Sequencing of the rpoB gene and flanking spacers for molecular identification of Acinetobacter species

Abstract

Acinetobacter species are defined on the basis of several phenotypic characters, results of DNA-DNA homology, and more recently, similarities or dissimilarities in 16S rRNA gene sequences. However, the 16S rRNA gene is not polymorphic enough to clearly distinguish all Acinetobacter species. We used an RNA polymerase beta-subunit gene (rpoB)-based identification scheme for the delineation of species within the genus Acinetobacter, and towards that end, we determined the complete rpoB gene and flanking spacer (rplL-rpoB and rpoB-rpoC) sequences of the 17 reference strains of Acinetobacter species and 7 unnamed genomospecies. By using complete gene sequences (4,089 bp), we clearly separated all species and grouped them into different clusters. A phylogenetic tree constructed using these sequences was supported by bootstrap values higher than those obtained with 16S rRNA or the gyrB or recA gene. Four pairs of primers enabled us to amplify and sequence two highly polymorphic partial sequences (350 and 450 bp) of the rpoB gene. These and flanking spacers were designed and tested for rapid identification of the 17 reference strains of Acinetobacter species and 7 unnamed genomospecies. Each of these four variable sequences enabled us to delineate most species. Sequences of at least two polymorphic sequences should be used to distinguish Acinetobacter grimontii, Acinetobacter junii, Acinetobacter baylyi, and genomic species 9 from one another. Finally, 21 clinical isolates of Acinetobacter baumannii were tested for intraspecies relationships and assigned correctly to the same species by comparing the partial sequences of the rpoB gene and its flanking spacers.

FIG. 1.

Graphical representation of range site variability in rpoB gene and spacer sequences of Acinetobacter species in the present study using SVARAP software. The x axis indicates the positions of nucleotides, and the y axis indicates the percent variabilities for 50 nucleotides. Primers that amplified spacers rplL-rpoB and rpoB-rpoC and hypervariable partial sequences of rpoB bordered by conserved regions are shown.

FIG. 2.

Dendrogram representing phylogenetic relationships of Acinetobacter species using the neighbor-joining method. The tree was derived from the alignment of complete rpoB gene sequences. The support of each branch, as determined from 1,000 bootstrap samples, is indicated by the value at each node (in percent). Only bootstrap values of ≥75% are indicated.

FIG. 3.

Dendrogram representing phylogenetic relationships of Acinetobacter species and intraspecies relationships of A. baumannii strains using the neighbor-joining method. The tree was derived from the alignment of concatenated zone 1 and zone 2 of the partial rpoB gene sequences. The support of each branch, as determined from 1,000 bootstrap samples, is indicated by the value at each node (in percent). Only bootstrap values of ≥75% are indicated.