We thank Ms. Susan Staurovsky at the UConn Health Center for Cell Analysis and Modeling Microscopy Facility for her assistance with microscopy experiments. We thank Dr. Mona Wu Orr and the Essentials of Staphylococcal Genetics and Metabolism Workshop for their protocols and advice on cloning in P. aeruginosa and S. aureus, respectively. This work was supported by funding from the National Institutes of Health (NIH; DP2GM146456-01 and 1R01AI167886-01A1 to W.W.K.M., 1F30DE032598-01A1 to P.J.H., and 1F31DK136259-01A1 to T.J.L.). The funders had no role in the design of our experiments or preparation of this manuscript.

Visualizing Antibiotic Persistence in Bacterial Pathogens

<ol>
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The authors have no conflicts of interest to declare.

We have found that the success of a time-lapse microscopy experiment hinges on the quality of the agarose pads and their stability throughout the course of imaging. The stainless-steel chamber-enclosed agarose pads are relatively easy to prepare, resulting in consistently planar samples that can be stably imaged over dozens of hours. This enables the imaging of tens of thousands of cells in a single experiment and increases the likelihood of detecting rare phenotypic variants, like persister cells, in a population.
This agarose pad preparation method presents an easily implementable alternative to previously published methods. Our protocol does not require the technical precision of microfluidic device fabrication or the dexterous manipulations of agarose &#34;sandwich&#34; methods, making it easier to achieve consistent preparations from run to run14,16,36. Furthermore, the system is cost-effective. The stainless-steel chamber is sterilizable and reusable (unlike single-use plastic chambers) and the setup does not require specialized equipment16,37. The chamber is easily fit to different microscopy systems using commercially available stage inserts. Additionally, because bacteria are immobilized at the agarose-cover glass interface, we have had success with tracking highly motile bacteria such as P. aeruginosa while still allowing for morphological changes (Figure&#160;4, Supplementary Video 4). Other single-cell imaging techniques, such as the &#34;mother machine&#34;, confine cells to channels that preclude observation of morphological changes other than filamentation36.
For success with this protocol, there are some critical steps and parameters to keep in mind. For pad preparation, it is important to thoroughly heat and melt the agarose, as any remaining agarose crystals will cause light diffraction and affect image quality. Similarly, one must take care to pipette the agarose into the chamber without introducing air bubbles. To ensure that the thickness of the agarose pads remains consistent and to limit sample drift, it is important to allow the pad to equilibrate&#8212;typically for 15 min&#8212;in the humidified, temperature-controlled enclosure before imaging begins. Another factor that can cause poor image quality is humidity control: low humidity will result in the agarose pad dehydrating and shrinking, whereas high humidity (or improper sample warming in the chamber) could cause the warm air to condense on the sample and distort the imaging. An example of suboptimal time-lapse imaging due to condensation can be found&#160;Supplementary Video 8.
A limitation of the current set-up is that the culture media cannot be exchanged, which prevents continuous tracking of individual bacteria before, during, and after antibiotic treatment. We anticipate that coupling the agarose pad with flow cells or microfluidic devices that permit culture media exchange could enable populations to be tracked during nutritional or environmental change. Another parameter of the current design that could be improved is sample aeration. The O-ring seal, screw-top design of the interchangeable coverslip dish enables better sample aeration compared with set-ups that require the use of wax or grease-based sealant to seal the pads16. However, aeration in the sealed chamber may still be limited and may not support the growth of obligate aerobes, though this remains to be tested.
The time-lapse imaging sample preparation protocol that we present in this article enables thousands of bacteria to be tracked as they respond to or recover from antibiotic treatment. This method is also highly generalizable and has a variety of potential applications beyond persister biology. For example, the agarose pad and divider set-up allows for the seeding of spatially separated cell samples yet permits cell-cell communication via diffusion through the agarose pad. We are currently exploring this set-up&#39;s potential for testing how the exchange of secreted products affects cell growth in multispecies communities. We anticipate that this protocol will provide a low barrier of entry to time-lapse microscopy for the new investigator and limitless variations for the seasoned microbiologist to explore.

