# Multiphase antibiofilm potential of shrimp-shell–derived chitosan nanoparticles against Aeromonas hydrophila isolated from tropical aquaculture environments

## TL;DR

Chitosan nanoparticles from shrimp shells can effectively combat biofilms of Aeromonas hydrophila, a harmful bacteria in aquaculture, offering a sustainable alternative to antibiotics.

## Contribution

The study introduces shrimp-shell-derived chitosan nanoparticles as a novel, biodegradable antibiofilm agent effective against A. hydrophila in aquaculture.

## Key findings

- Chitosan nanoparticles significantly reduced biofilm adhesion and planktonic growth of A. hydrophila isolates.
- Mature biofilm degradation reached 63% at 45 µg mL-¹, comparable to antibiotic treatments.
- Mechanisms include electrostatic disruption and penetration of extracellular polymeric substances.

## Abstract

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.

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.

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.

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.