Abstract
Antibiotic resistance mediated by β-lactamases, encoded by bla genes, is a significant global health threat, necessitating systematic studies of their diversity and evolution, particularly among pathogenic bacteria lineages. Leveraging over 6,000 quality-filtered Vibrio genomes alongside six newly sequenced marine symbiotic strains representing 128 nominal and 57 unclassified Vibrio species, our study extends taxonomic breadth and resolution for investigating β-lactamase diversity. We identified 4,431 β-lactamases across 41 species, encompassing all four Ambler classes (A-D). Among these, carbenicillin-hydrolyzing Class A β-lactamases encoded by blaCARB family were the most prevalent (60.7%) and exhibited a clade-centric distribution particularly in Harveyi clade and V. cholerae, underscoring the influence of specific ecological and evolutionary pressures. We refined carbenicillin-hydrolyzing Class A β-lactamase classification into two subfamilies: CARB-17-like (blaCARB-17-like) confined to Harveyi clade and CARB-1-like (blaCARB-1-like) found exclusively outside Harveyi clade based on phylogenetic placement, sequence similarity, and inheritance patterns, providing a clearer framework for delineating their functional and phylogenetic nuances. Notably, blaCARB-17-like genes in nonpathogenic Harveyi Subclade II showed significantly relaxed selection, accompanied by unusual mutations within key conserved motifs especially catalytic serine residues, suggesting evolutionary drift that may compromise canonical enzymatic activity. Furthermore, blaCARB-17-like genes, present as a single copy, emerged as a core gene in Harveyi clade, showing promise as a diagnostic marker for clinically significant Harveyi clade species, despite limited yet significant interspecies genetic exchanges mediated by recombination or mobile genetic elements. Our study advances the understanding of β-lactamase evolution and genomic distribution in Vibrio, with broad implications for diagnostic applications and resistance management strategies.