Previous Article | Next Article 
Journal of Clinical Microbiology, September 2000, p. 3165-3173, Vol. 38, No. 9
Department of Pediatrics, University of
British Columbia and British Columbia's Children's Hospital, British
Columbia's Research Institute for Children's and Women's
Health,1 Molecular Diagnostics
Laboratory, British Columbia's Centre for Disease Control, and
Department of Pathology, University of British
Columbia,2 Terragen Diversity
Incorporated,3 Department of
Microbiology, University of British
Columbia,4 and Department of Medicine,
University of British Columbia,5 Vancouver,
British Columbia, Canada, and Laboratory for Microbiology,
Faculty of Science, University of Ghent, Ghent,
Belgium6
Received 17 February 2000/Returned for modification 28 April
2000/Accepted 12 June 2000
Bacteria of the Burkholderia cepacia complex consist of
five discrete genomic species, including genomovars I and III and three
new species: Burkholderia multivorans (formerly genomovar II), Burkholderia stabilis (formerly genomovar IV), and
Burkholderia vietnamiensis (formerly genomovar V). Strains
of all five genomovars are capable of causing opportunistic human
infection, and microbiological identification of these closely related
species is difficult. The 16S rRNA gene (16S rDNA) and recA
gene of these bacteria were examined in order to develop rapid tests
for genomovar identification. Restriction fragment length polymorphism
(RFLP) analysis of PCR-amplified 16S rDNA revealed sequence
polymorphisms capable of identifying B. multivorans and
B. vietnamiensis but insufficient to discriminate strains
of B. cepacia genomovars I and III and B. stabilis. RFLP analysis of PCR-amplified recA
demonstrated sufficient nucleotide sequence variation to enable
separation of strains of all five B. cepacia complex
genomovars. Complete recA nucleotide sequences were
obtained for 20 strains representative of the diversity of the B. cepacia complex. Construction of a recA phylogenetic
tree identified six distinct clusters (recA groups):
B. multivorans, B. vietnamiensis, B. stabilis, genomovar I, and the subdivision of genomovar III
isolates into two recA groups, III-A and III-B. Alignment
of recA sequences enabled the design of PCR primers for the
specific detection of each of the six latter recA groups. The recA gene was found on the largest chromosome within
the genome of B. cepacia complex strains and, in contrast
to the findings of a previous study, only a single copy of the gene was
present. In conclusion, analysis of the recA gene of the
B. cepacia complex provides a rapid and robust nucleotide
sequence-based approach to identify and classify this taxonomically
complex group of opportunistic pathogens.
The Burkholderia cepacia
complex is a very diverse group of bacteria (28). They are
important opportunistic human pathogens that cause devastating
infections in patients with cystic fibrosis (CF) (8, 13) and
in other vulnerable individuals (25). The ability of
B. cepacia to cause disease is not limited to the human
host, as these bacteria are also important plant pathogens (7). In addition, B. cepacia complex bacteria may
have commercially useful properties and have been used in agriculture
as biocontrol agents and in the bioremediation of toxic agents (9,
14). Current taxonomic classification divides isolates previously
classified as B. cepacia into five genomovars or discrete
genomic species, all of which may be isolated from clinical infection
(28). Strains of genomovar II have been proposed as the new
species Burkholderia multivorans (28), and
strains of genomovar V were found to be members of the proposed species
Burkholderia vietnamiensis (6, 28). Strains
within genomovar IV have recently been proposed as the new species
Burkholderia stabilis (29). The remaining genomovars, I and III, await species designation if differential phenotypic tests can be found (28). Determination of the
genomovar status of B. cepacia complex strains was based on
a polyphasic taxonomic approach which utilized phenotypic tests such as
whole-cell protein profile analysis and genotypic tests such as DNA-DNA
hybridization (27, 28). A single test for identification of
genomovar status is currently not available. Even conventional
phenotypic identification of B. cepacia and its
differentiation from closely related species are often not
straightforward (3, 10, 24, 30). Incorrect diagnosis of
infection, especially in patients with CF, may have serious clinical
ramifications (13). A simple means of B. cepacia complex species identification will enhance our understanding of the
pathogenesis and epidemiology of these opportunistic human pathogens.
Molecular diagnostic probes based on PCR provide a rapid and frequently
highly discriminatory means of microbial identification (2, 15,
24, 30). In order to develop rapid tests to determine the
genomovar status of B. cepacia complex isolates, we examined nucleotide sequence polymorphism in two genes, the 16S rRNA gene (16S
rDNA) and recA gene. Although widely used for bacterial
systematics, recent results demonstrate that 16S rDNA is limited in its
ability to differentiate the B. cepacia complex (15,
23). Nucleotide sequence variation within this gene is not
sufficient to enable all current genomovars within the complex to be
easily identified (2, 15, 23). The B. cepacia
complex recA (RecA is a protein essential for repair and
recombination of DNA [5, 11]) was chosen as an
alternative template gene for the following reasons. First, analysis of
recA has proven very useful in previous studies of molecular
systematics among closely related bacteria (5, 11). Second,
although only a limited number of B. cepacia genes have been
characterized, two recA sequences, each from a different strain, were available in the nucleotide sequence databases as a basis
to initiate analysis (19, 31). Finally, in B. cepacia, recA has been shown to be diploid, with a
single copy of the gene residing on each of the two large chromosomes
present in B. cepacia strain ATCC 25416 (21). If
both copies of recA are identical, they may form a more
robust platform upon which to base diagnostic PCR probes than the
multicopy rRNA genes, which are dispersed across the multiple replicons
constituting the genome of B. cepacia and are potentially
susceptible to genomic rearrangement (4, 12, 21). Diagnostic
tests for the determination of B. cepacia complex genomovar
status, developed using systematic genetic approaches, are described.
Bacterial strains.
B. cepacia complex strains were
cultured and biochemically identified as described previously (10,
16, 28). Bacteria were routinely grown on blood agar, Trypticase
soy agar, or Luria-Bertani broth (LB; 23) at 35°C
for 24 to 48 h until confluent growth was obtained (10,
16). Molecular identification approaches were developed using the
35 isolates listed in Table 1, of which 29 isolates were derived from a published panel of strains
representative of each genomovar (18). The sources of the
six additional strains initially screened in this study are also
provided in Table 1. The latter 35 isolates and 68 further B. cepacia complex isolates examined (103 total) were recovered from
various sources including patients with CF, patients with non-CF
infection, and the natural environment. Strains were selected to be
representative of the genetic diversity of B. cepacia
complex from previous studies (10, 16-18, 28). Additional
reference strains were obtained from the American Type Culture
Collection (ATCC, Manassas, Va.). Related bacterial species commonly
recovered from patients with CF and misidentified as B. cepacia were derived from previous studies (10, 16).
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
DNA-Based Diagnostic Approaches for Identification
of Burkholderia cepacia Complex, Burkholderia
vietnamiensis, Burkholderia multivorans,
Burkholderia stabilis, and Burkholderia
cepacia Genomovars I and III
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
B. cepacia complex strains used to develop
molecular diagnostic approaches
Genomovar status. The genomovar status of B. cepacia complex strains used to develop the molecular identification strategies was determined by whole-cell protein profile analysis and a polyphasic approach as described previously (27, 28).
PCR analysis.
PCR was performed essentially as described
previously (16, 17) however, PCR reagents were purchased
from Qiagen Inc. Canada, and tests were performed in the presence of
Qiagen Q solution, which enhances amplification of DNA templates which
are rich in G+C content (Qiagen Inc. [http://www.qiagen.com]).
Template DNA was prepared from fresh overnight cultures as previously
described (17), with modification of the lysis buffer to
include pronase at 0.5 mg/ml. DNA was quantitated by visualization on
agarose gels, and approximately 20 ng was incorporated into 25-µl
reactions which contained 1 U Taq DNA polymerase, 250 µM
(each) deoxynucleoside triphosphate, 1.5 mM MgCl2, and 1×
PCR buffer. Approximately 20 pmol of each appropriate oligonucleotide
primer (Table 2) was added to each
reaction, and amplification was carried out using a Gene Cycler
(Bio-Rad, Mississauga, Ontario, Canada) for 30 cycles of 30 s at
94°C, 45 s at the appropriate annealing temperature (Table 2),
and 60 s at 72°C; a final extension of 10 min at 72°C was
applied to all thermal cycles. The sequences and specificity of all PCR
primers used in this study are provided in Table 2.
|
Nucleotide sequence analysis. Sequencing reactions were prepared using ABI PRISM DyeDeoxy Terminator cycle sequencing kits with AmpliTaq FS DNA polymerase according to the manufacturer's instructions and analyzed using either an ABI PRISM model 373 Stretch or a model 377 DNA sequencer (Perkin-Elmer Applied Biosystems, Foster City, Calif.). Raw sequences from both strands of the PCR products were then aligned, and a consensus sequence was derived using DNASTAR software (DNASTAR Inc., Madison, Wis.). Sequence identity was confirmed by analysis using the basic local alignment sequence tool (BLAST) (1) at the National Center for Biotechnology Information (NCBI, Bethesda, Md.).
