Pseudomonas aeruginosa Bacteriophage PA1Ø Requires Type IV Pili
for Infection and Shows Broad Bactericidal and Biofilm Removal
Shukho Kim,aMarzia Rahman,aSung Yong Seol,aSang Sun Yoon,b,cand Jungmin Kima
Department of Microbiology, School of Medicine, Kyungpook National University, Daegu, Republic of Korea,aand Department of Microbiology and Immunology, Brain
Korea 21 Project for Medical Sciences,band Institute for Immunology and Immunological Diseases, Yonsei University College of Medicine,cSeoul, Republic of Korea
We isolated a new lytic Pseudomonas aeruginosa phage that requires type IV pili for infection. PA1Ø has a broad bactericidal
therapeutic agent for biofilm-related mixed infections with P. aeruginosa and Staphylococcus aureus.
Due to their ability to lyse bacteria, attempts have been made to
clinical promise as therapeutic antimicrobial agents (2, 11, 16).
However, phages are not commonly used therapeutically due to
phage preparations, poor stability or viability of phage prepara-
tions, and a lack of understanding of the heterogeneity and mode
spread of multidrug-resistant (MDR) pathogenic bacteria has led
to the revival of this therapeutic approach (12).
In this study, we isolated a novel lytic phage, PA1Ø, which is
capable of lysing MDR pathogenic bacteria, especially Pseudomo-
nas aeruginosa and Staphylococcus aureus. From sewage samples
collected from poultry and pig farms located in Gyeonggi Prov-
ince, South Korea, several phages specific to P. aeruginosa were
agated, and purified successfully according to the method of
Merabishvili et al. (13). Concentrated phage solutions were pre-
pared as previously described (5) using cesium chloride (CsCl)
was obtained and stored at 4°C for future use.
tail by transmission electron microscopy analysis. The lengths of
the icosahedral head and the long tail were approximately 58 and
ily Siphoviridae (1). To assess the host range of PA1Ø, spot tests
and plaque formation assays were performed with various species
on the lawns of P. aeruginosa, Shigella sonnei, S. aureus, Staphylo-
coccus epidermidis, Staphylococcus hominis, Streptococcus pneu-
ever, no lytic zones were found on the lawns of Escherichia coli,
Serratia marcescens, Enterobacter aerogenes, Acinetobacter bau-
mannii, or certain Streptococcus spp. In the plaque formation as-
says, PA1Ø formed distinct plaques only when used to infect P.
aeruginosa or S. sonnei. PA1Ø could not produce typical plaques
when used to infect S. aureus, although it formed distinct clear
zones on the lawn of S. aureus in spot test with lytic areas two to
or S. sonnei. This result indicates that PA1Ø can infect and prop-
agate in only P. aeruginosa and S. sonnei.
acteriophages (phages) are bacterial viruses that play an im-
portant role in bacterial biology, diversity, and evolution (6).
The adsorption rate of PA1Ø was determined. About 87% of
PA1Ø phage were adsorbed to P. aeruginosa PAO1 cells within 2
min. The rate of PA1Ø adsorption to S. sonnei was similar to that
for P. aeruginosa. A one-step growth experiment showed that the
latent period of PA1Ø was approximately 10 min. The first burst
be approximately 261 PFU/bacterium.
To identify the receptor molecule used by PA1Ø to infect P.
aeruginosa, we constructed and screened a library of random
transposon (Tn) insertion mutants of P. aeruginosa strain PAO1
insertion mutants of P. aeruginosa strain PAO1 was constructed
were pooled, and an aliquot of the pool (?108CFU/ml) was in-
growth at 37°C, cultures were diluted 100-fold in fresh LB con-
200 ?g/ml gentamicin (Gm) to isolate mutants that had poten-
tially acquired resistance to PA1Ø. Under these conditions, three
mutants were successfully identified as demonstrating normal
growth in the presence of 1 ? 1011PFU/ml PA1Ø, while
the growth of wild-type PAO1 was greatly diminished under the
same conditions; the optical cell density of these mutants in the
LB medium. Further sequence analysis revealed Tn insertion into
of these selected mutants. Since these genes encode core compo-
suggests that PA1Ø binds to the pilus to infect P. aeruginosa. The
Received 7 March 2012 Accepted 20 June 2012
Published ahead of print 29 June 2012
Address correspondence to Jungmin Kim, firstname.lastname@example.org, or Sang Sun Yoon,
S.K. and M.R. contributed equally to this work.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
aem.asm.orgApplied and Environmental Microbiologyp. 6380–6385September 2012 Volume 78 Number 17
TABLE 1 Results of spot tests and plaque formation assays
Shigella sonnei NIH1
Escherichia coliATCC 25922
Serratia marcescensATCC 8100
Acinetobacter baumanniiATCC 19606
Staphylococcus aureus ATCC 29213
Staphylococcus epidermidis KNUH-134
Staphylococcus hominis KNUH-328
Streptococcus salivarius 65
Streptococcus gordonii 143
Listeria monocytogenes 10403S
No Martin Wiedermann
a??, large clear lysis; ?, small clear lysis; ?, no lysis.
bATCC, American Type Culture Collection.
