|Year : 2015 | Volume
| Issue : 1 | Page : 6-12
Bacteriological evaluation of sampled sachet water sold in Samaru-Zaria, Kaduna-State, Nigeria
S Ugochukwu1, FJ Giwa2, A Giwa1
1 Department of Textile Science and Technology, Ahmadu Bello University, Zaria, Kaduna, Nigeria
2 Department of Medical Microbiology, Faculty of Science, Ahmadu Bello University, Zaria, Kaduna, Nigeria
|Date of Web Publication||8-May-2015|
F J Giwa
Department of Medical Microbiology, Faculty of Medicine, Ahmadu Bello University, Zaria
Source of Support: None, Conflict of Interest: None
Background: The quality of drinking water is an important environmental determinant of health. Widespread production and consumption of inadequately processed or contaminated packaged drinking water can lead to waterborne disease outbreaks. To safeguard public health, it is important that available packaged water is duly registered and regularly analysed. Aim: This study was carried out to determine the bacteriological quality of ten selected brands of sachet water produced and sold in Samaru Zaria metropolis. Materials and Methods: From the brands of sachet water being sold in samaru, ten most popular brands were identified and selected based on patronage by consumers and distributors. A total of 20 samples, two from each of the ten brands of sachet water were bought from water vendors every fortnight for 1 month (may - june 2013). Results: The total coliform count ranged from 2-18+/100 ml. Isolates were identified to be Klebsiella spp., 0[40%] Proteus spp., [30%] Pseudomonas spp. [20%] and Chromobacterium violaceum [10%]. All were negative for thermotolerant coliforms, Salmonella, Shigella and Vibrio cholera. Conclusion: The results of this study showed contamination with micro-organisms found naturally in water, soil or vegetation which indicates a problem with the overall quality of water production but showed no contamination with sewage or animal waste. Microbiological assessment of drinking water quality should be done periodically with the regulatory body National agency for food and drug administration and control (NAFDAC) ensuring good quality assurance and maintenance of internationally defined drinking water standards.
Keywords: Bacteriological evaluation, coliforms, drinking water, Kaduna state, sachet water, Samaru Zaria
|How to cite this article:|
Ugochukwu S, Giwa F J, Giwa A. Bacteriological evaluation of sampled sachet water sold in Samaru-Zaria, Kaduna-State, Nigeria. Niger J Basic Clin Sci 2015;12:6-12
|How to cite this URL:|
Ugochukwu S, Giwa F J, Giwa A. Bacteriological evaluation of sampled sachet water sold in Samaru-Zaria, Kaduna-State, Nigeria. Niger J Basic Clin Sci [serial online] 2015 [cited 2018 Jun 25];12:6-12. Available from: http://www.njbcs.net/text.asp?2015/12/1/6/156663
| Introduction|| |
Access to safe drinking water is essential to Health. It is a basic human right and a component of effective policy for health protection.  The quality of drinking water is a powerful environmental determinant of health. Water is vital to sustaining life and it needs to be treated that way. A satisfactory (adequate, safe and accessible) supply must be available to all.  Diseases related to contamination of drinking water constitute a major burden on human health, interventions to improve the quality of drinking water provides significant health benefits. Recently, the United Nations General Assembly declared the period from 2005 to 2015 as the International decade for action, 'Water for life'.  The standard industrialized world model for delivery of safe drinking water and sanitation technology is, however, not available in most of the developing world.  Safe and portable water supplies in urban centres in Nigeria are still inadequate despite four decades of independence and several efforts from various governments. 
The Urban Zaria Area is blessed with abundant water resource both ground and surface and the distribution of this resource have very little variation in both time and space amongst the sub-settlements.  There are two major river systems in the area, the Kubanni and Saye, joined at a confluence to form river Galma. These rivers together with their tributaries (Kamacha and Shika) drain the land area of Urban Zaria. 
