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DOI : http://doi.org/10.22438/jeb/39/5/MRN-713
Journal of Environmental Biology 693-701Vol. 392018September
Abstract
Aim :
Methodology :
Results :
Interpretation :
Potable water can be contaminated with variety of bacterial pathogens during the distribution
process. The aim of this study was to analyze the bacterial risks associated with drinking water supply
system of the city Kasur, Pakistan.
Water analysis was accomplished by following the most probable number (MPN) method.
Bacterial diversity was determined by 16S rRNA gene sequencing. Screening for O157 antigen was
conducted for all the identified strains of . Strains were also screened for antibiotic
susceptibility and biofilm formation.
Analysis showed 2400 MPN index for few water samples. 16S rRNAsequencing confirmed the
presence of bacterial strains belonging to the genera of
and
Serological analysis also detected the presence of pathogenic strains of O157. Antibiotic sensitivity
pattern showed the resistance of strains against nitrofurantoin, cephalexin, nalidixic acid and ampicillin. For
biofilm formation, DG1 and J4 were the most active biofilm producer in
single cultures.
Majority of the water samples were contaminated in terms of coliforms. Biofilm formation
by bacterial strains indicated that water distribution network might be colonized by potential human
pathogens.
Escherichia coli
Escherichia, Pseudomonas, Bacillus,
Exiguobacterium, Klebsiella, Acinetobacter, Aeromonas, Enterobacter, Citrobacter Shigella.
E. coli
Ex. artemiae An. haemolyticus
≥
*Corresponding Author Email :
Publication Info
Paper received : 05.08.2017Revised received : 07.12.2017Re-revised received : 08.03.2018Accepted : 29.03.2018
© , Lucknow (India)Triveni Enterprises
Authors Info
T. Ishaq and B. Ali*
Department of Microbiology andMolecular Genetics, University ofthe Punjab, Quaid-i-AzamCampus, Lahore-54590, Pakistan
Key words
Antibiotic resistanceDrinking water biofilms
O157Fecal coliformsPotable water
Escherichia coli
p-ISSN: 0254-8704e-ISSN: 2394-0379
CODEN: JEBIDP
P Dlagiarism etectorWhite Smoke
Just write.
Journal Home page : www.jeb.co.in« E-mail : [email protected]
Journal of Environmental Biology
Risk assessment and biofilm formation
of bacterial communities associated
with drinking water distribution system
Original Research
JEBTM
TM
Journal of Environmental Biology, September 2018
hides for leather production.Animal wastes that are discharged insewage system contribute bacterial pathogens to local drinkingwater supply system as a result of cross contamination.Therefore, in this work bacterial diversity associated with localwater supply system has been reported. Moreover, culturedependent analysis related to antibiotic susceptibility and biofilmformation was also conducted.
Samples of potable waterwere collected from different areas of city Kasur, Pakistan. Out often samples, one was ground water and others were collectedfrom water distribution systems. The tests were performedsequentially on each sample under analysis by following the mostprobable number (MPN) protocol of Cappuccino and Sherman(2002). This method can detect the presence of human coliformswhich is indicator of fecal contamination. For enumeration ofdifferent potential pathogenic bacteria selective broth media wereused. For instance, Buffered peptone broth was used for
Rappaport vassiliadis enrichment broth candetect MacConkey broth for alkalinepeptone water for and L-broth for Five milliliter ofeach broth was poured into test tubes and measured aliquots ofwater (5 ml) were used for testing. Culture tubes were incubatedfor 24 hrs at 37 C. Afterwards, inoculum was taken and streakedon selective agar plates.
To assess the final taxonomicstatus of bacterial diversity of drinking water, strains wereidentified by 16S rRNA gene sequencing. Genomic DNA frombacterial cultures was extracted by using Tissue Genomic DNAExtraction Mini Kit (Favorprep Favorgen). PCR amplification of16S rRNA gene was carried by using forward primer 27f (5'-AGAGTTTGATCCTGGCTCAG-3') and 1522r reverse primer (5'-AAGGAGGTGATCCA(AG)CCGCA-3') (Johnson, 1994).Amplification was done by using Dream Taq™ Green PCR MasterMix (Fermentas) as mentioned earlier (Akhtar andAli, 2011). PCRproducts were purified using Favorprep Gel Purification Mini Kit(Favorgen) and sequenced.
