Abstract：
Riemerella anatipestifer encodes an iron acquisition system, but whether it encodes the iron efflux pump and its role in antibiotic resistance are largely unknown.
To screen and identify an iron efflux gene in R. anatipestifer and determine whether and how the iron efflux gene is involved in antibiotic resistance.
In this study, gene knockout, streptonigrin susceptibility assay and inductively coupled plasma mass spectrometry were used to screen for the iron efflux gene ietA. The MIC measurements, scanning electron microscopy and reactive oxygen species (ROS) detection were used to verify the role of IetA in aztreonam resistance and its mechanism. Mortality and colonization assay were used to investigate the role of IetA in virulence.
The deletion mutant ΔietA showed heightened susceptibility to streptonigrin, and prominent intracellular iron accumulation was observed in ΔfurΔietA under excess iron conditions. Additionally, ΔietA exhibited increased sensitivity to H2O2-produced oxidative stress. Under aerobic conditions with abundant iron, ΔietA displayed increased susceptibility to the β-lactam antibiotic aztreonam due to heightened ROS production. However, the killing efficacy of aztreonam was diminished in both WT and ΔietA under anaerobic or iron restriction conditions. Further experiments demonstrated that the efficiency of aztreonam against ΔietA was dependent on respiratory complexes Ⅰ and Ⅱ. Finally, in a duckling model, ΔietA had reduced virulence compared with the WT.
Iron efflux is critical to alleviate oxidative stress damage and β-lactam aztreonam killing in R. anatipestifer, which is linked by cellular respiration.
To screen and identify an iron efflux gene in R. anatipestifer and determine whether and how the iron efflux gene is involved in antibiotic resistance.
In this study, gene knockout, streptonigrin susceptibility assay and inductively coupled plasma mass spectrometry were used to screen for the iron efflux gene ietA. The MIC measurements, scanning electron microscopy and reactive oxygen species (ROS) detection were used to verify the role of IetA in aztreonam resistance and its mechanism. Mortality and colonization assay were used to investigate the role of IetA in virulence.
The deletion mutant ΔietA showed heightened susceptibility to streptonigrin, and prominent intracellular iron accumulation was observed in ΔfurΔietA under excess iron conditions. Additionally, ΔietA exhibited increased sensitivity to H2O2-produced oxidative stress. Under aerobic conditions with abundant iron, ΔietA displayed increased susceptibility to the β-lactam antibiotic aztreonam due to heightened ROS production. However, the killing efficacy of aztreonam was diminished in both WT and ΔietA under anaerobic or iron restriction conditions. Further experiments demonstrated that the efficiency of aztreonam against ΔietA was dependent on respiratory complexes Ⅰ and Ⅱ. Finally, in a duckling model, ΔietA had reduced virulence compared with the WT.
Iron efflux is critical to alleviate oxidative stress damage and β-lactam aztreonam killing in R. anatipestifer, which is linked by cellular respiration.
摘要：
背景：鼠疫Riemerella编码铁采集系统，但是它是否编码铁外排泵及其在抗生素耐药性中的作用在很大程度上是未知的。
目的：筛选和鉴定鼠疫菌的铁外排基因，并确定铁外排基因是否以及如何参与抗生素耐药性。
方法：在本研究中，基因敲除,使用链霉素敏感性测定和电感耦合等离子体质谱法筛选铁外排基因ietA。MIC测量，扫描电镜和活性氧（ROS）检测验证了IetA在氨曲南耐药中的作用及其机制。死亡率和定植试验用于研究IetA在毒力中的作用。
结果：缺失突变体ΔietA对链霉素的易感性提高，在过量铁条件下，在ΔfuriΔietA中观察到显着的细胞内铁积累。此外,ΔietA对H2O2产生的氧化应激的敏感性增加。在富含铁的有氧条件下，由于ROS的产生增加，ΔietA对β-内酰胺抗生素氨曲南的敏感性增加。然而,在厌氧或铁限制条件下，WT和ΔietA中氨曲南的杀伤效力均降低。进一步的实验表明，氨曲南抗ΔietA的效率取决于呼吸复合物Ⅰ和Ⅱ。最后,在小鸭模型中，与WT相比,ΔietA具有降低的毒力。
结论：铁外排对减轻鼠疫菌的氧化应激损伤和β-内酰胺氨曲南的杀伤至关重要，与细胞呼吸有关。
目的：筛选和鉴定鼠疫菌的铁外排基因，并确定铁外排基因是否以及如何参与抗生素耐药性。
方法：在本研究中，基因敲除,使用链霉素敏感性测定和电感耦合等离子体质谱法筛选铁外排基因ietA。MIC测量，扫描电镜和活性氧（ROS）检测验证了IetA在氨曲南耐药中的作用及其机制。死亡率和定植试验用于研究IetA在毒力中的作用。
结果：缺失突变体ΔietA对链霉素的易感性提高，在过量铁条件下，在ΔfuriΔietA中观察到显着的细胞内铁积累。此外,ΔietA对H2O2产生的氧化应激的敏感性增加。在富含铁的有氧条件下，由于ROS的产生增加，ΔietA对β-内酰胺抗生素氨曲南的敏感性增加。然而,在厌氧或铁限制条件下，WT和ΔietA中氨曲南的杀伤效力均降低。进一步的实验表明，氨曲南抗ΔietA的效率取决于呼吸复合物Ⅰ和Ⅱ。最后,在小鸭模型中，与WT相比,ΔietA具有降低的毒力。
结论：铁外排对减轻鼠疫菌的氧化应激损伤和β-内酰胺氨曲南的杀伤至关重要，与细胞呼吸有关。
