A novel method for direct detection of Bacillus cereus toxin genes in powdered dairy products

Journal Pre-proof A novel method for direct detection of Bacillus cereus toxin genes in powdered dairy products Jennifer A. Sánchez-Chica, Margarita M. Correa, Angel E. Aceves-Diez, Laura M. Castañeda-Sandoval PII:

S0958-6946(19)30262-6

DOI:

https://doi.org/10.1016/j.idairyj.2019.104625

Reference:

INDA 104625

To appear in:

International Dairy Journal

Received Date: 23 August 2019 Revised Date:

5 December 2019

Accepted Date: 8 December 2019

Please cite this article as: Sánchez-Chica, J.A., Correa, M.M., Aceves-Diez, A.E., Castañeda-Sandoval, L.M., A novel method for direct detection of Bacillus cereus toxin genes in powdered dairy products, International Dairy Journal, https://doi.org/10.1016/j.idairyj.2019.104625. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

1

A novel method for direct detection of Bacillus cereus toxin genes in powdered dairy

2

products

3 4 5 6 7

Jennifer A. Sánchez-Chica a, Margarita M. Correa a, Angel E. Aceves-Diez b, Laura M.

8

Castañeda-Sandoval c,*

9 10 11 12 13

a

14

UdeA, Calle 70 No. 52-21, Medellín, Colombia.

15

b

16

546, Col. La Nogalera, Guadalajara, Jalisco, P.O. Box 44470, Mexico.

17

c

18

No. 52-21, Medellín, Colombia.

Grupo de Microbiología Molecular. Escuela de Microbiología, Universidad de Antioquia

Departamento de Investigación y Desarrollo, Laboratorios Minkab, Av. 18 de Marzo No.

Grupo BioMicro. Escuela de Microbiología, Universidad de Antioquia UdeA, Calle 70

19 20 21 22 23

Corresponding author: +57 4 2195492

24

E-mail address: [email protected] (L. Castañeda) 1

25

_________________________________________________________________________

26

ABSTRACT

27 28

Bacillus cereus is a major foodborne pathogen with the potential of causing emetic and

29

diarrhoeal food poisoning. This study reports on the development of a sensitive and reliable

30

method that involves direct DNA extraction from powdered milk and infant formula and a

31

multiplex PCR for detection of four B. cereus toxin genes (nheA, hblD, cytK2 and cesB).

32

Detection limits were 2 × 102 cfu g-1 for enterotoxin genes and 2 × 103 cfu g-1 for both the

33

enterotoxin and emetic genes. Determination of B. cereus toxin genes in powdered dairy

34

food samples revealed five toxigenic groups. Groups I to IV contained enterotoxin genes

35

while group V, enterotoxin and emetic genes. The results indicate that the molecular

36

method developed is a rapid and efficient diagnostic tool for direct detection and

37

monitoring of B. cereus toxin genes in powdered milk and infant formula and possibly in

38

other powdered foods.

39

_________________________________________________________________________

40

2

41

1.

Introduction

42 43

In public health, the foodborne diseases are a serious global problem due to

44

widespread foodborne pathogens causing outbreaks (Oliver, Jayarao, & Almeida, 2005).

45

Among the microorganisms able to produce toxins and responsible for foodborne diseases

46

are Staphylococcus aureus, Vibrio cholerae, Clostridium botulinum, Clostridium

47

perfringens, Escherichia coli O157 and Bacillus cereus (Fusco, Quero, Morea, Blaiotta, &

48

Visconti, 2011). Of these, the endospore forming B. cereus has the capacity to contaminate

49

foods (De Vos et al., 2009), causing two types of food poisoning, the emetic and diarrhoeal

50

syndromes (Logan, 2012). A pre-formed toxin, cereulide, causes the emetic syndrome that

51

occurs after ingestion of a contaminated food; this toxin is produced by a nonribosomal

52

peptide synthetase encoded by the 24-Kb cereulide synthetase (ces) gene cluster (Stenfors

53

Arnesen, Fagerlund, & Granum, 2008). Three different enterotoxins are associated with the

54

diarrhoeal syndrome, haemolysin BL (HBL), non-haemolytic enterotoxin (NHE) and

55

cytotoxin K (CytK). HBL has two lytic components, L1 and L2, and the binding protein B,

56

encoded by the hblC, hblD and hblA genes, respectively (Böhm, Huptas, Krey, & Scherer,

57

2015). NHE has also three components, encoded by the nheA, nheB and nheC genes

58

(Lindbäck, Fagerlund, Rødland, & Granum, 2004). These enterotoxin complexes are

59

organised in operons and require expression of all components for virulence (Granum,

60

2005). Cytotoxin K is a pore-forming toxin and two variants are known. CytK1 is encoded

61

by the cytK1 gene, it has a high cytotoxic activity, related to necrotic enteritis, and the most

62

common variant CytK2 is encoded by cytK2 gene (Økstad & Kolstø, 2011; Stenfors

63

Arnesen et al., 2008).

3

64

B. cereus is often found contaminating food samples such as pasta, rice, meat,

65

chicken, vegetables, fruits, grain, spices, powdered milk and other pulverised foods

66

(Kotiranta, Lounatmaa, & Haapasalo, 2000). B. cereus spores are highly resistant to

67

dehydrating and heating treatments and during food preparation, spores may germinate,

68

producing food spoilage or food poisoning (Logan, 2012). Vulnerable consumers such as

69

children and pregnant or lactating women are under serious risk of foodborne illness;

70

therefore, detecting pathogenic B. cereus is very important for food safety (Mintegi et al.,

71

2015). The accurate number of food poisoning cases produced by B. cereus in diverse

72

countries is unknown; this because the syndromes caused by this pathogen are self-limiting

73

and generally not severe, thus are not always diagnosed (Stenfors Arnesen et al., 2008). In

74

Colombia, despite limited reports (INS, 2007; González, González, Puerta, & Torres,

75

2014), it is estimated that B. cereus causes one third of the total food outbreaks (INS,

76

2011), and the pathogen was detected in foods such as cooked rice, sauces, sausages, dairy

77

products (Padilla, 2007) and ready-to-eat cereals (Forero, Galindo, & Morales, 2018).

