The core physiological function of the Gram-negative bacterial outer membrane is to act as a selective permeability barrier, preventing the entry of harmful molecules such as antibiotics, detergents, bile salts, and host defense peptides while permitting the absorption of necessary nutrients. The asymmetric structure of the outer membrane lipids is key to this barrier function. This feature not only enables bacteria to survive and colonize successfully in complex host environments but also makes it difficult for larger antibiotics such as vancomycin and daptomycin to penetrate, thereby exhibiting intrinsic resistance (Gray and Wenzel, 2020; Maher and Hassan, 2023).

The outer membrane’s barrier function is dynamically regulated and can adapt to environmental stress, such as pH fluctuations, osmotic pressure changes, and antimicrobial peptide attacks. For example, bacteria can modify the surface charge of the outer membrane by partially adding phosphoethanolamine or Ara4N groups to the lipid A or core oligosaccharides of LPS via two-component regulatory systems like PmrA/PmrB (Maria-Neto et al., 2015). This alteration reduces the affinity for cationic antimicrobial peptides (CAMPs). Moreover, environmental factors such as osmotic pressure and nutrient concentrations can regulate porin expression. For instance, high osmotic pressure can promote the expression of OmpC while inhibiting OmpF expression (Maher and Hassan, 2023). This adaptive remodeling of the outer membrane, while beneficial for bacterial survival, may also influence antibiotic permeability and alter antibiotic sensitivity. Studying the dynamic regulation mechanisms of the outer membrane helps to uncover new resistance mechanisms and may provide insights for developing therapeutic targets that interfere with bacterial adaptability or restore drug sensitivity.

Although the LPS layer in the outer membrane forms a robust barrier, bacteria still need to acquire essential small molecules through porins in the outer membrane. Nonspecific porins allow hydrophilic small molecules such as monosaccharides, amino acids, peptides, nucleosides, and inorganic ions to passively diffuse into the periplasmic space. These channels are also the primary routes for many clinically important hydrophilic antibiotics, such as β-lactams, tetracyclines, chloramphenicol, and some fluoroquinolones, to enter bacterial cells (Overly Cottom et al., 2025). Therefore, the type, expression level, pore size, and charge selectivity of porins directly determine the rate at which antibiotics can enter bacteria. Reduced expression or structural mutations of porins can restrict antibiotic entry and significantly increase bacterial resistance.

In the antibiotic resistance mechanisms of Gram-negative bacteria, the outer membrane barrier plays a fundamental and critical role. The barrier function of the outer membrane itself limits the entry of many antibiotics into the cell. The antibiotic resistance mediated by the outer membrane is primarily achieved through changes or loss of porins, overexpression or functional enhancement of efflux pump systems, and structural modifications of LPS (Kumari and Saraogi, 2025). For example, large molecules like vancomycin cannot penetrate the outer membrane, and therefore, Gram-negative bacteria have intrinsic resistance to them (Maher and Hassan, 2023). Similarly, hydrophilic β-lactam antibiotics must pass through outer membrane porins to enter the periplasmic space (Masi et al., 2022). If bacteria reduce porin expression or narrow the pore size through mutation, antibiotic uptake is significantly reduced. This entry barrier makes Gram-negative bacteria much less sensitive to large molecules and many hydrophilic antibiotics such as vancomycin and β-lactams, making them more difficult to affect by these antibiotics compared to Gram-positive bacteria (Maher and Hassan, 2023). The modification of LPS mainly impacts resistance to polymyxin antibiotics.

When this natural barrier is combined with mutations or regulatory changes in specific resistance genes, the level of bacterial resistance can increase significantly. For instance, in Enterobacteriaceae, a decrease in the expression of OmpC and OmpF is closely associated with resistance to β-lactam antibiotics, particularly when coexisting with β-lactamases (Davin-Regli et al., 2024). Furthermore, the loss of the CarO porin in A. baumannii often leads to carbapenem resistance (Siroy et al., 2005).

Efflux pumps like AcrAB-TolC and MexAB-OprM are key contributors to multidrug resistance in Gram-negative bacteria. Efflux pumps recognize and expel a wide range of antibiotics, including β-lactams, fluoroquinolones, and tetracyclines (Lorusso et al., 2022). This reduces the intracellular drug concentration, preventing effective antibacterial or bactericidal activity. The upregulation or functional enhancement of efflux pumps can significantly lower intracellular antibiotic levels, often leading to clinical multidrug resistance.

