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Listeria monocytogenes: incidence on vegetables
Food Control, Vol. 7, No. 415. pp. 223-228. Copyright Printed ELSEVIER
PII: SO956-7135(96)00039-4
in Great
0 1996 Elsevier Britain.
1996
Science
Ltd
All rights reserved
OYS6-71.15196
$15.00+0.00
PAPER
Listeria monocytogenes: incidence on vegetables Larry R. Beuchat Listeria monocytogenes is widely distributed on plant vegetation, including raw vegetables. Its presence on plant materials is likely due to contamination from decaying vegetation, animal faeces, soil, sur$ace, river and canal waters, or effluents from sewage treatment operations. The organism is known to survive in plant materials for IO-12 years. The presence of L. monocytogenes on many types of raw and minimally processed vegetables destined for human consumption has been clearly demonstrated in many countries but the role these vegetables play as vehicles for human infection is not known. It is certain, however; that if consumers are to continue to benefit from the essential contribution raw and minimally processed vegetables make to their nutritional well-being, they will also very likely continue to consume vegetables containing low numbers of L. monocytogenes. Copyright 0 1996 Elsevier Science Ltd. Keywords: Listeriu monocytogenes; vegetables; food-borne
pathogen
INTRODUCTION
OCCURRENCE IN NATURE
Given enough time and appropriate methodology for detection, Listeria monocytogenes can be found in or on a large number of species representative of every class of living things that swim, crawl, jump, walk or fly. Furthermore, the organism can also be isolated from many living things, namely plants, that do not possess within themselves these abilities of movement. Plants play a significant role in the dissemination of L. monocytogenes from natural habitats to the human food supply. This role may be indirect, for example by contamination of milk via forage or silage, or direct in the form of contaminated raw vegetables and perhaps fruits. Summarized here is information on the presence, survival and proliferation of L. monocyfogenes on vegetation in general and more specifically on raw and lightly processed vegetables.
Center for Food Safety and Quality Enhancement, ment of Food Science and Technology, Georgia, Griffin, Georgia 30223-l 797, USA.
DepartUniversity of
OF L. MONOCYTOGENES
Listeria monocytogenes
is present in many animals, including humans, so it is not too surprising that the organism can also be isolated from faeces of these animals, on the land they occupy, in sewage, in soils to which sewage is applied and on plants, including those producing vegetables, which grow in these soils. Some examples will serve to illustrate the cycle of L. monocytogenes between plants and humans (Figure 1). The presence of Listeria in sewage in many countries has been demonstrated. Watkins and Sleath (1981) analysed 52 sewage, river water and industrial effluents in England for the presence of potentially pathogenic microorganisms. Effluents were from abattoirs, cattle markets and poultry packing plants. Listeria monocytogenes was isolated from every sample. In many instances, populations of L. monocytogenes were higher than those for salmonellae, and in some instances L. monocytogenes was isolated when no salmonellae were detected. Application of sludge containing L. monocytogenes and salmonellae to soil revealed that L. monocytogenes could survive
Food Control 1996 Volume 7 Number 4/5
223
Listeria monocytogenes: incidence on vegetables: L.R. Beuchat
ANIMALS
VEGETABLES -
water -+-
\
\b!/
,
,.
7s
Sr
HUMANS
/. .
contamrnatron)
\
plants-
silage, feed -
meat, milk, eggs
’
Figure 1 Potential pathways for transmission of L. munocytugenes to humans via vegetables.
longer periods of time. Populations of L. monocytogenes in soil remained essentially unchanged during seven weeks following spraying. Welshimer (1960) demonstrated that L. monocytogenes could survive in soil for up to 295 days. Reports from other countries also indicate the presence of L. monocytogenes in sewage. The organism has been isolated from a sewage treatment plant in Baghdad, Iraq (Al-Ghazali and Al-Azawi, 1986). Treatment processes at the plant did not yield a sewage sludge cake or a final discharge free of Listetiu. It was concluded that the use of sewage as a fertilizer could be a route of contaminating vegetation destined for human consumption. Dewatering of sewage reduced the number of L. monocytogenes but it was suggested that long periods of exposure to sun would be needed to render the sewage sludge cake ‘safe’. MacGowan et al. (1994) examined sewage in England at two-month intervals in 1991-92 and found 84-100% contained L. monocytogenes and/or L. innocua. Welshimer and Donker-Voet (1971) isolated 27 strains of L. monocytogenes from soil and vegetation taken from 19 sites in The Netherlands. Vegetation samples were taken from the same sites in September and April of the following year. Listeria monocytogenes was isolated only from samples collected in April. Plant materials from which the organism were isolated included dead and decayed corn and soybean plants and wild grasses, indicating its preference to exist in nature as a saprophyte. A study of soil and domestic animal faeces in England has shown that Listeriu is more often present in July to September than in other months (MacGowan et al., 1994). L. monoqtogenes and L. innocua were predominant in
224
Food Control 1996 Volume 7 Number 4/5
faeces whereas L. ivanovi and L. seeligeri were most common in soil. Welshimer (1968) examined vegetation in a rural area in Virginia (USA) where clinical listeriosis of man and animals was rare. Dead soybean plant material and stalks, leaves and tassels of corn were collected in April following the previous planting year. Eight of twelve sampling sites yielded plant materials positive for L. monocytogenes. Only 25% of the strains were pathogenic for mice, which is a low frequency compared with the percentage of pathogenic strains isolated from listeric humans and animals in Virginia and the USA as a whole. These observations suggest that the predominance of certain serotypes of L. monocytogenes may be influenced by the nature of the host environment and that the majority of strains indigenous to decaying plant vegetation are incapable of causing illness in animals. In a survey conducted in Germany, Weiss and Seeliger (1975) isolated 154 strains of L. monocytogenes from soil and plants, 16 from faeces of deer and stag, nine from mouldy fodder and wildlife feeding grounds and eight from birds. Corn, wheat, oats, barley and potato plants and soils from fields in which they were growing were among the materials analysed. Nearly 10% of the corn plants and 13% of the grain plants were positive for L. monocytogenes. Plants from cultivated fields had a lower incidence of the organism (12.5%) compared to plants from uncultivated fields (44%). Twenty-three percent of samples collected from wildlife feeding grounds were positive for L. monocytogenes. Virulent strains were isolated from all locations. It was suggested that L. monocytogenes is a saprophyte which lives in a plantsoil environment and could therefore be contracted
Listeria monocytogenes:
by humans and other animals via many possible routes from many sources. It is very unlikely that birds and animals are the only sources responsible for the distribution of L. monocytogenes in nature and its presence on vegetables. The relationship between feeding silage to animals and listeriosis has been recognized since the early 1960s (Gray, 1963; Gray and Killinger, 1963). Consumption of mouldy or badly cured silage with with elevated pH values has been correlated outbreaks of listeriosis in cattle and sheep. The frequency of L. monocytogenes in silage increases with increasing pH. Gronstol (1979) isolated the organism from 22% of silage samples with pH ~4, from 37% with pH 4 to 5 and from 56% with pH >5.
