TB acknowledges a research fellowship from Cystic Fibrosis Canada.

Note that Research Ethics Board (REB) is required to collect and store sputum samples from human subjects. These studies were approved by the Hospital for Sick Children REB#1000019444.
1. Preparing CF Sputum Samples
<ol>
	<li>Collect sputum sample from patients during routine visits to the cystic fibrosis clinic and keep on ice.</li>
	<li>Transport sputum sample on ice within the first hour of collection, to the research laboratory, to undergo processing.</li>
</ol>
2. ...

<ol>
	<li>Beaudoin, T., Waters, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infections+with+biofilm+formation:+selection+of+antimicrobials+and+role+of+prolonged+antibiotic+therapy.">Infections with biofilm formation: selection of antimicrobials and role of prolonged antibiotic therapy.</a> Pediatr.Infect.Dis.J. , (2016).</li><li>Donlan, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilms:+microbial+life+on+surfaces.">Biofilms: microbial life on surfaces.</a> Emerg.Infect.Dis. 8 (9), 881-890 (2002).</li><li>Hobley, L., Harkins, C., MacPhee, C. E., Stanley-Wall, N. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+basis+for+Pseudomonas+aeruginosa+biofilm+antibiotic+resistance.">A genetic basis for Pseudomonas aeruginosa biofilm antibiotic resistance.</a> Nature. 426 (6964), 306-310 (2003).</li><li>Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biofilm-specific+antibiotic+resistance.">Biofilm-specific antibiotic resistance.</a> Future Microbiol. 7 (9), 1061-1072 (2012).</li><li>Beaudoin, T., Zhang, L., Hinz, A. J., Parr, C. J., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biofilm-specific+antibiotic+resistance+gene+ndvB+is+important+for+expression+of+ethanol+oxidation+genes+in+Pseudomonas+aeruginosa+biofilms.">The biofilm-specific antibiotic resistance gene ndvB is important for expression of ethanol oxidation genes in Pseudomonas aeruginosa biofilms.</a> J. Bacteriol. 194 (12), 3128-3136 (2012).</li><li>Beaudoin, T., Aaron, S. D., Giesbrecht-Lewis, T., Vandemheen, K., Mah, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+clonal+strains+of+Pseudomonas+aeruginosa+isolated+from+cystic+fibrosis+patients+in+Ontario,+Canada.">Characterization of clonal strains of Pseudomonas aeruginosa isolated from cystic fibrosis patients in Ontario, Canada.</a> Can. J. 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L., 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=Cystic+fibrosis-adapted+quorum+sensing+mutants+cause+hyperinflammatory+responses.">Cystic fibrosis-adapted quorum sensing mutants cause hyperinflammatory responses.</a> Sci.Adv. 1 (6), e1500199 (2015).</li><li>Staudinger, B. 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J., Waters, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+High-Dose+Antimicrobials+on+Biofilm+Growth+of+Achromobacter+Species+Isolated+from+Cystic+Fibrosis+Patients.">Effect of High-Dose Antimicrobials on Biofilm Growth of Achromobacter Species Isolated from Cystic Fibrosis Patients.</a> Antimicrob.Agents Chemother. 60 (1), 650-652 (2015).</li><li>Heydorn, A., 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=Quantification+of+biofilm+structures+by+the+novel+computer+program+COMSTAT.">Quantification of biofilm structures by the novel computer program COMSTAT.</a> Microbiology. 146 (Pt 10), 2395-2407 (2000).</li><li>Vorregaard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Informatics and Mathematical Modelling. , (2008).</li><li>Jurcisek, J. 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The methods described herein allow for visualization of bacterial biofilms grown in the presence of exogenous products. Not surprisingly, the production of the exoproducts is of importance when using this type of system. For instance, Dithiothreitol (DTT), is often used on human sputum samples to help liquefy the samples. However, the effect of DTT alone can decrease biofilm development and viability (data not shown). Thus, proper controls for all conditions are necessary. Furthermore, the addition of human sputum produc...

