Effects of storage and the presence of a beef microflora on the thermal resistance of Salmonella Typhimurium DT104 in beef and broth systems
Máiréad McCann, The Ashtown Food Research Centre, Teagasc, Ashtown, Dublin 15, Ireland. E-mail: email@example.com
Aims: To investigate the effects of storage and the presence of a beef microflora on the thermal resistance of Salmonella serotype Typhimurium DT104 on beef surfaces and in a broth system during subsequent heat treatments after extended low-temperature storage (4°C for 14 days) or mild temperature abuse (10°C for 7 days).
Methods and Results: Surviving Salm. Typhimurium DT104 cells were estimated after heating in a water bath (55°C) by plating beef and broth samples on tryptone soya agar and overlaying with xylose–lysine–deoxycholate agar. In beef and broth systems, D55 values for Salm. Typhimurium DT104 stored at 4°C or 10°C in the presence or absence of a beef microflora were significantly lower (P < 0·01) than the D values for this organism heat-treated immediately after inoculation. In beef systems, the D55 values were significantly lower (P < 0·05) in the presence of a beef microflora than the D55 values obtained in ‘pure’ culture under all temperature/storage combinations. However, in broth systems, there was no significant difference between the D55 values obtained in ‘pure’ culture and the D55 values obtained from systems containing beef microflora.
Conclusions: Storage of Salm. Typhimurium DT104 significantly reduced the thermal resistance of the pathogen in beef and broth systems. In the presence of high numbers of a Gram-negative beef microflora, the heat sensitivity of the pathogen was further increased on beef surfaces but not in broth.
Significance and Impact of the Study: Studies investigating the survival of Salm. Typhimurium DT104 in different food systems will help define safe food preservation processes and will aid in the elimination this pathogen from the food production environments.
Many food products are pasteurized to destroy foodborne zoonotic pathogens such as Salmonella spp. and to extend product shelf life by significantly reducing the number and impact of surviving spoilage micro-organisms. However, such treatments can have undesirable effects on a range of other important food characteristics such as colour, flavour and texture. Therefore, it is of considerable interest to industry and public health agencies to be able to accurately identify and ensure the consistent application of thermal treatments, which achieve the former (desirable) effects while avoiding or at least limiting the latter (undesirable) effects. The key factor in this delicate balance, i.e. the thermal resistance of pathogens, has been most widely studied in broth-based systems (Blackburn et al. 1997; Duffy et al. 1999a; Whiting and Golden 2002; Bacon et al. 2003), but it is becoming clearer that such models may not allow accurate predictions of thermal resistance in complex systems, such as meat (Humphrey et al. 1997; Quintavalla et al. 2001). In addition, most meat-based studies are carried out on minced meat, rather than solid meat, probably because of difficulties in measuring surface temperatures on solid meat pieces. Thus, most determinations of the thermal resistance of pathogens in meat use models are one or more stages removed from the conditions that prevail in solid meat systems. These may be subject to significant inaccuracies in terms of estimation of pathogen resistance in such systems leading to uncertainties in the assessment and control of associated risks. Considering the potential impact of heat treatment, i.e. ‘unsuccessful pasteurization’ in terms of pathogen survival and human infection, such uncertainties are a matter of concern.
Such concerns in relation to effective control of foodborne pathogens have been increased in recent years by the increasing emergence of antibiotic resistance. For example, challenges presented by members of the genus Salmonella, long recognized as internationally important human and animal pathogens, have been increased by the emergence of serotypes such as Salmonella serotype Typhimurium DT104, which has been shown to be resistant to at least five antibiotics (Humphrey 2001). Human infections with this pathogen have been linked to the consumption of contaminated beef, pork and poultry (Wall et al. 1994; Davies et al. 1996; Ethelberg 2005). Such contamination is frequent and Salm. Typhimurium is currently the predominant serotype isolated from Irish cattle and pigs (O'Hare et al. 2004). A study of Irish beef abattoirs demonstrated that 7·6% of hygienically produced beef carcasses were contaminated with Salmonella, the majority of which were multi-antibiotic-resistant strains of Salm. Typhimurium DT104 (Kerr and Sheridan 2002). It is therefore particularly disconcerting to note that this frequently occurring and multiple antibiotic-resistant serotype has also been reported to be more heat resistant than many other Salmonella strains (Humphrey 2001; Walsh et al. 2005), increasing the importance of investigations to derive accurate data and models to underpin the development and application of consistently efficient and effective thermal treatments for the pasteurization of meat and meat products.
