The authors would like to acknowledge the Cystic Fibrosis Foundation that provided funding for this research and MedImmune for their generous donation of anti-Psl0096 antibodies. For this study imaging was performed at the CAMiLoD imaging facility at the University of Toronto.

Research Ethics Board (REB) approval is required to collect and store sputum samples from human subjects. Studies presented herein were approved by the Hospital for Sick Children REB#1000058579.
1. Sputum Collection
<ol>
	<li>Store expectorated sputum in a sterile collection cup and immediately store at 4 &#176;C for a maximum of 24 h prior to the fixation. 
	NOTE: Leaving the sputum too long at 4 &#176;C without fixation can lead to cellular degradation, particularly degradation of whi...

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H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposing+the+three-dimensional+biogeography+and+metabolic+states+of+pathogens+in+cystic+fibrosis+sputum+via+hydrogel+embedding,+clearing,+and+rRNA+labeling.">Exposing the three-dimensional biogeography and metabolic states of pathogens in cystic fibrosis sputum via hydrogel embedding, clearing, and rRNA labeling.</a> mBio. 7 (5), 1-11 (2016).</li><li>Murray, M. P., Doherty, C. J., Govan, J. R. W., Hill, A. 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P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-Dose+Nitric+Oxide+as+Targeted+Anti-biofilm+Adjunctive+Therapy+to+Treat+Chronic+Pseudomonas+aeruginosa+Infection+in+Cystic+Fibrosis.">Low-Dose Nitric Oxide as Targeted Anti-biofilm Adjunctive Therapy to Treat Chronic Pseudomonas aeruginosa Infection in Cystic Fibrosis.</a> Molecular Therapy. 25 (9), 2104-2116 (2017).</li><li>Pestrak, M. 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The purpose of this protocol is to allow a glimpse into the in-situ organization of P. aeruginosa cells in sputum from CF patients. Sputum samples should be stored at 4 &#176;C until processed if they cannot be immediately fixed. It has been demonstrated that P. aeruginosa cell numbers in sputum do not change significantly if processed at 1 h, 24 h, or 48 h, when stored at 4 &#176;C, though if left at 25 &#176;C for 24 or 48 h, bacterial cell counts will significantly increase as a result of bacterial g...

In this study, experiments were designed to visualize the in vivo structure of Pseudomonas aeruginosa within the sputum of pediatric cystic fibrosis (CF) patients. P. aeruginosa infections becomes chronic in 30-40% of the pediatric CF population; once chronic infections become established, they are almost impossible to eliminate1. P. aeruginosa isolates from patients with early infection are generally more susceptible to antimicrobials, therefore, these are treated with anti-pseudomonal antibiotics to prevent the establishment of chronic infection2. Unfortunately, not all P. aeruginosa isolates are effectively cleared from the lung following antibiotic therapy. The precise mechanisms associated with antibiotic failure have not been fully elucidated. Previous studies have shown that variations in biofilm cell density, aggregation, and polysaccharide production can affect antibiotic efficacy3. P. aeruginosa produces three extracellular polysaccharides: Pel, Psl, and alginate4. Most strains of P. aeruginosa have the genetic capacity to express each of the exopolysaccharides, though often one type of polysaccharide is expressed predominantly5. The exopolysaccharide alginate is associated with chronic infections in the CF lung, resulting in a mucoid phenotype6,7. The polysaccharides Pel and Psl have multiple functions including aiding initial attachment and the maintenance of biofilm structure, and conferring antibiotic resistance8.
Methods aimed at visualizing in vivo structures of tissues have been developed for a variety of sample types9,10,11. More recently, they have been tailored to visualize in vivo microbial communities within sputum from CF patients12. The optimization of a tissue clearing protocol specifically for the identification of microbial communities within sputum was developed by DePas et al., 201612. The term MiPACT, which stands for microbial identification after Passive CLARITY technique was coined for the clearing of CF sputum11,12. For tissue clearing techniques, the specimens are first fixed, then rendered transparent while leaving their inherent architecture intact for staining and microscopic visualization11. Fixing and clearing CF sputum samples allow researchers to answer questions related to biofilm structure, bacterial cell density, polymicrobial associations, and associations between pathogens and host cells. The advantage of directly examining bacteria which have been preserved within the sputum is that they can be analyzed and visualized in a host-specific context. Although in vitro growth of clinical isolates in the laboratory for experimentation can be very informative, such methods are unable to fully recreate the CF lung environment, resulting in a disconnect between laboratory results and patient outcomes.
The methods presented here can be used to fix and clear sputum to visualize bacteria, whether from CF patients or patients with other respiratory infections. The specific type of staining and microscopic analysis described herein is fluorescence in situ hybridization (FISH), followed by anti-Psl-antibody binding within the hydrogel, and subsequent analysis via confocal laser scanning microscopy (CLSM). Following tissue clearing, other immunohistochemistry and microscopy methods can also be applied.

