基于菌群拮抗原理治疗多重耐药肺炎克雷伯菌感染的实验研究  

Experimental study on the treatment of multidrug-resistant Klebsiella pneumoniae infection based on the principle of microbiota antagonism

作　　者：陈燕 邓丽英 韦莹莹 戴佩如 吴晓梅 胡文 陈春林[1] Chen Yan;Deng Liying;Wei Yingying;Dai Peiru;Wu Xiaomei;Hu Wen;Chen Chunlin(Yichun College,School of Chemistry and Biological Engineering,Yichun 336000;Shanghai Pudong Huamu Community Health Service Center,Shanghai 201204;Yichun College of Medicine,Yichun 336000;Yichun City People's Hospital,Yichun 336000)

机构地区：[1]宜春学院化学与生物工程学院,宜春336000 [2]上海市浦东新区花木社区卫生服务中心,上海201204 [3]宜春学院医学院,宜春336000 [4]宜春市人民医院,宜春336000

出　　处：《中国抗生素杂志》2025年第2期205-215,共11页Chinese Journal of Antibiotics

摘　　要：目的 探究铜绿假单胞菌拮抗多重耐药肺炎克雷伯菌的作用及其机制,以期为临床治疗多重耐药肺炎克雷伯菌感染提供实验依据。方法 收集本院临床分离的4株铜绿假单胞菌(PA)和1株多重耐药肺炎克雷伯菌(MDR-KP8),建立两种菌共培养模型和平板互作模型,观察两者间的互作关系;收集菌株上清液,盐酸-氯仿、硫酸-蒽酮法对绿脓素和鼠李糖脂进行初步提取和定量,探究PA与MDR-KP8共培养的变化;硅胶吸附法和高效液相色谱法对绿脓素进行纯化和定量,肉汤稀释法测定绿脓素对MDR-KP8的最小抑菌浓度(MIC)和最小杀菌浓度(MBC),评价绿脓素的抗菌活性;碱性磷酸酶试剂盒、傅里叶红外光谱(FTIR)法探究绿脓素对MDR-KP8作用机理。结果 共培养4 d,4株PA菌落数均多于MDR-KP8菌落数;平板互作实验显示随着4株PA与MDR-KP8距离的增加,MDR-KP8菌落面积也在增加,而PA菌落面积未发生明显变化;定量结果显示4株PA共培养分泌的绿脓素和鼠李糖脂含量均多于单独培养时的含量。绿脓素对MDR-KP8最小抑菌浓度为128μg/mL;经绿脓素处理后,上清液中碱性磷酸酶、核酸、蛋白质等物质含量增加,蛋白质的二级结构发生变化。结论 PA与MDR-KP8间存在拮抗关系,PA分泌的绿脓素通过破坏细菌细胞壁,破坏蛋白质的二级结构,从而抑制MDR-KP8的生长繁殖。Objective To explore the effect and mechanism of Pseudomonas aeruginosa in antagonizing multidrug-resistant Klebsiella pneumoniae in order to provide an experimental basis for the clinical treatment of multidrug-resistant Klebsiella pneumoniae infection.Methods Four strains of Pseudomonas aeruginosa(PA)and one strain of multidrug-resistant Klebsiella pneumoniae(MDR-KP8)were collected from our hospital,the co-culture model and plate interaction model of the two strains were established,and the interaction between the two strains was observed.The supernatant of the strain was collected,the pyocyanin and rhamnolipid were preliminary extracted and quantified by the hydrochloric acid-chloroform and sulfate-anthone methods,and the changes of PA and MDR-KP8 co-culture were explored.The silica gel adsorption method and high-performance liquid chromatography were used to purify and quantify pyocyanin,the minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of pyocyanin against MDR-KP8 were determined by the broth dilution method,and the antibacterial activity of pyocyanin was evaluated.Alkaline phosphatase kit and Fourier transform infrared spectroscopy(FTIR)were used to explore the mechanism of action of pyocyanin on MDR-KP8.Results The results showed that the number of colonies in the four PA strains was higher than that of MDR-KP8 after 4 days.Plate interaction experiments showed that as the increase of the distance between the four PA strains and MDR-KP8 grew,so did the colony area of MDR-KP8.However,the colony area of PA did not change significantly.The quantitative results showed that the contents of pyocyanin and rhamnolipid secreted by the four PA co-cultures were higher than those in the separate culture.The minimum inhibitory concentration of pyocyanin against MDR-KP8 was 128µg/mL;After treatment with pyocyanin,the content of alkaline phosphatase,nucleic acid,protein and other substances in the supernatant increased,and the secondary structure of protein changed.Conclusion There was a

关 键 词：多重耐药肺炎克雷伯菌 铜绿假单胞菌 拮抗作用 绿脓素 细胞膜 

分 类 号：R965.1[医药卫生—药理学]