These noted difficulties in relation to modelling the thermal inactivation of pathogens in solid food systems are further complicated by the increasing recognition of the effects of the nature and numbers of other nonpathogenic bacteria present in food systems. Thus, a number of studies have revealed that the presence of other micro-organisms can influence the growth and death rates of pathogens (Duffy et al. 1995,1999b; Vold et al. 2000; Berry and Koohmaraie 2001; Tamplin 2002).
Duffy et al. (1995) confirmed that the presence of high concentrations (>8 log units) of other microbes in a test medium reduced the rate of thermal inactivation of Salmonellae. Similarly, Tamplin (2002) investigating another closely related enteric pathogen (Escherichia coli O157:H7) demonstrated that higher background microflora populations were associated with reduction in maximum potential growth rate of E. coli O157:H7 in raw ground beef stored at 10°C. Berry and Koohmaraie (2001) reported that the presence of the beef microflora appeared to delay the growth of E. coli O157:H7 on beef carcass tissue stored at 12°C, but that such effects did not occur in the same meat system at 4°C. Such results underline the complexity of interactions among pathogens, the wider beef microflora and other intrinsic and extrinsic factors including the nature/form of food matrices and environmental conditions in the development and persistence of pathogens in food products. They also confirm the need for improvement and validation of existing broth-based models for pathogen survival and growth in realistically complex food systems.
This study aimed to determine the effects of a number of these factors on the thermal resistance of Salm. Typhimurium DT104. The pathogen was inoculated into broth and a solid meat system, in the presence or absence of natural beef microflora, stored under simulated standard refrigeration temperatures (4°C for 14 days) or simulated conditions of mild thermal abuse (10°C for 7 days) and heat-treated at 55°C. Surviving pathogen numbers were estimated and used to calculate D55 values as indices of thermal sensitivity.
Materials and methods
Natural microflora from beefMusculus tensor fascia latae (knuckle from beef hind quarter) was collected from a local commercial abattoir 24 h post-slaughter, transported to the laboratory at 8–10°C within 1 h and stored in a chill at 4°C until use. Samples of the microflora of this material were recovered by an adaptation of the method of Berry and Koohmaraie (2001), in which 30 g excised samples were aseptically homogenized with 270 ml of brain–heart infusion broth (BHI; Oxoid, Basingstoke, UK) for 1 min in a Colworth stomacher (Model BA 6024; A.J. Steward & Company Ltd, London, UK) and incubated for 48 h at 25°C. Bacteria were recovered from the resultant culture by centrifugation at 4500 g for 10 min, washed three times and resuspended in 9 ml of maximum recovery diluent (MRD; Oxoid). This suspension was confirmed as Salmonella negative using standard ISO 6579 (Anon 2004). Aliquots (1 ml) were used to inoculate cryoprotective beads (Technical Service Consultants Ltd, Heywood, UK) and stored at −20°C prior to use.
Salmonella Typhimurium DT104 The Salm. Typhimurium DT104 isolate used in this study was from the Ashtown Food Research Centre culture collection. This bovine isolate was naturally resistant to ampicillin; streptomycin sulfate; sulfonamides; chloramphenicol; tetracycline; rifampicin and erythromycin. The culture was stored on cryoprotective beads at −20°C prior to use.
