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Abstract
Clostridium difficile is a nosocomial pathogen that causes a serious toxin-mediated enteric disease in humans. Reducing C. difficile toxin production could significantly minimize its pathogenicity and improve disease outcomes in humans. This study investigated the efficacy of two, food-grade, plant-derived compounds, namely trans-cinnamaldehyde (TC) and carvacrol (CR) in reducing C. difficile toxin production and cytotoxicity in vitro. Three hypervirulent C. difficile isolates were grown with or without the sub-inhibitory concentrations of TC or CR, and the culture supernatant and the bacterial pellet were collected for total toxin quantitation, Vero cell cytotoxicity assay and RT-qPCR analysis of toxin-encoding genes. The effect of CR and TC on a codY mutant and wild type C. difficile was also investigated. Carvacrol and TC substantially reduced C. difficile toxin production and cytotoxicity on Vero cells. The plant compounds also significantly down-regulated toxin production genes. Carvacrol and TC did not inhibit toxin production in the codY mutant of C. difficile, suggesting a potential codY-mediated anti-toxigenic mechanism of the plant compounds. The antitoxigenic concentrations of CR and TC did not inhibit the growth of beneficial gut bacteria. Our results suggest that CR and TC could potentially be used to control C. difficile, and warrant future studies in vivo.
Highlights
Clostridium difficile is a gram-positive, spore-forming, anaerobic bacterium that causes a toxin-mediated enteric disease in humans [1,2]
The sub-inhibitory concentrations (SICs) of CR and TC were found to be 0.60 mM (0.10 mg/mL) and 0.38 mM, (0.05 mg/mL), respectively. These concentrations of plant compounds did not inhibit the growth of all C. difficile isolates including CodY mutant and parental strains after 24 h incubation at 37 °C
This indicated that the concentrations of CR and TC used in this study were non-inhibitory to the growth of C. difficile (As shown in the Supplementary Figures S1–S3) as well as the seven selected beneficial bacteria
Summary
Clostridium difficile is a gram-positive, spore-forming, anaerobic bacterium that causes a toxin-mediated enteric disease in humans [1,2]. Prolonged antibiotic therapy results in the disruption of the normal enteric microflora, leading to the germination of C. difficile spores and pathogen colonization in the intestine with subsequent production of toxins [1,8]. The toxins, TcdA and TcdB, act as glucosyl transferases that inactivate the Rho family GTPases associated with F-actin regulation, and cause disruption of the cytoskeleton and intestinal epithelial tight junctions [9,10]. This leads to an inflammatory response with the release of cytokines and leukotrienes, causing pseudomembrane formation and watery diarrhea [6,7,11]. The genes tcdA and tcdB, which encode the C. difficile toxins
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