Part of this work was performed at the Marine Biological Lab with support from the Marine Biological Lab, DOE (DE-SC0016127), NSF (MCB1822263), HHMI (grant number 5600373), and a gift from the Simons Foundation.

This study received approval from the Partners Institutional Review Board (Protocol # 2018P002934). Inclusion criterion included adult patients with cystic fibrosis who provided written informed consent for the study. There was no exclusion criterion. According to protocol guidelines, all sputum samples were collected from patients with cystic fibrosis during a scheduled outpatient visit with their clinical provider.
1. Artificial Sputum Medium Preparation
NOTE: Quantities listed here are for the production of 1 L of final artificial sputum medium, and assume the specific reagents listed in the Materials Table. Numbers must be adjusted for other volumes or for the use of different reagents to ensure the same final product. See Table 1 for target concentrations.
<ol>
	<li>Artificial sputum chemical mix (ASCM) 
	NOTE: ASCM makes up 25% of the final medium volume. It is shelf-stable and can be prepared in bulk or in advance. If being prepared for later use, autoclave the chemical mix and safely store it at room temperature.
	<ol>
		<li>Mix the constituent chemical stock solutions.
		<ol>
			<li>Prepare 1 M NaCl stock: Add 58.44 g of NaCl per liter of sterile water.</li>
			<li>Prepare 1 M KCl stock: Add 74.55 g of KCl per liter of sterile water.</li>
			<li>Prepare 1 M MgSO4 stock: Add 246.47 g of MgSO4&#183;7H2O per liter of sterile water, or 120.37 g of anhydrous MgSO4 per liter of sterile water.</li>
			<li>Prepare 1 M glucose stock: Add 180.16 g of glucose per liter of sterile water.</li>
		</ol>
		</li>
		<li>Autoclave sterilize the chemical stock solutions, as well as an empty 250 mL bottle. Perform the autoclaving steps to at least standard values of 121 &#176;C and 15 PSI for 30 min.</li>
		<li>Add 80.59 mL of sterile water to the empty 250 mL bottle.</li>
		<li>Add 152.30 mL of 1 M NaCl stock to the mix.</li>
		<li>Add 15.8 mL of 1 M KCl stock to the mix.</li>
		<li>Add 610 &#181;L of 1 M MgSO4 stock to the mix.</li>
		<li>Add 700 &#181;L of 1 M glucose stock to the mix.</li>
	</ol>
	</li>
	<li>Artificial sputum mucin mix (ASMM) 
	NOTE: ASMM makes up 50% of the final medium volume. Ensure that it is prepared on the same day as the final medium batch.
	<ol>
		<li>Add 450 mL of sterile water to an empty 1 L bottle.</li>
		<li>Add 50 mL of 10x Phosphate-Buffered Saline (PBS) to the bottle.</li>
		<li>Add a disposable magnetic stir bar to the bottle.</li>
		<li>Autoclave the bottle containing PBS and the stir bar.</li>
		<li>Measure out 10 g of mucin powder and add it to the PBS.</li>
		<li>Shake the bottle vigorously for preliminary mixing.</li>
		<li>Place the bottle onto a hot plate with a magnetic stirrer. Set heat to medium-high targeting 50 &#176;C and stirring speed to 1100 rpm. Ramp up the speed gradually so that the bar does not fly off the magnet.
		<ol>
			<li>Allow to heat and stir for 15 min.</li>
			<li>Pick up the bottle with heat-resistant gloves. Observe to check if mucin powder settles out of the solution.</li>
			<li>If mucin powder is not fully dissolved, return the bottle to heat/stirrer for 5-min intervals until it is completely dissolved.</li>
		</ol>
		</li>
		<li>Allow the mucin mix to cool to room temperature.</li>
	</ol>
	</li>
	<li>Artificial sputum biological mix (ASBM) 
	&#8203;NOTE: ASBM is 25% of the final medium volume. Prepare it on the same day as the final medium batch, and unlike the other mixes, do not expose its components to any heat.
	<ol>
		<li>Thaw the 100x vitamin stock in a 4 &#176;C fridge or on ice. 
		&#8203;NOTE: Pre-portion the vitamin stock into 10 mL aliquots to minimize the number of freeze/thaw cycles.</li>
		<li>Add 124.24 mL of sterile water to the empty autoclaved 250 mL bottle.</li>
		<li>Add 25.76 mL of 50x essential amino acid stock to the mix.</li>
		<li>Add 80.14 mL of 100x non-essential amino acid stock to the mix.</li>
		<li>Add 10 mL of (thawed) 100x vitamin stock to the mix.</li>
		<li>Add 1 mL of 1000x trace metals stock to the mix.</li>
		<li>Add 8.33 mL of 30% egg yolk emulsion to the mix.</li>
		<li>Add 400 &#181;L of 10 g/L ferritin stock to the mix.</li>
		<li>Mix the solution well via manual shaking.</li>
	</ol>
	</li>
	<li>Artificial sputum medium (ASM)
	<ol>
		<li>Add 250 mL of ASCM to the 1 L bottle containing ASMM.</li>
		<li>Add 250 mL of ASBM to the medium bottle.</li>
		<li>Titrate the medium with basic MOPS buffer (1 M) to reach a pH of 6.3 on a narrow range pH paper. Prior to titration, the medium mix will be too acidic.</li>
		<li>Refrigerate the resulting artificial sputum medium at 4 &#176;C until it is ready for filtration.</li>
		<li>To start the filtration process, transfer 200 mL of unfiltered artificial sputum medium to a vacuum filtration system with a 0.22 &#181;m pore size filter.</li>
		<li>Connect the filtration system to the vacuum pump, turn on the vacuum pump, set it to 70 mbar, and then place the chamber on an orbital shaker shaking at 90 rpm in a cold room at 4 &#176;C.
		<ol>
			<li>Top off with an additional 150 mL of the medium as an appreciable amount is filtered. It takes 1-2 days to filter 1 L of medium completely.</li>
			<li>Repeat with additional chambers until all the media is filtered. 
			NOTE: Try not to filter more than 350 mL of the medium through the same 0.22 &#181;m filter since mucin will plug the filter over time.</li>
		</ol>
		</li>
		<li>Refrigerate filtered artificial sputum medium at 4 &#176;C until ready for use. Use ASM within one month of preparation for best results.</li>
	</ol>
	</li>
</ol>
2. Oxygen Sparging
<ol>
	<li>Sparging station setup 
	NOTE: This protocol should only need to be done in full once, after which point the setup can be maintained through simple maintenance as necessary. See Figure 1 for a visual schematic of the oxygen sparging system.
	<ol>
		<li>Obtain and properly secure the compressed air and oxygen tanks. 