Preparation of inocula
Beads (six) of Salm. Typhimurium DT104 were incubated in 30 ml of BHI broth at 37°C for 24 h, and subsequently inoculated (1 ml vols) into 12 × 99 ml vols of BHI broth. These were incubated at 37°C for 16 h and centrifuged at 4500 g for 10 min at 4°C. Salmonella Typhimurium DT104 cell pellets were washed three times in 10 ml vols of MRD, recovered by centrifugation as above, combined and resuspended in 10 ml of MRD. A bulk suspension of beef microflora was prepared by the same procedure. The beef microflora was grown at 37°C, to encourage the growth of the enteric bacteria present in the natural beef microflora, as these were previously found to inhibit the growth of E. coli O157:H7 (Duffy et al. 1999b). These suspensions were diluted in 9 ml vols of MRD to an approximate concentration of 9 log10 CFU ml−1 for both the Salm. Typhimurium DT104 and the beef microflora. Cell numbers in these suspensions were verified by spiral plating appropriate dilutions (Whitley Automatic Spiral Plater; Don Whitley Scientific, West Yorkshire, UK) on tryptone soya agar (TSA, Oxoid) plates. From these suspensions, inocula containing either Salm. Typhimurium DT104 (S) or Salm. Typhimurium DT104 combined with the beef microflora (SM) were prepared for inoculation of beef and broth.
Sample preparation and irradiation Chilled (4°C) beef samples were sliced using a Cookworks detachable slicer (Argos, Stafford, UK), adjusted to produce slices, which were approx. 2 mm thick. Meat samples (3 × 3 cm, approx. 1·0 g), cut from these meat slices using a sterile metal template and scalpel, were placed in vacuum bags (LDPE vacuum bags, Cryovac BB4; Cole-Palmer, London, UK, 12·5 × 7·7 cm), vacuum packed (Vac Star S220; Vacuum Packer, Sugiez, Switzerland, Level 5, Temperature 5, 15 psi). Vacuum packs were placed in sterile stomacher bags in batches of 100 samples. These were chilled to 4°C within 1 h and transported under refrigeration (<10°C) to an irradiation facility at Queen's University, Belfast. Meat samples were subjected to ionizing radiation (6·0 kGy h−1) by exposure to a gamma beam 650-irradiation unit containing cobalt-60 for 6 h. The irradiation temperature was 4°C and the average dose absorbed by the samples was calculated to be 29·0 kGy. Irradiated samples were returned to the Ashtown Food Research Centre under refrigeration within 6 h. Samples were tested for sterility by the random extraction and microbiological analysis of ten meat samples from each treated batch. Samples were pulsified (Filtoflex Ltd, Almonte, ON, Canada) in 10 ml MRD in a sterile stomacher bag for 1 min, spiral plated (Whitley Automatic Spiral Plater) in duplicate onto plate count agar (PCA; Oxoid) and incubated at 30°C for 72 h.
Inoculation and storage The seal of each irradiated vacuum-packaged beef sample was aseptically removed on a sterile plastic tray in a laminar flow cabinet (Nuaire, NU-425-400E; Unitech Ltd, Dublin, Ireland). Beef samples in vacuum packages were point-inoculated, with 100 μl of either inoculum S or SM, to give a surface count of approx. 8 log10 CFU cm−2 of Salm. Typhimurium DT104. Samples were left for 15 min at room temperature (21–23°C) to facilitate bacterial attachment to beef surfaces. Inoculated meat samples were re-vacuumed, chilled for 15 min in an ice water bath to approx. 4°C. Samples were heat-treated immediately as described in the following section or stored aerobically unsealed in sterile Petri dishes at 4°C for 14 days or 10°C for 7 days. After storage, aerobic meat samples were re-vacuumed, chilled and heat-treated as described.
Heat treatment Chilled vacuum-packaged meat samples were fully submerged in a temperature-controlled water bath (Grant Y28; Grant Instruments, Cambridge, UK) adjusted to 55°C ± 0·1°C. Two T-Type copper–constantan thermocouples were placed at each end of the water bath to monitor the temperature throughout the heat treatments. The average surface temperatures of meat samples were estimated by inserting thermocouples just below the surface of three uninoculated vacuum-packed meat samples. Temperature readings were recorded every second using a Grant Squirrel 1000 series Data Logger (Grant Instruments). Duplicate meat samples were removed at designated time intervals up to 14 min, cooled to 4°C within 60 s by submerging in iced water (0–1°C) and examined within 30 min as described in the following section.
