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Journal of Clinical Microbiology, December 2001, p. 4247-4255, Vol. 39, No. 12
Molecular Epidemiology Research Unit,
Northern Ireland Public Health Laboratory, Department of
Bacteriology,1 and Northern Ireland
Regional Adult Cystic Fibrosis Center,5 Belfast
City Hospital, Belfast, Northern Ireland, United Kingdom BT9 7AB;
Cardiff School of Biosciences, Cardiff University, Cardiff,
Wales, United Kingdom CF1 3US2; Bradbury
Adult Cystic Fibrosis Center, Wythenshawe Hospital, Manchester,
England, United Kingdom M23 9LT3; and
Biomolecular Sciences Group, School of Pharmacy, The Queen's
University of Belfast, Belfast, Northern Ireland, United Kingdom
BT9 7BL4
Received 22 June 2001/Returned for modification 5 August
2001/Accepted 8 September 2001
PCR amplification of the recA gene followed by
restriction fragment length polymorphism (RFLP) analysis was
investigated for the rapid detection and identification of
Burkholderia cepacia complex genomovars
directly from sputum. Successful amplification of the B.
cepacia complex recA gene from cystic
fibrosis (CF) patient sputum samples containing B.
cepacia genomovar I, Burkholderia multivorans, B. cepacia
genomovar III, Burkholderia stabilis, and
Burkholderia vietnamiensis was
demonstrated. In addition, the genomovar identifications determined
directly from sputum were the same as those obtained after selective
culturing. Sensitivity experiments revealed that
recA-based PCR could reliably detect B.
cepacia complex organisms to concentrations of
106 CFU g of sputum Patients with cystic fibrosis (CF)
are extremely susceptible to pulmonary infection with a range of
bacterial flora (29). Over the last 20 years,
Burkholderia cepacia has emerged as an opportunistic microbial pathogen in patients with CF as well as immunocompromised patients without CF (13, 25).
B. cepacia can be transmitted between patients,
is frequently resistant to a wide range of antimicrobial treatments,
and produces an increase in pulmonary symptoms and a decrease in
long-term survival (9, 10, 27, 28). In addition,
approximately 20% of all CF patients infected with B. cepacia succumb to cepacia syndrome, a necrotizing pneumonia with bacteremia which leads to an acute and frequently fatal
clinical decline (17). In response to these serious
problems, CF centers now segregate patients so that cross-infection
with the organism is reduced (12, 20).
The taxonomy of B. cepacia has proved to be very
complex. Initial phylogenetic investigations demonstrated that isolates
previously classified as B. cepacia comprised at
least five genotypically distinct genomovars, collectively referred to
as the B. cepacia complex (30).
B. cepacia genomovar V has been identified as Burkholderia vietnamiensis, a nitrogen-fixing
organism associated with rice roots (11), while
B. cepacia genomovar II and genomovar IV have
been proposed as the new species Burkholderia
multivorans and Burkholderia stabilis,
respectively (30, 31). At present, B. cepacia genomovar III awaits assignment of a binomial
species name pending the availability of suitable phenotypic
identification criteria. Strains of B. cepacia
genomovar I (which contains the type strain) will be known as
B. cepacia when taxonomic reappraisal is
complete. Very recently, the description of B. cepacia genomovar VI and genomovar VII (also known as
Burkholderia ambifaria sp. nov.) as members of
the B. cepacia complex has further demonstrated the extraordinary diversity of this group of organisms (6, 7,
16).
Ribotyping of serial B. cepacia complex strains
has revealed that CF patients are infected and colonized with a single
genomovar strain (3, 22). Although all species of the
B. cepacia complex have been cultured from CF
patients, the majority of infections result from strains of
B. cepacia genomovar III and B. multivorans (21, 30). B. cepacia complex organisms also show differences with respect
to transmissibility and virulence, with strains of B. cepacia genomovar III being responsible for most epidemic
outbreaks as well as cases of cepacia syndrome (8,
30).
