Toxigenic Potential of Bacillus cereus Strains Isolated from Retail Dairy Products in Egypt

Abstract

Little is known about the virulence in Bacillus cereus strains isolated from retail dairy products in the Middle East and particularly from Egypt. In this study, the occurrence of B. cereus in 290 samples of dairy products (raw milk, Ras cheese, pasteurized extended shelf life [ESL] milk) collected from retail shops was investigated. The potential of 126 selected isolates of B. cereus to possess genes encoding nonhemolytic enterotoxin, hemolysin BL, and cytotoxin K (cytK), and to grow at 7°C was verified. The highest occurrence of B. cereus was found in raw milk (85%, 85/100) followed by Ras cheese (10%, 10/100) and ESL milk samples (8.8%, 8/90). A large proportion of the B. cereus isolates from raw milk (48.9%, 48/99) and Ras cheese (71.4%, 10/14) had at least one complete set of toxin genes (nhe or hbl). Enterotoxin genes, nheA, nheB, nheC, hblA, hblD, and hblC, were detected in 38.4% (5/13), 53.8% (7/13), 61.5% (8/13), 46.1% (6/13), 46.1% (6/13), and 23.1% (3/13) of ESL milk isolates, respectively. cytK was identified in 42.4% (42/99), 50% (7/14), and 46.2% (6/13) of raw milk, Ras cheese, and ESL milk isolates, respectively. The psychrotrophic ability was observed in 22.2% and 15.3% of isolates recovered from raw milk and ESL milk, respectively. The toxigenic potential of B. cereus strains described in this study may pose a health risk to the consumer and, therefore, the presence of these bacteria in retail dairy products should be monitored to ensure consumers' safety.

Introduction

Bacillus cereus is a ubiquitous Gram-positive spore-forming bacterium that is widely spread throughout the environment. It causes a wide range of clinical infections such as endocarditis, endophthalmitis, bacteremia, septicemia, pneumonia, and gastritis in both immunocompromised and immunocompetent patients (Parihar, 2014). It is also a major cause of two foodborne illness syndromes, namely “diarrheal syndrome” and “emetic syndrome.”

The diarrheal syndrome is a type of “food toxicoinfection” because the toxin is formed in the small intestine during the growth of vegetative bacteria. Four different heat-labile enterotoxins can cause the diarrheal syndrome, including two protein complexes, nonhemolytic enterotoxin (NHE) and hemolysin BL (HBL), and two enterotoxin proteins, cytotoxin K (cytK) and enterotoxin T (Rajkovic, 2014). The coexpression of NHE and HBL has been reported in ∼40–60% of isolated strains (Jeßberger et al., 2014).

The increasing consumption of raw milk is a serious public health concern throughout the world. In some countries, health authorities do not prohibit the on-farm consumption of raw milk by farmers and their families, and in surveys in Canada and the United States, 35–88.7% of dairy farmers reported that they or their families consumed raw milk from their bulk tanks (Ontario Agency for Health Protection and Promotion, 2013). In Egypt, the milk sector is still largely traditional as the majority of dairy producers are small family farms and most of the population consume unpasteurized milk. Around 85% of the milk sold in Egypt is loose milk, which is that sold on the street from vendors or at dairy shops in plastic bags (Oxford Business Group, 2012). Consequently, contamination of milk with foodborne pathogens, particularly ubiquitous B. cereus spores, during production and distribution is more likely to occur.

In 2005, the Egyptian Organization for Standardization and Quality approved the production of pasteurized milk with a shelf life of not more than 15 days on condition that application of microfiltration or high hydrostatic pressure in production of this milk (Egyptian Standards, 2005). The pasteurized milk sector in Egypt is markedly growing to meet the current demand of fresh safe milk. However, it should be considered that either normal pasteurization temperatures or higher temperatures used in the production of ultrapasteurized milk or extended shelf life (ESL) milk could eliminate B. cereus spores (Hanson et al., 2005).

