Highly inclined light sheet allows volumetric super-resolution imaging of efflux pumps distribution in bacterial biofilms

Year: 2024

Authors: Vignolini T., Capitanio M., Caldini C., Gardini L., Pavone FS.

Autors Affiliation: LENS, European Lab Nonlinear Spect, Via N Carrara 1, I-50019 Sesto Fiorentino, Italy; Univ Florence, Dept Phys & Astron, Via G Sansone 1, I-50019 Sesto Fiorentino, Italy; CNR, Natl Inst Opt, Via N Carrara 1, I-50019 Sesto Fiorentino, Italy; Univ Paris Cite, Inst Pasteur, Paras RNA Biol Grp, F-75015 Paris, France.

Abstract: Bacterial biofilms are highly complex communities in which isogenic bacteria display different gene expression patterns and organize in a three-dimensional mesh gaining enhanced resistance to biocides. The molecular mechanisms behind such increased resistance remain mostly unknown, also because of the technical difficulties in biofilm investigation at the sub-cellular and molecular level. In this work we focus on the AcrAB-TolC protein complex, a multidrug efflux pump found in Enterobacteriaceae, whose overexpression is associated with most multiple drug resistance (MDR) phenotypes occurring in Gram-negative bacteria. We propose an optical method to quantify the expression level of the AcrAB-TolC pump within the biofilm volume at the sub-cellular level, with single-molecule sensitivity. Through a combination of super-resolution PALM with single objective light sheet and precision genome editing, we can directly quantify the spatial distribution of endogenous AcrAB-TolC pumps expressed in both planktonic bacteria and, importantly, within the bacterial biofilm volume. We observe a gradient of pump density within the biofilm volume and over the course of biofilm maturation. Notably, we propose an optical method that could be broadly employed to achieve volumetric super-resolution imaging of thick samples.

Journal/Review: SCIENTIFIC REPORTS

Volume: 14 (1)      Pages from: 12902-1  to: 12902-10

More Information: This work was supported by the European Union’s Horizon2020 research and innovation program under grant Agreement No 871124 Laserlab-Europe. EMPIR-Horizon 2020 under Grant Agreement 15HLT01, MetVBadBugs. The authors thank L. Piddock for providing us with the E. coli strain BW25113. They thank Kim Hardie and James Brown for training on biofilm culturing and insightful scientific discussion.
KeyWords: Escherichia-coli; Pseudomonas-aeruginosa; Antibiotic-resistance; Multidrug-resistance; Intrinsic Resistance; Codon Context; Expression; Genes; Tolc; Acrb
DOI: 10.1038/s41598-024-63729-x


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