Knowledge of the B. cepacia complex genomovar
species responsible for pulmonary infections is extremely important for
appropriate segregation and grouping of CF patients into cohorts. We
routinely use the recA-based diagnostic scheme recently
described by Mahenthiralingam et al. (23) to identify
B. cepacia complex isolates. Particular advantages of this multifaceted approach are its capacity to identify all genomovars of the B. cepacia complex and to
differentiate B. cepacia genomovar III isolates
into the two distinct recA cluster groups, known as III-A
and III-B. This diagnostic approach provides the clinical
microbiologist with a variety of experimental methods to identify
genomovar-specific polymorphisms within B. cepacia complex isolates. These include restriction fragment
length polymorphism (RFLP) analysis of the PCR-amplified
recA gene, the use of genomovar-specific recA
primers, and direct nucleotide sequencing of recA. Due to the flexibility of the approach, these tests can be used individually or, if desired, can be applied for multiple complementary analyses. We
now describe the novel application of recA-based PCR-RFLP
analysis for the rapid detection and identification of B. cepacia complex genomovars directly from CF sputum samples.
The ability, using a single PCR, to both detect and differentiate all
members of the B. cepacia complex in sputum may
prove particularly valuable for diagnostic laboratories.
Bacterial strains.
Table 1
lists the 25 B. cepacia complex reference strains
used for this study, as well as their original sources of isolation. The organisms were obtained from the Belgium Coordinated Collections of
Microorganisms/Laboratorium voor Microbiologie Ghent located at the
University of Ghent (http://www.belspo.be/bccm/), the Canadian B. cepacia Strain Repository located at the
University of British Columbia (16), and the American Type
Culture Collection (Manassas, Va.) (http://www.atcc.org/home.cfm). The
strains were selected to represent the different genomovar species of
the B. cepacia complex, as previously determined
by genotypic and phenotypic analyses (23, 30). Archived
bacterial strains of other organisms found in CF patients, namely,
Burkholderia gladioli, Ralstonia pickettii, Pseudomonas aeruginosa,
Staphylococcus aureus,
Stenotrophomonas maltophilia, and
Haemophilus influenzae, were isolated from the sputum of adult patients attending the CF clinic at Belfast City Hospital. All organisms were stored at
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.12.4247-4255.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
PCR-Based Detection and Identification of
Burkholderia cepacia Complex Pathogens in
Sputum from Cystic Fibrosis Patients
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
1. To fully assess the
diagnostic value of the method, sputum samples from 100 CF patients
were screened for B. cepacia complex
infection by selective culturing and recA-based PCR.
Selective culturing identified 19 samples with presumptive
B. cepacia complex infection, which was
corroborated by phenotypic analyses. Of the culture-positive sputum
samples, 17 were also detected directly by recA-based
PCR, while 2 samples were negative. The isolates cultured from both recA-negative sputum samples were subsequently
identified as Burkholderia gladioli. RFLP
analysis of the recA amplicons revealed 2 patients (12%) infected with B. multivorans, 11 patients (65%) infected with B. cepacia
genomovar III-A, and 4 patients (23%) infected with B.
cepacia genomovar III-B. These results demonstrate the potential of recA-based PCR-RFLP analysis for the rapid
detection and identification of B.
cepacia complex genomovars directly from sputum. Where
the sensitivity of the assay proves a limitation, sputum samples can be
analyzed by selective culturing followed by recA-based
analysis of the isolate.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
70°C in defibrinated horse blood (E & O Laboratories, Bonnybridge, Scotland).
TABLE 1.
B. cepacia complex strains used to establish
genomovar-specific RFLP patterns
Study population. For our clinical study, sputum samples were collected from patients attending the Bradbury Adult Cystic Fibrosis Center, Wythenshawe Hospital, Manchester, England. This group consisted of 100 adults (53 males; 47 females) with an age range of 17 to 50 years and a mean age of 25 years.
Processing of sputum samples. Expectorated sputum samples were collected postphysiotherapy and physically mixed with an equal amount of fresh Sputolysin (Calbiochem, La Jolla, Calif.) before incubation at 37°C for 30 min.
