A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

Summary

We describe a delayed inoculation protocol for generating chronic wound infections in immunocompetent mice.

Abstract

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of chronic wound infections in diabetic and hospitalized patients. There is an urgent need for chronic infection models to aid in the investigation of wound pathogenesis and the development of new therapies against this pathogen. Here, we describe a protocol that uses delayed inoculation 24 hours after full-thickness excisional wounding. The infection of the provisional wound matrix present at this time forestalls either rapid clearance or dissemination of infection and instead establishes chronic infection lasting 7–10 days without the need for implantation of foreign materials or immune suppression. This protocol mimics a typical temporal course of post-operative infection in humans. The use of a luminescent P. aeruginosa strain (PAO1:lux) allows for quantitative daily assessment of bacterial burden for P. aeruginosa wound infections. This novel model may be a useful tool in the investigation of bacterial pathogenesis and the development of new therapies for chronic P. aeruginosa wound infections.

Introduction

Pseudomonas aeruginosa (P. aeruginosa) is a Gram-negative rod-shaped bacterium with increasing relevance to human health and disease. It is responsible for extensive morbidity and mortality in nosocomial settings, particularly involving wound infections in immunocompromised patients^1,2. The emergence of multidrug-resistant strains of this pathogen has provided further impetus for investigation into factors contributing to P. aeruginosa virulence, mechanisms of P. aeruginosa antibiotic resistance, and new methods for prevention and treatment of this deadly infection

Protocol

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) at Stanford University.

1. Preparation and growth of bacteria

1. Conduct all work with P. aeruginosa and animals with BSL-2 precautions per the researcher's institutional biosafety committee and animal use committee guidelines. Do all steps described here involving P. aeruginosa, including mouse inoculation, in a biosafety cabinet.
2. The luminescent PAO1:lux strain of P. aeruginosa is available from our lab by request. Streak PAO1, stored as frozen glycerol stock, on Lysogeny Broth (LB) aga....

Results

Using a luminescent strain of PAO1 with a plasmid encoding the luxABCDE reporter system (PAO1:lux), we performed excisional wounding on mice, inoculated these wounds with planktonic P. aeruginosa 24 h later, and measured bacterial burden over time (Figure 1 and Figure 2). A representative image obtained using an imaging optical system demonstrates that this model results in detectable luminescence (F.......

Discussion

We have developed a novel delayed inoculation P. aeruginosa wound infection model. The strategy of delaying inoculation with bacteria until 24 h after excisional wounding enables the evaluation of wound infections over a 1-week timeframe. By using a luminescent strain of P. aeruginosa, it is possible to track infection progression throughout the infection course. The longer course of infection compared to other P. aeruginosa infection models will allow new opportunities for studying host-pathog.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.9% Sodium Chloride injection	Hospira	2484457
18 G x 1 sterile needle	BD	305195
25 G x 1 1/5 sterile needle	BD	305127
Alcohol swab	BD	326895
Aura Imaging Software	Spectral Instruments Imaging	n/a
Betadine	Purdue Frederick Company	19-065534
Buprenorphine SR LAB	Zoopharm	n/a
C57BL/6J male mice	The Jackson Laboratory	000664
Disposable biopsy punch, 6mm	Integra	33-36
Fine scissors - Tungsten Carbide	Fine Science Tools	14568-09
Glass Bead Dry Sterilizer	Harvard Apparatus	61-0183
Granulated Agar	Fisher BioReagents	BP9744
Heating Pad	Milliard	804879481218
Insulin syringe with 28 G needle	BD	329461
Lago X Imaging System	Spectral Instruments Imaging	n/a
LB broth	Fisher BioReagents	BP1426
Leur-Lok 1 mL syringe	BD	309628
Mini Arco Animal Trimmer	Wahl Professional	919152
Nair Hair Removal Lotion with Baby Oil	Church and Dwight	n/a	Available at any pharmacy
Octagon Forceps	Fine Science Tools	11041-08
Petri dish	Falcon	351029
Phosphate Buffered Saline (PBS) 1x	Corning	21-040-CV
Press and Seal Cling Wrap	Glad	n/a
SafetyGlide Insulin syringe with 30 G needle	BD	305934
Safetyglide Insulin syringe, 1/2 mL, 30 G x 5/16 TW	BD	305934
Scale	Ohaus Scout Pro	SP202
Supplical Nutritional Supplement	Henry Schein Animal Health	29908
Tegaderm, 6 cm x 7 cm	3M	1624W