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Journal of Clinical Microbiology, May 2000, p. 1876-1884, Vol. 38, No. 5
Eijkman-Winkler Institute for Microbiology,
Infectious Diseases and Inflammation, University Medical Centre
Utrecht, 3584 CX Utrecht,1 and
Department of Medical Microbiology and Infectious Diseases,
Erasmus University Medical Center Rotterdam, 3015 GD
Rotterdam,2 The Netherlands; Laboratory
for Microbiology, University of Gent, B-9000 Gent,
Belgium3; and Klinische Forscher
Gruppe, Medizinische Hochschule Hannover, 30623 Hannover,
Germany4
Received 22 November 1999/Returned for modification 2 February
2000/Accepted 11 February 2000
Several species belonging to the genus Burkholderia are
clinically relevant, opportunistic pathogens that inhabit major
environmental reservoirs. Consequently, the availability of means for
adequate identification and epidemiological characterization of
individual environmental or clinical isolates is mandatory. In the
present communication we describe the use of the Riboprinter microbial characterization system (Qualicon, Warwick, United Kingdom) for automated ribotyping of 104 strains of Burkholderia species
from diverse sources, including several publicly accessible
collections. The main outcome of this analysis was that all strains
were typeable and that strains of Burkholderia gladioli and
of each species of the B. cepacia complex, including
B. multivorans, B. stabilis, and B. vietnamiensis, were effectively discriminated. Furthermore, different ribotypes were discerned within each species. Ribotyping results were in general agreement with strain classification based on
restriction fragment analysis of 16S ribosomal amplicons, but the
resolution of ribotyping was much higher. This enabled automated molecular typing below the species level. Cluster analysis of the
patterns obtained by ribotyping (riboprints) showed that within B. gladioli, B. multivorans, and B. cepacia genomovar VI, the different riboprints identified always
clustered together. Riboprints of B. cepacia genomovars I
and III, B. stabilis, and B. vietnamiensis did
not show distinct clustering but rather exhibited the formation of
loose assemblages within which several smaller, genomovar-specific clusters were delineated. Therefore, ribotyping proved useful for
genomovar identification. Analysis of serial isolates from individual
patients demonstrated that infection with a single ribotype had
occurred, despite minor genetic differences that were detected by
pulsed-field gel electrophoresis of DNA macrorestriction fragments. The
automated approach allows very rapid and reliable identification and
epidemiological characterization of strains and generates an easily
manageable database suited for expansion with information on additional
bacterial isolates.
Bacterial species belonging to the
genus Burkholderia are renowned for their pathogenicity in
both plants and people. Although the details of their pathogenic
potential and their capacity to adapt to a potentially hostile host
environment remain elusive to a large extent (9), research
on their taxonomic classification is continuously ongoing. The
taxonomic positions of two of the current Burkholderia
species, B. cepacia and B. gladioli, have evolved
dynamically over the past few decades (7, 44). Still, accurate species-level identification is often difficult, and, especially in relation to their pathogenicity in man, this may introduce uncertainties in the clinical relevance of strains belonging to the different species. The importance of B. gladioli is
rising with the number of reports describing this bacterial species as an invasive human pathogen (2, 12, 17, 20, 21, 35). However,
B. gladioli remains controversial as a putative pathogen, since mere colonization of the lungs of cystic fibrosis (CF) patients without apparent pathology has also been noted for this bacterial species (6).
Similarly, strains of B. cepacia, presently referred to as
the B. cepacia complex, have been classified into five
genomic species, called genomovars (labeled I to V), some of them with attributed names, namely B. multivorans (genomovar II)
(39), B. stabilis (genomovar IV)
(40), and B. vietnamiensis (genomovar V)
(10). Recently, a sixth genomovar was identified (T. Coenye, J. J. LiPuma, D. Henry, B. Hoste, K. Vandemeulebroeke, M. Gillis, D. P. Speert, and P. Vandamme, submitted for publication).
Distinguishing between genomovars relies on DNA-DNA hybridization,
whole-cell protein pattern similarity, and phenotypic markers.
Classification of B. cepacia-like strains in multiple
species is stimulating the hypothesis that the genetic identities of
the infective strains influence the clinical outcome. As a matter of
fact, Burkholderia spp. pathogenicity can vary greatly,
particularly in CF patients (19). Strains of different
genomovars also greatly differ in their transmissibility
(11), stressing the importance of rapid and reliable strain
identification systems.