These resistance mechanisms do not exist in isolation but rather act synergistically to build a robust resistance barrier. In clinically isolated resistant strains, multiple mechanisms are typically detected simultaneously, such as overexpression of efflux pumps, decreased outer membrane permeability, increased production of antibiotic-inactivating enzymes, and mutations in antibiotic target sites. These mechanisms work together to enhance the bacteria’s resistance to multiple antibiotics. This multifaceted defense strategy can be conceptualized as a series of sequential barriers that an antibiotic must overcome to reach its target.

The stability of the Gram-negative bacterial outer membrane largely depends on divalent cations (primarily Mg²⁺ and Ca²⁺), which bind electrostatically to the core region of the LPS molecules and the phosphate groups of lipid A, maintaining the structural integrity of the outer membrane. Chelating agents disrupt this stability by removing these critical divalent cations, thereby breaking the connections between LPS molecules and disrupting the outer membrane structure, increasing its permeability to hydrophobic substances and antibiotics (Stephan et al., 2023).

Ethylenediaminetetraacetic acid (EDTA) is a classic chelating agent whose mechanism of outer membrane disruption has been extensively confirmed. EDTA effectively chelates the divalent cations in the outer membrane, disrupting the electrostatic crosslinks within the LPS layer and leading to the partial dissociation of the LPS molecular structure. This action destabilizes the outer membrane structure, significantly increasing its permeability to various compounds, including antibiotics that typically struggle to penetrate Gram-negative bacterial cells. For instance, EDTA has been shown to increase the sensitivity of Haemophilus influenzae biofilms to ampicillin and ciprofloxacin (Pakkulnan et al., 2024). Additionally, EDTA disrupts the electrostatic interactions between these divalent cations and extracellular DNA (eDNA), destabilizing the eDNA matrix and reducing the mechanical strength of the biofilm matrix, thus effectively inhibiting biofilm formation (Raad et al., 2008; Wang D. et al., 2025).

Although EDTA demonstrates good outer membrane disruption and synergistic enhancement in vitro, its clinical application as a systemic therapeutic agent is significantly limited. This is primarily due to EDTA’s ability to chelate divalent cations essential for host cell functions, potentially causing host cell dysfunction (Vervaet et al., 2017). Its rapid clearance from the body and potential renal toxicity further restrict its clinical safety and efficacy (Griffin et al., 2019). In addition to EDTA, other chelating agents have also shown similar abilities to enhance outer membrane permeability and synergize antimicrobial activity. For example, sodium hexametaphosphate, a potent Ca²⁺ chelator, enhances the antimicrobial activity of hydrophobic antibiotics against Gram-negative bacteria and increases the uptake of N-phenyl-1-naphthylamine, a fluorescent probe used to measure outer membrane permeability, indicating its ability to enhance outer membrane permeability (Vaara and Jaakkola, 1989). Citric acid and 2,3-dimercaptosuccinic acid, which is used clinically to treat lead poisoning, have also been reported to synergize with various antibiotics, with the mechanism involving increased outer membrane permeability (Jacinto et al., 2025). These different types of chelating agents all disrupt the outer membrane through similar mechanisms, further highlighting the vulnerability of the Gram-negative bacterial outer membrane to its dependence on divalent cations. However, translating this effective in vitro mechanism into a safe clinical drug remains a significant challenge, and future research needs to focus on developing more targeted and less toxic antimicrobial strategies.

Polymyxin class antibiotics, such as polymyxin B (PMB) and polymyxin E (colistin), are cyclic lipopeptides. These compounds are characterized by a decapeptide ring, composed of multiple positively charged L-α,γ-diaminobutyric acid (Dab) residues, along with a hydrophobic acyl chain, usually 6-methyl-octanoyl or octanoyl, attached to the peptide ring (Slingerland et al., 2022). The peptide ring structure of PMB and polymyxin E differs mainly in their acylation patterns, with PMB containing a D-Leu and polymyxin E containing a D-Phe at a key position, along with differences in their fatty acid side chains.

