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Open Access
ELUCIDATION OF FUNCTIONAL INTERVENTION OF HEAT SHOCK PROTEIN 70 ON EXPRESSION PROFILE OF SELECTED PATTERN RECOGNITION RECEPTORS DURING THERMAL STRESS IN CATTLE
(Dept. of molecular and cellular engineering jacob institute of biotechnology, 2019) Sengar, Gyanendra Singh; Kant, Dr. Rajiv
Impending climate change scenario is a unanimously accepted reality. The climate change has a complex impact on the domestic animal production. Heat stress is one of the major factor directly or indirectly influences on the health and productivity of dairy cattle population. The least tropically adapted and most susceptible dairy cattle population will severely be affected by heat stress in coming years. Apart from short-term managemental methods to alleviate the heat stress impacts, understanding the genetic differences and molecular mechanisms of the stress response for innate resilience and thermos tolerance will be a significant long-term strategy for selection of thermos tolerant animals for increasing productivity. The theme of the thesis is an attempt to understand the climate resilient nature of our indigenous cattle breed especially in the times when climate change is becoming reality. The objectives of the proposed studies are (i) To study the differential expression profile of HSP70 under different thermal stress condition among native and crossbred cattle; (ii) To elucidate the functional analysis of HSP70 gene on the differential expression profile of certain pathogen associated molecular patterns (TLRS, NLRS) during thermal stress in cattle and (iii) To identify microRNAs associated with thermal stress in cattle. The expression level of HSP70(heat shock protein-70) significantly altered during the thermal imbalance and higher concentration of the protein is noticed during such situations. Hence, measuring the quantity of HSPs in the cells of animals exposed to extreme environmental conditions could be used as a reliable indicator of the thermal stress condition in cattle. First of all, the present study was focused to characterize the HSP70 among native and crossbred cattle. Real Time PCR quantative assay and indirect enzyme linked immunosorbent assay (ELISA) revealed that, there was a significant (p<0.05) higher levels of expressed HSP70 specific mRNAs and proteins in the stress induced bovine PBMCs (peripheral blood mononuclear cell) of native X cattle compared to crossbred counterparts. This technique, however, has certain inherent disadvantages in terms of cost, time, sophisticated instruments, and highly trained manpower etc., which hinders its application in the majority of the laboratories. In view of the above facts, the present work was designed to develop a novel Reverse Transcriptase Loop-mediated isothermal amplification (RT-LAMP) technique for in vitro profiling of HSP70 in bovine PBMC cell culture model utilizing the absorbance level of magnesium pyrophosphate-a by-product of LAMP reaction. A set of bovine HSP70 specific RT-LAMP primers were designed to detect the differential absorbance level of magnesium pyrophosphate a by-product which signifies the degree of HSP70 amplification from cDNA of thermally induced cultured cells at different recovery periods. The study revealed significant (p<0.05) correlation between absorbance level and the fold change of HSP70 transcripts at different kinetic intervals of heat stress recovery in bovine PBMC cell culture models. RT-LAMP based absorbance assay can be used as an indicator to measure the degree of bovine HSP70 transcripts produced during thermal stress and can be used as an alternative to the traditional Real time PCR assay. Developed RT-LAMP assay can be used as a cost-effective method for profiling of bovine HSP70 gene. Despite of its chaperon and stress tolerance actions, HSP70 is also known to modulate immune status of an individual. The modulatory effects of HSP70 inducer (GGA) on the in vitro expression profile of bovine major pattern recognition receptors (PRRs) viz. TLR2/4 and NOD1/2 were studied. The expression levels in NOD1/2 and TLR2/4 in GGA induced groups were significantly (p<0.01) upregulated than the non -induced groups. However, among all the PRRs, a highest level of expression was observed in TLR4, followed by NOD2, TLR2 and NOD1. Further, it was observed that after treatment with HSP70 specific siRNAs on GGA induced bovine Peripheral Blood Mononuclear Cells (PBMC) and Madin- Darby Bovine Kidney (MDBK) cell lines, significantly (p<0.01) down regulates all the four receptors. Future understanding the basic molecular mechanisms of interaction between the PRRs and HSP70 will make it potential to realistically modulate immune