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97 Seiten, Note: 67.13
LIST OF TABLES
LIST OF FIGURES
LIST OF PLATES
ABBREVIATIONS AND ACRONYMS
CHAPTER ONE INTRODUCTION
1.1 Background of the study
1.2 Problem statement and justification
1.3 Research questions
1.4 Study hypotheses
1.5 Objectives of the study
1.5.1 General objective
1.5.2 Specific objectives
CHAPTER TWO LITERATURE REVIEW
2.1 Global overview of salmonellosis
2.2 Isolation of Salmonella
2.2.1 Raw cow milk
2.2.2 Animal intestinal wastes
2.2.3 Fruits and vegetable salads
2.2.4 Waste water
2.2.5 Water sources in Nakuru North Sub- County
2.3 Methods of water treatment
2.3.1 Boiling of water
2.3.4 Solar disinfection
2.4 Antimicrobial resistance
2.5 Global trends in resistance pattern
CHAPTER THREE MATERIALS AND METHODS
3.1 Study area
3.2 Sample size
3.3 Identification of predisposing factors to salmonellosis
3.4 Sample collection
3.5 Salmonella isolation procedure
3. 6 Analytical Profile Index (API 20E)
3.7 Serological test
3.8 Solar disinfection
3.9 Antimicrobial sensitivity testing
3.10 Data management and analysis
CHAPTER FOUR RESULTS
4.1 Predisposing factors of salmonellosis
4.1.1 Ages of the respondents
4.1.2 Education level of the respondents
4.1.3 Occupation of the respondents
4.1.4 Sex of the respondents
4.1.5 Storage of food
4.1.6 Cleaning of kitchen utensils
4.1.7 Hand washing practices
4.1.8 Human waste disposal
4.1.9 Animal waste disposal
4.1.10 Sewerage in the area
4.1.11 Waste water disposal
4.1.12 Sources of water for use in the households
4.1.13 Water treatment in the households
4.1.14 Methods of water treatment
4.2 Isolation of Salmonella
4.2.1 Salmonella load of raw milk
4. 2.2 Salmonella load from animal intestinal wastes
4.2.3 Salmonella load of raw fruits and vegetable salads
4.2.4 Salmonella load of waste water
4.2.5 Salmonella load of water sources
4.3 Salmonella load of boiled, chlorinated and filtered water
4.4 Solar disinfection
4.5 Serotyping of Salmonella
4.6 Antimicrobial sensitivity test
CHAPTER FIVE DISCUSSION, CONCLUSION AND RECOMMENDATIONS
5.1.1 Predisposing factors of salmonellosis infections
5.1.2 Salmonella load of samples
5.1.3 Effectiveness of water treatment methods
5.1.4 Serotyping of Salmonella
5.1.5 Antimicrobial resistance patterns of Salmonella
APPENDIX I: CONSENT FORM
APPENDIX II: QUANTITATIVE DATA COLLECTION TOOL (STRUCTURED QUESTIONNAIRE)
Table 4.1: Participants social-demographic data in Nakuru North Sub-County from May to September 2012
Table 4.2: Colony forming units of Salmonella, temperature and pH in raw milk samples from Maili 5, Bahati and Kabatini in Nakuru North Sub-County from May to September 2012
Table 4.3: Colony forming units of Salmonella, temperature and pH in sheep intestinal waste samples from Maili 5, Bahati and Kabatini in Nakuru North Sub-County from May to September 2012
Table 4.4: Colony forming units of Salmonella, temperature and pH in cattle intestinal waste samples from Maili 5, Bahati and Kabatini in Nakuru North Sub-County from May to September 2012
Table 4.5: Colony forming units of Salmonella in raw fruits and vegetable salads, storage time and pH from Maili 5, Bahati and Kabatini in Nakuru North Sub-County from May to September 2012
Table 4.6: Colony forming units of Salmonella from waste water, temperature and pH from Maili 5, Bahati and Kabatini in Nakuru North Sub-County from May to September 2012
Table 4.7: Colony forming units of Salmonella, temperature, turbidity and pH from water running in community taps from Maili 5, Bahati and Kabatini in Nakuru North Sub-County from May to September 2012
Table 4.8: Colony forming units of Salmonella, temperature, turbidity and pH in water from river Kandutura in Nakuru North Sub-County from May to September 2012
Table 4.9: Mean Salmonella colony forming units in Maili 5, Bahati and Kabatini waters after treatment in Nakuru North Sub-County from May to September 2012
Table 4.10: Mean Salmonella (cfu/100 ml) after solar disinfection in Nakuru North Sub-County from May to September 2012
Table 4.11: Salmonella serotypes isolated in Nakuru North Sub-County from May to September 2012
Table 4.12: Antimicrobial sensitivity test results for the seven antimicrobials in Nakuru North Sub-County from May to September 2012
Figure 3.1: Map of Nakuru North Sub-County (KFSSG, 2008).
