Isolation And Physiological Function Investigation Of Low-abundant Bacteroidales In Healthy Human Instestine

Bacteroidales is one of the core communities in the intestinal microbiota,which possesses considerate amount of genes encoding enzymes that are associated with carbohydrate utilizations and can be steadily switched according to the available carbon sources.Thus short-chain fatty acid and glycans are released during fermentation of resistant dietary fibers to facilitate the absorbance and utilization of nutrients,formation of mucosal vessels,maturation of immune system and maintenance of intestinal homeostasis.The syntrophic interactions and interference competition between Bacteroidales species contribute to better stability over Firmicutes.Bacteroidales species display anti-or pro-inflammatory functions to the host.However,due to the limitation of bacterial isolation method,there are more researches focused on the major Bacteroidales species.Thus this project is supposed to establish selective isolation methods for low-abundant Bacteroidales species from human intestine;to investigate the interactions of the low-abundant Bacteroidales species with the intestinal microbiota and the host using the model species of B.fragilis as control;and to explore the safety issues of low-abundant Bacteroides strains with potential beneficial functions as foundation for futher application.The results are summarized as follows:Two selective isolation methods for low-abundant Bacteroidales species were established based on regular Bacteroidales isolation medium according to the differentiation of carbohydrates utilization capabilities.The first method was to use medium with xylan(cf-xylan),rhanmnose(cf-rhamnose)or mannitol(cf-mannitol)as the sole carbon source for isolation.The results showed that all of the three medium were able to enrich the low-abundant species of Bacteroides ovatus.Furthermore,cf-xylan medium increased the isolation efficiency of Prevotella copri and B.xylanisolvens which was not detected by the traditional isolation medium.The second method was to incubate samples with medium using arabinose as the sole carbon source and supplemented with ampicillin(arab-amp)for 4 hours before traditional isolation,thus the low-abundant species which are not able to ferment arabinose,such as B.cellulosilyticus and B.fragilis,were enriched.B.ovatus was selected as the representative species for investigations on the interactions between low-abundant Bacteroidales and the intestinal microbiota based on healthy and inflamed mice models,B.fragilis was adopted as the reference species.The results revealed that the low-abundant species performed limited impact on gut microbiota,especially B.ovatus ELH-B2 displayed even less effects on the composition of gut microbiota and production of SCFA compared to B.fragilis.It is noteworthy that both B.ovatus and B.fragilis were able to recover the disturbed gut microbiota induced by LPS infection,particularly the tumorigenesis-related species were downregulated.B.fragilis HCK-B3decreased the production of some short-chain fatty acid.The NF-?B reporter cell line,healthy mice and inflamed mice models were used for the investigations of the interactions between the low-abundant Bacteroidales species and the host.The Bacteroidales strains exhibited differentiations of activation functio n on the NF-?B pathway of the Thp1-XBlue cells.B.ovatus displayed relatively weak activation on the NF-?B pathway and limited upregulation effects on the activated NF-?B pathway,while B.fragilis obviously enhanced the activated NF-?B pathway.B.ovatus ELH-B2 rarely influenced the cytokine production,Treg cells in spleen and NF-?B pathway activation in colon of healthy mice.B.fragilis HCK-B3 significantly upregulated the NF-?B pathway and increased the permeability of the colon in health mice,but ma intained the histological structure of the colon tissue.Bacteroides spp.was reported to be able to attenuate the disorders caused by pathogens and drugs like oxazolone,but the modification mechanism based on the LPS-induced inflammation remained unknown.Herein,B.ovatus ELH-B2 was able to restoring Treg/Th-17 balance of the LPS-inflammed mice through upregulating the number of splenic Treg cells.Additionally,B.fragilis HCK-B3 was capable of alleviate LPS infection via suppressing pro-inflammatory TNF-?and increasing anti-inflammatory IL-10.The increased intestinal permeability and the enhanced NF-?B signals of the LPS-infected mice without damaging the intestinal integrity by B.fragilis HCK-B3 was predicted to be the foundation of anti-inflammatory effects after passing through the peritoneum.Therefore,B.ovatus ELH-B2 and B.fragilis HCK-B3 could be considered as natural strains of next-generation probiotics due to the modulatory effects on the immune responses and disturbed gut microbiota of the LPS-inflamed mice.Safety evaluations of these two strains were conducted as basis for future industrial and clinical applications.Both strains were confirmed to be non-motile without flagella synthesized.B.fragilis HCK-B3 is negative for hemolysin,but B.ovatus ELH-B2 is slightly hemolytic.B.ovatus ELH-B2 and B.fragilis HCK-B3 is different from the existed Bacteroides strains after whole genome comparison,and neither the potential virulence genes nor pathogenecity islands have been identified.The critical genes for synthesis PSA are identified in B.fragilis HCK-B3 which is similar to the PSA-producing B.fragilis ATCC25285.Both Bacteroides strains are resistant to ampicillin,ceftriaxone,clindamycin,kanamycin,erythromycin and polymycin B,B.ovatus ELH-B2 is also resistant to ciprofloxacin and vancomycin.The antibiotic resistance features can hardly be transferred to other intestinal microorganisms as no plasmids or mobility-related genomic islands are identified.No intracorporal pathogenic properties of B.ovatus ELH-B2 and B.fragilis HCK-B3 were discovered in healthy mice.Immunosuppressed mice models were adopted for further safety assessment.Cyclophosphamide injection led to immuno-deficient symptoms of body weight loss,the reduction of immunocytes,platelets and hemoglobin concentration,and the destruction of spleen.However,daily dose of 10~9 cfu of B.ovatus ELH-B2 and B.fragilis HCK-B3 rarely impacted the body weight,hematological and liver parameters,cytokine production and the histological structure of liver,spleen and colon in the immunosuppressed mice,but partially restored the biochemical indicators.Anti-inflammatory cytokines were upregulated in the immunosuppressed mice intervened by B.fragilis HCK-B3.