Protocol for isolating single species of bacteria with swarming ability from human feces.

Understanding the specific movements of bacteria isolated from human feces can serve as a novel diagnostic and therapeutic tool for inflammatory bowel disease. Here, we present a protocol for a microbial swarming assay and to isolate the bacteria responsible for swarming activity. We describe steps for identifying bacteria using MALDI-TOF mass spectrometry and whole-genome sequencing. We then detail procedures for validating findings by observing the same swarming phenotype upon reperforming the swarming assay. For complete details on the use and execution of this protocol, please refer to De et al.1.

Enterobacter sp. Strain SM1_HS2B Manifests Transient Elongation and Swimming Motility in Liquid Medium.

Many species of bacteria change their morphology and behavior under external stresses. In this study, we report transient elongation and swimming motility of a novel Enterobacter sp. strain, SM1_HS2B, in liquid broth under a standard growth condition. When growing in the Luria-Bertani medium, HS2B cells delay their cell division and elongate. Although transient over a few hours, the average cell length reaches over 10 times that of the stationary-state cells. The increase is also cumulative following repeated growth cycles stimulated by taking cells out of the exponential phase and adding them into fresh medium every 2 hours. The majority of the cells attain swimming motility during the exponential growth phase, and then they lose swimming motility over the course of several hours. Both daughter cells due to division of a long swimming cell retain the ability to swim. We confirm that the long HS2B cells swim with rigid-body rotation along their body axis. These findings based on microscopic observation following repeated cycles of growth establish HS2B as a prototype strain with sensitive dependence of size and motility on its physical and biochemical environment. IMPORTANCE Bacteria undergo morphological changes in order to cope with external stresses. Among the best-known examples are cell elongation and hyperflagellation in the context of swarming motility. The subject of this report, SM1_HS2B, is a hyperswarming strain of a newly identified species of enterobacteria, noted as Enterobacter sp. SM1. The key finding that SM1_HS2B transiently elongates to extreme length in fresh liquid medium offers new insights on regulation in bacterial growth and division. SM1_HS2B also manifests transient but vigorous swimming motility during the exponential phase of growth in liquid medium. These properties establish HS2B as a prototype strain with sensitive dependence of size and motility on its physical and biochemical environment. Such a dependence may be relevant to swarming behavior with a significant environmental or physiological outcome.

An Inexpensive Imaging Platform to Record and Quantitate Bacterial Swarming.

Bacterial swarming refers to a rapid spread, with coordinated motion, of flagellated bacteria on a semi-solid surface (Harshey, 2003). There has been extensive study on this particular mode of motility because of its interesting biological and physical relevance, e.g., enhanced antibiotic resistance (Kearns, 2010) and turbulent collective motion ( Steager et al., 2008 ). Commercial equipment for the live recording of swarm expansion can easily cost tens of thousands of dollars (Morales- Soto et al., 2015 ); yet, often the conditions are not accurately controlled, resulting in poor robustness and a lack of reproducibility. Here, we describe a reliable design and operations protocol to perform reproducible bacterial swarming assays using time-lapse photography. This protocol consists of three main steps: 1) building a "homemade," environment-controlled photographing incubator; 2) performing a bacterial swarming assay; and 3) calculating the swarming rate from serial photos taken over time. An efficient way of calculating the bacterial swarming rate is crucial in performing swarming phenotype-related studies, e.g., screening swarming-deficient isogenic mutant strains. The incubator is economical, easy to operate, and has a wide range of applications. In fact, this system can be applied to many slowly evolving processes, such as biofilm formation and fungal growth, which need to be monitored by camera under a controlled temperature and ambient humidity.

Confinement discerns swarmers from planktonic bacteria.

