Effect of hypoxia on enterocyte endocytosis of enteric bacteria

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Abstract
To clarify the effect of hypoxia on bacteria-enterocyte interactions. Randomized. Research laboratory. Enteric bacterial and cultured human intestinal epithelial cells, HT-29 cells. The effect of hypoxia on bacterial internalization and intracellular survival was studied, using enterocytes cultured for 21 days in either 20%, 10%, or 5% oxygen. The effect of bacterial growth conditions on bacterial internalization by enterocytes was studied, using bacterial cells in either the log phase or stationary phase of aerobic growth, and using bacterial cells in stationary phase, grown either under low oxygen conditions or under anaerobic conditions. Individual strains of enteric bacteria were incubated with HT-29 cells for 1 hr. Numbers of internalized bacteria were subsequently quantified after enterocyte lysis. Bacterial growth conditions (anaerobic vs. aerobic and log-phase vs. stationary-phase bacterial cells) had no noticeable effect on the numbers of Salmonella typhimurium, Proteus mirabilis, and Escherichia coli internalized by enterocytes. Enterocytes cultivated in 20%, 10%, or 5% oxygen were >95% viable. Enterocytes cultivated in 20% oxygen were confluent, but those enterocytes cultivated in hypoxia were not confluent and were fewer in number compared with enterocytes cultivated in normoxia. Compared with enterocytes grown in normoxia, enterocytes cultivated in 5% and 10% oxygen internalized greater numbers of each of seven strains of enteric bacteria, including Listeria monocytogenes (two strains), Enterococcus faecalis (two strains), and P. mirabilis, E. coli (two strains), with statistically significant increases noted for five of these seven bacterial strains. Intracellular survival of L. monocytogenes and P. mirabilis was assayed. Both species survived intracellularly for 22 hrs, with no noticeable differences in the numbers of intracellular bacteria recovered from enterocytes cultivated in 20%, 10%, and 5% oxygen. These in vitro results suggest that augmented bacterial endocytosis by enterocytes might at least partially explain the increased frequency of bacterial translocation associated with tissue ischemia.