The prevalence and number of Salmonella in sausages and their destruction by frying, grilling or barbecuing

Transcription

1 Journal of Applied Microbiology 2002, 93, The prevalence and number of Salmonella in sausages and their destruction by frying, grilling or barbecuing K.L. Mattick, R.A. Bailey, F. Jørgensen and T.J. Humphrey PHLS Food Microbiology Research Unit, Heavitree, Exeter, UK : received 21 January 2002, revised 17 May 2002 and accepted 19 June 2002 K. L. M A T T I C K, R. A. B A ILEY, F. J Ø R G E N S E N A N D T.J. H U M P H R E Y Aims: To determine the prevalence and number of Salmonella and Campylobacter in sausages and to evaluate their destruction during cooking. Methods and Results: One hundred and sixty-two packs of uncooked economy or catering sausages, comprising 53 packs of frozen and 109 of chilled sausages, were purchased in Devon between March and July All were tested for the presence of Salmonella and 51 packs of chilled sausages were also examined for the presence of Campylobacter spp. To investigate the heat tolerance of Salmonella enterica serovar Typhimurium DT104 in sausage-meat, chilled, handmade and frozen sausages were inoculated with approx. 1Æ bacterial cells per sausage (300 cfu g )1 ) and then cooked by frying, grilling or barbecuing. The levels of creatinine kinase, lactate dehydrogenase and alkaline phosphatase in uncooked and cooked sausages were measured to evaluate their potential as indicators of adequate cooking and, therefore, pathogen elimination. Salmonella were detected in 7Æ5% of frozen and 9Æ1% of the chilled sausages (8Æ6% overall) but Campylobacter spp. were not isolated. After cooking, a visual assessment suggested that all of the sausages were thoroughly cooked. Despite this, barbecuing and frying sometimes allowed Salmonella cells to survive and the temperature profiles during cooking indicated that the lethal range was sometimes not reached. The enzyme levels tested were not reliable indicators of the inactivation of bacterial pathogens because Salmonella were sometimes isolated from sausages with low values of all three enzymes. Conclusions: Salmonella spp. are present in a significant proportion of sausages and are not always killed during the cooking process. Significance and Impact of the Study: These findings have clear implications for public health. INTRODUCTION In the year 2000, the average person in Great Britain ate 60 g of sausages per person per week (Anon. 2000). Assuming a population of approx. 57 million ( this corresponds to metric tons of sausages consumed each year. By law, a sausage must contain at least 50% meat and may also contain rusk (filler), water, salt, sugar, starch, non-meat protein, stabilisers, preservatives, antioxidants, colour and Correspondence to: T. Humphrey, Division of Food Animal Science, University of Bristol, Langford House, Lower Langford, Bristol BS40 5DU, UK ( tom.humphrey@bristol.ac.uk). flavourings. The term meat, however, can include fat, skin, gristle and sinew and can often include poultry meat that has been mechanically recovered from carcasses. Both poultry meat and pork can harbour human pathogens including Salmonella and Campylobacter (Anon. 1996; Uyttendaele et al. 1999; Davies et al. 2000). Between 1988 and 1994 there were 24 reported outbreaks of salmonellosis associated with sausages or sausage-meat in England and Wales, corresponding to more than 1000 cases of food poisoning (Nichols and de Louvois 1995). More recently, a case-control study of patients with gastroenteritis caused by Salmonella enterica serovar Typhimurium definitive type (DT) 104 found a significant association between human infection and the ª 2002 The Society for Applied Microbiology

2 542 K.L. MATTICK ET AL. consumption of sausages (Wall et al. 1994). A study of the microbiological safety of sausages detected Salmonella in 17% of uncooked fresh and frozen sausages in England and Wales (Nichols and de Louvois 1995). The main serovar isolated was Typhimurium. The frequency of contamination with Salmonella was inversely proportional to the price per kilogram and the higher prevalence in low cost sausages may reflect the use of cheap ingredients that may be more likely to contain this organism. In the cheapest sausages, Salmonella were isolated from 20% of frozen and 50% of fresh sausage samples. It has also been reported that sausages from large producers are more often contaminated with Salmonella (Roberts et al. 1975). These reports suggest that uncooked economy and catering sausages could be an important source of Salmonella infection in humans and