Improved in vitro detection of hemolysin BL from Bacillus cereus

International Journal of Food Microbiology 57 (2000) 1–8 www.elsevier.nl / locate / ijfoodmicro

Improved in vitro detection of hemolysin BL from Bacillus cereus 1

Christophe Fermanian , Amy C.L. Wong* Food Research Institute, University of Wisconsin — Department of Food Microbiology and Toxicology, 1925 Willow Dr., Madison, WI 53706, USA Received 8 September 1999; accepted 5 January 2000

Abstract The production of a discontinuous hemolysis pattern that is characteristic of hemolysin BL, an enterotoxic and dermonecrotic hemolysin previously described, was investigated with 114 Bacillus cereus, two B. thuringiensis and nine B. mycoides strains. Discontinuous patterns were monitored by a blood agar gel diffusion assay of 5 h and overnight culture supernatants, and by direct examination after colony growth on blood agar. For gel diffusion, the condition for optimal discontinuous pattern development included the use of sheep blood agar containing 1–2 mM EDTA, the addition of calf serum (8% v / v) to supernatants before loading the gels, and incubating at 278C; under these conditions the patterns generally appeared within 4 h of incubation. The final percent of strains exhibiting a pattern was 74–78%. For the colony system, two types of blood agar were used: nutrient agar (NA) and brain heart infusion agar with glucose (BHIG) containing 8% calf serum and 0.1–1 mM EDTA. Both NA and BHIG, containing 0.1 mM EDTA and incubated at 228C, gave the highest percent of strains exhibiting a pattern. The patterns generally appeared between 12 and 28 h. Used in combination, NA and BHIG gave 73–74% of positive B. cereus /thuringiensis. Previous results obtained with 68 strains (Beecher and Wong, 1994a,b) were confirmed; the gel diffusion system was improved regarding homogeneity of time range of development, clarity, stability and final yield of patterns; the conditions for the colony growth system were chosen to make monitoring compatible with a laboratory working schedule. In both cases central hemolytic zones, that can hamper the observation of the patterns, were decreased. It did not seem that B. cereus, B. thuringiensis and B. mycoides can be differentiated on the basis of production of hemolysin BL.  2000 Elsevier Science B.V. All rights reserved. Keywords: Hemolysin BL; Bacillus cereus; Gel diffusion; Colony growth; Blood agar

1. Introduction

*Corresponding author. Tel.: 1 1-608-263-1168; fax: 1 1-608263-1114. E-mail address: [email protected] (A.C.L. Wong) 1 Present address: Dr C. Fermanian, Laboratoire de biostatistique, ˆ ` Hopital Necker, 149 rue de Sevres, 75015 Paris, France.

Hemolysin BL (HBL) is a dermonecrotic diarrheal enterotoxin produced by Bacillus cereus (Beecher and Macmillan, 1991; Beecher and Wong, 1994a; Beecher et al., 1995a,b). It produces an unusual discontinuous hemolysis pattern (DH) on sheep or calf blood agar, which is diagnostic of HBL. Two

0168-1605 / 00 / $ – see front matter  2000 Elsevier Science B.V. All rights reserved. PII: S0168-1605( 00 )00226-9

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simple methods using blood agar media for detection of HBL in culture supernatants and from colony growth have been developed (Beecher and Wong, 1994b). However, in crude samples, in addition to the DH which develops a few millimeters away from the HBL source, a central continuous hemolytic pattern (CH) is frequently observed. With time, the CH will expand and may obscure the DH. The cause of the CH is currently unknown. B. cereus produces several other hemolysins and enzymes that individually or cooperatively may produce this effect. These include: (i) cereolysin or hemolysin I (Cowell et al., 1976), (ii) hemolysin II (Coolbaugh and Williams, 1978; Sinev et al., 1993), (iii) hemolysin III (Baida and Kuzmin, 1995), (iv) cereolysin AB, composed of phosphatidylcholinespecific phospholipase C (PC-PLC) and sphingomyelinase (SMase) (Gilmore et al., 1989), and (v) the lysin described by Matsuyama et al. (1995). Minimization of CH development would enhance DH detection, especially with weak producers of HBL. Our objective in this study was to modify the blood agar assays previously developed by Beecher and Wong (1994b) to optimize DH detection. Modifications included variation of blood agar composition and use of metal chelators and inhibitors of proteases, cereolysin and SMase. Other parameters such as incubation temperature and time, and time of harvest of supernatants for the gel diffusion assay, were also investigated.

