1.Relationship between Beijing genotypes of Mycobacterium tuberculosis and drug-resistant phenotypes.
Sheng-Fen WANG ; Yan-Lin ZHAO ; Hai-Rong HUANG ; Qiang LI ; Yang ZHOU ; Xi-Chao OU ; Yu-Hong FU
Acta Academiae Medicinae Sinicae 2009;31(4):427-431
OBJECTIVETo investigate the distribution of the Beijing genotypes of Mycobacterium tuberculosis (M. tuberculosis) and the relationships between Beijing genotype strains and drug-resistant phenotypes in China.
METHODSClinical isolates were collected during a 9-month research period from April to December in 2008 in six geographic regions of China. One isolate that had been biochemically confirmed to be a member of the M. tuberculosis complex was collected from each patient. The demographic data of the patients (eg. sex, age, and history of tuberculosis) as well as the drug resistance patterns and sources of the clinical isolates were collected. Drug susceptibility testing was performed using proportion method. Beijing genotypes of M. tuberculosis were identified by spacer oligonucleotide typing or insertion of IS6110 in the genomic dnaA-dnaN locus.
RESULTSAmong the 410 M. tuberculosis clinical isolates, 67.1% (275/410) isolates were Beijing genotypes of M. tuberculosis. Significantly larger proportions of tuberculosis patients were infected with Beijing genotypes in the northeastern regions of China than that of in the central-western regions (chi2 = 20.50, P = 0.000). No significant associations were found either between Beijing genotype strains and patients' age, sex, or treatment history. Multidrug-resistant isolates and rifampin-resistant isolates were more common among Beijing genotype strains than among non-Beijing strains (P = 0.002, P = 0.005).
CONCLUSIONSAbout two third of the clinical isolates of M. tuberculosis in China are Beijing genotypes. Beijing genotype strains are not correlated with patients' age, sex, treatment history. People living in the northeastern regions of China are more susceptible to Beijing genotypes than those living in the central-western of China. Beijing genotype strains tend to be rifampin-resistant or multidrug-resistant.
Antitubercular Agents ; pharmacology ; China ; Genotype ; Humans ; Mycobacterium tuberculosis ; classification ; genetics ; Phenotype ; Rifampin ; pharmacology ; Tuberculosis ; drug therapy ; Tuberculosis, Multidrug-Resistant ; genetics
2.Characterization of Mutations in Genes Related to Rifampicin and Isoniazid Resistance in Multidrug-resistant Mycobacterium tuberculosis Strains from Hangzhou, China.
Yin Yan HUANG ; Li XIE ; Yi Fei WU ; Qing Jun JIA ; Qing Lin CHENG ; Qing Chun LI ; Li Yun AI ; Xue Xin BAI
Biomedical and Environmental Sciences 2023;36(9):869-873
Humans
;
Isoniazid/pharmacology*
;
Mycobacterium tuberculosis/genetics*
;
Rifampin/pharmacology*
;
Antitubercular Agents/pharmacology*
;
Mutation
;
Microbial Sensitivity Tests
;
Tuberculosis, Multidrug-Resistant/microbiology*
;
Drug Resistance, Multiple, Bacterial/genetics*
;
Bacterial Proteins/genetics*
3.Quantitative proteomics analysis of ClpS-mediated rifampicin resistance in Mycobacterium.
Gulishana ADILIJIANG ; Shan FENG ; Kaixia MI ; Haiteng DENG
Chinese Journal of Biotechnology 2014;30(7):1115-1127
Adaptor protein ClpS is an essential regulator of prokaryotic ATP-dependent protease ClpAP, which delivers certain protein substrates with specific amino acid sequences to ClpAP for degradation. However, ClpS also functions as the inhibitor of the ClpAP-mediated protein degradation for other proteins. Here, we constructed the clpS-overexpression Mycobacterium smegmatis strain, and showed for the first time that overexpression of ClpS increased the resistance of M. smegmatis to rifampicin that is one of most widely used antibiotic drugs in treatment of tuberculosis. Using quantitative proteomic technology, we systematically analyzed effects of ClpS overexpression on changes in M. smegmatis proteome, and proposed that the increased rifampicin resistance was caused by ClpS-regulated drug sedimentation and drug metabolism. Our results indicate that the changes in degradation related proteins enhanced drug resistance and quantitative proteomic analysis is an important tool for understanding molecular mechanisms responsible for bacteria drug resistance.
