Evaluation of Microorganisms of Drinking Water of Rafha City, Northern Borders, Saudi Arabia

Evaluation of Microorganisms of Drinking Water of Rafha City, Northern Borders, Saudi Arabia Abdel Haleem AA1,2*, Hemida SK1,3 and Abdellatif MM1,4 1Department of Biology, Faculty of Arts & Science, Northern Border University, Rafha, Saudi Arabia 2Biological Department, Faculty of Education, Ain Shams University, Cairo, Egypt 3Botany Department, Faculty of Science, Assiut University, Egypt 4Department of Zoology, College of Veterinary Medicine, Nyala University, Sudan


Introduction
Water is scarce and valuable resource in Saudi Arabia. Thereby, well or ground water is still and will continue to be one of the main sources of drinking water in Saudi Arabia, especially in rural and border areas. The microbial contamination of drinking water constitutes a major issue worldwide, because it is still a major source of infection and mortality. Evaluation of the microbiological quality of drinking water aims to protect consumers from illness, that due to protozoon's parasites, bacteria, fungi, algae and viruses. Nowadays, prevention of contaminated drinking-water-related-illness is still an important challenge [1]. Rafha-citizens basically obtained their drinking water from treated wells stored in public tanks and re-distributed through waterborne-pipes to houses. However, citizens are usually avoid using tap water as a source of drinking water, owing to their dis-satisfaction of taste, odour and colour of this water. Instead, they prefer drinking water-consumption of desalinated groundwater obtained from some desalinating-private companies others drink industrially bottled water. The famous protozoan parasites contaminating drinking water are Giardia, Cryptosporidium, Entamoeba histolytica and Cyclospora, that cause severe gastrointestinal disorders; namely giardiasis, cryptosporidiosis, amoebiasis and cyclosporiasis respectively [2]. Indication of the presence of disease-causing bacteria in drinking water is the coliform bacteria, that are non-pathogenic but associated with many diseases; from which Proteus, Escherichia coli and Enterobacter; indicating faecal contamination of water. Proteus mirabilis is associated with urinary tract infections [3].
In this concept, the coliforms constitute common intestinal commensal bacteria. This group contains important pathogens, as E. coli, which is the most prominent and causes disease when the immune-system is suppressed [4]. E. coli considered as an indicator of fecal contamination when found in drinking water [5]. Enterobacter species act as opportunistic pathogens.
Some algal genera are pathogenic, as; Anabaena, Microcystis and Oscillatoria. These genera secrete toxins, as Anabaena and Microcystis that secrete microcystin-LR and cylindrospermopsin, leading to liver inflammation, pneumonia, dermatitis, kidney damage and tumor growth and anatoxin-a group-toxin from Anabaena and Oscillatoria that causes nervous disorders [14]. SASO (Saudi Arabian Standards Organization) continuously evaluates drinking water standards for bottled, tap and well-waters to define a quality of water that re-inforce healthy population. These standards set limits for the permissible and maximum contaminants Algae: Aliquots of 100 ml from each water sample were allowed to settle overnight in sterilized conical flasks. Then after, solid materials from the bottom of the flasks were pipetted for examination. The experiment had been repeated thrice. Algal species had been identified using binocular microscope (Zeiss) [22].

Statistical Methods
Analysis of Variance "ANOVA": The mean data of analysis of variance "ANOVA", between four categories, protozoans, bacteria, fungi and algae to obtain significant differences, using the statistical SPSS-program.

Bacteria
Coliform Bacteria: The present work reported four gram-negative coliform bacteria-species; facultative anaerobic Proteus mirabilis, Escherichia coli, Enterobacter cloacae and E. aerogenes. Table 1 displays mean counted numbers, as CFU/100 ml, where the highest values of P. mirabilis, E.cloacae and E. aerogenes had been found in well-water (1318.7, 34.2 and 92.5 respectively), beside the highest value of E. coli in El-Razaz Company-water (700).

Aerobic bacteria
Gram negative bacteria: Beside the previous coliform bacteria; aggregated gram negative Acinetobacter haemolyticus and coccobacillus gram-negative Pseudomonas aeruginosa resulted.
The mean counts, as CFU/100 ml, displays the highest values of A. haemolyticus and P. aeruginosa in well-water (16.7 and 100000 respectively).

