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.
1. Preparing transcytosis inserts with T84 cells (approximately 1-2 weeks before the experiment)
<ol>
	<li>Grow American Type Culture Collection (ATCC) T84 cells in tissue culture medium (TCM + antibiotics) consisting of Dulbecco&#39;s Modified Eagle Medium:Ham&#39;s F-12 nutrient mixture (1:1, final concentration: 50% each), 5% fetal bovine serum, and 1% (100 U/mL) penicillin/streptomycin dual antibiotic mixture. Incubate the cells at 37 &#176;C with 5% CO2. 
	NOTE: A variation of this medium formulation without penicillin/streptomycin (TCM w/o antibiotics) is used for the later steps (section 2 and onward) in the procedure. Ensure that the correct formulation is used for each step.</li>
	<li>Working inside a biosafety cabinet (BSC), seed the T84 cells into polyethylene terephthalate membrane cell culture transwell inserts with 3 &#181;m pores made for 24-well plates. Include transwell insert replicates for each desired experimental condition plus uninfected controls to monitor for possible contamination.
	<ol>
		<li>In a 24-well plate designed to hold transwell inserts, fill the desired number of collecting wells with 1 mL of TCM + antibiotics.</li>
		<li>In each well, place one transwell insert.</li>
		<li>Seed these inserts with 1 x 105 T84 cells suspended in 500 &#181;L of TCM + antibiotics. Quantify the number of cells using a hemocytometer with trypan blue stain or an automated cell counter17.</li>
		<li>Incubate the transwell plates containing seeded inserts in the same conditions that the cells were grown in.</li>
		<li>Verify with light microscopy that the monolayers have started to become confluent after seeding the insert, approximately 48 h after seeding.</li>
	</ol>
	</li>
	<li>Every 2 days after seeding, measure and record the transepithelial electrical resistance (TEER) using an epithelial volt/ohm meter (EVOM) to assess the maturity of the monolayer. Once the inserts reach a TEER of at least 1,000 &#937;&#183;cm2, they are deemed ready for the assay18. 
	NOTE: The inserts typically take 7-10 days to reach this TEER after seeding. The T84 cells will show 100% confluence under light microscopy once this resistance is reached.
	<ol>
		<li>Store the EVOM probe with the electrodes submerged in 0.15 M KCl when not in use.</li>
		<li>Prior to measuring the TEER, decontaminate the electrode probe by submerging it in 5 mL of 70% ethanol in a 50 mL conical tube for 10-15 min. Remove the probe, shake off the excess ethanol, and allow it to air-dry inside the BSC for 10 min. Retain the tube of ethanol.</li>
		<li>Test the EVOM and probe by placing the dry decontaminated probe in a sterile well containing 1 mL of TCM + antibiotics with a sterile insert inside containing 500 &#181;L of TCM + antibiotics. Ensure that the EVOM reading is &#60;200 &#937;. Record this blank value to use it in the later resistance calculations described in step 1.3.6.</li>
		<li>Remove the probe from the tube of TCM + antibiotics. Retain this tube for the storage of the probe throughout the experiment.</li>
		<li>Gently lower the probe into the first insert with the long electrode in the collecting well and the short electrode inside the insert. Allow the long electrode to touch the bottom of the collecting well, but do not push down as this may disrupt the epithelial monolayer.</li>
		<li>Repeat this process to measure and record the resistance in Ohms (&#937;) for every insert. When finished, decontaminate the probe in the ethanol by submerging it for another 10-15 min. Then, move the decontaminated probe back to the KCl solution for storage. Subtract the blank resistance obtained in step 1.3.3 from each value obtained from each insert containing T84 cells. Multiply the resulting resistance (&#937;) for each insert by the area of the bottom of each insert (cm2) to obtain the final TEER measurement (&#937;&#183;cm2).</li>
		<li>Once the TEER reaches at least 1,000 &#937;&#183;cm2, the epithelial monolayer is mature and ready for infection assays.</li>
	</ol>
	</li>
	<li>As the TEER matures, provide the cells with fresh medium every 1-2 days.
	<ol>
		<li>In a new 24-well plate, add 1 mL of TCM + antibiotics to one well for each seeded insert being prepared.</li>
		<li>Using sterile forceps, carefully transfer the inserts to the newly replenished wells.</li>
		<li>Replace the media in the inserts.
		<ol>
			<li>Remove the old media from the inserts by tilting the plate and using an in-house vacuum aspirator to gently remove the media with a pipette tip along the side of the insert. The aspirator allows the regulation of low-level suction to prevent the disruption of the cells. Do not allow the pipette tip to touch the bottom of the insert, as this will disrupt the developing epithelial monolayer.</li>
			<li>Add 500 &#181;L of TCM + antibiotics to the inserts. Visualize the monolayer with light microscopy to verify that it remains intact.</li>
		</ol>
		</li>
		<li>Every 1-2 days, measure the TEER across each insert as described above in steps 1.3.2-1.3.6.</li>
	</ol>
	</li>
</ol>
2. Preparing the T84 cells 1 day before the experiment using TCM w/o antibiotics
<ol>
	<li>Measure and record the TEER the day before the experiment.</li>
	<li>Replace the TCM in the same manner as during the prior cell preparation and maintenance. However, TCM w/o antibiotics is used instead in preparation for the infection (1 mL in the plate well and 500 &#181;L in the insert).</li>
