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Bacillus cereus group: Persistent and harmful toxin-producing microorganisms in the food industry

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The Bacillus cereus group, also known as B. cereus sensu lato or presumptive Bacillus cereus” consists of Gram-positive, rod-shaped, spore-forming bacilli

 

What is the Bacillus cereus group?

The Bacillus cereus group, also known as B. cereus sensu lato or “presumptive Bacillus cereus” consists of Gram-positive, rod-shaped, spore-forming bacilli, commonly isolated from soil, other environmental and food matrices.

The B. cereus Group is a subdivision of the Bacillus genus that comprises eight formally recognised species: B. cereus sensu stricto, B. anthracis, B. thuringiensis, B. weihenstephanensis, B. mycoides, B. pseudomycoides, B. cytotoxicus and B. toyonensis (EFSA, 2016). This current taxonomy of the B. cereus group and the status of separate species mainly rely on phenotypic characteristics established before the genomic erea. During the last 20 years, various genotypic methods including the lately introduced Whole Genome Sequencing (Zervas et al., 2020, Carroll et al. 2020) have revealed the existence of three broad phylogenetic clades within the Bacillus cereus group, further divided into seven phylogenetic groups (Priest et al., 2004, Guinebretiere et al., 2008, Okinaka et al., 2016; Bazinet, 2017). On these grounds, tentative new species within the Bacillus cereus group were proposed (Liu et al., 2017; BfR, 2020).

It also appeared that the traditional phenotypic classification and the phylogeny were not necessarily aligned mainly since the bacterial genomes do have real-life concrete plasticity. The assignment of a specific presumptive Bacillus cereus group’s isolate to a single species or phylogenic group is further complicated since the phylogeny is based on chromosomic genes while important virulence and phenotypic traits are plasmid-borne and can then be lost, horizontally transferred and inconsistent in their presence within a species (EFSA, 2016). Some strains affiliated for example to B. thuringiensis display a similar repertoire of potential virulence genes on the chromosome than Bacillus cereus sensu stricto but bear on plasmids other pivotal genes critical for biopesticides production (EFSA, 2016). Therefore, the relevance of the taxonomic segregation of the B. cereus group into separate species has been questioned (EFSA, 2016).

The pathogenicity of the B. cereus group bacteria is associated, apart from the life-threatening anthrax and some anthrax-like diseases caused by B. anthracis and a few B. cereus strains, to two disabling gastro-intestinal diseases, the emetic and diarrheal syndromes.

 

What are the risks for the consumers?

The emetic syndrome is caused by the toxin cereulide produced by B. cereus sensu lato isolates bearing plasmid-borne cereulide synthetase genes (Ehling-Schulz et al., 2006). Cereulide is commonly pre-formed in food resulting in rapid onset of vomiting (15 min to 6 h) after uptake of the contaminated food. Normally this disease is self-limiting, but occasionally, more severe intoxications are reported requiring hospitalization and intensive medical care. Due to its acid and heat stability, cereulide is usually not inactivated once it is formed in the normal food-processing environment.

The diarrheal syndrome of B. cereus food poisoning is characterized by abdominal pain and watery diarrhoea; It has notably been linked to three pore-forming chromosomally encoded enterotoxins. They are i) two three-component toxins, the nonhemolytic enterotoxin (Nhe), the hemolytic toxin (Hbl) and ii) the single protein cytotoxin (CytK). the enterotoxins are heat labil and are enzymatically digested during the stomach passage while B. cereus spores (and also some vegetative cells) survive Gastro-Intestinal crossing. Thus, it is generally assumed that diarrhoea is induced by enterotoxin production after 8 -16 hours outgrowth of spores in close proximity or direct contact to the intestinal epithelium cells. However, phospholipases, sphingomyelinases, hemolysins, proteinases and peptidases likely represent additional virulence factors involved in the syndrome. These enterotoxins and additional virulence factors are broadly distributed among the members of the B. cereus group (Ehling-Schulz et al., 2019).

