ISOLATION AND IDENTIFICATION OF BIOFILMS FORMING BACTERIA IN MOUAU FISH POND

ABSTRACT

A  biofilm is a complex aggregation of microorganism growing on a solid substrate. It act as defensive role for microorganism during various stress conditions. The proposed work was carried out to study the biofilm forming ability of microorganisms specifically  Escherichia coli, Staphylococcus aures, Streptococcous spp, Bacillus,  Pseudomonas,  Enterobacter,  Salmonella  and Listeria from fish pond in MOUAU. The bacteria were isolated using selective media morphological and biochemical characterization of isolates were carried out. All eight (8) isolates were found to be Gram positive and Gram negative pathogens bacteria. Biofilm forming ability was studied by Congo red agar method (CRA) Staphylococcus aureus, Bacillus and Pseudomonas occurrence at 100% (6) each are strong biofilm producer based on black colonies appearance on (CRA) While Streptococcus, Escherichia coli and Listeria spp are moderate biofilm producers occurrence at 66.7% (4) each and non-biofilm producers are Enterobacter and Salmonella spp. Occurrence at 50%(3) each. This present study will help to understand the biofilm formation of fish pond.

TABLE OF CONTENTS

Title page                                                                                                                                 i

Dedication                                                                                                                                ii

Acknowledgement                                                                                                                    iii

Certification                                                                                                                              iv

Declaration

Table of contents                                                                                                                       v

List of tables                                                                                                                             vii

Abstract                                                                                                                                    x

CHAPTER ONE

1.0 Introduction                                                                                                                         1-2

1.1 Background of study                                                                                                            2

1.2 Aim and objective of the study                                                                                             3

CHAPTER TWO

2.0 Literature review                                                                                                                 4                                                                                                         

2.1 General concept of biofilms                                                                                                 4

2.2Mechanism for the formation of biofilms                                                                               4

2.3 Different places where biofilms can be formed                                                                       5                  

2.4 Biofilms and infectious diseases                                                                                           6

2.5 Biofilms in the food industry                                                                                                7

2.6 Bacteria associated with farmed fish                                                                                     8

2.6.1 Vibrio Paraheamotyticus and other vibrios:                                                                         9-10

2.6.2 Escherichia coli                                                                                                                10-11

2.6.3 Salmonella Spp:                                                                                                                11

2.6.4 Staphylococcus Aureus:                                                                                                    11                                                                               

2.6.5 Listeria Monocytogenes:                                                                                                   12                                                                                           

2.6.6 Clostridium botulinum:                                                                                                     13                                                                   

2.6.7 Pseudomonas Aeruginosa:                                                                                                13                                                                                           

2.6.8 Aeromonas Species:                                                                                                          14                                                                                                       

2.6.9 Citrobacter Freundii:                                                                                                         14-15

2.7 Factors contributing to fish contamination                                                                             16-17

CHAPTER THREE      

3.0 Materials and methods                                                                                                         18                                                                                                                   

3.1 Collection and preparation of the fish pond water sample                                                       18                                                                                           

3.2 Preparation of media                                                                                                            18                                                       

3.3 Isolation of biofilm producing bacteria                                                                                  18

3.4 Characterization and identification of isolates                                                                        19

3.4.1 Gram Staining                                                                                                                  19                                                                                                       

3.5 Biochemical Tests                                                                                                              20                                                                                           

3.5.1 Catalase Test                                                                                                                    20

3.5.2 Urease Test                                                                                                                       20

3.5.3 Indole Test

3.5.4 Methyl Red-Voges Proskauer Test                                                                                     20-21                                                                                             

3.5.5 Citrate Utilization Test                                                                                                      21                                          

3.5.6 Sugar Fermentation Tests                                                                                                  21                                                                                                       

CHAPTER FOUR

4.0 Results                                                                                                                                22-24                                                                                                               

CHAPTER FIVE

5.1 Discussion                                                                                                                           25                                                                   

5.2 Conclusions                                                                                                                        26                                                                               

Reference                                                                                                                                  28-29

