Murine Models of Streptococcus pyogenes Infection

Samantha Roberts1, June R. Scott1, Linda K. Husmann2, Christine A. Zurawski3

1 Emory University School of Medicine, Atlanta, 2 The University of Tennessee at Martin, Martin, Tennessee, 3 Infectious Disease Solutions, Atlanta
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 9D.5
DOI:  10.1002/9780471729259.mc09d05s02
Online Posting Date:  September, 2006
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Abstract

This unit describes procedures for testing virulence of Streptococcus pyogenes in mice. S. pyogenes is an important human pathogen and causes one of the most common childhood diseases. The syndromes that result from S. pyogenes infection are diverse, ranging from mild, superficial throat or skin infection to severe, invasive disea/se that is often lethal. Thus, a greater understanding of the virulence factors of this bacterium and development of modalities to prevent or relieve the infections it causes are important. Since S. pyogenes is a strictly human pathogen (with the exception of a single strain), the value of all animal models is limited. This unit describes a model for longā€term throat colonization following the natural route of infection (inhalation), one for pneumonia and systemic dissemination following intratracheal inoculation, and one for systemic dissemination following the more natural route of skin infection. In addition, methods are presented for culturing S. pyogenes from tissues of the infected animal.

Keywords: animal model; mouse; Streptococcus pyogenes; inoculation routes; pharyngitis; scarlet fever; impetigo; erysipelas; cellulitis; necrotizing fasciitis; group A Streptococcus (GAS)

     
 
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Table of Contents

  • Basic Protocol 1: Throat Colonization Model of Streptococcus pyogenes
  • Basic Protocol 2: Pneumonia Model of Streptococcus pyogenes Infection (Intratracheal)
  • Basic Protocol 3: Subcutaneous Inoculation of CD1 Mice with Streptococcus pyogenes
  • Culturing of Various Mouse Organs and Fluids to Assay for Streptococcus pyogenes Strain B514‐SM
  • Basic Protocol 4: Preparing Cultures from the Meninges
  • Basic Protocol 5: Preparing Cultures from Trachea
  • Basic Protocol 6: Preparing Cultures from Spleen
  • Basic Protocol 7: Preparing Cultures from Lung
  • Basic Protocol 8: Preparing Cultures from Blood
  • Basic Protocol 9: Preparing Cultures from Nasal Passages
  • Basic Protocol 10: Collection of Saliva from Mice
  • Basic Protocol 11: Assay of Group A Streptococcus Colonization by Throat Culture from Mice
  • Reagents and Solutions
  • Commentary
  • Literature Cited
     
 
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Materials

Basic Protocol 1: Throat Colonization Model of Streptococcus pyogenes

  Materials
  • Streptococcus pyogenes strain B514‐Sm (Hook et al., )
  • 2× brain heart infusion (BHI)/2% (v/v) supplement B (see recipe)
  • 0.9% saline ( appendix 2A)
  • 7‐week‐old female (preferred) C57BL10/SnJ mice (Jackson Laboratories): place order to allow for acclimatization one week prior to inoculation at the age indicated (see )
  • Solution containing 1.5 mg/ml xylazine and 100 mg/ml ketamine
  • THY plates containing 1000 µg/ml streptomycin (see recipe)
  • 85 µg/ml hyaluronidase (optional)
  • 15‐ml screw‐cap conical tubes
  • Klett flasks, screw‐cap (Bellco)
  • Klett colorimeter with red filter
  • Refrigerated centrifuge (e.g., Beckman with JA‐14 rotor) and centrifuge bottles
  • Rodent ear punch
  • Balance accurate to 0.01 g
  • 20‐µl micropipettor and round‐tipped sequencing pipet tips
  • Additional reagents and equipment for counting cells ( appendix 4A) and for anesthesia (Donovan and Brown, )

Basic Protocol 2: Pneumonia Model of Streptococcus pyogenes Infection (Intratracheal)

