Genome-based classification of Burkholderia cepacia complex provides new insight into its taxonomic status

Abstract

Background: Accurate classification of different Burkholderia cepacia complex (BCC) species is essential for therapy, prognosis assessment and research. The taxonomic status of BCC remains problematic and an improved knowledge about the classification of BCC is in particular needed.

Methods: We compared phylogenetic trees of BCC based on 16S rRNA, recA, hisA and MLSA (multilocus sequence analysis). Using the available whole genome sequences of BCC, we inferred a species tree based on estimated single-copy orthologous genes and demarcated species of BCC using dDDH/ANI clustering.

Results: We showed that 16S rRNA, recA, hisA and MLSA have limited resolutions in the taxonomic study of closely related bacteria such as BCC. Our estimated species tree and dDDH/ANI clustering clearly separated 116 BCC strains into 36 clusters. With the appropriate reclassification of misidentified strains, these clusters corresponded to 22 known species as well as 14 putative novel species.

Conclusions: This is the first large-scale and systematic study of the taxonomic status of the BCC and could contribute to further insights into BCC taxonomy. Our study suggested that conjunctive use of core phylogeny based on single-copy orthologous genes, as well as pangenome-based dDDH/ANI clustering would provide a preferable framework for demarcating closely related species.

Reviewer: This article was reviewed by Dr. Xianwen Ren.

Keywords: Burkholderia cepacia complex; Classification; Genome; Single-copy orthologous genes; Taxonomy; dDDH and ANI.

Fig. 1

The phylogenetic relationships of BCC strains based on (A) 16S rRNA, (B) recA and (C) hisA sequences.The maximum-likelihood trees were constructed from the alignments of (a) full length 16S rRNA (1539 bp), (b) full length recA (1071 bp) and (c) full length hisA (756 bp) sequences. B. pseudomallei K96243, B. oklahomensis C6786^T and B. glumae LMG 2196^T were chosen as outgroups. Numbers below branches are bootstrap support values from 1000 replicates if equal to or larger than 50%. Type strains are printed in bold font as well as marked by a blue star (B. ubonensis MSMB22 is the representative genome of this species; because of its examined high-quality and unavailability of the whole genome of this species, we treated MSMB22 as the same as the type strain of B. ubonensis in this paper). Species with at least two members are colored except B. pseudomultivorans, B. latens, B. diffusa, B. stagnalis and B. seminalis in (a); species with single members (and outgroups) are black

Fig. 2

Comparison of MLSA and species tree. a Maximum-likelihood phylogenetic tree of 116 BCC genomes based on concatenated amino acid alignments of 1005 single-copy orthologous genes (274,980 AA) and rooted at node pointed out by MLSA phylogeny. Node support values were based on the Shimodaira-Hasegawa test. b Maximum-likelihood phylogenetic tree of the concatenated nucleotide sequences (2771 bp) from the seven housekeeping gene fragments [atpD (443 bp), gltB (400 bp), gyrB (454 bp), recA (393 bp), lepA (395 bp), phaC (385 bp) and trpB (301 bp)]. B. pseudomallei K96243, B. oklahomensis C6786^T and B. glumae LMG 2196^T were chosen as outgroups. Other display settings in (a) and (b) are the same as in Fig. 1

Fig. 3

Heat map of dDDH and ANI from pairwise genomes comparisons.The lower triangle displays the dDDH values, and the upper triangle displays the ANI values. Type strains are indicated by a superscript T

Fig. 4

Heat map of pairwise dDDH values for 116 genomes of BCC strains. Hierarchical clustering of the 116 tested strains is indicated in both axes. At the 70% dDDH threshold for species delineation, 36 clusters or species (labeled BCC01-BCC36) are classified. Boxed regions indicate inferred clusters with at least two members. The name of each strain was composed of cluster labels and the original infraspecific name

Fig. 5

Reclassification of 116 BCC genomes.The maximum-likelihood phylogenetic tree is the same tree as in Fig. 2a except the layout is a circle instead of a rectangular phylogram. Leaves are colored according to their affiliation with clusters. (i.e., Burkholderia cepacia complex, BCC) and with at least two members; clusters with single members have a white background. Type strains are printed in bold font as well as marked by a blue star. Node support values from the Shimodaira-Hasegawa test are not shown if below 50%. The backgrounds are colored according to the corresponding cluster