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Journal of Clinical Microbiology, September 1998, p. 2737-2741, Vol. 36, No. 9
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Molecular Procedure for Rapid Detection of
Burkholderia mallei and Burkholderia
pseudomallei
Adolf
Bauernfeind,*
Carsten
Roller,
Detlef
Meyer,
Renate
Jungwirth, and
Ines
Schneider
Max von Pettenkofer Institut, D-80336 Munich,
Germany
Received 10 November 1997/Returned for modification 21 March
1998/Accepted 11 June 1998
 |
ABSTRACT |
A PCR procedure for the discrimination of Burkholderia
mallei and Burkholderia pseudomallei was developed.
It is based on the nucleotide difference T 2143 C (T versus C at
position 2143) between B. mallei and B. pseudomallei detected in the 23S rDNA sequences. In
comparison with conventional methods the procedure allows more rapid
identification at reduced risk for infection of laboratory
personnel.
| |
TEXT |
Both Burkholderia
mallei and Burkholderia pseudomallei cause
severe infectious diseases in humans, namely, glanders or
melioidosis. B. pseudomallei is found in soil and water
(e.g., rice paddies). Humans can be infected by soil contamination of
skin abrasions, ingestion, or inhalation (5). Melioidosis is
endemic in Southeast Asia and northern Australia (14). Cases
in humans or animals occur sporadically throughout the world. The
mortality of untreated infections is high (95% [18]).
Glanders is primarily an infectious disease of the horse, mule, or
donkey. Glanders in humans is acquired from infected animals or by
contact with organisms causing human glanders via ingestion or
inhalation (7). Laboratory workers are at high risk to be
infected with glanders by aerosols (3). The outcome of
untreated infections (e.g., septicemia) is uniformly fatal (7,
18).
The detection and identification of B. mallei and B. pseudomallei entail a particular risk of infection for
laboratory personnel. We established a PCR procedure which allows
rapid, less dangerous, and specific identification and discrimination
of both species.
Organisms.
The strains used are specified in Table
1. Only nonviable material of B. mallei and B. pseudomallei was available.
Nucleic acid purification.
Genomic DNA was purified by using
the QiaAmp purification kit (Qiagen, Hilden, Germany).
PCR.
Custom oligonucleotide primers were purchased from MWG
Biotech, Ebersberg, Germany (Table 2).
Amplification reactions were performed in a 50-µl final volume
with 1 U of Taq polymerase (Boehringer, Mannheim, Germany),
5 µl of the reaction buffer supplied by the manufacturer
(diluted 1:10), a 10 µM concentration of each deoxynucleotide triphosphate, and a 50 pM concentration of each oligonucleotide primer.
To avoid reading mistakes, the Expand High Fidelity PCR system
(Boehringer) with a proofreading polymerase was used.
To enhance the specificity of
B. mallei
identification, a double concentration (100 pM) of a competitive
oligonucleotide probe,
which covers the respective 23S ribosomal
DNA (rDNA) primer binding
sites of all
Burkholderia
spp. except those of
B. mallei, was
used. Due to a
modification at its 3' end with an amino linker
(MWG Biotech) no
PCR products can be amplified with this probe.
Approximately 50 to 100 ng of DNA template was used in each
amplification. The PCR was performed in a GeneAmp PCR system 9600
(Perkin-Elmer Cetus) with an initial denaturation step of 5 min
at
95°C followed by 25 amplification cycles of 30 s at
95°C, 30
s at the primer-specific annealing
temperature (Table
2) and
45 s at 72°C. The samples were then
incubated at 72°C for another
7 min and cooled to 4°C.
Double-distilled, sterile water instead
of template DNA was used as the
negative control to exclude amplicon
contamination.
The amplification products were checked by agarose gel
electrophoresis and subsequently purified by using the PCR purification
kit (Qiagen) to desalt and remove excess primers.
Agarose gel electrophoresis.
