表征形态变化的议定书<em>艰难梭菌</em>应对抗生素治疗
Summary
Antibiotic efficacy is most commonly determined by conducting killing kinetic studies and measuring colony forming units (CFUs). By integrating scanning electron microscopy (SEM) with these standard methods, we can distinguish the pharmacological effects of treatment between different antibiotics.
Abstract
用针对厌氧菌的新药开发评估抗生素的作用是困难和技术上的要求。为了获得可能的MOA的了解，可以使用扫描电子显微镜（SEM）显现与抗生素暴露相关的形态学变化。将SEM成像与传统杀伤曲线整合可能会提高我们对药物作用的深入了解，推动药物开发过程。为了测试这个前提，用已知但不同的MOA（万古霉素和甲硝唑）的药物进行杀伤曲线和SEM研究。 艰难梭菌细胞（R20291）在有或没有抗生素存在下长达48小时生长。在48小时的间隔期间，在多个时间点收集细胞以确定抗生素的功效并在SEM上成像。与以前的报道一致，万古霉素和甲硝唑在通过菌落形成单位（CFU）计算的24小时治疗后具有显着的杀菌活性婷。使用SEM成像，我们确定甲硝唑对细胞长度有显着影响（与对照组和万古霉素相比，每种抗生素的细胞长度减少50％， P <0.05）。虽然以前没有以这种方式记录对药物治疗的表型反应，但是它们与药物的MOA一致，证明了成像和测量的通用性和可靠性以及该技术对其他实验化合物的应用。
Introduction
艰难梭菌是一种革兰氏阳性，形成细菌的细菌，每年在美国造成大约50万感染，被疾病控制和预防中心（CDC）认定为威胁级紧急病原体，这是最高的风险。 1过去十年来，针对艰难梭菌活性的抗微生物药物开展了相当大的药物开发 。 2，3 体外研究是药物开发过程的必要组成部分。 4传统上， 体外易感性和时间杀死研究用于验证未来动物和其他体内研究。
虽然这些方法在评估杀伤作用方面起着重要作用，但它们不能捕获细胞对药理学治疗的表型反应。通过纳入扫描电子显微镜（SEM）与标准d杀死动力学研究，可以更全面地表征抗生素的直接作用。 5，6，7这里，我们提供一种方法，其中SEM用作描述抗生素治疗功效的手段。
Protocol
Representative Results
Discussion
目前的研究目的是创建一种高通量的方法来分离艰难梭菌 ，并使用扫描电子显微镜（SEM）作为抗菌药物药理作用的更全面表征的手段。使用本文概述的方案，我们已经证明，成像细胞对抗生素治疗的表型反应可以揭示药物的药理作用的洞察力。总的来说，该方案的成像部分在收集细胞后的持续时间大约需要2小时，但比单独的典型的杀伤动力学研究更为歧视。在学习使用SEM的同时，技术上?…
Declarações
The authors have nothing to disclose.
Acknowledgements
These experiments have been supported by research grants from Merck and Co. and Summit, PLC.
Materials
| cotton gauze | Caring | PRM21408C | |
| NaCl | Macron | 7532 | |
| 50mL tubes | Falcon | 352098 | |
| Brain Heart Infusion (BHI) | Criterion | C5141 | |
| L-cysteine | Alfa Aesar | A10389 | |
| yeast extract | Criterion | C741 | |
| sodium taurocholate | Alfa Aesar | A18346 | |
| anaerobic chamber | Coy | vinyl anaerobic chamber | |
| cycloserinecefoxitin fructose agar (CCFA) plates | Anaerobe systems | AS-213 | |
| blood agar plates | Hardy diagnostics | A-10 | |
| latex agglutination reagent | Oxoid | DR1107A | C. diff test kit |
| microcentrifuge tubes | Eppendorf | 222363204 | |
| PBS | Gibco | 10010-031 | |
| 4% paraformaldehyde | Fisher Scientific | 50-259-98 | |
| microscope slides | J. Melvin freed brand | 7525M | 75x25mm |
| flow hood | Labconco | Class II type A2 | biosafety cabinet |
| desk sputtering machine | Denton Vacuum | Desk II | |
| tape | Plastic Core | 05072-AB | SPI Double Sided Adhesive Carbon Tape |
| gold | Denton Vacuum | TAR001-0158 | 2.375” Diameter x .002” Thick Gold foil |
| scanning electron microscope | FEI | XL-30 |
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