ISOLATION AND SCREENING OF SOIL MICRO ORGANISMS FOR ANTIBIOTIC PRODUCTION

ABSTRACT

The lack of new therapeutic options to replace many antimicrobial agents that are losing their efficacy against resistant bacteria is a growing concern. The search for new antimicrobials from rare and/or existing sources have thus become a worthwhile effort in recent years. Soil samples collected from Umudike were studied in an effort to screen for novel antibiotics produced by soil microbes. A total of 45 bacterial and 19 fungal isolates were successfully obtained from the soil samples. Antibiotic activity screening using the paper disc diffusion technique was used to evaluate the antagonistic potentials of the isolates against ATCC Strains of bacteria including Staphylococcus aureus (ATCC 25923), Salmonella typhi, Escherichia coli (ATCC 25922). Of the 64 isolates, 6.25% (4/64) were active against at least one of the test organisms with diameter zones of clearing that ranged from 10.0mm to 17.0mm. Isolate VC7 exhibited the strongest antibacterial activity against S. typhi and E. coli with diameter zones of clearing of 17mm and 11.0mm respectively followed by VC29 which recorded a zone of inhibition of 14mm and 10mm against E. coli and S. aureus respectively. VC7 was tentatively identified as an Enterobacter species based on their colonial morphology and biochemical tests results. Among the isolates which demonstrated antagonism against the test bacteria, VC18, VC26, and VC7 were identified as Bacillus spp., Penicillium spp and Enterobacter spp respectively.

TABLE OF CONTENTS

Title Page                                                                                                                                i

Certification                                                                                                                           iii

Dedication                                                                                                                              iv

Acknowledgement                                                                                                                  v

Table of Contents                                                                                                                   vi

List of Tables                                                                                                                          vii

Abstract                                                                                                                                  ix

CHAPTER ONE: INTRODUCTION                                                                                   1

1.1       Aims and Objectives                                                                                                  6

CHAPTER TWO: LITERATURE REVIEW                                                                      7

2.1       Antimicrobials                                                                                                            8

2.2       Antibiotics from Microorganisms                                                                              9

2.2.1    Actinomycetales                                                                                                         10

2.2.2    Actinomycetes as a novel source of antimicrobials                                                   10

2.2.2.1 Isolation of Actinomycetes                                                                                         13

2.2.3    True Bacteria                                                                                                              14

2.2.4    Fungi                                                                                                                           14

2.2.4    Biochemistry of Antibiotic Production                                                                      14

2.3       Natural Habitat of Antibiotic Producing Microorganisms                                         18

2.4       Antibiotic Producing Soil Microbes                                                                           20

2.5       Secondary Metabolites from Bacillus sp. in Soil                                                       21

2.6       Importance and Diversity of Secondary Metabolites Produced by Actinomycetes            23

CHAPTER THREE: MATERIALS AND METHODS                                                      24

3.1       Sample Collection                                                                                                      24

3.2       Isolation of Soil Microorganisms                                                                               24

3.3       Characterization and Identification of the Isolates                                                     25

3.3.1    Gram Staining                                                                                                            25

3.4       Biochemical Tests                                                                                                      25

3.4.1    Catalase Test                                                                                                               25

3.4.2    Indole Test                                                                                                                  25

3.4.3    Citrate Utilization Test                                                                                               26

3.4.5    Oxidase Test                                                                                                               26

3.4.6    Starch Hydrolysis                                                                                                       26

3.5       Identification of Fungal Isolates                                                                                 27

3.6       Preparation of Inoculums of Test Organisms                                                             27

3.7       Preparation of Inoculum of each Isolate and Antimicrobial Testing                                     28

CHAPTER FOUR: RESULTS                                                                                             29

CHAPTER FIVE: DISCUSSION AND CONCLUSION                                                    34

5.1       Discussion                                                                                                                   34

5.2       Conclusion                                                                                                                  37

            References

LIST OF TABLES

CHAPTER ONE

1.0     INTRODUCTION

Antibiotic production is a feature of several kinds of soil bacteria and fungi and may represent a survival mechanism whereby organisms can eliminate competition and colonize a niche. The potential of B. subtilis and so many other soil organisms to produce antibiotics has been recognized for more than 50 years. Peptide antibiotics represent the predominant class. However, systematic studies that survey the complete spectrum of antibiotic activities by different B. subtilis strains are rare (Pinchuk et al., 2002). Altogether, it seems to be that B. subtilis is outstanding in the genus Bacillus with regards to its potential to produce so many different antibiotics. However, B. subtilis is by far the most commonly investigated Bacillus genus, and the large number of known B. subtilis antibiotics might reflect the numerousness of natural isolates and studies. Also other Bacilli such as Bacillus brevis (brevistin, edeines, gramicidines, tyrocidin) or B. amyloliquefaciens (Koumoutsi et al., 2004) produce a couple of antibiotics.

