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Application of a bacteriophage cocktail to

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contamination on pig skin
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International Journal of Food Microbiology 151 (2011) 157163

Contents lists available at SciVerse ScienceDirect

International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Application of a bacteriophage cocktail to reduce Salmonella Typhimurium U288

contamination on pig skin
Steven P.T. Hooton, Robert J. Atterbury, Ian F. Connerton
University of Nottingham, School of Biosciences, Division of Food Sciences, Sutton Bonington Campus, Sutton Bonington, Loughborough LE12 5RD, United Kingdom

a r t i c l e

i n f o

Article history:
Received 3 June 2011
Received in revised form 15 August 2011
Accepted 16 August 2011
Available online 22 August 2011
Keywords:
Bacteriophage (phage)
Phage therapy
Salmonella Typhimurium U288
Biosanitization

a b s t r a c t
Multidrug-resistant Salmonella Typhimurium U288 is a signicant pathogen of pigs, accounting for over half
of all outbreaks on UK pig production premises. The potential of this serovar, and other salmonellae, to enter
the food chain during the slaughtering process requires that efforts be made to reduce the prevalence of these
bacteria at both the pre- and post-harvest stages of production. A bacteriophage cocktail (PC1) capable of lysing various Salmonella enterica serovars was designed using the broad host-range phage Felix 01, and three
phages isolated from sewage. PC1 applied to pig skin experimentally-contaminated with U288 achieved signicant reductions (P b 0.05) in Salmonella counts when stored at 4 C over 96 h. Reductions of N1 log10 unit
were observed when the ratio of phage applied was in excess of the bacterial concentration. The treatment
was found to be effective at a multiplicity of infection (MOI) of 10 or above, with no signicant reductions
taking place when the MOI was less than 10. Under these conditions U288 counts of log10 4.14.3 CFU
were reduced to undetectable levels following the application of PC1 to pig skin (N 99% reduction). These
data suggest phage cocktails could be employed post-slaughter as a means to reduce Salmonella contamination of pig carcasses.
2011 Elsevier B.V. All rights reserved.

1. Introduction
The biocontrol of bacterial pathogens via the application of virulent bacteriophages (phages) has gained increasing credibility as an
alternative to traditional antibiotic therapies (Cairns et al., 2009;
Housby and Mann, 2009; Garca et al., 2008). Phages are ubiquitous
in the biosphere (an estimated 10 31 particles), which places them as
the most abundant biological entity on Earth (O' Flaherty et al.,
2009). By outnumbering their bacterial counterparts by 10:1 in a diverse range of environments, phages and their often virulent lifecycles are implicated in destroying half of the global bacterial
population every 48 h (Hendrix, 2002; Fischetti et al., 2006). Phage
therapy has been proposed as a potential solution (Payne and Jansen,
2000; Skurnik et al., 2007; Mann, 2008) to deal with the problems
posed by the increasing number of multidrug-resistant (MDR) bacterial pathogens (Fluit, 2005; Beutin, 2006). MDR bacteria can enter the
human food chain from the use of antibiotics in farm animals, therefore in the EU, the use of antibiotics has been limited to therapeutic
applications, and their use as growth promoters is banned (Vigre et
al., 2008). However, even in the absence of the selective pressure of
antibiotics, swine reared in antibiotic-free production systems are
continually exposed to persistent MDR S. Typhimurium (Boyen et
Abbreviations: MOI, multiplicity of infection; CFU, colony forming unit; PFU, plaque
forming unit; MDR, multi-drug resistant.
Corresponding author. Tel.: + 44 115 9516119; fax: + 44 115 9516162.
E-mail address: ian.connerton@nottingham.ac.uk (I.F. Connerton).
0168-1605/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ijfoodmicro.2011.08.015

