ISOLATION AND SCREENING OF ANTIBIOTIC-PRODUCING SOIL MICROORGANISMS AROUND FISH POND ENVIRONMENT IN UMUDIKE

ABSTRACT

The investigation was carried out to isolate antibiotics producing microorganisms around fish pond environment in Umudike. Soil samples were collected from 20 different locations of the fish pond for the isolation of potential antibiotic producing microorganisms. Spread plate technique was used for isolation and screening of microorganism. The media used for the isolation were Sabouraud dextrose agar, Tryptone soy agar and Nutrient agar. Sensitivity test using Staphylococcus aureus, Enterococcus feacalis, Pseudomonas aeruginosa, E.coli, as test organisms was employed  to investigate the antimicrobial activity of the isolates .Out of forty eight microorganisms  isolated, four of the isolates showed evidence of antimicrobial property against any of the bacteria test organisms and  they include; FPC1 which after biochemical screening was identified to be a Streptomyces species.,., FPA2 a Bacillus species., FPD2 a Bacillus species., and FPF3 a  Clostridium species., all the microbial isolates were inhibitory to the growth of atleast one of  the test bacteria . FPC1 produce inhibition zone of 9mm against P. areuginosa ,FPA2 produce inhibition zone of 8mm against  staphylococcus aureus ,  FPD2 produce inhibiton zone of 7mm  against E.faecalis and FPF3 produce inhibition of 6mm against P.aeruginosa. The result of this investigation demonstrated that strains of antibiotics producing microorganism are present in soil around fish pond environment  in  Umudike  and could be harnessed by pharmaceutical industries for the production of antibiotic from local sources.

                                                TABLE OF CONTENTS

CERTIFICATION  i                    

DEDICATION                                                                                                              ii

ACKNOWLEDGEMENTS                                                                                          iii

TABLE OF CONTENTS                                                                                              iv

LIST OF TABLES                                                                                                        vi

ABSTRACT                                                                                                                   vii

CHAPTER ONE

1.0 INTRODUCTION 1

1.1 Aims and Objectives                                                                                4

CHAPTER TWO

2.0 Literature Review  5       

2.1 Factors affecting antibiotics production by microorganism                          7

2.2 Past advances made in search for antibiotics producing

              microorganisms in novel  environment                                                         11

2.3 Techniques employed in screening for antibiotics producing

             Microorganism 15

CHAPTER THREE

Materials and Method 18

3.0 Sample Collection and Preparation 18                                                            

3.1 Media Preparation 18

3.2 Sample Preparation 18

3.3 Inoculation 19

3.4 Sub Culturing 19

3.5 Gram Staining 19

3.6 Biochemical Test 20

3.6.1 Hydrogen Sulfide test 20

3.6.2 Catalase Test 20

3.6.3 Starch Hydrolysis Test 20

3.7 Screening for Antimicrobial Activity 20

CHAPTER FOUR

4.1 RESULT 22

CHAPTER FIVE

5.0 Discussion 26

5.1 Conclusion 27

5.2 Recommendation 28

 REFRENCE

CHAPTER ONE

INTRODUCTION

The term antibiotics means against life in our every day usage however we use the world to describe a set of chemical that inhibit or kill bacteria .antibiotics are the most important commercially exploited secondary metabolites produced by the bacteria and employed in a wide range .on the whole last 55 years have seen the discovery of more than 12,000 antibiotics (Goldstein et al.,2008;Badosa et al ., 2004)

Antibiotics are one of the pillars of modern medicine ( Ball, 2004) but the rate of loss of efficacy of old antibiotics is outstripping their replacement with new ones for many species of pathogenic bacteria ( Hancock, 2007 ). The emergence of antibiotic resistant bacteria is a problem of growing significance in dermatological and surgical wound infections ( Colsky et al ., 1998 ; Giacometti et al ., 2000 ). In general, the most important resistance problems in the management of wounds have been observed with S. aureus and coagulase-negative Staphylococci among the Gram-positive species and with E. coli , Klebsiella pneumoniae and P.aeruginosa among the Gram-negative species( Filius and Gyssens, 2002 ).

