Methods to Inhibit Bacterial Pyomelanin Production and Determine the Corresponding Increase in Sensitivity to Oxidative Stress

Summary

Bacterial pyomelanin production results in increased resistance to oxidative stress and virulence. We report on techniques that can be used to determine inhibition of pyomelanin production and assay the resulting increase in sensitivity to oxidative stress in bacteria, as well as determine antibiotic minimum inhibitory concentration (MIC).

Abstract

Pyomelanin is an extracellular red-brown pigment produced by several bacterial and fungal species. This pigment is derived from the tyrosine catabolism pathway and contributes to increased oxidative stress resistance. Pyomelanin production in Pseudomonas aeruginosa is reduced in a dose dependent manner through treatment with 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC). We describe a titration method using multiple concentrations of NTBC to determine the concentration of drug that will reduce or abolish pyomelanin production in bacteria. The titration method has an easily quantifiable outcome, a visible reduction in pigment production with increasing drug concentrations. We also describe a microtiter plate method to assay antibiotic minimum inhibitory concentration (MIC) in bacteria. This method uses a minimum of resources and can easily be scaled up to test multiple antibiotics in one microtiter plate for one strain of bacteria. The MIC assay can be adapted to test the affects of non-antibiotic compounds on bacterial growth at specific concentrations. Finally, we describe a method for testing bacterial sensitivity to oxidative stress by incorporating H₂O₂ into agar plates and spotting multiple dilutions of bacteria onto the plates. Sensitivity to oxidative stress is indicated by reductions in colony number and size for the different dilutions on plates containing H₂O₂ compared to a no H₂O₂ control. The oxidative stress spot plate assay uses a minimum of resources and low concentrations of H₂O₂. Importantly, it also has good reproducibility. This spot plate assay could be adapted to test bacterial sensitivity to various compounds by incorporating the compounds in agar plates and characterizing the resulting bacterial growth.

Introduction

Pseudomonas aeruginosa is a Gram negative bacterium that produces a variety of pigments including pyomelanin, a red-brown pigment that helps provide protection from oxidative stress^1-4 and binds a variety of compounds, including aminoglycoside antibiotics^5-7. Pyomelanin production is caused by a defect in the tyrosine catabolism pathway^4,8, either through deletions or mutations of the gene encoding homogentisate 1,2-dioxygenase (HmgA)^1,9 or through imbalances in the various enzymes in the pathway¹⁰. Homogentisate accumulates due to inactivation of HmgA, and is secreted and oxidized to form pyomelanin¹¹. Production of pyomelanin can be abolished or reduced in a dose dependent manner through treatment with the herbicide 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC)¹², which inhibits 4-hydroxyphenylpyruvate dioxygenase (Hpd) in the tyrosine catabolism pathway¹³. Hpd is required for the formation of homogentisate, and therefore pyomelanin¹¹.

We describe in detail three techniques that were important in our studies of NTBC treatment of pyomelanin producing strains of P. aeruginosa. These techniques include titration of NTBC to determine the concentrations that will abolish or reduce pyomelanin production in laboratory and clinical pyomelanin producing strains, determination of the minimum inhibitory concentration (MIC) of antibiotics when bacteria are treated with NTBC, and the resulting sensitivity to oxidative stress with NTBC treatment.

The titration assay we developed serves two purposes. First, the assay will allow the user to determine if NTBC can abolish or reduce pyomelanin production in the bacterium being studied and at which concentrations. This will allow the user to determine sensitivity to NTBC, since different strains of bacteria may have different sensitivities to this compound, as observed in P. aeruginosa¹². Second, the NTBC titration assay will allow the user to determine the appropriate concentration of NTBC to use in subsequent assays, such as antibiotic MIC and oxidative stress response assays, if the goal is to abolish or reduce pyomelanin production and determine the effects of pigment reduction.

The titration assay works because a visible difference in pyomelanin production can be seen in strains treated with NTBC and the differences in pyomelanin production are dose dependent¹². Additionally, this technique can be applied to the study of other compounds that may eliminate or enhance pigment production in bacteria.

