Name: Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients (Video) | JoVE Journal
Uploaded: 2020-07-16T15:00:00.000+00:00
Duration: 7 min 25 s
Description: Learn to visualize Pseudomonas aeruginosa and Psl polysaccharide in cystic fibrosis sputum using tissue clearing and confocal microscopy. This method reveals bacterial spatial organization and biofilm formation.

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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 white blood cells. Fixation as soon as possible is preferred.</li>
	<li>Transfer the sputum samples to a sterile 15 mL tube.</li>
	<li>Add an equal volume of 4% paraformaldehyde (PFA) to the sputum samples. For example, if the sputum sample is 0.5 mL, add 0.5 mL of 4% PFA. Mix by gentle inversion.</li>
	<li>Incubate the sputum overnight at 4 &#176;C.</li>
	<li>Wash the sample by adding 5 mL of phosphate buffered saline (PBS) to each 2 mL of fixed sputum. 
	NOTE: There is no centrifugation required for this washing step.</li>
	<li>Carefully remove the supernatant with a pipette. 
	NOTE: Avoid sucking up the sputum with the pipette by pointing the tip away from the sputum and very slowly aspirate off the surface liquid. The washing step does not involve centrifugation to not disturb the structural integrity of the sputum plugs.</li>
	<li>Repeat steps 1.6-1.7 two more times.</li>
	<li>Resuspend the pellet in 2x the volume of PBS with 0.01 % (w/v) sodium azide.</li>
	<li>Store the sputum at 4 &#176;C.</li>
</ol>
2. MiPACT (tissue clearing technique) processing of sputum
<ol>
	<li>Degas the hydrogel components (30% 29:1 acrylamide:bis-acrylamide, hardner, and PBS) in a sealed container containing an anaerobic pack prior for 72 h. 
	NOTE: The anaerobic pack and sealed container are necessary because oxygen inhibits acrylamide polymerization. Alternatively, oxygen can be removed by performing this step in an anaerobic hood, by applying a vacuum to a sealed container, or by bubbling N2 gas through the acrylamide mixture.
	<ol>
		<li>Add 2 mL of a 30% 29:1 acrylamide:bis-acrylamide solution to a 15 mL tube with the cap off inside the sealed anaerobic container.</li>
		<li>Make a concentrated stock of 10% (w/v) hardener by adding 0.5 g of hardener to 5 mL of PBS in a 15 mL tube. Leave the cap off the tube and store in the sealed anaerobic container.</li>
		<li>Leave a few mL of sterile PBS in a tube, with the cap off, inside the anaerobic container.</li>
	</ol>
	</li>
	<li>Make 5 mL solution of hydrogel with a final concentration of 0.2% hardener and 4% 29:1 acrylamide:bis-acrylamide in PBS in a 15 mL tube. Mix by inversion and filter sterilize.</li>
	<li>Remove sputum samples from the fridge, then cut into small sections (roughly 5 mm in diameter) under sterile conditions with a scalpel. 
	NOTE: If the sputum is quite fluid then this step can still be performed, however, it takes increased patience and practice to remove the sputum from the storage solution with tweezers prior to using the scalpel to separate the desired fractions.</li>
	<li>Place the cut sputum samples inside a well of an 8 chambered coverglass slide.</li>
	<li>Add 300 &#181;L of filter sterilized hydrogel solution from step 2.2 to each of the wells that contain sputum.</li>
	<li>Place the 8 chambered coverglass inside a sealed container containing an anaerobic pack for 3 h at 37 &#176;C. 
	NOTE: Once polymerized the hydrogel with embedded sputum should be the consistency of firm gel.</li>
	<li>Transfer the solidified hydrogel sputum samples to a 15 mL culture tube containing 5 mL of 8% sodium dodecyl sulfate (SDS), pH 8, and allow samples to clear for 3-14 days at 37 &#176;C (with or without shaking), until the sputum becomes transparent. 
	NOTE: Clearing times depend on sputum composition and can generally be decreased with shaking. However, take care to not increase the shaking speed to the point of disrupting sample integrity. More DNA-rich samples take longer to clear compared to mucus rich samples.</li>
	<li>Decant the 8% SDS solution into a waste collection container. Using sterile tweezers transfer each hydrogel-embedded sample to a sterile 50 mL conical tube. Add 10 mL of PBS to each of the 50 mL conical tubes to wash the hydrogels and let the solution sit for 30 min to 1 h before decanting. Repeat this 2x more times. 
