CNKI, Weipu and Wanfang databases from 1990 to 2010 was conducted. A total of 525 articles exploring the relationship of Beijing genotype of MTB and drug resistance were found through literature search. Following the inclusion and exclusion criteria, a Meta-subgroup analysis was conducted in Beijing genotype of MTB and drug resistance.

RESULTSA total of 38 articles were selected, including 22 articles on isoniazid resistance, 24 articles on rifampin resistance, 19 articles on ethambutol resistance, 18 articles on ethambutol resistance, 26 articles on multi-drug resistance (MDR). Meta-subgroup analysis showed that in China, there was an association between Beijing genotype and resistance to rifampin, ethambutol and MDR: rifampin (OR = 1.62, 95%CI: 1.13 - 2.31), ethambutol (OR = 1.67, 95%CI: 1.16 - 2.40), MDR (OR = 1.79, 95%CI: 1.20 - 2.68); in Russia, there was an association between Beijing genotype and resistance to isoniazid, rifampin, ethambutol and MDR: isoniazid (OR = 4.82, 95%CI: 3.19 - 7.29), rifampin (OR = 4.84, 95%CI: 3.84 - 6.10), ethambutol (OR = 3.32, 95%CI: 2.51 - 4.40), MDR (OR = 5.42, 95%CI: 3.36 - 8.74); in Vietnam, there was an association between Beijing genotype and resistance to isoniazid, rifampin, ethambutol and MDR: isoniazid (OR = 2.12, 95%CI: 1.55 - 2.91), rifampin (OR = 4.71, 95%CI: 3.01 - 7.36), ethambutol (OR = 3.78, 95%CI: 1.63 - 8.77), MDR (OR = 4.21, 95%CI: 1.58 - 11.18); in other countries, there was an association between Beijing genotype and resistance to isoniazid, rifampin, ethambutol and MDR: isoniazid (OR = 1.69, 95%CI: 1.19 - 2.42), rifampin (OR = 2.48, 95%CI: 1.92 - 3.19), ethambutol (OR = 3.04, 95%CI: 2.13 - 4.33), MDR (OR = 2.36, 95%CI: 1.52 - 3.68).

CONCLUSIONBeijing genotype of MTB was positively associated with three kinds of first-line anti-tuberculosis drugs (isoniazid, rifampin, ethambutol) and MDR, and the relationship intensity was different in different countries.

Antitubercular Agents ; pharmacology ; China ; DNA, Bacterial ; Drug Resistance, Multiple, Bacterial ; Genotype ; Humans ; Mycobacterium tuberculosis ; drug effects ; genetics ; isolation & purification ; Russia ; Tuberculosis, Multidrug-Resistant ; genetics ; microbiology ; Vietnam

OBJECTIVETo understand the characteristics of pncA gene in multidrug-resistant Mycobacterium tuberculosis isolates and its correlation with drug resistance to pyrazinamide.

METHODSA total of 127 clinical isolates of multidrug-resistant mycobacterium tuberculosis were collected from Shenzhen from year 2007 to 2009. PZA susceptibility was determined by the BACTEC MGIT 960 PZA method. Pyrazinamidase (PZase) activity testing and pncA gene sequencing were performed in all the isolates. The type and frequency of mutations in pncA were determined. Correlation analysis among PZA susceptibility and PZase activity, pncA mutation was performed.

RESULTSAmong the 127 isolates, 62 isolates (48.8%) were found resistance to PZA. Among the 62 PZA resistant isolates, 45 isolates which had various pncA mutations were negative for PZase. Mutation rate was 77.4% (48/62) in total PZA resistance isolates. Different types of 48 resistant isolates were identified in the pncA gene, including base substitution (33 isolates), frame shift mutation (12 isolates) and codon mutation (3 isolates). No mutations except one isolate (N11D) existed in all PZA-susceptibility isolates which were positive for PZase. A total of 5 mutations which have not been described previously were found as follows: H57P, P62Q, G108R, D110Y and G162V. The correlation among the PZA susceptibility and the PZase activity (r = 0.895, P < 0.05), the pncA mutation (r = 0.779, P < 0.05) were significant in 127 multidrug-resistant isolates.