Figure 4.1: Distribution of the respondents according to sex in Nakuru North Sub-County from May to September 2012.
Figure 4.2: Distribution of the respondents according to food storage in Nakuru North Sub-County from May to September 2012.
Plate 3.1: Membrane filtration of liquid samples.
Plate 4.1: Salmonella in Salmonella-Shigella agar.
Plate 4.2: Serotyping using slide agglutination test.
Plate 4.3: Sensitivity test using Karby Bauer disk diffusion test.
Salmonellosis is one of the most common and widely distributed group of enteric diseases in the world today. It causes high mortality and morbidity especially in developing countries. This study was aimed at identifying the factors that predispose people to salmonellosis. In addition the study was meant to isolate Salmonella, and determine its antimicrobial sensitivity and test the effectiveness of water treatment for the control of salmonellosis. To identify the predisposing factors of salmonellosis, a questionnaire was used. For the isolation of Salmonella, a total of 630 samples were collected from raw cows’ milk, sheep and cattle intestinal wastes, raw fruit and vegetable salads, waste water, water sources and water that had been treated by the study population through boiling, chlorination and filtration. Samples were also collected from water that was treated through solar disinfection. The samples were pre-enriched using peptone water then selectively enriched using Selenite F broth and incubated at 37 oC for 24 hrs and subcultured in xylose lysine desoxycholate (XLD) agar and Salmonella- Shigella agar in five replicates. Typical Salmonella colonies were confirmed by biochemical test using API E-20 and the species serotyped. The isolated serovars were tested against seven antibiotics; cephalexin, nalidixic acid, chloramphenicol, ciprofloxacin, gentamicin, amoxycillin and sulfa-trimethoprim. The results were analyzed by the use of chi-square test, correlation test and Anova using Statistical Package for Social Sciences (SPSS version 11.50) software. Level of education, occupation, method of food storage, cleaning of kitchen utensils, hand washing, human waste disposal, animal wastes, presence or absence of sewers, waste water, tap water, river water and water treatment were significantly associated with salmonellosis while sex, well water and method of water treatment were not. There was no significant difference between the microbial load of Salmonella isolates from milk, sheep and cattle intestinal wastes, waste water, fruits and vegetable salads in Maili 5, Bahati and Kabatini. However, River Kandura’s water mean Salmonella isolates varied significantly from upstream to downstream. Of the 105 Salmonella isolates Salmonella enterica serovar Typhimurium were (45.7 %), S. enterica serovar Typhi (22.9 %), S. enterica serovar Enteritidis (21.9 %) and S. enterica serovar Dublin (9.5 %). All the serovars were susceptible to gentamicin with a minimum of at least 2 Salmonella being resistant. All the samples that turned out positive were highly contaminated with Salmonella apart from isolates from the upstream of river Kandutura, the most effective method of water treatment being used in the study area was chlorination. Solar disinfection is effective upon continous exposure of water to sunlight for 3 to 5 h. In addition, Salmonella isolates from Nakuru North Sub-County are sensitive to gentamicin. There is need to educate people on the predisposing factors of the disease, regular food inspection by the authorities concerned, sensitization of the entire population on the need of proper use of antimicrobials and use of gentamicin for the treatment of salmonellosis.
Salmonella are well-known pathogens, highly adaptive and capable of causing disease in humans and/or animals. Salmonella infections are capable of producing serious infections that are often food borne and present as gastroenteritis. However, a small percentage of these infections may become invasive and result in bacteremia and extra intestinal disease (Fluit, 2005). The main reservoirs for non-typhoidal Salmonella are animals such as poultry, livestock, pets and reptiles. Salmonella enterica serovar Typhi and Salmonella enterica serovar Paratyphi colonize only humans and are often acquired through feacally contamined food or water, a person who has typhoid fever, or from chronic carriers (CIDRAP, 2006).
