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Development and validation of a sensitive, high throughput bioassay of radiolabeled E. coli adhesion to uroepithelial cell in vitro
Author(s) -
Mathison Bridget D,
Kimble Lindsey L,
Kaspar Kerrie L,
Khoo Christina,
Chew Boon P
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.644.8
Subject(s) - chemistry , adhesion , bioassay , escherichia coli , cell adhesion , in vitro , ic50 , serial dilution , incubation , biochemistry , chromatography , food science , microbiology and biotechnology , biology , medicine , genetics , alternative medicine , organic chemistry , pathology , gene
Anti‐adherent properties of cranberry bioactives have been used to prevent uropathogenic E. coli adherence to uroepithelial cells (UEC). We developed and validated an assay to assess anti‐adhesion activity of cranberry extracts and urine. P‐fimbriated E. coli (CFT073) were labeled with 3 H‐uridine then co‐incubated with HTB‐4 UEC at a 400:1 ratio. Cranberry extract (0–20 mg PACs/mL) was pre‐incubated with 3 H‐labeled E. coli yielding a sensitive inhibition curve; LLD and IC50 were 0.5 mg and 1.85 mg PAC equivalents/mL, respectively; intra‐and inter‐assay CV were <10% and <15%, respectively. NIST cranberry extract showed identical inhibition of E. coli adhesion. Serial dilutions of urine from human subjects who consumed cranberry beverages showed a linear decrease in anti‐adhesion activity in the assay. Adding extract to UEC prior to or at the time of bacteria:UEC co‐incubation produced similar anti‐adhesion activity. Mannose had no inhibitory effects indicating little to no Type I‐fimbriation. Anti‐adhesion assays conducted with urine from a pilot human cranberry intervention study showed good agreement with results obtained using the hemagglutination assay. Therefore, we have developed a sensitive, high throughput, biologically relevant anti‐adhesion assay using 3 H‐ E. coli co‐incubated with UEC which can be used for studying the action of cranberry bioactives. Funded by Ocean Spray Cranberries Inc.