In contrast, there is an increased production and consumption of raw milk cheeses on a global basis. In some countries, the sale of cheese made from raw milk is allowed as long as the cheese has been aged >60 days (FDA, 2017; Ganz et al., 2020). However, despite the potential health risks associated with B. cereus isolated from raw milk cheeses all over the world, little is still known about the risks associated with the consumption of hard rennet-coagulated cheeses made from raw milk. Ras cheese is one of the popular raw milk hard cheeses in the Mediterranean countries, particularly in the Middle East. It is valued for its high nutrient contents, rich flavor, and long shelf life. It is made from cow's milk or a mixture of cow and buffalo's milk. The coagulation process is carried out using rennet and no starter cultures are added. After a ripening period of 3–4 months at the ambient temperature, the cheese develops a sharp pungent flavor and open texture (Ayad et al., 2004).

The food poisoning caused by B. cereus differs from country to country. In Europe and North America, the frequency of diarrheal syndrome is dramatically higher than that of the emetic syndrome, whereas in Japan emetic syndrome is reported ∼10 times more often than diarrheal syndrome (Gibbs, 2009). Understanding the frequency and toxigenic potential of B. cereus in dairy products would help in estimating the risk of food poisoning by B. cereus, establishing the basis for research concerning its survival and multiplication in these products, designing reliable tracking procedures, and developing control measures. This study aimed to check the presence of B. cereus in dairy food available for retail sale in Egypt and to investigate the virulence profiles of obtained strains.

Materials and Methods

Isolation and identification of B. cereus strains

Samples of raw milk (n = 100), Ras cheese (n = 100), and pasteurized ESL milk (n = 90) were collected from different supermarkets in the El-Menoufia governorate, Egypt. All raw milk samples were collected on the same day of their production and pasteurized ESL milk samples were collected within the 1st week of their production. Ras cheese samples were of the old type (ripened for >2 months). All the samples were immediately placed on ice, transported to our laboratory, and stored at 4°C until being analyzed within 24 h after purchase. In the laboratory, 25 g of Ras cheese samples was placed in sterile pages containing 225 mL of 2% sodium citrate buffer, and the mixtures were homogenized for 10 min in a stomacher. Regarding pasteurized ESL and raw milk samples, 25 mL of each sample was transferred into sterile wide-mouth jars containing 225 mL of sterile buffered peptone water (Oxoid, Basingstoke, England) before plating. Then, 100 μL from each diluted sample was spread on Polymyxin Pyruvate Egg-Yolk Mannitol Bromothymol Blue Agar (PEMBA) (Oxoid) plates, a medium developed by Holbrook and Anderson (1980) that showed a high selective power for isolation of B. cereus from dairy products (Schulten et al., 2000). After incubation of PEMBA plates at 30°C for 24 h, typical colonies, with turquoise to peacock blue color surrounded by egg yolk precipitate, were picked. A maximum of three typical colonies from each sample were subcultured on nutrient agar (BD, Sparks, MD) and confirmed as B. cereus with the biochemical assays described in the FDA Bacteriological Analytical Manual (2020). Then, each colony was transferred into cryotubes contained tryptic soy broth (Oxoid) and further incubated for 24 h at 30°C. The cultures were frozen at −80°C after the addition of glycerol, at a final concentration of 15%.

Molecular characterization of B. cereus isolates

Total genomic DNA was prepared from the isolated strains with the GeneJET Genomic DNA Purification Kit (Thermo Scientific, Waltham, MA). The quality and quantity of extracted DNA were checked using NanoDrop Spectrophotometer 2000 (Thermo Scientific). The concentration of DNA was adjusted to 25 ng/μL. The PCR mixture (25 μL) contained 1 × GoTaq G2 Green Master Mix (Promega, USA) (GoTaq G2 DNA polymerase, reaction buffer [pH 8.5], 200 μM of each deoxynucleoside triphosphate, and 1.5 mM MgCl₂), 50 ng of target DNA, and 1 μM of each primer. Additional MgCl₂ was added to reach a final concentration of 3 mM. Nuclease-free water was added to a final volume of 25 μL. The PCR protocols are described in Supplementary Table S1.

