A total of 12 strains of Coxiella burnetii (8 Greek isolates from acute Q-fever patients, two reference strains-Nine Mile and Q212-and two pefloxacin-resistant laboratory strains) were examined for the presence of point mutations in the quinolone resistance determining region (QRDR) of gyrA gene by direct DNA sequencing of the polymerase chain reaction (PCR)-amplified fragments. The gene sequences of all eight Greek isolates and the two reference strains Nine Mile and Q212 [minimal inhibitory concentration (MIC)</= 4 microg/ml] were identical. Direct DNA sequencing of the in vitro-selected resistant strains (MICs to pefloxacin, 8-32 microg/ml) revealed a transition (G-->A) at the corresponding codon 87 of E. coli. This mutation lead to the substitution of Glu (codon GAG) by Lys (codon AAG ). Restriction maps of amplified gyrA gene sequences were determined by GCG Wisconsin PACKAGE, and the MnlI restriction enzyme was found to cut only the sensitive strains sequences and not the resistant ones. The present PCR-RFLP analysis has proved to be a simple, rapid, and useful method for the detection of Coxiella burnetii and, at the same time, for the diagnosis of quinolone-resistant Coxiella burnetii strains.
Copyright 2000 Wiley-Liss, Inc.