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PROJECT TOPIC AND MATERIAL ON ISOLATION AND CHARACTERIZATION OF ANTIBIOTIC PRODUCING ACTINOMYCETES IN A RHIZOSPHERE ENVIRONMENT
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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
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.
1.3. LITERATURE REVIEW
1.4. DEFINITION OF TERMS
Actinobacteria is a phylum of Gram-positive bacteria with high guanine and cytosine content in their DNA (Ventura. et al., 2007). The G+C content of Actinobacteria can be as high as 70%, though some may have a low G+C content (Ghai et al.,2012). They can be terrestrial or aquatic (Servin et al., 2008). Although understood primarily as soil bacteria, they might be more abundant in freshwaters (McMahon et al 2011) Actinobacteria is one of the dominant bacterial phyla and contains one of the largest of bacterial genera, Streptomyces (Michael, 2010). Analysis of glutamine synthetase sequence has been suggested for phylogenetic analysis of Actinobacteria (Hayward et al., 2009).
Although some of the largest and most complex bacterial cells belong to the Actinobacteria, the group of marine Actinomarinales has been described as possessing the smallest free-living prokaryotic cells (Ghai et al., 2013).
The term “antibiotic” was first used in 1942 by Selman Waksman and his collaborators in journal articles to describe any substance produced by a microorganism that is antagonistic to the growth of other microorganisms in high dilution. This definition excluded substances that kill bacteria but that are not produced by microorganisms (such as gastric juices and hydrogen peroxide). It also excluded synthetic antibacterial compounds such as the sulfonamides.
The term “antibiotic” derives from anti + βιωτικός (biōtikos), “fit for life, lively” which comes from βίωσις (biōsis), “way of life” and that from βίος (bios), “life”(Liddell et al., 2002)
The term “antibacterial” derives from Greek ἀντί (anti), “against” βακτήριον (baktērion), diminutive of βακτηρία (baktēria), “staff, cane”, because the first ones to be discovered were rod-shaped (Liddell et al., eds)
Antibiotics, also called antibacterials, which are types of antimicrobial (News Ghana, 2015) used in the treatment and prevention of bacterial infection (NHS, 2014)’.They may either kill or inhibit the growth of bacteria. A limited number of antibiotics also possess antiprotozoal activity (John and sons 2012). Antibiotics are not effective against viruses such as the common cold or influenza, and may be harmful when taken inappropriately.
In 1928, Alexander Fleming identified penicillin, the first chemical compound with antibiotic properties. Fleming was working on a culture of disease-causing bacteria when he noticed the spores of little green mold in one of his culture plates. He observed that the presence of the mold killed or prevented the growth of the bacteria.
Antibiotics revolutionized medicine in the 20th century, and have together with vaccination led to the near eradication of diseases such as tuberculosis in the developed world. Their effectiveness and easy access led to overuse, especially in livestock raising, prompting bacteria to develop resistance. This has led to widespread problems with antimicrobial and antibiotic resistance, so much as to prompt the World Health Organization to classify antimicrobial resistance as a “serious threat [that] is no longer a prediction for the future, it is happening right now in every region of the world and has the potential to affect anyone, of any age, in any country”(WHO,2014)
The era of antibacterial chemotherapy began with the discovery of arsphenamine, first synthesized by Alfred Bertheim and Paul Ehrlich in 1907, used to treat syphilis(William,2009) The first systemically active antibacterial drug, prontosil was discovered in 1933 by Gerhard Domagk (Goodman et al.,1941;Aminov,2010) . All classes of antibiotics in use today were first discovered prior to the mid-1980s (Galarza et al., 2013).
Sometimes the term antibiotic is used to refer to any substance used against microbes, synonymous to antimicrobial, leading to the widespread but incorrect belief that antibiotics can be used against viruses (Brooks and Megan, 2015).
