Journal of Bioscience and Bioengineering

VOL. xxx No. xxx, xxx, xxxx

www.elsevier.com/locate/jbiosc

Characterization of Pseudomonas lytic phages and their application as a cocktail

with antibiotics in controlling Pseudomonas aeruginosa

Soo Peng Ong,1 Aa Haeruman Azam,2 Teppei Sasahara,2 Kazuhiko Miyanaga,1 and Yasunori Tanji1, *

School of Life Science and Technology, Tokyo Institute of Technology, 4259 J2-15 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan1 and Division of Bacteriology, Department of
Infection and Immunity, Faculty of Medicine, Jichi Medical University, Yakushiji, Shimotsuke 329-0498, Japan2

Received 20 September 2019; accepted 1 February 2020

Available online xxx
Pseudomonas aeruginosa is an opportunistic pathogen that causes nosocomial disease among immunocompromised
and chronic cystic fibrosis (CF) patients. We characterized two newly isolated Pseudomonas phages, fPA01 and fPA02,
with different host spectra, and examined their effect as a cocktail with antibiotics against P. aeruginosa, to indicate the
possibility of combining a phage cocktail and antibiotics in treating pseudomonal infection. Phages fPA01 (66,220 bp)
and fPA02 (279,095 bp) belong to the genus Pbunalikevirus and Phikzlikevirus, respectively. No virulence or lysogenic
associated gene was found in their genomes, thus they are potentially safe for phage therapy. We generated respective
phage-resistant strains to investigate cross-resistance between two phages. Slight cross-resistance to fPA02 in fPA01-
resistant strain was observed, while fPA02-resistant strain remained susceptible to fPA01. A fPA01 resistant strain that
was cross-resistant to fPA02 appeared in round 5 (R5-PA01R), revealed frameshift mutation in phosphoglucomutase
(algC), which is important for the synthesis of core lipopolysaccharide (LPS). Knockout of algC was resistant to both
phages. Complementation of DalgC restored phages’ infectivity, suggesting that LPS as host receptor. Phage cocktail
suppressed the growth of P. aeruginosa for longer (20 h) hour compared with single phage (8e9 h), further suggesting
their potential to be used as a phage cocktail. Furthermore, application of the phage cocktail with ciprofloxacin (0.25 mg/
ml) and meropenem (2 mg/ml), respectively managed to suppress the growth of P. aeruginosa up to 96 h. Our results
show the potential application of fPA01 and fPA02 as phage cocktail together with antibiotics for treatment of P.
aeruginosa.
Ó 2020, The Society for Biotechnology, Japan. All rights reserved.

[Key words: Antibiotics; Phage therapy; Phikzlikevirus; Pbunalikevirus; Pseudomonas aeruginosa]

