Research in Bragonzi&#8217;s laboratory has been funded by the Italian Cystic Fibrosis Foundation (CFaCore) and EU-F7-2009-223670. Part of this work was carried out in ALEMBIC, an advanced microscopy laboratory, and mouse histopathology was performed in the Unit of Pathological Anatomy (San Raffaele Scientific Institute).

1. Preparing Bacteria for Chronic Infection (Three and Two Days prior to Mouse Challenge)
<ol>
	<li>Select the appropriate P. aeruginosa strain to be tested.</li>
	<li>Inoculate a loopful of P. aeruginosa from a -80 &#176;C stock culture to a Trypticase Soy Agar (TSA) plate and incubate at 37 &#176;C overnight.</li>
	<li>Pick a single colony and inoculate into 5 ml Trypticase Soy Broth (TSB) in a 15 ml snap-capped tube and incubate at 37 &#176;C overnight in a shaking incubator at 200 rpm.</li>
</ol>
2. Embedding&#160;Bacteria in Agar Beads for Chronic Infection (One Day prior to Infection)
<ol>
	<li>Take a small aliquot of bacterial overnight culture, dilute 1:50 in phosphate buffered saline (PBS) and measure the optical density (OD) at 600 nm.</li>
	<li>Dilute the overnight bacterial culture by adding 2 OD in a new snap-capped tube containing 20 ml of fresh TSB.</li>
	<li>Incubate at 37 &#176;C for approximately 3-4 hr to log phase in a shaking incubator at 200 rpm, until a total of 10-15 OD is reached.</li>
	<li>In the meantime, prepare the TSA, made of TSB with 1.5% agar, autoclave and equilibrate at 50 &#176;C in a water bath. Equilibrate 150 ml of preautoclaved heavy mineral oil in an Erlenmeyer flask at 50 &#176;C in a water bath.</li>
	<li>Once P. aeruginosa reaches the log phase, collect the bacterial cells by centrifugation at 2,700 x g for 15 min at 4 &#176;C and discard the supernatant.</li>
	<li>Resuspend the bacterial pellet in 1 ml of sterile PBS and vortex thoroughly to resuspend the pellet completely.</li>
	<li>Mix 1 ml bacterial suspension with 9 ml of liquid TSA pre-equilibrated at 50 &#176;C.</li>
	<li>Add the 10 ml TSA-P. aeruginosa mixture to heavy mineral oil (prewarmed at 50 &#176;C) and immediately stir for 6 min at room temperature. The agitation must produce a visible vortex in the oil.</li>
	<li>Cool the mixture to 4 &#176;C, stirring at the minimum speed for 35 min&#160;(Figure 1).</li>
	<li>Rest the agar-beads-oil mixture in ice for an additional 20 min.</li>
	<li>Transfer the agar-beads into 50 ml Falcon tubes and centrifuge at 2,700 x g for 15 min at 4 &#176;C.</li>
	<li>Accurately remove mineral oil and wash with sterile PBS six times, as described in step 2.11. After three washes, the beads can be pelleted by gravity instead of using the centrifuge. After the last wash, resuspend the agar-beads in 20-30 ml PBS.</li>
	<li>Take an aliquot of the beads (approximately 0.5 ml) and aseptically homogenize.</li>
	<li>Take 100 &#181;l of the homogenized beads and dilute in 900 &#181;l of sterile PBS. Serially dilute 1:10 down to 10-6.</li>
	<li>Plate serial dilution on TSA plates, including undiluted sample down to 10-6 and incubate plates at 37 &#176;C.</li>
	<li>Measure the bead diameter using an inverted light microscope in several fields. The bead diameter must be between 100-200 &#181;m (Figure 1).</li>
	<li>Store the beads overnight at 4 &#176;C.</li>
</ol>
<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/51019/51019fig1highres.jpg" src="/files/ftp_upload/51019/51019fig1.jpg" width="500px" /> 
Figure 1. Overview of the agar beads preparation and mouse infection. P. aeruginosa cells are resuspended in 1 ml of PBS and added to 9 ml of liquid TSA (50 &#176;C). This mixture is added to 150 ml heavy mineral oil at 50 &#176;C in a flask and stirred at high speed for 6 min at room temperature. When the flask is cooled to 4 &#176;C with a slow stirring for 35 min, the agar solidifies creating beads, and bacteria present in the mixture are embedded into the agar beads. Detail of an agar bead containing bacterial cells is shown (A). After removing the mineral oil with several washes using sterile PBS, the agar-beads suspension is ready for inoculation in the lungs of mice by an intratracheal injection (B). <a href="https://www.jove.com/files/ftp_upload/51019/51019fig1highres.jpg" target="_blank">Click here to view larger image</a>.