1.5. BRIEF HISTORY OF ANTIBIOTICS
Antibiotics are probably the most successful family of drugs so far developed for improving human health. Besides this fundamental application, antibiotics (antimicrobials at large) have also been used for preventing and treating animals and plants infections as well as for promoting growth in animal farming (McManus et al., 2002; Smith et al., 2002; Cabello, 2006). All these applications made antibiotics to be released in large amounts in natural ecosystems. Little is known on the overall effects of antibiotics on the population dynamics of the micro biosphere (Sarmah et al., 2006). However, the effect of antibiotics used for treating infections or for farming purposes in the selection of antibiotic-resistant microorganisms, which can impact human health has been studied in more detail (Witte, 1998; Ferber, 2003; Singer et al., 2003). As stated by the World Health Organization, the increasing emergence of antibiotic resistance in human pathogens is a special concern, not only for treating infectious disease, but also for other pathologies in which antibiotic prophylaxis is needed for avoiding associated infections. In this regard, the spread of antibiotic-resistant bacteria “means that commonplace medical procedures once previously taken for granted could be conceivably consigned to medical limbo. The repercussions are almost unimaginable’’ (WHO, 2000). It is important to remark that several antibiotics are produced by environmental microorganisms. Conversely, antibiotic resistance genes, acquired by pathogenic bacteria through Horizontal Gene Transfer (HGT) have been originated as well in environmental bacteria (Davies, 1997), although they can evolve later on under strong antibiotic selective pressure during the treatment of infections (Martinez and Baquero, 2000; Martinez et al., 2007). To understand in full the development of resistance, we will thus need to address the study of antibiotics and their resistance genes, not just in clinics but in natural non-clinical environments also (Martinez, 2008). The situation concerning antibiotics and their resistances resembles in some aspects to heavy metal contamination. Like antibiotics, heavy metals are natural compounds present in different ecosystems. However, their utilization by humans has increased their bioavailability, leading to dramatic changes in polluted ecosystems. Differing to heavy metals that challenge all forms of life, antimicrobials mainly alter the micro biosphere and probably because of this, the consequences of antibiotic pollution on the biodiversity have received less attention. Understanding heavy metal resistance in natural ecosystems may help as well to understand antibiotic resistance in the environment. The elements involved in the resistance to heavy metals are encoded in the chromosomes of bacteria like Ralstonia, which are well adapted for surviving in naturally heavy metals-rich habitats (e.g. volcanic soils). However, strong selective pressure due to anthropogenic pollution has made that these chromosomally-encoded determinants are now present in gene-transfer units, so that they can efficiently spread among bacterial populations (Silver and Phung, 1996, 2005; Nies, 2003). Similarly, antibiotic resistance genes that were naturally present in the chromosomes of environmental bacteria (D’Acosta et al., 2006; Wright, 2007; Fajardo et al., 2008) are now present in plasmids that can be transferred to human pathogens. It has been highlighted that the contact of bacteria from human-associated micro biota with environmental microorganisms in sewage plants or in natural ecosystems is an important feature to understand the emergence of novel mechanisms of resistance in human pathogens (Baquero et al., 2008). A key issue for this emergence will be the integration of antibiotic resistance genes in gene-transfer elements (e.g. plasmids), a feature that is favored by the release of antibiotics in natural ecosystems (Cattoir et al., 2008).
1.6. FUNCTIONAL ROLE OF ANTIBIOTICS AND ANTIBIOTIC RESISTANT ELEMENTS IN BACTERIAL NATURAL ECOSYSTEMS
Since antibiotics are efficient inhibitors of bacterial growth produced by environmental microorganisms, it has been widely accepted that their role in nature will be to inhibit microbial competitors. Conversely, antibiotic resistance determinants should serve to avoid the activity of antibiotics, in such a way that they would be a good example of the Darwinian struggle for life.
Although this can be true in some occasions, an alternative hypothesis stating that antibiotics could be signal molecules that shape the structure of microbial communities has been proposed (Linares et al., 2006; Yim et al., 2007).Under this view, the antibiotics will have a hermetic effect, beneficial at low concentrations likely found in most natural ecosystems, and harmful at the high concentrations used for therapy (Calabrese,2004, 2005; Davies et al., 2006).Similarly, it has been stated that some elements that serve to resist high concentrations of antibiotics, have disparate functional roles (e.g. cell homeostasis, signal trafficking, metabolic enzymes) in their original hosts (Martinez et al., 2007). The strong Increase of antibiotic concentrations in natural ecosystems as the consequence of human activities (human therapy, farming) shifts the original functions of antimicrobials and resistance elements to the weapon/shield roles they play in hospitals or farms (Martinez, 2008). These changes might influence, not just the selection of antibiotic-resistant microorganisms, but also the structure of the natural microbial populations and may alter the physiology of microorganisms as well. Besides selecting antibiotic-resistant mutants and favoring the acquisition of antibiotic resistance determinants by gene-transfer elements that can spread among the environmental micro biota, antibiotic pollution can enrich the population of intrinsically resistant microorganisms, and reduce the population of susceptible micro biota. For instance Cyanobacteria, which are responsible of more than a third of total free O2 production and CO2 fixation, are susceptible to antibiotics. There is not at the moment any indication that the Cyanobacteria population is suffering the impact of antibiotic