Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be...

The Bacillus cereus sensu lato group contains diverse Gram-positive spore-forming bacteria that can cause gastrointestinal diseases and severe eye infections in humans. They have also been incriminated in a multitude of other severe, and frequently fatal, clinical infections, such as osteomyelitis, septicaemia, pneumonia, liver abscess and meningitis, particularly in immuno-compromised patients and preterm neonates. The pathogenic properties of this organism are mediated by the synergistic effects of a number of virulence products that promote intestinal cell destruction and/or resistance to the host immune system. This review focuses on the pore-forming haemolysins produced by B. cereus: haemolysin I (cereolysin O), haemolysin II, haemolysin III and haemolysin IV (CytK). Haemolysin I belongs to the cholesterol-dependent cytolysin (CDC) family whose best known members are listeriolysin O and perfringolysin O, produced by L. monocytogenes and C. perfringens respectively. HlyII and CytK are oligomeric ß-barrel pore-forming toxins related to the α-toxin of S. aureus or the ß-toxin of C. perfringens. The structure of haemolysin III, the least characterized haemolytic toxin from the B. cereus, group has not yet been determined.

Trypan blue is a dye that has been widely used for selective staining of dead tissues or cells. Here, we show that the pore-forming toxin HlyII of Bacillus cereus allows trypan blue staining of macrophage cells, despite the cells remaining viable and metabolically active. These findings suggest that the dye enters viable cells through the pores. To our knowledge, this is the first demonstration that trypan blue may enter viable cells. Consequently, the use of trypan blue staining as a marker of vital status should be interpreted with caution. The blue coloration does not necessarily indicate cell lysis, but may rather indicate pore formation in the cell membranes and more generally increased membrane permeability.

The virulence ofBacillus cereusrequires that bacteria have the capacity to colonize their host, degrade specific tissues, and circumvent the host immune system. To study this aspect of pathogenesis, we focused on three metalloproteases, InhA1, InhA2, and InhA3, which share more than 66% identity. The expression of these metalloprotease genes was assessed by transcriptional fusions with alacZreporter gene. The expression profiles suggest a complementary time course of InhA production. Indeed, the genes are simultaneously expressed but are oppositely controlled during stationary phase. We constructed single and multipleinhAmutants and assessed the bacterial locations of the proteins as well as their individual or additive roles in macrophage escape and toxicity, antibacterial-peptide cleavage, and virulence. InhA1, a major component of the spore exosporium, is the only InhA metalloprotease involved in bacterial escape from macrophages. A mutant lackinginhA1,inhA2, andinhA3shows a strong decrease in the level of virulence for insects. Taken together, these results show that the InhA metalloproteases ofB. cereusare important virulence factors that may allow the bacteria to counteract the host immune system.

Bacillus cereus EntFM displays an NlpC/P60 domain, characteristic of cell wall peptidases. The protein is involved in bacterial shape, motility, adhesion to epithelial cells, biofilm formation, vacuolization of macrophages, and virulence. These data provide new information on this, so far, poorly studied toxin and suggest that this protein is a cell wall peptidase, which we propose to rename CwpFM.

Bacillus cereusis found in food, soil, and plants, and the ability to cause food-borne diseases and opportunistic infection presumably varies among strains. Therefore, measuring harmful toxin production, in addition to the detection of the bacterium itself, may be key for food and hospital safety purposes. All previous studies have focused on the main known virulence factors, cereulide, Hbl, Nhe, and CytK. We examined whether other virulence factors may be specific to pathogenic strains. InhA1, NprA, and HlyII have been described as possibly contributing toB. cereuspathogenicity. We report the prevalence and expression profiles of these three new virulence factor genes among 57B. cereusstrains isolated from various sources, including isolates associated with gastrointestinal and nongastrointestinal diseases. Using PCR, quantitative reverse transcriptase PCR, and virulencein vivoassays, we unraveled these factors as potential markers to differentiate pathogenic from nonpathogenic strains. We show that thehlyIIgene is carried only by strains with a pathogenic potential and that the expression levels ofinhA1andnprAare higher in the pathogenic than in the nonpathogenic group of strains studied. These data deliver useful information about the pathogenicity of variousB. cereusstrains.