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 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.
Protocol
Representative Results
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 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 |
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