The gyrB gene was PCR amplified to confirm the B. cereus group strains (Wei et al., 2018). The enterobacterial repetitive intergenic consensus (ERIC)-PCR was performed using primer pairs ERIC-F and ERIC-R designed by Versalovic et al. (1991) to exclude clonal isolates. The gene encoding production of cytK was amplified as previously described by Ehling-Schulz et al. (2006). All strains were analyzed with a multiplex PCR for the presence of the three genes encoding the HBL enterotoxin, hblA, hblD, and hblC, and the three genes encoding the NHE, nheA, nheB, and nheC, using the multiplex primers described by Hansen and Hendriksen (2001). B. cereus ATCC 14579 was used as a positive control for PCR assays. The primer sequences and PCR conditions are shown in Supplementary Table S1.

Detection of enterotoxin production

Confirmed B. cereus isolates that carried the three components of the HBL or NHE operons were analyzed for the production of HBL or NHE using the reverse passive latex agglutination test kit (Oxoid), and the Bacillus Diarrheal Enterotoxin Visual Immunoassay kit (Tecra Diagnostics, Rosewille, Australia), according to the manufacturer's instructions, respectively.

Visualization of virulence genes profiles

ComplexHeatmap (v2.6.2) R package (Gu et al., 2016) was used to plot a summary heatmap for the presence or absence of virulence genes in the isolated strains.

Psychrotrophic/mesophilic discrimination by growth profiles and PCR

The strains were grown on nutrient agar plates at 30°C. The bacterial cultures were transferred to the nutrient broth and grown with shaking at 7°C for 14–21 days. All the cultures that could grow at 7°C were defined as psychrotrophic strains, and the others as mesophilic strains (Zhou et al., 2010). The gene encoding the B. cereus major cold-shock protein, cspA, was PCR amplified as described previously (Francis et al., 1998).

Results and Discussion

Little is known about the virulence in B. cereus strains isolated from retail dairy products in the Middle East. In this study, the distribution and proportions of seven toxin genes were revealed providing a comprehensive reference on the occurrence and characteristics of B. cereus isolated from dairy products in Egypt. Ninety-nine strains were presumptively identified as B. cereus group strains from 85 raw milk samples (85%, 85/100) based on phenotypic and biochemical characteristics. The gyrB gene, an efficient target for the identification of members of the B. cereus group, was positively amplified in all these strains. Our result was higher than those reported by Yobouet et al. (2013) (41%), Aouadhi et al. (2014) (47.5%), or Hassan et al. (2010) (30%), but similar to that reported by Gundogan and Avci (2014) (90%). Although spore-forming bacteria are ubiquitous and can find their way into milk from different sources, including equipment and water, recent studies have shown that dirty udders are the main source of contamination with these bacteria (Vissers et al., 2007; Masiello et al., 2014). Dirt attached to the exterior of teats, whether collected during grazing or housing, usually rinses off during milking. Our results indicate the high occurrence of B. cereus in retail raw milk, which emphasizes the need for stricter hygiene measures during milking and the handling of raw milk during small-scale production.

In contrast, even though raw milk cheeses are categorized as “risky foods,” the occurrence of potentially toxigenic B. cereus in hard rennet-coagulated cheeses made from raw milk has been rarely addressed in the literature (Berthold-Pluta et al., 2019; Abdeen et al., 2020). We recovered 20 presumptive Bacillus isolates from Ras cheese. Fourteen isolates were positively identified as B. cereus from 10 samples (10%, 10/100). Our result is much lower than those reported by Berthold-Pluta et al. (2019) (43.4%) but similar to that reported by Abdeen et al. (2020) (8.5%). Interestingly, a high proportion of the Egyptian Ras cheese is produced under artisan production, small factories, and in rural areas. It is worth mentioning that vegetative B. cereus cells can be inactivated by several synergistic factors during the ripening period such as the presence of competitor microorganisms that produce antimicrobial agents, the existence of salt, drop in pH, and shift in the oxidation–reduction potential during the production process (Rukure and Bester, 2001). However, our finding indicated that aging raw milk cheese for 60 days [obligated in some international standards such as FDA (2017)], which is proposed to make raw milk cheese safe for consumption, may not eliminate B. cereus in raw milk cheese, thus posing a potential hazard to consumers.

