Name: kinetic visualization of single-cell interspecies bacterial interactions
Uploaded: 2020-08-05T13:30:00
Description: Watch this Scientific Journal Video about Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions at JoVE.com

This work was supported by funding from the Cystic Fibrosis Foundation Postdoc-to-Faculty Transition Award LIMOLI18F5 (DHL), Cystic Fibrosis Foundation Junior Faculty Recruitment Award LIMOLI19R3 (DHL), and NIH T32 Training Grant 5T32HL007638-34 (ASP). We thank Jeffrey Meisner, Minsu Kim, and Ethan Garner for sharing initial protocols and advice for imaging and making pads.

NOTE: A full description and catalog numbers for all supplies in this protocol can be found in the Table of Materials.
1. Preparation of M8T minimal media
<ol>
	<li>Prepare 1 L of M8 minimal salts base (5x) by dissolving 64 g of Na2HPO4&#183;7H2O, 15 g of KH2PO4, and 2.5 g NaCl in 800 mL of ultrapure water (UPW, resistivity 18 M&#937;/cm) in a 2 L bottle. pH to 7.6. Complete volume to 1 L with UPW. Autoclave for 45 min.</li>
	<li>Prepare...

<ol>
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R., Lawrence, J. R., Sutton, B., Caldwell, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+laminar+flow+velocity+on+the+kinetics+of+surface+recolonization+by+Mot++and+Mot-+Pseudomonas+fluorescens.">Effect of laminar flow velocity on the kinetics of surface recolonization by Mot+ and Mot- Pseudomonas fluorescens.</a> Microbial Ecology. 18, 1-19 (1989).</li><li>Lawrence, J. R., Korber, D. R., Caldwell, D. 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The methods presented here describe a protocol for live-cell imaging of bacterial species interactions at the single-cell level with modifications for other applications including cell tracking and monitoring cell viability. This method opens new avenues for studying single-cell behaviors of microorganisms in coculture with other species over time. Specifically, the protocol demonstrates the usefulness of this coculture method in observing bacterial surface behaviors, particularly when studying organisms that have both s...

