Animal Model Of Antibiotic-associated Diarrhea

Background and ObjectivesOur gastrointestinal tract is home to over 1000 types of micro-organisms, with a total weight of 1-2 kilograms and a total number of 1014, which is 10 times the number of the cells of a human being. These micro-organisms constitutes a huge and complex micro-ecosystem in human’s gastrointestinal tract.Though there is a wide variety of intestinal flora,They mainly belong to four groups:Bacteroides, Firmicutes, Proteobacteria and Actinobacteria. Mutual antagonism and mutual coordination take place in different types of intestinal flora, forming a dynamic equilibrium in terms of quality and quantity to form the human intestinal barrier in the biological barrier section. Thereby the micro-organisms play an important role in human body:fighting against bacteria and keeping the integrity of the mucosal barrier; adjusting immune system, including systemic and partial immune system; degrading food debris and compounding nutrients.The composition, distribution and quantity of intestinal microflora is in a stable condition due to their ability of autogenous regulation. However, once the balance is broken, certain pathophysiological changes is seen and eventually leads to multiple diseases such as antibiotic-associated colitis,Inflammatory bowel disease(IBD), Irritable bowel syndrome(IBS), cancer or even obesity, diabetes and coronary heart disease.Antibiotic-associated diarrhea (ADD), refers to the iatrogenic diarrhea caused by the alteration of intestinal flora after the application of antibiotics, which is one of the usual adverse reactions to a variety of antibiotics.The major clinical manifestations of mild AAD includes abnormal frequency or consistency of defecation while the severe AAD mainly result in mass propagation of bacteria,causing intestinal mucosal injury and inflammation, and finally the inflammation diarrhea. Clostridium difficile associated diarrhea (CDAD) accounts for 10% -25% of AAD, and other pathogenic bacterium include Salmonella, Clostridium perfringens, Klebsiella oxytoca, Candida and Staphylococcus aureus etc. Factors which bring risks of antibiotic-associated diarrhea contains the followings:in the host factor respect, the age over 65, female, reduced immunity and combination with severe underlying diseases are included; in the antibiotic factor respect, almost all antibiotics can cause antibiotic-associated diarrhea, but its effect on intestinal microecology and the probability of diarrhea mainly connects with the antibacterial spectrum, pharmacokinetics, route of administration and its concentration when reaching enteric cavity of the antibiotics. Broad-spectrum antibiotic, especially those aim at enteric bacilli, anaerobion, and vestigital antibiotics of high concentration in enteric cavity—also means the antibiotic absorbs little or incomplete secretion of bile, would lead to large changes of intestinal bacteria. Thus, the occurrence rate of diarrhea caused by broad-spectrum antibiotic, cephalosporins and clindamycin,etc. is quite high.Available studies suggest that the pathogenesis of AAD:antibiotic inhibits the growth of normal dominant bacterial community, and the clinical manifestations may be asymptomatic, or normal bacterial community will restore in the short term after stopping application. To some patients, the occurrence of imbalance of normal flora will lead to excessive growth of opportunistic pathogens such as difficile Clostridium, and afterwards diarrhea or even pseudomembranous colitis happen.In addition, dysbacteriosis caused by antibiotics also destroys the metabolism of micro-ecology, and the absorption of carbohydrates is reduced, and finally the osmotic diarrhea occurs; or the production of short-chain fatty acid is reduced, and less liquid is absorbed by colon culminating in secretory diarrhea. On the other hand, the antibiotic’s own allergic reaction and toxicity can directly cause intestinal damage and atrophy of intestinal epithelial cilia, leading to malabsorption diarrhea. Finally, certain antibiotics (such as macrolides) are motilin receptor agonist, and motilin gastrointestinal peptides can stimulate antral and duodenal contractions, causing changes in bowel movements, causing diarrhea, intestinal cramps and vomiting.Currently microecologics including probiotics, prebiotics and synbiotics have been widely used as a novel means of treating antibiotic-associated diarrhea, and its fundamental purpose is to restore and stabilize the original micro-ecotope system, but research on their specific effectiveness and mechanism is still relatively scarce. Preparation of antibiotic-associated diarrhea model is in favor of research on cure effects of microecologics against antibiotic-associated diarrhea as well as mechanism. Besides, it is also good for promotion of the application of microecologics. There are lots of methods to establish a model in which the imbalance of intestinal flora is caused by antibiotics, and roughly can be divided into two categories:Type A. Application of antibiotics only. Various types of antibiotics is used, including ceftriaxone, lincomycin or clindamycin, ampicillin, polymyxin, and even two or triple therapy; and the dosage of gavage is different too, ranging from 1 to 20 times within 1 to 10 days or so. For example, Li Jing and other people use ampicillin to gavage mice with 0.5ml (0.45g/ml) each time, two times one day, and last for 5 days to form the model in which the imbalance of intestinal flora is caused by antibiotics. Type B. Bacteria plus pathogenic bacteria. After the application of antibiotic gives rise to dysbacteriosis, the animal is given common pathogenic bacterium factitiously, leading to a model of diarrhea, for example, Xiong Dexin and some other people use (107—109CFU/ml,0.2ml/t)Shigella,E.coli, Salmonella typhi to attack the animal model dealed with ampicillin sodium(15--40g/kg).Though there are many methods,lots of shortcomings exist:related records do not form an animal model standard of judging antibiotic-associated diarrhea, while most of them predicate base on concept of dysbacteriosis. Some records do not mention whether there is symptom of diarrhea, and some just briefly mention diarrhea rate without definite evaluation criterion; moreover, there is still lack of a clear unified standard of the antibiotic, its dosage and time when building the model.In this study, we sought to develop standards of antibiotic-associated diarrhea according