Name: culture of small colony variant of pseudomonas aeruginosa and quantitation of its alginate
Uploaded: 2020-02-22T12:00:00
Description: Watch this Scientific Journal Video about Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate at JoVE.com

This work was supported by the National Institutes of Health (NIH) grants R44GM113545 and P20GM103434.

1. SCV Growth Conditions and Physiological Activation of the Salvage Pathway
<ol>
	<li>Detection of SCV.
	<ol>
		<li>Streak the P. aeruginosa strains PAO1, PAO1&#916;pyrD, PAO581, and PAO581&#916;pyrD on prewarmed Pseudomonas isolation agar (PIA) plates and grow at 37 &#176;C for 48 h. On the growth plate identify a single colony isolate that has the SCV phenotype (colony size of 1&#8722;3 mm as opposed to the normal 3&#8722;5 mm colony size).</li>
		<li>Repeat step 1.1.1 to obtain a...

<ol>
	<li>Govan, J. R., Deretic, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+pathogenesis+in+cystic+fibrosis:+mucoid+Pseudomonas+aeruginosa+and+Burkholderia+cepacia.">Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia.</a> Microbiological Reviews. 60 (3), 539-574 (1996).</li><li>Hogardt, M., Heesemann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptation+of+Pseudomonas+aeruginosa+during+persistence+in+the+cystic+fibrosis+lung.">Adaptation of Pseudomonas aeruginosa during persistence in the cystic fibrosis lung.</a> International Journal of Medical Microbiology. 300 (8), 557-562 (2010).</li><li>Evans, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+colony+variants+of+Pseudomonas+aeruginosa+in+chronic+bacterial+infection+of+the+lung+in+cystic+fibrosis.">Small colony variants of Pseudomonas aeruginosa in chronic bacterial infection of the lung in cystic fibrosis.</a> Future Microbiology. 10 (2), 231-239 (2015).</li><li>Johns, B. E., Purdy, K. J., Tucker, N. P., Maddocks, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+and+Genotypic+Characteristics+of+Small+Colony+Variants+and+Their+Role+in+Chronic+Infection.">Phenotypic and Genotypic Characteristics of Small Colony Variants and Their Role in Chronic Infection.</a> Microbiology Insights. 8, 15-23 (2015).</li><li>Pestrak, 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=Pseudomonas+aeruginosa+rugose+small-colony+variants+evade+host+clearance,+are+hyper-inflammatory,+and+persist+in+multiple+host+environments.">Pseudomonas aeruginosa rugose small-colony variants evade host clearance, are hyper-inflammatory, and persist in multiple host environments.</a> PLoS Pathogones. 14 (2), e1006842 (2018).</li><li>Al Ahmar, R., Kirby, B. D., Yu, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrimidine+Biosynthesis+Regulates+Small+Colony+Variant+and+Mucoidy+in+Pseudomonas+aeruginosa+Through+Sigma+Factor+Competition.">Pyrimidine Biosynthesis Regulates Small Colony Variant and Mucoidy in Pseudomonas aeruginosa Through Sigma Factor Competition.</a> Journal of Bacteriology. 201 (1), e00575-e00618 (2019).</li><li>Ramsey, D. M., Wozniak, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+the+control+of+Pseudomonas+aeruginosa+alginate+synthesis+and+the+prospects+for+management+of+chronic+infections+in+cystic+fibrosis.">Understanding the control of Pseudomonas aeruginosa alginate synthesis and the prospects for management of chronic infections in cystic fibrosis.</a> Molecular Microbiology. 56 (2), 309-322 (2005).</li><li>Mathee, K., McPherson, C. J., Ohman, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posttranslational+control+of+the+algT+(algU)-encoded+sigma22+for+expression+of+the+alginate+regulon+in+Pseudomonas+aeruginosa+and+localization+of+its+antagonist+proteins+MucA+and+MucB+(AlgN).">Posttranslational control of the algT (algU)-encoded sigma22 for expression of the alginate regulon in Pseudomonas aeruginosa and localization of its antagonist proteins MucA and MucB (AlgN).</a> Journal of Bacteriology. 179 (11), 3711-3720 (1997).</li><li>Schurr, M. J., Yu, H., Martinez-Salazar, J. M., Boucher, J. C., Deretic, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+AlgU,+a+member+of+the+sigma+E-like+family+of+stress+sigma+factors,+by+the+negative+regulators+MucA+and+MucB+and+Pseudomonas+aeruginosa+conversion+to+mucoidy+in+cystic+fibrosis.">Control of AlgU, a member of the sigma E-like family of stress sigma factors, by the negative regulators MucA and MucB and Pseudomonas aeruginosa conversion to mucoidy in cystic fibrosis.</a> Journal of Bacteriology. 178 (16), 4997-5004 (1996).</li><li>Rehm, B. H. A., Rehm, B. H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alginate+Production:+Precursor+Biosynthesis,+Polymerization+and+Secretion.">Alginate Production: Precursor Biosynthesis, Polymerization and Secretion.</a> Alginates: Biology and Applications. , 55-71 (2009).</li><li>Remminghorst, U., Rehm, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+alginates:+from+biosynthesis+to+applications.">Bacterial alginates: from biosynthesis to applications.</a> Biotechnology Letters. 28 (21), 1701-1712 (2006).</li><li>Potvin, E., Sanschagrin, F., Levesque, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sigma+factors+in+Pseudomonas+aeruginosa.">Sigma factors in Pseudomonas aeruginosa.</a> FEMS Microbiology Reviews. 32 (1), 38-55 (2008).</li><li>Damron, F. H., Goldberg, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolytic+regulation+of+alginate+overproduction+in+Pseudomonas+aeruginosa.">Proteolytic regulation of alginate overproduction in Pseudomonas aeruginosa.</a> Molecular Microbiology. 84 (4), 595-607 (2012).</li><li>Bowness, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+the+carbazole+reaction+to+the+estimation+of+glucuronic+acid+and+flucose+in+some+acidic+polysaccharides+and+in+urine.">Application of the carbazole reaction to the estimation of glucuronic acid and flucose in some acidic polysaccharides and in urine.</a> The Biochemical Journal. 67 (2), 295-300 (1957).</li><li>Fazio, S. A., Uhlinger, D. J., Parker, J. H., White, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimations+of+uronic+acids+as+quantitative+measures+of+extracellular+and+cell+wall+polysaccharide+polymers+from+environmental+samples.">Estimations of uronic acids as quantitative measures of extracellular and cell wall polysaccharide polymers from environmental samples.</a> Applied Environmental Microbiology. 43 (5), 1151-1159 (1982).</li><li>Knutson, C. A., Jeanes, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+modification+of+the+carbazole+analysis:+application+to+heteropolysaccharides.">A new modification of the carbazole analysis: application to heteropolysaccharides.</a> Analytical Biochemistry. 24 (3), 470-481 (1968).</li></ol>