Besides its food poisoning potential, B. cereus sensu lato is also increasingly recognized as a nosocomial pathogen causing life-threatening infections in immunocompromised patients but there are also case reports available of B. cereus infections in immunocompetent patients. Among the highest risk groups are neonates or patients with indwelling catheters (EFSA, 2016).

Public Health impacts of food-borne illnesses due to Bacillus cereus are largely underreported (EU or USA) as only outbreaks are possibly investigated, not the sporadic cases (Scallan et al, 2011).

Key figures

In the EU, in 2019 (EFSA & ECDC, 2021), toxins produced by B. cereus were :

- responsible for 155 outbreaks (against 75 outbreaks due to C. perfringens and 74 outbreaks to S. aureus), 1,636 cases, 44 hospitalizations, 7 deaths (14 deaths for all bacterial toxins which correspond to a high proportion of all fatal cases reported in 2019 in FBOs (23.3%)).

- the toxigenic agents most frequently reported In the USA, the Center of Disease Control (CDC) estimated (Scallan et al., 2011) the annual number of sporadic & outbreaks cases associated to Bacillus cereus at 63,400 (with 0,4% of hospitalization and 0% of death rates).

Most cases of food-borne outbreaks caused by the B. cereus group have been associated with bacterial concentrations above 105 CFU/ g of foodstuff. Some cases of both emetic and diarrhoeal illness involved between 103 and 105 CFU/g of B. cereus (EFSA, 2016; FDA, 2012).

 

How is Bacillus cereus transmitted?

Soil is the primary source of contamination of raw foods with spores of B. cereus. Soil can contain between 103 and 105 spores of B. cereus per gram. In complex foods, some ingredients have been identified as important source of contamination with B. cereus spores, such as texturing agents, liquid eggs, herbs and spices. Spores can survive intense processing steps such as dehydration and subsequently contaminate diverse foodstuffs via dehydrated ingredients. Additional contamination during food processing may occur because spores of B. cereus which have strong adhesion properties, might form biofilms and may persist on the surface of processing equipment.

Storage of the processed product or use of raw materials in complex foods with conditions suitable for B. cereus (for instance spices in recipe dishes, or liquid eggs in refrigerated custards or cream caramel) permit spore germination and growth of B. cereus to numbers that might represent a hazard for consumers. Most foods with high humidity and not acidified would support its growth. Growth is however limited by refrigeration; Below 10°C, only a minority of the B. cereus strains present in a food product will be able to grow (EFSA, 2005).

 

What common industries are affected by B. cereus?

Then, a wide variety of foods, including meats, milk, vegetables, and fish have been associated with the diarrheal-type food poisoning. The vomiting-type outbreaks generally have been associated with rice products; however, other starchy foods, such as potato, pasta, and cheese products also have been implicated. Food mixtures, such as sauces, puddings, soups, casseroles, pastries, and salads, frequently have been linked with food-poisoning outbreaks (FDA, 2012).

 

How can Bacillus cereus be prevented and controlled in the food industry?

Broad requirements for prevention of adulteration of foodstuffs have been embedded in US laws (FDA Food Safety Modernization Act; USDA-FSIS Food Safety Acts for Meat, Poultry & Egg Products) and EU enforced regulations (EU Regulation 178/2002). All food business operators i.e., producers, manufacturers, processors, distributors, importers, retailers, etc. are expected to comply and are liable if they do not distribute safe foods.

Moreover, US and EU regulations impose that food business operators must analyze the specific food safety risks associated to their foodstuffs and processes and manage them through risk-based safety plans defined according to the HACCP principles (FDA 21CFR117 et al. - FSIS Pathogen reduction and HACCP regulation 9 CFR 304 et al. - EU Regulation 2073/2005).

Mandatory recalls of any tainted foodstuff further strengthen the regulatory framework applicable to the management of the foodborne risks throughout the food chain (FDA Food Safety Modernization Act, FSIS 9 CFR 304 et al., EU Regulation 178/2002).