al., 2008). Bacteriophages offer the prospect of a sustainable alternative antimicrobial treatment against such pathogens since they are
compatible with food use, with the exibility that they can be applied
therapeutically or for biosanitization purposes (Skurnik and Strauch,
2006; Hanlon, 2007). In the USA, generally recognized as safe
(GRAS) status has been granted for the use of a number of phage
products as biosanitization agents on ready-to-eat foods (Monk et
al., 2010). Under EU legislation, phages are under consideration as
processing aids (Directive 89/107/EEC) and could be applied during
the manufacturing process providing any treatment residues do not
have any technological effect on the nished product. However, the
responsibility for safety in this case lies with the manufacturer as Regulation (EC) No. 178/2002 states it is their responsibility to ensure the
nal product is safe for human consumption (von Jagow and Teufer,
2007).
Virulent bacteriophages are natural predators of their bacterial
hosts that complete their lifecycle by lysis of the infected bacterium,
and it is these that are utilized in a therapeutic context (Grski and
Weber-Dabrowska, 2005; Leverentz et al., 2003; Guenther et al.,
2009,). This is in contrast to temperate bacteriophages that can form
a stable genetic relationship with the host during the process of lysogeny. The culmination of lysogeny is the integration of the bacteriophage genome into that of the host creating a stable genetic element
known as a prophage (St-Pierre and Endy, 2008; Koch, 2007). These
undesirable traits are not associated with virulent bacteriophages
which actively replicate at the expense of the bacterial population
(Summers, 2001; Allison, 2007; Villafane et al., 2008). Therefore, as

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S.P.T. Hooton et al. / International Journal of Food Microbiology 151 (2011) 157163

long as the prevailing environmental conditions permit active infection/replication cycles, then bacterial numbers should decline while
the phage population increases (Johnson et al., 2008; Matsuzaki et
al., 2005). Although bacterial resistance to infection is a welldocumented phenomenon associated with phage predation (Scott et
al., 2007a, 2007b; Cairns et al., 2009; Labrie et al., 2010), the use of a
number of different phages in combination a phage cocktail can
overcome this. Phage cocktails not only potentially provide a means
to circumvent resistance to a single phage (Cairns and Payne, 2008;
Kunisaki and Tanji, 2010) they also allow the treatment of multiple
pathogens simultaneously (Merabishvili et al., 2009).
Phage intervention strategies have been used to control various Salmonella serovars including Enteritidis and Typhimurium, with experiments highlighting their potential use for biosanitization (Sillankorva
et al., 2010; Bigwood et al., 2009; Carey-Smith et al., 2006; Goode et
al., 2003) and for phage therapy of infected animals (Fiorentin et al.,
2005; Toro et al., 2005; Atterbury et al., 2007; Wall et al., 2010). These
studies have shown that phages can be effectively utilized against
these pathogens. Most recently, a 23 log10 CFU reduction of S. Typhimurium 4232 was achieved following application of a phage cocktail
designed to reduce S. Typhimurium 4232 levels in articiallyinfected market weight swine (Wall et al., 2010). An earlier study involving broiler chickens reported reductions of S. Typhimurium 4/74
(N2.19 log10 CFU reduction) and S. Enteritidis P125109 (N4.2 log10
CFU reduction) following phage application (Atterbury et al., 2007).
Salmonella enterica serovar Typhimurium U288 is a MDR pathogen
of livestock and has consistently been identied as the most prevalent
serovar on UK pig production premises (VLA, 2009a). Also, several
deaths have been documented following an S. Typhimurium U288
outbreak in elderly patients in Denmark during 2008 (Bruun et al.,
2009). The antibiotic resistance prole of S. Typhimurium U288
covers a wide spectrum of the classes that are currently utilized by
human and veterinary medicine. A core resistance to ampicillin
(Am), chloramphenicol (C), streptomycin (S), sulphonamides (SU),
tetracycline (T) and trimethoprim (TM) AmCSSuTTm was observed
in 76% of isolates submitted to the Veterinary Laboratory Agency in
the UK in 2008 (VLA, 2009b), and as such represents a reservoir of antibiotic resistance within pig production units. The resistance prole
of S. Typhimurium U288 is similar to that of the signicant human
pathogen S. Typhimurium DT104 (AmCSSuT), which has been a
major global cause for concern since its emergence a few decades
ago (Cooke et al., 2008; Perron et al., 2007).
This study involves the application of a phage cocktail targeted to
reduce S. Typhimurium U288 levels on articially-contaminated pig
skin. The S. Typhimurium U288 strain was identied during screening
of a pig production farm known to be contaminated with Salmonella
(H. Davies, personal communication). The phage cocktail comprises
four distinct anti-Salmonella phages SH17, SH18, SH19, and
Felix 01. Felix 01, a member of the Myoviridae, was originally isolated
by Felix and Callow (1943) and has been successfully utilized in previous Salmonella phage therapy studies (O'Flynn et al., 2006; Wall et
al., 2010). Pig production environments have proved a rich source
of bacteriophage (Callaway et al., 2010), and accordingly SH bacteriophages were isolated from environmental sources (water, sewage,
pig feces/caecal content) during the course of this study.

