貂源铜绿假单胞菌的分离鉴定及其环介导等温扩增检测方法的建立
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摘要
水貂出血性肺炎是由铜绿假单胞菌感染引起的一种以发病水貂呼吸困难、口鼻流血为主要特征的烈性传染病，发病急、死亡快、呈地方性流行，已成为危害我国水貂养殖业最严重的细菌性传染病之一，给广大水貂养殖户造成了巨大的经济损失，因此开展对此病的研究迫在眉睫。
铜绿假单胞菌曾称绿脓杆菌，是一种人兽共患性病原菌，属于革兰氏阴性短杆菌，有鞭毛，能单向运动。该菌对外界环境的抵抗力较强，在污染的环境及土壤中可长时间存活，对很多化学消毒剂和抗生素有抵抗力。
本实验室从威海送检的发病水貂肺脏中分离到一株细菌，通过培养特性、生化试验和PCR方法鉴定为铜绿假单胞菌，命名为PA-WH-0808。致病性试验结果表明，8.0×1010CFU/mL的菌液10倍系列稀释后，感染60日龄健康昆明系小鼠，半数致死量和最小致死量分别为1.4×106.0CFU和8.0×105.0CFU。死亡小鼠剖检可见皮下和肺脏出血,并能从死亡小鼠的肺脏分离出与感染菌株形态特征完全一致的菌落。药敏试验结果显示，该分离菌株对庆大霉素、环丙沙星、诺氟沙星等较为敏感，而对青霉素、链霉素不敏感。系统发育分析结果表明，该分离菌株与从小麦中分离出的铜绿假单胞菌的亲缘关系较近，因此猜测该发病水貂可能是食用了污染的饲料引起的。
我们采用环介导等温扩增技术(loop-mediated isothermal amplification, LAMP)，建立了一种可视化的快速检测铜绿假单胞菌的实验方法。该方法针对铜绿假单胞菌16SrDNA基因的保守区域设计了4条引物，在Bst DNA聚合酶的作用下，可以实现DNA的梯状等温扩增。优化LAMP的反应体系，并检验其灵敏性、特异性。PA-LAMP检测方法从核酸抽提到检测结果出现仅需60min，该方法具有良好的特异性，与感染毛皮动物的常见菌如大肠杆菌、沙门氏菌等无交叉反应，灵敏度是常规PCR的100倍。试验结果证明，PA-LAMP方法可快速、灵敏、特异地检测铜绿假单胞菌，在实验室和现场应用都具有良好的应用前景。
Hemorrhagic pneumonia of mink is an acute infectious disease caused byPseudo mo na s a e rugin o sa, clinical signs are various for bleeding from the nose ormouth, dyspnoea and sudden death.This infectious disease makes the mosteconomic loss for the farmed mink in China, therefore it is necessary to carry outthis study of the disease.
P se ud o mo n a s a erugino sa is a common bacterium that can cause disease inanimals and humans. It is a Gram-negative, rod-shaped bacterium with unipolarmotility by monotrichate flagellum. Pseudomonas aeruginosa has strong resistanceto the external environment, and can survive in a polluted environment and soil fora long time, and is resistant to many chemical disinfectants and antibiotics.
A Pseudomonas aeruginosa was isolated from the lung of a dead mink fromWeihai of Shan Dong Province.We identified the Pseudomonas aeruginosa basedon colony characteristics,biochemical tests and PCR. Pathogenicity test showedthat the LD50for mice is1.4×106.0CFU,and the MLD is8.0×105.0CFU.Wesuccessfully isolated the same bacteria as infectious bacteria from the infectiousmice.Drug susceptibility test showed that the isolated was sensitive to some drugssuch as gentamicin,Ciproflaxin and norfloxacin,but resistant to penicillin andstreptomycin. On the basis of the bacterial16srRNA gene sequence phylogeneticanalysis and comparison of this gene sequence with sequence in RNA sequencedatabase, it was considered that the isolated was closely related to me mbers of thePseudo mo na s aerugino sa from the wheat,so we suepected that the isolated wasfrom the contaminated feed.
