Extraction of Coelomic Fluid from the Earthworm Polypheretima elongata for the Antibacterial Activities

It is proved that earthworm coelomic fluid is having antibacterial activity. It was our interest to know the antibacterial activity of the coelomic fluid of Polypheretima elongata against pathogenic bacteria like Escherichia coli, Staphylococcus aureus, Pseudomonas, Proteus and Bacillus. Polypheretima elongata is a local species of earthworm present in Gulbarga. The antibacterial activity of the coelomic fluid of the earthworm was tested in comparison with the commercial antibiotics available in the market. The antibacterial activity was determined by the zone of inhibition by coelomic fluid of earthworm Polypheretima elongata against bacterial strains. The zone of inhibition varies from species to species, it is species specific perticular bacteria. The best inhibitory effect of coelomic fluid of Polypheretima elongata on the growth of Staphylococcus aureus is 10mm, Escherichia coli and Proteus is 9mm, Bacillus is 7mm and Pseudomonas is 5mm. I) Introduction: Invertebrates exhibit different immune mechanisms against environmental pathogens. In earthworms, coelomocytes (leucocytes) located in coelomic cavity are responsible for innate cellular immune functions such as phagocytosis and encapsulation against parasites and pathogens. The coelomocyte population has been divided into different subpopulations. The coelomocytes possess immuno-defense related biological functions. The effector cells participate mainly in cellular mechanism but chloragocytes and the granulocytes of coelomocytes may produce humoral factors which may mediate the cellular and humoral responses as well. Microbial killing results from the combined action of the phagocytic process with humoral immune factors such as agglutinin (viz., lactin), lysosomal enzymes (viz., acid phosphatases) and various cytotoxic and antimicrobial molecules. Invertebrates have developed innate immune mechanism that detects pathogens by recognizing conserved molecular patterns. Molecules responsible for the recognition of foreign material have been named as pattern recognition proteins (PRPs) (Medzhitov and Janeway, 1997) because the host’s primitive effector cells would recognize molecular pattern rather than particular structure of the invading microorganisms. Examples of pathogen–associated molecules, which are not found in the multicelluar organisms, are lipopolysaccharides (LPS) or peptidoglycans from bacterial cell walls β-1, 3-glucan of fungal cell walls and double stranded RNA of viruses. Immune mechanism of earthworms includes both cellular and humoral components. Earthworm coelomocytes respond to the presence of pathogens by phagocytosis, encapsulation/ brown body formation and N K cells activity (Cooper et al., 2001). Humoral components include lectin, antimicrobial peptides, pore forming proteins, phenoloxidases and proteases. They include sequestration of antigenic material by agglutination, cytotoxicity and antibacterial activity. Cytolisin of Eisenia fetida coelomic fluid, named eiseniapore, was found to cause pore like structure at the target membranes. Formation of pore proteins is the result of oligomarization of eiseniapore monomers (Lange et al., 1999). There are many investigations on the presence of antibacterial substances in coelomic fluid of earthworms since bacterolytic molecules were identified as lysozyme like molecules, active only against gram positive bacteria (Jolles and Zuili, 1960; Scherbert and Messner, 1997; Lassegues, 1986). The coelomic fluid of earthworm, Eisenia fetida (Oligochaeta: Lumbricidae) was demonstrated to possess an antibacterial activity directed against earthworm pathogenic bacteria –namely: Gram negative Aeromonas hydrophila and Grampositive Bacillus megaterium by Valembois et al. (1992). Our present study is aimed to know the antibacterial activity of coelomic fluid (crude form) of the tropical earthworms Polypheretima elongata on pathogenic bacteria like Escherichia coli, Staphylococcus aureus, Pseudomonas, Proteus, and Bacillus. This antibacterial activity of the coelomic is fluid compared with that of commercially available antibiotics like Streptomycin, Norfloxacin, Ciprofloxacin, Gentamicin and Amoxillin. II)Materials and Methods: a)Bactericidal studies The bacterial strains used for determining antibacterial activity are Escherichia coli, Staphylococcus aureus, Pseudomonas, Proteus and Bacillus which were obtained from Department of Microbiology, Gulbarga University, Gulbarga. The nutrient agar media was prepared in the laboratory by adding agar to the nutrient broth (Hi-Media make). Whatman no. 1 filter paper was used to prepare the discs. Commercially available antibiotic discs (Streptomycin, Norfloxacin, Ciprofloxacin, Gentamicin and Amoxillin) were purchased from Hi-media. Sterile swabs were purchased from local medical distributors. Nutrient agar media was purchased from Hi-media. b) Method of Extraction of Coelomic Fluid for Antibacterial Activities : Earthworms were washed, kept on soft paper to absorb surface body moisture and then transferred to Petri dish, excited with 6 volt electrical stimulation, which induced them to extrude coelomic fluid through epidermal dorsal pores. The fluid extracted was collected in the small tubes and used for antibacterial tests. Sterilized blotting paper (about 6 mm diameter), free from any antibacterial activity, were impregnated with 20 μl of the fraction to be tested and placed on agar dishes inoculated with bacterial strain. c) Disk method: The target bacterial species were separately cultured in the nutrient broths to yield 10-7 cfu/ml and 18-24 hr cultures were separately spread plated on nutrient agar plates. Then, the 6 mm paper disks impregnated with 20 μl coelomic fluid fraction and incubated upright at 370C in an incubator for 24 h. The bactericidal activity of the fractions was measured in


