In Vitro And In Vivo Antibacterial Activity Of Palmatine Alone Or In Combination With Metronidazole Against Clostridium Difficile

ObjectiveClostridium difficile is an anaerobic,Gram-positive,spore-forming bacillus that causes disease ranging from self-limiting diarrhoea to severe pseudomembranous colitis.C.difficile infection(CDI)commonly affects hospitalised patients and is increasingly identified in patients in the community with no hospital contact.For the last 15 years the incidence of CDI worldwide has been rising,especially in the northern hemisphere.The yearly average number of hospitalizations as a result of this disease is estimated to be over a quarter of a million per year in the United States alone.The main risk factor for CDI is exposure to antimicrobials that affect the gut microflora and,paradoxically,the most common treatments for CDI are the antimicrobials,metronidazole and vancomycin.However,the increasing frequency of highly virulent C.difficile strains,antimicrobial treatment failures,hospital outbreaks,patients with severe complications and cases with multiple recurrences have driven the search for new therapies.Palmatine is an isoquinoline alkaloid derived from the dry rattan of Fibraurea recisa Pierre.Palmatine contained in the 2015 edition of " Chinese Pharmacopoeia" and "Chinese Veterinary Pharmacopoeia",has detoxification effect,treating gynecological inflammation,dysentery,enteritis,respiratory tract and urinary tract infections,surgical infection,conjunctivitis.Modern pharmacological studies show that palmatine has antibacterial,anti-inflammatory,enhance immune function.Palmatine can inhibit a variety of fungi,gram positive and gram negative bacteria.Therefore,it is called as a plant antibiotic.The aim of this study is to study the in vitro and in vivo antibacterial activity of palmatine alone or in combination with metronidazole against C.difficile,so as to provide a reference for the development of palmatine as a new veterinary drug,and to provide a scientific basis for the safe and effective application of palmatine.Searching for natural alternatives,including plant extracts and natural products,and combine them with antibiotics to reduce the use of antibiotics and even replace the current antibiotics.This study contains two parts:In vitro antibacterial activity of palmatine alone or in combination with metronidazole and vancomycin against Clostridium difficile;Protective effect of palmatine alone or in combination with metronidazole in C57BL/6 mice model of Clostridium difficile infection(CDI).Methods20 worldwide isolates of Clostridium difficile were collected from Dena Lyras s laboratory at Monash University.The ribotype was analyzed by PCR and 6 representative strains were selected for the follow-up study.The solvent toxicity test was used as the reference standard for the subsequent drug preparation.Through literature review,selected 10 different types of natural compounds with different antibacterial activity(palmatine,oxymatrine,matrine,arecoline,baicalin and polydatin,anemonin,andrographolide,ursolic acid,honokiol)and 2 antibiotics(metronidazole,vancomycin)of Clostridium difficile early sieve against Clostridium difficile activity in vitro,According to the preliminary screening results of antibacterial activity,palmatine’s follow-up experiments were carried out to analyze the drug resistance of 20 Clostridium difficile isolates from palmatine,metronidazole and vancomycin,and to detect the MIC and MBC of 6 representative strains.A chessboard method was used to test the drug sensitivity of palmatine with metronidazole,palmatine and vancomycin,metronidazole and vancomycin.According to the results of checkerboard experiment,3 strains(reference strain 630,hypervirulent strain M7404 and clinical strain CD72)with synergistic and additive effects were selected for bactericidal experiment in vitro,and time-kill curves were drawn,and the combined effect of drugs was evaluated from another aspect.The combination of palmatine and metronidazole and the combination of palmatine and vancomycin were selected for the combined drug sensitivity test.The toxin production and sporulation assays using hypervirulent Clostridium difficile strain M7404 were carried out in vitro.Palmatine was administered through gavage to C57BL/6 mice with established CDI-induced intestinal injury and colitis.The disease activity index(DAI),mean relative weight,histopathology scores,and levels of toxins A and B in fecal samples were measured.An Illumina sequencing-based analysis of 16S rRNA genes was used to determine the overall structural change in the microbiota in the mouse ileocecum.Results20 isolates of C.difficile isolated from different parts of the world were analyzed by PCR-ribotype,and the ribotypes were 027,087,012,014,002,etc.Under the experimental concentration,only palmatine,metronidazole and vancomycin showed the antimicrobial activity against C.difficile and the other compounds had no bacteriostasis.Although palmatine also showed strong bacteriostasis activity,it was far away from two antibiotics.Checkerboard assay showed synergistic effect of palmatine and metronidazole on 1 strains and additive effect on 5 strains of C.difficile;additive effect of palmatine and vancomycin on 1 strains of C.difficile:additive effect of metronidazole and vancomycin on 5 strains of C.difficile.Time-kill assay showed synergistic effect of palmatine+metronidazole and palmatine+vancomycin on 3 strains of C.difficile.The single or combined use of sub inhibitory concentration of palmatine,metronidazole and vancomycin had no obvious inhibitory effect on the growth of M7404.Subinhibitory concentration of palmatine or metronidazole alone inhibited the production of C.difficile M7404 toxin in a dose-dependent manner.However,the combined use of the two can also inhibit the production of toxins,but the effect is not as good as single use.The effect of subinhibitory concentration of vancomycin on the toxin production of Clostridium difficile M7404 is little,and it seems to show the phenomenon of promoting the production of toxin.Subinhibitory concentration of palmatine combined with vancomycin can inhibit the production of toxin,but the effect after combined use is not as good as that of single use.Subinhibitory concentrations of palmatine,metronidazole and vancomycin alone or in combination could not inhibit the sporulation of M7404,a highly virulent strain of Clostridium difficile,and the sporulation rate was 100%.Palmatine administration significantly promoted the restoration of the intestinal microbiota by inhibiting the expansion of members of the family Enterobacteriaceae and counteracting the side effects of metronidazole treatment.Therapy consisting of metronidazole and palmatine combined prevented weight loss,improved the DAI and the histopathology scores,and effectively decreased the mortality rate.Palmatine prevented CDIs from relapsing and significantly improved survival in the mouse model of CDI.Combination of palmatine and metronidazole is more effective than metronidazole alone for treating CDI.One of the possible mechanisms by which palmatine prevents a CDI relapse is through modulation of the gut microbiota.Conclusion1.The antibacterial activity of palmatine against C.difficile in vitro was weak,but metronidazole showed strong antibacterial activity against C.difficile.2.The combination of palmatine and metronidazole has synergistic or additive effects against C.difficile in vitro.3.Subinhibitory concentrations of palmatine and metronidazole can inhibit the toxin production of C.difficile M7404 in a dose-dependent manner.4.Palmatine administration significantly promoted the restoration of the intestinal microbiota by inhibiting the expansion of members of the family Enterobacteriaceae and counteracting the side effects of metronidazole treatment.Also,palmatine prevented CDIs from relapsing and significantly improved survival in the mouse model of CDI.One of the possible mechanisms by which palmatine prevents a CDI relapse is through modulation of the gut microbiota.Therapy consisting of metronidazole and palmatine combined prevented weight loss,improved the DAI and the histopathology scores,and effectively decreased the mortality rate.