the antimicrobial effect of lactic acid bacteria (30), and there have been only very few reports on the interactions of other microorganisms in foods. Pseudomonas ...
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1993, p. 2197-2203
Vol. 59, No. 7
0099-2240/93/072197-07$02.00/0 Copyright © 1993, American Society for Microbiology
Inhibitory Effect against Pathogenic and Spoilage Bacteria of Pseudomonas Strains Isolated from Spoiled and Fresh Fish LONE GRAM
Technological Laboratory, Danish Ministry of Fisheries, Technical University, Building 221, DK-2800 Lyngby, Denmark Received 12 August 1992/Accepted 16 April 1993
The antibacterial effects of 209 Pseudomonas strains isolated from spoiled iced fish and newly caught fish assessed by screening target organisms in agar diffusion assays. One-third (67 strains) inhibited the growth of one or several of six target organisms (Escherichia coli, ShewaneUla putrefaciens, Aeromonas sobria, Pseudomonasfluorescens, Listeria monocytogenes, and Staphylococcus aureus), of which S. aureus and A. sobria were the most sensitive. The inhibitory action was most pronounced among the strains producing siderophores, and the presence of iron eliminated the antibacterial effect of two-thirds of the inhibitory strains. Siderophoremediated competition for iron may explain the inhibitory activity of these strains. All but nine of the inhibiting strains were found to inhibit the growth of 38 psychrotrophic S. putrefaciens strains isolated from spoiling fish and fish products. Siderophore-containing Pseudomonas culture supernatants inhibited growth of S. putrefaciens, as did the addition of iron chelators (ethylenediamine dihydroxyphenylacetic acid [EDDHA]). In particular, Pseudomonas strains isolated from newly caught and spoiled Nile perch (Lates niloticus) inhibited S. putrefaciens. This suggests that microbial interaction (e.g., competition or antagonism) may influence the selection of a microflora for some chilled food products. were
Interactions between microorganisms are well-known phenomena, and substrate competition and antagonism are believed to be important in the selection of a microflora in a given ecological niche (10). Studies of such interactions in certain niches, e.g., in the rhizosphere (32), have been very detailed, but in other niches, such as in food products, they have received less attention. Studies of microbial interactions in foods have mostly been limited to investigations of the antimicrobial effect of lactic acid bacteria (30), and there have been only very few reports on the interactions of other microorganisms in foods. Pseudomonas species are important spoilage organisms in many chilled food products, such as milk (40), chicken (39), meat (7), and fish (34), in which they become the dominant microflora during chill storage. This dominance is assumed to be attributable exclusively to their rapid growth at chill
enhanced the subsequent growth of lactic acid bacteria (3), possibly because of increased availability of nutrients arising from the hydrolytic activity of Pseudomonas spp. The interaction between Pseudomonas spp. and Listena monocytogenes has been described, but results are contradictory, showing both enhancement of growth of L. monocytogenes by Pseudomonas spp. and an inhibition of its growth (9, 11, 33). One study (11) showed that four strains of Pseudomonas spp. isolated from plants or foods inhibited the growth of both gram-positive and gram-negative bacteria in laboratory media. This inhibitory action was enhanced on King's agar B, on which production of fluorescent siderophores is stimulated, compared with the inhibitory action observed in assays with brain heart infusion agar. Studies of microbial interaction in foods have focused on the potential inhibition of pathogenic organisms, and only one study (11) has related the findings to the changes in microflora during storage. Moreover, only a limited number of strains (13 in total) isolated from plants and food were studied. The present study was undertaken to determine the antibacterial potential of Pseudomonas spp. isolated from fish, to evaluate the role of interaction in selection of a microflora, and to assess the potential of the aquatic Pseudomonas spp. for production of secondary metabolites with antibiotic activity (27, 37). Special notice is paid to Pseudomonas spp. isolated from spoiling fish and to their possible interaction with another psychrotrophic fish spoilage bacterium, Shewanella putrefaciens.
temperature.