3. Mice Challenge with Agar-beads
Ethics Statement: This protocol and experimentation follow the guidelines from the animal care and ethics committee of San Raffaele Scientific Institute.
<ol>
	<li>Count the number of Colony Forming Units (CFU) on the TSA plates to determine the number of CFU/ml in the agar-beads suspension. Dilute the agar-beads with sterile PBS to 2-4 x 107 CFU/ml to reach an optimal inoculum of 1-2 x 106 in 50 &#181;l.</li>
	<li>Anesthetize C57Bl/6NCr (20-22 g, 6-8 weeks old) male mice with ketamine (50 mg/ml) and xylazine (5 mg/ml) in 0.9 % NaCl administered at a volume of 0.002 ml/g body weight by intraperitoneal injection. 
	NOTE: Anesthesia is considered adequate when the animal stays still quietly, is unresponsive to external stimuli, and has constant heart and respiratory rates.</li>
	<li>Place the mouse in supine position. Disinfect the coat of the mouse with 70% ethanol.</li>
	<li>Expose the trachea by a vertical cut of the skin and intubate the trachea with a sterile, flexible 22 G&#160;0.9 mm x 25 mm I.V. catheter, keeping attention to remove the stylette while moving down into the trachea. Insert the catheter not too deep into the trachea. Stop before reaching the carina (bifurcation).</li>
	<li>Immediately take a volume of 50 &#181;l of agar bead suspension by a 1 ml syringe and attach it to the catheter. Gently push the plunger of the syringe, allowing the beads to be implanted into the lung. Close the incision using suture clips.</li>
	<li>Place the animal on a heating pad until fully awake.</li>
</ol>
4. Mice Evaluation
<ol>
	<li>Observe the mice daily for clinical signs including coat quality, posture, ambulation, and hydration status. Monitor daily body weight. Mice that lose &#8805;20% body weight must be euthanized.</li>
	<li>Follow Point 5 for collection of bronchoalveolar lavage fluid (BAL) and Points 6 or 7 for the collection of lungs and the analysis of total CFU, histological analysis, inflammatory response in terms of total and differential cell count in the BAL, cytokine analysis, and myeloperoxidase (MPO) activity.</li>
</ol>
5. BAL Fluid Collection and Analysis
<ol>
	<li>Euthanize the mice by CO2 inhalation.</li>
	<li>Place the mouse in the supine position. Disinfect the coat of the mouse with 70% ethanol.</li>
	<li>Expose the trachea and the thoracic cage by a vertical cut of the skin. Expose the lungs by cutting the diaphragm.</li>
	<li>Insert a suture thread under the trachea using tweezers and intubate the trachea with a sterile, flexible 22&#160;g 0.9 mm x 25 mm I.V. catheter. Pull the two ends of the suture thread to bind the catheter to the trachea and knot the thread around the trachea.</li>
	<li>Take a volume of 1 ml of RPMI 1640 using a 1 ml syringe and attach it to the catheter. Push the plunger of the syringe to wash the lungs and immediately recover the liquid, storing it in a 15 ml tube. 
	NOTE: If cytokines are to be analyzed, add protease inhibitors to RPMI 1640.</li>
	<li>Repeat this step three times with a total of 3 ml of RPMI. From now on, store the BAL fluid on ice. Go to step 6 for the collection and analysis of lungs. 
	NOTE: Please note that not all of the liquid will be retrieved (2.8 ml maximum).</li>
	<li>For quantification of bacteria present in the BAL fluid, sample a small aliquot (300 &#181;l), serially dilute 1:10 in sterile PBS, plate on TSA plates and incubate at 37 &#176;C overnight.</li>
