The authors would like to acknowledge the University of Lille and the Pasteur Institute of Lille, especially Thierry Chassat and Jean-Pierre Decavel, responsible for animal housing breeding safety and husbandry. This work was supported by the "Soci&#233;t&#233; de Pathologies Infectieuses de Langue Fran&#231;aise" (SPILF).

The regional ethics regional committee for animal experiments has approved this method, in accordance with national and international animal care and use in investigational research guidelines.
1. Sample Collection
<ol>
	<li>Sample storage
	<ol>
		<li>Collect all samples and immediately store at - 20 &#176;C or on ice until freezer storage to avoid deterioration. Place sterile phosphate buffered saline (PBS) on ice to improve broncho-alveolar lavages (BAL) performance.</li>
	</ol>
	</li>
	<li>Surgery
	<ol>
		<li>Sterilize all surgical equipment using an autoclave. 
		NOTE: If possible, it is recommended to use two different sets of instruments for abdominal and thoracic steps to avoid cross-contamination. Required dissection equipment is detailed in Figure 4A</li>
	</ol>
	</li>
</ol>
2. Mice, Bacterial and Yeast Strains
<ol>
	<li>House mice in compliance with local use of animals in research committee guidelines in a ventilated rack without exceeding 5 mice per cage, with food and water ad lib, in a biosafety level 2 housing facility due to the use of biosafety level 2 micro-organisms : P. aeruginosa and C. albicans.</li>
	<li>Keep the bacterial strains at -80 &#176;C in 40% glycerol medium.
	<ol>
		<li>Add bacteria directly from frozen stock into culture tube containing 3 ml of sterile Luria-Bertani broth using a 10 &#181;l inoculation loop. Leave O/N&#160;at 37 &#176;C with orbital shaking (400 rpm) the day before instillation.</li>
		<li>Harvest bacteria by centrifugation at 2,000 x g for 5 min.</li>
		<li>Aspirate the supernatant into an appropriate closed biohazard waste disposal. Observe white adherent pellet at the bottom of culture tube.</li>
		<li>Wash and suspend the pellet using 5 ml of PBS.</li>
		<li>Repeat steps 2.2.2 to 2.2.3 a second time to perform a second wash.</li>
		<li>Resuspend the pelleted bacteria using 1 ml of PBS and brief vortexing.</li>
		<li>Determine the inoculum density using optical densitometer at 600 nm using an Optical Density Meter. A density of 0.9 corresponds to 109 CFU/ml for PAO1, dilute accordingly. 
		NOTE: This result has to be obtained for each used strain by determining density of successive dilutions of a calibrated inoculum.</li>
		<li>Verify the inoculum by serial logarithmic dilutions and plate 100 &#181;l of each dilution on bromocresol purple agar (BCP) plates and O/N culture. Administer each mouse intranasally 50 &#181;l of the solution containing 1 x 108 to 2 x108 CFU per ml (5 x 106 to 1 x107 CFU per mouse).</li>
	</ol>
	</li>
	<li>Use C. albicans SC5314 as a reference strain. Conserve the strain in 40% glycerol medium at -80&#176;C.
	<ol>
		<li>Supplement Yeast-Peptone-Dextrose broth with 0.015% amikacin to avoid bacterial contamination and facilitate further count.</li>
		<li>Add yeast using 10 &#181;l inoculation loop into prepared YPD-broth supplemented with amikacin O/N at 37&#176;C.</li>
		<li>Harvest yeast by centrifugation at 2,000 x g for 5 min.</li>
		<li>Remove supernatant into an appropriate bioharzard waste disposal. White adherent pellet should be observed in the bottom of culture tube.</li>
		<li>Wash and suspend the pelleted bacteria using 5 ml of PBS and brief vortexing.</li>
		<li>Repeat steps 2.2.2 to 2.2.3 a second time to perform a second wash.</li>
		<li>Resuspend the pellet using 1 ml of PBS and brief vortexing.</li>
		<li>Determine the size of the inoculum by counting on a Mallassez hematocytometer using a standard microscope at 40x magnification. 
		NOTE: Concentration (in CFU/ml) is obtained using the following formula: (number of yeast x 105)/(number of counted grid rectangles on the Mallassez hematocytometer).</li>
		<li>Verify by serial logarithmic dilution to 10-5 and 10-6 to confirm that solution contains 2 x 106 CFU/ml.</li>
		<li>Plate on YPD agar plates supplemented with 0.015% amikacin.</li>
	</ol>
	</li>
</ol>
3. Airways Colonization by C. albicans
NOTE: After environmental adaptation, mice are weighed twice a day.
<ol>
	<li>Under a fume hood, deposit 500 &#181;l of Sevoflurane onto a 4 cm x 4 cm gauze (open-drop technique)14.
	<ol>
		<li>Immediately place the gauze on the floor of an approximately 750 ml induction chamber. Immediately place a mesh-raised platform above the gauze to avoid direct contact between the animal and the gauze.</li>
		<li>Close airtight lid and wait 1 to 2 min to allow diffusion of Sevoflurane in the chamber.</li>
