Determination of Minimum Inhibitory Concentrations (MICs)

MIC experiments were carried out in 96-well microplates. Ten antibiotics (ampicillin, chloramphenicol, ciprofloxacin, erythromycin, gentamicin, tetracycline, kanamycin, streptomycin, clindamycin, and spectinmycin) were selected among those frequently used to test the antibiotic susceptibility of LAB and five antibiotics (ofloxacin, sulfadiazine, furacillin, furazolidone, and sulfamethazine) were selected because of their frequent use in the culturing of marine animals. A 2-fold serial dilution was prepared for each antibiotic in the respective solvents (the solvents of ampicillin, gentamicin, kanamycin, streptomycin, and spectinmycin were deionized water; the solvents of chloramphenicol, erythromycin and tetracycline were ethanol; the solvent of ofloxacin was 0.85% sodium chloride solution; the solvent of ciprofloxacin was acetonitrile; the solvent of ofloxacin was 0.1M HCl; the solvent of furazolidone was chloroform; the solvents of sulfadiazine, furacillin and sulfamethazine were dimethyl sulfoxide) and the final antibiotic concentrations were between 0.5 and 512 mg/L. T. muriaticus strains were cultured in MRS broth containing 6% NaCl until they reached 10⁸ CFU/mL. The final inoculum density in each well was 5 × 10⁵ CFU/mL. The plates were then incubated at 36°C for 20 h under static conditions. The MIC for each antibiotic was recorded as the lowest concentration at which no turbidity was observed in the wells. Resistance to a particular antibiotic was defined as the point at which the MIC value for a tested antibiotic was higher than its recommended breakpoint value as defined by the European Committee on Antimicrobial Susceptibility Testing (EUCAST)¹ in 2015 and 2020. Five independent experiments were conducted.

For the genus Tetragenococcus, the breakpoint values of the antibiotics have not been defined by the EFSA. Thus we referred to the breakpoint values provided by the EUCAST for Enterococcus in 2015 and 2020. Microbiological breakpoints are generally set by studying the distribution of the MICs of the chosen antimicrobial agents in a bacterial population belonging to a single taxonomical unit (species or genus). Strains with MICs higher than the breakpoints are considered resistant (EFSA, 2008).