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The adhesion of Bacillus cereus spores to epithelial cells might be an additional virulence mechanism
International Journal of Food Microbiology 39 (1998) 93–99
The adhesion of Bacillus cereus spores to epithelial cells might be an additional virulence mechanism Annika Andersson
a ,b ,
a *, Per Einar Granum b , Ulf Ronner ¨
a
Department of Food Science, SIK, The Swedish Institute for Food and Biotechnology, Department of Food Science, 402 29 Gothenburg, Sweden b Department of Pharmacology, Microbiology and Food Hygiene, Norwegian College of Veterinary Medicine, Oslo, Norway Received 25 March 1997; received in revised form 3 November 1997; accepted 3 November 1997
Abstract Four out of ten Bacillus cereus strains produced spores able to adhere to monolayers of Caco-2 cells (human epithelial cells). One of these strains has been involved in an outbreak of food poisoning where the symptoms were more severe and persisted for longer than a normal B. cereus food poisoning. The hydrophobicity of the spores is a contributing factor for the adhesion to occur. The spores are able to germinate in an environment similar to that of the small intestine and then the vegetative cells can produce the enterotoxin directly at the target place. A concentrated and active form of the enterotoxin will be taken up by the epithelial cells in the small intestine. Spore adhesion could be an important virulence factor for some B. cereus strains. 1998 Elsevier Science B.V. Keywords: Bacillus cereus; Spores; Food poisoning
1. Introduction Bacillus cereus causes two different types of food poisoning: diarrhoeal syndrome and emetic syndrome (Kramer and Gilbert, 1989). The diarrhoeal type of food poisoning will normally have an incubation period of 8–16 h, and the duration is usually less than 1 day. In a few cases the incubation time has been longer with food poisoning of a more severe type (Granum, 1994). It is known that B. cereus spores are commonly adhesive to several *Corresponding author. Tel.: 1 46 31 355600; fax: 1 46 31 833782; e-mail: [email protected]
types of surfaces (Husmark, 1993) and compared with other Bacillus spp. spores, B. cereus spores are ¨ the most adhesive and the most hydrophobic (Ronner et al., 1990). The spore surfaces are also covered with appendages which may contribute to the adhe¨ sion (Husmark and Ronner, 1992). A case of B. cereus food poisoning, affecting 17 people, where three patients were hospitalised, one for 5 weeks, made us look for an explanation for the severity of this outbreak. The infective dose seemed to be as low as 5 3 10 4 spores (200 spores / g food) in this outbreak, based on cfu (colony forming units). A plausible explanation for the severity of this outbreak could be adhesion of B. cereus vegetative cells or
0168-1605 / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII S0168-1605( 97 )00121-9
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A. Andersson et al. / International Journal of Food Microbiology 39 (1998) 93 – 99
spores to the epithelium, at least for some patients. Growth and enterotoxin production in situ would be expected to cause a more severe form of the disease than if enterotoxin is secreted, diluted and even degraded in the middle of the small intestine, before it reaches the target cells. To test our hypothesis, we selected ten different B. cereus strains, out of which four had been involved in food poisoning outbreaks. We tested the hydrophobicity and adhesion properties of the spores of these ten strains. From the experiments, we have an indication that the B. cereus spores, at least from some strains, are able to adhere to the epithelial cells, germinate and grow out to vegetative cells and produce the enterotoxin on the target cells, and thus create more severe and persisting symptoms.
2.2. Production of spores Spores from ten different strains of B. cereus were produced (Table 1). The different strains were grown overnight in broth containing: 15 g / l tryptone (Bacto), 1.5 g / l yeast extract (Difco), 0.15 g / l CaCl 2 (Merck) and 0.25 g / l MgSO 4 (Merck) and then transferred to sporulation agar plates. The sporulation medium contained: 8 g / l nutrient broth (Difco), 0.25 g / l MgSO 4 , 0.97 g / l KCl (Merck), 0.15 g / l CaCl 2 , 2 3 10 23 g / l MnCl 2 (Merck), 0.3 3 10 23 g / l ˚ FeSO 4 (Merck) and 3% agar (Oxoid) (S. Stahl, Department of Microbiology, University of Lund, Sweden). After incubation for 5–7 days (308C) a good spore yield was obtained (85–100%). The spore crop was harvested, washed three times in 0.9% NaCl and stored at 2 408C.
2.3. Hydrophobic measurements 2. Materials and methods
2.1. Production of vegetative bacteria Vegetative bacteria from ten different strains were produced (Table 1). The bacteria were grown overnight in BHI broth (brain heart infusion, Oxoid) at 308C, harvested and washed three times in 0.9% NaCl.