Antibiotic MICs are used to determine the sensitivity of bacteria to antibiotics. There are several methods to determine MICs, including agar dilution plates and broth dilutions¹⁴. Broth dilutions can be performed in small test tubes or in a 96-well microtiter plate. The microtiter plate method of MIC determination described herein will allow the user to test a wide range of antibiotics using a minimum of resources. The assay provides reproducibility as well as flexibility in the number of antibiotics and strains tested by this method. Additionally, with the incorporation of NTBC in the assay, the user can determine if elimination or reduction of pyomelanin production alters antibiotic sensitivity in bacteria that produce pyomelanin.

Bacterial response to oxidative stress can be tested in several ways. The most common methods described are either viable counts of bacteria subjected to oxidative stress for a period of time¹, or oxidative stress disc diffusion assays¹⁵. These methods tend to use high concentrations of oxidative stressors to examine the effects of oxidative stress in bacteria and results can be quite variable between biological replicates. The viable count assay also tends to use more agar plates than the other methods. The spot plate assay we describe uses low concentrations of H₂O₂ and allows the user to test the oxidative stress response of multiple strains using a minimum of plates. The assay is also consistently reproducible between technical and biological replicates. As pyomelanin is involved in resistance to oxidative stress, the incorporation of NTBC in the assay allows the user to determine the effects of elimination of pyomelanin production on oxidative stress resistance.

Protocol

1. Preparation of Culture Media, Antibiotics, and 2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC)

1. Make LB broth (1% tryptone, 0.5% yeast extract, 0.25% NaCl in H₂O) and aliquot into appropriate volumes. Sterilize by autoclave. Store at room temperature.
2. Make 100 ml LB agar (1% tryptone, 0.5% yeast extract, 0.25% NaCl, 1.5% agar in H₂O) in 250 ml flasks. Sterilize by autoclave and store at room temperature. Ensure that the agar is melted before pouring into plates.
   NOTE: Flasks containing 100 ml of LB agar will yield 4 plates. The amount of LB agar can be altered to correspond to the number of plates needed for the assay.
3. Make PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄)¹⁶. Sterilize by autoclave or filtration and store at room temperature.
4. Prepare the antibiotic stock solutions for gentamicin, kanamycin, and tobramycin.
   1. Prepare appropriate antibiotic stock concentrations for P. aeruginosa strains containing 100 mg/ml gentamicin, 30 mg/ml kanamycin, and 10 mg/ml tobramycin. Dissolve the antibiotics in water, filter sterilize (0.2 µm), and store at 4 °C. Alter the antibiotics and concentrations depending on the bacterium studied.
5. Prepare the NTBC stock solutions. Dissolve 10 mg of NTBC in 400 µl of DMSO. This yields a concentration of 75.9 mM NTBC. Store NTBC stock solutions at -20 °C. Thaw solutions at room temperature as needed.
   NOTE: Different sources of NTBC have differences in solubility. Determine the appropriate vehicle in which to dissolve the NTBC based on the manufacturer’s recommendations and adjust Step 1.5 accordingly.

2. NTBC Titrations of Bacterial Strains

1. Set up overnight cultures of the strains to be tested. Add 2 ml LB broth to 16 x 150 mm test tubes (one per strain) and inoculate with 1 isolated colony from each strain. Incubate overnight at 37 °C with aeration on a tissue culture rotator in an air incubator.
2. The following day, prepare titrations of NTBC in LB broth. Use an initial range from 0 to 900 µM NTBC since different strains have differences in sensitivity to NTBC.
   1. Add 1 ml LB broth to 4 to 5 test tubes (16 x 150 mm) per strain.
   2. Add the NTBC stock solution (75.9 mM) to the test tubes (16 x 150 mm) in a range of concentrations. See Table 1 for NTBC concentrations and corresponding stock volumes to add to 1 ml of LB broth.
3. Measure the OD₆₀₀ of the overnight cultures. Wash cultures before taking OD₆₀₀ readings to eliminate pyomelanin present in the media.
   1. Wash the cultures by centrifuging 1 ml of culture in a microcentrifuge at 16,000 x g for 2 min. Remove the supernatant and any loosely pelleted cells with a micropipettor and resuspend the solid cell pellet in 1 ml LB.
4. Inoculate titration tubes at OD₆₀₀ 0.05. Calculate the amount of washed culture needed to inoculate the tubes.
   NOTE: Use the washed cultures for inoculations since pyomelanin should not be present.
5. Incubate the titration tubes for approximately 24 hr at 37 °C with aeration using a tissue culture rotator in an air incubator.
6. Photograph the titration tubes and compare pigment production within and between strains to determine the amount of NTBC to use for MIC and oxidative stress assays. Use OD₆₀₀ readings to determine the amount of pyomelanin in cell free culture supernatant and to determine cell density.
   NOTE: The OD₆₀₀ ratio of pyomelanin in culture supernatant to cells can be calculated to quantify differences in pyomelanin production after treatment with NTBC.