</ol>
3. E. coli cultures (started 1 day before the experiment)
CAUTION: Use Biosafety Level 2 (BSL-2) precautions when working with pathogenic clinical E. coli strains.
<ol>
	<li>Take a labeled 15 mL conical tube with 5 mL of sterile lysogeny broth (LB), and use a sterile loop to inoculate the broth with one colony from one bacterial strain (E. coli). Repeat this process, creating one overnight culture tube for each strain to be tested.</li>
	<li>Incubate the overnight culture, with the caps of the tubes loosened, in an incubator shaker (250 rpm, 37 &#176;C).</li>
</ol>
4. Preparing the E. coli inoculum, epithelial cells, and materials (on the morning of the experiment)
NOTE: Use TCM w/o antibiotics warmed up to 37 &#176;C from this point on.
<ol>
	<li>Add 250 &#181;L from each overnight LB culture to 25 mL of TCM w/o antibiotics in a 50 mL conical tube (one per individual strain). Keep the caps of the tubes loosened. Place these new culture tubes in the shaker at the same settings (250 rpm, 37 &#176;C) for exactly 2 h. Perform the remaining substeps while waiting.</li>
	<li>Measure the TEER across each insert, as described in steps 1.3.2-1.3.6. Record these as the TEERs at time (t) = 0 h.</li>
	<li>Move the inserts to wells in a new plate, and change the media using the technique described in step 1.4.3. However, this time, fill the new collecting wells with 500 &#181;L of TCM w/o antibiotics, and fill the inserts with 400 &#181;L of TCM w/o antibiotics. Keep the inserts inside the tissue culture incubator until the time of infection.</li>
	<li>Set out a sufficient number of square LB agar plates to warm to room temperature (RT) for later plating and bacterial quantification.</li>
</ol>
5. Inoculation of the cells (start of the experiment)
<ol>
	<li>After exactly 2 h, remove the morning bacterial cultures from the shaker, and centrifuge them for 10 min (1,900 x g, 4 &#176;C). 
	NOTE: For all the following steps, keep all the bacterial suspensions on ice to minimize growth.</li>
	<li>Resuspend the bacteria pellet in TCM w/o antibiotics. Use a spectrophotometer to adjust the optical density (OD) to 0.7-0.9, and further dilute with TCM w/o antibiotics to a concentration of 1 x 106 colony forming units (CFU)/mL (approximately 1:100 dilution). Use this bacterial suspension to infect each insert with 1 x 105 CFU per 100 &#181;L volume.</li>
	<li>Label the transwell plate, and infect each insert with 100 &#181;L of the OD-adjusted inoculum (total of 1 x 105 CFU per insert). The assay has now begun. Note the time, and record it as t = 0 h.</li>
	<li>Plate the inoculum bacterial suspension to quantify the CFU/mL using the track dilution method, plating 10 &#181;L aliquots on square LB agar plates19.</li>
</ol>
6. Quantifying transcytosis
<ol>
	<li>Every 30 min following inoculation, fill new wells with 500 &#181;L of TCM w/o antibiotics. Transfer the inserts to these new wells using a different set of sterile forceps for each different bacterial strain.</li>
	<li>Collect the media from the used collecting well for each insert into separate labeled tubes. Place these tubes on ice. Return the transwell plates to the incubator in between time points.</li>
	<li>For each insert, combine the collected media from t = 0.5 h, t = 1 h, t = 1.5 h, and t = 2 h, and vortex briefly. Plate the collected media on LB agar plates using the track dilution method to quantify the amount of bacteria transcytosed in the first 2 h of the experiment. 
	NOTE: Retrieving the bacteria every 30 min and keeping them on ice ensures the bacterial growth in the collecting wells is minimized and measurements are done on predominantly transcytosed bacteria.</li>
	<li>Place the labeled track dilution LB agar plates in the bacterial incubator at 37 &#176;C without supplemental CO2, and return the T84 transwell plate to the tissue culture incubator.</li>
	<li>At t = 4 h, repeat step 6.3 by combining the collected media from t = 2.5 h, t = 3 h, t = 3.5 h, and t = 4 h.</li>
	<li>At t = 6 h, repeat step 6.3 by combining the collected media from t= 4.5 h, t = 5 h, t = 5.5 h, and t = 6 h. Additionally, at t = 6 h, plate the media from the control wells.</li>
</ol>
7. End of the experiment
<ol>
	<li>Measure and record the TEER at the end of the experiment, t = 6 h. Use the procedure described in steps 1.3.2-1.3.6.</li>
	<li>Decontaminate the probe by submerging it in 70% ethanol for 10-15 min. Discard the inserts, or save/process them for additional applications, if desired. Allow the LB agar plates to incubate overnight, and disinfect and/or safely dispose of all the other used materials.</li>
	<li>After overnight incubation, count the bacterial colonies manually on the track dilution LB plates to determine the inoculum amount and the amount of E. coli transcytosis. Ensure that the control plates do not show any bacterial growth.</li>
</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><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&#039;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>EVOM2</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Fisher Scientific</td><td>10437028</td><td></td></tr><tr><td>Ham&#039;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 &amp;micro;m pores,&amp;nbsp; 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 ...

