The effect of β-lactamase evolution on biofilm formation in Vibrio cholerae

Abstract

Antimicrobial resistance (AMR) has emerged as one of the greatest threats to public health in the 21st century. Especially, the production of -lactamase enzymes has become the leading cause of -lactam resistance in Gram-negative pathogens. In addition, many of the pathogens associated with extensive antimicrobial resistance form biofilms, which further increases their persistence. Studies from different Gram-negative bacteria have demonstrated that -lactamases can inhibit biofilm formation. However, no study has investigated how the biofilm lifestyle affect -lactamase evolution. Thus, acquiring insights into the evolutionary relationship between biofilms and -lactamases could generate knowledge that has implications for combating AMR. Using a biofilm quantification assay, directed evolution based on mutational libraries, and biofilm evolution, this master project aimed to understand how the production and evolution of -lactamases affect biofilm formation in V. cholerae. Quantification of biofilm formation demonstrated that 7/8 tested -lactamases inhibited biofilm formation in V. cholerae. Applying strong biofilm selection on a mutational library of the -lactamase KPC-2 identified multiple KPC-2-harboring mutants with increased biofilm formation. Sequencing of the evolved mutants revealed the presence of single and double mutants sharing an amino acid change at the same position (N136x). Functional studies of the newly evolved variants, using a binding-deficient mutant (S70A), revealed that disruption of binding (S70A) led to reversal of the observed increase in biofilm formation. Thus, suggesting that the evolutionary changes in the KPC-2 mutants could be related to the evolution of a new enzymatic function rather than a loss-of-function. Taken together, this study shows that the evolution of -lactamases and biofilms affect each other. Comprehending evolutionary connections, as described in this study between biofilm and the evolution of blaKPC-2 in V. cholerae, may help to understand the spread and evolution of antimicrobial resistance genes.