to the definition of clinical diarrhea and dysbacteriosis, to build a stable, reproducible model of antibiotic diarrhea with different dosage of clindamycin or triple antibiotic. Besides, we try to monitor the common flora changes of rats dynamically during the whole molding by bacterial culture and fluorescent quatititive PCR, and to determine the natural course of medication.Methods1. Medication and drug schedule of antibiotic-associated diarrhea model respectively using 2ml solution of normal saline(A),25mg/ml clindamycin(B), 50mg/ml clindamycin(C),75mg/ml clindamycin(D), (25+28+14mg/ml) clindamycin+ampicillin+streptomycin(E), (50+56+28mg/ml) clindamycin+ ampicillin+streptomycin(F), (75+84+42mg/ml) clindamycin+ampicillin+ streptomycin, and low(G), to feed SD rats in control group(A),low(B), medium(C), high(D) clindamycin Group, and,low(E), medium(F),high(G) triple antibiotic group for seven days, and then enter an eight-day course of observation.2. to record the weight, food intake, water intake and feces morphology of rat, and weigh the total weight of feces for 2 hours in a fixed time during the day from 7th day to 14th day. Furthermore, drying the feces to calculate its dry-wet ratio and evaluate the diarrhea indicators of rats.3. When the experiment comes the first, fourth, eighth, eleventh, fourteenth day, we collect rat feces for each group, and then operate the common flora test through bacterial culture and PCR as well as contrastive analysis on the results of these two different methods.4. In 11th day and 15th day, half of the rats in each group are executed, and pathology test with ileum, cecum and colon tissue is operated, then analysis on cecal colonization of colon is on the way after collecting cecal feces.ResultI. Weight, food intake and water intake:Compared with the control group, the AAD groups showed no significant difference in weight and food intake. The water intake of clindamycin groups increases with the drug concentration but is not dose-dependent. While in combined antibiotic groups the water intake is related with the drug concentration and is dose-independent.2.Assessment of the diarrheaExcrement characters:Compared with the control group, loose stools was observed in the AAD groups (except group D) since the 2nd day and was most obvious in the 6th and 7th day. The symptoms lasted to the 14th day and showed relief in the 15th day. Diarrhea symptoms of group B, E, F, G was more obvious and stable compared with the rest groups.Dry-wet ratio of feces:Compared with the control group, the dry-wet ratio of feces in AAD groups (except group D) exhibited a dramatically drop from day 7 to day 14 and the result was relatively stable with statistical significance. Besides, in clindamycin groups the dry-wet ratio of feces was negatively related with the drug dose and in combined antibiotic groups no dose-response relationship was observed.The 2-hour feces amount:There is a great difference between the female and male rats. The 2-hour feces amount was increased in group B, C and F and was statistically significant when compared with the control group. Group D and the male rats in group G showed no statistical significance. In group E, the results fluctuate dramatically and no statistical significance was found.3. Bacterial flora changes over timeBacterial culture results showed the flora was stable during the whole period in the control group. While in the AAD group, a decrease was observed in lactobacillus, bifidobacterium and clostridium and a increase was seen in escherichia coli and saccharomycetes. The enterococcus, staphylococcus aureus bacteria and anaerobic bacteria was inhibited successively in AAD group and was recovered to some degree in 14th day. Besides, the changes was similar between clindamycin group and combined antibiotic group, nor was statistical significance was found among different doses.Fluorescence quantitative PCR also showed that the flora was stable in control group. In group C, the flora showed no obvious change as a whole, while bifidobacterium and lactobacillus decreased dramatically, escherichia coli increased obviously, enterococcus and bacteroid experienced a decrease at first and followed with an increase form day 8 to day 11 and still failed to reach the initial amount. In group F, the amount of bacteria decreased significantally in the 4th day, which manifest with a decrease of bacteroid, bifidobacterium, lactobacillus, escherichia coli and enterococcus.The amount of bifidobacterium and lactobacillus was still in a low level from day 8 to day 11 while the amount of escherichia coli and enterococcus increased obvilously.4.Comparison between fluorescence quantitative PCR and bacterial culture:The detection rate of bacteroid was higher in quantitative PCR while that of escherichia coli was higher in bacterial culture. As for lactobacillus, bifidobacterium and enterococcus, the detection rate of two techniques was similar.5. The result of cecum bacterial colonization shows that in the 11th day, the amount of total bacteria and bacteroid in group C and F showed no significant difference compared with the control group. In those two group, escherichia coli and enterococcus increased and formed the dominant bacterial community while lactobacillus and bifidobacterium decreased. In the 15th day, the total bacterium of group C and F showed no significant difference compared with the control group, though some recovery phenomenon was observed in lactobacillus and bifidobacterium the dominant bacterial community was still characterized by escherichia coli and enterococcus.6.Pathological result showed that in the 11th and 15th day, the HE stained ileum and colon of rats showed no inflammation by microscope when compared with the control group.While the cecum, all AAD groups showed mild to moderate inflammation and group B, F and D was more obvious than other groups.Conclusion1.Imbalance of the intestinal flora can be induced by different doses of clindamycin and combined antibiotics, while group B and F was considered as better models of AAD when concerning the diarrhea symptom.2.No dose dependent relationship was observed between antibiotics and the diarrhea symptom, imbalance of intestinal flora or the severity of inflammation.3.The model belongs to moderate diarrhea type and is mainly located in cecum, it can lasts over 1 week.4.A combination of fluorescence quantitative PCR and bacterial culture can be a better method in detecting the imbalance of flora.