Cinnamomum zeylanicum essential oil inactivates Shigella flexneri by inhibiting biofilm formation, disrupting cell membrane integrity, and suppressing virulence gene expression.

Shigella flexneri, a foodborne pathogen that causes shigellosis, poses a significant risk to human health and food safety. Cinnamomum zeylanicum essential oil (CZEO) is known to inhibit microbial growth, but its possible antibacterial and antibiofilm mechanisms against S. flexneri remain unclear. The chemical composition of CZEO was identified via GC-MS. The minimum inhibitory concentration of CZEO against S. flexneri was 200 μg/ml. CZEO-treated S. flexneri exhibited an extended lag phase, indicating delayed bacterial growth, and demonstrated potent bactericidal activity in fresh coconut water. CZEO has an antibacterial effect by disrupting cell membranes, leading to leakage of nucleic acids and proteins. Congo red agar, safranin staining and XTT assays confirmed the ability of CZEO to prevent biofilm formation and decrease the metabolic activity of S. flexneri cells. CZEO reduced hydrophobicity, aggregation, and S. flexneri motility, contributing to the antibiofilm effect. An annexin V-FITC/PI assay revealed that CZEO induced cell death in S. flexneri via apoptosis, whereas gene expression analysis revealed significant downregulation of biofilm-associated genes such as yebL, csgD, rcsB, rcsC, rfbA, rfbE, mdoC, SF2159, and waaG. Light, fluorescence, and scanning electron microscopy revealed that CZEO inhibited biofilms in a dose-dependent manner and was able to induce irreversible morphological damage to S. flexneri cells. Homology modelling and molecular docking demonstrated interactions between virulence proteins and major constituents of CZEO. The MTT assay revealed that CZEO was nontoxic to human embryonic kidney (HEK293) cells even at relatively high concentrations. The above results confirmed that CZEO could be used as a natural food additive to combat Shigella flexneri and its biofilm.