Genetic variability of Bacillus anthracis and related species

Abstract

We evaluated the abilities of pulsed-field gel electrophoresis (PFGE) and sequences of intergenic spacer regions (ISRs) between two highly conserved genes, 16S-23S rDNA and gyrB-gyrA ISRs, to detect variation in strains of Bacillus anthracis as well as two closely related species, B. cereus ATCC 14579 and B. mycoides ATCC 6462. For each restriction enzyme, (NotI, SfiI, and SmaI), the PFGE banding patterns for three B. anthracis strains (Ames, Vollum, and Sterne) were identical. However, closely related species could be differentiated from B. anthracis and from each other. PCR amplification of the 16S-23S rDNA ISR yielded a 143-to 144-bp fragment, showing identical sequences for B. anthracis strains, one nucleotide deletion between B. cereus and B. anthracis, and 13 nucleotide differences between B. mycoides and B. anthracis. The gyrase ISR sequences (121 bp) in B. anthracis strains were also identical, but those in B. cereus and B. mycoides differed from that in B. anthracis by 1 and 2 nucleotides, respectively, and from each other by only 1 nucleotide. Given the diverse geographic origins of these B. anthracis strains, this species is very homogenous. We conclude that methods such as PFGE and sequences of ISRs may be useful in separating B. anthracis from closely related species, but more sensitive methods are needed for strain identification of B. anthracis.

16S–23S rRNA internal transcribed spacers as molecular markers for the species of the 16S rRNA group I of the genus Bacillus

“…To discriminate the species of the group more clearly, the ITS sequences of B. cereus 31 and B. mycoides 2048 were determined. The sequences were identical to those obtained by Harrel et al [18] (data not shown). The presence of two restriction sites (DraI and HincII) in the sequence of B. mycoides (Fig.…”

“…The sequencing primers were the same as used for ITS-PCR ¢ngerprinting. The sequences obtained were manually aligned with those determined by Harrel et al [18], and a restriction map was determined with the Webcutter (http:// www.medkem.gu.se/cutter/). Restriction analysis of the shortest ITS of B. cereus group isolates was per-formed with the enzymes DraI and HincII (Amersham Pharmacia) in 20 Wl ¢nal volume, following the conditions suggested by the manufacturer.…”

Intriguing diversity ofBacillus anthracisin eastern Poland – the molecular echoes of the past outbreaks

“…Bacillus anthracis is undoubtedly one of the most monomorphous species known. Standard molecular typing methods such as pulsed-field gel electrophoresis (PFGE), PCR amplification of 16S-23S rRNA, as well as gyrA gyrB intergenic spacer regions (ISR) were unable to effectively distinguish B. anthracis isolates [2]. In 1996, the vrrA open reading frame (ORF) containing a variable number (2-6 copies) of 12-bp tandem repeat (VNTR) has been described [3].…”

Restriction site insertion-PCR (RSI-PCR) for rapid discrimination and typing of closely related microbial strains

“…The 16S rDNA [3,14], the 23S rDNA [4] and the spacer between gyrA and gyrB [6] were chosen as target sequences for the RSI-PCR. The primers were designed by taking into consideration the following nucleotide substitutions: (i) T/C (between psychrotolerant Bacillus weihenstephanensis/B.…”

“…mycoides [14] and mesophilic strains of the B. cereus group) at position 981 of the 16S rDNA [14], (iv) A/G (between B. cereus and B. anthracis) at position 1558 of the 23S rDNA [4], (v) G/A (between B. cereus/B. mycoides and B. anthracis) at position 24 of the spacer between gyrB and gyrA [6]. In order to detect length di¡erences between the cut and the un-cut PCR product, primers of at least 20 nucleotides were chosen to amplify fragments not longer than 350 bp.…”