Development of a Polymicrobial Colony Biofilm Model to Test Antimicrobials in Cystic Fibrosis

Our research focused on using complex polymicrobial models to understand the complex interactions between microbes in the cystic fibrosis environment and how this results in increased antimicrobial tolerance. We hope to use this knowledge to develop novel therapeutic interventions. Advancements within the biofilm field is being driven by visualization, coupled with quantification methodologies alongside biophysical approaches.

This includes techniques like 3D OrbiSIMS that give spatial resolution down to the micro and nanoscale. This research aims to create a polymicrobial model that maintains a level of complexity, representative of what is seen in the antibiotic treatments in CF patients. It will also allow for increased throughput, more relevant outputs and adaptability to other strains and conditions.

This protocol offers a relevant testing environment for CF pathogens. It's robust and has a relatively high throughput. It's also amenable to multiple endpoints, allowing for more complex studies to be undertaken.

This research has shown that an environment relevant to CF alters and microbial susceptibility. In addition, the type of fungi present can also alter the susceptibility of Pseudomonas aeruginosa, and this may have significant clinical relevant. To begin, take cultures of Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans grown to the exponential phase.

Using a micropipette, transfer one milliliter of each liquid culture to a separate sterile 1.5 milliliter tube. Centrifuge the tubes for two minutes at 8, 000 x g to pellet the bacteria or fungi. After that, using a pipette, aspirate the supernatant from each tube and resuspend the pellets in one milliliter of PBS.

Dilute the resuspended Pseudomonas aeruginosa, and Staphylococcus aureus cells one to 10 in PBS. Then using a spectrophotometer, measure the optical density at a wavelength of 600 nanometers. After diluting the washed samples of Candida albicans one to 100 in PBS, pipette 10 microliters of each sample into two individual chambers of a hemocytometer.

Then adjust the Candida albicans sample to 1 x 10 to the power of eight CFU per milliliter in PBS. To prepare the inoculum for addition to the biofilm, adjust the Pseudomonas aeruginosa, Candida albicans, and Staphylococcus aureus inoculum using PBS. For mono-species biofilms, pipette 10 microliters of the diluted microbe onto the center of a polycarbonate disc placed on the SCFM2 1.5%technical agar in 6-well plates.

Incubate the plate statically at 37 degrees Celsius for 24 hours before treatment or disruption. For polymicrobial biofilms, add 10 microliters of each Staphylococcus aureus and Candida albicans on top of one another on the same polycarbonate disc. Incubate the inoculated disc statically for 24 hours at 37 degrees Celsius.

Following incubation using sterile forceps, transfer the polycarbonate discs to fresh SCFM2 1.5%technical agar plates. Add 10 microliters of the 1 x 10 to the power of four CFU per milliliter dilution of Pseudomonas aeruginosa on top of the pre-established Staphylococcus aureus, Candida albicans biofilm. Incubate for another 24 hours at 37 degrees Celsius.

To begin, take solid air interface colony biofilm of Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans. Add ceramic beads with a diameter of 2.8 millimeters to the plate and UV crosslink the plate. Using flame sterilized forceps, transfer five ceramic beads to a sterile two milliliter homogenizer tube.

Pipette one milliliter of PBS into the two milliliter homogenization tube. Vortex the tubes for at least 10 seconds or until the biofilm has been removed from the polycarbonate disc as determined by visual inspection. Once removed, take out the polycarbonate disc, then place the tubes with the beads in the homogenizer and beat them two times for 10 seconds at six meters per second with a ten second interval between beats.

Now pour the disrupted biofilm from the homogenizer tube into a seven milliliter bijou containing four milliliters of PBS resulting in a final volume of five milliliters. Centrifuge the homogenizer tubes at 8, 000 x g for two minutes. Then transfer the remaining liquid to the seven milliliter bijou to ensure all liquid is transferred.

Add 200 microliters of the disrupted biofilm into a clear bottomed black 96-well plate, then pipette 10 microliters of 0.02%resazurin solution into each well. Incubate the resazurin-treated biofilm in a plate reader at 37 degrees Celsius. Apply orbital shaking every 30 minutes for 10 seconds at 200 RPM before taking any fluorescent readings.

Measure the fluorescence using an excitation wavelength of 540 nanometers and an emission wavelength of 590 nanometers. Mono-species biofilms required significantly lower concentrations of meropenem and tobramycin to achieve 50%killing compared to polymicrobial biofilms with a two log increase in antibiotic concentration necessary for the latter. Pseudomonas aeruginosa survival increased in polymicrobial biofilms with 64 micrograms per milliliter of tobramycin, whereas meropenem showed a decrease in survival under similar conditions.

In mono-species biofilms, a strong correlation was observed between the survival of Pseudomonas aeruginosa and its metabolic activity when treated with both meropenam and tobramycin.