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Effect of phage and host concentration on the inactivation of Escherichia coli O157:H7 on cooked and raw beef JA Hudson, C Billington, T Wilson and SLW On Food Science and Technology International published online 27 November 2013 DOI: 10.1177/1082013213513031 The online version of this article can be found at: http://fst.sagepub.com/content/early/2013/11/21/1082013213513031
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Consejo Superior de Investigaciones Científicas (Spanish Council for Scientific Research)
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Research Article
Effect of phage and host concentration on the inactivation of Escherichia coli O157:H7 on cooked and raw beef JA Hudson, C Billington, T Wilson and SLW On
Abstract A previously described phage infecting Escherichia coli O157:H7 was added to raw and cooked beef pieces at concentrations ranging from 101–108 plaque forming units/cm2 to either low (4 log10 CFU/cm2 at both temperatures could be achieved compared to phage-free controls. There was a trend for greater inactivation to occur with increasing phage concentration. While re-growth of surviving cells occurred in nearly all samples incubated for 24 h at 24 C, these conditions are not typical of those experienced by perishable foods. It was concluded that phages can be used to reduce the concentration of a bacterial pathogen on meat, but the concentration of phages needs to be high (>4–5 log10 plaque forming units/cm2) for reductions to occur. A concentration of the order 8 log10 plaque forming units/cm2 was needed to achieve a 4 log10 CFU/cm2 reduction. Keywords Escherichia coli O157:H7, bacteriophage, food safety, biocontrol Date received: 17 July 2013; revised: 21 October 2013
INTRODUCTION Escherichia coli O157:H7 is a significant foodborne pathogen as clinical consequences of infection can include kidney failure and death. In the UK, for example, there have been a number of high-profile outbreaks of infections caused by this organism associated with meat products. One in Scotland (Cowden et al., 2001) resulted in 512 cases among which there were 22 deaths, and another in Wales involved 157 cases and one death (Salmon and Outbreak Control Team, 2005). Control of this pathogen in food manufacture and distribution is therefore crucial for the protection of public health. One such control is the use of bacteriophages (phages) that infect and kill bacteria (Coffey et al., 2010).
Food Science and Technology International 0(0) 1–6 ! The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1082013213513031 fst.sagepub.com
The literature describing the application of phages to control the growth of foodborne pathogens is steadily growing, with work to date largely focusing on Campylobacter jejuni, Listeria monocytogenes, Salmonella and E. coli O157:H7 (Hagens and Loessner, 2010; Hudson et al., 2010). In many of these studies, reductions in the concentrations of bacterial pathogens were measured at the highest phage concentrations used, the inhibitory effect occurred quickly and at temperatures beneath the minimum growth temperature of the host bacterium (Lo´pez-Cuevas et al., 2012). Phage replication in situ was therefore unlikely to have occurred. The mechanism of action proposed for a food surface with a low concentration of bacterial cells is one where the phages must be applied at a sufficient Food Programme, ESR Ltd, Ilam, Christchurch, New Zealand Corresponding author: JA Hudson, Food Programme, ESR Ltd, Ilam, Christchurch, New Zealand. Email: [email protected]
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Food Science and Technology International 0(0) concentration for cell and phage to come into contact, and that once infection has occurred, then the cell will die on resumption of growth (Hagens and Offerhaus, 2008). Some information for the inactivation of L. monocytogenes on the surface of smoked salmon is presented by Hagens and Offerhaus (2008) and in a later paper reporting results for raw salmon (Soni and Nannapaneni, 2010), which showed increasing control of pathogen cells with increasing phage concentration. With respect to the treatment of E. coli O157:H7 on surfaces with phages, significant reductions have been reported on calf hide pieces, after 1-h incubation (Coffey et al., 2011), on chicken skin (Lo´pez-Cuevas et al., 2012), raw beef (O’Flynn et al., 2004) and fresh cut lettuces and cantaloupes (Sharma et al., 2009). The work described here sought to use a phageinfecting E. coli O157:H7, FAHEc1 (Hudson et al., 2013), to investigate the inactivation of its bacterial host on raw and cooked beef. The work was designed to investigate how a variety of phage concentrations might control E. coli O157:H7 cells present at low or high concentrations. It is more likely that this pathogen will be present on beef at a low concentration rather than a high one, and so it is important to investigate phage biocontrol under realistic conditions. The work was also conducted at two temperatures, one that permitted growth of E. coli O157:H7 (24 C) and one that did not (5 C).
