ORIGINAL RESEARCH
published: 21 March 2019
doi: 10.3389/fmicb.2019.00574
Therapeutic Characterization and
Efficacy of Bacteriophage Cocktails
Infecting Escherichia coli, Klebsiella
pneumoniae, and Enterobacter
Species
Prasanth Manohar 1 , Ashok J. Tamhankar 2,3* , Cecilia Stalsby Lundborg 2 and
Ramesh Nachimuthu 1*
1
Antibiotic Resistance and Phage Therapy Laboratory, Department of Biomedical Sciences, School of Bioscience
and Technology, Vellore Institute of Technology, Vellore, India, 2 Global Health-Health Systems and Policy: Medicines,
Focusing Antibiotics, Department of Public Health Sciences, Karolinska Institutet, Stockholm, Sweden, 3 Indian Initiative
for Management of Antibiotic Resistance, Mumbai, India
Edited by:
Robert Czajkowski,
University of Gdańsk, Poland
Reviewed by:
Hidetomo Iwano,
Rakuno Gakuen University, Japan
Alicja Wegrzyn,
˛
Polish Academy of Sciences, Poland
*Correspondence:
Ashok J. Tamhankar
ashok.tamhankar@ki.se;
ejetee@gmail.com
Ramesh Nachimuthu
drpnramesh@gmail.com
Specialty section:
This article was submitted to
Virology,
a section of the journal
Frontiers in Microbiology
Received: 21 October 2018
Accepted: 06 March 2019
Published: 21 March 2019
Citation:
Manohar P, Tamhankar AJ,
Lundborg CS and Nachimuthu R
(2019) Therapeutic Characterization
and Efficacy of Bacteriophage
Cocktails Infecting Escherichia coli,
Klebsiella pneumoniae,
and Enterobacter Species.
Front. Microbiol. 10:574.
doi: 10.3389/fmicb.2019.00574
Infections due to antibiotic resistant bacteria are increasing globally and this needs
immediate attention. Bacteriophages are considered an effective alternative for
the treatment of bacterial infections. The aim of this study was to isolate and
characterize the bacteriophages that infect Escherichia coli, Klebsiella pneumoniae,
and Enterobacter species. For this, clinical bacterial isolates of the mentioned
species were obtained from diagnostic centers located in Chennai, Tamil Nadu,
India. The bacteriophages were isolated from sewage water samples collected from
Tamil Nadu, India. Phage isolation was performed using enrichment method and
agar overlay method was used to confirm the presence of bacteriophages. All the
phages were characterized for their life cycle parameters, genome analysis, and
in vitro phage cocktail activity. The three bacteriophages exhibited broad host range
activity: Escherichia virus myPSH2311 infecting E. coli belonging to six different
pathotypes, Klebsiella virus myPSH1235 infecting K. pneumoniae belonging to four
different serotypes and Enterobacter virus myPSH1140 infecting four different species
of Enterobacter. Morphological observations suggested that the bacteriophages
belonged to, Phieco32virus (Escherichia virus myPSH2311), Podoviridae (Klebsiella
virus myPSH1235), and Myoviridae (Enterobacter virus myPSH1140). The life cycles
(adsorption, latent period, and cell burst) of Escherichia virus myPSH2311, Klebsiella
virus myPSH1235 and Enterobacter virus myPSH1140 were found to be 26, 40, and
11 min, respectively. Genomic analysis revealed that Escherichia virus myPSH2311
is closely related to Escherichia phage vB_EcoP_SU10, Klebsiella virus myPSH1235
is closely related to Klebsiella phage vB_KpnP_KpV48 and Enterobacter virus
myPSH1140 is closely related to Enterobacter phage PG7 and Enterobacter phage
CC31. When phage cocktail was used against multiple bacterial mixtures, there was a
reduction in bacterial load from 106 to 103 CFU/mL within 2 h. All the three characterized
phages were found to have a broad host range activity and the prepared phage cocktails
Frontiers in Microbiology | www.frontiersin.org
1
March 2019 | Volume 10 | Article 574
Manohar et al.
Antibacterial Activity of Characterization Bacteriophages
were effective against mixed bacterial population that are resistant to meropenem and
colistin, two last resort antibiotics. Infections caused by drug resistant bacteria will be a
serious threat in the future and the use of virulent bacteriophages in therapy may offer
an effective solution.
Keywords: bacteriophage genome, E. coli, Klebsiella pneumoniae, Enterobacter cloacae, phage cocktail, phage
therapy
INTRODUCTION
phages belonging to families Myoviridae, Siphoviridae, and
Podoviridae have been reported with complete genome
sequences1 . Genome sequenced phages against Klebsiella
(≈29) and Enterobacter (≈10) have been reported much lesser
in number2 . Here, we report characterization of three lytic
bacteriophages that showed promising ability to lyse E. coli,
K. pneumoniae, and Enterobacter species, report their host range
specificity and also efficacy of phage cocktails made using these
three phages in various permutations and combinations, in
effectively killing combinations of host bacteria using in vitro
phage killing assay.
Bacteriophages are the viruses of bacteria that live in the same
ecological niche, where their host bacteria are present (Rohwer,
2003). Phages are generally very specific (species-specific and
strain-specific) to their bacterial host but some phages are
polyvalent, and can infect more than one species or strain
of bacteria (Chibani-Chennoufi et al., 2004). Phage therapy
largely involves the treatment of bacterial infections using
bacteriophages/phages (Levin and Bull, 2004). Phages with
broad host range are mostly chosen for therapy, because of
their broad spectrum host-range activity against multiple
bacteria. The phages belonging to the order Caudovirales
(Family-Myoviridae, Siphoviridae, and Podoviridae) with
proteinaceous tail, that follow only lytic pathway, are
preferred for therapy (Gill and Hyman, 2010). The use of
bacteriophages for therapeutic purpose is an old concept
that is re-emerging after about a century (Sulakvelidze et al.,
2001). Antibiotic resistance has become a human health
concern globally as the infections caused by resistant bacteria
are becoming difficult to cure (World Health Organization
[WHO], 2014; Ventola, 2015). Phage therapy can be one of the
alternatives for combating antibiotic resistant bacterial infections
(Rios et al., 2016).
