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

doi:10.11843/j.issn.0366-6964.2021.07.021

畜牧兽医学报 ›› 2021, Vol. 52 ›› Issue (7): 1991-1999.doi: 10.11843/j.issn.0366-6964.2021.07.021

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

权衡¹, 陈启伟², 宫晓炜², 王燕萍², 秦明星¹, 朱雨佳¹, 郑福英^2*, 蔺国珍^1*

1. 西北民族大学生命科学与工程学院, 兰州 730030;
2. 中国农业科学院兰州兽医研究所, 家畜疫病病原生物学国家重点实验室, 兰州 730046

收稿日期:2020-11-04 出版日期:2021-07-23 发布日期:2021-07-23
通讯作者: 蔺国珍,主要从事细菌耐药机制和致病机制研究,E-mail:lgzh18@163.com;郑福英,主要从事细菌耐药机制和致病机制研究,E-mail:zhengfuying@caas.cn
作者简介:权衡(1996-),男,陕西宝鸡人,硕士生,主要从事细菌耐药机制和致病机制研究,E-mail:1214453647@qq.com
基金资助:
中央高校基本科研业务费专项资金（31920180007）；中国农业科学院中央级公益性科研院所基本科研业务费专项（1610312020001）

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

QUAN Heng¹, CHEN Qiwei², GONG Xiaowei², WANG Yanping², QIN Mingxing¹, ZHU Yujia¹, ZHENG Fuying^2*, LIN Guozhen^1*

1. College of Life Science and Engineering, Northwest Minzu University, Lanzhou 730030, China;
2. State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China

Received:2020-11-04 Online:2021-07-23 Published:2021-07-23

摘要/Abstract

摘要： 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。

关键词: 鸭疫里默氏杆菌, rant基因, 耐药性, 生长, 毒力

Abstract: 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 mutant Δrant 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.

Key words: Riemerella anatipestifer, rant gene, resistance, growth, virulence

中图分类号:

S852.61

权衡, 陈启伟, 宫晓炜, 王燕萍, 秦明星, 朱雨佳, 郑福英, 蔺国珍. 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[J]. 畜牧兽医学报, 2021, 52(7): 1991-1999.

QUAN Heng, CHEN Qiwei, GONG Xiaowei, WANG Yanping, QIN Mingxing, ZHU Yujia, ZHENG Fuying, LIN Guozhen. Construction of MFS Efflux Pump rant Gene Deletion Mutant of Riemerella anatipestifer and Its Contribution to Drug Resistance[J]. Acta Veterinaria et Zootechnica Sinica, 2021, 52(7): 1991-1999.

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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

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