The relative spore surface hydrophobicity was measured using the BATH assay (Rosenberg et al., 1980) with some modifications. This method involves partitioning a spore suspension between an aqueous phase and the aqueous–hydrocarbon interface based on the degree of spore surface hydrophobicity. Spore suspension in saline (3 ml, 10 8 spores / ml) and hexadecane (1 ml) was dispersed in
Table 1 The different strains used in this study, with results from cell toxicity assay, detection of different toxin components, and digestion of starch. Strain
Isolated from
Involved in food poisoning
Vero cell assay a
Tecra ELISA
Oxoid RPLA
B-component PCR
Digestion of starch
IAM 1110 NCTC 2599 NVH 61 NVH 80 NCTC 11145
Unknown Unknown Whipping cream Whipping cream Meat loaf
1 1 1 2 1
1 1 1 2 1
2 1 2 2 1
2 1 2 2 1
2 1 2 1 1
NVH 1230-88
Meat stew
1
1
1
1
1
NVH 75-95
Meat stew
1
1
2
2
2
NVH 100
Meat stew
1
1
2
2
2
SNT 90 SNT 100
Whipping cream Whipping cream
No No No No Yes, Ref. Taylor and Gilbert, 1975 Yes, Ref. Granum et al., 1993 Yes, Ref. Lund and Granum, 1996 Yes, Ref. Granum et al., 1995 No No
2 2
2 2
2 2
2 2
1 1
a
All the positives gave more than 90% inhibition on protein synthesis.
A. Andersson et al. / International Journal of Food Microbiology 39 (1998) 93 – 99
a test tube, vigorously vortexed for 15 s and then allowed 15 min for phase separation. The absorbance (A 450nm , 1.0 cm) of the aqueous phase before and after treatment was measured spectrophotometrically (Beckman, DU-64) and the decrease in absorbance was recorded as the value of relative spore surface hydrophobicity. The mean value of five measurements was calculated.
2.4. Determination of the ability to digest starch The ability to digest starch was determined on plate count agar containing 0.5% starch. The bacteria were grown for 2 days (308C) and then Lugols solution was flooded over the plate. The bacteria that had the ability to digest starch produced a clear zone around the colony.
2.5. Enterotoxin production and PCR ( polymerase chain reaction) studies The B. cereus strains were cultured in BHIG broth (BHI medium, Oxoid, with an additional 10 g / l of glucose) with moderate agitation at 328C for 6 h. The enterotoxin was then concentrated, using 70% ammonium sulphate precipitation. The enterotoxin extract was dissolved in 20 mM phosphate buffer, pH 6.8. and dialysed. The toxicity was determined using a Vero cell assay (Sandvig and Olsnes, 1982). After 2 h incubation the reduced ability to incorporate [ 14 C]leucine was measured. In addition, the Bacillus diarrhoeal enterotoxin visual immunoassay (Tecra, Australia) and the BCET-RPLA kit (Oxoid, UK) were used. Haemolysin BL, composed of B, L 1 , L 2 , has been suggested to be a candidate for the principal virulence factor of B. cereus (Beecher et al., 1995). The B-component gene of the haemolysin BL (Heinrichs et al., 1993) was detected using PCR. PCR reactions were carried out in a Perkin Elmer Cetus DNA Thermal Cycler or a MJ Research MiniCycler TM using the following program: 30 cycles at 928C for 1 min, 508C for 1 min. and 728C for 1 min. The DyNAzyme DNA Polymerase kit (Cat. No. F550L, Espoo, Finland) was used according to the manufacturers instructions. The target DNA was from 1 ml of about 10 8 / ml cells of B. cereus culture (BHIG) or, if negative, purified DNA (5 ng) according to Wilson (1990), as modified in Heinrichs et al. (1993) was
95
used. The forward primer was 59-CCCTTACAAATTACTCGCTG-39 and the reverse primer was 59-CTCCTTGTAAATCTGTAATCCCT-39. The spores ability to germinate followed by vegetative growth and enterotoxin production in the gastrointestinal environment were investigated. 10 8 spores / ml Eagle medium were incubated with Vero cells in CO 2 atmosphere, 378C for 3 days and then the enterotoxin was checked for with the Bacillus diarrhoeal enterotoxin visual immunoassay.
2.6. Adhesion to human colon carcinoma cell line in culture The human colon cancer line Caco-2 (Pinto et al., ¨ Department 1983) (kindly provided by T. Wadstrom, of Medical Microbiology, University of Lund, Sweden) was cultured in Bio Whittaker’s RPMI 1640 (500 ml) with addition of 50 ml foetal calf serum, 0.3 ml gentamicin (100 mg / ml), 6 ml L-glutamine (200 mM) in eight well chamber slides (Nunc, Naperville, IL, USA) at 378C in 7% CO 2 . The cultures were used at postconfluence after 15 days of culture. Before the adhesion test, modified by Viboud et al. (1993), the cells were washed five times in culture medium. A suspension of 10 8 spores (determined with Helbers cell counter in 0.9% NaCl) were added to the tissue culture and the mixture was incubated at 378C for 1 h. After five washes with culture medium, the cells were examined by phasecontrast microscopy (Leitz Laborlux S) to determine the spore adherence. Bacillus subtilis spores were used as a negative control because these spores have ¨ a low hydrophobicity (Ronner et al., 1990). The number of adherent spores was determined by four countings, ten randomly chosen microscopic fields per count. The experiments were repeated twice. One microscopic field contains about 100 Caco-2 cells. The mean values and standard error of the mean [standard deviation /(number of counts)0.5 ] was calculated.
