Publication: From log-phase to biofilms: assessing the potential benefits of two non-antibiotic drugs in co-treatment therapies with classical antibiotics
Files
Date
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
The antibiotic resistance crisis demands novel therapeutic methods that can provide mechanisms of action to which bacteria have not yet become resistant. Here, we begin to characterize two drugs, fenobam and cisplatin, in their ability to effectively target log-phase and/or biofilm MRSA both independently and within a co-treatment using various classical antibiotics. These drugs were identified in a previous small molecule drug screen against non-growing MRSA in which compounds were applied to a 1:10^6 dilution of cells in PBS to assess for non-growing antibacterial activity. To assess the extent to which each drug is inhibited by the inoculum effect against non-growing MRSA, we applied ranging drug concentrations to various dilutions of cells in PBS. We found that while cisplatin exhibits activity against cell densities up to 3.189E+08 CFU/mL, fenobam experiences a larger inoculum effect, only being effective for up to 3.116E+07 CFU/mL. Furthermore, against MRSA in PBS fenobam was found to stimulate cell proliferation at concentrations 32 and 64 µg/mL. To begin the characterization of drug interactions between cisplatin and three different classical antibiotics (vancomycin, clindamycin, and trimethoprim), we applied ranging concentrations of cisplatin against ranging concentrations of classical antibiotics to log-phase MRSA in TSB. We found that instead of increasing the efficacy of any antibiotic tested, cisplatin stimulated cell proliferation in a dose-dependent way for antibiotic concentrations below MIC at earlier timepoints. Later in the incubation, cisplatin exhibited a dose-dependent OD600 reduction in cell density plateau. To examine the activity of fenobam both independently and within a co-treatment with vancomycin, mature MRSA biofilms were treated for 2 hours with subsequent cell viability assessment with alamarBlue. Fenobam exhibited activity against MRSA biofilms not only within a co-treatment with vancomycin, but also independently. Unfortunately, vancomycin and fenobam were found to hinder the ability of each other to target the biofilm, where the improved reduction of biofilm viability post-treatment was less than expected based on the individual effects artificially combined. To limit the variations in the result due to variations in biofilm maturation or inconsistent cell loss at wash steps, we began optimizing a live vs. dead staining protocol for fluorescence microscopy resulting in preliminary imaging. Lastly, we proposed a cyclical resistance protocol for the development of resistance mutations for non-growing specific antibacterial agents. These data help to inform the field on new possible directions to take antibiotic therapy development.