Pseudomonas aeruginosa is one of the main causes of chronic their survival, bacteria possess diverse defense mechanisms
lung infection among patients who suffer from cystic fibrosis (CF) against invasion by phage, including blocking of phage receptors,
(1). A high lethal rate among CF patients is commonly caused by restriction of virion DNA, degradation of viral mRNA and so on
multidrug-resistant (MDR) P. aeruginosa, especially among children (10). But phages are well known for their ability to adapt to these
who receive prophylactic antibiotic treatment (2). Besides, defense mechanisms (11) and evolved phages have become more
P. aeruginosa also causes urinary tract infection, bacteremia and virulent (12).
pneumonia in immunocompromised patients. Due to its intrinsic Pbunalikevirus and Phikzlikevirus phages can be found in diverse
resistance mechanism, only a few classes of antibiotics are effective geographical area and phages belongs to these two genera have
against P. aeruginosa, such as carbapenem, aminoglycoside, qui- distinct genome features (13,14). Phikzlikevirus phages have large
nolone and polymyxin B. Further resistance toward these antibi- genomic size (211 kbe317 kbp) with genes encoding tRNAs and
otics can be developed through acquisition of plasmid-containing carry independent transcriptional machinery, such as DNA depen-
resistance gene or through chromosomal mutation to upregulate dent RNA polymerase (RNAP) (15,16). In contrast to Phikzlikevirus,
expression of resistance mechanisms (3). the genomic size of Pbunalikevirus ranges from 64 to 67 kbp and is
Recently, successful treatment of a diabetic patient infected devoid of genes encoding tRNA and phage encoded RNAP. There-
with MDR Acinetobacter baumannii by phage cocktail has directed fore, the transcription of phage proteins is entirely dependent on
public attention towards phage therapy as an alternative to treat the host (16,17). Phages from these two genera are widely used in
MDR bacterial infection (4). Studies have been conducted in vivo therapeutic phage cocktail (18).
to assess the efficiency of phage therapy against P. aeruginosa Carbapenem and quinolone are antipseudomonal antibiotics
(5,6) also by using a CF zebrafish model (7). However, an evolu- that target penicillin-binding protein (19) and DNA gyrase (20),
tionary arms race between bacteria and phage is very common respectively. Complementing antibiotic treatment with phage can
and this gives rise to phage resistant bacteria (8,9). To ensure assert profound evolutionary selective pressure compared with
single treatment (21). However, only a certain combination of
phage and antibiotic can work, depending on the mechanism of
each antimicrobial agent (22,23).
* Corresponding author. Tel.: þ81 45 924 5763; fax: þ81 45 924 5818.
E-mail address: ytanji@bio.titech.ac.jp (Y. Tanji).

1389-1723/$ e see front matter Ó 2020, The Society for Biotechnology, Japan. All rights reserved.
https://doi.org/10.1016/j.jbiosc.2020.02.001

Please cite this article as: Ong, S. P et al., Characterization of Pseudomonas lytic phages and their application as a cocktail with antibiotics in
controlling Pseudomonas aeruginosa, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2020.02.001
2 ONG ET AL. J. BIOSCI. BIOENG.,

FIG. 1. TEM image of (A,B) fPA01 and (C,D) fPA02. Arrows show the contracted tail. Scale bars: (AeC) 50 nm; (D) 100 nm. Staining: (A, C) uranyl acetate; (C, D) EM stainer. One-step
growth curve of (E) fPA01 and (F) fPA02.

