Flow cytometric monitoring of antibiotic-induced injury in Escherichia coli using cell-impermeant fluorescent probes
Flow cytometric monitoring of antibiotic-induced injury in Escherichia coli using cell-impermeant fluorescent probes
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DOI：
10.1128/aac.44.3.676-681.2000
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发表时间：
2000-03-01
影响因子：
4.9
通讯作者：
Gant, VA
中科院分区：
文献类型：
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作者：
Mortimer, FC;Mason, DJ;Gant, VA
评价了三种荧光核酸结合染料-碘化丙啶、TO-PRO-1和SYTOX绿色，并比较了它们区分具有和不具有完整胞质膜的细菌细胞的能力。每种染料都能够容易地区分健康的和透化的大肠杆菌细胞，尽管SYTOX绿色在染色受损时显示出更大的荧光强度增强，与健康的相反，流式细胞仪检测E.大肠杆菌在暴露于几种抗菌剂后用这些染料染色，表明所有三种染料都能够检测抗菌作用。然而，值得注意的是，细胞相关荧光的强度与抗微生物剂的作用机制有关。对于所有染料，在β-内酰胺抗生素作用之后观察到荧光强度的大的变化，但是较小的变化在暴露于直接或间接作用于核酸合成的抗微生物剂后，观察到了细胞内的DNA含量（或无变化）。细胞相关的荧光与平板计数所确定的存活力的丧失无关。尽管提供了对抗菌剂作用机制的深入了解，但如果将菌落形成用作标准，则这些基本问题与快速抗菌剂敏感性测试的开发相关。
Three fluorescent nucleic acid binding dyes-propidium iodide, TO-PRO-1, and SYTOX green ere evaluated, and their abilities to distinguish between bacterial cells,vith and without an intact cytoplasmic membrane were compared, Each dye was readily able to discriminate between healthy and permeabilized cells of Escherichia coli, although SYTOX green showed a greater enhancement in fluorescence intensity on staining-compromised, as opposed to healthy, cells in log-phase growth, than either PI or TO-PRO-I, Flow cytometric analysis of E. coli stained with these dyes after exposing them to several antimicrobial agents showed that all three dyes were able to detect antimicrobial action. Notably, however, the intensity of the cell-associated fluorescence was related to the mechanism of action of the antimicrobial agent, Large changes in fluorescence intensity were observed for all the dyes subsequent to beta-lactam antibiotic action, but smaller changes (or no change) were seen subsequent to exposure to antimicrobials acting directly or indirectly on nucleic acid synthesis, Furthermore, cell-associated fluorescence did not relate to loss of viability as determined by plate counts, Despite offering much insight into antimicrobial mechanisms of action, these fundamental problems become relevant to the development of rapid antimicrobial susceptibility tests if colony formation is used as the standard.