Chapter 22

Murine Model of Pneumococcal Pneumonia

Eva Medina

Abstract

Respiratory tract infections remain among the most common clinical problems worldwide. Pneumoniaor inflammation of the lungs can be caused by infection with bacteria, viruses, and other organisms.Pneumonia management has been challenged by the widespread distribution of antibiotic-resistant strainsof Streptococcus pneumoniae, the commonest cause of community acquired pneumonia. Experimentalmodels of pneumonia have played a crucial role for testing the efficacy of antimicrobial agents as well asfor gaining a better understanding of the disease pathogenesis. These models have also received increasedattention as tools for deriving pharmacodynamic data and for determining the clinical significance of drugresistance.

Key words: Respiratory infection, Streptococcus pneumoniae , pneumonia, intranasal inoculation.

1. Introduction

Streptococcus pneumoniae is a major human pathogen thatcolonizes the upper respiratory tract and causes both life-threatening diseases such as pneumonia, sepsis, and meningi-tis and milder but common diseases, such as sinusitis and otitismedia (1). S. pneumoniae is the leading cause of bacterial pneu-monia. The burden of pneumococcal infections is particularlylarge among children and the elderly and is exacerbated by the ris-ing numbers of isolates resistant to antibiotics (2). Mouse modelsof pneumonia have been used to characterize the course of infec-tion by determining animal survival after infection, the bacterialloads in lungs and other organs, levels of inflammation, and his-tology of lung tissue (3). In addition, studies in the mouse modelof streptococcal pneumoniae have provided critical informationon antimicrobial efficacy that is directly relevant to the treatment

G. Proetzel, M.V. Wiles (eds.), Mouse Models for Drug Discovery, Methods in Molecular Biology 602,DOI 10.1007/978-1-60761-058-8 22, © Humana Press, a part of Springer Science+Business Media, LLC 2010

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of human infection (4, 5). Murine models of S. pneumoniae havebeen also essential for testing strategies of vaccination against thispathogen (6).

In the mouse model described here, pneumonia is inducedafter the intranasal instillation of S. pneumoniae. When using thismodel for evaluation of antimicrobial agents, therapeutic treat-ment should start after the infection has already been establishedin the lungs (6–12 h after bacterial inoculation).

2. Materials

1. Phosphate-buffered saline (PBS): 8 g NaCl, 0.2 g KCl,1.44 g Na2HPO4, and 0.24 g KH2PO4 in 800 ml of dis-tilled H2O. Adjust the pH to 7.4 with HCl. Add H2O to1 l. Sterilize by autoclave.

2. 70% (v /v) ethanol.3. Homogenator: POLYTRON R© System PT 4000 (KINE-

MATICA AG, Littau/Luzern, Switzerland).4. EasyClean Dispersing aggregates (KINEMATICA AG, Lit-

tau/Luzern, Switzerland).5. 1 ml syringes and 25 G needles.6. THY broth: Todd–Hewitt broth (Beckton Dickinson, MD,

USA) supplemented with 1% BactoTM yeast extract (Beck-ton Dickinson, MD, USA).

7. Blood agar plates (Beckton Dickinson, MD, USA; Cat. Nr.221165).

8. Glycerol (Roth, Karlsruhe, Germany).9. Isoflurane (Baxter, Germany).

10. Gauze or cotton.11. Closed glass container with raised floor.12. 10% neutral buffered formalin: 10% Formaldehyde in PBS

(see Note 1).13. Ketamine (Narketan R©, Ravensburg, Germany).14. Xylene (J.T. Baker Chemical Co, Phillipsburg, USA).15. Bacteria (see Note 2): the majority of the published studies

have used the serotype 2 S. pneumoniae (NCTC 7466) thatcan be obtained from the National Collection of Type Cul-tures, London, United Kingdom. Other strains suitable forthe mouse model are the serotype 3 S. pneumoniae (ATCC6303) and serotype 1 S. pneumoniae (ATCC 6301),both can be obtained from the American Type CultureCollection.

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16. Mice (see Note 3): several inbred strains of mice havebeen used in this infection model including BALB/c andCBA/Ca among others (see Note 4).

