Recommendation of an appropriate medium for in vitro drug susceptibility testing of the fish pathogen Tenacibaculum maritimum

In the present study, Anacker and Ordal agar, marine agar (MA), and Flexibacter maritimus medium (FMM) were compared with the dilute versions of Mueller-Hinton agar (DMHA) medium recommended by the National Committee for Clinical Laboratory Standards (NCCLS) for their use in disk diffusion tests with Tenacibaculum maritimum strains and to calculate the MICs of five drugs by the Etest method. Preliminary growth tests performed with 32 strains of this pathogen on each medium revealed that all strains failed to grow on DMHA, while the remaining media supported good growth of all isolates. In the susceptibility tests, which were carried out with the other three media, all strains were resistant to oxolinic acid and were highly susceptible to amoxicillin and trimethoprim-sulfamethoxazole, showing a good correspondence with the Etest values, which ranged from 0.064 to 0.75 and 0.006 to 1.5 μg/ml, respectively. Enrofloxacin and oxytetracycline produced significantly smaller inhibition zones and MICs on MA than on the other media assayed. However, fast, clear, and well-defined zones of inhibition were displayed for all strains at 24 h of incubation only on FMM by both the disk diffusion assay and Etest. In addition, FMM prepared with commercial sea salts instead of seawater was also suitable for bacterial isolation as well as for susceptibility testing. On the basis of these results, the use of FMM to determine the in vitro susceptibility of T. maritimum and its inclusion in a future revision of the NCCLS M42 report are recommended.