responses towards immunity or thermotolerance in cattle during summer stress. An increase in innate immune receptor expression when Hsp70 expression is also XI increased should suggest that immunity should not reduce during heat stress conditions. microRNAs (miRNAs) are a class of small non-coding RNAs that play key roles in post transcriptional gene regulation that influence various fundamental cellular processes, including the cellular responses during environmental stresses. In the present investigation a set of differentially expressed miRNAs during thermal stress among native (Sahiwal) and crossbred (Frieswal) dairy cattle breed were identified through Ion Torrent deep sequencing and CLC-genomic analysis. Most of the identified differentially expressed miRNAs were found to target heat shock responsive genes especially members of heat shock protein (HSP) family. Real-time quantification-based analysis of selected miRNAs revealed that bta-mir-1248, bta-mir-2332, bta-mir-2478, and bta-mir-1839 were significantly (p<0.01) over expressed while bta-mir-16a, bta-let-7b, bta-mir-142, and bta-mir-425 were significantly (p <0.01) under expressed during summer in comparison to winter in native cattle breed. While in crossbred cattle, bta-miR-103-2, bta-miR-2898, bta-miR-150, bta-miR-2478, and bta-miR-181b-2 were significantly (p< 0.01) upregulated during summer stress. The correlations between the differentially expressed miRNAs during thermal stress with specific expression of bovine HSP70 transcripts revealed that bta-mir-181b-2, bta-mir-142 and bta-mir-150 had higher correlation with HSP70 gene, indicating their possible role in the post transcriptional regulation of HSP70. However, on the other hand, bta-mir-103-2 and bta-mir-2311 had higher negative correlation with HSP70 indicting their insignificant role in regulating the HSP70 gene in heat stress conditions in crossbred cattle. Being greater in their expression profile during peak summer, bta-miR-2898 was chosen for reporter assay to identify its effect on the target HSPB8 gene in heat stressed bovine PBMC cell cultured model. This study provides the ground work for uncovering the role of miRNA in thermal stress that may be helpful for development of miRNA-based molecular biomarker for cellular thermotolerance in dairy cattle.
Open Access
Phytochemical and Molecular assessment of In vitro raised Ephedra gerardiana: an endangered medicinal plant
(Department of Molecular and Cellular Engineering Jacob Institute of Biotechnology and Bioengineering Sam Higginbottom University of Agriculture, Technology and Sciences Allahabad – 211007, 2019) RAUTELA, INDRA; Mishra, Dr. Pragati
Ephedra gerardiana is an important medicinal plant used as Somalata in Indian Ayurvedic system of medicine and as traditional Chinese medicine since several thousand years. Plant is known to contain valuable secondary metabolite such as ephedrine and pseudo ephedrine. These secondary metabolites are utilized for several medicinal purposes. The present study describes in vitro regeneration of Ephedra gerardiana using tissue culture technique. Experiments were conducted to study the effect of various combinations of auxins (IBA) and cytokinins (BAP) alone or in different combinations on in vitro culture of nodal segments. Shoot induction and elongation from cultured nodal segments were obtained onto MS medium supplemented with various concentrations of BAP and 15 μM Kinetin. MS+IBA media was utilized for in vitro root induction. Among different media combinations utilized MS+ 5 μM BAP+ 15 μM kinetin was found best for shooting and ¼ MS+ 20 μM IBA was most appropriate for rooting. Molecular marker analysis was conducted to screen genetic fidelity among in vitro raised plantlets compare with mother plant of Ephedra gerardiana. Genetic fidelity of regenerated plants was assessed using Random Amplified Polymorphic DNA (RAPD) and Simple Sequence Repeat (SSR) Primers. A total of 50 RAPD primers and 30 SSR primers were utilized in the present study to analyze genetic fidelity of mother plant and among tissue culture raised plants of Ephedra gerardiana. Out of 50 RAPD primers, 19 primers exhibited DNA amplification in all the DNA samples and out of 30 SSR primers, 18 were showed amplification. The amplified products of the regenerated plants showed similar banding patterns to that of the mother plant thus demonstrated the homogeneity of the micropropagated plants. The banding pattern ruled out presence of any kind of somaclonal variation. Thus, the results revealed genetic fidelity exist between the micropropagated and mother plant in Ephedra gerardiana and supports the suitability of tissue culture technique for generation of genetically similar plants.
