Smegmatis And Mycobacterium Mutant Strain Library To Create And Resistance To Capreomycin Mutants And Nature Study

Tuberculosis is a common and deadly infectious disease caused by Mycobacteria tuberculosis.Over one-third of the world's population is infected with M.tuberculosis. According to WHO, there are twenty million new cases of clinical tuberculosis and an estimated 1.5 million people died from TB in 2006. In addition, the association between HIV and Multidrug-resistant tuberculosis (MDR-TB) may be higher death rate. In 2006, another 200,000 people with HIV died from HIV-associated TB.China, as a developing country with huge population pressure, has a very high TB burden.At now, there are four primary drugs in the clinic: rifampicin (RIF), isoniazid (INH), ethambutol (EMB) and streptomycin (STR).But resistance of these drugs makes tuberculosis difficult to treat and severely threaten public health control efforts. Furthermore, some of African countries, Extensively drug resistant TB (XDR-TB) was found.For countries facing high proportions of drug resistance, high burden countries carrying the largest absolute burden of MDR-TB, and countries with a population heavily co infected with HIV, developing rapid detection and management of drug resistant cases is of great urgency.The numbers of MDR cases and persister are tested for the appropriate second-line anti-TB drugs .Capreomycin is ideal second-line TB drugs. It is also recognized as an attractive template to develop more peptide antibiotics. Accordingly, there is need for the monitoring of resistance to some of the key second-line anti-TB drugs and a better understanding of the biosynthesis gene cluster of capreomycin, the action mechanism unveiled by transcriptome .Transposon mutagenesis is a useful tool to search for nonessential genes encoding products that alter a drug target, are involved in drug transport, or convert a prodrug to its active form. The aim of present study is to investigate capreomycin resistance in mycobacteria, libraries of transposon mutants from M. smegmatis was utilized. Characterization of transposon revealed that none had mutations in the region of the 16S rRNA gene (rrs) associated with resistance to aminoglycosides. But the rRNA methyltransferase (tlyA) gene which confers capreomycin resistance in mycobacteria can't be indentificated. Further work is needed to discover the basis of resistance in capreomycin-resistant strains that do not have mutations in tlyA or rrs. We extracted mutants genome and digested it with BamH1 & EcoR1, self-ligated and transformed into Escherichia coli DH5a/λpir. We sended the positive transformant to sequence by single specific primer of transposon and geted the flanking sequence.The four mutants were analyzed by many biochemistry methods: Comparison of growth in the presence and absence of capreomycin;Cross-resistance between kanamycin(Kan), rifampicin (RIF), streptomycin (STR) and capreomycin;SDS-PAGE analysis of M. smegmatis transformants;Analysis of the whole cell C9-C20 fatty acid ingredients from mutants. All the results reported here demonstrate that cell physiological changes can confer capreomycin resistance.