We would like to acknowledge Dr. George A. O&#39;Toole and Dr. Thomas H. Hampton for their significant role in the design and development of the in vitro polymicrobial community model. We thank Dr. Sophie Robitaille for her helpful comments on the manuscript. This work was supported by a Cystic Fibrosis Foundation grant JEAN21F0 to F.J-P. Figure 1 of the manuscript was created using BioRender.

The details of all the reagents and equipment are listed in the Table of Materials.
1. Preparing artificial sputum medium
NOTE: Throughout this protocol, artificial sputum medium (ASM) is defined as the CF-relevant medium modified from SCFM2, previously published by Turner and colleagues17. Below is a description of the steps to prepare this medium. See Table of Materials to make stock solutions and storage conditions. This version of ASM differs from Turner et al.,17 as the buffering capacity in the original version does not allow for a stable pH over time. That is, fermentation activity carried out by Streptococcus spp. and anaerobes like Prevotella spp. results in the acidification of the growth medium (unpublished observations). Therefore, the 3-morpholinopropane-1-sulfonic acid (MOPS) concentration has been adjusted from 10 mM to 100 mM.
NOTE: Preparing a 2x ASM base (ASMb) stock solution facilitates the addition of components such as mucin, agar, water, etc., without impacting the final concentration of molecules present in ASM10,18. The 2x ASMb can be prepared in advance and stored at 4 &#176;C for up to two weeks in a dark place. If the medium turns yellow, discard.
<ol>
	<li>To prepare 2x ASM base stock, in a clean beaker containing 400 mL of diH2O, add the following while stirring:
	<ol>
		<li>Add 6.50 mL of a 0.2 M NaH2PO4.H2O stock; 6.25 mL of a 0.2 M Na2HPO4 stock; 348 &#181;L of a 1 M KNO3 stock; 1.084 mL of a 0.25 M K2SO4 stock.</li>
		<li>Add 4.0 g of Yeast synthetic dropout without Tryptophan; 3.032 g of NaCl; 20.92 g of MOPS; 1.116 g of KCl and 0.124 g of NH4Cl.</li>
		<li>Add 9.30 mL of a 1 M L-Lactic acid stock; 2.70 mL of a 1.1 M glucose stock; 1.75 mL of a 1 M CaCl2.2H2O stock; 1.20 mL of a 0.25 M N-acetylglucosamine stock and 1.00 mL of a 3.6 mM FeSO4.7H2O stock.</li>
		<li>Add 660 &#181;L of a 0.1 M Tryptophan stock; 606 &#181;L of a 1 M MgCl2.6H2O stock; 0.60 g of deoxyribonucleic acid from herring sperm; 400 &#181;L of a 250 mg/mL 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) stock.</li>
		<li>Once all the components have been added, adjust the pH to 6.80 with a 5 M NaOH stock. Complete to 500 mL with diH2O. Sterilize using a 0.22 &#181;m filter, and store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>To prepare a 10 mg/mL (2x) mucin stock, use a clean glass bottle containing 250 mL of diH2O and while stirring:
	<ol>
		<li>Add 5.0 g of mucin from porcine stomach - Type II. Mix the suspension for 10 min. Complete to 500 mL with diH2O.</li>
		<li>Sterilize by autoclaving at 121 &#176;C for 15 min. Keep at 4 &#176;C until utilization.</li>
	</ol>
	</li>
	<li>Reconstitute the ASM. 
	NOTE: This step needs to be performed on the day of the experiment.
	<ol>
		<li>Mix a 1:1 volume ratio of the 2x ASMb stock and the 10 mg/mL mucin stock.</li>
		<li>Vortex thoroughly before use.</li>
	</ol>
	</li>
</ol>
2. Preparing polymicrobial community selective media
NOTE: Below are the quantities necessary to prepare a volume of 1 L of media.
<ol>
	<li>To prepare Mannitol Salt Agar (MSA) and Pseudomonas Isolation Agar (PIA), follow the manufacturer&#39;s instructions.</li>
	<li>To prepare Prevotella Selective Agar (PSA), in a clean glass bottle, add the following:
	<ol>
		<li>30.0 g of tryptic soy broth (TSB). 15.0 g of agar. 5.00 g of Yeast extract. 0.50 g of L-cysteine hydrochloride.</li>
		<li>10.0 mL of a 0.5 mg/mL hemin stock. 100 &#181;L of a 10 mg/mL menadione stock.</li>
		<li>Add 940 mL of diH2O and stir. Sterilize by autoclaving at 121 &#176;C for 15 min.&#160;</li>
