We're trying to understand how microbes behave when grown in conditions mimicking real life conditions, with the goal of determining the mechanisms driving phenotypic changes in multi-species communities compared to monoculture growth. Through the utilization of this in vitro system, we have characterized a molecular pathway driving the recalcitrants of Pseudomonas aeruginosa, a notorious pathogen in the context of cystic fibrosis airway infections. Due to its 96-well plate format and with growth conditions reflecting the ones observed in the cystic fibrosis lung, we can explore a wide range of microbiome interactions with ease.
We have gained insights into the mechanisms behind microbes'alter susceptibility to antimicrobials in a fully microbiome context. Our model serves as a bridge between clinical observations and individual lab work in cystic fibrosis research, offering a valuable tool for studying various microbiome interactions. To begin, prepare all the required reagents and media.
For the co-culture experiment, centrifuge the bacterial overnight cultures, and wash the cells with sterile PBS. After centrifuging, discard the supernatant carefully and resuspend each pellet in one milliliter of 1X water diluted artificial sputum medium or ASM base. Measure the optical density of 1 to 10 diluted samples at 600 nanometers in a spectrophotometer or in a plate reader.
Dilute each bacterial sample to a final optical density at 600 nanometers of 0.01 and vortex thoroughly for five seconds. Add 100 microliters of the monoculture and co-culture suspensions to three separate wells of a sterile plastic flat bottom 96-well plate and incubate the plate for 24 hours at 37 degrees Celsius in anoxic conditions. Then using a multi-channel pipette, remove the unattached planktonic cells and replenish the preformed biofilms with 100 microliters of fresh ASM or the desired treatment reagent.
After aspirating the planktonic cells, gently wash the biofilms two times with 125 microliters of sterile PBS and discard the wash solution. Add 50 microliters of sterile PBS. And using a 96-pin replicator, gently scraped the cells off from the plate.
Transfer the resuspended biofilm cells to row A of a new sterile 96-well plate and perform a 10X serial dilution. Plate three to five microliters of each dilution sample onto the agar plates. After the inoculation spots dry, incubate the plates appropriately.
Several phenotypes were observed, including a reduction in the number of viable Pseudomonas aeruginosa and Staphylococcus aureus cell counts when grown in a mixed planktonic community compared to the monoculture. An increase in polymicrobial growth of Streptococcus sanguinis and mixed community growth of Prevotella melaninogenica was observed.
