| Objective:To understand the mutation of Delamanid-resistant genes associated with Mycobacterium Tuberculosis（MDR-TB）and the correlation with drug-resistant phenotype in vitro of the new drug delamanid for the treatment of MDR-TB,and to provide laboratory data for the treatment of MDR-TB with Delamanid.Methods:Collected in our hospital in 20172018 tuberculosis patients clinical sputum specimens of this acid fast stain,mycobacterium tuberculosis culture for mycobacterium tuberculosis in clinical isolates,using the method of CTAB extraction strains of DNA,identified as mycobacterium tuberculosis PCR,to carry on the whole genome sequencing,ddn,fgd1,fbiA,fbiB and fbiC of delamanid resistance related gene is analysed,and contrast with the standard strains of mycobacterium tuberculosis H37Rv sequences.The clinical isolates of mycobacterium tuberculosis were tested by the liquid method for the phenotypic drug sensitivity test and the MIC test,and the phenotypic drug sensitivity test for the first-line and second-line anti-tuberculosis drugs was carried out by the proportion method.To analyze and compare the relationship between delamanid resistance related gene mutation and drug susceptibility phenotype,as well as the correlation between delamanid and rifampin resistance.Results:In this study,a total of 124 clinical isolates of Mycobacterium Tuberculosis were obtained.DNA was extracted by CTAB method for whole-genome sequencing,and 123 whole-genome sequencing data were successfully obtained.Ddn,fgd1,fbiA,fbiB and fbiC of five delamanid drug-resistant genes were found in a total of 10non-synonymous mutation sites in 123 clinical isolates of mycobacterium tuberculosis,as follows:there were 3 mutation points in ddn,G158A（Gly53Asp）,C160T（Arg54Cys）and G421A（Asp141Asp）were non-synonymous mutations,and the non-synonymous mutation rate was 2.4%（3/123）,T960C（Phe320Phe）was found in fgd1.There were 5mutation points in fbiA,among which G595A（Ala199Thr）,G616A（Ala206Thr）,C584G（Ala195Gly）were non-synonymous mutations,C337T（Leu113Leu）and G315A（Gly105Gly）were synonymous mutations,and the non-synonymous mutation rate was 3.3%（4/123）.One non-synonymous mutation site C997T（Arg333Cys）was found in fbiB,and the mutation rate was 1.6%（2/123）.There were 5 mutation sites in fbiC,of which C1571G（Ala524Gly）and A1711G（Lys571Glu）were non-synonymous mutations,T1640C（Val547Ala）,C2427G（Gly809Gly）and G630C（Ser210Ser）were synonymous mutations,and the non-synonymous mutation rate was4.1%（5/123）.The drug sensitivity test（liquid method）and MIC for the phenotype of delamanid were conducted,with concentrations ranging from 0.0021.024μg/ml,MIC500 is 0.064μg/ml,MIC900 is 0.512μg/ml,and H37Rv is 0.016μg/ml and 0.008μg/ml respectively.It was found that 6 cases of delamanid MIC>1.024μg/ml,among which1 case had mutation points fbiA-C337T（Leu113Leu）and fbiA-G315A（Gly105Gly）,1case had mutation points fbiB-C1571G（Ala524Gly）and fbiC-C2427G（Gly809Gly）,and 1 case had mutation points fbiB-C1571G（Ala524Gly）.Mutation points fbiA-C337T（Leu113Leu）and fgd1-T960C（Phe320Phe）were found in 1 case and 2 cases had fgd1-T960C（Phe320Phe）.Among the 69 strains with a MIC of0.0041.024μg/ml,the MIC of fbiA-G616A（Ala206Thr）and fbiA-C584G（Ala195Gly）mutated strains was 0.512μg/ml,and the MIC of fbiC-T1640C（Val547Ala）was0.256μg/ml.Fgd1-T960C was found in 50%of delamanid resistant strains and 59.4%of delamanid sensitive strains,showing no significant correlation with delamanid resistance.The MIC of the mutated strains of ddn-G158A（Gly53Asp）and C160T（Arg54Cys）was 0.004μg/ml.The MIC of delamanid in 2 cases with fbiB-C997T was0.008μg/ml.52 cases of rifampicin resistant strains（2 cases of rifampicin alone,2cases of multidrug resistance,44 cases of multidrug resistance,and 4 cases of extensive drug resistance）and 72 cases of rifampicin sensitive strains（68 cases of total sensitivity,4 cases of multidrug resistance）were obtained after the sensitivity test of first-line and second-line drugs against tuberculosis（solid proportion method）.In addition,in rifampicin resistant strains,the drug resistance rate of delamanid（MIC>1.024μg/ml）was 15%（5/33）,and in rifampin sensitive strains,the drug resistance rate of delamanid was 2.4%（1/42）.Conclusion:In this study,whole-genome sequencing results of 123 clinical isolates of mycobacterium tuberculosis were successfully obtained.Mutational sites were found in ddn,fgd1,fbiA,fbiB and fbiC genes related to drug resistance of delamanid.It was speculated that non-synonymous mutational sites of fbiA and fbiC genes fbiA-C584G（ALa195Gly）,fbiA-G616A（Ala206Thr）,fbiC-C1571G（Ala524Gly）and fbiC-T1640C（Val547Ala）might be related to resistance of mycobacterium tuberculosis to delamanid.The mutation sites of fbiB and ddn genes fbiB-C997T（Arg333Cys）,ddn-G158A（Gly53Asp）and ddn-C160T（Arg54Cys）did not significantly lead to the increase of delamanid MIC,so it is necessary to further expand the sample size for indepth study on whether it is related to drug resistance.In this study,G421A,G158A and C160T mutations of ddn gene,G616A,G595A and C584G mutations of fbiA gene,C997T mutations of fbiB gene and C1571G,T1640C and A1711G mutations of fbiC gene have not been reported,and their significance remains to be further studied.There was no significant correlation between rifampin resistance and delamanid resistance. |
