Replicates within experiments are expressed as a mean for a single experiment. ANOVA and unpaired Student’s t-test were conducted using InStat3 (GraphPad, San Diego, CA). Means were compared using ANOVA and Tukey’s post-hoc test. Results AIEC infection decreases TER in T84 and MDCK-I epithelial cell monolayers Similar to EHEC O157:H7, apical infection for 16 h with AIEC, strain

LF82 caused a 46% reduction in TER in human colonic T84 cells (Figure 1A; ANOVA: p < 0.01, compared with uninfected sham controls). When the pathogen was introduced into the basolateral aspect of monolayers there was an 81% reduction in TER, relative to sham control monolayers, with AIEC infection (p < 0.001), compared to a 50% reduction with EHEC BEZ235 in vivo infection (p < 0.01; t test of AIEC vs. EHEC: p = 0.052). In contrast, both apical and basolateral infection of T84 monolayers with non-pathogenic E. coli, strain HB101 did not lead to a reduction in TER (N = 2). Figure 1 AIEC, strain LF82 disrupts the integrity of polarized selleck inhibitor epithelial monolayers. Model epithelial cell monolayers [T84 (Panel A) and MDCK-I

(Panels B & C)] grown in Transwells were infected with either E. coli, strain LF82 (AIEC) or EHEC O157:H7 – employed as a positive control – for 16 h at 37°C. Both apical (black bar histograms) and basolateral (gray bars) infections of human intestinal T84 monolayers caused a reduction in TER (Panel A; N = 4–6). Similar effects of infection on monolayer integrity were observed when MDCK-I cell monolayers were infected with AIEC, strain LF82 (Panel B), together with an increase in permeability to a macromolecular (10-kilodalton) dextran probe, indicating barrier disruption (Panels C; N = 2–4). HK denotes heat-killed bacteria. ANOVA: * p < 0.05; ** p < 0.01; *** p < 0.001. Apical and basolateral infections of canine kidney-derived MDCK-I polarized monolayers with EHEC and AIEC caused a comparable reduction of 53–73% in TER (Figure 1B; ANOVA: p < 0.01). Live bacteria were required, because there was no drop in TER with either heat-inactivated

or formaldehyde-fixed Rho bacteria (Figure 1B). The effects were not due to the metabolic activity of bacteria on epithelial cells, since incubation with tissue culture medium corrected to pH 6 (the pH of medium after 16 h of infection) did not reduce TER (N = 2). Macromolecular permeability increases following AIEC infection of MDCK-I monolayers Transcytosis of a 10-kDa dextran probe across monolayers supported the TER results. Consistent with previous reports [26], EHEC O157:H7 caused a dramatic increase in permeability to dextran, indicating breakdown of the epithelial barrier. Infection with AIEC also resulted in increased dextran permeability in MDCK-I cells (ANOVA: p < 0.05 for basolateral AIEC infection) comparable to findings seen with EHEC infection (Figure 1C; p > 0.05). There was a similar, but more modest, increase in permeability of T84 monolayers infected with AIEC (data not shown).