Survival and germination of Bacillus cereus spores during in vitro simulation of gastrointestinal...

Title 1

Survival and germination of Bacillus cereus spores during in vitro simulation of 2

gastrointestinal transit occurred without outgrowth and enterotoxin production 3

4

Authors 5

Siele Ceuppens1,2, Mieke Uyttendaele1, Katrien Drieskens1,2, Marc Heyndrickx3,4, Andreja 6

Rajkovic1,5, Nico Boon2, Tom Van de Wiele2* 7

8

Affiliations 9

1 Ghent University, Faculty of Bioscience Engineering, Laboratory of Food Microbiology and 10

Food Preservation (LFMFP), Ghent, Belgium. 11

2 Ghent University, Faculty of Bioscience Engineering, Laboratory of Microbial Ecology and 12

Technology (LabMET), Ghent, Belgium. 13

3 Ghent University, Faculty of Veterinary Sciences, Department of Pathology, Bacteriology 14

and Poultry Diseases, Merelbeke, Belgium. 15

4 Institute for Agricultural and Fisheries Research (ILVO), Technology and Food Science 16

Unit, Melle, Belgium. 17

5 Belgrade University, Faculty of Agriculture, Department of Food Safety and Food Quality 18

Management, Belgrade-Zemun, Serbia. 19

20

* Correspondence to: Tom Van de Wiele; Ghent University, Faculty of Bioscience 21

Engineering, Laboratory of Microbial Ecology and Technology (LabMET), Coupure Links 22

653, B-9000 Gent, Belgium; phone: +32 (0)9 264 59 12; E-mail: [email protected]; 23

Webpage: http://www.labmet.ugent.be/. 24

25

Copyright © 2012, American Society for Microbiology. All Rights Reserved.Appl. Environ. Microbiol. doi:10.1128/AEM.02142-12 AEM Accepts, published online ahead of print on 24 August 2012

ABSTRACT 26

To study the gastrointestinal survival and enterotoxin production of the food-borne pathogen 27

Bacillus cereus, an in vitro simulation experiment was developed to mimic gastrointestinal 28

passage in 5 phases: 1) the mouth, 2) the stomach with gradual pH decrease and fractional 29

emptying, 3) the duodenum with high concentrations of bile and digestive enzymes, 4) 30

dialysis to ensure bile re-absorption and 5) the ileum with competing human intestinal 31

bacteria. Four different B. cereus strains were cultivated and sporulated in mashed potato 32

medium to obtain an inoculum of 7.0 log spores/mL. The spores showed survival and 33

germination during the in vitro simulation of gastrointestinal passage, but vegetative 34

outgrowth of the spores was suppressed by the intestinal bacteria during the final ileum phase. 35

No bacterial proliferation nor enterotoxin production was observed, despite the high inoculum 36

levels. Only small strain variability was observed: except for the psychrotrophic food isolate, 37

the spores of all strains survived well throughout the gastrointestinal passage. The in vitro 38

simulation experiments investigated the survival and enterotoxin production of B. cereus in 39

the gastrointestinal lumen. The obtained results support the hypothesis that localized 40

interaction of B. cereus with the host's epithelium is required for diarrhoeal food poisoning. 41

42

INTRODUCTION 43

According to the current hypothesis, B. cereus diarrhoeal food poisoning is caused by 44

destruction of epithelial cells in the small intestine due to enterotoxin production by 45

vegetative cells (16, 44). Those B. cereus cells originate from ingested vegetative cells that 46

survive gastric passage and/or from ingested spores, which germinate in the small intestine. 47

During vegetative growth, B. cereus produces various enterotoxins and virulence factors 48

implicated in diarrhoeal food poisoning, such as non-haemolytic enterotoxin (Nhe), 49

haemolysin BL (Hbl), cytotoxin K (CytK), enterotoxin FM (entFM), phospholipases C, 50

haemolysins, collagenases and cereolysins (6). The Nhe, Hbl and CytK toxins possess 51

haemolytic and cytotoxic activity due to pore-formation in the cell membrane (4, 14, 19, 25). 52

Phospholipases C damage human epithelial cells and the sub-epithelial matrix and also cause 53

haemolysis by enzymatic degradation of the cell membrane (15, 41). The enterotoxin EntFM, 54

haemolysins and degradative enzymes are not directly cytotoxic, but they contribute to the 55

cytotoxic and haemolytic activity of B. cereus and its adhesion to epithelial cells (2, 3, 26, 56

37). 57

In vitro experiments that simulate the physicochemical conditions, enzymatic digestive 58

activity and microbiological interactions in the gastrointestinal tract were developed (29, 30, 59

