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Toxin production potential and the detection of toxin genes among strains of the Bacillus cereus group isolated along the dairy production chain
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International Dairy Journal 17 (2007) 1201–1208 www.elsevier.com/locate/idairyj
Toxin production potential and the detection of toxin genes among strains of the Bacillus cereus group isolated along the dairy production chain Birgitta Svenssona, Amanda Montha´na, Marie-He´le`ne Guinebretie`reb, Christophe Nguyen-The´b, Anders Christianssona, a Swedish Dairy Association, Research and Development, Scheeleva¨gen 18, SE 223 63 Lund, Sweden Institut National de la Recherche Agronomique, UMR A408 Se´curite´ et Qualite´ des Produits d’Origine Ve´ge´tale, INRA, Domaine Saint-Paul, Site Agroparc, F 84914 Avignon Cedex 9, France
b
Received 7 July 2006; accepted 4 March 2007
Abstract Isolates of the Bacillus cereus group (396 in total) from farms, silo tanks and production lines for pasteurised milk were tested for toxin production potential, and by polymerase chain reaction (PCR) for the presence of toxin genes. Comparison between the tests indicated the presence of gene polymorphisms. Highly toxigenic strains, based on production of subunit A of the nonhemolytic enterotoxin, NHE (NheA) and subunit C of the haemolytic enterotoxin, HBL (HblC), were less common among dairy isolates compared with farm and silo isolates. No producer of high levels of both toxins was found among 156 psychrotrophic dairy isolates (B. weihenstephanensis) and only 3% of all psychrotrophs were high producers of NheA. Psychrotrophic B. cereus from pasteurised milk appeared to have a low enterotoxin production potential, and they were not producers of emetic toxin or cytotoxin K and therefore may be less likely to cause illness than mesophilic strains. r 2007 Elsevier Ltd. All rights reserved. Keywords: Bacillus cereus; Enterotoxins; Emetic toxin; Toxin production; Psychrotrophic; Milk
1. Introduction Bacillus cereus is a common cause of two types of foodborne illnesses, characterized by abdominal cramps and diarrhoea 8–16 h following ingestion (the diarrhoeal type), and by vomiting 1–5 h after ingestion of the incriminated food (the emetic type); (Granum, 2001; Schoeni & Wong, 2005). The diarrhoeal disease is caused by several enterotoxins, i.e. the haemolytic toxin (HBL) the nonhemolytic toxin (NHE) and by cytotoxin K. HBL and NHE are both multicomponent protein toxins, each consisting of three immunologically distinct subunits. Cytotoxin K consists of only one protein. These toxins Corresponding author. Tel.: +46 46 19 25 81; fax: +46 46 13 70 40.
E-mail address: [email protected] (A. Christiansson). 0958-6946/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2007.03.004
are heat labile and are probably formed during bacterial growth in the small intestine. The emetic toxin is a small ring-forming peptide, cereulide, which acts as a potassium ionophore, and is extremely heat stable. It is produced during growth in food, that is stored at an inappropriate temperature. For reviews see Granum (2001) and Schoeni and Wong (2005). Methods to detect the toxin genes have been proposed in terms of polymerase chain reaction (PCR) primers for toxin (sub)units, once the toxins were cloned and sequenced (Granum, OSullivan, & Lund, 1999; Heinrichs, Beecher, Macmillan, & Zilinskas, 1993; Lund, De Buyser, & Granum, 2000; Ryan, MacMillan, & Zilinskas, 1997). However, these primers have not undergone stringent validation on a large variety of strains. Two commercial immunological kits were available for subunit C of the haemolytic enterotoxin (HblC), the Bacillus cereus
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enterotoxin reverse passive latex agglutination (RPLA) kit (Oxoid, Basingstoke, England), and for subunit A of the nonhemolytic enterotoxin (NheA), the Bacillus diarrhoeal enterotoxin visual immunoassay (Tecra Diagnostics, Roseville, NSW, Australia). Some authors have also used proprietary antibodies. For the emetic toxin, a rapid test based on mobility of boar sperm cells has been developed (Andersson et al., 2004). For an overall estimation of toxicity, various cell cytotoxicity tests based on Vero, HEp2 and Caco cells, for example, can be used (Beattie & Williams, 1999; Lund & Granum, 1996; Pru¨ss, Dietrich, Nibler, Ma¨rtlbauer, & Scherer, 1999). However, the cytotoxicity tests are difficult to perform and are not appropriate for screening large number of strains. Bacillus cereus is a common contaminant of milk (Christiansson, 2003). Some investigations have addressed the occurrence of toxin genes and toxin production of strains in milk and dairy products (Beattie & Williams, 1999; Granum, Brynestad, & Kramer, 1993; In’t Veld et al., 2001). Milk strains have been included in other investigations (Andersen Borge, Skeie, Sørhaug, Langsrud, & Granum, 2001; Granum et al., 1996; Pru¨ss et al., 1999; Schoeni & Wong, 1999; Stenfors & Granum, 2001; Stenfors, Mayr, Scherer, & Granum, 2002), but none monitored all toxins simultaneously. However, the tested strains were generally from pasteurised dairy products and not from earlier parts of the production chain. It is known that the major contamination of milk by B. cereus occurs at the farm, and additional contamination can occur in the dairy plant (Christiansson, Bertilsson, & Svensson, 1999; Lin, Schraft, Odumeru, & Griffiths, 1998; Svensson, Ekelund, Ogura, & Christiansson, 2004; Te Giffel, Beumer, Bonestroo, & Rombouts, 1996). However nothing is known about the properties of strains originating from the farm in comparison with the strains in the final product. In order to obtain a better characterization of the hazard posed by B. cereus, the occurrence of toxigenic strains along the whole chain should be addressed. Previously, we have investigated the routes of contamination of spores of B. cereus to milk at the farm, in silo tanks at dairy plants and along the production line for pasteurised milk (see Svensson et al. (2004) and references therein). Based on a selection of strains (characterized by random amplified polymorphic DNA (RAPD) PCR) from these investigations, we tested 396 strains in this work for the presence of toxin genes and toxin production potential. Particular emphasis was given to the properties of psychrotrophic strains as compared to mesophilic strains since there is a selection for psychrotrophs in the production chain due to the low temperature at which milk is stored.
and site of isolation. We did not try to differentiate between species/subspecies within the B. cereus group since the presence of toxin genes has been demonstrated in all group members (Hansen & Hendriksen, 2001; Stenfors et al., 2002). Thus B. cereus, B. thuringiensis, B. mycoides, B. pseudomycoides and B. weihenstephanensis all might have been included in the collection. However, strains that grow at low temperatures and possess the cspA-gene are generally found to be B. weihenstephanensis (Francis, Mayr, von Stetten, Stewart, & Scherer, 1998; Stenfors et al., 2002). One hundred strains from nine different dairy farms were selected from a collection of 950 isolates. The strains had been isolated from 84 different samples of milk, water, used bedding material, manure, feed and air during earlier investigations of B. cereus in the in-door housing environment (Christiansson, Magnusson, Nilsson, Ekelund, & Samuelsson, 1997). From dairy silo tanks, 100 strains were selected from a collection of 1384 strains. The strains had been isolated by filtration (Christiansson, Ekelund, & Ogura, 1997) of milk from eight different dairy plants (Svensson et al., 2004). The 100 selected isolates came from 46 different samples. During investigations of contamination routes for B. cereus in dairy plants (Eneroth, Svensson, Molin, & Christiansson, 2001; Svensson, Eneroth, Bredehaug, & Christiansson, 1999; Svensson, Eneroth, Brendehaug, Molin, & Christiansson, 2000) approximately 2800 strains were isolated from the dairy production chain. From this, collection 196 strains were selected from two different dairies, and were isolated from 102 samples collected at six different sampling points in the dairy. B. cereus spores were isolated from unpasteurised milk samples by filtration after heat treatment. Pasteurised milk samples were incubated at 7 1C for 7–11 d to simulate anticipated storage conditions and shelf life, and B. cereus were isolated on blood agar plates. 2.2. DNA preparation and analysis of mesophilic and psychrotrophic B. cereus
2. Materials and methods
B. cereus strains were cultured on tryptone glucose yeast extract agar (AB Kemikalia, Lund, Sweden) plates for 24–72 h. DNA was prepared as described earlier by freezing and boiling the cells (Nilsson, Svensson, Ekelund, & Christiansson, 1998). DNA preparations from strains that were difficult to lyse were made with the DNeasys kit from Qiagen (Qiagen Nordic AB, Solna, Sweden) according to the manufacturer’s manual for Gram-positive bacteria. Both preparation methods worked equally well for PCR. Analysis of mesophilic and psychrotrophic B. cereus was performed using PCR, based on a coldshock gene, according to Francis et al. (1998).