3. Results Of the ten different B. cereus strains used in this study, seven were enterotoxigenic and three nonenterotoxigenic based on the Vero cell assay (Table 1). Four of the strains have been reported to cause food
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poisoning. Of the seven enterotoxigenic strains, all were positive for the Tecra enzyme-linked immunoabsorbent assay (ELISA), while only three were positive for both the B- and the L 2 -component of the Haemolysin BL enterotoxin. Lack of the ability to digest starch is a common feature for strains that cause the emetic type of food poisoning (Nishikawa et al., 1996). Four of the strains are therefore also possible emetic toxin producers (Table 1). The results from the adhering assay are shown in Fig. 1. Four out of the ten strains adhered with more than 60 spores / 1000 Caco-2 cells. Ten microscopic fields contain about 1000 Caco-2 cells. The remaining six strains adhered with , 20 spores / 1000 Caco2 cells. Fig. 1 also shows the results of the relative hydrophobicity of the ten strains. The relative hydrophobicity ranged from about 30% to above 80%. The adhesion of B. cereus spores to Caco-2 cells is correlated with the hydrophobicity of the spores, although adhesion is not explained by hydrophobicity alone (Fig. 1). B. cereus spores contain appendages (Fig. 2) which probably contribute sig-
Fig. 2. Transmission electron micrograph of a spore from Bacillus cereus strain NVH 100 (8500 3 magnification).
nificantly to the adhesion, in addition to hydrophobicity. The B. subtilis spores (negative control) did not adhere the Caco-2 cells at all. Neither did vegetative cells from the different strains. The adhesion experiments were followed for 1 h, which was long enough for the spores to germinate. The contact with the epithelial cells somehow triggered germination since spores added to the growth medium, without the Caco-2 cells, incubated for 1 h at 378C did not germinate. After germination the
Fig. 1. The relative hydrophobicity and adhesion to the Caco-2 cells for spores from ten different Bacillus cereus strains. (j) Hydrophobicity, (relative %), standard error is given on the bars; (h) adhesion of spores to Caco-2 cells, standard error of the mean is given on the bars.
A. Andersson et al. / International Journal of Food Microbiology 39 (1998) 93 – 99
Fig. 3. The adhesion of spores from Bacillus cereus strain NVH 100 to Caco-2 cells (1000 3 magnification).
vegetative bacteria produced cytotoxin (enterotoxin) on the Caco-2 cells (Fig. 3). Visually, following the Caco-2 cells over time incubated with 10 8 spores of the adhering strain NVH 100, showed that after 8 h the epithelial cells close to the B. cereus layer had mainly disintegrated, indicating production of enterotoxin. Visually, following the Vero cells, incubated with 10 8 spores of the ten different strains, over time showed that all the positive strains made the cells disintegrate. This is indicative of enterotoxin production. Indeed the ELISA kit showed that all the enterotoxin positive strains (Table 1) were able to produce at least one of the components of the nonhaemolytic enterotoxin (Lund and Granum, 1996) in environments similar to those of the small intestine.
4. Discussion It has just been shown that B. cereus can produce a nonhaemolytic enterotoxin (Lund and Granum, 1996) in addition to the haemolytic enterotoxin (HBL) (Heinrichs et al., 1993). One of the three components of each of the enterotoxin complexes can be detected by the Oxoid RPLA and the Tecra ELISA (Lund and Granum, 1996). The ability of the ten strains to produce the two different enterotoxins was investigated (Table 1). Seven out of ten strains were enterotoxin producing strains based on the Vero
97
cell assay. All seven strains produced the nonhaemolytic enterotoxin, while only three of them produced the haemolytic enterotoxin (HBL), using the Oxoid kit and PCR detection of the B-component gene (Heinrichs et al., 1993). The results were confirmed by immunoblot analysis (results not shown). Our findings confirm what has been shown by Christiansson (1993), that cytotoxicity tests and the results testing for enterotoxin production by the Tecra ELISA kit are in agreement. Only strain NCTC 2599, NCTC 11145 and NVH 1230-88 produced the haemolytic enterotoxin. Strains unable to digest starch have recently been shown to correlate well with production of emetic toxin (Nishikawa et al., 1996). Although none of the ten strains used in this study have been involved in emetic outbreaks, four of the strains may also have the ability to produce the emetic toxin. The B. cereus spores are known to be more hydrophobic than spores from other Bacillus species ¨ (Husmark, 1993; Ronner et al., 1990). It is believed that the hydrophobicity is one of the main reasons for the adhesion problems caused by B. cereus spores in the dairy industry (Andersson et al., 1995). Fig. 1 shows that the hydrophobicity varies from 30% to 80% relative hydrophobicity. All the strains that adhere well to the Caco-2 cells are highly hydrophobic ( . 63). The spore appendages are probably also responsible for the adhesion, since the hydrophobicity alone cannot explain the adhesion (Fig. 1). Too little is yet known about the appendages (Kozuka and Tochikubo, 1985) to give any explanation to the differences in adhesion properties possibly caused by these. Six out of 11 tested pathogenic gram-negative bacteria are reported to express high cell surface hydrophobicity, which is associated with the bacterial binding to the host cell. These six species are Escherichia coli, Shigella spp., Yersinia enterocolitica, Vibrio cholera, Bordetella pertussis and Pseudomonas aeruginosa (Lachica, 1990). Although our experiments are not conclusive on whether spore adhesion to epithelial cells is an additional virulence mechanism, the food poisoning outbreaks some of the strains have been involved in is supportive of such a theory. Three of the strains have been involved in food poisoning outbreaks in Norway (NVH 1230-88, NVH 100 and NVH 75-95). The infective doses were low in all the three cases;
