Rhodomyrtone-rich fractions from Rhodomyrtus tomentosa (Aiton) Hassk. leaves: A potent antimicrobial compound for Staphylococcus pseudintermedius.

Abstract

Background and aim: Staphylococcus pseudintermedius is an opportunistic zoonotic pathogen frequently implicated in skin and wound infections in companion animals. Its ability to form biofilms complicates treatment by increasing antibiotic resistance. Rhodomyrtone, a potent acylphloroglucinol isolated from Rhodomyrtus tomentosa, exhibits promising antibacterial activity against Gram-positive bacteria. This study aimed to develop rhodomyrtone-rich fractions and evaluate their antibacterial and antibiofilm activities against S. pseudintermedius.

Materials and methods: Ethanolic extracts of R. tomentosa leaves were subjected to acetone partitioning followed by quick column chromatography, yielding fractions F1-F15. Fractions F3-F7 were selected based on thin-layer chromatography and ¹H nuclear magnetic resonance for rhodomyrtone content and quantified by high-performance liquid chromatography. Antibacterial activity against Staphylococcus aureus American Type Culture Collection (ATCC) 25923, S. pseudintermedius ATCC 49444, and 10 clinical S. pseudintermedius isolates was assessed using broth microdilution to determine minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values. Fraction F4, with the highest rhodomyrtone content, was further investigated using time-kill kinetics, scanning electron microscopy (SEM), and a crystal violet assay for biofilm inhibition.

Results: Fraction F4 contained the highest rhodomyrtone concentration (489.08 mg/g) and demonstrated the most potent antibacterial activity, with MIC and MBC values ranging from 0.5 to 2 µg/mL and 2 to 8 µg/mL, respectively, against clinical isolates. The time-kill study revealed a 4-log reduction (99.99%) in bacterial load within 8 h at 2× MIC. Biofilm formation by all tested S. pseudintermedius isolates was significantly inhibited at sub-MIC concentrations of F4 (p < 0.05). SEM analysis showed notable morphological disruptions in bacterial cells treated with F4, suggesting membrane damage as a possible mechanism of action.

Conclusion: Fraction F4 from R. tomentosa leaf extract exhibited strong antibacterial and antibiofilm activity against S. pseudintermedius, comparable to that of pure rhodomyrtone and superior to doxycycline. These findings support the potential use of rhodomyrtone-rich fractions as standardized herbal antibacterial agents in veterinary medicine, providing an effective alternative for treating drug-resistant staphylococcal infections.