部分铜绿假单胞菌临床分离株中整合子的分布、耐药性分析及其分子生物学特征
|
摘要
目的:
了解整合子在部分铜绿假单胞菌临床分离株中的分布及耐药性情况,分析整合子与铜绿假单胞菌耐药和多重耐药的相关性。
了解整合子可变区携带的耐药基因型,研究其分子生物学特征,探讨整合子在细菌耐药性产生和传播扩散中的作用。
材料与方法:
1.菌株来源:
1997年至2003年间江苏省人民医院收集并随机抽取的非重复性并具有临床意义的铜绿假单胞菌菌株,共47株。
2.方法:
以煮沸法提取47株铜绿假单胞菌的总DNA为模板,根据1、3类整合子各自整合酶基因保守区设计的引物,PCR扩增以筛选出各自阳性菌株;再将整合子阳性菌株与铜绿假单胞菌PU21共孵,行转移接合试验;采用琼脂平皿稀释法对47株菌株以及转移接合子进行MIC测定,分析药敏情况。
对1类整合子阳性菌株,根据1类整合子5'和3'保守区设计的通用可变区引物,PCR扩增其可变区。
将可变区PCR产物胶回收纯化后,进行序列分析,结果与Genbank序列进行比对,确定可变区所含耐药基因盒。
以1类整合子阳性菌株与其对应接合子的总DNA为模板,根据1类整合子可变区测序结果设计所含耐药基因盒对应的引物,PCR扩增每一个耐药基因以确认克隆测序结果及初步检验整合子是否处于质粒上。
采用碱裂解法抽取整合子阳性菌株与其对应接合子的质粒。
采用脉冲场凝胶电泳(pulsed field gel electrophoresis,PFGE)分析整合子阳性菌株的同源性。
结果:
47株铜绿假单胞菌经PCR法29株检测到含有1类整合酶,占所有分离菌株的61.7%(29/47),3类整合子未检出。药敏结果显示1类整合子阳性菌株较阴性菌株耐药率明显升高;整合子阳性菌株对哌拉西林、头孢他啶、阿米卡星、庆大霉素、氯霉素、链霉素的耐药率最高,达到100%。29株1类整合子阳性菌株均转移接合成功;转移接合子与野生株比较,对哌拉西林/他唑巴坦、亚胺培南、环丙沙星均敏感,MIC_(90)分别下降了8、128和128倍。
29株携带的1类整合子可变区扩增产物均约为4.3kb。
序列分析结果表明:发现该可变区包含4个耐药基因盒,形成一个新的组合形式aac(6')-Ⅱ-aadA13-cmlA8-oxa-10,已提交Genbank,序列号为EU182575。其中aadA13首次在铜绿假单胞菌中发现。同时第三个开放阅读框的序列未见报道,与已知的cmlA5同源性最高为95%,将其命名为cmlA8。
无论野生株或接合子经PCR均分别扩增出上述4个耐药基因盒,序列测定与克隆测序一致。
无论野生株或接合子均抽提出大小一致,大于10Kb的耐药性质粒。
脉冲场凝胶电泳的分析结果显示,所有1类整合子阳性菌株具有高度同源性,存在克隆传播和可能相关关系。
结论:
本院铜绿假单胞菌临床分离株中有1类整合子的流行。1类整合子阳性的菌株较阴性菌株对多种抗生素更容易产生耐药。结合药敏表型及整合子携带耐药基因的基因型,整合子与铜绿假单胞菌耐药和多重耐药相关。和野生株相比,转移接合子对哌拉西林/他唑巴坦、亚胺培南、环丙沙星均敏感。
该1类整合子可变区基因相同,携带有新的耐药基因盒组合形式。整合子位于接合性质粒。这些1类整合子阳性菌株间存在着水平或垂直传播的可能性。
Objective:
To investigate the disseminaton of integons carried by Pseudomonas aeruginosa clinical isolates from our hospital and to analyze the correlation between integrons and the multi-drug resistance of Pseudomonas aeruginosa.
To study the genotypes of variable region and the biochemical properties of integrons and to disclose its action in the acquisition, transmission or dissemination of antibiotic resistance.
Materials and Methods:
1. Isolates:
Totally 47 strains of Pseudomonas aeruginosa were collected between 1997 and 2003 from infection cases in Jiangsu Province Hospital and randomly chosen for study.
2. Method:
All the isolates were candidates for screening class 1 and class 3 integrons by using specific primers located on integron-encoded integrase gene, the intI1 gene and intI3 gene, respectively; conjugation experiments was performed between integron positive isolates and PU21 to transfer resistance plasmid; agar dilution method was used to determine MICs of 9 antibiotics against 47 isolates and the transconjugants.
The variable regions of class 1 integrons were amplified by using universal primers located on 5' and 3' conserved segments.
The PCR amplicons were purified. Then DNA sequencing and blastn program was used to ascertain the genotype of the variable region of class 1 integrons.
Every gene cassette in wild-type isolates and transconjugants were amplified by primers designed according to the results of DNA sequencing of variable regions in class 1 integrons, respectively. The DNA sequence of PCR products was determined by direct sequencing.
Plasmid DNA was extracted from wild-type isolates and transconjugants by alkaline lysis.
Pulsed-field gel electrophoresis method was performed to analyze the homology of integron positive isolates.
Results:
Of 47 isolates, 29 strains of Pseudomonas aeruginosa were found containing class 1 integron, and no class 3 integron was detected. All class 1 integron isolates were successfully conjugated to the recipient cell. Susceptibility testing revealed that the antibiotic resistance rate of the integron positive strains was higher than that of the integron negative strains. All class 1 integron positive wild-type isolates and the corresponding transconjugants exhibited an overall resistant property to piperacillin, ceftazidime, gentamicin, amikacin, chloramphenicol and streptomycin. Compared with the wild-type Pseudomonas aeruginosa, the transconjugants were all susceptible to piperacillin-tazobactam, ciprofloxacin and imipenem, and MIC_(90) values of those antimicrobial agents decreased about 8 times, 128 times and 128 times, respectively.
