Emetic toxin formation of Bacillus cereus is restricted to a single evolutionary lineage of closely related strains

Abstract

An in-depth polyphasic approach was applied to study the population structure of the human pathogen Bacillus cereus. To assess the intraspecific biodiversity of this species, which is the causative agent of gastrointestinal diseases, a total of 90 isolates from diverse geographical origin were studied by genetic [M13-PCR, random amplification of polymorphic DNA (RAPD), multilocus sequence typing (MLST)] and phenetic [Fourier transform Infrared (FTIR), protein profiling, biochemical assays] methods. The strain set included clinical strains, isolates from food remnants connected to outbreaks, as well as isolates from diverse food environments with a well documented strain history. The phenotypic and genotypic analysis of the compiled panel of strains illustrated a considerable diversity among B. cereus connected to diarrhoeal syndrome and other non-emetic food strains, but a very low diversity among emetic isolates. Using all typing methods, cluster analysis revealed a single, distinct cluster of emetic B. cereus strains. The isolates belonging to this cluster were neither able to degrade starch nor could they ferment salicin; they did not possess the genes encoding haemolysin BL (Hbl) and showed only weak or no haemolysis. In contrast, haemolytic-enterotoxin-producing B. cereus strains showed a high degree of heterogeneity and were scattered over different clusters when different typing methods were applied. These data provide evidence for a clonal population structure of cereulide-producing emetic B. cereus and indicate that emetic strains represent a highly clonal complex within a potentially panmictic or weakly clonal background population structure of the species. It may have originated only recently through acquisition of specific virulence factors such as the cereulide synthetase gene.Abbreviations: FTIR, Fourier transform Infrared; Hbl, haemolysin BL; MLST, multilocus sequence typing; Nhe, non-haemolytic enterotoxin; RAPD, random amplification of polymorphic DNA.The GenBank/EMBL/DDBJ accession numbers for the sequences of the internal gene fragments used for MLST and for the sporulation stage III AB genes reported in this paper are AY762151-AY762213 and AY578317-AY578349, respectively.

Phylogenetic Analysis of Bacillus cereus Isolates from Severe Systemic Infections Using Multilocus Sequence Typing Scheme

“…Several typing methods have been developed to study this group. These include 16S rRNA and 23S rRNA analysis (5,9), fluorescent amplified fragment length polymorphism analysis (33), multilocus enzyme electrophoresis (14,29), random amplification of polymorphic DNA (11), rep-PCR fingerprinting (10) and multilocus sequence typing (15,19,25,30). These publications and whole genome analysis studies (3,16,18,26,27) showed that the phenotypically distinct species are genetically very closely related.…”

Genomics of Bacteria from an Ancient Marine Origin: Clues to Survival in an Oligotrophic Environment

“…Pan-genomes help us determine when other genome dynamics are shaping interesting phenotypes. For example, when comparing strains of the Bacillus cereus-anthracis group (Anderson et al 2005;Ehling-Schulz et al 2005;Helgason et al 2004), it was found that they were very clonal, and that their central pathogenic traits were the result of horizontal gene transfer in mobile elements. A surprising finding in the many available microbial genomes was the high number of genes that had been acquired by exchange of DNA between microbes.…”

“…This has led to the hypothesis that bacteria in the CCB water systems are descendants of marine bacteria from Pre-Cambrian (Souza et al 2006). Systematic studies have also been used to describe new species of bacteria using current criteria (Anderson et al 2005;Cerritos et al 2008;Ehling-Schulz et al 2005;Escalante et al 2009;Helgason et al 2004) as well as by attempting to apply multivariate analysis and physical-chemical analyses in order to determine special relationships amongst the bacteria (Cerritos et al 2011). At the genomic scale, 2 genomes (Alcaraz et al 2010;Alcaraz et al 2008) and 3 metagenomes (Breitbart et al 2009;Desnues et al 2008;Dinsdale et al 2008) had been described, in which genes that allow for adaptation to the oligotrophic environment of Cuatro Cienegas have been identified.…”

Bacillus cereus, an unusual cause of fulminant liver failure: diagnosis may prevent liver transplantation

“…To demonstrate the toxigenic potential of B. cereus isolates, genes encoding emetic-toxin cereulide (ces) and enterotoxins (nhe, hbl and cytK) were analysed by multiplex PCR, as previously described (Ehling-Schulz et al, 2006). Strains isolated from each stool showed the same toxin gene pattern, ces + nhe + hbl 2 cytK 2 , characteristic of cereulide-producing strains (Ehling-Schulz et al, 2005Pó sfay-Barbe et al, 2008). Moreover, these strains were not capable of producing amylase, a feature highly suggestive of cereulide-producing strains (Ehling-Schulz et al, 2004).…”