Due to increasing reports of multidrug-resistant (MDR) Vibrio cholerae O1, the goal of this study was to characterize the in vitro antimicrobial activity of chitosan microparticles (CMs) to evaluate their potential as a novel therapeutic agent for cholera. We examined the antimicrobial activity of CMs against toxigenic V. cholerae O1 using direct enumeration, microscopy, and fluorescence microplate assays. Bacterial viability kinetics were measured with different concentrations of CMs, solution pH, and salt content using a live/dead staining technique. Growth inhibition of CM-exposed V. cholerae strains was conducted using a redox-sensitive stain and compared between wild-type and isogenic outer membrane (OM) mutants. CM concentrations above 0.1 wt % were sufficient to kill V. cholerae O1 suspensions with approximately 108 CFU/mL within 3 h. The nonviable cells demonstrated increased OM permeability that corresponded to gross morphological changes observed through scanning electron microscopy. CMs exhibited dose-dependent bactericidal activity that increased predictably at lower pH and decreased with salt addition. V. cholerae O1 strains lacking O-antigen were twice as susceptible to growth inhibition by CMs, whereas those with glycine modification to lipid A were ten times more resistant. We propose that CMs exert vibriocidal activity via electrostatic surface interactions between their positively charged amine groups and the negatively charged Gram-negative bacterial OM, resulting in disruption, increased permeability, decreased redox metabolism, and subsequent loss of cellular viability. Further research should be conducted in vivo to evaluate the efficacy of CMs as luminal agents to treat infections caused by MDR, toxigenic V. cholerae and other diarrheal pathogens.
Keywords: Vibrio cholerae O1; antimicrobial agents; chitosan microparticles; cholera treatment; multidrug resistance (MDR).