Mh agar

Antimicrobial susceptibility testing using disc diffusion method and minimal inhibitory concentrations (MICs) using the E-test technique (bioMérieux, Marcy L’Etoile, France) were determined on Mueller-Hinton (MH) agar (Bio-Rad, Marnes-La-Coquette, France), and interpreted according to EUCAST guidelines, as updated in 2016 (http://www.eucast.org). Clinical carbapenem breakpoints for susceptibility/resistance were ≤ 2/>8 mg/l for imipenem and meropenem and ≤0,5/>1 mg/l for ertapenem.

The minimum inhibitory concentration (MIC) of black currant leaf extract was determined according to the recommendations of EUCAST [36 (link)]. Mueller–Hinton (MH) agar (Bio-Rad, Hercules, CA, USA) was used as a nonselective medium for the growth of tested microorganisms (Table 2). For lactic acid bacteria (LAB), 10 g/L glucose (Sigma-Aldrich, Poznań, Poland) was added to the MH medium. The bacterial suspensions were prepared using overnight cultures. Aqueous black currant leaf extract was diluted with a molten MH medium to prepare tested concentrations (1–5 mg/mL). After solidification of the MH agar, the tested strain with the adjusted density of 10⁴ colony-forming units (cfu)/mL was spread on the medium. Then, the samples were incubated at 37 °C for 24 h. The positive control consisted of MH agar inoculated with the test bacteria without the extract, while uninoculated plates containing black currant leaf extract served as the negative control. When the visible growth inhibition was observed (judged by the naked eye), regardless of the appearance of a single colony or a thin haze, the MIC of the extract was determined.

Two Gram-positive (G⁺) [Staphylococcus aureus ATCC 25,923 and Listeria monocytogenes ATCC 19,117] and two Gram-negative (G⁻) [Salmonella enterica ATCC 43,972 and Escherichia coli ATCC 25,922] bacteria stains were used to evaluate the antibacterial efficacy of LmPS. The bacterial strains origin as well as cell culture were previously reported by Ben Hsouna [5 (link)]. Briefly, bacteria were obtained from both the international collections of the American Type of Culture Collection (ATCC) and the local collection of cultures from the Center of Biotechnologie of Sfax (Tunisia). The bacterial progeny was grown on MH agar (Bio-Rad, Marnes-la-Coquette, France) at a temperature of 37 °C for 12–14 h. The inoculum preparation was carried out using an overnight broth culture by dilution in saline solution to 10⁶ colony-forming units (CFU/mL).