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Biofilms are microbial communities that represent a highly abundant form of microbial life on Earth. Inside biofilms, phenotypic and genotypic variations occur in three-dimensional space and time; microscopy and quantitative image analysis are therefore crucial for elucidating their functions. Here, we present BiofilmQ—a comprehensive image cytometry software tool for the automated and high-throughput quantification, analysis and visualization of numerous biofilm-internal and whole-biofilm properties in three-dimensional space and time., BiofilmQ is an image cytometry software tool that enables the visualization, quantification and analysis of biofilm properties, providing insights into their structure and function.

Bacterial biofilms are widespread in nature and in medical settings and display a high tolerance to antibiotics and disinfectants. Extracellular vesicles have been increasingly studied to characterise their origins and assess their potential for use as a versatile drug delivery system; however, it remains unclear whether they also have antibiofilm effects. Outer membrane vesicles are lipid vesicles shed by Gram-negative bacteria and, in the case of myxobacteria, carry natural antimicrobial compounds produced by these microorganisms. In this study, we demonstrate that vesicles derived from the myxobacteria Cystobacter velatus Cbv34 and Cystobacter ferrugineus Cbfe23 are highly effective at inhibiting the formation and disrupting biofilms by different bacterial species.

Significance Most living systems, from individual cells to tissues and swarms, display collective self-organization on length scales that are much larger than those of the individual units that drive this organization. A fundamental challenge is to understand how properties of microscopic components determine macroscopic, multicellular biological function. Our study connects intracellular physiology to macroscale collective behaviors during multicellular development, spanning five orders of magnitude in length and six orders of magnitude in time, using bacterial swarming as a model system. This work is enabled by a high-throughput adaptive microscopy technique, which we combined with genetics, machine learning, and mathematical modeling to reveal the phase diagram of bacterial swarming and that cell–cell interactions within each swarming phase are dominated by mechanical interactions., Coordinated dynamics of individual components in active matter are an essential aspect of life on all scales. Establishing a comprehensive, causal connection between intracellular, intercellular, and macroscopic behaviors has remained a major challenge due to limitations in data acquisition and analysis techniques suitable for multiscale dynamics. Here, we combine a high-throughput adaptive microscopy approach with machine learning, to identify key biological and physical mechanisms that determine distinct microscopic and macroscopic collective behavior phases which develop as Bacillus subtilis swarms expand over five orders of magnitude in space. Our experiments, continuum modeling, and particle-based simulations reveal that macroscopic swarm expansion is primarily driven by cellular growth kinetics, whereas the microscopic swarming motility phases are dominated by physical cell–cell interactions. These results provide a unified understanding of bacterial multiscale behavioral complexity in swarms.

Three-dimensional fluorescence-based imaging of living cells and organisms requires the sample to be exposed to substantial excitation illumination energy, typically causing phototoxicity and photobleaching. Light sheet fluorescence microscopy dramatically reduces phototoxicity, yet most implementations are limited to objective lenses with low numerical aperture and particular sample geometries that are built for specific biological systems. To overcome these limitations, we developed a single-objective light sheet fluorescence system for biological imaging based on axial plane optical microscopy and digital confocal slit detection, using either Bessel or Gaussian beam shapes. Compared to spinning disk confocal microscopy, this system displays similar optical resolution, but a significantly reduced photobleaching at the same signal level. This single-objective light sheet technique is built as an add-on module for standard research microscopes and the technique is compatible with high-numerical aperture oil immersion objectives and standard samples mounted on coverslips. We demonstrate the performance of this technique by imaging three-dimensional dynamic processes, including bacterial biofilm dispersal, the response of biofilms to osmotic shocks, and macrophage phagocytosis of bacterial cells.

Bacteriophages can be trapped in the matrix of bacterial biofilms, such that the cells inside them are protected. It is not known whether these phages are still infectious and whether they pose a threat to newly arriving bacteria. Here, we address these questions using Escherichia coli and its lytic phage T7. Prior work has demonstrated that T7 phages are bound in the outermost curli polymer layers of the E. coli biofilm matrix. We show that these phages do remain viable and can kill colonizing cells that are T7-susceptible. If cells colonize a resident biofilm before phages do, we find that they can still be killed by phage exposure if it occurs soon thereafter. However, if colonizing cells are present on the biofilm long enough before phage exposure, they gain phage protection via envelopment within curli-producing clusters of the resident biofilm cells.

