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>

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<li>Nseir, S., Ader, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pseudomonas+aeruginosa+and+Candida+albicans%3A+do+they+really+need+to+stick+together%5BTitle%5D)+AND+%22Crit.+Care+Med%22%5BJournal%5D)">Pseudomonas aeruginosa and Candida albicans: do they really need to stick together.</a> Crit. Care Med. 37 (3), 1164-1166 (2009).</li>
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<li>Gibbons, D. L., Spencer, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+and+human+intestinal+immunity%3A+same+ballpark%2C+different+players%3B+different+rules%2C+same+score%5BTitle%5D)+AND+%22Mucosal+Immunol%22%5BJournal%5D)">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/pubmed?term=((Differences+in+the+repertoire%2C+regulation+and+function+of+Toll-like+Receptors+and+inflammasome-forming+Nod-like+Receptors+between+human+and+mouse%5BTitle%5D)+AND+%22Curr.+Opi.+Micro.%22%5BJournal%5D)">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/pubmed?term=((Ventilator-Induced+Lung+Injury%5BTitle%5D)+AND+%22NEJM%22%5BJournal%5D)">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/pubmed?term=((Medically+important+bacterial-fungal+interactions%5BTitle%5D)+AND+%22Nat.+Rev.+Micro%22%5BJournal%5D)">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/pubmed?term=((Candida+albicans+impairs+macrophage+function+and+facilitates+Pseudomonas+aeruginosa+pneumonia+in+rat%5BTitle%5D)+AND+%22Crit.+Care+Med%22%5BJournal%5D)">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/pubmed?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%5BTitle%5D)+AND+%22Infect.+Immun%22%5BJournal%5D)">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/pubmed?term=((Short+term+Candida+albicans+colonization+reduces+Pseudomonas+aeruginosa+load+and+lung+injury+in+a+mouse+model%5BTitle%5D)+AND+%22Crit.+care%22%5BJournal%5D)">Short term Candida albicans colonization reduces Pseudomonas aeruginosa load and lung injury in a mouse model.</a> Crit. care. , 1-33 (2009).</li>
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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 interaction are young adults aged 6 to 10 weeks with a mature and unaltered immune response. When focusing on the innate immune response, C57Bl6/J background mice are preferred. To avoid an effect of sex and hormonal cycle (particularly estrogen) on the immune response, males are therefore the best choice. To achieve statistical significance, groups must have at least 5 individuals at the end of the experiment, but as suggested by all animal experimentation guidelines, the number of animals used should be reduced and refined to a strict minimum.
Transport from the breeder providing the animals to the research facility induces stress in mice. The consequence is an increased secretion of inflammatory cytokines that can alter subsequent experiments such as ours. Moreover, a new environment and new &#34;cage mates&#34; contribute to stress. Consequently, mice must be acclimated for at least seven days prior to study in their new housing environment. This housing environment has to be controlled, providing standard food and water ad-lib, a day/night cycle, and appropriate stable humidity and temperature.
Both lung colonization and lung infection models require practice and dexterity. Instillation can be performed intra-nasally or intra-tracheally. The latter is more difficult and requires greater expertise through training due to a high risk of anoxic cardiac arrest. Indeed, the procedure requires to successfully intubate a mouse in less than 15 sec and therefore required deeper anesthesia. Our choice of intra-nasal instillation is easier to perform since the route of administration is accessible, less risky requiring lighter anesthesia and is therefore more reproducible.
Boutoille et al already described our acute lung injury model, particularly the assessment of lung injury through measuring alveolar-capillary barrier permeability using FITC-labeled albumin16. To reduce the number of mice per experiment, this method was adapted and coupled with bronchoalveolar lavage (BAL). In the studies of Boutoille et al, we used the comparison between the fluorescence of FITC-labeled albumin in the lung homogenate supernatants and the blood supernatants17.
