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 ....

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 solution.......

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.