Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Phages of Pseudomonas aeruginosa: response to

environmental factors and in vitro ability to inhibit
bacterial growth and biofilm formation
P. Knezevic1, D. Obreht1, S. Curcin1, M. Petrusic1, V. Aleksic1, R. Kostanjsek2 and O. Petrovic1
1 Department of Biology and Ecology, Faculty of Sciences, University of Novi Sad, Novi Sad, Vojvodina, Serbia
2 Department of Biology, Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia

Keywords
bacteriophage(s), biocontrol, pseudomonads.
Correspondence
Petar Knezevic, Department of Biology and
Ecology, Faculty of Sciences, University of
Novi Sad, Trg Dositeja Obradovica 2, 21 000
Novi Sad, Vojvodina, Serbia.
E-mail: petar.knezevic@dbe.uns.ac.rs

2011 0222: received 7 February 2011,

revised and accepted 27 April 2011
doi:10.1111/j.1365-2672.2011.05043.x

Abstract
Aims: To examine effects of various environmental factors on adsorption and
inactivation of Pseudomonas aeruginosa-specific phages: d (family Podoviridae),
J-1, r-1 and 001A (family Siphoviridae) and their ability to inhibit bacterial
growth and biofilm formation.
Methods and Results: The phages examined in the study were clonally different, as revealed by RFLP. The temperature in the range 744C had no influence on the adsorption of Podoviridae, but did affect Siphoviridae adsorption,
particularly 001A. All phages were significantly stable at pH 59, and phages
d and 001A even at pH 3. Most of the examined carbohydrates and exopolysaccharides of the original host efficiently inactivated phage d, while phages r-1
and J-1 were inactivated considerably only by the amino acid alanine. Silver
nitrate efficiently inactivated all the phages, while Siphoviridae were more resistant to povidone-iodine. Serum of nonimmunized rats had no influence on
phage inactivation and adsorption. Only phage d showed ability to effectively
inhibit in vitro bacterial growth and biofilm formation.
Conclusions: The examined environmental parameters can significantly influence the adsorption and viability of Ps. aeruginosa-specific phages. The phage
d is a good candidate for biocontrol of Ps. aeruginosa.
Significance and Impact of the Study: The study provides important data on
Ps. aeruginosa-specific phage adsorption, inactivation and in vitro lytic efficacy.

Introduction
Pseudomonas aeruginosa is an opportunistic pathogen of a
particular medical interest, being able to cause various
infections. This organism possesses intrinsic and acquired
mechanisms of resistance and can grow in the form of a
biofilm, which aggravates its eradication by means of
conventional antimicrobial agents. The phenomenon has
significantly increased an interest in Ps. aeruginosa-specific
phages as potential alternative antimicrobial agents during
the last decade.
At the beginning of last century, one of the main reasons
of phage therapy failure was incomprehension of phage
biology (Sulakvelidze and Kutter 2005). Prior to phage
application in vivo, there are several prerequisites that
should be met: the first and the most important is that

each phage intended for therapy should be well characterized (Skurnik and Strauch 2006). Unfortunately, phage
biological characteristics have been neglected in most studies dealing with Ps. aeruginosa-specific phages therapeutic
properties. In the majority of prominent studies, only
in vivo antimicrobial characteristics of isolated phages are
examined (Soothill 1992; Wang et al. 2006; Watanabe
et al. 2007; Heo et al. 2009), without a detailed examination of their interaction with environmental factors and or
in vitro lytic efficacy. Consequently, the data on Ps. aeruginosa-specific phage adsorption and inactivation are
scarce, although they are of great interest not only for valid
selection of those viruses that can be successfully applied
as anti-Pseudomonas agents in specific environmental
conditions, but also for better understanding the fundamentals of phage biology and phagehost interactions.

2011 The Authors

Journal of Applied Microbiology 111, 245254 2011 The Society for Applied Microbiology

245

Pseudomonas-specific phages

P. Knezevic et al.

Recently, a number of Ps. aeruginosa-specific phages

have been isolated, some of them possessing a broad
activity range against various strains of this species
(Jensen et al. 1998; Knezevic et al. 2009). This characteristic is desirable from the aspect of phage application as
biocontrol agents, as phages are generally restricted in
their interactive range (Carlton 1999). However, further
examination is needed to evaluate conditions in which
the phages can be applied most efficiently.
This study was undertaken to investigate Ps. aeruginosa-specific Podoviridae and Siphoviridae phage adsorption
and inactivation under various environmental conditions and to determine their ability to inhibit bacterial
growth and biofilm formation.

