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Keywords:

  • Bacillus cereus;
  • enterotoxin;
  • food;
  • growth;
  • porcine bile;
  • small intestine;
  • survival

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Aims:  To determine the effects of porcine bile (PB) on Bacillus cereus vegetative cells and Haemolysin BL (HBL) enterotoxin production in reconstituted small intestine media (IM).

Methods and Results:  The effects of PB on the growth of B. cereus vegetative cells in reconstituted IM at PB concentrations ranging between 0 and 3·0 g l−1 were examined. Four gastric media (GM) named GM-J broth (JB), GM-chicken, GM-milk and GM-pea were prepared by mixing equal volumes of a gastric electrolyte solution containing pepsin with JB, chicken, semi-skimmed milk and pea soup, respectively. Bacillus cereus was inoculated at approx. 2 × 104 CFU ml−1 into each GM at pH 5·0 for 30 min at 37°C, then mixed to the same volume of double-strength JB (IM) and PB to give concentrations of between 0 and 3·0 g of PB per litre at pH 6·5 and incubated at 37°C. The diarrhoeal B. cereus strain F4430/73 grew in IM-JB, IM-chicken and IM-milk at PB concentrations of up to 0·6, 1·5 and 1·2 g l−1, respectively. Growth was observed in IM-pea at all concentrations tested. The highest PB concentrations allowing a 3 log B. cereus increase in IM-JB, IM-chicken, IM-milk and IM-pea after a 7–10 h incubation period were 0·3, 0·9, 0·9 and 3·0 g l−1, respectively. The effect of PB on B. cereus cells was strongest in IM-JB, followed by IM-chicken, IM-milk and IM-pea. Haemolysin BL enterotoxin was detectable in IM-chicken, IM-whole milk, IM-semi-skimmed milk and IM-pea up to PB concentrations of only 0·6, 0·6, 0·3 and 0·9 g l−1, respectively. The diarrhoeal B. cereus strain F4433/73 behaved similarly to B. cereus strain F4430/73, whereas the food strain TZ415 was markedly more susceptible to bile.

Conclusions:  The tolerance of B. cereus cells to PB strongly depends on the type of food contained in the IM. Bile tolerance is also subject to strain variation.

Significance and Impact of the Study:  The probability that B. cereus cells will grow in the small intestine, produce toxins and cause diarrhoea is likely to depend on the food they are ingested with, on the bile tolerance of the B. cereus strain, and on bile concentration.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The pathogenic bacteria, Bacillus cereus, is a major cause of foodborne poisoning in many countries (Kramer and Gilbert 1989; Todd 1992; Todd 1996; Bean et al. 1997; Granum and Baird-Parker 2000; Lindqvist et al. 2000; Haeghebaert et al. 2002). Most food poisoning cases are due to the production of several diarrhoeal toxins (Lund and Granum 1997) in the small intestine that cause diarrhoeal syndrome (Kramer and Gilbert 1989; Granum 1994). High numbers of cells (>105 CFU g−1) probably have to be ingested along with the foods before they can cause disease (Kramer and Gilbert 1989) because the spores or vegetative cells have to survive stomach acidity before being able to multiply in the small intestine. High numbers of B. cereus vegetative cells are able to survive gastric transit (Clavel et al. 2004), particularly when mixed with foods buffering stomach acidity to a pH of up to 5·0. Cells surviving the gastric passage may be stressed by volatile fatty acids, an increase in pH after gastric transit, low oxygen and low redox potential, competition with commensal flora and high osmolarity when entering the small intestine (Chowdhury et al. 1996). Furthermore, bile salts (BS) contained in the bile secreted by the liver into the intestinal tract are also a major cause of bacterial stress (Begley et al. 2005). The ability of B. cereus to tolerate bile is likely to be a major factor for survival and multiplication in the small intestine and may thus influence the production of enterotoxins.