Open Access
Molecular characterization of outer capsid protein genes and study of neutralization behavior of bluetongue virus serotype – 16
(Department of Molecular and Cellular Engineering Jacob Institute of Biotechnology and Bioengineering Sam Higginbottom University of Agriculture Technology and Sciences Prayagraj (Allahabad), U.P. – 211007, INDIA, 2019) Saxena, Arpit; Naskar, Dr. Jishnu
Bluetongue (BT) is the most common, economically important vector-borne viral disease of domestic and wild ruminants. The causative agent of BT is Bluetongue virus (BTV), which belongs to the family Reoviridae within the genus Orbivirus. BTV contains ten double-stranded RNA (dsRNA) genome segments, that encode seven structural (VP1-VP7) and four non-structural (NS1-NS4) proteins. The outer capsid proteins VP2 and VP5 determine the serotype of virus and induce a host protective immune response by eliciting neutralizing antibodies. In the present research work, VP2 and VP5 gene based molecular characterization and cross neutralization studies have been conducted with the idea to determine the genetic and antigenic relationship among Indian BTV-16 isolates. The phylogenetic analysis revealed that isolates make monophyletic cluster with eastern topotypes. Isolates recovered from Uttarakhand (PDP2/13/IND and MUK1/12/IND) were found close to the virus (G53/ABT/HSR) reported from Gujarat (western India) as evident from phylogenetic analyses. It is possible that these viruses might have evolved from the common ancestor. However, BTV-16 isolates, recovered from southern part of India (SRL69/IND and KAR50/IND), segregated in separate cluster indicating that the viruses evolved separately from the viruses recovered from northern and western part of the country. Indian BTV-16 isolates share maximum nucleotide sequence similarity with the viruses of Mediterranean BTV, indicating probable origin of the virus from the ancestors of Mediterranean basin. vii Isolates were found to share 91.5% to 97.5% nucleotide similarities with previously reported BTV-16 isolates reported from India, whereas 74.1% to 97.0% nucleotide similarities were observed with BTV-16 reported from all over the world. Cross neutralization study indicates presence of minor subtype antigenic variations among isolates recovered from Uttarakhand (PDP2/13/IND and MUK1/12/IND) with evidence of 64.96% antigenic similarity. Higher magnitudes of antigenic relationship (rmean 0.80 to 0.90) of Indian BTV-16 isolates observed in ex-vivo neutralization study with hyper-immune serum against GNT7/07/IND and PDP2/13/IND suggests that vaccine preparation containing any of these isolates may provide a wide range of protection. Future studies targeting the complete genome sequence and recovery of more number of the viruses from different geographic origin of country and their genetic characterization are required to select a better vaccine candidate.
Open Access
Molecular characterization and development of sensitive diagnostics for Citrus yellow mosaic badnavirus and Citrus greening bacterium (Candidatus Liberibacter spp.)
(Department of Molecular and Cellular Engineering Sam Higginbottom University of Agriculture, Technology and Sciences Allahabad – 211007, U.P. India 2019, 2019) Motghare, Manali; Shukla, Dr Pradeep
Citrus yellow mosaic badnavirus (CMBV) is the causal agent of citrus yellow mosaic disease, a quarantine pathogen which is till to date restricted only in India. The disease is prominently present in southern part of the country and affects mostly Sathgudi sweet orange (Citrus sinensis). CMBV is a plant pararetrovirus, belongs to one of the class bacilliform badnaviruses. Another dreadful disease of citrus is citrus greening aka Huanglongbing (HLB) which affects citrus industry worldwide. It is responsible for huge crop reduction which ultimately leads to economical loss. The causal bacterium belongs to alpha subdivision of proteobacteria, genus Candidatus Liberibacter (Ca. L.) in the family Rhizobiaceae. Till today, there is no cure available for both diseases and therefore accurate disease diagnosis and removal of infected plant is the best remedy. Therefore pathogen specific primers for conventional as well as qPCR were designed based on coat protein region for CMBV and 16S rRNA region for CLas. For early diagnosis, right choice of target sample is very crucial and knowledge of pathogen distribution within a plant system will help to choose the tissue sample. Thus relative distribution of CMBV was studied using SYBR green real-time PCR. Primers were designed based on capsid protein gene of the virus, and conventional and real-time PCR were performed using different tissue samples including shoot tip, symptomatic leaf, asymptomatic leaf, senescent leaf, thorn, stem and feeder root. Highest virus concentration found in feeder roots followed by stem, symptomatic leaf, asymptomatic leaf and senescent leaf whereas lowest