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bioRxiv · 10.1101/2025.04.03.647129

Single-cell bacterial culturing and antibiotic susceptibility testing using permeable hydrogel-shelled microcapsules

Pratt, S. L.·Williamson, K. S.·Franklin, M. J.·Chang, C. B.
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Abstract

Microbial communities, such as biofilms, consist of bacteria that exhibit cell-to-cell heterogeneity in their physiological properties, including enzyme activity and gene expression. This single-cell heterogeneity influences the communitys overall metabolic activity and contributes to stress tolerance, such as antimicrobial resistance. To study the impact of single-cell heterogeneity on population-level behaviors, methods have been developed to isolate and characterize bacteria at the single-cell level. One such method includes water-in-oil drop-based microfluidics. However, a limitation of this approach is that the droplet contents cannot be exchanged during experiments, making it difficult to investigate how cells respond to changing environmental conditions. To address this limitation, we developed a drop-based microfluidic technique called Bioflex, which creates permeable hydrogel-shell microcapsules that act as growth chambers for individual bacterial cells. The microcapsules are formed by crosslinking a shell made from a PEG-based hydrogel (4-arm PEG-maleimide) around a dextran core. After cross-linking, the dextran core diffuses out of the capsules and is replaced with buffer or growth medium. By adjusting fluid flow rates during the process, we can control the size and shell thickness of the microcapsules, allowing the production of different capsule architectures. The Bioflex capsules are biocompatible, supporting the encapsulation and growth of Pseudomonas aeruginosa by allowing nutrient transport across the capsule shell. Moreover, the capsules remained permeable to antimicrobial treatments introduced during P. aeruginosa incubation. For example, P. aeruginosa cells in Bioflex capsules responded to externally delivered ciprofloxacin treatments, with their responses varying depending on the timing of the antibiotic introduction. In summary, Bioflex capsules provide a novel, high-throughput platform for isolating single-cells and testing how single-cell derived communities react to time-dependent stresses, such as antibiotic treatments. Graphical AbstractBacterial cultivation using permeable microscale Bioflex capsules. (A) P. aeruginosa PAO1 expressing eGFP cultured in a Bioflex capsule starting from a single cell. (B) Bioflex capsules are permeable to nutrient and waste transport. (C) The capsules allow for exchange of media to study the responses of single cells or small populations of cells within the drops using time-lapse imaging. O_FIG O_LINKSMALLFIG WIDTH=101 HEIGHT=200 SRC="FIGDIR/small/647129v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@69d8bforg.highwire.dtl.DTLVardef@11507b6org.highwire.dtl.DTLVardef@1ad6649org.highwire.dtl.DTLVardef@1167ae0_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Pratt, S. L., Williamson, K. S., Franklin, M. J., Chang, C. B.. 2025-04-09. Single-cell bacterial culturing and antibiotic susceptibility testing using permeable hydrogel-shelled microcapsules. https://doi.org/10.1101/2025.04.03.647129

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