Lehtinen Janne, Järvinen Suvi, Virta Marko, Lilius Esa-Matti
Department of Biochemistry and Food Chemistry, University of Turku, Arcanum, Vatselankatu 2, 20014 Turku, Finland.
J Microbiol Methods. 2006 Sep;66(3):381-9. doi: 10.1016/j.mimet.2006.01.002. Epub 2006 Feb 17.
Kinetic measurements of the bacteriostatic, bactericidal and bacteriolytic activities of six model antibiotics (ampicillin, erythromycin, nalidixic acid, polymyxin B, tetracycline, and trimethoprim) against Escherichia coli as target bacteria were performed by bioluminescence, fluorescence, and optical density based real-time assay. Additionally, plate counting was used as a control measurement. The gfp and insect luciferase (lucFF) genes were cloned into cells used for measurements to enable fluoro-luminometric detection. Bacteria were exposed to antibiotics for 10 h, and the effects of antimicrobial agents were established. Inhibitory concentration of 50% (IC(50)), minimum bactericidal concentration (MBC), and bactericidal concentration of 50% (BC(50)) of each antibiotic were calculated from these procedures. Bacteriostatic, bactericidal or bacteriolytic actions of each antibiotic, as well the time interval from exposure to visible effect, were readily observed from kinetic data. No significant differences were observed between data obtained with the different methods employed. Ampicillin and polymyxin B were clearly bacteriolytic, nalidixic acid and tetracycline showed bactericidal effects, and erythromycin and trimethoprim were bacteriostatic drugs. The assay has the advantage of speed and accurately discerns between lytic, cidal and static compounds. Thus, this reliable and fully automated novel kinetic assay with high sample capacity offers new possibilities for real-time detection, making it suitable for diverse high throughput screening (HTS) applications.
通过基于生物发光、荧光和光密度的实时测定法，对六种模型抗生素（氨苄青霉素、红霉素、萘啶酸、多粘菌素B、四环素和甲氧苄啶）针对目标细菌大肠杆菌的抑菌、杀菌和溶菌活性进行了动力学测量。此外，平板计数用作对照测量。将绿色荧光蛋白（gfp）基因和昆虫荧光素酶（lucFF）基因克隆到用于测量的细胞中，以实现荧光-发光检测。将细菌暴露于抗生素10小时，确定抗菌剂的效果。从这些程序中计算出每种抗生素的50%抑制浓度（IC(50)）、最低杀菌浓度（MBC）和50%杀菌浓度（BC(50)）。从动力学数据中可以很容易地观察到每种抗生素的抑菌、杀菌或溶菌作用，以及从暴露到可见效果的时间间隔。采用的不同方法所获得的数据之间未观察到显著差异。氨苄青霉素和多粘菌素B明显具有溶菌作用，萘啶酸和四环素显示出杀菌效果，而红霉素和甲氧苄啶是抑菌药物。该测定法具有速度快的优点，能够准确区分溶菌、杀菌和抑菌化合物。因此，这种可靠且全自动的新型动力学测定法具有高样本容量，为实时检测提供了新的可能性，使其适用于各种高通量筛选（HTS）应用。
