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Abstract

Host-specific bacteriophages against some enteric human pathogens were isolated from Ganga water collected from Har Ki Pauri of Haridwar. Development of clear plaques on the host bacteria, i.e., Escherichia coli ATCC27 853, Salmonella typhi MTCC 733 and Klebsiella pneumoniae MTCC 432, indicated the presence of lytic bacteriophages. All the three bacteriophages infected their respective host and formed plaques characterized with different sizes, shapes and numbers showing their host-specificity. Cross-infectivity test showed that each phage infected only its host bacteria but not the others. Number of plaques formed on lawn of S. typhi was the maximum and that on K. pneumoniae was the minimum. However, the plaques formed on lawn of K. pneumoniae were the largest in size. Scanning electron microscopy revealed deformities in the Phage-infected bacterial cells due to lysis by phages and by damaging the structure of the cell wall and cell membrane.
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Scientific Transactions in Environment and Technovation
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J. Sci. Trans. Environ. Technov. 12(1), 2018 1
Scientific Transactions in
Environment and
Technovation
Isolation of host-specific bacteriophages from Ganga water against some
enteric bacterial pathogens of human
Shivani Tyagi and R.C. Dubey*
Department of Botany and Microbiology, Gurukula Kangri Vishwavidyalaya, Haridwar- 249404, India.
Abstract
Host-specific bacteriophages against some enteric human pathogens were isolated from Ganga water collected from
Har Ki Pauri of Haridwar. Development of clear plaques on the host bacteria, i.e., Escherichia coli ATCC27 853,
Salmonella typhi MTCC 733 and Klebsiella pneumoniae MTCC 432, indicated the presence of lytic bacteriophages. All
the three bacteriophages infected their respective host and formed plaques characterized with different sizes, shapes
and numbers showing their host-specificity. Cross-infectivity test showed that each phage infected only its host bacte-
ria but not the others. Number of plaques formed on lawn of S. typhi was the maximum and that on K. pneumoniae was
the minimum. However, the plaques formed on lawn of K. pneumoniae were the largest in size. Scanning electron
microscopy revealed deformities in the Phage-infected bacterial cells due to lysis by phages and by damaging the
structure of the cell wall and cell membrane.
Key words: Ganga River, Bacteriophages, Host-specificity, Scanning electron microscopy
Received : April 2017 Revised and Accepted : July 2018
*Corresponding Author :
email: profrcdubey@gmail.com
J. Sci. Trans. Environ. Technov. 2018, 12(1) : 1-5
1
INTRODUCTION
Ganga River is a lifeline of millions of people residing
on its bank and regarded as the most respectful pious
rivers of Hindus. The Ganga water is frequently used
for drinking, cooking, and bathing purposes due to
ancient knowledge that ‘Ganga water does not putrefy
even after long periods of storage’. Most religious
beliefs involve some ceremonial use of ‘holy’ water,
and in India the water of the river Ganga is treated
with such reverence. The river Ganga is mentioned in
Rigveda with Yamuna and Saraswati (Rigveda 10.75.
5-6). Ganga water has been used since ancient time for
remedial purposes also. During 500 BC, Hippocrates
wrote about the wound healing properties of Ganga
water. However, the role of the bath in the treatment of
leprosy lead to believe that water was used for curative
effects (Kloss, 1939). Since time immemorial, Hindus
have been burning the dead bodies due to religious
belief and miraculous non-putrefying properties
irrespective of any infectious or non-infectious
diseases.
The Hindus have been storing Ganga water in their
homes for the purpose of cleaning and rituals for
centuries. Antibacterial properties of the waters of
Ganga and Yamuna had been characterized by
Hankin (1896) long before the genesis of the concept
of bacteriophages developed by d’Herelle (1917).
Hankin (1896) reported that the cure of diarrhea and
cholera by using raw Ganga water. Also, Nautiyal
(2009) found the presence of self-purificatory
properties in Ganga water due to its fluidity and
presence of some unknown heat-labile peptides that
kill the pathogenic Escherichia coli O157:H7.