2.1. Selection of strains
2.3. Multiplex PCR on enterotoxin genes
The 396 strains were selected to provide maximum diversity between strains based on RAPD-PCR fingerprints
The prevalence of two enterotoxin genes, nheA and hblC was studied by a multiplex PCR. Reactions were
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performed in a volume of 50 mL with final concentrations of 1 PCR buffer (Applied Biosystems, Stockholm, Sweden), 200 mM each of dNTPs (Applied Biosystems), 2 mM MgCl2, nheA-primers (Stenfors & Granum, 2001) 0.6 mM each, and hblC-primers (Hansen & Hendriksen, 2001) 0.8 mM each (Table 1), Taq DNA polymerase (Applied Biosystems) 2.5 units per reaction and with 1 mL DNA-preparation as template. The PCRs were run with the following cycling conditions: initial denaturation at 95 1C for 1 min, 30 cycles with denaturation at 95 1C for 1 min, annealing at 52 1C for 1 min and elongation at 72 1C for 1 min, and with a final cycle at 72 1C for 7 min. 2.4. PCR detection of cytK The presence of the cytK gene was studied with degenerated primers (Table 1). PCR was performed in 50 mL volumes with final concentrations of 1 PCR buffer (Applied Biosystems), 200 mM each of dNTPs (Applied Biosystems), 2 mM MgCl2, 0.25 mM of cytK-primers each (Table 1), Taq DNA polymerase (Applied Biosystems) 2.5 units per reaction and with 1 mL template DNA. The temperature cycling profile was initial denaturation at 95 1C for 1 min, 30 cycles with denaturation at 95 1C for 1 min, annealing at 58 1C for 1 min and elongation at 72 1C for 1 min, and with a final cycle at 72 1C for 7 min (EhlingSchultz et al., 2006). 2.5. Toxin production analysis
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Cultivation at 30 1C has previously been shown to be suitable for detection of toxin production among psychrotrophic milk isolates (Christiansson, Naidu, Nilsson, Wadstro¨m, & Pettersson, 1989) as well as pathogenic strains (Guinebretie`re, Broussolle, & Nguyen-The´, 2002). A sample of each culture was taken out aseptically, diluted 1:10 and the absorbance measured at 550 nm. If the value of A550 was less than 0.4 in the non-diluted sample, the cultures were left for another 24 h, otherwise they were processed further (not less than 1 108 cfu mL1 was required). The tests were performed according to the instructions provided with the RPLA and Tecra kits. A semi-quantitative estimation of the product formed was made visually on a 1–5-grade scale as described in the manufacturer’s instructions for use of the kits. Strains giving an index of 4 or 5 on supernatants diluted 1:10 were considered to be high producers of toxin. 2.6. Bioassay for emetic toxin The strains were surface streaked on TSA (Trypticase Soy Agar, Oxoid/Becton Dickinson, Boule Nordic, Huddinge, Sweden) plates and incubated at room temperature for 48–72 h. A loopful (1 mL size) of colony was added to 200 mL of methanol and boiled for 20 min. A sperm motility assay was performed to identify emetic toxin producing strains (Andersson et al., 2004). 2.7. Statistical analysis
Production of enterotoxin component NheA was measured with Bacillus diarrhoeal enterotoxin visual immunoassay (TECRAs, diarrhoeal enterotoxin immunoassay, Bioenterprises Pvt. Ltd., Roseville, New South Wales, Australia) and production of enterotoxin component HblC was measured with Bacillus cereus Enterotoxin Test Kit (BCET-RPLA; Oxoid, Basingstoke, UK). The strains were cultured in 10–15 mL of Brain Heart Infusion broth (Oxoid) with 0.1% glucose for 24 h at 30 1C with shaking.
Statistical analyses for significant differences between proportions (e.g. the prevalence of highly toxigenic producers of toxin among farm and dairy isolates) was performed by the Pearson w2 test using SYSTATs (SPSS Inc., Chicago, IL, USA) ver. 9 for Windows. 3. Results and discussion 3.1. Mesophilic and psychrotrophic strains
Table 1 Primers used for PCR detection of enterotoxin genes Primer
Sequence
Fragment size
nheA 7173 F (Forward)a nheA 7174 R (Reverse)a hblC L2A (Forward)b hblC L2B (Reverse)b CytK F2c CytK R5c
50 -GCTCTATGAACTAGCAGGAAAC-30
560 bp
a
50 -GCTACTTACTTGATCTTCACCG-30 50 -AATGGTCATCGGAACTCTAT-30 50 -CTCGCTGTTCTGCTGTTAAT-30 50 -ACAGATATCGGICAAAATGC-30 50 -CAAGTIACTTGACCIGTTGC-30
Stenfors and Granum (2001). Hansen and Hendriksen (2001). c Ehling-Schultz et al. (2006). b
749 bp
421 bp
All strains were analysed for the major cold-shock protein using the PCR method described by Francis et al. (1998). The overall proportion of strains with the psychrotrophic cspA signature was 58% (Table 2). Twenty-five percent of the farm isolates (it is normal to have 25% psychrotrophic isolates during in-door conditions); 49% of the silo tank isolates and 80% among the dairy isolates were psychrotrophic according to PCR. This increase was expected since most of the dairy isolates from the production line of pasteurised milk were isolated from milk samples following storage at 7 1C. Thus psychrotrophic strains were selected for. Generally there was a good agreement between the PCR detection of cspA signatures and growth in TG-broth at 8 1C (data not shown). Almost all psychrotrophic strains were therefore considered to be B. weihenstephanenis.
