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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新型盒式测定可同时定量耐碳青霉烯的肺炎克雷伯菌和阴沟肠杆菌中五个β-内酰胺的膜透过性。

通过革兰氏阴性细菌的外膜（OM）渗透不良是抗生素发展的主要障碍。尽管β-内酰胺抗生素通常用于抗肺炎克雷伯菌和阴沟肠杆菌，但关于OM渗透性的数据有限，尤其是在肺炎克雷伯菌中。在这里，我们开发了一种新型盒式分析，可以同时量化碳青霉烯中5种β-内酰胺的OM渗透性耐药的肺炎克雷伯菌和阴沟肠杆菌两种临床分离株均带有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（“离散测定”）和同时（“盒式测定”）研究每种抗生素的OM渗透性。我们的K 肺炎分离株对多粘菌素具有抗性，而阴沟肠杆菌对多粘菌素敏感。对于两种分离物，亚胺培南穿透OM的速度比美罗培南至少快7倍。与氨曲南，头孢吡肟和头孢他啶相比，亚胺培南穿透阴沟肠杆菌的速度至少快258倍，而肺炎克雷伯菌的速度快150倍。对于我们的β-内酰胺类，阴沟肠杆菌的OM渗透性比肺炎克雷伯菌分离株（氨曲南除外）高得多。如蛋白质组学所确定，这与阴沟肠杆菌中较高的OmpC孔蛋白产量相关。盒式和离散测定显示出可比的结果，表明在通过OM孔蛋白流入期间竞争有限或没有竞争。这种盒式分析首次使我们能够有效地量化耐碳青霉烯的肺炎克雷伯氏菌和大肠杆菌中多个β-内酰胺的OM渗透性。泄殖腔表征OM渗透性对战胜抗药性耐药性危机至关重要，并使我们能够合理地优化使用β-内酰胺类抗生素。重要说明抗药性正在引起全球人类健康危机，并影响所有抗生素类别。虽然β-内酰胺类药物通常用于抗击肺炎克雷伯菌和阴沟肠杆菌的易感分离株，但耐碳青霉烯的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-内酰胺更完全地失活其靶受体。尽管如此，关于β-内酰胺通过许多革兰氏阴性病原体的外膜的渗透性的有限的有限数据。这项研究提出了一种新颖的盒式检测方法，可以同时表征五种β-内酰胺在耐多药临床分离株中的渗透性。我们显示，碳青霉烯类，尤其是亚胺培南，比非卡巴培南β-内酰胺渗透速度更快，穿透肺炎克雷伯菌和阴沟肠杆菌的外膜的速度更快。有效表征外膜通透性的能力对于优化β-内酰胺的使用和对抗耐碳青霉烯的菌株至关重要。