3. Antibiotic Minimum Inhibitory Concentration (MIC) Assay in 96-well Plates

1. Set up overnight cultures of the strains to be tested in LB with and without NTBC.
   NOTE: This protocol is described using the representative level of 300 µM NTBC. The appropriate level of NTBC to be used is determined in Step 2.6.
   1. Add 300 µM NTBC to 2 ml LB. Add an equivalent volume of vehicle (DMSO) to 2 ml LB for the no NTBC condition.
   2. Using sterile toothpicks, inoculate tubes with one isolated colony of bacteria. There will be one culture with NTBC and one culture without NTBC for each strain. Incubate overnight at 37 °C with aeration using a tissue culture rotator in an air incubator.
2. The following day, make LB + NTBC and LB + DMSO master solutions for setting up the MIC assay. Add NTBC at a concentration of 600 µM as this will be diluted two fold when inoculum is added, yielding a final concentration of 300 µM.
   1. To test one antibiotic for one strain, add 600 µM NTBC to 2 ml LB and mix to make the NTBC master solution. Add an equivalent volume of vehicle (DMSO) to 2 ml LB and mix to make the no NTBC master solution. Use these solutions for creating antibiotic stock solutions as well as for setting up the dilution series in 96-well plates.
      NOTE: The master solution formulations will yield extra solution to account for pipetting errors. The master solutions can be scaled up or down as required depending on the number of antibiotics and strains tested.
3. Prepare the antibiotic solutions in the LB + NTBC or LB + DMSO master solutions.
   NOTE: The antibiotic concentration in these solutions should be double the final desired concentration. Enough solution should be made to transfer 100 µl to four wells in a 96-well plate.
   1. Prepare gentamicin +/- NTBC stock solution at 64 µg/ml. To make this solution, add 0.288 µl of gentamicin stock (100 mg/ml) to 450 µl LB + NTBC or LB + DMSO master solution.
      NOTE: The maximum concentration of gentamicin for P. aeruginosa PAO1 is 32 µg/ml.
   2. Make the kanamycin +/- NTBC stock solution at 256 µg/ml. To make this solution, add 3.84 µl of kanamycin stock (30 mg/ml) to 450 µl LB + NTBC or LB + DMSO master solutions.
      NOTE: The maximum concentration of kanamycin for P. aeruginosa PAO1 is 128 µg/ml.
   3. Prepare tobramycin +/- NTBC stock solution at 8 µg/ml. To make this solution, add 0.36 µl of tobramycin stock (10 mg/ml) to 450 µl LB + NTBC or LB + DMSO master solutions.
      NOTE: For P. aeruginosa PAO1, the maximum concentration of tobramycin is 4 µg/ml.
      NOTE: The antibiotics and concentrations can be adjusted for the bacteria to be tested.
4. Add 100 µl of each 2x antibiotic solution to four wells in a 96-well plate. Place these solutions in row A. For example, gentamicin should be placed in A1 through A4, kanamycin should be placed in A5 through A8, and tobramycin should be placed in A9 through A12. See Figure 1A for a diagram of a 96-well plate set up.