Powered by flagella, many bacterial species exhibit collective motion on a solid surface commonly known as swarming. As a natural example of active matter, swarming is also an essential biological phenotype associated with virulence, chemotaxis, and host pathogenesis. Physical changes like cell elongation and hyper-flagellation have been shown to accompany the swarming phenotype. Less studied, however, are the contrasts of collective motion between the swarming cells and their counterpart planktonic cells of comparable cell density. Here, we show that confining bacterial movement in circular microwells allows distinguishing bacterial swarming from collective swimming. On a soft agar plate, a novel bacterial strain Enterobacter sp. SM3 in swarming and planktonic states exhibited different motion patterns when confined to circular microwells of a specific range of sizes. When the confinement diameter was between 40 µm and 90 µm, swarming SM3 formed a single-swirl motion pattern in the microwells whereas planktonic SM3 formed multiple swirls. Similar differential behavior is observed across several other species of gram-negative bacteria. We also observed 'rafting behavior' of swarming bacteria upon dilution. We hypothesize that the rafting behavior might account for the motion pattern difference. We were able to predict these experimental features via numerical simulations where swarming cells are modeled with stronger cell-cell alignment interaction. Our experimental design using PDMS microchip disk arrays enabled us to observe bacterial swarming on murine intestinal surface, suggesting a new method for characterizing bacterial swarming under complex environments, such as in polymicrobial niches, and for in vivo swarming exploration.

Bacterial Swarmers Enriched During Intestinal Stress Ameliorate Damage.

BACKGROUND AND AIMS: Bacterial swarming, a collective movement on a surface, has rarely been associated with human pathophysiology. This study aims to define a role for bacterial swarmers in amelioration of intestinal stress. METHODS: We developed a polymicrobial plate agar assay to detect swarming and screened mice and humans with intestinal stress and inflammation. From chemically induced colitis in mice, as well as humans with inflammatory bowel disease, we developed techniques to isolate the dominant swarmers. We developed swarm-deficient but growth and swim-competent mutant bacteria as isogenic controls. We performed bacterial reinoculation studies in mice with colitis, fecal 16S, and meta-transcriptomic analyses, as well as in vitro microbial interaction studies. RESULTS: We show that bacterial swarmers are highly predictive of intestinal stress in mice and humans. We isolated a novel Enterobacter swarming strain, SM3, from mouse feces. SM3 and other known commensal swarmers, in contrast to their mutant strains, abrogated intestinal inflammation in mice. Treatment of colitic mice with SM3, but not its mutants, enriched beneficial fecal anaerobes belonging to the family of Bacteroidales S24-7. We observed SM3 swarming associated pathways in the in vivo fecal meta-transcriptomes. In vitro growth of S24-7 was enriched in presence of SM3 or its mutants; however, because SM3, but not mutants, induced S24-7 in vivo, we concluded that swarming plays an essential role in disseminating SM3 in vivo. CONCLUSIONS: Overall, our work identified a new but counterintuitive paradigm in which intestinal stress allows for the emergence of swarming bacteria; however, these bacteria act to heal intestinal inflammation.

An expanding bacterial colony forms a depletion zone with growing droplets.

Many species of bacteria have developed effective means to spread on solid surfaces. This study focuses on the expansion of Pseudomonas aeruginosa on an agar gel surface under conditions of minimal evaporation. We report the occurrence and spread of a depletion zone within an expanded colony, where the bacteria laden film becomes thinner. The depletion zone is colocalized with a higher concentration of rhamnolipids, the biosurfactants that are produced by the bacteria and accumulate in the older region of the colony. With continued growth in population, dense bacterial droplets occur and coalesce in the depletion zone, displaying remarkable fluid dynamic behavior. Whereas expansion of a central depletion zone requires activities of live bacteria, new zones can be seeded elsewhere by adding rhamnolipids. These depletion zones due to the added surfactants expand quickly, even on plates covered by bacteria that have been killed by ultraviolet light. We explain the observed properties based on considerations of bacterial growth and secretion, osmotic swelling, fluid volume expansion, interfacial fluid dynamics involving Marangoni and capillary flows, and cell-cell cohesion.

Discovery of oscillations in rotational speed of body-tethered Caulobacter crescentus.

Swarmer cells of Caulobacter crescentus have been found to tether to glass at a point on the cell body. The rolling of the freely rotating flagellum near the glass surface causes the cell body to rotate. We describe the discovery of damped oscillations in the rotational speed of these cell bodies. We show that the damped oscillations are robust over multiple cells and that they depend more on the cell's accumulated rotation angle than on time. We also find that their phase is determined by the moment the flagellar motor changes the direction of its rotation. The oscillations occur only for one direction of cell rotation, when the flagellum is in pulling mode. We discuss possible explanations for these oscillations, including fluctuations in flagellar motor torque and periodic changes in flagellar orientation, and illustrate both of these cases using simplified computer models. Finally, we present the hypothesis that the oscillations are the result of fluctuations in the proton motive force, initiated by a sudden change in proton current that occurs when the motor switches rotation direction.