		<li>Transfer the mouse from cage to mesh platform and close lid. Light anesthesia with conserved spontaneous breathing should be achieved in 30 to 45 sec.</li>
		<li>Monitor for hypotonia by observing loss of righting reflex at which point the mouse can be removed from the box and instilled.</li>
	</ol>
	</li>
	<li>Intranasal instillation (Can be performed in 10 sec by a trained operator).
	<ol>
		<li>Hold the mouse one-handed nestled on its back held upright (Figure 4B).</li>
		<li>Using index finger, support the head and use the thumb to keep the jaw closed to avoid expectoration (Figure 4C).</li>
		<li>As described at step 2.3.8, ensure that the prepared C. albicans solution contains 2 x106 CFU per ml for an 50 &#181;l instilled volume. 
		NOTE: The second critical point is the instilled volume. A volume lower than 50 &#181;l could result in an insufficient colonization or inhomogeneous instillation of airway, a larger volume may induce drowning/suffocation and death.</li>
		<li>Instil the mouse intra-nasally by approaching the pipette to the nostrils.</li>
		<li>Pipette a 50 &#181;l drop forming a bubble containing the solution on the nostrils, subsequently inhaled by the spontaneously breathing mouse.</li>
		<li>Place mouse in a recovery area (e.g., a large well-aerated bare cage with an overhead heating lamp). Mice must be monitored until complete awakening. Do not leave an animal unattended until it has regained sufficient consciousness to maintain sternal recumbency and righting reflex. At this point, the mouse can be returned to a normal housing cage.</li>
	</ol>
	</li>
</ol>
4. P. aeruginosa-induced Acute Lung Infection
NOTE: Mice are weighed during the four following days. Normally, mice gain weight during C. albicans-mediated airway colonization (Figure 2A).
<ol>
	<li>Prepare the suspension containing P. aeruginosa the day of the instillation after O/N growth (section 2.2).</li>
	<li>Anesthetize briefly using inhaled Sevoflurane as described above (section 3.1). 
	NOTE: To perform acute lung infection, recommended bacterial burden are suggested in Table 1.</li>
	<li>Instil the mouse as described above (section 3.2) with particular attention to post-instillation recovery.</li>
</ol>
5. Measure of Lung Injury Index
<ol>
	<li>Prepare a solution containing FITC-labeled albumin. Inject this solution 2 hr before animal euthanasia.
	<ol>
		<li>Weigh 0.2 mg of albumin-FITC with the appropriate equipment.</li>
		<li>Add 0.2 mg into 1 ml PBS. Briefly vortex. If not used immediately, place the solution in foil to avoid exposure to ambient light.</li>
		<li>Inject intra-peritoneally 200 &#181;l of FITC-labeled albumin solution to each mouse.</li>
	</ol>
	</li>
	<li>Euthanasia
	<ol>
		<li>Weigh the mice for the last weight data.</li>
		<li>Euthanize a single mouse in accordance with local use of animals in research committee guidelines using one intra-peritoneal injection of a lethal overdose of pentobarbital : 300 &#181;l of 5.47% pentobarbital.</li>
		<li>Remove mouse from the cage and receives lethal injection by operator.</li>
		<li>Following injection, transfer the mouse alone to another cage, hidden from any other animals. Observe the mouse until absence of movement. Confirm death is by absence of movement, particularly respiratory movement, lack of pulse.</li>
		<li>Perform surgical collection of samples on dead animals, therefore without anesthetics nor analgesics.</li>
	</ol>
	</li>
	<li>Surgical sample collection: Thoracic stage. 
	NOTE: To maintain sterile conditions, all surgery is performed using sterile equipment in a biosafety level 2 environment.
	<ol>
		<li>Apply ethanol to the skin. Perform a midline skin incision from sternum to mid abdomen with scissors. From midline incise along the ribcage on either side. Fold back the skin on either side of the thorax to visualize the rib cage.</li>
		<li>Perform a vertical incision of the ribcage on either side going up towards the clavicles in order to be able to recline the entire anterior chest wall with the sternum allowing the perfect visualization of heart and lungs (Figure 5A, 5B).</li>
		<li>Collect blood using a pre-heparined syringe by puncturing the heart next to the interventricular artery. Withdraw a minimum of 500 &#181;l&#160;to obtain at least 100 &#181;l of plasma. Place the blood sample on ice.</li>
		<li>Perform a midline cervical incision to visualize the trachea (Figure 5B and 5C). Carefully dissect the fascia around the trachea. Place a suture behind the trachea (Figure 5C and 5D). Subsequently the suture will be closed around the cannulating needle to ensure proper lavage.</li>
