| Background and ObjectionClostridium difficile (CD) is a gram positive, facultative anaerobic thick bacillus, under the harsh living environment could formed spore at time of extreme, mainly between the person and person through fecal-oral transmission and colonized in the gut. The asymptomatic carrier rate was 5-15%, and the rate of the patients in hospital was 57%, and the rate of Clostridium difficile at infant was 84.4%, higher than that of adults. Clostridium difficile is the main pathogenic bacteria in hospital acquired diarrhea, and it is also an important pathogen of community acquired diarrhea.In 1978, the relationship of Clostridium difficile and Pseudomembranous were found for the first time. Under normal circumstances, Clostridium difficile could be found in the intestinal tract, which does not cause disease. When long-term use of a large number of broad-spectrum antibiotics or other reasons caused by intestinal homeostasis and then Clostridium difficile overgrowth, which result in Clostridium difficile associated infection (CDI) occur that could lead to Clostridium difficile associated diarrhea (CDAD). CDI is the cause of antibiotic associated diarrhea (AAD) in 15%-25%, and is also the cause of almost all antibiotic associated pseudo membranous colitis.Most clinic patients who infected with Clostridium difficile are asymptomatic which called healthy carriers; clinical symptoms can appear from mild to severe watery diarrhea, outbreak of enteritis, pseudomembranous colitis even infectious shock and other systemic symptoms. The process of diarrhea often occurs in the use of antibiotics or discontinuation of antibiotics in a short term, occasionally stopped after months of onset. All antibiotics can be caused by Clostridium difficile infection, which caused by clindamycin, cephalosporins and quinolones at higher risk of infection, and piperacillin/tazobactam, rarely trigger CDI. Currently CDI standard treatment regimen is metronidazole or vancomycin, but up to 25% of patients could relapse in the use of these 2 kinds of antibiotics within 30 days, the recurrence rate can be increased to 40-65% after first recurrence. Recurrent CDI is not only difficult to treat, but also affects the patient for months or even years, reduces the quality of life, and increases the economic burden.Due to broad-spectrum antibiotics abuse, the appearance of high virulence strains, resistant strain rate increase and other factors, the incidence of CDI rate gradually increased, especially in the recent history of hospitalization or someone who living in long-term care facilities in the elderly population. Europe and the United States have occurred many times the outbreak of the epidemic, the prevalence of mortality increased significantly, resulting in increased health care costs. In order to study the pathogenesis of Clostridium difficile infection, the preparation of vaccine and the evaluation of the new treatment plan, establishing a stable and reliable animal model of CDAD is urgent.Hamsters, guinea pigs, germ free rats, rats and mice have been used as models of CDAD. The most widely used animal in the past is the hamster model, but because the symptoms of hamster infected with Clostridium difficile, which is a kind of explosive, deadly disease process. This is different from the common outcome of human infection with Clostridium difficile in China, which could not completely represent the human infection process. In recent years, the application of CDI model in mice was increased because of the new method increased susceptibility of mice symptom and the improvement of the specific reagents that can be applied to analysis of mouse host tissues. Chen et al found that C57BL/6 mice could successfully infect Clostridium difficile and show up diarrhea after mixed antibiotic and clindamycin,, which infection process was similar to humans, thus became the mainly CDAD animal models. However, our group have found that the C57BL/6 model is difficult to control the severity of infection in mice, most of the mice with mild diarrhea, self-healing, may limit its application in the study of refractory and recurrent Clostridium infections.This research choose three commonly used strains of mice in domestic research, constructed of CDAD model of different strains of mice, than assessed by clinical and pathological indexes screened out the most representative of the clinical and stable animal model, which could provide reliable experimental tools for Clostridium difficile infection related research.Fecal microbiota transplantation (FMT) is transplanting the function flora in healthy human feces to gastrointestinal tract of patients and reconstructing normal function of