嗜水气单胞菌不同分离株外膜蛋白图谱分析及其与耐药性的关系
|
摘要
嗜水气单胞菌(Aeromonas hydrophila,Ah)普遍存在于淡水、土壤、人畜粪便等各种环境中,可引起多种水产动物的败血症以及畜禽和人类腹泻。嗜水气单胞菌是重要的水生病原菌,给水产养殖业造成极大的经济损失,由于抗生素在渔业方面的滥用,导致嗜水气单胞菌产生耐药性,对水产养殖业的进一步发展产生影响.外膜蛋白作为革兰氏阴性菌外膜的重要结构对细菌耐药性的形成有一定的影响.
对43株嗜水气单胞菌的外膜蛋白(OMP)进行SDS-PAGE分析,根据不同菌株蛋白质电泳条带之间的差异性可以将其分成多个型：OMP I-OMPⅥ.使用统计软件SPSS13.0进行聚类分析,以期在流行病学调查方面提供进一步了解。采用微量双倍稀释法测定43株嗜水气单胞菌最小抑菌浓度。药敏试验结果参照美国NCCLS标准判定各菌株是否耐药。在所选的7种抗生素中,环丙沙星、恩诺沙星、氟苯尼考对临床分离的43株嗜水气单胞菌的作用较好,抑菌率分别达97.67%(42／43)、97.67%(42／43)和95.35%(41／43),而其它几种抗生素的抑菌效果较差,红霉素、泰乐菌素,抑菌率只有9.30%(4／43)、6.98%(3／43),所有43株菌株对氨苄西林和磺胺间甲基嘧啶则完全耐药。多数菌株具有多重耐药性,两个与多重耐药输出泵相关的基因AHA_0858和aheABC在嗜水气单胞菌各菌株中广泛分布,检出率分别为77%(33／43)和81%(35／43)。实验中发现嗜水气单胞菌的外膜蛋白电泳图谱与细菌的耐药性有一定的关系,外膜蛋白型相同的菌株在耐药性也基本一致.同其他菌株相比,菌株NB-1,A.Pun和MR-1于30kD～45kD处部分外膜蛋白条带缺失,并且其对氟苯尼考(florfenicol)耐药性也大幅增强,提示部分外膜蛋白的缺失可能与嗜水气单胞菌的耐氟苯尼考有一定的关系.同时发现部分具有完全一致OMP型的菌株在某些抗生素的耐药性上也存在差异,表明嗜水气单胞菌的耐药性有其他因素影响.
为了对嗜水气单胞菌的耐药机理进行更进一步的研究,本实验通过对嗜水气单胞菌J-1株(诺氟沙星敏感株)利用半数最小抑菌浓度的方法诱变形成稳定的诺氟沙星耐药株,出发菌株J-1对诺氟沙星的最小抑菌浓度为0.78μg／mL,诱变后形成的诺氟沙星耐药菌株NJ-1最小抑菌浓度为90μg／mL.利用比较蛋白质组学的方法对嗜水气单胞菌J-1和诺氟沙星耐药诱变株NJ-1进行二维电泳的研究,软件分析比较两者的蛋白质电泳图谱的差异蛋白点。通过双向电泳、质谱测定肽质量指纹图谱,鉴定出嗜水气单胞菌外膜蛋白中4个与嗜水气单胞菌诺氟沙星耐药性形成相关的蛋白,包括寡聚ABC转运器蛋白、玻璃酸酶蛋白、OmpAⅡ和二氢吡啶二羧酸合酶。细菌对诺氟沙星耐药性的形成可能与细菌外膜通透性改变从而减少抗生素的摄入以及产生灭活酶或钝化酶修饰抗生素有关.本研究为进一步揭示嗜水气单胞菌外膜蛋白与耐药性相关性提供了理论依据.
Aeromonas hydrophila is a ubiquitous organism of many environments such as fresh water, sewage and soil which can cause the septicemia of aquatic animals as well as the diarrhea of human. A. hydrophila is the causative agent of fatal hemorrhagic septicemia in fish. A. hydrophila gained the resistance to antibiotics since its abuse in the fishery which would have the impact to further development. As the important structure of outer membrane of gram-negative bacteria, outer membrane protein has certain correlation with the resistance of bacteria.
The SDS-PAGE profiles of outer membrane proteins (OMP) of 43 A. hydrophila strains showed diverse patterns and they could at least be divided into several types:OMP I-OMPVI. Cluster analysis of the OMP profiles was carried out by SPSS 13.0 to get further information about the epidemiologic survey of A. hydrophila. The minimum inhibitory concentrations (MIC) of 43 strains were determined by double microdilution method. The results were determined by referring to NCCLS. Ciprofloxacin, enrofloxacin and florfenicol had the better antibacterial effect to the 43 clinical isolates of A. hydrophila than the other 4 antibiotics of all 7 antibiotics selected. And the inhibitory rate of them was 97.67 %(42/43),97.67%(42/43) and 95.35%(41/43). The inhibitory rate of erythromycin and tylosin was 9.30%(4/43),6.98%(3/43). All the strains of 43 A. hydrophila are uniformly resistant to ampicillin and sulfamonomethoxine. A large number of isolates were resistant to more than one antibiotic. Positive rate of AHA_0858 and aheABC, two genes related to multiple drug-efflux pump, were 77% (33/43) and 81%(35/43) which means these two genes were widely distributed in A. hydrophila. The OMP profiles had a correlation with the resistance of A. hydrophila to a certain extent, and the strains with the similar SDS-PAGE profiles had similar resistances. Compared with other strains, the strains NB-1, A.Pun and MR-1 have lost part of their outer membrane proteins with the molecular weights between 30kD and 45kD and their resistance to florfenicol substantially increased, which suggest that the loss or reduction of OMP might have relationship with resistance to florfenicol. Some strains which showed completely identical OMP types had some differences in their resistance to antibiotics, which indicate that there might be other factors that affect the drug-resistance of A. hydrophila.