78

Food pathogen detection and enumeration constitute important steps to ensure food

79

safety. Conventional culture methods are currently used to detect foodborne pathogenic

80

bacteria. They have the advantage of being inexpensive but their limitations include their

81

dependence on microbial cell viability to growth in artificial media and the requirement of

82

biochemical identification/confirmation tests that take up to a week to complete. In

83

addition, some microorganisms may need pre-enrichment media to grow, increasing

84

processing time (Mandal, Biswas, Choi, & Pal, 2011).

85

In Colombia, detection and enumeration of presumptive B. cereus in foods is

86

performed according to Colombian Technical Norm NTC 4679 (ICONTEC, 2006), which

87

is a translated version of the ISO 7932:2004 method (ISO, 2004). However, this culture4

88

based method is time-consuming and does not allow detection of toxin genes; therefore, the

89

toxigenic potential of the B. cereus isolates cannot be stablished (Bhunia, 2008). Thus, the

90

aim of this work was to develop a simple, rapid, sensitive and specific method to detect

91

hblC, nheA, cytK2, and cesB toxin genes from B. cereus by multiplex PCR, using DNA

92

directly extracted from powdered milk and infant formula. This approach helps to

93

overcome the limitations of conventional methods and avoids isolating bacteria in pure

94

cultures.

95 96

2.

Materials and methods

2.1.

Bacterial strains

97 98 99 100

Eleven reference strains were used to evaluate the method, and included two B.

101

cereus emetic strains, F4810/72 and NVH 1257, and nine B. cereus enterotoxic strains,

102

ATCC 14579, ATCC 10987, ATCC 21281, ATCC 27348, ATCC 6464, NVH 1230/88,

103

NVH 0075/95, F4094/73 and BC307. To determine the detection limit of this methodology,

104

the emetic F4810/72 and enterotoxic ATCC 14579 B. cereus strains were used to

105

artificially contaminate powdered dairy food samples. The foodborne pathogens,

106

Salmonella typhimurium ATCC14028, Shigella sonnei ATCC 25931, E. coli ATCC25922,

107

Staph. aureus ATCC6538 and Listeria monocytogenes ATCC 19118 were used as

108

reference toxin-negative controls for mPCR.

109 110

2.2.

DNA extraction

111 5

For DNA extraction from B. cereus reference strains, bacteria were grown in Luria

112 113

Bertani (LB) broth and incubated at 37 °C for 18 h. DNA from B. cereus spores and cells

114

and negative control strains was extracted as previously described (D’Alessandro, Antunez,

115

Piccini, & Zunino, 2007), with some modifications. Cultures containing spores and cells

116

were centrifuged at 13,000 × g for 2 min and the pellets were resuspended in 500 µL of

117

lysis buffer (0.1 M DTT, 0.1 M NaOH, 0.1 M NaCl, 1% SDS, pH 10.8) and subjected to

118

mechanical disruption with glass beads, followed by incubation at 80 °C for 90 min, with a

119

vortex step every 5 min. Subsequently, cellular components were treated with lysozyme

120

(1.5 g L-1) at 37 °C for 60 min and proteinase K (0.2 g L-1) at 56 °C for 60 min. Next, 30

121

µL of 8 M ammonium acetate were added and the suspension was centrifuged at 13,000 × g

122

for 30 min. The supernatant was recovered and the DNA was purified and concentrated by

123

centrifugation using 99.5% isopropanol and 70% ethanol, and finally, resuspended in 30 µL

124

sterile Milli-Q water (Merck Millipore, Darmstadt, Germany).

125 126

2.3.

Detection limit of the method using artificially contaminated powdered dairy foods

127 128

To establish the detection limit of the method for direct detection of B. cereus toxin

129

genes, powdered milk and infant formula samples were tested according to NTC 4679

130

(ICONTEC, 2006) and also by applying the molecular method to ensure the absence of

131

viable B. cereus and of its toxin genes. These food samples were artificially inoculated with

132

B. cereus reference strains. Twenty-five grams of each powdered dairy food sample were

133

dissolved in 225 mL of sterile distilled water using glass beads to produce a uniform food

134

homogenate that was filtered through a 11 µm pore size Whatman N°1 (Sigma-Aldrich,

135

Inc, St. Louis, MO, USA). Six replicates of this procedure were performed per sample. The 6

136

filtrate of the first bottle was recovered and inoculated with one colony of both emetic

137

F4810/72 and enterotoxigenic ATCC 14579 B. cereus strains. This suspension was serially

138

diluted with the filtrate of the remaining bottles to obtain final dilutions ranging from 10-1

139

to 10-6. The dilutions were centrifuged at 6000 × g for 30 min and from these pellets, the

140

DNA was extracted as previously described. To establish the detection limit of the method,

141

or the minimum number of B. cereus cells in which the toxin genes could be detected, 0.1

142

mL of each obtained dilution was inoculation onto LB agar plates, by triplicate, incubated

143

at 37 °C for 48 h.

144 145

2.4.

