The Horizontal Transfer And The Function Evaluation Of HPI In Avian E.coli

High pathogenicity island (HPI) is a region of clustered genes found in Yersiniabacteria chromosome, which plays a key role in the toxicity of mice and has a closerelationship with the mouse lethal phenotype. HPI mainly includes virulence geneclusters that associated with iron uptake, as yersiniabactin (Ybt, siderophore) geneand its regulation genes, and plays a function of iron uptake. The structure featuresand the wide distribution of HPI in intestinal coli suggest that it may have horizontaltransfers, but the horizontal transfer of HPI in avian Escherichia coli by the way ofco-culture at low temperature and low iron liquid medium has not been reported.Studies of recent years have shown that Escherichia coli HPI mainly plays a role inuptake and regulation function of iron and make bacteria have virulence andpathogenicity. But the change of the host bacteria’s function after the acquisition ofHPI is still unreported.In order to study the feature of the distribution and the transfer of HPI in avianEscherichia coli and its function in the acquired strain, the distribution of HPI inavian Escherichia coli clinical isolates was analyzed. A transfer positive strain wasscreened after the co-culture of appropriate donor and acquired strains of HPI in thecondition of low temperature and iron. Biological activity and pathogenicity changesof the acquired strain were compared in the condition of the HPI transfer. Wepreliminarily discussed the function of HPI in avain Escherichia coli, provide somebasis to further clarification of characteristics of the transfer HPI in avain Escherichiacoli and the relationship of the avian Escherichia coli pathogenicity with HPI. Thespecific content and results are as follows:1. Analysis of characteristics of the distribution of HPI in avian Escherichiacoli clinical isolates and the correlation of HPI+isolatesGenomic DNAs of avian Escherichia coli strains were extracted as templates,specific primers were designed and synthesized for irp2, fyuA, intB and ERIC genes.The positive rate of HPI was detected by the established PCR detection methods andthe genotype correlation of clinical isolates of HPI positive was analyzed, then thecorrelation of irp2and intB genes of them was analyzed. The results showed thatthere were40avian Escherichia coli strains, the positive rate was17.5%, and therewas a fyuA deletion strains in7HPI positive strains. there was a big differencebetween the genotypes of7HPI positive Escherichia coli strains, indicating that HPI can exist in multiple genotypes of Escherichia coli irp2and intB of HPI positivestrains was highly conservative, the homology of these genes among same strains wasnot always higher.2. The HPI transfer in avian Escherichia coli clinical isolates and theidentification of its donor and receptorSuitable HPI donors and acquired strains were co-cultured in LB liquid mediumcontaining0.2mM with the proportion of10%after activated inoculation of plant, theinoculation temperature was4℃. The HPI transfer strain was screened with thefollowing methods, chloramphenicol resistance plate, irp2gene PCR and ERIC-PCR.The acquired strain of HPI transfer was determined by the analsis of the gene typedifference of the transfer strain and acquired strains. The donor strain of HPI transferwas detemined by PCR amplification of intB gene of transfer strain and donor strains.The results showed HPI positive colony was obtained from chloramphenicolresistance plate, comparative analysis of ERIC-PCR after the discovery, the genotypesimilarity between the strain and a acquired strain, named AE-a1,was100%, thedifference with other donor strains were more than20%. The int gene in AE-t andAE-d2was the same, unlike AE-d1.3. Effects on growth characteristics and the pathogenicity of the receptorwith the transfer of HPIThe effects on the HPI acquired strain with the transfer of HPI were detected asfollows: liquid LB was added into96holes cell culture plate and the tested bacteriawas inoculated and cultured, the OD600of bacterium liquid was detected each2hoursand the growth curve was made. Treated bacterial suspension was inoculated on theCAS solid plate, and then cultured untill the colony diameter did not change. Adifferent dosage of chelating agent was added into the LB medium，then equalbacteria liquids was inoculated into each hole，histograms were drawn to compare thelow tolerance of two strains. The invasion and adhesion to DF-1cell and the lethalability on of7day-old chicks of the acquired strain and the transfer strain wasdetected to compare virulence difference between them. The growth curve showed thefirst4growth period of AE-a1and AE-t was obvious, the growth speed of AE-a1wasbetter than AE-t. The CAS test showed, siderophore production capacity of AE-t wasthe highest. The growth status of AE-a1was better than that of AE-t in none ironchelator medium, but the growth status of AE-t was better than that of AE-a1when theiron chelator concentration was higher than0.128mM, and significant difference in first24h. The result of cell adhesion and invasion test and animal toxicity test showedthe virulence of AE-a1was stronger than that of AE-t Very significantly.The above results showed that, the HPI transfer strain was obtained in thecondition of low temperature and iron, and the donor and acquired strain wereconfirmed. So the horizontal transfer of HPI exists in Escherichia coli, and thetransfer may be mainly based on the homologous recombination in the HPI-adjacentsequences. The results of biological activity and pathogenicity changes of theacquired strain indicated that the HPI and the pathogenicity of avian Escherichia colihas no direct relationship and the nutritional role of HPI in avian Escherichia coli maybe the main function.