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 phenotype^6,7. 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 types^9,10,11. 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 sputum^11,12. 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

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 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.
4. Incubate the sputum overnight at 4 °C.
5. 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.
6. 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.
7. Repeat steps 1.6-1.7 two more times.
8. Resuspend the pellet in 2x the volume of PBS with 0.01 % (w/v) sodium azide.
9. Store the sputum at 4 °C.

2. MiPACT (tissue clearing technique) processing of sputum

1. 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.
   1. 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.
   2. 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.
   3. Leave a few mL of sterile PBS in a tube, with the cap off, inside the anaerobic container.
2. 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.
3. 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.
4. Place the cut sputum samples inside a well of an 8 chambered coverglass slide.
5. Add 300 µL of filter sterilized hydrogel solution from step 2.2 to each of the wells that contain sputum.
6. Place the 8 chambered coverglass inside a sealed container containing an anaerobic pack for 3 h at 37 °C.
   NOTE: Once polymerized the hydrogel with embedded sputum should be the consistency of firm gel.
7. 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 °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.
8. 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.
9. Store washed samples in PBS with 0.01% (w/v) sodium azide and 1x RNase inhibitor at 4 °C.

3. Hydrogel fluorescent in situ hybridization (FISH) protocol

1. 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).
2. Using a sterile scalpel cut the hydrogels into ~ 1 mm thick slices.
3. Place the 1 mm sections of hydrogel inside sterile 1.5 mL tubes.
4. 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.
5. 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 °C¹³.
6. Add 200-500 µL of hybridization buffer to each ~1 mm section of hydrogel and ensure the entirety of the hydrogel sample is submerged.
7. Allow the PseaerA probe to hybridize with the hydrogel samples by placing them in the dark for ~ 18-24 h, at 46 °C, without shaking.
8. Decant the hybridization buffer into a waste collection container.
9. 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).
10. Add 1 mL of fresh wash buffer to the tubes, then incubate the samples in the dark for 6 h at 48 °C, without shaking.

4. Hydrogel and Psl0096 antibody binding

1. Sterilely remove the wash buffer using a 1 mL pipettor.
2. Rinse the samples with a 2% BSA (w/v) in PBS solution by adding and removing 1 mL of the 2% BSA solution.
3. Add 500 µL 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.
4. Sterilely remove the blocking solution using a 1 mL pipette.
5. Prepare the Psl0096-Texas Red antibody solution by diluting the antibody to a final concentration of 0.112 µg/mL in 500 µL of fresh 2% BSA/PBS.
6. Add the 500 µL antibody solution to the hydrogel samples and incubate them at RT for 6 h, protected from light, without shaking.

5. DAPI (4′,6′-diamidino-2-phenylindole) staining

1. Prepare the refractive index matching solution (RIMS) by adding 40 g of non-ionic density gradient medium, 30 µL of Tween20, 3 µg 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.
2. Filter sterilize the solution into a 50 mL conical tube using a 10 mL syringe and a sterile 0.2 µm filter.
   NOTE: The solution can be stored at 4 °C for several months.
3. Remove the Psl0096-Texas Red antibody solution with a sterile 1 mL pipette.
4. Rinse the hydrogel samples by adding and then removing 1 mL of PBS.
5. Incubate the hydrogel samples with 250 µL of RIMS solution and 10 µg/mL of DAPI at RT with gentle shaking, in the dark, overnight.
6. 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.

6. Imaging

1. Perform confocal laser scanning microscopy imaging using standard techniques at 25x, 40x, 63x, or 100x magnifications.

Representative 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 antibody binding protocols are summarized. The FISH and antibody staining protocols take 4 days to complete but should be completed and confocal images taken once started. Using the above protocols, high resolution 3D images of P. aeruginosa cells can be obtained with their in-situ structure within sputum visualized.

The clearing of sputum and subsequent application of fluorescent stains used in this protocol allows for detailed visualization of P. aeruginosa within the samples. The sputum samples shown in Figure 3 and Figure 4 were collected from a pediatric CF patient (17 years old) with a new-onset P. aeruginosa infection. In Figure 3A, an aggregate of cells was seen within a sputum sample; the appearance of yellowish-green rods was due to the overlap of all three fluorophores. Although we do not have cell counts for this sputum sample, we found the probe detection limit to be 10⁴ cells/mL (see Supplemental Figure 1). In Figure 3B, individual rod-shapes were seen in green from the species-specific binding of the PseaerA-488 probe to P. aeruginosa cells. The Psl0096-Texas Red antibody seen in red illustrates where the pseudomonal exopolysaccharide was located within the sputum; in this case the Psl0096-antibody appeared to overlap mostly with the P. aeruginosa cells (Figure 3C). This method also allowed for the visualization of pseudomonal cells within the sputum in relation with other bacterial cells and host structures. In Figure 4, a cluster of P. aeruginosa cells was seen phagocytosed within a eukaryotic cell and other small coccal cell clusters were observed in the vicinity. It has been demonstrated that the Psl0096 antibody can also bind to Psl produced form planktonic P. aeruginosa (see Supplemental Figure 2).

