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Journal of Clinical Microbiology, April 1999, p. 964-970, Vol. 37, No. 4
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Identification of Mycobacterium kansasii
by Using a DNA Probe (AccuProbe) and Molecular Techniques
Elvira
Richter,1,*
Stefan
Niemann,1
Sabine
Rüsch-Gerdes,1 and
Sven
Hoffner2
Forschungszentrum Borstel, National Reference
Center for Mycobacteria, Borstel, Germany,1
and Swedish Institute for Infectious Disease Control,
Stockholm, Sweden2
Received 17 September 1998/Returned for modification 9 November
1998/Accepted 8 December 1998
 |
ABSTRACT |
The newly formulated Mycobacterium kansasii AccuProbe
was evaluated, and the results obtained with the new version were
compared to the results obtained with the old version of this test by
using 116 M. kansasii strains, 1 Mycobacterium
gastri strain, and 19 strains of several mycobacterial species.
The sensitivity of this new formulation was 97.4% and the specificity
was 100%. Still, three M. kansasii strains were missed by
this probe. To evaluate the variability within the species, genetic
analyses of the hsp65 gene, the spacer sequence between the
16S and 23S rRNA genes, and the 16S rRNA gene of several M. kansasii AccuProbe-positive strains as well as all
AccuProbe-negative strains were performed. Genetic analyses of the one
M. gastri strain from the comparative assay and of two
further M. gastri strains were included because of the
identity of the 16S rRNA gene in M. gastri to that in
M. kansasii. The data confirmed the genetic heterogeneity
of M. kansasii. Furthermore, a subspecies with an
unpublished hsp65 restriction pattern and spacer sequence
was described. The genetic data indicate that all M. kansasii strains missed by the AccuProbe test belong to one
subspecies, the newly described subspecies VI, as determined by the
hsp65 restriction pattern and the spacer sequence. Since the M. kansasii strains that are missed are rare and all
M. gastri strains are correctly negative, the new
formulated AccuProbe provides a useful tool for the identification of
M. kansasii.
| |
INTRODUCTION |
Mycobacterium kansasii is
one of the most important causes of pulmonary disease resulting from
nontuberculous mycobacteria. Although the clinical picture of patients
with M. kansasii infection resembles that of patients with
tuberculosis, treatment of M. kansasii infection differs
from that of regular tuberculosis. Due to this, a rapid means of
identification of this species is essential.
Routine laboratory identification of M. kansasii relies upon
growth characteristics and several biochemical tests, like
photochromogenicity, Tween hydrolysis, nitrate reduction, and the
pattern of susceptibility to several antituberculosis drugs. Several
weeks are required to verify the results.
For the prompt identification of mycobacteria, molecular methods are
gaining increasing importance due to their rapidity and, in most cases,
unequivocal results. PCR of the hsp65 gene followed by
restriction enzyme analysis (PCR-restriction fragment length polymorphism [PCR-RFLP] analysis) is a rapid technique for the identification of mycobacteria (14). However, difficulties
arise from the small sizes of the fragments and marginal differences between the several mycobacterial species. Furthermore, the PCR-RFLP patterns of unknown species are not published and may correspond to the
patterns of other species. Sequencing of the hsp65 gene (6), the 16S rRNA gene (7), the spacer region
between the 16S rRNA and the 23S rRNA genes (5, 12, 18), or
the 32-kDa protein gene (13) are powerful techniques for the
identification of mycobacterial species. Yet, these techniques are
laborious and require large-scale technical equipment. Tests with
commercially available DNA probes (AccuProbe; GenProbe, San Diego,
Calif.) are easy to perform and make possible rapid identifications.
They are developed for the identification of the M. tuberculosis complex, the M. avium complex, M. avium, M. intracellulare, M. gordonae, and
M. kansasii. Several studies have shown the high degrees of sensitivity of the DNA probes for the M. tuberculosis
complex, the M. avium complex, and M. gordonae
and have shown that their use allows the rapid and correct
identification of these frequently isolated species. In contrast,
AccuProbes for M. kansasii have been reported to have a low
sensitivity and miss from 7 up to 56% of the strains (8, 11,
15, 16; unpublished observations).
Genotypic heterogeneity among M. kansasii isolates has been
reported from several studies (1, 4, 19, 20). PCR-RFLP analysis of the hsp65 gene led to the classification of five
subspecies of M. kansasii (3, 9). Furthermore,
differences in the sequence of the 16S-23S rRNA gene spacer region have
been shown to exist (2).