Phage DNA extraction and RFLP profiling

The purified phage suspensions were treated with DNase
I (10 U) at 37C for 2 h. After DNase I inactivation
(65C, 1 h), the capsids were disintegrated using proteinase K, EDTA and SDS. The phage DNAs were extracted
by phenol chloroform method (Sambrook and Russell
2001) and precipitated by standard ethanol procedure.
The isolated DNAs were digested by EcoRI, EcoRV and
BamHI (Fermentas, Burlington, Canada) following the
manufacturers recommendations. The obtained fragments were visualized after 1% agarose gel electrophoresis
with ethidium bromide and illumination by UV light.
Adsorption at various temperatures

Material and methods

Bacterial hosts and culture conditions
Pseudomonas aeruginosa reference strain ATCC 9027 as
well as two environmental strains designated as PA-4U
and PA-M2 was used as phage original hosts. The strain
PA-4U was isolated from activated carbon (Knezevic and
Petrovic 2008) and PA-M2 from river water. The bacteria
were stocked in LuriaBertani broth (LB) containing glycerol (v v 10%) at )70C. For all the experiments, they
were cultivated in LB at 37C, stored at 4C and periodically transferred.
Phages
Four Ps. aeruginosa-specific phages, previously isolated
from water samples and partially characterized, were
examined in the study. The phages were designated as d,
J-1, r-1 and 001A. The phage d belonged to family
Podoviridae and was isolated using PA-4U strain while
the phages J-1, r-1 and 001A exhibited characteristics of
family Siphoviridae. Two of these phages, J-1 and r-1,
were propagated on ATCC 9027, while 001A was isolated
using a Ps. aeruginosa cocktail and further multiplied on
the strain PA-M2, considered as its original host in this
study. The phages d and 001A possessed a broad activity
range when tested against 33 Ps. aeruginosa strains, phage
r-1 exhibited a moderate and J-1 showed a narrow lytic
spectrum (Knezevic et al. 2009). The phages were propagated on appropriate bacterial host lawns, concentrated
by NaCl and PEG 6.000 precipitation and purified by
CsCl equilibrium ultracentrifugation at 110 000 g for
24 h in a Beckman Ti50 rotor (Sambrook and Russell
2001). After dialysis, phage counts were determined using
double-agar overlay method (Carlson 2005) and the prepared stocks were stored at 4C. The phage final count in
the experiments was 1 106 ml)1 unless stated otherwise.
246

Prior to the adsorption experiments, phage survival at 7,

18, 26, 37 and 44C for 30 min in SM buffer
(50 mmol l)1 Tris-HCl [pH 75], 01 mol l)1 NaCl,
8 mmol l)1 MgSO4, 0012 w v gelatin) was determined
using double-agar overlay method and their growth
parameters, including latent periods, were estimated by
single-step growth method (Carlson 2005). As phages survived the temperature treatments without significant PFU
reduction and their latent periods were approx. 60 min
for phage d, 75 min for r-1 and J-1 and 90 min for
phage 001A, their adsorption rates at the above temperatures were determined as follows. The phages and bacterial suspensions were mixed in SM buffer at a multiplicity
of infection (MOI) 001 and incubated at various temperatures for 30 min. The mixtures were centrifuged
(10 000 g, 15 min), and unabsorbed phage counts in
supernatant were determined. Phage adsorption rates
were expressed as percentages of adsorbed phages in relation to the initial phage counts. The data were plotted
and fitted with exponential curves using software Origin
6.0 (Microcal Software Inc., Northampton, MA).
Inactivation at various pH values
Phage suspensions were added in SM buffer whose pH was
adjusted to 15, 3, 5, 7 and 9 and incubated at 37C for
30 min. After incubation, the phage suspensions were
immediately serially diluted in SM buffer (pH 74) and
phage titres were determined. The phage survival rates were
expressed as percentages of viable phages in suspensions.
Phage neutralization by carbohydrates and amino acids
To determine phage potential receptors, the modified
method of neutralization was used (Valyasevi et al.
1990). Briefly, phages were incubated at 37C for
30 min in SM buffer amended with glucose, rhamnose,

2011 The Authors

Journal of Applied Microbiology 111, 245254 2011 The Society for Applied Microbiology

P. Knezevic et al.

mannose, galactose, glucosamine, glutamine or alanine

(final concentrations 500 mmol l)1). Phages incubated
in SM buffer without any compound served as a
control. The mixtures were diluted, assayed for plaques,
and the percentages of phage neutralization were calculated. A compound was considered as a component of
phage receptors if it neutralized at least 50% of bacteriophages (PhI50) in comparison with the corresponding control.
Phage neutralization by exopolysaccharides (EPS) and
lipopolysaccharides (LPS)
EPS of host strains were isolated after 10-day incubation
on MacConkey plates with 5% glycerol using a slightly
modified method described by May and Chacrabarty
(1994). Briefly, the biomass was suspended in 09% KCl,
vortexed and centrifuged (3000 g for 20 min at 4C). The
supernatant was treated by absolute ethanol, and after
overnight precipitation at 4C, EPS were pelleted
(17 000 g 1 h). The EPS were resuspended in 70% ethanol, centrifuged, air dried and finally freeze-dried. For
LPS isolation, previously prepared and lyophilized host
cell walls were treated by hot phenol (45%) at 74C for
20 min (Bartell et al. 1971). Following the procedure, the
mixture was subsequently cooled on ice, centrifuged
(2000 g, 1 h at 4C), the aqueous phase was dialysed for
4 days at 4C and lyophilized.
For neutralization experiments, the weight of dried EPS
and LPS was measured and suspended in sterile distilled
water to obtain twofold concentrations from 019 to
200 lg ml)1. The phages were incubated at 37C for
30 min with appropriate concentration of the original
host cell components and titred. Phages incubated in SM
buffer without any compound served as a control. The
cell components were considered receptors if the applied
concentration reached PhI50.
Phage inactivation by silver nitrate and povidone-iodine
The influence of silver nitrate and povidone-iodine on
phage viability was determined by mixing phage suspensions in SM buffer with appropriate volumes of 1% silver
nitrate or 10% povidone-iodine to obtain the final
concentration of silver nitrate 0003, 00165, 003, 01, 02
and 05% and povidone-iodine 05, 1, 25, 5 and 75%.
The mixtures were incubated for 30 min at 37C, neutralized by sodium thiosulfate sodium thioglycolate solution
for 20 min (Tilton and Rosenberg 1978) and serially
diluted in SM buffer for phage titring. The logit values of
phage survival rates were plotted against the log10 of the
compound concentrations using software Origin 6.0, and
PhI50 values were calculated.