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Research

JoVE Journal

Immunology and Infection

1.2K Views.  University of Missouri-Kansas City School of Medicine. Deze methode maakt het mogelijk om de transcytosecapaciteiten van verschillende geëgaliseerde stammen te vergelijken over gepolariseerde intestinale epitheelcellen met volwassen juncties. Dit is een relatief eenvoudige techniek die geen uitgebreid gebruik van middelen of tijd vereist en het mogelijk maakt om een belangrijke stap in de pathogenese van neonatale E.coli invasieve ziekte te onderzoeken. De procedure wordt gedemonstreerd door Josh Wheatley, een onderzoeksassistent in mijn laborat...

Video: Assessment of Intestinal Transcytosis of Neonatal Escherichia coli Bacteremia Isolates

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

Biochemistry

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell culture assays are performed under aerobic conditions, these in vitro models may not accurately represent the in vivo environment in which the host-pathogen interactions take place. We have developed an in vitro model of infection that permits the coculture of bacteria and host cells under different medium and gas conditions. The Vertical Diffusion Chamber (VDC) model mimics the conditions in the human intestine where bacteria will be under conditions of very low oxygen whilst tissue will be supplied with oxygen from the blood stream. Placing polarized intestinal epithelial cell (IEC) monolayers grown in Snapwell inserts into a VDC creates separate apical and basolateral compartments. The basolateral compartment is filled with cell culture medium, sealed and perfused with oxygen whilst the apical compartment is filled with broth, kept open and incubated under microaerobic conditions. Both Caco-2 and T84 IECs can be maintained in the VDC under these conditions without any apparent detrimental effects on cell survival or monolayer integrity. Coculturing experiments performed with different C. jejuni wild-type strains and different IEC lines in the VDC model with microaerobic conditions in the apical compartment reproducibly result in an increase in the number of interacting (almost 10-fold) and intracellular (almost 100-fold) bacteria compared to aerobic culture conditions1. The environment created in the VDC model more closely mimics the environment encountered by C. jejuni in the human intestine and highlights the importance of performing in vitro infection assays under conditions that more closely mimic the in vivo reality. We propose that use of the VDC model will allow new interpretations of the interactions between bacterial pathogens and host cells.

Enteric Bacterial Invasion Of Intestinal Epithelial Cells In Vitro Is Dramatically Enhanced Using a Vertical Diffusion Chamber Model

Performing cell culture assays for investigating bacterial adhesion and invasion under aerobic conditions is usually unrepresentative of the in vivo environment. A Vertical Diffusion Chamber model allows study of interactions of the human pathogen Campylobacter jejuni with intestinal epithelial cells under more in vivo-like conditions, resulting in enhanced bacterial invasion.

The interactions of bacterial pathogens with host cells have been investigated extensively using in vitro cell culture methods. However as such cell ...

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils.&#160;This process has the potential to be destructive to tissues if excessive or held in an unresolved state.&#160; Cocultured in vitro models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration.&#160;This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil.&#160;Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface.&#160;The in vitro model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa (PAO1).&#160;Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli (MC1000).&#160; The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls.&#160;This in vitro model system can be manipulated and applied to other mucosal surfaces.&#160;Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections.&#160;A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.