Pre-enrichment of sorbitolnegative strains of was accomplished in Tryptic Soy Broth(TSB) at 37°C for 24 hrs. Further, a selective enrichment in TSBsupplemented with novobiocin for 6 hrs was given to each strainat 42°C in order to reduce any background microflora. Afterincubation, serial dilutions of selective enrichment culture wasprepared and plated on cefixime-tellurite sorbitol MacConkeymedium which was used as a selective and differential medium.Plates were incubated at 37°C for 24 hrs. Cefixime and potassiumtellurite serves as selective agents and inhibit the growth of nonO157 and other non-sorbitol fermenters. After incubation,colorless colonies were selected for further processing.
Sorbitol negative strains ofwere tested for the presence of antigen O157 by using Prolex
Materials and Methods
Sample collection and analysis :
16S rRNA gene sequencing :
Pre-enrichment for O157 :
Latex agglutination test :
Pseudomonas,Salmonella, Shigella,
Vibrio Bacillus.
E. coli
E. coli
E. coli
o
TM
TM
TM
E. coli
Introduction
Water is the medium of life and most abundant compoundon our planet. About 2.6% of global supply of water on earth isfresh water that is mainly used for drinking (Szewzyk 2000;Coffey 2007). The source of drinking water production ismainly surface fresh water which is used by most of the people.Water quality may decline dramatically after leaving the freshwater storage tank. The reasons of drop in microbial water qualitymay be credited to system deficits such as cross-connections,broken water pipes and pollution during major storage andpreservations (Anita 2016). Moreover, another majorproblem is the formation of microbial biofilms within the pipe linesfrom which cells may be detached into the water stream(Bhagobaty 2015; Proctor and Hammes, 2015).
Many microbes like
andhave been reported to be transmitted by water (Zhang2012; Zareen 2014). Due to the hazards associated withpublic health, it is important to understand the pathogen ecologyand identification of the source of these pathogens (Coffey2007; Lebaron 2015). is found in all human and animalfeces at higher concentrations. Therefore, it was chosen as thebiological indicator of water quality (Edberg 2000). Theoccurrence of coliform in potable water indicates the presence ofother potential pathogenic bacteria in water (Cappuccino andSherman, 2002; Zareen 2014; Shrestha 2017). Todetermine the treatment efficacy of water treatment plants, totalcoliforms are one of the best indicators (Tallon 2005).
Majority of terrestrial microorganisms live in biofilmswhich are communities associated to surfaces (O'Toole, 2011).Allsolid surfaces that are in contact with water can be colonized bymicroorganisms in drinking water distribution system (Wingenderand Flemming, 2004). Biofilms in potable water arepredominantly formed by indigenous microflora irrespective oftheir association to human health. However, drinking waterbiofilms can be colonized with opportunistic human pathogenswhich can cause diseases, especially in immunocompromisedpeople. Pathogens survived after treatment could colonize anexisting biofilm; where, they could grow and later on release intothe bulk flow (September 2007). Bacterial activity in drinkingwater network is increased due to the presence of iron corrosionproduct that also triggers both free living bacteria and formation ofmicrobial biofilm. A large gathering of bacterial communitiesobserved on environmental ferric surfaces and on putrefiedmetallic structures. It may be partially ascribed to the surfaceproperties of iron oxides and their surface charge may enhanceattachment and colonization by microorganisms (Appenzeller
2002; Douterelo 2014).
The present study aims to determine the bacterial risksassociated with drinking water distribution network of the cityKasur, Pakistan. Kasur is well known for the processing of animal
et al.,et al.,
et al.,
et al.,
Campylobacter, Escherichia coli,Pseudomonas, Legionella, Enetrobacter, Aeromonas,Helicobacter, Salmonella, Shigella, Vibrio Cryptosporidium
et al.,et al.,
et al.,et al., E. coli
et al.,
et al., et al.,
et al.,
et al.,
etal., et al.,
694 T. Ishaq and B. Ali
Journal of Environmental Biology, September 2018
Bacterial diversity in drinking water 695
single, double and triple bacterial cultures in the presence of ironand chromium. The culture combinations were processed in TSBsupplemented with 0, 5, 10, 15 µg ml concentrations of iron andchromium. Single, double or triple cultures were used insupplemented broths and processed for biofilm formation asmentioned above.