Inoculation, storage and heat treatment BHI broth (22 ml vols) were prepared, dispensed into glass test tubes with rubber stoppers and were sterilized by autoclaving at 121°C for 15 min. Sterility of BHI broth was tested by spiral plating duplicate samples onto PCA and incubated at 30°C for 72 h.
BHI broth samples were either
- •Equilibrated to 55°C ± 0·1°C in a temperature-controlled water bath, inoculated with 100 μl of either inoculum S or SM (to give an initial count of approx. 8 log10 CFU ml−1 of Salm. Typhimurium DT104), stirred with a sterile inoculating loop to ensure adequate mixing and held at 55°C ± 0·1°C for up to 14 min. The average temperature of inoculated broth was monitored as described using thermocouples being inserted into three uninoculated BHI samples. Duplicate 1 ml samples of inoculated broth were aseptically removed and transferred into sterile centrifuge tubes at designated time intervals up to 14 min, cooled to 4°C within 60 s by placing centrifuge tubes in ice water (0–1°C ± 1°C) and examined within 30 min, as described in the following.
- •Inoculated at room temperature (21°C–23°C) with 100 μl of either inoculum S or SM and stored aerobically at 4°C for 14 days or at 10°C for 7 days. Bacteria in inoculated BHI broth were recovered after storage by centrifugation at 4500 g for 10 min at 4°C. The resulting pellets from each treatment were resuspended in 100 μl of MRD, inoculated into 22 ml vols sterile BHI broth preheated to 55°C ± 0·1°C, heat-treated and chilled and examined within 30 min, as described in the following section.
Monitoring changes in Salmonella Typhimurium DT104 and the beef microflora during storage
Non-heat-treated beef and broth samples, inoculated with either inoculum S or SM, were monitored for changes in bacterial counts during storage. Beef and broth samples were examined at 0, 3, 7, 9 and 14 days during storage at 4°C for 14 days and at 0, 3 and 7 days during storage at 10°C for 7 days.
Recovery and enumeration of surviving bacteria
Heat-treated, chilled meat samples and non-heat-treated samples were placed in 10 ml vols of MRD in a BagPage sterile filter stomacher bag (Interscience, Saint-Nom-La-Bretèche, France) and homogenized for 1 min using a pulsifier (Filtoflex Ltd).
Meat homogenates and chilled broth (heat-treated and non-heat-treated) samples were serially diluted in 9 ml vols of MRD, and bacterial numbers for samples inoculated with inoculum S or SM were estimated using the selective overlay resuscitation technique (Doyle and Schoeni 1984; Duffy et al. 1999a; Byrne et al. 2002). This procedure, which facilitated the recovery of injured and uninjured micro-organisms involved spiral plating appropriate dilutions onto TSA (Oxoid) followed by resuscitation for 2 h at 25°C. After resuscitation, plates were overlaid with 10 ml of xylose–lysine–deoxycholate agar (XLD; Oxoid) supplemented with antibiotics (ampicillin, 10 μg ml−1; streptomycin, 10 μg ml−1; chloramphenicol, 30 μg ml−1) to assist in the selective isolation of the pathogen and incubated at 37°C for 24 h. Typical Salmonella colonies, i.e. red colonies with a black centre were counted using an Acolyte colony counter (Synbiosis, Cambridge, UK).
The beef microflora was examined for the presence of total viable counts (TVC) by spiral plating appropriate dilutions on PCA plates and incubating at 30°C for 72 h. Pseudomonas spp. were enumerated by spiral plating appropriate dilutions on Pseudomonas CFC selective agar (Oxoid) plates and incubating at 25°C for 48 h. The Enterobacteriaceae were determined by adding 0·1 ml aliquots of appropriate dilutions to 10 ml of molten violet red bile glucose agar (VRBGA; Oxoid) in sterile Petri dishes, mixing thoroughly and when the agar solidified, over-poured with a further 10 ml of molten VRBGA and incubating for 24 h at 37°C.
