Inhibition of RND-mediated efflux attenuates antibiotic resistance and virulence in hypervirulent <i>Klebsiella pneumoniae</i>

<i>Klebsiella pneumoniae</i> (Kp) is a major human pathogen causing hospital-acquired and community-acquired infections with emerging hypervirulent strains (hvKp) posing a significant threat due to its ability to cause severe invasive infections in healthy individuals. In addition to antimicrobial resistance, virulence factors including capsule production, biofilm formation, and iron acquisition systems are critical for hvKp pathogenesis. In this study, we investigated how resistance-nodulation-division (RND)-family efflux systems contribute to antimicrobial resistance and virulence in hvKp strain KPPR1 using the RND-specific inhibitor phenyl-arginine β-naphthylamide (PAβN). We found that PAβN treatment rendered KPPR1 more susceptible to multiple antibiotics while simultaneously attenuating virulence factor production. PAβN significantly reduced capsule biosynthetic gene expression, resulting in decreased uronic acid levels, hypermucoviscosity, and biofilm formation. PAβN also impaired growth under iron-limited conditions, suggesting RND-mediated efflux contributes to iron acquisition. PAβN-dependent virulence attenuation was demonstrated through reduced KPPR1 adherence to cultured intestinal enterocytes and decreased pathogenicity in the <i>Galleria mellonella</i> infection model compared to untreated controls. Collectively, these results demonstrate that RND-mediated efflux is critical for both antimicrobial resistance and virulence in hvKp strain KPPR1. Our findings establish RND efflux inhibitors as promising dual-target therapeutics that can simultaneously combat antibiotic resistance and attenuate virulence in hvKp infections.