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Previous Article | Next Article 
Journal of Clinical Microbiology, April 2001, p. 1595-1599, Vol. 39, No. 4
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.4.1595-1599.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Use of Spoligotyping To Study the Evolution of the
Direct Repeat Locus by IS6110 Transposition in
Mycobacterium tuberculosis
Eric
Legrand,
Ingrid
Filliol,
Christophe
Sola, and
Nalin
Rastogi*
Unité de la Tuberculose et des
Mycobactéries, Institut Pasteur de Guadeloupe, 97165 Pointe-à-Pitre Cedex, Guadeloupe
Received 12 October 2000/Returned for modification 19 December
2000/Accepted 30 January 2001
 |
ABSTRACT |
Based on the variability of 43 spacers within the direct repeat
(DR) locus of Mycobacterium tuberculosis complex organisms, spoligotyping is a rapid method that aids in the study of the epidemiology of tuberculosis. It was recently hypothesized that despite
its presence in the DR locus, spacer 31 could not be amplified in
M. tuberculosis clinical isolates belonging to spoligotype 50 due to the insertion of an extra copy of IS6110 between
spacers 31 and 32 that could lead to an asymmetrical split of the
primer targets (I. Filliol, C. Sola, and N. Rastogi, J. Clin.
Microbiol. 38:1231-1234, 2000). In the present investigation, previous
observations were extended to 25 clinical isolates of type 50 showing
that the primer set IS6-DRb that selectively amplified the left and central DR regions was indeed able to demonstrate the presence of
spacer 31. IS6110-restriction fragment length polymorphism (RFLP) and DR-RFLP showed that type 50 isolates were characterized by
the presence of two copies of IS6110 associated with the DR locus and an additional double IS6110 band of 1.4 kb. The
primer set IS3-IS6 was then used to selectively amplify a 750-bp
inter-IS6110 fragment within the DR locus. The sequencing
of the central DR region corroborated our previous findings and showed
that the absence of spacer 31 among the type 50 isolates was due to the asymmetric insertion of an extra copy of IS6110 between
spacers 31 and 32, leading to an unequal split of the DRa-DRb target
into two portions, of 6 and 30 bp, respectively. These results show that the DR locus constitutes an ideal IS6110 preferential
locus (ipl), permitting the insertion of two or more copies
of IS6110, and provide new clues for epidemiological and
phylogenetic interpretation of changes in IS6110-RFLP and
spoligotyping profiles.
| |
TEXT |
Among DNA fingerprinting techniques
that permit the investigation of patient-to-patient transmission of
tubercle bacilli, both IS6110-restriction fragment length
polymorphism (RFLP) and spoligotyping constitute important molecular
tools to study tuberculosis epidemiology (15). However, in
contrast to IS6110-RFLP, which remains a cumbersome
technique requiring large amounts of bacterial DNA, PCR-based
spoligotyping is an easy and rapid method that can be easily applied in
a routine microbiological laboratory and is useful for both detection
and strain differentiation within the Mycobacterium
tuberculosis complex (11). Based on the variability of the inter-direct repeat (DR) spacers, this methodology was originally described for a set of 43 spacers (37 from M. tuberculosis H37Rv and 6 from Mycobacterium bovis BCG
[11]). Among 51 new spacers evaluated recently, 26 were
found to be specific to Mycobacterium canettii
(17), which is a newly recognized species in the M. tuberculosis complex (13, 19).
In a previous work we showed that two of the most prevalent
spoligotypes in the world differed only by the absence or presence of
spacer 31 (spoligotype patterns 50 and 53, respectively); we hypothesized that despite its presence among the isolates of type 50, spacer 31 may not have been amplified with the primer set DRa-DRb
because of the insertion of an extra copy of IS6110 within the DR locus leading to its asymmetrical split and the subsequent lack
of the DRa target (6). Using appropriate primer
combinations and subsequent sequencing of the central DR region within
the DR locus, the present investigation confirms the postulated
mechanism by which asymmetric insertion of IS6110 in a DR
can eliminate a primer site for the adjacent spacer and thus alter the spoligotype.
Spoligotyping.
