Efficacy of allyl isothiocyanate in killing enterohemorrhagic Escherichia coli O157:H7 on alfalfa seeds

Efficacy of allyl isothiocyanate in killing enterohemorrhagic Escherichia coli O157:H7 on alfalfa seeds

International Journal of Food Microbiology 56 (2000) 13–20 www.elsevier.nl / locate / ijfoodmicro Efficacy of allyl isothiocyanate in killing enteroh...

342KB Sizes 0 Downloads 31 Views

International Journal of Food Microbiology 56 (2000) 13–20 www.elsevier.nl / locate / ijfoodmicro

Efficacy of allyl isothiocyanate in killing enterohemorrhagic Escherichia coli O157:H7 on alfalfa seeds C.M. Park, P.J. Taormina, L.R. Beuchat* Center for Food Safety and Quality Enhancement, Department of Food Science and Technology, University of Georgia, 1109 Experiment Street, Griffin, GA 30223 -1797, USA Received 26 June 1999; received in revised form 13 November 1999; accepted 22 November 1999

Abstract Volatile compounds occurring in the essential oil of plants were tested for their efficacy in killing Escherichia coli O157:H7. Experiments using an agar disk assay revealed that exposure of the pathogen to 50 ml of eugenol, carvacrol, linalool, or methyl jasmonate in a 950-cc jar at 20, 37 or 478C for up to 48 h failed to inhibit colony formation. However, exposure to 8 ml of allyl isothiocyanate (AIT) (equivalent to 8.4 ppm in the air within the jar, if completely volatilized) resulted in more than a 7-log 10 reduction in population of E. coli O157:H7 at 378C within 48 h; significant (P # 0.05) reduction in populations also occurred in the presence of 4 ml of AIT compared to 2 ml, which had no lethal affect. At 208C, the lethality of AIT was substantially less, although significant reduction occurred when disks were exposed to 8 or 10 ml of AIT compared to 4 or 6 ml and when exposed to 4 or 6 ml compared to 2 ml. Treatment with 10 ml of AIT for 5 h at 478C resulted in death of 6 log 10 of E. coli O157:H7. The efficacy of AIT in killing E. coli O157:H7 on dry and wet alfalfa seeds was investigated. The pathogen, at an initial population of 2.7 log 10 cfu / g of seed, was not recovered by direct plating ( , 0.7 log 10 cfu / g) or enrichment of wet seeds exposed to 50 ml of AIT / 950-cc jar for 24 h at 37 or 478C. Exposure of dry seeds containing 2.9 log 10 cfu of E. coli O157:H7 per g to an atmosphere containing 100 ml of AIT / 950-cc jar (ca. 105 ppm AIT if completely volatilized) for 24 h at 478C did not eliminate viable E. coli O157:H7 cells. Unfortunately, the enhanced effectiveness of AIT in killing the pathogen on wet alfalfa seeds is offset by a dramatic reduction in seed viability. Nevertheless, the use of AIT as an alternative to chlorine for the purpose of killing E. coli O157:H7 and perhaps other pathogens on alfalfa seed holds promise. Factors that may influence conditions rendering increased sensitivity of E. coli O157:H7 to AIT without compromising seed viability should be investigated.  2000 Elsevier Science B.V. All rights reserved. Keywords: Escherichia coli O157:H7; Alfalfa seeds; Allyl isothiocyanate

1. Introduction *Corresponding author. Tel.: 1 1-770-412-4740; fax: 1 1-770229-3216. E-mail address: [email protected] (L.R. Beuchat)

Vegetable seeds harbor microorganisms from the time they are harvested through production of mature sprouts. Contamination with pathogenic bacteria can