Given the dynamics and controversies mentioned above, novel diagnostic
tests for B. cepacia, including its different genomovars, and B. gladioli have been reported. These assays include
culture approaches (15, 16, 42), enzyme-linked immunosorbent
assays (27), and, of course, PCR (3, 4, 23, 43).
Some of these assays have the capacity to discriminate reliably among the various members of the B. cepacia complex (3, 4,
23). Here we present evidence that the automated Riboprinter
microbial characterization system can be used to distinguish between
isolates of the B. cepacia complex species and B. gladioli and at the same time provide information on subspecific
genetic heterogeneity.
Bacterial isolates.
The strains analyzed in the present
study were gathered from diverse sources, including several publicly
accessible reference collections (41). In addition to the
collection described in reference 41, strains from
the BCCM/LMG Bacterial Collection (Ghent, Belgium) and from the
collection of the Medizinische Hochschule (Hannover, Germany) were
included. A single strain of B. plantarii and three strains
of Ralstonia pickettii (formerly Burkholderia pickettii [45]) were included for reasons of
comparison and control. Serial isolates derived from individual
patients were included. Table
1 describes the origins
of the strains and provides information on the geographical and
environmental or clinical sources of several of the isolates. For the
strains included in reference 41, PCR-restriction
fragment length polymorphism (RFLP) characterization profiles are also
indicated. The four-letter codes presented in Table 1 identify the
types as established by using the restriction enzymes AluI,
CfoI, MspI, and DdeI.
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Distinguishing Species of the Burkholderia
cepacia Complex and Burkholderia gladioli by
Automated Ribotyping
ABSTRACT
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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.
Origin, characterization, and identification of the
Burkholderia strains analyzed
Automated ribotyping. Strains were grown overnight on Brucella blood agar medium (bioMerieux, Marcy l'Étoile, France). Automated ribotyping was performed under the conditions recommended by the manufacturer of the Riboprinter microbial characterization system (Qualicon Europe Ltd., Warwick, United Kingdom) (34). Restriction enzymes that were validated with respect to their usefulness were EcoRI and PvuII. A large DNA probe harboring the genes for both the small-subunit rRNA and the large-subunit rRNA of Escherichia coli was employed. The ribotypes were categorized in different ribogroups by the Riboprinter. The banding patterns were compared by using the GelCompar software (Applied Maths, Ghent, Belgium). Clustering was performed by the unweighted pair group method with arithmetic averages (UPGMA) method based on the Pearson correlation coefficient, using an optimization coefficient of 1.2% (identical to that used by the Riboprinter analysis system).
Whole-cell protein electrophoresis. Whole-cell protein electrophoresis was used to determine the species of representative strains of most ribogroups. Bacteriological purity was determined by plating and examining living cells by phase-contrast microscopy and by Gram staining. Strains were grown on nutrient agar (CM3; Oxoid, Haarlem, The Netherlands) supplemented with 0.04% (wt/vol) KH2PO4 and 0.24% (wt/vol) Na2HPO4 · 12H2O. Agar plates were incubated aerobically at 28°C. After 48 h, whole-cell protein extracts were prepared and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed as described previously (39). The densitometric analysis, normalization, and interpolation of the protein profiles, including numerical analyses, were performed by using the Pearson product moment correlation coefficient and are expressed as percent similarity values. Data for referral have been presented on previous occasions (39, 40; Coenye et al., submitted). Only a representative set of isolates, described in Table 1, was analyzed in this way.
PFGE. Strains from the Hannover region of Germany were analyzed by pulsed-field gel electrophoresis (PFGE) in accordance with experimental protocols described previously (32). Total SpeI digests of agarose-embedded genomic DNA were separated in 1% agarose gels in 0.5× TBE buffer (45 mM Tris, 45 mM boric acid, 1 mM EDTA [pH 8.3]) by using a Bio-Rad CHEF-DR III apparatus (two linear ramps [17 h, 3 to 35 s; 20 h, 5 to 80 s], 120° reorientation angle, 210 V). After electrophoresis, gels were stained with ethidium bromide and photographed by using Polaroid equipment.
Epidemic-marker PCR. PCR amplification to test for the presence of the B. cepacia epidemic strain marker was performed as described elsewhere (26). Control experiments to ensure the quality of DNA were performed by amplifying part of the 16S RNA gene region, using primers BuRa-16-1 and BuRa-16-2 (4).