	<li>Count total cells using an inverted light optical microscope diluting an aliquot of the BAL fluid 1:2 with Tuerk solution in a Burker cell count chamber.</li>
	<li>Centrifuge the remaining BAL fluid at 330 x g for 8 min at 4 &#176;C. Take the supernatant for ELISA cytokine analysis, storing it at -80 &#176;C. Follow steps 5.10-5.13 for differential cell count by cytospin.</li>
	<li>If the pellet is red, lyse the erythrocytes resuspending the pellet in 250-300 &#181;l of RBC lysis buffer diluted 1:10 in ultra-pure distilled water for 3 min. Neutralize with 2 ml PBS and centrifuge at 330 x g for 8 min at 4 &#176;C.</li>
	<li>Discard the supernatant and resuspend the pellet in RPMI 10% fetal bovine serum (FBS). Use a volume that will provide 1 x 106 cells/ml, based on the total cell count.</li>
	<li>Place microscope slides and filters into appropriate slots in the cytospin with the cardboard filters facing the center of the cytospin. Pipette 150 &#181;l of each sample into the appropriate wells of the cytospin and centrifuge in a cytocentrifuge at 300 x g for 5 min.</li>
	<li>Stain slides by Romanowsky staining using a commercial kit, according to the manufacturer&#39;s instructions and as described previously12. Follow steps 5.14-5.17 for MPO activity analysis.</li>
	<li>Centrifuge the remaining volume of BAL at 380 x g for 5 min at 4 &#176;C. Discard the supernatant and resuspend the pellet in 250 &#181;l of Hexadecyltrimethylammonium chloride 0.5% in ultra-pure distilled water to lyse the cells. The suspension can be frozen at -20 &#176;C for several days before performing the assay.</li>
	<li>Centrifuge at 16,000 x g for 30 min at 4 &#176;C and use the supernatant to perform MPO assay in 96-well plates, adding the sample in duplicate to each well and if needed, also proper dilutions of the sample.</li>
	<li>Add to each sample in the wells an equal volume of 3,3&#39;,5,5&#39;-tetramethylbenzidine (TMB) as a substrate for peroxidase. Allow the reaction to take place in the dark for at least 5 min and until there is no further development in the color.</li>
	<li>Stop the reaction by adding 2 M H2SO4 and measure the OD at 450 nm. The OD value will be directly proportional to peroxidase activity.</li>
</ol>
6. Measurement of Bacterial Load in Lung and Cytokine Analysis
<ol>
	<li>Immediately after BAL fluid collection, excise lungs from the mouse, rinse them in sterile PBS, separate lobes, put them in a round-bottom tube with 2 ml of sterile PBS and store on ice. 
	NOTE: If cytokines are to be analyzed, add protease inhibitors to PBS to the tubes.</li>
	<li>Aseptically homogenize lungs, take a small aliquot (300 &#181;l) from the homogenate, serially dilute 1:10 in PBS, plate on TSA plates and incubate at 37 &#176;C overnight. Please note that the total airway bacterial load will be the sum of the CFUs found in BAL fluid and lung.</li>
	<li>Centrifuge the remaining homogenate at 16,000 x g for 30 min at 4 &#176;C. Take the supernatant for ELISA cytokine analysis, and store at -80 &#176;C.</li>
</ol>
7. Histological Examination
<ol>
	<li>Perform histological analysis only on the lungs in which BAL fluid has not been collected, to preserve lung aspect and properties.</li>
	<li>Euthanize the mouse by CO2 inhalation.</li>
	<li>Expose the thoracic cage by a vertical cut of the skin, expose the lungs by cutting the diaphragm.</li>
	<li>Excise lungs, rinse them in PBS, separate the lobes, put them in a tube containing 5-10 ml 10% neutral buffered formalin (4% formaldehyde) and store at 4 &#176;C protected from light.</li>
	<li>Embed lungs in paraffin, using standard procedures.</li>
	<li>Cut 5 &#181;m&#160;thick sections using a microtome.</li>
	<li>Stain slides with hematoxylin and eosin and cover the slides with a coverslip, according to standard procedures.</li>
	<li>Examine slides using an inverted brightfield microscope and acquire images by connecting the microscope to a camera.</li>
</ol>

