Streptomycin

The antibiotic susceptibilities of three probiotic candidates were assayed using the minimum inhibitory concentration (MIC) test strip method. Nine antibiotic strips were used for testing the bacterial strains, namely, ampicillin, chloramphenicol, clindamycin, erythromycin, gentamicin, kanamycin, streptomycin, tetracycline, and vancomycin (Liofilchem, Abruzzi, Italy). The bacteria were grown for 18 h at 37 °C in MRS medium. The cells were harvested via centrifugation at 3470× g for 5 min, washed twice with PBS (pH 7.0), and resuspended in PBS to a McFarland turbidity of 0.5. The cell suspensions were inoculated on BHI agar using swabs. The plates were dried for 15 min, and the MIC test strips were placed on the agar surface according to the manufacturer’s instructions. The plates were then incubated at 37 °C, and the results were assessed after 20 h of inoculation, according to the European Food Safety Authority (EFSA) guidelines [23 (link)].

Iodine (≥99.0%), copper iodide (CuI) and Mueller Hinton Broth (MHB) were obtained from Sigma Aldrich (Gillingham, UK). 1,4,7,10-Tetraoxacyclododecan (12-crown-4) was received from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Disposable sterilized Petri dishes with Mueller Hinton II agar and McFarland standard sets were bought from Liofilchem Diagnostici (Roseto degli Abruzzi (TE), Italy). The bacterial strains E. coli WDCM 00013 Vitroids, P. aeruginosa WDCM 00026 Vitroids, K. pneumoniae WDCM00097 Vitroids, C. albicans WDCM 00054 Vitroids, and Bacillus subtilis WDCM0003 Vitroids were received from Sigma-Aldrich Chemical Co. S. pneumoniae ATCC 49619, S. aureus ATCC 25923, E. faecalis ATCC 29212, S. pyogenes ATCC 19615, P. mirabilis ATCC 29906 were purchased from Liofilchem. Gentamicin (9125, 30 µg/disc), cefotaxime (9017, 30 µg/disc), chloramphenicol (9128, 10 µg/disc), streptomycin (SD031, 10 µg/disc), and nystatin (9078, 100 IU/disc) were obtained from Liofilchem. Methanol (analytical grade) was received from EMSURE (Merck KGaA, Darmstadt, Germany). Acetonitrile (analytical grade) was purchased from MTEDIA (TEDIA Company, Fairfield, OH, USA). Sterile filter paper discs with a diameter of 6 mm were bought from Himedia (Mumbai, India). Ultrapure water was utilized and all reagents were of analytical grade and used as received.

The determination of bacterial sensitivity to selected antibiotics was performed according to the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST). The method was slightly modified to adapt to the tested strain.
Bacterial culture of Lpb. plantarum DB2, grown in MRS broth, was separated from the medium by centrifugation at 6440× g for 10 min and resuspended in sterile water, with OD values adjusted to 0.5 ± 0.05. The cell suspension was applied to MRS agar plates by uniformly swabbing a cotton swab, previously immersed in the suspension, in three directions. After inoculation, discs of the tested antibiotics (Liofilchem S.r.l., Roseto degli Abruzzi, Italy) were placed on the inoculated MRS agar. The plates were then incubated at 37 °C, and after 24 h, the appearance of inhibition zones was observed, indicating the sensitivity of the tested strain to the antibiotic.
In this study, the sensitivity of lactic acid bacteria isolate DB2 to the following antibiotics was investigated: ampicillin (10 µg), erythromycin (15 µg), gentamicin (10 µg), kanamycin (30 µg), clindamycin (10 µg), chloramphenicol (10 µg), streptomycin (10 µg), tetracycline (30 µg), and vancomycin (30 µg) (Liofilchem S.r.l., Roseto degli Abruzzi, Italy).