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Journal of Clinical Microbiology, March 2000, p. 1231-1234, Vol. 38, No. 3
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Detection of a Previously Unamplified Spacer
within the DR Locus of Mycobacterium tuberculosis:
Epidemiological Implications
Ingrid
Filliol,
Christophe
Sola, and
Nalin
Rastogi*
Unité de la Tuberculose et des
Mycobactéries, Institut Pasteur, F-97165 Pointe à Pitre
Cedex, Guadeloupe
Received 28 September 1999/Accepted 8 December 1999
 |
ABSTRACT |
Spoligotyping, a method based on the variability of distribution of
the 43 inter-direct repeat (DR) spacers of Mycobacterium tuberculosis and Mycobacterium bovis BCG, is useful
to study the molecular epidemiology of bovine and human tuberculosis.
Recently, a major family of M. tuberculosis clinical
isolates named the Haarlem family, which did not contain spacers 31 and
33 to 36, was reported in a multicenter study. Independently, a data
bank containing all the published spoligotypes showed that the two most
prevalent spoligotypes in the world differed only by the presence or
absence of spacer 31. A careful analysis of the DR locus sequence led
us to hypothesize that spacer 31 may not have been amplified in some
isolates with the primer sets DRa and DRb currently used for
spoligotyping. Consequently, a modified spoligotyping method based on
different combinations of the 36-bp DR and IS6110 primers
was devised that was able to discriminate between the left and the
right parts of the DR locus and demonstrated the presence of the
previously unamplified spacer 31 for some of the clinical isolates. By
analogy, we suggest that a single-spacer difference in some
epidemiologically linked cases of tuberculosis may simply arise due to
the insertion of an extra copy of IS6110 within the DR
locus, leading to its asymmetrical disruption and subsequent lack of
the DRa or DRb targets. The influence of the IS6110
preferential insertion sites within the DR locus on spoligotyping results should be further investigated.
| |
INTRODUCTION |
Originally described in
Mycobacterium bovis (12), the direct repeat (DR)
locus of the Mycobacterium tuberculosis complex genome
consists of a variable number of copies of a 36-bp DR interspersed with
specific spacer sequences, which has been used to develop a rapid
molecular fingerprinting technique called spoligotyping (14), often associated with the IS6110
restriction fragment-length polymorphism (RFLP) for epidemiological
purposes (9, 10). A recent multicenter study involving
comparison of various molecular markers for the study of epidemiology
of M. tuberculosis concluded that, along with other
techniques, spoligotyping was a method of choice for reproducible
PCR-based genotyping (15). In parallel, this study also
underlined that several separate genotype families within the M. tuberculosis complex could be recognized on the basis of multiple
genetic markers, namely, the Beijing, Africa, and Haarlem families
(15). A careful examination of these families showed that
the Haarlem family was always characterized by the lack of spacer 31. This observation, along with the fact that epidemiologically related
M. tuberculosis clinical isolates may sometimes differ by a
single spacer (14), and our independent observations that
the two most prevalent spoligotypes around the world (named types 53 and 50 in Fig. 2) differ by the presence or absence of spacer 31 (18), prompted us to have a closer look at this spacer by
using a modified spoligotyping method.
Bacterial isolates.
All of the experimentally studied M. tuberculosis isolates were obtained from the collection of the
Pasteur Institute of Guadeloupe and were isolated and identified
locally by standard mycobacteriological procedures.
Spoligotyping.
Standard spoligotyping was performed with the
DRa and DRb primers as described previously (14). For the
left-right (L-R) spoligotyping, primers IS6 and IS3 (4, 5)
were used in combination with biotinylated DRb or biotinylated DRa to
amplify either the left or the right part of the IS6110
boundaries or the central part of the DR locus for strains harboring
two IS6110 copies within the DR locus. The following primers
were used: DRa, GGTTTTGGGTCTGACGAC (14); DRb,
CCGAGAGGGGACGGAAAC (14); IS3,
GCTGCCTACTACGCTCAAC (4, 5); and IS6,
CAAGTAGACGGGCGACCTC (4, 5). The primer combinations are shown in Table 1. The
PCR protocol and the conditions for membrane preparation,
hybridization, and detection were the same as those reported earlier
(14), except for the elongation time, which was set to 3 min.
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TABLE 1.
Primer combinations used in this study and expected
results according to the number of IS6110 copies inserted
within the DR locus of M. tuberculosis
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|
IS6110 and DRr hybridization.
IS6110
and DRr RFLPs were performed according to published procedures
(12, 21) by either direct or indirect labeling. Detection by
the enhanced chemoluminescence (ECL) systems was performed according to
the supplier's protocols (Amersham, Buckinghamshire, United Kingdom).
Modified LR spoligotyping: basis for development and results.
