Study On Quinolone Resistance Gene Of Mycobacterium Tuberculosis

Background and ObjectiveTuberculosis is a serious infectious disease causing 9.2 million new cases and 1.8 million deaths worldwide in 2006. This disease is difficult to treat and requires regimens containing several drugs administered over a prolonged period. Relatively few drugs are effective against Mycobacterium tuberculosis (MTB), so drug-resistant tuberculosis, especially multidrug-resistant tuberculosis (MDR-TB) is becoming an increasing public health problem and poses a further threat to global TB control.The quinolones have a broad spectrum of activity and improved pharmacokinetics, so they gradually become second-line drugs recommended to treat MDR-TB, even could be ideal first-line drugs potentially improving the treatment of drug-susceptible TB. Unfortunately, the increasing use including the misuse in the sense of unnecessary use and the use of quinolones with poor activity in some developing countries has led to emergence of quinolone-resistant MTB. Although previous studies found that the intrinsic resistance of MTB to quinolones was generally attributed to mutations of gyrA and gyrB genes, there are also some differences in the findings of different researchers on mutation codons and frequencies. Thus, further research on this topic is still needed.In present study, we selected levofloxacin-resistant MTB clinical isolates to systematically investigate the internal mechanisms of quinolone resistance in MTB. We first determined the phenotype of susceptibility of each strain by the absolute concentration method and detected the sequences of the QRDR ( quinolone resistance-determining region) in gyrA and gyrB genes with DNA direct sequencing technique. Then we analyzed the molecular characterization of gyrA and gyrB genes in quinolone-resistant MTB clinical isolates by comparing our results with others'. MethodsA total of 67 MTB clinical strains were originally isolated from patients with pulmonary tuberculosis. The phenotype of susceptibility of each strain was determined by the absolute concentration method and the sequences of the QRDR in gyrA and gyrB genes were detected with DNA direct sequencing technique.Results1. 47 of 64(73.4%) levofloxacin-resistant MTB clinical isolates, including 20 of 22(90.9%) high-level resistant strains and 27 of 42(64.3%) low-level resistant strains, had mutation in QRDR of gyrA gene. Among the 47 mutated isolates, 45 mutated only in one codon and 2 mutated in double codons. The mutation in single codon was found in codon 70, 89, 90, 91 or 94 and the mutation in double codons was found in codons 90 and 94. Frequency of the mutation in codon 94 was higher than that in other codons and amounted to 63.8%. The mutation patterns in our study involved seven patterns of single codon mutation (H70R, D89N, A90V, S91A, D94G, D94A or D94N) and one pattern of double codons mutation (A90V with D94A). Among the seven patterns of single codon mutation in our data, two ones (H70R and D89N) only developed high-level resistance to levofloxacin. In contrast with the single codon mutation, the double codons mutation is relatively rare and frequently correlated to high-level resistance to levofloxacin.2. Among 64 levofloxacin-resistant MTB clinical isolates, only one strain mutated in codon 511 of gyrB gene and resulted in a change of ACC→AAC (Thr→Asn).ConclusionsOur results confirm that mutations in gyrA and gyrB genes are the major mechanism of quinolone resistance in MTB and suggest that different mutation can lead to different level of quinolone resistance. Nevertheless, mutations in the gyrA and gyrB genes could not account for all quinolone-resistant MTB isolates. Some other mechanisms such as the presence of drug efflux pumps need to be further studied.