To get further knowledge about the mechanism of the resistance of A. hydrophila, the strain J-1 has been mutated by using the method of half minimum inhibit concentration with norfloxacin. The MIC of J-1 to norfloxacin was 0.78μg/mL while the resistant strain NJ-1 was 90μg/mL. A comparative proteomics study has been carried out to find out the mechanism of A. hydrophila's resistance to norfloxacin. Four proteins which have a certain correlation with the resistance to norfloxacin have been identified by the method of 2-DE and peptides mass fingerprint (PMF), including oligopeptide ABC transporter, ABC transporter permease protein,OmpAⅡand dihydrodipicolinate synthase. The change of outer membrane permeability and the generation of inactive enzyme or modifying enzyme might be the reason that bacteria have the resistance to norfloxacin. The research was aim to know more about the correlation between A. hydrophila outer membrane protein and the resistance.
对43株嗜水气单胞菌的外膜蛋白(OMP)进行SDS-PAGE分析,根据不同菌株蛋白质电泳条带之间的差异性可以将其分成多个型：OMP I-OMPⅥ.使用统计软件SPSS13.0进行聚类分析,以期在流行病学调查方面提供进一步了解。采用微量双倍稀释法测定43株嗜水气单胞菌最小抑菌浓度。药敏试验结果参照美国NCCLS标准判定各菌株是否耐药。在所选的7种抗生素中,环丙沙星、恩诺沙星、氟苯尼考对临床分离的43株嗜水气单胞菌的作用较好,抑菌率分别达97.67%(42／43)、97.67%(42／43)和95.35%(41／43),而其它几种抗生素的抑菌效果较差,红霉素、泰乐菌素,抑菌率只有9.30%(4／43)、6.98%(3／43),所有43株菌株对氨苄西林和磺胺间甲基嘧啶则完全耐药。多数菌株具有多重耐药性,两个与多重耐药输出泵相关的基因AHA_0858和aheABC在嗜水气单胞菌各菌株中广泛分布,检出率分别为77%(33／43)和81%(35／43)。实验中发现嗜水气单胞菌的外膜蛋白电泳图谱与细菌的耐药性有一定的关系,外膜蛋白型相同的菌株在耐药性也基本一致.同其他菌株相比,菌株NB-1,A.Pun和MR-1于30kD～45kD处部分外膜蛋白条带缺失,并且其对氟苯尼考(florfenicol)耐药性也大幅增强,提示部分外膜蛋白的缺失可能与嗜水气单胞菌的耐氟苯尼考有一定的关系.同时发现部分具有完全一致OMP型的菌株在某些抗生素的耐药性上也存在差异,表明嗜水气单胞菌的耐药性有其他因素影响.
为了对嗜水气单胞菌的耐药机理进行更进一步的研究,本实验通过对嗜水气单胞菌J-1株(诺氟沙星敏感株)利用半数最小抑菌浓度的方法诱变形成稳定的诺氟沙星耐药株,出发菌株J-1对诺氟沙星的最小抑菌浓度为0.78μg／mL,诱变后形成的诺氟沙星耐药菌株NJ-1最小抑菌浓度为90μg／mL.利用比较蛋白质组学的方法对嗜水气单胞菌J-1和诺氟沙星耐药诱变株NJ-1进行二维电泳的研究,软件分析比较两者的蛋白质电泳图谱的差异蛋白点。通过双向电泳、质谱测定肽质量指纹图谱,鉴定出嗜水气单胞菌外膜蛋白中4个与嗜水气单胞菌诺氟沙星耐药性形成相关的蛋白,包括寡聚ABC转运器蛋白、玻璃酸酶蛋白、OmpAⅡ和二氢吡啶二羧酸合酶。细菌对诺氟沙星耐药性的形成可能与细菌外膜通透性改变从而减少抗生素的摄入以及产生灭活酶或钝化酶修饰抗生素有关.本研究为进一步揭示嗜水气单胞菌外膜蛋白与耐药性相关性提供了理论依据.
Aeromonas hydrophila is a ubiquitous organism of many environments such as fresh water, sewage and soil which can cause the septicemia of aquatic animals as well as the diarrhea of human. A. hydrophila is the causative agent of fatal hemorrhagic septicemia in fish. A. hydrophila gained the resistance to antibiotics since its abuse in the fishery which would have the impact to further development. As the important structure of outer membrane of gram-negative bacteria, outer membrane protein has certain correlation with the resistance of bacteria.
The SDS-PAGE profiles of outer membrane proteins (OMP) of 43 A. hydrophila strains showed diverse patterns and they could at least be divided into several types:OMP I-OMPVI. Cluster analysis of the OMP profiles was carried out by SPSS 13.0 to get further information about the epidemiologic survey of A. hydrophila. The minimum inhibitory concentrations (MIC) of 43 strains were determined by double microdilution method. The results were determined by referring to NCCLS. Ciprofloxacin, enrofloxacin and florfenicol had the better antibacterial effect to the 43 clinical isolates of A. hydrophila than the other 4 antibiotics of all 7 antibiotics selected. And the inhibitory rate of them was 97.67 %(42/43),97.67%(42/43) and 95.35%(41/43). The inhibitory rate of erythromycin and tylosin was 9.30%(4/43),6.98%(3/43). All the strains of 43 A. hydrophila are uniformly resistant to ampicillin and sulfamonomethoxine. A large number of isolates were resistant to more than one antibiotic. Positive rate of AHA_0858 and aheABC, two genes related to multiple drug-efflux pump, were 77% (33/43) and 81%(35/43) which means these two genes were widely distributed in A. hydrophila. The OMP profiles had a correlation with the resistance of A. hydrophila to a certain extent, and the strains with the similar SDS-PAGE profiles had similar resistances. Compared with other strains, the strains NB-1, A.Pun and MR-1 have lost part of their outer membrane proteins with the molecular weights between 30kD and 45kD and their resistance to florfenicol substantially increased, which suggest that the loss or reduction of OMP might have relationship with resistance to florfenicol. Some strains which showed completely identical OMP types had some differences in their resistance to antibiotics, which indicate that there might be other factors that affect the drug-resistance of A. hydrophila.
