The Screening And Characterization Of Virulent And Avirulent Strains Of Pseudomonas Aeruginosa Isolated From Mink And Their Virulence Factors Identification

Pseudomonas aeruginosa is one of the important pathogens which can cause mink hemorrhagic pneumonia.Compared to the well documented epidemiology of Pseudomonas aeruginosa,the virulence genes of the pathogens are poorly studied.The aim of this study was to investigate the virulence genes of Pseudomonas aeruginosa and to study the pathogenicity of the gene to host cells.Based on the results of the epidemiological investigation of PA within Shandong province,representative strains were chosen for pathogenicity tests for screening of virulent and avirulent strains.Differentially expressed genes correlated with the pathogenicity of Pseudomonas aeruginosa from mink were screened by comparative transcriptome and Real-time PCR.The gene knockout mutants were constructed for the analysis of the correlation of the selected gene and the pathogenicity of PA.The current study is composed of the following sections:1.The epidemiological investigation of Pseudomonas aeruginosaA total of 78 Pseudomonas aeruginosa isolates originated from mink?n=60?,human?n=16?and sheep?n=2?were analyzed by O-antigen serotyping,H-antigen flagella typing,and pulse field gel electrophoresis typing,respectively,which were selected from Pseudomonas aeruginosa isolates in different collection areas of Shandong Province.Our results showed that PA infection of mink was highly correlated with isolates of serotype G and flagella A?P<0.01?,while PA infection of human was closely related with isolates of serotype E and flagella A?P<0.01?.Two isolates originated from sheep were both serotype I and flagella B.The PFGE typing showed the characteristic with complexity and diversity among strains from different sources.Some isolates originated from mink showed a unrelated relationship of each other.2.Selection of Pseudomonas aeruginosa virulent and avirulent strainsThe representative strains were selected from 78 Pseudomonas aeruginosa isolates based on the epidemiological survey.PA infected mouse models were established in the current study to analyze the virulence of selected PA isolates based on the mortality and bacterial count from organs of the dead mice.The results showed the mortality of mice infected with PA of serotypes G and B?over 80%?were higher than that of serotypes M and G?less than20%?under the same infection dose.Two virulent strains 11092618,11092304 and two avirulent strains 11082616,QT1 were selected based on the results above.3.The screening and identification of virulence genes in Pseudomonas aeruginosa isolated from minkThe comparative transcriptome studies were used for virulent and avirulent Pseudomonas aeruginosa isolates which obtained from mice pathogenicity test.Library preparation were carried out using Illumina Truseq^TMM RNA sample prep Kit.Sequencing were conducted using Illumina HiSeq 2500,which produced 2×100 bp paired-end reads.30differentially expressed genes were screened by GO and KEGG annotation.Real-time PCR were used for the confirmation of differentially expressed genes.The 16S rDNA with constant copy number were selected as the internal reference.The default amplification efficiency E=2 was used to determine the up-regulation and down-regulation of the gene.9differentially expressed genes related to virulence were finally selected.4.The study of pyoS5 gene on the virulence in Pseudomonas aeruginosaThe pyoS5 gene?pyoS5,PA0985?which encodes the pyocyanin of PA is highly related to the virulence of PA and selected for the study of its function and correlation with PA virulence.pyoS5 gene knockout DNA fragment was cloned to the suicide plasmid pCVD442.Using the homologous fragment at both ends of the deleted gene,the integration site of the suicide plasmid was localized.We also used PCR amplification the target fragments,DNA sequencing and Real-time PCR methods were used to the verification of the accuracy of the gene knockout.In order to further study the virulence of the pyoS5 gene.The biological characteristics and pathogenicity of pyoS5 gene knockout mutant were analyzed and showed the relationship between 11092618?pyoS5 and 11092618.11092618?pyoS5 had no significant difference compared with wild strain 11092618 in growth rate and the mutant did not transcribe the pyoS5 gene?pyoS5,PA0985?.In the pathogenicity test of mice with different doses,the LD₅₀0 of the mutant was lower than that of the wild strain;the virulent of mutant was decreased.The mink mortality of the mutant was also lower than that of the wild strain in the same dose,those results indicated that the pyoS5 gene?pyoS5,PA0985?was an important virulence gene of Pseudomonas aeruginosa.