摘要
结核分枝杆菌(Mycobacterium tuberculosis,MTB)是结核病的病原菌。全世界
每年约有200余万人死于结核病。近年,由于耐药结核菌的出现,特别是多重耐药
结核菌的出现以及人类免疫缺陷病毒/艾滋病的流行,使这一全球性的疾病卷土重
来,其感染率、发病率和死亡率均居高不下。目前临床常用的一线抗结核药物主要
为INH、RFP、SM、PZA和EMB,但均已出现耐药菌,使得药物治疗效果不断下
降,而且耐药菌比例仍在不断上升。耐药结核菌使得临床治疗工作变得十分困难,
更增加了结核病的危险性,引起世界各国对结核菌耐药问题的极度重视。本实验在
对结核菌耐药性基因检测的基础上,筛选对耐药结核菌的耐药性有抑制作用或可增
强一线抗结核药物作用效果的中草药,为耐药结核菌的防治提供实验依据。
本实验通过BACTEC-TB460检测系统和MTT微量板法从临床分离出38株结核
菌,并确定分离菌株的耐药谱,从中筛选到17株耐利福平结核菌,9株耐链霉素结
核菌。根据结核菌耐利福平基因rpoB和耐链霉素基因rpsL的核酸序列设计合成引
物,对耐药基因进行PCR扩增、克隆及核酸序列测定,分析其耐药基因突变,检测
到3种耐利福平基因rpoB的突变,2种耐链霉素基因rpsL的突变,从分子水平确定
耐药产生的原因。
根据药性选择部分中草药通过煎煮、浸渍、蒸馏等方法制备成中草药粗提物。
采用MTT微量板法检测所制备中草药粗提物的敏感性对耐利福平、耐链霉素的结核
菌的最小抑菌浓度(MIC),结果大部分中草药粗提物高浓度可抑菌或杀菌,低浓度
无作用。将上述耐药菌在含药培养基上培养及传代,用MTT法检测其对RFP、SM
的耐药性,通过分析菌株耐药性的变化,确定中草药对菌株耐药性的影响,获得2
种中草药(黄连、百部)对细菌SM耐药性有抑制作用,同时还发现有5种中草药
可以不同程度增强链霉素的作用效果;但未发现中草药对耐RFP菌耐药性有抑制作
用。根据细菌耐药机制,对中草药抑制耐药性的作用机制进行分析,相对应地检测
了中草药作用作用前后上述耐药菌的菌体形态,菌体蛋白图谱和SM耐药基因rpsL
的耐药突变变化情况。结果表明,细菌菌体形态和菌体蛋白图谱无明显变化。SM耐
药基因rpsL序列发生突变,突变点有4处。
Mycobacterium tuberculosis (MTB) is the pathogenic bacterium of tuberculosis (TB). One-third of the global population is sure to be infected with bacteria of the Mycobacterium tuberculosis complex. More than 8 million new cases of tuberculosis occur annually leading to 2 million deaths. Nowadays, because of occurrence of drug resistance tuberculosis, especially multiple-drug resistance tuberculosis (MDR-TB) and outbreak in association with human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS), tuberculosis have became more dangerous. Therapeutic efficacy of the anti-tuberculosis drugs commonly used in clinical, such as INH RFP SM PZA and EMB, is on descent. There have been ample warnings that MDR-TB will continue to emerge if countries do not strengthen their control of TB. The aim of this study was to detect drug-resistance genes and screen herbs that could inhibit drug resistance of MTB or enhance the anti-tuberculosis drugs' effect in order to prevent and cure MTB.
