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

Summary

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.

Abstract

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.

Introduction

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 outcomes^2,3. In clinical studies of pwCF, supplemental oxygen therapy has been associated with more advanced disease^4,5, 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 vivo^8,9,10, 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 sputum^11,12,13. 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 mixes^14,15. 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.

Protocol

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.

1. 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.
   1. Mix the constituent chemical stock solutions.
      1. Prepare 1 M NaCl stock: Add 58.44 g of NaCl per liter of sterile water.
      2. Prepare 1 M KCl stock: Add 74.55 g of KCl per liter of sterile water.
      3. Prepare 1 M MgSO₄ stock: Add 246.47 g of MgSO₄·7H₂O per liter of sterile water, or 120.37 g of anhydrous MgSO4 per liter of sterile water.
      4. Prepare 1 M glucose stock: Add 180.16 g of glucose per liter of sterile water.
   2. 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 °C and 15 PSI for 30 min.
   3. Add 80.59 mL of sterile water to the empty 250 mL bottle.
   4. Add 152.30 mL of 1 M NaCl stock to the mix.
   5. Add 15.8 mL of 1 M KCl stock to the mix.
   6. Add 610 µL of 1 M MgSO₄ stock to the mix.
   7. Add 700 µL of 1 M glucose stock to the mix.
2. 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.
   1. Add 450 mL of sterile water to an empty 1 L bottle.
   2. Add 50 mL of 10x Phosphate-Buffered Saline (PBS) to the bottle.
   3. Add a disposable magnetic stir bar to the bottle.
   4. Autoclave the bottle containing PBS and the stir bar.
   5. Measure out 10 g of mucin powder and add it to the PBS.
   6. Shake the bottle vigorously for preliminary mixing.
   7. Place the bottle onto a hot plate with a magnetic stirrer. Set heat to medium-high targeting 50 °C and stirring speed to 1100 rpm. Ramp up the speed gradually so that the bar does not fly off the magnet.
      1. Allow to heat and stir for 15 min.
      2. Pick up the bottle with heat-resistant gloves. Observe to check if mucin powder settles out of the solution.
      3. If mucin powder is not fully dissolved, return the bottle to heat/stirrer for 5-min intervals until it is completely dissolved.
   8. Allow the mucin mix to cool to room temperature.
3. Artificial sputum biological mix (ASBM)
   ​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.
   1. Thaw the 100x vitamin stock in a 4 °C fridge or on ice.
      ​NOTE: Pre-portion the vitamin stock into 10 mL aliquots to minimize the number of freeze/thaw cycles.
   2. Add 124.24 mL of sterile water to the empty autoclaved 250 mL bottle.
   3. Add 25.76 mL of 50x essential amino acid stock to the mix.
   4. Add 80.14 mL of 100x non-essential amino acid stock to the mix.
   5. Add 10 mL of (thawed) 100x vitamin stock to the mix.
   6. Add 1 mL of 1000x trace metals stock to the mix.
   7. Add 8.33 mL of 30% egg yolk emulsion to the mix.
   8. Add 400 µL of 10 g/L ferritin stock to the mix.
   9. Mix the solution well via manual shaking.
4. Artificial sputum medium (ASM)
   1. Add 250 mL of ASCM to the 1 L bottle containing ASMM.
   2. Add 250 mL of ASBM to the medium bottle.
   3. 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.
   4. Refrigerate the resulting artificial sputum medium at 4 °C until it is ready for filtration.
   5. To start the filtration process, transfer 200 mL of unfiltered artificial sputum medium to a vacuum filtration system with a 0.22 µm pore size filter.
   6. 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 °C.
      1. 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.
      2. 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 µm filter since mucin will plug the filter over time.
   7. Refrigerate filtered artificial sputum medium at 4 °C until ready for use. Use ASM within one month of preparation for best results.

2. Oxygen Sparging

1. 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.
   1. 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.
   2. 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.
   3. 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.
   4. Connect the tubing from the regulators to a Y-connector to combine the gas flow from the two tanks.
   5. Connect the output of the Y-connector to the central T-junction valve.
   6. Connect one side of the central T-junction valve to a gas pressure gauge.
   7. Connect the other side of the gas pressure gauge to a 25 mm diameter sterile syringe filter with a 0.22 µm pore size.
   8. Attach a second 25 mm diameter syringe filter to a syringe without a plunger to be used as a gas release during sparging.
   9. Connect the final side of the central T-junction valve to a second T-junction valve for the oxygen monitor.
   10. Connect a 25 mm diameter syringe filter to one side of this second T-junction valve, along with tubing to attach 18 G needles.
   11. Connect the final side of the second T-junction valve to the oxygen monitoring apparatus.
   12. 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 .
   13. For the maintenance of the system and to keep it working at optimal performance, the following practices are beneficial.
       1. 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.
       2. Keep combined flow rate under 10 L/min to mitigate maximum pressure and prevent failures.
       3. 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).
       4. 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.
       5. Calibrate the oxygen monitor to 21% oxygen compressed air prior to carrying out measurements.
       6. Upon completion of the system use, turn off the tanks and bleed the excess gas from the regulators until the flow completely stops.
2. Serum bottle culture sparging
   1. Label 500 mL autoclaved serum bottles with sample identifiers, date/time of inoculation, and target oxygen percentage.
   2. In a biological hood, add 24 mL of the artificial sputum medium to each serum bottle being set up.
   3. 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.
   4. Using sterile tweezers, place the autoclaved rubber stoppers onto the top of each serum bottle.
   5. Press down the rubber stoppers, take care not to touch the underside of the stopper with hands.
   6. Remove the bottles from the hood, apply and crimp the aluminum seals. Remove the center piece from the seals.
   7. Wipe down the top of bottles with an alcohol wipe and pass them through a Bunsen burner flame.
   8. Affix a sterile 18-G needle to a plunger-less syringe with a filter. Insert this gas release into the bottle first.
   9. Affix a sterile 18-G needle to the gas output from the system and insert the gas output needle into the bottle as well.
   10. 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.
   11. 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.
   12. 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.
   13. Remove the gas release 18-G needle.
   14. 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.
   15. Place the serum bottle into a 37 °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.
3. Outflow oxygen measurement
   1. Calibrate the oxygen meter to 21% compressed air, and then turn off the tank.
   2. Route the serum bottle intake through the oxygen monitor and affix a sterile needle to the end.
   3. Insert the needle through the rubber stopper into the serum bottle.
   4. 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%).
   5. If performing multiple readings, flush the system with compressed air between readings.

Results

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'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 of e...

Discussion

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...

Materials

List of materials used in this article

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