Assessing Transcytosis of E. coli Across Intestinal Epithelial Monolayers

Begin with an E. coli culture isolated from newborns with bloodstream infections.

Centrifuge to pellet the bacteria, discard the supernatant, and resuspend the pellet in an antibiotic-free medium.

Take a plate with a transwell insert containing an intestinal epithelial cell monolayer.

Measure electrical resistance across the cell layer with a probe to confirm monolayer integrity.

Add the bacterial suspension into the insert and incubate.

Bacteria adhere to epithelial cells, are internalized within vesicles, and are transported across the cells to the lower well via a process called transcytosis.

Measure electrical resistance again; a stable value confirms an intact monolayer during transcytosis.

Transfer the insert to a fresh well and collect the medium from the previous lower well.

Plate the collected medium on an agar plate and Incubate to develop the bacterial colonies.

Count the colonies to determine the extent of E. coli transcytosis across the intestinal epithelial barrier.

Every 30 minutes following the inoculation, fill new wells with 500 microliters of TCM without antibiotics, and transfer the inserts to these new wells. Collect the media from the used collecting well for each insert into a separate labeled tube and place the tube on ice. Return the Transwell plate to the incubator between time points.

For each insert, combine the collected media from different time points and vortex briefly. Plate the collected media on LB auger plates using the track dilution method to quantify the number of bacteria transcytose in the first two hours of the experiment. Collect the media at four and six hours.

Additionally, at six hours, plate the media from the control wells. At six hours, measure and record the TEER. After overnight incubation, count the bacterial colonies manually on the track dilution LB plates.