Study On The PncA Gene Mutation In Multidrug-resistant Mycobacterium Tuberculosis

Objective: To understand the association between pnc A gene mutation and Multidrug-resistant mycobacterium tuberculosis,the correlation between pnc A gene mutation and the genotype of the strains,and the relationship between pnc A gene mutation and the primary/retreated tuberculosis,and provide laboratory data for the molecular detection of pyrazinamide-resistant tuberculosis and its resistance mechanism.Methods: Collect the clinical sputum samples of patients with pulmonary tuberculosis to perform anti-acid bacilli smearing,Mycobacterium culture,identification of Mycobacterium tuberculosis species and the drug susceptibility test.DNA was extracted from Mycobacterium tuberculosis clinical isolates and pnc A gene was amplified by PCR technique and the products of PCR were sequenced and blasted with H37 Rv in NCBI database.The identification of the strains' genotype were performed by the method of deletion-targeted multiplex PCR(DTM-PCR)and then analyzed.Results: A total of 103 isolates of multidrug-resistant mycobacterium tuberculosis(MDR group)and 110 strains(sensitive group)which were all sensitive to 4 kinds of first anti-tuberculosis drugs(HRSE)detected.In the MDR group,the primary tuberculosis accounted for 28.2% and the retreated tuberculosis accounted for 71.8%;the Beijing genotype strains accounted for 70.9%,while the non-Beijing genotype strains accounted for 29.1%;48 strains mutated with the mutation rate of 46.6%.Among the 48 mutated strains,primary tuberculosis with pnc A gene mutation accounted for 25% and retreated tuberculosis with pnc A gene mutation accounted for 75%,and the mutated Beijing species accounted for 75%,while the mutated non-Beijing genotype species accounted for 25%.52 mutant types were detected in the group,in which 18 types were reported to confer pyrazinamide-resistant tuberculosis at high confidence,18 types were recorded in literatures,and 16 mutations are considered as the new mutant types and has been submitted to the US Genbank for further verification,including FSC46(Del G),CCG54 GCG,TTC58GTC,CCG62 TCG,TCC65CCC,GGT97 CGT,GAA107CAA,GAT126 AAT,GAT126CAT,GTC131 TTC,ACC135GCC,GTG155 GAG,GGT162CGT,GCC170 CCC,GTC180CTC and GAG181 AAG.In the sensitive group,87 cases were primary tuberculosis,accounting for 79.1% and 23 cases were retreated tuberculosis,accounting for 20.9%;the Beijing genotype strains accounted for 52.7%,while the non-Beijing genotype strains accounted for 47.3%;only 3 strains were detected to have mutant with the mutation rate of 2.7%.Among the 3 pnc A mutated isolates,2 strains were the primary tuberculosis and 1 strain was retreated tuberculosis,and 2 strains were Beijing genotype,while 1strain was non-Beijing genotype.Conclusions: The multidrug-resistant phenotype was correlated to the pnc A gene mutation,and combination of MTB multidrug-resistant phenotype and pnc A gene mutation may predict the possibility of pyrazinamide-resistance.Beijing genotype strain may be still the mainly epidemic strain of tuberculosis and multidrug-resistant tuberculosis,which could be more easily resistant to PZA.The pnc A gene mutation rate increase significantly in the retreated tuberculosis.