大肠杆菌O157喹诺酮类耐药相关机制的研究
摘要
大肠杆菌0157:H7是医学和兽医学临床感染中常见的病原菌之一,建立快速检测方法对食品安全和人类健康具有重要意义。大肠杆菌0157对多种抗菌药物耐药主要与菌体中存在的外输泵有关。一般认为AcrAB-To1C外输泵是最主要的外输泵,能够输出多种抗菌药物、化疗试剂、洗涤剂和染料类。如果使该系统失活,大肠杆菌0157可从多重耐药状态变为相对敏感状态。本实验通过建立大肠杆菌0157:H7的快速检测方法,研究大肠杆菌0157:H7对氟喹诺酮类药物的基因突变耐药机制,检测临床分离的动物源性大肠杆菌0157的acrA、acrB基因的mRNA和蛋白的表达水平,寻找acrA、acrB基因的表达水平与大肠杆菌0157多重耐药水平的相关性。
研究快速、特异、灵敏的检测肠出血性大肠杆菌0157:H7的多重PCR、荧光定量PCR方法并比较单一PCR和多重PCR、荧光定量PCR对检测灵敏度的影响。选择0157:H7的O抗原、H鞭毛抗原及SLT1和SLT2毒素基因特异的4对引物,分别或共同进行PCR扩增,检测24株0157:H7和非0157:H7菌株,将细菌稀释后比较PCR的检测灵敏度。同时设计TaqMan荧光探针进行荧光定量PCR进行灵敏度检测。所有0157:H7菌株均在497bp和625bp处出现0157抗原基因和H7抗原基因产物,其产毒株在484bp和(或)210bp处出现SLT1和(或)SLT2基因产物,非0157:H7菌株PCR结果均为阴性;单一PCR检测,灵敏度为150CFU/PCR反应,多重PCR为>1500CFU/PCR反应,而荧光定量PCR为14CFU/PCR反应。
选用临床分离的4株耐药菌、实验室诱导培养获得的3株耐药菌和1株敏感菌,提取其染色体DNA,PCR扩增gyrA和parC基因片段,并将其克隆和测序。结果表明,无论是临床分离的还是实验室诱导的耐药菌,gyrA基因在其编码第83位或第87位氨基酸处均发生突变,parC基因在其编码第80位或第84位氨基酸处发生突变,而敏感菌ES在2个基因位点上均未发生突变。其中2株低度耐药菌株的gyrA基因出现单一突变,使其编码的氨基酸发生改变,分别为Ser83-Leu或Asp87-Asn,但在parC基因上却未发生突变;其余5株高度耐药菌gyrA基因突变导致氨基酸发生改变:Ser83-Leu,Asp87-Asn和Tyr,其中两株parC基因突变导致氨基酸改变:Ser80-Ile和GIu84-Lys。这2个基因的突变均与文献报道的突变相同,表明gyrA基因Ser83和Asp87突变以及parC基因Ser80和G1u84突变可能与大肠杆菌0157的氟喹诺酮类药耐药机制有关,且低度耐药只在gyrA基因上出现单一位点突变,当高度耐药时,才同时在parC基因上出现突变,gyrA , parC是大肠杆菌0157:H7耐氟喹诺酮类药物的基因标识。
多重耐药大肠杆菌0157acrA、acrB基因的mRNA的水平比较,将耐药株的反转录产物量相当的内参稀释度与大肠杆菌0157 EDL933的反转录产物量相当的内参稀释度做比较的结果表明:高水平耐药与EDL933菌株的acrA、acrB的mRNA水平相比较提高4-8倍;中度耐药的大肠杆菌0157的acrA、acrB的mRNA水平与EDL933的acrA、acrB的mRNA水平相比提高1-2倍,低水平耐药与EDL933的acrA、acrB的mRNA水平相当。与EDL933的acrA、acrB基因的mRNA的水平相比,同一菌株的acrA、acrB基因的mRNA的水平基本一致,可能是由于二者由一个操纵子调控的原因。acrA、acrB是大肠杆菌0157:H7多重耐药的基因标识,其mRNA转录水平与大肠杆菌0157:H7的多重耐药水平呈正相关性。
本研究结果表明,多重PCR和荧光定量PCR可对大肠杆菌0157:H7进行定性定量检测:大肠杆菌0157:H7gyrA基因、Ser83和Asp87突变以及parC基因Ser80和G1u84突变可能与大肠杆菌0157的喹诺酮类药耐药机制有关,且低度耐药只在gyrA基因上出现单一位点突变;当高度耐药时,才同时在parC基因上出现突变。大肠杆菌0157:H7同一菌株的acrA与acrB基因的mRNA的水平是基本一致的:与敏感株相比,acrA的mRNA水平与其蛋白表达水平基本一致;多重耐药大肠杆菌O157的mRNA和蛋白表达水平与其耐药水平有一定的相关性。
E.coli O157 is one of bacteria in medical science and veterinary clinically. Swif-detected method has important significance to foods safety and human health. AcrAB efflux pump plays a major role in the antibiotics resistance phenotype of Esherichia coli O157,multitiple antiobiotics resistance mutant.AcrAB-ToIC system can efflux out an extraordinarily wide variety of antibiotics, chemotherapeutic agent, detergent and dyes.The coordinated operation of the inner membrane transporler acrB and outer channel To1C is thought to be mediatd by acrA. Here we will establish a swif-detected method, study gene mutant mechanism of E.coli 0157 resistance to quinolone, determine the level of mRNA of acrA and acrB and level of acrA protein expresion.
To develop methods of multiplex PCR and real-time fluorescence PCR to detect E.coli O157:H7 rapidly, specifically and sensitively. To compare their sensitivity with conventional PCR. Four pairs of primers were designed from O antigen and H flagellar antigen and Shiga一like Toxin 1 and 2 genes. 24 strains of O157:H7 and non O157:H7 were detected by conventional PCR and multiplex PCR amplification.At the same time, we design TaqMan fluorescence probe to put up real-time fluorescence PCR .The sensitivities of PCR were estimated when the strain was diluted .O antigen and H flagellar antigen genes amplification generated amplicons of both 497bp and 625bp in all EHEC 0157:H7 strains, SLTl and (or) SLT2 genes amplification generated amplicons of 210bp and(or)484bp in toxinogenic O157:H7. Other non O157:H7 failed to yield any amplicon under comparable conditions. The sensitivity of detection by conventional PCR and multiplex PCR were shown to be at least 150CFU per PCR and1500 CFU per PCR. The sensitivity of detection by real-time fluorescence PCR was 14CFU per PCR.
Chromosome DNAs from 8 strains of E.coli O157 were extracted, among which 4 strains were collected from clinical samples, 3 strains were got by induction with different concentration of quinolone and 1 susceptible strain. The fragments of gyrA and parC genes of the 8 strains were amplified, cloned and sequenced by PCR. Mutaions were found at residues 83 and 87 in gyrA gene and 80 and 84 in parC gene in all the quinolone-resistant strains,while no mutations were found in the susceptible strain. Only one mutation was found in gyrA gene in 2 low resistant strains,which resulted in Ser83→Leu or Asp87→Asn, and no mutation was found in parC gene. Doublemutations were found in both gyrA and parC genes in 5 high resistant strains. Mutations in gyrA gene resulted in Ser83→Leu and Asp 87→Asn or Tyr , and in parC resulted in Ser83→Ile and Glu84→L ys.The mutations are identical with data from the published references. The results revealed that mutations of gyrA and parC genes are involved in the quinolone-resistant mechanism of E scherichia coli. And the low-resistant E.coli exist only single mutation in gyrA gene, the high resistant E.coli existmutations in both parC and gyrA genes at the same t ime.
The level of mRNA of multiple-antibiotic resistance in Escherichia coli O157 gene acrA and acrB compares,the internal standard dilution of retroposon production from drug-resistant strain compares with that of E.coli O157 EDL933.The result is that the content of mRNA transcribing from acrA and acrB in the high-drug-resistant E.coli is 4or 8 times higher than in the EDL933, in the moderate-drug-resistant E.coli ,it is 1 or 2 times higher than in the EDL933, and the low- drug-resistant E.coli ,it is as same as in the EDL933.The content of mRNA describing from acrA is similar to acrB in the same bacterium. It probably because that acrA and acrB are controlled by one operon. So acrA and acrB are the multi-drug-resistant gene-marker in E.coli O157:H7.The contant of mRNA describing from acrA and acrB is direct-ratio to the drug-resistant level in E.coli O157:H7.