CONCLUSIONA high diversity of pncA gene mutation was found among PZA resistant strains of MTB. This study revealed five new mutations of the pncA gene that were not previously described, which scattered in the hot-spot regions located in the metal coordination site and active site of the enzyme. Mutations had a high correlation with the PZA resistance.

Antitubercular Agents ; pharmacology ; DNA, Bacterial ; genetics ; Humans ; Mutation ; Mycobacterium tuberculosis ; drug effects ; genetics ; isolation & purification ; Pyrazinamide ; pharmacology ; Tuberculosis, Multidrug-Resistant ; genetics ; microbiology

OBJECTIVETo understand the characteristics of embB gene mutation of Mycobacterium tuberculosis (MTB) isolates from tuberculosis patients in Chongqing, and the value of embB306 as a molecular marker used to diagnose ethambutol (EMB)-resistant MTB strains.

METHODSDirect sequencing was used to analyze the polymorphism of embB mutation in 51 EMB-resistant MTB strains and 50 EMB-sensitive MTB strains. And diagnostic testing was used to evaluate the value of embB306 as a molecular marker of EMB -resistant MTB strains as compared with the traditional sensitivity test.

RESULTSAll 34 of 51 EMB-resistant strains (66.7%) and 3 of 51 EMB-sensitive strains (6%) had had embB306 mutation. The embB306 mutation rate in EMB-resistant strains coming from previously treated case was 87.5%, showing significantly higher than that from new cases (48.1%, P < 0.01); embB306 mutation rate was increased with the number of the resistant drugs; embB306 mutation serving as a marker to diagnose EMB-resistant MTB strains comparing with the traditional sensitivity test, had the rate of sensitivity = 66.7%, specificity = 94.0%, accuracy = 80.2% and Youden index = 60.7%.

CONCLUSIONembB306 mutation should be the main mechanism of MTB resistance to EMB in Chongqing, showing an association with the history of the treated and numbers of the resistant drugs. embB306 mutation should be a good marker to diagnose EMB-resistant MTB strains.

China ; DNA Mutational Analysis ; DNA, Bacterial ; genetics ; Genes, Bacterial ; Humans ; Mutation ; Mycobacterium tuberculosis ; drug effects ; genetics ; isolation & purification ; Pentosyltransferases ; genetics ; Tuberculosis, Multidrug-Resistant ; microbiology

OBJECTIVETo induce Mycobacterium tuberculosis (MTB) resistance with ofloxacin (Ofx) of stepwise increasing concentration in vitro, investigate stability to fluoroquinolone (FQs) antibiotic of MTB, and analyze the molecular mechanism and mutation specialty of drug resistance preliminarily.

METHODSMTB Standard strain H37RV and 24 clinical isolates susceptible to Ofx were selected and experimentally serially subcultured in liquid culture medium containing increasing concentration of Ofx and induced the drug resistance to Ofx. Variety of Minimal Inhibitory Concentrations (MICs) to FQs drugs were detected by microwell-MIC-test method. Mutations of quinolone resistance determining region (QRDR) of gyrA gene were sequenced and identified. Relationship of different mutation sites and drug resistant degree were analyzed. A total of 6 MTB clinical isolates resistant to Ofx and induced drug resistant isolates in vitro were serially subcultured in liquid culture medium without drug. Variety of drug resistant stability, including MIC and mutation of gyrA gene were detected.

RESULTSMIC values of 21 Ofx susceptible isolates after induction were eight times higher than before, which were induced to drug resistant strains successfully and also resistant to Lfx and Mfx. Hot mutations of QRDR of gyrA gene were detected by sequencing, except one strain. Mutation of codon 94 occurred in 60% (12/20) of the strains with mutations and corresponding value of 50% Minimal Inhibitory Concentrations(MIC50) was ≥ 8 µg/ml. In all, 4 of 6 MTB clinical isolates resistant to Ofx harbored mutation of codon 90 (67%) , but the corresponding value of MIC50 was 2 µg/ml. After 21 serially subcultured in liquid culture medium without drug, MIC values of 6 clinical isolates resistant to Ofx were not changed obviously and mutations were also not changed. After 11 times serially subcultured in culture medium without drug, MIC values of induced drug resistant strains were also not changed obviously, but new mutations were detected in QRDR of 3 isolates.