<table>
	<tbody>
		<tr>
			<td>29:1 acrylamide bisacrylamide, 30 % solution</td>
			<td>BioRad</td>
			<td>161-0146</td>
			<td></td>
		</tr>
		<tr>
			<td>8-Chambered Coverglass Nunc Lab-Tek</td>
			<td>ThermoFischer Scientific</td>
			<td>155411</td>
			<td></td>
		</tr>
		<tr>
			<td>Anaerogen2.5L</td>
			<td>Oxid Inc.</td>
			<td>35108</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverwell perfusion chambers</td>
			<td>Electron Microscopry Sciences</td>
			<td>70326 -12/-14</td>
			<td></td>
		</tr>
		<tr>
			<td>HistoDenz</td>
			<td>Sigma</td>
			<td>D2158</td>
			<td></td>
		</tr>
		<tr>
			<td>Protect RNA Rnase Inhibitor</td>
			<td>Sigma</td>
			<td>R7387</td>
			<td></td>
		</tr>
		<tr>
			<td>PseaerA - GGTAACCGTCCCCCTTGC</td>
			<td>Eurofins</td>
			<td></td>
			<td>Order Details: Product: Modified DNA Oligo; Name: PseaerA; Sequence: [Alexa488]GGTAACCGTCCCCCTTGC; Synthesis: 50 nmol; Purification: HPLC; Ship state: Full yield (dry)</td>
		</tr>
		<tr>
			<td>Psl0096-Texas Red</td>
			<td>Medimmune</td>
			<td></td>
			<td>The Psl0096-Texas red antibodies were a gift kindly provided by Medimmune and the company should be contacted for order inquiries.</td>
		</tr>
		<tr>
			<td>VA-044 Hardener</td>
			<td>Wako</td>
			<td>27776-21-21</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The overall design of the experiment is summarized in Figure 1 and Figure 2. Figure 1 provides a summary of the sputum processing and sputum clearing protocols. Sputum processing and clearing may take up to 17 days. Though, the protocol may be stopped, and samples can be stored after fixation with PFA (day 2) or following tissue clearing (days 5-17 depending on clearing time). In Figure 2, the FISH and ...

Watch this Scientific Journal Video about Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients at JoVE.com

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.

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 ...