Aliquots of PCR products were
diluted 10:1 in serving buffer (20% Ficoll, 50 mM EDTA) and
electrofocused in a 1% agarose gel (BIOzym, Oldendorf, Germany) on a
horizontal electrophoresis apparatus (Gibco BRL, Eggenstein, Germany)
at 100 V and 150 mA. Gels were stained with ethidium bromide as
described by Sambrook et al. (17) and documented digitally
with EASY Image Plus, version 4.13 (Herolab, Wiesloch, Germany).
Lengths of the PCR products were compared with those of internal PCR
product standards and DNA molecular weight markers (Boehringer).
Sequence determination.
Sequence analyses of the
in-vitro-amplified rDNA genes were performed as described previously
(10) with gene-specific primers (10, 16). The two
strands of the DNA were sequenced from different PCR products.
Sequencing was performed by the dideoxy chain termination procedure
using an automatic sequencer (373A; Applied Biosystems, Weiterstadt,
Germany).
Analysis of the sequence data.
The nucleotide sequences were
aligned with reference rDNA sequences provided in the noncommercial
program package ARB (beta-version 2.4; e-mail address:
arb{at}mikro.biologie.tu-muenchen.de). The secondary structure analysis
was performed as described by Ludwig et al. (11).
16S and 23S rDNA sequences.
The nucleotide sequences of the
16S rDNAs of B. mallei and B. pseudomallei (data
from the literature [6, 20, 24] and our own sequence
data) were found to be completely identical. So there is no possibility
to differentiate B. mallei from B. pseudomallei
at the 16S rDNA level.
So the 23S rDNA was analyzed for sequence deviations appropriate
to discriminate between the two species. As no sequence data
were
available at the time, the complete 23S rDNA gene sequences
(2,882 bp)
of two
B. mallei strains (ATCC 23344
T and
ATCC 15310) and three
B. pseudomallei type culture strains
were determined (Table
1). The 23S rDNA gene sequences of the
two
B. mallei strains were completely identical (Fig.
1). This
was in contrast to the 23S rDNA
gene sequences of the
B. pseudomallei strains, which turned
out to be heterogeneous. Nucleotide substitutions
in comparison with
the
B. mallei sequence were identified: for
B. pseudomallei 2, C 541 G (C versus G at position 541), T 542
C, T
543 A, C 544 A, T 1521 G, C 1522 A, C 1526 A, G 1529 T, and
A 1530 C;
for the
B. pseudomallei type strain, T 1521 G, C 1523
T, T
1524 C, C 1526 A, and A 1530 C; for
B. pseudomallei B, no
difference except C 2143 T. These substitutions were, however,
inadequate for species-specific primers, as they are variable
among
different strains of
B. pseudomallei.
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FIG. 1.
23S rDNA gene sequence of B. mallei ATCC
23344T. The T at position 2120 (corresponding to 2143 in
the E. coli numbering system described by Brosius et al.
[2]), which is different from the corresponding
nucleotide for all other Burkholderia species, is in
boldface.
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|
A comparison of the 23S rDNA nucleotide sequences of
B. mallei,
B. pseudomallei, and other
Burkholderia species demonstrated
that all three
B. mallei strains carry a thymidine (T) at position
2143 of the 23S
rDNA (Fig.
2) in contrast to a cytosine
(C) in
all strains of the other
Burkholderia species
investigated. So
this substitution, C 2143 T, appears unique for
B. mallei within
the
Burkholderia/Ralstonia
sublineage (Table
1). This finding
provides a possible means for the
molecular discrimination of
B. mallei from
B. pseudomallei.
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FIG. 2.
Alignment of the 23S rDNA within the helix 78 region. In
the sequences of all three B. mallei strains a T is located
at position 2143 instead of C in the B. pseudomallei
strains. Underlined nucleotides show the target signature for the
B. mallei-specific PCR primer (M 23-2). Bm, B. mallei; Bp, B. pseudomallei; Bv, B. vietnamiensis; Bc, B. cepacia; Bg, Burkholderia
gladioli DSM 4285.
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|
Definition of species-specific oligonucleotide primers.