	NOTE: This washing step does not involve centrifugation. Excess fluid and supernatant are carefully removed with a pipette.</li>
	<li>Store washed samples in PBS with 0.01% (w/v) sodium azide and 1x RNase inhibitor at 4 &#176;C.</li>
</ol>
3. Hydrogel fluorescent in situ hybridization (FISH) protocol
<ol>
	<li>Remove the hydrogel samples from their storage solution using sterile tweezers and place the samples on a sterile surface (such as a glass slide or Petri dish).</li>
	<li>Using a sterile scalpel cut the hydrogels into ~ 1 mm thick slices.</li>
	<li>Place the 1 mm sections of hydrogel inside sterile 1.5 mL tubes.</li>
	<li>Prepare 1 mL the hybridization buffer (25% formamide, 0.9 M NaCl, 20 mM Tris-HCl [pH 7.6], 0.01% SDS, purified and deionized H2O) in a 1.5 mL tube.</li>
	<li>Add the fluorescently labeled PseaerA probe (150.7 nM12) to the hybridization buffer and mix by inversion. 
	NOTE: Formalin solution should be kept away from an open flame. The hybridization buffer can be prepared in advanced and stored in aliquots at -20 &#176;C13.</li>
	<li>Add 200-500 &#181;L of hybridization buffer to each ~1 mm section of hydrogel and ensure the entirety of the hydrogel sample is submerged.</li>
	<li>Allow the PseaerA probe to hybridize with the hydrogel samples by placing them in the dark for ~ 18-24 h, at 46 &#176;C, without shaking.</li>
	<li>Decant the hybridization buffer into a waste collection container.</li>
	<li>Rinse samples once with filter sterilized wash buffer (337.5 mM NaCl, 20 mM Tris-HCl, 5 mM EDTA (Ethylenediaminetetraacetic acid) [pH 7.2], 0.01% SDS, and purified and deionized H2O) by adding 1 mL of wash buffer to each of the 1.5 mL tubes and then remove it. 
	NOTE: The wash buffer can be made in advance and stored at room temperature (RT).</li>
	<li>Add 1 mL of fresh wash buffer to the tubes, then incubate the samples in the dark for 6 h at 48 &#176;C, without shaking.</li>
</ol>
4. Hydrogel and Psl0096 antibody binding
<ol>
	<li>Sterilely remove the wash buffer using a 1 mL pipettor.</li>
	<li>Rinse the samples with a 2% BSA (w/v) in PBS solution by adding and removing 1 mL of the 2% BSA solution.</li>
	<li>Add 500 &#181;L of the 2% BSA/PBS solution to the hydrogel samples to block non-specific protein binding. Then incubate the samples overnight, in the dark, at RT, without shaking.</li>
	<li>Sterilely remove the blocking solution using a 1 mL pipette.</li>
	<li>Prepare the Psl0096-Texas Red antibody solution by diluting the antibody to a final concentration of 0.112 &#181;g/mL in 500 &#181;L of fresh 2% BSA/PBS.</li>
	<li>Add the 500 &#181;L antibody solution to the hydrogel samples and incubate them at RT for 6 h, protected from light, without shaking.</li>
</ol>
5. DAPI (4&#8242;,6&#8242;-diamidino-2-phenylindole) staining
<ol>
	<li>Prepare the refractive index matching solution (RIMS) by adding 40 g of non-ionic density gradient medium, 30 &#181;L of Tween20, 3 &#181;g sodium azide, and 30 mL of PBS to a flask containing a magnetic stir bar. Stir the solution for 15 min on a magnetic stirrer, or until completely dissolved.</li>
	<li>Filter sterilize the solution into a 50 mL conical tube using a 10 mL syringe and a sterile 0.2 &#181;m filter. 
	NOTE: The solution can be stored at 4 &#176;C for several months.</li>
	<li>Remove the Psl0096-Texas Red antibody solution with a sterile 1 mL pipette.</li>
	<li>Rinse the hydrogel samples by adding and then removing 1 mL of PBS.</li>
	<li>Incubate the hydrogel samples with 250 &#181;L of RIMS solution and 10 &#181;g/mL of DAPI at RT with gentle shaking, in the dark, overnight.</li>
	<li>Prior to confocal imaging mount the samples onto 0.9 mm or 1.7 mm perfusion chambers and seal with a glass coverslip. 
	NOTE: After FISH and/or immunohistochemistry and before immersion in RIMS, fluorescent lectin stains can be applied if visualization of sputum mucous is desired12.</li>
</ol>
6. Imaging
<ol>
	<li>Perform confocal laser scanning microscopy imaging using standard techniques at 25x, 40x, 63x, or 100x magnifications.</li>
</ol>

