Virus Research 211 (2016) 199208

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Virus Research
journal homepage: www.elsevier.com/locate/virusres

Potential of phage cocktails in the inactivation of Enterobacter

cloacaeAn in vitro study in a buffer solution and in urine samples
S. Pereira a , C. Pereira a , L. Santos a , J. Klumpp b , A. Almeida a,
a
b

Department of Biology and CESAM, University of Aveiro, Campus Universitrio de Santiago, 3810-193 Aveiro, Portugal
Institute of Food, Nutrition and Health, ETH Zurich, Schmelzbergstr. 7, 8092 Zurich, Switzerland

a r t i c l e

i n f o

Article history:
Received 27 July 2015
Received in revised form 27 October 2015
Accepted 29 October 2015
Available online 2 November 2015
Keywords:
Bacterial-phage inactivation
Phage cocktails
Enterobacter cloacae
Multidrug resistant bacteria
Urinary tract infections
Phenotypic resistance

a b s t r a c t
The objective of this study was to compare the dynamics of three previously isolated phages for Enterobacter cloacae in order to evaluate their ability to treat urinary tract infections (UTI). The phages
genomes, survival, host range, were characterized, and the host-phage dynamics was determined in
culture medium and urine samples. The presence of prophages in bacteria, host recovery and development of resistance to phage after treatment was also evaluated. The growth of the E. cloacae was inhibited
by the three phages, resulting in a decrease of 3 log. The use of cocktails with two or three phages was
signicantly more effective (decrease of 4 log). In urine, the inactivation was still effective (2 log). Both
phages were considered safe to inactivate the bacteria (no integrase and toxin codifying genes). Some
bacteria remained viable in the presence of the phages, but their colonies were smaller than those of
the non-treated control and were visible only after 5 days of incubation (visible after 24 h in the control). A high bacterial inactivation efciency with phage cocktails combined with the safety of the phages
and their long periods of survival, even in urine samples, paves the way for depth studies, especially
in vivo studies, to control urinary tract infection and to overcome the development of resistances by the
nosocomial bacterium E. cloacae.
2015 Elsevier B.V. All rights reserved.

1. Introduction
Enterobacter cloacae is a Gram-negative pathogen responsible
for respiratory tract, urinary tract and intra-abdominal infections, endocarditis, septic arthritis, osteomyelitis and skin and soft
tissue infections and these bacteria are responsible for healthcareassociated infections or nosocomial infections (Mezzatesta et al.,
2012). Nosocomial infections are acquired during hospital admission or medical procedures and have been increasing due to the
overuse of antibiotics in human and veterinary medicine. According to the latest European Centre for Disease Prevention and Control
report, from a total of 231,459 patients from 947 hospitals, the
prevalence of patients with at least one nosocomial infection was
6.0%. Of a total of 264 infections by E. cloacae reported during 2011,
94 were multi-drug resistant (ECDC, 2013).
Urinary tract infection (UTI) associated with urethral catheters
are the most common infections occurring during hospitalization, accounting for up to 40% of all nosocomial infections (Kunin,
1997). There are reports indicating a relation between catheter-

Corresponding author. Fax: +351 234372587.

E-mail address: aalmeida@ua.pt (A. Almeida).
http://dx.doi.org/10.1016/j.virusres.2015.10.025
0168-1702/ 2015 Elsevier B.V. All rights reserved.

ized patients and infections caused by E. cloacae (Tambyah and

Maki, 2000). Moreover, E. cloacae infections have the highest mortality rate compared to other Enterobacter infections (Kanemitsu
et al., 2007). Phage therapy (use of lytic phages to inactivate bacteria) can be used as an alternative to control this nosocomial
infection.
Bacteriophages or phages are viruses that are capable to infect
exclusively bacteria. Until the advent of antibiotics, phage therapy
was widely used, especially in the Eastern Europe countries. It fell
into disuse and now, due to the overuse of antibiotics and consequent appearances of resistant bacteria, a growing interest in this
therapy approach can be noted (Ackermann, 2003; Hanlon, 2007;
Sulakvelidze et al., 2001).
The selection of the appropriate bacteriophage is a critical factor to the success of the phage therapy treatment. Among the main
criteria required to select viruses for phage therapy are (i) host
range, (ii) survival in the environment; (iii) no potential for lysogenic induction and conversion and/or generalised transduction
and, of course, (iv) the efciency of bacterial inactivation (Skurnik
and Strauch, 2006).
Therapeutic phages should have a very broad host range, which
means that phages should be able to lyse the majority of the strains
of a given bacterial species (Almeida et al., 2009). Some studies that

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S. Pereira et al. / Virus Research 211 (2016) 199208

have focused on the host range of individual phages have shown

a signicant variation in phage specicity, both within and across
bacterial species. In fact, some phages that appear to be broader,
in the sense that they can infect different genera of bacteria, fail to
infect a subset of strains or species within the same genus. This
is most likely the result of both specic phage adaptations and
the subsequent evolution of bacterial resistance in some lineages,
including via transfer of plasmids, which makes difcult to decipher
specic rules concerning phage host range (Koskella and Meaden,
2013). For instance, Jensen et al. (1998) show that nine of ten phages
of Escherichia coli, Pseudomonas aeruginosa and Sphaerotilus natans
presented a broad-host-range, infecting their hosts and bacteria
from another orders (such as, Pseudomonadales, Burkholderiales
or Enterobacteriales) (Jensen et al., 1998). However, the jumbo

phage RaK2, isolated using Klebsiella sp. as host by Simoli

et al.
unas
(2013), was tested on 40 bacterial strains from different genera.
However, 39 of these bacterial strains were resistant to the phage