32). These in vitro simulations enable, amongst others, to study the bio-accessibility of food 60

contaminants and the effect of pre- and probiotics on the gastrointestinal microbiota (17, 38, 61

39). Although host interactions are excluded, these in vitro experiments can provide valuable 62

information regarding the survival of probiotic bacteria in different formulations during 63

gastrointestinal passage (27, 34). Similarly, in vitro experiments were used to assess the 64

survival and behaviour of food pathogens in the gastrointestinal lumen of the host after 65

ingestion. For example, subsequent batch incubation of B. cereus spores in gastric and 66

intestinal simulation media at 37 °C showed germination and growth for the majority of the 67

tested strains (43). However, this former study did not assess the enterotoxin production. 68

Other batch incubation studies with B. cereus under gastrointestinal conditions revealed an 69

important influence of the added food type and the bile concentration on the growth and 70

survival of B. cereus (10, 11). In contrast to the average gastric conditions simulated in batch 71

incubation, the in vivo gastric pH and residence time are highly variable parameters (7, 9, 13). 72

Similarly, digestive secretions in the proximal small intestine result in initially high bile and 73

enzyme concentrations in the duodenum followed by very low concentrations in the ileum due 74

to removal and re-absorption (33). These aspects were included in the current study by 75

developing a dynamic in vitro simulation experiment by continuous acid secretion and 76

fractional emptying of the gastric vessel and dialysis of the intestinal vessel. Moreover, 77

competition with intestinal microbiota was included during the ileum phase after dialysis, 78

since the indigenous microbial community has been shown to impact the intestinal survival of 79

B. cereus (8). 80

The aim of the present study was to investigate the survival and intestinal enterotoxin 81

production of B. cereus spores produced in mashed potato medium during dynamic in vitro 82

simulation of gastrointestinal passage with exclusion of host signals and influences. 83

84

MATERIALS AND METHODS 85

Bacillus cereus strains, cultivation and enumeration 86

The B. cereus strains (Table 1) were cultivated in Tryptone Soya Broth (TSB, Oxoid) for 24 h 87

at 30 °C and subcultured before inoculation in mashed potato medium. Mashed potato 88

medium was a mixture (50:50, w/w) of food solution (Table 2) and mashed potato flakes 89

(Mousline classic, Maggi) reconstituted with whole milk according to the manufacturer’s 90

recipe. Approx. 3 log CFU/mL vegetative B. cereus cells of the subculture were inoculated in 91

83 mL mashed potato medium in stomacher bags and incubated for 1 to 2 weeks at 30 °C 92

until 7 to 8 log spores/mL were produced, conditions depending on the strain. The day before 93

the simulation experiment, the spore concentration was adjusted to 7.0 log spores/mL with 94

fresh mashed potato medium and this spore inoculum was stored at 2 °C until use. Total B. 95

cereus concentrations were determined by plating the appropriate dilutions in Physiological 96

Peptone Salt solution (PPS, 8.5 g/L NaCl (Fluka) and 1 g/L neutralized bacteriological 97

peptone (Oxoid)) on Tryptone Soya Agar (TSA). Spore concentrations were determined by 98

plating on TSA after heating samples for 10 min at 80 °C. In the presence of intestinal 99

bacteria, i.e. during the ileum phase of the gastrointestinal simulation experiment, total B. 100

cereus numbers were monitored by quantitative real-time PCR (qPCR), because plate count 101

enumeration was no longer possible due to overgrowth of the plates by the intestinal bacteria 102

(5). 103

104

Simulation media 105

The composition of the simulation media was based on the media from the RIVM (National 106

Institute for Public Health and Environment, the Netherlands) in vitro digestion model 107

representing the fed state (18) and the Simulator of the Human Intestinal Microbial Ecosystem 108

(SHIME) reactor feed (35). The composition of all simulation media is presented in Table 2. 109

The volume ratio of the food / saliva / gastric / intestinal simulation medium was 1,5 / 1 / 2 / 110

4,5. 111

112

Gastrointestinal simulation experiments 113

The gastrointestinal simulation experiment comprised five phases: 1) the mouth, 2) the 114

stomach, 3) the duodenum, 4) dialysis and 5) the ileum (Figure 1). The stomach phase took 115

place in the gastric vessel, while the duodenum phase, dialysis and the ileum phase all 116

occurred subsequently in the intestinal vessel. The gastric and intestinal vessels were two 117

