2011 Phages of Pseudomonas Aeruginosa Response to Environmental Factors and in Vitro Ability to Inhibit Bacterial Growth and Biofilm Formation

ORIGINAL ARTICLE

Phages of Pseudomonas aeruginosa: response toenvironmental factors and in vitro ability to inhibitbacterial growth and biofilm formationP. 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

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 character-

ized (Skurnik and Strauch 2006). Unfortunately, phage

biological characteristics have been neglected in most stud-

ies 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 examina-

tion of their interaction with environmental factors and orin vitro lytic efficacy. Consequently, the data on Ps. aeru-

ginosa-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 funda-

mentals of phage biology and phagehost interactions.

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: [email protected]

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 bacterialgrowth and biofilm formation.

Methods and Results: The phages examined in the study were clonally differ-

ent, as revealed by RFLP. The temperature in the range 744C had no influ-ence 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 exopoly-saccharides of the original host efficiently inactivated phage d, while phages r-1and J-1 were inactivated considerably only by the amino acid alanine. Silver

nitrate efficiently inactivated all the phages, while Siphoviridae were more resis-

tant to povidone-iodine. Serum of nonimmunized rats had no influence on

phage inactivation and adsorption. Only phage d showed ability to effectivelyinhibit in vitro bacterial growth and biofilm formation.

Conclusions: The examined environmental parameters can significantly influ-

ence 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.

Journal of Applied Microbiology ISSN 1364-5072

2011 The AuthorsJournal of Applied Microbiology 111, 245254 2011 The Society for Applied Microbiology 245

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 characteris-

tic 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. aerugin-

osa-specific Podoviridae and Siphoviridae phage adsorption

and inactivation under various environmental condi-

tions and to determine their ability to inhibit bacterial

growth and biofilm formation.

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 glyc-

erol (v v 10%) at )70C. For all the experiments, theywere cultivated in LB at 37C, stored at 4C and periodi-cally 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 familyPodoviridae and was isolated using PA-4U strain while

the phages J-1, r-1 and 001A exhibited characteristics offamily 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 activityrange when tested against 33 Ps. aeruginosa strains, phage

r-1 exhibited a moderate and J-1 showed a narrow lyticspectrum (Knezevic et al. 2009). The phages were propa-

gated 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 pre-

pared stocks were stored at 4C. The phage final count inthe experiments was 1 106 ml)1 unless stated otherwise.

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 protein-ase K, EDTA and SDS. The phage DNAs were extracted

by phenol chloroform method (Sambrook and Russell2001) 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 frag-

ments were visualized after 1% agarose gel electrophoresis

with ethidium bromide and illumination by UV light.

Adsorption at various temperatures

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 determinedusing double-agar overlay method and their growth

parameters, including latent periods, were estimated by

single-step growth method (Carlson 2005). As phages sur-

vived 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 forphage 001A, their adsorption rates at the above tempera-

tures were determined as follows. The phages and bacte-

rial suspensions were mixed in SM buffer at a multiplicity

of infection (MOI) 001 and incubated at various temper-atures 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 rela-

tion 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 for30 min. After incubation, the phage suspensions were

immediately serially diluted in SM buffer (pH 74) andphage 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 for30 min in SM buffer amended with glucose, rhamnose,

Pseudomonas-specific phages P. Knezevic et al.

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

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 calcu-

lated. A compound was considered as a component of

phage receptors if it neutralized at least 50% of bacte-

riophages (PhI50) in comparison with the correspond-

ing 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). Thesupernatant 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% etha-

nol, 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 for20 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 for4 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 to200 lg ml)1. The phages were incubated at 37C for30 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 suspen-

sions 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, 02and 05% and povidone-iodine 05, 1, 25, 5 and 75%.The mixtures were incubated for 30 min at 37C, neutral-ized by sodium thiosulfate sodium thioglycolate solutionfor 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.