		<li>When cooled down (i.e., the bottle can be held barehanded), add while stirring: 50.0 mL of defribrinated sheep&#39;s blood. 2.00 mL of a 50 mg/mL kanamycin stock. 150 &#181;L of a 50 mg/mL vancomycin stock. 500 &#181;L of a 10 mg/mL polymixin B stock.</li>
	</ol>
	</li>
	<li>To prepare Streptococcus Selective Agar (SSA), in a clean glass bottle, add the following:
	<ol>
		<li>30.0 g of TSB. 15.0 g of agar.</li>
		<li>Add 950 mL of diH2O and stir. Sterilize by autoclaving at 121 &#176;C for 15 min.&#160;</li>
		<li>When cooled down (i.e., the bottle can be held barehanded), add while stirring: 50.0 mL of defribrinated sheep&#39;s blood. 1.00 mL of a 10 mg/mL polymixin B stock. 1.00 mL of a 10 mg/mL oxolinic acid stock. 
		NOTE: Pour in plates and keep at 4 &#176;C for a maximum of 1 month. Before using, let the plates dry at room temperature up to 24 h in advance before they are inoculated. PSA has been validated with P. melaninogenica and Prevotella intermedia. SSA has been validated with S. sanguinis, and the Streptococcus milleri group (Streptococcus constellatus, Streptococcus intermedius, and Streptococcus anginosus, denoted as SMG). It is strongly recommended to de novo validate these selective media if new strains are to be tested.</li>
	</ol>
	</li>
</ol>
3. Bacterial liquid media preparation and growth conditions
<ol>
	<li>To routinely grow P. aeruginosa and S. aureus, use a sterile rich medium (e.g., lysogeny broth (LB) or TSB).
	<ol>
		<li>Start liquid overnights by picking a single colony previously grown on an LB/TSB agar plate for 20-24 h at 37 &#176;C.</li>
		<li>Grow the broth cultures at 37 &#176;C overnight with shaking at 250 rpm for 16-18 h.</li>
	</ol>
	</li>
	<li>To grow Streptococcus spp., use sterile Todd Hewitt Broth supplemented with 0.5 % yeast extract (THB-YE).
	<ol>
		<li>Start liquid culture overnight from a single colony previously grown on a TSB agar plate supplemented with 5% defibrinated sheep&#39;s blood (blood agar plate) for 24 h at 37 &#176;C + 5% CO2.</li>
		<li>Grow the THB-YE cultures anaerobically or at 37 &#176;C + 5% CO2 overnight without shaking for 18-22 h.</li>
	</ol>
	</li>
	<li>To grow Prevotella spp., prepare 1 L of Prevotella growth medium (PGM) by adding the following to a clean bottle:
	<ol>
		<li>30.0 g of TSB. 5.00 g of yeast extract. 0.50 g of L-cysteine hydrochloride. 10.0 mL of a 0.5 mg/mL hemin stock and 100 &#181;L of a 10 mg/mL menadione stock.</li>
		<li>Add 990 mL of diH2O. Sterilize by autoclaving at 121 &#176;C for 15 min. Wrap the bottle in aluminum foil to protect it from light.</li>
		<li>Grow Prevotella spp. colonies on a blood agar plate incubated anaerobically at 37 &#176;C for 48 h.</li>
		<li>Inoculate a single colony in PGM and grow overnight without shaking at 37 &#176;C anaerobically for 24 h. 
		NOTE: PGM can be kept at room temperature for up to two weeks. Performing sterility tests on each growth medium is strongly recommended before use. If not using an anaerobic chamber, it might be necessary to start liquid overnights of Prevotella spp. from a patch of colonies.</li>
	</ol>
	</li>
</ol>
4. Co-culture experiment preparation
NOTE: Figure 1 depicts a summarized experimental workflow. Setting up the experiment can be done in oxic conditions if the preparation time takes less than 1 h. If it is expected to take longer than that, it is strongly recommended to perform the experiment using an anaerobic chamber (with 10% CO2, 10% H2, and 80% N2 mixed gas). Anaerobic jars can also be used to incubate the experimental samples.
<ol>
	<li>Preparation of the monocultures and co-cultures 
	NOTE: Perform all the centrifugation steps at 10,000 x g for 2 min at room temperature. For the co-cultures, mix the bacterial samples just before inoculating the 96-well plates. Typically, a volume of 1 mL for P. aeruginosa and S. aureus strains are collected from the overnight cultures. For Streptococcus spp. and Prevotella spp., the overnight volumes may vary between 2-4 mL. These volumes can be adjusted depending on the number of conditions to be tested.
	<ol>
