Name: culture of small colony variant of pseudomonas aeruginosa and quantitation of its alginate
Uploaded: 2020-02-22T12:00:00.000+00:00
Duration: 9 min 6 s
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 pure isolate of the SCV. 
		NOTE: Growth may require up to 72 h due to the slow growth rate of the SCV colonies.</li>
	</ol>
	</li>
	<li>Physiological activation of the salvage pathway.
	<ol>
		<li>Using a sterile inoculation loop, pick the SCV colony from the PIA plate and streak on a prewarmed PIA supplemented with 0.1 mM of uracil. Grow plate at 37 &#176;C for 24&#8722;48 h. 
		NOTE: All PIA plates used in this protocol contain 20 mL/L of glycerol added to the mixture before autoclaving. After autoclaving and after mixture temperature is below 55 &#176;C add 11.21 mg/L of uracil (0.1 mM) to the liquid media before pouring plates. This method would help identify pyrimidine mutations that cause SCV phenotype in P. aeruginosa. If the SCV phenotype is a result of said mutation, then normal colony growth and size (3&#8722;5 mm) would be observed on the uracil supplemented PIA plates. If strains had the ability to produce alginate, then the mucoid phenotype will also return upon uracil supplementation.</li>
	</ol>
	</li>
</ol>
2. Uronic Acid Carbazole Assay
<ol>
	<li>From a pure culture of desired strain to be tested, identify a single colony. Using a sterile toothpick, pick the colony, and place the toothpick in a culture test tube containing 5 mL of Pseudomonas isolation broth (PIB). Grow in a shaker incubator at 37 &#176;C for 24 h. 
	NOTE: The following strains PAO581 (PAO1mucA25), PAO581carA, PAO581carB, and PAO581pyrD were grown on PIA plates or PIA plates supplemented with 0.1 mM uracil to harvest alginate for measurement.</li>
	<li>Onto a prewarmed PIA plate add 150 &#181;L of cultured PIB broth (for 15 mm x 100 mm plates) or 450 &#181;L of broth (for 15 mm x 150 mm plates). Using a sterile cell spreader, spread the broth over the plate. Grow the plate at 37 &#176;C for 24 h. 
	NOTE: Aspirate any excess fluid, if any, from the plate using a pipet by tipping the plate to one side. Both PIB and PIA plates used in the protocol contain 20 mL/L of glycerol to be used by cells as a carbon source to aid in alginate production.</li>
	<li>Using a pipette controller and a sterile 50 mL pipette, add 0.85% NaCl to the lawn grown and collect sample by scrapping the plate using a cell spreader. Aspirate the sample using a fresh 50 mL pipet into a 50 mL collection tube. Vortex the sample on high to mix and place the samples on ice. 
	NOTE: The volume of added 0.85% NaCl varies depending on the size of the plate. For 15 mm x 100 mm plates, use 10&#8722;30 mL, and for 15 mm x 150 mm plates, use 20&#8722;50 mL.</li>
	<li>Measure the optical density at 600 nm (OD600) of the samples by adding 1 mL of the sample to a disposable cuvette and reading the OD using a spectrophotometer. Repeat this step 2x to obtain a triplicate of reads for each sample.</li>
	<li>Add 3 mL of the sulfuric acid/borate solution into culture tubes and let sit on ice. Add 350 &#181;L of the collected sample SLOWLY to the acid mix in the test tubes and vortex on low briefly.
	<ol>
		<li>Prepare borate stock by adding 10.099 g of potassium hydroxide (KOH) powder to 45 mL of water. Add 24.74 g of boric acid (H3BO3) to the mixture and bring volume to 100 mL.</li>
		<li>Place 1 L bottle in a sink with ice and fill bottle with 500 mL of concentrated sulfuric acid. SLOWLY add 15 mL of the prepared borate stock. Allow bottle to cool.</li>