SHIME reactor vessels, i.e. double jacketed glass vessels of 1 L kept at 37 °C with continuous 118

stirring. The headspace of the gastric vessel consisted of normal atmospheric air, but the 119

intestinal vessel with the intestinal simulation medium was flushed for 30 min with pure 120

nitrogen gas to obtain anaerobic conditions. The experiments were performed in triplicate 121

with different B. cereus inocula on different days. 122

The B. cereus inoculum was produced in the mashed potato medium (see above). The mouth 123

phase consisted of mixing the mashed potato medium containing the spores (83 mL 124

containing 7.0 log spores/mL) with 56 mL saliva medium (37 °C, pH 6.5) by stomaching for 125

1 min (Stomacher Lab Blender 400, Seward). At the beginning of the stomach phase, the pH 126

in the gastric vessel was manually set at exactly 5.00 (± 0.02). Then, the pH was decreased 127

from 5.0 to 3.0 during the first 90 min by automatically and continuously added acid (0.18 M 128

HCl) at 0.2 mL/min (Masterflex L/S Economy Digital Drive with 0.51 mm tubing (PharMed 129

BPT, Saint-Gobain Performance Plastics)) and at 0.1 mL/min during the last 90 min to reach 130

pH 2.0. Gastric emptying was initiated 30 min after the start of the gastric phase by 131

discontinuous pumping (Masterflex L/S Economy Digital Drive, 2 mL/min). The contents of 132

the gastric vessel were transferred to the intestinal vessel in 5 fractions (indicated as grey 133

areas in Figure 2) by discontinuous pumping in such a way that approx. 25 % of the gastric 134

content was removed after 1 h, 50 % after 2 h and 75 % after 3 h. The fractional gastric 135

emptying resulted in a 150 min overlap between the stomach and duodenum phase, in which 136

the B. cereus inoculum was divided in subpopulations which were subjected to various 137

different incubation times in the stomach phase (min. 30 min, max. 180 min) and duodenum 138

phase (min. 10 min, max. 160 min). The duodenal pH fluctuates randomly between 5.0 and 139

6.0 in healthy people who consumed a solid meal (13). Therefore, the pH in the intestinal 140

vessel was automatically adjusted by a pH controller (FerMac 260, Electrolab) to remain at 141

pH 5.0 and at pH 6.0 during the first 45 min and the last 115 min of the duodenum phase 142

respectively, at pH 6.5 during the dialysis and at pH 7.3 during the ileum phase. An overview 143

of the dynamically changing parameters during the stomach and duodenum phase and the 144

dialysis is presented in Figure 2. 145

The consumption of food induces bile secretion in the small intestine in the range of 7 to 15 146

mM bile salts, which corresponds with 5 to 10 g/L bile (18, 29, 33). Dialysis to ensure ≥ 90 % 147

(w/v) bile removal (from 5 g/L to ≤ 0.5 g/L) was performed by repeated filtration of the 148

duodenal content with a Diacap® Polysulfone high-flux dialyser (Diacap HiFlo18, B. Braun) 149

during 75 min at 40 mL/min with fresh dialysis medium in counter flow at 80 mL/min 150

(Masterflex L/S Economy Digital Drive). The quantification of the bile concentration in 151

intestinal medium was done by measuring the OD (optical density) at 350 nm (VersaMax™ 152

Absorbance Microplate Reader, Molecular Devices) of 300 µL samples. The OD350 values 153

were converted to bile concentrations with the linear standard curve (R² = 0.997) generated by 154

a dilution series of bile (0.5 to 10.0 g/L oxgall, Difco) in intestinal medium. The lower limit of 155

this bile quantification method was 0.5 g/L bile (results not shown). 156

After the dialysis, the ileum phase (4 h) was started by the addition of 1 mL human intestinal 157

bacteria (8 log CFU/mL) obtained from the colon ascendens vessel of a SHIME reactor, 158

started up with faecal material of a healthy 27-year-old male, fed with standard reactor feed 159

and kept under the standard reactor conditions (32, 35). In the human ileum, the resident 160

microbiota originates from bacteria in the ingested food and caecal reflux (45). Therefore, the 161

intestinal bacteria used to simulate competition in the ileum were taken from the colon 162

ascendens vessel from SHIME to simulate reflux inoculation. According to PCR-DGGE and 163

metabolite analysis, the SHIME contains stable bacterial communities which resemble the 164

human microbiota of the colon in bacterial numbers and species composition (35). The exact 165

composition of the intestinal microbiota depends on both host-specific characteristics and 166

diet. It was previously shown that differences in SHIME reactor feed (simulating different 167

diets) affected the competition kinetics of B. cereus with the intestinal microbiota and thus its 168

survival within the intestinal community (8). During these experiments, the variation in the 169

intestinal bacterial community was minimized by using colon ascendens bacteria from the 170

same SHIME reactor maintained under standard conditions with the standard feed. This 171

allows the comparison of the intestinal survival of the different B. cereus strains relative to 172

each other. The growth of the intestinal bacteria during the ileum phase in the absence of B. 173

cereus was monitored by plating. Total (facultative) aerobic bacteria, staphylococci, total 174

coliforms, enterococci, and total anaerobes were counted according to Possemiers et al. 175