To fully perform this comparison, hemoglobin levels and hematocrit levels are also required. This method was adapted to allow the concomitant analysis of host response by dedicating one lung to homogenization and assessment of lung injury and the other to lavage and study of host response. Indeed, BAL fluid can be used to assess cytokine levels, protein secretion and cell recruitment. Our adaptation provides more results from a single animal experiment, reducing the cost and the required number of mice, particularly when using knock-out mice17 as recommended in animal experimentation guidelines. Moreover, a part of the lung is kept at -80 &#176;C to perform total RNA extraction and quantitative polymerase chain reaction or histologic analysis. Comparison between the previous method and the new adapted method coupled with BAL shows comparable results (Figure 3A) to assess lung injury index.
In the study of Mear et al, using the same procedures, flow cytometer analysis was performed on BAL cells obtained by centrifugation of BAL fluid. Similar analyses were performed on total pulmonary cells from the lungs12. In this case, lung injury assessment with FITC-labeled albumin cannot be performed concomitantly, due to artefacts induced by FITC (same channel than green fluorescence protein). Therefore, if flow cytometry is required, experiments must be planned accordingly.
The timing of euthanasia is critical. A time course of the acute lung infection model over the first 72 hr is presented Figure 3. In this model, the acme of lung injury and host response occurs between 24 and 36 hr (Figure 3). Thus, in our design the 24 hr end-point was chosen as a readout for 3 reasons: first, it is easy to organize in the laboratory, second, to avoid loss of mice due to mortality between 24 and 36 hr, and finally, because host response was maximal at this time point.
The first step of our interaction study is the colonization of the airway with C. albicans. Using an instillation of 105 CFU per mouse, a 4-day persistence model is obtained without any lung injury. In fact, mice gained weight over the time course of this phase. Weight gain is a useful indicator of the absence of injury in line with colonization as opposed to infection. Indeed, an increased initial load (over 106 CFU per mouse) induced lung injury (Figure 2C) and a deleterious host-response far beyond priming, in which case mice lost weight (Figure 2D). On the contrary, using a smaller initial load (for example 104 CFU per mouse) an airway persistence model could not be obtained. Thus, to obtain the observed priming of host immunity described by Mear et al12, calibration of the instilled fungal burden is critical to the success and monitoring the weight curve is a key control.
The same precision is required for P. aeruginosa to induce an acute infection. An insufficient inoculum does not induce lung injury and P. aeruginosa is rapidly cleared from the airways by an appropriate host-response. Using an overly high initial P. aeruginosa surpasses the capacities of host defense or even induces an inappropriate and derleterious response resulting in load leads to massive acute injury and death erasing all differences between groups. The optimal inoculum to induce acute injury depends of the presence of a functional type-3 secretion system (T3SS) and the production of exotoxin U, a toxin translocated by the T3SS into the host cell cytoplasm. These two strain-specific attributes have to be considered in the choice of the initial bacterial burden. Initial bacterial burdens are suggested in this article as examples of lower and upper limits to induce acute lung injury with T3SS-negative or positive strain, and strains producing exotoxin U or not (Table 1). When studying involvement of T3SS, strain has to be grown O/N and revived with new LB medium 3 hr before the preparation of the inoculum to obtain optimal activity of T3SS.
The determination of bacterial and fungal burden 24 hr after P. aeruginosa-induced lung infection requires specific considerations discussed in this section. Indeed, as shown, when mice are infected with 5 x 106 CFU T3SS-positive strain, bacterial burden in the lungs at 24 hr will decreased to about 1 log (Figure 3C). Serial dilutions of samples must be performed up to 5-log dilution and plated on BCP-agar plates. Concerning C. albicans in the lungs, at 72 hr, fungal burden is decreased to about 2 log and samples must to be plated on YPD-agar supplemented with amikacin to facilitate colony identification. Finally, determination of bacterial dissemination can be assessed by plating 100 &#181;l of blood sample on BCP-agar or by plating 100 &#181;l of spleen homogenates on the same medium. The two methods were already been compared and spleen culture seems to be more accurate. Indeed, the spleen is an organ that &#34;filters&#34; whole blood and may &#34;concentrate&#34; and conserve bacteria having disseminated onto the blood. Thus, spleen homogenates reflect the systemic bacterial dissemination during acute lung infection with a higher sensitivity than blood. Blood samples represent bacterial dissemination at a very specific given time and may not truly reflect the phenomenon of bacterial dissemination as a whole. Therefore spleen homogenate cultures are preferred.
