Anthrax, caused by the bacterium Bacillus anthracis, is a disease of historical and current importance that is found throughout the world. The basis of its historical transmission is anecdotal and ...its true global population structure has remained largely cryptic. Seven diverse B. anthracis strains were whole-genome sequenced to identify rare single nucleotide polymorphisms (SNPs), followed by phylogenetic reconstruction of these characters onto an evolutionary model. This analysis identified SNPs that define the major clonal lineages within the species. These SNPs, in concert with 15 variable number tandem repeat (VNTR) markers, were used to subtype a collection of 1,033 B. anthracis isolates from 42 countries to create an extensive genotype data set. These analyses subdivided the isolates into three previously recognized major lineages (A, B, and C), with further subdivision into 12 clonal sub-lineages or sub-groups and, finally, 221 unique MLVA15 genotypes. This rare genomic variation was used to document the evolutionary progression of B. anthracis and to establish global patterns of diversity. Isolates in the A lineage are widely dispersed globally, whereas the B and C lineages occur on more restricted spatial scales. Molecular clock models based upon genome-wide synonymous substitutions indicate there was a massive radiation of the A lineage that occurred in the mid-Holocene (3,064-6,127 ybp). On more recent temporal scales, the global population structure of B. anthracis reflects colonial-era importation of specific genotypes from the Old World into the New World, as well as the repeated industrial importation of diverse genotypes into developed countries via spore-contaminated animal products. These findings indicate humans have played an important role in the evolution of anthrax by increasing the proliferation and dispersal of this now global disease. Finally, the value of global genotypic analysis for investigating bioterrorist-mediated outbreaks of anthrax is demonstrated.
Bacillus anthracis is a bacterial pathogen of great importance, both historically and in the present. Despite this importance, many questions remain regarding defending against its use as a ...biological weapon, the bacteria's variation in virulence, and its epidemiology in nature. Using Etest strips (AB BIODISK, Solna, Sweden) to measure the minimum inhibition concentrations (MICs), 25 genetically diverse isolates of B. anthracis were tested to determine their susceptibility to seven clinically relevant antimicrobial agents. Using the National Committee for Clinical Laboratory Standards (NCCLS) MIC breakpoints for staphylococci, three isolates were found to be resistant to penicillin and negative for beta-lactamase production. From a group of investigations, results indicated B. anthracis virulence is related to clonality and the copy numbers per cell of the virulence plasmids, pXO1 and pXO2. Isolates were characterized with respect to their plasmid copy number (pXO1/2) using a novel method of quantitative PCR and the numbers differ greatly from previous reports. Anthrax Vaccine with Adjuvant (AVA) vaccinated guinea pigs were challenged with 20 B. anthracis strains representative of worldwide genetic diversity. A virulence model was constructed by combining the survival, plasmid copy number, and genotyping (based on multilocus variable number tandem repeat analysis typing) data of each isolate. The model obtained was validated using a randomly chosen set of 12 B. anthracis isolates and verified model robustness. Two carcass disposal methods, incineration and burial, are recommended to decrease or prevent environmental spore contamination. The extent of contamination from an anthrax carcass is almost totally unknown in either method of disposal. Studies of environmental contamination by spores of B. anthracis from infected carcasses have only recently been possible because of new technologies. A method utilizing real-time quantitative PCR was developed to quantitate B. anthracis in environmental samples. Absolute quantitation was made possible by the use of clones. This method has allowed the evaluation of the environmental contamination by the different carcass disposal methods and by scavenging of the carcass. The results support the complete burning of a carcass soon after death as the method choice to decrease environmental contamination for the disposal of anthrax affected carcasses.
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