   NOTE: Multiple antibiotics can be tested in one plate, but only one strain should be tested per plate to eliminate the potential for cross-contamination from other strains.
5. Add 50 µl of the LB + NTBC or LB + DMSO master solution to rows B through H of the 96-well plate. Ensure that one plate is LB + NTBC and one plate is LB + DMSO. See Figure 1A.
   1. Use LB + NTBC for the antibiotics in LB + NTBC. Use LB + DMSO for the antibiotics in LB + DMSO.
6. Using a micropipettor perform two fold serial dilutions of the antibiotics by transferring 50 µl of the solution from row A to row B. Mix the solution, change the pipet tips, and transfer 50 µl of the solution from row B to row C. Repeat for the remaining rows. After diluting row G, remove 50 µl of the solution from that row and discard. Use row H as a no antibiotic control for bacterial growth. See Figure 1B.
   NOTE: Each well in rows A through G now contains 50 µl of antibiotic in LB + NTBC or LB + DMSO at 2X the final desired concentration. Row H contains LB + NTBC or LB + DMSO with no antibiotics.
7. Measure the OD₆₀₀ of the overnight cultures. Wash all cultures before taking OD₆₀₀ readings to eliminate pyomelanin present in the media.
   1. Wash the cultures by centrifuging 1 ml of culture in a microcentrifuge at 16,000 x g for 2 min. Remove the supernatant with a micropipettor and resuspend the cell pellet in 1 ml LB.
8. Dilute the overnight cultures to 2.75x10⁵ CFU/ml in LB.
   NOTE: Assume that one OD₆₀₀ unit is the equivalent of 1x10⁹ CFU/ml for P. aeruginosa. OD to CFU/ml conversions may be different in other bacteria.
9. Add 50 µl of the diluted bacterial culture to the appropriate well.
   NOTE: Cultures grown in NTBC should be added to the wells containing NTBC and cultures grown in DMSO should be added to the wells containing DMSO. See Figure 1B.
   1. Add bacteria to three wells for each strain and antibiotic concentration. Add 50 µl of LB to the fourth well to act as a control for bacterial contamination. See Figure 1B.
   2. Use a multi-channel micropipettor to inoculate the wells. Ensure that pipet tips are near the bottom of the wells when adding inoculum to prevent contamination of neighboring wells.
      NOTE: Adding bacterial culture to the wells will dilute the antibiotic and NTBC concentrations two fold.
10. Cover the 96-well plates with parafilm and incubate approximately 24 hr at 37 °C. Incubate 96-well plates statically, in an air incubator.
11. Examine the plates for bacterial growth in the wells. The MIC is the lowest concentration of antibiotic in which no bacterial growth is seen for all three replicates of each strain.
    1. Visually examine the plate for growth or read using a plate reader set to OD₆₀₀.

