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>

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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 growth14. For this study, sputum samples were stored at 4 &#176;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 &#176;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 analysis11,16. 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 cells19,20.
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.

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 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).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61631/61631fig01.jpg" /> 
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 &#730;C or used with the FISH and antibody binding protocol. This image was created with BioRender.com. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61631/61631fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61631/61631fig02.jpg" /> 
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. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61631/61631fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61631/61631fig03.jpg" /> 
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. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61631/61631fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61631/61631fig04.jpg" /> 
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). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61631/61631fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Fluorescence in situ hybridization of a planktonic culture of PAO1 with the PseaerA-488 probe. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61631/61631supfig01.jpg" target="_blank">Please click here to download this figure.</a>
Supplementary Figure 2: P. aeruginosa stained with DAPI and the Psl0096-Texas Red antibody. (A) Strain PAO1-&#916;psl, and (B) strain PAO1. <a href="https://cloudflare-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/61631/61631supfig02.jpg" target="_blank">Please click here to download this figure.</a>

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 ...

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

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

3.0K Views.  Hospital for Sick Children. This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.

Video: Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

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.

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 cytotoxicity.

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to 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 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.

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 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 ...

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

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the development and persistence of many health related infections. One of the most well described and studied biofilms in human disease occurs in chronic pulmonary infection of cystic fibrosis patients. When studying biofilms in the context of the host, many factors can impact biofilm formation and development. In order to identify how host factors may affect biofilm formation and development, we used a static chambered coverglass method to grow biofilms in the presence of host-derived factors in the form of sputum supernatants. Bacteria are seeded into chambers and exposed to sputum filtrates. Following 48 hr of growth, biofilms are stained with a commercial biofilm viability kit prior to confocal microscopy and analysis. Following image acquisition, biofilm properties can be assessed using different software platforms. This method allows us to visualize key properties of biofilm growth in presence of different substances including antibiotics.

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.

Biofilms consist of groups of bacteria encased in a self-secreted matrix. They play an important role in industrial contamination as well as in the ...

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

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

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

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.

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...

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

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a poor correlation between antibiotic susceptibility testing (AST) results using standard diagnostic methods (e.g., broth microdilution, disk diffusion, or Etest) and clinical outcomes after antibiotic treatment. Attempts to improve AST by the use of off-the-shelf biofilm growth platforms show little improvement in results. The limited ability of in vitro biofilm systems to mimic the physicochemical environment of the CF lung and, therefore bacterial physiology and biofilm architecture, also acts as a brake on the discovery of novel therapies for CF infection. Here, we present a protocol to perform AST of CF pathogens grown as mature, in vivo-like biofilms in an ex vivo CF lung model comprised of pig bronchiolar tissue and synthetic CF sputum (ex vivo&#160;pig lung, EVPL).
Several in vitro&#160;assays exist for biofilm susceptibility testing, using either standard laboratory medium or various formulations of synthetic CF sputum in microtiter plates. Both growth medium and biofilm substrate (polystyrene plate vs. bronchiolar tissue) are likely to affect biofilm antibiotic tolerance. We show enhanced tolerance of clinical Pseudomonas aeruginosa and Staphylococcus aureus isolates in the ex vivo model; the effects of antibiotic treatment of biofilms is not correlated with the minimum inhibitory concentration (MIC) in standard microdilution assays or a sensitive/resistant classification in disk diffusion assays.
The ex vivo platform could be used for bespoke biofilm AST of patient samples and as an enhanced testing platform for potential antibiofilm agents during pharmaceutical research and development. Improving the prescription or acceleration of antibiofilm drug discovery through the use of more in vivo-like testing platforms could drastically improve health outcomes for individuals with CF, as well as reduce the costs of clinical treatment and discovery research.

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

This workflow can be used to perform antibiotic susceptibility testing using an established&#160;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.

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...

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

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a large proportion of the infecting bacteria form biofilms within airway sputum. Pa biofilms isolated from CF sputum have been shown to grow in small, dense aggregates of ~10-1,000 cells that are spatially organized and exhibit clinically relevant phenotypes such as antimicrobial tolerance. One of the biggest challenges to studying how Pa aggregates respond to the changing sputum environment is the lack of nutritionally relevant and robust systems that promote aggregate formation. Using a synthetic CF sputum medium (SCFM2), the life history of Pa aggregates can be observed using confocal laser scanning microscopy (CLSM) and image analysis at the resolution of a single cell. This in vitro system allows the observation of thousands of aggregates of varying size in real time, three dimensions, and at the micron scale. At the individual and population levels, having the ability to group aggregates by phenotype and position facilitates the observation of aggregates at different developmental stages and their response to changes in the microenvironment, such as antibiotic treatment, to be differentiated with precision.