RaK2 (Simoli
unas
et al., 2013).
The success of phage therapy to control pathogenic bacteria
depends on viral survival and viability in the environment, maintaining their lytic attributes. Although there are some data available
about the study of the mechanisms and rates of mortality or loss
of infectivity of phages, little is known about their time of survival
in the environment. De Paepe and Taddei (2006) by comparing life
history traits of 16 phages infecting the bacterium E. coli, showed
that their inactivation rate is constant with time and negatively
correlated with their multiplication rate in the bacterial host. The
authors showed that the capsid thickness and the density of the
packaged genome account for 82% of the variation in the inactivation rate (De Paepe and Taddei, 2006). Tsonos et al. (2013) used
a cocktail of four different phages to cure avian pathogenic E. coli
(APEC) infected chickens. Although the phages in the cocktail were
able to efciently lyse the APEC strain in vitro, treated chickens did
not show a signicant decrease in mortality, lesion scores or weight
loss compared to untreated groups, even though the APEC-specic
phages could be re-isolated from the lung and heart of chickens that
were euthanized. Moreover, the re-isolated bacteria from infected
chickens had remained sensitive to the phage cocktail (Tsonos et al.,
2013).
To select phages with therapeutic potential it is essential to
assure that there is none potential for lysogenic induction and
lysogenic conversion in order to maintain their lytic characteristics, inactivating the pathogenic bacteria efciently and to avoid
the expression of genes that encoding toxins. The selection of
phages must also have in consideration a potential ability to perform generalised transduction, i.e. the transmission of resistance
genes between bacteria (Skurnik and Strauch, 2006).
Another major concern regarding the use of phages to control
infections is the emergency of phage-resistant mutants (Hyman
and Abedon, 2010). The development of phage-resistant bacteria
has been attributed to genetic changes. Genetic resistance may
result in the alteration or loss of the bacterial cell surface receptors, inhibition of phage DNA penetration, production of restriction
endonucleases, which degrade the phage DNA, clustered regularly
interspaced short palindromic repeats (CRISPR) system, among
others (Deveau et al., 2010). However, nowadays, there is a growing
recognition that bacterial populations may maintain their viability
in the presence of phage due to phenotypic resistance, remaining
genetically sensitive to the phages (Bull et al., 2014; Vieira et al.,
2012). Phenotypic resistance may be (i) inducedthe products of
phage-lysed bacteria result in a change in uninfected bacterial gene
expression, reducing adsorption; (ii) intrinsicreduced adsorption
is due to a physiological or gene expression state that exists prior to
the phage introduction; and (iii) dynamicdegradation or blocking
of bacterial receptors by phage proteins released during cell lysis
(Bull et al., 2014).

It has been stated that phage-resistance development can be

overcome by the combined use of two or more phages or phage
cocktails (Chan et al., 2013). The mutation induced by phages may
also lead to loss of pathogenic properties (Filippov et al., 2011).
Another advantage of the use of phage cocktails is the ability to
treating multiple pathogens, this feature broaden the spectrum
of action of phage therapy (Cairns et al., 2009; Chan et al., 2013;
Kunisaki and Tanji, 2010).
The objective of this work was to evaluate the effectiveness and
safety of phage cocktails in order to control UTI caused by E. cloacae.
Nowadays, a major concern regarding the use of phages to control
infections is the emergency of phage-resistant mutants and it has
been stated that resistance can be overcome by the combined use of
two or more phages. In this sense, phage cocktails were tested, and
the development of phage-resistant mutants was evaluated after
treatment. In order to evaluate the potential use of these phages to
treat UTI, the best phage cocktail was also tested in human urine
samples.
2. Material and methods
2.1. Bacterial strains and growth conditions
The bacterial strain E. cloacae used in study as phage host
was previously isolated in our laboratory (Pereira, 2014). The bacterial strains used in host range studies are listed in Table 1.
Salmonella enterica serovar Typhimurium (Salmonella Typhimurium)
(ATCC 13311 and ATCC 14028), E. coli (ATCC 25922), Aeromonas
hydrophila (ATCC 7966), Vibrio scheri (ATCC 49387), Staphylococcus aureus (ATCC 6538 and DSM 25,693), Vibrio parahaemolyticus
(DSM 27657), Vibrio anguillarum (DSM 215 97), Photobacterium
damselae damselae (DSM 7482), Shigella exreni (DSM 4782), Listeria innocua (NCTC 11288), Listeria monocytogenes (NCT C1194)
and Aeromonas salmonicida (CECT 894) were purchased from ATCC,
DSM, NCTC and CECT collection, respectively. Four S. aureus enterotoxic strains (2065 MA, 2153 MA, 2095 MA and 2043 MA) were
isolated from food (Baptista et al., 2015). Five strains of Salmonella
enterica serovar Enteriditis (Salmonella Enteriditis) were isolated
from food given by Controlvet. The other bacterial strains used
in this study were isolated in other study from water collected
in Ria Aveiro (Louvado et al., 2012). The isolate were maintained in Tryptic Soy Agar medium (TSA; Liolchem, Italy) at
4 C. Before each assay, one isolated colony was transferred to
10 mL of Tryptic Soy Broth medium (TSB; Liolchem, Italy) and
was grown overnight at 37 C. An aliquot of this culture (100 L)
was aseptically transferred to 10 mL of fresh TSB medium and
grown overnight at 37 C to reach an optical density (O.D. 600)
of 0.8.
2.2. Phage isolation and phage host range determination
Three phages (E-2, E-3 and E-4) were isolated from wastewater collected at a secondary-treated sewage plant near the
city of Aveiro (station EEIS9 of SIMRIA Multi Sanitation System of Aveiro) using E. cloacae as host according. The sewage
water was ltered sequentially by 3 m and then 0.45 m-poresize polycarbonate membranes (Millipore, Billerica, USA). The
ltrate was added to a fresh bacterial culture in double concentrated TSB (Liolchem, Italy). The mixture was incubated at
37 C for 5 h at and then centrifuged (10,000 g, 10 min). The
phage titer was determined by the double-layer method (Adams,
1959) using the centrifuged supernatant as phage suspension
and TSA (Liolchem, Italy) as culture medium. The plates were
incubated at 37 C and examined for lysis plaques after 12 h.
Two more successive single-plaque isolations were performed to