Regarding pasteurized ESL milk, eight samples (8.9%, 8/90) contained Bacillus spp. We recovered 24 presumptive Bacillus isolates from pasteurized ESL milk. Thirteen isolates were positively identified as B. cereus from eight samples. Overall, compared with previous surveys in other countries, the occurrence of B. cereus in our pasteurized ESL milk samples was low, similar to that reported in Tunisia (7.5%), Germany, Austria, Switzerland (8.8%), and China (10%) (Schmidt et al., 2012; Aouadhi et al., 2014; Zhao et al., 2020). Such low occurrence of B. cereus in ESL milk highlighted the efficacy of control measures applied by the ESL milk producers in Egypt to produce safe milk.

PCR amplification of the nonhemolytic and hemolytic enterotoxin genes revealed PCR products of 480, 754, 564, 320, 430, and 750 bp for nheA, nheB, nheC, hblA, hblD, and hblC genes, respectively. The distribution of virulence genes among 126 B. cereus group strains is given in Table 1. Visualization of virulence genes profiles of all strains was conducted using a heatmap (Fig. 1). Overall, strains from raw milk, Ras cheese, and ESL milk were clustered into 34 clusters. Stains isolated from raw milk samples were clustered into 31 clusters. Of note, 89.9% (89/99) of the isolated strains from raw milk carried one or more genes from the nhe operon. The three individual components of the nhe operon, nheA, nheB, and nheC, were detected in 72.7% (72/99), 72.7% (72/99), and 54.5% (54/99) of isolates, respectively. Several studies have suggested that NHE is the predominant diarrheal toxin secreted by most B. cereus strains. Because all three components of the NHE are necessary for its maximal biological activity, 38.4% (38/99) of the strains isolated from raw milk in this study were potentially toxigenic because they carried the complete nhe operon (Fig. 1). Similar results were obtained by Svensson et al., (2007). In contrast, as presented in Figure 1, 58.6% (58/99), 57.6% (57/99), and 33.3% (33/99) of the isolates were PCR positive for the three individual genes of the hbl operon, hblA, hblD, and hblC, respectively. Interestingly, 33.3% (33/99) of the isolates could potentially produce the HBL toxin because they carried the complete hbl operon.

FIG. 1.

Heatmap showing virulence profiles of isolates strains. Virulence genes were visualized using the ComplexHeatmap (Gu et al., 2016). Clustering and distance methods were “ward.D2” and “binary”, respectively. The black color of the box indicates the presence of the gene while the white color indicates the absence of the gene. The dendrogram on the left reflects the hierarchical clustering of virulence genes in the isolated strains. The numbers on the dendrogram (1 to 34) indicate the numbers of clusters. The dendrogram on the top reflects the hierarchical clustering of screened virulence genes. The numbers of strains isolated from raw milk (RM-1 to RM-99), Ras cheese (SC-1 to SC-14), and ESL (extended shelf life) milk (PM-1 to PM-13) were written on the left of the heat map.

Table 1.

Distribution of Enterotoxin Genes in Bacillus cereus Group Strains Isolated from Dairy Products

Toxigenic genes	Number of strains (%) ^a positive for target gene(s)
Toxigenic genes	Raw milk (n = 99)	Ras cheese (n = 14)	ESL milk (n = 13)
NHE gene complexes
nheA	72 (73)	14 (100)	5 (38)
nheB	72 (73)	10 (71)	7 (54)
nheC	54 (55)	14 (100)	8 (62)
nheA + nheB	55 (56)	10 (71)	0 (0)
nheA + nheC	45 (45)	13 (93)	5 (38)
nheB + nheC	46 (46)	10 (71)	3 (23)
nheA + nheB + nheC	38 (38)	10 (71)	0 (0)
Nondetected	9 (9)	0 (0)	1 (8)
HBL gene complexes
hblA	58 (59)	7 (50)	6 (46)
hblC	33 (33)	7 (50)	3 (23)
hblD	57 (58)	7 (50)	6 (46)
hblA + hblC	33 (33)	7 (50)	0 (0)
hblA + hblD	54 (55)	7 (50)	6 (46)
hblC + hblD	33 (33)	7 (50)	0 (0)
hblA + hblC + hblD	33 (33)	7 (50)	0 (0)
Nondetected	38 (38)	7 (50)	4 (31)
Cytotoxin K
cytk	42 (42)	7 (54)	6 (46)

Percentages have been converted to the nearest whole numbers.