3. Methods

3.1. InoculumPreparation

1. Stock bacteria cultures are maintained in THY broth plus20% glycerol at –70◦C.

2. Plate a small amount of stock onto blood agar the daybefore infection and incubate overnight at 37◦C in 5%CO2.

3. Inoculate a fresh colony from a blood agar plate into THYmedium.

4. Incubate at 37◦C until mid-log phase (OD600 = 0.3–0.4).5. Harvest pneumococci by centrifugation and wash twice

with sterile PBS.6. Adjust the inoculum to the desired concentration in ster-

ile PBS (see Note 5). For standardization of S. pneumoniaeinoculum, a regression curve should be constructed for theselected strain by plotting the log number of serial bacterialdilutions against the percentage optical density of the sus-pensions read at 600 nm wavelength in a spectrophotome-ter (Novospec R© II, Pharmacia). Optical density of uninoc-ulated THY from the same batch is used as blank.

7. The inoculum concentration should be verified by plating10-fold serial dilutions onto blood agar and counting afterincubating for 24 h at 37◦C (see Note 6).

3.2. IntranasalInfection Procedure

1. Anesthetize mice by inhalation of isoflurane in order to facil-itate aspiration (see Note 7):1.1 Pour isoflurane on gauze or cotton placed in the bottom

of a closed glass container.1.2 Place a raised floor over the soaked material to

allow isoflurane to vaporize without impregnating themouse fur.

1.3 Place the mouse in the container and close the lid.1.4 Keep the animal inside until reaching slow and regular

breathing.1.5 Remove the animal from the container to perform

infection.

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2. Holding the mice vertically, deliver a 40 �l volume of bacte-rial inoculum to the nostrils to induce aspiration pneumonia.Inoculate control mice with sterile PBS.

3.3. Bacterial Countsin the Lungs asParameter to MonitorInfection

1. Euthanize mice at selected time intervals following infec-tion by cervical dislocation. Remove the lungs asepticallyand place them in a 10 ml homogenization tube containing4.5 ml of sterile saline.

2. Homogenize the lungs and perform 10-fold serial dilutionsof lung homogenate in sterile PBS.

3. Apply 100 �l of each dilution to a blood agar plate and gen-tly swirl the plate.

4. Incubate plates at 37◦C for 24 h.5. Determine the bacterial load within the lungs using the fol-

lowing formula:

CFU/lungs = C × Vt

Vp × DF

where C is the colony counts, Vt is the total volume of the lunghomogenate, VP is the volume plated, and DF is the dilution fac-tor of the sample.

3.4. HistologicalExamination of LungTissue to MonitorInfection

1. At chosen intervals after bacterial inoculation, mice are euth-anized by an overdose of ketamine (see Note 8).

2. Remove lungs and fix them with neutral buffered formalinfor 48 h at 4◦C.

3. Wash lungs with dH2O and dehydrate the tissue by serialimmersion in ethanol (50, 70, 95, and 100%) 1 h each, fin-ishing with incubation for 1 h in 100% xylene.

4. Embed lung tissue in paraffin and cut 5 �m thickness sec-tions.

5. Dewax tissue section starting with 100% xylene for 1 h andcontinuing with serial immersion in ethanol (100, 95, 70,and 50%) 1 h each finishing with dH2O.

6. Stain sections and mount (see Note 9).7. Examine by light microscopy.

3.5. LeukocyteRecruitment into theLungs as Parameterto Monitor Infection

The recruitment of leukocytes into the lungs following infectionwith S. pneumoniae is analyzed by broncho-alveolar lavage (BAL)fluid counts:

1. Euthanize mice by an overdose of ketamine (see Note 8).2. Disinfect the neck region with 70% ethanol.

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3. Make a middle incision in the neck area and retract the skinwith forceps.

4. Cut away the muscles overlying the trachea carefully to notaffect the aorta.

5. Make a small incision in the upper part of the trachea andinsert a polyethylene catheter attached to a 25 G needle ona 1 ml tuberculin syringe (see Note 10).

