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Journal of Clinical Microbiology, June 2001, p. 2102-2109, Vol. 39, No. 6
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.6.2102-2109.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Differentiation of Mycobacterial Species by
PCR-Restriction Analysis of DNA (342 Base Pairs) of the RNA
Polymerase Gene (rpoB)
Bum-Joon
Kim,1
Keun-Hwa
Lee,2,3,4
Bo-Na
Park,2,3,4
Seo-Jeong
Kim,5
Gill-Han
Bai,6
Sang-Jae
Kim,6 and
Yoon-Hoh
Kook2,3,4,*
Department of Microbiology, Cheju National
University College of Medicine, Cheju 690-7561,1
Department of Microbiology, Institute of Endemic Diseases,
SNUMRC,2 Cancer Research Center, Seoul
National University College of Medicine,3 and
Clinical Research Institute, Seoul National University
Hospital,4 Seoul 110-799, Department of
Pediatrics, Pundang CHA General Hospital, Pochun CHA Medical School,
Kyonggi-do Sungnam 463-670,5 and The
Korean Institute of Tuberculosis, The Korean National Tuberculosis
Association, Seoul 137-140,6 Korea
Received 16 October 2000/Returned for modification 2 January
2001/Accepted 8 April 2001
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ABSTRACT |
PCR amplification-restriction analysis (PRA) of rpoB
DNA (342 bp), which comprises the Rifr region, was used for
the differential identification of 49 mycobacteria. The DNA had been
used previously for the identification of mycobacterial species by
comparative sequence analysis (B. J. Kim et al., J. Clin.
Microbiol. 37:1714-1720, 1999). Digestion with four restriction enzymes (HaeIII, HindII, MvaI, and
AccII), which were selected on the basis of
rpoB DNA sequences, generated distinctive PRA patterns that
allowed not only the reference strains but also the clinical isolates
of mycobacteria to be distinguished. Both rapidly and slowly growing
mycobacteria were distinctly differentiated by HaeIII
digestion of the amplified rpoB DNA. By HindII
digestion the Mycobacterium tuberculosis complex was
distinguished from the other mycobacteria. Furthermore, six subspecies
of Mycobacterium kansasii (subspecies I to VI) as well as
the closely related Mycobacterium gastri, and other closely
related species, were distinguished by simultaneous digestion of
MvaI and AccII. According to the rpoB PRA scheme, 240 strains of clinical isolates could be
identified. It was also possible to detect and identify M. tuberculosis directly from sputa and bronchoalveolar lavage
specimens. These results suggest that PRA of rpoB DNA is a
simple and feasible method not only for the differentiation of culture
isolates but also for the rapid detection and identification of
pathogenic mycobacteria in primary clinical specimens.
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INTRODUCTION |
The genus Mycobacterium
comprises more than 70 species, some of which are pathogenic or
potentially pathogenic to humans and animals, and some of which are
saprobes. Human infections are mainly caused by slowly growing
mycobacteria such as Mycobacterium tuberculosis, M. avium
complex (MAC), and M. kansasii. Recently, reports of
infections due to mycobacteria other than M. tuberculosis complex (MOTT) have been increasing. Because of their clinical importance, in terms of strategies for treatment and the
epidemiological implications, the rapid differentiation of causative
mycobacteria is important. However, isolation and identification
procedures usually require several weeks.
Various PCR-mediated methods had been applied for the rapid detection
and identification (or differentiation) of mycobacterial species. Among
these different methods PCR-restriction analysis (PRA) is preferred, as
a simple and cost-effective method that does not involve radioisotopes.
It has been applied to several genes, such as 16S ribosomal DNA (rDNA)
(6, 9, 10, 33), hsp65 (2, 4, 8, 18, 19,
22, 28, 30, 31), and dnaJ (29), for the
rapid differentiation of closely related clinical isolates, such as
Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis
(8), and subspecies of M. kansasii
(18). Previously we reported that comparative sequence
analysis of the RNA polymerase gene (rpoB) DNA (342 bp), encompassing the region related to rifampin resistance
(Rifr) in M. tuberculosis, is useful for the
identification of mycobacterial species (12). The present
study demonstrates that the same region can be directly used for the
differential identification of mycobacteria in sputa and culture
isolates. PRA of amplified rpoB DNA can differentiate the
two naturally divided mycobacterial groups (rapidly growing and slowly
growing mycobacteria) and can distinguish M. tuberculosis from MOTT. According to the identification scheme established in this
study, most of the human pathogens of MOTT can be differentiated.
