The protocol described here makes it possible to identify conditions that can significantly impact bacterial persistence in a high-throughput manner. This method is readily adaptable to screen for various areas, such as drug panels, gene libraries and more. As the method includes various sub steps, which are to be conducted simultaneously, visual demonstration can aid researchers to manage their timing to obtain precise results.
Begin by preparing microarray cell cultures. Transfer 250 microliters of exponential phase cells to 25 milliliters of fresh modified LB medium in a 50 milliliter centrifuge tube. Then gently mix the cell suspension to make it homogenous.
Transfer the diluted cell suspension into a sterile 50 milliliters reservoir. Using a multi-channel pipette, transfer 150 microliters of the cell suspension to each well of a microarray plate containing various chemicals. Microarrays can also be generated manually following the method described in the text manuscript.
Cover the microarray plate with a gas permeable ceiling membrane and incubate it in an orbital shaker at 37 degrees Celsius and 250 RPM for 24 hours. To make persister assay plates, prepare 25 milliliters of modified LB medium containing five micrograms per milliliter of Ofloxacin in a 50 milliliter centrifuge tube and transfer this medium to a sterile reservoir. Transfer 190 microliters of the modified LB medium into each well of a generic flat bottom 96 well plate.
After the 24 hour incubation, remove the microarray from the shaker and transfer 10 microliters of cell cultures from the microarray to the wells of the persister assay plate containing modified LB medium with Ofloxacin. Take 10 microliters of cell suspensions from the persister essay plate and serially dilute it three times in 290 microliters of PBS solution using a round bottom 96 well plate and a multi-channel pipette. Then spot 10 microliters of all serially diluted cell suspensions on antibiotic free fresh agar plates.
Cover the persister assay plate with a gas permeable ceiling membrane and incubate it in an orbital shaker at 37 degrees Celsius and 250 RPM for six hours. After the six hour incubation, repeat the serial dilution and spotting on agar plates. Incubate the agar plates for 16 hours at 37 degrees Celsius.
Then count the colony forming units or CFUs. Use the CFU levels before and six hours after the antibiotic treatment to calculate the persister fraction in each well. The CFU counts before the OFX treatment also help assess the effects of osmolites on e coli viability.
Transfer 250 microliters of exponential phase cells to 25 milliliters of fresh modified LB medium containing the osmolite identified from the microarray screening. Incubate the flask in an orbital shaker at 250 RPM and 37 degrees Celsius for 24 hours. After 24 hours, remove the flask from the shaker and transfer 250 microliters of the cell culture to 25 milliliters of fresh modified LB medium in a 250 milliliter baffled flask.
Add 25 microliters of Ofloxacin stock solution to the cell suspension and shake the flask gently to make the assay culture homogenous. Incubate the flask in a shaker at 37 degrees Celsius and 250 RPM. At every hour during the treatment, including a time point before addition of Ofloxacin, transfer one milliliter of the assay culture from the flask to a 1.5 milliliter micro centrifuge tube and centrifuge it at 17, 000 times G for three minutes.
Remove 950 microliters of supernatant and wash the cells three times with 950 microliters of PBS. After the final wash, re-suspend the cell pellet in 100 microliters of PBS solution. Take 10 microliters of the cell suspension and serially dilute it six times with 90 microliters of PBS inside a 96 well round bottom plate.
Spot 10 microliters of the diluted cell suspensions on an antibiotic free agar plate. To increase the limit of detection, plate the remaining 90 microliters of cell suspension on a fresh agar plate. Incubate the plates at 37 degrees Celsius for 16 hours, then count CFUs.
A representative image of agar plates used to determine CFU levels of cell cultures before and after OFX treatment is shown here. The first column is the control group. The second column has cells that were cultured in 100 millimolar sodium nitrate and the third column contains cells that were cultured in 60 millimolar sodium nitrate.
For simplicity, only two osmolites are focused on here. A graphical representation of the CFU data from the plates and persister fractions of the cell cultures tested in 96 well plates are shown here. To calculate the fractions, persister counts were normalized to the cell counts obtained before the antibiotic treatment.
To generate byphasic kill curves, the cells were cultured in 25 milliliters of modified LB medium with the indicated osmolites and baffled flasks for 24 hours, then transferred to persister assay flasks for persister enumeration. When attempting this protocol, keep in mind that spotting the cells on agar plate is labor intensive and requires focus. An error during the step can lead to cross-contamination among the conditions being tested.
So it is important to practice spotting multiple samples simultaneously prior to the experiment. Following this protocol, drug panels and mutant cell libraries can be screened to study the impacts on the persistence. With this screening, we have identified a number of osmolites and PS conditions that have a significant impact on bacterial persistence.