intestinal flora to treat intestinal and extraintestinal diseases. Over the past ten years, because of the outstanding prominent effect in the treatment of CDI, fecal microbiota transplantation receives more and more attention domestic and overseas. All over the world thousands of cases of application of fecal bacteria transplantation have been reported at present, these cases including gastrointestinal diseases (such as difficult Clostridium difficile infection, inflammation bowel disease, irritable bowel syndrome etc.) and non gastrointestinal diseases (such as fatigue syndrome, idiopathic thrombocytopenic purpura).The effective of fecal microbiota transplantation in the treatment of refractory Clostridium difficile infection is up to 80%-90%, compared to the application of vancomycin and other treatment options, FMT treatment is short, cost less. In February 2013, the United States Food and Drug Administration have been written FMT to the Treatment Guide of CDI, which recommend at third times relapse after the treatment of intermittent vancomycin, could consider the use of FMT.Although FMT has been used in the clinical treatment of a variety of diseases, but as a clinical treatment FMT is not fully mature, like its long-term safety issues, which is lack of adequate clinical and laboratory information. The main adverse reactions include belch, abdominal distension, abdominal cramps and other gastrointestinal symptoms; and part of the patients after the FMT can be symptoms of fever, diarrhea increased small intestinal bacterial overgrowth, the symptoms of multi within 3 days after the FMT disappear. FMT greater risk may be based on changes in intestinal flora, induced chronic disease. At present, it is known that intestinal flora is related to some diseases occur, including obesity, diabetes, atherosclerosis, inflammatory bowel disease, colon cancer, non alcoholic fatty liver disease, irritable bowel syndrome, asthma and autism etc. Changing the structure of the host’s original intestinal flora may increase the risk of these diseases.There is not enough research to evaluate the safety of FMT, this study intends to verify the effective of FMT in the treatment of CDI by using the mouse model of CDAD, and make a preliminary study on effects of FMT on host activity, metabolism, immunity, than provide a basis for the safety evaluation of FMT.Materials and Results1. Establish a stable model of Clostridium infection of mice, select the ideal model animal50 strains of clinical isolates in Nanfang Hospital were recovered and cultured, and the methods of colony morphology, microscopic morphology, lactate dehydrogenase detection, biochemical identification and toxin detection were identified. According to ribotyping, select one of the most common as the experimental strains.Select C57BL/6, BALB/c, Kunming mice,3 strains of mice each 24, according to the principle of random grouping is divided into 4 groups (control group, low concentration group, middle concentration group, high concentration group), each group of 6. The experimental group after induced by antibiotics, were to be clinically isolated strains of different concentrations (108CFU/ml～1010CFU/ml) suspension gavage, and histological changes were observed in the different strains of mice diarrhea, systemic and colon tissue pathology.1.1. The diarrhea symptoms of BALB/c mice were concentration dependent.Low concentration group (108CFU/mL) in C57BL/6 mice (n=6) did not appear diarrhea; BALB/c mice 3 appear severe diarrhea symptoms; KM mice 1 appear mild diarrhea symptoms.Concentration group (109CFU/mL) in C57BL/6 mice 1 only mild diarrhoea; BALB/c mice may only appear severe diarrhea, wet tail bounds than the low concentration group slightly larger; KM mice 4 different levels of diarrhea, which a death, to observe the end survival rate was 83.3%.High concentration group (1O1OCFU/mL) in C57BL/6 mice 3 only appeared severe diarrhea; BALB/c mice 6 developed diarrhea, mice were killed, to observe the end survival rate was 83.3%; KM mice 4 diarrhea symptoms, found no dead.After infection with average weight of mice decreased significantly, we also observed that the mice decreased activity, hair loss, serious person appear arched. 