The competitive or antagonistic activity of Pseudomonas in the rhizosphere has been very well described (1, 2, 29). Thus, the antimicrobial activity of Pseudomonas spp. has been recognized as a major factor in the suppression of many root pathogens. Several antibiotic-like substances have been identified, including bacteriocins (notably pyocin from Pseudomonas aeruginosa), a phenazine antibiotic (17), and non-nitrogen-containing compounds such as 2,4-diacetylphloroglucinol (43). However, one of the most important mechanisms responsible for the suppression of plant disease by Pseudomonas spp. is siderophore-mediated competition for iron (18, 29, 31). Apart from depleting the environment of iron, some siderophores may also act as antibiotics (36). A few studies have also described the antimicrobial effect of aquatic Pseudomonas spp. (28), but only rarely has the inhibitory mechanism been elucidated (48). The possible interaction between Pseudomonas spp. and other microorganisms in foods has received limited attention, and the results are not consistent. Early experiments showed that preinoculation of milk with Pseudomonas spp. spp.
MATERIALS AND METHODS Bacterial target organisms. Six target organisms were screened for the inhibitory effect of Pseudomonas spp.: Escherichia coli ATCC 25922, Aeromonas sobria (a clinical isolate, M2, obtained from S. Kn0chel [23]), S. putrefaciens ATCC 8071, L. monocytogenes Scott A isolated from an outbreak of listeriosis (obtained from Campden Food and 2197
2198
GRAM
Drink Association, Chipping Campden, United Kingdom), Staphylococcus aureus (received from the Royal Veterinary and Agricultural University, Frederiksberg, Denmark), and a Pseudomonas fluorescens isolate from spoiled Nile perch (Lates niloticus) (15). Pseudomonas strains showing reproducible effects against one or several of the six target organisms were tested for their inhibitory effect on the growth of 38 strains of S. putrefaciens, 35 isolated from spoiled chilled fish and fish products and 3 (NCMB 1733, NCMB 1738, and ATCC 8071) obtained from culture collections. Bacterial inhibitor strains. A total of 209 Pseudomonas strains were screened for antibacterial activity in an agar diffusion assay. Of these, 72 were isolated from fresh and spoiled iced Nile perch (15) and 39 were isolated from spoiled iced sole, sardinella, and sea bream from Senegal (13). A further 33 strains were isolated in our laboratory from spoiled iced cod (5a), and 16 were isolated from newly caught farm-reared trout. Another 33 strains were received from Mario Gennari, Milan, Italy. All strains were tentatively identified by the following reactions: Gram staining in 3% KOH (16), oxidase reaction using Bactident oxidase strips (13300; Merck), and catalase activity by exposure to 3% H202. Motility and shape were observed by phasecontrast microscopy, and glucose metabolism was tested in modified Hugh and Leifson medium (19) in which 0.04% (NH4)2HP04 was substituted for 0.2% peptone to avoid alkalization due to breakdown of peptone. Reduction of trimethylamine oxide (TMAO) was observed in a TMAO medium (14), and production of H2S was tested in iron agar tubes (14). Arginine dihydrolase activity was tested in Falkow's medium (8). Gram-negative, motile rods with positive catalase and oxidase reactions, an oxidative glucose metabolism, and arginine dihydrolase activity were identified as Pseudomonas spp. if they did not reduce TMAO or produce H2S (5, 26). Bacteriological media. Nutrient broth (veal infusion broth; Difco), iron agar (CM 867; Oxoid), and iron agar supplemented with 5% blood were used for culturing of bacteria. Agar diffusion assays to screen for antibiotic activity were done by using a modified iron agar (MIA), omitting ferric citrate, sodium thiosulfate, and L-cysteine (2% Bacto Peptone, 0.3% yeast extract, 0.3% Lab-Lemco powder [L 29; Oxoid], 0.5% NaCl, 1.2% agar [pH 7.0]). Inhibitory activity in broth culture was tested with a modified iron broth (MIB) (MIA with agar omitted). MIA and MIB were supplemented with iron by addition of 0.3% ferric citrate (product no. 28381; BHD Chemicals). King's agar B (10991; Merck) (21) was used for testing of fluorescence. The production of siderophores was tested on solid medium by using CAS [chrome azurol S, iron(III), hexadecyltrimethylammonium bromide] medium (42) and in liquid medium (2% sucrose, 0.2% L-asparagine, 0.1 g of K2HPO4, 0.05% MgSO4. 