From Lab to Life: Validating and Studying P. mirabilis’ Crystalline Biofilms in an in-vitro Bladder Model

Abstract Purpose: Proteus mirabilis forms crystalline biofilms that cause catheter encrustation and blockage. This study aimed to develop and validate an in vitro bladder model to study crystalline biofilms of P. mirabilis on urinary catheters. Methods: Double-walled glass vessel was used as a bladder model (BM), maintained at 37°C by circulating water. Artificial urine was supplied at 0.7 mL/min via a peristaltic pump, and a Foley catheter connected to a drainage bag formed a sterile closed system. Four catheterized BMs were connected in series. Model validation included calcium and magnesium leaching over 5 days and reproducibility based on catheter encrustation after 2 weeks. Artificial urine medium(AUM) was validated by comparing uropathogen growth with human urine. BM1 contained P. mirabilis–inoculated AUM (positive control), BM2 uninoculated AUM (negative control), and BM3–BM4 AUM inoculated with clinical P. mirabilis strains (n=6). Models were evaluated at 2,7, 14 days and until catheter blockage. Biofilm quantification and visualization were done using MTT, CV assays, scanning electron microscopy-energy dispersive X-ray analysis, atomic absorption spectroscopy. A quantitative PCR was performed to detect expression of biofilm-associated virulence genes. Results: All P. mirabilis strains formed strong biofilms after 14 days. The increase in catheter encrustation with calcium and magnesium over time was significant (p<0.05). SEM-EDX revealed biofilm architecture, microcolonies, calcium, and magnesium deposits. UreC, SpeA, MrpA, and RsbA genes showed significant expression after biofilm formation compared to initial stage. Conclusion:The developed bladder model provides a valid platform to study CAUTIs, biofilm formation, and develop novel approaches for controlling infections.