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The authors declare that they have no competing financial interests.

The critical steps in the P. aeruginosa-beads preparation and mouse challenge are reported below.
The P. aeruginosa strain used for mice challenge is critical. Mortality, chronic infection or clearance may differ significantly depending on the bacterial strain used for the challenge. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chron...

Cystic fibrosis (CF) is a genetic disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. This gene encodes for a chloride channel expressed on the membrane of most epithelial cells. Bronchiectasis, mucus plugging and parenchymal destruction caused mainly by Pseudomonas aeruginosa infections progressively lead to severe lung disease and mortality in most of the CF patients1. Understanding CF pathogenesis and further development of novel therapies rely on animal model with characteristic features of CF. Several mice, genetically modified for the Cftr gene, have been generated, but limitations in the ability of these species to recapitulate CF-like lung disease and several other organ abnormalities seen in CF patients have been widely documented2.
	
	Development of infection is one of the major challenges in CF animal model. The literature clearly suggests that a chronic infection lasting more than one month can be achieved only if mice are inoculated with bacteria embedded in an immobilizing agent such as agar, agarose, or seaweed alginate3-5. These immobilizing agents provide the microaerobic/anaerobic conditions that allow bacteria to grow in the form of microcolonies, similarly to the growth in the mucus of CF patients6. This model of chronic infection leads to the persistence of the bacteria in the lungs causing airway inflammation and damage7. However, depending on the method used, the bacterial strain and the dose inoculated in the lungs, the percentage of chronic infected mice and the bacterial load recovered in the lungs at different time points can differ considerably. In particular, the main challenge for long-term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host. By selecting the P. aeruginosa RP73 clinical strain from a collection of CF isolates8 we successfully obtained low mortality, more severe lesions, and high percentage of chronic infection with a stable bacterial load up to one month in C57Bl/6NCrl mice.
	
	This paper details the methodology for embedding P. aeruginosa in the agar beads; we have infected mice by intratracheal instillation, measured the bacterial load and cytokines in lungs, collected BAL fluid and performed histological examination. Overall, this protocol will aid researchers in addressing fundamentally important questions on pathogenesis8,9 and testing novel therapies against P. aeruginosa chronic infection10,11.

<table border="0" cellpadding="0" cellspacing="0" width="605">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Bacto Tryptic Soy Broth</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:143px;">211823</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Difco Agar, granulated</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:143px;">214510</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Heavy mineral oil</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:143px;">330760-1L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">S-(+)-Ketamine hydrochloride</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:143px;">K1884</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylazine hydrochloride</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:143px;">X1251</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:272px;">1 ml syringe 25 G 5/8&#39;&#39;&#160;0.5 x 16 mm</td>
			<td style="width:191px;">PIC</td>
			<td style="width:143px;">3071250300350</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:272px;">Catheter 22GA 0.9 x 25 mm</td>
			<td style="width:191px;">Becton Dickinson</td>
			<td style="width:143px;">381223</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:272px;">Graefe Forceps - 0.5 mm Tips Curved</td>
			<td style="width:191px;">Fine Science Tools</td>
			<td style="width:143px;">11152-10</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:272px;">Scissors, Iris, 11 cm, straight</td>
			<td style="width:191px;">World Precision Instruments</td>
			<td style="width:143px;">501758</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Suture clips</td>
			<td style="width:191px;">Fine Science Tools</td>
			<td style="width:143px;">12040-01</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Suture thread</td>
			<td style="width:191px;">Fine Science Tools</td>
			<td style="width:143px;">18020-40</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">RPMI 1640</td>
			<td style="width:191px;">Lonza</td>
			<td style="width:143px;">BE12-167F</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Complete protease inhibitor cocktail</td>
			<td style="width:191px;">Roche</td>
			<td style="width:143px;">11836145001</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:272px;">Fast-Read 102 Burker disposable chamber</td>
			<td style="width:191px;">Biosigma</td>
			<td style="width:143px;">390497</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Tuerk solution</td>
			<td style="width:191px;">Fluka</td>
			<td style="width:143px;">93770</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">RBC lysis buffer</td>
			<td style="width:191px;">Biolegend</td>
			<td style="width:143px;">420301</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Fetal bovine serum</td>
			<td style="width:191px;">Lonza</td>
			<td style="width:143px;">DE14-801F</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">EZ cytofunnel</td>
			<td style="width:191px;">Thermo Scientific</td>
			<td style="width:143px;">A78710021</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:272px;">Superfrost ultra plus microscope slides</td>
			<td style="width:191px;">Thermo Scientific</td>
			<td style="width:143px;">J3800AMNZ</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Diff-Quik Romanowsky staining set</td>
			<td style="width:191px;">Medion Diagnostics</td>
			<td style="width:143px;">130832</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:272px;">Hexadecyltrimethylammonium chloride</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:143px;">52366-10G</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">96-well EIA/RIA plate</td>
			<td style="width:191px;">Costar</td>
			<td style="width:143px;">3590</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">3,3&#8217;,5,5&#8217;- tetramethylbenzidine</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:143px;">T8665-1L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Sulfuric acid</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:143px;">320501-1L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">10% neutral buffered formalin</td>
			<td style="width:191px;">Bio-optica</td>
			<td style="width:143px;">05-01005Q</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Harris haematoxylin non Papanicolau</td>
			<td style="width:191px;">Bio-optica</td>
			<td style="width:143px;">05-M06004</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:272px;">Eosin plus alcoholic solution</td>
			<td style="width:191px;">Bio-optica</td>
			<td style="width:143px;">05-M11007</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
<td>[header]</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;"></td>
			<td style="width:191px;"></td>
			<td style="width:143px;"></td>
<td>Equipment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Shaking incubator</td>
			<td style="width:191px;">Amerex Instruments</td>
			<td style="width:143px;">Steady Shake 757</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Water bath</td>
			<td style="width:191px;">Grant</td>
			<td style="width:143px;">SUB14</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Homogenizer</td>
			<td style="width:191px;">Ystral</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Precision balance</td>
			<td style="width:191px;">KERN</td>
			<td style="width:143px;">440-47N</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Cytocentrifuge</td>
			<td style="width:191px;">Thermo Scientific</td>
			<td style="width:143px;">A78300003</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Low Cost Heating Pad</td>
			<td style="width:191px;">2biol</td>
			<td style="width:143px;">LCHP</td>
		</tr>
		<tr height="21">
			<td height="42" rowspan="2" style="height:42px;width:272px;">Homogenization probe</td>
			<td rowspan="2" style="width:191px;">Ystral</td>
			<td style="width:143px;">2366931(great)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:143px;">2366925(small)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Inverted optical microscope</td>
			<td style="width:191px;">Zeiss</td>
			<td style="width:143px;">Axioplan2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Camera (microscope)</td>
			<td style="width:191px;">Zeiss</td>
			<td style="width:143px;">Axiocam MRc5</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:272px;">Rotary microtome</td>
			<td style="width:191px;">Leica</td>
			<td style="width:143px;">RM2255</td>
		</tr>
	</tbody>
</table>

