Linezolid and levofloxacin: an uncommon pairing that suppresses evolution of resistance in <i>E. faecalis</i>

Drug combinations offer one potential strategy for slowing the evolution of antibiotic resistance. In this work, we investigate the evolution of drug resistance (strictly, the decrease in susceptibility) in populations of <i>Enterococcus faecalis</i>, an opportunistic human pathogen, exposed to different concentrations of the rarely used combination of linezolid (LZD) and levofloxacin (LEV). Using continuous culture bioreactors, we measured the two-dimensional dose-response surface of ancestral populations of <i>E. faecalis</i>, revealing that the LZD-LEV combination is strongly antagonistic, resulting in increased growth as LZD concentration is increased at a (sufficiently high) fixed concentration of LEV. Next, we performed multiday (50+ hour) evolution experiments by continually exposing populations to four different fixed-concentration combinations and then characterized isolates from adapted populations using whole genome sequencing and phenotypic dose-response curves. To control for differences in inhibition levels for different concentration combinations, we chose LZD-LEV combinations that fall along a single contour of constant growth in the two-drug space of LZD-LEV concentrations. Despite similar levels of initial inhibition across the four concentration combinations, we found that adaption is markedly different, with the most rapid adaptation and highest levels of evolved resistance occurring at combinations that include a high concentration of one drug and low concentration of the other. By contrast, we found almost no adaptation when both drugs were used at high concentrations, an initially surprising result that can be explained by simple drug-rescaling arguments applied to the antagonistic dose-response surface. Our results underscore the potential utility of a nonstandard (and strongly antagonistic) drug combination in suppressing resistance.