0168-1605 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 99 )00202-0

14

C.M. Park et al. / International Journal of Food Microbiology 56 (2000) 13 – 20

occur at any point, from the field to the sprouting process, and during subsequent handling of sprouts until they are consumed. Populations of Salmonella (Jaquette et al., 1996) and Escherichia coli O157:H7 (Taormina and Beuchat, 1999a) have been reported to reach 10 6 –10 7 cfu / g of sprouts produced from contaminated seeds. Treatment of alfalfa seeds containing 10 2 –10 3 cfu of Salmonella Stanley per g with 1010 ppm chlorine failed to eliminate the pathogen (Jaquette et al., 1996). Taormina and Beuchat (1999b) examined the efficacy of a broad range of sanitizers, including chlorine, as well as heat treatment, in killing E. coli O157:H7 on alfalfa seeds. Treatment with 20,000 ppm chlorine failed to eliminate the pathogen on seeds containing 2.7 log 10 cfu / g. They concluded that removal of E. coli O157:H7 from alfalfa seeds subjected to chemical treatments is not completely successful because of the inaccessibility of treatment solutions to cells in crevices or cracks on the seed surface. Outbreaks of illness caused by Bacillus cereus (Portnoy et al., 1976; Harmon et al., 1987) and salmonellae (O’Mahony et al., 1989; Jerngklinchan and Saitanu, 1993; Ponka et al., 1995; Mahon et al., 1997; Van Beneden et al., 1999) have been associated with raw seed sprouts. Outbreaks of E. coli O157:H7 infection in Japan have been associated with radish sprouts (Gutierrez, 1997; National Institutes of Infectious Diseases and Infectious Disease Control Division, Ministry of Health and Welfare of Japan, 1997). More recently, E. coli O157:H7 infections have been linked to alfalfa sprouts (Centers for Disease Control and Prevention, 1997). Infections associated with eating seed sprouts were recently reviewed by Taormina et al. (1999). Essential oils and essences of many herbs and spices are known to have antimicrobial activity. Inhibition of growth of E. coli by thyme oil has been reported (Farag et al., 1989; Manou et al., 1998; Smith-Palmer et al., 1998). Eugenol, alone or combined with monolaurin and sodium citrate (Blaszyk and Holley, 1998), and C 6 compounds such as (E)-2hexenal (Deng et al., 1993) inhibit the growth of E. coli. o-Ethylphenol inhibits verotoxin production by verotoxigenic E. coli (Sakagami et al., 1999). Treatment of strawberries (Moline et al., 1997), celery, and peppers (Buta and Moline, 1998) with vapors of methyl jasmonate, which occurs widely in the plant kingdom (Sembder and Parthier, 1993), extends

shelf life by inhibiting microbial growth. Fumigation of coleslaw (Delaquis et al., 1997) and stonefruit (Sholberg and Gaunce, 1996) with vaporized acetic acid has been shown to be effective in controlling the growth of microorganisms. Allyl isothiocyanate (AIT) results from hydrolysis of glucosinolates by myrosinase in cruciferous plants, including mustard and horseradish. Although the antimicrobial activity of AIT varies widely (Delaquis and Mazza, 1995), the volatile compound has been shown to inhibit the growth of E. coli (Isshiki et al., 1992; Kyung and Fleming, 1997), including serotype O157:H7 (Delaquis and Sholberg, 1997). Vaporized essential oil of horseradish strongly inhibited Pseudomonas and Enterobacteriaceae on roast beef (Delaquis et al., 1999). The hypothesis that hydrophobic aqueous solutions of chemicals are minimally effective in killing human pathogens on alfalfa seed because they do not come in contact, at least in an active form, with cells lodged in cracks and crevices gave impetus to evaluating volatile plant components for their efficacy in killing E. coli O157:H7. It was surmised that volatile compounds, particularly AIT, which are potentially lethal to microorganisms, could more easily reach cells of E. coli O157:H7 in areas otherwise protected from contact with aqueous solutions. Results of these experiments are presented here.

2. Materials and methods

2.1. Bacterial strains used Four strains of E. coli O157:H7 (932, human isolate; 994, salami isolate; E0018, calf fecal isolate; and H1730, human isolate) were used. Each strain was grown in 10 ml of tryptic soy broth (TSB, pH 7.3; Difco, Detroit, MI, USA) at 378C for 24 h. Cells used in seed studies were adapted to grow in TSB containing nalidixic acid (50 mg / ml) (Sigma, St. Louis, MO, USA) to minimize colony development by naturally occurring microflora on enumeration media. A mixture of approximately equal populations of each strain was used in experiments to evaluate the efficacy of volatile plant essences in killing the pathogen on agar disks and alfalfa seeds.

