Department of Chemistry & Biochemistry, University of Oklahoma, Norman, Oklahoma, USA.
Chemical Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, New York, USA.
Antimicrob Agents Chemother. 2023 Feb 16;67(2):e0137722. doi: 10.1128/aac.01377-22. Epub 2023 Jan 30.
Gram-negative bacteria are notoriously more resistant to antibiotics than Gram-positive bacteria, primarily due to the presence of the outer membrane and a plethora of active efflux pumps. However, the potency of antibiotics also varies dramatically between different Gram-negative pathogens, suggesting major mechanistic differences in how antibiotics penetrate permeability barriers. Two approaches are used broadly to analyze how permeability barriers affect intracellular accumulation of antibiotics. One compares the antibacterial activities of compounds, while the other measures the total intracellular concentrations of compounds in nongrowing cells, with both approaches using strains harboring wild-type or genetically modified efflux systems and permeability barriers. Whether the two assays provide similar mechanistic insights remains unclear. In this study, we analyzed the intracellular accumulation and antibacterial activities of antibiotics representative of major clinical classes in three Gram-negative pathogens of high clinical importance, Pseudomonas aeruginosa, Escherichia coli, and Acinetobacter baumannii. We found that both assays are informative about properties of permeability barriers, but there is no quantitative agreement between the assays. Our results show that the three pathogens differ dramatically in their permeability barriers, with the outer membrane playing the dominant role in E. coli and P. aeruginosa but efflux dominating in A. baumannii. However, even compounds of the same chemotype may use different permeation pathways depending on small chemical modifications. Accordingly, a classification analysis revealed limited conservation of molecular properties that define compound penetration into the three bacteria.
革兰氏阴性菌对抗生素的耐药性明显高于革兰氏阳性菌，这主要是由于其外膜的存在和大量活跃的外排泵。然而，不同革兰氏阴性病原体对抗生素的敏感性也有很大差异，这表明抗生素穿透渗透屏障的机制存在重大差异。目前广泛采用两种方法来分析渗透屏障如何影响抗生素在细胞内的积累。一种方法比较化合物的抗菌活性，另一种方法测量非生长细胞中化合物的总细胞内浓度，这两种方法都使用携带野生型或基因修饰的外排系统和渗透屏障的菌株。这两种测定方法是否提供类似的机制见解尚不清楚。在这项研究中，我们分析了三种具有高度临床重要性的革兰氏阴性病原体（铜绿假单胞菌、大肠杆菌和鲍曼不动杆菌）中代表主要临床类别的抗生素的细胞内积累和抗菌活性。我们发现，这两种测定方法都能提供关于渗透屏障特性的信息，但两种方法之间没有定量一致性。我们的研究结果表明，这三种病原体的渗透屏障差异很大，大肠杆菌和铜绿假单胞菌的外膜起主要作用，而鲍曼不动杆菌则主要依赖外排。然而，即使是同一化学类型的化合物也可能根据微小的化学修饰而使用不同的渗透途径。因此，分类分析显示，定义化合物穿透三种细菌的分子特性的保守性有限。