<ol><li>Banerjee, D., Stableforth, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+treatment+of+respiratory+pseudomonas+infection+in+cystic+fibrosis%3A+What+drug+and+which+way%5BTitle%5D)+AND+%22Drugs%22%5BJournal%5D)">The treatment of respiratory pseudomonas infection in cystic fibrosis: What drug and which way.</a> Drugs. 60 (5), 1053-1064 (2000).</li>
<li>Rosenfeld, M., Ramsey, B. W., Gibson, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pseudomonas+acquisition+in+young+patients+with+cystic+fibrosis%3A+Pathophysiology%2C+diagnosis%2C+and+management%5BTitle%5D)+AND+%22Current+Opinion+in+Pulmonary+Medicine%22%5BJournal%5D)">Pseudomonas acquisition in young patients with cystic fibrosis: Pathophysiology, diagnosis, and management.</a> Current Opinion in Pulmonary Medicine. 9 (6), 492-497 (2003).</li>
<li>Ciofu, O., Tolker-Nielsen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Tolerance+and+Resistance+of+Pseudomonas+aeruginosa+Biofilms+to+Antimicrobial+Agents-How+P.+aeruginosa+Can+Escape+Antibiotics%5BTitle%5D)+AND+%22Frontiers+in+Microbiology%22%5BJournal%5D)">Tolerance and Resistance of Pseudomonas aeruginosa Biofilms to Antimicrobial Agents-How P. aeruginosa Can Escape Antibiotics.</a> Frontiers in Microbiology. 10, 913(2019).</li>
<li>Billings, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+Extracellular+Matrix+Component+Psl+Provides+Fast-Acting+Antibiotic+Defense+in+Pseudomonas+aeruginosa+Biofilms%5BTitle%5D)+AND+%22PLoS+Pathogens%22%5BJournal%5D)">The Extracellular Matrix Component Psl Provides Fast-Acting Antibiotic Defense in Pseudomonas aeruginosa Biofilms.</a> PLoS Pathogens. 9 (8), 1003526(2013).</li>
<li>Franklin, M. J., Nivens, D. E., Weadge, J. T., Lynne Howell, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biosynthesis+of+the+Pseudomonas+aeruginosa+extracellular+polysaccharides%2C+alginate%2C+Pel%2C+and+Psl%5BTitle%5D)+AND+%22Frontiers+in+Microbiology%22%5BJournal%5D)">Biosynthesis of the Pseudomonas aeruginosa extracellular polysaccharides, alginate, Pel, and Psl.</a> Frontiers in Microbiology. 2, 167(2011).</li>
<li>Yang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polysaccharides+serve+as+scaffold+of+biofilms+formed+by+mucoid+Pseudomonas+aeruginosa%5BTitle%5D)+AND+%22FEMS+Immunology+and+Medical+Microbiology%22%5BJournal%5D)">Polysaccharides serve as scaffold of biofilms formed by mucoid Pseudomonas aeruginosa.</a> FEMS Immunology and Medical Microbiology. 65 (2), 366-376 (2012).</li>
<li>Wozniak, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Alginate+is+not+a+significant+component+of+the+extracellular+polysaccharide+matrix+of+PA14+and+PAO1+Pseudomonas+aeruginosa+biofilms%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Alginate is not a significant component of the extracellular polysaccharide matrix of PA14 and PAO1 Pseudomonas aeruginosa biofilms.</a> Proceedings of the National Academy of Sciences of the United States of America. 100 (13), 7907-7912 (2003).</li>
<li>Baker, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exopolysaccharide+biosynthetic+glycoside+hydrolases+can+be+utilized+to+disrupt+and+prevent+Pseudomonas+aeruginosa+biofilms%5BTitle%5D)+AND+%22Science+Advances%22%5BJournal%5D)">Exopolysaccharide biosynthetic glycoside hydrolases can be utilized to disrupt and prevent Pseudomonas aeruginosa biofilms.</a> Science Advances. 2 (5), 1-9 (2016).</li>
<li>Yang, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single-cell+phenotyping+within+transparent+intact+tissue+through+whole-body+clearing%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">Single-cell phenotyping within transparent intact tissue through whole-body clearing.</a> Cell. 158 (4), 945-958 (2014).</li>
<li>Chung, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structural+and+molecular+interrogation+of+intact+biological+systems%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Structural and molecular interrogation of intact biological systems.</a> Nature. 497 (7449), 332-337 (2013).</li>
<li>Treweek, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole-body+tissue+stabilization+and+selective+extractions+via+tissue-hydrogel+hybrids+for+high-resolution+intact+circuit+mapping+and+phenotyping%5BTitle%5D)+AND+%22Nature+Protocols%22%5BJournal%5D)">Whole-body tissue stabilization and selective extractions via tissue-hydrogel hybrids for high-resolution intact circuit mapping and phenotyping.</a> Nature Protocols. 10 (11), 1860-1896 (2015).</li>
