318
Anthrax (Bacillus anthracis), Molecular Biology of
isolates. Looking at a specifc gene impor-
tant For virulence, Price et al. Found only 5
single base pair changes in
pagA
between
26
B. anthracis
strains,
using
complete
DNA sequence data For the gene. ±urther,
LR REP-PCR analysis oF 105
B. anthracis
strains oF diverse geographical origins al-
lowed the strains to be classifed into only
fve groups. A Few explanations For the
monomorphic nature oF
B. anthracis
have
been proposed. It may be that anthrax has
a slow rate oF evolution and divergence, al-
though this is thought to be unlikely. Other
possible explanations include a very recent
origin For this species, giving it little time
to evolve and diverge, or a population bot-
tleneck that reduced its genetic diversity in
the recent past.
So Far, the best screening method For dis-
tinguishing
B. anthracis
strains has been
MLVA.
In
one
study,
MLVA
oF
426
B. anthracis
isolates using 8 VNTR loci
identifed 89 distinct genotypes that could
be studied in terms oF their genetic re-
latedness. MLVA was used For molecular
subtyping oF
B. anthracis
in the 2001 an-
thrax outbreak. All 135 isolates From the
outbreak had identical MLVA character-
istics. Rather than being useFul For dis-
tinguishing this strain From others, MLVA
characterization turned out to be useFul For
the confrmation that each case was caused
by the same
B. anthracis
strain and For sep-
arating these results From environmental
contamination. During this investigation,
it became clear that MLVA was not suFf-
cient to determine the origin oF the attack
strain. On the basis oF MLVA analysis,
the strain oF
B. anthracis
isolated in the
attacks appeared to be identical to the
Ames strain that is used by many research
labs, so this technique did not provide
the resolution necessary For the genetic
investigation.
As the
B. anthracis
genome sequence
was nearing completion at this time, re-
search
turned
to
comparing
the
DNA
sequence oF the
B. anthracis
strain isolated
From a patient who contracted anthrax in
±lorida with that oF the Porton isolate
oF the Ames strain, which was used to
generate the standard
B. anthracis
genome
sequence. Even at this level oF resolution,
only Four diFFerences were Found between
the chromosomal sequences oF the ±lorida
and Porton isolates. As For plasmid se-
quences, the Porton isolate has been cured
oF its plasmids, so the pXO1 and pXO2
sequences oF the attack isolate were com-
pared with those oF the Sterne (pXO1) and
Pasteur (pXO2) strains. A total oF 38 sin-
gle nucleotide polymorphisms, 8 VNTRs,
3 insertion/deletion polymorphisms, and
2 inversions were identifed. One oF the
inversions was Found on pXO1 and was
centered on the pathogenicity island. In
Fact, the ±lorida isolate contained a mix-
ture oF both orientations oF this plasmid
segment, confrming the Fact that this
DNA segment can invert, as had been
suggested previously.
The polymorphisms identifed in this
sequence
comparison
were
then
used
in an attempt to distinguish fve addi-
tional Ames isolates that previously had
been indistinguishable. When plasmid se-
quences were compared, one laboratory
isolate and a strain isolated From an in-
Fected goat did have a small number oF
plasmid sequence diFFerences that allowed
them to be classifed separately. The poly-
morphisms identifed in this study also
enabled researchers to readily distinguish
two non-Ames
B. anthracis
strains From
the Ames strains. However, the remaining
three Ames lab isolates and the ±lorida
strain could not be distinguished From
each other, on the basis oF either the chro-
mosomal or plasmid sequences.
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