Sequencing of the <i>rpoB</i> Gene and Flanking Spacers for Molecular Identification of <i>Acinetobacter</i> Species

<jats:title>ABSTRACT</jats:title> <jats:p> <jats:italic>Acinetobacter</jats:italic> 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 <jats:italic>Acinetobacter</jats:italic> species. We used an RNA polymerase β-subunit gene ( <jats:italic>rpoB</jats:italic> )-based identification scheme for the delineation of species within the genus <jats:italic>Acinetobacter</jats:italic> , and towards that end, we determined the complete <jats:italic>rpoB</jats:italic> gene and flanking spacer ( <jats:italic>rplL</jats:italic> - <jats:italic>rpoB</jats:italic> and <jats:italic>rpoB</jats:italic> - <jats:italic>rpoC</jats:italic> ) sequences of the 17 reference strains of <jats:italic>Acinetobacter</jats:italic> 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 <jats:italic>gyrB</jats:italic> or <jats:italic>recA</jats:italic> gene. Four pairs of primers enabled us to amplify and sequence two highly polymorphic partial sequences (350 and 450 bp) of the <jats:italic>rpoB</jats:italic> gene. These and flanking spacers were designed and tested for rapid identification of the 17 reference strains of <jats:italic>Acinetobacter</jats:italic> 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 <jats:italic>Acinetobacter grimontii</jats:italic> , <jats:italic>Acinetobacter junii</jats:italic> , <jats:italic>Acinetobacter baylyi</jats:italic> , and genomic species 9 from one another. Finally, 21 clinical isolates of <jats:italic>Acinetobacter baumannii</jats:italic> were tested for intraspecies relationships and assigned correctly to the same species by comparing the partial sequences of the <jats:italic>rpoB</jats:italic> gene and its flanking spacers. </jats:p>