- Email: [email protected]
Microbial competition: effect of Pseudomonas fluorescens on the growth of Listeria monocytogenes
Food Microbiology, 1999, 16, 523^529 Available online at http://www.idealibrary.com on
Article No. fmic.1998.0264
ORIGINAL ARTICLE
Microbial competition: e¡ect of Pseudomonas £uorescens on the growth of Listeria monocytogenes R. L. Buchanan* and L. K. Bagi
Listeria monocytogenes Scott A was cultured alone and in coculture with Pseudomonas £uorescens ATCC 33231 to characterize quantitatively the e¡ects of microbial competition on the growth of this psychrotrophic pathogen. The bacteria were cultured in brain±heart infusion broth (BHI), using a 3636362 complete factorial design to assess the impact of temperature (4,12,198C), initial pH (50, 60, 70), and sodium chloride content (5, 25, 45 g l71) on the interaction between the two microorganisms. Samples were periodically plated on BHI agar and Vogel Johnson agar to obtain total counts and L. monocytogenes counts, respectively. Growth curves were generated by ¢tting the data to the Gompertz equation, and the derived growth kinetics were compared. When P. £uorescens did in£uence the growth of L. monocytogenes, the primary e¡ect was a suppression of the maximum population density (MPD) reached by the pathogen. Suppression of L. monocytogenes was generally associated with low incubation temperatures (48C) and sodium chloride levels (5 and 25 g l71). Slight increases (510 log cfu ml71) in the MPD attained by L. monocytogenes were observed when grown in the presence of P. £uorescens at higher temperatures (12 and 198C) and sodium chloride levels (25 and 45 g l71) when the pH was 50.The current study supports earlier work that indicates that reliance on microbial competition as a barrier to control L. monocytogenes in refrigerated foods will require detailed knowledge of how the interaction between the pathogen and the micro£ora is a¡ected by environmental and food characteristics such as storage temperature, pH, and water activity. # 1999 Academic Press
Introduction The psychrotrophic pathogen, Listeria monocytogenes, can be isolated routinely in low numbers from a variety of foods, but is seldom present at elevated levels. A variety of environmental factors, such as pH and water activity, * Corresponding author. Current address: U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, 200 C-Street, SW, Washington, DC, USA. 0740 - 0020/99/050523 + 07 $30.00/0
in£uence the rate and extent of L. monocytogenes growth, and these factors have been studied extensively. An additional factor that is often discussed, but for which there are few quantitative data, is the impact of competing micro-organisms. Recently, our laboratory studied how Carnobacterium piscicola, a common lactic acid bacterium that appears to share the same niche with Listeria in refrigerated foods of animal origin, a¡ected the growth of L. monocytogenes, and how that interaction was in£uenced by temperature, pH, and water # 1999 Academic Press
Received: 8 October1998 USDA ARS Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, Pennsylvania19038, USA
524 R. L. Buchanan and L. K. Bagi
activity of the environment (Buchanan and Bagi 1997). The spoilage micro-organism was able to suppress the maximum population density achieved by L. monocytogenes. The magnitude of the competitive e¡ect was in£uenced strongly by cultural conditions, particularly temperature. The mode of inhibition appeared to be associated with the relative growth rates of the two micro-organisms under any speci¢c set of environmental conditions. The objective of the current study was to determine if this competitive e¡ect occurs with a second psychrotrophic spoilage microorganism under the various combinations of temperature, pH, and sodium chloride content used in the earlier study. Pseudomonas £uorescens was selected as an example of a psychrotrophic, Gram-negative spoilage microorganism that L. monocytogenes would likely encounter in a variety of refrigerated foods. This species was also selected because of the di¡erences in the e¡ect of P. £uorescens on L. monocytogenes that have been observed by various investigators. In dairy products, chicken meat, and microbiological media, P. £uorescens isolates have been reported to stimulate the growth of L. monocytogenes (Farrag and Marth 1989b, Marshall and Schmidt 1989, 1991, Marshall et al. 1992), inhibit the growth of L. monocytogenes (Cheng et al. 1995, Freedman et al. 1989, Farrag and Marth 1989c), or have no e¡ect of L. monocytogenes growth (Farrag and Marth 1989a, Marshall et al. 1992). Activity appears to be strain dependent. For example, Gram (1993) reported that only eight of 209 Pseudomonas spp. isolated from ¢sh had activity against L. monocytogenes.
Materials and Methods Experimental design The e¡ects of pH (50, 60, 70), sodium chloride (5, 25, 45 g l71), and temperature (4, 12, 198C) on the coculture growth of L. monocytogenes and P. £uorescens were studied using a 3636362 complete factorial design, with each pH/NaCl/ temperature combination being tested on two separate occasions.
Micro-organisms Listeria monocytogenes Scott A and P. £uorescens ATCC 33231 were used throughout the study. The Pseudomonas isolate was initially screened and then selected on the basis of its inability to produce a detectable bacteriocin (Buchanan and Bagi 1997) or a siderophore (Cheng et al. 1995) against L. monocytogenes Scott A. Stock cultures of L. monocytogenes and P. £uorescens were obtained from the Eastern Regional Research Center stock culture collection and maintained in brain±heart infusion broth (BHI, Difco, Detroit, Michigan, USA) incubated for 18±24 h at 378C and 288C, respectively, and then stored at 48C. These working stock cultures were transferred monthly. Prior to the initiation of a coculture trial, a loopful of the P. £uorescens working stock culture was transferred to individual 250 -ml Erlenmeyer £asks containing 50 ml of BHI. The £ask was then incubated for 24 h at 288C on a rotary shaker (150 rpm). L. monocytogenes was cultured in the same manner except the culture was incubated at 378C.
Cocultures Cocultures and controls were prepared and sampled using the techniques described by Buchanan and Bagi (1997). BHI was prepared, supplemented with sodium chloride as required to achieve target levels of 5, 25, or 45 g l71, the pH adjusted to 50, 60 or 70 with concentrated hydrochloric acid, and brought up to volume. The broth was transferred in 100 -ml portions to 250 -ml Erlenmeyer £asks, which were sealed with foam plugs and sterilized by autoclaving for 20 min at 1218C. The pH of the medium was veri¢ed after autoclaving to ensure that it remained within + 01 pH units of the target value. The £asks were inoculated with 01 ml of appropriate dilutions of the 24 -h cultures of L. monocytogenes and P. £uorescens to achieve inoculum levels of approximately 103 cfu ml71. Flasks inoculated with only L. monocytogenes were used as controls. The £asks were then incubated on rotary shakers at 4, 12 or 198C.