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 37Cfor 30 min, as well as in serum with previously inacti-

vated system of complement at 56C for 30 min. Follow-ing 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 tem-

peratures. 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 96-

well microtitre plates were filled with 100 ll of inoculateddouble strengthen LB and 100 ll of prepared phage dilu-tions in SM buffer in such a way to provide final bacterial

count 5 106 CFU ml)1 in each well and to obtainphage 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 eachphagehost combination at specific MOIs was performed

in triplicate. The controls of plate sterility, phage suspen-

sion sterility and bacterial growth without phage addition

were also included. The plates were incubated at 37C for18 h, each well was amended with 200 ll ml)1 of TTCand 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 for18 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 meth-

anol for 15 min. Upon removal of fixative and plate

P. Knezevic et al. Pseudomonas-specific phages


drying, biofilm was stained with 200 ll of 04% crystalviolet 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 inhi-

bition, 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 corre-

sponding controls without phages.

Results

Phage genome characteristics

The restriction analysis of phage DNAs is presented in

Fig. 1. The phage d DNA was sensitive to all threeenzymes, and its size was approx. 371 79 kb. Therestriction of phage r-1 DNA revealed that it was resis-tant to enzyme BamHI, having size of approx.

634 21 kb. The restriction pattern of phage 001ADNA showed partial similarity to phage r-1. Its DNA wasresistant to BamHI and was the largest (779 60 kb).The phage J-1 genome was cut by all of the used restric-

tion enzymes, having size of approx. 612 45 kb.

Influence of temperature on phage adsorption

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 adsorptionwere observed. The first pertained to the examined

Podoviridae, whose adsorption rate was not influenced by

temperature and was almost linear, ranging from

9234 168% at 7C to 9784 021% at 44C. Thephages r-1 and J-1 showed the second adsorption pattern the minimum of adsorption was obtained at 7C as6503 020% of J-1 and 6811 221% of r-1 virionswere adsorbed to host cells, while their adsorption rates

were considerably higher at 37C, reaching maximum at44C. The adsorption of phage 001A, in contrast to theother phages, was significantly affected by temperature, as

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 phage001A. None of the examined phages was able to survive

the concentration of 03% of this compound during30 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, washighly sensitive to this chemical, as even the lowest con-

centration completely inactivated them for 30 min at

37C. The second pattern of viability loss was observed inphages r-1 and J-1 that were significantly resistant to it.The determined PhI50 values for r-1 and J-1 inactivationby povidone-iodine were 465 and 488%, respectively.The povidone-iodine concentration higher than 5%

reduced more than a half of the phage viability. Neverthe-

less, even 75% was not sufficient for complete inactiva-tion 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 main-

tained viability ranged from 6599 717% for phage J-1to 9776 388% for phage r-1, while in the serum withinhibited complement more than 94% of phages survived,

except for the phage J-1 (8375 1794%). However,KruskalWallis test showed that difference between phage

10 20 30 40

40

60

80

100

Adso

rbed

pha

ge (%

)

Temperature (C)

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 SDof three determinations.

1 2 3 4 5 6 7 8 9 100

20

40

60

80

100

Phag

e su

rviva

l (%)

pH

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 threedeterminations.

Table 1 Phage inactivation with components of Pseudomonas

aeruginosa cell surface

Compound*

Phage neutralization (%)

d r-1 J-1 001A

Glucose 8917 134 4823 191 3032 472 N.D.

Rhamnose 8472 491 4037 357 0 N.D.

Glucosamine 8597 610 3404 215 0 N.D.

Mannose 7912 794 2093 112 0 N.D.

Alanine 8561 1334 7583 707 5630 758 N.D.

Galactose 0 037 053 1757 387 N.D.

Glutamine 0 1206 456 2005 478 N.D.

LPS 0 2135 470 2673 421 N.D.

EPS 9420 410 2342 519 4123 274 N.D.

Values are the mean SD of three determinations.

N.D., not determined; LPS, lipopolysaccharides; EPS, exopolysaccha-

rides.

*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

Phage

PhI50*

AgNO3 (%) Povidone-iodine (%)

d 0070

inactivation in serum with and without the complement

was not statistically significant (P = 0096). The adsorp-tion 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 (theresults 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-

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 bacterialgrowth only at very high MOIs and had no effect on

biofilm formation.

