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.

A randomized, double-blind, placebo-controlled pilot study to assess bacterial anti-adhesive activity in human urine following consumption of a cranberry supplement

“…Previous studies have shown that cranberry polyphenolic compounds such as proanthocyanidins, 13,14 flavonols and their glycosides (e.g. myricetin, quercetin), 15,16 condensed tannins (e.g. procyanidins, prodelphinidin), 15,17 and phenolic acids (e.g.…”

“…myricetin, quercetin), 15,16 condensed tannins (e.g. procyanidins, prodelphinidin), 15,17 and phenolic acids (e.g. benzoic acid) 18 were able to inhibit the in vitro adherence of P-type E. coli to the glycoprotein receptor on epithelial cells or red blood cell analogues.…”

A high‐throughput assay for the measurement of uropathogenic Escherichia coli attachment to urinary bladder cells

“…, ; Kimble et al . ). This method requires extemporaneous labelling before each assay and can lead to batch‐to‐batch variations in fluorescence intensity.…”

“…In some of them, the co‐incubation of bacteria and cells was performed in suspension (Kimble et al . ) or a centrifugation step was used to enhance the contact of bacteria with the cells monolayer (Scharenberg et al . ; Rafsanjany et al .…”

“…In our protocol, bacteria are allowed to attach to solid-immobilized, physiologically relevant cells without centrifugation, to be as close as possible to the natural conditions of infection in a bladder full of urine. In some studies, bacteria were labelled using chemical coupling to a fluorophore (Rafsanjany et al 2013(Rafsanjany et al , 2015Kimble et al 2014). This method requires extemporaneous labelling before each assay and can lead to batchto-batch variations in fluorescence intensity.…”