Name: rna isolation of pseudomonas aeruginosa colonizing the murine gastrointestinal tract
Uploaded: 2011-09-28T12:00:00
Duration: 8 min 52 s
Description: Watch this Scientific Journal Video about RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract at JoVE.com

This work was funded by the National Institutes of Health grants AI62983 (AYK), AI22535 (GPB).

1. Murine Model of P. aeruginosa GI Colonization and Dissemination
<ol>
<li>C3H/HeN mice (6-8 wks old, female, Harlan) are treated with oral antibiotics to deplete commensal flora and then mono-colonized with PA as previously described4.</li>
</ol>
2. Harvesting Murine Cecal Luminal Contents
<ol>
<li>Immerse a cleaned stainless steel mortar into a liquid nitrogen bath.</li>
<li>Euthanize mice by carbon dioxide asphyxiation.</li>
<li>Secure mouse carcass on a styrofoam board and shower with 95% ethanol. Make a midline longitudinal incision through the skin from the sternum to the perineum. Reflect the skin and peritoneum to expose the abdominal cavity.</li>
<li>Resect the entire cecum. Hold the cecum with forceps over the stainless steel mortar. Snip both ends of the cecum with dissection scissors.</li>
<li>Insert a P1000 pipette tip filled with 1 ml of cecal flushate buffer (10 mM TrisHCl, 1 mM EDTA and 200 mM NaCl)5 into the proximal end of the cecum. Flush the cecal flushate buffer and cecal luminal contents into the stainless steel mortar. 1 ml of cecal flushate buffer will be sufficient for flushing all of the cecal contents. Cecal flushate contents will freeze immediately upon coming into contact with the mortar.</li>
<li>Grind the cecal flushate contents with a sterile pestle.</li>
<li>Place ground frozen cecal luminal contents into a 50 ml polypropylene conical tube submerged in a dry ice/ethanol bath.</li>
<li>Repeat Steps 2.2 through 2.7 for each additional mouse.</li>
<li>Store at -80&deg;C.</li>
</ol>
3. Bacterial RNA Isolation
<ol>
<li>Warm 1 volume (based on final volume of two cecal luminal contents, approximately 3 ml) of acid phenol/chloroform (5:1, v/v) contained in a 50 ml Oak Ridge centrifuge tube with the cap secured in parafilm in a 65&deg;C water bath.</li>
<li>Boil 0.5 volume of lysis buffer (2% SDS, 16 mM EDTA and 200 mM NaCl)6 contained in a 50 ml polypropylene conical tube for 5 minutes.</li>
<li>Add boiling lysis buffer to cecal luminal contents. Homogenize. Boil for 5 minutes with periodic vortexing.</li>
<li>Add the 100&deg;C cecal contents/lysis sample to the 65&deg;C acid phenol/chloroform in the Oak Ridge centrifuge tube. Seal cap with parafilm.</li>
<li>Incubate at 65&deg;C for 10 minutes with periodic vortexing (every 2 minutes).</li>
<li>Centrifuge sample at 2,500 g at 4&deg; for 15 minutes.</li>
<li>Carefully transfer aqueous phase (avoiding any of the white interface) to a fresh 50 ml Oak Ridge centrifuge. The volume of the aqueous phase will be approximately 50% of the total volume of cecal contents, lysis buffer, and acid phenol/chloroform. Add an equal volume of acid phenol/chloroform.</li>
<li>Seal cap with parafilm and mix well by vortexing at high speed.</li>
<li>Centrifuge sample at 2,500 g at 4&deg; for 15 minutes.</li>
<li>Repeat Steps 3.7 to 3.9 until there is no visible white interface between the aqueous and organic phases.</li>
<li>Carefully transfer aqueous phase to a fresh 50 ml Oak Ridge centrifuge. Add an equal volume of chloroform/isoamyl alcohol (24:1, v/v).</li>
<li>Seal cap and mix well by vortexing.</li>
<li>Centrifuge sample at 2,500 g at 4&deg; for 15 minutes. Remove aqueous phase (volume will be approximately 50% of the total reaction volume) to 15 ml polypropylene conical tube and add an equal volume of isopropanol.</li>
<li>Incubate at -20&deg; for at least 2 hours or overnight.</li>
<li>Centrifuge sample at 2,500 g at 4&deg; for 45 minutes. A gel-like pellet will form at the bottom of the tube. Remove supernatant.</li>
<li>Wash the pellet with 1 ml of ice-cold 70% ethanol. Vortex. Centrifuge sample at at 10,000 g at 4&deg; for 5 minutes.</li>
<li>Remove the supernatant and invert the tube to let the pellet dry at room temperature for 10 minutes.</li>
<li>Resuspend the RNA pellet in 200 &mu;l of RNase-free water.</li>
</ol>
4. DNase Treatment (Turbo DNA-Free, Applied Biosystems)
<ol>
<li>Add 20 &mu;l of 10X Turbo DNase buffer and 2 &mu;l Turbo DNase and mix gently.</li>
<li>Incubate at 37&deg; for 20 minutes.</li>
<li>Add 20 &mu;l of resuspended DNase inactivation reagent and mix well.</li>
<li>Incubate 5 minutes at room temperature, mixing occasionally.</li>
<li>Centrifuge at 10,000 g at room temperature for 1.5 minutes and transfer the supernatant to a new microfuge tube.</li>
<li>Add one volume of cold (4&deg;) acid phenol/chloroform and mix thoroughly by vortexing for 1 minute.</li>
<li>Centrifuge at 12,500 g at room temperature for 2 minutes.</li>
<li>Transfer the aqueous phase to a new tube and add 1 volume of chloroform/isoamyl alcohol (49:1 v/v), vortex.</li>
<li>Centrifuge at 12,500 g at room temperature for 2 minutes.</li>
<li>Transfer the aqueous phase (approximately 50% of the total reaction volume) to a new tube.</li>
<li>Add 0.1 volume of 3M sodium acetate, pH 5.5 and 2.5 volumes of ice-cold 100% ethanol. Vortex. </li>
<li>Incubate at -20&deg; for at least 2 hours or at -80&deg; for 30 minutes.</li>
<li>Centrifuge at 12,000 g for 30 minutes at 4&deg;</li>
<li>Remove supernatant.</li>
<li>Wash the pellet with 1 ml of ice-cold 70% ethanol by centrifuging for 5 minutes at 10,000 g at 4&deg;.</li>
<li>Remove the supernatant and air dry for 10 minutes.</li>
<li>Resuspend the pellet in 100 &mu;l of RNase-free water. May need to place in 65&deg; water bath to resuspend and dissolve the pellet.</li>
<li>To test the efficacy of the DNase treatment, a standard RT-PCR reaction for the amplification of a ribosomal protein (or other reference gene) could be performed for treated and non-treated samples, using no RT controls.</li>
</ol>
5. RNA Cleanup Step (Qiagen, RNeasy Kit)
<ol>
<li>Refer to pages 56-57 of Qiagen RNeasy Mini Handbook (4th Edition, April 2006). Follow as indicated. </li>
</ol>
6. Representative Results
The amount of bacterial total RNA recovered by using this protocol is approximately 2-3 &mu;g from two cecums. The RNA recovered is of sufficient quantity and quality for subsequent qPCR, transcription profiling, and RNA Seq experiments. RNA purity is routinely assessed by measuring the 260nm/280nm ratio7 of the sample, yet this method provides no information about RNA integrity. The Agilent Bioanalyzer is a microfluidics-based platform for sizing, quantification and quality control of DNA, RNA, proteins and cells; and utilizes an RNA integrity metric known as RNA Integrity Number (RIN)8. RNA extracted with this protocol produces 260/280 ratios ranging from 1.7 to 2.0 and RIN values &ge; 7.0. An example of an Agilent Bioanalyzer analysis of the bacterial RNA recovered by this protocol is shown in Figure 1. 
<img src="/files/ftp_upload/3293/3293fig1.jpg" alt="Figure 1"/> Figure 1. Agilent Bioanalyzer electropherogram and gel-like image of Pseudomonas aeruginosa total RNA sample isolated and recovered from murine cecal contents. RIN 8.0.

