Isolation and Characterization of Aeromonas SPP. from Amarantus SPP. and Irrigation Water

ABSTRACT

This study was carried out to isolate, characterize and identify Aeromonas spp. from Irrigation water and vegetables. Water and vegetable samples were collected and analyzed for physicochemical and microbiological analysis.

 Physicochemical parameters such as pH, Temperature, Total Dissolved Solid (TDS), Dissolved Oxygen and Biological Oxygen Demand were determined using standard methods for water samples only. Microbiological parameters such as Total Coliform count (TC), Faecal Colifom count (FC) and Heterotrophic Plate Count (HPC) were determined using spread plate for both water and vegetable samples. Antibiotic Susceptibility pattern were determined using double disc diffusion method.

Heterotrophic plate count, total coliform count and faecal coliform count were analyzed using R2A, m-Endo and m-Fc agar respectively. Spread plate technique was employed for isolation of Aeromonas spp. from water and vegetable samples using Aeromonas agar base. Aeromonas spp. isolated were identified using conventional biochemical tests. Antibiotic susceptibility pattern of the isolates were determined using disc diffusion method. A total of 13 water and vegetable samples were collected. Physicochemical studies shows that the highest mean pH of 7.6±0.00 was recorded in the first sampling while the lowest pH value of 6.55±0.05 was recorded in the second sampling. Total Dissolved Solid (TDS) had the highest value of 201±0.00mg/mL in the fourth sampling at Ede road and the lowest TDS value recorded was 152±0.31mg/mL in the second sampling, also at Ede road. The highest mean temperature was 36.00±0.00OC recorded three times, twice at Ede road, in the second and fifth sampling and once at Opa in the second sampling. The lowest mean DO recorded was 2.50 0.06mg/L from the first sampling while the highest mean DO recorded was 6.50mg/L from the first sampling in Opa and Ede road respectively. The lowest mean BOD recorded was 1.97 0.05mg/mL recorded at fifth sampling at Ede road while the highest mean BOD recorded was 2.78±0.57mg/mL from Ede road during the third sampling. The mean HPC of the water samples obtained at the Ede road range from 0.97 0.27 to 3.40±0.05CFU/mL, for vegetable sample, HPC range was between 1.23±0.15 to 3.4±0.58CFU/mL while that of the water samples obtained from Opa were between the range of 2.10±1.20 CFU/mL and 3.77±0.05 CFU/mL, for vegetable sample, the HPC range was between 0.9±0.51to 3.33CFU/mL. The mean TC of the water samples obtained from Ede road range from 1.97±0.07CFU/mL to 2.95±CFU/mL and between 0.10±0.12 to 1.86±0.54CFU/mL for vegetable samples, while the TC obtained from Opa was between the range of 0.05±0.01 CFU/mL and 3.09±0.47 CFU/mL, and between 0.12±0.00 to 3.30±0.44CFU/mL for vegetable samples. The mean of the FC obtained at the Ede road range from 1.10±0.15CFU/mL and 2.39±0.05CFU/mL. For vegetable samples, FC range was between 0.48±0.12 to 3.44±0.84CFU/mL while that of the water samples obtained from Opa were between the range of 0.15±0.04 CFU/mL and 3.33±0.01CFU/mL. For vegetable sample, FC range was between 0.1±0.00 to 3.45±0.04, which is higher than the acceptable standard. A total of 17 Aeromonas isolates were obtained from water and vegetable samples, 94%, 94%, 94%, 29%, 94%, 94%, 94% of different aeromonas isolates were resistant to Cefuroxime, Nitroimidazole, Gentamycin, Ciprofloxacin, Cefaroxine and Cetazidine respectively. 100%, 6%, were susceptible to Ofloxacin and Augmentin respectively. This study reveals that’s both irrigation water sources (Ede road and Opa) are contaminated and not suitable for irrigation purposes.