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Technical issues.
Pilot experiments performed with a limited
number of strains revealed that EcoRI provides a higher
level of resolution than PvuII (Fig.
1). Indeed, two groups of three strains
each that were indistinguishable with PvuII were each
separated into two different ribogroups with EcoRI. However,
since the use of PvuII resulted in a sharper clustering of
all of the B. multivorans strains tested, this enzyme
appeared potentially more useful for genomovar identification.
Therefore, the entire collection of strains was tested with
PvuII. In total, the strains analyzed exhibited 39 distinct
patterns, or ribotypes (Fig. 2).
Adequate reproducibility of the tests was indicated by the fact
that the duplicate reference strains generated identical patterns
(CCUG12691 equals ATCC 25416, CCUG2115 equals ATCC 19302, and CCUG1782
equals ATCC 10248). The only exception was the duplicate ATCC
17616-LMG17588, which was categorized into two distinct ribogroups by
the Riboprinter (Table 1). However, the two patterns were very
similar (Fig. 2), and the difference could be attributed to a slightly
incomplete digestion of the DNA of strain LMG17588. The pattern of this
strain presented a number of faint bands as well as a
high-molecular-weight band not evident when strain ATCC 17616 was
analyzed. This result indicates that when strains are being typed,
Riboprinter analysis results must be checked by visual inspection of
the patterns, and differences in the high-molecular-weight range should
be interpreted with caution. Importantly, the differences between the
patterns of these two samples did not affect their clustering (Fig. 2).
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Concordance between genomovar classification and ribotype identification. Figure 2 surveys all of the different ribotypes that were identified in the present set of strains. Only one isolate per patient or per duplicate strain is shown (except for ATCC 17616-LMG17588 [see above]). Table 1 shows the number of occurrences of each ribotype and indicates the number of samples tested for each patient and duplicate strain. One or several representative strains of most ribogroups were identified at the species level by whole-cell protein electrophoresis. These strains, considered as ribogroup-specific reference strains, are indicated in Table 1.
When the riboprints were clustered by the UPMGA method, several interesting subsets of strains were observed (Fig. 2). First, all B. gladioli strains fell into six ribogroups which clustered into a single branch of the dendrogram. Similarly, strains belonging to the species R. pickettii, as well as the single strain of B. plantarii, segregated separately. Second, distinctions between the genomovars were evident since strains belonging to different genomovars never showed the same riboprint pattern. Several ribotypes were distinguished within each of the genomovars. For example, among nine strains in genomovar III, eight ribogroups were found. Moreover, within this genomovar, the two strains (C5424 and LMG16656) previously characterized by multilocus enzyme electrophoresis as belonging to electrophoretic type ET12 (39) had clearly distinct riboprints. Genomovar VI could not be separated into different ribogroups, but only two strains with this genomovar type were analyzed. These two isolates (LMG18941 and LMG18942) were clearly distinct from all other strains, in agreement with their recent distinction as a separate genomovar (Coenye et al., submitted). With respect to the potential of ribotyping for genomovar identification, it was very interesting that all of the B. multivorans isolates (formerly genomovar II) clustered into one group. Moreover, B. multivorans strains could be distinguished from the other B. cepacia complex strains on the basis of their unique riboprints. Generally, these patterns contained only three or four bands restricted to a narrow range of molecular weights, whereas most of the other strains showed patterns with more hybridizing fragments and more variation in their size. In contrast to the strains of B. multivorans and of genomovar VI, genomovar I, III, IV, and V strains did not cluster into separate branches (Fig. 2). However, a tendency of strains of a given genomovar to cluster was observed. For example, all strains of B. stabilis (genomovar IV) clustered in the lower branch of the dendrogram. More interestingly, however, smaller clusters specifically comprised several ribotypes of a single genomovar (e.g., strains LMG16656, C5424, and C6433 of genomovar III, CCUG9631 and SKMM of B. vietnamiensis, and CEP0521 and LMG18821 of genomovar I).Concordance between 16S PCR-RFLP classification and ribotype identification. The clustering obtained on the basis of ribotyping was in good agreement with 16S PCR-RFLP analysis data (Fig. 2). For example, most strains of the upper branch of the dendrogram showed the AAAB type, although they belonged to three distinct genomovars (I, III, and V). In genomovar III, the only analyzed strain (patE-4) that did not cluster with the other strains of that genomovar was also the only one with a different RFLP type (AAAA, versus AAAB in the others).
