Amikacin Protection Assay for Quantification and Visualization of Escherichia coli Cell Invasion

We focus on characterizing how adherent invasive E.coli invade intestinal epithelial cells and identifying bacterial factors that contribute to this process. Gentamycin-resistant isolates limit traditional invasion assays. This amikacin-based protocol allows for accurate quantification of intracellular bacteria.

To begin, gather all reagents and media required for the invasion assay. Next, retrieve the frozen glycerol stock of adherent-invasive Escharicia colli, or AIEC, strain from the 80 degrees Celsius freezer. Using a sterile wooden stick or pipette tip, gently scrape bacteria from the stalk and streak it onto a Luria-Bertani, or LB, auger plate to obtain single colonies.

Incubate the plate at 37 degrees Celsius overnight. The next day, using a sterile wooden stick or pipette tip, pick a single colony from the auger plate. Inoculate the colony into 3 milliliters of LB broth and incubate the culture at 37 degrees Celsius at 200 revolutions per minute overnight.

Then perform a 1 to 10 dilution of the overnight culture in 1 milliliter of LB broth. Measure the optical density at 600 nanometers of the diluted culture using a spectrophotometer. Transfer 100 microliters of the overnight culture into a 1.5 milliliter microcentrifuge tube and centrifuge at 6, 000 G for 10 minutes at 4 degrees Celsius.

Aspirate the supernatant carefully without disturbing the cell pellet and re-suspend the pellet in 1 milliliter of serum and antibiotic-antimycotic-free media. Calculate the required volume of bacterial suspension to achieve a multiplicity of infection or MOI of 10 using the provided formula. To prepare the infection media, transfer the calculated volume of bacterial suspension into a sterile 1.5 milliliter microcentrifuge tube and make up the volume to 1 milliliter with serum and antibiotic-antimycotic-free media.

Mix gently by pipetting up and down several times. Prepare a 96-well plate to determine the colony forming units per milliliter of the diluted overnight bacterial culture by performing tenfold serial dilutions in sterile 1X PBS. Transfer 200 microliters of the diluted overnight culture into the first well of the plate.

Pipette 20 microliters of the culture into the next row containing 180 microliters of 1X PBS. Mix by pipetting up and down 10 times and repeat the dilution process until reaching 10 to the power of 6 using a fresh pipette tip each time. Now plate 10 microliters of spot dilutions in triplicate from the 10 to the power of 2 to 10 to the power of 6 dilutions onto LB auger plates.

Allow the spots to absorb into the auger near a flame. Incubate the plates overnight at 27 to 30 degrees Celsius to prevent colony overgrowth and obtain clear individual colonies. Remove the healthy confluent Caco-2 epithelial cell monolayers cultured in a collagen coated 24-well culture plate from the incubator and aspirate the complete media from each well.

Pipette one milliliter of serum and antibiotic-antimycotic-free media into each well. Aspirate the media to remove traces of media and repeat the wash one more time for a total of two washes. Now add 1 milliliter of the previously prepared infection media containing AIEC into each well of the 24-well plate containing the Caco-2 cells.

Centrifuge the 24-well plate at 500G for 15 minutes at room temperature to synchronize bacterial contact with the epithelial monolayer and incubate the plate at 37 degrees Celsius with 5%carbon dioxide for three hours to allow bacterial invasion to occur. Prepare antibiotic-containing media by adding 240 micrograms per milliliter of amikacin to pre-warmed serum and antibiotic-antimycotic-free media. After the infection period, aspirate the media from the Caco-2 cell monolayers and wash each well with 1 milliliter of serum and antibiotic-antimycotic-free media.

Aspirate the media and repeat the wash once more for a total of two washes. Then pipette one milliliter of 240 micrograms per milliliter amikacin containing media into each well. In parallel, treat another set of cells with 100 micrograms per milliliter gentamicin for comparison and incubate the cells at 37 degrees Celsius with 5%carbon dioxide for 1 hour to eliminate extracellular bacteria.

After incubation, aspirate the media from each well. Add 1 milliliter of PBS to wash away adherent bacteria. Aspirate the PBS and repeat the wash one more time for a total of two washes.

Now add 200 microliters of 1%Triton X100 diluted in PBS into each well and incubate on an orbital shaker at 90 revolutions per minute for 10 minutes to lyse epithelial cells. Pipette up and down approximately 30 times per well to detach the cells from the plate. Next, perform tenfold serial dilutions of the cell lysates in a 96-well plate using PBS.

Plate 10 microliters of spot dilutions in triplicate from the undiluted to 10 to the power of 3 dilutions onto LB auger plates. After the spots dry, incubate the plates overnight at 27 to 30 degrees Celsius to obtain clear individual colonies. Following incubation, remove the plates and count well-isolated colonies from the appropriate spot dilutions containing more than seven colonies per spot across the triplicates.

Finally, calculate the inoculum and retrieved colony-forming units per milliliter and percent invasion using the equations. Treatment with either gentamicin or amikacin following infection of Caco-2 cell monolayers with the invasive AIEC strain NRG857c, or the non-invasive Escherichia coli DH5 alpha control strain resulted in similar invasion profiles. These findings indicated that amikacin was as effective as gentamicin in eliminating extracellular bacteria and accurately reflected intracellular bacterial burden.

Treatment of gentamicin-resistant clinical Escherichia coli isolates obtained from patients with inflammatory bowel disease with gentamicin resulted in highly variable bacterial recovery values ranging from around 21 to 2, 788%In contrast, amikacin treatment dramatically reduced bacterial recovery across all isolates, demonstrating its effectiveness and enabling more reliable invasion measurements. This protocol enables accurate quantification of intracellular bacteria and assessment of bacterial invasion in epithelial cells. Additional methods such as immunofluorescence microscopy can be used to further validate bacterial invasion and validate intracellular localization.

Future studies can identify novel virulence factors and investigate host pathways that are targeted during adherent invasive E.coli infection.