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Table 2 Incidence of toxin genes (nheA, hblC and cytK) and expression of gene products (NheA and HblC) among Bacillus cereus isolates from the dairy production chain Origin
Number of strains
Percent psychrotrophic strainsa
Percent nheA positive strainsb
Percent NheA positive strainsc
Percent hblC positive strainsb
Percent HblC positive strainsd
Percent cytK positive strainsb
Farm Silo Dairy All strains
100 100 196 396
25 49 80 58
94 76 84 84
78 72 72 74
46 51 54 51
70 77 74 74
43 27 7 21
a
PCR positive for cold-shock protein. PCR. c TECRA kit, 1/10 dilution. See Section 2 for details. d BCET-RPLA kit, 1/10 dilution. See Section 2 for details. b
3.2. Enterotoxins NHE and HBL—PCR analyses and antibody tests In total, 84% of all strains were positive in PCR for the nheA-gene, 51% were PCR-positive for the hblC-gene (Table 2) and 91% positive for at least one gene. Expression of nheA and hblC, for cells grown at 30 1C, was analysed using the TECRA- and the BCET-RPLAkits. The PCR and the antibody test did not always match. Some strains were negative in PCR but positive in the antibody tests, and vice versa. Fewer strains were positive in the TECRA antibody test (74%) than in PCR (84%), for all isolates together (Table 2). This discrepancy is most likely due to some strains producing too little toxin to be detectable in diluted samples. Te Giffel, Beumer, Granum, and Rombouts (1997) found that for a substantial fraction of milk isolates, toxin production was not detectable without concentration. In other investigations, supernatants were concentrated 5–20 times before testing (Granum et al., 1996; Schoeni & Wong, 1999; Stenfors & Granum, 2001). On the other hand, the nhe genes seem to be present in a majority of strains of B. cereus (Granum, 2001). The comparatively low figure of 84% PCR-positive in our investigation therefore points to the existence of strains that were not detected with the primers used. The expression of hblC at 30 1C among all strains, as analysed by BCET-RPLA, gave more positive strains (74%) than in the PCR analysis (51%). These tendencies were similar for all strain sets (Table 2). A similar pattern was seen for both mesophilic and psychrotrophic strains (data not shown). Guinebretie`re et al. (2002) found that for both hbl and nhe, a substantial number of strains were not detected based on PCR with published primers compared with Southern blotting. This was true among isolates from cooked chilled foods and vegetables as compared with food poisoning strains, which indicates a higher degree of polymorphism in the food-related strains (Guinebretie`re et al., 2002). This also seems to be the case among our milk strains. The low percentage of hblC-positive strains by PCR and the much higher percentage found using the BCET-RPLA in our investigation is in line with their
observation. They also found that clearly positive TECRA and BCET-RPLA tests always corresponded to strains carrying the nhe and hbl genes based on Southern blotting (Guinebretie`re et al., 2002). Our results indicate that the primers used for nheA and hblC were not able to detect some strains with gene polymorphisms. Therefore, the PCR results cannot be interpreted separately. They can be used to find strains that are TECRA or BCET-RPLA negative but do have the gene, i.e., are potentially toxigenic. New primers for the nhe and hbl genes have been developed and validated after this investigation as a result of the observations of gene polymorphisms (Ehling-Schultz et al., 2006). Based on the findings of Guinebretie`re et al. (2002) the results from the antibody tests, although probably not perfect (some toxins may not have been detected by the antibodies), can be used for the purpose of this investigation. As the culture supernatants were diluted tenfold, false positive results with the tests are very unlikely. Furthermore, the results will represent the most toxigenic strains, i.e. those of importance for food safety. Guinebretie`re et al. (2002) found that food-poisoning strains gave high positive test scores with the kits. In the following, only results from antibody tests for NHE and HBL are discussed. Both gene products were detected at 30 1C in more than half of the strains (58%), as shown by the antibody tests. Sixteen percent of the strains expressed either NheA or HblC and not the other gene. For 10% of the strains, none of the gene products were detected (data not shown). There was no significant difference (P40.1) within the strain sets. Furthermore, there was no significant difference between the proportion of mesophilic and psychrotrophic strains within the different gene combinations (P40.05). Fiftyeight percent of both the psychrotrophic and the mesophilic isolates expressed both genes under the conditions tested. The production of the toxin components was further evaluated semi-quantitatively based on a 1–5 index as suggested in the instructions provided with the RPLA and Tecra kits. Fourteen percent and 39% of all strains were high producers of NheA and HblC (index 4–5),
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respectively. Ten percent produced high levels of both proteins. There were more high producers of HblC than of NheA in all of the strain sets (Fig. 1). This was true for both mesophilic and psychrotrophic isolates (data not shown). The prevalence of high producers of both toxins, among isolates from dairies, were significantly fewer (Po0.01) than for farm or silo tank strains. Among the psychrotrophic isolates, a high expression of both toxin genes was rare (Fig. 2a). This explains why there were fewer high-toxin producers among the dairy isolates. Only 3% of all psychrotrophic strains produced high levels of both proteins. Thirty-one percent were high producers of HblC as compared with 59% among the mesophilic strains. The proportion of psychrotrophic strains that 60 Farms Silo tanks Dairies
50
% isolates
40 30 20 10 0 NheA
HblC
NheA+HblC
Fig. 1. Prevalence of B. cereus strains, among isolates from farms, silo tanks and dairies, that produced high levels of enterotoxin components NheA (subunit A of the nonhemolytic enterotoxin NHE) and HblC (subunit C of the haemolytic enterotoxin HBL) following growth at 30 1C in brain heart infusion broth with 0.1% glucose. High toxin level was defined as index 4–5 on a semi-quantitative scale, for cell supernatants diluted 1:10, when tested with the RPLA and Tecra immunological kits (see Section 2 for details).
70 60
A
B
Mesophilic Psychrotrophic
% isolates
50
1205
produced high levels of both toxins was higher among the silo tank isolates (five of 49) than for the other two strain sets; one of 25 farm isolates and notably none (out of 156) among the dairy isolates (data not shown). 3.3. Prevalence of the cytK gene In total, 84 (21%) of the 396 examined strains were cytK+ (Table 2), with a decreasing incidence along the production chain. All positive isolates were mesophilic (Fig. 2). In total, 52% of the mesophilic isolates were positive for cytK. Fifty-seven percent, 53% and 36% for mesophilic farm, silo tank and dairy isolates were cytK+, respectively. Guinebretie`re et al. (2002) found the cytKgene among 37% of 51 food-related strains and 73% of diarrhoeal strains. None of the positive strains in our investigation was psychrotrophic. Likewise, no cytK+ psychrotolerant B. cereus were found in sandy loam (Hendriksen, Munk Hansen, & Johansen, 2006). As for the nhe and hbl genes, the cytK gene shows some polymorphism (Fagerlund, Ween, Lund, Hardy, & Granum, 2004; Guinebretie`re et al., 2002). Therefore degenerated primers where developed to maximise the detection of cytK, and were validated (Ehling-Schultz et al., 2006). No method was available to evaluate the toxicity of CytK in our strains. 3.4. Emetic toxin-producing strains All strains were tested for emetic toxin production but only two were positive in the assay, one isolate from a milk sample collected at a farm and one isolate from a dairy silo tank. The very low prevalence (0.5%) is in the same range as in a previous investigation along the milk production chain (Svensson et al., 2006). The strains were HblCnegative and BCET-RPLA test negative, negative for cytK by PCR and gave a strong signal in the TECRA-test. They had the same phenotypic and genotypic traits as earlier reported for emetic strains of B. cereus (Ehling-Schulz, Svensson, Guinebretie`re, Lindba¨ck, Andersson, Schulz et al., 2005). Both strains also lacked the major cold shock protein and were not psychrotrophic. 3.5. Toxin-production potential of B. cereus strains in the dairy chain
40 30 20 10 0 NheA
HblC
NheA+HblC
cyt K gene
Fig. 2. Prevalence of mesophilic and psychrotrophic strains (% of strains in each category) that produced high levels of enterotoxin components NheA (subunit A of the nonhemolytic enterotoxin NHE) and HblC (subunit C of the haemolytic enterotoxin HBL) or both at 30 1C (a) and the prevalence of strains with the cytK gene (b).
