In Vitro Persister Assay: A Method to Evaluate the Effect of Osmolytes on Bacterial Persistence

On exposure to harmful environment conditions, a subset of bacteria enter a state of low metabolic activity, evading the detrimental effects. These bacteria, called persisters, can tolerate transient exposure to high concentrations of antibiotics.

To study the effect of osmolytes - small organic solutes - on bacterial persistence, first, obtain an exponential phase bacterial cell culture with pre-existing persisters below the limit of detection, suspended in media.

Next, transfer the suspension into osmolyte-containing wells of a microarray plate. Keep a set of wells without osmolyte as control. Seal the plate with a gas-permeable membrane to allow rapid gas diffusion for cell growth and incubate.

The osmolytes in the medium at appropriate concentrations induce osmotic stress, triggering some cells to reprogram their cellular metabolism from rapid to slow growth, forming persisters.

Now, transfer the osmolyte-treated and control cells to an antibiotic-containing persister assay plate. Seal the persister assay plate and incubate.

In the presence of antibiotics, normal cells undergo cell death, whereas persisters - being antibiotic-tolerant - survive. Last, collect a small fraction of the cells, serially dilute with buffer, and spot onto an antibiotic-free agar plate. Incubate the plate.

In the absence of antibiotics, the persisters resume growth and proliferate, forming colonies. Count the number of colonies on the agar plate.

A higher number of colonies than no-osmolyte control post-antibiotic treatment suggests a positive effect of osmolytes on persister formation.

Begin by preparing microarray-cell cultures.

Transfer 250 microliters of exponential-phase cells to 25 milliliters of fresh modified LB medium, in a 50-milliliters centrifuge tube. Then, gently mix the cell suspension to make it homogeneous.

Transfer the diluted cell suspension into a sterile 50-milliliter 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 sealing 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 5 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 assay plate, and serially dilute it 3 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 sealing membrane, and incubate it in an orbital shaker at 37 degrees Celsius, and 250 RPM for six hours.

After the 6-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 osmolytes on E. coli viability.