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

摘要/Abstract

摘要： 目的探讨莫西沙星（MXF）与左氧氟沙星(LVF)对结核分枝杆菌的交叉耐药性及耐药基因突变位点，为临床用药提供理论依据。方法选取结核分枝杆菌标准株H₃₇Rv和73株左氧氟沙星耐药的耐多药结核分枝杆菌（MDR-TB）临床分离株以及5株对左氟沙星敏感的MDR-TB临床分离株，以试管二倍稀释法采用7H9培养基，测定以上菌株对MXF和LVF的MIC；通过直接测序法测定gyrA基因喹诺酮类药物耐药决定区（QRDR）320bp的基因突变位点。结果MXF的MIC比LVF低4～8倍；H₃₇Rv未发生gyrA基因突变；78株MDR-TB临床分离株均有Ser95Thr突变；73株LVF耐药的MDR-TB菌株中，35株（47.94%）对LVF低耐药（MIC为1～8μg/ml）的菌株和31株（42.46％）对LVF高耐药株中，除4株只有Ser95Thr突变外，其他菌株同时有Ala90Val、Asp94His、Asp94Asn、Asp94Gly、Asp94Ala、Asp94Tyr突变。结论莫西沙星较左氧氟沙星的抗结核活性强4～8倍，为不完全交叉耐药；gyrA基因90位和94位突变与MDR-TB菌株对LVF、MXF耐药有关，低耐药与高耐药的突变氨基酸种类有差别，有望将测序作为临床左氟沙星分子药敏试验方法。

关键词: 分枝杆菌, 结核, 抗药性, 多种, 细菌, 莫西沙星, 氧氟沙星

Abstract: ObjectiveTo investigating the cross resistance of moxifloxacin and levofloxacin in M. tuberculosis, gene mutations of quinolone-resistant isolates to provide a theoretical basis for clinical treatment. MethodsM. tuberculosis strain H₃₇Rv and 78 multidrug-resistant M. tuberculosis clinical isolates (73 LVF-resistant and 5 LVF-sensitive) were studied. The MICs of these isolates to moxifloxacin and levofloxacin were determined by the tube doubling dilution method on 7H9 media. The quinolone resistance-determining region (QRDR) mutations of gyrA genes from M. tuberculosis isolates were detected with DNA sequencing. ResultsThe MICs of MXF were lower 4 to 8 fold than that of LVF. The strain H₃₇Rv wasn’t found any gyrA gene mutation. 78 MDR-TB isolates have Ser95Thr gyrA mutations. Of 73 MDR-TB isolates with LVF resistance, 35（47.94%）were low-resistant to LVF （MICs 1 to 8μg/ml）, 31 （42.46%）were high-resistant to LVF. Four isolates have only gyrA Ser95Thr mutation, the other isolates had combinative gene mutations as follow: Ala90Val, Asp94His, Asp94Asn, Asp94Gly, Asp94Ala, and Asp94Tyr. ConclusionsThe MXF and LVF presented a incomplete cross-resistance. The mutation of gyrA gene correlates with the LVF and MXF resistances in M. tuberculosis.

Key words: Mycobacterium tuberculosis, Drug resistance,multiple,bacterial, moxifloxacin, Ofloxacin

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.

Zhao Weijie，Li Peng，Lu Yu. Study on the cross-resistance of moxifloxacin and levofloxacin in Mycobacterium tuberculosis[J]. Chinese Journal of Antituberculosis, 2009, 31(8): 469-472.