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only between 2 3 10 2 –2 3 10 5 bacteria or spores per gram were isolated from the implicated foods (meat stews). Strain NVH 1230-88 and NCTC 11145 were due to outbreaks with normal incubation period ( , 12 h) and symptoms (watery diarrhoea lasting for about 12 h). Strain NVH 75-95 caused an outbreak during the Norwegian junior ski championship in 1995, where 152 out of 252 persons were affected (Granum et al., 1995). The incubation period was in some cases more then 24 h and the duration of the disease was from one to several days. Strain NVH 100 has, however, caused the most severe food poisoning outbreak in connection with a 80-years birthday party, 17 out of 24 persons who ate meat stew became ill (Granum, 1994). Three people were ill for more than 1 week, and one of them (63 years old female) was treated intravenously for 5 weeks. An interesting feature with this outbreak was that severity of the disease increased with increasing incubation time. This special outbreak could be due to special characteristics of the spores, which needed time to adhere to the intestine, germinate, followed by growth and enterotoxin production in situ. We showed that the spores were able to germinate and produce enterotoxin incubated with Vero cells. It has earlier been shown that B. cereus can grow and produce enterotoxin anaerobically (Granum et al., 1993). Three of the strains that adhere well to Caco-2 cells have not been involved in food poisoning outbreaks. Strain NVH 100 adhere quite well. Strain NVH 75-95 could possibly adhere, although not as well as strain NVH 100. Even for this strain (NVH 75-95) the symptoms came rather late in some patients and lasted longer than usually. One difference of strain NVH 100 and also to some extent NVH 75-95 compared to the other strains, is that the spores of these strains form aggregates. Therefore it is rather an aggregate of several spores and not a single spore that will adhere and there will be an even higher concentration effect of the enterotoxin. This ability of some B. cereus spores to adhere to the epithelial cells of the small intestine, grow and produce enterotoxin in situ could be an important virulence mechanism. If our theory is correct ingestion of spores with binding abilities would be of importance since it might cause a much more severe types of B. cereus food poisoning.
Acknowledgements The authors would like to thank Dr. K. Tochikubo and Dr. S. Kozuka, Nagoya City University Medical School, for kindly providing the transmission electron micrograph of strain NVH 100. We also thank Kristin O’Sullivan, Norwegian College of Veterinary Medicine for technical assistance. This research was supported by a grant (50.0045 / 95) from the Swedish Council for Forestry and Agricultural Research.
References ¨ Andersson, A., Ronner, U., Granum, P.E., 1995. What problems does the food industry have with the sporeforming pathogens Bacillus cereus and Clostridium perfringens?. Int. J. Food Microbiol. 28, 145–155. Beecher, D.J., Schoeni, J.L., Wang, A.C.L., 1995. Enterotoxin activity of hemolysin from Bacillus cereus. Infect. Immun. 63, 4423–4428. Christiansson, A., 1993. Enterotoxin production in milk by Bacillus cereus: a comparison of methods for toxin detection. Neth. Milk Dairy J. 47, 79–87. Granum, P.E., Brynestad, S., O’Sullivan, K., Nissen, H., 1993. Enterotoxin from Bacillus cereus: production and biochemical characterization. Neth. Milk Dairy J. 47, 63–70. Granum, P.E., 1994. Bacillus cereus as a food pathogen. Norsk Vet. Tidskr., 106, 911–915 (in Norwegian). Granum, P.E., Næstvold, A., Nordli Gundersby, K., 1995. An outbreak of Bacillus cereus food poisoning during the Norwegian ski championships for juniors. Norsk Vet. Tidskr.. 107, 945–948 (in Norwegian; English abstract). Heinrichs, J.H., Beecher, D.J., Macmillan, J.D., Zilinskas, B.A., 1993. Molecular cloning and characterization of the hblA gene encoding the B component of hemolysin BL from Bacillus cereus. J. Bacteriol. 175, 6760–6766. ¨ Husmark, U., Ronner, U., 1992. The influence of hydrophobic, electrostatic and morphologic properties on the adhesion of Bacillus spores. Biofouling 5, 335–344. Husmark, U., 1993. Adhesion mechanisms of bacterial spores to solid surfaces. Ph.D. thesis, Department of Food Science, Chalmers University of Technology and SIK, The Swedish ¨ Institute for Food Research, Goteborg, Sweden. Kramer, J.M., Gilbert. R.J., 1989. Bacillus cereus and other Bacillus species. In: Doyle, M.P. (Ed.), Foodborne Bacterial Pathogens. Marcel Dekker, New York and Basel, pp. 21–70. Kozuka, S., Tochikubo, K., 1985. Properties and origin of filamentous appendages on spores of Bacillus cereus. Microbiol. Immunol. 29, 21–35. Lachica, R.V., 1990. Significance of hydrophobicity in the adhesiveness of pathogenic gram-negative bacteria. Microbial cell surface hydrophobicity. American Society for Microbiology, Washington, DC, pp. 297–313.