ESL, extended shelf life; HBL, hemolysin BL; NHE, nonhemolytic enterotoxin.

Ras cheese isolates showed four toxigenic patterns as shown in Figure 1. They were clustered into four clusters (1, 2, 22, and 29). Among diarrheal enterotoxin genes, detection rates of nheA, nheB, and nheC were 100% (14/14), 71.4% (10/14), and 100% (14/14) of isolates, respectively. Of interest, 71.4% (10/14) of the isolates carried the three nonhemolytic genes, nheA, nheB, and nheC, whereas 50% (7/14) of the isolates carried the three hemolytic genes, hblA, hblD, and hblC. Regarding ESL milk isolates, they were clustered into four clusters (7, 18, 28, and 32). Nonhemolytic genes nheA, nheB, and nheC were detected in 38.5% (5/13), 53.8% (7/13), and 61.5% (8/13) of isolates, respectively, whereas hblA, hblD, and hblC were detected in 46.1% (6/13), 46.1% (6/13), and 23.1% (3/13) of isolates, respectively. However, none of the strains isolated from pasteurized ESL milk carried complete nheABC and hblACD gene clusters. Toxin production in all strains carried the three components of hblACD and/or nheABC gene clusters was confirmed using a HBL latex agglutination assay and an NHE immunoassay, respectively. Our results are a cause for public health concern because a large proportion of the B. cereus isolates from raw milk (48.9%, 48/99) and Ras cheese (71.4%, 10/14) had at least one complete set of toxin genes (nhe or hbl), so they were potentially toxigenic.

cytK is a major gene frequently detected in B. cereus causing diarrheal-type food poisoning. It was detected in 1998 in B. cereus strain NVH 391/98, which was responsible for a severe food-poisoning outbreak, with fatal bloody diarrhea, which killed three people in France (Lund et al., 2000). In this study, 42.4% (42/99), 50% (7/14), and 46.2% (6/13) of our isolated strains from raw milk, Ras cheese, and ESL milk carried cytK in combination with one or more components of the nhe and/or hbl operon(s), respectively. Only one strain (RM-79) isolated from raw milk carried the cytK gene alone (cluster 20). Interestingly, Guinebretière and Broussolle (2002) reported that the B. cereus strains isolated by them displayed all three nhe and all three hbl genes, as well as the cytK gene, indicating a toxin gene profile typical of food poisoning strains. As presented in the first cluster in Figure 1, this pattern was detected in 16 (16.2%, 16/99) and 4 (28.5%, 4/14) strains from raw milk and Ras cheese samples, respectively, indicating their high toxigenic potential.

The combination of psychrotrophic and enterotoxigenic properties in the B. cereus strains contaminating milk constitutes a great health hazard for consumers of milk and dairy products. In this study, 22.2% (22/99) of our isolates from raw milk grew at 7°C, whereas 15.3% (2/13) of our isolates from pasteurized ESL milk grew at the same temperature. Conversely, none of the isolated strains from Ras cheese grew at 7°C. Retail milk is usually exposed to temperature fluctuations during its transportation and storage, which may favor the growth of mesophilic strains. Of the 22 psychrotrophic strains isolated from raw milk, 12 carried cytK and both HBL and NHE operons, 4 carried both HBL and NHE operons, 3 carried only HBL operon, and 3 carried cytK and NHE operon. None of the emetic strains was psychrotrophic. Our results confirm that psychrotrophic strains of B. cereus are common and may contain the full complement of enterotoxin genes (Stenfors Arnesen et al., 2007). Moreover, all the psychrotrophic strains carried the cspA gene, encoding a cold-shock protein. Noteworthy, in developing countries, the milk produced by small producers in villages is transported to the market without refrigeration.

Conclusions

This study provides comprehensive insights into the virulence potential of B. cereus strains associated with dairy products in Egypt. The virulence profile of B. cereus strains suggests the potential to constitute a public health hazard. The minimum aging period (60 days) of raw milk cheese should be revised to ensure consumers' safety.