  Materials
  • S. pyogenes strain B514‐Sm (Hook et al., )
  • 2× BHI/2% supplement B (see recipe)
  • 0.9% saline ( appendix 2A)
  • 5‐week‐old female C3HeB/FeJ mice (Jackson Laboratories; http://www.jax.org): place order to allow for acclimatization one week prior to inoculation at the age indicated (see )
  • Solution containing 1.5 mg/ml xylazine and 100 mg/ml ketamine
  • THY plates containing 1000 µg/ml streptomycin (see recipe)
  • 85 µg/ml hyaluronidase (optional)
  • 15‐ml screw‐cap conical tubes
  • Klett flask, screw‐cap (Bellco)
  • Klett colorimeter with red filter
  • Refrigerated centrifuge (e.g., Beckman with JA‐14 rotor) and centrifuge bottles
  • Rodent ear punch
  • Balance accurate to 0.01 g
  • 22‐G straight feeding needle (Popper and Sons) and 1‐cc tuberculin syringe
  • Additional reagents and equipment for counting cells ( appendix 4A) and for anesthesia (Donovan and Brown, )

Basic Protocol 3: Subcutaneous Inoculation of CD1 Mice with Streptococcus pyogenes

  Materials
  • 6‐week‐old female CD1 (outbred) mice, 21 to 23 g (Charles River Laboratories; http://www.criver.com): place order to allow for acclimatization one week prior to inoculation at the age indicated (see )
  • THY medium (see recipe)
  • Group A Streptococcus (GAS) strain, preferably streptomycin‐resistant (e.g., AM3; Stamp and Hendry, )
  • 0.9% saline ( appendix 2A), 4°C, sterile
  • 2.5% Avertin (see recipe)
  • Depilatory agent (e.g., Nair)
  • THY plates containing 1000 µg/ml streptomycin (see recipe)
  • 85 µg/ml hyaluronidase (optional)
  • Isoflurane
  • 15‐ml conical tube
  • Klett flask (Bellco)
  • Klett colorimeter and with red filter
  • Refrigerated centrifuge (e.g., Beckman with JA‐20 rotor)
  • Balance accurate to 0.01 g
  • Rodent ear punch
  • Cotton‐tipped applicators
  • Gauze pads
  • Alcohol wipes
  • 27‐G needle and 1‐ml insulin/tuberculin syringe
  • Calipers (optional)
  • Urogenital swab, single‐use (Puritan)
  • Dissecting tools, sterile
  • Dounce tissue homogenizer
  • Additional reagents and equipment for counting cells ( appendix 4A), removal of spleen ( protocol 6, steps to ), and anesthesia (Donovan and Brown, )

Basic Protocol 4: Preparing Cultures from the Meninges

  Materials
  • Mouse infected with S. pyogenes strain B514‐SM ( protocol 1, protocol 22, or protocol 33), sacrificed
  • THY medium (see recipe)
  • Surgical scissors, sterile
  • Sterile swab, calcium alginate urogenital

Basic Protocol 5: Preparing Cultures from Trachea

  Materials
  • Mouse infected with S. pyogenes strain B514‐SM ( protocol 1, protocol 22, or protocol 33), sacrificed
  • THY medium and plates (see recipe)
  • 0.9% saline ( appendix 2A)
  • Surgical scissors and forceps, sterile

Basic Protocol 6: Preparing Cultures from Spleen

  Materials
  • Mouse infected with S. pyogenes strain B514‐SM or AM3 ( protocol 1, protocol 22, or protocol 33), sacrificed
  • THY medium and plates (see recipe)
  • Surgical scissors and forceps, sterile
  • Dounce tissue homogenizer, sterile

Basic Protocol 7: Preparing Cultures from Lung

  Materials
  • Mouse infected with S. pyogenes strain B514‐SM (Basic Protocols protocol 11 or protocol 22), sacrificed
  • THY medium (see recipe)
  • 0.9% saline ( appendix 2A)
  • Surgical scissors and forceps, sterile
  • Dounce tissue homogenizer

Basic Protocol 8: Preparing Cultures from Blood

  Materials
  • Mouse infected with S. pyogenes strain B514‐SM (Basic Protocols protocol 11, protocol 22, or protocol 33), sacrificed
  • THY medium (see recipe)
  • Surgical scissors, sterile
  • 200‐µl micropipettor and appropriate tips
  • 15‐ml screw‐cap conical tube

Basic Protocol 9: Preparing Cultures from Nasal Passages

  Materials
  • Mouse infected with S. pyogenes strain B514‐SM (Basic Protocols protocol 11 or protocol 22), sacrificed
  • THY medium (see recipe)
  • 200‐µl micropipettor and appropriate tips
  • 1.5‐ml snap‐cap tube

