Invasion of bovine epithelial cells by verocytotoxin-producing Escherichia coli O157: H7

Abstract

The ability of verocytotoxin‐producing Escherichia coli (VTEC) O157: H7 to enter selected human (RPMI‐4788 and HeLa) and bovine (MAC‐T, mammary secretory; MDBK, kidney) epithelial cell lines was evaluated. All VTEC evaluated efficiently entered RPMI‐4788 and MAC‐T cell lines. VTEC entered MDBK cells at approximately 4% of MAC‐T cells. VTEC were not able to invade HeLa cells. Presence of plasmid had no influence on efficiency of entry, nor did production of shiga‐like toxin (SLT I or SLT II). Internalization required microfilaments, but not microtubules. Two types of adherence, localized and diffuse, were exhibited depending on isolate and cell line evaluated. Ability of VTEC to invade bovine mammary epithelial cells may be important in pathogenesis in the bovine, may indicate a route by which raw milk may potentially become contaminated, and may provide a reservoir of bacteria for the contamination of workers, equipment and carcass at time of slaughter.

Internalization of Escherichia Coli O157:H7 by Bovine Rectal Epithelial Cells

“…This suggests that E. coli O157:H7 uptake by the bovine epithelial cells was through a microfilament-dependent pathway. These data are in agreement with previous studies using immortal cell lines (Matthews et al, 1997) and indicate that EPEC and EHEC exploit distinct host cell processes to stimulate their uptake.…”

“…The uptake of ATCC 43895 by the PBRE cells was 19.38% of the original inoculum. This level is ∼100-fold higher than the other E. coli O157:H7 strains tested (Table 3) and was at a level similar to previous studies done with a variety of cell lines (Matthews et al, 1997; Dibb-Fuller et al, 2001; Uhlich et al, 2002, 2009; Abu-Ali et al, 2010). Also, ATCC 43895 was more proficient at forming biofilm compared to the other strains tested.…”

“…To identify host cell factors involved in internalization of 43895 and 43894, PBRE cells were treated with eukaryotic cell inhibitors known to block bacterial invasion (Rosenshine et al, 1992b; Matthews et al, 1997; Martinez and Hultgren, 2002). Treatment with cytochalasin D, an inhibitor of actin microfilaments, did not modify the number of cell-associated bacteria ( P  > 0.05; Figure 5A).…”

“…The bacterial internalization by the PBRE cells was measured using a standard gentamicin protection assay (Small et al, 1987; Matthews et al, 1997). Monolayers of the PBRE cells were washed twice with HBSS and infected with 1 ml of the cell culture medium without antibiotics and FBS (internalization medium) at a MOI of 100 bacteria/epithelial cell.…”

“…To assess the effect of eukaryotic cell inhibitors on bacterial internalization, the PBRE cell monolayers were incubated with cytochalasin D (1 μg/ml, Sigma), colchicine (1.25 μM, Sigma), compactin (25 μM, Sigma), or genistein (50 μM, Sigma) prior to addition of bacteria as described previously (Matthews et al, 1997). Cytochalasin D and colchicine were added to the monolayers 30 min prior to inoculation, genistein 15 min prior to inoculation, and compactin 18 h prior to inoculation.…”