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MATERIALS AND METHODS |
Mycobacteria and sputa.
Forty-nine reference strains and 240 clinical isolates of mycobacteria, as well as 50 sputum specimens and 3 specimens from bronchoalveolar lavage (BAL) from patients suspected of
having mycobacterial infections, were provided by the Korean Institute of Tuberculosis (KIT), Department of Clinical Pathology, and the Department of Internal Medicine, Seoul National University Hospital. Five heat-killed reference strains of M. kansasii
(subspecies I to V) and two strains of subspecies VI were kindly
provided by Veronique Vincent (TB and Mycobacteria Lab, Institut
Pasteur, Paris, France) and Elvira Richter (Forschungszentrum Borstel, National Reference Center for Mycobacteria, Borstel, Germany) respectively. Clinical isolates were identified by growth
characteristics, conventional biochemical tests, and molecular
biological methods (Table 1).
All sputa which had been requested for the culture of
M. tuberculosis or MOTT were processed by 1% NaOH liquefaction,
decontamination,
and sedimentation (at 12,000 ×
g for
15 min) (
17). Sediments
were resuspended in 1.5 ml of
phosphate buffer (pH 6.8). DNAs
were isolated from 1.0 ml of the
sediments, and 0.5 ml of the
residual sediments were inoculated onto
Löwenstein-Jensen media.
Culture isolates of
M. tuberculosis were simultaneously identified
by the methods listed
in Table
1 by KIT. Results obtained from
cultures were compared to the
results of
rpoB PRA (
HindII) and
IS
6110 PCR by blind
testing.
Preparation of DNA.
Mycobacterial DNAs from the cultures and
sediments of sputa were prepared by the method previously described
(12). A loopful of culture of each mycobacterium or
sediment was suspended with 200 µl of TEN buffer (10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl [pH 8.0]), placed in a 2.0-ml screw-cap
microcentrifuge tube filled with 100 µl (packed volume) of glass
beads (diameter, 0.1 mm; Biospec Products, Bartlesville, Okla.) and 100 µl of phenol-chloroform-isopropyl alcohol (50:49:1). To disrupt the
mycobacteria, the tube was oscillated on a Mini-Bead beater (Biospec
Products) for 1 min, and to separate phases, the tube was centrifuged
(at 12,000 × g for 5 min). After the aqueous phase was
transferred into another clean tube, 10 µl of 3 M sodium acetate and
250 µl of ice-cold ethanol were added; to enable the DNA to
precipitate, the mixture was kept at 
20°C for 10 min. The DNA
pellet was then washed with 70% ethanol, dissolved in 60 µl of TE
buffer (10 mM Tris-HCl-1 mM EDTA [pH 8.0]) and used as a template
for PCR. DNAs from sputa were prepared as above and dissolved in 60 µl of TE buffer (pH 8.0).
Amplification of rpoB and IS6110
DNA.
A set of mycobacterium-specific primers (MF and MR) which had
been used for the sequence analysis of rpoB DNA (342 bp)
(12) was also used in this study. Template DNA (50 ng for
the culture and 5 to 10 µl for the sputa) and 20 pmol of each primer
were added to a PCR mixture tube (AccuPower PCR PreMix; Bioneer,
Choongbuk, Korea) which contained 1 U of Taq DNA polymerase,
250 µM each deoxynucleoside triphosphate, 50 mM Tris-HCl (pH 8.3), 40 mM KCl, 1.5 mM MgCl2, and gel loading dye, and the volume
was adjusted to 20 µl with distilled water. The reaction mixture was
subjected to 30 cycles of amplification (30 s at 95°C, 30 s at
60°C, and 45 s at 72°C) followed by a 5-min extension at
72°C (Thermocycler model 9600; Perkin-Elmer Cetus). M. tuberculosis IS6110 DNAs (536 bp) (32)
were separately amplified using an IS6110 kit (catalog no.
