Proanthocyanidin-enriched extract from Cranberry (Vaccinium macrocarpon Aiton) exhibits anti-invasive and not anti-adhesive activity against uropathogenic Escherichia coli (UPEC)

Urinary tract infections (UTIs) are one of the most common infectious diseases. 80% of UTIs are caused by UPEC. Rising antibiotic resistance and high recurrence rates are major problems of UTIs in clinical praxis. Therefore, the need for alternative therapies and prophylaxis is evident. Extracts from Cranberry fruits are traditionally used for prevention of recurrent UTIs and show considerably activity in clinical studies. The common opinion on the mode of action implies that A-type proanthocyanidins inhibit the adherence of UPEC to bladder epithelium by interacting with P-fimbriae and type 1 fimbriae (mannose specific) as determined by isolated adhesins and protein-ligand assay. Only limited data are available with meaningful bacteria-cell assays. Therefore the present study aimed to investigate the claimed antiadhesive effects of Cranberry in advanced cell-based assays.

Methods: Adhesion assay was performed with FITC-labeled UPEC (strain 2980, DSM 10791) on T24 human bladder epithelial cells (ATCC-No. HTB-4). Bacteria and T24 cells were coincubated for 2h with Cranberry EtOH extract (Frutarom Belgium). The interaction of UPEC/T24 was quantified by FACS. Confocal laser scanning microscopy (CLSM) was done by labeling F-actin (Texas Red®-X Phalloidin), nucleus (DAPI) and UPEC (FITC). For invasion assay bacteria and T24 cells were coincubated with test extract for 3h; gentamicin was added for removal of extracellular bacteria, incubation continued for 1h, cells were lysed, plated onto agar and CFU counted after 20h.

Results: Within an adhesion assay Cranberry extract exhibited significant higher fluorescence than the untreated control, indicating strong increased bacterial adhesion. This is in total contrast to the common hypothesis that Cranberry decreases bacterial adhesion to bladder cells. Also CLSM indicated significantly increased adhesion of UPEC and showed agglutination of UPEC also at low concentrations of 1 µg/ml. Additionally it was visible that the bacteria clusters were exclusively located on the surface of the bladder cells, while the inside was almost completely free of UPEC. Scanning Electron Microscopy confirmed this finding and clearly indicated the cluster formation on the epithelial outside (Fig. 1 – 3).

Fig. 1: Adhesion of untreated UPEC to bladder cells. SEM 5000 x

Fig. 2: Cluster of UPEC after treatment with Cranberry extract (25 μg/ml) and adhesion to T24 bladder cells. SEM 5000 x

Fig. 3: Cluster of UPEC after treatment with Cranberry extract (25 μg/ml) and adhesion to T24 bladder cells. SEM 10000 x

Invasion assay demonstrated that as a consequence of the agglutination, UPEC-clusters are not internalized any more into the epithelial cells: the inhibition of internalization by test extract (50 µg/ml) was 88%. From these data it can be clearly deduced that Cranberry extract exhibits an increase in bacterial adhesion to bladder cells, but inhibits the internalization into the cells.