Preliminary Characterization and in Vitro Antimicrobial Activity of Hirudo Verbana Secretions Against Selected Multidrug-Resistant Bacteria

Abstract

Naser Matlabi, Erdal Polat and Khadija Narimanova

Introduction: The global rise in multidrug-resistant bacteria necessitates the exploration of novel antimicrobial sources. Leech secretions, known for their complex bioactive composition, represent a promising avenue for discovery. This study investigated the in vitro antimicrobial activity of Hirudo verbana secretions against a panel of bacterial and fungal strains and aimed to provide preliminary characterization of the active components.

Methods: Hirudo verbana secretions were collected using L-Arginine stimulation. Antimicrobial activity was assessed against nine bacterial strains (Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Klebsiella pneumoniae, Enterococcus faecalis, Morganella morganii, Streptococcus pyogenes, Enterobacter cloacae,Acinetobacter baumannii) and one fungal strain (Candida albicans) using the disk diffusion method. Minimum Inhibitory Concentration (MIC) was also determined using a broth microdilution assay. Secretions were subjected to protein analysis (SDS- PAGE), a preliminary functional assay to check for protease activity, and High-Performance Liquid Chromatography (HPLC) to isolate fractions with antimicrobial activity.

Results: H. verbana secretions exhibited antimicrobial activity against E. coli and S. aureus in disk diffusion assays, with mean inhibition zones of 5.2 mm ± 0.4 mm and 4.1 mm ± 0.3 mm, respectively. The MIC for E. coli was 6.25 mg/ mL and for S. aureus was 12.5 mg/mL. No significant inhibition was observed against other bacterial strains or Candida albicans in disk diffusion. SDS-PAGE analysis revealed a complex protein profile, and the zymography assay showed protease activity. HPLC analysis identified two major peaks at retention times of 5.3 and 9.1 min, with the fraction corresponding to the 5.3 min peak demonstrating antimicrobial activity against E. coli.

Discussion: Our findings demonstrate that H. verbana secretions possess in vitro antimicrobial activity against E. coli and S. aureus, supported by both disk diffusion and MIC data. The secretions also exhibited protease activity. HPLC analysis indicated that specific compounds within the secretion may be responsible for the observed antimicrobial activity. The limited spectrum of activity in disk diffusion could be due to factors like the high molecular weight of active compounds, which hinders their diffusion in agar. The specific mechanism of action and the reason for the specificity of the secretion for certain bacterial targets require further investigation.

Conclusion: This study provides evidence that H. verbana leech secretions contain antimicrobial components with selective in vitro activity against E. coli and S. aureus. Preliminary characterization using HPLC identified a potentially bioactive fraction. These findings warrant further investigation into H. verbana secretions as a potential source of novel antimicrobial agents.

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