鸭源鸡杆菌ompW基因缺失菌株的构建及其药物敏感性分析

Construction of ompW Gene Deletion Strain of Gallibacterium anatis and Its Susceotibility to Antibacterial Agents

探索OmpW在鸭源鸡杆菌耐药性形成中的作用。运用M-IV培养基诱导鸭源鸡杆菌自然感受态细胞,用含有氯霉素抗性(Cmpr)筛选标记和同源臂的线性DNA打靶片段替换目的基因;以PCR、SDS-PAGE及Western blot鉴定阳性转化子ΔompW。通过微量稀释法测定12种抗菌药物对鸭源鸡杆菌亲本菌株RU和突变株ΔompW的最小抑菌浓度(MIC)。结果显示:鸭源鸡杆菌阳性转化子缺失了ompW,成功构建了ompW缺失突变菌株ΔompW;相对于RU,土霉素和四环素对ΔompW的MIC值分别降低到1/8(16/128,P<0.01)和1/32(4/128,P<0.01),其他药物的MIC值无明显变化。本研究首次利用自然转化法构建了鸭源鸡杆菌ompW缺失菌株ΔompW;OmpW在鸭源鸡杆菌抗四环素类药物中发挥重要作用。

The study was conducted to explore the role of ompW gene in the drug resistance of Gallibacterium anatis（G.anatis）.The ompW gene knock-out mutant of G.anatis was constructed by natural transformation.The competent cells of G.anatis were induced following transfer of cells to M-IV medium from colonies on a blood agar plate.Then,transformation was performed by incubating the M-IV-compentent cells together with linear DNA fragment consisting of homologous arms and cmpr gene.The transformants were identified by using PCR,SDS-PAGE and Western blot analysis.The MIC-determinations of 12 kinds of antibacterial agents were performed for both wild type and mutantΔompW G.anatis using Mueller Hinton-II.The ompW gene deletion strainΔompW was successfully constructed.Comparing with wild type G.anatis,the MICs of terramycin and tetracycline forΔompW were decreased to 1/8-fold（16/128）and1/32-fold（4/128）,respectively.The MICs of other drugs in this study showed no obvious change.In conclusion,ompW deletion strain of G.anatis was constructed by natural transformation for the first time in this study.OmpW plays an important role in the formation of tetracyclines resistance phenotype of G.anatis strains.