Both SCV and alginate are important disease markers implicated in several chronic infections. Therefore, the ability to grow SCV as well as study the regulation and production of alginate by P. aeruginosa is integral to the discovery of novel treatments for these chronic illnesses.
SCV strains are notoriously difficult to grow due to their slow growth rate4 as compared to other P. aeruginosa strains, which aids in their antimicrobial resistance<sup cla...

Chronic lung infections with Pseudomonas aeruginosa are a major cause of morbidity and mortality in patients with cystic fibrosis (CF). During early childhood, patients are colonized by multiple bacterial pathogens including nonmucoid isolates of P. aeruginosa1,2. Emergence of the small colony variant (SCV) isolates as well as mucoid isolates is a marker for the onset to chronic infections. SCV isolates are highly drug resistant3 due to their slow growth rates4, which renders them a severe deterrent in the treatment regiments and other chronic infections5 by P. aeruginosa. Work by Al Ahmar et al.6 showed a link between SCV and mucoidy linked by de novo pyrimidine biosynthesis. Pyrimidine starvation, due to mutations in genes involved with pyrimidine production, resulted in SCV phenotype in the nonmucoid reference strain PAO1 and the mucoid derivative, PAO581 (PAO1mucA25).
Even though alginate overproduction is an important disease marker for chronic lung infections in CF, it is not clear whether there is a direct correlation between the amount of alginate and lung pathology, and it is unclear if alginate can be used as a prognosis marker for treatment7. Alginate production is mainly regulated by two operons, a regulatory operon (algUmucABCD)8,9 and the biosynthetic operon (algD operon)10,11. Alginate production is tightly regulated by the sigma factor AlgU9,12 (also known as AlgT) and the degradation of the anti-sigma factor MucA13. The ability to monitor the production of alginate in situ from the patients&#39; sputum specimens can aid in the development of novel therapeutic options.
Here, we describe a growth condition that detects the presence of SCV caused by mutants that cannot synthesize the pyrimidine de novo. Supplementation of uracil and/or cytosine, the nitrogenous base of pyrimidine nucleotide, to the medium activates the salvage pathway, thus restoring the normal growth in mutants. This growth method for these specific SCV mutants may be used as a screening method to identify pyrimidine mutations in patient samples. In addition, we discuss two methods for detection and measurement of alginate produced and secreted by P. aeruginosa. The first is the traditional method14,15,16 of degrading the polysaccharide using a high concentration of acid and then adding a colorimetric indicator to quantitate the concentration in the sample. The second method, developed in our laboratory, utilizes the Enzyme-Linked Immunosorbent Assay (ELISA) using an anti-alginate monoclonal antibody (mAb) developed by QED Biosciences. The ELISA method proves to be more specific and sensitive than the uronic acid assay and allows for safer use due to the avoidance of the highly concentrated sulfuric acid. With the ability of the ELISA to be used directly on patient sputum samples to measure alginate, it can be developed as a monitoring diagnostic tool to follow the amount of alginate present in the lungs at different periods of the infection.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-Step Ultra TMB-ELISA</td>
			<td>Thermo Scientific</td>
			<td>34028</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Absolute Ethanol (200 Proof)</td>
			<td>Fisher Scientific</td>
			<td>BP2818-4</td>
			<td>Molecular Bio-grade</td>
		</tr>
		<tr>
			<td>Accu Block Digital Dry Bath</td>
			<td>Labnet</td>
			<td>NC0205808</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Assay Plates 96-well</td>
			<td>CoStar</td>
			<td>2021-12-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Bench Top Vortex-Genie 2</td>
