Endres Bradley, Bassères Eugénie, Rashid Tasnuva, Chang Long, Alam M Jahangir, Garey Kevin W
Department of Pharmacy Practice and Translational Research, University of Houston College of Pharmacy.
Department of Electrical and Computer Engineering, University of Houston.
J Vis Exp. 2017 May 25(123):55383. doi: 10.3791/55383.
Assessment of antibiotic action with new drug development directed towards anaerobic bacteria is difficult and technically demanding. To gain insight into possible MOA, morphologic changes associated with antibiotic exposure can be visualized using scanning electron microscopy (SEM). Integrating SEM imaging with traditional kill curves may improve our insight into drug action and advance the drug development process. To test this premise, kill curves and SEM studies were conducted using drugs with known but different MOA (vancomycin and metronidazole). C. difficile cells (R20291) were grown with or without the presence of antibiotic for up to 48 h. Throughout the 48 h interval, cells were collected at multiple time points to determine antibiotic efficacy and for imaging on the SEM. Consistent with previous reports, vancomycin and metronidazole had significant bactericidal activity following 24 h of treatment as measured by colony-forming unit (CFU) counting. Using SEM imaging we determined that metronidazole had significant effects on cell length (> 50% reduction in cell length for each antibiotic; P< 0.05) compared to controls and vancomycin. While the phenotypic response to drug treatment has not been documented previously in this manner, they are consistent with the drug's MOA demonstrating the versatility and reliability of the imaging and measurements and the application of this technique for other experimental compounds.
评估针对厌氧菌的新药开发中的抗生素作用既困难又对技术要求很高。为了深入了解可能的作用机制，可以使用扫描电子显微镜（SEM）观察与抗生素暴露相关的形态学变化。将SEM成像与传统的杀菌曲线相结合，可能会增进我们对药物作用的了解，并推动药物开发进程。为了验证这一前提，我们使用了具有已知但不同作用机制的药物（万古霉素和甲硝唑）进行了杀菌曲线和SEM研究。艰难梭菌细胞（R20291）在有或没有抗生素存在的情况下培养长达48小时。在整个48小时期间，在多个时间点收集细胞，以确定抗生素疗效并用于SEM成像。与先前的报道一致，通过菌落形成单位（CFU）计数测量，万古霉素和甲硝唑在治疗24小时后具有显著的杀菌活性。通过SEM成像，我们确定与对照和万古霉素相比，甲硝唑对细胞长度有显著影响（每种抗生素处理后细胞长度减少>50%；P<0.05）。虽然此前尚未以这种方式记录药物治疗的表型反应，但它们与药物的作用机制一致，证明了成像和测量的通用性和可靠性，以及该技术在其他实验化合物中的应用。