The aim of this study was to investigate the effect of porcine bile (PB) on B. cereus cells and Haemolysin BL (HBL) enterotoxin production in reconstituted small intestine media (IM) following acid stress in a simulated gastric transit. The PB concentrations tested were in the range of those reported in clinical studies (Gilliland et al. 1984; Goldin and Gorbach 1992; Armand et al. 1994; Armand et al. 1996). The food items added to the IM were selected to simulate different food components: chicken for protein, peas for starch and fibre, milk for various concentrations of fat and J broth (JB) for no food additives.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Bacterial strains

Bacillus cereus strain F4430/73 (B4ac) isolated from a pea soup (Spira and Goepfert 1975) and strain F4433/73 isolated from meat loaf (Melling et al. 1976) were gifts from Professor P.E. Granum of The Norwegian School of Veterinary Science, Oslo, Norway. Both these strains have been involved in a diarrhoeal foodborne outbreak. Bacillus cereus strain INRAAV TZ415 was isolated from vegetable-containing, cooked, chilled food at the Institut National de la Recherche Agronomique, Avignon, France (Choma et al. 2000). Stock cultures of spores and vegetative cells were kept at −20°C in water and in a 20% glycerol solution (v/v), respectively.

Growth media

Working cultures of B. cereus were grown in JB (5 g l−1 peptone, 15 g l−1 yeast extract, 3 g l−1 K2HPO4, 2 g l−1 glucose, adjusted to pH 7·2) or in food media. JB is recommended for the culture of Bacillus species (Claus and Berkeley 1986) and has been used to successfully grow B. cereus in our laboratory. Glucose was added aseptically after autoclaving (20 min, 121°C) using a 0·22 μm filter-sterilized glucose solution. The food media used were commercial whole milk, semi-skimmed milk, skimmed milk (each UHT sterilized), split pea purée and chicken meat. Split pea purée was obtained by mixing split pea and demineralized water (1/10 w/v), then autoclaving at 121°C for 20 min. Chicken meat medium was obtained by boiling chicken breast in demineralized water (1/2 w/v), then blending and filtering through a 0·7-mm nylon mesh. Chicken media aliquots were stored at −20°C until use. J agar (JA) containing JB plus agar (15 g l−1) was used for plate counts.

Inocula

Vegetative cell inocula were prepared from stock culture purified on JA. One pure colony was transferred into 250-ml flasks containing 40 ml of either JB or food media and cultivated at 30°C. Unless otherwise specified, B. cereus cultures were grown for 18 h under vigorous shaking (130 rev min−1) to achieve stationary growth phase.

Concentrations of vegetative cells were determined by duplicate plating of serial dilutions on JA.

Gastric media and gastric treatment

Gastric media (GM) were prepared as previously reported by Clavel et al. (2004). Briefly, 50 ml of a sterile gastric electrolyte solution (4·8 g l−1 NaCl, 1·56 g l−1 NaHCO3, 2·2 g l−1 KCl, 0·22 g l−1 CaCl2) (Ganzle et al. 1999) and 50 ml of either JB or each food media were sterilized by autoclaving at 121°C for 20 min. Each sterile GM was supplemented with 500 U l−1 of a 0·22 μm filter-sterilized pepsin solution (P6887, Sigma-Aldrich, Saint-Quentin-Fallavier, France) in water. The four GM are named GM-JB, GM-chicken, GM-milk (i.e. semi-skimmed milk unless stated otherwise) and GM-pea.

The pH of the GM was adjusted to 5·0 using sterile HCl at 6 mol l−1. The pH of each GM was measured at the beginning of each experiment with a Schott-Geräte 6820 electrode calibrated using freshly prepared buffers at pH 4·0 and pH 7·0 and a CG825 pH meter (Hofheim, Germany). Bacillus cereus vegetative cells were added to the GM to obtain target-starting populations of 2 × 104B. cereus CFU ml−1. The GM were then incubated for 30 min at 37°C under shaking (130 rev min−1) to simulate human stomach conditions. This gastric treatment does not modify B. cereus concentrations (Clavel et al. 2004).

