Previous Article | Next Article 
Journal of Clinical Microbiology, September 1998, p. 2471-2476, Vol. 36, No. 9
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Specific Differentiation between
Mycobacterium bovis BCG and Virulent Strains of the
Mycobacterium tuberculosis Complex
Juana
Magdalena,
Philip
Supply, and
Camille
Locht*
Laboratoire de Microbiologie
Génétique et Moléculaire, INSERM U447, Institut
Pasteur de Lille, F-59019 Lille Cedex, France
Received 10 April 1998/Returned for modification 12 May
1998/Accepted 29 May 1998
 |
ABSTRACT |
A PCR procedure based on the intergenic region (IR) separating two
genes encoding a recently identified mycobacterial two-component system, named SenX3-RegX3, was developed and was shown to be suitable for identifying Mycobacterium bovis BCG. The
senX3-regX3 IR contains a novel type of repetitive
sequence, called mycobacterial interspersed repetitive units
(MIRUs). All tested BCG strains exclusively contained 77-bp MIRUs
within the senX3-regX3 IR, whereas all non-BCG
M. tuberculosis complex strains contained a 53-bp MIRU, in
addition to the 77-bp MIRUs. All 148 strains analyzed so far could be
divided into eight different groups according to the copy
numbers of the 77-bp MIRU and to the presence or absence of the 53-bp
MIRU. BCG strains contained either one, two, or three 77-bp MIRUs. The
other strains contained one to five 77-bp MIRUs invariably followed by
a 53-bp MIRU. The consistent absence of the 53-bp MIRU in BCG strains
and its presence in virulent strains allowed us to develop an
enzyme-linked immunosorbent assay using specific capture
oligonucleotide probes to distinguish between BCG and other M. tuberculosis complex strains.
| |
INTRODUCTION |
Mycobacterium
tuberculosis is the most devastating human pathogen, and yet, its
virulence mechanisms are poorly understood. It is estimated that
one-third of the world's population is infected with M. tuberculosis and that 3 million people die of tuberculosis each
year (22). In addition to M. tuberculosis sensu
stricto, other members of what is generally referred to as the M. tuberculosis complex can also cause this disease. The other
species of this complex are Mycobacterium africanum,
Mycobacterium microti, and Mycobacterium bovis.
The latter species includes the Bacille de Calmette et Guérin
(BCG) derived from M. bovis. Its virulence has been
attenuated through 230 successive passages on media impregnated with
beef bile between 1908 and 1921 (3). BCG is today the only
available vaccine against tuberculosis.
Since its introduction for clinical use as a tuberculosis vaccine
(18) and for immunotherapy against bladder cancer
(2), the original BCG vaccine strain has been subcultured
many times and has been distributed to a large number of production
centers throughout the world. As a consequence, several substrains of the original vaccine strain are now in circulation, and there is
evidence that they vary in certain characteristics, such as their
antigenic structures (9), their mycolic acid patterns (21), their secreted protein profiles (1), and
their IS986 copy numbers (6). The three most
commonly used BCG vaccine strains are the Pasteur strain, the Japanese
strain, and the Glaxo strain. Other daughter strains include the Moreau
(Brazilian), the Montreal, the Russian, the Prague, and the Danish
strains.
Although largely considered nonpathogenic, in certain circumstances BCG
can nevertheless cause disease in humans (16, 17). This risk
may be substantial for immunocompromised hosts (4). However,
the prevalence of BCG infection is not known, because most laboratories
cannot quickly differentiate between BCG and other members of the
M. tuberculosis complex. Several DNA fingerprinting methods which differentiate M. tuberculosis strains are
available, but none of them segregate BCG from other M. tuberculosis strains.
We have recently identified an operon coding for new mycobacterial
two-component system called SenX3-RegX3 (24). The
senX3-regX3 intergenic region (IR) was found to contain
mycobacterial interspersed repetitive units (MIRUs) of 77 bp and, in
certain strains, MIRUs of 53 bp (19, 24). This
senX3-regX3 IR can be used for the detection of
M. tuberculosis complex strains by PCR since it was found in all members of the M. tuberculosis complex but
in none of the other mycobacterial species tested (19). In
the study described in this report, we showed that the
senX3-regX3 IR can also be used to differentiate between BCG
and virulent strains of the M. tuberculosis complex,
because all the virulent strains contained the 53-bp MIRU, whereas none
of the BCG strains did.
| |
MATERIALS AND METHODS |
Bacterial strains.
Eight M. bovis strains
were used. Four M. bovis strains (strains 60 and 63 isolated from goats and strains 76 and 78 isolated from cows) were
kindly provided by C. Martin (University of Zaragoza, Zaragoza, Spain).
M. bovis AN5 was obtained from G. Marshall (Institut Pasteur, Paris, France). Two M. bovis mtp40-containing
strains (strains 89936 and 88367) were provided by B. B. Plikaitis
from the Centers for Disease Control and Prevention (Atlanta, Ga.). One
M. bovis strain (strain 1) was obtained from the Centre
Hospitalier Régional de Lille (Lille, France).