		<li>Catheterize the trachea using the 20-G modified gavage needle (Figure 5D and 4A). Tie a surgical knot around the cannulated trachea with the previously placed suture.</li>
		<li>To perform bronchoalveolar lavages (BAL), gently and progressively inject and draw 500 &#181;l of ice-cold PBS into/from the lung. Place the sample on ice to avoid cellular lysis.</li>
		<li>Repeat step 5.3.6, 3 times to obtain a total of 1,500 &#181;l BAL fluid and pool lavage samples into a 2 ml centrifuge tube (Figure 5E).</li>
		<li>Remove the lungs from the chest. Place a lung segment (size should correspond to the half of one lung or a lobe) into 1.5 ml centrifuge tube and store rapidly at - 80&#176;C.</li>
		<li>Place a lung segment in a pre-weighed hemolysis tube containing PBS to determine bacterial burden and place it on ice.</li>
	</ol>
	</li>
	<li>Surgical sample collection: abdominal stage.
	<ol>
		<li>Perform another incision on the left-side of the abdomen. Observe the spleen through the peritoneum.</li>
		<li>Remove the spleen and place into a second hemolysis tube containing 1 ml PBS and place on ice.</li>
	</ol>
	</li>
	<li>Lung injury index 
	NOTE: Alveolar-capillary membrane permeability is assessed by measuring FITC-labeled-albumin leakage from the vascular compartment to the alveolar-interstitial compartment.
	<ol>
		<li>Centrifuge blood sample and BAL fluid for 10 min at 1,500 x g. Collect the supernatants into new centrifuge tubes. The pellets correspond to recruited plasma or BAL cells and should be placed on ice.</li>
		<li>Add 100 &#181;l of each blood supernatant (plasma, yellow) or BAL supernatant to a 96-well transparent plate (300 &#181;l wells). Place a foil on the plate if not used immediately.</li>
		<li>Measure fluorescence levels in plasma and BAL supernatants using a fluorescence microplate reader (excitation, 487 nm; emission, 520 nm).</li>
		<li>Determine the lung injury index by calculating the fluorescence ratio [(BAL supernatant/blood supernatant) x 100].</li>
	</ol>
	</li>
	<li>Bronchoalveolar lavage (BAL) differential cell count. 
	NOTE: Use the cell pellet obtained from centrifugation of BAL fluid at step 5.5.1.
	<ol>
		<li>If needed, use red-cell lysis buffer. Add 500 &#181;l of red-cell lysis buffer into the centrifuge tube containing the cell pellet. Briefly vortex and leave 10 min on ice. Add 500 &#181;l PBS to stop red-cell lysis.</li>
		<li>Harvest cells by centrifugation for 10 min at 1,500 x g. Remove supernatant and suspend the cell pellet into 1 ml of sterile PBS. Enumerate cells on a Mallassez hematocytometer. Using a Hemacytometer to Count Cells. Concentrate cells on a slide with a cytospin.</li>
		<li>Stain cells using coloration kit allowing cell identification and count (macrophages, lymphocytes, neutrophils).</li>
	</ol>
	</li>
	<li>Lung bacterial burden and bacterial dissemination 
	NOTE: To assess lung bacterial burden and bacterial dissemination, lungs and spleen were respectively collected and stored into pre-weighed hemolysis tubes containing 1 ml of PBS (step 5.3.9).
	<ol>
		<li>Weigh hemolysis tubes containing 1 ml PBS and either lung or spleen. Homogenize the samples with a tissue homogenizer to obtain lung homogenates and spleen homogenates.</li>
		<li>Deposit 100 &#181;l of tissue homogenates into centrifuge tubes containing 900 &#181;l of sterile PBS to obtain serial logarithmic dilutions.</li>
		<li>Plate the two last appropriate diluted samples (10-3 and 10-2) on either BCP-agar for P. aeruginosa or YPD-amikacin-supplemented agar for C. albicans lung and spleen burden determination.</li>
		<li>Incubate the plates O/N at 37 &#176;C. The following day, enumerate the colonies on plates.</li>
		<li>Index the result to lung weight to obtain a CFU per gram of lung. Lung sample sizes are not the same, the results should be expressed in CFU per gram of lung. 
		Formula for the index is: [CFU] x [weight of hemolysis tube and lung] - [weight of hemolysis tube].</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Casadevall, A., Pirofski, L. -. 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+damage-response+framework+of+microbial+pathogenesis.">The damage-response framework of microbial pathogenesis.</a> Nat. Rev. Micro. 1 (1), 17-24 (2003).</li><li>Eddens, T., Kolls, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+defenses+against+bacterial+lower+respiratory+tract+infection.">Host defenses against bacterial lower respiratory tract infection.</a> Curr. Opi. Immunol. , (2012).</li><li>Beck, J. M., Young, V. B., Huffnagle, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microbiome+of+the+lung.">The microbiome of the lung.</a> Translational research : J. Lab. Clin Med. 160 (4), 258-266 (2012).