The result is that E.coli O157:H7 can be quantitatively and qualitatively analyzed by multiplex PCR and fluorescence quantitative PCR. The mutation of E.coli O157:H7, gyrA,Ser83 and Asp87,parC gene, Ser80 and G1u84 is related with the drug-resistant mechanism of E.coli on the quinolone. There is a single mutation on the gyrA gene in low-drug-resistant strain .In high-drug-resistant strain, there also have another mutation on the parC gene. The level of mRNA describing from acrA is similar to acrB in E.coli O157:H7. The level of mRNA describing from acrA is same to the level of protein expressing from acrA. The level of mRNA and protein has relative relationship with drug-resistance in E.coli O157.
研究快速、特异、灵敏的检测肠出血性大肠杆菌0157:H7的多重PCR、荧光定量PCR方法并比较单一PCR和多重PCR、荧光定量PCR对检测灵敏度的影响。选择0157:H7的O抗原、H鞭毛抗原及SLT1和SLT2毒素基因特异的4对引物,分别或共同进行PCR扩增,检测24株0157:H7和非0157:H7菌株,将细菌稀释后比较PCR的检测灵敏度。同时设计TaqMan荧光探针进行荧光定量PCR进行灵敏度检测。所有0157:H7菌株均在497bp和625bp处出现0157抗原基因和H7抗原基因产物,其产毒株在484bp和(或)210bp处出现SLT1和(或)SLT2基因产物,非0157:H7菌株PCR结果均为阴性;单一PCR检测,灵敏度为150CFU/PCR反应,多重PCR为>1500CFU/PCR反应,而荧光定量PCR为14CFU/PCR反应。
选用临床分离的4株耐药菌、实验室诱导培养获得的3株耐药菌和1株敏感菌,提取其染色体DNA,PCR扩增gyrA和parC基因片段,并将其克隆和测序。结果表明,无论是临床分离的还是实验室诱导的耐药菌,gyrA基因在其编码第83位或第87位氨基酸处均发生突变,parC基因在其编码第80位或第84位氨基酸处发生突变,而敏感菌ES在2个基因位点上均未发生突变。其中2株低度耐药菌株的gyrA基因出现单一突变,使其编码的氨基酸发生改变,分别为Ser83-Leu或Asp87-Asn,但在parC基因上却未发生突变;其余5株高度耐药菌gyrA基因突变导致氨基酸发生改变:Ser83-Leu,Asp87-Asn和Tyr,其中两株parC基因突变导致氨基酸改变:Ser80-Ile和GIu84-Lys。这2个基因的突变均与文献报道的突变相同,表明gyrA基因Ser83和Asp87突变以及parC基因Ser80和G1u84突变可能与大肠杆菌0157的氟喹诺酮类药耐药机制有关,且低度耐药只在gyrA基因上出现单一位点突变,当高度耐药时,才同时在parC基因上出现突变,gyrA , parC是大肠杆菌0157:H7耐氟喹诺酮类药物的基因标识。
多重耐药大肠杆菌0157acrA、acrB基因的mRNA的水平比较,将耐药株的反转录产物量相当的内参稀释度与大肠杆菌0157 EDL933的反转录产物量相当的内参稀释度做比较的结果表明:高水平耐药与EDL933菌株的acrA、acrB的mRNA水平相比较提高4-8倍;中度耐药的大肠杆菌0157的acrA、acrB的mRNA水平与EDL933的acrA、acrB的mRNA水平相比提高1-2倍,低水平耐药与EDL933的acrA、acrB的mRNA水平相当。与EDL933的acrA、acrB基因的mRNA的水平相比,同一菌株的acrA、acrB基因的mRNA的水平基本一致,可能是由于二者由一个操纵子调控的原因。acrA、acrB是大肠杆菌0157:H7多重耐药的基因标识,其mRNA转录水平与大肠杆菌0157:H7的多重耐药水平呈正相关性。
本研究结果表明,多重PCR和荧光定量PCR可对大肠杆菌0157:H7进行定性定量检测:大肠杆菌0157:H7gyrA基因、Ser83和Asp87突变以及parC基因Ser80和G1u84突变可能与大肠杆菌0157的喹诺酮类药耐药机制有关,且低度耐药只在gyrA基因上出现单一位点突变;当高度耐药时,才同时在parC基因上出现突变。大肠杆菌0157:H7同一菌株的acrA与acrB基因的mRNA的水平是基本一致的:与敏感株相比,acrA的mRNA水平与其蛋白表达水平基本一致;多重耐药大肠杆菌O157的mRNA和蛋白表达水平与其耐药水平有一定的相关性。
E.coli O157 is one of bacteria in medical science and veterinary clinically. Swif-detected method has important significance to foods safety and human health. AcrAB efflux pump plays a major role in the antibiotics resistance phenotype of Esherichia coli O157,multitiple antiobiotics resistance mutant.AcrAB-ToIC system can efflux out an extraordinarily wide variety of antibiotics, chemotherapeutic agent, detergent and dyes.The coordinated operation of the inner membrane transporler acrB and outer channel To1C is thought to be mediatd by acrA. Here we will establish a swif-detected method, study gene mutant mechanism of E.coli 0157 resistance to quinolone, determine the level of mRNA of acrA and acrB and level of acrA protein expresion.
To develop methods of multiplex PCR and real-time fluorescence PCR to detect E.coli O157:H7 rapidly, specifically and sensitively. To compare their sensitivity with conventional PCR. Four pairs of primers were designed from O antigen and H flagellar antigen and Shiga一like Toxin 1 and 2 genes. 24 strains of O157:H7 and non O157:H7 were detected by conventional PCR and multiplex PCR amplification.At the same time, we design TaqMan fluorescence probe to put up real-time fluorescence PCR .The sensitivities of PCR were estimated when the strain was diluted .O antigen and H flagellar antigen genes amplification generated amplicons of both 497bp and 625bp in all EHEC 0157:H7 strains, SLTl and (or) SLT2 genes amplification generated amplicons of 210bp and(or)484bp in toxinogenic O157:H7. Other non O157:H7 failed to yield any amplicon under comparable conditions. The sensitivity of detection by conventional PCR and multiplex PCR were shown to be at least 150CFU per PCR and1500 CFU per PCR. The sensitivity of detection by real-time fluorescence PCR was 14CFU per PCR.
Chromosome DNAs from 8 strains of E.coli O157 were extracted, among which 4 strains were collected from clinical samples, 3 strains were got by induction with different concentration of quinolone and 1 susceptible strain. The fragments of gyrA and parC genes of the 8 strains were amplified, cloned and sequenced by PCR. Mutaions were found at residues 83 and 87 in gyrA gene and 80 and 84 in parC gene in all the quinolone-resistant strains,while no mutations were found in the susceptible strain. Only one mutation was found in gyrA gene in 2 low resistant strains,which resulted in Ser83→Leu or Asp87→Asn, and no mutation was found in parC gene. Doublemutations were found in both gyrA and parC genes in 5 high resistant strains. Mutations in gyrA gene resulted in Ser83→Leu and Asp 87→Asn or Tyr , and in parC resulted in Ser83→Ile and Glu84→L ys.The mutations are identical with data from the published references. The results revealed that mutations of gyrA and parC genes are involved in the quinolone-resistant mechanism of E scherichia coli. And the low-resistant E.coli exist only single mutation in gyrA gene, the high resistant E.coli existmutations in both parC and gyrA genes at the same t ime.
The level of mRNA of multiple-antibiotic resistance in Escherichia coli O157 gene acrA and acrB compares,the internal standard dilution of retroposon production from drug-resistant strain compares with that of E.coli O157 EDL933.The result is that the content of mRNA transcribing from acrA and acrB in the high-drug-resistant E.coli is 4or 8 times higher than in the EDL933, in the moderate-drug-resistant E.coli ,it is 1 or 2 times higher than in the EDL933, and the low- drug-resistant E.coli ,it is as same as in the EDL933.The content of mRNA describing from acrA is similar to acrB in the same bacterium. It probably because that acrA and acrB are controlled by one operon. So acrA and acrB are the multi-drug-resistant gene-marker in E.coli O157:H7.The contant of mRNA describing from acrA and acrB is direct-ratio to the drug-resistant level in E.coli O157:H7.
