It is therefore of utmost importance to gain insight into the processes and determinants

that promote intestinal colonization of nosocomial E. faecium strains. Only then we will be able to impede subsequent spread of these nosocomial clones. Methods Bacterial strains and growth conditions In this study E. faecium strains E135, E1162 and E1162Δesp were used. E135 is an esp negative community surveillance feces isolate, while strain E1162 is a hospital-acquired blood isolate, positive for Esp ��-Nicotinamide research buy expression. The isogenic Esp-deficient mutant, E1162Δesp, was previously constructed by introduction of a chloramphenicol resistance cassette (cat) resulting in an insertion-deletion mutation of the esp gene [21].E. faecium strains were grown in either Todd-Hewitt (TH) or Brain Heart Infusion (BHI)

broth or on Tryptic Soy Agar (TSA) with 5% sheep red blood cells (Difco, Detroit, MI). Slanetz and Bartley (SB) agar plates were used to selectively grow enterococci. E. faecium strain E1162 and its isogenic mutant are high-level resistant to ceftriaxone (minimum inhibitory concentration > 32 μg/ml). Caco-2 cell cultures Human colorectal adenocarcinoma cells, Caco-2 cells, were obtained from the American Type Culture Collection (HTB-37, ATCC, USA) and were cultured in Dulbecco’s Modified Eagle Medium Cediranib in vivo (DMEM; Gibco, Invitrogen, Paisley, UK) supplemented with 10% heat-inactivated fetal calf serum (Integro B.V, Zaandam, The Netherlands), 1% non-essential amino acids (Gibco), 2 mM glutamine (Gibco), and 50 μg/ml gentamicin (Gibco). Cells were collected every 7th day by washing the monolayer twice with 0.022% disodium-ethylenediamine tetra acetic acid (di-Na-EDTA; Acros Organics, Morris Plains, NJ) in PBS and trypsinizing the cells using 50 μg/ml trypsine (Gibco), in 0.022% di-Na-EDTA in PBS. Cells

were seeded at 1 × 106 cells in 10 ml DMEM in 75 cm2 culture bottles (Costar, Corning, NY) and incubated in a humidified, 37°C incubator with 5% CO2. The culture medium was refreshed every 4th day after passage of the cells. Differentiated Caco-2 cells were prepared by seeding cells from passage 25 to 45 in 12-wells HM781-36B tissue culture plates (Costar) at 1.6 × 105 cells/ml in DMEM. To each well 1 ml of this suspension Carbohydrate was added and plates were incubated at 37°C with 5% CO2 for 14–16 days before use to allow the Caco-2 cells to differentiate. The medium in the wells was replaced by fresh medium three times a week. Adherence assay Overnight-grown cultures of E135, E1162 and E1162Δesp in BHI broth were diluted (1:50) and grown at 37°C to an OD660 of 0.4, while shaking. Bacteria were harvested by centrifugation (6,500 × g; 3 min) and resuspended in DMEM to a concentration of 1 × 107 CFU/ml. For each strain, 1 ml bacterial suspension was added to the wells (100 bacteria to 1 Caco-2 cell). Plates were centrifuged (175 × g; 1 min) and incubated for 1 h at 37°C in 5% CO2 to allow adherence to the Caco-2 cells.