Curr Microbiol (2009) 58:2529

DOI 10.1007/s00284-008-9260-3

Self-Purificatory Ganga Water Facilitates Death of Pathogenic

Escherichia coli O157:H7
Chandra Shekhar Nautiyal

Received: 25 June 2008 / Accepted: 25 August 2008 / Published online: 23 September 2008
Springer Science+Business Media, LLC 2008

Abstract Concern over the prevalence of active pharmaceutical agents and subsequent occurrence of antimicrobial
resistance in the environment is increasing. Incorruptible
ability of Ganga water was evaluated using fresh, 8-yearold, and 16-year-old Ganga water samples spiked with
pathogenic Escherichia coli serotype O157:H7. Survival of
E. coli O157:H7 over the course of the experiment was 3, 7,
and 15 days for fresh, 8-year-old, and 16-year-old Ganga
waters, respectively. On the contrary, in Milli Q water the
decline in viable count of E. coli O157:H7 up to 30 days was
only 2 log units. Survival of E. coli O157:H7 was greater in
boiled water compared with water after passage through a
0.2-lm-pore-size membrane filter, indicating involvement
of heat-labile agents influencing survival of E. coli O157:H7
in Ganga water, which seems to indicate the role of antimicrobial peptides. Functional diversity of Ganga waters
native microbial community structure as assessed with
Biolog Eco plates was not affected even in the presence of a
5-fold log units higher pathogenic load of E. coli O157:H7.
These findings suggest that Ganga water has certain novel
antimicrobial attributes, besides its remarkable fluidity,
which may provide a much-needed basis for the development of new antimicrobial compounds.

Introduction
The water of the River Ganga is frequently used for
drinking, cooking, and bathing purposes due to ancient
C. S. Nautiyal (&)
Division of Plant Microbe Interactions, National Botanical
Research Institute, Rana Pratap Marg, Lucknow 226001, India
e-mail: csn@nbri.res.in

knowledge that Ganges water does not putrefy, even after

long periods of storage. Water has been used from time
immemorial for remedial purposes. Most religious beliefs
involve some ceremonial use of holy water, and in India
the water of the River Ganga is treated with such reverence. Under the continuous Saraswati-Indus civilization
going back to *7500 BC, the River Ganga is mentioned in
Rigveda [16]. Hippocrates, going back to *500 BC, wrote
about the healing of disease with water. Bathing held a
prominent place in the law that was prepared by Moses
under divine instruction for the government of the Hebrew
nation. The role of the bath in the treatment of leprosy also
would lead one to believe that water was used for curative
effects [11]. Outbreaks of acute diarrheal disease have been
identified as causes of fatal disease dating back as far as the
Sanskrit literature and during Hippocratic times [15].
Ernest Hankin, a British bacteriologist, reported in 1896 on
the presence of marked antibacterial activity against Vibrio
cholerae, which he observed in the water of the River
Ganga river India, and he suggested that it might help to
decrease the incidence of cholera in people using water
from the Ganges. Though invisible, it was possible to show
that this principle was particulate and DHerelle called it
bacteriophage [6]. Thus in a way the world owes the
discovery of bacteriophages to the Ganges water.
Overuse in human medicine and for agricultural purposes has become a recognized medical problem, and
scientists have become increasingly concerned about the
occurrence of antibacterial resistance in the environment.
To curtail the development and spread of antimicrobial
resistance will require both the preservation of current
antimicrobials through their appropriate use and the discovery and development of new agents. Technologies for
accessing and screening new sources of badly needed and
novel antibiotics have improved dramatically during the

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past decade [10, 12, 14, 22]. This study was conducted to
validate our ancient knowledge about the antimicrobial
effect of Ganga water and to evaluate the potential of
Ganga water in our endeavor to explore the possibility of
using it as a novel source of antimicrobial compounds.
Enterohemorrhagic Escherichia coli is a worldwide cause
of infection in humans and animals. E. coli O157:H7 is a
major enteropathogen responsible for causing outbreaks of
hemorrhagic colitis and hemolytic uremic syndrome [1].
The human infectious dose is very low, and ingestion of as
few as 10 cells is thought to be sufficient to cause illness
[3]. The objective of this study was to evaluate the incorruptible self-purificatory characteristic and microbial
community structure of Ganga water when spiked with E.
coli O157:H7.

