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Abstract River Nile as a main source of drinking water in Egypt‟ ‟ has become a matter of interest and one of the most important national goals. Rosetta branch has a great importance since it is one of River Nile branches and the main source of water in the Delta region. Unfortunately, it receives a variety of wastes coming from sewage, agricultural and industrial drainage that affect its water quality. From this point, the present study aims to study the biodiversity of bacteria and viruses resulting from some physical, chemical and biological factors to know the differentiation between the autochthonous and allochthonous microorganisms. On the light of the previously mentioned objective, water samples were collected in July 2010. These samples were subjected to physicochemical and microbiological analysis. The results of the study can be summarized as follows: Ecodiversity of water Physical factors 1- Temperature values ranged from (30°C-33°C) with note the absence of any source of thermal pollution. 2- pH values for all collected water samples are within the permissible limits of law 48/1982 (7.0–8.5). 3- Turbidity values ranged from in 6.08-37.5 and 8.6–86 NTU in both of Rosetta branch and drainage water, respectively. Chemical factors 1- EC values for water samples collected from drains outfalls were recorded elevated, where they fluctuated between (632-1484 μmhos/cm) in drains outfalls and from (333-582 μmhos/cm) in Rosetta branch, respectively. 2- TDS concentrations for water samples collected from Rosetta branch are within the permissible limits of law 48/1982 (˂ 500 mg/l) except for Rosetta branch, upstream El-Tahreer drain recorded 530 mg/l. On contrast, TDS concentrations for water samples collected from drains outfalls exceeded the permissible limits where they ranged from (523-950 mg/l) except for Zawiet El-Bahr drain outlet was recorded 405 mg/l and within the permissible limits. 3- Ammonia concentrations for water samples collected from Rosetta branch and drains outfalls exceeded the permissible limits of the law 48/1982 (˂ 0.5 mg/l), where it ranged from (1.3664-7.517 mg/l in Rosetta branch) and (1.888-22.54mg/l in drains outfalls). Except for Rosetta branch, upstream El-Rahawy drain was recorded (0.324mg/l) and within the permissible limits. 4- DO values for water samples collected from Rosetta branch were variable compared to the permissible limits of law 48/1982 (>5 mg/l) where they fluctuated between (2.13-7.3 mg/l). On the other hand, DO values for water samples collected from drains outfalls violated that limit where they ranged from (0.18-4.12mg/l). 5- BOD concentrations for water samples collected from Rosetta branch were higher than the permissible limits in the law 48/1982 (˂ 6 mg/l) and ranged between (2-50mg/l). On the other hand, BOD concentrations for water samples collected from drains outfalls were high and exceeded the permissible limits in the law 48/1982 (˂ 10 mg/l) and ranged between (2-110mg/l) particularly for El-Rahawy and Sabal drains. Except for El-Tahreer and Zawiet El-Bahr drains outlet were recorded (2 & 8mg/l, respectively) and within the permissible limits. 6- COD concentrations for water samples collected from Rosetta branch were higher than the permissible limits in the law 48/1982 (˂ 10 mg/l) and ranged between (19-70mg/l). On the other hand, BOD concentrations for water samples collected from drains outfalls were high and exceeded the permissible limits in the law 48/1982 (˂ 15 mg/l) and ranged between (16-214mg/l) particularly for El-Rahawy and Sabal drains. 7- All Concentrations of nitrate (NO3 -) and phosphate (PO4 -3) were within the permissible limits in the Law 48/1982. The quantitative biodiversity for microorganisms 1- Standard plate count (SPC) bacteria at 22°C and 37°C in all collected water samples recorded high values and varied regionally. The highest value was recorded at El-Rahawy drain outlet. 2- Bacterial indicators (total coliform, fecal coliform and fecal streptococci) in all collected water samples exceeded the international permissible limits. 3- Salmonella sp. strains were isolated from only drains outlets.The qualitative biodiversity for microorganisms The water quality index (WQI) The water quality index (WQI) revealed that, El-Rahawy and Sabal drains have a very bad water quality, while its upstream El-Rahawy drain water was medium. The other studied drains as well as the rest of selected points in Rosetta branch were evaluated as being of bad quality and suffer mainly from bacteriological pollution. Isolation and identification of bacteria found in water samples Identification of bacteria involved isolation of “225” isolates, out of which “212” isolates were identified into “7” different species belonging to “3” main bacterial groups. These species were: Citrobacter freundii, Enterococcus