Detection and phylogenic analysis of one anthrax virulence plasmid pXO1 conservative open reading frame ubiquitous presented within Bacillus cereus group strains.
ABSTRACT The presence of one of the anthrax virulence plasmid pXO1 conserved fragments was analyzed in 24 Bacillus cereus and B. thuringiensis strains, including 6 B. thuringiensis subspecies, by polymerase chain reactions. Twelve out of 24 strains showed PCR-positive for an ORF101 homologous sequence. Two pXO1-ORF101-like fragments from a B. cereus B-4ac and a commercial B. thuringiensis kurstaki HD1 were cloned, sequenced and expressed in Escherichia coli. Toxicity assays revealed that the product encoded by the pXO1-ORF101-like fragment had no impact on either Vero cells or Chinese Hamster Ovary cells, suggesting that this fragment probably not contribute to enterotoxic activity. Sequence alignment of the pXO1-ORF101 from three Bacillus anthracis and ORF101-like fragments from other 12 B. cereus group isolates indicated high identity (more than 90%) and the presence of subgroup- and strain-specific SNPs among these fragments.
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ABSTRACT: The awareness of the threat of Bacillus anthracis, the causative agent of the disease anthrax, as a biowarfare and bioterrorism weapon has revived the development of new technologies for rapid and accurate detection of virulent isolates in environmental and clinical samples. Here we explore the utility of molecular beacon real-time PCR technology for detection of virulent Bacillus anthracis strains. Molecular beacons are nucleic acid probes with high specificity, that act as switches by emitting fluorescence when bound to their nucleotide sequence targets by means of altering their conformation. In this study, five molecular beacons targeting Bacillus anthracis capA, capB, capC, lef, and pag alleles were designed and used in five uniplex assays. Another molecular beacon targeting the Bacillus group chromosomal 16s rRNA allele was designed for use in a duplex assay with an internal PCR amplification control. The molecular beacons were used in a real-time PCR assay for the detection of and differentiation between virulent B. anthracis and other members of the B. cereus group at the molecular level. Various B. anthracis samples as well as other bacterial and human samples were used to demonstrate the sensitivity and specificity of this assay. Use of the molecular beacon real-time PCR technology should accelerate current efforts to swiftly detect B. anthracis strains and its virulence plasmids in clinical and environmental samples and may extend to the development of additional molecular beacon-based assays for the identification of other pathogenic agents or the identification of B. anthracis directly from clinical samples.Journal of microbiological methods 05/2009; 78(1):45-53. · 2.43 Impact Factor
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ABSTRACT: Bacillus anthracis is a member of the Bacillus cereus group species (also known as the "group 1 bacilli"), a collection of Gram-positive spore-forming soil bacteria that are non-fastidious facultative anaerobes with very similar growth characteristics and natural genetic exchange systems. Despite their close physiology and genetics, the B. cereus group species exhibit certain species-specific phenotypes, some of which are related to pathogenicity. B. anthracis is the etiologic agent of anthrax. Vegetative cells of B. anthracis produce anthrax toxin proteins and a poly-d-glutamic acid capsule during infection of mammalian hosts and when cultured in conditions considered to mimic the host environment. The genes associated with toxin and capsule synthesis are located on the B. anthracis plasmids, pXO1 and pXO2, respectively. Although plasmid content is considered a defining feature of the species, pXO1- and pXO2-like plasmids have been identified in strains that more closely resemble other members of the B. cereus group. The developmental nature of B. anthracis and its pathogenic (mammalian host) and environmental (soil) lifestyles of make it an interesting model for study of niche-specific bacterial gene expression and physiology.Molecular Aspects of Medicine 09/2009; 30(6):386-96. · 10.38 Impact Factor
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ABSTRACT: Bacillus anthracis is the cause of anthrax, and two large plasmids are essential for toxicity: pXO1, which contains the toxin genes, and pXO2, which encodes a capsule. B. anthracis forms a highly monomorphic lineage within the B. cereus group, but strains of Bacillus thuringiensis and B. cereus exist that are genetically closely related to the B. anthracis cluster. During the past five years B. cereus strains that contain the pXO1 virulence plasmid were discovered, and strains with both pXO1 and pXO2 have been isolated from great apes in Africa. Therefore, the presence of pXO1 and pXO2 no longer principally separates B. anthracis from other Bacilli. The B. anthracis lineage carries a specific mutation in the global regulator PlcR, which controls the transcription of secreted virulence factors in B. cereus and B. thuringiensis. Coevolution of the B. anthracis chromosome with its plasmids may be the basis for the successful development and uniqueness of the B. anthracis lineage.Annual review of microbiology 07/2009; 63:451-76. · 12.80 Impact Factor