VEGETABLES LISTERIOSIS
AS VEHICLES
FOR
As early as 1967, Blenden and Szatalowicz, noting that 731 cases of human listeriosis had been reported between 1933 and 1966, stated that it seemed feasible that plant products such as lettuce or other fresh vegetables may be contaminated with L. monocytogenes and perhaps be responsible for causing infection. Evidence supporting this hypothesis has subsequently emerged. An outbreak of L. monocytogenes infection in 1979 involving 23 patients from eight Boston hospitals was reported by Ho et al. (1986). Twenty (87%) of the isolates were L. monocytogenes serotyped during the 26 months preceding the outbreak. Case patients differed from patients with sporadic Listeria infection in that more of the case patients had hospital-acquired infection (75% v 22%), had received antacids or cimetidine before the onset of listeriosis (60% v 17%), and had gastrointestinal tract symptoms that began at the same time as fever (85% v 22%). Tuna fish, chicken salad and cheese were preferred by case patients more frequently than by control patients, but the only common foods served with these foods were raw celery, tomatoes and lettuce. It was concluded that the consumption of these raw vegetables could have caused the listeriosis outbreak. No attempt was made to isolate L. monocytogenes from the vegetables at the time of the outbreak. In a report from another laboratory, however, Hofer (1975), in a study of Listeria species on vegetables suitable for consumption, isolated three Listeria strains from lettuce. Human infection associated with the consumption of raw cabbage containing L. monocytogenes occurred in Canada in 1981 (Schlech et al., 1983). Thirty-four cases of perinatal listeriosis and seven cases of adult disease occurred between 1 March and 1 September of that year. During May, June and July 1981, the attack rate for perinatal listeriosis at a maternity hospital was approximately 1.3%. A survey revealed that cases were more likely than controls to have consumed coleslaw in the three months before the
incidence
on vegetables:
L.R. Beuchat
onset of illness. Coleslaw obtained from the refrigerator of a patient was positive for L. monocytogenes serotype 4b, which was the serotype of the epidemic strain and the strain isolated from the patient’s blood. The coleslaw had been commercially prepared with cabbage and carrots obtained from wholesalers and local farmers. After prolonged cold enrichment, two unopened packages of coleslaw purchased from two different supermarkets yielded L. monocytogenes, serotype 4b. An investigation of the sources of raw vegetables identified a farmer who, in addition to raising cabbage, also maintained a flock of sheep. A review of the farmer’s agronomic practices revealed that both composted and raw sheep manure had been applied to fields in which cabbage was grown. From the last harvest in October through the winter and early spring, cabbage was kept in a cold-storage shed. A shipment of cabbage during the period of the outbreak was traced to the implicated coleslaw processor. Information obtained in this investigation strongly suggests that the source of this outbreak was coleslaw. A statistical association of listeriosis with coleslaw was supported by identification of the epidemic strain in the implicated food. The coleslaw outbreak heightened interest in determining the prevalence of L. monocytogenes in fresh produce. Sizmur and Walker (1988) detected L. monocytogenes in four of 60 prepacked, ready-to-eat salads in the UK. Vegetables included in the two types of contaminated salads were cabbage, celery, carrots, lettuce, cucumber, onion, leeks, watercress and fennel. Listeria innocua was isolated from 13 samples representing five salad varieties. incubation of salads containing L. monocytogenes at 4°C for four days resulted in a roughly two-fold increase in the population of the organism. A survey of 1000 samples of 10 types of fresh produce at the retail level in the USA has revealed the presence of L. monocytogenes on cabbage, cucumbers, potatoes and radishes (Heisick et al., 1989b). In another survey, prepared mixed salads and two individual salad ingredients were found to contain L. monocytogenes (Velani and Roberts, 1991). The higher rate of contamination of prepared salads was attributed to cross contamination of the pathogen during chopping, mixing and packaging. Beckers et al. (1989) detected L. monocytogenes in 11 of 25 samples of fresh cut vegetables in The Netherlands and Harvey and Gilmour (1993) reported that 7 of 66 samples of salad vegetables and prepared salads produced in Northern Ireland contained the organism. Farber et al. (1989), on the other hand, did not detect Listeria species in 110 samples of vegetables, including lettuce, celery, tomatoes and radishes analysed in Canada. Likewise, Petran et al. (1988) failed to detect L. monocytogenes in market samples of fresh vegetables in the United States. Some reports documenting the presence of L. monocytogenes in vegetables are listed in Table 1.
Food Control 1996 Volume 7 Number 4/5
225
Listeriamonocytogenes: incidence on vegetables:
Table 1
L.R. Beuchat
Raw vegetables from which L. monocyfogenes has been isolated
Vegetable
Country
Prevalence’
Reference
Bean sprouts Cabbage
Malaysia Canada USA Malaysia Pakistan USA Malaysia USA USA Northern Ireland UK USA USA Germany Northern Ireland The Netherlands Pakistan Italy Spain Taiwan UK
6/7 (85.7%) 2/92 (2.2%) l/92 (1.1%) 4/5 (80%) l/15 (6.7%) 2/92 (2.2%) 5/22 (22.7%) 19/70 (27.1%) 28/132 (21.2%) 3/21 (14.3%) 4/60 (13.3%) 25/68 (36.8%) 19/132 (14.4%) 6/263 (2.3%) 7/66 (10.6%) 1 l/25 (44%) 2/15 (13.3%) 7/102 (6.9%) 8/103 (7.8%) 6/49 (12.2%) 4/64 (6.2%)
Arumugaswdmy et al. (1994) Schlech et al. (1983) Heisick et al. (1989a) Vahidy (1992) Heisick et al. (1989b) Arumugaswamy et al. (1994) Arumugaswamy et al. (1994) Heisick et al. (1989a) Heisick et al. (1989b) Harvey and Gilmour (1993) Sizmur and Walker (1988) Heisick et al. (1989a) Heisick et al. (1989b) Breer and Baumgartner (1992) Harvey and Gilmour (1993) Beckers et al. (1989) Vahidy (1992) Gola et al. (1990) de Simon et al. (1992) Wong et al. (1990) MacGowan et al. (1994)
Cucumber
Leafy vegetables Potatoes Prepacked salads Radish Salad vegetables
Tomato Vegetables
“Prevalence in all vegetables was 170/1495 (11.4%).