A length of 4,300bp variable region was found in all the class 1 integron positive strains by PCR amplification. The result of DNA sequencing revealed that it was comprised of a new array of aac(6')- II -aadA13-cmlA8-oxa-10 gene cassettes, and the Genbank number is EU182575. According to the database in Genbank, aadA13 was previously unreported cassette in Pseudomonas aeruginonsa; chloramphenicol efflux pumping determinant, named as cmlA8 shared 95% nucleotide similarity to cmlA5 gene.
All four gene cassettes harbored by class 1 integron in wild-type isolates and transconjugants were detected by PCR amplification, respectively. The direct sequencing results were identical to the results of DNA sequencing of variable region.
The length of plasmids DNA extracted from all wild-type isolates and transconjugants were identical to each other and larger than 10Kb.
All those class 1 integron positive isolates showed high homology by PFGE pattern analysis after digestion with Spe I .
Conclusion:
Class 1 integron was wildspread in Pseudomonas aeruginosa clinical isolates from our hospital. Susceptibility testing revealed that the antibiotic resistance rate of the integron positive strains to many antimicrobial agents was significantly higher than that of the integron negative strains. The resistance phenotype and genotype of the variable region harbored by integons suggested that the presence of integron was closely associated with resistance and multi-drug resistance of Pseudomonas aeruginosa. Compared to the wild-type isolates, the correlative transconjugants were susceptible to piperacillin-tazobactam, ciprofloxacin and imipenem.
Class 1 integrons disseminated among some clinical isolates of Pseudomonas aeruginosa in our hospital possessed novel array of gene cassettes and located on transferable plasmid. Horizontal or clonal (vertical) transmission might exist between these Class 1 integrons positive isolates.
了解整合子在部分铜绿假单胞菌临床分离株中的分布及耐药性情况,分析整合子与铜绿假单胞菌耐药和多重耐药的相关性。
了解整合子可变区携带的耐药基因型,研究其分子生物学特征,探讨整合子在细菌耐药性产生和传播扩散中的作用。
材料与方法:
1.菌株来源:
1997年至2003年间江苏省人民医院收集并随机抽取的非重复性并具有临床意义的铜绿假单胞菌菌株,共47株。
2.方法:
以煮沸法提取47株铜绿假单胞菌的总DNA为模板,根据1、3类整合子各自整合酶基因保守区设计的引物,PCR扩增以筛选出各自阳性菌株;再将整合子阳性菌株与铜绿假单胞菌PU21共孵,行转移接合试验;采用琼脂平皿稀释法对47株菌株以及转移接合子进行MIC测定,分析药敏情况。
对1类整合子阳性菌株,根据1类整合子5'和3'保守区设计的通用可变区引物,PCR扩增其可变区。
将可变区PCR产物胶回收纯化后,进行序列分析,结果与Genbank序列进行比对,确定可变区所含耐药基因盒。
以1类整合子阳性菌株与其对应接合子的总DNA为模板,根据1类整合子可变区测序结果设计所含耐药基因盒对应的引物,PCR扩增每一个耐药基因以确认克隆测序结果及初步检验整合子是否处于质粒上。
采用碱裂解法抽取整合子阳性菌株与其对应接合子的质粒。
采用脉冲场凝胶电泳(pulsed field gel electrophoresis,PFGE)分析整合子阳性菌株的同源性。
结果:
47株铜绿假单胞菌经PCR法29株检测到含有1类整合酶,占所有分离菌株的61.7%(29/47),3类整合子未检出。药敏结果显示1类整合子阳性菌株较阴性菌株耐药率明显升高;整合子阳性菌株对哌拉西林、头孢他啶、阿米卡星、庆大霉素、氯霉素、链霉素的耐药率最高,达到100%。29株1类整合子阳性菌株均转移接合成功;转移接合子与野生株比较,对哌拉西林/他唑巴坦、亚胺培南、环丙沙星均敏感,MIC_(90)分别下降了8、128和128倍。
29株携带的1类整合子可变区扩增产物均约为4.3kb。
序列分析结果表明:发现该可变区包含4个耐药基因盒,形成一个新的组合形式aac(6')-Ⅱ-aadA13-cmlA8-oxa-10,已提交Genbank,序列号为EU182575。其中aadA13首次在铜绿假单胞菌中发现。同时第三个开放阅读框的序列未见报道,与已知的cmlA5同源性最高为95%,将其命名为cmlA8。
无论野生株或接合子经PCR均分别扩增出上述4个耐药基因盒,序列测定与克隆测序一致。
无论野生株或接合子均抽提出大小一致,大于10Kb的耐药性质粒。
脉冲场凝胶电泳的分析结果显示,所有1类整合子阳性菌株具有高度同源性,存在克隆传播和可能相关关系。
结论:
本院铜绿假单胞菌临床分离株中有1类整合子的流行。1类整合子阳性的菌株较阴性菌株对多种抗生素更容易产生耐药。结合药敏表型及整合子携带耐药基因的基因型,整合子与铜绿假单胞菌耐药和多重耐药相关。和野生株相比,转移接合子对哌拉西林/他唑巴坦、亚胺培南、环丙沙星均敏感。
该1类整合子可变区基因相同,携带有新的耐药基因盒组合形式。整合子位于接合性质粒。这些1类整合子阳性菌株间存在着水平或垂直传播的可能性。
Objective:
To investigate the disseminaton of integons carried by Pseudomonas aeruginosa clinical isolates from our hospital and to analyze the correlation between integrons and the multi-drug resistance of Pseudomonas aeruginosa.
To study the genotypes of variable region and the biochemical properties of integrons and to disclose its action in the acquisition, transmission or dissemination of antibiotic resistance.
Materials and Methods:
1. Isolates:
Totally 47 strains of Pseudomonas aeruginosa were collected between 1997 and 2003 from infection cases in Jiangsu Province Hospital and randomly chosen for study.
2. Method:
All the isolates were candidates for screening class 1 and class 3 integrons by using specific primers located on integron-encoded integrase gene, the intI1 gene and intI3 gene, respectively; conjugation experiments was performed between integron positive isolates and PU21 to transfer resistance plasmid; agar dilution method was used to determine MICs of 9 antibiotics against 47 isolates and the transconjugants.