Orbiting of Flagellated Bacteria within a Thin Fluid Film around Micrometer-Sized Particles.

Bacterial motility under confinement is relevant to both environmental control and the spread of infection. Here, we report observations on Escherichia coli, Enterobacter sp., Pseudomonas aeruginosa, and Bacillus subtilis when they are confined within a thin layer of water around dispersed micrometer-sized particles sprinkled over a semisolid agar gel. In this setting, E. coli and Enterobacteria orbit around the dispersed particles. The liquid layer is shaped like a shallow tent with its height at the center set by the seeding particle, and the meniscus profile set by the strong surface tension of water. The tent-shaped confinement and the left handedness of the flagellar filaments result in exclusively clockwise circular trajectories. The thin fluid layer is resilient because of a balance between evaporation and reinforcement of fluid that permeated out of the agar. The latter is driven by the Laplace pressure caused by the concave meniscus. In short, we explain the physical mechanism of a convenient method to entrap bacteria within localized thin fluid film near a permeable surface.

Buckling Causes Nonlinear Dynamics of Filamentous Viruses Driven through Nanopores.

Measurements and Langevin dynamics simulations of filamentous viruses driven through solid-state nanopores reveal a superlinear rise in the translocation velocity with driving force. The mobility also scales with the length of the virus in a nontrivial way that depends on the force. These dynamics are consequences of the buckling of the leading portion of a virus as it emerges from the nanopore and is put under compressive stress by the viscous forces it encounters. The leading tip of a buckled virus stalls and this reduces the total viscous drag force. We present a scaling theory that connects the solid mechanics to the nonlinear dynamics of polyelectrolytes translocating nanopores.

Capillary flow and mechanical buckling in a growing annular bacterial colony.

A growing bacterial colony is a dense suspension of an increasing number of cells capable of individual as well as collective motion. After inoculating Pseudomonas aeruginosa over an annular area on an agar plate, we observe the growth and spread of the bacterial population, and model the process by considering the physical effects that account for the features observed. Over a course of 10-12 hours, the majority of bacteria migrate to and accumulate at the edges. We model the capillary flow induced by imbalanced evaporation flux as the cause for the accumulation, much like the well-known coffee stain phenomenon. Simultaneously, periodic buckles or protrusions occur at the inner edge. These buckles indicate that the crowding bacteria produce a jam, transforming the densely packed population at the inner edge to a solid state. The continued bacterial growth produces buckles. Subsequently, a ring of packed bacteria behind the inner edge detach from it and break into pieces, forming bacterial droplets. These droplets slowly coalesce while they continually grow and collectively surf on the agar surface in the region where the colony had previously spread over. Our study shows a clear example of how fluid dynamics and elasto-mechanics together govern the bacterial colony pattern evolution.

Nanopore Measurements of Filamentous Viruses Reveal a Sub-nanometer-Scale Stagnant Fluid Layer.

We report measurements and analyses of nanopore translocations by fd and M13, two related strains of filamentous virus that are identical except for their charge densities. The standard continuum theory of electrokinetics greatly overestimates the translocation speed and the conductance associated with counterions for both viruses. Furthermore, fd and M13 behave differently from one another, even translocating in opposite directions under certain conditions. This cannot be explained by Manning-condensed counterions or a number of other proposed models. Instead, we argue that these anomalous findings are consequences of the breakdown of the validity of continuum hydrodynamics at the scale of a few molecular layers. Next to a polyelectrolyte, there exists an extra-viscous, sub-nanometer-thin boundary layer that has a giant influence on the transport characteristics. We show that a stagnant boundary layer captures the essential hydrodynamics and extends the validity of the electrokinetic theory beyond the continuum limit. A stagnant layer with a thickness of about half a nanometer consistently improves predictions of the ionic current change induced by virus translocations and of the translocation velocity for both fd and M13 over a wide range of nanopore dimensions and salt concentrations.

Influence of Physical Effects on the Swarming Motility of Pseudomonas aeruginosa.

