Identification of pathways to high-level vancomycin resistance in Clostridioides difficile that incur high fitness costs in key pathogenicity traits.

Clostridioides difficile is an important human pathogen, for which there are very limited treatment options, primarily the glycopeptide antibiotic vancomycin. In recent years, vancomycin resistance has emerged as a serious problem in several gram-positive pathogens, but high-level resistance has yet to be reported for C. difficile, although it is not known if this is due to constraints upon resistance evolution in this species. Here, we show that resistance to vancomycin can evolve rapidly under ramping selection but is accompanied by fitness costs and pleiotropic trade-offs, including sporulation defects that would be expected to severely impact transmission. We identified 2 distinct pathways to resistance, both of which are predicted to result in changes to the muropeptide terminal D-Ala-D-Ala that is the primary target of vancomycin. One of these pathways involves a previously uncharacterised D,D-carboxypeptidase, expression of which is controlled by a dedicated two-component signal transduction system. Our findings suggest that while C. difficile is capable of evolving high-level vancomycin resistance, this outcome may be limited clinically due to pleiotropic effects on key pathogenicity traits. Moreover, our data identify potential mutational routes to resistance that should be considered in genomic surveillance.

Tracking bacteria at high density with FAST, the Feature-Assisted Segmenter/Tracker.

Most bacteria live attached to surfaces in densely-packed communities. While new experimental and imaging techniques are beginning to provide a window on the complex processes that play out in these communities, resolving the behaviour of individual cells through time and space remains a major challenge. Although a number of different software solutions have been developed to track microorganisms, these typically require users either to tune a large number of parameters or to groundtruth a large volume of imaging data to train a deep learning model-both manual processes which can be very time consuming for novel experiments. To overcome these limitations, we have developed FAST, the Feature-Assisted Segmenter/Tracker, which uses unsupervised machine learning to optimise tracking while maintaining ease of use. Our approach, rooted in information theory, largely eliminates the need for users to iteratively adjust parameters manually and make qualitative assessments of the resulting cell trajectories. Instead, FAST measures multiple distinguishing 'features' for each cell and then autonomously quantifies the amount of unique information each feature provides. We then use these measurements to determine how data from different features should be combined to minimize tracking errors. Comparing our algorithm with a naïve approach that uses cell position alone revealed that FAST produced 4 to 10 fold fewer tracking errors. The modular design of FAST combines our novel tracking method with tools for segmentation, extensive data visualisation, lineage assignment, and manual track correction. It is also highly extensible, allowing users to extract custom information from images and seamlessly integrate it into downstream analyses. FAST therefore enables high-throughput, data-rich analyses with minimal user input. It has been released for use either in Matlab or as a compiled stand-alone application, and is available at https://bit.ly/3vovDHn, along with extensive tutorials and detailed documentation.

Steering self-organisation through confinement.

Self-organisation is the spontaneous emergence of spatio-temporal structures and patterns from the interaction of smaller individual units. Examples are found across many scales in very different systems and scientific disciplines, from physics, materials science and robotics to biology, geophysics and astronomy. Recent research has highlighted how self-organisation can be both mediated and controlled by confinement. Confinement is an action over a system that limits its units' translational and rotational degrees of freedom, thus also influencing the system's phase space probability density; it can function as either a catalyst or inhibitor of self-organisation. Confinement can then become a means to actively steer the emergence or suppression of collective phenomena in space and time. Here, to provide a common framework and perspective for future research, we examine the role of confinement in the self-organisation of soft-matter systems and identify overarching scientific challenges that need to be addressed to harness its full scientific and technological potential in soft matter and related fields. By drawing analogies with other disciplines, this framework will accelerate a common deeper understanding of self-organisation and trigger the development of innovative strategies to steer it using confinement, with impact on, e.g., the design of smarter materials, tissue engineering for biomedicine and in guiding active matter.

Microbial competition in porous environments can select against rapid biofilm growth.

Microbes often live in dense communities called biofilms, where competition between strains and species is fundamental to both evolution and community function. Although biofilms are commonly found in soil-like porous environments, the study of microbial interactions has largely focused on biofilms growing on flat, planar surfaces. Here, we use microfluidic experiments, mechanistic models, and game theory to study how porous media hydrodynamics can mediate competition between bacterial genotypes. Our experiments reveal a fundamental challenge faced by microbial strains that live in porous environments: cells that rapidly form biofilms tend to block their access to fluid flow and redirect resources to competitors. To understand how these dynamics influence the evolution of bacterial growth rates, we couple a model of flow-biofilm interaction with a game theory analysis. This investigation revealed that hydrodynamic interactions between competing genotypes give rise to an evolutionarily stable growth rate that stands in stark contrast with that observed in typical laboratory experiments: cells within a biofilm can outcompete other genotypes by growing more slowly. Our work reveals that hydrodynamics can profoundly affect how bacteria compete and evolve in porous environments, the habitat where most bacteria live.