			<td>Scientific Industries</td>
			<td>G560</td>
			<td></td>
		</tr>
		<tr>
			<td>Boric Acid</td>
			<td>Research Products International Corp.</td>
			<td>10043-35-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Cabinet Incubator</td>
			<td>VWR</td>
			<td>1540</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbazole</td>
			<td>Sigma</td>
			<td>C-5132</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbonate-Bicarbonate Buffer</td>
			<td>Sigma</td>
			<td>C3041</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge Tubes (50 ml)</td>
			<td>Fisher Scientific</td>
			<td>05-539-13</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Culture Test Tubes</td>
			<td>Fisher Scientific</td>
			<td>14-956-6D</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Cuvette Polystyrene (1.5 ml)</td>
			<td>Fisher Scientific</td>
			<td>14955127</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Cytosine</td>
			<td>Acros Organics</td>
			<td>71-30-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Diposable Inoculation Loops</td>
			<td>Fisher Scientific</td>
			<td>22-363-597</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Mannuronic Acid Sodium</td>
			<td>Sigma Aldrich</td>
			<td>SMB00280</td>
			<td></td>
		</tr>
		<tr>
			<td>FMC Alginate</td>
			<td>FMC</td>
			<td>2133</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fisher Scientific</td>
			<td>BP906-5</td>
			<td>For Molecular Biology</td>
		</tr>
		<tr>
			<td>Mouse Anti-Alginate Monoclonal Antibody</td>
			<td>QED Biosciences</td>
			<td>N/A</td>
			<td>Lot # :15725/15726</td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline Powder (PBS)</td>
			<td>Sigma</td>
			<td>P3813</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce Goat Anti-Mouse Poly-HRP Antibody</td>
			<td>Thermo Scientific</td>
			<td>32230</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Potassium Hydroxide</td>
			<td>Fisher Scientific</td>
			<td>1310-58-3</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Prism 7</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pseudomonas Isolation Agar (PIA)</td>
			<td>Difco</td>
			<td>292710</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Pseudomonas Isolation Broth (PIB)</td>
			<td>Alpha Biosciences</td>
			<td>P16-115</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Round Toothpicks</td>
			<td>Diamond</td>
			<td></td>
			<td>Any brand</td>
		</tr>
		<tr>
			<td>Seaweed alginate (Protanal CR 8133)</td>
			<td>FMC Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Skim Milk</td>
			<td>Difco</td>
			<td>232100</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>SmartSpec Plus Spectrophotometer</td>
			<td>BioRad</td>
			<td>170-2525</td>
			<td>or preferred vendor</td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>Sigma</td>
			<td>S-5886</td>
			<td></td>
		</tr>
		<tr>
			<td>SpectraMax i3x Multi-mode MicroPlate Reader</td>
			<td>Molecular Devices</td>
			<td>i3x</td>
			<td>or preferred vendor</td>
		</tr>
		<tr>
			<td>Sterile Petri Dish 100mm x 15mm</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td>via Fisher Scientific</td>
		</tr>
		<tr>
			<td>Sulfuric Acid</td>
			<td>Fisher Scientific</td>
			<td>A298-212</td>
			<td>Technical Grade</td>
		</tr>
		<tr>
			<td>Sulfuric Acid (2 Normal -Stop Solution)</td>
			<td>R&#38;D Systems</td>
			<td>DY994</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Sigma</td>
			<td>P2287</td>
			<td></td>
		</tr>
		<tr>
			<td>Uracil</td>
			<td>Acros Organics</td>
			<td>66-22-8</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Figure 1 shows plates of PAO1 and PAO581 with or without in-frame deletion in the pyrD gene (a gene in the pyrimidine biosynthesis pathway) that results in SCV6. The PAO1 SCV mutant was restored to normal growth in response to uracil supplementation (Figure 1A,B). Furthermore, the PAO581&#916;pyrDSCV mutant was returned to mucoidy with the same uracil treatment, because t...

