Fecal Microbiota Transplantation And Probiotics Effectively Reduce The Intestinal Colonization Of Clostridium Difficile In A Mouse Model

Objective:To evaluate the effect of fecal microbiota transplantation(FMT)and novel probiotics on Clostridium difficile infection(CDI),we determined the colon colonization of C.difficile and alteration of flora microbiota after treatment by FMT and protiotics in a mouse model of CDI by using the quantitative method for detecting C.difficile in the faeces.Methods:1.Specific primers and probes were designed to establish a sensitive and specific detection method for C.difficile;determine the accurate standard curve for detection the C.difficile in the mouse faeces.2.Establishment of CDI mouse model:The mice were administered by the mixture of antibiotics(antibiotic cocktail)prior to infection with C.difficile.Female C57BL mice aged 6-8 weeks were used in all experiments,and the mice were infected with C.difficile NAP 1/027 strain.The symptoms of the infected mice including the weight and the colonization amount of C.difficile in the fecas were monitored everyday.3.FMT treatment in CDI mouse modle:Two different subgroups(6N,6J)of C57BL healthy mice were selected as donors for FMT.The mice were treated with FMT the next day after C.difficile infection,one group was treated with faeces from the same subgroup(FMT-C57N),and the other group was treated with faeces of different subgroup(FMT-C57J).The effect of different source of FMT on the colonization of C.difficile in mose model was compared and the changes of intestinal microflora structure in mice was characterise4.Probiotic treatment in CDI mouse model:By using the established CDI model,109 CFU probiotics were administered daily to each mouse after infection,and the amount of C.difficile colonization in mouse intestinal tract was monitored.The amounts of C.difficile colonization in the faeces was compared between model group and probiotics-treated group.At the end of the experiment,mice were sacrificed by cervical dislocation,the colon tissues were collected and the mRNA expression of various genes in the colon tissue of mice was detected by fluorescence quantitative PCR.results:1 Quantitative detection of C.difficile by Taqman real-time PCR:(1)The TaqMan real-time PCR detection has an excellent liner range,and the detection limit for C.difficile is 101 CFU/PCR;(2)The specificity and sensitivity of the TaqMan real-time PCR detection were high;as well as the accuracy;(3)The same concentration of C.difficite in faeces of mice and pure bacteria was detected by fluorescence quantitative PCR and the standard curves were compared,there was no significant difference between them.2 The CDI mouse model:The weight of mice dropped rapidly on the first 2 days post infection,accompanied with symptoms such as diarrhea(wet tail).On the fifrth day post infection,the weight of the mice had completely recovered to the normal weight,with an average body weight of(101.53±0.24)%.The C.difficile successfully colonized the first day post infection,and the average amount of C.difficile colonized in mice intestine was 6.17±0.25,there was no remarkable change in the number of bacterial colonization within 5 days after infection.3 FMT regulated the intestinal flora of CDI mice and reduced the colonization of C.difficile in mouse colon:(1)FMT can effectively reduce the colonization of C.difficile in mouse intestine.Fecal transplantation with faeces from different strains of mice was more effective in reducing the colonization of C.difficile.(2)FMT can effectively influenced intestinal microflora in mice.At the phylum level,level,the relative abundance of Bacteroideae in the negative control group was(41.15±11.66)%,which is much higher than that of the positive control group,FMT-C57J,and FMT-C57N group,and the positive control group with the lowest relative abundance(0.66±0.26)%.The abundance of Proteobacteria in the positive control group,FMT-C57J,and FMT-C57N groups were all increased,and the positive control group increased the most to(17.89±2.66)%,while the abundance of Proteobacteria in the negative control group was only(0.66±0.26)%.The abundance of Verrucomicrobia in the positive group was(9.18±6.58)%,which was decreased in the positive control group and the FMT-C57N group,but increased in the FMT-C57J group,At the genus level,the relative abundance of Akkermansia in the positive control group,FMT-C57J,and FMT-C57N groups were all decreased,In the positive control group and FMT-C57J group,Bacteroides accounted for the largest proportion((49.1 5±18.63)and(18.27±18.63))%respectively,and in the FMT-C57N group,the Prevotella accounted for the largest proportion(13.64±6.59)%.In the positive control group,Bacteroides was significantly increased,in the FMT-C57J group,Clostridium had a significant increase and in the FMT-C57N group,Akkermansia decreased significantly.4 Probiotics treatment in CDI mouse model:(1)Mixed probiotics can effectively reduce the colonization of C.difficile in the intestinal of mice,at the fifth day after treatment with probiotics,The colonization of C.difficile was significantly lower than that of the positive control group((7.15±0.34)vs.(7.78±0.06)),and the amount of C.difficile colonized in the faeces continued to decrease along with the persist intervention of the probiotics in CDI mice.At ninth day after the treatment of probiotics,the amount of C.difficile in mice faeces was reduced by approximately 102 CFU per gram fecel((5.64±0.62)vs.(7.41±0.20)).(2)On the 10th day after CDI,the expression of related genes in colon tissue was detected.The results showed that the relative expression levels of Reg3-β and IL-12 in the positive control mice were significantly higher than those in the probiotic group.There was no significant difference in the relative expression of CXCL1,Reg3-γ,Ccl2,Muc2,IL-1b,CSF,TNF between the positive control group and the probiotic group.Conclusions:1 Taqman real-time fluorescence quantitative PCR can quickly and effectively detect the amount of C.difficile in faeces,and the amount of C.difficile in mouse faeces can be quantified by the standard curve of C.difficile pure bacteria.2 Antibiotic cocktail mouse model mirrors key features of CDI in humans,including diarrhoea,weight loss and histological damage,the model is stable and reliable.3 The FMT can regulate the intestinal flora of CDI mice and reduce the colonization of C.difficile,and the FMT from C57J mice was much effective than that from C57N mice.4 Mixed probiotics can effectively reduce the colonization of C.difficile in the intestinal of CDI mice.At the fifth day after the intervention of probiotics,the amount of C.difficile in the intestinal of mice was significantly reduced.The colonization of C.difficile continued to decrease as probiotics intervention went on.The relative expression of Reg3-β and IL-12 were decrease in probiotics group implying that probiotic intervention may affect the host immune response.