The main mechanism of action of polymyxins is their specific interaction with LPS on the outer membrane of Gram-negative bacteria. The positively charged Dab residues on the polymyxin molecule interact electrostatically and bind with high affinity to the negatively charged phosphate groups on the lipid A region of the LPS molecules (McInerney et al., 2016). This binding competitively displaces the divalent cations (Mg²⁺, Ca²⁺) that originally stabilize the LPS layer structure, leading to a disorganized arrangement of the LPS molecules and destabilization of the outer membrane. The disruption of the outer membrane increases its permeability, and the hydrophobic acyl chain of polymyxin can effectively insert into the damaged areas of the outer membrane, facilitating the entry of the molecule into the periplasmic space (Khadka et al., 2018). The polymyxin then interacts with the cytoplasmic membrane, causing damage to it and leading to leakage of cellular contents. Clinically, despite its significant nephrotoxicity and neurotoxicity, polymyxin is still used as a last-resort drug to treat multidrug-resistant Gram-negative bacterial infections, particularly carbapenem-resistant strains, when other antibiotics fail (Ballı et al., 2024).

To overcome the toxicity issues of polymyxins while retaining their ability to disrupt the outer membrane, researchers have developed various derivatives. One important milestone is polymyxin B nonapeptide (PMBN). PMBN is a derivative of PMB obtained by the proteolytic cleavage of its N-terminal fatty acid-acylated tripeptide side chain. This structural modification removes the hydrophobic tail required for inner membrane insertion, thereby abrogating direct bactericidal activity and significantly reducing toxicity (Tsubery et al., 2001). However, PMBN retains the polycationic cyclic peptide structure, enabling it to bind avidly to anionic Lipid A. This interaction competitively displaces the divalent cations (Mg²⁺, Ca²⁺) essential for stabilizing the LPS leaflet, leading to outer membrane disorganization (Figure 1). Consequently, PMBN serves as a powerful outer membrane permeabilizer. By compromising membrane integrity, it acts as a potentiator for antibiotics generally ineffective against Gram-negative bacteria, such as hydrophobic rifampin, macrolides, and certain beta-lactams (Slingerland et al., 2022). Furthermore, studies indicate that PMBN not only lowers the minimum inhibitory concentration (MIC) of these agents synergistically but also improves efficacy against bacterial persister cells and reduces the frequency of resistant mutant strains (Wesseling and Martin, 2022).

Antimicrobial peptides (AMPs), particularly CAMPs, represent a promising new generation of antibiotics. Naturally occurring CAMPs are a key component of the host defense system and possess broad-spectrum antimicrobial activity. They are typically cationic amphipathic molecules consisting of 12–40 amino acids, rich in cationic amino acids such as lysine and arginine, and hydrophobic amino acids. AMPs exert their antibacterial effect by interacting electrostatically and hydrophobically with bacterial membranes, especially the outer membrane of Gram-negative bacteria, which is rich in LPS, and the inner membrane, which is rich in phosphatidylglycerol or cardiolipin (Gagandeep et al., 2024). This interaction destabilizes the membrane structure, forms transmembrane pores, and dissipates the membrane potential, thereby increasing the membrane’s permeability to other molecules and ultimately leading to bactericidal activity.

Antimicrobial peptides typically act rapidly and are less likely to induce bacterial resistance. This is because they can nonspecifically disrupt bacterial membrane structures or target multiple sites simultaneously, making it difficult for bacteria to develop resistance through single mutations or simple metabolic changes (Yang et al., 2024). In addition to natural CAMPs, numerous synthetic peptide mimics with optimized characteristics, such as enhanced stability, higher antimicrobial activity, and lower host toxicity, are also being developed. These mimics aim to replicate the key physicochemical properties of natural CAMPs while overcoming certain drawbacks (Yadav and Chauhan, 2024).

A typical example of a natural AMP is LL-37, which is produced by the human body. LL-37 not only has direct antimicrobial and antibiofilm activity but also has immunomodulatory functions, modulating chemokine expression, bidirectionally regulating inflammatory responses depending on context, enhancing macrophage phagocytosis, and stimulating the release of inflammatory mediators (Pahar et al., 2020). Its antimicrobial mechanism primarily involves binding to bacterial membranes and perturbing their structural integrity, possibly leading to transient pore-like disruptions or membrane destabilization, as well as targeting multiple intracellular sites (Chen et al., 2023). In addition to membrane disruption, some AMPs can translocate into the cytoplasm and interfere with key intracellular processes, such as inhibiting DNA, RNA, or protein synthesis and enzymatic activity; although interference with protein folding has also been suggested, the underlying mechanisms remain unclear. For example, buforin II and indolicidin target nucleic acid or protein synthesis (Cardoso et al., 2019). Many AMPs possess multi-target characteristics, acting on both the cell membrane and intracellular targets. This multi-target feature substantially lowers the likelihood of rapid resistance development, although bacterial resistance to AMPs can still emerge under prolonged or sub-lethal exposure through mechanisms such as surface charge modification, efflux pumps, or proteolytic degradation.