Bacterial biofilms are groups of microorganisms that are attached to one another and encased in a self-secreted matrix.1,2 Classically, they represent bacteria physically attached to an abiotic or biotic surface formed under conditions of flow. Biofilms have also been shown to grow in static conditions (absence of flow) and distal from surfaces, such as at the air-liquid interface of thermal pools or pellicles formed in test tubes. These biofilms have long been recognized in the environment and are a major detriment to industrial processes, as they can form in water reservoirs or in pipes, resulting in biofouling, corrosion and blockages.3,4
Biofilms are also critical in healthcare settings, as they have been shown be involved in catheter related infections, pulmonary infections in cystic fibrosis patients, as well as in numerous other infections.5,6 One of the hallmarks of biofilm infections is the decreased susceptibility of bacteria to antibiotics and impaired clearance by the innate immune system.7-9 The most well studied, clinically relevant scenarios involving biofilm-based infection occurs in patients with cystic fibrosis (CF), who are chronically infected with Pseudomonas aeruginosa biofilms. P. aeruginosa can undergo a number of changes during establishment of chronic infection that make it very difficult to treat.10,11 Biofilms can differentially activate innate immunity and drive inflammation.12-14 As these infections lead to increased morbidity and mortality in CF patients, it is crucial to understand factors that can affect biofilm development in this context.
A recent study suggests that host-factors are critical in the formation of P. aeruginosa biofilm aggregates.15 These biofilms contribute to reduced susceptibility to antibiotics and host defense mechanisms. The presence of host-derived factors, such as neutrophil elastase, as well as secreted products from microorganisms present in the CF lung, have the potential to greatly modulate biofilm formation and development.16 Additionally, biofilms interact with the host to modulate expression of numerous pathways and initiate inflammation. While high throughput methods, such as the standard crystal violet assay, can provide some information with regards to the biofilm process, visualization of the biofilm in response to these factors provide more in-depth information.
In this manuscript we describe a method for using factors from the sputum of patients with CF to study the development of biofilms in vitro. This method allows for rapid visualization of biofilms exposed to sputum containing host factors using a commercial biofilm viability kit. This technique can be used to visually identify changes that occur during biofilm growth in the presence of exogenous products, and represents an improved method to analyze the changes in biofilm development under various conditions.

<table border="0" cellpadding="0" cellspacing="0" width="1270">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Lab-Tek II Chambered coverglass, #1.5 borosilicate, 8-well</td>
			<td style="width:159px;">Thermo Sicher Scientific</td>
			<td style="width:136px;">155409</td>
			<td style="width:729px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Filmtracer Live/Dead Biofilm Viabilty Kit</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:136px;">L10316</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Blood agar plates</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td style="width:136px;">R10215</td>
			<td>Confirming viability via CFU counts or selecting colonies for innoculation</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">COMSTAT</td>
			<td style="width:159px;">Availble software online</td>
			<td style="width:136px;"></td>
			<td>COMSTAT is software to analyze biofilm images. Available www.comstat.dk&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Millers LB Broth</td>
			<td style="width:159px;">Thermo Fisher Scientific</td>
			<td>12780-052</td>
			<td style="width:729px;">Standard media for overnight gowth/biofilm growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Millex-GV Syringe Filters</td>
			<td style="width:159px;">Millipore</td>
			<td>SLGV013SL</td>
			<td>Filtering of sputum supernants</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Phosphate Buffered Saline (Dulbecco A)</td>
			<td style="width:159px;">Oxoid</td>
			<td style="width:136px;">BR0014G</td>
			<td>Washing of biofilm chambers after media removal</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zeiss AxioVert 200M</td>
			<td style="width:159px;">Carl Zeiss</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hamamatsu C9100-13 EM-CCD</td>
			<td style="width:159px;">QS Technologies Inc.</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spectral Borealis</td>
			<td style="width:159px;">Qs Technologies Inc.</td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Perkin Elmer Volocity</td>
			<td style="width:159px;">QS Technologies Inc.</td>
			<td style="width:136px;"></td>
			<td>Instructions for this software can be found at: http://cellularimaging.perkinelmer.com/pdfs/manuals/VolocityuserGuide.pdf</td>
		</tr>
	</tbody>
</table>

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.