Bacterial pathogens can evade the effects of antibiotics through two primary mechanisms: antibiotic resistance, which involves genetic changes, and phenotypic tolerance, which involves non-genetic changes. Antibiotic resistance is a genetically encoded phenomenon that confers the ability of a given bacterial cell to not only survive but also replicate in the presence of an antibiotic1. Phenotypic tolerance, which can encompass antibiotic-tolerant or antibiotic-persistent bacteria, occurs when cells withstand bactericidal antibiotic treatment without gaining the ability to replicate in the presence of an inhibitory concentration of the antibiotic1,2. What differentiates tolerance from persistence is that tolerance refers to the ability of the whole population to survive treatment, whereas persistence refers to a subset of an isogenic but phenotypically heterogeneous population that survives antibiotic treatment. When a clonal culture is treated with bactericidal antibiotics and survivors that remain in the culture are plotted against time on a log-linear scale, a biphasic curve is usually detected when persisters are present. On these curves, the first phase shows that the majority of the population is killed relatively quickly, and the second phase indicates that an antibiotic persistent fraction is killed at a slower rate or not at all1,2.
Antibiotic persistence presents a major burden on global healthcare systems. For instance, Staphylococcus aureus and Pseudomonas aeruginosa persisters, which are the focus of this article, are thought to cause antibiotic-recalcitrant infections, including recurrent airway infections in patients with cystic fibrosis and chronic wound infections3,4. Therefore, further elucidating persister cell biology and phenotypic programs is critical. While progress has been made in understanding how persisters form and resuscitate, critical knowledge gaps pertaining to the coordination of metabolic reprogramming and molecular events in individual cells that underlie persistence remain5,6,7,8.
Effectively studying persistence has proven to be a technical challenge. Since persistence is only observable in a small subset of a bacterial population, techniques that sample bulk bacterial populations often fail to capture relevant biological information1,2,8,9,10. Furthermore, since phenotypic changes that underlie persistence are transient and not heritable, tracking the fate of persister cells can be complex1,8,9,10,11. Once bacterial persisters resume growth, they can divide and give rise to both persisters and non-persisters, which makes it impossible to enrich for pure persister populations by culturing. These challenges highlight the need for techniques that can fulfill the following criteria: 1) the ability to capture biological information of live, single cells and 2) the ability to be used in tandem with fluorescent dyes, probes, sensors, and reporters that allow phenotypes of individual cells in heterogeneous populations to be interrogated over time.
Recent advancements in single-cell technologies have provided an avenue to effectively investigate bacterial heterogeneity and overcome these hurdles in studying persistence12,13. Some of these techniques include fluorescence microscopy, flow cytometry/fluorescence-activated cell sorting, microfluidics, and single-cell RNA sequencing12,13. Here, we describe protocols for elucidating single-cell persister physiology using epifluorescence time-lapse microscopy of transcriptional or translational reporter strains. Fluorescence microscopy is a powerful technique that fulfills the criteria for studying persister phenotypes, namely, the ability to identify which individual cells in a large population propagate after antibiotic removal and can thus be defined as persisters. With the introduction of automated camera technologies and incubated chambers, capturing live bacterial cells is widely accessible across the field of microbiology. Crucially, time-lapse microscopy offers the ability to visualize single cells in real-time, over the course of hours and even days, which makes it possible to track bacteria before, during, and after antibiotic treatment14,15,16. Insights from these investigations harnessing time-lapse microscopy have immense potential to generate insight into the complex mechanisms of persister biology.

