tm jeb tm · journal of environmental biology, september 2018 bacterial diversity in drinking water...

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 :

[email protected]

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

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TM

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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


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



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



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




SH3 Shahbaz road SH3 98 KT027800





BHS1 Bazar Kasur BHS1 99 KT027769




DG1 Din Gharh DG1 98 KT027762







KP1 Kot peeran KP1 97 KT027772





BS1 Bhatta sohn din BS1 98 KT027791





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



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

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b

aa

b



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


(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


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



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


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


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

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b



Str

ain

An

tib

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cs

FC

NC

LN

OR

AK

NA

AM

SC

IPC

TE

Zo

ne

S/R

(mm

)

DG

-7

BH

S-1

DG

-6

SH

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RA

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J-4

KP

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Q-4

BS

-5

BH

S-2

JP-4

DG

-1

DG

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Zo

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S/R

Zo

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S/R

Zo

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S/R

Zo

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S/R

Zo

ne

S/R

Zo

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S/R

Zo

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S/R

Zo

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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

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27S

19S

0R

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13I

26S

0R

0R

0R

16S

0R

28S

20S

0R

0R

12I

28S

0R

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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

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esis

tant

; I i

nter

med

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; F N

itrof

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toin

; CN

Gen

tam

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; CL

Cep

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xin;

NO

R N

orflo

xaci

n;A

KA

mox

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in; N

A N

alid

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aci

d;A

MA

mpi

cilli

n; S

Str

epto

myc

in;

CIP

Cip

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Chl

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Tetr

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Tab

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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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