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Applied and Environmental Microbiology, November 2003, p. 6740-6749, Vol. 69, No. 11
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.11.6740-6749.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Usefulness of rpoB Gene Sequencing for Identification of Afipia and Bosea Species, Including a Strategy for Choosing Discriminative Partial Sequences

Atieh Khamis, Philippe Colson, Didier Raoult, and Bernard La Scola*

Unité des Rickettsies, CNRS UPRESA 6020, Faculté de Médecine, Université de la Méditerrannée, 13385 Marseille Cedex 05, France

Received 23 June 2003/ Accepted 18 August 2003

Bacteria belonging to the genera Afipia and Bosea are amoeba-resisting bacteria that have been recently reported to colonize hospital water supplies and are suspected of being responsible for intensive care unit-acquired pneumonia. Identification of these bacteria is now based on determination of the 16S ribosomal DNA sequence. However, the 16S rRNA gene is not polymorphic enough to ensure discrimination of species defined by DNA-DNA relatedness. The complete rpoB sequences of 20 strains were first determined by both PCR and genome walking methods. The percentage of homology between different species ranged from 83 to 97% and was in all cases lower than that observed with the 16S rRNA gene; this was true even for species that differed in only one position. The taxonomy of Bosea and Afipia is discussed in light of these results. For strain identification that does not require the complete rpoB sequence (4,113 to 4,137 bp), we propose a simple computerized method that allows determination of nucleotide positions of high variability in the sequence that are bordered by conserved sequences and that could be useful for design of universal primers. A fragment of 740 to 752 bp that contained the most highly variable area (positions 408 to 420) was amplified and sequenced with these universal primers for 47 strains. The variability of this sequence allowed identification of all strains and correlated well with results of DNA-DNA relatedness. In the future, this method could be also used for the determination of variability hot spots in sets of housekeeping genes, not only for identification purposes but also for increasing the discriminatory power of sequence typing techniques such as multilocus sequence typing.

* Corresponding author. Mailing address: Unité des Rickettsies, CNRS UPRESA 6020, Faculté de Médecine, Université de la Méditerrannée, 27 Blvd. Jean Moulin, 13385 Marseille Cedex 05, France. Phone: 33.04.91.38.55.17. Fax: 33.04.91.83.03.90. E-mail: bernard.lascola{at}medecine.univ-mrs.fr.

Applied and Environmental Microbiology, November 2003, p. 6740-6749, Vol. 69, No. 11
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.11.6740-6749.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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