Antibacterial Effects of Sulfated Chitosan Against <em>Piscirickettsia salmonis</em>: A Biomimetic Strategy as an Antimicrobial Alternative in Aquaculture for Cell Membrane Disruption and Antibiofilm Activity

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 (P. salmonis), the etiological agent of Salmonid Rickettsial Septicemia (SRS), represents the main driver of antibiotic use in Chilean aquaculture. In this study, the in vitro antibacterial activity of ChS against P. salmonis was evaluated. Elemental characterization by SEM-EDS confirmed successful sulfation of the polymer, with a degree of sulfation ranging from 0.92 to 0.95. 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 &lt; 0.05) in the Green/Red fluorescence ratio of 37–46% between 5h and 96h of exposure, corresponding to alteration of 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 native chitosan. Also, ChS reduced (p &lt; 0.05) biofilm formation (&gt;50% at day 6 and 34.8% at day 8). These results demonstrated that ChS disrupts cell membrane and reduces biofilm formation in P. salmonis, which in consequence affects viability. This is currently the first report of the antibacterial effect of ChS as a HS analogue on P. salmonis.