TABLE OF CONTENT
CHAPTER
ONE
INTRODUCTION AND LITERATURE
REVIEW
1.0
Introduction
Aims
and Objectives
1.1 Literature Review
1.1.1 Water
1.1.2 Pollution of Water
1.1.3 Quality of Water
1.1.3.1 Physicochemical parameters
1.1.3.2 Microbiological Quality
1.1.4 Escherichia
coli as an indicator of faecal contamination
1.1.5 Characteristics of Indicator Organisms
1.1.6
Antibiotics
CHAPTER TWO
MATERIALS AND METHODS
2.0 LIST OF MATERIALS
2.1 STUDY AREA DESCRIPTION
2.1.1 STUDY AREA SELECTION
2.2 SAMPLE COLLECTION
2.3 Sterilization of glass wares and media,
and disinfection
2.4 Media and Preparation
2.5 PHYSICO-CHEMICAL ANALYSIS OF WATER
SAMPLES
2.6 Bacteriological Analysis of Water samples
2.6.1 Serial dilution
2.6.2 Standard Plate count
2.6.3 Standard Multiple Tube Fermentation
Technique
2.7 CHARACTERIZATION AND IDENTIFICATION OF ISOLATES
2.7.1 Cultural Characteristics of Isolates
2.7.2Morphological
Characteristics of Isolates
2.7.3 Microscopy
2.8 Biochemical Tests
2.8.1 Oxidase
Test
2.8.2 Sugar
Fermentation
2.8.3 Indole
Test
2.8.4
Methyl
Red- Voges Proskauer Test (MRVP)
2.8.5 Citrate Utilization Test
2.8.6 Catalase
Test
2.9 ANTIBIOTIC SENSITIVITY
CHAPTER THREE
3.0
RESULTS
3.1 Physicochemical analysis of sample
3.2 Results
of Most probable number
3.3 Results
of HPC, TC and FC count
3.4 Probable
identities of bacteria isolates
3.5 Antibiotic
Sensitivity
CHAPTER
FOUR
DISCUSSION
CHAPTER FIVE
5.0 CONCLUSION
REFERENCES
APPENDIX
I
CHAPTER
ONE
INTRODUCTION AND LITERATURE
REVIEW
1.0
Introduction
Water
is a natural resource and is essential to sustain life. Accessibility and
availability of fresh clean water does not only play a crucial role in economic
development and social welfare (Odonkorand Ampofo,2013) but also, it is an
indispensable liquid. Many do not have access to safe and clean water and many
die of waterborne bacterial infections.Hence, having it available in sufficient
quantity and quality contributes to the maintenance of health (Nougang et al., 2011).According to WHO (2004),
about 80% of all diseases and over one third of deaths in developing countries
are caused by drinking contaminated water.
Water
is essential to human life, for basic health and survival, as well as food
production and economic activities. Presently, theworld is facing a global
emergency in which over one billion people lack access to a basic supply of
clean water and over 2billion do not have access to adequate sanitation which
is the primary cause of waterborne disease (WHO, 2003).Water aids in digestion
and is essential in almost all other body processes. It makes up more than two
thirds of human body weight, and without water, there is no life. The human
brain is made up of 95% water, blood is 82%, and lungs 90%.Water helps in maintaining
the moisture of internal organs of the body, the normal volume and consistency
of fluids such as blood and lymph as wellas in the regulation of body
temperature, removal ofpoisons or toxins from the body through
urine, sweat and breathing; and the regulation of the normal structure and
functions of the skin. The body loses about four litres of water every day. It
is therefore necessary to replenish this volume daily by drinking at least the
equivalent amount of quality water. In developing countries with deteriorating
environments, the demand for clean drinking water supply is growing rapidly in
recent times(Odonkor and Ampofo,2013).