This protocol is significant because it allows for the direct visualization of Pseudomonas aeruginosa and the exopolysaccharide Psl within cystic fibrosis sputum. This technique helps to answer questions about how the spatial organization of P.aeruginosa such as aggregation or biofilms affects host clearance and antimicrobial susceptibility. Store the expectorated sputum in a sterile collection cup at four degrees Celsius for a maximum of 24 hours prior to fixation.When ready to fix the samples, transfer them to a sterile 15 milliliter tube, and add an equal volume of 4%PFA, then incubate the sputum overnight at four degrees Celsius. Wash the samples by adding five milliliters of PBS for every two milliliters of fixed sputum. Carefully remove the supernatant with a pipette, and repeat the wash two more times.Resuspend the pellet in twice the volume of PBS with 0.01%sodium azide, and store it at four degrees Celsius. Add two milliliters of a 30%29 to one acrylamide/bisacrylamide solution to a three milliliter tube with the cap off inside a sealed anaerobic container, then add 0.5 grams of hardener to two milliliters of PBS in a three milliliter tube. Leave the cap off the tube, and store it in the sealed anaerobic container.Leave a few milliliters of sterile PBS in the tube with the cap off inside the anaerobic container. Degas the hydrogel components in the sealed container with an anaerobic pack for 72 hours. Make a 2.5 milliliter solution of hydrogel with a final concentration of 0.2%hardener and 4%acrylamide/bisacrylamide in PBS in a 15 milliliter tube, and mix the hydrogel by inversion, then filter sterilize the solution.Cut the sputum samples into two small sections with a scalpel, then place each sample in a well of an eight chambered covered glass slide. Add 300 microliters of the filter sterilized hydrogel solution to each well that contains sputum, and place it inside a sealed container with an anaerobic pack for three hours at 37 degrees Celsius. Transfer the solidified hydrogel sputum samples to a 15 milliliter culture tube with five milliliters of 8%SDS at pH eight, and allow the samples to clear for three to 14 days at 37 degrees Celsius until the sputum becomes transparent.Use sterile tweezers to transfer each hydrogel embedded sample to a 50 milliliter conical tube, and add 10 milliliters of PBS to wash the hydrogel. Let the solution sit for 30 to 60 minutes before decanting, then repeat the wash two more times. Store the washed samples in PBS with 0.01%sodium azide and RNase inhibitor at four degrees Celsius.Remove the hydrogel samples from their storage solution, and place the samples on a sterile surface. Use a sterile scalpel to cut the hydrogels into approximately one millimeter thick slices, and place the sections into 1.5 milliliter tubes. Prepare one milliliter of hybridization buffer in a 1.5 milliliter tube, then add the fluorescently labeled Seer A-probe, and mix by inversion.Add 200 to 500 microliters of hybridization buffer to each section, making sure that the entirety of the hydrogel sample is submerged. Allow the Seer A-probe to hybridize with the hydrogel samples by placing them in the dark for approximately 18 to 24 hours at 46 degrees Celsius without shaking, then add one milliliter of fresh wash buffer to the tubes, and incubate them in the dark for six hours at 48 degrees Celsius. Add 500 microliters of the 2%BSA and PBS to the hydrogel samples to block non-specific protein binding, then incubate the samples overnight in the dark.On the next day, prepare the Psl0096-Texas Red antibody solution by diluting the antibody to a final concentration of 0.112 micrograms per milliliter in 500 microliters of fresh 2%BSA in PBS. Add the antibody solution to the hydrogel samples, and incubate them in the dark for six hours. Next, stain the hydrogel samples with DAPI by incubating them with 250 microliters of RIMs solution and 10 micrograms per milliliter of DAPI overnight in the dark with gentle shaking.After staining, mount the samples onto 0.9 or 1.7 millimeter perfusion chambers, and seal them with a glass coverslip. Perform confocal laser scanning microscopy with 25, 40, 63, or 100 times magnifications. This protocol was used to obtain the high resolution 3D images of P.aeruginosa cells and their NC2 structure within sputum.An aggregate of cells within a sputum sample is shown here. Individual rod shapes were seen in green from the species-specific binding of the Seer A-488 probe to P.aeruginosa cells. The Psl0096-Texas Red antibody shows that the Pseudomonal exopolysaccharide within the sputum was mostly overlapping with the P.aeruginosa cells.This method was also used for the visualization of Pseudomonal cells within the sputum in relation to other bacterial cells and host structures. A cluster of P.aeruginosa cells was seen to phagocytose within a eukaryotic cell while other small coccal cell clusters were observed in the vicinity. It was also demonstrated that the PSL 0096 antibody could bind to PSL produced from planktonic P.Aeruginosa.The main advantage of this technique is that it implements a tissue clearing approach followed by fluorescent in situ hybridization, antibody staining, and confocal microscopy. This enables researchers to visualize and use image processing techniques to quantitatively measure differences in P.aeruginosa colonization structures within CF sputum in relation to patient health outcomes.

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Immunology and Infection

2.9K 조회수.  Hospital for Sick Children. This protocol is significant because it allows for the direct visualization of Pseudomonas aeruginosa and the exopolysaccharide Psl within cystic fibrosis sputum. This technique helps to answer questions about how the spatial organization of P.aeruginosa such as aggregation or biofilms affects host clearance and antimicrobial susceptibility. Store the expectorated sputum in a sterile collection cup at four degrees Celsius for a maximum of 24 hours prior to fixation.When ready to fix th...