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Behaviour of Listeria monocytogenes in the presence of Listeria innocua during storage of minced beef under vacuum or in air at 0°C and 10°C
Food Microbiology, 2000, 17, 571^578 Available online at http://www.idealibrary.com on
doi:10.1006/fmic.2000.0354
ORIGINAL ARTICLE
Behaviour of Listeria monocytogenes in the presence of Listeria innocua during storage of minced beef under vacuum or in air at 08C and 108C G. Du¡y1, D.Walsh1*, J. J. Sheridan1, C. M. Logue1, D. Harrington2, I. S. Blair3 and D. A. McDowell3
Minced beef was inoculated with low levels (1?2^1?7 log10 cfu g71) of Listeria monocytogenes or Listeria innocua, or a combination of the two strains. Inoculated samples were stored at 0 or 108C under two packaging atmospheres (aerobic and vacuum) for up to 28 days and surviving organisms recovered on Palcam Agar. The only signi¢cant increases in numbers of Listeria spp. occurred in samples held at 108C under aerobic conditions. In vacuum packs, growth of both strains was inhibited. Under aerobic conditions meat pH increased from an initial value of pH 5?85 to c. 8?85 within 28 days.The pH of vacuum packaged meat declined to c. 4?95 during the same period. These di¡erences in pH may be related to di¡erences in the nature and e¡ects of di¡erent background micro£ora that were observed to develop under each of these packaging conditions. Pseudomonas spp. predominated in aerobically stored beef, whereas in vacuum packed beef lactic acid bacteria predominated. No signi¢cant di¡erences were observed between the growth rates of Listeria spp. inoculated into beef mince in pure and mixed culture. This suggests that the more frequent prevalence of Listeria innocua than Listeria monocytogenes in meat and meat products is not due to overgrowth or inhibition of the pathogen (Listeria monocytogenes) by the non-pathogen (Listeria innocua) during low-temperature storage. # 2000 Academic Press
Introduction Of the seven species of Listeria, only Listeria monocytogenes is considered pathogenic for humans, and it is recognized that contaminated foods are the main source of listeriosis (WHO 1988, McLauchlin 1996). While the disease is rare, it has a high mortality rate,
*Corresponding author: Fax: 353 1 8059550. E-mail: [email protected] 0740 -0020/00/060571 + 08 $35.00/0
and an accurate infective dose level has yet to be established (Skinner 1996). Overall, concentrations of Listeria spp. in foods tend to be low (Sheridan et al. 1994, Jay 1996, Walsh et al. 1998). Thus, it is important that detection methods are e¡ective in isolating L. monocytogenes from foods containing (a) other Listeria spp., and (b) much higher concentrations of other potentially competitive microorganisms. Some studies have reported that the incidence of L. innocua, a closely related but non-pathogenic species (Jones 1990) is # 2000 Academic Press
Received: 5 September1999 1
The National Food Centre,Teagasc, Dunsinea, Castleknock, Dublin 15, Ireland 2 Teagasc, Statistics Department,19 Sandymount Avenue, Dublin 4, Ireland 3 University of Ulster, Jordanstown, Newtownabbey, Co. Antrim BT37 OQB, Northern Ireland
572 G. Du¡y et al.
considerably higher than the incidence of L. monocytogenes (Walsh et al. 1998, McEvoy et al. 1998). It has yet to be established whether this is due to a naturally higher prevalence of L. innocua on food entering storage, or if this predominance is the result of more rapid growth of the non-pathogenic L. innocua during storage. Alternatively, the higher frequency of recovery of L. innocua may be the result of preferential selection during laboratory and isolation procedures. Some authors have noted that L. innocua and L. monocytogenes have di¡erent growth rates in some enrichment media (Du¡y et al. 1994, MacDonald and Sutherland 1994). In experiments where foods were inoculated with both L. monocytogenes and L. innocua and enriched in selective enrichment broths, L. innocua was detected more often, while L. monocytogenes was only isolated when it was at a higher level than L. innocua (Curiale and Lewus 1994). The growth and survival of L. monocytogenes in beef has been documented (Grau and Vanderlinde 1990, Kaya and Schmidt 1991, Grau and Vanderlinde 1992, Barbosa et al. 1995) and has been shown to be highly dependent on storage temperature, meat pH and packaging environment. The e¡ect of competition between L. innocua and L. monocytogenes in beef during refrigerated storage, and their subsequent growth and recovery during enrichment procedures has however, received little attention to date. In this work, the e¡ect of competitive micro£ora, packaging, and storage temperature on the growth of mixed cultures of L. monocytogenes and L. innocua in minced beef is examined.
Organisms Listeria innocua L3036C, resistant to 50 mg ml71 tetracycline hydrochloride, was obtained from Dr Jim McLaughlin at the Food Hygiene Laboratory, PHLS Central Public Health Laboratory, London. Listeria monocytogenes serotype 4b (NCTC 11994) was obtained from the National Collection of Typed Cultures, PHLS Central Public Health Laboratory, London. This strain was rendered resistant to 1000 mg ml71 streptomycin using the method described by Blackburn and Davies (1994). Brie£y, the wild type L. monocytogenes (NCTC1 1994) was incubated in 100 ml Nutrient Broth (Oxoid, UK) at 308C for 24 h. A 100 ml fresh nutrient broth containing 2000 mg ml71 of streptomycin was added to the culture to give a ¢nal streptomycin concentration of 1000 mg ml71. The broth was incubated at 308C for a further 24 h. Aliquots (0?5 ml) of the resulting culture were spread onto the surface of Nutrient Agar (NA) (Oxoid) plates containing 1000 mg ml71 of streptomycin. The plates were incubated at 308C for up to 5 days, and any colonies that developed were subcultured on fresh NA. Resistance was tested by culturing on NA containing 1000 mg ml71 streptomycin. The growth kinetics of the mutant strain was compared to the wild type strain in Bu¡ered Peptone Water (BPW) and UVM Medium (UVM) (Oxoid) to ensure that growth characteristics were not signi¢cantly di¡erent. After many attempts, an antibiotic-resistant mutant was isolated and labeled L. monocytogenes M63. Both L. monocytogenes M63 and L. innocua L3036C were stored on ceramic beads (Protect Bacterial Preservers, UK) at 7208C.