In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration

Neutrophil trans-epithelial migration in response to mucosal bacterial infection contributes to epithelial injury and clinical disease.&#160;An in vitro model has been developed that combines pathogen, human neutrophils, and polarized human epithelial cell layers grown on transwell filters to facilitate investigations towards unraveling the molecular mechanisms orchestrating this phenomenon.

Mucosal surfaces serve as protective barriers against pathogenic organisms.&#160;Innate immune responses are activated upon sensing pathogen leading to ...

Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli

Conjugation represents one of the main mechanisms facilitating horizontal gene transfer in Gram-negative bacteria. This work describes methods for the study of the mobilization of naturally occurring conjugative plasmids, using two naturally-occurring plasmids as an example. These protocols rely on the differential presence of selectable markers in donor, recipient, and conjugative plasmid. Specifically, the methods described include 1) the identification of natural conjugative plasmids, 2) the quantification of conjugation rates in solid culture, and 3) the diagnostic detection of the antibiotic resistance genes and plasmid replicon types in transconjugant recipients by polymerase chain reaction (PCR). The protocols described here have been developed in the context of studying the evolutionary ecology of horizontal gene transfer, to screen for the presence of conjugative plasmids carrying antibiotic-resistance genes in bacteria found in the environment. The efficient transfer of conjugative plasmids observed in these experiments in culture highlights the biological relevance of conjugation as a mechanism promoting horizontal gene transfer in general and the spread of antibiotic resistance in particular.

Detection of Horizontal Gene Transfer Mediated by Natural Conjugative Plasmids in E. coli

Conjugation mediates horizontal gene transfer by mobilizing plasmid DNA across two different cells, facilitating the spread of beneficial genes. This work describes a widely-used method for the efficient detection of conjugative plasmid transfer, based on the use of differential markers in the conjugative plasmid, donor, and recipient to detect transconjugation.

Conjugation represents one of the main mechanisms facilitating horizontal gene transfer in Gram-negative bacteria. This work describes methods for the ...

Genetics

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal features of the natural infection. This protocol describes procedures for the establishment and evaluation of systemic infection due to neuropathogenic Escherichia coli K1 in the neonatal rat. Colonization of the gastrointestinal tract leads to dissemination of the pathogen along the gut-lymph-blood-brain course of infection and the model displays strong age dependency. A strain of E. coli O18:K1 with enhanced virulence for the neonatal rat produces exceptionally high rates of colonization, translocation to the blood compartment and invasion of the meninges following transit through the choroid plexus. As in the human host, penetration of the central nervous system is accompanied by local inflammation and an invariably lethal outcome. The model is of proven utility for studies of the mechanism of pathogenesis, for evaluation of therapeutic interventions and for assessment of bacterial virulence.

Non-Invasive Model of Neuropathogenic Escherichia coli Infection in the Neonatal Rat

Here, a procedure is described for the establishment of systemic infection in the neonatal rat with cultures of Escherichia coli K1. This non-invasive procedure permits colonization of the gastrointestinal tract, translocation of the pathogen to the systemic circulation, and invasion of the central nervous system at the choroid plexus.

Investigation of the interactions between animal host and bacterial pathogen is only meaningful if the infection model employed replicates the principal ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

A Neonatal Imaging Model of Gram-Negative Bacterial Sepsis

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a protocol for studying the pathogenesis of E. coli O1:K1:H7, a serotype responsible for high mortality rates in neonates. Our method utilizes intravital imaging of neonatal pups at different time points during the progression of infection. This imaging, paralleled by measurement of bacteria in the blood, inflammatory profiling, and tissue histopathology, signifies a rigorous approach to understanding infection dynamics during sepsis. In the current report, we model two infectious inoculums for comparison of bacterial burdens and severity of disease. We find that subscapular infection leads to disseminated infection by 10 h post-infection. By 24 h, infection of luminescent E. coli was abundant in the blood, lungs, and other peripheral tissues. Expression of inflammatory cytokines in the lungs is significant at 24 h, and this is followed by cellular infiltration and evidence of tissue damage that increases with infectious dose. Intravital imaging does have some limitations. This includes a luminescent signal threshold and some complications that can arise with neonates during anesthesia. Despite some limitations, we find that our infection model offers an insight for understanding longitudinal infection dynamics during neonatal murine sepsis, that has not been thoroughly examined to date. We expect this model can also be adapted to study other critical bacterial infections during early life.