Phylogenetic analysis. Evolutionary relationships between recA genes were determined using the Data Analysis in Molecular Biology software (DAMBE; http://web.hku.hk/~xxia/software/software.htm). After recA sequence determination, multiple sequence alignments were performed using CLUSTAL W (26). Phylogenetic trees were drawn from the resulting alignments using the genetic distance-based neighbor-joining algorithms of DAMBE. Paralinear and Jukes-Cantor-based algorithms were evaluated and found to demonstrate identical phylogenies for the strains examined (data not shown). Trees constructed using Jukes-Cantor distance matrices are presented in this report. Sequence input order was randomized, and 100 data sets were examined by bootstrapping resampling statistics for each analysis.
PFGE and Southern hybridization. Macrorestriction and pulsed-field gel electrophoresis (PFGE) genomic fingerprinting of B. cepacia strains were performed as described previously (18). Multiple replicons which constitute the B. cepacia genome were separated as previously described (4, 21). Southern blot transfer of the DNA to nylon membranes was allowed to proceed for 48 h and probed with a digoxigenin-labeled recA PCR probe as previously described (17).
Nucleotide sequence accession numbers. The complete recA nucleotide sequences were determined for the 20 B. cepacia complex strains listed in Table 1. Sequences were submitted to GenBank as an aligned set, and each was assigned an accession number as shown in Table 1.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Analysis of 16S rRNA genes of the B. cepacia complex
and closely related species.
Amplified-rDNA restriction analysis
(ARDRA; 23, 27) was performed to examine sequence
polymorphism of the 16S rRNA gene in strains representative of the
B. cepacia complex. The primer pair used for ARDRA analysis
of the 16S rDNA in this study is novel (UNI2 and UNI5 [Table 2]) and
has proven effective for amplification of the gene from all bacterial
species tested to date (S. K. Byrne, unpublished data). To
evaluate the efficacy of both the 16S rRNA gene (and recA
[see below]) for systematic differentiation of the B. cepacia complex, a panel of 35 (Table 1) strains representative of
all five genomovars of the B. cepacia complex
(18) was initially examined. The 16S rDNA 1-kb amplicon was
successfully amplified with primers UNI2 and UNI5 from all 35 strains.
The identity of the amplified DNA was confirmed to encode 16S rDNA by
direct nucleotide sequence analysis of PCR products (data not shown).
To detect nucleotide sequence variation within the amplified gene,
several restriction endonucleases expected to cleave frequently within
bacterial rDNA were screened for their ability to reveal
genomovar-specific RFLPs (data not shown). The enzyme DdeI
was found to produce the most discriminatory RFLPs for the B. cepacia complex (Fig. 1A). Three 16S
rDNA ARDRA patterns were found among the 35 strains (Table 1): type 1, B. vietnamiensis; type 2, B. cepacia genomovars I
and III and B. stabilis; and type 3, B. multivorans (Fig. 1A). Polymorphism detected by ARDRA analysis of
the 16S rDNA was consistent with the taxonomic classification of the
new species B. multivorans and B. vietnamiensis
(28) but was not sufficient to separate B. cepacia genomovars I and III (28) and the new species
B. stabilis (29).
|
PCR amplification of B. cepacia complex recA gene. Two recA sequences, each from a different strain of B. cepacia, were available within GenBank. The genomovar status of B. cepacia strain JN25, from which one recA sequence (GenBank accession no. D90120) was derived, was not known; it was an isolate of clinical origin characterized in Japan in 1990 (19). The second recA sequence (accession no. U70431) was derived from the B. cepacia reference strain ATCC 17616 (31); this strain has been classified as the new species B. multivorans (28). Each sequence was aligned using BLAST (1). PCR primers BCR1 and BCR2 (Table 2) were designed from homologous sequences at the 5' and 3' ends of the recA open reading frame. These primers amplified a single 1-kb amplicon from all 35 strains representative of the B. cepacia complex (Table 1). The identity of the 1-kb fragment was subsequently confirmed to be recA by direct nucleotide sequence analysis of PCR products (see below). A further 68 isolates, each with biochemical- (10) and genomovar-specific (28) properties characteristic of the B. cepacia complex, also tested positive with these recA primers. PCR with primers BCR1 and BCR2 failed to amplify PCR products from the following species: B. gladioli, R. picketti, C. meningosepticum, S. maltophilia, C. acidovorans, Escherichia coli, and Pseudomonas aeruginosa (data not shown).
RFLP analysis of the B. cepacia complex
recA gene.
RFLP analysis with AluIII,
BsaWI, MnlI, and HaeIII was
investigated. RFLP types generated by digestion with HaeIII
were the most discriminatory among the four restriction enzymes (data
not shown). Ten distinct HaeIII RFLP patterns were found
among the 35-isolate B. cepacia complex panel initially
examined, and each pattern was assigned an alphabetical code (Fig.
2; Table 1). HaeIII RFLP
analysis was capable of discriminating among all five genomovars (Fig.
2A) except for RFLP pattern J, which was shared by the genomovar III
strain PC184 and all of the B. stabilis strains examined. To distinguish genomovar III and B. stabilis
strains with this RFLP type, analysis with an additional enzyme (such as MnlI [Fig. 2B]) was required.
|
|
Nucleotide sequence analysis of recA. To confirm the sequence variation detected by RFLP analysis of the amplified recA gene and facilitate the design of PCR primers specific to each B. cepacia genomovar, complete nucleotide sequence analysis of the gene was performed for 20 strains representative of each recA RFLP type (Table 1). The complete recA gene was sequenced in two 500-bp segments using the combinations of PCR primers BCR1, BCR2, BCR3, and BCR4 described in Table 2. The sequence of each segment was combined to produce the full-length recA. Sequence analysis of the recA amplicon of B. multivorans ATCC 17616 was performed as a control for the sequencing strategy; the sequence determined in this study was identical to the published sequence for this strain (31). BLAST analysis (1) of each nucleotide sequence confirmed that each encodes RecA with homology to the published B. cepacia sequences (19, 31). Computational analysis of the HaeIII restriction sites within each nucleotide sequence matched those HaeIII sites determined by RFLP analysis of the complete recA amplicon for each strain. Sequence-based restriction mapping of recA from genomovar III strain PC184 and the B. stabilis strains demonstrated the presence of closely overlapping HaeIII sites. However, seven cleavage sites were present in PC184 and six in the B. stabilis recA, and each of these recA sequences was quite different (see phylogenetic analysis below). The minor differences in the HaeIII fragment sizes were not distinguished under the electrophoresis conditions used for RFLP analysis (Fig. 2A).
Phylogenetic analysis of recA.
Construction of a
recA phylogenetic tree of the B. cepacia complex
was carried out as described in Materials and Methods. The
Bordetella pertussis recA gene (accession no. X53457) was used to root the tree because previous molecular systematic analysis of
bacterial recA genes had demonstrated that it was closely
related to B. cepacia recA and had placed both bacteria
within the 
-subgroup of the Proteobacteria (5,
11). The P. aeruginosa (accession no. X05691),
Methylobacillus flagellatum (accession no. M35525), and
Xanthomonas campestris (accession no. U49086)
recA sequences were also included in the phylogenetic
analysis as indicators of the ability of the analysis to differentiate
among unrelated species. The resulting nucleotide sequence-based
phylogenetic tree is shown in Fig. 3. All
five previously determined genomovars (28) formed distinct
arms within the tree, consistent with their proposal as new species
(28, 29) or unnamed genomic species awaiting formal binomial
classification (28). In total, six distinct phylogenetic
groups were present within the recA tree and were given the
following designations as recA groups (Fig. 3): I, B. cepacia genomovar I; III-A and III-B, B. cepacia
genomovar III recA clusters A and B, respectively;
Bs, B. stabilis; Bm, B. multivorans; and Bv, B. vietnamiensis. These
B. cepacia complex phylogenies were also distinct from the
unrelated bacterial species chosen as out groups for the tree (Fig. 3).
