| Escherichia coli (E.coli) is a kind of very important zoonotic pathogen with widespread in nature, complexed serotypes and varied pathogenicity. According to the genetic inheritance and pathogenicity feature, Escherichia coli can divide into three parts: commensal bacteria, intraintestinal pathogenic Escherichia coli and extraintestinal pathogenic Escherichia coli. Extraintestinal pathogenic Escherichia coli (ExPEC) are a group of E.coli which can colonize in intestinal tract but cann’t cause diarrhea. Because of ExPEC’s special virulence factors, they can cause many diseases both to human and animals, including neonatal septicaemia and urinary tract infections for human; pneumonia, nephritis, meningitis and septicemia for pigs; airsacculitis, synovitis, peritonitis and septicemia for birds.Iron is a very important nutrient element. Metal ions are involved in many biological processes. As heme is one of the three main ironsources, so the uptake ability of heme is related with the survivability of E.col. In this research, we choosed the hmuU and hmuV genes which are participated in the heme acquisition system to constructe three mutants of ExPEC PCN033AhmuU, AhmuV and AhmuU/AhmuV. Carried out the research on its’ biological characteristics and virulence test in mice. The main research was described as follows:1. Construction and identification of PCN033strain mutants AhmuU, AhmuV and AhmuU/AhmuVConstruct the recombination plasmid pREAhmuU, pREAhmuV and pREAhmu with the suicide plasmid pRE112. The recombination plasmid pREAhmuU and pREAhmuV were transformed into the E. coil donor strainX7213, and then conjugated with the recipient PCN033. Then the two mutant AhmuU and AhmuV were selected. The AhmuU/AhmuV mutant with the deletion of both hmuU and hmuV gene was selected with the foundation of mutant AhmuV. The basic way is the same.2. Genetic stability and growth feature of the mutants AhmuU, AhmuV and AhmuU/AhmuVThe hereditary stability of the three mutants were evaluated by serial subcultivation, the PCR test of20generations showed it had a stable ability. Under the condition of complete nutrition, the growth curve of the three mutants and the parent strain are the same, without obvious difference. Under the condition of iron limit, the growth curve of the mutants showed slower than the parent strain PCN033. The AhmuU/AhmuV mutant grew showed slowest.3. The resistant phagocytosis and conglutination and invasion of the mutants AhmuU,△hmuV and AhmuU/AhmuVThe PAM cell and RAW cell were used to detecte the resistant phagocytosis of the mutants, and the PK15cell was used to detecte the conglutination and invasion of the mutants. The results were recorded and analyzed statistically. The mutants and tne parent strain has a significant difference in PAM (P<0.01) cell and RAW (P<0.05) cell but has no significant difference in PK15(P>0.05) cell. The result showed that both the ablilty of resistant phagocytosis and conglutination and invasion of the mutants were weaker than that of the partent strain.4. Virulence in mice and the ability of colonization in tissues60mice were divided into4groups, in which3small groups were divided with5mice each small group. The virulence of the mutants and parent strain were detected in4-week old KunMing mice and the LD50were analysed. The results showed that the LD50of△hmuU/△hmuV, AhmuU,△hmuV and the parent strain were1.5×107CFU,1.5×106CFU,1.5×106CFU and9.5×105CFU, which indicated the virulence of△hmuU/△hmuV was decreased about16times compared with the parent strain, both△hmuU and AhmuV were decreased about2times.Anatomied of mice which died12hours after infection, then isolated E.coli from liver, heart, spleen, lung, kidney and brain. Counted the amount of bacteria in5.0×106CFU and5.0×107CFU dose groups. The data showed that the ability of colonization in tissues for mutants were weaker than that of the parent strain for about5-10times. The△hmuU/△hmuV was much weaker than both△hmuU and△hmuV. The virlence of PCN033was declined after delection of gene hmuU and hmuV. |
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