(2004) (35). Fecal lactobacilli, clostridia and bifidobacteria were determined on LamVab agar 176

(20), Tryptose Sulfite Cycloserine (TSC) agar (Merck) and Raffinose-Bifidobacterium agar 177

(RB) (21) with 1 % (w/v) bromocresol purple (Merck), respectively. 178

179

Toxin production 180

The intestinal production of enterotoxins by B. cereus was assessed hourly during the ileum 181

phase by testing 1 mL samples after filtration (0.2 µm syringe filters, Whatman) with the 182

Duopath® Cereus Enterotoxins (Merck) according to the manufacturers’ instructions. The 183

detection limits of the Duopath® kit are not provided by the manufacturer, but they have been 184

estimated at 6 ng/mL for Nhe-B and 20 ng/mL Hbl-L2 (23). 185

186

RESULTS 187

Gastrointestinal simulation experiments with B. cereus spores 188

Mashed potato medium containing 7.0 log spores/mL was subjected to in vitro simulation of 189

the gastrointestinal passage. The spores of all B. cereus strains (Table 1) were unaffected by 190

the mouth, stomach and duodenum phase, during which the entire population remained viable 191

without germinating (Figure 3). The slight increase of total and spore counts during the 192

duodenum phase was solely the consequence of the gradual transfer of the gastric content to 193

the intestinal vessel (Figure 2). At the end of the duodenum phase and during the dialysis, the 194

spore germination had started, noticeable as the decreasing spore percentage of the B. cereus 195

population during those phases (Table 3). Subsequently, the competing intestinal bacteria 196

were added to the intestinal vessel at the final concentration of 5.5 log CFU/mL. During the 197

ileum phase, plating on MYP was no longer possible for B. cereus discrimination from the 198

ileal bacteria, so qPCR was applied (5). This analysis revealed stable DNA levels of all B. 199

cereus strains, except for the psychrotrophic food isolate B. cereus LFMFP 710. These results 200

suggest spore survival, but further germination to vegetative cells without outgrowth or slight 201

inactivation of the spores cannot be ruled out, since these scenarios all occur under stable 202

DNA concentrations. The concentration of B. cereus LFMFP 710 decreased more than 10-203

fold during the 4 h ileum phase, namely - 1.36 log copy numbers/mL. This indicates 204

inactivation of this strain followed by DNA degradation. In comparison, the concentration of 205

the other strains decreased only slightly with 0.30 and 0.32 log copy numbers/mL for 206

mesophilic strains B. cereus NVH 1230-88 and B. cereus RIVM 9903295-4 respectively, and 207

with 0.45 log copy numbers/mL for the diarrhoeal psychrotropic strain B. cereus LFMFP 381. 208

As a general feature, the psychrotropic food isolate B. cereus LFMFP 710 consistently 209

showed lower total counts than spore counts (Table 3). This suggests that the spores of this 210

strain required the heat treatment prior to the spore count to ensure outgrowth of all spores on 211

the plates, resulting in lower CFU numbers in the total counts due to the lack of outgrowth of 212

non-heat activated spores. 213

When no competing intestinal bacteria were added during the ileum phase, the germination of 214

B. cereus spores was followed by vegetative outgrowth to approx. 7.0 log CFU/mL (Figure 215

4). The remaining spore concentration during the ileum phase was constant throughout one 216

experiment, but highly variable among the four replicate experiments, namely 4.5 log 217

spores/mL, 5.5 log spores/mL, 3.8 log spores/mL and none detected % (< 1.0 log spores/mL). 218

Although large variation in germination was observed, ranging from 94 % to 100 % of the 219

population, it was always followed by significant outgrowth of the vegetative cells to approx. 220