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

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.

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a ...

Adaptable CF Model for Antimicrobial Testing and Microbial Dynamics Analysis

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

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as colony-forming units or metabolic activity. Furthermore, many biofilm models do not reflect the biological and physiochemical properties of the human host environment. This is an important issue in many conditions, but most noticeably in cystic fibrosis (CF). A large proportion of people with CF suffer from both chronic and intermittent infections, and in vitro, antibiotic susceptibility testing poorly correlates with patient treatment outcomes. Some biofilm models incorporate CF lung-relevant media, including synthetic sputum mimics, but do not consider the polymicrobial nature of the environment, which alters biofilm architecture, physiology, and the way microbes respond to treatment. The solid-air interface colony biofilm model described here is highly adaptable and incorporates both CF-relevant media and a polymicrobial context. This model can also be used for mid-throughput screening of antimicrobials and to study their effect on polymicrobial dynamics. Output measurements from the model can be colony-forming units, metabolic activity, and confocal microscopy analysis. The model can easily be adapted to different microorganisms, media, temperatures, and variable oxygen conditions and can be used to test a wide range of chemical, biological, and physical treatments.

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.

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as ...

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

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. Microbes have traditionally been classified based on their ability to use or tolerate oxygen. Supplemental oxygen is a common medical therapy administered to people with cystic fibrosis (pwCF); however, existing studies on oxygen and the airway microbiome have focused on how hypoxia (low oxygen) rather than hyperoxia (high oxygen) affects the predominantly aerobic and facultative anaerobic lung microbial communities. To address this critical knowledge gap, this protocol was developed using an artificial sputum medium that mimics the composition of sputum from pwCF. The use of filter sterilization, which yields a transparent medium, allows optical methods to follow the growth of single-celled microbes in suspension cultures. To create hyperoxic conditions, this model system takes advantage of established anaerobic culturing techniques to study hyperoxic conditions; instead of removing oxygen, oxygen is added to cultures by daily sparging of serum bottles with a mixture of compressed oxygen and air. Sputum from 50 pwCF underwent daily sparging for a 72-h period to verify the ability of this model to maintain differential oxygen conditions. Shotgun metagenomic sequencing was performed on cultured and uncultured sputum samples from 11 pwCF to verify the ability of this medium to support the growth of commensal and pathogenic microbes commonly found in cystic fibrosis sputum. Growth curves were obtained from 112 isolates obtained from pwCF to verify the ability of this artificial sputum medium to support the growth of common cystic fibrosis pathogens. We find that this model can culture a wide variety of pathogens and commensals in CF sputum, recovers a community highly similar to uncultured sputum under normoxic conditions, and creates different culture phenotypes under varying oxygen conditions. This new approach might lead to a better understanding of unanticipated effects induced by the use of oxygen in pwCF on airway microbial communities and common respiratory pathogens.

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 communities.

Airway microbial communities are thought to play an important role in the progression of cystic fibrosis (CF) and other chronic pulmonary diseases. ...

Medicine

In Vitro Polymicrobial Interactions in Cystic Fibrosis Lung Infections

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

Most in vitro models lack the capacity to fully probe bacterial phenotypes emerging from the complex interactions observed in real-life environments. This is particularly true in the context of hard-to-treat, chronic, and polymicrobial biofilm-based infections detected in the airways of individuals living with cystic fibrosis (CF), a multiorgan genetic disease. While multiple microbiome studies have defined the microbial compositions detected in the airway of people with CF (pwCF), no in vitro models thus far have fully integrated critical CF-relevant lung features. Therefore, a significant knowledge gap exists in the capacity to investigate the mechanisms driving the pathogenesis of mixed species CF lung infections. Here, we describe a recently developed four-species microbial community model, including Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica grown in CF-like conditions. Through the utilization of this system, clinically relevant phenotypes such as antimicrobial&#160;recalcitrance of several pathogens were observed and explored at the molecular level. The usefulness of this in vitro model resides in its standardized workflow that can facilitate the study of interspecies interactions in the context of chronic CF lung infections.

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.

Most in vitro models lack the capacity to fully probe bacterial phenotypes emerging from the complex interactions observed in real-life environments. This ...

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

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Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

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

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

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

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

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

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

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

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

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