The authors would like to thank Dr. Jeffery Weiser of the University of Pennsylvania for his gift of the clinical strains of Streptococcus pneumoniae. This work was funded by the Canadian Institutes for Health Research. CV was funded by a M. G. DeGroote fellowship and a fellowship from the Canadian Thoracic Society. This work was funded by the Ontario Lung Association and Canadian Institutes of Health Research (CIHR). Work in the Bowdish laboratory is supported in part by he Michael G. DeGroote Centre for Infectious Disease Research and the McMaster Immunology Research Centre.

Before you begin: all steps are done in a Biohazard Level 2 (BSL2) Biological Safety Cabinet (BSC) unless otherwise stated. Please ensure that you have obtained the appropriate Biohazard Approval for use of infectious bacterial pathogens as per institutional guidelines prior to initiation of the experiments. Additionally, please ensure that you have all the materials and reagents necessary to conduct the procedure prepared beforehand. Mice used in these experiments have included female C57BL/6 mice from Jackson Laboratories, Charles River or Taconic and were 10-14 weeks of age (although we have not found any gender-dependent significant differences in kinetics of nasal colonization clearance or infection). All mice used in these experiments were bred and maintained under specific-pathogen free conditions, and were free of common viruses, (LCMV, MNV,MPV, reovirus ECTV, and other) bacteria (e.g. H. pylori) and parasites (e.g. pinworm, ectoparasites) by fecal sample testing as well as frequent anatomical assessment of sentinel mice cohoused within their facility rooms. When conducting these experiments, we recommend using control mice no younger than 10-12 weeks of age and no older than 6 months of age. Mice younger or older than this age range are more susceptible to longer nasopharyngeal carriage duration and increased likelihood of disseminating infection. Mouse background is another important consideration that may impact the outcomes of a colonization experiment, as several groups have demonstrated that mice of different genetic backgrounds have different susceptibilities to the S. pneumoniae D39 (serotype 2) strain9,10. S. pneumoniae is not a naturally occurring murine pathogen and its only natural reservoir is the human nasopharynx. Transmission occurs via respiratory droplets, and as mice do not produce respiratory secretions, individual mice cannot transmit the bacterium to other mice, so there is no concern for mouse-to-mouse transmission11. For a visual overview of the procedures described within this manuscript, please refer to Figure 1.
1. Preparation of S. pneumoniae Culture
<ol>
	<li>Inoculate 5 ml of tryptic soy agar for the suspension growth of Streptococcus pneumoniae.</li>
	<li>Culture under static conditions at 37 &#176;C in 5% CO2 until the bacterial inoculum reaches log phase growth with a corresponding liquid density of 108 CFU/ml as determined by an odometer set to 600 nm. The exact reading corresponding to this CFU will differ depending on the specific selected bacterial strain; for most strains of S. pneumoniae this corresponds to an OD600 range of 0.45-0.55. Typically, S. pneumoniae strains in liquid culture will grow to this density within 1.5-2.5 hr under the recommended conditions, with no need for subculturing. The culture should not be allowed to overgrow (beyond an OD reading of 0.75) as this represents the point at which the bacteria are no longer in log phase growth and are undergoing extensive autolysis.</li>
	<li>Each mouse will be inoculated with approximately 107 bacteria. Therefore, for every 9 mice to be colonized, pipette 1 ml of inoculum into an Eppendorf tube and spin at 15,000 x g for 1 min. A whitish pellet should be visible. Remove the supernatant, being careful not to disturb the pellet and resuspend the bacteria in 100 &#956;l of phosphate buffered saline (PBS), thus increasing the concentration to 109 CFU/ml. At this stage, the bacteria should remain viable, but will not readily replicate.</li>
	<li>If using multiple aliquots, combine into one tube to control for slight inter-sample variations in bacterial density.</li>
	<li>Keep bacteria on ice until ready for inoculation, for a maximum of 1 hr.</li>
	<li>To obtain an exact bacterial count, perform log-wise serial dilution series starting with neat bacterial inoculum. Dilute serially 10-fold, adding 10 &#956;l of to 90 &#956;l of sterile PBS.</li>