7.0 log CFU/mL. Therefore, the lack of vegetative outgrowth in the presence of human 221

intestinal bacteria (Figure 3) can be attributed to competition and/or inhibition of the 222

indigenous microbiota. 223

No enterotoxins (Nhe nor Hbl) were detected during the ileum phase of any of the simulation 224

experiments (Figure 3 and 4), not even when approximately 7 log CFU/mL growing 225

vegetative cells were present in absence of competing bacteria. 226

227

Dialysis 228

To mimic the approx. 95 % bile salt re-absorption in the human ileum (33), dialysis was 229

performed between the duodenum and the ileum phase. At the beginning of the duodenum 230

phase, the pure intestinal medium contained the maximal bile concentration of 10 g/L oxgall, 231

corresponding with approx. 15 mM bile salts (27), which gradually decreased to 5 g/L due to 232

dilution with the content of the gastric vessel (Figure 2). Dialysis of intestinal medium 233

containing 5 g/L and 10 g/L bile showed that resp. 90 % and 95 % of the bile was removed 234

after resp. 60 and 80 min (results not shown). 235

236

Intestinal bacteria 237

At the start of the ileum phase, 5.5 log CFU/mL human intestinal bacteria were added to 238

simulate competition of B. cereus with the indigenous intestinal microbiota. These bacteria 239

proliferated to approx. 8 log CFU/mL at the end of the ileum phase with the preservation of 240

their relative abundances (Figure 5). 241

242

DISCUSSION 243

B. cereus spores survived throughout the simulation of the gastrointestinal passage. Realistic 244

but worst-case conditions were selected for the gastrointestinal simulation experiment, namely 245

the high inoculum concentration of 7 log spores/mL and the slow kinetics of gastric 246

acidification (from pH 5.0 to 2.0 over 3 h time). However, the gastic acidification profile was 247

previously shown to only affect vegetative B. cereus cells, since the spores were fully 248

resistant to pH values between 5.0 and 2.0 (7). The food source was highly contaminated with 249

7.0 log spores/mL, which corresponds with a total infective dose of 8.9 log spores. This is far 250

more than the currently postulated infective dose of 5 to 8 log viable B. cereus for diarrhoeal 251

food poisoning (16). It was previously hypothesized that B. cereus induced diarrhoea is 252

caused by enterotoxin production in close proximity of the intestinal epithelium cells (16, 44). 253

This study reinforces this hypothesis by showing that no B. cereus proliferation and toxin 254

production occurred in the intestinal lumen during in vitro gastrointestinal passage of mashed 255

potatoes heavily contaminated with B. cereus spores. 256

257

No toxin production was detected by any of the strains during the ileum phase, not even when 258

high concentrations (approx. 7 log CFU/mL) of growing vegetative cells were present. 259

However, the lack of enterotoxin detection does not necessarily mean that toxin production 260

did not occur, because the enterotoxins are very susceptible to degradation by the digestive 261

host enzymes. These enzymes were added in high concentrations to the simulation media to 262

mimic the fed state of the host. It is possible that these digestive host enzymes were partially 263

removed during the dialysis for bile removal, since the sizes of the enzymes fall in the range 264

of the cut-off value of the polysulfone membrane of 10 to 60 kDa (personal communication 265

with the product manager of Vedefar NV; UniProt database, 02/04/2012). However, dialysis 266

was performed to remove approx. 95 % of the much smaller conjugated bile acids (approx. 267

0.5 kDa), so the majority of the digestive proteases is presumable still present in the 268

simulation broth after dialysis. 269

270

The indigenous microbiota prevented B. cereus outgrowth during the ileum phase, so this 271

study shows inhibition of pathogen outgrowth by the intestinal bacterial community during in 272

vitro simulation of gastrointestinal passage. It was already known that the indigenous 273

intestinal microbiota plays an important role in the maintenance of gastrointestinal 274

homeostasis and the host’s intestinal health (47). Commensal and probiotic intestinal bacteria 275

are frequently reported to play a protective role against enteric disease by several 276

mechanisms, including competitive exclusion and production of antimicrobial compounds. 277

For example, pre-incubation of epithelium cells with probiotic Lactobacillus strains could 278

reduce the Campylobacter jejuni infection, depending on the specific combination of the 279

bacterial strains and the eukaryote cell line (47). Antimicrobial compounds produced by 280

Lactobacillus acidophilus provided protection of intestinal epithelium cells against a range of 281

enteric pathogens such as B. cereus, Staphylococcus aureus, Listeria monocytogenes, 282

Salmonella typhimurium, Shigella flexneri, Escherichia coli (12, 24). More specifically, the 283

indigenous microbial community was found to inhibit the growth of newly arriving bacterial 284

species and prevent their establishment in the community (22). 285

286

The gastrointestinal survival of the four B. cereus strains used in this study was remarkably 287

similar, although the strains were selected to represent the diversity of B. cereus in growth 288

temperature (psychrotropic – mesophilic) and origin of isolation (clinical – food) (Table 1). 289