Inoculation
Materials and Methods Samples Samples of beef diaphragm muscle (6^8 kg) were purchased from a local abattoir and trimmed of membranes and excess fat. The meat was cut into chunks of 50^100 g size, portioned into 1-kg lots, and stored at 08C overnight in sterile plastic bags.
Inocula were prepared from overnight cultures of L. monocytogenes and L. innocua incubated in 30 ml brain^heart infusion broth (BHIB, Oxoid) at 308C. Cells were aseptically recovered from the 24-h cultures by centrifugation at 5000 rpm for 5 min at 48C, resuspended in 10 ml of fresh sterile BHIB and mixed. Cell numbers were estimated using an acridine orange direct count (AODC) technique (Du¡y et al. 1991) and adjusted by the addition of sterile
Listeria monocytogenes and Listeria innocua in beef 573
¢ltered MRD to produce a 2 l solution containing 1000^2000 cells ml71 of either L. monocytogenes or L. innocua species. Mixed inocula were prepared by adding 1 l of L. innocua suspension to 1 l of the L. monocytogenes suspension and mixing well. Thus, three inocula were prepared; L. monocytogenes only, L. innocua only, and a mixed inoculum containing both L. monocytogenes and L. innocua. Each of the three batches of prepared beef pieces was inoculated by immersing for 5 s in one of the inocula prepared above. Excess liquid was allowed to drain o¡ the meat, which was then double-minced through a sterile 10 -mm plate using a Crypto-Peerless (UK) mincing machine. This resulted in an inoculation level of approximately 102 g71 Listeria cells in the meat as estimated from previous experiments, and from Day 0 results in this experiment.
Packaging The inoculated, double-minced meat was distributed in 20+l g quantities (a) onto Plastic food trays (Dynopack, Norway) covered with cling ¢lm to reduce drying, or (b) into small pouches made from Cryovac (W.R. Grace, Ireland) vacuum bags. These pouches were sealed using a vacuum-packaging machine (Swissvac, UK).
Storage Packaged samples were divided into two lots, and stored at 08C or 108C. Four packs of each type (air/vacuum packed) were removed from each storage temperature on each of days 0, 7, 14 and 28 and examined as described below.
Microbiological analysis A 5+0?5 g sample from each pack was weighed into a stomacher bag ¢tted with integral ¢lter (Seward Medical, UK) and diluted 1:10 using an automatic gravimetric dilutor (Watson^ Marlow, UK). The bags were stomached for 1 min in a laboratory stomacher (Seward), and the resultant ¢ltrate serially diluted in MRD. Aliquots (1?0 ml) of serial dilutions were plated out onto Palcam Agar plates (Oxoid) containing 50 mg ml71 tetracycline hydrochloride
(T-3383, Sigma) UK (PATC) and Palcam Agar containing 1000 mg ml71 streptomycin sulphate (S-9137, Sigma) (PASS). Both sets of plates were then incubated at 308C for 48 h and examined for growth of Listeria. Samples of the recovered ¢ltrate were also examined for members of the background spoilage micro£ora, i.e., Pseudomonas spp. (Pseudomonas Agar CFC, at 258C for 72 h), Enterobacteriaceae (VRBG Agar at 378C for 24 h), Brocothrix thermosphacta (STAA Agar at 228 C for 72 h), Lactic acid bacteria (MRS Agar at 308C for 72 h), and Total Viable Counts (SPC Agar at 308C for 72 h) (all media obtained from Oxoid).
pH measurement A 2 g sample from each pack was added to 10 ml iodoacetate bu¡er (sodium iodoacetate 5 mM; BDH Chemicals, UK, and potassium chloride 150 mM; AnalaR, BDH). The sample was homogenised (Silverson Machines, UK) for two 15 -s intervals with a 15 s interval between treatments, and the pH of the homogenate measured.
Statistical analysis Three replicates of the above experiment were carried out. Results were analysed using ANOVA (Genstat 5, Rothamsted Experimental Station) to determine any signi¢cant di¡erences between the numbers (cfu g71) of L. monocytogenes and L. innocua during storage, and to establish if packaging type or storage temperature, had an e¡ect on the levels of either strain.
Results Initial numbers (cfu g71) on Day 0 ranged from 1?12^1?88 log10 cfu g71 and in Table 1 it can be seen that after 28 days storage the numbers of both Listeria strains have decreased, ranging from 0^1?95 log10 cfu g71. No signi¢cant di¡erences were noted between the levels of L. monocytogenes and L. innocua detected in samples held for the same period under similar storage conditions. No signi¢cant di¡erences were observed between the numbers of each species of
574 G. Du¡y et al.
Table 1. Populations (log10 cfu g71) of Listeria innocua and Listeria monocytogenes in minced beef after 28 days storage at di¡erent temperatures and using air or vacuum packaging Storage temperature Listeria spp. L. monocytogenes Single inoculum Mixed inoculum L. innocua Single inoculum Mixed inoculum
Packaging environment
08C
108C
Vacuum Air
1?25 0?80
0?00 1?13
Vacuum Air
1?02 0?81
0?55 1?45
Vacuum Air
0?74 0?94
0?08 1?94
Vacuum Air
0?67 0?26
0?65 1?84
Standard error of di¡erence between means = 0?635, degrees of freedom = 30.
Figure 1. Growth of Listeria innocua and Listeria monocytogenes in minced beef during storage under vacuum or air packaging and at 0 or 108C. L. monocytogenes single (^^^), L. monocytogenes mixed (^&^), L. innocua single (^~^), L. innocua mixed (^*^). (a) = 08C, vacuum packed; (b) = 108C, vacuum packed, (c) = 08C, air packed, (d) = 108C, air packed.
