Document Type : Original Article(s)

Authors

• Sirous Banafi ¹
• Mohammad Hossein Marhamatizadeh ¹
• Nader Tanideh ^{2, 3}
• Ebrahim Rahimi ¹
• Afshin Zare ⁴
• Ramazon Safarzoda Sharoffidin ⁵
• Amin Tamadon ^{6, 2, 7}

¹ Department of Food Hygiene, School of Veterinary Medicine, Kazerun Branch, Islamic Azad University, Kazerun, Iran

² Stem Cells Technology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran

³ Department of Pharmacology, School of Medicine, Shiraz University of Medical Sciences, Shiraz, Iran

⁴ International PhD Program in Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan

⁵ Department of Pharmaceutical Technology, Avicenna Tajik State Medical University, Dushanbe, Tajikistan

⁶ Department of Natural Sciences, West Kazakhstan Marat Ospanov Medical University, Aktobe, Kazakhstan

⁷ PerciaVista R&D Co., Shiraz, Iran

Abstract

Background: Clostridium perfringens (C. perfringens) is an opportunistic anaerobic pathogen associated with severe soft tissue and gastrointestinal infections and increasing antimicrobial resistance. This study aimed to evaluate the in vitro antibacterial activity of human umbilical cord mesenchymal stem cell (hUC-MSC)-derived exosomes against C. perfringens and to explore potential exosome–enzyme interactions using in silico protein–protein docking.
Methods: This study was conducted at Shiraz University of Medical Sciences, Shiraz, Iran, in 2025. In this experimental in vitro and in silico study, human umbilical cord mesenchymal stem cells (hUC-MSCs) were isolated from term umbilical cords (UCs), characterized, and induced into osteogenic and adipogenic differentiation. Exosomes were isolated using a commercial precipitation kit and characterized by scanning electron microscopy (SEM), dynamic light scattering (DLS), and flow cytometry for CD63 expression. The antibacterial activity of exosomes against C. perfringens ATCC 2592 was evaluated by a broth microdilution minimum inhibitory concentration (MIC) assay under anaerobic conditions. Optical density (OD600) values were recorded and compared across exosome concentrations. Protein–protein docking was performed between exosomal proteins and four essential C. perfringens enzymes using ClusPro. OD600 values were compared across exosome concentrations using one-way analysis of variance (ANOVA) (P<0.05).
Results: hUC-MSCs displayed typical spindle-shaped morphology, tri-lineage differentiation potential, and a mesenchymal immunophenotype. Isolated exosomes were spherical nanovesicles (~30–100 nm) with high CD63 positivity. UC-MSC-derived exosomes inhibited C. perfringens growth in a dose-dependent manner. At 500 µg/mL, exosomes achieved 95.1% inhibition with an OD600 of 0.12±0.03 vs. 2.45±0.10 in the growth control (n=3, P<0.001). Docking analysis suggested favorable in silico interactions between Hsp70 and C. perfringens RNA polymerase and ATP synthase, and between cathelicidin and DNA gyrase, with lower but consistent docking scores for Annexin A1 across targets.
Conclusion: hUC-MSC-derived exosomes exerted significant in vitro antibacterial effects against C. perfringens and exhibited favorable in silico interactions with key bacterial enzymes.

Highlights

Sirous Banafi (Google Scholar)
Mohammad Hossein Marhamatizadeh (Google Scholar)

Keywords

• Exosomes
• Mesenchymal stem cells
• Umbilical cord
• Clostridium perfringens
• Anti-bacterial agents