C.M. Park et al. / International Journal of Food Microbiology 56 (2000) 13 – 20

2.2. Inoculation of alfalfa seeds with E. coli O157: H7 The procedure for inoculation of alfalfa seeds with a four-strain mixture of nalidixic acid-resistant E. coli O157:H7 was described by Taormina and Beuchat (1999b). Inoculated dried seeds were stored at 48C for at least 5 days before being used in studies to determine the bactericidal effect of AIT.

2.3. Determination of bactericidal activity of volatile plant essences using an agar disk assay AIT, eugenol, carvacrol, linalool, and methyl jasmonate (Sigma) were examined for their ability to kill E. coli O157:H7 inoculated onto agar disks using a modified procedure described by Delaquis and Sholberg (1997). Tryptic soy agar (TSA, pH 7.3, Difco) disks (13 mm diameter 3 3 mm) were prepared using a sterile cork borer and placed on a sterile glass microscope slide. A four-strain mixture of E. coli O157:H7 was serially diluted in sterile 0.1% peptone water. Diluted cell suspensions (10 ml) were deposited on the top of agar disks. Two glass slides, each containing seven agar disks, were then placed in a sterile 950-cc glass jar. Up to 50 ml of canola oil (control) or volatile plant compounds in sterile aluminum cups (10 mm diameter 3 7 mm) were immediately placed in separate jars which were then sealed with lids. Disks incubated at 20, 37 or 478C for 2, 5, 9, 24 or 48 h were examined for colonies of E. coli O157:H7. Disks on which colonies were not detected were placed in covered petri dishes and incubated in the absence of volatile plant compounds at 378C for an additional 48 h. Disks were again examined for colonies of E. coli O157:H7. Counts before or after the additional 48-h incubation period were used to calculate bactericidal activity as log 10 (Nc 2 Nt ), where Nc is the number of colonies on the control agar disk and Nt is the number colonies formed on disks exposed to test compounds.

2.4. Procedure to determine effectiveness of AIT in killing E. coli O157: H7 and mesophilic aerobic microorganisms on alfalfa seeds Inoculated dry (ca. 6.8% moisture) seeds (2 g) and wet (ca. 22.5% moisture) seeds were used. Wetting

15

was achieved by submerging 2 g of inoculated dry seeds in 300 ml of sterile deionized water (238C) for 10 s. The dry or wet seeds were placed in a single layer in a 950-cc glass jar. Sterile aluminum cups containing up to 50 ml of AIT or canola oil (control) were placed in separate jars which were then sealed with lids. Jars were held at 20, 37 or 478C for 6, 9, 12 or 24 h. On termination of treatment, jars from which lids were removed were placed in a laminar flow hood for 10 min before seeds were removed for microbiological analysis and testing for germination. Treated seeds (1.8 g) were combined with 10 ml of modified TSB (mTSB) (Padhye and Doyle, 1991) supplemented with nalidixic acid (50 mg / ml) (mTSBN) in a stomacher bag, pummeled for 1 min, allowed to set for 10 min, and again pummeled for 1 min. The mTSBN wash was surface plated in duplicate (0.1 ml) and quadruplicate (0.25 ml), and serially diluted in sterile 0.1% peptone water and plated in duplicate (0.1 ml) on sorbitol MacConkey agar supplemented with nalidixic acid (50 mg / ml) (SMAN) and TSA to enumerate E. coli O157:H7 and aerobic microorganisms, respectively. Presumptive colonies of E. coli O157:H7 were counted after incubating SMAN plates at 378C for 24 h; colonies formed on TSA were counted after incubating plates at 308C for 48 h. The pummeled mixture of seeds and mTSBN was incubated at 378C for 24 h. The resulting culture was streaked on SMAN and the plates were incubated at 378C for 24 h. Selected presumptive E. coli O157:H7 colonies were subjected to E. coli O157 latex agglutination test (Unipath– Oxoid, Columbia, MD, USA) and API 20E diagnostic tests (BioMerieux, Hazelwood, MO, USA) for confirmation.

