The Microbial Identification Of Phocaeicola Sp.FXJYN30E22 And The Evaluation Of Its Effects Against Colitis In Mice

Bacteroidaceae,mainly composed of Bacteroides and Phocaeicola,is a type of symbiotic bacteria widely presenting in the intestines of humans and animals,and play an important role in the maintenance of host health.Many studies have shown that there was a close interaction between Bacteroidaceae related microorganisms and intestinal microbiota,intestinal immune,and intestinal barrier function in the host.Therefore,Bacteroidaceae has the potential to be the next generation of probiotics.To date,52 species of Bacteroidaceae have been isolated and identified.Although most species of Bacteroidaceae had been identified before 2010,six species have been discovered successively in the past five years.In the process of Bacteroidaceae screening in fecal samples of Chinese population,a strain named FXJYN30E22 belonging to Phocaeicola genus was isolated in our research group.The 16 S r RNA sequencing results revealed that the 16 S r RNA sequence similarity between the strain FXJYN30E22 and its closest recognized species was only 95.37%,which was much lower than the threshold of species classification based on 16 S r RNA sequence(98.7%).This result indicated that this strain might be a potential new species of Phocaeicola genus.For further research and application,we conducted the microbial taxonomic study to identify the taxonomic status of this strain,and further analyzed its role in intestinal immune regulation.First of all,the genome information of the strain FXJYN30E22 and 11 type strains of Phocaeicola species was compared by comparative genomics analyses,including the G+C contents,digital DNA-DNA hybridization(d DDH),average nucleotide identity(ANI),COG functional genes and carbohydrate-active enzyme genes.Our results showed that d DDH and ANI between strain FXJYN30E22 and its closest recognized species was only 41.9% and91.1%,respectively,which was far below the threshold of species classification according to d DDH(70%)and ANI(95%).These results provided evidence for the status of the strain FXJYN30E22 as a new species of the genus Phocaeicola.Next,we analyzed the distribution of the strain FXJYN30E22 in the intestine of healthy human from different countries or with different diet compositions based on metagenomic data,and the results showed that the abundance of this strain in the intestines of the Chinese population was significantly higher than that of the European population.This suggested that there was host differences in the distribution of strain FXJYN30E22 in the intestine of healthy people.On the basis of genome comparison results,the taxonimic status of FXJYN30E22 was further determined by the analyses of phylogenetics,morphology characteristics,physiological and biochemical characteristics,and chemical characteristics.First,the phylogenetic tree were constructed based on 16 S r RNA sequence or orthologous genes,and we determined that Phocaeicola massiliensis(P.massiliensis)JCM 13223 has the closest genetic relationship with the strain FXJYN30E22.Then,the physiological,biochemical and chemical characteristics of the above two strains were detected.The results revealed that there were differences between the strain FXJYN30E22 and P.massiliensis JCM 13223 in the characteristics including gelatin hydrolysis,activities of histidine arylamidase,tyrosine arylamidase and glycine arylamidase,and the major cellular fatty acids composition.Based on the above results,we determined and proposed FXJYN30E22 as a new species of Phocaeicola genus.The effect of the strain FXJYN30E22 on the intestinal immunoregulation of mice was further analyzed by constructing a mouse model of ulcerative colitis.Bacteroides fragilis(B.fragilis)NCTC 9343,which was proved to relieve the enteritis,was selected to be the reference strain of efficacy evaluation on colitis.Our results showed that strain FXJYN30E22 could alleviate colitis,manifested as restored body weight,reduced disease activity index(DAI),increased colon length,attenuated damage of colon tissue,enhanced intestinal barrier integrity and reduced levels of pro-inflammatory factors.Moreover,these protective roles of the strain FXJYN30E22 on alleviating colitis were close to that of B.fragilis NCTC 9343.In addition,further analyses based on short-chain fatty acids(SCFAs)and microbiota composition in intestine revealed that the beneficial effects of the strain FXJYN30E22 might attribute to its ability to increase the concentrations of acetate and butyrate and decrease the abundance of Alistipes and Butyricimonas.To sum up,with the combination of comparative genomics analysis,phylogenetic analysis,morphological analysis,physiological and biochemical characteristics analysis and chemical characteristics analysis,we proved that the strain FXJYN30E22 was a new species of Phocaeicola genus.Based on in vivo colitis model,we found that this strain could protect mice against colitis by regulating the intestinal microbiota,increasing the production of SCFAs,promoting the integrity of the intestinal barrier and inhibiting the inflammatory response.