BACTERIOLOGICAL AND PHYSICOCHEMICAL QUALITY ASSESSMENT OF PACKAGED AND OTHER DRINKING WATER

TABLE OF CONTENTS

Cover Page………………………………………………………………………………i

Fly Leaf………………………………………………………………………………….ii

Title Page…………………………………..……………………………………………iii

Table of Contents ………..............................................................................................viii

List of Figures …….........................................................................................................xv

viii

2.1.6          Heavy metals...................................................................................................................................... 5

2.1.7        Cadmium................................................................................................................................................. 6

2.1.8         Lead......................................................................................................................................................... 6

2.1.9        Iron............................................................................................................................................................. 6

2.1.10    Chromium............................................................................................................................................... 7

2.2         Microbiological Quality of Drinking Water........................................................................ 7

2.2.1      Coliforms (Total and Faecal Coliforms)................................................................................... 7

2.3         Antibiotics Resistance in Bacteria.............................................................................................. 8

2.4     Water Sources in Zaria ….............................................................................................. 9

2.5     Production of Packaged Drinking Water............................................................................... 9

2.6      Microorganisms Associated With Packaged and Other Drinking Water

Sources …………………………………………………………………...... 9

2.6.1     Escherichia coli ………................................................................................................................... 10

2.6.2    Enterococci …………….………...………………………………………….…12

2.6.3   Enteric viruses........................................................................................................................................ 14

2.7      Current Methods and Emerging Approaches for the Detection and

Enumeration of Coliforms in Drinking Water..................................................... 14

CHAPTER THREE....................................................................................................................................... 16

3.0    MATERIALS AND METHODS............................................................................................... 16

3.1    The Study Area …......................................................................................................... 16

3.2    Sampling Area …….......................................................................................................................... 16

3.3    Study Design............................................................................................................................................ 16

3.4    Sample Size............................................................................................................................................... 16

3.5    Collection of Samples......................................................................................................................... 18

3.6    Physicochemical Analysis of Water Samples …............................................................... 18

3.6.1    Temperature, pH_, electrical conductivity and total dissolved solids........................ 18

3.6.2    Dissolved oxygen and biological oxygen demand............................................................ 19

3.6.3    Nitrate …................................................................................................................................................ 19

3.6.4    Sulphate................................................................................................................................................... 20

3.6.5    Determination of heavy metals in the water samples........................................................ 20

ix

3.7 Bacteriological Analysis of Water Samples Using Membrane Filter (MF)

Technique ………………………………………………………………...21

3.7.1     Enumeration of Escherichia coli using membrane lactose glucuronide agar

(MLGA)………………...…………………………………………….........21

3.7.2    Enumeration of total coliforms ……......................................................................................... 22

3.7.3    Enumeration of faecal coliforms ….......................................................................................... 22

3.7.4    Enumeration of thermotolerant (faecal) Escherichia coli …......................................... 23

3.7.5    Enumeration of enterococci........................................................................................................... 23

3.7.6    Enumeration of total heterotrophic bacteria or total viable count (TVC).............. 23

3.7.7    Isolation of Escherichia coli O157:H7 from water Samples ...................................... 24

3.8    Biochemical Characterization of Isolates by Conventional Methods................... 25

3.8.1    Indole test…….................................................................................................................................... 25

3.8.2    Methyl red-voges proskaeur (MR-VP) test........................................................................... 25

3.8.3    Citrate test ……................................................................................................................................... 25

3.8.4    Litmus milk decolorization test to identify Enterococcus spp..................................... 26

3.8.5    Aesculin hydrolysis test to identify Enterococcus spp.................................................... 26

3.8.6    Oxidase test............................................................................................................................................ 26

3.9 Biochemical Identification of presumptive Escherichia coll isolates using

Microgen™ GnA-ID Kit …….................................................................................... 26

3.10 Biochemical Identification of presumptive Enterococcus species isolates using

Microgen™ STREP-ID...................................................................................................... 27

3.11    Serological Identification of Presumptive E. coli O157............................................... 28

3.12    Antibiotics Susceptibility Testing …..................................................................................... 28

