Name: studying microbial communities in vivo: a model of host-mediated interaction between candida albicans and pseudomonas aeruginosa in the airways
Uploaded: 2016-01-13T14:00:00
Description: 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

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>
	<l...

<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>

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.
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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 ...

The overall goal of this procedure is to build a model to study the interactions between an individual bacterium and a single fungus. This message can help to answer key questions in the field of pathogen interactions, such as what factors are involved in cross-talk and interspecies dialect. The main advantage of this technique is the data generated from this in vivo model have clinical relevance.To intranasally install the fungus, first dilute two times ten to the sixth CFU of C.albicans per milliliter in sterile PBS. Next, after confirming the loss of the righting reflex in an adult hypotonic mouse, aspirate at least 50 microliters of the fungal suspension into a 200 microliter pipette. Now, grasp the mouse with one hand and turn the animal so that it is nestled on its back with its abdomen facing upright.When the mouse is in position, use the index finger of the hand holding the mouse to support the head, and the thumb to keep the jaw closed, and move the pipette tip close to the animal's nose. Then, carefully dispense a 50 microliter drop of fungus onto the nostrils, allowing the drop to be inhaled by the spontaneous breathing of the mouse. And place the mouse alone in a cage until it is fully recovered.Four days after C.albicans installation, shave the abdomen of the euthanized animal, followed by ethanol sterilization of the exposed skin. Next, use scissors to open the skin from the sternum to the mid-abdomen. Then, extend the incision along the rib cage on either side of the midline and fold back the skin on either side of the thorax.When the rib cage is visible, make a vertical incision on either side of the chest toward the clavicles, and use the sternum to recline the entire anterior chest wall to visualize the heart and lungs. Then, using a pre-heparinized syringe, puncture the heart next to the intraventricular artery, and withdraw a minimum of 500 microliters of blood. Place the blood sample on ice.Then, make a midline cervical incision to reveal the trachea. Carefully dissect the fascia around the trachea, followed by the placement of a suture under the trachea. Then, use a 20-gauge modified gavage needle to catheterize the trachea, securing the cannula with the previously placed suture.To perform a bronchoalveolar lavage, gently dispense 500 microliters of ice-cold PBS into the lung. Then, aspirate the lavage fluid and transfer the fluid into a two milliliter centrifuge tube on ice. After collecting two more 500 microliter samples, remove the lungs from the chest, and place a piece of lung into a 1.5 milliliter centrifuge tube for immediate storage at minus 80 degrees Celsius.Then, place another piece of lung tissue into a pre-weighed hemolysis tube containing PBS on ice. Finally, make an incision in the left side of the abdomen. Harvest the spleen into a second hemolysis tube containing one milliliter of PBS and place the tube on ice.A four days persistence model is obtained by intranasal installation of five times ten to the fifth CFU of C.albicans per mouse. During these four days, mice gain weight, but do not exhibit lung injury. As the CFU of the inoculum increases, however, so does the lung injury.With the maximum lung injury observed between 24 to 36 hours after infection. The bacterial burden, by contrast, demonstrates a one log CFU per milliliter decrease every 24 hours, with a cumulative bacterial dissemination increase observed over each day. In the bronchoalveolar lavage harvested from infected mice, neutrophils are widely recruited, with the differential cell count demonstrating a 90 percent neutrophil, 10 percent macrophage lymphocyte infiltrating immune cell population.Following this procedure, other methods like analysis of the bronchoalveolar lavage can be performed to answer additional questions about the characterization of the inflammatory response. After watching this video, you should have a good understanding of how to build a colonization Pseudomonas. Don't forget that working with luminescent Candida can be extremely hazardous, and the precautions, such as wearing protective gear to avoid droplet contamination, should always be taken while performing this procedure.

Research

JoVE Journal

Immunology and Infection

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

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

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

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 ...

Pseudomonas aeruginosa Induced Lung Injury Model

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. ...

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

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Clarifying and Imaging Candida albicans Biofilms

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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

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

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

Antibiotic Efficacy Testing in an Ex vivo Model of Pseudomonas aeruginosa and Staphylococcus aureus Biofilms in the Cystic Fibrosis Lung

The effective prescription of antibiotics for the bacterial biofilms present within the lungs of individuals with cystic fibrosis (CF) is limited by a ...

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Tools for the Real-Time Assessment of a Pseudomonas aeruginosa Infection Model

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a ...