BEHAVIOUR
ON
VEGETABLES
It was not until after the Canadian coleslaw outbreak that much research attention was directed toward examining survival and growth characteristics of L. monocytogenes on vegetables. The effects of temperature, pH and NaCl on growth and survival of L. monocytogenes in cabbage have been studied (Conner et al., 1986). Initial studies involved the use of heatsterilized cabbage juice. At 3O”C, populations of the L. monocytogenes strain isolated from the coleslaw outbreak increased in juice containing O-1.5% NaCl; a decrease in population occurred in juice containing 22% NaCl. The organism grew well in cabbage juice at an initial pH of 5.0-6.1. In another study, the growth of L. monocytogenes in raw, shredded cabbage prepared in a manner similar to that followed for preparing coleslaw was investigated (Beuchat et al., 1986). The organism increased from 1.6 x lo4 to 2.6 x 10’ CFU/g of cabbage stored at 5°C for 25 days. Extended storage to 64 days resulted in only a slight decrease in viable population. In contrast, the viable population of L. monocytogenes steadily decreased in heat-sterilized shredded cabbage stored at 5°C. This suggests that heat treatment either decreases the availability of certain nutrients or results in constituents which inhibit growth or are toxic. Survival and growth characteristics of L. monocytogenes on lettuce and in lettuce juice were studied by Steinbruegge et al. (1988). Behaviour of the organism was variable. In most trials, the population of L. monocytogenes on lettuce stored at 5 and 25°C increased by several logs during 14 days of storage, while in some trials the organism was not detected at the end of the storage period. Growth was observed in lettuce juice held at 5°C. The effects of shredding, chlorine treatment and modified atmosphere packaging on survival and
226
Food Control 1996 Volume 7 Number 4/5
growth of L. monocytogenes on lettuce stored at 5°C and 10°C were studied by Beuchat and Brackett (1990b). With the exception of shredded lettuce that had not been chlorine treated, no significant changes in populations of L. monocytogenes were detected during the first eight days of incubation at 5°C. However, significant increases occurred between 8 and 15 days. Significant increases occurred within three days when lettuce was stored at 10°C; after 10 days, populations reached lox-10’ CFU/g. Chlorine treatment, modified atmosphere (3% O,, 97% NJ and shredding did not influence growth of L. monocytogenes. It was concluded that L. monocytogenes is capable of growing on lettuce subjected to commonly used packaging and distribution procedures used in the food industry. The behaviour of L. monocytogenes on raw broccoli, cauliflower and asparagus stored under air and modified atmospheric gas packaging conditions at 4-15°C has been studied (Berrang et al., 1989). Growth at 15°C resulted in populations as high as 6.0 x 10’ CFU/g of broccoli florets, 3.7 x 10’ CFU/g of cauliflower florets and 1.1 x 10’ CFU/g of asparagus before these vegetables would be considered inedible by subjective evaluation. Modified atmosphere appears to have little, if any, effect on the rate of growth of L. monocytogenes on these vegetables. Rates of death and growth of L. monocytogenes inoculated onto raw whole tomatoes and into chopped tomatoes were studied by Beuchat and Brackett (1991). Growth occurred on whole tomatoes held at 21°C but not at lO”C, while death occurred in chopped tomatoes stored at these temperatures. Raw carrot juice appears to have a lethal affect on L. monocytogenes (Nguyen-the and Lund, 1990). Populations of L. monocytogenes have been shown to decrease upon contact with whole and shredded raw carrots but not cooked carrots (Beuchat and
Listeria monocytogenes:
Brackett, 1990a). Small populations detected on whole carrots immediately after inoculation were essentially non-detectable after seven days of storage at 5 or 15°C. Beuchat et al. (1994) investigated the influence of pH and sodium chloride on survival and growth of L. monocytugenes in carrot juice. Lethal and inhibitory effects over a 48 h period were greatest in a pH range of 5.0-6.4. Inhibition was enhanced by reducing the incubation temperature from 20°C to 12°C and again from 12°C to 5°C. A concentration of 5% sodium chloride protected L. monocytogenes against inactivation in carrot juice. Washing shredded lettuce with carrot juice significantly reduces populations of L. monocytogenes and retards growth during subsequent storage at 5°C (Beuchat and Doyle, 1995). The efficacy of chlorine for killing L. monocytugenes on inoculated Brussels sprouts has been evalu1987). While the organism was ated (Brackett, relatively sensitive to chlorine in the absence of organic matter, it remained viable on Brussels sprouts dipped for 10 s in water containing 200 @ml chlorine. The viable population was reduced by about 2 log,,, CFU/g from an initial population of about 6 log,,, CFU/g. The effectiveness of chlorine dips in inactivating L. monocytogenes on other raw vegetables has not been thoroughly investigated. Based on observations, however, the lethal effect of chlorine may be minimal. Brackett (1987) concluded that hypochlorite was ineffective in removing L. monocytogenes from contaminated vegetables.
CONCLUSIONS The per capita consumption of raw or minimally processed vegetables in the USA as well as in several other countries is steadily increasing. This trend stems from the desire of an increased percentage of the population to reduce the consumption of foods from animal origin and increase the consumption of grains, legumes, vegetables and fruits. The fresh produce industry has accommodated this demand by utilizing distribution and packaging technologies for delivering high-quality products to the consumers. The availability of numerous ready-to-eat salad items in supermarkets, the proliferation of salad bars in restaurants and the use of salad vegetables as integral parts of a wide range of types of sandwiches and salads offered by the food service industry are also on the increase. Innovative technologies and techniques for maintaining sensory qualities of raw salad vegetables are continuously being explored and introduced in the marketplace. While the ecology of L. monocy@genes on vegetables is not fully understood, one thing is certain. If consumers are to continue to benefit from the essential contribution raw and minimally processed vegetables make to their nutritional well-being, they will also continue to consume
vegetables
incidence on vegetables: L.R. Beuchat
containing
low
numbers
of
L.
monocytogenes.
