Reconfirmation of antimicrobial activity in the coelomic fluid of the earthworm Eisenia fetida andrei by colorimetric assay

A novel tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) was used in the assessment of antimicrobial activity in earthworm in the presence of phenazine methosulphate (PMS) as an electron coupling reagent. This activity was purified from the coelomic fluid of the earthworm (ECF), Eisenia fetida andrei (Oligochaeta, Lumbricidae, annelids) using a series of column chromatography techniques and was tested against three Gram-negative strains of Escherichia coli, Pseudomonas aeruginosa, Aeromonas hydrophila and three Gram-positive strains of Staphylococcus aureus, Bacillus megaterium, Arthrobacter sp., respectively. Only the pigment-free eluate of coelomic fluid of the earthworm (ECFPE) showed activity against B. megaterium amongst three isolated active fractions. The anion (DEAE-52) exchange effluent of the ECFPE was reported to have the strongest activity against P. aeruginosa amongst the three active fractions. The 20% acetonitrile eluate (AE) by Sep-Pak C-18 cartridge was also tested and showed fair resistance against E. coli, P. aeruginosa and Arthrobacter sp., respectively.