Apart from the enforced regulatory requirements, it should be stressed that food business operators should also integrate in their food safety management plans the possible impacts that recalls, or legal prosecutions could have on their businesses.

At the EU level, “presumptive Bacillus cereus” has been defined as a regulatory Process Hygiene criterion for Dried infant formulae and dried dietary foods for special medical purposes intended for infants below six months of age (Regulation1441/2007). The upper acceptability limit has been set at 500 UFC / g considering the potentially fragile profile of the targeted customers. 

However, beyond this explicit regulatory criterion, since Bacillus cereus sensu lato and its toxins have been widely recognized as a public health concern and linked to a large range of food sectors and foodstuffs, all corresponding US and EU Food business operators have then, in terms of compliance to regulations, to integrate the B. cereus risk in their food safety plans to control the public health risk for their downstream customers. 

 

How can the presence of B. cereus be detected/numerated in the food industry?

Both EU and US regulatory frames require that food business operators shall perform microbial testing as appropriate when they are validating or verifying on a routine basis the correct functioning of their HACCP-based control procedures and good hygiene practices (EU Regulation 2073/2005 - FDA Food Safety Modernization Act).

Apart from advanced testing for epidemiological purposes (for a complete review see EFSA, 2016 paragraph 3.3), simple enumeration or detection methods of “presumptive Bacillus cereus” have proven to fit the needs for routine verification of effectiveness of the industrial safety control plans. Various standard methods (ISO 7932, ISO 21871, FDA-BAM, USDA/FSIS- MLG) or validated (AOAC OMA or RI, EN/ISO 16140-2) rapid methods have been described. 

bioMérieux provides the food safety managers with proven standardized or validated methods for the management of the Bacillus cereus group & its toxins risk along the Food Chain.

Jean-Pierre Facon
作者:
Jean-Pierre FACON

博士, 生物技术顾问

Picture Isabelle DESFORGES
作者:
Isabelle DESFORGES

Global Marketing Scientific Manager / Scientific Affairs

Food Business Industry Unit, bioMérieux SA, France

French Delegate of Food Microbiology Standardization committees
(AFNOR V08B, ISO/TC 34/SC 9 and CEN/TC 463)

bioMérieux solutions and products

Sample and culture media preparation:
- DILUMAT® gravimetric diluter
- SMASHER®  lab blender
- MASTERCLAVE® automated media preparator

Culture media:
- large range of traditional culture media
- Alternative method: BACARATM chromogenic culture medium for enumeration of Bacillus cereus in food and feed products (ISO 16140-2 certificate AES 10/10-07/10) 

Quality indicators automated TEMPO® enumeration platform:
- TEMPO® BC (Enumeration of Bacillus cereus) (ISO 161420-2 certificate 2014LR47)(AOAC-RI certificate PTM 071401)

Identification:
- Biochemical panels: API® 50CH panel (50 CHB/E medium)
- Automated biochemical identification system: VITEK® 2 BCL
- Maldi-Tof mass spectrometry: VITEK® MS

References

Bazinet A.L. Pan-genome and phylogeny of Bacillus cereus sensu lato. BMC Evol. Biol. 2017, 17.

BfR (German Federal Institute for Risk Assessment). Bacillus cereus bacteria in foodstuffs may cause gastrointestinal diseases. Opinion No. 048/2020. 2020: 18p.

Carroll L.M., et al. Proposal of a Taxonomic Nomenclature for the Bacillus cereus Group Which Reconciles Genomic Definitions of Bacterial Species with Clinical and Industrial Phenotypes., 2020, 11: 1-15.

EFSA J. Opinion of the Scientific Panel on Biological Hazards on Bacillus cereus and other Bacillus spp in foodstuffs. 2005: 175,1-48.

EFSA J. Scientific opinion on the risks for public health related to the presence of Bacillus cereus and other Bacillus spp. including Bacillus thuringiensis in foodstuffs. 2016;14(7):4524, 93 pp.

