| Limosilactobacillus fermentum is gram-positive and is pervasive in environments such as fermented vegetable and meet material and the intestinal tract of humans and animals.L.fermentum was included in the Qualified Presumption of Safety(QPS)list of the European Food Safety Authority(EFSA)in 2009 and listed as a “generally recognized as safe”(GRAS)organism by the US Food and Drug Administration(FDA)in 2013.An increasing number of studies have indicated that L.fermentum can have a positive effect on the health of its host.It has been shown to antagonize pathogenic bacteria,improve intestinal dynamics,regulate lipid metabolism and have anti-oxidation and immunoregulatory roles in disease progression.To promote the application of L.fermentum in food fermentation and probiotics,it is necessary to further explore functional genes and the molecular evolution of L.fermentum strains.So far,genomes of L.fermentum strains can now be accessed from the National Coalition Building Institute database are little and these L.fermentum are mainly isolated from various fermented foods.Studies on the genetic differences of L.fermentum are still very few.Therefore,the objective of this study includes three parts.First,to develop a more selective medium for screening L.fermentum strains from human fecal samples and screen L.fermentum strains from human fecal samples from China.Second,to investigated the intestinal abundance of L.fermentum in human subjects from different regions in China through a unit-specific gene sequencing and Mi Seq high-throughput sequencing approach.Third,to evaluate the alleviating effects of L.fermentum with different genetic backgrounds on colitis and functional constipation,and candidate genes that may contribute to the probiotic effects of specific L.fermentum strains was identified.The main research results are as follows:An efficient screening method for L.fermentum in human intestinal tract was established.According to the literature reports over the years,22 strains of lactic acid bacteria(including Lactobacillus,Pediococcus and Weissella)were most common in the human intestinal tract and were the carbohydrate utilization and antibiotic sensitivity of them were tested.The result showed that the carbohydrate utilization capacity and antibiotic tolerance of varied lactic acid bacteria were different.L.fermentum strains could grow well in medium containing arabinose,mannose,trehalose,or ribose as the carbon source but not in medium containing xylose or cellobiose as the carbon source.L.fermentum is susceptible to clindamycin,inhibited at low concentrations of tetracycline,erythromycin,chloramphenicol,ampicillin,and penicillin G,and exhibits a high resistance to streptomycin gentamicin and vancomycin.Combined with the genotype and phenotype for carbohydrate metabolism,we found that L.fermentum can decompose arabinose into D-Ribulose-5P with genes for ara A.The streptomycin and gentamicin resistance of Lactobacillus showed a high spearman correlation coefficient with desR,tlrC,oleB,mdtD,mpr F,mur A and adeC.Therefore,arabinose was selected as the only carbon source in our optimized LAMVAB medium,and 64-mg/L gentamicin and 256-mg/L streptomycin were selected as the antibiotics for addition in the medium as they could inhibit most other bacterial communities.The final new medium was named Limosilactobacillus fermentum medium with arabinose and three antibiotics or,briefly,LFMATA.The rate of L.fermentum isolation on LFMATA(82%±2%)was significantly higher than that on the basic LAMVAB medium(6%±4%).The distribution of L.fermentum in intestinal tract of Chinese population was studied and the genetic differences of L.fermentum isolated from Chinese human gut were analyzed.The relative abundance of Lactobacillus in human fecal samples among different regions in China is detected through a unit-specific gene sequencing and Mi Seq high-throughput sequencing approach and a total of 51 Lactobacillus species were detected in 200 human fecal samples.Among the entire Lactobacillus flora of the human intestinal tract,the most abundant Lactobacillus are L.mucosae,L.ruminis,L.crispatus,L.gasseri,L.plantarum and L.delbrueckii.Although L.fermentum occurres very frequently in the human fecal samples(over 90%),its relative abundance in people is generally less than 1%.164 isolates of L.fermentum strains were separated from fecal samples of populations in different regions in China using LFMATA medium and their draft genomes were sequenced.Phylogenetic and genomic differences analyse of 224 L.fermentum strains(164 were isolated from the gut of humans in our lab and 60 were obtained online)are conducted.Result showed that these L.fermentum strains separated into six clades in phylogenetic tree and no significant adaptive evolution between the genomes of L.fermentum strains and the geographical location,sex and age of the hosts.However,L.fermentum isolated from different sources displayed a different evolution trend.High number of genes related to orthologous protein,carbohydrate metabolism and antibiotic resistance were variable in L.fermentum strains from different phylogenetic clades.The effect of L.fermentum with genetic differences on DSS induced colitis mice was evaluated and related functional genes were also analyzed.Four L.fermentum strains(XJ61,CECT5716,WX115 and GD121)from different branches of the phylogenetic tree were selected and their antiinflammatory activities in a dextran sulphate sodium(DSS)-induced colitis mouse model were further investigated.Both L.fermentum XJ61 and L.fermentum CECT5716 significantly alleviated UC by reducing all colitis-associated histological indices,maintaining mucosal integrity,and stimulating replenishment of short-chain fatty acids(SCFAs),while the other two strains failed to offer similar protection.The anti-inflammatory mechanisms of L.fermentum XJ61 and L.fermentum CECT5716 were related to the inhibition of nuclear factor kappa-B(NF-?B)signaling pathway activation.Comparative genomic analysis of these strains identified 11 candidate genes that may contribute to the anti-inflammatory effects of L.fermentum strains.Among these genes,gene for the synthesis of alcohol dehydrogenase(adh4)involved in the metabolism of alcohol to acetic acid is predicted to be involved in the protection effect of L.fermentum XJ61 on DSS induced colitis mice.Further research shows that when the gene adh4 of L.fermentum is knocked out,the alleviating effect of L.fermentum XJ61 on DSS induced colitis mice was significantly weakened.The effect of L.fermentum with genetic differences on alleviating loperamide induced constipation mice was evaluate and related functional genes were also analyzed.Five L.fermentum strains(XJ61,CECT5716,WX115,GD121 and YN54)from different branches of the phylogenetic tree were selected for the evaluation of their effects against constipation induced by loperamide in mice.L.fermentum YN54 significantly alleviated UC,while the other four L.fermentum strains failed to offer similar protection.Comparative genomic analysis of these strains identified 12 candidate genes that may contribute to the protective effects of L.fermentum strains on constipation.These specific genes can be divided into three categories according to their gene functions.Genes related to fatty acid hydrase,polysaccharide and cell membrane synthesis(function gene 1),genes related to inorganic ions,carbohydrate transport and metabolism(function gene 2)and genes related to function unknown,general or conserved(function gene 3)are classified.Further research shows that L.fermentum FJ12(with function gene 1 and 2)and L.fermentum GX51(with function gene 1 and without function gene 2)have protective effects on constipation mice,while L.fermentum ZH1010(with function gene 2 and without function gene 1)does not have protective effects on constipation mice,which indicate that genes for fatty acid hydrase,polysaccharides and cell membrane synthesis are high associated with the protective effects of L.fermentum on constipation mice. |
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