Critical Reviews™ in Eukaryotic Gene Expression, 28(1):1–12 (2018)

Antibacterial Activity of Novel Strains of

Bacteriophages: An Experimental Approach
Muhammad Imran Qadira,b,* & Zunera Chauhdaryb
Institute of Molecular Biology and Biotechnology, Bahauddin Zakariya University, Multan, Pakistan; bFaculty of
a

Pharma­ceutical Sciences, Governament College University, Faisalabad, Pakistan

*Address all correspondence to: Muhammad Imran Qadir, Institute of Molecular Biology and Biotechnology, Bahauddin Zakariya University,
Multan, Pakistan, E-mail: mrimranqadir@hotmail.com

ABSTRACT: The evolution of antibiotic resistance in bacteria has increased research in the development of alterna-
tive therapies to conventional drugs. In this study, isolated phages were characterized and antibacterial activity was
determined by standard agar disc diffusion method. The phages showed maximum propagation at 37°C to 40°C and
highest viability at pH 7. Sugars influenced the bacteriophage viability. Sodium chloride decreased the phage propa-
gation. Calcium chloride and magnesium chloride increased the phage propagation up to a certain limit. SDS-PAGE
analysis confirmed the presence of protein cover and showed the various bands ranging from 10 to 200 kDa. Nucleic
acid analysis confirmed the presence of RNA with a size of approximately 20 kb. Transmission electron microscopy
indicated that the phages belong to Siphoviridae, Leviviridae, and Podoviridae families.

KEY WORDS: bacteriophages, characterization, antibacterial activity

I. INTRODUCTION performed to check whether the phage samples could

The evolution of antibiotic resistance in bacteria has
stimulated research into the development of alter-
native therapies to conventional drugs. One of the
emerging approaches that can be used as an alterna-
tive to antibiotics is bacteriophage therapy. Although
researchers are trying to find alternatives with bacte-
rial derivatives, the humankind is again entering the
preantibiotic era.1 To solve the problem of bacterial
resistance, new techniques are required for thera-
peutic treatment of resistant bacteria. Among the most
credible modern approaches, the most promising
technique is bacteriophage therapy.2 Bacteriophages
are a class of viruses that infect bacteria. They are in-
tracellular parasites that multiply inside bacteria and
make use of their biosynthetic machinery.
The objective of the present study was to iso-
late, characterize, and evaluate the antibacterial ac-
tivity of some local strains of bacteriophages.
II. MATERIALS AND METHODS
A. Isolation of Bacteriophages
infect bacterial strains. The water samples were centri-
fuged at 3,500 ×g for 25 min at 10ºC. The supernatant
was separated by micropipettes and filtered through
filter paper of 0.45 µm pore size. The filtrates were
divided into three sterilized Falcon tubes, and 1 mL of
Luria-Bertani (LB) broth was added to each tube. Then
100 ml (~108 CFU) of Escherichia coli, Pseudomonas
aeruginosa, and Streptococcus pneumoniae strains
were added separately. The tubes were incubated at
37ºC for 18 hr. These were centrifuged at 10,000 ×g
for 10 min; bacteria and debris were removed by fil-
tering the supernatant with filter paper of 0.22 mm
pore size. The plaque assay was performed for deter-
mination of bacteriophage titer. Plates producing more
than 30 plaques were selected for further studies.
B. Characterization of Bacteriophages
The isolated phages were characterized according to
methods used by Yang, et al.3
1. Multiplicity of Infection (MOl)

Sewage water samples were collected from GM- The average number of bacteriophages per bac­
Abad Hospital, Faisalabad, Pakistan. Spot tests were terium infected is called multiplicity of in­
fec-

