Correlation between levels of ofloxacin resistance and gyrA mutations in Mycobacterium tuberculosis
LIU Zhi-guang1, 2, GUO Qian1, 2, 3, WEI Jian-hao1, 2, 4, ZHANG Jing-rui1, 2, ZHAO Li-li1, 2, JIANG Yi1, 2, ZHAO Xiu-qin1, 2, WAN Kang-lin1, 2, LI Gui-lian1, 2
1 State Key Laboratory for Infectious Disease Prevention and Control National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, , Beijing 102206, China; 2 Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Hangzhou 310003, China; 3 Pathogenic Biology Institute, South of China University, Hengyang 421000, Hunan, China; 4 Wenzhou Medical College, Key Lab of Laboratory Medicine, Ministry of Education, Wenzhou 325035, Zhejiang,China
Abstract：Objective To determine the correlation between ofloxacin resistance levels and mutations in gyrA in Mycobacterium tuberculosis. Methods Microplate alamar blue assay was used to determine the ofloxacin MIC in 39 ofloxacin resistant and 32 ofloxacin susceptible M. tuberculosis clinical isolates, and direct sequencing was used to detect the mutations of gyrA, chi-square test was used to evaluate the association between ofloxacin resistance levels and mutations in gyrA. ResultsThe MIC of ofloxacin against 32 ofloxacin susceptible M. tuberculosis was <2.000μg/ml and carried wild type of gyrA. Among ofloxacin resistant isolates with ofloxacin MIC≥4.000 μg/ml and MIC≤2.0000 μg/ml, 18 out of 24 and 5 out of 15 isolates carried mutations at codon 94 of gyrA respectively, the P value was 0.010. The ofloxacin MIC of five out of 7 isolates carried single condon mutation of gyrA 91 were all 2.000 μg/ml. Two isolates carried combined mutations of codons 94 and 91 or 90 showed high level resistance of ofloxacin and had ofloxacin MIC of 16.000μg/ml and 64.000μg/ml, respectively. ConclusionMutations of gyrA 94 and combined mutations in gyrA were correlated with high level resistance of ofloxacin,mutation of gyrA91 was associated with low level resistance of ofloxacin.
[1] Takiff H E, Salazar L, Guerrero C, et al. Cloning and nucleotide sequence of Mycobacterium tuberculosis gyrA and gyrB genes and detection of quinolone resistance mutations [J]. Antimicrob Agents Chemother, 1994, 38(4): 773-780. [2] 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. [3] Zhao L L, Chen Y, Liu H C, et al. Molecular characterization of multidrug-resistant Mycobacterium tuberculosis isolates from China [J]. Antimicrob Agents Chemother, 2014, 58(4): 1997-2005. [4] World Health Organization. Policy guidance on drug-susceptibility testing (DST) of second-line antituberculosis drugs (WHO/HTM/TB/2008.392). World Health Organization, Geneva,2008. [5] 李桂莲, 张敬蕊, 赵秀芹, et al. 微孔板Alamar blue显色法检测结核分枝杆菌利福平和异烟肼耐药性的研究 [J]. 中国人兽共患病学报, 2012, 28(4): 319-322. [6] Van Embden J D, Cave M D, Crawford J T, et al. Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology [J]. J Clin Microbiol, 1993, 31(2): 406-409. [7] Zhu C, Zhang Y, Shen Y, et al. Molecular characterization of fluoroquinolone-resistant Mycobacterium tuberculosis clinical isolates from Shanghai, China [J]. Diagn Microbiol Infect Dis, 2012, 73(3): 260-263. [8] Rodwell T C, Valafar F, Douglas J, et al. Predicting extensively drug-resistant Mycobacterium tuberculosis phenotypes with genetic mutations [J]. J Clin Microbiol, 2014, 52(3): 781-789. [9] Wang H, Zhang X, Luo T, et al. Prediction of XDR/pre-XDR tuberculosis by genetic mutations among MDR cases from a hospital in Shandong, China [J]. Tuberculosis (Edinb), 2014, 94(3): 277-281. [10] Kunz B A, and Kohalmi S E. Modulation of mutagenesis by deoxyribonucleotide levels [J]. Annu Rev Genet, 1991, 25339-359. [11] Hu Y, Hoffner S, Wu L, et al. Prevalence and genetic characterization of second-line drug-resistant and extensively drug-resistant Mycobacterium tuberculosis in Rural China [J]. Antimicrob Agents Chemother, 2013, 57(8): 3857-3863. [12] 李国利, 陈澎, 孙昌文, et al. 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. [13] Chen J, Chen Z, Li Y, et al. Characterization of gyrA and gyrB mutations and fluoroquinolone resistance in Mycobacterium tuberculosis clinical isolates from Hubei Province, China [J]. Braz J Infect Dis, 2012, 16(2): 136-141. [14] Yin X, and Yu Z. Mutation characterization of gyrA and gyrB genes in levofloxacin-resistant Mycobacterium tuberculosis clinical isolates from Guangdong Province in China [J]. J Infect, 2010, 61(2): 150-154. [15] De Rossi E, Ainsa J A, and Riccardi G. Role of mycobacterial efflux transporters in drug resistance: an unresolved question [J]. FEMS Microbiol Rev, 2006, 30(1): 36-52. [16] Siddiqi N, Das R, Pathak N, et al. Mycobacterium tuberculosis isolate with a distinct genomic identity overexpresses a tap-like efflux pump [J]. Infection, 2004, 32(2): 109-111. [17] Bhatt K, Banerjee S K, and Chakraborti P K. Evidence that phosphate specific transporter is amplified in a fluoroquinolone resistant Mycobacterium smegmatis [J]. Eur J Biochem, 2000, 267(13): 4028-4032.
2015, Vol. 22 