P. £uorescens vs L. monocytogenes 525
Periodically, 40 -ml samples of each coculture and each control culture were removed, diluted appropriately in sterile 01% peptone water, and surface plated in duplicate on BHI agar (BHIA; Difco) and Vogel Johnson agar (VJA; Difco) plates using a spiral platter (Model D, Spiral Biotech, Bethesda, Maryland, USA).The BHIA plates were incubated for 24 h at 288C and the VJA plates were incubated for 24 h at 378C. The plates were then counted using an automated counter (Laser Counter, Spiral Biotech) and converted to log values. The BHIA counts re£ected the total counts of both L. monocytogenes and P. £uorescens colonies, whereas VJA only supported the growth of L. monocytogenes. The counts obtained for the Listeria only controls on BHIA (data not shown) and VJA were virtually identical. In those instances were P. £uorescens grew more rapidly in coculture than L. monocytogenes, the number of P. £uorescens could be estimated by the BHIA count.
Curve ¢tting Growth curves were generated by ¢tting the data to the Gompertz equation as previously described (Gibson et al. 1988, Buchanan et al. 1989), and used to calculate lag phase durations (LPD), exponential growth rates (EGR), generation times (GT), and maximum population densities (MPD). The time to a 10 -fold increase (T10) was also calculated using the Gompertz equation.This value provided a measure of the combined LPD and EGR. A 10 -fold increase was selected because, in some instances, the increase in population density above the inoculum was less than 100 fold.
Results The growth kinetics of L. monocytogenes when cultured alone and in coculture with P. £uorescens at various combinations of temperature, initial pH and sodium chloride content are summarized in Table 1. The growth kinetics for the total bacterial population of the cocultures (L. monocytogenes + P. £uorescens) are also provided. Four responses were observed. At 48C, L. monocytogenes did not grow at pH 50, and
P. £uorescens only grew at that pH/temperature combination at the lowest sodium chloride level. Likewise, neither organism grew at 128C at the highest sodium chloride level when the pH was 50. At 12 and 198C, the growth of L. monocytogenes at pH 60 and 70 was similar in monoculture and coculture, particularly with higher NaCl levels. With these combinations of environmental conditions, P. £uorescens had no discernable e¡ect on the growth of L. monocytogenes. However, at the lower temperatures and salt concentrations, P. £uorescens suppressed the MPD reached by L. monocytogenes by as much as four log cycles. Finally, the pH 50 cocultures of L. monocytogenes grew to somewhat higher (510 log cycle) MPDs than the corresponding monocultures with three temperature/sodium chloride combinations: 128C 25 g71 l71 NaCl, 198C 25 g71 l71 NaCl, and 198C 45 g71 l71 NaCl.This may re£ect a small stimulation of growth by P. £uorescens under these conditions. Examples of growth curves derived from the experimental data for environmental conditions where P. £uorescens suppressed, increased, or did not a¡ect MPD values for L. monocytogenes are provided in Fig. 1.The di¡erences in the MPD values reached by L. monocytogenes when grown in monoculture and coculture are summarized graphically in Fig. 2. In our earlier studies, the magnitude of the suppression of L. monocytogenes MPD by C. piscicola was related to the relative growth rates of the two species, as determined by comparison of the MPD di¡erences between L. monocytogenes monocultures and cocultures vs the ratio of the T10 values based on the counts on BHI agar and Vogel Johnson agar (Buchanan and Bagi 1997). Such a comparison contrasts the di¡erence in the extent of growth of L. monocytogenes in monoculture and coculture against the relative time it took L. monocytogenes and the competing micro-organism to initiate exponential growth (i.e. complete lag phase and increase by one log cycle). Increasing MPD di¡erentials with increasing T10 values would suggest that growth suppression by a competing micro-organism was due primarily to the competitor being able to grow faster than L. monocytogenes. This relationship was less clear cut when the P. £uorescens cocultures
526 R. L. Buchanan and L. K. Bagi
Table 1. Growth kinetics of L. monocytogenes Scott A when cultured as a monoculture and as a coculture with P. £uorescens ATCC 33231 Culture conditions Temp (8C)
L. monocytogenes: monoculture control MPD LPD [log(cfu (h) ml71)]
L. monocytogenes: coculture MPD GT LPD [log(cfu (h) (h) ml71)]
T10 (h)
Total coculture: Pseudomonas + Listeria MPD GT (LPD) [log(cfu (h) (h) ml71)]
pH
NaCl (gl71)
GT (h)
19 19 19 19 19 19 19 19 19
50 50 50 60 60 60 70 70 70
5 25 45 5 25 45 5 25 45
7?2a 9?8 24?9 2?1 2?2 2?8 1?4 1?6 2?9
264 429 699 106 131 191 143 142 154
83 82 76 97 97 95 93 88 99
494 22 741 88 1508 234 165 20 195 22 275 27 184 16 191 15 235 27
418 558 927 106 139 191 117 148 144
74 90 85 89 97 96 92 88 98
490 15 831 96 1665 220 167 18 203 22 271 28 164 12 195 16 220 27
286 519 1161 134 140 202 166 157 185
97 98 84 108 100 97 100 88 99
333 801 1860 182 204 283 200 207 263
12 12 12 12 12 12 12 12 12
50 50 50 60 60 60 70 70 70
5 25 45 5 25 45 5 25 45
15?4 1182 13?7 1111 NGb Ð 4?5 217 4?3 304 5?3 279 3?3 104 4?3 222 4?7 325
78 82 Ð 99 97 96 83 99 99
1678 155 536 1551 57 1589 Ð NG Ð 339 36 191 428 45 304 438 50 301 209 31 113 339 50 268 457 45 339
75 89 Ð 63 92 99 81 90 99
1043 11 1764 55 Ð NG 308 30 438 42 451 53 213 29 412 43 465 44
670 1618 Ð 199 336 298 149 333 386
102 98 Ð 106 101 99 109 104 99
701 1782 Ð 275 456 459 228 444 514
4 4 4 4 4 4 4 4 4
50 50 50 60 60 60 70 70 70
5 25 45 5 25 45 5 25 45
NG Ð NTc Ð NT Ð 14?4 879 13?6 951 16?0 1267 9?9 565 9?8 789 12?8 1118
Ð Ð Ð 95 95 84 98 97 96
Ð Ð Ð 1317 1363 1776 859 1073 1491
Ð Ð Ð 52 69 85 67 74 97
Ð Ð Ð 1114 1149 1577 793 1046 1536
63 1063 NT Ð NT Ð 65 494 79 938 161 1045 49 499 83 824 118 1264
102 Ð Ð 101 107 87 102 102 97
1250 Ð Ð 689 1159 1554 642 1059 1611
NG Ð NT Ð NT Ð 124 696 144 671 164 1057 87 505 104 707 120 1186
T10 (h)
T10 (h)
a
Average of two independent trials. Individual values were consistently within 10% of mean. No growth. c Not tested. GT, Generation time; LPD, lag phase duration; MPD, maximum population density attained by culture; T10, time to a 10 -fold increase in population density. b
were evaluated (Fig. 3). While the environmental conditions that lead to suppression of L. monocytogenes were generally those that favored the growth of P. £uorescens, there were several instances where rapid growth of P. £uorescens did not result in a suppression of L. monocytogenes MPD (Table 1).
