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IJFM U288 Therapy Paper
IJFM U288 Therapy Paper
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Steve Hooton
Robert J Atterbury
Novolytics
University of Nottingham
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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
158
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
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
+++
+++
+++
+++
(+++)
+++
+++
+++
(++)
(+)
(++)
+++
+++
+++
+++
+++
(+++)
(++)
+++
(++)
(++)
(+)
+++
+++
+++
+++
+++
(++)
(++)
+++
(++)
(++)
(++)
(++)
(++)
(++)
(++)
(++)
(++)
+++
(++)
(+++)
159
160
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
105
104
10
1000
10,000
0.1
10
100
0.01
1
10
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
5.4
5.2
5
4.8
4.6
4.4
4.2
4
161
3.8
1
24
48
72
96
120
144
168
192
216
5.4
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)
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
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
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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
Centre, Nottingham, UK) for assistance with TEM imaging. Many
thanks to Dr Andrew Timms for assistance during manuscript
preparation.
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