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Volume 13, Number 12—December 2007

Letter

Bartonella australis sp. nov. from Kangaroos, Australia

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To the Editor: During April–May 1999, 3 Bartonella isolates (AUST/NH1, AUST/NH2, AUST/NH3) were cultivated and established from the blood of 5 Macropus giganteus gray kangaroos from central coastal Queensland, Australia. We used multigene sequencing to evaluate whether these Bartonella isolates fulfill the minimum requirements for classification as a new species.

DNA from each Bartonella isolate was extracted by using the QIAamp tissue kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. Partial PCR amplification and sequencing of the genes encoding the 16S rDNA (rrs), citrate synthase (gltA), β-subunit of the RNA polymerase (rpoB), and cell division protein (ftsZ), as well as for the 16S–23S rDNA intergenic spacer (ITS) were attempted by using previously described primers and conditions (1). Bartonella sp. isolates AUST/NH1 to AUST/NH3 exhibited identical sequences for all 4 genes and the spacer studied, and isolate AUST/NH1 was selected as type strain among kangaroo isolates. Similarity rates between strain Aust/NH1 and validated Bartonella species (Appendix Table [PDF - 13 KB - 1 page]) ranged from 84.7% to 91.6%, from 97.5% to 98.5%, from 79.6% to 87.2%, from 85.4% to 95.0%, and from 83.5% to 87.1% for the ITS and rrs, gltA, rpoB, and ftsZ genes, respectively. Therefore, for each of these 4 genes or the spacer, strain AUST/NH1 exhibited similarity rates with all other species lower than the cutoffs published to classify Bartonella isolates within a validated species (1). It may thus be regarded as a new species.

To estimate the genomic G+C content of strain AUST/NH1, we amplified and sequenced its ftsY gene as described (2) by using the BartftsyF (5′-ATGACAAAAYCYTTTATMAA-3′) and BartftsyR (5′-TCATGAGTGTCTTCCTGC-3′) primers. The ftsY G+C content was 37.7%; the calculated genomic G+C content was 39.51%. The ftsY sequence was deposited in GenBank under accession no. DQ538398.

Figure

Thumbnail of Unrooted dendrogram showing the phylogenetic position of Bartonella sp. strain AUST/NH1 among Bartonella species inferred from the comparison of concatenated sequences from the rrs, gltA, intergenic spacer, rpoB, and ftsZ genes by the neighbor-joining method. We included only species for which all 5 genes were available. Bootstrap values are indicated at the nodes. The scale bar indicates nucleotide sequence divergence of 0.5%.

Figure. Unrooted dendrogram showing the phylogenetic position of Bartonella sp. strain AUST/NH1 among Bartonella species inferred from the comparison of concatenated sequences from the rrs, gltA, intergenic spacer, rpoB, and ftsZ genes...

The phylogenetic relationships among the studied bartonellae were inferred from sequence alignments of each gene and from concatenated gene sequences by using the maximum parsimony and neighbor-joining methods within the MEGA version 2.1 software package (3) and the maximum-likelihood method within the PHYLIP software package (4). Using rrs, gltA, and rpoB sequences, the phylogenetic position of strain AUST/NH1 was supported by bootstrap values <70%. In contrast, by using the ITS, ftsZ, and concatenated sequences, strain AUST/NH1 clustered with a group of B. tribocorum, B. grahamii, and B. elizabethae, with elevated bootstrap values according to the 3 analysis methods (Figure).

The Bartonella strains we describe are the first, to our knowledge, obtained from kangaroos and, more generally, from marsupials. Before this study, the only 2 Bartonella species found in Australia were B. henselae (5) and B. quintana (6). We demonstrated that strain AUST/NH1 was reliably associated with a well-established cluster, including the rodent-associated B. elizabethae, B. grahamii, and B. tribocorum (7). Therefore, we are confident that the phylogenic position of the new Bartonella, which was similar according to 3 analysis methods and supported by high bootstrap values, is reliable. Although B. grahamii (8) and B. elizabethae (9), members of the same phylogenetic cluster as strain AUST/NH1, cause human infections, the pathogenicity of B. tribocorum is as yet unknown. Its pathogenicity should therefore be investigated, especially for persons who come in contact with kangaroos.

B. australis is a facultative intracellular gram-negative bacterium. It grows on Columbia agar with 5% sheep blood at 32°C to 37°C in a moist atmosphere containing 5% CO2. A primary culture was obtained after 7 days, and subculture was obtained after 4 days under the same conditions. Colonies are homogeneous, smooth, round, and gray-white. The 3 strains tested were oxidase negative, catalase negative, and nonmotile. Pathogenicity for humans is, as yet, unknown.

