ANTIBIOGRAM OF BACTERIA ISOLATED FROM FRUITS AND VEGETABLES

ABSTRACT

This study determined the antibiogram of fruits and vegetables sold in Umuahia metropolis. A total of Twenty (20) fruit (watermelon, pawpaw) and vegetable samples were purchased randomly from five different markets in Umuahia, Abia State. The bacterial species were identified by morphological characteristics, pigmentation on media, microscopy, biochemical and sugar fermentation methods. The major bacteria species isolated include; Escherichia coli, Staphylococcus aurues, Salmonella species and Shigella species respectively. The total bacteria mean counts recorded in this study was found higher in Gate six 8.3x105cfu/ml (vegetables), while the least viable bacteria count was recorded for Gate six (2.2x105 cfu/ml), (watermelon). From the findings in this study, it was observed that Staphylococcus aureus was the most frequently occurred isolate with a high percentage occurrence of 10(33.3%) followed by Escherichia coli at 9(30.0%). Antibiotics susceptibility test indicated that all the isolated bacteria recorded highest sensitivity with Ciprofloxacin and Levofloxacin while the highest resistance occurred when the antibiotics Amoxil and Ampiclox were used. However, the bacteria species isolated indicated a public health concern as they showed high counts and occurrence on the samples which suggested heavy bacterial contamination of the fruit and vegetables by handling, processing equipment, environment, packaging materials and the personnel’s in the production process. Therefore, it is recommended that retailers and sellers of fruits and vegetables should be properly educated and sensitized on different hygiene practices.

TABLE OF CONTENTS

Title Page                                                                                                                                i

Certification                                                                                                                           iii

Dedication                                                                                                                              iv

Acknowledgements                                                                                                                v

Table of Contents                                                                                                                   vi

List of Tables                                                                                                                          viii

List of Figures                                                                                                                         ix

Abstract                                                                                                                                  x

CHAPTER ONE

1.0       Introduction                                                                                                                1

1.1       Aim and Objectives                                                                                                    3

CHAPTER TWO

2.0       Literature Review                                                                                                       5

2.1       General Aspects of Microbiological Safety of Food and Agricultural Produce  5

2.2       Microbiological Safety of Fruits and Vegetables                                                       6

2.2.1    Overview of Produce-Related Human Infections                                                      6

2.3       Sources and Mechanism of Contamination of Fruits and Vegetables                  6

2.3.1    Production Environment                                                                                            7

2.3.2    Postharvest Handling                                                                                                  8

2.3.3    Human Hygiene                                                                                                          9

2.4       Potential Food-Borne Illness Outbreaks Associated with Consumption of

            Fruits and Vegetables                                                                                                 10

2.5       Shelf Life of Fresh-Cut Fruits and Vegetable                                                            11

2.6       Impact of Microbiological Spoilage                                                                           13

2.6.1    Microbiological Spoilage Mechanisms in Fresh-Cut Fruit and Vegetable                    14

2.6.2    Prevention and Control of Microbial Spoilage                                                           16

2.7       Antibiotics Resistance Mechanisms                                                                           17

2.7.1    Resistance Based on Altered Receptors for a Drug                                                    18

2.7.2    Decreased Entry of Antibiotics                                                                                  19

2.7.3    Synthesis of Resistance or Alternative Pathway                                                        20

2.8       Modes of Action of Antibacterial Agents                                                                  20

2.8.1    Inhibition of Cell-Wall Synthesis                                                                               21

2.8.2    Inhibition of Protein Synthesis                                                                                   22

2.8.3    Injury to the Plasma Membrane                                                                                 23

2.8.4    Inhibition of Nucleic Acid Synthesis                                                                         23

2.8.5    Inhibition of Essential Metabolites                                                                            23

CHAPTER THREE

3.0       Materials and Methods                                                                                               25

3.1       Source/Sample Collection                                                                                          25

3.2       Materials and Media Used                                                                                          25

3.2.1    Sterilization of Materials                                                                                            25

3.3       Processing of Samples                                                                                                26

3.4       Preparation of Culture Media                                                                                     26

3.4.1    Mannitol Salt Agar (MSA)                                                                                         26

3.4.2    Nutrient Agar (NA)                                                                                                    26

3.4.3    MacConkey Agar (MA)                                                                                              27

3.4.4    Salmonella and shiegella Agar (SSA)                                                                        27

3.5       Microbiological Studies                                                                                             27

3.5.1    Inoculation and Enumeration of Microorganisms                                                      27

3.5.2    Colonial Morphology of the Bacterial Isolates                                                          27

3.6       Microbial Characterization and Identification                                                           28

3.6.1    Gram Staining                                                                                                             28

3.6.2    Biochemical Tests                                                                                                      28

3.6.2.1 Indole Test                                                                                                                  28

3.6.2.2 Carbohydrate Utilization Analysis                                                                             28

3.6.2.3 Catalase Test                                                                                                               29

3.6.2.4 Oxidase Test                                                                                                               29

3.6.2.5 Coagulase Test                                                                                                           29

3.6.2.6 Citrate Utilization Test                                                                                               29

3.6.2.7 Motility Test                                                                                                               29

3.6.2.8 Methyl Red Test                                                                                                         30

3.6.2.8 Sugar Utilization Test                                                                                                 30

3.6.3    Colonial Morphology of the Bacterial Isolates                                                          30

3.7       Antibiotic Sensitivity Testing                                                                                     30

CHAPTER FOUR

4.0       Results                                                                                                                        32

CHAPTER FIVE

5.0       Discussion, Conclusion and Recommendations                                                        45

5.1       Discussion                                                                                                                   45

5.2       Conclusion                                                                                                                  51

5.3       Recommendations                                                                                                      51

            References

LIST OF TABLES

LIST OF FIGURE

CHAPTER ONE

1.0       INTRODUCTION

Despite foods being an important source of nutrients to consumers, it serves as an excellent medium for microbial Food-borne illnesses are reported to be a major international problem and continue to be a public health concern, especially in developing countries where food standards, regulations and safety policies are not well established or are seldom in place (Mensah et al., 2002). Despite the health risk and widespread concern of food-borne infections, only a fraction of them are diagnosed and reported, or can be traced to a definite source (Lukinmaa et al., 2004).

