Bacteriological Quality Assessment of Borehole Water in Ogbaru Communities, Anambra State, Nigeria

Ogbaru Communities are usually submerged by intense flood during the rainy season, but such flood recedes during the dry season. This natural occurrence makes the quality of the borehole water in the area questionable. The total bacterial, total coliform, faecal coliform and Vibrio cholerae counts of samples from fifteen boreholes in the communities were determined during both seasons using standard analytical methods. The values were 100-270 cfu/100ml;10-42cfu/100ml;0-28 cfu/100ml and 0-13cfu/100ml for total bacterial, total coliform, faecal coliform and Vibrio cholerae counts respectively during the dry season and 130-450 cfu/100ml; 25-86 cfu/100ml; 0-75 cfu/100ml and 0-18 cfu/100ml for the total bacterial, total coliform, faecal coliform and Vibrio cholerae counts respectively during the rainy season. Salmonella typhi (53.3%), Enterobacter aerogenes (53.3%), Pseudomonas aeruginosa (46.7%), Proteus vulgaris (46.7%), klebsiella variicola (26.7%), Escherichia coli (26.7%), Staphylococcus aureus (13.3%) and Vibrio cholerae (33.3%) were isolated during the dry season while S. typhi (60.0%), E. aerogenes (60.0%), P. aeruginosa (53.3%), P. vulgaris (46.7%). K. variicola (33.3%), E. coli (26.7%), S. aureus (13.3%), V. cholerae (46.7%) and Providencia sneebia (6.7%) were recovered during the rainy season. S. typhi occurred most frequently during both seasons. Total bacterial, total coliform and Vibrio cholerae counts were significant at 5% significance level using t-distribution. The boreholes analysed were polluted by bacteria and need adequate treatment such as sand filtration, chlorination and boiling before drinking to avert a public health hazard.


Introduction
Ground water is an increasingly important resource all over the world. It is the subsurface water that occurs beneath the water table in soils and geological formation that are usually saturated. It supports drinking water supplies, livestock needs, irrigation, industrial and many commercial activities. Ground water is generally less susceptible to contamination and pollution when compared to surface water bodies [1].Ground water pollution occurs widely from diverse sources such as water disposal facilities, industrial pollution, agricultural practices, atmosphere fallout, clearing of vegetation, over abstraction of ground water and excavation below the water table [2]. It not only affects water quality but also threatens human health, economic development and social prosperity [3].Microbiological health risks are associated with many aspects of water use, including drinking water in developing countries, irrigation, reuse of treated wastewater and recreational water use [4]. It has been reported that drinking water supplies have a long history of association with a wide spectrum of microbial infections.Some microorganisms are native or adapted to saturated sediment and rock and are present in significant numbers in most water supply aquifers and even deep geoclinal formations. Biofilm formation sometimes encourages the growth of bacteria in wells and groundwater. Events which occur between and within bacteria and plankton populations also affect water quality [5].
A lot of bacterial, viral and protozoan pathogens are causing waterborne infections. Some are primarily the enteric bacterial pathogens such as Vibrio cholerae, Salmonella spp, Shigella spp and recognized pathogens from faecal sources such as E. coli [6]. Several new bacterial pathogens such as legionella spp, Pseudomonas aeruginosa and Mycobacterium spp are found in drinking water usually in low numbers and grow within the distribution system biofilms [7].The evaluation of potable water supplies for coliform bacteria is important in determining the sanitary quality of drinking water. High coliform counts indicate a contaminated source,inadequate treatment or post treatment deficiencies. Many developing countries suffer from either chronic shortages of fresh water or the readily accessible water resources are heavily polluted [8].Ogbaru communities are located along the coast of River Niger in Anambra State of Nigeria. Agriculture including fish farming is the major occupation of the inhabitants of the area. The communities are usually submerged by flood during the rainy season .The flood recedes during the dry season leaving behind organic and inorganic materials including bacteria. It is therefore important that the bacteriological quality of water from the boreholes sited in the communities should be assessed to determine their potability during the dry and rainy seasons, which was the aim of this work.

