Multiparameter flow cytometric analysis of antibiotic effects on membrane potential, membrane permeability, and bacterial counts of Staphylococcus aureus and Micrococcus luteus
Multiparameter flow cytometric analysis of antibiotic effects on membrane potential, membrane permeability, and bacterial counts of Staphylococcus aureus and Micrococcus luteus
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DOI：
10.1128/aac.44.4.827-834.2000
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发表时间：
2000-04-01
影响因子：
4.9
通讯作者：
Shapiro, HM
中科院分区：
文献类型：
--
作者：
Novo, DJ;Perlmutter, NG;Shapiro, HM
虽然流式细胞术已被用于研究抗生素对细菌膜电位（MP）和膜通透性的影响，流式细胞术的结果并不总是很好地相关的细菌计数的变化。使用新的，精确的技术，我们同时测量MP，膜渗透性，和颗粒计数的抗生素处理和未处理的金黄色葡萄球菌和微球菌黄体细胞。MP由二乙基氧碳菁[DiOC（2）（3）]的红色和绿色荧光的比率计算。由核酸染料TO-PRO-3的远红色荧光和绿色DiOC（2）（3）荧光的比率计算归一化渗透性参数。通过向样品中加入已知浓度的聚苯乙烯珠来计算细菌计数。阿莫西林在45分钟内增加渗透性，在< 1 μ g/ml的浓度下，一些生物体显示出增加的渗透性，但MP正常; 4小时后该群体消失，而细菌计数增加。当阿莫西林浓度高于1 μ g/ml时，MP不可逆地降低，颗粒计数不增加。四环素和红霉素引起较小的，剂量和时间依赖性的减少MP。四环素浓度< 1 μ g/ml时不改变渗透性，而四环素浓度为4 μ g/ml时可使50%的细菌渗透;红霉素浓度为4 μ g/ml时可使20%的细菌渗透。链霉素大幅降低MP，对渗透性无影响;氯霉素不改变渗透性或MP。红霉素预处理细菌防止链霉素和阿莫西林的影响。流式细胞术提供了一种敏感的手段，监测细菌暴露于抗菌药物中发生的动态细胞事件;然而，期望单个细胞参数的变化足以确定所有物种对所有药物的敏感性可能过于简单。
Although flow cytometry has been used to study antibiotic effects on bacterial membrane potential (MP) and membrane permeability, flow cytometric results are not always well correlated to changes in bacterial counts. Using new, precise techniques, we simultaneously measured MP, membrane permeability, and particle counts of antibiotic-treated and untreated Staphylococcus aureus and Micrococcus luteus cells. MP was calculated from the ratio of red and green fluorescence of diethyloxacarbocyanine [DiOC(2)(3)]. A normalized permeability parameter was calculated from the ratio of far red fluorescence of the nucleic acid dye TO-PRO-3 and green DiOC(2)(3) fluorescence. Bacterial counts were calculated by the addition of polystyrene beads to the sample at a known concentration. Amoxicillin increased permeability within 45 min, At concentrations of < 1 mu g/ml, some organisms showed increased permeability but normal MP; this population disappeared after 4 h, while bacterial counts increased. At amoxicillin concentrations above I mu g/ml, MP decreased irreversibly and the particle counts did not increase. Tetracycline and erythromycin caused smaller, dose- and time-dependent decreases in MP. Tetracycline concentrations of < 1 mu g/ml did not change permeability, while a tetracycline concentration of 4 mu g/ml permeabilized 50% of the bacteria; 4 mu g of erythromycin per ml permeabilized 20% of the bacteria. Streptomycin decreased MP substantially with no effect on permeability; chloramphenicol did not change either permeability or MP. Erythromycin pretreatment of bacteria prevented streptomycin and amoxicillin effects. Flow cytometry provides a sensitive means of monitoring the dynamic cellular events that occur in bacteria exposed to antibacterial agents; however, it is probably simplistic to expect that changes in a single cellular parameter will suffice to determine the sensitivities of all species to all drugs.