Escherichia coli, Klebsiella pneumoniae, and Enterobacter
cloacae are Gram-negative bacteria that belong to the family
Enterobacteriaceae. All the three are enteric pathogens causing
serious opportunistic infections in humans (Morens et al.,
2004; Schlossberg, 2015). They cause hospital acquired and
community acquired infections such as diarrhea, meningitis,
urinary tract infections (UTIs), bacteremia, pneumonia, surgical
site infections, and sepsis (Schlossberg, 2015). The increasing
reports of resistance to carbapenem and colistin, two last
resort drugs, among Enterobacteriaceae world over (World
Health Organization [WHO], 2014) and more particularly
so in the developing countries (Ventola, 2015) is a serious
threat to their therapeutic use, which prompts search for
alternative treatment options. Studies using bacteriophages
as an antibacterial agent have shown promising outcomes in
both in vitro and in vivo studies, and therefore phage therapy
is being studied as a candidate to cure bacterial infections
(McVay et al., 2007; Hung et al., 2011; Cao et al., 2015;
Mirzaei and Nilsson, 2015). Phage cocktails have shown broad
spectrum activity against many bacterial strains (Mendes et al.,
2014; Yen et al., 2017). The characterization of phages for
therapeutic purpose involves isolation of potential lytic phages,
multi-step in vitro characterization, cocktail preparation and
purification, dosing and in vivo studies. More than 50 Escherichia
Frontiers in Microbiology | www.frontiersin.org
MATERIALS AND METHODS
Isolation of Clinical Bacterial Strains for
the Study
This study does not include any human subjects; therefore,
ethical approval was not required for this study according to
national and institutional guidelines. The clinical isolates of
Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae,
E. hormaechei, E. asburiae, and E. aerogenes used in this
study were collected from diagnostic centers in Chennai,
Tiruchirappalli, and Madurai located in the state of Tamil
Nadu in India, during December 2014- September 2016. All the
isolates were preserved in 30% glycerol stocks at −20◦ C. The
clinical samples used for bacterial isolation were urine, sputum,
pus, blood, wound swab, and bronchial aspirate. Bacterial
identification was performed using VITEK identification system
(bioMèrieux, Inc., United States) and 16S rRNA analysis.
Universal primers, 27F (5′ -AGAGTTTGATCCTGGCTCAG-3′ )
and 1492R (5′ -GGTTACCTTGTTACGACTT-3′ ), were adopted
for gene amplification and sequences of 16S rRNA genes
were deposited in GenBank. All the clinical isolates were
studied for resistance against meropenem and colistin, two
last resort antibiotics, using microbroth dilution method
following Clinical and Laboratory Standards Institute
[CLSI], 2018. For the study, a total of 150 non-repetitive,
Gram-negative bacterial isolates belonging to three genera
Escherichia, Klebsiella, and Enterobacter were used. The
isolated clinical pathogens included 80 E. coli isolates, 44
Klebsiella pneumoniae isolates and in the case of Enterobacter
isolates, there were four different species namely E. cloacae
(n = 15), E. hormaechei (n = 4), E. asburiae (n = 4), and
E. aerogenes (n = 3) (Table 1). The results for meropenem and
1
2
2
https://www.ncbi.nlm.nih.gov/genome/?term=escherichia+phage
https://www.ncbi.nlm.nih.gov/
March 2019 | Volume 10 | Article 574
Manohar et al.
Antibacterial Activity of Characterization Bacteriophages
TABLE 1 | Host range infection and efficiency of plating (EOP) of the phages Escherichia virus myPSH2311, Klebsiella virus myPSH1235, and Enterobacter
virus myPSH1140.
Bacteria
Spot test (%)
High
(EOP ≥ 0.5)
Moderate
(EOP > 0.1− < 0.5)
Low
(EOP ≤ 0.1)
No activity
(EOP < 0.001)
Sum of EOP
values
Escherichia virus myPSH2311 lytic activity against different pathotypes of E. coli
EPEC (n = 12)
10 (83%)
5
1
1
3
5.30
EHEC (n = 10)
6 (60%)
2
0
1
3
1.91
ETEC (n = 8)
4 (50%)
3
0
0
1
3.10
EIEC (n = 11)
9 (82%)
5
3
0
1
5.55
EAEC (n = 14)
10 (71%)
6
0
2
2
5.86
UPEC (n = 21)
17 (81%)
11
0
1
5
10.75
2 (50%)
2
0
0
0
1.60
58 (73%)
34
4
5
15
34.07
Unknown (n = 4)
Total (n = 80)
Klebsiella virus myPSH1235 lytic activity against different serotypes of K. pneumoniae
Serotype K1 (n = 3)
2 (67%)
1
1
0
0
1.20
Serotype K2 (n = 7)
5 (71%)
2
1
1
1
2.85
Serotype K5 (n = 9)
6 (67%)
2
0
2
2
2.10
Unknown (n = 25)
10 (40%)
5
1
1
3
Total (n = 44)
23 (52%)
10
3
4
6
5.85
12.0
Enterobacter virus myPSH1140 lytic activity against different species of Enterobacter
E. cloacae (n = 15)
15 (100%)
7
3
1
4
E. hormaechei (n = 4)
3 (75%)
2
0
0
1
8.50
2.72
E. asburiae (n = 4)
2 (50%)
2
0
0
0
2.10
E. aerogenes (n = 3)
2 (67%)
2
0
0
0
1.44
Values represent the lytic activity of the respective phage against the target bacterium. EPEC (enteropathogenic E. coli), EHEC (enterohemorrhagic E. coli), ETEC
(enterotoxigenic E. coli), EIEC (enteroinvasive E. coli), EAEC (enteroaggregative E. coli), UPEC (uropathogenic E. coli). EOP was calculated using double agar overlay
method. EOP was deemed as ‘High’ if the test bacterium had at least 50% activity (EOP ≥ 0.5) compared to the host bacterium, ‘Moderate’ if between >10 and <50%
(EOP > 0.1− < 0.5), anything < 10% (EOP ≤ 0.1) was reported as ‘Low.’
and the phage titer was determined. For spot test method, the
bacterial (using exponentially grown host bacterial culture) lawn
was prepared in LB agar plates and 10–50 µL of phage filtrate
was placed as a spot over the target bacterial lawn to evaluate
the phage activity. The development of clear spots indicated the
phage activity and the time taken for bacterial clearance indicated
the lytic activity of phages. The bacterium initially used for phage
isolation was deemed as a host bacterium against the phage.
Multiplicity of infection (MOI) was calculated using the number
of phage particles against the potent host bacteria (PFU/CFU).
colistin resistance screening of these isolates are presented in
Supplementary Table S1.
Phage Isolation and Enrichment
Bacteriophages were isolated from water samples collected from
Ganges River near Varanasi in Northern part of India and sewage
water treatment plants (secondary treatment stage) from different
locations in Chennai, Bangalore, Tirupathi, Vellore, Karur, and
Trichy in Southern part of India. Initially, the isolated bacterial
strains (one isolate at a time) were grown in Luria-Bertani broth
(Himedia, India) medium and were used as a host for phage
isolation. Briefly, to a 5 mL of exponentially grown bacterial
culture (optical density at 600 nm = 0.6), 10 mL of phage
containing water samples was added and incubated at 37◦ C
for 24 h in shaking incubator to enrich the phages against
the host bacterium. This mixture was centrifuged for 15 min
at 12,000 × g and the supernatant was filtered in 0.22-µm
pore-sized membrane syringe filters for separation of phages
from other contaminants. The filtrate was used for double-agar
overlay method (Li et al., 2016a). Briefly, to the exponentially
grown host bacterial culture (400 µL), the phage filtrate (200 µL)
was added and incubated for 15 min. To the mixture, 3 mL
of molten soft agar (0.75% agar) was added and over-laid onto
prepared LB agar plate (1.5% agar). The plates were incubated
at 37◦ C for 10 h and the appearance of clear plaques indicated
the presence of phages against the host bacterium. The phage
plaques were picked-up from the plate for further purification
Frontiers in Microbiology | www.frontiersin.org
Purification of Lytic Phages
The isolated phage lysates were prepared in high titers using
phage multiplication strategy (propagation on host bacterium).
