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目的调查我院2003-2005年临床分离多重耐药绿脓假单胞菌的氨基糖苷类修饰酶基因、β内酰胺酶编码基因和外膜通道蛋白oprD2基因存在状况;并研究整合子参与绿脓假单胞菌多重耐药的机制。方法纸片扩散法测定绿脓假单胞菌对14种抗菌药物的药物敏感性,并筛选出14株多重耐药菌株;聚合酶链反应检测氨基糖苷类修饰酶基因、β内酰胺酶编码基因和外膜通道蛋白oprD2基因;聚合酶链反应检测整合子5′保守区的整合酶基因和3′保守区的qacE△1-sulI基因,对整合酶基因的阳性扩增产物的限制性片段长度多态性分析进行整合子分类,整合子可变区扩增并测序。结果14株绿脓假单胞菌对14种抗菌药(哌拉西林等)的耐药率为14·3%~100·0%;氨基糖苷类修饰酶基因ant(2”)-Ⅰ、aac(3)-Ⅱ、aac(6’)-Ⅱ、aac(6’)-Ⅰ、ant(3”)-Ⅰ和aac(3)-Ⅰ检出率分别为78·6%、57·1%、57·1%、14·3%、7·1%、0;β内酰胺酶编码基因TEM和IMP的检出率分别为92·9%和42·9%,未检出VIM、OXA、PER、GES和SHV基因,1株外膜通道蛋白oprD2基因缺失;整合酶基因及qacE△1-sulI基因检出率分别为85·7%和78·6%,整合子可变区扩增有3种片段:1000、1300和1700,测序证实分别为aadA2、aadA6-orfD和dfrⅫ-orfF-aadA2,其中aadA2是首次在绿脓假单胞菌的整合子中检出,aadA6-orfD是一种新型的整合子可变区基因盒组合形式,Genbank号分别为DQ091178和DQ091179。结论我院多重耐药绿脓假单胞菌对β内酰胺类抗生素的耐药主要与TEM和IMP型耐药基因有关,对氨基糖苷类抗生素的耐药主要与氨基糖苷类修饰酶基因ant(2”)-Ⅰ、aac(3)-Ⅱ和aac(6’)-Ⅱ有关;整合子参与了绿脓假单胞菌的耐药和多重耐药。
Objective To investigate the existence of aminoglycoside-modifying enzyme gene, β-lactamase-encoding gene and oprD2 gene of Pseudomonas aeruginosa isolated clinically from 2003 to 2005 in our hospital. Mechanism of multidrug resistance in Pseudomonas. Methods The drug susceptibility of Pseudomonas aeruginosa to 14 antimicrobial agents was determined by disk diffusion method and 14 multidrug-resistant strains were screened. Polymerase chain reaction was used to detect the aminoglycoside-modifying enzyme gene and β-lactamase-encoding gene And the outer membrane channel protein oprD2 gene. Polymerase chain reaction was used to detect the integrase gene in the 5 ’conserved region of the integron and the qacE △ 1-sulI gene in the 3’ conserved region. The restriction fragment length of the positive amplification product of the integrase gene Polymorphism analysis was carried out by sub-classification, and the integron variable region was amplified and sequenced. Results The resistance rates of 14 strains of Pseudomonas aeruginosa to 14 antibiotics (piperacillin and others) were 14.3% -100.0%. The aminoglycoside modified enzyme gene ant (2 “) - Ⅰ, aac The detection rates of (3) -Ⅱ, aac (6 ’) - Ⅱ, aac (6’) - Ⅰ, ant (3 ”) - Ⅰ and aac (3) Ⅰ were 78.6% and 57.1% , 57.1%, 14.3%, 7.1%, 0 respectively. The detection rates of TEM and IMP of β-lactamase gene were 92.9% and 42.9% respectively. VIM, OXA, PER, GES and SHV genes, and one of the outer membrane channel oprD2 genes were deleted. The detection rates of integrase gene and qacE △ 1-sulI gene were 85.7% and 78.6% Three fragments: 1000, 1300 and 1700 were sequenced and confirmed to be aadA2, aadA6-orfD and dfrⅫ-orfF-aadA2, respectively. Among them, aadA2 was first detected in integrons of Pseudomonas aeruginosa and aadA6-orfD The new integron variable region gene cassette combinations, Genbank numbers were DQ091178 and DQ091179. Conclusion The resistance of multi-drug resistant Pseudomonas aeruginosa to β-lactam antibiotics in our hospital is mainly related to TEM and IMP-type resistance genes. The resistance to aminoglycoside antibiotics is mainly related to aminoglycoside-modifying enzyme gene ant ( 2 ") - Ⅰ, aac (3) - Ⅱ and aac (6 ’) - Ⅱ. The integron participates in the resistance and multidrug resistance of Pseudomonas aeruginosa.