All 25 strains (isolates 94072, 94098, 94126, 95013, 95048, 95069, 96020, 96027, 96042, 96060, 96106, 96110, 96121, 96128, 96135, 96136, 96137, 96139, 98042, 98045, 98049, IPC14, IPC22, IPC26, and IPC107) used in this study were isolated at the Institut Pasteur from clinical specimens from patients residing in Guadeloupe or
were received for identification and drug-susceptibility testing from
neighboring Martinique and French Guiana. The isolates were grown on
Löwenstein-Jensen slants at 37°C, and DNA was prepared using
the cetyl-trimethyl-ammonium bromide method (18). Standard spoligotyping was performed by using biotinylated (biot) DRa and DRb.
The left-right (LR) spoligotyping was performed by using primer sets
(biot)DRa-IS3, (biot)IS6-DRb, and (biot)IS6-IS3, which resulted in
the amplification of the right and central DR spacers, the left and
central DR spacers, and the central DR spacers, respectively. The
primers (sequences) used were DRa (GGTTTTGGGTCTGACGAC), DRb (CCGAGAGGGGACGGAAAC), IS3 (GCTGCCTACTACGCTCAAC),
and IS6 (CAAGTAGACGGGCGACCTC).
IS6110- and DR-RFLP.
The separation of
PvuII-digested DNA restriction fragments was performed on a
0.8% agarose gel (Gibco-BRL Life Technologies, Cergy-Pontoise, France)
in 1× Tris-acetate-EDTA buffer (40 mM Tris-acetate, 1 mM EDTA, pH 8.0)
followed by Southern blot hybridization using the IS6110 and
the DR-right (DR-r) probes as described previously (10,
18). The chemiluminescence-based detection of bands was performed using the ECL RPN 3000 kit for IS6110-RFLP and the
ECL RPN 2130 kit for DR-RFLP (Amersham, Buckinghamshire, United
Kingdom), according to the manufacturer's instructions.
Inter-IS6110 PCR and sequencing.
Inter-IS6110 PCR using primers IS3 and IS6 was performed as
previously described (3, 4). The PCR product was loaded on
a 2% (wt/vol) low-melting-temperature agarose gel (Gibco-BRL Life
Technologies), run in 1× Tris-acetate-EDTA buffer, and detected by
ethidium bromide staining. A 100-bp ladder (Pharmacia-Biotech, Uppsala,
Sweden) served as an external molecular size marker. The images were
video captured and analyzed using Gel-Analyst software (Bioprobe
Systems, Montreuil, France). A specific IS3-IS6 PCR-amplified fragment
of about 750 bp was purified on Qiagen columns (QIAquick Gel Extraction
Kit; Qiagen S.A., Courtaboeuf, France) and used for both standard and
LR spoligotyping. Direct sequencing of the purified 100-ng PCR product
was performed using the IS3 and IS6 primers and Thermo-Sequenase dye
terminator kits Cy5.0 and Cy5.5 (AP-Biotech, Piscataway, N.J.) on an
Opengene Long Read Tower sequencing system (Visible Genetics Inc.,
Toronto, Canada) or, alternatively, by using the BigDye Terminators
premix on an Applied Biosystems 373XL sequencer according to the
manufacturer's instructions (Applied Biosystems, Foster City, Calif.).
In the latter case, the sequencing was subcontracted to Genome-Express (Genome-Express, Grenoble, France).
Results and discussion.
First described for M. bovis and later for the members of the M. tuberculosis
complex (10), the DR locus is composed of multiple 36-bp
DR copies interspersed by nonrepetitive short sequences (inter-DR or
spacers) of about equal length (11). The DR locus may
contain one or two IS6110 sequences that usually occur in tandem (2, 8, 9). A careful examination of the DR locus sequencing data by Groenen et al. (8) revealed that an
IS6110 copy may be inserted either symmetrically, with a
resulting split of the DR flanking IS6110 into two equal
portions, or asymmetrically, with portions of 6 and 30 bp. As we will
see below, this little detail may have important consequences on the
spoligotyping patterns of one of the most widely represented groups of
M. tuberculosis clinical isolates (type 50) which is
ubiquitous throughout the world (16).
