ISOLATION AND IDENTIFICATION OF ESCHERICHIA COLI AS AN INDICATOR OF FAECAL POLLUTION IN STREAMS ON OBAFEMI AWOLOWO UNIVERSITY (O.A.U) CAMPUS, ILE-IFE, OSUN STATE

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 ?-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).