Genetic Evolution Analysis And Comparative Genomics Of Pseudomonas Aeruginosa Isolated From Mink

Mink hemorrhagic pneumonia is an acute infectious disease caused by Pseudomonas aeruginosa?PA?,which is characterized by hemorrhagic pneumonia.To elucidate the epidemiological features,genetic evolution,drug resistance,genomic characteristics of mink-derived PA in China.PA isolates 107 PA strains isolated from minks died of hemorrhagic pneumonia and 8 PA strains isolated from mink farm soil from2007 to 2017 were studied.The following results are obtained.The biological characteristics and pathogenic characteristics were elucidated.The results showed that the serotype of mink isolates were 86 of G,7 of B,3 of I,1 of C,1 of D,9 serotypes was undifferentiated in107 PA mink isolates.The serotype of mink farm soil isolates were 4 of G,4 of A,1 of E,1 of D and 1serotype was undifferentiated.PA mink isolates have strong sporting ability and biofilm forming ability,There were 45 in 46 strains producing brown or pink pigments;Pathogenic model of PA to mink was established,except for two PA avirulent strains,LD₅₀ of all isolates were distributed between 10⁵ CFU-10⁷CFU for mice and minks.Histopathological examination showed that there were serious pathological changes in tissues of infected mink.Immunohistochemical observation showed that PA was distributed in trachea,alveolar wall epithelial cells.The results indicated that mink PA strains had strong virulence,invasive and infective ability,In order to elucidate the population structure characteristics and genetic evolution of PA isolates were analysised by pulsed field electrophoresis?PFGE?and multilocus sequence typing?MLST?;and the Core-SNP and Core-Pan evolutionary trees of 46 isolates were constructed,The PFGE results showed that PA strains of the same outbreak were all the same clone.The MLST results showed that the population structure of genotype of PA mink-origin isolates were diversity,the STs of isolates were the same as human isolates.MCG cluster tree showed that the evolutionary distance of PA human isolates and mink isolates was far,The homology 27 strains from mink?including one mink-field soil isolate?was 100%,indicating that the strains were transmitted between these areas in different years.The soil strains of mink farm were also the source of infection.The drug resistance and genes of PA isolates were detected.The results showed that nine strains were resistant to quinolones?MIC:16?g/ml-256?g/ml?,two strains resistant to fosfomycin?MIC=1024?g/ml?,quinolone resistance was caused by mutations of gyrA?Thr-83 Ile?and ParC?Ser-87 Leu?;Fosfomycin resistance was caused by glpt deletion(T₂₂₀CGCCT_226,Ile74-Ala75);The distribution of drug resistance genes were analyzed by Whole genome sequencing.A total of 87 drug resistance genes located on chromosomes were detected in 46 mink-derived PA isolates,The results showed that drug-resistance of PA in mink was resistance gene loci mutation in chromosome.In order to elucidate the genome differences between mink-derived PA and human-derived high virulent PA,Comparative analysis of Core-pan,secretory systems and collinearity were done for 46 PA mink-origin isolates and human isolates LESB58.The results showed that the specific genes,SNP mutations,repeat sequences,RNA,genome islands,secreted proteins and secondary metabolites of mink and environmental isolates were lower than those of LESB58.Isolates from mink and soil lacked the key genes vgrG1 and vgrG2 in T6SS,indicating that T6SS secretion was weak.The translocation and inversion of genes were found in the collinear comparison between the high virulent strains WH10.3 and D15 and the low virulent strain WD15.3.It was speculated that the attenuated virulence of the low virulent strain might be related to the insertion and deletion of large fragments.The results indicated that the big genomic differences between PA mink-origin and human-origin,The simple in metabolism and function of mink isolates and soil isolates,which may be the result of evolution of environmental singularity of strain located.The genes of PA5155 and ygfA of strain HY-3 were knocked out and their functions were studied.The results showed that comParEd with HY-3,the virulence(LD₅₀)of?PA5155 and?ygfA in mice were decreased by 2.9 times?P<0.05?and 2.6 times?P<0.01?,The adhesion ability of PA5155 to Mink lung epithelial cells?MLEC cells?decreased significantly?adhesion rate,14.6%?11.23%??P<0.01?;?PA5155and?ygfA could reduce resistance to chloramphenicol?CM?and cefotaxime?CEF??MIC:CM,64?g/ml?32?g/ml;CEF:256?g/ml?64?g/ml?,The results suggesting that PA5155 and ygfA have a certain effect on the virulence and drug resistance,while was not an important virulence factor of PA.In summary,the biological and pathogenic characteristics of PA mink and soil origins were detected for the first time in this study.The population structure and genetic evolution of PA were elucidated by PFGE,MLST and Core-SNP methods.The drug resistance of PA mink isolates were determined.The differences between PA mink and human PA genomes were clarified;.The biological function of genes of the ygfA and PA5155 were analyzed.The results clarified the pathogenic characteristics and transmission law of PA isolated from mink hemorrhagic pneumonia in China,and provided theoretical basis for the prevention and control of mink hemorrhagic pneumonia and the protection of human health.