Prothionamide-Resistant Gene In Clinical Specimens Of Tuberculosis

Objective:To understand the mutation of Prothionamide-resistant genes in clinical isolates of Mycobacterium tuberculosis and its correlation with drug-sensitive phenotype of Mycobacterium tuberculosis,and to provide a reference for the clinical application of prothionamide.Methods: Clinical Mycobacterium tuberculosis specimens from March 2015 to March 2018 were collected and cultured.And after Ziehl-Neelsen staining,Mycobacterium tuberculosis and Drug susceptibility testing were performed.170 clinical isolates of Mycobacterium tuberculosis were obtained,of which 111 cases were sensitive.59 cases were drug-resistant.The DNA of the strains was extracted using by CTAB method.Polymerase Chain Reaction was used to amplify and sequence inhA,ethA and ethR genes.The results were compared with H37 Rv sequences of standard strains in NCBI database.The mutation sites of inhA,ethA and ethR genes were analyzed and compared with drug susceptibility phenotype analysis of Mycobacterium tuberculosis to further understand relationship of the target genes' mutation and the drug susceptibility phenotype.Results: In this study,a total of 170 clinical isolates of Mycobacterium tuberculosis were obtained,111 cases were new cases,including 70 drug sensitive cases and 41 drug resistant cases(13 monoresistance,11 polyresitance,11 multi-drug resistant,6 extensively drug resistant),59 cases were previously treated cases,including 19 drug sensitive and 40 drug resistant cases(2 monoresistance,7 polyresitance,22 multi-drug resistant,9 extensively drug resistant).In this study,PCR amplification and gene sequencing analysis of prothionamide-resistance related genes of inhA,ethA and ethR were carried out respectively.InhA gene mutation was found in 4 cases of 170 clinical isolates,2 of them were nonsynonymous mutations and the others were synonymous mutations.The total mutation rate was 1.18(2/170),and one mutation was found in a new case of drug sensitive strain,the mutation rate in new cases was 1.1%(1/89),and the mutation form was GAA62GGA(Glu62Gly),and the other was found in a previously treated case of multi-drug resistant strain,the mutation rate in previously treated cases was 1.2%(1/81),and the mutant form was GCC190TCC(Ala190Ser).In the two strains of inhA gene GAA62 GGA and GCC190 TCC mutations,PTO drug sensitivity test showed drug sensitive,but the strain of inhA gene GCC190 TCC showed resistant to INH.The two mutation sites of GAA62 GGA and GCC190 TCC in inhA gene have not been previously reported,which are considered as novel mutation sites,and have been sent to the Genbank.No mutations in the ethA and ethR genes were detected in the 170 clinical isolates of M.tuberculosis.Conclusion: In this study,two new mutation sites of TCC190TCC(Ala190Ser)and GAA62GGA(Glu62Gly)in inhA genes were found,which has been reported to Gen Bank with MK674461 and MK674460.And no mutations of ethA and ethR genes were detected in the 170 mycobacterium tuberculosis clinical isolates.Mutations of inhA gene TCC190TCC(Ala190Ser)and GAA62GGA(Glu62Gly)in Mycobacterium tuberculosis have no significant relationship with the phenotype of PTO resistance,but GCC190 TCC mutation may be related to INH resistant.These findings could help to provide a reference for the clinical application of prothionamide and isoniazid.