N5811; Bioneer).
Restriction analysis.
Enzyme restriction sites for the
sizing of DNA fragments on each of the rpoB sequences
of mycobacteria (GenBank accession no. AF057449 to AF057493) were
generated by MapDraw, version 3.14 (DNASTAR, Madison, Wis.).
Restriction enzymes which produced the most discernible fragments
between clusters or strains were selected. Ten microliters of the
amplified PCR products was transferred to a fresh microcentrifuge tube
and digested with restriction enzymes, according to the supplier's
instructions. Following digestion, the mixtures were electrophoresed on
a 3% agarose gel (at 100 V for 25 min). DNA bands were visualized by
ethidium bromide staining and photographed. The results obtained from
the analysis of MAC and M. kansasii isolates were compared
with those of DT1-DT6 PCR (5) and PRA (BstEII
and HaeIII) of the hsp65 gene (2,
4), respectively.
Nucleotide sequencing.
The rpoB DNA sequences for
the five reference strains of M. kansasii (subspecies II to
VI) and clinical isolates were directly determined as previously
described (12). A 373A automatic sequencer and a BigDye
Terminator Cycle Sequencing kit (Perkin-Elmer Applied Biosystems,
Warrington, United Kingdom) were used. For the sequencing reaction, 60 ng of PCR-amplified DNA, 3.2 pmol of either the forward or the reverse
primer, and 8 µl of BigDye Terminator RR mix (Perkin-Elmer Applied
Biosystems; catalog no. 4303153) were mixed, and made up to 20 µl
with distilled water. The reaction was run with 5% (vol/vol) dimethyl
sulfoxide for 30 cycles of 15 s at 95°C, 10 s at
50°C, and 4 min at 60°C. The sequences determined were aligned by using the multiple alignment algorithm in the MegAlign package (Windows, version 3.12e; DNASTAR).
Nucleotide sequence accession numbers.
The rpoB
sequences of five M. kansasii subspecies (II to VI) were
deposited in GenBank (accession no. AF173084 to AF173088).
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RESULTS |
Restriction enzyme sites.
The nucleotide sequences of 49 rpoB DNAs were compared by computer-aided analysis. Four
useful restriction enzyme sites (HaeIII, HindII, MvaI, and AccII),
which produced the most discernible fragments among the tested
mycobacterial strains, were selected.
Differentiation of rapidly growing mycobacteria from slowly growing
mycobacteria.
Mycobacteria belonging to the natural division of
rapidly and slowly growing groups were differentiated by the PRA
patterns of HaeIII. All of the rapidly growing mycobacteria
had G467L468 encoded by
GGCCT(G/C) (12). HaeIII
specifically cleaves the rpoB DNAs of rapidly growing
mycobacteria, yielding a 61-bp DNA fragment, but does not cleave those
of slowly growing mycobacteria. In addition to the 61-bp fragment,
another useful marker DNA fragment for rapidly growing mycobacteria
upon HaeIII digestion was the 201-bp (or 202-bp) fragment.
Most rapidly growing mycobacteria produced the 201/202 bp fragment
except M. abscessus and M. fallax (168 bp) and
M. chitae (146 bp). Thus, M. abscessus, formerly Mycobacterium chelonae subsp. abscessus, was
easily distinguished from M. chelonae and M. fortuitum upon HaeIII digestion (Fig. 1). Nineteen clinical isolates (11 strains of M. fortuitum and 8 strains of M. chelonae) were confirmed as rapidly growing mycobacteria by PRA of
the rpoB DNA. Interestingly, eight strains of M. chelonae complex which had been identified by culture were
identified as M. abscessus by PRA of rpoB
(HaeIII and AccII), yielding characteristic bands
(Table 2). Furthermore, HaeIII
was also useful for distinguishing closely related species such as
M. avium-M. paratuberculosis-M. intracellulare and M. celatum type 1-M. celatum type 2. Five subspecies of
M. kansasii, except subspecies V, were differentiated from M. gastri.
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FIG. 1.