Watch this Scientific Journal Video about Culture of Small Colony Variant of Pseudomonas aeruginosa and Quantitation of its Alginate at JoVE.com

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.

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 ...

The small colony variant or SCV technique allows for selection and growth of small colony variants of Pseudomonas aeruginosa. The method is very definitive and allows for easier selection than normal methods. This protocol also details a safer, more sensitive ELISA method for alginate quantification as compared to the standard uronic acid carbazole method, enabling direct alginate quantification in samples without cautery or sample manipulation.Demonstrating the procedure will be Brandon Kirby, a lab technician, and Roy Al Ahmar, a graduate student from my laboratory. To detect the small colony variant or SCV, first streak the P.aeruginosa strain PAO1 delta PYRD on pre-warmed PIA plates. Grow the strains at 37 degrees Celsius for 48 hours.On the growth plate, identify a single colony isolate that has the SCV phenotype characterized by a colony size of one to three millimeters as opposed to the normal three to five millimeter colony size. Re-streak the selected colonies to obtain a pure isolate of the SCV. To perform physiological activation of the salvage pathway, use a sterile inoculation loop to pick the SCV colony from the PIA plate.Streak the selected colony on a pre-warmed PIA plate supplemented with 0.1 millimolar uracil. Grow the plate at 37 degrees Celsius for 24 to 48 hours. To begin the uronic acid carbazole assay, identify a single colony from a pure culture of the desired strain to be tested and pick the colony using a sterile toothpick.Place the toothpick in a culture test tube containing five milliliters of PIB. Grow in a shaker incubator at 37 degrees Celsius for 24 hours. Following incubation, add an aliquot of the cultured PIB broth onto a pre-warmed PIA plate.Using a sterile cell spreader, spread the broth over the plate and grow at 37 degrees Celsius for 24 hours. Using a pipette controller and a sterile 50 milliliter pipette, add 0.85%sodium chloride to the grownup lawn and collect the sample by scraping the plate using a cell spreader. Aspirate the sample using a fresh 50 milliliter pipette and transfer into a 50 milliliter collection tube.Vortex the sample on high to mix and place the samples on ice. To measure the OD600 of the samples, first blank the spectrophotometer using one milliliter of 0.85%sodium chloride. Then add one milliliter of the sample to a new disposable cuvette and read the OD.Repeat this step twice to obtain a triplicate of reads for each sample.Now add three milliliters of the sulfuric acid borate solution into culture tubes and let sit on ice. Add 350 microliters of the collected sample slowly to the acid mixture in the test tubes. After briefly vortexing on low, add 100 microliters of 0.1%carbazole and ethanol solution to the acid sample mixture.Cap the tube and vortex on the medium setting for five seconds. Then place in a dry bath at 55 degrees Celsius for 30 minutes. After incubation, vortex the tubes briefly on high and allow to cool for five minutes.Use the tube with 0.85%sodium chloride as a blank to the spectrophotometer. Then add one milliliter of the mixture to a clean cuvette and read the OD of the samples at 530 nanometers on a spectrophotometer. Prepare a standard curve by measuring the OD530 of serial dilutions of known concentrations of d-mannuronic acid.Repeat twice and extract a linear equation from these readings. Calculate the concentration of the alginate in each sample using the standard curve and divide the alginate concentration from linear equation by OD600 to obtain the total amount of alginate per OD600. Using a micropipette, add 50 microliters of the collected sample to an untreated 96-well plate.Then add 50 microliters of ELISA coating buffer to the wells. Incubate the plate at 37 degrees Celsius for two hours. Using a squirt bottle, wash the plate wells twice with PBS-T by filling the wells and then draining them by flipping the plate over.Add 200 microliters of blocking buffer to the wells with a micropipette and incubate at four degrees Celsius overnight. The next day, wash the plate twice with PBS-T as before. Then add 100 microliters of diluted primary antibody to the wells and incubate at 37 degrees Celsius for one