Bacteriophages play a key role in biotic environment
and generate diversity by showing specificity to their
hosts. They are highly host-specific having the ability
to infect and alter the composition of a microbial
community. Some hosts are more resistant to local
phages than the others or are better able to respond to
phage-mediated selection (Flores et al., 2011). All
bacteria are not infected by all phages but most phages
can infect a specific species of bacteria (Chatterjee
et al., 2012). Much work has been done on pollution
aspects of Ganga water. Jansen (1998) described the
prevalence of broad-host-range lytic bacteriophages
of Escherichia coli. This is the first report on isolation of
bacteriophages from Ganga water from Haridwar and
testing of their host-specificity by using pathogenic
enteric bacterial species of human.
MATERIALS AND METHODS
Bacterial Strains
Three standard cultures of human enteric pathogenic
bacteria viz., Escherichia coli ATCC 27853, Salmonella
typhi MTCC 733 and Klebsiella pneumoniae MTCC 432
were procured from the Culture Collection of the
Department of Botany and Microbiology, Gurukul
Kangri Vishwavidyalaya. The bacteria were separately
grown on Luria Bertani (LB) agar medium at pH 7.0
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2
and incubated at 37ºC for 16-18 h. The plates were
stored at 4ºC. Exponentially growing bacterial culture
was used in all the tests.
Isolation of Bacteriophages
Sample Collection
Water samples were collected from Har Ki Pauri,
Haridwar in sterile 50 mL conical tubes. The water
was mixed thoroughly at collection site and the
sediments were collected together with the overlying
water.
Enrichment of Bacteriophages
Bacteriophage enrichment method in the collected
samples was performed following the method of Twest
and Kropinski (2009) with slight modifications. The
samples were separately centrifuged at 6000 rpm for
10 min to remove large particulates and bacterial cells.
40 mL of the centrifuged samples were transferred into
a new sterile conical tube. The tubes were inoculated
with 10 mL broth culture of host bacterium (OD600 =
0.4-0.6) and mixed thoroughly. The tubes were then
incubated for specific phage-enrichment at 37ºC for 24
h. The contents of the tubes were centrifuged at 10,000
rpm for 15 min. The supernatant of the water sample
was filtered slowly through vacuum filtration
assembly with nylon membrane filter (0.45µ pore size,
47mm diam.). The filtered sample was carefully
transferred to a new sterile tube and stored at 4ºC.
Double Agar Overlay Plaque Assay
A modified method of the double agar overlay method
was adapted (Kropinski et al., 2009). A row of 4 sterile
microcentrifuge tubes was set up and each tube was
numbered with the appropriate sequential 100-fold
dilutions. Then 9 mL of LB broth as diluents was added
aseptically to each tube. 1 mL of undiluted phage
suspension was added to the first tube and mixed well.
Serial dilution was performed by transferring 1 mL of
suspension from first tube to the second tube and so
on. The control tube was devoid of any phage. The
third and fourth phage preparations were used for
plaque assay. A plate of bacterial control having no
phage was prepared as negative control.
The host bacteria were separately grown in 5 mL of LB
broth at 200 rpm and 37ºC until it reached the log phase
(OD600 = 0.4-0.6). 2 mL of this culture was separately
transferred into a sterile 20 mL conical tube. 1 ml of the
phage suspension was added into it. The tubes were
pre-incubated at 37ºC for 15 min to allow phage
adsorption onto host bacterium. After pre-incubation,
5 mL of molten (45ºC) soft agar (0.75%) previously
prepared from 1.5% of LB agar was added into the
tubes. The tubes were gently mixed for few times and
the contents were poured on Petri plates containing
LB bottom agar. The plates were swirled to spread the
mixture evenly over the medium. The plates were left
at room temperature for 10-15 min until the soft agar
had solidified and the plates were incubated at 37ºC
for 16-18 h. The plaques formed in each plate were
enumerated.
