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Bartonella sp. are intracellular bacteria associated with an increasing number of clinical manifestations but with few published data on in vitro susceptibility testing of antibiotics. Our objective was to evaluate in vitro antibiotic susceptibilities of 20 new Bartonella isolates from animals in Australia.MICs were determined using Etest assay on Columbia agar supplemented with 5% horse blood. The presence of mutations in the quinolone-resistance-determining region (QRDR) of gyrA was searched for after PCR amplification and DNA sequencing using specific oligonucleotide primers.Bartonella isolates from Australia were susceptible to rifampicin, tetracyclines, β-lactam and macrolide compounds but were resistant to vancomycin. We found heterogeneity of susceptibility for fluoroquinolones with ciprofloxacin being more effective (MICs from 0.06 to 0.5 mg/L) than ofloxacin (MICs from 0.5 to 4 mg/L). This heterogeneity was linked to a natural mutation Ser-83→Ala (Escherichia coli numbering) in the QRDR. Surprisingly, this mutation was also present in the QRDR of Bartonella henselae, Bartonella quintana and Bartonella bacilliformis.Etest is a sensitive and reliable assay for evaluation of antibiotic susceptibility in the genus Bartonella. The higher sensitivity of this method allowed us to detect heterogeneity of susceptibility among fluoroquinolones that was associated with natural mutation in the QRDR of the DNA gyrase. Because a high level of resistance to fluoroquinolones due to a second mutation may be obtained easily in vitro, we believe that fluoroquinolone compounds should be avoided for the treatment of any Bartonella-related diseases.