Phylogenetic trees based on the protein translation of each
recA nucleotide sequence also demonstrated the same
clustering of genomovars and new species (data not shown).
|
Genomovar-specific PCR. Using the nucleotide sequence alignment of the 20 novel recA genes determined above and the two within the databases (19, 31), specific PCR primers were designed to detect the six recA-derived clusters of the B. cepacia complex identified by phylogenetic analysis (see Fig. 3). Primer pairs for the identification of recA groups I, III-A, III-B, B. multivorans, B. stabilis, and B. vietnamiensis are listed in Table 2. Each primer pair was designed to match all sequences determined within each phylogenetic subgroup (see Fig. 3) and be mismatched at the 3' base (and as many other bases as possible) with all other B. cepacia complex recA sequences (Table 2). Each primer set was tested on all 103 B. cepacia complex isolates. Amplification products of the correct size (Table 2) were obtained from strains of the appropriate recA group with each specific primer pair (data not shown). No products of the predicted size were seen in strains outside the target group for each primer pair (data not shown). The identity of each recA group-specific PCR product was confirmed to be the appropriate portion of the recA gene by direct sequence analysis of the PCR product for each of the following strains: B. vietnamiensis PC259, B. multivorans ATCC 17616, B. stabilis LMG 7000, B. cepacia ATCC 25416 (I), B. cepacia C5424 (III-A), and B. cepacia C1394 (III-B) (data not shown). The recA-specific PCR, HaeIII recA RFLP analysis, and 16S ARDRA results obtained from all 103 B. cepacia complex isolates are summarized in Table 3.
The majority of 103 strains examined were genetically heterogeneous (66 out of 103) and possessed unique RAPD or PFGE fingerprints (Table 3). Each recA primer set was 100% specific for isolates which possessed recA HaeIII RFLP types (Table 1) characteristic of the respective new species, conventional genomovar, or recA phylogenetic subgroup for which they were designed (Table 3). RFLP types and specific PCR results remained stable for sequential or multiple isolates of a given genetic strain type. ARDRA profiles obtained with the 16S rDNA primers also correlated as expected with the recA-specific primer results (Table 3).Genomic location of recA.
Given the
multichromosomal structure of the B. cepacia complex genome
(4, 12, 21), it was important to determine the copy number
and genomic location of recA. Previous studies on B. cepacia genomovar I strain ATCC 25416 (Table 1) demonstrated the
presence of two copies of the gene, one on each large chromosome (21). Electrophoretic separation of linear replicons from
B. multivorans genomovars I, III-A, and III-B and B. stabilis strains is shown in Fig.
4A. Each genomovar possessed a
multireplicon structure, with between two and four large replicons,
ranging from approximately 0.6 to 3.8 Mb in size, being detected (data not shown for B. vietnamiensis). For each B. cepacia complex genomovar tested, only the largest chromosome
produced a positive signal when probed with recA by Southern
hybridization (Fig. 4B). Further analysis of the genomic location of
the recA gene using RFLP approaches was also consistent with
the presence of only one copy of the recA gene within the
B. cepacia complex genome (data not shown).
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
Systematic analysis of two conserved genes, 16S rDNA and recA, in strains of the B. cepacia complex demonstrated that each may have a use in the identification and classification of this important group of opportunistic pathogens. Although widely used for bacterial identification, analysis of the 16S rDNA of the B. cepacia complex by an ARDRA approach demonstrated that this gene was useful for discrimination of the two new species, B. vietnamiensis and B. multivorans, but insufficient to separate genomovars I and III and B. stabilis. Because of these discriminatory limitations, a novel PCR-RFLP analysis was developed based on the B. cepacia recA gene. Nucleotide sequence polymorphism was identified in recA which was sufficient to identify all five current B. cepacia complex genomovars defined by a polyphasic approach (28). This study validates the use of nonribosomal housekeeping genes in the development of alternative molecular diagnostic strategies, especially among taxonomically complex species such as B. cepacia. A complete nucleotide sequence-based identification approach based on this conserved, stable, and single-copy gene was developed.
Four approaches based on the B. cepacia complex recA gene may be used to identify the genomovar status of clinical isolates and, depending on the resources of the diagnostic laboratory, they may be applied as individual tests or multiple complementary analyses. First, amplification of recA with primers BCR1 and BCR2 can be used as an initial means of placing an isolate within the B. cepacia complex since cross-reaction of these primers with other species commonly found in CF sputum was not detected. In the future, direct testing and application of these B. cepacia complex-specific primers to CF sputum, which contains multiple bacterial species, may also be possible. Second, after successful amplification of recA, RFLP with HaeIII and MnlI may be used to place the isolate within a specific genomovar or recA group (Table 1). Third, the design of recA group-specific primers capable of identifying all of the genomovars within the current B. cepacia complex (28, 29) provides a means of genomovar identification in a single test. With further development such recA-based primer sets may be employed in a single multiplex PCR for rapid genomovar detection. Finally, nucleotide sequence determination of recA provides a powerful means of both identification and classification of these poorly defined bacteria. Overall, the availability of several complementary tests based on a single diagnostic gene provides a robust approach to B. cepacia complex identification.
Phylogenetic analysis of recA from a range of bacteria has demonstrated that the gene may be very useful for the separation of closely related species and may define evolutionary trees that are consistent with those observed for rRNA genes (5, 11). Its application herein confirms these observations and illustrates the benefits of examining protein-encoding DNA when systematic analysis of ribonucleotide-encoding sequences fails to yield discriminatory classifications among closely related species. Analysis of a single genomic locus, recA, in this study was consistent with the polyphasic taxonomic approach (27) originally used to define the B. cepacia complex genomovars, with the exception of the phylogenetic subdivision of organisms classified as genomovar III by DNA-DNA hybridization experiments (28). The taxonomic significance of the subdivision of genomovar III-classified strains into recA groups III-A and III-B awaits further research. In addition B. cepacia complex strains possessing novel recA RFLPs, not reported in this study, have recently been identified (E. Mahenthiralingam and P. Vandamme, unpublished data); preliminary polyphasic analysis (27) of these strains indicates that they are members of novel genomic species within the complex (Mahenthiralingam and Vandamme, unpublished data). The ability to use recA analysis as a means to assist taxonomic classification of the B. cepacia complex was demonstrated in the recent proposal of B. stabilis (formerly genomovar IV) as a new species (29) and is also corroborated by the data presented in this study.
Analysis of recA also revealed new insights into the epidemiology and pathogenesis of the B. cepacia complex. Concerns about commercial use of these bacteria (9, 14) are confirmed by the recA phylogenetic analysis. Strains isolated from clinical infection and those recovered from the natural environment cluster within the B. multivorans, B. vietnamiensis, and B. cepacia genomovar I arms of the evolutionary tree (Fig. 3). In addition, among the 103 isolates screened, there were clinical and environmental strains of B. cepacia genomovar III-B which shared the same RFLP type (Table 3) and hence would cluster phylogenetically if nucleotide sequence analysis were subsequently performed. Only within recA group III-A and B. stabilis were no environmental isolates found in this study. The results of recA analysis corroborate the taxonomic findings of Vandamme et al. (28) which demonstrate that all genomovars within the B. cepacia complex can cause human opportunistic infection. Phylogenetic distinctions between environmental and clinical strains do not appear to exist, and all strains within the complex appear to possess conserved traits which enable them to cause infection in vulnerable individuals.
Determination of the phylogeny of the B. cepacia complex enabled the association of known epidemiological features with each recA subgroup to be examined. The cable pilus gene was primarily associated with genomovar III strains of the ET12 lineage (22), such as K56-2 and C5424, which clustered in recA group III-A (Table 1; Fig. 3). However, one unique strain recovered from an individual CF patient at a center in Australia, which possessed DNA homologous to cblA, was identified as recA HaeIII RFLP type E and B. cepacia genomovar I by specific PCR (Table 3). Therefore, possession of cblA still appears to be a rare feature for the B. cepacia complex (17), but it is not solely possessed by genomovar III strains of the ET12 lineage (22). The prevalence of the BCESM within the B. cepacia complex recA phylogeny also appears more widespread that originally observed (17). The marker was associated with strains that had spread among patients with CF when it was originally described (17); however, unlike cblA, no specific virulence trait has been associated with the BCESM locus. From the results of this study, DNA homologous to the BCESM appears to be primarily associated with epidemic CF strains belonging to recA groups III-A and III-B (all classified as genomovar III) (18). However, BCESM was also found in B. vietnamiensis strain ATCC 29424, but none of the other B. vietnamiensis strains examined (Table 3), suggesting that it may occasionally occur outside of the highly transmissible genomovar III strains (16-18). The recA probes described herein provide an additional means of identifying strains with an epidemiological precedent (16-18) for patient-to-patient spread among individuals with CF. The recA group III-A- and III-B-specific primers detect strains which have caused several outbreaks (16-18) and may ultimately prove to be better diagnostic probes than the not-yet fully-defined BCESM DNA (17). Early diagnosis of infection by B. cepacia recA PCR may in future facilitate rapid-enactment infection control measures and aggressive therapy to improve the poor outcome associated with B. cepacia infection in patients with CF (13).
