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Survival of Campylobacter jejuni on beef and pork under vacuum packaged and retail storage conditions: Examination of the role of natural meat microflora on C. jejuni survival
Food Microbiology 28 (2011) 1003e1010
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Survival of Campylobacter jejuni on beef and pork under vacuum packaged and retail storage conditions: Examination of the role of natural meat microflora on C. jejuni survival Sampathkumar Balamurugan*, Frances M. Nattress, Lynda P. Baker, Bryan D. Dilts Agriculture & Agri-Food Canada, 6000 C & E Trail, Lacombe, Alberta, T4L 1W1 Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 November 2010 Received in revised form 25 January 2011 Accepted 27 January 2011 Available online 4 February 2011
The ability of Campylobacter jejuni ATCC 11168 to survive on beef and pork stored under chilled, vacuum packaged and retail display conditions were examined. In addition, the effect of natural microflora on commercial beef and pork on the survival of C. jejuni under these storage conditions was examined. When sterile cores of beef and pork were inoculated with w105 to 106 cfu cm2 C. jejuni, and were stored under aerobic or vacuum packaged conditions at 1.5 or 4 C, its numbers dropped significantly and C. jejuni could not be enumerated by direct plating after 21 d of the 6 wks study. In contrast, survival of C. jejuni on commercial vacuum packaged beef and pork was significantly enhanced, resulting in only 1 log cfu cm2 reduction at the end of 6 wks. During 7 d of display in a retail case, numbers of C. jejuni dropped quickly, but could be enumerated by direct plating even after the 7 d. The presence of high numbers of inoculated C. jejuni on beef and pork had no significant effect on the natural microflora numbers compared to uninoculated controls when the meat was stored either in vacuum or in a retail display case. These results show that natural microflora on vacuum packaged meat afford enhanced survival of C. jejuni present on the surfaces of both beef and pork when stored at refrigeration temperatures. Hence, strict hygienic practices or the implementation of decontamination technologies are recommended to ensure safety of meat with respect to this pathogen. Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
Keywords: Campylobacter jejuni Survival Vacuum packaging Retail display Natural meat microflora
1. Introduction Campylobacter jejuni is one of the leading zoonotic causes of human gastroenteritis in Europe and North America (CDC, 2010; EFSA, 2005; PHAC, 2005). It has a very low infectious dose (Black et al., 1988; Robinson, 1981) and in addition to the seldom fatal but severe gastroenteritis caused by this organism, the serious immunemediated Guillain-Barré syndrome, which is a chronic development in some previously infected individuals (van der Meche and van Door, 2000), make this organism a substantial public health concern. Based on collated epidemiological data from outbreaks, undercooked poultry is regarded as the major risk factor for foodborne campylobacteriosis (Fields and Swerdlow, 1999). In addition, outbreaks of infection due to C. jejuni have been linked to a number of sources including eggs (Finch and Blake, 1985), seafood (Feldhusen, 2000), unchlorinated water (Bopp et al., 2003; Jakopanec et al., 2008; Koenraad et al., 1997) and raw milk (Heuvelink et al., 2009; Peterson, 2003; Schildt et al., 2006). C. jejuni is often present in the
* Corresponding author. Tel.: þ1 403 782 8119; fax: þ1 403 782 6120. E-mail address: [email protected] (S. Balamurugan).
gastrointestinal tracts (Gill and Harris, 1982a; Hudson et al., 1999; Inglis and Kalischuk, 2003, 2004; Inglis et al., 2005; Van Laack et al., 1993) and on carcasses (Gill and Harris, 1982a; Kotula and Stern, 1984; Stern, 1981) of meat animals. It has also been isolated from bulk packed red meats (Vanderlinde et al., 1998) and retail ready meats (Wong et al., 2007). A large proportion of Canadian beef and pork is delivered to retail stores sealed in vacuum packages. The meat is subsequently opened at retail and cut into steaks, chops and roasts, which are typically repackaged in an oxygen permeable overwrapped film and placed in a retail display case. With the elimination of oxygen in vacuum packaged meat, the growth of aerobic spoilage organisms is significantly reduced, thereby extending product storage life (Lambert et al., 1991). The storage life of vacuum packaged fresh primals and subprimals is generally reported as approximately 35e45 d, while the expected shelf life of product packaged in oxygen permeable overwrap at retail is 3e7 d. In the meat industry, vacuum packaging and storage at strictly controlled temperatures of 1.5 C are widely used to store and export raw meat. A number of studies have raised concerns of the potential of these packaging conditions to increase the risk of campylobacteriosis by allowing the growth of C. jejuni (Gill and Harris,
0740-0020/$ e see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2011.01.012
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1982b; Hänninen et al.,1984; Van Laack et al.,1993). However, studies investigating the effect of vacuum and modified atmosphere packaging on the survival of C. jejuni on meat (Gill and Harris, 1982b; Hänninen et al., 1984; Van Laack et al., 1993) have collectively indicated that chill storage under vacuum or modified atmosphere is likely to increase the safety of meat. More recently in 2001, a study investigating the effect of these preservative packing of meat at a strictly controlled 1.5 C on survival of C. jejuni showed no significant changes in numbers of this pathogen (Dykes and Moorhead, 2001), raising causes for concern. C. jejuni is fastidious with respect to growth requirements, is sensitive to oxygen (Stern et al., 1992) and only grows in the temperature range of 30e44 C. Therefore, survival rather than growth on meat is the concern. While growth of C. jejuni will not occur at chill temperatures, it is necessary to understand the nature and extent of the effect of different meats, the natural bacterial populations on these meats and modern commercial meat packaging and storage practices on the survival of this pathogen. Studies to date have focused on the effect of storage parameters rather than the influence of the natural bacterial populations on beef and pork on the survival of C. jejuni in vacuum packaged and retail display conditions. Hilbert et al. (2010) noted the importance of the interaction between spoilage bacteria and C. jejuni. They demonstrated in vitro that coincubation of C. jejuni with Pseudomonas spp. enhanced the ability of C. jejuni to survive aerobic incubation at 35 C. Coincubation did not affect the growth of Pseudomonas spp. The aim of the present study was to examine the survival of C. jejuni on beef and pork under vacuum packaged and retail display conditions at normal meat storage temperatures. In addition, the effect of natural microflora on survival of C. jejuni on beef and pork was examined.
were then transferred to 800 600 commercial barrier bags (oxygen transmission rate of 40e50 cc m2 24 h1; Winpak Ltd., Winnipeg, MB Canada) for vacuum packing (Multivac AGI, Knud Simonsen Industries Ltd, Rexdale, ON Canada) The vacuum packaged cores were incubated at 1.5 or 4.0 C and C. jejuni numbers enumerated or recovered using enrichment over a 6 wks storage time. To enumerate C. jejuni, cores were removed aseptically from the vacuum packs, transferred to 90 ml 0.1% sterile peptone water and mixed in a Seward stomacher (Fisher Scientific) at medium setting for 2 min. Appropriate dilutions were plated on Campylobacter Blood-Free Selective Agar (mCCDA; Oxoid) plates and the plates were incubated under microaerophilic condition for 48 h at 42 C. Another core was transferred to Brucella broth containing Campylobacter Growth Supplement (Oxoid; SR0232) and enriched for Campylobacter spp. under microaerophilic conditions at 42 C for 48 h followed by swabbing onto mCCDA plates to detect presence or absence of Campylobacter spp. The mCCDA plates were incubated under microaerophilic conditions for 48 h at 42 C. To examine the effect of oxygen permeability through the packaging material on C. jejuni survival, the above study was repeated with inoculated and uninoculated cores that were overwrapped with oxygen permeable, polyvinyl chloride film (oxygen transmission rate of 8000 cc m2 24 h2; Vitafilm Choice Wrap, Goodyear Canada Inc., Toronto, ON) or vacuum packaged in oxygen impermeable barrier bags that had a very low oxygen transmission rate of 0.4 cc m2 24 h1 (Winpak). These studies were repeated 3 times and final bacterial counts are means of these 3 replications.