LAMP is a rapid and visible method to detect the Pseudomonas aeruginosa.Aset of four primers were designed based on the PA-16SrDNA gene sequence byPrimerExplorerV4software. The specificity and sensitivity are very good byoptimizing reaction system and reaction time.It was showed that the Pseudomonsaaeru ginoa s could be detected within60minutes by this LAMP, and the sensitivitywas100times higher than that of general PCR. Establishment of LAMP provides anew alternative method for the rapid detection of Pseudomonsa aeruginoas,and hasa good prospect in the labortary and clinical tests.
铜绿假单胞菌曾称绿脓杆菌，是一种人兽共患性病原菌，属于革兰氏阴性短杆菌，有鞭毛，能单向运动。该菌对外界环境的抵抗力较强，在污染的环境及土壤中可长时间存活，对很多化学消毒剂和抗生素有抵抗力。
本实验室从威海送检的发病水貂肺脏中分离到一株细菌，通过培养特性、生化试验和PCR方法鉴定为铜绿假单胞菌，命名为PA-WH-0808。致病性试验结果表明，8.0×1010CFU/mL的菌液10倍系列稀释后，感染60日龄健康昆明系小鼠，半数致死量和最小致死量分别为1.4×106.0CFU和8.0×105.0CFU。死亡小鼠剖检可见皮下和肺脏出血,并能从死亡小鼠的肺脏分离出与感染菌株形态特征完全一致的菌落。药敏试验结果显示，该分离菌株对庆大霉素、环丙沙星、诺氟沙星等较为敏感，而对青霉素、链霉素不敏感。系统发育分析结果表明，该分离菌株与从小麦中分离出的铜绿假单胞菌的亲缘关系较近，因此猜测该发病水貂可能是食用了污染的饲料引起的。
我们采用环介导等温扩增技术(loop-mediated isothermal amplification, LAMP)，建立了一种可视化的快速检测铜绿假单胞菌的实验方法。该方法针对铜绿假单胞菌16SrDNA基因的保守区域设计了4条引物，在Bst DNA聚合酶的作用下，可以实现DNA的梯状等温扩增。优化LAMP的反应体系，并检验其灵敏性、特异性。PA-LAMP检测方法从核酸抽提到检测结果出现仅需60min，该方法具有良好的特异性，与感染毛皮动物的常见菌如大肠杆菌、沙门氏菌等无交叉反应，灵敏度是常规PCR的100倍。试验结果证明，PA-LAMP方法可快速、灵敏、特异地检测铜绿假单胞菌，在实验室和现场应用都具有良好的应用前景。
Hemorrhagic pneumonia of mink is an acute infectious disease caused byPseudo mo na s a e rugin o sa, clinical signs are various for bleeding from the nose ormouth, dyspnoea and sudden death.This infectious disease makes the mosteconomic loss for the farmed mink in China, therefore it is necessary to carry outthis study of the disease.
P se ud o mo n a s a erugino sa is a common bacterium that can cause disease inanimals and humans. It is a Gram-negative, rod-shaped bacterium with unipolarmotility by monotrichate flagellum. Pseudomonas aeruginosa has strong resistanceto the external environment, and can survive in a polluted environment and soil fora long time, and is resistant to many chemical disinfectants and antibiotics.
A Pseudomonas aeruginosa was isolated from the lung of a dead mink fromWeihai of Shan Dong Province.We identified the Pseudomonas aeruginosa basedon colony characteristics,biochemical tests and PCR. Pathogenicity test showedthat the LD50for mice is1.4×106.0CFU,and the MLD is8.0×105.0CFU.Wesuccessfully isolated the same bacteria as infectious bacteria from the infectiousmice.Drug susceptibility test showed that the isolated was sensitive to some drugssuch as gentamicin,Ciproflaxin and norfloxacin,but resistant to penicillin andstreptomycin. On the basis of the bacterial16srRNA gene sequence phylogeneticanalysis and comparison of this gene sequence with sequence in RNA sequencedatabase, it was considered that the isolated was closely related to me mbers of thePseudo mo na s aerugino sa from the wheat,so we suepected that the isolated wasfrom the contaminated feed.
