INVASION OF INTESTINAL EPITHELIAL CELLS IN VITRO BYESCHERICHIA COLI IS INFLUENCED BY MICROAEROPHILIC CONDITIONS AND THE PRESENCE OF ibe GENES.

Abstract

96 Enteric bacteria are an important cause of sepsis in neonates, children with gastrointestinal diseases (such as short bowel syndrome), immunocompromised hosts, and children with central venous catheters. It is unclear what microbial factors may mediate the translocation of enteric bacteria across the intestinal epithelial barrier. E. coli RS218 is a K1 encapsulated clinical isolate from the CSF of a newborn infant with meningitis, which has been used to elucidate mechanisms of invasion of brain microvascular endothelial cells. The aims of our study were to evaluate (1) the ability of RS218 and TnphoA transposon mutants of RS218 to invade two intestinal epithelial cell lines and (2) the influence of growth conditions on RS218 invasion. Materials and Methods: Monolayers of Caco-2 and C2BBe1 cells were allowed to fully differentiate, expressing dome formation characteristic for transporting epithelia, and were infected at a minimum of 15 days post-confluency. Bacterial strains were grown overnight under either aerobic or microaerophilic conditions, and approximately 107 bacteria were added to each monolayer. Cells were incubated at 37 °C for 1.5 hours, washed, and then incubated with a gentamicin containing medium for one hour to eliminate extracellular bacteria. Cells were lysed and viable bacteria were enumerated by plating onto sheep blood agar plates. Results: RS218 was invasive in both Caco-2 and C2BBe1 cell lines. RS218 was 3 to 6.5-fold more invasive than HB101, a known noninvasive E. coli, in both cells lines. RS218 grown under microaerophilic conditions showed a 10-fold increase in invasion in both cell lines. TnphoA mutants, which have been shown to be noninvasive in brain microvascular endothelial cells, were at least 16 to 300-fold less invasive than RS218 in both cell lines. Conclusions: (1) The E. coli genes involved in the invasion of brain microvascular endothelial cells (such as ibe10) also play a role in the in vitro invasion of intestinal epithelial cells. (2) The microaerophilic environment enhances E. coli invasion of gut epithelial cells in vitro.