Gram positive bacteria:
Obligate aerobe Bacillus subtilis, rod level of parasites and the indicator-microorganism that endanger the health of consumers [15]. A substantial number of these standards are originated from the accurate World Health Organization internationalstandards for drinking water [16]. The main objective of this study is to assess the parasitological and microbiological status of drinkable water of Rafha City in Saudi Arabia.

Water sampling
The present studies were carried out from July to October, 2014, in Rafha City, Northern Borders, Saudi Arabia. The water-samples had been collected from three major water sources; ten wells, tap and three private desalinating companies; El-Shefaa, Bardy and El-Razaz. Aliquots of 100 ml from each water-sample were collected in sterilized conical flasks, provided with silica gel to keep dryness.

Enumeration of protozoans, bacteria, fungi and algae
Protozoan parasites: All samples preserved in 4% neutralized formalin solution, left to settle [17]. Then, supernatants were collected, filtered through 20 µm net mesh. Materials that retained by filter were then fixed with Lugol's solution. The protozoans were then counted by Sedwgwish Rafter counting method and identified (Cells/100 ml), using Olympus binocular compound microscope.
To confirm identification of Giardia lamblia and Cryptosporidium parvum, aliquots of 100 ml of water samples were filtered through a 47 mm diameter, 0.450 μm pore size membrane filter. Each material retained by the filter was fixed with Lugol iodine on a separate slide. Fresh preparations of 0.9% saline smear of samples were visually examined for parasitic cysts over approximately 100 fields and then subjected to cold acid-fast. Trichrome staining technique was then applied [18].

Bacteria-count
Coliform bacteria: Coliform bacteria were determined by incubation of samples into lactose broth as presumptive test. The test tubes are placed at 35°C for 24 hours for gas production. To confirm the presence of coliform, gas produced in incubation into Brilliant Green Bile broth at 35°C for 24 hours [19]. Water quality analysis was based on the most probable number of Cells/100 ml. The test had been repeated three times.

Aerobic and other facultative anaerobic bacteria:
One ml of each water sample was inoculated and spread on blood agar and nutrient agar plates. Then, plates were incubated at 37°C for 18 to 24 h for determination of bacterial cell count, as cells/100 mL, and for isolation and differentiation of various bacterial strains depending on their morphological descriptions. Different isolated bacteria were further identified by Bilog system (Biolog, Hayward, CA, USA). Experiment had been repeated three times [20].
Fungi: Sabouraud Dextrose Agar (SDA) was used as a culture medium to reveal the presence of fungi and chloramphenicol is used to increase selectivity against commensal microorganisms. For isolation of fungi from water-contaminated specimens, SDA-medium should be inoculated and the plates incubated at 28°C in an inverted position with increased humidity. For isolation of fungi, two sets of media should be inoculated at 28°C and a duplicate set at 35°C. All cultures should be examined 7-10 days for fungal growth, and should be held for 5 weeks before being reported as negative [21]. The test had been repeated three times.      El-Razaz Co. 0 0 0 0
It is worthy of mentioning that the fungus A. flavus appeared in all five water-sources followed by A. alternate in four sources. Concerning counts of the fungi, as CFU/100ml, the statistical analysis of variance "ANOVA" revealed low significant differences between the four sources; wells, tap and the three companies concerning Aspergillus fumigatus and Penicillium whereas rest of fungi show non-significant differences (Table 7).

Types of water
Respecting algal counts "cells/100 ml", the statistical analysis of variance revealed highly significant differences in the alga Merismopedia tenuissima between the present four sources (Table 7).