Culturing of organisms. Before analysis, B. cepacia complex reference strains were removed from storage and grown to confluence at 37°C for 48 h on Columbia agar (Oxoid Ltd., Basingstoke, England) supplemented with 5% (vol/vol) horse blood (E & O Laboratories) (blood agar). B. cepacia complex organisms in patient sputum samples were isolated by culturing at 37°C for 48 h on MAST selective agar (MAST Diagnostics, Liverpool, England). Culturing of organisms in nutrient broth (Oxoid) was performed overnight at 37°C.
Phenotypic analysis. Phenotypic analysis was performed using the multitest API 20NE identification system (bioMèrieux, Marcy l'Etoile, France) in accordance with the manufacturer's instructions.
Preparation of template DNA from bacterial cultures. Fresh cultures of the bacterial strains were suspended in 1 ml of 10 mM Tris-HCl buffer (pH 8.0) containing 1 mM EDTA (TE buffer) and centrifuged at 10,000 × g for 10 min. Supernatants were removed, and the resulting pellets of bacteria were resuspended in 0.2 ml of TE buffer. Genomic DNA was prepared using a high-purity PCR template kit (Roche Molecular Biochemicals, Lewes, England). In brief, samples were treated with 150 µg of lysozyme (Sigma-Aldrich) and incubated at 37°C for 30 min followed by incubation with 1 mg of proteinase K at 72°C for 10 min. Template DNA was precipitated with 100 µl of isopropanol and recovered from the samples by centrifugation in a membrane filter attached to an underlying collection tube. The DNA was then washed in 2 mM Tris-HCl (pH 7.5) containing 20 mM NaCl and 80% (vol/vol) ethanol before elution in 10 mM Tris (pH 8.5). Control samples consisting of 0.2 ml of sterile water (Biowhittaker, Walkersville, Md.) in place of the DNA samples were run in parallel. Successful isolation of bacterial genomic DNA was confirmed by electrophoresis in 0.7% (wt/vol) agarose gels (Life Technologies GIBCO BRL Products, Paisley, Scotland). The quantity and purity of the bacterial genomic DNA were assessed by measuring the absorbances at 260 and 280 nm.
Preparation of template DNA from CF patient sputum samples. Liquefied (Sputolysin-treated) sputum (1 ml) was centrifuged at 10,000 × g for 10 min. The resulting pellet was resuspended in 0.2 ml of TE buffer. Bacterial cells were fractured by snap-freezing in liquid nitrogen for 3 min followed by heating at 100°C for 1 min (34). This step was repeated three more times. Samples were also treated with 150 µg of lysozyme at 37°C for 30 min to ensure complete bacterial cell lysis. After treatment with proteinase K and precipitation with isopropanol, DNA was purified as described for bacterial cultures.
PCR analysis.
PCR analysis was performed with a DNA thermal
cycler (Cetus GeneAmp 9600; Perkin-Elmer Applied Biosystems, Foster
City, Calif.). The B. cepacia complex
recA gene (1,040 bp) was amplified using primers BCR1 and
BCR2 (Table 2), which target the 5' and
3' ends of the recA gene locus, respectively
(23). PCRs were performed with a total volume of 50 µl.
Samples contained 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 200 µM each
deoxynucleoside triphosphate (Amersham-Pharmacia Biotech, Little
Chalfont, England), 150 nM each BCR1 and BCR2, 1.5 mM
MgCl2, 5% (vol/vol) dimethyl sulfoxide (DMSO)
(Sigma-Aldrich), 2.5 U of AmpliTaq DNA polymerase
(Perkin-Elmer), and 100 ng of pure genomic DNA or 5 µl of sputum DNA.
Samples were initially heated at 96°C for 3 min before amplification
of the recA sequence using 35 cycles consisting of 1 min of
denaturation at 96°C, 1 min of annealing at 56°C, and 1.5 min of
extension at 72°C. The PCR was completed with a final extension step
at 72°C for 10 min.
