Ecotoxicology and Environmental Safety ] (]]]]) ]]]]]]

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Contamination of potable water by enterotoxigenic Escherichia coli:

qPCR based culture-free detection and quantication
C.B. Patel a, P. Vajpayee a, G. Singh a, R.S. Upadhyay b, R. Shanker a,n
a
b

Environmental Toxicology Group, CSIR-Indian Institute of Toxicology Research, Post Box 80, Mahatma Gandhi Marg, Lucknow 226001, UP, India
Department of Botany, Banaras Hindu University, Varanasi 221005, UP, India

a r t i c l e i n f o

abstract

Article history:
Received 4 April 2011
Received in revised form
20 July 2011
Accepted 23 July 2011

Tourists visiting to endemic zones may acquire Enterotoxigenic Escherichia coli (ETEC) infection
resulting into diarrhea due to consumption of contaminated potable waters. In this study, a qPCR
assay (SYBR Green), targeting LT1 and ST1 genes was designed to quantify ETEC in potable waters
derived from civic water supply. The assay could detect lowest 1 CFU/PCR targeting LT1/ST1 gene from
ten-fold diluted culture of the reference strain (E. coli MTCC 723) and is ten-fold more sensitive than the
conventional PCR. The quantication of the ETEC in potable waters collected from civic supply of a
major city of the northern India exhibiting high ow of tourists reveals that all the sites that ran along
sewage line were contaminated by the ETEC. Contamination was due to percolation of sewage. The
assay could be used for the regular monitoring of potable water in places exhibiting heavy ow of
tourists to prevent ETEC induced diarrhea.
& 2011 Elsevier Inc. All rights reserved.

Keywords:
Enterotoxigenic E. coli
Potable water
Quantitative PCR

1. Introduction
In India and some other countries, surface waters from rivers,
lakes and ponds are processed by alum treatment, ltration and
chlorination to be used as drinking water (Clever et al., 2000; Ram
et al., 2008b). Some recent studies reported the prevalence of multi
antimicrobial-resistant Escherichia coli isolates positive for virulence
determinants for Enterotoxigenic E. coli (ETEC) in surface waters that
are being used as raw water to supply drinking water (Begum et al.,
2005; Ram et al., 2007; Singh et al., 2010). ETEC induced watery
diarrhea worldwide in humans, affecting mainly children and travelers (Turner et al., 2006). In the developing world, an estimated 650
million cases of ETEC infections occur each year, resulting in 800,000
deaths, mostly in young children (WHO, 1999). ETEC secretes at least
one of two types of enterotoxins (heat-labile, LT; and heat-stable,
ST enterotoxins) encoded by LT1 and ST1 genes, respectively (Turner
et al., 2006). The heat-labile enterotoxins of E. coli are oligomeric
toxins classied into two major groups (LTI and LTII). The LT1 gene
commonly present in strains associated with human illness has been
frequently observed in ETEC strains recovered from surface waters of
India and other south Asian countries contaminated by fecal wastes
of human origin (Begum et al., 2005; Ram et al., 2008c). Hence, the
occurrence of ETEC in extensively used water resources is an
important health concern because a large population in India and
other developing countries depend on processed or unprocessed

surface waters for drinking (Lothigius et al., 2008). Recently, insufcient treatment of surface waters for the drinking water supply,
malfunctioning of sewage collection systems and defective water
distribution pipelines have led to contamination of potable water by
Enterohemorrhagic E. coli (EHEC) and other pathogenic bacteria
(Shrivastava et al., 2004; Bhatta et al., 2007; Ram et al., 2008b,
2011; Lienemann et al., 2010; Wullings et al., 2011). However, despite
recurrent incidences of water-borne diseases in India and other parts
of developing world, the potable water supplies have never been
investigated for the presence of ETEC. Further, worldwide drinking
water is routinely monitored for recent fecal contamination by testing
for fecal-indicator organisms E. coli, thermotolerant coliforms, and/or
intestinal enterococci to demonstrate microbial safety (Van der
Wielen and Medema, 2010). Although these indicator organisms
based microbial safety evaluation measures have been used for many
decades, they have certain limitations as these gives no idea about
the virulent form of these indicator organism (Singh et al., 2010).
Therefore, in this study for the rst time, culture- free quantitative
enumeration of ETEC in potable water collected from northern India
has been done by targeting LT1 and ST1 gene by SYBR Green based
quantitative PCR (qPCR).

2. Material and methods

2.1. Designing of primers specic to LT1 and ST1 genes for qPCR

Corresponding author. Fax: 91 0522 2611547.

E-mail address: rishishanker1@rediffmail.com (R. Shanker).

The complete coding sequences of ST1 (V00612, M58746, M25607) gene was
retrieved from NCBI GenBank database (http://www.ncbi.nlm.nih.gov) to design a
set of primers for specic detection of ETEC harboring ST1 gene in potable water.

