There has  been  noticeable  increase  in  the  number  of Drinking and recreational  water health related publication between 1980 and 2015. Research productivity on water related diseases from Asia and Africa have equally witnessed an upward increase in the last few years (Sweileh et al., 2016).

Waterborne diseases are rampant in sub Saharan Africa due to lack of access to clean water and poor sanitation. In Nigeria, contamination of drinking water with pathogens has also been reported in several towns (Olowe et al., 2016). Waterborne disease outbreaks occur either when public drinking water supplies are not adequately treated after contamination with surface water or when surface water contaminated with enteric pathogens is used for recreational purpose (Johnson et al., 2003).

In developing countries, the two main water problems man contends with are the quantity and quality of water (Adeniyi, 2004; Olajuyigbe, 2010a). Only about 58 per cent of Nigerians have access to safe water (UNICEF with WHO, 2012). Thus, most households have to resort to drinking water from wells and streams especially in the rural and suburban communities. These water sources are largely untreated and generally harbour pathogens causing waterborne diseases such as cholera, typhoid fever and hepatitis (Rahman et al., 2001; Adekunle, 2004; Fenwick, 2006). 

Waterborne diseases are caused by pathogenic microorganisms which are directly transmitted when contaminated fresh water is consumed. Contaminated fresh water, used in the preparation of food, can be the source of water borne diseases. Many rivers, streams and wells worldwide are affected by faecal contamination leading to increased health risks to persons exposed to the water (Simmons, 1994; Obiri-Danso et al., 2009). Pathogenic bacteria that may be associated with faecal contamination include pathogenic strains of Escherichia coli, Campylobacter species, Salmonella species, Shigella species and Vibrio cholerae. In addition to these organisms causing human diseases, resistance to antibiotics has made treatment of the diseases they cause more difficult (Lamikanra and Okeke, 1997; Okeke et al., 2007).

According to the Nigerian Bureau of Statistics (NBS), cholera with symptoms of vomiting, watery stool, dehydration, fatigue, renal failure and occasional muscle cramps has been an extreme burden on Nigerians and often occurs rapidly and progresses to a large scale of outbreak (NBS, 2010).Newspapers in 2011 reported the death of one person and the hospitalization of 20 others following a cholera outbreak in Osogbo, the capital of Osun state. Also in 2012, a cholera outbreak at the Ede north and South Local government areas of Osun State left three people dead and 42 others hospitalized (Moses, 2012).

In Ile-Ife, between 2008 and 2010, scores of water borne disease incidence were reported in various primary health  centers  across  the  city.  Following  the  reported cases, a high prevalence of typhoid was indicated with over 1000 cases in the city as well as other water borne diseases like diarrhorea with about 400 cases and dysentery with about 200 cases. However, only few cases of cholera were recorded in Ile-Ife within those periods (Olajuyigbe, 2010b).

This research investigated the bacteriological quality of water from the Opa River and how its specific usage affected public health. The objectives included identifying potential pathogens, evaluating pathogenic load in the river and assessing the prevalence of water borne diseases in the neighbouring communities. All of this was done to understand how the activities of people impacted the bacteriological quality of the water and how the quality of the water in turn affected health in the locations of interest. This would be used to create a model for sensitization of local residents on harmful practices that affect surface water quality and invariably, health.

Study site

The Opa River’s source is in Esa-Oke in Osun-State and flows through many towns and villages before emptying into the Osun River at Asejire. Five different points (Figure 1) along the Opa River which flows through Ile-Ife were purposively chosen for sampling based on the presence of bordering communities along the river banks and the fact that the water from the river along those points was being actively used for domestic activities such as cooking, bathing and washing, small scale industrial activities, recreational activities and agricultural activities. The sampling points were Ajebamidele community, Alakowe area of Ile – Ife, the Abattoir located at Ede road popularly called Odo- Eran and the Saw mill locally called the Iso-Pako or Oke-Opa area.