2. Materials and methods

2.1. Bacterial strains A total of 114 B. cereus, two B. thuringiensis and nine B. mycoides isolates were used. They were isolated from dairy and egg products, rice, vanilla pudding, potatoes, cattle feed, soil, diarrheal and emetic outbreaks, and nongastrointestinal infections. The suppliers were: D.J. Beecher, M.S. Bergdoll, C. Fermanian, E.B. Somers, University of Wisconsin, Madison, USA; J.A. Buswell, The Chinese University of Hong Kong, Hong Kong; M.S. Gilmore, University of Oklahoma, Oklahoma City, USA; Per Einar Granum, Norwegian College of Veterinary Medicine, Oslo, Norway; M.W. Griffiths, University

of Guelph, Guelph, Canada; J.L. Kornacki, Silliker Laboratories, Madison, USA; J.M. Kramer, Central Public Health Laboratory, London, England; P.-L. Makinen, University of Michigan, Ann Arbor, USA; T. Pirhonen, State Control Office for Dairy Products, Helsinki, Finland; K. Shinagawa, Iwate University, Iwate, Japan. Preliminary experiments to determine optimal conditions were performed with nine strains: F4433 / 73, B-4ac, 29.155 (diarrheal outbreaks); F837 / 76 (postoperative infection); S1C (soil); 3505M (steamheated potatoes); F4581 / 76L, F4581 / 76R (leg infection); and Soc67 (periodontitis). These strains have been described elsewhere (Beecher and Wong, 1994b; Fermanian et al., 1996). Culture stocks were maintained on tryptone soja agar (TSA; Oxoid, Basingstoke, UK). Seed cultures were prepared by transferring a loopful of culture to brain heart infusion (Difco Laboratories, Detroit, MI, USA) plus glucose 0.1% (BHIG) and incubating at 328C for 16–18 h. Seed cultures were used directly for the colony growth assay. For the gel diffusion assay, flasks containing 50 ml of BHIG were inoculated with 1% (v / v) of the appropriate seed cultures and incubated at 328C on a rotary shaker at 200 rpm. Cultures were harvested after 5 h of growth, cells were removed by centrifugation and the supernatants stored at 2 208C until testing by gel diffusion.

2.2. Colony growth assay Erythrocytes from defibrinated sheep, calf or pig blood (Crane Laboratories, Syracuse, NY, USA) were washed by centrifugation in 150 mM NaCl until the supernatant was colourless. Washed blood was resuspended in 150 mM NaCl to the initial volume of blood, and was added at a final concentration of 2.5 or 5% (v / v) to nutrient agar (Difco) supplemented with 150 mM NaCl. In some cases, the nutrient agar base was replaced by BHIG and 1.5% Bacto agar (Difco). When needed, calf serum (Sigma, St. Louis, MO, USA) was added to the blood agar base at a final concentration of 8% (v / v) unless specified otherwise. Cholesterol (final concentrations at 5, 20 and 80 mg / ml) was added as a 75 mg / ml solution in dioxane. Ethylenediamine tetraacetic acid, disodium salt (0.1, 0.5 and 1 mM, EDTA) was added to the agar before autoclaving; phenylmethylsulfonyl fluoride (200 mM, PMSF) was

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dissolved in 2-propanol and added to achieve a final concentration of 0.3 mM; parahydroxymercuribenzoate (10 mM, pHMB) was dissolved in 0.01 N NaOH and used at a final concentration of 0.1 mM. N-Ethyl-5-phenylisoxazolium-39-sulfonate (1 to 10 mM, Woodward’s reagent K; Tomita et al., 1993) was added as a 0.4 M solution in distilled water. All chemicals were obtained from Sigma. Blood agar plates were spot inoculated with seed cultures of the appropriate strains as described by Beecher and Wong (1994b) and incubated at 228C unless noted otherwise. Development of CH and DH was monitored beginning at 12 h after inoculation, and at specified intervals thereafter.