		<li>Add another 10 mL of borate stock for a total of 25 mL. Once bottle is cooled, bring the volume to 1 L. 
		CAUTION: This method relies on the use of highly concentrated sulfuric acid. Proper personal protective equipment should be used to ensure safety and proper disposal protocol of the sample should be followed. 
		NOTE: For a positive control a known concentration of D-mannuronic acid and/or a known bacterial alginate samples harvested and purified through alginate producing strains of P. aeruginosa (e.g.: PAO581-PAO1mucA25). For a negative control use 0.85% NaCl solution and/or PAO1&#916;algD (In-frame deletion of algD gene that renders the bacteria completely unable to produce alginate). Multiple tubes can be made from each sample to increase the number of technical repeats to aid in statistical analysis. Prepare 3&#8722;5 tubes of each sample.</li>
	</ol>
	</li>
	<li>Add 100 &#181;L of 0.1% carbazole in ethanol solution to the acid/sample mix. Cap the tube and vortex on medium setting for 5 s. Place in a dry bath at 55 &#176;C for 30 min.</li>
	<li>After incubation, remove the tubes and vortex briefly on high and allow to cool for 5 min. 
	NOTE: Color to be seen would be a shade of purple. Color will remain stable for measurement for 1&#8722;2 h.</li>
	<li>Read the OD530 of each tube by adding 1 mL of the mixture to a clean cuvette and reading the OD of the samples at 530 nm on a spectrophotometer. Use the tube with 0.85% NaCl as a blank to the spectrophotometer.</li>
	<li>Prepare a standard curve by measuring the OD530 of serial dilutions of known concentrations of D-Mannuronic acid (1,024 &#181;g/mL, 512 &#181;g/mL, 256 &#181;g/mL, 128 &#181;g/mL, 64 &#181;g/mL, 32 &#181;g/mL, 16 &#181;g/mL, and 8 &#181;g/mL). Repeat 2x. Extract a linear equation from these readings. 
	NOTE: Standard curve only needs to be done once. The linear equation extracted from it can be used for later testing.
	<ol>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. ELISA for Alginate Quantitation
<ol>
	<li>Repeat steps 2.1&#8722;2.4 to obtain samples for alginate measurement. 
	NOTE: Strains used were PAO1, PAO1&#916;algD, and PAO1 carrying pHERD20T-algU.</li>
	<li>Using a micropipette add 50 &#181;L of the collected sample to an untreated 96-well plate. Add 50 &#181;L of coating buffer (carbonate/bicarbonate buffer pH 9.6) to the wells. Incubate the plate at 37 &#176;C for 2 h. 
	NOTE: For a positive control, a known concentration of D-Mannuronic acid and/or a known stable alginate producing strain (e.g.: PAO581-PAO1mucA25) can be used. For a negative control use 0.85% NaCl solution and/or PAO1&#916;algD (in-frame deletion of algD gene renders the bacteria completely unable to produce any alginate). Multiple wells can be made from each sample to increase the number of technical repeats to aid in statistical analysis. Prepare 3&#8722;5 wells of each sample.</li>
	<li>Using a squirt bottle, wash the plate wells 2x with 1x phosphate buffer saline (PBS) with 0.05% Tween 20 (PBS-T) by filling the wells, and then draining them by flipping the plate over.</li>
	<li>Using a micropipette add 200 &#181;L of blocking buffer (10% skim milk in PBS-T) to the wells. Incubate at 4 &#176;C overnight. 
	NOTE: Wrap plate in paraffin film or shrink wrap to help avoid any evaporation in the refrigerator.</li>
	<li>Using a squirt bottle wash the plate wells 2x with PBS-T by filling the wells, and then draining them by flipping plate over.</li>
	<li>Using a micropipette add 100 &#181;L of diluted primary antibody (mouse anti-alginate monoclonal antibody) to the wells and incubate at 37 &#176;C for 1&#8722;2 h. 