A. Andersson et al. / International Journal of Food Microbiology 39 (1998) 93 – 99 Lund, T., Granum, P.E., 1996. Characterisation of a non haemolytic enterotoxin complex from Bacillus cereus isolated after a foodborne outbreak. FEMS Microbiol. Lett. 141, 151– 156. Nishikawa, Y., Kramer, J.M., Hanoka, H., Yasukawa, A., 1996. Evaluation of serotyping, biotyping, plasmid banding pattern analysis and HEp-2 vacuolation factor analysis in the epidemiological investigation of Bacillus cereus emetic-syndrome food poisoning. Int. J. Food Microbiol. 31, 149–159. Pinto, M., Robine-Leon, S., Appay, M.-D., Kedinger, M., Triadou, N., Dussaulx, E., Lacroix, B., Simon-Assmann, P., Haffen, K., Fogh, J., Zweibaum, A., 1983. Enterocyte-like differentiation and polarization of human colon carcinoma cell line Caco-2 in culture. Biol. Cell 47, 323–330. Rosenberg, M., Gutnick, D., Rosenberg, E., 1980. Adherence of bacteria to hydrocarbons: a simple method for measuring cell surface hydrophobicity. FEMS Microbiol. Lett. 9, 29–33.
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¨ Ronner, U., Husmark, U., Henriksson, A., 1990. Adhesion of Bacillus spores in relation to hydrophobicity. J. Appl. Bacteriol. 69, 550–556. Sandvig, K., Olsnes, S., 1982. Entry of the toxic proteins abrin, modeccin, ricin and diphteria toxin into cells. J. Biol. Chem. 257, 7495–7503. Taylor, A.J., Gilbert, R.J., 1975. Bacillus cereus food poisoning: a provisional serotyping scheme. J. Med. Microbiol. 8, 543–550. Viboud, G.I., Binsztein, N., Svennerholm, A.-M., 1993. A new fimbrial putative colonization factor, PCFO20, in human enterotoxigenic Escherichia coli. Infect. Immunol. 61, 5190– 5197. Wilson, K., 1990. Preparation of genomic DNA from bacteria. Current protocols in molecular biology, vol. 2, suppl. 9. Wiley, New York, pp. 2.4.1–2.4.5.
The adhesion of Bacillus cereus spores to epithelial cells might be an additional virulence mechanism Annika Andersson
a ,b ,
a *, Per Einar Granum b , Ulf Ronner ¨
a
Department of Food Science, SIK, The Swedish Institute for Food and Biotechnology, Department of Food Science, 402 29 Gothenburg, Sweden b Department of Pharmacology, Microbiology and Food Hygiene, Norwegian College of Veterinary Medicine, Oslo, Norway Received 25 March 1997; received in revised form 3 November 1997; accepted 3 November 1997
Abstract Four out of ten Bacillus cereus strains produced spores able to adhere to monolayers of Caco-2 cells (human epithelial cells). One of these strains has been involved in an outbreak of food poisoning where the symptoms were more severe and persisted for longer than a normal B. cereus food poisoning. The hydrophobicity of the spores is a contributing factor for the adhesion to occur. The spores are able to germinate in an environment similar to that of the small intestine and then the vegetative cells can produce the enterotoxin directly at the target place. A concentrated and active form of the enterotoxin will be taken up by the epithelial cells in the small intestine. Spore adhesion could be an important virulence factor for some B. cereus strains. 1998 Elsevier Science B.V. Keywords: Bacillus cereus; Spores; Food poisoning
1. Introduction Bacillus cereus causes two different types of food poisoning: diarrhoeal syndrome and emetic syndrome (Kramer and Gilbert, 1989). The diarrhoeal type of food poisoning will normally have an incubation period of 8–16 h, and the duration is usually less than 1 day. In a few cases the incubation time has been longer with food poisoning of a more severe type (Granum, 1994). It is known that B. cereus spores are commonly adhesive to several *Corresponding author. Tel.: 1 46 31 355600; fax: 1 46 31 833782; e-mail: [email protected]
types of surfaces (Husmark, 1993) and compared with other Bacillus spp. spores, B. cereus spores are ¨ the most adhesive and the most hydrophobic (Ronner et al., 1990). The spore surfaces are also covered with appendages which may contribute to the adhe¨ sion (Husmark and Ronner, 1992). A case of B. cereus food poisoning, affecting 17 people, where three patients were hospitalised, one for 5 weeks, made us look for an explanation for the severity of this outbreak. The infective dose seemed to be as low as 5 3 10 4 spores (200 spores / g food) in this outbreak, based on cfu (colony forming units). A plausible explanation for the severity of this outbreak could be adhesion of B. cereus vegetative cells or
0168-1605 / 98 / $19.00 1998 Elsevier Science B.V. All rights reserved. PII S0168-1605( 97 )00121-9
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A. Andersson et al. / International Journal of Food Microbiology 39 (1998) 93 – 99
spores to the epithelium, at least for some patients. Growth and enterotoxin production in situ would be expected to cause a more severe form of the disease than if enterotoxin is secreted, diluted and even degraded in the middle of the small intestine, before it reaches the target cells. To test our hypothesis, we selected ten different B. cereus strains, out of which four had been involved in food poisoning outbreaks. We tested the hydrophobicity and adhesion properties of the spores of these ten strains. From the experiments, we have an indication that the B. cereus spores, at least from some strains, are able to adhere to the epithelial cells, germinate and grow out to vegetative cells and produce the enterotoxin on the target cells, and thus create more severe and persisting symptoms.