LAMP is a rapid and visible method to detect the Pseudomonas aeruginosa.Aset of four primers were designed based on the PA-16SrDNA gene sequence byPrimerExplorerV4software. The specificity and sensitivity are very good byoptimizing reaction system and reaction time.It was showed that the Pseudomonsaaeru ginoa s could be detected within60minutes by this LAMP, and the sensitivitywas100times higher than that of general PCR. Establishment of LAMP provides anew alternative method for the rapid detection of Pseudomonsa aeruginoas,and hasa good prospect in the labortary and clinical tests.
引文
1.蔡正求，宋未，东秀珠，等，细菌种属特异性的16SrDNA寡核苷酸探针数据库的初步构建[J].首都师范大学学报，2004，25：110～115.
2.蔡宝祥，家畜传染病学[M].北京：中国农业出版社，2001,86～87.
3.常小斌,刘洵,程天印，等，环介导等温基因扩增检测技术及其应用研究进展[J].畜牧兽医科技信息，2006，12：10～12.
4.陈蕾，猪瘟病毒RT-LAMP快速诊断试剂盒的研制与应用[硕士学位论文].北京：中国兽医药品监察所，2009.
5.高玉伟,夏咸柱,胡桂学，等，细菌分离和基因扩增确诊水貂出血性肺炎[J].黑龙江畜牧兽医，2006，4：53～54.
6.关中湘，王树志，朴厚坤，等，水貂出血性肺炎病原体探讨[J].吉林农业大学学报，1984，12（2）：3～5.
7.金美玲，周宏德，朱家新，等，一日龄雏鸡败血性绿脓杆菌病诊治[J].中国家禽，1995，6（4）：14～16.
8.韩青松，简永利，涂宜强，等，绿脓杆菌研究进展[J].畜牧与饲料科学,2012，33（1）：122～
124.
9.刘永生，丁耀忠，张杰，等环介导等温扩增（LAMP）技术的应用研究[J].中国农学通报，2010，26（8）：87～89.
10.李瑞华.鸡大肠杆菌和绿脓杆菌混合感染的诊疗[J].河南畜牧兽医杂志,2000，8（21）:32～
36.
11.陆承平，兽医微生物[M].北京：中国农业出版社，2001,274-275.
12.彭大新，张如宽，徐圻，等，山羊绿脓杆菌感染的诊断[J].中国兽医科技，1997，（2）：37～38.
13.彭永刚，孙恩成，刘飞，等，环介导等温扩增技术在畜牧兽医领域的应用[J].中国兽医科学，2010，40（05）：543～546.
14. R.E.布坎南，N.E.吉本斯.伯杰细菌鉴定手册[M].8版，北京：科学出版社，1983.
15.唐珂心，牛钟相，何元龙，等，山羊慢性化脓性肺炎病原菌的分离鉴定[J].中国兽医杂志，2001，37（6）：20～21.
16.吴阳升，罗淑萍，一种新的高效快速核酸恒温扩增方法—LAMP[J].生物技术，2004，14（4）：76～82.
17.闫新华，程世鹏，闫喜军，等，毛皮动物疾病诊疗原色图谱[M].北京：中国农业出版社，2008，58～61.
18.闫新华，水貂出血性肺炎[J].中国兽医杂志，1988，14（4）：22.
19.杨正时，兽医微生物学(3版)［M］．北京:中国农业出版社，2007：662～675．
20.叶应妩，王毓三，全国临床检验操作规程(2版)［M］．南京:东南大学出版社，1997：553～
562．
21.于柏峰，张慧云，刘冰，等，铜绿假单胞菌致病力和致病机理研究进展[J].微生物学杂志，24(1)：52～53.
22.于静泉，段晓坤，刘双，等，雏鸡大肠杆菌性脑炎与绿脓杆菌病混合感染的诊治[J].AnimalHusbandry&Veterinary Medicine2003,35(2)：44～46.
