The Nomenclature Of Escherichia Marmotae And Analysis Of Its General Feature,Complete Genome And Virulent Genes

A group of seven closely related lactose-negative enterobacterial strains were isolated from fresh fecal samples of Marmota himalayana collected from the Qinghai-Tibetan plateau,China,termed as HT07316,HT080401,HT080709,HT072503,HT073105,HT080118,HT080711,respectively.All the strains showed to be light purple colonies when growed on Salmonella selective agar after 18-24h,which were indicative of putative isolates of Salmonella based on the colour reaction on CHROMagarTM.However,after confirming by a polyphasic approach,the strains were recognized not Salmonella but represent a novel speciesof the genus Escherichia,which named as Escherichia marmotae.Escherichia marmotae Cells are facultative anaerobic,Gram-reaction-negative,non-sporulating,non-motile,short rods?0.5-1×1-2.5?m?.Colonies on LB agar were circular,semi-transparent,gray white,convex and-2 mm in diameter after incubation at 37?for 1 day.The strain can grow at 25-37? and in 0-6%NaCI.The optimum growth condition is 37?and 1%NaCl.It is positive for catalase and negative for oxidase and urease.By Kirby-Bauer disc diffusion testing,the strain is susceptible to 18 antibiotics tested including streptomycin,gentamicin,amikacin,ciprofloxacin,enrofloxacin,imipenem,ertapenem,ampicillin,cefazolin,cefotaxime,piperacillin/tazobactam,amoxicillin/clavulanate,cefotaxime/clavulanic acid,tetracycline,polymyxin,chloramphenicol,florfenicol,and trimethoprim/sulfamethoxazole.In API 20E tests,it is positive for lysine decarboxylase,but negative for(3-galactosidase,arginine dihydrolase,ornithine decarboxylase,citrate utilisation,H2S production,phenylalanine deaminase,indole production,Voges-Proskauer test and gelatin reaction.In API 50CH tests,it is positive for glycerol,D-arabinose,L-arabinose,RNA,D-xylose,galactose,glucose,fructose,mannose,rhamnose,mannito,sorbitol,N-acetyl glucosamine,maltose,trehalose,L-fucose,gluconate;negative for red versicolor alcohol,L-xylose,adonit,?-methyl-D-xyloside,sorbose,dulcitol,inositol,a-methyl-D-mannoside,a-methyl-D-glycoside,amygdalin,arbutin,esculin,salicin,cellobiose,lactose,melibiose.sucrose,inulin,melezitose,raffinose,starch,glycogen,xylitol,gentiobiose,D-turanose,D-lyxose,D-tagatose,D-fucose,D-arabitol,L-arabitol,2-keto-Gluconate,5-keto-gluconate.Escherichia marmotae can differentiated from Escherichia coli by negative reaction of ?-galactosidase,ornithine decarboxylase,indole,melibiose;Escherichia marmotae can differentiated from Salmonella by negative reaction ofarginine dihydrolase,ornithine decarboxylase,citrate utilisation,H2S,indole,melibiose;Escherichia marmotae can differentiated fromShigella by negative reaction of?-galactosidase and positive reaction of lysine decarboxylase,sorbitol,rhamnose.Phylogenetic analysis based on thel6S rRNA gene sequences showed that the seven strains formed a monophyletic group with five other member species of the genus Escherichia.By 16S rRNA gene sequences,the representativestrain,HT073016,showed highest similarity values with Escherichia fergusonii_ATCC35469 at 99.3%,Escherichia coli ATCC11775 at 99.2%,Escherichia albertii LMG₂0976at 98.9%,Escherichia hermannii CIP₁03176 at 98.4%,and Escherichia vulneris ATCC₃3821 at 97.7%.Digital DNA-DNA hybridization studies between strain HT073016 and five other species of the genus Escherichia showed that it shared less than 70%DNA-DNA relatedness values with all known species of Escherichia,supporting the novel species status of the strain.Escherichia marmotae displayed the highest DNA relatedness values,43.1%,with the type strain of Escherichia coli DSM 30083.TheDNA relatedness values of HT073016with the other Escherichia species were much lower,ranging from 20.9 to 38.1%.The homology of seven isolates of Escherichia marmotae was determined by pulsed field gel electrophoresis?PFGE?.Isolates of HT073016,HT08070 and HT080711 showed the same PFGE type,which were isolated from different sample collected in the same town of Haxiu.Isolates of HT073105,HT080118 and HT080401 show the same PFGE type,which were isolated from differnet sample collected in the same town of Dezuotan.The strain HT072503 was isolated from sample collected from Zhongda town,which showed a PFGE type.Genome analysis of HT073016 show that HT073016 genome size is 4.6Mbp and GC%is 50.65%,which contains 4438 genes including 961 hypothetical protein genes.The number of 16S,23S,5SrRNA is 7,7,7 respectively.It has 85 tRNA.Interestingly,HT073016 has two virulent related plasmids,termed as pMarmota₁48 and pMarmota₇6.The plasmid genome size is 148kbp,76kbp,respectively.GC%is 44.49,46.26,respectively.There are several virulent factors in pMarmota₁48,such as T3SS,sepA,yopJ,non-LEE effectors,and S fimbriae.The T3SS contained in pMarmota₁48 is similar with T3SS in Shigella.The plasmid pMarmota₇6 also contains T3SS effector IpaH.Phylogenetic analysis was performed between strains of Escherichia marmotae and A,B1,B2,D,and H groups of Escherichia coli,Escherichia Clade ?,?,?,V,Shigella,Escherichia albertii,Escherichia fergusonii,and Salmonella.Phylogenetic analysis based on the core gene sequences showed that Escherichia marmotae strains formed a monophyletic group,which contains Escherichia Clade V strains.Escherichia Clade V strains were recongnized as Escherichia marmotae.Genome comparision with commensal Escherichia coli MG1655,we detected 21 specific genomic island in,abbrivated GI1?21,in Escherichia marmotae genome HT073016.The size of GI is between 4k?30kbp and GC%is 30,50%?57.49%.GI1?6 were shared in marmot isolated strain HT073016 and Escherichia Clade V strain TW09308.GI7?21 were specifc in marmot isolated strain HT073016.GI 7 is T6SS related genomic islands.GI8 is LPS synthesis genomic island,which is the type 8 O antigen of Shigella dysenteriae.GI9,10,11,12,20,21,are associated with phage.GI14,18,are associated with the outer membrane structure.GI15 is associated with invasive protein.GI16 is flagella genomic island.Meanwhile,GI17,19 are associated with glucose metabolism.