The overall goal of this experiment is to demonstrate the efficacy of quorum-quenching enzymes in bacterial biofilm disruption. This method will help answer key questions in the field of anti-bacterial therapeutics, and to help understand the effect of quorum-quenching lactonases on pathogenic bacteria within a biofilm environment. The main advantage of this technique is that quorum-quenching lactonases can be directly added in an in vitro environment.

Thus, its effect on biofilm disruption are easily quantitated. Demonstrating this procedure is Terrence Tay a technician in our lab. To begin, grow a 5 mililiter culture of A.baumannii S1 in LB at 30 degrees Celsius in a shaking incubator for 16 hours.

Adjust the culture to an OD 600 of 0.8. Then use fresh LB containing 4 miligrams per mililiter of purified GKL enzyme to dilute the culture one to 100. And add 100 microliters to the wells of a 96-well plate.

Use the lids to cover the plates, and place them into a 10 liter plastic container. Seal the container and incubate at 30 degrees Celsius for three hours, before gently removing the medium. Add another 100 microliters of fresh LB medium to the wells, and incubate the plate at 30 degrees Celsius for 21 hours.

After gently removing the medium, use 200 microliters of sterile water to gently wash the planktonic bacteria cells. Position the pipette tip at the bottom of the well and carefully remove the liquid medium to ensure minimal perturbations on the biofilm foreign between the air and liquid interface. Then add 100 microliters of 1%crystal violet solution to each well, and incubate for 15 minutes at room temperature.

Remove the crystal violet solution by using 200 microliters of sterile water to wash the wells three times. Add 100 microliters of 33%acetic acid to each well and incubate with gentle shaking for 15 minutes to dissolve the dye. Quantitate the amount of biofilm by measuring the absorbance of crystal violet at 600 nanometers.

The amount of crystal violet is proportional to the amount of biofilm formed. To carry out confocal laser scanning microscopy, or CLSM, of A.baumannii S1 biofilms, grow a five mililiter culture of A.baumannii S1 in LB at 30 degrees Celsius in a shaking incubator for 16 hours. Adjust the culture of A.baumannii S1 to an OD 600 of 0.8.

Then use fresh LB containing 1.2 miligrams per milileter of purified GKL enzyme to dilute the culture, one to 100, and add one mililiter to a 35 mililiter glass-bottomed microdish. Cover the microdish with a lid and place it in a 10 liter plastic container and seal it before incubating the dish at 30 degrees Celsius for three hours. After gently removing the medium, add one mililter of fresh medium containing 30 microliters of purified GKL enzyme then incubate at 30 degrees Celsius for another 21 hours.

Gently remove the medium a second time and replace with another one mililiter of GKL enzyme in fresh medium. Incubate 30 degrees Celsius for 24 hours. After the incubation, gently remove the medium and add 500 microliters of five micrograms per mililiter of Alexa Fluor 488 conjugated wheat germ agglutinin dissolved in HBSS.

Incubate the microdish at 37 degrees Celsius for 30 minutes to stain the biofilms. To fix the biofilms, add 500 microliters of 3.7%formaldehyde dissolved in HBSS and incubate at 37 degrees Celsius for 30 minutes. Use two mililiters of HBSS to wash the microdish once, then remove the solution completely.

Store the dish in the dark at four degrees Celsius prior to CLSM imaging. For CLSM imaging and analysis, use a 63X objective to generate 97 stacks per image with an interval of 0.21 microns per stack. In the crystal violet quantitation experiment, Wild-type GKL and an improved GKL double-mutant quorum-quenching enzyme were used for lactonase activity against three hydroxydecanoyl l homoserine lactone or 3OHC10HSL, the major quorum molecule used by A.baumanii S1.As shown here, both enzymes were able to significantly reduce biofilms formation in pre-treated Wild-type A.baumanii S1.The extent of biofilms disruption by both enzymes is not proportional to their efficacy against 3OHC10HSL.

As seen in this table, the turnover rate of the mutant GKL is six times faster than Wild-type GKL against 3OHC10HSL. However, the difference in the extent of biofilms disruption between the enzymes is only twofold. In this experiment, the improved GKL mutant enzyme and its catecholy inactive enzyme were used for comparison.

This DIC image shows that treatment with the improved GKL mutant enzyme resulted in a decrease in biofilm size compared to the inactive enzyme. Analysis of the fluorescence images also revealed that there was a reduction in surface area, biomass, and average thickness of biofilms, when treated with the improved GKL mutant enzyme. Once mastered, this technique can be done in three to four hours excluding incubation time if it is performed properly.

While attempting this procedure, it is important to remember to minimize the removal and disruption of the biofilm during the washing steps. Following this procedure, other methods like cell counting and enzyme stability assays can be performed. This will answer additional questions like the effects of quorum-quenching lactonases on cell population within the biofilm as well as their duration of action.

After its development, this technique paved the way for researchers in the field of infectious disease treatment to explore the effects of potential quorum-quenching lactonases in the biofilm produced by pathogenic bacteria. After watching this video, you should have a good understanding on how to quantify biofilm disruption by crystal violet staining, and to assess morphological changes in the biofilm by confocal imaging. Don't forget that working with pathogenic material can be extremely hazardous.

Precautions, such as using proper biological risk-containment facilities should always be taken while performing this procedure.