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This study was designed to evaluate the occurrence of Bacillus cereus spores with a damaged exosporium and the consequences of such damages on spore adhesion. The analysis of nine strains sporulated under optimal conditions (Spo8-agar, 30 degrees C) revealed that damaged exosporia were systematically found in one strain (B. cereus D17) and occasionally in two others (B. cereus ATCC 14579T and B. cereus D6). The prevalence of spores with damaged exosporia increased when sporulation occurred under less favorable conditions (Spo8-broth or high temperature); for example, more than 50% of the B. cereus ATCC 14579T spores were damaged when sporulation occurred at 40 degrees C on Spo8-agar or at 30 degrees C in Spo8-broth. Furthermore, when subjected to shear stresses by circulation of spore suspensions through a peristaltic pump, the exosporium of a significant amount of spores became partially or totally shorn off (for example, 40% of the B. cereus ATCC 14579T spores). The ability of damaged spores to adhere to inert surfaces and to resist cleaning under shear stress was significantly affected when compared with intact spores, resulting in a decreased number of adhering spores (P < or = 0.004) and enhanced resistance to cleaning (P < or = 0.008). This study provides evidence that, under various conditions, the exosporium of B. cereus spores can be partly or wholly damaged, thereby affecting the ability of spores to contaminate the surfaces of food processing lines. The presence of spores devoid of exosporium will be of importance in determining the risk associated with B. cereus spores adherent to food processing line surfaces.

Bacillus cereus spores are surrounded by a loose-fitting layer called the exosporium, whose distal part is mainly formed from glycoproteins. The role played by the exosporium glycoproteins of B. cereus ATCC 14579 (BclA and ExsH) was investigated by considering hydrophobicity and charge, as well as the properties of spore adhesion to stainless steel. The absence of BclA increased both the isoelectric point (IEP) and hydrophobicity of whole spores while simultaneously reducing the interaction between spores and stainless steel. However, neither the hydrophobicity nor the charge associated with BclA could explain the differences in the adhesion properties. Conversely, ExsH, another exosporium glycoprotein, did not play a significant role in spore surface properties. The monosaccharide analysis of B. cereus ATCC 14579 showed different glycosylation patterns on ExsH and BclA. Moreover, two specific glycosyl residues, namely, 2-O-methyl-rhamnose (2-Me-Rha) and 2,4-O-methyl-rhamnose (2,4-Me-Rha), were attached to BclA, in addition to the glycosyl residues already reported in B. anthracis.The food-borne pathogen Bacillus cereus has been extensively isolated in the form of spores from various environments, including food contact surfaces (26,33). Spores of the B. cereus group (closely related species, such as B. cereus, B. anthracis, or B. thuringiensis) are characterized by the presence of an outer layer called the exosporium, which surrounds the spore and is suspected of playing a major role in the interface phenomena. Indeed, the exosporium has been shown to play a role in spore adhesion to abiotic surfaces (14) and to professional phagocytic cells (25), in the escape of spores from macrophages (27), and in spore germination (16). As revealed by electron microscopy, the exosporium is made of an external hair-like nap on top of a paracrystalline basal layer (18). The hair-like nap is mainly composed of the collagen-like glycoprotein BclA in B. anthracis (34). The BclA protein contains three domains: the N-terminal domain (NTD) of 44 amino acids (aa) is involved in targeting and anchoring the protein in the exosporium (38); the C-terminal domain (CTD) is composed of multiple ␤-strands (28); and the collagen-like region (CLR), with a triple-helix conformation (34), is composed of GXX repeats, mostly GPT, whose threonine residues provide putative glycosylation sites (8). The filament length is proportional to the number of GXX repeats (35). The presence of BclA at the surfaces of B. anthracis spores has been shown to have an impact on the overall hydrophobicity (6) and charge (7) of the spores. Furthermore, the presence of BclA affects the germination of B. anthracis spores and is involved in specific interaction with immune cells (4, 24) and surfactants (28). Genes of other collagen-like proteins are also present in the B. anthracis genome (22, 39), as well as in other Bacillus species, such as exsH, which encodes a protein with an NTD of 145 aa with putative helical secondary structures, or exsJ (40). However, their loc...