摘要
目的建立测定细胞内活菌含量的活菌核酸微定量技术,考察临床常用抗菌药物对胞内鲍曼不动杆菌感染的影响。方法采用抗菌药物-病原菌-上皮细胞共培养模型,以改良叠氮溴化丙锭-实时荧光定量聚合酶链反应(PMAxx-qPCR)法测定不同种类和不同浓度的抗菌药物对鲍曼不动杆菌侵袭人支气管上皮(HBE)细胞形成胞内菌数量的影响;显微镜下观察0.1倍最低抑菌浓度(MIC)庆大霉素对绿色荧光蛋白融合鲍曼不动杆菌(AB-GFP)侵袭HBE细胞的影响。结果与对照组比较,经0.1倍、1倍、10倍MIC的阿奇霉素、左氧氟沙星、头孢哌酮舒巴坦、替加环素,以及10倍、1倍MIC的多西环素、庆大霉素干预的鲍曼不动杆菌侵袭HBE细胞形成胞内菌的数量均显著降低(P<0.001);经0.1倍MIC的庆大霉素干预的胞内鲍曼不动杆菌的数量均显著升高(P<0.001)。在0.1倍MIC的庆大霉素作用下,AB-GFP侵袭进入HBE细胞的量显著增加。结论0.1倍、1倍、10倍MIC的阿奇霉素、左氧氟沙星、头孢哌酮舒巴坦、替加环素、庆大霉素、多西环素均不能有效杀灭胞内菌。该方法操作简便、结果准确,能快速地测定胞内鲍曼不动杆菌的浓度,可用于评价抗菌药物对胞内鲍曼不动杆菌感染的影响,以及胞内鲍曼不动杆菌对抗菌药物的敏感性。
Objective To establish a micro-quantification method of nucleic acid for live bacteria for detecting intracellular viable bacterial content,and to investigate the effect of commonly used antibiotics on the intracellular infection of Acinetobacter baumannii.Methods Through the improved azide bromide propidium real-time fluorescence quantitative polymerase chain reaction(PMAxx-qPCR)method,a co-culture model of antibiotics-pathogens-epithelial cells was adopted to determine the effect of different types and concentrations of antibiotics on the number of intracellular bacteria formed by Acinetobacter baumannii invading human bronchial epithelial(HBE)cells.The effect of 0.1 times minimum inhibitory concentration(MIC)of gentamicin on the HBE cells invaded by Acinetobacter baumannii with green fluorescent protein fusion(AB-GFP)was observed under the microscepe.Results Compared with those in the control group,the number of intracellular Acinetobacter baumannii invading epithelial cells significantly reduced after MIC intervention of azithromycin,levofloxacin,cefoperazone/sulbactam,tigecycline at concentrations of 0.1,1,10 times MIC respectively,and doxycycline and gentamicin at concentrations of 10,1 times MIC(P<0.001),the number of intracellular Acinetobacter baumannii significantly increased after the intervention of gentamicin at the concentration of 0.1 times MIC(P<0.001).Under the action of gentamicin at the concentration of 0.1 times MIC,the invasion of AB-GFP to HBE cells significantly increased.Conclusion 0.1,1,10 times MIC of azithromycin,levofloxacin,cefoperazone and sulbactam,tigecycline,gentamicin,doxycycline can not effectively kill intracellular bacteria.This method is simple and accurate,which can quickly determine the concentration of intracellular Acinetobacter baumannii.It can be used to evaluate the effect of antibiotics on intracellular infection of Acinetobacter baumannii and the sensitivity of Acinetobacter baumannii to antibiotics.
作者
翁邦碧
李玉良
罗丹
枉前
WENG Bangbi;LI Yuliang;LUO Dan;WANG Qian(The First Affiliated Hospital of the Army Medical University,Chongqing,China 400038;The 920th Hospital of Joint Logistics Support Force,Kunming,Yunnan,China 650032)
出处
《中国药业》
CAS
2023年第11期33-38,共6页
China Pharmaceuticals
基金
国家自然科学基金青年科学基金项目[81802117]
重庆市卫生健康委员会医学科研项目[2022WSJK013]。
