Backgrounds and aims: Clostridium difficile causes disease ranging from self-limiting diarrhoea to severe pseudomembranous colitis. Antimicrobial treatment failures and patients with multiple recurrences have driven the search for new therapies. Some natural products have broad-spectrum antimicrobial activity with several showing activity against C. difficile. Understanding the mechanism of action of these products is important to further characterise their efficacy. Thus, this study aimed to investigate the mechanism of action of five natural bactericidal products, cinnamon root powder, peppermint oil, trans-cinnamaldehyde, menthol and zingerone and four bacteriostatic products, fresh garlic bulb extract, garlic clove powder, allicin and Leptospermum honey against two C. difficile strains.
Methods and results: As determined by measuring the optical density at 620 nm, none of the products caused bacteriolysis. The time-kill assay showed a > 3 log10 reduction in C. difficile viable counts by all five bactericidal products after 24 h of exposure. An ATP-leakage assay showed that all five products at most concentrations significantly reduced the intracellular ATP after 1 h of incubation (P ≤ 0.01). Alterations in cell permeability were assessed by measuring the leakage of 260-nm absorbing materials, protein leakage using Bradford assay and the propidium iodide uptake assay. All five bactericidal products damaged the cell membrane as seen in two or more cell permeability assays. The effect of three bacteriostatic products on protein synthesis was determined using an Escherichia coli S30 extract system, and only Leptospermum honey (16% w/v) showed inhibition of prokaryotic protein synthesis (P < 0.01 ). None of the products showed elevated minimum inhibitory concentrations against strains of C. difficile harbouring DNA gyrase mutations, or conjugative transposons carrying ermB and tetM.
Conclusions: The findings indicate that damage to the cytoplasmic membrane may contribute to the mechanism of action of several natural products against C. difficile. Also, the absence of cross-over mechanisms of resistance between standard antibiotics and natural products are suggested. Further studies are required to determine the efficacy of these products in vivo.