Exploring the anti-virulent potential of pyridine derivatives against Vibrio cholerae

Vibrio cholerae, the causative agent of cholera, poses a continuous threat to global public health, especially in regions with poor sanitation. Its ability to form biofilms and rapidly acquire antimicrobial resistance (AMR) complicates therapeutic interventions. In this study, the quorum-sensing (QS) response regulator LuxO was targeted for anti-virulence therapy using a synthesized pyridine-based compound, QSI^py. Pyridine derivatives are explored as inhibitors that can target the LuxO protein, a key regulator in the QS pathway of V. cholerae. By inhibiting LuxO, these compounds reduced the virulence factor expression and biofilm formation, offering a novel antivirulence strategy without promoting resistance. Molecular docking showed that QSI^py exhibited a strong binding affinity, with a glide score of -5.046 at the ATP-binding domain of LuxO. In vitro evaluation revealed that QSI^py had no inhibitory effect on planktonic bacterial growth, indicating a non-bactericidal mechanism. However, it showed significant inhibition of biofilm formation, as confirmed by crystal violet assay and quantified through MBIC determination. Pellicle CFU enumeration demonstrated a reduction in viable biofilm-associated cells, particularly at the lowest concentrations of 15.6 µg mL^-1 and 31.2 µg mL^-1, while fluorescence microscopy validated the loss of pellicle integrity. Additionally, checkerboard synergy testing with azithromycin (AZM) revealed a high synergy score based on Bliss independence modelling, whereas no synergy was observed with ciprofloxacin or doxycycline. These results suggest that QSI^py potentiates macrolide antibiotic activity by interfering with quorum-sensing-regulated biofilm and virulence expression.