铁载体毒力基因entB对碳青霉烯类耐药肺炎克雷伯菌毒力的影响

The effect of siderophore virulence gene entB on the virulence of Carbapenem-resistant Klebsiella pneumoniae

目的明确铁载体毒力基因entB对碳青霉烯类耐药肺炎克雷伯菌(CRKP)毒力的影响。方法从昆明医科大学第一附属医院收集到的30株CRKP菌株中筛选出CRKP-27作为实验菌株,规律间隔成簇短回文重复序列(CRISPR)-Cas9基因敲除技术构建entB基因缺失株ΔentB、回补株C-ΔentB,并通过PCR验证敲除及回补是否成功。观察CRKP-27、ΔentB、C-ΔentB菌株的菌落形态并进行拉丝试验,初步了解entB对CRKP菌落形态和毒力表型的影响。绘制菌株生长曲线,测定entB对CRKP菌株生长的影响。铬天青S(CAS)检测液对菌株产铁载体能力进行定量检测。建立小鼠腹腔感染模型,观察小鼠生存率直观了解entB基因对CRKP毒力的影响。计量资料均采用平均数±标准差表示,服从正态分布的数据采用方差分析进行组间比较,不服从正态分布的数据采用Kruskal-Wallis秩和检验。小鼠生存分析用Log-Rank检验进行比较。P<0.05表示差异具有统计学意义。结果PCR结果显示缺失株和回补株构建成功。entB基因对CRKP的菌落形态、荚膜和毒力表型无明显影响。ΔentB组的生长速度明显快于CRKP-27组(P=0.008)和C-ΔentB组(P=0.001),说明entB基因削弱了CRKP菌株的生长能力。entB组与CRKP-27组(P=0.001)和C-ΔentB组(P=0.001)相比铁载体产量明显下降,分别下降11.7393%和11.9642%,说明entB基因显著增加了CRKP产铁载体能力。动物实验中,与CRKP-27组(P=0.023)和C-ΔentB组(P=0.024)相比,ΔentB组小鼠生存率明显增高,说明entB基因增加了CRKP菌株的毒力。结论铁载体毒力基因entB明显减弱了菌株的生长能力,但使CRKP产铁载体能力、毒力明显增强。

Objective To study the effect of the siderophore virulence gene entB on the virulence of carbapenem-resistant Klebsiella pneumonia(CRKP).Methods CRKP-27 was selected as the experimental strain from 30 CRKP strains collected from the First Affiliated Hospital of Kunming Medical University.The knockdown strain(ΔentB)and complementing strain(C-ΔentB)were constructed by the clustered regularly interspaced short palindromic repeat-Cas9 technology,and verified by polymerase chain reaction(PCR).In order to initially understand the effect of entB on CRKP colony morphology and virulence phenotype,the colony morphology of CRKP-27,ΔentB,and C-ΔentB strains were observed and string test were tested.Draw the growth curve of the strains and determine the effect of entB on the growth of the CRKP strains.The siderophores production ability of the strains were detected quantitatively using chrome azurol S(CAS)detection solution.Mice model of inflammation was established to observe the survival rate of mice and intuitively understand the effect of entB on CRKP virulence.Results The PCR results showed that theΔentB strain and C-ΔentB stranin were constructed successfully.The entB has no significant effect on the colony morphology,capsule and virulence phenotype of CRKP.The growth rate ofΔentB was significantly faster than that of CRKP-27(P=0.008)and C-ΔentB(P=0.001),which showed that entB weakened the growth ability of CRKP.Compared with CRKP-27(P=0.001)and C-ΔentB(P=0.001),the siderophore production ofΔentB was significantly decreased by 11.7393%and 11.9642%,indicating that entB gene increased the capacity of CRKP to produce siderophpres.In animal experiments,compared with CRKP-27(P=0.023)and C-ΔentB(P=0.024),the survival rate of mice in theΔentB group was significantly increased,indicating that the entB increased the virulence of the CRKP.Conclusion The siderophore virulence gene entB significantly weakened the growth ability of the strain,but clearly enhanced the siderophore production capacity and virulence of CRKP.