Amplification and Purification of Bacteriophages
Phage Selection and Amplification
The exponentially growing bacterial cultures were
inoculated into a microcentrifuge tube. On the basis of
size and clarity the phages were selected by using 100
µL pipette. The discrete plaques were selected by using
pipette tip inserted into centre. The soft agar containing
the bacteriophages was removed carefully. The phage-
containing plug was separately added into the
inoculated tubes and the contents were incubated at
37ºC for 24 h. The phage suspension was centrifuged
at 14,000 g for 15 min. The supernatant was transferred
into fresh, sterile microcentrifuge tube for further
analysis.
Concentration and Purification of Bacteriophages
The precipitation method was used with polyethylene
glycol (PEG) to concentrate and purify the phages
(Boulanger, 2009). The host bacterial strains were
separately cultured in 50 mL LB broth with constant
agitation (200 rpm) at 37ºC until OD600 of 0.4–0.6 was
achieved. The isolated phage particles were added into
the bacterial culture and agitated overnight at 37ºC
and 200 rpm. The bacteria and phage mixture was
centrifuged at 9,000 rpm for 15 min to remove the cell
debris. Supernatant containing phage was transferred
into a new conical tube (50 mL) and 10 mL of PEG/
NaCI was added. The mixture was agitated and
incubated on ice for at least 1 h in order to precipitate
phage particles. The precipitated phage was collected
by centrifugation at 9,000 rpm and 4ºC for 30 min and
the supernatant was carefully discarded. The conical
tube was turned over to drain away the remaining fluid
from the pellets for 5 min. The phage pellets were re-
suspended in 500 µL phosphate buffered saline (PBS)
and transferred to a sterile micro-centrifuge tube. The
phage particles were separated from co-precipitated
bacterial debris by centrifugation at 11,000 g for 15
min. Then the supernatant was collected into a sterile
micro-centrifuge tube. All lysates of phage were stored
at 4ºC.
Host Specificity Test
Bacterial susceptibility to different phage was assayed
based on the spot test method (Raya and Hébert, 2009)
with slight modification. The host ranges of the isolated
phages were determined using the standard culture of
E. coli, S. typhi and K. pneumoniae. The bacterial strains
were cultured in LB broth at 37ºC with constant
Shivani Tyagi and R.C.Dubey
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agitation at 200 rpm. A sterile swab was moistened
with the host cells and spread over the surface of LB
agar. The plate was previously marked to allow
identification of each inoculum. 1mL of each phage
suspension was spotted, dried and incubated at 37ºC
for 16-18 h. The host range of phages was determined
based on susceptibility of bacterial strains by observing
the plaques formed in the bacterial lawn.
Scanning Electron Microscopy (SEM)
SEM samples were prepared according to the method
of Drab (2016) with some modification to interpret the
effect of phages on the growth of bacterial species. The
area on the antibacterial plate showing plaques against
the selected bacterial species was excised with agar,
fixed by immersion in 2% glutaraldehyde in 0.1 M
phosphate buffer (pH 7.2) for at least 1 h. The samples
were drained out and placed in three consecutive
washes (1 h) of 0.1 M phosphate buffer. Samples were
then stored in fresh cold phosphate buffer for electron
microscopy at Wadia Institute of Himalayan Geology,
Dehradun (India). 20-100% acetone-water was used
in a series to dehydrate the samples for 15 min each
and at the critical-point they were dried with liquid
CO2. A thin layer of gold-palladium was used for
coating the sample. The morphological deformities in
bacterial cells were observed under scanning electron
microscope.
RESULTS
A total of three types of phages were isolated from
Ganga water by using the specific host bacterial strains
viz., E. coli, S. typhi and K. pneumoniae based on their
numbers, shape and sizes of plaque. The plaques
formed on the colonies of E. coli were small sized,
scanty and less in numbers, whereas that formed on S.
typhi were small sized, many, enumerable and
coalesced. In contrast, the plaques formed on lawn of
K. pneumoniae were very large in size, spherical and
least in numbers (Fig.1, Table 1).
Table.1. Characteristic features of plaques formed on
bacterial lawns.
