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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.

This study investigated the effects of the volatile terpene-rich oil from Melaleuca alternifolia (tea tree oil) on the formation of biofilms and the adhesion of C. albicans cells to both biotic and abiotic surfaces. Biofilm formation on polystyrene was significantly inhibited for 70% of the isolates at the lowest test concentration of 0.016% of tea tree oil (TTO) when quantified by XTT and 40% of isolates when measured by crystal violet staining. Adhesion to polystyrene, quantified by crystal violet staining, was significantly reduced for 3 isolates at 0.031%, 6 isolates at 0.062% and 0.125% and for all 7 isolates at 0.25% TTO. Reductions in adhesion were not due to loss of viability (at concentrations of ≤ 0.125%) or interactions between the TTO and polystyrene. Similarly, adhesion to buccal epithelial and HeLa cells was also significantly reduced in the presence of 0.016-0.062% TTO. Treatment with 0.125% TTO, but not 0.062%, decreased the cell surface hydrophobicity of C. albicans, indicating one potential mechanism by which adhesion may be reduced. These data demonstrate that sub-inhibitory TTO reduces the adhesion of C. albicans to both human cells and polystyrene, inhibits biofilm formation and decreases cell surface hydrophobicity.

The in vitro activity of Melaleuca alternifolia (tea tree) oil against dermatophytes (n = 106) and filamentous fungi (n = 78) was determined. Tea tree oil MICs for all fungi ranged from 0.004% to 0.25% and minimum fungicidal concentrations (MFCs) ranged from

The effect of tea tree oil (TTO) on the formation of germ tubes by Candida albicans was examined. Two isolates were tested for germ tube formation (GTF) in the presence of TTO concentrations (% v/v) ranging from 0.25% (1/2 minimum inhibitory concentration [MIC]) to 0.004% (1/128 MIC). GTF at 4 h in the presence of 0.004 and 0.008% (both isolates) and 0.016% (one isolate) TTO did not differ significantly (P > 0.05) from controls. At all other concentrations at 4 h, GTF differed significantly from controls (P < 0.01). A further eight isolates were tested for GTF in the presence of 0.031% TTO, and at 4h the mean GTF for all 10 isolates ranged 10.0-68.5%. Two isolates were examined for their ability to form germ tubes after 1 h of pre-exposure to several concentrations of TTO, prior to induction of germ tubes in horse serum. Cells pre-exposed to 0.125 and 0.25% TTO formed significantly fewer germ tubes than control cells at 1 h (P < 0.05), but only those cells pre-exposed to 0.25% differed significantly from control cells at later time points (P < 0.01). GTF by C. albicans is affected by the presence of, or pre-exposure to, sub-inhibitory concentrations of TTO. This may have therapeutic implications.

The antimicrobial activity of plant oils and extracts has been recognized for many years. However, few investigations have compared large numbers of oils and extracts using methods that are directly comparable. In the present study, 52 plant oils and extracts were investigated for activity against Acinetobacter baumanii, Aeromonas veronii biogroup sobria, Candida albicans, Enterococcus faecalis, Escherichia col, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella enterica subsp. enterica serotype typhimurium, Serratia marcescens and Staphylococcus aureus, using an agar dilution method. Lemongrass, oregano and bay inhibited all organisms at concentrations ofor = 2.0% (v/v). Six oils did not inhibit any organisms at the highest concentration, which was 2.0% (v/v) oil for apricot kernel, evening primrose, macadamia, pumpkin, sage and sweet almond. Variable activity was recorded for the remaining oils. Twenty of the plant oils and extracts were investigated, using a broth microdilution method, for activity against C. albicans, Staph. aureus and E. coli. The lowest minimum inhibitory concentrations were 0.03% (v/v) thyme oil against C. albicans and E. coli and 0.008% (v/v) vetiver oil against Staph. aureus. These results support the notion that plant essential oils and extracts may have a role as pharmaceuticals and preservatives.

The effect of some potentially interfering substances and conditions on the antimicrobial activity of Melaleuca alternifolia (tea tree) oil was investigated. Agar and broth dilution methods were used to determine minimum inhibitory and cidal concentrations of tea tree oil in the presence and absence of each potentially interfering substance. Activity was determined against Gram-positive and -negative bacteria, and Candida albicans. Minimum inhibitory or cidal concentrations differed from controls by two or more dilutions, for one or more organisms, where Tween-20, Tween-80, skim-milk powder and bovine serum albumin were assessed. These differences were not seen when assays were performed in anaerobic conditions, or in the presence of calcium and magnesium ions. The effect of organic matter on the antimicrobial activity of tea tree oil was also investigated by an organic soil neutralization test. Organisms were exposed to lethal concentrations of tea tree oil ranging from 1-10% (v/v), in the presence of 1-30% (w/v) dry bakers' yeast. After 10 min contact time, viability was determined. Ator = 1%, organic matter compromised the activity of each concentration of tea tree oil against Staphylococcus aureus and C. albicans. At 10% or more, organic matter compromised the activity of each tea tree oil concentration against Pseudomonas aeruginosa. Organic matter affected 1 and 2% tea tree oil, but not 4 and 8%, against Escherichia coli. In conclusion, organic matter and surfactants compromise the antimicrobial activity of tea tree oil, although these effects vary between organisms.

Thirty isolates of Pseudomonas aeruginosa, 15 isolates of Pseudomonas putida and 11 isolates of Pseudomonas fluorescens were tested for susceptibility to tea tree oil (TTO), the essential oil of Melaleuca alternifolia, and the components terpinen-4-ol, alpha-terpineol, cineole, gamma-terpinene and rho-cymene.MICs were determined by broth microdilution in Mueller-Hinton medium supplemented with 0.002% (v/v) Tween 80.The MIC90 of TTO for all isolates tested was 4% (v/v) or less. Susceptibility to components tested varied between species.Pseudomonas spp. are susceptible to TTO and some of its components although they are less susceptible than many other bacteria tested previously.

The susceptibility of 64 Malassezia furfur isolates to Melaleuca alternifolia oil was determined. The minimum inhibitory concentration for 90% of isolates was 0·25% by agar dilution and 0·12% by broth dilution. These data indicate that tea tree oil may be useful in the treatment of skin conditions involving M. furfur.