Abstract: The aim of this study was to evaluate bacterial quality and pathogenic organism presence in the Nile water (Blue, White and Main River Nile) as sources of drinking water in Sudan. The bacterial genera were isolated and identified using microbiological and biochemical method of evaluation. The most predominant aerobic microflora genera and species found in the Nile waters were Bacillus, Aerococcus, Micrococcus, Pseudomonas, Staphylococcus, Actinomycetes, Aeromonas and Streptococcus. Only two genera were identified to their species, Bacillus (B. cereus, B. subtilis, B. licheniformis and B. Penibacillus) and Streptococcus (S. faecalis, S. faecium). The most frequent coliforms isolated were Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae, Enterobacter agglomerans, Klebsiella oxytoca, Klebsiella planticola, Serratia spp. and Citrobacter spp. The results of bacterial genera isolated in this study indicate that, the Nile water was potentially contaminated with the pathogenic bacteria and therefore, presence of Escherichia coli could be public health risk.
INTRODUCTION
The Nile is probably the longest river of the world, although some sources say the Mississippi precedes it. The Nile, according to a popular wisdom in Sudan, is one of the cleanest rivers of the world, if not the cleanest. The Farmers lay on the Nile banks in north of Khartoum; they use irrigation water for bathing, food preparation of food, ablution and for drinking. If this water is not clean and free of microorganisms, it is almost certain to cause disease and the greatest microbial risks are associated with ingestion of water contaminated with human and animal excreta (Atiribom et al., 2007). It has been suggested most of the mortality and morbidity associated with water related disease especially in developing countries are due directly or indirectly to infectious agents who infect man (Obasohan et al., 2012).
Khartoum City, occupies a unique position in that it lies on the bank of the Nile at the junction of the Blue and the White Niles, the major tributaries of the most important river of the world. However, the growing city could have a negative effect on the pristine water of the Nile, because of increasing the urbanization on the Nile banks, a remarkable increase in industrial, agricultural and human activities and recreational activities. This situation increased the waste discharged into water bodies. Moreover, the wastes of buildings take their way into the Nile waters during the flood season. It has been observed that most factories located in Khartoum discharge their waste directly into Nile Rivers without any treatment. Therefore, microbiological monitoring is one of the most important methods to safeguard the river Nile. The microbiological quality of the water of the Blue Nile, White Nile and the Main Nile were provided in the mid-1980's. Abdel Magid et al. (1984), Mahgoub (1984), Mahgoub and Dirar (1986) and Dirar (1986) investigated the Nile water for faecal contamination and microbial pollution using samples from various points on the three rivers. The results of these studies indicated that the Nile water was contaminated with the indicators organisms, which reflected the contamination with human faeces. The aims of this study were to know the prevalence of coliform bacteria and bacterial genera in Nile water, so as to determine their risk in Nile water as one of the main sources of drinking water.
MATERIALS AND METHODS
Samples: A total number of 120 samples were collected from three Nile waters (Blue, White and Main River) from different locations on monthly basis for two years (2006-2007). Sterile screw-cap bottles were used to collect water samples from all sites. Then the samples were stored in ice containers and laboratory analysis began immediately after arrival.
Bacteriological analysis
Isolation and identification of dominant bacteria: The predominant bacterial
colonies were isolated from plate count agar done by pour plate method according
to Harrigan (1998); these isolates were purified by streaking
twice on nutrient agar and stored in a refrigerator, then further the identification
done through Biochemical tests Harrigan (1998). The genus
of Bacillus was identified to species level using BD BBL crystalTM
identification systems (Enteri/Nonfermenter ID Kit) according to the manufacturing
procedures, Germany.
Isolation and identification of Streptococci: Positive tubes of Azide dextrose test were selected and faecal streptococci were isolated by using Brain heart infusion agar (Barrow and Feltham, 1993).
Isolation and identification of coliform bacteria: Total coliforms and faecal coliform were determined by the Most Probable Number (APHA, 1995). Coliform bacteria were isolated by taking a loopful of positive BGB (Brilliant Green lactose-bile Broth) medium tubes (Oxoid) and streaking onto MacConkey agar, which was incubated at 37°C. Primary isolates were identified by Barrow and Feltham (1993) methods and then followed by IMViC tests for differentiation.
