A Small Molecule-Screening Pipeline to Evaluate the Therapeutic Potential of 2-Aminoimidazole Molecules Against Clostridium difficile

Abstract

Antibiotics are considered to be the first line of treatment for mild to moderately severe <i>Clostridium difficile</i> infection (CDI) in humans. However, antibiotics are also risk factors for CDI as they decrease colonization resistance against <i>C. difficile</i> by altering the gut microbiota and metabolome. Finding compounds that selectively inhibit different stages of the <i>C. difficile</i> life cycle, while sparing the indigenous gut microbiota is important for the development of alternatives to standard antibiotic treatment. 2-aminoimidazole (2-AI) molecules are known to disrupt bacterial protection mechanisms in antibiotic resistant bacteria such as <i>Pseudomonas aeruginosa, Acinetobacter baumannii</i>, and <i>Staphylococcus aureus</i>, but are yet to be evaluated against <i>C. difficile</i>. A comprehensive small molecule-screening pipeline was developed to investigate how novel small molecules affect different stages of the <i>C. difficile</i> life cycle (growth, toxin, and sporulation) <i>in vitro</i>, and a library of commensal bacteria that are associated with colonization resistance against <i>C. difficile</i>. The initial screening tested the efficacy of eleven 2-AI molecules (compound 1 through 11) against <i>C. difficile</i> R20291 compared to a vancomycin (2 μg/ml) control. Molecules were selected for their ability to inhibit <i>C. difficile</i> growth, toxin activity, and sporulation. Further testing included growth inhibition of other <i>C. difficile</i> strains (CD196, M68, CF5, 630, BI9, M120) belonging to distinct PCR ribotypes, and a commensal panel (<i>Bacteroides fragilis, B. thetaiotaomicron, C. scindens, C. hylemonae, Lactobacillus acidophilus, L. gasseri, Escherichia coli, B. longum</i> subsp. <i>infantis</i>). Three molecules compound 1 and 2, and 3 were microbicidal, whereas compounds 4, 7, 9, and 11 inhibited toxin activity without affecting the growth of <i>C. difficile</i> strains and the commensal microbiota. The antimicrobial and anti-toxin effects of 2-AI molecules need to be further characterized for mode of action and validated in a mouse model of CDI.