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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.
Scientific Transactions in Environment and Technovation
P - ISSN 0973 - 9157
E - ISSN 2393 - 9249
July to September 2018
J. Sci. Trans. Environ. Technov. 12(1), 2018 1
Scientific Transactions in
Environment and
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.
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 :
J. Sci. Trans. Environ. Technov. 2018, 12(1) : 1-5
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
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.
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
Scientific Transactions in Environment and Technovation
P - ISSN 0973 - 9157
E - ISSN 2393 - 9249
July to September 2018
J. Sci. Trans. Environ. Technov. 12(1), 2018
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
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
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
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
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).
Numbers Shape Size
Control - - - -
typhi MTCC
733 STP Many,
coalesced Irregular 2
Escherichia coli
ATCC 27853 ECP Scanty, less,
scattered Spherical 3
MTCC 432 KPP Least Spherical 9
Host bacteria Phage Characteristic of plaques
Isolation of host-specific bacteriophages. . .
Scientific Transactions in Environment and Technovation
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J. Sci. Trans. Environ. Technov. 12(1), 2018
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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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-
The authors thank the Head, Department of Botany
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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). ...
This study presents a novel coliphage-based approach for biocontrol of Multidrug-Resistant (MDR) Escherichia coli (E. coli) and coliforms in wastewater and improve the physicochemical water quality . Newly isolated coliphages and E. coli strains were isolated from the Rosetta branch of River Nile and outlet points of five associated drains. Coliphages were characterized by transmission electron microscopy and coat protein-gene analyses. The isolated coliphages PR01, PR02, and PR03 belonged to the Siphoviridae, Myoviridae, and Podoviridae families, respectively. They exhibited, separately or in a cocktail, a wide host range pattern against wild E. coli strains with higher specificity to strains isolated from drains (87%) than those of the Rosetta branch (72%). Phage-sensitive E. coli strains exhibited elevated antibiotics resistance patterns, suggesting that they are MDR strains. Interestingly, the efficacy of phages cocktail (PR01/PR02/PR03) controlled the growth of wild MDR E. coli strains and coliform populations under laboratories conditions. The kinetic trend in removal efficiency (%) for coliforms was gradually increasing in a time-dependent manner and peaked at 12 h post incubation with phages mixture. In all water samples, total and fecal coliforms revealed at least a 30-fold reduction post 12 h of treatment with a mixture of coliphages. Physicochemical analyses of phage-treated wastewater showed reducing levels in biological oxygen demand (BOD), chemical oxygen demand (COD), turbidity, electric conductivity (EC), ammonia (NH3), total dissolved solids (TDS) and nitrate (NO3), while rising in dissolved oxygen (DO) content compared to untreated wastewater. Overall, this study suggests that phage-based treatment could be an effective and cheap alternative approach for the reduction of water pollutants in drainage drainage and waste water and thus eventually enhance the physicochemical quality of water.
... There are various studies which states that Bacteriophages are in huge number found in the Ganga water. Some 3-4 times more quantity of phage is present in the River Ganga than there in the River Yamuna and Saraswati [1].These phages are deeply involved in providing the immunity to the body and helps in building the immune homeostasis [2]. In the present study, we hypothesize that SARS CoV-2 infection has been contained naturally by Gangetic phage. ...
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SARS CoV-2, pandemics have been in second to third wave in various countries, across the globe. With current infection rate in millions per day, we have been witnessing good recovery rates in Indian population. The paper focuses on the behaviour of disease and its interaction with the population of Varanasi, which is one of the oldest living cities of world. Being predominantly, Hindu population, city residents, use Ganga water for bathing and drinking. In previous scientific communication, Ganga River is considered to have rich variety and density of phage, which do have properties to resist growth of infectious organisms. In this research article, we have hypothesized, as do Ganga water playing role in better recovery in Indian population?
... Ours is the first report on bactericidal properties of Ganga water killing enteropathogenic bacteria in vivo in mice. Earlier we also reported the presence of three bacteriophages of these three pathogens in Ganga water [20]. Therefore, these two factors may be responsible for non-putrifying properties of Ganga water. ...
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River Ganga is considered as one of the most pious rivers of the world since the ancient time due to its self-cleansing property. Therefore, its water has traditionally been used for religious purposes. A total of 4×103 cfu/ml of residential microflora from Ganga water of Haridwar was screened on selective media. The three standard pathogenic bacteria viz., Escherichia coli, Salmonella typhi and Klebsiella pneumoniae were inoculated in different types of water such as fresh water, three-years stored water, boiled water and tap water. None of the isolated bacteria were found pathogenic among the residential population. The freshly collected Ganga water was more potential for gradual killing of E. coli and K. pneumoniae as compared to S. typhi in vitro and in vivo. Moreover, the fresh and stored filtered water possessed the growth inhibitory property against the pathogens. But this property got lost after boiling the Ganga water. Inhibitory effect of Ganga water on the antibiotic-resistant standard pathogens was also tested in vivo on albino mice. A decrease in growth of antibiotic resistant marker strains of pathogen in the fecal matter of test mice showed the bactericidal property of Ganga water. Some TLC-separated compounds of Ganga water imparted antibacterial property by killing the entero-pathogenic bacteria of human in vivo. Moreover, loss in antibacterial properties upon boiling of water indicated the presence of heat-labile antibacterial agents in Ganga water. This is the first report on bactericidal properties of Ganga water in vivo using albino mice.
