Effects of NaCl Concentration on Antimicrobial Susceptibility Testing

Antimicrobial resistance is a major One Health issue, and marine environments are increasingly recognized as important reservoirs of antibiotics and resistance genes.However, antimicrobial susceptibility testing for marine-derived microorganisms still relies largely on clinical standard protocols, and the effects of NaCl concentration on test results have not been sufficiently evaluated.In this study, we examined the effects of NaCl concentration on antimicrobial susceptibility testing using standard reference strains, including Escherichia coli and Enterococcus faecalis.Disk diffusion and minimum inhibitory concentration (MIC) assays were performed using Mueller-Hinton agar, Marine agar, and Mueller-Hinton media supplemented with 0.0-2.0%NaCl.Significant differences in inhibition zone diameters were observed between Mueller-Hinton agar and Marine agar, particularly for erythromycin, tetracycline, and trimethoprim/sulfamethoxazole.In both E. coli and E. faecalis, inhibition zones for streptomycin and erythromycin decreased as NaCl concentration increased.In MIC assays, most antibiotics remained relatively stable at NaCl concentrations up to 2.0%, whereas gentamicin showed greater sensitivity to strain, NaCl concentration, and incubation temperature.Additional validation with other standard reference strains also revealed condition-dependent variation in several strain-antibiotic combinations.These findings indicate that NaCl concentration can affect antimicrobial susceptibility testing and should be carefully considered when evaluating marine-derived microorganisms.