0147-6513/$ - see front matter & 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.ecoenv.2011.07.022

Please cite this article as: Patel, C.B., et al., Contamination of potable water by enterotoxigenic Escherichia coli: qPCR based culture-free
detection and quantication. Ecotoxicol. Environ. Saf. (2011), doi:10.1016/j.ecoenv.2011.07.022

C.B. Patel et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]]]]

Table 1
Sequences of primers used in the study.
a

Nucleotide sequence (50 30 )

Table 2
Specicity of SYBR Green based qPCR assays targeting LT1 and ST1 genes to detect
Enterotoxigenic Escherichia coli.
Position

Product size (bp)

9961145

150

Detection of genes specic to aETEC

by qPCRb,c (1  103 CFU)

327495

169

LT1

ST1




Isolate/strain identity and source

a

LT1 F: 50 -GGCAGGCAAAAGAGAAATGG-30
a
LT1 R: 50 -TTGGTCTCGGTCAGATATGTG-30
b
ST1F: 50 - TTTCCCCTCTTTTAGTCAGTCAAC-30
b
ST1R: 50 -TGGAGCACAGGCAGGATTAC-30
a
b

Adopted from Ram et al. (2008a).

The primers were designed for this study.

An alignment of the sequences retrieved for ST1 gene was created separately
using Clustal W program (www.ebi.ac.uk/clustalW) to determine the conserved
sequences. A set of primers for ST1 gene was designed in highly conserved region
of the gene using Beacon Designer 5.0, Premier Biosoft International (Table 1). The
specicity of primers was determined against known microbial genomes and
sequences by BLAST (Basic Local Alignment Search Tool) program (http://www.
ncbi.nlm.nih.gov/BLAST/) to ensure no homology was observed with known gene
sequences of other water-borne pathogens. A primer set designed in the highly
conserved region of the LT1 gene was adopted from Ram et al. (2008a) to detect
LT1 gene harboring strains of ETEC in potable water (Table 1). All the primers used
in this study were synthesized from Metabion (Gmbh, Germany).

2.2. Generation of standard curve for quantitative enumeration of ETEC

For preparation of standard curve reference strain (Enterotoxigenic E. coli
MTCC 723 exhibiting LT1 and ST1 gene procured from Microbial Type Culture
Collection at Institute of Microbial Technology (IMTECH), Chandigarh, India) was
grown in triplicate in 1 ml LuriaBertani (LB) broth for 12 h at 377 1 1C to
approximately optical density of 0.8 at 600 nm. The number of colony forming
units (CFU/ml) of cell suspension was determined by plating 100 ml of the three
fold diluted culture on to ve LuriaBertani agar plates. The average number of
CFU from the ve plates was used to calculate the concentration (CFU/ml) of cells
in the culture. Further, triplicate cultures of reference strain (optical density 0.8 at
600 nm) were serially diluted ten-fold to yield 107 down to 1 CFU/ml in phosphate
- buffered saline as estimated by standard plate count method. DNA template was
prepared from each dilution as per Jyoti et al. (2010) and qPCR assays for both the
genes were performed using a iCycler (BIO-RAD, USA) real-time PCR instrument
and Quantifast SYBR Green PCR kit (Qiagen, Germany). Briey, the reaction
mixture contained 0.2 mM of the forward and reverse oligonucleotide primer
(1 ml each) for LT1/ST1 gene, 12.5 ml of Quantifast SYBR Green PCR kit (Qiagen,
Germany), deionized water (5.5 ml nuclease free water provided by Qiagen,
Germany) and 5 ml DNA template (corresponding to 1061 CFU/PCR) in a nal
volume of 25 ml. In negative controls 5 ml sterilized Milli-Q water used as
template. The PCR amplication protocol for the assay for both the genes consisted
of initial denaturation of 5 min at 95 1C, followed by 45 cycles of three steps
consisting of 10 s at 95 1C, 20 s at 55.8 1C and 20 s at 72 1C. The uorescence
signals were measured at the end of each extension step. At the completion of PCR
amplication, melting temperature (Tm) analysis of the products was performed
by reducing the temperature to 65 1C and then heating to 95 1C at a rate of
0.2 1C/S. Fluorescence was monitored continuously to conrm amplication
specicity. The Tm peaks were calculated based on the initial uorescence curve
(F/T) by plotting the negative derivative of uorescence over temperature versus
temperature ( dF/dT versus T). The qPCR assays to generate standard curves for
both the genes were repeated thrice and average PCR efciencies and R2 values
were calculated to check the reproducibility of the assays.