ATP-Dependent Proteases
;
metabolism
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Drug Resistance, Bacterial
;
Endopeptidase Clp
;
metabolism
;
Mycobacterium smegmatis
;
drug effects
;
metabolism
;
Proteolysis
;
Proteomics
;
Rifampin
;
pharmacology
4.Establishment and drug susceptibility test of isoniazid resistant Mycobacterium smegmatis.
Ping-ping JIA ; Li-li ZHAO ; Xiao-yu LI ; Quan ZHANG ; Zhen-long LIU ; Xin WANG ; Li-yan YU ; Li-xun ZHAO ; Shan CEN
Acta Pharmaceutica Sinica 2011;46(4):377-382
With the emergence of drug resistant tuberculosis, it is very urgent to find novel anti-tuberculosis drugs, especially novel anti-drug-resistant tuberculosis drugs. Because of the slow growth and the need to work in a biosafty environment of Mycobacterium tuberculosis, the development of evaluation of drug effect is severely impeded. In order to solve these issues, non-pathogenic fast-growing Mycobacterium smegmatis is introduced as test organism. The inhA is one of a target of isoniazid (INH) overexpression or mutation of this gene in Mycobacterium tuberculosis conferring resistant to INH. A recombinant plasmid bearing inhA was constructed and electroporated into Mycobacterium smegmatis, using shuttle expression vector pMV261. Transformants were induced to express a protein of inhA, identified by SDS-PAGE. Results show that Mycobacterium smegmatis containing inhA plasmids exhibited 100-fold or greater increased resistance to INH, but it conferred no increased resistance to others first-line anti-tuberculosis drugs. Resazurin microtiter assay plate testing of Mycobacterium smegmatis susceptibility to drugs is a rapid, simple, and inexpensive method and could decrease color background of drugs by detecting fluorescence. It will be benefit for high-throughout screening of drugs of anti-isoniazid-resistant Mycobacteria.
Anti-Bacterial Agents
;
pharmacology
;
Antibiotics, Antitubercular
;
pharmacology
;
Antitubercular Agents
;
pharmacology
;
Bacterial Proteins
;
genetics
;
metabolism
;
Drug Resistance, Bacterial
;
Electroporation
;
Ethambutol
;
pharmacology
;
Isoniazid
;
pharmacology
;
Microbial Sensitivity Tests
;
Mycobacterium smegmatis
;
drug effects
;
genetics
;
metabolism
;
Oxidoreductases
;
genetics
;
metabolism
;
Plasmids
;
Rifampin
;
pharmacology
;
Streptomycin
;
pharmacology
5.In Vitro Bacteriostatic Effects of Rifampin on Orientia tsutsugamushi.
Jae Hyoung IM ; Ji Hyeon BAEK ; Jin Soo LEE ; Moon Hyun CHUNG ; Sun Myoung LEE ; Jae Seung KANG
Journal of Korean Medical Science 2014;29(2):183-189
We performed an in vitro cell culture experiment to ascertain whether rifampin exhibits bactericidal effects against Orientia tsutsugamushi, the causative agent of scrub typhus. ECV304 cells were infected with the Boryong or AFSC-4 strain of O. tsutsugamushi and then, the cultures were maintained in media with increasing concentrations of rifampin, azithromycin, doxycycline, or chloramphenicol for 4 days. On day 5, the media were replaced with fresh antibiotic-free medium and the cultures were maintained until day 28. On days 5, 13, and 28, immunofluorescence (IF) staining of O. tsutsugamushi was performed. IF staining on days 13 and 28 revealed increasing numbers of IF-positive foci in all cultures, even in cultures initially exposed to the highest concentration of rifampin (80 microg/mL), azithromycin (80 microg/mL), doxycycline (20 microg/mL), or chloramphenicol (100 microg/mL). The present study reveals that rifampin has no bactericidal effect against O. tsutsugamushi as observed for azithromycin, doxycycline, and chloramphenicol. A subpopulation of the bacteria that are not killed by high concentrations of the antibiotics may explain the persistence of O. tsutsugamushi in humans even after complete recovery from scrub typhus with antibiotic therapy.
