Bacteriological contamination of water and associated risk

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CHAPTER THREE RESEARCH DESIGN AND METHODOLOGY

Introduction

This chapter describes the research design and methodology used to provide data to investigate the research questions. The research was executed in three phases: Phase 1 involved gathering of information of drinking water sources in the study area, sampling sites observations, water sampling, coliforms and E. coli counts analysis and phenotypic characterization of bacteria. Phase II involved antimicrobial susceptibility testing, molecular characterization of E. coli and the determination of virulence genes associated with multiple antibiotic resistant E. coli. Phase III involved determination of the radiation sensitivity (D₁₀) of the multidrug resistant E. coli isolates. The chapter then ends with a description of data management and statistical analysis employed; finally, with consent and ethical considerations issues.

Demarcation of the study area

The Dangme West District is situated in the southeastern part of Ghana, lying between longitude 5° 45‘ south and 6° 05‘ north and longitude 0° 05‘ east and 0° 20‘ west. The District has a total land area of 1,442 square kilometers, making it the largest in the Greater Accra Region. The land size represents 41.5% of the regional land area. The District forms part of sixteen (16) metropolitan, municipal and districts in the Greater Accra Region of Ghana.
The administrative capital of the district is Dodowa. The Dangme West District is completely rural. It shares boundaries with North Tongu District to the northeast, Yilo and Manya Krobo Districts to the northwest, Akwapim North District to the west, Tema Metro to the southwest, Dangme East District to the east and the Gulf of Guinea to the south. The District has a 37 Km Coastline and a 17km stretch of the Volta River (TDWD, 2013). Figure 3.1 shows the map of the Dangme West District of Ghana.

Geographical description of the study area.

Topography and drainage

The district forms the central portions of the Accra plains. The relief is generally gentle and undulating, a low plain with heights not exceeding 70 metres. The plains are punctuated in isolated areas by a few prominent inselbergs, isolated hills, outliers and knolls scattered erratically over the area. Prominent relief features include the Yongua inselberg (427 metres) which appears conical in the air with a number of outliers close to the north of the district around Asutsuare and Osuwem areas; the Krabote inselberg also to the north and the Shai Hills (289 metres) found towards the western portions of the district (MLGRDE, 2008).
Large rock outcrops and boulders conspicuously stand in the vicinity of the hills in certain places. The rocky hills together with the large boulders provide immense potentials for stone quarrying, which is already a major pre-occupation in the district. The general pattern of drainage in the Dangme West District is dendritic with most of the streams taking their source from the Akwapim range, which also serves as a watershed and then flow in a northwest to southwest direction into lagoons on the coast. Flowing over a fairly low terrain, most of the streams have carved wide valleys for themselves, which are left dry for most parts of the year. Rainfall is generally very low with most of the rains, very erratic in nature and coming mostly between September and November. Mean annual rainfall increases from 762.5 milliliters on the coast to 1220 milliliters to the North and Northeast close to the foothills of Akwapim Range and on the summit. (MLGRDE, 2008)

Climate and Vegetation

The southeastern coastal plain of Ghana, which encompasses the Dangme West District, is one of the hottest and driest parts of the country. Temperatures are however subjected to occasional and minimal moderating influences along the coast and altitudinal influences affected by the Akwapim range in the northwest. Temperatures are appreciably high for most parts of the year with the highest during the main dry season (November – March) and lowest during the short dry season (July – August). They average a few degrees lower on the coast and close to the Akwapim range than they do over most of the plains. The absolute maximum temperature is 40 °C (MLGRDE 2008).
The predominant vegetation type found in the district is of the short grass savannah interspersed with shrubs and short trees, a characteristic of the Sub-Sahel in type. A large portion of vegetation, particularly towards the south, remains dry for most parts of the year except for the short rainy season. The ravaging effects of seasonal bushfires that sweep across most parts of the district especially during the dry season further depreciate the quality of the vegetation.
Along some stream courses, however, higher vegetation type ranging from thickets to light forest is common. Some light forest with tall trees is also found along the foothills of the Akwapim Ridge especially around Dodowa, Ayikuma and Agomeda areas. There is a Forest, Game and Wildlife Reserve around the Shai hills (MLGRDE, 2008).