23.云水丽，杨勇，王小波，等，Ⅱ型鸭疫里默氏杆菌的分离鉴定及致病性研究[J].中国预防兽医学报，2009，31（8）：605～609.
24.万忠海，何畅，江禹，等，重组绿脓杆菌外毒素A受体结合区蛋白动物免疫保护试验[J].中国兽医学报，2001，21（4）：359～361.
25.赵乃昕，马子行,假单孢菌属的分类现状.国外医学微生物学分册，1996,19（2）：20～23.
26.张道永，胡景韶，王文贵,等.绿脓杆菌研究进展[J].四川畜牧兽医，1995，9（3）：55～58.
27.邹玲，任慧英，水貂出血性肺炎的微生物学检查[J].黑龙江畜牧兽医，2009，1：92～93.
28.张道永，胡景韶，王文贵，等，绿脓杆菌研究进展[J].四川畜牧兽医，1995，9（3）：55～58.
29.许景云，王少林，黄世生，等，铜绿假单胞菌血清学分型及耐药性研究[J].邵阳医学院院报，1999，18（4）：210～213.
30. Allured VS,Collier RJ,Carroll SF,et al.,Structure of exotoxin A of Pseudomonas aeruginosa at3.0-angseron resolution[J].Proc Natl Acad Sci USA,1986,83(5):1320～1324.
31. Chen TY,Lin CP,Loa CC,et al.,A nontoxic Pseudomonas exotoxin A induces active immunityand passive protective antibody against Pseudomonas exotoxin A intoxication[J].J BiomedSci,1999,6(5):357～363.
32. Clarridge JE3rd, Impact of16S rRNA gene sequence analysis for identification of bacteria onclinical microbiology and infectious diseases［J］. Clin Microbial Res,2004,17（4）:840～862.
33. Coburn J,Wyatt R.T,Iglewski BH,Gill D.M et al.,Several GTP-binding proteins, including o24C-H-ras,are preferred substrates of Pseudomonas aeruginosa exoenzyme [J].J BiolChem,1989,264～268.
34. Cross AS,Sadoff JC,Iglewski BH,Sokol PA et al.,Evidence for the role of toxin A in thepathogenesis of infections with Pseudomonas aeruginosa in humans[J].Infect Dis1980,142:538~540.
35. Diekema DJ et al., Trends in antimicrobial susceptibility of bacterial pathogens isolated frompatients with bloodstream infections in the USA, Canada and Latin America. Clin InfectDis,1999,29:595.
36. Fine et al., Prognosis and outcomes of patients with community-acquired pneumonia[J]. Ameta-analysis,1996,275:134~136.
37. Gray G.L,Smith D.H,Baldrige J.S,et al.,Cloning, nucleotide sequence,and expression inEscherichia coli of the exotoxin A structural gene of Pseudomonas aeruginosa[J],Proc NatlAcad Sci USA,1984,81(9):2645
38. Harvey, R.A, Champe P.C., Fisher B.D et al.,Microbiology Lippincott's Williams&WilkinsHammer A S,Pedersen K,Andersen T H.Comparison of Pseudomonas aeruginosaisolates from mink by serotyping and pulsed-field gel electrophoresis[J].VeterinaryMicrobiology,2003,94(6):237～243.
39. Hassan R,Fitzgerald D.J,et al.,Immunotoxin therapyof cancer[J].Nature cancer,2006,6(7):559～
565.
40. Hommal.J.Y et al.,a class of serum-sensitive,nontypable strains deficient in lipopolysaccharideO side chain[J].Infect Immun,1980(50):149~152.
41. Hwang J,Fitzgerald D.J,Adhya S,et al.,Functional domains of Pseudomonas exotoxin identifiedby deletion analysis of the gene expressed in E.coli[J].Cell,1987,48(1):129～136.
42. Kawwakami M,Kawakami K,Puri RK.Interleukin-4-Pseudomonas exotoxin chimeric fusionprotein for malignant glioma therapy[J].J Neurooncol,2003,65(1):15～25.