To the best of our knowledge, this is the first investigation to simultaneously address the prevalence of all enterotoxins of B. cereus known to have been involved in food poisoning, and the emetic toxin, in a set of strains representing the entire dairy production chain from the farm to pasteurised milk. Previous investigations (see Section 1) are not readily comparable with the present one, since in these publications the strains were isolated with different methods, growth conditions, sample preparations, and the test conditions for PCR and cytotoxicity differed between investigations. B. cereus is a common contaminant
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in milk and the psychrotrophic strains can grow at refrigeration temperatures, causing spoilage of the milk. Presence of pathogenic traits in the psychrotrophic strains could therefore be a hazard to the consumers’ health. However, knowledge about toxigenicity of psychrotrophic strains in milk in comparison with mesophilic strains has been largely unknown. We found that there was no difference with respect to qualitative production of HblC and NheA between the strain sets (Table 2), and also with respect to psychrotrophic versus mesophilic strains. There were, however, important differences with respect to high toxin producers (Figs. 1 and 2). Our results indicate that psychrotrophic strains (mainly B. weihenstephanensis) are enriched in the milk during cold storage and that these strains have less toxin production potential than the mesophiles. The test methods that were used in this investigation are only indicative of the potential to produce toxins. In order to further assess the potential for toxigenicity, quantitative cell cytotoxicity tests will have to be performed. This was not possible for us, considering the number of strains. Choma et al. (2000) concluded that the most psychrotrophic isolates of B. cereus from commercial cooked chilled foods containing vegetables were the least cytotoxic. Cytotoxicity was not detectable for most of those strains without concentration of the culture supernatant fluid. Stenfors et al. (2002) investigated the pathogenic potential of 50 psychrotolerant B. cereus strains (B. weihenstephanensis) and found that 72% were not cytotoxic, an unusually high figure compared with B. cereus in general, according to the authors. Six strains were however highly cytotoxic in their semi-quantitative assay. A similarly low figure was found by Pru¨ss et al. (1999), i.e., nine out of 10 B. weihenstephanensis displayed a low cytotoxicity titre. These results are confirmed by the large strain set in this investigation. Twenty-six out of 37 isolates from Dutch pasteurised milk obtained from refrigerators were cytotoxic, but only for 17 of them following 20 concentration of the culture supernatant, i.e., 75% of the isolates were non-toxic or produced low levels of toxin (Te Giffel et al., 1997). With respect to foodborne strains in general, the percentage of strains where toxin genes are detected is therefore presumably less important than the potential amount of toxins that can be produced (Moravek et al., 2006). There is not yet a simple method to predict true toxicity by B. cereus in humans. Use of improved immunological methods with better antibodies (Moravek et al., 2006) as well as improved PCR methods that take into account the gene polymorphisms (Ehling-Schultz et al., 2006) will give better predictions. At present, a combination of immunology and PCR is needed to find the maximal number of potentially toxigenic strains. Kits for cytK will also be needed. Cytotoxicity on Vero cells was demonstrated to be dominated by Nhe rather than HBL, indicating that the former toxin may have the highest diarrhoeic potential (Moravek et al., 2006). This is interesting, considering the
very few high Nhe toxin producers among the psychrotrophic strains found in our study. Although B. cereus is commonly isolated from pasteurised milk, surprisingly few cases of foodborne illness caused by milk have been described (Christiansson et al., 1989). Notermans et al. (1997) estimated that 7% of the milk portions consumed in the Netherlands may contain more than 105 cfu mL1 at the time of consumption, indicating a substantial exposure of the Dutch population to psychrotrophic B. cereus from milk. In a double blind experiment, 34 healthy human volunteers consumed pasteurised milk that was stored at 7.5 1C for 3–14 d (Langeveld, Van Spronsen, Van Beresteijn, & Notermans, 1996). Only 18 cases of non-specific gastrointestinal complaints were observed upon 259 milk exposures, most of which occurred upon consumption of more than 1 107 B. cereus. Furthermore, Wijnands, Dufrenne, Zwietering, and Van Leusden (2006) found that spores of mesophilic B. cereus germinated better and grew faster in simulated gastro-intestinal conditions than spores from psychrotrophic strains. The slower growth rates of psychrotrophic strains may make them less important than mesophiles in the onset of diarrhoeal illness if consumed in equal numbers. 4. Conclusions Among the species in the B. cereus group, B. weihenstephanensis dominated in pasteurised milk stored at 7 1C by natural selection. Mesophilic isolates of B. cereus from the farm, in silo tanks and pasteurised milk were more frequently high producers of enterotoxin Hbl and Nhe, whereas B. weihenstephanensis were less toxigenic with respect to HblC, and very few produced high levels of NheA (recently indicated to be the major determinant of cytotoxicity in B. cereus). These observations may be important when considering microbiological criteria for B. cereus in pasteurised milk and possibly in other foods stored at low temperature. Acknowledgements This work has been carried out with the financial support from the Fifth European Community Framework Programme, under the Quality of Life and Management of Living Resources specific programme, Contract QLK12001-00854, Preventing Bacillus cereus food-borne poisoning in Europe—Detecting hazardous strains, tracing contamination routes and proposing criteria for foods. We thank Henrik Nilsson for developing the multiplex PCR-method. References Andersen Borge, G. I., Skeie, M., Sørhaug, T., Langsrud, T., & Granum, P. E. (2001). Growth and toxin profiles of Bacillus cereus isolated from
ARTICLE IN PRESS B. Svensson et al. / International Dairy Journal 17 (2007) 1201–1208 different food sources. International Journal of Food Microbiology, 69, 237–246. Andersson, M. A., Ja¨a¨skela¨inen, E. L., Shahen, R., Pirhonen, T., Wijnands, L. M., & Salkinoja-Salonen, M. S. (2004). Sperm bioassay for rapid detection of cereulide producing Bacillus cereus in food and related environments. International Journal of Food Microbiology, 15, 175–183. Beattie, S. H., & Williams, A. G. (1999). Detection of toxigenic strains of Bacillus cereus and other Bacillus spp. with an improved cytotoxicity assay. Letters in Applied Microbiology, 28, 221–225. Choma, C., Guinebretie`re, M. H., Carlin, F., Schmitt, P., Velge, P., Granum, P. E., et al. (2000). Prevalence, characterization and growth of Bacillus cereus in commercial cooked chilled foods containing vegetables. Journal of Applied Microbiology, 88, 617–625. Christiansson, A. (2003). Bacillus cereus. In H. Roginsky, J. W. Fuquay, & P. F. Fox (Eds.), Encyclopedia of dairy sciences (pp. 123–128). Amsterdam, The Netherlands: Academic Press. Christiansson, A., Bertilsson, J., & Svensson, B. (1999). Bacillus cereus spores in raw milk: Factors affecting the contamination of milk during the grazing period. Journal of Dairy Science, 82, 305–314. Christiansson, A., Ekelund, K., & Ogura, H. (1997). Membrane filtration method for enumeration and isolation of spores of Bacillus cereus from milk. International Dairy Journal, 7, 743–748. Christiansson, A., Magnusson, M., Nilsson, J., Ekelund, K., & Samuelsson, O. (1997). The occurrence of Bacillus cereus-spores in bulk tank milk during the indoor winter season. Rep. no. 114. SLU-JBT, Alnarp, Sweden. Christiansson, A., Naidu, A. S., Nilsson, I., Wadstro¨m, T., & Pettersson, H.-E. (1989). Toxin production by Bacillus cereus dairy isolates in milk at low temperatures. Applied and Environmental Microbiology, 55, 2595–2600. Ehling-Schultz, M., Guinebretie`re, M.-H., Montha´n, A., Berge, O., Fricker, M., & Svensson, B. (2006). Toxin gene profiling of enterotoxic and emetic Bacillus cereus. FEMS Microbiology Letters, 260, 232–240. Ehling-Schulz, M., Svensson, B., Guinebretie`re, M. H., Lindba¨ck, T., Andersson, M., Schulz, A., et al. (2005). Emetic toxin formation in Bacillus cereus is restricted to a single evolutionary lineage of closely related strains. Microbiology, 151, 183–197. Eneroth, A˚., Svensson, B., Molin, G., & Christiansson, A. (2001). Contamination of pasteurized milk by Bacillus cereus in the filling machine. Journal of Dairy Research, 68, 189–196. Fagerlund, A., Ween, O., Lund, T., Hardy, S. P., & Granum, P. E. (2004). Genetic and functional analysis of the cytK family of genes in Bacillus cereus. Microbiology, 150, 2689–2697. Francis, K. P., Mayr, R., von Stetten, F., Stewart, G. S. A. B., & Scherer, S. (1998). Discrimination of psychrotrophic and mesophilic strains of the Bacillus cereus group by PCR targeting of major cold shock protein genes. Applied and Environmental Microbiology, 64, 3525–3529. Granum, P. E. (2001). Bacillus cereus. In M. P. Doyle (Ed.), Food microbiology: Fundamentals and frontiers (pp. 373–381). Washington, DC, USA: ASM Press. Granum, P. E., Andersson, A., Gayther, C., Te Giffel, M., Larsen, H., Lund, T., et al. (1996). Evidence for a further enterotoxin complex produced by Bacillus cereus. FEMS Microbiology Letters, 141, 145–149. Granum, P. E., Brynestad, S., & Kramer, J. M. (1993). Analysis of enterotoxin production by Bacillus cereus from dairy products, food poisoning incidents and non-gastrointestinal infections. International Journal of Food Microbiology, 17, 269–279. Granum, P. E., OSullivan, K., & Lund, T. (1999). The sequence of the non-haemolytic enterotoxin operon from Bacillus cereus. FEMS Microbiology Letters, 177, 225–229. Guinebretie`re, M.-H., Broussolle, V., & Nguyen-The´, C. (2002). Enterotoxigenic profiles of food-poisoning and food-borne Bacillus cereus strains. Journal of Clinical Microbiology, 40, 3053–3056. Hansen, B. M., & Hendriksen, N. B. (2001). Detection of enterotoxic Bacillus cereus and Bacillus thuringiensis strains by PCR analysis. Applied and Environmental Microbiology, 67, 185–189.