Pseudomonas-specific phages

Ex vivo experiments
Ex vivo experiments were carried out using polled sera of
12 nonimmunized, female Wistar rats 52 days old. The
phage inactivation was examined in native serum at 37C
for 30 min, as well as in serum with previously inactivated system of complement at 56C for 30 min. Following the phage incubation in the serum, the suspensions
were serially diluted in SM buffer and phage titres were
determined. In parallel, the phage adsorption to bacterial
host cells was determined in mixture of bacteria and
phages in native serum and the serum with inactivated
complement, as described for adsorption at various temperatures. The statistical difference of the obtained results
was estimated using KruskalWallis test, and the null
hypothesis was that there was no significant difference
between phage inactivation in the native serum and the
serum with inhibited complement. The null hypothesis
was rejected if P 005.
Inhibition of bacterial growth and biofilm formation by
phages
In vitro lytic efficacy of the Ps. aeruginosa-specific phages
was examined against their original hosts. The phage
ability to inhibit bacterial growth was determined using
microtitre plate method with 2,3,5-triphenytetrazolium
chloride (TTC), while the inhibition of biofilm formation
by phages was examined by means of microtitre plate
method with crystal violet as described previously
(Knezevic and Petrovic 2008). Briefly, for the purpose of
bacterial growth inhibition assay, wells of flat bottom 96well microtitre plates were filled with 100 ll of inoculated
double strengthen LB and 100 ll of prepared phage dilutions in SM buffer in such a way to provide final bacterial
count 5 106 CFU ml)1 in each well and to obtain
phage counts 5 102, 5 103, 5 104, 5 105, 5 106
and 5 107 PFU ml)1 in various wells. Consequently,
phage in vitro lytic efficacy was examined at several doses
of MOI (00001, 0001, 001, 01, 1 and 10), and each
phagehost combination at specific MOIs was performed
in triplicate. The controls of plate sterility, phage suspension sterility and bacterial growth without phage addition
were also included. The plates were incubated at 37C for
18 h, each well was amended with 200 ll ml)1 of TTC
and the plates were incubated for additional 3 h. To
examine phage ability to inhibit bacterial biofilm
formation, plates were prepared in the same way as for the
bacterial growth inhibition test and incubated at 37C for
18 h. After incubation, planktonic cells were removed and
the plates were washed twice with phosphate-buffered
saline, air dried and biofilm was fixed with absolute methanol for 15 min. Upon removal of fixative and plate

2011 The Authors

Journal of Applied Microbiology 111, 245254 2011 The Society for Applied Microbiology

247

Pseudomonas-specific phages

P. Knezevic et al.

drying, biofilm was stained with 200 ll of 04% crystal

violet solution. The plates were washed, air dried and the
stain was diluted in 250 ll 33% acetic acid for 20 min.
For bacterial growth inhibition and biofilm formation inhibition, the absorbance was measured using a microtitre
plate reader (Multiskan EX; Thermo-Labsystem, Vantaa,
Finland) at 540 and 595 nm, respectively. The data
obtained in three independent experiments were calculated
and expressed as mean percentages of bacterial growth or
biofilm formation inhibition in relation to the corresponding controls without phages.

Podoviridae, whose adsorption rate was not influenced by

temperature and was almost linear, ranging from
9234 168% at 7C to 9784 021% at 44C. The
phages r-1 and J-1 showed the second adsorption pattern
the minimum of adsorption was obtained at 7C as
6503 020% of J-1 and 6811 221% of r-1 virions
were adsorbed to host cells, while their adsorption rates
were considerably higher at 37C, reaching maximum at
44C. The adsorption of phage 001A, in contrast to the
other phages, was significantly affected by temperature, as
<50% of virions adsorbed to the host cells at 7C.

Results

Influence of pH on phage viability

The ability of the phages to adsorb to original host cells

at various temperatures for 30 min is shown in Fig. 2. All
phages were able to adsorb within the temperature range
744C, and three different patterns of phage adsorption
were observed. The first pertained to the examined

BamHI

II

001-A
EcoRV

EcoRI

BamHI

II

J-1
EcoRV

EcoRI

BamHI

II

-1
EcoRV

EcoRI

The results of phage neutralization by various chemical

compounds are presented in Table 1.
The phage d was considerably neutralized by glucose,
rhamnose, glucosamine, mannose and alanine, as well as
cellular EPS. The estimated PhI50 for EPS was 308 lg ml)1
(the result is not shown). Phages r-1 and J-1 showed
mutually similar pattern of neutralization, being significantly neutralized only by the amino acid alanine.

