Original Article

In vitro investigations of coelomic fluid of Eisenia fetida: protein analysis, antioxidant activities and antibacterial effects on diabetic wounds' bacteria

Abstract

Background and Objectives: Diabetes is a metabolic disorder characterized by elevated glucose levels, leading to complications such as infections and impaired wound healing. Diabetic wounds are prone to bacterial infections, with common pathogens including Staphylococcus, Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa. Coelomic fluid of Eisenia fetida (CFEF) exhibits antimicrobial properties, making it a potential alternative to traditional antibiotics. This study aims to evaluate the in vitro antibacterial effects of CFEF on diabetic wound pathogens, alongside analyzing its protein content and antioxidant activities.
Materials and Methods: This study used bacterial strains Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Bacillus subtilis ATCC 19659, and Pseudomonas aeruginosa ATCC 27853. CFEF was extracted using warm water and electric shock methods. Protein concentration was determined using the Bradford method, and protein analysis was conducted via Tricine SDS-PAGE. Antioxidant activities were evaluated using DPPH, FRAP, superoxide dismutase, and catalase assays. Antibacterial activities were tested by disc diffusion, MIC, and MBC methods.
Results: The study showed that CFEF exhibited significant antibacterial and antioxidant activities against common bacteria found in diabetic wound infections. The warm water shock method yielded superior results compared to the electric shock method.
Conclusion: CFEF demonstrates promising antibacterial and antioxidant properties, suggesting its potential as a natural alternative for treating diabetic wound infections. Further research is needed to evaluate its clinical application and safety.

