We describe a method for analyzing and quantifying the movement pattern of 1 µm carboxylate beads through heterogeneous bacterial biofilms. Comparison of the movement patterns can be used to quantitate differences in material properties of biofilms.
Differences in the material properties of bacterial biofilms have been observed in biofilms of different bacterial species, within the same species under different growth conditions and after treatment with matrix modifying molecules. To better quantitate the material properties of 3D biofilms, an experimental and computational workflow was developed and applied to examine differences between Enterococcus faecalis, Salmonella enterica serotype Typhimurium and Escherichia coli biofilms as well as the role of the amyloid curli in confirming rigidity to Enterobacteriaceae biofilms. The spatio-temporal dynamics of 1 µm carboxylate beads in biofilms were tracked in 20 µm 3D biofilms over 20 minutes. The 4D image stacks were processed using the Mosaic plugin in ImageJ to produce 3D trajectory data of bead movement. This trajectory data was analyzed with a newly developed Bead Evaluator toolbox, where movement data, including trajectory lifespans, bead velocities, cell densities along trajectories, and bounding box information were computed and stored in csv-files. This paper presents the workflow from experimental setup and image recording to bead trajectory computation and analysis. The structure of curli-containing biofilms resulted in more stable bead interactions and less bead movement than in curli-mutant and Enterococcal biofilms. Bead movement did not appear strongly dependent on cell density when measuring the bead velocity and trajectory bounding box volume, supporting the hypothesis that other material properties of the biofilms control the bead dynamics. This technique is widely applicable to quantitating differences in biofilms of different matrix compositions as well as biofilms before and after matrix-modifying treatments.
Bacterial biofilms are ubiquitous as part of human microbiota and continuously interact with molecules. These molecules range in size from 1 nm antibiotics and 1-3 mm bacteria to larger particles of fiber in the gastrointestinal tract. The composition of single- or multispecies biofilms affects the material properties and thus the movement pattern of particles through biofilms1,2,3,4,5. One example is bacterial amyloids, which have a conserved, fibrillar cross-beta sheet structure6. Amyloid curli is expressed by enteric bacteria such as Escherichia coli and Salmonella enterica serotype Typhimurium and genes have been detected in multiple other bacterial phylum7. Various material properties of biofilms are affected by curli8,9. Curli directly interacts with other components of the matrix such as extracellular DNA (eDNA) and cellulose10,11. Curli surrounds the cells and affects cellular membrane rigidity12 and the overall viscoelastic properties of the biofilm13. Curli mediates increased tensile strength by binding to fibronectin, resulting in an increase in strong glass-surface attachment14. Incoming bacteriophages bind to curli and limit phage invasion into biofilms15.
When using multitest coated well slides to analyze roughly 20 µm thick Enterococcus faecalis, E. coli, and S. Typhimurium biofilms using confocal microscopy, clear differences between E. coli, S. Typhimurium10,16 and E. faecalis biofilms (current study) could be observed. While Enterobacteriaceae species biofilms had a high level of rigidity and areas with low cellular density were easy to image, obtaining clear high-resolution pictures of E. faecalis biofilms using line and frame averaging required the application of pressure to the slide to induce sufficient surface tension for cell stability during the imaging process. Bacterial amyloids such as curli form highly ordered structures, suggesting they may be relatively rigid17. This motivated the hypothesis that amyloid curli could be inducing rigidity in E. coli and S. Typhimurium biofilms. There was no clear evidence that E. faecalis was expressing amyloids under the conditions studied. The protein Esp, a pilin gene associated with more pathogenic strains of E. faecalis, was recently shown to produce amyloid structures18; however, using blastn and blastp searches, this gene was not detected in the E. faecalis commensal type strain OG1RF used in these studies. The pheromone cOB1, produced by OG1RF, can form amyloid-like structures19. However, with the given biofilm growth conditions and amyloid detection methods previously used for S. Typhimurium amyloid staining10 de E. faecalis, OG1RF amyloids could not be detected (data not shown). A new four-dimensional (4D) image technique was developed to compare the overall material properties amongst the viscous E. faecalis, E. coli and S. Typhimurium as well as to determine the contribution of amyloid to Enterobacteriaceae biofilms using amyloid mutants of S. Typhimurium and E. coli.
In the past, fluorescent beads were successfully used to analyze the material properties of biofilms in two dimensions (2D) using microrheology20,21,22,23,24,25. This can be applied to a three-dimensional biofilm by studying 2D optic slices at various depths in the biofilm26. The current technique was developed to track 1 µm microscale beads in 3D over time for use in 4D modeling. Part of the rationale was the overarching concept of using 4D modeling to understand movement of plasmids through gastrointestinal microbiota communities. Fluorescently charged carboxylate beads with a 1 µm diameter were used since these correspond well, with respect to size and charge, to E. faecalis, the chosen model organism for plasmid movement and maintenance27, 28. A 4D assay to quantify the physical properties of biofilms was developed (Figure 1A). In the devised methodology, beads were added to biofilms and their spatio-temporal trajectories were recorded through 10-20 µm thick biofilms over the course of 10-20 minutes. Bead trajectories in 3D were then quantified in terms of trajectory length, bead velocity, trajectory bounding box volume (minimal box containing the trajectory), and bounding box cellular density using a newly developed toolbox. The following protocol can be employed to generate 4D image data of bacteria and bead containing biofilms, to preprocess the data with ImageJ29 and the plugin Mosaic, and to analyze bead trajectories with a Bead Evaluator toolbox.