However, the psychrotropic food isolate was probably inactivated during the final ileum stage 290

of the gastrointestinal passage simulation. This observation corresponds with the finding that 291

the germination and growth of mesophilic B. cereus strains in the intestinal environment is 292

better in comparison to that of psychrotrophic strains (43). 293

294

The plethora of enterotoxins and virulence factors with cytotoxic and pore-forming properties 295

produced by vegetative B. cereus cells suggests that the host’s epithelial cells may be targeted 296

by diarrhoeal B. cereus in the small intestine. Moreover, in vitro research demonstrated that 297

the presence of human intestinal cells (Caco-2 cells) specifically induced spore germination 298

(1, 42). Subsequent outgrowth and toxin production of vegetative cells resulted in detachment 299

of the epithelial cells, microvilli damage, decreased mitochondrial activity, membrane damage 300

and cell lysis (31, 36). These reports from literature and the data obtained in the current study 301

support the hypothesis that ingested B. cereus spores do not multiply in the intestinal lumen 302

after germination, but first adhere to the intestinal mucosa, followed by outgrowth and 303

enterotoxin production, which damages the host epithelium and eventually results in 304

diarrhoea. Future research should study the influence of inter-host variability in intestinal 305

communities on the intestinal behaviour of B. cereus, since it is known that different 306

microbiota show different degrees of growth inhibition towards B. cereus (8). Moreover, the 307

influence and interactions with the host should be assessed by extending the in vitro 308

simulation experiments with modules that allow bacterial adhesion to mucin layers and 309

interactions with eukaryote cells (28, 40). 310

311

CONCLUSIONS 312

B. cereus spores were able to survive throughout gastrointestinal passage as simulated by the 313

in vitro experiment, except for the psychrotrophic food isolate. No bacterial outgrowth and no 314

enterotoxin production were observed in the intestinal lumen, despite the high inoculum 315

concentration of 7 log spores/mL in the mashed potato food matrix. It is hypothesized that B. 316

cereus induced diarrhoea is not caused by massive B. cereus proliferation and toxin 317

production in the intestinal lumen, but by localized growth and enterotoxin production at the 318

host’s mucus layer or epithelial surface. Moreover, our results indicate that the host’s 319

intestinal microbiota is an important defence barrier to control B. cereus outgrowth in the 320

small intestine and may thus play an important role in the host’s susceptibility to diarrhoeal 321

food poisoning. 322

323

ACKNOWLEDGEMENTS 324

This work was supported by the Special Research Funds of Ghent University as a part of the 325

project ‘Growth kinetics, gene expression and toxin production by Bacillus cereus in the 326

small intestine’ B/09036/02 fund IV1 31/10/2008 - 31/10/2012, by the Federal Public Service 327

(FOD) Health, Food Chain Safety and Environment project RT09/2 Bacereus and by the 328

Research Foundation Flanders (FWO) postdoctoral mandate of dr. Andreja Rajkovic. 329

330

CAPTIONS OF THE FIGURES AND TABLES 331

Figure 1: The setup of the gastrointestinal simulation experiment and the details of its five 332

phases: 1) mouth, 2) stomach, 3) duodenum, 4) dialysis and 5) ileum. 333

334

Figure 2: Above: the pH (■) and the volume (◊) in the gastric vessel, which was gradually 335

emptied, resulting in 5 gastric fractions (indicated as light grey areas). Below: the pH (■), the 336

volume (◊) and the bile concentration (○) in the intestinal vessel, which was gradually 337

supplied with the gastric content in 5 fractions. After complete transfer of the gastric vessel’s 338

content, dialysis was performed (indicated as dark grey area) to reduce the bile levels ≤ 90 % 339

of the initial concentration. 340

341

Figure 3: The survival of B. cereus spores during the gastrointestinal simulation experiment 342

with B. cereus NVH 1230-88 (A), B. cereus RIVM 9903295-4 (B), B. cereus LFMFP 381 (C) 343

and B. cereus LFMFP 710 (D); open symbols indicate the total count and filled symbols the 344

spore count in mashed potato medium (□), mouth phase (◊), stomach phase (Δ), duodenum 345

phase and dialysis (○) and qPCR enumeration during the ileum phase (×); the average values 346

and standard deviation of triplicate experiments on are presented. 347

348

Figure 4: The survival of B. cereus LFMFP 381 spores during the gastrointestinal simulation 349

experiment without the addition of intestinal bacteria during the ileum phase; open symbols 350

indicate the total count and filled symbols the spore count in the mashed potato medium (□), 351

the mouth phase (◊), stomach phase (Δ), duodenum phase, dialysis and ileum phase (○); the 352

average values and standard deviation of quadruple experiments are presented. 353

