A Mouse Model of Streptococcus pyogenes Necrotizing Skin Infection

Summary

This protocol describes a mouse subcutaneous infection model for Streptococcus pyogenes that enables independent quantification of bacterial burden and lesion progression. The method provides a standardized and reproducible platform for evaluating the roles of bacterial virulence factors, host immune responses, or therapeutic interventions aimed at reducing tissue damage.

Abstract

Streptococcus pyogenes is a human pathogen that causes severe necrotizing soft tissue infections characterized by rapid and extensive tissue destruction. A key challenge in studying these infections is the lack of experimental models that allow independent assessment of bacterial replication and host-mediated tissue damage. This article presents a standardized and reproducible murine subcutaneous model of S. pyogenes necrotizing skin infection designed to overcome this limitation. The protocol describes the preparation of a log-phase bacterial inoculum, subcutaneous infection of mice, and longitudinal monitoring of lesion development. Key steps include sonication of bacterial cultures to standardize chain length, digital imaging of lesions for quantitative analysis of ulcer area, and determination of bacterial burden through tissue homogenization and plating. This versatile model can be readily adapted to evaluate the effects of specific bacterial genes, host factors, or therapeutic interventions on infection progression and tissue pathology. By enabling reproducible and quantitative assessment of both bacterial and host parameters, this method provides a robust platform for studying pathogenesis and testing strategies to limit tissue damage during invasive streptococcal infection.

Introduction

Streptococcus pyogenes (Group A Streptococcus, GAS) is a significant human pathogen that causes necrotizing soft tissue infections (NSTIs)¹, resulting in rapid destruction of skin and underlying tissues^2,3,4. Studying the pathogenesis of these infections requires animal models that recapitulate localized tissue injury while allowing independent assessment of bacterial replication and host-mediated pathology⁵. Many traditional infection models, such as systemic inoculation or in vitro cell culture syste....

Protocol

All animal experiments were performed in compliance with the Marshall University School of Medicine Institutional Animal Care and Use Committee (IACUC) and Animal Resource Facility. All procedures involving Streptococcus pyogenes were conducted in the PHS-assured, AAALAC-accredited animal facility under Biosafety Level 2 (BSL-2) containment in accordance with institutional biosafety protocols. The experimental workflow is illustrated in Figure 1.

NOTE: Specific details for reagents, materials, and equipment are provided in the Table of Materials.

1. Pr....

Results

The mouse subcutaneous infection model for S. pyogenes consistently generates a localized, progressive necrotic ulcer, enabling simultaneous quantification of bacterial burden and tissue pathology. Representative results from this standardized protocol are as follows.

Experimental groups typically consist of 6-10 mice to provide sufficient statistical power (e.g., 80-90%) for detecting significant differences in lesion size and bacterial burden, based on prior studies

Discussion

The mouse subcutaneous S. pyogenes necrotizing skin infection model described here provides a robust and reproducible system for dissecting the mechanisms underlying severe soft tissue infection. Its primary value lies in the ability to decouple bacterial burden from tissue pathology, enabling mechanistic studies of how host and pathogen factors independently contribute to disease progression. This distinction is particularly important in S. pyogenes infection, where necrotizing fasciitis severity often.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1 mL Syringe	BD	309628	For subcutaneous injection
28-G Needle	BD	305109	For subcutaneous injection
Bacterial Hemocytometer / Counting Chamber	Hausser Scientific	02-671-6	For bacterial counting pre-inoculation
BD BBL GasPak EZ Anaerobic Container System	Becton, Dickinson and Company (BD)	260001	For anaerobic incubation
C57BL/6J mice	The Jackson Laboratory	664	8-12 weeks old
Carbon Dioxide (CO2) Tank	Local supplier	N/A	For euthanasia
Cup-Horn Sonicator (e.g., Q700)	Qsonica	431C2	For standardizing bacterial chain length
Digital Camera	N/A	N/A	For lesion imaging
Electric Clippers	Wahl	e.g., 8655-200	For shaving mouse flank
FastPrep-24 Homogenizer	MP Biomedicals	116005500	For tissue homogenize
ImageJ Software	National Institutes of Health (NIH)	N/A	https://imagej.nih.gov/ij/
Isoflurane	Baxter	1001936060	For anesthetic
Isoflurane Anesthesia System (Chamber & Vaporizer)	VetEquip or similar	N/A	For mouse anesthesia
Peptone	Becton, Dickinson and Company (BD)	211677	For preparation of C medium
Phosphate-Buffered Saline (PBS)	Gibco	10010023	Sterile, pH 7.4
S. pyogenes strain HSC5	Caparon lab		M1T1 clinical isolate
SKH1 mice	Charles River Laboratories	477	8-12 weeks old, hairless
Stainless steel beads, 2.8 mm	Universal Medical	D1133-28	Sterile
Sterile Surgical Scissors and Forceps	Fine Science Tools or similar	N/A	For aseptic tissue collection
Todd Hewitt Broth	Becton, Dickinson and Company (BD)	249240	For bacterial subculture
Yeast Extract (for C medium)	Becton, Dickinson and Company (BD)	212750	For preparation of C medium (same as for broth supplement)