Molecular identi ﬁ cation of biological contaminants in different drinking water resources of the Jazan region, Saudi Arabia

Drinking water quality plays a remarkable role in human infections and diseases. This study used polymerase chain reaction (PCR) techniques to detect bacterial pathogens. In addition, a physicochemical analysis was performed on drinking water samples from several sources. A total of 123 drinking water samples were collected from different areas in the Jazan region in Saudi Arabia: ground water (40 samples), bottled water (15 samples), tap water (52 samples), and water puri ﬁ cation shops(16samples).Toisolatethebacterialpathogens,thewatersampleswerespreadonNutrientand MacConkey agar media, and the grown pathogens were then identi ﬁ ed by the 16S ribosomal RNA technique. In 87 (70.7%) of the 123 drinking water samples, there was no pathogen growth on the two-culture medium. However, 36 (29.3%) of the samples were found to be contaminated with bacteria. The physicochemical analysis indicated that the water samples were within the Saudi drinking water standards. The bacteria were resistant to Cefotaxime, Cefotaxime/Clavulanic acid, Erythromycin, Penicillin G, Rifampin and Sulfamethoxazole – Trimethoprim, respectively. The ﬁ ndings suggest that in Jazan, bottled water is a safer source of potable water than tap water. The contamination in the water may be occurring at the reservoirs rather than the water sources. Drinking water quality plays a remarkable role in human infections and diseases. This study


INTRODUCTION
Access to safe drinking water can improve health, productivity, and the ability to earn a living. Sustainable development and poverty reduction can be achieved by the availability and accessibility of clean, fresh water (Al-Bratty et al. ). The most suitable water for human use originates from rivers, ground water, aquifers, lakes, and waterways. Because water is in short supply in Saudi Arabia, groundwater is the main source of the water that is suitable for human ingestion (Alhababy & Al-Rajab ).
Jazan is located in the southwestern part of Saudi Arabia (16 53 0 21″N 42 33 0 40″E). It is approximately 13,457 km 2 , and according to the 2010 census, the population is 1,365,110. It is considered to be the most densely populated area in Saudi Arabia (Al-Hatim et al. ). In Jazan, the potable options are mainly bottled water or water available in plastic containers after being processed at private water treatment stations (hereafter referred to as water purification shops). In addition, the local government in Jazan supplies treated desalinated sea water mixed with rain water to homes through pipes. Several studies have reported on the physical and chemical contamination of groundwater by wastewater, pesticides, fertilizers, and industrial waste (Ashbolt ). The results of that study showed that the water quality of the samples was within the World Health Organization's (WHO's) guidelines.
Human enteric pathogens are known to cause waterborne diseases. Enteric pathogens fall into three major groups: viruses, bacteria, and protozoa (Desouky et al. ). Their fates can take many potential routes in the water environment.
Bacteria are considered to be among the major microorganisms responsible for waterborne disease outbreaks. In many habitats, they are the most successful forms of life. A wide variety of bacterial pathogens have been detected in water sources, and many are enteric in origin. They include Campylobacter jejuni, enterotoxigenic Escherichia coli, Shigella spp., Vibrio cholerae O1, Salmonella typhi, enteropathogenic E. coli, Aeromonas spp., V. cholerae O139, and enterotoxigenic Bacteroides fragilis (Fricker ).
The common assessment of the microbial quality of drinking water has been based exclusively on culture techniques (Gantzer et al. ). Because these methods do not allow for the detection of specific water pathogens, 'indicator' bacteria exhibiting the possible presence of pathogens are monitored. Through the indicator method, which usually requires cultivation on Nutrient media, a reliable result can be obtained in less than 1 day (usually 3-7 days are required). However, by the time the results are available, the pathogens might have spread throughout the water distribution system. Therefore, the development of methods for the efficient monitoring of water supplies in order to detect the presence of microbial pathogens is essential for protecting public health and maintaining consumer confidence (Strauba & Chandlerb ).
Because of its high sensitivity and specificity, the polymerase chain reaction (PCR) is the most commonly employed molecular tool. It was developed for the detection of strictly opportunistic pathogenic bacteria (Salmonella, enterohaemorrhagic Escherichia coli, and Aeromonas hydrophilia) in raw and treated water (Tekpor et al. ). Compared to the traditional culture techniques, the PCR method has enhanced specificity, sensitivity, simplicity, and speed. The results are available in 24-48 h (WHO ).
Through the use of PCR techniques, the study aimed to develop a rapid, sensitive, and specific method for detecting specific pathogenic bacteria in the drinking water from sources such as groundwater, commercially bottled water, tap water, residential water tanks, and water purification shops in Jazan. A review of the literature suggests that the present study is the first microbiological investigation of multiple drinking water sources in Jazan.