</li><li>Hogan, D. A., Kolter, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas-Candida+interactions:+an+ecological+role+for+virulence+factors.">Pseudomonas-Candida interactions: an ecological role for virulence factors.</a> Science. 296 (5576), 2229-2232 (2002).</li><li>Nseir, S., Ader, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pseudomonas+aeruginosa+and+Candida+albicans:+do+they+really+need+to+stick+together.">Pseudomonas aeruginosa and Candida albicans: do they really need to stick together.</a> Crit. Care Med. 37 (3), 1164-1166 (2009).</li><li>Hibbing, M. E., Fuqua, C., Parsek, M. R., Peterson, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+competition:+surviving+and+thriving+in+the+microbial+jungle.">Bacterial competition: surviving and thriving in the microbial jungle.</a> Nat. Rev. Micro. 8 (1), 15-25 (2010).</li><li>Gibbons, D. L., Spencer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+and+human+intestinal+immunity:+same+ballpark,+different+players;+different+rules,+same+score.">Mouse and human intestinal immunity: same ballpark, different players; different rules, same score.</a> Mucosal Immunol. 4 (2), 148-157 (2011).</li><li>Ariffin, J. K., Sweet, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+the+repertoire,+regulation+and+function+of+Toll-like+Receptors+and+inflammasome-forming+Nod-like+Receptors+between+human+and+mouse.">Differences in the repertoire, regulation and function of Toll-like Receptors and inflammasome-forming Nod-like Receptors between human and mouse.</a> Curr. Opi. Micro.. , (2013).</li><li>Slutsky, A. S., Ranieri, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ventilator-Induced+Lung+Injury.">Ventilator-Induced Lung Injury.</a> NEJM. 369 (22), 2126-2136 (2013).</li><li>Peleg, A. Y., Hogan, D. A., Mylonakis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medically+important+bacterial-fungal+interactions.">Medically important bacterial-fungal interactions.</a> Nat. Rev. Micro. 8 (5), 340-349 (2010).</li><li>Roux, D., Gaudry, S., 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=Candida+albicans+impairs+macrophage+function+and+facilitates+Pseudomonas+aeruginosa+pneumonia+in+rat.">Candida albicans impairs macrophage function and facilitates Pseudomonas aeruginosa pneumonia in rat.</a> Crit. Care Med. 37 (3), 1062-1067 (2009).</li><li>Mear, J. B., Gosset, P., 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=Candida+albicans+Airway+Exposure+Primes+the+Lung+Innate+Immune+Response+against+Pseudomonas+aeruginosa+Infection+through+Innate+Lymphoid+Cell+Recruitment+and+Interleukin-22-Associated+Mucosal+Response.">Candida albicans Airway Exposure Primes the Lung Innate Immune Response against Pseudomonas aeruginosa Infection through Innate Lymphoid Cell Recruitment and Interleukin-22-Associated Mucosal Response.</a> Infect. Immun. 82 (1), 306-315 (2013).</li><li>Ader, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+term+Candida+albicans+colonization+reduces+Pseudomonas+aeruginosa+load+and+lung+injury+in+a+mouse+model.">Short term Candida albicans colonization reduces Pseudomonas aeruginosa load and lung injury in a mouse model.</a> Crit. care. , 1-33 (2009).</li><li>Risling, T. E., Caulkett, N. A., Florence, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open-drop+anesthesia+for+small+laboratory+animals.">Open-drop anesthesia for small laboratory animals.</a> Can Vet J. 53 (3), 299-302 (2012).</li><li>Stover, C. K., Pham, X. Q., 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=Complete+genome+sequence+of+Pseudomonas+aeruginosa+PAO1,+an+opportunistic+pathogen.">Complete genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen.</a> Nature. 406 (6799), 959-964 (2000).</li><li>Boutoille, D., Marechal, X., Pichenot, M., Chemani, C., Guery, B. P., Faure, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FITC-albumin+as+a+marker+for+assessment+of+endothelial+permeability+in+mice:+comparison+with+125I-albumin.">FITC-albumin as a marker for assessment of endothelial permeability in mice: comparison with 125I-albumin.</a> Exp. Lung Res. 35 (4), 263-271 (2009).</li><li>Faure, E., Mear, J. -. 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=Pseudomonas+aeruginosa+type-3+secretion+system+dampens+host+defense+by+exploiting+the+NLRC4-coupled+inflammasome.">Pseudomonas aeruginosa type-3 secretion system dampens host defense by exploiting the NLRC4-coupled inflammasome.</a> American Journal of Respiratory and Critical Care Medicine. 189 (7), 799-811 (2014).</li><li>Peleg, A. Y., Hogan, D. A., Mylonakis, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medically+important+bacterial-fungal+interactions.">Medically important bacterial-fungal interactions.</a> Nat. Rev. Micro. 8 (5), 340-349 (2010).</li></ol>

