Isolation And Identification Of A Gut Commensal Bacteria Romboutsia Lituseburensis Sp. Nov. And Its Function In Glycometabolism Of Tilapia

As one of the three essential nutrients,carbohydrate is a crucial source of energy of fish feed.However,fish have limited capability to utilize carbohydrates compared to mammals.How to improve the capability of fish to use carbohydrates has become an important area of study in fish nutrition.Gut microbiota plays an important role in carbohydrate metabolism.Romboutsia,as gut commensal bacteria,can widely utilize carbohydrates.The main research contents were as follows:(1)The 16 S r RNA gene sequencing technique was used to investigate the intestinal flora structure of tilapia,especially on the genus Romboutsia.(2)We isolated a strain of Romboutsia from healthy tilapia intestines.Its physiological and biochemical characteristics were analyzed.(3)The whole genome sequencing of the isolate was performed,and its carbohydrate metabolism-related genes were identified.(4)The content of short-chain fatty acids(SCFAs)and carbohydrate in feed fermented with strain MY01 was analyzed.Tilapia were cultured with feed fermented with MY01 for a month to study its effect on growth and physiology of tilapia.The results were as follows:(1)The intestinal flora of11 days post-fertilization(dpf)tilapia was dominated by Gemmata.When tilapia was fed with artemia,the genus Gemmobacter and Cetobacterium were the dominant bacteria of tilapia intestinal flora.When tilapia was fed with plant-based diet,the genus Cetobacterium were the dominant bacteria and the relative abundance of the genus Romboutsia increased to 9.27-9.90% in tilapia gut.(2)A strain of the genus Romboutsia was isolated from the intestine of tilapia It was designated as MY01.The colony morphology of MY01 on the culture medium was circle,flat,white or light grey,translucent with smooth and shiny surface,1-2 mm in diameter,gram positive,and obligately anaerobic.The cells are obligately anaerobic,short rods and 3.5-3.8μm in size.MY01 was positive in urease reaction and gelatin test,but negative in catalase reaction and indole production.It could utilize D-glucose and D-sucrose but could not use D-mannitol and D-lactose.(3)The MY01 had a size of 3,925,608 bp genome,consisted of 6 contigs and had a GC content of 28.4%.The number of CDS(Coding sequence),r RNA genes,t RNA genes and sn RNA and si RNA was 3659,59,119,18,respectively.Two CRISPRs were predicted and genomic islands were not predicted.The number of genes were 2927,2089,2683,1705 and 3534 which were annotated by Pfam,GO,COG,KEGG and Refseq databases,respectively.Furthermore,ANI and DDH were calculated by comparing MY01 to other Romboutsia strains.The results showed that MY01 was a novel candidate species of Romboutsia.(4)The content of isobutyric acid,isovaleric acid and acetic acid in feed fermented with MY01 was significantly higher than in the control(P<0.05).The content of mannose,glucose,galactose,arabidose and fucose in feed fermented with MY01 were obviously lower than in the control(P<0.05).(5)Feed fermented with MY01 significantly increased tilapia condition factor(P<0.05),reduced blood sugar content of tilapia(P<0.05)and increased insulin content(P<0.05).In summary,a novel species of the genus Romboutsia,designated as MY01,was isolated and identified in the present study.Whole genome sequencing of MY01 was carried out.Its role in glycometabolism of tilapia was investigated.The result showed MY01 consumed carbohydrate and generated SCFAs during fermentation.The feed fermented with MY01 promoted glucose utilization in tilapia.The research provided a foundation for the further application of Romboutsia in aquaculture.