Listeria recovered from minced beef samples inoculated with single or mixed inocula. Temperature had an e¡ect on the nature and rate of growth of Listeria in stored minced beef. In samples held at 108C, signi¢cantly higher (P50?001) numbers of both Listeria strains were recovered from aerobic packs than from
vacuum packs. In samples held at 08C, signi¢cantly higher (P50?001) numbers of both Listeria strains were recovered from vacuum packs than from aerobic packs. In Fig. 1 (d) it can be seen that at 108C in aerobically packed samples, numbers of both Listeria stains had increased by 1?5^2 log10 cfu g71
Listeria monocytogenes and Listeria innocua in beef 575
between day 0 and day 7, but levels decreased between days 14 and 28. On days 7 and 14 numbers of Listeria in minced beef samples stored at 108C in air were signi¢cantly higher (P50?001) than in samples stored under the other storage conditions. Figure 2 shows the pattern of changes in pH values in inoculated minced beef during storage. In samples held at 108C in vacuum packs the pH declined from 5?85 to c. 5?2 by day 7, and thereafter decreased more slowly, achieving a pH of 5?1 after 28 days. In samples held at 108C in air the pH values increased to 7?8 by day 7 and continued to increase, achieving a pH value of 8?7 by day 28. In samples held at 08C a similar pattern was observed, although the changes noted were smaller. pH values
decreased in vacuum packed samples, and increased in air packed samples. Table 2 displays the counts for members of the resident spoilage micro£ora, which developed on samples of inoculated minced beef during storage for 28 days. At 08C in aerobically packed minced beef, Pseudomonas spp., Brocothrix thermosphacta and total viable counts were higher than those counts in samples stored under the other conditions. At 108C in aerobically packed minced beef, Enterobacteriaceae counts were higher than in samples stored under the other conditions. At 08C in vacuum packed minced beef, total viable counts and counts of all the other bacteria examined, except Brocothrix thermosphacta, were lower than under the other storage conditions. At 108C in vacuum packed minced beef, counts of Brocothrix thermosphacta were lower than under the other storage conditions. This is the only member of the resident spoilage micro£ora examined that decreased in counts during the storage period, though the decrease only occurred in samples held at 108C in vacuum packs. Counts of Lactic Acid Bacteria were higher at 108C in vacuum packed minced beef than under the other storage conditions.
Discussion
Figure 2. Changes in pH of minced beef during storage at 08C or 108C, under vacuum or in air. Vacuum, 08c (^^^), Vacuum, 108C (^*^), air, 08C (^~^), air, 108C (^&^).
In this study, Listeria spp. did not grow in vacuum packed samples stored at 08C or 108C. In aerobically packed samples Listeria spp. grew at 108C but not at 08C.The observation that this genus can grow in air under conditions of low
Table 2. Populations (log10 cfu g71 ) of some spoilage bacteria in minced beef after 28 days storage at di¡erent temperatures and using air or vacuum packaging. Packaging Storage temperature Total viable count Enterobacteriaceae Pseudomonads Lactic acid bacteria Brochothrix thermosphacta a
(5?66) (2?25) (4?59) (4?88) (4?74)
a
Vacuum
Air
08C
108C
08C
108C
d.f.
s.e.
7?80 4?46 4?90 6?12 4?91
9?71 6?05 5?75 8?84 4?25
11?1 5?97 11?1 6?5 8?94
10?2 7?14 9?33 5?84 8?65
94 93 91 94 89
0?68 0?73 0?84 0?62 0?99
= count at day 0, d.f. = degrees of freedom, s.e. = standard error of di¡erence between means.
576 G. Du¡y et al.
temperature abuse (108C) is in agreement with Sheridan et al. (1995) who found that L. monocytogenes can grow on air-packed lamb at 58C. Growth was found to be slow under these conditions, which is in agreement with previous reports that Listeria spp. did not grow well in meat at low temperatures (Sheridan et al. 1994, Barbosa et al. 1995. Farkas and Andrassy 1996). The packaging atmosphere had a signi¢cant e¡ect on growth rate. Listeria did not grow in vacuum packs, even at the higher temperature (108C). Sheridan et al. (1995) reported that Listeria spp. did not grow in meat (lamb) in vacuum packs at 58C. While Listeria spp. are psychrotrophic, the optimum temperature for growth is between 30 and 378C (Johnson et al. 1990), so it would be expected that the growth of the Listeria spp. in meat would increase as storage temperature increased. This study revealed that growth did not occur under temperature abuse (108C) in vacuum packaged beef, con¢rming the importance of packaging atmosphere in controlling growth rates in this genus. This study showed that the numbers of naturally occurring micro£ora on beef were quite high, regardless of storage conditions, and this may have contributed to the inhibition of Listeria growth. This study found di¡erent patterns of pH change during storage, dependent on the packaging atmosphere, and that storage atmosphere is more important than storage temperature in determining gross pH changes. Thus, pH values increased in aerobically stored samples at both temperatures examined, and decreased in vacuum packaged samples at both temperatures. Under aerobic storage conditions the pH of the meat increased by more than two pH units over the storage period. In this experiment, growth of both Listeria spp. was highest in the meat with the highest pH (at 108C in air), suggesting that pH has a signi¢cant e¡ect on Listeria growth.This is in agreement with several workers who have reported that Listeria spp. grew better in meat of high pH than on low pH meat (Kaya and Schmidt 1991, Grau and Vanderlinde 1993, Barbosa et al. 1995). However, this study also noted that the rate of growth of Listeria spp. declined between day 14 and day 28, although meat pH continued to increase, albeit more slowly, dur-