ETEC
E. coli MTCC 723 (cMTCC, Chandigarh)
R2A, (dITRC: River Ganga)
M2E dITRC (River Ganga)
L2C dITRC (River Ganga)
R3D dITRC (River Ganga)
M3D dITRC (River Ganga)
M3E dITRC (River Ganga)
L3D dITRC (River Ganga)
L1A dITRC (River Ganga)
L1B dITRC (River Ganga)
L1C dITRC (River Ganga)
L1D dITRC (River Ganga)
M5CdITRC (River Ganga)
L5A dITRC (River Ganga)
L6A dITRC (River Ganga)
L6B dITRC (River Ganga)
1 dITRC (River Gomti)
2 dITRC (River Gomti)
26 dITRC (River Gomti)
30 dITRC (River Gomti)
31 dITRC (River Gomti)
38 dITRC (River Gomti)
50dITRC (River Gomti)
11b dITRC (River Gomti)
68 dITRC (River Gomti)
69 dITRC (River Gomti)
75 dITRC (River Gomti)
77 dITRC (River Gomti)
78 dITRC (River Gomti)
80 dITRC (River Gomti)
86 dITRC (River Gomti)
89 dITRC (River Gomti)
129 dI.T.R.C. (River Gomti)
133 dITRC (River Gomti)

Non-Enterotoxigenic Escherichia coli isolates

NA
97 dITRC (River Gomti)
98 dITRC (River Gomti)

99 dITRC (River Gomti)

M5E dITRC (River Ganga)

d
R6D ITRC (River Ganga)

XRC1 dITRC (River Saryu)

d
XMA3 ITRC (River Saryu)

XLC3 dITRC (River Saryu)










Other bacteria
Enterococcus faecalis eATCC 51299
Enterococcus durans cMTCC 3031
Enterococcus faecium eATCC 51559
Vibrio cholerae eATCC 51394
Salmonella typhimurium
Salmonella typhimurium eATCC 14028















2.3. Specicity and sensitivity of the qPCR assays

a

34 E. coli strains containing LT1 gene (29 positive for both LT1 and ST1 gene and
ve positive for only LT1 gene), three strains positive for only ST1 gene, two strains of
Vibrio cholerae exhibiting ctx gene and ve bacterial strains negative for LT1 and ST1
genes were used for evaluating the specicity of the qPCR primers (Table 2). To test
the specicity of the developed qPCR assays for detection of target genes in presence
of non-specic DNA, assays were performed in presence or absence of background of
DNA of a non-pathogenic E. coli DH5a (107 CFU/ml or 106 CFU/PCR). In brief, E. coli
(MTCC 723) exhibiting both the genes was grown in LB broth for 12 h at 37711C
(optical density 0.8 at 600 nm). Sterile Milli-Qs water (10 ml) in triplicate was
spiked by a ten-fold serially diluted culture of E. coli MTCC 723 (1  107 down to
10 CFU/ml to get 1051 CFU/PCR in 25 mL qPCR reaction) in the presence or absence
of non-pathogenic E. coli DH5a (107 CFU/ml or 106 CFU/PCR). DNA was prepared
from 1 ml spiked samples by extracting total DNA (50 mL) as described by Jyoti et al.
(2010). 5 ml DNA from each concentration was used in 25 mL qPCR reaction to get
1051 CFU/PCR. Bacterial recovery from each spiked sample was assayed by qPCR as
described in Section 2.2. The sensitivity of qPCR assay designed for detection
and quantitative enumeration of ETEC in potable water was also compared with

ETEC: Enterotoxigenic Escherichia coli.

The Observed product Tm by melt curve analyses for LT1 gene and ST1 gene
were 82.5 7 0.244 and 79.5 70.189 1C, respectively.
c
MTCC: Microbial Type Culture Collection at Institute of Microbial Technology
(IMTECH), Chandigarh, India.
d
ITRC: Industrial Toxicology Research Centre (renamed as Indian Institute of
Toxicology Research).
e
ATCC: American Type Culture Collection, USA.
b

conventional PCR using same primer set for each targeted gene and PTC-150 Mini
cycler (MJ Research, USA). In brief, the reaction mixture in a nal volume of
50 ml comprised of dNTP (0.2 mM, 1 ml), TaqDNA polymerase (1.5 units, 1 ml),
10  reaction buffer (5 ml), MgCl2 (1.5 mM, 3 ml), primers (0.2 mM, 1 ml each) for
LT1/ST1 gene, 33.0 ml nuclease free water and DNA template (5 ml) from spiked
water samples (sterilized water spiked by serial ten-fold dilutions from 1  107
down to 10 CFU/ml (105 down to1 CFU/PCR in 50 mL reaction) of E. coli MTCC 723 in
presence or absence of 107 CFU/ml or 106 CFU/PCR of E. coli DH5a) as described

Please cite this article as: Patel, C.B., et al., Contamination of potable water by enterotoxigenic Escherichia coli: qPCR based culture-free
detection and quantication. Ecotoxicol. Environ. Saf. (2011), doi:10.1016/j.ecoenv.2011.07.022