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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. Journal of Bacteriology, 175, 6760–6766. Hendriksen, N. B., Munk Hansen, B., & Johansen, J. F. (2006). Occurrence and pathogenic potential of Bacillus cereus group bacteria in sandy loam. Antonie van Leeuwenhoek, 80, 239–249. In’t Veld, P. H., Ritmeester, W. S., Delfgou-van Asch, E. H. M., Dufrenne, J. B., Wernars, K., Smit, E., et al. (2001). Detection of genes encoding for enterotoxins and determination of the production of enterotoxins by HBL blood plates and immunoassay of psychrotrophic strains of Bacillus cereus isolated from pasteurised milk. International Journal of Food Microbiology, 64, 63–70. Langeveld, L. P. M., Van Spronsen, W. A., Van Beresteijn, E. C., & Notermans, S. H. W. (1996). Consumption by healthy adults of pasteurized milk with a high concentration of Bacillus cereus: A double blind study. Journal of Food Protection, 59, 723–726. Lin, S., Schraft, H., Odumeru, J. A., & Griffiths, M. W. (1998). Identification of contamination sources of Bacillus cereus in pasteurized milk. International Journal of Food Microbiology, 43, 159–171. Lund, T., De Buyser, M. L., & Granum, P. E. (2000). A new cytotoxin from Bacillus cereus that may cause necrotic enteritis. Molecular Microbiology, 38, 254–261. Lund, T., & Granum, P. E. (1996). Characterisation of a non-haemolytic enterotoxin complex from Bacillus cereus isolated after a foodborne outbreak. FEMS Microbiology Letters, 141, 151–156. Moravek, M., Dietrich, R., Buerk, C., Brousolle, V., Guinebretie`re, M.L., Granum, P. E., et al. (2006). Determination of the toxic potential of Bacillus cereus isolates by quantitative enterotoxin analyses. FEMS Microbiology Letters, 257, 293–298. Nilsson, J., Svensson, B., Ekelund, K., & Christiansson, A. (1998). A RAPD-PCR method for large-scale typing of Bacillus cereus. Letters in Applied Microbiology, 27, 168–172. Notermans, S., Dufrenne, J., Teunis, P., Beumer, R., te Giffel, M., & Peeters Weem, P. (1997). A risk assessment study of Bacillus cereus present in pasteurized milk. Food Microbiology, 14, 143–151. Pru¨ss, B. M., Dietrich, R., Nibler, B., Ma¨rtlbauer, E., & Scherer, S. (1999). The hemolytic enterotoxin HBL is broadly distributed among species of the Bacillus cereus group. Applied and Environmental Microbiology, 65, 5436–5442. Ryan, P. A., MacMillan, J. D., & Zilinskas, B. A. (1997). Molecular cloning and characterization of the genes encoding the L1 and L2 components of hemolysin BL from Bacillus cereus. Journal of Bacteriology, 179, 2551–2556. Schoeni, J. L., & Wong, A. C. L. (1999). Heterogeneity observed in the components of hemolysin BL, an enterotoxin produced by Bacillus cereus. International Journal of Food Microbiology, 53, 159–167. Schoeni, J. L., & Wong, A. C. L. (2005). Bacillus cereus food poisoning and its toxins. Journal of Food Protection, 68, 636–648. Stenfors, L. P., & Granum, P. E. (2001). Psychrotolerant species from the Bacillus cereus group are not necessarily Bacillus weihenstephanensis. FEMS Microbiology Letters, 197, 223–228. Stenfors, L. P., Mayr, R., Scherer, S., & Granum, P. G. (2002). Pathogenic potential of fifty Bacillus weihenstephanensis strains. FEMS Microbiology Letters, 215, 47–51. Svensson, B., Ekelund, K., Ogura, H., & Christiansson, A. (2004). Characterisation of Bacillus cereus isolated from milk silo tanks at eight different dairy plants. International Dairy Journal, 14, 17–27. Svensson, B., Eneroth, A˚., Bredehaug, J., & Christiansson, A. (1999). Investigation of Bacillus cereus contamination sites in a dairy plant with RAPD-PCR. International Dairy Journal, 9, 903–912. Svensson, B., Eneroth, A˚., Brendehaug, J., Molin, G., & Christiansson, A. (2000). Involvement of a pasteurizer in the contamination of milk by Bacillus cereus in a commercial dairy plant. Journal of Dairy Research, 67, 455–460. Svensson, B., Montha´n, A., Shaheen, R., Andersson, M. A., SalkinojaSalonen, M., & Christiansson, A. (2006). Occurrence of emetic toxin
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pasteurised milk in household refrigerators in the Netherlands. International Journal of Food Microbiology, 34, 307–318. Wijnands, L. M., Dufrenne, J. B., Zwietering, M. H., & Van Leusden, F. M. (2006). Spores from mesophilic Bacillus cereus strains germinate better and grow faster in simulated gastro-intestinal conditions than spores from psychrotrophic strains. International Journal of Food Microbiology, 112, 120–128.
International Dairy Journal 17 (2007) 1201–1208 www.elsevier.com/locate/idairyj
Toxin production potential and the detection of toxin genes among strains of the Bacillus cereus group isolated along the dairy production chain Birgitta Svenssona, Amanda Montha´na, Marie-He´le`ne Guinebretie`reb, Christophe Nguyen-The´b, Anders Christianssona, a Swedish Dairy Association, Research and Development, Scheeleva¨gen 18, SE 223 63 Lund, Sweden Institut National de la Recherche Agronomique, UMR A408 Se´curite´ et Qualite´ des Produits d’Origine Ve´ge´tale, INRA, Domaine Saint-Paul, Site Agroparc, F 84914 Avignon Cedex 9, France
b
Received 7 July 2006; accepted 4 March 2007
Abstract Isolates of the Bacillus cereus group (396 in total) from farms, silo tanks and production lines for pasteurised milk were tested for toxin production potential, and by polymerase chain reaction (PCR) for the presence of toxin genes. Comparison between the tests indicated the presence of gene polymorphisms. Highly toxigenic strains, based on production of subunit A of the nonhemolytic enterotoxin, NHE (NheA) and subunit C of the haemolytic enterotoxin, HBL (HblC), were less common among dairy isolates compared with farm and silo isolates. No producer of high levels of both toxins was found among 156 psychrotrophic dairy isolates (B. weihenstephanensis) and only 3% of all psychrotrophs were high producers of NheA. Psychrotrophic B. cereus from pasteurised milk appeared to have a low enterotoxin production potential, and they were not producers of emetic toxin or cytotoxin K and therefore may be less likely to cause illness than mesophilic strains. r 2007 Elsevier Ltd. All rights reserved. Keywords: Bacillus cereus; Enterotoxins; Emetic toxin; Toxin production; Psychrotrophic; Milk
1. Introduction Bacillus cereus is a common cause of two types of foodborne illnesses, characterized by abdominal cramps and diarrhoea 8–16 h following ingestion (the diarrhoeal type), and by vomiting 1–5 h after ingestion of the incriminated food (the emetic type); (Granum, 2001; Schoeni & Wong, 2005). The diarrhoeal disease is caused by several enterotoxins, i.e. the haemolytic toxin (HBL) the nonhemolytic toxin (NHE) and by cytotoxin K. HBL and NHE are both multicomponent protein toxins, each consisting of three immunologically distinct subunits. Cytotoxin K consists of only one protein. These toxins Corresponding author. Tel.: +46 46 19 25 81; fax: +46 46 13 70 40.