Lung injury index is a sensitive assessment of lung injury resulting from infection and/or inappropriate host response and the effect of potential therapeutics on these components. Additionally, this in&#160;vivo model allows the collection of several different samples to study host response. Lung can be used for RNA extraction and analysis of gene transcription. Lung samples can also be placed into paraformaldehyde (PFA) to further histological observations. BAL fluid can be used for assessment of protein secretion such as inflammatory cytokines. Finally, as discussed above the protocol can be adapted to provide samples for flow cytometry analysis.
Finally, this protocol can be adapted to modelize C. albicans-microbes interaction involved in ventilator-associated pneumonia such as Staphylococcus aureus18 or Enterobacteriae. Another adaptation could be airway colonization using bacteria instead of C. albicans to modelize bacterial interaction in chronic suppurative lung disease such as bronchiectasis. To this end, immunocompromised mice have to be used, because bacterial clearance in immune competent mice does not allow a persistent airway colonization.
For instillation, the position of the hand holding the animal is critical. As already underlined in the previous section, when intra-nasally instilling the mouse, the operator must ensure that mouth is perfectly closed to avoid expectoration of the solution. The thumb supports the jaw and maintains the mouth closed during the entire instillation procedure (Figure 4B). Then, with the other hand, the pipette is deposited on a nostril (Figure 4C) and solution progressively and gently instilled without air to prevent bubble formation. Obviously, instillation must be performed in a level 2-biosafety cabinet.
Collection of samples requires certifed small-animal surgical training of the operator. At each step, samples must be placed on ice to avoid cell lysis and preserve proteins from denaturation. Basic surgical instruments are required (Figure 4A). They must to be sterilized by autoclaving before use. It is recommended that different surgical instrument sets be used for abdominal and thoracic surgical steps, avoiding cross-contamination of lung samples. The different critical steps of surgical dissection are presented (Figure 5A-5E). Position of the mouse is critical; the animal should be immobilized flat on it&#39;s back head opposite from the operator and straight. Ethanol should be liberally used to prep before the first incision to avoid the spread of hair in the samples and potential bacterial contamination from the skin. Skin is well vascularized and must be retracted carefully to avoid bleeding (Figure 5A).
Then the ribcage is reclined following a lateral chest incision during which the ribcage should be constantly maintained in the tweezers grip to avoid injury to the heart and lungs during the incision. Lung and Heart are exposed (Figure 5B). Uninfected lung appear whitish (Figure 5B). Dissection of the cervical area exposes the trachea and a suture is carefully placed behind the upper trachea (Figure 5C). Cannulating the trachea must be performed with caution. Do not use an oversized cannula (maximum size is 20 G). A small anterior incision of the membraneous trachea between two cartilaginous rings allows insertion of the cannula. When the cannula is observed by transparency through the trachea above the carina (Figure 5D), the suture is tightly tied around the trachea securing it around the cannula (Figure 5E). The cannula is connected to a 3-way male Luer-lock valve with 2 syringes connected to the female ports. One syringe contains ice-cold PBS for bronchoalveolar lavage; the other is empty to draw back the BAL fluid. These syringes should be chaged between groups.
In conclusion, priming prior to acute lung injury model is a relevant and powerful model to explore in&#160;vivo host-mediated interactions between pathogens. The use of animals is the main limitation and should be carefully weighed against the information obtainable in&#160;vitro.