MATERIALS AND METHODS Phage isolates and host bacterium used Phage FAHEc1 has been previously described (Hudson et al., 2013), and some aspects of its ability to control host cells on meat are described in a preliminary manner. The E. coli O157:H7 host used was New Zealand Reference Culture Collection, Medical Section (NZRM) 3647, a non-toxigenic strain. Preparation of high-titre phage stocks In brief, Luria Broth Base (LB; Invitrogen, Carlsbad, CA, USA) was inoculated with E. coli O157:H7 and incubated overnight at 37 C. A subculture was then made to the same medium and incubated for 3–4 h prior to 100 ml volumes being inoculated into 3 ml volumes of molten LB soft agar overlay (LB broth þ 0.7% agar) held at 46 C. Tubes of soft agar were also inoculated with 100 ml volumes of diluted phage FAHEc1. Inoculated soft agar was poured as overlays onto preprepared LB agar (1.5% agar) plates and, after setting, incubated at 37 C overnight. SM buffer (5 ml; 0.05 M Tris, 0.1 M NaCl, 0.008 M MgSO4, 0.01% (w/v) gelatin pH 7.5) was added to plates with almost confluent plaques. The plates were gently shaken for 2–3 h, and the
liquid was transferred to a centrifuge tube. The remaining agar overlay was stomached for 2 min in 10 ml of SM buffer and then centrifuged at 4000 g for 10 min to pellet the agar. The phage-containing SM buffer samples were pooled and filtered through a 0.22-mm pore-size filter (Millipore, Cork, Ireland) into a sterile foil-covered universal bottle. The concentration of phages in the suspension was subsequently determined (Carlson, 2005). Experiments with meat The phage was applied to raw and cooked ready-to-eat beef in the presence of E. coli O157:H7 at two temperatures (5 C and 24 C) to represent refrigeration and room temperatures. Escherichia coli O157:H7 host cells were inoculated at low (
Effect of phage and host concentration on the inactivation of Escherichia coli O157:H7 on cooked and raw beef JA Hudson, C Billington, T Wilson and SLW On Food Science and Technology International published online 27 November 2013 DOI: 10.1177/1082013213513031 The online version of this article can be found at: http://fst.sagepub.com/content/early/2013/11/21/1082013213513031
Published by: http://www.sagepublications.com
On behalf of:
Consejo Superior de Investigaciones Científicas (Spanish Council for Scientific Research)
Additional services and information for Food Science and Technology International can be found at: Email Alerts: http://fst.sagepub.com/cgi/alerts Subscriptions: http://fst.sagepub.com/subscriptions Reprints: http://www.sagepub.com/journalsReprints.nav Permissions: http://www.sagepub.com/journalsPermissions.nav
>> OnlineFirst Version of Record - Nov 27, 2013 What is This?
Downloaded from fst.sagepub.com at TEXAS SOUTHERN UNIVERSITY on November 10, 2014
XML Template (2013) [22.11.2013–9:47am] //blrnas3/cenpro/ApplicationFiles/Journals/SAGE/3B2/FSTJ/Vol00000/130127/APPFile/SG-FSTJ130127.3d
(FST)
[1–6] [PREPRINTER stage]
Research Article
Effect of phage and host concentration on the inactivation of Escherichia coli O157:H7 on cooked and raw beef JA Hudson, C Billington, T Wilson and SLW On
Abstract A previously described phage infecting Escherichia coli O157:H7 was added to raw and cooked beef pieces at concentrations ranging from 101–108 plaque forming units/cm2 to either low (4 log10 CFU/cm2 at both temperatures could be achieved compared to phage-free controls. There was a trend for greater inactivation to occur with increasing phage concentration. While re-growth of surviving cells occurred in nearly all samples incubated for 24 h at 24 C, these conditions are not typical of those experienced by perishable foods. It was concluded that phages can be used to reduce the concentration of a bacterial pathogen on meat, but the concentration of phages needs to be high (>4–5 log10 plaque forming units/cm2) for reductions to occur. A concentration of the order 8 log10 plaque forming units/cm2 was needed to achieve a 4 log10 CFU/cm2 reduction. Keywords Escherichia coli O157:H7, bacteriophage, food safety, biocontrol Date received: 17 July 2013; revised: 21 October 2013
INTRODUCTION Escherichia coli O157:H7 is a significant foodborne pathogen as clinical consequences of infection can include kidney failure and death. In the UK, for example, there have been a number of high-profile outbreaks of infections caused by this organism associated with meat products. One in Scotland (Cowden et al., 2001) resulted in 512 cases among which there were 22 deaths, and another in Wales involved 157 cases and one death (Salmon and Outbreak Control Team, 2005). Control of this pathogen in food manufacture and distribution is therefore crucial for the protection of public health. One such control is the use of bacteriophages (phages) that infect and kill bacteria (Coffey et al., 2010).
