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

Summary

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.

Abstract

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.

Introduction

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. aeruginosa^1,2. 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 AlgU^9,12 (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' 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 method^14,15,16 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.

Protocol

1. SCV Growth Conditions and Physiological Activation of the Salvage Pathway

1. Detection of SCV.
   1. Streak the P. aeruginosa strains PAO1, PAO1ΔpyrD, PAO581, and PAO581ΔpyrD on prewarmed Pseudomonas isolation agar (PIA) plates and grow at 37 °C for 48 h. On the growth plate identify a single colony isolate that has the SCV phenotype (colony size of 1−3 mm as opposed to the normal 3−5 mm colony size).
   2. 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.
2. Physiological activation of the salvage pathway.
   1. 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 °C for 24−48 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 °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 (3−5 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.

2. Uronic Acid Carbazole Assay

1. 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 °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.
2. Onto a prewarmed PIA plate add 150 µL of cultured PIB broth (for 15 mm x 100 mm plates) or 450 µL 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 °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.
3. 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 10−30 mL, and for 15 mm x 150 mm plates, use 20−50 mL.
4. 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.
5. Add 3 mL of the sulfuric acid/borate solution into culture tubes and let sit on ice. Add 350 µL of the collected sample SLOWLY to the acid mix in the test tubes and vortex on low briefly.
   1. 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.
   2. 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.
   3. 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 PAO1ΔalgD (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 3−5 tubes of each sample.
6. Add 100 µL 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 °C for 30 min.
7. 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 1−2 h.
8. 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.
9. Prepare a standard curve by measuring the OD₅₃₀ of serial dilutions of known concentrations of D-Mannuronic acid (1,024 µg/mL, 512 µg/mL, 256 µg/mL, 128 µg/mL, 64 µg/mL, 32 µg/mL, 16 µg/mL, and 8 µg/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.
   1. 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.

3. ELISA for Alginate Quantitation

1. Repeat steps 2.1−2.4 to obtain samples for alginate measurement.
   NOTE: Strains used were PAO1, PAO1ΔalgD, and PAO1 carrying pHERD20T-algU.
2. Using a micropipette add 50 µL of the collected sample to an untreated 96-well plate. Add 50 µL of coating buffer (carbonate/bicarbonate buffer pH 9.6) to the wells. Incubate the plate at 37 °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 PAO1ΔalgD (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 3−5 wells of each sample.
3. 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.
4. Using a micropipette add 200 µL of blocking buffer (10% skim milk in PBS-T) to the wells. Incubate at 4 °C overnight.
   NOTE: Wrap plate in paraffin film or shrink wrap to help avoid any evaporation in the refrigerator.
5. Using a squirt bottle wash the plate wells 2x with PBS-T by filling the wells, and then draining them by flipping plate over.
6. Using a micropipette add 100 µL of diluted primary antibody (mouse anti-alginate monoclonal antibody) to the wells and incubate at 37 °C for 1−2 h.
   NOTE: Primary antibody (mouse anti-alginate monoclonal antibody) was provided at a concentration of 0.5 µg/mL (dilution of 1:2,000 of 1 mg/mL stock) in antibody diluent solution (1% skim milk in 1x PBS).
7. Using a squirt bottle wash the plate wells 3x with PBS-T by filling the wells, and then draining them by flipping plate over.
8. Using a micropipette add 100 µL of diluted secondary antibody to the wells and incubate at 37 °C for 1−2 h.
   NOTE: Secondary antibody used was pierce goat anti-mouse poly-HRP antibody to a concentration of 0.25 µg/mL (dilution of 1:2,000 of 0.5 mg/mL stock) in antibody diluent solution.
9. Using a squirt bottle wash the plate wells 3x with PBS-T by filling the wells, and then draining them by flipping plate over.
10. Using a micropipette, add 100 µL 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.
11. Using a micropipette, add 100 µL 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.
12. Using a plate reader, read the OD at 450 nm.
13. Produce a standard curve by measuring the OD₄₅₀ of serial dilutions of known concentrations of D-Mannuronic acid (1,024 µg/mL, 512 µg/mL, 256 µg/mL, 128 µg/mL, 64 µg/mL, 32 µg/mL, 16 µg/mL, and 8 µg/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.
    1. 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.

4. Statistical Analysis of Alginate Measurement

1. 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.
2. 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.
3. Perform statistical analysis using preferred statistical software (e.g., GraphPad Prism 7.02).
4. Open the software. Choose Column under New Table & Graph and choose the one-way ANOVA data set.
5. Name each column with the strain tested and add the alginate concentrations underneath.
6. 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 α value (normally α value is set for p < 0.01).
7. 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.

Results

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 PAO581ΔpyrDSCV mutant was returned to mucoidy with the same uracil treatment, because the pa...

Discussion

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

Materials

List of materials used in this article

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