2. Materials and methods
Fresh vacuum packed beef and pork loins were obtained from a commercial meat plant. They were transported to the Lacombe Research Centre under refrigerated conditions and were stored at 1.5 C overnight. These loins were cut into pieces weighing w500 g (w750 cm2) each using sterile knives and placed in commercial barrier bags. The pH values of muscle tissue were determined before inoculation for each incubation temperature. A 7.5 ml volume of the C. jejuni suspension (prepared as described in Section 2.1) was inoculated onto each of the pieces to a final level of w105e106 cfu cm2. The inoculum was massaged onto the pieces before vacuum packing. The vacuum packed bags were incubated at 1.5 or 4.0 C for up to 6 wks. Uninoculated control pieces were similarly processed by adding 7.5 ml of sterile water. Enumeration of Campylobacter spp. and background bacterial counts was performed on both the inoculated and uninoculated samples over the 6 wks storage period. To enumerate Campylobacter spp. and background bacterial counts, two 10 cm2 cores were removed aseptically from the meat at times 0, 2, 5, 7, 9, 12,14, 21, 28, 35 and 42 d. They were transferred to 90 ml 0.1% sterile peptone water and mixed in a stomacher at medium settings for 2 min. Appropriate dilutions were plated on selective and non-selective media. Campylobacter spp. was enumerated using mCCDA, incubated under microaerophilic conditions at 42 C for 48 h. Selective media described by Baird et al. (1987) were used to enumerate Brochothrix spp., Pseudomonas spp., lactic acid bacteria and Enterobacteriaceae. Total aerobes were enumerated using TSA plates incubated for 48 h at 25 C. Streptomycin thallous acetate agar (STAA; Oxoid) and cephaloridine fucidin cetrimide agar (CFC; Oxoid) was used to enumerate Brochothrix spp. and Pseudomonas spp., respectively, by incubating the plates at 25 C for 48 h. Enumeration of presumptive lactic acid bacteria (LAB) was done on de Man, Rogosa and Sharpe agar (MRSA; Difco) with a 72e96 h anaerobic incubation at 25 C. Enterobacteriaceae were enumerated by plating on Violet Red Bile Glucose agar (VRBGA; BD-Difco, BD Canada, Mississauga, ON Canada) with 18e24 h anaerobic incubation at 35 C. Anaerobic conditions were
2.1. Bacteria and culture media C. jejuni NCTC 11168 (ATCC 700819) was used in this study. The C. jejuni culture was maintained at 80 C and resuscitated by streaking on tryptic soy agar plates (TSA, Oxoid, Nepean, ON Canada) at 42 C for 24 h under microaerophilic conditions generated using CampyGenÔ (Oxoid) gas generation sachets. An isolated colony was transferred to 75 ml tryptic soy broth (TSB, Oxoid) containing Campylobacter Growth Supplement (Oxoid, SR0117) and incubated under microaerophilic conditions for 48 h at 42 C. Subsequently, 750 ml of this culture was transferred to 75 ml of TSB with Campylobacter Growth Supplement and incubated under microaerophilic conditions for 24 h at 42 C. Cells were harvested by centrifugation and resuspended in 0.1% sterile peptone water (same volume as supernatant discarded). This culture was used to inoculate meat in all our studies. 2.2. C. jejuni inoculation and survival on sterile beef and pork Fresh boneless beef and pork loins (Longissimus dorsi) of normal muscle quality were obtained from the Lacombe Research Centre abattoir. Sterile beef and pork muscle tissue discs (10 cm2) were aseptically excised from the meat as previously described (Greer and Dilts, 1995). The pH values of muscle tissue discs were determined. Surface pH was measured using an Accumet AP61 portable pH meter (Fisher Scientific, Ottawa, ON Canada) equipped with a flat surface electrode (Accumet 13-620-289, Fisher Scientific). Each core was placed individually on a styrofoam tray (Scott National Food Service, Edmonton, AB Canada) and was inoculated with 100 ml volume of the C. jejuni suspension (prepared as described in section 2.1) to a final level of w105 to 106 cfu cm2. The bacteria were allowed to adhere for 5 min. The trays with the inoculated or uninoculated control cores
2.3. C. jejuni inoculation and survival on commercial vacuum packaged beef and pork
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established using a BBL anaerobic gas pack system (BD-BBL, BD Canada). Psychrotrophic bacteria were enumerated by plating on TSA and incubating plates at 4 C for 10 d. These studies were repeated 3 times and final bacterial counts are means of these 3 replications. 2.4. C. jejuni inoculation and survival under retail display storage conditions Vacuum packed fresh beef and pork loins were obtained from a commercial meat plant and stored at 1.5 C overnight. These loins were prepared as described in 2.3. At 2 wks intervals (0, 2, 4, and 6 wks), 5 meat samples were removed from their vacuum packages, placed on styrofoam trays and overwrapped with oxygen permeable, polyvinyl chloride film (oxygen transmission rate of 8000 cc m2 24 h2; Vitafilm Choice Wrap, Goodyear Canada Inc., Toronto, ON) and placed in a retail display case (Williams Restaurant Equipment, Winnipeg, MB Canada) set to operate at 4 C. At days 0, 2, 5 and 7 one sample was removed. Bacterial numbers were determined as described in Section 2.3 and final pH was measured. These studies were repeated 3 times and final bacterial counts are means of these 3 replications. 2.5. Statistical analysis Numbers of bacteria were transformed to log10 cfu cm2 for statistical analysis. The limit of detection by the direct plate counting
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method was 2 log cfu cm2. When cells could not be detected by the direct plate counting method but were recovered on plates following enrichment of the samples for C. jejuni, the cell numbers were regarded as 1 (0 log cfu cm2) but when the cells became unrecoverable even after enrichment the cell numbers were regarded as 0.1 (1 log cfu cm2). General Linear Models Analysis of Variance Procedure (ANOVA) of the Statistical Analysis System (SAS, 1989) was applied to the significance of differences, if any, between bacterial counts, temperatures, meat types and packaging conditions. Significance was based on a level of 5.0% (P < 0.05). 3. Results 3.1. C. jejuni on sterile beef and pork Changes in numbers of C. jejuni cells inoculated on sterile beef and pork cores or commercial beef and pork, packaged and stored under different conditions are presented in Fig. 1. The numbers of C. jejuni inoculated on sterile beef and pork cores stored under aerobic or vacuum packaged conditions at 1.5 or 4 C dropped significantly (P < 0.05) over the time of the study. C. jejuni could not be recovered from beef cores by direct plating after 7 and 9 d during aerobic storage at 1.5 and 4 C, respectively, and were unrecoverable by enrichment after 14 d at the two storage temperatures. C. jejuni could not be recovered from beef cores by direct plating after 9 and 14 d when the beef was vacuum packaged using commercial barrier bags and stored at 1.5 and 4 C, respectively,
Fig. 1. Numbers of C. jejuni surviving on sterile beef and pork cores inoculated with C. jejuni and packaged by overwrapping using oxygen permeable polyvinyl chloride film (aerobic; C), vacuum packaged using commercial (-) or oxygen impermeable barrier bags (A), and on commercial beef and pork inoculated with C. jejuni and vacuum packaged using commercial barrier bags (:) and stored at 1.5 or 4 C. Data are least square means of bacterial numbers. Standard error (SE) ¼ 0.36.
