Session J13: Physics of Biofilms II

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In your gut, as in the gut of every animal, legions of microbes cooperate, compete, and influence their host’s health. Conventional approaches for studying the intestinal microbiome reveal its constituent species and their constituent genes, and suggest biochemical networks that govern microbial activities. While such approaches have yielded profound insights, they are largely blind to the spatial structure, individual behaviors, and physical forces present in the gut -- factors that, as in every ecosystem, should be major determinants of function. To address this, my lab applies light sheet fluorescence microscopy, an optical technique that enables high-speed, high-resolution three-dimensional imaging, to larval zebrafish, a model organism that enables a high degree of experimental control. I will describe this approach and experiments that have revealed how bacteria can manipulate intestinal mechanics, how antibiotics can cause collapses in gut populations by altering bacterial community morphology, and how the host immune system might sense bacterial activity. In all these cases, the physical structure of microbial groups, especially their aggregation state and its coupling to intestinal transport, emerges as a major determinant of microbial population dynamics.   

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Bacteria use intercellular signaling, or quorum sensing (QS), to share information and respond collectively to aspects of their surroundings. The autoinducers that carry this information are exposed to the external environment; consequently, they are affected by factors such as removal through fluid flow, a ubiquitous feature of bacterial habitats ranging from the gut and lungs to lakes and oceans. Here, we develop and apply a general theory that identifies the conditions required for QS activation in fluid flow by linking cell- and population-level genetic and physical processes. We predict that, when a subset of the population meets these conditions, cell-level positive feedback promotes a robust collective response by overcoming flow-induced autoinducer concentration gradients. By accounting for a dynamic flow in our theory, we predict that positive feedback in cells acts as a low-pass filter at the population level in oscillatory flow, allowing a population to respond only to changes in flow that occur over slow enough timescales. Our theory is readily extendable, and provides a framework for assessing the functional roles of diverse QS network architectures in realistic flow conditions.   

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Biofilms are colonies of sessile bacteria attached to surfaces. They are often encountered in confined geometries where also an external flow is present. Biofilms can be detrimental, e.g. in filtration systems, or beneficial, e.g. for remediation of brittle materials by microbially induced calcite precipitation (MICP). In any case, the interplay between bacterial motility, biofilm growth and external flow in confined geometries needs to be understood, starting at the scale of individual cells.

We present a bacterial model based on coupling single cells to a surrounding flow. Bacteria are simulated as molecular dynamics particles, the fluid dynamics problem is solved using the lattice Boltzmann method. The two algorithms are connected via a point-friction coupling.
We are able to determine likely locations for biofilm initiation by explicitly taking into account bacterial motility. During the biofilm growth phase, the external flow field determines the shape of the biofilm. On the other hand, the growing biofilm restricts the fluid flow.
By applying our model to a simple porous structure, we show how flow paths can be altered or even blocked.   

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Approximately 75% of all hospital-acquired UTIs are associated with urinary catheters. Pseudomonas aeruginosa is a biofilm-forming bacteria that is frequently responsible for these infections. P. aeruginosa attachment to a surface can be altered by varying the surface stiffness. This study focuses on the development of experimentation methods used to measure bacterial attachment to surfaces of different stiffnesses over time. The first study analyzed bacterial attachment to 0.5 to 3% concentrated agar gel at 2 and 24 hours after inoculation using a vortex mixer to prepare the attached bacteria and gel for dilution. The second study used a bead homogenizer in place of the vortex. Due to the pandemic, this study switched to a related project that analyzes previously-collected data on attachment of P. aeruginosa to PEGDA gel surfaces from 0.5 to 3 hours after inoculation. Developing a high throughput method to measure surface attachment is incredibly important for future analysis of different species of bacteria under the same growth conditions, and multiple types of bacteria competing for the same surface. This study analyzes growth patterns and collection methods to recommend methods for measuring attachment and surface conditions to minimize bacterial growth.   