The variable regions of class 1 integrons were amplified by using universal primers located on 5' and 3' conserved segments.
The PCR amplicons were purified. Then DNA sequencing and blastn program was used to ascertain the genotype of the variable region of class 1 integrons.
Every gene cassette in wild-type isolates and transconjugants were amplified by primers designed according to the results of DNA sequencing of variable regions in class 1 integrons, respectively. The DNA sequence of PCR products was determined by direct sequencing.
Plasmid DNA was extracted from wild-type isolates and transconjugants by alkaline lysis.
Pulsed-field gel electrophoresis method was performed to analyze the homology of integron positive isolates.
Results:
Of 47 isolates, 29 strains of Pseudomonas aeruginosa were found containing class 1 integron, and no class 3 integron was detected. All class 1 integron isolates were successfully conjugated to the recipient cell. Susceptibility testing revealed that the antibiotic resistance rate of the integron positive strains was higher than that of the integron negative strains. All class 1 integron positive wild-type isolates and the corresponding transconjugants exhibited an overall resistant property to piperacillin, ceftazidime, gentamicin, amikacin, chloramphenicol and streptomycin. Compared with the wild-type Pseudomonas aeruginosa, the transconjugants were all susceptible to piperacillin-tazobactam, ciprofloxacin and imipenem, and MIC_(90) values of those antimicrobial agents decreased about 8 times, 128 times and 128 times, respectively.
A length of 4,300bp variable region was found in all the class 1 integron positive strains by PCR amplification. The result of DNA sequencing revealed that it was comprised of a new array of aac(6')- II -aadA13-cmlA8-oxa-10 gene cassettes, and the Genbank number is EU182575. According to the database in Genbank, aadA13 was previously unreported cassette in Pseudomonas aeruginonsa; chloramphenicol efflux pumping determinant, named as cmlA8 shared 95% nucleotide similarity to cmlA5 gene.
All four gene cassettes harbored by class 1 integron in wild-type isolates and transconjugants were detected by PCR amplification, respectively. The direct sequencing results were identical to the results of DNA sequencing of variable region.
The length of plasmids DNA extracted from all wild-type isolates and transconjugants were identical to each other and larger than 10Kb.
All those class 1 integron positive isolates showed high homology by PFGE pattern analysis after digestion with Spe I .
Conclusion:
Class 1 integron was wildspread in Pseudomonas aeruginosa clinical isolates from our hospital. Susceptibility testing revealed that the antibiotic resistance rate of the integron positive strains to many antimicrobial agents was significantly higher than that of the integron negative strains. The resistance phenotype and genotype of the variable region harbored by integons suggested that the presence of integron was closely associated with resistance and multi-drug resistance of Pseudomonas aeruginosa. Compared to the wild-type isolates, the correlative transconjugants were susceptible to piperacillin-tazobactam, ciprofloxacin and imipenem.
Class 1 integrons disseminated among some clinical isolates of Pseudomonas aeruginosa in our hospital possessed novel array of gene cassettes and located on transferable plasmid. Horizontal or clonal (vertical) transmission might exist between these Class 1 integrons positive isolates.
引文
1. National Nosocomial Infection Surveillance System. National Nosocomial Infection Surveillance (NNIS) System report, data summary from January 1992 through June 2004, issued October 2004. Am. J. Infect. Control. 2004, 32:470-485.
2. Navon-Venezia S, Ben-Ami R, Carmeli Y. Update on Pseudomonas aeruginosa and Acinetobacter baumannii infections in the healthcare setting. Cur Opin Infect Dis. 2005, 18(4):306-313.
3. Quale, J., S. Bratu, J. Gupta, and D. Landman. Interplay of efflux system, ampC, and oprD expression in carbapenem resistance of Pseudomonas aeruginosa clinical isolates. Antimicrob Agents Chemother. 2006, 50:1633-1641.
4. Pai H, Kim J W, Kim J ,et al . Carbapenem Resistance Mechanism in Pseudomonas Aeruginosa Clinical Isolates. Antimicrob Agents Chemother. 2001, 45:480-484.
5. Mac Gowan AP, Bowker KE. Mechanism of fluoroquinolone resistance is an important factor in determining the antimicrobial effect of gemifloxacin against Streptococcus pneumoniae in an in vitro pharmacokinetic model. Antimicrob Agents Chemother. 2003, 47(3):1096.
6. Sobel ML, Hocquet D, Cao L, et al. Mutations in PA3574 (na1D) lead to increased MexAB-OprM expression and multidrug resistance in laboratory and clinical isolates of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2005, 49(5): 1782-1786.
7. Mah TF, O'Toole GA. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol. 2001, 9(1): 34-39.
8. Docquier JD, Riccioml, Mugnaiolic, et al. IMP212, a new plasmid encoded metallo-beta-lactamase from Pseudomonas putide clinical isolate. Antimicrob Agents Chemother. 2003, 47(5): 1522-1528.
9. Verdet C, Arlet G, Barnaud G, et al. A novel integron in Salmonella enterica serovar Enteritidis, carrying the bla(DHA-l) gene and its regulator gene ampR, originated from Morganella morganii. Antimicrob Agents Chemother. 2000, 44: 222-225.
10.Stokes H W, Hall R M. A novel family of potentially mobile DNA elements encoding site-specific gene integration functions: integrons. MolMicrobiol. 1989, 3 (12): 1669-1683.
11.Hall R M, Stokes H W. Integrons: novel DNA elements which capture genes by site-specific recombination. Genetica. 1993, 90(223): 115-132.
12.Brown H J, Srokes H W, Hall R M. The integrons InO, In2, and In5 are defective transposon derivatives. J Bacteriol, 1996, 178 (15): 4429-4437.
13.Fluit A C, Schmitz F J. Resistance integrons and super-integrons. ClinMicrobiol Infect. 2004, 10 (4): 272-288.
