Department of Basic Medical Sciences, Purdue University, West Lafayette, IN, 47907, USA.
Bindley Bioscience Center, Purdue University, West Lafayette, IN, 47907, USA.
Appl Microbiol Biotechnol. 2024 Jul 3;108(1):406. doi: 10.1007/s00253-024-13232-0.
The proliferation and dissemination of antimicrobial-resistant bacteria is an increasingly global challenge and is attributed mainly to the excessive or improper use of antibiotics. Currently, the gold-standard phenotypic methodology for detecting resistant strains is agar plating, which is a time-consuming process that involves multiple subculturing steps. Genotypic analysis techniques are fast, but they require pure starting samples and cannot differentiate between viable and non-viable organisms. Thus, there is a need to develop a better method to identify and prevent the spread of antimicrobial resistance. This work presents a novel method for detecting and identifying antibiotic-resistant strains by combining a cell sorter for bacterial detection and an elastic-light-scattering method for bacterial classification. The cell sorter was equipped with safety mechanisms for handling pathogenic organisms and enabled precise placement of individual bacteria onto an agar plate. The patterning was performed on an antibiotic-gradient plate, where the growth of colonies in sections with high antibiotic concentrations confirmed the presence of a resistant strain. The antibiotic-gradient plate was also tested with an elastic-light-scattering device where each colony's unique colony scatter pattern was recorded and classified using machine learning for rapid identification of bacteria. Sorting and patterning bacteria on an antibiotic-gradient plate using a cell sorter reduced the number of subculturing steps and allowed direct qualitative binary detection of resistant strains. Elastic-light-scattering technology is a rapid, label-free, and non-destructive method that permits instantaneous classification of pathogenic strains based on the unique bacterial colony scatter pattern. KEY POINTS: • Individual bacteria cells are placed on gradient agar plates by a cell sorter • Laser-light scatter patterns are used to recognize antibiotic-resistant organisms • Scatter patterns formed by colonies correspond to AMR-associated phenotypes.
抗菌药物耐药菌的增殖和传播是一个日益严重的全球性挑战，主要归因于抗生素的过度或不当使用。目前，检测耐药菌株的金标准表型方法是琼脂平板培养，这是一个耗时的过程，涉及多个传代步骤。基因型分析技术快速，但它们需要纯起始样品，并且不能区分活的和非活的生物体。因此，需要开发一种更好的方法来识别和防止抗生素耐药性的传播。这项工作提出了一种通过结合细菌检测的细胞分选仪和细菌分类的弹性光散射方法来检测和识别抗生素耐药菌株的新方法。细胞分选仪配备了处理病原体的安全机制，并能够将单个细菌精确地放置在琼脂平板上。图案形成在抗生素梯度平板上进行，其中在抗生素浓度高的部分中菌落的生长证实了存在耐药菌株。抗生素梯度平板也使用弹性光散射装置进行了测试，其中记录了每个菌落的独特菌落散射模式，并使用机器学习对其进行分类，以快速识别细菌。使用细胞分选仪在抗生素梯度平板上对细菌进行分选和图案化减少了传代步骤的数量，并允许直接定性二元检测耐药菌株。弹性光散射技术是一种快速、无标记且非破坏性的方法，可根据独特的细菌菌落散射模式即时对致病菌株进行分类。关键点：
通过细胞分选仪将单个细菌细胞放置在梯度琼脂平板上。
激光光散射图案用于识别具有抗药性的生物。
菌落形成的散射图案与 AMR 相关表型相对应。
