Poster U15 - Development of an In Vitro Model of Recurrent Urinary Tract Infections

Introduction: Recurrent urinary tract infections (RUTI) are defined as three episodes of a urinary tract infection within 12 months or two episodes within six months, caused either by a reinfection or persistent infection. RUTIs place a burden on the healthcare system and reduce the patient’s quality of life. Data collected from 79 RUTI patients showed a high population (82%) with a plasminogen activator inhibitor 1 (PAI-1) polymorphism. PAI-1 is a protein that inhibits the conversion of plasminogen into plasmin, preventing the degradation of fibrin. A mutation in the SERPINE1 gene causes the PAI-1 4G/4G and PAI-1 4G/5G polymorphisms, which upregulates PAI-1 activity and causes a buildup of fibrin in the system. Urine and vaginal swabs from the same 79 RUTI patients were sequenced, and it was found that the most prevalent bacteria in urine were Escherichia coli and Enterococcus faecalis. Based on the patient data we developed an in vitro model of RUTI using a fibrin gel to mimic the PAI mutation in the patient’s bladder, on which E. coli and E. faecalis biofilms were grown. We hypothesized that the presence of a fibrin gel would increase E. coli and E. faecalis growth and resistance to treatments.

Materials and

Methods: Fibrin gels were prepared to a final fibrinogen concentration of 3.3 mg/mL by combining human fibrinogen, 1 μL 2M CaCl2, and 0.5 U thrombin per mg fibrinogen in phosphate buffered saline (PBS). Immediately after mixing, the solution was pipetted into 24 well plates and allowed to polymerize for 1 hour at 37oC. E. coli (ATCC 53103 or ATCC 11775-mCherry) or E. faecalis (ATCC 19433) were seeded (103 CFU/mL) into 24 well plates with or without 3D fibrin gels and cultured for 2 days in BHI medium, at 37oC, under aerobic conditions. On day two, biofilms were exposed to medium alone (control), ciprofloxacin (4 mg/L), Biofilm Defense (an oral supplement, 42.5 mg/L) and/or 100% hypochlorous acid (Vashe) for a period of 24 hours. Biofilms were then harvested, homogenized, serially diluted, and drop plated to quantify viability. Bacteria were also fixed with 4% paraformaldehyde (PFA) in 1x PBS for 30 min at RT on day 3 of culture and visualized with confocal microscopy, either directly (E. coli – expressing mCherry) or after immunostaining (E. faecalis). Immunostaining was performed by blocking with 2% bovine serum albumin (BSA), permeabilized with 0.1% Triton-X 100 solution, incubated at room temperature for 2 hours with the Enterococcus polyclonal antibody (1:100, Life Technologies), incubated for 1 hour with Alexa Fluor 568 goat anti-rabbit IgG secondary antibody (1:100), washed with 1X PBS, and imaged using a confocal microscope.

Results, Conclusions, and Discussions: Results
The presence of fibrin did not result in an increase in the cells numbers of E. coli or E. faecalis biofilms. In the presence of fibrin, E. coli cells translocated across the fibrin gel while E. faecalis remained at the surface of the gel (Figure 1). Biofilm Defense did not affect the viability of E. coli, but significantly increased the viability of E. faecalis. Compared to controls, the presence of fibrin resulted in an increased resilience to killing when exposed to Vashe, 3-Log decrease compared to 8-Log, but no change during ciprofloxacin challenge. In contrast, E. coli biofilms in the presence of fibrin were more resilient to ciprofloxacin exposure, with a 3-Log decrease compared to 4-Log.
Discussion
Ciprofloxacin is a fluoroquinolone antibiotic that targets DNA gyrase in bacteria. It is used as an oral medication to treat many different bacterial infections and is a superior treatment for Gram-negative bacteria such as E. coli. Ciprofloxacin is currently an antibiotic used for UTI treatment clinically. Biofilm Defense is an oral supplement that contains proteases along with other enzymes that dissolve the fibrin components. Vashe wound cleanser is a sterile, hypochlorous acid (HOCl) solution that replicates the body’s natural defense against bacteria. Exposure of E. coli biofilms to ciprofloxacin, and E. faecalis biofilms to Vashe, in the absence of fibrin, demonstrated the greatest decrease in bacterial viability. However, resistance to treatments was demonstrated by the diminished decrease in viable counts for these treatments in the presence of fibrin. Overall, all of the treatments demonstrated an increase in viability once the treatment was removed after 24 hours and allowed to recover, demonstrating the bacteria’s ability to regenerate when the treatment is removed.
Conclusion
The goal of this work was to create a RUTI in vitro model that would enable the testing and development of established or novel treatments for RUTI. Results with the in vitro model indicate that infections in RUTI patients that have an increase of fibrin and are colonized with E. coli and E. faecalis may have a more resilient infection.

Acknowledgements (Optional): This research was funded by NSF REU #2150229.