Bacteria often live in the context of spatially restricted groups held together by a self-secreted, adhesive extracellular matrix. These groups, termed biofilms, are likely where many phage-bacteria encounters occur. A number of recent studies have documented that phages can be trapped in the outer matrix layers of biofilms, such that the bacteria inside are protected from exposure. It is not known, however, what might happen after this: are the trapped phages still viable on the biofilm exterior? If so, do they pose a threat to newly arriving cells that might otherwise colonize the existing biofilm? Here we set out to address these questions using a biofilm-producing strain ofEscherichia coliand its lytic phage T7. Prior work has demonstrated that T7 phages are trapped in the outermost layers of curli polymers within theE. colimatrix. We show that these phages do remain viable and kill incoming colonizing cells so long as they are T7-susceptible. If colonizing cells arrive to the outside of a resident biofilm before phages do, they can still be killed by phage exposure if it occurs soon thereafter. However, if colonizing cells are present on the biofilm long enough before phage exposure, they gain phage protection via envelopment within curli-producing clusters of the resident biofilm cells. This work establishes that phages trapped in the outer matrix layers of a resident biofilm can be incidentally weaponized as a mode of protection from competition by newly arriving cells that might otherwise colonize the biofilm exterior.

Biofilms are now considered to be the most abundant form of microbial life on Earth, playing critical roles in biogeochemical cycles, agriculture, and health care. Phenotypic and genotypic variations in biofilms generally occur in three-dimensional space and time, and biofilms are therefore often investigated using microscopy. However, the quantitative analysis of microscopy images presents a key obstacle in phenotyping biofilm communities and single-cell heterogeneity inside biofilms. Here, we present BiofilmQ, a comprehensive image cytometry software tool for the automated high-throughput quantification and visualization of 3D and 2D community properties in space and time. Using BiofilmQ does not require prior knowledge of programming or image processing and provides a user-friendly graphical user interface, resulting in editable publication-quality figures. BiofilmQ is designed for handling fluorescence images of any spatially structured microbial community and growth geometry, including microscopic, mesoscopic, macroscopic colonies and aggregates, as well as bacterial biofilms in the context of eukaryotic hosts.

Bacterial cells in nature are frequently exposed to changes in their chemical environment1,2. The response mechanisms of isolated cells to such stimuli have been investigated in great detail. By contrast, little is known about the emergent multicellular responses to environmental changes, such as antibiotic exposure3–7, which may hold the key to understanding the structure and functions of the most common type of bacterial communities: biofilms. Here, by monitoring all individual cells in Vibrio cholerae biofilms during exposure to antibiotics that are commonly administered for cholera infections, we found that translational inhibitors cause strong effects on cell size and shape, as well as biofilm architectural properties. We identified that single-cell-level responses result from the metabolic consequences of inhibition of protein synthesis and that the community-level responses result from an interplay of matrix composition, matrix dissociation and mechanical interactions between cells. We further observed that the antibiotic-induced changes in biofilm architecture have substantial effects on biofilm population dynamics and community assembly by enabling invasion of biofilms by bacteriophages and intruder cells of different species. These mechanistic causes and ecological consequences of biofilm exposure to antibiotics are an important step towards understanding collective bacterial responses to environmental changes, with implications for the effects of antimicrobial therapy on the ecological succession of biofilm communities. Single-cell imaging analysis shows that translation-inhibiting antibiotics disrupt Vibrio cholerae biofilm structure and enable entry of bacteriophages and intruder cells.

Biofilms are microbial communities that represent a highly abundant form of microbial life on Earth. Inside biofilms, phenotypic and genotypic variations occur in three-dimensional space and time; microscopy and quantitative image analysis are therefore crucial for elucidating their functions. Here, we present BiofilmQ-a comprehensive image cytometry software tool for the automated and high-throughput quantification, analysis and visualization of numerous biofilm-internal and whole-biofilm properties in three-dimensional space and time.

