6‐Aminoflavone Inhibits Planktonic Growth, Disrupts Biofilm Architecture, and Regulates Quorum Sensing in Klebsiella pneumoniae

Abstract

Antibiotic resistance has emerged as a major global health challenge, particularly in biofilm‐forming pathogens that exhibit enhanced tolerance to antimicrobial therapies. K. pneumoniae, a multidrug‐resistant pathogen, is a leading cause of hospital‐acquired infections, including pneumonia, septicemia, and device‐associated infections. Its robust biofilm‐forming capacity facilitates immune evasion, restricts antibiotic penetration, and contributes to recurrent infections. Therefore, the identification of agents capable of targeting both planktonic bacterial growth and biofilm architecture is of considerable therapeutic importance. In the present study, the antibacterial, antibiofilm, and antivirulence potential of 6‐Aminoflavone was investigated against K. pneumoniae. Antibacterial activity was evaluated using growth inhibition assays, time–kill kinetics, and clonogenic survival analysis. Mechanistic investigations revealed significant intracellular ROS accumulation, increased oxidative stress susceptibility, and induction of apoptosis‐like cell death, suggesting ROS‐mediated antibacterial activity. In addition to suppressing bacterial proliferation, 6‐Aminoflavone exhibited promising antibiofilm efficacy, inhibiting biofilm formation by 86.23% and eradicating 84.77% of established biofilms. These effects were associated with a substantial reduction in cell surface hydrophobicity (∼45.39%), and EPS levels were reduced to ∼2.33% as compared to untreated controls, indicating severe destabilization of the biofilm matrix. Furthermore, eDNA, a key structural component of the biofilm scaffold, exhibited a maximum ∼2.13‐fold reduction following treatment. Confocal microscopy confirmed marked disruption and collapse of biofilm architecture. Additionally, quorum sensing (QS)–regulated virulence factors, including urease (∼51.92%), protease (∼55.18%), and lipase (∼44.90%), were significantly attenuated. These findings demonstrate that 6‐Aminoflavone exerts antimicrobial activity by inducing oxidative stress–mediated apoptosis‐like bacterial death, disrupting biofilm structural components, and suppressing QS‐regulated virulence in K. pneumoniae.