An experiment consisted of obtaining thermal death data at 55°C for beef and broth samples inoculated with Salm. Typhimurium DT104 or Salm. Typhimurium DT104 and a beef microflora heat-treated immediately or after storage at 10°C for 7 days or 4°C for 14 days. Experiments were carried out in duplicate on three separate occasions. Mean surviving Salm. Typhimurium DT104 numbers (beef: log10 CFU cm−2, broth: log10 CFU ml−1) were plotted against time (min) and the slope and standard error (SE) were obtained for each plot using linear regression analysis. In addition, the data were also fitted to a number of other models including quadratic, exponential, logistic and Gompertz functions. D values were calculated from the linear slopes, where D = −1/slope. The t-test was used to compare treatments by analysing differences between the mean slope, and P < 0·05 was considered significant.
Differences between mean counts on beef surfaces and BHI broth samples immediately after inoculation and counts recovered after chilled holding were compared using the t-test at a 95% confidence interval. All statistical analyses were performed using Genstat 5 (Statistics Department, Rothamsted Experimental Station, Hertfordshire, UK).
The beef microflora had TVC counts of 9·02–9·25 log10 CFU ml−1, Enterobacteriaceae counts of 8·93–9·26 log10 CFU ml−1 and Pseudomonas spp. were not detected. There was no significant difference in the Enterobacteriaceae and TVCs, indicating that the Enterobacteriaceae constituted the majority of the microflora.
Beef system Initial levels of Salm. Typhimurium DT104 in beef samples ranged from 8·00 to 8·20 log10 CFU cm−2 in the absence of a beef microflora (S) and 7·90–8·20 log10 CFU cm−2 in the presence of a beef microflora (SM). There were no significant differences between initial numbers of Salm. Typhimurium DT104 on beef surfaces and the numbers recovered after storage (4°C for 14 days or 10°C for 7 days) in the presence or absence of a beef microflora. During the heat treatments, the heat-up times (i.e. the time for the thermocouple at the beef surfaces to reach 55°C) were constant at approx. 30 s.
Figure 1 shows the inactivation curves of Salm. Typhimurium DT104 on beef surfaces in the presence and absence of a beef microflora, during heat treatment at 55°C, before and after storage. Linear regressions were used to determine the D values for comparison of the different heat treatments. The R2 values for the inactivation curves of the pathogen ranged from 0·86 to 0·99 on beef surfaces (Table 1). In two instances, however, nonlinear regressions give a better fit to the data than linear regression and these are indicated in Table 1.
Table 1. D values (min) for Salmonella Typhimurium DT104 in the presence or absence of beef microflora on the surface of beef and in brain–heart infusion broth at 55°C
The D55 values for Salm. Typhimurium DT104 stored at 4°C for 14 days and 10°C for 7 days in the presence or absence of a beef microflora were always significantly lower (P < 0·01) than the D values for this organism heat-treated prior to storage (Table 1). D55 values for Salm. Typhimurium DT104 stored at 4°C for 14 days were significantly lower than the D55 values for cells stored at 10°C for 7 days, and this was observed in the presence (P < 0·001) or absence (P < 0·01) of the beef microflora.
The D55 values for Salm. Typhimurium DT104 in the presence of a beef microflora were always significantly lower (P < 0·05) than the values calculated in the absence of microflora, at all temperatures and storage conditions investigated.
Broth system Initial levels of Salm. Typhimurium DT104 in broth ranged from 7·50 to 7·72 log10 CFU ml−1 in the absence of a beef microflora (S) and 7·66–8·20 log10 CFU ml−1 in the presence of a microflora (SM). There were no significant differences between initial numbers of Salm. Typhimurium DT104 in broth and the numbers recovered after storage at 4°C for 14 days in the presence or absence of a beef microflora. However, there were significant increases (P < 0·001) in Salm. Typhimurium DT104 numbers following storage at 10°C for 7 days in the presence and absence of a beef microflora. These increases in pathogen numbers occurred between day 3 and day 7 of storage.
