This work was supported by National Institutes of Health grants HL105947-01 (YL), HL07829-16 (AM), HL090152 (AM).

The animal experiments were approved by the Animal Care Committee and Institutional Biosafety Committees of the University of Illinois at Chicago.
NOTE: All procedures involving pseudomonas should be performed with Biosafety Level 2 (BSL2) practices, which include but are not limited to: mask, eye protection, gown or jumpsuit, and double gloves. Work in certified Biosafety Cabinet. Treat instruments in contact with bacteria with bleach or chlorine dioxide based disinfectant. Use a sealed box for transport samples.
1. P. aeruginosa Culture and Growth
<ol>
	<li>Store P. aeruginosa PA103 as a bacterial stock in a screw cap cryovial at -80 &#176;C.</li>
	<li>Place the vial in a rack in a box with 70% ethanol wetted paper towels and take to the Biosafety Level 2 (BSL2) laboratory.</li>
	<li>Streak the bacteria onto sheep blood agar plates and grow at 37 &#176;C for ~15 hr in an incubator.</li>
	<li>In a BSL2 hood, scratch the bacteria from plate with bacteria loop and resuspend in 5 ml PBS.</li>
	<li>Store the stock at 4 &#176;C for up to 3 months. However, determine the titer every 2 weeks.</li>
	<li>Serially dilute the bacteria in PBS (usually from 1:104 to 1:107) and plate out the known dilution on sheep blood agar plates.</li>
	<li>Incubate the plates for ~15 hr. Count colonies and calculate Colony Forming Unit (CFU) to determine bacteria concentration.</li>
	<li>Resuspend the appropriate concentrations (~5 x 103 CFU/&#181;l) in 0.5 ml PBS in 1.5 ml sterile screw cap cryovials. Seal the cryovials and place them in a cryovial rack on disinfectant laden paper towels in a snap lid box.</li>
	<li>Transport the box to BSL2 animal facility. Once there, open the box in the BSL2 cabinet.</li>
</ol>
2. P. aeruginosa Instillation
<ol>
	<li>Perform survival surgery aseptically (sterile gloves, sterile instruments, and aseptic techniques). Use a sterile drape to provide a working surface for sterile (autoclaved) surgical instruments. Sterilize instruments using a hot bead sterilizer between each tracheal instillation procedure.</li>
	<li>Weigh mice prior to anesthetization. Anesthetize mice with ketamine (100 mg/kg), xylazine (5 mg/kg), in 0.1 - 0.2 ml PBS intraperitoneally (i.p.). Determine the effectiveness of the anesthetic by non-responsiveness to toe pinch. Use a vet ointment on eyes to prevent dryness under anesthesia.</li>
	<li>Restrain mice on a surgical board in BSL2 cabinet.</li>
	<li>Identify the area for the cut down. Shave this area and prepare the skin using alternating alcohol and povidone iodine swabs 3 times.</li>
	<li>Treat the incision area with local anesthetic (lidocaine) as this anesthesia is sufficient for a minor surgery such as a skin incision to access the trachea.</li>
	<li>Make a small incision (approximately 5 mm) at the midline of the neck, and use blunt forceps to gently move the muscle for access to the trachea. Expose the trachea by surgical dissection.</li>
	<li>Draw bacteria solution into a 1 ml disposable syringe with 27 G needle. For each mouse, administer 20-30 &#181;l of bacteria at the appropriate concentration (up to 105 CFU each mouse).</li>
	<li>Insert needle into the trachea. Inject solution slowly into the tracheal.</li>
	<li>Ensure that the animal gasps, which usually indicates that solution has reached into the lung.</li>
	<li>Close the wound with sterile sutures (6-0 monofilament) under aseptic conditions.</li>
	<li>Use 0.1 mg/kg buprenorphine by subcutaneous injection as post-op analgesia to control post-surgical pain.</li>
	<li>Ensure that the time required from initial anesthetic induction to incision closure is less than 15 min.</li>
	<li>After injection, dispose of syringes and needles in an appropriate biohazard sharps container.</li>
	<li>Treat the surgical instruments with chlorine dioxide based disinfectant for 15 min, rinse and return to the lab for sterilization and reuse as necessary.</li>
	<li>House the mice singly in clean cages at warm environment.</li>
	<li>Check on the animal every 30 min until it regains consciousness and starts to move; and later at 12 hr intervals for the first 3 days post-surgery.</li>
	<li>Keep the mice in the animal BSL2 facility throughout the experiment. Ensure that only tissue contained in sealed box leaves the animal BSL2 facility. If the mice express any of the moribund behaviors (defined as respiratory distress, lethargy, failure to ambulate in response to gentle stimulation) at any point in the study, euthanize the mice by CO2 inhalation from a bottled source followed by cervical dislocation.</li>
