一株对3种碳青霉烯类抗菌药物均耐药的肺炎克雷伯菌耐药机制研究

Study on the resistance mechanism of an isolate of Klebsiella pneumoniae resistant to 3 kinds of carbopenems

目的研究临床分离的一株肺炎克雷伯菌(K30)对碳青霉烯类抗菌药物耐药的机制。方法采用琼脂稀释法测定肺炎克雷伯菌K30对13种抗菌药物的最低抑菌浓度(MIC);用改良Hodge试验检测碳青霉烯酶;聚合酶链反应(PCR)检测A类碳青霉烯酶(KPC)、B类碳青霉烯酶(NDM、IMP、VIM、SIM)、超广谱β-内酰胺酶(ESBLs,CTX、TEM、SHV)、头孢菌素酶(AmpC,FOX、EBC、ACC、DHA、CIT、MOX)基因和Ⅰ类整合子;实时荧光定量PCR分析肺炎克雷伯菌膜孔蛋白基因ompK35、ompK36的mRNA表达;利用质粒接合试验研究肺炎克雷伯菌K30耐药基因的水平传播能力。结果药物敏感性试验显示,肺炎克雷伯菌K30对包括碳青霉烯类抗菌药物在内的11种抗菌药物均耐药,仅对头孢西丁钠中介,对阿米卡星敏感。肺炎克雷伯菌K30的改良Hodge试验为阳性。PCR扩增A类碳青霉烯酶基因KPC和2种ESBLs基因CTX、SHV为阳性,其PCR产物经测序后同GenBank数据库比对证实分别为KPC-2、CTX-M3和SHV-38。其余耐药基因和Ⅰ类整合子扩增均为阴性。与肺炎克雷伯菌(ATCC 700603)相比,膜孔蛋白基因ompK35、ompK36的表达没有降低。质粒接合试验未成功获取结合子。结论临床分离的一株对碳青霉烯类抗菌药物耐药的肺炎克雷伯菌主要耐药机制与产A类碳青霉烯酶KPC-2合并产ESBLs有关,且KPC-2可能不由质粒介导。

Objective To investigate the resistance mechanism of an isolate of Klebsiella pneumoniae （K30 ） resistant to carbopenems.Methods Minimum inhibition concentrations （MIC）of Klebsiella pneumoniae K30 to 1 3 antibiotics were determined by agar dilution method.Modified Hodge test was used to detect carbopenems.Class A carbopenem （KPC）,Class B carbopenem （NDM,IMP,VIM and SIM）,extended spectrum beta-lactamases [ESBLs （CTX,TEM and SHV）],AmpC beta-lactamases [Amp C （FOX,EBC,ACC,DHA,CIT and MOX）]and Class Ⅰintegron were amplified by polymerase chain reaction （PCR）.Real-time fluorescence quantitation PCR was carried out to investigate the mRNA expression levels of porin genes（ompK35 and ompK36）.Plasmid conjugation experiment was subjected to reveal the transferability of Klebsiella pneumoniae K30 resistant genes. Results The antimicrobial susceptibility test showed that Klebsiella pneumoniae K30 was resistant to 1 1 antibiotics,but kept intermediary to cefoxitin sodium and susceptible to amikacin.Modified Hodge test was positive in Klebsiella pneumoniae K30.Class A carbopenem KPC gene and 2 ESBLs CTX and SHV genes were positive by PCR amplification.The genes were conformed as KPC-2,CTX-M3 and SHV-38 by sequencing and comparing in GenBank.No other resistance gene Class Ⅰ integron was detected.The porin gene ompK35 and ompK36 expression levels did not decrease comparing with those of Klebsiella pneumoniae （ATCC 700603 ）.Plasmid conjugation experiment was failed in the study.Conclusions The main resistance mechanism of the isolate of Klebsiella pneumoniae resistant to carbopenems might be associated with the combined producing KPC-2 and ESBLs,and KPC-2 may be not mediated by plasmid.