		CAUTION: High pressure makes the tanks extremely dangerous when mishandled. Ensure that the tanks are completely sealed and secured, there are no leaks when the tank is closed, and that all handling personnel is fully trained in their use.</li>
		<li>Attach an air regulator to the compressed air tank with a wrench. For optimal flow reading on the regulator, attach the regulator as close as possible to an upright position.</li>
		<li>Attach an oxygen regulator to the compressed oxygen tank, attaching as close as possible to an upright position. Depending on the oxygen tank, one may need to invert the direction to tighten.</li>
		<li>Connect the tubing from the regulators to a Y-connector to combine the gas flow from the two tanks.</li>
		<li>Connect the output of the Y-connector to the central T-junction valve.</li>
		<li>Connect one side of the central T-junction valve to a gas pressure gauge.</li>
		<li>Connect the other side of the gas pressure gauge to a 25 mm diameter sterile syringe filter with a 0.22 &#181;m pore size.</li>
		<li>Attach a second 25 mm diameter syringe filter to a syringe without a plunger to be used as a gas release during sparging.</li>
		<li>Connect the final side of the central T-junction valve to a second T-junction valve for the oxygen monitor.</li>
		<li>Connect a 25 mm diameter syringe filter to one side of this second T-junction valve, along with tubing to attach 18 G needles.</li>
		<li>Connect the final side of the second T-junction valve to the oxygen monitoring apparatus.</li>
		<li>Connect a cut-off tube to the other side of the oxygen monitoring apparatus to be used as a gas release during monitoring. 
		CAUTION: When testing/using the oxygen sparging system, take careful note of the position of the T-junctions and ensure it matches the intended path through the system. Failure to do so will result in pressure buildup inside the system and cause components to fail and come apart .</li>
		<li>For the maintenance of the system and to keep it working at optimal performance, the following practices are beneficial.
		<ol>
			<li>Reinforce the connections with liberal amounts of Teflon tape to greatly improve their seal and reduce the chance of components coming apart from the internal pressure.</li>
			<li>Keep combined flow rate under 10 L/min to mitigate maximum pressure and prevent failures.</li>
			<li>If a leak is suspected, use a detergent solution such as commercially available liquid leak detectors to identify its location easily, as it will bubble above any gas leaks. Patch leaks using a Polytetrafluoroethylene tape (e.g., Teflon).</li>
			<li>Replace the 25 mm diameter syringe filters in the oxygen sparging system bi-weekly, but this varies with use frequency. Over time, particles caught in the filter reduce the gas flow rate and cause pressure buildups.</li>
			<li>Calibrate the oxygen monitor to 21% oxygen compressed air prior to carrying out measurements.</li>
			<li>Upon completion of the system use, turn off the tanks and bleed the excess gas from the regulators until the flow completely stops.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Serum bottle culture sparging
	<ol>
		<li>Label 500 mL autoclaved serum bottles with sample identifiers, date/time of inoculation, and target oxygen percentage.</li>
		<li>In a biological hood, add 24 mL of the artificial sputum medium to each serum bottle being set up.</li>
		<li>Add 1 mL of the patient sputum homogenized with an 18-G needle (diluted with sterile saline if necessary to obtain sufficient volume of sample for each culture condition) to each serum bottle.</li>
		<li>Using sterile tweezers, place the autoclaved rubber stoppers onto the top of each serum bottle.</li>
		<li>Press down the rubber stoppers, take care not to touch the underside of the stopper with hands.</li>
		<li>Remove the bottles from the hood, apply and crimp the aluminum seals. Remove the center piece from the seals.</li>
		<li>Wipe down the top of bottles with an alcohol wipe and pass them through a Bunsen burner flame.</li>
		<li>Affix a sterile 18-G needle to a plunger-less syringe with a filter. Insert this gas release into the bottle first.</li>
		<li>Affix a sterile 18-G needle to the gas output from the system and insert the gas output needle into the bottle as well.</li>
		<li>Route the T-junctions from the tanks through the oxygen monitor. Verify that the target oxygen concentration is flowing through the system. Target approximately 5 L/min of gas flow.</li>
		<li>Reroute the T-junctions from the tanks through to the gas output. The gas starts to flow through the serum bottle. 
		CAUTION: Pay close attention to the pressure gauge during oxygen sparging. If pressure increases unexpectedly, shut the system off immediately.</li>
		<li>Run oxygen sparge through the serum bottle for 1 min. At 5 L/min, this allows for 10 air exchanges and ensures the internal atmosphere reaches the desired partial pressure.</li>
		<li>Remove the gas release 18-G needle.</li>
		<li>Allow the pressure in the serum bottle to build to +1 atmosphere (2 atmospheres at sea level) and then immediately remove the gas output needle. 
		NOTE: Maintaining pressure aids retention of hyperoxic conditions over time.</li>
		<li>Place the serum bottle into a 37 &#176;C incubator shaker at 150 RPM. Incubate the samples for three 24-h intervals. At each 24-h interval, take an aliquot for downstream analysis, re-sparge the samples and return them to incubation for a total incubation time of 72 h.</li>
	</ol>
	</li>
	<li>Outflow oxygen measurement
	<ol>
		<li>Calibrate the oxygen meter to 21% compressed air, and then turn off the tank.</li>
		<li>Route the serum bottle intake through the oxygen monitor and affix a sterile needle to the end.</li>
		<li>Insert the needle through the rubber stopper into the serum bottle.</li>
		<li>Wait for the outflow reading to stabilize. A low flow rate out of the serum bottles means this may take up to 2 min. Report the peak difference from room air (number furthest from 21%).</li>
		<li>If performing multiple readings, flush the system with compressed air between readings.</li>
	</ol>
	</li>
</ol>

