Isolation And Identification Of Lactic Acid Bacteria From Breast Milk And Polyphasic Taxonomy Of New Species Of Streptococcus

In this thesis,the microbial diversity of 12 breast milk samples collected from Hulunbuir area was studied by pure culture method and 16S r RNA gene sequence,and three suspected new species（IMAU99125,IMAU99674,and IMAU99199）were identified for acaurate taxonomic status.The results of the study were as follows:A total of 191 strains of lactic acid bacteria were isolated from 12 breast milk samples,which were divided into 5 genera and 9 species by 16S r RNA gene sequence alignment.The dominant bacterial genus was Staphylococcus,and the dominant bacterial species was Staphylococcus epidermidis,accounting for 35.11%of the flora structure.There were 3strains were identified as uncultured biological clones.The results of 16S r RNA gene sequence,housekeeping genes（gyr B,rpo B）and whole genome phylogenetic tree showed that the three suspected new species of closely related bacteria were Streptococcus mitis ATCC 49456^T and Streptococcus pseudopneumoniae ATCC BAA-960^T,in which IMAU99125 and IMAU99674 were clustered in the same species with 99.93%homology,and the homology of IMAU99199 with IMAU99125 and IMAU99674 is 97.00%.The full-length genomes of IMAU99125,IMAU99674 and IMAU99199 were 2027143 bp,2007344 bp and 2003636 bp,respectively.The DNA G+C%contents were 40.04 mol%,39.95 mol%and 39.83 mol%,which were consistent with the DNA G+C%of Streptococcus range of 33.79 mol%-43.40 mol%.The ANI of IMAU99125 and IMAU99674 were 99.98%and d DDH was 99.70%.The ANI of IMAU99199 about IMAU99125 and IMAU99674 were 94.03%and 94.02%,respectively.The d DDH values were 55.40%and 55.50%,which were lower than the identification thresholds at the bacterial species（ANI>95%,d DDH>70%）.The ANI values of IMAU99125,IMAU99674 and IMAU99199 for Streptococcus mitis ATCC 49456^T and Streptococcus pseudopneumoniae ATCC BAA-960^T were 93.28%and 92.74%,93.28%and 92.73%,92.92%and 92.44%respectively,d DDH values were 53.30%and 50.30%,53.10%and 50.20%,52.50%and 49.80%,respectively,which were far below the threshold for bacterial species-level identification.Physiological and biochemical reaction results showed that IMAU99125,IMAU99674 and IMAU99199 were Gram-positive,catalase-negative,α-hemolytic Streptococci with paired or chain-arranged cells.The main fatty acid are C₁₆,without ubiquinone and menaquinone.IMAU99125 and IMAU99674 were contain a small amount of diphosphatidylglycerol（DPG）and unidentified Glycolipids（GL1-3）,but IMAU99199wsa traces of diphosphatidylglycerol（DPG）,phosphatidylglycerol（PG）,phosphatidylethanolamine（PE）,unknown aminoglycolipid（AGL）and unknown Glycolipid（GL1）.In addition,the physiological and biochemical reaction results of these three strains were significantly different from other closely related Streptococcus species.In conclusion,through multiple taxonomic studies,IMAU99125,IMAU99674 and IMAU99199 are new species of Streptococcus.IMAU99125 and IMAU99674 were a new species of Streptococcus,named Streptococcus humanilactis sp.nov.,and the type strain is IMAU99125^T（=GDMCC 1.1876^T=KCTC 21157^T）.The optimal growth temperature of the strain is 37°C,it was not tolerant to Na Cl,can grow in liquid medium with p H value of6.0-8.0.IMAU99199 is another new species of Streptococcus,named Streptococcus hohhotenis sp.nov.,and the type strain is IMAU99199^T（=GDMCC 1.1874^T=KCTC21155^T）.The strain grew better in anaerobic conditions and could grow in liquid medium containing 2%（w/v）Na Cl.The reaction withβ-galactosidase,α-glucosidase and N-acetyl-glucosaminidase was positive.