1045-4403/18/$35.00 © 2018 Begell House, Inc. www.begellhouse.com 1

2 Qadir & Chauhdary
tion (MOl). It was determined by the following
formula:
MOI = (Volume in ml × pfu / ml)/
(Volume in ml × cfu/ml)
2. One-Step Growth
This experiment was performed to determine the la-
tent period, rise period, and burst size of all the bac-
teriophages. The elapsed time between adsorption
of a phage particle to its host cell and lysis of that
cell with the release of phage progeny is called the
latent period. The interval from the end of the latent
period until all phages are extracellular is known as
the rise period. The mean number of phage particles
liberated per infected bacterium is called the burst
size. Burst size was calculated from the following
formula:
Burst size = (Final PFU – Initial PFU) /
Number of infected bacterial cells
The bacteriophages of MOI 5 were incubated
at 37°C for 5 min for adsorption. These were cen-
trifuged at 10,000 ×g for 5 min and diluted with
LB broth. These were incubated at 37°C. After
every 10-min interval, aliquots of 4 mL were taken
from each tube and serially diluted. The soft agar
overlay method was used to determine the phage
titer. These studies were continued for 4 hr. After
incubation at 37ºC for 12 hr, plaques were counted
and the latent period, rise period, and burst size
were determined.
3. Phage Adsorption
This experiment was performed using the same
method previously described, except free bacterio-
phages particles were isolated by filtration instead
of centrifugation by using syringe filters of 0.22 mm.
At zero-time interval, a 5 mL aliquot was taken for
the control titer.
Percentage of adsorption = (Mean of control titer
– Mean of residual titer)/Mean of residual titer
4. Broth Clearance
This experiment was performed to check the clearing
of bacterial strains. The bacterial strains were inoc-
ulated into LB broth. These tubes were incubated
at 37°C until the optical density at 600 nm was 1.
The phage lysate was added into respective labeled
tubes at MOI of 5. Three control tubes were also
maintained. Readings were taken from all tubes at
600 nm with a UV spectrophotometer.
5. Effect of Different Factors on Phage
Propagation
Effect of different factors on phage propagation,
such as temperature, pH, and sugar, NaCl, calcium
chloride, and magnesium chloride concentrations,
were estimated to characterize the phages.
6. SDS-PAGE Analysis
The gel was prepared and was placed in SDS-
PAGE apparatus. The protein samples and marker
were loaded and the gel was run at 80 V until the
sample entered the resolving gel. Then, the current
was increased to 100 V. The run was stopped when
the dye front reached 1 cm above the lower end of
the glass plate. The gel was removed and stained in
the staining solution. The gel was destained till the
bands became clear.
7. Nucleic Acid Analysis
Phage nucleic acids were isolated by using com-
mericially available kits and were digested with
restriction endonucleases. RNase A was used to con-
firm whether it was ssRNA. The restriction reaction
was analyzed by agarose gel electrophoresis on 1%
agarose gel run at 70 V for 3 hr to estimate the size
of the nucleic acids.
8. Electron Microscopy
Purified samples of bacteriophages were subjected
to transmission electron microscopy at magnifica-
tion of ×100,000.
Critical Reviews™ in Eukaryotic Gene Expression
Antibacterial Activity of Novel Strains of Bacteriophages 3
C. Antibacterial Assay
Antibacterial activity was determined by the stan-
dard agar disc diffusion method. The prepared discs
of the phages were placed on the surface of inocu-
lated agar plates of the bacterial strains with sterile
forceps. A standard disc was also placed along with
test discs to compare inhibition of bacterial growth.
These plates were incubated at 37°C. After 24 hr, the
plates were examined for zones of inhibition.4
III. RESULTS
A. Isolation of Bacteriophages
The bacteriophages isolated from sewage water
collected from GM-Abad Hospital, Faisalabad,
Pakistan by using Escherichia coli, Pseudomonas
aeruginosa, and Streptococcus pneumoniae were
named as QjA, QjB, and QjC, respectively (Qj:
Qadirphage). These were subjected to spot test; all
of these bacteriophages yielded positive spot tests
(Fig. 1). The plaque assay was performed to de-
termine bacteriophage titer. Plates producing more
than 30 plaques were selected for further studies.
B. Characterization of Bacteriophages
1. Multiplicity of Infection
MOI is the highest titer of bacteriophage obtained
under standard conditions, as it is the ratio of par-
ticles of virus infected to host cells. That titer of all
FIG. 1: Spot test for bacteriophages: a) QjA, b) QjB, and c) QjC.
Volume 28, Issue 1, 2018
bacteriophages was used for further propagation and
experimental work. The data was collected and in-
terpreted, and the optimal multiplicity of infection
of all bacteriophages was determined to be 5 PFU/
mL (Fig. 2).
2. One-Step Growth Curve
One-step growth curves for all bacteriophages were
obtained at MOI of 5 PFU/mL and temperature of
37°C. From these curves, we calculated the latent
period, rise period, and burst size of all the bacterio-
phages. Approximately all bacteriophages showed
a latent period of almost 40 min, and a rise period
of almost 50 min. Burst size was highest for QjC
and lowest for QjA. The burst size for QjC was 77
phages per cell, and for QjA it was 72 phages per
cell (Fig. 3).
3. Phage Adsorption
The adsorption rate showed that 65% adsorption oc-
curred within 15 min, and 80% adsorption occurred
within 35 min (Fig. 4).
4. Broth Clearance Experiment
The results showed that no pronounced clearance
was observed in any of the bacteriophages, even
when these were incubated at 37°C for 48 hours.
The optical densities were increased in case of all
bacteriophages from 0 to 2.3 and remain constant up
to 48 hours (Fig. 5).
4 Qadir & Chauhdary

FIG. 2: Multiplicity of infection (MOl) of the phages.

FIG. 3: One step growth of the phages.