Data for biofilm formation was subjected toanalysis of variance (ANOVA) by IBM SPSS Statistics 20software. Means of different values were compared by Duncan's
multiple range test (P 0.05).
Following incubation, test tube cultures were evaluatedfor turbidity and gas production. Positive gas production fromthree sets of tubes was counted to generate 3 digit codes thatwere compared with MPN index to determine total coliforms in100 ml water sample. Shahbaz Road (SR) and Din Gharh (DG)
samples showed 2400 MPN per 100 ml. Inoculum from
positive tubes was used to streak eosin methylene blue (EMB)agar for determining Presumptively positivewater samples recorded the presence of with metallicgreen sheen on EMB agar. Moreover, colonies of and
were also detected that were further confirmed byacid and gas production in MacConkey broth. Finally, 55bacterial strains were isolated and purified for finalidentification. According to water safety standards, orother coliforms should be absent in 100 ml of water sample(Cappuccino and Sherman, 2002).
Taxonomic status of 39 bacterial strains were confirmedafter sequence analysis of 16S rRNA gene. The sequences weresubmitted to GenBank and accession numbers of bacterialstrains were obtained (Table 1). Sequences of 5 bacterial strainsincluding S9, SH4, SH6, SH7 and DG8 showed homology withgenus Similarly, 17 bacterial strains showedsimilarity with genus Strains KP7, KP4, KP1, JP5showed affilication with genus Rest of the bacterialisolates showed resemblance with genus
orBy observing the diversity of bacterial strains, it was
concluded that majority of the organisms belonged toGammaproteobacteria. Williams (2010) also reported thepresence of bacterial groups that belonged to alpha, beta orgammaproteobacteria from drinking water. Similarly, a variety ofbacterial strains from genus
Enterococci and Staphylococci have been documented in drinkingwater supply system (Hamieh 2015; Shrestha 2017).
strains can be classified as sorbitol fermenters ornon-fermenters on the basis of their ability to ferment sorbitol.Strains that recorded pink coloration on sorbitol MacConkey agarwere considered negative for this test. However, strains withcolorless growth were categorized as non-sorbitol fermenter. One
-1
Statistical analysis :
Results and Discussion
≤
≥
Escherichia coli.E. coli
KlebsiellaEnterobacter
E. coli
Escherichia.Pseudomonas.
Bacillus.Acinetobacter, Klebsiella,
Exiguobacterium, Enterobacter, Aeromonas, CitrobacterShigella.
et al.
Salmonella, Listeria, Escherichia,Citrobacter, Serratia, Acinotobacteria, Arcobacter, Clostridium,
et al., et al.,
E. coli
Latex Agglutination Kit (Pro-Lab Diagnostics). This test involvedthe mixing of suspected pathogen with antiserum that contained theantibodies of O157. Positive strains of showedagglutination with homologous antiserum. This kit comprised of
latex reagent particles coated with antibodies against targetpathogen, positive and negative controls of O157. For test, purifiedcolonies were suspended in 0.2 ml normal saline and turbidityadjusted to 2 McFarland. One drop of O157 latex reagent wasmixed with one drop of the test organism on the provided test card.After 2 min, test card was examined for the presence ofagglutinationaccording to the manufacturer's instructions.
Bacterial strains were screenedfor sensitivity against variety of antibiotics. For antibiotic assay,plates of Mueller-Hinton (MH) agar were prepared and heavilyinoculated with target strains with sterile cotton swab to ensureuniform confluent growth on agar surface. The antibiotic discs(Bioanalyse ) for amoxicillin (20 µg), tetracycline (30 µg),ampicillin (10 µg), streptomycin (10 µg), nalidixic acid (30 µg),chloramphenicol (30 µg), ciprofloxacin (5 µg), norfloxacin (10µg), cephalexin (30 µg), nitrofurantoin (300 µg) and gentamicin(10 µg) were placed on the surface of agar plates at equaldistances. The plates were incubated for 24 hrs at 37°C and thenevaluated for zones of inhibition around the discs. Zones weremeasured (in millimeter) by using Inhibition Zone Ruler providedby the manufacturer. The zones were then compared with thestandardized chart provided by Clinical Laboratory StandardInstitute (Hombach 2013).