<li>DePas, W. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Exposing+the+three-dimensional+biogeography+and+metabolic+states+of+pathogens+in+cystic+fibrosis+sputum+via+hydrogel+embedding%2C+clearing%2C+and+rRNA+labeling%5BTitle%5D)+AND+%22mBio%22%5BJournal%5D)">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>Molecular Instruments HCR v3.0 protocol for bacteria in suspension. , Available from: 
 <a target="_blank" href="www.molecularinstruments.com">www.molecularinstruments.com</a> 1-6 (2019).</li>
<li>Murray, M. P., Doherty, C. J., Govan, J. R. W., Hill, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Do+processing+time+and+storage+of+sputum+influence+quantitative+bacteriology+in+bronchiectasis%5BTitle%5D)+AND+%22Journal+of+Medical+Microbiology%22%5BJournal%5D)">Do processing time and storage of sputum influence quantitative bacteriology in bronchiectasis.</a> Journal of Medical Microbiology. 59 (7), 829-833 (2010).</li>
<li>Efthimiadis, A., Jayaram, L., Weston, S., Carruthers, S., Hargreave, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induced+sputum%3A+Time+from+expectoration+to+processing%5BTitle%5D)+AND+%22European+Respiratory+Journal%22%5BJournal%5D)">Induced sputum: Time from expectoration to processing.</a> European Respiratory Journal. 19 (4), 706-708 (2002).</li>
<li>Chao, Y., Zhang, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Optimization+of+fixation+methods+for+observation+of+bacterial+cell+morphology+and+surface+ultrastructures+by+atomic+force+microscopy%5BTitle%5D)+AND+%22Applied+Microbiology+and+Biotechnology%22%5BJournal%5D)">Optimization of fixation methods for observation of bacterial cell morphology and surface ultrastructures by atomic force microscopy.</a> Applied Microbiology and Biotechnology. 92 (2), 381-392 (2011).</li>
<li>St-Laurent, J., Boulay, M. E., Prince, P., Bissonnette, E., Boulet, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+cell+fixation+methods+of+induced+sputum+specimens%3A+An+immunocytochemical+analysis%5BTitle%5D)+AND+%22Journal+of+Immunological+Methods%22%5BJournal%5D)">Comparison of cell fixation methods of induced sputum specimens: An immunocytochemical analysis.</a> Journal of Immunological Methods. 308 (1-2), 36-42 (2006).</li>
<li>Hogardt, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Specific+and+rapid+detection+by+fluorescent+situ+hybridization+of+bacteria+in+clinical+samples+obtained+from+cystic+fibrosis+patients%5BTitle%5D)+AND+%22Journal+of+Clinical+Microbiology%22%5BJournal%5D)">Specific and rapid detection by fluorescent situ hybridization of bacteria in clinical samples obtained from cystic fibrosis patients.</a> Journal of Clinical Microbiology. 38 (2), 818-825 (2000).</li>
<li>Hoffmann, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Erratum%3A+Novel+mouse+model+of+chronic+Pseudomonas+aeruginosa+lung+infection+mimicking+cystic+fibrosis%5BTitle%5D)+AND+%22Infection+and+Immunity%22%5BJournal%5D)">Erratum: Novel mouse model of chronic Pseudomonas aeruginosa lung infection mimicking cystic fibrosis.</a> Infection and Immunity. 73 (8), 5290(2005).</li>
<li>Nair, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Utility+of+Gram+staining+for+evaluation+of+the+quality+of+cystic+fibrosis+sputum+samples%5BTitle%5D)+AND+%22Journal+of+Clinical+Microbiology%22%5BJournal%5D)">Utility of Gram staining for evaluation of the quality of cystic fibrosis sputum samples.</a> Journal of Clinical Microbiology. 40 (8), 2791-2794 (2002).</li>
<li>Howlin, R. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Low-Dose+Nitric+Oxide+as+Targeted+Anti-biofilm+Adjunctive+Therapy+to+Treat+Chronic+Pseudomonas+aeruginosa+Infection+in+Cystic+Fibrosis%5BTitle%5D)+AND+%22Molecular+Therapy%22%5BJournal%5D)">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. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Treatment+with+the+Pseudomonas+aeruginosa+glycoside+hydrolase+PslG+combats+wound+infection+by+improving+antibiotic+efficacy+and+host+innate+immune+activity%5BTitle%5D)+AND+%22Antimicrobial+Agents+and+Chemotherapy%22%5BJournal%5D)">Treatment with the Pseudomonas aeruginosa glycoside hydrolase PslG combats wound infection by improving antibiotic efficacy and host innate immune activity.</a> Antimicrobial Agents and Chemotherapy. 63 (6), 00234(2019).</li>
</ol>