43. King E.O, Ward MK, Raney D.E. Two simple media for the demonstration of pyocyanin andfluorescin[J]. J Lab Clin Med,1954,44(2):301～307.
44. Krueger K.M,Barbieri J.T et al.,The family of bacterial ADP-ribosylating exotoxins[J].ClinMicrobiol Rev,1995,8(1):34～47.
45. Kubler-Kielb J,Majadly F,Wu Y,et al.,Long-lasting and transmission-blocking activity ofantibodies to Plasmodium falciparum elicited in mice by protein conjugates ofPfs25[J].Proceeding of the National Academy of Science of the United States ofAmerica,2007,104(1):293～298.
46. KureishiA, Bryan L.E.Pre-boiling high GC content,mixed primers with3′complementationallows the successful PCR amplification of Pseudomonas aeruginosa DNA[J].Nucleic AcidsRrsearch,1992,20(5):1155.
47. Lin PV,H sich H.Exotoxins of Pseudomonas aeruginsa.I Factors that influence the production ofexotoxin in A[J].J Infectious Diseases1973,128(4):506～513.
48. Lindstedt B A, Heir E, Nygard I, et al., Characterisation of class I integrons in clinical strains ofSalmonella enterica subsp. Enterica serovars typhimurium and enteritidis from norwegianhospitals [J]. J Med Microbiol,2003,52(2):141～149.
49. Liu P V,Hsieh H,Exooxins of Paeruginosa.Characteristic of antitoxin A[J].J InfectDis,1973,128:520～524.
50. Nikaido H,Rosenber EY,Foulds J.Porin channels in Esherichia coli:studies with lactams inintact cell[J].Journal of Bacteriology,1983,153:232～235.
51. Notomi T,Okayama H,Masubuchi H,et al., Loop-mediated isoehermal amplification ofDNA[J].Nuceic Acids Res,2000,28(12):E63.
52. Parson Y N, Panges S, Smartch, et al., use of subtractive hybridization to identify a diagnosticprobe for cystic fibrosis epidemic strain of Pseudomonas aeruginosa[J]. J Clin Microbiol,2002,40(12):4607～4611.
53. Paraskaki I,Epidemiology of community-acquired Psdudomonas aeruginosa infections inchildren[J].Eur J Clin Microbiol Infect Dis,1996,15:782～785.
54. Pollack M,Pseudomonas Aeruginosa.In:Mandell GL,Bennett JE,Dolin R,eds.Principles andPractice of Infectious Diseases.5th ed.New York,NY:Churchill Livingstone,2000,23:10～27.
55. Prithiviraj B et al.,Down regulation of virulence factors of Pseudomonas aeruginosa by salicylicacid attenuates its virulence on Arabidopsis thaliana and Caenorhabditis elegans[J]. InfectImmun,2005,73(9):5319～5328.
56. Robbins D.H，Margulies I,Stetler-Stevenson M,et al.,Hairy cell leukemia,a B-cell neoplasm thatis particularly sensitive to the cytotoxic effect of anti-Tac(Fv)-PE38(lmb-2)[J].Clin CancerRes,2000,6(2):693～700.
57. Ryan KJ,Ray CG (editors). Sherris Medical Microbiology [M].4th ed.McGraw Hill.2004，132～135.
58. Salazar D,Hernández Y,Palma S,González A,et al.,Antimicrobian activity of twelve drugsagainst Pseudomona aeruginosa strains from clinical isolates Bioquimia[J].2001,26(4):79～84.
59. Saint O, Romeyer F, Lebel P,et al.,Specificity of the Pseudomonas aeruginosa PAO1lipoproteinI gene as a DNA probe and PCR target region within the Pseudomonadaceae[J].J GeneralMicrobiol1992,138(4):733～741.
60. Schoni M,Macrolide antibiotic therapy in patients with cystic fibrosis.Swiss Med Wkly2003,133(21-22):297～301.
61. Sokol PA,Kooi C,Hodges R.S,et al.,Immunization with a Pseudomonas aeruginosa elastasepeptide reduces severity of experimental lung infections due to P.aeruginosa or Burkholderiacepacia[J].J Infect Dis,2000,181(5):1682～1692.