Materials and Methods

Sampling Stations
Water samples for the present study were collected form
the upper stretch of River Ganga (hilly region) at Rishikesh, having the geographical coordinates of longitude
78420 E and latitude 3070 N. The water samples were
collected from the same site in March 2000 and 2007. A
16-year-old sample was collected in 1991 from Gomukh in
the snout of the Gangotri Glacier, a vast expanse of ice
5 9 15 miles in the higher Himalaya, at an altitude of
3920 m, having the geographical coordinates of longitude
78540 E and latitude 30540 N. The samples thus collected
were stored at Lucknow in clean glass bottles fitted with
screw caps indoors in a cool dark place. Lucknow is
located in mid-Ganges plains at an altitude of 123 m,
having geographical coordinates of longitude 80590 E and
latitude 26550 N and a mean ambient temperature in the
range of 20 to 35C. A fresh Ganga water sample was
filtered with a sterilized filtration unit, through a 0.22-lmpore-size membrane filter (diameter, 47 mm; Millipore
Inc., Billerica, MA, USA). The purpose of this filtration
was to allow viruses to pass through the filter while
removing organic and inorganic chemicals, natural organic
matter, protozoans, algae, zooplankton, and free-living
aquatic bacteria. Boiled water was obtained by boiling a
fresh Ganga water sample for 20 min to kill micro-organisms [13].
Bacterial Analysis
Heterogeneous bacterial counts in the water were elucidated by serial dilution plating directly on nutrient agar
(from HI-MEDIA Laboratories Pvt. Ltd., Mumbai, India)
plates. After 72 h of incubation at 28C, the colonies that

123

C. S. Nautiyal: Self-Purificatory Ganga Water

developed on the plate were counted [17]. Strains of E. coli

were not detected in Ganga water. E. coli O157:H7 (ATCC
43895) was obtained from King George Medical University, Lucknow. Rainbow agar O157:H7 was used for
enhanced detection of E. coli O157:H7 (Biolog, Inc.,
Hayward, CA, USA).
Survival of E. coli O157:H7 in Water
Inoculation of E. coli in Ganga water and Milli-Q water
was carried out using overnight grown culture to assess the
impact of water on survival of E. coli O157:H7. Overnightgrown culture was centrifuged and the pellet was washed
three times using 0.85% sterile saline (w/v NaCl), then
inoculated in Ganga water and Milli-Q water to a starting
concentration of about 5 9 107 CFU/mL, and the mixture
placed in three replicate (sterile) polypropylene tubes at
30C under static conditions. E. coli O157:H7 surviving in
the water was quantified at the designated time up to
30 days, diluted, and plated on Hi-Crome ECC agar plates
(from HI-MEDIA Laboratories Pvt. Ltd.). All experiments
were performed independently at least three times.
Microbial Diversity Using the Carbon Source
Utilization Pattern
Biolog Eco plates (Biolog, Inc.) were used to determine the
effect of E. coli on carbon source utilization pattern after
different times of incubation in Ganga water and Milli-Q
water. Inoculation of E. coli in Ganga water and Milli-Q
water was carried out as described earlier [16]. Data were
recorded for days 17 at 590 nm. Microbial activity in each
microplate, expressed as average well color development
(AWCD), was determined as described by Garland [8].
Diversity and evenness indexes were calculated as described by Staddon et al. [21]. Principal component analysis
(PCA) was performed on data divided by the AWCD as
described by Garland and Mills [9]. Formulas used for
diversity calculations were described by Staddon et al.
[21]; data collected after day 5 were used. At least three
independent experiments were conducted for each treatment. Statistical analyses were performed using SPSS 16.0
and Windowstat 7.5.