While some serovars of Salmonella enterica such as Salmonella enterica serovar Typhi, S. enterica serovar Paratyphi, S. enterica serovar Enteritidis and S. enterica serovar Derby cause disease in humans and a variety of animals, other serovars are highly restricted to a specific host such as Salmonella enterica serovar Gallinarum in poultry and Salmonella enterica serovar Abortus-ovis in sheep. Salmonella infections range from gastrointestinal infections that are accompanied by inflammation of intestinal epithelia, diarrhoea and vomiting, to typhoid fever, a life threatening infection (Hensel, 2004). The outcome of Salmonella infections is determined by the host and the status of the bacterium. Whereas, age, genetic and environmental factors mainly determine the status of the host, for the bacterium it is determined by virulence factors (Alphons et al., 2005).
Serotypes adapted to man, such as Salmonella enterica serovar Typhi and Salmonella enterica serovar Paratyphi, usually cause severe diseases in humans as a septicaemic typhoidic syndrome (enteric fever). These serotypes are not usually pathogenic to animals. Serotypes that are highly adapted to animal hosts, such as Salmonella enterica serovar Gallinarum (poultry) or Salmonella enterica serovar Abortus-ovis (sheep) usually produce very mild symptoms in man (Fluit, 2005). However, Salmonella enterica serovar Choleraesuis which has the pig as a primary host also causes severe systemic illness. In the same way, Salmonella enterica serovar Dublin, which has a preference for bovines, is primarily responsible for the systemic form of salmonellosis in people. In young calves this disease causes high mortality, while in adult cattle it results in fever, reduced milk yield, diarrhoea, abortion and occasionally death. Ubiquitous serotypes, such as Salmonella enterica serovar Enteritidis or Salmonella enterica serovar Typhimurium, which affect both man and animals, generally cause gastrointestinal infections usually less severe than enteric fever. However, they also have the capacity to produce typhoid-like infections in mice and in humans, or asymptomatic intestinal colonization in chickens (Velge et al., 2005).
Salmonellosis is one of the most emerging and re-emerging infectious diseases in the world (WHO, 2004). The high prevalence of salmonellosis is attributed to lack of adequate water supply, poor sewage effluent disposal coupled with contaminated foods (Jones, 2005). About 1,195 outbreaks of salmonellosis were reported in Brazil in 2007, with 22.6 % of them provoked by the consumption of foods with raw eggs (Wray, 2001). It is estimated that 22 million typhoid cases are reported with 200, 000 deaths in each year. The estimates in Africa are very low because very few people seek medical attention. For example, 59/100, 000 in East Africa and 39/100, 000 cases in Kenya per year (Kariuki et al., 2012). The highest burden due to salmonellosis is felt mostly in the developing countries (Henson, 2003).
Salmonellosis is an important global health problem which causes substantial morbidity and thus it has a significant economic impact. In spite of the improvement in hygiene, food processing, education of food handlers and information to the consumers, food borne diseases are still prevalent and most important health problem in most countries (Dominguez et al., 2002). Many foods, particularly those of animal origin, have been identified as vehicles of transmission of these pathogens to human beings and spreading them to the processing and kitchen environment (Uyttendaele et al., 2002).
The major constraints for development in the food safety in Nakuru North Sub-County are the lack of basic infrastructure and the appropriate technology, and an acute shortage of trained personnel in the food safety and quality control. The food production system in Nakuru North Sub-County is still a livelihood enterprise, and commercial food producing companies are very few (KFSSG, 2008). Routine food inspection and quality control is a responsibility of public health officers under the Ministry of Health. A majority of the food inspectors are certificate holders with short-term training in food inspection (Moy and Schlundt, 2005). The laboratories are not fully equipped, both in terms of facilities as well as in manpower. Therefore, with the lack of resources such as skilled manpower and laboratory facilities to efficiently carry out the regulatory works cases of salmonellosis have escalated over the recent past (Kariuki et al., 2012).