Briefly, the phages were multiplied using host bacterium for 24 h
(day 1) and centrifuged at 6000 × g for 15 min. The collected
supernatant was mixed with exponentially grown host bacterium
(day 2) and allowed to multiply. Similar passages were carried
out for 5 days and evaluated for phage activity/ titer by spot test
and double agar overlay method against E. coli, K. pneumoniae,
and Enterobacter species. The obtained high titer phages were
precipitated using 10% PEG 6000 (polyethylene glycol) and 1 M
NaCl. Briefly, to the phage lysate 10% PEG 6000+1 M NaCl was
added, mixed gently (did not vortex) and the mixture was stored
at 4◦ C for 24 h. The precipitated phage particles were centrifuged
at 15,000 × g for 45 min and the obtained pellet was resuspended
in sterile SM buffer (For 1 L: 5.8 g, NaCl; 50 mL, 1 M Tris-HCl
3
March 2019 | Volume 10 | Article 574
Manohar et al.
Antibacterial Activity of Characterization Bacteriophages
Characterization: Adsorption Rate,
Latency Period, and Burst Size
[pH 7.5]; 2 g, MgSO4 .7H2 O; 5 mL, 2% gelatin). The extraction
was carried out by adding equal volume of chloroform and the
aqueous phase was sedimented by centrifugation at 18,000 × g
for 80 min. The obtained phage particles were dialyzed against
phosphate buffer saline (PBS) for 6 h by changing buffer every
2 h and the purified phage suspension was stored at 4◦ C for
further analysis.
Exponentially grown bacterial cells were mixed with the
respective phages at a MOI of 0.001 and incubated at 37◦ C.
Aliquots of 100 µL were removed after every 4 min interval for
40 min and diluted in 4.4 mL LB broth and 0.5 mL of chloroform
was added. After incubating the mixture for 30 min at 37◦ C, the
number of non-adsorbed phages was determined subsequently
using double agar overlay method. The adsorption curve was
constructed using the ratio of non-adsorbed phage particles at
different time intervals to the number of initial phages. One-step
growth experiment was performed to determine the latent period
and burst size (14). Briefly, the bacterial cells (108 CFU/mL) were
infected with the phage particles (MOI of 0.001) and allowed to
adsorb (based on the adsorption time determined previously) at
37◦ C. The mixture was then centrifuged at 12,000 × g for 5 min
and the pellet was resuspended in 10 mL of LB broth and the
incubation was continued at 37◦ C. The samples were taken at
5 min intervals for 80 min and titrated against the host bacterium.
The latent-period was calculated as the duration between the
phage adsorbed until the release of phage virions. The burst size
of the phage was calculated using the final number of free phage
particles to the initial number of phages.
Electron Microscopic Analysis
The purified phage particles (105 PFU/mL) were negatively
stained using phosphotungstic acid, PTA (2% [w/v], pH 7.0).
Briefly, 10 µL of phage lysate was added over the copper grid
and the liquid was allowed to absorb for 10 min. The remaining
liquid was removed using tissue paper and the prepared 2% PTA
solution (staining solution) was added. After allowing it to stain
for 5 min, the excess stain was removed and the grid was washed
twice with sterile water. The negatively stained phage particles
in the copper grid were allowed to dry at room temperature
for 20–30 min and visualized under Transmission Electron
Microscopy (FEI-TECNAI G2-20 TWIN, Bionand, Spain). The
phage morphology was determined and head/ tail lengths (10
measurements each) were measured using ImageJ software.
Host-Range Specificity Determination
and Efficiency of Plating (EOP)
Phage Stability Studies
The lytic activity of isolated phages was tested against the
target bacteria. Accordingly, Escherichia phage was tested
against 80 E. coli isolates belonging to pathotypes: EPEC
(enteropathogenic E. coli), EHEC (enterohemorrhagic E. coli),
ETEC (enterotoxigenic E. coli), EIEC (enteroinvasive E. coli),
EAEC (enteroaggregative E. coli), and UPEC (uropathogenic
E. coli). Klebsiella phage was tested against 37 K. pneumoniae
isolates that belonged to serotypes K1, K2, K5 and Enterobacter
phage was tested against 15 E. cloacae, 4 E. hormaechei, 4
E. asburiae, and 3 E. aerogenes isolates. Spot test was carried
out to assess the host-range specificity of phages against the
test bacteria and the resulting positive isolates were again
tested for their plaque forming ability in double agar overlay
method for calculating efficiency of plating (EOP) (Mirzaei and
Nilsson, 2015). EOP was calculated using the number of virus
particles infecting the test bacterium against the same titer of
virus particles infecting the host bacterium. Accordingly, all
the test bacterial strains were grown overnight (16 h) and the
concentration of 106 –109 (CFU/mL) was used for double agar
overlay method. For the assay, 200 µL of bacterial culture
was mixed with 100 µL of phage lysate (MOI = 0.01) and
EOP was determined using the formula, plaque forming units
(PFUs) on the test bacterium/PFU on the host bacterium,
evaluated by double agar overlay method. EOP was classified as
‘High,’ ‘Moderate,’ and ‘Low’ based on the productive infection
on the target bacterium. EOP was deemed as ‘High’ only if
the phage-bacterium combination against the test bacterium
had a productive infection of at least 50% (EOP ≥ 0.5)
compared to the host bacterium. EOP between >10 and <50%
(EOP > 0.1− < 0.5) was considered ‘Moderate’ and EOP < 10%
(EOP ≤ 0.1) was recorded as ‘Low’ (Mirzaei and Nilsson, 2015).
DNA Isolation, Genome Sequencing, and
Analysis
Frontiers in Microbiology | www.frontiersin.org
Stability studies were conducted at different pH and temperature.
For thermal stability tests, phage lysates (108 PFU/mL) were
incubated at 4, 20, 35, 45, 50, 55, 60, 70, and 80◦ C for 60 min in
temperature-controlled water bath and immediately transferred
to the ice cold condition (−20◦ C) which was further tested
for phage activity using double agar overlay method. The pH
stability studies were performed using SM buffer and pH was
adjusted using 1N NaOH and 1N HCl. The phage lysates (108
PFU/mL) were incubated at pH 1–14 for 60 min and the
aliquots were removed for stability analysis. The results were
expressed as phage viability in terms of percentage of initial viral
counts. All the stability studies were tested using E. coli ec311,
K. pneumoniae kp235, and E. cloacae el140, and the experiments
were repeated in triplicates.
The phage DNA was extracted from purified phage particles
using phenol-chloroform (24:1) method and precipitated using
ethanol (100%). The purified phage DNA was visualized on 0.8%
agarose gels and the PE libraries were prepared using Illumina
TruSeq Nano DNA library Prep kit. The prepared libraries
were sequenced using Illumina Nextseq 500 system (using
2 bp × 150 bp chemistry) at Eurofins Genomics, Bangalore,
India. The sequenced raw data was processed to obtain high
quality clean reads using Trimmomaticv 0.35 to remove adapter
sequences, ambiguous reads (reads with unknown nucleotides
“N” larger than 5%), and low-quality sequences [reads with
more than 10% quality threshold (QV) < 20 phred score].
