title: Development of a fluorometric microplate antiadhesion assay using uropathogenic Escherichia coli and human uroepithelial cells.
abstract: A fluorometric microplate assay has been developed to determine Escherichia (E.) coli adhesion to uroepithelial cells (UEC). P-fimbriated E. coli were labeled with BacLight Green and preincubated 30 min with human urine or standard. Fluorescent-E. coli were added to UEC in mircoplates at a 400:1 ratio, incubated 1 h, and washed, and the fluorescence intensity was measured. Specific labeling and adherence were confirmed by flow cytometry. A myricetin (1) standard curve (0-30 μg/mL) was developed; the lower limit of detection was 0.1 μg/mL, and half-maximal inhibitory concentration was 0.88 μg/mL (intra- and interassay coefficients of variance were <10% and <15%, respectively). Vaccinium macrocarpon (cranberry) extracts, quercetin (2), and procyanidins B1 (3), B2 (4), and C1 (5) showed similar inhibition. Antiadhesion activity of urine samples from subjects (n = 12) consuming placebo or V. macrocarpon beverage determined using this assay was positively correlated (R(2) = 0.78; p < 0.01) with a radiolabeled-E. coli assay.
authors.affiliation: School of Food Science, Washington State University , Pullman, Washington 99164-6376, United States.
authors.forename: Lindsey L
authors.initials: LL
authors.lastname: Kimble
authors.forename: Bridget D
authors.initials: BD
authors.lastname: Mathison
authors.forename: Kerrie L
authors.initials: KL
authors.lastname: Kaspar
authors.forename: Christina
authors.initials: C
authors.lastname: Khoo
authors.forename: Boon P
authors.initials: BP
authors.lastname: Chew
mesh_terms: D001422:Bacterial Adhesion / Q000187:drug effects; D044946:Biflavonoids / Q000032:analysis / Q000737:chemistry; D002392:Catechin / Q000032:analysis / Q000737:chemistry; D004847:Epithelial Cells / Q000187:drug effects*; D005419:Flavonoids / Q000032:analysis / Q000494:pharmacology; D005638:Fruit / Q000737:chemistry; D006801:Humans; D015394:Molecular Structure; D010936:Plant Extracts / Q000494:pharmacology; D044945:Proanthocyanidins / Q000032:analysis / Q000737:chemistry; D011794:Quercetin / Q000032:analysis / Q000494:pharmacology; D014556:Urine / Q000382:microbiology; D056689:Uropathogenic Escherichia coli / Q000187:drug effects*; D029799:Vaccinium macrocarpon / Q000737:chemistry*
chemical_list: D044946:Biflavonoids; D005419:Flavonoids; D010936:Plant Extracts; D044945:Proanthocyanidins; C542735:procyanidin A2; C479579:procyanidin B1; C040015:myricetin; D002392:Catechin; D011794:Quercetin