others (Anon. 1981, Anon. 1989). If sausages are cooked adequately prior to consumption, any Salmonella cells present would be killed and there would be no risk of food poisoning. Therefore, the delivery of adequate heat during the cooking of comminuted meat products is important to ensure food safety. To inactivate bacterial pathogens, it is recommended that a temperature of 70 C is achieved in all parts of the sausage for a minimum of 2 min or the equivalent (Gaze et al. 1989). Previous work at the PHLS Food Microbiology Research Unit, however, demonstrated that cooking by frying did not always eliminate Salm. Typhimurium DT104 cells in sausages, even when manufacturer s instructions were followed (Wilde et al. 2002). In addition, barbecuing is an increasingly popular cooking method and 25% of barbecued products, including sausages, are reported to be undercooked. The ability of Salmonella to withstand cooking may depend on many factors, including the bacterial strain, the level of injury of the bacterial cells and the amount of fat in the sausage. Salmonella Typhimurium DT104 cells are also capable of attachment to raw meat and this markedly increased heat tolerance (Humphrey et al. 1997). The muscle-associated enzymes lactate dehydrogenase, creatinine kinase and alkaline phosphatase have been suggested as possible indicators that adequate heat treatment has occurred (Wilde et al. 2002). With such an indicator, sausages that are sold precooked could rapidly and cheaply be assessed for the likelihood of pathogen presence. In this study, the prevalence and numbers of Salmonella and Campylobacter in retail economy and catering sausages were determined. The ability of Salmonella to survive cooking in sausages and the adequacy of manufacturer s instructions for cooking in terms of bacterial inactivation were also considered. Finally, enzymatic indicators of heat destruction and the thermal inactivation of Salmonella in cooked sausages were assessed. MATERIALS AND METHODS Source of sausages One hundred and sixty-two packs of economy or catering sausages were purchased from retail outlets and catering establishments in Devon, UK, during the period from March to July The study comprised 53 packs of frozen and 109 packs of chilled sausages. Economy sausages were defined as those that were the cheapest available from retailers with a wide selection of sausages. These were often labelled with ÔeconomyÕ or ÔvalueÕ on the packaging. Catering sausages were obtained from catering suppliers who sell only in bulk and not directly to the general public. Each sausage had an average weight of 50 g. Detection and enumeration of Salmonella Packs of sausages (n ¼ 162) were unwrapped aseptically and a 50-g sample, containing meat from both ends of each sausage in the pack, was removed. The sample was diluted in 450 ml buffered peptone water (BPW) and homogenized using a stomacher (Laboratory-Blender 400; Seward, London, UK) prior to incubation at 37 C for h. After incubation, a 0Æ1-ml aliquot of the broth was added to 10 ml Rappaport Vassiliadis soya peptone broth (RVS) and a further 1 ml added to 10 ml selenite cystine broth. The selective enrichment broths were incubated at 41Æ5 and 37 C, respectively, for 24 h. Aliquots (10 ll) of the selective enrichment broths were then plated for single colonies onto modified brilliant green agar and xylose lysine desoxycholate agar (XLD), with plate incubation at 37 C for 24 h. Suspect colonies were confirmed using triple sugar iron agar, lysine iron agar and urea broth (urea broth base containing 40% urea solution). During the enrichment procedure, the remaining sausage sample was held at 6 C. All media were from Oxoid (Basingstoke, UK), unless otherwise stated. Where Salmonella were detected by enrichment, cells were enumerated in the remaining meat of individual sausages using a nine-tube most probable number (MPN) method. For cooked sausages, a 25-g sample was removed from each individual sausage in the pack, diluted with 225 g BPW, homogenized and two further tenfold dilutions made in BPW; 1 ml of each of the dilutions was then added to each of three tubes containing 10 ml BPW. The tubes were incubated at 37 C for 24 h. After incubation, 0Æ1 ml was added to 10 ml RVS and incubated at 37 C for 24 h. A 10-ll loop of the RVS culture was spread for single colonies onto XLD agar. The procedure for uncooked sausages was as above but a further dilution was prepared and the three most dilute were used for the MPN. Therefore, the detection limit was 3 cells g )1 for cooked sausages and 30 cells g )1 for uncooked sausages.

