The Anti-tuberculosis Drugs Susceptibility And Fluoroquinolone Resistance Mechanism In Mycobacterium Tuberculosis In Wuhan

Tuberculosis is still a respiratory infectious disease that seriously threatens public health. In the practice of prevention from tuberculosis, drug resistance of mycobacterium tuberculosis, especially multidrug-resistant tuberculosis(MDR-TB) has become an important barrier to the effective control of tuberculosis. Fluoroquinolones are the pivotal second-line drugs, particularly in regimen for MDR-TB. Once MDR-TB patients are resistant to fluoroquinolones, they will have poor prognosis. DNA topoisomerases are a diverse set of essential enzymes responsible for maintaining chromosomes in an appropriate topological state. Fluoroquinolones inhibit DNA gyrase(topoisomerase II) and topoisomerase IV, resulting in microbial death for the block of microbial replication.DNA gyrase is a tetrameric A2B2 protein. The A subunit carries the breakage-reunion active site, whereas the B subunit promotes ATP hydrolysis. Mycobacterium tuberculosis has respectively gyrA and gyrB genes encoding the A and B subunits. A conserved region, the quinolone-resistance-determining region(QRDR) of gyrA (320bp) and gyrB(375bp), has been found to be the most important area involved in the exhibition of fluoroquinolone resistance in Mycobacterium tuberculosis. Mutations within the QRDR of gyrA or gyrB gene will result in the resistance to fluoroquinolones. In 1994, Takiff HE for the first time cloned and sequenced the gyrA and gyrB genes and studied resistance mechanisms of fluoroquinolones. Mutations within the QRDR of gyrA have been identified in clinical and laboratory-selected isolates of Mycobacterium tuberculosis, largely clustered at codon 90,91,94,with Asp 94 being relatively frequent.For clinical isolates,gyrB mutations appear to be of much rarer occurrence. Among some fluoroquinolone-resistant Mycobacterium tuberculosis, there are no mutation in gyrA and gyrB genes. More researches are needed to identify new resistance mechanisms.In this study, clinical isolates of Mycobacterium tuberculosis were collected from Wuhan and the susceptibility to six antituberculosis drugs were determined. To understand the effect of newer fluoroquinolones on the ofloxacin-resistant Mycobacterium tuberculosis, the MICs of four fluoroquinolones were determined by the resazurin colorimetric assay among the ofloxacin-resistant Mycobacterium tuberculosis. Mutations within the QRDR of gyrA and gyrB genes were detected by DNA direct sequencing to identify the types and frequency of mutations.Part I Analysis of drug susceptibility testing results of 1223 MTB strains from patients with smear-positive sputaObjective To understand the drug resistance of mycobacterium tuberculosis in patients with smear positive, and provide reasonable regimens to doctors.Methods From Janurary 2008 to December 2009,535 MTB isolates from previously treated patients with smear positive were collected and 688 MTB isolates from new cases with smear positive were also collected during resistance baseline survey in 2008. The drug resistance data were analyzed by drug susceptibility testing.Results The drug susceptibility tests showed that the total resistance rate was 31.3%, the initial and the acquired resistance rate was 18.0%and 48.4%. The total MDR-TB resistance rate was 12.7%, the initial and the acquired MDR-TB resistance rate was 4.5%and 23.2%, respectively. The resistance rate of drug susceptibility testing of streptomycin, isoniazid, rifampicin, ethambutol, ofloxacin and kanamycin was18.7%,21.1%,19.4%,8.3%,7.6%, 0.9%, respectively. Among the new patients, the resistance rate of drug susceptibility testing of streptomycin, isoniazid, rifampicin, ethambutol, ofloxacin and kanamycin was 12.3%,10.3%,5.2%,2.5%,3.6%,0.4%, respectively. Among the previously treated patients, The resistance rate of drug susceptibility testing of streptomycin, isoniazid, rifampicin, ethambutol, ofloxacin and kanamycin was 26.9%,35.0%,30.7%,15.5%, 12.9%,1.5%, respectively. The ofloxacin and kanamycin resistance rate was 27.7%(43/144) and 5.2%(8/155) among the MDR-TB strains.Conclusion The resistance rate is higher among previously treated patients, suggesting that treatment regimen should be changed according to the result of drug susceptibility testing.The high prevalence of drug resistance has been a major challenge for TB control.Part II Comparison of MICs of four fluoroquinolones for Mycobaterium tuberculosis by resazurin colometric assay.Objective To understand effects and cross-resistance of fluoroquinolones on mycobacterium tuberculosis, four fluoroquinolones'MICs for Mycobaterium tuberculosis were determined by resazurin colorimetric assay.Methods MICs of 65 ofloxacin-susceptible Mycobacterium tuberculosis strains and 60 ofloxacin-resistant clinical isolates were detected by resazurin colorimetric assay. The proportion method on L-J medium was used as a gold standard. Breakpoint concentration of ofloxacin were determined by ROC curve analysis.Results The best breakpoint value of drug susceptibility testing of Ofloxacin were 2μg/ml, sensitivity was 98.3%, specificity was 96.9%and accuracy was 97.6%. MIC50 and MIC90 of gatifloxacin and moxifloxacin for Mycobaterium tuberculosis were 4-8 times lower than ofloxacin and ciprofloxacin.Conclusion Newer fluoroquinolones such as moxifloxacin, gatifloxacin may be used to treat low degree ofloxacin-resistant MDR-TB. PartⅢAssociation of Mutations of gyrA/B and fluoroquinolone resistance in Mycobacterium tuberculosis.Objective The aim of this study was to observe gyrA and gyrB genes mutations and resistance mechnisms of ofloxacin-resistant Mycobacterium tuberculosis(MTB) isolates from Wuhan, China.Methods A total of 93 MTB clinical strains were originally isolated from patients with pulmonary tuberculosis. The phenotype of susceptibility of each strain was determined by the proportion method and the QRDR in gyrA and gyrB genes were sequenced with DNA direct sequencing technique. The MICs of ofloxacin and ciprofloxacin were determined at the presence of the efflux pump inhibitor reserpine.Results 54 of 61 (88.5%) ofloxacin-resistant MTB clinical isolates had mutations in the QRDR of gyrA gene. The mutation patterns involved seven patterns of single codon mutation (A90V,S91P,S91T,D94N, D94Y,D94G and D94A) and two patterns of double codons mutation (S91P with D94H, S91P with D94A). No mutation in gyrB gene was detected among all MTB strains. Among seven MTB strains without gyrA and gyrB genes, the MICs of ofloxacin and ciprofloxacin in one MTB strain decreased 8-fold at the presence of the efflux pump inhibitor reserpine. No differences were detected among strains with different amino acid mutations at codon 94 in the QRDR of gyrA gene(F=0.40, P=0.755). The MICs of all ofloxacin-resistant strains showed significant difference among strains with mutions at condons 90,91 or 94 in gyrA gene(F=11.70, P<0.0001).Conclusion Mutations of gyrA codons 90,91 and 94 constitute the primary mechanism of FQ resistance among Mycobacterium tuberculosis. Efflux pump system may play a role in some low level ofloxacin-resistant MTB strains. Mutations at codon 91 in gyrA gene may relate to low level resistance of ofloxacin.