Detection of enteric bacteria in two groundwater sources and associated microbial health risks

The microbial quality of two groundwater sources (well and borehole) and associated risks were quantitatively assessed. Water samples from the selected borehole and well were collected over a period of 12 weeks (n1⁄4 48). The concentrations of Escherichia coli, faecal coliforms, Salmonella, Shigella, Clostridium, Bifidobacterium and Campylobacter were determined using standard microbiological methods, which involve the use of a membrane filter technique. The water samples were filtered through a 0.45 μmmembrane filter using vacuum pump pressure and plated on selective agar for the bacteria under test. The number of colonies of the bacterial growth observed after the incubation period was counted and recorded. The physicochemical properties of the water were determined using standard methods. The risk of Salmonella, Shigella, Clostridium and Campylobacter infections resulting from the ingestion of water from the borehole and well was estimated. The results showed that the levels of enteric bacteria in the borehole were higher than those in the well. The mean levels of E. coli in water from the borehole and well were 3.3 and 1.7 log10 cfu/100 ml, respectively, and exhibited a negative relationship with salinity (r1⁄4 0.53). The estimated risks of infection associated with the pathogens in water from the borehole and well were greater than the acceptable risk limit of 10 4 and followed this order Clostridium< Salmonella< Campylobacter< Shigella. The findings of this study suggest recent and continuous faecal contamination of the two groundwater sources, thus exposing the residents relying on the water for drinking to potential risks of gastrointestinal infections.


INTRODUCTION
Groundwater is present in the underground layer of the Earth's crust and constitutes about 37% of the Earth's nonocean water (Trenberth et al. ). Water from groundwater sources represents approximately 50% of drinking water in the world, because it is usually less exposed to contamination, pathogen penetration and evaporation, thus making it more stable and suitable for supply when compared with surface waters (Zektser & Everett ). Organization. Coliform bacteria are always found in the gastrointestinal tracts of humans and animals, thus making them a candidate for assessing the quality of waters used for drinking and other domestic purposes (Kolarević et al. ). The detection of E. coli, Salmonella, Shigella or Clostridium perfringens in water from groundwater sources is sufficient evidence that the water is not safe for drinking, except when treated. The presence of these bacteria in water often reflects faecal pollution that may originate from different sources (Olajubu & Ogunika ; Olalemi & Dauda ).
Furthermore, the physicochemical properties of water such as temperature, pH, turbidity, electrical conductivity, salinity, total dissolved solids and dissolved oxygen (DO) are essential parameters to consider in the evaluation of water quality, because the intrinsic properties of water may be used to assess the nature and state of water in terms of quality (Kolarević et al. ). This study set out to determine the microbial quality of two groundwater sources (borehole and well) used for drinking and other domestic purposes in Akure, Nigeria. The objectives of the study were to determine the level of faecal contamination in the borehole and well; evaluate the relationship between the physicochemical properties of water from the borehole and well and the amount of enteric bacteria; and quantitatively assess the microbial risks of the pathogens in water from the borehole and well.

Sampling site and collection of samples
Akure is a city in south-western Nigeria and is the capital of Ondo State (Figure 1). Akure is situated at 7 18 0 29.8N and 7 12 0 46.5N latitude, 5 07 0 34.6E and 5 15 0 38.5E longitude and 396 m elevation above the sea level. The city has a human population of over 800,000. The borehole is located at Red-roof Akad junction, and the well is situated in Apatapiti; both groundwater sources are located around the waste dumpsites (about 20 m from the borehole and well).
The well was shallow and uncovered, which exposed it to various kinds of chemical contamination that make the physicochemical properties of water unstable, such as the pH and electrical conductivity. Also, the temperature of the well environment influences the temperature of the well water. Since the well was uncovered, rain water dropping into the well reduced its salinity and temperature and also added other chemical ions from the atmosphere into the water. The borehole was deeper, which caused the temperature to be low, and its storage tanks were not adequately maintained; the water pipes and storage tanks were not constantly washed to get rid of chemical deposits and biological contaminants, which influence the taste, acidic pH and other physicochemical properties of the borehole water. The temperature of the water was determined on-site during sample collection using a mercury-in-glass thermometer.
The pH, electrical conductivity, salinity, total dissolved solids, turbidity and DO of the water samples were determined using a multi-parameter analyzer (HI98194, PH/ ORP/EC/DO).