C.B. Patel et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]]]]

earlier in this section. The PCR program was as follows: initial denaturation at 95 1C
for 3 min and then 45 cycles at 95 1C for 20 s, 55.8 1C for 30 s, and 72 1C for 30 s. All
the assays were done in triplicate.
2.4. Quantitative enumeration of ETEC in potable water
For potable water supply in Lucknow (a major city of northern India) water is
pumped from the river Gomti at Gaughat, which is outside the city, and is sent
through a pipeline to Lucknow Jal Sansthan, Aishbagh, 4 km away (Fig. 1), where
the water is puried by alum treatment, ltration, and chlorination before being
released into the drinking water supply (Ram et al., 2008b). To test the possibility
of the contamination of potable waters by ETEC due to defective water distribution systems and insufcient treatment during production, we have collected
water samples (1 L) in triplicate for culture-free quantitative enumeration at nine
sites: site # 1, Gaughat (raw water intake point in the river Gomti); site
# 2 Aishbagh Waterworks (before water enters the distribution system); site #
3, Hussainganj; site # 4, Kaiserbagh (water distribution pipeline that neither
damaged nor ran along open drainage); site# 5, Nakkhas; site # 6, Shashtri Nagar,
site # 7, Saadatganj; site # 8, City Station; site # 9, Moti Nagar (damaged pipeline
ran along open drainage; Fig. 1). All the potable water samples were collected into
sterile glass bottles on the same day in densely populated areas across the urban
boundaries of Lucknow city (latitude, 26.281N; longitude, 80.241E; altitude,
126 m), stored on ice, and transported to the laboratory for analyses within 6 h
(APHA, 1998). The sites were numbered in order of the sample collection.
Bacteriological quality of the potable water samples collected in this study was
assayed to ascertain fecal contamination. Total coliform and fecal coliform levels
in potable water collected from each site were determined by most probable
number (MPN) method as per APHA (1998). qPCR for ETEC and MPN assays for
total coliform and fecal coliform were performed simultaneously in split samples
of potable water collected from each site.
For qPCR assays multigenomic DNA from water (500 ml) was extracted by boiling
prep as per procedure described by Jyoti et al. (2010) and quantication of ETEC
based on LT1 and ST1 gene was done by extrapolating the observed concentrations of
bacterium to ten-fold standard curve generated by diluting the culture of Enterotoxigenic E. coli MTCC 723 exhibiting the known number of cells.
2.5. Statistical analyses
For comparison of PCR amplication efciencies and detection sensitivities
among different experiments, slopes of the standard curves were calculated by
TM
performing a correlation and regression analysis through iCycler iQ Real-Time

Detection System Software Version 3.0 A. Amplication efciency (E) was estimated by using the slope of the standard curve and the formula E (10  1/slope)  1.
A reaction with theoretical 100% efciency will generate a slope of  3.322. Data
obtained from conventional and qPCR were compared by Wilcoxon Rank-SumTest.
ETEC counts retrieved in this study at different sites were analyzed using one way
analysis of variance (Gomez and Gomez, 1984).

3. Results
3.1. Sensitivity and specicity of the qPCR assays
The SYBR Green based qPCR assays targeting LT1 or ST1 gene
could detect directly (without any pre-concentration step) lowest
1 CFU/PCR (10 CFU/ml) from serially ten-fold diluted culture of the
E. coli MTCC 723. Characterization of the amplied products by melt
curve temperature analysis of independently repeated tests indicated that the average measured Tm values were in alignment with
the theoretical calculated values (ST1: 81.2, LT1: 81.0) for each of the
product and the variation between the observed product Tm (ST1:
79.5, LT1: 82.5) value and the calculated value was less than 2%. The
qPCR assays for both the genes were linear with average R2 value
0.99070.004 and 0.99470.003 (mean of three separate qPCR
runs for each gene7SD) for LT1 and ST1 gene, respectively. Further,
average PCR efciency for qPCR assays targeting LT1 and ST1 genes
were 99.170.96 and 98.270.59 (mean of three separate qPCR runs
for each gene7SD). CT values observed during three independent
qPCR assays for each gene were highly reproducible and variability
in CT values for both the genes (LT1 gene: 106: 16.470.459, 105:
19.570.585, 104: 23.270.812, 103 : 27.570.555, 102: 30.671.285,
101: 34.271.128, 1: 35.371.024; ST1 gene : 106: 17.170.394, 105:
20.470.735, 104: 24.070.984, 103: 27.871.068, 102: 31.170.870,
101: 33.471.169, 1: 34.371.063; CT values for each gene are mean
((n3)7SD) between three independent assays was less than 5%.
In this study, thirty four strains of E. coli reported to harbor LT1
gene were positive for the LT1 gene by the SYBR Green qPCR

Fig.1. Locations of sampling sites in the vicinity of the Lucknow City for collection of potable water to detect Enterotoxigenic E. coli through culture-free qPCR.

