临床分离志贺菌属细菌多重耐药机制的研究
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摘要
志贺菌属细菌又称痢疾杆菌，它是引起细菌性痢疾(菌痢)的主要病原。本病是全球流行的传染病，尤其在卫生条件相对落后的国家发病率较高。目前我国尽管在菌痢的防治工作中已取得了重大成就，但由于人口众多，经济不发达，且志贺菌菌型复杂，易变迁，各菌型间无交叉免疫等原因，菌痢发病率一直处于较高水平，是我国夏秋季常见的肠道传染病。菌痢自广泛应用抗菌药物治疗以来，志贺菌不断产生耐药株，并呈多重耐药，不仅耐药率高，且耐药产生快及广泛，给本病的防治带来很大困难，从而造成本病蔓延及流行。1996年痢疾志贺菌被WHO认定为耐药性日渐增长给人类带来巨大威胁的细菌。研究志贺菌属的多重耐药机制，克服细菌多重耐药是提高菌痢防治水平的重要课题。大肠杆菌研究发现：AcrAB-TolC多重耐药泵系统在大肠杆菌多重耐药过程中起到重要作用，其耐药机制称之为主动流出机制，该机制是由染色体介导的在一个调节位点控制下的多基因协作表型，即导致细菌多重耐药泵蛋白表达增加，该泵系统可将结构不同的药物泵出细胞膜外，致胞内药物浓度达不到有效浓度，从而使细菌产生多重耐药。已有的研究发现，类似的多重耐药泵在肠道杆菌中广泛存在。为了解志贺菌属细菌的多重耐药机制及探索解决其问题的途径，本研究对临床收集的志贺菌进
郑州大学硕士学位论文
临床分离志贺菌属细菌多重耐药机制的研究
行PCR扩增编码多重耐药泵蛋白的结构基因acrAB一tolC、运用PCR一SSCP对其突
变性进行检测、N。找hem印迹检测其表达情况，并在实验室研究泵蛋白抑制剂
MC207，1 10对志贺菌的作用。
方法
1.多重耐药株和敏感野生株的筛选:采用琼脂稀释法测定抗菌药物对志贺菌
的最低抑菌浓度(minlrnu工n inllibition coneentration，MIC)。以实验室耐四环素、
氯霉素、氨节青霉素、毗呱酸或环丙沙星的菌株为多重耐药株(multiPle antibiotic
resistant，Mar)，均敏感的菌株为敏感野生株(丽ld tyPe，WT)。
2.志贺菌细胞内环丙沙星的积聚:应用荧光法测定志贺菌细胞内环丙沙星的
积聚。积聚试验均于37’C水浴中进行，样品用预冷至0℃的0.1 mol几(pH 3.0)
的甘氨酸溶液处理。志贺菌细胞内环丙沙星浓度为OD60。=10时每升菌液细胞内所
含的环丙沙星毫克数表示。
3.多重耐药泵系统acrAB一olC基因的单链构象多态性(P CR一5 5 CP)分析:PCR
扩增acrAB一tolC基因，扩增产物经限制性内切酶EcoRI(Alul)消化(或不消
化)，变性后经中性聚丙烯酸胺凝胶电泳，根据单链带数目和位置判断有无基因
突变。
4.采用随机引物法标记探针:将待标记的DNA探针片段变性后与随机引物杂
交，然后以此杂交的寡核昔酸为引物，在DNA聚合酶I大片段的催化下，合成与
探针DNA互补的DNA链，反应液中含有DIG一dUTP，即形成地高辛标记的DNA
探针。
5.Northem印迹:提取细胞总RNA，行甲醛变性凝胶电泳，将RNA转移至尼
龙膜，在含有探针的杂交液中杂交，应用地高辛检测试剂盒检测杂交结果，根据
杂交条带密度半定量细胞中mRNA表达水平。
结果
郑州大学硕士学位论文
临床分离志贺菌属细菌多重耐药机制的研究
1.多重耐药株和敏感野生株的筛选:根据临床上分离的141株志贺菌对四环
素、氯霉素、氨节青霉素、毗呱酸或环丙沙星5种抗生素的药敏试验结果，筛选
出64株多重耐药株，6株敏感野生株。
2.志贺菌细胞内环丙沙星的积聚:敏感野生株细胞内环丙沙星稳态浓度约为
多重耐药株Marl的2.5倍，为多重耐药株MarZ的1 .5倍，差异有显著性
(P<0 .0001)。加入能量抑制剂CCCP后，多重耐药株Marl细胞内环丙沙星稳态
浓度上升了2.2倍，多重耐药株MarZ上升了0.8倍，敏感野生株上升了0.3倍，各
组之间比较差异有显著性(尸<0 .0001)。
3.32株志贺菌属多重耐药株和6株敏感野生株及标准株全部分别扩增出1697
bp、510 bp和1633 bp长度的片段，分别是aerA、aerB和tolc基因的扩增产物，
未发现多重耐药泵基因缺失株。
4.多重耐药泵系统acrAB一to1C基因的单链构象多态性(P CR-SsCP)分析:对38
株临床分离志贺菌属细菌研究，发现4株多重耐药株acrA基因突变，突变率为
103%;有1株敏感野生株tolC基因突变，突变率为2.6%;未发现acrB基因突变
株。
5.随机引物法标记探针:随机引物法标记的探针浓度分别约为:acrA基因为
990 ng/协l、acrB基因800 ng/协l和tolC基因900 ng/林l。
6.多重耐药泵系统acrAB一tolC表达水平结果分析:多重耐药株Marl、MarZ其
acrA基因表达水平显著高于敏感野生株(尸<0.05);多重耐药株Marl、MarZ其
acrB基因表达水平略高于敏感野生株，但统计学比较差异无显著性，F=0.92，
尸二 0.45;多重耐药株Marl、MarZ及敏感野生株其tolC基因表达水平经统计学比
较差异无显著性，F=0.39，尸二0.76。
7.多重耐药泵抑制剂MC一207，110对志贺菌的抑制作用结果分析:在多重耐
药泵基因高表达的一些菌株中作用明显，该抑制剂可通过抑制多重耐药泵从而使
郑州大学硕士学位论文
临床分离志贺菌属细菌多重耐药机制的研究
环丙沙星对志贺菌相?