Moreover, compared to traditional antibiotics that target a single molecular site, bacteria find it harder to evolve resistance against membrane physical disruption. AMPs offer unique advantages and great potential in treating resistant bacterial infections, as they are less likely to induce resistance (Zhu et al., 2025). However, their clinical translation faces numerous challenges, including insufficient understanding of their detailed mechanisms of action, lack of universal rational design principles, complex drug delivery routes, potential host cell toxicity, immunogenicity risks, poor in vivo stability, and high production costs (Cresti et al., 2024). Current research focuses on improving the stability, antibacterial activity and reducing the toxicity of AMPs through peptide engineering or synthetic mimics. Future development may focus more on developing peptide mimics that replicate AMP penetration mechanisms but offer higher in vivo stability, lower host toxicity, and greater economic feasibility, such as stabilized-modified peptides or novel non-peptide small molecules (Mwangi et al., 2023). For example, fusing bacteriolytic lysins that can degrade bacterial peptidoglycan with AMPs could significantly enhance their activity against Gram-negative bacteria (Abdelkader et al., 2022; Son et al., 2023). Some peptide-based permeabilizers, such as SPR741, a non-antibacterial derivative of PMB, are being developed as adjuvants to enhance outer membrane permeability and facilitate the efficacy of co-administered antibiotics. Several AMPs or their derivatives, including PL-5, DPK-060, brilacidin, and LTX-109, have already entered preclinical or various stages of clinical trials (Gan et al., 2021).

As the major component of the outer leaflet of the outer membrane, LPS functions as a potent endotoxin, enabling bacteria to resist external stressors and contributing to immune system activation during infection. Therefore, inhibiting LPS biosynthesis or blocking its transport from the inner membrane to the outer membrane can effectively disrupt the structural integrity of the Gram-negative bacterial outer membrane, thereby reducing bacterial survival. This represents an effective approach for the development of new antimicrobial drugs.

Lipoproteins play a crucial role in maintaining the stability and functionality of the cell envelope, including processes such as nutrient uptake, signal transduction, outer membrane biosynthesis, and homeostasis. The biosynthesis, maturation, and localization of lipoproteins is a complex, multi-step process that is strictly regulated by a series of specific enzymes and transport systems. These pathways, characterized by highly conserved protein sequences and structures in bacteria, are essential for bacterial survival and lack homologous counterparts in eukaryotes. Thus, they have become important targets for the development of selective antibacterial drugs.

Porins are the primary channels through which many small hydrophilic molecules, including antibiotics, enter the periplasmic space of Gram-negative bacteria. In general, porins allow small hydrophilic molecules with a molecular weight of approximately 600 Da or less to passively diffuse into the periplasm. This is the main route for the entry of β-lactam antibiotics, chloramphenicol, tetracyclines, and some fluoroquinolones across the outer membrane into the periplasmic space (Munita and Arias, 2016). When drug molecules exceed this size limit or exhibit strong hydrophobicity, their efficiency of permeation through porin channels is significantly reduced. Additionally, within the porin channel, there is a narrow region known as the “eyelet region,” which is composed of charged amino acid residues, creating a strong local electrostatic field that significantly affects the permeability of charged drugs. For example, the eyelet region of E. coli OmpF porin is rich in negatively charged amino acid residues, which generates a transverse electrostatic field that preferentially attracts and orients positively charged drug molecules, facilitating their translocation through the narrow channel (Maher and Hassan, 2023). However, when the charge of the drug molecule is repelled by the charge of the channel, its permeation efficiency is significantly reduced. Therefore, the structural size and electrochemical properties of porins jointly determine the effectiveness of drug passage through the outer membrane.

It is worth noting that the expression levels or structural mutations of porins are closely related to antibiotic resistance in Gram-negative bacteria. Under prolonged antibiotic selection pressure, pathogens often limit antibiotic entry by reducing porin expression or generating mutations in porin structure, thus significantly increasing resistance (Winterhalter, 2021). For example, downregulation or functional loss of OmpF or OmpC in E. coli can greatly reduce the ability of β-lactam antibiotics to enter the periplasmic space; P. aeruginosa decreases sensitivity to carbapenem antibiotics by downregulating OprD porin or undergoing structural mutations in the porin channel (Wang M. et al., 2025); similarly, impaired expression or function of the outer membrane porin CarO in A. baumannii shows a similar carbapenem resistance mechanism (Kyriakidis et al., 2021). These studies clearly show that the regulation and structural changes of porins are key mechanisms of antibiotic resistance in Gram-negative bacteria. Therefore, the development of antimicrobial drugs needs to fully consider the porin profile and expression patterns of the target bacterial species.