faecalis, Escherichia coli, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella sp., & Staphylococcus aureus. Statistical study revealed the predominance of E.coli. This was followed by E.faecalis, P.aeruginosa, P.vulgaris, S.aureus, Salmonella sp. (which isolated only from drains) while C.freundii was the least. The biodiversity of isolated bacteria from water using different antibiotics The antibiotic sensitivity test was performed using 20 commercially prepared antibiotic discs belonging to 11 different groups. The identified bacterial isolates either from drains or Rosetta branch showed multiple antibiotic resistance (MAR) against the individual antibiotics used with different percentages. Resistance was 100% against Cephalothin, Carbenicillin and Clindamycin and was least to Norfloxacin, Ofloxacin and Piperacillin. The results also reported the presence of Methicillin resistant Staphylococcus aureus (MRSA) in all studied sites and isolates of vancomycin resistant Enterococcus faecalis (VRE) were monitored in some sites. Also, none of the tested antibiotics was active against all tested isolates. On the other hand, the multiple antibiotic resistance index (MAR index) revealed values higher than the permissible limits (0.25) which indicate that these sites pose a high risk source of contamination. The most pronounced values were recorded for P.aeruginosa, followed by P.vulgaris, followed by Salmonella sp., E.faecalis, E. coli, C.freundii, and least for S.aureus.pathogenicity of isolated bacteria from water samples In order to monitoring virulence characteristics of wild isolated bacteria from aquatic environment, the virulence test was performed using congo red reaction. Virulence was 100% for C.freundii and Salmonella sp. This was followed by P.aeruginosa (98%), P.vulgaris (91%), E.coli (60%), E.faecalis (40%) and least for S.aureus (37%). The biodiversity of bacterial isolates using 16s rDNA gene Sequence The DNA isolated from the three P.aeruginosa isolates were amplified by PCR conventional method using specific primers sequences for 16s rDNA gene in P.aeruginosa. 1- The three contig showed different partial nucleotide sequences of 16s rDNA gene, and identified as P.aeruginosa_1 with length (1274bp), P.aeruginosa_2 (1280bp) and P.aeruginosa_3 (1286bp), respectively. 2- Based on comparison of partial nucleotide sequences for three isolates with bacterial species recorded on the Genebank and based on phylogenetic tree analysis were showed five clusters in which the P.aeruginosa_1 and P.aeruginosa_2 were found to be highly homologous with percentage (95%) while the P.aeruginosa_3 showed distant homology (38%). P.aeruginosa_3 and both of P.aeruginosa strains JQ796859.1; KC121042.1; HM067869.1 and JQ659920.1 were found to be highly homologous with percentage (94%), while percentage was (98%) between P.aeruginosa_3 and strains JQ796859.1; KC121042.1 and HM067869.1, also percentage was (99%) between P.aeruginosa_1 and P.aeruginosa_2 and strains KC121042.1; HM067869.1 and JQ659920.1. 3- Sequence analysis showed variability based on sequence alignment information could be derived by determining the sequence statistics such as variability of count of four types of nucleotides, secondary structure and G+C percentage for all isolates. The biodiversity of phages specific for some bacterial isolates 1- Water samples collected from Rosetta branch and drainage outlets were examined for the presence of phages specific for E.coli & P.aeruginosa. The obtained results showed that, presence of specific phage for E.coli strain B, 3 and 1 and P.aeruginosa strains B2 and 101 using the spot test technique.2- All isolated phages formed circular, clear plaques whose diameters ranged between 1mm to 5mm, where detected with different concentrations and isolated from different sites. 3- The virulent sixteen phage isolates specific for E.coli & P.aeruginosa were isolated from collected water samples by spot test and then were biologically purified by single plaque assay. The pure isolated E.coli & P.aeruginosa phages were propagated in liquid culture of its host to obtain large amount of the particles before the chemical purification by PEG, 6000. These phage isolates specific for E.coli were designated C1, C2, C3, C4, C5, C6, C7 and C8, while P.aeruginosa phages designated Ps1, Ps2, Ps3, Ps4, Ps5, Ps6, Ps7 and Ps8. 4- E.coli and P.aeruginosa bacteriophages infectivity were tested against different pH values from 4 to 12 and it was found that, the coliphages have activity and able to lyse its host at pH ranged from 6 to 10 while inhibited completely at pH 4, 5, 11 and 12. P.aeruginosa phages have activity and able to lyse its host at pH ranged from 6 to 9 while inhibited completely at pH 4, 5, 10, 11, 12 and found that viral stability to acidity and alkalinity different from type of phage to another. 