		<li>Using the bacterial liquid from the overnight cultures, perform the following steps:
		<ol>
			<li>For P. aeruginosa and S. aureus strains, wash twice using sterile phosphate-buffered saline (PBS).</li>
			<li>For Streptococcus spp. and Prevotella spp., wash once with sterile PBS. 
			NOTE: Use a pipette to carefully discard supernatants, as the pellets can easily be lost.</li>
			<li>After the washes, resuspend each pellet in 1 mL of 1x water-diluted ASMb.</li>
			<li>Make a 1:10 dilution of each sample in sterile PBS and measure the OD600.</li>
			<li>Adjust all the cultures to an OD600 of 0.2 in ASM.</li>
			<li>Using the OD600 = 0.2 suspensions, dilute in ASM the bacterial samples to a final OD600 of 0.01 (for each microbe) for the monocultures and co-cultures.</li>
			<li>Vortex thoroughly for 5 s. 
			NOTE: For example, to prepare a total of 1 mL of P. aeruginosa monoculture ASM suspension at OD600 = 0.01, add 50 &#181;L of P. aeruginosa at OD600 = 0.2 to a volume of 950 &#181;L of ASM. For the co-culture, take 50 &#181;L of each P. aeruginosa, S. aureus, Streptococcus spp. and Prevotella spp. suspensions at an OD600 = 0.2 and add to 800 &#181;L ASM.</li>
			<li>Using a pipette, add 100 &#181;l of the monoculture and co-culture suspensions in three separate wells of a sterile plastic flat bottom 96-well plate.</li>
			<li>Incubate the plate (without shaking) for 24 h at 37 &#176;C in anoxic conditions. 
			NOTE: Other oxygen tensions (e.g., microxia, normoxia) can also be used here. However, the model has been developed and validated with anoxic conditions. Confirm the starting inocula by serially diluting the mono and co-culture bacterial suspensions, and plating on PIA, MSA, SSA, and PSA is strongly recommended. The targeted starting concentration for each bacterial species is 1 &#215; 107 CFU/mL (P. aeruginosa), 3.5 &#215; 106 CFU/mL (S. aureus), 1.2 &#215; 106 CFU/mL (Streptococcus spp.), and 4.6 &#215; 106 CFU/mL (Prevotella spp.). Bacterial inocula can also be modified, as described by Jean-Pierre and colleagues10.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Replace the media after biofilm formation.
	<ol>
		<li>After 24 h of incubation, remove the unattached (planktonic) cells by aspiration using a multichannel pipette.</li>
		<li>Replenish the pre-formed biofilms with 100 &#181;L of fresh ASM or the desired treatment (e.g., antibiotics, metabolites, etc.).</li>
		<li>Incubate the plate for an additional 24 h at 37 &#176;C in anoxic conditions. 
		NOTE: These steps can be done aerobically if performed quickly (i.e., less than 1 min). Otherwise, please use an anaerobic chamber.</li>
	</ol>
	</li>
</ol>
5. Collecting and plating the samples
<ol>
	<li>After the additional 24 h of incubation, aspirate the non-attached (planktonic) cells with a multichannel pipette. 
	NOTE: The planktonic fraction can be kept, serially diluted, and plated on selective media or discarded.</li>
	<li>Gently wash the biofilms twice using 125 &#181;L of sterile PBS and discard the volumes.</li>
	<li>Add 50 &#181;L of sterile PBS and detach the biofilms by gently scraping the cells from the plate using a 96-pin replicator.</li>
	<li>Transfer the resuspended biofilm cells to row A of a new sterile 96-well plate. 
	NOTE: The 96-well plate will contain sterile PBS in rows B to H.</li>
	<li>Perform a 10x serial dilution of the planktonic (if needed) and biofilm fractions.</li>
	<li>Plate 3-5 &#181;L of each dilution sample onto PIA, MSA, SSA, and PSA.</li>
	<li>Allow the inoculation spots to dry.</li>
</ol>
6. Incubating the selective plates and colony forming unit (CFU) counting
<ol>
	<li>For P. aeruginosa and S. aureus, incubate at 37 &#176;C for 18-20 h.</li>
	<li>For Streptococcus spp, incubate at 37 &#176;C in anoxic conditions for 24 h.</li>
	<li>For Prevotella spp, incubate at 37 &#176;C in anoxic conditions for 48 h.</li>
	<li>After incubation, count the colonies (~10-35 per spot) and calculate the CFU per mL.</li>
	<li>Plot the data using a visualization tool.</li>
</ol>