The sequenced high quality reads were de novo assembled
using CLC Genomics Workbench version 9.5.2. Protein-coding
4
March 2019 | Volume 10 | Article 574
Manohar et al.
Antibacterial Activity of Characterization Bacteriophages
24 h to calculate the reduction in bacterial count. To test the
activity of phage cocktails, bacteria were also used in combination
similar to phage combinations (Supplementary Table S2). In
the case of control experiments, bacteriophage buffer alone was
used with the bacterial inoculum and bacterial growth was
determined. All the cocktail studies were tested using E. coli
ec311, K. pneumoniae kp235, and E. cloacae el140, and the
experiments were repeated in triplicates.
and tRNA genes were identified using the final assembly. The
transfer-RNA (tRNA) genes were predicted using tRNAscan-SE
2.0 web server while the protein coding genes (CDS) were
predicted using FGENESV web server. Functional annotation
of the predicted proteins was performed using the amino acid
sequences viaBLASTp program online against a custom database
of viral proteins in NCBI. Gene ontology (GO) annotations of the
genes were determined by the Blast2GO platform. Distribution of
GO terms across the categories – Biological Process, Molecular
Function and Cellular Component was obtained through
WEGOportal3 . The NCBI sequence was downloaded from NCBI2
for sequence comparison and the scaffolds were then subjected to
reference-based assembly via CONTIGuator2. The final assembly
generated by CONTIGuator was validated based on sequence
homology to known bacteriophage sequences in NCBI via BlastN.
RESULTS
Nomenclature and Morphological
Characterization of Phages
The phages were named as Escherichia virus myPSH2311,
Klebsiella virus myPSH1235, and Enterobacter virus myPSH1140
following the bacteriophage nomenclature guidelines (Krupovic
et al., 2016). Examination of phage morphology by TEM
analysis showed that Escherichia virus myPSH2311 had an
icosahedral head of 33 ± 3.0 nm, a non-contractile tail
length of 65 ± 2.5 nm and belonged to genus Phieco32virus,
Klebsiella virus myPSH1235 had the icosahedral head of
80 ± 4.5 nm and very short non-contractile tail that
showed the phage belonged to family Podoviridae and
Enterobacter virus myPSH1140 had an elongated head of
approximately 80 ± 2.0 nm and long contractile tail of
101 ± 3.5 nm in length indicating that it belonged to the family
Myoviridae (Figure 1).
Composition and Preparation of Phage
Cocktails
Phage cocktails containing different compositions of isolated
phages under study were prepared and evaluated for activity
against target species. For cocktail preparation, two or three
different phages were mixed together in equal proportions to
obtain a concentration of 106 PFU/mL. Briefly, cocktail EK1
contained Escherichia phage plus Klebsiella phage; KL2 contained
Klebsiella phage plus Enterobacter phage; EL3 contained
Escherichia phage plus Enterobacter phage and EKL4 contained
Escherichia phage plus Klebsiella phage plus Enterobacter phage.
The prepared cocktails were tested for in vitro phage-killing assay
against respective bacterial strains. The results were compared
to the activity of phages in cocktails to the activity of phages
alone, and one bacterium from each genus was used for this
study. Accordingly, 1 mL of host bacterium (6 × 107 CFU/mL)
was diluted in LB broth to yield a final concentration of
6 × 106 CFU/mL. For each study, 100 µL (each phage at the
concentration of 106 PFU/mL; MOI of 1.0) of bacteriophage
suspension was added and the mixture was incubated at 37◦ C
and the aliquots (100 µL) were removed at 0, 2, 4, 6, and
3
Host-Range Activity Determination and
Efficiency of Plating (EOP)
The spot test assay showed that the Escherichia virus myPSH2311
had lytic activity against 73% of the tested E. coli isolates,
Klebsiella virus myPSH1235 had activity against 52%
K. pneumoniae isolates and Enterobacter virus myPSH1140
showed activity against 15 E. cloacae (n = 15), 3 E. hormaechei
(n = 4), 2 E. asburiae (n = 4), and 2 E. aerogenes (n = 3)
isolates. In the case of double agar overlay method, Escherichia
virus myPSH2311 had plaques against 43/80 tested E. coli
http://wego.genomics.org.cn
FIGURE 1 | Transmission electron microscopy (TEM) images of (A) Escherichia virus myPSH2311, (B) Enterobacter virus myPSH1140, (C) Klebsiella virus
myPSH1235. The smaller images in (A,C) represent 100 nm.
Frontiers in Microbiology | www.frontiersin.org
5
March 2019 | Volume 10 | Article 574
Manohar et al.
Antibacterial Activity of Characterization Bacteriophages
against 2/4 E. hormaechei, 2/4 E. asburiae, and 2/3 E. aerogenes,
respectively (Table 1).
isolates, Klebsiella virus myPSH1235 had plaques against 17/44
K. pneumoniae isolates and Enterobacter virus myPSH1140
had plaques against 11/15 E. cloacae, 2/4 E. hormaechei, 2/4
E. asburiae, and 2/3 E. aerogenes isolates. The percentage activity
difference between the spot test and double agar overlay method
was found to be 29.7% for Escherichia virus myPSH2311, 30%
for Klebsiella virus myPSH1235 and for Enterobacter virus
myPSH1140, it was 30.7% for E. cloacae, 40% for E. hormaechei,
0% for both E. asburiae and E. aerogenes. The EOP analysis that
was used to differentiate the phage infectivity between spot test
and double agar overlay method, showed a different scenario
(Table 1). Though, spot test results showed that Escherichia
virus myPSH1311 produced clear zone (spot) against 58/80
tested E. coli isolates; the EOP analysis showed high productive
infection in 34/80 E. coli isolates whereas against 9 E. coli isolates
it was moderate or low productive infection and 15/80 E. coli
isolates had no infection. Klebsiella virus myPSH1235 had
high productive infection against 10/44 K. pneumoniae isolates
with 23/44 in spot test assay and the percentage difference
was 78.7%. Even if all the EOP results (High + Moderate +
Low) were considered for Klebsiella phage myPSH1235, the
number of isolates producing (17/44) plaques in double agar
overlay assay was still lower than the spot test (23/44) results.
Enterobacter virus myPSH1140 had high productive infection
against 7/15 tested E. cloacae isolates compared to 15/15 in spot
test assay. The same phage showed high productive infection
Phage Characterization: One-Step and
Stability Studies
The multiplication capacity of phages was determined by
one-step growth experiment to analyze the adsorption velocity,
latency period and burst size (Figure 2). Accordingly, for
Escherichia virus myPSH2311, the adsorption velocity was
1.1 × 10−9 mL/min with latency period of 26 min and the burst
size of approximately 110 phages/infected cell. The adsorption
velocity for Klebsiella virus myPSH1235 was 4.35 × 10−9 and
the latency period was 40 min with the burst size of 120
phages/infected cell. Enterobacter virus myPSH1140 had an
adsorption velocity of 2.8 × 10−9 , a very short latency period of
11 min and a burst size of 135 phages/infected cell (Figure 2).