Shigella called as dysenteriae bacillary is the main pathogen of shigellosis. It is an epidemic contagion throughout the world, especially in the countries with the laggard sanitation. These days, though our country has made a great achievement on the shigellosis of prevention and cure, the incidence of shigellosis was always high, because of much more population, underdevelopment economy in our country and because of the complex of the bacterial type, the easy variance and without the crossover immunity in Shigella. It is the familiar contagion in summer and autumn. Since antimicrobial agents were widely employed to treat shigellosis, antimicrobial resistance in Shigella spp .has been widely appeared and was multiple-antibiotic-resistance, which brought great hard to shigellosis of prevention and cure, consequently shigellosis spread and prevail. In 1996, Shigella spp. had been identified by the World Health Organization as one of the growing antibiotic-resistant bacteria posing a major threat to mankind. I
t is the important task increasing the level of shigellosis of prevention and cure to study the multiple-antibiotic-resistant mechanisms in Shigella spp.
and to overcome multiple-antibiotic-resistance (Mar) of bacterial .The study on Escherichia coli.suggested that AcrAB-TolC multidrug efflux pump played a main role in multiple-antibiotic-resistance E.coli. This mechanism is called as active efflux pump system, which is multiple genes collaborateing phenotype and is regulated by chromosomal. It makes the level of mar gene expression increased, which can transport different drugs outside the cell and decrease drug accumulating density inside the cell, thus the bacterial bring about the resistance to multiple antibiotic. It was found that these multidrug efflux pump genes widely lie in enterobacteriaceae. In order to know the multiple-antibiotic-resistant mechanisms in clinical strains of Shigella spp.and quest for the track to solve this problem, this study was planned to amplify multidrug efflux pump genes acrAB-tolC by polymerase chain reaction (PCR), single strand conformational polymorphism analysis (SSCP) were applied to all PCR products and the level of mar gene expression were measured by Northern blots. This study identified the role of the inhibitors of efflux pumps (MC-207, 110) to the multiple-antibiotic-resistance strains of Shigella spp. Methods
1. Filtrateing the Mar strains and WT: antimicrobial susceptibilities were tested by the agar doubling dilution method. The strains appearing resistance to Teracycline (TE), Chloramphenicol(C), Ampicillin (Am) and Pipemidic adcid (PI) or Ciprofloxacin (CIP) were called as Mar, susceptible called as wild type (WT).
2. Accumulation of CIP in clinical isolates of Shigella spp.:
accumulation of CIP in clinical isolates of Shigella spp. was measured by fluorometry. Test of accumulation was in the water of 37癈, the samples were resuspended in O.lmol/L (pH 3.0) ice-cold glycine hydrochloride. The concentration of CIP in Mar strains was shown with the CIP milligram in the cell per litre when the cell of ODeoo was 10.