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

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 ...

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 ...

visualization-pseudomonas-aeruginosa-within-sputum-cystic-fibrosis

Research

JoVE Journal

Immunology and Infection

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Source: Beaudoin, T., et al. Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model. J. Vis. Exp. (2016)This video demonstrates an in vitro method to assess how patient-derived cystic fibrosis sputum influences bacterial biofilm formation. Using chamber slides, fluorescent staining, and confocal microscopy, it reveals increased thickness, altered architecture, and higher viable biomass in biofilms grown with sputum, highlighting the role of host components...

Confocal Imaging of Bacterial Biofilms Induced by Sputum Filtrates from Cystic Fibrosis Patients

Source: Jackson, L., et al. Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients. J. Vis. Exp. (2020)This video demonstrates the in situ visualization of Pseudomonas aeruginosa within chemically cleared cystic fibrosis sputum using fluorescent labeling and confocal microscopy to reveal bacterial localization and biofilm structure.

Visualization of Pseudomonas aeruginosa in Sputum Samples from Cystic Fibrosis Patients

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

This protocol describes the visualization of biofilm development following exposure to host-factors using a slide chamber model. This model allows for direct visualization of biofilm development as well as analysis of biofilm parameters using computer software programs.

Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

This workflow can be used to perform antibiotic susceptibility testing using an established ex vivo model of bacterial biofilm in the lungs of individuals with cystic fibrosis. Use of this model could enhance the clinical validity of MBEC (minimal biofilm eradication concentration) assays.

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

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.

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

The protocol here demonstrates a fast and standardized microbiological rapid on-site evaluation (M-ROSE) workflow, including three steps: slide making, staining, and interpretation. This protocol will help physicians make rapid clinical decisions.

Microbiological Rapid On-Site Evaluation for Pulmonary Infectious Diseases

This protocol describes a cystic fibrosis (CF) lung-relevant four-species polymicrobial biofilm model that can be used to explore the impact of bacterial interspecies interactions.

Growing a Cystic Fibrosis-Relevant Polymicrobial Biofilm to Probe Community Phenotypes

This methodology allows for the establishment of a polymicrobial biofilm model in cystic fibrosis for antimicrobial sensitivity testing in research and clinical laboratories. This model provides accurate and reliable results over a range of outputs.

Development of a Polymicrobial Colony Biofilm Model to Test Antimicrobials in Cystic Fibrosis

Learn to visualize Pseudomonas aeruginosa and Psl polysaccharide in cystic fibrosis sputum using tissue clearing and confocal microscopy. This method reveals bacterial spatial organization and biofilm formation.

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

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