to two hours.Following incubation, wash the plate wells three times with PBS-T as before. Next, add 100 microliters of diluted secondary antibody to the wells and incubate at 37 degrees Celsius for one to two hours. After washing the plate again three times, use a micropipette to add 100 microliters of TMB ELISA solution.Incubate the plate at room temperature for 30 minutes in the dark by placing the plate into a drawer or wrapping in foil. Then add 100 microliters of stop solution. Use a plate reader to measure the OD at 450 nanometers.Produce a standard curve by measuring the OD450 of serial dilutions of known concentrations of d-mannuronic acid. Repeat the measurements twice and extract a linear equation. Calculate the concentration of the alginate in each sample using the standard curve and divide the alginate concentration from the linear equation by OD600 to obtain the total amount of alginate per OD600.Shown here are samples of PAO581 and PAO581 with mutations in genes regulating pyrimidine de novo biosynthesis grown on PIA and PIA supplemented with 0.1 millimolar of uracil. The data show that the presence of uracil in the media results in the conversion of the mutant strain back to mucoidy as seen by the restored alginate production. Here, results are shown for the anti-alginate monoclonal antibody-based ELISA.Colonies are grown on PIA plates with arabinose for the induction of the PBAD promoter and the pHERD-20T plasmid. Shown are PAO1, PAO1 carrying the expression plasmid pHERD-20T with the main alginate specific sigma factor algU, and PAO1 with an in-frame deletion of the algD gene in coding the key alginate biosynthetic enzyme GDP-mannose dehydrogenase. This data compares the non-mucoid levels of alginate measured for PAO1 and PAO1 delta algD versus the mucoid levels of alginate measured for PAO1 with pHERD-20T algU.Anti-alginate ELISA was also tested on patient sputum samples without prior growth on plates. Three CF sputum samples that had growth of mucoid P.aeruginosa showed detectable alginate compared with two patient sputum samples that contained either non-mucoid P.aeruginosa or no P.aeruginosa growth. The preparation of the standard curves for the alginate quantification is very crucial as this will allow for precise and accurate measurement of alginate within the sample.The acid solution used in the carbazole method are very dangerous so proper personal protective equipment and precautions must be used to ensure safety. After isolating SCVs, further genetic profiling can be done to better understand mutations occurring and the pathogenicity associated with such mutations. The ELISA method can be adapted to monitor chronic lung infection in patients with cystic fibrosis.Furthermore, our growth method could help diagnose infections caused by specific SCVs.

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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 ...

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 ...

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 ...

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa. To better understand alginate regulation, we describe a protocol using the mini-himar1 transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1 transposon (pFAC) from host E. coli SM10/&#955;pir into recipient P. aeruginosa PAO1 via biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE (PA4033) and kinB (PA5484), in strain PAO1 with a wild-type mucA encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, &#963;22). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.

Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Here we describe a protocol using the mini-himar1 mariner transposon-mediated mutagenesis for generating a high-density insertion mutant library to screen, isolate and identify novel alginate regulators in the prototypic Pseudomonas aeruginosa strain PAO1.

Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial ...

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. ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. The protocol utilizes cryopreservation of monocytes coupled with their bulk differentiation into macrophages. Then harvested macrophages can then be seeded into culture wells at required cell densities for carrying out experiments.

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation ...

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 ...