In Vitro Polymicrobial Interactions in Cystic Fibrosis Lung Infections

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The authors declare no disclosures.

The usefulness of this clinically informed in vitro co-culture system resides in its capacity to detect polymicrobial-specific bacterial functions. That is, through the utilization of this model we have reported microbial phenotypes ranging from community-specific growth of Prevotella spp. (Figure 2) to changes in susceptibility to front-line CF Abx of P. aeruginosa, S. aureus and S. sanguinis (Figure 3). Moreover, ev...

Strategies aimed at eradicating disease-causing microbes such as the ones detected in the cystic fibrosis (CF) airway, a multiorgan genetic disease, often fail1. That is, the presence of resilient biofilm-like microbial communities growing in the mucus-rich CF lung environment can cause chronic infections spanning multiple decades2. Furthermore, although the utilization of front-line antimicrobials (Abx) and modulators has resulted in improved outcomes in people with CF (pwCF), the &#34;one bug, one infection&#34; clinical approach typically employed against canonical CF pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus has been ineffective in resolving hard-to-treat infections detected in the lungs of these individuals3,4,5,6,7.
Over the last two decades, multiple studies have changed our understanding of chronic CF lung disease8. That is, reports indicate that infections detected in the airways of pwCF are not solely caused by a single pathogen but are rather polymicrobial in nature8. Furthermore, although the pathogenesis of mixed species CF lung infections is still poorly understood, clinical evidence shows that pwCF that are co-infected with both S. aureus and P. aeruginosa in their airways have worsened lung function than individuals colonized by either of these two pathogens9.
Interspecies interactions among CF pathogens are hypothesized to be a reason why polymicrobial biofilm-based infections are not readily eradicated through the utilization of CF therapeutics, ultimately impacting patient outcomes3. Supporting this, in vitro studies have demonstrated that interactions between microbes such as P. aeruginosa, S. aureus, and others can impact clinically relevant phenotypes such as Abx responsiveness to front-line CF drugs, including vancomycin and tobramycin6,10,11. Therefore, revisiting the current treatment strategies aimed at eradicating CF pathogens in the context of mixed-species infections remains to be achieved.
The gold standard in the management of microbial-based CF lung infections heavily relies on the utilization of antimicrobial susceptibility testing (AST) to guide clinical interventions12. However, AST is typically done using bacterial monocultures grown in rich and well-mixed cultures, which is not reflective of the microbial growth conditions detected in the CF airway3,13. Given the significant disconnect between patient outcomes and treatment success, clinical reports now advocate for the development of novel approaches integrating critical features of the CF lung12,14.
Few studies have developed CF-relevant bacterial multispecies in vitro systems containing more than two pathogens15,16. However, these models (1) do not entirely reflect the polymicrobial and biofilm-like nature of the CF lung and (2) do not use nutritional and environmental conditions approximating the ones detected in the CF airway15,16. Through the mining of large CF-lung derived 16S rRNA gene data sets and computation, Jean-Pierre and colleagues recently developed an in vitro co-culture model integrating the above-mentioned features10. This system includes P. aeruginosa, S. aureus, Streptococcus spp. and Prevotella spp. grown in CF airway-like conditions and stable for up to 14 days. The authors also reported community-specific growth of Prevotella spp. and several changes in the Abx responsiveness of these CF pathogens through mechanisms that remain to be identified10. This tractable in vitro system also offered the possibility to delve into mechanistically focused questions related to Abx recalcitrance of a common variant of P. aeruginosa frequently detected in the airways of pwCF10. Therefore, the goal of this protocol is to provide the CF research community with a standardized experimental workflow to grow an in vitro biofilm that has the potential to be further expanded to tackle a number of CF-relevant questions.

