Leptospira interrogans biofilms in acute canine infection and their in vitro interactions with antibiotics and p-coumaric acid.

Leptospirosis is a zoonotic disease that affects multiple hosts, with dogs being highly susceptible and frequently developing renal impairment. Leptospira biofilms have been documented in the environment, chronic hosts, and in vitro; however, their role in acute infections remains unclear. Given its association with antimicrobial tolerance and bacterial persistence, biofilm formation poses a One Health concern at the human-animal-environment interface. This study aimed to investigate Leptospira biofilms in the renal parenchyma of naturally infected dogs with acute leptospirosis and to evaluate the antimicrobial susceptibility of biofilm and planktonic forms. Histological staining (Alcian Blue and periodic acid-Schiff) and immunohistochemistry targeting LipL32 were used to identify biofilm components and bacterial cells. Four L. interrogans autochthonous strains (C20, C29, C51, and C82) isolated from these cases were tested for susceptibility to doxycycline, ciprofloxacin, and p-coumaric acid (p-CA) (0.02-1600 μg/mL) under planktonic and biofilm growth conditions (7- and 21-day). Scanning electron microscopy was performed to assess biofilm structural disruption after treatment. No extracellular matrix staining was detected in renal tissues; however, Leptospira aggregates resembling early-stage biofilms were identified. Biofilm maturation negatively impacted antimicrobial efficacy, with increased MICs observed as early as seven days post-formation. p-CA exhibited lower MICs than the tested antibiotics in both planktonic and biofilm forms. Despite structural disruption, intact bacterial cells persisted in all treatments. These findings reinforce the importance of early therapeutic intervention and further investigation into biofilm-associated Leptospira persistence, the specific mechanisms of p-CA action, and potential combination therapies to enhance treatment efficacy.