Basic Protocol 10: Collection of Saliva from Mice

  Materials
  • Carbamyl chloride (carbachol; Sigma‐Aldrich)
  • Soybean trypsin inhibitor (Sigma‐Aldrich)
  • Phenylmethylsulfonyl fluoride (PMSF; Sigma‐Aldrich)
  • BSA (Sigma‐Aldrich)
  • Sodium azide (NaN 3)
  • Mouse infected with S. pyogenes strain B514‐SM (Basic Protocols protocol 11 or protocol 22)
  • 22‐G straight feeding needle (Popper and Sons) and 1‐cc tuberculin syringe
  • 200‐µl micropipettor
  • −80°C freezer

Basic Protocol 11: Assay of Group A Streptococcus Colonization by Throat Culture from Mice

  Materials
  • Mouse infected with S. pyogenes strain B514‐SM (Basic Protocols protocol 11 or protocol 22)
  • Calcium alginate swab (urethro‐genital applicator; Baxter Healthcare)
  • THY plates and medium containing 1000 µg/ml streptomycin (see recipe)
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Figures

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Literature Cited

Literature Cited
   Ashbaugh, C.D., Moser, T.J., Shearer, M.H., White, G.L., Kennedy, R.C., and Wessels, M.R. 2000. Bacterial determinants of persistent throat colonization and the associated immune response in a primate model of human group A streptococcal pharyngeal infection. Cell Microbiol. 2:283‐292.
   Bisno, A.L. and Stevens, D.L. 1996. Streptococcal infections of skin and soft tissues. New Engl. J. Med. 334: 240‐245.
   Bunce, C., Wheeler, L., Reed, G., Musser, J., and Barg, N. 1992. Murine model of cutaneous infection with gram‐positive cocci. Infect. Immun. 60: 2636‐2640.
   Dalton, T.L., Hobb, R.I., and Scott, J.R. 2006. Analysis of the role of CovR and CovS in the dissemination of Steptococcus pyogenes in invasive skin disease. Microb. Pathogenesis. In press.
   Donavan, J. and Brown, P. 1995. Blood Collection. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, and W. Strober, eds.) pp. 1.7.1‐1.7.8. John Wiley & Sons, Hoboken, N.J.
   Donovan, J. and Brown, P. 1998. Anesthesia. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, and W. Strober, eds.) pp. 1.4.1‐1.4.5. John Wiley & Sons, Hoboken, N.J.
   Hollingshead, S.K., Simecka, J.W., and Michalek, S.M. 1993. Role of M protein in pharyngeal colonization by group A streptococci in rats. Infect. Immun. 61:2277‐2283.
   Hook, E.W., Wagner, R.R., and Lancefield, R.C. 1960. An epizootic in Swiss mice caused by a group A streptococcus, newly designed type 50. Am. J. Hyg. 72:111‐119.
   Husmann, L.K., Dillehay, D.L., Jennings, V.M., and Scott, J.R. 1996. Streptococcus pyogenes infection in mice. Microb. Pathogenesis 20:213‐24.
   Husmann, L.K., Yung, D.L., Hollingshead, S.K., and Scott, J.R. 1997. Role of putative virulence factors of Streptococcus pyogenes in mouse models of long‐term throat colonization and pneumonia. Infect. Immun. 65:1422‐1430.
   Limbago, B., Penumalli, V., Weinrick, B., and Scott, J.R. 2000. Role of streptolysin O in a mouse model of invasive group A streptococcal disease. Infect. Immun. 68:6384‐6390.
   Limbago, B., McIver, K.S., Penumalli, V., Weinrick, B., and Scott, J.R. 2001. Restoration of Mga function to a Streptococcus pyogenes strain (M Type 50) that is virulent in mice. Infect. Immun. 69:1215‐1220.
   Neely, M.N., Pfeifer, J.D., and Caparon, M. 2002. Streptococcus‐zebrafish model of bacterial pathogenesis. Infect. Immun. 70:3904‐3914.
   Piepmeier, E. Jr., Hammett‐Stabler, C., Price, M., Peters, J., Kemper, G., and Davis, M. Jr., 1995. Myositis and fasciitis associated with group A beta hemolytic streptococcal infections: Development of a rabbit model. J. Lab. Clin. Med. 126:137‐143.
   Stamp, T.C. and Hendry, E.B. 1937. The immunizing activity of certain chemical fractions isolated from haemolytic streptococci. Lancet 1:257‐259.
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