ing this period.This suggests that other factors are involved in the overall determination of the growth rates of Listeria spp. under aerobic storage conditions. These factors may include such gross e¡ects as the overall depletion of nutrients and/or the build up of metabolic waste products. Psuedomonas numbers in aerobic samples were very high by the end of the experiment (28 days).This group comprised a major component of the total £ora at this stage, and was probably responsible for the pH increases noted. In general terms, psuedomonads are widely recognized as having major e¡ects on the shelf-life of aerobically stored meats (Labadie 1999). Previous studies have reported the inhibition of food pathogens (including Listeria spp.) by Psuedomonads (Matilla-Sandholm and Skitta 1991, Liao and Sapers 1999). In vacuum packs however, lactic acid bacteria predominated within the developed £ora of stored beef mince. These organisms, previously shown as being favoured by vacuum packaging (Barbosa et al. 1995, Labadie 1999), can produce lactic acid, H2O2, and bacteriocins (Juven et al. 1998), and are probably responsible for the observed reductions in pH in beef mince stored under these conditions. It has also been reported by some workers that lactic acid bacteria can inhibit Listeria spp. (Buchanan and Klawitter 1990, Juven et al. 1998). This study did not reveal any signi¢cant differences between the numbers of L. innocua and L. monocytogenes that developed in beef mince inoculated with pure or mixed cultures of these species.This pattern was consistently observed under all storage conditions and storage atmospheres. These observations suggest that there is no signi¢cant competition between these species in stored minced beef. However, in other circumstances, di¡erences between the growth rates of these two closely related species have been reported. A previous study (Petran and Swanson 1993) reported that L. innocua grew more rapidly than L. monocytogenes in selective media.This pattern was not observed in non-selective media and in that environment growth rates were similar. The reasons for reported di¡erences in growth rates in selective media are not clear, although they have signi¢cant implications for
Listeria monocytogenes and Listeria innocua in beef 577
those involved in food safety surveillance. Listeria innocua may be better adapted to growth in selective media than L. monocytogenes, leading to overgrowth and preferential recovery of the non-pathogenic species. Thus Yokoyama et al. (1998) have reported that in laboratory media, L. innocua produces a bacteriocin-like substance that has an inhibitory e¡ect on the growth of L. monocytogenes. This e¡ect has been demonstrated only in laboratory media, and may not occur in foods. However, the ability of organisms to speci¢cally inhibit the growth of related species in laboratory media may signi¢cantly interfere with the recovery and detection of important target species. If, as in this case, widely occurring non-pathogenic species can interfere with the recovery and detection of related less frequently occurring pathogenic species, estimations of the frequency of occurrence of pathogens will be rendered less accurate, and causative agents of food poisoning may not be adequately detected. The main aim of this study was to examine why L. innocua is isolated more often than L. monocytogenes from foods. The results of this study suggest that this pattern of prevalence of L. innocua was not due to changes occurring in meat during storage in relation to (a) competition between the two strains, (b) inhibition of one strain by the other, or, (c) competition with, or inhibition by, other members of the normal bacterial £ora of meat. Further studies are required to ascertain whether competition between L. monocytogenes and L. innocua during growth in enrichment broth alters the relative frequency of recovery of these organisms from such media. As noted in the introduction to this paper, when L. monocytogenes is present in foods, populations are low. Thus any e¡ects or interactions between this pathogen, and more frequently occurring related species, and/or other members of the normal micro£ora of meat and meat products, during storage and during laboratory recovery procedures, may reduce the e⁄ciency of recovery of L. monocytogenes and reduce the accuracy of subsequent reported incidence levels. Such interactions have implications in relation to meat safety and quality surveillance.
References Barbosa, W. B., Sofos, J. N., Schmidt, G. R. and Smith, G. C. (1995). Growth potential of individual strains of Listeria monocytogenes in fresh vacuum-packaged refrigerated ground top rounds of beef. J. Food Prot. 58, 398^403. Blackburn, C. de W. and Davies, A. R. (1994) Development of antibiotic-resistant strains for the enumeration of foodborne pathogenic bacteria in stored foods. Int. J. Food Microbiol. 24, 125^136. Buchanan, R. L. and Klawitter, L. A. (1990). E¡ect of temperature history on the growth of Listeria monocytogenes Scott A at refrigeration temperatures. Int. J. Food Microbiol. 12, 235^245. Curiale, M. S. and Lewus, C. M. (1994). Detection of Listeria monocytogenes in samples containing Listeria innocua. J. Food Prot. 57, 1048^1051. Du¡y, G., Sheridan, J. J., McDowell, D. A., Blair, I. S. and Harrington, D. (1991). The use of Alcalase 2.5L in the acridine orange direct count technique for the rapid enumeration of bacteria in beef mince. Lett. Appl. Microbiol. 13, 198^201. Du¡y, G., Sheridan, J. J., Buchanan, R. L., McDowell, D. A., and Blair, I. S. (1994). The e¡ect of aeration, initial innoculum and meat micro£ora on the growth kinetics of Listeria monocytogenes in selective enrichment broths. Food Microbiol. 11, 429^438. Farkas, J. and Andrassy, E. (1996). Behaviour of Listeria monocytogenes in an extended shelf-life chilled product. Acta Alimentaria 25, 185^187. Grau F. H. and Vanderlinde, P. B. (1990). Growth of Listeria monocytogenes on vacuum-packaged beef. J. Food Prot. 53, 739^741. Grau F. H. and Vanderlinde, P. B. (1992). Occurrence, numbers and growth of Listeria monocytogenes on some vacuum-packaged processed meats. J. Food Prot. 55, 4^7. Grau F. H. and Vanderlinde, P. B. (1993). Aerobic growth of Listeria monocytogenes on lean beef and fatty tissue: Equations describing the e¡ects of temperature and pH. J. Food Prot. 56, 96^101. Jay, J. M. (1996). Prevalence of Listeria spp. in meat and poultry products. Food Control 7, 209^214. Johnson, J. L., Doyle, M. P., and Cassens, R. G. (1990). Listeria monocytogenes and other Listeria spp. in meat and meat products: a review. J. Food Prot. 53, 81^91. Jones, D. (1990). Foodborne Listeriosis. The Lancet, 336, 1171^1174. Juven, B. J., Barefoot, S. F., Pierson, M. D., McCaskill, L. H. and Smith, B. (1998). Growth and survival of Listeria monocytogenes in vacuumpackaged ground beef inoculated with Lactobacillus alimentarious FloraCarn L-2. J. Food Prot. 61, 551^556. Kaya, M. and Schmidt, U. (1991). Behaviour of Listeria monocytogenes on vacuum-packed beef. Fleischwirtsch 71, 424^426.