Reports of using phage cocktail and antibiotics to treat Characterization of phage growth and determination of phage host
P. aeruginosa are scarce. Therefore, we investigated the effect of a range A one-step growth curve was constructed to determine the burst size
and latent period as previously described, with modification (24). Briefly, the
combined treatment using phage cocktail together with antibiotics. phage was added to refreshed overnight culture of P. aeruginosa PAO1
We characterized two P. aeruginosa’s phages, fPA01 and fPA02, (OD660nm ¼1) at multiplicity of infection (MOI) 0.01, and incubated at 37  C for
isolated from sewage and assessed their potential to be used as a 10 min, with shaking at 120 rpm. Unbound phage was removed by centrifugation
cocktail by generating respective phage resistant mutants to and washed with chilled LB medium five times. Cells infected with phage were
incubated at 37  C for 1 h. The number of phage was tittered by double layer agar
investigate cross-resistance. Analysis of phage-resistant
method. The host range was determined using 58 strains of clinical P. aeruginosa
P. aeruginosa revealed distinct mutations in strain resistant to collected from Jichi Medical University Hospital (Tochigi, Japan), by dropping 1 mL
respective phage, suggesting the differences in host-resistant of phage lysate of 107 PFU/ml on P. aeruginosa mixed with 0.5% top agar (w/v). All
mechanism towards fPA01 and fPA02. Receptor of two phages the clinical strains were isolated from different sporadic cases, originated from
was revealed. Finally, we investigated the effect of phage cocktail patients admitted in different wards (Table S1). Results were scored as clear
(sensitive), turbid (sensitive) or no plaque (resistant).
treatment complemented with either ciprofloxacin or meropenem.
DNA extraction, sequencing, genome analysis, phage growth
characterization Genomic DNAs of fPA01 and fPA02 were extracted by phage
DNA isolation kit (Norgen Biotek Corp., Thorold, ON, Canada) and were submitted to
BGI (Hong Kong) for whole genome sequencing by Illumina HiSeq platform with
MATERIALS AND METHODS
genome coverage (sequencing depth) of 100-fold with 100-bp paired end. The
sequence was assembled using Velvet De Novo Assembler v1.2.10 (EMBL-EBI) (26).
Bacteria, culture media and growth condition Standard strain Open reading frames (ORFs) were predicted and annotated using the RAST server
P. aeruginosa PAO1 was used in all experiments, unless otherwise stated. All ex- (http://rast.nmpdr.org/) (27) and phage-carried tRNA genes were identified using
periments were conducted in LuriaeBertani (LB) broth (10 g polypeptone, 10 g tRNA Scan SE ver. 1.21 software (28). A phylogenetic tree was constructed using
sodium chloride and 5 g yeast extract per liter) at 37  C, with shaking at 120 rpm, VICTOR Virus Classification and Tree Building Online Resource (29) based on
unless otherwise stated. settings recommended for prokaryotic viruses (30) and was visualized with
Isolation and preparation of phage stock Two lytic phages, fPA01 and FigTree ver 1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/) based on a method
fPA02, were isolated from sewage influent obtained from the municipal wastewater described previously (31). Nucleotide and amino acid sequence were compared by
treatment plant in Tokyo using P. aeruginosa PAO1 as the host by double layer agar Basic Local Alignment Search Tool (BLAST) (32) or Clustal W (33).
plating method. Phages were propagated and purified by the method described Generation and isolation of phage resistant strains Phage resistant strains
elsewhere (24). Briefly, purified phage was propagated by mixing 1% of overnight were generated by repeated co-culturing of P. aeruginosa PAO1 and phage based on a
culture of PAO1 in liquid LB and incubated overnight. Host cells were removed by previous study (9). The co-culture was started by infecting PAO1 with phage at MOI
centrifugation (11,000 g, 20 min, 4  C) before phage concentration by 0.1 and cultured for 48 h before transferring 1% (v/v) cells at stationary phase to the
polyethylene glycol 6000-NaCl (PEG-NaCl) method and filtered with a 0.45 mm new medium. Serial transfer was continued for 4e5 rounds and phage-resistant
Millex-GP filter (Merck, Millipore, Darmstadt, Germany). colonies were isolated from each round by plating the stationary bacterial cell
TEM imaging of phages Phages were observed by transmission electron culture on LB plates. Each isolated resistant strain was assigned as PA0X-RY,
microscope (TEM) as described previously (25). Briefly, PEG-NaCl concentrated where 0X indicates the type of phage to which the bacteria is resistant to 01-
phage lysate was purified by cesium chloride (CsCl) step centrifugation (step fPA01, 02-fPA02, while Y indicates the batch number R1eR5. Cross-resistance of
densities, 1.46, 1.55 and 1.63 g/ml) and concentrated phage suspension 109 plaque phage-resistant strains was tested by spot test as described above using wild type
forming unit (PFU/ml) was spotted on top of a hydrophilic plastic-carbon-coated fPA01 and fPA02.
copper grid (Nissin EM Corporation, Tokyo, Japan). Phages were allowed to adsorb Molecular cloning, plasmids constructions and genome
for 1 min before removing excess sample. Next, 10 ml of distilled water was editing Knockout of algC (DalgC) was generated based on detailed method re-
spotted on the grid and removed after a short time. Phages were stained by 2% ported (34) using CRISPR/Cas9 system with modification. Primers and plasmids used
uranyl acetate or EM Stainer (Nissin EM Corporation). Excess stain was removed in this study are listed in Tables S2 and S3. In brief, spacers for target gene were
after 1 min, and the grid was allowed to air-dry for 30 min before observing with searched by online software CHOPCHOP (35). The double stranded spacer was
the JEOL JEM-1400Plus (TEM) operating at 80 kV. generated by phosphorylation using T4 polynucleotide kinase and was inserted to