3.12.1 Measurement of inhibition zone diameter............................................................................... 29

3.12.2 Multi-drug resistance index ……................................................................................................ 29

3.13   Molecular Characterization of Escherichia coli O157:H7 and Enterococcus

species……………………………………………………………………...29

3.13.1    DNA extraction using extraction kit …….......................................................................... 29

3.13.2    DNA quantification using NanoDrop.................................................................................... 29

3.13.3   DNA amplification and detection............................................................................................. 31

3.13.4   Electrophoresis of PCR amplicons............................................................................................ 31

x

3.14   Statistical Analysis …...................................................................................................................... 31

CHAPTER FOUR …….............................................................................................................................. 34

4.0 RESULTS ….............................................................................................................................................. 34

4.1 Physicochemical Properties of the Packaged and other Drinking Water Sources

in Zaria, Kaduna State, Nigeria..................................................................................... 34

4.2 Heavy Metal Content of the Drinking Water Sources and Packaged Water Sold

in Zaria, Kaduna State........................................................................................................ 44

4.3 Bacteriological Quality of the Packaged and other Drinking Water Types in

Zaria Kaduna State, Nigeria............................................................................................ 48

4.3.1   Effects of seasonal variations on the bacteriological quality of the packaged and

other drinking water sources in Zaria.............................................................................. 65

4.4 Biochemical Characterization and Identification of Isolates......................................... 69

4.5 Antibacterial Susceptibility Profile of Bacteria Isolates................................................... 74

4.6   Molecular Characterization of Isolates.................................................................................... 82

CHAPTER FIVE............................................................................................................................................. 91

5.0 DISCUSSION ……........................................................................................................... 91

5.1 Physicochemical Parameters of the Packaged and other Drinking Water

Sources in Zaria ………………………………...………………………..91

5.2   Heavy Metal Content of the Packaged and other Drinking Water Sources in

Zaria................................................................................................................................................ 94

5.3   Bacteriological Quality of the Packaged and other Drinking Water Sources in

Zaria................................................................................................................................................ 96

5.4   Antibacterial Susceptibility Profile of the Bacteria Isolates...................................... 100

5.5    Molecular Characterization of Isolates by the Polymerase Chain Reaction

(PCR) Technique................................................................................................................. 102

CHAPTER SIX …………....................................................................................................................... 104

6.0 SUMMARY, CONCLUSION AND RECOMMENDATIONS.............................. 104

6.1 Summary………………..….…………………………………………………….104

6.2 Conclusion…………………..……………………………………………………106

6.1   Recommendations............................................................................................................................... 107

REFERENCES …….............................................................................................................................. 110

APPENDICES ………............................................................................................................................. 117

xi

xii

Table                                                                                                                                                                     Page

xiii

Table                                                                                                                                                                     Page

xiv

xv

LIST OF PLATES

Plate                                                                                                                                                                     Page

I:                      Membrane Lactose Glucuronide Agar Plate of Sachet water brand B.......... 51

II:                     Agarose gel electrophoresis plate of the extracted DNA..................................... 83

III:                           Agarose gel electrophoresis plate showing the presence of ENTl conserved

region of the Enterococcus species      85

IV:                 Agarose gel electrophoresis plate showing the presence of VANR gene in

Enterococcus spp 645bp……............................................................................................... 86

V:                   Agarose gel electrophoresis plate showing the presence of extended-

spectrum β-lactamase CTX-M gene in Escherichia coli O157:H7. 894bp..87

VI:                   Agarose gel electrophoresis plate showing the presence of extended-

spectrum (β-lactamase TEM gene in Escherichia coli O157:H7 198bp......... 88

VII:                Agarose gel electrophoresis plate of Escherichia coli O157:H7 shiga toxin

STX1 Gene …..………….................................................................................................... 89

VIII:              Agarose gel electrophoresis plate of Escherichia coli O157:H7 shiga toxin

STX2 gene ……..................................................................................................................... 90

xvi

LIST OF APPENDICES

Appendix                                                                                                                                                              Page