In some instances, there have been incidences of Salmonella mass food poisoning such as in schools, picnics sites and villages in Nakuru North Sub-County, attributed to consumption of contaminated foods (ROSA, 2007). Although Salmonella in foods has been recognized as a major source of infection, no systematic studies have been carried out with respect to factors that predispose people to salmonellosis, Salmonella load of food and environmental samples collected from Nakuru North Sub-County, the effectiveness of water treatment methods and antimicrobial resistance patterns of isolates of Salmonella serotypes. Although efforts have been made to make people practice recognized standard operating procedures and processing standards like Hazard Analysis Critical Control Points (HACCP) and Safe Quality Food (SQF) (Press release, 2006), the regulations are rarely observed. The Sub-County also lack bulk food storage facilities as of now. Majority of the food selling shops in Nakuru North don’t have deep freezers for storage and the shops that sell foods are of the open type.
Little is known about the state of salmonellosis in Nakuru North Sub-County despite the observed cases of salmonellossis in the area (ROSA, 2007). Although epidemiological studies show 48.1 % cases of salmonellosis (Hlupheka, 2001) in Nakuru North Sub-County, there is still little information on the current state of affairs. The emergence of multiple-antibiotic resistant Salmonella, in another major problem in Nakuru North Sub-County (Okeke et al., 2005).
i) What are the factors that predispose people to salmonellosis in Nakuru North Sub- County?
ii) What is the Salmonella load of milk, intestinal wastes, fruits and vegetable salads, waste water and different water sources sampled in the area?
iii) What is the effectiveness of the methods of water treatment in controlling salmonellosis?
iv) Are the Salmonella serotypes isolated from the chosen samples sensitive to antibiotics?
i) There is no significant relationship between the predisposing factors of salmonellosis and infection of salmonellosis.
ii) There is no significant relationship between the methods of water treatment and control of salmonellosis.
To identify the factors that predispose people to salmonellosis, isolate Salmonella, carry out sensitivity to antibiotics and assess the effectiveness of water treatment methods in the control of salmonellosis in Nakuru North Sub-County.
i) To identify the factors that predispose people in Nakuru North Sub-County to salmonellosis.
ii) To determine the bacterial load of samples collected from milk, intestinal wastes, fruits and vegetable salads, different water sources and waste water.
iii) To determine the effectiveness of the water treatment methods in the control of salmonellosis.
iv) To determine antimicrobial resistance patterns of Salmonella serotypes isolates.
In majority of developing countries, the incidence of human Salmonella infection has continued to increase over the years. Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis have been implicated in causing most of these infections (Buck et al., 2003; Chiu et al., 2004; Sadeyen et al., 2004). By 2010, the cost linked to foodborne infections ranged between 551 million and 2.7 billion Euros in Europe, where Salmonella infections was estimated to cost 161 000 Euros (Newell et al., 2010). It is reported that the rate of salmonellosis in the United States is between 15 to 20 cases per 100 000 people annually. The Enter-net surveillance program reported Salmonella enterica serovar Enteritidis and Typhimurium as the most predominant organisms identified by the participating countries comprising of over 81 % of all isolates during the period of 2005-2011 (Eurosurveillance, 2011).
Salmonellosis is endemic to rural and urban Sub-Saharan Africa (Srikantiah et al., 2011). In rural Mozambique, the incidence of salmonellosis is 120 cases per 100,000 people annually (Crump and Mintz, 2010). The true incidence of salmonellosis is likely to be 2–3 times this figure, because the incidence of bacteremia among patients who die before reaching the district hospital has not been ascertained in either study (Cox and Pavic, 2010). In Uganda, occurrence of salmonellosisis 500 cases per 100,000 people per year (Gomez et al., 2011). In rural Kenya, the estimated minimum incidence of salmonellosis is 88 cases per 100,000 people per year (Kariuki et al., 2012).
With the increasing population in Nakuru North Sub-County, there is an increasing demand for food and water which will force the present resource driven system of these products to a demand driven system which will increase the Salmonella transmission (Zessin et al., 2006). Limited information regarding Salmonella in milk, sheep and cattle offal, salads, waste water and different water sources in Kenya is currently available which will assist in curbing the problem of salmonellosis.