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and were unrecoverable by enrichment after 21 and 28 d at the two storage temperatures. C. jejuni could not be recovered from beef cores by direct plating after 14 and 35 d when beef was vacuum packaged using oxygen impermeable barrier bags and stored at 1.5 and 4 C, respectively, and were recoverable by enrichment throughout the length of the study (42 d) at the two storage temperatures. Similarly, on pork cores, C. jejuni could not be recovered after 7 and 9 d by direct plating during aerobic storage at 1.5 and 4 C, respectively, and were unrecoverable by enrichment after 14 d at the two storage temperatures. During vacuum packaging using commercial barrier bags C. jejuni could not be recovered by direct plating after 21 and 14 d at 1.5 and 4 C, respectively, and were unrecoverable by enrichment after 28 and 21 d at the two storage temperatures. However, during vacuum packaging using oxygen impermeable barrier bags C. jejuni could not be recovered after 35 and 21 d at 1.5 and 4 C, respectively, by direct plating and was recoverable by enrichment throughout the length of the study for both temperatures. Use of oxygen impermeable barrier bags significantly (P < 0.05) increased survival of C. jejuni compared to aerobic and vacuum packaging using commercial barrier bags in both beef and pork cores at both temperatures tested. 3.1.1. C. jejuni on commercial beef and pork The examination of the effect of the presence of natural microflora on C. jejuni numbers on beef and pork vacuum packaged using commercial barrier bags revealed that the numbers remained high enough to be enumerated using direct plating (Fig. 1) and the rate of decline in numbers of C. jejuni was slow and was not affected by meat type or storage temperature (P < 0.05). During the entire
6 wks storage time, numbers of C. jejuni only dropped about 1 log cfu cm2. 3.1.2. Natural microflora on commercial beef and pork The numbers of the mesophilic and psychrotrophic meat microflora increased on both beef and pork vacuum packaged and stored at 1.5 and 4 C, but were generally higher on meat stored at 4 C at the end of 6 wks (data not shown). On beef, growth of Enterobacteriaceae was inhibited at 1.5 C while at 4 C their numbers increased approximately 3 log cfu cm2. On pork, their numbers increased about 4 log cfu cm2. Similarly, growth of presumptive lactic acid bacteria and Pseudomonas spp. was less at 1.5 C when compared to 4 C and numbers on pork were higher than those on beef (Fig. 2). On both beef and pork, numbers of Brochothrix spp. increased and reached a maximum at around 14e21 d following which numbers declined or showed no significant (P > 0.05) increase at both 1.5 and 4 C (Fig. 2). 3.2. C. jejuni on beef and pork under retail display storage conditions When meat was removed from vacuum packs, repackaged in oxygen permeable film and placed in a retail display case, numbers of C. jejuni dropped quickly. However, even after 7 d, they could still be enumerated by direct plating (Fig. 3). Presumptive lactic acid bacteria, Brochothrix spp., Pseudomonas spp. and Enterobacteriaceae all grew to higher numbers in response to the change in packaging conditions (Fig. 4). In general, numbers of all bacterial groups were lower when meat was removed after storage at 1.5 C than when it
Fig. 2. Numbers of Enterobacteriaceae [A; SE: 0.30 (beef) 0.37 (pork)], Brochothrix spp. [C; SE: 0.43 (beef) 0.56 (pork)], Pseudomonas spp. [-; SE: 0.45 (beef), 0.38 (pork)] and presumptive lactic acid bacteria [:; SE: 0.54 (beef), 0.41 (pork)] on commercial beef and pork in vacuum packages stored at 1.5 or 4 C. Data are least square means of bacterial numbers from samples that were and were not inoculated with C. jejuni since there was no effect of C. jejuni on growth of natural microflora (P > 0.05).
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was stored at 4 C but by the end of 7 d retention in a retail display case, all samples had similar numbers of each group of bacteria (Fig. 4). An analysis of the effect of natural meat microflora on C. jejuni survival on beef and pork stored under aerobic conditions revealed that C. jejuni survival was not significantly (P > 0.05) affected by the presence of the natural meat microflora compared to sterile meat inoculated with C. jejuni (Fig. 5). None of the bacterial groups numbers was affected by the presence of the high number of inoculated C. jejuni (P > 0.05) when stored either in vacuum or in retail display case compared to uninoculated controls (data not shown). Similarly, there was no effect of the presence of high numbers of C. jejuni on pH (P > 0.05) at any of the vacuum packaged and retail display sampling times (data not shown).
4. Discussion As indicated in the Introduction, studies investigating the effect of vacuum and modified atmosphere packaging on the survival of C. jejuni have collectively indicated that chill storage under vacuum or modified atmosphere is likely to increase the safety of meat (Gill and Harris, 1982b; Hänninen et al., 1984; Van Laack et al., 1993). However, it has to be pointed out that the chill temperatures examined in these studies were in the range of 1e4 C, while Gill and Harris (1982b) studied C. jejuni survival on fresh meat at 37 C and sterile meats at 1 or 18 C stored under aerobic conditions. These
Fig. 3. Numbers of C. jejuni surviving on commercial beef and pork inoculated with C. jejuni that were stored in vacuum packages at either 1.5 or 4 C for up to 6 wks, removed, wrapped in oxygen permeable polyvinyl chloride film and stored in retail display case for up to 7 d. 0 time (A), 2 wks (-), 4 wks (:) and 6 wks (C). Data are least square means of bacterial numbers.
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studies do not reflect the combination of parameters now widely used in the meat industry to store and export product, namely vacuum packaging at a strictly controlled 1.5 C. There are no studies to our knowledge that have examined the effect of storage conditions and chill storage on C. jejuni survival on meat lacking the native microflora. More recently, results showing no significant changes in numbers of C. jejuni during storage at 1.5 C on beef packaged under vacuum (Dykes and Moorhead, 2001) raised serious questions regarding the safety of current transport and storage regimes. The authors had indicated that the interaction between natural bacteria populations and Campylobacter spp. would be of interest, but did not examine this and instead focussed on the effect of storage parameters alone. This precisely was the focus of this study, and the results clearly demonstrate that the natural microflora on vacuum packaged beef and pork enhances the survival of C. jejuni. However, the mechanism involved in this enhanced survival is unclear. C. jejuni has been shown to survive better within preexisting biofilms, indicating survival due to a protective biofilm (Trachoo et al., 2002). Gill and Harris (1982b) hypothesized that “increasing numbers of spoilage bacteria may compete with, and reduce, numbers of Campylobacter spp., or alternatively make them difficult to recover”. This has not been the observation in the present study since C jejuni numbers could be enumerated by direct plating for the entire 6 wks of the study. It has been suggested that the reduction of oxygen concentrations by competitor microorganisms may protect associated pathogens from cold stress (Aldsworth et al., 1998). It could therefore be hypothesized that since C. jejuni is a microaerophile, the natural microflora growing on vacuum packaged meat sequesters the oxygen that manages to pass through the barrier bags, and provides a protective environment resulting in enhanced survival of C. jejuni. This was tested by repeating C. jejuni survival studies on sterile beef and pork cores vacuum packaged using oxygen impermeable barrier bags, and it was expected that C. jejuni survival would be similar to its survival observed in the presence of natural microflora. Although survival was significantly enhanced, compared to survival on sterile meat that was inoculated, in aerobic or vacuum packaging using commercial barrier bags (higher oxygen transmission), they were not similar to vacuum packaged (using commercial barrier bags) meat containing natural microflora (Fig. 1). These results indicate that only part of the protective effect of the natural microflora can be explained by protection of C. jejuni from oxygen. There may be other factors that remain to be explained. The microflora of vacuum packaged chill stored beef and pork has been extensively studied and it was found that lactic acid bacteria are the most predominant (Borch et al., 1996; Hammes et al., 1992; Jones, 2004; Sutherland et al., 1975). Vacuum packaging results in reduced oxygen availability and in combination with storage at 1.5 C restricts the growth of other spoilage bacteria like Pseudomonas spp., Brochothrix thermospacta and Enterobacteriaceae resulting in extending the shelf life of the product to more than 8 wks (Borch et al., 1996; Gill, 1996; Jones, 2004; Nissen et al., 1996). Increases in numbers of lactic acid bacteria have been shown to decrease growth of B. thermospacta (Russo et al., 2006) and have been implicated in controlling growth of some meatborne pathogens (Nissen et al.,1996; Vold et al., 2000). However, in the present study, although growth of Brochothrix was inhibited (Fig. 2) past 14 d under vacuum, the increase in lactic acid bacteria did not have an inhibitory effect on C. jejuni, but on the contrary was protective. In addition, the absence of a difference between numbers of different bacterial groups on meats inoculated with C. jejuni as compared to uninoculated meat indicates that the introduced C. jejuni also had no impact on the numbers and survival of different bacterial groups on vacuum packaged beef and pork. In retail, beef and pork is displayed in aerobic packs and also held at higher temperatures of 4e8 C, which results in the increase of
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Fig. 4. Numbers of Brochothrix spp., Enterobacteriaceae, presumptive lactic acid bacteria, and Pseudomonas spp. on commercial beef and pork that were stored in vacuum packages at either 1.5 or 4 C for up to 6 wks, removed, wrapped in oxygen permeable polyvinyl chloride film and stored in retail display case for up to 7 d. 0 time (A), 2 wks (-), 4 wks (:) and 6 wks (C). Data are least square means of bacterial numbers from samples that were and were not inoculated with C. jejuni since there was no effect of C. jejuni on growth of natural microflora (P > 0.05).
