Liselotte Jauffred

Biofilms are dynamic communities of microorganisms encased in a self-produced extracellular matrix. The mechanical behaviour of a biofilm is shaped by the heterogenous composition of its matrix, both spatially and chemically. In turn, these mechanics influence the biofilm’s architecture at micro- and macroscopic scales, driving its complexity and adaptability. Morphologically, this is reflected in mechanical deformations of the biofilm known as wrinkles. In nature, biofilms often host different species of bacteria, allowing for a great diversity of matrix components. Here, we use a combination of two Escherichia coli strains as a model for a multispecies biofilm in which each bacterial strain produces one of two complementary matrix fibres: an amyloid protein (curli) or a polysaccharide (phosphoethanolamine-cellulose). Using fluorescence microscopy, we confirm that the two bacterial strains rapidly segregate into isogenic sectors, decreasing local heterogeneity. Furthermore, we show how wrinkles form, both in the homogenous central region of the biofilm, as well as at the boundary between sectors (i.e. where the two matrix producers co-localize). Finally, we show that increasing strain intermixing via the addition of bacteriophages results in thicker, taller wrinkles, irrespective of whether the two fibres are produced by two different strains or co-produced by the same bacteria.

The survival of bacterial communities depends on complex dynamics at molecular, cellular, and ecosystem levels. Understanding antibiotic resistance requires a broader community context, as emergent dynamics can lead to unexpected outcomes, such as the persistence of susceptible populations or community collapse. We capture these behaviors by integration of microscopy and mathematical modeling to understand how bacterial interactions and spatial organization shape bacteriostatic antibiotic resistance in a two-strain community. We show that local chloramphenicol detoxification and mechanical pushing shape bacterial coexistence and spatial organization, promoting the survival and growth of otherwise susceptible bacteria. Additionally, the timing of antibiotic administration critically determines the growth dynamics, co-existence, local diversity of susceptible and resistant bacteria, and overall community resistance. Together, these insights highlight how community-level interactions fundamentally reshape antibiotic responses and open new avenues to understand and control bacterial resilience. SIGNIFICANCE STATEMENT: Antibiotic resistance is usually treated as a property of individual bacterial strains, yet bacteria typically grow in dense, spatially structured communities where physical interactions matter. We find that under bacteriostatic (growth pausing) antibiotic stress, resistant bacteria can create highly localized protective environments that allow sensitive cells to survive and proliferate. This protection arises not only from antibiotic detoxification, but also from growth–driven mechanical pushing that maintains close cell–cell proximity. As a result, antibiotic tolerance emerges as a collective, spatially dependent property rather than an intrinsic trait of single cells. These findings show that spatial organization, physical forces, and treatment timing can strongly reshape therapy outcomes, with implications for how resistance is understood and managed in microbial communities.

Biofilms are dynamic communities of microorganisms encased in a self-produced extracellular matrix. These resilient structures pose challenges across nearly all human activities, from healthcare to industry. The mechanical behaviour of a biofilm is shaped by the heterogenous composition of its matrix, both spatially and chemically. In turn, these mechanics influence the biofilm’s architecture at micro- and macroscopic scales, driving its complexity and adaptability. Morphologically, this is reflected in mechanical deformations of the biofilm known as wrinkles. In nature, biofilms often host different species of bacteria, allowing for a great diversity of matrix components. Here, we use a combination of two Escherichia coli strains as a model for a multispecies biofilm in which each bacterial strain produces one of two complementary matrix fibres: an amyloid protein (curli) or a polysaccharide (phosphoethanolamine-cellulose). Using fluorescence microscopy, we confirm that the two bacterial strains rapidly segregate into isogenic sectors, decreasing local heterogeneity. Furthermore, we show how wrinkles form, both in the homogenous central region of the biofilm, as well as at the boundary between sectors (i.e. where the two matrix producers co-localize). Finally, we show that increasing strain intermixing via the addition of bacteriophages results in thicker, taller wrinkles, irrespective of whether the two fibres are produced by two different strains or co-produced by the same bacteria.

Bacterial competition shapes community architecture, yet a universally conserved determinant remains elusive. We show that cell aspect ratio –a simple morphological feature– confers a competitive advantage. Using growth-based range expansion experiments, we show that longer bacteria conquer the expanding front, even when initially in minority. Using an agent-based model of dividing bacteria, to isolate the effect of aspect ratio, we reveal that the takeover mechanism is collective alignment: groups of locally aligned bacteria form “nematic arms” bridging the central region of the colony to the expanding front. Once at the front, bacteria align parallel to it and block shorter bacteria from access to nutrients and space. We confirm this observation with single-cell experiments and further generalise our findings by introducing a generic continuum model of alignment-dominated competition, explaining both experimental and cell-based model observations. Moreover, we extend our predictions to spherical range expansions and confirm the competitive advantage, even though the effect is less pronounced than in surface-attached colonies. Our results uncover a simple, yet hitherto overlooked, mechanical mechanism determining the outcome of bacterial competition, which is potentially ubiquitous among various bacteria. Current advances in genetic engineering enable aspect ratio tuning as a mechanism with broad implications for biofilm control.