Please cite this article as: Ong, S. P et al., Characterization of Pseudomonas lytic phages and their application as a cocktail with antibiotics in
controlling Pseudomonas aeruginosa, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2020.02.001
VOL. xxx, xxxx PHAGE COCKTAIL AND ANTIBIOTIC COMBINED TREATMENT 3

TABLE 1. Genomic characterization of FPA01 and FPA02 and comparison with

similar phages.

Phage (accession Genome Identity Query tRNA ORFs Reference

number) size (bp) (%) coverage (%) no. no.

fPA01 (AP19535) 66220 e e 0 92 This study

LBL3 (NC 64427 95 96 0 92 13
_011165.1)
PB1 (NC 65764 95 96 0 93 13
_011810.1)
KTN6 (NC 65994 95 97 0 91 17
_041865.1)
KPP12 (NC 64144 94 94 0 88 44
_019935.1)
fPA02 279095 e e 6 343 This study
(AP019418)
KZ (NC_004629.1) 280334 99 97 6 343 37
KTN4 279593 99 97 6 368 38
(KU521356.1)
PA7 (NC 266743 99 93 6 337 Unpublished
_042060.1)
SL2 (NC 279696 98 95 4 355 42
_042081.1)

plasmid pACRISPR by Golden Gate assembly. Homologous arms generated from

overlap PCR was digested by restriction enzyme and inserted to pACRISPR-spacer
ligated by T4 ligase. All enzymes were from New England Biolabs. FIG. 2. Cross-resistant analysis of fPA01 and fPA02 by spot test (left) and adsorption
Complementation of algC was done by cloning algC gene from wildtype PAO1 efficiencies (right) to phage resistant strains. Single and double asterisks indicate
using Taq polymerase (Takara Bio, Shiga, Japan) and ligated into plasmid pBBR1- statistical difference (P < 0.01) of fPA01 and fPA02, respectively.
MCS2 (36). PCR products and plasmids were purified using Nucleospin kit
(MachereyeNagel, Düren, Germany).
Adsorption assay of phage Adsorption efficiency of phages on wildtype large capsid with the diameter and height of 121  3 nm and 134
PAO1, DalgC mutant and phage resistant strains was measured by titrating free
 4 nm, respectively. This feature is similar to reported giant
phages present in the supernatant after 20 min of cell-phage contact at MOI of 0.01.
One hundred microliters of cell-phage solution were sampled and immediately
phages (37). The relaxed tail fiber of fPA02 was 206  7 nm long
added to 9.9 ml of chilled SM buffer. The solution was gently vortexed before and 25  1 nm wide. Meanwhile, the diameter and height of
taking 1 ml for centrifugation (10,000 g, 5 min, 4  C) in order to remove the fPA01’s capsid was 73  6 nm and 73  2 nm, respectively, with
bacterial cells before titration of phage concentration. Adsorption efficiency was a 148  3 nm long and 21  1 nm wide tail sheath, measured in
calculated by dividing the number of adsorbed phages by the initial number of
extended state. Its features are similar to phages from
phages. Statistical analysis was carried out using two-tailed student’s t-test.
Pbunalikevirus (13).
Treatment effect of phage cocktail and antibiotics combination In order
to examine the treatment effects of fPA01, fPA02, the phage cocktail and antibiotics Genomic characterization of novel phages It was revealed
combination, two sets of experiments were set up. In the first experiment, fPA01,
that fPA01 (66,220 bp) and fPA02 (279,095 bp) belonged to the
fPA02 or phage cocktail were added at 1 h (early logarithmic growth phase) after 1%
of stationary overnight culture was inoculated in 4 mL fresh LB broth and incubated genus Pbunalikevirus and Phikzlikevirus, respectively. Analysis via