The M. bovis BCG strains included eight vaccine strains
and 10 clinical isolates from BCG-osis patients. The Glaxo, Russian, Moreau (Brazilian), Danish, Montreal, and Prague M. bovis BCG vaccine strains were provided by M. Lagranderie
(Institut Pasteur, Paris, France), and the 1173P2 (Pasteur) and
Japanese strains were obtained from the World Health Organization
collection in Stockholm, Sweden. The 10 clinical isolates from BCG-osis
patients were from the Institut Pasteur de Lille (strains 1 to 5) and
the Centre Hospitalier Régional (Lille, France) (strains 6 to
10). The M. tuberculosis strains included the
following: three M. tuberculosis strains isolated in
Vietnam and lacking IS6110 (strains V.729, V.761, and V.808)
were kindly provided by G. Marshall (Institut Pasteur, Paris, France),
M. tuberculosis 2296207 was described previously
(19, 24), and six clinical isolates (strains 1033, 1035, 1036, 1037, 1038, and 1039) came from the Centre Hospitalier Régional (Lille, France).
Isolation of chromosomal DNA.
The mycobacterial genomic DNA
was isolated as described previously (19). Briefly, the
mycobacteria were grown in the medium described by Sauton
(23), and protoplasts were obtained by incubating the
mycobacteria in a lysozyme-containing solution. The protoplasts were
then lysed with proteinase K, and the DNA was extracted with phenol-chloroform, precipitated with isopropanol, resuspended, and
treated with RNase. The DNA was extracted again with phenol-chloroform and chloroform, precipitated with ethanol, and resuspended in double-distilled water. The final DNA concentration was approximately 0.33 µg/µl.
PCR amplification of the senX3-regX3 IR and analysis
of the PCR products.
The senX3-regX3 IRs of the various
mycobacterial strains were amplified by PCR as described previously
(19), with oligonucleotides C5
(5'-GCGCGAGAGCCCGAACTGC-3') and C3
(5'-GCGCAGCAGAAACGTCAGC-3') used as primers. The negative
controls contained the PCR mixture without template DNA. The positive
controls contained pRegX3Mt1 or pRegX3Bc1 (24) as template
DNA. The PCR products were analyzed by electrophoresis on a 2.5%
agarose gel and were visualized with ethidium bromide. The PCR products
were then cloned and sequenced with the Zero background/kan cloning kit
(Invitrogen Corporation, San Diego, Calif.) as described previously
(19).
Hybridization analysis of the senX3-regX3 IR PCR
products.
The senX3-regX3 IR PCR products were
also analyzed by hybridization with the digoxigenin
(DIG)-labeled senX3-regX3 IR of M. tuberculosis 2296207 isolated from pRegX3Mt2 as described
previously (19). Alternatively, specific DIG-labeled
oligonucleotides were used for hybridization. These oligonucleotides
were 3' end labeled by using the Boehringer Mannheim 3' end labeling
kit (catalog no. 1362372) as specified by the supplier (Boehringer
Mannheim, Mannheim, Germany) and had the following sequences:
5'-CCACTCCTCCTCATC-3' for oligonucleotide O53, which is
specific for the 53-bp MIRU present in the senX3-regX3 IR,
and 5'-GGGTGGTGCCCCCAC-3' for oligonucleotide O77, which is
specific for the 77-bp MIRU present in the senX3-regX3 IR.
ELISA.
For the detection of the 77-bp or the 53-bp MIRU by
enzyme-linked immunosorbent assays (ELISAs), two kits provided by
Boehringer Mannheim were used: the PCR ELISA DIG labeling kit (catalog
no. 1636120) and the PCR ELISA DIG detection kit (catalog no. 1636111). First, the senX3-regX3 IR was amplified by using the PCR
ELISA DIG labeling kit to obtain DIG-labeled PCR products. The second kit was used to immobilize biotinylated oligonucleotides (O53, O53R, or
O77) onto streptavidin-coated microtiter plates. The sequence of the
O53R oligonucleotide, specific for the 53-bp MIRU, was
5'-GCGCCACTCCTCCTCATC-3' and was thus three bases longer
than that of O53. The DIG-labeled senX3-regX3 IR PCR
products were added to the captured oligonucleotide probes in the
microtiter plates, and the plates were incubated at room temperature or
at 40°C. The bound hybrids were detected by incubation with an
anti-DIG-peroxidase conjugate and the colorimetric substrate
2,2'-azino-di(3-ethylbenzthiazoline sulfonate) (ABTS) (Boehringer
Mannheim) at 37°C for 1 to 4 h. The absorbence at 405 nm was
then measured with an automated microplate reader (model EL340; Bio-Tek
Instrument, Inc., Winooski, Vt.).
| |
RESULTS |
Amplification of the senX3-regX3 IR.