Differentiation of rapidly and slowly growing
mycobacteria by PRA of rpoB (HaeIII). Amplified
rpoB DNAs (342 bp) of reference strains were digested with
HaeIII and electrophoresed on a 3% agarose gel. DNA
fragments of 61 or 201 bp are observed in the lanes of rapidly growing
species (lanes 6 to 9). Lanes: M;  X174/RF DNA/HaeIII
digest; 1, M. tuberculosis; 2, M. avium; 3, M. intracellulare; 4, M. kansasii; 5, M. szulgai; 6, M. fortuitum; 7, M. abscessus;
8, M. chelonae; 9, M. chitae.
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Identification of M. tuberculosis complex.
Identification of M. tuberculosis complex was
simply performed with HindII. This restriction enzyme
was very useful for differentiating between M. tuberculosis
and MOTT. The rpoB DNA sequences of M. tuberculosis complex had only one HindII
restriction site
(GGG523TT
G524ACC525), which was
not found in MOTT and other bacteria (12). Thus, two fragments (232 and 110 bp) were generated from the DNAs of M. tuberculosis complex by HindII, while the MOTT type
strains were not cleaved (342 bp) (Fig.
2). When this procedure was applied to
134 strains of M. tuberculosis isolates (104 rifampin-susceptible and 30 rifampin-resistant strains), they were
easily identified (Table 3), showing
characteristic PRA patterns (data not shown).
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FIG. 2.
Differentiation of M. tuberculosis complex
from MOTT by the PRA of rpoB (HindII).
Amplified rpoB DNAs (342 bp) of 28 reference mycobacteria
were digested with HindII and electrophoresed on a 3%
agarose gel. Only DNAs from the members of M. tuberculosis
complex (A; lanes 1 to 4) were digested (232 and 110 bp), while those
of MOTT were not. Lanes: M, X174/RF DNA/HaeIII digest; 1, M. tuberculosis H37Rv; 2, M. bovis; 3, M. bovis BCG; 4, M. africanum; 5, M. avium; 6, M. paratuberculosis; 7, M. intracellulare; 8, M. scrofulaceum; 9, M. celatum; 10, M. xenopi; 11, M. kansasii; 12, M. gastri; 13, M. nonchromogenicum; 14, M. terrae; 15, M. triviale; 16, M. fortuitum; 17, M. chelonae;
18, M. gordonae; 19, M. szulgai; 20, M. ulcerans; 21, M. marinum; 22, M. simiae; 23, M. haemophilum; 24, M. malmoense; 25, M. smegmatis; 26, M. phlei; 27, M. vaccae; 28, M. genavense.
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PRA of
rpoB DNA (
HindII) and
IS
6110 PCR were directly applied to 50 sputa and 3 BAL
samples, and these results were compared
to the results of culture
(Table
4). Nothing was amplified from
the
culture-negative sputa, which were also smear negative for
acid-fast
bacilli (AFB). However,
rpoB DNAs (342 bp) were amplified
from the three smear-positive sputa identified as MOTT by culture.
rpoB PRA was compared with the IS
6110 PCR for the
ability to detect
the most common pathogen,
M. tuberculosis.
The sensitivity of
rpoB PRA for detection and identification
of
M. tuberculosis (93.2%)
was slightly lower than that of
IS
6110 PCR (97.7%). This discrepancy
possibly originates
from the different copy numbers of
rpoB and
IS
6110. However, it should be noted that the existence of
MOTT
in the three sputum samples was revealed by
rpoB PRA
(
HindII)
(Fig.
3A, lanes
9, 10, and 11), while it was not detected by IS
6110 PCR
(Fig.
3B, lanes 9, 10, and 11). Both methods specifically
detected
M. tuberculosis. M. tuberculosis was also detected and
cultivated from the three BAL samples.
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TABLE 4.
Comparison of the results obtained by two different
methods for detecting and identifying M. tuberculosis
from 50 sputa and 3 BAL specimens
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FIG. 3.
