In Vivo Animal Models: Quantitative Models Used for Identifying Antibacterial Agents

Jeffrey Fernandez1

1 Johnson & Johnson Pharmaceutical Research and Development, L.L.C., Raritan, New Jersey
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 13A.5
DOI:  10.1002/0471141755.ph13a05s34
Online Posting Date:  October, 2006
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Determining the in vivo bactericidal potential of novel agents is critical for selecting drug candidates. Described in this unit are two protocols that measure the amount of bacterial killing under very different conditions. The mouse pouch protocol is a rapid assay that provides efficacy data in an acute infection, whereas the rabbit tissue cage protocol assesses the ability of a compound to eradicate bacteria in a chronic abscess infection. Data from these tests, along with those from other qualitative and quantitative assays, are important for defining antibacterial efficacy in vivo.

Keywords: abscess; subcutaneous infection; tissue cage infection; mouse pouch

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Table of Contents

  • Basic Protocol 1: Murine Subcutaneous Pouch Model of Infection
  • Basic Protocol 2: Rabbit Subcutaneous Abscess Tissue Cage Infection
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Murine Subcutaneous Pouch Model of Infection

  • Female Swiss Webster mice, 20 to 25 g
  • Frozen bacteria
  • Brain heart infusion (DIFCO, Becton‐Dickinson) or other broth recommended for infecting microorganisms
  • 0.9% (w/v) NaCl (saline), ice cold
  • 7% mucin in 0.9% (w/v) NaCl (saline), Type II crude from porcine stomach (Sigma)
  • Test compound (antimicrobial agent) prepared in appropriate vehicle
  • Vehicle alone (without test compound)
  • 70% isopropyl alcohol
  • Hanging wire cages
  • Animal feeding needles (Popper and Sons)
  • 1‐ and 3‐ml syringes (Becton‐Dickinson)
  • 21‐, 25‐, and 27‐G needles (Becton‐Dickinson)
  • 37°C shaking incubator
  • Scale for weighing animals
  • 1.7‐ml microcentrifuge tubes
  • Spiral plate analyzer with CASBA 4 software (Spiral Biotech)
  • Additional reagents and equipment for CO 2 asphyxiation (Donavan and Brown, ) and plating bacteria (unit 13.3)

Basic Protocol 2: Rabbit Subcutaneous Abscess Tissue Cage Infection

  • Female New Zealand white rabbits, 3.0 to 4.0 kg
  • Ethylene oxide sterilization kit (Getinge)
  • 2% chlorhexidine diacetate (e.g., Novasan solution, Fort Dodge Animal Health)
  • Acepromazine malate
  • Ketamine⋅HCl
  • Xylazine
  • 3% isoflurane (e.g., IsoFlo, Abbott Laboratories)
  • Butorphanol (e.g., Torbugesic, Fort Dodge Laboratories)
  • Penicillin G‐procaine‐benzathine (e.g., PenG, Phoenix Pharmaceutical)
  • Frozen bacterial stock
  • Brain‐heart infusion broth (DIFCO, Becton‐Dickinson) or other broth recommended for infecting microorganisms
  • 0.9% (w/v) NaCl (saline), ice cold
  • 5% (w/v) mucin in saline, type II crude from porcine stomach (Sigma)
  • Test compound (antimicrobial agent) and vehicle
  • Todd‐Hewitt agar (Becton Dickinson) or other appropriate medium
  • Lithium heparin tubes (LH/1.3, Sarstedt)
  • Hanging wire cages
  • Golf practice wiffle ball (43‐mm diameter; Wilson Sporting Goods)
  • Oster animal clippers, no. 40 blade
  • Prolene sutures (Ethicon)
  • Surgical staples (e.g., Endo‐surgery staples, Ethicon)
  • 37°C shaking incubator
  • 18‐ and 23‐G needles (Becton‐Dickinson)
  • 1‐ and 3‐ml syringes (Becton‐Dickinson)
  • 1.7‐ml microcentrifuge tubes
  • HPLC series 1100 (Hewlett‐Packard)
  • Additional reagents and equipment for plating bacteria (unit 13.3)
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Literature Cited