2.3. Gel diffusion assay Erythrocytes were prepared as described above and used at a final concentration of 2.5 or 5% (v / v) in 1% noble agar (Difco) in 50 mM Tris–HCl, 150 mM NaCl (pH 7.4). Molten agar was poured into 10 cm Petri plates to 0.15 ml / cm 2 . When needed EDTA, PMSF, pHMB and Woodward’s reagent K were prepared and added as described above. In addition, the following reagents (all obtained from Sigma) were tested: ethyleneglycol-bis-(b-aminoethyl ether) tetraacetic acid (final concentrations 0.03 to 10 mM, EGTA) was added as a 100 mg / ml solution in 1 M NaOH; 1,10-phenanthroline (1 and 10 mM) was added as a 1 M solution in ethanol; Na citrate (0.1 to 30 mM) was added as an aqueous solution; Na iodoacetate (1 mM) as a 0.1 M solution in physiological saline; and calf serum was added to supernatant samples at 8% unless indicated otherwise. Samples (10 ml) were loaded in 3 mm wells in the blood agar. Gels were incubated at 278C (unless noted otherwise) and monitored for CH and DH development beginning 1 h after sample loading and at specified times thereafter.

3. Results

3.1. Colony growth on blood agar Sheep, calf and pig blood were tested at concentrations of 2.5 and 5% (v / v) in a nutrient agar base. No DH was detected on pig blood agar. DH developed around colonies on both sheep and calf

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blood agar plates, but the patterns were more distinct with sheep blood. When the sheep blood concentration was increased from 2.5 to 5%, the development of DH was more rapid and the discontinuous pattern more distinct. With calf blood, only the clarity of the pattern was improved. Sheep blood (5%) was used in subsequent experiments. Beecher and Wong (1994b) reported that addition of 2% calf serum to the blood agar enhanced the DH. We tested the addition of 0.5, 1, 2, 4, and 8%, and observed that the diameter of the CH was decreased with increasing calf serum concentration, with 8% giving the most distinct DH. Cholesterol, a serum component, is an inhibitor of cereolysin, which may contribute to the CH, hence we examined the effect of cholesterol on CH and DH development. At $ 80 mg / ml, cholesterol retarded CH formation. However, as cholesterol is not water soluble, it formed precipitates in the blood agar, and also initiated small zones ( , 1 mm diameter) of nonspecific lysis of the red blood cells. Hence, the use of cholesterol was not pursued further. Since several hemolysins and enzymes produced by B. cereus are known to require cations for activity, we tested a chelator, EDTA, for its ability to reduce CH. Addition of 1 mM EDTA to the blood agar base with or without 8% calf serum inhibited growth of B. cereus slightly. However, CH development was also inhibited, and the clarity of the DH was enhanced. Three incubation temperatures (22, 27 and 308C) were tested when EDTA was used alone or in conjunction with serum, and 228C was determined to be optimal for DH detection. At this temperature, DH was usually detectable within 36 h with minimal interference from CH development. At 278C, the CH developed too fast occasionally and masked the DH. The DH was the least distinct and most transient at 308C. To determine if the use of protease inhibitors would enhance the effect of EDTA, pHMB (0.1 mM) and PMSF (0.3 mM), inhibitors of cysteine and serine proteases, respectively, were added to blood agar containing 1 mM EDTA. A pattern-initiating effect was observed for some strains having very low level of HBL activity. Woodward’s reagent K, a specific SMase inhibitor, was also tested at 2.8 and 8.5 mM in blood agar. Its effect was found to be not as pronounced as the effect of EDTA. Results obtained so far with nine B. cereus isolates

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indicated that incorporation of 8% calf serum to the blood agar base inhibited CH and enhanced DH development. Addition of 1 mM EDTA further decreased CH formation, however, colony growth was inhibited. We therefore tested the effectiveness of 8% serum with three concentrations of EDTA (0.1, 0.5 and 1 mM) on DH detection in 114 B. cereus strains. A summary of the results are shown in Table 1. Using 0.1 mM EDTA permitted detection of the highest number of HBL producers. Of 114 isolates, 70, 52 and 46% produced the DH on blood agar containing 0.1, 0.5 and 1 mM EDTA, respectively. A concentration of 0.1 mM EDTA was less inhibitory for growth than the two higher concentrations but still inhibited CH development sufficiently for the DH to be easily detected. When BHIG instead of nutrient agar was used as the base, 62, 52 and 44% of the isolates produced the DH with 0.1, 0.5 and 1 mM EDTA, respectively. We also recorded the number of positive strains after grouping the methods. The cluster N 0.1 1 B 0.1 (media containing 0.1 mM EDTA) yielded a higher number of positive strains than the clusters N 0.5 1 B 0.5 and N 1 1 B 1 (Table 2).