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><tbody><tr><td>Sevorane, Sevoflurane</td><td>Abott</td><td>05458-02</td><td>250 ml plastic bottle</td></tr><tr><td>Fluorescence Reader Mithras&amp;nbsp; LB940</td><td>Berthold Technologies</td><td>reference in first column</td><td>no comment</td></tr><tr><td>Bromo-cresol purple agar</td><td>Biomerieux</td><td>43021</td><td>20x per unit</td></tr><tr><td>Pentobarbital sodique 5.47%</td><td>CEVA</td><td>6742145</td><td>100 ml plastic bottle</td></tr><tr><td>2-headed valve&amp;nbsp;</td><td>Distrimed</td><td>92831</td><td>no comment</td></tr><tr><td>Sterile inoculation loop 10 &amp;micro;l</td><td>Dutscher</td><td>10175</td><td>x1,000 conditioning</td></tr><tr><td>Insuline syringes 1 ml</td><td>Dutscher</td><td>30003</td><td>per 100 conditioning</td></tr><tr><td>2 positions Culture tube 8 ml</td><td>Dutscher</td><td>64300</td><td>no comment</td></tr><tr><td>Ultrospec 10&amp;nbsp;</td><td>General Electric life sciences</td><td>80-2116-30</td><td>no comment</td></tr><tr><td>Hemolysis tubes 13 x 75 mm&amp;nbsp;</td><td>Gosselin</td><td>W1773X</td><td>per 100</td></tr><tr><td>PBS - Phosphate-Buffered Saline</td><td>Life technologies</td><td>10010023</td><td>packaged in 500 ml</td></tr><tr><td>amikacin 1 g</td><td>Mylan</td><td>62516778</td><td>per 10&amp;nbsp;</td></tr><tr><td>Heparin 10,000 UI in 2 ml</td><td>Pan pharma</td><td>9128701</td><td>10x per unit</td></tr><tr><td>RAL 555 coloration kit</td><td>RAL Diagnostics</td><td>361550</td><td>3 flacons of 100 mL</td></tr><tr><td>1.5 ml microcentrifuge tube</td><td>Sarstedt</td><td>55.526.006</td><td>x 1000</td></tr><tr><td>Transparent 300 &amp;micro;l 96-well plate</td><td>Sarstedt</td><td>82 1581500</td><td>no comment</td></tr><tr><td>Yest-peptone-Dextrose Broth</td><td>Sigma</td><td>95763</td><td>in powder</td></tr><tr><td>FITC-albumin</td><td>Sigma</td><td>A9771</td><td>in powder</td></tr><tr><td>Luria Bertani Broth</td><td>Sigma</td><td>L3022</td><td>in powder</td></tr><tr><td>25-gauge needle</td><td>Terumo or unisharp</td><td>A231</td><td>x 100 conditioning</td></tr><tr><td>Cytocentrifuge</td><td>Thermo Scientific</td><td>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.
The first step is the preparation of C. albicans inoculum and intra-nasal instillation to obtain the airway colonization by C. albicans. A 4 days-persistence model is obtained by intranasal instillation of 5 x 105 CFU of C. albicans per mouse (Figure 2B). During these 4 days, mice gain weight (Figure 2A) and instillation of 5x105 CFU does not induce lung injury (Figure 2C). Although C. albicans may persist up to 4 days in this model, load decreases after 48 hr. Therefore, P. aeruginosa-induced acute lung infection is perfomed at 48 hr of C. albicans persistence.
P. aeruginosa strain PAO1 is a largely characterized laboratory strain comprising the major virulence factor, the type three-secretion system (T3SS), as in 75% of clinical lung isolates 15. For theses reasons, PAO1 is a relevant strain in animal models of acute lung infection. Lung injury is assessed through alveolo-capillar permeability measured by protein leak from the vascular compartment into the airway expressed as the lung injury index. Lung injury increases with the inoculum (Figure 3A). Here we report kinetics of the acute lung injury component of our model induced by PAO1 strain (5x106 CFU/mouse) (Figure 3B-3F) alone without prior C. albicans-mediated priming. Depending on strain and time course of the model, the choice of initial P. aeruginosa inoculum is discussed in the next section and is suggested in Table 1.
Five mice per group were used. Lung injury index (Figure 3B), bacterial burden in the lung (Figure 3C), bacterial burden in the spleen, reflecting bacterial dissemination (Figure 3D), BAL cellularity (Figure 3E) and differential cell count (Figure 3F) were determined every 12 hr. Lung injury was maximal between 24 hr and 36 hr after infection (Figure 3B). Bacterial burden showed a 1-log CFU/ml decrease every 24 hr (Figure 3C). Cumulative bacterial dissemination assessed by spleen homogenate cultures increased each day (Figure 3D). Finally, While BAL cellularity in uninfected mice is mainly composed (90%) of alveolar macrophages, in BAL from infected mice, neutrophils were widely recruited and differential cell count showed 90% neutrophils and 10% macrophages and lymphocytes (Figure 3E, 3F).
<img alt="Figure 1" src="/files/ftp_upload/53218/53218fig1.jpg" /> 