	<li>Plate out 3 drops of 10 &#956;l samples of dilutions 10-5 - 10-9, plus a PBS-only contamination control, onto separately labeled sections of tryptic soy agar (TSA) plate supplemented with 5% sheep&#8217;s blood (Figure 2). Ensure that pipette tips are changed for each step diluting from a higher CFU concentration to a lower CFU concentration to avoid carrying over excess bacteria and increasing variability of results. Human blood agar (HBA) plates may also be used in lieu of TSA. Since many strains of S. pneumoniae are resistant to neomycin (from 5-20 &#956;g/ml), this antibiotic may also be added to the agar medium of choice during the plate preparation phase. This facilitates enumeration as it eliminates nonresistant bacteria. The antibiotic susceptibility of each strain must be tested in advance to determine the optimal concentration of antibiotic to use for each bacterial strain.</li>
	<li>Allow to dry for 15-30 min uncovered, then cover plates and place upside down in bacterial incubator set to 37 &#176;C and 5% CO2. Grow up bacterial colonies on plate for 24 hr.</li>
	<li>Determine number of colony forming units and their corresponding concentration. Based on determinations of the OD600 value, concentration should be within the range of 1-4 x 109 CFU/ml. S. pneumoniae colonies should appear as small, circular colonies a yellowish-beige in color, with a small depression at the center giving them a donut-like appearance (Figure 3).</li>
</ol>
2. Murine Intranasal Colonization
<ol>
	<li>Restrain mice by placing them into a mouse restrainer apparatus (a modified 50 ml Falcon tube with the tip cut off to create an aperture) securing them by the base of their body with thumb so that their noses just emerge out of tapered end of the restrainer apparatus (Figure 4). Use of this apparatus allows for immobilization of the mouse&#8217;s head and segregation of its nares in a manner that minimizes movement as well as impedes attempts from the animal to masticate the pipette tip, allowing for complete delivery of the inoculum. Alternatively, mice can be immobilized via scuffing at the neck and manual restraint. We do not recommend anesthesia of the animals prior to intranasal inoculation. Administrating the inoculum to animals under anesthesia results in some of the inoculum spreading to the lungs12,13.</li>
	<li>Using a P10 or P20 pipette, inoculate each mouse by depositing 10 &#956;l of the prepared culture, distributing it evenly between both nares (allow inoculum to drip into nose by pulsing the inoculation gradually, taking time for mice to inhale inoculum). To achieve complete delivery of the inoculum, pause administration at any point the mouse begins to move its nose excessively. The entire inoculum may not be injected into the nares as the mice may expel some through the nose during exhalation; however, as the expelled amount tends to be minuscule, and the inoculum contains an extremely high amount of bacteria, this does not significantly affect the colonizing bacterial load. Additionally, the surface area available for colonization in the nasopharyngeal mucosa is limited and consequently we and others have found that the recommended 107 dose is sufficient to obtain consistent levels of bacteria in all mice, resulting in minimal variability in initial amounts of colonizing bacteria14,15 .</li>
	<li>Weigh mice if utilizing weight indicators as part of your end-point monitoring. Monitor mice every 12-24 hr for clinical symptoms, including lethargy, ruffled fur and weight loss. Mice typically will not show symptoms of illness until 3-5 days post-colonization, and these will be preceded by weight loss which may average around 5% of total body weight a day. As the mice become increasingly ill they will assume hunched postures and show decreased activity and decreased responsiveness to stimulation, including handling. At this stage, illness is typically indicative of sepsis and/or pneumonia and will likely be terminal, although mice may be treated with 1 ml of subcutaneous saline daily to improve outcomes. Surviving mice should start showing improvement after day 7 post-colonization, as evidenced by stabilization of weight followed by weight gain, although different strains of S. pneumoniae may induce illness more quickly and result in progression of clinical symptoms along a different time-line. Please see Figure 5 for a representative result of weight tracked in mice colonized with the P1547 strain.</li>
</ol>
3. Nasal Lavage Sample Collection
Before beginning: prepare cannulated needles using 1 ml syringes capped with 26 3/8 G beveled needles. Cut 2.5 cm pieces of PE20 polyethylene tubing with an inner diameter 0.38 mm, ensuring that each end has a beveled tip. Using forceps, slide a 2.5 cm long piece of PE20 polyethylene tubing (inner diameter 0.38 mm) on to the needle tip, avoiding puncturing the tubing side. Cannulated needles can be kept in 70% ethanol until needed.
<ol>
	<li>Euthanize experimental mice. As cervical dislocation can potentially damage the trachea, this method of euthanasia must be avoided. Our preferred method is isoflurane anesthesia followed by exsanguination, however, ensure you follow institutional guidelines when selecting mode of euthanasia.</li>