We have
previously shown that MIRUs are present in the senX3-regX3
IR of all 122 strains of the M. tuberculosis complex
tested so far but that their copy numbers vary between different
strains (19). The previous study principally focused on
clinical M. tuberculosis isolates, and only two BCG
strains were analyzed in that study. Interestingly, the
senX3-regX3 IRs of these two BCG strains were composed of
only 77-bp MIRUs, whereas those of the other strains of the
M. tuberculosis complex contained an additional 53-bp
MIRU (19, 24). To assess whether this difference could
generally distinguish BCG strains from other M. tuberculosis complex strains, the senX3-regX3 IRs of a
total of 26 M. bovis strains including 18 BCG strains
were analyzed by PCR. In all cases the senX3-regX3 IR PCR
gave rise to a DNA fragment, as detected by electrophoresis on a 2.5%
agarose gel, which hybridized with the senX3-regX3 IR of
M. tuberculosis 2296207, confirming the specificity of
the PCR products that were obtained. Results of electrophoresis and
hybridization analyses of representative samples are shown in Fig.
1. Although a PCR fragment was amplified
for each strain, their sizes varied somewhat. On the basis of this size
variation, as well as on the basis of this size variation, as well as
on the basis of that observed previously (19), all the
strains of the M. tuberculosis complex could be
subdivided into eight different groups (groups I to VIII, from the
longest to the shortest DNA fragment) (Table
1). In addition to the strains tested in
this study, Table 1 presents data for several M. tuberculosis strains that have been studied previously. The
majority of the M. tuberculosis strains are included in
group V, and strain 2296207 is shown as a representative. Three
M. tuberculosis strains lacking IS6110
correspond to groups I and II.
View larger version (61K):
[in this window]
[in a new window]
|
FIG. 1.
Agarose gel electrophoresis and Southern blot analysis
of senX3-regX3 IR PCR products. The PCR products were
analyzed by electrophoresis and ethidium bromide staining with a 2.5%
agarose gel (A) and by Southern blot hybridization with a DIG-labeled
221-bp DNA fragment of M. tuberculosis 2296207 containing the senX3-regX3 IR as a probe (B). The
senX3-regX3 IR PCR products were obtained from the Japanese
(lanes 1), Russian (lanes 2), Glaxo (lanes 3), Montreal (lanes 4), and
Prague (lanes 5) M. bovis BCG strains and from
non-BCG M. bovis strains 60 (lanes 6), 63 (lanes 7), 78 (lanes 8), AN5 (lanes 9), 76 (lanes 10), and 1 (lanes 11). Positive
(pRegX3Mt1 [lanes 12] and pRegX3Bc1 [lanes 13]) and negative
(lanes 14) controls are also shown, as are molecular size markers (1-kb
ladder; left and right lanes). The sizes of the PCR products that were
obtained are indicated in the left and right margins.
|
|
The 26
M. bovis strains tested in this study were
distributed throughout seven of the eight groups (groups II to VIII;
Table
1). The 18 BCG strains analyzed were restricted to three
different
groups (groups IV, VI, and VIII) (Fig.
1; Table
1). The
Japanese
BCG vaccine strain and six BCG strains isolated from patients
with BCG-osis (strains 1, 2, 3, 6, 7, and 8) yielded a 353-bp
PCR
fragment (group IV). Another group of nine BCG strains (the
1173P2,
Glaxo, Russian, Moreau, and Danish strains, as well as
the remaining
four BCG strains isolated from patients with BCG-osis)
yielded a
276-bp PCR fragment (group VI). Finally, two BCG strains
(strains
Prague and Montreal) yielded a PCR fragment of 199 bp
(group VIII).
The other eight
M. bovis strains which are not BCG fell
into four different classes (groups II, III, V, and VII) (Fig.
1;
Table
1). Four of these eight strains (strains 63, 78, AN5, and
89936) were
found in group III on the basis of the presence of
a 406-bp PCR
fragment. One
M. bovis strain (strain 60) was in
group
II and had a 483-bp fragment, two strains (strains 76 and
88367) were in group V and each had a 329-bp fragment, and one
strain (strain 1) was in group VII and had a 252-bp fragment.
Sequence analysis of the senX3-regX3 IR.
The
senX3-regX3 IR PCR products obtained from all 26 M. bovis strains were cloned and sequenced (Fig.
2; Table 1). The senX3-regX3 IR was always found to contain one or several copies of the 77-bp MIRU, whereas the 53-bp MIRU was present only in groups I, II, III, V, and VII. In all cases, the nucleotide sequences of the 77-bp
and the 53-bp MIRUs were identical to those reported previously (19, 24). Interestingly, all the BCG strains invariably fell within those groups (groups IV, VI and VIII) that did not contain the 53-bp MIRU. They only contained either one (group VIII),
two (group VI), or three (group IV) 77-bp MIRUs. All the other
M. bovis strains, as well as the previously analyzed
M. tuberculosis, M. microti, and
M. africanum strains, contained a 53-bp MIRU.
View larger version (17K):
[in this window]
[in a new window]
|
FIG. 2.
