多药耐药肺炎克雷伯菌β-内酰胺酶基因及膜孔蛋白突变检测

Detection of β-lactamase gene and porin mutation in multi-drug resistant Klebsiella pneumoniae

目的：了解多药耐药肺炎克雷伯菌耐药性及耐药基因的表达，为临床治疗肺炎克雷伯菌致呼吸机相关性肺炎（VAP）提供理论依据和指导。方法收集医院2011年7月-2012年6月入住儿童重症监护病房（PICU ）20例呼吸机相关性肺炎患儿下呼吸道痰液标本，通过药敏试验检测耐药率，PCR法及基因测序检测β-内酰胺酶和膜孔蛋白的表达。结果共20株肺炎克雷伯菌对头孢类抗菌药物均存在耐药，对头孢唑林、头孢呋辛、头孢他啶、头孢噻肟、头孢吡肟、头孢西丁耐药率分别为100．0％、95．0％、70．0％、70．0％、55．0％、30．0％；共检出6种β-内酰胺酶基因，分别为A 类 SHV、TEM、CTX-M-1、LA P ，C类 DHA-1和D类 OXA-1，ompK35膜孔蛋白基因检测10株为基因缺失和8株为基因突变，ompK36膜孔蛋白基因7株缺失和2株突变；6株非产 ESBLs株均同时存在om p K35／36的突变或缺失，且伴有β-内酰胺酶基因的表达。结论20株肺炎克雷伯菌均检出1～3种β-内酰胺酶基因；om p K35／36膜孔蛋白基因检测结果显示，该蛋白几乎均有缺陷；提示该组耐β-内酰胺类药物为产β-内酰胺酶和ompK膜孔蛋白缺陷的双重作用，特别是非产ESBLs菌株。

OBJECTIVE To explore drug resistance patterns and expression of drug resistant genes in multi-drug resistant K lebsiella pneumonia ,so as to provide theoretical basis and guidance for clinical treatment of K .pneu-monia induced ventilator-associated pneumonia （VAP） .METHODS Sputum specimens from lower respiratory tract in 20 patients in pediatric intensive care unit in the children hospital from Jul .2011 to Jun .2012 were collected . The drug resistance was tested by drug susceptibility test . The expression of β-lactamases and porins were detected by PCR and gene sequencing .RESULTS All strains were resistant to cephalosporins ,the resistance rates were 100 .0% ,95 .0% ,70 .0% ,70 .0% ,55 .0% ,30 .0% to cefazolin ,cefuroxime ,ceftazidime ,cefotaxime , cefepime ,cefoxitin respectively .In 20 strains of K . pneumoniae ,6 β-lactamase genes ,such as SHV ,TEM , CTX-M-1 ,LA P ,DHA and OXA-1 ,were detected .However ,the porin gene detection showed 10 strains lost and 8 strains mutated for ompK35 gene ,meanwhile 7 strains lost and 2 strains mutated for ompK36 gene .And 6 non-ESBLs producing strains co-presented with ompK35/36 absence or mutation accompanied with expression ofβ-lactamase genes .CONCLUSION The 20 strains were detected with 1 -3 kinds of β-lactamase genes .The test results of ompK35/36 porin gene showed that almost all the porins had defects .It suggested that the resistance toβ-lactamase of this group of K .pneumoniae is due to the double effect of β-lactamase production and omp porin defects ,especially the non-ESBLs producing strains .