S. Pereira et al. / Virus Research 211 (2016) 199208

201

Table 1
Bacterial sensitivity and efciency of plating of E. cloacae bacteriophages on 47 bacterial strains.
Species

Efcacy of plating (PFU mL1 )

Spot test
E-2

E-3

E-4

E-2

E-3

E-4

Salmonella typhimurium ATCC 14,028

Salmonella typhimurium ATCC13311
Salmonella Enteriditis CVA
S. Enteriditis CVB
S. Enteriditis CVC
S. Enteriditis CVD
S. Enteriditis CVE
E. coli ATCC 25922
E. coli ATCC 13706
E. coli BC30
E. coli AE11
E. coli AD6
E. coli AF15
E. coli AN19
E. coli AC5
E. coli AJ23
E. coli BN65
E. coli BM62
Shigella exneri DSM 4782
Citrobacter freundii 6F
C. freundii 10I
Providencia sp.
P. vermicola
Proteus vulgaris
Proteus mirabilis
K. pneumoniae
Enterococcus faecalis
Enterococcus faecium
S.aureus DSM 25693
S. aureus ATCC 6538
S. aureus 2065 MA
S. aureus 2153 MA
S. aureus 2043 MA
S. aureus 2043 MA
Listeria innocua NCTC 11288
L. monocytogenes NCTC 1194
Vibrio parahaemolyticus DSM 27657
V. anguillarum DSM 21597
V. scheri ATCC 49387
Photobacterium damselae damselae DSM 7482
Aeromonas hydrophila ATCC 7966
A. salmonicida CECT 894
Pseudomonas aeruginosa
P. uorescens
P. putida
P. segetis
P. gingeri

+
+
+
+

+
+
+

ND
ND
0
ND
ND
ND
ND
ND
0
ND
ND
ND
ND
9.21 106
ND
ND
ND
0
6.75 104
0
0
ND
ND
ND
0
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND

0
ND
0
ND
ND
ND
0
ND
ND
ND
ND
ND
ND
1.55 101
0
0
ND
ND
1.57 102
ND
ND
ND
ND
ND
0
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND

0
ND
0
ND
ND
ND
ND
ND
0
ND
ND
ND
ND
1.00 104
ND
ND
ND
ND
2.76 102
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND

Total number of strains infected

Clear lysis zone (+) and no lysis (). ND Not determined.

obtain pure phage suspensions. Phage suspensions (109 PFU.mL1)

were stored at 4 C with 1% chloroform (nal volume) (Scharlau,
Spain).
Phage host range was determined by the spot test according to Pereira et al. (2011). Ten microliters of concentrated
phage lysate (>109 PFU.mL1 ) were dropped onto a TSA plate
overlaid with E. cloacae (108 CFU.mL1 ). The plate was allowed
to dry and incubated for 812 h. According to the clarity
of the spot, bacteria were differentiated into two categories:
clear lysis zone (+), and no lysis (). The efciency of plating (EOP) was determined for bacteria with positive spot test
(occurrence of a clear lysis zone), using the double-layer agar
method (Adams, 1959). The EOP (average PFU on target bacteria/average PFU on host bacteria) was calculated according to
Kutter (2009). For each phage, three independent experiments
were done. Bacterial susceptibility to the bacteriophages was
assayed for 47 pathogenic bacterial strains (Table 1), included in 15
genera.

2.3. Phage survival to long-term storage

Phage survival was tested in phosphate buffered saline (PBS,
137 mM NaCl (Sigma), 2.7 mM KCl (Sigma), 8.1 mM Na2 HPO4
2H2 O, 1.76 mM KH2 PO4 (Sigma), pH 7.4) at 25 C. Phage suspensions of the three phages (E-2, E-3 and E-4) were added to PBS
(estimated nal concentration 107 PFU.mL1 ) and incubated at
25 C without shaking. The phage titer was determined as described
above, at time zero and at intervals of 12 h until day 1, 24 h until
day 5, 48 h until day 9, 72 h until day 12, 120 h until day 45, and
240 h until the end of the experiment (day 185), by the doublelayer method. The plates were incubated at 37 C and examined
for plaques after 48 h. For each phage, three independent experiments were done.
Phage DNA extraction
Bacteriophage suspensions (109 PFU.mL1 ) of the three phages
(E-2, E-3 and E-4) were centrifuged 3 times at 6.000 g for 10 min.
The phage lysates were ultracentrifuged (Beckman, Optima LE-