3.2. Gel diffusion in blood agar As observed in the colony growth method, a clearer DH was produced in sheep blood compared to calf blood, and increasing the blood concentration from 2.5 to 5% permitted a more rapid development of the DH. Calf serum (2, 4 and 8%) added to

Table 2 Colony growth — comparison of media containing 0.1, 0.5 or 1 mM EDTA (after clustering of the media for each concentration)a Cluster of media

N 0.1 1 B 0.1 N 0.5 1 B 0.5 N1 1 B1

Number of strains exhibiting a DH

Percent

Set 1

Set 2

Set 1

Set 2

86 66 58

85 65 56

74.1 56.9 50.0

73.3 56.0 48.3

a Strains and cultivation methods were as in Table 1. Within a given cluster, a given strain was recorded as positive if at least one of the methods clustered led to a dis-continuous hemolysis (DH). Otherwise, the strain was recorded as negative. For example in the cluster ‘‘N 0.1 1 B 0.1 ’’, if only medium N 0.1 gave a DH, the strain was assigned a positive response. DH, discontinuous hemolysis.

culture supernatants inhibited CH development, and enhanced the DH, with 8% serum being the most effective. When 1 or 2 mM EDTA was added to the gel diffusion agar, it was effective in inhibiting CH, but did not have any effect when added to the culture supernatant. The inhibition was more pronounced than that observed with 8% serum in the culture supernatant. A combination of 1 mM EDTA and 8% serum permitted the development of a very clear and stable DH. Two other chelators, EGTA (0.03–10 mM) and Na citrate (0.1–30 mM), also inhibited CH development, but to a lesser degree than EDTA. In addition to pHMB and PMSF, two other protease inhibitors, Na iodoacetate (1 mM) and 1,10 phenanthroline (1

Table 1 Colony growth — comparison of NA and BHIG agar containing 0.1, 0.5 or 1 mM EDTAa Nutrient base

[EDTA] (mM)

Name of the medium

Number of strains exhibiting a DH

Percent

Set 1

Set 2

Set 1

Set 2

NA

0.1 0.5 1

N 0.1 N 0.5 N1

84 56 53

78 64 55

72.4 48.3 45.7

67.2 55.2 47.4

BHIG

0.1 0.5 1

B 0.1 B 0.5 B1

73 60 48

71 61 53

62.9 51.7 41.4

61.2 52.6 45.7

a A total of 116 B. cereus /thuringiensis strains were used in both experimental sets (set 1 and set 2). In the case of set 2, but not set 1, strains underwent an acclimatization for ca. 4 weeks at room temperature before cultivation. The data for B. mycoides are not given because these strains exhibited rhizoid growth that probably hampered the detection of the discontinuous hemolysis (DH) patterns.

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and 10 mM), were used in conjunction with 1 mM EDTA or 8% serum. None of the protease inhibitor combinations increased the efficacy of EDTA or serum alone. Woodward’s reagent K (1, 5 and 10 mM) did not improve the clarity of the DH either, when compared to EDTA. Three incubation temperatures, 22, 27 and 308C, were examined for their effect on time of DH development when 1 mM EDTA, 8% serum or both were used in the assay. It was found that 278C was optimal, with the DH generally observed after 2–4 h. About 6 h was necessary to observe the DH at 228C. At 308C, the CH frequently developed too fast for the DH to be observed. Subsequently the concentration of 2 mM EDTA was found to increase DH

Table 3 Gel diffusion — influence of the sampling time on the DH patterns for three weak HBL producers in gels containing 1 mM EDTA Strain

Presence of the DH 5 h sampling a

3505M F4581 / 76L b F4581 / 76Rc

d

6 6d 2

7 h sampling

16 h sampling

1 6d 2

1 1 2

5

stability when the incubation occurred at 278C, the time of DH development being unchanged. We compared the detection of HBL producers grown in BHIG broth at 328C for 5, 7 and 16 h using gel diffusion agar containing 1 mM EDTA. In supernatants sampled at 5 and 7 h, the DH was not reproducible, especially for isolates known to be weak HBL producers, whereas 16 h samples developed clear and stable DH (Table 3). Using the optimal combination of 2 mM EDTA, 8% serum and an incubation temperature of 278C, we surveyed our collection of 114 B. cereus, two B. thuringiensis and nine B. mycoides strains. As we found that most of the B. thuringiensis and B. mycoides were positive for HBL, results are collectively reported. An average 65% of the cultures harvested at 5 h developed the DH, while ca. 72% of those harvested at 16 h produced the pattern (Table 4). This difference still existed when taking into account all media incubated with 5-h samples vs. all media incubated with overnight samples (clustered media) (Table 5).