Schematic representation of the mycobacterial
senX3-regX3 IR. (A) The 3.2-kb
EcoRI-BamHI fragment containing the
senX3-regX3 operon is shown by the thin line. The
senX3 and regX3 genes are indicated by the
thicker arrows. The IRs of the different strains can be divided into
eight groups. Five groups (groups I, II, III, V, and VII) correspond to
virulent, non-BCG strains and contain variable copies of the 77-bp
MIRU (thick arrows) and one copy of the 53-bp MIRU (thin
arrow). Three groups (groups IV, VI, and VIII) correspond to avirulent,
BCG strains and contain exclusively one to three 77-bp MIRUs. (B)
The nucleotide and amino acid sequences of the 77- and 53-bp MIRUs
are indicated by the single-letter code. The sequence differences
between the 77- and 53-bp MIRUs are circled. The O77
oligonucleotide specific for the 77-bp MIRU and O53 specific for
the 53-bp MIRU are boxed.
|
|
Differentiation between BCG and other strains of the M. tuberculosis complex by hybridization.
The invariable
presence of the 53-bp MIRU in all strains except BCG and its
invariable absence in BCG strains prompted us to use specific probes to
distinguish between the 53-bp and the 77-bp MIRUs in the
senX3-regX3 IR (Fig. 2). For this purpose oligonucleotides O53 and O77, specific for the 53-bp and the 77-bp MIRUs,
respectively, were designed for hybridization with the
senX3-regX3 IR PCR products used as a matrix (Fig. 2B). On
dot blotted PCR products, hybridization with O53 at 45 or 40°C,
followed by washing at room temperature or at 40°C, respectively,
yielded positive signals for PCR fragments obtained from non-BCG
strains only (data not shown). Hybridization and washing at 45°C were
too stringent, and the signal was lost. The strongest signal was
observed after hybridization at 45°C and washing at room temperature.
When the same conditions were used after Southern blotting of the
senX3-regX3 IR PCR products, O53 was also found to give a
positive signal only for PCR fragments obtained from non-BCG strains
(Fig. 3A).
View larger version (46K):
[in this window]
[in a new window]
|
FIG. 3.
Southern blot analysis of the senX3-regX3 IR
PCR products. DIG-labeled O53 (A) and O77 (B) were used as probes for
Southern blot analysis of the senX3-regX3 IR PCR products
obtained from M. tuberculosis V.808 (lane 1), V.729
(lanes 2), and 2296207 (lane 6); from non-BCG M. bovis
AN5 (lane 3), 76 (lane 4), 88367 (lane 5), and 1 (lane 7); and from the
Japanese (lane 8), 1173P2 (lane 9), Glaxo (lane 10), and Prague (lane
11) BCG strains. The group numbers and sizes of the amplified DNA
fragments are given in the left and right margins.
|
|
In contrast to O53, oligonucleotide O77 yielded strong signals for the
senX3-regX3 IR PCR products obtained from all strains
of the
M. tuberculosis complex tested (Fig.
3B) after
hybridization
at 55°C and washing at room temperature. The
combination of O53
and O77 therefore allowed us to develop a test which
includes
a positive control in itself, since hybridization with O77
yielded
a positive signal for each amplified
senX3-regX3 IR,
whereas hybridization
with O53 yielded a positive signal only for the
senX3-regX3 IR
PCR products of the non-BCG strains. This
allowed us to easily
distinguish between BCG and non-BCG strains of the
M. tuberculosis complex.
ELISA-based distinction between BCG and non-BCG strains.
Since the combined use of O77 and O53 allowed us to distinguish
between BCG and non-BCG strains, we developed an ELISA using specific
oligonucleotides as immobilized capture probes. When biotinylated
O77 was immobilized on streptavidin-coated microtiter plates,
DIG-labeled senX3-regX3 IR PCR fragments of all tested strains hybridized to the probe at room temperature or at 40°C, yielding optical density at 450 nm (OD450) values of 1.5 or
more for 10 µl of the PCR mixture (Fig.
4). In contrast, when the biotinylated oligonucleotide O53 was immobilized, only the DIG-labeled
senX3-regX3 IR PCR fragments of non-BCG strains
hybridized to the probe (data not shown). However, this DNA hybridized
only weakly at room temperature or at 40°C, which was not
sufficient for a clear distinction between BCG DNA and non-BCG DNA.
View larger version (80K):
[in this window]
[in a new window]
|
FIG. 4.
ELISA of the senX3-regX3 IR PCR products.
Biotinylated oligonucleotides O77 and O53R were immobilized on
streptavidin-coated microtiter plates and incubated with 10 µl of the
DIG-labeled senX3-regX3 IR PCR products obtained from
M. tuberculosis strains 1033 (wells 1), 1035 (wells 2),
1036 (wells 3), 1037 (wells 4), 1038 (wells 5), 1039 (wells 6), and
2296207 (wells 7) and the Japanese (wells 9), 1173P2 (wells 10), Glaxo
(wells 11), Russian (wells 12), Danish (wells 13), Prague (wells 14)
and Montreal (wells 15) BCG strains. Wells 8 contained no PCR product,
and wells 16 contained neither the PCR product nor the
oligonucleotides.
|
|
To increase the sensitivity of the assay, oligonucleotide
O53R, which had a higher melting temperature, was thus designed.