A new, improved version of the DNA probe for the identification of all
M. kansasii strains is now available (AccuProbe for M. kansasii; GenProbe).
In our study, we comparatively evaluated the sensitivity and the
specificity of the previous and the improved versions of the AccuProbe
M. kansasii test. To elucidate the combined two AccuProbe
results on a molecular basis, we characterized the hsp65 sequences and PCR-RFLP patterns, 16S rRNA gene sequences, and 16S-23S
rRNA gene spacer region of several strains.
| |
MATERIALS AND METHODS |
Strains analyzed.
A total of 116 M. kansasii
strains (strain ATCC 12478 and 115 clinical isolates), 1 strain of
M. gastri, and 19 strains of several mycobacterial species
(M. celatum, M. fortuitum, M. gilvum, M. gordonae, M. lentiflavum, M. marinum, M. paraffinicum, M. simiae, M. tuberculosis, and M. xenopi) were included in
this study. All strains were identified by using classical biochemical
tests, AccuProbes, and/or sequencing.
Samples of five further strains (three M. kansasii and two
M. gastri strains) with which only the new formulated
AccuProbe test for M. kansasii was performed were included.
For molecular biological analysis, 18
M. kansasii strains
were selected on the basis of the results of both the old and the
new
AccuProbe assays for the strains: 3 strains with negative
results by
both AccuProbe assays, 7 strains which were negative
with the old
detection kit but positive with the new one, 7 strains
with positive
results by both assays, as well as strain ATCC 12478.
Furthermore,
three
M. kansasii strains that were negative by the
new
AccuProbe assay but with which no old assays were performed
were
included. Moreover, three
M. gastri strains (one of which
was tested by both versions of the AccuProbe assay) were also
analyzed
on a genetic
basis.
AccuProbe assays.
M. kansasii AccuProbe assays were
performed according to the manufacturer's instructions. For the
comparative assays with the old and the new kits, two bacterial lysates
were prepared from each strain. After lysis, the samples were combined
and mixed. This sample (100 µl) was transferred to each of the
AccuProbe tubes containing the old or the new DNA probe. Hybridization
results, expressed as relative light units (RLUs), were determined with a Leader 50 luminometer (GenProbe). According to the manufacturer's instructions the cutoff value was 30,000 RLUs.
DNA for molecular biological analyses.
Bacterial DNA for
molecular biological analyses was obtained as follows. One loopful of
bacteria was suspended in distilled water, subjected to sonication for
15 min, and boiled for 15 min in a water bath. This suspension was
directly used for PCR analyses.
Determination of the sequence of a part of the 16S rRNA
gene.
A 590-bp fragment of the mycobacterial 16S rRNA gene was
amplified by PCR as described by Richter et al. (10).
Sequencing was performed with an automated DNA sequencer (ABI Prism
377; Applied Biosystems, Foster City, Calif.) with one of the PCR
primers (primer A) and one nested primer (primer 11 [5'-GACAAACCACCTACGAGCTC]) by using the RR DyeDeoxy
terminator sequencing kit (Applied Biosystems). Analyses and
comparisons of the sequences were done with DNasis software (Hitachi,
Olivet Cedex, France). The resulting DNA sequences were compared to the
sequences of the International Nucleotide Sequence Database
Collaboration (4a).
Determination of the sequence of the spacer region between the
16S and 23S rRNA genes.
The internal spacer region was amplified
with primers targeting the 3' end of the 16S rRNA gene (ITS1
[5'-GATTGGGACGAAGTCGTAAC]) or the 5' end of the 23S rRNA
gene (ITS2 [5'-AGCCTCCCACGTCCTTCATC]). PCR was performed
at an annealing temperature of 57°C for 35 cycles. The PCR product
was sequenced with primer ITS2. The resulting DNA sequences were
compared to the sequences of the International Nucleotide Sequence
Database Collaboration (4a).
Sequence determination and PCR-restriction fragment analysis of a
part of the hsp65 gene.
PCR of the hsp65
gene was performed as described by Telenti et al. (14). Both
strands of the PCR products were sequenced with the PCR primers. The
resulting sequences were aligned and were analyzed for restriction
sites with the restriction enzymes BstEII and
HaeIII. For restriction fragment analysis, 20 µl of the
PCR products was digested either with BstEII (Boehringer
Mannheim, Mannheim, Germany) or with HaeIII (Boehringer
Mannheim), and 20 µl of the restriction mixture was run on a 4%
NuSieve 3:1 agarose gel (Biozym, Hessisch-Oldendorf, Germany).