Cow’s milk has long been considered a highly nutritious and valuable human food and is consumed by millions daily in a variety of different products (Nusrat et al., 2012). It contains most of the nutrients such as carbohydrates, proteins, fats, vitamins and minerals required for the growth of microorganisms, combined with higher water activity. This makes it an ideal medium for bacterial growth and therefore it can be considered one of the most perishable agricultural products because it can also be very easily contaminated (Haeschen, 2000). Raw milk contains microorganisms that are capable of causing diseases such as salmonellosis (Headrick et al., 2001). Because of contamination of milk production systems, it is difficult to avoid contamination of milk with micro-organisms. Therefore determination of microorganisms in milk is a major feature in determining its quality (Rogelj, 2003).
Ruminants, such as cattle and sheep, are natural reservoirs for Salmonella. Salmonella is mostly present within the rumen of these animals and is shed through fecal matter, hence ending up in water sources and farms. Previous studies by Reicks et al. (2001) (73 %) and Kunze et al. (2007) (30.3 %) have reported differing proportions of Salmonella in intestinal samples. Cross contamination can begin during transport with other animals via direct body contact or indirectly via contact surfaces (McEvoy, 2004). Contaminants can be transferred to the digestive system during slaughter by initial cuts with knife and when the animal is feeding (Reid et al., 2000).
These contaminations have the risk of contaminating the entire production chain of the slaughter house. Several factors influence animal intestine contamination. These factors include but are not limited to transport conditions and duration, herd lot mingling, drinking water systems, hide cleanliness, and feed withdrawal, automated hide puller/debunging, extent/efficiency of abattoir cleaning and operative hygiene practices (McEvoy, 2004). Various Salmonella strains have been isolated from different animals. However, sheep and cattle are more prevalent carriers than nonruminant animals (Hussein, 2006). Due to the innate presence of Salmonella in the intestines, cattle hides have been identified as a central source of microbial contamination when animals lick one another or themselves (Hussein, 2006) especially on rainy season compared to dry season. In another study that investigated the seasonal prevalence of non-typhoidal Salmonella, it was established that the bacteria are more prevalent during the rainy season than in the dry season (Piddock, 2001).
Fruits and vegetables are an extraordinary dietary source of nutrients, micronutrients, vitamins and fibre for humans and are thus vital for health and well being. Well balanced diets, rich in fruits and vegetables, are especially valuable for their ability to prevent vitamin C and vitamin A deficiencies and are also reported to reduce the risk of several diseases (Kalia and Gupta, 2006). Fruits and vegetables are widely exposed to microbial contamination through contact with soil, dust and water and by handling at harvest or during postharvest processing. They therefore harbour a diverse range of microorganisms including plant and human pathogens (Carmo et al., 2004). Differences in microbial profiles of various fruits and vegetables result largely from unrelated factors such as resident microflora in the soil, application of nonresident microflora via animal manures, sewage or irrigation water, transportation and handling by individual retailers (Ray and Bhunia, 2007; Ofor et al., 2009).
Fresh produce such as fruit and vegetable salads are often consumed raw, putting consumers at risk of infection by contaminating organisms such as Salmonella. The fresh produce industry in many countries has responded to this by adopting various risk management practices designed to reduce the likelihood of contamination (Ofor et al., 2009). However, despite this, the number of reported illnesses linked to contaminated produce has increased (Sivapalasingam et al., 2004). Changes in agricultural processing and distribution practices that have enhanced both the supply and range of products such as triple-washing pre-packaged leafy greens using the same water may also have increased the risk for more widespread outbreaks (Greene et al., 2008).
Salmonella is the most commonly reported bacterial pathogen, accounting for nearly half of the outbreaks due to bacteria (Sivapalasingam et al., 2004). A wide spectrum of produce vehicles have been associated with Salmonella infections. Several large-scale outbreaks have been linked to consumption of tomatoes (Cummings et al., 2001; Gupta et al., 2007; Greene et al., 2008) and melons (Bowen et al., 2006; Munnoch et al., 2009). In 2008, jalapeño and serrano peppers were vehicles for a large multistate outbreak of Salmonella serovar Saintpaul infections (CDC, 2008). Examples of other outbreaks of Salmonella enterica linked to ready-to-eat plant produce include an outbreak in Scandinavia and the UK of serovar Thompson infections associated with consumption of rocket leaves (Nygard et al., 2008), an outbreak of serovar Anatum infections in Denmark linked to imported basil (Pakalniskiene et al., 2009) and an outbreak of serovar Senftenberg infection associated with imported Israeli basil affecting most countries in Sub-Saharan Africa (Pezzoli et al., 2008).