Samples Collection
Samples for analysis were collected from fifteen boreholes located in Ogbaru communities during the dry (January -March 2018) and rainy (May -July 2018) seasons. The samples were collected from each of the boreholes in triplicates. The boreholes were sited at the following locations within Ogbaru communities. a) Odunze Street Atani b) Abiribose Atani c) Ujadimegwu Atani d) Ochuche e) Akili-Ozizor f) Cathedral Road Atani g) Umundu Ohita h) Odekpe i) Iyiowa Odekpe j) Okoti Odekpe k) Okpotuno Odekpe l) Atani Road Okoti m) Okpoko n) Imeogbe o) Anibueze Odekpe The samples were collected in sterile one -litre plastic containers with screw caps and legibly labeled. The containers were transported to the laboratory in an ice box and the samples were analysed within 24 hours of collection. All the plastic containers were sterilized with 70% ethanol and initially rinsed with sterile distilled water and thereafter with the water samples from the boreholes.

Samples Preparation
The membrane filtration apparatus was used. It was placed in position and the vacuum pump connected. The funnel was removed and a sterile smooth-tipped forceps was used to collect the membrane filter paper which was thereafter placed on the porous disc of the filter base.The sterile funnel was after that carefully and securely replaced on the filter base.The water sample was mixed thoroughly by inverting the container for twenty five times. One hundred milliliters of the water sample were poured into the funnel and slowly filtered through the membrane filter paper which was then carefully removed with a sterile smooth-tipped forceps and placed with the grid side uppermost, on a selective culture medium contained in a Petri dish, ensuring that there were no air bubbles trapped under the membrane filter paper.

Bacteriological Analysis
The total bacterial, coliform, faecal coliform and Vibrio cholerae counts were determined as done by Onuorah et al. [9].

Total Bacterial Count
The membrane filter paper was carefully placed with the grid side uppermost on a sterile nutrient agar plate. Duplicate plates were prepared and incubated in an inverted position at 35 0 C for 24 hours after which the bacterial colonies that developed were counted. Each colony was subcultured and stored on a nutrient agar slant for characterization and identification.

Total Coliform Count
The membrane filter paper was aseptically placed with the grid side uppermost on MacConkey's agar plate. Duplicate plates were prepared and incubated at 28 0 C for 48 hours after which the coliform bacteria that developed were counted and each colony subcultured and stored on a sterile nutrient agar slant for further studies.

Faecal Coliform Count
The membrane filter paper was carefully placed with the grid side uppermost on Eosin methylene blue plate. Duplicate plates were prepared and incubation was carried out in an inverted position at 28 0 C for 48 hours after which the faecal coliform bacteria that grew were counted, subcultured and stored on nutrient agar slants for characterization and identification.

Vibrio Cholerae Count
The method described by Onuorah et al. [9] was used. The membrane filter paper was aseptically placed with the grid side uppermost on Thiosulphate Citrate Bile Sucrose agar plate. Incubation was in an inverted position at 28 0 C for 48 hours after which the colonies that developed were counted, subcultured and later stored on sterile nutrient agar slants for further studies.

Characterization and Identification of the Bacterial Isolates
The isolates were characterized on the basis of their morphological, biochemical and molecular characteristics. Gram staining, catalase, coagulase, motility, oxidase, citrate utilization, indole, vogesproskaeur, methyl red, urease and sugar (glucose, lactose and sucrose) fermentation tests were carried out as done by Onuorah et al. [9]. Molecular characterization was done using the 16S rDNA sequencing. The isolates were identified as done by

Statistical Analysis
Pearson correlation coefficient was used to determine the relationship between the data obtained for both seasons while the critical value was determined using the t-distribution table.
The bacterial counts of the borehole water investigated in Ogbaru communities during the rainy season are shown in Table 2. The total bacterial counts ranged from 130 to 450 cfu/100ml; total coliforms, 25 to 86 cfu/100ml; faecal coliforms, 0 to 75 cfu/100ml and Vibrio cholerae, 0-18 cfu/100ml. The morphological and biochemical characteristics of the bacterial isolates in the borehole water investigated in Ogbaru communities during the dry and rainy seasons are presented in Table 3 Table 5 showed the occurrence of the bacterial isolates in the borehole water investigated in Ogbaru communities during the rainy season. Salmonella typhi and Enterobacter aerogenes were each isolated from nine (60.0%), Pseudomonas aeruginosa from eight (53.3%), Proteus vulgaris from seven (46.7%),klebsiella variicola from five (33.3%), Escherichia coli from four (26.7%), Staphylococcus aureus from two (13.3%), Vibrio cholerae from seven (46.7%) and Providencia sneebia from one (6.7%) of the boreholes studied.
The frequency of occurrence of the bacterial isolates in the borehole water investigated in Ogbaru communities during the dry season is shown in Table 6. Salmonella typhi had the highest frequency of occurrence of 31.3% while Staphylococcus aureus had the lowest frequency of occurrence of 0.8%. The frequency of occurrence of the bacterial isolates in the borehole water investigated in Ogbaru communities during the rainy season is presented in Table 7. Salmonella typhi also had the highest frequency of occurrence of 26.2% while Providencia sneebia had the least frequency of occurrence of 0.5%.

