Erythromycin

Sponge-associated Bacillus sp. HS24 was routinely grown and maintained aerobically, on Difco marine agar/broth (MA/MB) (Difco 2216), at 30°C, unless otherwise stated. Luria-Bertani medium was routinely used for growth and maintenance of E. coli and B. subtilis 168.
Susceptibility to erythromycin was determined by spotting MB cultures onto Muller–Hinton (MH, Merck, Darmstadt, Germany) plates supplemented with different concentrations of erythromycin (Sigma-Aldrich, Munich, Germany) and incubated aerobically at 30°C. Initial tests were performed with plates supplemented with 0 to 0.5 mg ml⁻¹ erythromycin. The concentration range of erythromycin was subsequently expanded, with plates supplemented with 1.0, 1.5, 2.0, 2.5, 3.0 and 3.5 mg ml⁻¹. MIC values are defined as the minimal concentration of antibiotic able to inhibit the growth. As there are no specific established antibiotic breakpoints values for marine sponge Bacillus isolates, the breakpoint values used for categorizing strain HS24 as resistant were those recommended by EFSA [19] .

Samples of Cabrales cheese were taken during the manufacturing (3 days) and ripening (60 days) stages of production, homogenized in a Stomacher (Seward, Worthing, UK) with 2% (w/v) sodium citrate (Merck), and serially diluted in Ringer’s solution (Merck). Aerobic mesophilic bacteria and LAB resistant to tetracycline and erythromycin were enumerated by plating the dilutions on Plate Count Milk (PCM; Merck) and MRS agar, respectively, supplemented with tetracycline (25 µg mL⁻¹) or erythromycin (25 µg mL⁻¹) (both from Merck). Plates were incubated at 32 °C (PCM) or 37 °C (MRS) for 48 h. Colonies showing semi-confluent growth were harvested, suspended in Brain Heart Infusion (BHI) broth (Merck) without antibiotics, supplemented with 25% glycerol, and stored at −80 °C.

All swabs were placed in 2 mL of Frey Medium 4 (FM4) broth [66 (link)] supplemented with antimicrobials (Amphotericin B (Sigma-Aldrich) 25 μg/mL, Ampicillin (Sigma-Aldrich) 2 units/mL and Colistin (Sigma-Aldrich) 75 mg/mL) to obtain initial suspensions. Mycoplasmas were directly cultured by diluting 100 μL of initial suspension from each sample in 900 μL of FM4 broth supplemented with antimicrobials and serial dilutions up to 10^− 3 were performed. All dilutions were incubated at 37 +/− 2 °C until the culture developed an acid color change or for a maximum of 30 days. Since MS is naturally resistant to erythromycin [56 ], cultures were also performed in FM4 broth medium supplemented with different erythromycin (Sigma-Aldrich) concentrations ranging from 4 to 10 μg/mL when isolation of MS was made difficult due to the presence of other Mycoplasma species. Subcultures onto FM4 agar were performed after the development of an acid color change and agar plates were incubated at 37 +/− 2 °C with 5% CO₂ for 5–10 days. Clones were obtained by picking single Mycoplasma colonies under a stereomicroscope and growing them in FM4 broth as described above. Initial suspensions, cultures, and clones were stored at ≤ − 65 °C in 20% glycerol until further analysis.