Epistasis drives rapid divergence across multiple traits during the adaptive evolution of a carbapenemase

Interactions among beneficial mutations (i.e., epistasis) are often strong enough as to direct adaptation through alternative mutational paths. While alternative solutions should display similar fitness under the primary selective conditions, their properties across secondary environments may differ widely. The extent to which these cryptic differences are to be expected is largely unknown, despite their fundamental and practical importance, such as in the search for exploitable collateral sensitivities among antibiotic resistance mutations. Here we use directed evolution to characterize the diversity of mutational paths through which the prevalent carbapenemase KPC-2 can evolve high activity against the clinically-relevant antibiotic ceftazidime, an initially poor substrate. We identified 40 different substitutions, including many common clinical settings, spread along 18 different mutational trajectories. Initial mutations determined four major groups into which the trajectories can be classified, a signature of strong epistasis. Of note, despite minor variation in final ceftazidime resistance, groups diverged markedly across multiple phenotypic dimensions, from molecular traits such as stability and hydrolitic efficiency to macroscopic traits such as growth rate and activity against other {beta}-lactam antibiotics. Our results indicate that cryptic yet consequential phenotypic differences can readily accumulate under strong selective pressures, bearing implications for efforts to prevent unwanted evolution in microbes.