Identification of Bacterial Species in Kuwaiti Waters Through DNA Sequencing

With an objective of identifying the bacterial diversity associated with ecosystem of various Kuwaiti Seas, bacteria were cultured and isolated from 3 water samples. Due to the difficulties for cultured and isolated fecal coliforms on the selective agar plates, bacterial isolates from marine agar plates were selected for molecular identification. 16S rRNA genes were successfully amplified from the genome of the selected isolates using Universal Eubacterial 16S rRNA primers. The resulted amplification products were subjected to automated DNA sequencing. Partial 16S rDNA sequences obtained were compared directly with sequences in the NCBI database using BLAST as well as with the sequences available with Ribosomal Database Project (RDP).


Introduction
Bacteria are key microbes in marine microbiology that play fundamental roles in the ocean environment. Apart form being the base of the food chain, supplying more than half of the world's oxygen, controlling the role of marine nutrients, and being major processors of greenhouses gases, they are also rapidly responsive to changes in their ecosystem, making bacteria an ideal indictor of marine change [1] [2]. One such case is the microbialization of bacteria in contaminated waters over the past decade. The growing pathogenicity of marine microbes can cause disease in both marine and human life. Upon contact or consumption of specific bacteria, humans can experience cramps, nausea, diarrhea, tuberculosis, legionella pneumonia, typhoid, and cholera [3]. Bacterial diseases of fish include-but are not limited to: hemorrhagic septicemia, vibriosis, and columnaris [4]. The susceptibility of infection from pathogenic bacteria reinforces the importance of water purification before industrial, agricultural, or domestic applications. It is also ideal that water treatment processes use purification methods that limit leftover waste, as brine sediments often contain pathogens not killed off during treatment, and disposing them back into the ocean reintroduces the harmful bacteria into the aquatic environment in abundance.
There is growing interest in marine bacteria in the scientific world because pathogenic, or otherwise, their and identification serves many purposes. Firstly, extremophiles can be used as a measure of water pollution levels or aquatic condition because these species only grow in extreme environments. For example, halophilic bacteria only thrive in waters of high salt concentration, and are an indicator of overly high salinity. Next, being aware of pathogenic bacteria and locations were specific genera reside allows researchers, scientists and doctors to better prevent disease that could be contracted in the area-some of which can be very dangerous. Treatment plants can also better allocate purification methods for more effect water treatment with an understanding of the bacterial genera residing in specific marine regions. For example, bacteria killing methods of purification-such as electrochemical remediation, will be more efficient in treating water samples containing pathogens than other methods that do not eliminate bacteria. Employing target specific ways of water purification is more effective, efficient, and economical. Finally, bacterial identification is also important because many bacterial species such as: Marinobacter hydrocarbonoclasticus, Carboxydothermus hydrogenoformans, and Sphingomonas harbor properties that can be industrially useful in pharmaceutical, biotechnological, agricultural, chemical, and environmental fields. The purpose of this study is to investigate the diversity and properties of marine bacteria species from Salwa Beach (N1), North West (N2) and Sulaibikhat Beach (N3) by directly culturing seawater samples, PCR amplification of 16S rRNA gene, and DNA sequencing.

DNA Growth and Preparation
Bacteria from each beach was first grown by spreading 3μL of each sample onto separate marine agar plates, then incubated at 28°C in an bacteriological incubator for 24 hours. Grown bacteria cultures were then collected and mixed into cell pellets of 10μL of sterile distilled water. 48 samples of bacteria were collected from each beach.
Cultivation was also attempted on MacConkey, and nutritional agar plates. However, results indicated that bacteria could not be cultured on said plates, which made the identification of fecal coliforms based on culturing conditions and microscopical morphologies impossible.

PCR
Bacterial 16S rDNA was amplified from the extracted genomic DNA by using the following universal eubacterial 16S rRNA primers: forward primer 5' AGAGTTTGATCCTGGCTCAG 3' and reverse primer 5' ACGGCTACCT TGTTACGACTT 3'. An ABI PCR System 9700 was used to perform the PCR protocol in a volume of 24 μL of bacteria strain identification solution mix containing 10 x 2.5μL buffer, 1.25μL magnesium chloride, 1μL dNTP, 0.05μL primer 1, 0.05μL primer 2, 0.1μL enzyme taq, and 19.05μL of distilled water per 1μL of template bacteria. Thermal cycling consisted of a 10 minute initial denaturation at 95°C, 30 cycles of denaturation at 94°C for 30 seconds, 30 seconds of annealing at 60°C, and extension for 2 minutes at 72°C with a 10 minute final maintenance at 4°C [5].

