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Journal of Clinical Microbiology, March 2006, p. 827-832, Vol. 44, No. 3
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.3.827-832.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Sequencing of the rpoB Gene and Flanking Spacers for Molecular Identification of Acinetobacter Species{dagger}

Bernard La Scola,* Vijay A. K. B. Gundi, Atieh Khamis, and Didier Raoult

Unité des Rickettsies, CNRS UMR 6020, Faculté de Médecine, Université de la Méditerrannée, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 05, France

Received 17 October 2005/ Returned for modification 15 December 2005/ Accepted 3 January 2006

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 ß-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.

* Corresponding author. Mailing address: Unité des Rickettsies, CNRS UPRESA 6020, Faculté de Médecine, Université de la Méditerrannée, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France. Phone: 33 04 91 38 55 17. Fax: 33 04 91 83 03 90. E-mail: bernard.lascola{at}medecine.univ-mrs.fr.

{dagger} Supplemental material for this article may be found at http://jcm.asm.org/.

Journal of Clinical Microbiology, March 2006, p. 827-832, Vol. 44, No. 3
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.3.827-832.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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