Results and Discussion

Studies of factors affecting the survival of E. coli in
Ganges water is of great interest due to its importance as an
indicator of fecal pollution in natural waters. It is ancient
knowledge that Ganges water does not putrefy, even after
long periods of storage, thus water from the Ganges has for
millennia been regarded as incorruptible [4, 6, 19]. To

C. S. Nautiyal: Self-Purificatory Ganga Water

facilitate a fair assessment of the potential of its selfpurificatory and incorruptible abilities, Ganga water having
a resident bacterial population of 4.0 9 102 CFU/mL was
spiked with about 5.0 9 107 CFU/mL E. coli O157:H7.
The incorruptible nature of the water was studied in fresh,
8-year-old, and 16-year-old Ganga water samples spiked
with E. coli O157:H7. Figure 1 shows the decline in viable
counts of E. coli O157:H7 in fresh, 8-year-old, and 16year-old Ganga water during the course of the experiment.
In general, the number of culturable E. coli O157:H7
declined over time but tended to be greater in fresh water
than in 8- and 16-year-old water. Survival of E. coli
O157:H7 over the course of the experiment was 3, 7, and
15 days for fresh, 8-year-old, and 16-year-old Ganga
waters, respectively. On the contrary, in Milli-Q water the
decrease in the viable count of E. coli O157:H7 up to
30 days was 2.0 9 104 CFU/mL (Fig. 1).
Age of the water seems to influence survival of E. coli
O157:H7, thus its fate was further studied in boiled water
and after passage through a 0.2-lm-pore-size membrane
filter. To elucidate the involvement of active principals and
their sensitivity to high temperatures, the water was boiled.
Water samples thus prepared were spiked with E. coli
O157:H7 to evaluate the antibacterial ability of the water.
Boiling water at 100C kills microbes; filtration is
becoming increasingly the method of choice for sterilization of biologicals, especially when the product is heat
labile, because the filtration process is inherently nondestructive. In general, 0.2 lm will remove algae, protozoa,
and most bacteria, while a 0.01-lm filter is needed to

Fig. 1 Survival of E. coli O157:H7 in Milli-Q water and Ganga

water. Overnight-grown culture was inoculated into Ganga water and
Milli-Q water to an initial concentration of about 5 9 107 CFU mL-1
in polypropylene tubes before the experiment was carried out,
incubated under static conditions at 30C during the course of the
experiment, and plated on Hi-Crome ECC agar plates (HI-MEDIA
Laboratories Pvt. Ltd., Mumbai, India). E. coli O157:H7 surviving in
the water was quantified at the designated times up to 30 days. The
plotted data are averages from three independent experiments

27

remove viruses [13]. Overall, survival was higher in boiled

water (3.5 9 102 CFU/mL for up to 25 days; Fig. 1) than
in water after passage through a 0.2-lm-pore-size membrane filter (3.9 9 102 CFU/mL for up to 15 days; Fig. 1),
indicating that heat-labile agents influence the survival of
E. coli O157:H7 in Ganga water. An interesting observation was the ability of the 8- and 16-year-old Ganga water
to influence survival of E. coli O157:H7. Eight-year-old
water had a better ability to kill E. coli O157:H7 compared
with boiled water and water passed through a 0.2-lm-poresize membrane filter. While antibacterial activity of 16year-old water was better than that of boiled water and
almost comparable to that of water passed through a 0.2lm filter, indicating that a combination of factors controls
the rate of decline and does not let the water putrefy, even
after long periods of storage. Further studies should be
undertaken to establish which factors are the key regulators
influencing the death of E. coli O157:H7 in Ganga water.
To investigate the well-known self-purificatory characteristic of Ganga water, the impact of the addition of E. coli
O157:H7 on the microbial community structure in Milli-Q
water and Ganga water after incubation for 0, 3, 5, and
7 days was assessed, using Biolog Eco plates. Eco plates
are intended for environmental samples; they contain 31
different carbon sources in a triplicate pattern. Patterns of
carbon source utilization were used to calculate diversity
indexes of Shannon, Simpson, and McIntosh and related
evenness indexes. Analysis of carbon source utilization
patterns by microbial samples using Biolog plates shows
promise as a means of assessing microbial community
structure, which examines the functional capabilities of the
microbial population, and the resulting data can be analyzed using multivariate techniques to compare the
metabolic capability of communities. Biolog plates have
found application in the assessment of microbial metabolic
diversity in water [2]. We used the Biolog plates as a way
to estimate the metabolic diversity of the native heterotrophic bacteria in Ganga water in the absence and
presence of E. coli O157:H7. Such information allows
examination of the natural variation and diversity of
microbial communities. Most importantly, this technique
offers the potential to monitor changes in microbial
diversity caused by environmental fluctuations, management practices, and pollution. Variable significant
differences among treatments were noted for water samples
collected at 0, 3, 5, and 7 days using the McIntosh,
Shannon, and Simpson indexes (Table 1). Evenness calculated from both the Shannon and the McIntosh indexes,
however, was not significantly different among treatments
(Table 1). The principal components (PC) score plots
describe the characteristics of the samples and help to
clarify their distribution and clustering. The PC score plot
(PC-I and PC-II) shows the spatial distribution of the