Antibiotics, Antitubercular/*pharmacology
;
Cell Line, Tumor
;
Drug Resistance, Bacterial
;
Fluorescent Antibody Technique
;
Humans
;
Orientia tsutsugamushi/*drug effects/growth & development/metabolism
;
Rifampin/*pharmacology
6.Establishment and evaluation of nitrate reductase combined with mycobacteriophage assay.
Ai-Ying XING ; Zhong-Quan LIU ; Hong-Yan JIA ; Shu-Xiang GU ; Zong-De ZHANG
Acta Academiae Medicinae Sinicae 2009;31(4):413-416
OBJECTIVETo establish a rapid, inexpensive, and simple drug susceptibility test (DST) for Mycobacterium tuberculosis (M. tb) and evaluate its feasibility.
METHODWe used nitrate reductase combined with mycobacteriophage assay (PhaB-NRA) to test 49 clinical M. tb isolates of, and the results were compared with those of PhaB-NRA and traditional absolute concentration method.
RESULTSThe sensitivity, specificity, and accuracy of PhaB-NRA for rifampicin were 89.1%, 91.67%, and 89.8%; on the contrary, those of isonicotinyl hydrazide were 86.21%, 90.0%, and 87.8%, respectively. The coincidence between PhaB-NRA and traditional assay were 0.746 for rifampicin and 0.750 for isonicotinyl hydrazide.
CONCLUSIONSPhaB-NRA is an inexpensive, rapid, and simple DST method. It is a promising rapid screening technique for DST of M. tb.
Anti-Bacterial Agents ; pharmacology ; Biological Assay ; methods ; Humans ; Microbial Sensitivity Tests ; methods ; Mycobacteriophages ; physiology ; Mycobacterium tuberculosis ; drug effects ; Nitrate Reductase ; metabolism ; Rifampin ; pharmacology ; Sensitivity and Specificity
7.Multicenter Evaluation of the Molecular Line Probe Assay for Multidrug Resistant Mycobacterium Tuberculosis Detection in China.
Qiang LI ; Hai Yan DONG ; Yu PANG ; Hui XIA ; Xi Chao OU ; Zhi Ying ZHANG ; Jun Chen LI ; Jian Kang ZHANG ; Shi Tong HUAN ; Daniel P CHIN ; Kai Man KAM ; Yan Lin ZHAO
Biomedical and Environmental Sciences 2015;28(6):464-467
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
8.Rapid detection of rifampin-resistant clinical isolates of Mycobacterium tuberculosis by reverse dot blot hybridization.
Qian GUO ; ; Yan YU ; Yan Ling ZHU ; Xiu Qin ZHAO ; Zhi Guang LIU ; Yuan Yuan ZHANG ; Gui Lian LI ; Jian Hao WEI ; Yi Mou WU ; Kang Lin WAN ; ;
Biomedical and Environmental Sciences 2015;28(1):25-35
OBJECTIVEA PCR-reverse dot blot hybridization (RDBH) assay was developed for rapid detection of rpoB gene mutations in 'hot mutation region' of Mycobacterium tuberculosis (M. tuberculosis).
METHODS12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing (DST) and DNA sequencing to evaluate the RDBH assay.
RESULTSThe sensitivity and specificity of the RDBH assay were 91.2% (165/181) and 98.3% (117/119), respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the RDBH assay were 97.7% (293/300), 98.2% (164/167), and 97.0% (129/133), respectively. Furthermore, the results indicated that the most common mutations were in codons 531 (48.6%), 526 (25.4%), 516 (8.8%), and 511 (6.6%), and the combinative mutation rate was 15 (8.3%). One and two strains of insertion and deletion were found among all strains, respectively.
CONCLUSIONOur findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis.
Antitubercular Agents ; pharmacology ; Drug Resistance, Bacterial ; Genotype ; Immunoblotting ; methods ; Microbial Sensitivity Tests ; Mycobacterium tuberculosis ; drug effects ; genetics ; Polymerase Chain Reaction ; methods ; Rifampin ; pharmacology ; Sensitivity and Specificity ; Time Factors
9.Diagnostic accuracy of line probe assays for drug-resistant tuberculosis: a Meta-analysis.
