PDF(Pubmed)
Abstract：
Chromosomal and transferable AmpC β-lactamases represent top resistance mechanisms in different gram-negatives, but knowledge regarding the latter, mostly concerning regulation and virulence-related implications, is far from being complete. To fill this gap, we used Klebsiella pneumoniae (KP) and two different plasmid-encoded AmpCs [DHA-1 (AmpR regulator linked, inducible) and CMY-2 (constitutive)] as models to perform a study in which we show that blockade of peptidoglycan recycling through AmpG permease inactivation abolished DHA-1 inducibility but did not affect CMY-2 production and neither did it alter KP pathogenic behavior. Moreover, whereas regular production of both AmpC-type enzymes did not attenuate KP virulence, when blaDHA-1 was expressed in an ampG-defective mutant, Galleria mellonella killing was significantly (but not drastically) attenuated. Spontaneous DHA-1 hyperproducer mutants were readily obtained in vitro, showing slight or insignificant virulence attenuations together with high-level resistance to β-lactams only mildly affected by basal production (e.g., ceftazidime, ceftolozane/tazobactam). By analyzing diverse DHA-1-harboring clinical KP strains, we demonstrate that the natural selection of these hyperproducers is not exceptional (>10% of the collection), whereas mutational inactivation of the typical AmpC hyperproduction-related gene mpl was the most frequent underlying mechanism. The potential silent dissemination of this kind of strains, for which an important fitness cost-related contention barrier does not seem to exist, is envisaged as a neglected threat for most β-lactams effectiveness, including recently introduced combinations. Analyzing whether this phenomenon is applicable to other transferable β-lactamases and species as well as determining the levels of conferred resistance poses an essential topic to be addressed.IMPORTANCEAlthough there is solid knowledge about the regulation of transferable and especially chromosomal AmpC β-lactamases in Enterobacterales, there are still gaps to fill, mainly related to regulatory mechanisms and virulence interplays of the former. This work addresses them using Klebsiella pneumoniae as model, delving into a barely explored conception: the acquisition of a plasmid-encoded inducible AmpC-type enzyme whose production can be increased through selection of chromosomal mutations, entailing dramatically increased resistance compared to basal expression but minor associated virulence costs. Accordingly, we demonstrate that clinical K. pneumoniae DHA-1 hyperproducer strains are not exceptional. Through this study, we warn for the first time that this phenomenon may be a neglected new threat for β-lactams effectiveness (including some recently introduced ones) silently spreading in the clinical context, not only in K. pneumoniae but potentially also in other pathogens. These facts must be carefully considered in order to design future resistance-preventive strategies.
摘要：
染色体和可转移的AmpCβ-内酰胺酶代表了不同革兰氏阴性的最高抗性机制，但是关于后者的知识，主要是关于监管和毒力相关的影响，还远远没有完成。为了填补这个空白，我们使用肺炎克雷伯菌（KP）和两种不同的质粒编码的AmpC[DHA-1（AmpR调节子连接，诱导型）和CMY-2（组成型）]作为进行研究的模型，在该研究中，我们表明通过AmpG通透酶失活阻断肽聚糖再循环可以消除DHA-1的诱导性，但不影响CMY-2的产生，也没有改变KP的致病行为。此外，而定期生产两种AmpC型酶并没有减弱KP毒力，当blaDHA-1在ampG缺陷突变体中表达时，海绵铁的杀灭作用显着（但不是急剧减弱）。自发的DHA-1超生产者突变体很容易在体外获得，显示轻微或不显著的毒力减弱以及对β-内酰胺的高水平抗性，仅受到基础生产的轻度影响（例如，头孢他啶,头孢洛赞/他唑巴坦)。通过分析各种含有DHA-1的临床KP菌株，我们证明了这些超级生产者的自然选择并不例外(>10%的集合)，而典型的AmpC高产相关基因mpl的突变失活是最常见的潜在机制。这种菌株的潜在无声传播，似乎不存在与健身成本相关的重要争用障碍，被认为是对大多数β-内酰胺有效性的被忽视的威胁，包括最近推出的组合。分析这种现象是否适用于其他可转移的β-内酰胺酶和物种以及确定所赋予的抗性水平是需要解决的重要课题。重要事项虽然有关于肠杆菌中可转移尤其是染色体AmpCβ-内酰胺酶的调节的扎实知识，仍然有空白需要填补，主要与前者的调节机制和毒力相互作用有关。这项工作使用肺炎克雷伯菌作为模型来解决他们，深入研究一个几乎没有探索过的概念：获得质粒编码的诱导型AmpC型酶，其产量可以通过选择染色体突变来增加，与基础表达相比，耐药性急剧增加，但相关毒力成本较小。因此，我们证明临床肺炎克雷伯菌DHA-1高产菌株并不例外.通过这项研究,我们首次警告说，这种现象可能是β-内酰胺有效性（包括一些最近引入的）在临床背景下默默地传播的被忽视的新威胁，不仅在肺炎克雷伯菌中，还可能在其他病原体中。为了设计未来的抵抗预防策略，必须仔细考虑这些事实。
