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given species, hypervariable, and provides epidemiologically and/or phyloge-
netically robust data. Repetitive DNA sequences in bacterial genomes offer such
possibilities at both the species and subspecies level. In fact, repetitive DNA and
VNTRs in particular have been used since the early 1990s in human genetics
and forensic studies
(7,8)
. More recently, VNTR analysis has been employed
in a number of studies of pathogenic organisms, including
S. aureus
,
Mycobac-
terium tuberculosis
,
Bacillus anthracis,
and
Yersinia pestis
(9,10,11,12)
.
Numerous prokaryotic genomes contain monomeric sequences (repeat units)
that repeat periodically and are arranged in a head-to-tail configuration. These
DNA regions are catalogued on the basis of their repeat unit size, which ranges
from a single nucleotide to sequences >100 bp in length. Prokaryotic genomes
generally have microsatellite DNA that has repeat units ranging in size from
1-10 bp. These sequences are abundant throughout most bacterial genomes
and have been shown to play a significant role in both transcriptional and
translational control of gene expression. Many of the microsatellite sequences,
as well as larger minisatellite DNA (which has repeat units ranging in size
from 10 to 100), are commonly referred to as VNTRs. Such repeats have been
found in intergenic regions, gene expression control regions, and within open
reading frames
(13,14)
.
In
S. aureus
, minisatellite VNTRs have been identified in two well-conserved
genes: protein A and coagulase. These genes are unique to
S. aureus
and
contain an in-frame region composed of VNTRs. Protein A (
spa
) has a 24-bp
repeating unit near the 3´ terminus (
see
Fig. 1A
), and coagulase (
coa
) has
an 81-bp repeating unit in the 5´ region
(10,15)
. Genetic alterations in these
repetitive sequences include point mutations as well as intragenic recombination
events that presumably arise by slipped-strand mispairing during chromosomal
replication
(16)
and, in the case of protein A repeats, result in a high degree
of genetic polymorphism. In contrast to most VNTR analysis where the total
number of repeats is counted, in the case of coagulase and protein A, the repeat
units differ in DNA sequence, and the overall genotypic diversity is reflected
in repeat content, number of repeats, and organization
(10,15,17,18)
.
A comparative genotyping study conducted by the Centers for Disease
Control and Prevention evaluated 8 methods using a blinded collection of
S. aureus
strains from three separate outbreaks. The blinded samples included
59 staphylococci, of which 58 were
S. aureus
, and 37 were methicillin resistant;
29 isolates had been previously grouped into four identifiable clusters based on
sound epidemiological links
(10)
. When compared to PFGE, ribotyping, and
other genotyping methods,
spa
typing produced results better than the mean
score of 25 correct classifications and 5 misclassifications and showed that it
could correctly group epidemiologically related strains
(10,19)
. Analysis using
spa
and
coa
as target sequences to sub-speciate
S. aureus
isolates showed
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