PA1Ø Possesses Anti-Biofilm Activity
September 2012 Volume 78 Number 17 aem.asm.org 6381
adsorption assay using P. aeruginosa mutants revealed that PA1Ø
was unable to adsorb to the mutants, confirming that the type IV
pilus is the receptor molecule used by PA1Ø to infect P. aerugi-
midis KNUH-134 and KNUH-174). Overnight bacterial cultures
by a 10-fold dilution. For treatment of planktonic cultures with
phage, 100-?l aliquots (5 ? 106CFU) were inoculated into 96-
well microtiter plates, followed by inoculation of 100 ?l of PA1Ø
microliters of 0.1% triphenyltetrazolium chloride (TTC) was
added, and samples were further incubated at 37°C for 1 h, after
which absorbance at 540 nm was determined using an enzyme-
linked immunosorbent assay (ELISA) reader (VersaMax; Molec-
of an overnight bacterial culture (5 ? 106CFU) were inoculated
humidified incubator. After removal of planktonic cells and me-
?l of PA1Ø (1 ? 1010PFU) was inoculated into the biofilm. After
TTC was added and incubated for a further 1 h. Absorbance was
recorded at 540 nm. PA1Ø significantly inhibited the bacterial
6 h with PA1Ø and P. aeruginosa PAO1, the growth of PAO1 was
greatly inhibited in comparison with an untreated control. We
also measured PA1Ø’s biofilm removal activity (Fig. 2B). After
treatment of 24-hour-old biofilms of P. aeruginosa, S. aureus, S.
in phage-treated biofilms were greatly decreased in comparison
microscopy (FESEM) analysis clearly showed PA1Ø’s biofilm re-
the phage-untreated groups (Fig. 3A, C, E, and G).
The genome sequence of PA1Ø was deciphered and an-
nounced recently (8). The PA1Ø genome consists of 51 putative
mology to the D3112 phage genome in the study (8), we per-
formed Dot Matrix View and MAUVE genome alignment analy-
gene, was not found in the PA1Ø genome. Unmatched region 3
covers the coding regions of host nuclease inhibitor proteins in
both phages, and other unmatched regions correspond to genes
coding hypothetical proteins of both phages. Three highly con-
served regions between PA1Ø and D3112 cover hypothetical pro-
teins (regions A and C), the major head subunit (region B), and
the putative tail component (region C).
FIG 1 Three pilus mutants grow normally in the presence of PA1Ø. The
growth of three pilus-negative strains and wild-type PAO1 was monitored by
measuring the optical density (OD) at 600 nm. PAO1 growth in plain LB is
included as a control. *, P ? 0.001 versus all other growth.
with those of non-PA1Ø-treated controls (gray bars). 1, S. aureus ATCC 25923; 2, S. aureus WS-05; 3, S. aureus D43-a; 4, S. epidermidis KNUH-134; 5, S.
and values are means and standard deviations.
Kim et al.
aem.asm.orgApplied and Environmental Microbiology
In this study, we isolated a novel lytic bacteriophage, PA1Ø,
with a broad host range of P. aeruginosa and S. sonnei. Besides
these bacteria, PA1Ø could lyse a number of Gram-positive bac-
terial pathogens. Since PA1Ø could form plaques only when in-
fecting P. aeruginosa and S. sonnei, its bactericidal mechanism
against Gram-positive bacteria might be different from that
production of phage-associated lytic enzymes. In fact, Lai et al.
PA1Ø Possesses Anti-Biofilm Activity
September 2012 Volume 78 Number 17 aem.asm.org 6383
reported that Acinetobacter baumannii phage varphiAB2, which
shows broad bactericidal activity against Gram-positive and
Gram-negative bacteria, produced endolysin (LysAB2), which
destroy biofilms of P. aeruginosa, S. aureus, S. epidermidis, and S.
hominis. It could significantly lyse those bacterial cells in the es-
and biofilm cells exhibited by PA1Ø have never been demon-
strated in other phages.
which is a well-known temperate phage used for molecular biol-
ogy studies of P. aeruginosa (3, 19). Both phages are Pseudomonas
homologous to the genome of D3112 phage (GenBank accession
number AY394005.1), with overall nucleotide identity of 90%.
The difference of PA1Ø from D3112 is the absence of the un-
matched region 1 spanning first five ORFs of phage D3112. Be-
cause unmatched region 1 includes a c repressor-encoding gene,
known to play a critical role in the maintenance of the lysogenic
a lytic phage, not a temperate, transposable one.
phage that has a broad bactericidal spectrum covering various
species of Gram-positive and Gram-negative bacteria. Moreover,
PA1Ø can eradicate biofilm cells. Therefore, PA1Ø can be devel-
oped as an alternative antimicrobial agent that may be useful in
treatment of biofilm-related infections and mixed infections
caused by bacteria such as P. aeruginosa and S. aureus.
This study was supported by a grant from the Korea Healthcare Tech-
nology R&D Project, Ministry for Health, Welfare & Family Affairs,
Foundation of Korea (NRF), funded by the Korean Government
no. 2011-0016210 (to S.S.Y.).
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FIG 4 Alignment of the PA1Ø and D3112 genomes. This MAUVE-generated diagram shows the degree of sequence similarity between two phages. Both gene
genomic dissimilarity regions are marked with black bold lines and numbered, and affected genes in both phages are listed in numbered boxes. Regions A to C
show highly conserved regions of PA1Ø and phage D3112.
Kim et al.
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