Draft Screening Assessment for Bacillus cereus (ATCC 14579) Environment Canada Health Canada July 2013

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Table of contents

• Synopsis
• Introduction
• 1. Hazard Assessment
  • 1.1 Characterization
    • 1.1.1 Taxonomic Identification and Strain History
    • 1.1.2 Genetic Transfer
    • 1.1.3 Pathogenic and Toxigenic Characteristics
      • 1.1.3.1 Effects on Human Health
      • 1.1.3.2 Effects on the Environment
    • 1.1.4 Other Ecological Characteristics
  • 1.2 Hazard Severity
• 2. Exposure Assessment
  • 2.1 Sources of Exposure
  • 2.2 Exposure Characterization
    • 2.2.1 Environment
    • 2.2.2 Human
• 3. Risk Characterisation
• Conclusion
• References
• Appendices

Synopsis

Pursuant to paragraph 74(b) of the Canadian Environmental Protection Act, 1999 (CEPA 1999), the Ministers of Environment and of Health have conducted a screening assessment on a strain of Bacillus cereus (ATCC 14579). This strain was added to the Domestic Substances List (DSL) under subsection 105(1) of CEPA 1999 because it was manufactured in or imported into Canada between January 1, 1984 and December 31, 1986 and it entered or was released into the environment without being subject to conditions under CEPA 1999 or any other federal or provincial legislation.

B. cereus strains are generally considered ubiquitous, and have the ability to adapt to and thrive in many aquatic and terrestrial niches. B. cereus strains form endospores that permit survival under sub-optimal environmental conditions. They are resistant to a range of antibiotics and heavy metals. The ubiquity of B. cereus strains is partially explained by their minimal nutritional requirements and ability to grow over a wide range of temperatures and pH values. Various characteristics of B. cereus strains make them suitable for use as active ingredients in commercial and consumer products, including detergents, degreasers, additives for biodegradation and bioremediation, and in various industrial processes.

B. cereus ATCC 14579 is recognized as a Risk Group 2 animal pathogen by the Canadian Food Inspection Agency (Animal Pathogen Import Program). Generally, Risk Group 2 animal pathogens are any pathogens that can cause disease but under normal circumstances are unlikely to be a serious hazard to healthy organisms in the environment and from which effective treatment and preventive measures are available. For example, B. cereuscan cause mastitis in cattle that is treatable with specific veterinary antibiotics. There are no other cases where B. cereus has been shown to cause adverse effects to organisms in the Canadian environment in the scientific literature. There are scientific reports of B. cereus ATCC 14579 causing a reduced rate of reproduction in springtail (an arthropod), and decreased shoot and root length in red fescue (a plant). However, these were under specific laboratory conditions, which are not a concern under the current known exposure scenarios.

B. cereus ATCC 14579 is also recognized as a Risk Group 2 human pathogen by the Public Health Agency of Canada. Information from the scientific literature indicates that B. cereusATCC 14579 has pathogenic potential in both the otherwise-healthy general population and in susceptible groups (i.e., infants and the elderly, the immunocompromised and individuals with debilitating comorbidities). B. cereus is a gastrointestinal pathogen that can also cause other types of infection, including endophthalmitis and skin infections. As mentioned B. cereus is resistant to several clinical antibiotics, which could in some circumstances, compromise the effectiveness of treatment of B. cereus infections. B. cereusATCC 14579 produces a wide variety of extracellular enzymes and toxins that are important factors for its pathogenicity in humans.

This assessment considers human and environmental exposure to B. cereus ATCC 14579 from its deliberate use in consumer or commercial products or in industrial processes in Canada. B. cereus ATCC 14579 was nominated to the DSL based on its use in consumer and commercial products. Potential uses of B. cereus ATCC 14579 reported in the public domain include bioremediation of aquatic systems, biodegradation of organic and inorganic waste, bioleaching of metals in mining and waste management, treatment of sewage sludge, and uses in the pulp and paper and textile industries.

The government launched a mandatory information-gathering survey (Notice) under section 71 of CEPA 1999 as published in the Canada Gazette Part I on October 3rd, 2009.There were no reports of import or manufacture of B. cereus ATCC 14579, except for limited quantities for academic research, teaching, and research and development activities. Therefore the likelihood of exposure to this living organism in Canada resulting from commercial and consumer activity is low. It is therefore proposed to conclude that B. cereus ATCC 14579 does not meet the criteria under paragraph 64(a) or (b) of CEPA 1999 as it is not entering the environment in a quantity or concentration or under conditions that have or may have an immediate or long-term harmful effect on the environment or its biological diversity or that constitute or may constitute a danger to the environment on which life depends. It is also proposed to conclude that B. cereus ATCC 14579 does not meet the criteria under paragraph 64(c) of CEPA 1999 as it is not entering the environment in a quantity or concentration or under conditions that constitute or may constitute a danger in Canada to human life or health.

Therefore, it is proposed that B. cereus ATCC 14579 does not meet any of the criteria as set out in section 64 of CEPA 1999.

However, given the potential pathogenicity and toxicity of this strain to healthy and susceptible humans and to some susceptible non-human species, there is concern that this strain could meet the criteria as set out in section 64 of the Act should consumer, commercial or industrial activities resume. Therefore, it is recommended that the above substance be subject to the Significant New Activity provisions specified under subsection 106(3) of the Act, to ensure that any manufacture or import for a new use undergoes ecological and human health assessments as specified in section 10⁸ of the Act, prior to the organism being considered for re-introduction into Canada.

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Introduction

Pursuant to paragrph 74(b) of the Canadian Environmental Protection Act, 1999 (CEPA 1999), the Ministers of Environment and of Health are required to conduct screening assessments of those living organisms listed on the Domestic Substances List (DSL) to determine whether they present or may present a risk to the environment or human health (according to criteria as set out in section 64 of CEPA 1999). This living organism was nominated and added to the DSL under Section 105 of CEPA 1999 because it was manufactured in or imported into Canada between January 1, 1984 and December 31, 1986 and it entered or was released into the environment without being subject to conditions under CEPA 1999 or any other federal or provincial legislation.

Screening assessments examine scientific information and develop conclusions by incorporating a weight-of-evidence approach and precaution. This screening assessment included consideration of hazard information obtained from the public domain as well as from unpublished research data and from internal and external experts. Exposure information was also obtained from the public domain as well as from a mandatory CEPA 1999 s. 71 Notice published in the Canada Gazette Part I on October 3, 2009. Further details on the risk assessment methodology used are available in the “Framework on the Science-Based Risk Assessment of Micro-organisms under the Canadian Environmental Protection Act, 1999”, referred to in this document as the Risk Assessment Framework (which is available on the web).

Data that is specific to DSL-listed B. cereus ATCC 14579 is identified as such. Where data concerning this particular strain were not available, surrogate information from literature searches of both B. cereus and the genus Bacilluswas used. Surrogate organisms were identified to the taxonomic level provided by the source. Information identified as of September 2011 was considered for inclusion in this Report.

Disclaimer: A determination of whether one or more of the criteria of section 64 of CEPA 1999 are met is based upon an assessment of potential risks to the environment and/or to human health associated with exposure in the general environment. For humans, this includes, but is not limited to, exposure from air, water and the use of products containing the substance. A conclusion under CEPA 1999 may not be relevant to, nor does it preclude, an assessment against the criteria specified in the Controlled Products Regulations, which is part of the regulatory framework for the Workplace Hazardous Materials Information System (WHMIS) for products intended for workplace use. Individuals who handle Bacillus cereus ATCC 14579 in the workplace (i.e., laboratory and R&D facilities) should consult with their occupational health and safety representative about safe handling practices, applicable laws and requirements under WHMIS and the Laboratory Biosafety Guidelines.

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1. Hazard Assessment

A hazard assessment characterizes the micro-organism and identifies its potential adverse effects on the environment and/or human health and the extent and duration of those effects. Hazards may be posed by the micro-organism itself, its genetic material, toxins, metabolites or structural components.

1.1 Characterization

1.1.1 Taxonomic Identification and Strain History

The accurate taxonomic identification of a micro-organism is essential in distinguishing pathogenic from non-pathogenic species and strains. A polyphasic approach combining classical microbiological methods (such as culture-based methods) and molecular tools (such as genotyping and fatty acids analysis) is often required.

Bacillus cereus is a Gram-positive, facultatively anaerobic, spore-producing, motile, rod-shaped bacterium. B. cereus spores are ellipsoidal, subterminal and do not swell the sporangium. B. cereus cells tend to occur in chains and the stability of these chains determines the form of the colony, which may vary from strain to strain (Logan and De Vos 2009). Table 1 provides a comparison of colony morphologies of B. cereus from various sources.

B. cereus (sensu stricto)isa member of the B. cereus group, which consists of six very closely related species: B. cereus, B. thuringiensis, B. anthracis, B. weihenstephanensis, B. pseudomycoides and B. mycoides. Species differentiation within the B. cereus group is especially complex, and a polyphasic approach is required for clear identification.

B. cereus ATCC 14579 was first isolated from the air in a cow shed in the United Kingdom (Frankland and Frankland 1887). B. cereus ATCC 14579 is the type strain and has several accession numbers in other culture collections, including DSM 31 from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH and NCCB 75008 from the Netherlands Culture Collection of Bacteria.

The phenotypic characteristics summarized in Table 2 provide an overview of the metabolic capabilities of B. cereusATCC 14579 (the complete list of DSL strains) compared to other members of the B. cereus group. Data generated by Health Canada^[1], including growth in liquid media at different temperatures (Appendix 1A), growth on solid media and biochemical testing at 28ºC or 37ºC (Appendix 1B) and fatty acid methyl-ester (FAME) analysis (Appendix 1C) provided further confirmation of the identification. It should be noted that these techniques can not be used to differentiate the DSL-listed strain from other B. cereus strains. The discrepancies between data obtained by the nominator (submitted at the time of nomination to the DSL), Health Canada1, ATCC, and Bergey’s manual are within the range of acceptability for B. cereus, and may be due to variable culture conditions. The FAME analysis of B. cereus ATCC 14579 showed high similarity with B. thuringiensis,which is expected, given the genetic similarity among the B. cereus group members.

^[1]appearance on nutrient agar at 30°C

^[2] N/A not available.

^[3]appearance on TSB agar after 7 days of growth at room temperature

^[4]appearance on blood agar after 24-36 hours at 37°C

Genotypic methods, such as full genomic sequencing (Ivanova et al. 2003), amplified fragment length polymorphism (AFLP) (Ticknor et al. 2001), rep-PCR fingerprinting (Cherif et al. 2003),16S rDNA and 23S rDNA analysis (Ash et al. 1991), multi-locus enzyme electrophoresis (MLEE)(Ash and Collins 1992;Helgason et al. 2000b), multi-locus sequence typing (MLST) (Helgason et al.2004;Priest et al. 2004;Tourasse et al.2006) and suppression subtractive hybridization (SSH) (Radnedge et al. 2003), have been extensively used to demonstrate phylogenetic relationships and to understand the few genomic variations among the B. cereus group species. The genetic relatedness between members of the B. cereus group is so close that from a strictly phylogenetic point of view they can be seen as a single species.

The B. cereus group members are usually divided into three main phylogenetic clades; Clade I comprises B. anthracis and some B. cereus and B. thuringiensis, mostly from clinical sources; Clade II contains B. cereus ATCC 14579 and several other B. cereus strains, but is mostly composed of B. thuringiensis strains, few from clinical sources; and Clade III contains the non-pathogenic B. mycoides and B. weihenstephanensis (Didelot et al.2009;Helgason et al. 2000b;Kolsto et al.2009;Priest et al. 2004;Vassileva et al.2006)(see Appendix 2). Different lineages based on MLST have also emerged from Clades I and II. B.cereus ATCC 14579 belongs more specifically to the Tolworthi lineage (Barker et al. 2005; Priest et al. 2004;Vassileva et al.2006).

16S rDNA sequence analyses of the DSL B. cereus strain, conducted by Health Canada^[2], have shown 100% homology compared to B. cereus ATCC 14579 on the proprietary MicroSeq ® ID library and more than 99% homology compared to other members of the B.cereus group included on the database (B.thuringiensis ATCC 33679 and ATCC 10792, B. anthracis Ames and B. mycoidesATCC6462). This confirmed that the 16S rDNA from the DSL-listed strain obtained from the ATCC matched the published 16S rDNA sequence from B. cereus ATCC 14579. The DSL-listed B. cereus 16S rDNA sequence also showed the same high similarity when compared to published B. cereus sequences in NCBI.

Central to the identification of the various members of the B. cereus group is an analysis of phenotypic characteristics and pathogenicity traits, and of the presence of extra-chromosomal elements, which reflect the species’ virulence spectra. The extra-chromosomal elements that differ between members of the B. cereus group are presented in Appendix 3. The plasmids determining pathogenicity patterns in the B. cereus group include pXO1 and pXO2 of B. anthracis, which containthe anthrax pathogenicity island, pBtoxis of B. thuringiensis, coding for the insecticidal protein, and pCER270 of B. cereus, encoding an emetic toxin. Extrachromosomal elements can also differ between strains of the same species. While pXO1 has been found in some B. cereus strains, such as G9241, and others carry pCER270, these are not features of B. cereus ATCC 14579, which only contains one extrachromosomal element, pBClin15 (Ivanova et al. 2003). The plasmid pBClin15 does not contain genes associated with any known pathogenicity traits.

+ larger than 85%; - 0-15% positive; N/A indicates data not available; d, different strains give different reactions

^[1]Nominator data

^[2]based on information summarizing phenotype of several strains form various publications available in Bergey’s manual (Logan and De Vos 2009)

1.1.2 Genetic Transfer

Horizontal gene transfer has been recognized as one of the major mechanisms driving the evolution of micro-organisms and it plays a key role in their ability to adapt to new environments through acquisition of traits. The B. cereus group is a highly dynamic population and genetic transfer between the group members is an important evolutionary mechanism. Various plasmids are found in different strains of B. cereus (Appendix 3) and some harbour genes linked to pathogenicity or environmental adaptation (Helgason et al. 2000a;Hoffmaster et al.2004;Rasko et al. 2005;Rasko et al. 2007). Generally these plasmids are present in low copy number, and are not self transmissible, but they can be mobilized with the help of other plasmids carrying homologs of key components of a conjugative secretion system (Van der Auwera and Mahillon 2005). Transduction (phage-mediated horizontal gene transfer) is a potentially important mechanism of gene transfer in natural environments. Bacteriophage CP-51, a generalized transducing phage for B. cereus, B. anthracis and B. thuringiensis, mediates transduction of plasmid DNA (Ruhfel et al. 1984). The only plasmid found in B. cereus ATCC 14579 is linear plasmid pBClin15 (Ivanova, 2003), closely resembling the Bam35 phage, a common bacterial virus (Stromsten et al. 2003) but no transduction events have been associated with pBClin15 in the scientific literature.