To get further knowledge about the mechanism of the resistance of A. hydrophila, the strain J-1 has been mutated by using the method of half minimum inhibit concentration with norfloxacin. The MIC of J-1 to norfloxacin was 0.78μg/mL while the resistant strain NJ-1 was 90μg/mL. A comparative proteomics study has been carried out to find out the mechanism of A. hydrophila's resistance to norfloxacin. Four proteins which have a certain correlation with the resistance to norfloxacin have been identified by the method of 2-DE and peptides mass fingerprint (PMF), including oligopeptide ABC transporter, ABC transporter permease protein,OmpAⅡand dihydrodipicolinate synthase. The change of outer membrane permeability and the generation of inactive enzyme or modifying enzyme might be the reason that bacteria have the resistance to norfloxacin. The research was aim to know more about the correlation between A. hydrophila outer membrane protein and the resistance.
引文
[1]曹志传.抗生素耐药性机理及对策[J].医药产业资讯,2005,(223)：43-44.
[2]闻玉梅.现代微生物学[M].第1版,上海医科大学出版社,1999.6.193-208.
[3]糜租煌.细菌耐药的分子机制[J].临床儿科杂志,2005,23(7)：422-424.
[4]鲁景艳,王兴龙,雷连成.革兰氏阳性菌对β-内酰胺类抗生素耐药机制及基因同源性的分析[J].中国兽药杂志,2004,38(2)：34-37.
[5]蒲霞,曾繁荣,罗勇.细菌的耐药性与老年期感染的抗菌治疗[J].中国药业,2004,13(11)：23-25.
[6]谭艳,方治平.抗菌药物的作用机制及细菌耐药性机制的研究进展[J].国外医药：抗生素分册,2003,24(2)：65-69.
[7]蔡培泉,王春新,糜租煌.铜绿假单胞杆菌氨基糖苷类耐药性及其修饰酶基因的研究[J].中华微生物学和免疫学杂志,2006,26(4)：374.
[8]Farrow KA, LyrasD, Rood J I.2000. The macrolide-lincosamide-streptogramin B resistance determinant from Clostridium difficile 630 contains two erm(B) genes [J]. Antimicrobial Agents and Chemotherapy,44 (2):411-413,
[9]戚好文.喹诺酮类抗菌药的研究进展[J].中国抗生素杂志,1999,24(增刊)：41.
[10]张致平.喹诺酮类抗菌药研究的新进展[J].国外医药抗生素分册,2001,22(6)：241.
[11]Sanders CC, Bradford PA, Ehrhardt AF,et al. Penicillin-binding proteins and induction of AmpC beta-lactamase [J].Antimicrob Agents Chemother,1997,41(9):2-13
[12]Gimemo C, Navarro D, Savall F et al. Relationship between outer membrane protein profiles and resistance to ceftazidime, imipenem, and ciprofloxacin in Pseudomonas aeruginosa isolates from bacteremic patients [J]. Eur J Clin Microbio Infect Dis,1996,15 (1):82
[13]Nikaido H. Porins and specific diffusion channels in bacterial outer membrane [J]. J Bio Chem,1994; 269 (6):3905
[14]Fukuoka T, Ohya S, Narita T et al. Activity of the carbapenem panipenem and role of the OprD (D2) protein in its diffusion through the Pseudomonas aeruginosa outer membrane. Antimicrob Agents Chemother,1993; 37 (2):322
[15]Yamano Y, Nishikawa T, Komatsu Y. Outer membrane proteins responsible for the penetrat ion of β-lactams and quinolones in Pseudomonas aeruginosa [J]. J Antimicrob Chemother, 1990; 26 (2):175
[16]Yoneyama H, Nakae T. Mechanism of efficient elimination of protein D2 in outer membrane of imipenem-resistant Pseudomonas aeruginosa [J]. Antimicrob Agents Chemother,1993; 37 (11):2385,
[17]Tavio M, Vila J, Ruiz J, et al. Mechanisms involved in the development of resistance to fluoroquinolones in Escherichia coli isolates [J]. J Antimicrob Chemother,1999,44 (6):735-742.
[18]Pratt L A, Hsing K, Gibson E, et al. From acids to osmZ:multiple factors influence synthesis of the OmpF and OmpC porins in Escherichia coli [J]. Mol Microbiol,1996,20 (5):911-917.
[19]Levy SB. Active efflux mechanisms for antimicrobial resistance. Antimicrobial Agents and Chemotherapy,1992,36 (4):695-703
[20]Nikaido H. Multidrug efflux pumps of gram-negative bacteria [J]. J Bacteriol,1996, 178:5853-5859.
[21]Keith P. Multidrug resistance in gram-negative bacteria [J]. CurrOpin Microbiol,2004,4:500-508.
[22]Bush JA, Li G. Cancer chemo resistance:the relationship between P53 and Multidrug resistance. Int J Cancer,2002,98 (3):323-330.
[23]Mathee K, Ciofu O, Sernberg C, et al. Mucoid conversion of Pseudomonas aeruginosa by hydrogen peroxide:a mechanism for virulence activation in the cystic fibrosis lung. [J].Microbiology,1999,145 (6):13-49.
[24]Drenkard E, Ausubel F M. Pseudomonas biofilm formation and antibiotic resistance are linked to phenotypic variation [J]. Nature,2002,416:740.
[25]Delissalde F, Amabile O, Cuevas C F. Comparison of antibiotic susceptibility and plasmid content, between biofilm producing and non Oproducing clinical isolates of Pseudomonas aeruginosa [J]. Int J Antimicrob Agents,2004,24:405.
[26]Raymond D P, Pelletier S J, Crabtree T D, et al. Impact of rotating empiric antibiotic schedule on infectious mortality in an intensive care unit [J]. Crit Care Med,2001,29 (2): 1101-1108.
[27]David J.C. Knowles. New strategies for antibacterial drug design [J] Trends in Microbiology 1997,5 (10):379-383.
[28]Guerner-Takada C, Salavati R, Altman S. Phenotypic conversion of drug resistant bacteria to drug sensitivity [J]. Proc Natl Acad Sci USA,1997,94(16):8468-8472.