Fluorescent nanodiamonds (FNDs) with nitrogen-vacancy centers are promising candidates for long-term biolabeling and biosensing applications due to their biocompatibility and unique optomagnetic properties. The employment of nanomaterials in cancer therapy and diagnostics requires a deep understanding of how nanoparticles (NPs) interact with the three-dimensional (3D) tumor environment. We developed the “Tumor-in-a-Tube” platform, using 4D light-sheet microscopy to explore the spatiotemporal dynamics of FNDs with 3D tumor spheroids. By monitoring the real-time NP sedimentation, spheroid penetration, and cellular uptake of FNDs and polystyrene nanoparticles (PNPs), we marked the impact of the NP mass density on their spheroid interaction. Unlike PNPs, higher-density FNDs underwent rapid sedimentation, which minimized their effective concentration and hindered the FND–spheroid interactions. This results in constrained intratumoral accumulation and size-independent uptake and penetration. Longer FND effective-exposure time promotes size-dependent cell uptake, verified by FND treatment on 2D monolayers. Nonetheless, FNDs exhibited good biocompatibility and long-term spheroid labeling, allowing for cell isolation from different spheroid layers. Our results suggest the need for NP effective-exposure-time calibration in comparative NP assays, in 3D static models. Overall, our platform provides a valuable tool for bridging the gap between 2D and 3D static models in NP assessment, drug delivery, toxicology profiling, and translational research.

Fluorescent nanodiamonds (FNDs) with nitrogen-vacancy centers are promising candidates for long-term biolabeling and biosensing applications due to their biocompatibility and unique optomagnetic properties. The employment of nanomaterials in cancer therapy and diagnostics, requires a deep understanding of how nanoparticles (NPs) interact with the three-dimensional (3D) tumor environment. We developed a novel approach, the “Tumor-in-a-Tube” platform, using 4D light-sheet microscopy to explore the spatio-temporal dynamics of FNDs with 3D tumor spheroids. By monitoring the real-time NP sedimentation, spheroid penetration, and cellular uptake of FNDs and polystyrene nanoparticles (PNPs), we marked the impact of NP mass density on their spheroid interaction. Unlike PNPs, higher-density FNDs underwent rapid sedimentation, which minimized their effective concentration and hindered the FND – spheroid interactions. This results in constrained intratumoral accumulation, and size-independent uptake and penetration. Longer FND effective-exposure-time promotes size-dependent cell uptake, verified by FND treatment on 2D monolayers. Nonetheless, FNDs exhibited good biocompatibility and long-term spheroid labeling, allowing for cell isolation from different spheroid layers. Our results suggest the need for NP effective-exposure-time calibration in comparative NP assays, in 3D static models. Overall, our platform provides a valuable tool for bridging the gap between 2D and 3D static models in NP assessment, drug delivery, toxicology profiling and translational research.

Bacteria’s competition for nutrients and territory drives biofilm evolution (1 –4). The factors determining the outcome of competition among diverse bacterial species have a broad impact on a wide range of pathological (5), environmental (6), and microbiome interactions (7). While motility-related traits (8 – 11) and specific molecular mechanisms (12 , 13) have been identified as potential winning attributes in bacteria, a shared and universally conserved feature determining competition remains elusive. Here, we demonstrate that a simple morphological feature of individual bacteria, cell aspect ratio, provides a winning trait for the population. Using range expansion experiments (14), we show that relatively longer bacteria robustly conquer the expanding front, even when initially in minority. Using an agent based model of dividing bacteria, we reveal that the takeover mechanism is their emergent collective alignment: groups of locally aligned bacteria form "nematic arms" bridging the central region of the colony to the expanding front. Once at the front, bacteria align parallel to it and block the access of shorter bacteria to nutrients and space. We confirm this observation with single-cell experiments and further generalise our findings by introducing a generic continuum model of alignment-dominated competition, explaining both experimental and cell-based model observations. Moreover, we extend our predictions to spherical range expansions (15) and confirm the competitive advantage of being longer, even though the effect is less pronounced than in surface-attached colonies. Our results uncover a simple, yet hitherto overlooked, mechanical mechanism determining the outcome of bacterial competition, which is potentially ubiquitous among various bacteria. With the current advances in genetic engineering, varying aspect ratios can work as a simple tunable mechanism for the on-demand setting of the outcome of bacterial competitions with widespread implications for biofilm control.

Bacterial competition shapes community architecture, yet a universally conserved determinant remains elusive. We show that cell aspect ratio –a simple morphological feature– confers a competitive advantage. Using growth-based range expansion experiments, we show that longer bacteria conquer the expanding front, even when initially in minority. Using an agent-based model of dividing bacteria, to isolate the effect of aspect ratio, we reveal that the takeover mechanism is collective alignment: groups of locally aligned bacteria form “nematic arms” bridging the central region of the colony to the expanding front. Once at the front, bacteria align parallel to it and block shorter bacteria from access to nutrients and space. We confirm this observation with single-cell experiments and further generalise our findings by introducing a generic continuum model of alignment-dominated competition, explaining both experimental and cell-based model observations. Moreover, we extend our predictions to spherical range expansions and confirm the competitive advantage, even though the effect is less pronounced than in surface-attached colonies. Our results uncover a simple, yet hitherto overlooked, mechanical mechanism determining the outcome of bacterial competition, which is potentially ubiquitous among various bacteria. Current advances in genetic engineering enable aspect ratio tuning as a mechanism with broad implications for biofilm control.