I) Introduction:
Invertebrates exhibit different immune mechanisms against environmental pathogens. In earthworms, coelomocytes (leucocytes) located in coelomic cavity are responsible for innate cellular immune functions such as phagocytosis and encapsulation against parasites and pathogens. The coelomocyte population has been divided into different subpopulations. The coelomocytes possess immuno-defense related biological functions. The effector cells participate mainly in cellular mechanism but chloragocytes and the granulocytes of coelomocytes may produce humoral factors which may mediate the cellular and humoral responses as well. Microbial killing results from the combined action of the phagocytic process with humoral immune factors such as agglutinin (viz., lactin), lysosomal enzymes (viz., acid phosphatases) and various cytotoxic and antimicrobial molecules.
Invertebrates have developed innate immune mechanism that detects pathogens by recognizing conserved molecular patterns. Molecules responsible for the recognition of foreign material have been named as pattern recognition proteins (PRPs) (Medzhitov and Janeway, 1997) because the host's primitive effector cells would recognize molecular pattern rather than particular structure of the invading microorganisms. Examples of pathogen-associated molecules, which are not found in the multicelluar organisms, are lipopolysaccharides (LPS) or peptidoglycans from bacterial cell walls β-1, 3-glucan of fungal cell walls and double stranded RNA of viruses.
Immune mechanism of earthworms includes both cellular and humoral components. Earthworm coelomocytes respond to the presence of pathogens by phagocytosis, encapsulation/ brown body formation and N K cells activity (Cooper et al., 2001). Humoral components include lectin, antimicrobial peptides, pore forming proteins, phenoloxidases and proteases. They include sequestration of antigenic material by agglutination, cytotoxicity and antibacterial activity. Cytolisin of Eisenia fetida coelomic fluid, named eiseniapore, was found to cause pore like structure at the target membranes. Formation of pore proteins is the result of oligomarization of eiseniapore monomers (Lange et al., 1999).
There are many investigations on the presence of antibacterial substances in coelomic fluid of earthworms since bacterolytic molecules were identified as lysozyme like molecules, active only against gram positive bacteria (Jolles and Zuili, 1960; Scherbert and Messner, 1997; Lassegues, 1986). The coelomic fluid of earthworm, Eisenia fetida (Oligochaeta: Lumbricidae) was demonstrated to possess an antibacterial activity directed against earthworm pathogenic bacteria -namely: Gram negative Aeromonas hydrophila and Grampositive Bacillus megaterium by .
Our present study is aimed to know the antibacterial activity of coelomic fluid (crude form) of the tropical earthworms Polypheretima elongata on pathogenic bacteria like Escherichia coli, Staphylococcus aureus, Pseudomonas, Proteus, and Bacillus. This antibacterial activity of the coelomic is fluid compared with that of commercially available antibiotics like Streptomycin, Norfloxacin, Ciprofloxacin, Gentamicin and Amoxillin.