3 SALMONELLA IN SAUSAGES 543 Detection of Campylobacter spp. Packs of sausages (n ¼ 51) were unwrapped aseptically and a 25-g sample, containing material from both ends of each sausage in the pack, was removed and diluted in 225 g Exeter Campylobacter broth (made using nutrient broth DM180, FBP growth supplement SV61 and Campylobacter selective supplement SV59, all from Mast, Bootle, Merseyside, UK, and lysed defibrinated horse blood). The sample was homogenized and incubated at 37 C for 48 h. After incubation, a 10-ll loop of the selective enrichment broth was streaked for single colonies onto CCDA agar (CM739 with selective supplement SR155; Oxoid). The plates were incubated at 37 C for 48 h in a microaerobic environment. Preparation and inoculation of sausages for cooking trials The cooking trials were performed using artificially inoculated sausages (chilled, frozen or handmade) since the naturally contaminated sausages often contained numbers of Salmonella that were too low for reliable analysis. A stationary phase culture of Salm. Typhimurium DT104 strain 30 (originally isolated from cattle (Williams et al. 1998)), grown in BPW at 37 C, was diluted to a cell density of approx cells ml )1 using maximal recovery diluent. For chilled sausages, each sausage in a pack was injected into the centre with 0Æ5 ml of the diluted culture using a hypodermic needle (Microlance; Becton Dickinson, Franklin Lakes, NJ, USA) and 1-ml syringe (Terumo, Rome, Italy) to give a cell density of approx. 1Æ cells sausage )1. The inoculated chilled sausages were then stored overnight at 6 C to mimic the likely storage of naturally contaminated sausages in a domestic refrigerator prior to cooking. Frozen sausages were defrosted, inoculated and then refrozen. Handmade sausages were also prepared as this allowed the Salmonella cells and the sausage-meat to be mixed thoroughly, facilitating adherence of bacteria to the muscle tissue, a process that can increase the heat resistance of Salmonella under certain conditions (Humphrey et al. 1997). Stationary phase Salm. Typhimurium DT104 strain 30 cells, prepared as before, were diluted with maximal recovery diluent to approx cells ml )1.A0Æ5-ml aliquot of the culture was added to 100 g fresh minced pork meat, mixed thoroughly and then incorporated into 400 g sausage-meat obtained from a local sausage producer. This was fed into the sausage skins using a hand mixer fitted with a sausage-filling attachment. Each sausage weighed approx. 50 g. Cooking trials The sausages were cooked using three different methods: frying, grilling (both using a domestic electric cooker) and barbecuing (using a disposable barbecue), turning regularly during cooking. Sausages were fried over a medium heat for 10, 12 or 14 min or over a high heat for 5 min, using a small amount of vegetable oil and cooking three to four sausages at a time. The manufacturer s recommended cooking protocol was frying over a medium heat for 12 min. Sausages were grilled for 12, 14 and 16 min (with 14 min being the manufacturer s recommended cooking time) or barbecued for 6 min (the time suggested in the instructions for use of the disposable barbecue). All cooked sausages were analysed for the presence of Salmonella and samples testing positive by enrichment were enumerated using the MPN method. Temperature profiles for the different cooking methods were obtained by monitoring the internal temperature of sausages using a thermocouple inserted into the centre of the sausage prior to cooking (RS, Corby, Northamptonshire, UK). Measurement of enzyme activity Enzymes are denatured by high temperature and thus their destruction in meat could be a useful indicator that adequate cooking has occurred. Tenfold dilutions of the cooked and uncooked sausages were made in phosphate-buffered saline, homogenized and centrifuged at 3400 rev min )1 (2279 g) for 15 min (CR312; Jouan, Winchester, VA, USA). The supernatant fluid was filtered using a 0Æ2-lm filter. Levels of creatinine kinase, lactate dehydrogenase and alkaline phosphatase were determined using an automated dry slide analyser (Vitros AT Chemistry System; Johnson & Johnson Clinical Diagnostics, Rochester, NY, USA) using Vitros slides to perform discrete clinical chemistry tests. RESULTS Prevalence and levels of Salmonella and Campylobacter in retail sausages Salmonella were detected in 7Æ5% of frozen and 9Æ1% of chilled sausages, 8Æ6% overall (Table 1). Campylobacter spp. were not isolated from the 51 packs of chilled sausages tested. Salmonella were enumerated in the packs of sausages that were positive by enrichment and the cell concentration was always 210 cfu g )1 (Table 2). The concentration of Table 1 Prevalence of Salmonella in packs of uncooked sausages Type of sausage Salmonella-positive packs packs tested (%) Chilled Frozen All types (9Æ1) 4 53 (7Æ5) (8Æ6)