Data source and sample size determination

Data used for this study were obtained from water samples collected from the five sampling locations along the Opa River as well as the responses of residents along the 500 m buffer zone created using Arc GIS 10.1. This was within the bordering communities along which the water samples were collected. Questionnaires were used to elicit information on communal behavior prevalent in the bordering communities in terms of water usage, water management, waste and sewage disposal methods and the prevalence of water borne diseases in the community.

The sample size was determined using the equation:

Necessary Sample Size = (Z_α²*σ²)/d² for an unknown population size (Smith, 2013), where Z-Score used was 1.645 based on the confidence level of 90%; standard deviation of 0.5 was used and a margin of error (Confidence Interval) of 8% was used.

Sample size = ((1.645)² x 0.5(1-0.5)) / (0.08)²

(2.7060 x 0.25) / 0.0064

0.6765/0.0064=105.7

Study design and selection of sampling points

The  study  employed a cross-sectional study design. Five sampling points were selected purposively taking into consideration accessibility, availability of human settlements, and anthropogenic activities (domestic, small scale industrial and agricultural activities). The site locations and coordinates of the sampling points (Table 1) were determined using a portable global positioning system (GPS) set.

Sample collection

Twenty five water samples were collected from pre-selected sampling points (Table 1) over a period of five weeks from 25^th August 2015 to 22^nd September 2015. At each sampling point, sterile specimen bottles were used to collect water for bacteriological analysis. All surface water samples were collected directly by removing the cover cap and with the mouth facing upstream, plunging it downwards below the water surface. It was then slightly tilted upwards for the container to be completely filled with water. The water samples were placed in an insulated cold box and immediately transported to the microbiology laboratory for analysis.

A total of 100 questionnaires were distributed to residents to inquire about water use pattern and waste disposal practices in the study areas. Data from the questionnaires were analyzed using the SPSS statistical tool for descriptive and inferential analysis.

Enumeration of total heterotrophic bacteria

The number of viable bacteria in water sample was estimated based on standard techniques recommended in the Methods for the Examination of Water and Waste Water (APHA, 1998). A ten-fold serial dilution was done to thin out bacterial population in samples. 1 ml of serially diluted water samples was withdrawn and plated aseptically into properly labeled petri dishes. Then, about 20 ml of nutrient agar kept in molten form at 45°C in water bath to prevent heavy condensation was poured over the water sample and mixed properly by tilting and rotating the Petri dish on the work bench to ensure formation of a uniform layer as well as to prevent spilling. This was first done in a clock wise direction, then in an anti-clockwise direction, later to the left and to the right and finally, forwards and backwards to ensure that the microbes were well dispensed in the medium. The agar plates were allowed to set after which the plates were inverted and incubated at 35 ± 2°C for 24 h. After the incubation period, the plates were observed for growth and selected for count. The cultured plates in which the number of colonies fell within the statistically accepted range of number of colonies (that is 30 to 300 COLONIES per plate) and their respective duplicates were selected and counted. The average count per plate was multiplied by the reciprocal of the dilution factor at that dilution and expressed as the number of colony forming units (cfu)  per milliliter of the original water sample. This represented the viable cell count. Counting of colonies on plate was done manually.

Enumeration of total coliform bacteria

The most probable number (MPN) technique as described by American Public Health Association was employed for bacteria enumeration. The tubes in the first row held 10 ml of double-strength MacConkey broth as presumptive medium while the tubes in the second and third rows contained 10 ml of single-strength presumptive medium. With a sterile syringe, 10 ml of original water sample was aseptically inoculated into each of the five test tubes in the first row and 1 ml of water sample was also inoculated into each of the five tubes in the second row. For the third row of the last set of five test tubes, a 1:10 dilution was carried out on the water sample after which 1 ml of the 1:10 diluted sample was inoculated into each of the five test tubes in the third row.  Durham tubes were inserted in all test tubes and corked for possible gas collection. The cultured tubes were carefully agitated to mix and dispense the inocula within the broth medium. They were incubated at 35°C for 72 h. Each tube was observed for microbial growth which was indicated by gas production and cultured media turbidity. The combined numbers of positive tubes in each set of arranged test tubes in order of least diluted to the most diluted was read out from the standard 5 tube MPN table to obtain the estimated number of coliform cells present in 100 ml of the original water sample. Also, positive broth cultures were sub-cultured on MacConkey agar, incubated for 24 h to isolate pure culture of organisms (APHA, 1998).