prior to storage at 4 C. For the selection and enumeration of S. Typhimurium U288, XLD agar (Oxoid, UK) containing 50 g/mL kanamycin
(Fisher Scientic, UK) was used throughout.
2.2. Phage isolation

2. Materials & methods

SH17, SH18, and SH19 were identied during screening of

sewage efuent using S. Typhimurium WT (Rawlings) and S. Typhimurium DT104 (WT) as hosts. For phage isolation, sewage efuent
was ltered through 0.2 m Minisart lters (Sartorius Biotech, Germany) and the ltrate collected in sterile universals and stored at
4 C until required. For phage extraction from solid matter such as
pig feces, samples were used to create 10% (wt/vol) solutions in SM
buffer. To dissociate phage, the solution was incubated over night at
4 C with shaking and then processed as described above. For lawn
preparation, Salmonella 10 mL NZCYM broth cultures were prepared
and incubated overnight at 37 C with shaking. Following this, 50 L
of overnight culture was used to seed fresh 10 mL NZCYM broth
(10 mM MgSO4) which was then incubated for 2 h at 37 C with shaking. To 5 mL molten (tempered to ~50 C) NZCYM top agar (0.6% Bacteriological Agar No.1) 500 L of the required Salmonella was added,
followed by 500 L ltrate, and the mixture was poured onto
NZCYM agar plates. The plates were left to set on the bench for
20 min before being inverted then incubated overnight at 37 C. Any
plaques identied were picked using sterile pipette tips and resuspended in 500 L SM buffer (50 mM TrisHCl [pH 7.5], 100 mM
NaCl, 8 mM MgSO4:7H2O, 0.01% gelatin, pH 7.5), incubated at 37 C
for 1 h, then serial diluted in SM buffer. A 25 L volume of each dilution was then added to Salmonella/NZCYM top agar, and lawns were
prepared as described above. This process was repeated at least
three times for each phage in order to obtain single clonal isolates.
Novel phage isolates were then propagated to obtain high titre
stocks as described by Kocharunchitt et al. (2009). Briey, NZCYM
broth cultures of the required propagating host were prepared as described above. Following overnight incubation, 250 L of culture was
added to 25 mL NZCYM broth (10 mM MgSO4) in conical asks. To
each ask 25 L of the required phage was added, and asks were
then incubated overnight at 37 C with shaking. Following this, the lysate was drawn up using sterile syringes then passaged through
0.2 m Minisart lters. Aliquots of each phage stock were then 10fold serial diluted in SM buffer, followed by spotting of triplicate
10 L drops of each dilution on Salmonella lawns prepared as described above. Plates were enumerated the following day. All phage
stocks were stored at 4 C until required.
The efcacy of each phage isolate against a wide panel of Salmonella
serovars was determined as follows. Salmonella top agar lawns were
prepared as described above. To each Salmonella/NZCYM top agar
lawn, 20 L volumes of log107 PFU/mL dilutions of each puried phage
stock were applied. This approach delivered a routine test dilution of
105 PFU per spot. Following a sufcient drying period, plates were
inverted and incubated overnight at 37 C. The following day, the degree
of lysis of each phage was observed and the data obtained is presented in
Table 1. The salmonellae used during host-range determination encompass isolates from human and animal disease outbreaks, as well as NCTC
strains available for purchase. However, of note are the signicant
human (S. Typhimurium and S. Enteritidis serovars) and pig pathogens
(S. Derby, S. Kedougou, and S. Typhimurium).

2.1. S. Typhimurium U288 stock

2.3. Phage cocktail 1 (PC1)

S. Typhimurium U288 was cultured and maintained on solid

media containing NZCYM broth (Fisher Scientic, UK) supplemented
with 1.2% [wt/vol] Bacteriological Agar No.1 (Oxoid, UK). Working
stock plates were produced by streaking out a loopful of S. Typhimurium U288 15% glycerol stock (stored at 80 C) onto NZCYM agar. All
working stock plates were inverted and incubated overnight at 37 C,

High titre stocks of SH17, SH18, SH19, and Felix 01 were prepared and titrated as described in Section 2.2. The puried high titre
phage stocks were subsequently used to make a 20 mL phage cocktail
(PC1) in SM buffer with a combined titre of 10 8 PFU/mL. Any subsequent dilutions of PC1 were made in SM buffer and stored at 4 C
until required.