at 37  C, with shaking at 40 rpm. Phage was added at MOI of 1. In the second BLASTn showed that the genome sequence of fPA01 had highest
experiment, ciprofloxacin (CIP, 0.25 mg/ml), meropenem (MEM, 2 mg/ml), or com- similarities to phage LBL3 and PB1 (95%) (13). Meanwhile, fPA02
bination of CIP (0.25 mg/ml) and MEM (0.25 mg/ml) were added at the same con- had highest similarities to PhiKZ and KTN6 (99%) (37,38)
dition. Antibiotics’ concentrations were decided based on minimal inhibitory
concentration (MIC) of P. aeruginosa PAO1 determined in our lab. Growth of
(Table 1). The genome of fPA02 encodes 6 tRNAs specific for Leu
P. aeruginosa in each condition was monitored at 15-min intervals, for a minimum of (TAA), Pro (TGG), Ile (CAT), Asp (GTC), Asn (GTT) and Thr (TGT),
48 h based on optical density of 660 nm (OD660) using TVS062CA BioPhoto recorder but no tRNA encoding gene was found in fPA01. Virion associated
(Advantec, Tokyo, Japan). To observe propagation of each phage and number of RNAP (gp48, gp48, gp82 and gp156) and non-virion associated
surviving PAO1 cells in phage cocktail-antibiotic’s condition, the third experiment
RNAP (gp109, gp164, gp165, gp166 and gp185) were encoded in
was carried out in 20 ml LB in shake flask (due to volume limitation for sampling)
at 37  C with shaking at 100 rpm. One milliliter of medium was sampled each fPA02’s genome. However, such kind of transcriptional
time. The sample was centrifuged (10,000 g, 5 min, 4  C) and pelleted cells were machinery was not found in fPA01. Besides, no integrase and
washed three times with phosphate buffer saline (PBS) before plating for cell virulence genes were found in the genome of either phage. GC
count. Phage titer was determined using the supernatant from centrifugation. contents of fPA01 and fPA02 were 55.4% and 36.8%, respectively.
Titration of fPA01 and fPA02 in the cocktail experiment was done using fPA02-
resistant PAO1 generated in our lab and P. aeruginosa NBRC 3080 strain (resistant
fPA02’s genome encodes putative 343 ORFs, which is about four
to fPA01 but sensitive to fPA02), respectively. times larger than fPA01 (92 ORFs).
Accession number(s) The genome data of fPA01 and fPA02 were submitted Phage growth and host range The one-step growth curve
to the DNA Data Bank of Japan (DDBJ) database under the accession numbers
(Fig. 1E and F) showed that latent periods of fPA01 and fPA02
AP019535 and AP019418, respectively.
were 30 min and 35 min (in addition to 10 min of adsorption
time), each with a burst size of 32 and 49 phage particles per cell.
Phage fPA01 and fPA02 were able to lyse 36% and 47% of clinical
RESULTS P.aeruginosa, respectively, and showed different host spectra to
clinical isolates (Table S1.).
Morphology of phage fPA01 and fPA02 Based on the Isolation of phage-resistant strains and their cross-
morphology of fPA01 and fPA02 observed (Fig. 1AeD), they were resistance Phage-resistant strains were isolated from each
classified to the Myoviridae family. Both phages displayed a capsid batch of co-culture conducted up to four or five rounds. According
head connected to a long contractile tail (Fig. 1B and D, indicated to Fig. 2, phage-resistant mutants were resistant to their respective
by black arrows). fPA02 showed a distinct morphology with a phages used for infection, and adsorption efficiency of their