Molecular weight marker VIII (Boehringer Mannheim) served as an
external molecular size marker.
| |
RESULTS |
Results of the AccuProbe tests.
Of the 116 M. kansasii strains tested by the two versions of the AccuProbe
assay, 88 strains (including strain ATCC 12478) were identified with
the old kit, whereas 113 strains were positive with the new kit (Table
1). The three strains not detected with the new kit (1,615, 2,026, and 2,783 RLUs) were also negative with the
old kit. Neither the M. gastri strain (3,254 RLUs by the old
test; 2,699 RLUs by the new test) nor the other mycobacterial strains
had a false-positive AccuProbe result with the probes in both kits.
The sensitivity and specificity of the old
M. kansasii
AccuProbe test were 75.8 and 100%, respectively. In contrast, the
sensitivity
of the new formulated test reached 97.4%, and the
specificity
was 100%.
Molecular biological analyses.
To further analyze the genetic
heterogeneity of the M. kansasii strains and to compare it
to the features of M. gastri, we performed molecular
analyses of the hsp65 gene, the spacer sequence between the
16S and the 23S rRNA genes, and the 16S rRNA gene.
hsp65 gene.
Sequencing of the 443-bp fragment
resulting from the PCR and analysis of the restriction sites in this
fragment resulted in six groups that differed in their DNA sequences
and the restriction sites (Fig. 1 and
Table 2). On the basis of their
restriction patterns the groups were denominated as M. kansasii subspecies I to IV and M. gastri as described
by Picardeau et al. (9) (Fig.
2). Subspecies I includes strain ATCC
12478. However, the previously described M. kansasii
subspecies V (9) could not be detected in our study. In
addition to the strains classified as described above, six strains with
a unique DNA sequence were found (Fig. 1). BstEII digestion
of the PCR products of these strains resulted in a pattern identical to
that for subspecies III strains, whereas HaeIII digestion
yielded a moderately larger fragment compared to that found in
subspecies III strains (Fig. 2 and Table 2). Thus, these M. kansasii strains also exhibit unique restriction patterns.
However, this pattern is identical to the pattern for M. gastri, although the hsp65 sequences of both species
differ in stretches not affected by BstEII or
HaeIII digestion (Fig. 1). Subsequently, these M. kansasii strains are denominated subspecies VI.
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FIG. 1.
Alignment of the sequences of the hsp65 PCR
fragments of five M. kansasii subspecies and M. gastri. Marked sequences represent the recognition sites for
HaeIII (GGCC) and BstEII (GGTNACC).
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TABLE 2.
Fragment lengths of the hsp65 PCR products
after restriction with BstEII and HaeIII, deduced
from sequence analysis, and comparison to published data, deduced from
agarose gel electrophoresis
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FIG. 2.
BstEII and HaeIII restriction
analysis of the hsp65 PCR fragment of M. kansasii
subspecies I, II, III, IV, and VI and of M. gastri. Lanes:
m, molecular size marker (fragment lengths, 1,114, 900, 692, 501, 404, 320, 242, 190, 147, 124, 110, 67, 37, and 19 bp); 1, subspecies I; 2, subspecies II; 3, subspecies III; 4, subspecies IV; 5, subspecies VI;
6, M. gastri.
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Spacer sequence between 16S and 23S rRNA genes.
Sequencing of
the spacer region resulted in five different sequences for M. kansasii and another unique sequence for M. gastri (Fig. 3). Comparison of the sequences to
those of the International Nucleotide Sequence Database, the sequences
of subspecies I, III, and VI were identical to those stored in the
GenBank database (accession nos. L42262, L42263, and L42264,
respectively), whereas the sequence of subspecies IV showed 99%
identity to the sequence of one M. gastri strain (EMBL
database accession no. Y14182). The sequence of M. gastri
was the same as that of another strain of this species stored in the
EMBL database (accession no. X97633). No entry with a sequence
identical to the sequence of subspecies II was found.
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FIG. 3.
Alignment of the spacer sequences of five M. kansasii subspecies and M. gastri. For comparison, the
order was altered to M. kansasii subspecies I, II, III, VI,
and IV and M. gastri.
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The spacer sequence of the so far undescribed subspecies VI showed a
high degree of homology to the sequence of
M. kansasii subspecies III. In total, the sequences of subspecies III and
VI differ
at 13 bp (Fig.