II

BamHI

Influence of temperature on phage adsorption

Phage potential receptors

EcoRV

The restriction analysis of phage DNAs is presented in

Fig. 1. The phage d DNA was sensitive to all three
enzymes, and its size was approx. 371 79 kb. The
restriction of phage r-1 DNA revealed that it was resistant to enzyme BamHI, having size of approx.
634 21 kb. The restriction pattern of phage 001A
DNA showed partial similarity to phage r-1. Its DNA was
resistant to BamHI and was the largest (779 60 kb).
The phage J-1 genome was cut by all of the used restriction enzymes, having size of approx. 612 45 kb.

Although all phages were completely inactivated at pH 15

for 30 min and maintained maximal viability at pH 7,
their survival at pH 3, 5 and 9 was different (Fig. 3). The
phages r-1 and d were less susceptible to low pH than the
phages J-1 and 001A, because more than half of these virions survived at pH 3 and 5. Approximately half of J-1 and
001A virions maintained viability at pH 5. A high survival
level of all examined phages was observed at pH 9, and
the percentages were greater than those obtained at pH 5.

EcoRI

Phage genome characteristics

Figure 1 Restriction digestion of phage DNA with EcoRI, EcoRV and BamHI; I: k HindIII ladder; II: 1-kb ladder.

248

2011 The Authors

Journal of Applied Microbiology 111, 245254 2011 The Society for Applied Microbiology

P. Knezevic et al.

Pseudomonas-specific phages

Table 1 Phage inactivation

aeruginosa cell surface

100

with

components

of

Pseudomonas

Adsorbed phage (%)

Phage neutralization (%)

80

60

40

10

20
30
Temperature (C)

40

Figure 2 Phage adsorption rates at various temperatures for 30 min

at 37C: d (s) (R2 = 0995); J-1 (d) (R2 = 0984); r-1 (h)
(R2 = 0998); and 001A (n) (R2 = 0982). Values are the mean SD
of three determinations.

Compound*

Glucose
Rhamnose
Glucosamine
Mannose
Alanine
Galactose
Glutamine
LPS
EPS

8917
8472
8597
7912
8561
0
0
0
9420

J-1

r-1

134
491
610
794
1334

410

4823
4037
3404
2093
7583
037
1206
2135
2342

191
357
215
112
707
053
456
470
519

3032
0
0
0
5630
1757
2005
2673
4123

001A
472

758
387
478
421
274

N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.
N.D.

Values are the mean SD of three determinations.

N.D., not determined; LPS, lipopolysaccharides; EPS, exopolysaccharides.
*Concentration of 500 mmol l)1, except for LPS and EPS, for which
was used 200 lg ml)1

Table 2 Phage inactivation by silver nitrate and povidone-iodine

PhI50*

100

Phage survival (%)

80

60

Phage

AgNO3 (%)

Povidone-iodine (%)

d
r-1
J-1
001A

0070
0052
0058
0117

<05
465
488
<05

*Values are the mean of three determinations.

40

20

0
1

5
6
pH

10

Figure 3 Effect of pH on phage viability after 30 min at 37C: d (s);

J-1 (d); r-1 (h); and 001A (n). Values are the mean SD of three
determinations.

Influence of silver nitrate and povidone-iodine on phage

inactivation
Silver nitrate showed similar effect on all four phages
(Table 2). While silver nitrate did not significantly affect
phage viability at concentration 0003 and 003%,
significant reduction was observed with concentration
01%, except for phage 001A. PhI50 ranged from 0052%
of silver nitrate for phage r-1 to 0117% for the phage
001A. None of the examined phages was able to survive
the concentration of 03% of this compound during
30 min at 37C.

The examined phages showed two different patterns of

survival in the presence of povidone-iodine, as shown in
Table 2. The first group of phages, d and 001A, was
highly sensitive to this chemical, as even the lowest concentration completely inactivated them for 30 min at
37C. The second pattern of viability loss was observed in
phages r-1 and J-1 that were significantly resistant to it.
The determined PhI50 values for r-1 and J-1 inactivation
by povidone-iodine were 465 and 488%, respectively.
The povidone-iodine concentration higher than 5%
reduced more than a half of the phage viability. Nevertheless, even 75% was not sufficient for complete inactivation of J-1 and r-1 under the experimental conditions.
Effect of serum on phages
The level of phage inactivation by serum did not vary to
a great extent (Fig. 4). In the native serum, the maintained viability ranged from 6599 717% for phage J-1
to 9776 388% for phage r-1, while in the serum with
inhibited complement more than 94% of phages survived,
except for the phage J-1 (8375 1794%). However,
KruskalWallis test showed that difference between phage

2011 The Authors

Journal of Applied Microbiology 111, 245254 2011 The Society for Applied Microbiology

249

Pseudomonas-specific phages

P. Knezevic et al.

ited bacterial growth and biofilm formation for more

than a half at all MOIs. Phage J-1 was able to significantly
inhibit neither bacterial growth nor biofilm formation.
Similarly, phage r-1 significantly inhibited bacterial
growth only at very high MOIs and had no effect on
biofilm formation.