Single-cell twitching chemotaxis in developing biofilms.

Bacteria form surface-attached communities, known as biofilms, which are central to bacterial biology and how they affect us. Although surface-attached bacteria often experience strong chemical gradients, it remains unclear whether single cells can effectively perform chemotaxis on surfaces. Here we use microfluidic chemical gradients and massively parallel automated tracking to study the behavior of the pathogen Pseudomonas aeruginosa during early biofilm development. We show that individual cells can efficiently move toward chemoattractants using pili-based "twitching" motility and the Chp chemosensory system. Moreover, we discovered the behavioral mechanism underlying this surface chemotaxis: Cells reverse direction more frequently when moving away from chemoattractant sources. These corrective maneuvers are triggered rapidly, typically before a wayward cell has ventured a fraction of a micron. Our work shows that single bacteria can direct their motion with submicron precision and reveals the hidden potential for chemotaxis within bacterial biofilms.

Biofilm Formation As a Response to Ecological Competition.

Bacteria form dense surface-associated communities known as biofilms that are central to their persistence and how they affect us. Biofilm formation is commonly viewed as a cooperative enterprise, where strains and species work together for a common goal. Here we explore an alternative model: biofilm formation is a response to ecological competition. We co-cultured a diverse collection of natural isolates of the opportunistic pathogen Pseudomonas aeruginosa and studied the effect on biofilm formation. We show that strain mixing reliably increases biofilm formation compared to unmixed conditions. Importantly, strain mixing leads to strong competition: one strain dominates and largely excludes the other from the biofilm. Furthermore, we show that pyocins, narrow-spectrum antibiotics made by other P. aeruginosa strains, can stimulate biofilm formation by increasing the attachment of cells. Side-by-side comparisons using microfluidic assays suggest that the increase in biofilm occurs due to a general response to cellular damage: a comparable biofilm response occurs for pyocins that disrupt membranes as for commercial antibiotics that damage DNA, inhibit protein synthesis or transcription. Our data show that bacteria increase biofilm formation in response to ecological competition that is detected by antibiotic stress. This is inconsistent with the idea that sub-lethal concentrations of antibiotics are cooperative signals that coordinate microbial communities, as is often concluded. Instead, our work is consistent with competition sensing where low-levels of antibiotics are used to detect and respond to the competing genotypes that produce them.

Turbulent fluid acceleration generates clusters of gyrotactic microorganisms.

The motility of microorganisms is often biased by gradients in physical and chemical properties of their environment, with myriad implications on their ecology. Here we show that fluid acceleration reorients gyrotactic plankton, triggering small-scale clustering. We experimentally demonstrate this phenomenon by studying the distribution of the phytoplankton Chlamydomonas augustae within a rotating tank and find it to be in good agreement with a new, generalized model of gyrotaxis. When this model is implemented in a direct numerical simulation of turbulent flow, we find that fluid acceleration generates multifractal plankton clustering, with faster and more stable cells producing stronger clustering. By producing accumulations in high-vorticity regions, this process is fundamentally different from clustering by gravitational acceleration, expanding the range of mechanisms by which turbulent flows can impact the spatial distribution of active suspensions.

Microbial alignment in flow changes ocean light climate.

The growth of microbial cultures in the laboratory often is assessed informally with a quick flick of the wrist: dense suspensions of microorganisms produce translucent "swirls" when agitated. Here, we rationalize the mechanism behind this phenomenon and show that the same process may affect the propagation of light through the upper ocean. Analogous to the shaken test tubes, the ocean can be characterized by intense fluid motion and abundant microorganisms. We demonstrate that the swirl patterns arise when elongated microorganisms align preferentially in the direction of fluid flow and alter light scattering. Using a combination of experiments and mathematical modeling, we find that this phenomenon can be recurrent under typical marine conditions. Moderate shear rates (0.1 s(-1)) can increase optical backscattering of natural microbial assemblages by more than 20%, and even small shear rates (0.001 s(-1)) can increase backscattering from blooms of large phytoplankton by more than 30%. These results imply that fluid flow, currently neglected in models of marine optics, may exert an important control on light propagation, influencing rates of global carbon fixation and how we estimate these rates via remote sensing.