Please cite this article as: Patel, C.B., et al., Contamination of potable water by enterotoxigenic Escherichia coli: qPCR based culture-free
detection and quantication. Ecotoxicol. Environ. Saf. (2011), doi:10.1016/j.ecoenv.2011.07.022

C.B. Patel et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]]]]

(Table 2). Further, twenty nine E. coli isolates exhibiting both LT1
and ST1 genes were amplied by both the sets of primers
targeting LT1 or ST1 gene (Table 2). Three isolates harboring only
ST1 gene were positive for the ST1 gene specic primers only.
However, no amplication of the target genes was observed in
E. coli strains and other bacterial strains reported to be negative
for either LT1 or ST1 genes (Table 2) during qPCR assays. The Tm of
the products observed in the melt curve analysis for LT1 gene and
ST1 gene was 82.5 and 79.5 1C, respectively. Characterization of
the amplied products by melt curve temperature analysis of
independently repeated tests indicated that the average measured Tm values were in alignment with the theoretical calculated
values (ST1: 81.2, LT1: 81.0) for the products of LT1 and ST1 genes.
The variation between observed amplication product values
of LT1 and ST1 genes in different ETEC strains had a Standard
Deviation (SD) of 70.244 and 0.189 1C, respectively.
The qPCR assays targeting LT1 or ST1 gene individually were
capable of rapidly detecting 1 CFU/PCR (10 CFU/ml) in water
samples spiked by the reference organism (E. coli MTCC 723).
However, in the presence of 107 CFU/ml of a non-pathogenic
E. coli (E. coli DH5a), the lowest detection limit from spiked water
samples was 10 CFU/PCR (100 CFU/ml) for each gene during qPCR
assays for both the genes (Table 3). The melt curve analysis reveal
that Tm of the product for LT1 and ST1 gene were 79.5 (70.240)
and 82.5 1C ( 70.267), respectively.

The conventional PCR assays targeting LT1 and ST1 genes

were able to detect 103 CFU/ml (102 CFU/PCR) of E. coli MTCC 723,
respectively in spiked water samples (Table 4). However, the
presence of a non-pathogenic E. coli DH5a (107 CFU/ml or 106 CFU/
PCR) in spiked water samples reduced the sensitivity of the conventional PCR to 104 CFU/ml (103 CFU/PCR) of E. coli MTCC 723 for both
the genes (Table 4). The qPCR assays in SYBR Green format using the
same oligomers were ten-fold more sensitive than the conventional
PCR (Wilcoxon rank sum test, po0.05, Tables 3 and 4) for LT1 and
ST1 genes.
3.2. Bacteriological quality of potable water
Bacteriological quality of the potable water samples collected in
this study was assayed by determining total coliform and fecal
coliform levels (MPN method) to ascertain fecal contamination.
Potable water collected from sampling sites (site # 59) located in
water distribution system that ran along open drainage exhibit fecal
contamination (Table 5). Further, the water sample collected from
water intake point (site # 1: Gaughat in the river Gomti in urban
boundary of Lucknow city) for Aishbagh waterworks harbor signicantly (one way ANOVA, po0.05) high level of total and fecal
coliforms (Table 5). However, potable water samples collected from
Aishbagh waterworks i.e. potable water ready for civic supply and
samples collected from potable water distribution system where

Table 3
Detection of ETEC targeting LT1 and ST1 gene by SYBR Green based qPCR from water samples spiked by ten-fold serially diluted culture of Escherichia coli
MTCC 723 in presence or absence of 107 CFU/ml E. coli DH5a.
a

CFU/ml

10
105
104
103
102
10

Detection ETEC in spiked water samples by qPCRb,c

E. coli MTCC 723

E. coli MTCC 723 107 CFU/ml

(106 CFU/PCR) E. coli DH5a

E. coli MTCC 723

E. coli MTCC 723 107 CFU/ml

(106 CFU/PCR) E. coli DH5 a

LT1

LT1

ST1

ST1

(15.7 7 0.314 )
(18.9 7 0.378)
(22.9 7 0.687)
(26.5 7 0.761)
(29.5 7 0.885)
(32.2 7 1.288)

(16.27 0.486)
(19.57 0.390)
(23.27 0.835)
(27.67 0.439)
(30.57 0.915)
d
ND

(17.197 0.618)
(20.507 0.594)
(24.117 0.964)
(27.867 0.975)
(31.157 0.985)
(33.467 0.836)

(17.18 7 0.498 )
(21.4 70.428)
(25.2 70.957)
(29.1 70.698)
(32.35 7 0.808)
d
ND

a
DNA (5 mL) template from spiked water samples (106 down to 10 CFU/ml) in 25 mL qPCR assay corresponds to 105 down to 1 CFU/PCR . All the assays
were conducted in triplicates and Sterile Milli-Q water served as negative (no template) control.
b
Values inside parentheses are mean CT values7 SD (n 3).
c
Product Tm for LT1 and ST1 gene were 79.5 (7 0.240) and 82.5 1C (70.267), respectively.
d
ND: CT not available.