Discussion The interaction of bacterial species in food systems has been studied largely in relation to the production of di¡usable antimicrobial
agents (e. g. acids, bacteriocins, peroxides). In the absence of these agents, when a spoilage organism is present in the same environment as a pathogen, it can either not a¡ect, stimulate, or inhibit the rate or extent of growth of the pathogenic species. The current study suggests that P. £uorescens is capable of all three responses when growing in the presence of L. monocytogenes, depending on the environmental conditions. This, in part, may help to explain the array of reports that Pseudomonas spp. can inhibit (Cheng et al. 1995, Freedman et al. 1989, Farrag and Marth 1989c), stimulate (Farrag and Marth 1989b,
P. £uorescens vs L. monocytogenes 527
Figure 2. E¡ect of P. £uorescens ATCC 33231 on the maximum population density (MPD) reached by L. monocytogenes Scott A under various conditions of temperature, initial pH, and sodium chloride content. MPD di¡erential = log(MPDmonoculture)7 log(MPDcoculture). No growth of L. monocytogenes in cocultures or monocultures ($). [Note: as the di¡erence of two log values (and thus the ratio of two untransformed values), MPD di¡erentials are unitless.]
Figure 1. Examples of the types of growth re-
sponses observed when L. monocytogenes Scott A was grown alone or in coculture with P. £uorescens ATCC 33231. Growth curves were generated using the growth kinetics values provided in Table 1 in conjunction with the Gompertz equations; points depicted are not the experimental values. Listeria monocytogenes growth in monoculture control (&); L. monocytogenes growth in L. monocytogenes/P. £uorescens coculture (*); total count (L. monocytogenes + P. £uorescens) for L. monocytogenes/P. £uorescens coculture ($) (a) 48C Ð pH 60 Ð 5 g l71 NaCl. (b) 128C Ð pH 70 Ð 5 g l71 NaCl. (c) 198C Ð pH 50 Ð 25 g l71 NaCl.
Marshall and Schmidt 1989, 1991, Marshall et al. 1992), or have no e¡ect (Farrag and Marth 1989a, Marshall et al. 1992) on L. monocytogenes.
The ability of Pseudomonas spp. to inhibit the growth of L. monocytogenes (Cheng et al. 1995, Freedman et al. 1989, Gram 1993) and other spoilage (Gram 1993, Gram and Melchiorsen 1996) and pathogenic (Cheng et al. 1995, Freedman 1989, Gram 1993, Oberhofer and Frazier 1961, Seminiano and Frazier 1966) bacteria has been related to the production of siderophores that enhance the organism's acquisition of iron. However, the inhibition of L. monocytogenes in the current study is not likely to be related to siderophore production for two reasons. First, the P. £uorescens isolate used in the current study was selected because it did not produce, on routine screening, either a siderophore or a bacteriocin. Second, the use of BHI as a test system strongly depresses the impact of siderophore production by P. £uorescens, since the medium is high in iron (Freedman et al. 1989). However, this does not rule out depletion of another micronutrient such as a trace mineral; a mechanism that has been suggested for the inhibition of L. monocytogenes by C. piscicola (Buchanan and Bagi 1997). Further, the current study did not rule out the possibility that the P. £uorescens strain used only produces a siderophore or some other mechanism for sequestering trace minerals when incubated at low temperatures
528 R. L. Buchanan and L. K. Bagi
Figure 3. Relationship between the extent of maximum population density (MPD) suppression of L. monocytogenes Scott A and the relative growth rates of the L. monocytogenes and P. £uorescens ATCC 33231 MPD di¡erential = log(MPDmonoculture)7 log(MPDcoculture),T10 ratio = T10 based on coculture VJA counts 7T10 based on coculture BHIA counts, T10 = time to a 10 -fold increase in population density (see text).
(Table 1). Evaluation of this possibility will require additional studies. Additional studies will also be needed to con¢rm the small enhancement of growth under acidic conditions when L. monocytogenes was cocultured with P. £uorescens in BHI. It is not likely that it is due to P. £uorescens modifying the pH of the medium since such changes typically occur in the early stationary growth phase. At that point, L. monocytogenes had also achieved stationary growth (Fig. 1(c)). These observations are consistent with reports that this spoilage organism can stimulate growth of the pathogen in whole and skim milk (Farrag and Marth 1989b, Marshall and Schmidt 1989, 1991, Marshall et al. 1992). Growth of E. coli O157:H7 in skim milk has also been reported to be enhanced by P. £uorescens (Quinto et al. 1997). Stimulation of L. monocytogenes growth has been atributed to the hydrolysis of milk proteins by P. £uorescens. However, at the current time it is not clear how this explanation would account for stimulation of L. monocytogenes in the current study being restricted to only a few combinations of environmental conditions (Table 1). Reliance on microbial competition as one of the barriers that control L. monocytogenes growth in foods would require detailed knowledge of the food, its micro£ora, and how the
food is handled during distribution, marketing, and use. For example, the results of the current study indicate that holding a product with a pH of 60 and a sodium chloride content of 25 g l71 at 48C would suppress L. monocytogenes growth (Table 1). However, those same pH and sodium chloride values would o¡er no protection if the product was temperature-abused at 198C. Likewise, the identity of the principal members of the micro£ora would have to be constant if it were to provide consistent protection. For example, Thomas and Wimpenny (1996) used gradient plating techniques to study the e¡ects of temperature, pH, and sodium chloride content on the interaction between three Salmonella spp. and P. £uorescens or P. putida, two common spoilage species that compete for the same niche. They observed that at 308C, P. putida had no e¡ect on the salmonellae, and the pathogen inhibited P. £uorescens. At 208C, P. putida inhibited Salmonella but P. £uorescens had no activity. It is also important to note that additional factors that a¡ect the interaction between L. monocytogenes and competitive microorganisms would have to be identi¢ed and characterized. For example, P. putida inhibited Salmonella typhimurium in an aerobic environment, but not when oxygen was restricted (Thomas and Wimpenny 1996). Potentially, the use of modi¢ed atmospheres to control the growth of spoilage bacteria could drastically alter any protection provided by an indigenous micro£ora. A number of studies have indicated that the use of speci¢c modi¢ed atmosphere gases may inhibit the growth of spoilage microorganisms without a¡ecting L. monocytogenes, thus altering any protection provided by the micro£ora (Hendricks and Hotchkiss 1997, Marshall et al. 1991, 1992, Wimpfheimer et al. 1990). Likewise, factors such as strain di¡erences and the e¡ect of initial levels on the interaction of L. monocytogenes and a competitor would have to be taken into account (Buchanan and Klawitter 1992a, b). Considering the impact that food environment had on the interaction between L. monocytogenes and competing micro-organisms that was observed in our current and early study (Buchanan and Bagi 1997), it appears likely
P. £uorescens vs L. monocytogenes 529
that a speci¢c isolate with known activity against L. monocytogenes would have to be used if microbial competition was to be relied upon to provide an e¡ective hurdle. Selection of such an isolate would also have to take into account the impact of the micro-organism on the shelflife of the food. Using these criteria, it appears that C. piscicola would be a much better candidate as a biological control agent than P. £uorescens.