Most
of the infections (like cholera, typhoid, hepatitis, poliomyelitis etc.) in
developing countries can be attributed to lack of safe drinking water. This
owing to the fact that water is obtained from various untreated sources, among
which are streams, lakes, rivers, ponds, rain, springs and wells (Okonko et al., 2008). Large percentage of the
population in developing countries are not adequately supplied with potable
water and is thus compelled to use water from sources like shallow wel ls, boreholes, springs and streams
that render the water unsafe for domestic and drinking purposes due to high
possibilities of contamination (Welch et
al., 2000; Jamielson et al.,
2004; WHO, 2006). Faecal contamination of water is established by the presence
of faecal organisms because they do not occur freely in nature. The presence of
Escherichiacoli, Clostridiumperfringes and Streptococcusfaecalis
in water is sufficient evidence that the water is not safe, since enteric pathogens
are confirmed present (Ohanu et al.,
2012).
Aims
and Objectives
The
aim of the study is to isolate and identify Escherichia
coli as an indicator of faecal pollution in streams on Obafemi Awolowo
University (O.A.U) campus, Ile-Ife, Osun state.
Objectives
of the study
1. To
isolate and identify Escherichia coli
in O.A.U streams, Osun state, Nigeria.
2. To
determine the antimicrobial susceptibility pattern of the strains in the
samples analysed.
1.1 Literature Review
1.1.1 Water
Potable
water is defined as water that is free from disease-producing microorganisms
and chemical substances deleterious to health (Ihekoronye and Ngoddy, 1985).
Before water can be described as being potable, it has to comply with certain
physical, chemical and microbiological standards designed to ensure that the
water is potable and safe for drinking (Tebutt, 1983).
Ensuring
standard quality of environmental water used as a source of recreational or
drinking water is an important worldwide problem. Meanwhile, the presence of
these organisms may cause gastroenteritis in humans (Ratajczak et al., 2010). According to World Health
Organization and European guidelines, Escherichiacoli
indicates faecal contamination of water (Ratajczak et al., 2010).
1.1.2 Pollution of Water
Surface
water such as rivers, streams can be choked with sediments, hazardous
substances or poorly treated effluents accruing from industrial activities,
which in turn renders the water bodies unsuitable for use. Polluted surface
water can contain a wide variety of pathogenic microorganisms including
bacteria and viruses. Unfortunately, clean, pure and
safe water can exist only briefly in nature and immediately polluted by
prevailing environmental factors aided by human activities. Water from most
sources is therefore unfit for immediate consumption without some sort of
treatment (Okonko et al., 2008).
During
the early history of various countries, epidemics of diseases such as typhoid,
shigellosis, cholera and amoebiasis were common threats (Tyagi et al., 2006). It was subsequently
discovered that sewage was the primary source. Human faecal pollution enter water bodies in a number of ways- from
point sources (discharges from municipal sewage treatment plant and leaking
sewage pipes) and non-point sources (such as runoff, landfills, failing septic systems
and improper sewage disposal). Waste water effluents are a major source of faecal
contamination of aquatic ecosystems (George et
al., 2002).
The
presence of poisonous chemical substances, pathogenic organisms (infective and
parasitic agents), industrial or other wastes or sewage in water makes it
contaminated or polluted. To ensure safe consumption and use, water has to be
examined microbiologically to determine its sanitary and its suitability for
general use (Ohanu et al., 2012).
1.1.3 Quality of Water
The
quality of water influences the health status of any populace, hence, analysis
of water for physical, biological and chemical properties including trace
element contents are very important for public health studies. Shortage of
infrastructure for effective treatment and distribution of water accounts for
the incidence of high morbidity and mortality rate associated with water-borne
diseases in developing countries. One of the targets of the millennium
development goals (MDG) in terms of healthy living for the masses can be
achieved through the supply of safe and available water (Orewole et al., 2007). The availability of good
quality water sources is therefore getting more and more limited, and the
effect of water-borne pathogens on human health is expected to be of great
concern. It is therefore, important to understand the significance of natural
and drinking water contribution to transmission of pathogenic microorganisms
(Suresh and Smith, 2004).
The
quality of water may be described according to its physicochemical and microbiological
characteristics (Muniyan and Ambedkar, 2011).