1. Association AD. Diagnosis and classification of diabetes mellitus. Diabetes Care 2010; 33(Suppl 1): S62-S69.
2. Cho NH, Shaw JE, Karuranga S, Huang Y, da Rocha Fernandes JD, Ohlrogge A, et al. IDF Diabetes Atlas: Global estimates of diabetes prevalence for 2017 and projections for 2045. Diabetes Res Clin Pract 2018; 138: 271-281.
3. Drivsholm T, de Fine Olivarius N, Nielsen AB, Siersma V. Symptoms, signs and complications in newly diagnosed type 2 diabetic patients, and their relationship to glycaemia, blood pressure and weight. Diabetologia 2005; 48: 210-214.
4. Lipsky BA, Berendt AR, Deery HG, Embil JM, Joseph WS, Karchmer AW, et al. Diagnosis and treatment of diabetic foot infections. Plast Reconstr Surg 2006; 117(7 Suppl): 212S-238S.
5. Falanga V. Wound healing and its impairment in the diabetic foot. Lancet 2005; 366: 1736-1743.
6. Lipsky BA, Berendt AR, Cornia PB, Pile JC, Peters EJ, Armstrong DG, et al. 2012 Infectious Diseases Society of America clinical practice guideline for the diagnosis and treatment of diabetic foot infections. Clin Infect Dis 2012; 54(12): e132-e173.
7. Ousey K, Chadwick P, Jawień A, Tariq G, Nair HKR, Lázaro-Martínez JL, et al. Identifying and treating foot ulcers in patients with diabetes: saving feet, legs and lives. J Wound Care 2018; 27(Sup5): S1-S52.
8. Lipsky BA. Medical treatment of diabetic foot infections. Clin Infect Dis 2004; 39 Suppl 2: S104-S114.
9. Cunha BA. Antibiotic selection for diabetic foot infections: a review. J Foot Ankle Surg 2000; 39: 253-257.
10. Bowler PG, Duerden BI, Armstrong DG. Wound microbiology and associated approaches to wound management. Clin Microbiol Rev 2001; 14: 244-269.
11. Thanganadar Appapalam S, Muniyan A, Vasanthi Mohan K, Panchamoorthy R. A study on isolation, characterization, and exploration of multiantibiotic-resistant bacteria in the wound site of diabetic foot ulcer patients. Int J Low Extrem Wounds 2021; 20: 6-14.
12. Molnar L, Pollak E, Skopek Z, Gutt E, Kruk J, Morgan AJ, et al. Immune system participates in brain regeneration and restoration of reproduction in the earthworm Dendrobaena veneta. Dev Comp Immunol 2015; 52: 269-279.
13. Schenk S, Hoeger U. Annelid coelomic fluid proteins. Subcell Biochem 2020; 94: 1-34.
14. Permana S, Putri Fityanti R, Norahmawati E, Iskandar A, Anggraini Mulyadi ED, Tri Endharti A. Coelomic fluid of Eisenia fetida Ameliorates Cetuximab to reduce K-Ras and Vimentin expression through promoting RUNX3 in an AOM/DSS‐Induced Colitis Associated Colon Cancer. Evid Based Complement Alternat Med 2020; 2020: 9418520.
15. Chellathurai Vasantha N, Rajagopalan K, Selvan Christyraj JD, Subbiahanadar Chelladurai K, Ganesan M, Azhagesan A, et al. Heat-inactivated coelomic fluid of the earthworm Perionyx excavatus is a possible alternative source for fetal bovine serum in animal cell culture. Biotechnol Prog 2019; 35(4): e2817.
16. Ku H-K, Lim H-M, Oh K-H, Yang H-J, Jeong J-S, Kim S-K. Interpretation of protein quantitation using the Bradford assay: comparison with two calculation models. Anal Biochem 2013; 434: 178-180.
17. Kielkopf CL, Bauer W, Urbatsch IL. Bradford assay for determining protein concentration. Cold Spring Harb Protoc 2020; 2020: 102269.
18. Schägger H. Tricine–sds-page. Nat Protoc 2006; 1: 16-22.
19. Burgess RR. Protein precipitation techniques. Methods Enzymol 2009; 463: 331-342.
20. Miliauskas G, Venskutonis PR, Van Beek TA. Screening of radical scavenging activity of some medicinal and aromatic plant extracts. Food Chem 2004; 85: 231-237.
21. Hsieh C, Rajashekaraiah V. Ferric reducing ability of plasma: a potential oxidative stress marker in stored plasma. Acta Haematol Pol 2021; 52: 61-67.
22. Weydert CJ, Cullen JJ. Measurement of superoxide dismutase, catalase and glutathione peroxidase in cultured cells and tissue. Nat Protoc 2010; 5: 51-66.
23. Shangari N, O'Brien PJ. Catalase activity assays. Curr Protoc Toxicol 2006; Chapter 7: Unit 7.7.1-15.
24. Balouiri M, Sadiki M, Ibnsouda SK. Methods for in vitro evaluating antimicrobial activity: A review. J Pharm Anal 2016; 6: 71-79.
25. Patton T, Barrett J, Brennan J, Moran N. Use of a spectrophotometric bioassay for determination of microbial sensitivity to manuka honey. J Microbiol Methods 2006; 64: 84-95.
26. Hussain M, Liaqat I, Hanif U, Sultan A, Ara C, Aftab N, et al. Medicinal perspective of antibacterial bioactive agents in earthworms (Clitellata, Annelida): A comprehensive review. J Oleo Sci 2022; 71: 563-573.
27. Zhu Z, Deng X, Xie W, Li H, Li Y, Deng Z. Pharmacological effects of bioactive agents in earthworm extract: A comprehensive review. Animal Model Exp Med 2024; 7: 653-672.
28. Mustafa RG, Dr Saiqa A, Domínguez J, Jamil M, Manzoor S, Wazir S, et al. Therapeutic values of earthworm species extract from Azad Kashmir as anticoagulant, antibacterial, and antioxidant agents. Can J Infect Dis Med Microbiol 2022; 2022: 6949117.
29. Hua Z, Wang Y-H, Cao H-W, Pu L-J, Cui Y-D. Purification of a protein from coelomic fluid of the earthworm Eisenia foetida and evaluation of its hemolytic, antibacterial, and antitumor activities. Pharm Biol 2011; 49: 269-275.
30. Li C, Chen M, Li X, Yang M, Wang Y, Yang X. Purification and function of two analgesic and anti-inflammatory peptides from coelomic fluid of the earthworm, Eisenia foetida. Peptides 2017; 89: 71-81.
Files
IssueVol 17 No 1 (2025) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijm.v17i1.17814
Keywords
Anti-bacterial agents; Staphylococcus aureus; Escherichia coli; Bacillus subtilis; Pseudomonas aeruginosa

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
How to Cite
1.
Danafar N, Shokoohi M. In vitro investigations of coelomic fluid of Eisenia fetida: protein analysis, antioxidant activities and antibacterial effects on diabetic wounds’ bacteria. Iran J Microbiol. 2025;17(1):163-170.