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RFLP analysis of the B. cepacia complex recA gene. For RFLP analysis, B. cepacia complex recA amplicons were digested with HaeIII (Amersham-Pharmacia Biotech, St. Albans, England) and MnlI (New England Biolabs Inc., Hitchin, England) restriction endonucleases (23). Amplicons (5 to 15 µl) were added to the endonuclease along with the appropriate enzyme buffer, in accordance with the manufacturer's instructions, before incubation at 37°C for 2 h. When required, amplicons were concentrated using a QIAquick PCR purification kit (Qiagen Inc.; http://www.qiagen.com) to help increase the intensity of the digested recA DNA fragments. RFLP patterns were analyzed as described previously (23).
Detection of PCR and RFLP products. PCR-amplified products were routinely analyzed by electrophoresis in 2% (wt/vol) agarose gels (Life Technologies GIBCO BRL Products) containing 40 mM Tris buffer (pH 8.0) and 20 mM acetate. Restriction fragments were resolved in 3% (wt/vol) high-resolution Multiphore agarose gels (Flowgen, Lichfield, England). Molecular size markers (100-bp ladder; Life Technologies GIBCO BRL Products) were run in parallel on all gels. Resolved DNA products were stained with ethidium bromide and viewed under UV light. For clarity, the specific RFLP patterns obtained for strains of the different genomovars were classified as previously reported (23).
DNA sequence analysis. PCR amplicons were sequenced on an ABI PRISM apparatus (Perkin-Elmer) using DyeDeoxy Terminator chemistry and AmpliTaq FS DNA polymerase in accordance with the manufacturer's instructions. Nucleotide sequences were compared with previously published sequences using a basic local alignment sequence tool (BLAST) (1).
Detection limits.
To determine the minimum number of
B. cepacia complex organisms detectable in sputum
using recA-based PCR amplification, Sputolysin-treated sputum from a CF patient without B. cepacia
complex infection was inoculated with known concentrations of selected
reference strains serially diluted in one-quarter-strength Ringer's
solution (Oxoid). Experiments were performed using B. cepacia genomovar III-A strain C5424, B. cepacia genomovar III-B strain CEP511, and B. multivorans strains C5393 and LMG13010. These strains were selected because they represent the most prevalent genomovars recovered
from patients with CF (21, 30). The number of organisms added to each sputum sample was determined by colony counts on blood
agar plates inoculated in parallel. Cultures were incubated for 48 h at 37°C before enumeration. DNA was extracted from the inoculated
sputum samples and analyzed by PCR as described earlier. Results were
expressed as the number of CFU gram of sputum1.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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recA RFLP reference panel for B.
cepacia complex genomovar identification.
With
primers BCR1 and BCR2, the 1,040-bp recA gene product was
successfully amplified from the genomic DNAs of all 25 control organisms (Table 1). The recA amplicons from a sample of
eight strains, selected to represent all genomovars of our reference panel, were sequenced, and their identities were confirmed by BLAST
analysis (data not shown). A reference panel of genomovar-specific RFLP
patterns was created using restriction endonucleases HaeIII and MnlI (Fig. 1A and B,
respectively, and Table 1) as previously described (23).
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Specificity of primers BCR1 and BCR2. Since samples of sputum from CF patients contain a range of different bacterial flora, it was important to demonstrate that primers BCR1 and BCR2 did not react with other organisms commonly found in such samples. No recA amplicons were produced when the primers were tested against genomic DNAs prepared from B. gladioli, R. pickettii, P. aeruginosa, S. aureus, S. maltophilia, and H. influenzae isolates or sputum samples from 10 adult CF patients without B. cepacia complex infection.
recA-based PCR-RFLP detection and identification of
B. cepacia complex genomovars in
sputum.
To investigate if the B. cepacia
complex recA gene could be successfully amplified from
sputum DNA preparations, 15 samples from CF patients infected with
organisms of the B. cepacia complex were
analyzed. These sputum samples contained B. cepacia genomovar I (n = 1), B. multivorans (n = 4), B. cepacia genomovar III (n = 7), B. stabilis (n = 1), and B. vietnamiensis (n = 2). Upon PCR analysis
with recA primers BCR1 and BCR2, the 1,040-bp
recA gene was successfully amplified from all the samples,
demonstrating that recA-based detection of B. cepacia complex organisms directly from sputum DNA was
possible. Figure 2 shows the
recA gene product amplified from the sputum of patients
infected with different B. cepacia complex
genomovars. The amplified bands were clear and sharp, with no
background reaction due to nonspecific binding of the primers. PCR
amplification of the recA gene from sputum DNA also proved
very reproducible.