long term chronic pseudomonas aeruginosa airway infection in mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa clinical strains in the agar-beads in vitro, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.

When the protocol is done correctly, the P. aeruginosa agar-beads will measure between 100-200 &#181;m and can be observed with an inverted light microscope by pipetting a small volume of the agar-beads suspension on a slide. Single bacterial cells are visible in the agar beads, as shown in detail in Figure 1.
The choice of P. aeruginosa strain used in the agar-beads preparation is critical. Figure 2 and Table 1 show the data...

Watch this Scientific Journal Video about Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice at JoVE.com

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model ...

long-term-chronic-pseudomonas-aeruginosa-airway-infection-in-mice

Research

JoVE Journal

Immunology and Infection

22.3K Views.  San Raffaele Scientific Institute. We describe the agar-beads method to establish persistent long-term chronic Pseudomonas aeruginosa airway infection in the mouse model.

Video: Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living surfaces. In this paper, we describe protocols for forming Pseudomonas aeruginosa biofilms on human airway epithelial cells (CFBE cells) grown in culture. In the first method (termed the Static Co-culture Biofilm Model), P. aeruginosa is incubated with CFBE cells grown as confluent monolayers on standard tissue culture plates. Although the bacterium is quite toxic to epithelial cells, the addition of arginine delays the destruction of the monolayer long enough for biofilms to form on the CFBE cells. The second method (termed the Flow Cell Co-culture Biofilm Model), involves adaptation of a biofilm flow cell apparatus, which is often used in biofilm research, to accommodate a glass coverslip supporting a confluent monolayer of CFBE cells. This monolayer is inoculated with P. aeruginosa and a peristaltic pump then flows fresh medium across the cells. In both systems, bacterial biofilms form within 6-8 hours after inoculation. Visualization of the biofilm is enhanced by the use of P. aeruginosa strains constitutively expressing green fluorescent protein (GFP). The Static and Flow Cell Co-culture Biofilm assays are model systems for early P. aeruginosa infection of the Cystic Fibrosis (CF) lung, and these techniques allow different aspects of P. aeruginosa biofilm formation and virulence to be studied, including biofilm cytotoxicity, measurement of biofilm CFU, and staining and visualizing the biofilm.

Co-culture Models of Pseudomonas aeruginosa Biofilms Grown on Live Human Airway Cells

This paper describes different methods of growing Pseudomonas aeruginosa biofilms on cultured human airway epithelial cells. These protocols can be adapted to study different aspects of biofilm formation, including visualization of the biofilm, staining of the biofilm, measuring the colony forming units (CFU) of the biofilm, and studying biofilm cytotoxicity.

Bacterial biofilms have been associated with a number of different human diseases, but biofilm development has generally been studied on non-living ...