Isolation and identification of E. coli: Escherichia coli was isolated by taking a loopful of positive E. C medium tubes (Oxoid) and streaking onto MacConkey agar, which was incubated at 37°C. Primary E. coli isolates from MacConkey agar (pink colonies) were examined by using Eosin Methylene Blue Agar plate (EMB) (Oxoid) as a selective media for E. coli. Colonies with metallic green sheen were recorded as positive E. coli. Then metallic green sheen colonies were cultured into Colic Brilliance E. coli/Coliform selective media (Oxoid) for more confirmation. Blue or violet colonies were recorded as positive E. coli (Barrow and Feltham, 1993).
RESULTS
The predominant aerobic microflora genera isolated from Nile water samples are shown in Table 1 and Fig. 1-3.
In the Blue Nile, Fig. 1 illustrates that, Bacillus is the genus of the greatest proportion of isolates (78%) of Nile water samples. Aerococcus ranks second (67%), followed by Micrococcus (64%), Pseudomonas (47%) and Streptococcus (42%). Actinomycetes and Aeromonas (22% each) represented the smallest proportion.
| Fig. 1: | Predominant microorganisms isolates of the blue Nile water |
| Fig. 2: | Predominant microorganisms isolates of the white Nile water |
| Fig. 3: | Predominant microorganisms isolates of the main river Nile water |
In the White Nile, Fig. 2 illustrates that the genus which has recorded the highest proportion was also Bacillus (81%), followed by Aerococcus (73%), Micrococcus (62%) and Pseudomonas (38%). The genera Staphylococcus (26%), Streptococcus (22%) and Aeromonas (18%) recorded the lowest proportion. In the Main River Nile, Fig. 3 illustrates that, Bacillus gave the greatest proportion of the isolates (86%). Aerococcus was the second largest isolate.
| Table 1: | Morphological and biochemical identification tests of the aerobic microflora genera in Nile water |
| Table 2: | Results of morphological and biochemical tests of coliforms bacteria |
| Fig. 4: | Frequency isolates of Bacillus species of Nile water |
It was recorded (82%) of the isolates. Then followed by Micrococcus, Staphylococcus and Pseudomonas, they found in 62, 41 and 38% of isolates, respectively. The genera Streptococcus and Aeromonas were recorded the lowest proportion of the isolates. They are found in 24 and 13% of the isolates, respectively. The genus of Bacillus of three Nile water was identified to species level. Figure 4 shows that, B. cereus was the most common species (33%), followed by B. subtilis (27%), B. licheniformis (20%) and B. penibacillus (20%). The most frequent coliforms isolated from Nile water were Enterobacter aerogenes, Enterobacter cloacae, Enterobacter agglomerans, Klebsiella oxytoca, Klebsiella planticola, Serratia spp. and Citrobacter spp. (Table 2). Then the faecal coliform isolates were identified as two groups; Echerichia coli type I and type II using IMViC tests (Table 3).
| Table 3: | Results of morphological and biochemical tests of Echerichia coli isolates |
DISCUSSION
The results of this study indicate that, the most dominant three bacteria isolated from Nile water were Bacillus a typical soil inhabits organism, which represents the greater number of isolates and many species of this genus demonstrate a wide range of physiologic ability that allows them to live in many natural environments (Ahn et al., 2001). They are omnipresent in terrestrial and fresh water domain and are widely distributed in seawater (Ruger, 1989). Followed by Aerococcus, a typical airborne organism and have been isolated from different sources from air, vegetation, dust, hospital, water, human and animals (Ruoff, 2007; Vela et al., 2007; Volterra et al., 1986) and then Micrococcus a free organism of environmental existence (Brenner, 1984). Similar results were obtained by Ahmed (2005) and El-Tom (1997); they reported that Bacillus and Aerococcus were the two most common bacterial genera found in water. However, Elrofaei (2000) noticed that Bacillus and Micrococcus were the two most common genera found in drinking water. These results were in agreement with Oyeleke and Istifanus (2008) who reported that Gram-positive genera, especially the genus Bacillus were the most abundant bacteria isolated from Kaduna River in Nigeria. These results also agree with the findings of Hassan et al. (1995) who was found that Bacillus spp. in abundance in water. It thus reflects the predominance of spore formers in sands and soils. A among the other identified genera Pseudomonas is widespread in soil and water (Lightfoot, 2003). Streptococcus, Staphylococcus and Aeromonas were found in considerable numbers in water and they are non-faecal indicators of water quality. However, the existence of Aeromonas genus is the possible representatives of different sewage originating microbes in Nile water. Szczuka and Kaznowski (2004) were pointed that these bacteria are found in a wide range in the environment, particularly in surface water and sewage. Kueh et al. (1995) revealed that there is a significant relationship between Aeromonas levels and gastrointestinal illness.