3. PCR-SSCP of multidrug efflux pump genes acrAB-tolC: acrAB-tolC genes were amplified by PCR, the products of which were digested (or undigested) with restriction endonuclease EcoR I (Alu I ), this can detect a single nucleotide change as well as the loss or insertion of oligonucleotide by electrophoresis.
4. The label of the probe by random priming: the single DNA hybridized with random primer and the other single was synthesized with E. co/z'DNA polymerase I Klenow Fragment and DIG-11-dUTP was added to the new strand, the label of the probe was performed.
5. Northern blots: the total RNA of the cell was in the denaturing formaldehyde agarose gel and transferred to nitrogen nylon membrane, the blot was hybridized with the labeled probe.
Results
1. Filtrateing the Mar strains and WT: antimicrobial susceptibilities of 141 clinical isolates were tested by the agar doubling dilution method. 64 clinical isolate
郑州大学硕士学位论文
临床分离志贺菌属细菌多重耐药机制的研究
行PCR扩增编码多重耐药泵蛋白的结构基因acrAB一tolC、运用PCR一SSCP对其突
变性进行检测、N。找hem印迹检测其表达情况，并在实验室研究泵蛋白抑制剂
MC207，1 10对志贺菌的作用。
方法
1.多重耐药株和敏感野生株的筛选:采用琼脂稀释法测定抗菌药物对志贺菌
的最低抑菌浓度(minlrnu工n inllibition coneentration，MIC)。以实验室耐四环素、
氯霉素、氨节青霉素、毗呱酸或环丙沙星的菌株为多重耐药株(multiPle antibiotic
resistant，Mar)，均敏感的菌株为敏感野生株(丽ld tyPe，WT)。
2.志贺菌细胞内环丙沙星的积聚:应用荧光法测定志贺菌细胞内环丙沙星的
积聚。积聚试验均于37’C水浴中进行，样品用预冷至0℃的0.1 mol几(pH 3.0)
的甘氨酸溶液处理。志贺菌细胞内环丙沙星浓度为OD60。=10时每升菌液细胞内所
含的环丙沙星毫克数表示。
3.多重耐药泵系统acrAB一olC基因的单链构象多态性(P CR一5 5 CP)分析:PCR
扩增acrAB一tolC基因，扩增产物经限制性内切酶EcoRI(Alul)消化(或不消
化)，变性后经中性聚丙烯酸胺凝胶电泳，根据单链带数目和位置判断有无基因
突变。
4.采用随机引物法标记探针:将待标记的DNA探针片段变性后与随机引物杂
交，然后以此杂交的寡核昔酸为引物，在DNA聚合酶I大片段的催化下，合成与
探针DNA互补的DNA链，反应液中含有DIG一dUTP，即形成地高辛标记的DNA
探针。
5.Northem印迹:提取细胞总RNA，行甲醛变性凝胶电泳，将RNA转移至尼
龙膜，在含有探针的杂交液中杂交，应用地高辛检测试剂盒检测杂交结果，根据
杂交条带密度半定量细胞中mRNA表达水平。
结果
郑州大学硕士学位论文
临床分离志贺菌属细菌多重耐药机制的研究
1.多重耐药株和敏感野生株的筛选:根据临床上分离的141株志贺菌对四环
素、氯霉素、氨节青霉素、毗呱酸或环丙沙星5种抗生素的药敏试验结果，筛选
出64株多重耐药株，6株敏感野生株。
2.志贺菌细胞内环丙沙星的积聚:敏感野生株细胞内环丙沙星稳态浓度约为
多重耐药株Marl的2.5倍，为多重耐药株MarZ的1 .5倍，差异有显著性
(P<0 .0001)。加入能量抑制剂CCCP后，多重耐药株Marl细胞内环丙沙星稳态
浓度上升了2.2倍，多重耐药株MarZ上升了0.8倍，敏感野生株上升了0.3倍，各
组之间比较差异有显著性(尸<0 .0001)。
3.32株志贺菌属多重耐药株和6株敏感野生株及标准株全部分别扩增出1697
bp、510 bp和1633 bp长度的片段，分别是aerA、aerB和tolc基因的扩增产物，
未发现多重耐药泵基因缺失株。
4.多重耐药泵系统acrAB一to1C基因的单链构象多态性(P CR-SsCP)分析:对38
株临床分离志贺菌属细菌研究，发现4株多重耐药株acrA基因突变，突变率为
103%;有1株敏感野生株tolC基因突变，突变率为2.6%;未发现acrB基因突变
株。
5.随机引物法标记探针:随机引物法标记的探针浓度分别约为:acrA基因为
990 ng/协l、acrB基因800 ng/协l和tolC基因900 ng/林l。
6.多重耐药泵系统acrAB一tolC表达水平结果分析:多重耐药株Marl、MarZ其
acrA基因表达水平显著高于敏感野生株(尸<0.05);多重耐药株Marl、MarZ其
acrB基因表达水平略高于敏感野生株，但统计学比较差异无显著性，F=0.92，
尸二 0.45;多重耐药株Marl、MarZ及敏感野生株其tolC基因表达水平经统计学比
较差异无显著性，F=0.39，尸二0.76。
7.多重耐药泵抑制剂MC一207，110对志贺菌的抑制作用结果分析:在多重耐
药泵基因高表达的一些菌株中作用明显，该抑制剂可通过抑制多重耐药泵从而使
郑州大学硕士学位论文
临床分离志贺菌属细菌多重耐药机制的研究
环丙沙星对志贺菌相?
