Abstract：Our study investigated the phenotypic fluonoquinolones resistance and the molecular characteristics of mutation in gyrA in MDR Mycobacterium tuberculosis (Mtb) clinical strains in Fujian Province, and provided some reference for rapid molecular detection of fluonoquinolones resistance in Mtb. MDR isolates collected from drug resistant survey sites through the province in 2010-2011 and 2007-2008, and the strains were conducted FQs susceptibility testing by proportion method. The quinolone resistant determining region (QRDR) in gyrA was amplified by PCR and the PCR products were sequenced, the results of sequencing were blasted with H37Rv. Of 119 strains were collected and performed FQs susceptibility testing. The rate of resistant to FQs was 26.89% (Ofloxacin, Ofx), 25.21% (Levofloxacin, Lfx), and 11.76% (Moxifloxacin, Mfx) respectively. No mutation was found in gyrA in fluonoquinolones sensitive MDR strains. The mutation rate of gyrA in Ofx resistant MDR strains was 84.38% (27/32), the mutation rate of Lfx resistant MDR was 83.33% (25/30), and that of Mfx was 92.86% (13/14). The characteristic of gyrA mutation in our study was point mutation, with five mutation types, and mainly was Asp94Gly, Asp94Asn and Ala90Val. Thus, we conclude that gyrA mutation is the main reason of MDR strains resistant to flunoquinolones in Fujian Province. And the most common mutation is in the codon 94, codon 90, and codon 91.
[1] Zhao YL, Xu SF, Wang LX, et al. National survey of drug-resistant tuberculosis in China[J]. N Engl J Med, 2012, 366: 2161-2170. [2] Alangaden GJ, Manavathu EK, Vakulenko SB, et al. Characterization of fluoroquinolone resistant mutant strains of Mycobacterium tuberculosis selected in the laboratory and isolated from patients[J]. Antimicrob Agents Chemother, 1995, 39(8): 1711-1713. [3] Canetti G, Froman S, Grosset J, et al. Mycobacteria: laboratory methods for testing drug sensitivity and resistance[J]. Bull World Health Organ, 1963, 29: 565-578. [4] WHO. Guidelines for the programmatic management of drug-resistant tuberculosis[M]. WHO/HTM/TB/2008. 402. Geneva: WHO, 2008. [5] Zhao LL, Xia Q, Zhao XQ, et al. Quinolone resistance and gyr gene mutations in multi-drug resistant of Mycobacterium tuberculosis [J]. Chin J Zoonoses, 2011, 27(5): 390-393. doi:10.3969/j.issn.1002-2694.2011.05.03 (in Chinese) 赵丽丽,夏强,赵秀芹,等.耐多药结核分枝杆菌对喹诺酮类药物的耐药性与 gyr 基因突变的初步研究[J].中国人兽共患病学报,2011,27(5):390-393. [6] Chakravorty S, Aladegbami B, Thoms K, et al. Rapid detection of fluoroquinolone-resistant and heteroresistant Mycobacterium tuberculosis by use of sloppy molecular beacons and dual melting-temperature codes in a real-time PCR assay[J]. J Clin Microbiol, 2011, 49(3): 932-940. doi:10.1128/JCM.02271-10 [7] Li GL, Chen P, Sun CW, et al. The cross-resistance to levofloxacin and moxifloxacin in Mycobacterium tuberculosis and gene mutations in gyrA and gyrB [J]. Chin J Antituberculosis, 2010, 32(10): 616-621. (in Chinese) 李国利,陈澎,孙昌文,等. 结核分枝杆菌对左氧氟沙星与莫西沙星的交叉耐药性及 gyrA 和 gyrB 基因突变分析[J].中国防痨杂志,2010,32(10):616-621. [8] Zhao WJ, Li P, Lu Y. Study on the cross-resistance of moxifloxacin and levofloxacin in Mycobacterium tuberculosis [J]. Chin J Antituberculosis, 2009, 31(8): 469-472. (in Chinese) 赵伟杰,李芃,陆宇. 莫西沙星与左氧氟沙星对结核分枝杆菌的交叉耐药性研究[J].中国防痨杂志,2009,31(8):469-472. [9] Mokrousov I, Otten T, Manicheva O, et al. Molecular characterization of ofloxacin-resistant Mycobacterium tuberculosis strains from Russia[J]. Antimicrob Agents Chemother, 2008, 52(8): 2937-2939. doi:10.1128/AAC.00036-08 [10] Avalos E, Catanzaro D, Catanzaro A, et al. Frequency and geographic distribution of gyrA and gyrB mutations associated with fluoroquinolone resistance in clinical Mycobacterium tuberculosis isolates: A systematic review[J]. PLoS ONE, 10(3): e0120470. doi:10.1371/journal.pone.0120470 [11] Huang WL, Lin T, Wu MH, et al. Performance assessment of the GenoType MTBDRsl/test and DNA sequencing for detection of second-line and ethambutol drug resistance among patients infected with multidrug-resistant Mycobacterium tuberculosis[J]. J Clin Microbiol, 2011, 49(7): 2502-2508. doi:10.1128/JCM.00197-11 [12] Hu Y, Hoffner S, Wu LL, et al. Prevalence and genetic characterization of second-line drug-resistant and extensively drug-resistant Mycobacterium tuberculosis in rural China[J]. Antimicrob Agents Chemother, 57(8): 3857-3863. doi:10.1128/AAC.00102-13 [13] Aubry A, Sougakoff W, Bodzongo P, et al. First evaluation of drug-resistant Mycobacterium tuberculosis clinical isolates from Congo Revealed Misdetection of fluoroquinolone resistance by line probe assay due to a double substitution T80A-A90G in GyrA[J]. PLoS ONE, 9(4): e95083. doi:10.1371/journal.pone.0095083 [14] Zhao LL, Chen Y, Liu HC, et al. Molecular characterization of multidrug-resistant Mycobacterium tuberculosis isolates from China[J]. Antimicrob Agents Chemother, 58(4): 31997-2005. doi:10.1128/AAC.01792-13 [15] Chen J, Chen Z, Li Y, et al. Characteristization of gyrA and gyrB mutations and fluoquinolone resistance in Mycobacterium tuberculosis clinical isolates from Hubei Province, China[J]. Braz J Infect Dis, 2012, 16(2): 136-141. [16] Chen QY, Pang Y, Liang QF, et al. Molecular characteristics of MDR Mycobacterium tuberculosis strain isolated in Fujian, China[J]. Tuberculosis, 2014, 94: 159-161.