1.2. Isolated Clostridium difficile from the feces of diarrhea miceRandomly selected 3 kinds of diarrhea mice feces smear, staining, we can see the different degrees of intestinal flora imbalance and gram positive bacillus. The diarrhea mice were inoculated to the CCFA-HT culture medium and inoculated to the anaerobic culture of the intestinal contents, and the typical colony morphology of Clostridium was found. Karyotype analysis confirmed that the isolated strain of feces of mice was consistent with the experimental strains.1.3. Colonic pathological score was correlated with the severity of symptomsHealthy control group 0, the low concentration group in C57BL/6 mice for 0.3±0.5, BALB/c mice for 1.5±0.5, KM mice as 0.7±0.5.C57BL/6 mice in the medium concentration group were 0.7±0.5, BALB/c mice to 2.0±0.9, KM mice for 1.8±1.7. High concentration group in C57BL/6 mice for 1.0±0.6, BALB/c mice was 3.2± 1.2 KM mice for 1.8±1.0.2. Fecal microbiota transplantation could treat Clostridium difficile infection in mice A total of 60 BALB/c mice were divided into 4 groups according to the principle of random grouping,12 groups,5 rats in each group. According to the different treatment were divided into blank control group, irrigation bacteria group, single FMT group,3 FMT group,4 large groups,3 experimental points (1 days after FMT,1 weeks after FMT, a mouth after FMT) were executed 1 groups. Every time before being killed before modeling and field test.2.1. FMT treatment group did not appear severe diarrheaAfter infection, filling the bacteria group were 5 had mild diarrhea,10 severe diarrhea, which 2 died on day 3 after bacteria injection; single FMT mice 5 had mild diarrhea, no death; 3 times FMT mice only 3 appear mild diarrhea.2.2. FMT can reduce intestinal inflammation in mice2.2.1. Inhibition of colon shortening in infected miceThe colon length of the mice in the irrigation group was shortened, and the congestion and edema of the intestinal lumen were 2 days after infection, filling the bacteria group mice colon length (8.5±0.46cm) was significantly lower than that in the control group were (10.9±0.30cm,p< 0.01), single FMT mice (9.5± 0.64cm,p< 0.01) and 3 times FMT group mice (10.1+0.72cm, p < 0.01). The 8th day post infection, filling the bacteria group mice colon length (8.4± 0.24cm) was significantly lower than that of the control group mice (11.5±0.41cm,/>< 0.01), single FMT mice (9.8±0.48cm, p< 0.01) and 3 times FMT group mice (10.5±0.73cm, p< 0.01).2.2.2. Improve the pathological changes of intestinal inflammationIn the severe diarrhea mice (D2), inflammatory cells were seen in the whole mucous membrane and even in the lamina. The mucosal edema and the epithelial cells were disrupted and detached. After treatment of FMT mice intestinal visible part of the epithelium shedding, adjacent normal mucosa, submucosa inflammatory cells.2.2.3. Inhibiting the expression of inflammatory cytokinesCompared with the blank control group, FMT treatment group and treatment group, the expression levels of different inflammatory cytokines (IL-6, TNF-α, IL-1β) were increased, and the number of FMT was increased, and the expression of inflammatory cytokines could not be decreased. By ELISA method to detect the serum inflammatory cytokines found irrigation bacteria group of 3 kinds of cytokines were lower than those in the control group, FMT in the treatment group were added, and increase the number of FMT, cytokines was not reduced, and intestinal inflammatory cytokines expression of the same.2.3. fecal microbiota transplantation has no significant effect on the behavior and metabolism of miceBefore the start of the experiment, mice locomotor activity, anxiety level had no difference (P> 0.05); one day after treatment of FMT, total movement distance, time for exercise, edge distance, central distance of single FMT mice group compared with the blank control group were decreased (P< 0.05); one month after treatment, mouse total movement distance (P< 0.01), movement time (P< 0.05), edge distance (P< 0.01) of three times FMT group compared with the blank control group were decreased, and the single FMT group’s mouse total movement distance (P< 0.05) compared with the blank control group.The blood glucose, triglyceride, cholesterol, high density lipoprotein and low density lipoprotein in FMT group were not significantly different (P> 0.05) compared with the control group and the control group after 1 days,7 days and 1 months after treatment.Conclusion1. Treated with gentamicin and metronidazole, polymyxin, vancomycin, card that mildew element mixed antibiotic solution by intragastric administration of 9 days in+clindamycin intraperitoneal injection as pretreatment,24h after treated with bacterial 1010CFU/ml suspension gavage,BALB/c mice could be successfully established stable and representative CDAD model;2. increase in the number of fecal microbiota transplantation did not significantly increase the efficacy;3. fecal microbiota transplantation has no significant effect on the behavior and metabolism of mice in a long term, which could be considered as a safe and effective treatment method. |
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