Polymicrobial communities are common&#160;in nature, spanning a variety of environmental1,2,3 and human niches4,5. Some of the most notorious polymicrobial infections in human disease include dental biofilms6, chronic wounds7,8, and respiratory infections in individuals with chronic obstructive pulmonary disease9, ventilator-associated pneumonia10, and the genetic disease cystic fibrosis (CF)11,12. These infections are often composed of diverse microbial species; however, a recent focus on the interactions between the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Pseudomonas aeruginosa has emerged. Studies including patients coinfected with these organisms and in vitro analyses reveal both competitive and cooperative interactions that can have profound and unexpected influences on disease progression, microbial survival, virulence, metabolism, and antibiotic susceptibility (reviewed in detail by Hotterbeekx et al. 201713 and Limoli and Hoffman 20193).
The growing interest in interspecies interactions during infection has yielded a variety of methods for studying polymicrobial community behaviors. Typically, these studies have utilized plate or broth-based assays to investigate the phenotypic differences between monoculture and coculture. For example, coculturing P. aeruginosa and S. aureus on solid surfaces has allowed for visualization of growth inhibition or changes in colony phenotype, pigment, or polysaccharide production14,15,16. Mixed species biofilms, on biotic or abiotic surfaces, as well as coculturing bacterial species in liquid culture also has enabled measurement of changes in growth, metabolism, antibiotic tolerance, competition and viability, in addition to gene and protein expression17,18. While these bulk culture experiments have revealed insight into how P. aeruginosa and S. aureus might influence one another on the community-scale, they are unable to answer important questions about the interactions that take place at the single-cell level. The method presented here adds to the approaches for studying interspecies interactions by focusing on the changes in movement, cell viability, and gene expression of single cells within a cocultured community over time.
Single-cell interactions can widely differ from the interactions that take place in a bulk community of cells. Through single-cell analysis, heterogeneity within a community can be quantified to study how spatial placement of cells influence community dynamics or how gene and protein expression levels change within individual members of a group. Tracking cells can also provide insight into how single cells move and behave over time, through multiple generations. By tracking cell movement and changes in gene expression concurrently, correlations can be made between gene fluctuations and corresponding phenotypes. Previous protocols for studying individual bacterial species at the single-cell level have been described. These studies take advantage of live-imaging cells over time in a single plane, and have been useful for observing phenotypes like cell division and antibiotic susceptibility19,20. Additional live-imaging microscopy has been utilized to monitor growth, motility, surface colonization and biofilm formation of single bacterial species21,22,23. However, while these studies have been insightful for understanding the physiology of bacteria in monoculture, experiments for observing single-cell behavior of multiple bacterial species over time in coculture are limited.
Here, previous protocols used for single-species imaging have been adapted to study interactions between P. aeruginosa and S. aureus. These organisms can be differentiated under phase contrast based on their cell morphologies (P. aeruginosa are bacilli&#160;and S. aureus are cocci). Development of this method recently enabled the visualization of previously undescribed motility behaviors of P. aeruginosa in the presence of S. aureus24. P. aeruginosa was found to be capable of sensing S. aureus from a distance and responding with increased and directional single-cell movements towards clusters of S. aureus cells. P. aeruginosa movement towards S. aureus was found to require the type IV pili (TFP), hair-like projections whose coordinated extension and retraction generate a movement called twitching motility25.
These studies demonstrate the utility of this method for interrogating earlier interactions between species. However, imaging at high cell densities at later interaction time points is difficult given that single layers of cells can no longer be identified, which mostly poses issues during post-imaging analysis. Given this limitation, the method is best suited for earlier interactions that could then be followed up with traditional macroscopic assays at higher cell densities representative of later interactions. Additional limitations of this method include the low-throughput nature, since only one sample can be imaged at a time and the cost of the microscope, camera, and environmental chamber. Moreover, fluorescence microscopy poses risks to the bacterial cells like phototoxicity and photobleaching, therefore limiting the frequency that fluorescence images can be acquired. Lastly, the agarose pads used in this method are highly susceptible to changes in the environment, making it critical to control for conditions like temperature and humidity, given that the pads can start to shrink or expand if the conditions are not correct. Finally, while this method does not mimic the host environment, it provides clues for how different bacterial species respond on surfaces, which can be followed-up in assays designed to mimic environmental/host conditions.
This method differs from previous studies tracking single-cell movement, in that cells are inoculated between a coverslip and agarose pad, restricting cells to the surface. This enables cell tracking over time in a single plane; however, limits cycles of transient surface engagement observed when cells are submerged in liquid26. An additional benefit of imaging bacteria under an agarose pad is that it mimics macroscopic plate-based sub-surface inoculation assays classically used to examine P. aeruginosa twitching motility25. In this assay, bacterial cells are inoculated between the bottom of a Petri dish and the agar, keeping the cells in a single plane as they move across the bottom of the dish outward from the point of inoculation, much like this microscopy protocol.
The time-lapse microscopy protocol for visualizing interspecies interactions presented here consists of 1) preparing the bacterial sample and agarose pad, 2) selecting microscope settings for imaging acquisition and 3) post-imaging analysis. Detailed visualization of cell movement and tracking can be performed by acquisition of images at short time intervals by phase contrast. Fluorescence microscopy can also be utilized to determine cell viability or gene expression over time. Here, we show one example of adaptation for fluorescence microscopy through the addition of viability dyes to the agarose pads.