14.许宏涛,宣天芝,陶凤荣,等。3种非发酵革兰阴性杆菌5年的分离率及耐药趋势回顾。中国临床药理学杂志。2004,20:219-221。
15.植志全,何志恒,江鹏,等。多重耐药绿脓假单胞菌β-内酰胺类氨基糖苷类耐药相关基因研究。中华检验医学杂志。2005,28(11):1211-1214。
16.Gu B,Tong M Q,Zhao W J,et al.Prevalence and characterization of class Ⅰ integrons among Pseudomonas aeruginosa and Acinetobacter baumannii isolates from patients in Nanjing,China.J Clin Microbiol.2007,45:241-243.
17.Falagas ME,Bliziotis IA,Kasiakou SK,et al.Outcome of infections due to pandrug resistant(PDR)gram-negative bacteria.BMC Infect Dis.2005,5:24.
1.Levesque C,Brassard S,Lapointe J,et al.Diversity and relative strength of tandem p romoters for the antibiotic-resistance genes of several integrons.Gene.1994,142(1):49-54.
2.Fluit A C,Schmitz F J.Class 1 integrons,gene cassettes,mobility and epidemiology.Eur J Clin Microbiol Infect Dis.1999,18(11):761-770.
3.Collis C M,Hall R M.Expression of antibiotic resistance genes in the integrated cassettes of integrons.Antimicrob Agents Chemother.1995,39(1):155-162.
4.Shmara A,Weinsetel N,Dery K J,et al.Systematic analysis of a conserved region of the Aminoglycoside 6'-N-Acetyltransferase type Ib. Antimicrob Agents Chemother. 2001, 45:3287-3292.
5. Kehrenberg C, Catry B, Haesebrouck F, et al. Novel Spectinomycin/ Streptomycin resistance gene, aadA14, from Pasteurella multocida. Antimicrob Agents Chemother. 2005, 49:3046-3049.
6. Kadlec K, Kehrenberg C, Schwarz S. Efflux-mediated resistance to florfenicol and/or chloramphenicol in Bordetella bronchiseptica: identification of a novel chloramphenicol exporter. J Antimicrob Chemother. 2007, 59:191-196.
7. Bissonnette L, Champetier S, Buisson J P, et al. Characterization of the nonenzymatic Chloramphenicol Resistance (cm1A) gene of the In4 integron of Tnl696: similarity of the product to transmembrane transport proteins. J Bacterial. 1991, 173:4493-4502.
8. Ploy M C, Courvalin P, Lambert T. Characterization of In40 of Enterobacter aerogenes BM2688, a class 1 integron with two new gene cassettes, cmlA2 and qacF. Antimicrob Agents Chemother. 1998, 42:2557-2563.
9. Danel F, Hall L M C, Livermore D M. Laboratory mutants of OXA-10 (3-lactamase giving ceftazidime resistance in Pseudomonas aeruginosa. J Antimicrob Chemother. 1999, 43:339-344.
10. Coudron PE, Ellen SM, Kenneth ST. Occurrence and detection of AmpC Beta-lactamases among Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates at a Veterans Medical Center. J Clin Microbiol. 2000, 38: 1791-1796.
11. Guerra B, Soto S M, Arguelles J M, et al. Multidrug resistance is mediated by large plasmids carrying a class 1 integron in the emergent Salmonella enterica serotype [4,5,12:i:2]. Antimicrob Agents Chemother. 2001, 45:1305-1308.
12. Kaneko K, Okamoto R, Nakano R, et al. Gene mutations responsible for overexpression of AmpC beta-lactamase in some clinical isolates of Enterobacter cloacae. J Clin Microbiol. 2005, 43 (6):2955-2958.
13. Jean-Paul Pirnay, Daniel De Vos, Dimitris Mossialos. Analysis of the Pseudomonas aeruginosa oprD gene from clinical and environmental isolates. Environmental Microbiology. 2002, 4(12):872-882.
14. Mac Gowan AP, Bowker KE. Mechanism of fluoroquinolone resistance is an important factor in determining the antimicrobial effect of gemifloxacin against Streptococcus pneumoniae in an in vitro pharmacokinetic model. Antimicrob Agents Chemother. 2003, 47(3): 1096.
15. Tenover FC, Arbeit RD, Goering RV, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol. 1995, 33 (9):2233 -2239.
1. National Nosocomial Infection Surveillance System. National Nosocomial Infection Surveillance (NNIS) System report, data summary from January 1992 through June 2004, issued October 2004. Am. J. Infect. Control. 2004, 32:470-485.
2. Carmeli, Y., N. Troillet, G. Eliopoulos, M. H. Samore. Emergence and of antibiotic-resistant Pseudomonas aeruginosa: comparison of risks associated with different antipseudomonal agents. Antimicrob. Agents Chemother. 1999,43:1379-1382.
3. Cosgrove, S. E., and Y. Carmeli. The impact of antimicrobial resistance on health and economic outcomes. Clin. Infect. Dis. 2003, 36:1433-1437.
4. Stokes H W, Hall R M. A novel family of potentially mobile DNA elements encoding site-specific gene integration functions: integrons. MolMicrobiol. 1989, 3 (12): 1669-1683.
5. Barbolla R, Catalano M, Orman BE, et al . Class 1 integrons increase trimethoprim-sulfamethoxazole MICs against epidemiologically unrelated Stenotrophomonas maltophilia isolates. Antimicrob Agents Chemother. 2004, 48 (2):666-669.
6. Hall R M, Stokes H W. Integrons: novel DNA elements which capture genes by site-specific recombination. Genetica. 1993, 90(223): 115-132.
7. Brown H J, Srokes H W, Hall R M. The integrons InO, In2, and In5 are defective transposon derivatives. J Bacteriol. 1996, 178 (15): 4429-4437.
8. Hall RM, Collis CM. Mobile gene cassettes and integrons: capture and spread of genes by site-specific recombination. Mol Microbiol. 1995, 15: 593-600.
9. Collis CM, Hall RM. Expression of antibiotic resistance genes in the integrated cassettes of integrons. Antimicrob Agents Chemother. 1995, 39: 155-162.