REFERENCES Al-Ghazali, M.R. and Al-Azawi, S.K. (1986) Detection and enumeration of Listeria monocyfogenes in a sewage treatment plant in Iraq. J. Appl. Bacterial. 60, 251-254 Arumugaswamy, R.K., Ali, G. Rusul Rahamat and Hamid, Nadzriah Bte Abd (1994) Prevalence of Listeriu monocytogenes in foods in Malaysia. ht. J. Food Microbial. 23, 117-121 Beckers, H.J., in’t Veld, RH., Soentoro, P.&S. and Delfgou-van Asch, E.H.M. (1989) The occurrence of Listeria in food. In Foodborne Listeriosis: Proceedings of a Symposium, September 7, Wiesbaden, Germany. 85-97 Berrang, M.E., Brackett, R.E. and Beuchat, L.R. (1989) Growth of Listeria monocytogenes on fresh vegetables stored under a controlled atmosphere. J. Food Prot. 52, 702-705 Beuchat, L.R. and Brackett, R.E. (1990a) Inhibitory effects of raw carrots on Listeria monocytogenes. Appl. Environ. Microbial. 56, 1734-1742
Beuchat, L.R. and Brackett, R.E. (1990b) Growth of Listeria monocytogenes on lettuce as influenced by shredding, chlorine treatment, modified atmosphere packaging, temperature and time. J. Food Sci. 55, 755-758,870 Beuchat, L.R. and Brackett, R.E. (1991) Behavior of Listeria monocytogenes inoculated into raw tomatoes and processed tomato products. Appi. Environ. Microbiof. 57. 1367-1371 Beuchat, L.R., Bracket& R.E. and Doyle, M.R (1994) Lethality of carrot juice to Listeriu monocytogenes as affected by pH, sodium chloride and temperature. J. Food Prot. 57, 470-474 Beuchat, L.R., Brackett, R.E., Hao, D.Y.-Y. and Conner, D.E. (1986) Growth and thermal inactivation of Listeria monocytogenes in cabbage and cabbage juice. Can J. Microbiot. 32, 791-795
Beuchat, L.R. and Doyle, M.l? (1995) Survival and growth of Listeria monocytogenes in foods treated or supplemented with carrot juice. Food Microbial. 12, 73-80 Blenden, D.C. and Szatalowicz, ET. (1967) Ecological aspects of listeriosis. J. Am. I/et. Med. Assn. 151, 1761-1766 Brackett, R.E. (1987) Antimicrobial
effect of chlorine on Listeriu
monocytogenes. J. Food Prot. 50, 999-1003
Breer, C. and Baumgartner, A. (1992) Occurrence and behavior of Listeriu monocytogenes in salads, vegetables and fresh vegetable juices. Archiv. Lebensmittel hyg. 43, 108-l 10 Conner, D.E., Brackett, R.E. and Beuchat, L.R. (1986) Effect of temperature, sodium chloride, and pH on growth of Listeriu monocytogenes in cabbage juice. Appl. Environ. Microbial. 52, 59-63
de Simon, M., Tarrago, C. and Ferrer, M.D. (1992) Incidence of Listeria monocytogenes in fresh foods in Barcelona (Spain). Int. J. Food Microbial. 16, 153-146
Farber, J.M., Sanders, G.W. and Johnston, M.A. (1989) A survey of various foods for the presence of Listeriu species. J. Food Prot. 52, 456-458
Gola, S., Previdi, MJ?, Mutti, R and Belloli, S. (1990) Microbiological investigation of frozen vegetables, incidence of Listeriu and other psychrotrophic pathogens. Industria Conserve 65( 1), 36-38
Gray, M.L. (1963) Epidemiological
aspects of listeriosis. Am. J.
Public Health 53, 554-563
Gray, M.L. and Killinger, A.H. (1966) Listeriu monocytogenes and listeric infections. Bucteriol. RPY 30, 309-382 Gronstol, H. (1979) Listeriosis in sheep. Isolation of Listeriu monocytogenes from grass silage. Actu Vet. Scund. 20, 492-497
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Listeria monocytogenes: incidence
on vegetables:
LA. Beuchat
Harvey, J. and Gilmour, A. (1993) Occurrence and characteristics of Listeria in foods produced in Northern Ireland. ht. J. Food Microbial. 19, 193-205
Heisick, J.E., Harrell, EM., Peterson, E.H., McLaughlin, S., Wagner, D.E., Wesley, I.\ and Bryner, J. (1989a) Comparison of four procedures to detect Listeria spp. in foods. J. Food Prot. 52, 154-157
Heisick, J.E., Wagner, D.E., Niermand, M.L. and Peeler, J.T. (1989b) Listeria spp. found on fresh market produce. Appl. Environ. Microbial. 55, 1925-1927
Ho, J.L., Shands, K.N., Friedland, G., Eckind, I? and Fraser, D.W. (1986) An outbreak of type 4b Listeria monocytogenes infection involving patients from eight Boston hospitals. Arch. Intern. Med. 146,520-524
King, S.H., Nichols, E.S. and Broome, C.V. (1983) Epidemic listeriosis - Evidence for transmission by food. N. Engl. J. Med. 308,203-206
Steinbruegge, E.G., Maxcy, R.B. and Liewen, M.B. (1988) Fate of Listeria monocytogenes on ready to serve lettuce. J. Food Prot. 51,596-599 Sizmur, K. and Walker, C.W. (1988) Listeria in prepacked Lancet 1 8595: 1167
salads.
Vahidy, R. (1992) Isolation of Listeria tnonocytogenes from fresh fruits and vegetables. (Abstract) HortScience 27, 62 Velani, S. and Roberts, D. (1991) Listeria monocytogenes and other Listeria spp. in prepacked salad mixes and individual salad ingredients. PHLS Microbial. Digest (UK) 8, 21-22
Hofer, E. (1975) Study of Listeria species on vegetables suitable for human consumption. Gong. Braz. Microbial., Salvador, 27-31 July, 192. (Portuguese)
Watkins, J. and Sleath, K.P. (1981) Isolation and enumeration of Listeria monocytogenes from sewage sludge and river water.