Thus despite their nutritional and health benefits, outbreaks of human infections associated with the consumption of fresh or minimally processed fruits and vegetables have increased in recent years (Beuchat, 2002). Enteric pathogens such as Salmonella are among the greatest concerns during food-related outbreaks (Buck et al., 2003). Several cases of typhoid fever outbreak have been associated with eating contaminated vegetables grown in or fertilized with contaminated soil or sewage (Beuchat, 2002). These increases in fruits and vegetables-borne infections may have resulted from increased consumption of contaminated fruits and vegetables outside the home as most people spend long hours outside the home. In Kenya for instance, street vending of handy ready-to-eat sliced fruit and vegetables has recently become very common and the market is thriving (Muinde and Kuria, 2005). All of the above mentioned studies however failed to quantify the pathogen with reference to the storage pH and time so as to compare their values with the World Health Organization recommended values in fruits and vegetables.
Waste water play the main role in the transmission of Salmonella. It’s estimated that 2.4 billion people have no access to right infrastructure for safe disposal of waste water (Smith, 2002). Thus in many parts of the world, particularly Africa, Latin America, Caribbean and Asia, a greater percentage of waste water gets discharged to the environment without treatment (WHO, 2000). Surface water is often contaminated by urban wastewater, by effluents of meat industries and by wastewater from livestock ranches, involving different serotypes of Salmonella (Rusin et al., 2000). The serotypes isolated from human samples do not always coincide with the serotypes isolated from wastewater. In Spain, Salmonella enterica serovar Enteritidis (50.7 %), S. enterica serovar Typhimurium (23.2 %), and S. enterica serovar Hadar (4.7 %) were the most frequent serotypes isolated from clinical human samples in the year 2000 while S. enterica serovar Anatum was the one most often isolated from water samples (Usera et al., 2001).
Although wastewater is treated to eliminate pathogenic microorganisms and reduce waterborne transmission, numerous studies indicate that conventional waste water treatment does not guarantee their complete elimination given differences in pH and temperature of the waste water sample under study (Ngari et al., 2011). According to Howard et al. (2004), treated waste water contains MPN of 45/100 ml Salmonella. The survival of Salmonella despite treatment implies the possibility of selection of the most resistant strains, or the acquisition of resistance through the transference of genetic material. A further study was done by Amoah et al. (2005) involving the association of salmonellossis occurrences and waste water. Their study indicated a significant association between the two. With all these studies in place none quantified Salmonella pathogens in waste water in Nakuru North Sub-County.
In Kenya, salmonellosis is among the major illnesses affecting people living in urban and peri-urban areas. Nakuru North Sub-County has several water sources which include ground water, community taps and river water (APHRC, 2002). Where ground water is used as a source of domestic water, use of pit latrines is not recommended because the two are incompatible unless the water table is extremely low and soil characteristics are not likely to contribute to contamination of ground water. Where ground water and pit latrines coexist, it is difficult to give a general rule for all soil conditions. However, the commonly used guideline is that the well should be located in an area higher than and at least 15 m from the pit latrines and should be at least 2 m above the water table. Available evidence shows that increased lateral separation between the source of pollution and groundwater supply reduces the risk of faecal pollution (ARGOSS, 2001).
Pollution of river waters with Salmonella is on a steady increase in the recent past (Niyogi, 2005; Abraham, 2010). The major source of microbes in river water is faeces from human and other mammals. Entry of pathogens into rivers can occur either from a point source, non- point sources or both. Non-point source microbial pollution of rivers occurs from rainwater surface run-offs, storm sewer spillages or overflow, while point-source pollution comes from discharge of untreated or partially treated effluents from wastewater treatment plants (Petersen et al., 2005; Donovan et al., 2008). The impact of river pollution on human health depends mainly on the water uses, as well as the concentration of Salmonella in the water (Niyogi, 2005).
Water from community taps is a reliable source of water supply, because it is often unpolluted due to restricted movement of pollutants in the soil profile. However, they are most susceptible to contamination when cracks develop on them leading to contamination with Salmonella (Nassinyama et al., 2000; Adejuwon, 2011). Thus, contamination of drinking water from community taps is of primary importance due to the danger and risk of water related diseases (WHO, 2011).
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