7H20; +0.01% FeCl3 6H20) (pH 7.0) (41). All assays in liquid media were done at 25°C at 150 rpm. Siderophore assay. Pseudomonas spp. were spot inoculated (10 ,ul) onto CAS-agar plates, and the plates were incubated at 25°C for 2 days. Siderophore-producing strains showed orange halos around the colony (42). For some strains, siderophore production was also tested by the CAS spectrophotometric assay, in which 1.5 ml of culture supernatant was mixed with 1.5 ml of CAS assay solution (42). Sterile medium plus CAS assay solution served as the reference. TheA630 was read, and cultures with absorbances close to zero were recorded as siderophore negative [Fe(III) was not
APPL. ENvIRON. MICROBIOL.
removed from the blue complex]. Supernatants were also divided in two portions of 3 ml, and FeCl3 was added to one portion. The supernatants were scanned at 300 to 500 nm with the portion to which Fe(III) was added as a sample. Siderophore-positive cultures peaked at approximately 400 nm, at which the Fe(III)-siderophore complex absorbs (41). Iron-starved and iron-supplemented cultures were compared in both spectrophotometric assays. Agar bioassay. Cultures were pregrown in nutrient broth at 25°C for 2 days or at SOC for 10 days for 25°C and lowtemperature assays, respectively. A 10-,ul volume of target culture was mixed with 10 ml of sterile MIA, which had been melted and cooled to 45°C. The inoculated agar was poured into petri dishes, and wells were punched with a 3-mmdiameter gel puncher. A 12-,ul volume of the antagonistic cultures was pipetted into each well. An empty well and a well inoculated with sterile nutrient broth were always used as negative controls. Two Pseudomonas strains isolated from spoiled iced Nile perch served as positive controls. Plates were incubated for 1 day at 5°C followed by 1 day at 25°C. Inhibition of target organisms was seen as a clearing zone around the well. An isolate was classified as positive if activity against one or several of the six target organisms was found in two independent trials. Influence of culture conditions on inhibitory activity. Temperature effects on inhibitory activity were assessed by parallel incubation of plates at 0, 5, 10, and 25°C. The influence of availability of iron was assessed by comparing inhibitory activity in agar assays on MIA and MIA plus Fe. Broth bioassay. Inhibitory activity of Pseudomonas spp. against S. putrefaciens and A. sobnia was assessed by growing the organisms mixed in liquid culture. The growth of cultures was monitored by counting colonies in iron agar, in which Pseudomonas spp. (non-H2S producers) appear as white colonies and the two target strains (H2S producers) appear as black colonies. Samples were withdrawn at intervals, at approximately 10 points during a growth cycle. Curves were obtained for the target organisms grown alone and in combination with an inhibitory Pseudomonas strain. All trials were done in duplicate. Sterile filtered supernatants of Pseudomonas spp. grown under conditions of limited and adequate iron were tested for inhibitory activity against S. putrefaciens grown in MIB. A 5-ml volume of Pseudomonas supernatant was added to 5 ml of double-strength MIB, and tubes containing this mixture were inoculated with approximately 105 S. putrefaciens organisms per ml. Iron-depleted conditions were made by supplementing MIB with 1.0 mM ethylenediamine dihydroxyphenylacetic acid (EDDHA). Growth was measured by reading the A600 RESULTS Inhibition of growth of six target organisms in agar bioassay. Almost one-third of the Pseudomonas strains (67 strains) inhibited the growth of one or several of the six target organisms when assayed in MIA (Table 1). S. aureus and A. sobria were especially sensitive, each being inhibited by three-fourths of the strains (53 and 45 strains, respectively). One-third of the 67 strains inhibited the growth of only one target organism, whereas 5 strains (all isolated from Nile perch) inhibited five of six target organisms (Table 2). Proportions of antagonistic strains varied within the different groups of Pseudomonas spp., and strains isolated from newly caught or spoiled fish species from tropical waters had especially high inhibitory scores (Table 2 and
ANTIBACTERIAL EFFECTS OF PSEUDOMONAS SPP.