Determination of porcine bile tolerance in different intestinal media

Intestinal media were formed using 50 ml of inoculated GM and 50 ml of double-strength JB (2 × JB). The pH of the IM was adjusted to 6·5 using sterile HCl at 6 mol l−1. The pH of each IM was measured at the beginning and at the end of each experiment. The tolerance of B. cereus to PB was determined by tracking changes in B. cereus counts in IM containing concentrations of PB ranging from 0 g l−1 to 3·0 g l−1. Each IM was supplemented with 10 ml of an autoclaved PB solution in water (B8631, Sigma-Aldrich). The four IM are named IM-JB, IM-chicken, IM-milk and IM-pea. IM made of whole, semi-skimmed and skimmed milk were prepared in order to test the effect of milk fat content on the tolerance of B. cereus to PB.

After gastric treatment, the inoculated GM was mixed with 2 × JB to obtain IM at target-starting populations of 104B. cereus CFU ml−1. IM (100 ml) was incubated at 37°C in 125 ml closed flasks without shaking. One millilitre volumes were sampled at regular time points and serially diluted in 0·2 mol l−1 sodium phosphate buffer at pH 7·0, and then spread on duplicate plates of JA using a spiral plate apparatus (Spiral système, Intersciences, Saint-Nom-la-Bretèche, France). For low bacterial concentrations, samples were manually spread onto JA. Cell concentrations are expressed as CFU ml−1. The limit of detection was taken as one colony on the lowest dilution plate, i.e. 10 CFU ml−1. Changes in B. cereus counts were tracked for each IM and PB concentration in at least duplicate samples.

Enterotoxin detection

Detection of the HBL produced in the IM selected for the experiment was performed 6 h after inoculation using the B. cereus enterotoxin reverse passive latex agglutination test kit (BCET-RPLA TD950, Oxoid, Dardilly, France), which detects the L2 component of HBL encoded by hblC (Beecher and Wong 1994; Buchanan and Schultz 1994). According to the manufacturer, test sensitivity is to 2 ng ml−1. When the test index was negative at a particular bile concentration, enterotoxin production was not examined at higher bile levels.

Modelling of growth curves and determination of growth parameters

The parameters of growth in the IM, i.e. lag time (Lag), time to a 1000-fold increase in initial count (T1000) and standard growth rate (μ), were determined using the model described by Baranyi (Baranyi et al. 1993).

When modelled curves did not closely fit the data, growth parameters were estimated graphically by the following procedure. Lag was estimated as the time leading up to the point at which the tangent to the exponential growth phase crossed the level of the lowest concentration. The μ value was derived from the slope of the tangent to the exponential growth phase. T1000 was estimated as the time taken for a 3 log CFU ml−1 increase in the initial B. cereus concentration. When no growth was detected, Lag was estimated by default as the duration of the experiment, with μ = 0.

Statistical analysis

Results were subjected to analysis of variance using the general linear model procedure (Systat, version 9, SPSS, Chicago, IL, USA) to test the effects of PB concentration, growth medium, milk fat content and strain, and their interactions on Lag, T1000 and μ.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In IM-chicken, B. cereus F4430/73 vegetative cells were able to grow at concentrations of up to 1·5 g of PB per litre (Fig. 1); no growth was observed for higher PB concentrations. The increase in PB concentration from 0 to 1·5 g l−1 extended Lag from 0 to 4 h, and extended T1000 from 4 to 12 h (Fig. 2).

image

Figure 1.  Changes in counts of a vegetative cell inoculum of Bacillus cereus strain F4430/73 in intestinal medium (IM)-chicken (see Materials and methods) at the following porcine bile (PB) concentrations: 0·0 g l−1 (bsl00001), 0·3 g l−1 (◆), 0·6 g l−1 (bsl00066), 0·9 g l−1 (×), 1·2 g l−1 (+), 1·5 g l−1 (bsl00043), 1·8 g l−1 (bsl00000) and 2·0 g l−1 (bsl00084). All curves were done from one inoculum preparation. The replicate experiment (same range of PB concentration and time of analysis, different inocula) gave similar results.