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Labadie, J. (1999) Consequences of packaging on bacterial growth. Meat is an ecological niche. Meat Science, 52, 299^305. Liao, C-H., and Sapers, G. M. (1999). In£uence of soft rot bacteria on growth of Listeria monocytogenes on potato tuber slices. J. Food Prot. 62, 343^348. MacDonald, F., and Sutherland, A. D. (1994). Important di¡erences between the generation times of Listeria monocytogenes and Listeria innocua in two Listeria enrichment broths. J. Dairy Res. 61, 433^436. McEvoy, J. M., Doherty, A. M., Sheridan, J. J., and McGuire, L. (1998). The incidence of Listeria spp. and Escherichia coli O157:H7 on beef carcasses. In Proceedings 44th ICoMST, 346^347, Barcelona, Spain A43. McLauchlin, Institute for Food and Agricultural Research and Technology (IRTA) (1996). Molecular and conventional typing methods for Listeria monocytogenes: the UK approach. J. Food Prot. 59, 1102^1105. Matilla-Sandholm, T., and Skytta, E. (1991). The e¡ect of spoilage £ora on the growth of food pathogens in minced meat stored at chilled temperatures. Lebensm-wiss-u-Technol, 24, 116^120. Petran, R. L. and Swanson, K. M. J. (1993). Simultaneous growth of Listeria monocytogenes and Listeria innocua. J. Food Prot. 56, 616^618.
Sheridan, J. J., Du¡y, G., Buchanan, R. L., Mc Dowell, D. A., and Blair, I. S. (1994). The use of selective and non-selective enrichment broths for the isolation of Listeria spp. from meat. Food Microbiol., 11, 439^446. Sheridan, J. J., Doherty, A., Allen, P., McDowell, D. A., Blair, I. S. and Harrington, D. (1995). Investigations on the growth of Listeria monocytogenes on lamb packaged under modi¢ed atmospheres. Food Microbiol. 12, 259^266. Skinner, R. (1996) Listeria: the state of the science Rome 29^30 June 1995 Session IV: country and organizational postures on Listeria monocytogenes in food Listeria: UK government’s approach. Food Control, 7, 245^247. Walsh, D., Du¡y, G., Sherida, J. J., Blair, I. S. and McDowell, D. A. (1998). Comparison of selective and non-selective media for the isolation of Listeria species from retail foods. J. Food Safety 18, 85^89. World Health Organisation (WHO) Working Group. (1988). Foodborne Listeriosis. Bull WHO 66, 421^428. Yokoyama, E., Maruyama, S. and Katsube, Y. (1998). Production of bacteriocin-like substance by Listeria innocua against Listeria monocytogenes. Int. J. Food Microbiol. 40, 133^137.
doi:10.1006/fmic.2000.0354
ORIGINAL ARTICLE
Behaviour of Listeria monocytogenes in the presence of Listeria innocua during storage of minced beef under vacuum or in air at 08C and 108C G. Du¡y1, D.Walsh1*, J. J. Sheridan1, C. M. Logue1, D. Harrington2, I. S. Blair3 and D. A. McDowell3
Minced beef was inoculated with low levels (1?2^1?7 log10 cfu g71) of Listeria monocytogenes or Listeria innocua, or a combination of the two strains. Inoculated samples were stored at 0 or 108C under two packaging atmospheres (aerobic and vacuum) for up to 28 days and surviving organisms recovered on Palcam Agar. The only signi¢cant increases in numbers of Listeria spp. occurred in samples held at 108C under aerobic conditions. In vacuum packs, growth of both strains was inhibited. Under aerobic conditions meat pH increased from an initial value of pH 5?85 to c. 8?85 within 28 days.The pH of vacuum packaged meat declined to c. 4?95 during the same period. These di¡erences in pH may be related to di¡erences in the nature and e¡ects of di¡erent background micro£ora that were observed to develop under each of these packaging conditions. Pseudomonas spp. predominated in aerobically stored beef, whereas in vacuum packed beef lactic acid bacteria predominated. No signi¢cant di¡erences were observed between the growth rates of Listeria spp. inoculated into beef mince in pure and mixed culture. This suggests that the more frequent prevalence of Listeria innocua than Listeria monocytogenes in meat and meat products is not due to overgrowth or inhibition of the pathogen (Listeria monocytogenes) by the non-pathogen (Listeria innocua) during low-temperature storage. # 2000 Academic Press
Introduction Of the seven species of Listeria, only Listeria monocytogenes is considered pathogenic for humans, and it is recognized that contaminated foods are the main source of listeriosis (WHO 1988, McLauchlin 1996). While the disease is rare, it has a high mortality rate,
*Corresponding author: Fax: 353 1 8059550. E-mail: [email protected] 0740 -0020/00/060571 + 08 $35.00/0
and an accurate infective dose level has yet to be established (Skinner 1996). Overall, concentrations of Listeria spp. in foods tend to be low (Sheridan et al. 1994, Jay 1996, Walsh et al. 1998). Thus, it is important that detection methods are e¡ective in isolating L. monocytogenes from foods containing (a) other Listeria spp., and (b) much higher concentrations of other potentially competitive microorganisms. Some studies have reported that the incidence of L. innocua, a closely related but non-pathogenic species (Jones 1990) is # 2000 Academic Press
Received: 5 September1999 1
The National Food Centre,Teagasc, Dunsinea, Castleknock, Dublin 15, Ireland 2 Teagasc, Statistics Department,19 Sandymount Avenue, Dublin 4, Ireland 3 University of Ulster, Jordanstown, Newtownabbey, Co. Antrim BT37 OQB, Northern Ireland
572 G. Du¡y et al.
considerably higher than the incidence of L. monocytogenes (Walsh et al. 1998, McEvoy et al. 1998). It has yet to be established whether this is due to a naturally higher prevalence of L. innocua on food entering storage, or if this predominance is the result of more rapid growth of the non-pathogenic L. innocua during storage. Alternatively, the higher frequency of recovery of L. innocua may be the result of preferential selection during laboratory and isolation procedures. Some authors have noted that L. innocua and L. monocytogenes have di¡erent growth rates in some enrichment media (Du¡y et al. 1994, MacDonald and Sutherland 1994). In experiments where foods were inoculated with both L. monocytogenes and L. innocua and enriched in selective enrichment broths, L. innocua was detected more often, while L. monocytogenes was only isolated when it was at a higher level than L. innocua (Curiale and Lewus 1994). The growth and survival of L. monocytogenes in beef has been documented (Grau and Vanderlinde 1990, Kaya and Schmidt 1991, Grau and Vanderlinde 1992, Barbosa et al. 1995) and has been shown to be highly dependent on storage temperature, meat pH and packaging environment. The e¡ect of competition between L. innocua and L. monocytogenes in beef during refrigerated storage, and their subsequent growth and recovery during enrichment procedures has however, received little attention to date. In this work, the e¡ect of competitive micro£ora, packaging, and storage temperature on the growth of mixed cultures of L. monocytogenes and L. innocua in minced beef is examined.
