The Biofilm Lifestyle Shapes the Evolution of β-Lactamases

Abstract

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.

Keywords: Vibrio cholerae; AMR; biofilm; evolution; β-lactamases.

Graphical abstract

Fig. 1.

Biofilm lifestyle shapes the evolution of β-lactamases in V. cholerae. a. We first explored the influence of β-lactamase gene expression on the V. cholerae biofilm phenotype (left). Second, by subjecting a mutant library of KPC-2 to experimental evolution, we revealed how the biofilm lifestyle affects β-lactamase evolution (right). b. The expression of β-lactamase genes from Ambler classes A to D (top) significantly hindered biofilm formation in V. cholerae compared to the control vector. c. Our mutational library of KPC-2 (>5,000 mutants) exhibited significantly enhanced biofilm formation compared to wild-type KPC-2 (wtKPC-2) (****; one-way ANOVA, P < 0.0001), although it remained less than the vector control (**; one-way ANOVA, P = 0.001). d. Differences in biomass production related to V. cholerae's ability to form pellicles. While our control displayed signs of pellicle formation after 24 h incubation, the presence of KPC-2 completely suppressed biofilm pellicle development. In contrast, the presence of our KPC-2 mutational library resulted in a well-structured biofilm pellicle. e. While wtKPC-2 led to reduced biofilm capacity, N136K and Δ1-48/N136D/M152I/L167P, which were selected from and were enriched in the pellicle, demonstrated significant improvement in biofilm formation (P values reported in Table 2). β-Lactam binding-deficient (serine-to-alanine at position 70) variants of wtKPC-2 and Δ1-48/N136D/M152I/L167P strongly reduced the biofilm phenotype. On the contrary, S70A/N136K maintained high levels of biofilm formation compared to the evolved variant N136K. Deconvolution of mutations within Δ1-48/N136D/M152I/L167P displayed that, in contrast to N136D and L167P, the deletion Δ1-48 did not significantly increase biofilm formation compared to wtKPC-2 (P values reported in Table 2). f. Location of mutational sites compared to the key active site residues S70 and E166. g. Pearson correlation (R² = 0.63, P = 0.059) between ampicillin resistance and biofilm formation for wtKPC-2 (black), control (gray), and evolved mutants Δ1-48/N136D/M152I/L167P, N136K, N136D, and L167P (blue). Each datapoint in b, c, and e represents a biological replicate, and error bars display 95% confidence intervals.