Fidaxomicin

The MEGx library (AnalytiCon Discovery, Postdam, Germany) consisting of 1,000 natural products was screened as described before [20 (link)]. Briefly, the library was screened (screening concentration-3 μM) by dispensing 180 nL of a 1 mM stock solution into 384-well plates using an Echo acoustic dispenser. Vancomycin (Gold Biotechnology, Olivette, MO), metronidazole (Alfa Aesar), and fidaxomicin (Cayman Chemicals, Ann Arbor, MI) (screening concentration- 10 μM) were used as positive controls along with DMSO at the same concentration which served as the negative control. Data were recorded as mentioned previously [20 (link)]. The Z’ value for the assay was calculated as per the equation Z’ = 1-[(3σ_p+ 3σ_n)/(μ_p- μ_n)], where σ is the standard deviation, μ is the mean, p is the antibiotic-treated control, and n indicates the DMSO negative control. The plates with Z’<0.5 were repeated [21 (link)]. The inhibition of C. difficile ATCC BAA 1870 growth (%) was plotted using GraphPad Prism software version 8.0.

The bacterial isolates were obtained from the American Type Culture Collection (ATCC), the CDC, and the Biodefense and Emerging Infections Research Resources Repository (BEI Resources, Manassas, VA). C. difficile strains were grown in Mueller-Hilton broth II (Beckton, Dickinson, and Company, MD) or supplemented brain heart infusion agar plates (BHIS, Beckton, Dickinson, and Company, MD) at 37°C in an anaerobic chamber (Coy Laboratory Products Inc., Grass Lake, MI). Vancomycin hydrochloride (Sigma-Aldrich, St. Louis, MO) and fidaxomicin (Cayman Chemicals, Ann Arbor, MI) were purchased from commercial vendors. TRIzol Max Bacterial RNA Isolation Kit, Turbo DNA-free Kit, SuperScript III First-Strand Synthesis Kit (Invitrogen, Carlsbad, CA), and iTaq Universal SYBR Green One-Step Kit were purchased from commercial vendors (Bio-Rad Laboratories, Inc., Hercules, CA).

All the chemicals and reagents used in this study were of analytical grade. GPI0363 was obtained from the chemical library of the Drug Discovery Initiative at the University of Tokyo and its purity was confirmed by ultra-performance liquid chromatography-high resolution mass spectrometry (m/z: 343.1968 [M + H]⁺; calculated for C₁₉H₂₄N₄O: 343.1934) (ESI Fig. 7†). Rifampicin (potency: 1029 μg mg⁻¹), ammonium acetate, and HPLC grade acetonitrile were obtained from Fujifilm Wako Pure Chemical Industries, Ltd., Osaka, Japan. Fidaxomicin (purity: ≥95%) was obtained from Cayman Chemical (Ann Arbor, MI, USA). Salmon sperm DNA, actinomycin D (purity: ∼98%), and phenol : chloroform : isoamyl alcohol (125 : 24 : 1, v/v/v) were obtained from Sigma Aldrich, Tokyo, Japan. Tryptone, tryptic soy broth (TSB), yeast extract, and Mueller-Hinton broth (MHB) were obtained from Becton, Dickinson and Company (Franklin Lakes, NJ, USA). RNase inhibitor was obtained from Applied Biosystems (Beverly, MA, USA). ATP, CTP, GTP, UTP, and yeast tRNA were obtained from Ambion (Austin, TX, USA). [α-³²P] UTP was obtained from PerkinElmer, Waltham, MA, USA. The E. coli RNAP holoenzyme and core enzyme were obtained from New England Biolabs (Ipswich, MA, USA), and TALON® magnetic beads were obtained from Takara Bio USA Inc. (Mountain View, CA, USA).