Evaluation of different methods to discriminate Bacillus anthracis from other bacteria of the Bacillus cereus group

Abstract

Aims: To evaluate different methods that are useful for rapid and definitive discrimination of Bacillus anthracis from other bacteria of the Bacillus cereus group in environmental samples like letters claimed to contain anthrax spores. Methods and Results: Characterized strains and bacteria from environmental samples were analysed by microbiological and molecular methods (PCR and restriction analysis). Environmental isolates often shared several microbiological features with B. anthracis, e.g. lack of b-haemolysis and phospholipase C activity, and only the gamma phage assay was specific for B. anthracis. PCR assays targeting markers from the virulence plasmids exclusively detected B. anthracis, but other PCR targets were also detected in nonanthrax isolates. Additionally, the restriction pattern in an AluI restriction analysis of the SG-749 fragment is not 100% specific. The loci used for multiple-locus variable-number tandem repeat analysis of B. anthracis are also present in other members of the B. cereus group, but amplicon sizes are usually different. Conclusions: Environmental samples often contain borderline isolates closely related to B. anthracis both on microbiological and genetic levels. Real-time PCR targeting plasmidal and chromosomal markers should be used for rapid and definitive exclusion of a virulent strain of B. anthracis in such samples. Significance and Impact of the Study: This study gives an overview of the current microbiological and molecular methods used for identification of B. anthracis and shows that most assays have limits when borderline isolates present in environmental samples are analysed.

Characterization ofBacillus anthracis-Like Bacteria Isolated from Wild Great Apes from Côte d'Ivoire and Cameroon

“…strain 2617, and Bacillus sp. strain 153 (26). Sequencing of the SG-749 fragment of B. anthracis strains CI and CA revealed six nucleotide differences compared to classic B. anthracis strains (data not shown), confirming the restriction pattern through the identification of an additional AluI restriction site, which results in restriction fragments of 496 bp, 166 bp, and 89 bp compared to fragments of 662 bp and 89 bp for classic B. anthracis strains.…”

“…AluI restriction of the SG-749 fragment, a randomly amplified polymorphic DNA marker specific for the B. cereus complex, revealed a unique restriction type for all classic B. anthracis strains tested (9,26). However, in B. anthracis strains CI and CA, a different restriction pattern was found, which was identical to the patterns found in the environmental isolates B. cereus Hohenheim, Bacillus sp.…”

“…the rpoB marker had been observed previously for some strains of the B. cereus group (14,26) and was explained by residual nonspecific priming of the B. anthracis-specific primers on the closely related B. cereus genomic sequences.…”

“…It is able to produce a capsule in vivo and in vitro under appropriate conditions (53). However, not a single diagnostic trait appears to be consistent for all B. anthracis isolates (6,30,36), and atypical isolates of B. anthracis and other members of the B. cereus group have been described previously (26). Therefore, the application of molecularly based methods like PCR has become increasingly important for the diagnosis of B. anthracis (9,14,43,46).…”

Rapid and reliable detection of bacterial endospores in environmental samples by diagnostic electron microscopy combined with X-ray microanalysis

“…To check for the universality of the results that were obtained by using spores from Bacillus subtilis, spores of the following bacterial strains were analysed: Bacillus anthracis 527, Bacillus anthracis Stamatin Sokol, Bacillus anthracis Sterne strain 34F2, Bacillus anthracis UDIII-7 (see Klee et al, 2006 for a molecular typology of the different anthrax strains), Bacillus atrophaeus ATCC 9372, Bacillus cereus ATCC 10987, Bacillus stearothermophilus (gke Steri-Record; gke GmbH, Germany), Bacillus thuringiensis (DSM 350), Clostridium botulinum NCTC 7272, and Clostridium difficile NCTC 13366. Spores were produced according to a European standard procedure (prEN 14347:2001 [D]) and stored at 4 °C in double distilled water until use.…”