Table 4
Detection of ETEC targeting LT1 and ST1 gene by conventional PCR from water samples spiked by ten-fold serially diluted culture of Escherichia coli MTCC
723 in presence or absence of 107 CFU/ml E. coli DH5a.
a

CFU/ml

Detection ETEC in spiked water samples by conventional PCR (No. of positive replicates/no. of replicates analyzed)b

LT1

E. coli MTCC 107 CFU/ml

(106 CFU/PCR) E. coli DH5a
LT1

ST1

E. coli MTCC 723 107 CFU/ml

(106 CFU/PCR) E. coli DH5 a
ST1

(3/3)
(3/3)
(3/3)
(3/3)
(3/3)
c
ND

(3/3)
(3/3)
(3/3)
(3/3)
c
ND
c
ND

(3/3)
(3/3)
(3/3)
(3/3)
(3/3)
c
ND

(3/3)
(3/3)
(3/3)
(3/3)
c
ND
c
ND

E. coli MTCC 723

10
105
104
103
102
10
a
b
c

E. coli MTCC 723

DNA (5 ml) template from spiked water samples (106 down to 10 CFU/ml) in 50 ml conventional PCR assay corresponds to 106 down to 1 CFU/PCR.
All the assays were conducted in triplicates. Sterile Milli-Q water served as the negative (no template) control.
ND: Not Detected.

Please cite this article as: Patel, C.B., et al., Contamination of potable water by enterotoxigenic Escherichia coli: qPCR based culture-free
detection and quantication. Ecotoxicol. Environ. Saf. (2011), doi:10.1016/j.ecoenv.2011.07.022

C.B. Patel et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]]]]

Table 5
Quantitative enumeration of coliforms in the drinking water distribution system.
Sampling sites

Most probable Number/100 mla,b

Total coliforms

Fecal coliforms

5.3 E077 1.72 E02

d
ND
d
ND
d
ND
5.30 E 027 19
5.6 E027 25
9.5 E057 4.8 E04
1.600 E037 64
3.30 E027 13
d
ND

4.3 E057 9.2 E03

d
ND
d
ND
d
ND
3.30 E027 9
4.90 E027 20
5.9 E047 2.36 E02
9.50 E027 48
2.10 E027 8
d
ND

distribution system where pipeline neither damaged nor ran along

open drainage were negative for ETEC by LT1 and ST1 genes
targeting SYBR Green based qPCR assay (Table 6).

4. Discussion
Site:1 Gaughat
Site:2 Aishbagh Waterworks
Site: 3 Hussainganj
Site:4 Kaiserbagh
Site:5 Nakkhas
Site:6 Shashtri Nagar
Site:7 Saadatganj
Site:8 City Station
Site:9 Moti Nagar
c
Negative Control
a

Mean of three observations.

One way ANOVA, p o 0.05 for each column.
Sterile Milli-Q (Millipore, Bedford, MA, USA) water served as the negative
control.
d
ND, none detected.
b
c

Table 6
Culture-free detection and quantitative enumeration of ETEC in potable water
by qPCR.
a

Sampling sites

Site:1 Gaughat
Site:2 Aishbagh Waterworks
Site: 3 Hussainganj
Site:4 Kaiserbagh
Site:5 Nakkhas
Site:6 Shashtri Nagar
Site:7 Saadatganj
Site:8 City Station
Site:9 Moti Nagar

Potable water (ETEC/100 ml)b,c

Targeting LT1 gene

Targeting ST1 gene

1.75E 05 7 4.3E 04
d
ND
d
ND
d
ND
100 74
41 7 0.826
87 7 3.045
261 7 5.22
74.4 7 1.484

1.352 E03 758

d
ND
d
ND
d
ND
257 1
677 2.019
6430 7289.35
557 1.939
33.8 71.014

a
Site 1: raw water intake point in the river Gomti, site 2: Aishbagh Waterworks before water enters the distribution system; sites 34: water distribution
pipeline that neither damaged nor ran along open drainage; sites 59: pipeline
that damaged and ran along open drainage.
b
Sterile Milli-Q water used as negative control.
c
One way ANOVA, p o 0.05 for each column.
d
ND: not detected.

pipeline neither damaged nor ran along open drainage were not
contaminated by fecal contaminants (Table 5).
3.3. Quantitative enumeration of ETEC in potable water
The SYBR Green based qPCR assays targeting LT1 and ST1 genes
could detect quantitatively ETEC in the potable water samples
collected from urban boundaries of a city located in northern
India. A signicant variation in ETEC contamination at different
sites was observed (one way ANOVA, po0.05) Potable water
collected from sampling sites (site # 59) located in water
distribution system that ran along open drainage exhibit ETEC
contamination due to percolation of fecal contaminants from
damaged pipeline (Table 6). The ETEC levels at site # 59 targeting
LT1 gene were 10074, 4170.826, 8773.045, 26175.22 and
7471.484, respectively. However, the ETEC levels at site # 59
targeting ST1 gene were 2571, 6772.019, 64307289.35,
5571.939 and 3471.014, respectively (Table 6). The ETEC level
in raw water of the river Gomti used at Aishbagh waterworks
targeting LT1 and ST1 genes was 1.75E 0574.3 E04 and
1.352E 03758, respectively (Table 5). However, potable water
samples collected from Aishbagh waterworks i.e. potable water
ready for civic supply and samples collected from potable water