	<li>Using 70% aqueous ethanol, sterilize the superoanterior fur of the animal, particularly the neck, taking care to prevent ethanol from accessing the nares.</li>
	<li>Make a single longitudinal cut along the midline of the neck of the animal, and two horizontal cuts on either end, creating an opening to envision the trachea.</li>
	<li>Carefully peel back skin to either side, revealing neck tissue beneath.</li>
	<li>Trachea should be visible, surrounded by longitudinal muscles on either side. Carefully snip these to provide a clear view of the trachea itself, taking care not to sever the surrounding vasculature.</li>
	<li>If the vasculature was cut and blood is present, prior to proceeding, allow bleeding to stop, and then cleanse the area several times by dispensing sterile PBS and using sterile gauze to gently soak up excess moisture in the area.</li>
	<li>Once trachea is properly exposed, make a transverse, semilunar cut in the trachea about half-way up (Figure 6).</li>
	<li>Draw up 1,000 &#956;l of sterilized PBS into previously prepared cannulated needle.</li>
	<li>Insert cannula into the trachea towards the nose, keeping the beveled edge pointing downwards for ease of insertion (Figure 7). Once needle is in place, rotate it 180&#176;, and gently probe upwards until you feel light resistance.</li>
	<li>Place Eppendorf designated for sample collection just beneath the nose of the mouse.</li>
	<li>Test correct placement of needle by dispensing a minimal (~20 &#956;l) amount of PBS lavage fluid - drop of fluid should form around the nares of the mouse; if this is the case, proceed to step 3.13).</li>
	<li>If test PBS emerges directly out the mouth of the animal, pull the cannula back, and reposition by again gently probing forward until very slight resistance is felt - take care not to push cannula too far past this resistance, as you will move it past the nasal palate and through to the oral cavity.</li>
	<li>Dispense contents of needle rapidly to help displace and collect maximal amount of cells - contents should flow out through the nares of the mouse and into collection tube. Place sample immediately on ice.</li>
	<li>To collect samples for RNA analysis, repeat steps 3.8-3.13) using a cannulated needle containing 500 &#956;l of RNA lysis buffer on the same mouse. This will allow the collection of lysate sample from the remaining cell populations, largely composed of the nasopharyngeal mucosal epithelium, as nonadherent cells should have been removed following the initial PBS lavage. Please note that the RNA lysis buffer will denude the epithelium and destroy surrounding tissue, so care must be taken to avoid contact with organs such as the lungs, if retention of these tissues is desired. Once collected, place sample in RNA lysis buffer directly on dry ice to snap-freeze. Once in lysis buffer, samples can be stored as per the manufacturer&#8217;s instructions, and are stable typically at -70 &#176;C for several months.</li>
</ol>
4. Determination of Bacterial Load in the Nasopharynx
<ol>
	<li>Quantitate bacteria by preparing a serial dilution series for each murine nasal lavage sample. In general, bacterial load can be expected to be between 0-104 CFU, therefore conduct three 10-fold serial dilutions. Add 10 &#956;l of the neat nasal lavage sample (100 CFU/ml) to the first tube to a concentration of 10-1 CFU/ml. Vortex thoroughly.</li>
	<li>Divide bacteriological plate into quadrants, and label quadrants each with a member of the dilution series (100-10-3 CFU/ml). Plate out 3 drops of 10 &#956;l samples of the 3 dilutions and the neat sample on tryptic soy agar plates supplemented with 5% sheep&#8217;s blood, as in Figure 2.</li>
	<li>Allow to dry for 15-30 min uncovered, then cover plates and place upside down in bacterial incubator with optimal conditions for bacterial growth (typically 37 &#176;C and 5% CO2).</li>
	<li>Grow up bacterial colonies on plate for 18-24 hr.</li>
	<li>Determine number of colonizing bacteria by averaging the colonies formed on plate for each dilution (Figure 3). Figure 8 demonstrates bacterial density during different timepoints, as determined by culture of nasal lavages, in mice colonized with 3 different strains of S. pneumoniae for up to 21 days.</li>
</ol>
5. Preparation of Samples for Flow Cytometery
Before beginning: Prepare mix of antibodies. For quantification of leukocyte populations, we recommend the following mix at the specified dilutions: PE-Ly6G (clone 1A8, 1 &#956;g/ml), FITC-Ly6C (clone AL-21,1 &#956;g/ml), eFluor 450-CD45 (clone 30-F11, 2.67 &#956;g/ml), APC-F4/80 (clone PM8 RUO, 0.67 &#956;g/ml), PerCP-Cy5.5-CD11c (clone N418 RUO, 0.5 &#956;g/ml), PE-Cy7-CD11b (clone M1/70, 0.33 &#956;g/ml), Alexa Fluor 700-CD3 (clone 1782, 4 &#956;g/ml), eFluor 605NC-CD4 (clone GK1.5, 6.67 &#956;g/ml). Please note that this mix is 2x concentration (see step 5.5). All antibodies should be diluted in FACs Wash buffer (0.5% fetal calf serum, 2mM EDTA, 0.1% sodium azide in PBS) which should also be prepared beforehand. In a mixture of isotype matched control antibodies, ideally from the same supplier as the labeled antibodies and at the same concentrations as the specific antibodies, should be prepared. The samples treated with the isotype control antibodies will function as the negative control. Any fluorescence observed in the samples treated with the isotype control antibodies should be considered background.
