多西环素联合左氧氟沙星对耐药肺炎克雷伯菌的体内外抗菌活性研究  

In vitro and in vivo antibacterial activity of doxycycline combined with levofloxacin against carbapenem⁃resistant Klebsiella pneumoniae

作　　者：吴凡 王保贵 胡文 陈春林[2] WU Fan;WANG Bao-gui;HU Wen;CHEN Chun-lin(Jiangxi Drug Inspection Center,Nanchang 330000,China;School of Chemical and Biological Engineering,Yichun University,Yichun 336000,China;Yichun People's Hospital,Yichun 336000,China)

机构地区：[1]江西省药品检查员中心,南昌330000 [2]宜春学院化学与生物工程学院,宜春336000 [3]宜春市人民医院,宜春336000

出　　处：《中国新药杂志》2023年第23期2425-2433,共9页Chinese Journal of New Drugs

摘　　要：目的:探究多西环素联合左氧氟沙星对耐碳青霉烯类肺炎克雷伯菌(carbapenem resistant Klebsiella pneumoniae, CRKP)的体内外抗菌活性。方法:于宜春市人民医院收集临床分离的8株CRKP,体外采用微量肉汤稀释法测定多西环素和左氧氟沙星对8株CRKP的最小抑菌浓度(minimal inhibition concentration, MIC),棋盘稀释法判断两药联用的抑菌效果,时间-杀菌曲线法进一步评价两药联合的杀菌作用。结晶紫染色半定量生物膜法测定两药联用对生物膜形成的抑制作用和消除作用。体内利用鼻腔滴注CRKP菌液建立小鼠肺部急性感染模型,记录小鼠精神状态及肺部组织荷菌量、血清炎性因子C反应蛋白(C-reactive protein, CRP)、白细胞介素(IL)-6的水平和病理形态学改变等指标评估,分析多西环素联合左氧氟沙星对CRKP的体内抗菌活性。结果:多西环素对8株CRKP的MIC为4~256 ug·mL^(-1),左氧氟沙星对8株CRKP的MIC为16~256 ug·mL^(-1)。棋盘法显示多西环素联合左氧氟沙星对8株CRKP具有80%的协同或相加作用;时间-杀菌曲线显示,多西环素联合左氧氟沙星对CRKP2和CRKP7作用24 h后使细菌数较初始细菌数降低≥2 lg CFU·mL^(-1),呈现抑菌或杀菌作用;结晶紫染色法结果表明,多西环素与左氧氟沙星联用对CRKP生物膜具有抑制和破坏作用;动物实验表明,与模型组相比,联合组小鼠精神状态良好,肺部组织荷菌量及CRP,IL-6水平显著降低(P<0.001);HE染色结果表明,给药组较模型组肺泡结构清晰,炎性细胞减少,充血面积降低,差异均有统计学意义(P<0.001)。结论:体外实验表明,多西环素联用左氧氟沙星对CRKP具有协同抗菌或杀菌作用,体内实验表明多西环素联用左氧氟沙星可降低炎性指标,有效治疗肺组织细菌感染,有较好的体内抗菌作用。Objective:To investigate the in vivo and in vitro antibacterial activity of doxycycline combined with levofloxacin on carbapenem⁃resistant Klebsiella pneumoniae(CRKP).Methods:Eight clinically isolated CRKP strains were collected in Yichun People's Hospital,and the minimum inhibitory concentration(MIC)of 8 CRKPs was determined using microbroth dilution method in vitro.The antibacterial effect of the two drugs was judged by checkerboard dilution method. The time⁃sterilization curve was further applied to evaluate the combinedbactericidal effect of the two drugs. Crystal violet staining semi⁃quantitative biofilm method was used to determinethe inhibitory effect and elimination effect of the combination of two drugs on biofilm formation. In vivo, a model ofacute lung infection of mice was established by nasal instillation of CRKP bacteria, and the mental state of mice wasrecorded. The bacterial load of lung tissues, the levels of serum inflammatory factor C⁃reactive protein (CRP), IL⁃6and pathological morphological changes were evaluated and analyzed. Results: The MIC of doxycycline to 8 CRKPstrains was 4 ~ 256 ug·mL^(-1), levofloxacin to 8 strains of CRKP MIC of 16 ~ 256 ug·mL^(-1). The checkerboardmethod showed that doxycycline combined with levofloxacin had an 80% synergistic or additive effect on 8 CRKPstrains. The time⁃sterilization curve showed that after the treatment of doxycycline combined with levofloxacin onCRKP2 and CRKP7 for 24 hours, the number of bacteria decreased by ≥2 lg CFU·mL^(-1) compared with the initialbacterial number, showing bacteriostatic or bactericidal effects. The results of crystal violet staining showed that thecombination of doxycycline and levofloxacin had inhibitory and destructive effects on CRKP biofilms. Animalexperiments showed that compared with the model group, the combined group mice had a good mental state, andthe loading levels of lung tissue, C⁃reactive protein (CRP) and IL⁃6 significantly reduced (P <0.001). The resultsof HE staining showed that the a

关 键 词：耐碳青霉烯类肺炎克雷伯菌 多西环素 左氧氟沙星 联合药敏 生物膜 动物实验 

分 类 号：R965[医药卫生—药理学]