Globally, 41.1 billion people drink unsafe water and a vast

majority of diarrheal diseases are attributable to unsafe water,
sanitation and hygiene (WHO, 2002). Outbreaks of waterborne
diarrheal diseases due to consumption of contaminated drinking
water still pose a serious health threat worldwide, despite the fact
that drinking water is one of the most closely monitored and
strictly regulated resources. In countries of developing world
including India, a large population depends on processed surface
waters for drinking and other domestic usage.
In the present study, we found potable water samples to be
contaminated by coliform and fecal coliform bacteria. As per the
water quality norms provided by WHO (2002), the level of E. coli
or thermotolerant bacteria should be zero in a 100 ml sample of
water directly intended for drinking or in treated water entering a
distribution system. However, a few drinking water standards
allow the presence of ten coliforms/100 ml in drinking water
(Bureau of Indian Standards (BIS), 1991). All the potable water
samples positive for fecal contamination in the present study
exceeded the standard permissible limits recommended by various regulatory bodies for drinking water (BIS, 1991; WHO, 1993).
The observations made in this study suggest the possibility of
contamination of water distribution system by fecal contaminants due to percolation of sewage from the open drainage.
Earlier, it has been observed that leaking sewage lines, human
and animal excreta owing into open drains are potential sources
of contamination of pathogenic E. coli in defective drinking water
distribution systems (Ram et al., 2008b). Detection of fecal coliforms in a culturable metabolic state during the present study is
indicative of the recent contamination of the water distribution
system by human and/or animal feces due to defective sewage
lines and storage tanks. In India and some other countries
drinking water supplies contaminated by pathogenic bacteria
due to defective sewage lines has been reported (Schets et al.,
2005; Ram et al., 2008b). The drinking water quality standards
across the world depend entirely on the presence/absence of total
coliform and fecal coliform bacteria (WHO, 1993). Analysis for
fecal-indicator bacteria by MPN (most probable number) procedures provides an indication of fecal pollution in drinking water
supplies (WHO, 2002). However, 48 h are required to conrm
fecal pollution in a sample. However, this provides no insight
about the pathotypes of the E. coli or any other coliform bacteria
(Ram et al., 2011). Further, it has been reported that a value of
total coliform in an environmental sample consists of E. coli,
Citrobacter, Enterobacter and Klebsiella spp. (E. coli: Citrobacter/
Enterobacter: Klebsiella spp.; 94:4:2) and E. coli contributes 94% to
the total coliform value of an environmental sample (Dufour,
1977 ; Leclerc et al., 2001). E. coli is common constituent of gut
ora. Certain strains of E. coli, responsible for enteric and diarrheal
diseases, urinary tract infections and sepsis/meningitis are classied into eleven recognized pathotypes on the basis of their
distinct virulence properties and clinical symptoms (Kaper et al.,
2004). Thus, samples positive by MPN method lacking pathotypes
of E. coli sometimes may not be harmful to a population
consuming water for their daily requirements. Therefore, monitoring protocols should be inclusive of the assays capable for
detection of pathotypes of E. coli. The MPN based methods are
only applicable for primary screening of water to ascertain the
presence of bacteria exhibiting fecal origin. Enterotoxigenic E. coli
is one in eleven recognized pathotypes of E. coli (Kaper et al.,

Please cite this article as: Patel, C.B., et al., Contamination of potable water by enterotoxigenic Escherichia coli: qPCR based culture-free
detection and quantication. Ecotoxicol. Environ. Saf. (2011), doi:10.1016/j.ecoenv.2011.07.022

C.B. Patel et al. / Ecotoxicology and Environmental Safety ] (]]]]) ]]]]]]