[29]Barriere J C, Bouanchand D H, Paris J M, et al. Antimicrobial activity against Staphylococciaureus of semisynthetic in jectabale strep-tograwins RP59500 and related compounds [J]. J Antimicrob Chemother,1992,30:1
[1]胡志远,贺福初.蛋白质组研究进展[J].生物化学与生物物理进展,1999,26(3)：202-05.
[2]Wasinger V C, Cordwell S J, Cerpa Poljak A, et al. Progress withgene-product mapping of the Mollicutes:Mycoplasma genitalium [J]. Electrophoresis,1995,16:1090-1094.
[3]Zhou G, Li H M, De Camp D, et al.2D differential in gel electrophoresis for the identification esophageal scans cancer specific protein markers [J]. Mol Cell Proteomics,2002, (1):117-124.
[4]赵旭东.蛋白质组学研究[J].国外医学：分子生物学分册,2002,24(2)：90-93.
[5]Reynolds T. For proteomics research, a new race has begun [J]. J Natl Cancer Inst,2002,94 (8):552-554.
[6]Banks R E, Dunn M J, Hochstrasser D F, et al. Proteomics:new perspectives, new biomedical opportunities [J]. Lancet,2000,356:1749-1756.
[7]O'Farrell PH. High resolution two-dimensional electrophoresis of proteins. The Journal of biological chemistry,1975 May,25; 250(10):4007-4021.
[8]Manabe Takashi. Combination of electrophoretic techniques for comprehensive analysis of complex protein systems [J]. Electrophoresis,2000,21 (6):1116-1122.
[9]郭尧君.蛋白质电泳实验技术[M].北京：科学出版社,1996.161.
[10]Hanash S. Disease proteomics [J]. Nature,2003,422:226-232.
[11]王岚,刘骁勇,张华宁等.生物质谱技术在蛋白质组学研究中的应用[J].生物技术通讯,2007,18(1)：166-168.
[12]Mann M, Hendrickson R C, Pandey A. Analysis of proteins and proteomes by mass spectrometry [J]. Annu Rev Biochem,2001,70:437-473.
[13]曾嵘,夏其昌.蛋白质组学研究进展与趋势[J].中国科学院院刊,2002,17(3)：166-169.
[14]Ono M, Shitashige M, Honda K, et al. Label-free quantitative proteomics using large peptide data sets generated by nanoflow liquid chromatography and mass spectrometry [J]. Mol Cell Proteomics,2006,5(7):1338-1347.
[15]Jennifer S D, Matt hew E, Qian W J, et al. Advances in proteomics data analysis and display using an accurate mass and time tag approach [J]. Mass Spectrum Rev,2006,25 (3):450-482.
[16]Yates Ⅲ John R. Mass spectrometry and the age of the proteome[J]. Journal of Mass Spectrometry,1998,33(1):1-19.
[17]Godovac-Zimmermann Jasminka, Brown Larry R. Perspectives for mass spectrometry and functional proteomics [J]. Mass Spectrometry Reviews,2001,20(1):1-57.
[18]Mann Matthias, Hendrickson Ronald C, Pandey Akhilesh. Analysis of proteins and proteomes by mass spectrometry[J]. Annual Review of Biochemistry,2001,70:437-473.
[19]Kicman A T, Parkin M C, Iles R K. An introduction to mass spectrometry based proteomics detection and characterization of gonadotropinsand related molecules [J]. Mol Cell Endo crinol,2007 (2):212-227.
[20]韩俊,盛龙生,相秉仁等.基质辅助激光解吸离子化质谱法及其在生物大分子分析中的应用[J].药物分析杂志,1997,18(2)：125-132。
[21]Zhou G, Li H M, DeCamp D, et al.2D-differential in-gel electrophoresis for the identification esophageal scans cancer specific protein markers [J]. Mol Cell Proteomics,2002,1: 117-124
[22]Lee K, Kye M,Jang J S,et al. Proteomic analysis revealed a strong association of a high level of alpha 1 anti trypsin in gastric juice with gastric cancer [J]. Proteomics,2004,4 (11): 3343-3352.
[23]Ahram M,Best C J, Flaig M J, et al. Proteomic analysis of human prostate cancer [J]. Mol Carcinog,2002,33 (1):9-15.
[24]PR Jungblut, UE Schaible and HJ Mollenkopf. Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains:towards functional genomics of microbial pathogens [J].Mol Microbiol,1999,33 (6):11-31.
[25]Xu C, Lin X, Ren H, Zhang Y, Wang S, Peng X. Analysis of outer membrane proteome of Escherichia coli related to resistance to ampicillin and tetracycline [J]. Proteomics.2006 Jan; 6(2):462-473.
[26]陈川,王三英,彭宣宪.嗜水气单胞菌耐四环素的蛋白质组学初步研究[J].微生物学报,2004,44(3)：396-398.
[27]Diaz J I, Cazares L H, O J Semmes. Tissue sample collection for proteomics analysis [J]. Methods Mol Biol,2008,428 (1):43
[28]Orvisky E, Drake S K, Martin B M, et al. Enrichment of low molecular weight fraction of serum for MS analysis of peptides associated with hepatocellular carcinoma. Proteomics, 2006,6(9):2895
[29]Hood B L, Lucas D A, Kim G, et al. Quantitative analysis of the low molecular weight serum proteome using 180 stable isotope labeling in a lung tumor xenograft mouse model. J Am Soc Mass Spectrum,2005,16 (8):1221
[30]Chromy B A, Gonzales A D, Perkins J, et al. Proteomic analysis of human serum by two-dimensional differential gel electrophoresis after depletion of high-abundant proteins [J]. J Proteome Res,2004,3 (6):1120
[31]Boguski, M. S., McIntosh, M. W. Biomedical informatics for proteomics [J]. Nature 2003, 422 (6928):233-237.
[1]Nielsen ME, Hoi L. Schmidt AS, et al. Is Aeromonas hydrophila the dominant motile Aeromonas species that causes disease outbreaks in aquaculture production in the Zhejiang Province of China [J], Diseases of Aquatic Organisms.2001,46 (1):23-29.
[2]赵香汝,杨汉春.细菌外膜蛋白的研究现状[J].中国兽医杂志,1997,23(12)：41-42.
