亚抑菌浓度抗生素对高毒力肺炎克雷伯菌毒力基因表达的影响  

Study on effect of sub-inhibitory concentration of antibiotics on virulence gene expression of hypervirulent Klebsiella pneumoniae

作　　者：宋国滨 王刚[2] 黄颖[2] 吴文娟[1] 徐元宏[2] Song Guobin;Wang Gang;Huang Ying(Dept of Laboratory Medicine,Dongfang Hospital,Tongji University,Shanghai 200123;Dept of Laboratory,The First Affiliated Hospital of Anhui Medical University,Hefei 230022)

机构地区：[1]同济大学附属东方医院检验科,上海200123 [2]安徽医科大学第一附属医院检验科,合肥230022

出　　处：《安徽医科大学学报》2021年第3期352-357,共6页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金(编号:81171606)。

摘　　要：目的探究在亚抑菌浓度抗生素作用下,高毒力肺炎克雷伯菌毒力基因表达水平的变化。方法采用纸片扩散法、Vitek-2 compact法测定菌株对临床常见抗菌药物的敏感性,并用微量肉汤稀释法检测菌株对头孢西丁和阿米卡星的MIC值。聚合酶链式反应(PCR)对荚膜血清型(K1、K2、K5、K20、K54、K57)及毒力相关基因(rmpA、aerobactin、kfu、alls、magA、wcaG、fimH、mrkD)进行检测。根据菌株对两种药物的MIC值,配制1/2MIC、1/4MIC、1/8MIC亚抑菌浓度抗生素梯度。运用qRT-PCR检测1/2MIC、1/4MIC、1/8MIC浓度头孢西丁、阿米卡星对菌株处理后毒力基因相对表达量的改变,SPSS软件16.0版本对其差异进行比较分析,通过基质辅助激光解吸电离飞行质谱(MALDI-TOF MS)及ERIC-PCR检测菌株之间的同源性。结果共收集57株高毒力肺炎克雷伯菌,对大多数临床常用抗生素保持敏感,PCR结果显示K1型有17株,K2型21株,K20型2株,K57型8株,有9株未检出血清型。rmpA、aerobactin基因检测全部阳性,wcaG、kfu、magA 3个基因分别检出17株、21株、16株。fimH、mrkD全部阳性。亚抑菌浓度头孢西丁及阿米卡星作用下,与对照组相比,3种浓度(1/2MIC、1/4MIC、1/8MIC)的2个抗生素对2种毒力基因均有明显的抑制作用(P<0.05),头孢西丁的抑制程度最高。3种浓度抑制程度相比,以1/2MIC抗生素抑制效果最强。结论高毒力肺炎克雷伯菌对临床常用抗生素有较高的敏感性,血清型K1、K2是高毒力肺炎克雷伯菌的常见血清型,在亚抑菌浓度头孢西丁、阿米卡星作用下,rmpA、aerobactin毒力基因表达下降。Objective To study the expression level changes of virulence genes of hypervirulent Klebsiella pneumoniae under the action of antibiotics with subinhibitory concentration.Methods Disk diffusion method and ViteK2 instrument method were used to determine the susceptibility of the strains to common clinical antibiotics,and the MIC values of the strains to cefoxitin and amikacin were detected by broth dilution method.The serotypes of capsule(K1,K2,K5,K20,K54,K57)and virulence related genes(rmpA,aerobactin,kfu,alls,magA,wcaG,fimH,mrkD)were detected by polymerase chain reaction(PCR).According to the MIC value of the two drugs,the 1/2MIC,1/4MIC and 1/8MIC subantibiotic concentration gradient of antibiotics was prepared.qRTPCR was used to detect the change of the relative expression of virulence genes in 1/2MIC,1/4MIC,1/8MIC concentrations of cefoxitin and amikacin,and the difference was analyzed by SPSS software.The homology between the strains was detected by MALDI-TOF MS and ERIC-PCR.Results A total of 57 strains of Klebsiella pneumoniae were collected,which were sensitive to most of the commonly used antibiotics.PCR results showed that there were 17 strains of K1,21 strains of K2,2 strains of K20,8 strains of K57,and 9 strains of unknown haemorrhagic type.RmpA and aerobactin genes were all positive.17,21 and 16 strains of wcaG,kfu and magA genes were detected respectively.FimH and mrkD were all positive.Under the action of subinhibitory concentration of cefoxitin and amikacin,the two antibiotics of three concentrations(1/2 MIC,1/4 MIC,1/8 MIC)had obvious inhibitory effects on the two virulence genes(P<0.05),and the inhibition degree of cefoxitin was the highest.The inhibitory effect of 1/2 MIC was the strongest among the three concentrations.Conclusion Hypervirulent Klebsiella pneumoniae is highly sensitive to commonly used antibiotics.Serotypes K1 and K2 are common serotypes of hypervirulent Klebsiella pneumoniae than other serotypes.The virulence gene expression of rmpA and aerobactin decreased under the action of

关 键 词：亚抑菌浓度 高毒力肺炎克雷伯菌 毒力基因 

分 类 号：R446.5[医药卫生—诊断学]