IRPA的整合子分布及IMP-1基因表达的研究

发布时间：2018-04-14 01:33

本文选题：耐亚胺培南铜绿假单胞菌 + 金属β-内酰胺酶　；参考：《天津医科大学》2009年硕士论文

【摘要】：目的：铜绿假单胞菌是引起医院内感染常见的条件致病菌之一,其对抗菌药物的耐药机制非常复杂,获得并表达金属β-内酰胺酶(metallo-β-lactamase,MBL)是铜绿假单胞菌对碳青霉烯类抗生素耐药的主要机制之一。具有捕获及表达外来基因能力的整合子-基因盒系统在金属酶基因的快速播散中意义重大,其介导的多种耐药基因也是造成铜绿假单胞菌多重耐药的重要原因之一。本课题选取天津地区四所三级甲等医院临床分离的耐亚胺培南铜绿假单胞菌(imipenem-resistant Pseudomonas aeruginosa, IRPA)为研究对象,分析其对常见抗菌药物的耐药性,研究金属β-内酰胺酶和Ⅰ类整合子的流行情况,并对铜绿假单胞菌所携带的IMP-1基因进行定位,确定其是否位于整合子上;最后从IMP-1型金属酶基因mRNA表达的角度,对铜绿假单胞菌产金属酶情况和细菌对亚胺培南耐药性之间的关系进行分析。方法：选取2007年1月-12月天津市四所三级甲等医院分离出的67株耐亚胺培南铜绿假单胞菌为受试菌,采用K-B法对所有菌株进行药敏试验,并比较其对不同抗菌药物的耐药性；用EDTA纸片协同抑制试验对亚胺培南耐药株进行金属酶表型筛选,并用PCR方法扩增IMP和VIM型金属酶基因：采用PCR方法扩增I类整合酶基因,对整合酶阳性株进行可变区的PCR扩增并将PCR产物进行测序,将测序结果在Genbank上行同源性分析,确定可变区携带的基因盒；采用PCR方法对IMP-1基因进行定位；最后利用实时荧光定量RT-PCR的方法,检测IMP-1mRNA的表达量,分析其表达量与细菌对亚胺培南MIC之间的关系。结果： 1.67株临床分离的耐亚胺培南铜绿假单胞菌均为多重耐药菌株,药敏结果示：受试菌耐药情况严重,对亚胺培南、美罗培南的耐药率接近100%：对庆大霉素、氯霉素和环丙沙星的耐药率超过了70%,对阿米卡星的耐药率接近60%,对头孢他啶、头孢哌酮/舒巴坦的耐药率在40%左右,对氨曲南的耐药率最低,为31.34%。 2.PCR结果显示,在67株耐亚胺培南铜绿假单胞菌中,有19株携带IMP-1型金属酶基因,检出率接近28%,其中有16株金属酶表型筛选实验阳性；在这19株IMP-1基因阳性株中,有15株携带的IMP-1基因位于Ⅰ类整合子上,与aadB氨基糖苷耐药基因共同位于整合子可变区中。 3.在67株耐亚胺培南铜绿假单胞菌中,检出46株铜绿假单胞菌携带I类整合酶基因,阳性率68.65%,其中有2株检出可变区结构,1株(PA60)可变区为aadB和cm1A基因,其序列与Genbank中目标基因(DQ266448)中两基因序列100%一致；另1株(PA27)可变区为aac(6')-Ⅱ-cm1A8-OXA-10,确定为一种新型耐药基因盒组合形式,GenBank登录号为EU708817。 4.实时荧光定量RT-PCR和统计结果显示：Group1(低MIC组)和Group2(高MIC组)两组样本IMP-1基因mRNA相对表达量的差别有统计学意义(P=0.026), Group1(低MIC组)的(?)mRNA相对表达量显著低于Group2(高MIC组),亚胺培南耐药与IMP-1表达存在一定的相关性。结论：67株耐亚胺培南铜绿假单胞菌可选择抗生素有限,对氨曲南和头孢他啶的耐药性较低,分别为31.34和40.30%；19株携带IMP-1型金属β-内酰胺酶(28%),与国内外其他地区报道相近,其中大多数菌株携带的IMP-1基因位于I类整合子上,表明整合子在金属酶基因的快速播散中意义重大；同时I类整合酶基因广泛分布在耐亚胺培南铜绿假单胞菌中,此外本研究还检出1株携带新型基因盒组合形式I类整合子的铜绿假单胞菌,表明了整合子-基因盒系统的高度异质性；通过对IMP-1基因mRNA目对表达量的检测,发现细菌对亚胺培南的MIC与该基因mRNA的表达升高有关。
[Abstract]:Objective: Pseudomonas aeruginosa is a common nosocomial infection caused by pathogens of the antimicrobial resistance mechanism is very complex, and the expression of metallo beta lactamase (metallo- beta -lactamase, MBL) is one of the main mechanisms of Pseudomonas aeruginosa resistant to carbapenems. With rapid spread integron - ability to capture and express foreign genes gene cassette system in metal enzyme gene is significant, one of the important reasons of multiple drug resistance genes mediated is caused by multi drug resistant Pseudomonas aeruginosa. Imipenem resistant Pseudomonas aeruginosa the selected clinical isolates in Tianjin area from four to three hospitals (imipenem-resistant Pseudomonas aeruginosa, IRPA) as the research object, analysis of their resistance to common antibiotics, research of MBLS and integron epidemic situation, and The location of IMP-1 gene carried by Pseudomonas aeruginosa was located to determine whether it was located on integron. Finally, the relationship between metalloenzyme and Pseudomonas aeruginosa resistance to imipenem was analyzed from the angle of IMP-1 metalloproteinase gene mRNA expression.
