Mechanisms of quinolone resistance and clonal relationship among Aeromonas salmonicida strains isolated from reared fish with furunculosis

The mechanisms of resistance to quinolone and epidemiological relationships among A. salmonicida strains isolated from diseased fish in French marine farms from 1998 to 2000 were investigated. The quinolone resistance-determining regions of the gyrA and parC genes of 12 clinical A. salmonicida isolates with different levels of quinolone susceptibility were sequenced. MICs were determined in the presence of the efflux pump inhibitor (EPI) Phe-Arg beta-naphthylamide and E-max values (MIC without EPI/MIC in the presence of EPI) were calculated. Isolates fell into two classes: (i) those that had a wild-type gyrA gene with oxolinic acid MIC less than or equal to 0.5, flumequine MIC less than or equal to 1 and ciprofloxacin MIC less than or equal to 0.25 mug ml(-1); and (ii) those that had a single mutation in gyrA encoding Asp-87 --> Asn with oxolinic acid MIC greater than or equal to 2, flumequine MIC greater than or equal to 4 and ciprofloxacin MIC greater than or equal to 0.125 mug ml(-1). No mutations were found in parC. High E-max values obtained for flumequine and oxolinic acid (up to 16 and 8, respectively, for the most resistant isolates of the two classes) indicated an important contribution of efflux to the resistance phenotype. Flumequine accumulation experiments confirmed that high E-max values were associated with a much lower level of accumulation. PCR/RFLP assays conducted on 34 additional isolates showed the presence of a mutation at codon 87 of gyrA in nearly all the quinolone-resistant isolates. This finding, together with PFGE typing results, strongly suggests a common clonal origin of these quinolone-resistant isolates.