S.P.T. Hooton et al. / International Journal of Food Microbiology 151 (2011) 157163
Table 1
Lytic spectrum of Felix01, SH17, SH18, and SH19 on salmonellae.
Salmonella

S. Agama
S. Amina
S. Amsterdam
S. Atlanta NCTC 9986
S. Burielly NCTC 8745
S. Derby WT
S. Enteritidis SA025 PT4
S. Enteritidis SA029
S. Enteritidis WT (Harrison)
S. Enteritidis WT (Hood)
S. Enteritidis WT (Platten)
S. Hadar WT
S. Infantis NCTC 6903
S. Kedougou BP
S. Kedougou PI
S. Kubacha WT
S. Montevideo NCTC 5747
S. Montevideo WT
S. Muenster/Orion
S. Senftenburg WT
S. Thompson NCTC 2252
S. Tobga WT
S. Typhimurium DT104
S. Typhimurium LT2
S. Typhimurium WT (Rawlings)
S. Typhimurium WT (Turner)
S. Typhimurium U288
S. Virchow WT

Phage
Felix01

SH17

SH18

SH19

+++
+++
+++
+++

(+++)

+++
+++
+++

(++)

(+)

(++)
+++
+++
+++
+++
+++

(+++)
(++)
+++
(++)
(++)

(+)

+++
+++
+++
+++
+++

(++)
(++)
+++
(++)
(++)

(++)
(++)
(++)
(++)
(++)

(++)

(++)
+++
(++)

(+++)

+++ conuent lysis, ++ semi-conuent lysis, + individual plaques, () opalescent lysis.

2.4. Sizing of phage genomes

Pulse eld gel electrophoresis (PFGE) was used to determine phage
genome sizes. Briey, 1 mL of phage stock (10 910 11 PFU/mL) was
centrifuged for 2 h at 34,900 g using a JA18.1 rotor and Beckman
J2-21 centrifuge. Following this the supernatant was carefully decanted and the pellet resuspended in 50 L phage stock. To each tube,
10 L of 10 mg/mL proteinase K (Sigma Aldrich, UK) was added followed by mixing with an equal volume of molten 1.2% PFGE grade agarose (Biorad, USA) in TE (10 mM TrisHCl [pH 7.5], 1 mM EDTA)
buffer. The suspension was thoroughly mixed before being dispensed
into PFGE plug molds (Biorad, USA) and allowed to solidify. Each plug
was then incubated in 1 mL lysis buffer (50 mM TrisHCl [pH 8],
50 mM EDTA, 1% N-lauroyl sarcosine, 100 g/mL proteinase K) at
55 C overnight. Plugs were then washed 3 times for 15 min in 1 mL
wash buffer (20 mM TrisHCl [pH 8], 50 mM EDTA) at 55 C. A 3 mm
slice from each plug was loaded onto a 1% PFGE agarose gel (100 mL
1 TAE buffer) along with 0.1200 kb and 501000 kb DNA pulse
markers (Sigma Aldrich, UK), and the lanes sealed with molten 1%
PFGE grade agarose. A CHEF-DR II system was used to run the gel
with a switch time of 1030 s over 17 h at 6 V/cm, with 1 TAE as
the running buffer (circulating at 14 C). After electrophoresis the
gel was stained for several hours with ethidium bromide (1 g/mL)
and visualized with a Biorad Image Capture (Biorad, USA). A standard
curve of migration of marker DNA was used to approximately calculate the genome size of each phage.
2.5. Transmission electron microscopy (TEM) of phage
Freshly-prepared high titre phage suspensions of each phage were
sedimented at 34,900 g for 2 h (4 C). Following centrifugation, the
supernatant was decanted and each phage pellet was washed twice
with 0.1 M ammonium acetate for 1 h at 25,000 g. The wash solution was discarded and 2 mL SM buffer added to each centrifuge
tube. Phage pellets were recovered following overnight incubation