<table><tbody><tr><td>BaSiC</td><td>GitHub</td><td>https://github.com/marrlab/BaSiC</td><td></td></tr><tr><td>Certified Molecular Biology Agarose</td><td>Biorad</td><td>1613101</td><td></td></tr><tr><td>Fiji-ImageJ</td><td>NIH</td><td>https://imagej.net/software/fiji/downloads</td><td></td></tr><tr><td>Interchangeable Coverglass Dish</td><td>Bioptechs</td><td>190310-35</td><td>35 mm ICD for preparing agarose pads; comes with 30 mm (#1.5) coverslips</td></tr><tr><td>Lumencor Spectra 7 LED light engine</td><td>Lumencor</td><td>https://lumencor.com/products/spectra-light-engine</td><td>Spectra 7 LED light engine</td></tr><tr><td>MetaMorph</td><td>Molecular Devices</td><td>Premier version 7.10.5</td><td></td></tr><tr><td>pco.edge 4.2 bi sCMOS camera</td><td>Excelitas</td><td>https://www.excelitas.com/product/pcoedge-42-bi-usb-scmos-camera</td><td>sCMOS camera</td></tr><tr><td>PeCon live cell incubation chamber</td><td>peCon</td><td>https://www.pecon.biz/</td><td></td></tr><tr><td>Thomas Scientific Round cover glass, #1.5 thickness, 25 mm, 100 pack</td><td>Fisher Scientific</td><td>NC1272770</td><td>25 mm (#1.5) coverslips</td></tr><tr><td>Zeiss Axiovert 200M microscope</td><td>Zeiss</td><td>https://www.zeiss.com/microscopy/en/products/light-microscopes/widefield-microscopes/axiovert-for-materials.html</td><td>Inverted microscope with Plan-Apochromat 63x/1.40 Oil Ph3 M27 objective, Lumencor Spectra 7 LED light engine, and pco.edge 4.2 bi sCMOS camera (6.5 mm pixel size)</td></tr></tbody></table>

time-lapse epifluorescence microscopy imaging of pseudomonas aeruginosa and staphylococcus aureus heterogeneous phenotypes

Antibiotic persistence is a phenomenon in which a small number of bacterial cells in a genetically susceptible population survive antibiotic treatment that kills the other genetically identical cells. Bacterial persisters can resume replication once antibiotic treatment ends and are commonly thought to&#160;underlie clinical treatment failure.&#160;Recent work harnessing the power of time-lapse fluorescence microscopy, in which bacteria are labeled with fluorescent transcriptional reporters, translational reporters, and/or dyes for a variety of cellular features, has advanced our understanding of Escherichia coli persisters beyond what could be learned from population-level antibiotic survival assays. Such single-cell approaches, rather than bulk population assays, are essential for delineating the mechanisms of persister formation, damage response, and survival. However, methods for studying persisters in other important pathogenic species at this level of detail remain limited.
This study provides an adaptable approach for time-lapse imaging of Pseudomonas aeruginosa (a gram-negative rod) and Staphylococcus aureus (a gram-positive coccus)&#160;during antibiotic treatment and recovery. We discuss molecular genetic approaches to introduce fluorescent reporters into these bacteria. Using these reporters, as well as dyes, we can track the phenotypic changes, morphological features, and fates of individual cells in response to antibiotic treatment. Additionally, we are able to observe the phenotypes of individual persisters as they resuscitate following treatment. In all, this work serves as a resource for those interested in tracking the survival and gene expression of individual antibiotic-treated cells, including persisters, both during and after treatment, in clinically important pathogens.