E-mail address: [email protected] (A. Christiansson). 0958-6946/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2007.03.004
are heat labile and are probably formed during bacterial growth in the small intestine. The emetic toxin is a small ring-forming peptide, cereulide, which acts as a potassium ionophore, and is extremely heat stable. It is produced during growth in food, that is stored at an inappropriate temperature. For reviews see Granum (2001) and Schoeni and Wong (2005). Methods to detect the toxin genes have been proposed in terms of polymerase chain reaction (PCR) primers for toxin (sub)units, once the toxins were cloned and sequenced (Granum, OSullivan, & Lund, 1999; Heinrichs, Beecher, Macmillan, & Zilinskas, 1993; Lund, De Buyser, & Granum, 2000; Ryan, MacMillan, & Zilinskas, 1997). However, these primers have not undergone stringent validation on a large variety of strains. Two commercial immunological kits were available for subunit C of the haemolytic enterotoxin (HblC), the Bacillus cereus
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enterotoxin reverse passive latex agglutination (RPLA) kit (Oxoid, Basingstoke, England), and for subunit A of the nonhemolytic enterotoxin (NheA), the Bacillus diarrhoeal enterotoxin visual immunoassay (Tecra Diagnostics, Roseville, NSW, Australia). Some authors have also used proprietary antibodies. For the emetic toxin, a rapid test based on mobility of boar sperm cells has been developed (Andersson et al., 2004). For an overall estimation of toxicity, various cell cytotoxicity tests based on Vero, HEp2 and Caco cells, for example, can be used (Beattie & Williams, 1999; Lund & Granum, 1996; Pru¨ss, Dietrich, Nibler, Ma¨rtlbauer, & Scherer, 1999). However, the cytotoxicity tests are difficult to perform and are not appropriate for screening large number of strains. Bacillus cereus is a common contaminant of milk (Christiansson, 2003). Some investigations have addressed the occurrence of toxin genes and toxin production of strains in milk and dairy products (Beattie & Williams, 1999; Granum, Brynestad, & Kramer, 1993; In’t Veld et al., 2001). Milk strains have been included in other investigations (Andersen Borge, Skeie, Sørhaug, Langsrud, & Granum, 2001; Granum et al., 1996; Pru¨ss et al., 1999; Schoeni & Wong, 1999; Stenfors & Granum, 2001; Stenfors, Mayr, Scherer, & Granum, 2002), but none monitored all toxins simultaneously. However, the tested strains were generally from pasteurised dairy products and not from earlier parts of the production chain. It is known that the major contamination of milk by B. cereus occurs at the farm, and additional contamination can occur in the dairy plant (Christiansson, Bertilsson, & Svensson, 1999; Lin, Schraft, Odumeru, & Griffiths, 1998; Svensson, Ekelund, Ogura, & Christiansson, 2004; Te Giffel, Beumer, Bonestroo, & Rombouts, 1996). However nothing is known about the properties of strains originating from the farm in comparison with the strains in the final product. In order to obtain a better characterization of the hazard posed by B. cereus, the occurrence of toxigenic strains along the whole chain should be addressed. Previously, we have investigated the routes of contamination of spores of B. cereus to milk at the farm, in silo tanks at dairy plants and along the production line for pasteurised milk (see Svensson et al. (2004) and references therein). Based on a selection of strains (characterized by random amplified polymorphic DNA (RAPD) PCR) from these investigations, we tested 396 strains in this work for the presence of toxin genes and toxin production potential. Particular emphasis was given to the properties of psychrotrophic strains as compared to mesophilic strains since there is a selection for psychrotrophs in the production chain due to the low temperature at which milk is stored.
and site of isolation. We did not try to differentiate between species/subspecies within the B. cereus group since the presence of toxin genes has been demonstrated in all group members (Hansen & Hendriksen, 2001; Stenfors et al., 2002). Thus B. cereus, B. thuringiensis, B. mycoides, B. pseudomycoides and B. weihenstephanensis all might have been included in the collection. However, strains that grow at low temperatures and possess the cspA-gene are generally found to be B. weihenstephanensis (Francis, Mayr, von Stetten, Stewart, & Scherer, 1998; Stenfors et al., 2002). One hundred strains from nine different dairy farms were selected from a collection of 950 isolates. The strains had been isolated from 84 different samples of milk, water, used bedding material, manure, feed and air during earlier investigations of B. cereus in the in-door housing environment (Christiansson, Magnusson, Nilsson, Ekelund, & Samuelsson, 1997). From dairy silo tanks, 100 strains were selected from a collection of 1384 strains. The strains had been isolated by filtration (Christiansson, Ekelund, & Ogura, 1997) of milk from eight different dairy plants (Svensson et al., 2004). The 100 selected isolates came from 46 different samples. During investigations of contamination routes for B. cereus in dairy plants (Eneroth, Svensson, Molin, & Christiansson, 2001; Svensson, Eneroth, Bredehaug, & Christiansson, 1999; Svensson, Eneroth, Brendehaug, Molin, & Christiansson, 2000) approximately 2800 strains were isolated from the dairy production chain. From this, collection 196 strains were selected from two different dairies, and were isolated from 102 samples collected at six different sampling points in the dairy. B. cereus spores were isolated from unpasteurised milk samples by filtration after heat treatment. Pasteurised milk samples were incubated at 7 1C for 7–11 d to simulate anticipated storage conditions and shelf life, and B. cereus were isolated on blood agar plates. 2.2. DNA preparation and analysis of mesophilic and psychrotrophic B. cereus
2. Materials and methods
B. cereus strains were cultured on tryptone glucose yeast extract agar (AB Kemikalia, Lund, Sweden) plates for 24–72 h. DNA was prepared as described earlier by freezing and boiling the cells (Nilsson, Svensson, Ekelund, & Christiansson, 1998). DNA preparations from strains that were difficult to lyse were made with the DNeasys kit from Qiagen (Qiagen Nordic AB, Solna, Sweden) according to the manufacturer’s manual for Gram-positive bacteria. Both preparation methods worked equally well for PCR. Analysis of mesophilic and psychrotrophic B. cereus was performed using PCR, based on a coldshock gene, according to Francis et al. (1998).
2.1. Selection of strains
2.3. Multiplex PCR on enterotoxin genes
The 396 strains were selected to provide maximum diversity between strains based on RAPD-PCR fingerprints
The prevalence of two enterotoxin genes, nheA and hblC was studied by a multiplex PCR. Reactions were
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performed in a volume of 50 mL with final concentrations of 1 PCR buffer (Applied Biosystems, Stockholm, Sweden), 200 mM each of dNTPs (Applied Biosystems), 2 mM MgCl2, nheA-primers (Stenfors & Granum, 2001) 0.6 mM each, and hblC-primers (Hansen & Hendriksen, 2001) 0.8 mM each (Table 1), Taq DNA polymerase (Applied Biosystems) 2.5 units per reaction and with 1 mL DNA-preparation as template. The PCRs were run with the following cycling conditions: initial denaturation at 95 1C for 1 min, 30 cycles with denaturation at 95 1C for 1 min, annealing at 52 1C for 1 min and elongation at 72 1C for 1 min, and with a final cycle at 72 1C for 7 min. 2.4. PCR detection of cytK The presence of the cytK gene was studied with degenerated primers (Table 1). PCR was performed in 50 mL volumes with final concentrations of 1 PCR buffer (Applied Biosystems), 200 mM each of dNTPs (Applied Biosystems), 2 mM MgCl2, 0.25 mM of cytK-primers each (Table 1), Taq DNA polymerase (Applied Biosystems) 2.5 units per reaction and with 1 mL template DNA. The temperature cycling profile was initial denaturation at 95 1C for 1 min, 30 cycles with denaturation at 95 1C for 1 min, annealing at 58 1C for 1 min and elongation at 72 1C for 1 min, and with a final cycle at 72 1C for 7 min (EhlingSchultz et al., 2006). 2.5. Toxin production analysis
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Cultivation at 30 1C has previously been shown to be suitable for detection of toxin production among psychrotrophic milk isolates (Christiansson, Naidu, Nilsson, Wadstro¨m, & Pettersson, 1989) as well as pathogenic strains (Guinebretie`re, Broussolle, & Nguyen-The´, 2002). A sample of each culture was taken out aseptically, diluted 1:10 and the absorbance measured at 550 nm. If the value of A550 was less than 0.4 in the non-diluted sample, the cultures were left for another 24 h, otherwise they were processed further (not less than 1 108 cfu mL1 was required). The tests were performed according to the instructions provided with the RPLA and Tecra kits. A semi-quantitative estimation of the product formed was made visually on a 1–5-grade scale as described in the manufacturer’s instructions for use of the kits. Strains giving an index of 4 or 5 on supernatants diluted 1:10 were considered to be high producers of toxin. 2.6. Bioassay for emetic toxin The strains were surface streaked on TSA (Trypticase Soy Agar, Oxoid/Becton Dickinson, Boule Nordic, Huddinge, Sweden) plates and incubated at room temperature for 48–72 h. A loopful (1 mL size) of colony was added to 200 mL of methanol and boiled for 20 min. A sperm motility assay was performed to identify emetic toxin producing strains (Andersson et al., 2004). 2.7. Statistical analysis
Production of enterotoxin component NheA was measured with Bacillus diarrhoeal enterotoxin visual immunoassay (TECRAs, diarrhoeal enterotoxin immunoassay, Bioenterprises Pvt. Ltd., Roseville, New South Wales, Australia) and production of enterotoxin component HblC was measured with Bacillus cereus Enterotoxin Test Kit (BCET-RPLA; Oxoid, Basingstoke, UK). The strains were cultured in 10–15 mL of Brain Heart Infusion broth (Oxoid) with 0.1% glucose for 24 h at 30 1C with shaking.
