Name: assessment of intestinal transcytosis of neonatal escherichia coli bacteremia isolates
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Description: Watch this Scientific Journal Video about Assessment of Intestinal Transcytosis of Neonatal Escherichia coli Bacteremia Isolates at JoVE.com

This work was supported by a Sarah Morrison student grant issued by the University of Missouri-Kansas City School of Medicine to A.I.

NOTE: Perform all the manipulations of the T84 cells, bacteria, plates, and reagents in a Biosafety Level 2 (BSL-2) safety cabinet to avoid contamination. Use separate areas and incubators for all the work involving sterile T84 cells, infected T84 cells, and E. coli. The clinical E. coli isolates tested with the methods described here were obtained following the guidelines of the Institutional Review Board at our institution1,16.
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<ol>
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This method is derived from techniques used in gastroenterology and infectious disease20. In vitro models of the intestinal epithelial barrier have been used to elucidate the mechanisms by which the luminal contents interact with this relevant component of innate immunity6,8. The host-pathogen interactions of invasive neonatal E. coli have also been separately characterized through genetic analysis, studies of antimicrobi...

Escherichia coli is the most common cause of early-onset sepsis in newborns1,2,3. The mortality rate of neonatal E. coli bacteremia can reach 40%, and meningitis is a possible complication that is associated with severe neurodevelopmental disabilities2. The ingestion of maternal E. coli strains by the newborn can produce neonatal bacteremia; this process has been replicated in animal models2,4. Once ingested, pathogenic bacteria travel from the neonatal gut lumen across the intestinal barrier and enter the bloodstream, causing septicemia. Neonatal invasive E. coli strains that produce bacteremia vary in their ability to invade intestinal epithelial cells1,5. However, their ability to transcytose the intestinal epithelium after invasion has not been completely characterized.
This intestinal transcytosis model is a useful in vitro method to emulate bacterial passage across the intestinal epithelium. The overall goal of the methods presented in this manuscript is to compare the ability of neonatal E. coli isolates to transcytose the intestinal epithelium. The model described here utilizes T84 cells, which are immortalized human intestinal adenocarcinoma cells6,7. T84 cells are grown to confluence on a semipermeable membrane with two separate compartments. The rationale for using this technique is that, as happens in vivo, these intestinal cells polarize and develop mature tight junctions6,8. The side in contact with the membrane becomes the basal side. The opposite side of the cells becomes the apical side, resembling the intestinal lumen where ingested pathogens adhere and invade. The transwell membrane is permeable to bacteria, but the polarized intestinal cells form tight junctions, which impair bacterial paracellular movement9. Thus, this method provides the advantage of a controlled in vitro environment utilizing a human cell line to study the process of bacterial transcytosis, including the transcellular route. While other methods exist to investigate the transcytosis of bacteria across the intestinal epithelium, the transwell method presented here provides greater ease and accessibility. Alternative techniques, such as those utilizing ex vivo samples set up in Ussing chamber systems, are available. However, they utilize tissue specimens that may not be easily accessible, particularly if the research intends to study human physiology10. Intestinal organoids represent another example of an in vitro alternative for studying host-bacteria interactions11. While organoid monolayers can also be used in the transwell system to study bacterial transcytosis, they require the isolation and growth of stem cells and the use of specific growth factors to induce differentiation12. Thus, their use is more time-consuming and associated with greater costs as compared to the transwell method described in this manuscript.
The assessment of bacterial passage across the intestinal epithelium using this in vitro transwell system has been successfully performed for various pathogens. These studies have shown the utility of the transwell system using T84 cells to characterize the transcytosis of bacteria across the polarized intestinal epithelium13,14,15. However, the application of this transwell method to compare the transcytosis ability of bacteremia-producing neonatal E. coli strains has not been described in detail. This manuscript provides other researchers with a standard transwell protocol that is reliable and easy to use and does not require resources that are too expensive.
To compare the ability of neonatal invasive E. coli strains to transcytose the intestinal epithelium, the apical side of the intestinal epithelial monolayer can be infected with a known number of bacterial cells. After incubation, the medium on the basal side of the epithelium can be collected and the bacteria quantified to determine the amount of bacterial transcytosis over time. In this manuscript, the methods presented are utilized to study the transcytosis ability of neonatal E. coli clinical strains recovered from newborns hospitalized with bacteremia. The inclusion criteria for the selection of these neonatal clinical isolates for transcytosis studies have been published previously1,2,16. When this method is performed using different E. coli strains, their transcytosis abilities can be compared. Through this process, the intestinal transcytosis model provides valuable data to characterize the virulence factors of E. coli that contribute to the multistep process that culminates in the development of neonatal bacteremia.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10,000 U/ mL Penicillin/Streptomycin Mixture</td>
			<td>Fisher Scientific</td>
			<td>15-140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL sterile conical tubes</td>
			<td>MidSci</td>
			<td>C15B</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL microcentrifuge tubes</td>
			<td>Avant</td>
			<td>AVSS2000</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL sterile polypropylene conical tubes</td>
			<td>Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Aspirator</td>
			<td>Corning</td>
			<td>4930</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety Cabinets</td>
			<td>Labconco</td>
			<td>30441010028343</td>
			<td>Three of these are used in the method: one for sterile tissue work, one for infected tissue work, and one for bacterial work.</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Sorvall</td>
			<td>Legend RT</td>
			<td></td>
		</tr>
		<tr>
			<td>Disposable inoculation loops</td>
			<td>Fisherbrand</td>
			<td>22363605</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Gibco</td>
			<td>11965-084</td>
			<td></td>
		</tr>
		<tr>
			<td>Epithelial Volt/Ohm Meter</td>
			<td>World Precision Instruments</td>
			<td>EVOM</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Fisher Scientific</td>
			<td>10437028</td>
			<td></td>
		</tr>
		<tr>
			<td>Ham&#39;s F-12 Nutrient Mixture</td>
			<td>Gibco</td>
			<td>11765-047</td>
			<td></td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Sigma Aldrich, Bright Line</td>
			<td>Z359629</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator shaker</td>
			<td>New Brunswick</td>
			<td>Innova 4080</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubators</td>
			<td>Thermo Scientific</td>
			<td>51030284</td>
			<td>Three of these are used in the method: one for sterile tissue culturing, one for infected tissue culturing, and one for bacterial incubation.</td>
		</tr>
		<tr>
			<td>Lysogeny broth</td>
			<td>Difco</td>
			<td>244610</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysogeny broth agar</td>
			<td>IBI Scientific</td>
			<td>IB49101</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon Eclipse TS2R Microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Unico</td>
			<td>1100RS</td>
			<td></td>
		</tr>
		<tr>
			<td>T84 Intestinal Cells</td>
			<td>American Tissue Culture Collection</td>
			<td>CCL248</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture inserts, with polyethylene trephthalate membrane, 3 &#181;m pores,&#160; 24 well format</td>
			<td>Falcon</td>
			<td>353096</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture plate, 24 wells</td>
			<td>Falcon</td>
			<td>353504</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue stain</td>
			<td>Fisher Scientific</td>
			<td>T10282</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of intestinal transcytosis of neonatal escherichia coli bacteremia isolates