Shigella called as dysenteriae bacillary is the main pathogen of shigellosis. It is an epidemic contagion throughout the world, especially in the countries with the laggard sanitation. These days, though our country has made a great achievement on the shigellosis of prevention and cure, the incidence of shigellosis was always high, because of much more population, underdevelopment economy in our country and because of the complex of the bacterial type, the easy variance and without the crossover immunity in Shigella. It is the familiar contagion in summer and autumn. Since antimicrobial agents were widely employed to treat shigellosis, antimicrobial resistance in Shigella spp .has been widely appeared and was multiple-antibiotic-resistance, which brought great hard to shigellosis of prevention and cure, consequently shigellosis spread and prevail. In 1996, Shigella spp. had been identified by the World Health Organization as one of the growing antibiotic-resistant bacteria posing a major threat to mankind. I
t is the important task increasing the level of shigellosis of prevention and cure to study the multiple-antibiotic-resistant mechanisms in Shigella spp.
and to overcome multiple-antibiotic-resistance (Mar) of bacterial .The study on Escherichia coli.suggested that AcrAB-TolC multidrug efflux pump played a main role in multiple-antibiotic-resistance E.coli. This mechanism is called as active efflux pump system, which is multiple genes collaborateing phenotype and is regulated by chromosomal. It makes the level of mar gene expression increased, which can transport different drugs outside the cell and decrease drug accumulating density inside the cell, thus the bacterial bring about the resistance to multiple antibiotic. It was found that these multidrug efflux pump genes widely lie in enterobacteriaceae. In order to know the multiple-antibiotic-resistant mechanisms in clinical strains of Shigella spp.and quest for the track to solve this problem, this study was planned to amplify multidrug efflux pump genes acrAB-tolC by polymerase chain reaction (PCR), single strand conformational polymorphism analysis (SSCP) were applied to all PCR products and the level of mar gene expression were measured by Northern blots. This study identified the role of the inhibitors of efflux pumps (MC-207, 110) to the multiple-antibiotic-resistance strains of Shigella spp. Methods
1. Filtrateing the Mar strains and WT: antimicrobial susceptibilities were tested by the agar doubling dilution method. The strains appearing resistance to Teracycline (TE), Chloramphenicol(C), Ampicillin (Am) and Pipemidic adcid (PI) or Ciprofloxacin (CIP) were called as Mar, susceptible called as wild type (WT).
2. Accumulation of CIP in clinical isolates of Shigella spp.:
accumulation of CIP in clinical isolates of Shigella spp. was measured by fluorometry. Test of accumulation was in the water of 37癈, the samples were resuspended in O.lmol/L (pH 3.0) ice-cold glycine hydrochloride. The concentration of CIP in Mar strains was shown with the CIP milligram in the cell per litre when the cell of ODeoo was 10.
3. PCR-SSCP of multidrug efflux pump genes acrAB-tolC: acrAB-tolC genes were amplified by PCR, the products of which were digested (or undigested) with restriction endonuclease EcoR I (Alu I ), this can detect a single nucleotide change as well as the loss or insertion of oligonucleotide by electrophoresis.
4. The label of the probe by random priming: the single DNA hybridized with random primer and the other single was synthesized with E. co/z'DNA polymerase I Klenow Fragment and DIG-11-dUTP was added to the new strand, the label of the probe was performed.
5. Northern blots: the total RNA of the cell was in the denaturing formaldehyde agarose gel and transferred to nitrogen nylon membrane, the blot was hybridized with the labeled probe.
