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...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+acquisition+in+young+patients+with+cystic+fibrosis:+Pathophysiology,+diagnosis,+and+management.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tolerance+and+Resistance+of+Pseudomonas+aeruginosa+Biofilms+to+Antimicrobial+Agents-How+P.+aeruginosa+Can+Escape+Antibiotics.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Extracellular+Matrix+Component+Psl+Provides+Fast-Acting+Antibiotic+Defense+in+Pseudomonas+aeruginosa+Biofilms.">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. 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B., 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=Whole-body+tissue+stabilization+and+selective+extractions+via+tissue-hydrogel+hybrids+for+high-resolution+intact+circuit+mapping+and+phenotyping.">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/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. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do+processing+time+and+storage+of+sputum+influence+quantitative+bacteriology+in+bronchiectasis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+sputum:+Time+from+expectoration+to+processing.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+fixation+methods+for+observation+of+bacterial+cell+morphology+and+surface+ultrastructures+by+atomic+force+microscopy.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+cell+fixation+methods+of+induced+sputum+specimens:+An+immunocytochemical+analysis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+and+rapid+detection+by+fluorescent+situ+hybridization+of+bacteria+in+clinical+samples+obtained+from+cystic+fibrosis+patients.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Erratum:+Novel+mouse+model+of+chronic+Pseudomonas+aeruginosa+lung+infection+mimicking+cystic+fibrosis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+Gram+staining+for+evaluation+of+the+quality+of+cystic+fibrosis+sputum+samples.">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/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. J., 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=Treatment+with+the+Pseudomonas+aeruginosa+glycoside+hydrolase+PslG+combats+wound+infection+by+improving+antibiotic+efficacy+and+host+innate+immune+activity.">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

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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Research

JoVE Journal

Immunology and Infection

2.9K vues.  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...

Vidéo: 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...

use of artificial sputum medium to test antibiotic efficacy against pseudomonas aeruginosa in conditions more relevant to 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

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...

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

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

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

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Pseudomonas aeruginosa&#160;produces the rhamnolipid biosurfactants. Thin-layer chromatography detects and determines the proportion of mono- and di-rhamnolipids produced by each strain. Quantification of total rhamnolipids involves assessing rhamnose equivalents present in these biosurfactants extracted from the culture supernatants using the orcinol...

Comparative Analysis of Rhamnolipid Production in &lt;i&gt;Pseudomonas aeruginosa&lt;/i&gt; Strains

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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.

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A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described.  The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments.  This technique can be adapted for RNA isolation of other intestinal...

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Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Here, we describe a growth condition to culture the small colony variant of Pseudomonas aeruginosa. We also describe two separate methods for the detection and quantitation of the exopolysaccharide alginate produced by P. aeruginosa using a traditional uronic acid carbazole assay and an alginate-specific monoclonal antibody (mAb) based...

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

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Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant...

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

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Quantification of Rhamnolipids Present in the Biosurfactant of Pseudomonas aeruginosa

purification of the cystic fibrosis transmembrane conductance regulator protein expressed in saccharomyces cerevisiae

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

pseudomonas aeruginosa induced lung injury model

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

Pseudomonas aeruginosa Induced Lung Injury Model

design and development of a model to study the effect of supplemental oxygen on the cystic fibrosis airway microbiome

Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome

The goal of this protocol is to develop a model system for the effect of hyperoxia on cystic fibrosis airway microbial communities.  Artificial sputum medium emulates the composition of sputum, and hyperoxic culture conditions model the effects of supplemental oxygen on lung microbial...

in vitro investigation of the mexab efflux pump from pseudomonas aeruginosa

In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

We present a protocol for the in vitro investigation of efflux pumps from Pseudomonas aeruginosa. This protocol allows for the generation of a robust, reversible, and tunable proton gradient within the liposome membrane and hence should be adaptable to any membrane protein energized by the protomotive...

In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

co-culture models of pseudomonas aeruginosa biofilms grown on live human airway cells

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...

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.

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 ...

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 ...

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

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.

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

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

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...