致呼吸系统感染中铜绿假单胞菌整合子与耐药性的相关性研究
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摘要
目的:分析致呼吸系统感染的铜绿假单胞菌的耐药性,调查铜绿假单胞菌中Ⅰ类、Ⅱ类和Ⅲ类整合子的存在状况,以及整合子中基因盒的种类和排列;分析细菌携带基因盒与耐药表型的关系以及整合子与铜绿假单胞菌多重耐药性的关系,探讨整合子介导的铜绿假单胞菌多重耐药的机制。方法应用全自动细菌分析仪bioMerieux VITEK 2,测定97株铜绿假单胞菌对16种抗菌药物的敏感性;应用简并引物PCR方法,同时扩增整合子5’保守区的I类、Ⅱ类和Ⅲ类整合酶基因,对阳性PCR产物用限制性内切酶Hinf I作限制片段长度多态性(RFLP)分析进行整合子分类;对整合酶阳性标本进行整合子可变区的扩增并对大小相同的整合子进行酶切分析;对纯化后的整合子进行DNA测序。结果97株铜绿假单胞菌对16种抗菌药物的耐药率为4.2%~100%。72株(74.2%)细菌为多重耐药菌(耐受3种以上抗菌药物)。20.6%(20/97)的铜绿假单胞菌中检出整合子,均为I类整合子,未检出Ⅱ类和Ⅲ类整合子。20株整合酶阳性的铜绿假单胞菌中有17株含有基因盒,基因盒大小有3种类型:979bp、1833bp和2337bp。测序结果显示有二氢叶酸还原酶基因dfrl7和dhfrXII;氨基糖苷类修饰酶基因aadA2、aadA5、aadB和aac(6)-Ⅱ;羧基青霉素类耐药基因blaCARB-8。I类整合子阳性的菌株对抗菌药物的耐药率普遍比整合子阴性的菌株高,I类整合子与细菌多重耐药的发生有关。结论致呼吸系统感染的铜绿假单胞菌的耐药性强;铜绿假单胞菌I类整合子中的耐药基因盒,与早期使用的氨基糖苷类、甲氧苄啶等传统抗菌药物有着密切的联系;整合子参与了铜绿假单胞菌的耐药和多重耐药。
Objective To survey the antibiotic-resistance of isolates of Pseudomonas aeruginosa from respiratory infection and the distribution of classⅠ,ⅡandⅢintegrons in Pseudomonas aeruginosa and to investigate the characterization of integrons and gene cassette arrays. To analyze the association between gene cassettes and resistance phenotype as well as between integrons and multi-drug resistance and to explore the mechanism of integron-associated multi-drug resistance in these strains. Methods Antibiotic susceptibility of 97 strains of Pseudomonas aeruginosa were performed by using bioMerieux VITEK 2.Integrons were detected by PCR using degenerate primers targeted to 5 'conserved regions of classⅠ,ⅡandⅢintegrase genes, The class of the integron was determined by analyzing integrase PCR products by restriction fragment length polymorphism(RFLP) following digestion using Hinf I restriction enzyme. The variable region of integron was amplified by long fragment PCR ,and the PCR products were restricted with Hinf I. Three representative products were purified and sequenced. Results Resistant rates of 97 strains of Pseudomonas aeruginosa against 16 kinds of antibiotic ranged from 4.2% to 100%, 72(74.2%) isolates were multi-drug resistant(resistance to over 3 antibiotics). Integron was detected in 20/97(20.6%) Pseudomonas aeruginosa,which all were classⅠintegrons as PCR-RFLP analysis, no classⅡa ndⅢintegrons were detected. Seventeen of the 20 strains contained three kinds of gene cassettes:979bp,1833bp and 2337bp.These cassettes included dfrl7 and dhfrXII(genes for dihydrofolate reductase) ,aadA2, aadA5, aadB and aac (6) -Ⅱ(which were aminoglycoside-modifying enzyme genes), blaCARB-8(which was carboxyl Penicillin Resistance genes). The rate of drug resistance in isolates of integron positive was generally higher than that of in the integron negative strains. Integrons were significantly associated with multi-drug resistance to certain antibiotic. Conclusions High drug resistance was found in isolates of Pseudomonas aeruginosa from respiratory infection; Gene Cassetes in integron are significantly associated with old antimicrobial such as trimethoprim and early aminoglycosides. Integrons are associated with antibiotic-resistance and multi-drug resistance in Pseudomonas aeruginosa.