<ol>
	<li>Bodey, G. P., Bolivar, R., Fainstein, V., Jadeja, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infections+caused+by+Pseudomonas+aeruginosa.">Infections caused by Pseudomonas aeruginosa.</a> Rev Infect Dis. 5, 279-279 (1983).</li><li>Bertrand, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endemicity,+molecular+diversity+and+colonisation+routes+of+Pseudomonas+aeruginosa+in+intensive+care+units.">Endemicity, molecular diversity and colonisation routes of Pseudomonas aeruginosa in intensive care units.</a> Intensive Care Med. 27, 1263-1268 (2001).</li><li>Koh, A. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+in+vivo+transcription+profiling+for+identifying+Pseudomonas+aeruginosa+genes+needed+for+gastrointestinal+colonization+and+dissemination.">Utility of in vivo transcription profiling for identifying Pseudomonas aeruginosa genes needed for gastrointestinal colonization and dissemination.</a> PLoS One. 5, e15131-e15131 (2010).</li><li>Koh, A. Y., Priebe, G. P., Pier, 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=Virulence+of+Pseudomonas+aeruginosa+in+a+murine+model+of+gastrointestinal+colonization+and+dissemination+in+neutropenia.">Virulence of Pseudomonas aeruginosa in a murine model of gastrointestinal colonization and dissemination in neutropenia.</a> Infect Immun. 73, 2262-2272 (2005).</li><li>Alexander, R. J., Raicht, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+total+RNA+from+human+stool+samples.">Purification of total RNA from human stool samples.</a> Dig Dis Sci. 43, 2652-2658 (1998).</li><li>Fitzsimons, N. A., Akkermans, A. D., de Vos, W. M., Vaughan, E. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+gene+expression+detected+in+human+faeces+by+reverse+transcription-PCR.">Bacterial gene expression detected in human faeces by reverse transcription-PCR.</a> J Microbiol Methods. 55, 133-140 (2003).</li><li>Manchester, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+UV+methods+for+measurement+of+protein+and+nucleic+acid+concentrations.">Use of UV methods for measurement of protein and nucleic acid concentrations.</a> Biotechniques. 20, 968-970 (1996).</li><li>Schroeder, 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+RIN:+an+RNA+integrity+number+for+assigning+integrity+values+to+RNA+measurements.">The RIN: an RNA integrity number for assigning integrity values to RNA measurements.</a> BMC Mol Biol. 7, 3-3 (2006).</li><li>Turnbaugh, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+core+gut+microbiome+in+obese+and+lean+twins.">A core gut microbiome in obese and lean twins.</a> Nature. 457, 480-484 (2009).</li></ol>