Assay wasperformed with 12 bacterial strains. Each bacterial strain wasgrown in TSB at 37°C under agitation for 24 hrs. The opticaldensity of cell suspension was adjusted to 0.5 (600 nm) withspectrophotometer. Biofilm formation of individual, double andtriple bacterial cultures was determined by following the protocolof Stepanovi (2000). For single cultures, each well wasfilled with 20 µl of bacterial culture in 180 µl of TSB. In doublecultures, 10 µl of each strain and for three 7 µl of each strain wasadded to 180 µl of TSB in a sterile 96 wells sterile flat bottom plate.Three wells were used for each strain or treatment during theexperiment. Biofilm formation was induced by incubating plates at150 rpm on shaker at 37°C for 72 hrs. The liquid cultures fromeach well were then discarded and plates were washed thricewith distilled water to remove loosely attached cells. The platewas dried for 30 min; to analyze the remaining attached bacteriain terms of biomass adhered on the inner walls of the wells.Negative control was prepared by adding only TSB without anybacterial suspension. Biofilm in each well was fixed with 250 µl of98% methanol for 15 min. Afterwards, wells were allowed to airdry and stained with 200 µl of crystal violet for 5 min. Crystal violetfrom wells was solubilized with 33% glacial acetic acid and opticaldensity (570 nm) was recorded with Epoch microplatespectrophotometer (BioTek).
The abovementioned protocol was modified to monitor biofilm formation of
E. coli E. coliE.
coli i.e.,
E. coli
et al.,
et al.
Antibiotic sensitivity testing :
Microtiter plate assay for biofilm formation :
Effect of iron and chromium on biofilm formation :
®
ć
Journal of Environmental Biology, September 2018
696 T. Ishaq and B. Ali
showed sensitivity against gentamicin, norfloxacin andciprofloxacin with maximum zone of inhibition of 20 mm, 30 mmand 34 mm. Variable patterns were recorded forchloramphenicol, streptomycin and amoxicillin. In case oftetracycline, majority of the strains showed intermediatesensitivity. It has been shown that water treatment process mayalso increase antibiotic resistance in bacteria that enable themto survive in water distribution network. Such bacteria can actas an important source for spreading drug resistance toopportunistic pathogens that are present in contaminated water(Schwartz 2003; Xi 2009). In another study,
has been shown to disseminate antibioticresistance in treated water samples (Su 2018).
et al., et al.,Pseudomonas
et al.,
strain (SH7) gave colorless growth and were considered as non-sorbitol fermenter. Presumptively, positive O157 strainwas screened with Agglutination Kit that recorded positiveresult for SH7. In one study conducted in Lahore, Pakistan, thepresence of O157 from drinkig water supply system hasbeen reported (Zareen 2014). Similarly, Ngwa(2013) also showed the presence of enterohemorrhagicO157 from processed water samples.
Antibiotic sensitivity profile of selected strains wasanalyzed by disc diffusion procedure (Table 2). Overall, number ofbacterial isolates recorded resistance against nalidixic acid,cephalexin, ampicillin and nitrofurantoin. However, strains