All of the type 50 isolates were characterized by the absence of spacer
31 after routine spoligotyping using primers (biot)DRa and DRb
(Fig. 1A); however, spacer 31 was
effectively revealed by LR spoligotyping using primers (biot)IS6 and
DRb (Fig. 1B). IS6110-RFLP showed that all of the type 50 isolates were characterized by the presence of a double band of 1.4 kb,
a single copy at 1.8 kb, and nearly always two other copies, at 3.6 and
4.3 kb (Fig. 1C). To investigate the number of IS6110
elements within or near the DR locus, the
PvuII-IS6110 blots were rehybridized with
oligonucleotide probe DR-r as described previously (10).
In all the strains tested, the DR-r probe uniformly hybridized with the
1.8-, 3.6-, and 4.3-kb PvuII fragments. We conclude that the
type 50 isolates do not differ with regard to the chromosomal position
of the insertion element and the copy number of the DRs. Since the
restriction of a sequence at two digestion sites provides three
fragments, these results showed that the presence of three
PvuII fragments giving a uniform DR hybridization was indeed
compatible with two PvuII sites being located within the DR
locus of type 50 isolates. Thus, the two IS6110 copies
should allow us to selectively amplify the central part of the DR locus
that contains the direct variable repeats (DVRs) numbered 25 to 31, a
DVR being defined as the association of one DR and its adjacent spacer
(8, 17). Lastly, despite the fact that all of the strains
of type 50 represented a unique clade of organisms on the basis of
spoligotyping and also the fact that they did not differ in regard to
the chromosomal position of the IS6110 element (as revealed
by the DR-r probing of the PvuII-digested DNAs), there was
significant variation in the IS6110-RFLP patterns obtained
(Fig. 1C). Indeed, except for 5 isolates that were clonal (isolates
96139, IPC22, 96110, 96121, and 96027) and 2 isolates that were closely
related (94126 and 95048), the remaining 11 isolates were unrelated by
IS6110-RFLP (Fig. 1C). This observation suggests that the
type 50 isolates belong to a spoligotyping-defined ubiquitous family
whose members are not necessarily epidemiologically related.
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FIG. 1.
Spoligotyping and IS6110-RFLP results. (A)
Schematic representation of spoligotyping using (biot)DRa and DRb
primers for type 50 isolates of M. tuberculosis. (B)
Schematic representation of spoligotyping pattern using (biot)IS6 and
DRb primers. (C) Schematic representation of the IS6110-RFLP
results. The 1.4-kb band drawn in bold indicates the double band
observed, whereas the three bands at 1.8, 3.6, and 4.3 kb show the
PvuII fragments that hybridized with the DR-r probe in
strains 94072, 94126, 95013, 95048, 95069, 96027, and IPC22 (shown in
the figure) and also in strains 94098, 98049, 98042, 98045, IPC14,
IPC26, and IPC107 (not represented here). (D) Spoligotyping of the
750-bp inter-IS6110 fragment and whole DNA controls using
primers (biot)IS6 and -IS3. Numbers 1 to 43 show the 43 spacers used
for spoligotyping; numbers 1 and 2 on the right-hand side show the
results for total DNA from control isolates M. tuberculosis
H37Rv and M. bovis BCG, whereas numbers 3 to 9 show the
results for the 750-bp inter-IS6110 fragment from the
following strains: 3, isolate 94072; 4, isolate 94126; 5, isolate
95013; 6, isolate 95048; 7, isolate 95069; 8, isolate 96027; 9, isolate
IPC22. (E) Spoligotyping of the 750-bp inter-IS6110 fragment
and whole DNA controls using primers (biot)DRa and -DRb.
|
|
Based on the above observations and previous DR sequencing data
(8), we decided to selectively amplify the central DR
region within the DR locus using primers IS3 and IS6 in order to use the resulting PCR product for both routine and LR spoligotyping as well
as for sequencing. The parallel controls included primer sets IS3-IS3
and IS6-IS6 and a negative control. The results obtained with seven
randomly selected isolates showed no amplification with the negative
control and primers IS3 and IS3, whereas many bands were obtained with
primers IS6 and IS6, indicating that inversed IS6110 copies
may exist elsewhere in the bacterial choromosome (results not shown).