[3]孙建和,严亚贤,陈怀青等.温度对嗜水气单胞菌外膜蛋白表达的影响[J].中国兽医学报,1999,19(6)：555～558.
[4]Lee SY, Yin Z, Ge R, Sin YM. Isolation and characterization of fish Aeromonas hydrophila adhesins important for in vitro epithelial cell invasion [J], J Fish Dis.1997,20(1):69-75.
[5]Buchley JT, Howard SP. The Cytotoxic enterotoxin of Aeromonas hydrophila is aerolysin [J], Infect Immun,1999,67 (1):466-467.
[6]Aronoff SC. Outer membrane permeability in Pseudomonas cepacia:diminished porin content in a beta-lactam-resistant mutant and in resistant cystic fibrosis isolates [J], Antimicrob Agents Chemother.1988.32:1636-1639.
[7]Filip C, Fletcher G, Wulff JL, et al. Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate [J], J Bacteriol.1973,115(3):717-722.
[8]Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning:a Laboratory Manual Second Edition New York[M], Cold Spring Harbor Laboratory Press, U.S.1987:880-885
[9]Performance Standards for Antimicrobial Susceptibility Testing; Seventeenth Informational Supplement [S].CLSI documents M100-S17 Vol.27(1):1-68
[10]Dawson C E, Stubberfield E J, Perrett L L, et al. Phenotypic and molecular characterisation of Brucella isolates from marine mammals [J]. BMC Microbiol.2008; 17(8):224.
[11]Dassouli-Mrani-Belkebir A, Contrepois M, Girardeau J P, et al. Characters of Escherichia coli 078 isolated from septicaemic animals [J]. Vet Microbiol.1988; 17(4):345-56.
[12]Nikaido H. Molecular Basis of Bacterial Outer Membrane Permeability Revisited [J], Microbiol Mol Biol Rev.2003,67(4):593-656.
[13]Nikaido H. Porins and specific channels of the bacterial outer membrane [J], Mol Microbiol. 1992,6:435-442.
[14]Martinez-Martinez L, Hemandez-Alles S, Alberti S, Tomas JM, Benedi VJ, Jacoby GA. In vivo selection of porin-deficient mutants of Klebsiella pneumoniae with increased resistance to cefoxitin and expanded-spectrum-cephalosporins [J], Antimicrob Agents Chemother.1996, 40(2):342-348.
[15]Mallea M, Chevalier J, Bornet C, Eyraud A, Davin-Regli A, Bollet C, Pages JM. Porin alteration and active efflux:two in vivo drug resistance strategies used by Enterobacter
aerogenes [J], Microbiology.1998,144:3003-3009.
[16]Coulton JW, Mason P, Dorrance P. The permeability barrier of Haemophilus influenzae type b against beta-lactam antibiotics [J], J Antimicrob Chemother.1983,12(5):435-449.
[17]Pangon, B., C. Toro, C. S., S. R. Lobos, I. Calderon, M. Rodriguez, and G. C. Mora. Clinical isolate of a porin-less Salmonella typhi resistant to high levels of chloramphenicol [J]. Antimicrob Agents Chemother.1990.34:1716-1719.
[18]Seshadri R, Joseph S W, Chopra A K, et al. Genome sequence of Aeromonas hydrophila ATCC 7966T:jack of all trades[J], J Bacteriol.2006;188(23):8272-82.
[19]Morita Y, Kodama K, Shiota S, et al. NorM, a putative multidrug efflux protein, of Vibrio parahaemolyticus and its homolog in Escherichia coli [J]. Antimicrob Agents Chemother. 1998;42(7):1778-82.
[1]董传甫,林天龙.嗜水气单胞菌研究进展综述[J].福建农业学报,2003,18(4)：243-248.
[2]陆承平.兽医微生物[M],中国农业出版社,第四版,2007：31-32
[3]陈怀青,陆承平.家养鲤科鱼爆发性传染病病原研究[J],南京农业大学学报,1991,14(4)：81-91.
[4]Colwell R R, Macdonell MT, Deley J. Proposal to recognize the family Aeromonadaceae fam [J]. International Journal of Systemic Bacteriology,1986,34:473-477.
[5]郑国兴,周凯.嗜水气单胞菌欧洲鳗皮肤溃疡分离株的耐药性[J].中国水产科学,1999,6(3)：69.
[6]陆承平.致病性嗜水气单胞菌综述及其所致鱼病[J].水产学报,1992,16(3)：282-288
[7]赵香汝,杨汉春.细菌外膜蛋白的研究现状[J].中国兽医杂志,1997,23(12)：41-42.
[8]Tavio MM, Vila J, Ruiz J, et al. Mechanisms involved in the development of resistance to fluoroquinolones in Escherichia coli isolates [J]. J Antimicrob Chemother,1999,44 (6):735-742.
[9]苏林光,贾杰,潘光华.次抑菌浓度的药物诱导细菌耐药与交叉耐药[J].中国抗生素杂志,1997,22(4)：301-303.
[10]Filip C, Fletcher G, Wulff JL, et al. Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate [J], J Bacteriol.1973,115(3): 717-722.
[11]Robicsek A, Strahilevitz J, Jacoby G A, et al. Fluoroquinolone-modifying enzyme:a new adaptation of a common amino glycosideacetyl transferase [J]. Nat Med,2006,12 (1):83
[12]Mouneimne H, Robert J, et al. Type II topoisomerase mutations in ciprofloxacin resistant strains of Pseudomonas aeruginosa [J]. Antimicrob Agents Chemother,1999,43(1):62-66.
[13]Li XZ. Quinolone resistance in bacteria:emphasis on plasmid-mediated mechanisms [J]. Int J Antimicrob Agents,2005,25 (6):453
[14]Robicsek A, Jacoby G A, Hooper D C. The world wide emergence of plasmid-mediated quinolone resistance [J]. Lancet Infect Dis,2006,6(10):629
[15]Tran J H, Jacoby G A, et al. Interaction of the plasmid-encoded quinolone resistance protein Qnr with Escherichia coli DNA gyrase [J]. Antimicrob Agents Chemother,2005,49 (7):118
[16]Sugawara E, Nikaido H. OmpA protein of Escherichia coli outer membrane occurs in open and closed channel [J]. J Biol Chem,269 (1994):17981-7.
