Antimicrobial Activity Of Tannic Acid On Clostridioides Difficile In Vitro

Objective: Clostridioides difficile is an obligate anaerobic gram-positive bacillus and the main pathogen causing antibiotic-related diarrhea in hospitals.Tannic acid is a natural phenolic substance derived from plants.It has been widely used in the medical field because of its anti-oxidation,anti-cancer,anti-mutagenic,and antibacterial effects.This study aimed to evaluate the in vitro inhibitory effects of tannic acid on the biological functions of C.difficile spore formation,toxin production and biofilm formation.Methods:1.Measure the minimum inhibitory concentration(MIC)of tannic acid and vancomycin against the test strain by agar dilution method.2.Cultivate C.difficile with tannic acid(1/2MIC,1/4MIC)and vancomycin(1/2MIC,1/4MIC)for 5 days.Take 1 ml of the culture product everyday and heat to kill the vegetative cells.After washing,the obtained liquid was diluted in multiples and spread evenly on enhanced Brucella blood agar.After culturing for 48 hours,the growing colonies were counted and the number of spores formed was calculated.3.Cultivate C.difficile with tannic acid(1/2MIC,1/4MIC)and vancomycin(1/2MIC,1/4MIC)for 3 days.Take 1 ml of the culture product every day and use the ELISA method to determine the toxins Tcd A and Tcd B produced during the culture of C.difficile,and draw a standard curve,and calculate the toxin production based on the standard curve.4.The C.difficile were incubated with tannic acid(1/2MIC,1/4MIC)and vancomycin(1/2MIC,1/4MIC)for 48 hours,and the amount of biofilm formation was quantified by crystal violet staining.5.C.difficile was co-cultured with 1/4MIC tannic acid and 1/4MIC vancomycin,RNA was extracted at 6,12,and 24 h,and c DNA was obtained by reverse transcription using universal primers.Then,qPCR was used to analyze tcdA,tcdB and spo0A mRNA accumulation.Results: 1.The MIC of tannic acid to the test strains ATCC BAA-1870 and ATCC BAA-1382 were both 64 mg/L.The MIC of vancomycin for ATCC BAA-1870 is 1 mg/L,and the MIC for ATCC BAA-1382 is 0.5 mg/L.2.Compared with the untreated control group,the number of spores formed by C.difficile in the tannic acid treatment group was significantly reduced(P<0.05),while the vancomycin treatment group failed to decrease.3.Compared with the untreated control group,the toxin production of C.difficile in the tannic acid treatment group was significantly reduced(P<0.05),while the vancomycin treatment group did not decrease.4.Compared with the untreated control group,the biofilm formation of C.difficile in the tannic acid treatment group was significantly reduced,which was equivalent to vancomycin(P<0.05).5.Compared with the untreated control group,tannic acid has some inhibitory effect on the mRNA expression of test strains tcdA,tcdB and spo0A,while vancomycin failed to show an inhibitory effect.Conclusions:Tannic acid has a significant inhibitory effect on the spore formation,toxin production and biofilm formation of C.difficile,and has some inhibitory effect on the toxin encoding genes tcdA,tcdB and the key regulator of spore formation,spo0A.