Population and Single-Cell Analysis of Antibiotic Persistence in Escherichia coli

The protocol described here provides an integrative method combining classical microbiology assays with single-cell live imaging to characterize Escherichia coli persister cells after high ofloxacin treatment. The main advantage of this technique is to analyze persistence phenomenon at the population and single-cell level. Combining population and single-cell analysis allows for the molecular and cellular characterization of the persistence phenotype.

To begin, streak the bacterial strain of interest from a frozen glycerol stock on an LB agar plate, and incubate at 37 degree Celsius overnight for a period of 15 to 19 hours to obtain single colonies. Inoculate an isolated colony in a glass tube containing five milliliters of MOPS growth medium supplemented with 0.4%glucose, and, if required, add a selective antibiotic into the medium. Place the tube in a shaking incubator at 37 degrees Celsius and 180 rpm overnight.

The following day, centrifuge one milliliter of culture at 2, 300 g for three minutes to pellet the cells, and gently resuspend the pellet with the same volume of PBS. Measure the OD at 600 nanometers, and calculate the volume needed for an initial OD600 of 0.01 in a final volume of two milliliters. Next, add two milliliters of MOPS glycerol 0.4%medium into a well of a transparent-bottom, 24-well plate, and inoculate with the calculated overnight culture volume.

Place the 24-well plate into an automated microplate reader to monitor the OD600 for 24 hours. Set the microplate reader to measure the OD600 every 15 minutes at a temperature of 37 degrees Celsius and with a high orbital rotation of 140 rpm. Melt 100 milliliters of LB agar, and stock the flask at 55 degrees Celsius to avoid solidification.

Prepare six small glass flasks, and pipette five milliliters of liquid LB agar medium into each flask using a sterile pipette. Take 10 microliters of the five milligrams per milliliter ofloxacin stock solution, and dilute in 90 microliters of ultrapure water. Add increasing volumes of the diluted ofloxacin to each of the six glass flasks containing LB agar medium to get a final concentration of zero to 0.1 micrograms per milliliter of ofloxacin.

Mix the solution by rotating the flasks several times, and pour the antibiotic-added LB agar media into a six-well culture plate. Let the agar cool down until solidification, and dry the plate before using. Dilute an overnight bacterial culture to a final cell density of one times 10 to the seventh CFU per milliliter with PBS.

Spot two microliters of diluted culture onto every well of the dried six-well plate. Let the spots dry before placing the plate into an incubator at 37 degrees Celsius overnight. The next day, check at which ofloxacin concentration the growth of bacteria colonies is inhibited.

Pour approximately 25 milliliters of the prepared LB agar into a Petri dish. Then add five to eight sterile glass beads to each solidified and dried plate. Invert and label the plates.

Next, prepare tenfold serial dilution glass tubes containing 900 microliters of 0.01-molar magnesium sulfate solution. In a glass tube, dilute an overnight culture in fresh and temperature-adjusted medium to a final OD600 of approximately 0.001, and allow the culture to grow overnight in an incubator. The following day, when the OD600 reaches 0.3, transfer 100 microliters of the culture for dilution according to the spot assay data.

Perform a tenfold serial dilution of the bacterial culture. Then plate 100 microliters of the diluted culture on the prepared LB agar plates. Add the desired concentration of ofloxacin into the bacterial culture, and continue to incubate at 37 degrees Celsius while shaking.

At relevant time points, withdraw 100 microliters of the culture, and dilute the culture accordingly to the spot assay data. Plate 100 microliters of the diluted culture on the LB agar plates. The next day, count the colonies of the overnight incubated plates at the two highest dilutions for which colonies can be detected.

Calculate the survival ratio by normalizing the CFU per milliliter at each time point at the CFU per milliliter at T0, and plot the log base 10 normalized value as a function of time. Remove the conservation solution from every well of the microfluidic plate, and replace it with fresh culture medium. Seal the microfluidic plate with the manifold system by clicking on the Seal button or through the microfluidic software.

Next, on the microfluidic software interface, perform a first priming sequence by clicking on Run Liquid Priming Sequence. Incubate the plate in a thermostatically controlled cabinet of the microscope at 37 degrees Celsius for a minimum of two hours before starting the microscopy imaging. Then start a second Run Liquid Priming Sequence before beginning the experiment.

Seal off the microfluidic plate by clicking on Unseal Plate on the microfluidic software interface. Replace the medium in the wells with 200 microliters of fresh medium, antibiotic containing fresh medium, and 0.01 OD diluted culture sample in the fresh medium. After sealing the microfluidic plate as demonstrated before, place the plate onto the microscope objective inside the microscope cabinet.

In the microfluidic software, click on the Run Cell Loading Sequence to allow the loading of the cells into the microfluidic plate. Set an optimal focus using the transmitted light mode, and select several regions of interest where an appropriate cell number of up to 300 cells per field can be observed. On the microfluidic software, edit the Run a Custom Sequence to program the injection of fresh medium at 6.9 kilopascals for six hours to wells one and two, followed by the medium containing antibiotic at 6.9 kilopascals or six hours to well three, and finally the fresh medium at 6.9 kilopascals for 24 hours to wells four and five.

Perform microscopy imaging in time-lapse mode with one frame every 15 minutes using transmitted light and the excitation light source for the fluorescent reporter, and start the microfluidic program. Open the ImageJ or Fiji software on the computer, and drag the hyperstack time-lapse microscopy images into the Fiji loading bar. Use Image, followed by Color, and then Make Composite to fuse the different channels of the hyperstack.

Use Image, Color, and then Arrange Channels if the channels do not correspond to the desired color. Open the MicrobeJ plugin, and detect the bacterial cells using the manual editing interface. Delete the automatically detected cells, and manually outline the persister cells of interest frame by frame.

After detection, use the Result icon in the MicrobeJ manual editing interface to generate a ResultJ table. Use the ResultJ table to gain insights into different parameters of interest of the single-cell analysis, and save the ResultJ file. The MIC of ofloxacin for both strains was determined to be 0.06 micrograms per milliliter, indicating that the hupA-mCherry fusion did not affect the sensitivity of ofloxacin in comparison with the isogenic wild-type strain.

The spot assay showed isolated clones at appropriate dilutions over time, for example, 10 to the negative fifth dilution at time zero. In the time-kill assay, a typical biphasic curve was observed where the first slope reflects the rapid killing of the non-persister population. The second phase showed a slower killing rate, revealing the presence of drug-tolerant persister cells.

Notably, the time-kill curve shows that the hupA-mCherry fusion protein did not affect the time-kill kinetics. In the microfluidic experiment, the first growth phase indicates that cells were viable and dividing before the ofloxacin treatment. After this first growth phase, cell division was blocked as soon as the antibiotic reached the cells.

After ofloxacin treatment, on perfusion with fresh medium, the vast majority of the cells were unable to resume growth. In contrast, a small subpopulation could elongate and generate filamentous cells, which are defined as the persister cells. The dividing persister filament generated multiple daughter cells, most of which started to grow and divide similarly to untreated cells.

The increase in cell length correlated with an increase in the total mCherry fluorescence intensity, which reflects replication restart and an increase in nucleoid abundance. When using a strain containing an intracellular reporter to study persistence, it is imperial that the presence of the reporter does not interfere with growth, MIC, and killing as compared to a wild-type strain. The procedure described here can be applied to other conditions and bacteria species to monitor cellular responses to changing environments or stresses.

By using other fluorescent reporters in an identical setup, new insights into the physiology of persister cells can be probed.