pollution, and the risks for this situation are likely very low. However, the dramatic effect that eliminating Cyanobacteria as the consequence of antibiotic pollution might have for the biosphere reinforce the idea that the release of antibiotics in natural environments have relevant consequences not just in terms of resistance but for the maintenance of the global activity of the micro biosphere also. One example of this situation is a study in which the effect of the antibiotic ciprofloxacin on natural algal freshwater communities was tested upstream and downstream a wastewater treatment plant. Significant differences were observed in the final biomass yield, in the structure of suspended and attached algae, in the nutrient processing capacity and in the natural food web of the ecosystems (Halling-Sorensen et al., 1998; Hirsch et al., 1999). A similar study has demonstrated that tetracycline have a negative impact on the functional diversity of soil microbial communities (Kong et al., 2006). Antibiotics at much higher concentrations that usually found in natural ecosystems can be found in water (e.g. sewage waters) and soils (e.g. soils treated with manure and farm soils). However, these high concentrations are usually concentrated to areas of human activity, whereas pristine environments usually have low concentrations of antibiotics. Risk assessments might thus take into consideration mainly those areas with high antibiotic load and containing human-associated microorganisms (reactors for evolution of resistance, see Baquero et al., 2008) for analyzing the effect of antibiotic pollution on natural ecosystems. A different situation occurs for antibiotic resistance genes. It has been stated that acquisition of an antibiotic resistance phenotype produces a metabolic burden (Andersson and Levin, 1999; Morosini et al., 2000; Andersson, 2006), and it was predicted that in the absence of selective pressure, resistance would disappear. Unfortunately, this situation is not always true. Acquisition of antibiotic resistance may produce specific changes in the bacterial metabolism that can be even beneficial for bacterial growth in some habitats (Sanchez et al., 2002; Linares et al., 2005; Luo et al., 2005). Antibiotic resistance genes are found worldwide, as can be predicted due to their origin in environmental bacteria (Davies, 1994; Alonso et al., 2001). However, the wide dissemination of genes frequently present in human pathogens in places without a high antibiotic load (Pallecchi et al., 2008) indicates that, once those elements are present in gene-transfer platforms, the probability for their maintenance in natural ecosystems can be high. For this reason, antibiotic resistance genes are being considered as pollutants themselves. Since antibiotic resistance genes are naturally located in the chromosomes of environmental bacteria (D’Acosta et al., 2006; Wright, 2007; Martinez, 2008), only those elements that are present in gene-transfer elements and thus can be transferred and enriched under antibiotic selection, should be considered as bona fide pollutants. Contamination by antibiotics is relevant, not just for its impact on bacteria that can infect humans. As discussed along the review, the release of antibiotics together with antibiotic resistance bacteria can impact as well the environmental micro biota.
Different consequences of antibiotic pollution and antibiotic resistance pollution Antibiotic utilization for clinical or farming purposes select resistant microorganisms (Teuber, 2001; Livermore, 2005). It is thus predictable that residues from hospitals or farms will contain both types of pollutants: antibiotics and resistance genes. Nevertheless, the fate of both types of pollutants is likely different. Several antibiotics are natural compounds that have been in contact with environmental micro biota for millions of years and are thus biodegradable, and even serve as a food source for several microorganisms (Dantas et al., 2008). Synthetic antibiotics (e.g. quinolones) can be more refractory to biodegradation. However, they are still degraded at different rates in natural environments. It has been shown that ciprofloxacin present in river water samples is completely degraded after 3 months, whereas only 20% of oxolinic acid present in these samples is degraded after five months (Turiel et al., 2005). Recent work has shown that the binding of quinolones to soil and sediments delays their biodegradation. Nevertheless, wastewater treatment of quinolones-polluted waters efficiently removes these antibiotics by processes that include, not just biodegradation, but photo degradation also (Sukuland Spiteller, 2007). Consistent with these results, it has been shown that most antibiotics are usually below detectable limits in ground water samples, although they are more stable upon adsorption to sediments (Halling-Sorensen et al., 1998; Hirsch et al., 1999). For this reason, sediment samples from antibiotic-polluted environments have higher antibiotic concentrations than water samples from the same site (Kim and Carlson, 2007).The fact that antibiotics are degraded in natural ecosystems does not mean that they are not relevant pollutants. For instance the degradation process is slow at low temperatures in winter (Dolliver and Gupta, 2008), and the composition and moisture of the soil clearly impact antibiotic degradation (Stoob et al., 2007). More important, some ecosystems suffer a constant release of antibiotics (e.g. hospital effluents, farms residues), so that they are constantly polluted irrespectively of antibiotic degradation. As stated by Lindberg et al. Ecotoxicity tests are usually performed using very high concentrations of antibiotics for short periods of time, whereas in these types of environments, the organisms are continuously exposed to antibiotics at sub-therapeutic levels (Lindberg et al., 2007). Since sub-inhibitory concentrations of antibiotics trigger specific transcriptional responses in bacteria (Tsui et al., 2004; Linares et al.,2006; Yim et al., 2007;Fajardo and Martinez, 2008), the presence of antibiotics will necessarily modify the metabolic activity of the micro biota present in these polluted environments.
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