Genomic Characterization Of Enterococcus Hirae From Tibetan Gazelle And Identification Of New Species In Genus Lactobacillus From Marmota Himalayan In Qinghai-Tibet Plateau

ObjectiveThe research focuses on the theme of"pathogens of emerging infectious diseases carried by wild animals".Among the emerging infectious diseases in today's society,some pathogens come from wild animals,especially wild animals in the special geographical environment and climate such as the plateau and the tropics.These wild animals may carry unknown pathogens,which could cause severe public health issues if they infect and spread among human.The study of unknown bacteria and pathogens from wild animal is not only conducive to discovering new pathogens and studying the transmission routes and transmission mechanisms of animal infectious sources,but also helpful to broaden our cognizance on the diversity of wild animal intestinal flora on the Qinghai-Tibet Plateau,which is an important bacterial resource.Based on the above reasons,the genomic analysis of Enterococcus hirae isolated from Tibetan gazelle was carried out in this research to study its potential transmission capacity and transmission mechanism in the population,as well as to identify and name the Lactobacillus novel species isolated from Marmota Himalayan on the Qinghai-Tibet Plateau.Methods1.Using the selective medium KF streptococcus medium to isolate the enterococci from Tibetan gazelle.The enterococci were identified by biochemical identification,16S rDNA and rpoA gene sequence analyses.K-B paper method was used to antibiotics susceptibility test.Genomic characterization was applied by using COGs,SwissProt,CARD and VFDB databases.MUMmer and Tree Best software were used to construct the SNP phylogenetic tree.2.Two suspected new species of genus Lactobacillus were isolated on Colombian blood medium,and the identification and taxonomic status were determined by phylogenetic analyses,morphology,physiological biochemistry and cellular chemical composition analysis.Results1.Combined biochemical analysis,16S rDNA,rpoA gene sequence and genome hybridization,two strains isolated from 15 fecal samples of Tibetan gazelle were identified as E.hirae.The results of antibiotics susceptibility tests showed that two strains of E.hirae were not resistant to 13 commonly clinical antibiotics.Genomic analysis exhibited no antibiotics resistance genes existed in the two strains of E.hirae,but they contained a polysaccharide capsular gene cluster composed of 11 genes.The analysis of SNP phylogenetic tree showed that the two strains of E.hirae were the most closely related to animal-derived strains,but they belonged to two branches,respectively.2.The two strains appeared white,rough,irregular,round,convex colonies on the Columbia blood medium.Under optical microscope and transmission electron microscope,cells presented gram-strained positive,long-rod shape.Strain HT111-2^T 16S rDNA sequence(1546 bp)had the highest similarity with Lactobacillus hamsteri JCM 6256^T(97.3%)and Lactobacillus amylolyticus DSM11664^T(97.2%),and the similarity of 16S rDNA sequence between HT111-2^Tand HT170-2 was 99.0%.The similarities with L.amylolyticus DSM 11664^T and L.hamsteri JCM 6256^T rpoA and pheS gene were 91.4%,92.4%,82.3%and84.2%,respectively.Phylogenetic analyses of 16S rDNA,rpoA and pheS all showed that HT111-2^T and HT170-2 belonged to genus Lactobacillus.The genome hybridization(DDH)values of HT111-2^T with all known species of genus Lactobacillus were below the threshold 70%,and the DDH value of HT111-2^Tand HT170-2 was 99.2%.The G+C contents of HT111-2^T and HT170-2 were38.80%.The main fatty acids of two strain above were C_16:0?C_18:0?C_18:1?9c.The main amino acids of peptidoglycan were alanine(Ala),lysine(Lys),glutamate(Glu)and aspartic acid(Asp).The genome sizes of HT111-2^T and HT170-2 were 2,215,750 bp and 2,286,450 bp,respectively,with 2,082 and 2,145encoding genes.The results of amino acid sequence encoding by genes blast with COG and KEGG databases show that the genes and pathway of strain HT111-2^Tenriched in translation,ribosomal structure and biogenesis and metabolism of cell life.By amino acid sequence comparison with NR database,it was found that six genes within HT111-2^T genome related to lactic acid producing process,and five genes related to bacterial production with one gene related to cholylglycine hydrolase.Based on the analysis results of morphology,biochemistry and phylogenetics,strains HT111-2^T and HT170-2 were identified as novel species of genus Lactobacillus,named Lactobacillus xujianguonis,with the type strain was HT111-2^T.Conclusion1.Two strains of E.hirae isolated from Tibetan gazelle which are opportunistic pathogen were different from the ones isolated from clinical environment.Two strains of E.hirae are not resistant to 13 antibiotics,and possess a polysaccharide capsule gene cluster composed of 11 genes.2.Strains HT111-2^T and HT170-2 were identified as a novel species of genus Lactobacillus,named Lactobacillus xujianguonis,with the type strain was HT111-2^T.The genome of strains above comprised a bile acid hydrolase(BSH)gene and several bacteriocin-related genes,and six genes related to lactic acid producing.The two strains can produce lactic acid,and their biological values were needed to be explored.