TABLE OF CONTENTS

Title Page                                                                                                                    i

Certification                                                                                                                ii

Dedication                                                                                                                  iii

Acknowledgement                                                                                                       iv

List of Tables                                                                                                               v

List of Figures                                                                                                              vi

Abstract                                                                                                                      vii

Table of Content                                                                                                         viii

CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW                                                            1

1.1 Introduction                                                                                                                      1

   1.1.1 Aims and Objectives                                                                                       3

1.2 Literature Review                                                                                                 4

      1.2.1 Irrigation                                                                                                      4

      1.2.2 Problems of Irrigation                                                                                  4

      1.2.3 Vegetables                                                                                                   5  

      1.2.4 Aeromonas species                                                                                                                           6

   1.2.5 Clinically Important Aeromonas species                                                        7

   1.2.6 Epidemiology of Aeromonas species                                                              8

   1.2.7 Clinical Infections caused by Aeromonas spp.                                               9

      1.2.7.1 Gastrointestinal Infections                                                                        9

      1.2.7.2 Extraintestinal Infections                                                                          10

CHAPTER TWO

MATERIAL AND METHODS

2.1 Materials                                                                                                               11

2.2 Culture                                                                                                                  11

2.3 Reagents                                                                                                               11

2.4 Preparation of Media and Sterilization of Materials                                             11

2.5 Sample Collection                                                                                                 12

      2.5.1 Description of Study Area                                                                           12

       2.5.2 Water Collection                                                                                         13

         2.5.3 Vegetables Collection                                                                               13

         2.5.4 Handling and Transportation                                                                    13

2.6 On-Site Parameters                                                                                               13

        2.6.1 Surface water temperature                                                                         13

         2.6.2 pH                                                                                                             14

       2.6.3 Total Dissolved Solids (TDS)                                                                     14

2.7 Off-Site Assessment Parameters                                                                          14  

         2.7.1 Dissolved Oxygen (DO)                                                                           15 

        2.7.2 Biological Oxygen Demand (BOD)                                                          16

2.8 Microbiological Analysis                                                                                      17

       2.8.1 Enrichment of Medium                                                                               17

        2.8.2 Water                                                                                                         17

        2.8.3 Vegetable Sample                                                                                      17

        2.8.4 Spread plate techniques                                                                             18

        2.8.5 Heterotrophic plate count                                                                          18

        2.8.6 Total Coliform                                                                                           18

        2.8.7 Faecal Coliform                                                                                         19

2.9 Biochemical Tests                                                                                                 19

     2.9.1 Gram staining                                                                                                19

     2.9.2 Oxidase Test                                                                                                 20

     2.9.3 Salt Tolerance Test                                                                                        20

     2.9.4 Indole Test                                                                                                    20

     2.9.5 Urease Test                                                                                                   20

     2.9.6 Catalase Test                                                                                                 20

     2.9.7 Simmons Citrate Test                                                                                    21

     2.9.8 Motility Test                                                                                                 21

     2.9.9 Gas of Glucose                                                                                             21

     2.9.10 Voges Proskauer Test                                                                                 22

     2.9.11 Lysine decarbohyxylase Test                                                                      22

     2.9.12 Arginine decarbohyxylase Test                                                                   22  

3.0 Antiobiotic Sensitivity                                                                                          22

CHAPTER THREE

3.1 Sample Collection and Site                                                                                   24

3.2 Sanitary Survey                                                                                                     24

3.3 Physico-Chemical Parameters of Water Collected from both Sampling Sites     24

      3.3.1 pH                                                                                                                 24

       3.3.2 Temperature                                                                                                 24

       3.3.3 Total Dissolved Solid                                                                                 24

       3.3.4 Biological Oxygen Demand                                                                       25

3.4 General Bacteriological Analysis of Water                                                           27

     3.4.1 Heterotrophic Plate Count                                                                            27

     3.4.2 Total Coliform                                                                                              27

     3.4.3 Faecal Coliform                                                                                            27

3.5 Morphological and Biochemical Characteristics of Isolates                                 30

3.6 Antibiotic Susceptibility Pattern of Isolates                                                         30

3.7 Correlation between HPC in Water and in Vegetable ae Opa                             33

3.8 Correlation between HPC in Water and in Vegetable at Opa                              33

3.9 Percentage Occurrence of Isolates                                                                        36

CHAPTER FOUR

DISCUSSION AND CONCLUSION

4.1 Discussion                                                                                                             40

4.2 Conclusion                                                                                                             44

4.3 Recommendation                                                                                                  44

4.4 Appendix I                                                                                                            52

4.5 Appendix II                                                                                                           53

4.6 Appendix III                                                                                                         57