No conflicts of interest declared.

The RNA extraction method described here allows for recovery of sufficient quantities of high-quality Pseudomonas aeruginosa total RNA harvested from the murine GI tract. This method is not restricted to P. aeruginosa and can potentially be applied to other bacteria. The recovery of sufficient microbial organisms from the intestine will vary significantly from organism to organism. In our murine model, P. aeruginosa typically colonizes the murine GI tract at levels between 5 x 107 to 5 x 108 cfu/g feces4. Since the recovered cecal contents are roughly 0.5 gram, the estimated number of P. aeruginosa recovered from two cecums is between 5 x 107 to 5 x 108 cfu. If other microorganisms are used, it would be prudent to verify GI colonization levels and then calculate the number of cecums needed to recover the targeted number of cfu. It is also important to note that when using this particular murine model, antibiotic-treated mice not infected with PA have no quantifiable amounts of RNA isolated from their cecal contents. 
Our murine model of Pseudomonas aeruginosa gastrointestinal colonization and dissemination attempts to emulate the pathogenesis of P. aeruginosa bacteremia in cancer and stem cell transplant patients. In this patient population, commensal flora is often depleted secondary to antibiotic or chemotherapeutic treatment (e.g the antibiotic depletion of GI commensal flora) resulting in overgrowth of pathogenic microbes (e.g. mono-association with P. aeruginosa) and then subsequent dissemination after immune suppression. The advantages and limitations of this murine model have already been addressed previously4. The purpose of this current study is to provide a methodology for isolating microbial total RNA from the GI tract. This protocol can easily be adapted to other murine models that study other aspects of microbial pathogenesis in the GI tract (i.e. commensal flora interactions, bacterial effects on inflammatory bowel disease, etc.). 
One advantage of this method is the incorporation of multiple lysis steps including freeze/thaw, mechanical disruption (pulverizing with mortar/pestle, homogenization), boiling, and chemical lysis (e.g. SDS). Despite the multitude of lysis steps, some microorganisms (notably Gram-positive bacteria and fungi/yeast) may require additional mechanical disruption. After the hot lysis/acid phenol-chloroform incubation (Step 3.5), the addition of beads (0.1 mm for bacteria and/or 0.5-0.7 mm for yeast) and a subsequent bead-beating step should be sufficient to lyse these organisms9, 10. 
Given the complex nature of materials recovered from the murine cecum, the repeated cold acid-phenol/chloroform extractions (Step 3.7 to 3.9) are absolutely required in order to achieve acceptable RNA quality and integrity for further downstream reactions. Between 3-5 cold acid-phenol/chloroform extractions may be required before the white interface between the aqueous and organic phases is eliminated. Finally, the combination of both the DNase treatment (Step 4) and RNeasy Cleanup Protocol (Step 5) are essential for removing contaminating DNA and small non-mRNA (5s and tRNA). As stated previously, the RNA recovered by utilizing this protocol is of sufficient quantity and quality for subsequent transcription profiling 3, qPCR (unpublished), and RNA Seq (unpublished) experiments.

