Antibacterial Activity and Mechanism of Protocatechuic Acid Against Pathogens Isolated from Canine Endometritis

Canine endometritis is commonly associated with bacterial infections caused by Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and Streptococcus canis (S. canis), leading to reproductive disorders in dogs. With increasing concern regarding antimicrobial resistance, alternative therapeutic strategies are needed. This study evaluated the in vitro antibacterial activity and underlying mechanisms of protocatechuic acid (PCA) against clinical isolates of these pathogens obtained from dogs diagnosed with endometritis. The antibacterial efficacy of PCA was assessed by determining minimum inhibitory concentrations (MICs), minimum bactericidal concentrations (MBCs), and bacterial growth curves. PCA inhibited the growth of all three pathogens, with MIC values of 4 mg/mL for E. coli and S. aureus and 2 mg/mL for S. canis. The MBCs for E. coli and S. aureus were equal to their MICs, while the MBC for S. canis was twice the MIC, indicating bactericidal activity. Mechanistic analyses demonstrated that PCA disrupted bacterial membrane integrity, induced membrane depolarization, reduced intracellular ATP levels, and increased reactive oxygen species (ROS) accumulation. These effects were supported by SYTO9/PI fluorescence staining and scanning electron microscopy. In conclusion, PCA exhibits notable in vitro antibacterial activity against key pathogens associated with canine endometritis and represents a promising natural antimicrobial candidate.