	<li>Euthanize the experimental subject by exsanguination under anesthesia.
	<ol>
		<li>Collect lung tissue from euthanized mice. Carry out these procedures in a BSL2 hood. If needed, transfer the sample using sealed tubes placed in a rack on disinfectant laden paper towels in a snap lid box for further process.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Ware, L. B., Matthay, M. 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+acute+respiratory+distress+syndrome.">The acute respiratory distress syndrome.</a> N Engl J Med. 342, 1334-1349 (2000).</li><li>Matthay, M. A., Ware, L. B., Zimmerman, G. 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+acute+respiratory+distress+syndrome.">The acute respiratory distress syndrome.</a> J Clin Invest. 122, 2731-2740 (2012).</li><li>Shimabukuro, D. W., Sawa, T., Gropper, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Injury+and+repair+in+lung+and+airways.">Injury and repair in lung and airways.</a> Crit Care Med. 31 (8), 524-531 (2003).</li><li>Wansleeben, C., Barkauskas, C. E., Rock, J. R., Hogan, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+of+the+adult+lung:+Their+development+and+role+in+homeostasis,+regeneration,+and+disease.">Stem cells of the adult lung: Their development and role in homeostasis, regeneration, and disease.</a> Wiley Interdiscip Rev Dev Biol. 2, 131-148 (2013).</li><li>Singleton, P. A., 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=Dynamin+2+and+c-abl+are+novel+regulators+of+hyperoxia-mediated+nadph+oxidase+activation+and+reactive+oxygen+species+production+in+caveolin-enriched+microdomains+of+the+endothelium.">Dynamin 2 and c-abl are novel regulators of hyperoxia-mediated nadph oxidase activation and reactive oxygen species production in caveolin-enriched microdomains of the endothelium.</a> J Biol Chem. 284, 34964-34975 (2009).</li><li>Sadikot, R. T., Blackwell, T. S., Christman, J. W., Prince, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogen-host+interactions+in+pseudomonas+aeruginosa+pneumonia.">Pathogen-host interactions in pseudomonas aeruginosa pneumonia.</a> Am J Respir Crit Care Med. 171, 1209-1223 (2005).</li><li>Liu, Y., 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=Foxm1+mediates+the+progenitor+function+of+type+ii+epithelial+cells+in+repairing+alveolar+injury+induced+by+pseudomonas+aeruginosa.">Foxm1 mediates the progenitor function of type ii epithelial cells in repairing alveolar injury induced by pseudomonas aeruginosa.</a> J Exp Med. 208, 1473-1484 (2011).</li><li>Kurahashi, K., 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=Pathogenesis+of+septic+shock+in+pseudomonas+aeruginosa+pneumonia.">Pathogenesis of septic shock in pseudomonas aeruginosa pneumonia.</a> J Clin Invest. 104, 743-750 (1999).</li><li>Kumar, P. A., 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=Distal+airway+stem+cells+yield+alveoli+in+vitro+and+during+lung+regeneration+following+h1n1+influenza+infection.">Distal airway stem cells yield alveoli in vitro and during lung regeneration following h1n1 influenza infection.</a> Cell. 147, 525-538 (2011).</li><li>Delano, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sepsis+induces+early+alterations+in+innate+immunity+that+impact+mortality+to+secondary+infection.">Sepsis induces early alterations in innate immunity that impact mortality to secondary infection.</a> J Immunology. 186, 195-202 (2011).</li><li>Lange, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+murine+model+of+sepsis+following+smoke+inhalation+injury.">A murine model of sepsis following smoke inhalation injury.</a> Biochem Biophys Res Commun. 391 (3), 1555-1560 (2010).</li><li>Kobayashi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+chemokines+in+neutrophil+biology.">The role of chemokines in neutrophil biology.</a> Front Biosci. 13, 2400-2407 (2008).</li><li>Matute-Bello, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+acute+lung+injury.">Animal models of acute lung injury.</a> Am J Physiol Lung Cell Mol Physiol. 295, L379-L399 (2008).</li><li>Sadikot, R. T., 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=Targeted+immunomodulation+of+the+nf-kappab+pathway+in+airway+epithelium+impacts+host+defense+against+pseudomonas+aeruginosa.">Targeted immunomodulation of the nf-kappab pathway in airway epithelium impacts host defense against pseudomonas aeruginosa.</a> J Immunol. 176, 4923-4930 (2006).</li><li>Pinto, I. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+experimental+model+of+neutrophilic+pulmonary+response+induction+in+mice.">Development of an experimental model of neutrophilic pulmonary response induction in mice.</a> J Pneumologia. 29, 213-214 (2003).</li></ol>