159

at 4 C with gentle shaking. A small drop of washed phage suspension

was spotted onto a carbon-coated copper mesh grid and allowed to
sit for 3 min. Excess phage suspension was then removed with lter
paper. For negative staining one drop of phosphotungstic acid [pH
7.4] was added to each grid, and excess stain was removed 1 min
later with lter paper. Each grid was then covered and allowed to
dry for 15 min. Images were taken with a Fei Tecnai Biotwin TEM
(Fei Company, USA).
2.6. Porcine sample preparation
Skin sections from each 25 day-old pig (7.5 to 8 kg) carcasses
were removed post-mortem using an ethanol ame-sterilized scalpel.
Large sections of pig skin were then cut into approximately 4 cm 2
pieces which were subsequently placed in sterile 95 mm Petri dishes
(Sarstedt, Germany). A SALMOTYPE Pig Screen ELISA (Labor Diagnostik, Germany) was performed as per manufacturer's instructions
to conrm that the pigs were serologically Salmonella negative at
slaughter.
2.7. Survival and recovery of S. Typhimurium U288 on articiallycontaminated pig skin stored under fresh (4 C) and frozen (20 C)
storage conditions
To determine the effect of fresh (4 C) and frozen ( 20 C) storage conditions on the survival and recovery of S. Typhimurium
U288 on articially-contaminated pig skin, a small-scale sampling regime was prepared. Briey, triplicate 4 cm 2 pieces of pig skin were inoculated with log10 5.4 CFU/cm 2 S. Typhimurium U288 and the
inoculum was uniformly spread over each sample using sterile disposable spreaders. A drying period of 1 h was allowed before the
rst set of triplicate pieces of pig skin were added to 20 mL volumes
of MRD (Oxoid, UK) in sterile lter stomacher bags (Seward, UK). At
this point all remaining samples were stored under the appropriate
storage conditions (4 C or 20 C) until required. Samples were
subjected to stomaching for 3 min at 300 rpm using a Seward Lab
Blender 400. Serial dilutions of the stomachate were then performed
using MRD, prior to spread plating triplicate 100 L volumes of each
dilution onto XLD agar (50 g/mL kanamycin). Following overnight
incubation at 37 C, mean levels of S. Typhimurium U288 recovered
were recorded and standard deviations calculated. This process was
repeated every 24 h for the duration of the experiment. The data
obtained was used to plot a graph (Fig. 2A and B) of mean log10
CFU/cm 2 recovered (SD) of S. Typhimurium U288 over time, for
fresh and frozen storage conditions.
2.8. Survival and recovery of PC1 on articially-contaminated pig skin
stored under fresh (4 C) and frozen (20 C) storage conditions
A series of 4 cm 2 pieces of pig skin were inoculated with log10 6.4
PFU/cm 2 of PC1 which was then spread out evenly using sterile disposable spreaders. A drying period of 1 h was allowed before the samples were transferred to the appropriate storage conditions (4 C or
20 C). To recover PC1 from pig skin sections, samples were placed
in sterile lter stomacher bags containing 20 mL SM buffer prior to
stomaching for 1 min at 300 rpm (Seward Lab Blender 400). One mL
of stomachate was removed and ltered through 0.2 m Minisart lters, and the ltrate collected in a sterile tube. The ltrate was then
serial diluted in SM buffer and plaque assays were performed as follows (adapted from Santos et al., 2009). Molten 5 mL NZCYM/5% glycerol top agar (0.6% Bacteriological Agar No.1) tempered to ~ 50 C was
inoculated with 100 L S. Typhimurium U288/NZCYM broth overnight culture. Diluted ltrate (100 L) was then added to the top
agar and the mixture poured onto NZCYM/5% glycerol plates. Each
plate was rotated gently to ensure an even bacterial lawn, allowed
to dry on the bench for 20 min, then inverted and incubated overnight

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S.P.T. Hooton et al. / International Journal of Food Microbiology 151 (2011) 157163

Table 2
A 3 3 matrix used to create a range of MOIs to monitor the therapeutic effect of PC1
on 4 cm2 pig skins articially-contaminated with S. Typhimurium U288.
S. Typhimurium
U288 inoculum (CFU)
6