spoilage bacteria like Pseudomonas spp. B. thermospacta and Enterobacteriaceae due to the availability of oxygen (Blixt and Borch, 2002; Borch et al., 1996; Gill and Newton, 1977). These observations agree with results from this study and the higher numbers of inoculated C.
jejuni had no impact on survival or numbers of these predominant bacteria compared to uninoculated controls during retail display. In retail stores, meat is removed from vacuum packs and repackaged in oxygen permeable film and placed in a retail display
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Fig. 5. Numbers of C. jejuni surviving on beef (A) and pork (B) stored under aerobic conditions at 4 C. Sterile cores (A; data from Fig. 1) or commercial meat (-; data from 0 time in Fig. 3) were inoculated with C. jejuni. Data are least square means of bacterial numbers.
case. The enhanced survival of C. jejuni on vacuum packaged beef raises questions about the safety of meat in retail display cases. This is the first study that has examined the survival of C. jejuni by simulating the transfer of meat from vacuum packs to retail display. Although C. jejuni numbers decrease rapidly during retail display storage, the results are cause for concern since C. jejuni could be enumerated even after 7 d (retail shelf life of beef and pork). It might be argued that the levels of inoculum used in the present study were relatively high compared to levels of natural contamination and that this may affect the results. However, since all previous studies on C. jejuni survival on meat used similar levels, this is unlikely to be the case. A recent study by Hilbert et al. (2010) examined the survival of C. jejuni in the presence of meat spoilage bacteria and extended survival when incubated aerobically with various species of Pseudomonas was reported. Pseudomonas spp. is the most predominant bacteria present on retail beef and pork and numbers were in the range of 7e8.5 log cfu cm2 at the end of 7 days in retail in the present study. However, our results do not show significant differences (>0.05) in the survival of C. jejuni on meat incubated aerobically either in the presence or absence of natural meat microflora. It is necessary to point out that studies by Hilbert et al. (2010) were performed in broth (neutral pH) and at 35 C. These conditions do not reflect meat (acidic) or meat storage temperatures (1.5 or 4 C) which were examined in the present study. The results of the present study might be criticized that they are from a single strain. Strain specific variations in the survival of Campylobacter spp. from natural environment, farm animals, clinical and laboratory strains on preservative packaged chill stored meat have been previously reported (Dykes and Moorhead, 2001; Gill and Harris, 1982b; Hänninen et al., 1984). However, since C. jejuni cannot grow at the conditions examined (Stern et al., 1992) in the present study, the role of the natural meat microflora on the survival of C. jejuni on preservative packaged chill stored meat is nevertheless established. The low infective dose, the frequent contamination of poultry, and the increasing numbers of reports of the occurrence on other meats, and the apparent ability of C. jejuni to survive commercial meat storage conditions, suggest that the current methods of fresh meat preservation will not add a significant margin of safety with respect to C. jejuni. Instead, control of C. jejuni to protect the consumer will require an integrated approach throughout the meat production, processing, and sale continuum by adapting improved hygienic practices or through decontamination technologies which currently are the only way to avoid food safety concerns arising with respect to this pathogen.
Acknowledgments Funding for this research provided by the Alberta Livestock Industry Development Fund and Alberta Chicken Producers is acknowledged. References Aldsworth, T.G., Sharman, R.L., Dodd, C.E.R., Stewart, G.S.A.B., 1998. A competitive microflora increases the resistance of Salmonella typhimurium to inimical processes: evidence for a suicide response. Appl. Environ. Microbiol. 64, 1323e1327. Baird, R.M., Corry, J.E.L., Curtis, G.D.W., 1987. Pharmacopoeia of culture media for food microbiology. Int. J. Food Microbiol. 5, 187e299. Black, R.E., Levine, M.M., Clements, M.L., Hughes, T.P., Blaser, M.J., 1988. Experimental Campylobacter jejuni infection in humans. J. Infect. Dis. 157, 472e479. Blixt, Y., Borch, E., 2002. Comparison of shelf life of vacuum-packed pork and beef. Meat Sci. 60, 371e378. Bopp, D.J., Sauders, B.D., Waring, A.L., Ackelsberg, J., Dumas, N., Braun-Howland, E., Dziewulski, D., Wallace, B.J., Kelly, M., Halse, T., Musser, K.A., Smith, P.F., Morse, D.L., Limberger, R.J., 2003. Detection, isolation, and molecular subtyping of Escherichia coli O157:H7 and Campylobacter jejuni associated with a large waterborne outbreak. J. Clin. Microbiol. 41, 174e180. Borch, E., Kant-Muermans, M.L., Blixt, Y., 1996. Bacterial spoilage of meat and cured meat products. Int. J. Food Microbiol. 33, 103e120. CDC, 2010. FoodNet Facts and Figures e Numbers of Infections and Incidence per 100,000 Persons. http://www.cdc.gov/foodnet/factsandfigures/incidence.html. Dykes, G.A., Moorhead, S.M., 2001. Survival of Campylobacter jejuni on vacuum or carbon dioxide packaged primal beef cuts stored at 1.5 C. Food Contr. 12, 553e557. EFSA, 2005. The community summary report on trends and sources of zoonoses, zoonotic agents and antimicrobial resistance in the European Union in 2004. EFSA J. 12. http://www.efsa.europa.eu/en/efsajournal/scdoc/310ar.htm. Feldhusen, F., 2000. The role of seafood in bacterial foodborne diseases. Microb. Infect. 2, 1651e1660. Fields, P.I., Swerdlow, D.L., 1999. Campylobacter jejuni. Clin. Lab. Med. 19, 489e504. Finch, M.J., Blake, P.A., 1985. Foodborne outbreaks of campylobacteriosis: the United States experience, 1980e1982. Am. J. Epidemiol. 122, 262e268. Gill, C.O., 1996. Extending the storage life of raw chilled meats. Meat Sci. 43, S99eS109. Gill, C.O., Harris, L.M., 1982a. Contamination of red meat carcasses by Campylobacter fetus subsp. jejuni. Appl. Environ. Microbiol. 43, 977e980. Gill, C.O., Harris, L.M., 1982b. Survival and growth of Campylobacter fetus subsp. jejuni on meat and in cooked foods. Appl. Environ. Microbiol. 44, 259e263. Gill, C.O., Newton, K.G., 1977. The development of aerobic spoilage flora on meat stored at chill temperatures. J. Appl. Microbiol. 43, 189e195. Greer, G.G., Dilts, B.D., 1995. Lactic acid inhibition of the growth of spoilage bacteria and cold tolerant pathogens on pork. Int. J. Food Microbiol. 25, 141e151. Hammes, W.P., Weiss, N., Holzapfel, W.H., 1992. Lactobacillus and Carnobacterium. In: Balows, A., Truper, H.G., Dworkin, M., Harder, W., Schleifer, K.H. (Eds.), The Prokaryotes. Springer Verlag, New York, pp. 15335e15394. Hänninen, M.L., Korkeala, H., Pakkala, P., 1984. Effect of various gas atmospheres on the growth and survival of Campylobacter jejuni on beef. J. Appl. Microbiol. 57, 89e94. Heuvelink, A.E., van Heerwaarden, C., Zwartkruis-Nahuis, A., Tilburg, J.J., Bos, M.H., Heilmann, F.G., Hofhuis, A., Hoekstra, T., de Boer, E., 2009. Two outbreaks of
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campylobacteriosis associated with the consumption of raw cows’ milk. Int. J. Food Microbiol. 134, 70e74. Hilbert, F., Scherwitzel, M., Paulsen, P., Szostak, M.P., 2010. Survival of Campylobacter jejuni under conditions of atmospheric oxygen tension with the support of Pseudomonas spp. Appl. Environ. Microbiol. 76, 5911e5917. Hudson, J.A., Nicol, C., Wright, J., Whyte, R., Hasell, S.K., 1999. Seasonal variation of Campylobacter types from human cases, veterinary cases, raw chicken, milk and water. J. Appl. Microbiol. 87, 115e124. Inglis, G.D., Kalischuk, L.D., 2003. Use of PCR for direct detection of Campylobacter species in bovine feces. Appl. Environ. Microbiol. 69, 3435e3447. Inglis, G.D., Kalischuk, L.D., 2004. Direct quantification of Campylobacter jejuni and Campylobacter lanienae in feces of cattle by real-time quantitative PCR. Appl. Environ. Microbiol. 70, 2296e2306. Inglis, G.D., Kalischuk, L.D., Busz, H.W., Kastelic, J.P., 2005. Colonization of cattle intestines by Campylobacter jejuni and Campylobacter lanienae. Appl. Environ. Microbiol. 71, 5145e5153. Jakopanec, I., Borgen, K., Vold, L., Lund, H., Forseth, T., Hannula, R., Nygard, K., 2008. A large waterborne outbreak of campylobacteriosis in Norway: the need to focus on distribution system safety. BMC Infect. Dis. 8. http://www. biomedcentral.com/1471-2334/8/128. Jones, R.J., 2004. Observations on the succession dynamics of lactic acid bacteria populations in chill-stored vacuum-packaged beef. Int. J. Food Microbiol. 90, 273e282. Koenraad, P.M.F.J., Rombouts, F.M., Notermans, S.H.W., 1997. Epidemiological aspects of thermophilic Campylobacter in water-related environments: a review. Water Environ. Res. 69, 52e63. Kotula, A.W., Stern, N.J., 1984. The importance of Campylobacter jejuni to the meat industry: a review. J. Anim. Sci. 58, 1561e1566. Lambert, A.D., Smith, J.P., Dodds, K.L., 1991. Shelf life extension and microbiological safety of fresh meat e a review. Food Microbiol. 8, 267e297. Nissen, H., Sørheim, O., Dainty, R., 1996. Effects of vacuum, modified atmospheres and storage temperature on the microbial flora of packaged beef. Food Microbiol. 13, 183e191. Peterson, M.C., 2003. Campylobacter jejuni enteritis associated with consumption of raw milk. J. Environ. Health 65, 20e21.
