| 1. Isolation and identification of avian pathogenic Escherichia coli Samples of the birds died of colisepticemia were collected from the veterinary stations and breeding fields in and around the JiangSu province. Bacteria were isolated and identified by biochemistry identification in the lab. Finally, 195 APEC ( avian pathogenic Escherichia coli ) strains were identified. The O serotyping of these bacteria strains were performed by slide agglutination and cuvette agglutination using the standard O antiserum. The results of O serotype test showed that all the APEC strains isolated from poultries belonging to 53 serotypes, which exceed the number of O serotypes of the APEC strains isolated from poultries reported before.Moreover, the predominant O serotypes were O78,O88,O9,O93,O24,O109,O86. There existed some differences between APEC strains isolated from different areas and different poultry hosts. Although the O78 is the main serotype in most of the places where the bacteria strains isolated, there was no one APEC strain belonging to this serotype in the poultries from Jiangxi and Yangzhou. Moreover, the prominant O serotype of the APEC strains were O78, O109, O88 and O86 from chicken, O9,O93 from gooses. Furthermore, O114, O118, O123, O141, O23 and O73 were only found in gooses. The O143 was only found in ducks, and O109 and some other 28 serotypes were existed in the APEC strains isolated from chicken. O11 and O107 were found in all the three hosts.2. The drug sensitivity test for 195 APEC bacteria strains Nineteen antibiotic scrips popular in clinic were selected to the drug sensitivity test. By the K-B round scrips method, 195 APEC bacteria strains were detected the antibiotic sensitive. And the data were analysed according to the standard recommended by NCCLS ( National committee of clinical and laboratory standard). Results showed that antibiotic-resistance of the APEC strains is very popular. There were more than 70% of the strains resistant to NOR, CIP, ENNO, SXT, SPT, ,S, TE, AZI and RF. About 90% of the strains resist to more than eight drugs in our test. Comparatively, some new and special antibiotics such as Fosfomycin, CRO and CTX and so on have good effect to the APEC bacteria strains. There were some differences in the resistance to the antibiotics among those APEC strains isolated from different areas. Arrangeing the antibiotics in a certain sequence (antibiotic resistance pattern), we found that there was some relationship between the patterns of antibiotic resistance of the strains and the areas of the strains. Reversely, no evident relation between the pattern and the hosts of the strains were found.3. The molecular epidemiology on the virulent factors of APEC strainsAccording to the virulence related gene sequences available in the GenBank and some research reports, thirteen pairs of specific primers against different virulence genes of APEC bacteria strains were designed (the details of the primers were showed in latter chapters). The sequences of the thirteen virulence associated genes were acquired by PCR and all the sequences were analysed comparing to the standard sequences. Those genes which have a high homology to the standard sequences (above 95%) were set as the positive strains to the test. After optimizing the conditions, we built the PCR methods to get the epidemiological datas of 195 APEC strains. Results of the epidemiology showed that the genes were detected by different positive ratio respectively. The fimC and iucD gene which belonging to the colonization system and the iron acquisition-related system were detected with high rate, 93.8%,71.8% separately. It is suggested that colonization and metallic ion are important factors to the pathogenicity of the APEC. The irp2 and fyuA gene which are the structural genes of HPI Pathogenicity Island, have the same detection rate in the same strains, which is 45.1%. Comparatively, the papC and hlyE gene share a relative low positive ratio, 7.2% and 4.1%. The detection ratio to Stx2f, Vat, astA, colV, Tsh and Iss genes were hoist, which were 15.4%,26.2%,30.3%,34.9%,50.8% and 60.0% respectively. However, the eae gene which belonging to the LEE pathogenicity island was not detected any more.The irp2 and fyuA gene were detected with a higher positive rate which is 94.7% in the strains belonging to the O78 serotype than any other stains. The same evidence was found in the stx2f and vat genes, they existed in the strains of O11 serotype with a higher rate 66.7% than in any other strains. Moreover, we found that the papC gene existed in the O2 and O24 serotypes mainly. After arranging the virulence related genes in a certain pattern, we found that the fimC-iucD-irp2-fyuA-iss-colV-tsh combination pattern was shared in 32 APEC strains, about 16.4% to the total strains. APEC strains belonging to the predominant serogroups keep less patterns than those strains belonging to the other serogroups, which suggested that the patterns of virulence related genes have some relationship with the O serotypes.4. Mechanism of resistance to fosfomycin in E.coli field strainsAccording to the drug sensitivity test of the 195 APEC strains, three APEC strains were found the resistance to fosfomycin. They were JE1 strain, IF7 strain and CD11 strain. The MIC of strain JE1 and CD11 to fosfomycin were very high, up to 125000μg /ml and 51200μg /ml respectively. The other strain IF11 was a little low comparatively, the MIC value for it was 1280μg /ml. To get the information of the bio-transport system for fosfomycin in the cell wall and the uptaking ability to fosofmycin of the APEC strains, the three bacteria strains were cultured in minimal salts medium to measure their growth condition. The intracellular active fosfomycin level were determined by microbiological assay. The results showed that there were some mutations in the glpT transport system of IF7 strain, which decreased the transportion rate for fosfomycin into the bacterial cells and caused the resistant to fosfomycin with a little low level. From the test of intracellular active fosfomycin level, we found that the uptake capacity to fosfomycin of the CD11strain was very low comparing to the negative control strain BL21. It may contribute to the high resistant level for the CD11 strain. In contrast, the uptake capacity of the IF7 strain wassome lower than BL21. It caused by the mutation of the glpT transport system at a certain extent.The target of fosfomycin is a catalysis enzyme which encoded by the murA gene on the chromosome DNA. According to the sequence of the standard murA gene, a pair of specific primers against the gene were designed for the PCR assay. After amplifing the murA gene, the nucleotide sequence of the gene from the three APEC strains were get and analysised with the standard murA gene sequence. Moreover, the products of the PCR assay were purified and inserted into the prokaryotic expressional pET32a vector, the recombinant vector was then transported into the BL21(DE3) strain to construct the recombinant bacteria strains. The MIC value of the three recombinant strains to fosomycin were acquired to determine whether there are some relationship between the mutation on the murA gene and the fosfomycin resistance phenotype. From the results of the sequencing of the murA gene, we found that there existing a mutation T116A on the amino acid level of the CD11 strain, and the MIC to fosfomycin of the recombinant bacteria using the murA gene of CD11strain is very high. We can conclude that the mutation on murA of CD11 contribute to the high resistant level of CD11 strain. Interestingly, we found that although there is none mutation on the murA gene of JE1 on the amino acid level, but the recombinant bacteria using it murA gene resistant to fosfomycin at a high level.To get the expression level of the murA gene of the three different APEC srains, the total RNA of them were isolated and were transfered into cDNA by RT-PCR assay. Then the cDNA were used as templates to the relative quantification Real-Time PCR using the SYBR Green as indicator and the expression level of 16sRNA of the bacteria strains as endogenous control. The results of the assay suggested that all the expression level of the three fosfomycin resistant APEC strains were higher to the negative control bacteria strain BL21. Especially, the expression level of JE1 strain was the highest among the three APEC strains, about twice to IF7 and CD11 and quadplex to BL21 strain. It suggested that the increase expression of the gene may contribute to the high resistant level to fosfomycin partly. At the same time, we also found that both of the IF7 and CD11 strain sharing the nearly expression level of the murA gene which about triple to the BL21 strain. These results indicated that the mechanisms to fosfomycin resistance shared by the three field APEC strains are different and the mechanism exsiting in the bacteria strains is complex. |
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