Poor penetration through the outer membrane (OM) of Gram-negative bacteria is a major barrier of antibiotic development. While β-lactam antibiotics are commonly used against Klebsiella pneumoniae and Enterobacter cloacae, there are limited data on OM permeability especially in K. pneumoniae Here, we developed a novel cassette assay, which can simultaneously quantify the OM permeability to five β-lactams in carbapenem-resistant K. pneumoniae and E. cloacae Both clinical isolates harbored a bla KPC-2 and several other β-lactamases. The OM permeability of each antibiotic was studied separately ("discrete assay") and simultaneously ("cassette assay") by determining the degradation of extracellular β-lactam concentrations via multiplex liquid chromatography-tandem mass spectrometry analyses. Our K. pneumoniae isolate was polymyxin resistant, whereas the E. cloacae was polymyxin susceptible. Imipenem penetrated the OM at least 7-fold faster than meropenem for both isolates. Imipenem penetrated E. cloacae at least 258-fold faster and K. pneumoniae 150-fold faster compared to aztreonam, cefepime, and ceftazidime. For our β-lactams, OM permeability was substantially higher in the E. cloacae compared to the K. pneumoniae isolate (except for aztreonam). This correlated with a higher OmpC porin production in E. cloacae, as determined by proteomics. The cassette and discrete assays showed comparable results, suggesting limited or no competition during influx through OM porins. This cassette assay allowed us, for the first time, to efficiently quantify the OM permeability of multiple β-lactams in carbapenem-resistant K. pneumoniae and E. cloacae Characterizing the OM permeability presents a critical contribution to combating the antimicrobial resistance crisis and enables us to rationally optimize the use of β-lactam antibiotics.IMPORTANCE Antimicrobial resistance is causing a global human health crisis and is affecting all antibiotic classes. While β-lactams have been commonly used against susceptible isolates of Klebsiella pneumoniae and Enterobacter cloacae, carbapenem-resistant isolates are spreading worldwide and pose substantial clinical challenges. Rapid penetration of β-lactams leads to high drug concentrations at their periplasmic target sites, allowing β-lactams to more completely inactivate their target receptors. Despite this, there are limited tangible data on the permeability of β-lactams through the outer membranes of many Gram-negative pathogens. This study presents a novel, cassette assay, which can simultaneously characterize the permeability of five β-lactams in multidrug-resistant clinical isolates. We show that carbapenems, and especially imipenem, penetrate the outer membrane of K. pneumoniae and E. cloacae substantially faster than noncarbapenem β-lactams. The ability to efficiently characterize the outer membrane permeability is critical to optimize the use of β-lactams and combat carbapenem-resistant isolates.

中文翻译：

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新颖的盒式测定同时量化耐碳青霉烯类肺炎克雷伯菌和阴沟肠杆菌中五种 β-内酰胺的外膜渗透性。


革兰氏阴性菌外膜（OM）渗透性差是抗生素开发的主要障碍。虽然 β-内酰胺抗生素通常用于对抗肺炎克雷伯菌和阴沟肠杆菌，但有关 OM 渗透性的数据有限，尤其是肺炎克雷伯菌。在这里，我们开发了一种新型盒式测定法，可以同时量化碳青霉烯类中 5 种 β-内酰胺的 OM 渗透性耐药肺炎克雷伯菌和阴沟肠杆菌这两种临床分离株均含有 bla KPC-2 和其他几种 β-内酰胺酶。通过多重液相色谱-串联质谱分析确定细胞外β-内酰胺浓度的降解，分别研究每种抗生素的 OM 渗透性（“离散测定”）和同时研究（“盒式测定”）。我们分离的肺炎克雷伯菌对多粘菌素具有抗性，而阴沟肠杆菌对多粘菌素敏感。对于两种分离株，亚胺培南渗透 OM 的速度至少比美罗培南快 7 倍。与氨曲南、头孢吡肟和头孢他啶相比，亚胺培南渗透阴沟肠杆菌的速度至少快 258 倍，渗透肺炎克雷伯菌的速度至少快 150 倍。对于我们的 β-内酰胺，阴沟肠杆菌中的 OM 渗透性明显高于肺炎克雷伯菌分离株（氨曲南除外）。根据蛋白质组学的测定，这与阴沟肠杆菌中较高的 OmpC 孔蛋白产量相关。盒式分析和离散分析显示出可比较的结果，表明在通过 OM 孔蛋白流入期间竞争有限或没有竞争。这种盒式测定使我们首次能够有效量化耐碳青霉烯类肺炎克雷伯菌和大肠杆菌中多种 β-内酰胺的 OM 渗透性。 阴沟菌 OM 渗透性的表征对于应对抗菌素耐药性危机具有重要贡献，并使我们能够合理优化 β-内酰胺抗生素的使用。 重要性 抗菌素耐药性正在引发全球人类健康危机，并影响所有抗生素类别。虽然β-内酰胺通常用于对抗肺炎克雷伯菌和阴沟肠杆菌的敏感分离株，但碳青霉烯类耐药分离株正在全球范围内传播，并带来了巨大的临床挑战。 β-内酰胺的快速渗透导致其周质靶位点的药物浓度较高，从而使β-内酰胺能够更完全地灭活其靶受体。尽管如此，关于 β-内酰胺通过许多革兰氏阴性病原体外膜的渗透性的具体数据仍然有限。这项研究提出了一种新颖的盒式测定，可以同时表征多重耐药临床分离株中五种β-内酰胺的渗透性。我们发现碳青霉烯类，尤其是亚胺培南，穿透肺炎克雷伯菌和阴沟肠杆菌外膜的速度明显快于非碳青霉烯类β-内酰胺类。有效表征外膜渗透性的能力对于优化 β-内酰胺的使用和对抗碳青霉烯类耐药菌株至关重要。