4. Spot Plate Assay for Oxidative Stress Response

1. Set up overnight cultures of the strains to be tested in LB with and without NTBC as described in step 3.1.
2. The next day, prepare LB agar plates containing H₂O₂ as an oxidative stressor. A range of H₂O₂ concentrations from 0 to 1 mM is a good starting point.
   1. Melt the LB agar flasks. Cool media to approximately 50 °C at room temperature.
   2. Add H₂O₂ directly to the cooled media at the desired concentrations. Swirl flasks to mix. See Table 2 for concentrations of H₂O₂ and volumes of concentrated H₂O₂ to add. These values are based on 100 ml of LB agar.
   3. Pour plates immediately after adding H₂O₂ and flame the surface to remove bubbles. The yield is 4 plates per 100 ml of LB agar. Mark the plates with the H₂O₂ concentration.
   4. Place the plates uncovered in a biological flow hood with the fan running for 30 min to remove excess moisture from the plates.
      NOTE: Use the plates the same day they are prepared. Failure to do so may result in inconsistent data.
      NOTE: Oxidative stressors such as paraquat can be substituted for H₂O₂ in this assay. The concentrations used for other oxidative stressors may be different than those used for H₂O₂.
3. Wash and measure the OD₆₀₀ of the overnight cultures as described in step 3.7.
4. Normalize the OD₆₀₀ of all the overnight cultures to the lowest value for the set of strains being tested. P. aeruginosa generally has an OD₆₀₀ of approximately 2.5 when grown overnight in LB + NTBC or LB + DMSO.
   1. Determine the volume of culture needed to dilute the culture to the lowest OD₆₀₀ in a total volume of 1 ml. For example, if a culture has an OD₆₀₀ of 3 and the lowest OD₆₀₀ for the set of strains is 2.5, perform the following calculation: (2.5)(1 ml) = (3)(x). x = 0.833 ml. 0.833 ml of culture will be placed in a microfuge tube.
   2. Calculate the amount of LB + NTBC (300 µM) or LB + DMSO needed to bring the culture volume to 1 ml. For the example in step 4.4.1, the amount of LB + NTBC or LB + DMSO added to the culture would be 0.167 ml (1 ml total volume – 0.833 ml culture). Make stock solutions of LB + NTBC and LB + DMSO to use for these dilutions based on the volume needed for diluting all strains.
   3. Mix the culture and LB + NTBC or LB + DMSO by vortexing.
5. To maintain cultures in a constant concentration of NTBC or DMSO, perform ten fold serial dilutions of the normalized overnight cultures in PBS + NTBC or PBS + DMSO.
   1. Make stock solutions of PBS + NTBC and PBS + DMSO. For one set of dilutions for one strain, mix 300 µM NTBC or an equivalent volume of DMSO with PBS to yield a total volume of 720 µl. Scale these stocks up or down depending on how many strains are tested.
   2. Label microfuge tubes for 10^-1 through 10^-7 serial dilutions. Add 90 µl of PBS + NTBC or PBS + DMSO to the appropriate tubes. Use PBS + NTBC for strains grown in LB + NTBC and use PBS + DMSO for strains grown in LB + DMSO.
   3. Add 10 µl of culture to the appropriate 10^-1 dilution tube. Mix by vortexing and transfer 10 µl of the 10^-1 dilution to the 10^-2 dilution tube. Repeat until all dilutions have been performed. Change pipet tips between dilutions.
6. Spot 5 µl of the 10^-3 through 10^-7 dilutions on LB + H₂O₂ plates in duplicate for each strain. Use one pipet tip if spots are plated from most dilute to least dilute (10^-7 to 10^-3). Do not tip or tilt the plate until the liquid has dried into the plate.
7. Incubate the plates for 24-48 hr at 37 °C (air incubator), depending on the strain.
   NOTE: P. aeruginosa PAO1 will have good sized colonies on LB after 24 hr of incubation. Incubate strains until they have colonies approximately the same size as PAO1.
8. Photograph the plates using a CCD camera above a transluminator. Optionally, edit the photos for contrast and crop to the same size. Count the number of colonies in each spot to determine changes in sensitivity to oxidative stress.

Results

NTBC titrations

The NTBC titrations were used to determine if NTBC was able to reduce pyomelanin production in P. aeruginosa, and also identify the concentration of NTBC that eliminates or reduces pyomelanin production for use in additional assays. There may be variations in the levels of pyomelanin produced in different replications, but general trends remain constant. The NTBC titration assay could also be modified to test other compounds that may affect pigment production in other ...

Discussion

The NTBC titration method described in this protocol will allow the user to determine if NTBC can reduce or eliminate pyomelanin production in bacteria, and determine the concentration of NTBC required. The most critical step in the NTBC titration assay is determining the range of NTBC concentrations to use in the assay. Different strains of P. aeruginosa have different sensitivities to NTBC, and laboratory strains may be more sensitive to NTBC than clinical isolates¹² (Figure 2). The...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
2-[2-nitro-4-(trifluoromethyl)benzoyl]-1,3-cyclohexanedione (NTBC)	Sigma-Aldrich	SML0269-50mg	Also called nitisinone.  Soluble in DMSO.
H2O2	Sigma-Aldrich	216763-100ML	30 wt. % in H2O.  Stabilized.
Gentamicin	Gold Bio	G-400-100	Soluble in H2O.  Filter sterilize.
Kanamycin	Fisher Scientific	BP906-5	Soluble in H2O.  Filter sterilize.
Tobramycin	Sigma-Aldrich	T4014-100MG	Soluble in H2O.  Filter sterilize.