ABSTRACT
This research work was carried out to isolation and characterization of antibiotic producing actinomycetes in rhizosphere environments using the standard microbiological method, crowded plate method, streak plate method and pour plate method. Starch casein agar and Nutrient agar were used for the characterization of the growth organisms and tests such as gram staining, starch hydrolysis, casein hydrolysis, lipid hydrolysis, citrate, methyl red, catalase and indole were carried out. After culturing only 5 out of the 13 isolates (from a total of 25 soil samples) showed visible growth and had antimicrobial activity on selected organisms(Stapylococus aureus, Bacillus subtilis and Escherichia coli), the total count for the colony forming units ranged from 3.9×106– 5.2×106. The five isolates gotten from this work had four of them from the genus Streptomycetes (denoted as B, F, H and M) and the other from the genus Nocardia (L). Isolate B was active against Escherichia coli with a zone of inhibition measuring 26mm, isolate F, H, L and M were active against Staphylococcus aureus with zones of inhibition measuring 8mm, 9mm16mm and 9mm respectively, while isolate B, F, H, L and M were active against Bacillus subtitis with zones of inhibition measuring 5mm, 11mm, 11mm, 19mm and 7mm respectively. This study shows that Streptomyces are the most prevalent antibiotic producing actinomycetes in the soil. Therefore antibiotics should be taken only when needed to avoid antibiotic resistance by certain organisms and stored at appropriate conditions (temperature and pressure) hence, further purification, elucidation, and characterization are recommended to know the quality and novelty and commercial values of antibiotics.
Keywords:actinomycetes, rhizosphere, antimicrobial ,isolation,characterization
CHAPTER ONE
1.0. INTRODUCTION
1.1 BACKGROUND OF STUDY
Rhizosphere is the narrow region of soil that is directly influenced by root secretion and associated soil microorganisms (Bacteria, fungi, protozoa etc). The rhizosphere by definition is the soil region in close contact with the plant root. The term “rhizophere soil” generally refers to thin layer of soil adhering to a root system after the loose soil has been removed by shaking (Atlas 1981) the rhizosphere is basically divided into two general areas, the inner rhizosphere at the very root surface and the outer rhizosphere embracing the immediate adjacent soil.
Soil which is not part of rhizosphere is known as bulk soil, with the recent description and ecological characterization of plant growth –promoting rhizosphere –bacteria (PGPR) the use of bacteria from the root zone to enhance plant growth has been given attention to Kloepper (1980) showed that certain rhizosphere bacteria can metabolize seed exudates actively at cool temperature and that these bacteria can encourage seedling emergence in field soil. Kloepper (1980) also revealed that root colonization by some PGPR strains displaced native root micro flora thereby enhancing crop growth.
Alexander (1977) reported that the interactions between these microorganisms in the root of plant can have a considerable significance for crop production and soil fertility hence providing food for man and feed for animals.
The microbial population is more in the inner zone where the biochemical interaction between organisms are most pronounced. In the rhizoplane which is the surface directly covering the root within the rhizosphere and rhizoplane, the inner organism contribute excretory products. Some of the interactions (such as mutualisms) are beneficial to the plant while some (like parasitism) are detrimental.
An antibiotic (against life) is a compound or substances that kills or slow down the growth of bacteria. Antibiotics include a chemically heterogeneous group of small organic molecules of microbial origin that, at low concentration, are deleterious to the growth and metabolic activities.
The discovery and application of antibiotics in the treatment of bacterial diseases had been a noteworthy medical success of the 20th century. However, gradual emergence and spreading of antibiotics resistance among bacterial population due to misuse or overuse of antibiotics has led to the development of public health problems. Antibiotic resistance in bacterial isolates was recorded since the first use of antibacterial agents. Penicillin-resistant Escherichia coli were the first to be discovered in 1940 to possess penicillinases that inactivated the drug penicillin, followed by discovery of penicillin-resistant Staphylococcus aureus in 1944. In 2008, the NDM-1 gene, encoding novel beta-lactamase enzyme capable of hydrolyzing penicillins, cephalosporins and carbapenems was discovered in Klebsiella pneumoniae. Bacteria possessing the gene were found to be resistant for most of the tested antibacterial agents (Moellering, 2010). Although there are advances in drug discovery and development in recent years, the world is not keeping pace with bacterial ability in adapting and resisting antibiotics. In addition, many bacteria gain resistance to the newly launched drugs that were modifications of the existing antibiotics. Hence, it is highly essential to search for new antimicrobial compounds particularly from microorganisms to combat the threat of increasing population of antibiotic-resistant bacteria.