Statistical analyses for significant differences between proportions (e.g. the prevalence of highly toxigenic producers of toxin among farm and dairy isolates) was performed by the Pearson w2 test using SYSTATs (SPSS Inc., Chicago, IL, USA) ver. 9 for Windows. 3. Results and discussion 3.1. Mesophilic and psychrotrophic strains
Table 1 Primers used for PCR detection of enterotoxin genes Primer
Sequence
Fragment size
nheA 7173 F (Forward)a nheA 7174 R (Reverse)a hblC L2A (Forward)b hblC L2B (Reverse)b CytK F2c CytK R5c
50 -GCTCTATGAACTAGCAGGAAAC-30
560 bp
a
50 -GCTACTTACTTGATCTTCACCG-30 50 -AATGGTCATCGGAACTCTAT-30 50 -CTCGCTGTTCTGCTGTTAAT-30 50 -ACAGATATCGGICAAAATGC-30 50 -CAAGTIACTTGACCIGTTGC-30
Stenfors and Granum (2001). Hansen and Hendriksen (2001). c Ehling-Schultz et al. (2006). b
749 bp
421 bp
All strains were analysed for the major cold-shock protein using the PCR method described by Francis et al. (1998). The overall proportion of strains with the psychrotrophic cspA signature was 58% (Table 2). Twenty-five percent of the farm isolates (it is normal to have 25% psychrotrophic isolates during in-door conditions); 49% of the silo tank isolates and 80% among the dairy isolates were psychrotrophic according to PCR. This increase was expected since most of the dairy isolates from the production line of pasteurised milk were isolated from milk samples following storage at 7 1C. Thus psychrotrophic strains were selected for. Generally there was a good agreement between the PCR detection of cspA signatures and growth in TG-broth at 8 1C (data not shown). Almost all psychrotrophic strains were therefore considered to be B. weihenstephanenis.
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Table 2 Incidence of toxin genes (nheA, hblC and cytK) and expression of gene products (NheA and HblC) among Bacillus cereus isolates from the dairy production chain Origin
Number of strains
Percent psychrotrophic strainsa
Percent nheA positive strainsb
Percent NheA positive strainsc
Percent hblC positive strainsb
Percent HblC positive strainsd
Percent cytK positive strainsb
Farm Silo Dairy All strains
100 100 196 396
25 49 80 58
94 76 84 84
78 72 72 74
46 51 54 51
70 77 74 74
43 27 7 21
a
PCR positive for cold-shock protein. PCR. c TECRA kit, 1/10 dilution. See Section 2 for details. d BCET-RPLA kit, 1/10 dilution. See Section 2 for details. b
3.2. Enterotoxins NHE and HBL—PCR analyses and antibody tests In total, 84% of all strains were positive in PCR for the nheA-gene, 51% were PCR-positive for the hblC-gene (Table 2) and 91% positive for at least one gene. Expression of nheA and hblC, for cells grown at 30 1C, was analysed using the TECRA- and the BCET-RPLAkits. The PCR and the antibody test did not always match. Some strains were negative in PCR but positive in the antibody tests, and vice versa. Fewer strains were positive in the TECRA antibody test (74%) than in PCR (84%), for all isolates together (Table 2). This discrepancy is most likely due to some strains producing too little toxin to be detectable in diluted samples. Te Giffel, Beumer, Granum, and Rombouts (1997) found that for a substantial fraction of milk isolates, toxin production was not detectable without concentration. In other investigations, supernatants were concentrated 5–20 times before testing (Granum et al., 1996; Schoeni & Wong, 1999; Stenfors & Granum, 2001). On the other hand, the nhe genes seem to be present in a majority of strains of B. cereus (Granum, 2001). The comparatively low figure of 84% PCR-positive in our investigation therefore points to the existence of strains that were not detected with the primers used. The expression of hblC at 30 1C among all strains, as analysed by BCET-RPLA, gave more positive strains (74%) than in the PCR analysis (51%). These tendencies were similar for all strain sets (Table 2). A similar pattern was seen for both mesophilic and psychrotrophic strains (data not shown). Guinebretie`re et al. (2002) found that for both hbl and nhe, a substantial number of strains were not detected based on PCR with published primers compared with Southern blotting. This was true among isolates from cooked chilled foods and vegetables as compared with food poisoning strains, which indicates a higher degree of polymorphism in the food-related strains (Guinebretie`re et al., 2002). This also seems to be the case among our milk strains. The low percentage of hblC-positive strains by PCR and the much higher percentage found using the BCET-RPLA in our investigation is in line with their
observation. They also found that clearly positive TECRA and BCET-RPLA tests always corresponded to strains carrying the nhe and hbl genes based on Southern blotting (Guinebretie`re et al., 2002). Our results indicate that the primers used for nheA and hblC were not able to detect some strains with gene polymorphisms. Therefore, the PCR results cannot be interpreted separately. They can be used to find strains that are TECRA or BCET-RPLA negative but do have the gene, i.e., are potentially toxigenic. New primers for the nhe and hbl genes have been developed and validated after this investigation as a result of the observations of gene polymorphisms (Ehling-Schultz et al., 2006). Based on the findings of Guinebretie`re et al. (2002) the results from the antibody tests, although probably not perfect (some toxins may not have been detected by the antibodies), can be used for the purpose of this investigation. As the culture supernatants were diluted tenfold, false positive results with the tests are very unlikely. Furthermore, the results will represent the most toxigenic strains, i.e. those of importance for food safety. Guinebretie`re et al. (2002) found that food-poisoning strains gave high positive test scores with the kits. In the following, only results from antibody tests for NHE and HBL are discussed. Both gene products were detected at 30 1C in more than half of the strains (58%), as shown by the antibody tests. Sixteen percent of the strains expressed either NheA or HblC and not the other gene. For 10% of the strains, none of the gene products were detected (data not shown). There was no significant difference (P40.1) within the strain sets. Furthermore, there was no significant difference between the proportion of mesophilic and psychrotrophic strains within the different gene combinations (P40.05). Fiftyeight percent of both the psychrotrophic and the mesophilic isolates expressed both genes under the conditions tested. The production of the toxin components was further evaluated semi-quantitatively based on a 1–5 index as suggested in the instructions provided with the RPLA and Tecra kits. Fourteen percent and 39% of all strains were high producers of NheA and HblC (index 4–5),
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respectively. Ten percent produced high levels of both proteins. There were more high producers of HblC than of NheA in all of the strain sets (Fig. 1). This was true for both mesophilic and psychrotrophic isolates (data not shown). The prevalence of high producers of both toxins, among isolates from dairies, were significantly fewer (Po0.01) than for farm or silo tank strains. Among the psychrotrophic isolates, a high expression of both toxin genes was rare (Fig. 2a). This explains why there were fewer high-toxin producers among the dairy isolates. Only 3% of all psychrotrophic strains produced high levels of both proteins. Thirty-one percent were high producers of HblC as compared with 59% among the mesophilic strains. The proportion of psychrotrophic strains that 60 Farms Silo tanks Dairies
50
% isolates
40 30 20 10 0 NheA
HblC
NheA+HblC
Fig. 1. Prevalence of B. cereus strains, among isolates from farms, silo tanks and dairies, that produced high levels of enterotoxin components NheA (subunit A of the nonhemolytic enterotoxin NHE) and HblC (subunit C of the haemolytic enterotoxin HBL) following growth at 30 1C in brain heart infusion broth with 0.1% glucose. High toxin level was defined as index 4–5 on a semi-quantitative scale, for cell supernatants diluted 1:10, when tested with the RPLA and Tecra immunological kits (see Section 2 for details).