<ol>
	<li>Prechill a centrifuge capable of spinning 1.5 ml Eppendorf tubes to 4 &#176;C.</li>
	<li>Centrifuge nasal lavage samples at 2,000 x g for 10 min at 4 &#176;C. Carefully pipette out supernatant and reserve. Note: due to the small amount of cells within the nasopharynx, the cell pellet will not be visible unless there is undesired red blood cell contamination, which will bright red. If this is seen, sample should be discarded.</li>
	<li>Resuspend sample in 50 &#956;l of Fc?RIIb/CD16-2 (2.4G2) antibody (which binds Fc receptors and reduces nonspecific antibody binding) in FACs Wash Buffer at a concentration of 4 &#956;g/ml.</li>
	<li>Incubate sample on ice for 30 min.</li>
	<li>Add 50 &#956;l of preprepared 2x concentrated fluorescent antibody mix to sample. Set aside a representative sample from each experimental group to act as an isotype control. Add the isotype antibody mix to this sample in lieu of stain mix.</li>
	<li>Incubate sample on ice for 1 hr.</li>
	<li>Centrifuge samples at 2,000 x g for 10 min at 4 &#176;C. Discard supernatant and resuspend in 200 &#956;l of PBS.</li>
	<li>Repeat step 5.7.</li>
	<li>After the second wash, centrifuge samples again at 2,000 x g for 10 min at 4 &#176;C.</li>
	<li>Resuspend in either in PBS (if running sample immediately) or 2% paraformaldehyde (if running samples 1-3 days post-staining).</li>
	<li>When conducting flow cytometry collect the maximum amount of events per sample or until the entire sample has been aspirated. In uninfected, healthy, young mice, this will be as little as 1,000-2,000 total events; During a bacterial colonization event, this number may increase more than 2-to-5-fold&#160;dependeing on disease status in animal and factors such as age and genetic background. Figure 9 shows representative flow cytometry results collected from a 3-laser Becton Dickenson LSRII flow cytometer using a Forward Scatter of 450 and Side Scatter of 300, although we recommend optimizing parameters for the specific flow cytometer you intend to use prior to sample collection. Note: if a sample contains even trace amounts of blood contamination, the total events collected will be significantly higher than expected and the sample should be discounted from analysis.</li>
</ol>
6. Quantitative PCR (qPCR) Analysis of Nasal Lavages
<ol>
	<li>Thaw cell lysates from step 3.14 room temperature.</li>
	<li>Follow the recommended protocol provided with the preferred RNA extraction of choice.</li>
	<li>After completing RNA extraction procedure as instructed, quantify the amount of RNA using a spectrophotometer or electrophoresis based method (Figure 10). We routinely obtain between 975 and 3,250 ng total RNA per sample with a 260/280 nm ratio of &#62;1.7 or an RNA integrity number (RIN) around, 8.1&#177;0.13.</li>
	<li>Transcribe cDNA using the M-MULV Reverse Transcriptase according to the manufacturer&#8217;s protocol with 1,000 ng of RNA (maximum 13 &#956;l).</li>
	<li>Dilute resulting cDNA samples 8x, and aliquot equally into 4 separate tubes for longterm storage at -20 or -80 &#176;C.</li>
	<li>To measure gene expression by qPCR, prepare 25 &#956;l samples reactions in triplicate on ice or cold block containing: 12.5 &#956;l of 2x qPCR master mix from qPCR kit of your choice, 0.25 &#956;l reference dye, 2 &#956;l of diluted cDNA (step 7.5), 1 &#956;l of mixed forward and reverse primers (400 nM final), 9.25 &#956;l of RNAse-DNAse free water. This protocol is an adaptation of previously published methods16.</li>
	<li>In general we find that a two-step qPCR amplification ( 95 &#176;C for&#160;10 min followed by up to 40 cycles x [95 &#176;C x 15 sec, 60 &#176;C x 1 min]) is effective (Figure 11a); however, each primer pair must be optimized. Dissociation (melting) curves must be performed following amplification to ensure that no nonspecific amplification occurred. amplification (Figure 11b)</li>
	<li>We routinely run standard curves for each gene analyzed as well as a standard calibrator (derived from lung or spleen homogenate) for each 96-well plate analyzed. Relative transcript amounts are obtained by first normalizing raw cycle threshold (Ct) values by the reference dye and transforming the resultant values through the respective standard curve. These relative amounts are subsequently normalized to the standard calibrator and a housekeeping gene, as applicable.</li>
</ol>