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image

Figure 2.  Changes in lag time (Lag) (a), and changes in the time to a 1000-fold increase in initial number (T1000) (b) of Bacillus cereus strain F4430/73 in intestinal media (IM)-JB (◆), IM-chicken (×), IM-milk (bsl00066) and IM-pea (bsl00041) (see Materials and methods) at porcine bile (PB) concentration ranging between 0 and 3·0 g l−1. Each symbol represents data from one experiment. Solid line represents the mean of experimental data for each IM. When no growth was detected, Lag was estimated by default as the duration of the experiment (dash line).

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In IM-pea, growth was observed at all the PB concentrations tested, i.e. between 0·3 and 3·0 g of PB per litre. No growth was observed in IM-milk at 1·5 g of PB per litre or higher, or in IM-JB at 0·9 g of PB per litre or higher (Fig. 2a). Increasing PB concentrations resulted in an overall increase in both Lag (< 0·001) and T1000 (< 0·001) (Fig. 2) together with a decrease in μ (< 0·001) (data not shown). The effect of PB on growth was strongly dependent on the nature of the IM-food. At PB concentrations between 0·6 and 1·2 g l−1, B. cereus growth in IM-pea was markedly stronger (lower Lag and T1000) than in IM-chicken and IM-milk, followed by IM-JB. There was a strong interaction (< 0·001) between media and PB concentration; an increase in PB concentration had a much stronger effect on B. cereus cells in IM-JB than in IM-chicken and in IM-milk, whereas it had only a marginal effect on IM-pea. Milk fat content had a slight effect on Lag (< 0·05) but no significant effect on T1000 or μ (> 0·05) (data not shown). After 6 h incubation in the presence of PB, the B. cereus concentration was as follows: IM-whole milk > IM-semi-skimmed milk > IM-skimmed milk (Table 1).

Table 1.   Haemolysin BL (HBL) enterotoxin detection and Bacillus cereus F4430/73 concentrations in six intestinal media (IM-pea, IM-whole milk, IM-semi-skimmed milk, IM-skimmed milk, IM-chicken and IM-JB) (see Materials and methods) after 6 h incubation at 37°C
Intestinal media (IM)Porcine bile g l−1 in IM
00·30·60·91·21·51·82·02·53·0
  1. nt, not tested; ngo, no growth observed; nso, no survival observed.

  2. *The indices correspond to the last supernatant dilution (in two-fold serial dilutions) for which enterotoxin remained detectable, with the Oxoid test kit. Strains with an index of 0 were considered negative. A single test (n = 1) in each culture was done.

IM-pea
 B. cereus (log10 CFU ml−1)8·4, 8·78·5, 8·48·3, 8·17·5, 8·16·5, 7·86·5, 7·55·5, 7·25·0, 6·14·9, 5·24·1, 4·7
 HBL toxin test index*64, 6464, nt64, nt2, 160, 0ntntntntnt
IM-whole milk
 B. cereus (log10 CFU ml−1)8·1, 8·77·1, 8·47·4, 7·96·6, 7·64·3, 3·83·3, 2·8nsonsonsonso
 HBL toxin test index64, 6464, 321, 320, 00, 0ntntntntnt
IM-semi-skimmed milk
 B. cereus (log10 CFU ml−1)8·6, 8·47·7, 8·27·4, 7·17·1, 5·74·1, 4·6ngonsonsonsonso
 HBL toxin test index4, nt2, nt0, ntnt0, ntntntntntnt
IM-skimmed milk
 B. cereus (log10 CFU ml−1)7·4, 8·36·9, 7·95·8, 7·44·3, 5·61·9, 4·6ngonsonsonsonso
 HBL toxin test index64, 640, 00, 0ntntntntntntnt
IM-chicken
 B. cereus (log10 CFU ml−1)8·2, 7·86·9, 7·76·8, 7·55·3, 5·03·8, 2·93·1, 1·52·5, 1·82·1, 2·3ngongo
 HBL toxin test index64, 321, 161, 20, 00, 0ntntntntnt
IM-JB
 B. cereus (log10 CFU ml−1)8·36·0ngongonsonsonsonsonsonso
 HBL toxin test index20ntntntntntntntnt

