Quantification, Isolation And Identification Of Srb In The Gut Of Obese Rodents Induced By High Fat Diet

Obesity is a medical condition in which excess body fat hasaccumulated to the extent that it may have an adverse effect on health,leading to reduced life expectancy and/or increased healthy problems.Overweight and obesity are the sixth potential healthy risk contributing tothe burden of disease worldwide. Obesity is associated with variousdisease, such as cardiovascular disease, type2diabetes and coloncarcinoma. According to the World Health Organization statistics, morethan1.4billion adults were overweight by2008and whithin which over500million were obese. WHO predicted there will be2.3billion adultsoverweighted by2015,7million of them will be obese.Recently, accumulating research findings suggest that the change ofgut microbiota populations correlates with obesity. Sulfate-reducingbacteria (SRB) is one member of gut microbiota in human and animals, itis a group of anaerobic microorganisms that use organic/inorganiccompounds as electron donor to reduce sulfate and produce sulfide.Hydrogen sulfide is a cytotoxic compound which leads to impairment ordeath of colonic epithelial cells, and thus consequently causes chronicinflammation. And Lipopolysaccharides (LPS), which are produced bygram-negative SRB, are a motivation of obesity. Thus, it is worthy to studySRB in the gut of experimental mice and rats.In this stude, first, real-time qPCR was employed to quantify thenumber of SRB in gut of fat mice and rats using aprA gene as a molecularmarker.20Wistar rats were divided into2groups randomly. One groupwas fed with normal chow died (NCD), the other was fed with high-fat diet (HFD). Fecal samples and bodyweight data were collected at0th,8thand18thweek.24C57BL/6J mice were also divided into2groups randomly.One group was fed with NCD, the other was fed with HFD. Fecal samplesand bodyweight data were collected at0th,6thand16thweek. Afterquantification of SRB in all fecal samples and statistics of bodyweight andquantification data, we found the body weight gain of HFD group animalswas significantly higher than the ones fed with NCD. At the same time, thenumber and ratio of SRB in the gut of HFD group were significantlyincreased along with increased body weight. This research suggested theassociation between SRB and obesity.To continue a further study on the function of SRB in the gut, SRBwere isolated and identified from the gut of NCD and HFD feeding mice.149SRB isolates were picked out from the gut of healthy and fatC57BL/6J mice. Amplified Ribosomal DNA Restriction Analysis(ARDRA) and Enterobacterial Repetitive Intergenic Consensus SequencePCR (ERIC-PCR) fingerprint were employed to study the diversity ofisolates. Only one type of ARDRA pattern was detected. ERIC-PCRfingerprints divided149isolates into7types.16S rRNA gene sequencingand phylogenetic analyses using ARB program package of7ERIC typicalstrains showed they are all homologous to Desulfovibrio.To investigate if SRB can lead to obesity of animals, an inoculationexperiment (inoculating SRB into animal gut) is necessary. In order tolabel the exogenous SRB strain, FB8, a strain isolated from fat mice gut,was induced to resist rifampicin. Finally,3rifampicin resistant mutantswere obtained.In summary, to study the relationship between SRB and Obesity,real-time qPCR was firstly employed to quantify the change of SRB in gutof fat mice and rats. The number and ratio of SRB in the gut of fat rats andmice were significantly increased along with increased body weight. Itsuggested correlation between SRB and obesity. Then, in order to conductthe SRB inoculation experiment (inoculating SRB into animal gut) to investigate if SRB can lead to obesity of animals,149SRB isolates wereisolated from the gut of C57BL/6J mice and one was induced to resistrifampicin. These results provide a basis for animal experiment.