1.1.3.1 Physicochemical parameters
The
pH is the measurement of the acid/base activity in solution. In natural waters,
the pH scale runs from 0 to 14 and it is the most important parameter in
determining the corrosive nature of water. The lower the pH value, the higher
the corrosive nature of water (Gupta, 2009). The water temperature plays an
important role in the solubility of salts and gases. It is one of the most
significant parameters which control inherent physical qualities of water
(Hamaidi-Chergui et al., 2013).The
TDS are the total concentration of dissolved solids in water, and sometimes
also influences the salinity behaviour of river water. It is composed of
inorganic salts and some inorganic materials as well as dissolved organic
matter. The presence of these minerals in the water would come from a number of
natural sources as well as from the result of human activities.
1.1.3.2 Microbiological Quality
The
microbiological quality of treated wastewater is a concern to customers, water
suppliers, regulators and public health authority alike (Odonkor and Ampofo
2013). The increasing industrialization and the growing water demand has led to
a global deterioration of surface water quality (Tyagi et al., 2006). Thus, the need to assess the microbiological safety
of these waters by analysing them for the presence of specific pathogens and,
directing efforts to the removal of indicator microbes of faecal origin (George
et al., 2002; Tyagi et al., 2006).
Maintenance
of the microbiological quality and safety of water systems used for drinking, recreation,
and in the harvesting of seafood is imperative, as contamination of these
systems can exert high risks to human health as well as result in significant
economic losses due to closures of beaches and shellfish harvesting areas.
Water contaminated with human faeces are generally considered as a greater risk
to human health, as they are more likely to contain human-specific enteric
pathogens, including Salmonellaenterica
serovar Typhi, Shigella spp., hepatitis A virus, and Norwalk-group viruses.
Animals can also serve as reservoirs for a variety of enteric pathogens (various
serotypes of Salmonella, Escherichiacoli, and Cryptosporidium spp.). Understanding the
origin of faecal pollution is paramount in assessing associated health risks as
well as the actions necessary to remedy the problem while it still exists
(Griffin et al., 2000; Scott et al., 2002).
Monitoring
the microbiological quality of drinking water relies largely on the examination
of indicator bacteria such as coliforms, Escherichia
coli and Pseudomonas aeruginosa
(Odonkor and Ampofo, 2013). The presence of Escherichia
coli in water is a strong indication of recent sewage contamination. It is
important to note that Escherichia coli
and wastes can get into water in many different ways. For example, during
rainfall and snow melt, Escherichia coli
may be washed into creeks, rivers, streams, lakes or groundwater (Griffith et al., 2003; Roslev and Bukh, 2011)
from a land surface (Rock and Rivera, 2014).
1.1.4 Escherichia
coli as an indicator of faecal contamination
Various
bacteria are found in the digestive tracts and faeces of wild and domestic
animals as well as humans. Some of these bacteria, i.e.E. coli (a predominant member of the faecal coliform group), and Enterococcus spp., are used as
indicators of faecal contamination in natural waters (Whitlock et al.,2002). Few studies have focused on the identification
of specific characteristics of Escherichia
coli in the flow of bacteriological pollutants(Nougang et al., 2011). Its presence in humans and animals as a normal
inhabitant of the gastrointestinal tract creates opportunities for
contamination if proper hygiene is not well practised.Hence, they only infer
that pathogens may be present (Odonkor and Ampofo, 2013).
Escherichia coli
(E. coli) are gram negative bacteria
and are a type of faecal coliform bacteria commonly found in the
gastrointestinal tract of animals and humans. E. coli are so small they can’t be seen without a microscope;
however, their growth can be seen as colonies on agar media under special
conditions (Ingerson and Reid, 2011). They are considered a more specific
indicator of faecal contamination than faecal coliforms since the more general
test for faecal coliforms also detects thermotolerant non faecal coliform
bacteria. The E.coli test recommended
by the United States Environmental Protection agency (EPA) confirms presumptive
faecal coliforms by testing for the lack of an enzyme which is selective for E.coli. This test separates Escherichiacoli from non-faecal
thermotolerant coliforms (Odonkor and Ampofo, 2013).