Organisms Listeria innocua L3036C, resistant to 50 mg ml71 tetracycline hydrochloride, was obtained from Dr Jim McLaughlin at the Food Hygiene Laboratory, PHLS Central Public Health Laboratory, London. Listeria monocytogenes serotype 4b (NCTC 11994) was obtained from the National Collection of Typed Cultures, PHLS Central Public Health Laboratory, London. This strain was rendered resistant to 1000 mg ml71 streptomycin using the method described by Blackburn and Davies (1994). Brie£y, the wild type L. monocytogenes (NCTC1 1994) was incubated in 100 ml Nutrient Broth (Oxoid, UK) at 308C for 24 h. A 100 ml fresh nutrient broth containing 2000 mg ml71 of streptomycin was added to the culture to give a ¢nal streptomycin concentration of 1000 mg ml71. The broth was incubated at 308C for a further 24 h. Aliquots (0?5 ml) of the resulting culture were spread onto the surface of Nutrient Agar (NA) (Oxoid) plates containing 1000 mg ml71 of streptomycin. The plates were incubated at 308C for up to 5 days, and any colonies that developed were subcultured on fresh NA. Resistance was tested by culturing on NA containing 1000 mg ml71 streptomycin. The growth kinetics of the mutant strain was compared to the wild type strain in Bu¡ered Peptone Water (BPW) and UVM Medium (UVM) (Oxoid) to ensure that growth characteristics were not signi¢cantly di¡erent. After many attempts, an antibiotic-resistant mutant was isolated and labeled L. monocytogenes M63. Both L. monocytogenes M63 and L. innocua L3036C were stored on ceramic beads (Protect Bacterial Preservers, UK) at 7208C.
Inoculation
Materials and Methods Samples Samples of beef diaphragm muscle (6^8 kg) were purchased from a local abattoir and trimmed of membranes and excess fat. The meat was cut into chunks of 50^100 g size, portioned into 1-kg lots, and stored at 08C overnight in sterile plastic bags.
Inocula were prepared from overnight cultures of L. monocytogenes and L. innocua incubated in 30 ml brain^heart infusion broth (BHIB, Oxoid) at 308C. Cells were aseptically recovered from the 24-h cultures by centrifugation at 5000 rpm for 5 min at 48C, resuspended in 10 ml of fresh sterile BHIB and mixed. Cell numbers were estimated using an acridine orange direct count (AODC) technique (Du¡y et al. 1991) and adjusted by the addition of sterile
Listeria monocytogenes and Listeria innocua in beef 573
¢ltered MRD to produce a 2 l solution containing 1000^2000 cells ml71 of either L. monocytogenes or L. innocua species. Mixed inocula were prepared by adding 1 l of L. innocua suspension to 1 l of the L. monocytogenes suspension and mixing well. Thus, three inocula were prepared; L. monocytogenes only, L. innocua only, and a mixed inoculum containing both L. monocytogenes and L. innocua. Each of the three batches of prepared beef pieces was inoculated by immersing for 5 s in one of the inocula prepared above. Excess liquid was allowed to drain o¡ the meat, which was then double-minced through a sterile 10 -mm plate using a Crypto-Peerless (UK) mincing machine. This resulted in an inoculation level of approximately 102 g71 Listeria cells in the meat as estimated from previous experiments, and from Day 0 results in this experiment.
Packaging The inoculated, double-minced meat was distributed in 20+l g quantities (a) onto Plastic food trays (Dynopack, Norway) covered with cling ¢lm to reduce drying, or (b) into small pouches made from Cryovac (W.R. Grace, Ireland) vacuum bags. These pouches were sealed using a vacuum-packaging machine (Swissvac, UK).
Storage Packaged samples were divided into two lots, and stored at 08C or 108C. Four packs of each type (air/vacuum packed) were removed from each storage temperature on each of days 0, 7, 14 and 28 and examined as described below.
Microbiological analysis A 5+0?5 g sample from each pack was weighed into a stomacher bag ¢tted with integral ¢lter (Seward Medical, UK) and diluted 1:10 using an automatic gravimetric dilutor (Watson^ Marlow, UK). The bags were stomached for 1 min in a laboratory stomacher (Seward), and the resultant ¢ltrate serially diluted in MRD. Aliquots (1?0 ml) of serial dilutions were plated out onto Palcam Agar plates (Oxoid) containing 50 mg ml71 tetracycline hydrochloride
(T-3383, Sigma) UK (PATC) and Palcam Agar containing 1000 mg ml71 streptomycin sulphate (S-9137, Sigma) (PASS). Both sets of plates were then incubated at 308C for 48 h and examined for growth of Listeria. Samples of the recovered ¢ltrate were also examined for members of the background spoilage micro£ora, i.e., Pseudomonas spp. (Pseudomonas Agar CFC, at 258C for 72 h), Enterobacteriaceae (VRBG Agar at 378C for 24 h), Brocothrix thermosphacta (STAA Agar at 228 C for 72 h), Lactic acid bacteria (MRS Agar at 308C for 72 h), and Total Viable Counts (SPC Agar at 308C for 72 h) (all media obtained from Oxoid).
pH measurement A 2 g sample from each pack was added to 10 ml iodoacetate bu¡er (sodium iodoacetate 5 mM; BDH Chemicals, UK, and potassium chloride 150 mM; AnalaR, BDH). The sample was homogenised (Silverson Machines, UK) for two 15 -s intervals with a 15 s interval between treatments, and the pH of the homogenate measured.