Chinese Journal of Epidemiology 2018;39(11):1491-1495
Objective: To evaluate the diagnostic accuracy of line probe assays for drug- resistant tuberculosis (TB) in China. Methods: Chinese databases (CNKI, Wanfang, SinoMed, VIP Information) and English databases (PubMed, Embase, Cochrane Library) were used to retrieve the literatures regarding the accuracy of line probe assays in the diagnosis of drug-resistant tuberculosis in China between January 1, 2000 and September 1, 2017. Quality Assessment of Diagnostic Accuracy Studies-2 was used to evaluate the quality of the included studies. Sensitivity and specificity in different studies (using drug sensitivity test or gene sequencing as gold standard) were combined by Meta-analysis using bivariate or univariate model. In addition, subgroup analysis (GenoType MTBDRplus, GenoType MTBDRsl and Reverse dot blot hybridization) and sensitivity analysis were also carried out. Results: A total of 24 literatures involving 82 studies were included in the final analysis. The sensitivity and specificity of line probe assays for rifampicin resistant TB were 0.91(0.88-0.94) and 0.98 (0.97-0.99), respectively. The sensitivity and specificity of line probe assays for isoniazid resistant TB were 0.80 (0.77-0.83) and 0.98 (0.96-0.99), respectively. The sensitivity and specificity of line probe assays for multidrug-resistant TB were 0.81 (0.76-0.85) and 0.99 (0.99-1.00), respectively. The sensitivity and specificity of line probe assays for quinolone resistant TB were 0.92(0.88-0.95) and 0.94 (0.91-0.97), respectively. The sensitivity and specificity of line probe assays for second-line injectable drug resistant TB (including kanamycin, Capreomycin, amikacin) were 0.79(0.58-0.91) and 0.98 (0.90-1.00), respectively. The sensitivity and specificity of line probe assays for extensively drug-resistant TB were 0.46 (0.19-0.75) and 1.00 (0.98-1.00), respectively. Subgroup analysis showed that the overall diagnostic accuracy of GenoType MTBDRplus and GenoType MTBDRsl was higher than that of Reverse dot blot hybridization. According to the results of sensitivity analysis, the results of this study were robust. Conclusion: The diagnostic accuracy of line probe assays for drug-resistant TB is high.
Antitubercular Agents/therapeutic use*
;
Biological Assay/methods*
;
China
;
Humans
;
Isoniazid/pharmacology*
;
Microbial Sensitivity Tests/methods*
;
Mycobacterium tuberculosis/isolation & purification*
;
Rifampin/pharmacology*
;
Sensitivity and Specificity
;
Tuberculosis, Multidrug-Resistant/drug therapy*
10.Evaluation of pyrosequencing for the detection of rpoB gene mutation in Mycobacterium tuberculosis.
Rui-juan ZHENG ; Jie WANG ; Lian-hua QIN ; Jun-mei LU ; Zhong-yi HU
Chinese Journal of Preventive Medicine 2008;42(2):77-80
OBJECTIVETo detect the mutations of rpoB gene in Mycobacterium tuberculosis by pyrosequencing and to evaluate the values on detection of rifampin resistance in clinical isolates.
METHODSUsing the new technology of pyrosequencing, the mutations in the rifampin resistance determining region (RRDR) of rpoB gene were analyzed. The results were compared with those obtained from methods of the absolute concentration and the minimum inhibitory concentration (MIC).
RESULTSAmong the 150 Mycobacterium tuberculosis clinical isolates, 84 were susceptible and 66 resistant to RIF. 54 of the 66 resistant isolates were multidrug-resistant (MDR) strains. Ser531Leu and His526Asp or Tyr, including twelve different genotypes and six codons, were the most common mutations. In the drug susceptibility testing, the accordance rates of the pyrosequencing and the absolute concentration method as well as MIC were 92.7% and 97.8% respectively.
CONCLUSIONNot only is the pyrosequencing technology a fast, sensitive and high throughput method in detecting rifampin resistance in Mycobacterium tuberculosis, but also a useful tool in the research of rifampin resistance mechanism.
Bacterial Proteins ; genetics ; DNA-Directed RNA Polymerases ; Drug Resistance, Bacterial ; genetics ; Humans ; Microbial Sensitivity Tests ; Mutation ; Mycobacterium tuberculosis ; drug effects ; genetics ; Phosphoric Acids ; Polymerase Chain Reaction ; Rifampin ; pharmacology