Figure 1.&#160;Timeline of the Acute Lung Injury Model to Explore Host-mediated Interaction between C. albicans and P. aeruginosa. 
Graphic representation of the entire procedure. The first step is environmental adaptation of mice the housing facility. The Second step is C. albicans mediated airway colonization. Finally, the third step is the acute lung infection mediated by P. aeruginosa. <a href="https://www.jove.com/files/ftp_upload/53218/53218fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/53218/53218fig2.jpg" /> 
Figure 2.&#160;C. albicans airway Colonization. 
(A,B) Mice are intranasally instilled with 105 CFU C. albicans (strain SC5314). Mice gain weight during C. albicans-mediated airway colonization (A). Colonization of the airway can be prolonged to 3-4 days with only one initial instillation. In a previous study, priming of innate immunity takes place between 24 and 48 hr. (n = 5 per group), error bars represent means &#177; SD. (C,D) mice are intranasally instilled 5 x 105 or 5 x 106 CFU of C. albicans. Lung injury index (C) assessed by alveolar capillary barrier permeability at 24 hr. Weight gain (D) expressed as percent of initial weight (n=5 per group). Error bars represent means &#177; SD. <a href="https://www.jove.com/files/ftp_upload/53218/53218fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53218/53218fig3.jpg" /> 
Figure 3. Model of Acute Lung Injury Induced by P aeruginosa. 
(A) C57Bl/6J mice are intra-nasally infected with increasing loads of P. aeruginosa (from 1 x 106 to 5 x 107 CFU per mouse) (n=5 per group), error bars represent means &#177; SD. Mice are euthanized at 24 hr. Lung injury index is assessed by alveolar-capillary barrier permeability that increases proportionally with bacterial burden. Comparison of lung injury index obtained using old method with lung homogenates supernatants (black bars) and new combined-method using bronchoalveolar lavage supernatants (grey bars) (B-F) mice are intra-nasally infected with 5 x106 CFU per mouse. Mice are euthanized every 12 to 48 hr to acute injury model kinetics. Lung injury (B), lung bacterial burden (C), spleen bacterial burden (D), bronchoalveolar lavage (BAL) cellularity (E) and BAL differential cell count (F) are also assessed. (n=5) per group, error bars represent means &#177; SD. <a href="https://www.jove.com/files/ftp_upload/53218/53218fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53218/53218fig4.jpg" /> 
Figure 4. Surgical Equipment and Intranasal Instillation. 
(A)&#160;Surgical equipment required to perform acute injury model and bronchoalveolar lavage. Here Tracheal cannula (20 G) and the two 1 ml syringes are connected to a Luer-lock 3-way valve. One syringe to inject water into the lungs, one to draw the bronchoalveolar fluid back out from the lungs. (B,C)&#160;Position of the mouse in the hand to perform the intra-nasal instillation. In this photo, the thumb under the jaw ensures a closed mouth during instillation. <a href="https://www.jove.com/files/ftp_upload/53218/53218fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53218/53218fig5.jpg" /> 
Figure 5. Surgery and Broncho-alveolar Lavage. 
The chest is widely opened (A), and rib cage is opened laterally to avoid injury to the heart (B). Following blood collection, the cervical area is dissected to expose the trachea (C). Dental floss is used as a suture and is passed behind trachea (C,D). The trachea is then cannulated with the 20-G cannula combined (D) mounted on the syringe and 3-way valve. The trachea should be tightly secured around the cannula by tying a surgical knot using the suture in place behind the trachea. Finally 500 &#181;l of PBS are gently instilled in the lungs and then the BAL is gently drawn out. (E) Fluid-instilled lungs. <a href="https://www.jove.com/files/ftp_upload/53218/53218fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table>
	<tbody>
		<tr>
			<td></td>
			<td>Minimal Instilled Burden</td>
			<td>Maximal Instilled Burden</td>
		</tr>
		<tr>
			<td>T3SS-</td>
			<td>5 x 107</td>
			<td>1 x 108</td>
		</tr>
		<tr>
			<td>T3SS+</td>
			<td>5 x 106</td>
			<td>1 x 107</td>
		</tr>
		<tr>
			<td>T3SS+ exoU+</td>
			<td>5 x 104</td>
			<td>1 x 105</td>
		</tr>
	</tbody>
</table>
Table 1. P. aeruginosa Inocula Used in Acute Lung Infection Models. 
Suggested optimal intranasal concentrations of inocula to induce acute lung injury according to strains.