E. coli
strains have been discovered to cause diseases in countries with more advanced
public health and health care systems, and can remain viable for several months
in water and stream sediments. Trying to detect disease-causing bacteria and
other pathogens in water is expensive and may pose potential health hazards
(Alade, 2014).
The
use of E. coli as an indicator organism is somewhat restricted by the fact
that E. coli is not a single specie; certain genera of the coliform group
such as Proteus and Aerobacter are normally found outside
the human intestinal tract in soil; other organisms found in water that do not
represent faecal pollution possess some of the characteristics attributed to E. coli and E. coli identical to that found in humans is also found in the
intestinal tract of other warm-blooded animals. However, primarily, studies
have shown that E. coli is a much
better indicator of disease risk than other faecal coliforms, EPA has therefore
recommended that E. coli be used as a
criteria for classifying waters for fresh water contact recreation. Another
weakness of the faecal coliform test and perhaps any indicator organism test
geared to human waste is that there are some bacterial pathogens which are
unrelated to human wastes. To the degree that naturally occurring microbial
pathogens become a significant public health concern, completely new test
procedures may have to be developed (Odonkor and Ampofo, 2013).
1.1.5 Characteristics of Indicator Organisms
Indicator
organisms are not by themselves, usually a health concern for healthy
individuals, but their presence in water indicate an increased risk.
Historically, faecal indicator bacteria including total and faecal coliforms
have been used in many countries as monitoring tools for microbiological
impairment of water and for prediction of presence of bacterial, viral and
protozoan pathogens. These microorganisms are of faecal origin from higher
mammals and birds, and their presence in water may indicate faecal pollution
and possible association with enteric pathogens.
However,
numerous limitations associated with their application including short survival
in water bodies (Savichtcheva and Okabe, 2006), non-faecal source (Scott et al., 2002; Simpson et al., 2002), ability to multiply after
release into water column (Desmarais et
al., 2002; Solo-Gabriele et al.,
2000), great weakness to disinfection
process (Hurst et al., 2002),
inability to identify the source of faecal contamination (point and non-point),
low levels of correlation with the presence of pathogens and low sensitivity of
detection methods have been widely reported (Horman et al., 2004; Winfield and Groisman,2003).
The
indicator organisms presently used for monitoring the efficiency of wastewater
treatment facilities and surface water resources in developing countries are
total coliforms and faecal coliforms, although reliance on indicator organisms
as the main source of information about the safety of reclaimed water for
public health is under review in many jurisdictions.
Faecal
coliform bacteria include members of the genera E.coli, which are faecal in origin as well as organisms that are
found in both faecal and non-faecal environments such as Enterobacter, Klebsiella
and Citrobacterspp. (APHA et al., 2005).
Heterotrophic
plate count bacteria are also used as indicators of the genera microbiological
water quality (Nala et al., 2003).
These organisms use organic compounds for most or all of their carbon requirements (Singleton and Sainsbury,
2001).
The
term “Total coliforms” refer to a large group of Gram negative rod shaped
bacteria that share several characteristics. The group includes thermotolerant
coliforms and bacteria of faecal origin, as well as some bacteria that may be
isolated from environmental stress (Bartram et
al., 1996). Total coliform presence can be used to indicate that the
groundwater source may be vulnerable to contamination. There is some research
supporting a link between the presence of pathogens and total coliforms in
ground waters (Abbaszadegan et al.,
2003; Locas et al., 2007), although, because
total coliforms only indicate a vulnerability to contamination, they may be
present without pathogens being detected (Borchardt, 2003; Marrero-Ortiz,
2009). However, the absence of total coliforms in a single water sample does
not necessarily that the groundwater is less vulnerable to faecal
contamination. There is some research that suggests groundwater sources should
be sampled multiple times to determine their sanitary status (Atherholt, 2003).
Total
coliforms are generally considered unreliable indicators of faecal
contamination because many are capable of growth in both the environment and in
drinking water distribution systems. It was found that 61% of the total numbers
examined over 1000 strains of coliforms were non-faecal in origin (Tallon et al., 2005). The total coliform and
faecal coliform counts can occur from the presence of a variety of bacterial
group including Escherichia, Klebsiella, Citrobacter and Enterobacter
(not considered in faecal coliforms group.). On the contrary, many coliform
bacteria originate from soil, vegetation and aquatic environments totally
unrelated to faecal pollution. Klebsiella,
Enterobacter and Citrobacter have been the predominant environmental coliforms
worldwide (Leclerc et al., 2001).
