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Classification and identification of the Burkholderia cepacia complex: Past, present and future

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Abstract

The Burkholderia cepacia complex is a group of closely related species with conflicting biological properties. Triggered by the devastating effect of pulmonary infections in cystic fibrosis patients, the scientific community generated an unusually large amount of taxonomic data for these bacteria during the past 15 years. This review presents the polyphasic, multilocus and genomic methodology used for the classification and identification of these bacteria. The current state-of-the-art demonstrates that present day taxonomists can replace traditional DNAā€“DNA hybridizations for species level demarcation and 16S rRNA sequence analysis for studying phylogeny by superior whole genome sequence-based parameters within the framework of polyphasic taxonomic studies.

Introduction

Burkholderia cepacia complex bacteria are closely related species that share a high (>97.5%) level of 16S rRNA gene sequence similarity and moderate (30ā€“60%) DNAā€“DNA hybridization values [21], [83]. They are a challenging group of organisms with conflicting biological characteristics, which make them both friend and foe to humans [36]. Their unusually large genomes (7.5ā€“8.5Ā Mb) with a DNA GĀ +Ā C base composition of approximately 67Ā mol% consist of multiple replicons and provide them not only with unsurpassed metabolic capacities, but also with genotypic and phenotypic characteristics that defy our need to classify bacteria in well delineated groups. This review presents the polyphasic, multilocus and genomic methodology for the classification and identification of these bacteria. Table 1 gives an overview of the 17 validly named species within the complex and their isolation sources.

Section snippets

Historical background

B. cepacia complex bacteria were first isolated in the 1940s by Walter Burkholder who reported on ā€œsour skinā€ or ā€œslippery skinā€, a disease of onion bulbs [11]. Burkholder based his description of the causative agent of this bacterial rot disease on seven isolates from decaying onion bulbs obtained in 1947 and 1948, and proposed to name them Pseudomonas cepacia. Later, two additional pseudomonads were considered to represent the same species [5], [64], [68], [71]: one consisted of a group of

A species complex

From the early 1990s, a marked heterogeneity among strains identified as B. cepacia was noted using traditional and molecular identification approaches, including growth on selective media, classical biochemical tests and commercial biochemical microtest systems, whole-cell fatty acid analysis and several PCR-based techniques [7], [12], [33], [67], [77], [99]. This heterogeneity made correct identification problematic and evaluation of the techniques used showed that they were either not very

Biochemical identification of B. cepacia complex species

The progress made in recent years in refining the taxonomy of these bacteria has provided the basis for improved identification algorithms [90]. Nevertheless, reliable differentiation of these species from other related taxa, such as Ralstonia, Cupriavidus, Pandoraea, Achromobacter, Brevundimonas, Comamonas and Delftia species is challenging. Differentiation of species within the B. cepacia complex can be particularly problematic, even with an extended panel of biochemical tests [38], as they

Whole cell protein electrophoresis

The rationale for using whole-cell protein electrophoresis in bacterial systematics is that closely related bacteria are likely to have a highly similar protein content when grown in standardized conditions [61]. By comparison of the results of numerical analysis of whole cell protein profiles with those of DNAā€“DNA hybridization experiments, the use of this method for accurate species level identification of many bacterial species has been validated [82]. Vandamme et al. [83] reported that

Whole-cell fatty acid analysis

The high degree of automation, the relative simplicity and the low cost of whole-cell fatty acid analysis made it a valuable technique for rapid identification of isolates in clinical laboratories [95]. However, although cellular fatty acid methyl ester analysis appeared useful for identification of Burkholderia strains at the genus level, it was of limited utility for identification of individual B. cepacia complex species and did not differentiate B. gladioli [15], [83]. For several years,

16S rRNA gene-based analyses

When comparing 16S rRNA gene sequences of B. cepacia complex reference strains, high similarity values (typically above 98%) are measured [21], [90], [88], indicating that B. cepacia complex species are phylogenetically very closely related. In addition, as these species also exhibit up to 2% intraspecies diversity in their 16S rRNA gene sequences, they cannot reliably be identified at the species level by means of simple comparison of complete 16S rRNA gene sequences. Early efforts to develop

recA gene-based analyses

Since the rRNA genes have limited taxonomic resolution, Mahenthiralingam et al. [53] explored the utility of the recA gene for species identification in the B. cepacia complex. In several studies, they demonstrated that the recA genes of these bacteria typically showed 94ā€“95% similarity between different B. cepacia complex species and 98ā€“99% similarity within B. cepacia complex species [53], [90], [88]. Remarkably, B. cenocepacia appeared to comprise four subpopulations (referred to as B.

Multilocus sequence analysis (MLSA)

One of the particularly interesting developments in bacterial taxonomy is multilocus sequence analysis [32]. In contrast to multilocus sequence typing (MLST), a specific tool designed for molecular epidemiology and for defining strains within named species, whereby similarities and differences are usually measured as differences in allelic profiles, MLSA instead employs phylogenetic procedures based on the nucleotide sequences of the alleles in order to reveal similarities between strains

Other methods

A range of other methods, such as amplified fragment length analysis of genomic DNA [18], Fourier transform infrared spectroscopy [8], [23], fur and hisA gene sequence analysis [52], [60], and matrix assisted laser desorption ionization time of flight mass spectroscopy [27], [89] have been used for the identification of B. cepacia complex bacteria. Although several of these techniques appear promising, a more thorough evaluation of their usefulness in the taxonomy of B. cepacia complex bacteria

Whole-genome studies

For more than two decades, bacterial taxonomists considered whole-genome similarity the standard for determining taxonomy. The number of whole-genome sequences is rapidly increasing and allows assessment of genome level variation within and between species. There is a growing interest in using these whole-genome sequences to study evolutionary relationships among prokaryotic species and a range of novel approaches for assessing taxonomic relationships within and between species has become

A complex species?

Over the past two decades, B. cepacia has been transformed into a species complex which has currently been dissected into 17 validly named species. Published and unpublished results from recA sequencing and MLSA studies demonstrate that there is still a considerable number of unnamed B. cepacia complex species waiting in the wings. It is interesting, in this respect, to look at some of the historical strain collections and data in order to reflect upon our actions. As mentioned above, studies

Acknowledgements

This article is based on a lecture presented at the symposium ā€œRecent Advances in Microbial Taxonomyā€ by the ā€œDeutsche Akademie der Naturforscher Leopoldinaā€ in ZĆ¼rich, Switzerland, March 30ā€“31, 2009. This is the fourth and last of a series of minireviews from this Leopoldina Symposium that has also included contributions from Klenk and Gƶker [43], Schleifer [65], and van Regenmortel [81].

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