<table border="1">
 <tbody>
 <tr>
 <th>Name of the reagent</th>
 <th>Company</th>
 <th>Catalogue number</th>
 </tr>
 <tr>
 <td>Mortar and Pestle</td>
 <td>Fisher</td>
 <td>12-947-1</td>
 </tr>
 <tr>
 <td>Homogenizer</td>
 <td>Omni</td>
 <td>TM-125</td>
 </tr>
 <tr>
 <td>Oak Ridge centrifuge tubes (50 ml)</td>
 <td>Nalgene</td>
 <td>3119-0050</td>
 </tr>
 <tr>
 <td>Acid Phenol/Chloroform</td>
 <td>Ambion</td>
 <td>AM9720</td>
 </tr>
 <tr>
 <td>Chloroform</td>
 <td>Sigma-Aldrich</td>
 <td>C2432</td>
 </tr>
 <tr>
 <td>Isoamyl Alcohol</td>
 <td>Sigma-Aldrich</td>
 <td>W205702</td>
 </tr>
 <tr>
 <td>Isopropanol</td>
 <td>Sigma-Aldrich</td>
 <td>190764</td>
 </tr>
 <tr>
 <td>Diethyl pyrocarbonate (DEPC)</td>
 <td>Sigma-Aldrich</td>
 <td>40718</td>
 </tr>
 <tr>
 <td>DNase</td>
 <td>Ambion</td>
 <td>AM2238</td>
 </tr>
 <tr>
 <td>RNeasy Kit</td>
 <td>Qiagen</td>
 <td>74104</td>
 </tr>
 <tr>
 <td>3M Sodium Acetate</td>
 <td>Ambion</td>
 <td>AM9740</td>
 </tr>
 <tr>
 <td>100% Ethanol</td>
 <td>Sigma-Aldrich</td>
 <td>E7023</td>
 </tr>
 </tbody>
 </table>

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.

Watch this Scientific Journal Video about RNA Isolation of Pseudomonas aeruginosa Colonizing the Murine Gastrointestinal Tract at JoVE.com

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.

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 ...

rna-isolation-pseudomonas-aeruginosa-colonizing-murine

Research

JoVE Journal

Immunology and Infection

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 ...

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 ...

Pseudomonas aeruginosa Induced Lung Injury Model

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

Pseudomonas aeruginosa Induced Lung Injury Model

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

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

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

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

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

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

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

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa is able to form biofilms, develop antibiotic resistance, produce virulence factors, and rapidly evolve in the course of a chronic infection. Thus P. aeruginosa can cause both acute and chronic, difficult to treat infections, resulting in significant morbidity in certain patient populations. P. aeruginosa strain PA14 is a human clinical isolate with a conserved genome structure that infects a variety of mammalian and nonvertebrate hosts making PA14 an attractive strain for studying this pathogen. In 2006, a nonredundant transposon insertion mutant library containing 5,459 mutants corresponding to 4,596 predicted PA14 genes was generated. Since then, distribution of the PA14 library has allowed the research community to better understand the function of individual genes and complex pathways of P. aeruginosa. Maintenance of library integrity through the replication process requires proper handling and precise techniques. To that end, this manuscript presents protocols that describe in detail the steps involved in library replication, library quality control and proper storage of individual mutants.

Replication of the Ordered, Nonredundant Library of Pseudomonas aeruginosa strain PA14 Transposon Insertion Mutants

Pseudomonas aeruginosa infection causes significant morbidity in vulnerable hosts. The nonredundant transposon insertion mutant library of P. aeruginosa strain PA14, designated as PA14NR Set, facilitates analysis of gene functionality in numerous processes. Presented here is a protocol to generate high-quality copies of the PA14NR Set mutant library.

Pseudomonas aeruginosa is a phenotypically and genotypically diverse and adaptable Gram-negative bacterium ubiquitous in human environments. P. aeruginosa ...

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 ...

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 ...

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 ...