Many species of bacteria can spread over a moist surface via a particular form of collective motion known as "surface swarming". This form of motility is typically studied by inoculating bacteria on a gel formed by 0.4-1.5% agar, which contains essential nutrients for their growth and proliferation. Using Pseudomonas aeruginosa and its pili-less mutant, ΔPilA, we investigate physical factors that either facilitate or restrict the swarming motility, measured by the rate of increase in area covered by a spreading bacterial colony, i.e., a swarm. The wild-type colony spreads over the agar surface in highly branched structures. The pili-less mutant fills up the area more fully as it spreads, but it also produces numerous and fragmented branches, or tendrils, at the swarm front. Whereas additional surfactants enhance swarming, increasing the agar percentage, adding extra salt or sugar or incorporating viscous agents in the agar matrix all decrease swarming, supporting the conclusion that swarming motility is restricted by the surface tension at the swarm front and swarm growth is limited by the rate of water supply from within the agar gel. The physical basis elaborated through this study provides a useful framework for understanding the swarming behavior of numerous species of bacteria.

Measurements of fluid viscosity using a miniature ball drop device.

This paper describes measurement of fluid viscosity using a small ball drop device. It requires as little as 100 µl of fluid. Each measurement can be performed in seconds. The experiment is designed to yield reliable viscosity values by operating at properly chosen tilt angles and with calibration using well-characterized Newtonian fluids such as mixtures of glycerol and water. It also yields dynamical viscosity of non-Newtonian fluids at moderate shear rates. The device is easy to assemble and it allows for the measurement of viscosity even when the fluid samples are too small to measure using most commercial viscometers or rheometers. Therefore, the technique is particularly useful in characterizing biological fluids such as solutions of proteins, DNA, and polymers frequently used in biomaterial applications.

The Aerotactic Response of Caulobacter crescentus.

Many motile microorganisms are able to detect chemical gradients in their surroundings to bias their motion toward more favorable conditions. In this study, we observe the swimming patterns of Caulobacter crescentus, a uniflagellated bacterium, in a linear oxygen gradient produced by a three-channel microfluidic device. Using low-magnification dark-field microscopy, individual cells are tracked over a large field of view and their positions within the oxygen gradient are recorded over time. Motor switching events are identified so that swimming trajectories are deconstructed into a series of forward and backward swimming runs. Using these data, we show that C. crescentus displays aerotactic behavior by extending the average duration of forward swimming runs while moving up an oxygen gradient, resulting in directed motility toward oxygen sources. Additionally, the motor switching response is sensitive both to the steepness of the gradient experienced and to background oxygen levels, exhibiting a logarithmic response.

Flagellar Motor Switching in Caulobacter Crescentus Obeys First Passage Time Statistics.

A Caulobacter crescentus swarmer cell is propelled by a helical flagellum, which is rotated by a motor at its base. The motor alternates between rotating in clockwise and counterclockwise directions and spends variable intervals of time in each state. We measure the distributions of these intervals for cells either free swimming or tethered to a glass slide. A peak time of around one second is observed in the distributions for both motor directions with counterclockwise intervals more sharply peaked and clockwise intervals displaying a larger tail at long times. We show that distributions of rotation intervals fit first passage time statistics for a biased random walker and the dynamic binding of CheY-P to FliM motor subunits accounts for this behavior. Our results also suggest that the presence of multiple CheY proteins in C. crescentus may be responsible for differences between its switching behavior and that of the extensively studied E. coli.

Describing directional cell migration with a characteristic directionality time.

Many cell types can bias their direction of locomotion by coupling to external cues. Characteristics such as how fast a cell migrates and the directedness of its migration path can be quantified to provide metrics that determine which biochemical and biomechanical factors affect directional cell migration, and by how much. To be useful, these metrics must be reproducible from one experimental setting to another. However, most are not reproducible because their numerical values depend on technical parameters like sampling interval and measurement error. To address the need for a reproducible metric, we analytically derive a metric called directionality time, the minimum observation time required to identify motion as directionally biased. We show that the corresponding fit function is applicable to a variety of ergodic, directionally biased motions. A motion is ergodic when the underlying dynamical properties such as speed or directional bias do not change over time. Measuring the directionality of nonergodic motion is less straightforward but we also show how this class of motion can be analyzed. Simulations are used to show the robustness of directionality time measurements and its decoupling from measurement errors. As a practical example, we demonstrate the measurement of directionality time, step-by-step, on noisy, nonergodic trajectories of chemotactic neutrophils. Because of its inherent generality, directionality time ought to be useful for characterizing a broad range of motions including intracellular transport, cell motility, and animal migration.

Altered motility of Caulobacter Crescentus in viscous and viscoelastic media.

