Our research develops an aggregate biofilm model in artificial sputum to replicate lung infections in cystic fibrosis patients. This model allows us to analyze gene expression changes and understand why bacteria become more resistant to therapeutics in these conditions compared to standard drug testing environments. Developing a complex biofilm model has highlighted the challenges of recapitulating the hostile lung environment, making it challenging to predict if in vitro antimicrobial effects align with in vivo outcomes.
The patient specific nature of CF lung infections further complicates the development of effective laboratory models for testing antimicrobials. Throughout this research, we've successfully created a polymicrobial aggregate biofilm model, which provides a platform to test antimicrobials in a realistic environment, shows the level of resistance that can be observed in these biofilms, and highlights the potential for anti-virulence therapies targeting components such as alginate. Having an antimicrobial testing model designed to mimic the CF lung environment will bridge the gap that exists between in vivo and in vitro testing.
Furthermore, assessing the virome of bacteria within an environment that more closely mimics a CF lung will allow for the development and testing of anti-virulence therapeutics. To begin, streak a single bead of pseudomonas aeruginosa PAO1 from frozen bead stock cultures onto lysogeny broth auger. Incubate the plate for 24 hours at 37 degrees Celsius.
For single-species biofilm preparation, prepare overnight cultures of pseudomonas aeruginosa PAO1 with a single colony from the streak plate and incubate overnight for 18 hours. Then, dilute the culture to an optical density of 0.05 at 600 nanometers, equivalent to 1 times 10 to the power of 8 colony forming units per milliliter. Further, dilute this culture 1 to 100 in SCFM2 medium.
Next, add 180 microliters of the inoculum to the wells of a round-bottomed 96-well microtiter plate. Incubate the plate at 37 degrees Celsius while shaking at 75 revolutions per minute for 24 hours. Revive pseudomonas aeruginosa PAO1 and staphylococcus aureus SH1000 from frozen bead stock cultures as demonstrated earlier.
Revive candida albican CAF 2.1 as a single bead streak onto sabouraud dextrose agar, and incubate the plates for 24 hours at 37 degrees Celsius. Prepare overnight cultures of staphylococcus aureus and candida albicans with single colonies from their respective streak plates in 5 milliliters of lysogeny broth. Dilute the standardized cultures to form a single inoculum containing both species at required concentrations in SCFM2.
Then, add 162 microliters of the mixed inoculum to the wells of a 96-well microtiter plate. Incubate the plate at 37 degrees Celsius while shaking at 75 revolutions per minute for 24 hours to allow the formation of multi-species biofilms. Next, add 14.2 microliters of the prepared overnight culture of pseudomonas aeruginosa to wells of the 96-well microtiter plate, and incubate the plate again to allow polymicrobial biofilm formation.
To begin, obtain single-species and multi-species biofilms in 96-well microtiter plates. For disrupting the biofilm, add 8.81 microliters of DNase 1 stock directly to the wells containing the biofilms, achieving a final concentration of 50 micrograms per milliliter. Incubate the plate for one hour at 37 degrees Celsius while shaking at 75 revolutions per minute.
Obtain an antibiotic stock solution of meropenem at 2.56 milligrams per milliliter. Perform serial dilutions by a factor of two to achieve a concentration range of 0.01 milligrams per milliliter to 2.56 milligrams per milliliter. Now, add 20 microliters of each miropenem dilution to the biofilms, achieving a dosage range of 1 microgram per milliliter to 256 micrograms per milliliter.
Seal the 96-well microtiter plate with transparent film, and incubate it at 37 degrees Celsius for 24 hours while shaking it 75 revolutions per minute. The single-species pseudomonas biofilms showed no significant decrease in viable cells when treated with meropenem at any dosage up to 256 micrograms per milliliter. Pseudomonas aeruginosa recovery was 0.74 log 10 colony forming units per milliliter higher in single-species settings than in polymicrobial environments without antibiotics.
To begin, prepare the single-species and multi-species biofilms for RNA extraction. After incubation, transfer the biofilms from each well to a one milliliter micro centrifuge tube pooling the biofilm replicates for each sample. Centrifuge the tube at 16, 000 G for five minutes.
If RNA extraction is performed later, remove the supernatant, and store the pellet in 250 microliters of RNA stabilization solution at 80 degrees Celsius. Later, thaw the tube with pellet at room temperature and centrifuge at 16, 000 G for five minutes. Resuspend the pellet in 600 microliters of TRIzol reagent and manually disrupt it using a 0.2 millimeter needle attached to a two milliliter syringe, aspirating 5 to 10 times, or until the pellet is completely disrupted.
Follow the manufacturer's guidelines to purify the RNA from the disrupted biofilms. To quantify the purified RNA, prepare the working solution with 199 microliters of buffer and one microliter of reagent for each sample. Mix 190 microliters of the working solution and 10 microliters of standard one and standard two into separate tubes.
Then, add 199 microliters of the working solution and 1 microliter of the RNA sample into individual tubes. Vortex for 30 seconds, and store in a dark location for five minutes. Next, on the fluorimeter, select RNA, followed by Broad Range RNA, and follow the onscreen instructions.
After taking the blank reading, pipette one microliter of RNA onto the sample holder of the spectrophotometer, and measure the absorbance at 260 by 280 nanometers. For complimentary DNA synthesis, prepare the reaction mix in tubes. Place the tubes in a thermal cycler and run the program.
Use the CDNA immediately, or store it at 80 degrees Celsius. algD expression in single-species pseudomonas aeruginosa biofilms did not vary significantly across meropenem concentrations. In polymicrobial biofilms, algD expression was significantly higher at 256 micrograms per milliliter indicating increased alginate production in the presence of co-colonizing organisms.