Statistical analysis Three replicates of the above experiment were carried out. Results were analysed using ANOVA (Genstat 5, Rothamsted Experimental Station) to determine any signi¢cant di¡erences between the numbers (cfu g71) of L. monocytogenes and L. innocua during storage, and to establish if packaging type or storage temperature, had an e¡ect on the levels of either strain.
Results Initial numbers (cfu g71) on Day 0 ranged from 1?12^1?88 log10 cfu g71 and in Table 1 it can be seen that after 28 days storage the numbers of both Listeria strains have decreased, ranging from 0^1?95 log10 cfu g71. No signi¢cant di¡erences were noted between the levels of L. monocytogenes and L. innocua detected in samples held for the same period under similar storage conditions. No signi¢cant di¡erences were observed between the numbers of each species of
574 G. Du¡y et al.
Table 1. Populations (log10 cfu g71) of Listeria innocua and Listeria monocytogenes in minced beef after 28 days storage at di¡erent temperatures and using air or vacuum packaging Storage temperature Listeria spp. L. monocytogenes Single inoculum Mixed inoculum L. innocua Single inoculum Mixed inoculum
Packaging environment
08C
108C
Vacuum Air
1?25 0?80
0?00 1?13
Vacuum Air
1?02 0?81
0?55 1?45
Vacuum Air
0?74 0?94
0?08 1?94
Vacuum Air
0?67 0?26
0?65 1?84
Standard error of di¡erence between means = 0?635, degrees of freedom = 30.
Figure 1. Growth of Listeria innocua and Listeria monocytogenes in minced beef during storage under vacuum or air packaging and at 0 or 108C. L. monocytogenes single (^^^), L. monocytogenes mixed (^&^), L. innocua single (^~^), L. innocua mixed (^*^). (a) = 08C, vacuum packed; (b) = 108C, vacuum packed, (c) = 08C, air packed, (d) = 108C, air packed.
Listeria recovered from minced beef samples inoculated with single or mixed inocula. Temperature had an e¡ect on the nature and rate of growth of Listeria in stored minced beef. In samples held at 108C, signi¢cantly higher (P50?001) numbers of both Listeria strains were recovered from aerobic packs than from
vacuum packs. In samples held at 08C, signi¢cantly higher (P50?001) numbers of both Listeria strains were recovered from vacuum packs than from aerobic packs. In Fig. 1 (d) it can be seen that at 108C in aerobically packed samples, numbers of both Listeria stains had increased by 1?5^2 log10 cfu g71
Listeria monocytogenes and Listeria innocua in beef 575
between day 0 and day 7, but levels decreased between days 14 and 28. On days 7 and 14 numbers of Listeria in minced beef samples stored at 108C in air were signi¢cantly higher (P50?001) than in samples stored under the other storage conditions. Figure 2 shows the pattern of changes in pH values in inoculated minced beef during storage. In samples held at 108C in vacuum packs the pH declined from 5?85 to c. 5?2 by day 7, and thereafter decreased more slowly, achieving a pH of 5?1 after 28 days. In samples held at 108C in air the pH values increased to 7?8 by day 7 and continued to increase, achieving a pH value of 8?7 by day 28. In samples held at 08C a similar pattern was observed, although the changes noted were smaller. pH values
decreased in vacuum packed samples, and increased in air packed samples. Table 2 displays the counts for members of the resident spoilage micro£ora, which developed on samples of inoculated minced beef during storage for 28 days. At 08C in aerobically packed minced beef, Pseudomonas spp., Brocothrix thermosphacta and total viable counts were higher than those counts in samples stored under the other conditions. At 108C in aerobically packed minced beef, Enterobacteriaceae counts were higher than in samples stored under the other conditions. At 08C in vacuum packed minced beef, total viable counts and counts of all the other bacteria examined, except Brocothrix thermosphacta, were lower than under the other storage conditions. At 108C in vacuum packed minced beef, counts of Brocothrix thermosphacta were lower than under the other storage conditions. This is the only member of the resident spoilage micro£ora examined that decreased in counts during the storage period, though the decrease only occurred in samples held at 108C in vacuum packs. Counts of Lactic Acid Bacteria were higher at 108C in vacuum packed minced beef than under the other storage conditions.
Discussion
Figure 2. Changes in pH of minced beef during storage at 08C or 108C, under vacuum or in air. Vacuum, 08c (^^^), Vacuum, 108C (^*^), air, 08C (^~^), air, 108C (^&^).
In this study, Listeria spp. did not grow in vacuum packed samples stored at 08C or 108C. In aerobically packed samples Listeria spp. grew at 108C but not at 08C.The observation that this genus can grow in air under conditions of low
Table 2. Populations (log10 cfu g71 ) of some spoilage bacteria in minced beef after 28 days storage at di¡erent temperatures and using air or vacuum packaging. Packaging Storage temperature Total viable count Enterobacteriaceae Pseudomonads Lactic acid bacteria Brochothrix thermosphacta a
(5?66) (2?25) (4?59) (4?88) (4?74)
a
Vacuum
Air
08C
108C
08C
108C
d.f.
s.e.
7?80 4?46 4?90 6?12 4?91
9?71 6?05 5?75 8?84 4?25
11?1 5?97 11?1 6?5 8?94
10?2 7?14 9?33 5?84 8?65
94 93 91 94 89
0?68 0?73 0?84 0?62 0?99
= count at day 0, d.f. = degrees of freedom, s.e. = standard error of di¡erence between means.