Antibiotics are chemical substances produced by microorganisms that in small amount selectively inhibit or kill other microorganisms (Anejia, 2005; Tortora et al., 2010). They are secondary metabolites that inhibit other competing cells to give a competitive advantage for the microorganisms that produce them. Microorganisms especially bacteria and actinomycetes are virtually unlimited sources of novel compounds with many therapeutic applications. Actinomycetes among them hold a prominent position due to diversity and proven ability to produce new structures. Actinomycetes are widely distributed in terrestrial and aquatic ecosystems. Especially in soil, actinomycetes play a crucial role in the recycling of refractory biomaterials by decomposing complex mixtures of polymers in dead plant, animal and fungal materials and capable of producing several secondary metabolites (Hopwood, 2007). Actinomycetes are Gram-positive bacteria that produce over 60% of bacterial metabolites, known by the year 2000 (Sosio et al., 2000).  A significant portion of these organisms’ genome (between 5% and 10%) is used in the production of these metabolites (Baltz, 2008), among which antimicrobials stand out.

Antimicrobial agents are natural products and produced by various types of bacteria and fungi. Hundreds of these natural products have been identified, and developed as therapeutic agents against many infectious diseases (Berdy, 2005). Microbial natural metabolites still appear as the most promising sources of antibiotic in the future (Fernando, 2006). Some of the important antibiotic producing microorganisms belong to Streptomyces, Bacillus, Cephalosporium and Penicillium that have been studied continuously for their ability to produce antibiotics (Ceylan et al., 2008).

From all the known microbes, actinomycetes are the most important source of biologically active microbial products, including many medically and commercially important antibiotics (Dhanasekaran et al., 2009). The broad spectrum antibiotic, vancomycin the potent antimicrobial agent against methicillin resistant Staphylococcus aureus and rifampcin, the effective drug against tuberculosis and leprosy were derived from several species of actinomycetes (Berdy, 2005).

The genus Streptomyces has been widely recognized as industrially important microorganism because of its ability to produce and secrete a large variety of secondary metabolites (Saadoun and Gharaibeh, 2003; Pandey et al., 2004). These include aminoglcosides, macrolides, β-lactams, peptides, polyenes, teteraclines, anthracycline antibiotics and nucleosides (Vijayakumar et al., 2007). It is estimated that more than 80% of the antibiotics are obtained from Streptomycetes (Vijayakumar et al., 2007). Micromonospora is the runner up with less than one-tenth as many as Streptomyces (Saadoun and Gharaibeh, 2003; Arifuzzaman et al., 2010).

Concerns over different infectious diseases caused by drug-resistant bacteria and fungi are a major worldwide problem (Alanis, 2005). There is a need to find new antimicrobial agents to combat them. Actinomycetes are an important source of bioactive substances that are important for both medical and economic values, particularly in biotechnology (Mitchell et al., 2004). About two-thirds of antibiotics are from actinomycetes, most of which were produced by various Streptomyces species. This group of bacteria is interesting because it has complex life cycle and its members are antibiotic producers with a number of species (Bentley et al., 2002; Willey et al., 2006; Nguyen et al., 2007). Rising numbers of antibiotic unresponsive infectious disease agents confront patients worldwide (Livermore, 2003) and consensus has emerged that it is essential that novel antibiotic classes be developed as part of the strategy to control the emerging drug-resistant pathogens (Abbanat et al., 2003). In response, there is a renewed interest in discovering novel classes of antibiotics that have different mechanisms of action.

This necessitates the screening of microorganisms for antimicrobial activity for the production of new and novel drugs. Hence, intensive search for new antibiotics has become imperative worldwide especially from new actinomycetes (Oskay et al., 2004; Parungao et al., 2007).

It has been observed that on screening programs using highly selective procedures which allows detection and isolation of effective antibiotic producing microorganisms (Rondon et al., 2000; Oskay et al., 2004; Parungao et al., 2007) from soil, which is the largest source of microorganisms and a natural reservoir for microorganisms and their antimicrobial products (Dancer, 2004; Hackl et al., 2004).

