Fosfomycin

The tested antimicrobials were daptomycin (Cubist, Pharmaceuticals, Lexington, MA), fosfomycin (Sigma-Aldrich, St. Louis, USA), gentamicin (Sigma-Aldrich), linezolid (Pfizer, New York, USA), oxacillin (Sigma-Aldrich), and rifampicin (USP; Twinbrook Parkway, Rockville, MD, USA), and they were prepared according to the manufacturers’ instructions. MIC values were determined by broth microdilution in cation-adjusted Mueller-Hinton broth with an inoculum of 5 × 10⁵ CFU/ml in the wells of the microplates. The results were interpreted according to the CLSI guidelines [16 ]. MIC values represent the lowest drug concentration at which complete inhibition of visible microbial growth is observed. Calcium was added to the growth media containing daptomycin to a final concentration of 50 μg/mL, whereas 25 μg/mL glucose-6-phosphate was added to the medium containing fosfomycin [16 ]. One hundred MRSA isolates were randomly selected from those with MIC of 1 mg/L to daptomycin using a random digital table for the subsequent experiments. The MIC₅₀ and MIC₉₀ of and the susceptibilities to each tested drug except daptomycin were calculated.

RCC299 and CCMP1545 were grown under a 14 h/10 h light/dark cycle in L1 media in artificial seawater (as above) at 220 μE m⁻² s⁻¹ PAR. Both strains were maintained in light-acclimated, mid-exponential growth before experiment initiation. Two days before the experiment start cultures of each species were split into duplicates A and B. Ten mM Penicillin G (final concentration, i.e., 6000 Units ml⁻¹) and 10 mM Fosfomycin (Sigma-Aldrich) were added 1 h after lights on (at T₀). At each time point cells were fixed in 0.25 % glutaraldehyde (final concentration) for 30 min in the dark and frozen in liquid N₂. Cells were measured using an Influx flow cytometer (BD Biosciences) and analyzed using Winlist (version 7.1, Verity Software House). Forward angle light scatter (FALS) and SSC were normalized using 0.75 μm diameter YG beads (Polysciences Inc.) and chlorophyll fluorescence (692 ± 40 nm band pass) was normalized to 2 μm diameter Polychromatic Red beads (Polysciences, Inc.).
To measure RCC299 for the morphological description, >10,000 cells from a mid-exponential phase, axenic culture were measured live on a Coulter Multisizer II approximately midway through the light period. Cells were grown on a 14 h/10 h light/dark cycle in K medium in artificial seawater maintained in mid-exponential growth for >10 generations after acclimatization to 21 °C and 90 μmol photons m² sec⁻¹ PAR.

Fosfomycin[(−)-(1R,2S)-(1,2-Epoxypropyl)phosphonic acid,C₃H₅Na₂O₄P] from Sigma-Aldrich (St. Louis, MI, USA) was used for the surface modification of polymer nonwovens.

Meropenem (Tokyo Chemical Industry Co. Ltd., Tokyo, Japan), sulbactam (Acros Organics, Lot: A0405260, Shanghai, China) and fosfomycin (Wako Pure Chemical Industries, Ltd., Osaka, Japan) were obtained from their respective manufacturers. Stock solutions of these antibiotics were prepared in sterile Milli-Q water, filter-sterilised with a 0.22 µm polyvinylidene difluoride (PVDF) syringe filter, aliquoted and stored at −80 °C until required. Before each susceptibility test, an aliquot of the drug was thawed and diluted to the desired concentrations with cation-adjusted Mueller-Hinton II (CA-MH) broth. Broth or agar containing fosfomycin was supplemented with 25 mg/L of glucose-6-phosphate (Sigma-Aldrich, St. Louis, MO, USA).

Chloramphenicol (CHL), Erythromycin (ERY), Ciprofloxacin (CIP), Doxycycline (DOX), Rifampicin (RIF), Fusidic acid (FA), Fosfomycin (FOF), Aztreonam (ATM), Clindamycin (CLI), Tigecycline (TGC), Imipenem (IPM), Colistin (CST), Gentamycin (GEN), Piperacillin (PIP), Tazobactam (TZB), Trimethoprim (TMP), Sulfamethoxazole (ST), Daptomycin (DAP), Linezolid (LZD), Oxacillin (OXA), Vancomycin (VAN), and Ceftazidime (CAZ) were purchased from Sigma (Darmstadt, Germany) and Cayman Chemicals (Ann Arbor, MI, USA). All stock solutions of 10 mg/mL were prepared in recommended solvents in non-transparent Eppendorf tubes and kept in −20 °C until use.

Antibacterial activities of fosfomycin and β-CLA were carried out by tube-dilution method. In brief, bacterial isolates were cultured overnight at 36±0.5°C on nutrient agar medium, and then suspended in sterile buffer saline and used as inoculate within one hour after adjustment. Bacterial inoculate were added to serial dilutions of fosfomycin (Sigma-Aldrich) or β-CLA(Sigma-Aldrich) containing tubes, with 1.5×10⁶ CFU /mL as the final concentration, by adjusting with No. 0.5 of Mc Farland’s Turbidity Standards. MICs and MBCs of fosfomycin and β-CLA against each isolate were determined by preparing serial dilutions using Mueller Hinton Broth (BBL). Previous studies (9 (link), 13 (link)) have reported the concentration ranges for determining MIC and MBC values for fosfomycin and β-CLA to be 0.25–128 mg/L and 0.0625 −1 mM respectively. Experiments were carried out in triplicate. The MIC and MBC values were defined as the lowest antibiotic concentration that completely prevented turbidity in broth or colony growth on agar media after incubation, respectively at 37°C for 24 hours (14 (link)).