Mueller hinton broth (mhb)

The antimicrobial performance of the composite filaments was examined using disk diffusion tests. We utilized the following bacteria strains in the study: Escherichia coli ATCC 11,775 (E. coli), Staphylococcus aureus ATCC 12,600 (SA), Pseudomonas aeruginosa ATCC 9027 (PA), and Methicillin-resistant Staphylococcus aureus ATCC 33,591 (MRSA). We cultivated these bacteria strains on blood agar and stored them on Mueller Hinton agar (MHA) (Condalab, Madrid, Spain) plates in the refrigerator at 4 °C for later use. When the experiments were performed, the bacteria strains were activated in Mueller Hinton broth (MHB) (Scharlau, Barcelona, Spain); after measuring their turbidity (0.1 ± 0.02), they were spread on the MHA plates via swab cotton. The composite filaments were prepared in a disk shape and then fixed on the surface of the MHA plates containing the bacteria strain beside a standard 30 μg cefoxitin antibiotic disc (FOX) from Mast (Mast Group Ltd., Bootle, UK). Then, all MHA plates were incubated at 37 °C for 24 h. Finally, we measured the diameters of the bacteria growth inhibition zone; the antibacterial performances of the prepared composites were compared with the standard antibiotic FOX disks.

Lipoid S75 (S75), a mixture of soybean phospholipids (70% phosphatidylcholine, 9% phosphatidyletanolamine and 3% lysophosphatidylcholine), triglycerides and fatty acids, was purchased from Lipoid GmbH (Ludwigshafen, Germany). RPMI 1640 medium, DMEM medium, glucose, phosphate-buffered saline (PBS), tryptone water, methylene blue, dimethylsulfoxide (DMSO), and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma–Aldrich (Madrid, Spain). Mueller–Hinton agar and methanol were purchased from VWR Chemicals (Barcelona, Spain). Mueller–Hinton broth, 2-propanol, and hydrochloric acid 37% were supplied by Scharlau (Valencia, Spain). Sabouraud Dextrose Chloramphenicol Agar (SDCA) was obtained from Becton Dickinson (Madrid, Spain).

Ampicillin trihydrate MIC was determined by the broth microdilution method according to the CLSI guidelines [32 ]. In each case, the MIC was determined for AT, PAM-18Na polymer and the mix of PAM-18Na and AT, in a 1:1 ratio (based on the copolymer unit), as previously described. The concentrations evaluated were 0.0625, 0.125, 0.25, 0.35, 0.5, 2, 8, 32, 128, 192, 256 µg/mL, respectively. The assays were performed in 96 round bottom well plates (BD) using 500 µL of the appropriate S. aureus strain grown in Mueller-Hinton broth (Scharlau^®) at a turbidity of 0.1 absorbance units. Twenty-four replicates were performed for each concentration and visually inspected for the presence of a bacterial cell pellet.

Salmonella enterica CECT 7160, Escherichia coli CECT 405, Shigella sonnei CECT 457, Pseudomonas aeruginosa CECT 116, Listeria monocytogenes CECT 4032, Staphylococcus aureus CECT 239, Bacillus cereus CECT 8168, Zygosaccharomyces bailii CECT 11997 and Aspergillus niger CECT 2090 were obtained from the Spanish Collection of Type Cultures (CECT). Enterococcus faecalis S-47, Candida sake DMC03 and Penicillium expansum DMC01 were donated by the DMC Research pathogen collection. Mueller–Hinton broth (Scharlau, Barcelona, Spain) was used for the growth of bacteria [27 ] and RPMI-1640 medium with L-glutamine for yeast and fungi [28 ].
Minimum Biocidal Concentration (MBC) determination was carried out using the broth microdilution method established by the CLSI [27 ,28 ]. Decreasing concentrations (1:2) of blackberry extract from 100 to 0.78 mg/mL were prepared in the corresponding liquid culture medium. Next, they were inoculated with a 10⁵ CFU/mL microbial cell suspension. Dilutions were incubated overnight and cultured in agar plates. The lowest extract concentration that completely inhibited microbial growth was considered as the MBC.

PVP—polyvinylpyrrolidone, average Mw ~1,300,000, ethyl alcohol (≥99.5%) sodium chloride (≥99.5%), sodium phosphate dibasic (≥99.0%), potassium chloride (99.0–100.5%), and potassium phosphate monobasic (≥99.0%) were purchased from Sigma-Aldrich (St. Louis, MO, USA) Halicin (5-[(5-Nitro-2-thiazolyl) thio]-1,3,4thiadiazol-2-amine) was bought from Carbosynth Ltd. (Compton, UK). Mueller–Hinton broth was obtained from Scharlab, S.L (Barcelona, Spain). Deionized water (DW) was generated through Milli Q, Millipore (Billerica, MA, USA), and has been used throughout the study.

Bacterial growth and biofilm formation were quantified in nine different media: Marine Broth (MB) (Conda); Mueller-Hinton Broth (Scharlau) supplemented with NaCl to give a final concentration of 2% (MH2); cation-adjusted MH2 (CAMH2), that consisted in MH2 supplemented with 55 mg/l CaCl₂ and 40 mg/l MgCl₂; Brain Heart Infusion (Scharlau) supplemented with NaCl to a final concentration of 2% (BHI2); Tryptic Soy Broth (BD) supplemented with NaCl to a final concentration of 2% (TSB2); Luria Marine Broth (LMB); Supplemented Artificial Seawater (SASW); Väätänen Nine-Salt Solution (VNSS); and Marine Minimal Medium (MMM). LMB and SASW were prepared according to Lang et al. [35 (link)], NSS and VNSS followed the recipe described by Mårdén et al. [64 (link)]; and MMM was prepared as described by Östling et al. [65 (link)]. A summary of the composition of each medium is provided as additional information (Additional file 1: Table S1).