This technique has multiple applications for examining material properties as well as tracking particle and bacterial movement in three dimensions. For example, an early version of this technique was used to characterize the effect of monoclonal antibodies directed against curli on the structural integrity of biofilms16. The full version has multiple tools to provide a more detailed analysis of the biofilm material properties and is continuing to be used to examine effects of monoclonal antibody treatment on biofilms. Particles of different charges can be used to examine the material charge properties of the biofilms and movement of particles through biofilms with different matrix compositions. This could be used to compare the results from 2D microrheology that reveal the material properties responsible for movement of beads we observed in biofilms that were not under flow. This technique could also be used on mixed species biofilms with regions of different biofilm composition. Biofilms can be live imaged under flow in microfluidic devices and flow cells to examine changes in the material properties between static and flow biofilms as well as the effect of flow on movement of particles. The techniques can also be applied to fluorescently labeled bacteria to characterize the movement of exogenous bacteria through a biofilm community. Using three colors, fluorescently labeled donor bacteria, fluorescently labelled recipient bacteria and fluorescently labeled plasmids could be used to track movement, docking, and transfer of plasmids.
Critical steps and troubleshooting
The biggest challenge of this technique is using a mounted coverslip with a very viscous biofilm like E. faecalis. The coverslip needs to be carefully and accurately placed on the multiwell slide without repositioning it. During the sealing step, care needs to be used to prevent pushing down on the coverslip or accidently pushing/sliding it across the slide surface. Any movement or pressure may create surface tension and block movement of a viscous biofilm. If possible, comparing biofilm material properties by imaging a biofilm on an optic bottom well to a coverslip mount will allow technique assessment. When correctly performed, a coverslip mount very closely resembled a biofilm in an optic bottom plate for E. faecalis.
In addition, when using a mounted coverslip, imaging of the interfaces of the biofilm with the coverslip at the bottom or the slide at the very top should be avoided. When using an inverted scope, with the coverslip at the bottom, there can be trapped beads at the base of the biofilm against the coverslip. These beads pass through the biofilm and become trapped against the coverslip even after gentle washing. They have x, y and z coordinates of 0 and bounding box coordinates of 0. However, for certain applications, such as examining biofilm integrity after treatment, these data points can be used as a tool. The ability of beads to penetrate through a thick region of biofilm to the bottom of coverslip can be used to assess biofilm integrity after treatment (manuscript in preparation in collaboration with Tükel laboratory). At the top of the biofilm, in a viscous biofilm like E. faecalis we had some evidence of compaction imposed by the coverslip. This limited the movement of some beads at the glass slide interface and may have introduced some density dependence to the bead movement analysis.
The washing steps were necessary for the biofilms because the growth medium has strong autofluorescence in the green channel. We choose to use excess beads and remove unassociated beads by washing to maximize the associated beads to get the most accurate characterization of the observed regions.
The number of beads and washes needed to obtain desired data sets needs to be determined empirically. The presence of too many beads in a biofilm generates impossibly large data sets that are hard to analyze. The presence of too few beads does not generate a thorough sampling of the biofilm environments. However, control of the number of beads added (2×107 beads in 1 mL of PBS) and the use of wash steps, resulted in a relatively consistent number of beads (40-140) associating with the biofilm depending on its structure, spatial arrangement, and composition.
When studying biofilms with mixed viscous and rigid regions, beads can become trapped in the rigid regions over time. In this case, imaging needs to be started immediately after addition of the beads. This often cannot be accomplished using coverslips but requires optic bottom plates or flow cells where imaging can be done immediately after addition of the beads and the wash step(s).
Modifications and Future Applications
Use of microfluidic devices. In our studies, the optimal conditions established for the study of the Enterobacteriaceae biofilms required growth of the biofilm as a pellicle at the air-liquid interface. This limited the use of optic bottom plates and microfluidic devices in the studies. However, when biofilm formation conditions permit, the biofilms can be grown in microfluidic chambers or flow cells. The biofilms could then be washed, and beads introduced through the microfluidic device with minimal disruption of the biofilm.
Addition of beads during biofilm growth. We chose to add excess beads to the biofilms, and then remove unassociated beads by gentle washing to optimize the number of beads present during the analysis. In the viscous E. faecalis biofilms, it is possible that the beads disassociated and reassociated with during the 20-minute imaging time. If a low number of beads are added at different times during biofilm growth, it might be possible to trap the beads in the biofilm, allowing more accurate characterization of the biofilm movement in more viscous biofilms.
