耐碳青霉烯类肺炎克雷伯菌耐药机制研究

Analysis of resistance mechanism of carbapenem-resistant Klebsiella pneumoniae

目的研究临床分离的耐亚胺培南肺炎克雷伯菌的分子流行病学及耐药机制,为制定预防控制该类细菌传播提供系统的试验数据和参考依据。方法收集并鉴定碳青霉烯类抗菌药物耐药肺炎克雷伯菌40株,肠杆菌基因间重复性一致性序列-PCR(ERIC-PCR)分析菌株的分子流行病学;药敏试验采用琼脂稀释法,改良Hodge方法检测碳青霉烯酶,EDTA-Na2双纸片协同法检测金属β-内酰胺酶,PCR及耐药基因克隆测序方法研究细菌的耐药机制,质粒结合试验确定耐药基因的转移性。结果 40株肺炎克雷伯菌仅对多黏菌素和替加环素显示了较好的敏感性;改良Hodge试验阳性30株,金属酶检测试验结果均为阴性;ERIC-PCR将40株肺炎克雷伯菌分为4型,以Ⅰ型为主;PCR结果显示,KPC-2型32株、CTX-M-9型27株、SHV型22株、TEM型19株,其中有25株同时携带≥3种的耐药基因;质粒接合试验显示,3株分离菌株成功传递了对碳青霉烯类的耐药性。结论医院存在质粒介导的碳青霉烯类耐药性的水平传播;耐碳青霉烯类肺炎克雷伯菌耐药机制主要是产KPC-2碳青霉烯酶,CTX-M-9、SHV和TEM型β-内酰胺酶同时参与其多药耐药机制。

OBJECTIVE To investigate the molecular epidemiology and drug resistance mechanisms of imipenem-resistant Klebsiella pneumoniae so as to provide systematic experimental data and reference basis for preventing and controlling the spread of this kind of bacteria.METHODS Totally 40 strains of carbapenem resistant K.pneumoniae were collected and identified.Susceptibility testing for antibiotics was performed by the disc diffusion method.Carbapenemase production was confirmed by Modified Hodge test and MBL production by IPM/IPM-EDTA combined disc test.The resistant mechanism of bacteria were detected by PCR and followed by DNA sequencing.Plasmid conjugation experiments were performed to determine the transferability of the resistant gene.RESULTS ERIC-PCR results showed that 40 strains of K.pneumoniae were divided into 4subtypes,and the main type was typeⅠ.PCR results also showed that,32 strains were type KPC-2,27 strains were type CTX-M-9,22 strains were type SHV,and 19 strains were type TEM.Among them,25 of them were carrying 3or more drug resistant genes at the same time.Plasmid conjugation test showed that 3strains were successfully delivered the resistance of carbapenems.CONCLUSION Horizontal transmission of plasmid mediated carbapenem resistance was found in the hospital.And KPC-2was the main reason to cause the bacterial resistance to carbapenems,type CTX-M-9,type SHVand type TEM β-lactamases are also involved in the mechanism of multidrug resistance.