In conclusion, nucleotide sequence analysis of recA is a rapid and reproducible means of identifying all of the current genomovars and new species within the B. cepacia complex. Rapid identification of bacteria recovered from patients with CF and from other vulnerable individuals is vital if we are to further understand the clinical risks posed by each genomovar and new species within the B. cepacia complex.
| |
ACKNOWLEDGMENTS |
|---|
We thank David Speert for his support and helpful discussion and for providing access to the Canadian B. cepacia Strain Repository. Deborah Henry is acknowledged for assistance in biochemical analysis and identification of B. cepacia strains. We thank Gary Probe, Richard Parkes, and Julie Fadden for excellent technical assistance.
This work was funded by grants from the Canadian Cystic Fibrosis Foundation and UK Cystic Fibrosis Trust (E.M., project grant PJ472) and the Fund for Scientific Research, Belgium (P.V.). E.M. acknowledges the British Columbia Lung association for provision of a Career Development Award and the British Columbia Research Institute for Children's and Women's Health for an Investigator Establishment Award.
| |
FOOTNOTES |
|---|
* Corresponding author. Present address: Cardiff School of Biosciences, Main Building, Cardiff University, P.O. Box 915, Cardiff CF10 3TL, United Kingdom. Phone: 44 (029) 20875875. Fax: 44 (029) 20874305. E-mail: MahenthiralingamE{at}cardiff.ac.uk.
Present address: Department of Medical Genetics, University of
Alberta, Edmonton, Alberta, Canada.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
|
|---|
| 1. |
Altschul, S. F.,
T. L. Madden,
A. A. Schäffer,
J. Zhang,
Z. Zhang,
W. Miller, and D. L. Lipman.
1997.
Gapped BLAST and PSI-BLAST: a new generation of protein database search programs.
Nucl. Acids Res.
25:3389-3402 |
| 2. |
Bauernfeind, A.,
I. Scheider,
R. Jungwirth, and C. Roller.
1998.
Discrimination of Burkholderia species detectable in cystic fibrosis patients by PCR.
J. Clin. Microbiol.
36:2748-2751 |
| 3. | Burdge, D. R., M. A. Noble, M. E. Campbell, V. L. Krell, and D. P. Speert. 1995. Xanthomonas maltophilia misidentified as Pseudomonas cepacia in cultures of sputum from patients with cystic fibrosis: a diagnostic pitfall with major clinical implications. Clin. Infect. Dis. 20:445-448[Medline]. |
| 4. |
Cheng, H.-P., and T. G. Lessie.
1994.
Multiple replicons constituting the genome of Pseudomonas cepacia 17616.
J. Bacteriol.
176:4034-4042 |
| 5. | Eisen, J. A. 1995. The RecA protein as a model molecule for systematic studies of bacteria: comparison of trees of RecAs and 16S rRNAs from the same species. J. Mol. Evol. 41:1105-1123[Medline]. |
| 6. |
Gillis, M.,
T. V. Van,
R. Bardin,
M. Goor,
P. Hebbar,
A. Willems,
P. Segers,
K. Kersters,
T. Heulin, and M. P. Fernandez.
1995.
Polyphasic taxonomy in the genus Burkholderia leading to an emended description of the genus and proposition of Burkholderia vietnamiensis sp. nov. for N2-fixing isolates from rice in Vietnam.
Int. J. Syst. Bacteriol.
45:274-289 |
| 7. | Gonzalez, C. F., E. A. Pettit, V. A. Valadez, and E. M. Provin. 1997. Mobilization, cloning and sequence determination of a plasmid encoded polygalacturonase from a phytopathogenic Burkholderia (Pseudomonas) cepacia. Mol. Plant-Microbe Interact. 10:840-851[Medline]. |
| 8. |
Govan, J. R. W., and V. Deretic.
1996.
Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aerugionosa and Burkholderia cepacia.
Microbiol. Rev.
60:539-574 |
| 9. |
Govan, J. R. W., and P. Vandamme.
1998.
Agricultural and medical microbiology a time for bridging gaps.
Microbiology
144:2373-2375 |
| 10. | Henry, D. A., M. E. Campbell, J. J. LiPuma, and D. P. Speert. 1996. Identification of Burkholderia cepacia from patients with cystic fibrosis and a new selective medium for its isolation. J. Clin. Microbiol. 35:614-619[Abstract]. |
| 11. |
Karlin, S.,
G. M. Weinstock, and V. Brendel.
1995.
Bacterial classifications derived from RecA protein sequence comparisons.
J. Bacteriol.
177:6881-6893 |
| 12. | Lessie, T. G., W. Hendrickson, B. D. Manning, and R. Devereux. 1996. Genomic complexity and plasticity of Burkholderia cepacia. FEMS Microbiol. Lett. 144:117-128[CrossRef][Medline]. |
| 13. |
LiPuma, J. J.
1998.
Burkholderia cepaciamanagement issues and new insights.
Clin. Chest Med.
19:473-486[CrossRef][Medline].
|
| 14. | LiPuma, J. J., and E. Mahenthiralingam. 1999. Commercial use of Burkholderia cepacia: are there additional threats? Emerg. Infect. Dis. 5:5-6. |
| 15. |
LiPuma, J. J.,
B. J. Dulaney,
J. D. McMenamin,
P. W. Whitby,
T. L. Stull,
T. Coenye, and P. Vandamme.
1999.
Development of rRNA-based PCR assays for the identification of Burkholderia cepacia complex isolates recovered from cystic fibrosis patients.
J. Clin. Microbiol.
37:3167-3170 |
| 16. | Mahenthiralingam, E., M. E. Campbell, D. A. Henry, and D. P. Speert. 1996. Epidemiology of Burkholderia cepacia infection in patients with cystic fibrosis: analysis by random amplified polymorphic DNA (RAPD) fingerprinting. J. Clin. Microbiol. 34:2914-2920[Abstract]. |
| 17. | Mahenthiralingam, E., D. A. Simpson, and D. P. Speert. 1997. Identification and characterization of a novel DNA marker associated with epidemic strains of Burkholderia cepacia recovered from patients with cystic fibrosis. J. Clin. Microbiol. 35:808-816[Abstract]. |
| 18. |
Mahenthiralingam, E.,
T. Coenye,
J. Chung,
D. P. Speert,
J. R. W. Govan,
P. Taylor, and P. Vandamme.
2000.
Diagnostically and experimentally useful panel of strains from the Burkholderia cepacia complex.
J. Clin. Microbiol.
38:910-913 |
| 19. | Nakazawa, T., M. Kimoto, and M. Abe. 1990. Cloning, sequencing and transcriptional analysis of the recA gene of Pseudomonas cepacia. Gene 94:83-88[CrossRef][Medline]. |
| 20. | Revets, H., P. Vandamme, A. Van Zeebroeck, K. De Boeck, M. J. Streulens, J. Verhaegen, J. P. Ursi, G. Verschaegen, H. Franckx, A. Malfroot, I. Dab, and S. Lauwers. 1996. Burkholderia (Pseudomonas) cepacia and cystic fibrosis: the epidemiology in Belgium. Acta Clin. Belg. 51:222-230[Medline]. |
| 21. | Rodley, P. D., U. Römmling, and B. Tümmler. 1995. A physical genome map of the Burkholderia cepacia type strain. Mol. Microbiol. 17:57-67[Medline]. |
| 22. |
Sajjan, U. S.,
L. Sun,
R. Goldstein, and J. F. Forstner.
1995.
Cable (Cbl) type II pili of cystic fibrosis-associated Burkholderia (Pseudomonas) cepacia: nucleotide sequence of the cblA major subunit pilin gene and novel morphology of the assembled appendage fibers.
J. Bacteriol.
177:1030-1038 |
| 23. |
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloninga laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 24. |
Segonds, C.,
T. Heulin,
N. Marty, and G. Chabanon.
1999.
Differentiation of Burkholderia species by PCR-restriction fragment length polymorphism analysis of 16S rRNA gene and application to cystic fibrosis isolates.