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S. Pereira et al. / Virus Research 211 (2016) 199208

80K) at 100,000 g for 2 h at 10 C. Five hundred microliters of SM

buffer were added to the pellet and was allowed to rest for 2 h.
The suspension was then treated with DNase I (nal concentration 10 g/mL ) and RNase A (nal concentration 20 g/mL ) at
37 C for 20 min to remove any free nucleic acids contamination.
Nucleic acid extraction from phage particles was performed as
described by Grifths et al. (2000). Extraction was performed by
the addition of 0.5 mL of 2% hexadecyltrimethylammonium bromid (CTAB), 100 mM TrisHCl [pH 8], 20 mM EDTA, 1.4 M NaCl,
0.2% -mercaptoethanol, 0.1 mg/mL proteinase K (SigmaAldrich,
St. Louis, MO, USA) extraction buffer and 0.5 mL of phenolchloroform-isoamyl alcohol (25:24:1, pH 8.0. SigmaAldrich) to
the sample. The sample was lysed for 30 s in a FastPrep FP120
(BIO 101/Savant) at 5.5 m.s1 and centrifuged (16.000 g) for 5 min
at 4 C. The supernatant was pipetted into a clean vial, mixed 1:1
with chloroform-isoamyl alcohol (SigmaAldrich) and centrifuged
(16.000 g) for 5 min at 4 C. The supernatant was removed to a clean
tube and the nucleic acids were precipitated with two volumes of
30% (wt/v) polyethylene glycol 60001.6 M NaCl for 2 h at 25 C.
This mixture was centrifuged (18.000 g) at 4 C for 10 min, the pellet
washed in ice cold 70% (v/v) ethanol, centrifuged (18.000 g) at 4 C
for 10 min and then the pellet was air dried prior to re-suspension
in 30 L Tris-EDTA buffer. Nucleic acid yield was quantied in the
Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA, USA). The resulting product was electrophoresed through 0.8 % agarose gel at 80 V
for 40 min.
2.4. Whole genome sequencing and bioinformatics analyses
Ten g of DNA of each puried phage were subjected to a
Roche/454 pyrosequencing approach on a GS FLX device with
Titanium reagents. The six samples were barcoded and pooled
on 1/4 of the sequencing plate. Reads were assembled using
CLC Genomics Workbench version 7 (CLCbio, Aarhus, Denmark)
and Roche GSAssembler version 2.7 (Roche, Switzerland). Phage
genomes were automatically annotated using RAST (Aziz et al.,
2008; Overbeek et al., 2014). The homology level between the 3
phages was determined taking into account the values correspond
to percent nucleotide identity, using blast (bl2seq) on NCBI homepage. The genome sequences were deposited in GenBank under the
accession numbers KP791805 (Phage E-2), KP791806 (Phage E-3)
and KP 791,807 (Phage E-4).
2.5. Kill curves in phosphate buffered saline (PBS)
The phages E-2, E-3 and E-4 were separately tested and then
phage cocktails were tested (two or three phages mixed together
at the same concentration). The tested phage cocktails were: S-2/S3, S-2/S-4, S-3/S-4 and S-2/S-3/S-4 phages. In all assays E. cloacae
was used as host at a multiplicity of infection (MOI) of 100 (based in
preliminary studies using different MOI: 1, 10, 100 and 1000, data
not shown). All assays were performed in PBS. In order to obtain a
MOI of 100, 2.5 L of E. cloacae culture (108 CFU.mL1 ) and 20 L
of phage suspension (109 PFU.mL1 ) were added to 30 mL of TSB
medium and incubated at 37 C without agitation. For each assay,
two control samples were included: the bacterial control (BC) and
the phage control (PC). The BC was not inoculated with the phages
and the PC was inoculated with the phage(s) but without the bacteria. All controls were incubated exactly in the same conditions
as the test samples. One milliliter of test samples and of bacterial
and phage control were collected after 0, 2, 4, 6, 8, 10, 12 and 24 h
of incubation. For all assays, the phage titer was determined, in
duplicate, by the double agar layer method, after an incubation
period of 48 h at 37 C. The bacterial concentration was determined, in duplicate, in TSA medium after an incubation period of