Footnotes

Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by a Grant-in-Aid for Scientific Research to T.S. from Japan Society for the Promotion of Science (Grant No. 25460532).

Supplementary Material

Supplementary Table S1

References

Abdeen

, Hussien

, Hadad

GAE

, Mousa

. Prevalence of virulence determinants among Bacillus cereus isolated from milk products with potential public health concern. Pak J Biol Sci, 2020; 23:206–212.

Aouadhi

, Maaroufi

, Mejri

. Incidence and characterisation of aerobic spore-forming bacteria originating from dairy milk in Tunisia. Int J Dairy Technol, 2014; 67:95–102.

Ayad

EHE

, Awad

, El Attar

, de Jong

, El-Soda

. Characterisation of Egyptian Ras cheese. 2. Flavour formation. Food Chemistry, 2004; 86:553–561.

Berthold-Pluta

, Pluta

, Garbowska

, Stefanska

. Prevalence and toxicity characterization of Bacillus cereus in food products from Poland. Foods, 2019; 8:269.

Egyptian Standards. Pasteurized milk. Egyptian Organization for Standardization and Quality Control, ES, 2005, pp. 1616–2005.

Ehling-Schulz

, Guinebretiere

, Monthán

, Berge

, Fricker

, Svensson

. Toxin gene profiling of enterotoxic and emetic Bacillus cereus . FEMS Microbiol Lett, 2006; 260:232–240.

FDA Bacteriological Analytical Manual, 2020. Chapter 14: Bacillus cereus. Available at: http://www.fda.gov/Food/FoodScienceResearch/LaboratoryMethods/ucm070875.htm, accessed October 29, 2020 .

[FDA] Food and Drug Administration. Food and Drug Administration CFR—Code of Federal Regulations Title 21. Available at: https://wwwaccessdatafdagov/scripts/cdrh/cfdocs/cfcfr/cfrsearchcfm?cfrpart=133, accessed October 29, 2020 .

Francis

, Mayr

, von Stetten

, Stewart

, Scherer

. Discrimination of psychrotrophic and mesophilic strains of the Bacillus cereus group by PCR targeting of major cold shock protein genes. Appl Environ Microbiol, 1998; 64:3525–3529.

10.

Ganz

, Yamamoto

, Hardie

, Hum

, Hussein

, Locas

, Steele

. Microbial safety of cheese in Canada. Int J Food Microbiol, 2020; 321:108521.

11.

Gibbs

Characteristics of spore-forming bacteria. In: Blackburn

, McClure

, eds. Foodborne Pathogens: Hazards, Risk Analysis and Control, Volume 2. Boca Raton, FL: CRC Press, 2009.

12.

, Eils

, Schlesner

. Complex heatmaps reveal patterns and correlations in multidimensional genomic data. Bioinformatics, 2016; 32:2847–2849.

13.

Guinebretière

M-H

, Broussolle

. Enterotoxigenic profiles of food-poisoning and food-borne Bacillus cereus strains. J Clin Microbiol, 2002; 40:3053–3056.

14.

Gundogan

, Avci

. Occurrence and antibiotic resistance of Escherichia coli, Staphylococcus aureus and Bacillus cereus in raw milk and dairy products in Turkey. Int J Dairy Technol, 2014; 67:562–569.

15.

Hansen

, Hendriksen

. Detection of enterotoxic Bacillus cereus and Bacillus thuringiensis strains by PCR analysis. Appl Environ Microbiol, 2001; 67:185–189.

16.

Hanson

, Wendorff

, Houck

. Effect of heat treatment of milk on activation of Bacillus spores. J Food Prot, 2005; 68:1484–1486.

17.

Hassan

, Al-Ashmawy

MAM

, Meshref

AMS

, Afify

. Studies on enterotoxigenic Bacillus cereus in raw milk and some dairy products. J Food Saf, 2010; 30:569–583.

18.

Holbrook

, Anderson

. An improved selective and diagnostic medium for the isolation and enumeration of Bacillus cereus in foods. Canadian J Microbiol, 1980; 26:753–759.

19.

Jeßberger

, Dietrich

, Bock

, Didier

, Märtlbauer

. Bacillus cereus enterotoxins act as major virulence factors and exhibit distinct cytotoxicity to different human cell lines. Toxicon, 2014; 77:49–57.