Results
1. Filtrateing the Mar strains and WT: antimicrobial susceptibilities of 141 clinical isolates were tested by the agar doubling dilution method. 64 clinical isolate
引文
1. Navia MM, Capitano L, Ruiz J, et al. Typing and characterization of mechanisms of resistance of Shigella Spp. isolated from feces of children under 5 years of age from Ifakara, Tanzania. J Clin Microbiol, 1999, 37(10): 3113-3117
2． 裴红生．北京市1990-1997年细菌性痢疾流行病学分析．疾病监测，1998(11)：408-412
3． 朱静嫒．志贺菌属细菌耐药性及其耐喹诺酮类药物机制的研究．2002年硕士毕业论文
4. Davies J. Bacteria on the rampage. Nature, 1996, 383: 219-222
5． 李风云，韩俊，唐素兰，等．痢疾杆菌R质粒的检测、消除及药物敏感试验．蚌埠医学院学报，2000，25(3)：161-162
6． 丁建强，马亦林，龚正，等．志贺菌流行菌株耐药性及R质粒的研究．中华传染病杂志，1998，16(3)：61-163
7. Thinmarayanan MA, Jesudason MV, Jacob John T. Resistance of shigella to nalidixic acid and fluorinated quinolones. J Med Res [A], 1993, 11:239-241
8. Courvalin P. Plasmid-mediated 4-quinolone resistance: a real or apparent absence? J Antimicrob Chemother, 1990, 34: 681-684
9. Anindya SG, Ahamed J, Kamlesh K, et al. Involvement of an efflux system in high level fluoroquinolone resistance of Shigella dysenteriae. Biochemical and Biophysical Research Communications, 1998, 242(1): 54-56
10. Fralick JA. Evidence that TolC is required for functioning of the Mar/AcrAB efflux pump of Escherichia coli. J Bacteriol, 1996, 178(19): 5803-5805
11. Mark CS, Chad H, Cramer C, et al. Antibiotic susceptibility profiles of Escherichia
colis trains lacking multidrug efflux pump gene. Antimicrob Agents Chemother, 2001,45(4) : 1126-1136
12． Ma D, Cook DN, Alberti M,et al. Genes acrA and acrB encode a stress-induced efflux system of Escherichia coli. Mol Microbiol, 1995,16: 45-55
13． Klein J, Henrich B and Plapp R. Molecular analysis and nucleotide sequence of the envCD operon of Escherichia coli .Mol Genet, 1991,230: 230-240
14． Paulsen I, Brown MH and Skurray RA. Proton-dependent multidrug efflux systems. Microbiol Rev, 1996, 60:575-608
15． Nakamura H. Novel acriflavin resistance genes, acrC and acrD, in Escherichia coli. J Bacteriol, 1979,139: 8-12
16． Paulsen I. Microbiol genome analyses: global comparisons of transport capabilities based on phylogenies,bioenergetics and substrate specificities.J Mol Biol, 1992, 277:573-592
17． Naroditskaya V, Schlosser MJ, Fang NY, et al. An Escherichia coli gene emrD is involved in adaptation to low energy shock. Biochem Biophys Commun,1993, 196: 803-809
18． Edgar R, and Bibi E. MdfA, an Escherichia coli multidrug resistance protein with an extraordinarily broad spectrum of drug recognition. J Bacteriol, 1997, 179: 2274-2280
19． Lomovskaya O, and Lewis K. emr, an Escherichia coli locus for multidrug resistance. Proc Natl Acad Sci USA, 1992, 89: 8938-8942
20． Lewis K. Multidrug resistance pumps in bacteria: variation on a theme. Trends Biochem Sci, 1994, 19: 119-224
21． Turner R, Taylor DE and Weiner JH. Expression of Escherichia coli TehA gives
resistance to antiseptics and disinfectants similar to that conferred by multidrug resistance efflux pumps. Antimicrob Agents Chemother,1997, 41:440-448
22. Okusu H, Ma D, Nikaido H. AcrAB effux pumps plays a major role in the antibiotic resistance phenotype of mutiple-antibiotic-resistance (Mar) mutants. J Bacteriol, 1996, 178:306-308
23. Cohen SP, Yan W, Levy SB, et al. A multidrug resistance regulatory chromosomal locus is widespread among enteric bacteria. J Infect Dis, 1993 Aug, 168(2): 484-493
24. Renau TE, Leer R, Flamme EM, et al. Inhibitors of efflux pumps in pseudomonas aeruginosa potentiate the activity of the fluoroquinolone antimicrob. J. Med. Chem, 1999, 42:4928-4931