When sensitivity of phages to different pH conditions was
determined by exposing them to varying range of pH from 1
to 14 for 60 min, all the three phages were found to be viable
from pH 4 up to pH 11, but the phages were inactivated at
pH ≤ 3 and ≥ 12 (Figure 3). In the case of thermal stability,
all the phages were found to uphold their activity up to 55◦ C
and reduction in activity was observed at higher temperatures
(Figure 3). The complete characterization report is available in
Supplementary Table S3.
FIGURE 2 | (A) Adsorption rate of phages to their bacterial host, (A1) Escherichia virus myPSH2311, (A2) Enterobacter virus myPSH1140, (A3) Klebsiella virus
myPSH1235, and (B) One-step growth curve experiment of phages, (B1) Escherichia virus myPSH2311, (B2) Enterobacter virus myPSH1140, (B3) Klebsiella virus
myPSH1235. Legend within figure B: L-latency period; B-burst size.
Frontiers in Microbiology | www.frontiersin.org
6
March 2019 | Volume 10 | Article 574
Manohar et al.
Antibacterial Activity of Characterization Bacteriophages
FIGURE 3 | (A) Stability of phages at varying temperatures; (A1) Escherichia virus myPSH2311, (A2) Enterobacter virus myPSH1140, (A3) Klebsiella virus
myPSH1235 and (B) Stability of phages at varying pH at 37◦ C, (B1) Escherichia virus myPSH2311, (B2) Enterobacter virus myPSH1140, (B3) Klebsiella virus
myPSH1235.
Genomic Analysis and Annotation
virus myPSH1140 was having a genome size of 172,614 bp with
a GC content of 39.9%. The gene annotation studies showed 102
proteins with known function and 138 proteins were hypothetical
proteins. The obtained 1.22 Gb data was assembled into scaffolds
using CLC workbench version 9.5.2, and the assembly size was
5,193,726 bp with the average scaffold size of 6,059 bp. The
complete genome was having 92% similarity with Enterobacter
phage CC31 and 90% similarity with Enterobacter phage PG7
(Figure 4C and Supplementary Figures S1, S2C). The NCBI
accession number is MG999954. The complete list of all the
proteins is available in Supplementary Table S6.
The genome of Escherichia virus myPSH2311 measured
68,712 bp in size with a GC content of 42.4%. The genome
contains 89 proteins or coding sequences (CDS) and it includes
27 proteins of known putative function and 62 hypothetical
proteins. A total of 1.01 Gb data was assembled into scaffolds
using CLC workbench version 9.5.2, and the assembly size was
5,945,203 bp with the average scaffold size of 12,133 bp. The
arranged complete genome of Escherichia virus myPSH2311 is
closely related to Escherichia phage vB_EcoP_SU10 (88%)
and Escherichia virus phiEco32 (72%) (Figure 4A and
Supplementary Figures S1, S2A). The NCBI accession number
for this sequence is MG976803. The complete list of all the
proteins is available in Supplementary Table S4. Klebsiella virus
myPSH1235 was found to have a genome size of 45,135 bp with a
GC content of 53.7%. The genome contains 49 proteins or CDS,
of which 21 were found to have known putative function and
28 were hypothetical proteins. The obtained 1.37 Gb data was
assembled into scaffolds using CLC workbench version 9.5.2,
and the assembly size was 5,740,807 bp with the average scaffold
size of 1,321 bp. The genome was closely related to Klebsiella
phage vB_KpnP_KpV48 (95%) (Figure 4B and Supplementary
Figures S1, S2B). The complete genome of Klebsiella virus
myPSH1235 is free of toxins or toxin-related genes, and none
of the proteins representing a temperate or lysogenic lifestyle
was detected (Supplementary Table S4). The NCBI accession
number for this phage is MG972768. The complete list of all the
proteins is available in Supplementary Table S5. Enterobacter
Frontiers in Microbiology | www.frontiersin.org
In vitro Activity of Phage Cocktail
Phage cocktails were prepared to evaluate the activity of phages
against multiple bacterial strains. When the phage cocktail
containing all the three phages was tested against the three
meropenem and colistin resistant test bacteria, the growth
declined after 2 h from 106 to <105 CFU/mL and at the end
of 24 h the bacterial density reached to zero with no viable
cells. For EK1 cocktail, >2 fold decrease in bacterial cell count
(both E. coli and K. pneumoniae) was observed after 2 h, for
KL2 cocktail, the bacterial cell count (both K. pneumoniae
and E. cloacae) decreased from 106 to 103 CFU/mL within
2 h and for EL3 cocktail, twofold reduction of bacterial cells
(both E. coli and E. cloacae) was observed after 4 h. In phage
cocktail containing all the three phages, EKL4, a twofold decrease
in bacterial count (E. coli, K. pneumoniae, E. cloacae) was
observed in 2 h as comparable to the phages alone. In the
7
March 2019 | Volume 10 | Article 574
Manohar et al.
Antibacterial Activity of Characterization Bacteriophages
FIGURE 4 | Phylogenetic tree of (A) Escherichia virus myPSH2311, (B) Klebsiella virus myPSH1235, and (C) Enterobacter virus myPSH1140 constructed based on
the complete genome sequences of selected bacteriophages in NCBI-BLAST. The tree was produced using BLAST pairwise alignment using Neighbor-Joining
method. The query sequence is highlighted in yellow.
Frontiers in Microbiology | www.frontiersin.org
8
March 2019 | Volume 10 | Article 574
Manohar et al.
Antibacterial Activity of Characterization Bacteriophages
FIGURE 5 | In vitro phage activity of prepared phage cocktails. (A) activity of Escherichia virus myPSH2311 and Klebsiella virus myPSH1235 and their combination
(EK1); (B) activity of Klebsiella virus myPSH1235 and Enterobacter virus myPSH1140 and their combination (KL2); (C) activity of Escherichia virus myPSH2311 and
Enterobacter virus myPSH1140 and their combination (EL3); (D) activity of Escherichia virus myPSH2311, Klebsiella virus myPSH1235, Enterobacter virus
myPSH1140 and their combination (EKL4) (Control for all the experiments-bacterial growth without antibacterial agents and phages).
bacteriophages infecting Gram-negative bacteria, E. coli,
K. pneumoniae, and Enterobacter species.
In the present study, we characterized the phenotypic,
morphological, and genomic properties of three bacteriophages
that are independently specific in their activity against pathogenic
E. coli, K. pneumoniae, and Enterobacter species. All the three
characterized phages belong to order Caudovirales that are lytic
phages which are generally considered for therapy (Podoviridae
and Myoviridae). There are earlier studies on Escherichia phages
that showed many of the studied phages are specific to a strain
of E. coli (e.g., Escherichia phage OSYSP infecting only E. coli
O157:H7) or had a narrow host range activity (Carter et al.,
2012; Dalmasso et al., 2016; Baig et al., 2017; Lin et al., 2017).