Inspite of its potentials for good groundwater storage, there is incessant water shortage in the Urban Zaria Area, because the distribution and supply system of treated water are very poor.  This has made over 90% of the population reliant on water provision from sources other than the Zaria water scheme.  Many people depend on water vendors for provision of water for domestic and daily needs and this has led to the advent of locally sourced low cost alternatives sachet water called 'Pure water' becoming a major source of drinking water. The production, marketing and consumption of sachet water has increased tremendously with several brands being marketed in Nigeria and other developing nations. ,
The Nigerian National Agency for Food and Drug Administration and Control (NAFDAC) is mandated to enforce compliance with internationally defined drinking water guidelines, but regulation of the packaged water industry aimed at good quality assurance has remained a challenge to the agency. 
Water in sachets are readily available and affordable but there are concerns about their purity. The integrity of the hygienic environment and the conditions where the majority of the water in sachets are produced have also been questioned.  Although nationally documented evidence is rare, there are claims of past outbreaks of water borne illnesses that ensued from consumption of polluted water in sachets.  An understanding of their microbiological quality and safety are therefore imperative and should be a cause of concern to consumers, water suppliers, regulators and public health authorities. 
Disease-causing micro-organisms transmitted via drinking water are predominantly of faecal origin and are referred to as enteric pathogens. , The World Health Organization (WHO) estimates that about 1.1 billion people globally drink unsafe water  and the vast majority of diarrhoeal disease in the world [88%] is attributable to unsafe water, sanitation and hygiene.  Poor water quality, sanitation and hygiene accounts for 1.7 million deaths a year worldwide [3.1% of annual deaths].
The WHO standards  state that drinking water should not contain any micro-organisms known to be pathogenic or any bacteria indicative of faecal pollution. The concept of faecal indicator bacteria in determining the sanitary quality of water was first proposed in the 1880s when workers began to use bacteriologic media to assess microbial presence in water and food commodities.  Bacterial contamination cannot be detected by sight, smell or taste. A basic laboratory test is the best way to tell if Coliform organisms are present as they can be there with no appearance or taste difference. 
When water is tested for Faecal or Total Coliform, the results are usually given as the number of colony-forming units per 100 millilitres (CFU/100 ml) of water sampled. No sample should contain Faecal Coliform or E. coli, and ideally there should be no Total Coliform. 
Coliform bacteria have been used as indicators of unsanitary conditions in water and foods for over a century. The concept of coliform bacteria emerged in 1892 when Shardinger proposed the use of E. coli as an indicator of faecal contamination.  This was based on the premise that E. coli is abundant in human and animal faeces and is not usually found in other niches, and could be easily detected by its ablity to ferment lactose.  It was also easier to isolate than other known gastrointestinal pathogens, hence the presence of E. coli in food or water became accepted as an 'indicator' of faecal contamination and the possible presence of other frank pathogens found in stool like parasites, viruses, bacteria like Salmonella typhi, Shigella, etc. 
Coliform bacteria generally belong to four genera of the Enterobacteriaceae family, Citrobacter, Enterobacter, Esherichia and Klebsiella. Escherichia coli are the most well-known coliform.  Coliforms are easy to detect, but their association with faecal contamination can be questionable because some coliforms are found naturally in environmental samples (soil, water and vegetation).  This led to the introduction of faecal coliforms as an indicator of contamination. Faecal coliform, first defined based on the works of Eijkman  are a subset of total coliforms that grow and ferment lactose at elevated incubation temperature, 44.5-45.5°C and produce acid and gas from lactose within 48 hours. 
Almost all the methods used to detect E. coli, total coliforms or faecal coliforms are enumeration methods that are based on lactose fermentation. 
Studies conducted on the microbiological quality of pure water in Nigeria has shown various levels of contamination. ,,, Enforcing compliance by the requlator body NAFDAC to ensure good quality assurance and the maintenance of internationally defined drinking water standards has also remained a challenge  as many are not registered and even those that are registered do not always meet the standards required.