MacCowan, AR, Bowker, K., McLauchlin, J., Bennett, P.M. and Reeves, D.S. (1994) The occurrence and seasonal changes in the isolation of Listeria spp. in shop bought food stuffs, human faeces, sewage and soil from urban sources. Int. J. Food Micro-
Weiss, J. and Seeliger, H.P.R. (1975.) Incidence of Listeria monocytogenes in nature. Appl. Microbial. 29, 29-32
biol. 21, 325-334
J. Appl. Bacterial. 50, l-9
Welshimer, H.J. (1960) Survival of Listeria monocytogenes in soil. J. Bacterial. 80, 316-320
Nguyen-the, C. and Lund, B.M. (1990) The lethal effect of carrot on Listeria species. .I. Appl. Bacterial. 70, 479-488
Welshimer, H.J. (1968) Isolation of Listeria monocytogenes vegetation. .I. Bacterial. 95, 300-303
Petran, R.L., Zottola, E.A. and Gravini, R.B. (1988) Incidence of Listeria monocytogenes in market samples of fresh and frozen vegetable. J. Food Sci. 53, 1238-1240
Welshimer, H.J. and Donker-Voet, J. (1971) Listeria monocytogenes in nature. Appf. Microbial. 21, 516-519
Schlech, W.E, Lavigne, P.M., Bortolussi, R.A., Allen, A.C., Haldane, W.V., Wort, A.J., Hightower, A.W., Johnson, S.E.,
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from
Wong, H.-C., Chao, W.-L. and Lee, S.-J. (1990) Incidence and characterization of Listeria monocytogenes in foods available in Taiwan. Appl. Environ. Microbial. 56, 3101-3104
PII: SO956-7135(96)00039-4
in Great
0 1996 Elsevier Britain.
1996
Science
Ltd
All rights reserved
OYS6-71.15196
$15.00+0.00
PAPER
Listeria monocytogenes: incidence on vegetables Larry R. Beuchat Listeria monocytogenes is widely distributed on plant vegetation, including raw vegetables. Its presence on plant materials is likely due to contamination from decaying vegetation, animal faeces, soil, sur$ace, river and canal waters, or effluents from sewage treatment operations. The organism is known to survive in plant materials for IO-12 years. The presence of L. monocytogenes on many types of raw and minimally processed vegetables destined for human consumption has been clearly demonstrated in many countries but the role these vegetables play as vehicles for human infection is not known. It is certain, however; that if consumers are to continue to benefit from the essential contribution raw and minimally processed vegetables make to their nutritional well-being, they will also very likely continue to consume vegetables containing low numbers of L. monocytogenes. Copyright 0 1996 Elsevier Science Ltd. Keywords: Listeriu monocytogenes; vegetables; food-borne
pathogen
INTRODUCTION
OCCURRENCE IN NATURE
Given enough time and appropriate methodology for detection, Listeria monocytogenes can be found in or on a large number of species representative of every class of living things that swim, crawl, jump, walk or fly. Furthermore, the organism can also be isolated from many living things, namely plants, that do not possess within themselves these abilities of movement. Plants play a significant role in the dissemination of L. monocytogenes from natural habitats to the human food supply. This role may be indirect, for example by contamination of milk via forage or silage, or direct in the form of contaminated raw vegetables and perhaps fruits. Summarized here is information on the presence, survival and proliferation of L. monocyfogenes on vegetation in general and more specifically on raw and lightly processed vegetables.
Center for Food Safety and Quality Enhancement, ment of Food Science and Technology, Georgia, Griffin, Georgia 30223-l 797, USA.
DepartUniversity of
OF L. MONOCYTOGENES
Listeria monocytogenes
is present in many animals, including humans, so it is not too surprising that the organism can also be isolated from faeces of these animals, on the land they occupy, in sewage, in soils to which sewage is applied and on plants, including those producing vegetables, which grow in these soils. Some examples will serve to illustrate the cycle of L. monocytogenes between plants and humans (Figure 1). The presence of Listeria in sewage in many countries has been demonstrated. Watkins and Sleath (1981) analysed 52 sewage, river water and industrial effluents in England for the presence of potentially pathogenic microorganisms. Effluents were from abattoirs, cattle markets and poultry packing plants. Listeria monocytogenes was isolated from every sample. In many instances, populations of L. monocytogenes were higher than those for salmonellae, and in some instances L. monocytogenes was isolated when no salmonellae were detected. Application of sludge containing L. monocytogenes and salmonellae to soil revealed that L. monocytogenes could survive
Food Control 1996 Volume 7 Number 4/5
223
Listeria monocytogenes: incidence on vegetables: L.R. Beuchat
ANIMALS
VEGETABLES -
water -+-
\
\b!/
,
,.
7s
Sr
HUMANS
/. .
contamrnatron)
\
plants-
silage, feed -
meat, milk, eggs
’
Figure 1 Potential pathways for transmission of L. munocytugenes to humans via vegetables.