Isolation and identification of Vibrio cholerae

Double strength alkaline peptone water was used as enrichment medium for Vibrio Cholerae. After preparation, 5 ml of the double strength alkaline peptone water was dispensed into MacCartney bottles and sterilized at 121°C for 15 min. The peptone water was standardized to the required pH of 8.6 and 5 ml of the water sample to be cultured was then dispensed into each MacCartney bottle containing the sterile double strength enrichment medium using sterile syringe and incubated at 35°C for 24 h. After incubation, sub culture was made on thiosulfate-citrate-bile salts-sucrose (TCBS) agar plates and plates were incubated at 35°C for 24 h. Suspicious yellow colonies were gram stained and tested for motility and oxidase production according to the methods of  Theron et al. (2000).

Anti-microbial susceptibility test

Sensitivity of pathogenic bacteria to different  classes  of  antibiotics was assessed by disc diffusion method using ABTEK DT NEG-1 sensitivity discs.  Sterile Diagnostic Sensitivity Test (DST) Agar was prepared along with a 0.5 MacFarland equivalent standard of an 18-24 h old pure culture of the test organisms.  The standard organism solution (in peptone water) was flooded through the surface of the agar plate and allowed to dry for 15 to 20 min under the laminar air flow to pre-diffuse before sterile Gram negative antibiotics disc containing: Augmentin (30 µg), Ofloxacin (5 µg), Gentamicin (10 µg), Nalidixic acid (30 µg), Nitrofurantoin (200 µg), Cotrimoxazole (25 µg), Amoxycillin (25 µg) and Tetracycline (25 µg) were placed gently on the surface of agar plates with sterile forceps to avoid deformation of the agar. The plates were incubated at 35°C for 24 h, after which the clear zone of inhibition was observed and measured using a millimeter rule. The values obtained were then compared with a recent inhibition standard table and strains were classified as sensitive, intermediate or resistant (Cowan and Steel, 1993).

All identified potential pathogens were either coliforms, which are indicators of feacal contamination, or other potentially  pathogenic  bacteria capable of causing water borne illnesses. This agrees with the work of Abdu et al. (2013) who also reported the contamination of Opa River by coliforms and other pathogens after microbiologically analyzing water from five locations along Opa River.

E. coli, as observed in this study showed the highest frequency of occurrence (23.73%) across the five sampling points. The high occurrence of E. coli in water samples at all points indicates recent and probably continuous fecal contamination of the water from River Opa by human activities and unhygienic behaviours of people living at the river banks which were identified by questionnaires specifically as excreting and urinating by the river bank due to lack of proper sewage disposal methods in the bordering areas. Ihejirika et al. (2011), in a similar study, attributed the isolation of E.coli in relatively high percentages from the Imo River to human activities. S. typhi showed the second highest frequency of occurrence (12.71%) across all sampling points. it is also corroborated by the findings of Arvanitidou et al. (2005) in Northern Greek waters that were used for recreational purposes.  

V. cholerae was isolated from 8% of the water samples and its presence in water samples may have been due to animal contamination from birds, frogs, toads, crabs and fishes usually present in aquatic environments. The pathogen may have been introduced into the water from human feaces due to defecation along the river bank by dwellers of the bordering areas as reported by Ali et al. (2001). The presence of Shigella spp. (10.17%) in water samples might be due to the unsanitary condition of the areas  bordering   the   Opa   River   most    especially   in Ajebamidele and Ede Road Abattoir and secondary fecal contamination from intermediary sources. Ihejirika et al. (2011) reported a 100% isolation of Shigella spp from water sources in Ahiazu Mbaise and the implication of such result was reported as a possible outbreak of shigellosis  Emch et al. (2008).  