When
this oligonucleotide was used, hybridization with the PCR
fragments
from 10 µl of the reaction mixture of the non-BCG strains
gave
OD
450 values of more than 1.5 at 40°C, whereas the values
for BCG strains were less than 0.2 (Fig.
4). However, at room
temperature, this assay could not sufficiently discriminate BCG
strains
(OD
450, 0.8) from non-BCG strains (OD
450, 1.5).
Thus,
by using O77 and O53R in parallel at a hybridization temperature
of 40°C with as little as 10 µl of the PCR mixture, the ELISA
includes a positive control to permit the discrimination between
BCG
and non-BCG strains.
The OD
450 values were routinely measured 1 to 4 h after the addition of the ABTS substrate. Overnight incubation
with the
substrate did not change the sensitivity or the specificity of
the test.
| |
DISCUSSION |
BCG is one of the world's most widely used vaccines. It is also
used as one of the most effective medications in the immunotherapeutic treatment of human bladder carcinoma. Although considered relatively safe, BCG can occasionally cause disease such as sepsis, osteitis, and
meningitis (15-17). Therefore, timely and unequivocal
diagnosis of BCG infection is of importance in order to provide
appropriate therapy for BCG-mediated illness. However, it has been
notoriously difficult to distinguish BCG from the other members of the
M. tuberculosis complex. This distinction may be
particularly important for patients suffering from cellular
immunodeficiencies (4) or for patients undergoing bladder
cancer treatment (2). It has been estimated that
approximately 5% of BCG-treated cancer patients experience adverse
reactions to the treatment (15).
BCG strains can be distinguished from other M. tuberculosis complex strains on the basis of differences in
certain biochemical and growth characteristics and on the basis of the
resistance and sensitivity to cycloserine. However, these methods and
other, nonstandardized methods (5, 8, 12, 13) are
time-consuming, may be laborious, sometimes require specialized and
expensive equipment, and do not always provide definitive proof that a
clinical isolate is BCG. The insertion sequence IS1081 has
been proposed as a useful tool that can be used to recognize BCG by
restriction fragment length polymorphism analysis (26).
However, some M. tuberculosis strains have recently
been shown to contain IS1081 also (11). Mahairas
et al. (20) have identified genetic differences between BCG
and the other members of the M. tuberculosis complex, and on the basis of these differences Talbot et al. (25)
have developed a multiplex PCR method for the identification of BCG. However, the method has not been tested with mycobacteria that are not
part of the M. tuberculosis complex. Frothingham
(7) has used sequevar differences within the major
polymorphic tandem repeats to distinguish BCG from other M. tuberculosis complex strains, but these repeats can also be found
in mycobacteria that are not part of the complex (10).
In this study, we have developed a PCR method based on the
senX3-regX3 IR, which we have recently demonstrated to be
specific for M. tuberculosis complex strains
(19). This region is composed of two types of novel
repetitive sequences, named MIRUs (24). Our results show
that BCG strains exclusively contained 77-bp MIRUs in the
senX3-regX3 IR, whereas the other strains of the M. tuberculosis complex contained both 77-bp and 53-bp
MIRUs. This difference allowed us not only to distinguish BCG from
other strains on the basis of size differences of the PCR-amplified DNA
fragments but also to develop a discriminative ELISA using two
synthetic oligonucleotide probes.
Although all BCG strains contained only the 77-bp MIRU in the
senX3-regX3 IR, the copy number of this MIRU varied
somewhat between BCG strains. They could therefore be classified into
three different groups on the basis of the sizes of the PCR products. These products contained three, two, or one 77-bp MIRU,
respectively. None of the other M. tuberculosis complex
strains analyzed in this study or in a previous study (19)
fell within any of these three groups. On the basis of MIRU copy
numbers, all 148 strains of the M. tuberculosis complex
analyzed so far could be divided into eight distinct groups, two more
than was reported previously (19). Figure
5 depicts the relative frequency of the
strains within each group and shows that the majority of the strains
fell by far within group V. All the BCG strains fell in group IV,
(three 77-bp MIRUs), VI (two 77-bp MIRUs), or VIII (one 77-bp
MIRU), whereas all the other strains fell in group I, II, III, V,
or VII, each containing one 53-bp MIRU in addition to variable
numbers of copies (from one to five) of the 77-bp MIRU.
View larger version (12K):
[in this window]
[in a new window]
|
FIG. 5.
Distribution of the various M. tuberculosis complex strains among eight different groups. The
MIRU copy numbers of a total of 148 strains of the M. tuberculosis complex were determined in this study and in a
previous study (19). The distributions of the strains are
indicated as percentages for each group. Groups IV, VI, and VIII (black
bars) contain only BCG strains. Groups I, II, III, V, and VII (white
bars) contain only non-BCG strains.
|
|
The original BCG strain was initially derived from virulent
M. bovis between 1908 and 1921 and was subsequently
distributed and cultured under different conditions with various growth
media (3, 18). This resulted in the generation of several
BCG substrains which differ in antigenic and biochemical properties
(20, 21), as well as in their contents of certain
insertion sequences (6). Two subgroups have been
distinguished (1, 6, 21). Group A comprises the Japanese,
Russian, and Moreau strains, derived from the original Pasteur BCG
strain prior to 1925, and group B comprises the 1173P2, Glaxo, Danish,
Montreal, and Prague strains (Table 2).