	NOTE: Primary antibody (mouse anti-alginate monoclonal antibody) was provided at a concentration of 0.5 &#181;g/mL (dilution of 1:2,000 of 1 mg/mL stock) in antibody diluent solution (1% skim milk in 1x PBS).</li>
	<li>Using a squirt bottle wash the plate wells 3x with PBS-T by filling the wells, and then draining them by flipping plate over.</li>
	<li>Using a micropipette add 100 &#181;L of diluted secondary antibody to the wells and incubate at 37 &#176;C for 1&#8722;2 h. 
	NOTE: Secondary antibody used was pierce goat anti-mouse poly-HRP antibody to a concentration of 0.25 &#181;g/mL (dilution of 1:2,000 of 0.5 mg/mL stock) in antibody diluent solution.</li>
	<li>Using a squirt bottle wash the plate wells 3x with PBS-T by filling the wells, and then draining them by flipping plate over.</li>
	<li>Using a micropipette, add 100 &#181;L of TMB-ELISA solution (Table of Materials) and incubate at room temperature for 30 min in the dark. 
	NOTE: The color of a positive reaction would be a shade of blue.</li>
	<li>Using a micropipette, add 100 &#181;L of stop solution (2 N sulfuric acid). 
	NOTE: The color will turn from blue to yellow when the stop solution is added. Color is stable for up to 30 min after the reaction is stopped.</li>
	<li>Using a plate reader, read the OD at 450 nm.</li>
	<li>Produce a standard curve by measuring the OD450 of serial dilutions of known concentrations of D-Mannuronic acid (1,024 &#181;g/mL, 512 &#181;g/mL, 256 &#181;g/mL, 128 &#181;g/mL, 64 &#181;g/mL, 32 &#181;g/mL, 16 &#181;g/mL, and 8 &#181;g/mL). Repeat 2x. From these readings extract a linear equation. 
	NOTE: The standard curve needs to be only done once. The linear equation extracted from it can be used for later testing.
	<ol>
		<li>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.</li>
	</ol>
	</li>
</ol>
4. Statistical Analysis of Alginate Measurement
<ol>
	<li>In a graphical software sheet, set a column with the linear equation derived from the standard curve. Set the y-axis results as the alginate concentration and the x-axis results as the OD530 for uronic acid assay and OD450 for ELISA to obtain the alginate concentrations in each sample.</li>
	<li>To normalize the amount of alginate in each sample to the amount per 5.5 x108 CFU/mL (1 OD600), divide the concentration of each sample obtained above by its respective OD600 value. If multiple OD600 were measured for each sample, divide by the mean OD600.</li>
	<li>Perform statistical analysis using preferred statistical software (e.g., GraphPad Prism 7.02).</li>
	<li>Open the software. Choose Column under New Table &#38; Graph and choose the one-way ANOVA data set.</li>
	<li>Name each column with the strain tested and add the alginate concentrations underneath.</li>
	<li>After inputting all the data click on Analyze in the top menu. This will open an analysis settings window. Choose the appropriate parameters for testing and indicate whether any multiple comparisons need to be run. 
	NOTE: After analysis the data set will contain a p-value that would help determine the statistical significance of the data. Data would be considered statistically significant if the p-value is less than the set &#945; value (normally &#945; value is set for p &#60; 0.01).</li>
	<li>Under the graph tab choose the Bar Graph type. This will automatically chart the data with the indicated title from the input data sheet. Indicate statistical significance of the data as needed by showing the * symbol above connective lines between the bars of interest.</li>
</ol>

