Mouse Models of Acinetobacter baumannii Infection

Greg Harris1, Rhonda KuoLee1, H. Howard Xu2, Wangxue Chen3

1 Human Health and Therapeutics, National Research Council Canada, Ottawa, Ontario, 2 Department of Biological Sciences, California State University, Los Angeles, California, 3 Department of Biological Sciences, Brock University, St. Catharines, Ontario
Publication Name:  Current Protocols in Microbiology
Unit Number:  Unit 6G.3
DOI:  10.1002/cpmc.36
Online Posting Date:  August, 2017
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Abstract

This unit describes basic protocols for infecting mice through intranasal and intraperitoneal routes with Acinetobacter baumannii to induce associated pneumonia and sepsis, the two most common manifestations of clinical infections with this pathogen. By selecting the appropriate protocols and bacterial strains of different virulence, these mouse models provide an opportunity to study the infection pathogenesis and host‐immune responses, and to evaluate the efficacies of prophylactic and therapeutic anti‐A. baumannii candidates. © 2017 by John Wiley & Sons, Inc.

Keywords: Acinetobacter; animal models; mouse; pneumonia; sepsis

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Growth of A. baumannii LAC‐4 for Inoculation
  • Basic Protocol 2: Intranasal Inoculation of Mice with A. baumannii LAC‐4
  • Basic Protocol 3: Intraperitoneal Inoculation of Mice with A. baumannii LAC‐4
  • Alternate Protocol 1: Intraperitoneal Inoculation of Mice with A. baumannii Mixed with Porcine Mucin
  • Basic Protocol 4: Determination of Tissue and Blood Bacterial Burdens
  • Support Protocol 1: Preparation of A. baumannii LAC‐4 Bacterial Stocks
  • Support Protocol 2: Bronchoalveolar Lavage for Differential Cell Counts
  • Support Protocol 3: Peritoneal Lavage for Differential Cell Counts
  • Support Protocol 4: Collection of Various Fluid Samples for Determination of Antibodies and Cytokines
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Growth of A. baumannii LAC‐4 for Inoculation

  Materials
  • A. baumannii frozen stock (H. Howard Xu, Department of Biological Sciences, California State University)
  • Cystine heart agar (CHA) plates (see recipe)
  • 0.85% (w/v) saline (see recipe)
  • Tryptic soy broth (TSB) (see recipe)
  • Brain heart infusion (BHI) plates (see recipe)
  • 1‐ml glass pipets
  • 37°C water‐jacketed incubator (no special gas requirements)
  • 10‐µl inoculating loops
  • 15‐ml conical tubes
  • Vortex
  • Cell strainers (70‐µm), optional
  • 250‐ml baffled‐bottom flask and foam stopper
  • Spectrophotometer (Eppendorf BioPhotometer or similar) and microcuvettes
  • 37°C shaking incubator
  • 30‐ml round‐bottom, high‐speed centrifugation tubes
  • Petroff‐Hausser sperm/bacteria counting chamber
  • Microscope (40× magnification)

Basic Protocol 2: Intranasal Inoculation of Mice with A. baumannii LAC‐4

  Materials
  • Freshly grown A. baumannii bacteria (see protocol 1)
  • 0.85% (w/v) saline (see recipe)
  • 8‐ to 10‐week‐old male or female mice (Balb/c, C57BL/6, or other strains)Isoflurane inhalation anesthetic (AErrane, Baxter)
  • 30% medical‐grade oxygen
  • Brain heart infusion (BHI) or cystine heart agar (CHA) plates (see recipe)
  • Isoflurane vaporizer (Cyprane Isotec 4 or similar)
  • Mouse housing

Basic Protocol 3: Intraperitoneal Inoculation of Mice with A. baumannii LAC‐4

  Materials
  • Cultured A. baumannii LAC‐4 (see protocol 1)
  • 0.85% (w/v) saline (see recipe)
  • 8‐ to 10‐week‐old male or female mice (Balb/c, C57BL/6, or other suitable strains)
  • Brain heart infusion (BHI) or cystine heart agar (CHA) plates (see recipe)
  • 1‐ml sterile disposable syringes
  • Syringe needles (26‐G, 3/8‐in.)
  • Mouse housing