J. Clin. Microbiol.
37:2201-2208 |
| 25. | Speert, D. P., M. Bond, R. C. Woodman, and J. T. Curnutte. 1994. Infection with Pseudomonas cepacia in chronic granulomatous disease: role of nonoxidative killing by neutrophils in host defense. J. Infect. Dis. 170:1524-1531[Medline]. |
| 26. |
Thompson, J. D.,
D. G. Higgins, and T. J. Gibson.
1994.
CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice.
Nucleic Acids Res.
22:4673-4680 |
| 27. |
Vandamme, P.,
B. Pot,
M. Gillis,
P. de Vos,
K. Kersters, and J. Swings.
1996.
Polyphasic taxonomy, a consensus approach to bacterial systematics.
Microbiol. Rev.
60:407-438 |
| 28. |
Vandamme, P.,
B. Holmes,
M. Vancanneyt,
T. Coenye,
B. Hoste,
R. Coopman,
H. Revets,
S. Lauwers,
M. Gillis,
K. Kersters, and J. R. W. Govan.
1997.
Occurrence of multiple genomovars of Burkholderia cepacia in cystic fibrosis patients and proposal of Burkholderia multivorans sp. nov.
Int. J. Syst. Bacteriol.
47:1188-1200 |
| 29. |
Vandamme, P.,
E. Mahenthiralingam,
B. Holmes,
T. Coenye,
B. Hoste,
P. De Vos,
D. Henry, and D. P. Speert.
2000.
Identification and population structure of Burkholderia stabilis sp. nov. (formerly Burkholderia cepacia genomovar IV).
J. Clin. Microbiol.
38:1042-1047 |
| 30. | van Pelt, C., C. M. Verduin, W. H. F. Goessens, M. C. Vos, B. Tümmler, C. Segonds, F. Reubsaet, H. Verbrugh, and A. van Belkum. 1999. Identification of Burkholderia spp. in the clinical microbiology laboratory: comparison of conventional and molecular methods. J. Clin. Microbiol. 37:3167-3170. |
| 31. | van Waasbergen, L. G., S. P. Kidambi, and R. V. Miller. 1998. Construction of a recA mutant of Burkholderia (formerly Pseudomonas) cepacia. Appl. Microbiol. Biotechnol. 49:59-65[CrossRef][Medline]. |
Journal of Clinical Microbiology, September 2000, p. 3165-3173, Vol. 38, No. 9
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Hogardt, M., Ulrich, J., Riehn-Kopp, H., Tummler, B.
(2009). EuroCareCF Quality Assessment of Diagnostic Microbiology of Cystic Fibrosis Isolates. J. Clin. Microbiol.
47: 3435-3438
[Abstract] [Full Text] -
Wainwright, C E, France, M W, O'Rourke, P, Anuj, S, Kidd, T J, Nissen, M D, Sloots, T P, Coulter, C, Ristovski, Z, Hargreaves, M, Rose, B R, Harbour, C, Bell, S C, Fennelly, K P
(2009). Cough-generated aerosols of Pseudomonas aeruginosa and other Gram-negative bacteria from patients with cystic fibrosis. Thorax
64: 926-931
[Abstract] [Full Text] -
Otto-Karg, I., Jandl, S., Muller, T., Stirzel, B., Frosch, M., Hebestreit, H., Abele-Horn, M.
(2009). Validation of Vitek 2 Nonfermenting Gram-Negative Cards and Vitek 2 Version 4.02 Software for Identification and Antimicrobial Susceptibility Testing of Nonfermenting Gram-Negative Rods from Patients with Cystic Fibrosis. J. Clin. Microbiol.
47: 3283-3288
[Abstract] [Full Text] -
Springman, A. C., Jacobs, J. L., Somvanshi, V. S., Sundin, G. W., Mulks, M. H., Whittam, T. S., Viswanathan, P., Gray, R. L., LiPuma, J. J., Ciche, T. A.
(2009). Genetic Diversity and Multihost Pathogenicity of Clinical and Environmental Strains of Burkholderia cenocepacia. Appl. Environ. Microbiol.
75: 5250-5260
[Abstract] [Full Text] -
Behrends, V., Ebbels, T. M. D., Williams, H. D., Bundy, J. G.
(2009). Time-Resolved Metabolic Footprinting for Nonlinear Modeling of Bacterial Substrate Utilization. Appl. Environ. Microbiol.
75: 2453-2463
[Abstract] [Full Text] -
Vanlaere, E., Baldwin, A., Gevers, D., Henry, D., De Brandt, E., LiPuma, J. J., Mahenthiralingam, E., Speert, D. P., Dowson, C., Vandamme, P.
(2009). Taxon K, a complex within the Burkholderia cepacia complex, comprises at least two novel species, Burkholderia contaminans sp. nov. and Burkholderia lata sp. nov.. Int. J. Syst. Evol. Microbiol.
59: 102-111
[Abstract] [Full Text] -
Degand, N., Carbonnelle, E., Dauphin, B., Beretti, J.-L., Le Bourgeois, M., Sermet-Gaudelus, I., Segonds, C., Berche, P., Nassif, X., Ferroni, A.
(2008). Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Nonfermenting Gram-Negative Bacilli Isolated from Cystic Fibrosis Patients. J. Clin. Microbiol.
46: 3361-3367
[Abstract] [Full Text] -
Pimentel, J. D., MacLeod, C.
(2008). Misidentification of Pandoraea sputorum Isolated from Sputum of a Patient with Cystic Fibrosis and Review of Pandoraea Species Infections in Transplant Patients. J. Clin. Microbiol.
46: 3165-3168
[Abstract] [Full Text] -
Murray, S., Charbeneau, J., Marshall, B. C., LiPuma, J. J.
(2008). Impact of Burkholderia Infection on Lung Transplantation in Cystic Fibrosis. Am. J. Respir. Crit. Care Med.
178: 363-371
[Abstract] [Full Text] -
Bosch, A., Minan, A., Vescina, C., Degrossi, J., Gatti, B., Montanaro, P., Messina, M., Franco, M., Vay, C., Schmitt, J., Naumann, D., Yantorno, O.
(2008). Fourier Transform Infrared Spectroscopy for Rapid Identification of Nonfermenting Gram-Negative Bacteria Isolated from Sputum Samples from Cystic Fibrosis Patients. J. Clin. Microbiol.
46: 2535-2546
[Abstract] [Full Text] -
Vanlaere, E., LiPuma, J. J., Baldwin, A., Henry, D., De Brandt, E., Mahenthiralingam, E., Speert, D., Dowson, C., Vandamme, P.
(2008). Burkholderia latens sp. nov., Burkholderia diffusa sp. nov., Burkholderia arboris sp. nov., Burkholderia seminalis sp. nov. and Burkholderia metallica sp. nov., novel species within the Burkholderia cepacia complex. Int. J. Syst. Evol. Microbiol.
58: 1580-1590
[Abstract] [Full Text] -
Jacobs, J. L., Fasi, A. C., Ramette, A., Smith, J. J., Hammerschmidt, R., Sundin, G. W.
(2008). Identification and Onion Pathogenicity of Burkholderia cepacia Complex Isolates from the Onion Rhizosphere and Onion Field Soil. Appl. Environ. Microbiol.
74: 3121-3129
[Abstract] [Full Text] -
van Hal, S., Stark, D., Marriott, D., Harkness, J.
(2008). Achromobacter xylosoxidans subsp. xylosoxidans prosthetic aortic valve infective endocarditis and aortic root abscesses. J Med Microbiol
57: 525-527
[Abstract] [Full Text] -
Jorda-Vargas, L., Castaneda, N. C., Centron, D., Degrossi, J., D'Aquino, M., Valvano, M. A., Procopio, A., Galanternik, L.
(2008). Prevalence of Indeterminate Genetic Species of Burkholderia cepacia Complex in a Cystic Fibrosis Center in Argentina. J. Clin. Microbiol.
46: 1151-1152
[Full Text] -
Lynch, K. H., Dennis, J. J.
(2008). Development of a Species-Specific fur Gene-Based Method for Identification of the Burkholderia cepacia Complex. J. Clin. Microbiol.
46: 447-455
[Abstract] [Full Text] -
McDowell, A., Perry, A. L., Lambert, P. A., Patrick, S.
(2008). A new phylogenetic group of Propionibacterium acnes. J Med Microbiol
57: 218-224
[Abstract] [Full Text] -
Baldwin, A., Mahenthiralingam, E., Drevinek, P., Pope, C., Waine, D. J., Henry, D. A., Speert, D. P., Carter, P., Vandamme, P., LiPuma, J. J., Dowson, C. G.