4 544 K.L. MATTICK ET AL. Table 2 Numbers of Salmonella present in individual sausages from packs No. of Salmonella (cfu g )1 ) Sausage replicate no. Pack no Average (S.D.) 1 (chilled) (15) 2 (chilled) (50) 3 (chilled) (20) 4 (chilled) (23) 5 (chilled) (18) 6 (chilled) (16) 7 (chilled) (16) 8 (frozen) (4Æ0) 9 (chilled) (5Æ0) 10 (chilled) <30* <30* <30* <30* <30* <30* <30 11 (chilled) <30* <30* <30* <30* <30* <30* <30 12 (frozen) <30* <30* <30* <30* <30* <30* <30 13 (frozen) <30* <30* <30* <30* <30* <30* <30 14 (frozen) <30* <30* <30* <30* <30* <30* <30 *Positive by enrichment of 25 g. Salmonella cells was often lower in frozen than in chilled sausages (Table 2). Cooking trials of sausages inoculated with Salmonella After frying, grilling or barbecuing, sausages appeared to be cooked by visual inspection (Fig. 1). The temperatures reached in the centre of the sausages during cooking, however, indicated that Salmonella might have been able to survive some of the cooking regimes (Table 3) and the bacteria were isolated by enrichment following frying over a high heat for 5 min, a medium heat for 10 min (frozen sausages only) or barbecuing for 6 min (Table 4). Similar results were achieved when Salmonella were enumerated from sausages using the MPN technique. After frying at high heat or barbecuing, there were surviving Salmonella cells in sausages after all cooking times tested, whether the products were cooked fresh or from frozen (Table 5). After frying at medium heat for 10 min, however, there were only surviving cells in the frozen sausages (Table 5). When sausages were fried over a medium heat for 12 min (the manufacturer s recommended cooking time for chilled or frozen sausages) the temperature of the chilled or handmade sausages reached approx. 100 C, whereas the frozen sausages reached 77 C (Table 3). Salmonella were not recovered from sausages subjected to these cooking protocols (Table 4). When frozen sausages were fried over a medium heat for 10 min, the temperature of the sausage only reached approx. 50 C and Salmonella were recovered (Table 4). Fig. 1 Sausages cooked by frying over a high heat for 5 min from (a) frozen or (b) chill or by barbecuing for 6 min from (c) frozen or (d) chill. Salmonella were detected in all of these sausages Salmonella cells were consistently recovered from sausages fried for 5 min over a high heat (Table 4). With this heat treatment, the internal temperature of chilled and handmade

5 SALMONELLA IN SAUSAGES 545 Table 3 Internal sausage temperature profiles during different cooking regimes Treatment Internal temperature of sausage ( C) Time (min) Frozen Chilled Frying at medium heat 0 ) Frying at high heat 0 ) Æ Grilling at high heat 0 ) Barbecuing 0 ) Handmade, chilled sausages only reached approx. 50 C and frozen sausages reached 1Æ5 C in the centre (Table 3). Sausages grilled for 12 min or more reached temperatures > 75 C (Table 3) and Salmonella were not recovered (Table 4). After barbecuing for 6 min, the internal temperature was 58 C for frozen sausages and 69 or 66 C for chilled sausages, respectively (Table 3) and Salmonella were recovered from all sausage types (Table 4). Enzyme levels in cooked and uncooked sausages Uncooked sausages and those cooked by frying, grilling or barbecuing were assayed for the activity of three enzymes. For ease of analysis, the levels of enzymes were divided into low medium and high enzyme levels. Low, medium and high enzyme levels were defined as 14, 15 and 16 Units l )1 for alkaline phosphatase, 399, and 1000 International Units (IU) l )1 for creatinine kinase and 99, and 1000 IU l )1 for lactic dehydrogenase. Enzyme levels did alter during cooking, with more sausages having low levels after heat treatment (Table 6), but there were still some sausages with high enzyme levels Table 5 Numbers of Salmonella in sausages before and after cooking, using various methods Sausage type Average cfu g )1 sausage (S.D.) Uncooked Fried at medium heat 10 min Fried at high heat 5 min Barbecued 6 min Chilled 420 (190) <3 29 (10) 23 (9Æ0) Handmade 280 (200) <3 29 (10) 17 (4Æ0) Frozen 300 (200) 33 (20) 16 (7Æ0) 28 (10) Table 6 The percentage of uncooked and cooked sausages showing given levels of three enzymes Percentage of sausages Low enzyme level* Medium enzyme level* Alkaline phosphatase Uncooked Cooked Creatinine kinase Uncooked Cooked Lactic dehydrogenase Uncooked Cooked *See Results section for definitions of these levels. High enzyme level* after cooking. In addition, sausages with low alkaline phosphatase or creatinine kinase levels could be positive for Salmonella (Table 7). DISCUSSION In this study, Salmonella were detected in 7Æ5% of frozen and 9Æ1% of chilled sausages (8Æ6% overall), indicating that there may have been a fall in the prevalence of Salmonella in economy or catering sausages when compared with published data. A study carried out by the PHLS Food Microbiology Research Unit between December 1996 and Table 4 Surviving Salmonella cells in sausages immediately after cooking Cooking method and duration (min) Fried (medium heat) Fried (high heat) Grilled Barbecued Sausage type Chilled Handmade Frozen Values show the number of Salmonella-positive sausages as a fraction of the total number of sausages cooked.

