The bacterial and fungal strains (S2 Table in S1 File ) used in this study were clinical isolates obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA), the Centers for Disease Control and Prevention (CDC) and Biodefense and Emerging Infections Research Resources Repository (BEI Resources) (Manassas, VA, USA). RPMI 1640 (Thermo Fisher Scientific, Waltham, MA), YPD broth, YPD agar, cation adjusted Mueller-Hinton broth, brain heart infusion broth, and lactobacilli MRS broth (Becton, Dickinson and Company, Franklin Lakes, NJ) were purchased from commercial vendors. Phosphate buffered saline was purchased from Fisher Scientific (Waltham, MA). Yeast extract, L-cysteine, vitamin K, 3-(N-Morpholino)propanesulfonic acid (MOPS) and hemin were obtained from Sigma-Aldrich (St. Louis, MO). Human colorectal adenocarcinoma epithelial cells (Caco-2) (ATCC HTB-37), and monkey kidney epithelial cells (Vero) (ATCC CCL-81-VHG) were obtained from the American Type Culture Collection (ATCC) (Manassas, VA, USA).
Ypd agar
Manufactured by BD
Sourced in United States, Macao
YPD agar is a general-purpose growth medium used for the cultivation of yeast and other microorganisms. It contains yeast extract, peptone, and dextrose as the primary nutrients. This agar-based medium provides a suitable environment for the growth and maintenance of a variety of yeast species.
Automatically generated - may contain errors
Lab products found in correlation
3 n morpholino propanesulfonic acid mops, by Merck Group (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Caco 2, by American Type Culture Collection (1 mentions)
Yeast extract, by Merck Group (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Brain heart infusion broth, by BD (1 mentions)
Hemin, by Merck Group (1 mentions)
L cysteine, by Merck Group (1 mentions)
Ypd broth, by BD (1 mentions)
Lactobacilli mrs broth, by BD (1 mentions)
Cation adjusted mueller hinton broth, by BD (1 mentions)
Atcc htb 37, by American Type Culture Collection (1 mentions)
Vitamin k, by Merck Group (1 mentions)
Calcusyn program, by Biosoft (1 mentions)
Butyric acid, by Merck Group (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Gentamicin, by Thermo Fisher Scientific (1 mentions)
Vancomycin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Atlanta Biologicals (1 mentions)
Cefoperazone, by Merck Group (1 mentions)
5 protocols using ypd agar
1
Bacterial and Fungal Strain Characterization
2
Fungal Strain Cultivation and Characterization
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Fungal strains used in this study are listed in Supplementary Table 4 . C. albicans clinical isolates TWO7241 and TWO7243 were obtained from professor Theodor White (UMKC). SC5314 mutant derivatives SC-MRR1P683S and SC-TAC1G980E, containing gain-of-function alleles (MRR1P683S and TAC1G980E), were obtained from professor David Rogers (University of Tennessee Health Science Center). RPMI 1640 powder with glutamine, but without NaHCO3, was purchased from Thermo Fisher Scientific (Waltham, MA). 3-(N-Morpholino) propanesulfonic acid (MOPS) was obtained from Sigma Aldrich (St. Louis, MO). YPD broth medium and YPD agar were obtained from Becton, Dickinson Company (Franklin Lakes, NJ).
3
Antifungal Compound Screening Protocol
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Compounds were dissolved in DMSO and 3-fold dilutions in DMSO were prepared by serial dilution.
Aliquots (1 μL) of each dilution were transferred to wells of flat-bottomed 96-well microtiter plates, with three replicate wells for each treatment, and DMSO alone in control wells. Wells then received 100 μL of YPEth broth (10 g of yeast extract, 20 g of Bacto peptone and 2% (v/v) of ethanol per liter). Cells taken from overnight cultures grown on YPD agar (Becton, Dickinson and Co., Sparks, MD, USA) at 30 o C were suspended in YPEth broth at a cell density of 2 × 10 5 cells mL -1 . Wells were inoculated with 100 μL of cell suspension and the plates were incubated at 30 o C for 45 h. After mixing well contents to provide homogeneous cell suspension, growth was measured using a NEPHELOstar Galaxy plate reader (BMG Labtech, Cary, NC, USA) and 50% effective concentration (EC50) values for growth inhibition were determined using the CALCUSYN program (Biosoft, Cambridge, UK). Mutants were tested in two or more independent experiments and average EC50 values ± SEM determined across all replicates.
Aliquots (1 μL) of each dilution were transferred to wells of flat-bottomed 96-well microtiter plates, with three replicate wells for each treatment, and DMSO alone in control wells. Wells then received 100 μL of YPEth broth (10 g of yeast extract, 20 g of Bacto peptone and 2% (v/v) of ethanol per liter). Cells taken from overnight cultures grown on YPD agar (Becton, Dickinson and Co., Sparks, MD, USA) at 30 o C were suspended in YPEth broth at a cell density of 2 × 10 5 cells mL -1 . Wells were inoculated with 100 μL of cell suspension and the plates were incubated at 30 o C for 45 h. After mixing well contents to provide homogeneous cell suspension, growth was measured using a NEPHELOstar Galaxy plate reader (BMG Labtech, Cary, NC, USA) and 50% effective concentration (EC50) values for growth inhibition were determined using the CALCUSYN program (Biosoft, Cambridge, UK). Mutants were tested in two or more independent experiments and average EC50 values ± SEM determined across all replicates.
4
Candida albicans Infection and Treatment Protocol
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Candida albicans ATCC 10231 was purchased from ATCC. Candida albicans SC5314 was kindly provided by Dr. Andrew Koh from University of Texas Southwestern Medical Center2 (link). Media used in this study included RPMI 1640 (Gibco, MA), MOPS (Sigma, MO), YPD Agar (BD Biosciences, CA), and Fetal Bovine Serum (Atlanta Biologicals, GA). Short-chain fatty acids (acetic acid, butyric acid, and propionic acid) were purchased from Sigma Aldrich (MO). Mice were purchased from The Jackson Laboratory (ME). Cefoperazone was purchased from Sigma Aldrich (MO). Other materials were purchased as indicated: mouse oral gavages (Kent Scientific, MA), vancomycin (Alfa Aesar, MA), gentamicin (Acros Organics, NJ), paraformaldehyde (Alfa Aesar, MA), and glycerol (DOT Scientific, MI).
5
Fungal Strain Cultivation and Synthetic Media
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