2.5. Procedure for determining viability of seeds Approximately 100 seeds exposed to AIT were placed between two pieces of water-saturated filter paper in a petri dish and incubated at 308C for 3 days. The filter paper was moistened daily by application of water. The number of seeds developing a hypocotyl was counted and the percentage of seeds that germinated was calculated. Seeds were then enriched for E. coli O157:H7 by placing in 10 ml of mTSBN in a stomacher bag, pummeling for 1 min, and incubating for 24 h at 378C. Confirmation of presumptive colonies on SMAN plates streaked

16

C.M. Park et al. / International Journal of Food Microbiology 56 (2000) 13 – 20

with the enriched culture was done as described above.

2.6. Statistical analysis At least two replicates of each experiment were conducted and duplicate samples were plated on recovery media. Data from experiments using the agar disk method were subjected to the Statistical Analysis System (SAS Institute, Cary, NC, USA) for analysis of variance and to the Duncan’s multiple range test.

3. Results and discussion The number of colonies formed by E. coli O157:H7 on agar disks was not inhibited by exposure to 50 ml of eugenol, carvacrol, linalool, or methyl jasmonate in a 950-cc jar. The combined presence of 50 ml each of eugenol, carvacrol, and linalool also failed to inhibit colony development on agar disks. Other researchers have observed that eugenol inhibits the growth of E. coli O157:H7 (Blaszyk and Holley, 1998) and other gram-negative bacteria (Karapinar and Aktug, 1987). Carvacrol, a major volatile component in the essential oil of thyme and oregano, has been shown to inhibit E. coli O157:H7 (Kim et al., 1995) and Bacillus cereus (Ultee et al., 1998), and linalool, a main volatile

component in sweet basil oil, is active against a wide range of foodborne microorganisms (Lachowicz et al., 1998; Wan et al., 1998). Differences in the effectiveness of plant essences against microorganisms may be due to different test methods used in various laboratories. The concentration of plant compounds used in our study was low compared to concentrations used by other researchers. If 50 mg of test compound completely volatilized in the 950-cc jar, a concentration of 53 mg of AIT / cc (53 ppm) in the atmosphere surrounding the seeds would result. Thus, the ineffectiveness of eugenol, carvacrol, linalool, and methyl jasmonate in killing E. coli O157:H7 may have been due to their low concentrations. AIT, on the other hand, was lethal to E. coli O157:H7 inoculated onto agar disks. Fig. 1 shows the number of E. coli O157:H7 killed when cells were inoculated onto TSA disks and exposed to 4, 6, 8 or 10 ml of AIT in a 950-cc jar at 20 or 378C for 48 h. If completely volatilized, these volumes would result, respectively, in 4.2, 6.3, 8.4 and 10.5 mg of AIT / cc or air in the jar. Incubation of disks at 378C in a 950-cc jar containing 8 ml of AIT resulted in more than a 7-log 10 reduction in population of E. coli O157:H7. Significant reductions were also observed when disks were sealed in jars containing 4 or 6 ml of AIT compared to 2 ml (not shown), which was not lethal to the pathogen. At 208C, the lethality of AIT was substantially less, although significant

Fig. 1. Lethal effect of allyl isothiocyanate (AIT) on E. coli O157:H7. Inoculated agar disks were exposed to 4, 6, 8 or 10 ml of AIT in a 950-cc jar at 20 or 378C for 48 h. Within each incubation temperature, bars noted with the same letter are not significantly different (P # 0.05).