1:                                Biochemical Characterization and Identification of Presumptive Isolates of

Escherichia coli O1517:H7 using the Microgene® GnA-ID system........... 117

II:                              Biochemical Characterization and Identification of Enterococcus spp isolated from water samples in Zaria using the microgene Strep-ID system ……………………………………………………………………118

III :                Microgen™ GnA-ID Kit For the identification of Escherichia coli .......... 119

IV:                  Microgen™ STREP-ID for the identification of Enterococcus species...... 119

V:                                Work in progress for the Heavy Metal Analysis at the Multipurpose

laboratory of  Ahmadu Bello University, Zaria....................................................... 120

VI:                   Site 5 Children drinking well water.............................................................................. 121

VII:                Site 2 Children fetching water from hand-operated borehole ….................. 121

VIII:               Site 5 Filtration units in a plastic bagged sachet drinking water production

Factory………………………………………….………………... ………121

xvii

ABSTRACT

Continuous increase in the sale and indiscriminate consumption of packaged drinking water and untreated water types such as borehole and well waters in Nigeria is of public health significance. Three hundred samples comprising 120 sachets, 60 bottled water brands, 60 borehole and 60 well water samples from five sampling sites in Zaria, North Western Nigeria were analysed microbiologically and physicochemically using standard procedures. Membrane filter technique was used to detect the presence of bacterial indicators of water quality as well as specific pathogens such as Escherichia coli O157:H7 and Enterococcus spp. The research was conducted between June 2014 to February 2015 during the wet and dry seasons. Isolated pathogens were characterized conventionally, using Microgen^TM ID Kits and molecularly by Polymerase Chain Reactions (PCR). The nitrate level in samples of the wells (100%) and borehole (40%) water were above the United States Environmental Protection Agency (USEPA) minimum contamination level of 10mg/l. Cadmium (Cd) and Lead (Pb) contents of boreholes, wells, and brands of sachet waters were above the minimum permissible limits set by NAFDAC (0.003mg/l and 0.01mg/l respectively). However, Lead (Pb) was not detected in the bottled water brands sampled. Total coliforms and Escherichia coli were detected in sachet water brands (80%), bottled water brands (20%), borehole (100%) and well water (100%). Enterococci were recovered from sachet water brands (70%), borehole (100%) and well water (100%). There were no statistically significant differences (P≤0.05) between the total coliform counts of the sachet water brands and borehole water in Zaria, therefore, the purity of sachet water as claimed by the manufacturers is doubtful. Bacteriological counts were higher during the wet season than dry season. Antibiogram of Escherichia coli O157:H7 (80%) and Enterococcus spp (37.6%) isolates showed multiple antibiotics resistance (MAR) with MAR indices of 0.3 and above. Polymerase chain reaction (PCR) amplified some housekeeping genes such as tuf gene of Enterococcus genus and antibiotics resistance genes such as glycopeptides Vancomycin and Teicoplanin resistance gene VANR. Escherichia coli O157:H7 extended spectrum β-lactamase genes: bla-TEM and bla-CTX-M genes. Most of the sachet water brands fell below NAFDAC and WHO drinking water standards and are therefore of doubtful quality and need strict adherence to standards. Efforts need to be intensified in the monitoring of activities in this rapidly expanding industry with a view to raising standards while government at all levels in Nigeria should take the issue of supply of adequately treated water to the public as an essential public service.

xviii

CHAPTER ONE

1.0                                                                         INTRODUCTION

Water is a simple molecule, consisting of two hydrogen atoms bonded to an oxygen atom with molecular formula H₂O (Bassem, 2013). It is one of the most important chemical substances for the sustenance of life and is vital for all known forms of life on Earth; It constitutes about 75% of the Earth`s surface (UN, 2005; EL-Jakee et al., 2009; Hongyue et al., 2013). In terms of sheer volume, About 97.5% of all the water on Earth is salt water, only 2.5% of all the water on Earth is fresh water and 98.8% of that fresh water is frozen in Antarctica and Greenland icecaps or lies too deep underground to be accessible; only 1.2% of the Earth’s freshwater is available for withdrawal and human use (Shiklomanov, 2000; UN, 2005; Bassem, 2013). However, this is continually being polluted by various anthropogenic activities thereby further reducing the available freshwater for human use. The drinking water of most communities including Zaria in Nigeria is obtained from various sources: boreholes, rivers, streams, and well waters. Source water contamination poses a risk to public health and increases the cost of drinking water treatment.

