磷霉素联合亚胺培南对携带blaNDM的耐碳青霉烯类肠杆菌目细菌的体外抗菌活性  

In vitro antibacterial activity of fosfomycin combined with imipenem against blaNDM-positive carbapenem-resistant Enterobacterales strains

作　　者：林佳瑶 朱婕 闻奕丞 王靓 翟雅轩 钱艳 杜鸿[1] Lin Jiayao;Zhu Jie;Wen Yicheng;Wang Liang;Zhai Yaxuan;Qian Yan;Du Hong(Department of Clinical Laboratory,the Second Affiliated Hospital of Soochow University,Suzhou 215004)

机构地区：[1]苏州大学附属第二医院医学检验中心,苏州215004

出　　处：《中国抗生素杂志》2024年第10期1121-1130,共10页Chinese Journal of Antibiotics

基　　金：江苏省科技项目(No.BE2023704)。

摘　　要：目的探究磷霉素与亚胺培南联合使用对携带blaND00M的耐碳青霉烯类肠杆菌目细菌(carbapenem-resistant Enterobacterales,CRE)的体外抗菌活性。方法收集2021—2022年苏州大学附属第二医院医学检验中心临床分离的非重复CRE,并通过聚合酶链式反应(polymerase chain reaction,PCR)筛选出携带bla_(NDM)的耐碳青霉烯类肺炎克雷伯菌(carbapenem-resistant Klebsiella pneumoniae,CR-KP)和耐碳青霉烯类大肠埃希菌(carbapenem-resistant Escherichia coli,CR-E.coli)。通过微量肉汤稀释法测定菌株对常用抗生素的最低抑菌浓度(minimum inhibitory concentration,MIC)和琼脂稀释法测定菌株对磷霉素的MIC;采用棋盘法分别测定磷霉素与6种抗生素联用的部分抑菌浓度指数(fractional inhibitory concentration index,FICI)以判定药物联合效果;通过时间杀菌实验判断磷霉素与亚胺培南体外联合杀菌效果;采用结晶紫染色法检测菌株生物膜形成能力;通过荧光定量PCR检测不同抗生素条件下菌株bla_(NDM)、fosA、mrkA和murA基因的转录水平。结果PCR筛选出24株携带bla_(NDM)基因的CR-KP(12/24)和CR-E.coli(12/24)。棋盘试验显示磷霉素联合亚胺培南的协同作用(FICI≤0.5)相较其他抗生素效果最好(70.8%)。时间杀菌实验显示,磷霉素联合亚胺培南作用24 h内细菌数降低≥3 log_(10)CFU/mL,呈现出较强的杀菌效应。结晶紫染色结果显示磷霉素与亚胺培南联合可以抑制细菌生物膜形成。荧光定量PCR结果显示,磷霉素与亚胺培南联合处理后,生物膜形成与Ⅲ型菌毛合成重要基因mrkA表达相较对照组和亚胺培南组显著下调,而细胞壁形成重要基因murA表达相较其他两组上调。结论磷霉素与亚胺培南联用可以抑制细菌生长,其可能通过减少生物膜形成、影响菌毛和细胞壁形成等方面对携带bla_(NDM)的CR-KP与CR-E.coli发挥协同抗菌或杀菌作用。Objective To explore the in vitro antibacterial activity of fosfomycin combined with imipenem against bla_(NDM)-positive carbapenem-resistant Enterobacterales(CRE)strains.Methods Non-duplicate CRE strains were collected from the Department of Clinical Laboratory of the Second Affiliated Hospital of Soochow University during 2021-2022.And carbapenem-resistant Klebsiella pneumoniae(CR-KP)and carbapenem-resistant Escherichia coli(CR-E.coli)carrying bla_(NDM) were screened by polymerase chain reaction(PCR).Minimum inhibitory concentrations(MIC)of the strains against common antibiotics were determined by the microbroth dilution method,and the MIC of fosfomycin was determined by the agar dilution method.The effects of fosfomycin combined with other 6 antibiotics were described by fractional inhibitory concentration index(FICI)via checkerboard methods.A time-kill assay was conducted to observe the bactericidal effects of the combination of fosfomycin and imipenem in vitro.The ability of the biofilm formation was determined by crystal violet staining.Moreover,the transcription levels of bla_(NDM),fosA,mrkA,and murA genes under different antibiotic conditions were detected by fluorogenic quantitative PCR.Results A total of 24 strains of CR-KP(12/24)and CR-E.coli(12/24)carrying the bla_(NDM) gene were screened out by PCR.The checkerboard tests showed the best synergistic effect(FICI≤0.5)in the combination of fosfomycin with imipenem(70.8%)compared with other antibiotics.The time-kill assays showed that the combination of fosfomycin with imipenem reduced the number of bacteria to at least 3 log_(10)CFU/mL within 24 hours,indicating a strong bactericidal effect.Crystal violet staining showed that fosfomycin combined with imipenem could inhibit the formation of bacterial biofilm.Fluorogenic quantitative PCR showed that mrkA,an important gene for biofilm and typeⅢfimbriae formation,was significantly down-regulated under the combination of fosfomycin and imipenem compared with control groups and imipenem groups,while the im

关 键 词：磷霉素 亚胺培南 联合用药 肺炎克雷伯菌 大肠埃希菌 

分 类 号：R969.3[医药卫生—药理学]