Objective To survey the antibiotic-resistance of isolates of Pseudomonas aeruginosa from respiratory infection and the distribution of classⅠ,ⅡandⅢintegrons in Pseudomonas aeruginosa and to investigate the characterization of integrons and gene cassette arrays. To analyze the association between gene cassettes and resistance phenotype as well as between integrons and multi-drug resistance and to explore the mechanism of integron-associated multi-drug resistance in these strains. Methods Antibiotic susceptibility of 97 strains of Pseudomonas aeruginosa were performed by using bioMerieux VITEK 2.Integrons were detected by PCR using degenerate primers targeted to 5 'conserved regions of classⅠ,ⅡandⅢintegrase genes, The class of the integron was determined by analyzing integrase PCR products by restriction fragment length polymorphism(RFLP) following digestion using Hinf I restriction enzyme. The variable region of integron was amplified by long fragment PCR ,and the PCR products were restricted with Hinf I. Three representative products were purified and sequenced. Results Resistant rates of 97 strains of Pseudomonas aeruginosa against 16 kinds of antibiotic ranged from 4.2% to 100%, 72(74.2%) isolates were multi-drug resistant(resistance to over 3 antibiotics). Integron was detected in 20/97(20.6%) Pseudomonas aeruginosa,which all were classⅠintegrons as PCR-RFLP analysis, no classⅡa ndⅢintegrons were detected. Seventeen of the 20 strains contained three kinds of gene cassettes:979bp,1833bp and 2337bp.These cassettes included dfrl7 and dhfrXII(genes for dihydrofolate reductase) ,aadA2, aadA5, aadB and aac (6) -Ⅱ(which were aminoglycoside-modifying enzyme genes), blaCARB-8(which was carboxyl Penicillin Resistance genes). The rate of drug resistance in isolates of integron positive was generally higher than that of in the integron negative strains. Integrons were significantly associated with multi-drug resistance to certain antibiotic. Conclusions High drug resistance was found in isolates of Pseudomonas aeruginosa from respiratory infection; Gene Cassetes in integron are significantly associated with old antimicrobial such as trimethoprim and early aminoglycosides. Integrons are associated with antibiotic-resistance and multi-drug resistance in Pseudomonas aeruginosa.
引文
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4. White PA, McIver CJ, Rawlinson WD. Integrons and gene cassettes in the entero- bacteriaceae[J]. Antimicrob Agents Chemother 2001:45(9):2658-2661.
5. Stokes HW, HallRM.A novel family of potentially mobile DNA elements encod- ing site-specific gene-integration functions: integrons[J]. Mol Microbiol. 1989 ;3(12): 1669-1683.
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7. Fluit A C,Schmitz F J.Resistance integrons and super-integrons[J].Clin Microbiol Infect,2004,10(4):272-288.
8. Rowe-Magnus D A,Mazel D.The role of integrons in antibiotic reistanee gene capture[J].Int J Med Microbiol,2002,292(2):115—125.
9. Fluit AC, Schmitz FJ. Class 1 integrons, gene cassettes, mobility, and epid- emiology [J]. Eur J Clin Microbiol Infect Dis. 1999;18(11):761-770.
10. 夏涵,府伟灵,陈鸣,等.快速提取细菌DNA方法的研究[J].现代预防医学,2005,32(5):571-573.
11. Daly M,BuckleyJ,Power,E,et al.Molecular characterization of Irish Salmonella enterica serotype typhimurium:detection of class I integrons and assessment of gene- tic relationships by DNA amplification fingerprinting[J]. Appl Environ Microbiol.2000; 66(2):614-619.
12. Didier M,Broderick D, Vera A, et al. Antibiotic Resistance in the ECOR Collection: Integrons and Identification of a Novel aad Gene[J]. Antimicrob Agents Chemother.2000;44(6):1568–1574.
13. Guerra B ,Soto S ,Cal S ,Mendoza MC. Antimicrobial resistance and spread of class 1 integrons among Salmonella serotypes[J]. Antimicrob Agents Chemother. 2000; 44(8): 2166- 2169 .