Insertion sequences (IS) are another type of mobile element that can be involved in horizontal gene transfer. IS elements are composed of inverted repeats flanking a transposase gene (De Palmenaer et al. 2004) and have been found in various members of the B. cereus group. IS231 is one such element. Variants of IS231 have been identified in the chromosomes and plasmids of B. cereus group members, including B. cereus ATCC 14579 (De Palmenaer et al. 2004). IS231 has been implicated in the translocation of mobile insertion cassettes which may contain genes involved in antibiotic resistance or adaptation to the environment (Chen et al. 1999;De Palmenaer et al. 2004). The IS231 variant identified in B. cereusATCC 14579 is composed of two putative genes; one is 60% identical to a haloacid dehalogenase and the other is 55% identical to an acetyltransferase.

Group II introns were also identified in the genome of B. cereus group members (chromosome and plasmids), including one copy in B. cereus ATCC 14579. Even though these do not contain any pathogenicity genes, they are self-splicing, mobile retro-elements implicated in genetic transfer (Tourasse and Kolsto 2008). Other elements that can facilitate gene transfer may also be present in B. cereus. Økstad et al (2004) identified a DNA repeated element specific to the B. cereus group, bcr1. This element is present in B. cereus 14579 in 54 copies and possesses the characteristics of a mobile element. Therefore, bcr1 could be implicated in horizontal gene transfer within the B. cereus group. Furthermore, the full genome analysis of the sequence of B. cereus ATCC 14579 (Ivanova et al. 2003) revealed the presence of 28 transposase genes, which could be involved in horizontal gene transfer (Kolsto et al. 2009).

Gene transfer is possible and could increase the hazard potential of B. cereus, as occurred when strain G9241 acquired the B. anthracis pXO1 plasmid carrying the anthrax pathogenicity island. However, the presence of vegetative cells seems to be essential for conjugation (Santos et al., 2010). Since B. anthracis exists in the natural environment mainly as dormant spores, and its vegetative cells survive poorly outside the host, the acquisition of B. anthracis plasmids by other members of the B. cereusgroup is extremely rare, and may be restricted to, or at least be more common in, areas where anthrax is endemic (Hoffmaster et al., 2006). Moreover, growth of B. anthracis outside a host usually leads to loss of virulence (reviewed in Dragon and Rennie 1995). In a laboratory setting, the conjugal transfer of an insecticidal plasmid of B. thuringiensis to B. anthracis was observed at a ratio ranging from 6.9×10^-4 to 1.9×10^-7, but no naturally occurring insecticidal B. anthracis isolates have yet been reported (Yuan et al. 2010). Conjugation of B. thuringiensis plasmid pAW63 and pXO16 to one strain of B. cereus and between B. thuringiensis strains has been reported in food matrices under laboratory conditions (Van der Auwera et al. 2007). Conjugal transfer of plasmid pHT73- EM^R from B. thuringiensis var. kurstaki to B. cereus ATCC 14579 had frequencies of 1.1 ±0.90 × 10^-9 on nitrocellulose filter and was not detected on LB broth or on Bombyx mori larvae (Santos et al., 2010). While it is possible that B. cereus ATCC 14579 could acquire virulence plasmids from pathogenic relatives, the probability of such an occurrence is no higher than for other naturally occurring strains of B. cereus. The DSL strain does not contain plasmids bearing virulence factors, so it cannot be implicated in the conjugal transfer of virulence factors to other bacteria in the environment.

1.1.3 Pathogenic and Toxigenic Characteristics

The ability of B. cereus to produce infections in both human and non-human species is attributed to a wide array of mechanisms, including adherence, invasion, evasion of host defences and damage to host cells.

B. cereus can cause food poisoning and various opportunistic and nosocomial infections. It can cause two types of food poisoning, one resulting in vomiting through the action of the emetic toxin cereulide and the other resulting in diarrhea through the action of various enterotoxins (Granum 2001;Kotiranta et al. 2000;Stenfors Arnesen et al. 2008). Cereulide is a peptide toxin, that must be present in the food at the time of ingestion to cause vomiting (Agata et al. 2002). Live cells are not required to cause the emesis syndrome. For diarrheal syndrome, it is unclear if the enterotoxins are present in food or are produced in the small intestine by the live bacteria. However, enterotoxins are unstable at pH less than 4 and can be degraded by pepsin, trypsin and chymotrypsin (Granum 1994), so it is most likely that they are produced in the small intestine. Five toxins have been proposed as potential causes for the diarrheal syndrome: HBL, NHE, BceT, EntFM and CytK, but only three (HBL, NHE and CytK) have been related to food borne outbreaks (Agata et al.1995a;Lund et al. 2000;Lund and Granum 1997; Stenfors Arnesen et al. 2008; Schoeni and Lee Wong, 2005).

B. cereus ATCC 14579 produces several different toxins including enterotoxins (hemolysin BL [HBL], nonhemolytic enterotoxin [Nhe], hemolysins (hemolysin II [HlyII] and III [HlyIII]) and phospholipase C (PLC) of which three variants are recognized: phosphotidylinositol hydrolase (PIH), phosphotidylcholine hydrolase (PC-PLC) and sphingomyelinase (SMase) (see Appendix 4) (Ivanova et al. 2003). Data generated by Health Canada by PCR-analysis confirmed the presence of hBL, plC and hly-III in the chromosome of B. cereus ATCC 14579 (Seligy et al. 2004). The emetic toxin-encoding gene is located on a plasmid, pCER270, which is not carried by the DSL-strain ATCC 14579, making it unlikely to produce cereulide (Haggblom et al. 2002). Phospholipase C and hemolysins produced by B. cereus are necrotic toxins that mimic the effects of some staphylococcal or clostridial toxins, resulting in invasive, non-gastrointestinal infections (Turnbull and Kramer 1983).

Adherence of enteropathogens to the intestinal epithelium is the essential first step required for colonization. Attachment of the bacterium is linked to the presence of fimbriae, which recognize a specific site on the enterocytes. A crystalline cell surface protein (S-layer) has also been implicated in attachment, but was not detected on the cell surface of B.cereus ATCC 14579 (Kotiranta et al. 1998). The enterotoxin components are either expressed on the bacterial cell membrane or secreted into the intestinal lumen. There, the toxins cause diarrhea by perturbing the exchange of water and electrolytes (Belaiche 2000). It has been suggested that B. cereus HBL, Nhe and CytK enterotoxins form pores in the membrane of intestinal epithelial cells, which causes osmotic lysis (Beecher and Wong 1997;Hardy et al. 2001; Haug et al. 2010).

Other virulence factors specific to B. cereus include proteases, notably metalloproteases (Cadot et al., 2010; Guillemet et al. 2010), and other degradative enzymes play a role in the establishment and development of infection, and in circumventing the host immune system (Appendix 5). Some of these have been implicated in both human and non-human target infections (see Appendices 6A and 6B). The transcription factor PlcR is seen as a virulence factor as it is involved in the expression of most known virulence factors in B. cereus, including HBL, Nhe, CytK, PLCs and several proteases in the DSL strain and may be in part responsible for the variability of virulence amoung B. cereurs stains (Gohar et al. 2008). The ability of the B. cereus ATCC 14579 strain to grow at 37°C, as shown in Appendix 1A, is another concern from a human health standpoint.

Data generated by Health Canada^[3] with B. cereus ATCC 14579 (cells and culture filtrates) showed cytotoxic activity towards a human colon cancer cell line and a mouse macrophage cell line37°C that is consistent with findings from other laboratories. Also, strain ATCC 14579 showed high cytotoxicity on Vero cells when grown at 37°C and 15°C in BHIG (L. P. Stenfors Arnesen, personal communication^[4]). Linbäck et al. (1999) demonstrated the cytopathogenic effect of B. cereus ATCC 14579 (supernatant) on Vero cells and strong haemolytic activity against sheep erythtocytes, both at 37°C. Although cytotoxicity is evident in these studies, the results vary depending on the growth temperatures.

Due to the high genetic similarity among B. cereusgroup members, clinical isolates sharing the toxins known to be present in B. cereus ATCC 14579 are considered good surrogates for characterizing the potential human health hazard of B. cereus ATCC 14579, as long as it is recognized that B. cereus ATCC 14579 differs from the highly pathogenic strains of the B. cereus group in that it does not carry the virulence plasmids that are associated with the emetic syndrome or anthrax (Didelot et al. 2009;Helgason et al. 2000b; Kolsto et al. 2009; Rasko et al. 2005; Vassileva et al. 2006). B. cereus 14579 can also be distinguished from the highly pathogenic strains of the B. cereus group based on its genomic sequence and its phylogenetic position in Clade II of the B. cereus group. Clade II comprises the majority of B. thuringiensis isolates (Priest et al. 2004), which also include clinical isolates (Barker et al. 2005;Hoffmaster et al. 2008), whereas the highly pathogenic strains (B. anthracis and B. cereusemetic strains) are grouped in Clade I. Recently, Guinebretière et al. (2008) proposed a new classification of the B. cereus group based on AFLP. This new classification includes seven groups, each of which is associated with a particular temperature growth range and potential for pathogenicity. Under this scheme, B. cereus ATCC 14579 belongs to group IV, which includes those B. cereus and B. thuringiensis strains that grow at 37ºC and are implicated in food poisoning (Guinebretière et al. 2008; Guinebretière et al. 2008).

1.1.3.1 Effects on Human Health

Gastrointestinal illnesses are the most common infections associated with B. cereus. In healthy individuals the symptoms are generally mild, but complications can lead to more serious disease, or even death (Ginsburg, 2003; Girish et al. 2003; Lund et al. 2000; Shiota 2010; Dierick et al. 2005). B. cereus gastrointestinal outbreaks have been reported around the world (Appendix 7B). It is implicated in 1 to 33% of cases of food poisoning (Anonymous, 2005) with varying degrees of severity. The true number of cases is likely underestimated since most foodborne diseases are underreported. In Canada, B. cereus-related diseases are not notifiable and outbreaks are investigated at the local Health Unit level (J. Greig, personal communication). Only one foodborne outbreak has been reported for this species (J. Greig, personal communication^[5], McIntyre et al. 2008). There have been no reported laboratory-acquired infections to date.

B. cereus also causes non-gastrointestinal illness (review in Bottone, 2010; Drowbnieski, 1993). Non-gastrointestinal B. cereus outbreaks (Appendix 7A) are less frequent, and most are identified as nosocomial in origin. Endophthalmitis is a severe infection caused by the introduction of bacteria into the eye following trauma or surgery. Case reports of B. cereus endophthalmitis or panophthalmitis have been reported in the literature (Al-Jamali et al.2008;Altiparmak et al. 2007;Chan et al.2003;Martinez et al. 2007;Tobita and Hayano 2007;Zheng et al. 2008). Among the organisms that infect the eye, B. cereus has one of the most rapidly evolving courses of infection (Brinton et al. 1984) and is one of the most destructive (Levin and D'Amico 1991;Parke 1986;Pflugfelder and Flynn 1992). An experiment conducted on rabbits by Callegan et al. 2003showed reproducible endophthalmitis caused by B. cereus strain ATCC 14579. Despite aggressive drug and/or surgical intervention, B. cereus endophthalmitis typically results in migration of bacteria throughout the eye and a remarkably rapid progression to a severe intraocular inflammatory response, resulting in loss of functional vision within 24h to 48h (Davey and Tauber 1987;Vahey and Flynn 1991).

B. cereus can produce a variety of skin and soft tissue infections, including cellulitis (Dancer et al.2002;Latsios et al. 2003) and necrotizing cellulitis (Darbar et al. 2005;Hutchens et al. 2010; Sada et al. 2009). Wound infections, mostly in otherwise healthy persons, have been reported following trauma, surgery and burns (Crane and Crane 2007;Dubouix et al. 2005;Moulder et al. 2008;Pillai et al. 2006;Ribeiro et al. 2010;Shimoni et al. 2008;Stansfield and Caudle 1997). Catheter use was often associated with B. cereusinfection (Crane and Crane 2007;Flavelle et al. 2007;Koch and Arvand 2005;Monteverde et al. 2006;Ruiz et al. 2006;Srivaths et al. 2004).

B. cereus endocarditis is a rare condition that is associated with intravenous devices or recreational drug injections (Abusin et al. 2008). Morbidity and mortality associated with B. cereus endocarditis are high among patients with valvular heart disease (Cone et al. 2005;Steen et al. 1992).

Some cases of B. cereus meningoencephalitis (Evreux et al. 2007;Lebessi et al. 2009;Lequin et al. 2005;Manickam et al. 2008;Puvabanditsin et al. 2007) and bacteraemia (Girisch et al.2003;Hilliard et al. 2003;John et al.2007;Tuladhar et al. 2000;Van Der Zwet et al.2000) have been reported in neonates. Neonatal meningoencephalitis caused by B. cereus is rare, but it carries a high mortality. Cases of infection are often associated with medical equipment or devices.

Some cases of B. cereus pneumonia have been reported. Pulmonary infections due to B. cereus are rare compared to those attributed to B. anthracis, but can be just as life threatening in immunocompromised persons. The majority of cases were in metalworkers and immunocompromised patients who have greater susceptibility to infection. Avashia et al. (2007) reported the cases of two healthy metalworkers who died from B. cereus pneumonia. Another case of death, in an area where anthrax occurs naturally in herbivores, was also reported in a metalworker (Hoffmaster et al. 2006). However, in all of those cases, plasmid pX01 (but not pX02) was found in all B. cereus samples and the route of exposure was suspected to be inhalation. Cases of B. cereus pneumonia in cancer patients were reported by Frankard et al. (2004), Fredrick et al. (2006), Katsuya et al. (2009), and Sotto et al. (1995). In most cases, the route of infection was unknown but linked to existing B. cereus infections in the patients. In all but one case, the infection resulted in death.

Two studies in BALB/c mice showed that inhalation of either B. cereus ATCC 14579 spores or vegetative cells had adverse effects. Salimatou et al. (2000) reported that ninety percent of mice died after 24h after nasal instillation of 10⁷ spores, while all died after administration of 6 × 10⁶ vegetative cells. The cause of death was not determined but did not seem to depend on the growth of bacteria in the mice. Flaws in the study make its results questionable. The experiment was done only once, and no results for control mice were presented. The instillation of a large dose volume could have been the cause death by asphyxiation and pulmonary hemorrhage. Tayabali et al. (2010) reported no toxicological effects in BALB/c mice exposed to 10⁷ spores of B. cereus ATCC 14579 one week after endotracheal instillation. However, severe shock-like symptoms (lethargy, hunched appearance, ruffled fur, and respiratory distress) occurred 4 hours after exposure to 10⁵ or 10⁶ vegetative cells. An increase of inflammation cytokine levels was observed in the blood and lungs, but not in all mice, resulting in a high standard deviation. Post-testing revealed an intermediate cytokine response after exposure to 10⁴ and no response to lower vegetative cell exposure (10² and 10³) (A. Tayabali, personal communication). In comparison to the Salimatou study, the Tayabali study was better controlled and better standardized the production of spores and vegetative cells. Pre-study work on methodology was also done to limit the effect of the instillation procedure in the final results.