Successful introduction of reporter plasmids into P. aeruginosa and S. aureus is indicated by growth on the correct selective antibiotics and can be confirmed by colony PCR and/or sequencing. The modified strains should be verified as phenotypic reporters by subjecting them to conditions in which the gene of interest is known to be induced, and the resultant fluorescence can be measured by flow cytometry, spectrophotometry, or epifluorescence microscopy (Figure 1).
To facilitate the selection of an antibiotic dose(s) that will be used for subsequent experiments, perform concentration-dependent antibiotic persister assays for the P. aeruginosa or S. aureus strains of interest. Concentration-dependent assays typically result in a biphasic curve with a steep initial slope at lower antibiotic concentrations and a plateau or less steep slope at higher concentrations. However, for some antibiotic-species pairs, a distinct biphasic curve may not result. For example, the curve for S. aureus delafloxacin curve is clearly biphasic (Figure 3A), but the P. aeruginosa levofloxacin curve is not (Figure 3B)15. In this scenario, we would choose a concentration that is at least 10x the MIC (e.g.,&#160;5&#160;&#181;g/mL, which is about 15x the MIC for P. aeruginosa)15. However, because 15x levofloxacin MIC results in only ~0.001% P. aeruginosa survivors, we use 1 &#181;g/mL levofloxacin treatment if we want to see persisters when imaging cells as they recover on antibiotic-free agarose pads (Supplementary Video 6); otherwise, the number of fields of view needed to image multiple persisters becomes prohibitive.
At the start of imaging, the ideal sample and agarose pad preparation should appear planar throughout the field of view, free of large debris, wrinkles, or air bubbles, and with evenly distributed single cells. Obtaining well-distributed single cells may require optimization of the sample dilution or resuspension. For S. aureus, cells tend to form small clusters and need to be vortexed thoroughly before seeding to the agarose pad (Figure 4 and Figure 5). For P. aeruginosa, cells may form aggregates encased in a sticky extracellular matrix in suspension; it is necessary to pipette these samples thoroughly and disrupt the aggregates for imaging single cells. 
After an imaging experiment&#39;s conclusion, a successful time-lapse of images will appear in focus, stably illuminated, and with minimal drift in the x-y plane throughout the experiment. Supplementary Video 7 represents an optimal image acquisition: t is the phase channel of Supplementary Video 4 before shade or drift correction. Loss of focus can occur if condensation (from over-humidification or insufficient sample warming) causes water droplets to form on the top 25 mm coverslip, distorting the light and pushing the focal plane outside of the autofocus algorithm&#39;s maximal search range (Supplementary Video 8). Variation in illumination usually indicates insufficient immersion oil at the time of imaging. If the stage moves too quickly, the oil on the objective may drag behind and still be catching up when the images are acquired. This can be mitigated by adjusting the acquisition controls to slow the speed of movement or adding a pause between the movement to the next position and the image acquisition. Major sample drift will look like many cells streaking across the field of view while some stay in place (Supplementary Video 9). This typically occurs later in experiments because the agarose pad has dehydrated due to insufficient humidity control. The agarose pad preparation presented in this paper was designed to facilitate sample stability, but properly warming/humidifying the sample and its surrounding environment is necessary for optimal image acquisition.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67617/67617fig1.jpg" /> 
Figure 1: Fluorescent reporter strains illuminate the expression of a gene of interest. (A) S. aureus was transduced with a GFP transcriptional reporter for a gene of interest per Protocol 1. The reporter strain was treated for 24 h with antibiotic, washed with PBS, and then seeded onto an agarose pad made from CA-MHB plus propidium iodide (1.6 &#181;M) and chloramphenicol (10 &#181;g/mL for reporter plasmid maintenance) for imaging during recovery (Supplementary Video 1). (B) P. aeruginosa was transformed with a plasmid bearing a mScarlet-linked translational reporter for a protein of interest26. The reporter strain was treated for 5 h with antibiotic, washed with PBS, and then seeded onto an agarose pad made from BSM plus Tet (75 &#181;g/mL; for reporter plasmid maintenance) for imaging during recovery (Supplementary Video 2). <a href="https://www.jove.com/files/ftp_upload/67617/67617fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67617/67617fig2.jpg" /> 