BACKGROUND: Motility of flagellated bacteria depends crucially on their organelles such as flagella and pili, as well as physical properties of the external medium, such as viscosity and matrix elasticity. We studied the motility of wild-type and two mutant strains of Caulobacter crescentus swarmer cells in two different types of media: a viscous and hyperosmotic glycerol-growth medium mixture and a viscoelastic growth medium, containing polyethylene glycol or polyethylene oxide of different defined sizes. RESULTS: For all three strains in the medium containing glycerol, we found linear drops in percentage of motile cells and decreases in speed of those that remained motile to be inversely proportional to viscosity. The majority of immobilized cells lost viability, evidenced by their membrane leakage. In the viscoelastic media, we found less loss of motility and attenuated decrease of swimming speed at shear viscosity values comparable to the viscous medium. In both types of media, we found more severe loss in percentage of motile cells of wild-type than the mutants without pili, indicating that the interference of pili with flagellated motility is aggravated by increased viscosity. However, we found no difference in swimming speed among all three strains under all test conditions for the cells that remained motile. Finally, the viscoelastic medium caused no significant change in intervals between flagellar motor switches unless the motor stalled. CONCLUSION: Hyperosmotic effect causes loss of motility and cell death. Addition of polymers into the cell medium also causes loss of motility due to increased shear viscosity, but the majority of immobilized bacteria remain viable. Both viscous and viscoelastic media alter the motility of flagellated bacteria without affecting the internal regulation of their motor switching behavior.

Osmotic pressure in a bacterial swarm.

Using Escherichia coli as a model organism, we studied how water is recruited by a bacterial swarm. A previous analysis of trajectories of small air bubbles revealed a stream of fluid flowing in a clockwise direction ahead of the swarm. A companion study suggested that water moves out of the agar into the swarm in a narrow region centered ∼ 30 µm from the leading edge of the swarm and then back into the agar (at a smaller rate) in a region centered ∼ 120 µm back from the leading edge. Presumably, these flows are driven by changes in osmolarity. Here, we utilized green/red fluorescent liposomes as reporters of osmolarity to verify this hypothesis. The stream of fluid that flows in front of the swarm contains osmolytes. Two distinct regions are observed inside the swarm near its leading edge: an outer high-osmolarity band (∼ 30 mOsm higher than the agar baseline) and an inner low-osmolarity band (isotonic or slightly hypotonic to the agar baseline). This profile supports the fluid-flow model derived from the drift of air bubbles and provides new (to our knowledge) insights into water maintenance in bacterial swarms. High osmotic pressure at the leading edge of the swarm extracts water from the underlying agar and promotes motility. The osmolyte is of high molecular weight and probably is lipopolysaccharide.

Helical motion of the cell body enhances Caulobacter crescentus motility.

We resolve the 3D trajectory and the orientation of individual cells for extended times, using a digital tracking technique combined with 3D reconstructions. We have used this technique to study the motility of the uniflagellated bacterium Caulobacter crescentus and have found that each cell displays two distinct modes of motility, depending on the sense of rotation of the flagellar motor. In the forward mode, when the flagellum pushes the cell, the cell body is tilted with respect to the direction of motion, and it precesses, tracing out a helical trajectory. In the reverse mode, when the flagellum pulls the cell, the precession is smaller and the cell has a lower translation distance per rotation period and thus a lower motility. Using resistive force theory, we show how the helical motion of the cell body generates thrust and can explain the direction-dependent changes in swimming motility. The source of the cell body precession is believed to be associated with the flexibility of the hook that connects the flagellum to the cell body.

Stiff filamentous virus translocations through solid-state nanopores.

The ionic conductance through a nanometer-sized pore in a membrane changes when a biopolymer slides through it, making nanopores sensitive to single molecules in solution. Their possible use for sequencing has motivated numerous studies on how DNA, a semi-flexible polymer, translocates nanopores. Here we study voltage-driven dynamics of the stiff filamentous virus fd with experiments and simulations to investigate the basic physics of polymer translocations. We find that the electric field distribution aligns an approaching fd with the nanopore, promoting its capture, but it also pulls fd sideways against the membrane after failed translocation attempts until thermal fluctuations reorient the virus for translocation. fd is too stiff to translocate in folded configurations. It therefore translocates linearly, exhibiting a voltage-independent mobility and obeying first-passage-time statistics. Surprisingly, lengthwise Brownian motion only partially accounts for the translocation velocity fluctuations. We also observe a voltage-dependent contribution whose origin is only partially determined.