In nature, bacteria primarily live in surface-attached, multicellular communities, termed biofilms 1–6 . In medical settings, biofilms cause devastating damage during chronic and acute infections; indeed, bacteria are often viewed as agents of human disease 7 . However, bacteria themselves suffer from diseases, most notably in the form of viral pathogens termed bacteriophages 8–12 , which are the most abundant replicating entities on Earth. Phage–biofilm encounters are undoubtedly common in the environment, but the mechanisms that determine the outcome of these encounters are unknown. Using Escherichia coli biofilms and the lytic phage T7 as models, we discovered that an amyloid fibre network of CsgA (curli polymer) protects biofilms against phage attack via two separate mechanisms. First, collective cell protection results from inhibition of phage transport into the biofilm, which we demonstrate in vivo and in vitro. Second, CsgA fibres protect cells individually by coating their surface and binding phage particles, thereby preventing their attachment to the cell exterior. These insights into biofilm–phage interactions have broad-ranging implications for the design of phage applications in biotechnology, phage therapy and the evolutionary dynamics of phages with their bacterial hosts. At late stages of biofilm development, Escherichia coli cells express the curli polymer CsgA. CsgA assembles into a fibre network that protects biofilms from attack by lytic phages.

Summary Bacteria can generate benefits for themselves and their kin by living in multicellular, matrix-enclosed communities, termed biofilms, which are fundamental to microbial ecology and the impact bacteria have on the environment, infections, and industry [1, 2, 3, 4, 5, 6]. The advantages of the biofilm mode of life include increased stress resistance and access to concentrated nutrient sources [3, 7, 8]. However, there are also costs associated with biofilm growth, including the metabolic burden of biofilm matrix production, increased resource competition, and limited mobility inside the community [9, 10, 11]. The decision-making strategies used by bacteria to weigh the costs between remaining in a biofilm or actively dispersing are largely unclear, even though the dispersal transition is a central aspect of the biofilm life cycle and critical for infection transmission [12, 13, 14]. Using a combination of genetic and novel single-cell imaging approaches, we show that Vibrio cholerae integrates dual sensory inputs to control the dispersal response: cells use the general stress response, which can be induced via starvation, and they also integrate information about the local cell density and molecular transport conditions in the environment via the quorum sensing apparatus. By combining information from individual (stress response) and collective (quorum sensing) avenues of sensory input, biofilm-dwelling bacteria can make robust decisions to disperse from large biofilms under distress, while preventing premature dispersal when biofilm populations are small. These insights into triggers and regulators of biofilm dispersal are a key step toward actively inducing biofilm dispersal for technological and medical applications, and for environmental control of biofilms., Highlights • Cells in V. cholerae biofilms decide to disperse by combining two sensory mechanisms • Quorum sensing and RpoS provide information on different environmental parameters • Integration of both sensory inputs yields robust and optimal dispersal decisions, Triggers and mechanisms of biofilm dispersal are poorly understood. Singh et al. show that biofilm dispersal of Vibrio cholerae is regulated by combining individual and collective cell-level sensing mechanisms, via the RpoS-mediated general stress response and quorum sensing, respectively, for making robust dispersal decisions.

It was recently shown that haloarchaeal strains of different genera are able to adhere to surfaces and form surface-attached biofilms. However the surface structures mediating the adhesion were still unknown. We have identified a novel surface structure with Halobacterium salinarum strain R1, crucial for surface adhesion. Electron microscopic studies of surface-attached cells frequently showed pili-like surface structures of two different diameters that were irregularly distributed on the surface. The thinner filaments, 7 - 8 nm in diameter, represented a so far unobserved novel pili-like structure. Examination of the Hbt. salinarum R1 genome identified two putative gene loci (pil-1 and pil-2) encoding type IV pilus biogenesis complexes besides the archaellum encoding fla gene locus. Both pil-1 and pil-2 were expressed as transcriptional units, and the transcriptional start of pil-1 was identified. In silico analyses revealed that the pil-1 locus is present with other euryarchaeal genomes whereas the pil-2 is restricted to haloarchaea. Comparative real time qRT-PCR studies indicated that the general transcriptional activity was reduced in adherent versus planktonic cells. In contrast, the transcription of pilB1 and pilB2, encoding putative type IV pilus assembly ATPases, was induced in comparison to the archaella assembly/motor ATPase (flaI) and the ferredoxin gene. Mutant strains were constructed that incurred a flaI deletion or flaI/pilB1 gene deletions. The absence of flaI caused the loss of the archaella while the additional absence of pilB1 led to loss of the novel pili-like surface structures. The ΔflaI/ΔpilB1 double mutants showed a 10-fold reduction in surface adhesion compared to the parental strain. Since surface adhesion was not reduced with the non-archaellated ΔflaI mutants, the pil-1 filaments have a distinct function in the adhesion process.