The diarrhoeal B. cereus strain F4433/73 behaved similar to B. cereus strain F4430/73 in IM-milk, whereas the food strain TZ415 was markedly more susceptible to PB (effect of the ‘strains’ factor on Lag, T1000 and μ was significant at < 0·001) (Fig. 3). Bacillus cereus strain TZ415 was unable to grow in IM-milk at PB concentrations higher than 0·2 g of PB per litre, while B. cereus strains F4430/73 or F4433/73 were still able to grow in IM-milk containing up to 1·2 g of PB per litre.

image

Figure 3.  Changes in lag time (Lag) (a), and changes in the time to a 1000 fold-increase in initial number (T1000) (b) of Bacillus cereus strains F4430/73 (bsl00066), F4433/73 (×) and INRAAV TZ415 (bsl00041) in IM-milk (see Materials and methods) at porcine bile (PB) concentration ranging between 0 and 1·5 g l−1. Each symbol represents data from one experiment. Solid lines represent the mean of experimental data for each B. cereus strain. When no growth was detected, Lag was estimated by default as the duration of the experiment (dash line).

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The HBL enterotoxin was detected in IM-pea, IM-whole milk, IM-semi-skimmed milk and IM-chicken at PB concentrations of up to 0·9, 0·6, 0·3 and 0·6 g l−1, respectively, while no enterotoxin was detected in IM-skimmed milk or IM-JB at any of the PB concentrations tested (Table 1). The HBL enterotoxin test index was positively correlated with B. cereus concentrations. However, at similar B. cereus concentrations in the absence of PB, there were between-food differences in the HBL test index, which reached 64 in IM-pea, IM-whole milk, IM-skimmed milk and IM-chicken but no more than 4 in IM-semi-skimmed milk and IM-JB.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Foodborne pathogens have to resist the action of bile if they are to survive in the human small intestine and subsequently cause disease. Bile reacts with cell membrane phospholipids and proteins and disrupts cellular homeostasis (Begley et al. 2005). In this study, we have shown that B. cereus cells are able to tolerate PB and that this tolerance strongly depends on the type of food they are in contact with. Bacillus cereus grew in all three test foods (chicken, milk and pea) containing PB at concentrations of up to 1·5 g of PB per litre. This concentration would correspond to approx. 3·0 mmol l−1, assuming that the PB solutions used in this study contained exclusively glycocholate or taurocholate, which are the two major conjugated human BS (Mallory et al. 1973). Hence, this concentration of PB is close to the 5·0 mmol of BS per litre in the human small intestine as reported by Hoffmann et al. (1983) and Begley et al. (2002).

In the present study, the minimal inhibitory concentration (MIC) of PB was higher than 0·9 g l−1, regardless of the IM. In a previous study, the MIC of deoxycholate and taurodeoxycholate on B. cereus strain Bactisubtil® was cited at 0·08 and 0·1 g l−1, respectively (Spinosa et al. 2000). These differences could be explained by: (i) a different composition of the PB, which contains several conjugated and unconjugated bile acids, among other components (Begley et al. 2005); (ii) a different method of estimating tolerance to PB, in particular, a possible stress resistance induced by the simulated gastric transit before exposure to PB or (iii) variability in BS tolerance between different strains of B. cereus. Furthermore, it has previously been reported that three probiotic B. cereus strains showed very different susceptibilities to 2 g of BS per litre in a simulated intestinal fluid (Duc et al. 2004). Generally speaking, B. cereus appears to have higher susceptibility to BS than other gram-positive or gram-negative bacteria (Gomez et al. 1997; Hyronimus et al. 2000; Spinosa et al. 2000).