In our study, the characterized Escherichia virus myPSH2311 was
found to have broad host range activity infecting E. coli isolates
that belonged to six different pathotypes EPEC, EHEC, ETEC,
EIEC, EAEC, and UPEC besides some unknown pathotypes.
This is one of the rarest reports where an Escherichia phage
infecting six different pathotypes of E. coli was characterized.
But another study also report the broad host range activity
of Escherichia phage against multiple pathotypes of E. coli
case of prepared phage cocktails, all the combinations had
similar to better activity in comparison to the phages alone
(Figure 5). Our experiment proved the activity of prepared
phage cocktails against multiple bacterial genera and showed
promising results against meropenem and colistin resistant
bacteria in vitro.
DISCUSSION
Phage therapy is currently gaining attention in clinical medicine,
because the infections caused by ‘superbugs – multi-drug
resistant (MDR) bacteria’ are almost impossible to treat,
using available antibiotics (Chan et al., 2013). From clinical
point-of-view, bacteriophages are likely to be used in treatment,
only when the patient is infected with multi-drug resistant
bacteria or during the antibiotic crisis of non-curable infections
(Lin et al., 2017). The increasing studies on bacteriophages
and phage therapy could put forth phage therapy as one of
the alternative treatment options to treat ‘superbug’ infections
(Rios et al., 2016). This study is one such effort to characterize
Frontiers in Microbiology | www.frontiersin.org
9
March 2019 | Volume 10 | Article 574
Manohar et al.
Antibacterial Activity of Characterization Bacteriophages
of progeny phage particles (burst size) largely depends on the
availability of host bacterial cells (Dalmasso et al., 2016). Life
cycle parameters of bacteriophages will play significant role in
determining both in vitro and in vivo phage activities (during
therapy), because phage multiplication is directly proportional to
reduction in bacteria.
The genomes of all the three characterized phages showed
70–100% similarities to the already existing phage genomes in
the database. Escherichia virus myPSH2311 (68,712 bp) was
found to have 88% sequence similarity with Escherichia phage
vB_EcoP_SU10 (77,327 bp). The endolysin gene (ORF 12)
was found to have 98% sequence similarity with Escherichia
virus phiEco32 and 97% sequence similarity with Escherichia
phage vB_EcoP_SU10. The DNA injection protein (ORF 17)
was detected to have 98% sequence similarity with Escherichia
phage vB_EcoP_SU10. The genome does not have toxins
or toxin-related genes, and none of the genomic markers
representing a temperate or lysogenic lifestyle was detected
(Supplementary Table S4). Escherichia phage vB_EcoP_SU10 is a
C3 morphotype phage but Escherichia virus myPSH2311 does not
have any elongated regions in its morphological characterization
(Mirzaei et al., 2014) and related to T4-phages (Petrov et al.,
2010). Klebsiella virus myPSH1235 was found to have 80%
similarity with Klebsiella phage vB_KpnP_KpV48. Klebsiella
virus myPSH1235 has a genome size of 45,135 bp with 49 CDS
compared to Klebsiella phage vB_KpnP_KpV48, which has a
genome size of 44,623 bp with 57 CDS. According to the genome
morphology, phylogenetic analysis and sequence similarities,
the Klebsiella virus myPSH1235 is classified within the genus
Kp34virus, subfamily Autographivirinae, family Podoviridae.
Klebsiella phage vB_KpnP_KpV48 has no capsular specificity
and similarly, Klebsiella virus myPSH1235 was also found to
infect K. pneumoniae of capsular types K1, K2 and K5 (Solovieva
et al., 2018). From clinical point of view, the K. pneumoniae
capsular types K1 and K2 are the most virulent strains and that
the infections caused by these strains can be eliminated using
Klebsiella virus myPSH1235 is a promising result, which needs
further exploration. Enterobacter virus myPSH1140 was found
to have a large genome size of 172,614 bp with 240 CDS with
92% similarity with Enterobacter phage CC31 (165,540 bp and
279 CDS) and 90% similarity to Enterobacter phage PG7 (173,276
and 294 bp). The genome contains 240 proteins or CDS and all
the identified proteins were found to have 90–100% sequence
similarity with the Enterobacter phage CC31 and Enterobacter
phage PG7. Similarities in the phage genome showed that there
is abundance of these phages in the environment and therefore
they may be isolated for therapeutic purpose.
Earlier studies on phage cocktails showed promising results
in reducing the bacterial load in both in vitro and in vivo
models (Lin et al., 2017; Pan et al., 2017). In our in vitro
experiments, a phage cocktail that was prepared using the three
newly isolated bacteriophages infecting three different genera of
bacteria (Escherichia virus myPSH2311 against E. coli, Klebsiella
virus myPSH1235 against K. pneumoniae and Enterobacter virus
myPSH1140 against Enterobacter species) showed promising
results in totally suppressing the bacterial load within 24 h. The
phage cocktails reported in this study consisting of multiple
(Manohar et al., 2018b). The studied Klebsiella virus myPSH1235
showed broad host range activity against K. pneumoniae
belonging to serotypes K1, K2, K5 besides some unknown
serotypes. An earlier study showed that a multi-host Klebsiella
phage φK64-1 was capable to infect Klebsiella capsular types K1,
KN4, KN5, K11, K21, K25, K30, K35, K64, and K69. It is stated
that the multi-host infectivity of the bacteriophages is due to
the presence of multiple depolymerases in the tail-fibers (Pan
et al., 2017). The role of polyvalent phages in phage therapy needs
more detailed investigation. The studies on Enterobacter phage
thus far are very minimal or reports of established Enterobacter
phages capable of infecting various strains or species are scarce (Li
et al., 2016a; Pereira et al., 2016). The characterized Enterobacter
virus myPSH1140 in our study belonging to Myoviridae family
showed activity against four species of Enterobacter E. cloacae,
E. hormaechei, E. asburiae, and E. aerogenes. Our study and those
of some others strongly suggest that there are bacteriophages
that can have broad host range activity against different strains
or species or genera of bacteria (Hamdi et al., 2017). A recent
study showed that bacteriophages SH6 and SH7 were capable
of infecting bacteria from different genera – E. coli O157:H7,
Shigella flexneri, Salmonella paratyphi, and Shigella dysenteriae
(Hamdi et al., 2017). There are studies that showed ‘narrow’ host
range specificity of bacteriophages mainly because of the use of
standard isolation procedure where single host strain of bacteria
is used (Li et al., 2016b; Ross et al., 2016; Hamdi et al., 2017).
Future studies should focus on testing the host range activity of
bacteriophages but it is also believed that host range activity is
not a fixed property of bacteriophages and can evolve overtime
(Ross et al., 2016; Hamdi et al., 2017). It should also be noted that
we found dissimilarities in the results obtained between spot test
and plaque assay in host range activity determination, therefore
plaque assay is recommended in order to obtain productive
infection for the determination of host range activity of phages
(Mirzaei and Nilsson, 2015).