The range of reported non-gastrointestinal infections is wider in immunocompromised individuals. Necrotizing meningitis, subarachnoid hemorrhage and brain abscesses have been reported with systemic B. cereus infections in patients with leukemia (Gaur et al. 2001;Nishikawa et al. 2009). Other local and systemic B. cereus infections have also been reported in patients with compromised immunity (Akiyama et al. 1997;El Saleeby et al. 2004;Hernaiz et al. 2003; Hirabayashi et al. 2010;Kiyomizu et al. 2008;Kobayashi et al. 2005;Le Scanff et al. 2006;Musa et al. 1999;Nishikawa et al.2009).

Clinical reports demonstrate that the severity of B. cereus infection significantly correlates with its ability to synthesize toxins (Beecher et al. 2000; Ghelardi et al. 2002) and depends on the immune competence of the host and the virulence of the microbe. As mentioned in section 1.1.3, genes encoding for hemolysin BL, nonhemolytic enterotoxin (Nhe), hemolysins (hemolysin II and III), and phospholipase C (phosphotidylinositol hydrolase, phosphotidylcholine hydrolase and sphingomyelinase) are present in B. cereus ATCC 14579, but no data is available on the levels of expression of toxins or virulence factors. Hemolysin II and metalloproteases InHA1 and NprA can also serve as indicators of pathogenicity (Cadot et al. 2010), however it is impossible to predict which B. cereus strains are able to cause gastrointestinal disease based solely on the presence of these virulence factors (Anonymous 2005) since not all strains containing these factors cause adverse effects.

Treatment of B. cereus human infections is hampered by resistance to antimicrobial drugs. Antibiotic sensitivity tests showed that the resistance of B. cereus to different antibiotics is widely variable between strains (Bernhard et al. 1978;Weber et al. 1988). Most strains of B. cereus produce ß-lactamase and are therefore considered to be resistant to ß-lactam antimicrobial agents (Coonrod et al. 1971). Most B. cereus strains have been found to be resistant to penicillin, semisynthetic penicillin, cephalosporin (Stretton and Bulman 1975;Weber et al. 1988), ampicillin, colistin, polymyxin, kanamycin, tetracycline, bacitracin and cephaloridine (Bernhard et al. 1978;Wong et al. 1988). Mols et al, 2007 reported that B. cereus ATCC 14579 is resistant to antibiotics targeting cell wall components such as cefazolin, ketoprofen and moxalactam. Even with appropriate antibiotic regimens, there are reports in the literature presenting refractory B. cereus infection leading to fatal outcomes (Musa, 1999; Tuladhar, 2000). Antibiotic susceptibility tests conducted by Health Canada on 10 classes of antibiotics have shown that B. cereus ATCC 14579 is highly resistant to amoxycillin, aztreonam and trimethoprim, that it had intermediate sensitivity to cephotaxim and nalidixic acid but that it is sensitive to doxycyline, erythromycin, gentamicin and vancomycin (Table 3).

^[1] Work conducted using TSB-MTT liquid assay method (Seligy et al.1997). The reported values are based on a minimum of three independent experiments. Values correspond to the minimal inhibitory concentration (μg/ml) for B. cereus ATCC 14579 (20, 000 CFU/well) grown in the presence of antibiotic for 72 hrs at 37°C.

1.1.3.2 Effects on the Environment

B. cereus, as a species,is recognized as a Risk Group 2 pathogen by the Canadian Food Inspection Agency (Animal Pathogen Import Program). Generally, a Risk Group 2 pathogen is any pathogen that can cause disease, but under normal circumstances is unlikely to be a serious hazard to organisms in the environment. Effective treatment and preventive measures are available, and the risk of spread is limited. However, B. cereus can have a range of effects on non-human species, depending on the host and method of exposure. Some examples include diarrhea (monkeys), mastitis (cattle), inflammation (rabbits) and death (range of organisms) (see Appendices 6A and 6B). With the exception of mastitis, all of the data are from experimental studies. In the studies cited below, those that utilized B. cereus ATCC 14579 are indicated by an asterix.

Four cases involved mastitis in cattle, which were lethal in some cases (Appendix 6B). However, it is known that with the appropriate treatment, animals can survive such infection (Schiefer et al., 1976). With respect to invertebrates, two studies reported that B. cereus isolate WGPSB-2 has potential as a biocontrol agent against white grubs (Selvakumar et al.2007;Sushil et al. 2008). A number of experimental studies (outside of what were considered natural settings) used B. cereus on a variety of target organisms. These included invertebrates (Lepidopteran*, Blattarian* and Coleopteran insects and crustaceans) and mammals (guinea pigs, rabbits, mice*, cattle, monkeys and cats). Some of the methods of exposure included free ingestion or gavage, injection (intravenous, intrahaemocoelic, intracoelomic, intradermal, intravitreal, intraperitoneal, subcutaneous), nasal instillation, or dermal exposure to cultures or cell-free supernatants.

The objectives of the studies varied and included some of the following: characterization of the role of specific genes in virulence, investigation of the opportunistic properties of B. cereus, the suitability of specific organisms as an oral infection model for entomobacterial pathogens, investigation of natural pathogens for different pests, pathogenicity testing to characterize cause of larval death, purification and identification of a soil bacterial exotoxin, and models for human B. cereus pathogenicity.

The results of the studies referred to above also varied, depending on the target organisms, the strains of B. cereus used and the method of exposure. In many of the studies on lepidopteran invertebrates, B. thuringiensisinsecticidal crystal toxin (Cry1C) was co-administered with spores of B. cereus 14579. B. cereus spores contributed synergistically to mortality in these studies, and mortality in the absence of Cry1C was low. Nevertheless, a specific strain of B. cereus was identified as a lepidopteran pathogen by Koch’s postulates in Trichoplusia ni, and B. cereus sphingomyelinase* was demonstrated to be toxic to silkworms and cockroaches. Elm bark beetle larvae suspended in B. cereus cultures showed 63.6% mortality. B. cereus was also pathogenic toward orally inoculated Southern pine beetle larvae and showed varying degrees of toxicity and mortality when freely ingested by Boll weevil and Black Bean aphids (but not by Egyptian cotton leafworm). Water fleas exposed to B. cereus cultures died within 8 to 16 days. Pathogenicity and toxicity testing on terrestrial organisms were also performed at Environment Canada laboratories. Results of chronic testing with B. cereus ATCC 14579 using the invertebrate species Folsomia candida (springtail) demonstrated no effect on adult mortality, but a depression in juvenile reproduction at 10⁸ cfu/g soil (Princz, 2010).

In vertebrates, effects reported from various sources included necrotic inflammation at the site of subcutaneous injection, fluid accumulation in a rabbit ileal loop model, increased vascular permeability, presence of abscesses and/or nodules following intradermal injection, calcification and necrotic skin ulcers following intramuscular injection in rabbits, diarrhea following ingestion in monkeys, abortion in cattle and sheep injected intravenously with high doses, and mortality in mice. Specific details of these experiments are provided in Appendix 6A. Based on the available information, it is worth noting that the pathogenic effects noted in Appendix 6A are not expected to occur to biota in the environment given that the route of administration bypassed natural physical barriers to infection and/or the concentrations of bacteria used were higher than would be expected in the natural environment.

Pathogenicity and toxicity tests of B. cereus ATCC 14579 on plants were performed at Environment Canada laboratories. Plant testing using Festuca rubra (red fescue) demonstrated a significant decrease in shoot length (21% reduction relative to control response), root length (28% reduction) and root dry mass (42% reduction), but no effect on shoot dry mass in comparison to control growth in conducted tests (Princz, 2010). Despite the observed adverse effect on red fescue at the concentration tested, this strain is not suspected to be a frank plant pathogen nor is it expected to be used at this concentration in any industrial or consumer application to plants.

1.1.4 Other Ecological Characteristics

B. cereus has minimal nutritional requirements, grows over a range of temperatures and pH and has the ability to form spores; therefore, its vegetative cells have the capacity to colonize a variety of niches and its spores to persist indefinitely in many environments (Kotiranta et al. 2000). B. cereus forms endospores that permit survival under sub-optimal environmental conditions. These have a tough outer keratin-like layer which is heat-, chemical-, radiation-, disinfectant- and desiccation-resistant, often withstanding temperatures of 126°C for more than 90 minutes (Pillai et al. 2006). The spores are found in many types of soil and in sediments, dust and plant material, are described as having a ubiquitous presence in nature (Stenfors Arnesen et al, 2008) and may passively spread in the environment. The spores are not easily destroyed by means that eliminate vegetative cells and may germinate when in contact with organic matter, or once inside insect or animal hosts (Stenfors Arnesen et al. 2008).

Nevertheless, conditions required for growth and survival vary with the strain (Bassen et al. 1989; Gibriel and Abd-el Al 1973; Jaquette and Beuchat 1998; Rizk and Ebeid 1989; Rossland et al. 2003). The optimal growth temperature for most strains is between 30ºC and 37ºC, normally with no growth above 55ºC or below 5ºC. The optimal pH depends on the growth medium used (Andersson 1995), with no growth seen in media of pH lower than 4.3 or higher than 10.5.

Biofilm formation is in general associated with pathogenicity and increased resistance to antimicrobial agents. The species B. cereus is reported to have the ability to form biofilms; however, no biofilm formation was observed for B. cereus ATCC 14579 after incubation of an exponential phase culture at an OD (600 nm) of 0.01 into L-Broth medium in a 96-well polyvinylchloride microliter plate during 72 h at 30°C, whereas biofilm formation was observed under the same conditions in B. cereus ATCC 10987 (Auger et al. 2006). In another study, B. cereus ATCC 14579 formed biofilms on Y1 medium after 24 h at 20°C and 30°C, but after 48h the biofilms dispersed (Wijman et al., 2007). The conclusion of this study was that bioflm production was found to be strongly dependent on incubation time, temperature, and medium, as well as the strain used.

It has been shown that B. cereus ATCC 14579 is able to produce a bacteriocin-like inhibitory substance (BLIS) that is highly active against closely related Bacillus spp.(Risoen et al. 2004). However, there are currently no published reports or research articles indicating that B. cereus ATCC 14579 is harmful to microbiota in the environment at the population level (for the purpose of this assessment, this indicates a significant number of organisms of the same species inhabiting a given area).

B. cereus is most likely involved in biogeochemical nutrient cycling, as it produces a wide range of extracellular enzymes and can grow on decaying organic matter (Borsodi et al. 2005).Therefore, it is capable of playing a role in the decomposition and recycling of soil organic matter, when it appears in a vegetative state, however its widespread occurrence in soils is notably as spores. B. cereus is also capable of reducing nitrate to nitrite and ammonium and can thereby play a role in the nitrogen cycle (Andersson 1995).

1.2 Hazard Severity

The environmental hazard severity for B. cereus ATCC 14579 is estimated to be medium^[6]. Considerations that may result in a finding of medium hazard include that the micro-organism is known as an opportunistic pathogen, has some adverse but reversible effects, in the intermediate term, and effective treatments or mitigation measures are available. B. cereus is recognized as a Risk Group 2 animal pathogen by the Canadian Food Inspection Agency (Animal Pathogen Import Program). Such pathogens, under certain conditions can pre-dispose the host to infection, cause a range of symptoms that will debilitate the host and could kill it. In cases where it has been studied, such as in infections caused by B. cereus in an agricultural setting (mastitis in cattle), treatment with veterinary antibiotics allowed for survival of affected animals. Generally, in the absence of conditions that pre-dispose the host (which can include invasive routes of exposure), infection is unlikely to occur. This is consistent with the observation that there is no evidence in the scientific literature to suggest any adverse ecological effects at the population level.

The human hazard severity for B. cereus ATCC 14579 is estimated to be medium6. Considerations that resulted in a finding of medium hazard include: 1) cases of severe disease or fatality were limited to susceptible sub-populations (the immunocompromised) or were rare, localized and rapidly self-resolving in healthy humans; 2) there is little potential for transmission of infection to other humans; 3) effects in laboratory animal models of human infection were not lethal, and were limited to invasive exposure routes (i.e., endotracheal instillation) or were mild and rapidly self-resolving.

Information from the scientific literature indicates that this micro-organism has pathogenic potential in both otherwise healthy and immunocompromised humans. B. cereus is recognized by the Public Health Agency of Canada (PHAC) as a Risk Group 2 human pathogen. It produces a wide variety of extracellular enzymes and toxins that are important factors for its pathogenicity in susceptible and in healthy individuals. The vast majority of B. cereus-related diseases in healthy humans are mild, self-resolving and usually treatable. There are some reports of death related to B. cereus related infections in humans; however, the strains implicated in those cases contained important virulence plasmids that are not present in B. cereus ATCC 14579. No information was found to indicate that B. cereus ATCC 14579 has the ability to spread and acquire antibiotic resistance genes; however, the treatment of B. cereus ATCC 14579 infections could be hampered by its resistance to several antimicrobial drugs (refer to Table 3).

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2. Exposure Assessment

An exposure assessment identifies the mechanisms by which a micro-organism is introduced into a receiving environment (Section 2.1) and qualitatively and/or quantitatively estimates the magnitude, likelihood, frequency, duration, and/or extent of human and environmental exposure (Section 2.2). The exposure to the micro-organism itself, its genetic material, toxins, metabolites or structural components is assessed in this section.

2.1 Sources of Exposure

B. cereus is commonly found in the environment. It is widely distributed in nature and is able to survive and grow in a wide variety of environments, including soil, airborne dust, water, sediments, on plants and decaying matter (Logan and De Vos2009;Stenfors Arnesen et al.,2008). Humans and non-human species are regularly exposed to B. cereus. Nevertheless, the purpose of this section is to characterize the exposure to the DSL-listed strain, B. cereus ATCC 14579, from its deliberate addition to consumer or commercial products or its use in industrial processes in Canada.

B. cereus as a species has properties that make it of commercial interest in a variety of industries. B. cereusATCC 14579 was nominated to the DSL based on its past use in consumer and commercial products. A search of the public domain (internet, patent databases) suggests multiple potential uses, including food processing, pharmaceuticals, pulp and paper and textile processing, biochemical and enzyme production, bioremediation and biodegradation, bioleaching and biomining, and municipal and industrial wastewater treatment. For agricultural applications, some B.cereus strains have been used as livestock probiotics and as microbial pest control agents (Lodemann et al., 2008).

