Abstract
Biofilms are microbial communities of aggregated cells encased in extracellular matrix that are a pressing healthcare concern. Since biofilms have complex metabolic dynamics, in this study a new approach for studying biofilm metabolism is developed that employs optical-photothermal infrared (O-PTIR) spectroscopy imaging combined with 13C stable isotope probing and cryosectioning to track the carbon metabolism of cells at different depths of the biofilm. This approach demonstrated that metabolic gradients can be visualised using O-PTIR imaging, revealing a core of cells with low metabolic activity at the centre of the biofilm, with outer regions showing significantly higher metabolic activity. By incorporating the heavy stable isotope of carbon into bacterial biomass, we monitored the metabolic activity of gentamicin-resistant Salmonella Typhimurium within the biofilm structure upon exposure to various antibiotics. O-PTIR imaging revealed altered metabolic responses at various depths of the biofilm, with variations that depend on the bacterial antibiotic susceptibility profile.
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Data availability
Data presented in this study can be obtained in the supplementary data, with the underlying data for graphs in Figs. 1–2 found in supplementary data 1, the underlying data for graphs in Figs. 3–4 found in supplementary data 2, and the underlying data for graphs in Figs. 5–6 found in supplementary data 3. All other data are available from the corresponding author on reasonable request.
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Acknowledgements
We would like to express our gratitude to the University of Liverpool for providing funding (DS, HM and RG). HM would also like to thank the Analytical Chemistry Trust Fund (ACTF) and Community for Analytical Measurement Science (CAMS) for funding and support (600310/22/09). RR is grateful for funding from the BBSRC and Innovate UK via the UK National Biofilm Innovation Centre (grant numbers BB/R012415/1, BB/X002950/1). DS would also like to thank the University of Liverpool Shared Research Histology Core Facility, RRID:SCR_026606 for their help and support with developing the cryosectioning protocol.
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D.S.: Experimental design, sample collection and preparation, O-PTIR data collection, data analysis and interpretation, manuscript writing; X.Z.: preparation of Salmonella Typhimurium from strain collection, engineering resistance into strains and manuscript preparation; J.H.: provision of Salmonella Typhimurium from strain collection, experimental design and manuscript preparation; R.R.: supervision, experimental design and manuscript preparation; R.G. provision of O-PTIR & Bioscreen instruments for analysis and manuscript preparation; H.M.: principal investigator, experimental design, data interpretation and manuscript preparation. All authors contributed to the project and viewed the final manuscript.
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Smaje, D., Zhu, X., Hinton, J.C.D. et al. Investigating Salmonella biofilm responses to antibiotic treatment using optical photothermal infrared spectroscopy. Commun Biol (2026). https://doi.org/10.1038/s42003-026-09655-2
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DOI: https://doi.org/10.1038/s42003-026-09655-2