The three characterized bacteriophages had different growth
profile, which is one of the important characteristics of lytic
bacteriophages. In this study, Escherichia virus myPSH2311
(Podovirus) had a growth profile with the latent period of
26 min and burst size of 110 phages/infected cell, which is
beneficial for therapy. Earlier studies using Podoviridae phages
showed latency period of ≈15–25 min and burst size of ≈50–80
phages/infected cell (Dalmasso et al., 2016). An earlier study
on Podoviridae phages infecting Klebsiella species was found
to have a growth profile with the latency period of 15 min
and burst size of ≈50–60 phages/infected cell (Chaturongakul
and Ounjai, 2014). In our study, Klebsiella virus myPSH1235
belonging to Podoviridae family was found to have a latent
period of 40 min and burst size of 120 phages/infected cell,
which is beneficial for therapy. It was very clear from this
study that the growth profile of bacteriophages is not completely
dependent on either the family or the host these bacteriophages
infect. The bacteriophages belonging to Myoviridae family are
known to have shorter life cycles and similarly the characterized
Myoviridae phage (Enterobacter phage myPSH1140) had a latent
period of 11 min and burst size of 135 phages/infected cell.
It was also noted in this and earlier studies that the number
Frontiers in Microbiology | www.frontiersin.org
10
March 2019 | Volume 10 | Article 574
Manohar et al.
Antibacterial Activity of Characterization Bacteriophages
DATA AVAILABILITY
phage types could thus prove to be effective as clinical therapeutic
agents (Chan et al., 2013; K˛esik-Szeloch et al., 2013). Ours is
one of the rarest studies to use phage cocktails against different
genera of bacteria. In vivo activity of these phage cocktails also
showed promising results and the results are published elsewhere
(Manohar et al., 2018a).
The strength of our study is that we established the lytic
effectiveness of the phages and their cocktail against clinical
bacterial isolates of three genera that are known to be pathogenic.
Further we also demonstrated the lytic effectiveness of these
phages and their cocktail against pathogenic bacteria that were
resistant to two last resort antibiotics, meropenem, and colistin.
Thus further experiments can be taken up for in vivo studies
in search of a therapeutic treatment against bacterial infections
resistant to these drugs.
All the data that support the findings of this study have been
deposited in GenBank with the accession numbers MG976803
(Escherichia phage myPSH2311), MG972768 (Klebsiella phage
myPSH1235), and MG999954 (Enterobacter phage myPSH1140).
AUTHOR CONTRIBUTIONS
PM and RN collected the isolates and did bacteriophage
experiments, undertook the laboratory work, and wrote the initial
manuscript. AT, CL, and RN interpreted the data. CL and AT
revised and edited the manuscript. All authors approved the final
version of the manuscript.
FUNDING
CONCLUSION
The authors would like to thank Vellore Institute of Technology
(VIT) for providing research facilities to complete the work.
This research was partly supported by Swedish Research Council
with grants to CL (Grant Nos. 2012-02889 and 2017-01327).
The authors would also like to thank Council of Scientific and
Industrial Research (CSIR) for providing financial assistance
(SRF) to support this research.
The emergence of multi-drug resistant bacterial strains is a global
threat which needs to be addressed by using alternative therapies,
such as phage therapy. The three highlighted bacteriophages
(Escherichia virus myPSH2311 against E. coli, Klebsiella virus
myPSH1235 against K. pneumoniae and Enterobacter virus
myPSH1140 against Enterobacter species) and their cocktails
showed promising in vitro results that may be extended for
use as biocontrol agents in some clinical applications. The
development of phage cocktails could be considered for the
effective treatment of bacterial infections consisting of broad
targets. Hopefully, in future, availability of increased repertoire
of phages may allow the development of multi-phage cocktail
therapy possible.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fmicb.
2019.00574/full#supplementary-material
REFERENCES
potential for phage therapy. PLoS One 11:e0156773. doi: 10.1371/journal.pone.
0156773
Gill, J. J., and Hyman, P. (2010). Phage choice, isolation, and preparation for phage
therapy. Curr. Pharma. Biotechnol. 11, 2–14. doi: 10.2174/138920110790725311
Hamdi, S., Rousseau, G. M., Labrie, S. J., Tremblay, D. M., Kourda, R. S.,
Slama, K. B., et al. (2017). Characterization of two polyvalent phages infecting
Enterobacteriaceae. Sci. Rep. 7:40349. doi: 10.1038/srep40349
Hung, C. H., Kuo, C. F., Wang, C. H., Wu, C. M., and Tsao, N. (2011). Experimental
phage therapy in treating Klebsiella pneumoniae-mediated liver abscesses and
bacteremia in mice. Antimicrob. Agents Chemother. 55, 1358–1365. doi: 10.
1128/AAC.01123-10
K˛esik-Szeloch, A., Drulis-Kawa, Z., Weber-Dabrowska,
˛
B., Kassner, J., MajkowskaSkrobek, G., Augustyniak, D., et al. (2013). Characterising the biology of novel
lytic bacteriophages infecting multidrug resistant Klebsiella pneumoniae. Virol.
J. 10:100. doi: 10.1186/1743-422X-10-100
Krupovic, M., Dutilh, B. E., Adriaenssens, E. M., Wittmann, J., Vogensen, F. K.,
Sullivan, M. B., et al. (2016). Taxonomy of prokaryotic viruses: update from the
ICTV bacterial and archaeal viruses subcommittee. Arch. Virol. 161, 1095–1099.
doi: 10.1007/s00705-015-2728-0
Levin, B. R., and Bull, J. J. (2004). Population and evolutionary dynamics of phage
therapy. Nat. Rev. Microbiol. 2, 166–73. doi: 10.1038/nrmicro822
Li, E., Wei, X., Ma, Y., Yin, Z., Li, H., Lin, W., et al. (2016a). Isolation and
characterization of a bacteriophage phiEap-2 infecting multidrug resistant
Enterobacter aerogenes. Sci. Rep. 6:28338. doi: 10.1038/srep28338
Li, E., Zhao, J., Ma, Y., Wei, X., Li, H., Lin, W., et al. (2016b). Characterization of
a novel Achromobacter xylosoxidans specific siphoviruse: phiAxp-1. Sci. Rep.
6:21943. doi: 10.1038/srep21943
Baig, A., Colom, J., Barrow, P., Schouler, C., Moodley, A., Lavigne, R., et al. (2017).
Biology and genomics of an historic therapeutic Escherichia coli bacteriophage
collection. Front. Microbiol. 8:1652. doi: 10.3389/fmicb.2017.01652
Cao, F., Wang, X., Wang, L., Li, Z., Che, J., Wang, L., et al. (2015). Evaluation of the
efficacy of a bacteriophage in the treatment of pneumonia induced by multidrug
resistance Klebsiella pneumoniae in mice. Biomed. Res. Int. 2015:752930. doi:
10.1155/2015/752930
Carter, C. D., Parks, A., Abuladze, T., Li, M., Woolston, J., Magnone, J.,
et al. (2012). Bacteriophage cocktail significantly reduces Escherichia coli
O157: H7 contamination of lettuce and beef, but does not protect against
recontamination. Bacteriophage 2, 178–185. doi: 10.4161/bact.22825
Chan, B. K., Abedon, S. T., and Loc-Carrillo, C. (2013). Phage cocktails and the
future of phage therapy. Future Microbiol. 8, 769–783. doi: 10.2217/fmb.13.47
Chaturongakul, S., and Ounjai, P. (2014). Phage–host interplay: examples from
tailed phages and Gram-negative bacterial pathogens. Front. Microbiol. 5:442.
doi: 10.3389/fmicb.2014.00442
Chibani-Chennoufi, S., Bruttin, A., Dillmann, M. L., and Brüssow, H. (2004).
