Analysis Of The Diversity Of Sulfate Reducing Bacteria In Gut

Sulfate reducing bacteria （SRB）are those microorganisms that reduce sulfate(SO₂^-4) to hydrogen sulfide （H2S） while obtaining energy by oxidizing organiccompounds or molecular hydrogen （H2）. SRB not only inhibit butyrate production butalso consume butyrate, besides they inhibit butyrate oxidation by the colon cells. SRBproduce H2S which can regulate insulin release, inhibit mitochondrial cytochrome coxidase, induce DNA and cell damage. The lipopolysaccharide （LPS） of gram-negativeSRB displayed greater biological potency in inducing proinflammatory cytokinesecretion. These suggest that SRB might be a kind of functional gut bacteria whichhave effect on the health of their hosts. Other researches showed that SRB wereassociated with colitis, colon cancer, irritable bowel syndrome, metabolism syndromeetc.In first part of this thesis, a group-specific PCR-based DGGE method foranalyzing sulfate-reducing bacteria in human gut was developed. Two primer sets（AprA-3-FW/APS-RV and AprA-1-FW/AprA-5-RV） based onadenosine-5’-phosphosulfate reductase alpha subunit gene （aprA） were selected. TheaprA sequences of thirteen bacteria from the family Desulfovibrionaceae weredownloaded from GenBank. Clustal X and Simulated PCR（SPCR）programs were usedto compare the specificity of two primer sets targeted SRB in feces. Analysis of ClustalX and SPCR both implied the primer set AprA-3-FW/APS-RV was better thanAprA-1-FW/AprA-5-RV. The results of real PCR reaction also showed low efficiencyand non-specific amplification of primer AprA-1-FW/AprA-5-RV. DGGE system wasdeveloped and the fingerprints of10human individuals suggested that each individualharbored1-5SRB phylotypes. The results showed that DGGE targeted aprA gene is aneffective technique for analyzing the composition of SRB in human gut. In second part of this thesis, SRB strains were intended to isolate from humanintestinal samples and the diversity of SRB in these samples were also analyzed. Freshfecal samples of one patient with type2diabetes （D）, one healthy elder and one healthyyoung adult （H） were collected. By using anaerobe pure culture technique,150SRBisolates were obtained from the diabetic patient and were classed to4ERIC types.96SRB isolates were obtained from the healthy elder and were classed to2ERIC types.No SRB isolates were obtained from the healthy young adult. All isolates wereidentified as Desulfovibrio according to full-length16S rRNA gene sequence andpartial aprA gene sequence. This research also analyzed the diversity of SRB in thethree subjects’ gut using DGGE and clone library profiling techniques based on aprAgene. The results showed that strains isolated from the diabetes have99%similaritywith D. intestinalis and are the dominant type of SRB in the diabetic gut. Strainsisolated from the healthy elderhave99%similarity with D. desulfuricans and are maintype of SRB in the healthy elder gut. Although no SRB strain was isolated from thehealthy young adult, D. desulfuricans were detected in this sample by moleculartechniques. Compared with pure culture technique, DGGE and clone library techniquesare more efficient to analyze the diversity of SRB.In conclusion, a SRB group-specific PCR-based DGGE technique was developedand used to analyze the diversity of SRB in human gut combining with anaerobe pureculture and clone library techniques. The results showed that each individual harbored3-6SRB phylotypes. The dominant SRB strains in a diabetes and a healthy elder wereobtained. The study provided a method to analyze SRB structure in human gut and laidfoundations to further study the physiological characteristics and its relationship withdiabetes of SRB.