Individual bacterial cells can use spatial sensing of chemical gradients to direct chemotaxis on surfaces.

Swimming bacteria navigate chemical gradients using temporal sensing to detect changes in concentration over time. Here we show that surface-attached bacteria use a fundamentally different mode of sensing during chemotaxis. We combined microfluidic experiments, massively parallel cell tracking and fluorescent reporters to study how Pseudomonas aeruginosa senses chemical gradients during pili-based 'twitching' chemotaxis on surfaces. Unlike swimming cells, we found that temporal changes in concentration did not induce motility changes in twitching cells. We then quantified the chemotactic behaviour of stationary cells by following changes in the sub-cellular localization of fluorescent proteins as cells are exposed to a gradient that alternates direction. These experiments revealed that P. aeruginosa cells can directly sense differences in concentration across the lengths of their bodies, even in the presence of strong temporal fluctuations. Our work thus overturns the widely held notion that bacterial cells are too small to directly sense chemical gradients in space.

Two codependent routes lead to high-level MRSA.

Methicillin-resistant Staphylococcus aureus (MRSA), in which acquisition of mecA [which encodes the cell wall peptidoglycan biosynthesis component penicillin-binding protein 2a (PBP2a)] confers resistance to ß-lactam antibiotics, is of major clinical concern. We show that, in the presence of antibiotics, MRSA adopts an alternative mode of cell division and shows an altered peptidoglycan architecture at the division septum. PBP2a can replace the transpeptidase activity of the endogenous and essential PBP2 but not that of PBP1, which is responsible for the distinctive native septal peptidoglycan architecture. Successful division without PBP1 activity requires the alternative division mode and is enabled by several possible chromosomal potentiator (pot) mutations. MRSA resensitizing agents differentially interfere with the two codependent mechanisms required for high-level antibiotic resistance, which provides opportunities for new interventions.

Suicidal chemotaxis in bacteria.

Bacteria commonly live in surface-associated communities where steep gradients of antibiotics and other chemical compounds can occur. While many bacterial species move on surfaces, we know surprisingly little about how such antibiotic gradients affect cell motility. Here, we study the behaviour of the opportunistic pathogen Pseudomonas aeruginosa in stable spatial gradients of several antibiotics by tracking thousands of cells in microfluidic devices as they form biofilms. Unexpectedly, these experiments reveal that bacteria use pili-based ('twitching') motility to navigate towards antibiotics. Our analyses suggest that this behaviour is driven by a general response to the effects of antibiotics on cells. Migrating bacteria reach antibiotic concentrations hundreds of times higher than their minimum inhibitory concentration within hours and remain highly motile. However, isolating cells - using fluid-walled microfluidic devices - reveals that these bacteria are terminal and unable to reproduce. Despite moving towards their death, migrating cells are capable of entering a suicidal program to release bacteriocins that kill other bacteria. This behaviour suggests that the cells are responding to antibiotics as if they come from a competing colony growing nearby, inducing them to invade and attack. As a result, clinical antibiotics have the potential to lure bacteria to their death.

Reconfigurable Microfluidic Circuits for Isolating and Retrieving Cells of Interest.

Microfluidic devices are widely used in many fields of biology, but a key limitation is that cells are typically surrounded by solid walls, making it hard to access those that exhibit a specific phenotype for further study. Here, we provide a general and flexible solution to this problem that exploits the remarkable properties of microfluidic circuits with fluid walls─transparent interfaces between culture media and an immiscible fluorocarbon that are easily pierced with pipets. We provide two proofs of concept in which specific cell subpopulations are isolated and recovered: (i) murine macrophages chemotaxing toward complement component 5a and (ii) bacteria (Pseudomonas aeruginosa) in developing biofilms that migrate toward antibiotics. We build circuits in minutes on standard Petri dishes, add cells, pump in laminar streams so molecular diffusion creates attractant gradients, acquire time-lapse images, and isolate desired subpopulations in real time by building fluid walls around migrating cells with an accuracy of tens of micrometers using 3D printed adaptors that convert conventional microscopes into wall-building machines. Our method allows live cells of interest to be easily extracted from microfluidic devices for downstream analyses.

Gyrotaxis in a steady vortical flow.