The threats posed by deteriorating water quality caused by among other things, the contamination of potable water sources have led the public to seek for alternative potable water sources. Although access to safe and reliable sources of drinking water is a global challenge, it is particularly acute in developing countries, including Nigeria (Ivey et al., 2006).

The production, sale and consumption of plastic bagged drinking water has grown tremendously over the years in many developing countries such as Nigeria. The plastic bagged drinking water was introduced into the Nigerian market as a less expensive means of accessing drinking water than bottled water.

1

1.1         Statement of Research Problem

Estimate of the global burden of water associated human diseases provide a simple index hiding a complex reality. World Health Organization (WHO) estimates that worldwide some 2.2 million people die each year from diarrhoeal related diseases. For an estimated 88% of diarrhoeal cause, the underlying cause is unsafe water, inadequate sanitation and poor hygiene (WHO, 2008; Mosier et al., 2012). Water borne diseases continue to be one of the major health problems in developing nations, including Nigeria especially on the issues of safe drinking water quality (Mead et al., 1999). Water borne diseases account for one third of the intestinal infection worldwide (Hunter and Syed, 2001; Offre et al., 2011).

The high prevalence of diseases such as diarrhoea, typhoid fever, cholera, and bacillary dysentery among the populace have been linked to the consumption of unsafe water and unhygienic drinking water production practices (Mead et al., 1999; Park et al., 2010). Pathogens such as Salmonella species, Shigella species, Vibrio cholerae and E. coli being seen in human and animal faeces ultimately find their way into water supply through seepage of improperly treated sewage into ground water, (Dipaola, 1998; Wei et al., 2011).

1.2     Justification for the Study

Most bottle water manufacturers in Nigeria also engage in sachet water packaging and obtain their raw water mostly from local, municipal piped or well water, however, adherence to production and analytical standards are doubtful as most of the factories are observed to lack the appropriate technology for achieving these (Oyedeji et al., 2013). Diseases related to contamination of drinking-water constitute a major burden on human health and interventions to improve the quality of drinking-water provide significant benefits to health (UNDP, 2006). The potential health consequences of microbial contaminations are such that its control must always be of paramount importance and must never be compromised (Mead et al., 1999).

2

1.3 Research Hypothesis

H_O: Packaged and other drinking water sources in Zaria do not meet the bacteriological quality standard.

H_O: Drinking water sources do not contain Escherichia coli O157:H7.

H_O: The target pathogenic bacteria are not resistant to the antibiotics tested.

H_O: Escherichia coli O157:H7 and Enterococcus spp isolated do not possess the virulence shigatoxin genes (Stx1 and Stx2) and antibiotics resistance genes (CTX-M, TEM) and vanr.

1.4    Aim

The aim of this study was to carry out bacteriological and physicochemical quality assessment of packaged and other drinking water sources in Zaria, Kaduna State, Nigeria.

1.5     Objectives were to:

1.       Determine the bacteriological quality and physicochemical properties of packaged and other drinking water sources.

2.       Isolate and characterize Escherichia coli O157:H7 and Enterococcus spp in the water samples

3.       Determine the antibiotic susceptibility patterns of the isolates to commonly used antibiotics and screen for the presence of multiple antibiotic resistance strains.

4.       Screen the Escherichia coli O157:H7 for the possession of extended spectrum β-lactamases genes (bla-CTX-M and bla-TEM) and virulence shigatoxin genes (Stx1 and Stx2) using the polymerase chain Reaction (PCR) technique.

5.       Confirm Enterococcus using genus specific primer and detect the Vancomycin

resistance gene (VANR) in Enterococcus spp. isolated.