The authors have nothing to disclose.

Animal models, particularly mammals, are useful to elucidate complex mechanisms of host-pathogen interaction in the fields of immunity. Of course, the need for information obtainable only from animal models must be essential; otherwise, use of animals must be replaced by in vitro models. This animal model illustrates the insight that can only be provided by an animal model since the interaction between pathogens is mediated by a multi-component host response. Mice currently used to study this host-pathogen inter...

Animal models, especially mice, have been extensively used to explore immune responses against pathogens. Although innate and acquired immunity differ between rodents and humans7, the ease in breeding and the development of knockouts for numerous genes, make mice an excellent model to study immune responses8. The immune response is complex and results from the interaction of a pathogen, the resident microbial flora and several immune (lymphocytes, neutrophils, macrophages) and non-immune (epithelial cells, endothelial cells) cellular types2. In vitro studies do not allow observing these complex interactions and mainly focus on unique cell-pathogen interactions. While animal models must be used with caution and limited to very specific and relevant questions, mouse models provide a good insight into the mammal immune response in vivo and may address parts of important clinical questions7.
In the airways, the microbial community is complex associating a large number of different microorganisms6. While what constitutes a &#34;normal&#34; airway microbiome remains to be determined, resident communities are frequently polymicrobial, and originate from diverse ecological sources. Patients with suppurative chronic lung disease (cystic fibrosis, bronchectasis) or mechanically ventilated patients exhibit a particular flora due to colonization of the airways by environmentally-acquired microorganisms9. Pseudomonas aeruginosa and Candida albicans are both problem pathogens5, frequently isolated together from tracheobronchial samples, and responsible of severe opportunistic infection in these patients, especially in the intensive care unit (ICU)4.
Isolation of these microorganisms during acute pneumonia in ICU results in anti-microbial treatment against P. aeruginosa but yeast are usually not considered pathogenic at this site5. In vitro interactions between P. aeruginosa and C. albicans have been widely reported and showed that these microorganisms can affect the growth and the survival of each other but studies could not conclude if the presence of C. albicans is detrimental or beneficial for the host10. Mouse models were developed to address this relevance of P. aeruginosa&#160;and C. albicans&#160;in vivo, but the interaction between microorganisms was not the key point. Indeed, the model was established to evaluate the involvement of C. albicans in host immune response, and outcome.
A previous model established by Roux et al already used an initial colonization with C. albicans followed by an acute lung infection induced by P. aeruginosa. Using their model, the authors found a deleterious role of prior C. albicans colonization11. However Roux et al used a high load of C. albicans in their model with 2 x 106 CFU/mouse during 3 consecutive days. We established a 4-day model of C. albicans airway colonization, or at least persistence without lung injury, In this model C. albicans was retrieved up to 4 days after a single instillation of 105 CFU per mouse (Figure 2B) 12,13. After 4 days, no evidence of inflammatory cell recruitment, inflammatory cytokine production nor epithelial damage was observed. At 24 - 48 hr, at the peak presence of C. albicans, even though a cellular and cytokine innate immune response was observed, there was no evidence of lung injury. Surprisingly, mice thus colonized with C. albicans 48 hr prior to intranasal instillation of P. aeruginosa had attenuated infection compared to mice with P. aeruginosa infection alone. Indeed, mice exhibited lesser lung injury and decreased bacterial burden12,13.
Several hypotheses could explain this beneficial effect of prior colonization with C. albicans on P. aeruginosa-mediated acute lung infection. First, an interspecies cross-talk involving each microorganisms quorum-sensing systems, the homoserinelactone-based P. aeruginosa system and the farnesol-based C. albicans system, were evaluated. Second, C. albicans acting as a &#34;decoy&#34; target for P. aeruginosa diverting the pathogen from lung epithelial cells was studied. Both hypotheses were invalidated (unpublished data). The third hypothesis was that of a &#34;priming&#34; of the innate immune system by C. albicans responsible for an enhanced subsequent innate response against P. aeruginosa. This last hypothesis was confirmed. Indeed C. albicans colonization led to a priming of innate immunity through IL-22, mainly secreted by innate lymphoid cells, resulting in increased bacterial clearance and reduced lung injury12.
In conclusion, the host is a central actor in the interaction between microorganisms modulating the innate immune response and involving different inflammatory cell types. While these complex immune interactions can be dissected in vitro the initial hypotheses can only be provided by appropriate in&#160;vivo models. The following protocol provides an example of in vivo study of host-mediated pathogen interaction that may be adapted to others microorganisms.

