ANTIBIOTIC SCREENING OF MICROORGANISMS ISOLATED FROM DUMPSITES IN UMUAHIA FOR ANTIBIOTIC PRODUCTION

ABSTRACT

Antibiotic is one of the most important commercially exploited secondary metabolites produced by bacteria, fungi and Streptomyces and employed in a wide range. Most of the antibiotics used today are from the microbes. The aim of the study is to screen microorganisms isolated from different dumpsites in Umuahia, Metropolis for antibiotic production. A total of fifteen (15) waste dump soil samples were collected randomly from three (3) different waste dumpsites which includes about 2cm depth of the soil samples were taken with the aid of soil auger into a sterile plastic container and was transported to the laboratory for microbiology analysis. Serial dilution technique was employed in the inoculation of the soil samples. Each of the samples was diluted in the 10-fold serial dilution technique. Six test tubes were set up in a rack and filled with 9ml of sterile distilled water labelled stock, 10-1, 10-2, 10-3, 10-4, 10-5 and 10-6 respectively. One gram of each soil sample was added to the tube labelled stock and properly mixed together. An Aliquot (1ml) was transferred from this tube into the one labelled 10-1 and carefully mixed. Similarly, 1ml was drawn up from the 10-1 tube and transferred to the 10-2 tube. This was continued up to the last tube labelled 10-6 from where 1ml was discarded. Dilutions from 10-3, 10-5 and 10-6 were inoculated onto freshly Nutrient agar and Sabouraud Dextrose Agar Plates. The Spread plate method of inoculation  was used where 0.1ml of the respective dilutions(10-3 10-5 and 10-6) were plated on various agar plates and evenly spread over the entire plate using a flame sterilized glass rod. The inoculated plates were incubated at 350C for 24hrs for bacteria and at room temperature (25±20C) for fungi. A total of 45 bacterial and 19 fungal isolates were obtained from the soil samples. Antibiotic activity screening using the agar well diffusion was used to evaluate the inhibitory activity of  isolates against bacteria strains including Staphylococcus aureus, Salmonella typhi, Escherichia coli. Of the 64 isolates, (4/64) were active against at least one of the test organisms with diameter zones of inhibition ranging from 10.0mm to 17.0mm. Isolate AUG7 exhibited the strongest antibacterial activity against S. typhi and E. coli with diameter zones of clearing of 17mm and 11.0mm respectively followed by AUG29 which recorded a zone of inhibition of 14mm and 10mm against E. coli and S. aureus, respectively. AUG7 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, AUG18, AUG26, and AUG7 were identified as Bacillus spp., Penicillium spp and Enterobacter species respectively. In conclusion, search of new antibiotics, relatively simple and rapid methods have been developed for screening microorganisms for antibiotic producing ability. Soil samples are commonly employed in the isolation of antibiotic producing organism. The  detection  of  these  antagonistic  substances  revealed interesting properties that justify its importance and its study on the potential  application  in  biological  control  of  pathogenic microorganisms and spoilage  food. For these reasons, the biochemical nature and the best conditions to production  of the substances  studied  in  this  work  are  being  investigated  to  further purification experiments.

TABLE OF CONTENTS

Title Page                                                                                                                                i

Certification                                                                                                                            ii

Dedication                                                                                                                               iii

Acknowledgements                                                                                                                iv

Table of Contents                                                                                                                   v

List of Tables                                                                                                                          vii

Abstract                                                                                                                                   ix

CHAPTER ONE

1.0 INTRODUCTION                                                                                                          1

1.1  Aim And Objectives                                                                                                   4

 1.2 Objectives                                                                                                                        4

CHAPTER TWO

2.0 LITERATURE REVIEW                                                                                              5

2. 1 Wastes Generation and Management                                                                              5

2.1.1 Waste Dumps or Landfills                                                                                             6

2.1.2 Solid Waste Disposal                                                                                                     7

2.1.3 Microbial Degradation of Solid Waste                                                                          8

2.2   Antibiotic Producing Microorganisms from Soil                                                           9

2.3 Antibiotics from Microorganisms                                                                                    11