62. Spilker T,Coenye T,Vandamme P, et al., PCR based assay for differentiation of Pseudomonasaeruginosa from other Pseudomonas species recovered from cystic fibrosis Patients[J]. J ClinMicrobiol,2004,42(5):2074～2079.
2.蔡宝祥，家畜传染病学[M].北京：中国农业出版社，2001,86～87.
3.常小斌,刘洵,程天印，等，环介导等温基因扩增检测技术及其应用研究进展[J].畜牧兽医科技信息，2006，12：10～12.
4.陈蕾，猪瘟病毒RT-LAMP快速诊断试剂盒的研制与应用[硕士学位论文].北京：中国兽医药品监察所，2009.
5.高玉伟,夏咸柱,胡桂学，等，细菌分离和基因扩增确诊水貂出血性肺炎[J].黑龙江畜牧兽医，2006，4：53～54.
6.关中湘，王树志，朴厚坤，等，水貂出血性肺炎病原体探讨[J].吉林农业大学学报，1984，12（2）：3～5.
7.金美玲，周宏德，朱家新，等，一日龄雏鸡败血性绿脓杆菌病诊治[J].中国家禽，1995，6（4）：14～16.
8.韩青松，简永利，涂宜强，等，绿脓杆菌研究进展[J].畜牧与饲料科学,2012，33（1）：122～
124.
9.刘永生，丁耀忠，张杰，等环介导等温扩增（LAMP）技术的应用研究[J].中国农学通报，2010，26（8）：87～89.
10.李瑞华.鸡大肠杆菌和绿脓杆菌混合感染的诊疗[J].河南畜牧兽医杂志,2000，8（21）:32～
36.
11.陆承平，兽医微生物[M].北京：中国农业出版社，2001,274-275.
12.彭大新，张如宽，徐圻，等，山羊绿脓杆菌感染的诊断[J].中国兽医科技，1997，（2）：37～38.
13.彭永刚，孙恩成，刘飞，等，环介导等温扩增技术在畜牧兽医领域的应用[J].中国兽医科学，2010，40（05）：543～546.
14. R.E.布坎南，N.E.吉本斯.伯杰细菌鉴定手册[M].8版，北京：科学出版社，1983.
15.唐珂心，牛钟相，何元龙，等，山羊慢性化脓性肺炎病原菌的分离鉴定[J].中国兽医杂志，2001，37（6）：20～21.
16.吴阳升，罗淑萍，一种新的高效快速核酸恒温扩增方法—LAMP[J].生物技术，2004，14（4）：76～82.
17.闫新华，程世鹏，闫喜军，等，毛皮动物疾病诊疗原色图谱[M].北京：中国农业出版社，2008，58～61.
18.闫新华，水貂出血性肺炎[J].中国兽医杂志，1988，14（4）：22.
19.杨正时，兽医微生物学(3版)［M］．北京:中国农业出版社，2007：662～675．
20.叶应妩，王毓三，全国临床检验操作规程(2版)［M］．南京:东南大学出版社，1997：553～
562．
21.于柏峰，张慧云，刘冰，等，铜绿假单胞菌致病力和致病机理研究进展[J].微生物学杂志，24(1)：52～53.
22.于静泉，段晓坤，刘双，等，雏鸡大肠杆菌性脑炎与绿脓杆菌病混合感染的诊治[J].AnimalHusbandry&Veterinary Medicine2003,35(2)：44～46.
23.云水丽，杨勇，王小波，等，Ⅱ型鸭疫里默氏杆菌的分离鉴定及致病性研究[J].中国预防兽医学报，2009，31（8）：605～609.
24.万忠海，何畅，江禹，等，重组绿脓杆菌外毒素A受体结合区蛋白动物免疫保护试验[J].中国兽医学报，2001，21（4）：359～361.