24 h at 37 C. Three independent experiments were performed for

each condition.
2.6. Kill curves in urine
The efcacy of the three single phage suspensions and of the
most effective phage cocktail was tested in urine instead of PBS,
at a MOI of 100, in order to evaluate if this approach can be used
to treat Enterobacter cloacae UTI. The urine samples were ceded
by Laboratory of Clinical Analysis Avelab (Aveiro, Portugal). The
study was approved by the Ethical Committee of the Clinical Analysis Laboratory Avelab. Early urine samples were collected, using
the Avelab Laboratory protocol, by midstream clean-catch technique after patient daily hygiene. The initial and the end portion of
the micturition were discarded and the middle jact was collected
directly into the sterile recipient. The urine samples presented a pH
of about 6, a density of 1.021 and did not contain proteins, epithelial
cells or bacteria. The urine was previously centrifuged (10.000 g,
10 min) and ltered through a 0.45 m-pore-size polycarbonate
membrane. (Millipore, Billerica, USA). In order to obtain a MOI of
100, 2.5 L of 108 CFU.mL1 of the overnight E. cloacae culture and
20 L 109 PFU.mL1 of the phage suspensions were inoculated in
sterilized glass erlenmeyers with 30 mL of TSB medium and incubated at 37 C without agitation (test samples, samples added of
phages). For each assay, two control samples were included: BC
and PC. All controls were incubated exactly in the same conditions
as the test samples. Aliquots of test samples and of the bacterial
and phage control were collected after 0, 2, 4, 6, 8, 10 and 12 h
of incubation. For all phage therapy assays, the phage titer was
determined, in duplicate, by the double agar layer method, after an
incubation period of 48 h at 37 C. The bacterial concentration was
determined, in duplicate, in TSA medium (Liolchem, Italy) after an
incubation period of 24 h at 37 C. Three independent experiments
were performed for each condition.
2.7. Determination of the rate of emergence of bacterial mutants
resistant to bacteriophages.
Bacteria and phages were plated by the double layer agar
method and plates were incubated for 24 h. Resistant bacteria,
which grew inside the lysis plaque, were used to determine the
rate of emergence of bacterial mutants. Ten isolated colonies were
picked, inoculated into ten tubes with TSB medium, grown at 37
C for 24 h. Spontaneous mutants of E. cloacae resistant to three
phages were determined according to Filippov et al. (2011). A bacterial culture not added of phages was used as control. The averaged
colony number of mutants (obtained from the ten isolated colonies)
in 1 mL of culture (prepared from the culture with phages) was
divided by the averaged colony number of the control (prepared
from the culture without phages) (Filippov et al., 2011).
2.8. Prophage detection in the host bacterium after phage
addition
In order to evaluate if the phage was capable of lysogenic induction (i.e. has the ability to incorporate its own genome in the
bacterial genome), a test using mitomycin C was applied.
The petri plates used to determine the spontaneous E. cloacae
mutants resistant to bacteriophages were used. An isolated colony
was picked out from the lysis plaque, inoculated into tubes with TSB
medium and stress-induced with mitomycin C (Sigma Chemical, St.
Louis, MO, USA) at a nal concentration of 1 g per mL1 . Cells with
temperate phages usually result in the release of the phage (after
inducing it by mitomycin C). The samples were incubated overnight
at 37 C and centrifuged (10.000 g, 10 min). The supernatant was
checked for the presence of phages by applying the spot test. The

S. Pereira et al. / Virus Research 211 (2016) 199208

203

Fig. 1. Survival of E-2, E-3 and E-4 phages inphosphate buffered saline. Values represent the mean of three independent experiments; error bars represent the standard
deviation.

absence of a clear zone after stress inducing indicates that bacteria

have no prophages in their genome. Three independent assays were
performed.

higher with phage E-4 (2.76 102 PFU mL1 ). Detailed results are
described in Table 1.
3.2. Phage survival determination

2.9. Detection of host sensitivity to bacteriophages after one cycle

of phage contact
The petri plates used to determine the spontaneous E. cloacae
mutants resistant to bacteriophages were used. An isolated colony
was picked out from the lysis plaque, inoculated in TSB medium
and incubated for 24 h. After incubation the culture was used to do
the spot test and was also streak-plated on TSA solid medium. An
isolated colony in TSA was selected and the procedure was repeated
more 4 times. Overall 5 streak-plating steps on solid medium were
done. Three independent assays were done.

We assayed the survival of the three phages (E-2, E-3 and E-4)
in PBS to obtain stability data for long-term storage of the potential therapeutic. The results of the survival of E. cloacae phages on
PBS are represented in Fig. 4. The concentration of E-2 decreased
by two orders of magnitude in the rst 105 days. E-3 concentration decreased by two orders of magnitude in the rst 20 days
and reached a plateau until 77 days. Afterwards, the phage titer
decreased by three orders of magnitude until 156 days. E-4 concentration only decreased by one order of magnitude after 255 days
(Fig. 1).

2.10. Statistical analysis

3.2. Genome sequencing

Statistical analysis was performed using SPSS (SPSS 20.0 for

Windows, SPSS Inc., USA). Normal distributions were checked
by ShapiroWilk test. Homogeneity of variance was checked by
Levene test. The existence of signicant differences among the different phage therapy conditions was assessed by one-way analysis
of variance (ANOVA) model. For each situation, the signicance
of the differences was done by comparing the results obtained in
the test samples with the results obtained for the correspondent
control samples for the different times of each of the three independent assays. A value of p < 0.05 was considered to be statistically
signicant.

The phage genomes were sequenced with a Roche/454 pyrosequencing approach. A total of 23,955 reads were produced, with
460 bp average read length, resulting in a total of 110,342,246 bases.
Specically, 18082 reads were obtained for E-4, 115,491 reads for
E-2 and 31,628 reads for E-3. Phage genomes were de novo assembled using CLC Genomics Workbench 7 and Roche GSAssembler 2.7.
Phages E-2 (1367-fold coverage, 36,275 bp genome), E-3 (404-fold
coverage 31,522 bp genome) and E-4 (235-fold coverage, 39,142 bp
genome) were assembled with the GSAssember software.
All three genomes were very similar on nucleotide level (E2/E-3: 98%, E-2/E-4: 100%, E-3/E-4: 94%), with differences only
in small insertions and deletions (SI 1 of Supplementary information). The genomes were annotated using RAST and predicted
coding sequences were manually inspected (SI 2 of Supplementary
information). 42 coding sequences were predicted for E-2, 36 for
E-3 and 55 for E-4. None of the genomes featured any lysogenyrelated genes and it can be safely assumed that the three phages
feature a virulent lifestyle. No genes encoding toxins, virulence factors or antibiotic resistance genes were identied based on amino
acid sequence homology searches. The three phages have dsDNA
and feature signicant nucleotide homologies to Yersinia phages

