In vitro comparative evaluation of disinfectant-loaded nanoparticles against biofilm-forming Vibrio spp. isolated from gilthead seabream (Sparus aurata).

This study evaluated the in vitro efficacy of antimicrobial and antibiofilm of three disinfectant-loaded nanoparticles (AgNPs-H₂O₂, CuNPs-Virkon S, and CuNPs-TH4) against Vibrio alginolyticus and Vibrio fluvialis, isolated from naturally infected gilthead seabream in Egypt. Qualitative Congo red assay showed that V. fluvialis isolates were strong biofilm producers, whereas V. alginolyticus showed limited phenotypic variation. Quantitative assay results, analyzed with a Mann-Whitney U test, showed no statistically significant difference in overall biofilm biomass between V. alginolyticus and V. fluvialis isolates (U = 157.0, p = 0.175). Seasonal variation affected biofilm formation, peaking in summer. CuNPs-TH4 exhibited the highest antibiofilm effect, significantly reducing the biomass (p < 0.01). The Minimum Inhibitory Concentration (MIC) was significantly different between disinfectants (p < 0.001), with CuNPs-TH4 having the highest MIC values, followed by AgNPs-H₂O₂ and CuNPs-Virkon S. Minimum bactericidal concentration (MBC) values showed that AgNPs-H₂O₂ had the most consistent bactericidal activity in 85.4% of isolates tested (defined by MBC/MIC ≤ 4). A significant association was observed between MIC values and Vibrio species (p = 0.002). Molecular detection of antimicrobial resistance (AMR) genes revealed a high prevalence of sul, cat, and mphA, with V. alginolyticus exhibiting a broader resistance profile than V. fluvialis. These findings highlight the in vitro potential of nanotechnology-enhanced disinfectants, within the limitations of nanoparticle-only testing, as exploratory alternatives for controlling biofilm-associated Vibrio infections in aquaculture.