VOL. 59, 1993
2199
TABLE 1. Inhibitory activity of 209 Pseudomonas strains against six target organisms Isolation
No. of
isolates
temp (OC)
Pseudomonas strains isolated
S. aureus
Newly caught fish Trout Sardines' Nile perch
4 20-25 25
16 10 6
8 3 5
1 1 3
Spoiled fish MAP codb Sardinesc Sole, sea bream, and sardinellad Nile perche
0 0 0
33 33 39
10 2 8
0
72 209
Total
No. of
No. of Pseudomonas strains with inhibitory activity against:
Origin of Pseudomonas
monocytogenes
inhibitory strains
E. coli
S. putrefaciens
P. fluorescens
1 2 4
0 0 3
0 3 4
0 1 0
8 4 5
0 0 0
3 3 12
2 2 0
2 1 11
5 1 7
11 6 12
17
3
20
8
14
0
21
53
8
45
15
35
14
67
L.
A. sobria
a Mediterranean sardines. b MAP cod, cod fillets packaged in a modified atmosphere. Stored at 0'C for 16 to 20 days. c Mediterranean sardines. Stored 4 to 8 days in ice. d Stored 21 days in ice. e Fish from Lake Victoria. Stored 32 days in ice.
Fig. 1). The salinity of the original environment of the strains did not seem to affect their inhibitory profile. One hundred non-Pseudomonas strains (Moraxella, Acinetobacter, and Flavobacterium strains) isolated from Nile perch and Mediterranean sardines were also tested for antibacterial activity. Of these, only one isolate (a Bacillus sp. isolated from fresh Nile perch) showed inhibitory activity
(data not shown). Influence of availability of iron on inhibitory activity. Addition of iron to the substrate eliminated the inhibitory activity of two-thirds of the inhibitory strains. The activity of 21 strains was, except in a few strains, decreased in the presence of iron. Elimination of inhibitory activity by iron was most pronounced among the isolates from Nile perch: 25 of 26 isolates lost inhibitory activity when iron was added to the substrate. Siderophore production. Most of the inhibitory strains (80%) produced siderophores, whereas the majority (86%) of the noninhibitory strains did not produce these iron chelators (Table 3). In particular, Pseudomonas spp. isolated from Nile perch and Senegalese fish produced large orange halos on the CAS medium. The siderophore-positive Pseudomonas spp. produced spectra as shown in Fig. 2 after
3 days of incubation at 25°C. When supernatants of these Pseudomonas cultures were mixed with CAS solution, an absorbance of -1.000 was reached compared with the absorbance when CAS was added to the sterile medium. Broth bioassay. Three inhibitory isolates were tested against S. putrefaciens (at 2 and 0°C) and A. sobnia (at 25°C) in liquid culture. In neither of the experiments were any effects of the Pseudomonas strain on the growth of the two hydrogen sulfide producers detected (data not shown). Inhibition of S. putrefaciens. The majority of strains (59 of 67) inhibited one or several S. putrefaciens strains in agar assay (Fig. 1). Inhibitory strains isolated from Nile perch (either spoiled or newly caught) inhibited the growth of more than half of the S. putrefaciens