Development of a species-specific fur gene-based method for identification of the Burkholderia cepacia complex

Abstract

Burkholderia is an important bacterial genus with a complex taxonomy that contains species of both ecological and pathogenic importance, including nine closely related species collectively termed the Burkholderia cepacia complex (BCC). Unfortunately, 16S rRNA gene analysis has proven to be not sensitive enough to discriminate between species of the BCC. Alternative species identification strategies such as recA-based PCR followed by restriction fragment length polymorphism analysis, although initially useful, have proven to be inaccurate with the increasing species diversity of the BCC. recA gene sequence analysis is more discriminatory and corroborates other biochemical and polyphasic means of taxonomic differentiation. However, it is limited by the fact that certain BCC species are subdivided into discrete recA sequence subgroups that may confuse clinical diagnoses. In this study, an effective approach is described for the rapid differentiation of BCC species from both environmental and clinical sources by means of a single-locus sequencing and PCR assay using fur as a target gene that provides sequence phylogenies that are species specific and, with few exceptions, not divided into subspecies clusters. This assay is specific and can be used to correctly determine the species status of BCC strains tested following sequencing and amplification of the fur gene by both general and species-specific primers. Based on our results, this typing strategy is simpler than and as sensitive as established tests currently in use clinically. This assay is useful for the rapid, definitive identification of all nine current BCC species and potentially novel species groups.

FIG. 1.

Phylogenetic tree comparing sequences of a 429-bp fur fragment from strains and isolates of the BCC. The phylogenies are rooted due to the assumption of a common ancestor and biological clock. Genetic distance is shown on the scale, with demarcations representing 2% estimated substitutions. The genomovar status (I through IX) of each BCC species is shown in parentheses following the strain or isolate name. Abbreviations are as follows: Bg, Burkholderia gladioli; Bp, Burkholderia pseudomallei; Bsp, Burkholderia species; Bx, Burkholderia xenovorans.

FIG. 2.

Gel electrophoresis of fur PCR products for the BCC species B. vietnamiensis, B. dolosa, B. ambifaria, B. anthina, and B. pyrrocinia. PCR products were electrophoresed on a 0.8% agarose gel prior to visualization with ethidium bromide. Molecular weight standards (MW) are shown in lane 1, with the corresponding base pair sizes shown at the left of the gel. Shown below each BCC species name is the fur primer set used and the size of the obtained PCR product (in base pairs) with the following template DNAs: lane 2, PC259/LMG 18835; lane 3, G4; lane 4, ATCC 29424; lane 5, STM1441/LMG 21443; lane 6, CEP021/LMG 21819; lane 7, E12/LMG 21820; lane 8, L06; lane 9, CEP0996/LMG 19467; lane 10, M53; lane 11, J2552/LMG 16670; lane 12, C1765/LMG 20983; lane 13, AU1293/LMG 21821; lane 14, ATCC 39277/LMG 21822; lane 15, BC011/LMG 21823; lane 16, C1469/LMG 21824.