School of Life Sciences, Anhui Agricultural University, Hefei, Anhui 230036, China.
School of Life Sciences, Anhui Agricultural University, Hefei, Anhui 230036, China.
Poult Sci. 2020 Jul;99(7):3675-3687. doi: 10.1016/j.psj.2020.03.064. Epub 2020 Apr 24.
Avian pathogenic Escherichia coli (APEC) is a specific group of extraintestinal pathogenic E. coli that causes a variety of extraintestinal diseases in chickens, ducks, pigeons, turkeys, and other avian species. These diseases lead to significant economic losses in the poultry industry worldwide. However, owing to excessive use of antibiotics in the treatment of infectious diseases, bacteria have developed antibiotic resistance. The development of multidrug efflux pumps is one important bacterial antibiotic resistance mechanism. A multidrug efflux pump, MdtH, which belongs to the major facilitator superfamily of transporters, confers resistance to quinolone antibiotics such as norfloxacin and enoxacin. LsrR regulates hundreds of genes that participate in myriad biological processes, including mobility, biofilm formation, and antibiotic susceptibility. However, whether LsrR regulates mdtH transcription and then affects bacterial resistance to various antibiotics in APEC has not been reported. In the present study, the lsrR mutant was constructed from its parent strain APECX40 (WT), and high-throughput sequencing was performed to analyze the transcriptional profile of the WT and mutant XY10 strains. The results showed that lsrR gene deletion upregulated the mdtH transcript level. Furthermore, we also constructed the lsrR- and mdtH-overexpressing strains and performed antimicrobial susceptibility testing, antibacterial activity assays, real-time reverse transcription PCR, and electrophoretic mobility shift assays to investigate the molecular regulatory mechanism of LsrR on the MdtH multidrug efflux pump. The lsrR mutation and the mdtH-overexpressing strain decreased cell susceptibility to norfloxacin, ofloxacin, ciprofloxacin, and tetracycline by upregulating mdtH transcript levels. In addition, the lsrR-overexpressing strain increased cell susceptibility to norfloxacin, ofloxacin, ciprofloxacin, and tetracycline by downregulating mdtH transcript levels. Electrophoretic mobility shift assays indicated that LsrR directly binds to the mdtH promoter. Therefore, this study is the first to demonstrate that LsrR inhibits mdtH transcription by directly binding to its promoter region. This action subsequently increases susceptibility to the aforementioned four antibiotics in APECX40.
禽致病性大肠杆菌（APEC）是一种特定的肠外致病性大肠杆菌，可引起鸡、鸭、鸽、火鸡和其他禽类的多种肠外疾病。这些疾病导致全球家禽业遭受重大经济损失。然而，由于在传染病治疗中过度使用抗生素，细菌产生了抗生素耐药性。多药外排泵的发展是细菌产生抗生素耐药性的一个重要机制。一种多药外排泵 MdtH 属于转运蛋白的主要易化子超家族，使其对诺氟沙星和恩诺沙星等喹诺酮类抗生素产生耐药性。LsrR 调节数百个参与多种生物学过程的基因，包括运动性、生物膜形成和抗生素敏感性。然而，LsrR 是否调节 mdtH 的转录，进而影响 APEC 中各种抗生素的细菌耐药性尚未有报道。在本研究中，从其亲本菌株 APECX40（WT）构建了 lsrR 突变体，并对 WT 和突变体 XY10 菌株的转录谱进行了高通量测序分析。结果表明，lsrR 基因缺失上调了 mdtH 转录水平。此外，我们还构建了 lsrR 和 mdtH 过表达菌株，并进行了药敏试验、抗菌活性测定、实时逆转录 PCR 和电泳迁移率变动分析，以研究 LsrR 对 MdtH 多药外排泵的分子调控机制。lsrR 突变和 mdtH 过表达菌株通过上调 mdtH 转录水平降低了细胞对诺氟沙星、氧氟沙星、环丙沙星和四环素的敏感性。此外，lsrR 过表达菌株通过下调 mdtH 转录水平增加了细胞对诺氟沙星、氧氟沙星、环丙沙星和四环素的敏感性。电泳迁移率变动分析表明，LsrR 直接结合 mdtH 启动子。因此，本研究首次证明 LsrR 通过直接结合其启动子区域抑制 mdtH 转录。这种作用随后增加了 APECX40 对上述四种抗生素的敏感性。