Fig.1. Plaques formed by different bacteriophage on
colonies of different bacterial species; (A) Salmonella
typhi MTCC 733; (B) Escherichia coli ATCC 27853; (C)
Klebsiella pneumoniae MTCC 432; (D) Scanning Electron
micrograph (SEM) of Salmonella typhi MTCC 733; (E)
SEM of Escherichia coli ATCC 27853; (F) SEM of
Klebsiella pneumoniae MTCC 432. (a) Areas indicating
maximum changes in bacterial growth; (b) the
unaffected area (control). Arrows indicate the
morphological changes in bacterial colonies.
Scanning electron microscopy imaging analysis of the
plaques formed on bacterial colonies revealed the
morphological damages of the cell membrane and cell
wall structure. Distinct signs of deformities in E. coli
cells were visualized, such as detached cell wall and
cell burst caused by bacteriophage (Fig. 1D-a).
However, such morphological changes were not
observed in the cells present in the unaffected region
(Fig. 1D-b). Similarly, the effect of lytic activity of the
phages of S. typhi and K. pneuminiae was shown by
cell blisters on the surface as compared to unaffected
cells (Fig. 1E-F).
Plaque
Numbers Shape Size
(mm)
Control - - - -
Salmonella
typhi MTCC
733 STP Many,
enumerable,
coalesced Irregular 2
Escherichia coli
ATCC 27853 ECP Scanty, less,
scattered Spherical 3
Klebsiella
pneumoniae
MTCC 432 KPP Least Spherical 9
Host bacteria Phage Characteristic of plaques
Isolation of host-specific bacteriophages. . .
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Results of host-specificity test indicated that all the
three isolated phage were host-specific i.e. the
bacteriophages infected only their respective host
bacterium but not the others (Table 2).
Table.2. Cross-infectivity of isolated phages against
pathogenic bacteria.
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DISCUSSION
Ganga water possesses bacteriophages that kill
bacterial species resulting in formation of plaques of
different size and shapes. Differences in plaque size
and shapes could be attributed to the virulence of
bacteriophages infecting the enteric bacterial species.
Different types of phages of enteric bacteria have also
been isolated from fresh water with different levels of
faecal pollution (Araujo et al., 1997) and high
abundance of viruses found in aquatic environments
(Bergh, 1989). The results of this study do not claim for
the absence of other types of bacteriophages in Ganga
water. SEM-based observations confirm the
bacteriophages as the natural infectants of specific
bacterial species and a site for their multiplication.
Three bacteriophages isolated in the present study
showed specificity to their respective bacterial host. It
may be explained to be due to the presence of certain
molecular elicitors complementary to that present on
the surface of the host (Wang et al., 2000, Dubey and
Maheshwari, 2013). Since the enteric bacterial
pathogens are associated with human diseases,
drinking of Ganga water containing bacteriophages
have increased the purity of water by killing the
pathogens causing human diseases. The role of bath
in Ganga water for treatment of leprosy (Kloss, 1939)
and suppurative skin infection (Cislo, 1986) has also
been described. It may be concluded that the Ganga
water contains phages specific to bacterial species that
play a role in making Ganga water pious and non-
putrifying.
Acknowledgements
The authors thank the Head, Department of Botany
and Microbiology for laboratory facilities.
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Isolation of host-specific bacteriophages. . .
... In addition, aquatic coliphages capable of encountering their specific E. coli hosts due to their continual movements in water (Yahya et al., 2015). In agreement with our observation, coliphages detected in drainage and surface water were in a titer ranged between 10 5 -10 15 PFU mL − 1 and showing different plaque morphologies and diameters (Amarillas et al., 2017;Tyagi and Dubey, 2018). Our isolated plaques were circular and regular in shape and with sizes range of 1-6 mm in diameter against E. coli (ATCC 10798) and E. coli (ATCC 8739) as phage hosts. ...
... Similarly, ECA2 and phT4A coliphages with plaques of 1-5 mm and 0.5-2 mm in diameters, respectively, against E. coli (ATCC 13706) were isolated from sewage water (Pereira et al., 2017). Another coliphage, isolated from Ganga River, formed spherical plaques with diameters of 3 mm against E. coli (ATCC 27853) (Tyagi and Dubey, 2018). ...
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