Polyphasic Identification Of 7 Novel Species Of Lactic Acid Bacteria And Reclassification Of Enterococcus Xinjiangensis

There are many kinds of natural bacteria in the traditional fermented pickles products of Northeast region,the intestinal contents of bees and the yoghourt of Tibet Autonomous Region,PR China,which can be used as the source of isolating and screening the excellent lactic acid bacteria species.In this paper,the research status of isolating and screening excellent strains f rom different habitats at home and abroad was reviewed,and the application prospect of these strains as the starter and the disease resistance factors to prevent the foulbrood of honeybee larvae in Europe and the United States was prospected.In this stud y,five potential new species of lactic acid bacteria isolated from Northeast pickles,one potential new species of lactic acid bacteria isolated from Tibet yoghurt and one potential new species of lactic acid bacteria isolated from bee gastrointestinal tract were analyzed by a polyphasic approach.The aim is to identify their species and determine their taxonomic status.The polyphasic classification approach used in this study,including 16S r RNA gene sequence analysis and sequence analysis of three housekeeper genes(phe S gene,rpo A gene,rec A gene),determination of DNA G+C content,Average Nucleotide Identity(ANI)analysis,in digital DNA–DNA hybridisation(d DDH),fatty acid methyl ester(FAME)analysis and an analysis of phenotypic features.Five potential new species of lactic acid bacteria were isolated from rural households in Harbin,Heilongjiang Province,China.Strains 241-2-2^T,63-4^T and 190-7^T were closely related to the type strains of E.devriesei,E.viikkiensis,E.pseudoavium,E.xiangfangensis,E.avium,E.malodoratus,E.raffinosus and E.gilvus,having 99.1?99.9%16S r RNA gene sequence similarities,81.8?90.3%phe S gene sequence similarities,94.2?98.9%rpo A gene sequence similarities.Strain 94-2^T was distantly related to the type strains of E.phoeniculicola,E.rivorum and E.faecalis,having 95.2?96.1%16S r RNA gene sequence similarities,76.5?78.9%phe S gene sequence similarities,80.9?82.2%rpo A gene sequence similarities.Strain 85-4^T was distantly related to the type strains of E.casseliflavus,E.gallinarum,E.dispar,E.canintestini,E.saigonensis,E.diestrammenae,E.asini,E.cecorum and E.columbae,having 95.7?97.8%16S r RNA gene sequence similarities,76.2?84.7%phe S gene sequence similarities,85.9?87.6%rpo A gene sequence similarities.Less than 91.5%ANI and 45.3%d DDH values between strains241-2-2^T,63-4^T,190-7^T,94-2^T,85-4^T and type strains of phylogenetically related species showed that they represent five new species within the genus Enterococcus.Three strains of lactic acid bacteria,designated X0750^T,X0278 and X0401,isolated from traditional yogurt in Tibet Autonomous Region,PR China.Strin X0750^T was identical to strains X0401 and X0278 by MEGA 5.0 alignment,and the 16S r RNA and phe S genes were found to be similar and in the same phylogenetic position.The results showed that strains X0750^T,X0401 and X0278 were the same new species of lactic acid bacteria.Strain X0750^T was phylogenetically related to the type strains of W.hellenica,W.bombi,W.paramesenteroides,W.jogaejeotgali,W.thailandensis,W.oryzae,W.cibaria and W.confusa,having 94.4?100%16S r RNA gene sequence similarities,76.7?90.0%phe S gene sequence similarities,and 77.6?92.8%rec A gene sequence similarities,respectively.ANI and d DDH values between strain X0750^T and type strains of phylogenetically related species were less than 90.4%and 40.9%respectively,confirming that strain X0750^T represents a novel species within the genus Weissella.A potential new species of lactic acid bacteria,F79-211-2^T,was isolated from the gastrointestinal tract of bees in a beekeeping farm in Yichang,Hubei Province,China.Strain F79-211-2^T was phylogenetically related to the type strains of S.hailunensis,S.silagei,S.pentosiphilus,S.mixtipabuli,S.silagincola,S.similis and S.odoratitofui,having 99.1?99.6%16S r RNA gene sequence similarities,85.3?87.9%phe S gene sequence similarities,87.4?96.8%rpo A gene sequence similarities.Less than 83.2%ANI and 26.1%d DDH values between st rain F79-211-2^Tand type strains of phylogenetical related species showed that strain F79-211-2^Trepresents a new species within the genus Secundilactobacillus.The DNA G+C mol%of strains 241-2-2^T,63-4^T,190-7^T,94-2^T,85-4^T,X0750^T and F79-211-2^T were 40.1%,37.8%,40.3%,41.4%,39.7%,36.7%and 44.2%,respectively.The main fatty acids of strains 241-2-2^T,63-4^T,190-7^T and X0750^T were C_16:0,C_18:1?9c and Sum of C_19:1?6c/C_19:0 cyclo?10c.The main fatty acids of strain 94-2^T were C_16:0 and C_18:1?9c.The main fatty acids of strain 85-4^T are C_16:0,C_18:1?9c and Sum of C_18:1?6c/C_18:1?7c.The main fatty acids of strain F79-211-2^T are C_16:0,C_18:1?9c and Sum of C_19:0?6/unknown 18.846/C_19:0cyclo?10c.Seven new species of lactic acid bacteria were identified based upon the polyphasic classification approach in the study,and their taxonomic status was determined.Seven novel species were named as follows 241-2-2^T(Enterococcus pingfangensis sp.nov.),63-4^T(Enterococcus dongliensis sp.nov.),190-7^T(Enterococcus hulanensis sp.nov.),94-2^T(Enterococcus nangangensis sp.nov.),85-4^T(Enterococcus songbeiensis sp.nov.),X0750^T(Weissella sagaensis sp.nov.)and F79-211-2^T(Secundilactobacillus yichangensis sp.nov.).In addition,this study re-evaluated the relationship between E.lactis and E.xinjiangensis.In the 16S r RNA gene phylogeny,E.xinjiangensis was closely related to E.lactis and E.faecium.E.xinjiangensis and phylogenetically related to the type strain of E.lactis,having 99.8%16S r RNA gene sequence similarity,98.5%phe S sequence similarity,99.9%rpo A sequence similarity,98.7%average nucleotide identity value and 88.9%digital DNA-DNA hybridization values between E.xinjiangensis and phylogenetically related to the type strain of E.lactis showed that E.xinjiangensis and E.lactis represent the same species.Meanwhile,the type strains of E.xinjiangensis was phylogenetically related to the type strain of E.faecium,having 99.8%16S r RNA gene sequence similarity,97.1%phe S sequence similarity and 99.6%rpo A sequence similarity,94.7%ANI and 59.1%d DDH values between E.xinjiangensis and phylogenetically related to the type strain of E.faecium indicated that they represent two different species.On the basis of the results present here,we propose Enterococcus xinjiangensis Ren et al.2020 as a heterotypic synonym of Enterococcus lactis Morandi et al.2012.