Phage survival (%)

100

80

60

Discussion
40

20

J-1

001A

Phage
Figure 4 Phage inactivation by serum of nonimmunized rats with (h)
and without (n) complement for 30 min at 37C. Values are the
mean SD of three determinations.

inactivation in serum with and without the complement

was not statistically significant (P = 0096). The adsorption of the survived phages in serum, regardless of the
complement activity, was very high and ranged from
9968% for phage J-1 to 100% for phages d and r-1 (the
results are not shown).
Bacterial growth and biofilm formation inhibition
The results of bacterial growth and biofilm formation
inhibition are shown in Fig. 5. Phages d and 001A inhib-

Four Pseudomonas-specific bacteriophages analysed in the

study were previously morphologically characterized, and
their lytic activity was determined, indicating that the
phages were different from one another (Knezevic et al.
2009). This has been confirmed in this study by obtaining
distinctive restriction patterns of their DNAs, implying
that the phages were not clonally identical. The data are
important for understanding similarities and differences
of phage response to environmental factors and their lytic
activity in vitro.
Pseudomonas aeruginosa as a euritherm organism grows
in temperature range from 10 to 44C, with an optimum
around 35C (Pitt and Simpson 2006). Accordingly, it can
be expected that the highest adsorption rates of its phages
are also in this range. It was noticed for the examined
Siphoviridae, but the absorption rates of the phage from
family Podoviridae were very high and almost identical at
both 7 and 37C. The phenomenon has already been
observed in Lactobacillus phages from family Siphoviridae
(Quiberoni et al. 2004; Capra et al. 2006), but similar
reports on Ps. aeruginosa-specific Podoviridae phages have
been absent. According to the adsorption rates at various

(a)

(b)

100

Biofilm formation inhibition (%)

100

Growth inhibition (%)

80

60

40

20

80

60

40

20

104 103 102 101 100 101

104 103 102 101 100 101

MOI (log scale)

Figure 5 Bacterial growth inhibition (a) and inhibition of biofilm formation by (b) Pseudomonas aeruginosa-specific phages; bacterial initial count
approx. 5 106 CFU ml)1; phage d against strain PA-4U (s); phage J-1 against strain ATCC 9027 (d); phage r-1 against strain ATCC 9027 (h);
and phage 001A against strain PA-M2 (n). Values are the mean of three determinations.

250

2011 The Authors

Journal of Applied Microbiology 111, 245254 2011 The Society for Applied Microbiology

P. Knezevic et al.

temperatures, all of the examined phages can be useful

as in vivo therapeutic agents, while phage d can be
considered for application as an anti-Pseudomonas agent
in environments where the temperature cannot be strictly
controlled (hospital environments, water supplies, etc.).
Despite the scarce data on Ps. aeruginosa-specific
phages viability at various pH values, the results obtained
in this study are not very surprising, because the host
bacterium grows at pH between 56 and 8, with an optimum at 72. Generally, better survival of phages at neutral
and alkaline pH, in comparison with acid pH, has already
been reported (Adams 1959). However, some phages,
such as Salmonella-specific st104 (family Siphoviridae)
and Felix 01 (family Myoviridae), can maintain viability
at 37C during 120 min in porcine gastric juice (pH 25)
without significant reduction of their titre (OFlynn et al.
2006). Although the phages examined in this study were
sensitive to acid pH in general, phages d and 001A
showed considerable stability at pH 3 for 30 min. It is
interesting to mention that the examined phage d showed
different patterns of survival at various pH in comparison
with the previously characterized temperate Ps. aeruginosa
phage F116 from the same family, which rapidly lost viability outside the pH range 411 and was more stable at
pH 5 than pH 9 (Amin and Day 1988). Accordingly, all
of the examined Ps. aeruginosa-specific phages can be
considered for topical skin wound treatment and intravenous application, with skin and blood pH being around
55 and 74, respectively. The results suggest that oral
administration of the phages should be avoided.
The examined Ps. aeruginosa-specific phages belonging
to Podoviridae and Siphoviridae varied in their inactivation and or neutralization by the examined chemical
compounds.
Pseudomonas aeruginosa phages mainly use LPS, pili or
both of these cell components as receptors (reviewed in
Hertveldt and Lavigne 2008). EPS, core oligosaccharide
and O-side chains of LPS consist of various carbohydrates, while pili are proteinaceous structures (Rocchetta
et al. 1999; Seltmann and Holst 2001). Accordingly, when
the carbohydrate compounds were tested separately, it
could be assumed that LPS may be receptors for d phage
examined in this study. However, considering the inability of PA-4U LPS to neutralize this phage and the fact
that PA-4U EPS inactivate it, we can conclude that receptor molecules for phage d are rather EPS than LPS. The
low PhI50 obtained for EPS strongly supports these
findings. Although there are reports on EPS nature of
receptors for some phages (Baker et al. 2002; Sutherland
et al. 2004; Stummeyer et al. 2005), none of them
pertains to Ps. aeruginosa-specific phages. The significant
neutralization of phages r-1 and J-1 only by amino acid
alanine, along with the lack of inactivation by carbohy-

