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

Summary

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.

Abstract

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 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.

Introduction

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 "one bug, one infection" 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 individuals^3,4,5,6,7.

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 tobramycin^6,10,11. 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 airway^3,13. 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 lung^12,14.

Few studies have developed CF-relevant bacterial multispecies in vitro systems containing more than two pathogens^15,16. 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 airway^15,16. 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.

Protocol

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 ASM^10,18. The 2x ASMb can be prepared in advance and stored at 4 °C for up to two weeks in a dark place. If the medium turns yellow, discard.

1. To prepare 2x ASM base stock, in a clean beaker containing 400 mL of diH₂O, add the following while stirring:
   1. Add 6.50 mL of a 0.2 M NaH₂PO₄.H₂O stock; 6.25 mL of a 0.2 M Na₂HPO₄ stock; 348 µL of a 1 M KNO₃ stock; 1.084 mL of a 0.25 M K₂SO₄ stock.
   2. 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.
   3. 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.
   4. Add 660 µL of a 0.1 M Tryptophan stock; 606 µL of a 1 M MgCl₂.6H₂O stock; 0.60 g of deoxyribonucleic acid from herring sperm; 400 µL of a 250 mg/mL 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) stock.
   5. 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 µm filter, and store at 4 °C.
2. To prepare a 10 mg/mL (2x) mucin stock, use a clean glass bottle containing 250 mL of diH₂O and while stirring:
   1. Add 5.0 g of mucin from porcine stomach - Type II. Mix the suspension for 10 min. Complete to 500 mL with diH₂O.
   2. Sterilize by autoclaving at 121 °C for 15 min. Keep at 4 °C until utilization.
3. Reconstitute the ASM.
   NOTE: This step needs to be performed on the day of the experiment.
   1. Mix a 1:1 volume ratio of the 2x ASMb stock and the 10 mg/mL mucin stock.
   2. Vortex thoroughly before use.

2. Preparing polymicrobial community selective media

NOTE: Below are the quantities necessary to prepare a volume of 1 L of media.

1. To prepare Mannitol Salt Agar (MSA) and Pseudomonas Isolation Agar (PIA), follow the manufacturer's instructions.
2. To prepare Prevotella Selective Agar (PSA), in a clean glass bottle, add the following:
   1. 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.
   2. 10.0 mL of a 0.5 mg/mL hemin stock. 100 µL of a 10 mg/mL menadione stock.
   3. Add 940 mL of diH₂O and stir. Sterilize by autoclaving at 121 °C for 15 min. 
   4. When cooled down (i.e., the bottle can be held barehanded), add while stirring: 50.0 mL of defribrinated sheep's blood. 2.00 mL of a 50 mg/mL kanamycin stock. 150 µL of a 50 mg/mL vancomycin stock. 500 µL of a 10 mg/mL polymixin B stock.
3. To prepare Streptococcus Selective Agar (SSA), in a clean glass bottle, add the following:
   1. 30.0 g of TSB. 15.0 g of agar.
   2. Add 950 mL of diH₂O and stir. Sterilize by autoclaving at 121 °C for 15 min. 
   3. When cooled down (i.e., the bottle can be held barehanded), add while stirring: 50.0 mL of defribrinated sheep'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 °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.

3. Bacterial liquid media preparation and growth conditions

1. To routinely grow P. aeruginosa and S. aureus, use a sterile rich medium (e.g., lysogeny broth (LB) or TSB).
   1. Start liquid overnights by picking a single colony previously grown on an LB/TSB agar plate for 20-24 h at 37 °C.
   2. Grow the broth cultures at 37 °C overnight with shaking at 250 rpm for 16-18 h.
2. To grow Streptococcus spp., use sterile Todd Hewitt Broth supplemented with 0.5 % yeast extract (THB-YE).
   1. Start liquid culture overnight from a single colony previously grown on a TSB agar plate supplemented with 5% defibrinated sheep's blood (blood agar plate) for 24 h at 37 °C + 5% CO₂.
   2. Grow the THB-YE cultures anaerobically or at 37 °C + 5% CO₂ overnight without shaking for 18-22 h.
3. To grow Prevotella spp., prepare 1 L of Prevotella growth medium (PGM) by adding the following to a clean bottle:
   1. 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 µL of a 10 mg/mL menadione stock.
   2. Add 990 mL of diH₂O. Sterilize by autoclaving at 121 °C for 15 min. Wrap the bottle in aluminum foil to protect it from light.
   3. Grow Prevotella spp. colonies on a blood agar plate incubated anaerobically at 37 °C for 48 h.
   4. Inoculate a single colony in PGM and grow overnight without shaking at 37 °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.