longer periods of time. Populations of L. monocytogenes in soil remained essentially unchanged during seven weeks following spraying. Welshimer (1960) demonstrated that L. monocytogenes could survive in soil for up to 295 days. Reports from other countries also indicate the presence of L. monocytogenes in sewage. The organism has been isolated from a sewage treatment plant in Baghdad, Iraq (Al-Ghazali and Al-Azawi, 1986). Treatment processes at the plant did not yield a sewage sludge cake or a final discharge free of Listetiu. It was concluded that the use of sewage as a fertilizer could be a route of contaminating vegetation destined for human consumption. Dewatering of sewage reduced the number of L. monocytogenes but it was suggested that long periods of exposure to sun would be needed to render the sewage sludge cake ‘safe’. MacGowan et al. (1994) examined sewage in England at two-month intervals in 1991-92 and found 84-100% contained L. monocytogenes and/or L. innocua. Welshimer and Donker-Voet (1971) isolated 27 strains of L. monocytogenes from soil and vegetation taken from 19 sites in The Netherlands. Vegetation samples were taken from the same sites in September and April of the following year. Listeria monocytogenes was isolated only from samples collected in April. Plant materials from which the organism were isolated included dead and decayed corn and soybean plants and wild grasses, indicating its preference to exist in nature as a saprophyte. A study of soil and domestic animal faeces in England has shown that Listeriu is more often present in July to September than in other months (MacGowan et al., 1994). L. monoqtogenes and L. innocua were predominant in
224
Food Control 1996 Volume 7 Number 4/5
faeces whereas L. ivanovi and L. seeligeri were most common in soil. Welshimer (1968) examined vegetation in a rural area in Virginia (USA) where clinical listeriosis of man and animals was rare. Dead soybean plant material and stalks, leaves and tassels of corn were collected in April following the previous planting year. Eight of twelve sampling sites yielded plant materials positive for L. monocytogenes. Only 25% of the strains were pathogenic for mice, which is a low frequency compared with the percentage of pathogenic strains isolated from listeric humans and animals in Virginia and the USA as a whole. These observations suggest that the predominance of certain serotypes of L. monocytogenes may be influenced by the nature of the host environment and that the majority of strains indigenous to decaying plant vegetation are incapable of causing illness in animals. In a survey conducted in Germany, Weiss and Seeliger (1975) isolated 154 strains of L. monocytogenes from soil and plants, 16 from faeces of deer and stag, nine from mouldy fodder and wildlife feeding grounds and eight from birds. Corn, wheat, oats, barley and potato plants and soils from fields in which they were growing were among the materials analysed. Nearly 10% of the corn plants and 13% of the grain plants were positive for L. monocytogenes. Plants from cultivated fields had a lower incidence of the organism (12.5%) compared to plants from uncultivated fields (44%). Twenty-three percent of samples collected from wildlife feeding grounds were positive for L. monocytogenes. Virulent strains were isolated from all locations. It was suggested that L. monocytogenes is a saprophyte which lives in a plantsoil environment and could therefore be contracted
Listeria monocytogenes:
by humans and other animals via many possible routes from many sources. It is very unlikely that birds and animals are the only sources responsible for the distribution of L. monocytogenes in nature and its presence on vegetables. The relationship between feeding silage to animals and listeriosis has been recognized since the early 1960s (Gray, 1963; Gray and Killinger, 1963). Consumption of mouldy or badly cured silage with with elevated pH values has been correlated outbreaks of listeriosis in cattle and sheep. The frequency of L. monocytogenes in silage increases with increasing pH. Gronstol (1979) isolated the organism from 22% of silage samples with pH ~4, from 37% with pH 4 to 5 and from 56% with pH >5.
VEGETABLES LISTERIOSIS
AS VEHICLES
FOR
As early as 1967, Blenden and Szatalowicz, noting that 731 cases of human listeriosis had been reported between 1933 and 1966, stated that it seemed feasible that plant products such as lettuce or other fresh vegetables may be contaminated with L. monocytogenes and perhaps be responsible for causing infection. Evidence supporting this hypothesis has subsequently emerged. An outbreak of L. monocytogenes infection in 1979 involving 23 patients from eight Boston hospitals was reported by Ho et al. (1986). Twenty (87%) of the isolates were L. monocytogenes serotyped during the 26 months preceding the outbreak. Case patients differed from patients with sporadic Listeria infection in that more of the case patients had hospital-acquired infection (75% v 22%), had received antacids or cimetidine before the onset of listeriosis (60% v 17%), and had gastrointestinal tract symptoms that began at the same time as fever (85% v 22%). Tuna fish, chicken salad and cheese were preferred by case patients more frequently than by control patients, but the only common foods served with these foods were raw celery, tomatoes and lettuce. It was concluded that the consumption of these raw vegetables could have caused the listeriosis outbreak. No attempt was made to isolate L. monocytogenes from the vegetables at the time of the outbreak. In a report from another laboratory, however, Hofer (1975), in a study of Listeria species on vegetables suitable for consumption, isolated three Listeria strains from lettuce. Human infection associated with the consumption of raw cabbage containing L. monocytogenes occurred in Canada in 1981 (Schlech et al., 1983). Thirty-four cases of perinatal listeriosis and seven cases of adult disease occurred between 1 March and 1 September of that year. During May, June and July 1981, the attack rate for perinatal listeriosis at a maternity hospital was approximately 1.3%. A survey revealed that cases were more likely than controls to have consumed coleslaw in the three months before the
incidence
on vegetables:
L.R. Beuchat
onset of illness. Coleslaw obtained from the refrigerator of a patient was positive for L. monocytogenes serotype 4b, which was the serotype of the epidemic strain and the strain isolated from the patient’s blood. The coleslaw had been commercially prepared with cabbage and carrots obtained from wholesalers and local farmers. After prolonged cold enrichment, two unopened packages of coleslaw purchased from two different supermarkets yielded L. monocytogenes, serotype 4b. An investigation of the sources of raw vegetables identified a farmer who, in addition to raising cabbage, also maintained a flock of sheep. A review of the farmer’s agronomic practices revealed that both composted and raw sheep manure had been applied to fields in which cabbage was grown. From the last harvest in October through the winter and early spring, cabbage was kept in a cold-storage shed. A shipment of cabbage during the period of the outbreak was traced to the implicated coleslaw processor. Information obtained in this investigation strongly suggests that the source of this outbreak was coleslaw. A statistical association of listeriosis with coleslaw was supported by identification of the epidemic strain in the implicated food. The coleslaw outbreak heightened interest in determining the prevalence of L. monocytogenes in fresh produce. Sizmur and Walker (1988) detected L. monocytogenes in four of 60 prepacked, ready-to-eat salads in the UK. Vegetables included in the two types of contaminated salads were cabbage, celery, carrots, lettuce, cucumber, onion, leeks, watercress and fennel. Listeria innocua was isolated from 13 samples representing five salad varieties. incubation of salads containing L. monocytogenes at 4°C for four days resulted in a roughly two-fold increase in the population of the organism. A survey of 1000 samples of 10 types of fresh produce at the retail level in the USA has revealed the presence of L. monocytogenes on cabbage, cucumbers, potatoes and radishes (Heisick et al., 1989b). In another survey, prepared mixed salads and two individual salad ingredients were found to contain L. monocytogenes (Velani and Roberts, 1991). The higher rate of contamination of prepared salads was attributed to cross contamination of the pathogen during chopping, mixing and packaging. Beckers et al. (1989) detected L. monocytogenes in 11 of 25 samples of fresh cut vegetables in The Netherlands and Harvey and Gilmour (1993) reported that 7 of 66 samples of salad vegetables and prepared salads produced in Northern Ireland contained the organism. Farber et al. (1989), on the other hand, did not detect Listeria species in 110 samples of vegetables, including lettuce, celery, tomatoes and radishes analysed in Canada. Likewise, Petran et al. (1988) failed to detect L. monocytogenes in market samples of fresh vegetables in the United States. Some reports documenting the presence of L. monocytogenes in vegetables are listed in Table 1.