2.2. Production of spores Spores from ten different strains of B. cereus were produced (Table 1). The different strains were grown overnight in broth containing: 15 g / l tryptone (Bacto), 1.5 g / l yeast extract (Difco), 0.15 g / l CaCl 2 (Merck) and 0.25 g / l MgSO 4 (Merck) and then transferred to sporulation agar plates. The sporulation medium contained: 8 g / l nutrient broth (Difco), 0.25 g / l MgSO 4 , 0.97 g / l KCl (Merck), 0.15 g / l CaCl 2 , 2 3 10 23 g / l MnCl 2 (Merck), 0.3 3 10 23 g / l ˚ FeSO 4 (Merck) and 3% agar (Oxoid) (S. Stahl, Department of Microbiology, University of Lund, Sweden). After incubation for 5–7 days (308C) a good spore yield was obtained (85–100%). The spore crop was harvested, washed three times in 0.9% NaCl and stored at 2 408C.
2.3. Hydrophobic measurements 2. Materials and methods
2.1. Production of vegetative bacteria Vegetative bacteria from ten different strains were produced (Table 1). The bacteria were grown overnight in BHI broth (brain heart infusion, Oxoid) at 308C, harvested and washed three times in 0.9% NaCl.
The relative spore surface hydrophobicity was measured using the BATH assay (Rosenberg et al., 1980) with some modifications. This method involves partitioning a spore suspension between an aqueous phase and the aqueous–hydrocarbon interface based on the degree of spore surface hydrophobicity. Spore suspension in saline (3 ml, 10 8 spores / ml) and hexadecane (1 ml) was dispersed in
Table 1 The different strains used in this study, with results from cell toxicity assay, detection of different toxin components, and digestion of starch. Strain
Isolated from
Involved in food poisoning
Vero cell assay a
Tecra ELISA
Oxoid RPLA
B-component PCR
Digestion of starch
IAM 1110 NCTC 2599 NVH 61 NVH 80 NCTC 11145
Unknown Unknown Whipping cream Whipping cream Meat loaf
1 1 1 2 1
1 1 1 2 1
2 1 2 2 1
2 1 2 2 1
2 1 2 1 1
NVH 1230-88
Meat stew
1
1
1
1
1
NVH 75-95
Meat stew
1
1
2
2
2
NVH 100
Meat stew
1
1
2
2
2
SNT 90 SNT 100
Whipping cream Whipping cream
No No No No Yes, Ref. Taylor and Gilbert, 1975 Yes, Ref. Granum et al., 1993 Yes, Ref. Lund and Granum, 1996 Yes, Ref. Granum et al., 1995 No No
2 2
2 2
2 2
2 2
1 1
a
All the positives gave more than 90% inhibition on protein synthesis.
A. Andersson et al. / International Journal of Food Microbiology 39 (1998) 93 – 99
a test tube, vigorously vortexed for 15 s and then allowed 15 min for phase separation. The absorbance (A 450nm , 1.0 cm) of the aqueous phase before and after treatment was measured spectrophotometrically (Beckman, DU-64) and the decrease in absorbance was recorded as the value of relative spore surface hydrophobicity. The mean value of five measurements was calculated.
2.4. Determination of the ability to digest starch The ability to digest starch was determined on plate count agar containing 0.5% starch. The bacteria were grown for 2 days (308C) and then Lugols solution was flooded over the plate. The bacteria that had the ability to digest starch produced a clear zone around the colony.
2.5. Enterotoxin production and PCR ( polymerase chain reaction) studies The B. cereus strains were cultured in BHIG broth (BHI medium, Oxoid, with an additional 10 g / l of glucose) with moderate agitation at 328C for 6 h. The enterotoxin was then concentrated, using 70% ammonium sulphate precipitation. The enterotoxin extract was dissolved in 20 mM phosphate buffer, pH 6.8. and dialysed. The toxicity was determined using a Vero cell assay (Sandvig and Olsnes, 1982). After 2 h incubation the reduced ability to incorporate [ 14 C]leucine was measured. In addition, the Bacillus diarrhoeal enterotoxin visual immunoassay (Tecra, Australia) and the BCET-RPLA kit (Oxoid, UK) were used. Haemolysin BL, composed of B, L 1 , L 2 , has been suggested to be a candidate for the principal virulence factor of B. cereus (Beecher et al., 1995). The B-component gene of the haemolysin BL (Heinrichs et al., 1993) was detected using PCR. PCR reactions were carried out in a Perkin Elmer Cetus DNA Thermal Cycler or a MJ Research MiniCycler TM using the following program: 30 cycles at 928C for 1 min, 508C for 1 min. and 728C for 1 min. The DyNAzyme DNA Polymerase kit (Cat. No. F550L, Espoo, Finland) was used according to the manufacturers instructions. The target DNA was from 1 ml of about 10 8 / ml cells of B. cereus culture (BHIG) or, if negative, purified DNA (5 ng) according to Wilson (1990), as modified in Heinrichs et al. (1993) was
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used. The forward primer was 59-CCCTTACAAATTACTCGCTG-39 and the reverse primer was 59-CTCCTTGTAAATCTGTAATCCCT-39. The spores ability to germinate followed by vegetative growth and enterotoxin production in the gastrointestinal environment were investigated. 10 8 spores / ml Eagle medium were incubated with Vero cells in CO 2 atmosphere, 378C for 3 days and then the enterotoxin was checked for with the Bacillus diarrhoeal enterotoxin visual immunoassay.