... It has been found that the quantity of SARS-CoV-2 particles is significantly higher in wastewater [12]. Researchers have suggested that in the case of sewage, a single test is sufficient to determine if the whole population has been infected or not [13]. Coronavirus genetic material (RNA) remains stable as long as it is protected with the capsid, i.e., in the form of a complete virus particle. ...
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An outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in Wuhan City, China, in December 2019. Since then, the outbreak has grown into a global pandemic, and neither a vaccine nor a treatment for the disease, termed coronavirus disease 2019 (COVID-19), is currently available. The slow translational progress in the field of research suggests that a large number of studies are urgently required. In this context, this review explores the impact of bacteriophages on SARS-CoV-2, especially concerning phage therapy (PT). Bacteriophages are viruses that infect and kill bacterial cells. Several studies have confirmed that in addition to their antibacterial abilities, bacteriophages also show antiviral and antifungal properties. It has also been shown that PT is effective for building immunity against viral pathogens by reducing the activation of NF kappa B; additionally, phages produce the antiviral protein phagicin. The Ganges river in India, which originates from the Himalayan range, is known to harbor a large number of bacteriophages, which are released into the river gradually by the melting permafrost. Water from this river has traditionally been considered a therapeutic agent for several diseases. In this review, we hypothesize that the Ganges river may play a therapeutic role in the treatment of COVID-19.
Aim: Bacteriophages are effective natural antimicrobial agents against drug-resistant pathogens. Therefore, identification and detailed characterization of bacteriophages become essential to explore their therapeutic potential. This study aims to isolate and characterize a lytic bacteriophage against drug-resistant Pseudomonas aeruginosa. Methods and results: The Pseudomonas phage AIIMS-Pa-A1, isolated from the river Ganga water against drug-resistant P. aeruginosa, showed clear lytic zone on spot assay. The phage revealed icosahedral head (58.20 nm diameter) and small tail (6.83 nm) under transmission electron microscope. The growth kinetics showed adsorption constant of 1.5×10-9 phage particles cell-1 ml-1 minute-1 and latent period of approximately 15 minutes with the burst size of 27 phages per infected cell. The whole genome sequencing depicted a GC-rich genome of 40.97kb having a lysis cassette of holin, endolysin, and Rz protein, with features of the family Autographiviridae. The comparative genome analysis, Ortho-average nucleotide identity value, and phylogenetic analysis indicated the novelty of the phage AIIMS-Pa-A1. Conclusions: The study concludes that the Pseudomonas phage AIIMS-Pa-A1 is a novel member of the Autographiviridae family, truly lytic in nature for drug-resistant P. aeruginosa. Significance and impact of study: The Pseudomonas phage AIIMS-Pa-A1 is having promising potential for future therapeutic intervention to treat drug-resistant P. aeruginosa infections.
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The initial step of viral infection is the binding of a virus onto the host cell surface. This first viral-host interaction would determine subsequent infection steps and the fate of the entire infection process. A basic understating of the underlining mechanism of initial virus-host binding is a prerequisite for establishing the nature of viral infection. Bacteriophage λ and its host Escherichia coli serve as an excellent paradigm for this purpose. λ phages bind to specific receptors, LamB, on the host cell surface during the infection process. The interaction of bacteriophage λ with the LamB receptor has been the topic of many studies, resulting in wealth of information on the structure, biochemical properties and molecular biology of this system. Recently, imaging studies using fluorescently labeled phages and its receptor unveil the role of spatiotemporal dynamics and divulge the importance of stochasticity from hidden variables in the infection outcomes. The scope of this article is to review the present state of research on the interaction of bacteriophage λ and its E. coli receptor, LamB.