Please cite this article as: Ong, S. P et al., Characterization of Pseudomonas lytic phages and their application as a cocktail with antibiotics in
controlling Pseudomonas aeruginosa, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2020.02.001
4 ONG ET AL. J. BIOSCI. BIOENG.,

respective phages were significantly reduced. fPA01-resistant

mutants remained slightly sensitive to fPA02, except R5-PA01R,
which became resistant to both phages.
Meanwhile, fPA02-resistant mutants remained sensitive to
fPA01. Adsorption efficiency of fPA01 toward fPA02-resistant
mutants reduced significantly, except R1-PA02, compared with
wildtype. Adsorption efficiency of 4PA02 towards 4PA01-resistant
mutants (R1-R4) remained at around 73e98%, with decreased
infectivity. Adsorption of fPA02 to R5-PA01R dropped to 39%.
Strain R5-PA01R was sequenced to reveal spontaneous mutation
that confer resistance to both phages. Adsorption of both phages to
R5-PA01R was significantly decreased (Fig. 2). Nucleotide deletion
of one base pair A at position 1284 was found in algC (PA5322) that
encodes an enzyme with dual functions: phosphoglucomutase
(PGM) and phosphomannomutase (PMM). PGM synthesize
glucose-1-phosphate, which is an intermediate in core lipopoly-
saccharide (LPS) and rhamnolipid synthesis (39,40) Formation of
mannose-1-phospate through PMM activity is necessary in alginate
synthesis (41). Partial deletion spanning across two genes were
found in in R4-PA01R: a putative glycosyltransferase, ssg (PA5001)
and a hypothetical protein (PA5002) (Fig. S2A). Meanwhile, strain
R4-PA02R showed one-point mutations in the following genes: fliF
(PA1101), opmB (PA2525), rocS1 (PA3946) and a hypothetical pro-
tein (PA5148). Detailed mutations are summarized in Fig. S2B.
These mutations found were not reflected in R5-PA01R strain.
Mutation in algC blocked adsorption of fPA01 and fPA02 to
FIG. 4. Growth curve of PAO1 (A) treated with single and phage cocktail. Phage were
host A nucleotide deletion leads to frameshift mutation which
added at 1 h at MOI: 1. (B) Growth curve of PAO1 treated with 0.25 mg/ml ciprofloxacin
resulted in premature stop codon, was found in gene algC (PA5322) (CIP), 2.0 mg/ml meropenem (MEM) or a combination of both.
in R5-PA01R. Truncated protein with 472 amino acids (aa) was
produced compared to wildtype (868 aa) (Fig. 3B). This enzyme is
important in the initial steps of core LPS synthesis. Strain of DalgC
P. aeruginosa up to 10 h and 8 h, respectively (Fig. 4A). Since cross-
was resistant to fPA01 and fPA02. Adsorption rate of fPA01 and
resistance to both phages was not observed except in R5-PA01R,
fPA02 decreased significantly to 0 % and 39 %, respectively we combined fPA01 and fPA02 as a phage cocktail to treat
compared to wildtype (Fig. 3A), indicating that adsorption of
P. aeruginosa. The phage cocktail was able to suppress the
phages to resistant strain was hindered. Complementation of algC
growth of bacteria up to 20 h. Based on Fig. 4B, an antibiotic
restored the sensitivity and adsorption of both phages to DalgC
resistant mutant appeared around 20e22 h when treated with
mutant.
CIP (0.25 mg/ml) or MEM (2.0 mg/ml). The resistant mutant
Effect of phage, phage cocktails and antibiotic in PAO1’s appeared around 50 h and 94 h when treated with a
growth Phage fPA01 and fPA02 suppressed the growth of combination of CIP and MEM. Furthermore, when infected with

FIG. 3. Analysis of host receptor. (A) Spot test and adsorption analysis of fPA01 and fPA02 to wildtype, algC knockout and algC complemented strain. Single and double asterisks
indicate statistical difference (P < 0.01) of fPA01 and fPA02, respectively. (B) Amino acid alignment between wildtype and mutant protein of AlgC. Premature stop codon indicated
by black arrow. Shaded area showed truncated part of AlgC.