strains. Also, the strains isolated from Senegalese fish had a high inhibitory score against S. putrefaciens. The S. putrefaciens strains differed in sensitivity, but this did not correlate with the origin of the strains; e.g., no difference in sensitivity between isolates from fresh fish and preserved fish products was observed. Mixtures of inhibitory Pseudomonas spp. were found to inhibit mixtures of S. putrefaciens spp. (data not shown). Pseudomonas spp. inhibited growth of S. putrefaciens also at 0°C, although temperature did influence the inhibitory
TABLE 2. Inhibitory score measured as number of six target organisms inhibited by 67 Pseudomonas strains No. of
No. of Pseudomonas strains inhibiting indicated no. of target organisms
Origin of Pseudomonas isolatesa
1/6
2/6
3/6
4/6
5/6
6/6
strains
6 0 0
2 2 0
0 1 1
0 1 1
0 0 3
0 0 0
8 4 5
6 4 1 3
1 2 3 5
2 0 4 5
2 0 4 6
0 0 0 2
0 0 0 0
11 6 12 21
20
15
13
14
5
0
67
Newly caught fish Trout
Sardines Nile perch Spoiled fish MAP cod Sardines Sole, sea bream, and sardinella Nile perch Total a
inhibitory
Fish preservation and storage conditions were as described in the footnotes to Table 1.
2200
GRAM
APPL. ENVIRON. MICROBIOL. 20 co (D
Sole
3
15
Fresh Nile perch 5 strains
Fresh sardines 4 strains
Fresh trout 8 strains
0
sea
bream
sardinella
Sardine 6 strains
MAP cod 1 1 strains
12 strains
Nile perch 21 strains
_.
ce C. .0 C .1
co,
-I-
I
10
-K-
Cu
.co L-
C,)
40
0)
o z o
o
04 IV-,
O
0
XV
0
C
r
cV
Go
Cf!)
cul
CM
c'J
.1.. 0
0
CN es
OD
0
0
OD Ch
0
0
OD
0
Cr
CO
MCMc
CM
CO)
m-
CMl
CMJ
N'
CM
CO
CM
-
No. of S.putrefaciens inhibited
FIG. 1. Inhibitory activity of 67 antagonistic Pseudomonas strains against 38 strains of S. putrefaciens. Fish preservation and storage conditions were as described in the footnotes to Table 1.
activity (Table 4). Of 11 antagonistic strains, 7 inhibited a lower number of S. putrefaciens strains at 0 and 5°C than at 10 and 25'C, whereas 2 strains inhibited all eight S. putrefaciens strains at all four temperatures. The anti-Shewanella effect of two of the strains did not vary systematically with temperature.
Growth of S. putrefaciens was enhanced by supplementation of MIB with iron, whereas the addition of 1 mM EDDHA inhibited growth completely (Fig. 3). The addition of supernatant from an iron-starved P. fluorescens strain (AH2-Fe) inhibited growth to the same extent as EDDHA. Supematant from the same Pseudomonas strain grown in the
presence of iron (AH2+Fe) did not affect the growth of S. putrefaciens (Fig. 3).
DISCUSSION The present study shows that Pseudomonas spp. isolated from spoiled iced fish and newly caught fish have a broad antibacterial spectrum, inhibiting gram-negative as well as gram-positive bacteria. The inhibitory ability seems to be more widespread among Pseudomonas spp. than among other psychrotrophic bacteria isolated from fish. Similar activities have been reported for aquatic Pseudomonas spp.
