Antimicrobial resistance is an emerging threat that demands continuous development of new classes of antibiotic agents. In the interest of efficiently optimizing viable chemical targets, it is imperative to have experimental techniques capable of characterizing not only the efficacy of an antibiotic, but also the specific mechanism-of-action (MoA). We have recently demonstrated the surface-sensitive nonlinear optical technique, second-harmonic generation light scattering (SHS), for quantifying transmembrane molecular transport in living cells(1). We now show that SHS can be applied as a sensitive probe of membrane permeability in living bacteria. Specifically, by monitoring the uptake response of an SHS-active probe molecule following administration of an antibacterial agent, perturbations in the measured transport response reveal time- and concentration-dependent changes in the permeability of the bacterial membranes, thus permitting real-time characterization of the MoA. As an initial proof-of-principle, we apply SHS for deducing the sequential MoA of the antimicrobial, Bricilidin (Bn). Bn is a synthetic arylamide foldamer exhibiting amphiphilic topology similar to that of cell-penetrating peptides, and was designed to disrupt bacterial membranes(2). Using malachite green (MG) as an SHS-active probe, we characterize the MoA of Bn in Escherichia coli. In general, for low concentrations ( 15min), Bn begins to disrupt the CM, resulting in an apparent decreased permeability, likely indicating depolarization of the membrane.REFERENCES:1. Wilhelm, M.J., M. Sharifian Gh., and H.-L. Dai. 2015. Chemically Induced Changes to Membrane Permeability in Living Cells Probed by Nonlinear Light Scattering. Biochemistry. 54: 4427–4430.2. Mensa, B., G.L. Howell, R. Scott, and W.F. DeGrado. 2014. Comparative mechanistic studies of brilacidin, daptomycin, and the antimicrobial peptide LL16. Antimicrob. Agents Chemother. 58: 5136–5145.