6 546 K.L. MATTICK ET AL. Table 7 The level of viable Salmonella in cooked sausages when grouped by the enzyme levels in Table 6 ÔCookedÕ sausages containing Salmonella Alkaline phosphatase Low Medium High Creatinine kinase Low Medium High Lactic dehydrogenase Low Medium High Percentage January 1997 (Wilde et al. 2002) found that 15 of 32 samples (47%) of low priced sausages were contaminated with Salmonella. Mechanically recovered chicken meat is thought to be a major source of the contamination of sausage-meat with Salmonella. The number of sausages containing a high proportion of recovered chicken meat may have decreased due to a decrease in the price of pork (D. Lanning, personal communication) and this could explain the lower prevalence. Salmonella were also enumerated in the current study and the levels ranged from < 30 to 120 cfu g )1 on average approx cfu sausage )1 ). Campylobacter spp. were not isolated from the 51 packs of chilled sausages tested. In this study, slightly lower numbers of Salmonella were recovered from frozen than chilled sausages, suggesting that the freezing process may have a detrimental effect on the viability of Salmonella in sausage-meat. In an earlier study, Salmonella were isolated from 20% of frozen and 50% of fresh sausage samples in the cheapest category (Nichols and de Louvois 1995). Salmonella are usually killed by temperatures > 50 C, with the death rate increasing as the temperature increases (Doyle and Mazzotta 2000). When the chilled and handmade sausages were fried over a medium heat, all Salmonella cells were killed, even when the sausages were removed from the heat 2 min before the end of the cooking time recommended by the manufacturer. This result was consistent with the temperature profile since the centre of the sausage reached 80 C for approx. 2 min. When frozen sausages were fried over a medium heat, Salmonella were recovered after 10 min cooking but not after 12 or 15 min, suggesting that the manufacturer s instructions were adequate but gave little margin for error. After frozen sausages had been fried for 10 min, the temperature in the centre had only reached 40 C but a 1 log 10 reduction in cell numbers was observed, probably due to cell death in regions of high temperature near the sausage surface. When the chilled, handmade and frozen sausages were fried at a high heat for 5 min, the sausages looked cooked but there was only a relatively small decrease in Salmonella cell numbers. After this treatment, the internal temperature of the sausage was approx. 55 C for chilled and handmade sausages and approx. 1 C for frozen sausages. From our results, it seems that frying at high heat may not achieve the internal temperatures required to kill Salmonella by the time the sausage looks cooked and this may explain why the manufacturers recommend frying over a medium heat instead. Grilling the sausages for 12 min killed all Salmonella. After 6 min on the barbecue, the sausages looked cooked but there was usually only a 1 log 10 reduction in Salmonella numbers. The temperature profiles indicated that barbecuing for 8 or 10 min would be sufficient to inactivate Salmonella but the sausages might then be considered burnt. The cooking trials indicate that cooking sausages at a lower temperature for a longer time is the safer method in order to eliminate Salmonella. Uncooked sausages and those cooked by frying, grilling or barbecuing were assayed for the activity of three enzymes that have been suggested as potential indicators of heat processing. The levels of alkaline phosphatase were largely unchanged by cooking. The levels of creatinine kinase and lactate dehydrogenase changed significantly during cooking but Salmonella could still be recovered in some of the cooked sausages that had very low enzyme levels. It, therefore, appears that the enzymes tested were unsuitable as reliable indicators of adequate cooking, in terms of bacterial inactivation. To avoid human illness through the consumption of Salmonella-contaminated sausages, four main steps can be taken. It may be possible to reduce the prevalence of Salmonella in sausages by using meat