Macrolides: mechanisms, resistance, and boosting activity against Gram-negative bacteria

Macrolide antibiotics have been a mainstay of human medicine for more than seventy years, yet a comprehensive new review published in The Journal of Antibiotics argues that these widely prescribed drugs are being held back by three intertwined problems: rising resistance, a poorly understood phenomenon known as tolerance, and an intrinsic inability to penetrate and kill many Gram-negative bacteria. The review, authored by Umar A. Aulia, Sungwan Jung, Lukas I. Kronenberg and colleagues, weaves together the history, biochemistry and clinical realities of the macrolide class and lays out a roadmap for restoring their potency, primarily through the use of adjuvant compounds delivered alongside the antibiotics themselves.

Macrolides are defined chemically by a large macrocyclic lactone ring, typically a fourteen-, fifteen- or sixteen-membered ring decorated with sugars and other substituents that are essential for biological activity. The prototype of the class, erythromycin, was first isolated as a natural product from Streptomyces species, soil-dwelling bacteria renowned for their extraordinary capacity to produce bioactive secondary metabolites. The macrocyclic lactone scaffold is assembled inside the producing organism by modular polyketide synthase enzymes, gigantic multi-protein assembly lines that stitch together small carboxylic acid building blocks in a stepwise fashion. This biosynthetic logic, in which each module of the polyketide synthase corresponds to one elongation cycle, has not only fascinated biochemists for decades but also provided the synthetic blueprint for engineered and semisynthetic derivatives with improved pharmacological properties.

The clinical success of macrolides rests on a deceptively simple molecular mechanism: the inhibition of bacterial protein synthesis. Structural and biochemical studies have shown that macrolides bind within the nascent peptide exit tunnel of the bacterial 50S ribosomal subunit, the channel through which a growing polypeptide chain must pass as it emerges from the ribosome’s catalytic core. By parking themselves in this tunnel, macrolides physically obstruct elongation of the nascent chain, halting translation and ultimately depriving the bacterium of the proteins it needs to survive and replicate. The precise binding site involves nucleotides of the 23S ribosomal RNA, and the position of the drug within the tunnel determines which peptide sequences can still be synthesized and which are blocked. This mechanism explains why macrolides are bacteriostatic against most organisms, arresting growth rather than rapidly killing cells, although certain derivatives display more bactericidal behavior against specific pathogens.

That mechanism has proven clinically valuable across a remarkable range of infections. Macrolides are among the most commonly prescribed outpatient antibiotic classes in the world, and physicians reach for them to treat community-acquired pneumonia, sexually transmitted diseases including infections caused by Chlamydia and other atypical pathogens, and a variety of gastrointestinal infections. Their favorable oral bioavailability, extensive tissue penetration and accumulation inside cells such as macrophages make them particularly attractive for intracellular pathogens, while their relatively mild side-effect profile compared with older broad-spectrum agents has cemented their place in primary care. Successive generations of semisynthetic macrolides, including the second-generation azithromycin and clarithromycin and the third-generation telithromycin and ketolides, were developed specifically to overcome early resistance and to improve acid stability and pharmacokinetics.

Yet the review emphasizes that clinical successes have been tempered by a growing list of failures, and it organizes these failures into mechanistically distinct categories. The first and most visible is true resistance, the genetically encoded, heritable capacity of a bacterium to grow at antibiotic concentrations that would normally be inhibitory. For macrolides, the dominant resistance mechanism is enzymatic modification of the ribosomal target itself. Methyltransferases encoded by erm genes methylate a specific adenine residue in the 23S rRNA within the drug binding site, sterically blocking macrolide binding and often producing cross-resistance to other antibiotics that target the same ribosomal region, such as lincosamides and streptogramins, a phenotype known as the MLSb resistance pattern. Efflux pumps constitute a second major mechanism: membrane transporters of the Mef and Msr families actively pump the drug out of the cell, lowering the intracellular concentration below therapeutic thresholds. A third mechanism involves enzymatic inactivation, in which phosphotransferases, glycosyltransferases or esterases chemically modify or cleave the macrolactone ring or its sugar substituents. Finally, mutations in the 23S rRNA or in ribosomal proteins can alter the binding pocket directly, a route that becomes particularly important in organisms with few ribosomal RNA operons.