concentration found in shoot tip and thorn. It was found that for CMBV screening, symptomatic leaves lamella is the best option and if it is found negative, one can take small portion of secondary roots or green peel of the stem, to further confirm the results. Furthermore, for simultaneous detection of CMBV and CLas a duplex PCR technique using a simple nitrocellulose membrane (NCM) based DNA isolation protocol was developed. The protocol is simple, rapid and inexpensive thus can be used for largescale pathogen indexing. Additionally spotted membrane can be stored for few days before being used or can be transported to the places far away from collection site. The technique will be helpful to screen the samples from remote places. CLas, a causal agent of citrus greening disease is responsible for heavy crop loss. During the infection process, the bacterium produces several pathogenesis-related proteins that are crucial for its survival and multiplication in plant phloem tissue and insect vectors. There is a tremendous potential of developing antimicrobial inhibitors against these proteins to reduce pathogen population in plants and vectors. In an effort to identify potential drug (inhibitory molecules) targets towards achieving novel management strategies for citrus greening disease, four crucial protein genes of CLas was characterized. Proteome analysis reveals that these four proteins are unique, essential and conserved in pathogen population across India. Thus have a potential to act as target for developing inhibitory drug against the pathogen. The predicted structure of protein gives idea about active sites which are perfect target sites for blocking. This study will help to develop effective disease control strategies
Open Access
“Development of recombinant E6 protein of human papilloma virus type 16 and cervix cell line for study of cytopathic changes with HPV infection”
(Department of Molecular and Cellular Engineering Jacob Institute of Biotechnology and Bioengineering Sam Higginbottom University of Agriculture Technology and Sciences Allahabad – 211007, INDIA 2019, 2019) Ibrahim, Rahela; Ramteke, Prof. (Dr.) P.W.
Human papillomaviruses (HPVs) belong to the class of DNA viruses which are known to cause different human cancers. Development of new diagnostic methods and tools to study virus biology and mechanism of infection had been strongly felt in order to prevent HPV infections. In the present study we have designed new set of primers from E6 gene of HPV for its detection using PCR with an increased sensitivity of upto 0.2 pg. HPVs are known to infect basal undifferentiated cervical cells. Optimal annealing temperature of primers was found to be 57.7°C. Hence, we established and characterized a new cervical epithelial cell line designated as CEC to be used as an in vitro tool for studying viral biology in order to prevent HPV infections. The cell line was authenticated for its origin using chromosomal analysis which revealed a modal chromosome value of 46. The cell line was optimally maintained in L-15 medium supplemented with 10 % FBS at 37 °C for over 53 passages. CEC cells were evaluated for its revival efficiency which was exhibited 70-80 % revivability following cryopreservation. An increase in plating efficiency of CEC cells from 4.97±1.57 to 18.27±1.47 was observed when seeding density was increased from 100 to 1000 cells per flask. Epithelial morphology was confirmed through positive reaction observed with monoclonal antibody directed against cytokeratin. 30-35% transfection efficiency using pEGFP vector established its potential for genetic manipulation studies. The interaction of HPV with CEC cells resulted in its neoplastic transformation and cytological changes such as multinucleation, nuclear irregularity, nuclear enlargement and perinuclear halos. CEC cell line was thus successfully established as an in vitro model system for studying interaction of HPV with squamous cervical epithelial cells. These high risk HPV types, usually HPV-16 can be found in over 80% of cervicalcarcinomas. E6 is a significant toxin secreted by HPV-16, which contributes pathogenicityof to women. The complete ORF of E6 gene (2482 bp) was amplified using PCR. It wascloned in TA and sub-cloned in pET28a vector then transformed into Escherichia coliBL21DE3) codon plus RP cells expressed by the induction with 1.0 mM of IPTG. Theexpected size of expressed protein was 68.0 kDa estimated by migration in 12% SDS–PAGE. Anti-His monoclonal antibodies were used to sub- stantiate the recombinant proteinby Western blotting. The percent similarity between E6 of HPV-16 with other HPVs E6toxins revealed that the E6 sequence varied from 99.35 to 50.40%. Homology modelingwas used to construct 3-D structure of E6 of HPV-16 with the known crystal 3-D structure(PDB: 1PRE). This protein can be used for immunoassays and it is suitable for vaccinecandidate against HPV-16 infection.