3).
The sequences of
M. kansasii type IV and
M. gastri were very similar, differing at only 6 bp (Fig.
3).
For all strains the grouping into subspecies made on the basis of the
spacer sequences was concordant to the grouping made
on the basis of
the
hsp65 sequences.
16S rRNA gene.
Sequencing of the first 600 bp of the 16S rRNA
gene, including the species-specific region V2 of helix 10 as well as
the variable V3 region of the helix 18, was performed. All M. kansasii and all M. gastri strains investigated in this
study by molecular methods were characterized by the M. kansasii- and M. gastri-specific sequence at bases 121 to 139 and 176 to 255 (according to the numbering for Escherichia
coli) (7). However, in 12 strains a different, shorter
sequence comprising bases 83 to 96 and, furthermore, single base
substitutions in the V3 region were found (Fig.
4). These strains are characterized by
the presence of the hsp65 sequence and the spacer sequences
of subspecies II, III, and VI. The V3 regions of M. kansasii
subspecies VI strains were not identical to each other (Fig. 4B). Yet,
this variability could not be seen in the spacer sequence and was
detectable for only 1 bp (bp 354; R = A or T; Fig. 1) in the
hsp65 gene. M. kansasii subspecies I and IV as
well as M. gastri have sequences identical to those in the
database.
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FIG. 4.
Alignment of two parts of the 16S rRNA gene of the five
M. kansasii subspecies and M. gastri. (A) Bases
83 to 96 (according to the numbering for E. coli); (B) part
of the variable region V3 of helix 18 (bases 458 to 469 according to
the numbering for E. coli).
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Comparison of the results of the genetic analyses with the
AccuProbe results.
The new AccuProbe version clearly identifies
M. kansasii subspecies I, II, III, and IV, (Table
3). However, strains of subspecies VI
remain unidentified with this version. Those strains could be
identified by a positive nitrate reaction and a positive
photochromogenic reaction, or by sequencing. All M. gastri
strains were negative by the AccuProbe assay.
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TABLE 3.
Summary of results of molecular analyses of the
hsp65 PCR fragment, the 16S-23S spacer sequence, and the 16S
rRNA gene as well as the AccuProbe results
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|
No discrepant results concerning genetic features and AccuProbe assay
results within a subspecies of
M. kansasii and
M. gastri were
observed.
| |
DISCUSSION |
Identification of M. kansasii.
Identification of
M. kansasii can be performed with a high sensitivity with
the newly formulated AccuProbe kit. In our comparative study, 113 of
116 M. kansasii strains were correctly identified with this
kit. The specificity of the test with the old version of the test kit
was already excellent, and it remains so with the new version. We found
no strains with false-positive results.
Similar results were reported by Tortoli et al. (
17). In
contrast to our study, they observed no false-negative results,
although they tested a large number of isolates. Furthermore,
they
reported that two
M. gastri strains had false-positive
results.
Maybe these strains were mistyped as
M. gastri and
were actually
subspecies IV
M. kansasii strains which are
negative with the
old version of the test kit but positive with the new
version
of the AccuProbe test
kit.
In our study six strains remained unidentified with the new kit,
namely, strains of subspecies VI. This raises the question
of whether
these strains are really
M. kansasii. However, all
biochemical test results as well as the presence of the specific
16S
rRNA gene and the high degree of similarity of the subspecies
VI spacer
sequence to that of the subspecies III spacer sequence
confirm that
these strains are
M. kansasii. Since they were isolated
from
clinical specimens (respiratory specimens and blood) they
are
presumably pathogenic for humans. Thus, rapid identification
of these
strains is
necessary.
Frequency of occurrence of M. kansasii subspecies in
the present study.
Since the strains used in the present study
were derived from a routine laboratory, conclusions on the frequency of
occurrence of the different M. kansasii subspecies can be
drawn. The relative distributions of the different subspecies may be
estimated by the combined AccuProbe assay and sequencing results.
Strains belonging to subspecies I and III are characterized by a
positive result by the old AccuProbe test (Table 3). Thus, all 88 strains (75%) positive with the old kit belong to these two
subspecies. In our molecular analysis, eight of these strains have been
analyzed, and only one could be shown to belong to subspecies III.
Hence, the frequency of occurrence of this subspecies in the present study may be low, whereas most strains are subspecies I strains. This
is in accordance with the results reported from other studies (2).
