In this study, four different strains of H. pylori were used, including the reference 26695 and three clinical multiple drug resistant (MDR) strains, which had been previously isolated and banked [5,6,7]. Antimicrobial susceptibility was interpreted according to the EUCAST breakpoints, with resistance defined as MIC > 0.5 µg mL for clarithromycin, MIC > 0.125 µg mL for amoxicillin, and MIC > 1.0 µg mL for levofloxacin [16]. Based on these criteria, the MDR strains were characterized as follows: (1) Cla/LVX (MDR-1), (2) AMX/LVX (MDR-2), and (3) Cla/AMX/LVX (MDR-3). Bacterial strains were grown on Brucella agar medium (Merck, Germany) enriched with 10% defibrinated sheep blood, trimethoprim (5 mg L), vancomycin (10 mg L), and amphotericin B (8 mg L). The cultures were incubated at 37 °C for 3 to 5 days under microaerobic conditions (10% CO, 5% O, and 85% N) [17]. The effect of olorofim (a gift from F2G Ltd., UK) on H. pylori growth was assessed using the agar dilution method [18]. Serial twofold dilutions of olorofim were prepared in aforementioned agar media (50-55 °C) to yield final concentrations ranging from 0 to 10 µg mL. PEG-400 is a solvent for olorofim; therefore, the equivalent volumes of PEG-400 used to prepare different olorofim concentrations were tested in parallel as negative controls. Pre-cultured H. pylori strains were harvested from plates and suspended in sterile Brain Heart Infusion (BHI) to achieve an OD of 0.7 (equivalent to 4 McFarland and 10 CFU mL). Ten microliters of the diluted bacterial suspension were then plated onto the drug-containing and control plates. The plates were then incubated under previously described conditions at 37 °C. After 4 days of incubation, bacterial growth was visually inspected. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of olorofim at which no visible bacterial growth was observed, where the corresponding volume of PEG-400 alone did not inhibit growth. Each test was performed in triplicate.

To further evaluate the bactericidal activity of olorofim against H. pylori, the reference strain was cultured and resuspended in Brucella broth, containing 0.2% beta-cyclodextrin, until having reached an OD of 0.3. Subsequently, different concentrations of olorofim 0.6 and 1.2 µg mL were added. Clarithromycin (0.25 µg mL) was used as a positive control for bactericidal activity [19]. The liquid culture flasks were then incubated under microaerophilic conditions in a shaker incubator at 37 °C, 120 rpm, for 3 days. OD was determined at 24, 48, and 72 h, post inoculation. At each time point, 10 µL of each sample was plated to determine viability and rule out potential contaminations. The data are presented as mean ± standard deviation of duplicates.

The reversal assays were performed by supplementing the culture media with exogenous uridine and uracil in both solid and liquid cultures. Brucella agar plates containing olorofim (0.3 µg mL) were supplemented with uridine (5 mM, Sigma, USA) and uracil (10 mM, Sigma, USA) [14]. Control plates included bacteria without olorofim and those with olorofim but without uridine/uracil supplementation. Bacterial (H. pylori strain 26695) inoculation and incubation were performed as described in the MIC determination section. Growth recovery in the presence of uridine and uracil was compared with olorofim-only plates and visually assessed after 4 days. For the liquid broth reversal assays, H. pylori strain 26695 was precultured, then resuspended in Brucella broth supplemented with 0.2% β-cyclodextrin until reaching an OD of 0.3. Subsequently, olorofim (0.3 µg mL), uridine (5 mM), and uracil (10 mM) were added [14]. An untreated culture containing only the starting inoculum (without any supplements or drug) was used as control. The OD of all groups was measured at baseline (time zero) and at subsequent time points up to 72 h. All tests were performed in duplicate.

The general toxicity of olorofim was assessed against Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, and nonpathogenic E. coli strains. The bacteria were grown on Mueller-Hinton agar plates with and without olorofim (1 µg mL). The plates were then incubated at 37 °C overnight.

To reconfirm the cytotoxic effect of olorofim on eukaryotic cells [20] in our setting, a cell viability assay was conducted. J774.1 murine macrophage cells (Cell Bank, Pasteur Institute of Iran) were seeded onto a 96-well plate at a density of 2 × 10 cells per well. Olorofim was added at various concentrations ranging from 1 to 4.0 nM (0.5 to 2 µg mL). Following a 24-h incubation period at 37 °C and 5% CO, 100 µL of FBS-free RPMI medium was added to the wells. Subsequently, MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (Sigma, USA)] was added to each well at a final concentration of 0.83 µg mL. Following a 3-hour incubation at 37 °C and 5% CO, 100 µl of DMSO (dimethyl sulphoxide) was added to each well and placed on a shaking incubator for 30 min, in the dark. The optical density was then measured at 570 nm using a microplate spectrophotometer (Biotek, USA). Drug-free and PEG-400-containing samples served as negative controls. The data are presented as mean ± standard deviation of triplicates.

A substrate reduction assay was optimized to determine the minimum concentrations of the enzyme and coenzyme, in order to assess the inhibitory effect of olorofim. The recombinant H. pylori DHODH used in these studies was previously developed (submitted for publication). The reactions were conducted in an assay buffer (50 mM Tris-base/HCl, 150 mM KCl, 0.1% Triton X-100, pH 8.0) mixed with recombinant DHODH and dispensed into 96-well plates. The reactions were initiated by adding a freshly prepared substrate mixture [0.05 mM CoQ10 (Sigma, USA), 1 mM DHO (Sigma, USA), 0.12 mM DCPIP (Merck, Germany)]. Various concentrations of DHODH (0.5-64 µM) and CoQ10 (3.125-50 µM) were tested. Absorbance changes were measured at 600 nm, using a microplate spectrophotometer (Biotek, USA) at 30-s intervals, for up to 5 min. Each sample was run in duplicate, and the resulting means ± standard deviations of ΔOD (OD of test samples minus OD of the assay buffer were reported. For the inhibition test, olorofim was incubated with the recombinant enzyme at final concentrations of 62.5 µM and 125 µM, respectively. Following a 30-minute incubation period at ambient temperature, reactions were initiated by addition of the substrate mixture, as mentioned above. 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO), a known DHODH inhibitor that has previously been shown to inhibit recombinant H. pylori DHODH with an IC of 1.75 µM (submitted for publication), was used as positive control.

The amino acid sequences were obtained from the UniProt database (https://www.uniprot.org). The UniProt ID codes for the sequences used were as follows: B0XSD3 (Aspergillus fumigatus, strain CEA10), O25655 (H. pylori-26695) and B5Z6I2 (Helicobacter pylori-G27). The amino acid sequences of their DHODH enzymes were aligned using CLUSTAL Omega (1.2.4) pairwise sequence alignment.