Discussion
The total bacterial counts of the borehole water were 100-270 cfu/100ml and 130-450 cfu/100ml during the dry and rainy seasons respectively (Tables 1 and 2). Two (13.3%) of the boreholes examined complied with the World Health Organization permissible limit of 100ml cfu/100ml during the dry season while none of them met the standard during the rainy season. This result agreed with the work of Onuorah et al. [9] that reported the total bacterial counts of 107-261and 119-275 cfu/100ml for the public hand-pump borehole water they studied in Onueke, Ezza South Local Government Area of Ebonyi State, Nigeria during the dry and wet seasons respectively.
The total coliforms isolated from the samples ranged from 10 to 42 cfu/100ml during the dry season and 25 to 86 cfu/100ml during the rainy season (Tables1 and 2). One (6.7%) of the boreholes analysed complied with the WHO standard of total coliforms of 10 cfu/100ml for potable water during the dry season while all the boreholes did not meet the standard during the rainy season. Similar results were obtained by Mustafa et al. [11] and Ngele et al. [12] who reported total coliform counts of 6x10 3 -145x10 3 MPN/100ml and 280-540MPN/100ml respectively for the borehole water samples they analysed.
The faecal coliforms ranged from 0 to 28 cfu/100ml and 0 to 75 cfu/100ml during the dry and rainy seasons respectively (Tables 1 and 2). Eleven (73.3%) of the boreholes sampled met the WHO standard of faecal coliforms of 0cfu/100ml for drinking water during both seasons. However, Onuorah et al. [9]; Abdullahi et al. [13]; Olajuba and Ogunika [14] and Nkamare et al. [15] reported the absence of faecal coliforms in the borehole water samples they examined in Onueke, Ebonyi State; the Science Department and Staff school of Nigeri State Polytechnic Zungeru Campus; AkungbaAkoko, Ondo State and Okutukutu, Bayelsa State respectively, all Nigeria.
The Vibrio cholerae counts of the boreholes were 0-13cfu/100ml and 0-18cfu/100ml during the dry and rainy seasons respectively (Tables 1 and 2). Ten (66.7%) of the boreholes studied complied with the WHO standard of Vibriocholerae of 0cfu/100ml for potable water during the dry season while eight (53.3%) met the standard during the rainy season. However, Onuorah et al. [9] reported that 86.7% of the boreholes they analysed in Onueke, Ebonyi State, Nigeria during the dry season complied with the WHO standard while 66.7% met the standard during the wet season.
The isolates from the boreholes during both seasons were Salmonella typhi, Enterobacter aerogenes, Pseudomonas aeruginosa, Proteus vulgaris, Klebsiella variicola, Escherichia coli, Staphylococcus aureus, Vibrio cholerae and Providencia sneebia. (Table 3) Onuorah et al. [9] isolated Klebsiella oxytoca, Proteus vulgaris, Vibrio cholerae and Pseudomonas aeruginosa from the borehole water they examined in Onueke, Ebonyi State, Nigeria while Abdullahiet al. [13] isolated E.coli, Klebsiella and Salmonella from the Staff school, Science Department and female hostel boreholes in Niger State Polytechnic, Zungeru campus.
Ibe and Okpalenye [17] detected Escherichia coli, Klebsiellasp, Enterobacter sp, Pseudomonas sp and Staphylococcus aureus in the borehole water they analysed in Uli, Anambra State, Nigeria while Uhuoet al. [18] isolated Escherichia coli, Klebsiella aerogenes and Salmonella sp from the borehole water in Peri-Urban areas of Abakaliki, Ebonyi State, Nigeria. Ukpong and Okon [19] also analysed public and private borehole water supply sources in Uruan Local Government Area of AkwaIbom State and isolated Escherichia coli, Proteus vulgaris, Klebsiella aerogenes and Staphylococcus aureus in the samples.
The isolates were detected in more of the boreholes studied during the rainy that the dry season. (Tables 4 and  5). This may be attributed to the prevailing environmental conditions during the rainy season which must have been more favourable to the isolates and flood water that must have introduced some organic and inorganic matters including bacteria into the boreholes.Salmonella typhi was the predomiant bacterium isolated from the boreholes during both seasons (Tables 6 and 7) indicating that the environmental conditions were most favourable to the organism than the other isolates. However, Pseudomonas aeruginosa and Escherichia coli were the predominant bacteria isolated by Onuorah et al. [9] and Uhuoet al. [18] respectively from the borehole water samples they analysed. The variation showed that any organism favoured most by the available nutrients and environmental conditions will predominate over others.