The result is that E.coli O157:H7 can be quantitatively and qualitatively analyzed by multiplex PCR and fluorescence quantitative PCR. The mutation of E.coli O157:H7, gyrA,Ser83 and Asp87,parC gene, Ser80 and G1u84 is related with the drug-resistant mechanism of E.coli on the quinolone. There is a single mutation on the gyrA gene in low-drug-resistant strain .In high-drug-resistant strain, there also have another mutation on the parC gene. The level of mRNA describing from acrA is similar to acrB in E.coli O157:H7. The level of mRNA describing from acrA is same to the level of protein expressing from acrA. The level of mRNA and protein has relative relationship with drug-resistance in E.coli O157.
引文
[1].鞠洪涛,大肠杆菌耐庆大霉素基因定位.解放军军需大学[D],1999,2-6
[2].恽时锋,潘震寰,郑明球,鸡大肠杆菌病研究进展.畜牧与兽医[J].1998,30(6):227-230
[3].Yoshida H,Bogaki M,Nakamura M,etal..Quinolone resistance-determining region in the DNA gyrase gyrA gene of E.coli.Antimicrobial Agents and Chemotherpy[J]. 1990,10,1271-1272
[4].Claude Soussy,John Wolfson,Eva YNG.Limitations of plasmid complementation tese for detetmination of quinolone resistance due to changes in the gurase A protein and identification of conditional quinolone resistance locus. Antimicrobial Agents and Chemotherpy[J].1993,37(12):2588-2592
[5].Simone b,Volker H,Bernd W etal, Impact lf gyrA and parCon Quinolone resistance,doubling time,and supercoiling degree of Escherichia coli . Antimicrobial Agents and Chemotherpy[J]. 1999,8,868-875
[6].Cambau E,Bordon F,Collatz E, etal. Novel gyrApoint mutation in strain lf E.coli resistant to fluoroquinolones but not to nalidixic acid, Antimicrobial Agents and Chemotherpy[J]. 1993,7,1247-1256
[7].Piddock LJ.Mechanisms lf fluroquinolone resistance:an update 1994–1998.Drugs[J],1999,58(2):11-18
[8].Hallet P,Maxwell A..Novel quinolone resistance mutation of the E.coli DNA gyrase A protein:enzymatic analysis of the mutant protein. Antimicrobial Agents and Chemotherpy[J]. 1991,6,335-340
[9].Hiroaki Yohida.Quinolone Resistance-determining region in the DNA Gyrase gyrB gene of Escherichia coli . Antimicrobial Agents and Chemotherpy. [J] 1991,35(8):1647–1650
[10].Heisig P,.Genetic evidence for a role of parC in development of high-level fluoquinolone resistance in E.coli . Antimicrobial Agents and Chemotherpy[J]. 1996,3,879-885
[11].Vila H,Ruiz J,Goni P,etal.Detection of mutation in parC in quinolone resistant clinical isolates of E.coli. Antimicrobial Agents and Chemotherpy[J],1995,8,491-493
[12].David G White,Charlene Hudson,John J.Maurer etal.Characterization of chloramphenicol and florfenicol resistance in associated with Bovine diarrhea.Journal of Microbioligy[J],2000,38(12):4593-4598
[13].Gayatri Vedantam,Gordon G.Guay,Natasha E.Austria,etal.Nixhols Characrerization of mutations contributing to sulfthiazole resistance in Escherichia coli. Antimicrob-Agents-Chemother[J].1998,42(1):88-93
[14].Whiteway J,Koziarz,P,Veall,J,Sandhu N,Kumar P, etal.Oxygen-insensitive nitroreductases: analysis of the roles of nfsA and nfsB in development of resistance to nitrofuran derivatives in Escherichia coli.Bacteriol. [J].1998,180(21):5529-5539
[15].Bakai S,Fonshtein M Kharakter povrezhdenii DNK iikh likvidatsii prideistvii dioksidina nabakterii. [The nature of DNA damage and its repair after treatment of bacteria with dioxidine] Mol-Gen-Mikrobiol-Virusol[J]. 1987,(4): 35-39
[16].Douthwaite S,Hansen LH,Mauvais P Macrolide-ketolide inhibition of MLS–resistant ribosomes is improved by alternative drug interaction with domain II of 23SrRNA. Mol-Microbiol[J].. 2000,36(1): 183-193
[17]Jovanovic M,Lilic M,Janjusevic;etal, tRNA synthetase mutants of Escherichia coli K-12 are resistant to the gyrase inhibitor novobiocin.Bacteriol.[J]. 1999,181(9): 2979-2983
[18].Rakonjac J,Milic M,SavicDJ,cysB and cysE mutants of Escherichia coli K12 show increased resistance to novobiocin. Mol-Gen-Genet.[J].1991,228(1-2): 307-11
[19].George A.Jacoby,Antone A.Medeiros.More extended-spectrumβ-Lactamases. Antimicrob Agents Chemother[J].. 1991,35(9): 1697-1704
[20].Marie P Mingeot Ll,Youri G,etal.Minireview Aminoglycosides:activity and resistance. Antimicrob Agents Chemother[J].. 1999 ,43(4):727-737
[21].Hadfield C, CashmoreA,Meacock P, Sequence and expression characteristics of a shuttle chloramphenicol-resistance marker for Saccharomyces cerevisiae and Escherichia coli. Gene. [J].1987,52(1): 59-70
[22].Morozov G,Nosova L,Biketov F etal, Biokhimicheskie osnovy effekta sovmeshcheniia genov ustoichivostik kanamitsinui nitrofuranam vkletkakhEscherichia coli. [Biochemical bases for the effect of combining kanamycin and nitrofuran resistance genes in Escherichia coli cells] Mol-Gen-Mikrobiol-Virusol[J]1994,(2):11-14
[23].Noguchi N,Emura A,Matsuyama H, Nucleotide sequence and characterization of erythromycin resistance determinant that encodes macrolide 2'-phosphotransferase I in Escherichia coli. Antimicrob Agents Chemother[J]. 1995 ,39(10): 2359-2363
[24].TI Kono M,Ohara K, Ebisu T,Purification and characterization of macrolide 2'-phosphotransferase type II from a strain of Escherichia coli highly resistant to macrolide antibiotics. FEMS Microbiol Lett[J].1992 ,76(1-2): 9389-9460.