2.6. Adhesion to human colon carcinoma cell line in culture The human colon cancer line Caco-2 (Pinto et al., ¨ Department 1983) (kindly provided by T. Wadstrom, of Medical Microbiology, University of Lund, Sweden) was cultured in Bio Whittaker’s RPMI 1640 (500 ml) with addition of 50 ml foetal calf serum, 0.3 ml gentamicin (100 mg / ml), 6 ml L-glutamine (200 mM) in eight well chamber slides (Nunc, Naperville, IL, USA) at 378C in 7% CO 2 . The cultures were used at postconfluence after 15 days of culture. Before the adhesion test, modified by Viboud et al. (1993), the cells were washed five times in culture medium. A suspension of 10 8 spores (determined with Helbers cell counter in 0.9% NaCl) were added to the tissue culture and the mixture was incubated at 378C for 1 h. After five washes with culture medium, the cells were examined by phasecontrast microscopy (Leitz Laborlux S) to determine the spore adherence. Bacillus subtilis spores were used as a negative control because these spores have ¨ a low hydrophobicity (Ronner et al., 1990). The number of adherent spores was determined by four countings, ten randomly chosen microscopic fields per count. The experiments were repeated twice. One microscopic field contains about 100 Caco-2 cells. The mean values and standard error of the mean [standard deviation /(number of counts)0.5 ] was calculated.
3. Results Of the ten different B. cereus strains used in this study, seven were enterotoxigenic and three nonenterotoxigenic based on the Vero cell assay (Table 1). Four of the strains have been reported to cause food
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poisoning. Of the seven enterotoxigenic strains, all were positive for the Tecra enzyme-linked immunoabsorbent assay (ELISA), while only three were positive for both the B- and the L 2 -component of the Haemolysin BL enterotoxin. Lack of the ability to digest starch is a common feature for strains that cause the emetic type of food poisoning (Nishikawa et al., 1996). Four of the strains are therefore also possible emetic toxin producers (Table 1). The results from the adhering assay are shown in Fig. 1. Four out of the ten strains adhered with more than 60 spores / 1000 Caco-2 cells. Ten microscopic fields contain about 1000 Caco-2 cells. The remaining six strains adhered with , 20 spores / 1000 Caco2 cells. Fig. 1 also shows the results of the relative hydrophobicity of the ten strains. The relative hydrophobicity ranged from about 30% to above 80%. The adhesion of B. cereus spores to Caco-2 cells is correlated with the hydrophobicity of the spores, although adhesion is not explained by hydrophobicity alone (Fig. 1). B. cereus spores contain appendages (Fig. 2) which probably contribute sig-
Fig. 2. Transmission electron micrograph of a spore from Bacillus cereus strain NVH 100 (8500 3 magnification).
nificantly to the adhesion, in addition to hydrophobicity. The B. subtilis spores (negative control) did not adhere the Caco-2 cells at all. Neither did vegetative cells from the different strains. The adhesion experiments were followed for 1 h, which was long enough for the spores to germinate. The contact with the epithelial cells somehow triggered germination since spores added to the growth medium, without the Caco-2 cells, incubated for 1 h at 378C did not germinate. After germination the
Fig. 1. The relative hydrophobicity and adhesion to the Caco-2 cells for spores from ten different Bacillus cereus strains. (j) Hydrophobicity, (relative %), standard error is given on the bars; (h) adhesion of spores to Caco-2 cells, standard error of the mean is given on the bars.
A. Andersson et al. / International Journal of Food Microbiology 39 (1998) 93 – 99
Fig. 3. The adhesion of spores from Bacillus cereus strain NVH 100 to Caco-2 cells (1000 3 magnification).
vegetative bacteria produced cytotoxin (enterotoxin) on the Caco-2 cells (Fig. 3). Visually, following the Caco-2 cells over time incubated with 10 8 spores of the adhering strain NVH 100, showed that after 8 h the epithelial cells close to the B. cereus layer had mainly disintegrated, indicating production of enterotoxin. Visually, following the Vero cells, incubated with 10 8 spores of the ten different strains, over time showed that all the positive strains made the cells disintegrate. This is indicative of enterotoxin production. Indeed the ELISA kit showed that all the enterotoxin positive strains (Table 1) were able to produce at least one of the components of the nonhaemolytic enterotoxin (Lund and Granum, 1996) in environments similar to those of the small intestine.