C.M. Park et al. / International Journal of Food Microbiology 56 (2000) 13 – 20

reduction occurred when disks were exposed to 8 or 10 ml of AIT compared to 4 or 6 ml, and when exposed to 4 or 6 ml compared to 2 ml. At 8 ml, AIT did not kill all of the E. coli O157:H7 cells inoculated on agar disks; subsequent incubation at 378C for 48 h in an atmosphere free of AIT resulted in growth of the pathogen. Colonies developed slowly, however, indicating that exposure of cells to AIT may have caused sublethal injury. A test volume of 2 ml inhibited colony development at 20 or 378C for 48 h, but was not lethal. Fig. 2 shows the lethal activity of AIT (10 ml in a

17

950-cc jar) on E. coli O157:H7 on agar disks as influenced by exposure time. Exposure of E. coli O157:H7 for up to 5 h at 378C or 24 h at 208C resulted in reductions in numbers of viable cells by less than 1 log 10 . Exposure to 10 ml of AIT in a 950-cc jar for 24 h at 378C resulted in more than a 7-log 10 reduction. Lethality was greatly diminished at 208C, where only a 2-log 10 reduction occurred after exposure to 10 ml AIT for 48 h. The effect of temperature on lethal activity of AIT (10 ml in a 950-cc jar) is further illustrated in Fig. 3. Less than a 1-log 10 reduction occurred after 2 or 5 h

Fig. 2. Lethal effect of allyl isothiocyanate (AIT) on E. coli O157:H7. Inoculated agar disks were exposed to 10 ml of AIT at 20 or 378C for 2, 5, 7, 24 or 48 h. Within each incubation temperature, bars noted with the same letter are not significantly different (P # 0.05).

Fig. 3. Lethal effect of allyl isothiocyanate (AIT) on E. coli O157:H7. Inoculated agar disks were exposed to 10 ml of AIT at 20, 37 and 478C for 2 or 5 h. Within each incubation time, bars noted with the same letter are not significantly different (P # 0.05).

C.M. Park et al. / International Journal of Food Microbiology 56 (2000) 13 – 20

18

at 20 or 378C, although lethality was significantly greater after 2 h at 378C compared to 2 h at 208C. Treatment for 5 h at 478C resulted in death of 6.1 log 10 cfu on agar disks. Inactivation of E. coli O157:H7 on alfalfa seeds as affected by moisture content and temperature was investigated. Initial populations of E. coli O157:H7 on inoculated dry and wet seeds were 2.9 and 2.7 log 10 cfu / g, respectively. Table 1 lists populations of E. coli O157:H7 on dry and wet seeds exposed to 50 ml of AIT in a 950-cc jar at 25, 37 and 478C for 24 h. The pathogen was not recovered by direct plating ( , 0.70 log 10 cfu / g) or enrichment of wet seeds held at 37 or 478C. However, E. coli O157:H7 was recovered from wet seeds held at 258C, supporting observations that AIT activity diminishes with decreased temperatures (Figs. 1–3). Reduced activity may be due, in part, to lower amounts of AIT being volatilized at decreased temperatures. AIT is clearly more effective in killing E. coli O157:H7 on wet seeds compared to dry seeds. Enrichment of all samples of treated and untreated dry seeds revealed the presence of E. coli O157:H7. Even treatment with 100 ml of AIT at 478C for 24 h did not eliminate E. coli O157:H7 from dry seeds (data not shown). Unfortunately, exposure of wet seed to an atmosphere containing 50 ml of AIT per 950 cc also

resulted in a drastic reduction in the percentage of seeds capable of germinating (Table 1). The lethal mode of action of AIT against microbial cells is thought to involve respiratory inhibition (Kojima and Ogawa, 1971). AIT may also adversely affect respiratory mechanisms of the alfalfa seed. Table 2 shows populations and observations on presence / absence of E. coli O157:H7 on wet alfalfa seeds treated at 37 or 478C with AIT for up to 24 h. While treatment with either 20 or 40 ml of AIT in a 950-cc jar reduced populations to undetectable levels, consistent differences in effectiveness of 40 versus 20 ml of AIT are not evident. At 40 ml, AIT did not eliminate an initial population of 2.7 log 10 cfu / g of E. coli O157:H7 per g within 24 h, regardless of incubation temperature. Treatment with AIT tended to reduce seed germination percentage; reductions were greater in seeds treated with 40 ml compared to 20 ml of AIT (Table 2) but not as great as in wet seeds treated with 50 ml AIT (Table 1). Populations of aerobic microorganisms were not markedly decreased upon exposure to AIT (Table 2). The inconsistent lethal effects of 20 and 40 ml of AIT on E. coli O157:H7 inoculated onto seeds compared to those on agar disks are attributed in part to inaccessibility of volatile AIT to E. coli O157:H7 cells on seeds and to differences in moisture content and chemical composition of agar and seed. In