The review then turns to a subtler and less clinically appreciated phenomenon: antibiotic tolerance. Unlike resistance, tolerance does not allow bacteria to proliferate in the presence of the drug. Instead, tolerant cells survive transient exposure to otherwise lethal concentrations without growing, resuming normal division once the antibiotic is removed. Tolerance is frequently linked to slow growth, dormancy, nutrient limitation, stress responses and the formation of persister cells, subpopulations within an apparently susceptible culture that remain metabolically quiescent and thereby evade the bacteriostatic and bactericidal consequences of translation arrest. Because macrolides are primarily bacteriostatic and rely on active bacterial growth to express their full effect, metabolically dormant cells are disproportionately able to survive macrolide treatment. Tolerance, the authors stress, does not raise the minimum inhibitory concentration measured in standard susceptibility tests, which helps explain why it can go undetected in the laboratory while still compromising treatment outcomes in patients, particularly in chronic and biofilm-associated infections where nutrient gradients and slow growth are the norm.

The third barrier, and arguably the most consequential for the future of the class, is the limited activity of macrolides against Gram-negative bacteria. Gram-negative organisms, including the notorious Enterobacterales and non-fermenting pathogens such as Pseudomonas aeruginosa, are shielded by an outer membrane that functions as a molecular sieve. The lipid bilayer of this membrane excludes hydrophobic molecules, while embedded porin channels admit only small, water-soluble compounds, and macrolides, which are large, bulky and lipophilic, fit poorly through these gates. Compounding this physical barrier is the formidable armory of efflux pumps that Gram-negative bacteria maintain, transporters that recognize and expel macrolides from the periplasm and cytoplasm before the drugs can reach the ribosome in sufficient quantities. As a result, even though the ribosomal target inside Gram-negative bacteria is fundamentally the same as in susceptible Gram-positive species, the drug rarely arrives at its destination at concentrations high enough to inhibit translation. This pharmacological inaccessibility has largely excluded macrolides from the treatment of infections caused by multidrug-resistant Gram-negative pathogens, precisely the organisms for which new therapeutic options are most urgently needed.

The central argument of the review is that these barriers need not be permanent. The authors survey recent work aimed at identifying adjuvant compounds, molecules that are co-delivered with macrolides to disable the defenses that limit their activity. Adjuvants can act at several points of attack. Inhibitors of efflux pumps can cripple the transporters responsible for expelling the antibiotic, restoring intracellular concentrations to effective levels; this strategy is particularly relevant for Gram-negative pathogens whose efflux systems are major contributors to intrinsic macrolide resistance. Permeabilizers, including certain membrane-active agents, can transiently disrupt the outer membrane, widening the path through which the bulky macrolide molecule must pass to reach the periplasm and cytoplasm. Other adjuvant concepts target tolerance rather than resistance, for example by interfering with stress responses or metabolic states that keep persister cells dormant, thereby re-sensitizing these subpopulations to translation arrest. Combinations of macrolides with compounds that potentiate ribosomal binding or block protective modification of the target have also been explored. The review highlights that adjuvant strategies carry the additional appeal of extending the usable lifespan of existing drugs, sidestepping some of the cost and timeline pressures that have historically slowed the development of entirely new antibiotic scaffolds.

Underlying all of these efforts is the sobering epidemiological context. Macrolide resistance has climbed steadily worldwide, driven in part by the sheer volume of outpatient prescribing and by macrolide usage in agriculture, and resistance determinants such as erm and mef genes now circulate readily among clinical isolates and commensal organisms alike. Azithromycin resistance in Neisseria gonorrhoeae and in Salmonella serovars has become a particular concern, narrowing therapeutic options for diseases that were once trivially treatable. At the same time, the pipeline of new antibiotics active against Gram-negative bacteria remains thin, and the pharmaceutical industry’s retreat from antibiotic development has left clinicians increasingly dependent on older drug classes, their derivatives and their combinations. In this landscape, a rigorous re-examination of the macrolide class is both timely and pragmatic.

What emerges from the review is a picture of a drug class whose core mechanism is sound and whose clinical niche is secure, but whose full potential has been artificially constrained by cellular barriers and microbial ingenuity that are, in principle, pharmacologically addressable. The path forward proposed by Aulia, Jung, Kronenberg and their colleagues is not a single breakthrough but a coordinated campaign: better structural understanding of ribosome binding across resistant and Gram-negative targets, systematic screening for adjuvants that dismantle efflux and permeability barriers, deeper investigation of the metabolic determinants of tolerance, and careful clinical evaluation of combination regimens that can translate laboratory synergy into patient benefit. If those efforts succeed, one of the oldest and most trusted antibiotic families in medicine may yet regain relevance against pathogens that currently lie beyond its reach, offering a template for how rational combination therapy can rejuvenate established drugs in the era of antimicrobial resistance.