The type strain is strain AUST/NH1. The new species is distinguished from other Bartonella species by its 16S rRNA, gltA, rpoB, ftsZ gene sequences, as well as its 16S–23S rRNA ITS sequence. The estimated G+C content is 38%. The type strain exhibits a specific serotype (10) and was susceptible to amoxicillin, ceftriaxone, imipenem, erythromycin, clarithromycin, ofloxacin, ciprofloxacin, rifampin, and tetracycline (unpub. data). The type strain AUST/NH1 has been deposited in the Collection of the World Health Organization Collaborative Center for Rickettsioses, Borrelioses and Tick-borne Infections (CSUR), Marseille, France, under reference CSUR B1; in the Collection de l’Institut Pasteur (CIP) under reference CIP 108978T; and in the Culture Collection of the University of Göteborg (CCUG), Sweden, under reference CCUG 51999. The strains AUST/NH2 and AUST/NH3 have been deposited in CSUR under references CSUR B2 and CSUR B3, in the CIP under references CIP 108980 and CIP 108979, and in CCUG under references CCUG 52000 and CCUG 52001, respectively.

Pierre-Edouard Fournier*, Carmel Taylor†, Jean-Marc Rolain*, Lina Barrassi*, Greg Smith†, and Didier Raoult*Comments to Author 
Author affiliations: *Université de la Méditerranée, Marseille, France; †Queensland Health Scientific Services, Coopers Plains, Queensland, Australia;

Acknowledgment

We thank Annick Bernard, Laetitia Dorey, and Laurence Delaunay for technical help.

References

  1. La Scola B, Zeaiter Z, Khamis A, Raoult D. Gene-sequence-based criteria for species definition in bacteriology: the Bartonella paradigm. Trends Microbiol. 2003;11:31821. DOIPubMed
  2. Fournier PE, Suhre K, Fournous G, Raoult D. Estimation of prokaryote genomic G+C content by sequencing universally-conserved genes. Int J Syst Evol Microbiol. 2006;56:12059.
  3. Kumar S, Tamura K, Jakobsen IB, Nei M. MEGA2: molecular evolutionary genetics analysis software. Bioinformatics. 2001;17:12445. DOIPubMed
  4. Retief JD. Phylogenetic analysis using PHYLIP. Methods Mol Biol. 2000;132:24358.PubMed
  5. Fournier PE, Robson J, Zeaiter Z, McDougall R, Byrne S, Raoult D. Improved culture from lymph nodes of patients with cat scratch disease and genotypic characterization of Bartonella henselae isolates in Australia. J Clin Microbiol. 2002;40:36204. DOIPubMed
  6. Rathbone P, Graves S, Miller D, Odorico D, Jones S, Hellyar A, Bartonella (Rochalimaea) quintana causing fever and bacteremia in an immunocompromised patient with non-Hodgkin's lymphoma. Pathology. 1996;28:803. DOIPubMed
  7. Houpikian P, Raoult D. Molecular phylogeny of the genus Bartonella: what is the current knowledge? FEMS Microbiol Lett. 2001;200:17. DOIPubMed
  8. Kerkhoff FT, Bergmans AMC, van der Zee A, Rothova A. Demonstration of Bartonella grahamii DNA in ocular fluids of a patient with neuroretinitis. J Clin Microbiol. 1999;37:40348.PubMed
  9. Daly JS, Worthington MG, Brenner DJ, Moss WC, Hollis DG, Weyant RS, Rochalimaea elizabethae sp. nov. isolated from a patient with endocarditis. J Clin Microbiol. 1993;31:87281.PubMed
  10. Bonhomme CJ, Nappez C, Raoult D. Microarray for serotyping of Bartonella species. BMC Microbiol. 2007;7:59. DOIPubMed

Figure

Table

Suggested citation for this article: Fournier P-E, Taylor C, Rolain J-M, Barrassi L, Smith G, Raoult D. Bartonella australis sp. nov. from kangaroos, Australia [letter]. Emerg Infect Dis [serial on the Internet]. 2007 Dec [date cited]. Available from http://wwwnc.cdc.gov/eid/article/13/12/06-0559.htm

DOI: 10.3201/eid1312.060559

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Table of Contents – Volume 13, Number 12—December 2007

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Didier Raoult, Unité des Rickettsies, IFR 48, CNRS UMR 6020, Faculté de Médecine, Université de la Méditerranée, 27 Boulevard Jean Moulin, 13385 Marseille CEDEX 05, France;





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