Methods: January 2007 -12 month four Tianjin City three hospitals 67 strains of imipenem resistant Pseudomonas aeruginosa as tested bacteria, the drug susceptibility tests were performed on all strains by K-B method, and compare the drug resistance to different antibiotics; EDTA synergy inhibition test of metal enzyme phenotype screening the imipenem resistant strains, and amplified by PCR IMP and VIM metal enzyme gene amplification: class I integrase gene by PCR method of variable region of the integrase positive strains were amplified by PCR and the PCR products were sequenced. The sequencing results in analysis of Genbank homologous gene upstream, to determine the variable region carrying box by using PCR method; the localization of IMP-1 gene; finally, using the method of real-time fluorescence quantitative RT-PCR, detect the expression of IMP-1mRNA and analyze its expression and the relationship between bacteria to imipenem and MIC.
Result:
1.67 strains of imipenem resistant Pseudomonas aeruginosa strains clinical isolates were multi drug resistant strains and drug sensitivity showed that the tested strains resistant to imipenem, meropenem resistance rate is close to 100% of gentamicin, chloramphenicol and ciprofloxacin resistance rate of more than 70% of Amikacin's drug resistance rate close to 60% of cephalosporins ceftazidime, Cefoperazone / sulbactam in 40%, resistant to aztreonam was the lowest, 31.34%.
2.PCR results showed that the imipenem resistant Pseudomonas aeruginosa in 67 strains, 19 strains carrying IMP-1 metal enzyme gene, the detection rate of close to 28%, of which 16 strains of metal enzyme phenotype screening test positive; in the 19 strains of IMP-1 gene positive strains, 15 strains carrying the IMP-1 gene in class I integron aadB, Co located with the aminoglycoside resistance gene of integron in the variable regions.
3. of the 67 strains of imipenem resistant Pseudomonas aeruginosa, were detected in 46 strains of Pseudomonas aeruginosa I gene, the positive rate was 68.65%, of which 2 strains of variable region structure, 1 strains (PA60) for aadB and cm1A variable region gene, target gene and the sequence of Genbank (DQ266448). The two gene sequences of 100%; the other 1 strains (PA27) variable region (6') - AAC II -cm1A8-OXA-10, identified as a new resistance gene cassette combinations, GenBank accession number is EU708817.
4. real time fluorescence quantitative RT-PCR and statistical results showed that the Group1 (low MIC group) and Group2 (high MIC group) of two samples of IMP-1 gene mRNA expression was statistically significant difference (P=0.026), Group1 (low MIC group) of the (?) the relative expression of mRNA was significantly lower than that of Group2 (high MIC group). There is a correlation between the expression of imipenem resistance and IMP-1.
Conclusion: 67 strains of imipenem resistant Pseudomonas aeruginosa can choose antibiotics limited, low resistance to aztreonam and ceftazidime, respectively 31.34 and 40.30%; 19 strains carrying IMP-1 metallo (28%), with similar reports of other regions at home and abroad, the majority of strains carrying IMP-1 gene in the class I integron, showed that the rapid spread of integron in metal enzyme gene in great significance; at the same time the class I integrase gene is widely distributed in imipenem resistant Pseudomonas aeruginosa in addition, this study also detected 1 strains of Pseudomonas aeruginosa carrying new array of gene cassettes type I integron. Show the high heterogeneity of the integron gene cassette system; based on the IMP-1 gene to detect the expression of mRNA, that the higher expression of bacteria to imipenem and the MIC gene of mRNA.

【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R378.991

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