title: Antibiotic Susceptibility Testing of the Gram-Negative Bacteria Based on Flow Cytometry.
abstract: Rapidly treating infections with adequate antibiotics is of major importance. This requires a fast and accurate determination of the antibiotic susceptibility of bacterial pathogens. The most frequently used methods are slow because they are based on the measurement of growth inhibition. Faster methods, such as PCR-based detection of determinants of antibiotic resistance, do not always provide relevant information on susceptibility, particularly that which is not genetically based. Consequently, new methods, such as the detection of changes in bacterial physiology caused by antibiotics using flow cytometry and fluorescent viability markers, are being explored. In this study, we assessed whether Alexa Fluor® 633 Hydrazide (AFH), which targets carbonyl groups, can be used for antibiotic susceptibility testing. Carbonylation of cellular macromolecules, which increases in antibiotic-treated cells, is a particularly appropriate to assess for this purpose because it is irreversible. We tested the susceptibility of clinical isolates of Gram-negative bacteria, Escherichia coli and Pseudomonas aeruginosa, to antibiotics from the three classes: β-lactams, aminoglycosides, and fluoroquinolones. In addition to AFH, we used TO-PRO®-3, which enters cells with damaged membranes and binds to DNA, and DiBAC4 (3), which enters cells with depolarized membranes. We also monitored antibiotic-induced morphological alterations of bacterial cells by analyzing light scattering signals. Although all tested dyes and light scattering signals allowed for the detection of antibiotic-sensitive cells, AFH proved to be the most suitable for the fast and reliable detection of antibiotic susceptibility.
authors: Institut National de la Santé et de la Recherche Médicale, Sorbonne Paris Cité, Faculté de Médecine Paris Descartes, Université Paris Descartes Paris, France.
authors: Claude
authors: C
authors: Saint-Ruf
authors: Institut National de la Santé et de la Recherche Médicale, Sorbonne Paris Cité, Faculté de Médecine Paris Descartes, Université Paris Descartes Paris, France.
authors: Steve
authors: S
authors: Crussard
authors: Microbiology Unit, R&D Microbiology, BioMérieux SA La Balme Les Grottes, France.
authors: Christine
authors: C
authors: Franceschi
authors: Microbiology Unit, R&D Microbiology, BioMérieux SA La Balme Les Grottes, France.
authors: Sylvain
authors: S
authors: Orenga
authors: Institut National de la Santé et de la Recherche Médicale, Sorbonne Paris Cité, Faculté de Médecine Paris Descartes, Université Paris Descartes Paris, France.
authors: Jasmine
authors: J
authors: Ouattara
authors: Microbiology Unit, R&D Microbiology, BioMérieux SA La Balme Les Grottes, France.
authors: Mahendrasingh
authors: M
authors: Ramjeet
authors: Institut National de la Santé et de la Recherche Médicale, Sorbonne Paris Cité, Faculté de Médecine Paris Descartes, Université Paris DescartesParis, France; Microbiology Unit, R&D Microbiology, BioMérieux SALa Balme Les Grottes, France.
authors: Jérémy
authors: J
authors: Surre
authors: Institut National de la Santé et de la Recherche Médicale, Sorbonne Paris Cité, Faculté de Médecine Paris Descartes, Université Paris Descartes Paris, France.
authors: Ivan
authors: I
authors: Matic
keywords: antibiotic susceptibility testing; bacteria; flow cytometry; fluorescent hydrazide; protein carbonylation