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C. S. Nautiyal: Self-Purificatory Ganga Water

Table 1 Diversity/evenness index of E. coli O157:H7 sample in Milli-Q water (MQW) and Ganga water (GW) incubated for different time
periods
Sample no.

Treatment/
incubation
time (days)

McIntosh

MQW (0)

0.912 0.001a

2
3
4
5

GW (0)
MQW (3)
GW (3)
MQW (5)

0.951 0.001

bc

0.951 0.001

bc

0.954 0.004

bc

0.930 0.026

ab
c

McIntosh evenness

Shannon

0.935 0.038a

3.013 0.015a

3.186 0.002

de

3.205 0.004

3.137 0.032

bc

3.125 0.001

0.967 0.001

3.254 0.004

0.952 0.001

0.951 0.001

0.958 0.001

0.957 0.000

Shannon evenness

Simpson

0.881 0.001a

0.964 0.004a

0.983 0.002a

0.982 0.001a

0.983 0.001a

0.982 0.001a

0.946 0.005

0.987 0.002a

0.927 0.001
0.932 0.001
0.926 0.002
0.928 0.002

GW (5)

0.965 0.004

MQW (7)

0.951 0.001bc

0.955 0.001a

3.172 0.001cde

0.932 0.001b

0.982 0.002a

bc

bcd

0.965 0.030a

GW (7)

0.947 0.002

0.951 0.001

3.160 0.000

0.927 0.002

Note: Biolog Eco plates (Biolog, Inc., Hayward, CA, USA) were used to determine the diversity/evenness index of E. coli O157:H7 samples in
Milli-Q water (nos. 1, 3, 5, and 7) and Ganga water (nos., 2, 4, 6, and 8) incubated for 0, 3, 5, and 7 days, respectively. Data on Biolog Eco plates
were recorded for up to 7 days after a regular interval of 24 h at 590 nm with an automated microplate reader (Bio-Tek Instruments Inc., USA).
Microbial activity in each microplate, expressed as average well color development (AWCD), was determined as described by Garland [8]. Blank
subtracted data on day 5 divided by AWCD as described by Garland and Mills [9] was used to calculate diversity indexes of Shannon, Simpson,
and McIntosh and related evenness indexes. Formulas used for diversity calculations are described by Staddon et al. [21]. Letters indicate
significant differences revealed by Tukey test, at p [ 0.05. Error bars are standard deviation (n = 3). Statistical analyses were performed
using Microsoft Office 2003 and SPSS 16.0

