Reduction in the in-vitro activity of flumequine against Aeromonas salmonicida in the presence of the concentrations of Mg 2+ and Ca 2+ ions found in sea water

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The effect of an ionic representation of sea water on the kinetics of inhibition and killing of Aeromonas salmonicida isolates by flumequine was investigated. The minimum inhibitory concentration (MIC), the minimum bactericidal concentration (MBC) and the percent bioactivity all varied with respect to time. The concentration of flumequine required to inhibit growth over 24 h was 4 μg ml −1 in Tryptone Soya Broth (TSB) and 128 μg ml −1 in the same medium supplemented with sea water ions. The concentrations required to inhibit growth over 72 h were higher; 16 μg ml −1 and 256 μg ml −1, respectively. This increase in the MIC over time was shown to be due to the emergence, during the assay, of cells with elevated resistance to flumequine. These strains also showed reduced sensitivity to a number of unrelated antimicrobial agents. The MBC of flumequine at 24 h was 16 μg ml −1 in Tryptone Soya Broth (TSB) and 2048 μg ml −1 in the same medium supplemented with sea water ions. At 72 h the MBC determined in TSB increased to 32 μg ml −1, and in media supplemented with sea water ions the MBC decreased to 256 μg ml −1. Thus, the percentage reduction in the bioactivity of flumequine resulting from the presence of sea water ions varied not only with time but also with respect to whether MIC or MBC data was considered. There was no effect by sea water or sea water ions on the HPLC assay of flumequine over the range 1–4096 μg ml −1. It is argued that HPLC analysis will necessarily overestimate the concentrations of biologically active flumequine in the marine environment. The data presented in this paper indicates some of the factors that must be considered in the design of a valid and relevant biological assay of flumequine in this environment.