Figure 1: Flow diagram illustrating the sputum processing and MiPACT protocols.
In summary, sputum is fixed prior to being embedded within a polyacrylamide solution. Once cleared the hydrogel can either be stored at 4 ˚C or used with the FISH and antibody binding protocol. This image was created with BioRender.com. Please click here to view a larger version of this figure.

Figure 2: Flow diagram denoting the fluorescence in situ hybridization and antibody staining protocols.
Once the sputum has completely cleared within the hydrogel matrix staining methods can be applied. This image was created with BioRender.com. Please click here to view a larger version of this figure.

Figure 3: Immunofluorescence image of a sputum sample collected from a patient with an early P. aeruginosa infection embedded into a hydrogel matrix.
The hydrogel sample was hybridized with a PsearA-Alexa488 probe (green), a Psl0096-Texas Red antibody (red), and DAPI (blue). (A) sputum sample viewed under all 3 channels, (B) sputum under only the green channel indicating where the PseaerA-488 probe bound, and (C) sputum under only the red channel where the Psl0096-Texas red binding occurred. Images were taken at 100x magnification. Please click here to view a larger version of this figure.

Figure 4: Immunofluorescence image of a sputum sample collected from a patient with an early P. aeruginosa infection embedded into a hydrogel matrix.
The hydrogel sample was hybridized with a PsearA-Alexa488 probe (green), a Psl0096-Texas Red antibody (red), and DAPI (blue). Please click here to view a larger version of this figure.

Supplementary Figure 1: Fluorescence in situ hybridization of a planktonic culture of PAO1 with the PseaerA-488 probe. Please click here to download this figure.

Supplementary Figure 2: P. aeruginosa stained with DAPI and the Psl0096-Texas Red antibody. (A) Strain PAO1-Δpsl, and (B) strain PAO1. Please click here to download this figure.

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 growth¹⁴. For this study, sputum samples were stored at 4 °C up to a maximum of 24 h after expectoration. It should be noted that inflammatory cell counts have been shown to decrease in sputum if left at 4 °C and processed more than 9 h later¹⁵. Therefore, it is important to consider the specific cells and markers one wishes to visualize in sputum when deciding on a cut-off time for sample processing.

Sample processing in this method begins with the fixation of sputum samples in 4 % PFA. Paraformaldehyde will cross-link bacterial cells and their extracellular matrix, preserving their structures for microscopic visualization and analysis^11,16. Unfortunately, if the aim is to get total cell count on certain inflammatory cells in sputum, PFA has been shown to decrease the counts of these cells thus other fixatives should be considered¹⁷. Another limitation of this study is that it can be time consuming and may take over two weeks to perform. Thus, it may not be suitable for development into a diagnostic method requiring time sensitive treatment decisions. Furthermore, for understanding the total microbial diversity within sputum samples, this method would not be suitable, but could be paired with other high-throughput microbial detection methods such a qPCR.

The composition of the acrylamide hydrogel can be altered depending on tissue type and application¹¹. For unstable specimens like CF sputum, it is necessary to provide structural support with a 29:1 acrylamide:bis-acrylamide mixture (instead of just acrylamide). Including paraformaldehyde in the hydrogel can further stabilize the structure, with the trade-off of longer incubation times to allow diffusion of probes and antibodies⁹. Adding formaldehyde to the hydrogel can also prevent tissue swelling during the clearing process if that effect is undesirable¹¹.

The current method specifically targets P. aeruginosa cells within the sputum of CF patients. Alternative methods and modifications to this protocol can be considered to guide optimization of visualization of other bacteria. By applying species and genus-specific FISH and hybridization chain reaction (HCR) probes, other CF pathogens such as Staphylococcus aureus, Streptococcus sp., and Achromobacter xylosoxidans can be identified¹². In our study, we targeted the pseudomonal exopolysaccharide Psl. Other targets, including alginate or Pel can be examined with fluorescent antibodies specific for these exopolysaccharides in future experiments. Applying the MiPACT method along with FISH and antibody staining for CLSM takes a couple weeks to complete. If the research question does not concern the 3-dimensional spatial visualization of the sputum, there are more rapid methods to visualize bacteria present. Previous methods used to visualize bacteria within sputum samples utilize thin sectioning or smearing and include: FISH¹⁸, Gram stain, and immunohistochemistry techniques that apply primary antibodies and counterstains to allow the visualization of biofilm exopolysaccharides and bacterial cells^19,20.

There are several potential future applications of these types of imaging techniques. The ability to visualize different bacterial organisms and their interaction with host cells, such as phagocytes, may further our understanding of why some P. aeruginosa strains are effectively cleared from CF airways whereas other strains are not. Imaging bacteria within respiratory specimens may also be used as a measure of antimicrobial efficacy and as a study outcome for new anti-biofilm drugs²¹. In addition, visualizing the spatial relationship between P. aeruginosa and other organisms within the CF lung microbiome, such as Staphylococcus aureus, may help to elucidate the role of co-infection/colonization in the pathogenesis of pulmonary exacerbations, and their response to antibiotic treatment. In vivo imaging of bacteria can be applied to other infections as well, including those with ventilator-associated pneumonias or chronic wound infections²². The insights gained can thus be used to guide future therapeutic development.

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