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.

1. 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.
   1. Using the bacterial liquid from the overnight cultures, perform the following steps:
      1. For P. aeruginosa and S. aureus strains, wash twice using sterile phosphate-buffered saline (PBS).
      2. 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.
      3. After the washes, resuspend each pellet in 1 mL of 1x water-diluted ASMb.
      4. Make a 1:10 dilution of each sample in sterile PBS and measure the OD₆₀₀.
      5. Adjust all the cultures to an OD₆₀₀ of 0.2 in ASM.
      6. 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.
      7. 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 µL of P. aeruginosa at OD₆₀₀ = 0.2 to a volume of 950 µL of ASM. For the co-culture, take 50 µL of each P. aeruginosa, S. aureus, Streptococcus spp. and Prevotella spp. suspensions at an OD₆₀₀ = 0.2 and add to 800 µL ASM.
      8. Using a pipette, add 100 µl of the monoculture and co-culture suspensions in three separate wells of a sterile plastic flat bottom 96-well plate.
      9. Incubate the plate (without shaking) for 24 h at 37 °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 × 10⁷ CFU/mL (P. aeruginosa), 3.5 × 10⁶ CFU/mL (S. aureus), 1.2 × 10⁶ CFU/mL (Streptococcus spp.), and 4.6 × 10⁶ CFU/mL (Prevotella spp.). Bacterial inocula can also be modified, as described by Jean-Pierre and colleagues¹⁰.
2. Replace the media after biofilm formation.
   1. After 24 h of incubation, remove the unattached (planktonic) cells by aspiration using a multichannel pipette.
   2. Replenish the pre-formed biofilms with 100 µL of fresh ASM or the desired treatment (e.g., antibiotics, metabolites, etc.).
   3. Incubate the plate for an additional 24 h at 37 °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.

5. Collecting and plating the samples

1. 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.
2. Gently wash the biofilms twice using 125 µL of sterile PBS and discard the volumes.
3. Add 50 µL of sterile PBS and detach the biofilms by gently scraping the cells from the plate using a 96-pin replicator.
4. 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.
5. Perform a 10x serial dilution of the planktonic (if needed) and biofilm fractions.
6. Plate 3-5 µL of each dilution sample onto PIA, MSA, SSA, and PSA.
7. Allow the inoculation spots to dry.

6. Incubating the selective plates and colony forming unit (CFU) counting

1. For P. aeruginosa and S. aureus, incubate at 37 °C for 18-20 h.
2. For Streptococcus spp, incubate at 37 °C in anoxic conditions for 24 h.
3. For Prevotella spp, incubate at 37 °C in anoxic conditions for 48 h.
4. After incubation, count the colonies (~10-35 per spot) and calculate the CFU per mL.
5. Plot the data using a visualization tool.

