Antimicrobial peptides (AMPs) are a promising alternative to classical antibiotics in the fight against multi-resistant bacteria. They are produced by organisms from all domains of life and constitute a nearly universal defense mechanism against infectious agents. No drug can be approved without information about its mechanism of action. In order to use them in a clinical setting, it is pivotal to understand how AMPs work. While many pore-forming AMPs are well-characterized in model membrane systems, non-pore-forming peptides are often poorly understood. Moreover, there is evidence that pore formation may not happen or not play a role in vivo. It is therefore imperative to study how AMPs interact with their targets in vivo and consequently kill microorganisms. This has been difficult in the past, since established methods did not provide much mechanistic detail. Especially, methods to study membrane-active compounds have been scarce. Recent advances, in particular in microscopy technology and cell biological labeling techniques, now allow studying mechanisms of AMPs in unprecedented detail. This review gives an overview of available in vivo methods to investigate the antibacterial mechanisms of AMPs. In addition to classical mode of action classification assays, we discuss global profiling techniques, such as genomic and proteomic approaches, as well as bacterial cytological profiling and other cell biological assays. We cover approaches to determine the effects of AMPs on cell morphology, outer membrane, cell wall, and inner membrane properties, cellular macromolecules, and protein targets. We particularly expand on methods to examine cytoplasmic membrane parameters, such as composition, thickness, organization, fluidity, potential, and the functionality of membrane-associated processes. This review aims to provide a guide for researchers, who seek a broad overview of the available methodology to study the mechanisms of AMPs in living bacteria.

抗菌肽 (AMP) 是对抗多重耐药细菌的经典抗生素的有前途的替代品。它们由生命各个领域的生物体产生，构成了针对传染源的几乎普遍的防御机制。如果没有有关其作用机制的信息，任何药物都无法获得批准。为了在临床环境中使用它们，了解 AMP 的工作原理至关重要。虽然许多成孔 AMP 在模型膜系统中得到了很好的表征，但对非成孔肽通常知之甚少。此外，有证据表明孔隙形成可能不会发生或不会发挥作用体内。因此，有必要研究 AMP 如何与其目标相互作用体内从而杀死微生物。这在过去很困难，因为现有的方法没有提供太多的机械细节。特别是，研究膜活性化合物的方法一直很缺乏。最近的进展，特别是显微镜技术和细胞生物标记技术的进展，现在允许以前所未有的细节研究 AMP 的机制。这篇评论概述了可用的体内研究AMPs抗菌机制的方法。除了经典的作用模式分类分析之外，我们还讨论全局分析技术，例如基因组和蛋白质组方法，以及细菌细胞学分析和其他细胞生物学分析。我们介绍了确定 AMP 对细胞形态、外膜、细胞壁和内膜特性、细胞大分子和蛋白质靶标影响的方法。我们特别扩展了检查细胞质膜参数的方法，例如组成、厚度、组织、流动性、潜力和膜相关过程的功能。本综述旨在为寻求广泛概述现有方法来研究活细菌中 AMP 机制的研究人员提供指导。