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Antibacterial Activity of Novel Strains of Bacteriophages 5

FIG. 4: Bacteriophage adsorption.

FIG. 5: Broth clearance experiment.

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6 Qadir & Chauhdary

5. Effect of Temperature on Phage 6. Effect of pH on Phage Propagation

Propagation
The results showed that all bacteriophages showed
maximum propagation at 37°C to 40°C and min-
imum at 70°C to 100°C (Fig. 6).
There was minimal survival at pH 2 to 3 at 37°C. All
the bacteriophages showed highest viability at pH 7.
Viability of bacteriophages was gradually decreased
toward alkaline pH (Fig. 7).

FIG. 6: Effect of temperature on phage propagation.

FIG. 7: Effect of pH on phage propagation.

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Antibacterial Activity of Novel Strains of Bacteriophages 7

7. Effect of Sugars on Phage Propagation 8. Effect of NaCl on Phage

The sugars greatly influenced bacteriophage via-
bility, and the rate of inhibition ranged from 80%
to 100%. The greatest inhibition was observed with
rhamnose, which was 94% with QjA (Fig. 8).
Propagation
The results showed that an increasing concentration
of NaCl remarkably decreased bacteriophage via-
bility (Fig. 9).

FIG. 8: Effect of sugars on phages propagation.

FIG. 9: Effect of Sodium chloride on phages propagation.

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8 Qadir & Chauhdary

9. Effect of Calcium Chloride on Phage 10. Effect of Magnesium Chloride on

Propagation Phage Propagation

Almost all the bacteriophages showed maximum Almost all the bacteriophages showed maximum
propagation within 20 to 30 mm concentration of propagation within 20 to 30 mM concentration of
calcium chloride (Fig. 10). magnesium chloride (Fig. 11).

FIG. 10: Effect of calcium chloride on phage propagation.

FIG. 11: Effect of magnesium chloride on phage propagation.

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Antibacterial Activity of Novel Strains of Bacteriophages 9

11. SDS-PAGE Analysis for Proteins 12. Phage Nucleic Acids

Different number and size of protein bands were ob-
served on the gel, with molecular weights ranging
from 10 to 200 kDa (Fig. 12). All the phages were
enveloped.
Phage nucleic acids were not digested by restriction
endonucleases, indicating that all the phages did not
contain dsDNA. RNase A was used to confirm that
the nucleic acid was ssRNA. Size of the nucleic acid
for all the phages was approximately 20 kb (Fig. 13).

FIG. 12: SDS-PAGE analysis of the bacteriophages (M: Marker).

FIG. 13: Nucleic acid analysis of the phages and their size.

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10 Qadir & Chauhdary

13. Morphology and Classification of C. Antibacterial Activity

the Isolated Phages
QjA belongs to the family Siphoviridae, as its tail
was long and noncontractile,5 but it was not a pre-
viously described phage originating from E. coli.
Known phages (T2, T4, or T6) contain dsDNA, but
it was an ssRNA phage with size of 20 kb, 20 nm
wide, and 50 nm long (Fig. 14).
QjB belongs to the family Leviviridae, because
it is an ssRNA virus; but it was not previously de-
scribed. Leviviridae phages are nearly 20 nm wide,5
but its size was almost 8 nm.
QjC belongs to the family Podoviridae, be-
cause its tail is short.5 But it is not an already de-
scribed phage originating from S. pneumoniae,
because these known phages contain DNA,6 but
QjC is an ssRNA phage.
The antibacterial activity of QjA, QjB, and QjC
against E. coli, P. aeruginosa and S. pneumoniae
as compared to ampicillin (10 µg) by disc diffusion
method is given in Table 1.
IV. DISCUSSION
In this study, we isolated bacteriophages from
sewage water. Spot tests were performed for all the
bacteriophages, which were positive with respect to
all bacteriophages producing small cleared areas.
A plaque assay was performed for further purifica-
tion of all bacteriophages, which produced white,
fibrous, and turbid plaques.
A single plaque was picked and propagated
until all plaques of equal size were obtained, and the

FIG. 14: Transmission electron microscopy for (a) QjA; (b) QjB; and (c) QjC.

