Sulfated Chitosan Induces Membrane Disruption, Aggregation, and Antibiofilm Activity in Piscirickettsia salmonis: A Biomimetic Strategy as an Antimicrobial Alternative in Aquaculture

Background: Sulfated chitosan (ChS) is a chemically modified polysaccharide derived from chitin that mimics heparan sulfate (HS) structures and has emerged as a promising antimicrobial biomaterial. Piscirickettsia salmonis, the etiological agent of Salmonid Rickettsial Septicemia (SRS), represents the main driver of antibiotic use in Chilean aquaculture. Objective: In this study, the in vitro antibacterial activity of ChS against P. salmonis was evaluated. Methods: Elemental characterization by SEM-EDS and FTIR analysis confirmed successful sulfation of the polymer, with a degree of sulfation ranging from 0.92 to 0.95. Additionally, X-ray diffraction (XRD) analysis revealed a reduction in polymer crystallinity, indicating a transition toward a more amorphous structure associated with increased molecular flexibility and functional group accessibility. Results: Antibacterial assays revealed a minimum inhibitory concentration (MIC) of 1500 µg/mL and a minimum bactericidal concentration (MBC ≥ 1500 µg/mL). LIVE/DEAD™ fluorescence imaging showed the formation of bacterial aggregates with increasing size, frequency, and red fluorescence compared to controls over the exposure to ChS, indicating progressive membrane damage. This was supported by a reduction (p < 0.05) in the Green/Red fluorescence ratio of 37–46% between 5 h and 96 h of exposure, corresponding to alteration of the cell membrane. Scanning electron microscopy revealed pronounced morphological alterations by ChS, including surface disruption and loss of cellular integrity. This was more severe compared to commercial chitosan (ChC). Also, ChS reduced (p < 0.05) biofilm formation (>50% at day 6 and 34.8% at day 8). Conclusions: These results demonstrated that ChS disrupts the cell membrane and reduces biofilm formation in P. salmonis, thereby affecting viability. This is the first report of the antibacterial effect of ChS, an HS analogue, against P. salmonis.