In 2009, the government launched a mandatory information-gathering survey (Notice) under section 71 of CEPA 1999 as published in the Canada Gazette I on October 3rd, 2009 (hereafter “the Notice”). The Notice applied to any persons who, during the 2008 calendar year, manufactured or imported B. cereus ATCC 14579, whether alone, in a mixture, or in a product. Anyone meeting these reporting requirements was legally obligated to respond. Respondents were required to submit information on the industrial sector, uses and any trade names associated with products containing this strain, as well as the quantity and concentration of the strain imported or manufactured in the 2008 calendar year. No commercial or consumer activities using B. cereus ATCC 14579 were reported in response to the Notice. B. cereus ATCC 14579 was reported to be used in very small quantities for research and development (R&D), and teaching activities.

2.2 Exposure Characterization

The exposure characterization is based on activities reported in the Notice (R&D and teaching). As B. cereus ATCC 14579 is a Risk Group 2 human and animal pathogen, measures to reduce human and environmental exposure from its use in research and teaching laboratories are in place under the PHAC’s Laboratory Biosafety Guidelines and the CFIA’s Containment Standards for Veterinary Facilities. These include specific laboratory design, operational practices and physical requirements. For example, all material must be contained and is decontaminated prior to disposal or reuse in such a way as to prevent the release of an infectious agent, and equipment for emergency and decontamination response must be readily available and maintained for immediate and effective use. Arrangements for shipping of B. cereus ATCC 14579 must also meet requirements under Canada’s Transportation of Dangerous Goods Act and Regulations. These measures are designed to prevent any human or environmental exposure to the organism in the laboratory setting. Human and environmental exposure to B. cereus ATCC 14579 through R&D and teaching uses reported under the Notice is therefore expected to be low.

2.2.1 Environment

The environmental exposure for B. cereus strain ATCC 14579 is estimated to be low^[7] based on responses to the Notice, which indicate that this strain is no longer used in consumer or commercial products or for industrial processes in Canada.

Nevertheless, environmental exposure scenarios, in the event that consumer, commercial or industrial activities with B. cereus ATCC 14579 resume, have been considered along with persistence and survival properties of this micro-organism.

The magnitude of non-human species exposure to this micro-organism will depend on the persistence and survival of B.cereus ATCC 14579 in the environment. Persistence test data were obtained by Environment Canada on B. cereusATCC 14579 in agricultural soil. After inoculation of soil with live cells, samples from various time points were collected and the presence of B. cereus ATCC 14579 DNA was tested by specific AFLP PCR. DNA from this strain was detected for 127 days post-inoculation (Xiang et al., 2010). No testing for the presence of live cells was done. Another study (West et al., 1985) artificially inoculated natural (un-autoclaved) dry soil with 10⁴ spores of B. cereus and the population level increased to 10⁵ over the span of the experiment (64 days). This is consistent with the finding that spores can be maintained in the environment and are resistant to some of the factors that cause vegetative cell numbers to decline after artificial inoculation. Therefore, it is reasonable to believe that repeated releases of B. cereus spores into the natural environment could lead to increased numbers of spores being maintained in those environments. 

B. cereus is a persistent micro-organism that is frequently isolated from natural environments. However, studies in the scientific literature that contain data on population levels of B. cereus in the natural environment are very limited. One study (Tucker and McHugh, 1991) showed that, in various soils containing varying floral populations, the concentration of B. cereus reached 6 × 10⁴ CFU/g of soil. As well, no relevant reports concerning environmental persistence of toxins produced by B. cereus have been found. While large inputs of B. cereus ATCC 14579 into the environment could result in concentrations greater than background levels of B. cereus, high numbers of vegetative cells are unlikely to be maintained in water and in soil due to competition (Leung et al. 1995) and microbiostasis (van Veen et al. 1997), which is an inhibitory effect of soil, resulting in the rapid decline of populations of introduced bacteria. Nevertheless, B. cereus sporesare likely to persist and accumulate in the environment, as indicated by the information presented above. No reports documenting elimination of B. cereus spores following environmental contamination were found in the literature.

The potential exposure scenarios are based on former and probable future uses as described in Section 2.1 Sources of Exposure. Former and potential uses are likely to introduce B. cereus ATCC 14579 to both aquatic and terrestrial ecosystems. For example, use of B. cereus ATCC 14579 in wastewater treatment or its discharge in wastes from industrial applications, such as pulp and paper processing, textile manufacturing and biochemical production, could introduce B. cereus ATCC 14579 into aquatic ecosystems. Similarly, its use in bioremediation and biodegradation as well as in livestock probiotics and pest control agents could introduce B. cereus ATCC 14579 into terrestrial ecosystems.

In the event that consumer, commercial or industrial activities resume the environmental exposure to B. cereus strain ATCC 14579 could change based on the exposure scenarios described above.

2.2.2 Human

The human exposure to B. cereus ATCC 14579 is estimated to be low^[8] based on responses to the Notice, which indicate that this strain is no longer used in consumer or commercial products or for industrial processes in Canada.

Nevertheless, human exposure scenarios in the event that consumer, commercial or industrial activities with B. cereus ATCC 14579 resume have been considered. These are based on former and probable future uses as described in Section 2.1 Sources of Exposure. Workplace exposure to B. cereus ATCC 14579 is not considered in this assessment^[9].

Human exposure would be expected during the direct use and application of consumer or commercial products containing B. cereus ATCC14579. Skin and eye contact, inadvertent ingestion and inhalation of aerosolized droplets or particles are likely routes of direct user and bystander exposure. The use of such products in food preparation areas could result in the contamination of surfaces and foods at the time of product application. Subsequent lapses in proper food handling practices could allow B. cereus ATCC 14579 to proliferate in foods, possibly resulting in the ingestion of large numbers of cells.

Human exposure may also be temporally distant from the time of application. Subsequent to application, B. cereus ATCC 14579 is expected to remain viable and establish communities where organic matter accumulates (for example: countertops, drains, sinks, grease traps and kitchen garbage disposals). From such reservoirs, aerosols containing B. cereus ATCC 14579 could inoculate surfaces and foods. As above, subsequent lapses in proper food handling practices could allow the organism to proliferate in foods and result in the ingestion of large numbers of cells and lead to adverse effects.

Certain uses may introduce B. cereus ATCC 14579 into bodies of water, as described in section 2.2.1. Nevertheless, human exposure to the strain through the environment is expected to be low. Moreover, drinking water treatment processes are expected to effectively eliminate these micro-organisms and so limit their ingestion through drinking water.

In the event that consumer, commercial or industrial activities resume, the human exposure to B. cereusstrain ATCC 14579 could change based on the exposure scenarios described above.

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3. Risk Characterisation

Based on the current level of exposure inferred from responses to the Notice and notwithstanding the potential hazards to human health or to the Canadian environment known to be associated with this organism, the risk is estimated to be low^[10] to both the environment and human health from the DSL-listed strain B. cereusATCC 14579.

Nevertheless, resumption of the import, manufacture or use of B. cereus ATCC 14579 could result in an increased level of human and environmental exposure, as described in Section 2.2, which would increase the estimation of risk. Therefore, with respect to future importation and manufacturing activities, and taking into account the known and potential uses of B. cereus ATCC 14579 in various industries, the exposure to the environment could change.

Non-human species are expected to be exposed to B. cereus ATCC 14579 primarily through water and soil. Specifically, terrestrial and aquatic species can come into contact with this organism mainly from its release from industrial or manufacturing activities. Uses involving introduction into terrestrial environments could become problematic, as it has been shown that high (10⁷-10⁸ CFU per gram of dry soil) concentrations of B. cereus ATCC 14579 can cause adverse effects in springtail and red fescue and there is a lack of information on the potential adverse effects of B. cereuson aquatic species.

In the event that consumer, commercial or industrial activities resume and result in increased environmental exposure to B. cereus strain ATCC 14579, the associated risk of adverse effects to the environment could increase. Therefore, it is recommended that any new activities with this organism be assessed to ensure that any new uses do not present additional new risks.

The risk to human health will depend on the route of exposure. Of all routes identified, exposure through ingestion is of primary concern since B. cereus is primarily a gastrointestinal pathogen. B. cereus ATCC 14579 is known to produce important pathogenic factors (e.g., extracellular enzymes and toxins) implicated in gastrointestinal disease. The infectious dose of B. cereus is reported to range from 10² to 10⁵ cfu per gram of food or water and it is generally believed that any food containing concentrations of B. cereus exceeding 10³ to 10⁵cells or spores per gram is not safe for consumption (Anonymous, 2005; Haggblom et al. 2002; Stenfors Arnesen et al. 2008). The use of products containing B.cereusATCC 14579 in food preparation areas could result in the inoculation of foods and subsequent lapses in proper food handling practices could allow bacteria to proliferate. Cycles of reheating and inadequate refrigeration are particularly problematic for spore-forming bacteria like B. cereus, because spores are not inactivated during heating, and vegetative cells can germinate, multiply and re-sporulate between heating cycles. In this way, the number of viable cells in food increases in exponential fashion, eventually reaching a level that can lead to human gastrointestinal infection.

Skin and eye contact have been identified as potential routes of exposure, but these are less likely to result in adverse health effects. Wound infections have been documented for B. cereus in otherwise-healthy individuals; however, these are rare and there is no indication that B. cereusATCC 14579 could penetrate intact skin to cause dermal infection. Since skin is a natural barrier to microbial invasion of the human body, infections are likely to occur only if the skin was damaged through abrasions or burns (Dubouix et al. 2005). Similarly, although B.cereus is highly virulent in the eye, infection is likely only in cases of prior injury to the eye.

Inhalation of B. cereus ATCC 14579 cells or spores aerosolized through mechanical or air disturbances, either during or subsequent to product application, could lead to pulmonary exposure to spores or vegetative cells, but the number of inhaled spores or cells is unlikely to reach an infectious dose in healthy individuals. One survey conducted in the USA reported that a variety of B. cereus subgroup species are commonly present in urban aerosols across all seasons in 11 major American cities, but the reported incidence of respiratory infection due to B. cereus is extremely low in the USA. Exposure through inhalation is therefore not of concern (Merrill et al. 2006).

In the event that consumer, commercial or industrial activities resume and result in increased human exposure to B. cereusstrain ATCC 14579, the associated risk of adverse health effects in humans could increase. Therefore it is recommended that any new activities with this organism be assessed to ensure that they do not present additional risks.

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Conclusion

Based on responses to the Notice, it is concluded that B. cereus 14579 is not entering the environment in a quantity or concentration or under conditions that have or may have an immediate or long-term harmful effect in the environment or its biological diversity; constitute or may constitute a danger to the environment on which life depends; or constitute or may constitute a danger in Canada to human life or health. Therefore, it is proposed that this substance does not meet the criteria as set out in section 64 of the CEPA 1999.

Nevertheless, given the hazardous properties of B. cereus ATCC14579, reintroduction into Canada through import, manufacture or use could lead to this substance meeting the criteria set out in section 64 of the Act. Therefore, it is recommended that B. cereus ATCC 14579 be subject to the Significant New Activity (SNAc) provisions specified under subsection 106(3) of the Act, to ensure that any new activity involving this organism is notified and undergoes appropriate environmental and human health risk assessments as specified in section 10⁸ of the Act, prior to the organism being re-introduced into Canada. Under CEPA 1999, the effect of the SNAc will be to channel any new activity not covered by Acts listed in Schedule 4 (Pest Control Products Act, Heatlh of Animals Act, Feeds Act, and the Fertilizers Act) into CEPA 1999 risk assessment so that a determination of risk can be made for new activities not addressed in this report.

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References

Abul Nasr, S. E., Tawfik, M. F. S., Ammar, E. D.  and Farrag, S. M.(1978) Occurrence and causes of mortality among active and resting larvae of Pectinophora gossypiella (Lepidoptera, Gelechiidae) in Giza, Egypt. Zeitschrift für Angewandte Entomol  86, 403-414.

Abusin, S., Bhimaraj, A. and Khadra, S. (2008) Bacillus Cereus Endocarditis in a permanent pacemaker: a case report. Cases J 1, 95.

Ackermann, H.W., Roy, R., Martin, M., Murthy, M.R. and Smirnoff, W.A. (1978) Partial characterization of a cubic Bacillusphage. Can J Microbiol 24, 986-993.

Agata, N., Mori, M., Ohta, M., Suwa, S., Ohtani, I. and Isobe, M. (1994) A novel dodecadepsipeptide, cereulide, isolated from Bacillus cereus causes vacuole formation in HEp-2 cells. FEMS Microbiol Lett 121, 31-34.

Agata, N., Ohta, M., Arakawa, Y. and Mori, M. (1995a) The bceT gene of Bacillus cereus encodes an enterotoxic protein. Microbiology 141, 983-988.

Agata, N., Ohta, M., Mori, M. and Isobe, M. (1995b) A novel dodecadepsipeptide, cereulide, is an emetic toxin of Bacillus cereus. FEMS Microbiol Lett 129, 17-19.

Agata, N., Ohta, M. and Yokoyama, K. (2002) Production of Bacillus cereus emetic toxin (cereulide) in various foods. Int J Food Microbiol 73, 23-27.

Akiyama, N., Mitani, K., Tanaka, Y., Hanazono, Y., Motoi, N., Zarkovic, M., Tange, T., Hirai, H. and Yazaki, Y. (1997) Fulminant septicemic syndrome of Bacillus cereus in a leukemic patient. Intern Med 36, 221-226.

Al-Jamali, J., Felmerer, G., Alawadi, K., Kalash, Z. and Stark, G.B. (2008) Flexor tendon sheath infection due to Bacillus cereus after penetrating trauma. Eur J Plastic Surg31, 201-203.

Alouf, J.E. (2000) Cholesterol-binding cytolytic protein toxins. Int J Med Microbiol 290, 351-356.

Altiparmak, U.E., Ozer, P.A., Ozkuyumcu, C., Us, A.D., Aslan, B.S. and Duman, S. (2007) Postoperative endophthalmitis caused by Bacillus cereus and Chlamydia trachomatis. J Cataract Refract Surg 33, 1284-7.

Andersson, A. (1995) Bacillus cereus, strategy for survival.  SIK-report 1995.

Andreeva, Z.I., Nesterenko, V.F., Yurkov, I.S., Budarina, Z.I., Sineva, E.V. and Solonin, A.S. (2006) Purification and cytotoxic properties of Bacillus cereus hemolysin II. Protein Expr Purif 47, 186-193.

Andreeva, Z.I., Nesterenko, V.F., Fomkina, M.G., Ternovsky, V.I., Suzina, N.E., Bakulina, A.Y. , Solonin, A.S. and Sineva, E.V. (2007) The properties of Bacillus cereus hemolysin II pores depend on environmental conditions. Biochim Biophys Acta(BBA) - Biomembranes 1768, 253-263.