[17]Chenia HY, Pillay B, Pillay D. Analysis of the mechanisms of fluoroquinolone resistance in urinary tract pathogens [J]. J Antimicrob Chemother.2006; 58(6):1274-8
[2]闻玉梅.现代微生物学[M].第1版,上海医科大学出版社,1999.6.193-208.
[3]糜租煌.细菌耐药的分子机制[J].临床儿科杂志,2005,23(7)：422-424.
[4]鲁景艳,王兴龙,雷连成.革兰氏阳性菌对β-内酰胺类抗生素耐药机制及基因同源性的分析[J].中国兽药杂志,2004,38(2)：34-37.
[5]蒲霞,曾繁荣,罗勇.细菌的耐药性与老年期感染的抗菌治疗[J].中国药业,2004,13(11)：23-25.
[6]谭艳,方治平.抗菌药物的作用机制及细菌耐药性机制的研究进展[J].国外医药：抗生素分册,2003,24(2)：65-69.
[7]蔡培泉,王春新,糜租煌.铜绿假单胞杆菌氨基糖苷类耐药性及其修饰酶基因的研究[J].中华微生物学和免疫学杂志,2006,26(4)：374.
[8]Farrow KA, LyrasD, Rood J I.2000. The macrolide-lincosamide-streptogramin B resistance determinant from Clostridium difficile 630 contains two erm(B) genes [J]. Antimicrobial Agents and Chemotherapy,44 (2):411-413,
[9]戚好文.喹诺酮类抗菌药的研究进展[J].中国抗生素杂志,1999,24(增刊)：41.
[10]张致平.喹诺酮类抗菌药研究的新进展[J].国外医药抗生素分册,2001,22(6)：241.
[11]Sanders CC, Bradford PA, Ehrhardt AF,et al. Penicillin-binding proteins and induction of AmpC beta-lactamase [J].Antimicrob Agents Chemother,1997,41(9):2-13
[12]Gimemo C, Navarro D, Savall F et al. Relationship between outer membrane protein profiles and resistance to ceftazidime, imipenem, and ciprofloxacin in Pseudomonas aeruginosa isolates from bacteremic patients [J]. Eur J Clin Microbio Infect Dis,1996,15 (1):82
[13]Nikaido H. Porins and specific diffusion channels in bacterial outer membrane [J]. J Bio Chem,1994; 269 (6):3905
[14]Fukuoka T, Ohya S, Narita T et al. Activity of the carbapenem panipenem and role of the OprD (D2) protein in its diffusion through the Pseudomonas aeruginosa outer membrane. Antimicrob Agents Chemother,1993; 37 (2):322
[15]Yamano Y, Nishikawa T, Komatsu Y. Outer membrane proteins responsible for the penetrat ion of β-lactams and quinolones in Pseudomonas aeruginosa [J]. J Antimicrob Chemother, 1990; 26 (2):175
[16]Yoneyama H, Nakae T. Mechanism of efficient elimination of protein D2 in outer membrane of imipenem-resistant Pseudomonas aeruginosa [J]. Antimicrob Agents Chemother,1993; 37 (11):2385,
[17]Tavio M, Vila J, Ruiz J, et al. Mechanisms involved in the development of resistance to fluoroquinolones in Escherichia coli isolates [J]. J Antimicrob Chemother,1999,44 (6):735-742.
[18]Pratt L A, Hsing K, Gibson E, et al. From acids to osmZ:multiple factors influence synthesis of the OmpF and OmpC porins in Escherichia coli [J]. Mol Microbiol,1996,20 (5):911-917.
[19]Levy SB. Active efflux mechanisms for antimicrobial resistance. Antimicrobial Agents and Chemotherapy,1992,36 (4):695-703
[20]Nikaido H. Multidrug efflux pumps of gram-negative bacteria [J]. J Bacteriol,1996, 178:5853-5859.
[21]Keith P. Multidrug resistance in gram-negative bacteria [J]. CurrOpin Microbiol,2004,4:500-508.
[22]Bush JA, Li G. Cancer chemo resistance:the relationship between P53 and Multidrug resistance. Int J Cancer,2002,98 (3):323-330.
[23]Mathee K, Ciofu O, Sernberg C, et al. Mucoid conversion of Pseudomonas aeruginosa by hydrogen peroxide:a mechanism for virulence activation in the cystic fibrosis lung. [J].Microbiology,1999,145 (6):13-49.
[24]Drenkard E, Ausubel F M. Pseudomonas biofilm formation and antibiotic resistance are linked to phenotypic variation [J]. Nature,2002,416:740.
[25]Delissalde F, Amabile O, Cuevas C F. Comparison of antibiotic susceptibility and plasmid content, between biofilm producing and non Oproducing clinical isolates of Pseudomonas aeruginosa [J]. Int J Antimicrob Agents,2004,24:405.
[26]Raymond D P, Pelletier S J, Crabtree T D, et al. Impact of rotating empiric antibiotic schedule on infectious mortality in an intensive care unit [J]. Crit Care Med,2001,29 (2): 1101-1108.
[27]David J.C. Knowles. New strategies for antibacterial drug design [J] Trends in Microbiology 1997,5 (10):379-383.
[28]Guerner-Takada C, Salavati R, Altman S. Phenotypic conversion of drug resistant bacteria to drug sensitivity [J]. Proc Natl Acad Sci USA,1997,94(16):8468-8472.
[29]Barriere J C, Bouanchand D H, Paris J M, et al. Antimicrobial activity against Staphylococciaureus of semisynthetic in jectabale strep-tograwins RP59500 and related compounds [J]. J Antimicrob Chemother,1992,30:1
[1]胡志远,贺福初.蛋白质组研究进展[J].生物化学与生物物理进展,1999,26(3)：202-05.
[2]Wasinger V C, Cordwell S J, Cerpa Poljak A, et al. Progress withgene-product mapping of the Mollicutes:Mycoplasma genitalium [J]. Electrophoresis,1995,16:1090-1094.