Study area and water sample collection
Twelve locations in Jazan were selected for this study ( Figure 1). One hundred and twenty-three samples were collected from several sources: ground water, water purification shops, commercially bottled water, and tap water from mosques, offices, and houses. All of the water samples were collected in sterile plastic containers as reported by the American Public Health Association (APHA) () and transported in field coolers to the laboratory. The samples were immediately analysed and/or stored at 4 C for up to 2 days until use.

Physicochemical analysis of the water samples
The pH and water conductivity of the water samples were measured using a (Jenway, UK) electrode. Chloride and fluoride concentrations were estimated with a Sherwood chloride analyzer and EXTECH fluoride meter, respectively.

Isolation and count of bacteria
A total of 100 μl of water samples was spread on Nutrient and MacConcky agar as described by Abada et al. (, ).
The plates were incubated at 37 C for 24 h; the grown bacterial colonies were counted as CFU/ml, and a single colony was streaked on Nutrient agar slants and kept at 4 C.

Isolation of chromosomal DNA
One bacterial colony was suspended in 1 ml of lysis buffer and 15 μl of proteinase K (200 μg/ml), then the mixture was vortexed. The mixture was incubated at 56 C for 30 min, followed by 95 C for 10 min. An equal volume of ice-cold isopropanol was used to precipitate the DNA. Then, the DNA pellet was washed twice with 70% ethanol, dried, and resuspended in 50 μl of TE buffer (Abada et al. , ).

16S rRNA identification of bacterial pathogens
The 16S rRNA universal primers named 27F 5 0 (AGAGTTT-GATCCTGGCTCAG)3 0 and 1492R 5 0 (TACGGTTACCTTG TTACGACTT)3 0 were used to amplify the genomic DNA of the bacterial isolates. The PCR was preformed according to the instruction manual of Qiagen (PCR Kit). The PCR programme was 1 min of denaturation at 94 C, followed by 25 cycles of 96 C for 1 min, 55 C for 1 min, and 72 C for 1 min, with a final expansion under 72 C for 10 min. The PCR product was analysed on 1% (w/v) agarose gels. A 100 kb marker was used as a DNA marker using TBE as a buffer. Finally, the PCR product was purified using a QIAquick PCR purification kit (Qiagen) and eluted in 50 μl Tris-HCl before the sequence (Abada et al. , ).

Sequencing of the PCR product and gene homology
The purified PCR products were sequenced using a PRISM BigDye Terminator v3.1 Cycle Sequencing Kit. The sequencing primers were 785F 5 0 (GGATTAGATACCCTGGTA)3 0 Hi-Di formamide. The mixture was incubated at 95 C for 5 min, followed by 5 min on ice and then analysed by the ABI Prism 3730XL DNA Analyzer (Applied Biosystems).
The gene homology and related sequences were carried out by public databases BLAST at the NCBI server (http:// www.ncbi.nlm.nih.gov/blast/) (Abada et al. , ).