Microbial risk assessment
Salmonella, Shigella, Campylobacter and Clostridium are considered as pathogens that may be responsible for gastrointestinal illness in humans. Salmonella is the aetiological agent of typhoid fever and non-typhoidal salmonellosis.
Humans may be infected through the ingestion of 10 3 and 10 5 cells (Mahendra & Uma ). Shigella causes shigellosis in humans, with symptoms ranging from mild to severe and acute diarrhoea. The ingestion of 10 to 100 viable organisms may result in infection (Crockett et al. ).
Campylobacter is the aetiological agent of Campylobacteriosis, also known as Campylobacter enteritis, which presents symptoms such as fever, headache, abdominal pain, muscle ache, nausea, vomiting and diarrhoea. C. jejuni has been reported to be responsible for more than 80% of Cam- The beta-Poisson model (Equation (1)) was utilized to determine the probability of infection associated with exposure to Salmonella, Campylobacter and Shigella in water from the borehole and well, while the exponential model (Equation (2)) was utilized to determine the probability of infection associated with exposure to Clostridium in water from the borehole and well. The annual probability of infection (Equation (3)) as a result of consuming water from the borehole and well was also evaluated.
where P i is the probability of infection; α is a parameter defining the dose-response curve, β is an alternative parameter defining the dose-response curve, r is the rate of natural increase in the population of organism and N is the exposure (colony-forming unit); and P A is the annual probability of infection.
For exposure assessment, the minimum human health risks from using the water from the borehole and well for drinking and other domestic purposes were estimated. An intake of 100 ml was assumed for intentional consumption and intakes of 1 and 10 ml were assumed for accidental consumption as the goal for estimating the minimum human health risks from the ingestion of water from the borehole and well.

Statistical analysis
Data obtained were transformed to log 10 and examined

RESULTS
Detection of enteric bacteria in water from the borehole and well The mean concentrations of E. coli in water from the borehole and well were 3.7 and 1.7 log 10 cfu/100 ml, respectively, while those of faecal coliforms in water from the borehole and well were 4.0 and 2.4 log 10 cfu/100 ml, respectively. Similarly, the mean levels of Salmonella in water from the borehole and well were 3.2 and 2.3 log 10 cfu/100 ml, respectively, while those of Shigella in water from the borehole and well were 3.3 and 2.3 log 10 cfu/100 ml, respectively. In addition, the mean concentrations of Bifidobacterium in water from the borehole and well were 4.3 and 2.4 log 10 cfu/100 ml, respectively, while those of Campylobacter in water from the borehole and well were 3.7 and 2.8 log 10 cfu/100 ml, respectively. The mean concentrations of Clostridium in water from the borehole and well were 3.2 and 3.1 log 10 cfu/100 ml, respectively.
Of all the bacteria, Bifidobacterium had the highest concentration in water from the borehole, whereas Clostridium had the highest concentration in water from the well. On the other hand, Salmonella and Clostridium had the least concentrations in water from the borehole, whereas E. coli had the least concentration in water from the well ( Figure 2).

Physicochemical characteristics of water from the borehole and well
The mean values of temperature of water from the borehole and well were 24.3 and 28.1 C, respectively, while those of pH of water from the borehole and well were 7.2 and 6.6, respectively. Similarly, the mean values of electrical conductivity in water from the borehole and well were 137.6 and 163.5 μS/cm, respectively, whereas those of total dissolved solids in water from the borehole and well were 70.1 and 76.7 mg/l, respectively. In addition, the mean values of salinity in water from the borehole and well were 0.16 and 0.07 ppt, respectively, whereas those of DO in water from the borehole and well were 2.3 and 3.6 mg/l, respectively.
The mean values of turbidity of water from the borehole and well were 1.9 and 2.5 NTU, respectively (Table 2).