The overall design of the experiment is represented in Figure 1. The use of this protocol provides a convenient method to visualize the changes in biofilms grown for different periods of time (e.g., 24, 48 or 72 hr). Importantly, exogenous signals, such as sputum filtrates, can be added to visualize the changes in biofilm development. As seen in Figure 2, the presence of 10% sputum filtrates can change the architecture of the biofilm (Fig...

Watch this Scientific Journal Video about Visualizing the Effects of Sputum on Biofilm Development Using a Chambered Coverglass Model at JoVE.com

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

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 ...

The overall goal of this procedure is to visualize and quantify changes to biofilms in response to sputum and antibiotics. This method can be used by researchers to understand key questions, such as how the efficacy of various antibiotics against bacterial biofilms can be impacted by the presence of sputum. The main advantage of this methodology is it allows individuals to see changes occurring in biofilms in response to different factors.To begin the experiment, record the volume of the previously obtained sputum sample. Add phosphate-buffered saline, or PBS, add two times the volume of the sample. Next, use a transfer pipette to thoroughly mix the sample.Vortex the sample on the highest setting for one minute to mix it completely. Aliquot one milliliter of the mixture into 1.5 milliliter microcentrifuge tubes. Then spin down the tubes.Following centrifugation, save the supernatant and discard the pellet. Filter sterilize the supernatant through a 0.22 micron filter and collect it in a clean microcentrifuge tube. Store the sputum supernatant at 80 degrees Celsius for future use.Place 40 microliters of previously prepared culture into four milliliters of fresh Luria broth, or LB media, and grow the bacteria to obtain a culture with an optical density of 0.55 at 600 nanometers. Next, dilute 100 microliters of 0.5 OD culture into 400 microliters of prepared 10%sputum filtrates in LB.Use 200 microliters of the dilution to seed the wells of the slide chambers. Allow the bacteria to attach for four hours at 37 degrees Celsius without shaking.After four hours, remove the media and gently wash the biofilm with 1X fresh LB.Replace the remaining media with 200 microliters of fresh LB.Allow the biofilms to grow at 37 degrees Celsius without shaking. Replace the media every 12 hours without washing until the biofilm is ready for microscopy. It's very important to gently wash the slides so as not to disrupt any attached bacteria.Following growth, remove the media from the chamber wells and gently wash each chamber two times with 300 microliters of sterile PBS. Next, to prepare the biofilm staining mixture, add one microliter of each dye provided in the viability kit to each milliliter of solution needed and mix the solution. The optimal amount of dye and staining time is determined empirically for each organism.Add 200 microliters of the staining mixture to each well of the chambered cover glass. Incubate the cover glass. After incubation, remove the staining mixture from the chambers and wash each well with 300 microliters of sterile PBS.Replace the PBS with fresh media. Finally, proceed with visualization via confocal microscopy. Representative images of Burkholderia cepacia complex, or BCC clinical isolates, following 48 hours of growth in chambered cover glass with media alone or in the presence of 10%sputum filtrates demonstrate that their presence can change the architecture of the biofilm.The images were analyzed with Comstat software to obtain key biofilm parameters, including the average thickness of the biofilm, which was increased when treated with 10%sputum. In order to appreciate the different factors'effects on biofilm growth, clinical isolates were grown in media only or with sputum and either was treated with or without antibiotic. After watching this video, you should have a good understanding of how to form and treat biofilms on chambered cover glass and prepare them for staining and confocal analysis.

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Research

JoVE Journal

Immunology and Infection

8.3K Görüntüleme.  Hospital for Sick Children. The overall goal of this procedure is to visualize and quantify changes to biofilms in response to sputum and antibiotics. This method can be used by researchers to understand key questions, such as how the efficacy of various antibiotics against bacterial biofilms can be impacted by the presence of sputum. The main advantage of this methodology is it allows individuals to see changes occurring in biofilms in response to different factors.To begin the experiment, record the volume of t...