Fluorescent d‑amino Acid-Based Approach\nEnabling Fast and Reliable Measure of Antibiotic Susceptibility in\nBacterial Cells

The\nthreat of multidrug-resistant bacteria has been increasing\nsteadily in the past century, posing a major health risk (Organización\nMundial de la Salud. Directrices Sobre Componentes Básicos\nPara Los Programas de Prevención y Control de Infecciones a\nNivel Nacional y de Establecimientos de Atención de Salud Para\nPacientes Agudos; Organización Mundial de la Salud: Ginebra, <b>2017</b>). Even though every year, 226 million antibiotics are\nprescribed in the United States alone, 50% of these prescriptions\nare inappropriate for the patient’s condition (CDC. Get Smart\nabout Antibiotics Week; Centers for Disease Control and Prevention. <b>2016</b>,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\nhas resulted in the rise of antibiotic resistance at an alarming rate.\nIn a clinical setting, timely and accurate recognition of the pathogen\nallows for the most effective choice of treatment, highlighting the\nneed for novel, fast, and reliable antibiotic susceptibility testing.\nTraditional susceptibility testing techniques require costly and complex\nexperimental setups or extended cell incubation periods, delaying\na timely treatment response to the infection. Herein, we report that\na short-pulse fluorescent d-amino acid (FDAA)-based approach\nprovides insight not only into bacterial antibiotic susceptibility\nbut also into the mechanism of action of the antibiotic. Using the\nFDAA-labeling signal as a reflection of peptidoglycan (PG) integrity\nafter antibiotic treatment, we observed that drugs targeting PG biosynthesis\nresulted in a significant decrease in fluorescence, while antimicrobials\naffecting other cellular targets resulted in no fluorescence changes.\nOur method was validated and optimized via fluorescence microscopy\nand spectrofluorometry, shortening the required procedure time to\n15 min and providing reliably reproducible results. Significantly,\nwe demonstrate that our protocol can be used to identify β-lactam-resistant\nbacterial strains, further demonstrating the utility of these valuable\nmolecular tools.