The spoligotyping results for M. tuberculosis isolates from
our laboratory were pooled with those published previously to construct
a database containing 993 individual patterns. The nomenclature of
specific spoligotypes numbered 1 to 69 was recently described (18), to which new types 70 to 72 were added during the
course of this investigation. The description of these spoligotypes is available on request. Statistical analysis of spacer distribution on
these 993 isolates underlined a region (spacers 21 to 32) that is
characterized by successive decreases for some of the spacers, particularly spacer 31 (results not shown). Based on the available sequence of the DR locus (11), a physical map was redrawn
for a strain containing two copies of IS6110 (Fig.
1), with one copy being inserted after
the spacer 31. As shown in Fig. 1, a second copy inserted after spacer
31 may lead to an unequal split of the DR, leading to the consequent
loss of the DRa primer target, which in turn will not permit the
amplification of spacer 31 with primers DRa and DRb, since there was no
target for primer DRa at the right side of this spacer. Inversely, the
bordering spacers 24 and 25 would be detected (Fig. 1). To
experimentally verify this hypothesis, we searched in our collection
for isolates lacking spacer 31 after routine spoligotyping (types 3, 50, and 67 [Fig. 2]). Type 50, which represents the second most
frequent spoligotype in our database and is overrepresented in our
region, differs only by the lack of spacer 31 as compared to type 53, another very common spoligotype (18). The
PvuII-DR RFLPs of these types of isolates lacking spacer 31 (spoligotype patterns 3, 50, and 67) mostly showed the presence of
three DRr-hybridizing bands (Fig. 2),
suggesting the presence of a second IS6110 copy within the
DR locus, very likely inserted asymmetrically after spacer 31 (Fig. 1).
This assumption is strengthened by the observation that nearly 82% of
the strains of type 53 (universal type; presence of all spacers except
33 to 36) (18) harbored only two DRr-hybridizing bands (Fig.
2). Assessment of the percentage of strains of spoligotype 50 harboring
a second copy of IS6110 inserted asymmetrically and located
between spacers 31 and 25 of the DR locus is currently in progress.
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FIG. 1.
Schematic representation of the DR locus of M. tuberculosis with an enlargement of the two IS6110
insertion sites. The top of the figure shows the general organization
of the DR locus, whereas the bottom of the figure is an enlargement
showing the flanking regions of IS6110 insertions. In the
bottom part, the left-hand copy of IS6110 is inserted
exactly in the middle of the DRr sequence between spacers 24 and 25, splitting the DRr into two equal 18-bp fragments which serve as PCR
targets for primers DRa and DRb and give a positive hybridization
signal for spacers 24 and 25. On the other hand, an asymmetrical
disruption of the DRr sequence due to the insertion of a second copy of
IS6110 in the right-hand side of the DR locus between
spacers 31 and 25 results in two unequal parts in such a way that only
an 8-bp fragment of the DRa target is conserved toward spacer 31, not
enough for successful amplification and subsequent detection of this
spacer.
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FIG. 2.
Correlation between spoligotypes and PvuII-DR
RFLP results showing a link between the absence of spacer 31, the
presence of a third DRr band, and the consequent presence of a second
copy of IS6110 inserted within the DR locus.
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|
The modified L-R spoligotyping method, which is theoretically able to
selectively amplify the right or the left border of
the
IS
6110 sequence(s) of the DR locus, was initially tested on
reference strains of
M. tuberculosis (H37Rv) and
M. bovis BCG
(BCG-Pasteur), known to contain only a single
IS
6110 copy linked
to the DR. As expected, each of the
reference strains was split
into two distinct left and right
spoligotyping patterns by L-R
spoligotyping (Fig.
3 [data for BCG not shown]), confirming
that
the IS
6110 copy was indeed inserted immediately after
spacer 24.
Accordingly, the primer combinations C and D resulted in a
strongly
positive spoligotyping result in all cases, each distinctly
covering
half of the positive control as amplified with primer set A. Primer
set B always gave negative results (Fig.
3), whereas primer set
E produced no signals or weak and nonreproducible signals (results
not
shown). These results conclusively showed that primer sets
C and D were
able to discriminate the right and the left parts
of the DR region in
the reference
M. tuberculosis and
M. bovis BCG
strains. However, due to the lesser sensitivity of the L-R
spoligotyping technique (explained by the smaller amount of PCR
targets
[Fig.
1]), spacers located on the extremities of the DR
locus were
sometimes undetected (results not shown).
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FIG. 3.
Schematic representation of results obtained by L-R
spoligotyping of M. tuberculosis H37Rv and by the routine
spoligotyping method of representative clinical isolates that lacked
spacer 31. The primer sets A, B, C, and D have been detailed in the
text and in Table 1.