We show that gyrotactic motility within a steady vortical flow leads to tightly clustered aggregations of microorganisms. Two dimensionless numbers, characterizing the relative swimming speed and stability against overturning by vorticity, govern the coupling between motility and flow. Exploration of parameter space reveals a striking array of patchiness regimes. Aggregations are found to form within a few overturning time scales, suggesting that vortical flows might be capable of efficiently separating species with different motility characteristics.

Chain formation can enhance the vertical migration of phytoplankton through turbulence.

Many species of motile phytoplankton can actively form long multicellular chains by remaining attached to one another after cell division. While chains swim more rapidly than single cells of the same species, chain formation also markedly reduces phytoplankton's ability to maintain their bearing. This suggests that turbulence, which acts to randomize swimming direction, could sharply attenuate a chain's ability to migrate between well-lit surface waters during the day and deeper nutrient-rich waters at night. Here, we use numerical models to investigate how chain formation affects the migration of phytoplankton through a turbulent water column. Unexpectedly, we find that the elongated shape of chains helps them travel through weak to moderate turbulence much more effectively than single cells, and isolate the physical processes that confer chains this ability. Our findings provide a new mechanistic understanding of how turbulence can select for phytoplankton with elongated morphologies and may help explain why turbulence triggers chain formation.

Microfluidics with fluid walls.

Microfluidics has great potential, but the complexity of fabricating and operating devices has limited its use. Here we describe a method - Freestyle Fluidics - that overcomes many key limitations. In this method, liquids are confined by fluid (not solid) walls. Aqueous circuits with any 2D shape are printed in seconds on plastic or glass Petri dishes; then, interfacial forces pin liquids to substrates, and overlaying an immiscible liquid prevents evaporation. Confining fluid walls are pliant and resilient; they self-heal when liquids are pipetted through them. We drive flow through a wide range of circuits passively by manipulating surface tension and hydrostatic pressure, and actively using external pumps. Finally, we validate the technology with two challenging applications - triggering an inflammatory response in human cells and chemotaxis in bacterial biofilms. This approach provides a powerful and versatile alternative to traditional microfluidics.The complexity of fabricating and operating microfluidic devices limits their use. Walsh et al. describe a method in which circuits are printed as quickly and simply as writing with a pen, and liquids in them are confined by fluid instead of solid walls.

Turbulence drives microscale patches of motile phytoplankton.

Patchiness plays a fundamental role in phytoplankton ecology by dictating the rate at which individual cells encounter each other and their predators. The distribution of motile phytoplankton species is often considerably more patchy than that of non-motile species at submetre length scales, yet the mechanism generating this patchiness has remained unknown. Here we show that strong patchiness at small scales occurs when motile phytoplankton are exposed to turbulent flow. We demonstrate experimentally that Heterosigma akashiwo forms striking patches within individual vortices and prove with a mathematical model that this patchiness results from the coupling between motility and shear. When implemented within a direct numerical simulation of turbulence, the model reveals that cell motility can prevail over turbulent dispersion to create strong fractal patchiness, where local phytoplankton concentrations are increased more than 10-fold. This 'unmixing' mechanism likely enhances ecological interactions in the plankton and offers mechanistic insights into how turbulence intensity impacts ecosystem productivity.

Thin phytoplankton layers: characteristics, mechanisms, and consequences.

For over four decades, aggregations of phytoplankton known as thin layers have been observed to harbor large amounts of photosynthetic cells within narrow horizontal bands. Field observations have revealed complex linkages among thin phytoplankton layers, the physical environment, cell behavior, and higher trophic levels. Several mechanisms have been proposed to explain layer formation and persistence, in the face of the homogenizing effect of turbulent dispersion. The challenge ahead is to connect mechanistic hypotheses with field observations to gain better insight on the phenomena that shape layer dynamics. Only through a mechanistic understanding of the relevant biological and physical processes can we begin to predict the effect of thin layers on the ecology of phytoplankton and higher organisms.

Disruption of vertical motility by shear triggers formation of thin phytoplankton layers.

Thin layers of phytoplankton are important hotspots of ecological activity that are found in the coastal ocean, meters beneath the surface, and contain cell concentrations up to two orders of magnitude above ambient concentrations. Current interpretations of their formation favor abiotic processes, yet many phytoplankton species found in these layers are motile. We demonstrated that layers formed when the vertical migration of phytoplankton was disrupted by hydrodynamic shear. This mechanism, which we call gyrotactic trapping, can be responsible for the thin layers of phytoplankton commonly observed in the ocean. These results reveal that the coupling between active microorganism motility and ambient fluid motion can shape the macroscopic features of the marine ecological landscape.