Interestingly, the BCG strain that possesses the longest senX3-regX3 IR belongs to group A, and the BCG strains
possessing the smallest senX3-regX3 IR belong to group B. The BCG strain with an intermediate senX3-regX3 IR length
belongs either to group A or to group B.
Different BCG strains have also recently been evaluated for their
capacity to survive in mice and to trigger immune responses. The
1173P2, Glaxo, and Russian strains were found to multiply actively in
mice and to persist for several months, whereas the Japanese and Prague
strains did not (Table 2). These differences were attributed to
differences in the genetic characteristics of the various BCG strains
(14). We observed that the more persistent and immunogenic
strains (1173P2, Russian, and Glaxo) contained two 77-bp MIRUs in
the senX3-regX3 IR, whereas the less persistent strains
contained either three (Japanese) or one (Prague) 77-bp MIRU.
However, there is no evidence of any direct link between the number of
MIRUs and persistence or immunogenicity, and this observation may
be fortuitous.
In conclusion, the MIRUs contained within the
senX3-regX3 IR can be used as a target to differentiate BCG
from other strains of the M. tuberculosis complex
either by size analysis of the PCR products or by ELISA with specific
capture oligonucleotides.
| |
ACKNOWLEDGMENTS |
We thank G. Delcroix, B. B. Plikaytis, G. Marshall, M. Lagranderie, C. Martin, and A. Vachée for the gifts of bacterial
strains and E. Fort for photography.
The work was supported by INSERM, Institut Pasteur de Lille,
Région Nord-Pas de Calais, and Ministère de la Recherche.
J.M. held a fellowship from the Fondation Recherche et Partage and Sidaction, and P.S. is a researcher of CNRS.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: INSERM U447,
Institut Pasteur de Lille, 1, rue du Prof. Calmette, F-59019
Lille Cedex, France. Phone: (33) 3 20 87 11 51. Fax: (33) 3 20 87 11 58. E-mail: camille.locht{at}pasteur-lille.fr.
| |
REFERENCES |
| 1.
|
Abou-Zeid, C.,
I. Smith,
J. Grange,
J. Steele, and G. Rook.
1986.
Subdivision of daughter strains of bacille Calmette-Guérin (BCG) according to secreted protein patterns.
J. Gen. Microbiol.
132:3047-3053[Abstract/Free Full Text].
|
| 2.
|
Brosman, S. A.
1992.
Bacillus Calmette-Guérin immunotherapy.
Urol. Clin. N. Am.
19:557-564[Medline].
|
| 3.
|
Calmette, A.,
G. Guérin,
L. Nègre, and A. Boquet.
1926.
Prémunition des nouveaux-nés contre la tuberculose par le vaccin BCG, 1921-1926.
Ann. Inst. Pasteur (Paris)
40:89-133.
|
| 4.
|
Edwards, K. M., and D. S. Kernolde.
1996.
Possible hazards of routine bacillus Calmette-Guérin immunization in human immunodeficiency virus-infected children.
Pediatr. Infect. Dis. J.
15:836-838[Medline].
|
| 5.
|
Floyd, M. M.,
V. A. Silcox,
W. D. Jones, Jr.,
W. R. Butler, and J. O. Kilburn.
1992.
Separation of Mycobacterium bovis BCG from Mycobacterium tuberculosis and Mycobacterium bovis by high-performance liquid chromatography of mycolic acids.
J. Clin. Microbiol.
30:1327-1330[Abstract/Free Full Text].
|
| 6.
|
Fomukong, N. G,
J. W. Dale,
T. W. Osborn, and J. M. Grange.
1992.
Use of gene probes based on the insertion sequence IS986 to differentiate between BCG vaccine strains.
J. Appl. Bacteriol.
72:126-133[Medline].
|
| 7.
|
Frothingham, R.
1995.
Differentiation of strains in Mycobacterium tuberculosis complex by DNA sequence polymorphisms, including rapid identification of M. bovis BCG.
J. Clin. Microbiol
33:840-844[Abstract].
|
| 8.
|
Frothingham, R., and K. H. Wilson.
1994.
Molecular phylogeny of the Mycobacterium avium complex demonstrates clinically meaningful divisions.
J. Infect. Dis.
169:305-312[Medline].
|
| 9.
|
Harboe, M., and S. Nagai.
1984.
MPB70 a unique antigen of Mycobacterium bovis BCG.
Am. Rev. Respir. Dis.
129:444-452[Medline].
|
| 10.
|
Hermans, P. W. M.,
D. van Soolingen, and J. D. A. van Embden.
1992.
