Isolation,Identification And Comparative Genomics Study Of The Bacteria Of Families Francisellaceae And Fastidiosibacteraceae

ObjectiveThe families Francisellaceae and its neighboring bacteria of the order Fastidiosibacteraceae have potential pathogenicity.Some strains have strong pathogenic ability,have many natural hosts,have diverse transmission routes,and pose great harm to society;therefore,understanding the species diversity,genomic characteristics and microbiological characteristics of these groups are particularly important for clinical diagnosis.In order to research the species diversity of these groups of bacteria,the study intends to establish a set of methods for enrichment and isolation of the bacteria of two families,culture medium and the preparation methods of the medium.Besides,the comparative genomics method is used to further explore genomic characteristics of the bacteria of two families to lay the foundation for its pathogenicity and transmissibility.MethodsIn the chapter on selective isolation and identification of novel species of the families Francisellaceae and Fastidiosibacteraceae,the water samples from Shuangyue Bay and Xunliao Bay in Huizhou were collected,enriched,and cultured on BCYEα-2216E-GVPC medium and BHI-2216E-GVPC medium at 25℃.For the potential novel species of the families Francisellaceae and Fastidiosibacteraceae,the following analyses were performed to its taxonomic status,namely phenotypic,physiological and biochemical analysis.In order to identify their further taxonomic status,one of potential new genus and species(SYSU WZ-2T)and four potential new species(SYSU SYW-1T,SYW-2,SYW-3 and XLW-1)were analyzed using polyphasic taxonomy.The phenotypic determination were carried out by culture methods,gram staining,scanning electron microscopy and other methods.The traditional physiological and biochemical determinations were observed using catalase,oxidase,indole,urease,API NH,API ZYM,antibiotic susceptibilities testing.Besides,The polar lipids,fatty acid profile(FAME analysis)and respiratory quinones were analysed.The protein profile was revealed by the Mass spectrometer,The genetic analyses were examined by the 16S rRNA gene sequencing,the average nucleotide identity(ANI)and the phylogenetic tree based on the whole genome sequencing.The protein profile was revealed by the Mass spectrometer.In the chapter on comparative genomics on the family Francisellaceae,the whole genome data of the family Francisellaceae in the NCBI database is selected as the research objects.Based on comparative genomics research,functional cluster analysis and species evolution analysis were performed on 104 whole-genome data.Meanwhile,specific primers were designed for Francisella philomiragia associated with aquatic animal diseases and the highly pathogenic Francisella tularensis.ResultsThe addition of seawater 2216E medium can simulate a good original ecological environment for the growth of microorganisms in the seawater environmental samples.Among the 23 samples,10 were positive,with a positive rate of 43.5%.There were 29 potential new taxa,and even 19 potential new families of Fastidiosibacteraceae were isolated.Cells of strain SYSU WZ-2T were observed to be aerobic,non-motile Gram-negative,non-spore forming and coccobacilli.Catalase activity was weakly-positive and oxidase-negative.As for strain SYSU WZ-2T,ubiquinone(Q8)was the only respiratory.C18:0 3-OH,C18:1 ω9c,C16:0 and C14:0 were the major fatty acids.Diphosphatidylglycerol,phosphatidylcholine,phosphatidylethanolamine,an unidentified aminolipid,two unidentified aminophospholipids and two unidentified phospholipids were the major polar lipids,which were different from the members of the family Francisellaceae.SYSU WZ-2T had the highest 16S rRNA gene sequence similarity with strain‘Francisella frigiditurris’CA97-1460(99.9%),while the similarities with other Francisella and Allofrancisella were 94.8-95.9%and 93.8-94.2%,respectively.The phylogenetic tree based on the 16S rRNA gene also showed that SYSU WZ-2T and‘Francisella frigiditurris’ CA97-1460 clustered into a clade,which distinct from the two major clades of the genera Francisella and Allofrancisella.The MALDI-TOF MS analysis showed that the characteristic ion peak of SYSU WZ-2T was 5129 m/z,which was different from the characteristic ion peak of Francisella:5180m/z.The dendrogram based on MALDI-TOF MS also showed that SYSU WZ-2T formed a distinct lineage separated group in Francisillaceae.The ANI values of strain SYSU WZ-2T compared with the genera Francisella and Allofrancisella were 72-73%.The phylogenetic dendrograms tree revealed the strain SYSU WZ-2T formed an independent clade among the members of the family Francisellaceae.SYSU SYW-1T,SYW-2,SYW-3 and XLW-1 were isolated from the seawater in Huizhou.Cells of these four strains were Gram-negative,strictly aerobic,non-motile and non-spore forming.As for strain SYSU SYW-1T,the respiratory quinone was ubiquinone(Q8).The predominant cellular fatty acids were C18:0 3-OH,C14:0,C10:0 and C18:1 ω9c and the main polar lipids were diphosphatidylglycerol,phosphatidylcholine,phosphatidylethanolamine and an unidentified phospholipid which were consistent with those of members of genus Francisella in compositions as well as proportions.The comparison of 16S rRNA gene sequence similarity showed that the four strains were a new taxon of Francisella,and the similarity of 16S rRNA gene sequence with all species in Francisella was less than 98.8%,and the ANI value of the genome was less than 95%.Phylogenetic dendrograms according to 16S rRNA gene and the genomic sequences both indicated that the strains were clustered together with ’Francisella salina’TX07-7308 and’Francisella marina’E95-16,but formed an monophyletic clade in the genus Francisella which was different from other strains.MALDI-TOF MS analysis showed that the peaks of SYSU SYW-1T were 5180 m/z,which was consistent with the characteristic ion peak of Francisella.The phylogenetic tree based on the whole genome showed that Francisella tularensis clustered individually into a group of large branches adjacent to Francisella hispaniensis and Francisella novicida.Francisella philomiragia is also clustered separately,similar to’Francisella marina’ and ’Francisella salina’.According to the results of functional clustering,specific primers for Francisella philomiragia and Francisella tularensis were designed.ConclusionBased on the above analysis results,we established a set of methods for the enrichment and separation of the bacteria of families Francisellaceae and Fastidiosibacteraceae.Potential new families were obtained through this method.We determined the taxonomic status of SYSU WZ-2T was potential new genus of the families Francisellaceae,and the four strains(SYSU SYW-1T,SYW-2,SYW-3 and XLW-1)were potential new strians of the genus Francisella.SYSU WZ-2T was named Pseudofrancisella aestuarii and the model strain was SYSU WZ-2T(KCTC 52557T=CGMCC 1.13718T).The four strains(SYSU SYW-1T,SYW-2,SYW-3 and XLW-1)were named Francisella salimarina and the model strain was SYSU SYW-1T(CGMCC 1.17031T=NBRC 113781T).Based on comparative genomics,4 pairs of primers were designed for Francisella philomiragia and Francisella tularensis,but it is recommended to use them in combination to achieve higher accuracy.