The fitness landscape of TEM-1 β-lactamase is stratified and inverted by sublethal concentrations of cefotaxime

Adaptive evolutionary processes are constrained by the availability of mutations which cause a fitness benefit - a concept that may be illustrated by 'fitness landscapes' which map the relationship of genotype space with fitness. Experimentally derived landscapes have demonstrated a predictability to evolution by identifying limited 'mutational routes' that evolution by natural selection may take between low and high-fitness genotypes. However, such studies often utilise indirect measures to determine fitness. We estimated the competitive fitness of each mutant relative to all of its single-mutation neighbours to describe the fitness landscape of three mutations in a b-lactamase enzyme at sub-lethal concentrations of the antibiotic cefotaxime in a structured and unstructured environment. We found that in the unstructured environment the antibiotic selected for higher-resistance types - but with an equivalent fitness for subsets of mutants, despite substantial variation in resistance - resulting in a stratified fitness landscape. In contrast, in a structured environment with low antibiotic concentration, antibiotic-susceptible genotypes had a relative fitness advantage, which was associated with antibiotic-induced filamentation. These results cast doubt that highly resistant genotypes have a unique selective advantage in environments with sub-inhibitory concentrations of antibiotics, and demonstrate that direct fitness measures are required for meaningful predictions of the accessibility of evolutionary routes.