<table border="0" cellpadding="0" cellspacing="0" width="729">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sevorane, Sevoflurane</td>
			<td style="width:185px;">Abott</td>
			<td style="width:161px;">05458-02</td>
			<td style="width:167px;">250 mL plastic bottle</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fluorescence Reader Mithras&#160; LB940</td>
			<td style="width:185px;">Berthold Technologies</td>
			<td style="width:161px;">reference in first column</td>
			<td>no comment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bromo-cresol purple agar</td>
			<td style="width:185px;">Biomerieux</td>
			<td align="right" style="width:161px;">43021</td>
			<td>x20 per unit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pentobarbital sodique 5,47%</td>
			<td style="width:185px;">CEVA</td>
			<td align="right" style="width:161px;">6742145</td>
			<td>100 mL plastic bottle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-headed valve&#160;</td>
			<td style="width:185px;">Distrimed</td>
			<td align="right" style="width:161px;">92831</td>
			<td>no comment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile inoculation loop 10 &#181;L</td>
			<td style="width:185px;">Dutscher</td>
			<td align="right" style="width:161px;">10175</td>
			<td>x1000 conditioning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Insuline syringes 1 mL</td>
			<td style="width:185px;">Dutscher</td>
			<td align="right" style="width:161px;">30003</td>
			<td>per 100 conditioning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2 positions Culture tube 8 mL</td>
			<td style="width:185px;">Dutscher</td>
			<td align="right" style="width:161px;">64300</td>
			<td>no comment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultrospec 10&#160;</td>
			<td style="width:185px;">General Electric life sciences</td>
			<td style="width:161px;">80-2116-30</td>
			<td>no comment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hemolysis tubes 13 x 75 mm&#160;</td>
			<td style="width:185px;">Gosselin</td>
			<td style="width:161px;">W1773X</td>
			<td>per 100</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">PBS - Phosphate-Buffered Saline</td>
			<td style="width:185px;">Life technologies</td>
			<td align="right" style="width:161px;">10010023</td>
			<td>packaged in 500 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">amikacin 1g</td>
			<td style="width:185px;">Mylan</td>
			<td align="right" style="width:161px;">62516778</td>
			<td>per 10&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Heparin 10 000 UI in 2 mL</td>
			<td style="width:185px;">Pan pharma</td>
			<td align="right" style="width:161px;">9128701</td>
			<td>x 10 per unit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RAL 555 coloration kit</td>
			<td style="width:185px;">RAL Diagnostics</td>
			<td align="right" style="width:161px;">361550</td>
			<td>3 flacons of 100 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1,5 mL microcentrifuge tube</td>
			<td style="width:185px;">Sarstedt</td>
			<td style="width:161px;">55.526.006</td>
			<td>x&#160; 1000</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Transparent 300 &#181;L 96-well plate</td>
			<td style="width:185px;">Sarstedt</td>
			<td style="width:161px;">82 1581500</td>
			<td>no comment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Yest-peptone-Dextrose Broth</td>
			<td style="width:185px;">Sigma</td>
			<td align="right" style="width:161px;">95763</td>
			<td>in powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FITC-albumin</td>
			<td style="width:185px;">Sigma</td>
			<td style="width:161px;">A9771</td>
			<td>in powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Luria Bertani Broth</td>
			<td style="width:185px;">Sigma</td>
			<td style="width:161px;">L3022</td>
			<td>in powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">25-gauge needle</td>
			<td style="width:185px;">Terumo or unisharp</td>
			<td style="width:161px;">A231</td>
			<td>x100 conditioning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cytocentrifuge</td>
			<td style="width:185px;">Thermo Scientific</td>
			<td style="width:161px;">A78300003</td>
			<td>no comment</td>
		</tr>
	</tbody>
</table>