2.3.1 Actinomycetes                                                                                                               11

2.2.3 Bacteria                                                                                                                          11

2.3.3 Fungi                                                                                                                              12

2.4 Antibiotic Production by Microorganisms                                                                       12

2.4.1  Factors affecting antibiotic production                                                                         13

2.4.2 Different Types of Antibiotics produced by Microorganisms                                     14

CHAPTER THREE

3.0 MATERIALS AND METHODS                                                                                   16

3.1 Soil Sample Collection                                                                                                     16

3.2 Media Used                                                                                                                       16

3.3 Sterilization                                                                                                                      16

3.4 Enumeration of Microorganisms from Soil Samples                                                       17

3.5 Identification of Isolates                                                                                                   17

3.5.1 Gram Staining                                                                                                                17

3.5.2 Motility Test                                                                                                                  18

3.6 Biochemical and Cultural Characteristics                                                                        18

3.6.1 Catalase Test                                                                                                                  18

3.6.2 Citrate Test                                                                                                                    18

3.6.3 Indole Test                                                                                                                     19

3.6.4 Oxidase Test                                                                                                                  19

3.6.5 Starch hydrolysis test                                                                                                     19

3.6.6 Hydrogen Sulphide (H₂S) Production Test                                                                   20

3.7 Identification of Fungal Isolates                                                                                       20

3.8 Preparation of Inoculum of Test Organisms                                                                    20

3.9 Screening of Antibiotic Production by Agar Well Diffusion Method                                      21

CHAPTER FOUR

4.0 RESULTS                                                                                                                        22

CHAPTER FIVE

5.0 DISCUSSION, CONCLUSION AND RECOMMENDATION                                    27

5.1       Discussion                                                                                                                   27

5.2       Conclusion                                                                                                                  30

5.3       Recommendation                                                                                                        30        

References                                                           31

LIST OF TABLES

Table                           Title                                               Page

1      Inhibitory activity of isolates against the test organisms                                           23

2      Cultural and Microscopic Features of the Fungal Isolates                                         24

3      Percentage of Occurrence of Isolates from the Soil Samples                                     25

4      Colonial Morphology and Biochemical Characterization of the Microorganisms          26

 from the Soil Samples

CHAPTER ONE

1.0   INTRODUCTION

Waste dumps refer to areas or land where material wastes from several sources and processes are deposited. Solid waste disposal is crucial for the maintenance of both human and environmental health. Notwithstanding, the waste dumps are indiscriminately placed in developing countries (Odeyemi, 2012). It is needless to say that waste management has been rigorously assessed in terms of the potential danger to the immediate environment and public health. Efficient waste management is fundamental to the sustenance of physical and social infrastructure as well as the enhancement of the socio-economic wellbeing of any community (Osunwoke and Kuforiji, 2012). However, poor sanitation continues to be a leading cause of health problems especially in developing countries where it is estimated that about 80% of ill health are water and sanitation related; and about 15% of all child deaths under the age of 5years results from diarrheal diseases. Many things are discarded everyday ranging from ordinary rubbish to old newspapers, packaging, cleaning materials, and many different kinds of junk (Osunwoke and Kuforiji, 2012). With urban industrialization, social development and population increases, solid waste production is growing rapidly, making garbage pollution a serious problem (Mbata, 2018). Microorganisms such as bacteria and fungi rapidly populate materials using their components as their sources of nutrition for growth and multiplication, many of these microorganisms have been found to be harmful to man. Indiscriminate waste dumping breeds these microorganisms and causes a health hazard to the human population. Trends towards the use of waste dump as a preferred method of waste management might be altered if risks were adequately addressed and analyzed. Massive waste dump as an efficient, safe and economic method of solid waste is based on the supposition that such wastes are removed regularly and that the waste stream can be effectively managed.  An observable fact is that, as the wastes lie, decomposition takes place and microbial pathogens of all kinds thrive therein. Odeyemi  et al. (2012) discovered that bacterial pathogens in wastes that lack leachaete collection system are source of pathogens to the soil. These pathogens when increased in population, pose great risk to human health (Gurung  et al., 2019).  The isolation of pathogenic bacteria from soils of waste dumps connotes a serious health risk to waste handlers, scavengers and people living in the vicinity of the dumps. This risk is further exacerbated by the widely reported cases of waste dumps bacterial pathogens resistant to several antibiotics. For example, in a study conducted on the antibiogram status of bacterial isolates from air around dump sites, all the isolated organisms were resistant to most of the antibiotics against which they were tested. Pathogenic bacteria that may be associated with faecal contamination include pathogenic strains of Escherichia coli, Salmonella species, Shigella species and Vibrio cholerae. In addition to these organisms causing human disease, resistance to antibiotics has complicated the health problem (Ajayi and Akonai, 2013). Most microorganisms found in the waste can be transmitted by inhalation and contact (exposure to the dumpsite), with infection occurring in the respiratory tract, in skin lesion and mucus membrane (Awisan  et al., 2011).

