Application of NaCl-supplemented antimicrobial susceptibility testing conditions to marine bacterial isolates

Antimicrobial resistance (AMR) in marine environments remains difficult to assess under standard antimicrobial susceptibility testing conditions, which may not adequately reflect the physiological requirements of marine-derived bacteria. In this study, we applied NaCl-supplemented susceptibility testing conditions to bacterial isolates recovered from coastal environments and marine organism guts to evaluate their practical applicability and limitations. A total of 927 isolates were obtained from lagoon, seawater, soil, and gut samples of abalone, crab, eel, halibut, and salmon. Of these, 365 isolates grew on standard Mueller-Hinton (MH) medium, whereas 562 isolates were not evaluable under standard MH conditions. Taxonomic analysis showed that the isolate collection was dominated by Proteobacteria and Firmicutes, with Vibrio as the most abundant genus. Disk diffusion assays of the 365 MH-grown isolates revealed differences in screening-level reduced susceptibility profiles among sample groups, and putative multidrug resistance was frequently observed in Vibrio isolates from abalone and seawater. For isolates not evaluable under standard MH conditions, minimum inhibitory concentration (MIC) analysis was performed using 2% NaCl-supplemented MH broth. Under these conditions, 115 isolates showed stable growth and distinct antibiotic-dependent MIC profiles. Chloramphenicol and tetracycline inhibited most isolates at relatively low concentrations, whereas reduced susceptibility to penicillin was relatively high in isolates from seawater, abalone, and eel. Overall, these findings suggest that standard MH conditions alone may be insufficient for evaluating antibiotic responses in marine- and brackish-origin bacteria, and that 2% NaCl-supplemented MH broth can serve as a practical supplementary condition for selected marine-derived isolates.