【作者】 武渊；

【导师】 黄祖瑚；

【作者基本信息】 南京医科大学 ， 内科学， 2008， 硕士

【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,MIC₉₀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。更多还原

【Abstract】 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 MIC₉₀ 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.更多还原