This study determines the genetic background of the change in antimicrobial susceptibility to enrofloxacin of Ornithobacterium rhinotracheale (ORT) isolates with increased MIC values, isolated either from the field or from turkeys treated with enrofloxacin under experimental challenge conditions. In the field strains of ORT that were either less susceptible or, occasionally, resistant to enrofloxacin, point mutations had occurred in amino acids at positions 83 (serine) or 87 (aspartic acid) of the GyrA subunit. In the isolates showing reduced susceptibility following experimental enrofloxacin treatment (increase in MIC from < or =0.03 to 0.25 microg/ml), molecular analysis revealed a constantly recurring point mutation (G-->T) at nucleic acid position 646 (E. coli numbering) of gyrA resulting in an amino acid change from aspartic acid to tyrosine at position 87 of the GyrA subunit, which is a known hot spot for fluoroquinolone resistance. This study indicates that a single course of enrofloxacin treatment may contribute to the selection of the first mutant with reduced fluoroquinolone susceptibility in ORT. Acquired fluoroquinolone resistance is commonly encountered in ORT isolates. This is the first time that the causal mechanism of fluoroquinolone resistance in ORT has been investigated.