# RND-mediated efflux couples antimicrobial resistance and hypervirulence in contemporary Vibrio cholerae

## TL;DR

Recent Vibrio cholerae strains have evolved to be both highly drug-resistant and more virulent, thanks to a protein called VexB that links these traits.

## Contribution

VexB efflux pump is identified as a novel molecular mechanism that couples antimicrobial resistance and hypervirulence in modern V. cholerae.

## Key findings

- Contemporary V. cholerae isolates show enhanced colonization and multidrug resistance compared to early pandemic strains.
- VexB deletion reduces both antibiotic resistance and virulence by impairing adherence, motility, and membrane integrity.
- Laboratory evolution under antibiotic pressure alone does not produce the dual resistance-virulence phenotype seen in clinical isolates.

## Abstract

The prevailing view in bacterial pathogenesis is that antimicrobial resistance and virulence are constrained by evolutionary trade-offs, with resistance mechanisms imposing fitness costs that attenuate pathogenic potential. Herein we document that contemporary Vibrio cholerae clinical isolates from the ongoing seventh pandemic have circumvented this paradigm by coupling multidrug resistance with hypervirulence. We examined five geographically diverse Wave 3 isolates collected between 2017 and 2019 and compared them to early pandemic strains. These contemporary isolates exhibited both broad-spectrum antimicrobial resistance and markedly enhanced colonization capacity in the infant mouse model. Phylogenetic analysis of 67 O1 El Tor genomes spanning 1960–2019 confirmed that the isolates cluster within a representative Wave 3 sublineage. We identified the VexB RND efflux pump as a mediator of this coupled phenotype. Elevated vexB expression in the contemporary isolates conferred resistance to multiple antibiotic classes, while vexB inactivation simultaneously impaired resistance and colonization. This dual function was not observed in early pandemic strains, consistent with a recent evolutionary adaptation. VexB-mediated hypervirulence occurred through multiple pathways independent of cholera toxin and toxin-coregulated pilus production levels. VexB deletion impaired bacterial adherence to intestinal epithelial cells, impaired motility, and increased susceptibility to membrane-active antimicrobials. In contrast, laboratory evolution under antibiotic pressure alone generated resistant but avirulent strains, demonstrating that complex selective forces in nature enabled the co-optimization of resistance and virulence. These findings establish VexB as a molecular link between antimicrobial resistance and hypervirulence in pandemic V. cholerae, highlighting efflux pumps as dual-function therapeutic targets whose inhibition could both restore antibiotic activity and attenuate disease.

Antimicrobial resistance is often assumed to come at the cost of virulence, with resistant bacteria paying a fitness price that limits their ability to cause disease. Our work shows that this assumption does not hold for contemporary Vibrio cholerae, the pathogen responsible for cholera. We studied recent clinical isolates from the ongoing pandemic and found that they are both highly multidrug-resistant and unusually virulent, causing enhanced intestinal colonization compared to earlier pandemic strains. We identified the VexB efflux pump as the key factor linking these traits. In contemporary strains, mutating vexB simultaneously reduced antibiotic resistance and impaired colonization by decreasing bacterial adherence to intestinal cells, reducing motility, and increasing susceptibility to gut antimicrobials. This indicates that VexB has acquired new functions that allow modern isolates to optimize both traits at once. Laboratory evolution under antibiotic pressure produced resistant but weakened strains, showing that additional natural pressures are needed to drive this dual adaptation. These findings highlight efflux pumps as promising therapeutic targets that could both restore antibiotic activity and lessen disease severity.