Blank disk

Fosfomycin susceptibility was determined in Mueller–Hinton (MH) by agar dilution (gold standard) and disk diffusion methods according to CLSI/EUCAST guidelines [14 ,15 ]. For the agar dilution method, bacterial cultures were adjusted to the 0.5 MacFarland standard and diluted 1:10 in saline solution. Then, 2 μL (approximately 10⁴ CFU) were spotted onto the agar surface. Once the spots on the agar were absorbed, the plates were incubated at 35 ± 2 °C for 16 h. The MIC was defined as the lowest antibiotic concentration that inhibited visible growth. Strains with a MIC value for the clinical strains of ≤4 μg/mL were considered fosfomycin-susceptible, between >4 and ≤32 μg/mL as LLFR (still susceptible), and >32 μg/mL as fosfomycin-resistant. For the disk diffusion method, disks containing 200 μg of fosfomycin with 50 μg, 25 μg, 15 μg, and without G6P were used. Blank disks (Oxoid) were impregnated with 25 μL of each fosfomycin–G6P concentration. Diameter (in millimeters) of the zone of complete inhibition was determined after 16–20 h of incubation at 35 ± 2 °C. MICs and disk diffusion tests were repeated 3 times for each strain. Final data are the modal value from 3 repetitions. E. coli ATCC 25922 (laboratory collection) was used as the MIC control strain.

Antimicrobial susceptibility assays were performed according to a method described in our recent studies [9 (link),10 (link)]. Blank disks (6 mm, Oxoid, Basingstoke, UK) and Mueller–Hinton (MH) were purchased from Oxoid, Basingstoke, UK. An aliquot of 10 μL crude extract (500 μg/mL) was added onto each disk and the bacteriostatic effect on the corresponding strains evaluated by measuring the diameter of the inhibition zone after incubation at 37 °C for 12 h. A gentamicin disk (10 μg, Oxoid, Basingstoke, UK) was used as a positive control, while the methanol phase with water and chloroform phase with anhydrous ethanol were used as negative controls.
Broth dilution testing (microdilution) was carried out to determine MICs of the extracts according to the standard method issued by the Clinical and Laboratory Standards Institute, USA (CLSI, M100-S28, 2018). The standard solution of gentamicin (100 µg/mL) was purchased from the National Standard Material Information Center, Beijing, China [9 (link)].

The antimicrobial activity of each solution was evaluated by the agar diffusion method [32 (link)]. Each solution was combined with the emulsifier polysorbate 80 in the proportion of 1:10 (polysorbate 80: natural compound solution). Blank disks (Oxoid, Basingstoke, United Kingdom) were immersed in each solution and left submerged for 30 min to absorb the solution. Sterile swabs were immersed in each inoculum suspension (prepared as described in Section 2.4.1) and spread in Müeller-Hinton Agar (MHA, Biokar diagnostics) plates. Disks containing the solutions were added to the top of the culture medium, and plates were incubated at 30 °C for 24 h. Disks adsorbed with distilled water, polysorbate 80, acetic acid (PanReac), and ethanol (Sigma) were used as controls.