(2008). Elucidating Global Epidemiology of Burkholderia multivorans in Cases of Cystic Fibrosis by Multilocus Sequence Typing. J. Clin. Microbiol.
46: 290-295
[Abstract] [Full Text] -
Kutty, P. K., Moody, B., Gullion, J. S., Zervos, M., Ajluni, M., Washburn, R., Sanderson, R., Kainer, M. A., Powell, T. A., Clarke, C. F., Powell, R. J., Pascoe, N., Shams, A., LiPuma, J. J., Jensen, B., Noble-Wang, J., Arduino, M. J., McDonald, L. C.
(2007). Multistate Outbreak of Burkholderia cenocepacia Colonization and Infection Associated With the Use of Intrinsically Contaminated Alcohol-Free Mouthwash. Chest
132: 1825-1831
[Abstract] [Full Text] -
Mendes, R., Pizzirani-Kleiner, A. A., Araujo, W. L., Raaijmakers, J. M.
(2007). Diversity of Cultivated Endophytic Bacteria from Sugarcane: Genetic and Biochemical Characterization of Burkholderia cepacia Complex Isolates. Appl. Environ. Microbiol.
73: 7259-7267
[Abstract] [Full Text] -
Pimentel, J. D., Dubedat, S. M., N. Dodds, E. L., Benn, R. A. V.
(2007). Identification of Isolates within the Burkholderia cepacia Complex by a Multiplex recA and 16S rRNA Gene Real-Time PCR Assay. J. Clin. Microbiol.
45: 3853-3854
[Full Text] -
Turton, J. F., Arif, N., Hennessy, D., Kaufmann, M. E., Pitt, T. L.
(2007). Revised Approach for Identification of Isolates within the Burkholderia cepacia Complex and Description of Clinical Isolates Not Assigned to Any of the Known Genomovars. J. Clin. Microbiol.
45: 3105-3108
[Abstract] [Full Text] -
Brown, A. R., Govan, J. R. W.
(2007). Assessment of Fluorescent In Situ Hybridization and PCR-Based Methods for Rapid Identification of Burkholderia cepacia Complex Organisms Directly from Sputum Samples. J. Clin. Microbiol.
45: 1920-1926
[Abstract] [Full Text] -
Ortega, X. P., Cardona, S. T., Brown, A. R., Loutet, S. A., Flannagan, R. S., Campopiano, D. J., Govan, J. R. W., Valvano, M. A.
(2007). A Putative Gene Cluster for Aminoarabinose Biosynthesis Is Essential for Burkholderia cenocepacia Viability. J. Bacteriol.
189: 3639-3644
[Abstract] [Full Text] -
Cunha, M. V., Pinto-de-Oliveira, A., Meirinhos-Soares, L., Salgado, M. J., Melo-Cristino, J., Correia, S., Barreto, C., Sa-Correia, I.
(2007). Exceptionally High Representation of Burkholderia cepacia among B. cepacia Complex Isolates Recovered from the Major Portuguese Cystic Fibrosis Center. J. Clin. Microbiol.
45: 1628-1633
[Abstract] [Full Text] -
Denis, M. St., Ramotar, K., Vandemheen, K., Tullis, E., Ferris, W., Chan, F., Lee, C., Slinger, R., Aaron, S. D.
(2007). Infection With Burkholderia cepacia Complex Bacteria and Pulmonary Exacerbations of Cystic Fibrosis. Chest
131: 1188-1196
[Abstract] [Full Text] -
Weinberg, J. B., Alexander, B. D., Majure, J. M., Williams, L. W., Kim, J. Y., Vandamme, P., LiPuma, J. J.
(2007). Burkholderia glumae Infection in an Infant with Chronic Granulomatous Disease. J. Clin. Microbiol.
45: 662-665
[Abstract] [Full Text] -
Estivariz, C. F., Bhatti, L. I., Pati, R., Jensen, B., Arduino, M. J., Jernigan, D., LiPuma, J. J., Srinivasan, A.
(2006). An Outbreak of Burkholderia cepacia Associated With Contamination of Albuterol and Nasal Spray.. Chest
130: 1346-1353
[Abstract] [Full Text] -
Elborn, J S
(2006). Practical Management of Cystic Fibrosis. Chronic Respiratory Disease
3: 161-165
-
Vonberg, R.-P., Haussler, S., Vandamme, P., Steinmetz, I.
(2006). Identification of Burkholderia cepacia complex pathogens by rapid-cycle PCR with fluorescent hybridization probes. J Med Microbiol
55: 721-727
[Abstract] [Full Text] -
Bosshard, P. P., Zbinden, R., Abels, S., Boddinghaus, B., Altwegg, M., Bottger, E. C.
(2006). 16S rRNA Gene Sequencing versus the API 20 NE System and the VITEK 2 ID-GNB Card for Identification of Nonfermenting Gram-Negative Bacteria in the Clinical Laboratory. J. Clin. Microbiol.
44: 1359-1366
[Abstract] [Full Text] -
Assaad, W., Magalhaes, M., Plesa, M., Hart, C. A., Cornelis, P., Winstanley, C.
(2006). Identical Burkholderia cepacia complex strain types isolated from multiple patients attending a hospital in Brazil. J Med Microbiol
55: 247-249
[Full Text] -
Gingues, S., Kooi, C., Visser, M. B., Subsin, B., Sokol, P. A.
(2005). Distribution and Expression of the ZmpA Metalloprotease in the Burkholderia cepacia Complex. J. Bacteriol.
187: 8247-8255
[Abstract] [Full Text] -
Mahenthiralingam, E, Vandamme, P
(2005). Taxonomy and pathogenesis of the Burkholderia cepacia complex. Chronic Respiratory Disease
2: 209-217
[Abstract] -
Campana, S., Taccetti, G., Ravenni, N., Favari, F., Cariani, L., Sciacca, A., Savoia, D., Collura, A., Fiscarelli, E., De Intinis, G., Busetti, M., Cipolloni, A., d'Aprile, A., Provenzano, E., Collebrusco, I., Frontini, P., Stassi, G., Trancassini, M., Tovagliari, D., Lavitola, A., Doherty, C. J., Coenye, T., Govan, J. R. W., Vandamme, P.
(2005). Transmission of Burkholderia cepacia Complex: Evidence for New Epidemic Clones Infecting Cystic Fibrosis Patients in Italy. J. Clin. Microbiol.
43: 5136-5142
[Abstract] [Full Text] -
Baldwin, A., Mahenthiralingam, E., Thickett, K. M., Honeybourne, D., Maiden, M. C. J., Govan, J. R., Speert, D. P., LiPuma, J. J., Vandamme, P., Dowson, C. G.
(2005). Multilocus Sequence Typing Scheme That Provides Both Species and Strain Differentiation for the Burkholderia cepacia Complex. J. Clin. Microbiol.
43: 4665-4673
[Abstract] [Full Text] -
Middleton, P. G., Kidd, T. J., Williams, B.
(2005). Combination aerosol therapy to treat Burkholderia cepacia complex. Eur Respir J
26: 305-308
[Abstract] [Full Text] -
Wellinghausen, N., Kothe, J., Wirths, B., Sigge, A., Poppert, S.
(2005). Superiority of Molecular Techniques for Identification of Gram-Negative, Oxidase-Positive Rods, Including Morphologically Nontypical Pseudomonas aeruginosa, from Patients with Cystic Fibrosis. J. Clin. Microbiol.
43: 4070-4075
[Abstract] [Full Text] -
Payne, G. W., Vandamme, P., Morgan, S. H., LiPuma, J. J., Coenye, T., Weightman, A. J., Jones, T. H., Mahenthiralingam, E.
(2005). Development of a recA Gene-Based Identification Approach for the Entire Burkholderia Genus. Appl. Environ. Microbiol.
71: 3917-3927
[Abstract] [Full Text] -
Coenye, T., Spilker, T., Reik, R., Vandamme, P., LiPuma, J. J.
(2005). Use of PCR Analyses To Define the Distribution of Ralstonia Species Recovered from Patients with Cystic Fibrosis. J. Clin. Microbiol.
43: 3463-3466
[Abstract] [Full Text] -
Drevinek, P., Vosahlikova, S., Cinek, O., Vavrova, V., Bartosova, J., Pohunek, P., Mahenthiralingam, E.