In this study, we obtained 38 MTB isolations from clinical by BACTEC-TB 460 system. Using of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and microtiter plate, we assayed the minimal inhibitory concentration (MIC) of rifampin and streptomycin to the isolations. There are 17 rifampin-resistance isolations and 9 streptomycin-resistance isolations. According to the drug-resistance gene sequences of rpoB and rpsL, we designed 2 pairs of primers for PCR. By PCR and DNA sequencing, we analyzed the rifampin- and streptomycin-resistance isolations. The results were including 2 mutations identified in the rpsL gene and 3 mutations in the rpoB gene.
we made some crude preparation of Chinese medicine herb selected on the basis of nature of a drug, we detected crude preparation susceptibility to the rifampin- and streptomycin-resistance isolations by MTT and microtiter plate, resulting in most of non-sensitivity. Analyzing the changes of MIC of those resistance strains after cultivation and transferring of culture, we found 2 herbs could decrease the streptomycin-resistance of isolations and 5 herbs could enhance the drug's effect. But none could take effect on rifampin-resistance isolations. Based on mechanisms of drug resistance of MTB, we analyzed the changes of the morphology, protein electrophoresis map and drug-resistance genes occurred in those isolations. The results showed that no changes occurred in the two formers and the later changed for 4 sites by contrasted with the primordial drug-resistant isolatons.
引文
[1] World Health Organization. Anti-tuberculosis drug resistance in the world report, no.2. The W.H.O/IUATLD global project on anti-tuberculosis drug resistance surveillance 1997-2000. World Health Orgarfization, 2000, Genera, Switzerland.
[2] Lu PL, Lee YW, Peng CF, et al. The decline of high drug resistance rate of pulmonary Mycobacterium tuberculosis isolates from a southern Taiwan medical centre, 1996-2000. Int J Antimicrob Agents, 2003, 21(3): 239-243.
[3] D.Y.Kunimoto, M.S.Peppler, J.Talbot, et al. Anakysis of Mycobacterium avium Complex Isolates from Blood Sample of AIDS Patients by Pulsed-Field Gel Eleetrophoresis J Clin Microbiol, 2003, 41(1): 498-499.
[4] Ferdinand S, Sola C, Verdol B, et al. Molecular characterization and drug resistance patterns of strains of Mycobacterium tuberculosis isolated from patients in an AIDS counseling center in Port-au-Prince, Haiti: al-year study. J Clin Microbiol, 2003,41(2): 694-702.
[5] Samman Y, Krayem A, Haidar M, et al. Treatment outcome of tuberculosis among Saudi nationals: role of drug resistance and compliance. Clm Microbiol Infect, 2003,9(4): 289-94.
[6] Dahle UR, Sandven P, Heldal E, et al. Deciphering an Outbreak of Drng-Resistant Mycobacterium tuberculosis. J Clin Microbiol, 2003, 41(1): 67-72.
[7] Telenfi A, Imboden P, Marchesi F, et al. Detection ofrifampin-resistant mutations in Mycobacterium tuberculosis. Lancet, 1993, 341: 647-650.
[8] Donnabella V, Martiniuk F, Kinney D, et al. Isolation of the gene for the β subunit of RNA polymerase from Rifampicin-resistant M. tuberculosis and identification of new mutations. Am J Respir Cell Mol Biol, 1994, 11: 639-643.
[9] Williams D, Waguespack C, Eisenach K, et al. Characterization of rifampin resistance in pathogenic mycobacteria. Antimicrob Agents Chemothcr, 1994, 38: 2380-2386.
[10] Altanfirano M, Marostenmaki J, Wong A, et al. Mutations in file catalase-peroxidase gene from isoniazid-resistant Mycobacterium tuberculosis isolates. J lnfectDis, 1994, 169: 1162-1165.
[11] Zhang Y, Heym B, Allen B, et al. The catalase-peroxidase gene and isoniazid resistance of Mycobacterium tuberculosis. Nature, 1992, 358: 591-593.
[12] Heym B, Zhang Y, Poulet S, et al. Characterization of the KatG gene encoding a catalase-peroxidase required for the isoniazid susceptibility of Mycobacterium tuberculosis. J Baeteriol, 1993, 175: 4255-4259.
[13] Goto M, Oka S, Tachikawa N, et al. KatG sequence deletion is not the major cause of isoniazid resistance in Japanese and Yemeni Mycobacteriurn tuberculosis isolates. Mol Cell Probes, 1995, 9: 433-439.
[14] 倪语星,洪秀华主编.细菌耐药性监测与抗感染治疗.北京:人民军医出版社,2002,4.
[15] 张小刚,何秀云,李书琳等.结核分枝杆菌耐链霉素耐药基因的检测.实用医学杂志,2001,17(10):1005-1006.
