| ObjectivePhylogenetic analysis of E.coli ST1193 was carried out to explore the evolutionary trajectory of strains from different geographical sources,which was helpful to better understand the transmission trend of this sequence type.Comparative analysis of pathogenicity and survivability among different sequence types was conducted to evaluate whether E.coli ST1193 lineage had population advantage,to explore the possibility of ST1193 being the dominant clone and revealed the reasons for its spread in clinical environment.Methods1.Bacterial Strains:51 E.coli ST1193 and 15 E.coli ST131 were isolated from Fujian Medical University Union Hospital.The whole genome sequences of 25 non-Chinese E.coli ST1193 genomes involved in phylogenetic analysis were from the National Center for Biotechnology Information.2.Phylogenetic analysis:51 strains of E.coli ST1193 were submitted to whole genome sequence（Illumina）.The phylogenetic trees were constructed by extracting the core genome and accessory genome of 76 E.coli ST1193.The clade-specific genes were analyzed by comparative genomic analysis and subsequent submitted to GO enrichment analysis.3.Virulence genes,plasmid replicon types and antibiotics resistance genes of E.coli ST1193 were detected by CGE server and CARD database.4.Pathogenicity of E.coli ST1193:T24 cells were used for adhesion and invasion assays to compare the adhesion and invasion abilities among different sequence type’s strains.Crystal violet staining was used to detecte the biofilm forming ability among different sequence type’s strains.Macrocolony formation was assessed on macrocolony formation plates（containing congo red and coomassie brilliant blue）and the differences of biofilm formation ability among different sequence type’s strains were analyzed by observing the differences of the appearance of colonies formed.5.Survivability of E.coli ST1193:Serum resistance assay was used to compare the capacity to resist the serum bactericidal activity among different sequence type’s strains.Hydrogen peroxide resistance assay was used to compare the hydrogen peroxide resistance among different sequence type’s strains.RAW264.7 cells were used for anti-phagocytosis assay to compare the phagocytosis resistance among different sequence type’s strains.6.Statistical analysis:The experimental results were analyzed by SAS 9.4 software and the data were plotted by Graphpad Prism 7.0 software.Single factor analysis of variance（ANOVA）was used for comparison between groups,and Student’s t test was used for comparison between the two groups.The experimental results were expressed as mean±standard deviation（variance±SD）.A two-sided test P<0.05 was considered statistically significant.Results1.Phylogenetic analysis:All E.coli ST1193 were divided into two clades（clade A and B）in core genome phylogenetic tree.Clade A included 25 non-Chinese E.coli ST1193and clade B contained all Chinese isolates,respectively.The comparative genomics results indicated that Indels were identified in a total of 150 clade-specific genes and more than 70%genes were enriched into biological process and molecular function.All E.coli ST1193 were divided into seven clades（clade 1 to 7）in accessory genome phylogenetic tree.A high degree of correlation was observed between accessory genome clusters（clade 3 and 6）and core genome phylogeny in clades A.2.A total of 24 virulence genes were detected.Some virulence genes were identified more than 70%among E.coli ST1193,such as chu A,fyu A,yfc V,vat,sat,iha,gad and sen B.There were significant differences in virulence genes carried between clade A and clade B,including cia,neu C,gad and tra T（p<0.05）.A total of 19 types of plasmid replicators were identified.98.7%E.coli ST1193 carried at least one plasmid replicon types,covering 19 different known plasmid incompatibility types.96.1%E.coli ST1193 contained two types of F-type replicons（Inc FIA and Inc FIB）.There were significant differences in plasmid replicon types carried between clade A and clade B,including Colp VC,Inc Q1,Col156 and Inc B/O/K/Z（p<0.05）.A total of 71 antibiotics resistance genes were found.Among E.coli ST1193,more than 50%isolates carried acquired antimicrobial resistance genes which included bla TEM-1B,sul2,APH（6）-Id,dfr A17 and mph A.There were significant differences in antibiotics resistance genes carried between clade A and clade B,including bla CTX-M-55,bla TEM-1,sul2,tet（B）,tet（R）,APH（6）-ID and AAC（3）-iid（p<0.05）.3.bla CTX-M-55with different genetic environments:bla CTX-M-55located on downstream of a homologous tract of ORF477.Different mobile elements were detected on downstream or upstream of bla CTX-M-55,including ISEc9,Tn3 and integrase gene.bla TEM-1located on upstream of bla CTX-M-55in two Chinese isolates.4.Pathogenicity of E.coli ST1193:Compared with the negative control,E.coli ST1193 showed stronger adhesion capability,invasive capability and biofilm formation capability.Compared to E.coli ST131,E.coli ST1193 showed similar adhesion capability,invasive capability and biofilm formation capability（p<0.05）.The results of macrocolony formation showed that 66.7%of E.coli ST1193 possessed no curli/no cellulose.Compared to ST131,E.coli ST1193 exhibited a statistical difference in curli only（p<0.05）.5.Survivability of E.coli ST1193:Compared with the negative control,E.coli ST1193showed stronger serum resistance capability,hydrogen peroxide resistance capability and anti-phagocytic capability（p<0.05）.Compared to E.coli ST131,E.coli ST1193had a similar serum resistance,hydrogen peroxide resistanc and anti-phagocytic capability（p>0.05）.Conclusions1.The distinct phylogenetic characteristics of the spread of E.coli ST1193 in China and non-China region,.revealing that the different phylogenetic characteristics of the spread of E.coli ST1193 in China and non-China region.That different phylogenetic relationship supports both global transmission and localized lineage expansion of this lineage following specific introductions into a geographic locality.2.There were significant differences in the distribution of virulence genes,plasmid replicon types and antibiotics resistance genes between Chinese and non-Chinese E.coli ST1193（p<0.05）.3.Similar to ST131,E.coli ST1193 has the possibility of becoming a dominant clone. |
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