Comparative in vitro screening identifies beta-hairpin antimicrobial peptide leads against Piscirickettsia salmonis, supported by membrane-level molecular dynamics

Background Salmonid rickettsial septicemia caused by Piscirickettsia salmonis remains a major driver of antimicrobial use in Chilean aquaculture. Antimicrobial peptides are plausible alternatives, but their activity may depend on peptide scaffold and on the unusual membrane composition predicted for P. salmonis . Results We screened 13 compounds against P. salmonis LF-89 at 16.5 °C over a 7-day incubation period, including 11 cationic antimicrobial peptides together with two non-peptidic reference compounds, florfenicol and chlorhexidine. The assay was performed with three independent biological replicates initiated from separately seeded LF-89 cultures. Because the readout quantified percent inhibition of net OD600 growth rather than conventional complete visible growth inhibition, activity is reported as IC80 and IC90 growth-inhibition endpoints. Among the peptides, the two beta-hairpin peptides in the panel showed the strongest activity. Tachyplesin I showed IC80 and IC90 values of 2 and 16 µg/mL, respectively, whereas Protegrin-1 reached both endpoints at 32 µg/mL. Among the linear peptides, the strongest responses were observed for SMAP-29 and Cecropin B, both with IC80 values of 64 µg/mL. To examine whether the two most active peptide leads showed distinct membrane-interaction behavior, we performed three independent 300 ns all-atom molecular dynamics trajectories per peptide on a phosphatidylethanolamine-biased bilayer. Across replicate trajectories, both peptides adsorbed to the membrane and showed distinct interfacial behavior: Tachyplesin I remained more conformationally compact, whereas Protegrin-1 formed more peptide-lipid contacts and hydrogen bonds, underscoring that monomeric adsorption metrics and growth-inhibition potency are not interchangeable. Conclusions Within this 13-compound panel, disulfide-stabilized beta-hairpin peptides showed the strongest growth-inhibitory activity against P. salmonis . The molecular dynamics results support scaffold-dependent differences in membrane interaction and prioritize Tachyplesin I and Protegrin-1 as membrane-active leads for follow-up studies against this pathogen. Together, these data provide a robust comparative screening and replicate MD analysis that identifies Tachyplesin I and Protegrin-1 as the leading beta-hairpin candidates in this panel; dedicated safety, serum-stability, host-cell selectivity, and in vivo efficacy studies are warranted as the next stage of translational evaluation.