泛耐药肺炎克雷伯菌血液分离株耐药机制研究

Investigation of resistant mechanism of extensively drug-resistant Klebsiella pneumonia isolated from blood

目的探讨泛耐药肺炎克雷伯菌血液分离株耐药机制。方法对我院ICU住院患者血液分离的泛耐药肺炎克雷伯菌株,采用K-B纸片法药敏试验;采用聚合酶链反应(PCR)方法对该菌株可能携带的β-内酰胺酶编码基因及膜孔蛋白编码基因、与氨基糖苷类药物耐药相关的氨基糖苷类修饰酶编码基因、与氟喹诺酮类药物耐药相关基因以及与利福平耐药相关的aar基因进行检测,并对阳性产物进行测序,测序结果作BLAST对比分析。结果该株泛耐药肺炎克雷伯菌株检出TEM、SHV、DHA3种β-内酰胺酶编码基因,OmpK35膜孔蛋白编码基因检测呈阳性,OmpK36膜孔蛋白编码基因检测呈阴性,即该菌OmpK36膜孔蛋白编码基因为缺失型;aac(6')-Ⅰb氨基糖苷类修饰酶编码基因阳性;氟喹诺酮类药物作用靶位gyrA基因第83位密码子存在TCC→ATC有义突变(氨基酸序列S→I)、parC基因第80位密码子存在AGC→ATC有义突变(氨基酸序列S→I),且氟喹诺酮类药物作用靶位保护蛋白编码基因qnrB检测阳性;利福平获得性耐药基因arr检测阳性。结论泛耐药肺炎克雷伯菌同时存在多种耐药机制,从而对不同类抗菌药物形成耐药,给临床治疗带来极大的挑战;加强对该类菌株的监控、减少其在医院的传播、积极探寻对该类菌株的有效治疗措施迫在眉睫。

Objective To explore the resistant mechanism of extensively drug-resistant Klebsiella pneumoniae （XDR-KPN）. Methods The XDR Klebsiella pneumoniae strain isolated from the blood of patients in ICU of the Second Affiliated Hospital of Nanjing Medical University were tested by K-B method. Polymerase Chain Reaction （PCR） was applied for the detection of the resistance genes carried possibly by the the strain, including the genes encoding β-1actamase, porin, aminoglycoside-modifying enzymes, and the genes associated with fluoroquinolones or rifampicin resistance. Then, positive productions were characterized by DNA sequencing and BLAST searches. Results Three types of β-1actamase gene, TEM, SHV and DHA, and ompK35 encoding porin were identified in the strain. However, the detection of ompK36 in the strain was negative. The presence of aac （6＇） - I b, a gene encoding aminoglycoside-modifying enzymes, was confirmed. The sense mutations of TCC→ATC at codon83 （amino acid sequence S→I） and AGC→ATC at codon80 （amino acid sequence S→I） were found in gyrA and parC, respectively, and the qnrB gene was also detected. In addition, the rifampicin acquired drug resistance, arr, was positive. Conelusion The coexistence of multiple mechanisms in the XDR Klebsiella pneumoniae strain result in resistance to a variety of antimicrobials, which bring about a great challenge to clinical treatments. Strengthening the surveillanceon extensively drug-resistant klebsiella pneumoniae isolates, controlling its spread in hospital and searching for effective treatments are urgently needed.