Agarose Gel Electrophoresis
From each PCR product, an aliquot of 15μL was subject to 1.0% agarose gel electrophoresis containing 0.5 x TBE buffer (pH 8.0), and 5μL of ethidium bromide was used to stain the gel. Electrophoresis was conducted at 100 Volt, 400mA for 20-30 minutes with 0.8μL of 100bp DNA marker. After, the DNA bands were observed under ultraviolet light using gel documentation system [5].

Sequencing of 16S rRNA Genes
Sequencing reaction was carried out using ABI PRISM 310 Genetic Analyzer (PE applied Biosystems). For the sequencing reaction, Big Dye Ready Reaction DyeDeoxy Terminator Cycle Sequencing kit (Perkin-Elmer) was employed.

Data Analysis
Sequence analysis was performed with sequences in the NCBI database using BLAST as well as with sequences available with Ribosomal Database Project (RDP)  infections [7]. N1-021 Spirometra erinaceieuropaei: is a tapeworm that causes a parasitic infection called sparganosis [8]. N1-023 Hydatigera taeniaeformis: a parasitic tapeworm with cats as their primary definitive hosts [9]. N1-025 Schistosoma rodhaini: commonly known as blood-flukes, they are parasitic flatworms that cause schistosomiasis in humans-an infection considered by the World Health Organization to be the second most socioeconomically devastating parasitic disease infecting hundreds of millions worldwide [10]. N1-030 Xanthomonas oryzae pv. Oryzae: causes bacterial blight, a serious disease for rice, grass, and sedges worldwide [11]. N1-038 Diphyllobothrium latum: is the largest human tapeworm and the principle species of Diphyllobothrium causing diphyllobothriosis It infects humans through the somsuptiono of raw or undercooked seafood, but can also affect fish and other mammals [12]. N1-041 Schistosoma rodhaini: refer to N1-025 N1-045 Spirometra erinaceieuropaei: is a tapeworm that infects humans and domestic animals. In humans, is causes sparganosis [8]. N1-047 Plasmodium falciparum: is a protozoan parasite that is the deadliest of five human malaria species, being responsible for more than 85 % of all malaria cases, and has the highest mortality rate amongst all malaria species [13]. It can be inferred that Salwa beach is home to an abundance of tapeworms, as 6 out of 8 pathogenic bacteria identified were of such. Tapeworms, which generally reside in areas of inadequate sanitation is an indication of the area's contamination levels. Salwa beach is also a harbor. Many tapeworms could dwell inside the intestines of fish, so extra persuasions should be taken when eating undercooked seafood to prevent infections. Table.3. Bacteria with industrial uses found in Salwa Beach isolates after data analysis.

Isolate
Description N1-020 Sphingomonas: typically found in environments polluted with toxic compounds, they have the ability to utilize the contaminants as nutrients. Their biodegradative and biosynthetic capabilities have been utilized for numerous biotechnological applications including bioremediation of environmental contaminants for the production of extracellular polymers such as sphingans [7]. It is also effective in degrading degrade alkylated polyaromatic hydrocarbon (2-methylphenanthrene), commonly known as polystyrene plastic [14]. N1-031 Pseudoalteromonas sp. F497: capable of synthesizing biologically active molecules that produce compounds beneficial to eukaryotic organisms [15]. N1-046 Tetrapisispora phaffii: killer yeast that secretes a glycoprotein known as Kpkt that has antimicrobial activity with spoilage yeasts under winemaking conditions. It can be used to combat contaminating wild yeasts in food, to control pathogenic fungi in plants, and to develop novel antimycotics for the treatment of human and animal fungal infections in the medical field [16]. There is no pattern to the useful bacterial species identified in Salwa beach, but the particularly useful Sphingomonas was cultured and identified. Its unique ability to synthesize and degrade toxic compounds in water could be applied as an effective organism in water purification-a function that can be utilized in Kuwait's many water treatment plants.

North West Beach
Total bacteria counts: 112 should be taken into consideration, as it is the closest to a direct polluting source (industrial power plant) with notably less fish than in popular fishing regions such a Salwa Beach. This could possibly explain the sample's lack of pathogenic bacteria, as many species of pathogens reside in marine life. Table.6. Bacteria with industrial uses found in North West Beach isolates after data analysis.
Isolate Description N2-006 Marinobacter hydrocarbonoclasticus: are important to bioremediation because they can effectively degrade hydrocarbons-especially those found in alkanes and major components of oil as they are tolerant of high salinity and can grow aerobically and anaerobically [18]. The marinobacter hydrocarbonoclasticus' distinctive ability makes it ideal for treating recalcitrant pollutants [19]. N2-015 Bacillus pumilus: is used as an active ingredient in agricultural fungicides because its growth on plant roots prevents rhizoctonia and fusarium spores from germinating [20]. Bacillus pumilus proteases are also used in chemical, detergent, food, and leather industries as antimicrobials and antifungals. N2-019 Marinobacter hydrocarbonoclasticus: refer to N2-006 N2-029 Erythrobacter citreus: is a potential candidate for removing poisons because it can resist and reduce the toxic compound tellurite [21]. N2-030 Marinobacter hydrocarbonoclasticuss: refer to N2-006 3 isolates of Marinobacter hydrocarbonoclasticus was found out of the 48 bacteria samples taken, suggesting that the species is abundant in North West Beach waters. Its properties are very useful in purifying Kuwaiti water, as it is able to degrade hydrocarbons found in oil. Oil is an abundant resource in Kuwait that frequently pollutes the county's water sources, but is difficult to remove once contaminated. Therefore, the potential of using bacteria to purify this recalcitrant pollutant is exciting and could perhaps be more cost efficient than currently employed methods.