Simultaneous detection and identification of M. tuberculosis in sputa by rpoB PRA
(HindII). (A) rpoB DNAs (342 bp)
amplified from sputa were digested with HindII. Nine
specimens (lanes 1 to 8 and lane 12) showed two fragments (232 and 110 bp), while the others (lanes 9 to 11) remained intact, showing that the
former originated from M. tuberculosis and the latter from
MOTT. (B) For comparison with the rpoB PRA results,
IS6110 DNA (536 bp) was amplified from the same specimens.
Nothing was observed in three lanes (lanes 9 to 11). Lane M, X174/RF
DNA/HaeIII digest; lane Tb, M. tuberculosis
H37Rv; lane Av, M. avium.
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Differentiation of MOTT.
Many of the species pathogenic for
humans and animals belong to the slowly growing mycobacteria. These
could be differentiated by the simultaneous digestion of
rpoB DNA with MvaI, HaeIII, and AccII. For example M. avium, M. paratuberculosis,
and M. intracellulare are closely related and are not easy
to differentiate, but they were easily differentiated by digestion with
MvaI. Two DNA fragments of different sizes were produced
from the reference strains of M. avium (237 and 105 bp) and
M. intracellulare (294 and 48 bp). rpoB DNA from
M. scrofulaceum was not digested (342 bp) (Table 2). Only
HaeIII could distinguish M. avium (146, 117, 54, and 25 bp) from M. paratuberculosis (146, 117, and 79 bp).
Forty clinical isolates of MAC showed one of the PRA (MvaI)
patterns (data not shown) and thus were identified as either M. avium (8 strains) or M. intracellulare (32 strains)
(Table 3). The results were concordant with those obtained with
DT1-DT6 PCR.
M. terrae, M. nonchromogenicum, and
M. triviale,
grouped as the Terrae complex, members of which are rarely associated
with
human disease, were also easily distinguished (data not shown).
Other closely related pairs of clades, such as types 1 and 2 of
M. celatum, and
M. marinum and
M. ulcerans, could be differentiated
by
MvaI and
AccII.
Differentiation of M. kansasii subspecies and M. gastri.
The rpoB DNA sequences of five subspecies
of M. kansasii (subspecies II to VI) were determined in this
study and deposited in GenBank (accession no. AF173084 to AF173088).
There were marked variations (90.5 to 98.4% similarity) in the
rpoB sequences of six type strains. Subspecies VI of
M. kansasii showed low levels of similarity with the other
five subspecies (90.5 to 93.1%). However, it was clustered with five
other subspecies and M. gastri in the phylogenetic tree
constructed by either the neighborhood joining method or the UPGMA
method (data not shown). M. gastri, with a 16S rDNA sequence
identical with subspecies I, IV, and V, was most similar to subspecies
IV with respect to its rpoB sequence (95.8% similarity).
PRA was applied on the basis of these
rpoB sequences from
six type strains of subspecies. Although subspecies I of
M. kansasii was simply differentiated from
M. gastri by
HaeIII digestion,
MvaI and
AccII could
distinguish the six subspecies of
M. kansasii and
M. gastri (Table
2 and Fig.
4). This
procedure was also applied
to 47 clinical isolates of
M. kansasii. All strains showed identically
one of the fragment
polymorphisms of subspecies I (45 strains)
or II (2 strains) (Table
3).
This result was concordant with
those of the sequence analysis and PRA
of
hsp65 (data not shown).
The
rpoB sequences of
two subspecies II strains were identical,
and sequence variations among
the wild-type strains were less
than 0.6%.
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FIG. 4.
Differentiation of M. kansasii subspecies and
M. gastri by PRA of rpoB DNA. Amplified DNAs from
six subspecies of M. kansasii (lanes I to VI) and M. gastri (lane G) were digested with MvaI (A) and
AccII (B) and electrophoresed on a 3% agarose gel. Lanes
M1, X174/RF DNA/HaeIII digest; lanes M2, 25-bp DNA
ladder.
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Based on the above results, we constructed a scheme for the
differential identification of mycobacteria, including almost
all of
the major human pathogenic species, by
rpoB PRA (Fig.
5).
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FIG. 5.