studying microbial communities in vivo: a model of host-mediated interaction between candida albicans and pseudomonas aeruginosa in the airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of the host depends on the involvement of an adapted immune response against the pathogen1. Immune response is complex and results from interaction of the pathogens and several immune or non-immune cellular types2. In vitro studies cannot characterise these interactions and focus on cell-pathogen interactions. Moreover, in the airway3, particularly in patients with suppurative chronic lung disease or in mechanically ventilated patients, polymicrobial communities are present and complicate host-pathogen interaction. Pseudomonas aeruginosa and Candida albicans are both problem pathogens4, frequently isolated from tracheobronchial samples, and associated to severe infections, especially in intensive care unit5. Microbial interactions have been reported between these pathogens in vitro but the clinical impact of these interactions remains unclear6. To study the interactions between C. albicans and P. aeruginosa, a murine model of C. albicans airways colonization, followed by a P. aeruginosa-mediated acute lung infection was performed.

As seen previously during the protocol description, the experiment needs 5 day to complete (Figure 1: experiment timeline). One operator is solicited during the entire run of the experiment and can handle the processes up to a maximum of 10 mice. If more animals are required, two persons are needed particularly for surgical sample collection. Indeed all samples must be collected in under 2 hr to avoid an increased passive alveolar-capillary leakage of FITC-labeled albumin in the last mice.
<p class="...

Watch this Scientific Journal Video about Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways at JoVE.com

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

Studying host-pathogen interaction enables us to understand the underlying mechanisms of the pathogenicity during microbial infection. The prognosis of ...

studying-microbial-communities-vivo-model-host-mediated-interaction

Research

JoVE Journal

Immunology and Infection

8.4K Views.  Université Lille. While in vitro study of host-pathogen interactions allow the characterization of specific immune responses, in vivo models are required to observe the effects of complex responses. Using Candida albicans exposure followed by Pseudomonas aeruginosa-mediated lung infection, we established a murine model of microbial interactions involved in ventilator-associated pneumonia pathogenicity.

