Carbohydrate metabolism differences between subgroup A1 and B2 strains of Bacillus anthracis as assessed by comparative genomics and functional genetics.

In France, Bacillus anthracis subgroup B2 strains do not metabolize starch or glycogen but can use gluconate, whereas subgroup A1 strains show the inverse pattern. Functional genetic analysis revealed that mutations in the amyS and gntK genes encoding an alpha-amylase and a gluconate kinase, respectively, were responsible for these phenotypes.

Phylogeography of Francisella tularensis: global expansion of a highly fit clone.

Francisella tularensis contains several highly pathogenic subspecies, including Francisella tularensis subsp. holarctica, whose distribution is circumpolar in the northern hemisphere. The phylogeography of these subspecies and their subclades was examined using whole-genome single nucleotide polymorphism (SNP) analysis, high-density microarray SNP genotyping, and real-time-PCR-based canonical SNP (canSNP) assays. Almost 30,000 SNPs were identified among 13 whole genomes for phylogenetic analysis. We selected 1,655 SNPs to genotype 95 isolates on a high-density microarray platform. Finally, 23 clade- and subclade-specific canSNPs were identified and used to genotype 496 isolates to establish global geographic genetic patterns. We confirm previous findings concerning the four subspecies and two Francisella tularensis subsp. tularensis subpopulations and identify additional structure within these groups. We identify 11 subclades within F. tularensis subsp. holarctica, including a new, genetically distinct subclade that appears intermediate between Japanese F. tularensis subsp. holarctica isolates and the common F. tularensis subsp. holarctica isolates associated with the radiation event (the B radiation) wherein this subspecies spread throughout the northern hemisphere. Phylogenetic analyses suggest a North American origin for this B-radiation clade and multiple dispersal events between North America and Eurasia. These findings indicate a complex transmission history for F. tularensis subsp. holarctica.

Genotyping of Bacillus anthracis strains based on automated capillary 25-loci multiple locus variable-number tandem repeats analysis.

BACKGROUND: The genome of Bacillus anthracis, the etiological agent of anthrax, is highly monomorphic which makes differentiation between strains difficult. A Multiple Locus Variable-number tandem repeats (VNTR) Analysis (MLVA) assay based on 20 markers was previously described. It has considerable discrimination power, reproducibility, and low cost, especially since the markers proposed can be typed by agarose-gel electrophoresis. However in an emergency situation, faster genotyping and access to representative databases is necessary. RESULTS: Genotyping of B. anthracis reference strains and isolates from France and Italy was done using a 25 loci MLVA assay combining 21 previously described loci and 4 new ones. DNA was amplified in 4 multiplex PCR reactions and the length of the resulting 25 amplicons was estimated by automated capillary electrophoresis. The results were reproducible and the data were consistent with other gel based methods once differences in mobility patterns were taken into account. Some alleles previously unresolved by agarose gel electrophoresis could be resolved by capillary electrophoresis, thus further increasing the assay resolution. One particular locus, Bams30, is the result of a recombination between a 27 bp tandem repeat and a 9 bp tandem repeat. The analysis of the array illustrates the evolution process of tandem repeats. CONCLUSION: In a crisis situation of suspected bioterrorism, standardization, speed and accuracy, together with the availability of reference typing data are important issues, as illustrated by the 2001 anthrax letters event. In this report we describe an upgrade of the previously published MLVA method for genotyping of B. anthracis and apply the method to the typing of French and Italian B. anthracis strain collections. The increased number of markers studied compared to reports using only 8 loci greatly improves the discrimination power of the technique. An Italian strain belonging to the B branch was described, and two new branches, D and E, are proposed. Owing to the upgrading achieved here, precise genotyping can now be produced either by automated capillary electrophoresis, or by the more accessible but slower and for some markers slightly less accurate agarose gel methodology.

Diversity among French Bacillus anthracis isolates.

While outbreaks of animal anthrax zoonoses still regularly occur in France, little is known about the epidemiology links between them. We have used the eight-locus multilocus variable-number tandem repeat analysis typing technique against a collection of 50 Bacillus anthracis isolates from France. There were eight distinct genotypes belonging to two dissimilar genetic clusters. Regional strain patterns were observed, with the B2 genotypes prevalent in southern France and the A1a genotypes found only in northern France.

Antibiotic susceptibilities of 96 isolates of Bacillus anthracis isolated in France between 1994 and 2000.

Ninety-six isolates of Bacillus anthracis recovered in France between 1994 and 2000 were tested for their susceptibilities to 25 different antibiotics. Resistance to penicillin G and amoxicillin was 11.5%. All of the isolates were resistant to cotrimoxazole and susceptible to doxycycline, ciprofloxacin, pefloxacin, levofloxacin, teicoplanin, vancomycin, clindamycin, imipenem, and rifampin.

Variation in rRNA operon number as revealed by ribotyping of Bacillus anthracis strains.

Ribotyping of various Bacillus strains with one restriction enzyme (AccI) revealed significant similarity between Bacillus anthracis strains, Bacillus thuringiensis and Bacillus cereus strains, which are all members of the Bacillus cereus group. A further ribotyping study of 10 virulent and 8 attenuated B. anthracis strains, using 4 endonucleases and both 23S and 16S probes independently, was performed. The discrimination index D of Hunter and Gaston showed that the best combination for future large-scale ribotyping studies would be either the combination of AccI and 23S, or that of EcoRI and 16S. Depending on the B. anthracis strain analyzed 10 or 11 rRNA operons were found. In all cases, many strains were grouped into 2 to 3 patterns. Attenuated strains, including a laboratory-cured strain, yielded aberrant patterns.