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological properties compared to free living microorganisms. Biofilms in nature are often difficult to investigate and reside under poorly defined conditions1. Using a transparent substratum it is possible to device a system where simple biofilms can be examined in a non-destructive way in real-time: here we demonstrate the assembly and operation of a flow cell model system, for in vitro 3D studies of microbial biofilms generating high reproducibility under well-defined conditions2,3.
The system consists of a flow cell that serves as growth chamber for the biofilm. The flow cell is supplied with nutrients and oxygen from a medium flask via a peristaltic pump and spent medium is collected in a waste container. This construction of the flow system allows a continuous supply of nutrients and administration of e.g. antibiotics with minimal disturbance of the cells grown in the flow chamber. Moreover, the flow conditions within the flow cell allow studies of biofilm exposed to shear stress. A bubble trapping device confines air bubbles from the tubing which otherwise could disrupt the biofilm structure in the flow cell.
The flow cell system is compatible with Confocal Laser Scanning Microscopy (CLSM) and can thereby provide highly detailed 3D information about developing microbial biofilms. Cells in the biofilm can be labeled with fluorescent probes or proteins compatible with CLSM analysis. This enables online visualization and allows investigation of niches in the developing biofilm. Microbial interrelationship, investigation of antimicrobial agents or the expression of specific genes, are of the many experimental setups that can be investigated in the flow cell system.

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Protocol describing the application of a flow cell system for growing and analyzing microbial biofilms for Confocal Laser Scanning Microscopy (CLSM).

Many microbial cells have the ability to form sessile microbial communities defined as biofilms that have altered physiological and pathological ...

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with leukemia, severe burn wounds, or organ transplants1. In patients at high-risk for developing PA bloodstream infections, the gastrointestinal (GI) tract is the main reservoir for colonization2, but the mechanisms by which PA transitions from an asymptomatic colonizing microbe to an invasive, and often deadly, pathogen are unclear. Previously, we performed in vivo transcription profiling experiments by recovering PA mRNA from bacterial cells residing in the cecums of colonized mice 3 in order to identify changes in bacterial gene expression during alterations to the host&#x2019;s immune status.
As with any transcription profiling experiment, the rate-limiting step is in the isolation of sufficient amounts of high quality mRNA. Given the abundance of enzymes, debris, food residues, and particulate matter in the GI tract, the challenge of RNA isolation is daunting. Here, we present a method for reliable and reproducible isolation of bacterial RNA recovered from the murine GI tract. This method utilizes a well-established murine model of PA GI colonization and neutropenia-induced dissemination4. Once GI colonization with PA is confirmed, mice are euthanized and cecal contents are recovered and flash frozen. RNA is then extracted using a combination of mechanical disruption, boiling, phenol/chloroform extractions, DNase treatment, and affinity chromatography. Quantity and purity are confirmed by spectrophotometry (Nanodrop Technologies) and bioanalyzer (Agilent Technologies) (Fig 1). This method of GI microbial RNA isolation can easily be adapted to other bacteria and fungi as well.

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

A reliable method for the RNA isolation of Pseudomonas aeruginosa recovered from murine cecums is described. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. This technique can be adapted for RNA isolation of other intestinal microbes.

Pseudomonas aeruginosa (PA) infections result in significant morbidity and mortality in hosts with compromised immune systems, such as patients with ...

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

There is growing concern about the relevance of in vitro antimicrobial susceptibility tests when applied to isolates of P. aeruginosa from cystic fibrosis (CF) patients. Existing methods rely on single or a few isolates grown aerobically and planktonically. Predetermined cut-offs are used to define whether the bacteria are sensitive or resistant to any given antibiotic1. However, during chronic lung infections in CF, P. aeruginosa populations exist in biofilms and there is evidence that the environment is largely microaerophilic2. The stark difference in conditions between bacteria in the lung and those during diagnostic testing has called into question the reliability and even relevance of these tests3.
 Artificial sputum medium (ASM) is a culture medium containing the components of CF patient sputum, including amino acids, mucin and free DNA. P. aeruginosa growth in ASM mimics growth during CF infections, with the formation of self-aggregating biofilm structures and population divergence4,5,6. The aim of this study was to develop a microtitre-plate assay to study antimicrobial susceptibility of P. aeruginosa based on growth in ASM, which is applicable to both microaerophilic and aerobic conditions.
 An ASM assay was developed in a microtitre plate format. P. aeruginosa biofilms were allowed to develop for 3 days prior to incubation with antimicrobial agents at different concentrations for 24 hours. After biofilm disruption, cell viability was measured by staining with resazurin. This assay was used to ascertain the sessile cell minimum inhibitory concentration (SMIC) of tobramycin for 15 different P. aeruginosa isolates under aerobic and microaerophilic conditions and SMIC values were compared to those obtained with standard broth growth. Whilst there was some evidence for increased MIC values for isolates grown in ASM when compared to their planktonic counterparts, the biggest differences were found with bacteria tested in microaerophilic conditions, which showed a much increased resistance up to a &gt;128 fold, towards tobramycin in the ASM system when compared to assays carried out in aerobic conditions.
 The lack of association between current susceptibility testing methods and clinical outcome has questioned the validity of current methods3. Several in vitro models have been used previously to study P. aeruginosa biofilms7, 8. However, these methods rely on surface attached biofilms, whereas the ASM biofilms resemble those observed in the CF lung9 . In addition, reduced oxygen concentration in the mucus has been shown to alter the behavior of P. aeruginosa2 and affect antibiotic susceptibility10. Therefore using ASM under microaerophilic conditions may provide a more realistic environment in which to study antimicrobial susceptibility.