Table 1 Combined effects of AIT (50 ml in 950-cc jar), temperature, and moisture content on inactivation of E. coli O157:H7 on alfalfa seeds held at 25, 37 or 478C for 24 h Temperature (8C)

Seed condition

AIT (ml)

25

Dry

0 50

Wet

37

47

1

Population (log 10 cfu / g)

Enriched seed 1

Germination (%)

1.39 1.00

2 2

88 79

0 50

4.38 , 0.70

2 1

90 3

Dry

0 50

1.24 , 0.70

2 2

84 80

Wet

0 50

3.72 , 0.70

2 0

88 1

Dry

0 50

, 0.70 , 0.70

2 2

77 76

Wet

0 50

, 0.70 , 0.70

2 0

83 0

Number of samples out of two analyzed from two replicate trials in which E. coli O157:H7 was recovered by enrichment.

C.M. Park et al. / International Journal of Food Microbiology 56 (2000) 13 – 20

19

Table 2 Efficacy of AIT in killing aerobic microorganisms and E. coli O157:H7 on wet alfalfa seed Exposure time (h) 6

9

Exposure temperature (8C)

AIT (ml)

37

47

Enriched seeds 2

Germination (%)

nd / nd 3 1.5 / nd 1.2 / nd

1/1 1/1 1/1

89 82 80

4.1 a 3.0 a 2.9 a

1.3 / nd 1.0 / nd nd / nd

1/1 1/1 2/2

86 83 57

2/2

0 20 40

5.8 a 4.2 b 4.1 b

nd / 1.9 nd / 1.0 nd / nd

1 1/1 2/2

89 70 60

2/1

0 20 40

4.9 a 3.0 b 3.9 ab

nd / nd nd / nd nd / nd

1/1 1/1 2/2

91 85 68

2/1

37

0 20 40

4.7 a 3.2 b 3.6 ab

nd / 1.7 nd / 0.7 nd / 1.0

1 1/1 1/1

87 79 83

47

0 20 40

4.0 a 3.1 a 2.1 a

nd / 0.7 nd / nd nd / nd

1/1 1/2 2/2

85 84 68

2 2/1

0 20 40

3.4 b 4.4 a 2.5 c

nd / nd nd / nd nd / nd

1/1 1/2 2/2

83 75 57

1 2/2

0 20 40

3.6 a 2.7 a 2.0 a

nd / nd nd / nd nd / nd

1/1 2/2 1/2

82 65 78

2/2 2

37

47

12

24

37

47

Population (log 10 cfu / g) Aerobic microorganisms 1

E. coli O157:H7

0 20 40

5.3 a 4.9 a 4.3 a

0 20 40

1 Mean values in the same column, within the same exposure time and significantly different (P # 0.05); detection limit was 25 (log 10 1.4) cfu / g of 2 E. coli O157:H7 was detected ( 1 ) or not detected (2) by enrichment of indicates that enrichment was not done. 3 Not detected by direct plating [ , 5 ( , log 10 0.7) cfu / g of seed]; values

addition to moisture content of seeds, exposure temperature, treatment time, and factors such as physiological age of E. coli O157:H7 cells may influence their sensitivity to AIT. Factors that affect the efficacy of AIT in killing E. coli O157:H7 on alfalfa seeds warrant further investigation.

References Blaszyk, M., Holley, R.A., 1998. Interaction of monolaurin, eugenol and sodium citrate on growth of common meat spoilage and pathogenic organisms. Int. J. Food Microbiol. 39, 175–183.

Enriched germinated seeds 2

temperature, that are followed by the same letter are not seed. seeds in two separate trials (trial 1 / trial 2); absence of data are from two trials (trial 1 / trial 2).

