Skip to main content
2024 BMES Annual Meeting Main banner
Icon Legend
This session is not in your schedule.
This session is in your schedule. Click again to remove it.
Back

Womens Health

Women's Health - Poster Session A

Poster L17 - A 3D bacterial and bladder epithelium co-culture model of recurrent urinary tract infections

Thursday, October 24, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Urinary tract infections (UTIs) are the most common type of infections reported in adult women and have a high rate of recurrence even with antibiotic treatment [1]. Recurrent urinary tract infections (RUTIs) reduce patient quality of life, place a significant burden on the healthcare system, and contribute to antimicrobial resistance [2]. Human data on the mechanism and/or molecular basis of RUTIs are limited. RUTIs have been attributed to bacterial biofilm formation in the urinary tract [3]. The role of bacterial biofilms in female RUTIs is an emerging area of research that could have a significant impact on clinical practice. Data collected from 79 RUTI patients showed that 82% have 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 resulting in a buildup of fibrin in the patient. Urine samples from the same patients showed that the most prevalent bacteria in urine were Escherichia coli and Enterococcus faecalis. The goal of this work is to develop an in vitro model of RUTI using bladder epithelial cells, a fibrin gel to mimic the PAI mutation in the patient’s bladder, and E. coli or E. faecalis biofilms.

Materials and

Methods:  HTB-5 bladder epithelial cells were seeded at 100,000 cells/cm2 into 1.9 cm2 growth area wells. The cells were grown for 72 hours at 37oC and 5% CO2 in EMEM+10% HI FBS, and then 3.3 mg/mL fibrin was gelled on top of the cell monolayer. After 24 hours, E. coli and E. faecalis were added to the HTB-5 cells and fibrin cultures at a concentration of 103 CFU/cm2 and further cultured for 96 hours. Controls were HTB-5 cells without fibrin gels and HTB-5 cells with fibrin gels, both without bacteria. After 48 hours of mammalian/bacteria co-culture, 30 μg/mL of ciprofloxacin was added to EMEM and added on top of the epithelial cell and bacterial cultures. Ciprofloxacin remained in the culture medium for 24 hours before it was removed and replaced with fresh EMEM. Every 24 hours after bacterial seeding, bacterial CFU quantification and LIVE/DEAD viability assays were performed.

Results, Conclusions, and Discussions: Results
Figure 1A shows HTB-5 viability following seeding with E. faecalis with and without fibrin present. Figure 1B shows E. faecalis viability for 96 hours after seeding into HTB-5 plates with and without fibrin. HTB-5 viability was significantly higher in the absence of fibrin at 48 and 72 hours (Figure 1A). E. faecalis viability was also increased in plates with fibrin (Figure 1B). These results were replicated in plates seeded with E. coli, although overall HTB-5 viability was lower in wells seeded with E. coli. There were approximately 2-log more E. coli cells in the presence of fibrin at 24- and 48-hour time points. Following treatment with ciprofloxacin the E. coli viability decreased by 3-log and 4.4-log when seeded onto fibrin or no fibrin respectively (not shown), while E. faecalis viability decreased by 0.5-log and 1.9-log, respectively (Figure 1B).

Discussion
The presence of fibrin supports bacterial growth, as shown in Figure 1B. Ciprofloxacin is currently used as an antibiotic for RUTI patients and is a superior treatment for Gram-negative bacteria. Once ciprofloxacin was added to the mammalian/bacterial co-cultures, at 48 hours, the bacterial viability decreased, while mammalian cell viability increased. The bacterial decrease in viability was greater for E. coli (Gram-negative) than E. faecalis (Gram-positive), and bacterial biofilms cultured with fibrin showed more resistance to ciprofloxacin. HTB-5 bladder epithelial cells remained viable in co-cultures with E. coli and E. faecalis for 96 hours. HTB-5 cells co-cultured with bacteria and no fibrin had significantly higher viability than those co-cultured with bacteria and fibrin, likely because bacteria viability was higher when cultured with fibrin.

Conclusion
We have developed a RUTI model that includes human bladder epithelial cells, clinically relevant bacterial biofilms, and fibrin, representing a PAI-1 polymorphism common in the RUTI patients investigated. The presence of fibrin in this RUTI cell culture model supported bacterial growth and resulted in resistance to treatment. Data on the mechanism of RUTI is limited, and this model provides a platform for investigating RUTI development, progression, and treatment.

Acknowledgements (Optional):  This work is supported by NSF REU 2150229 and the Binghamton Scholars Program.

References
[1] 1. Smith AL, Brown J, Wyman JF, Berry A, Newman DK, Stapleton AE. Treatment and Prevention of recurrent lower urinary tract infections in women: a rapid review with practice recommendations. The Journal of urology. 2018;200(6):1174-91. Epub 2018/06/26. doi: 10.1016/j.juro.2018.04.088. PubMed PMID: 29940246.
[2] Foxman B. Urinary tract infection syndromes: occurrence, recurrence, bacteriology, risk factors, and disease burden. Infectious disease clinics of North America. 2014;28(1):1-13. Epub 2014/02/04. doi: 10.1016/j.idc.2013.09.003. PubMed PMID: 24484571.
[3] Soto SM, Smithson A, Horcajada JP, Martinez JA, Mensa JP, Vila J. Implication of biofilm formation in the persistence of urinary tract infection caused by uropathogenic Escherichia coli. Clinical microbiology and infection: the official publication of the European Society of Clinical
Microbiology and Infectious Diseases. 2006;12(10):1034-6. Epub 2006/09/12. doi: 10.1111/j.1469-0691.2006.01543.x. PubMed PMID: 16961644.