4. Discussion Hemolysin BL (HBL) is composed of three distinct proteins, B, L1 and L2, which are nontoxic individually, but when combined are hemolytic, enterotoxigenic, dermonecrotic, and are cytotoxic to

a

Pattern observed in the presence of 0.1 mM pHMB. Pattern preferably seen in the absence of pHMB. c Pattern only seen in the presence of PMSF. d Variable. b

Table 4 Gel diffusion — influence of the sampling time on the percent of strains exhibiting a DH in gels containing 2 mM EDTAa Age of supernatants

Inhibitors added to the media

Number of strains exhibiting a DH

Percent

Set 1

Set 2

Set 1

Set 2

5h

– pHMB PMSF pHMB 1 PMSF

69 71 69 71

90 95 93 96

55.2 56.8 55.2 56.8

72.0 76.0 74.4 76.8

Overnight

– pHMB PMSF pHMB 1 PMSF

90 90 89 92

87 90 89 92

72.0 72.0 71.2 73.6

69.6 72.0 71.2 73.6

a A total of 125 strains (116 B. cereus /thuringiensis and nine B. mycoides) were used in both experimental sets (set 1 and set 2). In the case of set 2, but not set 1, strains underwent an acclimatization for ca. 4 weeks at room temperature before cultivation. All supernatants were supplemented with 8% calf serum. DH, discontinuous hemolysis pattern.

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Table 5 Gel diffusion — percent of strains exhibiting a DH when clustering the methods a Cluster of methods

5 h supernatants Overnight supernatants Medium without protease inhibitor Medium with pHMB 1 PMSF All media

Number of strains exhibiting a DH

Percent

Set 1

Set 2

Set 1

Set 2

74 93 94 96 97

96 92 93 97 97

59.2 74.4 75.2 76.8 77.6

76.8 73.6 74.4 77.6 77.6

a The strains and cultivation conditions were as in Table 4. Within a given cluster, a given strain was recorded as positive if at least one of the methods clustered led to a discontinuous hemolysis (DH). Otherwise, the strain was recorded as negative. For example, in the cluster ‘‘5-h supernatants’’, if only the medium containing pHMB 1 PMSF gave a DH, the strain was assigned a positive response.

a variety of cell and tissue types (Beecher and Macmillan, 1991; Beecher and Wong, 1994a,b). It has been shown to contribute to diarrheal food poisoning and necrotizing infections such as endophthalmitis (Beecher et al., 1995a,b). The gel diffusion and colony growth methods originally described by Beecher and Wong (1994b) permitted specific detection of HBL in complex samples such as culture supernatants, and on primary culture media. One of the major limitations is the development of a CH immediately adjacent to the well containing HBL or a B. cereus colony. The CH varies in size and rate of development depending on the strain, media composition and incubation conditions. With time, the CH expands and may obscure the DH. Hence an obvious strategy would be to limit the CH while enhancing the DH. The cause of the CH is unknown. However, several potential candidates produced by B. cereus may act individually or in combination to produce the CH. These include cereolysin, hemolysins II and III, and the lysin of Matsuyama et al. (1995), which are all hemolysins and therefore capable of lysing red blood cells. Cereolysin can be inactivated by cholesterol, while no inhibitors for hemolysins II or III have been identified. SMase is specific for sphingomyelin, and can lyse sheep or calf red blood cells, or other cell types with high sphingomyelin content. Calcium ions facilitate binding of SMase to cells, while Mg 11 activates hydrolysis of sphingomyelin (Yamada et al., 1988; Tomita et al., 1991). ¨ PC-PLC is a zinc metalloenzyme (Mollby, 1978; Bjørklid and Little, 1980; Ikeda et al., 1991) that preferentially acts on phosphatidylcholine, but is also active against phosphatidylethanolamine and phosphatidylserine. However, it is unable to hydrolyze