<table><tbody><tr><td>1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)</td><td>Fisher Scientific</td><td>NC0387928</td><td>Prepare a 250 mg/mL stock in chloroform directly in the bottle. Keep at -20&amp;deg;C. Final concentration 200 &amp;mu;g/mL in 2x ASM base.</td></tr><tr><td>3-morpholinopropane-1-sulfonic acid (MOPS)</td><td>Sigma</td><td>M1254</td><td>Final concentration 200 mM in 2X ASM base.</td></tr><tr><td>96-pin replicator</td><td>Fisher Scientific</td><td>05-450-9</td><td>Disposable replicators can also be used. Cat #NC1567338.</td></tr><tr><td>96-well sterile standard plate with lid</td><td>Fisher Scientific</td><td>62406-081</td><td></td></tr><tr><td>Agar</td><td>Fisher Scientific</td><td>DF0145-17-0</td><td></td></tr><tr><td>AnaeroPack 2.5 L Rectangular Jar</td><td>Fisher Scientific</td><td>23-246-385</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;*2H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Fisher Scientific</td><td>C79-500</td><td>Prepare a 1 M stock in water and filter sterilize with a 0.22 &amp;mu;m filter. Final concentration 3.5 mM in 2x ASM base.</td></tr><tr><td>Defribrinated sheep&#039;s blood</td><td></td><td></td><td>To prepare growth plates for Streptococcus and Prevotella.</td></tr><tr><td>Deoxyribonucleic acid from herring sperm</td><td>Sigma</td><td>D7290</td><td>Final concentration 1.2 mg/mL in 2x ASM base.</td></tr><tr><td>FeSO&lt;sub&gt;4&lt;/sub&gt;*7H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Fisher Scientific</td><td>AA1449830</td><td>Prepare a 1 mg/mL stock (3.6 mM) in water and filter sterilize with a 0.22 &amp;mu;m filter. Keep wrapped in aluminum foil. Final concentration 0.0072 mM in 2x ASM base.</td></tr><tr><td>GasPaks</td><td>Fisher Scientific</td><td>B260678</td><td></td></tr><tr><td>Glucose</td><td>Fisher Scientific</td><td>D16-3</td><td>Prepare as a 20% stock (1.1 M) in water and sterilize by autoclave. Final concentration 6 mM in 2x ASM base.</td></tr><tr><td>Hemin</td><td>Sigma</td><td>51280</td><td>Dissolve 100 mg of hemin in 2 mL of 1 M NaOH and then bring up volume to 200 mL with water. Store in an amber bottle or a regular bottle wrapped with aluminum foil at 4 &amp;deg;C.</td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>P304-500</td><td>Prepare a 0.25 M stock in water and filter sterilize with a 0.22 &amp;mu;m filter. Final concentration 0.54 mM in 2x ASM base.</td></tr><tr><td>Kanamycin</td><td></td><td></td><td>Prepare a 50 mg/mL stock solution in water. Filter sterilize with a 0.22 &amp;mu;m filter and store at 4 &amp;deg;C.</td></tr><tr><td>KCl</td><td>Fisher Scientific</td><td>P217-500</td><td>Final concentration 30.0 mM in 2x ASM base.</td></tr><tr><td>KNO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher Scientific</td><td>P263-500</td><td>Prepare a 1 M stock in water and filter sterilize with a 0.22 &amp;mu;m filter. Final concentration 0.70 mM in 2x ASM base.</td></tr><tr><td>L-cysteine hydrochloride</td><td>Fisher Scientific</td><td>AAA1038922</td><td></td></tr><tr><td>L-Lactic acid&amp;nbsp;</td><td>Sigma</td><td>L1750</td><td>Prepare a 1 M stock in water and filter sterilize with a 0.22 &amp;mu;m filter. pH adjust to 7.0. Final concentration 18.6 mM in 2x ASM base.