Multiplex PCR

146 147

Four specific primers pairs were used to detect hblC, nheA, cytK2, and cesB toxin

148

genes from B. cereus (Table 1). To check for inhibitory compounds in the PCR mixture that

149

may produce false-negative results, a fragment of the internal transcribed spacer (ITS1)

150

situated between the genes coding for the ribosomal RNA (rRNA) small and large subunits

151

was used as internal amplification control (IAC). Multiplex PCR was carried out in a 16 µL

152

PCR reaction mixture containing 0.2 µM B. cereus toxin genes forward and reverse primers

153

and 0.1 µM primers for IAC, 0.6 mM dNTPs, 1.3 U of PlatinumTM Taq DNA polymerase

154

(Invitrogen, Waltham, MA, USA), 4 mM MgCl2 in 1.6 µL 10× PCR buffer and 2 µL DNA

155

template (from artificially contaminated powdered dairy foods or samples). Amplification

156

consisted of initial denaturation step at 94 °C for 5 min followed by 35 cycles of 94 °C for

157

1 min, 50 °C for 40 s, 72 °C for 2 min and a final extension at 72 °C for 5 min; this was

158

performed in a G-Storm GS482 thermocycler (Labtech, Heathfield, East Sussex, UK). The

159

PCR products were subjected to 2% (w/v) agarose gel electrophoresis (Amresco, 7

160

Cleveland, OH, USA). In addition, the amplicons from B. cereus reference strains were

161

sequenced and their sequences were compared with B. cereus genome sequences available

162

in GenBank using nucleotide Basic Local Alignment Search Tool (BLAST).

163 164

2.5.

Powdered dairy food samples evaluated

165

The samples evaluated were powdered milk (n = 75) and infant formula (n = 75).

166 167

These were collected in public and private educational institutions, bakeries and powdered

168

food companies located in Medellín, Colombia.

169 170

3.

Results and discussion

3.1.

Multiplex PCR

171 172 173 174

In this study samples were evaluated to detect viable B. cereus cells according to

175

NTC 4679 (ICONTEC, 2006) and then analysed to detect B. cereus toxin genes by a

176

molecular method that included direct DNA extraction and mPCR. Initially, the mPCR

177

conditions were optimised to achieve simultaneous amplification of all B. cereus toxin

178

genes from emetic and enterotoxic reference strains; no amplicons were obtained from non-

179

target strains and the IAC amplified in all PCR reactions (Table 2). Sequence comparison

180

with the GenBank database allowed determination that amplicons corresponded to B.

181

cereus toxin genes with a nucleotide identity ranging between 95 and 100%. The optimised

182

mPCR has the advantage of allowing the amplification of all toxin genes simultaneously.

183

Also, it includes relevant controls not found in previously reported multiplex PCR, which 8

184

do not meet requirements for routine diagnostics as they are missing an IAC and/or do not

185

include DNA sequence from non-target strains (Forghani, Seo, & Oh, 2014; Ombui, Gitahi,

186

& Gicheru, 2008). These features are mandatory for diagnostics, because they allow

187

detecting false-negative or false-positive results.

188 189

3.2.

Sensitivity and specificity of mPCR

190 191

In the mPCR assay, the detection limit of B. cereus enterotoxin genes in both

192

artificially contaminated powdered milk and infant formula was 102 cfu g-1 (13.3 cfu per

193

reaction). At this detection limit, the band corresponding to cesB gene was not visualised

194

(Fig. 1), but it appeared at counts of 103 cfu g-1 (133 cfu per reaction), this value being the

195

detection limit for B. cereus emetic and enterotoxin genes tested simultaneously. Although,

196

the detection limit of traditional methods is of approximately 10 cfu g-1 B. cereus cells, the

197

developed method allows detecting B. cereus toxin genes in only one day with optimal

198

detection limits. Moreover, there is no need for a pre-enrichment step, which translates in

199

processing time reduction. These results constitute an important contribution to researchers

200

aiming to detect B. cereus enterotoxin genes using this faster and relatively easy to perform

201

method. In the literature there are reports of mPCR assays that detect B. cereus toxin genes

202

in foods such as cooked rice, milk, cheese (Ombui et al., 2008), meat and meat products

203

(Rather, Aulakh, Gill, Rao, & Hassan, 2011; Razei et al., 2017). Different from the mPCR

204

assay described here, these assays include incubation of the food in a selective pre-

205

enrichment broth for 15–18 h, which significantly increases processing time. A 104 cfu g-1

206

detection limit for the emetic and enterotoxin genes was reported for cooked rice and 103

207

cfu mL-1 for milk and cheese (Ombui et al., 2008). Compared with those results, this work 9

208

reports a better detection limit (103 cfu g-1), with the advantage that the DNA extraction is

209

performed directly from the foods, which results in time and effort savings. Furthermore, B.

210

cereus cell counts in foods associated with foodborne diseases are typically in the range of

211

105 to 108 cfu g-1 (Fricker, Messelhauber, Scherer, & Ehling-Schulz, 2007). According to

212

the international standard, foods with counts lower than 103 cfu g-1 are considered safe for

213

consumers (ISO, 2004). Hence, the sensitivity of the molecular methodology developed is

214

enough to detect minimum contamination levels, even those legally permitted in food

215

samples.

216

Among the studies that report on molecular methodologies for B. cereus detection,

217

some do not include the detection of B. cereus toxin genes responsible for food poisoning

218

(Fernández-No et al., 2011; Martínez-Blanch, Sánchez, Garay, & Aznar, 2010, 2011; Priha,

219

Hallamaa, Saarela, & Raaska, 2004), others exclusively detect B. cereus enterotoxin genes

220

but do not target the emetic gene (Martínez-Blanch, Sánchez, Garay, & Aznar, 2009;

221

Ombui et al., 2008). Compared with those, the molecular method developed here, which

222

simultaneously detects enterotoxin and emetic genes, has the advantage of allowing

223

assessing the risk associated to enterotoxic and/or emetic food poisoning.

224

Foods are complex matrixes that may contain nucleases, cations, proteases, fatty

225

acids and other PCR inhibitors (Forghani et al., 2014). The amplification of an IAC in the

226

mPCRs performed indicated that no inhibitors were affecting the mPCR. It is assumed that

227

steps in the DNA extraction directly from powdered dairy foods, which involved successive

228

lysis, DNA purification and concentration steps, contributed to obtain high quality DNA. It

229

is possible that in studies in which DNA extraction only includes heating of the bacterial

230

suspension (80 °C, 10 min), followed by centrifugation (Ombui et al., 2008), the lack of

10

231

additional purification steps could be contributing to a lower sensitivity, even when

232

performing a pre-enrichment step.