3. Results
3.1. Phage host range
The three isolated phages (E-2, E-3 and E-4) on E. cloacae presented a positive spot test for E.coli and S. exneri, members of the
Enterobactereaceae family, but not for the other bacteria belonging
to the other families tested. For E. coli the EOP was higher with
phage E-2 (1.55 101 PFU mL1 ) and for S. exneri the EOP was

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S. Pereira et al. / Virus Research 211 (2016) 199208

Fig. 2. Inactivation of E. cloacae by the three phages (E-2, E-3 and E-4) in phosphate buffered saline at a MOI of 100 during the 24 h. A. Bacterial concentration: BCBacteria
control; B + PBacteria plus phage .B. Phage concentration: PCphage control; B + PBacteria plus phage. Values represent the mean of three independent experiments;
error bars represent the standard deviation.

YeO3-12, YpP-Y, YpP-R, YpsP-g, Y, G and vB YenP AP5 as well as

Salmonella phages phiSG-JL2 and Vi06 and Enterobacteria phages
T3 and T7.
3.3. E. cloacae inactivation in PBS
3.3.1. E. cloacae inactivation by single-phage suspensions
E. cloacae was challenged with the three phages, separately and
in a cocktail, in order to assess their potential as therapeutic agents.
The maximum of bacterial inactivation with the E-2 phage was 3.4
log after 4 h of incubation and after 12 h was still signicantly higher
(1.6 log) relatively to the bacterial control (p > 0.05) (Fig. 2A). The
phage concentration was constant (p > 0.05) during the 24 h of the
experiments, however, when the phage was incubated in the presence of the host, a signicant increase in phage concentration was
observed after 24 h relatively to phage control (1.1 log) (p > 0.05)
(Fig. 2B).
When the E-3 phage was tested, a maximum bacterium inactivation was observed after 4 h (3.8 log) and after 12 h the rate of
inactivation was still signicantly high (2.3 log) relatively to the
bacterial control (p > 0.05) (Fig. 2A). A signicant increase of 1.2 log
in phage concentration was observed after 24 h relatively to phage
control (p > 0.05). Phage control concentration remained constant
during the treatment from the beginning of the treatment (p > 0.05)
increasing signicantly, 1.2 log, in the presence of the host after 24 h
relatively (p > 0.05) (Fig. 2B).
The maximum of bacterium inactivation by the E-4 phage was
3.8 log after 6 h (p > 0.05) relatively to the bacterial control. After
12 h the bacterium inactivation was still high (2.5 log) relatively to
the bacterial control (p > 0.05) (Fig. 2A). The phage control remained

constant since the beginning of the treatment (p > 0.05), but the
phage concentration increased considerably in the presence of the
host, by 1.8 log, (p > 0.05) (Fig. 2B).
Phages E-2 and E-3 inactivation was similar during the treatment (p > 0.05). Compared to phage E-2 and phage E-3 the
inactivation of the bacteria by the phage E-4 was less efcient until
4 h of treatment but after 6 h it was signicantly more efcient than
the others (p > 0.05) (Fig. 2A).
3.3.2. Cloacae inactivation by phage cocktails
When the E-2/E-4 phage cocktail was used, the maximum of
bacterial inactivation was 4.3 log after 4 h of incubation. After
12 h the inactivation was 3.6 log. These results are signicantly
higher from the values obtained for the phage E-2 alone (p > 0.05)
(Fig. 3A ). The phage concentration was constant during study
period (p > 0.05) and in the presence of its host phage cocktail concentration increased signicantly (p > 0.05) (Fig. 3B) by 1 log.
When the phage cocktail E-2/E-3 was tested, the maximum of
bacterial inactivation was 3.7 and 3.5 log, respectively, after 4 h
and 12 h of incubation. The inactivation was signicantly higher
from the value obtained when phage E-2 was used alone (p > 0.05).
However, when compared to E-3 inactivation the results were not
signicantly different (p > 0.05) (Fig. 3A). The phage concentration was constant during study period in the absence of the host
(p > 0.05) and in the presence of its host the phage concentration
increased signicantly (p > 0.05) by 1 log (Fig. 3B).
Using the phage cocktail E-3/E-4 the maximum rate of the bacterial inactivation occurred within 4 h of incubation and was about 3
log. After 12 h of incubation the inactivation rate was 1.3 log. These
results were signicantly different from results obtained in the

S. Pereira et al. / Virus Research 211 (2016) 199208

205

Fig. 3. Inactivation of E. cloacae by phage cockails (E-3/E-4, E2/E3, E2/E4 and E2/E3/E4) in phosphate buffered saline at a MOI of 100 during the 24 h. A. Bacterial concentration:
BC Bacteria control; B + PBacteria plus phage .B. Phage concentration: PCphage control; B + PBacteria plus phage. Values represent the mean of three independent
experiments; error bars represent the standard deviation.

assays with both phages separately (Using the phage 0.05) (Fig. 3A).
The phages titer during the study period was constant (p > 0.05) and
there was an increasing in phage concentration in the presence of
the host during the study time (p > 0.05) of 1.3 log (Fig. 3B).
With the phage cocktail E-2/E-3/E-4 the maximum of bacterial
inactivation was 3.5 log achieved after 4 h of incubation and of 1 log
after 12 h. These results are signicantly different from the results
obtained using E-3 and E-4 alone (p > 0.05), but are similar with the
results obtained using E-2 phage (p > 0.05) (Fig. 3A). The phage titer
was constant during the study period (p > 0.05) and an increase of
1 log in phage concentration in the presence of bacterial host was
observed (p > 0.05) (Fig. 3B).
The inactivation of the bacteria by the phage cocktails E2/E3, E-2/E-4 and E-2/E-3/E-4 was similar (p > 0.05) (reductions of
3.54.3 log after 4 h of phage addition) but the phage cocktail E-3/E4 was less effective (reductions of 3.0 log after 4 h of phage addition)
than the other 3 to inactivate the bacteria (p > 0.05) (Fig. 3A).