Video: Studying Microbial Communities In Vivo: A Model of Host-mediated Interaction Between Candida Albicans and Pseudomonas Aeruginosa in the Airways

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. ...

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for macrophages. This predatory organism, similar to macrophages, employs phagocytosis to kill internalized cells. With the aid of a confocal laser-scanning microscope, images depicting interactive moments between cryptococcal cells and amoeba are captured. The resolution power of the electron microscope also helps to reveal the ultrastructural detail of cryptococcal cells when trapped inside the amoeba food vacuole. Since phagocytosis is a continuous process, quantitative data is then integrated in the analysis to explain what happens at the timepoint when an image is captured. To be specific, relative fluorescence units are read in order to quantify the efficiency of amoeba in internalizing cryptococcal cells. For this purpose, cryptococcal cells are stained with a dye that makes them fluoresce once trapped inside the acidic environment of the food vacuole. When used together, information gathered through such techniques can provide critical information to help draw conclusions on the behavior and fate of cells when internalized by amoeba and, possibly, by other phagocytic cells.

Complementary Use of Microscopic Techniques and Fluorescence Reading in Studying Cryptococcus-Amoeba Interactions

This paper details a protocol for preparing a co-culture of cryptococcal cells and amoebae that is studied using still, fluorescent images and high-resolution transmission electron microscope images. Illustrated here is how quantitative data can complement such qualitative information.

To simulate Cryptococcus infection, amoeba, which is the natural predator of cryptococcal cells in the environment, can be used as a model for ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

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 ...

Clarifying and Imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 &#181;m biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

Clarifying and Imaging Candida albicans Biofilms

To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm.

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important role in the dissemination and pathogenesis of this opportunistic fungal pathogen. However, methods to visualize fungal hyphae in the GI tract in vivo are challenging which limits the understanding of environmental signals in controlling this morphogenesis process. The protocol described here demonstrates a novel ex vivo method for visualization of hyphal morphogenesis in gut homogenate extracts. Using an ex vivo assay, this study demonstrates that cecal contents from antibiotic treated mice, but not from untreated control mice, promote C. albicans hyphal morphogenesis in the gut content. Further, adding back specific groups of gut metabolites to the cecal contents from antibiotic-treated mice differentially regulates hyphal morphogenesis ex vivo. Taken together, this protocol represents a novel method to identify and investigate the environmental signals that control C. albicans hyphal morphogenesis in the GI tract.

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

The ex vivo assay described in this study using gut homogenate extracts and immunofluorescence staining represents a novel method to examine the hyphal morphogenesis of Candida albicans in the GI tract. This method can be utilized to investigate the environmental signals regulating morphogenetic transition in the gut.

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important ...

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic infection. The development of chronic P. aeruginosa infections is associated with a decline in lung function and increased morbidity. Therefore, there is a great interest in elucidating the reasons for the failure to eradicate P. aeruginosa with antibiotic therapy which occurs in approximately 10-40% of pediatric patients. One of many factors that can affect host clearance of P. aeruginosa and antibiotic susceptibility is variations in spatial organization (such as aggregation or biofilm formation) and polysaccharide production. Therefore, we were interested in visualizing the in situ characteristics of P. aeruginosa within the sputum of CF patients. A tissue clearing technique was applied to sputum samples after embedding the samples into a hydrogel matrix to retain the 3D structures relative to host cells. After tissue clearing, fluorescent labels and dyes were added to allow visualization. Fluorescence in situ hybridization was performed for the visualization of bacterial cells, binding of fluorescently labeled anti-Psl-antibodies for the visualization of the exopolysaccharide and DAPI staining to stain host cells to obtain structural insight. These methods allowed for the high-resolution imaging of P. aeruginosa within the sputum of CF patients via confocal laser scanning microscopy.

Visualization of Pseudomonas aeruginosa within the Sputum of Cystic Fibrosis Patients

This protocol provides methods for visualization of bacterial cells and polysaccharide synthesis locus (Psl) polysaccharide within the sputum of cystic fibrosis patients.