10
104
103

Phage inoculum (PFU) and MOI

107

105

104

10
1000
10,000

0.1
10
100

0.01
1
10

30 min later. Control samples of non phage-treated S. Typhimurium

U288 (10 6, 10 4, 10 3 CFU) were also prepared to allow comparisons
with the phage-treated samples. Following phage inoculation a
30 minute drying period was allowed prior to samples being placed
in storage at 4 C. Skin sections were then processed to determine
the number of S. Typhimurium U288 in each sample, as described
above. The data obtained was log10 transformed and analyzed using
a one-tailed t test from the data analysis kit in Microsoft Excel 2007.
3. Results

at 37 C. Following overnight incubation, recoverable mean PFU (SD)

was calculated for the triplicate samples. This process was repeated
every 24 h for the duration of the experiment. The data obtained
was used to plot a graph (Fig. 2A and B) of mean log10 PFU/cm 2
recovered (SD) of PC1 over time, for fresh and frozen storage
conditions.
2.9. Bacteriophage-mediated biosanitization of articially-contaminated
pig skin
A 3 3 matrix (Table 2) of PFU against CFU was designed to cover
a range of multiplicities of infection (MOIs). MOI is used to describe
the ratio of phage to bacteria and in this instance ratios ranging
from 0.01 to 10,000 were employed. The matrix was then used to analyze the effects of administering various phage titres (10 7, 10 5, and
10 4 PFU) to a range of S. Typhimurium U288 concentrations (10 6,
10 4, and 10 3 CFU). Sampling took place at three separate time points
over a 96 hour period (1 h, 42 h, and 96 h) with the initial sampling
taking place 1 h post-inoculation. Triplicate 4 cm 2 pig skin samples
were prepared as described in Sections 2.7 and 2.8 with S. Typhimurium U288 being administered to the samples rst, followed by PC1

3.1. Selection and characterization of bacteriophage

A number of novel phage isolates were tested to identify potential
candidates for use as biosanitization agents against Salmonella. The
ability of each bacteriophage to lyse a panel of Salmonella in vitro
allowed the selection of three phages SH17, SH18, and SH19
(Table 1). These phages displayed broad activity against a range of
Salmonella, and along with Felix 01 (noted for its anti-Salmonella
properties) were selected for use in a phage cocktail (PC1). All three
SH phages were found to be capable of lysing a number of different
serovars: SH17 12/28, SH18 11/28, and SH19 11/28. The
genome sizes of the component bacteriophage of PC1 were then estimated by PFGE: SH17, SH18, and SH19 were found to be ~40 kb,
~48.5, and ~ 155 kb, respectively, while an estimate of 86.1 kb for
Felix 01 was consistent with the sequenced genome of 86,155 bp
(Whichard et al., 2010). TEM images of SH17 and SH18 (Fig. 1)
revealed a siphovirus type structure (icosahedral heads and exible
non-contractile tails), whereas SH19 had structure indicative of
the Myoviridae family (icosahedral head with contractile tail) that includes T4 and the T4-like phages. Phage isolates not selected for this

Fig. 1. TEM images of SH17 (A) at 87,000 magnication bar= 200 nm, SH18 (B) at 160,000 magnication bar = 100 nm, and SH19 (C and D) at 135,000 magnication
bar = 100 nm.

S.P.T. Hooton et al. / International Journal of Food Microbiology 151 (2011) 157163

study were discarded on the following criteria: the inability to clear

plaque on relevant hosts; deviations from the efciency of plating
using the routine test dilution of 10 7 PFU/mL.
3.2. Recovery of S. Typhimurium U288 and PC1 from pig skin stored at
4 C and 20 C
The survival and recovery of S. Typhimurium U288 on
experimentally-contaminated pig skin sections stored at 4 C and
20 C were monitored over a 9-day period. S. Typhimurium U288
recovery from an initial inoculum of log10 5.4 CFU/cm2 was followed
by a minor decrease in recoverable U288 on days 23, and thereafter
remained relatively stable throughout the remainder of the experiment (Fig. 2A and B). Evidently any S. Typhimurium U288 that remain
post-slaughter or that re-contaminate the skin surface will remain a
source of contamination throughout retail, and therefore a potential
hazard to the consumer. The stability and viability of the bacteriophage preparation were also monitored over a 9-day period at 4 C
and 20 C. From an initial titre of log10 6.4 PFU/cm 2, the phage fell
to log10 4.2 PFU/cm 2 1 h post-inoculation, and thereafter remained
stable with recoverable phage titres ranging between log10 4.1
4.4 PFU/cm 2.
3.3. Bacteriophage biosanitization of pig skin
PC1 was applied to a range of S. Typhimurium U288 concentrations
on pig skin at various MOIs (Table 2), and monitored over a 96-hour
period at 4 C. The data obtained was used to analyze reductions on
phage-treated skin sections, as compared with untreated S. Typhimurium U288-only controls. S. Typhimurium U288 could not be detected
below 10 2 CFU without prior enrichment. Therefore, XLD (50 g/mL