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The authors have no conflicts of interest to disclose.

In this study, an in vitro model was developed to study the effect of hyperoxia on lung microbial communities. This model, based on artificial sputum medium and daily sparging of serum bottles, maintains elevated oxygen concentrations and supports the growth of microbes identified in sputum from pwCF.
There are several critical steps of this approach. First is the choice to use filter-sterilization rather than heat-sterilization of the artificial sputum medium. Filter-sterilization pr...

Cystic fibrosis (CF) is a genetic disease characterized by an inability to clear thick mucus from the lungs, leading to repeated infections and progressive lung function decline that often results in the need for lung transplantation or death. The airway microbiome of people with cystic fibrosis (pwCF) appears to track disease activity1, with a reduction in microbial diversity associated with adverse long-term outcomes2,3. In clinical studies of pwCF, supplemental oxygen therapy has been associated with more advanced disease4,5, though traditionally, the use of oxygen therapy has been viewed as simply a marker for disease severity6. Recent studies from a clinical trial of patients with respiratory failure have shown that higher patient oxygen levels are paradoxically associated with an increase in serious bacterial infections and higher mortality7, suggesting that supplemental oxygen may contribute to disease pathogenesis. The effect of supplemental oxygen on the cystic fibrosis lung microbiome and associated lung and airway microbial communities has not been well studied.
Mechanistic studies often cannot be performed directly on human subjects due to logistical difficulties and potential ethical issues associated with interventions of unknown medical benefit or harm. Translational approaches that integrate human biospecimens into model systems can offer important biological insights in these cases. While the ability to use or tolerate oxygen has traditionally been an important component of microbial classification, little is known about how the therapeutic introduction of supplemental oxygen to the environment might perturb airway microbial communities. To shed light on the unknown effects of supplemental oxygen on the airway microbiomes of pwCF, we needed to address two major challenges; first, the creation of a culture medium that physiologically approximates the composition of CF sputum; second, the creation of a model system that allows the maintenance of elevated oxygen concentrations in culture over extended periods of time.
Artificial sputum media (ASM) are widely used to emulate lung sputum ex vivo8,9,10, but there is no clear consensus on a specific recipe. This protocol describes an artificial sputum medium recipe and preparation strategy carefully designed to physiologically approximate sputum from pwCF. Table 1 outlines the chosen recipe values based on published literature. Basic chemical components and pH were matched to values identified by studies of human CF sputum11,12,13. Low concentration physiological nutrients were added using egg yolk, which was included as 0.25% of the final volume10, as well as vitamin and trace metal mixes14,15. Mucin, the key component of sputum16, was included at 1% w/v14. Although more labor-intensive, filter sterilization was chosen over the more conventional practice of heat sterilization to reduce potential problems from heat-induced denaturation of essential media components10. An additional benefit of filter sterilization is that it generates media that are transparent (heat-sterilization can create turbid media due to precipitation and coagulation of salts and proteins), allowing this artificial sputum media to be used to follow microbial growth based on increases in turbidity.
This model system for the hyperoxic culture is based on anaerobic culturing techniques where oxygen is added rather than removed, creating a model for the effect of supplemental oxygen use for pwCF. Figure 1 and the associated oxygen sparging protocol outlines the components of an oxygen sparging system, which can be obtained at low cost from general laboratory and hospital suppliers. This system enables the mixing of compressed oxygen and air to fixed concentrations ranging from 21%-100% oxygen. The integration of an oxygen sensor allows for the verification of the concentration of the output gas mixture, as well as checking the outflow gas composition of previously sparged serum bottles to verify that the oxygen conditions have been maintained within the desired range.
This protocol outlines procedures to create an artificial sputum medium, the construction and use of an oxygen sparging system, and the application of both to culture CF sputum under differential oxygen conditions.