Infection of neonatal mice with bioluminescent E. coli O1:K1:H7 results in a septic infection with significant pulmonary inflammation and lung pathology. Here, we describe procedures to model and further study neonatal sepsis using longitudinal intravital imaging in parallel with enumeration of systemic bacterial burdens, inflammatory profiling, and lung histopathology.

Neonates are at an increased risk of bacterial sepsis due to the unique immune profile they display in the first months of life. We have established a ...

Protocols for Analyzing Shigella Infection in Colonic Epithelial Cells

Epithelial Cell Infection Analyses with Shigella

The human-adapted enteric bacterial pathogen Shigella causes millions of infections each year, creates long-term growth effects among pediatric patients, and is a leading cause of diarrheal deaths worldwide. Infection induces watery or bloody diarrhea as a result of the pathogen transiting the gastrointestinal tract and infecting the epithelial cells lining the colon. With staggering increases in antibiotic resistance and the current lack of approved vaccines, standardized research protocols are critical to studying this formidable pathogen. Here, methodologies are presented to examine the molecular pathogenesis of Shigella using in vitro analyses of bacterial adherence, invasion, and intracellular replication in colonic epithelial cells. Prior to infection analyses, the virulence phenotype of Shigella colonies was verified by the uptake of the Congo red dye on agar plates. Supplemented laboratory media can also be considered during bacterial culturing to mimic in vivo conditions. Bacterial cells are then used in a standardized protocol to infect colonic epithelial cells in tissue culture plates at an established multiplicity of infection with adaptations to analyze each stage of infection. For adherence assays, Shigella cells are incubated with reduced media levels to promote bacterial contact with epithelial cells. For both invasion and intracellular replication assays, gentamicin is applied for various time intervals to eliminate extracellular bacteria and enable assessment of invasion and/or the quantification of intracellular replication rates. All infection protocols enumerate adherent, invaded, and/or intracellular bacteria by serially diluting infected epithelial cell lysates and plating bacterial colony forming units relative to infecting titers on Congo red agar plates. Together, these protocols enable independent characterization and comparisons for each stage of Shigella infection of epithelial cells to study this pathogen successfully.

Author Spotlight: Advancements in Understanding and Combatting Shigella Infections

The present protocol describes infection assays to interrogate Shigella adherence, invasion, and intracellular replication using in vitro epithelial cell lines.

The human-adapted enteric bacterial pathogen Shigella causes millions of infections each year, creates long-term growth effects among pediatric patients, ...

Testing Epithelial Permeability in Fetal Tissue-Derived Enteroids

Human fetal tissue-derived enteroids are emerging as a promising in vitro model to study intestinal injuries in preterm infants. Enteroids exhibit polarity, consisting of a lumen with an apical border, tight junctions, and a basolateral outer layer exposed to growth media. The consequences of intestinal injuries include mucosal inflammation and increased permeability. Testing intestinal permeability in vulnerable preterm human subjects is often not feasible. Thus, an in vitro fetal tissue-derived intestinal model is needed to study intestinal injuries in preterm infants. Enteroids can be used to test changes in epithelial permeability regulated by tight junction proteins. In enteroids, intestinal stem cells differentiate into all epithelial cell types and form a three-dimensional structure on a basement membrane matrix secreted by mouse sarcoma cells. In this article, we describe the methods used for establishing enteroids from fetal intestinal tissue, characterizing the enteroid tight junction proteins with immunofluorescent imaging, and testing epithelial permeability. As gram-negative dominant bacterial dysbiosis is a known risk factor for intestinal injury, we used lipopolysaccharide (LPS), an endotoxin produced by gram-negative bacteria, to induce permeability in the enteroids. Fluorescein-labeled dextran was microinjected into the enteroid lumen, and serial dextran concentrations leaked into the culture media were measured to quantify the changes in paracellular permeability. The experiment showed that apical exposure to LPS induces epithelial permeability in a concentration-dependent manner. These findings support the hypothesis that gram-negative dominant dysbiosis contributes to the mechanism of intestinal injury in preterm infants.

This protocol details the establishment of enteroids, a three-dimensional intestinal model, from fetal intestinal tissue. Immunofluorescent imaging of epithelial biomarkers was used for model characterization. Apical exposure of lipopolysaccharides, a bacterial endotoxin, using microinjection technique induced epithelial permeability in a dose-dependent manner measured by the leakage of fluorescent dextran.

Assessment of Intestinal Transcytosis of Neonatal Escherichia coli Bacteremia Isolates

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