Pseudomonas-specific phages

drates, LPS and EPS, implies that receptors for these

phages are probably proteins.
Silver compounds are frequently used as topical antimicrobials, particularly for burn treatment, and possible
combination of phages and silver ion can be of great
interest. The effect of silver ion on RNA coliphage MS2
(family Leviviridae) has been largely examined, and the
studies have shown its high sensitivity (Butkus et al.
2004; Kim et al. 2008). The high level of Ps. aeruginosaspecific phages sensitivity to silver ion was also noticed
in this study and can be explained by the fact that the
antiviral effect of silver is a result of its interaction with
both thiol groups (-SH) of cysteine in proteins (Thurman and Gerba 1989; Russell and Hugo 1994) and
guanine in DNA structure (Arakawa et al. 2001). As silver nitrate is used for the topical treatment of Ps. aeruginosa infections as 05% solution (Moyer et al. 1965),
and all the phages are sensitive to this concentration, a
potential phage combination with this antimicrobial
agent should not be considered.
Similarly, iodine is frequently used as an antiseptic, and
inactivation of viruses has mainly been examined using
RNA bacteriophages as a model for human enteroviruses.
Brion et al. (2004) examined survival of MS2 phage, GA
phage (family Leviviridae), lipid containing PRD1 (family
Tectiviridae) and UX174 (family Microviridae) in the
presence of 115 mg l)1 iodine and found that MS2
phage was the most sensitive, while PRD1 proved to be
the most resistant. Even a filamentous coliphage fd was
examined from this aspect and shown to be highly sensitive to iodine that changed structure of viral capsid proteins (Olivieri et al. 1975). However, there are no available
data on Ps. aeruginosa-specific Podoviridae and Siphoviridae inactivation by iodine. The present results with povidone-iodine indicate that the phages d and 001A were
extremely sensitive even to a minimal concentration of
this disinfectant, while phages r-1 and J-1 were more
resistant to it, with at least ten times greater PhI50 values.
Although the results may seem surprising, it should be
taken into consideration that conformational changes of
phages caused by iodine can be reversible and phages can
maintain infectivity after iodine removal (Brion and Silverstein 1999). The difference in iodine activity against the
examined viruses can be additionally explained by iodine
mode of action the disinfectant attacks sulfhydryl groups
of amino acids, tyrosine and histidine rings and oxidize
tryptophan (Hsu et al. 1966; Olivieri et al. 1975; Cramer
et al. 1976). According to our results, it seems that phages
r-1 and J-1 possess less of these amino acids in their capsids or in the structure of their adhesins. It is also worth
noting that Brion et al. (2004) suggest that MS2 phage
widely used as a model for human viruses is inferior in
relation to the others, for instance GA. As model viruses

2011 The Authors

Journal of Applied Microbiology 111, 245254 2011 The Society for Applied Microbiology

251

Pseudomonas-specific phages

P. Knezevic et al.

should be reconsidered for their applicability, phages r-1

and J-1 appear as potential candidates for the examination
of iodine effect on DNA viruses. Finally, although phage
survival in the presence of silver nitrate and povidoneiodine is not encouraging, considering their combination
for therapeutic purposes, the results are significant from
the aspect of a potential use of these chemicals in cases
when phages applied as biocontrol agents should be
removed from the environment.
The experiments with the serum of nonimmunized animals showed insignificant phage inactivation both with
the native serum and the serum with inhibited complement. The results indicate that the animals had not been
exposed to the examined phages and thus did not possess
corresponding antibodies. Similarly, KucharewiczKrukovska and Slopek (1987) detected a low level of
Staphylococcus aureus-specific phages inhibition by serum
of nonimmunized humans the specific antibodies were
found in 21% of patients suffering from staphylococcal
infections and only 11% in healthy people. The low level
of inactivation observed in this study is probably the
result of nonspecific phage neutralization with serum
components, and the complement did not play a significant role in this phenomenon. The findings contribute to
furthering the scarce knowledge on phage interaction with
macro-organisms (Letarov and Kulikov 2009) and are
encouraging for intravenous phage application for therapeutic purposes.
The examination of in vitro phage lytic efficacy
showed that only Podoviridae phage d attained more
than 95% of bacterial reduction at MOI 10, for both
bacterial planktonic growth and growth in the form of
biofilm. It is interesting to notice that the determined
receptors for this phage are EPS, which accumulating on
the bacterial cell surface, contribute to biofilm formation
(Flemming et al. 2007). This finding is of great interest,
as Ps. aeruginosa biofilms enhance bacterial resistance to
conventional antibiotics, and some of the reasons for
this lie in the fact that EPS cause antibiotic inactivation
and cell impermeability (Gilbert et al. 1997). Accordingly, the phage d shows very good potential as a
Ps. aeruginosa biofilm formation control agent. In contrast to the phage d, the other phages did not show significant potential to inhibit either bacterial growth or
biofilm formation. Although phage 001A obtained better
reduction in bacterial count in comparison with J-1 and
r-1, it failed to reach phage d efficacy. The experimental
results of phagebacteria challenge test showed that the
final outcome of lysis was more dependent on a specific
phagehost system than on the applied MOIs. Thus, it
appears necessary to examine phage in vitro lytic efficacy
for each particular phagehost system in the studies
dealing with phage application as potential biocontrol
252