233 234

3.3.

Detection of B. cereus cells and its toxin genes in powdered dairy food samples

235 236

In the powdered dairy food samples from the various institutions, B. cereus detected

237

by standard microbiological procedures accounted for 14.7% (11 of 75) of powdered milk

238

samples and 10.7% (8 of 75) of infant formula samples (Table 3). Phenotypic determination

239

showed that after a 48-h incubation period, B. cereus isolates produced typical colonies that

240

retained the colour of phenol red because of their inability to ferment mannitol. In addition,

241

a white ring of precipitated egg yolk due to lecithin degradation surrounded the colonies.

242

Cell morphology, checked microscopically, evidenced Gram-positive rods and some of

243

them presented subterminal spores and poly-β-hydroxy-butyrate inclusions. The identity of

244

these isolates was further confirmed by mobility, positive reactions for catalase, Voges-

245

Proskauer, hydrolysed starch, casein and gelatin, a β-haemolytic pattern on the blood agar

246

plates, a positive glucose and negative xylose and arabinose fermentations, and variable

247

nitrate to nitrite reduction. All of these are typical B. cereus phenotypic features.

248

The molecular method developed detected B. cereus toxin genes in 78% (117 of

249

150) of powdered dairy foods; specifically, in 85.3% (64 of 75) of powdered milk samples

250

and in 70.7% (53 of 75) of infant formula (Table 3). In contrast, following standard

251

microbiological procedures B. cereus was detected only in 12.7% (19 of 150) of the

252

samples. These results demonstrate the high sensitivity of the molecular method as

253

compared with culture methods. This is not unexpected, given that previous studies have

254

demonstrated that PCR assays have better sensitivity than culture-based approaches (Lee et 11

255

al., 2014; Melendez et al., 2010). The lower sensitivity of culture methods may be

256

explained by the fact that during the drying steps in food processing some cells could be

257

injured, impairing further development and growth in culture (Wu, 2008), and limiting

258

pathogen detection. It is important to consider that viable organisms are the target of culture

259

methods, while the molecular method detects DNA corresponding to the toxin genes from

260

viable and non-viable B. cereus cells. The latter, and the fact that in the PCR there is an

261

exponential amplification of the target sequence, explain the higher sensitivity of the mPCR

262

method as compared with culture detection. All these issues should be considered when

263

selecting the method for B. cereus detection. In addition to the high sensitivity of the

264

molecular method, another advantage is the simultaneous detection of B. cereus emetic and

265

enterotoxin genes, which allows identification of the strain type contaminating the foods.

266 267

3.4.

Frequency of B. cereus toxin genes in powdered dairy food samples

268 269

The molecular methodology allowed the detection of B. cereus hblC, nheA, cytK2

270

and cesB toxin genes in 117 powdered dairy food samples. Overall, hblC was the most

271

frequently detected toxin gene (n = 114; 97.4%), followed by nheA (n = 41; 35%) and

272

cytK2 (n = 40; 34.2%). In particular, the cesB gene was only detected in three powdered

273

milk samples (2.6%) (Table 4). Similar to these frequencies, a study in Argentina reported

274

that hblC was also the most frequently detected gene (64%) in B. cereus isolated from

275

honey, followed by nheA (56%) and cytK (53%) (López, & Alippi, 2010). Of note, the

276

distribution of toxin gene frequencies in these two Latin American countries, mainly

277

considering that available reports from the United States (Ankolekar, Rahmati, & Labbé,

278

2009), Europe (Samapundo, Heyndrickx, Xhaferi, & Devlieghere, 2011) and Asia 12

279

(Forghani et al., 2014; Park et al., 2009; Rather et al., 2011), show that nheA is usually the

280

gene found in higher frequencies in foods. Moreover, in Brazil the nhe genes were found in

281

higher frequency among B. cereus strains isolated during three decades, from the 1980s to

282

2000s (Chaves, Pires, & Vivoni, 2011). It is possible that the epidemiology of toxigenic B.

283

cereus changes over time and depends on geographical location, as is documented for other

284

pathogens (Hawkey & Jones, 2009).

285

Regarding the detection of cesB gene from powdered dairy foods, only three

286

powdered milk samples (2.6%) were positive (Table 4). In general, and according to the

287

literature, emetic strains in foods are less frequent than enterotoxic strains (Altayar &

288

Sutherland, 2006); this is because the emetic gene is in a plasmid carried by a particular B.

289

cereus lineage (Økstad & Kolstø, 2011). The low frequency of emetic strains in powdered

290

milk samples is similar to the values registered for other food types. such as rice, cooked

291

chicken, baby food and raw milk, in countries like Argentina, England, Germany, Sweden

292

and South Korea (Ehling-Schulz, Fricker, & Scherer, 2004; Forghani et al., 2014; López,

293

Minnaard, Pérez, & Alippi, 2015; Park et al., 2009).

294

The results of this study, as those of previous studies with other foods, show a high

295

frequency of B. cereus enterotoxin genes and their worldwide distribution, a finding that

296

suggests a risk for vulnerable population to suffer the diarrhoeal syndrome. On the

297

contrary, the emetic genes appear in very low frequency, mainly in Asian countries, in

298

foods based on rice (Logan, 2012), where, in addition to the diarrhoeal, there is a risk for

299

the emetic syndrome. All the above indicates a broad distribution of B. cereus toxin genes,

300

that depends on the food type and geographical location, and suggests the need of

301

conducting surveys to evaluate the particular epidemiological situation of specific towns or

302

regions. 13

303 304

3.5.