Discussion
The present work revealed several pathogenic microorganisms in three sources of water; 10 wells, tap and three private desalinating water-companies; including three parasitic protozoan-cysts, three genera of Coliform bacteria-group and some aerobic and anaerobic bacteria, five genera of fungi as well as three genera of algae.
In this respect, results of Al-Turki [23] reinforced the present results where he revealed that microbiological water quality results showed that 20% of the samples examined are contaminated with coliform bacteria (Escherichia coli, and Enterobacter aerogenes), indicating the necessity of water-sanitation of Hael's water prior to use.
WHO estimated that 1.8 million people die each year as a result of severe diarrhea caused by drinking contaminated water, where developing countries have the prevalence of giardiasis in patients with diarrhea is about 20% [24].
Concerning parasitic cysts of Giardia lamblia, Cryptosporidium parvum and Cyclospora cayatenensis, they contaminate water-sources causing severe gastrointestinal disorders. The maximum score of the present parasitic protozoans is for C. parvum and minimum count for C. cayetanensis.
Giardia-cysts resist chlorine leads to giardiasis, its symptoms are flu and severe gastrointestinal disorders, detected in 81% of raw water samples and 17% of filtered water samples in the United States [25]. In this respect, WHO declared that chlorine disinfection of drinking-water has limitations against the protozoan pathogens, in particular Cryptosporidium [26]. C. parvum is protozoan parasite causes Cryptosporidiosis in humans. Oocysts of C. parvum in 87% of raw water samples and 27% of drinking water samples in 15 Canadian regions [25]. Le Chevallier et al. reinforced the present counts of C. parvum-cysts which is higher than those of G. lamblia in all watersources [25]. Cyclospora cayetanensis is cyst-forming coccidian protozoan that causes a self-limiting diarrhea named cyclosporiasis, with symptoms range from watery, loose stool, weight loss, cramping, fatigue, vomiting, fever and nausea [26].
The maximum score of coliform bacteria are for Proteus and the minimum for Enterobacter. Proteus species causes wound infections, septicemia and pneumonia whereas E. coli produce potentially lethal toxins and causes food poisoning. Moreover, Uro-pathogenic E. coli is responsible for 90% of urinary tract infections [27]. Enterobacter causes opportunistic infections, where the urinary and respiratory tracts are the sites of infection [28].
The studies revealed 5 pathogenic fungal species isolated. The maximum count of fungal-species are for Aspergillus flavus and minimum for Cladosporium cladosporioides. The most common pathogenic species are Aspergillus fumigatus and A. flavus. The latter species produces aflatoxin-toxin that contaminating foods and considers carcinogen leads to allergic disease while A. fumigatus causes allergic disease leads to chronic pulmonary infections [33].
Alternaria alternate causes respiratory infections and asthma in humans with compromised immunity [34]. Hyalo-hyphomycosis that caused by Acremonium includes arthritis, osteomyelitis, peritonitis, endocarditis, pneumonia, cerebritis, and subcutaneous infection [35]. Exophiala jeanselmei causes mycetoma, localized cutaneous infections, subcutaneous cysts, endocarditis, cerebral and disseminated infections, beside phaeohyphomycosis [36]. The highest value of the algae are for Merismopedia tenuissima and minimum for Anabaena sphaerica. Anabaena sphaerica and Microcystis aeruginosa secrete both microcystin-LR and cylindrospermopsin toxins that leading to liver inflammation, pneumonia, dermatitis, kidney damage and tumor growth. Anatoxin-a group 3 -toxin is also secreted by Anabaena and Oscillatoria limnetica, that causes nervous disorders [14]. Concerning standard levels of microorganisms in drinking water, EPA's (Environment Protection Agency) MCLG declared that detection of protozoans, especially Cryptosporidium, is difficult and not technically feasible for routine analysis of human drinking water. The recommendation considered the proper way to control pathogenic protozoans is using an effective water treatment technique, such as reverse osmosis or ozonation.
According to EPA MCL, coliform must be less than one/100 mL. In this concern, the microbiological guidelines and standards for drinking water for E. coli are zero CPU count/100 ml [37]. In this respect, average count/100 mL of all water sources of the present work revealed considerable higher levels than those of EPA MCL and an apparent risk on Rafha's citizens-health.
The present investigation indicated that water sources of Rafha city are important contributor to transmission of contaminants to consumers.The present work recommends a future-field-application of selected specialized anti-parasites and anti-microbial to control the present resulting contaminators to gain considerable safe levels of drinking water.