3.4. E. cloacae inactivation in urine

We wanted to assess the properties of the phages in question
in the clinically relevant setting, therefore the killing assays were
repeated in urine. The best results were obtained with phage cocktail E-2/E-4. The maximum bacterial inactivation was 2 log 4 h after
the beginning of treatment. These results are signicantly different
from the results obtained with experiments in PBS buffer (p > 0.05)
(Fig. 4A). The phage titer was constant during the 24 h of treatment
(p > 0.05) and increased signicantly, by 1 log, in the presence of
the host (Fig. 4B).
The maximum bacterial inactivation for E-2 in urine samples
was 2 log after 4 h of treatment, these results are signicantly dif-

ferent from those obtained in PBS (reduction of 3.4 log) (p > 0.05)
(Fig. 4A). The phage titer was constant during study period
(p > 0.05), and increased signicantly (1 log) in the presence of the
host after 12 h of treatment (p > 0.05) (Fig. 4B). The bacteria inactivation by the phage E-4 was signicantly lower compared to the
results obtained using the phage cocktail E-2/E-4 and the E-2 phage
in urine (1 log after 4 h) (p > 0.05) (Fig. 4A). The phage titer was constant during the experiments (p > 0.05) and phage concentration
increased signicantly in the presence of the host bacteria (p > 0.05)
(Fig. 4B). The inactivation of the bacteria in urine samples by the
phage cocktail E-2/E4 and phage E-2 was similar (2.2 log) (p > 0.05)
but the phage E-4 (1 log) was less effective than the cocktail and
the phage E-2 (p > 0.05) (Fig. 4A).

3.5. Rate of emergence of bacterial mutants resistant to

-bacteriophages.
The frequency of emergence of resistant mutants was 3.9 104
for E-2, 5.3 104 for E-3, 4.9 104 for E-4 and 2.5 104 for E2/E-4 cocktail (Table 2). The bacterial colonies were smaller than
those of the non-phage added control and were visible only after 5
days of incubation. In the control colonies were visible after 24 h of
incubation in the same conditions.

3.6. Prophage detection in the host after phage addition

No clear lysis zones were observed after the addition of mitomycin C to the mixture of bacteria and phages, indicating that
lysogeny did not occur during the experiment.

206

S. Pereira et al. / Virus Research 211 (2016) 199208

Fig. 4. Inactivation of E. cloacae by phage E-2 and E-4 and the cocktail E2/E4 in urine at a MOI of 100 during the 12 h. (A) bacterial concentration: BCBacteria control;
B + PBacteria plus phage. (B) Phage concentration: PCphage control; B + PBacteria plus phage. Values represent the mean of three independent experiments; error bars
represent the standard deviation.

Table 2
Frequencies of E. cloacae spontaneous phage-resistant mutants.
Control sample (CFU mL1 ) 1st day
E-2
E-3
E-4
E-2/E-4

1.1
9.4
1.6
1.2

0.4 10
2.4 107
0.3 108
0.5 108
8

Sample treated with phages (CFU mL1 ) 5th day

3.9
4.3
7.9
3.1

3.7. Host sensitivity to bacteriophages after one cycle of phage

contact
After incubation in the presence of phages and at least three
streak-plating steps on solid medium, host bacterium was sensitive
to phage infection and maintained this sensitivity for more two
streak-plating steps. All isolated colonies collected after one cycle
of phage contact and three streak-plating steps on solid medium
were sensitive to the phage and showed a clear zone of lysis on TSA
medium. For the rst two streak-plating steps on TSA medium no
clear lysis zones were observed for each of the three phages.
4. Discussion
The emergence of antibiotic resistant bacteria directed more
research interest to alternative therapies. Several studies have
demonstrated that phage therapy has great potential in the inactivation of infections caused by bacteria. E. cloacae infections are of
primary importance as a cause of urinary tract infections difcult to
control (Zhanel et al., 2005). This work is the rst report on phage
inactivation of E. cloacae in the urine.
The selection of suitable phages (effective and safe) and the
demonstration of their feasibility in vivo, are among the most

3.5 104
3.9 104
9.0 104
4.2 104

Mutation frequencies
3.9 104
5.3 104
4.9 104
2.5 104

important current challenges to evaluate the potential of bacterial

inactivation by phages.
The results of this study indicate that the three phages tested
in this study were effective to inactivate Enterobacter cloacae in a
buffer solution when used individually or in phage cocktails. Nevertheless, with the exception of the cocktail E-3/E-4, the efciency
of bacterial inactivation was signicantly higher when phage cocktails were used. These results can be explained by the presence of
phages with different receptors. As phage cocktail E-3/E-4 was not
more effective to inactivate the bacteria than single phage suspensions and, this cocktail was not so effective to inactivate the host as
the other three phage cocktails, probably phage E-3 uses the same
receptor of phage E-4 to infect E. cloacae. This indicate, as suggested
before (Chan et al., 2013) that when phage cocktails are prepared
to inactivate pathogenic bacteria, it is important to consider the
receptors used by the phage to infect the host to improve bacterial
inactivation effectiveness.
One of the major advantages of phage treatment is the phage
specicity, but a terapeutic phage should be able to lyse the majority of the strains of a given bacterial specie (Almeida et al., 2009).
The phages isolated in this study, beside its host, infect also two
non-Enterobacter strains (E. coli AN19 and Shigella exneri DSM
4782), which belong to different genera of bacteria, but both per-