References Buchanan, R. L. and Bagi, L. K. (1997) Microbial competition: e¡ect of culture conditions on the suppression of Listeria monocytogenes Scott A by Carnobacterium piscicola. J. Food Protect. 60, 254±261. Buchanan, R. L. and Klawitter, L. A. (1992a) Characterization of a lactic acid bacterium, Carnobacterium piscicola LK5, with activity against Listeria monocytogenes. J. Food Safety 12, 199±217. Buchanan, R. L. and Klawitter, L. A. (1992b) E¡ectiveness of Carnobacterium piscicola LK5 for controlling the growth of Listeria monocytogenes Scott A in refrigerated foods. J. Food Safety 12, 219±236. Buchanan, R. L., Stahl, H. G. and Whiting, R. C. (1989) E¡ects and interactions of temperature, pH, atmosphere, sodium chloride, and sodium nitrite on the growth of Listeria monocytogenes. J. Food Protect. 52, 844±851. Cheng, C.-M., Doyle, M. P. and Luchansky, J. B. (1995) Identi¢cation of Pseudomonas £uorescens strains isolated from raw pork and chicken that produce siderophores antagonistic towards foodborne pathogens. J. Food Prot. 58, 1340±1344. Farrag, S. A. and Marth, E. H. (1989a) Variation in initial populations of Pseudomonas £uorescens a¡ects behavior of Listeria monocytogenes in skim milk at 7 or 138C. Milchwissenschaft 46, 718±721. Farrag, S. A. and Marth, E. H. (1989b) Growth of Listeria monocytogenes in the presence of Pseudomonas £uorescens at 7 or 138C in skim milk. J. Food Prot. 52, 852±855. Farrag, S. A. and Marth, E. H. (1989c) Behavior of Listeria monocytogenes when incubated together with Pseudomonas species in tryptose broth at 7 and 138C. J. Food Prot. 52, 536±539. Freedman, D. J., Kondo, J. K. and Willrett, D. L. (1989) Antagonism of foodborne bacteria by Pseudomonas spp.: a possible role for iron. J. Food Prot. 52, 484±489.
Gibson, A., Bratchell, N. and Roberts, T. A. (1988) Predicting microbial growth: growth responses of salmonellae in a laboratory medium as a¡ected by pH, sodium chloride, and storage temperature. Int. J. Food Microbiol. 6, 155±178. Gram, L. (1993) Inhibitory e¡ect against pathogenic and spoilage bacteria of Pseudomonas strains isolated from spoiled and fresh ¢sh. Appl. Environ. Microbiol. 59, 2197±2203. Gram, L. and Melchiorsen, J. (1996) Interaction between ¢sh spoilage bacteria Pseudomonas spp. and Shewanella putrefaciens in ¢sh extracts and on ¢sh tissue. J. Appl. Bacteriol. 80, 589±595. Hendricks, M. T. and Hotchkiss, J. H. (1997) E¡ect of carbon dioxide on the growth of Pseudomonas £uorescens and Listeria monocytogenes in aerobic atmospheres. J. Food Prot. 60, 1548±1552. Marshall, D. L. and Schmidt, R. H. (1989) Growth of Listeria monocytogenes at 108C in milk preincubated with selected pseudomonads. J. Food Prot. 51, 277±282. Marshall, D. L. and Schmidt, R. H. (1991) Physiological evaluation of stimulated growth of Listeria monocytogenes by Pseudomonas species in milk. Can. J. Microbiol. 37, 594±599. Marshall, D. L., Wiese-Lehigh, P. L., Wells, J. H. and Farr, A. J. (1991) Comparative growth of Listeria monocytogenes and Pseudomonas £uorescens on precooked chicken nuggets stored under modi¢ed atmospheres. J. Food Prot. 54, 841±843, 851. Marshall, D. L., Andrews, L. S.,Wells, J. H. and Farr, A. J. (1992) In£uence of modi¢ed atmosphere packaging on the competitive growth of Listeria monocytogenes and Pseudomonas £uorescens on precooked chicken. Food Microbiol. 9, 303±309. Oberhofer,T. R. and Frazier,W. C. (1961) Competition of Staphylococcus aureus with other organisms. J. Milk Food Technol. 24, 172±175. Quinto, E. J., Franco, C. M., Fente, C. A., Vazquez, B. I. and Cepeda, A. (1997) Growth of Escherichia coli O157:H7 in the presence of Pseudomonas £uorescens in skimmed milk at 7 or 258C. J. Food Safety 16, 273±285. Seminiano, E. N. and Frazier, W. C. (1966) E¡ect of pseudomonads and Achromobacteraeae on growth of Staphylococcus aureus. J. Milk Food Technol. 29, 161±164. Thomas, L.V. and Wimpenny, J.W. T. (1996) Competition between Salmonella and Pseudomonas species growing in and on agar, as a¡ected by pH, sodium chloride concentration and temperature. Int. J. Food Microbiol. 29, 361±370. Wimpfheimer, L., Altman, N. S. and Hotchkiss, J. H. (1990) Growth of Listeria monocytogenes Scott A, serotype 4 and competitive spoilage organisms in raw chicken packaged under modi¢ed atmospheres and in air. Int. J. Food Microbiol. 11, 205±214.
