Isolation And Identification Of Desulfovibrio From Human Gut And Metagenomic Study Of Sulfate-reducing Bacteria Community

Sulfate-reducing bacteria(SRB)widely exist in anaerobic environments and obtain energy through degradation of organic matters.In addition,SRB are able to reduce sulfate to sulfide and therefore become the major H₂S-producing bacteria in intestine,which play an important role in regulating intestinal homeostasis and inflammation.Desulfovibrio(DSV)is the dominant genus of SRB in human intestine.Here,DSVs were isolated and identified from the intestines of 6 healthy volunteers(HV)and 6 patients with liver cirrhosis(LC).The desulfurization capability,H₂S production capacity,intestinal colonization capability and antibiotic sensitivity of the isolates were compared.Furthermore,the effect of different neutraceuticals on the growth and desulfurization capability of Desulfovibrio desulfuricans ATCC 29577 were studied.Lastly,the metagenomic study of SRB communities from HV was preliminarily performed.The main work is as follows:(1)Isolation and identification of DSVs from different populations.The desulfurization capability,H₂S production capacity,intestinal colonization capability and antibiotic sensitivity of the isolates were compared.53 DSV isolates were obtained from HV and 11 DSV isolates were obtained from patients with LC.The DSV isolates from patients with LC were all identified as D.desulfuricans.The DSV isolates from HV were mainly D.desulfuricans which accounts for 54%of the total,followed by D.intestinalis(15%),D.simplex(11%),D.piger(9%),D.legallii(5%),D.oxamicus(5%)and D.fairfieldensis(1%).Evaluation of desulfurization capability and H₂S production capacity of DSVs from HV showed that D.fairfieldensis(JN-SRB-45)and D.piger(JN-SRB-12,18,20,21,44)had the highest desulfurization capability and H₂S production capacity,followed by D.intestinalis(JN-SRB-3,4,7,9,23,37,49,50);in addition,D.legallii(JN-SRB-32,40)and D.oxamicus(JN-SRB-5,14)had the lowest desulfurization capability and H₂S production capacity.The same studies conducted on DSVs from patients with LC demonstrated that the desulfurization capability and H₂S production capacity of DSVs from patients with LC was stronger than that of DSVs from HV.Evaluation of intestinal colonization showed that in DSVs from HV,D.fairfieldensis and D.piger were more tolerant to simulated intestinal fluid(SIF)and simulated gastric fluid(GIF)compared with other isolates.There was no difference in tolerance to SIF and GIF between DSVs isolated from patients with LC.While DSVs from patients with LC were more tolerant to SIF than DSVs from HV,which may indicate they can colonize and proliferate in the intestinal tract.Study on the antibiotic sensitivity demonstrated that DSVs were highly sensitive to metronidazole and kanamycin sulfate.In addition,DSVs except D.desulfuricans and D.fairfieldensis were highly sensitive to ampicillin.(2)Effects of neutraceuticals on D.desulfuricans ATCC 29577.The results demonstrated that xylo-oligosaccharide,mannan-oligosaccharide and chitosan-oligosaccharide inhibited the growth of D.desulfuricans ATCC 29577 in a dose-dependent manner.Xylo-oligosaccharide and mannan-oligosaccharide significantly inhibited the growth of D.desulfuricans ATCC29577 at dose of 1000-2000?g/m L and 500-2000?g/m L,respectively(^***p<0.001).Chitosan-oligosaccharide extremely inhibited the growth of D.desulfuricans ATCC 29577 at dose of 500-2000?g/m L.However,the inhibitory effect was weakened at 250?g/m L(^***p<0.001)and 125?g/m L(^**p<0.01).The fermentation filtrate of Paecilomyces hepiali inhibited the growth of D.desulfuricans ATCC 29577 at dose of 500-2000?g/m L in a dose-dependent manner.(3)Metagenomic analysis of SRB communities.SRB communities in vitro were obtained after passaging the bacteria isolated from treated fecal samples from HV for 10generations using DSV-selective culture media.Results of the metagenomic analysis showed that the microbial composition of the SRB communities(n=5)were similar.Proteobacteria,Bacteroidetes and Fusobacteria were the dominated phyla;Citrobacter,Escherichia,Desulfovibrio,Parabacterioides and Fusobacterium were the dominated genera.The mutual microorganisms in 5 sample communities accounted for 77.78%.Gene prediction of SRB communities demonstrated an average of 19154 genes.Referring to the GO database and COG database annotation,the genes and proteins with high abundance were mainly involved in amino acid transport and metabolism,carbohydrate transport and metabolism,energy generation and transformation,and inorganic ion transport and metabolism.There was no significant difference between communities.The analysis results revealed that the SRB community isolated from intestinal tract in this experiment gradually formed a stable community after serially passaging in vitro,which provided a theoretical basis for the subsequent study of SRBs at the community level.