Scheme for the differential identification of
mycobacteria by PRA of rpoB DNA (342 bp) using four
restriction enzymes (HaeIII, HindII,
MvaI, and AccII).
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DISCUSSION |
The most common mycobacterial infection in the world is caused by
M. tuberculosis. There are also increasing reports of
nontuberculous mycobacterial infection not only in the
immunocompromised but also in immunocompetent hosts (25,
27). Among those species that are currently recognized,
slowly growing nontuberculous mycobacteria such as MAC and
M. kansasii and rapidly growing species such as M. fortuitum, M. chelonae, and M. abscessus are
frequently encountered and are clinically important. To identify these
mycobacteria, rapid, simple, and sensitive methods based on molecular
biology have been introduced. Methods employing PCR are feasible for
the specific detection and identification or differentiation of
mycobacteria. PRA is one of these methods and is simple and convenient.
A scheme that was derived from the PRA of hsp65 has been
widely used. Many reports have shown that this method is useful for
species identification from the cultures of type strains, clinical
isolates (2, 4, 8, 19, 22, 28), and isolates growing in
BACTEC cultures (30, 31). It also allowed the elucidation
of genetic heterogeneity of many M. kansasii strains by
grouping them into five subspecies (4, 18, 31). However,
although the use of the BACTEC system reduced the culturing period, all
reports published to date have characterized only the cultured mycobacteria.
In this study, we developed a PRA method using rpoB DNA (342 bp), which comprises the region related to rifampin resistance (Rifr) in M. tuberculosis. This DNA was
previously used for the identification of mycobacteria and the
determination of the rifampin susceptibility of M. tuberculosis by sequence analysis (12). Compared to
the other methods PRA of rpoB has unique characteristics,
which are practical enough for routine use in the clinical laboratory.
First of all, it can not only detect mycobacteria but also determine their natures early in the laboratory process, and it proved to be very
useful for identifying rapidly growing mycobacteria and M. tuberculosis. The growth of mycobacteria could be predicted by the
PRA of rpoB as by the sequence analysis (12).
According to the deduced amino acid sequences, only the M. terrae complex had the marker amino acid (underlined) for rapidly
growing mycobacteria, i.e., G467L468
[GGG(T/C)TG] and not
G467M468 [GGC(T/A)TG],
which was found only in slowly growing mycobacteria (12).
So HaeIII (GGCCTG) could not cleave the site in
the M. terrae complex as it did in the rapidly growing
mycobacteria. Because other bacteria also have L468, it is
reasonable to use the unique DNA fragment (61 and/or 201 bp) to
identify the rapidly growing mycobacteria by HaeIII
digestion. Some of the rapidly growing mycobacteria were cultivated
from skin biopsy specimens, but unfortunately the biopsy materials were
not available. However, considering that only a few members of the
rapidly growing mycobacteria cause human infections, PRA of
rpoB could be applied directly to biopsy materials as with
sputa and BAL specimens.
Second, the differentiation of M. tuberculosis and MOTT
could be performed on sputa as well as on primary cultures. Thus, the
rapid detection and identification of M. tuberculosis or
MOTT should provide a prompt differential diagnosis and settle proper management issues depending on the diagnosis. By testing sputa and BAL
specimens, we were able to demonstrate the existence of mycobacteria
and to identify the mycobacteria detected. Identification of M. tuberculosis in sputa proved to be very useful. For the specific
detection of M. tuberculosis, several genes had been used.
Of these, IS6110, a multicopy gene in M. tuberculosis, has been most widely used (20).
Although several cases of false-positive results due to amplification
from MOTT (11, 15, 16) and false-negative results due to
the absence of IS6110 in certain strains of M. tuberculosis had been reported (35), M. tuberculosis is efficiently detected by the amplification of
IS6110 (32). The sensitivity of
IS6110 PCR for the detection of M. tuberculosis in sputa was slightly higher than that of rpoB PRA in this
study. This was possibly due to the different copy numbers of
rpoB and IS6110. However, it is noteworthy that
the existence of MOTT in sputa was revealed not by IS6110
PCR but by PRA of rpoB (HindII). Because
IS6110 PCR can detect only M. tuberculosis, MOTT
was not detected in sputa (Fig. 3). Considering the increasing
incidence of MOTT infection, the detection of MOTT and its
differentiation from M. tuberculosis should be of value. So
far, of the bacteria which reside in the respiratory tract, only
M. tuberculosis is known to have a HindII
restriction site in the 342-bp region.