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The author Hongwei D. Yu is the Chief Science Officer and Co-founder of Progenesis Technologies, LLC.

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><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>&lt;em&gt;Diposable Inoculation Loops&lt;/em&gt;</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>&lt;em&gt;Pseudomonas&lt;/em&gt; Isolation Agar (PIA)</td><td>Difco</td><td>292710</td><td>via Fisher Scientific</td></tr><tr><td>&lt;em&gt;Pseudomonas&lt;/em&gt; 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 100 mm x 15 mm</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&amp;amp;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...

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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 ...

culture-small-colony-variant-pseudomonas-aeruginosa-quantitation-its

Research

JoVE Journal

Immunology and Infection

8.4K Views.  Marshall University. De kleine kolonievariant of SCV techniek maakt selectie en groei van kleine kolonievarianten van Pseudomonas aeruginosa mogelijk. De methode is zeer definitief en maakt gemakkelijker selectie dan normale methoden. Dit protocol beschrijft ook een veiligere, meer gevoelige ELISA-methode voor alginaat kwantificering in vergelijking met de standaard uronic acid carbazole-methode, waardoor directe alginaatkwantificering mogelijk is in monsters zonder cautery of monstermanipulatie.Het aanton...

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

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 ...

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 ...

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 ...

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. ...

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

Pseudomonas aeruginosa (Pa) is one of the most common opportunistic pathogens associated with cystic fibrosis (CF). Once Pa colonization is established, a large proportion of the infecting bacteria form biofilms within airway sputum. Pa biofilms isolated from CF sputum have been shown to grow in small, dense aggregates of ~10-1,000 cells that are spatially organized and exhibit clinically relevant phenotypes such as antimicrobial tolerance. One of the biggest challenges to studying how Pa aggregates respond to the changing sputum environment is the lack of nutritionally relevant and robust systems that promote aggregate formation. Using a synthetic CF sputum medium (SCFM2), the life history of Pa aggregates can be observed using confocal laser scanning microscopy (CLSM) and image analysis at the resolution of a single cell. This in vitro system allows the observation of thousands of aggregates of varying size in real time, three dimensions, and at the micron scale. At the individual and population levels, having the ability to group aggregates by phenotype and position facilitates the observation of aggregates at different developmental stages and their response to changes in the microenvironment, such as antibiotic treatment, to be differentiated with precision.

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

Synthetic cystic fibrosis sputum medium (SCFM2) can be utilized in combination with both confocal laser scanning microscopy and fluorescence-activated cell sorting to observe bacterial aggregates at high resolution. This paper details methods to assess aggregate populations during antimicrobial treatment as a platform for future studies.

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

Isolation of Total RNA from Pseudomonas aeruginosa within Biofilms for Measuring Gene Expression

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes infections in the airways of cystic fibrosis (CF) patients. P. aeruginosa is known for its ability to form biofilms that are protected by a matrix of exopolysaccharides. This matrix allows the microorganisms to be more resilient to external factors, including antibiotic treatment. One of the most common methods of biofilm growth for research is in microtiter plates or chambered slides. The advantage of these systems is that they allow for the testing of multiple growth conditions, but their disadvantage is that they produce limited amounts of biofilm for RNA extraction. The purpose of this article is to provide a detailed, step by step protocol on how to extract total RNA from small amounts of biofilm of sufficient quality and quantity for high throughput sequencing. This protocol allows for the study of gene expression within these biofilm systems.

Isolation of Total RNA from Pseudomonas aeruginosa within Biofilms for Measuring Gene Expression

This protocol presents a method to isolate RNA from Pseudomonas aeruginosa biofilms grown in chamber slides for high throughput sequencing.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that causes infections in the airways of cystic fibrosis (CF) patients. P. aeruginosa is ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

Biology

Adaptable CF Model for Antimicrobial Testing and Microbial Dynamics Analysis

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

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

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

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

In Vitro Polymicrobial Interactions in Cystic Fibrosis Lung Infections

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

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

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

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

Visualizing Antibiotic Persistence in Bacterial Pathogens

Time-Lapse Epifluorescence Microscopy Imaging of Pseudomonas aeruginosa and Staphylococcus aureus Heterogeneous Phenotypes

Antibiotic persistence is a phenomenon in which a small number of bacterial cells in a genetically susceptible population survive antibiotic treatment that kills the other genetically identical cells. Bacterial persisters can resume replication once antibiotic treatment ends and are commonly thought to&#160;underlie clinical treatment failure.&#160;Recent work harnessing the power of time-lapse fluorescence microscopy, in which bacteria are labeled with fluorescent transcriptional reporters, translational reporters, and/or dyes for a variety of cellular features, has advanced our understanding of Escherichia coli persisters beyond what could be learned from population-level antibiotic survival assays. Such single-cell approaches, rather than bulk population assays, are essential for delineating the mechanisms of persister formation, damage response, and survival. However, methods for studying persisters in other important pathogenic species at this level of detail remain limited.
This study provides an adaptable approach for time-lapse imaging of Pseudomonas aeruginosa (a gram-negative rod) and Staphylococcus aureus (a gram-positive coccus)&#160;during antibiotic treatment and recovery. We discuss molecular genetic approaches to introduce fluorescent reporters into these bacteria. Using these reporters, as well as dyes, we can track the phenotypic changes, morphological features, and fates of individual cells in response to antibiotic treatment. Additionally, we are able to observe the phenotypes of individual persisters as they resuscitate following treatment. In all, this work serves as a resource for those interested in tracking the survival and gene expression of individual antibiotic-treated cells, including persisters, both during and after treatment, in clinically important pathogens.

In this manuscript, we provide a comprehensive protocol for assessing antibiotic survival for Pseudomonas aeruginosa and Staphylococcus aureus, transforming plasmids into P. aeruginosa and S. aureus to create reporter strains and visualize&#160;phenotypic variants, such as persisters, by time-lapse epifluorescence&#160;microscopy.

Antibiotic persistence is a phenomenon in which a small number of bacterial cells in a genetically susceptible population survive antibiotic treatment ...

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

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