Dixneuf Sophie, Chareire-Kleiberg Anne-Coline, Mahé Pierre, El Azami Meriem, Kolytcheff Chloé, Bellais Samuel, Guyard Cyril, Védrine Christophe, Mallard Frédéric, Josso Quentin, Rol Fabian
BIOASTER Technology Research Institute, Lyon, France.
bioMérieux SA, La Balme-les-Grottes, France.
Front Microbiol. 2023 Aug 4;14:1232250. doi: 10.3389/fmicb.2023.1232250. eCollection 2023.
In this study, we assess the scattering of light and auto-fluorescence from single bacterial cells to address the challenge of fast (<2 h), label-free phenotypic antimicrobial susceptibility testing (AST). Label-free flow cytometry is used for monitoring both the respiration-related auto-fluorescence in two different fluorescence channels corresponding to FAD and NADH, and the morphological and structural information contained in the light scattered by individual bacteria during incubation with or without antibiotic. Large multi-parameter data are analyzed using dimensionality reduction methods, based either on a combination of 2D binning and Principal Component Analysis, or with a one-class Support Vector Machine approach, with the objective to predict the Susceptible or Resistant phenotype of the strain. For the first time, both (Gram-negative) and (Gram-positive) isolates were tested with a label-free approach, and, in the presence of two groups of bactericidal antibiotic molecules, aminoglycosides and beta-lactams. Our results support the feasibility of label-free AST in less than 2 h and suggest that single cell auto-fluorescence adds value to the Susceptible/Resistant phenotyping over single-cell scattering alone, in particular for the + (i.e., resistant) strains treated with oxacillin.
在本研究中，我们评估单个细菌细胞的光散射和自发荧光，以应对快速（<2小时）、无标记的表型抗菌药敏试验（AST）的挑战。无标记流式细胞术用于监测两个不同荧光通道中与呼吸相关的自发荧光，这两个通道分别对应黄素腺嘌呤二核苷酸（FAD）和烟酰胺腺嘌呤二核苷酸（NADH），以及在有或无抗生素孵育期间单个细菌散射光中包含的形态和结构信息。使用降维方法分析大量多参数数据，该方法基于二维分箱和主成分分析的组合，或采用一类支持向量机方法，目的是预测菌株的敏感或耐药表型。首次采用无标记方法对革兰氏阴性菌和革兰氏阳性菌分离株进行测试，并使用两组杀菌抗生素分子，即氨基糖苷类和β-内酰胺类。我们的结果支持在不到2小时内进行无标记AST的可行性，并表明单细胞自发荧光相对于仅单细胞散射，为敏感/耐药表型分析增加了价值，特别是对于用苯唑西林治疗的革兰氏阳性（即耐药）菌株。