Anonymous. (2005). Opinion of the scientific panel on biological hazards on Bacillus cereus and other Bacillus in foodstuffs. The EFSA Journal 1-48.

Asano, S.I., Nukumizu, Y., Bando, H., Iizuka, T. and Yamamoto, T. (1997) Cloning of novel enterotoxin genes from Bacillus cereus and Bacillus thuringiensis. Appl Environ Microbiol 63, 1054-1057.

Ash, C. and Collins, M.D. (1992) Comparative analysis of 23S ribosomal RNA gene sequences of Bacillus anthracis and emetic Bacillus cereus determined by PCR-direct sequencing. FEMS Microbiol Lett 73, 75-80.

Ash, C., Farrow, J.A., Dorsch, M., Stackebrandt, E. and Collins, M.D. (1991) Comparative analysis of Bacillus anthracis, Bacillus cereus, and related species on the basis of reverse transcriptase sequencing of 16S rRNA. Int J Syst Bacteriol 41, 343-346.

Auger, S., Krin, E., Aymerich, S. and Gohar, M. (2006) Autoinducer 2 affects biofilm formation by Bacillus cereus. Appl Environ Microbiol 72, 937-941.

Avashia, S.B., Riggins, W.S., Lindley, C., Hoffmaster, A., Drumgoole, R., Nekomoto, T., Jackson, P.J., Hill, K.K., Williams, K., Lehman, L. (2007) Fatal pneumonia among metalworkers due to inhalation exposure to Bacillus cereus Containing Bacillus anthracis toxin genes. Clin Infect Dis44, 414-416.

Baida, G.E. and Kuzmin, N.P. (1995) Cloning and primary structure of a new hemolysin gene from Bacillus cereus. Biochim Biophys Acta 1264, 151-154.

Baida, G.E. and Kuzmin, N.P. (1996) Mechanism of action of hemolysin III from Bacillus cereus. Biochim Biophys Acta 1284, 122-124.

Barker M, Thakker B, Priest FG. (2005) Multilocus sequence typing reveals that Bacillus cereus strains isolated from clinical infections have distinct phylogenetic origins. FEMS Microbiol Lett. 245, 179-84.

Barth, H., Aktories, K., Popoff, M.R. and Stiles, B.G. (2004) Binary bacterial toxins: biochemistry, biology, and applications of common Clostridium and Bacillus proteins. Microbiol Mol Biol Rev 68, 373-402.

Bartoszewicz, M., Hansen, B.M. and Swiecicka, I. (2008) The members of the Bacillus cereus group are commonly present contaminants of fresh and heat-treated milk. Food Microbiol 25, 588-596.

Bassen, M. K., Gupta, L. K., Jolly, L. and Tewari, R. P.(1989) Thermal Resistance of Bacillus cereus spores in custard preparations. World J Microbiol  Biotechnol5, 511-516.

Beecher, D.J. and Macmillan, J.D. (1991) Characterization of the components of hemolysin BL from Bacillus cereus. Infect Immun 59, 1778-1784.

Beecher, D.J. and Wong, A.C. (1994a) Improved purification and characterization of hemolysin BL, a hemolytic dermonecrotic vascular permeability factor from Bacillus cereus. Infect Immun  62, 980-986.

Beecher, D.J. and Wong, A.C. (1994b) Identification of hemolysin BL-producing Bacillus cereus isolates by a discontinuous hemolytic pattern in blood agar. Appl Environ Microbiol60, 1646-1651.

Beecher, D.J. and Wong, A.C. (1994c) Identification and analysis of the antigens detected by two commercial Bacillus cereusdiarrheal enterotoxin immunoassay kits. Appl Environ Microbiol  60, 4614-4616.

Beecher, D.J., Pulido, J.S., Barney, N.P. and Wong, A.C. (1995a) Extracellular virulence factors in Bacillus cereusendophthalmitis: methods and implication of involvement of hemolysin BL. Infect Immun 63, 632-639.

Beecher, D., Schoeni, J. and Wong, A. (1995b) Enterotoxic activity of hemolysin BL from Bacillus cereus. Infect. Immun. 63, 4423-4428.

Beecher, D.J. and Wong, A.C. (1997) Tripartite hemolysin BL from Bacillus cereus. Hemolytic analysis of component interactions and a model for its characteristic paradoxical zone phenomenon. J Biol Chem 272, 233-239.

Beecher, D.J. and Wong, A.C. (2000) Cooperative, synergistic and antagonistic haemolytic interactions between haemolysin BL, phosphatidylcholine phospholipase C and sphingomyelinase from Bacillus cereus. Microbiology146, 3033-3039.

Beecher, D.J., Olsen, T.W., Somers, E.B. and Wong, A.C. (2000) Evidence for contribution of tripartite hemolysin BL, phosphatidylcholine-preferring phospholipase C, and collagenase to virulence of Bacillus cereus endophthalmitis. Infect Immun 68, 5269-5276.

Belaiche.J  (2000) Pathophysiology of acute infectious diarrhea. Acta Endoscopia  30, 177-184.

Bernhard, K., Schrempf, H. and Goebel, W. (1978) Bacteriocin and antibiotic resistance plasmids in Bacillus cereus and Bacillus subtilis. J Bacteriol133, 897-903.

Berry, C., O'Neil, S., Ben-Dov, E., Jones, A.F., Murphy, L., Quail, M.A., Holden, M.T., Harris, D., Zaritsky, A. and Parkhill, J. (2002) Complete sequence and organization of pBtoxis, the toxin-coding plasmid of Bacillus thuringiensis subsp. israelensis. Appl Environ Microbiol 68, 5082-5095.

Borsodi, A.K., Micsinai, A., Rusznyak, A., Vladar, P., Kovacs, G., Toth, E.M. and Marialigeti, K. (2005) Diversity of alkaliphilic and alkalitolerant bacteria cultivated from decomposing reed rhizomes in a Hungarian soda lake. Microbial Ecology50, 9-18.

Bottone EJ. (2010) Bacillus cereus, a volatile human pathogen. Clin Microbiol Rev. 23, 382-98.

Brillard, J. and Lereclus, D. (2004) Comparison of cytotoxin cytK promoters from Bacillus cereus strain ATCC 14579 and from a B. cereus food-poisoning strain. Microbiology 150, 2699-2705.

Brinton, G.S., Topping, T.M., Hyndiuk, R.A., Aaberg, T.M., Reeser, F.H. and Abrams, G.W. (1984) Posttraumatic endophthalmitis. Arch Ophthalmol 102, 547-550.

Burdon, K.L., Davis, J.S. and Wende, R.D. (1967) Experimental infection of mice with Bacillus cereus: studies of pathogenesis and pathologic changes. J Infect Dis117, 307-316.

Cadot, C., Tran, S.L., Vignaud, M.L., De Buyser, M.L., Kolsto, A.B., Brisabois, A., Nguyen-The, C., Lereclus, D., Guinebretiere, M.H., and Ramarao, N. (2010) InhA1, NprA, and HlyII as candidates for markers to differentiate pathogenic from nonpathogenic Bacillus cereus strains. J Clin Microbiol48, 1358-1365.

Callegan, M.C., Kane, S.T., Cochran, D.C., Gilmore, M.S., Gominet, M. and Lereclus, D. (2003) Relationship of plcR-regulated factors to Bacillus endophthalmitis virulence. Infect Immun 71, 3116-24.

Callegan, M. C., Booth, M. C., Jett, B. D. and Gilmore, M. S. (1999) Pathogenesis of Gram-Positive Bacterial Endophthalmitis. Infect. Immun. 67, 3348-3356.

CDC (2005) Outbreak of cutaneous Bacillus cereusinfections among cadets in a university military program--Georgia, August 2004. MMWR Morb Mortal Wkly Rep54, 1233-1235.

Chan, W.M., Liu, D.T., Chan, C.K., Chong, K.K. and Lam, D.S. (2003) Infective endophthalmitis caused by Bacillus cereusafter cataract extraction surgery. Clin Infect Dis37, e31-34.

Chen, Y., Braathen, P., Leonard, C. and Mahillon, J. (1999) MIC231, a naturally occurring mobile insertion cassette from Bacillus cereus. Mol Microbiol32, 657-668.

Cherif, A., Brusetti, L., Borin, S., Rizzi, A., Boudabous, A., Khyami-Horani, H. and Daffonchio, D. (2003) Genetic relationship in the 'Bacillus cereus group' by rep-PCR fingerprinting and sequencing of a Bacillus anthracis-specific rep-PCR fragment. J Appl Microbiol 94, 1108-1119.

Choma, C. and Granum, P.E. (2002) The enterotoxin T (BcET) from Bacillus cereus can probably not contribute to food poisoning. FEMS Microbiol Lett 217, 115-119.

Christiansson, A., Naidu, A. S., Nilsson, I., Wadstrom, T. and Pettersson, H. E. (1989) Toxin production by Bacillus cereus dairy isolates in milk at low temperatures. Appl. Environ. Microbiol. 55, 2595-2600.

Clark, F. E. (1937) The relation of Bacillus siamensisand similar pathogenic spore-forming bacteria to Bacillus cereus. J. Bacteriol. 33, 435-443.

Cone, L.A., Dreisbach, L., Potts, B.E., Comess, B.E. and Burleigh, W.A. (2005) Fatal Bacillus cereus endocarditis masquerading as an anthrax-like infection in a patient with acute lymphoblastic leukemia: case report. J Heart Valve Dis14, 37-39.

Coonrod, J.D., Leadley, P.J. and Eickhoff, T.C. (1971) Antibiotic susceptibility of Bacillus species. J Infect Dis 123, 102-105.

Crane, P.K. and Crane, H.M. (2007) Indwelling intravenous catheter-associated Bacillus cereus sepsis in an immunocompetent patient. Infect Med 24, 40-42.

Dancer, S.J., McNair, D., Finn, P. and Kolsto, A.B. (2002) Bacillus cereus cellulitis from contaminated heroin. J Med Microbiol 51, 278-281.

Darbar, A., Harris, I.A. and Gosbell, I.B. (2005) Necrotizing infection due to Bacillus cereus mimicking gas gangrene following penetrating trauma. J Orthop Trauma19, 353-355.

Davey, R.T. Jr and Tauber, W.B. (1987) Posttraumatic endophthalmitis: the emerging role of Bacillus cereusinfection. Rev Infect Dis 9, 110-123.

De Palmenaer, D., Vermeiren, C. and Mahillon, J. (2004) IS231-MIC231 elements from Bacillus cereus sensu lato are modular. Mol Microbiol 53, 457-467.

Didelot, X., Barker, M., Falush, D. and Priest, F.G. (2009) Evolution of pathogenicity in the Bacillus cereus group. Syst Appl Microbiol 32, 81-90.

Dierick, K., Van Coillie, E., Swiecicka, I., Meyfroidt, G., Devlieger, H., Meulemans, A., Hoedemaekers, G., Fourie, L., Heyndrickx, M. and Mahillon, J. (2005) Fatal family outbreak of Bacillus cereus-associated food poisoning. J Clin Microbiol 43, 4277-4279.

Dragon, D.C. and Rennie, R.P. (1995) The ecology of anthrax spores: tough but not invincible. Can Vet J36, 295-301.

Drobniewski FA. (1993) Bacillus cereus and related species. Clin Microbiol Rev. 6, 324-338.

Drysdale, M., Heninger, S., Hutt, J., Chen, Y., Lyons, C.R. and Koehler, T.M. (2005) Capsule synthesis by Bacillus anthracis is required for dissemination in murine inhalation anthrax. EMBO J 24, 221-227.

Dubouix, A., Bonnet, E., Alvarez, M., Bensafi, H., Archambaud, M., Chaminade, B., Chabanon, G. and Marty, N. (2005) Bacillus cereus infections in Traumatology-Orthopaedics Department: retrospective investigation and improvement of healthcare practices. J Infect 50, 22-30.

Edwards, P.R. and Fife, M.A. (1961) Lysine-iron agar in the detection of Arizona cultures. Appl Microbiol9, 478-480.

Ehling-Schulz, M., Guinebretiere, M.-H., Monthán, A., Berge, O., Fricker, M. and Svensson, B. (2006) Toxin gene profiling of enterotoxic and emetic Bacillus cereus. FEMS Microbiol lett  260, 232-240.

El Emmawie, A.H., Aly, N.Y.A. and Al-Sawan, R. (2008) Bloodstream infection due to Bacillus cereus in a preterm neonate associated with necrotizing enterocolitis: A case report. Kuwait Med J 40, 140-142.

El Saleeby, C.M., Howard, S.C., Hayden, R.T. and McCullers, J.A. (2004) Association between tea ingestion and invasive Bacillus cereus infection among children with cancer. Clin Infect Dis 39, 1536-1539.

Evreux, F., Delaporte, B., Leret, N., Buffet-Janvresse, C. and Morel, A. (2007) Méninginte néonatale à Bacillus cereus, à propos d'un cas. Archives de Pédiatrie14, 265-368.

Fagerlund, A., Lindback, T., Storset, A.K., Granum, P.E. and Hardy, S.P. (2008) Bacillus cereus Nhe is a pore-forming toxin with structural and functional properties similar to the ClyA (HlyE, SheA) family of haemolysins, able to induce osmotic lysis in epithelia. Microbiology 154, 693-704.

Fagerlund, A., Ween, O., Lund, T., Hardy, S.P. and Granum, P.E. (2004) Genetic and functional analysis of the cytK family of genes in Bacillus cereus. Microbiology150, 2689-2697.

Fedhila, S., Buisson, C., Dussurget, O., Serror, P., Glomski, I. J., Liehl, P., Lereclus, D. and Nielsen-LeRoux, C. (2010) Comparative analysis of the virulence of invertebrate and mammalian pathogenic bacteria in the oral insect infection model Galleria mellonella. J. Invertebr Pathol 103, 24-29.

Flavelle, S., Tyrrell, G.J. and Forgie, S.E. (2007) A 'serious' bloodstream infection in an infant. Can J Infect Dis Med Microbiol 18, 311-312.

Frankard, J., Li, R., Taccone, F., Struelens, M.J., Jacobs, F. and Kentos, A. (2004) Bacillus cereus pneumonia in a patient with acute lymphoblastic leukemia. Eur J Clin Microbiol Infect Dis 23, 725-728.

Frankland, G. C. and Frankland, P. F. (1887) Studies on some new micro-organisms obtained from air. Philos Trans R Soc Lond B Biol Sci 178, 257–287.

Fredricks, D. N. and Myerson, D. (2006) Pneumonia in a neutropenic patient with leukemia. Inf Dis Clin Pract14, 107-109.

Fujii, S., Yoshida, A., Sakurai, S., Morita, M., Tsukamoto, K., Ikezawa, H. and Ikeda, K. (2004) Chromogenic assay for the activity of sphingomyelinase from Bacillus cereus and its application to the enzymatic hydrolysis of lysophospholipids. Biol Pharm Bull 27, 1725-1729.

