Cryptic phenotypic variation emerges rapidly during the adaptive evolution of a carbapenemase.

Interactions among beneficial mutations (that is, epistasis) are often strong enough to direct adaptation through alternative mutational paths. Although 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 importance-for example, in identifying exploitable collateral sensitivities among mutations conferring antibiotic resistance. Here we use directed evolution to characterize the diversity of mutational paths through which the prevalent carbapenemase Klebsiella pneumoniae carbapenemase-2 can evolve high activity against the clinically relevant antibiotic ceftazidime, an initially poor substrate. We identified 40 different substitutions-including many that are common in clinical settings-spread among 18 different mutational trajectories. Initial mutations determined four major groups into which the trajectories can be classified, a signature of strong epistasis. Despite similar final ceftazidime resistance, groups diverged markedly across multiple phenotypic dimensions, from molecular traits, such as in-cell stability and catalytic efficiency, to macroscopic traits, such as growth rate and activity against other β-lactam antibiotics. Our results indicate that cryptic yet consequential phenotypic differences can accumulate rapidly under strong selection, unpredictably shaping the long-term success of resistance enzymes in their journey across hosts and environments.