25.赵乃昕，马子行,假单孢菌属的分类现状.国外医学微生物学分册，1996,19（2）：20～23.
26.张道永，胡景韶，王文贵,等.绿脓杆菌研究进展[J].四川畜牧兽医，1995，9（3）：55～58.
27.邹玲，任慧英，水貂出血性肺炎的微生物学检查[J].黑龙江畜牧兽医，2009，1：92～93.
28.张道永，胡景韶，王文贵，等，绿脓杆菌研究进展[J].四川畜牧兽医，1995，9（3）：55～58.
29.许景云，王少林，黄世生，等，铜绿假单胞菌血清学分型及耐药性研究[J].邵阳医学院院报，1999，18（4）：210～213.
30. Allured VS,Collier RJ,Carroll SF,et al.,Structure of exotoxin A of Pseudomonas aeruginosa at3.0-angseron resolution[J].Proc Natl Acad Sci USA,1986,83(5):1320～1324.
31. Chen TY,Lin CP,Loa CC,et al.,A nontoxic Pseudomonas exotoxin A induces active immunityand passive protective antibody against Pseudomonas exotoxin A intoxication[J].J BiomedSci,1999,6(5):357～363.
32. Clarridge JE3rd, Impact of16S rRNA gene sequence analysis for identification of bacteria onclinical microbiology and infectious diseases［J］. Clin Microbial Res,2004,17（4）:840～862.
33. Coburn J,Wyatt R.T,Iglewski BH,Gill D.M et al.,Several GTP-binding proteins, including o24C-H-ras,are preferred substrates of Pseudomonas aeruginosa exoenzyme [J].J BiolChem,1989,264～268.
34. Cross AS,Sadoff JC,Iglewski BH,Sokol PA et al.,Evidence for the role of toxin A in thepathogenesis of infections with Pseudomonas aeruginosa in humans[J].Infect Dis1980,142:538~540.
35. Diekema DJ et al., Trends in antimicrobial susceptibility of bacterial pathogens isolated frompatients with bloodstream infections in the USA, Canada and Latin America. Clin InfectDis,1999,29:595.
36. Fine et al., Prognosis and outcomes of patients with community-acquired pneumonia[J]. Ameta-analysis,1996,275:134~136.
37. Gray G.L,Smith D.H,Baldrige J.S,et al.,Cloning, nucleotide sequence,and expression inEscherichia coli of the exotoxin A structural gene of Pseudomonas aeruginosa[J],Proc NatlAcad Sci USA,1984,81(9):2645
38. Harvey, R.A, Champe P.C., Fisher B.D et al.,Microbiology Lippincott's Williams&WilkinsHammer A S,Pedersen K,Andersen T H.Comparison of Pseudomonas aeruginosaisolates from mink by serotyping and pulsed-field gel electrophoresis[J].VeterinaryMicrobiology,2003,94(6):237～243.
39. Hassan R,Fitzgerald D.J,et al.,Immunotoxin therapyof cancer[J].Nature cancer,2006,6(7):559～
565.
40. Hommal.J.Y et al.,a class of serum-sensitive,nontypable strains deficient in lipopolysaccharideO side chain[J].Infect Immun,1980(50):149~152.
41. Hwang J,Fitzgerald D.J,Adhya S,et al.,Functional domains of Pseudomonas exotoxin identifiedby deletion analysis of the gene expressed in E.coli[J].Cell,1987,48(1):129～136.
42. Kawwakami M,Kawakami K,Puri RK.Interleukin-4-Pseudomonas exotoxin chimeric fusionprotein for malignant glioma therapy[J].J Neurooncol,2003,65(1):15～25.
43. King E.O, Ward MK, Raney D.E. Two simple media for the demonstration of pyocyanin andfluorescin[J]. J Lab Clin Med,1954,44(2):301～307.
44. Krueger K.M,Barbieri J.T et al.,The family of bacterial ADP-ribosylating exotoxins[J].ClinMicrobiol Rev,1995,8(1):34～47.