10.Radstrom P, Skold O. Transposon Tn5090 of plasmid R751, which carries an integron, is related to Tn7, Mu, and the retroelements. J Bacteriol. 1994, 176: 3257-3268.
11.Arakawa Y, Murakami M, Suzuki K, et al. A novel integron-like element carrying the metallo-beta-lactamase gene blaIMP. Antimicrob Agents Chemother. 1995, 39: 1612-1615.
12.Senda K, Arakawa Y, Ichiyama S, et al. PCR detection of metallo-beta-lactamase gene (blaIMP) in gram-negative rods resistant to broad-spectrum beta-lactams. J Clin Microbiol. 1996, 34: 2909-2913.
13.Fluit A C, Schmitz F J. Resistance integrons and super-integrons. ClinMicrobiol Infect. 2004, 10 (4): 272-288.
14.Dalsgaard A, Forslund A, Serichantalergs O, Sandvang D.Distnbution and content of class 1 integrons in different Vibrio cholerae O-serotype strains isolated in Thailand. Antimicrob Agents Chemother. 2000,44: 1315-1321.
15.Nesvera J, Hochmannova J, Patek M. An integron of class 1 is present on the plasmid pCG4 from gram-positive bacterium Corynebacterium glutamicum. FEMS Microbiol Lett. 1998,169: 391-395.
16.Martin C, Timm J. Transposition of an antibiotic resistance element in mycobacteria. Nature. 1990, 345: 739-743.
17.Fluit A C, Schmitz F J. Class 1 integrons, gene cassettes, mobility and epidemiology.Eur J Clin Microbiol Infect Dis.1999,18 (11) :761-770.
18.Hansson K, Sundstrom L, Pelletier A, et al. Int I 2 integron integrase in Tn7. JBacteriol. 2002, 184(6): 1714- 1721.
19.Hall RM, Collis CM, Kim MJ, et al. Mobile gene cassettes and integrons in elolution. Ann N Y Acad Sci. 1999, 18 (5): 68-80.
20.A. Gassama - Sowa, M. H. Dialloa , C. S. Boyeb , et al. Class 2 integron - associated antibiotic resistance in Shigella sonnei isolates in Dakar , Senegal. International Journal of Antimicrobial Agents. 2006, 27 (3): 267-270.
21.Miko A, Pries K, SchroeterA, et al. Multiple drug resistance in D-tartrate-positive salmonella enterica serovar paratyphiB isolates from poulty is mediated by class 2 integrons inserted into the bacterial chromosome.Antimicrob Agents Chemother. 2003,47(11) :3640-3643.
22.Collis CM, Kim MJ, Partridge SR, et al. Characterization of the class 3 integron and the site- specific recombination system it determines.J Bacteriol. 2002, 184(11): 3017- 3026.
23.Mazel D, Dychinco B , Webb V A ,et al . A distinctive class of integron in the Vibrio cholerae genome. Sience. 1998, 280:605 - 608.
24.Rowe Magnus D A, Guerout A M, Mazel D. Super integrons. Res Microbiol. 1999, 150:641 - 651.
25.Rowe MagnusD A, GueroutAM, Ploncard P, et al. The evolutionary history of chromosomal super-integrons provides an ancestry for multiresistant integrons. Proc Natl Acad Sci USA. 2001, 98 (2): 652-657.
26.Rowe Magnus D A, Guerout A M, Mazel D. Bacterial resistance evolution by recruitment of super-integron gene cassettes. Mol Microbiol. 2002, 43 (6): 1657-1669.
27.Recchia G D, Hall R M. Origins of the mobile gene cassettes found in integrons. TrendsMicrobiol. 1997, 5 (10): 389-394.
28.Stokes H W, OpGorman D B, Recchia G D, et al Structure and function of 59-base element recombination sites associated with mobile gene cassettes. MolMicrobiol. 1997, 26 (4): 731-745.
29.Doublet B, Boyd D, Mulvey MR, et al. The Salmonella genomic island1 is an integrative mobilizable element. MolMicrobiol. 2005, 55(6):1911 - 1924.
30.Collis C M, Hall R M. Site-specific deletion and rearrangement of integron insert genes catalyzed by the integron DNA integrase. J Bacteriol. 1992, 174(5): 1574-1585.
31.Collis C M, Hall R M. Expression of antibiotic resistance genes in the integrated cassettes of integrons. Antimicrob Agents Chemother. 1995, 39(1):155-162.
32.Levesque C, Brassard S, Lapointe J, et al. Diversity and relative strength of tandem p romoters for the antibiotic-resistance genes of several integrons. Gene. 1994, 142 (1): 49-54.
33.Maguire AJ, Brown DF, Gray JJ, Desselberger U. Rapid screening technique for class 1 integrons in Enterobacteriaceae and nonfermenting gram-negative bacteria and its use in. molecular epidemiology. Antimicrob Agents Chemother. 2001, 45: 1022-1029.
34.Girlich D, Poirel L, Leelaporn A, et al. Molecular epidemiology of the integron-located VEB-1 extendedspectrum beta-lactamase in nosocomial enterobacterial isolates in Bangkok, Thailand. J Clin Microbiol. 2001, 39: 175-182.
35.Partridge SR, Collis CM, Hall RM. Class 1 integron containing a new gene cassette, aadA10, associated with Tnl404 from R151. Antimicrob Agents Chemother. 2002, 46: 2400-2408.
36.Wal sh TR, Toleman MA, Poirel L , et al . Metallo-beta-lactamases: the quiet before t he storm. Clin Microbiol Rev. 2005, 18(2): 306-325.
37.Docquier JD,Riccioml,Mugnaiolic,et al.IMP212,a new plasmid encoded metallo-beta-lactamase from Pseudomonas putide clinical isolate.Antimicrob Agents Chemother.2003,47(5):1522-1528.
38.Verdet C,Arlet G,Barnaud G,et al.A novel integron in Salmonella enterica serovar Enteritidis,carrying the bla(DHA-1)gene and its regulator gene ampR,originated from Morganella morganii.Antimicrob Agents Chemother.2000,44:222-225.