Use of Artificial Sputum Medium to Test Antibiotic Efficacy Against Pseudomonas aeruginosa in Conditions More Relevant to the Cystic Fibrosis Lung

Current diagnostic antimicrobial susceptibility testing relies on the planktonic growth of isolates in nutrient rich, aerobic conditions. Here, we employ an alternative artificial sputum medium to study antimicrobial susceptibility of Pseudomonas aeruginosa biofilms under both aerobic and microaerophilic conditions more representative of the cystic fibrosis lung.

There  is growing concern about the relevance of in vitro antimicrobial  susceptibility tests when applied to isolates of P. aeruginosa from  cystic ...

Pseudomonas aeruginosa Induced Lung Injury Model

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa (P. aeruginosa or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.

Pseudomonas aeruginosa Induced Lung Injury Model

We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant.

In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. ...

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. Chronic mouse wounds models have been used to understand the underlying interactions between the microorganisms and the host. The models developed to date rely on the use of haired animals and terminal collection of wound tissue for determination of viable bacteria. While significant insight has been gained with these models, this experimental procedure requires a large number of animals and sampling is time consuming. We have developed a novel murine model that incorporates several optimal innovations to evaluate biofilm progression in chronic wounds: a) it utilizes hairless mice, eliminating the need for hair removal; b) applies pre-formed biofilms to the wounds allowing for the immediate evaluation of persistence and effect of these communities on host; c) monitors biofilm progression by quantifying light production by a genetically engineered bioluminescent strain of Pseudomonas aeruginosa, allowing real-time monitoring of the infection thus reducing the number of animals required per study. In this model, a single full-depth wound is produced on the back of STZ-induced diabetic hairless mice and inoculated with biofilms of the P. aeruginosa bioluminescent strain Xen 41. Light output from the wounds is recorded daily in an in vivo imaging system, allowing for in vivo and in situ rapid biofilm visualization and localization of biofilm bacteria within the wounds. This novel method is flexible as it can be used to study other microorganisms, including genetically engineered species and multi-species biofilms, and may be of special value in testing anti-biofilm strategies including antimicrobial occlusive dressings.

Come to the Light Side: In Vivo Monitoring of Pseudomonas aeruginosa Biofilm Infections in Chronic Wounds in a Diabetic Hairless Murine Model

Here we describe a novel diabetic murine model utilizing hairless mice for real-time, non-invasive, monitoring of biofilm wound infections of bioluminescent Pseudomonas aeruginosa. This method can be adapted to evaluate infection of other bacterial species and genetically modified microorganisms, including multi-species biofilms, and test the efficacy of antibiofilm strategies.

The presence of bacteria as structured biofilms in chronic wounds, especially in diabetic patients, is thought to prevent wound healing and resolution. ...

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary tissue during pneumonia results in the production of long-lived cytotoxins that can lead to subsequent end organ failure. We have developed in vitro assays to test the hypothesis that cytotoxins are produced during pulmonary infection. Isolated rat pulmonary endothelial cells and the bacterium Pseudomonas aeruginosa are used as model systems, and the production of cytoxins following infection of the endothelial cells by the bacteria is demonstrated using cell culture followed by direct quantitation using lactate dehydrogenase assays and a novel microscopic method utilizing ImageJ technology. The amyloid nature of these cytotoxins was demonstrated by thioflavin T binding assays and by immunoblotting and immunodepletion using A11 anti-amyloid antibody. Further analyses using immunoblotting demonstrated that oligomeric tau and A&#946; were produced and released by endothelial cells following infection by P. aeruginosa. These methods should be readily adaptable to analyses of human clinical samples.