This study was carried out to evaluate the bacteriological quality of some sachet water sold in Samaru Zaria, Kaduna state to find out the degree to which they meet the standards set out by WHO.
| Materials and methods|| |
This study was carried out in Samaru, Sabon Gari local government area of Kaduna state. Samaru is situated on latitude 112 o 12" N and longitude 07 o 37" E, at an altitude of 550-700 metres. It is about 13 km from Zaria city on the Sokoto road, 8 km to Shika and 7 km from Bassawa. According to the 1991 population census, Samaru had 12,978 people. Based on the 3% growth rate of the 1991 census, Samaru population growth was projected to be about 18,039 by 2009. Samaru evolved from a small colonial farming settlement to become a large community, a melting pot, often referred to as 'the University village'. It is cosmopolitan in nature, drawing and fusing people of divergent national and international backgrounds. It is also one of the major settlements that make up the Urban Zaria. It is an educational and administrative settlement which brought about the establishment of new settlement for non residents of Zaria City. It has grown as a result of the establishment of Ahmadu Bello University, which was established in 1962 and other institutes like Nigeria Institute of Leather and Science Technology, Federal Institute for Chemical and Leather Research, Federal College of Aviation, Nigerian Research Institute and Chemical Technology and Nigerian Institute of Transport Technology.
The Zaria water treatment plant at Shika Dam Zaria commissioned in 1975 cannot adequately meet the demands of the growing Population.  The water taps have remained dried for no less than 5 years with alternatives being wells and boreholes and purchased water from wheel-truck pushers for domestic use.  This has made the community heavily reliant on sachet water for drinking purposes due to the inadequacy both in quantity and quality of public water supply.
The brands of sachet water being sold in Samaru are 16. Out of these, ten most popular brands were identified and selected based on patronage by consumers and distributors. A total of 20 samples, two each from each of the ten brands of sachet water were bought from water vendors every fortnight for 1 month (May-June 2013).
Samples were labelled and transported in ice packs to the laboratory and examined for turbidity, colour, odour, taste and ph, NAFDAC registration number, manufacturing and expiry date and analysed within two hours of collection. Prior to analysis, the water in each sachet was thoroughly mixed and a portion of the sachet wiped with cotton wool soaked in ethanol before piercing with sterile needle and syringe.
Multiple Tube Fermentation Technique/MPN
The technique used for water analysis was the MPN.  In this technique, a total amount of 100 ml water sample was taken from each sachet as (five 10 ml amounts and one 50 ml amount) and innoculated into bottles of sterile double strength MacConkey broth of the same amount each containing an inverted Durhams tube for gas collection and detection. This was incubated aerobically at 35°C for 18-24 hours. This was the presumptive test for total coliform. After incubation at 37°C, the number of bottles in which lactose fermentation with acid and gas production had occurred were counted. Lactose fermentation and acid production was evidenced by a change in the color of MacConkey broth from purple to yellow and gas production was seen by the displacement of broth in the durhams tube with bubble. In the confirmatory test for faecal coliform, a loopful of broth from the positive tubes in the presumptive test was transferred into EC (elevated coliform) broth and incubated at 44.5°C for 24 hours. Production of gas in the tube after 24 hours is positive. After incubation at 44.5°C, there was no gas production in any of the tubes incubated at 44.5°C and streaking a loopful of the broth on Mac Conkey agar and incubating at 35°C for 18-24 hours yielded no growth. By reference to probability tables, the most probable number of coliforms in the 100 ml water sample was estimated. The positive tubes from the presumptive test were cultured on MacConkey agar, incubated aerobically at 35°C for 18-24 hours. The isolates were further identified by colonial morphology, gram stain, motility and biochemical tests.
Isolation of Salmonella, Shigella and Vibrio Cholerae
One millilitre was taken from each of the sachet water with a sterile syringe and inoculated into 5 mls of selenite F enrichment broth and incubated at 37°C for 8 hours. A loopful of this broth was then streaked onto Salmonella Shigella agar and incubated at 37°C for 18-24 hours.
One millilitre water sample was taken from each of the sachets and also inoculated into 5 ml double strength alkaline peptone water at 37°C for 4-6 hours. This was then inoculated on Thiosulphate Citrate Bile Salt Sucrose Agar and incubated at 37°C for 18-24 hours.
Identification of Isolates
A loopful of broth from the positive tubes in the presumptive test was streaked onto Mac Conkey agar plates and incubated aerobically at 37°C for 18-24 hours. Isolates were further identified by colonial morphological characteristics, gram stain, motility and biochemical tests which includes test for oxidase production, catalase, indole, citrate utilization, urease production and triple sugar iron agar test as described by Koneman. 
Data was analysed using Statistical Package of Social Sciences (SPSS) version 16.
| Results|| |
All the water samples tested were not turbid and had no taste or smell. None of the samples met the other labelling requirements stipulated by NAFDAC apart from the NAFDAC registration number.
