Expression of Biofilm Forming Virulence Genes in Proteus mirabilis: Insights from an in-vitro Bladder Model

Abstract

Background: Proteus mirabilis forms crystalline biofilms that lead to catheter encrustation and blockage, contributing to the chronicity and recurrence of catheter-associated urinary tract infections (CAUTIs), especially in long-term catheterized patients. Understanding the expression of biofilm-forming virulence genes of P. mirabilis is critical for developing strategies to prevent CAUTIs. Objective: To investigate the differential expression of biofilm-associated virulence genes (ureC, rsbA, mrpA, and speA) in P. mirabilis before and after biofilm formation using a validated in-vitro bladder model Methods: To establish an in-vitro bladder model, sterile double-walled glass vessels, aspirator bottle, catheters, and drainage bags were utilised, while a water bath and peristaltic pump were also incorporated into the setup. Tube ends were sealed in foil to further maintain sterility. The peristaltic pump maintained a steady urine flow, and a water bath with circulating water (37 °C) around the double-walled vessel regulated body temperature. The model was inoculated with a Proteus mirabilis quality control strain and clinical isolates (n=6), and functioned until the catheter became blocked. Expression levels of virulence genes were assessed before and after biofilm formation using quantitative real-time PCR (qPCR). Results: All four virulence genes showed a notable increase in expression after biofilm formation. Before biofilm formation, the relative expression levels of ureC, mrpA, rsbA, and speA ranged from 0.01-3.53, 0.02-2.26, 0.01-2.77, and 0.02-2.82, respectively. However, these values increased after biofilm formation, with expressions ranging from 0.57-34.77, 1.13-65.69, 0.13- 53.51, and 0.20-120.25 for ureC, mrpA, rsbA and speA, respectively. The fold change in gene expression further confirmed the upregulation, with ureC and rsbA showing the highest (1574- fold) and least (0.05-fold) increase, respectively. Expression of ureC and mrpA genes showed a significant increase following biofilm formation (Wilcoxon signed-rank test; p=0.036). Conclusions: The marked upregulation of ureC, rsbA, mrpA, and speA genes after biofilm formation highlights their key role in the persistence and development of P. mirabilis biofilms in catheterized environments.