Mutations in the quinolone resistance determining region conferring resistance to fluoroquinolones in Mycoplasma agalactiae.

M. agalactiae is the main causative agent of contagious agalactia, against which antimicrobial treatment is the main applied control measure. Quinolones are an effective group of antimicrobials inhibiting the growth of M. agalactiae, but in the last years, various reports have demonstrated an increase of resistance in field isolates due to its massive use. Nevertheless, the molecular mechanisms involved in the acquisition of fluoroquinolones resistance in M. agalactiae have not been elucidated yet. Therefore, the aim of this work was to analyze the presence of DNA variations that could be related to changes in fluoroquinolone susceptibility. For this purpose, three M. agalactiae strains were selected to obtain in vitro resistant mutants against enrofloxacin, marbofloxacin and moxifloxacin and afterwards, partial sequences of their gyrA, gyrB, parC and parE genes were analyzed. In addition, a set of field isolates with different MIC values were also studied. Changes related to variations in fluoroquinolones susceptibility were found in gyrB, parC and parE. Specifically, gyrB genes were affected at the predicted amino acid position 424, four amino acid changes were detected in parC (positions 78, 79, 80 and 84) and two substitutions were reported in parE (amino acid positions 429 and 459). Mutations at predicted positions 424 of gyrB and 429 of parE are novel DNA changes which had not been previously described and, on the whole, parC was the first gene showing alterations when changes in susceptibility to fluoroquinolones occurred. Thus, this gene is the most suitable target for a rapid study of fluoroquinolone resistance in field isolates of M. agalactiae.