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Interactions between bacteria and the viruses that infect them (i.e., phages) have profound effects on biological processes, but despite their importance, little is known on the general structure of infection and resistance between most phages and bacteria. For example, are bacteria-phage communities characterized by complex patterns of overlapping exploitation networks, do they conform to a more ordered general pattern across all communities, or are they idiosyncratic and hard to predict from one ecosystem to the next? To answer these questions, we collect and present a detailed metaanalysis of 38 laboratory-verified studies of host-phage interactions representing almost 12,000 distinct experimental infection assays across a broad spectrum of taxa, habitat, and mode of selection. In so doing, we present evidence that currently available host-phage infection networks are statistically different from random networks and that they possess a characteristic nested structure. This nested structure is typified by the finding that hard to infect bacteria are infected by generalist phages (and not specialist phages) and that easy to infect bacteria are infected by generalist and specialist phages. Moreover, we find that currently available host-phage infection networks do not typically possess a modular structure. We explore possible underlying mechanisms and significance of the observed nested host-phage interaction structure. In addition, given that most of the available host-phage infection networks examined here are composed of taxa separated by short phylogenetic distances, we propose that the lack of modularity is a scale-dependent effect, and then, we describe experimental studies to test whether modular patterns exist at macroevolutionary scales.
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Concentration and purification of infectious particles are prerequisites for structural and functional characterization of bacteriophages. The methods detailed in the first part of this chapter outline the protocols commonly used to obtain purified phages: the concentration of phage particles by precipitation with polyethylene glycol and their purification by centrifugation in CsCl step gradients and subsequently by equilibrium centrifugation. This sequence of procedures, if carried out as a whole, ensures a purification of high quality, which is well suited for most analytical techniques used to characterize bacteriophage particles. The second part of this chapter describes the preparation of “ghosts” or DNA-less bacteriophages. These particles should be preferred to the entire bacteriophages for one-dimensional SDS-PAGE analysis of phage structural proteins, since running of the phage proteins through the gel is not disturbed by the presence of the phage DNA. This allows an optimal resolution, which is necessary for proteomic approaches such as \(N\)-terminal protein sequencing or mass spectrometry using proteins isolated from distinct gel bands.
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Classical bacterial enrichment devised by Sergius Winogradsky (1856-1953) and Martinus Beijerinck (1851-1931) can be modified to enrich for bacteria-specific viruses. In this chapter simple protocols are presented for the enrichment of phages from water samples, such as sewage, and soil.
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Vibrio bacteriophages were isolated by enrichment from 177 of 643 samples of marine molluscan shellfish, crustaceans, seawater, and sediments. The predominant bacteriophage types isolated were specific for some strains of Vibrio parahaemolyticus. A high frequency of phage isolations was also observed with strains of agar-digesting vibrios (21 of 56) and psychrophilic vibrios (14 of 72) that were originally isolated from non-shellfish growing areas. No bacteriophages were isolated against V. alginolyticus and only rarely for V. anguillarum even though these were the two most abundant species found in near-shore environments. No V. cholerae phages were isolated. It was also determined from quantitative studies on the Pacific oyster (Crassostrea gigas) obtained from two environments in Washington and Oregon that the titers of V. parahaemolyticus bacteriophages increased with increasing seasonal water temperatures and that this was proportional to the increase in numbers of mesophilic vibrios and not with the incidence of V. parahaemolyticus. Titers of V. parahaemolyticus bacteriophages occasionally exceeded 10(6) per g of oyster during the summer months. Specific V. parahaemolyticus bacteriophages were also isolated from market seafoods and other marine samples that originated in cold environments where no mesophilic vibrios are expected to be found. The possibility that V. parahaemolyticus bacteriophages originate from Vibrio spp. other than V. parahaemolyticus and the role of these bacteriophages in the ecology of marine vibrios are discussed.
We report on a robust method for chemical element-sensitive imaging by scanning electron microscopy (SEM). The commercial Auriga FE-SEM microscope (Carl Zeiss, Oberkochen, Germany), equipped with an energy-selective grid detector (EsB) as a part of the experimental setup, was applied for generation of chemical contrast at low accelerating voltages, which is gentle for sensitive samples. The EsB-grid detector, conceptually adapted by us as an energy retarding field analyzer (RFA), was used to detect the two-dimensional (2D) energy spectrum for the first time. The electron energy spectrum measured by sweeping the retarding grid potential revealed thresholds corresponding to electronic transitions in the specimen, followed by 2D-derivation treatment applied just at the observed thresholds. This allowed chemical mapping by SEM. In this report the 273 eV Auger transition in carbon deposited onto the Si(100) sample was chosen as a source for chemical contrast in the SEM image. In addition to Auger electrons, we expect analogous energy-selective contrast enhancement for inelastically scattered electrons, for example, in plasmonic contrast and elastically scattered electrons, for example in phase contrast, our method, proved for carbon, is expected to apply to a broader list of elements as a general capability of chemical mapping, at several-fold better lateral resolution when compared with energy dispersive spectroscopy (EDS).
Most bacterial cells carry prophage genomes either integrated into the host DNA or present as repressed plasmids. Methods are described for the induction of prophages using Mitomycin C, and for the isolation of prophage-cured bacterial cell lines.
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-year-old, 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-microm-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 water's 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.