الفهرس 
Water quality assessment
Genetic variability of P.aeroginosa isolatesOnly 14 pages are availabe for public view
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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.