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Dis. 187, 1424-1432 (2003).</li><li>Mohler, J., Azoulay-Dupis, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+pneumoniae+strain-dependent+lung+inflammatory+responses+in+a+murine+model+of+pneumococcal+pneumonia.">Streptococcus pneumoniae strain-dependent lung inflammatory responses in a murine model of pneumococcal pneumonia.</a> Intensive Care Med. 29, 808-816 (2003).</li><li>Wu, H. Y., Virolainen, A., Mathews, B., King, J., Russell, M. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+Streptococcus+pneumoniae+nasopharyngeal+colonization+model+in+adult+mice.">Establishment of a Streptococcus pneumoniae nasopharyngeal colonization model in adult mice.</a> Microb. Pathog. 23, 127-137 (1997).</li><li>Zhang, Z., Clarke, T. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+effectors+mediating+Th17-dependent+clearance+of+pneumococcal+colonization+in+mice.">Cellular effectors mediating Th17-dependent clearance of pneumococcal colonization in mice.</a> J. Clin. Invest. 119, 1899-1909 (2009).</li><li>Parker, D., Martin, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptococcus+pneumoniae+DNA+initiates+type+I+interferon+signaling+in+the+respiratory+tract.">Streptococcus pneumoniae DNA initiates type I interferon signaling in the respiratory tract.</a> MBio. 2, (2011).</li><li>Haya, D. L., Camilli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+identification+of+serotype+4+Streptococcus+pneumoniae+virulence+factors.">Large-scale identification of serotype 4 Streptococcus pneumoniae virulence factors.</a> Mol. Microbiol. 45, 1389-1406 (2002).</li><li>Nakamura, S., Favis, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+stimulation+of+type+I+interferons+during+influenza+virus+coinfection+promotes+Streptococcus+pneumoniae+colonization+in+mice.">Synergistic stimulation of type I interferons during influenza virus coinfection promotes Streptococcus pneumoniae colonization in mice.</a> J. Clin. Invest. 121, 3657-3665 (2011).</li><li>Kim, J. O., Weiser, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+intrastrain+phase+variation+in+quantity+of+capsular+polysaccharide+and+teichoic+acid+with+the+virulence+of+Streptococcus+pneumoniae.">Association of intrastrain phase variation in quantity of capsular polysaccharide and teichoic acid with the virulence of Streptococcus pneumoniae.</a> J. Infect. Dis. 177, 368-377 (1998).</li><li>Roche, A. M., King, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+attenuated+Streptococcus+pneumoniae+strains+induce+serotype-independent+mucosal+and+systemic+protection+in+mice.">Live attenuated Streptococcus pneumoniae strains induce serotype-independent mucosal and systemic protection in mice.</a> Infect. Immun. 75, 2469-2475 (2007).</li><li>Cohen, J. M., Khandavalli, S., Camberlein, E., Hyams, C., Baxendale, H. E., Brown, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+contributions+against+invasive+Streptococcus+pneumoniae+pneumonia+of+antibody+and+Th17-Cell+responses+to+nasopharyngeal+colonisation.">Protective contributions against invasive Streptococcus pneumoniae pneumonia of antibody and Th17-Cell responses to nasopharyngeal colonisation.</a> PLoS One. 6 (10), (2011).</li><li>Cohen, J. M., Khandavalli, S., Camberlein, E., Hyams, C., Baxendale, H. E., Brown, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+contributions+against+invasive+Streptococcus+pneumoniae+pneumonia+of+antibody+and+Th17-Cell+responses+to+nasopharyngeal+colonisation.">Protective contributions against invasive Streptococcus pneumoniae pneumonia of antibody and Th17-Cell responses to nasopharyngeal colonisation.</a> PLoS One. 6 (10), (2011).</li><li>Richards, L., Ferreira, D. M., Miyaji, E. N., Andrew, P. W., Kadioglu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immunising+effect+of+pneumococcal+nasopharyngeal+colonisation;+protection+against+future+colonisation+and+fatal+invasive+disease.">The immunising effect of pneumococcal nasopharyngeal colonisation; protection against future colonisation and fatal invasive disease.</a> Immunobiology. , 215-251 (2010).</li><li>Lanie, J. A., Ng, W. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+sequence+of+Avery's+virulent+serotype+2+strain+D39+of+Streptococcus+pneumoniae+and+comparison+with+that+of+unencapsulated+laboratory+strain+R6.">Genome sequence of Avery's virulent serotype 2 strain D39 of Streptococcus pneumoniae and comparison with that of unencapsulated laboratory strain R6.</a> J. Bacteriol. 189, 38-51 (2007).</li><li>Robertson, G. T., Ng, W. L., Foley, J., Gilmour, R., Winkler, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+transcriptional+analysis+of+clpP+mutations+of+type+2+Streptococcus+pneumoniae+and+their+effects+on+physiology+and.">Global transcriptional analysis of clpP mutations of type 2 Streptococcus pneumoniae and their effects on physiology and.</a> 184, 3508-3520 (2002).</li><li>Orihuela, C. J., Gao, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+contributions+of+pneumococcal+virulence+factors+to+pathogenesis.">Tissue-specific contributions of pneumococcal virulence factors to pathogenesis.</a> J. Infect. Dis. 190, 1661-1669 (2004).</li><li>Orihuela, C. J., Gao, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ-specific+models+of+Streptococcus+pneumoniae+disease.">Organ-specific models of Streptococcus pneumoniae disease.</a> Scand. J. Infect. D. 35, 647-652 (2003).</li><li>Swirski, F. K., Nahrendorf, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+splenic+reservoir+monocytes+and+their+deployment+to+inflammatory+sites.">Identification of splenic reservoir monocytes and their deployment to inflammatory sites.</a> Science. 325, 612-616 (2009).</li></ol>