agents. The failure of bacterial eradication after phage

treatment in some in vivo experiments may be a consequence of similar in vitro experiments absence, confirming usefulness of applied phages for a specific host. To
make further results on phage lytic efficacy comparable
to other studies of this kind, and therefore more valid,
we recommend the examination of in vitro lytic efficacy
for each phagebacterial system at MOI 10 and bacterial
count on the order of 106 CFU ml)1.
Finally, the examined Ps. aeruginosa-specific phages,
even those genetically related, exhibited different adsorption and inactivation patterns under various environmental conditions and in vitro lytic abilities. Taking into
consideration all of the examined characteristics, the bacteriophage d is an interesting potential anti-Ps. aeruginosa
therapeutic and sanitation agent that should be further
examined.
Acknowledgements
This study was supported by the Ministry of Science and
Technological Development of Republic of Serbia, Grant
OI 172058. We thank Emilija Nikolic-Doric, M.Sc. (Faculty of Agriculture, University of Novi Sad) for contributing to the statistical analysis, Laboratory for Animal
Physiology (Faculty of Sciences, University of Novi Sad)
for Wistar rat serum providing and Ljiljana Knezevic,
M.A. for English revision (Faculty of Sciences, University
of Novi Sad).
References
Adams, M.H. (1959) Bacteriophages. London: Interscience
Publisher, Inc.
Amin, M.K. and Day, M.J. (1988) Influence of pH value on
viability and transduction frequency of Pseudomonas
aeruginosa phage F116. Lett Appl Microbiol 6, 9396.
Arakawa, H., Neault, J.F. and Tajmir-Riahi, H.A. (2001)
Silver(I) complex with DNA and RNA studied by Fourier
transform infrared spectroscopy and capillary electrophoresis. Biophys J 81, 15801587.
Baker, J.R., Dong, S. and Pritchard, D.G. (2002) The hyaluronan lyase of Streptococcus pyogenes bacteriophage H4489A.
Biochem J 365, 317322.
Bartell, P.F., Orr, T.E., Reese, J.F. and Imaeda, T. (1971) Interaction of Pseudomonas bacteriophage 2 with the slime
polysaccharide and lipopolysaccharide of Pseudomonas
aeruginosa. J Virol 8, 311317.
Brion, G.M. and Silverstein, J. (1999) Iodine disinfection of a
model bacteriophage, MS2, demonstrating apparent
rebound. Water Res 33, 169179.
Brion, G.M., OBanion, N.B. and Marchin, G.L. (2004)
Comparison of bacteriophages for use in iodine

2011 The Authors

Journal of Applied Microbiology 111, 245254 2011 The Society for Applied Microbiology

P. Knezevic et al.

inactivation: batch and continuous flow studies. J Water

Health 2, 261266.
Butkus, M.A., Labare, M.P., Starke, J.A., Moon, K. and Talbot,
M. (2004) Use of aqueous silver to enhance inactivation of
coliphage MS-2 by UV disinfection. Appl Environ Microbiol
70, 28482853.
Capra, M.L., Quiberoni, A. and Reinheimer, J. (2006) Phages
of Lactobacillus casei paracasei: response to environmental
factors and interaction with collection and commercial
strains. J Appl Microbiol 100, 334342.
Carlson, K. (2005) Working with bacteriophages: common
techniques and methodological approaches. In
Bacteriophages: Biology and Applications ed. Kutter, E. and
Sulakvelidze, A. pp. 437487. Boca Raton, FL: CRC Press.
Carlton, R.M. (1999) Phage therapy: past history and future
prospects. Arch Immunol Ther Exp 47, 267274.
Cramer, W.N., Kawata, K. and Kruse, W.K. (1976) Chlorination and iodination of poliovirus and f2. J Water Pollut
Control Fed 48, 6176.
Flemming, H.-C., Neu, T.R. and Wozniak, D.J. (2007) The
EPS matrix: the house of biofilm cells. J Bacteriol 189,
79457947.
Gilbert, P., Das, J. and Folez, I. (1997) Biofilm susceptibility to
antimicrobials. Adv Dent Res 11, 160167.
Heo, Y.-J., Lee, Y.-R., Jung, H.-H., Lee, J., Ko, G. and Cho,
Y.-H. (2009) Antibacterial efficacy of phages towards
Pseudomonas aeruginosa infections in mice and Drosophila
melanogaster. Antimicrob Agents Chemother 53, 2469
2474.
Hertveldt, K. and Lavigne, R. (2008) Bacteriophages of Pseudomonas. In Pseudomonas Model Organism, Pathogen, Cell
Factory ed. Rehm, B.H.A. pp. 255292. Weinheim: WileyVCH, Verlag GmbH & co. KGaA.
Hsu, Y.-C., Nomura, S. and Kruse, C.W. (1966) Some bactericidal and virucidal properties of iodine not affecting infectious RNA and DNA. Am J Epidemiol 82, 317328.
Jensen, E.C., Schrader, H.S., Rieland, B., Thompson, T.L., Lee,
K.W., Nickerson, K.W. and Kokjohn, T.A. (1998) Prevalence of broad-host-range lytic bacteriophages of
Sphaerotilus natans, Escherichia coli and Pseudomonas
aeruginosa. Appl Environ Microbiol 64, 575580.
Kim, J.Y., Leea, C., Choa, M. and Yoon, J. (2008) Enhanced
inactivation of E. coli and MS-2 phage by silver ions
combined with UV-A and visible light irradiation. Water
Res 42, 356362.
Knezevic, P. and Petrovic, O. (2008) A simple microtiter-plate
method for evaluation of phage effect on Pseudomonas
aeruginosa biofilm. J Microbiol Methods 72, 114118.
Knezevic, P., Kostanjsek, R., Obreht, D. and Petrovic, O.
(2009) Isolation of Pseudomonas aeruginosa specific bacteriophages with broad activity spectra. Curr Microbiol 59,
173180.
Kucharewicz-Krukovska, A. and Slopek, S. (1987) Immunogenic effect of bacteriophage in patients subjected to phage
therapy. Arch Immunol Ther Exp (Warsz) 35, 553561.