Choice of the region to image. For studies on material properties, it is best to choose thick and thin regions of the biofilm. However, when studying changes in the material properties of a treated biofilm, thick confluent regions may be imaged to determine changes in viscoelastic properties and bead penetration in those regions. In this case, looking for beads that penetrated the biofilm and ended up trapped against the coverslip are a useful measure of biofilm disruption.
Imaging under flow. Using optic glass flow cells or microfluidic devices, movement of beads or bacteria in a biofilm under flow can be imaged. This can be done in different ways. It can be done by injection of beads into the whole chamber followed by brief incubation to allow association of the beads with the biofilm. The unassociated beads can be removed by washing and the biofilm imaged with or without flow. Conversely, a small number of beads can be introduced into one side of the chamber and their movement through and in the biofilm can be tracked under flow. When using flow, caution will need to be used in choosing the Mosaic bead tracking settings (Step 4.5). In the current studies the dynamics setting was Brownian. MSD calculations confirmed that the movement was likely to be diffusive, making Brownian the appropriate setting.
Matrix staining. In the current studies, staining with Syto9 examines cellular density and not biofilm structure density. For example, the presence of amyloids likely increases the density of the matrix material of the biofilm. The dependence of movement on the amyloid density could be determined by using fluorescent matrix stains in lieu of Syto9.
Fluorescently labelled bacteria. Fluorescently labelled bacteria can be used to track movement of exogenous bacteria through biofilms (e.g., plasmid-containing bacteria). The challenge with fluorescently labelled bacteria, such as Enterococci, is that they form singles, diplococci, and short chains, which complicates the ability to accurately track the bacteria. This process would be easier if the bacteria have a single-cell morphology.
Limitations
Limitations in trajectory visualization and stitching.
One limitation of the method is trajectory visualization and stitching. Reconstructed and analyzed trajectories consist of x, y, z point coordinates, where subsequent points define the linear path between these points. Visualization of such piecewise linear trajectories can be achieved by various tools. One approach was to use Python and Jupyter notebooks together with the Python plugins, Pandas and Matplotlib. While it was possible to visualize individual existing trajectories in the Journal of Bacteriology article where this technique was originally published34, there were still significant limitations that are being addressed in future research.
Currently, the number of reconstructed trajectories is larger than the number of beads in the biofilm, meaning multiple trajectories may correspond to one bead. This can be caused by a weak confocal signal in one frame where Mosaic will terminate a trajectory and initiate a second. This may register as multiple shorter trajectories for one bead, especially in less viscous biofilms. Another cause for the large number of trajectories is the lack of trajectory stitching. Especially in E. faecalis optic bottom well biofilms, beads visually remain associated with the biofilm during imaging (Supplemental Video 2). However, there were no trajectories longer than 10 and over 90% of the trajectories had a lifespan of 5 frames or less (Figure 3D). If the software is used to analyze only trajectories above a defined length (e.g., when tracing cells that are capable of transferring plasmids), shorter trajectories can automatically be removed from the data set. However, there are other purposes for which stitching the trajectories may be very important. Finally, the inability to track rapid bead movement as a single trajectory could result in more trajectories in Enterobacteriaceae biofilms due to the rapid movement in the Z direction resulting in elliptical-shaped beads (Figure 2G). The possibility of stitching trajectories disrupted by rapid anisotropic movement will be important to study the effect of the curli amyloid matrix in Enterobacteriaceae.
Significance
A computational workflow was developed to study bead trajectories to compare the material properties of 3D biofilms. The workflow enables researchers to identify critical parameters that can be used in computational modeling of fluid dynamics in heterogeneous biofilms. With the help of this open-source bead evaluator, the effect bacterial amyloid curli on the material properties could be studied, showing increased biofilm matrix rigidity due to curli. In a more general context, the evaluator can be used to study changes in biofilm structure induced by biofilm treatment or different environmental conditions, such as flow. For example, the tool is being used to analyze the effect of monoclonal antibody treatment on the disruption of biofilm structures in collaboration with the Tükel laboratory (LKSOM Temple University). The bead evaluator toolbox is fully adaptable and extendable in a modular fashion using VRL-Studio to further enhance and extend its functions.
The authors have nothing to disclose.
Work in the GQ and BAB labs received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. The authors acknowledge Isaac Klapper, Ph.D (Department of Mathematics, Temple University) for helpful discussion and Çagla Tükel (Department of Microbiology and Immunology, Temple University) for Enterobacteriaceae expertise in the initial publication of containing this technique.
96-well plates, No. 1.5 Uncoated Coverslip, 5 mm Glass Diameter | MatTek | P96G1.55F | |
Fisherbrand Cover Glasses: Circles | Fisher Scientific | 12-293-232P | 1.5 optic glass coverslip |
Invitrogen Syto 9 Green Fluorescent Nucleic Acid Stain | Invitrogen | S34854 | |
Molecular Probes FluoSpheres Carboxylate-modified Microspheres, 1 um, crimson fluorescent (625/645) | Molecular Probes | F8816 |
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