The authors have nothing to disclose.

In this study we presented detailed methods for the intranasal colonization of mice using a clinical isolate strain of Streptococcus pneumoniae and the subsequent isolation and characterization of the immune cells recruited to nasopharynx in response to the bacteria. We demonstrated how a bacterial inoculum can be cultured in nutrient-rich media and used to establish a colonization event in mice, which is initially restricted to the nasopharynx. We then showed how responding immune cell types that are recruited ...

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characterization of inflammatory responses during intranasal colonization with streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Figure 1 represents an overview schematic summarizing the main steps of the protocol. Figures 2-3 provide visualization of the microbiological methodology inherent to the protocols described herein. Figure 4 represents proper positioning of a mouse to perform an intranasal colonization, while Figure 5 depicts typically changes in weight of mice colonized with S. pneumoniae strain P1547. Figures 6-7 represent specific stages of t...

Watch this Scientific Journal Video about Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae at JoVE.com

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

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Immunology and Infection

12.1K Views.  McMaster University. Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Video: Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin synthesis. They also supply the host with a considerable amount of nutrients, making this ecosystem an essential metabolic organ. In the context of increasing evidence of the link between the gut flora and the metabolic syndrome, understanding the metabolic interaction between the host and its gut microbiota is becoming an important challenge of modern biology.1-4
 Colonization (also referred to as normalization process) designates the establishment of micro-organisms in a former germ-free animal. While it is a natural process occurring at birth, it is also used in adult germ-free animals to control the gut floral ecosystem and further determine its impact on the host metabolism. A common procedure to control the colonization process is to use the gavage method with a single or a mixture of micro-organisms. This method results in a very quick colonization and presents the disadvantage of being extremely stressful5. It is therefore useful to minimize the stress and to obtain a slower colonization process to observe gradually the impact of bacterial establishment on the host metabolism.
 In this manuscript, we describe a procedure to assess the modification of hepatic metabolism during a gradual colonization process using a non-destructive metabolic profiling technique. We propose to monitor gut microbial colonization by assessing the gut microbial metabolic activity reflected by the urinary excretion of microbial co-metabolites by 1H NMR-based metabolic profiling. This allows an appreciation of the stability of gut microbial activity beyond the stable establishment of the gut microbial ecosystem usually assessed by monitoring fecal bacteria by DGGE (denaturing gradient gel electrophoresis).6 The colonization takes place in a conventional open environment and is initiated by a dirty litter soiled by conventional animals, which will serve as controls. Rodents being coprophagous animals, this ensures a homogenous colonization as previously described.7
 Hepatic metabolic profiling is measured directly from an intact liver biopsy using 1H High Resolution Magic Angle Spinning NMR spectroscopy. This semi-quantitative technique offers a quick way to assess, without damaging the cell structure, the major metabolites such as triglycerides, glucose and glycogen in order to further estimate the complex interaction between the colonization process and the hepatic metabolism7-10. This method can also be applied to any tissue biopsy11,12.

Assessing Hepatic Metabolic Changes During Progressive Colonization of Germ-free Mouse by 1H NMR Spectroscopy

A progressive colonization procedure is described to further assess its impact on the host hepatic metabolism. Colonization is monitored non invasively by evaluating the urinary excretion of microbial co-metabolites by NMR-based metabolic profiling while hepatic metabolism is assessed by High Resolution Magic Angle Spinning (HR MAS) NMR profiling of intact biopsy.