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Detection limit of the recA-based PCR assay.
Based on the success of these experiments, we examined the sensitivity
of recA-based PCR for the detection of B. cepacia complex organisms in sputum. The detection limit of
the PCR assay was investigated with four different B. cepacia complex genomovar strains, representing
B. cepacia genomovars III-A and III-B and B. multivorans. In the presence of DMSO, the
method could reliably detect 106 CFU g of
sputum1 for all four strains. The DMSO adjuvant
significantly enhanced amplification of the recA gene
compared to other additives examined, including bovine serum albumin,
glycerol, Taq Extender (Stratagene, La Jolla, Calif.), and
DyNAzyme EXT (Finnzymes, Espoo, Finland).
Clinical evaluation of recA-based PCR-RFLP analysis
for detection and identification of B.
cepacia complex genomovars in sputum.
To fully
assess the diagnostic potential of the recA-based PCR-RFLP
method, we screened 100 CF sputum samples for the presence of
B. cepacia complex infection. Sputum samples were
initially screened for the presence of B. cepacia
complex organisms by culturing on MAST selective agar followed by
phenotypic analysis of the isolates. Upon examination, growth was
observed on 19 plates (Table 3). All 19 isolates were identified as B. cepacia upon
phenotypic analysis.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Due to their taxonomic complexity, identification of B. cepacia complex pathogens has proved a challenging task for the clinical microbiologist. Currently, commercial phenotypic identification systems display significant variations in their capacity to accurately identify B. cepacia complex isolates and do not differentiate between individual genomovars (16, 19, 32). Not surprisingly, investigations have found that CF treatment centers frequently misidentify B. cepacia complex genomovars recovered from patients' sputum (24). Although phenotypic tests have now been described for analysis of the B. cepacia complex, it is still not possible to accurately differentiate all genomovars based on this approach (16).
In an attempt to resolve these problems, molecular biological approaches have been developed to facilitate accurate detection and identification of B. cepacia complex isolates (2, 21, 23, 26, 33). In addition, to aid laboratory diagnosis, a number of studies have also investigated the utility of PCR for the rapid detection of B. cepacia complex pathogens directly from CF sputum. For example, Campbell et al. (4) described a PCR assay for the detection of B. cepacia in sputum using primers PSL1 and PSR1. However, these primers were designed from published 16S rDNA sequences of B. cepacia strain ATCC 25416 (now known to be B. cepacia genomovar I) before taxonomic reappraisal initially identified five distinct genomovar species. Recent studies have now highlighted potential caveats associated with the use of PSL1 and PSR1, primarily their poor specificity for B. multivorans and B. vietnamiensis as well as the capacity to cross-react with other non-B. cepacia complex organisms (2, 5, 21). Karpati and Jonasson (18) similarly described a PCR assay for the detection of B. cepacia in sputum (also before taxonomic reappraisal) using primers directed to 16S rDNA sequences. However, 20% of B. cepacia strains analyzed with these primers failed to yield any amplified product, while other Burkholderia species, such as B. gladioli, Burkholderia caryophylli, and Burkholderia solanacearum, cross-reacted with the primers. More recently, Whitby et al. (34) described a PCR assay for the detection of B. cepacia complex pathogens in sputum based on primers G1 and G2 (also known as PC-SSF and PC-SSR), specific for the 16S-23S spacer region of the rRNA operon. However, since these primers reacted with B. cepacia genomovars I and III and B. stabilis only, they could not detect and identify all B. cepacia complex genomovars in sputum. We investigated whether it was possible to rapidly detect and identify B. cepacia complex organisms directly from crude sputum based on analysis of the recA gene. Our diagnostic algorithm used a single PCR step, with the B. cepacia complex recA primers BCR1 and BCR2, to detect the presence of B. cepacia complex species in sputum and, upon RFLP analysis of the amplicon, to identify the genomovar.
