Development of a fluorometric microplate anti‐adhesion assay using uropathogenic E. coli and human uroepithelial cells

Abstract

No sensitive, high throughput fluorometric assays are available to study anti‐adhesion mechanisms of cranberry bioactives. We developed a fluorometric microplate assay to determine E. coli adhesion to human uroepithelial cells (UEC). P‐fimbriated E. coli were labeled for 30 min with 400 μM BacLight Green and pre‐incubated with urine or standard for 30 min. Fluorescent‐E. coli were added to confluent HTB‐4 UEC in mircoplates at a ratio of 400:1, incubated 1 h, washed and fluorescent intensity measured (480 abs/516 em). Specific labeling and cell adhesion were confirmed by flow cytometry. A standard curve was developed using myricetin (0–30 μg/mL) with intra‐assay CV <10% and interassay CV <15%; LLD was 0.1 μg/mL and IC50 was 0.88 μg/mL. Standard curves using quercetin and procyanidins B1, B2, and C1 showed similar inhibition. The correlation coefficient of the anti‐adhesion activity of urine samples (n = 216) from human subjects who consumed a placebo or cranberry beverage was highly significant (R2 = 0.78; P < 0.01) when this assay was compared to a 3H‐E. coli microplate assay. Anti‐adhesion activity of human urine from a pilot cranberry intervention study (n = 167) also showed good agreement with a hemagglutination assay. Therefore, we successfully developed a biologically relevant high‐throughput assay for the determination of E. coli anti‐adhesion activity in human urine. Funded by Ocean Spray Cranberries Inc.