Utilizing Second Harmonic Scattering to Investigate Antibiotic Resistance Mechanisms in Gram-Positive Bacterial Membranes

The living Gram-positive lipid bilayer is a complex system that mediates the entry and transport of essential ions, while also enacting mechanisms to resist harmful exogenous substances. Through repeated exposure to these dangerous molecules, bacteria can develop and facilitate additional efforts to promote their survival. One such method is efflux pumps, where bacteria can actively remove small molecules from their membrane to mitigate damage. Second harmonic scattering (SHS) is an interface-specific technique that can provide information about the local populations of small molecules between individual leaflets in the lipid bilayer. As such, exploiting SHS as a means to develop our understanding of antibacterial resistance mechanisms enacted by Gram-positive bacteria provides unique advantages. First, polarization-resolved SHS was utilized to monitor FM 2-10 dye orientational alterations within living bacterial membranes. Monitoring the efflux of the probe FM 1-43 was conducted with time-resolved SHS on Gram-positive bacteria after exposure to the antibiotic tetracycline. To further expand this investigation of carrier-mediated transport, a proton motive force disruptor, CCCP, was applied to cells to observe the impact of antibiotic resistance on its ability to facilitate probe inner leaflet transport. Lastly, probes within the FM dye series were applied to resistant and non-resistant bacteria to ascertain the structural specificity of the efflux protein responsible for their transport.