Log reduction in viable spores Effect of natural products on the sporulation cycle of Clostridium difficile in vitro Niloufar Roshan a, Thomas V. Riley a-d, Katherine A. Hammer a a School of Biomedical Sciences, The University of Western Australia, Western Australia. b PathWest Laboratory Medicine, Queen Elizabeth II Medical Centre, Western Australia. c School of Veterinary & Life Sciences, Murdoch University, Western Australia. d School of Medical & Health Sciences, Edith Cowan University, Western Australia. INTRODUCTION RESULTS Clostridium difficile is an anaerobic, Gram-positive, spore-forming bacillus (Figure 1).1 Main virulence factors are toxin A (TcdA), toxin B (TcdB) and binary toxin (CDT).1 During C. difficile infection (CDI), it produces new endospores that both persist in the patient and are shed, thus increasing the chance of disease transmission and recurrence.2 Sporulation assay Of the 20 products, only onion juice (50% v/v) and coconut oil (32% v/v) inhibited spore formation in all four isolates by 50-90% compared to the untreated control. Ginger juice (50% v/v) was also inhibitory, although to a lesser extent. C. difficile ATCC 700057 Figure 1. Gram stain of C. difficile Clinical manifestations of CDI range from mild/self-limiting diarrhoea to fulminant colitis.1 Conventional treatments are metronidazole and vancomycin.2 C. difficile spores show resistance to antibiotics, the host immune system and bleach-free disinfectants.3 Figure 4. The percentage of spores after 48 h and 96 h growth in liquid tryptose yeast broth containing 0.5 × MIC of natural products or antibiotics determined by conventional spore recovery and microscopy. Onion juice (12.5% v/v); Ginger juice (50% v/v); Coconut oil (8% v/v) for C. difficile ATCC 700057, ATCC 43598 and UK 014; Coconut oil (16% v/v) for C. difficile ATCC 1366; Fidaxomicin (0.03 µg ml-1) for C. difficile ATCC 700057, ATCC 43598 and UK 014; Fidaxomicin (0.125 µg ml-1) for C. difficile ATCC 13366; dimethyl sulfoxide (DMSO) (2.5% v/v); * =P < 0.001; The remaining two isolates showed the same trend. C. difficile NCTC 13366 OBJECTIVE The purpose of this study was to examine the effect of several natural products on sporulation, spore germination and outgrowth of C. difficile, in addition to sporicidal activity. Spore germination and outgrowth assay C. difficile ATCC 700057 C. difficile NCTC 13366 METHODS Twenty natural products were selected for testing based on historical evidence, current popularity and feasibility against three reference isolates and one clinical isolate. Assays: 1. In vitro susceptibility: Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) by broth microdilution assay (Figure 2).4 Sporulation: (A) microscopy and (B) conventional spore recovery assay (Figure 3).2,5 Spore germination and outgrowth: (A) spectrophotometry and (B) phase contrast microscopy.6 Figure 5. Effect of supra-inhibitory concentrations of natural products on germination and outgrowth of C. difficile spores. DMSO (5% v/v), fidaxomicin (16 µg ml-1), artichoke (75 mg ml-1), onion juice (50% v/v), WA Leptospermum honey (32% w/v), allicin (75 mg ml-1). The remaining two isolates showed the same trend. No significant effect was observed on spore germination Four of the treatments (artichoke, onion juice, WA Leptospermum honey and allicin) altered the later stage of vegetative cell outgrowth. Figure 2. Broth microdilution Sporicidal activity assay Figure 3. Conventional plate assay Table 1. Sporicidal activity of selected natural products against C. difficile isolates. 4. Sporicidal activity: Plate recovery assay 3 Antimicrobial Agents (concentration)  Incubation time Log reduction in viable spores C. difficile ATCC 13366 C. difficile ribotype 014 ATCC 43598 C. difficile ATCC 700057 Cinnamon powder (75 mg ml-1) 24 h -0.05 ± 0.24 -0.21 ± 0.53 0.05 ± 0.23 -0.27 ± 0.24 48 h -0.02 ± 0.20 -0.19 ± 0.49 -0.17 ± 0.45 -0.39 ± 0.42 1 week 0.05 ± 0.22 -0.31 ± 0.45 -0.22 ± 0.11 -0.27 ± 0.22 Peppermint oil (32% v/v) 1.31 ± 0.82* 1.01 ± 0.56 0.88 ± 0.26* 0.83 ± 0.19 1.34 ± 0.41** 1.03 ± 0.70 1.39 ± 0.17** 1.03 ± 0.51* 1.39 ± 0.64** 1.18 ± 0.31* 1.11 ± 0.36** 1.59 ± 0.31*** Trans-cinnamaldehyde (32% v/v) 1.26 ± 0.41 1.04 ± 0.67 0.54 ± 0.01 1.07 ± 0.52* 1.18 ± 0.66* 1.01 ± 0.55 1.32 ± 0.08** 0.70 ± 0.20* 2.19 ± 0.09*** 1.56 ± 0.72* 1.20 ± 0.47 ** 1.14 ± 0.41*** Menthol 0.14 ± 0.24 -0.04 ± 0.47 0.12 ± 0.08 0.04 ± 0.22 0.28 ± 0.13 -0.17 ± 0.47 0.35 ± 0.18 -0.04 ± 0.35 0.40 ± 0.08 -0.08 ± 0.27 0.37 ± 0.10 0.10 ± 0.09 Zingerone 0.06 ± 0.34 0.07 ± 0.32 0.31 ± 0.18 0.02 ± 0.73 0.18 ± 0.12 -0.14 ± 0.41 0.37 ± 0.15 0.05 ± 0.43 0.41 ± 0.13 0.06 ± 0.45 0.38 ± 0.11 0.10 ± 0.18 RESULTS Peppermint oil (32% v/v) and trans-cinnamaldehyde (32% v/v) showed sporicidal activity with log10 reductions in C. difficile spores ranging from 0.54 to 2.19. Antimicrobial susceptibility testing All products except ginger juice showed activity against all four mid-exponential phase C. difficile isolates. Stationary phase C. difficile were comparatively less susceptible to the products. Products with inhibitory activity against stationary phase C. difficile were garlic preparations, cinnamon powder, WA Leptospermum honey, artichoke, peppermint oil, Aloe vera gel, allicin, trans-cinnamaldehyde, menthol and zingerone. Products with bactericidal activity against stationary phase C. difficile were cinnamon powder, peppermint oil, menthol, trans-cinnamaldehyde and zingerone. Sterile distilled water (SDW) and dimethyl sulfoxide (DMSO) were used as negative controls; h, hour. Statistical significance: * = P < 0.05, ** = P < 0.01, *** = P < 0.001. CONCLUSIONS REFERENCES Our data showed the potential of several natural products with inhibitory effects on sporulation and outgrowth of C. difficile and with sporicidal activity against C. difficile spores. In addition to the inhibition of sporulation, inhibiting the conversion of spores to vegetative cells can contribute to reducing the recurrence and relapses of CDI. Use of natural products to reduce the spore counts may be a powerful way to reduce the transmission of infection. Future studies are required to further understand the effect and mechanism of action of natural products on the life cycle of C. difficile spore and their potential effects in vivo. 1. Cheng AC, Ferguson JK, Richards MJ, Robson JM, Gilbert GL, et al. Australasian Society for Infectious Diseases guidelines for the diagnosis and treatment of Clostridium difficile infection. Med J Aust. 2011;194:353-358. 2. Garneau JR, Valiquette L, Fortier LC. 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