Isolate
Description N3-001 Carboxydothermus hydrogenoformans: is an extremely thermophilic bacteria that feeds almost entirely on toxic carbon monoxide and water, and produces hydrogen gas as part of its metabolism [27]. Its ability to rapidly and efficiently conduct this conversion makes it highly valuable for the future where hydrogen gas is being studied as potential biofuel [28]. N3-013 Exiguobacterium undae: According senior research engineer Wei-Min Wu in his report "Biodegradation and Mineralization of Polystyrene by Plastic-Eating Mealworms. 2. Role of Gut Microorganisms", injections of exiguobacterium undae into the gut of mealworms allows them to survive solely on a diet of polystyrene, and thus, can significantly help in the biodegradation and mineralization of plastic [29]. N3-014 Exiguobacterium undae: refer to N3-013 N3-015 Exiguobacterium undae: refer to N3-013 N3-034 Vibrio sp. B2-15-2 16S: is capable of producing biosurfactant that can be applied into environmental, technical, and potentially pharmaceutical industries [30].
A wide diversity of environmentally useful bacterial species was identified in Sulaibikhat Beach. The most abundant was Exiguobacterium undae. Its property of reverting mealworms to feed solely on polystyrene has enormous potential as an eco-friendly way of degrading plastic. Next, Carboxydothermus hydrogenoformans's diet of only toxic carbon monoxide and water, which later produces hydrogen gas, could provide an alterative for oil. Lastly, a useful species of Vibrio, sp. B2-15-2, was identified amongst its pathogenic counterparts and demonstrates the impressive biodiversity of marine bacteria and their respective properties.

Conclusion
The data analysis revealed that Kuwaiti water samples harbor extremely diverse bacteria belonging to over 53 bacterial genera. 24 genera were isolated in Salwa Beach, 17 in North West Beach, and 12 in Sulaibikhat Beach. In Salwa Beach samples, identified genera consisted primarily of Alteromonas (17.5% out of 40), North West Beach samples consisted of prominently Marinobacter (26.8% out of 41 sequences), and Sulaibikhat Beach samples consisted of mostly Vibrio (46.3% out of 41 sequences). Alteromonas and Marinobacter strains were found to be abundant and distributed in both Salwa and North West Beach, while none were cultured and identified in Sulaibikhat Beach.
Each sampled location exhibited specific patterns of cultivated pathogen bacterial species that reveals information regarding each beach's unique marine condition and life. In Salwa Beach, most pathogens were tapeworms, only Schistosoma mattheei were isolated from North West Beach, and 3 pathogenic species of Vibrio was found in Sulaibikhat Beach. Since tapeworms and Vibrio species are ubiquitous in aquatic environments copious in marine life, the intense mariculture in Salwa and Sulaibikhat Beach may be partially accountable for the high occurrence of these pathogens.
On the other hand, identified industrially useful bacteria demonstrated no pattern in accordance to sampled location, but similarities in valuable property. While a wide range of diverse, unrelated bacterial cultures were isolated in each beach, the majority of all had biodegradative and biosynthetic capabilities. For example, Sphingomonas found in Salwa Beach can utilize toxic compounds and contaminants as nutrients, Marinobacter hydrocarbonoclasticus found in North West Beach can effectively degrade hydrocarbons, and Carboxydothermus hydrogenoformans found in Sulaibikhat Beach can feed almost entirely on toxic carbon monoxide and water, while producing hydrogen gas. Upon further study, these species-along with the others identified, potentially offer incredible scientific breakthroughs in the usage of microorganisms in pharmaceutical, biotechnological, agricultural, chemical, and environmental fields.
In summery, the purpose of this study was to cultivate, isolate, identity, and determine properties of marine bacterial species in Kuwait from Salwa Beach (N1), North West (N2) and Sulaibikhat Beach (N3) through PCR amplification of 16S rRNA gene, and DNA sequencing of bacteria grown from collected water samples. The findings indicate towards a high level of macrobiotic diversity, and shed light on the ecological functions of both pathogenic and useful bacterial isolates-information that can be used towards precautionary causes or further inquiry and useful application.