2004). Diarrheal disease caused by ETEC was found to be

associated with high mortality (children below the age of ve
years) and morbidity in developing countries. ETEC may not be
detected by traditional microbiological screening by culturebased assays if samples exhibit high concentrations of sub lethally
injured cells. The sub lethally injured cells are viable but not
culturable metabolically active cells and could not be detected
due to their inability to grow on selective media. Therefore, in the
case of widespread diarrheal epidemics, rapid identication of
sources of contamination and causative agents is critical for early
intervention (Ram et al., 2011) The qPCR assays for a bacterium
including E. coli overcomes the limitations of the culture-based
assays that include length of time required (1896 h), potential
for false positive and negative results, loss of viability of bacteria
between the time of the sample collection and enumeration, and
lack of growth of viable but nonculturable bacteria (Khan et al.,
2007; Ram et al., 2008a). Therefore, in this study we have
monitored the potable water quality by a highly sensitive (Lowest
detection limit :1 CFU/PCR) SYBR Green based qPCR assay targeting two genes specic for Enterotoxigenic E. coli i.e. LT1 and ST1
genes encoding heat-labile and heat-stable toxins, respectively.
A comparison of the qPCR assays targeting LT1 and ST1 genes with
conventional PCR using the same set of PCR primers revealed that
the present assay is superior to conventional PCR in terms of the
sensitivity. The lowest detection limits for ETEC harboring LT1 or
ST1 gene observed in this study for SYBR Green based qPCR assay
and conventional PCR were 1 and 103 CFU/PCR, respectively. This
is in agreement with earlier studies which report that qPCR assays
for detection of ETEC are more rapid and sensitive in comparison
to conventional PCR (Reischl et al., 2004; Lothigius et al., 2008;
Ram et al., 2008a).
In this study, we could detect lowest 1 CFU/PCR or 10 CFU/ml
with DNA extracted through rapid boiling of the bacteria from the
spiked water samples followed by precipitation. In a previous assay
reported on SYBR Green based real-time PCR (qPCR) for detection of
ETEC from house hold surface water for drinking the detection limit
corresponds to 5  102 CFU/PCR with DNA extracted by rapid boiling
of the bacteria (Lothigius et al., 2008). Therefore, the present assay is
500 times more sensitive than earlier SYBR Green based assay
reported for detection of ETEC from house hold surface water for
drinking. Another Molecular Beacon based qPCR assay reported for
detection of ETEC targeting LT1 gene from surface water escapes
strains exhibiting only ST1 gene (Ram et al., 2008a).
In the present study, we could quantify as low as 1 CFU/PCR of
ETEC exhibiting LT1/ST1 gene in spiked water samples exhibiting
106 down to 10 CFU/ml (105 down to 1 CFU/PCR) of E. coli MTCC
723. However, presence of a high concentration of DNA from nonpathogenic E. coli reduced the detection limit ten-fold. Reduction
in lowest detection limit is probably due to competition generated by the non-specic DNA. Khan et al. (2007) reported a SYBR
green based assay targeting the distal and proximal conserved
anking regions of the 16 S rRNA gene, the internal transcribed
spacer region (ITS) region, and the 23 S rRNA gene that could
detect E. coli (10 CFU/ml) in agricultural watersheds. The lowest
detection limit of the assay reported by Khan et al. (2007) also
drops 10-fold in the presence of non-specic DNA. Further, the
assay reported by Khan et al. (2007) could not differentiate
between the pathogenic and non-pathogenic E. coli in real water
samples. Besides, these assays were not optimized for the detection
of ETEC from potable waters. In this study, we have detected ETEC
from potable water targeting both LT1 and ST1 genes. All the sites
exhibiting damaged pipeline that ran along sewage line were
contaminated by the ETEC due to percolation of the sewage. To
our knowledge we are reporting for the rst time contamination of
potable water distribution system by ETEC through qPCR in a south
Asian country exhibiting tourist attraction. Further, it is possible that

E. coli may transit between virulent and nonvirulent form by

acquiring or losing virulence genes encoded by plasmid(s) making
these forms indistinguishable from normal gut ora in individuals
including tourists consuming river water for daily needs (Hacker
et al., 1997; Ram et al., 2007). Therefore, the presence of E. coli
exhibiting plasmid encoded virulence genes (LT1 and ST1) in the
potable water may lead to emergence of new pathogenic variants.
Hence, the detection of LT1 and ST1 genes in multigenomic DNA
from potable water is always an indicative of health risk to human
population consuming contaminated water.

5. Conclusion
The present study concludes that the potable water distribution system ran along sewage line may acquire ETEC contamination. Low level of ETEC contamination of the potable water could
be detected by highly sensitive and rapid SYBR green assay
targeting LT1 or ST1 gene. The assay could be used for the regular
monitoring of potable water to prevent water-borne diarrhea
caused by ETEC in places exhibiting heavy ow of tourists.

Acknowledgments
We are thankful to Dr K.C. Gupta, Director, Indian Institute of
Toxicology Research for providing the necessary facilities for this
study. This work was supported by CSIR Network Project NWP-17.
The nancial assistance to C.B. (SRF), G.S. (SRF), and P.V. (WOS-A)
from CSIR and DST, Government of India, New-Delhi, respectively, is
acknowledged. We declare that we have no competing nancial
interests. IITR manuscript no. 2915.
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Please cite this article as: Patel, C.B., et al., Contamination of potable water by enterotoxigenic Escherichia coli: qPCR based culture-free
detection and quantication. Ecotoxicol. Environ. Saf. (2011), doi:10.1016/j.ecoenv.2011.07.022