Actinomycetes are filamentous bacteria that belong to the phyla actinobacteria and the order actinomycetales. Actinomycetes are known as the most invaluable prokaryotes in medical and biotechnology industries due to their ability in producing a vast number of bioactive molecules, particularly of the antibiotic compounds. Streptomyces, a representative genus of actinomycetes that is mainly of terrestrial soil origin, has accounted for the production of 60% of antibiotics which are useful in agricultural industries (Mellouli et al., 2003; Fguira et al., 2005; Singh et al., 2006; Thakur et al., 2007). The wide distribution of Streptomyces in soil and their proven ability to produce novel antibiotics and non-antibiotic lead molecules had caused these bacteria to be targeted in drug screening programme. Discovery of novel antibiotics from actinomycetes is important in helping to cope with the growing proportion of antibiotic-resistant bacterial infections that become untreatable. Hence, this investigation was conducted with the aim of isolating and screening for antibiotic-producing actinomycetes from rhizosphere soil. Selected antibiotic-producing actinomycetes were identified and effects of pH, temperature and concentration of sodium chloride on the growth of actinomycetes were also determined.
Secondary metabolites are produced by some organisms such as bacteria, fungi, plants, actinomycetes and so forth. Among the various groups of organisms that have the capacity to produce such metabolites, the actinomycetes occupy a prominent place (Berdy, 2005; Ramasamy et al., 2010; Sundaramoorthi et al., 2011). Actinomycetes are prokaryotes of Gram-positive bacteria but are distinguished from other bacteria by their morphology, DNA rich in guanine plus cytosine (G+C) and nucleic acid sequencing and pairing studies. They are characterized by having a high G+C content (>55%) in their DNA (Gonzalez-Franco et al., 2009).
Actinomycetes are of universal occurrence in nature and are widely distributed in natural and man-made environments. They are found in large numbers in soils, fresh waters, lake, river bottoms, manures, composts and dust as well as on plant residues and food products. However, the diversity and distribution of actinomycetes that produce secondary metabolites can be determined by different physical, chemical and geographical factors (Gurung et al., 2009; Ogunmwonyi et al., 2010).
Actinomycetes provide many important bioactive substances that have high commercial value. Their ability to produce a variety of bioactive substances has been utilized in a comprehensive series of researches in numerous institutional and industrial laboratories. This has resulted in the isolation of certain agents, which have found application in combating a variety of human infections (Retinowati, 2010). That is why more than 70% of naturally occurring antibiotics have been isolated from different genus of actinomycetes (Khanna et al., 2011). Out of these different genus, Streptomyces is the largest genus known for the production of many secondary metabolites(Maleki and Mashinchian,2011), which have different biological activities, such as antibacterial, antifungal, antiparasitic, antitumor, anticancer and immunosuppressive actions(Berdy,2005;Jemimah et al.,2011;Nonoh et al 2010).
Some antibiotics like penicillin, erythromycin, and methicillin which used to be one-time effective treatment against infectious diseases (Raja et al., 2010), are now less effective because bacteria have become more resistant to such antibiotics. Antibiotic resistant pathogens such as methicillin and vancomycin resistant strains of Staphylococcus aureus (S. aureus) and others cause an enormous threat to the treatment of serious infections. To avoid this happening, immediate replacement of the existing antibiotic is necessary (Ilic et al., 2005), and the development of novel drugs against drug resistant pathogens is significant for today.
Thus, finding and producing new antibiotics as well as using combined antibiotic therapy have been shown to delay the emergency of microbial resistance and can also produce desirable synergistic effects in the treatment of microbial infection. Antibiotic synergisms between known antibiotics and bioactive extracts are a novel concept and have an important activity against pathogens and host cells (Adwan and Mhanna, 2008).
Research in finding newer antibiotics and increasing productivity of such agents has been a very important activity (Sundaramoorthi et al., 2011). This is because some important drugs are expensive and/or have side effect to the host, some microbes have no successful antibiotics and others are developing multidrug resistance. This situation requires more attention to find solutions by searching and producing new and effective antibiotics from microbes like actinomycetes. However, there is no such scientific report on antibiotic producing actinomycetes from soil samples collected in Imo State University Owerri. Therefore, the objective of the present study was to isolate and screen antibiotic producing actinomycetes from soil samples. The outcome of this finding may be important to give direction for researchers and for future treatment of multidrug resistant human pathogens.
1.2. AIM AND OBJECTIVES
- To isolate and characterize antibiotic producing actinomycetes from a rhizosphere environment.
- To identify the microorganisms.
- To check for the antimicrobial activity of the isolated actinomycete(s) against various microorganism.
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