Alternate Protocol 1: Intraperitoneal Inoculation of Mice with A. baumannii Mixed with Porcine Mucin

  Materials
  • 0.85% (w/v) saline (see recipe)
  • Deionized, distilled water (ddH 2O)
  • Mice
  • 70% (v/v) ethanol (diluted in water)
  • BHI or CHA plates (see reciperecipes)
  • Homogenizer tubes and foam stoppers
  • 5‐ml culture tubes or similar (sample collection tubes)
  • Surgical scissors, fine‐tipped scissors, and forceps (various sizes)
  • 200‐μl pipet with sterile tips
  • Plastic petri dishes
  • Metal spatula
  • 10‐ml syringes (optional)
  • 70‐µm cell strainers (optional)
  • Hand‐held homogenizer with aerosol‐proof attachments (PRO Scientific or similar)
  • 37°C water‐jacketed incubator

Basic Protocol 4: Determination of Tissue and Blood Bacterial Burdens

  Materials
  • A. baumannii bacteria
  • Glycerol, sterilized
  • 15‐ml conical tubes
  • 1.8‐ml cryogenic vials
  • Additional reagents and equipment for bacterial culture (see protocol 1)

Support Protocol 1: Preparation of A. baumannii LAC‐4 Bacterial Stocks

  Materials
  • Ethylenediaminetetraacetic acid (EDTA)
  • Fetal bovine serum (FBS)
  • 0.85% (w/v) saline (see recipe)
  • Mice
  • 70% ethanol
  • Erythrocyte lysis buffer (ACK, see recipe)
  • Cell staining kit (PROTOCOL Hema‐3, Fisher Scientific) containing:
    • PROTOCOL Hema‐3 fixative
    • PROTOCOL Hema‐3 solution 1
    • PROTOCOL Hema‐3 solution 2
  • Mounting medium (Cytoseal 60, Fisher Scientific or similar)
  • Polyethylene tubing (0.51‐mm i.d.; 0.55‐mm o.d.) (Micro‐Line)
  • Scalpel or flat‐edged razor blade
  • 22‐G syringe needle (3/4‐in.)
  • Surgical scissors and forceps (fine‐tipped and regular)
  • 1‐ml sterile disposable syringes
  • Sample collection tubes (5‐ml culture tubes or similar)
  • Cell counting chamber (Reichart hemacytometer or similar)
  • Microscope with 20× magnification
  • CytoSep Cytospin funnels
  • Microscope slides
  • Cytospin centrifuge (Shandon)
  • Cell staining chambers and slide rack
  • Slide storage tray
  • Cover slips (18 × 18 mm)
  • Differential cell counter

Support Protocol 2: Bronchoalveolar Lavage for Differential Cell Counts

  Materials
  • Ethylenediaminetetraacetic acid (EDTA)
  • Fetal bovine serum (FBS)
  • 0.85% (w/v) saline (see recipe)
  • Mice
  • 70% ethanol
  • Forceps and surgical scissors
  • 10‐ml sterile disposable syringes
  • 21‐G syringe needle (1 1/2‐in.)
  • 15‐ml conical tubes

Support Protocol 3: Peritoneal Lavage for Differential Cell Counts

  Materials
  • Euthanized mice, BAL or PEL samples (see Support Protocols protocol 72 and protocol 83), or homogenized lungs for bacterial burden determination (see protocol 5)
  • Protease inhibitor cocktail tablets (Complete, Roche)
  • CHA plates (see recipe)
  • Blood collection tubes for serum separation (BD Microtainer or similar)
  • Refrigerated microcentrifuge
  • 1.5‐ml microcentrifuge tubes
  • Microcentrifugal filters with 0.22‐µm PVDF membrane (Millipore or similar)
  • Sample collection tubes (5‐ml culture tubes or similar)
  • Tube rotator
  • 10‐µl inoculating loops
  • 37°C water‐jacketed incubator (no special gas requirements)
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Figures

Videos

Literature Cited

Literature Cited
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