Probability of infection from the consumption of water from the borehole and well
The probability of Salmonella infection from the consumption of 1 ml of water from the well and borehole ranged   7.3 × 10 À11 to 3.4 × 10 À10 and from 3.6 × 10 À11 to 5.8 × 10 À10 , respectively. The consumption of 10 ml of water from the well and borehole revealed the probability of infection that ranged from 7.3 × 10 À10 to 3.4 × 10 À9 and from 3.6 × 10 À10 to 5.8 × 10 À9 , respectively. The probability of Clostridium infection from the consumption of 100 ml of water from the well and borehole ranged from 7.3 × 10 À9 to 3.4 × 10 À8 and from 3.6 × 10 À9 to 5.8 × 10 À8 , respectively. The mean probability of Clostridium infection from the consumption of 100 ml of water from the well was very low, at 2.4 × 10 À8 and 3.0 × 10 À8 from the borehole (Figure 3). The model predicts the probability of infection with exposure to a single CFU of Clostridium in water from the groundwater sources at 1.82 × 10 À11 . Based on these predictions, the risks of infection associated with the pathogens in water from the borehole and well were in this order Clostridium < Salmonella < Campylobacter < Shigella. In addition, the risks of infection from the consumption of water from the borehole were greater than those from the well.

DISCUSSION
The microbial quality of the two groundwater sources (borehole and well) used for drinking and other domestic purposes in Akure, Nigeria, was determined. The influence of the physicochemical characteristics of water on enteric bacteria detected in the water from the borehole and well was evaluated; and the risks of infection associated with the usage of the water from the borehole and well for drinking were quantitatively assessed. The concentration of E. coli in water from the well was lower than those observed by Osvalda et al.
() in water from 21 wells in Gargano, southern Italy. It was also higher than those observed in six wells (W1-6), but lower than those observed in three wells (W7-9) in the In the present study, an intake of 100 ml of water was assumed for intentional consumption, and intakes of 1 and 10 ml of water were assumed for accidental consumption as the goal for determining minimum human health risks.
The World Health Organization estimated the highest acceptable risk limit of enteropathogen infection in drinking water to be 10 À4 (WHO ). The mean probabilities of Salmonella, Shigella and Campylobacter infections from both accidental and intentional consumption of water from the borehole and well were higher than the acceptable risk limit, whereas the mean probability of Clostridium infection from accidental and intentional consumption of water from the borehole and well was lower than the acceptable risk limit. In the study area, a human population of approximately 1500 relies on the water from the borehole and well for drinking and other domestic activities. Assuming that there was uniform risk and if each person in the population was exposed to 1000 CFU (3 log) of Salmonella or Shigella or Campylobacter in the water from the borehole and well once in a year, the annual cases of infection associated with exposure to Salmonella would be predicted to be 20 cases; those associated with exposure to Shigella would be predicted to be 290 cases and those associated with exposure to Campylobacter would be predicted to be 240 cases. The risks of infection for Shigella and Campylobacter were 10 times greater than those for Salmonella. This is a result of the lower infective ability of Salmonella compared with those of Shigella and Campylobacter, since the population is predicted to be exposed to the same number of colonies of the bacteria.
In order to reduce or eliminate the risks of infection associated with the usage of water from the borehole and well for drinking, adequate treatment of the water must be a routine exercise. This will protect residents relying on the water sources from gastrointestinal illnesses. This will also reduce morbidity and ill health from diarrheal diseases in children under 5 years. Sinking boreholes and digging wells near septic tanks, sludge systems and waste dumpsites should be discouraged. In addition, boreholes and their storage tanks must be adequately maintained and wells must be covered properly.

CONCLUSION
The findings of this study demonstrated that the two groundwater sources (borehole and well) used for drinking and other domestic purposes do not have high microbial quality.
The physicochemical characteristics of water from the borehole and well have a correlation with the level of enteric bacteria in the water sources. The risk of Salmonella or Shigella or Campylobacter infections from the ingestion of water from the borehole and well was greater than the acceptable risk limit, thus exposing the residents relying on the water for drinking to potential risks of gastrointestinal illness. The high rates of enteric diseases reported in the communities where the borehole and well are located are the ones associated with Salmonella, Shigella and Campylobacter. These pathogens have a low infective dose, which reflects in the high cases of enteric diseases in these communities.