Gaulin, C., Viger, Y.B. and Fillion, L. (2002) An outbreak of Bacillus cereus implicating a part-time banquet caterer. Can J Public Health 93, 353-355.

Gaur, A.H., Patrick, C.C., McCullers, J.A., Flynn, P.M., Pearson, T.A., Razzouk, B.I., Thompson, S.J. and Shenep, J.L. (2001) Bacillus cereus bacteremia and meningitis in immunocompromised children. Clin Infect Dis32, 1456-1462.

Ghelardi, E., Celandroni, F., Salvetti, S., Barsotti, C., Baggiani, A. and Senesi, S. (2002) Identification and characterization of toxigenic Bacillus cereus isolates responsible for two food-poisoning outbreaks. FEMS Microbiol Lett 208, 129-134.

Gibriel, A.Y. and Abd-el Al, A.T. (1973) Measurement of heat resistance parameters for spores isolated from canned products. J Appl Bacteriol 36, 321-327.

Ginsburg A. S., Salazar L. G., True L. D., Disis M. L. (2003) Fatal Bacillus cereus sepsis following resolving neutropenic enterocolitis during the treatment of acute leukemia. Am J Hematol. 72 ,204-208.

Girisch, M., Ries, M., Zenker, M., Carbon, R., Rauch, R. and Hofbeck, M. (2003) Intestinal perforations in a premature infant caused by Bacillus cereus. Infection31, 192-193.

Glatz, B. A. and Goepfert, J. M. (1973) Extracellular factor synthesized by Bacillus cereus which evokes a dermal reaction in guinea pigs. Infect. Immun.8, 25-29.

Glatz, B. A., Spira, W. M. and Goepfert, J. M. (1974) Alteration of vascular permeability in rabbits by culture filtrates of Bacillus cereus and related species. Infect. Immun. 10, 299-303.

Goepfert, J.M. (1974) Monkey feeding trials in the investigation of the nature of Bacillus cereus food poisoning. Proc. IV. Internat. Congr. Food Sci. Technol. 3, 178-181.

Gohar, M., Okstad, O.A., Gilois, N., Sanchis, V., Kolsto, A.B., and Lereclus, D. (2002) Two-dimensional electrophoresis analysis of the extracellular proteome of Bacillus cereus reveals the importance of the PlcR regulon. Proteomics 2, 784-791.

Gohar M, Faegri K, Perchat S, Ravnum S, Økstad OA, Gominet M, Kolstø AB,Lereclus D. (2008) The PlcR virulence regulon of Bacillus cereus. PLoS One. 3, e2793.

Gorina, L.G., Fluer, F.S., Olovnikov, A.M. and Ezepcuk, YU. V. (1975) Use of the aggregate-hemagglutination technique for determining exo-Enterotoxin of Bacillus cereus. Appl. Environ. Microbiol. 29, 201-204.

Granum, P.E. (1994) Bacillus cereus and its toxins. Soc Appl Bacteriol Symp Ser 23, 61S-66S.

Granum, P.E., O'Sullivan, K. and Lund, T. (1999) The sequence of the non-haemolytic enterotoxin operon from Bacillus cereus. FEMS Microbiol Lett 177, 225-229.

Granum, PE. ( 2001) Bacillus cereus. In Food microbiology: fundamentals and frontiers ed. M. P. Doyle, L.R.B.a.T.J.M.e. pp. 327-336. Washington: American Society for Microbiology.

Guinebretiere, M.H., Fagerlund, A., Granum, P.E. and Nguyen-The, C. (2006) Rapid discrimination of cytK-1 and cytK-2 genes in Bacillus cereus strains by a novel duplex PCR system. FEMS Microbiol Lett 259, 74-80.

Guinebretière MH, Thompson FL, Sorokin A, Normand P, Dawyndt P, Ehling-Schulz  M, Svensson B, Sanchis V, Nguyen-The C, Heyndrickx M, De Vos P. (2008) Ecological diversification in the Bacillus cereus Group. Environ Microbiol. 10, 851-865

Guinebretière MH, Velge P, Couvert O, Carlin F, Debuyser ML, Nguyen-The C. (2010) Ability of Bacillus cereus group strains to cause food poisoning varies according to phylogenetic affiliation (groups I to VII) rather than species affiliation. J Clin Microbiol. 48, 3388-3391

Haggblom, M.M., Apetroaie, C., Andersson, M.A. and Salkinoja-Salonen, M.S. (2002) Quantitative analysis of cereulide, the emetic toxin of Bacillus cereus, produced under various conditions. Appl Environ Microbiol68, 2479-2483.

Hansen, B.M., Hiby, P.E., Jensen, G.B. and Hendriksen, N.B. (2003) The Bacillus cereus bceT enterotoxin sequence reappraised. FEMS Microbiol Lett 223, 21-24.

Hardy, S.P., Lund, T. and Granum, P.E. (2001) CytK toxin of Bacillus cereus forms pores in planar lipid bilayers and is cytotoxic to intestinal epithelia. FEMS Microbiol Lett197, 47-51.

Harvie, D.R., Vilchez, S., Steggles, J.R. and Ellar, D.J. (2005) Bacillus cereus Fur regulates iron metabolism and is required for full virulence. Microbiology151, 569-577.

Haug, T.M., Sand, S.L., Sand, O., Phung, D., Granum, P.E., and Hardy, S.P. (2010) Formation of very large conductance channels by Bacillus cereus Nhe in Vero and GH(4) cells identifies NheA + B as the inherent pore-forming structure. J Membr Biol 237, 1-11.

Helgason, E., Caugant, D. A., Olsen, I. and Kolsto, A.-B. (2000a) Genetic structure of population of Bacillus cereusand B. thuringiensis isolates associated with periodontitis and other human infections. J. Clin. Microbiol. 38, 1615-1622.

Helgason, E., Okstad, O.A., Caugant, D.A., Johansen, H.A., Fouet, A., Mock, M., Hegna, I. and Kolsto (2000b) Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis--one species on the basis of genetic evidence. Appl Environ Microbiol 66, 2627-2630.

Helgason, E., Tourasse, N.J., Meisal, R., Caugant, D.A. and Kolsto, A.B. (2004) Multilocus sequence typing scheme for bacteria of the Bacillus cereus group. Appl Environ Microbiol 70, 191-201.

Hernaiz, C., Picardo, A., Alos, J.I. and Gomez-Garces, J.L. (2003) Nosocomial bacteremia and catheter infection by Bacillus cereus in an immunocompetent patient. Clin Microbiol Infect 9, 973-975.

Hilliard, N.J., Schelonka, R.L. and Waites, K.B. (2003) Bacillus cereus bacteremia in a preterm neonate. J Clin Microbiol 41, 3441-3444.

Hirabayashi, K., Shiohara, M., Saito, S., Tanaka, M., Yanagisawa, R., Tsuruta, G., Fukuyama, T., Hidaka, Y., Nakazawa, Y., Shimizu, T., Sakashita, K., Koike, K. (2010) Polymyxin-direct hemoperfusion for sepsis-induced multiple organ failure. Pediatr Blood Cancer. 55, 202-205.

Hoffmaster, A.R., Ravel, J., Rasko, D.A., Chapman, G.D., Chute, M.D., Marston, C.K., De, B.K., Sacchi, C.T., Fitzgerald, C., Mayer, L.W., Maiden, M.C., Priest, F.G., Barker, M., Jiang, L., Cer, R.Z., Rilstone, J., Peterson, S.N., Weyant, R.S., Galloway, D.R., Read, T.D., Popovic, T. and Fraser, C.M. (2004) Identification of anthrax toxin genes in a Bacillus cereus associated with an illness resembling inhalation anthrax. Proc Natl Acad Sci U S A 101, 8449-8454.

Hoffmaster, A.R., Hill, K.K., Gee, J.E., Marston, C.K., De, B.K., Popovic, T., Sue, D., Wilkins, P.P., Avashia, S.B., Drumgoole, R., (2006) Characterization of Bacillus cereusisolates associated with fatal pneumonias: strains are closely related to Bacillus anthracis and harbor B. anthracis virulence genes. J Clin Microbiol44, 3352-3360.

Hoffmaster AR, Novak RT, Marston CK, Gee JE, Helsel L, Pruckler JM, Wilkins PP. (2008) Genetic diversity of clinical isolates of Bacillus cereus using multilocus sequence typing. BMC Microbiol. 6;8:191.

Horvath, G., Toth-Marton, E., Meszaros, J.M. and Quarini, L. (1986) Experimental Bacillus cereus mastitis in cows. Acta Vet Hung 34, 29-35.

Huang, C.C., Narita, M., Yamagata, T., Itoh, Y. and Endo, G. (1999) Structure analysis of a class II transposon encoding the mercury resistance of the Gram-positive Bacterium Bacillus megaterium MB1, a strain isolated from minamata bay, Japan. Gene 234, 361-369.

Hutchens, A., Gupte, A., McAuliffe, P.F., Schain, D., Soldevila-Pico, C., Knapik, J.A., Fujita, S., Mozingo, D.W., Richards, W.T. (2010) Bacillus cereus Necrotizing Fasciitis in a Patient with End-Stage Liver Disease. Surg Infect (Larchmt). 11, 469-474

Ikezawa, H., Mori, M. and Taguchi, R. (1980) Studies on sphingomyelinase of Bacillus cereus: hydrolytic and hemolytic actions on erythrocyte membranes. Arch Biochem Biophys 199, 572-578.

Inui, S., Kume, T., Hiramine, T. and Murase, N. (1979) Pathological survey of bovine mastitis.  Bull  Nat Institute Animal Health. 78, 25-38.

Ivanova, N., Sorokin, A., Anderson, I., Galleron, N., Candelon, B., Kapatral, V., Bhattacharyya, A., Reznik, G., Mikhailova, N., Lapidus, A., Chu, L., Mazur, M., Goltsman, E., Larsen, N., D'Souza, M., Walunas, T., Grechkin, Y., Pusch, G., Haselkorn, R., Fonstein, M., Ehrlich, S.D., Overbeek, R. and Kyrpides, N. (2003) Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis. Nature 423, 87-91.

Jaaskelainen, E.L., Teplova, V., Andersson, M.A., Andersson, L.C., Tammela, P., Andersson, M.C., Pirhonen, T.I., Saris, N.E., Vuorela, P. and Salkinoja-Salonen, M.S. (2003) In vitro assay for human toxicity of cereulide, the emetic mitochondrial toxin produced by food poisoning Bacillus cereus. Toxicol In Vitro 17, 737-744.

Jaquette, C.B. and Beuchat, L.R. (1998) Combined effects of pH, nisin, and temperature on growth and survival of psychrotrophic Bacillus cereus. J Food Prot 61, 563-570.

Jasper, D.E., Bushnell, R.B., Dellinger, J.D. and Stang, A.M. (1972) Bovine mastitis due to Bacillus cereus. J Am Vet Med Assoc 160, 750-756.

Jassim, H. K., Foster, H. A. and Fairhurst, C. P. (1990) Biological control of Dutch elm disease: Bacillus thuringiensis as a potential control agent for Scolytus scolytus and S. multistriatus. J. Appl Microbiol 69, 563-568.

John, A.B., Razak, E.A., Razak, E.E., Al-Naqeeb, N. and Dhar, R. (2007) Intractable Bacillus cereus bacteremia in a preterm neonate. J Trop Pediatr 53, 131-132.

Johnson, K.M. (1984) Bacillus cereus food-borne illness. An update. J Food Prot 47, 145-153.

Jones, T.O. and Turnbull, P.C. (1981) Bovine mastitis caused by Bacillus cereus. Vet Rec10⁸, 271-274.

Jucovic, M., Walters, F.S., Warren, G.W., Palekar, N.V. and Chen, J.S. (2008) From enzyme to zymogen: engineering Vip2, an ADP-ribosyltransferase from Bacillus cereus, for conditional toxicity. Protein Eng Des Sel21, 631-638.

Just, I., Schallehn, G. and Aktories, K. (1992) ADP-ribosylation of small GTP-binding proteins by Bacillus cereus. Biochem Biophys Res Commun 183, 931-936.

Katsuya, H., Takata, T., Ishikawa, T., Sasaki, H., Ishitsuka, K., Takamatsu, Y. and Tamura, K. (2009) A patient with acute myeloid leukemia who developed fatal pneumonia caused by carbapenem-resistant Bacillus cereus. J Infect Chemother 15, 39-41.

Kiyomizu, K., Yagi, T., Yoshida, H., Minami, R., Tanimura, A., Karasuno, T. and Hiraoka, A. (2008) Fulminant septicemia of Bacillus cereus resistant to carbapenem in a patient with biphenotypic acute leukemia. J Infect Chemother14, 361-367.

Klee, S.R., Brzuszkiewicz, E.B., Nattermann, H., Brüggemann, H., Dupke, S., Wollherr, A., Franz, T., Pauli, G., Appel, B., Liebl, W., Couacy-Hymann, E., Boesch, C., Meyer, F.D., Leendertz, F.H., Ellerbrok, H., Gottschalk, G., Grunow, R., Liesegang, H. (2010) The genome of a Bacillus isolate causing anthrax in chimpanzees combines chromosomal properties of B. cereuswith B. anthracis virulence plasmids. PLoS One. 5, e10986.

Kobayashi, K., Kami, M., Ikeda, M., Kishi, Y., Murashige, N., Tanosaki, R., Mori, S. and Takaue, Y. (2005) Fulminant septicemia caused by Bacillus cereus following reduced-intensity umbilical cord blood transplantation. Haematologica90, ECR06.

Koch, A. and Arvand, M. (2005) Recurrent bacteraemia by 2 different Bacillus cereus strains related to 2 distinct central venous catheters. Scand J Infect Dis37, 772-774.

Kolsto, A.B., Tourasse, N.J. and Okstad, O.A. (2009) What sets Bacillus anthracis apart from other Bacillusspecies? Annu Rev Microbiol 63, 451-476.

Kotiranta, A., Haapasalo, M., Kari, K., Kerosuo, E., Olsen, I., Sorsa, T., Meurman, J.H., and Lounatmaa, K. (1998) Surface structure, hydrophobicity, phagocytosis, and adherence to matrix proteins of Bacillus cereus cells with and without the crystalline surface protein layer. Infect Immun 66, 4895-4902.

Kotiranta, A., Lounatmaa, K. and Haapasalo, M. (2000) Epidemiology and pathogenesis of Bacillus cereusinfections. Microbes Infect 2, 189-198.

Kramer, J. M. and Gilbert, R. J. (1989) Bacillus cereusand other Bacillus species. In Foodborne Bacterial Pathogens ed. Doyle, M.P. pp. 21-70. New York: Marcel Dekker Inc.

