Department of Biotechnology and Laboratory Science in Medicine, National Yang-Ming University, Taipei, Taiwan.
Department of Pathology and Laboratory Medicine, Taipei Veterans General Hospital, Taipei, Taiwan.
J Antimicrob Chemother. 2021 May 12;76(6):1480-1486. doi: 10.1093/jac/dkab059.
Stenotrophomonas maltophilia, an opportunistic pathogen, is intrinsically resistant to most β-lactams except ceftazidime and ticarcillin/clavulanate, due to the inducibly expressed L1 and L2 β-lactamases. A two-component regulatory system (TCS) allows organisms to sense and respond to changes in different environmental conditions. The PhoPQ TCS of S. maltophilia plays regulatory roles in antibiotic susceptibility, physiology, stress adaption and virulence. Inactivation of S. maltophilia phoPQ increases β-lactam susceptibility.
To elucidate the PhoPQ-regulating mechanism for β-lactam resistance.
The candidate genes responsible for the ΔphoPQ-mediated β-lactam resistance compromise were identified by transcriptome analysis and verified by quantitative RT-PCR and complementation assay. Etest was used to assess β-lactam susceptibility. The phosphorylation level of the PhoP protein was determined by Phos-tag SDS-PAGE and western blotting. A β-lactam influx assay was used to investigate the influx efficiency of a β-lactam.
PhoPQ deletion down-regulated the expression of mltD1 and slt, attenuated the induced β-lactamase activity and then compromised the β-lactam resistance. Complementation of mutant phoPQ with mltD1 or slt genes partially reverted the induced β-lactamase activity and β-lactam resistance. The PhoPQ TCS was activated in logarithmically grown KJ cells and was further activated by low magnesium, but not by a β-lactam. However, low-magnesium-mediated PhoPQ activation hardly made an impact on β-lactam resistance enhancement. Furthermore, PhoPQ inactivation altered the outer membrane permeability and increased the influx of a β-lactam.
The PhoPQ TCS is activated to some extent in physiologically grown S. maltophilia. Inactivation of phoPQ attenuates the expression of mltD1 and slt, and increases β-lactam influx, both synergically contributing to β-lactam resistance compromise.
嗜麦芽窄食单胞菌是一种机会致病菌，由于可诱导表达的 L1 和 L2β-内酰胺酶，对大多数β-内酰胺类抗生素（除头孢他啶和替卡西林/克拉维酸外）固有耐药。双组分调控系统（TCS）使生物体能够感知和响应不同环境条件下的变化。嗜麦芽窄食单胞菌的 PhoPQ TCS 在抗生素敏感性、生理、应激适应和毒力方面发挥调节作用。嗜麦芽窄食单胞菌 phoPQ 的失活增加了β-内酰胺类抗生素的敏感性。
阐明 PhoPQ 调节β-内酰胺类抗生素耐药性的机制。
通过转录组分析确定了与ΔphoPQ 介导的β-内酰胺类抗生素耐药性相关的候选基因，并通过定量 RT-PCR 和互补实验进行了验证。Etest 用于评估β-内酰胺类抗生素的敏感性。通过 Phos-tag SDS-PAGE 和 Western blot 测定 PhoP 蛋白的磷酸化水平。通过β-内酰胺类抗生素流入实验研究β-内酰胺类抗生素的流入效率。
PhoPQ 缺失下调了 mltD1 和 slt 的表达，减弱了诱导的β-内酰胺酶活性，从而降低了β-内酰胺类抗生素的耐药性。用 mltD1 或 slt 基因互补突变体 phoPQ 部分恢复了诱导的β-内酰胺酶活性和β-内酰胺类抗生素的耐药性。PhoPQ TCS 在对数生长期的 KJ 细胞中被激活，并进一步被低镁激活，但不能被β-内酰胺激活。然而，低镁介导的 PhoPQ 激活对增强β-内酰胺类抗生素耐药性几乎没有影响。此外，PhoPQ 的失活改变了外膜通透性并增加了β-内酰胺类抗生素的流入。
在生理生长的嗜麦芽窄食单胞菌中，PhoPQ TCS 被一定程度地激活。PhoPQ 的失活减弱了 mltD1 和 slt 的表达，并增加了β-内酰胺类抗生素的流入，两者协同作用导致β-内酰胺类抗生素耐药性降低。