A total of 20 samples of sachet water was evaluated in 1 month. Every 2 weeks, a sample each of the branded sachet water was bought from water vendors, transported in ice packs and taken to the laboratory. The presumptive coliform count evaluated by the multiple tube fermentation technique and interpreted with the most probable number table from the ten sachet water is indicated in [Table 1] [Figure 1].
|Figure 1: Type of pathogen found in water sample The pathogens isolated from the positive tubes in the presumptive test were Klebsiella spp. 40%, Proteus spp. 30%, Pseudomonas spp. 20% and Chromobacterium violaceum 10%|
Click here to view
The MPN/100 ml for the sachet water ranged from 2 to 18+/100 ml. The least count was found in samples H and I. Samples C and F had a count of 3 and 6/100 ml, respectively, while the highest count of 18+/100 ml was found in samples A, B, D, E and J.
No faecal coliform was isolated after incubation at 44.5°C for 24-48 hours.
The subculture from selenite f broth on Salmonella Shigella agar and double strength alkaline peptone on Thiosulphate citrate bile salt agar yielded no growth of Salmonella, Shigella and Vibrio cholerae after incubation at 37°C for 24-48 hours.
| Discussion|| |
The results from this study showed sachet water contamination with total coliforms and heterotrophic bacteria but no contamination with faecal coliforms, Salmonella, Shigella and Vibrio cholerae. This is similar to studies done by ,, in Ogbomoso, Ibadan and Nnewi in Nigeria which showed contamination of the sachet water samples with total coliforms but absence of faecal coliforms.
The presence of total (non-faecal) coliforms may not indicate faecal contamination especially as no faecal coliform was isolated after incubation at a higher temperature. Their presence in drinking water most likely indicates contamination by organisms which occur naturally in surface waters, soil or vegetation from where there could be significant chemical or biological contamination, and is generally not harmful.  The presence or absence of these bacteria in treated water determines the quality of water and whether water treatment and disinfection was well done. 
The total coliform count of the sampled sachet water ranged from 2-18+/100 ml. This high level of coliform bacteria in the sachet water from the various brands sampled was inconsistent with the WHO guidelines for drinking water  which states that no sample should contain faecal coliform or E. coli and ideally there should be no total coliform. This finding compares with similar studies done in Lagos  and Nsukka  which found that the various brands of sachet water produced had higher coliform counts than that stipulated by WHO. This was however different from the findings of a study done in Osogbo Nigeria  which compared the bacteriological quality of sachet, well and tap water and found low coliform count of 0-1 coliform/100 ml for sachet water.
Klebsiella pneumonae, Proteus mirabilis, Pseudomonas aeruginosa and Chromobacterium violaceum made up ,,, % of the isolates found from the presumptive coliform count, respectively. Studies done in India  and Ibadan  found similar isolates in sachet water. All these organisms isolated are heterotrophic bacteria that can be found naturally in surface waters, soil or vegetation and their presence suggests problems with the processing of water which could be due to ineffective treatment,  or no treatment at all, as some unscrupulous producers just bag and seal well or pipe borne water without any form of treatment.  Intrusion of contaminated water into the portable water supply , or regrowth problems  could also be contributory. A study done in Ibadan metropolis and Ile Ife in South Western Nigeria  which examined the microbiological quality of packaged water found that problems with filteration and purification procedures, malfunctioning of equipment and poor quality control system could also result in contamination.
Improper storage conditions on uncemented floors with direct contact with soil could also lead to contamination with parasites as found in the study done by Egwari  where it was discovered that enteric pathogens and Escherichia coli were not isolated from the sachet water but contaminated the surface of the sachet water.
Escherichia coli is the definitive indicator of recent faecal contamination of water. It is the only member of the total coliform group that is found in the faeces of warm blooded animals and humans and not in the environment. The presence of Escherichia coli in water is nearly always associated with recent faecal pollution and it is the preferred indicator organism for this purpose.  Faeces can also be a source of pathogenic viruses, protozoa and helminths. The absence of Escherichia coli in drinking water indicates that the water is free from intestinal disease causing pathogens.
