Envelope proteome changes driven by RamA overproduction in Klebsiella pneumoniae that enhance acquired β-lactam resistance
Envelope proteome changes driven by RamA overproduction in Klebsiella pneumoniae that enhance acquired β-lactam resistance
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DOI：
10.1093/jac/dkx345
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发表时间：
2018-01-01
影响因子：
5.2
通讯作者：
Avison, Matthew B.
中科院分区：
文献类型：
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作者：
Jimenez-Castellanos, Juan-Carlos;Ismah, Wan Ahmad Kamil Wan Nur;Avison, Matthew B.
目的：在肺炎克雷伯菌中，RaMA 的过量产生导致包膜通透性降低和抗菌药物敏感性降低，但很少观察到临床相关耐药性。在这里，我们测试了 RamA 过量生产是否可以增强肺炎克雷伯菌的获得性 β-内酰胺耐药机制，并定义了在低渗透压和高渗透压培养基中生长期间 RamA 过量生产时包膜蛋白丰度的变化。方法：使用荧光染料积累测定法估计包膜通透性。使用纸片测试测量β-内酰胺敏感性。使用 LC-MS/MS 蛋白质组学对总包膜蛋白产量进行定量，并使用实时 RT-PCR 对转录物水平进行定量。结果：RaMA 过量产生增强了 β-内酰胺酶介导的 β-内酰胺抗性，在某些情况下显着增强，而不改变 β-内酰胺酶的产量。它增加了外排泵的产生并减少了 OmpK35 孔蛋白的产生，尽管 micF 过表达表明 OmpK35 的减少对包膜通透性几乎没有影响。对肺炎克雷伯菌血流分离株的调查显示，ramA 在 4 个碳青霉烯酶产生菌中的 3 个、21 个 CTX-M 产生菌中的 1 个和 19 个不携带 CTX-M 或碳青霉烯酶的菌株中的 2 个中过度表达。结论：虽然 RamA 本身并不是肺炎克雷伯菌抗生素耐药性的关键介质，但它对于增强获得性 β-内酰胺酶介导的 β-内酰胺耐药性谱系具有潜在重要意义。 LC-MS/MS 蛋白质组学分析表明，这种增强主要是通过激活外排泵产生来实现的。
Objectives: In Klebsiella pneumoniae, overproduction of RamA results in reduced envelope permeability and reduced antimicrobial susceptibility but clinically relevant resistance is rarely observed. Here we have tested whether RamA overproduction can enhance acquired beta-lactam resistance mechanisms in K. pneumoniae and have defined the envelope protein abundance changes upon RamA overproduction during growth in low and high osmolarity media.Methods: Envelope permeability was estimated using a fluorescent dye accumulation assay. beta-Lactam susceptibility was measured using disc testing. Total envelope protein production was quantified using LC-MS/MS proteomics and transcript levels were quantified using real-time RT-PCR.Results: RamA overproduction enhanced beta-lactamase-mediated beta-lactam resistance, in some cases dramatically, without altering beta-lactamase production. It increased production of efflux pumps and decreased OmpK35 porin production, though micF overexpression showed that OmpK35 reduction has little impact on envelope permeability. A survey of K. pneumoniae bloodstream isolates revealed ramA hyperexpression in 3 of 4 carbapenemase producers, 1 of 21 CTX-M producers and 2 of 19 strains not carrying CTX-M or carbapenemases.Conclusions: Whilst RamA is not a key mediator of antibiotic resistance in K. pneumoniae on its own, it is potentially important for enhancing the spectrum of acquired beta-lactamase-mediated beta-lactam resistance. LC-MS/MS proteomics analysis has revealed that this enhancement is achieved predominantly through activation of efflux pump production.