For
the differentiation of the B. mallei/B. pseudomallei group
from other Burkholderia species (1), sequence
deviations within the region of helices 9 and 10 (Fig.
3) of the 23S rDNA were used to design
sense primer VMP 23-1 specific for Burkholderia vietnamiensis, B. mallei, and B. pseudomallei (Table 2); those in the helix 45 region (Fig.
4) were used to design antisense primer
MP 23-2 specific for B. mallei and B. pseudomallei (Table 2). A PCR with this pair of primers results in
a product of 1,051 bp with template DNAs from B. mallei and
B. pseudomallei but not with template DNAs from other
Burkholderia species (Table 1).
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FIG. 3.
Alignment of the 23S rDNA within the region of helices 9 and 10. Underlined nucleotides show the target signature specific for
B. vietnamiensis, B. mallei, and B. pseudomallei (VMP 23-1). *, no nucleotide at this position.
Species abbreviations are as defined in the legend for Fig. 2.
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FIG. 4.
Alignment of the 23S rDNA within the helix 45 region.
Underlined nucleotides show the target signature specific for B. mallei and B. pseudomallei (MP 23-2). Species
abbreviations are as defined in the legend for Fig. 2.
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|
The substitution T 2143 C within the helix 78 region of the 23S rDNA,
which is unique for
B. mallei, was used for the
definition
of
B. mallei-specific antisense oligonucleotide
primer M 23-2
(Table
2). In combination with sense primer CVMP 23-1 (for
Burkholderia cepacia,
B. vietnamiensis,
B. mallei,
B. pseudomallei) it allows
the discrimination of
B. mallei from the other
Burkholderia species
investigated. To enhance the
specificity of the test, antisense
oligonucleotide primer CVP-23-2
(Table
2), appropriate for
B. cepacia,
B. vietnamiensis, and
B. pseudomallei but not for
B. mallei, was constructed and modified at its 3' end to
block the
initiation of PCR amplification (Fig.
5). With this procedure
PCR products of
the expected size (526 bp) were obtained for all
three
B. mallei strains investigated, while no amplification product
was detectable with templates from other
Burkholderia
species
(Table
1).
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FIG. 5.
PCR with a B. mallei-specific primer
combination. (A) PCR without a competitive B. pseudomallei-directed 3'-modified probe (primers CVMP 23-1 and M
23-2); (B) PCR with a competitive B. pseudomallei-directed
3'-modified probe (primers CVMP 23-1, M 23-2, and CVP-23-2
[3'-modified]). Lanes 1, 9, and 17, DNA molecular weight marker;
lanes 2 and 10, B. mallei ATCC 23344T; lanes 3 and 11, B. mallei ATCC 15310; lanes 4 and 12, B. mallei ATCC 10399; lanes 5 and 13, B. pseudomallei ATCC
23343T; lanes 6 and 14, B. pseudomallei ATCC
15682; lanes 7 and 15, clinical isolate of B. pseudomallei;
lanes 8 and 16, negative controls without template DNA. No difference
between the lanes with or without competitive primers is detectable.
|
|
B. mallei and
B. pseudomallei were assigned to
rRNA homology group II according to the results of DNA-rRNA
hybridization studies
(
13). The separation of
B. mallei and
B. pseudomallei into distinct
species is not
supported by the data from nucleic acid analysis.
DNA-DNA
hybridizations revealed DNA similarities of more than
80% between the
two species (
15), 10% above the threshold set
for the
separation of species (
19,
23). This close relationship
is
confirmed by comparison of the 16S and 23S rDNA sequences.
Our results
as well as the results of other authors (
6,
8,
20,
24)
indicate complete identity of the 16S rDNA of both
species. The
nucleotide difference detected within the 23S rDNA
at position 2143 (T
in
B. mallei, C in
B. pseudomallei) appears
to be
species specific as it was present in the 23S rDNA sequences
of all
three
B. mallei strains and was not detectable in any of
the
15
B. pseudomallei strains. The nucleotide exchange is
located
within the more conserved domain V of the 23S rDNA
(
12). It
therefore can be regarded as a stable
species-specific character.