TABLE 1: Zone of inhibition in mm (mean ± SE) by QjA, QjB, and QjC against Escherichia coli,
Pseudomonas aeruginosa, and Streptococcus pneumoniae
E. coli P. aeruginosa S. pneumoniae
Ampicillin (Standard) 25.00 ± 1.15 a
10.00 ± 1.15b
18.00 ± 0.58a
QjA 28.00 ± 2.52a 26.00 ± 1.15a 22.00 ± 0.58a
QjB 22.67 ± 0.88a 25.00 ± 1.53a 12.67 ± 1.20b
QjC 21.00 ± 0.58 a
18.00 ± 0.58a
15.00 ± 0.58a
Similar letter in a column are statistically nonsignificant (p > 0.05)

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Antibacterial Activity of Novel Strains of Bacteriophages 11
titer was calculated by tenfold serial dilution. The
plates that produced 30 to 300 plaques were selected
for further studies. From the plaque size and mor-
phology, we can determine the nature of the bacte-
riophage, because there are two types of life cycles:
lytic and lysogenic. If the plaque size is larger, then
it means a large number of bacteriophage, approx-
imately near 200, are produced every time or they
may have a longer time period on the plate and
consume more bacteria. Smaller plaques showed
the opposite behavior. If the plaque is very clear, it
means the bacteriophage follows the lytic form of
reproduction. If turbid plaques are produced, it is
evident that some bacteria can exist and it follows
the lysogenic life cycle. If the plaque shows areas of
both lytic and lysogenic types, then it is evident that
our bacteriophage is temperate.
The MOl resulting in highest phage titer under
standard conditions was considered as the optimal
MOI and used in subsequent large-scale phage pro-
duction. From the data obtained, the optimal multi-
plicity of infection was observed to be 5 PFU/mL
for all bacteriophages.
Previous research has revealed that the latent
period and burst size are very important, as these
show the ability of bacteriophage to survive in na-
ture.7 The results from this study revealed that all
these bacteriophages showed a latent period of al-
most 40 min, a rise period of almost 50 min, and a
burst size of 72 to 77. These results were quite dif-
ferent from other bacteriophages, as earlier research
has shown that it ranges from 5 to 610.8
The stability of all the phages were determined
by conducting an adsorption assay for 35 min.
Adsorption is a very important event in the life
cycle of bacteriophages; the burst size will be max-
imum if the adsorption of bacteriophage particles to
host cells at highly specific receptors is maximum
within a short period of time to start the lytic life
cycle.9
Temperature has a major role in the stability
of bacteriophages. In this study an experiment was
conducted to determine viability of bacteriophages.
It showed that all these bacteriophages can tolerate
temperatures up to 60°C, but the rate of bacterio-
phage survival was minimal in all bacteriophages at
70°C to 80°C.
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pH also plays a major role in the survival of bac-
teriophages, as the extreme pH can influence protein
structure. In this study, bacteriophages survive over
a wide range of pH, from pH 4 to 11. Suspensions
of all these bacteriophages were completely inacti-
vated at pH 2 to –3. The optimum pH for survival
of all the bacteriophages was neutral pH 7. Because
proteins are sensitive to pH change, most proteins
remain stable and retain their structure at neutral
pH, but at extreme pH, most proteins start to swell
and unfold.10 When phages attack a host bacterial
cell, it releases many lysozymes, which act on the
cell wall of the bacterium and cause it to lyse. Then
phage DNA is transferred to the bacterial cell. All
of these processes are decreased at extreme pH, be-
cause it can destroy the phage protein structure and
lysozymes.11
We also studied the effect of various sugars
on phage viability, which shows that these sugars
greatly inhibit bacteriophage propagation, ranging
from 80% to 94%, as these sugars are the part of
bacterial cell wall and receptors at which bacterio-
phages can attached to the bacterial cell surface.12
The effect of other salts, such as sodium chlo-
ride, magnesium chloride, and calcium chloride,
were also observed. Up to a certain limit, they in-
crease bacteriophage propagation; but at very high
concentrations, they inhibit the phage propagation.
SDS-PAGE of all bacteriophages showed var-
ious bands, some of which were major and others
that were minor protein bands. These protein bands
were in the range of 10 to –200 kDa. Nucleic acid
analysis showed the presence of ssRNA with an
approximate size of 20 kb. Morphology and other
characteristics revealed that these phages were dif-
ferent from previously described phages. It supports
the idea that at least 100 novel bacterial viruses are
described every year.5
Antibiotic resistance is a common issue all
over the world. E. coli cause hemolytic-uremic
syndrome, septicemia, gram-negative pneumonia,
urinary tract infections, and gastroenteritis leading
to diarrhea.13 P. aeruginosa is a causative agent of
pneumonia, septic shock, urinary tract infections,
gastrointestinal tract infections in premature infants,
and skin and soft tissue infections.14 S. pneumoniae
is a causative agent of pneumonia, otitis media,
12 Qadir & Chauhdary
sinusitis, meningitis, peritonitis, conjunctivitis, bac-
teremia, sepsis, and brain abscesses.15 This study
has proved the antibacterial activity of some novel
phages against the previously mentioned bacterial
strains. Therefore, these diseases just mentioned
may be controlled by the isolated phages.
V. CONCLUSION
The present study indicates that the isolated
novel phages may be used for antibacterial thera-
peutic purposes against E. coli, P. aeruginosa, and
S. pneumoniae.
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Critical Reviews™ in Eukaryotic Gene Expression