Antibiotic resistance poses a serious challenge to public health worldwide; however, the development of new antibiotic classes for combating bacterial infections, especially those caused by Gram-negative pathogens, has slowed in recent years. Dual-acting hybrid antibiotics with a metabolically non-cleavable covalent bond represent an emerging strategy for developing novel antibiotic classes to overcome antibiotic resistance. The covalent connection between two antibiotics results in a fixed pharmacokinetic profile of a single molecule and can impede bacterial efflux. However, as most antibiotics do not have membrane-destabilizing activity, the resulting increase in molecular weight by connection of two antibiotics could limit their activity against Gram-negative bacteria, whose outer membrane forms a strong barrier blocking the penetration of high-molecular weight antibiotics. Here, we review recent developments in dual-acting hybrid antibiotics targeting Gram-negative bacteria, with a focus on their antibacterial efficacy. We also discuss combination therapy strategies in which the underlying molecular mechanisms of synergy have been characterized. Finally, we outline future directions for the rational design of hybrid antibiotics against Gram-negative pathogens.

Porins in the outer membrane (OM) of Gram-negative bacteria play two main functions: passage of various extracellular molecules and maintenance of membrane integrity. OmpC, a non-specific porin, is involved in both functions; however, the exact mechanism of maintenance of membrane integrity remains unknown. In this study, we found that inhibiting cardiolipin biosynthesis partially restored the growth defect of the ompC mutant under envelope stress. Among the three enzymes involved in cardiolipin biosynthesis, ClsABC, this effect is primarily associated with ClsA. Notably, the deletion of ClsA also suppressed the similar phenotypes of an Escherichia coli mutant lacking YhdP, a transmembrane protein involved in phospholipid transport from the inner membrane to the OM. Collectively, these results imply that OmpC may contribute to membrane integrity, partially through mechanisms linked to transport or biosynthesis of phospholipids such as cardiolipin.