Food Science and Technology International 0(0) 1–6 ! The Author(s) 2013 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1082013213513031 fst.sagepub.com
The literature describing the application of phages to control the growth of foodborne pathogens is steadily growing, with work to date largely focusing on Campylobacter jejuni, Listeria monocytogenes, Salmonella and E. coli O157:H7 (Hagens and Loessner, 2010; Hudson et al., 2010). In many of these studies, reductions in the concentrations of bacterial pathogens were measured at the highest phage concentrations used, the inhibitory effect occurred quickly and at temperatures beneath the minimum growth temperature of the host bacterium (Lo´pez-Cuevas et al., 2012). Phage replication in situ was therefore unlikely to have occurred. The mechanism of action proposed for a food surface with a low concentration of bacterial cells is one where the phages must be applied at a sufficient Food Programme, ESR Ltd, Ilam, Christchurch, New Zealand Corresponding author: JA Hudson, Food Programme, ESR Ltd, Ilam, Christchurch, New Zealand. Email: [email protected]
Downloaded from fst.sagepub.com at TEXAS SOUTHERN UNIVERSITY on November 10, 2014
XML Template (2013) [22.11.2013–9:47am] //blrnas3/cenpro/ApplicationFiles/Journals/SAGE/3B2/FSTJ/Vol00000/130127/APPFile/SG-FSTJ130127.3d
(FST)
[1–6] [PREPRINTER stage]
Food Science and Technology International 0(0) concentration for cell and phage to come into contact, and that once infection has occurred, then the cell will die on resumption of growth (Hagens and Offerhaus, 2008). Some information for the inactivation of L. monocytogenes on the surface of smoked salmon is presented by Hagens and Offerhaus (2008) and in a later paper reporting results for raw salmon (Soni and Nannapaneni, 2010), which showed increasing control of pathogen cells with increasing phage concentration. With respect to the treatment of E. coli O157:H7 on surfaces with phages, significant reductions have been reported on calf hide pieces, after 1-h incubation (Coffey et al., 2011), on chicken skin (Lo´pez-Cuevas et al., 2012), raw beef (O’Flynn et al., 2004) and fresh cut lettuces and cantaloupes (Sharma et al., 2009). The work described here sought to use a phageinfecting E. coli O157:H7, FAHEc1 (Hudson et al., 2013), to investigate the inactivation of its bacterial host on raw and cooked beef. The work was designed to investigate how a variety of phage concentrations might control E. coli O157:H7 cells present at low or high concentrations. It is more likely that this pathogen will be present on beef at a low concentration rather than a high one, and so it is important to investigate phage biocontrol under realistic conditions. The work was also conducted at two temperatures, one that permitted growth of E. coli O157:H7 (24 C) and one that did not (5 C).
MATERIALS AND METHODS Phage isolates and host bacterium used Phage FAHEc1 has been previously described (Hudson et al., 2013), and some aspects of its ability to control host cells on meat are described in a preliminary manner. The E. coli O157:H7 host used was New Zealand Reference Culture Collection, Medical Section (NZRM) 3647, a non-toxigenic strain. Preparation of high-titre phage stocks In brief, Luria Broth Base (LB; Invitrogen, Carlsbad, CA, USA) was inoculated with E. coli O157:H7 and incubated overnight at 37 C. A subculture was then made to the same medium and incubated for 3–4 h prior to 100 ml volumes being inoculated into 3 ml volumes of molten LB soft agar overlay (LB broth þ 0.7% agar) held at 46 C. Tubes of soft agar were also inoculated with 100 ml volumes of diluted phage FAHEc1. Inoculated soft agar was poured as overlays onto preprepared LB agar (1.5% agar) plates and, after setting, incubated at 37 C overnight. SM buffer (5 ml; 0.05 M Tris, 0.1 M NaCl, 0.008 M MgSO4, 0.01% (w/v) gelatin pH 7.5) was added to plates with almost confluent plaques. The plates were gently shaken for 2–3 h, and the
liquid was transferred to a centrifuge tube. The remaining agar overlay was stomached for 2 min in 10 ml of SM buffer and then centrifuged at 4000 g for 10 min to pellet the agar. The phage-containing SM buffer samples were pooled and filtered through a 0.22-mm pore-size filter (Millipore, Cork, Ireland) into a sterile foil-covered universal bottle. The concentration of phages in the suspension was subsequently determined (Carlson, 2005). Experiments with meat The phage was applied to raw and cooked ready-to-eat beef in the presence of E. coli O157:H7 at two temperatures (5 C and 24 C) to represent refrigeration and room temperatures. Escherichia coli O157:H7 host cells were inoculated at low (