Please cite this article as: Ong, S. P et al., Characterization of Pseudomonas lytic phages and their application as a cocktail with antibiotics in
controlling Pseudomonas aeruginosa, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2020.02.001
VOL. xxx, xxxx PHAGE COCKTAIL AND ANTIBIOTIC COMBINED TREATMENT 5

FIG. 5. Phage propagation and bacteria growth in phage cocktail and antibiotic treatment. (A) Growth curve of PAO1 treated with phage cocktail and CIP or MEM measured by OD660.
(B) Killing curve of P. aeruginosa PAO1 with different treatments (MOI:1.0; CIP: 0.25 mg/ml; MEM: 2 mg/ml). (C, D) Phage titer of fPA01 and fPA02 with addition of CIP or MEM,
respectively. All phages and antibiotics were added at 1 h. Each condition was performed in triplicate and the means  standard errors are indicated.

the phage cocktail, together with CIP (0.25 mg/ml) or MEM (2.0 mg/ for RNAP were found (15). Five predicted ORFs which encode for
ml), OD of bacteria culture remained low at 0.1 OD660 up to 96 h virion associated RNAP and non-virion associated RNAP were
(Fig. 5A) and about 99.9% of viable cells were reduced within found in fPA02 but not in fPA01. This showed that fPA02 is
2 h (Fig. 5B). Treatment using the phage cocktail together with independent on host transcription mechanism compared to fPA01.
CIP reduced cells to 102 CFU/ml at around 6 h and bacteria
Cross-resistance of P. aeruginosa towards fPA01 and
regrew around 12 h. Based on Fig. 5B, approximately 105 CFU/ml
fPA02 The low adsorption of fPA01 and fPA02 to its resistant
of viable cells were detected at the end of the experiment
mutants might be due to the masking or loss of host receptor.
(96 h). Meanwhile, the phage cocktail treatment together with
Infection of fPA02 might be blocked in the post adsorption process
MEM further reduced viable cells up to 12 h, to less than
since it remained highly adsorbed to all fPA01 and fPA02-resistant
102 CFU/ml, while approximately 104 CFU/ml of viable cells were
mutants (73e98% and 49e63%, respectively), except R5-PA01R
detected at 96 h. Production of fPA01 and fPA02 progeny
(39.7%). In previous study, resistant strains of closely related PB1-
phages were observed in both treatments with CIP and MEM,
phages that utilized the same host receptor for infection showed
respectively (Fig. 5C and D). Phage titer of fPA01 and fPA02
cross-resistance (17). Thus, our results suggested that phages
peaked at 6 h after addition of phage and antibiotics at 1 h,
from distance lineage are potential phage cocktail candidates,
while the number of both phages decreased at 1.5 h (0.5 h after
whereby host resistant to both phages only appeared after 5th
phage addition). Titer of fPA01 decreased to about 5.0 x107 PFU/
batch of continuous co-culture.
ml at 96 h, while titer of fPA02 remained stable until the end of
Mutant of DalgC is devoid of A-band and B-band (O-specific
the experiment in both conditions. Phages’ propagation was not
antigen) and does not have complete core LPS (39). Noteworthy,
inhibited by either CIP or MEM. Our results clearly showed that
sequencing of R5-PA01R, which blocked adsorption of fPA01 and
applications of the phage cocktail together with CIP or MEM
fPA02 showed frameshift mutation that resulted in premature stop
were more effective in controlling P. aeruginosa compared with
codon in algC gene. By deletion and complementation of algC, we
the phage cocktail alone.
confirmed that both phages required LPS as their receptor. Previous
study showed that mutant of algC could not produce rhamnolipids
and alginates which are important in biofilm formation (40,41). The
DISCUSSION algC mutant was also found to be less virulent compared to wild-
type in burned mouse modal (45), showing that bacteria that
Genomic characterization of novel phages A well charac- become phage resistant would have trade-off in its virulence (46).
terized phage is one of the key factors determining the success of a Function of gene ssg is not well studied in P. aeruginosa. How-
phage therapy (18). Phages from the genera Pbunalikevirus and ever, transposon mutant of P.alkylphenolia in gene homologous to
Phikzlikevirus were able to control the growth and biofilm ssg had incomplete LPS without B-band (47), further supporting
formation of P. aeruginosa in vitro (42) and in vivo (43,44) in that host receptor of fPA01 was LPS, similar to its nearest phages
previous studies. Genomic analysis has revealed the absence of KTN6 (17) and KPP22 (48).
integrase, genes associated with toxin and virulence in fPA01 Point mutations found in R4-PA02 mutant were probably
and fPA02, suggesting that they are potentially safe candidates related to the latter steps in life cycle of phage after phage
for phage therapy (18). Previous study showed that fKZ protein adsorption, since adsorption of fPA02 to this strain remained
transcription was independent on its host and proteins encoding relatively high (Fig. 2). Bacteria can employ different strategy to