TABLE 3. Siderophore production by 67 inhibitory and 142 noninhibitory Pseudomonas strains isolated from fresh and spoiled fish No. of Pseudomonas strainsb No. of Origin of Pseudomonas isolatesa Pseudomonas Inhibitory Noninhibitory strains isolated
Siderophore positive
Siderophore negative
Siderophore positive
16 10 6
6 4 5
2 0 0
4 3 1
33 33 39 72
3 5 12 20
8d 1 0 1
1 3 2 6
21 24 25 45
209
55
12
20
122
Siderophore negative
Newly caught fish Trout
Sardines Nile perch
4 3c 0
Spoiled fish MAP cod Sardines Sole, sea bream, and sardinella Nile perch
Total
a Fish preservation and storage conditions were as described in the footnotes to Table 1. b Presence of siderophores determined by appearance of orange halos on CAS medium (42). c One strain produced fluorescent pigment on King's agar B. d Four strains produced fluorescent pigment on King's agar B.
ANTIBACTERIAL EFFECTS OF PSEUDOMONAS SPP.
VOL. 59, 1993
2201
absorbance at 600 nm
absorbance
0,7 *MIB - Fe
0,6 +PMIB + Fe 0,5
0,4
* MIB + EDDHA -wMIB + (AH2-Fe) * MIB. + (AH2+Fe)
0,3
-
D48
24 lambda,
72
96
time, hours at 25°C
nm
with
FIG. 3. Growth of S. putrefaciens under different iron availability conditions at 25°C.
(28, 35). The finding also corresponds to the widespread antibacterial activity of plant-associated Pseudomonas spp. (29, 31). Inhibitory activity was most pronounced among siderophore-producing strains, and as addition of iron eliminated the inhibitory activity of two-thirds of the strains, it is likely that the inhibitory mechanism of these strains is siderophore mediated. This mediation may be due exclusively to competition for iron (18), or it may be due to the antibiotic activity of the siderophores (36). Other studies have similarly shown that culturing Pseudomonas spp. on substrates inducing siderophore production enhances inhibitory activity (11, 22). In contrast, however, Jurkevitch et al. (20) showed that the sequestering of iron by siderophores was an advantage to other bacteria that were able to utilize the siderophores. In the present study, dense growth of L. monocytogenes and S. aureus was sometimes noted around the wells containing Pseudomonas strains, and the presumed antagonistic strains may have created a more advantageous nutritional composition, either through supply of iron (20) or by increasing the availability of low-molecular-weight nutrients (3). Similar observations have been made for L. monocytogenes and lactic acid bacteria (3, 24, 33). The antibacterial action of 21 strains was not completely
eliminated by iron supplementation, and their activity may be attributable to the production of several different bacterial inhibitors, such as antibiotics, bacteriocins, or HCN (2, 29, 43, 45, 46, 48). The present study does not allow any conclusions about these mechanisms to be made. The inhibitory action could not be detected when both target and test cultures were grown in liquid media, although all the test cultures assayed did produce siderophores in liquid media. This lack of inhibitory action could be due to cross feeding between the strains assayed or to the fact that Pseudomonas spp. should be present in high numbers to be effective. Also, a lack of production of antibiotic substances in liquid medium has been observed in some studies (43). The strong inhibition of A. sobria by some isolates suggests that some Pseudomonas spp. form part of the natural defense systems of fish. Organisms resembling A. sobria have been isolated from diseased cod (25). It should be noted, however, that the A. sobria used in this study was isolated from a human clinical source and may not be representative of the motile aeromonads pathogenic to fish. Several studies have shown that sequestering of iron by siderophores is essential for pathogenicity in both humans (44) and fish (4). It is thus likely that a nonpathogenic microflora serves as a protecting agent by causing an even
FIG. 2. Siderophore production by P. fluorescens and without iron at 25°C.
grown
TABLE 4. Inhibitory activity of 11 inhibitory Pseudomonas strains isolated from spoiled fish against eight strains of S. putrefaciens at four different temperatures
Origin of Pseudomonas strains MAP codb
Sole, sea bream, and sardinella
Nile perch
putrefaciens strains inhibited at:
No. of S.