Given that porin-mediated diffusion is both an important pathway for antibiotic entry into Gram-negative bacteria and a key regulatory point for bacterial resistance, current research is increasingly focused on optimizing the size, charge distribution, and hydrophilicity of drugs to better match the porin channel structure and enhance permeability (Prajapati et al., 2021). Moreover, with the rapid development of computational simulation technologies, researchers widely use techniques such as molecular docking and molecular dynamics (MD) simulations to reveal, at the atomic level, the interaction mechanisms between antibiotics and porins. Ekaterina et al. combined in vitro single-molecule permeation experiments with in silico MD simulations to analyze the diffusion dynamics of cephalosporin antibiotics in E. coli OmpF and OmpC porins, elucidating the energetic barriers, key amino acid interactions, and conformational dynamics that govern whether a drug successfully translocates or becomes trapped within the eyelet region (Nestorovich et al., 2002). Additionally, the integration of virtual screening with machine learning is being used to build robust predictive models that can rapidly evaluate the porin permeability potential of novel drug candidates and uncover key structural features influencing permeability. These advanced methods provide in-depth mechanistic insights into porin-mediated drug diffusion, effectively guiding the rational design and structural optimization of novel antimicrobial drugs.

The permeability of the outer membrane of Gram-negative bacteria is influenced not only by the electrostatic gradient formed by ion distribution across the membrane but also by outer membrane proteins and lipid structure. The outer membrane surface, particularly the inner core of LPS and the lipid A region, is rich in phosphate groups, giving it an overall negative charge. Additionally, the O-antigen region of LPS also contributes to the charge distribution of the outer membrane. These negatively charged groups form electrostatic bridges with divalent cations on the membrane surface, stabilizing the structure of LPS molecules, which, in concert with the lipid bilayer and membrane proteins, helps maintain the integrity of the outer membrane (Clifton et al., 2016). Furthermore, this negatively charged environment creates a Donnan potential across the membrane, effectively attracting and concentrating positively charged cationic molecules, which increases their local concentration on the outer membrane surface, accelerating their passage through porin channels or direct binding with LPS (Alegun et al., 2021).

Polycationic antibiotics, such as aminoglycosides and polymyxins, exert their antimicrobial effects through electrostatic interactions with LPS, as well as other mechanisms such as binding to bacterial ribosomes. These antibiotics, due to their high cationic charge density and strong electrostatic affinity, can competitively displace the electrostatic bridges between LPS molecules and divalent cations, thereby destabilizing the outer membrane structure. This increases the outer membrane permeability, making it easier for antimicrobial molecules to enter the bacterial cell. A large body of research has shown that appropriately increasing the number of cationic groups in antibiotic molecules or optimizing the distribution of these charges can significantly enhance electrostatic interactions between the drug and the bacterial outer membrane, thus improving drug penetration efficiency. However, this strategy may carry risks of resistance development (Trimble et al., 2016).

However, it is important to note that the outer membrane permeability of a drug is not only determined by its charge properties but also limited by the molecule’s size and hydrophilicity. Since Gram-negative bacterial porin channels impose strict size limitations on molecules, merely increasing the positive charge of the drug molecule without addressing its size limitation may not achieve the desired cellular uptake (Kojima and Nikaido, 2013). Therefore, optimizing the combination of drug molecule charge distribution, size, and hydrophobicity is a key strategy to enhance outer membrane penetration efficiency.

Although improving drug penetration through electrostatic interactions offers significant advantages, excessively strong electrostatic interactions may disrupt the stability of the outer membrane lipid bilayer, damage membrane protein structures, and potentially trigger host inflammatory responses (Vejzovic et al., 2022). Therefore, in the development of novel antimicrobial agents, it is crucial to optimize the drug’s molecular structure to precisely balance its antimicrobial efficacy with potential toxicity risks, especially in the context of electrostatic interactions. Future research should further employ quantitative analysis and MD simulations to elucidate how drug charge distribution affects outer membrane permeability, and explore the optimal balance between improving antimicrobial efficacy and ensuring host safety in the process of drug structure selection and optimization, aiming to develop more efficient and safer new antimicrobial drugs.

