应用脉冲式胶体电泳法分析猪只大肠杆菌基因型.doc

應用脈衝式膠體電泳法分析豬隻大腸桿菌基因型Application of Pulsed Field Gel Electrophoresis for Analysis of Escherichia coli from Swine 研究生：張惠卿 Chang, Hui-chingLiao, Ming-Huei 【摘要】 大腸桿菌 Escherichia coli 為革蘭氏陰性短桿菌，寄生於人類與溫血動物腸道內。大腸桿菌在豬場常被分離，造成原發性及繼發性不等程度的傷害。由於臨床上抗生素使用過量以至濫用之情況下，使得菌株產生抗藥性之問題更加嚴重，故細菌的治療已無法達到預期之療效。本試驗目的為經由發病豬隻所分離出之大腸桿菌，以脈衝式膠體電泳技術 pulsed-field gel electrophoresis, PFGE 分析菌株間之基因型，另檢測菌株是否帶有抗藥性基因及其對抗生素感受性之關係，提供養豬場於豬隻管理用藥時參考。從疑似感染大腸桿菌的下痢豬隻，經由生化鑑定及API20E確認為E. coli後，以聚合酶連鎖反應 polymerase chain reaction, PCR 、紙錠敏感試驗 disk susceptibility test 及脈衝式膠體電泳 PFGE 進行試驗。結果顯示PCR檢測之class I integron基因,可得產物分別為1009 bp及1913 bp，其中又以1913 bp 42%；45/108 最多。紙錠敏感試驗結果顯示，單一藥物抗藥性比例以Streptomycin 84.3%最高，其次為Tetracycline 83.3%；進一步以PFGE進行大腸桿菌基因分型，結果發現所收集下痢豬隻來源的大腸桿菌菌株其具高度的基因多樣性。關鍵詞：大腸桿菌、脈衝式膠體電泳、聚合酶連鎖反應E. coli are gram negative rod shaped bacteria that lives inside the intestinal tracts of humans and warm-blooded animals. E. coli is usually seen in piggeries, and cause primary and secondary infections. As a result of their common occurence antibiotics have been over used, resulting in increased drug resistance, making it very difficult to control and treat diseases caused by E. coli. The aim of this study bacterial strains isolated from pigs to identify the genes responsible for drug resistance and carry out antibiotic sensitivity tests to support drug use at the piggery. Using Pulsed Field Gel Electrophoresis PFGE it possible to recognize the genotype at the same time, understand the bacterial strain evolutional status. The isolated strains were detected using biochemistry and API20E techniques. This was followed by PCR, disk susceptibility test and PFGE. PCR was able to detect two integron class 1 genes one was 1009 bp while the other was 1913 bp. The 1913 bp product was most common in the isolates 42%; 45/108 . The disk susceptibility test results suggest that the isolates were highly resistant to Streptomycin 84.3% followed by Tetracycline 83.3% . PFGE was able to detect E. coli genotypes an