[25].Rachek L,Hines A,Tucker A,etal, Transformation of Rickettsia prowazekii to erythromycin re sistance encoded by the Escherichia coli ereB gene. Bacteriol[J] 2000,182(11): 3289-3293
[26].Seltmann G,Resistance of Escherichia coli to nourseothricin: sensitization of resistant strains by abolition of its outer membrane resistance. Zentralbl Bakteriol[J].1992,276(2): 143-151
[27].Matsuyama S,.Mizushima,Construction and characterization of a deletion mutant lacking micF ,aproposed regulatory gene fur OmpF synthesis in Escherichia col . Bacteriol[J]. 1985,162(3):1196-1202
[28].Cohen S,.McMurry,.Levy,marA locus causes decreased expression of OmpF porin in multiple-antobiotic-resistant mutants of Escherichia coli.J.Bacteriol[J].1988 , 170(12):5416-5422
[29].Hirai,K,Aoyama,Irikura,etal.Mitsuhashi.Differences in susceptibility to quinolones of outer membrane mutants of Salmonella typhimurium and Escherichia coli. Antimicrob Agents Chemother[J]. 1986,29(2):703-705
[30].George A,Jacoby,Lorraine Sutton.β-Lactamases andβ-Lactam resistance in Escherichia coli. Antimicrob Agents Chemother[J]. 1985,28(5):703-705
[31].Bissonnette L,Champetier S,Buisson J,etal, Characterization of the nonenzymatic chloramphenicol resistance gene of the In4 integron of Tn1696: similarity of the product to transmembrane transport proteins.Bacteriol[J].1991,173(14): 4493-4502
[32].Rahaman S,Mukherjee J,ChakrabartiA, Decreased membrane permeability in a polymyxin B-resistant Escherichia coli mutant exhibiting multiple resistance to beta-lactams as well as aminoglycosides.Microbiol[J]. 1998,161(2): 249-254
[33].Nummila K,Kilpelainen I,Zahringer,etal, Lipopolysaccharides of polymyxin B-resistant mutants of Escherichia coli are extensively substituted by 2-aminoethyl pyrophosphate and contain amino arabinose in lipid A. Mol-Microbiol[J]. 1995,16(2):271-278
[34].Seltmann G,Resistance of Escherichia coli to nourseothricin: reduced penetrability of the cell wall as an additional, possibly unspecific mechanism. Basic Microbiol[J].1989,29(7): 449-456
[35].Harke okusu,Dzwokai Ma, Hiroshi Nikaido.AcrAB efflux pump plays a major role in the antibiotic resistance phenotype of Escherichia coli muliple–antibiotic–resistance mutants.Journal of bacteriology[J]. 1996,178(3):306-308
[36].Ma,D,Cook,Hearst,ikaido. Efflux pumps and drug resistance in Gram-negative bacteria.Trends ,Microbiol[J]. 1994,(2):489-493
[37].Olga Lomovskaya,kim lewis,an Escherichia coli locus for multidrug resistance.Proc.Natl.Acad.Sci.[J]. 1992,(3):8938-8942
[38].Hagit Yerushalmi,Mario Libendiker,Shimon schuldiner,an Escherichia coli 12-Kda multidrug Transporter,exchanges toxic cation and H+ and is soluble in organic solvents. Journal of biological Chemistry[J]. 1995,270(5):6856-6863
[39].Bentley.Hyatt,.Ainley,etal.Cloning and sequence analysis of an Escherichia coli gene conferring bicyolomycin resistance.Gene[J]. 1993,127(6):117-120
[40].Edgar,R., E.Bibi. Mdfa,an Escherichia coli multidrug resistance protein with an extraordinarily brosd spectrum of drug recognition.Bacteriol[J]. 1997.179(6):2274-2280
[41].Nilsen,IW,Bakke,I,Vader,A, a novel Escherichiacoli chloramphenicol resistance gene encoding a putative efflux pump. Bacteriol[J]. 1996 ,178(11): 3188-3193
[42].Li,H.,Park,The periplasmic murein peptide-binding protein MppA is a negative regulator of multiple antibiotic resistance in Eschericha coli..Bacteriol[J]. 1997,181(2):4842-4847
[43].Kobayashi N,Nishino K,Yamaguchi ,A Novel macrolide-specific ABC-typeefflux transporter in Escherichia coli. Bacteriol[J]. 2001 ,183(19): 5639-4564
[44].Perreten V,Schwarz, V,Teuber M, a new efflux protein conferring multiple antibiotic resistance in Lactococcus lactis and Escherichia coli.Antimicrob Agents Chemother[J]. 2001 ,45(4):1109-1114
[45].Gregory S,Dahlberg A, Erythromycin resistance mutations in ribosomal proteins L22 and L4 perturb the higher order structure of 23 S ribosomal RNA.MolBiol. [J] 1999,289(4): 827-834
[46].ChittumH,ChampneyS,Ribosomal protein gene sequence changes in erythromycin-resistant mutants of Escherichia coli. Bacteriol[J].1994,176(20): 6192-6298
[47].Blot M,Heitman J,Arber W,Tn5-mediated bleomycin resistance in Escherichia coli requires the expression of host genes. Mol-Microbiol[J]. 1993 ,8(6): 1017-1024
[48].金少鸿,张凤凯编译,抗菌药物耐药性现状透视.国外医药药学分册[M].2000,27(2):74-79
[49]Reece R, Maxwell A,DNA gyrase,structure and function. CritRev Biochem Mol Biol[J],1991, 26(1):335~375.
[50]Wang J C,DNA topoisomerases. Annu Rev Biochem[J], 1996,65(5):635~692.
[51]Yoshida H, BogakiM, Nacamura M,etal.Quinolone-resistance-determining region inthe DNA gyrase gyrA gene of Escheroc ia coli. Antimicrob A gents Chemother[J], 1990, 34 (5) : 1271~1272.
[52]CullenME, WykeM W,Kuroda R, etal. Cloning and characterization of DNA gyraseAgene from Escherochia coli that confers clinicalresistance to 4 quino lones. Antimicrob A gents Chemother[J], 1989,33 (4):886~894.
[53]Heisig P, Schedletzky H, Mutations in the gyrA gene of a highly fluoroquino lone 2 resistant clinicalisolate of Escheroch ia coli. Antimicrob Agents Chemother[J], 1993,37(3):696～701.
[54]Oram M , Fisher L M. 42quino lone resistance mutations in the DNAgyrase ofEscherochia coli clinical iso lates identified by using thepo lymo rase chain reaction. Antimicrob Agents Chemother[J], 1991, 35(2):387~389.
[55]Weigel L M, Steward C D, Tenover F C. GyrA mutations associated with fluoroquino lone resistance in eight species of enterobacteriaceae. Antimicrob Agents Chemother[J].1998,42(10):2661~2667.
[56]GibreelA , Sjogren E, Kaijser B, et al. Rapid emergence of high-levelresistance to quino lones in campylobacter jejuni associated with mutantional changes in gyrA and parC. Antimicrob Agents Chemother[J], 1998,42(12): 3276~3278.
[57]Mouneimne H, Robert J, JarlierV, et al. Typeêtopo isomerasemutation in cip rofloxacin 2 resistant strains of pseudomonasaeruginosa. Antimicrob Agents Chemother, [J] 1999, 43(1):62~66.
[58]Babus S D, Bebear CM, Charron A , et al. Squencing of gyrase and topo isomerase I quino one-resistance-determ ining regions of chamydia trachomatis and characterization of quino lone-resistant mutants obtained in vitro. A ntim icrob Agents Chemother[J],1998,42(10):2474~2481.
[59]Belland R J, Mo rrison SG, Ison C, et al. Nesseria gono rrhoeaeaquiresmutations in analogous regions of gyrA and parC in fluonoquino lones-resistant iso lates, Mol Microbial[J],1994,14(2):371～380.
[60]Whillmot CJ,Maxw ellR. A sigle point mutation in theDNA gyraseA p rotein greatly reduces binding of fluo roquino lones to the gyrase2DNA complex.A ntimicrob Agents Chemother[J], 1993,37(1):126～127.
[61]Adams D E, Shekhtmann E M , Zechiedrich EL,et al. The role of topoisomerase I in partitioning bacterial rep licons and the structure of catenated intermediates in DNA rep lication. Cell[J],1992,71:277～288.
[62]Kato J, Nishimura Y, Imamura R, et al. New topoisomerase essential for chromo some segregation in Escheroch ia coli. Cell[J], 1990,63:393～404.
[63]M unozBellido J L , M anzanaresM A A , Guirao G Y, et al. In v itroactivities of 13 fluo roquino lones against staphylococcus aureus isolates w ith characterized mutations in gyrA ,gyrB ,grlA ,and norA and against wildtype isolates.Antimicrob A gents Chemother[J],1999,43(5):966～968.
[64]Tanaka M , O nodera Y, U chda Y, et al. Inh ibito ry activities of quino lones against DNA gyrase and topo isomerase I purified from staphylococcus aureus. Antim icrob Agents Chemother[J],1997,41(11):2362～2366.
[65]Li Zhiyu, Deguch i T, YasudaM , etal. Alteration in the GyrA sub-unit of DNAgyrase and the ParC subunit of DNA topo isomerase Iinquinolone2resistant clinical isolates of staphylococcus ep iderm idis.Antimicrob Agents Chemother[J], 1998,42(9) :3293～3295.
[66]TanakaM , Onodera Y, Uchida Y, etal. Quino lone resistance mutations in GrlB p rotein of staphylococcus aureus.Antimicrob Agents Chemother[J],1998,42(11):3 044～3046.
[67]Ferrero L , Cameron B, M anseB, et al. Cloning and p rimary structure of staphylococcus aureus DNA topo isomerase I : a p rimary target of fluo roquino lones. MolMicrobiol[J],1994,13:641～653.
[68]Kaatz GW , Seo SM , Ruble C A. M echanism of fluo roquino lone resistance s t a p h y l o c o c c u s a u r e u s . I n f e c t [ J ] , 1 9 9 0 , 1 6 3 : 1 0 8 0～1 0 8 6 .
[69]Kaatz GW , Seo SM,Ruble CA. Effluxmediated fluo roquinolone resistance inStaphy lococcus au reus . Antim icrob Agents Chemother[J],,1993,37(9):1086～1094.
[70]Ito H,Yoshida H, Bogak i2ShonaiM , e al. Quino lone resistance mutations in the DNA gyrase gyrA and gyrB genes of staphylococcus aureus.Antimicrob Agents Chemother[J],,1994,38(9):2014～2023.
[71]Eva YWNG, TruchsisM ,HooperD. Quinolone resistancemediated by norA : physio logic characterization and relationsh ip to flqB, aquino lone resistance locus on the staphylococcus aureus ch romo some[.Antimicrob Agents Chemother[J],,1994, 38 (6) : 1345～1355.