Figure 2: Propagating and harvesting bacteriophage. (A) The six plates show six different amounts of diluted phage stock on the lawns of S. aureus RN4220. The red outlines indicate the three plates that would be harvested, from the plate with the most clearing (bold red outline; 1 x109 PFU/mL) to the next two dilutions (1 x108 and 1 x 107 PFU/mL). The black arrows point to individual plaques. (B) To harvest phage from the plates, scrape the soft agar layer (left), transfer the slurry to the next dilution plate (center), and, after pooling the soft agar from all three plates together, combine into a conical tube for centrifugation (right). <a href="https://www.jove.com/files/ftp_upload/67617/67617fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67617/67617fig3.jpg" /> 
Figure 3: Representative concentration-dependent persister assays. Concentration-dependent fluoroquinolone persistence was assessed in stationary-phase (A)&#160;S. aureus (against delafloxacin) and (B)&#160;P. aeruginosa (against levofloxacin). Subsequent experiments utilize 5 &#181;g/mL delafloxacin (red circle) because S. aureus killing had plateaued at this concentration. A dosage of at least 1 &#181;g/mL levofloxacin (red circle) would be utilized for subsequent experiments with P. aeruginosa. Note that the bacterial killing does not plateau for P. aeruginosa, but there is still a less steep &#34;second phase&#34; of the biphasic curve that indicates a persistent subpopulation. Panel 3B has been adapted with permission from Hare et al.15. <a href="https://www.jove.com/files/ftp_upload/67617/67617fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67617/67617fig4.jpg" /> 
Figure 4: Imaging bacterial phenotypes during antibiotic treatment. Stationary-phase&#160;(A)&#160;S. aureus and&#160;(B)&#160;P. aeruginosa cells were seeded onto agarose pads containing fluoroquinolone antibiotics and monitored during treatment: 5 &#181;g/mL delafloxacin for S. aureus (Supplementary Video 3) and 5 &#181;g/mL levofloxacin for P. aeruginosa (Supplementary Video 4)15. Propidium iodide (PI; 16 &#181;M for P. aeruginosa, 1.6 &#181;M for S. aureus) was added to the pads to mark dead or dying cells. S. aureus cells remain largely intact and alive in the presence of the FQ, whereas most P. aeruginosa cells undergo drastic morphological changes, including forming round spheroplasts, before they lyse and die. <a href="https://www.jove.com/files/ftp_upload/67617/67617fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67617/67617fig5.jpg" /> 
Figure 5: Tracking persisters during recovery. (A)&#160;S. aureus and&#160;(B)&#160;P. aeruginosa populations were seeded onto agarose pads containing fresh media after they had been treated with fluoroquinolones (5 &#181;g/mL delafloxacin for S. aureus and 1 &#181;g/mL levofloxacin for P. aeruginosa) and monitored during their post-treatment recovery (Supplementary Video 5 and Supplementary Video 6). The perisisters seen are indicated with green arrows in the first two frames in each panel, and they remained intact and viable during antibiotic treatment. After an initial lag period, the persisters started to divide and gave rise to new progeny (indicated with green circles). <a href="https://www.jove.com/files/ftp_upload/67617/67617fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/67617/67617fig6.jpg" /> 
Figure 6: Microscope sample preparation. (A)&#160;Schematic of the sample preparation workflow using an interchangeable coverslip dish (&#34;chamber&#34;). (B) Picture of the disassembled chamber and its individual components. (C) Picture of the fully assembled chamber. <a href="https://www.jove.com/files/ftp_upload/67617/67617fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Video 1: S. aureus persister. Video file containing the images in Figure 1A. In brief, S. aureus bearing a GFP transcriptional reporter for a gene of interest was treated for 24 h with antibiotic, washed with PBS, then seeded onto an agarose pad made with CA-MHB plus propidium iodide (1.6 &#181;M) and chloramphenicol (10 &#181;g/mL) for imaging during recovery. <a href="https://www.jove.com/files/ftp_upload/67617/VideoS1_S-aureus_persister_video.avi" target="_blank">Please click here to download this video.</a>
