Choice of β-lactam resistance pathway depends critically on initial antibiotic concentration.

Antibiotic-resistance trajectories with different final resistance may critically depend on the first mutation due to epistatic interactions. Here, we study the effect of mutation bias and concentration-dependent fitness effects of two clinically important mutations in TEM-1 β-lactamase initiating alternative trajectories to cefotaxime resistance. We show that mutation R164S, conferring relatively low resistance, is competitively superior over larger-effect mutation G238S at low cefotaxime concentrations, highlighting a critical influence of antibiotic concentration on long-term resistance evolution.