Morpholinepropanesulfonic acid

The antifungal activity of the natural extract was evaluated according to the standard procedures of the Clinical and Laboratory Standards Institute (CLSI) M27-A3 [107 ]. The strains were grown on Sabouraud dextrose agar plates (SDA) (Oxoid, Milan, Italy) at 35 °C for 48 h. The cell suspensions were prepared in 5 mL of 0.145 M sterile saline solution and adjusted to 0.5 McFarland scale (1.5 × 10⁸ Colony Forming Units (CFUs)/mL) by a spectrophotometer (Bio-Tek Synergy HT Microplate Reader, Bio-Tek Instruments, Winooski, USA) at λ = 530 nm. For the antifungal susceptibility test, the culture medium bicarbonate-free Roswell Park Memorial Institute (RPMI) 1640 with l-glutamine, buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (Sigma-Aldrich, Milan, Italy), was used. OCLE was diluted in the 1:100 ratio in RPMI 1640 medium. Ten concentrations ranging from 0.57 to 293.55 µg/mL were obtained in sterile 96 U-well microplates (Corning, New York, NY, USA). The antifungal agent fluconazole, in concentrations ranging from 0.125 to 64.00 µg/mL, was used as the positive control. The final concentration of the inoculum was from 5 × 10² to 2.5 × 10³ cells/mL per well.
To determine MFC₅₀, 100 µL of sample were removed from the wells of the MIC₅₀ and subcultured in SDA plates. After incubation at 35 °C for 48 h, the CFUs were counted. Four independent experiments were performed.

Strains were preserved in storage media (10% glycerol, 1% NaCl, and 1% Tween 20) at −80 °C. They were revitalized and routinely grown on yeast peptone dextrose (YPD) agar plates (2% Bacto Peptone, 1% yeast extract, 2% dextrose, and 2% Bacto agar) at 35 °C, regularly sub-cultured before each experiment. Throughout the assays we also used YPD broth (2% Bacto Peptone, 1% yeast extract, 2% dextrose), Sabouraud dextrose agar (3% Sabouraud dextrose from Sigma-Aldrich, 2% Bacto agar), and RPMI (1.04% RPMI-1640 from Sigma-Aldrich, 3.453% morpholinepropanesulfonic acid from Sigma-Aldrich, pH adjusted to pH 7 with 10 M NaOH solution) (Adams et al., 1998 ).

Clove and cinnamon EOs (Sigma Aldrich, St. Louis, MO, USA) were diluted in 1% dimethyl sulfoxide (DMSO, Sigma) (Shahzad et al., 2014 (link)). The CFS of E. faecalis used in this study was purified using ammonium sulfate precipitation and dialysis from two strains in our previous study (Hassan et al., 2018 (link)). These strains were isolated from cheddar cheese (CFS1) and chicken intestine (CFS2) samples. The surfactants CTAB and SDS were purchased from Sigma Aldrich and stock solutions prepared in sterile distilled water and then sterilized through 0.22-μm filters (Dusane et al., 2012 (link)). FLC (Pfizer, Inc., New York, NY) was dissolved in RPMI 1640 adjusted to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (Sigma-Aldrich, St. Louis, Mo., USA) and used as a standard antifungal agent.

Wild type strains of C. albicans SC531418 (link), C.
glabrata CG46218 (link), C. tropicalis MYA340450 (link) and C. lusitaniae CL618 (link), were used in this
study. Frozen glycerol stock of the strain was regularly revived on YPD agar
medium (1% Bacto yeast extract, 2% Bacto peptone, 2% glucose and 2% Bacto agar).
For broth culture, strain was grown in YPD medium at 30 °C with
agitation (200 rpm). RPMI-1640 medium with L-glutamine without sodium
bicarbonate (Sigma) was buffered with 0.165 M morpholinepropanesulfonic
acid (Sigma) to a pH of 7. Stock solutions of extracted SL (supplementary material), AmB (Sigma) and FLZ
(Sigma) were prepared in dimethyl sulfoxide (DMSO, Sigma), and stored at
−20 °C until use.

Stock solutions of each agent were prepared using dimethyl sulfoxide (DMSO). Stock solutions of 1000 μg.mL^-1 were separated into aliquots and stored at -70ºC until they were used. RPMI 1640 liquid medium (Sigma) buffered to a pH 7.0 with 0.165 M morpholinepropanesulfonic acid (Sigma) was used to obtain final tested agent concentrations. To demonstrate that DMSO did not affect the growth of the studied strains, fungal colonies were grown in the presence of final (1% vol/vol) DMSO concentration and compared to growth in DMSO-naïve conditions.

The anti-biofilm activity of dendron 1 and dendron 2 was carried out as previously described [7 (link)]. An inoculum of C. glabrata was adjusted to 0.5 McFarland standard in RPMI 1640 medium (Sigma-Aldrich) with morpholine-propane-sulfonic acid (Sigma-Aldrich) and 2% glucose (RPMI + MOPS + GLU). Then, 50 μL of the suspension were inoculated in 96-well microtiter plates containing two-fold serial dilutions of the dendrons. Plates were incubated for 48 h at 37 °C. Candida controls free of compound, control of compound without inoculum, and control of medium free of compound and inoculum were included. The minimum biofilm inhibitory concentration (MBIC) was determined with resazurin colorimetric assay and has been previously defined as the lowest concentration in which no reduction in resazurin was observed (no growth, nor absorbance signal detected) in biofilm in formation [7 (link),8 (link),14 (link),15 (link)]. The minimum fungicidal concentration in biofilm (MFCB) was determined using the drop plate method and was defined as the lowest concentration capable of inducing the total death of the yeast population, avoiding the generation of the biofilm (0% cell viability) [7 (link),8 (link),14 (link),15 (link)]. These MFCB values were obtained by scraping the biofilm and plating 5 μL suspension of each well (drop plate method) [7 (link),8 (link)].

All reactions were carried out under an argon atmosphere. Chloroform was distilled under nitrogen from calcium hydride. 1,10-Phenanthroline was sublimated before use. Ampicillin and fluconazole were brought from Meilun Biotech Co. (Dalian, China), morpholinepropanesulfonic acid, and Salmon Sperm DNA were obtained from Sigma-Aldrich (St. Louis, MO, USA). Their stock solutions were freshly prepared, filter-sterilized, and used at indicated concentrations. SiI₄ was purchased from Alfa, and the other reagents were purchased from Sigma, Acros, or Strem and used without further purification. Column chromatography was performed with silica gel (230–400 mesh). ¹H- and ¹³C-NMR spectra were recorded on a Bruker Advance (400 MHz, Bruker Corporatio, Billerica, MA, USA) at ambient temperature.
In total, the following bacterial and fungal strains were used for this study: Cryptococcus neoformans H99, Candida albicans ATCC90028, Staphylococcus aureus ATCC 33591(MRSA), ATCC 25923(MSSA), Escherichia coli ATCC25922, Pseudomonas aeruginosa PAO1, and clinical isolated Acetobacter baumannii. S. aureus, E. coli, and P. aeruginosa were grown in TSB medium; C. neoformans and C. albicans were grown in YPD medium. These strains were stored as glycerin stock in −80 °C.