Lamanna, C. and Jones, L. (1963) Lethality for mice of vegetative and spore forms of Bacillus cereus and Bacillus cereus like insect pathogens injected intraperitoneally and subcutaneously. J. Bacteriol.85, 532-535.

Latsios, G., Petrogiannopoulos, C., Hartzoulakis, G., Kondili, L., Bethimouti, K. and Zaharof, A. (2003) Liver abscess due to Bacillus cereus: a case report. Clin Microbiol Infect 9, 1234-1237.

Le Scanff, J., Mohammedi, I., Thiebaut, A., Martin, O., Argaud, L. and Robert, D. (2006) Necrotizing gastritis due to Bacillus cereus in an immunocompromised patient. Infection34, 98-99.

Lebessi, E., Dellagrammaticas, H.D., Antonaki, G., Foustoukou, M. and Iacovidou, N. (2009) Bacillus cereus meningitis in a term neonate. J Matern Fetal Neonatal Med22, 458-461.

Lequin, M.H., Vermeulen, J.R., van Elburg, R.M., Barkhof, F., Kornelisse, R.F., Swarte, R. and Govaert, P.P. (2005) Bacillus cereus meningoencephalitis in preterm infants: neuroimaging characteristics. AJNR Am J Neuroradiol26, 2137-2143.

Leung, K., Trevors, J.T. and Lee, H. (1995) Survival of and lacZ expression in recombinant Pseudomonas strains introduced into river water microcosms. Can J Microbiol 41, 461-469.

Levin, M. R. and D'Amico, D. J. (1991) Traumatic endophthalmitis. In Eye trauma ed. B. J. Shingleton, P.S.H.a.K.R.K.ed. pp. 242-252. Missouri: Mosby Year Book.

Lindback, T., Fagerlund, A., Rodland, M.S. and Granum, P.E. (2004) Characterization of the Bacillus cereus Nhe enterotoxin.  Microbiology 150, 3959-3967.

Lindback, T., Okstad, O.A., Rishovd, A.L. and Kolsto, A.B. (1999) Insertional inactivation of hblC encoding the L2 component of Bacillus cereus ATCC 14579 haemolysin BL strongly reduces enterotoxigenic activity, but not the haemolytic activity against human erythrocytes. Microbiology145, 3139-3146.

Lindbäck, T. and Granum, P. E. (2006) Detection and Purification of Bacillus cereus Enterotoxins. In Food-Borne Pathogens pp. 15-26. Humana Press.

Lindbäck, T., Hardy, S.P., Dietrich, R., Sodring, M., Didier, A., Moravek, M., Fagerlund, A., Bock, S., Nielsen, C., Casteel, M., Granum, P. E. and Märtlbauer, E. (2010) Cytotoxicity of the Bacillus cereus Nhe enterotoxin requires specific binding order of its three exoprotein components. Infect Immun78, 3813-3821

Lodemann, U., Lorenz, B.M., Weyrauch, K.D., and Martens, H. (2008) Effects of Bacillus cereus var. toyoi as probiotic feed supplement on intestinal transport and barrier function in piglets. Arch Anim Nutr 62, 87-106.

Logan, N. A. and De Vos, P. (2009) Genus I. Bacillus. In Bergey's Manual of Systematic Bacteriology. Volume 3: The Firmicutes ed. De Vos, P., Garrity, G., Jones, D., Krieg, N.R., Ludwig, W., Rainey, F.A., Schleifer, K.-H. and Whitman, W.B.E. pp. 21-127. New York: Springer.

Lund, T., De Buyser, M.L. and Granum, P.E. (2000) A new cytotoxin from Bacillus cereus that may cause necrotic enteritis. Mol Microbiol 38, 254-261.

Lund, T. and Granum, P.E. (1996) Characterisation of a non-haemolytic enterotoxin complex from Bacillus cereusisolated after a foodborne outbreak. FEMS Microbiol Lett141, 151-156.

Lund, T. and Granum, P.E. (1997) Comparison of biological effect of the two different enterotoxin complexes isolated from three different strains of Bacillus cereus. Microbiology 143 , 3329-3336.

Mahler, H., Pasi, A., Kramer, J.M., Schulte, P., Scoging, A.C., Bar, W. and Krahenbuhl, S. (1997) Fulminant liver failure in association with the emetic toxin of Bacillus cereus. N Engl J Med 336, 1142-1148.

Manickam, N., Knorr, A. and Muldrew, K.L. (2008) Neonatal meningoencephalitis caused by Bacillus cereus. Pediatr Infect Dis J 27, 843-846.

Martinez, M.F., Haines, T., Waller, M., Tingey, D. and Gomez, W. (2007) Probable occupational endophthalmitis from Bacillus cereus. Arch Environ Occup Health62, 157-160.

McIntyre, L., Bernard, K., Beniac, D., Isaac-Renton, J.L., and Naseby, D.C. (2008). Identification of Bacillus cereusGroup Species Associated with Food Poisoning Outbreaks in British Columbia, Canada. Appl Environ Microbiol74, 7451-7453.

Melling, J., Capel, B. J., Turnbull, P. C. and Gilbert, R. J. (1976) Identification of a novel enterotoxigenic activity associated with Bacillus cereus. J Clin Pathol29, 938-940.

Merrill, L., Dunbar, J., Richardson, J., Kuske, C. R. (2006) Composition of Bacillus species in aerosols from 11 U.S. cities. J Forensic Sci. 51, 559-65.

Mikkola, R., Saris, N.-E. L., Grigoriev, P. A., Andersson, M. A. and Salkinoja-Salonenm, M. S. (1999) Ionophoretic properties and mitochondrial effects of cereulide. Eur J Biochem263, 112-117.

Miles, G., Bayley, H. and Cheley, S. (2002) Properties of Bacillus cereus hemolysin II: a heptameric transmembrane pore. Protein Sci 11, 1813-1824.

Miller, J.M., Hair, J.G., Hebert, M., Hebert, L., Roberts, F.J. Jr and Weyant, R.S. (1997) Fulminating bacteremia and pneumonia due to Bacillus cereus. J Clin Microbiol35, 504-507.

Mols, M., de Been, M., Zwietering, M.H., Moezelaar, R. and Abee, T. (2007) Metabolic capacity of Bacillus cereus strains ATCC 14579 and ATCC 10987 interlinked with comparative genomics. Environ Microbiol 9, 2933-2944.

Monteverde, M.L., Sojo, E.T., Grosman, M., Hernandez, C. and Delgado, N. (2006) Relapsing Bacillus cereus Peritonitis in a Pediatric Patient on Chronic Peritoneal Dialysis. Perit Dial Int 26, 715-716.

Moore, G.E. (1972) Pathogenicity of ten strains of bacteria to larvae of the southern pine beetle. J Invertebr Pathol 20, 41-45.

Moulder, E.H., Sharma, H.K. and Howell, F.R. (2008) Traumatic osteomyelitis of the femur treated with distraction osteogenesis without surgical bone resection: a case report. J Trauma65, E39-42.

Musa, M.O., Al Douri, M., Khan, S., Shafi, T., Al Humaidh, A. and Al Rasheed, A.M. (1999) Fulminant septicaemic syndrome of Bacillus cereus: three case reports. J Infect39, 154-6.

Narita, M., Matsui, K., Huang, C.C., Kawabata, Z. and Endo, G. (2004) Dissemination of TnMERI1-like mercury resistance transposons among Bacillus isolated from worldwide environmental samples. FEMS Microbiol Ecol 48, 47-55.

Nishikawa, T., Okamoto, Y., Tanabe, T., Kodama, Y., Shinkoda, Y. and Kawano, Y. (2009) Critical illness polyneuropathy after Bacillus cereus sepsis in acute lymphoblastic leukemia. Intern Med 48, 1175-1177.

Nishiwaki, H., Ito, K., Otsuki, K., Yamamoto, H., Komai, H. and Matsuda, K. (2004) Purification and functional characterization of insecticidal sphingomyelinase C produced by Bacillus cereus . Eur J Biochem  271, 601-606.

Okinaka, R.T., Cloud, K., Hampton, O., Hoffmaster, A.R., Hill, K.K., Keim, P., Koehler, T.M., Lamke, G., Kumano, S., Mahillon, J., Manter, D., Martinez, Y., Ricke, D., Svensson, R. and Jackson, P.J. (1999) Sequence and organization of pXO1, the large Bacillus anthracis plasmid harboring the anthrax toxin genes. J Bacteriol 181, 6509-6515.

Okstad, O.A., Tourasse, N.J., Stabell, F.B., Sundfaer, C.K., Egge-Jacobsen, W., Risoen, P.A., Read, T.D. and Kolsto, A.B. (2004) The bcr1 DNA repeat element is specific to the Bacillus cereus group and exhibits mobile element characteristics. J Bacteriol 186, 7714-7725.

Paananen, A., Mikkola, R., Sareneva, T., Matikainen, S., Hess, M., Andersson, M., Julkunen, I., Salkinoja-Salonen, M.S. and Timonen, T. (2002) Inhibition of human natural killer cell activity by cereulide, an emetic toxin from Bacillus cereus. Clin Exp Immunol 129, 420-428.

Parke, D. W. B. G. S. (1986) Endophthalmitis. In Infections of the eye ed. K. F. Tabbara, a.R.A.H.ed. pp. 563-585. Boston:  Little, Brown & Co.

Perchat, S., Buisson, C., Chaufaux, J., Sanchis, V., Lereclus, D. and Gohar, M. (2005) Bacillus cereus produces several nonproteinaceous insecticidal exotoxins. J Invertebr Pathol 90, 131-133.

Perrin, D., Greenfield, J. and Ward, G.E. (1976) Aucte Bacillus cereus mastitis in dairy cattle associated with use of a contaminated antibiotic. Can Vet J17, 244-247.

Pflugfelder, S.C. and Flynn, H.W. Jr (1992) Infectious endophthalmitis. Infect Dis Clin North Am6, 859-873.

Pillai, A., Thomas, S. and Arora, J. (2006) Bacillus cereus: the forgotten pathogen. Surg Infect (Larchmt)7, 305-308.

Priest, F.G., Barker, M., Baillie, L.W., Holmes, E.C. and Maiden, M.C. (2004) Population structure and evolution of the Bacillus cereus group. J Bacteriol186, 7959-7970.

Princz, J. (2010) Pathogenicity and Toxicity of Risk Group II Microbial Strains on Territrial Organisms. Special report prepared by Environment Canada. (Personal communication)

Puvabanditsin, S., Zaafran, A., Garrow, E., Diwan, R., Mehta, D. and Phattraprayoon, N. (2007) Bacillus cereusmeningoencephalitis in a neonate. HK J Paediatr12, 293-296.

Radnedge, L., Agron, P.G., Hill, K.K., Jackson, P.J., Ticknor, L.O., Keim, P. and Andersen, G.L. (2003) Genome differences that distinguish Bacillus anthracis from Bacillus cereus and Bacillus thuringiensis. Appl Environ Microbiol69, 2755-2764.

Rahmet-Alla, M. and Rowley, A.F. (1989) Studies on the pathogenicity of different strains of Bacillus cereus for the cockroach, Leucophaea maderae.  J Invert Pathol 53, 190-196.

Rasko, D.A., Altherr, M.R., Han, C.S. and Ravel, J. (2005) Genomics of the Bacillus cereus group of organisms. FEMS Microbiol Rev 29, 303-329.

Rasko, D.A., Ravel, J., Okstad, O.A., Helgason, E., Cer, R.Z., Jiang, L., Shores, K.A., Fouts, D.E., Tourasse, N.J., Angiuoli, S.V., Kolonay, J., Nelson, W.C., Kolsto, A.B., Fraser, C.M. and Read, T.D. (2004) The genome sequence of Bacillus cereusATCC 10987 reveals metabolic adaptations and a large plasmid related to Bacillus anthracis pXO1. Nucleic Acids Res 32, 977-988.

Rasko, D.A., Rosovitz, M.J., Okstad, O.A., Fouts, D.E., Jiang, L., Cer, R.Z., Kolsto, A.B., Gill, S.R. and Ravel, J. (2007) Complete sequence analysis of novel plasmids from emetic and periodontal Bacillus cereus isolates reveals a common evolutionary history among the B. cereus-group plasmids, including Bacillus anthracis pXO1. J Bacteriol189, 52-64.

Ribeiro, N.F., Heath, C.H., Kierath, J., Rea, S., Duncan-Smith, M. and Wood, F.M. (2010) Burn wounds infected by contaminated water: Case reports, review of the literature and recommendations for treatment. Burns 36, 9-22

Risoen, P.A., Ronning, P., Hegna, I.K. and Kolsto, A.B. (2004) Characterization of a broad range antimicrobial substance from Bacillus cereus. J Appl Microbiol96, 648-655.

Rizk, I.R. and Ebeid, H.M. (1989) Survival and growth of Bacillus cereus in Egyptian bread and its effect on bread staling.  Nahrung 33, 839-844.

Rossland, E., Andersen Borge, G.I., Langsrud, T. and Sorhaug, T. (2003) Inhibition of Bacillus cereus by strains of Lactobacillus and Lactococcus in milk. Int J Food Microbiol 89, 205-212.

Ruhfel, R.E., Robillard, N.J. and Thorne, C.B. (1984) Interspecies transduction of plasmids among Bacillus anthracis, B. cereus, and B. thuringiensis. J Bacteriol 157, 708-711.

Ruiz, S.R., Reyes, G.M., Campos, C.T., Jimenez, V.L., Rojas, R.T., de la Fuente, C.G. and Esteve, A.A. (2006) Relapsing Bacillus cereus peritonitis during automated peritoneal dialysis. Perit Dial Int 26, 612-613.

Sada, A., Misago, N., Okawa, T., Narisawa, Y., Ide, S., Nagata, M. and Mitsumizo, S. (2009) Necrotizing fasciitis and myonecrosis "synergistic necrotizing cellulitis" caused by Bacillus cereus. J Dermatol 36, 423-426.

Salamitou, S., Ramisse, F., Brehelin, M., Bourguet, D., Gilois, N., Gominet, M., Hernandez, E. and Lereclus, D. (2000) The plcR regulon is involved in the opportunistic properties of Bacillus thuringiensis and Bacillus cereus in mice and insects. Microbiology 146 , 2825-2832.

Santos, C.A., Vilas-Boas, G.T., Lereclus, D., Suzuki, M.T., Angelo, E.A. & Arantes, O.M. (2010) Conjugal transfer between Bacillus thuringiensis and Bacillus cereusstrains is not directly correlated with growth of recipient strains, J Invertebr Pathol 105, 171-175.

Schiefer, B., Macdonald, K.R., Klavano, G.G. and van Dreumel, A.A. (1976) Pathology of Bacillus cereus mastitis in dairy cows. Can Vet J 17, 239-243.

Schnepf, E., Crickmore, N., Van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D.R. and Dean, D.H. (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62, 775-806.