70 60
A
B
Mesophilic Psychrotrophic
% isolates
50
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produced high levels of both toxins was higher among the silo tank isolates (five of 49) than for the other two strain sets; one of 25 farm isolates and notably none (out of 156) among the dairy isolates (data not shown). 3.3. Prevalence of the cytK gene In total, 84 (21%) of the 396 examined strains were cytK+ (Table 2), with a decreasing incidence along the production chain. All positive isolates were mesophilic (Fig. 2). In total, 52% of the mesophilic isolates were positive for cytK. Fifty-seven percent, 53% and 36% for mesophilic farm, silo tank and dairy isolates were cytK+, respectively. Guinebretie`re et al. (2002) found the cytKgene among 37% of 51 food-related strains and 73% of diarrhoeal strains. None of the positive strains in our investigation was psychrotrophic. Likewise, no cytK+ psychrotolerant B. cereus were found in sandy loam (Hendriksen, Munk Hansen, & Johansen, 2006). As for the nhe and hbl genes, the cytK gene shows some polymorphism (Fagerlund, Ween, Lund, Hardy, & Granum, 2004; Guinebretie`re et al., 2002). Therefore degenerated primers where developed to maximise the detection of cytK, and were validated (Ehling-Schultz et al., 2006). No method was available to evaluate the toxicity of CytK in our strains. 3.4. Emetic toxin-producing strains All strains were tested for emetic toxin production but only two were positive in the assay, one isolate from a milk sample collected at a farm and one isolate from a dairy silo tank. The very low prevalence (0.5%) is in the same range as in a previous investigation along the milk production chain (Svensson et al., 2006). The strains were HblCnegative and BCET-RPLA test negative, negative for cytK by PCR and gave a strong signal in the TECRA-test. They had the same phenotypic and genotypic traits as earlier reported for emetic strains of B. cereus (Ehling-Schulz, Svensson, Guinebretie`re, Lindba¨ck, Andersson, Schulz et al., 2005). Both strains also lacked the major cold shock protein and were not psychrotrophic. 3.5. Toxin-production potential of B. cereus strains in the dairy chain
40 30 20 10 0 NheA
HblC
NheA+HblC
cyt K gene
Fig. 2. Prevalence of mesophilic and psychrotrophic strains (% of strains in each category) that produced high levels of enterotoxin components NheA (subunit A of the nonhemolytic enterotoxin NHE) and HblC (subunit C of the haemolytic enterotoxin HBL) or both at 30 1C (a) and the prevalence of strains with the cytK gene (b).
To the best of our knowledge, this is the first investigation to simultaneously address the prevalence of all enterotoxins of B. cereus known to have been involved in food poisoning, and the emetic toxin, in a set of strains representing the entire dairy production chain from the farm to pasteurised milk. Previous investigations (see Section 1) are not readily comparable with the present one, since in these publications the strains were isolated with different methods, growth conditions, sample preparations, and the test conditions for PCR and cytotoxicity differed between investigations. B. cereus is a common contaminant
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in milk and the psychrotrophic strains can grow at refrigeration temperatures, causing spoilage of the milk. Presence of pathogenic traits in the psychrotrophic strains could therefore be a hazard to the consumers’ health. However, knowledge about toxigenicity of psychrotrophic strains in milk in comparison with mesophilic strains has been largely unknown. We found that there was no difference with respect to qualitative production of HblC and NheA between the strain sets (Table 2), and also with respect to psychrotrophic versus mesophilic strains. There were, however, important differences with respect to high toxin producers (Figs. 1 and 2). Our results indicate that psychrotrophic strains (mainly B. weihenstephanensis) are enriched in the milk during cold storage and that these strains have less toxin production potential than the mesophiles. The test methods that were used in this investigation are only indicative of the potential to produce toxins. In order to further assess the potential for toxigenicity, quantitative cell cytotoxicity tests will have to be performed. This was not possible for us, considering the number of strains. Choma et al. (2000) concluded that the most psychrotrophic isolates of B. cereus from commercial cooked chilled foods containing vegetables were the least cytotoxic. Cytotoxicity was not detectable for most of those strains without concentration of the culture supernatant fluid. Stenfors et al. (2002) investigated the pathogenic potential of 50 psychrotolerant B. cereus strains (B. weihenstephanensis) and found that 72% were not cytotoxic, an unusually high figure compared with B. cereus in general, according to the authors. Six strains were however highly cytotoxic in their semi-quantitative assay. A similarly low figure was found by Pru¨ss et al. (1999), i.e., nine out of 10 B. weihenstephanensis displayed a low cytotoxicity titre. These results are confirmed by the large strain set in this investigation. Twenty-six out of 37 isolates from Dutch pasteurised milk obtained from refrigerators were cytotoxic, but only for 17 of them following 20 concentration of the culture supernatant, i.e., 75% of the isolates were non-toxic or produced low levels of toxin (Te Giffel et al., 1997). With respect to foodborne strains in general, the percentage of strains where toxin genes are detected is therefore presumably less important than the potential amount of toxins that can be produced (Moravek et al., 2006). There is not yet a simple method to predict true toxicity by B. cereus in humans. Use of improved immunological methods with better antibodies (Moravek et al., 2006) as well as improved PCR methods that take into account the gene polymorphisms (Ehling-Schultz et al., 2006) will give better predictions. At present, a combination of immunology and PCR is needed to find the maximal number of potentially toxigenic strains. Kits for cytK will also be needed. Cytotoxicity on Vero cells was demonstrated to be dominated by Nhe rather than HBL, indicating that the former toxin may have the highest diarrhoeic potential (Moravek et al., 2006). This is interesting, considering the
very few high Nhe toxin producers among the psychrotrophic strains found in our study. Although B. cereus is commonly isolated from pasteurised milk, surprisingly few cases of foodborne illness caused by milk have been described (Christiansson et al., 1989). Notermans et al. (1997) estimated that 7% of the milk portions consumed in the Netherlands may contain more than 105 cfu mL1 at the time of consumption, indicating a substantial exposure of the Dutch population to psychrotrophic B. cereus from milk. In a double blind experiment, 34 healthy human volunteers consumed pasteurised milk that was stored at 7.5 1C for 3–14 d (Langeveld, Van Spronsen, Van Beresteijn, & Notermans, 1996). Only 18 cases of non-specific gastrointestinal complaints were observed upon 259 milk exposures, most of which occurred upon consumption of more than 1 107 B. cereus. Furthermore, Wijnands, Dufrenne, Zwietering, and Van Leusden (2006) found that spores of mesophilic B. cereus germinated better and grew faster in simulated gastro-intestinal conditions than spores from psychrotrophic strains. The slower growth rates of psychrotrophic strains may make them less important than mesophiles in the onset of diarrhoeal illness if consumed in equal numbers. 4. Conclusions Among the species in the B. cereus group, B. weihenstephanensis dominated in pasteurised milk stored at 7 1C by natural selection. Mesophilic isolates of B. cereus from the farm, in silo tanks and pasteurised milk were more frequently high producers of enterotoxin Hbl and Nhe, whereas B. weihenstephanensis were less toxigenic with respect to HblC, and very few produced high levels of NheA (recently indicated to be the major determinant of cytotoxicity in B. cereus). These observations may be important when considering microbiological criteria for B. cereus in pasteurised milk and possibly in other foods stored at low temperature. Acknowledgements This work has been carried out with the financial support from the Fifth European Community Framework Programme, under the Quality of Life and Management of Living Resources specific programme, Contract QLK12001-00854, Preventing Bacillus cereus food-borne poisoning in Europe—Detecting hazardous strains, tracing contamination routes and proposing criteria for foods. We thank Henrik Nilsson for developing the multiplex PCR-method. References Andersen Borge, G. I., Skeie, M., Sørhaug, T., Langsrud, T., & Granum, P. E. (2001). Growth and toxin profiles of Bacillus cereus isolated from
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