<table>
	<tbody>
		<tr>
			<td>Agarose pads</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm Glass Bottom Dish with 20 mm Micro-well #1.5 Cover Glass</td>
			<td>Cellvis</td>
			<td>D35-20-1.5-N</td>
			<td>One for agarose pad molds, one for experiment</td>
		</tr>
		<tr>
			<td>KimWipes</td>
			<td>Kimberly-Clark Professional</td>
			<td>06-666A</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-Melt Agarose</td>
			<td>Nu-Sieve GTG/Lonza</td>
			<td>50081</td>
			<td>For making agarose pads</td>
		</tr>
		<tr>
			<td>Round-Bottom Spatulas</td>
			<td>VWR</td>
			<td>82027-492</td>
			<td></td>
		</tr>
		<tr>
			<td>Round-Tapered Spatulas</td>
			<td>VWR</td>
			<td>82027-530</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon Isolators, Press-to-Seal, 1 well, D diameter 2.0 mm 20 mm, silicone/adhesive</td>
			<td>Sigma-Aldrich</td>
			<td>S6685-25EA</td>
			<td>For agarose pad molds</td>
		</tr>
		<tr>
			<td>Sterile Petri Plates, 85 mm</td>
			<td>Kord-Valmark /sold by RPI</td>
			<td>2900</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>VWR</td>
			<td>89259-944</td>
			<td></td>
		</tr>
		<tr>
			<td>M8T Minimal Media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D (+) Glucose</td>
			<td>RPI</td>
			<td>G32045</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>RPI</td>
			<td>P250500</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Sigma-Aldrich</td>
			<td>208094</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>RPI</td>
			<td>S23025</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4.7H2O</td>
			<td>Sigma-Aldrich</td>
			<td>230391</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>BD Biosciences</td>
			<td>DF0123173</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Andor Sona 4.2B-11</td>
			<td>Andor</td>
			<td>77026135</td>
			<td>Camera. 4.2 Megapixel Back-illuminated sCMOS, 11 &#956;m pixel, 95% QE, 48 fps, USB 3.0, F-mount.</td>
		</tr>
		<tr>
			<td>Filter Cube GFP</td>
			<td>Nikon</td>
			<td>96372</td>
			<td>Filter cube</td>
		</tr>
		<tr>
			<td>Filter Cube TxRed</td>
			<td>Nikon</td>
			<td>96375</td>
			<td>Filter cube</td>
		</tr>
		<tr>
			<td>H201-NIKON-TI-S-ER</td>
			<td>Okolab</td>
			<td>77057447</td>
			<td>Stagetop incubator</td>
		</tr>
		<tr>
			<td>Nikon NIS-Elements AR with GA3 and 2D and 3D tracking</td>
			<td>Nikon</td>
			<td>77010609, MQS43110, 77010603, MQS42950</td>
			<td>Software for data analysis</td>
		</tr>
		<tr>
			<td>Nikon Ti2 Eclipse</td>
			<td>Nikon</td>
			<td>Model Ti2-E</td>
			<td>Microscope</td>
		</tr>
		<tr>
			<td>CFI Plan Apo &#411;20x objective (0.75NA)</td>
			<td>Nikon</td>
			<td>MRD00205</td>
			<td>Objective</td>
		</tr>
		<tr>
			<td>CFI Plan Apo &#411;100x oil Ph3 DM objective (1.45NA)</td>
			<td>Nikon</td>
			<td>MRD31905</td>
			<td>Objective</td>
		</tr>
		<tr>
			<td>ThermoBox with built-in fan heaters</td>
			<td>Tokai Hit</td>
			<td>TI2TB-E-BK</td>
			<td>Enclosure</td>
		</tr>
		<tr>
			<td>Bacterial Strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pseudomonas aeruginosa PA14 (WT)</td>
			<td></td>
			<td>PMID: 7604262</td>
			<td>Non-mucoid prototroph</td>
		</tr>
		<tr>
			<td>Pseudomonas aeruginosa PA14 (WT) pSMC21 (Ptac-GFP)</td>
			<td></td>
			<td>PMID: 9361441</td>
			<td></td>
		</tr>
		<tr>
			<td>Pseudomonas aeruginosa PAO1 (WT) pPrpoD-mKate2</td>
			<td></td>
			<td>PMID: 26041805</td>
			<td></td>
		</tr>
		<tr>
			<td>Staphylococcus aureus USA300 LAC (WT)</td>
			<td></td>
			<td>PMID: 23404398</td>
			<td>USA300 CA-Methicillin resistant strain LAC without plasmids</td>
		</tr>
		<tr>
			<td>Staphylococcus aureus USA300 LAC (WT) pCM29 (sarAP1-sGFP)</td>
			<td></td>
			<td>PMID: 20829608</td>
			<td></td>
		</tr>
		<tr>
			<td>Staphylococcus aureus USA300 LAC &#9651;agrBDCA</td>
			<td></td>
			<td>PMID: 31713513</td>
			<td></td>
		</tr>
		<tr>
			<td>Viability Stain</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Invitrogen</td>
			<td>L7012</td>
			<td>LIVE/DEAD&#8482; BacLight&#8482; Bacterial Viability Kit</td>
		</tr>
	</tbody>
</table>

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.

Successful use of the described method will result in a series of frames that generate a video in which the interspecies interactions can be observed over time. The schematic in Figure 1 provides a visual to highlight the key steps involved in preparing materials for imaging. Use of this method has allowed the demonstration of P. aeruginosa cells exhibiting different behaviors in monoculture versus in coculture with S. aureus. Compared to the P. aeruginosa cells in...

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Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

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 ...