<table><tbody><tr><td>BME Vitamins (100x) Solution</td><td>MilliporeSigma</td><td>B6891</td><td>Concentrated solution of supplemental vitamins.</td></tr><tr><td>Crimper, 30 mm</td><td>DWK Life Sciences</td><td>224307</td><td>Crimper for attaching aluminum seals to serum bottles.</td></tr><tr><td>D-(+)-Glucose</td><td>MilliporeSigma</td><td>G7021</td><td>Solid glucose powder (dextrorotatory isomer).</td></tr><tr><td>Diaphragm Pump ME 2 NT</td><td>VACUUBRAND</td><td>20730003</td><td>Vacuum pump for vacuum filtration.</td></tr><tr><td>Egg Yolk Emulsion</td><td>HiMedia</td><td>FD045</td><td>Sterile emulsion of 30% egg yolk in saline.</td></tr><tr><td>Ferritin, Cationized from Horse Spleen</td><td>MilliporeSigma</td><td>F7879</td><td>Ferritin (iron-storage protein) solution.</td></tr><tr><td>FIREBOY plus Safety Bunsen Burner</td><td>Integra Biosciences</td><td>144000</td><td>Bunsen burner with user interface and safety features.</td></tr><tr><td>Hydrion pH Paper (1.0&amp;ndash;14.0)</td><td>Micro Essential Laboratory</td><td>94</td><td>pH testing paper for the range of 1.0&amp;ndash;14.0.</td></tr><tr><td>Hydrion pH Paper (4.0&amp;ndash;9.0)</td><td>Micro Essential Laboratory</td><td>55</td><td>pH testing paper for the range of 4.0&amp;ndash;9.0.</td></tr><tr><td>Hydrion pH Paper (6.0&amp;ndash;8.0)</td><td>Micro Essential Laboratory</td><td>345</td><td>pH testing paper for the range of 6.0&amp;ndash;8.0.</td></tr><tr><td>Hypodermic Needle-Pro EDGE Safety Device, 18 G</td><td>Smiths Medical</td><td>401815</td><td>18 G needles with safety caps.</td></tr><tr><td>In-Line Pressure Gauge</td><td>MilliporeSigma</td><td>20469</td><td>Gas pressure gauge for monitoring bottle pressure.</td></tr><tr><td>Innova 42 Incubated Shaker</td><td>Eppendorf</td><td>2231000756</td><td>Combination incubator/orbital shaker.</td></tr><tr><td>Luer-Lok Syringe with Attached Needle</td><td>Becton Dickinson</td><td>309580</td><td>Combination 3 mL syringe and 18 G needle.</td></tr><tr><td>Luer Valve Assortment</td><td>World Precision Instruments</td><td>14011</td><td>Valves for gas flow tubing.</td></tr><tr><td>LSE Orbital Shaker</td><td>ThermoFisher Scientific</td><td>6780-NP</td><td>Orbital shaker to agitate media during filtration.</td></tr><tr><td>Magnesium Sulfate Heptahydrate</td><td>MilliporeSigma</td><td>M2773</td><td>Solid epsom salt (magnesium sulfate heptahydrate).</td></tr><tr><td>Medical Air Single Stage Regulator with Flowmeter</td><td>Western Enterprises</td><td>M1-346-15FM</td><td>Air flow rate regulator with 15 L/min meter.</td></tr><tr><td>MEM Amino Acids (50x) Solution</td><td>MilliporeSigma</td><td>M5550</td><td>Concentrated solution of essential amino acids.</td></tr><tr><td>MEM Non-Essential Amino Acids (100x) Solution</td><td>MilliporeSigma</td><td>M7145</td><td>Concentrated solution of non-essential amino acids.</td></tr><tr><td>Millex-GP Filter, 0.22 &amp;micro;m</td><td>MilliporeSigma</td><td>SLMP25SS</td><td>0.22 &amp;micro;m polyethersulfone membrane sterile syringe filter.</td></tr><tr><td>Milli-Q Academic</td><td>MilliporeSigma</td><td>ZMQS60E01</td><td>Milli-Q sterile water filtration system.</td></tr><tr><td>MiniOX 3000 Oxygen Monitor</td><td>MSA</td><td>814365</td><td>Gas flow oxygen percentage monitor.</td></tr><tr><td>MOPS Buffer (1 M, pH 9.0)</td><td>Boston BioProducts</td><td>BBM-90</td><td>MOPS buffer for adjusting media pH.</td></tr><tr><td>Mucin from Porcine Stomach</td><td>MilliporeSigma</td><td>M2378</td><td>Mucin (glycosylated gel-forming protein) powder.