Article No. fmic.1998.0264
ORIGINAL ARTICLE
Microbial competition: e¡ect of Pseudomonas £uorescens on the growth of Listeria monocytogenes R. L. Buchanan* and L. K. Bagi
Listeria monocytogenes Scott A was cultured alone and in coculture with Pseudomonas £uorescens ATCC 33231 to characterize quantitatively the e¡ects of microbial competition on the growth of this psychrotrophic pathogen. The bacteria were cultured in brain±heart infusion broth (BHI), using a 3636362 complete factorial design to assess the impact of temperature (4,12,198C), initial pH (50, 60, 70), and sodium chloride content (5, 25, 45 g l71) on the interaction between the two microorganisms. Samples were periodically plated on BHI agar and Vogel Johnson agar to obtain total counts and L. monocytogenes counts, respectively. Growth curves were generated by ¢tting the data to the Gompertz equation, and the derived growth kinetics were compared. When P. £uorescens did in£uence the growth of L. monocytogenes, the primary e¡ect was a suppression of the maximum population density (MPD) reached by the pathogen. Suppression of L. monocytogenes was generally associated with low incubation temperatures (48C) and sodium chloride levels (5 and 25 g l71). Slight increases (510 log cfu ml71) in the MPD attained by L. monocytogenes were observed when grown in the presence of P. £uorescens at higher temperatures (12 and 198C) and sodium chloride levels (25 and 45 g l71) when the pH was 50.The current study supports earlier work that indicates that reliance on microbial competition as a barrier to control L. monocytogenes in refrigerated foods will require detailed knowledge of how the interaction between the pathogen and the micro£ora is a¡ected by environmental and food characteristics such as storage temperature, pH, and water activity. # 1999 Academic Press
Introduction The psychrotrophic pathogen, Listeria monocytogenes, can be isolated routinely in low numbers from a variety of foods, but is seldom present at elevated levels. A variety of environmental factors, such as pH and water activity, * Corresponding author. Current address: U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, 200 C-Street, SW, Washington, DC, USA. 0740 - 0020/99/050523 + 07 $30.00/0
in£uence the rate and extent of L. monocytogenes growth, and these factors have been studied extensively. An additional factor that is often discussed, but for which there are few quantitative data, is the impact of competing micro-organisms. Recently, our laboratory studied how Carnobacterium piscicola, a common lactic acid bacterium that appears to share the same niche with Listeria in refrigerated foods of animal origin, a¡ected the growth of L. monocytogenes, and how that interaction was in£uenced by temperature, pH, and water # 1999 Academic Press
Received: 8 October1998 USDA ARS Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, Pennsylvania19038, USA
524 R. L. Buchanan and L. K. Bagi
activity of the environment (Buchanan and Bagi 1997). The spoilage micro-organism was able to suppress the maximum population density achieved by L. monocytogenes. The magnitude of the competitive e¡ect was in£uenced strongly by cultural conditions, particularly temperature. The mode of inhibition appeared to be associated with the relative growth rates of the two micro-organisms under any speci¢c set of environmental conditions. The objective of the current study was to determine if this competitive e¡ect occurs with a second psychrotrophic spoilage microorganism under the various combinations of temperature, pH, and sodium chloride content used in the earlier study. Pseudomonas £uorescens was selected as an example of a psychrotrophic, Gram-negative spoilage microorganism that L. monocytogenes would likely encounter in a variety of refrigerated foods. This species was also selected because of the di¡erences in the e¡ect of P. £uorescens on L. monocytogenes that have been observed by various investigators. In dairy products, chicken meat, and microbiological media, P. £uorescens isolates have been reported to stimulate the growth of L. monocytogenes (Farrag and Marth 1989b, Marshall and Schmidt 1989, 1991, Marshall et al. 1992), inhibit the growth of L. monocytogenes (Cheng et al. 1995, Freedman et al. 1989, Farrag and Marth 1989c), or have no e¡ect of L. monocytogenes growth (Farrag and Marth 1989a, Marshall et al. 1992). Activity appears to be strain dependent. For example, Gram (1993) reported that only eight of 209 Pseudomonas spp. isolated from ¢sh had activity against L. monocytogenes.
Materials and Methods Experimental design The e¡ects of pH (50, 60, 70), sodium chloride (5, 25, 45 g l71), and temperature (4, 12, 198C) on the coculture growth of L. monocytogenes and P. £uorescens were studied using a 3636362 complete factorial design, with each pH/NaCl/ temperature combination being tested on two separate occasions.
Micro-organisms Listeria monocytogenes Scott A and P. £uorescens ATCC 33231 were used throughout the study. The Pseudomonas isolate was initially screened and then selected on the basis of its inability to produce a detectable bacteriocin (Buchanan and Bagi 1997) or a siderophore (Cheng et al. 1995) against L. monocytogenes Scott A. Stock cultures of L. monocytogenes and P. £uorescens were obtained from the Eastern Regional Research Center stock culture collection and maintained in brain±heart infusion broth (BHI, Difco, Detroit, Michigan, USA) incubated for 18±24 h at 378C and 288C, respectively, and then stored at 48C. These working stock cultures were transferred monthly. Prior to the initiation of a coculture trial, a loopful of the P. £uorescens working stock culture was transferred to individual 250 -ml Erlenmeyer £asks containing 50 ml of BHI. The £ask was then incubated for 24 h at 288C on a rotary shaker (150 rpm). L. monocytogenes was cultured in the same manner except the culture was incubated at 378C.
Cocultures Cocultures and controls were prepared and sampled using the techniques described by Buchanan and Bagi (1997). BHI was prepared, supplemented with sodium chloride as required to achieve target levels of 5, 25, or 45 g l71, the pH adjusted to 50, 60 or 70 with concentrated hydrochloric acid, and brought up to volume. The broth was transferred in 100 -ml portions to 250 -ml Erlenmeyer £asks, which were sealed with foam plugs and sterilized by autoclaving for 20 min at 1218C. The pH of the medium was veri¢ed after autoclaving to ensure that it remained within + 01 pH units of the target value. The £asks were inoculated with 01 ml of appropriate dilutions of the 24 -h cultures of L. monocytogenes and P. £uorescens to achieve inoculum levels of approximately 103 cfu ml71. Flasks inoculated with only L. monocytogenes were used as controls. The £asks were then incubated on rotary shakers at 4, 12 or 198C.