(2005). Widespread clone of Burkholderia cenocepacia in cystic fibrosis patients in the Czech Republic. J Med Microbiol
54: 655-659
[Abstract] [Full Text] -
Lewis, D. A., Jones, A., Parkhill, J., Speert, D. P., Govan, J. R. W., LiPuma, J. J., Lory, S., Webb, A. K., Mahenthiralingam, E.
(2005). Identification of DNA Markers for a Transmissible Pseudomonas aeruginosa Cystic Fibrosis Strain. Am. J. Respir. Cell Mol. Bio.
33: 56-64
[Abstract] [Full Text] -
Reik, R., Spilker, T., LiPuma, J. J.
(2005). Distribution of Burkholderia cepacia Complex Species among Isolates Recovered from Persons with or without Cystic Fibrosis. J. Clin. Microbiol.
43: 2926-2928
[Abstract] [Full Text] -
Ramette, A., LiPuma, J. J., Tiedje, J. M.
(2005). Species Abundance and Diversity of Burkholderia cepacia Complex in the Environment. Appl. Environ. Microbiol.
71: 1193-1201
[Abstract] [Full Text] -
McDowell, A., Valanne, S., Ramage, G., Tunney, M. M., Glenn, J. V., McLorinan, G. C., Bhatia, A., Maisonneuve, J.-F., Lodes, M., Persing, D. H., Patrick, S.
(2005). Propionibacterium acnes Types I and II Represent Phylogenetically Distinct Groups. J. Clin. Microbiol.
43: 326-334
[Abstract] [Full Text] -
Jones, A M, Dodd, M E, Govan, J R W, Barcus, V, Doherty, C J, Morris, J, Webb, A K
(2004). Burkholderia cenocepacia and Burkholderia multivorans: influence on survival in cystic fibrosis. Thorax
59: 948-951
[Abstract] [Full Text] -
Brisse, S., Cordevant, C., Vandamme, P., Bidet, P., Loukil, C., Chabanon, G., Lange, M., Bingen, E.
(2004). Species Distribution and Ribotype Diversity of Burkholderia cepacia Complex Isolates from French Patients with Cystic Fibrosis. J. Clin. Microbiol.
42: 4824-4827
[Abstract] [Full Text] -
Souza, A. V., Moreira, C. R., Pasternak, J., Hirata, M. d. L., Saltini, D. A., Caetano, V. C., Ciosak, S., Azevedo, F. M., Severino, P., Vandamme, P., Magalhaes, V. D.
(2004). Characterizing uncommon Burkholderia cepacia complex isolates from an outbreak in a haemodialysis unit. J Med Microbiol
53: 999-1005
[Abstract] [Full Text] -
Vinion-Dubiel, A. D., Spilker, T., Dean, C. R., Monteil, H., LiPuma, J. J., Goldberg, J. B.
(2004). Correlation of wbiI Genotype, Serotype, and Isolate Source within Species of the Burkholderia cepacia Complex. J. Clin. Microbiol.
42: 4121-4126
[Abstract] [Full Text] -
McDowell, A., Mahenthiralingam, E., Dunbar, K. E.A., Moore, J. E., Crowe, M., Elborn, J. S.
(2004). Epidemiology of Burkholderia cepacia complex species recovered from cystic fibrosis patients: issues related to patient segregation. J Med Microbiol
53: 663-668
[Abstract] [Full Text] -
De Boeck, K., Malfroot, A., Van Schil, L., Lebecque, P., Knoop, C., Govan, J.R.W., Doherty, C., Laevens, S., Vandamme, P.
(2004). Epidemiology of Burkholderia cepacia complex colonisation in cystic fibrosis patients. Eur Respir J
23: 851-856
[Abstract] [Full Text] -
De Soyza, A, Morris, K, McDowell, A, Doherty, C, Archer, L, Perry, J, Govan, J R W, Corris, P A, Gould, K
(2004). Prevalence and clonality of Burkholderia cepacia complex genomovars in UK patients with cystic fibrosis referred for lung transplantation. Thorax
59: 526-528
[Abstract] [Full Text] -
Vermis, K., Coenye, T., LiPuma, J. J., Mahenthiralingam, E., Nelis, H. J., Vandamme, P.
(2004). Proposal to accommodate Burkholderia cepacia genomovar VI as Burkholderia dolosa sp. nov.. Int. J. Syst. Evol. Microbiol.
54: 689-691
[Abstract] [Full Text] -
Plesa, M., Kholti, A., Vermis, K., Vandamme, P., Panagea, S., Winstanley, C., Cornelis, P.
(2004). Conservation of the opcL gene encoding the peptidoglycan-associated outer-membrane lipoprotein among representatives of the Burkholderia cepacia complex. J Med Microbiol
53: 389-398
[Abstract] [Full Text] -
Manno, G., Dalmastri, C., Tabacchioni, S., Vandamme, P., Lorini, R., Minicucci, L., Romano, L., Giannattasio, A., Chiarini, L., Bevivino, A.
(2004). Epidemiology and Clinical Course of Burkholderia cepacia Complex Infections, Particularly Those Caused by Different Burkholderia cenocepacia Strains, among Patients Attending an Italian Cystic Fibrosis Center. J. Clin. Microbiol.
42: 1491-1497
[Abstract] [Full Text] -
Baldwin, A., Sokol, P. A., Parkhill, J., Mahenthiralingam, E.
(2004). The Burkholderia cepacia Epidemic Strain Marker Is Part of a Novel Genomic Island Encoding Both Virulence and Metabolism-Associated Genes in Burkholderia cenocepacia. Infect. Immun.
72: 1537-1547
[Abstract] [Full Text] -
Turton, J. F., Kaufmann, M. E., Mustafa, N., Kawa, S., Clode, F. E., Pitt, T. L.
(2003). Molecular Comparison of Isolates of Burkholderia multivorans from Patients with Cystic Fibrosis in the United Kingdom. J. Clin. Microbiol.
41: 5750-5754
[Abstract] [Full Text] -
O'Carroll, M R, Kidd, T J, Coulter, C, Smith, H V, Rose, B R, Harbour, C, Bell, S C
(2003). Burkholderia pseudomallei: another emerging pathogen in cystic fibrosis. Thorax
58: 1087-1091
[Abstract] [Full Text] -
Cunha, M. V., Leitao, J. H., Mahenthiralingam, E., Vandamme, P., Lito, L., Barreto, C., Salgado, M. J., Sa-Correia, I.
(2003). Molecular Analysis of Burkholderia cepacia Complex Isolates from a Portuguese Cystic Fibrosis Center: a 7-Year Study. J. Clin. Microbiol.
41: 4113-4120
[Abstract] [Full Text] -
Detsika, M. G., Corkill, J. E., Magalhaes, M., Glendinning, K. J., Hart, C. A., Winstanley, C.
(2003). Molecular Typing of, and Distribution of Genetic Markers among, Burkholderia cepacia Complex Isolates from Brazil. J. Clin. Microbiol.
41: 4148-4153
[Abstract] [Full Text] -
Drevinek, P., Cinek, O., Melter, J., Langsadl, L., Navesnakova, Y., Vavrova, V.
(2003). Genomovar distribution of the Burkholderia cepacia complex differs significantly between Czech and Slovak patients with cystic fibrosis. J Med Microbiol
52: 603-604
[Full Text] -
Liu, L., Spilker, T., Coenye, T., LiPuma, J. J.
(2003). Identification by Subtractive Hybridization of a Novel Insertion Element Specific for Two Widespread Burkholderia cepacia Genomovar III Strains. J. Clin. Microbiol.
41: 2471-2476
[Abstract] [Full Text] -
Langley, R., Kenna, D. T., Vandamme, P., Ure, R., Govan, J. R. W.
(2003). Lysogeny and bacteriophage host range within the Burkholderia cepacia complex. J Med Microbiol
52: 483-490
[Abstract] [Full Text] -
Ramisse, V., Balandreau, J., Thibault, F., Vidal, D., Vergnaud, G., Normand, P.
(2003). DNA-DNA hybridization study of Burkholderia species using genomic DNA macro-array analysis coupled to reverse genome probing. Int. J. Syst. Evol. Microbiol.
53: 739-746
[Abstract] [Full Text] -
Stryjewski, M. E., LiPuma, J. J., Messier, R. H. Jr., Reller, L. B., Alexander, B. D.
(2003). Sepsis, Multiple Organ Failure, and Death Due to Pandoraea pnomenusa Infection after Lung Transplantation. J. Clin. Microbiol.
41: 2255-2257
[Abstract] [Full Text] -
Coenye, T., LiPuma, J. J.