Methods for Detecting Cytotoxic Amyloids Following Infection of Pulmonary Endothelial Cells by Pseudomonas aeruginosa

Simple methods are described for demonstrating the production of cytotoxic amyloids following infection of pulmonary endothelium by Pseudomonas aeruginosa.

Patients who survive pneumonia have elevated death rates in the months following hospital discharge. It has been hypothesized that infection of pulmonary ...

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of these products are naturally produced by the organisms, other products require genetic engineering of the organism to increase the yields of production. Avirulent strains of Escherichia coli have traditionally been the preferred bacterial species for producing biopharmaceuticals; however, some products are difficult for E. coli to produce. Thus, avirulent strains of other bacterial species could provide useful alternatives for production of some commercial products. Pseudomonas aeruginosa is a common and well-studied Gram-negative bacterium that could provide a suitable alternative to E. coli. However, P. aeruginosa is an opportunistic human pathogen. Here, we detail a procedure that can be used to generate nonpathogenic strains of P. aeruginosa through sequential genomic deletions using the pEX100T-NotI plasmid. The main advantage of this method is to produce a marker-free strain. This method may be used to generate highly attenuated P. aeruginosa strains for the production of commercial products, or to design strains for other specific uses. We also describe a simple and reproducible mouse model of bacterial systemic infection via intraperitoneal injection of validated test strains to test the attenuation of the genetically engineered strain in comparison to the FDA-approved BL21 strain of E. coli.

Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection

Here, we describe a simple and reproducible protocol of mouse model of infection to evaluate the attenuation of the genetically modified strains of Pseudomonas aeruginosa in comparison to the United States Food and Drug Administration (FDA)-approved Escherichia coli for commercial applications.

Microorganisms are genetically versatile and diverse and have become a major source of many commercial products and biopharmaceuticals. Though some of ...

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype called mucoidy. Alginate is linked to chronic lung infections resulting in poor prognosis in patients with cystic fibrosis (CF). Understanding the pathways that regulate the production of alginate can aid in the development of novel therapeutic strategies targeting the alginate formation. Another disease-related phenotype is the small colony variant (SCV). SCV is due to the slow growth of bacteria and often associated with increased resistance to antimicrobials. In this paper, we first show a method of culturing a genetically defined form of P. aeruginosa SCV due to pyrimidine biosynthesis mutations. Supplementation of nitrogenous bases, uracil or cytosine, returns the normal growth to these mutants, demonstrating the presence of a salvage pathway that scavenges free bases from the environment. Next, we discuss two methods for the measurement of bacterial alginate. The first method relies on the hydrolysis of the polysaccharide to its uronic acid monomer followed by derivatization with a chromogenic reagent, carbazole, while the second method uses an ELISA based on a commercially available, alginate-specific mAb. Both methods require a standard curve for quantitation. We also show that the immunological method is specific for alginate quantification and may be used for the measurement of alginate in the clinical specimens.

Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate

Here, we describe a growth condition to culture the small colony variant of Pseudomonas aeruginosa. We also describe two separate methods for the detection and quantitation of the exopolysaccharide alginate produced by P. aeruginosa using a traditional uronic acid carbazole assay and an alginate-specific monoclonal antibody (mAb) based ELISA.

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterial pathogen, can overproduce an exopolysaccharide alginate resulting in a unique phenotype ...

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of chronic wound infections in diabetic and hospitalized patients. There is an urgent need for chronic infection models to aid in the investigation of wound pathogenesis and the development of new therapies against this pathogen. Here, we describe a protocol that uses delayed inoculation 24 hours after full-thickness excisional wounding. The infection of the provisional wound matrix present at this time forestalls either rapid clearance or dissemination of infection and instead establishes chronic infection lasting 7&#8211;10 days without the need for implantation of foreign materials or immune suppression. This protocol mimics a typical temporal course of post-operative infection in humans. The use of a luminescent P. aeruginosa strain (PAO1:lux) allows for quantitative daily assessment of bacterial burden for P. aeruginosa wound infections. This novel model may be a useful tool in the investigation of bacterial pathogenesis and the development of new therapies for chronic P. aeruginosa wound infections.

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

We describe a delayed inoculation protocol for generating chronic wound infections in immunocompetent mice.

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of ...