Rifampicin-resistant Analyses Of Phenotype And Genotype In Multidrug-resistant Mycobacterium Tuberculosis

Objective: To understand the correlation between rifampicin-resistant phenotype and genotype in multidrug-resistant(MDR)Mycobacterium tuberculosis(M.tb),and the mechanism of resistance to rifampicin.Methods: The acid-fast stain smear,bacterial culture,identification of bacterial types and drug susceptibilities were performed on clinical specimens from TB patients,to screen MDR M.tb isolates and gain resistant phenotype.DNA samples were extracted from isolates and used for PCR amplification and sequence analysis of rpo B,rpo C,rpo A and rpo Z entire coding regions respectively,to gain resistant genotype.The sequencing results were compared with the standard strain H37 Rv in the NCBI database and then analyzed.Results: In this study,65(91.5%)isolates had gene mutations related with rifampicin resistance in 71 isolates of MDR M.tb,including rpo B,rpo C and rpo A.Rpo Z didn't possess any mutations,6(8.5%)isolates didn't own the above 4 genes mutations.Mutations in rpo B appeared in all the 65 isolates,of which 63(88.7%)mutated in the rifampicin resistance-determining region(RRDR)and 531(54.9%),526(14.1%)and 516(9.9%)were the most common mutation sites;2(2.8%)harbored mutations outside the RRDR independently;7(9.6%)mutated within and outside the RRDR in 63 isolates.Among the65 isolates,27(38%)had mutations in rpo C and 3(4.2%)in rpo A.All of isolates with rpo C and rpo A mutations combined mutations with rpo B.11 new mutations were founded,including 7 mutations forms in rpo B: CCG126 TCG,CTG511CCC,TCG574 TAC,TCG663GCG,GGC747 GAC and combined mutation at GTG250GCG/GTC251 TAC,4types in rpo C: GAG49 GCG,GGC332TGC,CCG434 ACG and GCC710 AC.Conclusion: Mutations in rpo B is the main mechanism of M.tb resistant to rifampin(especially in RRDR),region in rpo C,rpo A and outside the RRDR in rpo B are possible belong to compensatory mutations of rpo B.The discovery of 11 novel mutations lay the laboratory foundation for further study of rifampicin resistant mechanism in M.tb.