P. £uorescens vs L. monocytogenes 525
Periodically, 40 -ml samples of each coculture and each control culture were removed, diluted appropriately in sterile 01% peptone water, and surface plated in duplicate on BHI agar (BHIA; Difco) and Vogel Johnson agar (VJA; Difco) plates using a spiral platter (Model D, Spiral Biotech, Bethesda, Maryland, USA).The BHIA plates were incubated for 24 h at 288C and the VJA plates were incubated for 24 h at 378C. The plates were then counted using an automated counter (Laser Counter, Spiral Biotech) and converted to log values. The BHIA counts re£ected the total counts of both L. monocytogenes and P. £uorescens colonies, whereas VJA only supported the growth of L. monocytogenes. The counts obtained for the Listeria only controls on BHIA (data not shown) and VJA were virtually identical. In those instances were P. £uorescens grew more rapidly in coculture than L. monocytogenes, the number of P. £uorescens could be estimated by the BHIA count.
Curve ¢tting Growth curves were generated by ¢tting the data to the Gompertz equation as previously described (Gibson et al. 1988, Buchanan et al. 1989), and used to calculate lag phase durations (LPD), exponential growth rates (EGR), generation times (GT), and maximum population densities (MPD). The time to a 10 -fold increase (T10) was also calculated using the Gompertz equation.This value provided a measure of the combined LPD and EGR. A 10 -fold increase was selected because, in some instances, the increase in population density above the inoculum was less than 100 fold.
Results The growth kinetics of L. monocytogenes when cultured alone and in coculture with P. £uorescens at various combinations of temperature, initial pH and sodium chloride content are summarized in Table 1. The growth kinetics for the total bacterial population of the cocultures (L. monocytogenes + P. £uorescens) are also provided. Four responses were observed. At 48C, L. monocytogenes did not grow at pH 50, and
P. £uorescens only grew at that pH/temperature combination at the lowest sodium chloride level. Likewise, neither organism grew at 128C at the highest sodium chloride level when the pH was 50. At 12 and 198C, the growth of L. monocytogenes at pH 60 and 70 was similar in monoculture and coculture, particularly with higher NaCl levels. With these combinations of environmental conditions, P. £uorescens had no discernable e¡ect on the growth of L. monocytogenes. However, at the lower temperatures and salt concentrations, P. £uorescens suppressed the MPD reached by L. monocytogenes by as much as four log cycles. Finally, the pH 50 cocultures of L. monocytogenes grew to somewhat higher (510 log cycle) MPDs than the corresponding monocultures with three temperature/sodium chloride combinations: 128C 25 g71 l71 NaCl, 198C 25 g71 l71 NaCl, and 198C 45 g71 l71 NaCl.This may re£ect a small stimulation of growth by P. £uorescens under these conditions. Examples of growth curves derived from the experimental data for environmental conditions where P. £uorescens suppressed, increased, or did not a¡ect MPD values for L. monocytogenes are provided in Fig. 1.The di¡erences in the MPD values reached by L. monocytogenes when grown in monoculture and coculture are summarized graphically in Fig. 2. In our earlier studies, the magnitude of the suppression of L. monocytogenes MPD by C. piscicola was related to the relative growth rates of the two species, as determined by comparison of the MPD di¡erences between L. monocytogenes monocultures and cocultures vs the ratio of the T10 values based on the counts on BHI agar and Vogel Johnson agar (Buchanan and Bagi 1997). Such a comparison contrasts the di¡erence in the extent of growth of L. monocytogenes in monoculture and coculture against the relative time it took L. monocytogenes and the competing micro-organism to initiate exponential growth (i.e. complete lag phase and increase by one log cycle). Increasing MPD di¡erentials with increasing T10 values would suggest that growth suppression by a competing micro-organism was due primarily to the competitor being able to grow faster than L. monocytogenes. This relationship was less clear cut when the P. £uorescens cocultures
526 R. L. Buchanan and L. K. Bagi
Table 1. Growth kinetics of L. monocytogenes Scott A when cultured as a monoculture and as a coculture with P. £uorescens ATCC 33231 Culture conditions Temp (8C)
L. monocytogenes: monoculture control MPD LPD [log(cfu (h) ml71)]
L. monocytogenes: coculture MPD GT LPD [log(cfu (h) (h) ml71)]
T10 (h)
Total coculture: Pseudomonas + Listeria MPD GT (LPD) [log(cfu (h) (h) ml71)]
pH
NaCl (gl71)
GT (h)
19 19 19 19 19 19 19 19 19
50 50 50 60 60 60 70 70 70
5 25 45 5 25 45 5 25 45
7?2a 9?8 24?9 2?1 2?2 2?8 1?4 1?6 2?9
264 429 699 106 131 191 143 142 154
83 82 76 97 97 95 93 88 99
494 22 741 88 1508 234 165 20 195 22 275 27 184 16 191 15 235 27
418 558 927 106 139 191 117 148 144
74 90 85 89 97 96 92 88 98
490 15 831 96 1665 220 167 18 203 22 271 28 164 12 195 16 220 27
286 519 1161 134 140 202 166 157 185
97 98 84 108 100 97 100 88 99
333 801 1860 182 204 283 200 207 263
12 12 12 12 12 12 12 12 12
50 50 50 60 60 60 70 70 70
5 25 45 5 25 45 5 25 45
15?4 1182 13?7 1111 NGb Ð 4?5 217 4?3 304 5?3 279 3?3 104 4?3 222 4?7 325
78 82 Ð 99 97 96 83 99 99
1678 155 536 1551 57 1589 Ð NG Ð 339 36 191 428 45 304 438 50 301 209 31 113 339 50 268 457 45 339
75 89 Ð 63 92 99 81 90 99
1043 11 1764 55 Ð NG 308 30 438 42 451 53 213 29 412 43 465 44
670 1618 Ð 199 336 298 149 333 386
102 98 Ð 106 101 99 109 104 99
701 1782 Ð 275 456 459 228 444 514
4 4 4 4 4 4 4 4 4
50 50 50 60 60 60 70 70 70
5 25 45 5 25 45 5 25 45
NG Ð NTc Ð NT Ð 14?4 879 13?6 951 16?0 1267 9?9 565 9?8 789 12?8 1118
Ð Ð Ð 95 95 84 98 97 96
Ð Ð Ð 1317 1363 1776 859 1073 1491
Ð Ð Ð 52 69 85 67 74 97
Ð Ð Ð 1114 1149 1577 793 1046 1536
63 1063 NT Ð NT Ð 65 494 79 938 161 1045 49 499 83 824 118 1264
102 Ð Ð 101 107 87 102 102 97
1250 Ð Ð 689 1159 1554 642 1059 1611
NG Ð NT Ð NT Ð 124 696 144 671 164 1057 87 505 104 707 120 1186
T10 (h)
T10 (h)
a
Average of two independent trials. Individual values were consistently within 10% of mean. No growth. c Not tested. GT, Generation time; LPD, lag phase duration; MPD, maximum population density attained by culture; T10, time to a 10 -fold increase in population density. b
were evaluated (Fig. 3). While the environmental conditions that lead to suppression of L. monocytogenes were generally those that favored the growth of P. £uorescens, there were several instances where rapid growth of P. £uorescens did not result in a suppression of L. monocytogenes MPD (Table 1).