CONCLUSIONMTB strains resistant to three kinds of FQs antibiotic were obtained by induction in vitro with Ofx. Codons 88, 94 mutations of QRDR of gyrA gene were related to the high level FQs drug resistance of MTB. Drug resistant stability of MTB to FQs was strong, and it is difficult for MTB to resume susceptibility.

Antitubercular Agents ; pharmacology ; DNA Gyrase ; genetics ; Drug Resistance, Bacterial ; genetics ; Microbial Sensitivity Tests ; Mycobacterium tuberculosis ; drug effects ; genetics ; isolation & purification ; Ofloxacin ; pharmacology

In order to evaluate the performance of a molecular Hain line probe assay (Hain LPA) for rapid detection of rifampicin and isoniazid resistance of Mycobacterium tuberculosis in China, 1612 smear positive patients were consecutively enrolled in this study. Smear positive sputum specimens were collected for Hain LPA and conventional drug susceptibility testing (DST). The sensitivity and specificity of Hain LPA were analyzed by using conventional DST as golden reference. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for rifampicin resistance detection were 88.33%, 97.66%, 81.54%, and 98.62%, respectively. The sensitivity, specificity, PPV and NPV for isoniazid resistance detection were 80.25%, 98.07%, 87.25%, and 96.78%, respectively. These findings suggested that Hain LPA can be an effective method worthy of broader use in China.
China ; Genotyping Techniques ; methods ; Humans ; Isoniazid ; pharmacology ; Mycobacterium tuberculosis ; drug effects ; genetics ; isolation & purification ; Rifampin ; pharmacology ; Tuberculosis, Multidrug-Resistant ; diagnosis ; microbiology

LysinB (LysB) in mycobacteriophage D29 was cloned and expressed and its enzymatic properties were analysed. The lysB gene was amplified by PCR from mycobacteriophage D29 genomic DNA and inserted into pET22b vector. The constructed recombinant plasmid was transformed into Escherichia coli BL21(DE3) to express fusion protein, which was purified by Ni-NTA column and enzymatic activity detected. The results showed that expression plasmid pET22b-lysB was constructed successfully. Highly purified recombination protein (His-LysB) was obtained 33.2 mg from 1 L LB culture medium. A screening for His-LysB activity on esterase and lipase substrates confirmed the lipolytic activity. With p-nitrophenyl butyrate as substrate, the thermal stability of the enzyme was poor when the temperature was above 30 degrees C. The enzyme exhibited higher stability at pH 5.0-9.5. The optimum temperature and pH for the lipolytic activity of His-LysB were 23 degrees C and 7.5 respectively. Under the optimum conditions, the specific activity of His-LysB was 1.3 U/mg. Zn2+, CU2+, Mg2+, Mn2+ and phenylmethane sulfonyl fruoride severely inhibited the lipolytic activity of His-LysB. The result provides a new option for tuberculosis drug research and development.
Cloning, Molecular ; Enzyme Stability ; Escherichia coli ; genetics ; metabolism ; Mycobacteriophages ; enzymology ; Mycobacterium tuberculosis ; drug effects ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; metabolism ; Viral Proteins ; biosynthesis ; genetics

OBJECTIVETo study the characteristics of drug-resistant genetic mutation of rpoB in multiple drugs resistant bacillus tuberculosis (MDR-TB) among patients of pneumoconiosis complicated with pulmonary tuberculosis.

METHODSA total of 114 clinical isolated strains of Mycobacterium tuberculosis were collected, MDR-TB were identified by conventional antimicrobial susceptibility test (AST). Their genomes DNA were extracted, the target genes were amplified by PCR, and the hot regions in the rpoB gene were analyzed by automated DNA sequenator.

RESULTSThe results by AST showed that there were 31 strains of MDR-TB in the 114 clinical isolated strains, the rate of drug resistance was 27.19% (31/114). No mutation of rpoB was identified in 10 rifampicin-sensitive strains that randomly chosen, while conformation changes were found in MDR-TB strains, and the mutation rate of rpoB was 93.55% (29/31) in resistant strains, mainly concentrated in codon 531 (45.16%, 14/31) and 526 (29.03%, 9/31), happened base substitutions, including 27 unit point mutation and 2 two point mutation.

CONCLUSIONSThe substitution of highly conserved amino acids encoded by rpoB gene results in the molecular mechanism responsible for RFP resistance in MDR-TB among patients of pneumoconiosis complicated with pulmonary tuberculosis. It also proves that rpoB gene is diversiform.