Toxigenic groups in powdered dairy foods

305

According to the presence of B. cereus toxin genes in the powdered dairy foods,

306 307

five toxigenic groups were established (Table 5, Figs. 2 and 3). Group I only contained the

308

hblC gene, this was the most frequent group in powdered milk (43.7%) and infant formulas

309

(47.2%); toxigenic group II was next, included hblC and cytK2 genes and was mostly

310

detected in infant formula samples. Toxigenic group III contained nheA and hblC genes,

311

while toxigenic group IV included nheA, hblC and cytK2. Both toxigenic groups were

312

mainly detected in powdered milk. Notably, toxigenic group V was the only group

313

containing the cesB and nheA genes, and it was only detected in powdered milk samples.

314

The B. cereus toxigenic groups were mainly formed by enterotoxin genes, with the emetic

315

gene found in lower frequency, following a similar distribution than B. cereus strains

316

isolated from foods worldwide (Chaves et al., 2011; Samapundo et al., 2011).

317 318

4.

Conclusions

319 320

The molecular methodology developed consists of DNA extraction directly from

321

powdered dairy foods coupled to a multiplex PCR. The strategy constitutes an efficient,

322

highly sensitive and time saving approach that eliminates the need of performing prolonged

323

processes such as a pre-enrichment steps, culture and colony purification. In addition, it

324

provides essential information about the toxigenic potential of B. cereus present in

325

powdered dairy foods and it has the possibility to be used for detecting pathogenic B.

326

cereus in other foods. These characteristics make of this approach an optimal application 14

327

for the food industry and the clinical setting. Finally, the finding of B. cereus toxin genes in

328

the powdered dairy foods points to the need for continuously monitoring the contamination

329

levels of these and other foods, mostly considering that products such as powdered milk

330

and infant formula are mainly consumed by children and pregnant or lactating women. The

331

results also indicate the importance of implementing best practices during handling,

332

cooking and storage of these products.

333 334

Acknowledgement

335 336

This research received support from a project funded by Universidad de Antioquia,

337

Convocatoria Programática, Project Code No. 2015-7543, to MMC, Medellin, Colombia. J.

338

A. Sánchez received financial support for her PhD studies from Colciencias, grant No. 647.

339

The authors are grateful to Niels Hendriksen (Department of Environmental Science,

340

Environmental Microbiology and Biotechnology, Roskilde, Denmark), Per Einar Granum

341

(The Norwegian School of Veterinary Science, Oslo, Norway), Lucas Wijnands

342

(Laboratory for Zoonoses and Environmental Microbiology, RIVM-Centre for Infectious

343

Disease Control, Bilthoven, The Netherlands) and Microbiología Industrial y Ambiental

344

Lab, for providing reference strains.

345 346

References

347 348

Altayar, M., & Sutherland, A. D. (2006). Bacillus cereus is common in the environment but

349

emetic toxin producing isolates are rare. Journal of Applied Microbiology, 100, 7–

350

14. 15

351

Ankolekar, C., Rahmati, T., & Labbé, R. G. (2009). Detection of toxigenic Bacillus cereus

352

and Bacillus thuringiensis spores in U.S. rice. International Journal of Food

353

Microbiology, 128, 460–466.

354

Bhunia, A. K (2008). Bacillus cereus and Bacillus anthracis. In D. R. Heldman (Ed.),

355

Foodborne microbial pathogens: Mechanisms and pathogenesis (pp. 135–147).

356

New York, USA: Springer

357

Böhm, M. E., Huptas, C., Krey, V. M., & Scherer, S. (2015). Massive horizontal gene

358

transfer, strictly vertical inheritance and ancient duplications differentially shape the

359

evolution of Bacillus cereus enterotoxin operons hbl, cytK and nhe. BMC

360

Evolutionary Biology, 15, Article 246.

361

Chaves, J. Q., Pires, E. S., & Vivoni, A. M. (2011). Genetic diversity, antimicrobial

362

resistance and toxigenic profiles of Bacillus cereus isolated from food in Brazil over

363

three decades. International Journal of Food Microbiology, 147, 12–16.

364

D’Alessandro, B., Antunez, K., Piccini, C., & Zunino, P. (2007). DNA extraction and PCR

365

detection of Paenibacillus larvae spores from naturally contaminated honey and

366

bees using spore-decoating and freeze-thawing techniques. World Journal of

367

Microbiology and Biotechnology, 23, 593–597.

368

De Vos, P., Garrity, G., Jones, D., Krieg, N. R., Ludwig, W., Rainey, F.A., et al. (2009).

369

Bergey’s manual of systematic bacteriology, Vol. 3. The firmicutes (2nd edn.). New

370

York, USA: Springer.

371

Ehling-Schulz, M., Fricker, M., & Scherer, S. (2004). Identification of emetic toxin

372

producing Bacillus cereus strains by a novel molecular assay. FEMS Microbiology

373

Letters, 232, 189–195.

16

374

Ehling-Schulz, M., Vukov, N., Schulz, A., Shaheen, R., Andersson, M., Martlbauer, E., et

375

al. (2005). Identification and partial characterization of the nonribosomal peptide

376

synthetase gene responsible for cereulide production in emetic Bacillus cereus.

377

Applied and Environmental Microbiology, 71, 105–113.

378

Fernández-No, I. C., Guarddon, M., Böhme, K., Cepeda, A., Calo-Mata, P., & Barros-

379

Velázquez, J. (2011). Detection and quantification of spoilage and pathogenic

380

Bacillus cereus, Bacillus subtilis and Bacillus licheniformis by real-time PCR. Food

381

Microbiology, 28, 605–610.

382 383

Forero, A., Galindo, M., & Morales, G. (2018). Aislamiento de Bacillus cereus en restaurantes escolares de Colombia. Biomédica, 38, 338–344.

384

Forghani, F., Seo, K., & Oh, D. (2014). Improved multiplex polymerase chain reaction

385

(PCR) detection of Bacillus cereus group and its toxic strains in food and

386

environmental samples. African Journal of Microbiology Research, 8, 3821–3829.