   Alder, J. and Clement, J.J. 2003. Comparative chemotherapeutic activity of temafloxacin, cefoxitin, clindamycin, imipenem and ampicillin/sulbactam against Bacteroides fragilis in a mouse subcutaneous abscess model. J. Antimicrob. Chemother. 31:303‐311.
   Arai, S., Kobayashi, S., Hayashi, S., and Sakaguchi, T. 1998. Distribution of cefpirome (HR 810) to exudates in the croton oil induced rat granuloma pouch and its therapeutic effects on experimental infections in the pouch. Antimicrob. Agents Chemother. 32:1396‐1399.
   Bamberger, D.M., Peterson, L.R., Gerding, D.N., Moody, J.A., and Fasching, C.E. 1986. Ciprofloxacin, azlocillin, ceftizoxime, and amikacin alone and in combination against Gram‐negative bacilli in an infected chamber model. J. Antimicrob. Chemother. 18:51‐63.
   Bamberger, D.M., Herndon, B.L., and Surarna, P.R. 1995. Azithromycin in experimental Staphylococcus aureus abscess model. J. Antimicrob. Chemother. 35:623‐629.
   Bush, K. 2004. Antibacterial drug discovery in the 21st century. Clin. Microbiol. Infect. 10:10‐17.
   Donavan, J. and Brown, P. 1997. Euthanasia. In Current Protocols in Immunology (J.E. Coligan, A.M. Kruisbeek, D.H. Margulies, and W. Strober, eds.) pp. 1.8.1‐1.8.5. John Wiley & Sons, Hoboken, N.J.
   Fernandez, J., Barrett, J.F., Licata, L., Amaratunga, D., and Frosco, M. 1999. Comparison of efficacies of oral levofloxacin and oral ciprofloxacin in a rabbit model of a staphylococcal abscess. Antimicrob. Agents Chemother. 43:667‐671.
   Fernandez, J.A., Goldschmidt, R., Hilliard, J.J., and Bush, K. 2001. Efficacy of RWJ‐54428 (MC‐02, 479) in a novel murine pouch model of infection caused by methicillin‐resistant (MRSA) and glycopeptide‐intermediate resistant (GISA) Staphylococcus aureus. 39th Interscience Conference on Antimicrobial Agents and Chemotherapy. Abstract 396.
   Gerding, D.N., Hall, W.H., Schierl, E.A., and Manion, R.E. 1976. Cephalosporin and aminoglycoside concentrations in peritoneal capsular fluid in rabbits. Antimicrob. Agents Chemother. 10:902‐911.
   Guhad, F. 2005. Introduction to the 3Rs (refinement, reduction and replacement). Contemp. Top. Lab. Anim. Sci. 44:58‐59.
   Knudsen, J.D. and Frinodt‐Møller, N. 2001. Animal models in bacteriology. Contrib. Microbiol. 9:1‐14.
   Li, C., Nicolau, D.P., Lister, P.D., Quintiliani, R., and Nightingale, C.H. 2004. Pharmacodynamic study of beta‐lactams alone and in combination with beta‐lactamase inhibitors against Pseudomonas aeruginosa possessing an inducible beta‐lactamase. J. Antimicrob. Chemother. 53:297‐304.
   Lorian, V. 2005. Antibiotics in Laboratory Medicine, 5th Edition. Lippincott, Williams, and Wilkins, Philadelphia, PA.
   Matsui, H. and Okuda, T. 1988. Penetration of cefpiramide and cefazolin into peritoneal capsular fluid in rabbits. Antimicrob. Agents Chemother. 32:33‐36.
   Mussalli, G.M., Brunnert, S.R., and Hirsch, E. 1999. A murine model of renal abscess formation. Clin. Diagn. Lab. Immunol. 6:273‐275.
   National Committee for Clinical Laboratory Standards (NCCLS). 1997. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically. Approved standard M7‐A4. National Committee for Clinical Laboratory Standards, Villanova, PA.
   Schaumann, R., Ackermann, G., Pless, B., Claros, M., and Rodloff, A.C. 1999. In vitro activities of gatifloxacin, two other quinolones and five nonquinolone antibacterials against obligately anaerobic bacteria. Antimicrob. Agents Chemother. 43:2783‐2786.