E. coli
E. coliet al., et al.
E. coli
Table 1 :
Strains Sample location Identified as Homology Accessions
16S rRNA gene sequencing of bacteria isolated from drinking water samples of Kasur, Pakistan
JP1 Jamaat pura JP1 98 KT027796
JP4 Jamaat pura JP4 99 KT027797
JP5 Jamaat pura JP5 99 KT027798
RA5 Railway Station RA5 98 KT027779
LA3 Lari adda LA3 99 KT027776
Q4 Quarters Q4 97 KT027784
Q5 Quarters Q5 98 KT027785
J2 Jinnah colony J2 99 KT027786
J3 Jinnah colony J3 99 KT027787
J4 Jinnah colony J4 99 KT027777
J5 Jinnah colony J5 99 KT027782
SH3 Shahbaz road SH3 98 KT027800
SH4 Shahbaz road SH4 99 KT027783
SH6 Shahbaz road SH6 99 KT027790
SH7 Shahbaz road SH7 99 KT027780
SH8 Shahbaz road SH8 99 KT027781
BHS1 Bazar Kasur BHS1 99 KT027769
BHS2 Bazar Kasur BHS2 99 KT027770
BHS3 Bazar Kasur BHS3 100 KT027771
BHS5 Bazar Kasur BHS5 99 KT027789
DG1 Din Gharh DG1 98 KT027762
DG2 Din Gharh DG2 96 KT027763
DG3 Din Gharh DG3 97 KT027764
DG5 Din Gharh DG5 99 KT027765
DG6 Din Gharh DG6 98 KT027766
DG7 Din Gharh DG7 96 KT027767
DG8 Din Gharh DG8 99 KT027768
KP1 Kot peeran KP1 97 KT027772
KP3 Kot peeran KP3 99 KT027773
KP4 Kot peeran KP4 98 KT027788
KP6 Kot peeran KP6 99 KT027774
KP7 Kot peeran KP7 99 KT027775
BS1 Bhatta sohn din BS1 98 KT027791
BS3 Bhatta sohn din BS3 99 KT027792
BS4 Bhatta sohn din BS4 99 KT027793
BS5 Bhatta sohn din BS5 99 KT027794
BS6 Bhatta sohn din BS6 98 KT027795
S9 Din Gharh S9 99 KT027799
S10 Kot peeran S10 99 KT027778
(%)
Pseudomonas mendocina
P. pseudoalcaligenes
Bacillus cereus
Aeromonas caviae
Shigella flexneri
P. monteilli
P. aeruginosa
Klebsiella oxytoca
P. aeruginosa
Acinetobacter haemolyticus
P. monteilli
P. otitidis
Escherichia coli
E. coli
E. coli
K. pneumoniae
Citrobacter freundii
P. aeruginosa
P. aeruginosa
P. stutzeri
Exiguobacterium artemiae
Ex. Sibricum
Ex. Sibricum
K. pneumonia
S. flexneri
Enterobacter cloacae
E. fergusonii
B. sublitis
Aci. Junii
B. tequilensis
P. mendocina
B. sublitis
P. mendocina
P. mendocina
P. aeruginosa
P. stutzeri
P. aeruginosa
E. coli
P. aeruginosa
Journal of Environmental Biology, September 2018
Bacterial diversity in drinking water 697
In triple cultures, LA3, DG5 andBHS1 showed good response (Fig. 1c). Recently, it has
been demonstrated that biofilms in drinking water distrubitionsystems were colonized by spp.,
spp. and variety of
S. flexneri K. pneumoniae Ct.freundii
E. coli, Campylobacter P.aeruginosa, Bacillus spp., Enterobacter
Biofilm formation for 12 bacterial strains was analyzed insingle, double and multiple cultures. In single culture,
DG1 and J4 were the most potentbiofilm former (Fig. 1a) In double culture, LA3 and
DG5 showed significant biofilm formation (Fig. 1b).