phospholipids in intact membranes. SMase and PCPLC, known as cereolysin AB (Gilmore et al., 1989) act synergistically to lyse red blood cells (Yamada et al., 1988; Tomita et al., 1991). In our study, calf serum was effective in inhibiting CH, which is in agreement with the findings of Beecher and Wong (1994b). Further, increasing the concentration from 2 to 8% enhanced the inhibitory effect. The specific cause is not known, but cholesterol, which is abundant in serum and is known to inhibit cereolysin, could be involved. Thuringiolysin and secondary hemolysin from B. thuringiensis were shown to be active against sheep erythrocytes by Pendleton et al. (1973). The corresponding enzymes from B. cereus could be similarly active. These authors used 200 mg / ml of cholesterol as a fine suspension to inactivate thuringiolysin. In the colony growth method we found that cholesterol added to the blood agar at $ 80 mg / ml was inhibitory to CH formation. Because of its lack of water solubility, cholesterol re-crystallized in the agar and caused small zones of nonspecific lysis. It was therefore not a useful agent for our assay systems. Based on the number of enzymes produced by B. cereus that require cations for activity, we tested the effects of several chelators on inhibition of the CH. Our results showed that EDTA had a major inhibitory effect on CH, both in the gel diffusion and colony growth methods. This implied that SMase and / or PC-PLC were involved in the CH development. Little and Oatness (1975) showed that PCPLC from B. cereus can be inactivated by EDTA or 1,10-phenanthroline. For the gel diffusion assay, incorporating 1 or 2 mM EDTA into the agar was effective, probably because a constant concentration of EDTA was available. In contrast, when EDTA

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was added to culture supernatants, its concentration was reduced as the sample diffused from the well, and no inhibitory effect was seen. For the colony growth method, EDTA incorporated into the blood agar was also effective in reducing CH, but a concentration of 1 mM was somewhat inhibitory to colony growth. A lower EDTA concentration of 0.1 mM, in addition to 8% calf serum, produced the desired effect. EDTA also appeared to improve the clarity of the DH. This was probably caused by increased stability of the HBL components due to the inhibition of metallo–proteases produced by B. cereus. However, when various protease inhibitors were added to our assays, the effect of EDTA was enhanced only in marginal cases. Ryan (1996) examined the effects of 10 protease inhibitors, including EDTA and PMSF used in our study, and found that EDTA was the most effective in inhibiting protease activity in culture supernatants of B. cereus. Incubation temperature is a major contributing factor in moderating the speed and extent of development of the CH. With the gel diffusion assay, HBL detection was optimal at 278C. This temperature permitted the DH to be detected in 2–4 h after the gels were loaded with samples, whereas ca. 6 h was necessary at 228C. At 308C, the CH developed too fast for the DH to be easily detected. With the colony growth method, 228C was the optimal temperature for reducing CH interference, and DH could be detected by 36 h. In conclusion, we have made simple but effective improvements on two previously described methods so that CH was inhibited and a stable DH was maintained for easy detection. The optimal conditions for the colony growth method included using a nutrient agar base with 5% sheep red blood cells, 8% calf serum and 0.1 mM EDTA and incubation at 228C for approximately 36 h. We must point out that weak HBL producers may sometimes appear as negative through this system, either because of subtle variations from one lot of blood to another, or because the DH is too transient to be detected. Two to three-hourly monitoring on the post-inoculation day may be necessary in order not to miss some of the patterns. For the gel diffusion assay, optimal conditions included addition of 5% sheep red blood cells and 1–2 mM EDTA, use of 16 h culture supernatants with the addition of 8% calf serum, and incubation at 278C for 2–4 h. Compared to previous

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results (Beecher and Wong, 1994b), the percent and the clarity of the DH was significantly increased with this method (McNemar’s test or ‘‘sign test’’, P , 0.01). All nine B. mycoides tested were positive after gel diffusion, and one out of the two B. thuringiensis strains was positive after gel diffusion whereas both were positive after colony growth. Production of HBL therefore did not allow to differentiate B. cereus from its close taxonomical relatives.

Acknowledgements ` from This work was supported by a Prix de These the Institut Franc¸ais pour la Nutrition (to C.F.), by grant 93-37201-9194 from the National Initiative Competitive Grants Program of the US Department of Agriculture (to A.C.L.W.), and by the College of Agricultural and Life Sciences, University of Wisconsin, Madison, USA.

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