Phage-host interaction: an ecological perspective. J. Bacteriol. 186, 3677–3686.
doi: 10.1128/JB.186.12.3677-3686.2004
Clinical and Laboratory Standards Institute [CLSI] (2018). Performance
Standards for Antimicrobial Susceptibility Testing; Twenty-Fifth Informational
Supplements. Approved Standard M100-S28. Wayne, PA: Clinical and
Laboratory Standards Institute [CLSI].
Dalmasso, M., Strain, R., Neve, H., Franz, C. M., Cousin, F. J., Ross, R. P., et al.
(2016). Three new Escherichia coli phages from the human gut show promising
Frontiers in Microbiology | www.frontiersin.org
11
March 2019 | Volume 10 | Article 574
Manohar et al.
Antibacterial Activity of Characterization Bacteriophages
Rios, A. C., Moutinho, C. G., Pinto, F. C., Del Fiol, F. S., Jozala, A., Chaud, M. V.,
et al. (2016). Alternatives to overcoming bacterial resistances: state-of-the-art.
Microbiol. Res. 191, 51–80. doi: 10.1016/j.micres.2016.04.008
Rohwer, F. (2003). Global phage diversity. Cell 113:141. doi: 10.1016/S00928674(03)00276-9
Ross, A., Ward, S., and Hyman, P. (2016). More is better: selecting for broad host
range bacteriophages. Front. Microbiol. 7:1352. doi: 10.3389/fmicb.2016.01352
Schlossberg, D. (ed.) (2015). Clinical Infectious Disease. Cambridge: Cambridge
University Press. doi: 10.1017/CBO9781139855952
Solovieva, E. V., Myakinina, V. P., Kislichkina, A. A., Krasilnikova, V. M.,
Verevkin, V. V., Mochalov, V. V., et al. (2018). Comparative genome analysis
of novel podoviruses lytic for hypermucoviscous Klebsiella pneumoniae of K1,
K2, and K57 capsular types. Virus Res. 243, 10–18. doi: 10.1016/j.virusres.2017.
09.026
Sulakvelidze, A., Alavidze, Z., and Morris, J. G. (2001). Bacteriophage therapy.
Antimicrob. Agents Chemother. 45, 649–659. doi: 10.1128/AAC.45.3.649-659.
2001
Ventola, C. L. (2015). The antibiotic resistance crisis: part 1: causes and threats.
Pharm. Therapeut. 40, 277–283.
World Health Organization [WHO] (2014). Antimicrobial Resistance: Global
Report on Surveillance 2014. Available at: http://www.who.int/drugresistance/
documents/surveillancereport/en/
Yen, M., Cairns, L. S., and Camilli, A. (2017). A cocktail of three virulent
bacteriophages prevents Vibrio cholerae infection in animal models. Nat.
Commun. 8:14187. doi: 10.1038/ncomms14187
Lin, D. M., Koskella, B., and Lin, H. C. (2017). Phage therapy: an alternative to
antibiotics in the age of multi-drug resistance. World J. Gastrointest. Pharmacol.
Therapeut. 8:162. doi: 10.4292/wjgpt.v8.i3.162
Manohar, P., Nachimuthu, R., and Lopes, B. S. (2018a). The therapeutic potential
of bacteriophages targeting gram-negative bacteria using Galleria mellonella
infection model. BMC Microbiol. 18:97. doi: 10.1186/s12866-018-1234-4
Manohar, P., Tamhankar, A. J., Lundborg, C. S., and Ramesh, N. (2018b). Isolation,
characterization and in vivo efficacy of Escherichia phage myPSH1131. PLoS
One 13:e0206278. doi: 10.1371/journal.pone.0206278
McVay, C. S., Velásquez, M., and Fralick, J. A. (2007). Phage therapy of
Pseudomonas aeruginosa infection in a mouse burn wound model. Antimicrob.
Agents Chemother. 51, 1934–1938. doi: 10.1128/AAC.01028-06
Mendes, J. J., Leandro, C., Mottola, C., Barbosa, R., Silva, F. A., Oliveira, M., et al.
(2014). In vitro design of a novel lytic bacteriophage cocktail with therapeutic
potential against organisms causing diabetic foot infections. J. Med. Microbiol.
63, 1055–1065. doi: 10.1099/jmm.0.071753-0
Mirzaei, M. K., Eriksson, H., Kasuga, K., Haggård-Ljungquist, E., and Nilsson,
A. S. (2014). Genomic, proteomic, morphological, and phylogenetic analyses
of vB_EcoP_SU10, a podoviridae phage with C3 morphology. PLoS One
9:e116294. doi: 10.1371/journal.pone.0116294
Mirzaei, M. K., and Nilsson, A. S. (2015). Isolation of phages for phage therapy:
a comparison of spot tests and efficiency of plating analyses for determination
of host range and efficacy. PLoS One 10:e0118557. doi: 10.1371/journal.pone.
0118557
Morens, D. M., Folkers, G. K., and Fauci, A. S. (2004). The challenge of
emerging and re-emerging infectious diseases. Nature 430, 242–249. doi: 10.
1038/nature02759
Pan, Y. J., Lin, T. L., Chen, C. C., Tsai, Y. T., Cheng, Y. H., Chen, Y. Y., et al.
(2017). Klebsiella phage 8K64-1 encodes multiple depolymerases for multiple
host capsular types. J. Virol. 91:e2457–6. doi: 10.1128/JVI.02457-16
Pereira, S., Pereira, C., Santos, L., Klumpp, J., and Almeida, A. (2016). Potential of
phage cocktails in the inactivation of Enterobacter cloacae-an in vitro study in
a buffer solution and in urine samples. Virus Res. 211, 199–208. doi: 10.1016/j.
virusres.2015.10.025
Petrov, V. M., Ratnayaka, S., Nolan, J. M., Miller, E. S., and Karam, J. D. (2010).
Genomes of the T4-related bacteriophages as windows on microbial genome
evolution. Virol. J. 7:292. doi: 10.1186/1743-422X-7-292
Frontiers in Microbiology | www.frontiersin.org
Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2019 Manohar, Tamhankar, Lundborg and Nachimuthu. This is an
open-access article distributed under the terms of the Creative Commons Attribution
License (CC BY). The use, distribution or reproduction in other forums is permitted,
provided the original author(s) and the copyright owner(s) are credited and that the
original publication in this journal is cited, in accordance with accepted academic
practice. No use, distribution or reproduction is permitted which does not comply
with these terms.
12
March 2019 | Volume 10 | Article 574