Results

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...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)	Fisher Scientific	NC0387928	Prepare a 250 mg/mL stock in chloroform directly in the bottle. Keep at -20°C. Final concentration 200 μg/mL in 2x ASM base.
3-morpholinopropane-1-sulfonic acid (MOPS)	Sigma	M1254	Final concentration 200 mM in 2X ASM base.
96-pin replicator	Fisher Scientific	05-450-9	Disposable replicators can also be used. Cat #NC1567338.
96-well sterile standard plate with lid	Fisher Scientific	62406-081
Agar	Fisher Scientific	DF0145-17-0
AnaeroPack 2.5 L Rectangular Jar	Fisher Scientific	23-246-385
CaCl2*2H2O	Fisher Scientific	C79-500	Prepare a 1 M stock in water and filter sterilize with a 0.22 μm filter. Final concentration 3.5 mM in 2x ASM base.
Defribrinated sheep's blood			To prepare growth plates for Streptococcus and Prevotella.
Deoxyribonucleic acid from herring sperm	Sigma	D7290	Final concentration 1.2 mg/mL in 2x ASM base.
FeSO4*7H2O	Fisher Scientific	AA1449830	Prepare a 1 mg/mL stock (3.6 mM) in water and filter sterilize with a 0.22 μm filter. Keep wrapped in aluminum foil. Final concentration 0.0072 mM in 2x ASM base.
GasPaks	Fisher Scientific	B260678
Glucose	Fisher Scientific	D16-3	Prepare as a 20% stock (1.1 M) in water and sterilize by autoclave. Final concentration 6 mM in 2x ASM base.
Hemin	Sigma	51280	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 °C.
K2SO4	Fisher Scientific	P304-500	Prepare a 0.25 M stock in water and filter sterilize with a 0.22 μm filter. Final concentration 0.54 mM in 2x ASM base.
Kanamycin			Prepare a 50 mg/mL stock solution in water. Filter sterilize with a 0.22 μm filter and store at 4 °C.
KCl	Fisher Scientific	P217-500	Final concentration 30.0 mM in 2x ASM base.
KNO3	Fisher Scientific	P263-500	Prepare a 1 M stock in water and filter sterilize with a 0.22 μm filter. Final concentration 0.70 mM in 2x ASM base.
L-cysteine hydrochloride	Fisher Scientific	AAA1038922
L-Lactic acid	Sigma	L1750	Prepare a 1 M stock in water and filter sterilize with a 0.22 μm filter. pH adjust to 7.0. Final concentration 18.6 mM in 2x ASM base.
L-Tryptophan	Sigma	T0254	Prepare a 0.1 M stock in 0.2 M NaOH and filter sterilize with a 0.22 μm filter. Keep refrigerated at 4 °C wrapped in aluminum foil. Final concentration 0.132 mM in 2x ASM base.
Mannitol Salt Agar	Fisher Scientific	B11407	Prepare using manufacturer's recommendations.
Menadione	Sigma	M5625	Prepare a 10 mg/mL stock solution dissolved in 96-100% ethanol. Conserve at 4 °C wrapped in aluminium foil. Vortex before use.
MgCl2*6H2O	Fisher Scientific	AA12288A9	Prepare a 1 M stock in water and filter sterilize with a 0.22 μm filter. Final concentration 1.21 mM in 2x ASM base.
Mucin from porcine stomach - Type II	Sigma	M2378	Prepare a 10 mg/mL (2x) stock in water; mix thoroughly before autoclaving. Keep at 4 °C.
Na2HPO4	Fisher Scientific	S375-500	Prepare a 0.2 M stock in water and filter sterilize with a 0.22 μm filter. Final concentration 2.5 mM in 2x ASM base.
N-acetylglucosamine	Sigma	A8625	Prepare a 0.25 M stock in water and filter sterilize with a 0.22 μm filter. Keep stock refrigerated at 4 °C. Final concentration 0.6 mM in 2x ASM base.
NaCl	Fisher Scientific	S271-500	Final concentration 103.7 mM in 2x ASM base.
NaH2PO4*H2O	Fisher Scientific	S369-500	Prepare a 0.2 M stock in water and filter sterilize with a 0.22 μm filter. Final concentration 2.6 mM in 2x ASM base.
NaOH	Fisher Scientific	S318-500	Prepare a 5 M stock solution. Use to adjust pH of 2x ASM base stock.
NH4Cl	Fisher Scientific	A661-500	Final concentration 4.6 mM in 2x ASM base.
Oxolinic acid			Prepare a 10 mg/mL stock solution 0.5 M NaOH. Filter sterilize with a 0.22 μm filter and store at 4 °C.
Polymixin B			Prepare a 10 mg/mL stock solution in water. Filter sterilize with a 0.22 μm filter and store at 4 °C.
Pseudomonas Isolation Agar	Fisher Scientific	DF0927-17-1	Prepare using manufacturer's recommendations.
Todd Hewitt Broth	Fisher Scientific	DF0492-17-6	Prepare using manufacturer's recommendations.
Tryptic Soy Broth	Fisher Scientific	DF0370-17-3	Prepare using manufacturer's recommendations.
Vancomycin			Prepare a 50 mg/mL stock solution in water. Filter sterilize with a 0.22 μm filter and store at 4 °C.
Yeast Extract	Fisher Scientific	B11929
Yeast Synthetic Dropout without Tryptophan	Sigma	Y1876	Final concentration 8.0 mg/mL in 2x ASM base.