Pseudomonas isolate
OOC
50C
1O°C
25°C
CN20-26 CN20-31 CN20-35 N100-31
8 0 8 1
8 1 8 0
8 3 8 3
8 5 8 7
L02 L04 L013 L017 L034
0 2
3
5 3
8 2 8 3 5
AH2 AH8
1 0
aAll fish were stored at O°C. b MAP cod, cod fillets packaged in a modified atmosphere. Stored at
4 0 5
0°C for 16 to 20 days.
6 8 0 7
8 1 7
4
6
1
6
7 6
2202
GRAM
stronger competition for iron. The probiotic effect of bacteria derived from fish and water has been suggested by several studies (6, 38, 47). The inhibitory effect of the Pseudomonas spp. implies that the selection of a microflora during storage of, e.g., a fish product is not exclusively determined by the growth rate as influenced by external chemical and physical parameters but is also influenced by microbial interaction. Thus, the strong anti-Shewanella activity found among the isolates from tropical fish may partly explain why Pseudomonas spp. become dominant during ice storage of certain tropical fish (12, 15). The fact that particularly strains from Nile perch produced large orange halos on the CAS medium may indicate that these strains produce siderophores with a very high affinity for iron. The large halos could also be explained by the production of smaller siderophores which diffuse more easily in the agar. The inhibition of growth of S. putrefaciens by EDDHA and the enhancement of its growth by iron (Fig. 3) indicates that this organism requires iron and is unable to produce chelators with a higher iron affinity than EDDHA has. As supernatants of Pseudomonas spp. grown under iron-limited conditions inhibit growth of S. putrefaciens, the mechanism of inhibition could be iron chelation by siderophores or another mechanism induced by ironlimited conditions. In conclusion, this report has documented that the psychrotrophic Pseudomonas spp. isolated from spoiled and fresh fish have a broad antibacterial potential. This effect is largely determined by the siderophore-mediated competition for iron. Studies of the role of the bioactive Pseudomonas spp. in the natural defense of fish against infection and in microflora selection in food products will be pursued. ACKNOWLEDGMENTS The skillful technical assistance of Jette Melchiorsen and Susanne Andersen is gratefully acknowledged. This project was financed by the Danish Biotechnology Programme 1991-1995 and carried out in collaboration with the Marine
Chemistry Group at Copenhagen University. REFERENCES 1. Bakker, A. W., and B. Schippers. 1987. Microbial cyanide production in the rhizosphere in relation to potato yield reduction and Pseudomonas spp.-mediated plant growth stimulation. Soil Biol. Biochem. 19:451-457. 2. Casida, L. E., Jr. 1992. Competitive ability and survival in soil of Pseudomonas strain 679-2, a dominant, nonobligate bacterial predator of bacteria. Appl. Environ. Microbiol. 58:32-37. 3. Cousin, M. A., and E. H. Marth. 1977. Lactic acid production by Streptococcus thermophilus and Lactobacillus bulgaricus in milk cultures precultured with psychrotrophic bacteria. J. Food Prot. 40:475-479. 4. Crosa, J. H. 1984. The relationship of plasmid-mediated iron transport and bacterial virulence. Annu. Rev. Microbiol. 38:6989. 5. Dainty, R. H., B. G. Shaw, C. D. Hardinger, and S. Michanie. 1979. The spoilage of vacuum packaged beef by cold tolerant bacteria, p. 83-110. In A. D. Russell and R. Fuller (ed.), Cold tolerant bacteria in spoilage and the environment. Academic Press, New York. Sa.Dalgaard, P. Unpublished data. 6. Dopazo, C. P., M. L. Lemos, C. Lodeiros, J. Bolinches, J. L. Barja, and A. E. Toranzo. 1988. Inhibitory activity of antibioticproducing marine bacteria against fish pathogens. J. Appl. Bacteriol. 65:97-101. 7. Edwards, R. A., R. H. Dainty, and C. M. Hibbard. 1987. Volatile compounds produced by meat pseudomonads and related reference strains during growth on beef stored in air and chill temperatures. J. Appl. Bacteriol. 62:403-412.
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