II)Materials and Methods: a)Bactericidal studies
The bacterial strains used for determining antibacterial activity are Escherichia coli, Staphylococcus aureus, Pseudomonas, Proteus and Bacillus which were obtained from Department of Microbiology, Gulbarga University, Gulbarga. The nutrient agar media was prepared in the laboratory by adding agar to the nutrient broth (Hi-Media make). Whatman no. 1 filter paper was used to prepare the discs. Commercially available antibiotic discs (Streptomycin, Norfloxacin, Ciprofloxacin, Gentamicin and Amoxillin) were purchased from Hi-media. Sterile swabs were purchased from local medical distributors. Nutrient agar media was purchased from Hi-media.

b) Method of Extraction of Coelomic Fluid for Antibacterial Activities :
Earthworms were washed, kept on soft paper to absorb surface body moisture and then transferred to Petri dish, excited with 6 volt electrical stimulation, which induced them to extrude coelomic fluid through epidermal dorsal pores. The fluid extracted was collected in the small tubes and used for antibacterial tests. Sterilized blotting paper (about 6 mm diameter), free from any antibacterial activity, were impregnated with 20 µl of the fraction to be tested and placed on agar dishes inoculated with bacterial strain.

c) Disk method:
The target bacterial species were separately cultured in the nutrient broths to yield 10 -7 cfu/ml and 18-24 hr cultures were separately spread plated on nutrient agar plates. Then, the 6 mm paper disks impregnated with 20 µl coelomic fluid fraction and incubated upright at 37 0 C in an incubator for 24 h. The bactericidal activity of the fractions was measured in RESEARCH PAPER terms of mm zone of inhibition. Antibacterial activity of the coelomic fluid of the Polypheretima elongata, was compared with the commercial antibiotics available in the market viz., Streptomycin, Norfloxacin, Ciprofloxacin, Gentamicin and Amoxillin.

III)Results:
The influence of coelomic fluid of Polypheretima elongata on the in vitro growth of bacterial cultures was evaluated against pathogenic bacteria such as Escherichia coli, Proteus, Pseudomonas, Staphylococcus aureus and Bacillus. The best inhibitory effect of coelomic fluid of Polypheretima elongata on the growth of Escherichia coli, Proteus and Staphylococcus aureus was seen to be 9 mm to 10 mm zone of inhibition. The inhibitory effect is comparatively less against Pseudomonas, and Bacillus, exhibiting 5 mm and 7 mm inhibitory zones in Pseudomonas and Bacillus respectively (Table -1, Plate 1 to 5).