Schoeni, J.L., and A.C. Wong. (2005) Bacillus cereus food poisoning and its toxins. J Food Prot 68, 636-648.

Seligy, V.L., Beggs, R.W., Rancourt, J.M. and Tayabali, A.F. (1997) Quantitative bioreduction assays for calibrating spore content and viability of commercial Bacillus thuringiensisinsecticides. J  Ind Microbiol Biotechnol18, 370-378.

Seligy, V. L., Shwed, P. S. and Tayabali., A. F. (2004)  Comparison of Macrophage Cell Models exposed to spores of Bacillus cereus and B.thuringiensis sspp.  Proc.12th Internat’l Immunol. Congress. Vol. ISBN 88-7587-0769-1 .Medimond S.r.l..

Selvakumar, G., Mohan, M., Sushil, S. N., Kundu, S., Bhatt, J. C. and Gupta, H. S. (2007) Characterization and phylogenetic analysis of an entomopathogenic Bacillus cereus strain WGPSB-2 (MTCC 7182) isolated from white grub, Anomala dimidiata (Coleoptera: Scarabaeidae). Biocontrol Sci Technol  17, 525-534.

Shimoni, Z., Mamet, Y., Niven, M., Mandelbaum, S., Valinsky, L. and Froom, P. (2008) Bacillus cereus peritonitis after Cesarean section. Surg Infect (Larchmt)9, 10⁵-10⁶.

Shinagawa, K., Ichikawa, K., Matsusaka, N. and Sugii, S. (1991a) Purification and some properties of a Bacillus cereusmouse lethal toxin. J Vet Med Sci 53, 469-474.

Shinagawa, K., Sugiyama, J., Terada, T., Matsusaka, N. and Sugii, S. (1991b) Improved methods for purification of an enterotoxin produced by Bacillus cereus. FEMS Microbiol Lett 64, 1-5.

Shinagawa, K., Konuma, H., Sekita, H. and Sugii, S. (1995) Emesis of rhesus monkeys induced by intragastric administration with the HEp-2 vacuolation factor (cereulide) produced by Bacillus cereus. FEMS Microbiol Lett130, 87-90.

Sineva, E. V., Kovalevskaya, Z. I. A. S. A. M., Gerasimov, Y. L., Ternovsky, V. I., Teplova, V. V., Yurkova, T. V. and Solonin, A. S. (2009)  Expression of Bacillus cereus hemolysin II in Bacillus subtilis renders the bacteria pathogenic for the crustacean Daphnia magna. FEMS Microbiol Lett299, 110-119.

Shiota, M., Saitou, K., Mizumoto, H., Matsusaka, M., Agata, N., Nakayama, M., Kage, M., Tatsumi, S., Okamoto, A., Yamaguchi, S., Ohta, M. and Hata, D. (2010) Rapid detoxification of cereulide in Bacillus cereus food poisoning. Pediatrics125, e951-955

Sotto, A., Porneuf, M., Gouby, A., Rossi, J.F., and Jourdan, J. (1995) Septicémie à Bacillus cereus et pneumopathie nécrosante au cours d’une leucémmie lymphoïque chronique. Méd Mal Infect 25, 1-3

Spira, W. M. and Goepfert, J. M. (1972) Bacillus cereus-Induced Fluid Accumulation in Rabbit Ileal Loops. Appl. Environ. Microbiol. 24, 341-348.

Srivaths, P.R., Rozans, M.K., Kelly, E. Jr and Venkateswaran, L. (2004) Bacillus cereus central line infection in an immunocompetent child with hemophilia. J Pediatr Hematol Oncol 26, 194-196.

Stansfield, R. and Caudle, S. (1997) Bacillus cereusand orthopaedic surgical wound infection associated with incontinence pads manufactured from virgin wood pulp. J Hosp Infect 37, 336-338.

Steen, M.K., Bruno-Murtha, L.A., Chaux, G., Lazar, H., Bernard, S. and Sulis, C. (1992) Bacillus cereus endocarditis: report of a case and review. Clin Infect Dis14, 945-946.

Stenfors Arnesen, L.P., Fagerlund, A. and Granum, P.E. (2008) From soil to gut: Bacillus cereus and its food poisoning toxins. FEMS Microbiol Rev 32, 579-606.

Stretton, R.J. and Bulman, R.A. (1975) Experimental infection of rabbits with Bacillus cereus. Zentralbl Bakteriol Orig A 232, 83-90.

Stromsten, N.J., Benson, S.D., Burnett, R.M., Bamford, D.H. and Bamford, J.K. (2003) The Bacillus thuringiensis linear double-stranded DNA phage Bam35, which is highly similar to the Bacillus cereus linear plasmid pBClin15, has a prophage state. J Bacteriol 185, 6985-6989.

Sushil, S. N., Mohan, M., Selvakumar, G., Bhatt, J. C. and Gupta, H. S. (2008) Isolation and toxicity evaluation of bacterial entomopathogens against phytophagous white grubs (Coleoptera: Scarabaeidae) in Indian Himalayan hills. Int J Pest Manage 54, 301-307.

Tayabali, A. F., Nguyen, K. C. and Seligy, V. L. (2010) Early murine immune responses from endotracheal exposures tu biotechnology-related Bacillus strains. Toxicol. Environ. Chem. DOI: 10.1080/02772248.2010.526784

Ticknor, L.O., Kolsto, A.B., Hill, K.K., Keim, P., Laker, M.T., Tonks, M. and Jackson, P.J. (2001) Fluorescent Amplified Fragment Length Polymorphism Analysis of Norwegian Bacillus cereusand Bacillus thuringiensis Soil Isolates. Appl Environ Microbiol 67, 4863-73.

To, W.N., Gudauskas, R.T. and Harper, J.D. (1975) Pathogenicity of Bacillus cereus isolated from Trichoplusia nilarvae. J Invertebr Pathol 26, 135-136.

Tobita, H. and Hayano, E. (2007) A fulminant case of endogenous endophthalmitis caused by gram-positive bacillus. Jpn J Cli Ophthalmol 61, 985-989.

Tourasse, N.J., Helgason, E., Okstad, O.A., Hegna, I.K. and Kolsto, A.B. (2006) The Bacillus cereus group: novel aspects of population structure and genome dynamics. J Appl Microbiol 101, 579-593.

Tourasse, N.J. and Kolsto, A.B. (2008) Survey of group I and group II introns in 29 sequenced genomes of the Bacillus cereus group: insights into their spread and evolution. Nucleic Acids Res 36, 4529-4548.

Tran, S.L., Guillemet, E., Ngo-Camus, M., Clybouw, C., Puhar, A., Moris, A., Gohar, M., Lereclus, D., Ramarao, N. (2010a) Hemolysin II is a Bacillus cereus virulence factor that induces apoptosis of macrophages. Cell Microbiol. 2010 Aug 23. [Epub ahead ofprint].

Tran S.L., Guillement, E., Gohar, M., Lereclus, D. and Ramarao, N. (2010b) CwpFM (EntFM) is a Bacillus cereus potential cell wall peptidase implicated in adhesion, biofilm formation, and virulence. J Bacteriol 192, 2638-2642

Tuladhar, R., Patole, S.K., Koh, T.H., Norton, R. and Whitehall, J.S. (2000) Refractory Bacillus cereus infection in a neonate. Int J Clin Pract 54, 345-347.

Turnbull, P. C. (1976) Studies on the production of enterotoxins by Bacillus cereus. J Clin Pathol29, 941-948.

Turnbull, P.C., Jorgensen, K., Kramer, J.M., Gilbert, R.J. and Parry, J.M. (1979) Severe clinical conditions associated with Bacillus cereus and the apparent involvement of exotoxins. J Clin Pathol 32, 289-293.

Turnbull, P.C. and Kramer, J.M. (1983) Non-gastrointestinal Bacillus cereus infections: an analysis of exotoxin production by strains isolated over a two-year period. J Clin Pathol 36, 1091-1096.

Turnbull, P., Kramer, J., Jorgensen, K., Gilbert, R. and Melling, J. (1979) Properties and production characteristics of vomiting, diarrheal, and necrotizing toxins of Bacillus cereus. Am J Clin Nutr 32, 219-228.

Usui, K., Miyazaki, S., Kaito, C. and Sekimizu, K. (2009) Purification of a soil bacteria exotoxin using silkworm toxicity to measure specific activity. Microb Pathog46, 59-62.

Vahey, J.B. and Flynn, H.W. Jr (1991) Results in the management of Bacillus endophthalmitis. Ophthalmic Surg22, 681-686.

Van der Auwera, G. and Mahillon, J. (2005) TnXO1, a germination-associated class II transposon from Bacillus anthracis. Plasmid 53, 251-257.

Van der Auwera, G.A., Timmery, S., Hoton, F. and Mahillon, J. (2007) Plasmid exchanges among members of the Bacillus cereus group in foodstuffs. Int J Food Microbiol113, 164-172.

Van Der Zwet, W.C., Parlevliet, G.A., Savelkoul, P.H., Stoof, J., Kaiser, A.M., Van Furth, A.M. and Vandenbroucke-Grauls, C.M. (2000) Outbreak of Bacillus cereus infections in a neonatal intensive care unit traced to balloons used in manual ventilation. J Clin Microbiol 38, 4131-4136.

van Veen, J.A., van Overbeek, L.S. and van Elsas, J.D. (1997) Fate and activity of microorganisms introduced into soil. Microbiol Mol Biol Rev 61, 121-135.

Vassileva, M., Torii, K., Oshimoto, M., Okamoto, A., Agata, N., Yamada, K., Hasegawa, T. and Ohta, M. (2006) Phylogenetic analysis of Bacillus cereus isolates from severe systemic infections using multilocus sequence typing scheme. Microbiol Immunol 50, 743-749.

Verheust, C., Fornelos, N. and Mahillon, J. (2005) GIL16, a new gram-positive tectiviral phage related to the Bacillus thuringiensis GIL01 and the Bacillus cereus pBClin15 elements. J Bacteriol 187, 1966-1973.

Virtanen, S.M., Roivainen, M., Andersson, M.A., Ylipaasto, P., Hoornstra, D., Mikkola, R. and Salkinoja-Salonen, M.S. (2008) In vitro toxicity of cereulide on porcine pancreatic Langerhans islets. Toxicon 51, 1029-1037.

Weber, D.J., Saviteer, S.M., Rutala, W.A. and Thomann, C.A. (1988) In vitro susceptibility of Bacillus spp. to selected antimicrobial agents. Antimicrob Agents Chemother32, 642-645.

Wijman, J.G., de Leeuw, P.P., Moezelaar, R., Zwietering, M.H., and Abee, T. (2007) Air-liquid interface biofilms of Bacillus cereus: formation, sporulation, and dispersion. Appl. Environ. Microbiol. 73; 1481-1488.

Wijnands, L.M., Dufrenne, J.B. and van Leusden, F.M. (2001) The pathogenic mechanisim of the diarrheal syndrome caused by Bacillus cereus. RIVM report 250912001/2002,1-18.

Wohlgemuth, K., Bicknell, E.J. and Kirkbride, C.A. (1972a) Abortion in cattle associated with Bacillus cereus. J Am Vet Med Assoc 161, 1688-1690.

Wohlgemuth, K., Kirkbride, C.A., Bicknell, E.J. and Ellis, R.P. (1972b) Pathogenicity of Bacillus cereus for pregnant ewes and heifers. J Am Vet Med Assoc 161, 1691-1695.

Wong, H.C., Chang, M.H. and Fan, J.Y. (1988) Incidence and characterization of Bacillus cereus isolates contaminating dairy products. Appl Environ Microbiol54, 699-702.

Yuan, Y., Zheng, D., Hu, X., Cai, Q. and Yuan, Z. (2010) Conjugative transfer of insecticidal plasmid pHT73 from Bacillus thuringiensis to B. anthracis and compatibility of this plasmid with pXO1 and pXO2. Appl Environ Microbiol 76, 468-473.

Zheng, X., Kodama, T. and Ohashi, Y. (2008) Eyeball luxation in Bacillus cereus-induced panophthalmitis following a double-penetrating ocular injury. Jpn J Ophthalmol52, 419-421.

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Appendices

• Appendix 1A: Growth of Bacillus cereus ATCC 14579 in liquid media at 28°C, 32°C, 37°C and 42 °C
• Appendix 1B: Characteristics of Bacillus cereus ATCC 14579-- Growth on Solid Media
• Appendix 1C: Characteristics of Bacillus cereus ATCC 14579 – Fatty Acid Methyl Ester (FAME) Analysis
• Appendix 2: Relationships within the Bacillus cereusgroup.
• Appendix 3: List of some Bacillus cereus groupmobile genetic elements and associated traits
• Appendix 4: Chromosomal genes coding for toxins in Bacillus cereusATCC 14579 as analysed by PCR
• Appendix 5: List of toxins produced by Bacillus cereus
• Appendix 6A: Pathogenicity to invertebrates and vertebrates
• Appendix 6B: Pathogenicity of Bacillus cereus to invertebrates and vertebrates in natural settings.
• Appendix 7A: Selected non gastrointestinal outbreaks caused by Bacillus cereus and reported in the literature.
• Appendix 7B: Reported Bacillus cereus Food-Related Outbreak
• Appendix 8: Considerations for Levels of Hazard Severity, Exposure and Risk as per Health Canada and Environment Canada’s “Framework for Science-Based Risk Assessment of Micro-organisms regulated under the Canadian Environmental Protection Act,1999”.

Footnotes

^[1] Environmental Health Science and Research Bureau
^[2] Enviromental Health Science and Research Bureau
^[3] Environmental Health Science and Research Bureau
^[4] Lotte Pia Stenfors Arnesen, Associate professor and head of the food microbiology laboratory at the Norwegian School of Veterinary Science (NVH), Dep. of Food Safety and Infection, Oslo, Norway
^[5] Judy Greig, Food Safety Microbiologist/Epidemiologist, Laboratory for Foodborne Zoonoses, Public Health Agency of Canada
^[6] see Appendix 8
^[7] see Appendix 8
^[8] see Appendix 8
^[9] DISCLAIMER: A determination of whether one or more of the criteria of section 64 of CEPA 1999 are met is based upon an assessment of potential risks to the environment and/or to human health associated with exposure in the general environment. For humans, this includes, but is not limited to, exposure from air, water and the use of products containing the substance. A conclusion under CEPA 1999 may not be relevant to, nor does it preclude, an assessment against the criteria specified in the Controlled Products Regulations, which is part of the regulatory framework for the Workplace Hazardous Materials Information System (WHMIS) for products intended for workplace use. Individuals who handle B.cereus ATCC14579 in the workplace should consult with their occupational health and safety representative about safe handling practices, applicable laws and requirements under WHMIS and the Laboratory Biosafety Guidelines.
^[10] See Appendix 8