Discussion The interaction of bacterial species in food systems has been studied largely in relation to the production of di¡usable antimicrobial
agents (e. g. acids, bacteriocins, peroxides). In the absence of these agents, when a spoilage organism is present in the same environment as a pathogen, it can either not a¡ect, stimulate, or inhibit the rate or extent of growth of the pathogenic species. The current study suggests that P. £uorescens is capable of all three responses when growing in the presence of L. monocytogenes, depending on the environmental conditions. This, in part, may help to explain the array of reports that Pseudomonas spp. can inhibit (Cheng et al. 1995, Freedman et al. 1989, Farrag and Marth 1989c), stimulate (Farrag and Marth 1989b,
P. £uorescens vs L. monocytogenes 527
Figure 2. E¡ect of P. £uorescens ATCC 33231 on the maximum population density (MPD) reached by L. monocytogenes Scott A under various conditions of temperature, initial pH, and sodium chloride content. MPD di¡erential = log(MPDmonoculture)7 log(MPDcoculture). No growth of L. monocytogenes in cocultures or monocultures ($). [Note: as the di¡erence of two log values (and thus the ratio of two untransformed values), MPD di¡erentials are unitless.]
Figure 1. Examples of the types of growth re-
sponses observed when L. monocytogenes Scott A was grown alone or in coculture with P. £uorescens ATCC 33231. Growth curves were generated using the growth kinetics values provided in Table 1 in conjunction with the Gompertz equations; points depicted are not the experimental values. Listeria monocytogenes growth in monoculture control (&); L. monocytogenes growth in L. monocytogenes/P. £uorescens coculture (*); total count (L. monocytogenes + P. £uorescens) for L. monocytogenes/P. £uorescens coculture ($) (a) 48C Ð pH 60 Ð 5 g l71 NaCl. (b) 128C Ð pH 70 Ð 5 g l71 NaCl. (c) 198C Ð pH 50 Ð 25 g l71 NaCl.
Marshall and Schmidt 1989, 1991, Marshall et al. 1992), or have no e¡ect (Farrag and Marth 1989a, Marshall et al. 1992) on L. monocytogenes.
The ability of Pseudomonas spp. to inhibit the growth of L. monocytogenes (Cheng et al. 1995, Freedman et al. 1989, Gram 1993) and other spoilage (Gram 1993, Gram and Melchiorsen 1996) and pathogenic (Cheng et al. 1995, Freedman 1989, Gram 1993, Oberhofer and Frazier 1961, Seminiano and Frazier 1966) bacteria has been related to the production of siderophores that enhance the organism's acquisition of iron. However, the inhibition of L. monocytogenes in the current study is not likely to be related to siderophore production for two reasons. First, the P. £uorescens isolate used in the current study was selected because it did not produce, on routine screening, either a siderophore or a bacteriocin. Second, the use of BHI as a test system strongly depresses the impact of siderophore production by P. £uorescens, since the medium is high in iron (Freedman et al. 1989). However, this does not rule out depletion of another micronutrient such as a trace mineral; a mechanism that has been suggested for the inhibition of L. monocytogenes by C. piscicola (Buchanan and Bagi 1997). Further, the current study did not rule out the possibility that the P. £uorescens strain used only produces a siderophore or some other mechanism for sequestering trace minerals when incubated at low temperatures
528 R. L. Buchanan and L. K. Bagi
Figure 3. Relationship between the extent of maximum population density (MPD) suppression of L. monocytogenes Scott A and the relative growth rates of the L. monocytogenes and P. £uorescens ATCC 33231 MPD di¡erential = log(MPDmonoculture)7 log(MPDcoculture),T10 ratio = T10 based on coculture VJA counts 7T10 based on coculture BHIA counts, T10 = time to a 10 -fold increase in population density (see text).
(Table 1). Evaluation of this possibility will require additional studies. Additional studies will also be needed to con¢rm the small enhancement of growth under acidic conditions when L. monocytogenes was cocultured with P. £uorescens in BHI. It is not likely that it is due to P. £uorescens modifying the pH of the medium since such changes typically occur in the early stationary growth phase. At that point, L. monocytogenes had also achieved stationary growth (Fig. 1(c)). These observations are consistent with reports that this spoilage organism can stimulate growth of the pathogen in whole and skim milk (Farrag and Marth 1989b, Marshall and Schmidt 1989, 1991, Marshall et al. 1992). Growth of E. coli O157:H7 in skim milk has also been reported to be enhanced by P. £uorescens (Quinto et al. 1997). Stimulation of L. monocytogenes growth has been atributed to the hydrolysis of milk proteins by P. £uorescens. However, at the current time it is not clear how this explanation would account for stimulation of L. monocytogenes in the current study being restricted to only a few combinations of environmental conditions (Table 1). Reliance on microbial competition as one of the barriers that control L. monocytogenes growth in foods would require detailed knowledge of the food, its micro£ora, and how the
food is handled during distribution, marketing, and use. For example, the results of the current study indicate that holding a product with a pH of 60 and a sodium chloride content of 25 g l71 at 48C would suppress L. monocytogenes growth (Table 1). However, those same pH and sodium chloride values would o¡er no protection if the product was temperature-abused at 198C. Likewise, the identity of the principal members of the micro£ora would have to be constant if it were to provide consistent protection. For example, Thomas and Wimpenny (1996) used gradient plating techniques to study the e¡ects of temperature, pH, and sodium chloride content on the interaction between three Salmonella spp. and P. £uorescens or P. putida, two common spoilage species that compete for the same niche. They observed that at 308C, P. putida had no e¡ect on the salmonellae, and the pathogen inhibited P. £uorescens. At 208C, P. putida inhibited Salmonella but P. £uorescens had no activity. It is also important to note that additional factors that a¡ect the interaction between L. monocytogenes and competitive microorganisms would have to be identi¢ed and characterized. For example, P. putida inhibited Salmonella typhimurium in an aerobic environment, but not when oxygen was restricted (Thomas and Wimpenny 1996). Potentially, the use of modi¢ed atmospheres to control the growth of spoilage bacteria could drastically alter any protection provided by an indigenous micro£ora. A number of studies have indicated that the use of speci¢c modi¢ed atmosphere gases may inhibit the growth of spoilage microorganisms without a¡ecting L. monocytogenes, thus altering any protection provided by the micro£ora (Hendricks and Hotchkiss 1997, Marshall et al. 1991, 1992, Wimpfheimer et al. 1990). Likewise, factors such as strain di¡erences and the e¡ect of initial levels on the interaction of L. monocytogenes and a competitor would have to be taken into account (Buchanan and Klawitter 1992a, b). Considering the impact that food environment had on the interaction between L. monocytogenes and competing micro-organisms that was observed in our current and early study (Buchanan and Bagi 1997), it appears likely
P. £uorescens vs L. monocytogenes 529
that a speci¢c isolate with known activity against L. monocytogenes would have to be used if microbial competition was to be relied upon to provide an e¡ective hurdle. Selection of such an isolate would also have to take into account the impact of the micro-organism on the shelflife of the food. Using these criteria, it appears that C. piscicola would be a much better candidate as a biological control agent than P. £uorescens.