45. Kubler-Kielb J,Majadly F,Wu Y,et al.,Long-lasting and transmission-blocking activity ofantibodies to Plasmodium falciparum elicited in mice by protein conjugates ofPfs25[J].Proceeding of the National Academy of Science of the United States ofAmerica,2007,104(1):293～298.
46. KureishiA, Bryan L.E.Pre-boiling high GC content,mixed primers with3′complementationallows the successful PCR amplification of Pseudomonas aeruginosa DNA[J].Nucleic AcidsRrsearch,1992,20(5):1155.
47. Lin PV,H sich H.Exotoxins of Pseudomonas aeruginsa.I Factors that influence the production ofexotoxin in A[J].J Infectious Diseases1973,128(4):506～513.
48. Lindstedt B A, Heir E, Nygard I, et al., Characterisation of class I integrons in clinical strains ofSalmonella enterica subsp. Enterica serovars typhimurium and enteritidis from norwegianhospitals [J]. J Med Microbiol,2003,52(2):141～149.
49. Liu P V,Hsieh H,Exooxins of Paeruginosa.Characteristic of antitoxin A[J].J InfectDis,1973,128:520～524.
50. Nikaido H,Rosenber EY,Foulds J.Porin channels in Esherichia coli:studies with lactams inintact cell[J].Journal of Bacteriology,1983,153:232～235.
51. Notomi T,Okayama H,Masubuchi H,et al., Loop-mediated isoehermal amplification ofDNA[J].Nuceic Acids Res,2000,28(12):E63.
52. Parson Y N, Panges S, Smartch, et al., use of subtractive hybridization to identify a diagnosticprobe for cystic fibrosis epidemic strain of Pseudomonas aeruginosa[J]. J Clin Microbiol,2002,40(12):4607～4611.
53. Paraskaki I,Epidemiology of community-acquired Psdudomonas aeruginosa infections inchildren[J].Eur J Clin Microbiol Infect Dis,1996,15:782～785.
54. Pollack M,Pseudomonas Aeruginosa.In:Mandell GL,Bennett JE,Dolin R,eds.Principles andPractice of Infectious Diseases.5th ed.New York,NY:Churchill Livingstone,2000,23:10～27.
55. Prithiviraj B et al.,Down regulation of virulence factors of Pseudomonas aeruginosa by salicylicacid attenuates its virulence on Arabidopsis thaliana and Caenorhabditis elegans[J]. InfectImmun,2005,73(9):5319～5328.
56. Robbins D.H，Margulies I,Stetler-Stevenson M,et al.,Hairy cell leukemia,a B-cell neoplasm thatis particularly sensitive to the cytotoxic effect of anti-Tac(Fv)-PE38(lmb-2)[J].Clin CancerRes,2000,6(2):693～700.
57. Ryan KJ,Ray CG (editors). Sherris Medical Microbiology [M].4th ed.McGraw Hill.2004，132～135.
58. Salazar D,Hernández Y,Palma S,González A,et al.,Antimicrobian activity of twelve drugsagainst Pseudomona aeruginosa strains from clinical isolates Bioquimia[J].2001,26(4):79～84.
59. Saint O, Romeyer F, Lebel P,et al.,Specificity of the Pseudomonas aeruginosa PAO1lipoproteinI gene as a DNA probe and PCR target region within the Pseudomonadaceae[J].J GeneralMicrobiol1992,138(4):733～741.
60. Schoni M,Macrolide antibiotic therapy in patients with cystic fibrosis.Swiss Med Wkly2003,133(21-22):297～301.
61. Sokol PA,Kooi C,Hodges R.S,et al.,Immunization with a Pseudomonas aeruginosa elastasepeptide reduces severity of experimental lung infections due to P.aeruginosa or Burkholderiacepacia[J].J Infect Dis,2000,181(5):1682～1692.
62. Spilker T,Coenye T,Vandamme P, et al., PCR based assay for differentiation of Pseudomonasaeruginosa from other Pseudomonas species recovered from cystic fibrosis Patients[J]. J ClinMicrobiol,2004,42(5):2074～2079.
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