Ex.artemiae An. haemolyticus
S. flexneri K.pneumoniae
Fig. 1 : Biofilm formation of bacterial cultures in TSB medium. (a) Single cultures; (b) Double cultures and (c) Triple cultures. Values are mean of three
replicates. Different letters on bars indicate significant difference between treatments using Duncan's multiple range test (P 0.05)≤
Op
tica
l den
sity
(a) 1.2
1
0.8
0.6
0.4
0.2
0
Control DG1 RA5 Q4 BHS1 LA3 DG5
Strains
J4 DG7 SH7 DG6 BS5 KP3
(b) 0.7
0.6
0.5
0.4
Op
tica
l den
sity
0.3
0.2
0.1
0
DG1;RA5 Q4;BHS1 LA3;DG5 J4;DG7 SH7;DG6 BS5;KP3
Strains (Double cultures)
KP3;DG1 BS5;RA5 DG6;Q4 SH7;BHS1 DG7;LA3 J4;DG5
Op
tical d
en
sit
y
(c) 0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0
DG1;RA5;Q4 LA3;DG5;BHS1 J4;DG7;SH7 BHS1;BS5;KP3 KP3;DG1;RA5 BS5;Q4;BHS1 LA3;DG5;DG6
Strains (Triple cultures)
a
e
c
bb
ab
d
e
c
ab
c
b
d
b
a
d
bb
aa a
b
a
bc
c
a
b
c
b
aa
b
Journal of Environmental Biology, September 2018
698 T. Ishaq and B. Ali
Enterococci or Stapylococci (Wingender and Flemming, 2011;Hamieh 2015; Güvensen 2017). All single, doubleor triple combinations were inoculated aseptically in a 96 wellssterile flat bottom plate supplemented with 0 µg, 5 µg, 10 µg
et al., et al.,and 15 µg of FeSO or K CrO . Biofilm forming ability of majority
of the strains was enhanced in the presence of FeSO ;
especially at lower concentrations. In single culture, SH7gave good response (Fig. 2a); whereas, SH7 and
4 2 4
4
E. coliE. coli En.
Op
tical d
en
sit
y
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
(a) 0 µg 5 µg 10 µg 15 µg
SH7 DG5 DG7 KP3 BHS1 DG6
Strains
0 µg 5 µg 10 µg 15 µg(b) 1.2
1
0.8
0.6
0.4
0.2
0
Op
tica
l den
sity
SH7;DG5 SH7;DG7 SH7;KP3 SH7;BSH1 SH7;DG6
Strains (Double cultures)
(c) 1.4
1.2
1
0.8
0.6
0.4
0.2
0
Op
tica
l den
sity
SH7;DG6;DG5 SH7;DG7;KP3 SH7;DG5;BHS1 BHS1;KP3;DG7
Strains (Triple cultures)
0 µg 5 µg 10 µg 15 µg
Fig. 2 : Effect of different concentrations of FeSO on biofilm formation of bacterial strains. (a) Single cultures, (b) Double cultures and (c) Triple cultures.
. Different letters on bars (within same color) indicate significant difference between treatments using Duncan's multiple
range test (P 0.05)
4
Values are mean of three replicates
≤
a b
c
a
c
a
cb
b
a
aa
b a
bc
b
b
b
aa
a
a a
a
b
a
bc
c
c
b b
c
b b
c
a aa
b
aa
b
aa
a a
aa
b b
a
b
a a
a
bb
c
Journal of Environmental Biology, September 2018
Bacterial diversity in drinking water 699
cloaceaeC. freundii An. junii
En. cloacae
DG7 were motst effective in double cultures at 5 µg ofFeSO (Fig. 2b). In triple cultures, BHS1,
KP3 and DG7 recorded significant biofilmformation (Fig. 2c). Variable response for biofilm was also
4
recorded when medium was supplemented with K CrO . In
single culture, BHS1, in double cultures, SH7and KP3 and in triple cultures, SH7,
DG5 and BSH1 gave the most effective
2 4
C. freundii E. coliAn. junii E. coli K.
penumoniae C. freundii
3.5(a)
3
2.5
2
1.5
1
0.5
0
Op
tica
l den
sity
0 µg 5 µg 10 µg 15 µg
SH7 DG5 DG6 KP3 BHS1
Strains
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
(b)
Op
tica
l den
sity
SH7;BHS1 SH7;DG5 SH7;DG6 SH7;KP3
Strains (Double cultures)
0 µg 5 µg 10 µg 15 µg
3(c)
2.5
2
1.5
1
0.5
0
Op
tica
l den
sity
SH7;DG6;DG5 SH7;KP3;BHS1 SH7;DG5;BHS1
Strains (Triple cultures)
0 µg 5 µg 10 µg 15 µg
Fig. 3 : Effect of different concentrations of K CrO on biofilm formation of bacterial strains. (a) Single cultures, (b) Double cultures and (c) Triple
cultures. Different letters on bars (within same color) indicate significant difference between treatments using
Duncan's multiple range test (P 0.05)
2 4
Values are mean of three replicates.
