In Advances in methods and concepts provide new insight into antibiotic fluxes across the bacterial membrane, we propose an original and integrated perspective on the antibiotic transport and accumulation in patient infectious sites using recent methods, concepts and tools.

Currently, the vast majority of studies investigating the relationship between antibiotic structure and antibacterial activity rely on MIC measurements (1,2,3). These MIC-based approaches are performed after extended incubation periods and neglect critical early and intermediate steps. These include penetration, accumulation, sensitivity to efflux pumps, and the molecule’s half-life near the target (1,3). Consequently, they fail to capture the molecular and kinetic specificities of the crucial early steps that modulate antibiotic action. To address this gap, we advocate an integrated approach combining in cellulo, in vitro and in silico methods. The aim is to provide a detailed characterization of the key steps involved in the action of antibiotics, ultimately identifying the strategic molecular profiles necessary to overcome membrane-associated bacterial resistance mechanisms.

Since the bacterial membrane is the first point of contact with external harmful compounds such as antibiotics, integrating these parameters is essential to optimize drug design (2,4,5). In particular, coordinating efforts to enhance antibiotic accumulation inside bacteria is a keystone for effective drug design. We further propose that efforts should include investigations of antibiotic and involved pathogen sampling at infectious sites.

The development of highly efficient tools such as mass spectrometry and advanced in vivo imaging techniques, has significantly expanded the potential of spatial biology (3,6). These technologies have improved the study of the in situ distribution of metabolites and antibiotics in infected tissues and have allowed for deeper analysis of their effects. We suggest combining these approaches to monitor in situ antibiotic accumulation, localization, and activity correlating these with pathogen metabolomic profiles. This approach is summarized in the Figure: From the sampling location (infectious site) a series of analyses is carried out, identification and quantification of antibiotics in situ, and analyses of bacterial phenotype (MIC, killing rates, drug transport). In parallel, in cellulo, in vitro and in silico assays are performed, using purified protein from isolates in artificial membrane, molecular and dynamics of transporters involved. The parameters collected from bacteria and molecules are used to highlight key membrane-associated resistance mechanisms for each antibiotic tested. Together, these analyses pave the way for rational approaches to circumvent membrane-associated resistance mechanisms.

Currently, only a limited number of publications reports the application of spatial biology to study the physiological state of pathogens within infectious sites (3,7). With recent advances in sample preservation methods (8), this approach could be extended to infections in specific tissue (e.g., respiratory tract or digestive tracts) and to pathogens requiring tailored therapeutic strategies. The comprehensive data generated through this integrative approach will greatly inform rational drug design and help clinicians select the most effective antibiotics or drug combinations.Finally, and most importantly, we emphasize that the ability of a drug to enter and accumulate in bacteria is a biological and chemical continuum and must be carefully considered during drug design and clinical use (1,3). With the development of spatial biology, this challenge may soon be addressed, allowing membrane-associated resistance mechanisms to be effectively overcome.

Reference

1. Vergalli, J. et al. Porins and small-molecule translocation across the outer membrane of Gram-negative bacteria. Nature Reviews Microbiology 18, 164–176 (2020).
2. Manrique, P. D., López, C. A., Gnanakaran, S., Rybenkov, V. V. & Zgurskaya, H. I. New understanding of multidrug efflux and permeation in antibiotic resistance, persistence, and heteroresistance. Annals of the New York Academy of Sciences 1519, 46–62 (2023).
3. Vergalli J, Réfrégiers M, Ruggerone P, Winterhalter M, Pagès JM. Advances in methods and concepts provide new insight into antibiotic fluxes across the bacterial membrane. Commun Biol. 2024 Nov 14;7(1):1508.
4. Darby, EM et al.Molecular mechanisms of antibiotic resistance revisited. Nat Rev Microbiol. 2023 May;21(5):280-295.
5. Geddes, E. J., Li, Z. & Hergenrother, P. J. An LC-MS/MS assay and complementary web-based tool to quantify and predict compound accumulation in coli. Nat Protoc 16, 4833–4854 (2021).
6. Richter, M. F. et al. Predictive compound accumulation rules yield a broad-spectrum antibiotic. Nature 545, 299–304 (2017).
7. Sollier, J. et al. Revitalizing antibiotic discovery and development through in vitro modelling of in-patient conditions. Nat Microbiol 9, 1–3 (2024).
8. Dannhorn, A. et al. Morphological and molecular preservation through universal preparation of fresh-frozen tissue samples for multimodal imaging workflows. Nat Protoc 19, 2685–2711 (2024).

Schematic representation of an integrated analytical workflow for the study of antibiotic resistance mechanisms.

From the infectious site, various tasks include in situ antibiotic identification and quantification and bacterial phenotypic characterizations (multi-omics assays). In parallel, in cellulo, in vitro and in silico assays are performed to measure drug transport across membranes of identified isolates. The collected data are integrated to identify key membrane-associated resistance mechanisms. This comprehensive approach illuminates rational strategies to circumvent bacterial membrane-associated mechanisms of resistance.