Advances in microscopy-based techniques applied to the antimicrobial resistance of foodborne pathogens

Background: Antimicrobial agents are effective weapons against foodborne pathogens; however, antimicrobial resistance (AMR) is increasing globally. The bacterial cell envelope is a key antimicrobial target, and understanding its structure and dynamics is crucial for detecting AMR pathogens and promoting rational antimicrobial design. Advanced microscopic techniques, such as cryo-electron microscopy (cryo-EM) and atomic force microscopy (AFM), provide unique real-time insights into cell envelope dynamics under antimicrobial stress, setting a benchmark for combating pathogens and AMR. Scope and approach: This review presents an overview of conventional and advanced microscopic techniques for bacterial cell envelope analysis, focusing, for each technique, the prospects, limitations, and current improvements. Key findings and conclusions: Conventional microscopic techniques, such as scanning electron microscopy, transmission electron microscopy, fluorescence microscopy, and confocal laser scanning microscopy, are predominant in foodborne bacterial analysis, mainly for morphological and ultrastructural observation. Advanced microscopic techniques, such as cryo-EM and AFM, are scarcely exploited owing to their expensive instrumentation, technical expertise requirements, and complex data processing. Nevertheless, cryo-EM reveals novel AMR mechanisms of numerous multidrug efflux pumps and resolves key membrane structures in foodborne pathogens. AFM captures antimicrobial action in bacterial membranes, providing insights into the structure-activity relationship of membrane-targeting antimicrobial agents. Additionally, cryo-EM and AFM can be integrated with artificial intelligence and machine learning tools, improving overall performance, reducing the need for technical expertise, and simplifying data processing. Therefore, advanced microscopic techniques are essential for developing novel strategies to overcome the threats of foodborne pathogens and AMR.