        The history of new drug discovery processes shows that skeletons have, in the majority of cases, come from natural sources (Bevan et al .,1995 ). Considerable research is being done in order to find new chemotherapeutic agents  solated from soil     ( Rondon et al., 2000; Crowe and Olsson, 2001; Courtois et al., 2003 ). Soil microbial communities are among the most complex, diverse and important assemblages of organisms in the biosphere and they participate in various biological activities. Accordingly, they are an important source for the search of novel antimicrobial agents and molecules with biotechnological importance ( Hackl et al ., 2004 ).

        Many groups of microorganisms like Gram-positive, Gram-negative bacteria and fungi have the ability of synthesizing antimicrobial agents and the top cultivable antimicrobial agent producers present in soils are the actinomycetes ( Pandey et al ., 2002).

On the whole, the last 55 years have seen the discovery of more than 12,000 antibiotics. The actinomycetes yielded about 70% of these and the remaining 30% are products of filamentous fungi and non-actinomycete bacteria ( Nanjwade et al., 2010). Actinomycetes are responsible for the production of about half of the discovered bioactive secondary metabolites ( Berdy, 2005 ), notably antibiotics ( Strohl, 2004 ). Actinomycetes encompass a wide range of bacteria. They have universal occurrence and  play an active part in the cycle of nature.

        They are a well defined group of Gram positive, free-living, saprophytic bacteria with high G+C content in their DNA ( Lo et al ., 2002 ). They are predominant in dry alkaline soil. Many of the presently used antibiotics such as streptomycin, gentamicin, rifamycin and erythromycin are the product of actinomycetes.

Most of the bioactive compounds from actinomycetes  sort into several major  structural classes such as aminoglycosides (example streptomycin ,kanamycin) , and ansamycins (example rifampin),anthracyclines (doxorubicin) macrolides (example erythromycin) and tetracycline(Brun et al .,2000). Penicillin, erythromycin and methicillin which used to one time effective treatment against infectious disease

Raja et al., 2010; Llic et al., tics resistant pathogen such as methicillin and vancomycin resistant strains of staphylococcus aureus and others cause an enormous threat to the treatment of serious infection. To avoid this happening immediate replacement to the existing antibiotic is (Llic et al.,2005) and the development of the novel drugs against drug resistant pathogen is significant for today. Finding and producing antibiotics as well as using combined antibiotic therapy have been showed to delay the emergency of microbial resistance and can also produce desirable synergistic effect in the treatment of microbial infection. Antibiotics synergisms between two antibiotics and bioactive extracts are a novel concept and have and have an important activity against pathogen and host cells (Adwan and Mhanne, 2008). Research in finding and extraction newer antibiotic and increasing productivity of such agent have been a very important activity (Sundara moorthi et al ., 2011.Retinowati , 2010).this is because some important plant are expensive and have side effect to the host , some microbes have no successful antibiotics and others  are developing multi drug resistance. Keeping in mind the above mentioned ideas   the pharmaceutical industries have employed radical method in searching for antibiotics from micro organism in novel environment. since the early 90’s this strategy appear sophisticated when compared with what obtainable before, but recently his proven ultimately less successful. nearly all antibiotics used today belong to the class discovered before 1970. They are products of a ‘Golden age’ of discovery lasting from 1945-1965, which sought naturally produced antibiotics from soil streptomyces and fungi. This process chit the law of diminishing returns by the 1960s with the same classes (particularly tetracyclines) being repeatedly discovered and few new ones emerging. Since 1970 the only indisputably new antibiotic classes to reach the market are the oxazolidinones (discovered in 1978, launched in 2000)(David , 2003).

Keeping in mind, the need for further screening of soil microorganism for new antibiotics production against the increasing emergence of antibiotic resistant pathogenic strain,  present study was to screen  soil   microbial isolates from pond sediments for antibacterial compounds against some  pathogenic and human clinical bacterial isolates.

1.2 AIMS AND OBJECTIVES

v To isolate, soil microorganism from soil samples collected from fish pond environment  in Michael Okpara University of Agriculture  Umudike,  Abia state.

v To screen the microbial isolates for antibiotic production by testing with selected micro organism.

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