PHAC, 2005. Notifiable Diseases On-Line. http://dsol-smed.hc-sc.gc.ca/dsol-smed/ ndis/c_indp_e.html. Robinson, D.A., 1981. Infective dose of Campylobacter jejuni in milk. Br. Med. J. 282, 1584. Russo, F., Ercolini, D., Mauriello, G., Villani, F., 2006. Behaviour of Brochothrix thermosphacta in presence of other meat spoilage microbial groups. Food Microbiol. 23, 797e802. SAS, 1989. SAS/STATÒ User’s Guide. SAS Publishing. Schildt, M., Savolainen, S., Hanninen, M.L., 2006. Long-lasting Campylobacter jejuni contamination of milk associated with gastrointestinal illness in a farming family. Epidemiol. Infect. 134, 401e405. Stern, N.J., 1981. Recovery rate of Campylobacter fetus ssp. jejuni on eviscerated pork, lamb, and beef carcasses. J. Food Sci. 46, 1291e1293. Stern, N.J., Patton, C.M., Doyle, M.P., Park, C.E., McCardell, B.A., 1992. Campylobacter. In: Vanderzant, C., Spittstoesser, D.F. (Eds.), Compendium of Methods for the Microbiological Examination of Foods. American Public Health Association, Washington, D.C. Sutherland, J.P., Patterson, J.T., Murray, J.G., 1975. Changes in the microbiology of vacuum-packaged beef. J. Appl. Bacteriol. 39, 227e237. Trachoo, N., Frank, J.F., Stern, N.J., 2002. Survival of Campylobacter jejuni in biofilms isolated from chicken houses. J. Food Prot. 65, 1110e1116. van der Meche, F.G., van Door, P.A., 2000. Guillain-Barré syndrome. Curr. Treat. Options Neurol. 2, 507e516. Van Laack, R.L.J.M., Johnson, J.L., Van Der Palen, C.J.N.M., Smulders, F.J.M., Snijders, J.M.A., 1993. Survival of pathogenic bacteria on pork loins as influenced by hot processing and packaging. J. Food Prot. 56, 847e851. Vanderlinde, P.B., Shay, B., Murray, J., 1998. Microbiological quality of Australian beef carcass meat and frozen bulk packed beef. J. Food Prot. 61, 437e443. Vold, L., Holck, A., Wasteson, Y., Nissen, H., 2000. High levels of background flora inhibits growth of Escherichia coli O157:H7 in ground beef. Int. J. Food Microbiol. 56, 219e225. Wong, T.L., Hollis, L., Cornelius, A., Nicol, C., Cook, R., Hudson, J.A., 2007. Prevalence, numbers, and subtypes of Campylobacter jejuni and Campylobacter coli in uncooked retail meat samples. J. Food Prot. 70, 566e573.
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Survival of Campylobacter jejuni on beef and pork under vacuum packaged and retail storage conditions: Examination of the role of natural meat microflora on C. jejuni survival Sampathkumar Balamurugan*, Frances M. Nattress, Lynda P. Baker, Bryan D. Dilts Agriculture & Agri-Food Canada, 6000 C & E Trail, Lacombe, Alberta, T4L 1W1 Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 5 November 2010 Received in revised form 25 January 2011 Accepted 27 January 2011 Available online 4 February 2011
The ability of Campylobacter jejuni ATCC 11168 to survive on beef and pork stored under chilled, vacuum packaged and retail display conditions were examined. In addition, the effect of natural microflora on commercial beef and pork on the survival of C. jejuni under these storage conditions was examined. When sterile cores of beef and pork were inoculated with w105 to 106 cfu cm2 C. jejuni, and were stored under aerobic or vacuum packaged conditions at 1.5 or 4 C, its numbers dropped significantly and C. jejuni could not be enumerated by direct plating after 21 d of the 6 wks study. In contrast, survival of C. jejuni on commercial vacuum packaged beef and pork was significantly enhanced, resulting in only 1 log cfu cm2 reduction at the end of 6 wks. During 7 d of display in a retail case, numbers of C. jejuni dropped quickly, but could be enumerated by direct plating even after the 7 d. The presence of high numbers of inoculated C. jejuni on beef and pork had no significant effect on the natural microflora numbers compared to uninoculated controls when the meat was stored either in vacuum or in a retail display case. These results show that natural microflora on vacuum packaged meat afford enhanced survival of C. jejuni present on the surfaces of both beef and pork when stored at refrigeration temperatures. Hence, strict hygienic practices or the implementation of decontamination technologies are recommended to ensure safety of meat with respect to this pathogen. Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
Keywords: Campylobacter jejuni Survival Vacuum packaging Retail display Natural meat microflora
1. Introduction Campylobacter jejuni is one of the leading zoonotic causes of human gastroenteritis in Europe and North America (CDC, 2010; EFSA, 2005; PHAC, 2005). It has a very low infectious dose (Black et al., 1988; Robinson, 1981) and in addition to the seldom fatal but severe gastroenteritis caused by this organism, the serious immunemediated Guillain-Barré syndrome, which is a chronic development in some previously infected individuals (van der Meche and van Door, 2000), make this organism a substantial public health concern. Based on collated epidemiological data from outbreaks, undercooked poultry is regarded as the major risk factor for foodborne campylobacteriosis (Fields and Swerdlow, 1999). In addition, outbreaks of infection due to C. jejuni have been linked to a number of sources including eggs (Finch and Blake, 1985), seafood (Feldhusen, 2000), unchlorinated water (Bopp et al., 2003; Jakopanec et al., 2008; Koenraad et al., 1997) and raw milk (Heuvelink et al., 2009; Peterson, 2003; Schildt et al., 2006). C. jejuni is often present in the
* Corresponding author. Tel.: þ1 403 782 8119; fax: þ1 403 782 6120. E-mail address: [email protected] (S. Balamurugan).
