Development of a fluorometric microplate antiadhesion assay using uropathogenic Escherichia coli and human uroepithelial cells

J Nat Prod. 2014 May 23;77(5):1102-10. doi: 10.1021/np400781y. Epub 2014 Apr 22.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Adhesion / drug effects
  • Biflavonoids / analysis
  • Biflavonoids / chemistry
  • Catechin / analysis
  • Catechin / chemistry
  • Epithelial Cells / drug effects*
  • Flavonoids / analysis
  • Flavonoids / pharmacology
  • Fruit / chemistry
  • Humans
  • Molecular Structure
  • Plant Extracts / pharmacology
  • Proanthocyanidins / analysis
  • Proanthocyanidins / chemistry
  • Quercetin / analysis
  • Quercetin / pharmacology
  • Urine / microbiology
  • Uropathogenic Escherichia coli / drug effects*
  • Vaccinium macrocarpon / chemistry*

Substances

  • Biflavonoids
  • Flavonoids
  • Plant Extracts
  • Proanthocyanidins
  • procyanidin A2
  • procyanidin B1
  • myricetin
  • Catechin
  • Quercetin