A number of different methods have been described for the preparation
of bacterial DNA from sputum. In particular, Campbell et al.
(4) and Whitby et al. (34) described the
lysis of bacterial cells in sputum by freeze fracturing in liquid
nitrogen followed by heating to 100°C. Upon centrifugation, the
resulting supernatant containing the released DNA was used directly for PCR. We have used a modification of this method for the preparation of
bacterial DNA from sputum. Rather than add a crude supernatant to the
PCR (which contains cytoplasmic and proteinaceous debris released
during cell lysis), we included an additional step in which the DNA is
precipitated with isopropanol and then purified before analysis. This
step served to remove any material that could potentially interfere
with or inhibit the PCR assay and also produced a high-quality DNA
template for amplification. Initial experiments demonstrated that
amplification of the B. cepacia complex
recA gene from genomovars in CF sputum was possible due to
the high specificity of the BCR1 and BCR2 primers for the B. cepacia complex. In addition, the genomovar results obtained
upon RFLP analysis of the recA gene amplified from sputum
were identical to those determined after culturing of the organisms.
These experiments highlighted the value of direct sputum detection and
identification of B. cepacia complex organisms,
which could be achieved within 1 day (if desired), as opposed to 3 to 4 days when a conventional selective culture step, which normally takes
between 48 and 72 h for good-quality growth (15), was
added. We further investigated the recA-based PCR-RFLP assay
by assessing the sensitivity of the method. Our results revealed
primers BCR1 and BCR2 could reliably detect B. cepacia complex organisms to concentrations of
106 CFU g of sputum1. The
detection limit observed with the recA-based PCR was higher than that reported with PCR assays based on analysis of 16S or 16S-23S
rDNA (4, 34). This reduced sensitivity likely reflects the
large size of the recA amplicon, in combination with only one copy of the gene (23) compared to the multiple copies
of the rRNA operon that exist within bacterial cells.
Within a clinical setting, the diagnostic potential of the recA-based PCR-RFLP method for the detection and identification of B. cepacia complex organisms directly from sputum was impressive. When applied to sputum from 100 CF patients, the recA-based PCR successfully detected 17 of 19 culture-positive sputum samples that were identified as containing B. cepacia by phenotypic analysis. Sputum samples from two culture-positive patients (M71 and M87) did not produce any reaction when analyzed with the recA primers. In addition, both samples were negative with all 16S or 16S-23S rDNA primers (except for PSL1 and PSR1), providing further evidence that these patients may not have been infected with a member of the B. cepacia complex but rather a closely related species which was biochemically indistinguishable on selective agar (16, 32). To date, the recA primers BCR1 and BCR2 remain highly specific for members of the B. cepacia complex only (7, 16, 23). Also, recent studies have shown that suspected B. cepacia complex isolates that are recA-based PCR negative belong to other closely related species that are not members of the current complex (16; E. Mahenthiralingam, unpublished data). Our studies confirmed this view, since further investigations did indeed reveal that the culture-positive, recA-negative sputum samples M71 and M87 were infected with B. gladioli and not organisms of the B. cepacia complex. Phenotypic misidentification of B. gladioli as B. cepacia is common with the API 20NE strip, which does not contain the taxon B. gladioli in its database.
The 16S rDNA primers PSL1 and PSR1 detected all 17 sputum samples containing members of the B. cepacia complex, including both samples with B. multivorans. It was interesting that both culture-positive sputum samples infected with B. gladioli also produced a very strong positive reaction when analyzed with these primers, although previous studies have not observed any cross-reactivity between PSL1 and PSR1 and B. gladioli isolates (4, 5). In addition, we found that a further 10 sputum samples from patients without any history of culturable B. cepacia infection similarly produced an amplicon of the correct molecular weight when analyzed with the primers. These samples did not show any reaction with primers G1 and G2, BC-GII and BC-R, or BC-GV and BC-R. These results would therefore appear to confirm recent studies that demonstrated the reaction of PSL1 and PSR1 with non-B. cepacia complex organisms (5, 21). On the basis of these data, we recommend that results obtained using these primers, especially from sputum, be interpreted with caution.
