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

Summary

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.

Abstract

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.

Introduction

The method described in this manuscript is called the WinCF system¹. The overall goal of WinCF is to provide an experimental setup capable of simulating the environment of a mucus-filled lung bronchiole. This will allow for a tractable system to study microbial pathogens of lung diseases with a mucus hypersecretion phenotype including cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), asthma and others. The procedure was designed specifically for the study of CF, which is characterized by mutations that cause lung secretions to become thick and hard to clear, eventually filling bronchioles and other small passageways with mucus....

Protocol

1. Preparation of Stocks for the Artificial Sputum Media

1. Create a 5% mucin solution. Add 1.0 g of dehydrated pig stomach mucin to 20 mL of deionized water. Autoclave the resulting solution.
   NOTE: The mucin sterilization will destroy its inherent structure; other methods to sterilize the mucin in its dry form include UV sterilization and irradiation. These methods have not been extensively used for the WinCF system however.
2. Add 2.2 g of KCl to 50 mL of deionized water and allow for dissolution. Add 5.0 g of NaCl to 50 mL of deionized water and allow for dissolution. Autoclave these two solutions.
3. Add 100 mg of salmon sperm DNA to 10....

Results

Microbiological growth across the various chemical conditions induced within the samples varied dramatically in some cases and more subtly in others. Many changes in activity were visual in nature, being readily apparent as soon as the incubation period ended. In the example of pH manipulation, the samples across the pH spectrum varied greatly as shown by multiple factors that became apparent after incubation. When no sputum samples were added to the media, the only change exhibited acros.......

Discussion

The microbiological makeup of a lung with CF contains a great variety of organisms, but the conditions within the lung likely have a significant influence on what kinds of microbes can survive and thrive^13,15. Specific mechanisms through which these conditions change and the exact effects they have on the lung microbiome are generally unclear at present. In this experimental method, we present an analysis of microbiological changes based upon manipulated chemical.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Color-Coded Capillary Tubes	Fisher Scientific	22-260943
Cha-seal Tube Sealing Compound	Kimble-Chase	43510
Mucin from porcine stomach	Sigma	M1778
Ferritin, cationized from horse spleen	Sigma	F7879
Salmon sperm DNA Sodium salt (sonified)	AppliChem Panreac	A2159
MEM Nonessential Amino Acids	Corning cellgro	25-025-CI
MEM Amino Acids	Cellgro	25-030-CI
Egg Yolk Emulsion, 50%	Dalynn Biologicals	VE30-100
Potassium Chloride	Fisher Scientific	P2157500
Sodium Chloride	Fisher Scientific	S271500
15 mL centriguge tubes with Printed Graduations and Flat Caps	VWR	89039-666
50 mL centrifuge tubes with Printed Graduations and Flat Caps	VWR	89039-656
1.5 mL microcentrifuge tubes	Corning	MCT-150-R
2.0 mL microcentrifuge tubes	Corning	MCT-200-C