Screening Of Bacteria Isolated From Earthworm Cast For Antibacterial Activities

1. 1. INTRODUCTION

The development of multidrug resistance in pathogenic organisms is a continuous problem faced by currently used antibiotics (Baltz, 2007). Therefore, the availability of potent drug is becoming particularly an acute problem (Neu, 1992). In this scenario, microorganisms have been intensively screened from soil as a source of therapeutically important molecules over a half century (Fenical, 1993) however; the frequency of discovery of structurally new compounds is apparently decreasing these years. This trend seems to imply that the easily accessible microorganisms in soil had been exhausted and there is a need to seek unutilized microorganisms from unexplored sources (Igarashi, 2004). It is likely that the diversity of secondary metabolites relies more or less on the isolation source, namely, the habitat of the producers (Igarashi, 2004). To cope with the demand for new pharmaceutical compounds and to combat the antibiotic resistant pathogens, researchers have been forced to look for novel microorganisms in unusual and unexplored environments.

The earthworm casting has rarely been explored for bacteria having antimicrobial activity and also industrial enzymes. The few times this site has been explored; the target organisms were actinomycetes. Actinomycetes is known to produce about 80% of the current antibiotics, this has aided the development of multidrug resistance in pathogenic bacteria. This study was focused on other bacteria genera, to be able to discover new secondary metabolites and a new line of antibiotics.

Extensive literature survey revealed that the casting activity led to nutrition and microbial enrichment (Sharon & Paul, 2001). Hence, the number of total bacteria, siderophore producing bacteria and fluorescent Pseudomonads were greater in casts as compare to the soil without casting

activity (Devliegher & Verstraete, 1998). The interaction between earthworm, beneficial soil microorganisms and root pathogens had been studied by Doube et al (1994) and found that earthworms act as vectors for beneficial soil bacteria. Hence, there is an immense possibility to identify new bacteria (other than actinomycetes) in the earthworm casting to discover novel bioactive compounds. Accordingly, the present study was aimed to screen important bacteria in the earthworm castings with the ultimate objective of discovering active antibacterial compounds.

1. 1. LITERATURE REVIEW

AIM AND OBJECTIVES

The aim of this study is to isolated bacteria species that have antibacterial characteristics from earthworm castings.

The objectives in this study includes;

• • • Getting samples (earthworm casts) from different sample sites

• • • culturing bacteria from earthworm castings

• • • Identifying bacteria isolated from samples and

• • • Screening bacterial isolates for antibacterial activity.

Antibiotics has been providing great assistance to the curing of diseases caused by bacteria, nevertheless the constant use of the same types or range of drugs have made bacterial strains develop multidrug resistance to these antibiotics. Hence, there is the need now to search for new lines of antibiotics from different sources and unusual organisms.

EARTHWORMS AND THEIR CASTINGS

Earthworms are the members of the class Oligochaeta of phylum Annelida. They are also tube- shaped, segmented worm. They have a world-wide distribution and are commonly found living in soil, feeding on live and dead organic matter. Amongst the many importance of earthworm, one of its major importance is its use in improving the soil to grow plants. It helps to make the soil more nutritious, improve water absorption and crop yield and it is used as an organic fertilizer. Studies have revealed that earthworms contribute significantly to soil structure and soil fertility. (Abbot & Parker, 1981) (Owa et al., 2003) (Owa et al., 2004) (Edwards & Bates 1992). reported that earthworms increased significantly the number, growth rate and yield of plants on inoculated sites.

Casting by earthworm is an important activity which, have been shown by several studies to have significant impact on soil fertility. A worm casting is a biologically active mound containing several bacteria, enzymes and remnants of plant materials and animal manure that were not digested by the earthworm (Appelhof, 1982). Castings has been shown to absorb water faster than soil and hold more water than equivalent amounts of soil thereby increasing moisture absorption and moisture availability to plants.

Furthermore, castings are known to absorb moisture from the air and hold it for plant use. Casting also holds nutrients for plant use as a non-pollutant natural fertilizer. Three major cast-types were observed to be produced by earthworms namely: Pellet casts (granular casts) produced by Eudrilus eugeniae, Agrotoreutus spp and Eutoreutus spp (Sims, 1971) (Segun, 1976). Turret casts (funnel/finger shaped) produced by Hyperiodrilus africanus and Ephyriodrilus afroccidentalis and the Mass (moldy) casts produced by Libyodrilus violaceus (Sims, 1971) (Beddard, 1981).

IDENTIFICATION AND ISOLATION OF BACTERIA

The identification of bacteria is a careful and systemic process that uses many different techniques to narrow down the types of bacteria that are present in an unknown culture. It produces benefits for many aspects of the research of microorganisms and helps physicians correctly treat patients. Bacteria can be identified and isolated morphologically, physiologically and through biochemical reactions.

Morphologically, the importance of this step is to isolate pure colonies of bacteria. The inoculation of the culture is made on the agar surface by either pour or streak plate method. This will make a dilution gradient across the agar plate. Upon incubation, individual colonies will arise from the biomass. The characteristic features of the colonies on solid agar media are then noted. This include shape (circular, rhizoid, or irregular), size, elevation (concave or convex), surface, edges, colour, structure (opaque, translucent or transparent).

Staining reactions are also used to identify bacteria morphology. The simple stain uses the basic dyes such as methylene blue or basic fuschin. Gram staining is a major type of staining reaction that imparts different colours to different bacterial structures. Usually it differentiates bacteria into two stain groups; gram positive and gram negative. The primary stain Cyrstal violet and mordent iodine form a strong CVI complex for most bacteria. Gram positive cells due to their thick peptidoglycan layer will retain the CVI complex even after it is subjected to decolourization with acetone or alcohol. Hence the counter stain safranin has no effect on gram positive cells. But in the case of gram negative, the thin peptidoglycan layer and more lipid contents would make them susceptible to the action of decolorizer and hence the gram-negative cells will absorb the colour of the counter stain safranin after the CVI complex is easily washed out. After gram staining, the gram-positive cells appear as purple and the gram-negative cells appear as pink. The staining techniques helps in preliminary identification of the isolate.

Biochemical reactions are reactions that identify bacteria through their response in a particular activity like sugar fermentation or enzyme production. There are different tests used for the differentiation of bacteria. Gram negative enteric bacilli play an important role in the contamination of food. Hence, they are the main causative agents of intestinal infections. Four specific tests have been identified to differentiate these gram-negative bacteria. They are indole,

methyl red, Voges Proskauer and citrate test; collectively known as IMViC series of reactions. There is also specific test for gram positive bacteria. They include catalase, coagulase, urease test, blood Agar plates, spirit blue agar etc.

SCREENING OF BACTERIA FOR ANTIBACTERIAL ACTIVITIES

Microbial screening or primary screening may be defined as detection and isolation of the desired microorganism based on its qualitative ability to produce the desired product like amino acid or enzyme. Hence bacterial screening id the detection and isolation of the desired bacteria based on its qualitative ability to produce antibiotics, enzymes amino acids etc.

Antibacterial activity of a molecule is completely associated with the compounds that provincially kill bacteria or slow down their rate of growth, without being extensively toxic to nearby tissues. Most recently discovered antibacterial agents are modified natural compounds. Antibacterial activity is the most important characteristic of medical textiles, to provide adequate protection against microorganisms, biological fluids, and aerosols, as well as disease transmission.

Screening bacteria for antibacterial activities is seen as isolating bacteria that have the ability to produce antibiotics and destroy other bacteria or suppress their growth, they can be classified as antibacterial agents. Antibacterial agents are the most important in fighting infectious diseases.