Obviously, the riboprints showed a higher degree of variability than RFLP typing. For instance, among the strains identified with PCR-RFLP code AAAA, eight different ribotypes were discerned. The increased heterogeneity among the riboprints was more obvious among the PCR-RFLP AAAB types; 11 different riboprint patterns were found. Finally, ribotyping distinguished between B. plantarii on the one hand and B. gladioli strains that shared the BBBC type with B. plantarii on the other hand. In no instance were different RFLP types found among the strains of a given ribogroup. In the B. cepacia complex, when considering only the 48 strains from different patients and from different environmental isolates, Simpson's index of diversity (used as an estimation of the discriminatory power [18]) for the PvuII characterization was 0.967. In comparison, RFLP discrimination among the 38 of such strains analyzed yielded a discriminatory power index of 0.647. Analysis of correspondence between genomovar classification and RFLP type showed that except for the patient E strains, RFLP type AAAA was specific for genomovar II. Moreover, RFLP type ABBB was characteristic of B. stabilis (genomovar IV). Although genomovar I strains were scattered across the dendrogram, all showed the AAAB type, but this type was also found among genomovar III and V strains.Serial samples from individual patients and geographic spread of
clones.
For several individual German or Dutch patients, three to
seven isolates collected at different times were included in the analysis. In all instances, strains belonged to a single ribogroup when
individual patients were considered. Consequently, persistent colonization by a single ribotype appears to be the most
prevalent situation. However, using PFGE, minor differences could be
detected between strains from the same patient (Fig.
3), suggesting evolution of the
PFGE pattern in the course of the infection.
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DISCUSSION |
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Materials and Methods
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Identification of Burkholderia species. Efforts to develop diagnostic tests for B. cepacia genomovars are presently intense (3, 4, 23, 33, 41). However, distinguishing between some genomovars, in particular genomovars I, III, and IV, has proven difficult or impossible, as was also illustrated for PCR-RFLP in the present study. Presently existing automated systems for bacterial identification, such as the Vitek and MicroScan systems, perform with varying but consistently insufficient accuracy for B. cepacia and B. gladioli identification, as was illustrated recently (41). Simple and reliable routine identification systems for Burkholderia species are currently lacking. In a previous study (41), PCR-RFLP analysis with four restriction enzymes was shown to be one of the best methods for Burkholderia species identification when done after screening by selective agar plate culture and performing API 20NE galleries to exclude all irrelevant bacterial species. Our results show that automated ribotyping has potential for identification of species of the B. cepacia complex.
Our results indicate that automated ribotyping with the Riboprinter has great potential for reliable distinction between strains of B. cepacia genomovars I, III, and VI, B. multivorans (genomovar II), B. stabilis (genomovar IV), B. vietnamiensis (genomovar V), and B. gladioli. Moreover, there is real potential for identification at the species level. First, strains belonging to different species or genomovars never showed the same ribotype. Therefore, the hypothesis that two strains belonging to the same ribogroup also belong to the same genomovar seems reasonable. Thus, determining the genomovar to which a strain belongs could be achieved by investigating whether its ribotype corresponds to one already present in the database. As with any library-based identification system, progressive expansion of the database of patterns with those of newly characterized strains will increase the probability of identifying a new strain. For this, any strain showing a new ribotype should be identified at the genomovar level by current methods. In addition, electronic communication between Riboprinters located in different laboratories or countries is now being initiated, making it possible to exploit fully the potential of library typing systems based on automated ribotyping. Second, clustering analysis could also be useful. Our results show that when PvuII is used, B. multivorans, B. gladioli, and B. cepacia genomovar VI each correspond to a single cluster. In these cases, a new ribotype that falls within such a cluster could also be considered as belonging to this species. For example, although strains from patient D showed a unique ribotype, it clustered with B. multivorans patterns and therefore probably corresponds to B. multivorans. This approach could also be used for the smaller, species-specific clusters identified in genomovars I, II, and V. As stated above, we were able to determine, based on the ribotyping analysis, the species of all strains in the study (Table 1) except the incubator water strain. This strain was not characterized by protein profiling and fell in a unique and unclustered ribogroup. As illustrated by this strain, two limitations to this approach exist. First, depending on the number of ribotypes existing in a given