Open Access
Structural bioinformatics analysis of potential inhibitors against microbial pathogens
(Department of Computational Biology and Bioinformatics Jacob Institute of Biotechnology and Bioengineering Sam Higginbottom University of Agriculture, Technology and Sciences (Formerly Allahabad Agricultural Institute) Prayagraj (Allahabad) – 211 007, INDIA, 2018) DHUSIA, KALYANI
The occurrence of microbial resistance to antibiotics is an unavoidable process that has nevertheless significantly been accelerated during recent decades by the massive use of antibiotics. For this reason, there is now an urgent need for both new generations of antibiotics with new targets, and for other novel strategies for overcome these pathogens. Serratia marcescens is one such MDR bacteria causing Hospital-acquired or nosocomial outbreaks. This gram-negative bacilli of the Enterobacteriaceae have been reported in neonatal care units in both developing and developed countries. Trichoderma genus are well-studied ascomycetous fungi commonly found in the agricultural, grassland, forest, saline, and desert soils that are widely marketed as biopesticides, bio-fertilizers and soil amendments, due to their ability to protect plants, enhance vegetative growth and control pathogen populations by various mechanisms such as the production of cell wall hydrolytic enzymes, antifungal metabolites, antibiotics, and mycoparasitism. Ergosterol is perceived as a foreign molecule by plant cells, triggering a series of reactions characteristic of nonspecific elicitors and recognised by plants as a Pathogen-Associated Molecular Pattern. Ornithine decarboxylase (ODC) enzyme, catalyses the decarboxylation of ornithine to form spermidine which is a committed step in the biosynthesis of polyamines. Here, in the present work, structure of ODC was modelled and studied for its active site. The stability of modelled structure was revalidated by the molecular dynamics simulation at 50 nanosecond time scale. 142 Natural products of Indofine Herbal Ingredient library from ZINC database were screened using Autodock Vina for the identification of potential leading herbal inhibitors. The results obtained from docking showed that Conessine is best inhibiting candidate with docking affinity of –9.7 Kcal/mol. Conessine is an alkaloid which proves its immense importance as metabolites. Thus, polyamine being harmful when synthesised in excess are necessary to be controlled at their genesis. Therefore, conessine might be a potential inhibitor for toxicity control in plant growth promoting rhizobacteria. In the present work, silver nanoparticles synthesized by ergosterol producing strains of Trichoderma were studied for their anti-pathogenic activity against phytopathogenic fungi namely Fusarium oxysporum f. sp. lycopersici and Colletotrichum gloeosporioides. Possible3 interaction of the silver nitrate with ergosterol has been investigated using computational approach. The size of formed silver were validated on the basis of the measurements of UV-VIS absorption using inter-atomic distance calculation via computational tool. Knowledge-based protein modeling and substrate docking experiments ii as well as structural and sequence comparisons were performed to identify potential activesite residues in ergestrol. The results obtained from docking software Glide showed the best pose which is derived from Schrodinger suite was -48.5747 with reranking score equal to - 40.228 at ergosterol’s active site positions for silver. Apart from the core of active site, four other positions have been occupied by nanoparticle sized silver. The interacting site surrounded by Cys339, Arg343, Lue365, Leu336 and Trp371 formed hydrophobic bonds with silver. Silver nanoparticle based formulations which are produced by antagonistic ergosterol producing strains of Trichoderma can be evaluated against different plant pathogen in further studies. The natural compound conessine is isolated from plant Holarrhena antidysenterica and it is studied against ODC of Serratia marcenses for its inhibitory potentials. This revealed unforeseen twisted position in root mean square fluctuation (RMSF) and ODC modelled conformation that influenced ligand binding. Both predicted model and ligand bound model were compared and found to be stable with Root Mean Square Deviation (RMSD) of approximately 7 nm and 0.25 nm to that of crystallographic structure over simulation time of 55 ns and 70 ns respectively. This work paves the way for future development of new drugs against nosocomial diseases caused by Serratia marcescens.
Open Access
Production , optimization and characterization of thermostable cellulase from aspergillus fumigatus AA001 and its application in production of reducing sugar from agriculture waste. "
(DEPARTMENT OF MOLECULAR AND CELLULAR ENGINEERING SAM HIGGINBOTTOM UNIVERSITY OF AGRICULTURE AND TECHNOLOGY SCIENCES (FORMERLY ALLAHABAD AGRICULTURE INSTITUTE) ALLAHABAD -211007, INDIA 2018, 2016) Srivastava, Neha; Ramteke, Dr. P. W.