As shown with lactic acid bacteria and Listeria monocytogenes, strains may have a high variability in their tolerance to BS (Chateau et al. 1994; Hyronimus et al. 2000; Begley et al. 2002). Furthermore, different methods of preparing the inocula may affect susceptibility: for instance, BS-adapted cultures show dramatically higher resistance than unadapted cultures (Leverrier et al. 2003).

Bacillus cereus growth was strongly affected by the food contained in the IM. This can be attributed to a protective effect of the food against the antibacterial activity of PB. In the present study, split pea purée showed a much higher protective effect against PB than chicken and milk. A protective effect of a food product against inactivation by BS has been previously observed on Bifidobacterium during growth in the presence of soymilk (Shimakawa et al. 2003) and on Lactobacillus curvatus during growth in the presence of meat (Ganzle et al. 1999), and was attributed to food components such as proteins. In the present study, a protective effect of proteins might explain the reduced activity of PB on B. cereus in IM-milk and IM-chicken that was not found in IM-JB. Begley et al. (2005) suggested that bacteria may not be exposed to bile in certain microenvironments created in vivo by the food matrix. Food components such as dietary fibre may bind BS and thus inhibit their toxic effects (Goel et al. 1998), and some are used as bile salt sequestrants because of their hypocholesterolemic properties. This phenomenon may be responsible for the strong protective effect observed in IM-pea, which is the food with the highest dietary fibre content among those tested (Souci et al. 2000). Finally, the antibacterial activity of BS may be lower in vivo than in vitro as BS complex in micelles may not be free to interact with bacteria. However, B. cereus growth in the pea-based food was much higher than in milk despite the observed protective effect of milk fat content. It should be noted that for gastric treatment the GM was at pH 5·0. Reports in the literature suggest that stomach acidity may vary with time within a day in relation to food ingestion, with age and some pathological situations. pH 5·0 was selected for the following reasons: (i) this pH is realistic in a number of situations reviewed in our previous paper (Clavel et al. 2004); in particular, pH 5·0 is observed in the human full stomach after ingestion of a meal (Armand et al. 1994) and (ii) this pH gives some chance to obtain a countable number of cells in IM, which is much more uncertain with lower test pHs. Future experiments are required to determine whether exposure to lower pHs will affect the bile tolerance of B. cereus.

This study is the first to report B. cereus enterotoxin production in the presence of PB. The HBL enterotoxin test results varied according to B. cereus concentration. However, at similar B. cereus concentrations, HBL production also varied between food media. Bacillus cereus HBL production is modulated by multiple factors including physiological state, temperature (Christiansson et al. 1989), redox potential (Zigha et al. 2006) and carbohydrate (Ouhib et al. 2006). Glucose is able to repress HBL toxin production, (Duport et al. 2004; Ouhib et al. 2006) which may explain the low concentrations obtained in IM-JB. However, for unexplained reason, the HBL index reached 64 in IM-whole milk and IM-skimmed milk while it reached only 4 in IM-semi-skimmed milk. The production of HBL is depending on growth, but whether the bile concentration has any effect cannot be determined in this experimental set-up. A recent study showed that rifampicin-resistant vegetative cells of B. cereus strain F4433/73R were unable to multiply or produce enterotoxins in the intestine of human-flora-associated rats, whereas the spores persisted (Wilks et al. 2006). However, this result cannot reliably be transposed to the human small intestine as the rat does not constitute an appropriate model for studying B. cereus virulence (Bishop et al. 1999).

Foods can modulate the risk of cellular multiplication and enterotoxin production after ingestion of a given number of B. cereus cells required to trigger the diarrhoeal syndrome. Therefore, the probability of B. cereus causing poisoning depends not only on the number and the form of cells ingested and stomach acidity at time of ingestion (Clavel et al. 2004) but also on the BS tolerance of the B. cereus population involved as well as bile secretion and the food ingested.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported by grants from the French Ministry of Research and the French Ministry of Agriculture as part of an ‘Aliment-Qualité-Sécurité 2000 R0013: Caractérisation de la virulence de Bacillus cereus’ contract.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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