| Objective Objective to obtain the minimum inhibitory concentration(MIC)of clinical isolates of Mycobacterium tuberculosis(MTB)to bedaquiline(Bdq)through drug sensitivity test(referred to as "drug sensitivity test"),and analyze the mechanism of resistance of MTB to bedaquiline from the gene level,so as to provide a theoretical basis for the generation of bedaquiline resistance.Method MTB strains were isolated from 151 patients with pulmonary tuberculosis in the cooperative Hospital of NDIP(new drug introduction protection)project.Among them,100 patients were only provided with baseline(within one week before taking the first dose of Bdq)culture positive strains,and 51 patients were provided with baseline strains and culture positive strains collected at 2,4,8,12,16,20 and 24 weeks after starting the treatment with Bdq.CMP1 BDQ 96 well drug sensitive microplate customized by Thermo Fisher company was used to detect the drug resistance of baseline MTB isolates to Bdq and other 11 kinds of anti tuberculosis drugs,and to detect the multi Bdq drug resistance of MTB clinical isolates in different periods after the patients started the treatment with Bdq regimen.The four genes,Rv0678,atp E,pep Q and Rv1979 c,were detected in the strains resistant to Bdq.The strains with no known mutation of resistance gene were sent to the whole genome for sequencing.The related genes that may cause resistance to bedaquiline were screened out.The transgenic experiment was designed in the model organism Mycobacterium smegmatis to verify the function of the new mutation gene,To explain the possible mechanism of drug resistance.The experimental results were expressed by median and quartile spacing.Chi square test was used to compare the differences of the rates.When P value was less than 0.05,the difference was significant.Results(1)More than 4/5 of the baseline MTB isolates(85.4%,129/151)had MIC values of 0.06 for Bdq0.03 μg/ml,MIC50 and MIC90 were 0.03 μg/ml and 0.12μg/ml;The primary resistance rate of baseline MTB isolates to Bdq was 2.0%(3 /151);The acquired resistance rate of Bdq was 2.0%(3/152).(2)The drug resistance rates of the baseline strains of 151 patients to the two drugs were 2.0%(3/151)and7.9%(12/151),respectively.The difference between the two groups was statistically significant(P= 0.003 < 0.05),that is,the drug resistance rates of the 151 patients to the two drugs were different.(3)The mutation of Rv0678 gene was detected in all the3 strains resistant to bedaquiline,but no mutation of atp E gene,pep Q gene and Rv1979 c gene was detected;Two of the three acquired drug-resistant strains had mutations in Rv0678 gene,but no mutations in pep Q,atp E and Rv1979 c genes were detected.Only one(1/7)of the mutations in Rv0678 gene was found to be resistant to bedaquiline.(4)After sequencing the whole genome of the drug-resistant strains without known drug-resistant gene mutation,the related gene Rv0020 c was screened out.Conclusions(1)In our country,the drug resistance of drug-resistant tuberculosis patients to bedaquiline is at a low level,and the use of bedaquiline in the treatment of drug-resistant tuberculosis is an effective choice.(2)Among the drug-resistant patients in this study,one patient with acquired drug resistance had no mutation in the known gene related to the resistance to bedaquiline,suggesting that there may be other mechanisms of resistance to bedaquiline,which needs further study. |
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