Establishment Of In Vitro Intestinal Microbial Ecosystem And Its Application In The Evaluation Of Intestinal Flora Toxicity Of Environmental Pollutants

BackgroundIntestinal microbes have been proved to be closely related to various physiological functions related to human health,and they are also susceptible to various factors.The in vitro digestion model is one of the methods to study the intestinal flora in recent years,and there are still very few studies on the direct interaction between environmental pollutants and intestinal microbes.Research on the toxicity of environmental pollutants to intestinal microbes mostly focuses on animal models or host fecal samples,and the results are difficult to directly extend to human intestinal microbes.ObjectiveThis study aims to establish an in vitro intestinal microbial ecosystem,the simulator of the human intestinal microbial ecosystem(SHIME),and expose the SHIME with environmental pollutants,Dechlorane plus(DP)and drinking water disinfection by-products(CBPs).Through 16S rRNA gene sequencing and metabolic analysis of short chain fatty acids(SCFAs)to evaluate the feasibility of applying the SHIME to the study of intestinal flora toxicity of environmental pollutants.Methods1.SHIME was established and fecal inoculum was prepared and inoculated into the SHIME for 21 days.The samples of Ascending colon,Transverse colon and Descending colon were collected daily and centrifuged at 4℃.DNA extraction,PCR amplification and 16S rRNA gene sequencing were performed to detect the bacterial community structure.SCFAs extraction and GC/MS concentration detection were performed to evaluate the stability of SHIME.2.In vivo experiment:12 pregnant SD rats were divided into control group and exposure group.The exposure group was given 5 mg/kg/D DP by gavage.After 21 days of gavage,the contents of cecum were dissected.In vitro experiment:in SHIME,5ml of bacterial culture medium was extracted from each colon and divided into solvent control group(S),low dose group(L),medium dose group(M)and high dose group(H).5ml of nutrient solution and corresponding concentration of DP were added.After 3 days of sealed culture,the bacterial structure and SCFAs concentration were analyzed.3.200 L water was collected from each of the three waterworks.XAD-2 macroporous resin was used to enrich the chlorination disinfection by-products,methanol/acetone mixture was used to elute,DMSO was used to fix the volume,and samples were sent for detection.According to the measured concentration,the mice were exposed to chloroform and N-nitrosodiethylamine in the SHIME,and the byproducts of corresponding concentration were added to the nutrient solution for 14 days.At the end of the exposure,the colon jar samples were centrifuged at low temperature to analyze the bacterial community structure and detect the concentration of SCFAs.Results1.The SHIME model ran until the 16th day when the flora structure was stable,and the SCFAs ran until 17-18 days when the SCFAs secretion was stable.The stabilized intestinal flora were mainly Bacteroidetes,Proteobacteria and Firmicutes.After stabilization,the original structure of the inoculation could be basically restored,and the main bacterial genera were retained.The ratio of acetic acid,propionic acid and butyric acid of SCFAs was about 0.55:0.3:0.15,which was close to the ratio of human fecal samples.2.The results of in vivo experiments and in vitro experiments show that the results were similar.Specifically,the abundance of Bacteroidetes and Proteobacteria decreased at the phylum level,and DP exposure increased the metabolism of SCFAs in both in vivo and in vitro.3.The structure of the intestinal flora changed after the compound exposure of the disinfection by-products,which was mainly manifested by the increase in the abundance of the opportunistic pathogens Citrobacter and Klebsiella,and the decline in the metabolism of SCFAs,but the difference was not statistically significant.ConclusionThe SHIME system as an in vitro model for studying intestinal flora has potential in the application of environmental pollutant intestinal flora toxicity studies.The results of its acute exposure to DP are similar to in vivo animal experiments,which are specifically manifested as changes in the structure of the flora and increased metabolism of SCFAs.Subacute compound exposure to disinfection by-products also changed the results of the intestinal flora of the SHIME and caused a decline in the metabolism of SCFAs.Therefore,the methods for acute and subacute exposure of environmental pollutants to the SHIME in this study are feasible.