Anti-bacterial effects of gallic acid-loaded graphene oxide nanocomposite (GAGO) against Vibrio spp., its potential mechanism of action and toxicity against Artemia nauplii

Vibriosis remains as the major constraint of aquaculture, intensified by antibiotic resistance and the limitations of conventional therapeutics. The present study investigates the antibacterial activity of gallic acid-loaded graphene oxide (GAGO) nanocomposite, elucidates its mechanism of action on several Vibrio spp., and investigates its safety profile in Artemia nauplii. The antibacterial properties were investigated through disk diffusion, colony formation unit (CFU) counting method and time-kill studies. The mechanisms of action for the antibacterial effect were elucidated through membrane integrity study, DNA and protein leakage, transmission electron microscopy (TEM) observations, zeta potential measurement, Fourier transform infrared (FTIR) analysis and reactive oxygen species (ROS) production. Transcriptomic analysis was also conducted on Vibrio parahaemolyticus upon exposure to GAGO. Toxicity test was carried out in Artemia nauplii. Over 70% of bacterial reduction were observed within 4 h of incubation with GAGO, increasing to 99% after 8 h, comparable to oxytetracycline (OTC). DNA and protein leakage, as well as membrane integrity loss were observed following treatment with GAGO. TEM revealed pronounced morphological alterations and cytoplasmic depletion in Vibrio spp. exposed to GAGO, more extensive than those treated with OTC. The increase in zeta potential value and lack of absorption spectra of FTIR indicate further damage to the bacterium’s lipopolysaccharide (LPS) layer. Transcriptomic profiling identified 2,266 differentially expressed genes, with significant enrichment of the ribosome, aminoacyl-tRNA biosynthesis, and microbial metabolism pathways, indicating broad translational and metabolic stress triggered in Vibrio parahaemolyticus by GAGO exposure. Importantly, GAGO showed improved toxicity in Artemia nauplii (LC₅₀ = 571.8 μg/mL at 24 h), in contrast to GO and GA, and was comparable to OTC. In conclusion, the findings have established GAGO as a potential anti-bacterial nanocomposite with multi-targeted mechanism of action and favourable safety profile for further development in the aquaculture industry.

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