The inactivation curves of Salm. Typhimurium DT104 in broth, in the presence or absence of a beef microflora, during heating at 55°C, before and after storage at 4°C for 14 days or 10°C for 7 days are presented in Fig. 2. The R2 values for these inactivation curves ranged from 0·90 to 0·98, in the broth system (Table 1). The D values for Salm. Typhimurium DT104 stored at both temperatures in the broth in the presence or absence of a beef microflora were always significantly lower (P < 0·01) than the values for this organism when heated prior to storage. D55 values for Salm. Typhimurium DT104 cells stored at 4°C for 14 days were significantly lower than the D55 values for cells stored at 10°C for 7 days, this occurred in the presence or absence (P < 0·001) of the beef microflora. There were no significant differences in D55 values for Salm. Typhimurium DT104 in broth in the presence or absence of the microflora, under all temperature/storage conditions investigated.
This study found that storage of Salm. Typhimurium DT104 at 10°C for 7 days or 4°C for 14 days significantly reduced the resistance of the pathogen to a subsequent thermal challenge at 55°C. This effect occurred on meat surfaces and in broth in the presence and absence of a beef microflora. Pathogen cells stored at 4°C for 14 days were more heat sensitive than those stored at 10°C for 7 days. In beef systems, the presence of a microflora significantly reduced the thermal resistance of this pathogen under all temperature/storage conditions, but this effect was not evident in the broth system.
The lower heat resistance of pathogen cells after storage at low temperatures, compared with samples without storage has been observed previously. In a study on Salmonella serotype Enteritidis PT4 cells (Humphrey 1990), stored in different broths at 4°C or 8°C for up to 12 days prior to heating at 55°C, resulted in a significant increase in heat sensitivity, compared with samples without storage. It was also observed that cells stored at 4°C were in general more heat sensitive than those stored at 8°C. It was noted that the increased heat sensitivity of cells reached its maximum after 24 h of storage.
A number of other authors have observed an increase in heat sensitivity of a range of Gram-positive and Gram-negative pathogens, as a result of low-temperature storage for shorter time periods (3–24 h) prior to heating (Katsui et al. 1982; Jackson et al. 1995; Humphrey 1998; Miller et al. 2000). Jackson et al. (1995) reported that stationary phase cells of E. coli O157:H7 heat-treated at 55°C in broth were more heat resistant than cultures that had been stored at 3°C prior to heating. Miller et al. (2000) found that cold shocking of Listeria monocytogenes cells at 0–15°C for 1–3 h increased the thermal sensitivity of this pathogen. The duration of this cold shock affected the thermal tolerance of the organism, more than the magnitude of the temperature downshift. In contrast, in experiments where Salmonella was cold-shocked at 4°C for 2 h, differences in heat resistance were not observed when compared with samples that were not cold shocked (Wesche et al. 2005).
Bacterial cells stored at low temperatures are subjected to a cold shock or stress, which results in increased heat sensitivity. Micro-organisms will respond to cold stress in a variety of ways when exposed to a temperature downshift near or below their minimum growth temperature. Cells can undergo alterations in protein synthesis, cell membranes and a variety of other cellular structures in an attempt to adapt to the new environmental conditions (Panoff et al. 1998; Rowbury 2003; Wilson and Nierhaus 2004). In the present study, during storage at 10°C, a significant level of growth of Salm. Typhimurium DT104 was observed in broth, although not on beef surfaces, in the presence or absence of a beef microflora. According to Scherer and Neuhaus (2005), when growth occurs at low temperatures (10°C), the mechanism of cold stress response will occur in two phases, a transient shock during which cold shock proteins are overexpressed, followed by a continuous acclimation response with the synthesis of cold acclimation proteins (Phadtare et al. 1999; Scherer and Neuhaus 2005). While this mechanism may have operated in broth, growth was not observed on cells attached to beef surfaces, but the heat sensitivity of the organism was similar in both situations. At 4°C, however, growth does not occur and under these conditions only cold shock proteins would have been induced (Scherer and Neuhaus 2005). These responses to cold shock at 4°C and 10°C may account for the differences in heat sensitivity observed at these temperatures. According to Bayles et al. (2000), cold shocking stationary phase L. monocytogenes cells from 37°C to 5°C or 0°C for 3 h increased the heat sensitivity of this pathogen and resulted in a significant decrease in the stability of ribosome structure at these temperatures, but this did not occur after cold shocking from 37°C to 10°C for the same time. While ribosomal stability was not affected at 10°C, thermal sensitivity was evident after cold shocking at this temperature.