The most abundant one genera (Bacillus) was identified up to the species-level Fig. 4 (B. cereus, B. subtilis, B. licheniformis and B. penibacillus) that deals with human food poisoning. It is reported that B. cereus was causes serious and potentially fatal non-gastrointestinal-tract infections and its pathogenicity, whether intestinal or non-intestinal, is closely associated to the production of tissue-destructive exoenzymes (Bottone, 2010). Other Bacillus spp. has more rarely been identified as agents of food borne diseases characterized by diarrhea and/or vomiting (Anonymous, 2005). However, B. subtilis has been involved in many cases of food poisoning (Gilbert et al., 1981; Kramer et al., 1982) as cited by Logan (1988) and therefore, the main symptom of food poisoning is acute vomiting, although diarrhea quite commonly follows. Similar species were identified by Bal et al. (2009) who reported that B. cereus, B. subtilis and B. licheniformis were the species identified from Bacillus genus isolated from seawater of Orissa coast in India. Contamination of water resources by microorganisms of faecal origin is a current world-wide public health concern. Coliform bacteria and faecal coliform bacteria were used as indicator bacteria of water contaminated with the faecal origin. In this study, the Nile water was faecal contaminated (Data not shown). Thus, the common genera coliform found were Enterobacter, Klebsiella, Citrobacter, Serratia and Echerichia. This finding agreement with LeChevallier et al. (1983) who stated that the common members of coliform bacteria are the genera of Escherichia, Enterobacter, Klebsiella and Citrobacter. However, the presence of Echerichia coli (type I and II) Table 3 indicated that the faeces of human and animal were taking place into the Nile water. Comparatively high numbers of coliform bacteria are discharged (2x109 coliforms/day/capita) in human and animal faeces (Bitton, 2005). Enterobacter species are important causing agents of hospital sand other infections (Iversen and Forsythe, 2003; Deal et al., 2007). Enterobacter infections are of concern because they are becoming increasingly common. Moreover, their antibiotic resistance is especially alarming (Anonymous, 2010). Faecal coliforms and fecal streptococci (including the sub-group enterococci) are traditionally used as the indicator organisms to guarantee microbiological safety of drinking water, natural water resources (Sidhu and Toze, 2009). Primary routes of coliform pollution of natural water sources comprises vegetation, flood, run-offs and untreated or partly treated waters, including domestic, agricultural and industrial wastes. Microbial contamination of the soil and natural water occurs from land application of sludge, which results from sewage and wastewater treatments (Sidhu and Toze, 2009). Although many coliform bacteria and other indicator organisms may not be pathogenic, their presence in water indicates the presence of potentially pathogenic agents, including viruses from the intestinal tract of warm-blooded animals (Haller et al., 2009; Noble et al., 2003). Thus, coliform bacteria, particularly faecal indicator bacteria, are used to evaluate the microbiological safety of water resources.
Bacteria levels in surface waters are frequently correlated with the levels of pathogenic organism in human, thus, those who interested with public health were concerned with the bacterial levels in surface waters. (USEPA, 1986). However, the Nile water was faecal contaminated and consumption of this water poses a risk to human health. So, further research is needed in Nile water for the presence of food borne pathogens.
CONCLUSION
From the results, it can be concluded that the Nile water (Blue, White and Main River Nile) were microbially contaminated. The presence of coliforms bacteria such as Enterobacter spp., Klebsiella spp., Serratia spp. and Citrobacter spp. indicates the existence of potentially pathogenic bacteria in water, especially Escherichia coli as the faecal indicator, could be risked for public health.