25. Armarita M, Yutaka T, Tiffany M, et al. High-level fluoroquinolone-resistant clinical isolates of Escherichia coli overproduce multidrug efflux protein AcrA. Antimicrob Agents Chemother, 2000, 44(12): 3441-3443
26. Chu YW, Houang TS, Lyon DJ, et al. Antimicrobial resistance in Shigella sonnei in Hong Kong, 1986 to 1995. Antimicrob Agents Chemother, 1998,42(2): 440-443
27. National Committee for Clinical Laboratory Standards.1997.Methods for dilution antimicrobial susceptibility. Tests for bacteria that grow aerobically, 4th ed. Approved standard. NCCLS document M7-A4. National Committee for Clinical Laboratory Standards, Wayne, Pa
28. Blattner, F.R., Plunkett, G.Ⅲ, Bloch, CA. The complete genome sequence of Escherichia coli K-12. Science. 1997,277 (5331): 1453-1474
29．谷志远主编．现代医学分子生物学．北京：人民军医出版社，1998，9：445-451．
30. Levy SB. The challenge of antibiotic resistance. (M) Scientific American
31．丁建强，马亦林，龚正，等．志贺茵流行菌株耐药性及R质粒的研究．中华传染病
杂志，1998，16(3)：61-163
32. Lima AAM, Lima NL, Pinho MCN, et al. High frequency of strains multiply resistant to ampicillin, trimethoprim-sulfamethoxazole, streptomycin, chloramphenicol, and tetracycline isolated from patients with shigellosis in northeastern Brazil during the period 1988 to 1993. Antimicrob Agents Chemother, 1995, 39:156-259
33. Wagner J, Hahn H. Increase of antimicrobial resistance in human baterial pathogens by resistance gene transfer from food of animal origin. [J] Berliner and Munchener Tierarztliche Wochenschrift, 1999, 112:10-11,380-384
34．包幼迪，俞树高，戴赓孙，等．痢疾杆菌耐药性变异的研究．中华微生物和免疫学杂志，1982，2：304-307
35．戴自英，刘裕昆，汪复．实用抗菌药物学(第2版)．上海．上海科学技术出版社，1999，12-28
36. Foster TJ. Microbiol Res, 1983, 47:361-368
37．聂青和．感染性疾病．人民卫生出版社，135-139
38. Nikaido H. Outer membrane barrier as a mechanism of antimicriobial resistance. Antimicrob Agents Chemother, 1989, 33:1831-1836
39. Levy SB. Active efflux mechanisms for antibacterial resistance. Antimicrob Agents Chemother, 1992, 36(4): 695-699
40. Giraud EA, Cloeckaert D, Kerboeuf, et al. Evidence for active efflux as the primary mechanism of resistance to ciprofloxacin in Salmonella enterica serovar typhimurium. Antimicrob Agents Chemother, 2000, 44: 1223-1229
41. Oonaka K, Fukuyama M, Tanaka, et al. Mechanism of resistance of Shigellaflexneri 2a resistant to new quinolone ntibiotics. Kansenshogaku Zasshi, 1998,72(4): 365-
370
42． Daikos GL, Lolans VT and Jackson GG. First-expoure adaptive resistance to aminoglycoside antibiotics in vivo with meaning for optimal clinical use. [J]. Antimicrob Agents Chemother, 1991,35: 117-122
43． Laura JV, Piddock, David G, et al. Evidence for an efflux mediating multiple antibiotic resistant in Salmonella enterica serovar typhimurium. Antimicrob Agents Chemother, 2000 ,44(11) : 3118-3121
44． 章建立，姚航平，周建英等．主动外排系统在临床分离肺炎克雷伯菌中的分布 和表达．中国抗感染杂志，2001，1(4) ：193-197
45． Ma D, Alberti M, Lynch C, et al. The local repressor AcrR plays a modulating role in the regulation of acrAB genes of Escherichia coli by global stresss signals. [J]. Mol Microbiol, 1996,19: 101-106
46． George AM and Levy SB. Gene in the major cotransduction gap of the Escherichia coliK-12 linkage map required for the expression of chromosomal resistance to tetracycline and other antibiotics. J Bacteriol,1983,155:541-546
47． Aono R, Tsudagoshi N, Yamamoto M. Involvement of outer membrane protein in TolC, a possible member of the mar sox regulon, in maintenance and improvement of organic solvent tolerance of the Escherichia coli K-12． J Bacteriol, 1998, 180:938-943
48． Paul F. Genetic relationship between soxRS and mar loci in promoting multiple-antibiotic-resistant in Escherichia coli. Antimicrob Agents Chemother, 1994, 38(8) 1773-1779
49． Mark S. Interscience conference on antimicrobiol agens and chemeotherapy-40th meeting (part Ⅴ). 17-20 September 2000, Toronto, Canada.