            Fruits and vegetables are an important part of a healthy and balanced diet in all societies around the globe. They are known to carry natural non-pathogenic epiphytic microbiota but can be contaminated with pathogens from human, animal or environmental sources during growth, harvest, transportation, processing and handling (Beuchat, 2002). While the prevalence of food-borne pathogens on dried fruits and vegetables and their involvement in food-borne outbreaks are not well documented, fresh fruits have been implicated in a number of documented outbreaks of food-borne illnesses particularly in Europe, Japan, United States and Canada (Erickson and Doyle, 2012). The major causes of the illnesses in the outbreaks were bacteria, viruses and parasites.

            Foods, such as fruits and vegetables, are sliced to increase surface-area to volume ratio for the loss of moisture during drying. Slicing is often done in the open with bare hands using a knife. The pieces are then dried in the open on the sun where they can come in contact with microorganisms, dirt, soil and insects and this subjects the products to possible microbial contamination. The food slices are left in full sun for some days until they have lost much of the moisture. Furthermore, some of the dried food products, especially fruits, are often consumed without any further processing steps such as cooking.

Fruits and vegetables are an extraordinary dietary source of nutrients, micronutrients, vitamins and fibre for humans and are thus vital for health and well being. Well balanced diets, rich in fruits and vegetables, are especially valuable for their ability to prevent vitamin C and A deficiencies and are also reported to reduce the risk of several diseases (Kalia and Gupta, 2006).

            Fruits and vegetables are widely exposed to microbial contamination through contact with soil, dust and water and by handling at harvest or during postharvest processing. They therefore harbour a diverse range of microorganisms including plant and human pathogens (Carmo et al., 2004). Differences in microbial profiles of various fruits and vegetables result largely from unrelated factors such as resident microflora in the soil, application of non-resident microflora via animal manures, sewage or irrigation water, transportation and handling by individual retailers (Ofor et al., 2009). In developing countries such as Nigeria, continued use of untreated waste water and manure as fertilizers for the production of fruits and vegetables is a major contributing factor to contaminations (Amoah et al., 2009). Thus despite their nutritional and health benefits, outbreaks of human infections associated with the consumption of fresh or minimally processed fruits and vegetables have increased in recent years (Beuchat, 2002).

            Various pathogens are associated with the contamination of fruits and vegetables, with different outbreaks of gastroenteritis related, associated with the consumption of contaminated vegetables have been recorded at various times (Jeddi et al., 2014). Pathogens implicated in contamination of fruits and vegetables include; Escherichia coli, Salmonella spp., Listeria monocytogenes, Aeromonas spp., Staphylococcus spp., Streptococcus spp., Vibrio spp. and Pseudomonas spp (Senjuti et al., 2014). In developing countries, food borne illness caused by contaminated fruits and vegetables are frequent but rarely properly documented and reported due to many reasons such as poor diagnostic facilities and lack of food borne disease investigation and surveillance. In Nigeria, Hazard Analytic Critical Control Point (HACCP) is not implemented thus, fruits and vegetables are grown with untreated human and animal wastes, transported and sold by local farmers or retail outlets for further processing by street vendors, by families at home or as part of meals eaten in restaurant and other eatery outlets (Eni et al., 2010), with this practice being pointed out to be one of the major contributing factors to high level of contamination of farm produce in Nigeria (Olayemi, 2007). Proper washing of fruits and vegetables is essential for decontamination. Water supplemented with varying concentrations of organic acids, such as acetic, citric and sorbic acids, has been shown to reduce microbial populations on fruits and vegetables. Previous studies revealed that a vinegar dip resulted in a 3 to 6 log10 decrease in the number of aerobic bacteria on parsley leaves, depending on vinegar concentration used and incubation time (Beuchat, 2002).

            The prevalence of antimicrobial resistance among food pathogens has increased during recent decades (Chui et al., 2002). Despite the extensive studies, the antibiotic sensitivity of theses bacteria has not been well studied especially in a developing country. Hence this study apparently determines the antibiogram of bacterial isolates from fruits and vegetables sold in Umuahia.

1.1       AIM

The aim of this study is to determine the antibiogram of fruits and vegetables sold in Umuahia metropolis.

1.1.1    Objectives

·       To determine the total bacterial counts on selected fruits and vegetables samples

·       To isolate various bacteria species associated with fruits and vegetable contamination.

·       To identify the isolated bacteria from fruits and vegetable sold in Umuahia, Abia State.

·       To determine the percentage of occurrence of isolate from fruits and vegetable samples sold in Umuahia, Abia State.

·       To determine the antibiotics susceptibility profile of the various isolates from the fruits and vegetable samples.

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