387

Fricker, M., Messelhauber, U., Scherer, S., & Ehling-Schulz, M. (2007). Diagnostic real-

388

time PCR assay for the detection of emetic Bacillus cereus strains in foods and

389

recent food-borne outbreaks. Applied and Environmental Microbiology, 73, 1892–

390

1898.

391

Fusco, V., Quero, G. M., Morea, M., Blaiotta, G., & Visconti, A. (2011). Rapid and reliable

392

identification of Staphylococcus aureus harbouring the enterotoxin gene cluster

393

(egc) and quantitative detection in raw milk by real time PCR. International Journal

394

of Food Microbiology, 144, 528–537.

395

Granum, P. E. (2005). Bacillus cereus. In P. M. Fratamico, A. K. Bhunia, & J. L. Smith,

396

(Eds.), Foodborne pathogens: Microbiology and molecular biology (pp. 409–419).

397

Norfolk, UK: Caister Academic Press. 17

398

González, G., González, G., Puerta, H., & Torres, Y. (2014). Intoxicación alimentaria por

399

Bacillus cereus en el Servicio de Neonatología del Hospital General de Medellín -

400

Colombia, 1977. Revista Facultad Nacional de Salud Pública 1974-2014 Antología,

401

32, 24–37.

402

Guinebretière, M. H., Broussolle, V., & Nguyen-The, C. (2002). Enterotoxigenic profiles of

403

food-poisoning and food-borne Bacillus cereus strains. Journal of Clinical

404

Microbiology, 40, 3053–3056.

405 406

Hawkey, P. M., & Jones, A. M. (2009). The changing epidemiology of resistance. Journal of Antimicrobial Chemotherapy, 64, i3–i10.

407

ICONTEC (2006). NTC 4679. Método horizontal para el recuento de Bacillus cereus

408

Técnica de recuento de colonias. Colombia: Instituto Colombiano de Normas

409

Técnicas.

410 411 412

INS. (2007). Informe de la vigilancia de las enfermedades transmitidas por alimentos. Colombia: Grupo funcional ETA-SVCSP. INS. (2011). Perfil de riesgo Bacillus cereus en alimentos listos para el consumo no

413

industrializados. Unidad de evaluación de riesgos para la inocuidad de los

414

alimentos, Ministerio de la Protección Social. Colombia: Instituto Nacional de

415

Salud.

416

ISO. (2004). Microbiology of food and animal feeding stuffs. Horizontal method for the

417

enumeration of presumptive Bacillus cereus. Colony-count technique at 30 °C ISO

418

7932:2004. Geneva, Switzerland: International Standardisation Organisation.

419

Kotiranta, A., Lounatmaa, K., & Haapasalo, M. (2000). Epidemiology and pathogenesis of

420

Bacillus cereus infections. Microbes and Infection, 2, 189–198.

18

421

Lindbäck, T., Fagerlund, A., Rødland, M. S., & Granum, P. E. (2004). Characterization of

422

the Bacillus cereus Nhe enterotoxin. Microbiology, 150, 3959–3967.

423

Lee, N., Kwon, K. Y., Oh, S. K., Chang, H. J., Chun, H. S., & Choi, S. W. (2014). A

424

multiplex PCR assay for simultaneous detection of Escherichia coli O157:H7,

425

Bacillus cereus, Vibrio parahaemolyticus, Salmonella spp., Listeria monocytogenes,

426

and Staphylococcus aureus in Korea ready-to-eat food. Foodborne Pathogens and

427

Disease, 11, 574–580.

428 429 430

Logan, N. A. (2012). Bacillus and relatives in foodborne illness. Journal of Applied Microbiology, 112, 417–429. López, A. C., & Alippi, A. M. (2010). Enterotoxigenic gene profiles of Bacillus cereus and

431

Bacillus megaterium isolates recovered from honey. Revista Argentina de

432

Microbiología, 42, 216–225.

433

López, A. C., Minnaard, J., Pérez, P. F., & Alippi, A. M. (2015). A case of intoxication due

434

to a highly cytotoxic Bacillus cereus strain isolated from cooked chicken. Food

435

Microbiology, 46, 195–199.

436

Mandal, P. K., Biswas, A. K., Choi, K., & Pal, U. K. (2011). Methods for rapid detection of

437

foodborne pathogens: an overview. American Journal of Food Technology, 6, 87–

438

102.

439

Martínez-Blanch, J. F., Sánchez, G., Garay, E., & Aznar, R. (2009). Development of a real-

440

time PCR assay for detection and quantification of enterotoxigenic members of

441

Bacillus cereus group in food samples. International Journal of Food Microbiology,

442

135, 15–21.

443 444

Martínez-Blanch, J. F., Sánchez, G., Garay, E., & Aznar, R. (2010). Evaluation of a realtime PCR assay for the detection and quantification of Bacillus cereus group 19

445

spores in food. Journal of Food Protection, 73, 1480–1485.

446

Martínez-Blanch, J. F., Sánchez, G., Garay, E., & Aznar, R. (2011). Detection and

447

quantification of viable Bacillus cereus in food by RT-qPCR. European Food

448

Research and Technology, 232, 951–955.

449

Melendez, J.H., Frankel, Y.M., An, A.T., Williams, L., Price, L.B., Wang, N.-Y., et al.

450

(2010). Real-time PCR assays compared to culture-based approaches for

451

identification of aerobic bacteria in chronic wounds. Clinical Microbiology and

452

Infection, 16, 1762–1769.

453

Mintegi, S., Esparza, M. J., González, J. C., Rubio, B., Sánchez, F., Vila, J. J., et al. (2015).

454

Recommendations for the prevention of poisoning. Anales de Pediatría, 83, Article

455

440.

456

Moravek, M., Wegscheider, M., Schulz, A., Dietrich, R., Burk, C., & Martlbauer, E.

457

(2004). Colony immunoblot assay for the detection of hemolysin BL enterotoxin

458

producing Bacillus cereus. FEMS Microbiology Letters, 238, 107–113.