[72]Yo sh ida H, Bogak iM ,N akamura S, et al. N ucleo tide sequence and characterization of the S tap hy lococcus au reus norA gene,w h ich confers resistance to quino lones.Bacteriol[J],,1990,172(9):6942～6959
[73]DudleyW A , M andler H D, Gilbert D, et al. Pharmacok inetics and pharmacodynam ics of intravenous cip rofloxacin. Am J Med[J],,1987, 82 ( supp l. 4A ) :363～368.
[74]Ferrero L , Cameroon B, Crouzet J. A nalysis of gy rA and g rlA mutations in stepw ise-selected cip rofloxacin2resistant mutants of staphylococcus aureus[.Antim icrobA gents Chemother[J],, 1995,39(7) :1554～1558.
[75]Chou J H, Greenberg J T,Demp le B. Posttranscrip tional rep ression of Escherichia coli OmpF protein in response to redox stress: po sitive contro l of the m icF antisense RNA by the soxRS locus.Bacteriol[J],,1993,175:1026～1031.
[76]Cohen S P, Hachler H, L evy S B. Genetic and functional analysis of the multip le antibio tic resistance locus in Escherich ia coli . Bacteriol[J],,1993,175: 1484～1492.
[77]Kohler T, MicheaHamzehpourM , P lestat P, etal. D ifferiential selection of multidrug efflux system s by quino lones in p seudomonasaerugino sa. A ntim icrob Agents Chemother[J],,1997,41(11):2540～2543.
[78]Lomovskaya O, L ew is K, M atin A. Em rR is a negative regulato r of the Escherich ia coli multidrug resistance pump Em rAB.Bacteriol[J],1995,177:2328～2334.
[79]V aerie Z, Claire J ,M arrie2Dom inique K, et al. A ctive efflux as a mechanism of resistance to cip rofloxacin in S trep tococcus p neum oniae. A ntimicrob Agents Chemother[J],1997,41 (9) :1973～1978.
[80]B renw ald N P, GillM J , W ise R. P revalence of a putative mechanism fluo roquino lone-resistant clinical iso lates of S trep tococcus p neum oniae.AntimicrobA gents Chemother[J],1999, 42 (8) : 2032～2035.
[81]GillM J ,B renw aldN P, W ise R. Identification of an efflux gene, pmrA , associated w ith fluo roquino lone resistance in S trep tococcus pneum oniae . Antim icrob Agents Chemother[J],, 1999, 43 (1) :187～189.
[82]N eyfakh A A. The multidrug efflux transpo rter of bacillus subtilis isa structural and functional homo log of the S tap hy lococcusNo rA p rotein.Antimicrob Agents Chemother[J],,1992,36(2) :484～485.
[83]Salyers AA , Amabile2Cuevas C F. W hy are antibio tic resistance genes so resistant to elim ination.AntimicrobAgents Chemother[J],1997,41 (11) :2321～2325.
[84]Courvalin P. Transfer of antibiotic resistance genes between grampositive and gramnegtive bacteria. Antim Icrob Agents Chemother[J],1994, 8(6):1447～1451.
[85]Kaatz GW , Seo SM. InducibleNorA mediatedmultidrug resistance in S taphy lococcus au reus . Antimicrob Agents Chemother[J],1995,39(12):2650～2655
[86]Paton, A.W.,J.C.Paton,P.N.Goldwater,andP.A.Manning.1993.Direct detection of Escherichia coli Shiga-like toxin genes in primary fecal cultures by polymerase chainreaction. Clin. Microbiol[J],.1994,31:3063～3067.
[87]Gannon, V. P., R. K. King, J. Y. Kim, and E. J. Thomas. Rapid and sensitive method for detection of Shiga-like toxin-producing Escherichia coli in ground beef using the polymerase chain reaction. Environ. Microbiol[J],1992,58(4):3809～3815
[88]Paton, A.W., and J.C. Paton.. Detection and characterization of Shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, enterohemorrhagic E. coli hlyA, rfbO111, and rfbO157.Clin. Microbiol[J],.1998 ,36:598～602
[89]Pollard D R, Johnson W M, Lior H, et al . Rapid and specific detection of verotoxin genes in Escherichia coli by the polymerase chain reacton.Clin Microbiol[J],1990,28:540～545
[90]Cannon V P J, D’souza S, Graham T, et al . Use of the flagellar H7 gene as a target in multiplex PCR assays and improved specificity in identification of enterohenmorrhagic Escherichia coli strain Clin Microbiol[J],, 1997,35:656～662
[91]周志江黄上媛郑明光等用PCR鉴定大肠杆菌O157:H7中国兽医学报[J] 1999,19(3):248～250
[92]Fratamico, P. M., S. K. Sackitey, M. Weidmann, and M. Yi Deng. Detection of Escherichia coli O157:H7 by multiplex PCR.Clin. Microbiol[J],. 1995,33: 2188～2191
[93]Nurmohammad Shaikh and Phillip I. Tarr Escherichia coli O157:H7 Shiga Toxin-Encoding Bacteriophages: Integrations, Excisions, Truncations, and Evolutionary Implications Journal of Bacteriology[J], 2003, 185: 12 3596～3605
[94]韩文瑜冯书章,现代分子病原细菌学[M] 2003吉林人民出版社221～238
[95]张小林、李家泰.主动外排系统acrAB在临床分离大肠杆菌中的分布和表达.中国临床药理学杂志[J],1999,15(3):171~174
[96]陈高明、王白凤、李春海.复合定量PCR检测谷光甘肽-S转移酶和多药耐药相关基因mRNA表达水平方法的建立和初步应用.临床检验杂志[J],.1999,17(4):197-199
[97]曹丽萍、王斌、郁知非.MRP基因定量RT-PCR内标准DNA模板和RNA模板的构建,中华血液学杂志[J],1998,15(5):56-59
[98]MazzariolA.,TokueY.,andKanegawaTM.,etal.High-level fluoroquinolone-resistantclinical isolates of Escherichia coli overproduce mulidrug efflux protein AcrA. Antimicob Agents Chemother[J],.2000,44(12):3441~3443
[99]Oethinger M,Kern WV,Jellen-Ritter AS.Ineffectiveeness of topoisomerase mutations in mediating clinically significant fluorlquinolone resistance innt fluoroquinolone resistance in of the AcrAB efflux pump. Antimicob Agents Chemother[J],.2000,44(1):10~13
[100]戴自英.临床抗菌药物学[M].北京:人民卫生出版社,1985.
[101]雷军,王浴生.国产氟喹诺酮药物体外耐药性研究[J].中国抗生素杂志[J],1993,(3):224-228.
[102] Michea HamzehpourM , Pechere, J C. Invitro stepw ise selectionofresistance to quinolones, Blactams and amikacin innosocomial Gramnegetive bacilli. Infection[J],1994,2(suppl):105.
[103]Swanberg S,W ang J C. Clonging and sequencing of the E.coli gyrA gene coding for theA subunit of DNA gyraseJMolecu Biol[J],1987,197:729-736.
[104]Junich i K, Yuk inobu N , Ryu I, et al. N ew topo isomerase essential for chromosome segregation in E.coli .Cell, [J],1990,63(2) :393-404.
[105]Cambau E, Gutmann L. M echanism s of Resistance to Quinolone Drugs[J],,1999, 45(Supp3):15-23.
[106]LauralV P. M echanism s of fluoroquinolone resistance:An Update 1994-1998. Drugs[J], 1999,58 (Supp l 2) : 11-18.
[107]Christopher J ,W illmott R,AnthonyM. A single point mutation in the DNA GyraseA protein greatly reduces binding of fluoroquinolones to the Gyrase DNA complex. Antimicrob Agents Chemotherapy[J],1993,37(1):126-127.
[108]JordiV,Joaquim R, Francesc M , etal. Association between double mutation in gyrA gene of ciprofloxacin resistance clinical isolates of Escherich ia coli . A ntimicrob A gents chemotherapy[J],1994,38(10):2477-2479.
[109]Peter H. Genetic evidence for a role of parC mutation in development of high-level fluoroquinolone resistance in E. coli .Antimicrob Agents Chemotherapy[J],1996,40(4):879-885.
[110]Martin J E, Yu FJ , Vito R, etal. Contributions of individual mechanisms to fluo roquinolone resistance in E scherich ia coli strains isolated from humans andanimals.AntimicrobAgents Chemotherapy[J],1996,40(10):2380-2386.
