Sequencing was performed at the Allan Wilson Centre Genome Service (Massey University,
Palmerston North, New Zealand), and traces were aligned using contigexpress selleck kinase inhibitor vector nti and the 16S rRNA gene sequences were compared with known bacterial sequences using the NCBI blast database. The EPEC O127:H6 (E2348/69) was obtained from Dr Roberto La Ragione at Veterinary Laboratories Agency, Weybridge, UK. Caco-2 cells (human colorectal adenocarcinoma cell line; ATCC HTB-37) were used as a model of the intestinal epithelial barrier because they differentiate spontaneously into polarized intestinal cells possessing apical brush borders and tight junctions. Caco-2 cells were seeded onto collagen membrane inserts (Cellagen™ Discs CD-24, MP Biomedicals, OH) and incubated in 12-well plates in M199 with 10% v/v foetal bovine serum, 1% v/v nonessential amino acids (MEM nonessential amino acids 100 × solution
and 1% v/v penicillin–streptomycin) (10 000 U penicillin G sodium salt Alectinib research buy and 10 000 μg streptomycin sulphate in 0.85% v/v saline). Caco-2 cells were grown at 37 °C in 5% CO2 for 5 days until confluent (undifferentiated) for the screening assays. Undifferentiated Caco-2 cells were used for the initial screening because of the ease of preparing undifferentiated Caco-2 cells compared with differentiated Caco-2 cells. This was necessary because of the high volume of assays that were carried out during the screening. Tangeritin The TEER assay measures the integrity of the tight junctions between epithelial cells, and as these tight junctions are already formed when Caco-2 cell monolayers reach confluence
(5 days), undifferentiated Caco-2 cells are often used to assess tight junction integrity. An additional TEER assay was carried out using differentiated Caco-2 cells (18 days old) to confirm the positive effects of the best selected isolates. Caco-2 monolayers were prepared the day before the TEER assay by removing the media, washing with PBS (pH 7.2) and adding M199 with 1% v/v nonessential amino acids (without foetal bovine serum and penicillin–streptomycin). In each experiment, control media (M199 with 1% nonessential amino acids) and a positive bacterial strain (either L. plantarum MB452 for commercially used probiotic strain testing or Lactobacillus rhamnosus HN001 for isolate testing) were included as controls. Overnight cultures of bacterial cells (MRS broth, 37 °C, 5% CO2) were collected by centrifugation (20 000 g for 5 min) and resuspended in M199 with 1% v/v nonessential amino acids to an OD600 nm of 0.9. After the initial resistance readings, the media were removed from the Caco-2 monolayers and replaced with treatment solutions. Each bacterial strain was tested in quadruplicate.