Fluorescent d-amino Acid-Based Approach Enabling Fast and Reliable Measure of Antibiotic Susceptibility in Bacterial Cells

The threat of multidrug-resistant bacteria has been increasing steadily in the past century, posing a major health risk (Organización Mundial de la Salud. Directrices Sobre Componentes Básicos Para Los Programas de Prevención y Control de Infecciones a Nivel Nacional y de Establecimientos de Atención de Salud Para Pacientes Agudos; Organización Mundial de la Salud: Ginebra, 2017 ). Even though every year, 226 million antibiotics are prescribed in the United States alone, 50% of these prescriptions are inappropriate for the patient’s condition (CDC. Get Smart about Antibiotics Week; Centers for Disease Control and Prevention. 2016, https://www.cdc.gov/media/dpk/antibiotic-resistance/antibiotics-week-2016/dpk-antibiotics-week-2016.html ). The increasing abuse of antibiotics in healthcare as well as agriculture has resulted in the rise of antibiotic resistance at an alarming rate. In a clinical setting, timely and accurate recognition of the pathogen allows for the most effective choice of treatment, highlighting the need for novel, fast, and reliable antibiotic susceptibility testing. Traditional susceptibility testing techniques require costly and complex experimental setups or extended cell incubation periods, delaying a timely treatment response to the infection. Herein, we report that a short-pulse fluorescent d -amino acid (FDAA)-based approach provides insight not only into bacterial antibiotic susceptibility but also into the mechanism of action of the antibiotic. Using the FDAA-labeling signal as a reflection of peptidoglycan (PG) integrity after antibiotic treatment, we observed that drugs targeting PG biosynthesis resulted in a significant decrease in fluorescence, while antimicrobials affecting other cellular targets resulted in no fluorescence changes. Our method was validated and optimized via fluorescence microscopy and spectrofluorometry, shortening the required procedure time to 15 min and providing reliably reproducible results. Significantly, we demonstrate that our protocol can be used to identify β-lactam-resistant bacterial strains, further demonstrating the utility of these valuable molecular tools.