Natural products show diverse mechanisms of action against Clostridium difficile

Aims To investigate the mechanisms of action of natural products with bactericidal (cinnamon root powder, peppermint oil, trans-cinnamaldehyde, menthol and zingerone) or bacteriostatic (fresh garlic bulb extract, garlic clove powder, Leptospermum honey and allicin) activity against two Clostridium difficile strains. Methods and Results Bactericidal products significantly reduced intracellular ATP after 1 h (P <= 0.01), quantified using the BacTiter-Glo reagent, and damaged the cell membrane, shown by the leakage of both 260-nm-absorbing materials and protein, and the uptake of propidium iodide. Bacteriolysis was not observed, determined by measuring optical density of treated cell suspensions at 620-nm. The effect of three bacteriostatic products on protein synthesis was quantified using an Escherichia coli S30 extract system, with Leptospermum honey (16% w/v) showing significant inhibition (P < 0.01). Lastly, no products showed elevated minimum inhibitory concentrations against antimicrobial-resistant C. difficile, determined by broth microdilution. Conclusions Cytoplasmic membrane damage was identified as a mechanism of action that may contribute to the activity of several natural products against C. difficile. Significance and Impact of the Study This study describes the possible mechanisms of action of natural products against C. difficile, yet the efficacy in vivo to be determined.