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
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PA1Ø Possesses Anti-Biofilm Activity
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