鸭疫里默氏杆菌MFS外排泵rant基因缺失株的构建及其介导的耐药性

Construction of MFS Efflux Pump rant Gene Deletion Mutant of Riemerella anatipestifer and Its Contribution to Drug Resistance

旨在构建鸭疫里默氏杆菌rant基因缺失株及回复株,明确MFS外排泵Rant对鸭疫里默氏杆菌耐药性的影响。本研究以鸭疫里默氏杆菌RA-LZ01株为研究对象,通过自杀质粒pRE112介导的同源重组,以红霉素抗性标记进行正向筛选,获得基因缺失株Δrant,并以大肠杆菌-鸭疫里默氏杆菌穿梭质粒pCPRA作为载体,构建回复株cΔrant。采用微量肉汤稀释法测定11类临床常用抗生素对野生株、缺失株及回复株的最小抑菌浓度(minimum inhibitory concentration,MIC),并测定3株菌的生长曲线,评价菌株对雏鸭的致病能力。结果表明,与野生株和回复株相比,四环素类抗生素对缺失株的MIC值明显下降,其他类抗生素的MIC值无变化;缺失株的生长速率明显下降;rant基因的缺失显著降低鸭疫里默氏杆菌的毒力。结果提示,鸭疫里默氏杆菌rant基因介导四环素类抗生素的外排,并可明显影响鸭疫里默氏杆菌的生长和毒力。

This study aimed to construct rant gene-deleted mutant and complemented strain to study the effect of MFS efflux pump Rant on the resistance of R.anatipestifer.R.anatipestifer RA-LZ01 strain was used as the parental strain.The deletion mutantΔrant was obtained by the suicide plasmid pRE112-mediated homologous recombination,and rant gene was replaced with erythromycin resistance cassette.The E.coli-R.anatipestifer shuttle plasmid pCPRA was used as the vector to construct the complemented strain cΔrant.The minimal inhibitory concentrations(MICs)of 11 classes of clinically common antibiotics against wild strain,deletion mutant and complemented strain were determined with micro broth dilution assay.The growth ability and virulence of the above 3 strains were also determined.The results showed that compared with the wild strain,the MIC value of tetracycline antibiotics to the deletion mutant was significantly reduced,and the MIC values of other antibiotics did not change.The growth rate of the deletion mutant was significantly reduced by comparison with that of the parental strain.Furthermore,the deletion of the rant gene significantly reduced the virulence of R.anatipestifer.These results indicate that rant gene of R.anatipestifer mediates the efflux of tetracycline antibiotics and can significantly affect the growth and virulence of R.anatipestifer.