The occurrence of Citrobacter spp, Klebsiella spp., Enterobacter sp. and other members of the family enterobacteriaceea in water samples from Opa River indicates further feacal contamination of the river as these pathogens are normally found in the gastrointestinal tracts of humans. While investigating the possible diarrheal disease potentials of water sources in Ahiazu Mbaise, Eastern Nigeria, Esomonu et al. (2012) identified similar organisms and reported their ability to cause infections in humans. All the pathogens isolated showed a pattern of multiple antibiotic resistance to the common antibiotics used in the susceptibility testing. S. typhi for instance recorded a 75% resistance to the following antibiotics: Augmentin, nalidixic acid, nitofuratoin, amoxicillin, tetracycline and cotrimazole. Isolated V.cholerae also showed a 100% resistance to all antibiotic used.

The pathogenic load recorded at each sampling point exceeded the World Health Organisation (WHO) and Nigeria Industrial Standard (NIS) permissible levels. Across all sampling points at all sampling periods, THB count showed a mean value of 6.68×10⁶ cfu/ml of water sample analysed while TCB count, showed a mean value of 969.36 per 100 ml of water examined. These values extremely  exceed  the   Nigerian   standard   for  drinking water quality (NIS 2007) and the WHO (2007) recommended limits for potable water. The Nigerian standard for drinking water recommends that the maximum permitted level (MPL) for human drinking water must not exceed 10 cfu/ml for heterotrophic bacteria count, 0 cfu/100 ml for total coliform count and 0 cfu/100 ml for E. coli, feacal Streptococcus and C. Perfringes spores, respectively (NIS, 2007). Also, freshwater quality criteria for domestic supply require that faecal bacteria levels should not exceed a geometric mean value of 100 cfu/100 ml while the drinking water criterion is 1 cfu/100 ml (WHO, 2001).     

A high THB count is generally regarded as an indicator of poor biological quality of water (USEPA, 2003). The high levels of faecal bacteria recorded in this study as well as the presence of pathogens in the river clearly indicate that the Opa River receives faecal contaminants on a continual basis. This may be due to the fact that its tributaries are highly impacted by commercial, semi-industrial and domestic activities but the Ajebamidele and Ede Road tributaries may be considered as the most impacted due to direct sewage discharge from most homes in the Ajebamidele community into the river body and waste discharge from the dressing of carcasses at the slaughter house at the Ede Road Abattoir respectively.  

All of these practices may predispose members of the bordering areas to water borne illnesses. However, members of the Ajebamidele community may be more vulnerable due to the fact that the highest pathogenic load values were recorded at Ajebamidele and most isolated coliforms and other pathogens capable of causing water borne diseases like E.coli, Salmonella typhi and Shigella spp. showed highest occurrence frequency at Ajebamidele than the other four bordering areas. This, coupled with the water usage system obtainable at Ajebamidele may make dwellers more vulnerable to water borne diseases than in the other four areas. Also, symptoms related to water borne illnesses were only observed at Ajebamidele. This is substantiated by the findings of Nwidu et al. (2008) which reported the frequent diagnosis of cholera, dysentery, typhoid fever and diarrhea in the Niger Delta region of Nigeria due to high levels of feacel contamination in River Amassoma.

Based on the findings from this study, it could be said that River Opa, which runs through many rural and semi-urban communities in Osun State, is contaminated with a wide array of antibiotic-resistant bacteria and potential pathogens that could predispose its users to water borne diseases. Therefore, water from the river may be unsuitable for domestic engagements without appropriate treatment measures as it could  serve  as  a  pathway  for human contamination, most especially to dwellers located along its banks.

The authors have not declared any conflict of interests.