5- Effect of phages on E.coli and P.aeruginosa reduction were studied by recorded O.D600 readings in liquid culture for hosts and mixtures contain the hosts and specific phages after 24 hrs. Data showed that phage infection produced a drastic decrease of E.coli and P.aeruginosa cultures as compared to control and a constant increase in O.D600 was seen after 16 and 18 hrs this is most probably due to growth resistant phages. 2% uranyl acetate 6- Electron microscopy of E.coli & P.aeruginosa bacteriophages were negatively stained with 2% uranyl acetate. Results revealed that the phage particles had an isometric head and long-contractile tail and some particles appeared containing short tail with full heads. Head of coliphage isolates number C1, C2 and C3 with diameter about 106.7, 113.3 and 80nm and length about 113.3, 113.3 and 80nm, respectively. While the length of tails about 1193.3, 180 and 100nm and diameter of tails about 20, 40 and 20nm, respectively. P.eruginosa phage isolates number Ps1, Ps2 and Ps3 have heads with length about 66.7, 106.7 and 86.7nm and head diameter about 66.7, 100 and 80nm, respectively. While the length of tails about 173.3, 186.7 and 46.7nm and diameter of tails about 13.3, 20 and26.7nm, respectively. The bacteriophages resemble those including in the Myoviridae, Siphoviridae and Podoviridae families. 7- Biological characteristics for isolated phages were determined and tested by the spot test method and it was found that phages specific for E.coli could lyses E.coli strains 1, 3, B (ATCC) but failed to lyses E.coli strain 2. While, phages specific for P.aeruginosa could lyses P.aeruginosa strains B2 and 101 but failed to lyses E.coli strain 1,2. 8- Restriction enzyme of the eight isolated coliphages (C1 to C8) by EcoRI, HindIII and BamHI showed the presence of dsDNA as well as heterogeneity among these phages. The results showed that EcoRI produced 8, 7, 5, 4, 4, 7, 9 and 5 unique fragments and HindIII produced 4, 3, 0, 0, 1, 6, 5 and 4 unique fragments while BamHI produced only 1, 1, 1, 0, 0, 3, 0 and 1 unique fragments, for the eight phage isolates, respectively. 9- Restriction enzymes technique showed the DNA diversity among eight phage isolates 79 DNA fragments were detected using three restriction enzymes EcoRI, HindIII and BamHI. Sixty four fragments appear polymorphic (specific fragment) amplified fragments represented (81%), one fragment appear monomorphic (common fragment) amplified fragment represented (10.13%), and eight unique fragment (genetic marker fragment) represented (10.13%) of all detected fragments for isolates number 2, 3, 6, 7 and 8. 10- The EcoRI enzyme induced the generation of 17 fragments with size ranged between 27-15kb. One fragment found common fragment (monomorphic), twelve fragments were specific fragments (polymorphic) for eight phages and four genetic markers (unique fragment) for isolates numbers 2, 3 and 8. While, HindIII enzyme induced the generation of 9 fragments with size ranged between 14.75-7kb. Seven fragments were polymorphic for eight phages, two unique fragments for isolates numbers 6 and 7. As well as, BamHI induced the generation of 4 fragments with size ranged between 3.5-0.5kb. Four fragments were polymorphic, one unique fragment for isolate number 6. 11- The phylogenetic tree for identified coliphage showed six clusters were belonging to two main groups: group (I) included two sub group and five subsubgroup concerning phage isolates C1, C2, C6, C7 and C8. group (II) included two sub group and two subsubgroup concerning C3, C4 and C5. Genetic distance between both of isolate number C7 & C1 and C2 & C8 and C3 & C5 recorded 3.5cm with percent 22%. The genetic distance between both of isolate number C6 and C1 & C7 and C4 and C3 & C5 recorded 7cm with percent 44%. As well as genetic distance between C2, C8 and C1, C6, C7 recorded 10.5cm with percent 66%. On the other hand, genetic distance between isolates number C1, C2, C6, C7, C8 and C3, C4 and C5 recorded 14cm with percent 88 %. 12- The similarity index values for individual coliphage isolates were recorded between C1 and other isolates (15.38, 53.85, 69.23, 61.54, 18.75, 7.14 and 23.08, respectively). This followed by C2 (45.45, 63.64, 54.55, 31.25, 21.43 and 9.09, respectively), C3 (33.33, 16.67, 62.5, 57.14 and 40, respectively), C4 (20, 75, 71.43 and 60, respectively), C5 (68.75, 64.29 and 50, respectively), C6 (12.5 and 37.5) and C7 (28.57). The biodiversity of enteroviruses in water using molecular biology techniques Our study was aimed to detect and quantitative of enteroviruses in collected water samples from Rosetta branch and drains outlet after ultrafiltered to concentrate enteric viruses. Out of fifteen sites two sites only and they are El-Rahawy drain outlet and Sabal drain outlet were found to be polluted with the enteroviruses with rate of 3.6X104 and 3.4X104 GC μl-1, respectively using real-time-qRT-PCR. |