A Mouse Model Of Clostridium Difficile-associated Disease

Background and ObjectivesClostridium difficile(C.difficile,CD)is a Gram-positive,spore-forming anaerobic bacterium,and it is an opportunistic infection pathogen that colonized in normal human intestines.C.difficile causes diarrhea mainly by secreting toxin A(TcdA)and toxin B(TcdB),leading to asymptomatic or mild diarrhea,even severe pseudomembranous colitis which induces intestinal perforation,abdominal infection,sepsis and so on.Nowadays,C.difficile is not only the most important pathogen of hospital-associated diarrhea,but also the crucial pathogen of community-acquired diarrhea.In addition,the emergence of the highly virulent strain ST-1(BI/NAP1/027)has led to an increase in the number of patients with seriously C.difficile-associated diseases(CDAD).C.difficile infection(CDI)has become a serious public health problem,posing a serious threat to global health and urgently requiring effective diagnosis and treatment.Scholars have developed many animal models that simulate the clinical process of CDI as much as possible to study the pathogenesis of CDI and to find new treatments.These animal models have limitations in terms of clinical representation and model stability.In order to find a stable,well-fitted,economical and easy to access animal model,we designed to use antibiotics and C.difficile spores induce a mouse model of CDADMethodsC57BL/6 mice was divided into three groups,including Blank(n=6),CDI model(n=8),and MTZ(n=6).Mice in the Blank group drunk autoclaved water and were reared without any treatment from beginning to end.Antibiotic cocktails are consisted of kanamycin(0.8 mg/mL),gentamicin(0.07 mg/mL),colistin(0.1135 mg/mL),metronidazole(0.43 mg/mL)and vancomycin(0.09 mg/mL).Antibiotic cocktails was provided for 7 consecutive days starting on Day-9,and then replaced with autoclaved water on Day-2.A dose of clindamycin(10 mg/kg)was given intraperitoneally at Day-1.The C.difficile spores were administered to the CDI model mice at a dose of 3× 108 colony forming units(cfu)by oral gavage from Day 0 to Day 2.The mice were observed every 4 hours,mainly for the presence of diarrhea and death after the spore challenge.Mice in MTZ were treated as described as CDI model and were given metronidazole(1 mg/day)orally from Day 0 to Day 4.Body weight was measured daily from Day-9 to Day 7.Cecal contents,cecum tissue and colon tissue samples were collected soon after the death of mice.The surviving mice were executed at Day 7.Tissue samples including cecum and colon tissues were collected for histopathologic and immunohistochemical analysis.Cecal contents were collected for 16S rRNA analysis.Result1.Weights showed increased slowly from Day-9 to Day1,but Mice in CDI model and MTZ had a weight loss from Day 2.CDI model mice developed diarrheic symptoms on Day 1,the diarrhea rate was 100%,and the survival rate was 87.5%.MTZ mice developed diarrheic symptoms on Day 2,the diarrhea rate was 66.7%,and the survival rate was 66.7%.2.The mucosa of the cecum and colon tissues in CDI model mice was incomplete,even shedding and gland structure was destructed,with infiltration of a large number of inflammatory cells.The mucosa and gland structure of the MTZ mice remained intact,but there are still many inflammatory cells infiltrated in the mucosa and submucosa.3.Immunohistochemistry showed that Muc-2 destruction was evident in the cecal and colonic epithelium of the CDI model,and there was statistical dif'ference between the Blank group and the MTZ group(cecum p<0.001,p<0.001;colon p<0.05,p<0.05).Destruction of ZO-1 in the colonic epithelium was significant,and there was a statistically difference between the Blank group and MlTZ group(p<0.05,p<0.01).4.The results of Western blot showed that the pro-apoptotic proteins Bax were not expressed in the Blank group but high expressed in the CDI modelh and expression of executor Caspase-3 was the same as Bax in the CDI model.The expression of anti-apoptotic protein Bcl-2 was inhibited in the CDI model which was normally expressed in the Blank mice.5.Blank group was dominated by Bacteroidetes and Firmicutes.The diversity and abundance of the model group were reduced,CDI model group was dominated by Firmicutes and Proteobacteria with a disappearance of Bacteroides and Verrucomicrobi.MTZ group showed a recover of gut microbiome with the dominant of Bacteroidetes.Conclusion1.A relatively stable mouse model of C.difficile-associated diseases was successfully induced by using 6 antibiotics and C.difficile spores.2.C.difficile infection promotes the mechanism of apoptosis in intestinal epithelial and destroys the gut barrier.3.The abundance and diversity of intestinal flora in mice were significantly decreased after C.difficile infection.Bacteroides and Verrucomicrobia disappeared,and Firmicutes and Proteobacteria became dominant bacteria.