Pseudomonas-specific phages

Letarov, A. and Kulikov, E. (2009) The bacteriophages in

human- and animal body-associated microbial communities. J Appl Microbiol 107, 113.
May, T.B. and Chacrabarty, A.M. (1994) Isolation and assay of
Pseudomonas aeruginosa alginate. Methods Enzymol 235,
295304.
Moyer, C.A., Brentano, L., Gravens, D.L., Magraf, H.W. and
Monafo, W.W. (1965) Treatment of large burns with 0.5%
silver nitrate solution. Arch Surg 90, 812867.
OFlynn, G., Coffey, A., Fizgerald, G.F. and Ross, R.P. (2006)
The newly isolated lytic bacteriophages st104a and st104b
are highly virulent against Salmonella enterica. J Appl
Microbiol 101, 251259.
Olivieri, V.P., Kruse, C.W., Hsu, Y.C., Griffiths, A.C. and
Kawata, K. (1975) The comparative mode of action of
chlorine, bromine, and iodine on f2 bacterial virus. In
DisinfectionWater and Wastewater ed. Johnson, J.D. pp.
145162. Ann Arbor, MI: Ann Arbor Science.
Pitt, T.L. and Simpson, J.H. (2006) Psudomonas and
Burkholderia spp. In Principles and Practice of Clinical
Bacteriology ed. Gillespie, S.H. and Hawkey, P.M. pp. 427
444. Chichester, UK: John Wiley & Sons, Ltd.
Quiberoni, A., Guglielmotti, D., Binetti, A. and Reinheimer, J.
(2004) Characterization of three Lactobacillus delbrueckii
subsp. bulgaricus phages and the physicochemical analysis
of phage adsorption. J Appl Microbiol 96, 340351.
Rocchetta, H.L., Burrows, L.L. and Lam, J.S. (1999) Genetics
of O-antigen biosynthesis in Pseudomonas aeruginosa.
Microbiol Mol Biol Rev 64, 523553.
Russell, A.D. and Hugo, W.B. (1994) Antimicrobial activity
and action of silver. Prog Med Chem 31, 351371.
Sambrook, J. and Russell, D.W. (2001) Molecular Cloning: A
Laboratory Manual, 3rd edn. New York: Cold Spring
Harbor Laboratory Press.
Seltmann, G. and Holst, O. (2001) Components outside the
cell wall. The Bacterial Cell Wall. Berlin, Heidelberg:
Springer.
Skurnik, M. and Strauch, E. (2006) Phage therapy: facts and
fiction. Int J Med Microbiol 296, 514.
Soothill, H.W. (1992) Treatment of experimental infections of
mice with bacteriophages. J Med Microbiol 37, 258261.
Stummeyer, K., Dickmanns, A., Muhlenhoff, M.,
Gerardy-Schahn, R. and Ficner, R. (2005) Crystal structure
of the polysialic acid-degrading endosialidase of bacteriophage K1F. Nat Struct Mol Biol 12, 9096.
Sulakvelidze, A. and Kutter, E. (2005) Bacteriophage therapy
in humans. In Bacteriophages Biology and Applications
ed. Kutter, E. and Sulakvelidze, A. pp. 381436. Boca
Raton, FL: CRC Press.
Sutherland, I.W., Hughes, K.A., Skillman, L.C. and Tait, K.
(2004) The interaction of phage and biofilms. FEMS
Microbiol Lett 232, 16.
Thurman, R.B. and Gerba, C.P. (1989) The molecular mechanisms of copper and silver ion disinfection of bacteria and
viruses. CRC Rev Environ Control 18, 295315.

2011 The Authors

Journal of Applied Microbiology 111, 245254 2011 The Society for Applied Microbiology

253

Pseudomonas-specific phages

P. Knezevic et al.

Tilton, R.C. and Rosenberg, B. (1978) Reversal of the silver

inhibition of microorganisms by agar. Appl Environ Microbiol 35, 11161120.
Valyasevi, R., Sandine, W.E. and Geller, B.L. (1990) The
bacteriophage kh receptors of Lactococcus lactis subsp.
cremoris KH is the rhamnose of the extracellular wall polysaccharide. Appl Environ Microbiol 56, 18821889.
Wang, J., Hu, B., Xu, M., Yan, Q., Liu, S., Zhu, X., Sun, Z.,
Reed, E. et al. (2006) Use of bacteriophage in the treat-

254

ment of experimental animal bacteremia from imipenemresistant Pseudomonas aeruginosa. Int J Mol Med 17, 309
317.
Watanabe, R., Matsumoto, T., Sano, G., Ishii, Y., Tateda, K.,
Sumiyama, Y., Uchiyama, J., Sakurai, S. et al. (2007) Efficacy of bacteriophages therapy against gut-derived sepsis
caused by Pseudomonas aeruginosa in mice. Antimicrob
Agents Chemother 51, 446452.

2011 The Authors

Journal of Applied Microbiology 111, 245254 2011 The Society for Applied Microbiology