Targeting Biofilm Associated Staphylococcus aureus Using Resazurin Based Drug-susceptibility Assay

The overall goal of this procedure is to easily, and reproducibly grow bacterial bio films in a 96-well plate format on lid-attached pegs for antibiotic susceptibility testing using a viability dye. This method can help answer key questions in the antibiotic discovery and development field by exploring activities on biofilm-associated cells which are often more representative of invivo infection states. The main advantage of this technique is the ability to easily challenge preformed biofilms in a medium three-part assay and determine inhibition using a common viability dye.

To begin, grow a culture of biofilm-producing organism in a nutrient-rich medium. Inoculate the Staphylococcus aureus strain Newman from a glycerol stalk in 10 milliliters of Muller-Hinton medium. Perform all work involving handling of S.aureus with gloves, and within a bio-safety cabinet.

Incubate for 16 hours at 37 degrees Celsius on a rotary shaker. Add the calculated culture volume to a centrifuge tube, and pellet cells by spinning for one minute at 10, 000 Gs.To prepare the biofilm inoculum, aspirate the spent growth medium and re-suspend the cell pellet in 20 milliliters of CRPMI. Using a 200 microliter multi-channel pipette, add 125 microliters of inoculum from a 25 milliliter reservoir to each well of rows A to G of the bottom plate.

Fill 125 microliters of sterile CRPMI medium in each well of row H as a sterility control. Take the peg lid and hold it above the plate bottom with the pegs facing downwards. Carefully align the individual pegs with their respective wells.

Avoid physical contact between the plate and the pegs. Once aligned, lower the lid carefully down. Seal the lid to the plate with paraffin film to prevent evaporation, and place it in a seal-able plastic bag, sealing tightly.

Keep the air volume in the bag as low as possible to minimize evaporation. Incubate the plate without shaking at 37 degrees Celsius for 48 hours, in a 5%Carbon Dioxide incubator. On the day of the biofilm challenge, take a fresh 96-well plate and add 100 microliters of CRPMI, equilibrated to room temperature, to each well of rows B to H.Dilute up to four test compounds separately, in 1.0 milliliter of CRPMI, to 2x the desired final concentration.

Add 200 microliters of compound 1 in wells A1-A3.200 microliters of compound 2 in wells A4-A6.200 microliters of compound 3 in wells A7-A9. And 200 microliters of compound 4 in wells A10-A12. Serially dilute the test compounds using a multi-channel pipette.

Begin by transferring 100 microliters from row A to row B, and mix. In that manner, continue to transfer 100 microliters well-to-well. Mix after each transfer, and stop in row F.After mixing, expel 100 microliters from row F into a waste container.

Do not transfer any liquid to rows G and H.Finally, add 100 microliters of CRPMI to each well of the plate using a multi-channel pipette, to bring the final volume to 200 microliters. Add 200 microliters of phosphate-buffered saline to a fresh, sterile, 96-well plate that features wells with dimensions identical to those of the biofilm initiation plate. Next, remove the plastic bag and paraffin film from the incubated biofilm initiation plate.

Lift the lid straight up, avoiding contact between the pegs and the plate bottom, as this may damage the biofilm. Keep the bottom part for subsequent OD600 analysis. Rinse off planktonic cells by placing the peg lid onto the plate containing PBS.

Submerge pegs for five seconds. Lift them out of PBS, and submerge once more, being careful not to hit the pegs against the well sides. Place the rinsed peg lid into the challenge plate.

Avoid unnecessary contact between pegs and the bottom plate, as this will damage the biofilm, leading to inconsistent results. Seal the plate with paraffin film, and place into a seal-able plastic bag as before. Incubate the plate, without shaking, for 24 hours at 37 degrees Celsius, in a 5%Carbon Dioxide incubator.

Take the bottom of the biofilm initiation plate and re-suspend the bacteria in each well using a multi-channel pipette. Measure the OD600 with a suitable microplate reader, to confirm growth occurring in all wells but the sterility controls in row H.Use a plate reader equipped with an incubation chamber and capable of taking kinetic fluorescence readings for detecting resazurin conversion. Set up a 24-hour kinetic fluorescence measurement using a 530 nanometer excitation, and 590 nanometer emission.

Set the chamber temperature to 37 degrees Celsius, and have the plate read every 20 minutes. Set the plate reader to measure from the bottom of the plate according to the manufacturer's instructions. Prepare to wash the plate by adding 200 microliters of PBS to each well of a sterile 96-well plate with a multi-channel pipette.

Then, prepare biofilm recovery medium, by adding 400 microliters of 0.8 milligram per milliliter resazurin stock solution to 20 milliliters of CRPMI medium, to a final concentration of 16 micrograms per miliiliter resazurin. Next, add 150 microliters of biofilm recovery medium with a multichannel pipette, to each well of a fresh 96-well plate. Transfer the challenge plate from the incubator into a bio-safety cabinet, and carefully remove the plastic bag and sealing film.

Remove the peg lid from the challenge plate, and rinse off planktonic cells in PBS as before, keeping the bottom of the challenge plate for subsequent OD600 analysis. After rinsing, place the peg lid into the plate bottom containing the biofilm recovery medium. Wrap the side of the plate with paraffin film, being careful not to obstruct the bottom of the perimeter wells.

Immediately after wrapping the plate, place it on the plate reader, and start a kinetic read over a 24-hour period, by initiating the previously made resazurin-kinetic protocol. To quantify growth of planktonic cells that may or may not occur during the challenge, read the OD600 of the entire challenge plate bottom using a microplate reader. Shown here is an example layout of a challenge plate, using two full dilutions to titrate compound concentrations.

Grown biofilms were treated with neocuproine, or its copper complex. OD600 readings of the challenge plate bottom after 24 hours revealed that the neocuproine copper complex, but not neocuproine alone, prevented shedding and subsequent growth of viable cells from treated biofilms at concentrations of 1.25 micromolar or above. The corresponding biofilms were then tested by the resazurin assay.

Visual inspection after 24 hours allows for a quick, yes-no answer if multiple plates are being run in parallel. Resazurin turns pink in wells with viable biofilms, but remains blue when the biofilm has been eradicated. A kinetic reading of resazurin conversion can uncover concentration-dependent effects on biofilm viability, as shown here with gentamicin.

The concentration-dependent delay in the onset of dye conversion, is indicative of a decrease in viability of the biofilm. While attempting this procedure, it's important to remember not to bump the pegs against the side of the wells.