Geology and soil

Ancient igneous rocks underlie the major part of the district. Strongly metamorphosed ancient sediments occur along the western boundary. There are also important areas of relatively young unconsolidated sediments in the south and southeast. Dahomeyan gneiss and schists occupy most of the plains proper. Basic gneiss forms a number of large inselbergs (isolated rocky hills) in the north and center of the belt. Small rock outcrops are also common in the north close to the inselbergs but are rare in south and southeast. The eastern belt of acidic gneiss consists mainly of the grained metamorphosed rocks rather richer in minerals than the rocks in the western belt (MLGRDE, 2008).
The predominant soil types in the district are the black clays classified as Akuse series and occupy the central to eastern parts of the district. The soils are highly elastic when wet but become hard and compact when dry and then crack vertically from the surface. This renders the soil unsuitable for land cultivation. The soil in the district consists of gray-brown soils loamy for about 15-30 centimeter of the surface.. The topsoil rapidly becomes draughty during the dry seasons. This type of soil fairly supports any level of crop production. Most parts of the area are, however, left for grazing purposes (MLGRDE, 2008).

Water sources and sanitation

One of the major bases for the experimental design of the current study is the water sources in the district. The supply of potable water in the district is woefully inadequate and only few sections of the district have regular supply of pipe-borne water. Analysis of the current water and sanitation in the district shows that more effort is needed to meet the 85% water and sanitation coverage. On the basis of the National Community Water and Sanitation Standards of 600 people per Stand pipe, 350 persons per borehole and 150 persons per hand dug well, the district has achieved about 66% coverage with 34% of the population lacking access to potable water supply. As it stands now, below 37 % of the district population have access to pipe-borne water or tanker services, whilst 6 % use well or borehole. The remaining population in the district depends on untreated water from sources such as ponds, dams, rivers, streams, dugouts and periodic harvesting of rainwater. This means that about 57 % of the settlements do not have access to potable water. Visually the water from the streams and dams are light brownish yellow caused by mostly decayed dead leaves. If turbulently disturbed it turns to deep brownish yellow and some suspended soils can be seen. With the projected population of 118,500 as at 2010, the district would have to increase investment in potable water supply by the provision of 67 Stand pipes or 115 boreholes or 268 hand dug wells to achieve one hundred percent potable water coverage in 2010 and provide 40,290 people with safe water (MLGRDE 2008).
In the area of improved sanitation, the coverage of not more than 10% in the district implies that the District Assembly in collaboration with development partners would have to double its effort to meet the needs of about 90% of the people. About 300 household latrines and hundreds of water closet toilets are needed to meet the sanitation needs of the people. This should also include hygiene and environmental education to address the behavioural needs of the people especially in the area of open defecation (MLGRDE, 2008

 Motivation for the selection of study site

To meet the study objectives, research was conducted at various sites within the Dangme West District of Ghana. The district was selected for a number of reasons. It is the largest district in the Greater Accra Region of Ghana. The district is a typical rural setting, whose composition and geography is largely representative of most rural settings in Ghana. The water source by which the research design is centered perfectly suits the conditions in the district. Majority of the population in the district depends on untreated water from sources such as dams, rivers, and streams. Increases in water scarcity are expected to grow in the district, due to the current district growth rate of 2.1% per annum. The district has recorded a number of water borne diseases, this allows for investigations of risk associated with bacteriological contaminants of water sources.

Quality assurance

Sterilization and disinfection

The basic function of an infection control procedure in an environment that is essentially non-sterile is to effectively break the chain of cross-contamination and cross-infection. Therefore, the definitive methods of avoiding microbial cross contamination are to sterilize and disinfect all equipment and materials used for laboratory based microbiological analysis. In pursuance of this, all materials were sterilized and disinfected using standardized procedures. All glassware such as petri dishes and test tubes were washed and rinsed with water and air-dried. They where then placed in a sterilizing oven, face up for two hours at 160 ⁰C. The working benches were also cleaned and disinfected, swabbing the working surface with 70% alcohol to prevent the introduction of contaminants. The laboratory benches were sterilized before and after use. It was ensured that nothing unsterile came into contact with the laboratory benches. Small pieces of equipment such as glass rods and metal tools were sterilized by dipping them in 70% ethanol (alcohol) and then flaming to burn off the alcohol. Sterilization of inoculating loops and needles were done by holding in a flame until red-hot.
During bacteriological cultures, inoculum transfers were aseptically carried out to prevent contamination of the media used. The inoculation loops were sterilized as described above before and after transfer of samples. The opening ends of all test tubes containing media to be used as well as those containing various samples were also flamed before and after transfers.

Equipment calibrations

All equipment used for the study were properly calibrated. These included pH meters, incubators, water bath, and autoclave. The pH electrodes were calibrated before every set of measurements by using any one (single-point calibration) of the WTW technical buffer solutions (pH values at 25 °C: 2.00 / 4.01 / 7.00 / 10.01). After the calibration, the electrode was thoroughly rinsed with deionized water before the sample measurements were taken.