References Buchanan, R. L. and Bagi, L. K. (1997) Microbial competition: e¡ect of culture conditions on the suppression of Listeria monocytogenes Scott A by Carnobacterium piscicola. J. Food Protect. 60, 254±261. Buchanan, R. L. and Klawitter, L. A. (1992a) Characterization of a lactic acid bacterium, Carnobacterium piscicola LK5, with activity against Listeria monocytogenes. J. Food Safety 12, 199±217. Buchanan, R. L. and Klawitter, L. A. (1992b) E¡ectiveness of Carnobacterium piscicola LK5 for controlling the growth of Listeria monocytogenes Scott A in refrigerated foods. J. Food Safety 12, 219±236. Buchanan, R. L., Stahl, H. G. and Whiting, R. C. (1989) E¡ects and interactions of temperature, pH, atmosphere, sodium chloride, and sodium nitrite on the growth of Listeria monocytogenes. J. Food Protect. 52, 844±851. Cheng, C.-M., Doyle, M. P. and Luchansky, J. B. (1995) Identi¢cation of Pseudomonas £uorescens strains isolated from raw pork and chicken that produce siderophores antagonistic towards foodborne pathogens. J. Food Prot. 58, 1340±1344. Farrag, S. A. and Marth, E. H. (1989a) Variation in initial populations of Pseudomonas £uorescens a¡ects behavior of Listeria monocytogenes in skim milk at 7 or 138C. Milchwissenschaft 46, 718±721. Farrag, S. A. and Marth, E. H. (1989b) Growth of Listeria monocytogenes in the presence of Pseudomonas £uorescens at 7 or 138C in skim milk. J. Food Prot. 52, 852±855. Farrag, S. A. and Marth, E. H. (1989c) Behavior of Listeria monocytogenes when incubated together with Pseudomonas species in tryptose broth at 7 and 138C. J. Food Prot. 52, 536±539. Freedman, D. J., Kondo, J. K. and Willrett, D. L. (1989) Antagonism of foodborne bacteria by Pseudomonas spp.: a possible role for iron. J. Food Prot. 52, 484±489.
Gibson, A., Bratchell, N. and Roberts, T. A. (1988) Predicting microbial growth: growth responses of salmonellae in a laboratory medium as a¡ected by pH, sodium chloride, and storage temperature. Int. J. Food Microbiol. 6, 155±178. Gram, L. (1993) Inhibitory e¡ect against pathogenic and spoilage bacteria of Pseudomonas strains isolated from spoiled and fresh ¢sh. Appl. Environ. Microbiol. 59, 2197±2203. Gram, L. and Melchiorsen, J. (1996) Interaction between ¢sh spoilage bacteria Pseudomonas spp. and Shewanella putrefaciens in ¢sh extracts and on ¢sh tissue. J. Appl. Bacteriol. 80, 589±595. Hendricks, M. T. and Hotchkiss, J. H. (1997) E¡ect of carbon dioxide on the growth of Pseudomonas £uorescens and Listeria monocytogenes in aerobic atmospheres. J. Food Prot. 60, 1548±1552. Marshall, D. L. and Schmidt, R. H. (1989) Growth of Listeria monocytogenes at 108C in milk preincubated with selected pseudomonads. J. Food Prot. 51, 277±282. Marshall, D. L. and Schmidt, R. H. (1991) Physiological evaluation of stimulated growth of Listeria monocytogenes by Pseudomonas species in milk. Can. J. Microbiol. 37, 594±599. Marshall, D. L., Wiese-Lehigh, P. L., Wells, J. H. and Farr, A. J. (1991) Comparative growth of Listeria monocytogenes and Pseudomonas £uorescens on precooked chicken nuggets stored under modi¢ed atmospheres. J. Food Prot. 54, 841±843, 851. Marshall, D. L., Andrews, L. S.,Wells, J. H. and Farr, A. J. (1992) In£uence of modi¢ed atmosphere packaging on the competitive growth of Listeria monocytogenes and Pseudomonas £uorescens on precooked chicken. Food Microbiol. 9, 303±309. Oberhofer,T. R. and Frazier,W. C. (1961) Competition of Staphylococcus aureus with other organisms. J. Milk Food Technol. 24, 172±175. Quinto, E. J., Franco, C. M., Fente, C. A., Vazquez, B. I. and Cepeda, A. (1997) Growth of Escherichia coli O157:H7 in the presence of Pseudomonas £uorescens in skimmed milk at 7 or 258C. J. Food Safety 16, 273±285. Seminiano, E. N. and Frazier, W. C. (1966) E¡ect of pseudomonads and Achromobacteraeae on growth of Staphylococcus aureus. J. Milk Food Technol. 29, 161±164. Thomas, L.V. and Wimpenny, J.W. T. (1996) Competition between Salmonella and Pseudomonas species growing in and on agar, as a¡ected by pH, sodium chloride concentration and temperature. Int. J. Food Microbiol. 29, 361±370. Wimpfheimer, L., Altman, N. S. and Hotchkiss, J. H. (1990) Growth of Listeria monocytogenes Scott A, serotype 4 and competitive spoilage organisms in raw chicken packaged under modi¢ed atmospheres and in air. Int. J. Food Microbiol. 11, 205±214.