Biofilm Formation and Para-cresol Production by Clostridioides difficile Under the Influence of Phytonutrients from Rhus chinensis.

In Asian countries, the decoction of Rhus chinensis berries is used in alleviation of gut related ailments, especially gastroenteritis. In the background of its ethnobotanical use, the aim of the study was to determine the inhibitory activity of berries against a gut pathobiont causing gut dysbiosis, namely Clostridioides difficile. The hot water extract of Rhus chinensis (RcHWE) indicated appreciable antimicrobial activity (60% to near 100%) against C. difficile ATCC 43,593, in the tested range of 0.5-5.5 mg/mL, evaluated by microbroth dilution and standard plate count technique. The minimum inhibitory concentration (MIC) was determined to be 3 mg/mL. There was visible inhibition of biofilm formation on filter membrane impregnated with varying concentration of RcHWE (0.25MIC - 10MIC). Results of flow cytometry with propidium iodide (PI) stained red fluorescence shift in extract supplemented cells, crystal violet staining with decrease in absorbance (34% to 100%) relative to unsupplemented control as well as confocal microscopy images (increase in PI-stained cells), across the tested range (0.25MIC- 10MIC), clearly indicated the antimicrobial and antibiofilm forming ability of RcHWE against C. difficile. Additionally, SEM results along with lactate dehydrogenase assay, clearly indicated extensive cell damage and destruction of cell integrity. Inhibition of p-cresol production (90-100%) with supplementation of RcHWE, was found to be both concentration and time dependent. UHPLC-HRMS/MS analysis indicated polyphenolic glycosides to be the predominant constituents of the extract. Findings highlights the modulatory potential of RcHWE against C. difficile and provides preliminary evidence for its use in gastric ailments especially due to gut dysbiosis.