The Biofilm Lifestyle Shapes the Evolution of β-Lactamases.

The evolutionary relationship between the biofilm lifestyle and antibiotic resistance enzymes remains a subject of limited understanding. Here, we investigate how β-lactamases affect biofilm formation in Vibrio cholerae and how selection for a biofilm lifestyle impacts the evolution of these enzymes. Genetically diverse β-lactamases expressed in V. cholerae displayed a strong inhibitory effect on biofilm production. To understand how natural evolution affects this antagonistic pleiotropy, we randomly mutagenized a β-lactamase and selected for elevated biofilm formation. Our results revealed that biofilm evolution selects for β-lactamase variants able to hydrolyze β-lactams without inhibiting biofilms. Mutational analysis of evolved variants demonstrated that restoration of biofilm development was achieved either independently of enzymatic function or by actively leveraging enzymatic activity. Taken together, the biofilm lifestyle can impose a profound selective pressure on antimicrobial resistance enzymes. Shedding light on such evolutionary interplays is of importance to understand the factors driving antimicrobial resistance.