It is well known that gut bacteria contribute significantly to the host homeostasis, providing a range of benefits such as immune protection and vitamin ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact cellular functions, including energetics and signal transductions. While both fluorescent and electrophysiological methods, including electrode usage and patch-clamping, have been well developed for measuring these events in eukaryotic cells, methodology for measuring similar events in microorganisms have proven more challenging to develop given their small size in combination with the more complex outer surface of bacteria shielding the membrane. During our studies of death-initiation in Streptococcus pneumoniae (pneumococcus), we wanted to elucidate the role of membrane events, including changes in polarity, integrity, and intracellular ion concentrations. Searching the literature, we found that very few studies exist. Other investigators had monitored radioisotope uptake or equilibrium to measure ion fluxes and membrane potential and a limited number of studies, mostly in Gram-negative organisms, had seen some success using carbocyanine or oxonol fluorescent dyes to measure membrane potential, or loading bacteria with cell-permeant acetoxymethyl (AM) ester versions of ion-sensitive fluorescent indicator dyes. We therefore established and optimized protocols for measuring membrane potential, rupture, and ion-transport in the Gram-positive organism S. pneumoniae. We developed protocols using the bis-oxonol dye DiBAC4(3) and the cell-impermeant dye propidium iodide to measure membrane depolarization and rupture, respectively, as well as methods to optimally load the pneumococci with the AM esters of the ratiometric dyes Fura-2, PBFI, and BCECF to detect changes in intracellular concentrations of Ca2+, K+, and H+, respectively, using a fluorescence-detection plate reader. These protocols are the first of their kind for the pneumococcus and the majority of these dyes have not been used in any other bacterial species. Though our protocols have been optimized for S. pneumoniae, we believe these approaches should form an excellent starting-point for similar studies in other bacterial species.

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Unlike that seen for eukaryotes, there is a paucity of studies that detail membrane depolarization and ion concentration changes in bacteria, primarily as their small size makes conventional methods of measurement difficult. Here, we detail protocols for monitoring such events in the significant Gram-positive pathogen Streptococcus pneumoniae utilizing fluorescence techniques.

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal epidemiology, capsular serotyping can provide useful information for vaccine efficacy and impact studies.&#160;The Quellung reaction is the gold standard method for pneumococcal capsular serotyping. The method involves testing a pneumococcal cell suspension with pooled and specific antisera directed against the capsular polysaccharide. The antigen-antibody reactions are observed microscopically. The protocol has three main steps: 1) preparation of a bacterial cell suspension, 2) mixing of cells and antisera on a glass slide, and 3) reading the Quellung reaction using a microscope. The Quellung reaction is reasonably simple to perform and can be applied wherever a suitable microscope and antisera are available.

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

The Quellung reaction is the gold standard technique for serotyping Streptococcus pneumoniae. This technique utilizes a microscope and specific pneumococcal antisera and is commonly used in reference and research laboratories worldwide.

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the &#8216;gold standard&#8217; Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. 
This method of production and quality control may also be suitable for other testing purposes.

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major ...

A Murine Model of Group B Streptococcus Vaginal Colonization

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of 10 - 30% of adults. In immune-compromised individuals, including neonates, pregnant women, and the elderly, GBS may switch to an invasive pathogen causing sepsis, arthritis, pneumonia, and meningitis. Because GBS is a leading bacterial pathogen of neonates, current prophylaxis is comprised of late gestation screening for GBS vaginal colonization and subsequent peripartum antibiotic treatment of GBS-positive mothers. Heavy GBS vaginal burden is a risk factor for both neonatal disease and colonization. Unfortunately, little is known about the host and bacterial factors that promote or permit GBS vaginal colonization. This protocol describes a technique for establishing persistent GBS vaginal colonization using a single &#946;-estradiol pre-treatment and daily sampling to determine bacterial load. It further details methods to administer additional therapies or reagents of interest and to collect vaginal lavage fluid and reproductive tract tissues. This mouse model will further the understanding of the GBS-host interaction within the vaginal environment, which will lead to potential therapeutic targets to control maternal vaginal colonization during pregnancy and to prevent transmission to the vulnerable newborn. It will also be of interest to increase our understanding of general bacterial-host interactions in the female vaginal tract.

A Murine Model of Group B Streptococcus Vaginal Colonization

The purpose of this protocol is to imitate human group B Streptococcus (GBS) vaginal colonization in a murine model. This method may be used to investigate host immune responses and bacterial factors contributing to GBS vaginal persistence, as well as to test therapeutic strategies.

