磷霉素对体外培养巨噬细胞吞噬和杀伤高毒力肺炎克雷伯菌的影响及其机制  

Effects of fosfomycin on phagocytosis and killing of highly virulent Klebsiella pneumoniae by cultured macrophages in vitro and study on its mechanism

作　　者：徐玉金 李培芬 黄加铭 孙细欢 林颖颖 张晓川 潘少敏[1] XU Yu-jin;LI Pei-fen;HUANG Jia-ming;SUN Xi-huan;LIN Ying-ying;ZHANG Xiao-chuan;PAN Shao-min(The Second Affiliated Hospital of Fujian Medical University,Quanzhou,Fujian 362000,China)

机构地区：[1]福建医科大学附属第二医院临床微生物实验室,福建泉州362000

出　　处：《中华医院感染学杂志》2023年第20期3041-3046,共6页Chinese Journal of Nosocomiology

基　　金：福建省自然科学基金资助项目(2020J01212);泉州市科技计划基金资助项目(2021N003S)。

摘　　要：目的探讨磷霉素对体外培养巨噬细胞吞噬和杀灭胞内高毒力肺炎克雷伯菌(hvKP)的影响及其作用机制。方法微量肉汤稀释法检测磷霉素的最低抑菌浓度(MIC)值;采用hvKP感染巨噬细胞(THP-1)建立感染模型,加入不同浓度的磷霉素孵育细胞,对照组加入PBS缓冲液,激光共聚焦显微镜观测处理4 h后细胞吞噬细菌的数量;单克隆形成实验检测不同浓度磷霉素处理12 h及8μg/ml磷霉素处理不同时间后胞内hvKP的数量;流式细胞术检测细胞内活性氧(ROS)水平。结果磷霉素对高毒力肺炎克雷伯菌的MIC>256,体外无抑菌活性;经磷霉素处理后,巨噬细胞中被吞噬的细菌数量高于空白对照组(P<0.05);高剂量组在感染12 h后胞内细菌的数量少于空白对照组(P<0.05),且少于低剂量组(P<0.05);随着处理时间的延长,磷霉素能抑制胞内hvKP的存活;与空白对照组相比较,低剂量和高剂量的磷霉素均增加了巨噬细胞中ROS的水平及氧化应激相关基因的表达(P<0.05)。结论磷霉素能促进巨噬细胞对hvKP的吞噬作用,且高剂量的磷霉素效果更加显著,呈现剂量依赖性;磷霉素能促进巨噬细胞对胞内hvKP的杀伤,其机制主要是通过增加细胞内ROS的释放水平来发挥作用。OBJECTIVE To investigate the effect of fosfomycin on macrophage phagocytosis and killing intracellular highly virulent Klebsiella pneumonia(hvKP)by cultured macrophages in vitro and its mechanism.METHODS The minimum inhibitory concentration(MIC)value of fosfomycin was detected by broth microdilution methods,the infection model was established by using hvKP-infected macrophages(THP-1),and the cells were incubated by adding different concentrations of fosfomycin,while the control group was treated with PBS buffer.The number of phagocytosis bacteria was observed by laser confocal microscope after 4hours of treatment.Monoclonal formation assay was performed to detect the number of intracellular hvKP after treatment with different concentrations of fosfomycin for 12hand 8μg/ml for different time.The intracellular reactive oxygen species(ROS)levels was detected by flow cytometry.RESULTS Fosfomycin had a MIC>256against highly virulent Klebsiella pneumoniae(hvKP)and had no antibacterial activity in vitro.The number of phagocytosed bacteria in macrophages was higher than that of the blank control group after treatment with fosfomycin(P<0.05).The number of intracellular bacteria in the high-dose group was less than that of the control group(P<0.05)and less than that of the lowdose group(P<0.05)at 12hours after infection.With prolonged treatment,fosfomycin inhabited the survival of intracellular hvKP,compared with the blank control group,both low and high doses of fosfomycin increased the levels of ROS and the expression of gene related to oxidative stress in macrophages(P<0.05).CONCLUSION Fosfomycin promoted the phagocytosis of hvKP by macrophages,and the effect of high dose of fosfomycin was more significant,showing a dose-dependence.Fosfomycin promoted the cytotoxicity of macrophages to intracellular hvKP,and its mechanism was mainly through increasing the level of intracellular ROS release.

关 键 词：磷霉素 高毒力肺炎克雷伯菌 巨噬细胞 活性氧 吞噬能力 杀菌实验 

分 类 号：R378[医药卫生—病原生物学]