S. Pereira et al. / Virus Research 211 (2016) 199208

taining to the Enterobactereaceae family, and are also frequently

implied in UTI (Linhares et al., 2013). These three phages do not
infect bacteria belonging to other families. Although the three
phages infect the E. cloacae strain tested in the present work, it
would be important to investigate if these phages were also able
to infect other strains of E. cloacae, namely strains isolated from
contaminated urine samples.
The success of phages to control pathogenic bacteria also
depends on viral survival and viability in environment to maintain their lytic attributes (Pereira et al., 2011). In this study, phages
maintained their high concentrations even after several months
(68), in buffer solution and, at least, 12 h in urine. These facts suggest these phages are well adapted to be used in acid conditions,
like in the urinary tract.
The genome sequence of the three phages isolated in this
study was obtained, and no genes encoding products such as toxins, antibiotic resistance and integrase enzymes were detected by
amino acid sequence comparisons. Consequently, from our current
state of knowledge, these phages can be considered safe to be used
in phage therapy.
Another concern of bacterial inactivation by phages is the emergence of phage-resistant bacteria (Levin and Bull, 2004; Vieira et al.,
2012). In fact, the results of this study indicate that phage-resistant
mutants emerged in the presence of the three tested phages, showing, however, low rates of inactivation as already observed for
other phages (Filippov et al., 2011; Silva et al., 2013). Besides,
the application of these phages as cocktails limits the development of resistance by the bacteria. However, the colony size of
the resistant bacteria was smaller than that of the non-resistant
ones and these bacteria growth slowly. Colonies formed by resistant bacteria were visible only after ve days of incubation but the
non-resistant bacterial colonies were visible after 24 h of incubation. These results indicate that phage-resistant bacteria tend to be
less t and, consequently, it is expected that they will be eliminated
from the environment fast than their wild-type relatives. Similar
results were already registered for other bacterial hosts (Mateus
et al., 2014; Silva et al., 2016).
After three streak-plating steps on solid medium, the resistant
bacteria showed positive spot test, which was not observed for
the rst two streak-plating steps. In a rst glance, it seems to be
due to lysogenic induction. However, the experiments of lysogenic
induction showed no evidence of lysogeny (results of the mitomycin test) was observed during the phage addition experiments.
Moreover, genes codifying to integrase were not detected in the
phages genomes. This behavior can be due some genomic or phenotypic modications in the resistant cells after phage-stress stop.
More studies of the research group are ongoing in order to clarify
this comportment.
The most effective phage cocktail in the PBS experiments (phage
cocktail E-2/E-4) and both phages included in this cocktail were also
tested in human urine samples in order to evaluate the potential
application of phages to inactivate bacteria causing UTI. The phage
cocktail was effective to inactivate E. cloacae in urine. The phages
E-2 and E-4 survived in the urine, maintained their concentration
in the absence of the host, and signicantly increased their titer in
the presence of the bacterium during the study period. However,
its effectiveness to inactivate the E. cloacae in urine was considerably lower compared to that observed in the experiment in PBS,
2.0 log against to 4.3 log after 4 h, respectively. The concentration
of the bacterium in the urine controls (in the absence of phages)
was constant during the experiment, indicating that the decrease
in bacterial inactivation was not due to the urine per se, but to the
presence of the phages. Although the phage concentration was constant during the incubation period in urine, and phage replication
in the presence of the host, has been similar to that observed in TSB,
the lower bacterial inactivation in urine seems to be due to a lower

207

viability of the phages. It has been shown that although phages survive in different values of pH, the efciency of phage to inactivate
bacteria is affected by low values of pH (Silva et al., 2013), which
explain the lower inactivation of E. cloacae in urine samples than in
buffer solution.
4. Conclusion
A high bacterial inactivation efciency with phage cocktails
combined with the safety of the phages and their long periods of
survival, even in urine samples, paves the way for depth studies,
especially in vivo studies, to control urinary tract infection and to
overcome the development of resistances by the nosocomial bacterium E. cloacae. However, as urine has a low pH, affecting the
viability of the phage, a high concentration of phages should be
used in order to obtain a more effective control of uropathogenic
bacteria. The results of this study highlight the importance of testing the efcacy of phages to inactivate bacteria in clinically relevant
setting.
Acknowledgements
work
was
supported
by
FEDER
through
This
COMPETEPrograma Operacional Factores de Competitividade and by national funding through Fundaco para a Cincia e
a Tecnologia (FCT). Thanks are also given to the Centre for Environmental and Marine Studies (project Pest-C/MAR/LA0017/2013)
and to the Department of Biology of the University of Aveiro.
Financial support to Pereira C (SFRH/BD/76414/2011) and Santos L
(BPD/CESAM/PTDC/MAR-EST/2314/2012). The authors declare no
conict of interest.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.virusres.2015.10.
025.
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