IV)Discussion:
Preparations made out of materials of animal origin (placenta, snake and bee poison, dags, sea and river hydrocoels) were used extensively in folk and modern medicine for treating many human diseases. These preparations are also gaining importance now-a-days, though some of them are not used in modern medicine because of resource deterioration. Renewable natural medical materials of animal origin and pharmaceutical preparations on their basis as well as biologically active food supplements are the future. This area of human activity and economics should be developed and improved in every possible way. In many South-East Asia countries earthworms have already been used for nearly 2300 years to cure different human diseases. In Vietnam a dry earthworm powder produced according to different techniques that guarantee its purity is the compulsory ingredient of pharmaceuticals or "magic medicines that save life with in 60 minutes". These pharmaceuticals are often used to treat various organ dysfunctions-the result of bacterial and viral infections.
Darwin too commented that tissue fluid of earthworm can dissolve fibrin, many Japanese researchers have extracted fibrin dissolving enzymes from Lumbricus rubellus.
Our results show that coelomic fluid of earthworm Polypheretima elongata applied in the certain concentrations could decrease growth of pathogenic bacteria.
In clinical experience, the inflection of skin wounds often occurs by Staphylococci causing a decrease in the proliferation of epithelial cells and fibroblasts in the wounds (Chang et al., 1997). Maja et al. (2005) have reported G-90; a glycolipoproteins mixture obtained from tissues homogenate of earthworm Eisenia fetida and has shown the antibacterial activity in vivo and in vitro in different concentrations of growth on non pathogenic and facultative pathogenic bacteria. The bacteriostatic effect of the G-90 was 21% stronger for facultative pathogenic bacteria than that observed for non-pathogenic bacteria.
Upon the inoculation of bacteria into the coelomic cavity of earthworm, the coelomocytes initiate the process of connecting with each other by their adhesive structures around the bacteria and form so called brown bodies Cooper et al., 1999). At the same time the coelomocytes intensively synthesize and secrete proteins that adhere to the bacteria, forming aggregations and may inhibit their further proliferation. One of these proteins is agglutination of 56 kDa molecular mass, which attaches to the lectin like monosaccharide of the cellular membrane of the bacteria.
Rejnek (1991), Tuckova (1991) and Valembois et al. (1993) demonstrated significant antibacterial activity of the earthworms and that they were assisted besides coelomocytes by the chloragocytes, i.e., cell from the intestinal tract of earthworms, in resistance mechanisms. The chloragocytes secrete two proteins with a molecular mass of 40 and 45 kDa, which share 35% similarities with immunoglobulins. They adhere to the bacteria making them suitable for phagocytosis by coelomocytes in coelomic fluid (Lassegues et al., 1997). The chloragocytes synthesis and secrete the protein lysenin (33 kDa), which binds specially to phospholipids of the cell membrane and causes cytolysis (Kobayashi et al., 2000;Ohit et al., 2000).
With the knowledge, it is difficult to define which molecule of coelomic fluid of earthworms is responsible for its antibacterial activity. Theoretically such activities could be ascribed to some of the following molecular masses: 33, 40, 42, 45 and 60 kDa, which are detected by SDS-PAGE in G-90 (Hrzenjak et al., 1992). By immunochemical analyses these proteins are shown to be belonging to the immunoglobulin super family (Popovic et al., 1998).
From the results of our study it is clear that antibacterial activity is exhibited at different levels by coelomic fluids of Polypheretima elongata. Coelomic fluid of Polypheretima elongata is exhibit highest antibacterial activity against Escherichia coli and Staphylococcus aureus.
These observations are made based on zone of inhibition in vitro. However, further investigations on the presence of antibacterial molecules in the coelomic fluid and/or coelomocytes are needed which may help to explore the medicinal value of coelomic fluids of local species of earthworms.

V) Summary and Conclusion
Earthworms are known for their medicinal value since ancient times all over the world. Earthworms have blood and the coelomic fluid that contains both haemocytes and coelomocytes in their fluids. Coelomic fluids as well as blood of the earthworm have coelomocytes and haemocytes which play an important role in fighting against pathogens. This quality of earthworms made us to find out the immunological and antibacterial activity. To know the antibacterial activity of the coelomic fluid of the earthworms Polypheretima elongata against pathogenic bacteria like E. coli, Proteus, Pseudomonas, S. aureus and Bacillus.Results reveal that crude form of coelomic fluids of all the species of earthworm exhibited antibacterial activity. Polypheretima elongata exhibit highest antibacterial activity against E. coli and S. aureus. These results are based on the zone of inhibition in vitro growth of bacterial cultures. However, further investigations on the presence of antibacterial molecules in coelomic fluid of earthworms are necessary.