[111]马红霞、邓旭明等.大肠杆菌多重耐药基因AcrA、AcrB内标准DNA的构建.吉林农业大学学报[J],,2002,24(6):68-71.
[2].恽时锋,潘震寰,郑明球,鸡大肠杆菌病研究进展.畜牧与兽医[J].1998,30(6):227-230
[3].Yoshida H,Bogaki M,Nakamura M,etal..Quinolone resistance-determining region in the DNA gyrase gyrA gene of E.coli.Antimicrobial Agents and Chemotherpy[J]. 1990,10,1271-1272
[4].Claude Soussy,John Wolfson,Eva YNG.Limitations of plasmid complementation tese for detetmination of quinolone resistance due to changes in the gurase A protein and identification of conditional quinolone resistance locus. Antimicrobial Agents and Chemotherpy[J].1993,37(12):2588-2592
[5].Simone b,Volker H,Bernd W etal, Impact lf gyrA and parCon Quinolone resistance,doubling time,and supercoiling degree of Escherichia coli . Antimicrobial Agents and Chemotherpy[J]. 1999,8,868-875
[6].Cambau E,Bordon F,Collatz E, etal. Novel gyrApoint mutation in strain lf E.coli resistant to fluoroquinolones but not to nalidixic acid, Antimicrobial Agents and Chemotherpy[J]. 1993,7,1247-1256
[7].Piddock LJ.Mechanisms lf fluroquinolone resistance:an update 1994–1998.Drugs[J],1999,58(2):11-18
[8].Hallet P,Maxwell A..Novel quinolone resistance mutation of the E.coli DNA gyrase A protein:enzymatic analysis of the mutant protein. Antimicrobial Agents and Chemotherpy[J]. 1991,6,335-340
[9].Hiroaki Yohida.Quinolone Resistance-determining region in the DNA Gyrase gyrB gene of Escherichia coli . Antimicrobial Agents and Chemotherpy. [J] 1991,35(8):1647–1650
[10].Heisig P,.Genetic evidence for a role of parC in development of high-level fluoquinolone resistance in E.coli . Antimicrobial Agents and Chemotherpy[J]. 1996,3,879-885
[11].Vila H,Ruiz J,Goni P,etal.Detection of mutation in parC in quinolone resistant clinical isolates of E.coli. Antimicrobial Agents and Chemotherpy[J],1995,8,491-493
[12].David G White,Charlene Hudson,John J.Maurer etal.Characterization of chloramphenicol and florfenicol resistance in associated with Bovine diarrhea.Journal of Microbioligy[J],2000,38(12):4593-4598
[13].Gayatri Vedantam,Gordon G.Guay,Natasha E.Austria,etal.Nixhols Characrerization of mutations contributing to sulfthiazole resistance in Escherichia coli. Antimicrob-Agents-Chemother[J].1998,42(1):88-93
[14].Whiteway J,Koziarz,P,Veall,J,Sandhu N,Kumar P, etal.Oxygen-insensitive nitroreductases: analysis of the roles of nfsA and nfsB in development of resistance to nitrofuran derivatives in Escherichia coli.Bacteriol. [J].1998,180(21):5529-5539
[15].Bakai S,Fonshtein M Kharakter povrezhdenii DNK iikh likvidatsii prideistvii dioksidina nabakterii. [The nature of DNA damage and its repair after treatment of bacteria with dioxidine] Mol-Gen-Mikrobiol-Virusol[J]. 1987,(4): 35-39
[16].Douthwaite S,Hansen LH,Mauvais P Macrolide-ketolide inhibition of MLS–resistant ribosomes is improved by alternative drug interaction with domain II of 23SrRNA. Mol-Microbiol[J].. 2000,36(1): 183-193
[17]Jovanovic M,Lilic M,Janjusevic;etal, tRNA synthetase mutants of Escherichia coli K-12 are resistant to the gyrase inhibitor novobiocin.Bacteriol.[J]. 1999,181(9): 2979-2983
[18].Rakonjac J,Milic M,SavicDJ,cysB and cysE mutants of Escherichia coli K12 show increased resistance to novobiocin. Mol-Gen-Genet.[J].1991,228(1-2): 307-11
[19].George A.Jacoby,Antone A.Medeiros.More extended-spectrumβ-Lactamases. Antimicrob Agents Chemother[J].. 1991,35(9): 1697-1704
[20].Marie P Mingeot Ll,Youri G,etal.Minireview Aminoglycosides:activity and resistance. Antimicrob Agents Chemother[J].. 1999 ,43(4):727-737
[21].Hadfield C, CashmoreA,Meacock P, Sequence and expression characteristics of a shuttle chloramphenicol-resistance marker for Saccharomyces cerevisiae and Escherichia coli. Gene. [J].1987,52(1): 59-70
[22].Morozov G,Nosova L,Biketov F etal, Biokhimicheskie osnovy effekta sovmeshcheniia genov ustoichivostik kanamitsinui nitrofuranam vkletkakhEscherichia coli. [Biochemical bases for the effect of combining kanamycin and nitrofuran resistance genes in Escherichia coli cells] Mol-Gen-Mikrobiol-Virusol[J]1994,(2):11-14
[23].Noguchi N,Emura A,Matsuyama H, Nucleotide sequence and characterization of erythromycin resistance determinant that encodes macrolide 2'-phosphotransferase I in Escherichia coli. Antimicrob Agents Chemother[J]. 1995 ,39(10): 2359-2363
[24].TI Kono M,Ohara K, Ebisu T,Purification and characterization of macrolide 2'-phosphotransferase type II from a strain of Escherichia coli highly resistant to macrolide antibiotics. FEMS Microbiol Lett[J].1992 ,76(1-2): 9389-9460.
[25].Rachek L,Hines A,Tucker A,etal, Transformation of Rickettsia prowazekii to erythromycin re sistance encoded by the Escherichia coli ereB gene. Bacteriol[J] 2000,182(11): 3289-3293
[26].Seltmann G,Resistance of Escherichia coli to nourseothricin: sensitization of resistant strains by abolition of its outer membrane resistance. Zentralbl Bakteriol[J].1992,276(2): 143-151
[27].Matsuyama S,.Mizushima,Construction and characterization of a deletion mutant lacking micF ,aproposed regulatory gene fur OmpF synthesis in Escherichia col . Bacteriol[J]. 1985,162(3):1196-1202
[28].Cohen S,.McMurry,.Levy,marA locus causes decreased expression of OmpF porin in multiple-antobiotic-resistant mutants of Escherichia coli.J.Bacteriol[J].1988 , 170(12):5416-5422
[29].Hirai,K,Aoyama,Irikura,etal.Mitsuhashi.Differences in susceptibility to quinolones of outer membrane mutants of Salmonella typhimurium and Escherichia coli. Antimicrob Agents Chemother[J]. 1986,29(2):703-705
[30].George A,Jacoby,Lorraine Sutton.β-Lactamases andβ-Lactam resistance in Escherichia coli. Antimicrob Agents Chemother[J]. 1985,28(5):703-705
[31].Bissonnette L,Champetier S,Buisson J,etal, Characterization of the nonenzymatic chloramphenicol resistance gene of the In4 integron of Tn1696: similarity of the product to transmembrane transport proteins.Bacteriol[J].1991,173(14): 4493-4502
[32].Rahaman S,Mukherjee J,ChakrabartiA, Decreased membrane permeability in a polymyxin B-resistant Escherichia coli mutant exhibiting multiple resistance to beta-lactams as well as aminoglycosides.Microbiol[J]. 1998,161(2): 249-254
[33].Nummila K,Kilpelainen I,Zahringer,etal, Lipopolysaccharides of polymyxin B-resistant mutants of Escherichia coli are extensively substituted by 2-aminoethyl pyrophosphate and contain amino arabinose in lipid A. Mol-Microbiol[J]. 1995,16(2):271-278
[34].Seltmann G,Resistance of Escherichia coli to nourseothricin: reduced penetrability of the cell wall as an additional, possibly unspecific mechanism. Basic Microbiol[J].1989,29(7): 449-456
[35].Harke okusu,Dzwokai Ma, Hiroshi Nikaido.AcrAB efflux pump plays a major role in the antibiotic resistance phenotype of Escherichia coli muliple–antibiotic–resistance mutants.Journal of bacteriology[J]. 1996,178(3):306-308
[36].Ma,D,Cook,Hearst,ikaido. Efflux pumps and drug resistance in Gram-negative bacteria.Trends ,Microbiol[J]. 1994,(2):489-493