Another difference between species was detected by Tyler et al.
(
20) in the 16S-23S spacer area common for both species
(G
in
B. mallei in comparison with T in
B. pseudomallei). This
signalizes a further possibility for a
B. mallei-specific signature
sequence. However, this
difference appears less appropriate as
it has to be regarded as less
stable due to the insignificance
of selective constraints within the
noncoding spacer region. This
difference between
B. mallei
and
B. pseudomallei was identified
by comparison of only one
strain of each species. Furthermore,
Kostman et al. (
9)
found a high level of variability within
the 16S-23S spacers of
different
B. cepacia strains.
The identification of three different 23S rDNA sequences within the
B. pseudomallei strains reveals a remarkable heterogeneity.
This observation is supported by the results of DNA hybridization
studies by Rogul et al. (
15) indicating genetic
heterogeneity
of
B. pseudomallei as well. A sequence
analysis of the 16S-23S
intercistronic spacers (
20)
demonstrated heterogeneity within
the same strain (
B. pseudomallei type strain). This heterogeneity
may be useful for
genotyping
B. pseudomallei strains from different
origins. It appears worthwhile to elucidate the degree of relationship
between different lines of descent within the species
B. pseudomallei.
The two-species concept for
B. mallei and
B. pseudomallei is based on major differences between them in their
phenotypes (e.g.,
biochemical activities) and in the clinical symptoms
and epidemiologies
of the diseases they cause. These differences
justify the definition
of
B. mallei and
B. pseudomallei as two distinct species in the
modern understanding
of taxonomy, which is polyphasic (
4,
21),
integrating
phenotypic, genotypic, and phylogenetic information.
In medical microbiology unequivocal identification of
B. mallei and
B. pseudomallei by
conventional biochemical reactions
is usually achieved. There is,
however, a remarkably high risk
of becoming infected while working with
living cultures of
B. mallei or
B. pseudomallei. This risk could be significantly reduced
by using
the identification procedure described. The speciation
part of the
laboratory work can then be performed with killed
bacteria or the
template DNA thereof. Apart from the reduction
of the risk of
infection, the time necessary for speciation can
be reduced to about 3 to 4 h in comparison with 2 days for conventional
identification.
Furthermore, the procedure can be adapted for
use for in situ
hybridization in clinical specimens (
22).
Nucleotide sequence accession numbers.
The complete 23S rDNA
gene sequences of the cited B. pseudomallei and
B. mallei strains will appear in the EMBL Database
under the accession no. Y17183 (B. mallei ATCC
23344T) and Y17184 (B. pseudomallei
ATCC 23343T).
| |
ACKNOWLEDGMENTS |
We thank D. Vidal, La Tronche, France, and E.-J. Fincke and H. Neubauer, Munich, Germany, and V. K. E. Lim, Kuala Lumpur, Malaysia,
for providing inactivated bacterial specimens for DNA isolation of
B. mallei and B. pseudomallei and B. Tümmler, Hannover, Germany, F. Ratjen, Essen, Germany, H. Bärmeier, Erlangen, Germany, N. Høiby, Copenhagen,
Denmark, J. Dankert, Amsterdam, The Netherlands, and D. P. Speert, E. Mahenthiralingam, and D. Henry, Vancouver, Canada for
strains of other Burkholderia species.
This work was supported by a grant from the German ministry of defense
(development of molecular procedures for diagnosis and epidemiology of
B. pseudomallei, B. mallei, and B. cepacia; Gesch. Z.: BA III 1/E/B31E/Q0343/Q5932).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Max von
Pettenkofer Institut, Pettenkoferstr. 9a, D-80336 Munich, Germany.
Phone: 0049-89-5160-5268. Fax: 0049-89-5160-5266. E-mail:
Adolf.Bauernfeind{at}mvp-bak.med.uni-muenchen.de.
| |
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Journal of Clinical Microbiology, September 1998, p. 2737-2741, Vol. 36, No. 9
0095-1137/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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