Supplementary Video 2: P. aeruginosa persister. Video file containing images in Figure 1B.&#160;In brief, P. aeruginosa bearing an mScarlet-linked translational reporter for a protein of interest was treated for 5 h with antibiotic, washed with PBS, then seeded onto an agarose pad made with BSM plus Tet (75 &#181;g/mL) for imaging during recovery26. <a href="https://www.jove.com/files/ftp_upload/67617/VideoS2_Paeru-fluorescent-persister.avi" target="_blank">Please click here to download this video.</a>
Supplementary Video 3: S. aureus during antibiotic treatment. A stationary-phase culture of S. aureus grown in rich chemically defined media was seeded to agarose pads made from the culture&#39;s cell-free conditioned media with propidium iodide (1.6 &#956;M) and delafloxacin (5 &#956;g/mL). <a href="https://www.jove.com/files/ftp_upload/67617/VideoS3_S-aureus_treatment.avi" target="_blank">Please click here to download this video.</a>
Supplementary Video 4: P. aeruginosa during antibiotic treatment. A stationary-phase culture of P. aeruginosa grown in BSM was seeded to agarose pads made from cell-free conditioned media from a culture of P. aeruginosa grown in BSM in parallel; the agarose pad also contained propidium iodide (16 &#181;M) and levofloxacin (5 &#181;g/mL). This video has been adapted with permission from Hare et al.15. <a href="https://www.jove.com/files/ftp_upload/67617/VideoS4_P-aeruginosa_treatment.avi" target="_blank">Please click here to download this video.</a>
Supplementary Video 5: S. aureus during post-antibiotic recovery. S. aureus was&#160;grown to&#160;stationary phase in rich chemically defined media. The stationary-phase cultures were treated with 5 &#181;g/mL delafloxacin in test tubes for 24 h, washed with PBS, and then seeded to antibiotic-free CA-MHB agarose pads containing propidium iodide (1.6 &#181;M) for imaging. <a href="https://www.jove.com/files/ftp_upload/67617/VideoS5_S-aureus_recovery.avi" target="_blank">Please click here to download this video.</a>
Supplementary Video 6: P. aeruginosa during post-antibiotic recovery. A stationary-phase culture&#160;of P. aeruginosa grown in BSM was treated with 1 &#181;g/mL levofloxacin in test tubes for 7 h, washed with PBS, then seeded to antibiotic-free BSM agarose pads containing propidium iodide (16 &#181;M) for imaging. <a href="https://www.jove.com/files/ftp_upload/67617/VideoS6_P-aeruginosa_recovery.avi" target="_blank">Please click here to download this video.</a>
Supplementary Video 7: Example of optimal image acquisition. This video is the phase channel of Supplementary Video 4 before image processing as an example of an optimal time-lapse acquisition. Note the minimal drift, stable illumination, and maintenance of focus throughout the experiment. <a href="https://www.jove.com/files/ftp_upload/67617/VideoS7_uncorrected-Paeru.avi" target="_blank">Please click here to download this video.</a>
Supplementary Video 8: Example of suboptimal image acquisition due to condensation. This video shows part of an experiment when the image acquisition was affected by poor focus, likely due to condensation on the chamber due to improper heating of the sample and/or over-humidification of the imaging environment. The sample being imaged was levofloxacin-treated P. aeruginosa during post-antibiotic recovery on a BSM agarose pad. <a href="https://www.jove.com/files/ftp_upload/67617/VideoS8_Suboptimal_Imaging_Condensation.avi" target="_blank">Please click here to download this video.</a>
Supplementary Video 9: Example of suboptimal image acquisition due to drift. This video shows part of an experiment when the image acquisition was affected by sample drift, likely due to dehydration and the shrinking/lifting of the agarose pad from the coverslip. The sample being imaged was P. aeruginosa on an agarose pad containing levofloxacin and propidium iodide. <a href="https://www.jove.com/files/ftp_upload/67617/VideoS9_Suboptimal_Drift.avi" target="_blank">Please click here to download this video.</a>
Supplementary File 1: 25mm-3D-divider-for-35mmBioptechs.stl <a href="https://www.jove.com/files/ftp_upload/67617/25mm-3D-divider-for-35mmBioptechs.stl" target="_blank">Please click here to download this file.</a>