Characterization of a major polymorphic tandem repeat in Mycobacterium tuberculosis and its potential use in the epidemiology of Mycobacterium kansasii and Mycobacterium gordonae.
J. Bacteriol.
174:4157-4165[Abstract/Free Full Text].
|
| 11.
|
Huh, Y. J.,
D. I. Ahn, and S. J. Kim.
1995.
Limited variation of DNA fingerprints (IS6110 and IS1081) in Korean strains of Mycobacterium tuberculosis.
Tubercle Lung Dis.
76:324-329[Medline].
|
| 12.
|
Jones, W. D., Jr.
1975.
Differentiation of known strains of BCG from isolates of Mycobacterium bovis and Mycobacterium tuberculosis by using mycobacteriophage 33D.
J. Clin. Microbiol.
1:391-392[Abstract/Free Full Text].
|
| 13.
|
Kristjansson, M.,
P. Green,
H. L. Manning,
A. M. Slutsky,
S. M. Brecher,
C. F. von Reyn,
R. D. Arbeit, and J. N. Maslow.
1993.
Molecular confirmation of bacillus Calmette-Guérin as the cause of pulmonary infection following urinary tract instillation.
Clin. Infect. Dis.
17:228-230[Medline].
|
| 14.
|
Lagranderie, M. R. R.,
A.-M. Balazuc,
E. Deriaud,
C. D. Leclerc, and M. Gheorghiu.
1996.
Comparison of immune responses of mice immunized with five different Mycobacterium bovis BCG vaccine strains.
Infect. Immun.
64:1-9[Abstract].
|
| 15.
|
Lamm, D. L.
1992.
Complications of bacillus Calmette-Guérin immunotherapy.
Urol. Clin. N. Am.
19:565-572[Medline].
|
| 16.
|
Lotte, A.,
O. Wasz-Hockert, and N. Poisson.
1988.
Second IUATLD study on complications included by intradermal BCG-vaccination.
Bull. Int. Union Tuberc.
63:47-59.
|
| 17.
|
Lotte, A.,
O. Wasz-Hockert,
N. Poisson,
N. Dumitrescu,
M. Verron, and E. Couvet.
1984.
BCG complications: estimates for the risks among vaccinated subjects and statistical analysis of their main characteristics.
Adv. Tuberc. Res.
21:107-193[Medline].
|
| 18.
|
Lugosi, L.
1992.
Theoretical and methodological aspects of BCG vaccine from the discovery of Calmette and Guérin to molecular biology. A review.
Tubercle Lung Dis.
73:252-261[Medline].
|
| 19.
|
Magdalena, J.,
A. Vachée,
P. Supply, and C. Locht.
1998.
Identification of new DNA region specific for members of Mycobacterium tuberculosis complex.
J. Clin. Microbiol.
36:937-943[Abstract/Free Full Text].
|
| 20.
|
Mahairas, G. G.,
P. J. Sabo,
M. J. Hickey,
D. C. Singh, and C. K. Stover.
1996.
Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis.
J. Bacteriol.
178:1274-1282[Abstract/Free Full Text].
|
| 21.
|
Minnikin, D. E.,
S. M. Minnikin,
G. Dobson,
M. Goodfellow,
F. Portaels,
L. van den Breen, and D. Sesardic.
1983.
Mycolic acid patterns of four vaccine strains of Mycobacterium bovis BCG.
J. Gen. Microbiol.
129:889-891[Abstract/Free Full Text].
|
| 22.
|
Raviglione, M. C.,
D. E. J. Snider, and A. Kochi.
1995.
Global epidemiology of tuberculosis-morbidity and mortality of a worldwide epidemic.
JAMA
273:220-226[Abstract/Free Full Text].
|
| 23.
|
Sauton, B.
1912.
Sur la nutrition minérale du bacille tuberculeux.
C. R. Acad. Sci.
155:860.
|
| 24.
|
Supply, P.,
J. Magdalena,
S. Himpens, and C. Locht.
1997.
Identification of novel intergenic repetitive units in a mycobacterial two-component system operon.
Mol. Microbiol.
26:991-1003[Medline].
|
| 25.
|
Talbot, E. A.,
D. L. Williams, and R. Frothingham.
1997.
PCR identification of Mycobacterium bovis BCG.
J. Clin. Microbiol.
35:566-569[Abstract].
|
| 26.
|
van Soolingen, D.,
P. W. M. Hermans,
P. E. W. de Haas, and J. D. A. van Embden.
1992.
Insertion element IS1081-associated restriction fragment length polymorphisms in Mycobacterium tuberculosis complex species: a reliable tool for recognizing Mycobacterium bovis BCG.
J. Clin. Microbiol.
30:1772-1777[Abstract/Free Full Text].
|
Journal of Clinical Microbiology, September 1998, p. 2471-2476, Vol. 36, No. 9
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Ashworth, M., Horan, K. L., Freeman, R., Oren, E., Narita, M., Cangelosi, G. A.
(2008). Use of PCR-Based Mycobacterium tuberculosis Genotyping To Prioritize Tuberculosis Outbreak Control Activities. J. Clin. Microbiol.