1.1.6
Antibiotics
Antibiotics
were originally defined as substances produced by one microorganism to inhibit
growth of other microorganisms (Berg et
al., 2002). The advent of synthetic antibiotics has however resulted in the
modification of this definition.Therefore, antibiotics now refer to substances
produced (wholly and partially)in low concentration by microorganisms or by
chemical synthesis to inhibit the growth or even destroy microorganisms (Berg
et al., 2002).
Classes
of antibiotics and Mechanism of action.
Different
classes of antibiotics (such as β-Lactam, Tetracyclines, Macrolide,
Aminoglycosides, Quinolones, Cyclic peptides, Lincosamides, Oxazolidinoes and
Sulfa antibiotics) have their mode of action, some of which are; inhibition of
cell wall synthesis, protein synthesis attack, plasma membrane attack, nucleic
acid synthesis attack and metabolites attack. These classes of antibiotics
affect microorganisms in several ways with variation from one antibiotic to the
other (Dubey and Maheshwari, 2005).
Antibiotic
drug resistance
Antimicrobial
drug resistance is the ability acquired by a microorganism to resist the
effects of an antimicrobial agent to which it is ordinarily susceptible.No
single antimicrobial agent has the ability to inhibit all microorganisms, and
some form of antimicrobial drug resistance is an inherent property of virtually
all microorganisms (Madigan et al.,
2012).
The
problem of rapid increase in antimicrobial resistance is a major public health
threat worldwide (Koplan, 2000) and of considerable medical significance (Khan
and Malik, 2001). Most of the antimicrobial resistance in microorganism have
emerged as a result of mutation or genetic material transfer between
microorganisms (Davies and Davies, 2010). Humans may be affected either
directly through consumption of water contaminated with the presence of
antimicrobial resistant bacteria or indirectly, through exposure to an
environment or food that has been contaminated by the water (Leclerc et al., 2002; Lee et al., 2002). Apart from the effects of microbial resistance to
antibiotics on human health, contamination of surface water bodies (especially
streams and rivers) with resistant bacterial strains from man activities and
livestock operations has also been reported (Harakeh et al., 2006). Enteric bacteria from human and animal faeces can be
found in surface waters; the faecal bacteria are introduced into aquatic
environments mainly through treated or untreated wastewater release, surface
runoffs and soil leaching (James et al.,
2003).
The
presence of pathogenic enteric microorganisms in aquatic environments can be a
source of disease when water is used for drinking, recreational activities or
irrigation. The sanitary risk is increased if the pathogenic enteric bacteria
present in water are antibiotic resistant because human infections caused by
such bacteria could be difficult to treat with drugs (Wenzel and Edmond, 2009).
In addition, faecal bacteria might be able to transmit antimicrobial resistance
to autochthonous bacteria through lateral transfer, when the resistance genes
are carried by transferable and mobile genetic elements such as plasmids and
thus contributing to the spread of antimicrobial resistance (Sayahet al., 2005). Bacteria with intrinsic
resistance to antibiotics are found in nature. Such organisms (native) may
acquire additional resistance genes from bacteria introduced into soil or
water, and the resident bacteria may be the reservoir of widespread resistant
organisms found in many environments (Ash et
al., 2002).
The
presence of antibiotics resistant bacteria in surface waters is of health
significance because of the danger of promoting multiple antibiotic resistant
organisms in humans through possible colonization of the gastrointestinal tract
and conjugal transfer of antibiotic resistance to the normal flora leading to
more multiple antibiotic resistant organisms (McKeon et al., 1995). The prevalence of drug resistant organisms poses a
great challenge to clinicians and consumption of water containing these
antibiotic resistant organisms may prolong the treatment of waterborne
diseases, and thus treatment would require new and mostly expensive antibiotics
(Tagoe et al., 2011).
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