576 G. Du¡y et al.
temperature abuse (108C) is in agreement with Sheridan et al. (1995) who found that L. monocytogenes can grow on air-packed lamb at 58C. Growth was found to be slow under these conditions, which is in agreement with previous reports that Listeria spp. did not grow well in meat at low temperatures (Sheridan et al. 1994, Barbosa et al. 1995. Farkas and Andrassy 1996). The packaging atmosphere had a signi¢cant e¡ect on growth rate. Listeria did not grow in vacuum packs, even at the higher temperature (108C). Sheridan et al. (1995) reported that Listeria spp. did not grow in meat (lamb) in vacuum packs at 58C. While Listeria spp. are psychrotrophic, the optimum temperature for growth is between 30 and 378C (Johnson et al. 1990), so it would be expected that the growth of the Listeria spp. in meat would increase as storage temperature increased. This study revealed that growth did not occur under temperature abuse (108C) in vacuum packaged beef, con¢rming the importance of packaging atmosphere in controlling growth rates in this genus. This study showed that the numbers of naturally occurring micro£ora on beef were quite high, regardless of storage conditions, and this may have contributed to the inhibition of Listeria growth. This study found di¡erent patterns of pH change during storage, dependent on the packaging atmosphere, and that storage atmosphere is more important than storage temperature in determining gross pH changes. Thus, pH values increased in aerobically stored samples at both temperatures examined, and decreased in vacuum packaged samples at both temperatures. Under aerobic storage conditions the pH of the meat increased by more than two pH units over the storage period. In this experiment, growth of both Listeria spp. was highest in the meat with the highest pH (at 108C in air), suggesting that pH has a signi¢cant e¡ect on Listeria growth.This is in agreement with several workers who have reported that Listeria spp. grew better in meat of high pH than on low pH meat (Kaya and Schmidt 1991, Grau and Vanderlinde 1993, Barbosa et al. 1995). However, this study also noted that the rate of growth of Listeria spp. declined between day 14 and day 28, although meat pH continued to increase, albeit more slowly, dur-
ing this period.This suggests that other factors are involved in the overall determination of the growth rates of Listeria spp. under aerobic storage conditions. These factors may include such gross e¡ects as the overall depletion of nutrients and/or the build up of metabolic waste products. Psuedomonas numbers in aerobic samples were very high by the end of the experiment (28 days).This group comprised a major component of the total £ora at this stage, and was probably responsible for the pH increases noted. In general terms, psuedomonads are widely recognized as having major e¡ects on the shelf-life of aerobically stored meats (Labadie 1999). Previous studies have reported the inhibition of food pathogens (including Listeria spp.) by Psuedomonads (Matilla-Sandholm and Skitta 1991, Liao and Sapers 1999). In vacuum packs however, lactic acid bacteria predominated within the developed £ora of stored beef mince. These organisms, previously shown as being favoured by vacuum packaging (Barbosa et al. 1995, Labadie 1999), can produce lactic acid, H2O2, and bacteriocins (Juven et al. 1998), and are probably responsible for the observed reductions in pH in beef mince stored under these conditions. It has also been reported by some workers that lactic acid bacteria can inhibit Listeria spp. (Buchanan and Klawitter 1990, Juven et al. 1998). This study did not reveal any signi¢cant differences between the numbers of L. innocua and L. monocytogenes that developed in beef mince inoculated with pure or mixed cultures of these species.This pattern was consistently observed under all storage conditions and storage atmospheres. These observations suggest that there is no signi¢cant competition between these species in stored minced beef. However, in other circumstances, di¡erences between the growth rates of these two closely related species have been reported. A previous study (Petran and Swanson 1993) reported that L. innocua grew more rapidly than L. monocytogenes in selective media.This pattern was not observed in non-selective media and in that environment growth rates were similar. The reasons for reported di¡erences in growth rates in selective media are not clear, although they have signi¢cant implications for
Listeria monocytogenes and Listeria innocua in beef 577
those involved in food safety surveillance. Listeria innocua may be better adapted to growth in selective media than L. monocytogenes, leading to overgrowth and preferential recovery of the non-pathogenic species. Thus Yokoyama et al. (1998) have reported that in laboratory media, L. innocua produces a bacteriocin-like substance that has an inhibitory e¡ect on the growth of L. monocytogenes. This e¡ect has been demonstrated only in laboratory media, and may not occur in foods. However, the ability of organisms to speci¢cally inhibit the growth of related species in laboratory media may signi¢cantly interfere with the recovery and detection of important target species. If, as in this case, widely occurring non-pathogenic species can interfere with the recovery and detection of related less frequently occurring pathogenic species, estimations of the frequency of occurrence of pathogens will be rendered less accurate, and causative agents of food poisoning may not be adequately detected. The main aim of this study was to examine why L. innocua is isolated more often than L. monocytogenes from foods. The results of this study suggest that this pattern of prevalence of L. innocua was not due to changes occurring in meat during storage in relation to (a) competition between the two strains, (b) inhibition of one strain by the other, or, (c) competition with, or inhibition by, other members of the normal bacterial £ora of meat. Further studies are required to ascertain whether competition between L. monocytogenes and L. innocua during growth in enrichment broth alters the relative frequency of recovery of these organisms from such media. As noted in the introduction to this paper, when L. monocytogenes is present in foods, populations are low. Thus any e¡ects or interactions between this pathogen, and more frequently occurring related species, and/or other members of the normal micro£ora of meat and meat products, during storage and during laboratory recovery procedures, may reduce the e⁄ciency of recovery of L. monocytogenes and reduce the accuracy of subsequent reported incidence levels. Such interactions have implications in relation to meat safety and quality surveillance.
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Sheridan, J. J., Du¡y, G., Buchanan, R. L., Mc Dowell, D. A., and Blair, I. S. (1994). The use of selective and non-selective enrichment broths for the isolation of Listeria spp. from meat. Food Microbiol., 11, 439^446. Sheridan, J. J., Doherty, A., Allen, P., McDowell, D. A., Blair, I. S. and Harrington, D. (1995). Investigations on the growth of Listeria monocytogenes on lamb packaged under modi¢ed atmospheres. Food Microbiol. 12, 259^266. Skinner, R. (1996) Listeria: the state of the science Rome 29^30 June 1995 Session IV: country and organizational postures on Listeria monocytogenes in food Listeria: UK government’s approach. Food Control, 7, 245^247. Walsh, D., Du¡y, G., Sherida, J. J., Blair, I. S. and McDowell, D. A. (1998). Comparison of selective and non-selective media for the isolation of Listeria species from retail foods. J. Food Safety 18, 85^89. World Health Organisation (WHO) Working Group. (1988). Foodborne Listeriosis. Bull WHO 66, 421^428. Yokoyama, E., Maruyama, S. and Katsube, Y. (1998). Production of bacteriocin-like substance by Listeria innocua against Listeria monocytogenes. Int. J. Food Microbiol. 40, 133^137.