Abstract

Background and Aim: Biofilm-forming Aeromonas hydrophila represents a critical constraint in aquaculture, driving recurrent infections, environmental persistence, and antimicrobial resistance. Sustainable alternatives to antibiotics are urgently needed. This study evaluated the multiphase antibiofilm activity of chitosan nanoparticles (ChNPs) synthesized from Litopenaeus vannamei shrimp shells against clinical A. hydrophila isolates from Indonesian gourami (Osphronemus gouramy), focusing on their effects during biofilm adhesion, planktonic proliferation, and mature biofilm degradation. 

Materials and Methods: Between February 2024 and March 2025, diseased gourami were sampled from aquaculture sites in Surabaya, Indonesia. Three wild-type A. hydrophila isolates (A1G1, A2G1, A3G1) were confirmed via biochemical and 16S rRNA sequencing. ChNPs were synthesized through ionic gelation of deacetylated chitosan with sodium tripolyphosphate and characterized by Scanning Electron Microscopy (SEM), dynamic light scattering, and Fourier Transform Infrared Spectroscopy (FTIR) analyses. Antibiofilm efficacy was tested at concentrations of 15–45 μg mL⁻¹ using crystal violet staining (optical density [OD]₅₉₅) for adhesion and degradation phases, and turbidity (OD₆₀₀) for planktonic inhibition. Data were analyzed using one- and two-way analysis of variance with Tukey’s post hoc test. 

Results: ChNPs exhibited spherical morphology (≈641 nm; ζ = +51 mV) and stable ionic crosslinking. They significantly inhibited adherent biomass formation (p < 0.05), reducing OD₅₉₅ from 0.787 to 0.317 in the most responsive strain A3G1 (> 59 % inhibition). Planktonic growth (OD₆₀₀) declined dose-dependently (63 % inhibition at 45 μg mL⁻¹), with significant strain–concentration interactions (p < 0.01). Mature biofilm degradation reached 63% at 45 μg mL⁻¹, approaching the level of the antibiotic-treated control. SEM and FTIR data supported electrostatic disruption and extracellular polymeric substance penetration as probable mechanisms. 

Conclusion: Shrimp-shell–derived ChNPs effectively suppressed A. hydrophila biofilms at multiple developmental stages, demonstrating a potent, biodegradable alternative for the control of aquaculture pathogens. Their integration into eco-friendly, antibiotic-free disease management aligns with circular bioeconomy and One Health frameworks. Further in vivo validation and formulation optimization are warranted. 

Keywords: Chitosan nanoparticles, Aeromonas hydrophila, biofilm inhibition, planktonic suppression, aquaculture biocontrol, One Health, circular bioeconomy.