Salmonella typhi which is chiefly spread through contaminated water is a gram negative bacterium and a strong pathogen that causes systemic infections and typhoid fever in humans. It is usually found in both warm and cold-blooded animals and has caused many deaths in developing countries with poor sanitation.Symptoms of the disease caused by this bacterium include a sudden onset of high fever, headache, nausea, loss of appetite, diarrhea and enlargement of the spleen [20].
Enterobacter aerogenes is a gram negative, rod-shaped bacterium and an opportunistic pathogen that is ubiquitous in nature. They are numerous in faecal materials and thatresults in their wide distribution in soil, water, sewage, plants and humans. The bacterium has been reported to cause a range of health conditions including meningitis, bacteremia, pneumonia, urinary tract infections and eye and skin infections [21].
Pseudomonas aeruginosa is a gram negative, rod shaped asporogenous bacterium and an opportunistic nosocomial pathogen of immune compromised persons which is found in the soil, water, plants, the skin on moist parts of a healthy human body and most man-made environments. The bacterium is associated with urinary tract infections, wound infections, blood infections, dermatitis, osteomyelitis and community acquired pneumonias [22]. Proteus vulgaris is a gram negative, rod shaped bacterium that can infect the lungs or wounds and frequently causes urinary tract infections, severe abcesses and nosocomial infections [23]. It inhabits the intestinal tracts of humans and animals and can be found in soil, water and faecal matter.
Klebsiella variicola is a gram negative, non-motile, rod shaped bacterium and an opportunistic pathogen that has been isolated from cows suffering from bovine mastitis [24]. The bacterium has been associated with diseases in humans and cattle.Escherichia coli is a gram negative, rod-shaped bacterium that resides in the intestinal tract of humans and animals and is the best indicator of faecal pollution of water [25]. Its presence is an indication of faecal contamination of water which may be of human or animal origin as well as the presence of pathogenic bacteria, viruses and protozoans [26]. The bacterium has been reported to cause diarrheal diseases, urethrocystitis, prostatis and pyelonephritis.
Staphylococcus aureus is a gram positive coccus that is frequently found in the nose, respiratory tract and on the skin of humans and has been reported to cause a variety of illness such as pimples, impetigo, boils, pneumonia, abcesses, meningitis, endocarditis, sepsis and osteomyelitis [27].Vibrio cholerae is a comma shaped, gram negative bacterium and a facultative human pathogen that is usually isolated from the estuarine and aquatic environments and is the causative agent of cholera which is responsible for significant mortality and economic damage particularly in the underdeveloped countries [28].
Providencia sneebia is a gram negative bacterium that is associated with humans, many other vertebrates and invertebrates including dogs and insects such as Drosophila flies. This organism has been reported to be pathogenic [29].Higher bacterial counts were obtained during the rainy than the dry season which may be attributed to flood which must have emptied its contents including nutrients and bacteria into the boreholes assessed. This result conformed to the work of Obiri-Danso et al. [30] that reported higher bacterial counts during the wet than the dry season for the borehole water samples they assessed in some peri-urban communities in Kumasi, Ghana.
The total bacterial, total coliform and Vibrio cholerae counts were significant at 5% significance level using t-distribution indicating that seasonal variation had pronounced effects on their growth and proliferation in the boreholes water assessed.

Conclusions
The water from the boreholes assessed in Ogbaru communities were of poor quality in terms of the bacteriological parameters. The detection of total bacteria, coliforms and faecal coliforms in significant numbers indicated a breach of the sanitary integrity of the boreholes. The existence of indicator bacteria demonstrated that there may be pathogenic bacteria in the water assessed, therefore it is necessary that the boreholes must be treated before human use. Fumigation of the area after each seasonal flooding must be carried out. Household treatment such as filtration and boiling before use of such water must be encouraged. In addition, periodic bacteriological analysis of the borehole water is recommended to determine their potability.