Bedaquiline Resistance Related Genes In Clinical Isolated Strains Of Mycobaterium Tuberculosis

Objective: To understand the mutation of bedaquiline-resistant associated genes of mycobacterium tuberculosis and the correlation with drug-resistant phenotype in vitro of the new drug bedaquiline on drug-resistant tuberculosis treatment,and to provide laboratory data for the treatment of drug-resistant tuberculosis with bedaquiline.Methods: Collected clinical sputum/BALF specimens of tuberculosis patients in our hospital from 2017 to 2020 for mycobacterium tuberculosis culture to obtain clinical isolates of mycobacterium tuberculosis and preserve the bacteria for follow-up research.Cetyltrimethylammonium bromide method(CTAB)was used to extract DNA of clinical isolates,and used PCR to verify the concentration and purity qualified samples.Then the whole genome sequencing was performed on the samples which identified as mycobacterium tuberculosis,and the sequencing result was aligned with the sequence of the standard strain of Mycobacterium tuberculosis H37 Rv to analyze whether there are mutations in bedaquiline resistance related genes Rv0678,atp E and pep Q.The above-mentioned bacteria-preserved specimens were resuscitated in batches.After successful resuscitation,bedaquiline phenotype susceptibility test and microplate MIC determination were performed to analyze and compare the relationship between the genotypes of bedaquiline resistance related genes and drug susceptibility phenotypes.Used the proportional method to conduct phenotypic drug sensitivity test of first-line anti-tuberculosis drugs to understand the correlation between bedaquiline and rifampicin resistance.Results: In this study,201 clinical isolates of Mycobacterium tuberculosis were obtained,and we obtained 200 whole genome sequencing data successfully.Non-synonymous and synonymous mutation sites were found in Rv0678,atp E and pep Q of bedaquiline respectively.Five non-synonymous mutation sites of Rv0678 gene were found in six clinical isolates of Mycobacterium tuberculosis: 1 case each of C98T(Thr33Leu),G149A(Arg50Glu),G256A(Ala86Thr)and C458T(Ala153Val),and 2cases of T469C(Thr157His).The nonsynonymous mutation rate was 3.0%(6/200).One synonymous mutation site C240T(Val80Val)of atp E was found in a Mycobacterium tuberculosis clinical isolate.There are two synonymous mutation sites of pep Q in 3 clinical isolates of Mycobacterium tuberculosis: 1 case of G865A(Gyl289Gyl)and 2 cases of G868A(Arg290Arg).Phenotypic drug sensitivity test(liquid method)and MIC determination of bedaquiline were performed on 137 isolated bacteria cultured successfully after resuscitation.The concentration range was0.004～2.048 μg/ml,MIC50 was 0.128 μg/ml,MIC90 was 0.512 μg/ml,and MIC of H37 Rv was 0.128 μg/ml.The MIC of bedaquiline was correlated with the mutation of drug-resistance gene.Among 137 strains with MICs of 0.016～1.024 μg/ml,Rv0678-(Thr157His)mutation occurred in the strains with MICs of 0.032～0.064 μg/ml.Rv0678-(Ala86Thr)mutation occurred in the strains with MICs of 0.064 μg/ml.Rv0678-(Thr33Leu)mutation occurred in the strains with MICs of 0.512 μg/ml.Rv0678-(Arg50Glu)and Rv0678-(Ala153Val)mutation occurred in the strains with MICs of 1.024 μg/ml.The first-line drug-tuberculosis drug sensitivity test(solid ratio method)was performed to determinate 37 cases of fully susceptible bacteria,2 cases of isoniazid resistant bacteria,3 cases of rifampicin resistant bacteria and 95 cases of multidrug-resistant bacteria.All the bedaquiline resistant bacteria were isolated from multidrug-resistant clinical isolates,and the resistance rate of bedaquinoline in multidrug-resistant bacteria was 5.3 %(5/95)(MIC>0.512 μg/ml).Conclusion: In this study,we successfully obtained the whole genome sequencing data of 200 clinical isolates of Mycobacterium tuberculosis.Five non-synonymous mutation sites were found in the bedaquiline resistance-related gene Rv0678,among which Rv0678-(Arg50Glu)and Rv0678-(Ala153Val)mutation may be related to bedaquiline resistance.The Rv0678-(Thr33Leu),Rv0678-(Ala86Thr)and Rv0678-(Thr157His)mutations did not increase the bedaquiline MIC significantly,so the relationship with bedaquiline resistance is unclear.The mutation has not been reported in the literature yet,and its significance needs to be further studied.In addition,the phenotypic resistance of rifampicin may be related to the phenotypic resistance of bedaquiline.