Antibacterial and antivirulence activities of auranofin against Clostridium difficile

Abstract

Clostridium difficile is a deadly, opportunistic bacterial pathogen. In the last two decades, C. difficile infections (CDIs) have become a national concern because of the emergence of hypervirulent mutants with increased capability to produce toxins and spores. This has resulted in an increased number of infections and deaths associated with CDI. The scarcity of anticlostridial drugs has led to unsatisfactory cure rates, elevated recurrence rates and permitted enhanced colonization with other drug-resistant pathogens (such as vancomycin-resistant enterococci) in afflicted patients. Therefore, both patients and physicians are facing an urgent need for more effective therapies to treat CDI. In an effort to find new anticlostridial drugs, we investigated auranofin, an FDA-approved oral antirheumatic drug that has recently been found to possess antibacterial activity. Auranofin exhibited potent activity against C. difficile isolates, inhibiting growth at a concentration of 1 µg/mL against 50% of all tested isolates. Auranofin inhibited both toxin production and spore formation, a property lacking in both vancomycin and metronidazole (the primary agents used to treat CDI). Auranofin had a direct protective activity against C. difficile toxin-mediated inflammation and inhibited the growth of vancomycin-resistant enterococci. Auranofin is a promising candidate that warrants further investigation as a treatment option for C. difficile infections.

Keywords: Anti-toxin; Antibacterial; Auranofin; C. difficile infection; Repurposing; Spores formation.

Fig. 1.. Toxin inhibition activity of auranofin and control anticlostridial drugs (vancomycin, metronidazole and fidaxomicin) against C. difficile.

Drugs, at concentrations of 1/8 ×, 1/4 × and1/2 × MIC were incubated with a hypervirulent, toxigenic strain of C. difficile (strain ATCC BAA-1870). The bacterial counts were determined for each sample, and the toxin levels were assessed in the supernatant using enzyme linked immune fluorescent assay (ELISA). Error bars represent standard deviation values from triplicate samples for each treatment.

Fig. 2.. Spore inhibition of activity of auranofin against C. difficile compared to the control anticlostridial drugs, vancomycin and metronidazole.

Drugs (1/2× and 1 × MIC) were incubated with bacteria for five days followed by serial dilution and plating to count both total bacterial count and heat resistant spores. Error bars represent standard deviation values from triplicate samples for each treatment. (*) denotes significant difference between the total and the spore counts.

Fig. 3.. Auranofin’s effect against C. difficile toxin-mediated inflammation of gut epithelial cells.

Human colorectal (Caco-2) epithelial cells were incubated with filtered C. difficile culture supernatant plus B. DMSO, 2.5%, C. vancomycin, 128 μg/mL, D. metronidazole, 128 μg/mL, fidaxomicin, 128 μg/mL, F. auranofin, 1 μg/mL or G. auranofin, 8 μg/mL for 24 hours and observed under the microscope. Reference wells were not treated with C. difficile supernatant but still treated with 2.5% DMSO (panel A). Cell rounding is an indication of inflammation.

Fig. 4.. Auranofin mediated IL-8 inhibition from gut cells treated with C. difficile toxins.

IL-8 level was assessed in Caco-2 cells treated with supernatant containing C. difficile toxins, with or without the addition of auranofin. OD₄₅₀ coincides with the level of IL-8 in the cell supernatant. Error bars represent standard deviation values from triplicate samples used for each test agent. An asterisk (*) indicates significant difference (P < 0.05) between cells exposed to supernatant containing toxin alone and cells treated with supernatant containing auranofin (1 µg/mL) using one-way (ANOVA) followed by Dunnett’s multiple comparisons test.