that is less likely to contain Salmonella or through consumer education to ensure that the general public (and particularly high risk groups) are aware that undercooked sausages could pose a risk to health. Auditing manufacturer s cooking instructions to ensure that there is an adequate safety margin to account for variation between cookers, grills and barbecues and improving the clarity of cooking instructions on packaging could also be helpful. Finally, new technologies are improving food safety, for example, high pressure processing in combination with moderate temperatures can bring about a significant reduction in pathogen burden without negatively affecting sausage quality (Yuste et al. 2000). Unfortunately, such techniques could prove expensive and, therefore, have a limited impact on the safety of sausages sold in the lower price bracket. An approach addressing all of the critical points identified, concurrently, is likely to be more successful in terms of improving the safety of sausages. In the meantime, it is important to be aware that uncooked sausages have a

7 SALMONELLA IN SAUSAGES 547 significant risk of containing harmful pathogens and should be thoroughly cooked prior to consumption. ACKNOWLEDGEMENTS The authors gratefully acknowledge funding from the Food Standards Agency and would particularly like to thank Paul Cook and Tabitha Dale for their involvement in both the project and the manuscript. REFERENCES Anon. (1981) Salmonellas from foods. Incidence in fresh pork sausages produced in the UK. Communicable Disease Report 81(28), 6. Anon. (1989) Salmonellas in pork sausages produced in the United Kingdom. Communicable Disease Report 89(39), 1. Anon. (1996) Survey of Salmonella contamination in UK-produced raw chicken on retail sale. In Report on Poultry Meat. Advisory Committee on the Microbiological Safety of Food, Vol. 5. London: HMSO. Anon. (2000) National Food Survey 2000, found at the following website: Davies, R., Paiba, G., Evans, S. and Dalziel, B. (2000) Surveys for Salmonella in pigs, cattle and sheep at slaughter in Great Britain. Veterinary Record 147, 695. Doyle, M.E. and Mazzotta, A.S. (2000) Review of studies on the thermal resistance of salmonellae. Journal of Food Protection 63, Gaze, J.E., Brown, G.D., Gaskell, D.E. and Banks, J.G. (1989) Heat Resistance of Listeria monocytogenes in Non-Dairy Food Menstrua. Technical Memorandum 523. Campden & Chorleywood Food Research Association. Chipping Campden. Humphrey, T.J., Wilde, S.J. and Rowbury, R.J. (1997) Heat tolerance of Salmonella typhimurium DT104 isolates attached to muscle tissue. Letters in Applied Microbiology 25, Nichols, G.L. and de Louvois, J. (1995) The microbiological quality of raw sausages sold in the UK. PHLS Microbiology Digest 12, Roberts, D., Boag, K., Hall, M.L. and Shipp, C.R. (1975) The isolation of salmonellas from British pork sausages and sausage meat. Journal of Hygiene 75, Uyttendaele, M., de Troy, P. and Debevere, J. (1999) Incidence of Salmonella, Campylobacter jejuni, Campylobacter coli, and Listeria monocytogenes in poultry carcasses and different types of poultry products for sale on the Belgian retail market. Journal of Food Protection 62, Wall, P.G., Morgan, D., Lamden, K., Ryan, M., Griffen, M., Threlfall, E.J. Ward, L.R. and Rowe, B. (1994) A case control study of infection with an epidemic strain of multi-resistant Salmonella typhimurium DT104 in England and Wales. Communicable Disease Report Review 4, R130 R135. Wilde, S., Jørgensen, F., Mattick, K.L. and Humphrey, T.J. (2002) Development and study of tests to differentiate between tolerant and sensitive isolates of Salmonella and Escherichia coli Project report code 206 written for the Department of Health. London: Food Standards Agency. Williams, A., Davies, A.C., Wilson, J., Marsh, P.D., Leach, S., and Humphrey, T.J. (1998) Contamination of the contents of intact eggs by Salmonella typhimurium DT104. Veterinary Record 143, Yuste, J., Pla, R. and Mor-Mur, M. (2000) Salmonella enteritidis and aerobic mesophiles in inoculated poultry sausages manufactured with high-pressure processing. Letters in Applied Microbiology 31,