(2003). Population structure analysis of Burkholderia cepacia genomovar III: varying degrees of genetic recombination characterize major clonal complexes. Microbiology
149: 77-88
[Abstract] [Full Text] -
(2002). Poster presentations. Thorax
57: iii48-94
[Full Text] -
Sajjan, U., Liu, L., Lu, A., Spilker, T., Forstner, J., LiPuma, J. J.
(2002). Lack of cable pili expression by cblA-containing Burkholderia cepacia complex. Microbiology
148: 3477-3484
[Abstract] [Full Text] -
VERMIS, K., COENYE, T., MAHENTHIRALINGAM, E., NELIS, H. J., VANDAMME, P.
(2002). Evaluation of species-specific recA-based PCR tests for genomovar level identification within the Burkholderia cepacia complex. J Med Microbiol
51: 937-940
[Abstract] [Full Text] -
Conway, B.-A. D., Venu, V., Speert, D. P.
(2002). Biofilm Formation and Acyl Homoserine Lactone Production in the Burkholderia cepacia Complex. J. Bacteriol.
184: 5678-5685
[Abstract] [Full Text] -
Ferroni, A., Sermet-Gaudelus, I., Abachin, E., Quesne, G., Lenoir, G., Berche, P., Gaillard, J.-L.
(2002). Use of 16S rRNA Gene Sequencing for Identification of Nonfermenting Gram-Negative Bacilli Recovered from Patients Attending a Single Cystic Fibrosis Center. J. Clin. Microbiol.
40: 3793-3797
[Abstract] [Full Text] -
Coenye, T., Spilker, T., Martin, A., LiPuma, J. J.
(2002). Comparative Assessment of Genotyping Methods for Epidemiologic Study of Burkholderia cepacia Genomovar III. J. Clin. Microbiol.
40: 3300-3307
[Abstract] [Full Text] -
Drevinek, P., Hrbackova, H., Cinek, O., Bartosova, J., Nyc, O., Nemec, A., Pohunek, P.
(2002). Direct PCR Detection of Burkholderia cepacia Complex and Identification of Its Genomovars by Using Sputum as Source of DNA. J. Clin. Microbiol.
40: 3485-3488
[Abstract] [Full Text] -
Miller, S. C. M., LiPuma, J. J., Parke, J. L.
(2002). Culture-Based and Non-Growth-Dependent Detection of the Burkholderia cepacia Complex in Soil Environments. Appl. Environ. Microbiol.
68: 3750-3758
[Abstract] [Full Text] -
MAHENTHIRALINGAM, E., BALDWIN, A., VANDAMME, P.
(2002). Burkholderia cepacia complex infection in patients with cystic fibrosis. J Med Microbiol
51: 533-538
[Abstract] [Full Text] -
Coenye, T., Goris, J., Spilker, T., Vandamme, P., LiPuma, J. J.
(2002). Characterization of Unusual Bacteria Isolated from Respiratory Secretions of Cystic Fibrosis Patients and Description of Inquilinus limosus gen. nov., sp. nov.. J. Clin. Microbiol.
40: 2062-2069
[Abstract] [Full Text] -
Brisse, S., Stefani, S., Verhoef, J., Van Belkum, A., Vandamme, P., Goessens, W.
(2002). Comparative Evaluation of the BD Phoenix and VITEK 2 Automated Instruments for Identification of Isolates of the Burkholderia cepacia Complex. J. Clin. Microbiol.
40: 1743-1748
[Abstract] [Full Text] -
Heath, D. G., Hohneker, K., Carriker, C., Smith, K., Routh, J., LiPuma, J. J., Aris, R. M., Weber, D., Gilligan, P. H.
(2002). Six-Year Molecular Analysis of Burkholderia cepacia Complex Isolates among Cystic Fibrosis Patients at a Referral Center for Lung Transplantation. J. Clin. Microbiol.
40: 1188-1193
[Abstract] [Full Text] -
Liu, L., Coenye, T., Burns, J. L., Whitby, P. W., Stull, T. L., LiPuma, J. J.
(2002). Ribosomal DNA-Directed PCR for Identification of Achromobacter (Alcaligenes) xylosoxidans Recovered from Sputum Samples from Cystic Fibrosis Patients. J. Clin. Microbiol.
40: 1210-1213
[Abstract] [Full Text] -
Moore, J E, Millar, B C, Xu, J, Crowe, M, Redmond, A O B, Elborn, J S
(2002). Misidentification of a genomovar of Burkholderia cepacia by recA restriction fragment length polymorphism. J. Clin. Pathol.
55: 309-311
[Abstract] [Full Text] -
Bevivino, A., Dalmastri, C., Tabacchioni, S., Chiarini, L., Belli, M. L., Piana, S., Materazzo, A., Vandamme, P., Manno, G.
(2002). Burkholderia cepacia Complex Bacteria from Clinical and Environmental Sources in Italy: Genomovar Status and Distribution of Traits Related to Virulence and Transmissibility. J. Clin. Microbiol.
40: 846-851
[Abstract] [Full Text] -
Hart, C A., Winstanley, C.
(2002). Persistent and aggressive bacteria in the lungs of cystic fibrosis children. Br Med Bull
61: 81-96
[Abstract] [Full Text] -
Waleron, M., Waleron, K., Podhajska, A. J., Lojkowska, E.
(2002). Genotyping of bacteria belonging to the former Erwinia genus by PCR-RFLP analysis of a recA gene fragment. Microbiology
148: 583-595
[Abstract] [Full Text] -
Yeager, C. M., Bottomley, P. J., Arp, D. J.
(2001). Requirement of DNA Repair Mechanisms for Survival of Burkholderia cepacia G4 upon Degradation of Trichloroethylene. Appl. Environ. Microbiol.
67: 5384-5391
[Abstract] [Full Text] -
McDowell, A., Mahenthiralingam, E., Moore, J. E., Dunbar, K. E. A., Webb, A. K., Dodd, M. E., Martin, S. L., Millar, B. C., Scott, C. J., Crowe, M., Elborn, J. S.
(2001). PCR-Based Detection and Identification of Burkholderiacepacia Complex Pathogens in Sputum from Cystic Fibrosis Patients. J. Clin. Microbiol.
39: 4247-4255
[Abstract] [Full Text] -
ARIS, R. M., ROUTH, J. C., LIPUMA, J. J., HEATH, D. G., GILLIGAN, P. H.
(2001). Lung Transplantation for Cystic Fibrosis Patients with Burkholderia cepacia Complex . Survival Linked to Genomovar Type. Am. J. Respir. Crit. Care Med.
164: 2102-2106
[Abstract] [Full Text] -
Coenye, T., Vandamme, P., Govan, J. R. W., LiPuma, J. J.
(2001). Taxonomy and Identification of the Burkholderia cepacia Complex. J. Clin. Microbiol.
39: 3427-3436
[Full Text] -
Agodi, A., Mahenthiralingam, E., Barchitta, M., Giannino, V., Sciacca, A., Stefani, S.
(2001). Burkholderia cepacia Complex Infection in Italian Patients with Cystic Fibrosis: Prevalence, Epidemiology, and Genomovar Status. J. Clin. Microbiol.
39: 2891-2896
[Abstract] [Full Text] -
WINSTANLEY, C., DETSIKA, M. G., GLENDINNING, K. J., PARSONS, Y. N., HART, C. A.
(2001). Flagellin gene PCR-RFLP analysis of a panel of strains from the Burkholderia cepacia complex. J Med Microbiol
50: 728-731
[Abstract] [Full Text] -
LiPUMA, J. J., SPILKER, T., GILL, L. H., CAMPBELL, P. W. III, LIU, L., MAHENTHIRALINGAM, E.
(2001). Disproportionate Distribution of Burkholderia cepacia Complex Species and Transmissibility Markers in Cystic Fibrosis. Am. J. Respir. Crit. Care Med.
164: 92-96
[Abstract] [Full Text] -
Berriatua, E., Ziluaga, I., Miguel-Virto, C., Uribarren, P., Juste, R., Laevens, S., Vandamme, P., Govan, J. R. W.
(2001). Outbreak of Subclinical Mastitis in a Flock of Dairy Sheep Associated with Burkholderia cepacia Complex Infection. J. Clin. Microbiol.
39: 990-994
[Abstract] [Full Text] -
Henry, D. A., Mahenthiralingam, E., Vandamme, P., Coenye, T., Speert, D. P.
(2001). Phenotypic Methods for Determining Genomovar Status of the Burkholderia cepacia Complex. J. Clin. Microbiol.
39: 1073-1078
[Abstract] [Full Text]
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»