 Media preparation

The various media used for the study were prepared from dehydrated stocks according to the manufacturer‘s instructions. All media were prepared with distilled water, and pH adjustments were made by using a pH meter (Hanna model). All solid media prepared were sterilized by autoclaving at 121⁰C for 15 minutes. It was then well mixed, dispensed aseptically into sterilized petri dishes and allowed to gel before sterility testing. Durham tubes were placed in, test tubes containing prepared broth (e.g. MacConkey) before autoclaving as described above

Media sterility testing

Media sterility test as a measure of quality assurance was carried out to verify freedom from contamination; demonstrate the correct performance of the medium used and ensure against physical or chemical imperfections. All prepared media were aseptically dispensed and incubated at 37⁰C for 24 hrs, before inspection. Inspection for physical imperfection included; uneven distribution of media; variable amounts of medium in petri dishes/tubes/bottles; gross deformation of surface of the media. All media that did not meet the required standards were discarded.

Dedication
Declaration 
Acknowledgement
Abstract 
CHAPTER ONE: GENERAL INTRODUCTION
1.1 Water quality and contamination
1.2 Biological contaminants of water
1.3 Antibiotics in water
1.4 Classification of antibiotics
1.5 Antibiotics and emerging resistances
1.6 Development of resistances
1.7 Mechanism of resistance
1.8 Spread of antibiotic resistance
1.9 Detection methods for waterborne pathogens and antibiotic resistance genes gemne genes
1.10 Antimicrobial resistance among indicator organisms and environmental pathogens
1.11 Radiation sensitivity (D10)
1.12 The statement of problem, rationale, and motivation
1.13 Research questions
1.14 Research objectives
1.14.1 General objectives
1.14.2 Specific objectives
1.15 The justification of the research
1.16 Expected outcome
CHAPTER TWO: LITERATURE REVIEW AND THEORETICAL FRAMEWORK
2.1 Introduction
2.2 Bacteriological contamination of water and associated risk
2.3 Bacteria resistance to antibiotics
2.4 Methods of detection and characterization of antibiotic resistance bacteria
2.5 Studies on radiation sensitivity (D10)
2.6 Theoretical framework of the study
CHAPTER THREE: RESEARCH DESIGN AND METHODOLOGY
3.1 Introduction
3.2 Demarcation of the study area
3.3 Geographical description of the study area
3.4 Motivation for the selection of study site
3.5 Quality assurance
3.6 Water sampling
3.7 Coliform populations analysis: MPN Technique
3.8 Storage of bacteria isolates
3.9 Phenotypic identification and characterization of bacteria isolates
3.10 Biochemical testing
3.11 API Analysis
3.12 Anti-bacteria susceptibility testing of E. coli
3.13 Identification of multiple antibiotic resistance (MDR)
3.14 MAR (multiple antibiotic resistance) index study
3.15 Genotypic characterization of E. coli
3.16 Determination radiation sensitivity (D10) of E. coli
3.17 Data management and analysis
3.18 Consent and ethical considerations
CHAPTER FOUR: RESULTS
4.1 Introduction
4.2 Bacteriological contamination of water and associated risk.
4.3 Antibiotic Resistant Profile of E. coli isolates
4.4 Multiple antimicrobial resistance (MAR) index profiles of E. coli isolates
4.5 Distribution and diversity of E. coli virulence factors
4.6 Comparison of methods for detection of E. coli
4.7 Association between antibiotic resistance and radiation sensitivity (D10)
CHAPTER FIVE: DISCUSSIONS
5.1 Introduction
5.2 Bactria contamination of water: coliforms and bacteriological profiles
5.3 Prevalence and susceptibility profiles of antibiotic resistant water-borne E. coli
5.4 Virulence genes associated multiple antibiotic resistant E. coli
5.5 Comparison of three laboratory based techniques for detection of E. coli
5.6 Association between antibiotic resistance and radiation sensitivity (D10)
CHAPTER SIX: CONCLUSIONS, LIMITATIONS OF THE STUDY AND RECOMMENDATIONS
6.1 Conclusions
6.2 Limitations of the study
6.3 Recommendations
REFERENCES 
APPENDIX
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RADIATION SENSITIVITY AND MOLECULAR CHARACTERIZATION OF WATER- BORNE MULTIDRUG RESISTANT ESCHERICHIA COLI