gastrointestinal tracts (Gill and Harris, 1982a; Hudson et al., 1999; Inglis and Kalischuk, 2003, 2004; Inglis et al., 2005; Van Laack et al., 1993) and on carcasses (Gill and Harris, 1982a; Kotula and Stern, 1984; Stern, 1981) of meat animals. It has also been isolated from bulk packed red meats (Vanderlinde et al., 1998) and retail ready meats (Wong et al., 2007). A large proportion of Canadian beef and pork is delivered to retail stores sealed in vacuum packages. The meat is subsequently opened at retail and cut into steaks, chops and roasts, which are typically repackaged in an oxygen permeable overwrapped film and placed in a retail display case. With the elimination of oxygen in vacuum packaged meat, the growth of aerobic spoilage organisms is significantly reduced, thereby extending product storage life (Lambert et al., 1991). The storage life of vacuum packaged fresh primals and subprimals is generally reported as approximately 35e45 d, while the expected shelf life of product packaged in oxygen permeable overwrap at retail is 3e7 d. In the meat industry, vacuum packaging and storage at strictly controlled temperatures of 1.5 C are widely used to store and export raw meat. A number of studies have raised concerns of the potential of these packaging conditions to increase the risk of campylobacteriosis by allowing the growth of C. jejuni (Gill and Harris,
0740-0020/$ e see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2011.01.012
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1982b; Hänninen et al.,1984; Van Laack et al.,1993). However, studies investigating the effect of vacuum and modified atmosphere packaging on the survival of C. jejuni on meat (Gill and Harris, 1982b; Hänninen et al., 1984; Van Laack et al., 1993) have collectively indicated that chill storage under vacuum or modified atmosphere is likely to increase the safety of meat. More recently in 2001, a study investigating the effect of these preservative packing of meat at a strictly controlled 1.5 C on survival of C. jejuni showed no significant changes in numbers of this pathogen (Dykes and Moorhead, 2001), raising causes for concern. C. jejuni is fastidious with respect to growth requirements, is sensitive to oxygen (Stern et al., 1992) and only grows in the temperature range of 30e44 C. Therefore, survival rather than growth on meat is the concern. While growth of C. jejuni will not occur at chill temperatures, it is necessary to understand the nature and extent of the effect of different meats, the natural bacterial populations on these meats and modern commercial meat packaging and storage practices on the survival of this pathogen. Studies to date have focused on the effect of storage parameters rather than the influence of the natural bacterial populations on beef and pork on the survival of C. jejuni in vacuum packaged and retail display conditions. Hilbert et al. (2010) noted the importance of the interaction between spoilage bacteria and C. jejuni. They demonstrated in vitro that coincubation of C. jejuni with Pseudomonas spp. enhanced the ability of C. jejuni to survive aerobic incubation at 35 C. Coincubation did not affect the growth of Pseudomonas spp. The aim of the present study was to examine the survival of C. jejuni on beef and pork under vacuum packaged and retail display conditions at normal meat storage temperatures. In addition, the effect of natural microflora on survival of C. jejuni on beef and pork was examined.
were then transferred to 800 600 commercial barrier bags (oxygen transmission rate of 40e50 cc m2 24 h1; Winpak Ltd., Winnipeg, MB Canada) for vacuum packing (Multivac AGI, Knud Simonsen Industries Ltd, Rexdale, ON Canada) The vacuum packaged cores were incubated at 1.5 or 4.0 C and C. jejuni numbers enumerated or recovered using enrichment over a 6 wks storage time. To enumerate C. jejuni, cores were removed aseptically from the vacuum packs, transferred to 90 ml 0.1% sterile peptone water and mixed in a Seward stomacher (Fisher Scientific) at medium setting for 2 min. Appropriate dilutions were plated on Campylobacter Blood-Free Selective Agar (mCCDA; Oxoid) plates and the plates were incubated under microaerophilic condition for 48 h at 42 C. Another core was transferred to Brucella broth containing Campylobacter Growth Supplement (Oxoid; SR0232) and enriched for Campylobacter spp. under microaerophilic conditions at 42 C for 48 h followed by swabbing onto mCCDA plates to detect presence or absence of Campylobacter spp. The mCCDA plates were incubated under microaerophilic conditions for 48 h at 42 C. To examine the effect of oxygen permeability through the packaging material on C. jejuni survival, the above study was repeated with inoculated and uninoculated cores that were overwrapped with oxygen permeable, polyvinyl chloride film (oxygen transmission rate of 8000 cc m2 24 h2; Vitafilm Choice Wrap, Goodyear Canada Inc., Toronto, ON) or vacuum packaged in oxygen impermeable barrier bags that had a very low oxygen transmission rate of 0.4 cc m2 24 h1 (Winpak). These studies were repeated 3 times and final bacterial counts are means of these 3 replications.
2. Materials and methods
Fresh vacuum packed beef and pork loins were obtained from a commercial meat plant. They were transported to the Lacombe Research Centre under refrigerated conditions and were stored at 1.5 C overnight. These loins were cut into pieces weighing w500 g (w750 cm2) each using sterile knives and placed in commercial barrier bags. The pH values of muscle tissue were determined before inoculation for each incubation temperature. A 7.5 ml volume of the C. jejuni suspension (prepared as described in Section 2.1) was inoculated onto each of the pieces to a final level of w105e106 cfu cm2. The inoculum was massaged onto the pieces before vacuum packing. The vacuum packed bags were incubated at 1.5 or 4.0 C for up to 6 wks. Uninoculated control pieces were similarly processed by adding 7.5 ml of sterile water. Enumeration of Campylobacter spp. and background bacterial counts was performed on both the inoculated and uninoculated samples over the 6 wks storage period. To enumerate Campylobacter spp. and background bacterial counts, two 10 cm2 cores were removed aseptically from the meat at times 0, 2, 5, 7, 9, 12,14, 21, 28, 35 and 42 d. They were transferred to 90 ml 0.1% sterile peptone water and mixed in a stomacher at medium settings for 2 min. Appropriate dilutions were plated on selective and non-selective media. Campylobacter spp. was enumerated using mCCDA, incubated under microaerophilic conditions at 42 C for 48 h. Selective media described by Baird et al. (1987) were used to enumerate Brochothrix spp., Pseudomonas spp., lactic acid bacteria and Enterobacteriaceae. Total aerobes were enumerated using TSA plates incubated for 48 h at 25 C. Streptomycin thallous acetate agar (STAA; Oxoid) and cephaloridine fucidin cetrimide agar (CFC; Oxoid) was used to enumerate Brochothrix spp. and Pseudomonas spp., respectively, by incubating the plates at 25 C for 48 h. Enumeration of presumptive lactic acid bacteria (LAB) was done on de Man, Rogosa and Sharpe agar (MRSA; Difco) with a 72e96 h anaerobic incubation at 25 C. Enterobacteriaceae were enumerated by plating on Violet Red Bile Glucose agar (VRBGA; BD-Difco, BD Canada, Mississauga, ON Canada) with 18e24 h anaerobic incubation at 35 C. Anaerobic conditions were
2.1. Bacteria and culture media C. jejuni NCTC 11168 (ATCC 700819) was used in this study. The C. jejuni culture was maintained at 80 C and resuscitated by streaking on tryptic soy agar plates (TSA, Oxoid, Nepean, ON Canada) at 42 C for 24 h under microaerophilic conditions generated using CampyGenÔ (Oxoid) gas generation sachets. An isolated colony was transferred to 75 ml tryptic soy broth (TSB, Oxoid) containing Campylobacter Growth Supplement (Oxoid, SR0117) and incubated under microaerophilic conditions for 48 h at 42 C. Subsequently, 750 ml of this culture was transferred to 75 ml of TSB with Campylobacter Growth Supplement and incubated under microaerophilic conditions for 24 h at 42 C. Cells were harvested by centrifugation and resuspended in 0.1% sterile peptone water (same volume as supernatant discarded). This culture was used to inoculate meat in all our studies. 2.2. C. jejuni inoculation and survival on sterile beef and pork Fresh boneless beef and pork loins (Longissimus dorsi) of normal muscle quality were obtained from the Lacombe Research Centre abattoir. Sterile beef and pork muscle tissue discs (10 cm2) were aseptically excised from the meat as previously described (Greer and Dilts, 1995). The pH values of muscle tissue discs were determined. Surface pH was measured using an Accumet AP61 portable pH meter (Fisher Scientific, Ottawa, ON Canada) equipped with a flat surface electrode (Accumet 13-620-289, Fisher Scientific). Each core was placed individually on a styrofoam tray (Scott National Food Service, Edmonton, AB Canada) and was inoculated with 100 ml volume of the C. jejuni suspension (prepared as described in section 2.1) to a final level of w105 to 106 cfu cm2. The bacteria were allowed to adhere for 5 min. The trays with the inoculated or uninoculated control cores