</td></tr><tr><td>Natural Polypropylene Barbed Fitting Kit</td><td>Harvard Apparatus</td><td>72-1413</td><td>Connectors for gas flow tubing.</td></tr><tr><td>Nextera XT DNA Library Preparation Kit</td><td>Illumina</td><td>FC-131-1096</td><td>Library preparation for identification during sequencing.</td></tr><tr><td>NovaSeq 6000 Sequencing System</td><td>Illumina</td><td>770-2016-025-N</td><td>Shotgun sequencing platform for generating sample reads.</td></tr><tr><td>Oxygen Single Stage Regulator with Flowmeter</td><td>Western Enterprises</td><td>M1-540-15FM</td><td>Oxygen flow rate regulator with 15 L/min meter.</td></tr><tr><td>Oxygen Tubing with 2 Standard Connectors</td><td>SunMed</td><td>2001-01</td><td>Tubing for connecting gas system components.</td></tr><tr><td>Phosphate buffered saline, 10x, pH 7.4</td><td>Molecular Biologicals International</td><td>MRGF-6235</td><td>Concentrated phosphate-buffered saline solution.</td></tr><tr><td>PC 420 Hot Plate/Stirrer</td><td>Marshall Scientific</td><td>CO-PC420</td><td>Combination hot plate/stirrer.</td></tr><tr><td>Potassium Chloride</td><td>MilliporeSigma</td><td>P9541</td><td>Solid potassium chloride salt.</td></tr><tr><td>PTFE Disposable Stir Bars</td><td>ThermoFisher Scientific</td><td>14-513-95</td><td>Disposable magnetic stir bars.</td></tr><tr><td>PTFE Thread Seal Teflon Tape</td><td>VWR</td><td>470042-938</td><td>Teflon tape for reinforcing gas system connections.</td></tr><tr><td>Q-Gard 2 Purification Cartridge</td><td>MilliporeSigma</td><td>QGARD00D2</td><td>Purification cartridge for Milli-Q system.</td></tr><tr><td>Reusable Media Storage Bottles</td><td>ThermoFisher Scientific</td><td>06-423A</td><td>Bottles for mixing and storing culture media.</td></tr><tr><td>Rubber Stopper, 30 mm, Gray Bromobutyl</td><td>DWK Life Sciences</td><td>224100-331</td><td>Rubber stoppers for serum bottles.</td></tr><tr><td>Serum Bottle with Molded Graduations, 500 mL</td><td>DWK Life Sciences</td><td>223952</td><td>Glass serum bottles for sealed culturing.</td></tr><tr><td>Small Bore Extension Set</td><td>Braun Medical</td><td>471960</td><td>Tubing extension with luer lock connectors.</td></tr><tr><td>Sodium Chloride</td><td>MilliporeSigma</td><td>S3014</td><td>Solid sodium chloride salt.</td></tr><tr><td>Spike-in Control I (High Microbial Load)</td><td>ZymoBIOMICS</td><td>D6320</td><td>Spike-in microbes (I. halotolerans and A. halotolerans) for absolute microbial load calculations</td></tr><tr><td>Stericup Quick Release Sterile Vacuum Filtration System</td><td>MilliporeSigma</td><td>S2GPU02RE</td><td>250 mL 0.22 &amp;micro;m vacuum filtration chamber.</td></tr><tr><td>Super Sani-Cloth Germicidal Disposable Wipes</td><td>Professional Disposables International</td><td>H04082</td><td>Disposable germicidal wipes for sterilization.</td></tr><tr><td>Trace Metals Mixture, 1000x</td><td>ThermoFisher Scientific</td><td>NC0112668</td><td>Concentrated solution of physiological trace metals.</td></tr><tr><td>Unlined Aluminum Seal, 30 mm</td><td>DWK Life Sciences</td><td>224187-01</td><td>Aluminum seals crimped over top of rubber stoppers.</td></tr><tr><td>USP Medical Grade Air Tank</td><td>Airgas</td><td>AI USP200</td><td>Compressed air tank for input to sparging system.</td></tr><tr><td>USP Medical Grade Oxygen Tank</td><td>Airgas</td><td>OX USP200</td><td>Compressed oxygen tank for input to sparging system.</td></tr></tbody></table>