39.Poirel L,Heritier C,Tolun V,et al.Emergence of oxacillinase mediated resistance to imipenem in Klebsiella pneumoniae.Antimicrob Agents Chemother.2004,48(1):15-22.
40.Dubois V,Poirel L,Marie C.Molecular characterization of a novel class 1 integron containing bla(GES-1)and a fused product of aac3-Ib /aac6'-Ib' gene cassettes in Pseudomonas aeruginosa.Antimicrob Agents Chemother.2002,46:638-645.
41.Martinez P,Fluit AC,Schmitz FJ,et al.Class Ⅰ integron in Gram-negtive isolates from different European hospitals and association with decreased susceptibility to multiple antibiotic compounds.J Antimicrob Chemother.1998,42(6):689-696.
42.顾兵,童明庆,等。南京地区铜绿假单胞菌的整合子流行性研究。临床检验杂志。2006,24(2):118-121。
43.Duijkeren EV,BoxAT,Schellen P,et al.Class 1 integrons in Enterobacteriaceae isolated from clinical infections of horses and dogs in The Netherlands. Microb Drug Resist. 2005, 11 (4): 383 -386.
44.Nemergut DR, Martin AP, Schmidt SK. Integron diversity in heavy-metal-contaminated mine tailings and inferences about integron evolution. Appl EnvironMicrobiol. 2004, 70 (2): 1160 -1168.
45.Rosser SJ, Young HK, Identification and characterization of class I integronsin bacteria from an aquatic environment. J Antimicrob Chemother. 1999,44(1): 11-18.
2. Navon-Venezia S, Ben-Ami R, Carmeli Y. Update on Pseudomonas aeruginosa and Acinetobacter baumannii infections in the healthcare setting. Cur Opin Infect Dis. 2005, 18(4):306-313.
3. Quale, J., S. Bratu, J. Gupta, and D. Landman. Interplay of efflux system, ampC, and oprD expression in carbapenem resistance of Pseudomonas aeruginosa clinical isolates. Antimicrob Agents Chemother. 2006, 50:1633-1641.
4. Pai H, Kim J W, Kim J ,et al . Carbapenem Resistance Mechanism in Pseudomonas Aeruginosa Clinical Isolates. Antimicrob Agents Chemother. 2001, 45:480-484.
5. Mac Gowan AP, Bowker KE. Mechanism of fluoroquinolone resistance is an important factor in determining the antimicrobial effect of gemifloxacin against Streptococcus pneumoniae in an in vitro pharmacokinetic model. Antimicrob Agents Chemother. 2003, 47(3):1096.
6. Sobel ML, Hocquet D, Cao L, et al. Mutations in PA3574 (na1D) lead to increased MexAB-OprM expression and multidrug resistance in laboratory and clinical isolates of Pseudomonas aeruginosa. Antimicrob Agents Chemother. 2005, 49(5): 1782-1786.
7. Mah TF, O'Toole GA. Mechanisms of biofilm resistance to antimicrobial agents. Trends Microbiol. 2001, 9(1): 34-39.
8. Docquier JD, Riccioml, Mugnaiolic, et al. IMP212, a new plasmid encoded metallo-beta-lactamase from Pseudomonas putide clinical isolate. Antimicrob Agents Chemother. 2003, 47(5): 1522-1528.
9. Verdet C, Arlet G, Barnaud G, et al. A novel integron in Salmonella enterica serovar Enteritidis, carrying the bla(DHA-l) gene and its regulator gene ampR, originated from Morganella morganii. Antimicrob Agents Chemother. 2000, 44: 222-225.
10.Stokes H W, Hall R M. A novel family of potentially mobile DNA elements encoding site-specific gene integration functions: integrons. MolMicrobiol. 1989, 3 (12): 1669-1683.
11.Hall R M, Stokes H W. Integrons: novel DNA elements which capture genes by site-specific recombination. Genetica. 1993, 90(223): 115-132.
12.Brown H J, Srokes H W, Hall R M. The integrons InO, In2, and In5 are defective transposon derivatives. J Bacteriol, 1996, 178 (15): 4429-4437.
13.Fluit A C, Schmitz F J. Resistance integrons and super-integrons. ClinMicrobiol Infect. 2004, 10 (4): 272-288.
14.许宏涛,宣天芝,陶凤荣,等。3种非发酵革兰阴性杆菌5年的分离率及耐药趋势回顾。中国临床药理学杂志。2004,20:219-221。
15.植志全,何志恒,江鹏,等。多重耐药绿脓假单胞菌β-内酰胺类氨基糖苷类耐药相关基因研究。中华检验医学杂志。2005,28(11):1211-1214。
16.Gu B,Tong M Q,Zhao W J,et al.Prevalence and characterization of class Ⅰ integrons among Pseudomonas aeruginosa and Acinetobacter baumannii isolates from patients in Nanjing,China.J Clin Microbiol.2007,45:241-243.
17.Falagas ME,Bliziotis IA,Kasiakou SK,et al.Outcome of infections due to pandrug resistant(PDR)gram-negative bacteria.BMC Infect Dis.2005,5:24.
1.Levesque C,Brassard S,Lapointe J,et al.Diversity and relative strength of tandem p romoters for the antibiotic-resistance genes of several integrons.Gene.1994,142(1):49-54.
2.Fluit A C,Schmitz F J.Class 1 integrons,gene cassettes,mobility and epidemiology.Eur J Clin Microbiol Infect Dis.1999,18(11):761-770.
3.Collis C M,Hall R M.Expression of antibiotic resistance genes in the integrated cassettes of integrons.Antimicrob Agents Chemother.1995,39(1):155-162.
4.Shmara A,Weinsetel N,Dery K J,et al.Systematic analysis of a conserved region of the Aminoglycoside 6'-N-Acetyltransferase type Ib. Antimicrob Agents Chemother. 2001, 45:3287-3292.
5. Kehrenberg C, Catry B, Haesebrouck F, et al. Novel Spectinomycin/ Streptomycin resistance gene, aadA14, from Pasteurella multocida. Antimicrob Agents Chemother. 2005, 49:3046-3049.