Streptococcus agalactiae (group B Streptococcus, GBS), is a Gram-positive, asymptomatic colonizer of the human gastrointestinal tract and vaginal tract of ...

Increased Recovery Time and Decreased LPS Administration to Study the Vagus Nerve Stimulation Mechanisms in Limited Inflammatory Responses

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release cytokines and other mediators of inflammation. For a long time, humoral and cellular mechanisms have been studied for their role in regulating the immune response, but recent advances in the field of immunology and neuroscience have also unraveled specific neural mechanisms with interesting therapeutic potential. The so-called cholinergic anti-inflammatory pathway (CAP) has been described to control innate immune responses and inflammation in a very potent manner. In the early 2000s, Tracey and collaborators developed a technique that stimulates the vagus nerve and mimics the effect of the pathway. The methodology is based on the electrical stimulation of the vagus nerve at low voltage and frequency, in order to avoid any side effects of overstimulation, such as deregulation of heart rate variability. Electrical devices for stimulation are now available, making it easy to set up the methodology in the laboratory. The goal of this research was to investigate the potential involvement of prostaglandins in the CAP. Unfortunately, based on earlier attempts, we failed to use the original protocol, as the induced inflammatory response either was too high or was not suitable for enzymatic metabolism properties. The different settings of the original surgery protocol remained mostly unchanged, but the conditions regarding inflammatory induction and the time point before sacrifice were improved to fit our purposes (i.e., to investigate the involvement of the CAP in more limited inflammatory responses). 
The modified version of the original protocol, presented here, includes a longer time range between vagus nerve stimulation and analysis, which is associated with a lower induction of inflammatory responses. Additionally, while decreasing the level of lipopolysaccharides (LPS) to inject, we also came across new observations regarding mechanistic properties in the spleen.

Vagus nerve stimulation has proven to have a strong efficacy for decreasing peripheral inflammation. Here, we present a modified vagus nerve stimulation protocol that allows for further examinations of the cholinergic anti-inflammatory mechanisms in limited inflammatory responses.

Inflammation is a local response to infection and tissue damage mediated by activated macrophages, monocytes, and other immune cells that release ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the pro-inflammatory capabilities of various factors is challenging and requires terminal procedures, such as bronchoalveolar lavage and the removal of lungs for in situ analysis, precluding longitudinal visualization in the same mouse. Here, lung inflammation is induced through the intratracheal instillation of Pseudomonas aeruginosa culture supernatant (SN) in transiently transgenized mice expressing the luciferase reporter gene under the control of a heterologous IL-8 bovine promoter. Luciferase expression in the lung is monitored by in vivo bioluminescent image (BLI) analysis over a 2.5- to 48-h timeframe following the instillation. The procedure can be repeated multiple times within 2 - 3 months, thus permitting the evaluation of the inflammatory response in the same mice without the need to terminate the animals. This approach permits the monitoring of pro- and anti-inflammatory factors acting in the lung in real time and appears suitable for functional and pharmacological studies.

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

The method described here allows for the visualization of IL-8 promoter-dependent inflammation activation in the lungs of mice through non-invasive bioluminescence imaging (BLI). The same animal can be subjected to BLI multiple times for up to two months from the time of delivery of the luciferase reporter construct.

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Ca2+ is a ubiquitous ion involved in all known cellular processes. While global Ca2+ responses may affect cell fate, local variations in free Ca2+ cytosolic concentrations, linked to release from internal stores or an influx through plasma membrane channels, regulate cortical cell processes. Pathogens that adhere to or invade host cells trigger a reorganization of the actin cytoskeleton underlying the host plasma membrane, which likely affects both global and local Ca2+ signaling. Because these events may occur at low frequencies in a pseudo-stochastic manner over extended kinetics, the analysis of Ca2+ signals induced by pathogens raises major technical challenges that need to be addressed.
Here, we report protocols for the detection of global and local Ca2+ signals upon a Shigella infection of epithelial cells. In these protocols, artefacts linked to a prolonged exposure and photodamage associated with the excitation of Ca2+ fluorescent probes are troubleshot by stringently controlling the acquisition parameters over defined time periods during a Shigella invasion. Procedures are implemented to rigorously analyze the amplitude and frequency of global cytosolic Ca2+ signals during extended infection kinetics using the chemical probe Fluo-4.

Imaging Ca2+ Responses During Shigella Infection of Epithelial Cells

Here, we present protocols to visualize calcium (Ca2+) responses elicited by HeLa cells infected by Shigella. By optimizing the parameters of bacterial infection and imaging with Ca2+ fluorescent probes, atypical global and local Ca2+ signals induced by bacteria over a large range of infection kinetics are characterized.