| Objective:Afa/Dr family adhesins express mainly in urinary pathogenic Escherichia coli and diffuse adhesion escherichia coli,which can cause urinary tract infection and chronic diarrhea of infant respectively.the Escherichia coli carrying Afa/Dr family adhesins has intimate relationship with urinary infect,diarrhea-relative infect,respiratory apparatus infect and some other systematic infect. This experiment took the clinical Escherichia coli as the research object,and deponded the method of molecular biology to analysis the afa/draC gene sequence and to make sure whether the gene has point mutation. Primer Premier 5 soft was used to find out the difference of amino acids with the standard bacteria.The last object was to carry out the epidemiological investigation of the Afa/Dr family adhesins of Escherichia coli isolated from urinary system,respiratory and digestive system. and to provide new ideas for the clinical infection caused by the Escherichia coli.Method:1,The strains of E.Coli were isolated from the urina of patients with UTI,sputum of patients with respiratory tract infection,the suitable of patients with acute diarrhea.2,amplification,T-A clone,sequence analysis of the afa/draC gene fragment:The afa/draC gene of the 20 clinical Escherichia coli were detected by PCR amplification taking the E.Coli IH11128(pCC90) as the positive controlling bacteria.Reclamating the PCR product by gel purification.After the T-A clone,we carryed out the PCR amplification again using the recombinant product as the template,and then had the product sequenced and analysised.3,epidemiological investigation of the Afa/Dr family adhesins:we amplified the afa/draC gene fragment of the 600 strains and use the SPSS statistical soft to compare the carrying rate of the afa/draC gene.Results:1,we collected 600 E.Coli strains in total which contained 261 strains from urina,22 strains from sputum,244 strains from suitable and the 73 strains from other systems. 2,amplification,T-A clone,sequence analysis of the afa/draC gene fragment: After the electrophoresis we found out that the clinical strains had the same product as the positive controlling strain; After T-A clone and the blue and white screening,we extracted the plasmid of the blue and the white clones, taking the two plasmids as template we carried out a PCR the same as before and then had the product sequenced.The sequence analysis showed that:There were 3-4 point mutation in the product of the clinical strains comparing with positive controlling strain on the GeneBank, and the homology was more than 99%. The point mutation was 284G→T,308T→C,600T→C and 620C→G.According to the gene sequence we got the amino acids sequence and found out that there were amino acids sequence change: Pro184→Ser and Gln190→His.3,epidemiological investigation:the positive strains of afa/draC gene was 402 out of the tatol 600 strains,among which 186 were from urine,169 were from diarrhea,13 strains were from sputamentum and 34 were from other specimen such as gastric fluid and the bring rate was 71.26%,69.55%,59.09%,46.58% separately. There were significant statistic difference at the carrying rate in two group, group 1: urine and other specimen,group 2:diarrhea and other specimen (p<0.01 and p=0.01 seperately).Conclusions:1,Afa/Dr family adhesins exists in Escherichia coli and the carrying rates were as follows Uropathogenic Escherichia coli is 71.26%, intestinal Escherichia.coli is 69.55%,respiratory intestine is 59.09%, other secretions of Escherichia coli is 46.58%.In contrast,Uropathogenic Escherichia coli and intestinal Escherichia coli isolates differed from the other secretions of Escherichia coli on statistics analysis.2,There are 3～4 point afa/draC gene mutations on Escherichia coli, and the measured strain have the same high degree of genetic variants.Gene mutations lead to the amino acid changes on the corresponding site,but whether the protein structure and function are changed needs to be further studied. |
PDF Full Text Request