Discussion

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 optimumaround 35C (Pitt and Simpson 2006). Accordingly, it canbe 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 beenobserved 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

0

20

40

60

80

100

Phage001AJ-11

Phag

e su

rviva

l (%)

Figure 4 Phage inactivation by serum of nonimmunized rats with (h)

and without (n) complement for 30 min at 37C. Values are themean SD of three determinations.

0

20

40

60

80

100(a)

101104 103

Gro

wth

inhi

bitio

n (%

)

0

20

40

60

80

100(b)

102 100 101 101104 103 102 100 101

Biof

ilm fo

rmat

ion

inhi

bitio

n (%

)

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.



temperatures, all of the examined phages can be useful

as in vivo therapeutic agents, while phage d can beconsidered 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 opti-mum at 72. Generally, better survival of phages at neutraland 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 001Ashowed considerable stability at pH 3 for 30 min. It is

interesting to mention that the examined phage d showeddifferent patterns of survival at various pH in comparison

with the previously characterized temperate Ps. aeruginosa

phage F116 from the same family, which rapidly lost via-

bility 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 intrave-

nous application, with skin and blood pH being around

55 and 74, respectively. The results suggest that oraladministration of the phages should be avoided.

The examined Ps. aeruginosa-specific phages belonging

to Podoviridae and Siphoviridae varied in their inactiva-

tion and or neutralization by the examined chemicalcompounds.

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 carbohy-

drates, 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 phageexamined in this study. However, considering the inabil-

ity of PA-4U LPS to neutralize this phage and the fact

that PA-4U EPS inactivate it, we can conclude that recep-

tor molecules for phage d are rather EPS than LPS. Thelow 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 acidalanine, along with the lack of inactivation by carbohy-

drates, LPS and EPS, implies that receptors for these

phages are probably proteins.

Silver compounds are frequently used as topical anti-

microbials, 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. aeruginosa-

specific 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 (Thur-

man and Gerba 1989; Russell and Hugo 1994) and

guanine in DNA structure (Arakawa et al. 2001). As sil-

ver nitrate is used for the topical treatment of Ps. aeru-

ginosa 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 thepresence of 115 mg l)1 iodine and found that MS2phage 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 sensi-

tive to iodine that changed structure of viral capsid pro-

teins (Olivieri et al. 1975). However, there are no available

data on Ps. aeruginosa-specific Podoviridae and Siphoviri-

dae inactivation by iodine. The present results with povi-

done-iodine indicate that the phages d and 001A wereextremely sensitive even to a minimal concentration of

this disinfectant, while phages r-1 and J-1 were moreresistant 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 Sil-

verstein 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 caps-ids 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



should be reconsidered for their applicability, phages r-1and 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 povidone-

iodine 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 ani-

mals showed insignificant phage inactivation both with

the native serum and the serum with inhibited comple-

ment. The results indicate that the animals had not been

exposed to the examined phages and thus did not possess

corresponding antibodies. Similarly, Kucharewicz-

Krukovska 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 signifi-

cant 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 thera-

peutic purposes.

The examination of in vitro phage lytic efficacy

showed that only Podoviridae phage d attained morethan 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). Accord-

ingly, the phage d shows very good potential as aPs. aeruginosa biofilm formation control agent. In con-

trast to the phage d, the other phages did not show sig-nificant 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 experimentalresults 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

agents. The failure of bacterial eradication after phage

treatment in some in vivo experiments may be a conse-

quence of similar in vitro experiments absence, confirm-

ing 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 adsorp-

tion and inactivation patterns under various environmen-

tal conditions and in vitro lytic abilities. Taking into

consideration all of the examined characteristics, the bac-

teriophage d is an interesting potential anti-Ps. aeruginosatherapeutic 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. (Fac-

ulty of Agriculture, University of Novi Sad) for contribut-

ing 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).

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2011 Phages of Pseudomonas Aeruginosa Response to Environmental Factors and in Vitro Ability to Inhibit Bacterial Growth and Biofilm Formation