Open Access
Study on potential antioxidant activity of Fisetin against oxidative stress in vivo
(DEPARTMENT OF MOLECULAR AND CELLULAR ENGINEERING SAM HIGGINBOTTOM UNIVERSITY OF AGRICULTURE AND TECHNOLOGY SCIENCES (FORMERLY ALLAHABAD AGRICULTURE INSTITUTE) ALLAHABAD -211007, INDIA 2018, 2018) SINGH, MANORAMA; Ramteke, Dr. P. W.
Oxidative stress is common mechanism that is implicated in the progression of various diseases. Oxidative stress is mainly induced by ROS. Relatively increased level of ROS damages the biomolecules such as DNA, protein and lipid. Lipid peroxidation is common damage induced by ROS thus preferably used as indicator for oxidative stress in research. Cell and mitochondria have antioxidant system to keep the ROS level under control. Altered activity of antioxidant enzymes and glutathione level causes oxidative stress .There are two established situations that are cause of oxidative stress. First, transcriptional suppression of expression of antioxidant enzymes and second is altered activity of antioxidant enzymes due to toxicity. Fisetin, a dietary flavonoid, has shown several chemopreventive effects against various ROS leads to disease. Structural evidences and in vitro studies suggested the antioxidant potential of fisetin. However, in vivo studies are largely less explored to suggest the mechanism of action in mitochondria and transcriptional suppression model. Thus, present study was conducted on above two situations to evaluate antioxidant potential of fisetin against oxidative stress in vivo. For First situation, CoCl2 induced cerebral cortex hypoxia model (40 mg/Kg body wt. for 15 days) was used which showed HIF mediated transcriptional suppression of SOD and CAT. Second, cisplatin (6mg/Kg body wt single dose) toxicity induced altered activity of antioxidant enzymes. In both the situations GSH level was also studied. In first situation, fisetin could able to prevent the transcriptional suppression mediated depletion of activity of SOD and CAT. Glutathione peroxidase activity was also enhanced by fisetin. In addition fisetin could able to increase the synthesis of GSH in fisetin alone group and similar increased level of GSH was observed in combined group also. In second situation, fisetin could able to restore the SOD, CAT, GPx and GR. In addition, fisetin I could able to restore the GSH level in mitochondrial matrix. Thus, in both the situations fisetin could able to modulate the antioxidant enzymes activity and restore GSH level in consequence reduced the level of ROS and LPO. However, further study is needed to confirm whether and how fisetin alter the HIF1α mediated transcription in cytosol and transport of GSH to mitochondria.
Open Access
Comparative Analysis of Bacterial Diversity in the Water Samples from Ganga River, Indian Ocean and Domestic wastewater through Metagenomics Technique
(DEPARTMENT OF MOLECULAR AND CELLULAR ENGINEERING JACOB INSTITUTE OF BIOTECHNOLOGY AND BIOENGINEERINSAM HIGGINBOTTOM UNIVERSITY OF AGRICULTURE, TECHNOLOGY AND SCIENCES, 2018) ZAED, JAMILAH ALI AHMED; TRIPATHI, DR.VIJAY
Microbial diversity in water is huge and everybody deals with it directly every day. The major question is, how can we deal enough with water safety to remove the organic and inorganic contaminates because it is directly related to the health problem. Here the main challenge is to know the biological diversity of water samples with the help of metagenomics techniques. Metagenomic is considered as a powerful method to study the bacterial diversity in environmental samples without using any cultural techniques. It provides a broader knowledge of bacterial diversity and avoid any risks. Till now only 1% bacterial species are cultivated but with the help of unculturable techniques we can study rest 99% of bacterial species. This study was conducted on 14 water sample collected from three different sources, including 03 samples from Ganga River and 04 samples from Indian Ocean, 07 samples from Domestic wastewater including 04 samples from Naini, Allahabad and 03 samples from Lucknow, India. The genomic of microbial content in those samples were extracted by DNA isolation kit and 16S rRNA nucleotide sequencing was performed by Sanger shotgun sequencing and analyze by bioinformatic tools. The results of this study identified 14 new strains that included 05 families: Bacillus, pseudomonas, Escherichia coli, Vibrio, Streptococcus. Mostly identified bacteria were pathogenic to humans. This study will help in reducing the water borne diseases which are to using several emerging bacterial pathogens.

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