Subspecies II and IV are characterized by a negative test result with
the old version of the AccuProbe kit but are positive
with the new
version. Thus, 25 strains (21%) investigated in the
present study with
the two versions of the AccuProbe kits belong
to these two subspecies.
Six of seven strains with a negative
result only by the old test
investigated on a molecular basis
were found to be subspecies II
strains. In conclusion, most of
the 25 strains negative by the old
assay will belong to subspecies
II.
Three strains negative by both versions of the assay represent
subspecies VI strains at a frequency of 2.5% in the comparative
study
of the two versions of the AccuProbe
kits.
Variability of M. kansasii strains.
The genetic
variability of the M. kansasii strains is reflected in the
differences observed in the DNA fragments investigated. Despite the
almost complete identity of the 16S rRNA sequences, subspecies III, IV,
and VI differ markedly from subspecies I and II in terms of their
spacer sequences. This confirms the differences observed by restriction
analysis with the hsp65 sequence, which led to the
subspecies classification (9). Furthermore, the negative
AccuProbe test result for subspecies VI strains indicates the sequence
differences in the part of the chromosome to which the probes
hybridize. Interestingly, despite the almost identical sequences of the
16S rRNA, the hsp65 gene, and the 16S to 23S spacers of
subspecies III and VI, the AccuProbe target sequence must be different
since subspecies VI strains are negative with the AccuProbe but
M. kansasii subspecies III strains are positive with the AccuProbe.
Considerably few differences occur between
M. kansasii
subspecies IV and
M. gastri. Yet, photochromogenicity and a
weakly
positive nitrate reaction for the subspecies IV strain identify
this strain as an
M. kansasii strain. However, the genetic
similarities
between these strains raise the question of whether
M. kansasii subspecies IV strains belong to the
M. kansasii group or in fact
are photochromogenic
M. gastri strains.
All the sequencing results presented here demonstrate a marked
variability among the
M. kansasii strains, resulting in
distinct
subspecies.
Implications for the identification of mycobacteria by molecular
methods.
RFLP analysis of a part of the hsp65 gene is
widely used for the differentiation of mycobacteria. The limits of this
technique are obvious in cases of variability within one species, as
was found for M. kansasii. The apparent discrepancy between
the real and the observed fragment lengths renders this method more difficult.
By 16S rRNA gene sequencing, all
M. kansasii strains can
clearly be identified. In addition,
M. kansasii subspecies
II, III,
and VI can be identified because they differ in the V3 region.
However,
M. gastri cannot be separated from
M. kansasii subspecies
I and IV by 16S rRNA gene sequencing. Thus, in
these cases, identification
of
M. gastri can be performed
either by sequencing of the
hsp65 gene or the spacer
sequence or by classical
tests.
Differences in the 16S rRNA gene sequences in a mycobacterial species
have been published, for example, for
M. gordonae and
M. fortuitum. For
M. kansasii, sequence variation
is documented
only for bases 71 to 84 (
2,
12). The variation
in the variable
stretch of the sequence of helix 18 is small but is
discriminatory
for subspecies II, III, and VI. Since several
mycobacterial species
vary only at one or two bases, these
substitutions should be taken
into account so that those
M. kansasii strains are not
missed.
In summary, the results of our study underline the observations of a
high degree of variability among
M. kansasii strains.
All
strains denominated as
M. kansasii are characterized by a
common 16S rRNA gene sequence. However, the presence of an identical
sequence in
M. gastri shows that this common sequence is not
sufficient
for species identification. Furthermore, variability in
other
parts of the genome of
M. kansasii strains strengthens
the differences
between several types. Due to the clinical importance
of
M. kansasii,
rapid and certain identification of all
types or subspecies is
essential. The newly formulated AccuProbe
provides a useful tool
for the identification of
M. kansasii
since all
M. gastri strains
are correctly negative and
M. kansasii strains are rarely
missed.
| |
ACKNOWLEDGMENTS |
We thank B. Schlüter and L. Klintz for excellent technical
work and GenProbe Inc. for providing reagents.
| |
FOOTNOTES |
*
Corresponding author. Mailing address:
Forschungszentrum Borstel, National Reference Center for Mycobacteria,
D-23845 Borstel, Germany. Phone: 49-4537-188658. Fax:
49-4537-188311. E-mail: erichter{at}fz-borstel.de.
| |
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Journal of Clinical Microbiology, April 1999, p. 964-970, Vol. 37, No. 4
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