Although soils have been screened by the pharmaceutical industry for about 50 years, only a small fraction of the surface of the earth has been sampled, and only a small fraction of actinomycetes taxa has been discovered (Baltz, 2007). This requires the employment of several strategies to explore for new compounds from microorganism such as actinomycetes from different ecological niches that may yield novel compounds with diverse antimicrobial properties (Pandey et al., 2004; Ningthoujam et al., 2009).

Search for new antibiotics effective against multi-drug resistant pathogenic bacteria is presently an important area of antibiotic research. Natural products having novel structures have been observed to possess useful biological activities. Soil is a natural reservoir for microorganisms and their antimicrobial products (Dancer, 2004). Filamentous soil bacteria belonging to the genus Streptomyces are widely recognized as industrially important microorganisms because of their ability to produce many kinds of novel secondary metabolites including antibiotics. In the course of screening for new antibiotics, several studies are oriented towards isolation of Streptomycetes from different habitats.

Because of the rapid dissemination of antibiotic resistance in pathogens, many of the antibiotics, which were highly effective earlier, became obsolete during the past few decades. The efficacy of antibiotic treatment is on the wane as a result of the emergence and dissemination of antibiotic resistance amongst the pathogens. This warrants the discovery of newer and more promising antibiotics with long-term efficacy against various life threatening infections.

Infections caused by bacteria are a leading cause of death. The mortality associated with severe infections is about 30%. Antibiotic treatment improves the survival (Silbiger et al., 2006). Waterer and Wunderink, (2001) reported that, certain gram-negative microorganisms are a particular problem in community-acquired infections, including Pseudomonas aeruginosa, Acinetobacter spp. and Enterobacteriaceae.

Bacterial pathogens have become increasingly resistant to commonly used antibiotics and antimicrobial resistance has become a major medical and public health problem as bacterial resistance often result in treatment failure, which can have serious consequences, especially in critically ill patients (Farrag et al., 2002; Tenover, 2006). Antibiotic resistance is a particular problem in gram-negative bacilli. Some strains showing high level resistance to aminoglycosides, β-lactam, and quinolones (Pitout et al., 2005).

The widespread use of broad-spectrum antibiotics has led to emergence of antibiotic resistant strains of many gram-negative organisms. Extensive antibiotic resistance has developed in gram-negative bacilli due to both innate resistance in some species and the fact that they are highly adapts acquiring antibiotic resistant determinants from each other. Certain gram-negative microorganisms are particular problems including Pseudomonas aeruginosa, Acinetobacter spp., and the Enterobacteriaceae. The new generation of antibiotics will face a persistent and increasing challenge from these pathogens (Al-Jasser, 2006).

Antagonistic compounds are widespread in sediments which is a treasure house for many useful micro-organisms and among them streptomycetes are considered the most important group of organisms capable of producing the widely used antibiotic substances. Over a thousand of antibiotic substances have so far been isolated from soil actinomycetes obtained from different type of sediments of the world.

Antibiotics are one of the most important commercially exploited secondary metabolites produced by the bacteria and employed in a wide range. Most of the antibiotic producers used today are the soil microbes. Fungal strains and Streptomyces members are extensively used in industrial antibiotic production (Farrag et al., 2002). Bacteria are easy to isolate, culture, maintain and to improve their strains. The microbial products of secondary metabolism carry an important role in human health, providing roadmaps for the biosynthesis of many synthetic and semi-synthetic drugs (Pitout et al., 2005). In nature, microbial bioactive products are present as mycotoxins or bacteriocins derived from filamentous and non-filamentous bacteria and fungi (Oskay et al., 2004). The soil-based actinomycetes have been the source of countless drugs, from streptomycin and actinomycin, to erythromycin and vancomycin. Natural soil harbors over 10⁹microorganisms/ gram and provides an ideal reservoir for bioactive microbiota, which springs virtually all clinical antibiotics used today. Over 500 antibiotics are found each year and over 80% of antibiotics in clinical use are obtained from soil isolates (Oskay et al., 2004). These bioactive microorganisms are most abundantly present at the top few inches of the soil, in soil containing straw and agricultural products (Ningthoujam et al., 2009).

The trend of search for antibiotics in the past and in recent times as a result of drug resistance by microbial species has required combing the earth for various sources of antibiotics including the soil. Nigeria, like some other tropical countries, is the site of easily spreading diseases caused by microbes. Hence there is the need to search for new antimicrobials.

1.1       Aims and Objectives

1. To isolate potential antibiotics-producing microbes from the soil.
2. To screen the isolated microbes for antagonistic activity against some test bacteria.

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