20.

Lund

, De Buyser

, Granum

. A new cytotoxin from Bacillus cereus that may cause necrotic enteritis. Mol Microbiol, 2000; 38:254–261.

21.

Masiello

, Martin

, Watters

, Galton

, Schukken

, Wiedmann

, Boor

. Identification of dairy farm management practices associated with the presence of psychrotolerant spore formers in bulk tank milk. J Dairy Sci, 2014; 97:4083–4096.

22.

Ontario Agency for Health Protection and Promotion. PHO (Public Health Ontario) Technical Report: Update on Raw Milk Consumption and Public Health: A Scientific Review for Ontario Public Health Professionals. Toronto, ON: Queen's Printer for Ontario, 2013. Available at: www.publichealthontario.ca/en/eRepository/PHO_Technical_Report_Raw_Milk_2013.pdf, accessed October 29, 2020 .

23.

Oxford Business Group. The milky way: Considerable scope for increasing milk production. 2012. Available at: https://oxfordbusinessgroup.com/analysis/milky-way-considerable-scope-increasing-milk-production, accessed January 21, 2020 .

24.

Parihar

. Bacillus cereus. In: Wexler

. ed. Encyclopedia of Toxicology. 3rd Edition, Volume 1. 2014, pp. 353–354.

25.

Rajkovic

. Microbial toxins and low level of foodborne exposure. Trends Food Sci Technol, 2014; 38:149–157.

26.

Rukure

, Bester

. Survival and growth of Bacillus cereus during Gouda cheese manufacturing. Food Control, 2001; 12:31–36.

27.

Schmidt

, Kaufmann

, Kulozik

, Scherer

, Wenning

. Microbial biodiversity, quality and shelf life of microfiltered and pasteurized extended shelf life (ESL) milk from Germany, Austria and Switzerland. Int J Food Microbiol, 2012; 154:1–9.

28.

Schulten

, in't Veld

, Nagelkerke

NJD

, Scotter

, de Buyser

, Rollier

, Lahellec

. Evaluation of the ISO 7932 standard for the enumeration of Bacillus cereus in foods. Int J Food Microbiol, 2000; 57:53–61.

29.

Stenfors Arnesen

, O'Sullivan

, Granum

. Food poisoning potential of Bacillus cereus strains from Norwegian dairies. Int J Food Microbiol, 2007; 116:292–296.

30.

Svensson

, Monthán

, Guinebretière

M-H

, Nguyen-Thé

, Christiansson

. Toxin production potential and the detection of toxin genes among strains of the Bacillus cereus group isolated along the dairy production chain. Int Dairy J, 2007; 17:1201–1208.

31.

Versalovic

, Koeuth

, Lupski

. Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes. Nucleic Acids Res, 1991; 19:6823–6831.

32.

Vissers

, Te Giffel

, Driehuis

, De Jong

, Lankveld

. Minimizing the level of Bacillus cereus spores in farm tank milk. J Dairy Sci, 2007; 90:3286–3293.

33.

Wei

, Chelliah

, Park

, Forghani

, Park

, Cho

, Park

, Oh

. Molecular discrimination of Bacillus cereus group species in foods (lettuce, spinach, and kimbap) using quantitative real-time PCR targeting groEL and gyrB . Microb Pathog, 2018; 115:312–320.

34.

Yobouet

, Kouamé-Sina

, Dadié

, Makita

, Grace

, Djè

, Bonfoh

. Contamination of raw milk with Bacillus cereus from farm to retail in Abidjan, Côte d'Ivoire and possible health implications. Dairy Sci Technol, 2013; 94:51–60.

35.

Zhao

, Chen

, Fei

, Feng

, Wang

, Ali

, Li

, Jing

, Yang

. Prevalence, molecular characterization, and antibiotic susceptibility of Bacillus cereus isolated from dairy products in China. J Dairy Sci, 2020; 103:3994–4001.

36.

Zhou

, Zheng

, Dou

, Cai

, Yuan

. Occurrence of psychrotolerant Bacillus cereus group strains in ice creams. Int J Food Microbiol, 2010; 137:143–146.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

0.02 MB