Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Summary

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

Abstract

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.

Introduction

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....

Protocol

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

1. Store expectorated sputum in a sterile collection cup and immediately store at 4 °C for a maximum of 24 h prior to the fixation.
   NOTE: Leaving the sputum too long at 4 °C without fixation can lead to cellular degradation, particularly degradation of white blood cells. Fixation as soon as possible is preferred.
2. Transfer the sputum samples to a sterile 15 mL tube.
3. Add an equal volum....

Results

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 .......

Discussion

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 °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 °C, though if left at 25 °C for 24 or 48 h, bacterial cell counts will significantly increase as a result of bacterial g.......

Materials

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