Time-Lapse Epifluorescence Microscopy Imaging of Pseudomonas aeruginosa and Staphylococcus aureus Heterogeneous Phenotypes

In this manuscript, we provide a comprehensive protocol for assessing antibiotic survival for Pseudomonas aeruginosa and Staphylococcus aureus, transforming plasmids into P. aeruginosa and S. aureus to create reporter strains and visualize&#160;phenotypic variants, such as persisters, by time-lapse epifluorescence&#160;microscopy.

Antibiotic persistence is a phenomenon in which a small number of bacterial cells in a genetically susceptible population survive antibiotic treatment ...

time-lapse-epifluorescence-microscopy-imaging-pseudomonas-aeruginosa

Research

JoVE Journal

Biology

692 Views.  UConn Health. In this manuscript, we provide a comprehensive protocol for assessing antibiotic survival for Pseudomonas aeruginosa and Staphylococcus aureus, transforming plasmids into P. aeruginosa and S. aureus to create reporter strains and visualize phenotypic variants, such as persisters, by time-lapse epifluorescence microscopy.

Video: Time-Lapse Epifluorescence Microscopy Imaging of Pseudomonas aeruginosa and Staphylococcus aureus Heterogeneous Phenotypes

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

Immunology and Infection

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of phenol soluble modulins (PSMs) and other toxins produced and secreted by Staphylococcus aureus on neutrophils. To study the effects of the PSMs on neutrophils we isolate fresh neutrophils using density gradient centrifugation. These neutrophils are loaded with a dye that fluoresces upon calcium mobilization. The activation of neutrophils by PSMs initiates a rapid and transient increase in the free intracellular calcium concentration. In a flow cytometry experiment this rapid mobilization can be measured by monitoring the fluorescence of a pre-loaded dye that reacts to the increased concentration of free Ca2+. Using this method we can determine the PSM concentration necessary to activate the neutrophil, and measure the effects of specific and general inhibitors of the neutrophil activation.	
	To investigate the expression of the PSMs in the intracellular space, we have constructed reporter fusions of the promoter of the PSM&alpha; operon to GFP. When these reporter strains of S. aureus are phagocytosed by neutrophils, the induction of expression can be observed using fluorescence microscopy.

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

We present methods to study the effect of PSMs and other toxins secreted by Staphylococcus aureus on neutrophils using flow cytometry and fluorescence microscopy.

We present methods to study the effect of phenol soluble modulins (PSMs)  and other toxins produced and secreted by Staphylococcus  aureus on neutrophils. ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Bioengineering

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence microscopy as observations made at a population level may not truly reflect what occurs at a single cell level. Live-cell timelapse microscopy allows investigators to monitor the changes in cell division or cell morphology which provide valuable insights regarding subcellular localization of proteins and timing of gene expression, as it happens, to potentially aid in answering important biological questions. Here, we describe our protocol to monitor phenotypic changes in Bacillus subtilis and Staphylococcus aureus using a high-resolution deconvolution microscope. The objective of this report is to provide a simple and clear protocol that can be adopted by other investigators interested in conducting fluorescence microscopy experiments to study different biological processes in bacteria as well as other organisms.

This article provides a step-by-step guide to investigate protein subcellular localization dynamics and to monitor morphological changes using high-resolution fluorescence microscopy in Bacillus subtilis and Staphylococcus aureus.

Investigations of factors influencing cell division and cell shape in bacteria are commonly performed in conjunction with high-resolution fluorescence ...

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method ...

