Toward understanding the antibacterial mechanism of chitosan: Experimental approach and in silico analysis

This study investigated the antibacterial mechanisms of chitosan (Cs), alkylated form (AlkCs), and chitosan nanoparticles (CsNPs) against Escherichia coli MG1655, a gram-negative model microorganism. CsNPs displayed a hemispherical shape with an average diameter of 169 nm using scanning electron microscopy (SEM) and a hydrodynamic diameter of 240 nm with a polydispersity index (PDI) of 0.154 based on dynamic light scattering (DSL) analysis. Energy Dispersive X-Ray (EDX), Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and UV–Vis spectroscopy (UV) techniques were employed to verify the presence of unique elements and characteristics in Cs, AlkCs, and CsNPs. AlkCs demonstrated the highest growth inhibition zone against E. coli MG1655 compared to Cs and CsNPs. Furthermore, AlkCs and CsNPs exhibited lower minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values compared to Cs. Flow cytometry showed Cs caused significant membrane permeability at 200 μg/mL, while AlkCs and CsNPs induced greater permeability at 20, 50, and 750 μg/mL. Exposure to Cs, AlkCs, and CsNPs caused significant changes in cell size, number, and morphology, as observed through light and atomic force microscopy (AFM) images, compared to control cells. Microarray analysis, gene ontology (GO), and VENN diagram analysis were employed to analyze the differential expression of target genes (DEGs) and predict the mechanism of action of chitosan-based compounds. The findings indicated that CsNPs and AlkCs have the potential to act as antibacterial agents by inhibiting outer membrane biosynthesis, metabolic activities, and membrane-related signaling pathways. This study provides insights into the antibacterial mechanism of chitosan and its derivatives.