International Journal of Antimicrobial Agents 32 (2008) 227–232 In vitro-selected resistance to fluoroquinolones in two Brucella strains associated with mutational changes in gyrA Aun Turkmani a,b,∗ , Anna Psaroulaki a , Athanasia Christidou a , Dimosthenis Chochlakis a , Darem Tabaa b , Yannis Tselentis a a Laboratory of Clinical Bacteriology, Parasitology, Zoonoses and Geographical Medicine, WHO Collaborating Center, Department of Social Medicines, Faculty of Medicine, University of Crete, P.O. Box 1393, TK 71409 Heraklion, Crete, Greece b Faculty of Veterinary, Al Baath University, Homs, Syria Received 11 January 2008; accepted 6 March 2008 Abstract In the present study, Brucella melitensis biovar Abortus 2308 and Brucella abortus 3196 biotype 5 reference strains, which are susceptible to fluoroquinolones, became in vitro-resistant to fluoroquinolones by culture in trypticase soy agar. The quinolone resistance-determining regions (QRDRs) of the gyrA and parC genes of the two reference strains were analysed by polymerase chain reaction sequencing analysis to obtain the wild-type sequence. These sequences were then compared with the corresponding sequences of four in vitro-selected fluoroquinolone-resistant mutants to characterise mutations associated with resistance. Sequencing of the ofloxacin-selected resistant mutant 2308 revealed a transition of GAT to AAT (corresponding to position 87 of Escherichia coli gyrA), leading to substitution of Asp91 → Asn, whilst at the same position the ciprofloxacin-selected resistant mutant 2308 revealed a transition of GAT to TAT (corresponding to the same position of E. coli as above), leading to substitution of Asp91 → Tyr. The ofloxacin-selected resistant mutant 3196 had a transition of GCT to GTT, generating an amino acid change of Ala87 → Val. Amino acid changes were detected in the portion of the Brucella gyrA gene (Ala71 to Gln110) corresponding to the E. coli gyrA QRDR region (Ala67 to Gln110). Amino acid changes were also detected in Ser83, corresponding to the region where fluoroquinolone-associated amino acid changes are most commonly found in other bacterial species. © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. Keywords: Brucella; Fluoroquinolones; QRDR; Quinolone-resistant mutants; Mutations 1. Introduction Infections caused by Brucella spp. are common zoonoses in many parts of the world [1]. Members of the genus Brucella are Gram-negative, facultatively intracellular, highly infec- tious bacteria that can spread through contact with infected animal products or through the air, making them a potential bioterrorism agent [2,3]. Human brucellosis is a severe febrile disease with a broad spectrum of clinical manifestations and is characterised by focal complications, chronic courses as ∗ Corresponding author at: Laboratory of Clinical Bacteriology, Parasitol- ogy, Zoonoses and Geographical Medicine, Faculty of Medicine, University of Crete, P.O. Box 1393, TK 71409 Heraklion, Crete, Greece. Tel.: +30 2810 394 743. E-mail address: auntur@gmail.com (A. Turkmani). well as relapse and therapeutic failures [1,4,5]. The disease results in increased morbidity as well as considerable loss of productivity in animal husbandry in the developing world [6]. Brucella infection is treated with a combination of antibi- otics. Combinations of doxycycline plus streptomycin or rifampicin are recommended by World Health Organiza- tion (WHO) for human brucellosis [7]. Complicated forms of brucellosis require prolonged treatment, whilst therapeu- tic failures have been reported regardless of the antibiotic regimen used. Quinolones have been intriguing candidates since their emergence, but most trials utilise small numbers of patients and are inconsistently planned, not being comparable with the officially accepted regimens. The limited existing data sup- port the equality, but not superiority, of quinolone-containing regimens. 0924-8579/$ – see front matter © 2008 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved. doi:10.1016/j.ijantimicag.2008.03.012