459

Ngamwongsatit, P., Buasri, W., Pianariyanon, P., Pulsrikarn, C., Ohba, M., Assavanig, A.,

460

et al. (2008). Broad distribution of enterotoxin genes (hblCDA, nheABC, cytK, and

461

entFM) among Bacillus thuringiensis and Bacillus cereus as shown by novel

462

primers. International Journal of Food Microbiology, 121, 352–356.

463

Økstad, O., & Kolstø, A. (2011). Bacillus cereus. In M. Wiedmann, & W. E. Zhang, (Eds.),

464

Genomics of foodborne bacterial pathogens (pp. 29–53). New York, USA:

465

Springer.

466

Oliver, S. P., Jayarao, B. M., & Almeida, R. A. (2005). Foodborne pathogens in milk and

467

the dairy farm environment: food safety and public health implications. Foodborne

468

Pathogens and Disease, 2, 115–129. 20

469

Ombui, J., Gitahi, J., & Gicheru, M. (2008). Direct detection of Bacillus cereus enterotoxin

470

genes in food by multiplex polymerase chain reaction. International Journal of

471

Integrative Biology, 2, 172–181.

472

Padilla J. (2007). Validación secundaria del método de recuento en placa en superficie de

473

Bacillus cereus y Staphylococcus aureus en muestras de alimentos en un laboratorio

474

de referencia (Bachelor dissertation). Bogotá, Colombia: Pontificia Universidad

475

Javeriana. Retrieved from

476

http://www.javeriana.edu.co/biblos/tesis/ciencias/tesis15.pdf

477

Park, Y. B., Kim, J. B., Shin, S. W., Kim, J. C., Cho, S. H., Lee, B. K., et al. (2009).

478

Prevalence, genetic diversity, and antibiotic susceptibility of Bacillus cereus strains

479

isolated from rice and cereals collected in Korea. Journal of Food Protection, 72,

480

612–617.

481

Priha, O., Hallamaa, K., Saarela, M., & Raaska, L. (2004). Detection of Bacillus cereus

482

group bacteria from cardboard and paper with real-time PCR. Journal of Industrial

483

Microbiology and Biotechnology, 31, 161–169.

484

Rather, M. A., Aulakh, R. S., Gill, J. P. S., Rao, T. S., & Hassan, M. H. (2011). Direct

485

detection of Bacillus cereus and its enterotoxigenic genes in meat and meat products

486

by Polymerase Chain Reaction. Journal of Advanced Veterinary Research, 1, 99–

487

104.

488

Razei, A., Sorouri, R., Mousavi, S.L., Nazarian, S., Amani, J., & Aghamollaei, H. (2017).

489

Presenting a rapid method for detection of Bacillus cereus, Listeria monocytogenes

490

and Campylobacter jejuni in food samples. Iranian Journal of Basic Medical

491

Sciences, 20, 1050–1055.

21

492

Samapundo, S., Heyndrickx, M., Xhaferi, R., & Devlieghere, F. (2011). Incidence,

493

diversity and toxin gene characteristics of Bacillus cereus group strains isolated

494

from food products marketed in Belgium. International Journal of Food

495

Microbiology, 150, 34–41.

496

Stackebrandt, E., & Liesack, W. (1992). The potential of rDNA in identification and

497

diagnostics. In C. Kessler (Ed.), Nonradioactive labeling and detection of

498

biomolecules (pp 232–239). Berlin, Germany: Springer.

499 500 501 502

Stenfors Arnesen, L. P., Fagerlund, A., & Granum, P. E. (2008). From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiology Reviews, 32, 579–606. Wu, C. H. (2008). A review of microbial injury and recovery methods in food. Food Microbiology, 25, 735–744.

22

Figure legends

Fig. 1. Agarose gel electrophoresis showing mPCR amplicons corresponding to B. cereus toxigenic genes detected in powdered milk artificially contaminated with both emetic F4810/72 and enterotoxigenic ATCC 14579 B. cereus reference strains at various dilutions. Lanes 1 and 9 are molecular size markers (1 Kb plus Invitrogen®) and lane 8 is the PCR negative control. Lanes 2–7 show the amplicons obtained from DNA extracted from pellets at various dilutions that correspond to the following bacterial counts: lane 2, 2 × 105 cfu g-1; lane 3, 2 × 104 cfu g-1 ; lane 3, 2 × 103 cfu g-1; lane 5, 2 × 102 cfu g-1; lane 6, 2 × 101 cfu g-1; lane 7, 2 × 100 cfu g-1.

Fig. 2. Agarose gel electrophoresis showing band patterns representing Bacillus cereus toxigenic groups detected in powdered milk. Lanes 1–10 show amplicons of B. cereus toxigenic genes from powdered milk (in duplicate): lanes 1 and 2, Group I; lanes 3 and 4, Group III; lanes 5 and 6, Group IV; lanes 7–10, Group V. Lane 11, gene products from B. cereus F4810/72 and ATCC 14579 reference strains (Positive control); lane 12, molecular size marker (GeneRuler 100 bp plus Thermo Scientific®).

Fig. 3. Agarose gel electrophoresis showing band patterns representing Bacillus cereus toxigenic groups detected in infant formula. Lanes 1–8 show amplicons of B. cereus toxigenic genes from infant formula (in duplicate): lanes 1 and 2, Group I; lanes 3 and 4, Group IV; lanes 5 and 6, Group II; lanes 7 and 8, Group III. Lane 9, DNA from B. cereus F4810/72 and ATCC 14579 reference strains (Positive control); lane 10, molecular size marker (1 Kb plus Invitrogen®).

Table 1

Description of primers used in the mPCR for detection of B. cereus toxin genes.

