Investigating the effect of prophylactic D-mannose treatment
on adhesion of Uropathogenic Escherichia coli to the human
bladder epithelium

Ahmed, S

Abstract

Urinary tract infection (UTI) is the second most common bacterial infection worldwide, affecting millions of people annually, mainly women, and primarily caused by Uropathogenic Escherichia coli (UPEC). Treatment of UTIs is challenged by the continued increase of antibiotic resistance, and alternative strategies are necessary
to reduce this disease burden. The pathology of UTI rapidly develops when microorganisms bind to and invade the lining of the bladder. Therefore, interventions to prevent UPEC adhesion have promising potential to reduce the occurrence of recurrent infections. Several studies have suggested that D-mannose reduces the
binding of UPEC to the lining of the bladder in vitro and in animal models. This study used a 3D bladder epithelial cell model to investigate UPEC binding in the presence
of urine samples from the women enrolled on a double-blind clinical trial of D-mannose. All human samples were collected as part of the MERIT trial with full ethical approval (REC 18/SW/0245; Protocol number PID13650-SP001-AC001; IRAS project ID 245539. 15th Aug 2019). Trial registration number ISRCTN 13283516.

The growth rate of three well characterised UPEC strains was compared. Strains UTI89, CFT073 and 536 were all able to grow in artificial urine medium (AUM), but at a slower rate than in the standard nutrient-rich bacterial growth medium, Luria Bertani (LB) broth. Based on growth rates, strains CFT073 and 536 appeared better adapted to AUM than UTI89. After 24 hours strains UTI89, CFT073 and 536 reached an optical density (OD) of 0.96, 1.41, 2.06 in AUM and 3.39, 3.22 and 4.88 in LB broth. Biofilm formation was assessed in AUM and LB broth using three different densities of CFT073 inoculum. After 24 h, all conditions resulted in
similar biofilm densities in AUM (OD600 0.30 - 0.37) and in LB broth (OD600 0.17-0.26).

Specialised epithelial cells, Long-Term Human Bladder Epithelial Cells (HBLAK), were grown and differentiated on porous membranes to establish a 3D model of the bladder epithelium. A gentamycin protection assay was used to quantify adhesion to and invasion of differentiated HBLAK cells by strain CFT073. Initial
experiments to optimise the assay found that HBLAK cells could withstand exposure to both urine and CFT073, but no invasion was detected. Adhesion assays in the presence of urine from D-mannose and placebo arms of a randomised double-blind
clinical trial detected no significant difference in CFT073 adhesion. A mean number of 2.55 x 107 C.F.U ml-1 adhered to differentiated HBLAK layer in the presence of urine from the treatment arm of the study D-mannose compared to 2.70 x 107 C.F.U ml-1 in the presence of urine from the placebo arm. This finding is consistent with results from the clinical trial that found no significant reduction in rUTI occurrence between the groups.

Several factors could have influenced the outcomes of the trial including different rates of metabolism or absorption of D-mannose in each trial participant, or different infecting UPEC strains that may have expressed different types of fimbriae. These points could be further investigated by measuring concentrations of D-mannose in
the urine samples, and isolating E. coli from samples before filtering and using western blotting to determine the types of fimbriae expressed. If there was access to a wider group of samples, this may have enabled stratification of samples for further
comparison.

Citation

bladder epithelium. (Dissertation). University of Salford

• Thesis Type: Dissertation
• Acceptance Date: Aug 22, 2022
• Deposit Date: Oct 5, 2022
• Award Date: Mar 3, 2022