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic infection. The development of chronic P. aeruginosa infections is associated with a decline in lung function and increased morbidity. Therefore, there is a great interest in elucidating the reasons for the failure to eradicate P. aeruginosa with antibiotic therapy which occurs in approximately 10-40% of pediatric patients. One of many factors that can affect host clearance of P. aeruginosa and antibiotic susceptibility is variations in spatial organization (such as aggregation or biofilm formation) and polysaccharide production. Therefore, we were interested in visualizing the in situ characteristics of P. aeruginosa within the sputum of CF patients. A tissue clearing technique was applied to sputum samples after embedding the samples into a hydrogel matrix to retain the 3D structures relative to host cells. After tissue clearing, fluorescent labels and dyes were added to allow visualization. Fluorescence in situ hybridization was performed for the visualization of bacterial cells, binding of fluorescently labeled anti-Psl-antibodies for the visualization of the exopolysaccharide and DAPI staining to stain host cells to obtain structural insight. These methods allowed for the high-resolution imaging of P. aeruginosa within the sputum of CF patients via confocal laser scanning microscopy.

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic ...

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a large proportion of the infecting bacteria form biofilms within airway sputum. Pa biofilms isolated from CF sputum have been shown to grow in small, dense aggregates of ~10-1,000 cells that are spatially organized and exhibit clinically relevant phenotypes such as antimicrobial tolerance. One of the biggest challenges to studying how Pa aggregates respond to the changing sputum environment is the lack of nutritionally relevant and robust systems that promote aggregate formation. Using a synthetic CF sputum medium (SCFM2), the life history of Pa aggregates can be observed using confocal laser scanning microscopy (CLSM) and image analysis at the resolution of a single cell. This in vitro system allows the observation of thousands of aggregates of varying size in real time, three dimensions, and at the micron scale. At the individual and population levels, having the ability to group aggregates by phenotype and position facilitates the observation of aggregates at different developmental stages and their response to changes in the microenvironment, such as antibiotic treatment, to be differentiated with precision.

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Synthetic cystic fibrosis sputum medium (SCFM2) can be utilized in combination with both confocal laser scanning microscopy and fluorescence-activated cell sorting to observe bacterial aggregates at high resolution. This paper details methods to assess aggregate populations during antimicrobial treatment as a platform for future studies.

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a ...

Time-lapse Imaging of Bacterial Swarms and the Collective Stress Response

Swarming is a form of surface motility observed in many bacterial species including Pseudomonas aeruginosa and Escherichia coli. Here, dense populations of bacteria move over large distances in characteristic tendril-shaped communities over the course of hours. Swarming is sensitive to several factors including medium moisture, humidity, and nutrient content. In addition, the collective stress response, which is observed in P. aeruginosa that are stressed by antibiotics or bacteriophage (phage), repels swarms from approaching the area containing the stress. The methods described here address how to control the critical factors that affect swarming. We introduce a simple method to monitor swarming dynamics and the collective stress response with high temporal resolution using a flatbed document scanner, and describe how to compile and perform a quantitative analysis of swarms. This simple and cost-effective method provides precise and well-controlled quantification of swarming and may be extended to other types of plate-based growth assays and bacterial species.

We detail a simple method to produce high-resolution time-lapse movies of Pseudomonas aeruginosa swarms that respond to bacteriophage (phage) and antibiotic stress using a flatbed document scanner. This procedure is a fast and simple method for monitoring swarming dynamics and may be adapted to study the motility and growth of other bacterial species.

Swarming is a form of surface motility observed in many bacterial species including Pseudomonas aeruginosa and Escherichia coli. Here, dense populations ...

Time-Lapse Epifluorescence Microscopy Imaging of Pseudomonas aeruginosa and Staphylococcus aureus Heterogeneous Phenotypes

Summary

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Chapters in this video

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

Studying Interactions of Staphylococcus aureus with Neutrophils by Flow Cytometry and Time Lapse Microscopy

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

Live-Cell Fluorescence Microscopy to Investigate Subcellular Protein Localization and Cell Morphology Changes in Bacteria

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Time-lapse Imaging of Bacterial Swarms and the Collective Stress Response