354

Figure 5: The survival and growth of human intestinal bacteria during ileum phase conditions 355

determined by plate count enumeration of enterococci (■), clostridia (■), staphylococci (■), 356

coliforms (■), and total (facultative) anaerobes (■); the average values and standard deviation 357

of duplicate experiments are presented. 358

359

Table 1: The origin and characteristics of the Bacillus cereus strains used in this study. 360

361

Table 2: The composition of the simulation media for food, saliva, gastric secretion, intestinal 362

secretion and dialysis. 363

364

Table 3: Presentation of the Bacillus cereus spore population during the gastrointestinal 365

simulation experiments, expressed as the percentage of the total B. cereus count, determined 366

by plating on TSA with and without prior heat treatment (10 min at 80 °C). 367

368

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Microbiol. 28:63-69. 512

513

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Tab

le 1

: The

ori

gin

and

char

acte

rist

ics o

f the

Bac

illus

cer

eus

stra

ins u

sed

in th

is st

udy.

Min

imal

gro

wth

te

mpe

ratu

re (°

C)

BC

ET

-RPL

A D

uopa

th D

uopa

thV

IA-B

DE

NV

H 1

230-

88

clin

ical

(hum

an fa

eces

)8

++

++

RIV

M 9

9032

95-4

cl

inic

al (h

uman

faec

es)

7+

-+

+

LFM

FP 3

81fo

od (d

ried

pota

to fl

akes

)>

10+

-+

-

LFM

FP 7

10fo

od (m

ashe

d po

tato

es)

7+

++

+

RIV

M =

Nat

iona

l Ins

titut

e fo

r Pub

lic H

ealth

and

Env

ironm

ent,

Bilt

hove

n, th

e N

ethe

rland

sLF

MFP

= L

abor

ator

y of

Foo

d M

icro

biol

ogy

and

Food

Pre

serv

atio

n, G

hent

Uni

vers

ity, G

hent

, Bel

gium

NV

H =

Nor

weg

ian

Scho

ol o

f Vet

erin

ary

Scie

nce,

Osl

o, N

orw

ay

B. c

ereu

s st

rain

Ori

gin

Hbl

pro

duct

ion

Nhe

pro

duct

ion

Food

solu

tion

Saliv

a m

ediu

mG

astr

ic m

ediu

m

Inte

stin

al m

ediu

m

Dia

lysi

s med

ium

Sa

lts2,

804,

154,

3212

,89

8,41

NaC

l2,

800,

302,

756,

438,

41N

aH2P

O4.2

H2O

-1,

150,

35-

-K

Cl

-0,

900,

820,

50-

NH

4Cl

-0,

31-

--

KSC

N-

0,20

--

-N

a 2SO

4.10H

2O-

1,29

--

-C

aCl 2.

2H2O

--

0,40

0,21

-K

H2P

O4

--

-0,

05-

MgC

l 2.6H

2O-

--

0,03

-N

aHC

O3

--

-5,

67-

Nut

rien

ts10

,50

0,00

0,00

0,00

0,00

Ara

bino

gala

ctan

(fro

m la

rch

woo

d, S

igm

a-A

ldric

h)2,

00-

--

-Pe

ctin

(fro

m a

pple

s, Si

gma-

Ald

rich)

2,00

--

--

Xyl

an (f

rom

birc

h w

ood,

Sig

ma-

Ald

rich)

2,00

--

--

Tab

le 2

: The

com

posi

tion

of th

e si

mul

atio

n m

edia

for

food

, sal

iva,

gas

tric

secr

etio

n, in

test

inal

secr

etio

n an

d di

alys

is.

Com

poun

dsC

once

ntra

tion

(g/L

)

y(

,g

),

Yea

st e

xtra

ct (O

xoid

)3,

00-

--

-Pr

oteo

se p

epto

n (O

xoid

) 0,

50-

--

-C

yste

ine

(non

-ani

mal

sour

ce, S

igm

a-A

ldric

h)

1,00

--

--

Hos

t fac

tors

0,00

0,27

10,1

918

,42

1,13

Am

ylas

e (�

-am

ylas

e fr

om B

acill

us sp

., Si

gma-

Ald

rich)