Buta, J.G., Moline, H.E., 1998. Methyl jasmonate extends shelf life and reduces microbial contamination of fresh-cut celery and peppers. J. Agric. Food Chem. 46, 1253–1256. Centers for Disease Control and Prevention, 1997. Outbreak of Escherichia coli O157:H7 infection associated with eating alfalfa sprouts – Michigan and Virginia, June–July 1997. Morbid. Mortal. Weekly Rpt. 46, 741–744. Delaquis, P.J., Graham, H.S., Hocking, R., 1997. Shelf-life of coleslaw made from shredded cabbage fumigated with vaporized acetic acid. J. Food Process. Preserv. 21, 129–140. Delaquis, P.J., Mazza, G., 1995. Antimicrobial properties of isothiocyanates and their role in food preservation. Food Technol. 49 (11), 73–84. Delaquis, P.J., Sholberg, P.L., 1997. Antibacterial activity of gaseous allyl isothiocyanate. J. Food Prot. 60, 943–947. Delaquis, P.J., Ward, S.M., Holley, R.A., Cliff, M.C., Mazza, G., 1999. Microbiological, chemical and sensory properties of

20

C.M. Park et al. / International Journal of Food Microbiology 56 (2000) 13 – 20

pre-cooked roast beef preserved with horseradish essential oil. J. Food Sci. 64, 519–524. Deng, W., Hamilton-Kemp, T.R., Nielsen, M.T., Anderson, R.A., Collins, G.B., Hilderbrand, D.F., 1993. Effects of six carbon aldehydes and alcohols on bacterial proliferation. J. Agric. Food Chem. 41, 506–510. Farag, R.S., Daw, Z.Y., Hewedi, F.M., El-Baroty, G.S.A., 1989. Antimicrobial activity of some Egyptian spice essential oils. J. Food Prot. 52, 665–667. Gutierrez, E., 1997. Japan prepares as O157 strikes again. Lancet 349, 1156. Harmon, S.M., Kautter, D.A., Solomon, H.M., 1987. Bacillus cereus contamination of seeds and vegetable sprouts in home sprouting kit. J. Food Prot. 50, 62–65. Isshiki, K., Tokuoka, K., Mori, R., Chiba, S., 1992. Preliminary examination of allyl isothiocyanate vapor for food preservation. Biosci. Biotech. Biochem. 56, 1476–1477. Jaquette, C.B., Beuchat, L.R., Mahon, B.E., 1996. Efficacy of chlorine and heat treatment in killing Salmonella stanley inoculated onto alfalfa seeds and growth and survival of the pathogen during sprouting and storage. Appl. Environ. Microbiol. 62, 2212–2215. Jerngklinchan, J., Saitanu, K., 1993. The occurrence of salmonellae in bean sprouts in Thailand. Southeast Asian J. Trop. Med. Pub. Health 24, 114–118. Karapinar, M., Aktug, S.E., 1987. Inhibition of foodborne pathogens by thymol, eugenol, menthol and anethole. Int. J. Food Microbiol. 4, 161–166. Kim, J.M., Marshall, M.R., Wei, C.I., 1995. Antibacterial activity of some essential oil components against five foodborne pathogens. J. Agric. Food Chem. 43, 2839–2845. Kojima, M., Ogawa, K., 1971. Studies on the effects of isothiocyanates and their analogues on microorganisms. I. Effect of isothiocyanates on the oxygen uptake of yeasts. J. Ferment. Technol. 49, 740–746. Kyung, K.H., Fleming, H.P., 1997. Antimicrobial activity of sulfur compounds derived from cabbage. J. Food Prot. 60, 71. Lachowicz, K.J., Jones, G.P., Briggs, D.R., Bienvenu, F.E., Wan, J., Wilcock, A., Conventry, M.J., 1998. The synergistic preservative effects of essential oils of sweet basil (Ocimum basilicum L.) against acid-tolerant food microflora. Lett. Appl. Microbiol. 26, 209–214. Mahon, B.E., Ponka, A., Hall, W.N., Komatsu, K., Dietrich, S.E., Sittonen, A., Cage, G., Hayes, P.S., Lambert-Fair, M.A., Bean, N.H., Griffin, P.M., Slutsker, L., 1997. An international outbreak of Salmonella infections caused by alfalfa sprouts grown from contaminated seeds. J. Infect. Dis. 175, 876–882. Manou, I., Bouillard, L., Devleeschouwer, M.J., Barel, A.O., 1998. Evaluation of the preservative properties of Thymus vulgaris essential oil in tropically applied formulations under a challenge test. J. Appl. Microbiol. 84, 368–376. Moline, H.E., Buta, J.G., Saftner, R.A., 1997. Comparison of three volatile natural products for the reduction of postharvest decay in strawberries. Adv. Strawberry Res. 16, 43–48. National Institute of Infectious Diseases and Infectious Disease