Please cite this article as: Ong, S. P et al., Characterization of Pseudomonas lytic phages and their application as a cocktail with antibiotics in
controlling Pseudomonas aeruginosa, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2020.02.001
6 ONG ET AL. J. BIOSCI. BIOENG.,

block phage infection at different step of phage cycle: adsorption, it in clinical setting also provides an important insight for future
phage DNA injection and protein translation (49). Gene fliF encodes clinical application. Further examination in vivo is needed before
the precursor forming M-ring at the base of flagella (50). Product of application to patients in order to improve the success rate of
gene opmB was found to be part of component in efflux pump (51) treatment.
while rocS1 regulates the expression of adhesins important for Supplementary data to this article can be found online at
biofilm formation (52). Tail fibers of fKZ were made up of at least https://doi.org/10.1016/j.jbiosc.2020.02.001.
32 proteins and their detailed mechanisms are still not well-known
(53). Mutation found in genes other than LPS showed that giant
ACKNOWLEDGMENTS
phage might possess a more complicated host recognition system
(54).
We are thankful to Professor Longzhu Cui, Dr. Shinya Watanabe
Distinct mutations found in each phage resistant mutant sug-
and Dr. Kitaro Kiga (Jichi Medical Hospital University, Department
gested that fPA01 and fPA02 have different phage infection
of Bacteriology) for kindly supporting us in conducting the exper-
mechanism. Even though both phages used LPS for host recogni-
iment using the clinical strains collection from Jichi Medical Uni-
tion, phage specifically recognized certain component in LPS as
versity Hospital. S.P. Ong received scholarship from Ministry of
host receptor (48,55) since LPS is a complex structure made up of
Education, Culture, Sports, Science and Technology of Japan during
different components (56). In Fig. 2, adsorption of fPA01 to R4-
her post graduate study. All authors declared that there are no
PA01R is lower compared to fPA02, showing that these phages
conflicts of interest in this article. This article does not contain any
recognize different host receptor for successful infection.
studies with human participants or animals.
Relation of mutations to phage resistance will be further
investigated in future work. In this work we focused on the appli-
cation of combined treatment using phage cocktail and antibiotic. References

1. Sousa, A. and Pereira, M.: Pseudomonas aeruginosa diversification during

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Please cite this article as: Ong, S. P et al., Characterization of Pseudomonas lytic phages and their application as a cocktail with antibiotics in
controlling Pseudomonas aeruginosa, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2020.02.001
VOL. xxx, xxxx PHAGE COCKTAIL AND ANTIBIOTIC COMBINED TREATMENT 7

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Please cite this article as: Ong, S. P et al., Characterization of Pseudomonas lytic phages and their application as a cocktail with antibiotics in
controlling Pseudomonas aeruginosa, J. Biosci. Bioeng., https://doi.org/10.1016/j.jbiosc.2020.02.001