-0,

73-

--

Muc

in (f

rom

por

cine

stom

ach

type

II, S

igm

a-A

ldric

h)-

0,05

3,00

--

Ure

a (S

igm

a-A

ldric

h)-

0,20

0,09

0,15

-U

ric a

cid

(Sig

ma-

Ald

rich)

-0,

02-

--

D-g

lucu

roni

c ac

id (F

luka

)-

-0,

02-

-D

-(+)-g

luco

sam

ineh

ydro

chlo

ride

(Sig

ma-

Ald

rich)

--

0,33

--

Bov

ine

seru

m a

lbum

in (f

ract

ion

V, M

erck

)-

-2,

251,

271,

13Pe

psin

(fro

m p

orci

ne st

omac

h m

ucos

a, S

igm

a-A

ldric

h)-

-4,

50-

-Pa

ncre

atin

(fro

m p

orci

ne p

ancr

eas,

Sigm

a-A

ldric

h)-

--

6,00

-Li

pase

(fro

m p

orci

ne p

ancr

eas,

Sigm

a-A

ldric

h)-

--

1,00

-B

ile (O

xgal

l, de

hydr

ated

fres

h bi

le, D

ifco)

--

-10

,00

- -

= c

ompo

und

not a

dded

to th

e m

ediu

m

Tab

le 3

:The

Bac

illus

cer

eus

spor

e po

pula

tion

duri

ng th

e ga

stro

inte

stin

al si

mul

atio

n ex

peri

men

ts, e

xpre

ssed

as t

he p

erce

ntag

e of

the

tota

l B. c

ereu

s

coun

t, de

term

ined

by

plat

ing

on T

SA w

ith a

nd w

ithou

t pri

or h

eat t

reat

men

t (10

min

at 8

0 °C

).

B. c

ereu

s

N

VH

12

30-8

8B.

cer

eus

RIV

M

9903

295-

4 B.

cer

eus

LFM

FP 3

81B.

cer

eus

LFM

FP 7

10B.

cer

eus

LFM

FP 3

81

with

out c

ompe

titio

nM

ashe

d po

tato

med

ium

0,0

60 (±

37)

89 (±

71)

64 (±

35)

458

(± 9

0)12

5 (±

113

)M

outh

pha

se0,

150

(± 3

7)75

(± 7

)30

(± 1

0)50

4 (±

173

)73

(± 2

0)St

omac

h ph

ase

0,3

81 (±

47)

91 (±

30)

51 (±

17)

500

(± 1

04)

85 (±

65)

Stom

ach

phas

e1,

348

(± 3

6)73

(± 7

)83

(± 2

7)45

2 (±

194

)10

2 (±

63)

Stom

ach

phas

e2,

375

(± 4

5)11

5 (±

26)

94 (±

26)

285

(± 2

26)

90 (±

29)

Stom

ach

phas

e3,

361

(± 3

7)77

(± 1

9)10

8 (±

18)

586

(± 6

6)85

(± 3

3)D

uode

num

pha

se1,

366

(± 4

1)99

(± 2

1)11

9 (±

17)

622

(± 2

48)

91 (±

86)

Duo

denu

m p

hase

2,3

60 (±

32)

91 (±

26)

83 (±

3)

2496

(± 3

642)

82 (±

56)

Duo

denu

m p

hase

3,3

28 (±

16)

43 (±

12)

69 (±

7)

411

(± 2

42)

N.A

.D

ialy

sis

3,4

30 (±

18)

33 (±

11)

40 (±

21)

403

(± 2

45)

36 (±

22)

Dia

lysi

s4,

719

(± 1

2)29

(± 5

)13

(± 8

)59

9 (±

513

)N

.A.

Ileum

pha

se4,

8N

.A.

N.A

.N

.A.

N.A

.54

(± 3

2)Ile

um p

hase

5,8

N.A

.N

.A.

N.A

.N

.A.

20 (±

21)

Ileum

phas

e6

8N

AN

AN

AN

A5

(±5)

Spor

es (%

of t

otal

pop

ulat

ion �

stan

dard

dev

iatio

n)Sa

mpl

eT

ime

(h)

Ileum

pha

se6,

8N

.A.

N.A

.N

.A.

N.A

.5

(± 5

)Ile

um p

hase

7,8

N.A

.N

.A.

N.A

.N

.A.

2 (±

3)

Ileum

pha

se8,

8N

.A.

N.A

.N

.A.

N.A

.2

(± 3

) N

.A. =

not

app

licab

le, t

he sp

ore

perc

enta

ge c

an n

ot b

e ca

lcul

ated

from

the

avai

labl

e da

ta.

Survival and germination of Bacillus cereus spores during in vitro simulation of gastrointestinal...

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