 Due to inadequate and lack of government authorized waste disposal infrastructure in most rural and urban areas, most of the population dump waste are in open spaces and drainage channels. These wastes are largely untreated and poorly managed and might serve as shelter and food source for rodents, flies and insect vectors. These are known to transmit various pathogenic agents of diseases including, amoebic and bacillary dysentery, typhoid fever, plague and cholera. A good percentage of these infections are caused by bacteria which can be found in these refuse dumps and may cause diseases (Mbata, 2018). The diversity of soil microorganisms was of great significance as a factor promoting the early discovery of antibiotics . Many types of microorganisms such as moulds, bacteria, protozoa and algae, all competing for limited nutrients in the soil, have to devise strategies to survive. Among these microbes are autotrophs, free living nitrogen fixers, thermophiles, acidophiles, pathogens and saprophytes. Support for the fact that antagonistic interrelationships occur among microorganisms can be traced back to Pasteur's observation that an injection of a mixed population of soil microbes, which included anthrax spores, was less infective for animals than injections of the anthrax organism alone (Ajayi and Akonai, 2013). Thereafter, the study of interactions among soil inhabiting microbes ceased in favour of screening for new antibiotic producers. Although the first commercially produced antibiotic, penicillin, was discovered by chance, most present day antibiotics are discovered by systematic searching. As the soil is a vast repository of microorganisms, many of which remain undetected, it is a potential source of many species with the ability to produce new antibiotics. Thus attention is most often turned to the soil whenever new antibiotic producers are being sought (Okoli  et al., 2012). One of the most important steps towards controlling bacterial infections in the 21st century is the development and the use of antibiotics (Obire  et al., 2012). There is worldwide concern about the appearance and rise of bacterial resistance to commonly used antibiotics. In this regard, programmes for monitoring resistance have been implemented in many countries for the purpose of protecting the health of humans as well as animals (Chatujinda  et al., 2010).

However, the subsequent appearance and spread of antibiotic resistance in pathogenic organisms had made many currently available antibiotics ineffective (Gurung  et al., 2019). Globally, antibiotic resistance among many bacterial isolates had increased a great deal in both developed and developing counties. For instance, high rates of resistance of Salmonella serovars, Escherichia coli and Vibrio cholerae strains to conventional antibiotics such as ampicillin, chloramphenicol, tetracycline, cotrimoxazole (trimethoprim-sulfamethoxazole), streptomycin and other newer antibiotics which were isolated from the soil have been reported with increasing frequency in many parts of the world (Heuer  et al., 2011).

1.1 AIM AND OBJECTIVES

 The aim of the study is to screen microorganisms isolated from different dumpsites in Umuahia, Metropolis for antibiotic production.

1.2 OBJECTIVES

1. To isolate and identify microorganisms from soils of different dumpsites in Umuahia, Metropolis, Abia State.

2. To determine the antibiotic production profile of different microorganisms isolated from soils of different dumpsites in Umuahia, Metropolis, Abia State.

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