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 ...

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Universidad Científica del Sur

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Immunology and Infection

8.8K 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

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 ...

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 ...

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.

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.

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 ...

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 ...

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of bacteria in lung models can re-adapt epithelial arrangement, while immune cells coordinate an inflammatory response against the bacteria in the microenvironment. While in vivo models have been the choice for testing new anti-infectives in the context of cystic fibrosis, they still do not accurately mimic the in vivo conditions of such diseases in humans and the treatment outcomes. Complex in vitro models of the infected airways based on human cells (bronchial epithelial and macrophages) and relevant pathogens could bridge this gap and facilitate the translation of new anti-infectives into the clinic. For such purposes, a co-culture model of the human cystic fibrosis bronchial epithelial cell line CFBE41o- and THP-1 monocyte-derived macrophages has been established, mimicking an infection of the human bronchial mucosa by P. aeruginosa at air-liquid interface (ALI) conditions. This model is set up in seven days, and the following parameters are simultaneously assessed: epithelial barrier integrity, macrophage transmigration, bacterial survival, and inflammation. The present protocol describes a robust and reproducible system for evaluating drug efficacy and host responses that could be relevant for discovering new anti-infectives and optimizing their aerosol delivery to the lungs.

P. aeruginosa Infected 3D Co-Culture of Bronchial Epithelial Cells and Macrophages at Air-Liquid Interface for Preclinical Evaluation of Anti-Infectives

We describe a protocol for a three-dimensional co-culture model of infected airways, using CFBE41o- cells, THP-1 macrophages, and Pseudomonas aeruginosa, established at the air-liquid interface. This model provides a new platform to simultaneously test antibiotic efficacy, epithelial barrier function, and inflammatory markers.

fDrug research for the treatment of lung infections is progressing towards predictive in vitro models of high complexity. The multifaceted presence of ...

Kinetic Visualization of Single-Cell Interspecies Bacterial Interactions

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method has been developed for observing interspecies interactions between two bacterial pathogens. The use of this method to interrogate interactions between a motile Gram-negative pathogen, Pseudomonas aeruginosa and a non-motile Gram-positive pathogen, Staphylococcus aureus is demonstrated here. This protocol consists of co-inoculating each species between a coverslip and an agarose pad, which maintains the cells in a single plane and allows for visualization of bacterial behaviors in both space and time.
Furthermore, the time-lapse microscopy demonstrated here is ideal for visualizing the early interactions that take place between two or more bacterial species, including changes in bacterial species motility in monoculture and in coculture with other species. Due to the nature of the limited sample space in the microscopy setup, this protocol is less applicable for studying later interactions between bacterial species once cell populations are too high. However, there are several different applications of the protocol which include the use of staining for imaging live and dead bacterial cells, quantification of gene or protein expression through fluorescent reporters, and tracking bacterial cell movement in both single species and multispecies experiments.

This live-bacterial cell imaging protocol allows for visualization of interactions between multiple bacterial species at the single-cell level over time. Time-lapse imaging allows for observation of each bacterial species in monoculture or coculture to interrogate interspecies interactions in multispecies bacterial communities, including individual cell motility and viability.

Polymicrobial communities are ubiquitous in nature, yet studying their interactions at the single-cell level is difficult. Thus, a microscopy-based method ...

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.

Early detection and eradication of Pseudomonas aeruginosa within the lungs of cystic fibrosis patients can reduce the chance of developing chronic ...