species, obtaining a comprehensive database including most of the representative ribotypes of this species may be difficult to achieve. Second, further research on more isolates will be needed to determine if any newly characterized strain of a given species will fall in its corresponding cluster. At the same time, it will have to be verified that no strain of any other species will by chance fall in this cluster. This risk is illustrated, for example, by the fact that strains ATCC 25416 and ATCC 17759 of genomovar I fell within the cluster containing all B. stabilis strains (Fig. 2). Both limitations might be overcome by searching for restriction enzymes with less discriminatory power. Indeed, this not only would reduce the number of different ribotypes within a species but also would result in patterns comprising more-conserved characters, including diagnostic ones, and therefore in a better species-specific clustering. For these reasons, we opted to use PvuII rather than EcoRI, because of the lower discriminatory power of the former enzyme. However, a search for even less-discriminatory enzymes might be warranted. The identification potential of enzymes HincII and SmaI is presently under investigation, since they proved to yield interesting clustering results for a wide range of pseudomonads (5).Characterization of Burkholderia spp. strains. Many different procedures have proven useful for discrimination of Burkholderia spp. strains below the species level. The technologies used generally identify polymorphism at the protein level or screen chromosomes for variability in restriction sites or PCR primer annealing sites (14, 24, 25, 39). The PCR-RFLP approach is one that can be used for monitoring the spread of bacterial strains as well as the dynamics of patient colonization and infection (8, 36). It must be emphasized, however, that the resolution of PCR-RFLP is lower than that of PFGE. German patients 1 and 2, for instance, were carrying strains that were both of the PCR-RFLP AAAA type (Table 1) but were clearly differentiated by PFGE (Fig. 3).
Conventional ribotyping is not a novel approach for the genetic characterization of bacteria in general or for Burkholderia spp. in particular. Ribotyping has been instrumental in the analysis of nosocomial outbreaks of infection (28), in the deciphering of the spread of infectious strains in restricted locales such as summer camps for CF patients (29), and for the identification of clones capable of extensive geographic spread (31, 37). Over the years, ribotyping has acquired a central position among the technologies available to the microbial epidemiologist. Due to its automation, the Riboprinter microbial characterization system is suited for rapid and high-throughput typing of bacterial strains. This approach was shown to be successful for the epidemiological study of strains belonging to species such as Pseudomonas aeruginosa, Listeria monocytogenes, and E. coli (1, 13, 30). An important advantage of this automated system is its standardization and easily manageable database. Therefore, it can be developed as a library typing system by which strains characterized at different times and various locations can be reliably compared (38). Whether there is a link between pathogenicity or epidemicity potential and ribotype cannot be assessed on the basis of the present data set, but the determination of such a link will certainly be facilitated by this characterization system. Using ribotyping, assessments of subspecific variation, suited for epidemiological and patient-related research, were also possible in this study. In agreement with the data of LiPuma et al. (22), long-term colonization with a single strain was documented in all patients investigated. Geographic dissemination was determined, and we showed that both local prevalence and extensive spread can be documented for different ribotypes. However, achieving the best discrimination between strains was not the goal of the present study, and PFGE typing proved to be more discriminatory than PvuII and EcoRI ribotyping, even on a limited test sample. However, due to its speed (8 h for characterization of strains), automated ribotyping might be useful for epidemiological investigations in a hierarchical approach combining a preliminary screening for ribotype differences with complementary, slower approaches when no discrimination is evidenced (30).| |
ACKNOWLEDGMENTS |
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We gratefully acknowledge Cindy van Pelt (Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center Rotterdam, Rotterdam, The Netherlands) for maintaining the culture collection of Burkholderia spp. and for help with culture of the strains. Helke van Dessel and Karlijn Kusters are thanked for practical assistance in performing part of the riboprint analyses.
Peter Vandamme is a postdoctoral research fellow of the Fund for
Scientific Research
Flanders (Belgium). Sylvain Brisse was supported by a European Communities TMR grant.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Medical Microbiology and Infectious Diseases, Erasmus University Medical Center Rotterdam (EMCR), Dr. Molewaterplein 40, 3015 GD Rotterdam, The Netherlands. Phone: 31-10-4635813. Fax: 31-10-4633875. E-mail: vanbelkum{at}bacl.azr.nl.
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Clinical Microbiology, May 2000, p. 1876-1884, Vol. 38, No. 5
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