The PCR product obtained using primers IS3 and IS6 showed bands similar
to those obtained using IS6 and IS6; however, a single specific band of
about 750 bp was found to be exclusively present with primers IS3 and
IS6 and corresponded to the theoretical length expected for the central
DR region (6, 8). When this fragment was purified and used
directly for LR spoligotyping using (biot)IS6-IS3, it revealed the
presence of the previously missing spacer 31 as well as spacers 25 to
30 (Fig. 1D). When the same fragment was used for the routine
spoligotyping method using (biot)DRa-DRb, only spacers 25 to 30 (and
not spacer 31) were revealed (Fig. 1E). One intriguing observation was
the fact that this 750-bp fragment always resulted in a high
hybridization signal for spacers 30 and 31 and a low hybridization
signal for spacers 25 to 29 (Fig. 1D). We have no valid explanation for
this observation for the time being.
Following these results, two clinical isolates (94072 and 94126) were
selected for sequencing experiments. Before sequencing, both the total
DNA and the 750-bp fragment from these two isolates were subjected to
spoligotyping using all the primer combinations tested; the results
obtained (Fig. 2A) unambiguously
confirmed that the 750-bp fragment did correspond to the central DR
region within the DR locus and strengthened our previous assumption
that spacer 31, although present, is not detected among type 50 M. tuberculosis isolates due to an asymmetric insertion of a
second copy of IS6110 between spacers 31 and 32 (6).
View larger version (45K):
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FIG. 2.
Spoligotyping and inter-IS6110 sequencing
results and schematic representation of the central region of the DR
locus. (A) Spoligotyping and LR spoligotyping of the total bacterial
DNA versus the 750-bp inter-IS6110 fragment for isolates
94072 and 94126. Primer sets: A, (biot)DRa-DRb; C, (biot)DRa-IS3; D,
(biot)IS6-DRb; F, (biot)IS6-IS3. (B) Sequencing results of the 750-bp
inter-IS6110 fragment obtained using primers IS3 and IS6;
the sequence shown was identical for isolates 94072 and 94126, and the
illustration shown is limited to the 560-bp portion within the
IS6110-flanking region that comprises spacers 25 to 31 as
shown below. The shaded areas show the corresponding spacer sequences,
and the underlined area corresponds to the DR repeats. The 3' side of
the first copy of IS6110 and the 5' side of the second copy
of IS6110 are shown in italics, with the inverted repeats
shown with dotted lines. (C) Schematic representation of the central
region of the DR locus for spoligotype 50 isolates of M. tuberculosis. In this figure, spacers 25 to 31 are flanked by two
tandem copies of IS6110. Note that on the 3' flanking side
of the first IS6110 copy, 20 bases of one DR are present in
DVR 25, whereas on the 5' flanking side of the second IS6110
copy, DVR 31 is split into two asymmetrical portions, resulting in only
a 6-bp portion of the DR bordering the second IS6110 copy,
which is not sufficient to allow hybridization with the 18-mer DRa
primer. Arrows on IS3, IS6, a (DRa), and b (DRb) show the sense of the
PCRs, whereas Rv2818c and Rv2819c show the hypothetical presence of an
additional ipl region adjacent to the DR locus. The targets
for hybridization with IS6110 and DR-r probes are shown with
bold lines; note that the DR-r probe hybridizes with all the DRs. DR*
represents target DR flanking the first copy of IS6110.
|
|
As expected, the sequencing results showed the presence of spacers 25, 26, 27, 28, 29, 30, and 31 flanked by two tandem copies of
IS6110 (Fig. 2B); on the 3' flanking side of the first
IS6110 copy, 20 bases of one DR are present before spacer
25, whereas on the 5' flanking side of the second IS6110
copy, DVR 31 is split into two asymmetrical portions, resulting in only
a 6-bp portion of the DR bordering the second IS6110 copy
(Fig. 2C). However, the 6-bp portion of the DR bordering the second
IS6110 copy is not sufficient to allow hybridization with
the 18-mer DRa primer, hence creating the inability to detect spacer 31 by routine spoligotyping using primers DRa and DRb (Fig. 2C). In our
opinion, this is the first definitive evidence showing that
IS6110 transposition may alter the spoligotyping profile,
and it provides new clues on one of the potential evolutionary
mechanisms of the DR locus. In an independent investigation, similar
results were recently reported by van Embden et al. (17)
for explaining single spacer differences between two M. bovis strains that were isolated during an epidemic of
multidrug-resistant tuberculosis in Spain.