Establishing a Mouse Model for Candida albicans-Associated Catheter-Related Infection

A Catheter-Related Candida albicans Infection Model in Mouse

Catheter-related infection (CRI) is a common nosocomial infection caused by candida albicans during catheter implantation. Typically, biofilms are formed on the outer surface of the catheter and lead to disseminated infections, which are fatal to patients. There are no effective prevention and treatment management in clinics. Therefore, it is urgent to establish an animal model of CRI for the preclinical screening of new strategies for its prevention and treatment. In this study, a polyethylene catheter, a widely used medical catheter, was inserted into the back of the BALB/c mice after hair removal. Candida albicans ATCC MYA-2876 (SC5314) expressing enhanced green fluorescent protein was subsequently inoculated on the skin's surface along the catheter. Intense fluorescence was observed on the surface of the catheter under a fluorescent microscope 3 days later. Mature and thick biofilms were found on the surface of the catheter via scanning electron microscopy. These results indicated the adhesion, colonization, and biofilm formation of candida albicans on the surface of the catheter. The hyperplasia of the epidermis and the infiltration of inflammatory cells in the skin specimens indicated the histopathological changes of the CRI-associated skin. To sum up, a mouse CRI model was successfully established. This model is expected to be helpful in the research and development of therapeutic management for candida albicans associated CRI.

Author Spotlight: Enhancing Candida albicans Detection in Catheter Infections Using Fluorescent Protein Tagging 

We establish a mouse model of C.albicans-associated catheter-related infection (CRI), in which biofilm forms on the catheter, and the interaction between C.albicans and host correlates well with the clinical CRI. This model helps screen therapies for C.albicans biofilm-associated CRI, laying a foundation for clinical transformation.

Catheter-related infection (CRI) is a common nosocomial infection caused by candida albicans during catheter implantation. Typically, biofilms are formed ...

Adaptable CF Model for Antimicrobial Testing and Microbial Dynamics Analysis

Development of a Polymicrobial Colony Biofilm Model to Test Antimicrobials in Cystic Fibrosis

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as colony-forming units or metabolic activity. Furthermore, many biofilm models do not reflect the biological and physiochemical properties of the human host environment. This is an important issue in many conditions, but most noticeably in cystic fibrosis (CF). A large proportion of people with CF suffer from both chronic and intermittent infections, and in vitro, antibiotic susceptibility testing poorly correlates with patient treatment outcomes. Some biofilm models incorporate CF lung-relevant media, including synthetic sputum mimics, but do not consider the polymicrobial nature of the environment, which alters biofilm architecture, physiology, and the way microbes respond to treatment. The solid-air interface colony biofilm model described here is highly adaptable and incorporates both CF-relevant media and a polymicrobial context. This model can also be used for mid-throughput screening of antimicrobials and to study their effect on polymicrobial dynamics. Output measurements from the model can be colony-forming units, metabolic activity, and confocal microscopy analysis. The model can easily be adapted to different microorganisms, media, temperatures, and variable oxygen conditions and can be used to test a wide range of chemical, biological, and physical treatments.

This methodology allows for the establishment of a polymicrobial biofilm model in cystic fibrosis for antimicrobial sensitivity testing in research and clinical laboratories. This model provides accurate and reliable results over a range of outputs.

A range of bacteria biofilm models exist for the testing of antibiotics. However, many of these are limited to a single experimental output, such as ...

In Vitro Polymicrobial Interactions in Cystic Fibrosis Lung Infections

Growing a Cystic Fibrosis-Relevant Polymicrobial Biofilm to Probe Community Phenotypes

Most in vitro models lack the capacity to fully probe bacterial phenotypes emerging from the complex interactions observed in real-life environments. This is particularly true in the context of hard-to-treat, chronic, and polymicrobial biofilm-based infections detected in the airways of individuals living with cystic fibrosis (CF), a multiorgan genetic disease. While multiple microbiome studies have defined the microbial compositions detected in the airway of people with CF (pwCF), no in vitro models thus far have fully integrated critical CF-relevant lung features. Therefore, a significant knowledge gap exists in the capacity to investigate the mechanisms driving the pathogenesis of mixed species CF lung infections. Here, we describe a recently developed four-species microbial community model, including Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenica grown in CF-like conditions. Through the utilization of this system, clinically relevant phenotypes such as antimicrobial&#160;recalcitrance of several pathogens were observed and explored at the molecular level. The usefulness of this in vitro model resides in its standardized workflow that can facilitate the study of interspecies interactions in the context of chronic CF lung infections.

This protocol describes a cystic fibrosis (CF) lung-relevant four-species polymicrobial biofilm model that can be used to explore the impact of bacterial interspecies interactions.

Most in vitro models lack the capacity to fully probe bacterial phenotypes emerging from the complex interactions observed in real-life environments. This ...

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

Summary

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