</td></tr><tr><td>L-Tryptophan</td><td>Sigma</td><td>T0254</td><td>Prepare a 0.1 M stock in 0.2 M NaOH and filter sterilize with a 0.22 &amp;mu;m filter. Keep refrigerated at 4 &amp;deg;C wrapped in aluminum foil. Final concentration 0.132 mM in 2x ASM base.</td></tr><tr><td>Mannitol Salt Agar</td><td>Fisher Scientific</td><td>B11407</td><td>Prepare using manufacturer&#039;s recommendations.</td></tr><tr><td>Menadione</td><td>Sigma</td><td>M5625</td><td>Prepare a 10 mg/mL stock solution dissolved in 96-100% ethanol. Conserve at 4 &amp;deg;C wrapped in aluminium foil. Vortex before use.</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;*6H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;</td><td>Fisher Scientific</td><td>AA12288A9</td><td>Prepare a 1 M stock in water and filter sterilize with a 0.22 &amp;mu;m filter. Final concentration 1.21 mM in 2x ASM base.</td></tr><tr><td>Mucin from porcine stomach - Type II&amp;nbsp;</td><td>Sigma</td><td>M2378</td><td>Prepare a 10 mg/mL (2x) stock in water; mix thoroughly before autoclaving. Keep at 4 &amp;deg;C.</td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>S375-500</td><td>Prepare a 0.2 M stock in water and filter sterilize with a 0.22 &amp;mu;m filter. Final concentration 2.5 mM in 2x ASM base.</td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-acetylglucosamine&amp;nbsp;</td><td>Sigma</td><td>A8625</td><td>Prepare a 0.25 M stock in water and filter sterilize with a 0.22 &amp;mu;m filter. Keep stock refrigerated at 4 &amp;deg;C. Final concentration 0.6 mM in 2x ASM base.</td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>S271-500</td><td>Final concentration 103.7 mM in 2x ASM base.</td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;*H2O</td><td>Fisher Scientific</td><td>S369-500</td><td>Prepare a 0.2 M stock in water and filter sterilize with a 0.22 &amp;mu;m filter. Final concentration 2.6 mM in 2x ASM base.</td></tr><tr><td>NaOH</td><td>Fisher Scientific</td><td>S318-500</td><td>Prepare a 5 M stock solution. Use to adjust pH of 2x ASM base stock.</td></tr><tr><td>NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Fisher Scientific</td><td>A661-500</td><td>Final concentration 4.6 mM in 2x ASM base.</td></tr><tr><td>Oxolinic acid</td><td></td><td></td><td>Prepare a 10 mg/mL stock solution 0.5 M NaOH. Filter sterilize with a 0.22 &amp;mu;m filter and store at 4 &amp;deg;C.</td></tr><tr><td>Polymixin B</td><td></td><td></td><td>Prepare a 10 mg/mL stock solution in water. Filter sterilize with a 0.22 &amp;mu;m filter and store at 4 &amp;deg;C.</td></tr><tr><td>&lt;em&gt;Pseudomonas &lt;/em&gt;Isolation Agar</td><td>Fisher Scientific</td><td>DF0927-17-1</td><td>Prepare using manufacturer&#039;s recommendations.</td></tr><tr><td>Todd Hewitt Broth</td><td>Fisher Scientific</td><td>DF0492-17-6</td><td>Prepare using manufacturer&#039;s recommendations.</td></tr><tr><td>Tryptic Soy Broth</td><td>Fisher Scientific</td><td>DF0370-17-3</td><td>Prepare using manufacturer&#039;s recommendations.</td></tr><tr><td>Vancomycin</td><td></td><td></td><td>Prepare a 50 mg/mL stock solution in water. Filter sterilize with a 0.22 &amp;mu;m filter and store at 4 &amp;deg;C.</td></tr><tr><td>Yeast Extract</td><td>Fisher Scientific</td><td>B11929</td><td></td></tr><tr><td>Yeast Synthetic Dropout without Tryptophan&amp;nbsp;</td><td>Sigma</td><td>Y1876</td><td>Final concentration 8.0 mg/mL in 2x ASM base.</td></tr></tbody></table>