2.3. C. jejuni inoculation and survival on commercial vacuum packaged beef and pork
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established using a BBL anaerobic gas pack system (BD-BBL, BD Canada). Psychrotrophic bacteria were enumerated by plating on TSA and incubating plates at 4 C for 10 d. These studies were repeated 3 times and final bacterial counts are means of these 3 replications. 2.4. C. jejuni inoculation and survival under retail display storage conditions Vacuum packed fresh beef and pork loins were obtained from a commercial meat plant and stored at 1.5 C overnight. These loins were prepared as described in 2.3. At 2 wks intervals (0, 2, 4, and 6 wks), 5 meat samples were removed from their vacuum packages, placed on styrofoam trays and overwrapped with oxygen permeable, polyvinyl chloride film (oxygen transmission rate of 8000 cc m2 24 h2; Vitafilm Choice Wrap, Goodyear Canada Inc., Toronto, ON) and placed in a retail display case (Williams Restaurant Equipment, Winnipeg, MB Canada) set to operate at 4 C. At days 0, 2, 5 and 7 one sample was removed. Bacterial numbers were determined as described in Section 2.3 and final pH was measured. These studies were repeated 3 times and final bacterial counts are means of these 3 replications. 2.5. Statistical analysis Numbers of bacteria were transformed to log10 cfu cm2 for statistical analysis. The limit of detection by the direct plate counting
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method was 2 log cfu cm2. When cells could not be detected by the direct plate counting method but were recovered on plates following enrichment of the samples for C. jejuni, the cell numbers were regarded as 1 (0 log cfu cm2) but when the cells became unrecoverable even after enrichment the cell numbers were regarded as 0.1 (1 log cfu cm2). General Linear Models Analysis of Variance Procedure (ANOVA) of the Statistical Analysis System (SAS, 1989) was applied to the significance of differences, if any, between bacterial counts, temperatures, meat types and packaging conditions. Significance was based on a level of 5.0% (P < 0.05). 3. Results 3.1. C. jejuni on sterile beef and pork Changes in numbers of C. jejuni cells inoculated on sterile beef and pork cores or commercial beef and pork, packaged and stored under different conditions are presented in Fig. 1. The numbers of C. jejuni inoculated on sterile beef and pork cores stored under aerobic or vacuum packaged conditions at 1.5 or 4 C dropped significantly (P < 0.05) over the time of the study. C. jejuni could not be recovered from beef cores by direct plating after 7 and 9 d during aerobic storage at 1.5 and 4 C, respectively, and were unrecoverable by enrichment after 14 d at the two storage temperatures. C. jejuni could not be recovered from beef cores by direct plating after 9 and 14 d when the beef was vacuum packaged using commercial barrier bags and stored at 1.5 and 4 C, respectively,
Fig. 1. Numbers of C. jejuni surviving on sterile beef and pork cores inoculated with C. jejuni and packaged by overwrapping using oxygen permeable polyvinyl chloride film (aerobic; C), vacuum packaged using commercial (-) or oxygen impermeable barrier bags (A), and on commercial beef and pork inoculated with C. jejuni and vacuum packaged using commercial barrier bags (:) and stored at 1.5 or 4 C. Data are least square means of bacterial numbers. Standard error (SE) ¼ 0.36.
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and were unrecoverable by enrichment after 21 and 28 d at the two storage temperatures. C. jejuni could not be recovered from beef cores by direct plating after 14 and 35 d when beef was vacuum packaged using oxygen impermeable barrier bags and stored at 1.5 and 4 C, respectively, and were recoverable by enrichment throughout the length of the study (42 d) at the two storage temperatures. Similarly, on pork cores, C. jejuni could not be recovered after 7 and 9 d by direct plating during aerobic storage at 1.5 and 4 C, respectively, and were unrecoverable by enrichment after 14 d at the two storage temperatures. During vacuum packaging using commercial barrier bags C. jejuni could not be recovered by direct plating after 21 and 14 d at 1.5 and 4 C, respectively, and were unrecoverable by enrichment after 28 and 21 d at the two storage temperatures. However, during vacuum packaging using oxygen impermeable barrier bags C. jejuni could not be recovered after 35 and 21 d at 1.5 and 4 C, respectively, by direct plating and was recoverable by enrichment throughout the length of the study for both temperatures. Use of oxygen impermeable barrier bags significantly (P < 0.05) increased survival of C. jejuni compared to aerobic and vacuum packaging using commercial barrier bags in both beef and pork cores at both temperatures tested. 3.1.1. C. jejuni on commercial beef and pork The examination of the effect of the presence of natural microflora on C. jejuni numbers on beef and pork vacuum packaged using commercial barrier bags revealed that the numbers remained high enough to be enumerated using direct plating (Fig. 1) and the rate of decline in numbers of C. jejuni was slow and was not affected by meat type or storage temperature (P < 0.05). During the entire
6 wks storage time, numbers of C. jejuni only dropped about 1 log cfu cm2. 3.1.2. Natural microflora on commercial beef and pork The numbers of the mesophilic and psychrotrophic meat microflora increased on both beef and pork vacuum packaged and stored at 1.5 and 4 C, but were generally higher on meat stored at 4 C at the end of 6 wks (data not shown). On beef, growth of Enterobacteriaceae was inhibited at 1.5 C while at 4 C their numbers increased approximately 3 log cfu cm2. On pork, their numbers increased about 4 log cfu cm2. Similarly, growth of presumptive lactic acid bacteria and Pseudomonas spp. was less at 1.5 C when compared to 4 C and numbers on pork were higher than those on beef (Fig. 2). On both beef and pork, numbers of Brochothrix spp. increased and reached a maximum at around 14e21 d following which numbers declined or showed no significant (P > 0.05) increase at both 1.5 and 4 C (Fig. 2). 3.2. C. jejuni on beef and pork under retail display storage conditions When meat was removed from vacuum packs, repackaged in oxygen permeable film and placed in a retail display case, numbers of C. jejuni dropped quickly. However, even after 7 d, they could still be enumerated by direct plating (Fig. 3). Presumptive lactic acid bacteria, Brochothrix spp., Pseudomonas spp. and Enterobacteriaceae all grew to higher numbers in response to the change in packaging conditions (Fig. 4). In general, numbers of all bacterial groups were lower when meat was removed after storage at 1.5 C than when it
Fig. 2. Numbers of Enterobacteriaceae [A; SE: 0.30 (beef) 0.37 (pork)], Brochothrix spp. [C; SE: 0.43 (beef) 0.56 (pork)], Pseudomonas spp. [-; SE: 0.45 (beef), 0.38 (pork)] and presumptive lactic acid bacteria [:; SE: 0.54 (beef), 0.41 (pork)] on commercial beef and pork in vacuum packages stored at 1.5 or 4 C. Data are least square means of bacterial numbers from samples that were and were not inoculated with C. jejuni since there was no effect of C. jejuni on growth of natural microflora (P > 0.05).
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was stored at 4 C but by the end of 7 d retention in a retail display case, all samples had similar numbers of each group of bacteria (Fig. 4). An analysis of the effect of natural meat microflora on C. jejuni survival on beef and pork stored under aerobic conditions revealed that C. jejuni survival was not significantly (P > 0.05) affected by the presence of the natural meat microflora compared to sterile meat inoculated with C. jejuni (Fig. 5). None of the bacterial groups numbers was affected by the presence of the high number of inoculated C. jejuni (P > 0.05) when stored either in vacuum or in retail display case compared to uninoculated controls (data not shown). Similarly, there was no effect of the presence of high numbers of C. jejuni on pH (P > 0.05) at any of the vacuum packaged and retail display sampling times (data not shown).