6. Kadlec K, Kehrenberg C, Schwarz S. Efflux-mediated resistance to florfenicol and/or chloramphenicol in Bordetella bronchiseptica: identification of a novel chloramphenicol exporter. J Antimicrob Chemother. 2007, 59:191-196.
7. Bissonnette L, Champetier S, Buisson J P, et al. Characterization of the nonenzymatic Chloramphenicol Resistance (cm1A) gene of the In4 integron of Tnl696: similarity of the product to transmembrane transport proteins. J Bacterial. 1991, 173:4493-4502.
8. Ploy M C, Courvalin P, Lambert T. Characterization of In40 of Enterobacter aerogenes BM2688, a class 1 integron with two new gene cassettes, cmlA2 and qacF. Antimicrob Agents Chemother. 1998, 42:2557-2563.
9. Danel F, Hall L M C, Livermore D M. Laboratory mutants of OXA-10 (3-lactamase giving ceftazidime resistance in Pseudomonas aeruginosa. J Antimicrob Chemother. 1999, 43:339-344.
10. Coudron PE, Ellen SM, Kenneth ST. Occurrence and detection of AmpC Beta-lactamases among Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis isolates at a Veterans Medical Center. J Clin Microbiol. 2000, 38: 1791-1796.
11. Guerra B, Soto S M, Arguelles J M, et al. Multidrug resistance is mediated by large plasmids carrying a class 1 integron in the emergent Salmonella enterica serotype [4,5,12:i:2]. Antimicrob Agents Chemother. 2001, 45:1305-1308.
12. Kaneko K, Okamoto R, Nakano R, et al. Gene mutations responsible for overexpression of AmpC beta-lactamase in some clinical isolates of Enterobacter cloacae. J Clin Microbiol. 2005, 43 (6):2955-2958.
13. Jean-Paul Pirnay, Daniel De Vos, Dimitris Mossialos. Analysis of the Pseudomonas aeruginosa oprD gene from clinical and environmental isolates. Environmental Microbiology. 2002, 4(12):872-882.
14. Mac Gowan AP, Bowker KE. Mechanism of fluoroquinolone resistance is an important factor in determining the antimicrobial effect of gemifloxacin against Streptococcus pneumoniae in an in vitro pharmacokinetic model. Antimicrob Agents Chemother. 2003, 47(3): 1096.
15. Tenover FC, Arbeit RD, Goering RV, et al. Interpreting chromosomal DNA restriction patterns produced by pulsed field gel electrophoresis: criteria for bacterial strain typing. J Clin Microbiol. 1995, 33 (9):2233 -2239.
1. National Nosocomial Infection Surveillance System. National Nosocomial Infection Surveillance (NNIS) System report, data summary from January 1992 through June 2004, issued October 2004. Am. J. Infect. Control. 2004, 32:470-485.
2. Carmeli, Y., N. Troillet, G. Eliopoulos, M. H. Samore. Emergence and of antibiotic-resistant Pseudomonas aeruginosa: comparison of risks associated with different antipseudomonal agents. Antimicrob. Agents Chemother. 1999,43:1379-1382.
3. Cosgrove, S. E., and Y. Carmeli. The impact of antimicrobial resistance on health and economic outcomes. Clin. Infect. Dis. 2003, 36:1433-1437.
4. Stokes H W, Hall R M. A novel family of potentially mobile DNA elements encoding site-specific gene integration functions: integrons. MolMicrobiol. 1989, 3 (12): 1669-1683.
5. Barbolla R, Catalano M, Orman BE, et al . Class 1 integrons increase trimethoprim-sulfamethoxazole MICs against epidemiologically unrelated Stenotrophomonas maltophilia isolates. Antimicrob Agents Chemother. 2004, 48 (2):666-669.
6. Hall R M, Stokes H W. Integrons: novel DNA elements which capture genes by site-specific recombination. Genetica. 1993, 90(223): 115-132.
7. Brown H J, Srokes H W, Hall R M. The integrons InO, In2, and In5 are defective transposon derivatives. J Bacteriol. 1996, 178 (15): 4429-4437.
8. Hall RM, Collis CM. Mobile gene cassettes and integrons: capture and spread of genes by site-specific recombination. Mol Microbiol. 1995, 15: 593-600.
9. Collis CM, Hall RM. Expression of antibiotic resistance genes in the integrated cassettes of integrons. Antimicrob Agents Chemother. 1995, 39: 155-162.
10.Radstrom P, Skold O. Transposon Tn5090 of plasmid R751, which carries an integron, is related to Tn7, Mu, and the retroelements. J Bacteriol. 1994, 176: 3257-3268.
11.Arakawa Y, Murakami M, Suzuki K, et al. A novel integron-like element carrying the metallo-beta-lactamase gene blaIMP. Antimicrob Agents Chemother. 1995, 39: 1612-1615.
12.Senda K, Arakawa Y, Ichiyama S, et al. PCR detection of metallo-beta-lactamase gene (blaIMP) in gram-negative rods resistant to broad-spectrum beta-lactams. J Clin Microbiol. 1996, 34: 2909-2913.
13.Fluit A C, Schmitz F J. Resistance integrons and super-integrons. ClinMicrobiol Infect. 2004, 10 (4): 272-288.
14.Dalsgaard A, Forslund A, Serichantalergs O, Sandvang D.Distnbution and content of class 1 integrons in different Vibrio cholerae O-serotype strains isolated in Thailand. Antimicrob Agents Chemother. 2000,44: 1315-1321.
15.Nesvera J, Hochmannova J, Patek M. An integron of class 1 is present on the plasmid pCG4 from gram-positive bacterium Corynebacterium glutamicum. FEMS Microbiol Lett. 1998,169: 391-395.
16.Martin C, Timm J. Transposition of an antibiotic resistance element in mycobacteria. Nature. 1990, 345: 739-743.
17.Fluit A C, Schmitz F J. Class 1 integrons, gene cassettes, mobility and epidemiology.Eur J Clin Microbiol Infect Dis.1999,18 (11) :761-770.