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.

As represented in Figure 2, several phenotypes were reported including (1) a reduction in the number of P. aeruginosa and S. aureus viable cell counts when grown a mixed planktonic communities compared to monoculture, (2) an increase in polymicrobial growth of S. sanguinis cells and, (3) mixed community growth of P. melaninogenica as previously reported by Jean-Pierre and colleagues10.
Furthermore, as dep...

Read this Scientific Article Protocol about Growing a Cystic Fibrosis-Relevant Polymicrobial Biofilm to Probe Community Phenotypes at JoVE.com

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

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 ...

growing-cystic-fibrosis-relevant-polymicrobial-biofilm-to-probe

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

460 Views.  Université de Sherbrooke. 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.

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

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only. 
The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. 
In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.

In this video we present the microfluidic probe1 (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer solution.

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. ...

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 ...

Immunology and Infection

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their mucus-plugged bronchioles create a favorable habitat for microbial colonization. Various pathogens thrive in this environment interacting with each other and driving many of the symptoms associated with CF disease. Like any microbial community, the chemical conditions of their habitat have a significant impact on the community structure and dynamics. For example, different microorganisms thrive in differing levels of oxygen or other solute concentrations. This is also true in the CF lung, where oxygen concentrations are believed to drive community physiology and structure. The methods described here are designed to mimic the lung environment and grow pathogens in a manner more similar to that from which they cause disease. Manipulation of the chemical surroundings of these microbes is then used to study how the chemistry of lung infections governs its microbial ecology. The method, called the WinCF system, is based on artificial sputum medium and narrow capillary tubes meant to provide an oxygen gradient similar to that which exists in mucus-plugged bronchioles. Manipulating chemical conditions, such as the media pH of the sputum or antibiotics pressure, allows for visualization of the microbiological differences in those samples using colored indicators, watching for gas or biofilm production, or extracting and sequencing the nucleic acid contents of each sample.

The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping project is to collect a tissue sample from each individual plant. The traditional approach to this task is to sample plants one-at-a-time. If one wishes to genotype hundreds or thousands of individuals, however, using this strategy results in a significant bottleneck in the genotyping pipeline. The Ice-Cap method that we describe here provides a high-throughput solution to this challenge by allowing one scientist to collect tissue from several thousand seedlings in a single day 1,2. This level of throughput is made possible by the fact that tissue is harvested from plants 96-at-a-time, rather than one-at-a-time.
The Ice-Cap method provides an integrated platform for performing seedling growth, tissue harvest, and DNA extraction. The basis for Ice-Cap is the growth of seedlings in a stacked pair of 96-well plates. The wells of the upper plate contain plugs of agar growth media on which individual seedlings germinate. The roots grow down through the agar media, exit the upper plate through a hole, and pass into a lower plate containing water. To harvest tissue for DNA extraction, the water in the lower plate containing root tissue is rapidly frozen while the seedlings in the upper plate remain at room temperature. The upper plate is then peeled away from the lower plate, yielding one plate with 96 root tissue samples frozen in ice and one plate with 96 viable seedlings. The technique is named "Ice-Cap" because it uses ice to capture the root tissue. The 96-well plate containing the seedlings can then wrapped in foil and transferred to low temperature. This process suspends further growth of the seedlings, but does not affect their viability. Once genotype analysis has been completed, seedlings with the desired genotype can be transferred from the 96-well plate to soil for further propagation. We have demonstrated the utility of the Ice-Cap method using Arabidopsis thaliana, tomato, and rice seedlings. We expect that the method should also be applicable to other species of plants with seeds small enough to fit into the wells of 96-well plates.

Ice-Cap: A Method for Growing Arabidopsis and Tomato Plants in 96-well Plates for High-Throughput Genotyping

The Ice-Cap method allows one to grow plants in 96-well plates and non-destructively harvest root tissue from each seedling. DNA extracted from this root tissue can be used for genotyping reactions. We have found that Ice-Cap works well for Arabidopsis thaliana, tomato, and rice seedlings.

It is becoming common for plant scientists to develop projects that require the genotyping of large numbers of plants. The first step in any genotyping ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

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.

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.

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic ...

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 ...

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

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