Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

Summary

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

Abstract

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.

This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.

This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

Introduction

Cystic fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. This gene encodes for a chloride channel expressed on the membrane of most epithelial cells. Bronchiectasis, mucus plugging and parenchymal destruction caused mainly by Pseudomonas aeruginosa infections progressively lead to severe lung disease and mortality in most of the CF patients¹. Understanding CF pathogenesis and further development of novel therapies rely on animal model with characteristic features of CF. Several mice, genetically modified for the Cftr gene, have been generated, but limitat....

Protocol

1. Preparing Bacteria for Chronic Infection (Three and Two Days prior to Mouse Challenge)

1. Select the appropriate P. aeruginosa strain to be tested.
2. Inoculate a loopful of P. aeruginosa from a -80 °C stock culture to a Trypticase Soy Agar (TSA) plate and incubate at 37 °C overnight.
3. Pick a single colony and inoculate into 5 ml Trypticase Soy Broth (TSB) in a 15 ml snap-capped tube and incubate at 37 °C overnight in a shaking incubator at 200 rpm.

2. Embedding Bacteria in Agar Beads for Chronic Infection (One Day prior to Infection)

1. Take a small aliquot of bacterial overnigh....

Results

When the protocol is done correctly, the P. aeruginosa agar-beads will measure between 100-200 µm and can be observed with an inverted light microscope by pipetting a small volume of the agar-beads suspension on a slide. Single bacterial cells are visible in the agar beads, as shown in detail in Figure 1.

The choice of P. aeruginosa strain used in the agar-beads preparation is critical. Figure 2 and Table 1 show the data obta.......

Discussion

The critical steps in the P. aeruginosa-beads preparation and mouse challenge are reported below.

The P. aeruginosa strain used for mice challenge is critical. Mortality, chronic infection or clearance may differ significantly depending on the bacterial strain used for the challenge. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chron.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bacto Tryptic Soy Broth	Becton Dickinson	211823
Difco Agar, granulated	Becton Dickinson	214510
Heavy mineral oil	Sigma-Aldrich	330760-1L
S-(+)-Ketamine hydrochloride	Sigma-Aldrich	K1884
Xylazine hydrochloride	Sigma-Aldrich	X1251
1 ml Syringe 25 G 5/8 in 0.5 mm x 16 mm	PIC	3071250300350
Catheter 22 G 0.9 mm x 25 mm	Becton Dickinson	381223
Graefe Forceps - 0.5 mm Tips Curved	Fine Science Tools	11152-10
Scissors, Iris, 11 cm, straight	World Precision Instruments	501758
Suture clips	Fine Science Tools	12040-01
Suture thread	Fine Science Tools	18020-40
RPMI 1640	Lonza	BE12-167F
Complete protease inhibitor cocktail	Roche	11836145001
Fast-Read 102 Burker disposable chamber	Biosigma	390497
Tuerk solution	Fluka	93770
RBC lysis buffer	Biolegend	420301
Fetal bovine serum	Lonza	DE14-801F
EZ cytofunnel	Thermo Scientific	A78710021
Superfrost ultra plus microscope slides	Thermo Scientific	J3800AMNZ
Diff-Quik Romanowsky staining set	Medion Diagnostics	130832
Hexadecyltrimethylammonium chloride	Sigma-Aldrich	52366-10G
96-well EIA/RIA plate	Costar	3590
3,3’,5,5’- Tetramethylbenzidine	Sigma-Aldrich	T8665-1L
Sulfuric acid	Sigma-Aldrich	320501-1L
10% Neutral buffered formalin	Bio-Optica	05-01005Q
Harris hematoxylin non Papanicolau	Bio-Optica	05-M06004
Eosin plus alcoholic solution	Bio-Optica	05-M11007
Equipment
Shaking incubator	Amerex Instruments	Steady Shake 757
Water bath	Grant	SUB14
Homogenizer	Ystral
Precision balance	KERN	440-47N
Cytocentrifuge	Thermo Scientific	A78300003
Low Cost Heating Pad	2biol	LCHP
Homogenization probe	Ystral	2366925(small)
Inverted optical microscope	Zeiss	Axioplan2
Camera (microscope)	Zeiss	Axiocam MRc5
Rotary microtome	Leica	RM2255