Development of a Fluorometric Microplate Antiadhesion Assay Using Uropathogenic Escherichia coli and Human Uroepithelial Cells

作者：Kimble Lindsey L; Mathison Bridget D; Kaspar Kerrie L; Khoo Christina; Chew Boon P^*

来源：Journal of Natural Products, 2014, 77(5): 1102-1110.

DOI：10.1021/np400781y

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 mu g/mL) was developed; the lower limit of detection was 0.1 mu g/mL, and half-maximal inhibitory concentration was 0.88 mu g/mL (intra- and interassay coefficients of variance were %26lt;10% and %26lt;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 %26lt; 0.01) with a radiolabeled-E. coli assay.

• 出版日期2014-5