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The opportunistic pathogen Pseudomonas aeruginosa explores surfaces using twitching motility powered by type IV pili (TFP). Single cells also use TFP to sense the surface, and respond by upregulating many genes associated with virulence. To twitch and sense surfaces, cells cyclically extend, attach and retract their TFP. Both TFP activity and mechanosensing depend on activation of a chemotaxis-like system called Chp. However, how TFP activates the Chp system and how this feeds back on TFP activity remains unknown. Here we show that Chp activation by TFP provides a positive feedback on its activity. We first demonstrate that surface contact increases twitching motility in a Chp-dependent manner. Using localization of fluorescent protein fusions and measurements of piliation by interferometric scattering microscopy, we highlight the mechanism by which the chemotaxis like system controls motility, independently of transcriptional feedback.   

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Bacteriophage (‘phage’) - viruses that infect and lyse bacteria - can be deployed to treat infections caused by bacterial pathogens. Most reported studies of the therapeutic potential of phage neglect the role of spatial heterogeneity in bacterial communities, e.g., in microcolonies and biofilms. In this study, we investigate the density-dependent dynamics arising from interactions between P. aeruginosa and phage. We utilize high throughput methods such as spectrophotometry and biofilm quantification assays to scan the large parameter space for outcomes. We find that in regions with high initial phage density, bacterial clearance is often followed by reemergence. We further examine the mechanism of phage propagation and their effect on biofilm population using high-resolution time-lapse imaging with spinning disk confocal microscopy. Through experiments and theoretical modeling, we probe the interplay between planktonic and biofilm sub-populations in modulating the outcome of phage-biofilm dynamics.   

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Studies in cellular aggregates such as cancer and microbial colonies have shown that the dynamics of evolution play out very differently in populations that have spatial structure as compared to well-mixed populations. However, much of the theoretical work on spatially organized populations has focused on 1D systems or other models with simple spatial structure, ignoring the effects of mechanics as well as metabolism, which can play an important role in aggregate morphology. We study a stochastic model for microbial colony morphology that includes nutrient dynamics and a growth instability, and observe how different colony morphologies affect genetic drift. We compare rough and flat colony fronts and find that rough fronts exhibit two regimes of diversity loss, a transient regime in which diversity is lost much quicker than flat fronts and a long-time regime in which the diversity loss is more similar.   

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The attachment of bacteria onto a surface, consequent signaling, and the accumulation and growth of the surface-bound bacterial population are key initial steps in the formation of pathogenic biofilms – yet whether, and how, this is impacted by the mechanics of the surface is not known. We use ultrathin and thick hydrogels coated on glass coverslips to create stiff and soft composite materials, respectively, with the same surface chemistry. We find that the accumulation, motility, and signaling of the opportunistic human pathogen Pseudomonas aeruginosa are sensitive to material stiffness. Just after attachment, the cell-surface-exposed protein PilY1 acts as a mechanosensor to discriminate between substrates of different stiffness. This PilY1-mediated mechanoresponse gives rise to differences in cyclic-di-GMP signaling that are linked to surface motility and detachment. Later, the pilus retraction motor PilT also acts as a mechanosensor to discriminate between substrates of different stiffness, giving rise to differences in the growth of the surface-bound population. To our knowledge, this is the first demonstration of a two-stage mechanoresponse in early biofilm formation.   