4. Discussion As indicated in the Introduction, studies investigating the effect of vacuum and modified atmosphere packaging on the survival of C. jejuni have collectively indicated that chill storage under vacuum or modified atmosphere is likely to increase the safety of meat (Gill and Harris, 1982b; Hänninen et al., 1984; Van Laack et al., 1993). However, it has to be pointed out that the chill temperatures examined in these studies were in the range of 1e4 C, while Gill and Harris (1982b) studied C. jejuni survival on fresh meat at 37 C and sterile meats at 1 or 18 C stored under aerobic conditions. These
Fig. 3. Numbers of C. jejuni surviving on commercial beef and pork inoculated with C. jejuni that were stored in vacuum packages at either 1.5 or 4 C for up to 6 wks, removed, wrapped in oxygen permeable polyvinyl chloride film and stored in retail display case for up to 7 d. 0 time (A), 2 wks (-), 4 wks (:) and 6 wks (C). Data are least square means of bacterial numbers.
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studies do not reflect the combination of parameters now widely used in the meat industry to store and export product, namely vacuum packaging at a strictly controlled 1.5 C. There are no studies to our knowledge that have examined the effect of storage conditions and chill storage on C. jejuni survival on meat lacking the native microflora. More recently, results showing no significant changes in numbers of C. jejuni during storage at 1.5 C on beef packaged under vacuum (Dykes and Moorhead, 2001) raised serious questions regarding the safety of current transport and storage regimes. The authors had indicated that the interaction between natural bacteria populations and Campylobacter spp. would be of interest, but did not examine this and instead focussed on the effect of storage parameters alone. This precisely was the focus of this study, and the results clearly demonstrate that the natural microflora on vacuum packaged beef and pork enhances the survival of C. jejuni. However, the mechanism involved in this enhanced survival is unclear. C. jejuni has been shown to survive better within preexisting biofilms, indicating survival due to a protective biofilm (Trachoo et al., 2002). Gill and Harris (1982b) hypothesized that “increasing numbers of spoilage bacteria may compete with, and reduce, numbers of Campylobacter spp., or alternatively make them difficult to recover”. This has not been the observation in the present study since C jejuni numbers could be enumerated by direct plating for the entire 6 wks of the study. It has been suggested that the reduction of oxygen concentrations by competitor microorganisms may protect associated pathogens from cold stress (Aldsworth et al., 1998). It could therefore be hypothesized that since C. jejuni is a microaerophile, the natural microflora growing on vacuum packaged meat sequesters the oxygen that manages to pass through the barrier bags, and provides a protective environment resulting in enhanced survival of C. jejuni. This was tested by repeating C. jejuni survival studies on sterile beef and pork cores vacuum packaged using oxygen impermeable barrier bags, and it was expected that C. jejuni survival would be similar to its survival observed in the presence of natural microflora. Although survival was significantly enhanced, compared to survival on sterile meat that was inoculated, in aerobic or vacuum packaging using commercial barrier bags (higher oxygen transmission), they were not similar to vacuum packaged (using commercial barrier bags) meat containing natural microflora (Fig. 1). These results indicate that only part of the protective effect of the natural microflora can be explained by protection of C. jejuni from oxygen. There may be other factors that remain to be explained. The microflora of vacuum packaged chill stored beef and pork has been extensively studied and it was found that lactic acid bacteria are the most predominant (Borch et al., 1996; Hammes et al., 1992; Jones, 2004; Sutherland et al., 1975). Vacuum packaging results in reduced oxygen availability and in combination with storage at 1.5 C restricts the growth of other spoilage bacteria like Pseudomonas spp., Brochothrix thermospacta and Enterobacteriaceae resulting in extending the shelf life of the product to more than 8 wks (Borch et al., 1996; Gill, 1996; Jones, 2004; Nissen et al., 1996). Increases in numbers of lactic acid bacteria have been shown to decrease growth of B. thermospacta (Russo et al., 2006) and have been implicated in controlling growth of some meatborne pathogens (Nissen et al.,1996; Vold et al., 2000). However, in the present study, although growth of Brochothrix was inhibited (Fig. 2) past 14 d under vacuum, the increase in lactic acid bacteria did not have an inhibitory effect on C. jejuni, but on the contrary was protective. In addition, the absence of a difference between numbers of different bacterial groups on meats inoculated with C. jejuni as compared to uninoculated meat indicates that the introduced C. jejuni also had no impact on the numbers and survival of different bacterial groups on vacuum packaged beef and pork. In retail, beef and pork is displayed in aerobic packs and also held at higher temperatures of 4e8 C, which results in the increase of
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Fig. 4. Numbers of Brochothrix spp., Enterobacteriaceae, presumptive lactic acid bacteria, and Pseudomonas spp. on commercial beef and pork that were stored in vacuum packages at either 1.5 or 4 C for up to 6 wks, removed, wrapped in oxygen permeable polyvinyl chloride film and stored in retail display case for up to 7 d. 0 time (A), 2 wks (-), 4 wks (:) and 6 wks (C). Data are least square means of bacterial numbers from samples that were and were not inoculated with C. jejuni since there was no effect of C. jejuni on growth of natural microflora (P > 0.05).
spoilage bacteria like Pseudomonas spp. B. thermospacta and Enterobacteriaceae due to the availability of oxygen (Blixt and Borch, 2002; Borch et al., 1996; Gill and Newton, 1977). These observations agree with results from this study and the higher numbers of inoculated C.
jejuni had no impact on survival or numbers of these predominant bacteria compared to uninoculated controls during retail display. In retail stores, meat is removed from vacuum packs and repackaged in oxygen permeable film and placed in a retail display
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Fig. 5. Numbers of C. jejuni surviving on beef (A) and pork (B) stored under aerobic conditions at 4 C. Sterile cores (A; data from Fig. 1) or commercial meat (-; data from 0 time in Fig. 3) were inoculated with C. jejuni. Data are least square means of bacterial numbers.
case. The enhanced survival of C. jejuni on vacuum packaged beef raises questions about the safety of meat in retail display cases. This is the first study that has examined the survival of C. jejuni by simulating the transfer of meat from vacuum packs to retail display. Although C. jejuni numbers decrease rapidly during retail display storage, the results are cause for concern since C. jejuni could be enumerated even after 7 d (retail shelf life of beef and pork). It might be argued that the levels of inoculum used in the present study were relatively high compared to levels of natural contamination and that this may affect the results. However, since all previous studies on C. jejuni survival on meat used similar levels, this is unlikely to be the case. A recent study by Hilbert et al. (2010) examined the survival of C. jejuni in the presence of meat spoilage bacteria and extended survival when incubated aerobically with various species of Pseudomonas was reported. Pseudomonas spp. is the most predominant bacteria present on retail beef and pork and numbers were in the range of 7e8.5 log cfu cm2 at the end of 7 days in retail in the present study. However, our results do not show significant differences (>0.05) in the survival of C. jejuni on meat incubated aerobically either in the presence or absence of natural meat microflora. It is necessary to point out that studies by Hilbert et al. (2010) were performed in broth (neutral pH) and at 35 C. These conditions do not reflect meat (acidic) or meat storage temperatures (1.5 or 4 C) which were examined in the present study. The results of the present study might be criticized that they are from a single strain. Strain specific variations in the survival of Campylobacter spp. from natural environment, farm animals, clinical and laboratory strains on preservative packaged chill stored meat have been previously reported (Dykes and Moorhead, 2001; Gill and Harris, 1982b; Hänninen et al., 1984). However, since C. jejuni cannot grow at the conditions examined (Stern et al., 1992) in the present study, the role of the natural meat microflora on the survival of C. jejuni on preservative packaged chill stored meat is nevertheless established. The low infective dose, the frequent contamination of poultry, and the increasing numbers of reports of the occurrence on other meats, and the apparent ability of C. jejuni to survive commercial meat storage conditions, suggest that the current methods of fresh meat preservation will not add a significant margin of safety with respect to C. jejuni. Instead, control of C. jejuni to protect the consumer will require an integrated approach throughout the meat production, processing, and sale continuum by adapting improved hygienic practices or through decontamination technologies which currently are the only way to avoid food safety concerns arising with respect to this pathogen.
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