2． 裴红生．北京市1990-1997年细菌性痢疾流行病学分析．疾病监测，1998(11)：408-412
3． 朱静嫒．志贺菌属细菌耐药性及其耐喹诺酮类药物机制的研究．2002年硕士毕业论文
4. Davies J. Bacteria on the rampage. Nature, 1996, 383: 219-222
5． 李风云，韩俊，唐素兰，等．痢疾杆菌R质粒的检测、消除及药物敏感试验．蚌埠医学院学报，2000，25(3)：161-162
6． 丁建强，马亦林，龚正，等．志贺菌流行菌株耐药性及R质粒的研究．中华传染病杂志，1998，16(3)：61-163
7. Thinmarayanan MA, Jesudason MV, Jacob John T. Resistance of shigella to nalidixic acid and fluorinated quinolones. J Med Res [A], 1993, 11:239-241
8. Courvalin P. Plasmid-mediated 4-quinolone resistance: a real or apparent absence? J Antimicrob Chemother, 1990, 34: 681-684
9. Anindya SG, Ahamed J, Kamlesh K, et al. Involvement of an efflux system in high level fluoroquinolone resistance of Shigella dysenteriae. Biochemical and Biophysical Research Communications, 1998, 242(1): 54-56
10. Fralick JA. Evidence that TolC is required for functioning of the Mar/AcrAB efflux pump of Escherichia coli. J Bacteriol, 1996, 178(19): 5803-5805
11. Mark CS, Chad H, Cramer C, et al. Antibiotic susceptibility profiles of Escherichia
colis trains lacking multidrug efflux pump gene. Antimicrob Agents Chemother, 2001,45(4) : 1126-1136
12． Ma D, Cook DN, Alberti M,et al. Genes acrA and acrB encode a stress-induced efflux system of Escherichia coli. Mol Microbiol, 1995,16: 45-55
13． Klein J, Henrich B and Plapp R. Molecular analysis and nucleotide sequence of the envCD operon of Escherichia coli .Mol Genet, 1991,230: 230-240
14． Paulsen I, Brown MH and Skurray RA. Proton-dependent multidrug efflux systems. Microbiol Rev, 1996, 60:575-608
15． Nakamura H. Novel acriflavin resistance genes, acrC and acrD, in Escherichia coli. J Bacteriol, 1979,139: 8-12
16． Paulsen I. Microbiol genome analyses: global comparisons of transport capabilities based on phylogenies,bioenergetics and substrate specificities.J Mol Biol, 1992, 277:573-592
17． Naroditskaya V, Schlosser MJ, Fang NY, et al. An Escherichia coli gene emrD is involved in adaptation to low energy shock. Biochem Biophys Commun,1993, 196: 803-809
18． Edgar R, and Bibi E. MdfA, an Escherichia coli multidrug resistance protein with an extraordinarily broad spectrum of drug recognition. J Bacteriol, 1997, 179: 2274-2280
19． Lomovskaya O, and Lewis K. emr, an Escherichia coli locus for multidrug resistance. Proc Natl Acad Sci USA, 1992, 89: 8938-8942
20． Lewis K. Multidrug resistance pumps in bacteria: variation on a theme. Trends Biochem Sci, 1994, 19: 119-224
21． Turner R, Taylor DE and Weiner JH. Expression of Escherichia coli TehA gives
resistance to antiseptics and disinfectants similar to that conferred by multidrug resistance efflux pumps. Antimicrob Agents Chemother,1997, 41:440-448
22. Okusu H, Ma D, Nikaido H. AcrAB effux pumps plays a major role in the antibiotic resistance phenotype of mutiple-antibiotic-resistance (Mar) mutants. J Bacteriol, 1996, 178:306-308
23. Cohen SP, Yan W, Levy SB, et al. A multidrug resistance regulatory chromosomal locus is widespread among enteric bacteria. J Infect Dis, 1993 Aug, 168(2): 484-493
24. Renau TE, Leer R, Flamme EM, et al. Inhibitors of efflux pumps in pseudomonas aeruginosa potentiate the activity of the fluoroquinolone antimicrob. J. Med. Chem, 1999, 42:4928-4931
25. Armarita M, Yutaka T, Tiffany M, et al. High-level fluoroquinolone-resistant clinical isolates of Escherichia coli overproduce multidrug efflux protein AcrA. Antimicrob Agents Chemother, 2000, 44(12): 3441-3443
26. Chu YW, Houang TS, Lyon DJ, et al. Antimicrobial resistance in Shigella sonnei in Hong Kong, 1986 to 1995. Antimicrob Agents Chemother, 1998,42(2): 440-443