≤
a
bb
bb b
bb
b
a aa a
a a a a a
c c
b
c
aa a
a
a a
b
a aa
a
b
b b
a a a aa a b
c
b
b
c
b
Journal of Environmental Biology, September 2018
700 T. Ishaq and B. Ali
Str
ain
An
tib
ioti
cs
FC
NC
LN
OR
AK
NA
AM
SC
IPC
TE
Zo
ne
S/R
(mm
)
DG
-7
BH
S-1
DG
-6
SH
-7
RA
-5
J-4
KP
-3
Q-4
BS
-5
BH
S-2
JP-4
DG
-1
DG
-5
Zo
ne
S/R
Zo
ne
S/R
Zo
ne
S/R
Zo
ne
S/R
Zo
ne
S/R
Zo
ne
S/R
Zo
ne
S/R
Zo
ne
S/R
Zo
ne
S/R
Zo
ne
S/R
(mm
)(m
m)
(mm
)(m
m)
(mm
)(m
m)
(mm
)(m
m)
(mm
)(m
m)
16I
15S
12R
16I
16R
0R
0R
16S
19R
23S
16I
19S
17S
12R
21R
18S
0R
0R
18S
19R
25S
16I
11R
16S
0R
21S
16R
18I
0R
16S
21S
20S
15I
12R
15S
11R
15I
13R
0R
0R
12I
15R
21S
15I
17S
16S
0R
21R
16R
0R
0R
15S
20R
24S
16I
0R
14I
0R
16I
15R
15I
0R
11R
21S
11R
16I
0R
16S
0R
29R
20S
0R
0R
15S
30S
12R
0R
0R
15S
0R
22S
19S
0R
0R
17S
22S
0R
11R
0R
16S
0R
27S
19S
0R
0R
13I
26S
0R
0R
0R
16S
0R
28S
20S
0R
0R
12I
28S
0R
0R
0R
20S
0R
30S
20S
20S
0R
18S
28S
0R
16I
8R
14R
14R
26S
26S
16I
0R
16S
34S
10R
14R
10R
18S
0R
20S
18S
20S
0R
14I
30S
20S
16I
Abb
revi
atio
ns :
S/R
: sen
sitiv
e/ r
esis
tant
; I i
nter
med
iate
; F N
itrof
uran
toin
; CN
Gen
tam
icin
; CL
Cep
hale
xin;
NO
R N
orflo
xaci
n;A
KA
mox
icill
in; N
A N
alid
ixic
aci
d;A
MA
mpi
cilli
n; S
Str
epto
myc
in;
CIP
Cip
roflo
xaci
n; C
Chl
oram
phen
icol
; TE
Tetr
acyc
line
::
::
::
::
::
::
Tab
le 2
:A
ntib
iotic
sen
sitiv
ity p
atte
rn o
f sel
ecte
d ba
cter
ial s
trai
ns is
olat
ed fr
om d
rinki
ng w
ater
dis
trib
utio
n sy
stem
Journal of Environmental Biology, September 2018
Bacterial diversity in drinking water 701
biofilm formation at 5 µg of K CrO (Fig. 3). In the present study,
microtiter plate assay was used to study microbial biofilms. It isconsidered an important tool for the study of early stages ofbacterial biofilm formation (O'Toole, 2011).
Isolation of coliforms particularly those of fecal origin fromdrinking water samples of city Kasur, Pakistan showed poor waterquality with respect to standard maximum check limits set byWorld Health Organization. Fecal coliforms such as
and indicated the possiblesewage mixing with distribution lines and inadequate disinfectiontreatment. Detection of O157 from one sample also revealsa possible health hazards to the local community. Bacterial strainsalso showed biofilm forming potential that may be more resistantto disinfection than suspended microflora. Therefore, biofilmsshould be eliminated from water distribution system to prevent thecolonization of pathogenic microbial flora.
We are thankful for the financial support of University ofthe Punjab, Lahore, Pakistan to conduct this research work(No.D/4112/Est.1).
2 4
E. coli, K.pneumoniae, S. flexneri En. cloaceae
E. coli
Acknowledgment
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