14. 万建华,张静萍,宋建,等.临床分离革兰阴性杆菌耐药性监测[J].中华医院感染学杂志,2005,15(3):336-338.
15. 王一兵,李卫光,朱其凤.山东省医院感染控制网下呼吸道感染病原菌分布及耐药分析[J].中华医院感染学杂志,2005,15(5):490-492.
16. 陈晓辉,魏衍超,温得良,等.危重病房的细菌耐药监测及分析[J].中华医院感染学杂志,2005,15(8):946-949.
17. 许宏涛,张秀珍.医院感染铜绿假单胞菌多重耐药机制的研究[J].中国抗感染化疗杂志,2005,5(3):141-145.
18. 杨凤华,孟祥东,周景欣,等.呼吸道感染中铜绿假单胞菌药物敏感性分析[J].大连医科大学学报,2007,29(3):301-302
19. Naohiro Shibata,Yohei Doi,Kunikazu Yamane,et a1.PCR Typing of Genetic Determinants for Metallo-β-Lactamases and Integrases Carried by Gram-Negative Bacteria Isolated in Japan,with Focus on the Class 3 Integron[J].Journal of Microbiology,Dec,2003,5 407-5 413.
20. 杜艳,陈端,单斌等.整合子相关的铜绿假单胞菌耐药性分析[J]检验医学,2005,20(6):556-558.
21. Martinez-Freijo P, Fluit A C, Schmitz F J,et al.Class I integrons in Gram-negative isolates from different European hospitals and association with decreased susceptibility to multiple antibiotic compounds[J]. J. Antimicrob. Chemother.1998,42:689-696.
22. Leverstein-van Hall MA, Box AT, Blok HE, et al. Evidence of transfer in tegron-mediated multidrug-resistant of extensive interspecies antimicrobial resistance genes among Enterobacteriaceae in a clinical setting[J]. Infect Dis. 2002:186(1):49-56.
23. MaguireA J,Brown DF,G ray JJ,et al. Rapid screening technique for class 1 integrons in Enterobacteriaceae and nonfermenting gram-negative bacteria and its use in molecular epidemiology[J].Antimicrob Agents Chemother.2001;45(4):1022-1029.
24. Schmitz FJ, Hafner D, Geisel R, et al. class I integrons in Escherichia coli Increased prevalence of Klebsiella species, and Enterobacter species isolates over a 7-year period in a German university hospital[J]. J Clin Microbiol.2001;39(10): 3724-3726.
25. Bert F.Branger C,Lambert-Zechovsky N.Identification of PSE and OXA B-laetamase genes in Pseudomonas aeruginosa using PCR-restriction fragment length polymorphism[J].J. Antimicrob. Chemother.2002,50(1):11—18.
26. Ribera A, Vila J, Fernandez-Cuenca F, et al. Type 1 integrons in epidemiologi- cally unrelated Acinetobacter baumannii isolates collected at Spanish hospitals[J]. Antimicrob Agents Chemother.2004:48(1):364-365.
27. Martinez-Freijo P, Fluit AC, Schmitz FJ, et al. Many class I integrons comprise distinct stable structures occurring in 41 differents pecies of Enterobacteriaceae isolated from widespread geographic regions in Europe[J]. Antimicrob Agents Chemother. 1999 ;43 (3):686-689.
28. Leverstein-Van Hall MA, Paauw A, Box AT, et al. Presence of integron- associated resistance in the community is Widespread and contributes to multidrug resistance in the hospital[J].J Clin Microbiol.2002;40(8):3038-3040.
29. Ploy MC,Denis F ,Courvalin P ,et al. Molecular characterization of integrons in Acinetobacter baumannii:description of ahybrid class 2 in tegron[J].Antimicrob Agents Chemother. 2000; 44(10):2684-2688.
30. Aarati N. Rao, Miriam Barlow, Leigh Ann Clark, et al. Class 1 Integrons in Resistant Escherichia coli and Klebsiella spp.,US Hospitals[J]. Emerging Infectious Diseases .2006;12( 6):1011-1014.
31. Rowe-Magnus DA, Mazel D. R esistance gene capture[J]. Curr Opin Microbiol. 1999;2(5):483-488
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