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|
This experimental approach was later extended to investigate the DR
loci of various clinical isolates lacking spacer 31 that
were typed in
parallel by
PvuII-DR RFLP; a total of 12 isolates
selected
comprised either strains found to harbor a single copy
of
IS
6110 within the DR locus (types 2, 72, 62, and 47) or
those
found to possess two copies within the DR locus (types 50, 3,
and
var3; the latter differed from type 3 by absence of a single
spacer,
spacer 40). Representative experimental results for some
of the
isolates are shown in Fig.
3; for types 2, 72, 62, and
47, the primer
sets C and D showed a split in position 24 resulting
in distinct right
and left amplifications of the DR locus starting
from the point of
insertion, whereas for types 50 and var3, an
additional overlapping of
spacers 25 to 31 by the primer set D
corresponded to the insertion of
the second IS
6110 copy (Table
1 and Fig.
3). The fact that
the left part of the DR pattern
was absent in the type 2 isolate when
primer set D was used suggests
that this portion was effectively
missing and that a single copy
of IS
6110 was inserted
immediately after spacer 31 (Fig.
1). Finally,
the observation that
previously undetected spacer 31 was revealed
with primer set D in types
50 and var3 was in agreement with an
asymmetric insertion of the second
copy of IS
6110 within the DR
immediately after spacer 31 (Fig.
1 and
3).
| |
DISCUSSION |
In M. tuberculosis, various repetitive loci have been
described, among which are numerous insertion sequences, such as
IS6110 (20), as well as the variable number
tandem repeats (8, 13), the DR (12), and the
polymorphic GC-rich sequence (16). All of these markers have
recently been used in a multicenter study of M. tuberculosis
epidemiology (15). Spoligotyping based on the variability of
inter-DR sequences has been proposed as a method of choice for
reproducible PCR-based genotyping (15). In this context, the
existence of preferential IS6110 insertion sites in the DR
locus of M. tuberculosis (4, 7) may indirectly contribute to the variability of spoligotyping results, which may have
important epidemiological consequences. Moreover, clinical isolates of
M. tuberculosis differing by a single DR spacer or a single
IS6110 copy that were either epidemiologically related (14) or were serial isolates from the same patient
(17) have been reported. Consequently, we decided to
investigate the fine structure of the DR locus in relation to the
insertion of IS6110 and its consequences in terms of
spoligotyping results.
In this investigation, a modified spoligotyping method based on
different combinations of DRr and IS6110 primers was able to
discriminate between the left and the right parts of the DR locus and
demonstrated the presence of previously undetected spacer 31 for some
of the clinical isolates studied. Although limited to spacer 31 in this
study, this interpretation of a missing spacer provides us with a new
interpretation for a single negative hybridization spot in
spoligotyping. However, this interpretation of the absence of spacer 31 does not mean that all single-spacer absences are caused by an
asymmetrical IS6110 insertion: e.g., the lack of spacers 3, 9, and 16 for M. bovis BCG is not linked to any
IS6110 insertion (11).
The study of preferential IS6110 transposition sites may
have important applications in the study of the evolutionary genetics of M. tuberculosis, particularly because the DR locus is
likely to evolve more slowly than IS6110, whose evolution
has recently been shown to be too fast for the evolution of
multi-drug-resistant strains of M. tuberculosis to be
monitored efficiently (1). A recent study has evaluated the
rate of change of IS6110 RFLP patterns in serial M. tuberculosis patient isolates as being 3.2 years (3).
Seen from an evolutionary point of view, various mechanisms, such as
the transposition of IS6110 and other mobile genetic
elements (2, 5, 19), homologous recombination (11), and slipped-strand mispairing (15), are
believed to drive genomic diversification in M. tuberculosis. Our study underlines the role of IS6110
transpositional events in the evolution of the DR locus. Alternatively,
a one-spacer difference may also be due to mutations in the spacer
sequence itself; however, if the DR locus is neutral (no environmental
and/or selective pressure), point mutations in this locus should be
extremely rare, as shown by the restricted allelic diversity shown in
M. tuberculosis (19). More likely,
IS6110-mediated deletions and insertions and homologous recombination may have driven the evolution of the DR locus (6, 11). Our experimental approach may increase the discriminatory power of spoligotyping to study isolates differing by a single missing
spacer and thus result in a better evaluation of strain relatedness in
epidemiological studies of tuberculosis.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Unité de
la Tuberculose et des Mycobactéries, Institut Pasteur, Morne
Jolivière, BP 484, F-97165 Pointe-à-Pitre Cedex,
Guadeloupe. Phone: 590-893-881. Fax: 590-893-880. E-mail:
rastogi{at}ipagua.gp.
| |
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Journal of Clinical Microbiology, March 2000, p. 1231-1234, Vol. 38, No. 3
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