[3]Zhou G, Li H M, De Camp D, et al.2D differential in gel electrophoresis for the identification esophageal scans cancer specific protein markers [J]. Mol Cell Proteomics,2002, (1):117-124.
[4]赵旭东.蛋白质组学研究[J].国外医学：分子生物学分册,2002,24(2)：90-93.
[5]Reynolds T. For proteomics research, a new race has begun [J]. J Natl Cancer Inst,2002,94 (8):552-554.
[6]Banks R E, Dunn M J, Hochstrasser D F, et al. Proteomics:new perspectives, new biomedical opportunities [J]. Lancet,2000,356:1749-1756.
[7]O'Farrell PH. High resolution two-dimensional electrophoresis of proteins. The Journal of biological chemistry,1975 May,25; 250(10):4007-4021.
[8]Manabe Takashi. Combination of electrophoretic techniques for comprehensive analysis of complex protein systems [J]. Electrophoresis,2000,21 (6):1116-1122.
[9]郭尧君.蛋白质电泳实验技术[M].北京：科学出版社,1996.161.
[10]Hanash S. Disease proteomics [J]. Nature,2003,422:226-232.
[11]王岚,刘骁勇,张华宁等.生物质谱技术在蛋白质组学研究中的应用[J].生物技术通讯,2007,18(1)：166-168.
[12]Mann M, Hendrickson R C, Pandey A. Analysis of proteins and proteomes by mass spectrometry [J]. Annu Rev Biochem,2001,70:437-473.
[13]曾嵘,夏其昌.蛋白质组学研究进展与趋势[J].中国科学院院刊,2002,17(3)：166-169.
[14]Ono M, Shitashige M, Honda K, et al. Label-free quantitative proteomics using large peptide data sets generated by nanoflow liquid chromatography and mass spectrometry [J]. Mol Cell Proteomics,2006,5(7):1338-1347.
[15]Jennifer S D, Matt hew E, Qian W J, et al. Advances in proteomics data analysis and display using an accurate mass and time tag approach [J]. Mass Spectrum Rev,2006,25 (3):450-482.
[16]Yates Ⅲ John R. Mass spectrometry and the age of the proteome[J]. Journal of Mass Spectrometry,1998,33(1):1-19.
[17]Godovac-Zimmermann Jasminka, Brown Larry R. Perspectives for mass spectrometry and functional proteomics [J]. Mass Spectrometry Reviews,2001,20(1):1-57.
[18]Mann Matthias, Hendrickson Ronald C, Pandey Akhilesh. Analysis of proteins and proteomes by mass spectrometry[J]. Annual Review of Biochemistry,2001,70:437-473.
[19]Kicman A T, Parkin M C, Iles R K. An introduction to mass spectrometry based proteomics detection and characterization of gonadotropinsand related molecules [J]. Mol Cell Endo crinol,2007 (2):212-227.
[20]韩俊,盛龙生,相秉仁等.基质辅助激光解吸离子化质谱法及其在生物大分子分析中的应用[J].药物分析杂志,1997,18(2)：125-132。
[21]Zhou G, Li H M, DeCamp D, et al.2D-differential in-gel electrophoresis for the identification esophageal scans cancer specific protein markers [J]. Mol Cell Proteomics,2002,1: 117-124
[22]Lee K, Kye M,Jang J S,et al. Proteomic analysis revealed a strong association of a high level of alpha 1 anti trypsin in gastric juice with gastric cancer [J]. Proteomics,2004,4 (11): 3343-3352.
[23]Ahram M,Best C J, Flaig M J, et al. Proteomic analysis of human prostate cancer [J]. Mol Carcinog,2002,33 (1):9-15.
[24]PR Jungblut, UE Schaible and HJ Mollenkopf. Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains:towards functional genomics of microbial pathogens [J].Mol Microbiol,1999,33 (6):11-31.
[25]Xu C, Lin X, Ren H, Zhang Y, Wang S, Peng X. Analysis of outer membrane proteome of Escherichia coli related to resistance to ampicillin and tetracycline [J]. Proteomics.2006 Jan; 6(2):462-473.
[26]陈川,王三英,彭宣宪.嗜水气单胞菌耐四环素的蛋白质组学初步研究[J].微生物学报,2004,44(3)：396-398.
[27]Diaz J I, Cazares L H, O J Semmes. Tissue sample collection for proteomics analysis [J]. Methods Mol Biol,2008,428 (1):43
[28]Orvisky E, Drake S K, Martin B M, et al. Enrichment of low molecular weight fraction of serum for MS analysis of peptides associated with hepatocellular carcinoma. Proteomics, 2006,6(9):2895
[29]Hood B L, Lucas D A, Kim G, et al. Quantitative analysis of the low molecular weight serum proteome using 180 stable isotope labeling in a lung tumor xenograft mouse model. J Am Soc Mass Spectrum,2005,16 (8):1221
[30]Chromy B A, Gonzales A D, Perkins J, et al. Proteomic analysis of human serum by two-dimensional differential gel electrophoresis after depletion of high-abundant proteins [J]. J Proteome Res,2004,3 (6):1120
[31]Boguski, M. S., McIntosh, M. W. Biomedical informatics for proteomics [J]. Nature 2003, 422 (6928):233-237.
[1]Nielsen ME, Hoi L. Schmidt AS, et al. Is Aeromonas hydrophila the dominant motile Aeromonas species that causes disease outbreaks in aquaculture production in the Zhejiang Province of China [J], Diseases of Aquatic Organisms.2001,46 (1):23-29.
[2]赵香汝,杨汉春.细菌外膜蛋白的研究现状[J].中国兽医杂志,1997,23(12)：41-42.
[3]孙建和,严亚贤,陈怀青等.温度对嗜水气单胞菌外膜蛋白表达的影响[J].中国兽医学报,1999,19(6)：555～558.
[4]Lee SY, Yin Z, Ge R, Sin YM. Isolation and characterization of fish Aeromonas hydrophila adhesins important for in vitro epithelial cell invasion [J], J Fish Dis.1997,20(1):69-75.