Food Control 1996 Volume 7 Number 4/5
225
Listeriamonocytogenes: incidence on vegetables:
Table 1
L.R. Beuchat
Raw vegetables from which L. monocyfogenes has been isolated
Vegetable
Country
Prevalence’
Reference
Bean sprouts Cabbage
Malaysia Canada USA Malaysia Pakistan USA Malaysia USA USA Northern Ireland UK USA USA Germany Northern Ireland The Netherlands Pakistan Italy Spain Taiwan UK
6/7 (85.7%) 2/92 (2.2%) l/92 (1.1%) 4/5 (80%) l/15 (6.7%) 2/92 (2.2%) 5/22 (22.7%) 19/70 (27.1%) 28/132 (21.2%) 3/21 (14.3%) 4/60 (13.3%) 25/68 (36.8%) 19/132 (14.4%) 6/263 (2.3%) 7/66 (10.6%) 1 l/25 (44%) 2/15 (13.3%) 7/102 (6.9%) 8/103 (7.8%) 6/49 (12.2%) 4/64 (6.2%)
Arumugaswdmy et al. (1994) Schlech et al. (1983) Heisick et al. (1989a) Vahidy (1992) Heisick et al. (1989b) Arumugaswamy et al. (1994) Arumugaswamy et al. (1994) Heisick et al. (1989a) Heisick et al. (1989b) Harvey and Gilmour (1993) Sizmur and Walker (1988) Heisick et al. (1989a) Heisick et al. (1989b) Breer and Baumgartner (1992) Harvey and Gilmour (1993) Beckers et al. (1989) Vahidy (1992) Gola et al. (1990) de Simon et al. (1992) Wong et al. (1990) MacGowan et al. (1994)
Cucumber
Leafy vegetables Potatoes Prepacked salads Radish Salad vegetables
Tomato Vegetables
“Prevalence in all vegetables was 170/1495 (11.4%).
BEHAVIOUR
ON
VEGETABLES
It was not until after the Canadian coleslaw outbreak that much research attention was directed toward examining survival and growth characteristics of L. monocytogenes on vegetables. The effects of temperature, pH and NaCl on growth and survival of L. monocytogenes in cabbage have been studied (Conner et al., 1986). Initial studies involved the use of heatsterilized cabbage juice. At 3O”C, populations of the L. monocytogenes strain isolated from the coleslaw outbreak increased in juice containing O-1.5% NaCl; a decrease in population occurred in juice containing 22% NaCl. The organism grew well in cabbage juice at an initial pH of 5.0-6.1. In another study, the growth of L. monocytogenes in raw, shredded cabbage prepared in a manner similar to that followed for preparing coleslaw was investigated (Beuchat et al., 1986). The organism increased from 1.6 x lo4 to 2.6 x 10’ CFU/g of cabbage stored at 5°C for 25 days. Extended storage to 64 days resulted in only a slight decrease in viable population. In contrast, the viable population of L. monocytogenes steadily decreased in heat-sterilized shredded cabbage stored at 5°C. This suggests that heat treatment either decreases the availability of certain nutrients or results in constituents which inhibit growth or are toxic. Survival and growth characteristics of L. monocytogenes on lettuce and in lettuce juice were studied by Steinbruegge et al. (1988). Behaviour of the organism was variable. In most trials, the population of L. monocytogenes on lettuce stored at 5 and 25°C increased by several logs during 14 days of storage, while in some trials the organism was not detected at the end of the storage period. Growth was observed in lettuce juice held at 5°C. The effects of shredding, chlorine treatment and modified atmosphere packaging on survival and
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Food Control 1996 Volume 7 Number 4/5
growth of L. monocytogenes on lettuce stored at 5°C and 10°C were studied by Beuchat and Brackett (1990b). With the exception of shredded lettuce that had not been chlorine treated, no significant changes in populations of L. monocytogenes were detected during the first eight days of incubation at 5°C. However, significant increases occurred between 8 and 15 days. Significant increases occurred within three days when lettuce was stored at 10°C; after 10 days, populations reached lox-10’ CFU/g. Chlorine treatment, modified atmosphere (3% O,, 97% NJ and shredding did not influence growth of L. monocytogenes. It was concluded that L. monocytogenes is capable of growing on lettuce subjected to commonly used packaging and distribution procedures used in the food industry. The behaviour of L. monocytogenes on raw broccoli, cauliflower and asparagus stored under air and modified atmospheric gas packaging conditions at 4-15°C has been studied (Berrang et al., 1989). Growth at 15°C resulted in populations as high as 6.0 x 10’ CFU/g of broccoli florets, 3.7 x 10’ CFU/g of cauliflower florets and 1.1 x 10’ CFU/g of asparagus before these vegetables would be considered inedible by subjective evaluation. Modified atmosphere appears to have little, if any, effect on the rate of growth of L. monocytogenes on these vegetables. Rates of death and growth of L. monocytogenes inoculated onto raw whole tomatoes and into chopped tomatoes were studied by Beuchat and Brackett (1991). Growth occurred on whole tomatoes held at 21°C but not at lO”C, while death occurred in chopped tomatoes stored at these temperatures. Raw carrot juice appears to have a lethal affect on L. monocytogenes (Nguyen-the and Lund, 1990). Populations of L. monocytogenes have been shown to decrease upon contact with whole and shredded raw carrots but not cooked carrots (Beuchat and
Listeria monocytogenes:
Brackett, 1990a). Small populations detected on whole carrots immediately after inoculation were essentially non-detectable after seven days of storage at 5 or 15°C. Beuchat et al. (1994) investigated the influence of pH and sodium chloride on survival and growth of L. monocytugenes in carrot juice. Lethal and inhibitory effects over a 48 h period were greatest in a pH range of 5.0-6.4. Inhibition was enhanced by reducing the incubation temperature from 20°C to 12°C and again from 12°C to 5°C. A concentration of 5% sodium chloride protected L. monocytogenes against inactivation in carrot juice. Washing shredded lettuce with carrot juice significantly reduces populations of L. monocytogenes and retards growth during subsequent storage at 5°C (Beuchat and Doyle, 1995). The efficacy of chlorine for killing L. monocytugenes on inoculated Brussels sprouts has been evalu1987). While the organism was ated (Brackett, relatively sensitive to chlorine in the absence of organic matter, it remained viable on Brussels sprouts dipped for 10 s in water containing 200 @ml chlorine. The viable population was reduced by about 2 log,,, CFU/g from an initial population of about 6 log,,, CFU/g. The effectiveness of chlorine dips in inactivating L. monocytogenes on other raw vegetables has not been thoroughly investigated. Based on observations, however, the lethal effect of chlorine may be minimal. Brackett (1987) concluded that hypochlorite was ineffective in removing L. monocytogenes from contaminated vegetables.