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.

These protocols were applied to 50 expectorated sputum samples from pwCF presenting for routine care to an outpatient cystic fibrosis clinic at Massachusetts General Hospital in Boston, Massachusetts. Each patient&#39;s sputum was cultured under 21%, 50%, and 100% oxygen conditions using the artificial sputum medium, with 0.5 mL aliquots taken from each culture at 24 h, 48 h, and 72 h of culture time for testing. Cultures were photographed when extractions were made to track visual changes. In addition, a 0.5 mL aliquot ...

Watch this Scientific Journal Video about Design and Development of a Model to Study the Effect of Supplemental Oxygen on the Cystic Fibrosis Airway Microbiome at JoVE.com

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

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. ...

design-development-model-to-study-effect-supplemental-oxygen-on

Research

JoVE Journal

Medicine

2.3K Views.  Massachusetts General Hospital. Deze protocollen zijn ontworpen om de in vitro effecten van aanvullend zuurstofgebruik op het longmicrobioom van cystische fibrosepatiënten te modelleren. Dit mediarecept is afgestemd op de fysiologische samenstelling van sputum voor mensen met cystische fibrose. Het spargingproces introduceert het vermogen om onder verschillende zuurstofomstandigheden te kweken.Veranderingen aan kunstmatig sputummedium maken het mogelijk om andere chronische longziekten na te bootsen en microbiële g...

