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

The overall goals of the following experiments are to determine the effects of NTBC on P mein production and sensitivity to oxidative stress, as well as antibiotic minimum inhibitory concentrations or mics. This is achieved by adding NTBC to bacterial cultures to determine the concentration that reduces p melanin production as a second step. Bacterial cultures are treated with NTBC and antibiotics, which will allow the user to determine if NTBC treatment has an effect on antibiotic M mics.

Mics next NTPC treated cultures are exposed to hydrogen peroxide in order to determine sensitivity to oxidative stress with NTPC treatment results are obtained that show treating genic bacteria with NTPC leads to a reduction in pigment production and increases sensitivity to oxidative stress while having no effect on antibiotic mics based on oxidative stress response spot plates and microtiter MIC assays respectively. The main advantage of the oxidative stress spot plate technique over existing methods like viable counts and disc diffusion assays, is that lower concentrations of hydrogen peroxide can be used rather than the high concentrations of hydrogen peroxide used in other assays. Generally individuals new to our method of determining antibiotic mics will struggle because a lack of precision in pipetting technique introduces variability between replicates.

This variability can make it difficult to accurately determine the antibiotic. MIC One day prior to the MIC assay. Prepare cultures of the strains to be tested in LB with and without.

Two two nitro, four trior ethyl benzoyl one three Cyclohexane Dion, or NTBC. This demonstration uses the representative level of 300 micromolar of NTBC. The titration method for determining the appropriate concentration of NTBC is described in the protocol text incubate the cultures overnight at 37 degrees Celsius with aeration on the following day.

Make LB plus NTBC and LB plus DMSO master solutions for the MIC assay to test one antibiotic for one strain at 600 micromolar of NTBC to two milliliters of LB and mix. To make the NTBC master solution. NTBC is added at a concentration of 600 micromolar because it will be diluted twofold when the inoculum is added.

Yielding a final concentration of 300 micromolar add an equivalent volume of the vehicle DMSO to two milliliters of LB and mix to make the NO NTBC master solution. These two master solutions will be used for creating antibiotic stock solutions as well as for setting up the dilution series in 96 well plates. Next, prepare the antibiotic solutions in the LB plus NTBC or LB plus DMSO Master solutions.

Prepare the Gentamycin plus or minus NTBC stock solution at 64 micrograms per milliliter. Make the KANAMYCIN plus or minus NTBC stock solution at 256 micrograms per milliliter. Prepare the tobramycin plus or minus NTB stock solution at eight micrograms per milliliter.

After the two x antibiotic solutions have been made at 100 microliters of each antibiotic solution to four wells in a 96 well plate in row A.For example, gentamycin should be placed in a one through a four kanamycin should be placed in a five through a eight, and tobramycin should be placed in a nine through a 12. Add 50 microliters of the LB plus NTBC or LB plus DMSO master solution to rose B through H of the 96 Well plate ensure that one plate is LB plus NTBC and one plate is LB plus DMSO. Using a micro pipetter, perform twofold serial dilutions of the antibiotics by transferring 50 microliters of the solution from row A to row B.Mix the solution, change the pipette tips and transfer 50 microliters of the solution from row B to row C.Repeat for the remaining rows after diluting row G.Remove 50 microliters of the solution from that row and discard.

Use row H as a no antibiotic control for bacterial growth. Retrieve the overnight cultures after washing all cultures to eliminate lanin Present in the media measure the OD 600 of the cultures dilute the overnight cultures in lb. Using a multi-channel micro pipetter.

Add 50 microliters of the diluted bacterial culture to the appropriate wells. Add bacteria to three wells for each strain and antibiotic concentration. Add 50 microliters of LB to the fourth well to act as a control for bacterial contamination.

Cover the 96 well plates with perfil and incubate for approximately 24 hours at 37 degrees Celsius on the following day. Examine the plates visually 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.

To begin this procedure set up overnight cultures of the strains to be tested in LB with and without NTBC as shown before on the following day. Prepare LB APLs containing hydrogen peroxide as an oxidative stressor. First melt the LB agro flasks and cool the media to approximately 50 degrees Celsius at room temperature.

Next, add hydrogen peroxide directly to the cooled media at the desired concentrations. Swirl the flask to mix a range of hydrogen peroxide concentrations from zero to one. Millimolar is a good starting point.

Pour the plates immediately and flame the surface to remove bubbles. The yield is four plates per 100 milliliters of lb agar. Mark the plates with the hydrogen peroxide concentration.

Place the plates uncovered in a biological flow hood with the fan running for 30 minutes to remove excess moisture from the plates. The plates should be used on the same day. They are prepared while the plates are drying, wash and measure.

The OD 600 of the overnight cultures normalize the OD 600 of all the overnight cultures to the lowest value for the set of strains being tested. To do this, determine the volume of culture needed to dilute the culture to the lowest OD 600 in a total volume of one milliliter. For example, if a culture has an OD 600 of three and the lowest OD 600 for the set of strains is 2.5, perform the following calculation, which will result in 0.833 milliliters as the volume of culture needed place 0.833 milliliters of culture in a micro fuge tube.

Next, calculate the amount of LB plus NTBC or LB plus DMSO needed to bring the culture volume to one milliliter. In this example, the amount would be 0.167 milliliters mix by vortexing to maintain cultures in a constant concentration of NTBC or DMSO perform tenfold serial dilution of the normalized overnight cultures in PBS plus NTBC or PBS plus DMSO make stock solutions of PBS plus NTBC and PBS plus DMSO scaling up or down, depending on how many strains are tested for one set of dilutions for one strain mix, 300 micromolar of NTBC or an equivalent volume of DMSO. With PBS teal, a total volume of 720 microliters add 90 microliters of PBS plus NTBC or PBS plus DMSO to the appropriate micro fuge tubes labeled for 10 to the minus one through 10 to the minus seven serial dilution.

Add 10 microliters of culture to the appropriate 10 to the minus one dilution tube mixed by vortexing and transfer 10 microliters of the 10 to the minus one dilution to the 10 to the minus two dilution tube. Changing pipette tips between dilution repeat until all dilution have been performed. Spot five microliters of the 10 to the minus three through 10 to the minus seven dilution on LV plus hydrogen peroxide plates in duplicate.

For each strain, use one pipette tip. If spots are plated from most dilute to least dilute, do not tip or tilt the plate until the liquid is dried into the plate. Incubate the plates at 37 degrees Celsius for 24 to 48 hours depending on the strain.

Once good sized colonies have appeared, photograph the plates using a CCD camera above a transluminator. NTBC titrations were used to determine if NTBC was able to reduce p melanin production in p aerogen and to identify the concentration of NTBC that eliminates or reduces p melanin production for use in additional assays as shown here. NTBC reduced PY melanin production in a dose-dependent manner in p melanin producers, but had no effect on pigment production in strains that do not produce p melanin.

This table shows MIC assay results for different p aerogen strains treated with and without NTBC and various antibiotics mics were recorded as the lowest concentration of antibiotic that inhibited bacterial growth. NDBC treatment had no effect on the antibiotic mics for the strains tested. A spot plate assay for oxidative stress response was performed on PIO melanin and non melanin producing strains treated with and without NDBC.

The zero millimolar hydrogen peroxide plate showed that all the strains were diluted properly and increased sensitivity to oxidative stress was observed when PIO melanin producing bacteria were treated with NTBC strains that do not produce PIO melanin were more sensitive to oxidative stress than strains that produce p melanin. After watching this video, you should have a good understanding of how to determine the effects of NTBC treatment on PIO melanin production by testing sensitivity to oxidative stress and antibiotic mics.