   Schechtman, L.M. 2002. Implementation of the 3Rs (refinement, reduction, and replacement): Validation and regulatory acceptance considerations for alternative toxicological test methods. Ilar J. 43:S85‐S94.
   Shapiro, M.A., Sesnie, J.C., Desaty, T.M., Griffin, T.J., and Heifetz, C.L. 1998. Comparative therapeutic efficacy of clinafloxacin in a Pseudomonas aeruginosa mouse renal abscess model. J. Antimicrob. Chemother. 41:403‐405.
   Stearne, L.E., Gyssens, I.C., Goessens, W.H., Mouton, J.W., Oyen, W.J., van der Meer, J.W., and Verbrugh, H.A. 2001. In vivo efficacy of trovafloxacin against Bacteroides fragilis in mixed infection with either Escherichia coli or a vancomycin‐resistant strain of Enterococcus faecium in an established‐abscess murine model. Antimicrob. Agents Chemother. 45:1394‐1401.
   Stearne, L.E., Buijk, S.L., Mouton, J.W., and Gyssens, I.C. 2002. Effect of a single percutaneous abscess drainage puncture and imipenem therapy, alone or in combination, in treatment of mixed‐infection abscesses in mice. Antimicrob. Agents Chemother. 46:3712‐3718.
   Tsuji, M., Takema, M., Miwa, H., Shimada, J., and Kuwahara, S. 2003. In vivo antibacterial activity of S‐3578, a new broad‐spectrum cephalosporin: Methicillin‐resistant Staphylococcus aureus and Pseudomonas aeruginosa experimental infection models. Antimicrob. Agents Chemother. 47:2507‐2512.
   Ueda, Y. and Sunagawa, M. 2003. In vitro and in vivo activities of novel 2‐(thiazol‐2‐ylthio)‐1beta‐methylcarbapenems with potent activities against multiresistant Gram‐positive bacteria. Antimicrob. Agents Chemother. 47:2471‐2480.
   Worlitzsch, D., Kaygin, H., Steinhuber, A., Dalhoff, A., Botzenhart, K., and Döring, G. 2001. Effects of amoxicillin, gentamicin, and moxifloxacin on the hemolytic activity of Staphylococcus aureus in vitro and in vivo. Antimicrob. Agents Chemother. 45:196‐202.
   Xuan, D., Zhong, M., Mattoes, H., Bui, K‐Q, McNabb, J., Nicolau, D., Quintilliani, R., and Nightingale, C.H. 2001. Streptococcus pneumoniae response to repeated moxifloxacin or levofloxacin exposure in a rabbit tissue cage model. Antimicrob. Agents Chemother. 45:794‐799.
Key References
   Fernandez et al., 1999. See above.
  This is the main publication for the rabbit abscess model and describes, in detail, the study with levofloxacin and ciprofloxacin. The pharmacokinetics for both drugs are also described.
   Fernandez et al., 2001. See above.
  This is the main publication for the mouse pouch assay. It contains data on levofloxacin and erythromycin against S. aureus Smith. It also describes efficacy data for linezolid, vancomycin, and RWJ‐54428 against MRSA and glycopeptide‐intermediate S. aureus (GISA) strains. There is also limited pharmacokinetic data for some of the compounds in plasma and pouch fluid.
   Li et al., 2004. See above.
  This reference describes a rabbit abscess model using P. aeruginosa as the infecting organism. The authors compare the pharmacokinetics and efficacy of piperacillin and piperacillin/tazobactam.
   Xuan et al., 2001. See above.
  This paper describes a rabbit tissue cage model with S. pneumoniae as the infecting organism. It contains the efficacy and pharmacokinetic data of moxifloxacin and levofloxacin in this model.
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