参考文献

[1] 中华人民共和国卫生部. 2000年全国结核病流行病学抽样调查资料汇编[M]. 北京：人民卫生出版社, 2003.
[2] 世界卫生组织. 耐药结核病规划管理指南[M].WHO/HTM/TB/2006：361.
[3] Takiff HE, Salazar L, Guerrero C, Philipp W, Huang WM, Kreiswirth B, Cole ST, Jacobs WR Jr, Telenti A.Cloning and Nucleotide Sequence of Mycobacterium tuberculosis gyrA and gyrB and Detection of Quinolone Resistance Mutations[J].Antimicrob Agents Chemother，1994,38(4):773-780.
[4] 萨姆布鲁克. 分子克隆实验指导(第二版)[M].北京:科学出版社,1998:955.
[5] Rodríguez JC, Cebrián L, López M, Royo G. Mutant prevention concentration: comparison of fluoquinolones and linezolid with Mycobacterium tuberculosis[J]. J Antimicrob Chemother,2004, 53(2):441-444.
[6] Guillemin I, Cambau E, Jarlier V. Sequences of Conserved Region in the A Subunit of DNA Gyrase from Nine Species of the genus Mycobacterium: phylogenetic analysis and implication for intrinsic susceptibility to quinolones[J]. Antimicrob Agents Chemother,1995,39(9):2145-2149.
[7] Ginsburg AS, Sun R, Calamita H, Scott CP, Bishai WR, Grosset JH. Emergence of fluoroquinolone resistance in Mycobacterium tuberculosis during continuously dosed moxifloxacin monotherapy in a mouse model[J]. Antimicrob Agents Chemother, 2005,49(9):3977-3979.
[8] Onodera Y, Tanaka M, Sato K. Inhibitory activity of quinolones against DNA gyrase of Mycobacterium tuberculosis[J]. J Antimicrob Chemother, 2001,47(4):447-450.
[9] Kam KM, Yip CW, Cheung TL, Tang HS, Leung OC, Chan MY. Stepwise decrease in moxifloxacin susceptibility amongst clinical isolates of multidrug-resistant Mycobacterium tuberculosis: correlation with ofloxacin susceptibility[J]. Microb Drug Resist, 2006,12(1):7-11.
[10] Cheng AF, Yew WW, Chan EW, Chin ML, Hui MM, Chan RC. Multiplex PCR amplimer conformation analysis for rapid detection of gyrA mutations in fluoroquinolone-resistant Mycobacterium tuberculosis clinical isolates[J]. Antimicrob Agents Chemother, 2004,48(2):596-601.
[11] Sulochana S, Narayanan S, Paramasivan CN, Suganthi C, Narayanan PR. Analysis of fluoroquinolone resistant in clinical isolates of Mycobacterium tuberculosis from India[J]. J Chemother，2007，19(2):166-171.
[12] Sun Z, Zhang J, Zhang X, Wang S, Zhang Y, Li C. Comparison of gyrA gene mutations between laboratory-selected ofloxacin-resistant Mycobacterium tuberculosis strains and clinical isolates[J]. Int J Antimicrob Agents，2008，31(2):115-121.
[13] 安慧茹，王巍，王天昊，何珂，刘真，李素梅. 结核分枝杆菌喹诺酮耐药基因的研究[J].医学临床研究，2005，22（1）:23－29.
[14] 赵明才，鲍朗，吴悦涵,龙洋，曾献武，张会东，谢益新. 四川地区结核分枝杆菌喹诺酮耐药基因突变研究[J].四川大学学报，2004，35（3）:313－315.

[1]	王庆枫，王隽，王敬，段鸿飞，戈启萍，黄学锐，杜亚东，韩喜琴，初乃惠. 含不同剂量左氧氟沙星方案的抗耐多药结核病疗效研究[J]. 中国防痨杂志, 2015, 37(2): 161-166.
[2]	王前，宋媛媛，逄宇，王玉峰，欧阳琴，赵雁林. 耐氧氟沙星结核分枝杆菌对五种氟喹诺酮类药物交叉耐药的研究[J]. 中国防痨杂志, 2014, 36(6): 453-457.
[3]	李史来，陈伟生，黄钥藩，林少云，吴少仪，蔡清河. 含左氧氟沙星联合利福布丁方案治疗复治肺结核患者的疗效观察[J]. 中国防痨杂志, 2014, 36(5): 375-379.
[4]	王庆徐东芳. 噬菌体生物扩增法检测结核分枝杆菌氧氟沙星耐药性研究[J]. 中国防痨杂志, 2011, 33(1): 43-46.
[5]	张廷梅, 熊敏, 蔡翠, 骆科文, 龙应林. 9例广泛耐药肺结核患者临床分析[J]. 中国防痨杂志, 2010, 32(5): 19-20.
[6]	刘琳, 王仲元, 张凯. 经皮肺穿刺注药治疗巨大肺结核空洞1例介绍[J]. 中国防痨杂志, 2010, 32(12): 845-846.
[7]	李国利, 陈澎, 孙昌文, 张灵霞, 李邦印, 赵雁林. 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): 14-19.
[8]	肖和平, . 耐药结核病化学治疗指南(2009)[J]. 中国防痨杂志, 2010, 32(04): 181-198.
[9]	闫丽萍,肖和平. 住院肺结核患者耐氧氟沙星情况分析[J]. 中国防痨杂志, 2009, 31(8): 477-480.
[10]	杨立娟. 含左氧氟沙星方案治疗复治肺结核临床疗效的Meta分析[J]. 中国防痨杂志, 2009, 31(7): 415-419.
[11]	徐金田. 含左氧氟沙星方案治疗溃疡型浅表淋巴结结核临床分析[J]. 中国防痨杂志, 2007, 29(3): 247-249.
[12]	李月龙,矫庆辉,王迓文,刘建木,. 左氧氟沙星联合奈替米星治疗耐多药肺结核临床疗效观察[J]. 中国防痨杂志, 2004, 26(6): 351-353.
[13]	高春荣,袁东运,徐立江,. 含阿米卡星及左氧氟沙星方案治疗耐多药肺结核近期疗效观察[J]. 中国防痨杂志, 2004, 26(3): 165-167.
[14]	王秀珍,孙永娟,石俊仕,. 含OFLX方案治疗结核性渗出性胸膜炎的观察[J]. 中国防痨杂志, 2003, 25(5): 311-313.
[15]	郭磊,马娟玫,原君平,. 左氧氟沙星治疗耐多药肺结核近期疗效观察[J]. 中国防痨杂志, 2003, 25(4): 254-256.