Assessment of Intestinal Transcytosis of Neonatal Escherichia coli Bacteremia Isolates

Summary

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

Abstract

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.

Introduction

Escherichia coli is the most common cause of early-onset sepsis in newborns^1,2,3. 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 models^2,4. Once ingested, pathogenic bacteria travel from the neonatal gut lumen acro....

Protocol

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 institution^1,16.

Discussion

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 immunity^6,8. The host-pathogen interactions of invasive neonatal E. coli have also been separately characterized through genetic analysis, studies of antimicrobi.......

Materials

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