Control Division, Ministry of Health and Welfare of Japan, Ministry of Health and Welfare of Japan, 1997. Verocytotoxinproducing Escherichia coli (enterohemorrhagic E. coli) infection, Japan, 1996–June 1997. Infect. Agents Surveillance Rep. 18, 153–154. O’Mahony, M., Cowden, J., Smyth, B., Lynch, D., Hall, M., Rowe, B., Teare, E.L., Tettmar, R.E., Rampling, A.M., Coles, M., Gilbert, R.J., Kingcott, E., Barlett, C.L.R., 1989. An outbreak of Salmonella Saint-Paul associated with beansprouts. Epidemiol. Infect. 104, 229–235. Padhye, N.V., Doyle, M.P., 1991. Rapid procedure for detecting enterohaemorrhagic Escherichia coli O157:H7 in food. Appl. Environ. Microbiol. 57, 2693–2698. Ponka, A., Anderson, Y., Sitonen, A., deJong, B., Jahkola, M., Haikala, O., Kuhmonen, A., Pakkala, P., 1995. Salmonella in alfalfa sprouts. Lancet 345, 462–463. Portnoy, B.L., Goepfert, J.M., Harmon, S.M., 1976. An outbreak of Bacillus cereus food poisoning resulting from contaminated vegetable sprouts. Am. J. Epidemiol. 103, 589–594. Sakagami, Y., Ichise, R., Kajimura, K., Yokoyama, H., 1999. Inhibitory effect of creosote and its main components on production of verotoxin of enterohaemorrhagic Escherichia coli O157:H7. Lett. Appl. Microbiol. 28, 118–120. Sembder, G., Parthier, B., 1993. The biochemistry and the physiological and molecular actions of jasmonates. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44, 569–589. Sholberg, P.L., Gaunce, A.P., 1996. Fumigation of stonefruit with acetic acid to control postharvest decay. Crop Prot. 15, 681– 686. Smith-Palmer, A., Stewart, J., Fyfe, L., 1998. Antimicrobial properties of plant essential oils and essences against five important food-borne pathogens. Lett. Appl. Microbiol. 26, 118–122. Taormina, P.J., Beuchat, L.R., 1999a. Behavior of enterohemorrhagic Escherichia coli O157:H7 on alfalfa sprouts during the sprouting process as influenced by treatment with various chemicals. J. Food Prot. 62, 850–856. Taormina, P.J., Beuchat, L.R., 1999b. Comparison of chemical treatments to eliminate enterohemorrhagic Escherichia coli O157:H7 on alfalfa seeds. J. Food Prot. 62, 318–324. Taormina, P.J., Beuchat, L.R., Slutsker, L., 1999. Infections associated with eating seed sprouts: an international concern. Emerg. Infect. Dis. 5, 626–634. Ultee, A., Gorris, L.G.M., Smid, E.J., 1998. Bactericidal activity of carvacrol towards the food-borne pathogen Bacillus cereus. J. Appl. Microbiol. 85, 211–218. Van Beneden, C.A., Keene, W.E., Strang, R.A., Weiker, D.H., King, A.S., Mahon, B., Hedberg, K., Bell, A., Kelly, M.T., Balan, V.K., MacKenzie, W.R., Fleming, D., 1999. Multinational outbreak of Salmonella entericia serotype Newport infections due to contaminated alfalfa sprouts. J. Am. Med. Assoc. 281, 158–162. Wan, J., Wilcock, A., Coventry, M.J., 1998. The effect of essential oils of basil on the growth of Aeromonas hydrophila and Pseudomonas fluorescens. J. Appl. Microbiol. 84, 152–158.