This protocol allows for visualization and tracking of interspecies bacterial interactions at the single cell level over time. This techniques main advantage is its applicability to studying bacterial interactions, including cell tracking, gene expression and viability staining. Additionally, the conditions can be modified to study different organisms.To begin, prepare agarose pads by melting 2%low melt agarose in 10 milliliters of bacterial growth media in a clean 50 milliliter Erlenmeyer flask. Microwave the flask in short intervals until the agarose is in solution, then let it cool in a 50 degree Celsius water bath for at least 15 minutes. Align a silicon cut out with the opening in a 35 millimeter dish, then flip the dish and lightly tap the silicon cut out with a spatula to secure it to the dish and to remove any air bubbles.Once the agarose has cooled, pipette 915 microliters of molten agarose into the mold and let the pad dry. Warm the microscope environmental chamber to the experimental temperature at least two hours prior to setting up the experiment. Prepare humidity wipes by tightly rolling up a lint-free wipe.Place the rolled wipe inside a sterile Petri dish and add 500 microliters of sterile water directly to the wipes. Warm the wipes, pads and a sterile 35 millimeter dish to the experimental temperature in order to equilibrate all materials. Once the pads are dry, pipette one microliter of co-culture evenly across the bottom of a pre-warmed sterile 35 millimeter glass coverslip dish.Remove the silicone cut out from the agarose mold using sterile tweezers, tilt it to the side and slip a slightly bent spatula under the edge of the agarose pad to drop it onto a sterile petriplate lid. Transfer the pad to the dish with the bacterial cells bottom side down by sliding a 90 degree angled spatula under the pad and placing it on top of the inoculated cover slip. Use the spatula to make the pad flush against the cover slip and gently press out any air bubbles.Remove excess moisture from the moist wipe and place it around the edge of the dish, making sure it does not touch the pad. The sample is now ready for imaging. Prior to inoculated pads, set up the microscope by performing color illumination to align all image frames.Once the inoculated dish is ready, view the dish with 100X objective and focus on the bacterial results. Click the phase option in the imaging software and adjust the percentage of DIA LED light emitted by selecting the light source and sliding the bar on the light percentage scale. Alternatively, manually enter an exposure time.Save the settings for the phase channel by right clicking on the phase channel and selecting the Assign Current Microscope Setting option. If using fluorescence, select the fluorescence channel of interest and set the percentage of fluorescence light emitted, then adjust the exposure time as previously described. Alternatively, change the bid depth to adjust the dynamic range by selecting one of the other options in the drop down menu.Save the settings for the fluorescence channel by right clicking on the fluorescence channel and selecting the Assign Current Microscope Setting option. In the XY tab, click the box in the point name column to save the selected XY position of interest. Then click the PFS box at the bottom of the tab to turn on the perfect focus system.Focus the cells and click the arrow in the PFS column to save the focus of the XY position. Repeat this process for all other XY positions. Save the settings for the XY positions under the phase tab as previously demonstrated.Click the time tab and check the box to select the acquisition interval, frequency and imaging duration. Then click the wave length tab and check the box to select channels to be used in imaging and channel interval frequency. When fished with the settings, click Run Now to initiate the time lapse imaging.Compare to the P.aeruginosa cells in monoculture that remain grouped in rafts when in co-culture with S.aureus, P.aeruginosa has increased single cell motility towards S.aureus colonies. P.aeruginosa single cells will surround and eventually invade S.aureus colonies. Too much or too little humidity during the experiment and incorrectly dried pads are two common problems that lead to drift due to the pad shifting and dragging the cells out of the field of view.Photo bleaching and photo toxicity can cause cells to first, loose their fluorescence then stop dividing and eventually die in subsequent frames. Cells that are initially too close in proximity may not have sufficient time to establish a gradient of secreted signals to which other species can respond. Bacterial cell viability can be visualized by adding propidium iodide to the agarose pads.Green cells indicate live cells actively expressing GFP, while red cells indicate dead propidium iodide stained cells after being treated with P.aeruginosa cell free supernatant. The movement of individual P.aeruginosa cells are tracked from the frame in which a cell leaves the raft through the frame in which the cells reaches the S.aureus cluster. The distance between the P.aeruginosa raft and the S.aureus cluster, provide the Euclidean distance, while the total track lengths provide the accumulated distance.Directedness of each cell is calculated as a ratio of the euclidean distance to the accumulated distance. Wild type P.aeruginosa moves toward wild type S.aureus with significantly higher directedness than toward S.Aureus lacking agr-regulated secreted factors which are necessary for directional motility. When attempting this protocol, it is important to keep in mind that each investigator will need to optimize conditions for drying agarose pads in live imaging based on geographical location, laboratory environment and their particular experiment.This technique reveal interactions between Pseudomonas aeruginosa and Staphylococcus aureus, previously obscured during our investigations of their behavior in bulk culture, bringing us closer to understanding how these organisms interact during infections.

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