Adult ; Aged ; Bacterial Proteins ; genetics ; DNA-Directed RNA Polymerases ; Drug Resistance, Multiple, Bacterial ; genetics ; Genes, Bacterial ; Humans ; Male ; Microbial Sensitivity Tests ; Middle Aged ; Mining ; Mutation Rate ; Mycobacterium tuberculosis ; drug effects ; genetics ; isolation & purification ; Pneumoconiosis ; microbiology ; Sequence Analysis ; Tuberculosis, Pulmonary ; microbiology

BACKGROUND: Tuberculosis (TB) remains an important cause of morbidity and mortality throughout the world. The surge of TB has been accompanied by an increase in multi-drug-resistant tuberculosis (MDR-TB). In this study, we developed a denaturing HPLC (DHPLC) method for detecting rpoB gene mutation as a rifampin resistance based on sequence. METHODS: In this study, we used 99 mycobacterial isolates grown in Ogawa media. At first, we used a PCR method that can amplify the 235 bp and 136 bp rpoB DNAs of Mycobacterium tuberculosis complex (MTB) and Non-tuberculous mycobacteria (NTM). And then, PCR-restriction fragment length polymorphism (RFLP) of rpoB DNA (342 bp), which comprises the Rif(T) region, was used for the differential identification of Mycobacteria. Finally, we detected these amplicons by DHPLC, compared to PCR-RFLP results, and performed sequencing. RESULTS: Among 99 mycobacterial isolates, 80 (81%) were MTB and 19 (19%) were NTM. NTM were identified to 7 different species by DHPLC and PCR-RFLP. rpoB mutation was detected in 9 (11%) of the MTB specimens. These results were confirmed by using sequencing. CONCLUSIONS: DHPLC provided a rapid, simple, and automatable performance for detection of rifampin resistant Mycobacterium tuberculosis complex and would be helpful as a supplemental method in high-throughput clinical laboratories.
Antibiotics, Antitubercular/*pharmacology ; Bacterial Typing Techniques ; Chromatography, High Pressure Liquid/*methods ; DNA, Bacterial ; Drug Resistance, Bacterial/genetics ; Humans ; Mutation ; Mycobacterium tuberculosis/*drug effects/genetics/*isolation & purification ; Rifampin/*pharmacology ; Tuberculosis/*microbiology

New anti-tubercular drugs and drug targets are urgently needed to reduce the time for treatment and also to identify agents that will be effective against Mycobacterium tuberculosis persisting intracellularly. Mycobacteria have a unique cell wall. Deletion of the gene for arylamine N-acetyltransferase (NAT) decreases mycobacterial cell wall lipids, particularly the distinctive mycolates, and also increases antibiotic susceptibility and killing within macrophage of Mycobacterium bovis BCG. The nat gene and its associated gene cluster are almost identical in sequence in M. bovis BCG and M. tuberculosis. The gene cluster is essential for intracellular survival of mycobacteria. We have therefore used pure NAT protein for high-throughput screening to identify several classes of small molecules that inhibit NAT activity. Here, we characterize one class of such molecules-triazoles-in relation to its effects on the target enzyme and on both M. bovis BCG and M. tuberculosis. The most potent triazole mimics the effects of deletion of the nat gene on growth, lipid disruption and intracellular survival. We also present the structure-activity relationship between NAT inhibition and effects on mycobacterial growth, and use ligand-protein analysis to give further insight into the structure-activity relationships. We conclude that screening a chemical library with NAT protein yields compounds that have high potential as anti-tubercular agents and that the inhibitors will allow further exploration of the biochemical pathway in which NAT is involved.
Antitubercular Agents ; chemistry ; isolation & purification ; pharmacology ; Arylamine N-Acetyltransferase ; antagonists & inhibitors ; chemistry ; Enzyme Inhibitors ; chemistry ; isolation & purification ; pharmacology ; High-Throughput Screening Assays ; Humans ; Mycobacterium bovis ; drug effects ; enzymology ; genetics ; Mycobacterium tuberculosis ; drug effects ; enzymology ; genetics ; Protein Conformation ; Structure-Activity Relationship ; Triazoles ; chemistry ; isolation & purification ; pharmacology

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