Inhibitory Effect Of Tannic Acid On Clostridioides Difficile And Its Spores In Vitro

Objective:Clostridioides difficile is a gram-positive spore-forming anaerobic bacillus,which is the main pathogen of antibiotic-associated diarrhea and nosocomial infection.The main pathogenic factors are tcd A and tcd B.Spores formed by C.difficile persist in the hospital environment and are the major form of C.difficile infection transmission or recurrence（re-infection）.As the preferred drug for CDI treatment,long-term use of antibiotics is prone to drug resistance and CDI recurrence.Therefore,rational use of antibiotics and active search for other treatment methods for CDI have become an urgent problem to be solved.Galla chinensis has been reported to have good bacteriostasis,and its main components are tannic acid and gallic acid.Our laboratory previous research showed that galla chinensis decoction had antibacterial effect on C.difficile,which provided a new idea for the treatment of CDI.By studying the antibacterial effect of galla chinensis main components on C.difficile and comparing the effects of galla chinensis,tannic acid and vancomycin on the spore germination of C.difficile,in order to find drugs that can inhibit C.difficile and its spores germination.Methods:One hundred and fifty-two strains of C.difficile strains from Hebei Provincial Bank for Medical Culture Collections（HBMCC）were selected by using a random number table.With reference to the anaerobic bacteria drug sensitive test method recommended by CLSI（M11-A7）,using agar dilution method determination of metronidazole,vancomycin,fidarimycin,rifaximin,levofloxacin,ciprofloxacin,ceftazidime,ceftriaxone,meropenem,erythromycin,clindamycin,tetracycline,chloramphenicol,gallic acid,and tannic acid on the minimal inhibitory concentrations（MIC）of C.difficile.The spore germination test was carried out on three standard strains ATCC1870,ATCC43598,ATCC1382 and two clinical strains C201010230006 and C201103040011.The MIC of five strains of C.difficile against vancomycin,tannic acid and galla chinensis decoction was first determined by agar dilution method.Then,the spores were prepared,and the change of absorbance OD₆₂₀of 1/2 MIC or MIC vancomycin,tannic acid and galla chinensis decoction on the germination of 5 strains of C.difficile spores was detected,and the final result was expressed by OD_{620（t/t0）},and painted into a curve.Results:1.MIC of tannic acid against 152 strains of C.difficile was concentrated at three concentrations of 16μg/ml,32μg/ml and 64μg/ml,and the cumulative inhibition percentage reached 100%at 64μg/ml.2.The MIC of gallic acid for 152 strains of C.difficile was>256μg/ml.3.The drug resistance rates of levofloxacin and ciprofloxacin in the toxigenic group and the non-toxigenic group were different,P<0.05;There was no statistical difference in the drug resistance rate between other antibiotics groups,P>0.05.4.Vancomycin and normal spore germination curves of 5 strains showed a downward trend,OD_{620（t/t0）}gradually decreased,and there was no statistical difference among three germination curves at each time point（P>0.05）.However,the germination curve of spore with tannic acid and galla chinensis decoction did not decrease,and there were statistical differences in three germination curves at each time point（P<0.05）.Only three curves of C201103040011 tannic acid group showed no statistical difference at 10min（P>0.05）,and three curves of ATCC43598 vancomycin group showed statistical difference at 20min（P<0.05）,but the overall trend of the curve was consistent with the expectation.Conclusions:1.Tannic acid has antibacterial effect on C.difficile,while gallic acid has no antibacterial effect on C.difficile.2.Tannic acid and galla chinensis decoction can inhibit the spore germination of C.difficile,while vancomycin could not inhibit the spore germination of C.difficile.