RFLP analysis of our B. cepacia complex recA amplicons identified the genomovar responsible for infection. Two patients were infected with B. multivorans, while the remaining patients were infected with B. cepacia genomovar III. Analysis of these samples with the rDNA primers BC-GII and BC-R (for B. multivorans) and G1 and G2 (for B. cepacia genomovars I and III and B. stabilis) confirmed these results, although one B. cepacia genomovar III sample did not react with G1 and G2. These data therefore provide further evidence for the prevalence of both B. multivorans and B. cepacia genomovar III strains within the CF community. RFLP analysis of our recA amplicons also revealed that the B. cepacia genomovar III-infected patients differed with respect to the recA cluster group to which their genomovar belonged. The taxonomic significance of recA groups III-A and III-B is, at the moment, not clear. Recent studies have not found any significant biochemical differences between these two recA subgroups, except for the ability to reduce nitrate (16). Also, it has not been established whether B. cepacia genomovars III-A and III-B have different effects on CF patient morbidity and mortality. However, after retrospective review of our patients' records, we have established that the B. cepacia genomovar III-A and III-B strains identified in our clinical study correspond to the epidemic B. cepacia genomovar III strains 1 (Edinburgh-Toronto epidemic) and 2 (Manchester epidemic), respectively, which were previously reported as the cause of epidemic infections in CF patients attending the Bradbury Adult Cystic Fibrosis Center, Wythenshawe Hospital (14). More interestingly, the patients infected with these two different strains appeared to show no difference in clinical outcome, suggesting that infection with B. cepacia genomovar III-A or III-B has no significant effect on prognosis. Further investigations will be required to confirm this observation.
Although the detection limit of the recA-based PCR was
higher than that of PCR assays based on the rRNA operon, the clinical results obtained with this method were excellent and comparable to
those obtained by conventional culturing. This finding may reflect the
fact that patients with B. cepacia complex
infection frequently have high genomovar concentrations in their sputum and saliva (13, 15). For such patients, the
recA-based PCR-RFLP method is an excellent way to rapidly
detect and identify the B. cepacia complex
genomovar present. When levels of infection are lower and the
sensitivity of the assay proves a limitation, sputum samples can be
analyzed by selective culturing followed by recA-based
confirmation and identification of the isolate. Indeed, we normally
perform a parallel culture step so that further analyses, such as
continued diagnostic investigation (if required), molecular typing
experiments, and susceptibility testing, can be carried out on the
isolate. Figure 4 illustrates the
experimental approach that we have adopted for routine
recA-based detection and identification of B. cepacia complex genomovars in CF sputum.
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In summary, rapid detection and identification of B. cepacia complex pathogens from CF sputum based on recA is possible. In addition, since the very recently described B. cepacia genomovar VI and genomovar VII (B. ambifaria) species of the B. cepacia complex also show reaction with the recA primers BCR1 and BCR2 and can be distinguished by RFLP analysis (7, 16; Mahenthiralingam, unpublished), it should be possible to detect and identify these organism in CF sputum also. At present, we are examining the diagnostic value of genomovar-specific recA primers for the detection and identification of individual B. cepacia complex organisms in sputum.
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ACKNOWLEDGMENTS |
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This work was supported by grants (PJ470 and PJ472) from the Cystic Fibrosis Trust, Bromley, United Kingdom.
We thank Tyrone Pitt and his staff at the Public Health Laboratory Service for assistance in identifying B. gladioli strains isolated from the sputum of our patients.
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FOOTNOTES |
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* Corresponding author. Present address: Biomolecular Sciences Group, School of Pharmacy, The Queen's University of Belfast, Medical Biology Center, 97 Lisburn Rd., Belfast, Northern Ireland, United Kingdom BT9 7BL. Phone: 44 (0)2890 272047. Fax: 44 (0)2890 247794. E-mail: a.mcdowell{at}qub.ac.uk.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Clinical Microbiology, December 2001, p. 4247-4255, Vol. 39, No. 12
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.12.4247-4255.2001
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