The authors have nothing to disclose.

The Pseudomonas mouse lung injury model that we describe here mimics the whole process of inflammation, lung injury, repair, and resolution that occur following acute lung injury or pneumonia. It has unique advantages comparing with several other injury models in that it is clinically relevant and relatively safe and easy to handle.
The critical step in the procedure is that the injection of bacteria solution needs to be very slow. If injection is too fast, the mice are likely to die by choke....

Lungs are exposed to environmental pathogens and are susceptible to inflammation and injury1-3. During pathological conditions such as pneumonia or Adult Respiratory Distress Syndrome (ARDS), pathogens as well as inflammatory factors released by leukocytes induce injury and death of alveolar cells1-3. It is important to develop animal models of acute lung injury to facilitate the study of pathology of injury as well as mechanism of repair.
Currently, most people use hyperoxia and bleomycin induced mouse lung injury models4. However, the mechanisms of hyperoxia caused injury are not the same as most common lung injuries that occur during pneumonia or ARDS5. Bleomycin induced acute injury is rare in a clinical context4. Here we report a mouse lung injury model using intra-tracheal injection of P. aeruginosa 6,7. This model is clinically relevant, and mimics the processes that happen following pneumonia8.
As an opportunistic, nosocomial pathogen of immunocompromised individuals, P. aeruginosa typically infects the pulmonary tract, urinary tract, burns, wounds, and also causes other blood infections6. The bacteria release virulence factor exotoxin A, multiply and trigger immune responses6. Intra-trachael administration of P. aeruginosa reflects the situation in human exposure to the bacteria which cause pneumonia and the pathology is likely to be different from the recently reported influenza virus H1N1 induced lung injury model9. Since P. aeruginosa is an opportunistic pathogen, it is relatively safe to handle as compared to some of the more virulent pathogens. &#160;Here we used intra-tracheal injection to administer the bacteria because we observed that this method introduced more bacteria into the distal alveoli region of the lung compared with some other procedures such as using a catheter via mouth.
Compared with other acute lung injury models, the P. aeruginosa model described here is suitable for studying lung injury induced by bacteria and by excessive inflammation. Unlike other animal models that use P. aeruginosa to induce sepsis10,11, here we use intra-tracheal injection of these bacteria to induce localized acute lung injury.