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

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 ...

Immunology and Infection

Capturing Actively Produced Microbial Volatile Organic Compounds from Human-Associated Samples with Vacuum-Assisted Sorbent Extraction

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the host&#39;s microbial community. To disentangle the origin of microbial VOCs, volatile headspace analysis of bacterial mono- and co-cultures of Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, and stable isotope probing in biological samples of feces, saliva, sewage, and sputum were performed. Mono- and co-cultures were used to identify volatile production from individual bacterial species or in combination with stable isotope probing to identify the active metabolism of microbes from the biological samples.
Vacuum-assisted sorbent extraction (VASE) was employed to extract the VOCs. VASE is an easy-to-use, commercialized, solvent-free headspace extraction method for semi-volatile and volatile compounds. The lack of solvents and the near-vacuum conditions used during extraction make developing a method relatively easy and fast when compared to other extraction options such as tert-butylation and solid phase microextraction. The workflow described here was used to identify specific volatile signatures from mono- and co-cultures. Furthermore, analysis of the stable isotope probing of human associated biological samples identified VOCs that were either commonly or uniquely produced. This paper presents the general workflow and experimental considerations of VASE in conjunction with stable isotope probing of live microbial cultures.

This protocol describes the extraction of volatile organic compounds from a biological sample with the vacuum-assisted sorbent extraction method, gas chromatography coupled with mass spectrometry using the Entech Sample Preparation Rail, and data analysis. It also describes culture of biological samples and stable isotope probing.

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the ...

Biochemistry

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

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 ...

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 ...

Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

Using the cystic fibrosis airway as an example, the manuscript presents a comprehensive workflow comprising a combination of metagenomic and metatranscriptomic approaches to characterize the microbial and viral communities in animal-associated samples.

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and ...

Biology

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 ...

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 ...

Anti-Infective Efficacy in Cystic Fibrosis with Aggregate Biofilm Models

Cystic Fibrosis Aggregate Biofilm Model to Study Infection-relevant Gene Expression

Standard pre-clinical testing methods for novel antimicrobial therapeutics used to treat chronic lung infections in people with cystic fibrosis do not reflect the environmental conditions of the hostile lung niche. Current reductionist testing conditions can lead to the progression of compounds along a preclinical pipeline without evidence of their activity under cystic fibrosis lung niche-appropriate conditions. Several approaches used to study traditional antimicrobials may not be suitable for antibiotic alternatives, including anti-virulence therapeutics like anti-quorum sensing agents and siderophore inhibitors. This protocol documents an aggregate biofilm model of Pseudomonas aeruginosa to compare resistance and infection-relevant gene expression in single-species and multi-species cultures (Staphylococcus aureus and Candida albicans), examining colony-forming unit (CFU) reductions and changes in gene expression, using algD as an exemplar. The model was optimized for small, static volumes of bacterial cultures to allow the study of novel compounds in the discovery phase of the drug development pipeline, where compound quantities may be limited. Single-species P. aeruginosa biofilms were formed in Synthetic Cystic Fibrosis Medium 2 (SCFM2) for 24 h before treatment with meropenem at different concentrations (1, 16, and 256 &#181;g/mL) for a further 24 h. Polymicrobial biofilms were established by growing Staphylococcus aureus and Candida albicans together in SCFM2, then inoculating with P. aeruginosa for an additional 24 h and treating with meropenem. The lack of a direct connection between compound efficacy measures in pre-clinical testing and clinical trial results has cast doubt on the applicability of current laboratory screening tools. This model allows us to understand the impact of relevant factors on P. aeruginosa gene expression, including genes contributing to resistance and virulence, thereby bridging this gap.

This study outlines a framework for developing an aggregate polymicrobial biofilm model and optimizing an RNA extraction protocol to assess Pseudomonas aeruginosa gene expression reflective of the cystic fibrosis lung environment. Applications include evaluating antimicrobial effects and studying antibiotic alternatives under conditions relevant to cystic fibrosis.

Standard pre-clinical testing methods for novel antimicrobial therapeutics used to treat chronic lung infections in people with cystic fibrosis do not ...

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

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