Gram-negative bacteria commonly possess a variety of multidrug efflux pumps composed of membrane transport proteins, which reduce intracellular drug concentrations and lead to resistance by actively expelling a wide range of harmful substances (Gaurav et al., 2023). Based on sequence homology, energy coupling mechanisms, and substrate specificity, bacterial efflux pumps are mainly classified into the ABC superfamily, major facilitator superfamily (MFS), multidrug and toxic compound extrusion (MATE) family, Resistance-Nodulation-Cell Division (RND) superfamily, small multidrug resistance (SMR) family, and proteobacterial antimicrobial compound efflux (PACE) family (Zack et al., 2024). Except for ABC superfamily pumps, which directly use ATP hydrolysis to drive efflux, all other types are secondary active transporters that rely on proton or sodium ion gradients for energy.

In Gram-negative bacteria, the RND-type efflux system is the most clinically important multidrug efflux pump, with typical representatives including E. coli’s AcrAB-TolC system and P. aeruginosa’s MexAB-OprM system (Blair and Piddock, 2009). These systems generally consist of three components: an RND transporter located in the inner membrane, a periplasmic membrane fusion protein such as AcrA or MexA, and an outer membrane channel protein such as TolC or OprM (Tikhonova et al., 2002). RND efflux pumps utilize proton motive force to induce conformational changes, working in coordination with membrane fusion proteins and outer membrane channel proteins to capture a wide range of substrates from the cell interior or periplasm and directly expel them through the outer membrane channel (Nikaido, 2011). Their substrate spectrum is extremely broad, efficiently expelling a variety of structurally diverse molecules, including tetracyclines, fluoroquinolones, chloramphenicol, macrolides, bile salts, and dyes. Due to the widespread presence and central role of RND efflux pumps in many important pathogens, they have become the primary targets for the development of efflux pump inhibitors (EPIs) (Duffey et al., 2024).

Apart from the RND family, Gram-negative bacteria also possess MFS and MATE-type efflux pumps. MFS-type efflux pumps typically exist as single-component, proton-dependent transporters and are relatively rare in Gram-negative bacteria, but, for example, the KpnGH pump in K. pneumoniae can effectively expel toxic substances such as dyes and disinfectants (Srinivasan et al., 2014). MATE pumps usually use Na⁺ or H⁺ gradients to drive transport; a representative member, PmpM in P. aeruginosa, can expel quaternary ammonium disinfectants, dyes, and some quinolone drugs (Zhang et al., 2023). The SMR family of efflux pumps has a compact and simple structure; for instance, EmrE in E. coli expels quaternary ammonium salts and dyes, among other hydrophobic cationic compounds (Bay and Turner, 2012). ABC superfamily efflux pumps use ATP hydrolysis as a direct driving force and mainly participate in the secretion of specific macromolecular toxins or certain antibiotics in Gram-negative bacteria, while broad-spectrum multidrug efflux is more commonly mediated by RND and MFS pumps (Zack et al., 2024). A typical ABC transporter is the E. coli MacAB-TolC system, which is involved in the export of macromolecules such as specific toxins and some macrolide antibiotics. The PACE family is a recently discovered group of efflux pumps, usually composed of a single protein, with a simpler structure than the complex multi-component RND efflux pumps. These pumps are widely found in Acinetobacter species and some Enterobacteriaceae, and can expel specific antimicrobial agents and disinfectants, such as chlorhexidine and PMB, thereby mediating low-level intrinsic resistance to these compounds (Chetri, 2023). Under environmental stress, the expression levels of PACE family members may increase and may synergize with other resistance mechanisms, such as RND pumps, to enhance overall resistance. Although no specific, highly efficient inhibitors of PACE family members have been reported, the mechanisms of action under certain conditions warrant further research, potentially providing a foundation for developing effective inhibitors.

The multidrug resistance of Gram-negative bacteria is largely attributed to their powerful efflux pump systems. Therefore, the development of EPIs to block efflux functions has become a potential adjunctive therapeutic strategy to restore the efficacy of existing antibiotics. EPIs increase the intracellular concentration of antimicrobial agents by inhibiting bacterial active efflux mechanisms, thereby enhancing the sensitivity of resistant strains to antibiotics (Pagès et al., 2005).