<table border="0" cellpadding="0" cellspacing="0" width="650">
	<tbody>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">Name</td>
			<td style="width:145px;">Company</td>
			<td style="width:145px;">Catalog number&#160;</td>
			<td style="width:145px;">Comments</td>
		</tr>
		<tr height="38">
			<td height="38" style="height:38px;width:215px;">Anesthetic: Ketamin, xylazine, lidocaine, buprenorphine</td>
			<td style="width:145px;"></td>
			<td style="width:145px;"></td>
			<td style="width:145px;">pharmaceutical grade</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">27g needle</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:145px;">1482648</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">syringe</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:145px;">14823434</td>
			<td style="width:145px;">1 ml</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">scissors</td>
			<td style="width:145px;">Fine Science tools</td>
			<td style="width:145px;"></td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">forceps</td>
			<td style="width:145px;">Fine Science tools</td>
			<td style="width:145px;"></td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;width:215px;">suture&#160;</td>
			<td style="width:145px;">Fisher</td>
			<td style="width:145px;">NC0147607</td>
			<td style="width:145px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Eye gauge, glove, gown</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">Biosafety Cabinet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">chlorine dioxide based sterilant&#160;</td>
			<td></td>
			<td></td>
			<td>Clidox</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:27px;">sheep blood agar plates&#160;</td>
			<td>Medex supply</td>
			<td>HL-1160</td>
			<td></td>
		</tr>
	</tbody>
</table>

pseudomonas aeruginosa induced lung injury model

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

Starting from 24 - 72 hr post P. aeruginosa injection, increased cellularity was observed in lung sections (Figure 1A-D). The lung started to recover from 96 hr post injury (Figure 1E). At 7 days post P. aeruginosa, normal alveoli morphology was largely restored (Figure 1F). Tunnel staining using lung sections prepared at 24 hr post P. aeruginosa showed cell death in alveoli cells (Figure 1G-I). In order to study the repair pro...

Watch this Scientific Journal Video about Pseudomonas aeruginosa Induced Lung Injury Model at JoVE.com

Pseudomonas aeruginosa Induced Lung Injury Model

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

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. ...

pseudomonas-aeruginosa-induced-lung-injury-model

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Immunology and Infection

18.4K 조회수.  University of Illinois at Chicago. We have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa. This model mimics lung injury during pneumonia and is clinically relevant. 

비디오: Pseudomonas aeruginosa Induced Lung Injury Model

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

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

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

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

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

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

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

Pseudomonas aeruginosa and Saccharomyces cerevisiae Biofilm in Flow Cells

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

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

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

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

RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract

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

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

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

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

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

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

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

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 ...

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.

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

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

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

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

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

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

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

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

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

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

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

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

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

A Delayed Inoculation Model of Chronic Pseudomonas aeruginosa Wound Infection

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

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

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes susceptibility to bacterial infections in the respiratory system. However, the effects of cigarette smoking on bacterial infections in human lung epithelial cells have yet to be thoroughly studied. Described here is a detailed protocol for the preparation of cigarette smoking extracts (CSE), treatment of human lung epithelial cells with CSE, and bacterial infection and infection determination. CSE was prepared with a conventional method. Lung epithelial cells were treated with 4% CSE for 3 h. CSE-treated cells were, then, infected with Pseudomonas at a multiplicity of infection (MOI) of 10. Bacterial loads of the cells were determined by three different methods. The results showed that CSE increased Pseudomonas load in lung epithelial cells. This protocol, therefore, provides a simple and reproducible approach to study the effect of cigarette smoke on bacterial infections in lung epithelial cells.

Studying Effects of Cigarette Smoke on Pseudomonas Infection in Lung Epithelial Cells

Described here is a protocol to study how cigarette smoke extract affects bacterial colonization in lung epithelial cells.

Cigarette smoking is the major etiological cause for lung emphysema and chronic obstructive pulmonary disease (COPD). Cigarette smoking also promotes ...