In a phylogenetic context, LR spoligotyping will permit discrimination
between isolates that really lack spacer 31 and those in which it is
present but not revealed due to the asymmetric disruption of the DRa
target due to IS6110 insertion. As shown in this paper, LR
spoligotyping may also constitute an interesting tool to study
IS6110 transpositional events. These results show that the
DR locus constitutes an ideal IS6110 preferential locus (ipl), permitting the insertion of two or more copies of
IS6110, and provide new clues for epidemiological and
phylogenetic interpretation of changes in IS6110-RFLP and
spoligotyping profiles. Indeed, other mycobacterial ipl loci
have been reported, e.g., the ipl locus, which has been
reported by Fang et al. (3, 4, 5) to form part of the
recently identified IS1547 insertion sequence, mapping to
two genomic locations represented by Rv0797 and Rv3327 within the
M. tuberculosis H37Rv genome (1). Another
insertional hot-spot region described in clinical isolates from South
Africa spans a 9.4-kb region containing seven IS6110
insertion points (14) and maps to different positions, in
region Rv1754c to Rv1765c (7). The presence of
IS6110 copies in tandem in the case of these South African
isolates may hypothetically be detected by PCR using primers IS3 and
IS6 (as shown in the present investigation to selectively amplify the
inter-insertion sequence region), e.g., sequencing of the region
Rv1754c to Rv1758c in M. tuberculosis H37Rv shows the
presence of two IS6110 copies in tandem separated by a
621-bp portion, with the second copy being inserted in plcD (Rv1755c), a phospholipase C homologue (7). Using
insertion site mapping, another study recently identified a 537-bp
dnaA-dnaN intergenic region as an IS6110 hot spot
(12).
Recently, in a study on the genome plasticity of M. tuberculosis that focused on IS6110-flanking regions,
an insertion site between the Rv2818c and Rv2819c genes was found to
lie close to the DR locus (20). A calculation based on the
genome sequence of M. tuberculosis (1)
suggested that it may correspond to the additional band of 4.3 kb
observed in the present investigation after DR-r probing of the
PvuII-IS6110 blots (Fig. 1C). On the other hand,
we do not yet have an explanation for the presence of a double band at
1.4 kb in all the type 50 isolates that we studied. Further studies on
the relative importance of IS6110 insertion, homologous
recombination, and replication slippage in guiding evolutionary changes
of the M. tuberculosis genome may help us to understand the
link between spoligotyping patterns and IS6110-RFLP.
| |
ACKNOWLEDGMENTS |
This work was supported through grants from the
"Délégation Générale au Réseau
International des Instituts Pasteur et Instituts Associés,"
Institut Pasteur, and Fondation Française Raoul Follereau, Paris, France.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Unité de
la Tuberculose et des Mycobactéries, Institut Pasteur de
Guadeloupe, Morne Jolivière, BP 484, F-97165 Pointe-à-Pitre
Cedex, Guadeloupe. Phone: 590-893-881. Fax: 590-893-880. E-mail:
rastogi{at}pasteur.gp.
| |
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Warren, R. M.,
S. L. Sampson,
M. Richardson,
G. D. van der Spuy,
C. J. Lombard,
T. C. Victor, and P. D. van Helden.
2000.
Mapping of IS6110 flanking regions in clinical isolates of Mycobacterium tuberculosis demonstrates genome plasticity.
Mol. Microbiol.
37:1405-1416[CrossRef][Medline].
|
Journal of Clinical Microbiology, April 2001, p. 1595-1599, Vol. 39, No. 4
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.4.1595-1599.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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