Although the importance of efflux pumps as antimicrobial targets is widely recognized, the path to translating effective EPIs into clinical applications is fraught with challenges, including issues such as pharmacokinetic properties, toxicity risks, and inconsistent efficacy in vivo and in vitro. Ideal EPIs should exhibit no independent antimicrobial activity, low toxicity, good outer membrane permeability, broad-spectrum inhibition of major efflux pump families such as RND, MFS, and MATE, and show significant synergy with various antibiotics (Rouvier et al., 2025). However, to date, no EPI has been approved for clinical use, primarily due to factors such as suboptimal pharmacokinetic properties of the compounds, high toxicity risks, and inconsistent efficacy in vivo and in vitro. Furthermore, prolonged use of EPIs could induce the upregulation of efflux pump expression or structural mutations, leading to the gradual development of resistance, further increasing the difficulty of development (Rouvier et al., 2025).

Classic small molecule EPIs are mostly synthesized or derived from existing drugs, typically working by competitively occupying the substrate-binding sites of efflux pumps or interfering with the energy supply required for pump function. An example is the dipeptide compound phenylalanine-arginine β-naphthylamide (PAβN), which effectively reduces the MIC of various antibiotics, including macrolides, fluoroquinolones, and chloramphenicol, by competitively binding to the substrate binding sites of RND efflux pumps (Sharma et al., 2019). However, the clinical translation of PAβN faces significant obstacles, including poor solubility, low stability, non-specific membrane permeability increase, potential toxicity, and poor pharmacokinetic properties. Therefore, current EPI development focuses more on optimizing molecular structure, improving drug selectivity, and enhancing pharmacokinetic properties to boost their clinical potential.

In recent years, research focus has gradually shifted toward the development of highly selective inhibitors targeting specific subcomponents of efflux pumps. For example, some novel inhibitors interfere with the functional rotation mechanism of the AcrB trimer or block the interaction between AcrA and AcrB/TolC components, inhibiting the overall assembly and function of the efflux pump by altering its conformational changes (Sharma et al., 2019). Among these, the recently developed BDM series of pyridine-piperazine compounds, such as BDM91288, can specifically bind to key sites in the AcrB structure, significantly blocking the AcrAB-TolC efflux system in K. pneumoniae, showing good synergistic effects with quinolones and other antibiotics, and demonstrating excellent oral bioavailability and pharmacokinetic properties (Vieira Da Cruz et al., 2024). This precise targeting mechanism effectively reduces non-specific toxicity risks and improves the selectivity and clinical potential of the drug.

Although EPI development faces numerous challenges such as compound permeability, toxicity, metabolic stability, and the risk of resistance development, the continuous deepening of our understanding of efflux pump structures and functional mechanisms, along with the rapid development of computational-aided drug design and high-throughput screening technologies, is making the discovery and optimization of new-generation EPIs increasingly possible. Future research should focus on addressing the issue of poor in vivo and in vitro efficacy of EPIs and explore optimized strategies for combination with novel antimicrobial drugs to facilitate the true clinical translation of EPIs.

The outer membrane of Gram-negative bacteria acts as a formidable selective barrier, significantly restricting the permeability of most antibiotics. Therefore, optimizing the physicochemical properties of drug molecules to improve their ability to penetrate the outer membrane and accumulate inside the cell has become a key strategy for enhancing antimicrobial activity (Cooper et al., 2018). This strategy represents a fundamental exercise in rational molecular design aimed at bypassing the specific physicochemical constraints imposed by the outer membrane architecture. To be effective, antibiotic molecules must navigate two primary biophysical hurdles: the strong electrostatic repulsion generated by the anionic LPS layer and the stringent steric restrictions of the narrow porin channels. In this context, the eNTRy rules proposed by Haloi et al. (2021) provide a seminal roadmap for overcoming these obstacles. Although initially established based on E. coli permeability data, the eNTRy framework highlights key physicochemical features that guide drug molecules efficiently into Gram-negative bacteria, even if its applicability may vary among species (Haloi et al., 2021). These features include: the presence of an ionizable amine group, which facilitates electrostatic interactions with the negatively charged LPS core oligosaccharide region; low globularity, which makes the drug more linear or flattened in structure, thereby enhancing its ability to traverse porin channels; and higher molecular rigidity, indicated by fewer rotatable bonds, which reduces entropy loss during transmembrane diffusion, thermodynamically favoring permeation.

