锌螯合剂多吡啶配体对产金属β-内酰胺酶细菌抗生素耐药性的抑制作用  

Inhibitory effect of zinc chelator polypyridine ligands on antibiotic resistance in metallo-β-lactamase-producing bacteria

作　　者：袁建平[1] 曾云清[2] 陈敏[3] 王婷 蒋宏[4] YUAN Jianping;ZENG Yunqing;CHEN Min;WANG Ting;JIANG Hong(Department of Allergy,Hunan Maternal and Child Health Hospital,Changsha 410000,China;Department of Neonatology,Hunan Maternal and Child Health Hospital;Department of Laboratory Medicine,Hunan Maternal and Child Health Hospital;Department of Infection Control,Hunan Maternal and Child Health Hospital)

机构地区：[1]湖南省妇幼保健院院感科,长沙410000 [2]湖南省妇幼保健院新生儿一科 [3]湖南省妇幼保健院检验科 [4]湖南省妇幼保健院感染控制科

出　　处：《山西医科大学学报》2022年第10期1294-1301,共8页Journal of Shanxi Medical University

摘　　要：目的探讨锌螯合剂多吡啶配体对产金属β-内酰胺酶细菌(MBL)抗生素耐药性的抑制作用。方法收集湖南省妇幼保健院检验科于2020年10月至2021年10月检测的住院患者β-内酰胺类耐药肠杆菌感染阳性的痰液标本35份,并分离3种产MBL临床菌株:产新德里金属β-内酰胺酶1(NDM-1)肺炎克雷伯菌、产L1型金属β-内酰胺酶嗜麦芽葡萄球菌和产VIM型金属β-内酰胺酶(VIM)梭状芽孢杆菌。利用乙二胺骨架设计合成锌配体:N,N′-二(2-吡啶甲基)-乙二胺(Bispicen)、N,N,N′-三(2-吡啶甲基)-乙二胺(Trispicen)、N,N′-二(2-吡啶甲基)-乙二胺-N′-乙酸(BispicenA)、N,N,N′-三(2-吡啶甲基)-乙二胺-N′-乙酸(TrispicenA)和N,N,N′-三(2-吡啶甲基)-乙二胺-N′-乙酰-D-丙氨酰-D-丙氨酰-D-丙氨酸甲酯(TrispicenDAla)。采用分光光度法测定并计算锌配体的稳定常数(K);核磁共振氢谱(1H NMR)检测TrispicenA与Zn^(2+)的络合作用;黄酰罗丹明B(SRB)法检测锌配体细胞毒性,计算其半数抑制浓度(IC_(50));标准微量肉汤稀释法测定合成配体对临床分离耐药菌的最低抑菌浓度(MIC)。棋盘法联合抑菌实验检测美罗培南(MEM)和锌配体之间的协同作用,并计算每种锌配体的分级抑制浓度指数(FICI)。结果5种锌配体K值范围为10.9~15.6,且均可与Zn^(2+)产生络合反应,具有稳定的化学结构。TPEN、Trispicen、TrispicenA和Bispicen对分离菌株显示出细胞毒性作用,IC_(50)范围为(4.91±0.12)μmol/L至(17.8±4.1)μmol/L,而BispicenA和TrispicenDAla未产生细胞毒性。标准微量肉汤稀释法结果显示,TPEN、TrispicenDAla、TrispicenA和BispicenA对3种菌株的MIC值较高,均分别为256 mg/L,>256 mg/L,512 mg/L,>512 mg/L。而Trispicen、Bispicen仅在产NDM肺炎克雷伯菌和产VIM梭状芽孢杆菌中观察到较高MIC值,分别为256 mg/L和64 mg/L。棋盘法联合抑菌实验结果显示,MEM和TrispicenDAla、TrispicenA、Trispicen、TPEN之间FICI均小于0.5,对所有测Objective To investigate the inhibitory effect of zinc chelator polypyridine ligands on the antibiotic resistance of metallo-β-lactamase-producing bacteria(MBL).Methods A total of 35 positive sputum samples ofβ-lactam-resistant Enterobacteriaceae infection were collected from inpatients in Hunan Provincial Maternal and Child Health Hospital from October 2020 to October 2021,and three MBL-producing clinical strains were isolated:New Delhi metallo-β-lactamase 1(NDM-1)-producing Klebsiella pneumoniae,L1 metallo-β-lactamase-producing Staphylococcus maltophilia,VIM-type metallo-β-lactamase(VIM)-producing clostridium.Zinc ligands were designed and synthesized using ethylenediamine skeleton:N,N′-di(2-pyridylmethyl)-ethylenediamine(Bispicen),N,N,N′-tris(2-pyridylmethyl)-ethylenediamine(Trispicen),N,N′-di(2-pyridylmethyl)-ethylenediamine-N′-acetic acid(BispicenA),N,N,N′-tris(2-pyridylmethyl)-ethylenediamine-N(TrispicenA)and N,N,N′-tris([2-Pyridylmethyl)-ethylenediamine-N′-acetyl-D-alanyl-D-alanyl-D-alanine methylester(TrispicenDAla).The stability constants(K)of zinc ligands were determined and calculated by spectrophotometry.Nuclear magnetic resonance spectra(1H NMR)was used to determine the complexation of TrispicenA with Zn^(2+).Sulforhodamine B(SRB)staining was used to detect the cytotoxicity of zinc ligands and calculate their half-inhibitory concentrations(IC_(50)).Standard microbroth dilution method was used to detect the minimum inhibitory concentration(MIC)of synthetic ligands against clinically isolated drug-resistant bacteria.The synergistic effect between meropenem(MEM)and zinc ligands was detected by the checkerboard method combined with antibacterial experiments,and the fractional inhibitory concentration index(FICI)of each zinc ligand was calculated.Results The K values of the five zinc ligands ranged from 10.9 to 15.6,and all of them had complex reactions with Zn^(2+) and had stable chemical structures.TPEN,Trispicen,TrispicenA and Bispicen showed cytotoxicity to isolated strains with IC_

关 键 词：多吡啶配体 产金属β-内酰胺酶细菌 抗生素耐药性 美罗培南 

分 类 号：R969[医药卫生—药理学]