CONCLUSIONS The per capita consumption of raw or minimally processed vegetables in the USA as well as in several other countries is steadily increasing. This trend stems from the desire of an increased percentage of the population to reduce the consumption of foods from animal origin and increase the consumption of grains, legumes, vegetables and fruits. The fresh produce industry has accommodated this demand by utilizing distribution and packaging technologies for delivering high-quality products to the consumers. The availability of numerous ready-to-eat salad items in supermarkets, the proliferation of salad bars in restaurants and the use of salad vegetables as integral parts of a wide range of types of sandwiches and salads offered by the food service industry are also on the increase. Innovative technologies and techniques for maintaining sensory qualities of raw salad vegetables are continuously being explored and introduced in the marketplace. While the ecology of L. monocy@genes on vegetables is not fully understood, one thing is certain. If consumers are to continue to benefit from the essential contribution raw and minimally processed vegetables make to their nutritional well-being, they will also continue to consume
vegetables
incidence on vegetables: L.R. Beuchat
containing
low
numbers
of
L.
monocytogenes.
REFERENCES Al-Ghazali, M.R. and Al-Azawi, S.K. (1986) Detection and enumeration of Listeria monocyfogenes in a sewage treatment plant in Iraq. J. Appl. Bacterial. 60, 251-254 Arumugaswamy, R.K., Ali, G. Rusul Rahamat and Hamid, Nadzriah Bte Abd (1994) Prevalence of Listeriu monocytogenes in foods in Malaysia. ht. J. Food Microbial. 23, 117-121 Beckers, H.J., in’t Veld, RH., Soentoro, P.&S. and Delfgou-van Asch, E.H.M. (1989) The occurrence of Listeria in food. In Foodborne Listeriosis: Proceedings of a Symposium, September 7, Wiesbaden, Germany. 85-97 Berrang, M.E., Brackett, R.E. and Beuchat, L.R. (1989) Growth of Listeria monocytogenes on fresh vegetables stored under a controlled atmosphere. J. Food Prot. 52, 702-705 Beuchat, L.R. and Brackett, R.E. (1990a) Inhibitory effects of raw carrots on Listeria monocytogenes. Appl. Environ. Microbial. 56, 1734-1742
Beuchat, L.R. and Brackett, R.E. (1990b) Growth of Listeria monocytogenes on lettuce as influenced by shredding, chlorine treatment, modified atmosphere packaging, temperature and time. J. Food Sci. 55, 755-758,870 Beuchat, L.R. and Brackett, R.E. (1991) Behavior of Listeria monocytogenes inoculated into raw tomatoes and processed tomato products. Appi. Environ. Microbiof. 57. 1367-1371 Beuchat, L.R., Bracket& R.E. and Doyle, M.R (1994) Lethality of carrot juice to Listeriu monocytogenes as affected by pH, sodium chloride and temperature. J. Food Prot. 57, 470-474 Beuchat, L.R., Brackett, R.E., Hao, D.Y.-Y. and Conner, D.E. (1986) Growth and thermal inactivation of Listeria monocytogenes in cabbage and cabbage juice. Can J. Microbiot. 32, 791-795
Beuchat, L.R. and Doyle, M.l? (1995) Survival and growth of Listeria monocytogenes in foods treated or supplemented with carrot juice. Food Microbial. 12, 73-80 Blenden, D.C. and Szatalowicz, ET. (1967) Ecological aspects of listeriosis. J. Am. I/et. Med. Assn. 151, 1761-1766 Brackett, R.E. (1987) Antimicrobial
effect of chlorine on Listeriu
monocytogenes. J. Food Prot. 50, 999-1003
Breer, C. and Baumgartner, A. (1992) Occurrence and behavior of Listeriu monocytogenes in salads, vegetables and fresh vegetable juices. Archiv. Lebensmittel hyg. 43, 108-l 10 Conner, D.E., Brackett, R.E. and Beuchat, L.R. (1986) Effect of temperature, sodium chloride, and pH on growth of Listeriu monocytogenes in cabbage juice. Appl. Environ. Microbial. 52, 59-63
de Simon, M., Tarrago, C. and Ferrer, M.D. (1992) Incidence of Listeria monocytogenes in fresh foods in Barcelona (Spain). Int. J. Food Microbial. 16, 153-146
Farber, J.M., Sanders, G.W. and Johnston, M.A. (1989) A survey of various foods for the presence of Listeriu species. J. Food Prot. 52, 456-458
Gola, S., Previdi, MJ?, Mutti, R and Belloli, S. (1990) Microbiological investigation of frozen vegetables, incidence of Listeriu and other psychrotrophic pathogens. Industria Conserve 65( 1), 36-38
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Gray, M.L. and Killinger, A.H. (1966) Listeriu monocytogenes and listeric infections. Bucteriol. RPY 30, 309-382 Gronstol, H. (1979) Listeriosis in sheep. Isolation of Listeriu monocytogenes from grass silage. Actu Vet. Scund. 20, 492-497
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on vegetables:
LA. Beuchat
Harvey, J. and Gilmour, A. (1993) Occurrence and characteristics of Listeria in foods produced in Northern Ireland. ht. J. Food Microbial. 19, 193-205
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Vahidy, R. (1992) Isolation of Listeria tnonocytogenes from fresh fruits and vegetables. (Abstract) HortScience 27, 62 Velani, S. and Roberts, D. (1991) Listeria monocytogenes and other Listeria spp. in prepacked salad mixes and individual salad ingredients. PHLS Microbial. Digest (UK) 8, 21-22
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