While Escherichia coli is a useful indicator, it has its Limitations. Its absence may however be insufficient to justify the purity of the analysed packaged water from infection with pathogenic viruses, protozoa and helminths  which are now increasingly transmitted through drinking water  and cannot be identified by the currently used method. Faecal coliforms are a reliable indicator of the survival of most bacterial pathogens, but are less reliable as an indicator for the presence of viruses and parasites.  Attempting to assess routinely viruses and protozoans of public health concern in the developing world is currently expensive. This calls for more scientific research aimed at developing low cost technologies for detection of these pathogens in drinking water. 
Other organisms which may be more appropriate indicators of persistent microbial hazards include spores of enterococci and Clostridium perfringes.
These pathogens may be naturally present in the environment but are generally not harmful to individuals with intact immune systems but may be able to cause disease in people with impaired local or general immune defence mechanisms, such as the elderly or the very young, patients with burns or extensive wounds, those undergoing immunosuppressive therapy or those with acquired immunodeficiency syndrome (AIDS).  If water used by such persons for drinking or bathing contains sufficient numbers of these organisms, they can produce various infections of the skin and the mucous membranes of the eye, ear, nose and throat. Examples of such agents are Pseudomonas aeruginosa and species of Flavobacterium, Acinetobacter, Klebsiella, Serratia, Aeromonas and certain 'slow-growing' (non-tuberculous) mycobacteria. 
In this study, Klebsiella spp. was the most predominant organism isolated, this was similar to the findings of Chinelo.  Klebsiella spp. are natural inhabitants of many water environments, and may multiply to high numbers in waters rich in nutrients, such as pulp mill wastes, textile finishing plants and sugarcane processing operations. , They are known to colonize washers in taps and can grow in water distribution systems. Klebsiella spp. are also excreted in the faeces of many healthy humans and animals, and are readily detected in sewage-polluted waters. Klebsiella spp. are not considered to represent a source of gastrointestinal illness in the general population through ingestion of drinking-water. Klebsiella spp. detected in drinking-water are generally biofilm organisms and are unlikely to represent a health risk. The organisms are reasonably sensitive to disinfectants, and entry into distribution systems can be prevented by adequate treatment. ,
Pseudomonas aeruginosa made up 20% of the isolates found in this study which was similar to the findings of Oladipo.  Pseudomonas is a common environmental organism and can be found in faeces, soil, water and sewage. It can multiply in water environments and also on the surface of suitable organic materials in contact with water. ,,
Although Pseudomonas aeruginosa can be significant in certain settings such as healthcare facilities, there is no evidence that normal uses of drinking-water supplies contaminated with Pseudomonas are a source of infection in the general population. , However, the presence of high numbers of P. aeruginosa in potable water, notably in packaged water, can be associated with complaints about taste, odour and turbidity. Pseudomonas aeruginosa is sensitive to disinfection, and entry into distribution systems can be minimized by adequate disinfection. ,
Chromobacterium violaceum is a common inhabitant of soil and water in tropical and subtropical regions.  Occasionally, it can act as an opportunistic pathogen in animals and humans and the initial skin lesion can lead to multiple liver and lung abscesses and fatal septicemia. In some of these cases, the route of entry was through the broken skin, following contamination with soil or water.  C. violaceum has been reported to have more severe effects on people in a state of malnourishment, immune deficiency and who are immunocompromised. 
Proteus mirabilis made up 30% of the isolates found, this was similar to the findings of Okonko.  Proteus mirabilis are widely distributed as free-living organisms in soil and water in the natural environment. In humans, Proteus is found as part of the normal flora of the gut. Its main pathological role is in infections of the urinary tract, but it can also cause wound infections and septicaemia. 
| Conclusion and recommendation|| |
The results of this study showed contamination with microorganisms found naturally in water, soil or vegetation which indicates a problem with the overall quality of water production which could be due to no treatment or inadequate treatment, but showed no contamination with sewage or animal waste as evidenced by the absence of faecal coliforms, Salmonella, Shigella and Vibrio cholera.
Microbiological assessment of drinking water quality should be done periodically with the regulatory body NAFDAC ensuring good quality assurance and maintenance of internationally defined drinking water standards. There should be regular monitoring, inspection and sanctions by regulatory bodies to enforce existing water regulations.
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