EFSA and ECDC (European Food Safety Authority and European Centre for Disease Prevention and Control). The European Union One Health 2019 Zoonoses Report. EFSA Journal 2021;19(2):6406, 286 pp.

EU Regulation 178/2002 laying down the general principles and requirements of food law, establishing the European Food Safety Authority and laying down procedures in matters of food safety.

EU Regulation 2073/2005 on microbiological criteria for foodstuffs.

EU Regulation 1441/2007 amending Regulation (EC) No 2073/2005 on microbiological criteria for foodstuffs.

Ehling-Schulz M., et al. Cereulide synthetase gene cluster from emetic Bacillus cereus: structure and location on a mega virulence plasmid related to Bacillus anthracis toxin plasmid pXO1. BMC Microbiol 2006, 6:20.

Ehling-Schulz M., et al. The Bacillus cereus Group: Bacillus Species with Pathogenic Potential. Microbiol. Spectr. 2019, 7.

FDA. Bad Bug Book. Foodborne Pathogenic Microorganisms and Natural Toxins. Bacillus cereus and other Bacillus species. 2012. 4p.

FDA Food Safety Modernization Act. Public Law - An Act to amend the Federal Food, Drug, and Cosmetic Act with respect to the safety of the food supply. 2011. 89p.

FDA. 21 CFR Parts 1, 11, 16, 106, 110, 114, 117, 120, 123, 129, 179, and 211. Current Good Manufacturing Practice, Hazard Analysis, and Risk-Based Preventive Controls for Human Food. 2015. 262p.

FDA - BAM (Bacteriological Analytical Manual). Chap 14. Bacillus cereus. 2020

Guinebretiere, M.H., et al. Ecological diversification in the Bacillus cereus Group. Environ. Microbiol. 2008, 10, 851–865. 

ISO (International Organization for Standardization), ISO 7932:2004. Microbiology of food and animal feeding stuffs – horizontal method for the enumeration of presumptive Bacillus cereus – Colony-count technique at 30 degrees C.

ISO 7932:2004/Amd.1:2020.  Microbiology of food and animal feeding stuffs – horizontal method for the enumeration of presumptive Bacillus cereus – Colony-count technique at 30 degrees C. AMENDMENT 1: Inclusion of optional tests.

ISO 21871:2006 Microbiology of food and animal feeding stuffs - Horizontal method for the determination of low numbers of presumptive Bacillus cereus – most probable number technique and detection method.

Liu Y., et al. Proposal of nine novel species of the Bacillus cereus group. Int. J. Syst. Evol. Microbiol. 2017. 67 :2499–2508.

Okinaka, R.T., et al. The Phylogeny of Bacillus cereus sensu lato. Microbiol. Spectr. 2016, 4. 

Priest, F.G.et al. Population structure and evolution of the Bacillus cereus group. J. Bacteriol. 2004, 186, 7959–7970. 

Scallan, E., et al. Foodborne illness acquired in the United States—major pathogens. Emerging Infectious Diseases 2011; 17:7–15.

USDA (US Department of Agriculture) - FSIS (Food Safety Inspection Service) – Food Safety Acts: Federal Meat Inspection Act – Poultry Products Inspection Act – Eggs Product Inspection Act. 

USDA. 9 CFR Parts 304, 308, 310, 320, 327, 381, 416, and 417. Pathogen Reduction; Hazard Analysis and Critical Control Point (HACCP) Systems; Final Rule.1996. 185p.

USDA – FSIS: Microbiology Laboratory Guidebook. Chap 12. Examination of Meat and Poultry for Bacillus cereus.1998. 6p.

Zervas A., et al. Identification and Characterization of 33 Bacillus cereus sensu lato isolates from Agricultural Fields from Eleven Widely Distributed Countries by Whole Genome Sequencing. Microorganisms 2020, 8 :1-17.

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