Mean log10 recoverable

CFU/PFU/cm2 (4C)

5.4
5.2
5
4.8
4.6
4.4
4.2
4

161

kanamycin) plates containing no recoverable S. Typhimurium U288

during sampling were assumed to be b2 log10 CFU. Table 3 shows
the S. Typhimurium U288 counts recovered throughout the duration
of the experiment with control inoculums standard deviations.
The application of PC1 produced signicant reductions 1 h postinoculation, with the largest reduction recorded the combination of
10 7 PFU PC1 and 10 4 CFU S. Typhimurium U288 (MOI 1000). This
combination resulted in a 1.2 log10 CFU reduction (log10 3.5 0.1
CFU) when compared with untreated controls (log10 4.7 0.2 CFU).
In this instance a 92.6% reduction in recoverable S. Typhimurium
U288 CFU (P b 0.01) was observed from the sampled pieces of pig
skin. A 1.0 log10 CFU reduction resulted from an MOI of 10 (10 5
PFU/10 4 CFU) reducing S. Typhimurium U288 numbers from log10
4.7 0.2 CFU (untreated controls) to 3.7 0.2 CFU recovered
(phage-treated samples) that corresponds with a 92.1% reduction of
recoverable S. Typhimurium U288 CFU (P b 0.01). By comparison
MOIs of 100 and 1000 with 10 3 CFU inoculums produced reductions
of 92.1% and 91.9% respectively in the recoverable S. Typhimurium
U288 counts (P b 0.05). No reductions were observed on any of the
samples with an MOI of less than 10.
At day 3, treatment with an MOI of 10 (10 7 PFU/10 6 CFU) resulted
in a log10 1.3 CFU reduction in recoverable S. Typhimurium U288
counts (log10 5.0 0.3 CFU) compared with untreated controls
(log10 6.3 0.1 CFU) that is equivalent to a 92.3% reduction
(P b 0.001). The application of PC1 at an MOI of 1000 (10 7 PFU/104
CFU) resulted in a reduction of log10 1.4 CFU (96.1%) from the control
count of log10 CFU 4.3 0.1 (P b 0.01). Notably the combination of 10 4
PFU and 10 3 CFU inoculated on pig skin resulted in no detectable S.
Typhimurium U288 on any of the triplicate samples, implying a
Nlog10 2 CFU reduction compared to the control value of log10 CFU
4.0 0.2. As with day 1, no signicant reductions were observed
when MOIs of less than 10 were used.
Day 5 had a number of reductions of log10 1.0 CFU or greater. No S.
Typhimurium U288 could be recovered from two combinations with
initial inoculums of 10 3 CFU and PC1 corresponding with MOIs of 10
and 100. The bacterial counts on these skin sections fell below the
level of detection implying reductions N99% compared with the
untreated controls (log10 CFU 4.3 0.2). Consistent with the results
of the previous time point only the sample containing 10 7 PFU produced a signicant reduction (P N 0.001) with the 10 6 CFU S. Typhimurium U288 inoculums, which corresponds with an MOI of 10. All
samples treated with an MOI of less than 10 showed no signicant reductions in S. Typhimurium U288 counts recovered compared to
controls.

3.8
1

24

48

72

96

120

144

168

192

216

Sample point (hours)

5.4

Mean log10 recoverable

CFU/PFU/cm2 (-20C)

5.2
5
4.8
4.6
4.4
4.2
4
1

24

48

72

96

120

144

168

192

Table 3
Mean log10 CFU counts ( standard deviation) of S. Typhimurium U288 recovered from
experimentally-contaminated 4 cm2 pig skin sections of control and PC1 treated samples (ND = not detectable; *P N 0.01 and **P N 0.001 show signicant differences compared to control values).
U288 inoculum
(CFU)

Phage inoculum (PFU)

Untreated
controls

Sample time:

107

105

104

1h
106
104
103

6.2 0.1
3.5 0.1*
3.8 0.1

6.1 0.2
3.7 0.2*
3.3 0.4

6.2 0.2
4.6 0.1
3.4 0.1*

6.2 0.1
4.7 0.2
4.2 0.2

48 h
106
104
103

5.0 0.1**
2.9 0.4*
3.6 0.2

5.9 0.2
3.9 0.1*
3.6 0.4

6.5 0.2
4.1 0.1
ND

6.3 0.1
4.3 0.1
4.1 0.2

96 h
106
104
103

5.5 0.2*
3.2 0.3*
2.8 0.7

6.6 0.2
3.4 0.2*
ND

6.7 0.1
4.1 0.4
ND

6.5 0.2
4.5 0.1
4.3 0.3

216

Sample point (hours)

Fig. 2. Mean log10 recoverable S. Typhimurium U288 CFU/cm2 () and PC1 PFU/cm2
() ( standard deviation) following storage at 4 C (A) and 20 C (B).

162

S.P.T. Hooton et al. / International Journal of Food Microbiology 151 (2011) 157163

4. Discussion

References

The data obtained from this phage therapy trial provides a proof of
principle that the application of a suitable phage cocktail (PC1) can reduce levels of S. Typhimurium U288 (the most prevalent serovar
found in pigs) on articially-contaminated pig skins. A number of
factors were identied during the trials which may be of signicance
during future studies. The use of MOIs in excess of the bacterial concentration appears to be of great relevance to the outcome of the treatment.
In each instance where a signicant reduction in S. Typhimurium U288
was observed, PC1 was administered in excesses ranging from 10 to
10,000. Reductions to below detectable levels were observed on a number of occasions when the MOI was between 10 and 100 and the initial
S. Typhimurium U288 inoculum was 103 CFU. PC1 shows greater efcacy against low levels of S. Typhimurium U288 contamination, as might
be anticipated for S. Typhimurium contaminations of post process pig
carcasses. Moreover the application of phage preparations like PC1
would complement biosecurity measures targeted to reduce the exposure of the consumer and the health of pigs in meat production. Pigs
can be susceptible to low levels of Salmonella exposure, for example, it
is reported that N103 S. Typhimurium HL10969 cells were required to
induce acute salmonellosis infection in pigs (Loynachan and Harris,
2005).
In summary, the application of a phage cocktail produced signicant reductions of S. Typhimurium U288 on experimentallycontaminated pig skin; however this appears to be linked to an MOI
in excess of the target bacterium. When applied at an MOI of 10 or
above, PC1 is capable of reducing S. Typhimurium U288 up to 2
log10 units over the course of 96 h. Little or no reductions were observed
when PC1 was applied at an MOI of 1 or less. This suggests that when
PC1 is used as a decontamination agent the therapeutic effect observed
is passive. Under these conditions the initial phage dose is sufciently in excess of the target bacterium population to cause reductions
without the need for the bacteriophage to replicate and complete
their life cycle. In contrast active therapy involves phage infection/
replication cycles to reduce the target bacterium. One of the major
advantages of passive therapy is a reduction in the likelihood of the
development of bacterial resistance. Over the course of active therapy, initial low numbers of resistant bacteria may rise in abundance
eventually replacing the original susceptible population. This process has been well-documented during in vitro and in vivo studies
(Cairns and Payne, 2008). However, the single-hit approach utilized
here greatly limits opportunities for the target bacterium to develop
resistance. Due to the commercial storage conditions adopted in this
study (4 C), the growth of Salmonella is greatly reduced or halted,
which will either prevent or at least slow phage replication. The
low temperature required for meat storage does not impede the passive action of the bacteriophage. This study has set the ground work
for in vivo trials to reduce S. Typhimurium U288 levels in pigs and
their production environments via the application of bacteriophage as
therapeutic and biosanitization agents. Of the bacteriophage examined
here SH19 and felix01 have broad spectrum activities, and conform to
the virulent phage families that are preferred for commercial application. Further in vitro phage therapy work will involve the application
of bacteriophage to reduce levels of S. Typhimurium U288 and
S. Typhimurium DT104 on various foods and fomites.

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Acknowledgments
This work was funded by the Food Standards Agency, UK (Postgraduate Scholarship Scheme PG1021). Many thanks to Helen Davies
(S. Typhimurium U288) and Melanie Le Bon for porcine materials
used during this study, and Dr Denise Christie (AMU, Queens Medical
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