[5]Buchley JT, Howard SP. The Cytotoxic enterotoxin of Aeromonas hydrophila is aerolysin [J], Infect Immun,1999,67 (1):466-467.
[6]Aronoff SC. Outer membrane permeability in Pseudomonas cepacia:diminished porin content in a beta-lactam-resistant mutant and in resistant cystic fibrosis isolates [J], Antimicrob Agents Chemother.1988.32:1636-1639.
[7]Filip C, Fletcher G, Wulff JL, et al. Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate [J], J Bacteriol.1973,115(3):717-722.
[8]Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning:a Laboratory Manual Second Edition New York[M], Cold Spring Harbor Laboratory Press, U.S.1987:880-885
[9]Performance Standards for Antimicrobial Susceptibility Testing; Seventeenth Informational Supplement [S].CLSI documents M100-S17 Vol.27(1):1-68
[10]Dawson C E, Stubberfield E J, Perrett L L, et al. Phenotypic and molecular characterisation of Brucella isolates from marine mammals [J]. BMC Microbiol.2008; 17(8):224.
[11]Dassouli-Mrani-Belkebir A, Contrepois M, Girardeau J P, et al. Characters of Escherichia coli 078 isolated from septicaemic animals [J]. Vet Microbiol.1988; 17(4):345-56.
[12]Nikaido H. Molecular Basis of Bacterial Outer Membrane Permeability Revisited [J], Microbiol Mol Biol Rev.2003,67(4):593-656.
[13]Nikaido H. Porins and specific channels of the bacterial outer membrane [J], Mol Microbiol. 1992,6:435-442.
[14]Martinez-Martinez L, Hemandez-Alles S, Alberti S, Tomas JM, Benedi VJ, Jacoby GA. In vivo selection of porin-deficient mutants of Klebsiella pneumoniae with increased resistance to cefoxitin and expanded-spectrum-cephalosporins [J], Antimicrob Agents Chemother.1996, 40(2):342-348.
[15]Mallea M, Chevalier J, Bornet C, Eyraud A, Davin-Regli A, Bollet C, Pages JM. Porin alteration and active efflux:two in vivo drug resistance strategies used by Enterobacter
aerogenes [J], Microbiology.1998,144:3003-3009.
[16]Coulton JW, Mason P, Dorrance P. The permeability barrier of Haemophilus influenzae type b against beta-lactam antibiotics [J], J Antimicrob Chemother.1983,12(5):435-449.
[17]Pangon, B., C. Toro, C. S., S. R. Lobos, I. Calderon, M. Rodriguez, and G. C. Mora. Clinical isolate of a porin-less Salmonella typhi resistant to high levels of chloramphenicol [J]. Antimicrob Agents Chemother.1990.34:1716-1719.
[18]Seshadri R, Joseph S W, Chopra A K, et al. Genome sequence of Aeromonas hydrophila ATCC 7966T:jack of all trades[J], J Bacteriol.2006;188(23):8272-82.
[19]Morita Y, Kodama K, Shiota S, et al. NorM, a putative multidrug efflux protein, of Vibrio parahaemolyticus and its homolog in Escherichia coli [J]. Antimicrob Agents Chemother. 1998;42(7):1778-82.
[1]董传甫,林天龙.嗜水气单胞菌研究进展综述[J].福建农业学报,2003,18(4)：243-248.
[2]陆承平.兽医微生物[M],中国农业出版社,第四版,2007：31-32
[3]陈怀青,陆承平.家养鲤科鱼爆发性传染病病原研究[J],南京农业大学学报,1991,14(4)：81-91.
[4]Colwell R R, Macdonell MT, Deley J. Proposal to recognize the family Aeromonadaceae fam [J]. International Journal of Systemic Bacteriology,1986,34:473-477.
[5]郑国兴,周凯.嗜水气单胞菌欧洲鳗皮肤溃疡分离株的耐药性[J].中国水产科学,1999,6(3)：69.
[6]陆承平.致病性嗜水气单胞菌综述及其所致鱼病[J].水产学报,1992,16(3)：282-288
[7]赵香汝,杨汉春.细菌外膜蛋白的研究现状[J].中国兽医杂志,1997,23(12)：41-42.
[8]Tavio MM, Vila J, Ruiz J, et al. Mechanisms involved in the development of resistance to fluoroquinolones in Escherichia coli isolates [J]. J Antimicrob Chemother,1999,44 (6):735-742.
[9]苏林光,贾杰,潘光华.次抑菌浓度的药物诱导细菌耐药与交叉耐药[J].中国抗生素杂志,1997,22(4)：301-303.
[10]Filip C, Fletcher G, Wulff JL, et al. Solubilization of the cytoplasmic membrane of Escherichia coli by the ionic detergent sodium-lauryl sarcosinate [J], J Bacteriol.1973,115(3): 717-722.
[11]Robicsek A, Strahilevitz J, Jacoby G A, et al. Fluoroquinolone-modifying enzyme:a new adaptation of a common amino glycosideacetyl transferase [J]. Nat Med,2006,12 (1):83
[12]Mouneimne H, Robert J, et al. Type II topoisomerase mutations in ciprofloxacin resistant strains of Pseudomonas aeruginosa [J]. Antimicrob Agents Chemother,1999,43(1):62-66.
[13]Li XZ. Quinolone resistance in bacteria:emphasis on plasmid-mediated mechanisms [J]. Int J Antimicrob Agents,2005,25 (6):453
[14]Robicsek A, Jacoby G A, Hooper D C. The world wide emergence of plasmid-mediated quinolone resistance [J]. Lancet Infect Dis,2006,6(10):629
[15]Tran J H, Jacoby G A, et al. Interaction of the plasmid-encoded quinolone resistance protein Qnr with Escherichia coli DNA gyrase [J]. Antimicrob Agents Chemother,2005,49 (7):118
[16]Sugawara E, Nikaido H. OmpA protein of Escherichia coli outer membrane occurs in open and closed channel [J]. J Biol Chem,269 (1994):17981-7.
[17]Chenia HY, Pillay B, Pillay D. Analysis of the mechanisms of fluoroquinolone resistance in urinary tract pathogens [J]. J Antimicrob Chemother.2006; 58(6):1274-8