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A biofilm is a community of bacteria bound together in a matrix of extracellular polymeric substances (EPS) that can be produced by constituent bacteria or incorporated from the environment. The biofilm matrix protects constituent bacteria from external threats such as antibiotics and the immune system. Understanding how the production of particular polymers contributes to the bulk mechanics of a biofilm could lead to developing new methods of compromising the matrix structure and rendering the biofilm more susceptible to antibiotic treatment and/or clearance by the immune system. We have previously shown that treatment of biofilms with enzymes matched to the dominant matrix polymer results in alterations of bulk mechanics. We predict that polymer-specific enzyme treatment could render biofilms more susceptible to phagocytic clearance by neutrophils, and will present early results from this investigation. Furthermore, we explore the extent to which emergent mechanical properties of biofilms can be predicted from single-cell interactions of bacteria in the planktonic state by simulating bacteria and EPS as spheres bound together by Hookean forces, which we estimated from the force required to separate cohered bacteria, measured using an AFM cantilever as a force transducer   

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Extensive research over decades has led to a relatively mature understanding of the molecular mechanisms of antibiotic resistance, but developing drugs to outpace the resistance threat is a major challenge. To complement this molecular approach, researchers have recently shifted their focus to longer length scales, where ecological and evolutionary dynamics of bacterial communities highlight new approaches for slowing resistance with currently available drugs. In this talk, I will discuss our ongoing work to understand how antibiotics shape, and are reshaped by, the spatial architecture of bacterial biofilms at the single-cell level. Biofilms dynamics differ significantly from that of well-stirred populations in liquid ("planktonic") communities. By combining confocal microscopy with simple mathematical models I will show how antibiotics impact not only the composition of the biofilm--specifically, the balance of susceptible and resistant cells in the community-- but also the way those cells are arranged in space. Our results suggest that in spatially structured populations, which may more accurately reflect natural bacterial communities, the selection of resistance is not a simple result of homogeneous selections but depends critically on the spatial arrangement of cells.   

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Recent work, from our group and others, has shown that bacteria can sense and respond to mechanical cues when they are in the single-cell state. We have also shown that the response to mechanical cues is linked to the production of cyclic-di-GMP (c-di-GMP), an intracellular signal that controls the transition of bacteria from the single-cell state to the biofilm state. Biofilms are communities of aggregated, interacting bacteria that resist antibiotics and the immune system and as a result cause most chronic, intractable bacterial infection. However, whether bacteria can respond to mechanical cues once they are part of a biofilm is not known and, to our knowledge, has not been investigated. If they are capable of such response, this could have implications for how biofilms respond to mechanically dynamic environments. To elucidate this, we want to examine two types of mechanical cues – deforming stress on a biofilm, and changes in biofilm elasticity. We use small (~millimeter-sized) hydrogels encapsulating bacteria to act as model biofilms. This allows us either to apply osmotic pressure to change the bulk volume of the bacteria-containing gel with the bacteria in situ or to controllably vary the elastic modulus of the encapsulating material.   

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Biofilms are communities of bacteria embedded in an extracellular matrix and are one of the dominant forms of bacterial life on earth. Recent studies have shown how bacteria grow from single cells to communities of many thousands of cells; however, this work has been limited to free-growing biofilms that are not confined by any external mechanical forces. Using Vibrio Cholerae as a model biofilm former, we examine the growth of biofilms confined at the interface of a glass and gel surface. At the macroscopic level, we find that the degree of mechanical confinement plays an important role in controlling the overall shape of the biofilm, and at the single-cell level, we find that increasing mechanical confinement drives long-range ordering consisting of bacteria radially aligned along the glass and gel surfaces. Finally, we use theoretical and computational tools to elucidate the fundamental physics driving the cellular ordering inside these biofilms.   

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Exposure to antibiotic at low doses has been shown to stimulate biofilm formation. To study how low-dose antibiotic can promote aggregation and biofilm formation, we developed an experimental assay where E. coli bacteria form aggregates in a shaken suspension upon exposure to a low dose of cell-wall targeting antibiotic. We quantified the dynamical transition from a planktonic culture to a suspension of aggregates using a combination of microscopic, spectroscopic and microbiological methods. Our results support a picture in which cluster formation happens primarily due to the release of DNA by antibiotic-induced cell lysis, with a secondary effect due to the presence of pili . This aggregation process happens even in a strongly shaken suspension, and we speculate that such aggregates might form precursors to biofilm formation.