Antibiotic sensitivity test
The antibacterial sensitivity test was performed by the disc diffusion method on Nutrient agar plates, as described by

RESULTS
A safe water supply is essential. It is one of the eight principal components in primary health care. However, the inaccessibility and insufficient supply of this precious resource are still a significant issue in several communities (WHO ). The goal of this study was the evaluation of the quality of the drinking water in Jazan.
PCR techniques were used for the detection of specific pathogenic bacteria in the potable water from sources such as groundwater, commercially bottled water, tap water, residential water tanks, water purification shops, and tap water in houses. Such information could facilitate the determination of the presence of contamination and the possible influence of drinking water on infection and disease in the community.

Physicochemical analysis
All of the water samples were investigated with regard to the physicochemical parameters, such as pH, water electrical conductivity, and chloride and fluoride concentrations.
The pH values were 6.91-8.33, and the electrical conductivity was 169-1,809 μS/cm. While the chloride content was as high as 25 mg/l, the fluoride content was in a range of 0.1-0.5 mg/l. The parameters measured in this study were within the guidelines of the WHO () and the Gulf Cooperation Council Standardization Organization ().

Plate count of bacterial isolate
One hundred and twenty-three water samples were collected from the following sources in the Jazan region:  (Table 1).

Antibiotic sensitivity test
The antibiotic susceptibility tests found various levels of antibiotic resistance in the bacterial isolates in the water samples. All of the bacterial isolates showed 100% resistance to CEC and P. Klebsiella pneumonia, Pseudomonas aeruginosa, and Shigella resinvorans exhibited 75% resistance. Aeromonas enteropelogenes, Chryseobacterium echinoideorum, Shigella flexneri and Sphingomonas ursincola showed 62.5% resistance. Acidovorax temperans, Acinetobacter junii, Acinetobacter sp., Pseudomonas alcaligenes, and Pseudomonas oryzae showed 50% resistance.
Cronobacter helveticus and Rheinheimera mesophila exhibited 37.5% resistance. Finally, the lowest rate of resistance, 12.5%, was observed for Bacillus indicus (Table 2).  Maintaining the quality of water sources and protecting them from contamination pose serious challenges (Aksever et al. ). In the present study, the 16S rRNA analysis of the commercially bottled water showed it to be free of bacteria.

DISCUSSION
The highest bacterial contamination, 11% of all samples, was found in the water purification shop samples. Five bacterial isolates were found to belong to the genera Acidovorax, Rheinheimera, Acinetobacter, and Pseudomonas. A study by  supply of these communities is routinely monitored for compliance with the drinking water standards, and it is generally However, the Jazan tap water, which comes from the government distribution system, is potable. It is in accordance with the WHO guidelines (unpublished report).
Currently, many government buildings, mosques, and houses have water tanks (reservoirs) for storage and as a solution to the problem of low water pressure. The water supplied through the government distribution pipes is not used directly. It is stored in tanks prior to use. Although these storage tanks have many benefits, they can become contaminated by bacteria if they are not cleaned or maintained appropriately. For example, the sediment that collects at the bottom of unmaintained tanks has been found to be biologically contaminated (Khan et al. ).  In the present study, the bacteriological quality of bottled water was found to be better than that The results indicate that the drinking water quality from the investigated sources was within the WHO recommended limits. The evaluation of total water quality will require additional investigations to determine the presence of other kinds of pollutants, such as pesticides, polycyclic aromatic hydrocarbons, pharmaceuticals, and personal care products. This study revealed the presence of antibioticresistant bacteria in the Jazan water sources. Because of the public health implications, stringent measures must be taken to prevent the outbreak of disease. Finally, the findings suggest that bottled drinking water is the safest source of drinking water in Jazan; therefore, regular monitoring studies are highly recommended.

ACKNOWLEDGEMENT
We thank the Deanship of Scientific Research, Jazan University, KSA, for the financial support of this work (project number: JUP8/000228; Molecular identification of biological contaminants in different drinking water resources of the Jazan region, Saudi Arabia).