samples. PCA of the carbon source utilization pattern on

Biolog Eco plates showed clustering among the Ganga
water samples incubated with E. coli for 0, 3, 5, and
7 days, while the samples from Milli-Q water samples
incubated with E. coli for 0, 3, 5, and 7 days distributed
separately from each other, at 34 and 77% on the PCA
vector 1 and 2 axis (Fig. 2). There was distinct resolution
of Milli-Q water microbial communities from Ganga water
microbial communities spiked with E. coli O157:H7
(Fig. 2). PCA thus indicated no impact of the addition of E.
coli O157:H7 on microbial community structure in Ganga
water, while the addition of E. coli O157:H7 in Milli-Q
water resulted in significant differences in the microbial
community structure.
The survival of fecal bacteria in aquatic environments is
in general dependent on their ability to tolerate a range of
biological, physical, and chemical stresses. These include
the influence of temperature, UV radiation, predation, and
nutrient availability [7]. For boiled and membrane-filtered
Ganga water in which survival of E. coli O157:H7 was
monitored, it appeared that biotic factors had a strong
influence on survival. These results attain further importance when one considers the fact that the resident bacterial
population of 4 9 102 CFU/mL Ganga water was spiked
with about 5 9 107 CFU/mL E. coli O157:H7. Thus even a
5-fold log units higher pathogenic load of E. coli O157:H7
did not affect the Ganga waters native microbial community structure in the studied environment. To the best of
our knowledge this is the first report demonstrating that the
functional diversity of Ganga water as assessed with Biolog
Eco plates was not affected even in the presence of a high

123

Fig. 2 Principal component analysis (PCA) of carbon source utilization pattern on Biolog Eco plates (Biolog, Inc., Hayward, CA,
USA) of E. coli O157:H7 samples in Milli-Q water (nos. 1, 3, 5, and
7) and Ganga water (nos. 2, 4, 6, and 8) incubated for 0, 3, 5, and
7 days, respectively, was carried out. Biolog Eco plates were used to
determine the effect of E. coli on carbon source utilization pattern at
different times of incubation in Ganga water and Milli-Q water. Data
on Biolog Eco plates were recorded every 24 h at 590 nm with an
automated microplate reader (BioTek Instruments Inc., USA). At 5th
day PCA was performed on blank subtracted data divided by the
average well color development (AWCD) as described by Garland
and Mills [9]. The plotted data are averages of three independent
experiments. PCA was performed using Windowstat 7.5

pathogenic load of E. coli O157:H7. The studies clearly

demonstrate that Ganga water indeed has certain novel
antimicrobial attributes, besides its remarkable fluidity and
adaptability in the presence of a heavy load of E. coli
O157:H7, thereby validating the rivers remarkable
magical self-cleansing properties. Although it is not
possible to extrapolate the behavior of a single strain of E.

C. S. Nautiyal: Self-Purificatory Ganga Water

coli O157:H7 under laboratory conditions to that of all

strains under environmental conditions, this study has
provided an insight into the impact of Ganga water on E.
coli O157:H7 survival. Technologies for accessing and
screening new sources of badly needed and novel antibiotics have improved dramatically during the past decade
[5, 18, 20]. Where combinatorial chemistry and genomics
have failed, could an exploration of untapped sources usher
in a second golden age of antibiotic discovery? The
involvement of heat-labile agents influencing survival of E.
coli O157:H7 in Ganga water seems to indicate a role of
antimicrobial peptides (AMPs). AMPs are part of the
innate immune system, and an important component of
immune defense. They are produced by plants, animals,
insects, and single-celled organisms, and possess antimicrobial properties. As such, they are an ideal target for
future antibiotic production [5, 18, 20]. The encouraging
results of these experiments demonstrating the Gangas
antimicrobial capacity, indicating involvement of heatlabile agents, if carefully developed, could eventually
provide a much-needed basis for the development of new
antimicrobial compounds.
Acknowledgments The author is deeply indebted to R.K. Trivedi,
former Governor of the State of Gujarat, India, for providing the 16year-old Ganga water sample; V. Sharma, King George Medical
University, Lucknow, for providing the strain of E. coli O157:H7; and
B. Staddon, Eastern Kentucky University, Richmond, USA for, his
generous help in the Diversity/Evenness Index analysis. Thanks are
due to the Director, National Botanical Research Institute, Lucknow,
for the necessary support. This work was supported by the New
Millennium Indian Technology Leadership Initiative (NMITLI) program and Task Force Grant NWP-0019 from the Council of Scientific
and Industrial Research, New Delhi.

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