The Study Of Identification And Characterization Of New Species Of Francisella Philomiragia-like Bacteria

ObjectivesFrancisella is a kind of facultative intracellular parasitic bacteria.Some strains have strong pathogenicity,many hosts,diverse transmission routes and serious social hazards.Previously,our team isolated and discovered new species of Francisella guangzhouesis,new genera of Allofrancisella,new genera of Cysteinphilum,and many unnamed new species and genera in clinic and environment.On the basis of previous studies,this study intends to further systematically classify the new strains of Francisella philomiragia-like isolated in earlier stage by using multiphase taxonomy method.Meanwhile,using the isolated and identified Francisella philomiragia,a database of MALDI-TOF mass spectra was established to study the new species of Francisella philomiragia-like and explore potential new species.MethodsTraditional and modern phenotypic identification methods such as culture,Gram staining and transmission electron microscopy were used;physiological and biochemical identification and drug sensitivity detection methods such as oxidase,contact enzyme,indole,API ZYM,API 20NE,Vitek 2 GN identification card,NH identification card,whole cell fatty acid determination,E-text drug sensitivity determination were used;16S rRNA gene sequencing analysis technology and matrix-assisted laser desorption electrolysis were used.Based on the method of Orthologous Average Nucleotide Identity(OrthoANI)analysis were used,to polyphasic taxonomy of Francisella philomiragia-like strain SYW-1,SYW-3 and SYW-9 was pre-isolated.The MALDI-TOF MS SuperMap database of Francisella philomiragia was constructed by using pre-isolated and identified strains QH-2,QH-3,QH-4,QH-5,QH-12,QH-15,1958,Zhangzhou,YJ-2 and standard strain ATCC 25015.The database was validated by other related strains such as Allofrancisella guangzhouesis NCTC 13053.ResultsThe experimental strains SYW-1,SYW-3 and SYW-9 are aerobic bacteria with strict nutritional requirements.In BCYE a medium containing L-cysteine,1 mm gray-white colony with smooth surface,round protuberance,neat edge,translucent and wet colony was formed in 48 hours.Gram staining was negative,and cocci were scattered in sand.The size ranges from 1.0 to 1.2 × 1.2 to 1.5 ?n.Biochemical and zymographic reactions were not active.Only a few tests,such as y-glutamyltransferase,L-proline aromatase,ornithine decarboxylase,leucine transaminase,phenylalanine aromatase and L-glutamine,had reactivity.Compared with ATCC 25015,some cells lack or contain less fatty acids.It was sensitive to chloramphenicol,doxycycline,levofloxacin,tetracycline,gentamicin and ciprofloxacin in vitro.According to 16S rRNA gene analysis,the taxonomic status of the tested strains belonged to the genus Francisella,and their genetic relationship was most closely related to Francisella philomiragia and Francisella noatunensis.Based on the average homologous nucleotide consistency of the whole genome sequence,the G+C content of the tested strains was 31.4%-32.9%,which was similar to that of Francisella.The OrthoANI ratio of internal strains was less than 95%.The MALDI-TOF MS SuperMap database of Francisella philomiragia was constructed successfully.ConclusionsFrancisella philomuiragia-like strains isolated in earlier stage belong to the genus Francisella from the molecular level.They are closely related to Francisella philomiragia and Francisella noatunensis,but the ANI ratio does not support any of them.According to the latest requirements of the International Commission on Prokaryotes,the Francisella phiomiragia-like test strains SYW-1,SYW-3 and SYW-9 are all new species of Francisella is preliminarily judged.