Newborns ingest maternal E. coli strains that colonize their intestinal tract around the time of delivery. E. coli strains with the ability to translocate across the gut invade the newborn's bloodstream, causing life-threatening bacteremia. The methodology presented here utilizes polarized intestinal epithelial cells grown on semipermeable inserts to assess the transcytosis of neonatal E. coli bacteremia isolates in vitro. This method uses the established T84 intestinal cell line that has the ability to grow to confluence and form tight junctions and desmosomes. After reaching confluence, mature T84 monolayers develop transepithelial resistance (TEER), which can be quantified using a voltmeter. The TEER values are inversely correlated with the paracellular permeability of extracellular components, including bacteria, across the intestinal monolayer. The transcellular passage of bacteria (transcytosis), on the other hand, does not necessarily alter the TEER measurements. In this model, bacterial passage across the intestinal monolayer is quantified for up to 6 h post-infection, and repeated measurements of TEER are made to monitor the paracellular permeability. In addition, this method facilitates the use of techniques such as immunostaining to study the structural changes in tight junctions and other cell-to-cell adhesion proteins during bacterial transcytosis across the polarized epithelium. The use of this model contributes to the characterization of the mechanisms by which neonatal E. coli transcytose across the intestinal epithelium to produce bacteremia.

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64241/64241fig01.jpg" /> 
Figure 1: T84 TEER over time. As the T84 cell layer matures on the insert, the electrical resistance of the monolayer increases. At a TEER of at least 1,000 &#937;&#183;cm2, the cell layer is sufficiently developed to decrease the paracellular bacterial transport and allow the measurement of primarily transcellular bacterial transit....

Watch this Scientific Journal Video about Assessment of Intestinal Transcytosis of Neonatal Escherichia coli Bacteremia Isolates at JoVE.com

Assessment of Intestinal Transcytosis of Neonatal Escherichia coli Bacteremia Isolates

Escherichia coli&#160;causes sepsis in neonates who ingest the bacteria around the time of birth. The process involved in&#160;E. coli&#8217;s&#160;ability to travel from the enteric tract to the bloodstream is poorly understood. This&#160;in vitro&#160;model assesses the ability of&#160;E. coli&#160;strains to travel through the intestinal epithelial cells.

Assessment of Intestinal Transcytosis of Neonatal Escherichia coli Bacteremia Isolates

Newborns ingest maternal E. coli strains that colonize their intestinal tract around the time of delivery. E. coli strains with the ability to translocate ...