While this report was being prepared, a method using rpoB
DNA was reported (14). However, it was not based on the
rpoB sequences of MOTT and nonmycobacteria. Thus, the
investigators had to use only the culture isolates and could not apply
PRA to sputa or other primary clinical specimens. The target
rpoB DNA differs from ours and does not comprise the
Rifr region. The target region we chose has very important
information on the rifampin resistance of M. tuberculosis
and other naturally rifampin resistant bacteria. Thus, the region can
be used for many purposes. Because HindII specifically
cleaves the PCR products from M. tuberculosis in sputa, the
cleaved fragments of rpoB DNA could be directly sequenced to
detect the mutations related to rifampin resistance (data not shown).
On occasion, the undigested DNA could be used for MOTT identification.
Therefore, we suggest that it would be very useful to apply PRA of
rpoB directly to the primary specimens early in the
diagnostic process. This would save the several weeks that are required
for primary culture. Amplification of rpoB DNA with
mycobacterium-specific primers (MF-MR) and the sequence information of
MOTT and rifampin-resistant M. tuberculosis would facilitate
the procedure.
The results of this study also demonstrated that PRA of rpoB
DNA usefully distinguished many closely related mycobacterial species.
Besides differentiating among M. abscessus-M. chelonae-M. fortuitum, M. avium-M. paratuberculosis-M. intracellulare, M. marinum-M. ulcerans, and M. celatum types 1 and 2, we
found that rpoB PRA allowed the differentiation of six
subspecies of M. kansasii. Although it may be isolated from
the environment (7, 13, 21), M. kansasii has
been increasingly reported to cause infection among patients infected
with human immunodeficiency virus (3). Molecular biology
methods are also feasible for the rapid identification of M. kansasii. However, such methods are not simple because of the
genetic heterogeneity of the species (1, 4, 34) and the
identical 16S rDNA sequences of M. kansasii and M. gastri (24). The existence of a genetic subspecies of
M. kansasii was suggested by information on 16S rDNA
(26), the hsp65 gene (4, 18), and
the spacer region between the 16S-23S rRNA genes (2). In
addition, several strains of M. kansasii have recently been proposed to constitute M. kansasii subspecies VI
(23). This report also showed that subspecies I, IV, and V
have the same 16S rDNA sequences as M. gastri; for this
reason the authors recommended sequencing the hsp65 gene or
spacer or performing classical tests to distinguish these species.
However, all reference strains and clinical isolates were simply
identified by PRA of rpoB. Not only do the results of
rpoB PRA correspond with those for the hsp65 gene, but they are also concordant with the fact that M. kansasii subspecies I is the most common human isolate
(2). Unfortunately, isolates of the other subspecies were
not available.
Results reaffirming the analytical advantages of rpoB DNA
have been demonstrated. Not only can rpoB PRA be applied to
culture isolates, but it is also applicable directly to sputa. Its use on sputa will save time and expense. Thus, this technique is suggested as a simple and useful method for the rapid detection and simultaneous identification of pathogenic mycobacteria in primary clinical specimens
or for the identification of culture isolates.
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ACKNOWLEDGMENTS |
This work was supported by grant 98-N1-02-01-A-08 from the
National Project for Medical Research, funded by the Ministry of Science and Technology (MOST) of Korea, by grant 1999 from Seoul National University College of Medicine and the Hospital Research Fund,
and in part by project BK21 for Medicine, Dentistry and Pharmacy.
We thank V. Vincent and E. Richter for providing the type strains of
M. kansasii.
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FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-799, Korea. Phone: (82) 2-740-8306. Fax: (82) 2-743-0881. E-mail:
yhkook{at}plaza.snu.ac.kr.
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Journal of Clinical Microbiology, June 2001, p. 2102-2109, Vol. 39, No. 6
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.6.2102-2109.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
