Use of Long-Range Repetitive Element Polymorphism-PCR To Differentiate Bacillus anthracis Strains

Abstract

The genome of Bacillus anthracis is extremely monomorphic, and thus individual strains have often proven to be recalcitrant to differentiation at the molecular level. Long-range repetitive element polymorphism-PCR (LR REP-PCR) was used to differentiate various B. anthracis strains. A single PCR primer derived from a repetitive DNA element was able to amplify variable segments of a bacterial genome as large as 10 kb. We were able to characterize five genetically distinct groups by examining 105 B. anthracis strains of diverse geographical origins. All B. anthracis strains produced fingerprints comprising seven to eight bands, referred to as "skeleton" bands, while one to three "diagnostic" bands differentiated between B. anthracis strains. LR REP-PCR fingerprints of B. anthracis strains showed very little in common with those of other closely related species such as B. cereus, B. thuringiensis, and B. mycoides, suggesting relative heterogeneity among the non-B. anthracis strains. Fingerprints from transitional non-B. anthracis strains, which possessed the B. anthracis chromosomal marker Ba813, scarcely resembled those observed for any of the five distinct B. anthracis groups that we have identified. The LR REP-PCR method described in this report provides a simple means of differentiating B. anthracis strains. Anthrax is often a fatal bacterial infection that results afterBacillus anthracis spores enter a suitable host either via inhalation or ingestion or through the contamination of a wound or abrasion (8). Since death occurs within a few days after the onset of symptoms, B. anthracis presents a very serious threat if deployed as a biological weapon (13). Naturally occurring human B. anthracis infections are rare and generally result from contact with contaminated animals or their products (5).Due to the highly monomorphic nature of B. anthracis, differentiation of strains from diverse origins has proven to be very difficult. DNA fingerprinting of B. anthracis strains using ribotyping has shown some strain-to-strain variations, presumably due to changes in the organization and number of rrn loci (18). However, due to insufficient numbers of variant strains, ribotyping was found to have limited discriminatory potential.Neither arbitrarily-primed PCR fingerprints (1) nor amplified fragment length polymorphisms fingerprints (15) have been useful in discriminating between B. anthracis strains, although some genetic diversity has been observed. In this case, the diversity was due to the presence of a variable-number tandem repeat (14) sequence in the open reading frame (ORF) vrrA. Recently, seven additional loci containing these sequences were discovered in B. anthracis, leading to the identification of six genetically distinct groups of B. anthracis (16).The presence of multiple copies of highly conserved repetitive extragenic palindromic (REP) sequences in DNA (12) has been identified in a number of microorganisms (28). The technique of REP-PCR takes advantage of the fact that multiple copies of these REP sequences...

PCR Assay of the groEL Gene for Detection and Differentiation of Bacillus cereus Group Cells

“…The results presented in this study demonstrated that B. anthracis is genetically closely related to several B. cereus food poisoning and environmental strains. Perhaps the difficulty in differentiating B. anthracis strains is because B. anthracis is actually a subspecies of the much more polymorphic B. cereus lineage (7). The phenotypic differences may be altered by horizontal transfer of plasmids (24).…”

Bacillus anthracis Diverges from Related Clades of the Bacillus cereus Group in 16S-23S Ribosomal DNA Intergenic Transcribed Spacers Containing tRNA Genes

“…These differences can be observed in the ribosomal operon organization (35,36,39) or at the level of the ITS, as in strain Davis TE702 (14,24,35) or the strains isolated from the Pyrenees and Alps (37) during the 1994 and 1997 outbreaks (14). The few variations observed in B. anthracis by ITS homoduplex-heteroduplex polymorphism analysis (14) and sequencing of ITS containing tDNA mirror the whole-genome polymorphisms identified by amplified fragment length polymorphism (27,28), long-range repetitive elements PCR (8), and, recently, whole-genome sequencing (42). These data on ITS sequences confirm the observation that B. cereus and B. thuringiensis cannot be distinguished easily, apart from the entomocidal genotype and phenotype.…”

Existence of Separate Domains in Lysin PlyG for Recognizing Bacillus anthracis Spores and Vegetative Cells

“…The experimenters were equipped with masks, gloves, and exposure suits. Bacillus anthracis A16 was cultured in standard LB medium and harvested as vegetative cells, as reported previously (2,7,50). The washed vegetative cells were heat treated for 5 min under boiling water before staining with the truncated proteins and the synthetic peptides.…”