27. National Committee for Clinical Laboratory Standards.1997.Methods for dilution antimicrobial susceptibility. Tests for bacteria that grow aerobically, 4th ed. Approved standard. NCCLS document M7-A4. National Committee for Clinical Laboratory Standards, Wayne, Pa
28. Blattner, F.R., Plunkett, G.Ⅲ, Bloch, CA. The complete genome sequence of Escherichia coli K-12. Science. 1997,277 (5331): 1453-1474
29．谷志远主编．现代医学分子生物学．北京：人民军医出版社，1998，9：445-451．
30. Levy SB. The challenge of antibiotic resistance. (M) Scientific American
31．丁建强，马亦林，龚正，等．志贺茵流行菌株耐药性及R质粒的研究．中华传染病
杂志，1998，16(3)：61-163
32. Lima AAM, Lima NL, Pinho MCN, et al. High frequency of strains multiply resistant to ampicillin, trimethoprim-sulfamethoxazole, streptomycin, chloramphenicol, and tetracycline isolated from patients with shigellosis in northeastern Brazil during the period 1988 to 1993. Antimicrob Agents Chemother, 1995, 39:156-259
33. Wagner J, Hahn H. Increase of antimicrobial resistance in human baterial pathogens by resistance gene transfer from food of animal origin. [J] Berliner and Munchener Tierarztliche Wochenschrift, 1999, 112:10-11,380-384
34．包幼迪，俞树高，戴赓孙，等．痢疾杆菌耐药性变异的研究．中华微生物和免疫学杂志，1982，2：304-307
35．戴自英，刘裕昆，汪复．实用抗菌药物学(第2版)．上海．上海科学技术出版社，1999，12-28
36. Foster TJ. Microbiol Res, 1983, 47:361-368
37．聂青和．感染性疾病．人民卫生出版社，135-139
38. Nikaido H. Outer membrane barrier as a mechanism of antimicriobial resistance. Antimicrob Agents Chemother, 1989, 33:1831-1836
39. Levy SB. Active efflux mechanisms for antibacterial resistance. Antimicrob Agents Chemother, 1992, 36(4): 695-699
40. Giraud EA, Cloeckaert D, Kerboeuf, et al. Evidence for active efflux as the primary mechanism of resistance to ciprofloxacin in Salmonella enterica serovar typhimurium. Antimicrob Agents Chemother, 2000, 44: 1223-1229
41. Oonaka K, Fukuyama M, Tanaka, et al. Mechanism of resistance of Shigellaflexneri 2a resistant to new quinolone ntibiotics. Kansenshogaku Zasshi, 1998,72(4): 365-
370
42． Daikos GL, Lolans VT and Jackson GG. First-expoure adaptive resistance to aminoglycoside antibiotics in vivo with meaning for optimal clinical use. [J]. Antimicrob Agents Chemother, 1991,35: 117-122
43． Laura JV, Piddock, David G, et al. Evidence for an efflux mediating multiple antibiotic resistant in Salmonella enterica serovar typhimurium. Antimicrob Agents Chemother, 2000 ,44(11) : 3118-3121
44． 章建立，姚航平，周建英等．主动外排系统在临床分离肺炎克雷伯菌中的分布 和表达．中国抗感染杂志，2001，1(4) ：193-197
45． Ma D, Alberti M, Lynch C, et al. The local repressor AcrR plays a modulating role in the regulation of acrAB genes of Escherichia coli by global stresss signals. [J]. Mol Microbiol, 1996,19: 101-106
46． George AM and Levy SB. Gene in the major cotransduction gap of the Escherichia coliK-12 linkage map required for the expression of chromosomal resistance to tetracycline and other antibiotics. J Bacteriol,1983,155:541-546
47． Aono R, Tsudagoshi N, Yamamoto M. Involvement of outer membrane protein in TolC, a possible member of the mar sox regulon, in maintenance and improvement of organic solvent tolerance of the Escherichia coli K-12． J Bacteriol, 1998, 180:938-943
48． Paul F. Genetic relationship between soxRS and mar loci in promoting multiple-antibiotic-resistant in Escherichia coli. Antimicrob Agents Chemother, 1994, 38(8) 1773-1779
49． Mark S. Interscience conference on antimicrobiol agens and chemeotherapy-40th meeting (part Ⅴ). 17-20 September 2000, Toronto, Canada.