[16] 吴雪琼,梁建琴,李洪敏等.结核分枝杆菌耐乙胺丁醇分离株embB基因突变的研究.中华检验医学杂志,2001,24(4):214-216.
[17] Drobniewski F, Balabanova Y, Ruddy M, et al. Rifampin- and multidrug-resistant tuberculosis in Russian civilians and prison mates: dominance of the beijing strain family. Emerg Infect Dis, 2002, 8(11): 1320-1326.
[18] Lemaitre N, Sougakoff W, TruffotPemot C, et al. Antimicrob Agents Chemother, 1999, 43: 761-763.
[19] Ying Zhang, Angelo Scorpio, Hiroshi Nikaido, et al. Role of Acid pH and Deficient Effiux of Pyrazinoic Acid in Unique Susceptibility of Mycobacterium tuberculosis to Pyrazinamide. Journal of Bacteriology, 1999, 181(7): 2044-2049.
[20] Morris S, Bai GH, Suffys P, et al. Molecular mechanisms of multiple drug resistance in clinical isolates of Mycobacterium tuberculosis. JID, 1995, 171 954-96O.
[21] Githui WA. Laboratory methods for diagnosis and detection of drug resistant Mycobacterium tuberculosis complex with reference to developing countries: a review. East Afr Med J, 2002, 79(5): 242-248.
[22] Drobniewski FA, Caws M, Gibson A, et al. Modem laboratory diagnosis of tuberculosis. Lancet Infect Dis, 2003, 3(3): 141-147.
[23] Robert N, Mshana, Genet Tadesse, et al. Use of 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-Diphenyl Tetrazolium Bromide for Rapid Detection of Rifampin-Resistant Mycobacterium tuberculosis. Journal of Clinical Microbiology, 1998, 36, 5): 1214-1219.
[24] Jahll B, Martin E, Stueben A, et al. Susceptibility testing of Candida albicans and Aspergillus species by a simple microtiter menadione— angmented 3—(4, 5—dimethyl— 2—thiazolyl)—2, 5— diphenyl— 2H—tetrazolium bromide assay. J Clin Microbicl, 1995, 33: 661-667.
[25] Bardarov S Jr, Dou H, Eisenach K, et al. Detection and drug-susceptibility testing of M.tuberculosis from sputum samples using luciferase reporter phage: comparison with the Mycobacteria Growth Indicator Tube (MGIT) system. Diagn Microbiol Infect Dis, 2003, 45(1): 53-61.
[26] 吕斌,徐顺清,陈志飞等.用重组噬菌体检测结核菌耐药性.中华检验医学杂志,2000,23:144.147.
[27] Mchugh M, Miner R C, Logan L H, et al. Simultaneous detection of antibodies to cytomegalovirus and herpes simplex virus by using flow cytometry and a microsphere-based fluotescence immunoassay. J Clin Microbiol, 1988, 26: 1957-1961.
[28] 金玲,康熙雄,鱼瑛等.银染单链多态性分析检测rpoB基因突变.中华医学检验杂志,1999,22:36-39.
[29] Siddiqi N, Shamim M, Hussain S, et al. Molecular characterization of multidrug-resistant isolates of Mycobacterium tuberculosis from patients in North India. Antimicrob Agents Chemother, 2002, 46(2): 443-450.
[30] Baptism IM, Oelemann MC, Opromolla DV, et al. Drug resistance and genotypes of strains of Mycobacterium tuberculosis isolated from human immunodeficiency virus-infected and non-infected tuberculosis patients in Bauru, So Paulo, Brazil. Mem Inst Oswaldo Cruz, 2002, 97(8): 1147-1152.
[31] Eltringham IJ, Wilson SM, Drobniewski FA, et al. J. Clin Microbiol, 1999, 37: 3528-3532.
[32] Mokrousov I, Filliol I, Legrand E, et al. Molecular characterization of multiple-drug-resistant Myeobacterium tuberculosis isolates from northwestern Russia and analysis of rifampin resistance using RNA/RNA mismatch analysis as compared to the line probe assay and sequencing of the rpoB gene. Res Microbiol, 2002, 153 (4) : 213-219.