These design principles have been successfully applied in the development of new antimicrobial drugs. A typical example is the new generation broad-spectrum fluoroquinolone delafloxacin, where the molecule’s rigidity and charge distribution were specifically optimized to improve its permeability and antimicrobial activity against various resistant Gram-negative bacteria, such as P. aeruginosa and E. coli (El-Sagheir et al., 2025). Another classic example is the structural optimization of deoxynybomycin. Deoxynybomycin, a natural DNA gyrase inhibitor, was originally only effective against Gram-positive bacteria but had difficulty penetrating the outer membrane of Gram-negative bacteria (Lewis, 2020). Based on the eNTRy rule, Gurvic and Zachariae (2024) introduced a primary amine group into the molecule’s structure, enhancing its hydrophilicity and positive charge without affecting its target binding ability, thereby expanding its antimicrobial spectrum to include Gram-negative bacteria and demonstrating the tremendous potential of rational drug design.

However, it should be noted that the permeability of the Gram-negative bacterial outer membrane is influenced by multiple factors. Differences in the LPS structure, porin expression, and membrane permeability across different species result in variability, meaning the universality of the eNTRy rule may differ between strains or species (Saxena et al., 2023). Additionally, other physicochemical features such as the molecular polar surface area, hydrogen bond donor/acceptor count, and dipole moment also play significant roles in drug transmembrane permeability (Gurvic and Zachariae, 2024). Furthermore, after crossing the outer membrane, antimicrobial molecules may become substrates for efflux pumps, thereby limiting intracellular accumulation and increasing uncertainty in their antimicrobial efficacy. Therefore, future drug design must simultaneously optimize drug permeability and its ability to evade recognition by efflux pumps. To achieve this, high-throughput screening technologies and computational methods should be utilized to quantitatively analyze the specific outer membrane composition characteristics of different bacterial species and their impact on drug permeation, thus guiding the design of personalized antimicrobial drugs to improve drug development success rates and clinical efficacy. In summary, the permeability of the Gram-negative bacterial outer membrane results from a synergy of multiple factors, and the development of antimicrobial drugs should consider factors such as drug molecule size, polarity, charge distribution, rigidity, and efflux pump substrate recognition features to effectively enhance drug accumulation within bacteria and therapeutic efficacy.

In addition to optimizing the physicochemical properties of the drug itself, the development of novel drug delivery systems using nanotechnology has become a cutting-edge strategy for enhancing antibiotic activity, especially for drugs with poor intrinsic permeability or those prone to degradation (Makabenta et al., 2021). Nanocarriers can encapsulate antibiotics within their core or adsorb them onto their surface, significantly improving the drug’s bioavailability by enhancing its solubility, stability, and release kinetics, while protecting it from bacterial enzymatic degradation (Ahsan et al., 2024). Moreover, nanocarriers can substantially increase the local concentration of drugs on the bacterial surface and, through special mechanisms such as membrane fusion, endocytic pathways, or localized disruption of the outer membrane structure, further promote drug permeation across the outer membrane of Gram-negative bacteria, thereby greatly enhancing the antimicrobial effect. Currently, various nanomaterials, including liposomes, polymeric nanoparticles, solid lipid nanoparticles, nanostructured lipid carriers, metallic nanoparticles, and carbon-based nanomaterials, have been widely explored for antimicrobial drug delivery (Su and Kang, 2020).

The mechanisms of action of nanocarriers are diverse. By altering the overall physicochemical properties of the drug, such as increasing its solubility and stability, nanocarriers can significantly improve the drug’s bioavailability. Many nanocarriers possess the ability to strongly interact with bacterial outer membranes (Modi et al., 2023). For example, cationic liposomes or polymeric nanoparticles adsorb onto the negatively charged LPS on the bacterial outer membrane through electrostatic interactions, leading to local disruption or even destruction of the membrane structure, which improves the drug’s transmembrane permeability. In some cases, this membrane-damaging mechanism of the nanocarriers themselves may also exert antimicrobial effects. Additionally, nanocarriers can be surface-modified with specific ligands, such as short peptides or small molecules that bind selectively to outer membrane porins, achieving a “Trojan horse” style active delivery mechanism, inducing bacterial uptake of drug-loaded nanoparticles (Huang et al., 2013). These carriers also protect the drugs from degradation by outer membrane and intracellular enzymes, and by increasing the intracellular drug concentration or local enrichment, they effectively reduce efflux pump-mediated drug expulsion, significantly improving drug penetration and distribution in complex infection environments, such as biofilms. Furthermore, through precise design, nanocarriers can achieve targeted responses to specific infection microenvironments, enabling targeted drug release and minimizing the non-specific toxicity to host cells. For example, research has developed polymeric nanocarrier systems capable of simultaneously loading multiple antibiotics, which release drugs upon contact with bacteria, achieving a multi-target synergistic effect and significantly enhancing antimicrobial efficacy (Meng et al., 2025).