Two New Species In Genus Neisseria Isolated From Intestional Contents Of Plateau Pika

Six hundred and seven strains were isolated from intestional eontents of Plateau Pika,which were identified into 19 genera,31 species.One hundred and sixty-six strains could not be identified by the existing methods.There are fifty-nine strains whose identity of full 16S rDNA lower than 98.7%may be new species including Arthrobacter,Bacillus,Bordetella,Microbacterium,Neisseria,Paenibacillus and Rhodococcus.Two new species were successfully obtained,which belonged to Neisseria whose number are 10009,10011,10022T and 10010,10023T.Through biochemical analysis,morphological observation,culture characteristics,16S rRNA sequence analysis,genome hybridization,rplF gene sequence analysis,ribosomal protein gene multi-locus sequence typing(rMLST),core-genome multi-locus sequence typing(cgMLST)and determination of fatty acids,ultimately our five strains were determined two new species belonging to Neisseria whose name Neisseria weixii sp.nov(10009,10011,10022T)and Neisseria chenwenguii sp.now(10010,10023T)are proposed.After 36-48 hours of culturing on Columbia blood plate,the colonies of Neisseria weixii were small,raised,moist,hoar with regular margins,low transparency,1-1.2mm in diameter.Cells stain Gram-negative,are single or double spherical.Under electron microscopy cells showed typical spherical,without flagella and with pili,about 0.6-0.9um for Neisseria weixii in diameter.Growth occurs at 22 ?,30?,37 ?,42 ?,but not at 4?,15 ? for Neisseria weixii.Neisseria weixii could grow on the media of Columbia blood agar,Nutrient agar,BHI,BHI with 5%defibrinated sheep blood,Chocolate plate,but not on Maconkey Agar.Main fatty acids of Neisseria weixii was C16:0,C16:l?7c/16:1?6c,and the types or contents of major fatty acid were different between Neisseria weixii and other known species in Neisseria.For example,Neisseria weixii does not contain C18:2?6,9c/C18:0 antei,while its close strains contain such fatty acids.Biochemical experiments showed that Neisseria weixii was oxidase-positive and can ferment sucrose,D-glucose,maltose with API NH.There are obvious differences in the biochemical characteristics between our new species and other known species in Neisseria.According to the evolution analysis of 16S rRNA gene,rplF gene,rMLST and cgMLST,we can see that Neisseria weixii was in independent branch of evolution.The analysis of whole genome sequencing showed that the circular chromosome of Neisseria weixii comprises 2,511,904bp with an overall G+C content of 49.22%.Putative virulence-associated factors including LPS,Capsule,Mtr CDE,KatA,LOS,indicating the pathogenic potential of Neisseria weixii.Digital DNA-DNA hybridization studies showed less than 70%DNA-DNA relatedness between our new species and other species of the genus Neisseria that had deposited in GenBank database,18.6%-43.3%for Neisseria weixii.After 36-48 hours of culturing on Columbia blood plate,the colonies of Neisseria chenwenguii was small,raised,moist,hoar with regular margins,0.6-1.omm in diameter.Cells stain Gram-negative,are single or double spherical.Under electron microscopy cells showed typical spherical,without fiagella and with pili,about 0.2-0.9um for Neisseria chenwenguii in diameter.Growth occurs at 22?,30?,37?,but not at 4?,15 C,42 C for Neisseria chenwenguii.Neisseria chenwenguii could grow on the media of Columbia blood agar,Nutrient agar,BHI,BHI with 5%defibrinated sheep blood,Chocolate plate,but not on Maconkey Agar.Main fatty acids of Neisseria chenwenguii was C16:0,C16:1?7c/16:1?6c,and the types or contents of major fatty acid were different between our new species and other known species in Neisseria.For example,Neisseria chenwenguii does not contain C16:1?7c and/or iso-15:0 2-OH which were the major fatty acids of its proximal strain.Compared with its related strains,the specific fatty acids of Neisseria chenwenguii are C18:2?6,9c/C18:0 antei.Biochemical experiments showed that our new species are oxidase-positive and can ferment D-gtucose,D-fructose,maltose and sucrose with API NH.There are obvious differences in the biochemical characteristics between our new species and other known species in Neisseria.According to the evolution analysis of 16S rRNA gene,rplF gene,rMLST and cgMLST,we can see that Neisseria chenwenguii was in independent branch of evolution.The analysis of whole genome sequencing showed that the circular chromosome of Neisseria chenwenguii contains 2,496,444bp with an overall G+C content of 54.02%.Putative virulence-associated factors including LPS,Capsule,Mtr CDE,KatA,LOS,indicating the pathogenic potential of Neisseria chenwenguii.Digital DNA-DNA hybridization studies showed less than 70%DNA-DNA relatedness between our new species and other species of the genus Neisseria that had deposited in GenBank database,18.9%-25%for Neisseria chenwenguii.