[33] 李国利,赵铭,庄玉辉,等.聚合酶链反应分子灯塔法检测结核分枝杆菌.中华检验医学杂志2001,24(02):82-83
[34] Piatek AS, Tyapi S, Poi AC, et al. Molecular beacon sequence analysis for detecting drug resistance in Mycobaterium tuberculosis.Nat Biotechnol, 1998,12:219-226
[35] Troesch A,Nguyen H,Muyada CG,et al. Mycobacterium species identification and rifampin resistance resting with high-density DNA probe arrays. J Clin Mierobiol, 1999. 37: 49-55.
[36] Stomakhin A,VASILISKOV V, TIMOFEEV E et al. DNA sequence analysis by hybridization with oligonucleotide microchip:MALDI mass spetrometry identification of 5mers contiguously stacked to microchip pligonucleotides. Nucl acids Res,2000,28;1193-1198
[37] MCDADE R. A unsiversal laboratory platform for the enw millennium. American Laboratory, 2001, 33: 26-30.
[38] 靳连群,基因芯片技术与微生物学.微生物学免疫学进展.2002,30(01):85-90.
[39] 徐伟文,李文全,毛裕民.表达谱基因芯片.生物化学与生物物理进展2001,28(6):822-827.
[40] 王嵩.中草药抗细菌感染的研究.北京中医杂志2002,21(4);249.
[41] 赵韶星,刘芬.对细菌耐药性的研究.山西医药杂志,2000,29(2):173.
[42] 李仝.中医药防治细菌耐药进展.中国医药学报,2000,16(3):29-32.
[43] 王志强,陈杖榴.兽用中草药与抗菌西药联合应用研究进展.中兽医医药杂志,2000,19(3):15-18.
[44] 祝衰菊,杨建策.中草药饲料抗菌保健剂的研究与应用.畜禽业,2001,9:56-60.
[45] 鞠洪涛,学位论文:大肠杆菌耐药性基因定位及耐药性和耐药质粒消除的研究, 解放军军需大学,1999,6.
[46] 谢兆霞,谭达人,谢俊明,等。“拮新康”的逆转耐药与抗肿瘤作用的研究[J].中国现代医学杂志,1996,6(4):18-19.
[47] 敖忠芳.夏薇,汉防己甲素逆转白血病细胞耐药的研究.中华血液学杂志1995,16(5):235-237.
[48] 梁蓉,杨平地,陈协群.川芎嗪逆转HL60/VCR细胞多药耐药的体外研究.第六届全国检验血液学会议论文摘要。1997,11:196-197.
[49] 何滇杨,盂凡宏,张鸿卿.粉防己碱和蝙蝠葛碱减低抗三尖杉酯碱的人白血病HL60细胞对阿霉素的抗性.中国药理学报,1996,17(2):179-181.
[50] 胡凯文,陈信义.中药活性成分抗耐药肿瘤细胞体外筛选研究.中国医药学报,1998,13(2):10-12.
[51] 骆和生.提高机体免疫力是清热药抗感染的重要途径.新中医,1985,17(7):51-52.
[52] 刘文富.抗感染中草药药理研究进展及其研究方法问题.药学通报,1982,17(1) 24-27.
[53] 夏中伟等,葛根芩连汤抗伤寒杆菌活性和体外配伍抗菌药的实验观察.实用中西医结合杂志,1996,9(7):394.
[54] 郭钧主编.结核病细菌学.沈阳:辽宁科学技术出版社,1983.
[55] Pedro E. A. Silva, Fabiana Bigi, et al. Characterization of P55, a Multidrug Efflux Pump in Mycobaeterium bovis and Mycobaeterium tuberculosis. Antimicrobial Agents and Chemotherapy, 2001, 45(3): 800-804.
[56] 施永初摘译.中草药在体外抗菌作用的比较.国外医学·中医中药分册,1987,9(1):26.
[57] 陈友梅主编.中药化学.山东科学技术出版社,1988.
[58] 曹春林主编.中药制剂汇编.人民卫生出版社,1983.
[59] 陈荣,温博海,钟敏等.序列分析重庆地区耐利福平结核分支杆菌的rpoB基因突变.中国人兽共患病杂志,2002,18(4):20-22.