Intracellular survival of Staphylococcus aureus within cultured enterocytes.

BACKGROUND: Little is known about the mechanisms involved in bacterial translocation from the intestinal lumen to extraintestinal sites. Because Staphylococcus aureus can colonize the intestinal tract, and because the intestinal tract is a reservoir for antibiotic resistant S. aureus, experiments were designed to clarify the interactions of S. aureus with cultured intestinal epithelial cells, and assays included measurements of bacterial internalization, enterocyte apoptosis, and epithelial barrier function. METHODS AND RESULTS: Mature, confluent enterocytes were incubated 1 h with S. aureus, and the gentamicin protection assay was used to quantify intracellular bacterial survival at various time intervals up to 120 h later. Enterocyte apoptosis was assessed using Annexin V, and the permeability of confluent enterocyte cultures was measured by transepithelial electrical resistance and by transmigration of Escherichia coli across confluent enterocytes.S. aureus was internalized by cultured enterocytes and remained viable for up to 120 h within both HT-29 and Caco-2 enterocytes. S. aureus intracellular survival was associated with enterocyte apoptosis and with decreased transepithelial electrical resistance across confluent Caco-2 enterocytes. S. aureus intracellular survival over time was also associated with increased E. coli transmigration across confluent Caco-2, but not HT-29, enterocytes. CONCLUSIONS: S. aureus appeared to survive within cultured enterocytes for prolonged time periods, up to several days. Survival of S. aureus within host eukaryotic cells, such as enterocytes, might facilitate persistence of S. aureus in infected tissue despite appropriate antibiotic therapy.

Effect of tumor necrosis factor alpha, interferon gamma, and interleukin-4 on bacteria-enterocyte interactions.

BACKGROUND: Little is known about the mechanisms involved in bacterial translocation from the intestinal lumen to extraintestinal sites. Because the cytokine cascade associated with sepsis, inflammation, and trauma has been shown to affect intestinal epithelial permeability, experiments were designed to clarify the effects of selected cytokines on bacterial adherence to and internalization by cultured HT-29 and Caco-2 enterocytes. METHODS: Mature, confluent enterocytes were pretreated 48 to 72 h with tumor necrosis factor alpha (TNF-alpha), interferon gamma, (IFN-gamma), or interleukin-4 (IL-4). Adherence of Listeria monocytogenes, Salmonella typhimurium, Proteus mirabilis, and Escherichia coli was measured by enzyme-linked immunosorbent assay and bacterial internalization was quantified by the gentamicin protection assay. Enterocyte permeability was measured by transepithelial electrical resistance and by flux of 40-kDa fluorescent dextran. Bacterial transmigration across confluent enterocytes was measured using enterocytes cultivated on permeable supports. RESULTS: TNF-alpha, IFN-gamma, and IL-4 had variable effects on bacterial adherence to HT-29 and Caco-2 enterocytes, although the most consistent finding was increased bacterial adherence associated with INF-gamma. However, none of these cytokines had a noticeable effect on bacterial internalization by either Caco-2 or HT-29 enterocytes. In addition, none of these cytokines had a noticeable effect on the permeability of confluent enterocytes as measured by transepithelial electrical resistance or dextran flux. Bacterial transmigration across confluent HT-29 enterocytes was not altered by TNF-alpha, IFN-gamma, or IL-4; however, IL-4 consistently decreased bacterial transmigration across confluent Caco-2 enterocytes. CONCLUSIONS: IFN-gamma may augment the epithelial adherence of selected species of enteric bacteria, and IL-4 may act as a barrier-sustaining agent to decrease bacterial migration across the intestinal epithelium.