18.Hansson K, Sundstrom L, Pelletier A, et al. Int I 2 integron integrase in Tn7. JBacteriol. 2002, 184(6): 1714- 1721.
19.Hall RM, Collis CM, Kim MJ, et al. Mobile gene cassettes and integrons in elolution. Ann N Y Acad Sci. 1999, 18 (5): 68-80.
20.A. Gassama - Sowa, M. H. Dialloa , C. S. Boyeb , et al. Class 2 integron - associated antibiotic resistance in Shigella sonnei isolates in Dakar , Senegal. International Journal of Antimicrobial Agents. 2006, 27 (3): 267-270.
21.Miko A, Pries K, SchroeterA, et al. Multiple drug resistance in D-tartrate-positive salmonella enterica serovar paratyphiB isolates from poulty is mediated by class 2 integrons inserted into the bacterial chromosome.Antimicrob Agents Chemother. 2003,47(11) :3640-3643.
22.Collis CM, Kim MJ, Partridge SR, et al. Characterization of the class 3 integron and the site- specific recombination system it determines.J Bacteriol. 2002, 184(11): 3017- 3026.
23.Mazel D, Dychinco B , Webb V A ,et al . A distinctive class of integron in the Vibrio cholerae genome. Sience. 1998, 280:605 - 608.
24.Rowe Magnus D A, Guerout A M, Mazel D. Super integrons. Res Microbiol. 1999, 150:641 - 651.
25.Rowe MagnusD A, GueroutAM, Ploncard P, et al. The evolutionary history of chromosomal super-integrons provides an ancestry for multiresistant integrons. Proc Natl Acad Sci USA. 2001, 98 (2): 652-657.
26.Rowe Magnus D A, Guerout A M, Mazel D. Bacterial resistance evolution by recruitment of super-integron gene cassettes. Mol Microbiol. 2002, 43 (6): 1657-1669.
27.Recchia G D, Hall R M. Origins of the mobile gene cassettes found in integrons. TrendsMicrobiol. 1997, 5 (10): 389-394.
28.Stokes H W, OpGorman D B, Recchia G D, et al Structure and function of 59-base element recombination sites associated with mobile gene cassettes. MolMicrobiol. 1997, 26 (4): 731-745.
29.Doublet B, Boyd D, Mulvey MR, et al. The Salmonella genomic island1 is an integrative mobilizable element. MolMicrobiol. 2005, 55(6):1911 - 1924.
30.Collis C M, Hall R M. Site-specific deletion and rearrangement of integron insert genes catalyzed by the integron DNA integrase. J Bacteriol. 1992, 174(5): 1574-1585.
31.Collis C M, Hall R M. Expression of antibiotic resistance genes in the integrated cassettes of integrons. Antimicrob Agents Chemother. 1995, 39(1):155-162.
32.Levesque C, Brassard S, Lapointe J, et al. Diversity and relative strength of tandem p romoters for the antibiotic-resistance genes of several integrons. Gene. 1994, 142 (1): 49-54.
33.Maguire AJ, Brown DF, Gray JJ, Desselberger U. Rapid screening technique for class 1 integrons in Enterobacteriaceae and nonfermenting gram-negative bacteria and its use in. molecular epidemiology. Antimicrob Agents Chemother. 2001, 45: 1022-1029.
34.Girlich D, Poirel L, Leelaporn A, et al. Molecular epidemiology of the integron-located VEB-1 extendedspectrum beta-lactamase in nosocomial enterobacterial isolates in Bangkok, Thailand. J Clin Microbiol. 2001, 39: 175-182.
35.Partridge SR, Collis CM, Hall RM. Class 1 integron containing a new gene cassette, aadA10, associated with Tnl404 from R151. Antimicrob Agents Chemother. 2002, 46: 2400-2408.
36.Wal sh TR, Toleman MA, Poirel L , et al . Metallo-beta-lactamases: the quiet before t he storm. Clin Microbiol Rev. 2005, 18(2): 306-325.
37.Docquier JD,Riccioml,Mugnaiolic,et al.IMP212,a new plasmid encoded metallo-beta-lactamase from Pseudomonas putide clinical isolate.Antimicrob Agents Chemother.2003,47(5):1522-1528.
38.Verdet C,Arlet G,Barnaud G,et al.A novel integron in Salmonella enterica serovar Enteritidis,carrying the bla(DHA-1)gene and its regulator gene ampR,originated from Morganella morganii.Antimicrob Agents Chemother.2000,44:222-225.
39.Poirel L,Heritier C,Tolun V,et al.Emergence of oxacillinase mediated resistance to imipenem in Klebsiella pneumoniae.Antimicrob Agents Chemother.2004,48(1):15-22.
40.Dubois V,Poirel L,Marie C.Molecular characterization of a novel class 1 integron containing bla(GES-1)and a fused product of aac3-Ib /aac6'-Ib' gene cassettes in Pseudomonas aeruginosa.Antimicrob Agents Chemother.2002,46:638-645.
41.Martinez P,Fluit AC,Schmitz FJ,et al.Class Ⅰ integron in Gram-negtive isolates from different European hospitals and association with decreased susceptibility to multiple antibiotic compounds.J Antimicrob Chemother.1998,42(6):689-696.
42.顾兵,童明庆,等。南京地区铜绿假单胞菌的整合子流行性研究。临床检验杂志。2006,24(2):118-121。
43.Duijkeren EV,BoxAT,Schellen P,et al.Class 1 integrons in Enterobacteriaceae isolated from clinical infections of horses and dogs in The Netherlands. Microb Drug Resist. 2005, 11 (4): 383 -386.
44.Nemergut DR, Martin AP, Schmidt SK. Integron diversity in heavy-metal-contaminated mine tailings and inferences about integron evolution. Appl EnvironMicrobiol. 2004, 70 (2): 1160 -1168.
45.Rosser SJ, Young HK, Identification and characterization of class I integronsin bacteria from an aquatic environment. J Antimicrob Chemother. 1999,44(1): 11-18.