[37].Olga Lomovskaya,kim lewis,an Escherichia coli locus for multidrug resistance.Proc.Natl.Acad.Sci.[J]. 1992,(3):8938-8942
[38].Hagit Yerushalmi,Mario Libendiker,Shimon schuldiner,an Escherichia coli 12-Kda multidrug Transporter,exchanges toxic cation and H+ and is soluble in organic solvents. Journal of biological Chemistry[J]. 1995,270(5):6856-6863
[39].Bentley.Hyatt,.Ainley,etal.Cloning and sequence analysis of an Escherichia coli gene conferring bicyolomycin resistance.Gene[J]. 1993,127(6):117-120
[40].Edgar,R., E.Bibi. Mdfa,an Escherichia coli multidrug resistance protein with an extraordinarily brosd spectrum of drug recognition.Bacteriol[J]. 1997.179(6):2274-2280
[41].Nilsen,IW,Bakke,I,Vader,A, a novel Escherichiacoli chloramphenicol resistance gene encoding a putative efflux pump. Bacteriol[J]. 1996 ,178(11): 3188-3193
[42].Li,H.,Park,The periplasmic murein peptide-binding protein MppA is a negative regulator of multiple antibiotic resistance in Eschericha coli..Bacteriol[J]. 1997,181(2):4842-4847
[43].Kobayashi N,Nishino K,Yamaguchi ,A Novel macrolide-specific ABC-typeefflux transporter in Escherichia coli. Bacteriol[J]. 2001 ,183(19): 5639-4564
[44].Perreten V,Schwarz, V,Teuber M, a new efflux protein conferring multiple antibiotic resistance in Lactococcus lactis and Escherichia coli.Antimicrob Agents Chemother[J]. 2001 ,45(4):1109-1114
[45].Gregory S,Dahlberg A, Erythromycin resistance mutations in ribosomal proteins L22 and L4 perturb the higher order structure of 23 S ribosomal RNA.MolBiol. [J] 1999,289(4): 827-834
[46].ChittumH,ChampneyS,Ribosomal protein gene sequence changes in erythromycin-resistant mutants of Escherichia coli. Bacteriol[J].1994,176(20): 6192-6298
[47].Blot M,Heitman J,Arber W,Tn5-mediated bleomycin resistance in Escherichia coli requires the expression of host genes. Mol-Microbiol[J]. 1993 ,8(6): 1017-1024
[48].金少鸿,张凤凯编译,抗菌药物耐药性现状透视.国外医药药学分册[M].2000,27(2):74-79
[49]Reece R, Maxwell A,DNA gyrase,structure and function. CritRev Biochem Mol Biol[J],1991, 26(1):335~375.
[50]Wang J C,DNA topoisomerases. Annu Rev Biochem[J], 1996,65(5):635~692.
[51]Yoshida H, BogakiM, Nacamura M,etal.Quinolone-resistance-determining region inthe DNA gyrase gyrA gene of Escheroc ia coli. Antimicrob A gents Chemother[J], 1990, 34 (5) : 1271~1272.
[52]CullenME, WykeM W,Kuroda R, etal. Cloning and characterization of DNA gyraseAgene from Escherochia coli that confers clinicalresistance to 4 quino lones. Antimicrob A gents Chemother[J], 1989,33 (4):886~894.
[53]Heisig P, Schedletzky H, Mutations in the gyrA gene of a highly fluoroquino lone 2 resistant clinicalisolate of Escheroch ia coli. Antimicrob Agents Chemother[J], 1993,37(3):696～701.
[54]Oram M , Fisher L M. 42quino lone resistance mutations in the DNAgyrase ofEscherochia coli clinical iso lates identified by using thepo lymo rase chain reaction. Antimicrob Agents Chemother[J], 1991, 35(2):387~389.
[55]Weigel L M, Steward C D, Tenover F C. GyrA mutations associated with fluoroquino lone resistance in eight species of enterobacteriaceae. Antimicrob Agents Chemother[J].1998,42(10):2661~2667.
[56]GibreelA , Sjogren E, Kaijser B, et al. Rapid emergence of high-levelresistance to quino lones in campylobacter jejuni associated with mutantional changes in gyrA and parC. Antimicrob Agents Chemother[J], 1998,42(12): 3276~3278.
[57]Mouneimne H, Robert J, JarlierV, et al. Typeêtopo isomerasemutation in cip rofloxacin 2 resistant strains of pseudomonasaeruginosa. Antimicrob Agents Chemother, [J] 1999, 43(1):62~66.
[58]Babus S D, Bebear CM, Charron A , et al. Squencing of gyrase and topo isomerase I quino one-resistance-determ ining regions of chamydia trachomatis and characterization of quino lone-resistant mutants obtained in vitro. A ntim icrob Agents Chemother[J],1998,42(10):2474~2481.
[59]Belland R J, Mo rrison SG, Ison C, et al. Nesseria gono rrhoeaeaquiresmutations in analogous regions of gyrA and parC in fluonoquino lones-resistant iso lates, Mol Microbial[J],1994,14(2):371～380.
[60]Whillmot CJ,Maxw ellR. A sigle point mutation in theDNA gyraseA p rotein greatly reduces binding of fluo roquino lones to the gyrase2DNA complex.A ntimicrob Agents Chemother[J], 1993,37(1):126～127.
[61]Adams D E, Shekhtmann E M , Zechiedrich EL,et al. The role of topoisomerase I in partitioning bacterial rep licons and the structure of catenated intermediates in DNA rep lication. Cell[J],1992,71:277～288.
[62]Kato J, Nishimura Y, Imamura R, et al. New topoisomerase essential for chromo some segregation in Escheroch ia coli. Cell[J], 1990,63:393～404.
[63]M unozBellido J L , M anzanaresM A A , Guirao G Y, et al. In v itroactivities of 13 fluo roquino lones against staphylococcus aureus isolates w ith characterized mutations in gyrA ,gyrB ,grlA ,and norA and against wildtype isolates.Antimicrob A gents Chemother[J],1999,43(5):966～968.
[64]Tanaka M , O nodera Y, U chda Y, et al. Inh ibito ry activities of quino lones against DNA gyrase and topo isomerase I purified from staphylococcus aureus. Antim icrob Agents Chemother[J],1997,41(11):2362～2366.
[65]Li Zhiyu, Deguch i T, YasudaM , etal. Alteration in the GyrA sub-unit of DNAgyrase and the ParC subunit of DNA topo isomerase Iinquinolone2resistant clinical isolates of staphylococcus ep iderm idis.Antimicrob Agents Chemother[J], 1998,42(9) :3293～3295.
[66]TanakaM , Onodera Y, Uchida Y, etal. Quino lone resistance mutations in GrlB p rotein of staphylococcus aureus.Antimicrob Agents Chemother[J],1998,42(11):3 044～3046.
[67]Ferrero L , Cameron B, M anseB, et al. Cloning and p rimary structure of staphylococcus aureus DNA topo isomerase I : a p rimary target of fluo roquino lones. MolMicrobiol[J],1994,13:641～653.
[68]Kaatz GW , Seo SM , Ruble C A. M echanism of fluo roquino lone resistance s t a p h y l o c o c c u s a u r e u s . I n f e c t [ J ] , 1 9 9 0 , 1 6 3 : 1 0 8 0～1 0 8 6 .
[69]Kaatz GW , Seo SM,Ruble CA. Effluxmediated fluo roquinolone resistance inStaphy lococcus au reus . Antim icrob Agents Chemother[J],,1993,37(9):1086～1094.
[70]Ito H,Yoshida H, Bogak i2ShonaiM , e al. Quino lone resistance mutations in the DNA gyrase gyrA and gyrB genes of staphylococcus aureus.Antimicrob Agents Chemother[J],,1994,38(9):2014～2023.
[71]Eva YWNG, TruchsisM ,HooperD. Quinolone resistancemediated by norA : physio logic characterization and relationsh ip to flqB, aquino lone resistance locus on the staphylococcus aureus ch romo some[.Antimicrob Agents Chemother[J],,1994, 38 (6) : 1345～1355.
[72]Yo sh ida H, Bogak iM ,N akamura S, et al. N ucleo tide sequence and characterization of the S tap hy lococcus au reus norA gene,w h ich confers resistance to quino lones.Bacteriol[J],,1990,172(9):6942～6959
[73]DudleyW A , M andler H D, Gilbert D, et al. Pharmacok inetics and pharmacodynam ics of intravenous cip rofloxacin. Am J Med[J],,1987, 82 ( supp l. 4A ) :363～368.