This method allows comparing the transcytosis abilities of various equalized strains across polarized intestinal epithelial cells with mature junctions. This is a relatively easy technique that does not require extensive use of resources or time, and allows investigating an important step in the pathogenesis of neonatal E.coli invasive disease. Demonstrating the procedure will be Josh Wheatley, a research assistant in my laboratory.Working inside a biosafety cabinet, seed T84 cells into polyethylene terephthalate membrane cell culture Transwell inserts. In a 24 well plate designed to hold Transwell inserts, fill the desired number of collecting wells with one milliliter of tissue culture medium or TCM with antibiotics. Seed these inserts with one times 10 to the fifth T84 cells suspended in 500 microliters of TCM with antibiotics.Approximately 48 hours after seeding, verify with light microscopy if the monolayers have started to become confluent. Every two days after seeding, measure and record the transepithelial electrical resistance or TEER using an epithelial volt per OM meter or EVOM. Before measuring the TEER, decontaminate the electrical probe by submerging it in five milliliters of 70%ethanol for 10 to 15 minutes.Remove the probe, shake off the excess ethanol, and let it air dry inside the biosafety cabinet for 10 minutes. Test the EVOM and probe by placing the dry decontaminated probe in a sterile well containing one milliliter of TCM with antibiotics with a sterile insert inside containing 500 microliters of TCM with antibiotics. Ensure that the EVOM reading is less than 200 OM.Record this blank value to use it in the later resistance calculations.Remove the probe from the tube of TCM with antibiotics. Gently lower the probe into the first insert with the long electrode in the collecting well. Allow the electrode to touch the bottom of the collecting well, but do not push down as this may disrupt the epithelial monolayer and the short electrode inside the insert.When finished, decontaminate the probe in the ethanol. Subtract the blank resistance value from each value obtained from each insert containing T84 cells. Then multiply the resulting resistance for each insert by the area of the bottom of each insert to obtain the final TEER measurement.Once the TEER reaches 1000 OMs per square centimeter, the epithelial monolayer is mature and ready for infection assays. As the TEER matures, provide the cells with a fresh medium every one to two days. In a new 24 well plate, add one milliliter of TCM with antibiotics to one well for each seeded insert.Using sterile forceps, carefully transfer the inserts to the newly replenished wells. Replace the media in the inserts. Remove the old media from the inserts by tilting the plate, and gently remove the media with a pipette tip along the side of the insert.Add 500 microliters of TCM with antibiotics to the inserts and visualize the monolayer to verify that it remains intact. The day before the experiment, measure and record the TEER. Replace the medium with one milliliter of TCM without antibiotics in the plate well, and 500 microliters in the insert.Take a labeled 15 milliliter conical tube with five milliliters of sterile lysogeny broth or LB, and use a sterile lube to inoculate the broth with one colony from one Escherichia coli strain. Incubate the culture overnight with the cap from the tube loosened. The next day, add 250 microliters from overnight LB culture to 25 milliliters of TCM without antibiotics in a 50 milliliter conical tube.Incubate the tube for two hours. Measure the TEER across each insert. Record these as the TEERs at the time T to zero hour.Move the inserts to wells in a new plate and change the media. Fill the new collecting wells with 500 microliters and inserts with 400 microliters of TCM without hand antibiotics. Keep the inserts inside the tissue culture incubator until the time of infection.After two hours, remove the morning bacterial cultures from the shaker and centrifuge. Resuspend the pellet in TCM without antibiotics. Then use a spectrophotometer to adjust the optical density or OD to 0.7 or 0.9 and further dilute to a concentration of 1 million colony forming units or CFU per milliliter.Infect each insert with 100 microliters of the OD adjusted inoculum. Note the time and record it as time zero hour. Place the inoculum bacterial suspension to quantity CFU per milliliter using the track dilution method plating 10 microliter aliquot on a square LB auger plate.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.During the proliferation of the sterile T84 cells, the TEERs across the T84 monolayers continuously rise over time, surpassing 1000 OMs per square centimeter approximately after seven days from seeding. Transcytosis of neonatal E.coli isolates over time is shown here. The comparisons of the mean transcytosis values demonstrated significant differences between the neonatal E.coli isolates one versus two at two hours post-infection.In contrast, the nonpathogenic E.coli strain DH5 alpha underwent minimal transcytosis. TEER results before and after infection with two neonatal E.coli clinical isolates with different abilities to transcytose intestinal epithelial cells are shown here. The rate and the amount of transcytosis vary among strains but the TEER significantly increases after infection with both pathogenic strains compared to the non-infected inserts.Performing TEER measurements in a consistent manner is crucial for obtaining reproducible results with this method. Following strict sterile techniques to avoid contamination is also very important.

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