Diagnosis of quinolone-resistant Coxiella burnetii strains by PCR-RFLP

A total of 12 strains of Coxiella burnetii (8 Greek isolates from acute Q-f ever patients, two reference strains-Nine Mile and Q212-and two pefloxacin- resistant laboratory strains) were examined for the presence of point mutat ions in the quinolone resistance determining region (QRDR) of gyrA gene by direct DNA sequencing of the polymerase chain reaction (PCR)-amplified frag ments. The gene sequences of all eight Greek isolates and the two reference strains Nine Mile and Q212 [minimal inhibitory concentration (MIC) less th an or equal to 4 mu g/ml] were identical. Direct DNA sequencing of the in v itro-selected resistant strains (MICs to pefloxacin, 8-32 mu g/ml) revealed a transition (G-->A) at the corresponding codon 87 of E. coli. This mutati on lead to the substitution of Glu (codon GAG) by Lys (codon AAG). Restrict ion maps of amplified gyrA gene sequences were determined by GCG Wisconsin PACKAGE, and the Mnn restriction enzyme was found to cut only the sensitive strains sequences and not the resistant ones. The present PCR-RFLP analysi s 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-res istant Coxiella burnetii strains, J. Clin, Lab. Anal. 14:59-63, 2000. (C) 2 000 Wiley-Liss, Inc.