[74]Ferrero L , Cameroon B, Crouzet J. A nalysis of gy rA and g rlA mutations in stepw ise-selected cip rofloxacin2resistant mutants of staphylococcus aureus[.Antim icrobA gents Chemother[J],, 1995,39(7) :1554～1558.
[75]Chou J H, Greenberg J T,Demp le B. Posttranscrip tional rep ression of Escherichia coli OmpF protein in response to redox stress: po sitive contro l of the m icF antisense RNA by the soxRS locus.Bacteriol[J],,1993,175:1026～1031.
[76]Cohen S P, Hachler H, L evy S B. Genetic and functional analysis of the multip le antibio tic resistance locus in Escherich ia coli . Bacteriol[J],,1993,175: 1484～1492.
[77]Kohler T, MicheaHamzehpourM , P lestat P, etal. D ifferiential selection of multidrug efflux system s by quino lones in p seudomonasaerugino sa. A ntim icrob Agents Chemother[J],,1997,41(11):2540～2543.
[78]Lomovskaya O, L ew is K, M atin A. Em rR is a negative regulato r of the Escherich ia coli multidrug resistance pump Em rAB.Bacteriol[J],1995,177:2328～2334.
[79]V aerie Z, Claire J ,M arrie2Dom inique K, et al. A ctive efflux as a mechanism of resistance to cip rofloxacin in S trep tococcus p neum oniae. A ntimicrob Agents Chemother[J],1997,41 (9) :1973～1978.
[80]B renw ald N P, GillM J , W ise R. P revalence of a putative mechanism fluo roquino lone-resistant clinical iso lates of S trep tococcus p neum oniae.AntimicrobA gents Chemother[J],1999, 42 (8) : 2032～2035.
[81]GillM J ,B renw aldN P, W ise R. Identification of an efflux gene, pmrA , associated w ith fluo roquino lone resistance in S trep tococcus pneum oniae . Antim icrob Agents Chemother[J],, 1999, 43 (1) :187～189.
[82]N eyfakh A A. The multidrug efflux transpo rter of bacillus subtilis isa structural and functional homo log of the S tap hy lococcusNo rA p rotein.Antimicrob Agents Chemother[J],,1992,36(2) :484～485.
[83]Salyers AA , Amabile2Cuevas C F. W hy are antibio tic resistance genes so resistant to elim ination.AntimicrobAgents Chemother[J],1997,41 (11) :2321～2325.
[84]Courvalin P. Transfer of antibiotic resistance genes between grampositive and gramnegtive bacteria. Antim Icrob Agents Chemother[J],1994, 8(6):1447～1451.
[85]Kaatz GW , Seo SM. InducibleNorA mediatedmultidrug resistance in S taphy lococcus au reus . Antimicrob Agents Chemother[J],1995,39(12):2650～2655
[86]Paton, A.W.,J.C.Paton,P.N.Goldwater,andP.A.Manning.1993.Direct detection of Escherichia coli Shiga-like toxin genes in primary fecal cultures by polymerase chainreaction. Clin. Microbiol[J],.1994,31:3063～3067.
[87]Gannon, V. P., R. K. King, J. Y. Kim, and E. J. Thomas. Rapid and sensitive method for detection of Shiga-like toxin-producing Escherichia coli in ground beef using the polymerase chain reaction. Environ. Microbiol[J],1992,58(4):3809～3815
[88]Paton, A.W., and J.C. Paton.. Detection and characterization of Shiga toxigenic Escherichia coli by using multiplex PCR assays for stx1, stx2, eaeA, enterohemorrhagic E. coli hlyA, rfbO111, and rfbO157.Clin. Microbiol[J],.1998 ,36:598～602
[89]Pollard D R, Johnson W M, Lior H, et al . Rapid and specific detection of verotoxin genes in Escherichia coli by the polymerase chain reacton.Clin Microbiol[J],1990,28:540～545
[90]Cannon V P J, D’souza S, Graham T, et al . Use of the flagellar H7 gene as a target in multiplex PCR assays and improved specificity in identification of enterohenmorrhagic Escherichia coli strain Clin Microbiol[J],, 1997,35:656～662
[91]周志江黄上媛郑明光等用PCR鉴定大肠杆菌O157:H7中国兽医学报[J] 1999,19(3):248～250
[92]Fratamico, P. M., S. K. Sackitey, M. Weidmann, and M. Yi Deng. Detection of Escherichia coli O157:H7 by multiplex PCR.Clin. Microbiol[J],. 1995,33: 2188～2191
[93]Nurmohammad Shaikh and Phillip I. Tarr Escherichia coli O157:H7 Shiga Toxin-Encoding Bacteriophages: Integrations, Excisions, Truncations, and Evolutionary Implications Journal of Bacteriology[J], 2003, 185: 12 3596～3605
[94]韩文瑜冯书章,现代分子病原细菌学[M] 2003吉林人民出版社221～238
[95]张小林、李家泰.主动外排系统acrAB在临床分离大肠杆菌中的分布和表达.中国临床药理学杂志[J],1999,15(3):171~174
[96]陈高明、王白凤、李春海.复合定量PCR检测谷光甘肽-S转移酶和多药耐药相关基因mRNA表达水平方法的建立和初步应用.临床检验杂志[J],.1999,17(4):197-199
[97]曹丽萍、王斌、郁知非.MRP基因定量RT-PCR内标准DNA模板和RNA模板的构建,中华血液学杂志[J],1998,15(5):56-59
[98]MazzariolA.,TokueY.,andKanegawaTM.,etal.High-level fluoroquinolone-resistantclinical isolates of Escherichia coli overproduce mulidrug efflux protein AcrA. Antimicob Agents Chemother[J],.2000,44(12):3441~3443
[99]Oethinger M,Kern WV,Jellen-Ritter AS.Ineffectiveeness of topoisomerase mutations in mediating clinically significant fluorlquinolone resistance innt fluoroquinolone resistance in of the AcrAB efflux pump. Antimicob Agents Chemother[J],.2000,44(1):10~13
[100]戴自英.临床抗菌药物学[M].北京:人民卫生出版社,1985.
[101]雷军,王浴生.国产氟喹诺酮药物体外耐药性研究[J].中国抗生素杂志[J],1993,(3):224-228.
[102] Michea HamzehpourM , Pechere, J C. Invitro stepw ise selectionofresistance to quinolones, Blactams and amikacin innosocomial Gramnegetive bacilli. Infection[J],1994,2(suppl):105.
[103]Swanberg S,W ang J C. Clonging and sequencing of the E.coli gyrA gene coding for theA subunit of DNA gyraseJMolecu Biol[J],1987,197:729-736.
[104]Junich i K, Yuk inobu N , Ryu I, et al. N ew topo isomerase essential for chromosome segregation in E.coli .Cell, [J],1990,63(2) :393-404.
[105]Cambau E, Gutmann L. M echanism s of Resistance to Quinolone Drugs[J],,1999, 45(Supp3):15-23.
[106]LauralV P. M echanism s of fluoroquinolone resistance:An Update 1994-1998. Drugs[J], 1999,58 (Supp l 2) : 11-18.
[107]Christopher J ,W illmott R,AnthonyM. A single point mutation in the DNA GyraseA protein greatly reduces binding of fluoroquinolones to the Gyrase DNA complex. Antimicrob Agents Chemotherapy[J],1993,37(1):126-127.
[108]JordiV,Joaquim R, Francesc M , etal. Association between double mutation in gyrA gene of ciprofloxacin resistance clinical isolates of Escherich ia coli . A ntimicrob A gents chemotherapy[J],1994,38(10):2477-2479.
[109]Peter H. Genetic evidence for a role of parC mutation in development of high-level fluoroquinolone resistance in E. coli .Antimicrob Agents Chemotherapy[J],1996,40(4):879-885.
[110]Martin J E, Yu FJ , Vito R, etal. Contributions of individual mechanisms to fluo roquinolone resistance in E scherich ia coli strains isolated from humans andanimals.AntimicrobAgents Chemotherapy[J],1996,40(10):2380-2386.
[111]马红霞、邓旭明等.大肠杆菌多重耐药基因AcrA、AcrB内标准DNA的构建.吉林农业大学学报[J],,2002,24(6):68-71.