46: 856-862
[Abstract]
[Full Text]
-
Supply, P., Allix, C., Lesjean, S., Cardoso-Oelemann, M., Rusch-Gerdes, S., Willery, E., Savine, E., de Haas, P., van Deutekom, H., Roring, S., Bifani, P., Kurepina, N., Kreiswirth, B., Sola, C., Rastogi, N., Vatin, V., Gutierrez, M. C., Fauville, M., Niemann, S., Skuce, R., Kremer, K., Locht, C., van Soolingen, D.
(2006). Proposal for Standardization of Optimized Mycobacterial Interspersed Repetitive Unit-Variable-Number Tandem Repeat Typing of Mycobacterium tuberculosis. J. Clin. Microbiol.
44: 4498-4510
[Abstract]
[Full Text]
-
Okazaki, T., Ebihara, S., Takahashi, H., Asada, M., Sato, A., Seki, M., Ohto, H., Sasaki, H.
(2005). Multiplex PCR-Identified Cutaneous Tuberculosis Evoked by Mycobacterium bovis BCG Vaccination in a Healthy Baby. J. Clin. Microbiol.
43: 523-525
[Abstract]
[Full Text]
-
Prabhakar, S., Mishra, A., Singhal, A., Katoch, V. M., Thakral, S. S., Tyagi, J. S., Prasad, H. K.
(2004). Use of the hupB Gene Encoding a Histone-Like Protein of Mycobacterium tuberculosis as a Target for Detection and Differentiation of M. tuberculosis and M. bovis. J. Clin. Microbiol.
42: 2724-2732
[Abstract]
[Full Text]
-
Broccolo, F., Scarpellini, P., Locatelli, G., Zingale, A., Brambilla, A. M., Cichero, P., Sechi, L. A., Lazzarin, A., Lusso, P., Malnati, M. S.
(2003). Rapid Diagnosis of Mycobacterial Infections and Quantitation of Mycobacterium tuberculosis Load by Two Real-Time Calibrated PCR Assays. J. Clin. Microbiol.
41: 4565-4572
[Abstract]
[Full Text]
-
Savine, E., Warren, R. M., van der Spuy, G. D., Beyers, N., van Helden, P. D., Locht, C., Supply, P.
(2002). Stability of Variable-Number Tandem Repeats of Mycobacterial Interspersed Repetitive Units from 12 Loci in Serial Isolates of Mycobacterium tuberculosis. J. Clin. Microbiol.
40: 4561-4566
[Abstract]
[Full Text]
-
Cowan, L. S., Mosher, L., Diem, L., Massey, J. P., Crawford, J. T.
(2002). Variable-Number Tandem Repeat Typing of Mycobacterium tuberculosis Isolates with Low Copy Numbers of IS6110 by Using Mycobacterial Interspersed Repetitive Units. J. Clin. Microbiol.
40: 1592-1602
[Abstract]
[Full Text]
-
Skuce, R. A., McCorry, T. P., McCarroll, J. F., Roring, S. M. M., Scott, A. N., Brittain, D., Hughes, S. L., Hewinson, R. G., Neill, S. D.
(2002). Discrimination of Mycobacterium tuberculosis complex bacteria using novel VNTR-PCR targets. Microbiology
148: 519-528
[Abstract]
[Full Text]
-
Mazars, E., Lesjean, S., Banuls, A.-L., Gilbert, M., Vincent, V., Gicquel, B., Tibayrenc, M., Locht, C., Supply, P.
(2001). High-resolution minisatellite-based typing as a portable approach to global analysis of Mycobacterium tuberculosis molecular epidemiology. Proc. Natl. Acad. Sci. USA
98: 1901-1906
[Abstract]
[Full Text]
-
Zahrt, T. C., Deretic, V.
(2000). An Essential Two-Component Signal Transduction System in Mycobacterium tuberculosis. J. Bacteriol.
182: 3832-3838
[Abstract]
[Full Text]
-
Behr, M. A., Schroeder, B. G., Brinkman, J. N., Slayden, R. A., Barry, C. E. III
(2000). A Point Mutation in the mma3 Gene Is Responsible for Impaired Methoxymycolic Acid Production in Mycobacterium bovis BCG Strains Obtained after 1927. J. Bacteriol.
182: 3394-3399
[Abstract]
[Full Text]
-
Kasai, H., Ezaki, T., Harayama, S.
(2000). Differentiation of Phylogenetically Related Slowly Growing Mycobacteria by Their gyrB Sequences. J. Clin. Microbiol.
38: 301-308
[Abstract]
[Full Text]
-
Sechi, L. A., Leori, G., Lollai, S. A., Duprè, I., Molicotti, P., Fadda, G., Zanetti, S.
(1999). Different Strategies for Molecular Differentiation of Mycobacterium bovis Strains Isolated in Sardinia, Italy. Appl. Environ. Microbiol.
65: 1781-1785
[Abstract]
[Full Text]
Top
Abstract
Introduction