6. Slowly inject 0.5 ml of sterile PBS through the catheter intothe mouse lungs recovering the resultant BAL.

7. Determine total leukocyte counts under optical microscopeusing a Neubauer chamber.

8. Determine differential cell counts on cytospin smears of lungwashes (3 min at 100×g) stained by the Wright–Giemsamethod.

3.6. Quantification ofInflammatoryCytokines inBroncho-alveolarLavage (BAL): AsParameter to MonitorInfection

1. Centrifuge lavage fluids for 10 min at 800×g.2. Remove supernatant with a pipette and use for quantification

of cytokines by ELISA (see Note 11).

4. Notes

1. Work with 10% buffered neutral formalin is recommendedto be executed in a well-ventilated area, wearing goggles,gloves, and lab coat. Storage areas should have appropriateventilation systems.

2. S. pneumoniae is considered to be a potential hazard to per-sonnel, and Biosafety Level 2 practices and facilities shouldbe used when working with this pathogen. All work withthis microorganism should have prior approval of the insti-tutional Biological Safety Department and should strictlyfollow the guidelines and regulations for the handling ofthis pathogen.

3. All protocols using live animals must first be reviewed andapproved by the corresponding Governmental and Institu-tional Animal Care and Use Committee and must conformto the regulations regarding the care and use of laboratoryanimals.

4. Different mouse strains strongly differ in their degree ofsusceptibility to S. pneumoniae (7). While CBA/Ca andSJL are highly susceptible, BALB/c is highly resistant andC3H/He, FVB/n, NIH, AKR, C57BL/6, and DBA/2exhibit intermediate resistance.

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5. Concentrations ranging between 5×105and 107CFU canbe used.

6. Due to propensity of S. pneumoniae to autolysis, the inocu-lum should never be vortexed or subjected to drasticshaking.

7. All procedures using isoflurane should be conducted ina fume hood that continuously exhausts anesthetic gasesaway from personnel.

8. CO2 asphyxiation is not recommended since it can causeperivascular edema in the lungs or alveolar hemorrhage.

9. The staining technique will depend on the type of cell ofinterest. Hematoxylin and eosin (H&E) stain is the mostwidely used method in histology. Using this technique,basophilic white blood cells stain dark blue, eosinophilicwhite blood cells stain bright red, and neutrophils stain aneutral pink.

10. The catheter should not be insert too far into the tracheasince this will result in the dispensing of PBS into only onelobe of the lungs.

11. The simplest method for determination of cytokines byELISA is to use commercially available kits with match-ing antibody pairs and standards for the specific cytokine.The reader can consult web sites such as http://www.biocompare.com/ to select form a wide range of commer-cially available kits with the corresponding manufacturesinstructions for use.

References

1. Austrian, R. (1999) The pneumococcus atthe millennium: not down, not out. J InfectDis. 179 Suppl 2, S338–S341.

2. Ortqvist, A., Hedlund, J., and Kalin, M.(2005) Streptococcus pneumoniae: epidemiol-ogy, risk factors, and clinical features. SeminRespir Crit Care Med. 26, 563–574.

3. Chiavolini, D., Pozzi, G., and Ricci, S.2008. Animal models of Streptococcus pneu-moniaedisease. Clin Microbiol Rev. 21,666–685.

4. Craig, W. A. (1998) Pharmacokinetic/pharmacodynamic parameters: rationale forantibacterial dosing of mice and men. ClinInfect Dis. 26, 1–10.

5. Moine, P., Vallee, E., Azoulay-Dupuis,E., Bourget, P., Bedos, J. P., Bauchet, J.,

and Pocidalo, J. J. (1994) In vivo effi-cacy of a broad-spectrum cephalosporin,ceftriaxone, against penicillin-susceptibleand -resistant strains of Streptococcus pneu-moniae in a mouse pneumonia model.Antimicrob Agents Chemother. 38, 1953–1958.

6. Steinhoff, M.C. (2007) Pneumococcal vac-cine animal model consensus group. Animalmodels for protein pneumococcal vaccineevaluation: a summary, Vaccine 25, 2465–2470.

7. Kadioglu, A. and Andrew, P. W. (2005) Sus-ceptibility and resistance to pneumococcaldisease in mice. Brief Funct Genomic Pro-teomic. 4, 241–247.