Target

Primer sequence (5’-3’)

gene hblC

Product size

Reference

(bp) F-5′-CGAAAATTAGGTGCGCAATC-3′

411

Moravek et al. (2004)

755

Guinebretière et al. (2002)

565

Ngamwongsatit et al. (2008)

1271

Ehling-Schulz et al. (2005)

1514

Stackebrandt and Liesack (1992)

R-5′-TAATATGCCTTGCGCAGTTG-3′ nheA

F-5′-ACGAATGTAATTTGAGTCGC-3′ R-5′-TACGCTAAGGAGGGGCA-3′

cytK2

F-5′-CGACGTCACAAGTTGTAACA-3′ R-5′-CGTGTGTAAATACCCCAGTT-3′

cesB

F-5′-GGTGACACATTATCATATAAGGTG-3′ R-5′-GTAAGCGAACCTGTCTGTAACAACA-3′

ITS1

F-5′-AGAGTTTGATCCTGGCTCA-3′ R-5′-CGGCTACCTTGTTACGAC-3′

Table 2 Gene profiles for emetic and enterotoxic B. cereus reference strains. a Species/ Strains

mPCR result

Origin/Source

cesB

cytK2

hblC

nheA

IAC

Bacillus cereus ATCC 14579

-

+

+

+

+

Niels Hendriksen

Bacillus cereus ATCC 10987

-

+

-

+

+

Niels Hendriksen

Bacillus cereus ATCC 6464

-

+

+

+

+

Niels Hendriksen

Bacillus cereus ATCC 21281

-

+

+

+

+

Niels Hendriksen

Bacillus cereus ATCC 27348

-

+

+

+

+

Niels Hendriksen

Bacillus cereus NVH 1230/88

-

+

+

+

+

Per E. Granum

Bacillus cereus NVH 0075/95

-

-

-

+

+

Per E. Granum

Bacillus cereus F4094/73

-

+

+

+

+

Niels Hendriksen

Bacillus cereus BC307

-

+

+

+

+

Lucas Wijnands

Bacillus cereus F4810/72

+

-

-

+

+

Niels Hendriksen

Bacillus cereus NVH 1257

+

-

-

+

+

Per E. Granum

Salmonella typhimurium ATCC 14028

-

-

-

-

+

MIA Lab

Shigella sonnei ATCC 25931

-

-

-

-

+

MIA Lab

Escherichia coli ATCC 25922

-

-

-

-

+

MIA Lab

Staphylococcus aureus ATCC 6538

-

-

-

-

+

MIA Lab

Listeria monocytogenes ATCC 19118

-

-

-

-

+

MIA Lab

a

Abbreviations are: ATCC, American Type Culture Collection, USA. NVH: Norwegian School of

Veterinary Science, Norway; MIA Lab, Laboratorio Microbiología Industrial y Ambiental. + indicates PCR product of the expected size was formed (gene present); - indicates no PCR product was formed (gene absent).

Table 3 Detection of B. cereus and toxin genes in dairy powdered foods. a

Type of dairy powdered food

According to NTC 4679

According to molecular method

Positive

Negative

Positive

Negative

Powdered milk

11 (14.7%)

64 (85.3%)

64 (85.3%)

11 (14.7%)

Infant formula

8 (10.7%)

67 (89.3%)

53 (70.7%)

22 (29.3%)

Total

19 (12.7%)

131 (87.3%)

117 (78%)

33 (22%)

a

Abbreviation: NTC, Norma Técnica Colombiana, Molecular method includes direct DNA

extraction from dairy powdered food and mPCR. Seventy-five samples of powdered milk and of infant formula (150 samples in total) were analysed. Values are numbers followed by percentage in parentheses.

Table 4 Frequencies of toxin genes in dairy powdered food samples. a

Toxin genes

Powdered milk

Infant formula

Total

(n = 64)

(n = 53)

(n = 117)

hblC

61 (95%)

53 (100%)

114 (97.4%)

nheA

35 (55%)

6 (11%)

41 (35%)

cytK2

13 (20%)

27 (51%)

40 (34.2%)

cesB

3 (5%)

ND

3 (2.6%)

a

Values are numbers followed by percentage in parentheses; ND, not detected.

Table 5 Toxigenic groups in dairy powdered food samples. a

Toxigenic group

Toxin genes

Powdered milk

Infant formula

Total

(n = 64)

(n = 53)

(n = 117)

I

hblC

28 (43.7%)

25 (47.2%)

53 (45.3%)

II

hblC, cytK2

1 (1.6%)

22 (41.5%)

23 (19.7%)

III

nheA, hblC

20 (31.3%)

1 (1.9%)

21 (17.9%)

IV

nheA, hblC, cytK2

12 (18.7%)

5 (9.4%)

17 (14.5%)

V

cesB, nheA

3 (4.7%)

ND

3 (2.6%)

a

Values are numbers followed by percentage in parentheses; ND, not detected.

Author Contributions Term Conceptualization

Authors Jennifer A. Sánchez-Chica, Margarita M. Correa, Angel E. Aceves-Diez, Laura M. Castañeda-Sandoval.

Methodology

Jennifer A. Sánchez-Chica, Angel E. Aceves-Diez, Laura M. Castañeda-Sandoval.

Formal analysis

Jennifer A. Sánchez-Chica, Margarita M. Correa, Angel E. Aceves-Diez, Laura M. Castañeda-Sandoval. Jennifer A. Sánchez-Chica, Margarita M. Correa, Angel E. Aceves-Diez, Laura M. Castañeda-Sandoval.

Investigation

Jennifer A. Sánchez-Chica, Laura M. Castañeda-Sandoval.

Resources Writing - Original Draft

Margarita M. Correa, Laura M. Castañeda-Sandoval.

Validation

Jennifer A. Sánchez-Chica, Laura M. Castañeda-Sandoval

Writing - Review & Jennifer A. Sánchez-Chica, Margarita M. Correa, Angel E. Aceves-Diez, Laura M. Editing Castañeda-Sandoval. Supervision Project administration

Laura M. Castañeda-Sandoval. Laura M. Castañeda-Sandoval.

Funding acquisition Margarita M. Correa, Laura M. Castañeda-Sandoval.