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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

Aims: To investigate the antimicrobial activity of various natural products against Clostridium difficile in vitro. Methods and Results: The antibacterial activity of 20 natural products was determined by the agar well diffusion and broth microdilution assays against four C. difficile strains, three comparator organisms and four gastrointestinal commensal organisms. Of the raw natural products, garlic juice had the highest activity. The most active processed products were peppermint oil and the four pure compounds trans-cinnamaldehyde, allicin, menthol and zingerone. Furthermore, Bacteroides species had similar susceptibility to C. difficile to most natural products; however, Lactobacillus casei was less susceptible. The combined effect of natural products with vancomycin or metronidazole was determined using the conventional checkerboard titration method and the fractional inhibitory concentration index was calculated. The results showed a possible synergism between trans-cinnamaldehyde and vancomycin and partial synergy between trans-cinnamaldehyde and metronidazole. Conclusions: The study indicates a range of antimicrobial activity of natural products against C. difficile and suggests that they may be useful as alternative or complementary treatments for C. difficile infection (CDI), particularly as most are able to be given orally. Significance and Impact of the Study: This study encourages further investigation of natural products for treatment of CDI.

Clostridium difficile infection is a toxin-mediated disease of the colon. C. difficile virulence is primarily attributed to the production of toxin A and toxin B; thus this study was aimed to investigate the effect of a range of natural products on the production and activity of C. difficile toxins in vitro. Twenty-two natural products were investigated against four C. difficile strains. The activity of products against toxins was determined using Vero and HT-29 cells cytotoxicity and neutral red uptake assays. The indirect effect of products on toxin-mediated cytotoxicity was determined using the same cell lines. The effect of seven products on toxin production by C. difficile was determined using ELISA. Zingerone (0.3 mg/ml) protected both cell lines from C. difficile cytopathic effects, confirmed by the neutral red uptake assay (P Leptospermum honeys (4% w/v), fresh onion bulb extract (12.5% v/v) and trans-cinnamaldehyde (0.005% v/v) all reduced toxin production and activity significantly (P ≤ 0.023). Garlic clove powder (4.7 mg/ml) only reduced toxin activity (P ≤ 0.047). Overall, several natural products had activity against C. difficile toxins in vitro encouraging further investigation against C. difficile toxins in vivo.

Clostridium difficile is an anaerobic, Gram-positive, spore-forming bacillus that causes disease ranging from self-limiting diarrhoea to severe pseudomembranous colitis. C. difficile infection (CDI) commonly affects hospitalised patients and is increasingly identified in patients in the community with no hospital contact. For the last 15 years the incidence of CDI worldwide has been rising, especially in the northern hemisphere. The yearly average number of hospitalizations as a result of this disease is estimated to be over a quarter of a million per year in the United States alone. The main risk factor for CDI is exposure to antimicrobials that affect the gut microflora and, paradoxically, the most common treatments for CDI are the antimicrobials, metronidazole and vancomycin. However, the increasing frequency of highly virulent C. difficile strains, antimicrobial treatment failures, hospital outbreaks, patients with severe complications and cases with multiple recurrences have driven the search for new therapies. Several novel or popular complementary and alternative therapies are self-prescribed for treatment of other diarrheal diseases, and these may also be appropriate for treating CDI. In general, complementary and alternative medicine (CAM) is mainly used by patients when conventional therapeutic agents show limited success against C. difficile and other antimicrobial-resistant bacteria. Among these alternative approaches, a number of treatments, such as herbal remedies, are embraced less by pharmaceutical and medical professions. This review summarises current knowledge of non-conventional antimicrobial and alternative therapies for treatment of CDI. As the demand for non-conventional antimicrobial therapies increases, further studies are required in the field of CAM, especially natural products, for the treatment of CDI.