In the present study, the thermal resistance of Salm. Typhimurium DT104 was reduced on beef surfaces in the presence of a beef microflora, but this effect was not apparent in the broth system. A number of studies have demonstrated that the presence of a competitive microflora can inhibit pathogen growth. Vold et al. (2000) observed that the growth of E. coli O157:H7 was inhibited by the presence of a competitive microflora from beef that had been added to ground beef samples. Duffy et al. (1999b) reported that Hafnia alvei significantly inhibited the growth of E. coli O157:H7 in broth, while other competitors, such as Pediococcus acidilactici only extended the lag phase. There have been few previous studies on the effect of a microflora on the thermal resistance of pathogenic bacteria. In experiments in which exponential phase cells of Salm. Typhimurium LT2 were grown in the presence of a competitive microflora, composed of E. coli, Citrobacter freundii and Pseudomonas fluorescens, pathogen growth was inhibited when competitor cells exceeded 5 log10 CFU ml−1. In addition, the D55 value increased from 0·34 to 2·09 min, but only when the competitor microflora was at a level of 8 log10 CFU ml−1, indicating a significant instantaneous protective effect by the competing microflora (Duffy et al. 1995). Duffy et al. (1995) proposed that the high concentration of competitors is a signal to the Salmonella cells of entry into stationary phase. It is well documented that stationary phase cells are more heat resistant than exponential phase cells and are therefore better able to withstand potentially lethal heat treatments (Jackson et al. 1995; Doyle and Mazzotta 2000; McMahon et al. 2000). Aldsworth et al. (1998) demonstrated that the presence of no less than 8 log10 CFU ml−1 of competitive microflora also protected exponential phase cells of Salm. Typhimurium from freezing. In the present study, the stationary phase cells of the pathogen studied were in contact with the competitive microflora for long periods (7 or 14 days), which may have eliminated the instantaneous protective effect from heat stress on the pathogens outlined earlier. On sterile beef surfaces, it was observed that the presence of a beef microflora increased the heat sensitivity of the pathogen, but this effect was not apparent in broth. On meat surfaces, the pathogen and beef microflora would have been in intimate contact and remained so over the entire storage period, while in broth such an intimate contact would not have existed. This contact on meat surfaces made the pathogen more heat sensitive, but the cause of this effect is not known. This effect has been observed previously, where the presence of a predominantly Gram-negative microflora increased the sensitivity of stationary phase cells of L. monocytogenes to an acid stress (Samelis and Sofos 2001).
In these experiments, while it was possible to demonstrate an effect from the microflora on the heat sensitivity of the pathogen on beef surfaces, such an effect is unlikely to occur under natural conditions because of the large numbers of microflora required. In preliminary experiments using the same microflora at levels of 5 log10 CFU ml−1, no effect on the heat sensitivity was observed on beef surfaces, indicating that a high level of microflora was required to sensitize the pathogen cells.
In conclusion, this study observed that long-term storage at low temperatures increased the heat sensitivity of Salm. Typhimurium DT104 on sterile beef surfaces and in broth. The presence of high numbers of a Gram-negative beef microflora further increased pathogen sensitivity to heat on beef surfaces but not in broth.
This study has been carried out with financial support from the Commission of the European Communities, specific RTD programme ‘Quality of Life and Management of Living Resources’, QLK1-2001-01415 ‘Bugdeath’. The authors thank Dr D. Harrington and Ms Paula Reid for statistical analysis of the data.