4. Discussion It has just been shown that B. cereus can produce a nonhaemolytic enterotoxin (Lund and Granum, 1996) in addition to the haemolytic enterotoxin (HBL) (Heinrichs et al., 1993). One of the three components of each of the enterotoxin complexes can be detected by the Oxoid RPLA and the Tecra ELISA (Lund and Granum, 1996). The ability of the ten strains to produce the two different enterotoxins was investigated (Table 1). Seven out of ten strains were enterotoxin producing strains based on the Vero
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cell assay. All seven strains produced the nonhaemolytic enterotoxin, while only three of them produced the haemolytic enterotoxin (HBL), using the Oxoid kit and PCR detection of the B-component gene (Heinrichs et al., 1993). The results were confirmed by immunoblot analysis (results not shown). Our findings confirm what has been shown by Christiansson (1993), that cytotoxicity tests and the results testing for enterotoxin production by the Tecra ELISA kit are in agreement. Only strain NCTC 2599, NCTC 11145 and NVH 1230-88 produced the haemolytic enterotoxin. Strains unable to digest starch have recently been shown to correlate well with production of emetic toxin (Nishikawa et al., 1996). Although none of the ten strains used in this study have been involved in emetic outbreaks, four of the strains may also have the ability to produce the emetic toxin. The B. cereus spores are known to be more hydrophobic than spores from other Bacillus species ¨ (Husmark, 1993; Ronner et al., 1990). It is believed that the hydrophobicity is one of the main reasons for the adhesion problems caused by B. cereus spores in the dairy industry (Andersson et al., 1995). Fig. 1 shows that the hydrophobicity varies from 30% to 80% relative hydrophobicity. All the strains that adhere well to the Caco-2 cells are highly hydrophobic ( . 63). The spore appendages are probably also responsible for the adhesion, since the hydrophobicity alone cannot explain the adhesion (Fig. 1). Too little is yet known about the appendages (Kozuka and Tochikubo, 1985) to give any explanation to the differences in adhesion properties possibly caused by these. Six out of 11 tested pathogenic gram-negative bacteria are reported to express high cell surface hydrophobicity, which is associated with the bacterial binding to the host cell. These six species are Escherichia coli, Shigella spp., Yersinia enterocolitica, Vibrio cholera, Bordetella pertussis and Pseudomonas aeruginosa (Lachica, 1990). Although our experiments are not conclusive on whether spore adhesion to epithelial cells is an additional virulence mechanism, the food poisoning outbreaks some of the strains have been involved in is supportive of such a theory. Three of the strains have been involved in food poisoning outbreaks in Norway (NVH 1230-88, NVH 100 and NVH 75-95). The infective doses were low in all the three cases;
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only between 2 3 10 2 –2 3 10 5 bacteria or spores per gram were isolated from the implicated foods (meat stews). Strain NVH 1230-88 and NCTC 11145 were due to outbreaks with normal incubation period ( , 12 h) and symptoms (watery diarrhoea lasting for about 12 h). Strain NVH 75-95 caused an outbreak during the Norwegian junior ski championship in 1995, where 152 out of 252 persons were affected (Granum et al., 1995). The incubation period was in some cases more then 24 h and the duration of the disease was from one to several days. Strain NVH 100 has, however, caused the most severe food poisoning outbreak in connection with a 80-years birthday party, 17 out of 24 persons who ate meat stew became ill (Granum, 1994). Three people were ill for more than 1 week, and one of them (63 years old female) was treated intravenously for 5 weeks. An interesting feature with this outbreak was that severity of the disease increased with increasing incubation time. This special outbreak could be due to special characteristics of the spores, which needed time to adhere to the intestine, germinate, followed by growth and enterotoxin production in situ. We showed that the spores were able to germinate and produce enterotoxin incubated with Vero cells. It has earlier been shown that B. cereus can grow and produce enterotoxin anaerobically (Granum et al., 1993). Three of the strains that adhere well to Caco-2 cells have not been involved in food poisoning outbreaks. Strain NVH 100 adhere quite well. Strain NVH 75-95 could possibly adhere, although not as well as strain NVH 100. Even for this strain (NVH 75-95) the symptoms came rather late in some patients and lasted longer than usually. One difference of strain NVH 100 and also to some extent NVH 75-95 compared to the other strains, is that the spores of these strains form aggregates. Therefore it is rather an aggregate of several spores and not a single spore that will adhere and there will be an even higher concentration effect of the enterotoxin. This ability of some B. cereus spores to adhere to the epithelial cells of the small intestine, grow and produce enterotoxin in situ could be an important virulence mechanism. If our theory is correct ingestion of spores with binding abilities would be of importance since it might cause a much more severe types of B. cereus food poisoning.
Acknowledgements The authors would like to thank Dr. K. Tochikubo and Dr. S. Kozuka, Nagoya City University Medical School, for kindly providing the transmission electron micrograph of strain NVH 100. We also thank Kristin O’Sullivan, Norwegian College of Veterinary Medicine for technical assistance. This research was supported by a grant (50.0045 / 95) from the Swedish Council for Forestry and Agricultural Research.
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