LIST OF TABLES

Table 1: Confirmation of Biofilm Formation

Table 2: occurrence of bacteria Isolate in mouau fish pond

Table 3:characterization of bacteria isolates from mouau fish pond

CHAPTER ONE

1.0. Introduction

The cheapest source of animal protein in Africa is fish (Clucas and Ward 1996). Fish products could incubate pathogens from the natural micro-flora of fish ponds. The risks of food borne disease associated with products from aquaculture are related to inland or coastal ecosystems, where the potential of environmental contamination is greater when compared with capture fisheries. Most of the food safety hazards associated with products from aquaculture can be controlled by good fish farm management practices and appropriate consumer education regarding such risks as eating raw or partially cooked products that may contain pathogenic bacteria. The estimated annual mortality of food and water-borne infectious diseases in developing countries amounts to high death rates, mainly of infants and children.

Biofilm is a population of microbial cells that are enclosed within a matrix made of primarily polysaccharide material which have the ability to stick to surfaces (Donlan, 2002). There is a compromise of food safety especially in minimally processed foods and raw foods due to the presence of biofilms on food and food contact surfaces (Frank and Chmielewski, 2001).

 Microorganism can colonize to form biofilms on solid surfaces when adequate nutrients, minerals, and organic matter are present in fish processing environment. Free living bacteria are more susceptible to sanitizers and antimicrobial cleaning agents than bacteria in biofilms. Biofilms are difficult to remove from food contact surfaces and equipment by normal cleaning, thus posing a food safety risk (Kariyawasam and Asooriya, 2006). The presence and multiplication of opportunistic pathogens such as Escherichia coli, Listeria monocytogenes and Staphylococcus aureus is a concern in food processing and storage. The main serotype of enterohsemorrhagic Escherichia coli (EHEC) is Escherichia coli O157:H7. Escherichia coli O157:H7isthe common cause of sporadic and multi-person out breaks of bloody diarrhoea in the U.S.( Dean-Nystrom et al.,2003). The ability to attach, colonize, and form biofilms on various surfaces has been shown by Escherichia coli O157:H7 (Uhlich et al.2006).

The main cause of staphylococcal infections is mainly Staphylococcus aureus. This pathogen is responsible for mild skin infections, invasive diseases and toxin mediated diseases and it is resistant to a wide range of commonly used antibiotics Recently a report of about 40% resistance which increases yearly was made to methicillin. The adhesion ability of Staphylococcus aureus on catheters and other indwelling medical devices and form biofilm on polymeric surfaces have been reported (Cramton et al., 1999).The adhesion capacity depends production of polysaccharide intercellular adhesion(PIA) encoded by the intercellular adhesion gene cluster icaADBC (Cramton et al., 1999). Listeria monocytogenesis an important hardy food borne pathogen(USFDA-CFSAN, 2006) responsible for major outbreaks associated with dairy and other food products (Farber and Peterkin, 1991, Fleming, 1998, Adetunji et al., 2003). The organism is ubiquitous throughout nature and is frequently isolated from food processing environment (Hood and Zottola, 1997; Destro et al., 1996; Johansen et al., 1997). It has also been reported that Listeria monocytogenes has ability to form biofilms on food contact surfaces (Adetunji and Adegoke, 2008; Adetunji and Isola, 2011).

Information on biofilm forming ability of bacteria isolates of fish is scarce.

1.1  Background of study

A biofilm is any group of microorganisms in which cells stick to each other and often these cells adhere to a surface. These adherent cells are frequently embedded within a self- produced matrix of extracellular polymeric substance (EPS). Biofilm extracellular polymeric substance, which is also referred to as slime (although not everything described as slime is a biofilm), is a polymeric conglomeration generally composed of extracellular DNA, proteins, and polysaccharides. Biofilms may form on living or non-living surfaces and can be prevalent in natural, industrial and hospital settings. The microbial cells growing in a biofilm are physiologically distinct from planktonic cells of the same organism, which, by contrast, are single-cells that may float or swim in a liquid medium. Microbes form a biofilm in response to many factors, which may include cellular recognition of specific or non- specific attachment sites on a surface, nutritional cues, or in some cases, by exposure of plank tonic cells to sub- inhibitory concentrations of antibiotics.

1.2 Aims and objective of the study

The aim and objective of this study is to specifically isolate and identify different biofilms forming bacteria present in MOUAU fish ponds

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