Decoding Proteus mirabilis biofilms: expression of selected virulence genes and association with antibiotic resistance

Abstract

Background: Proteus mirabilis is a uropathogens with a strong ability to form resilient crystalline biofilms, particularly on urinary catheters, contributing to its persistence and antibiotic resistance. As biofilm-driven virulence is key in complicated urinary tract infections, understanding its virulence genes and resistance mechanisms is crucial for improving treatment strategies. We investigated the presence, and expression of key virulence genes (ureC, mrpA, speA, and rsbA) in biofilm-forming P. mirabilis strains sourced from both urine (n = 26) and non-urine specimens, such as pus, wounds, and blood isolates (n = 26) and analyzed their association with antimicrobial resistance profiles. The presence and expression of P.mirabilis's virulence genes were detected using conventional PCR and Quantitative real-time PCR assays (qPCR), respectively. Antibiotic susceptibility test (AST) was conducted using the Kirby-Bauer method, adhering to Clinical and Laboratory Standards Institute (CLSI) guidelines. Statistical analysis was performed using the R language.

Results: Virulence genes (ureC, mrpA, speA, and rsbA) exhibited high prevalence (> 92% in urine, > 84% in non-urine isolates) with no significant differences (Cochran's Q test; p = 0.801). Multidrug resistance (MDR) and extensively drug-resistant (XDR) were detected in 100% and 57.69% of urine isolates, and 96.15% and 65.38% of non-urine isolates, respectively. Gene combinations are strongly linked to higher resistance rates. mrpA exhibited the highest expression in urine-derived strains, followed by rsbA, and ureC, with speA having the lowest. Post-hoc analysis revealed a significant variation in the rsbA expression compared to speA (p = 0.029) and ureC (p = 0.007). speA showed the highest expression in non-urine isolates, followed by rsbA, ureC, and mrpA, with significant differences among all gene pairs (Conover's all-pairs test; p > 0.05) except rsbA vs. speA. XDR status showed no significant effect or interaction on gene expression in urine and non-urine isolates (p = 0.290; p > 0.05).

Conclusion: The virulence genes ureC, mrpA, speA, and rsbA are consistently found in P. mirabilis strains from both urine and non-urine specimens; however, their expression varies significantly, likely due to host or environmental factors. The presence of high multidrug-resistant (MDR) and extensively drug-resistant (XDR) P. mirabilis strains, potentially driven by the combination of these virulence genes, suggests an increase in virulence.

Keywords: Antibiotic resistance; Biofilm forming Proteus mirabilis; Expression; Genes; Urinary tract infections; Virulence.

Fig. 1

Distribution and co-occurrence of virulence genes in P. mirabilis clinical isolates *Image A represents isolates obtained from urine specimens (n = 26), while the image B represents isolates from non-urine clinical specimens (n = 26). The left, horizontal bar charts (blue) show the total number of strains positive for all four genes (mrpA, ureC, speA, rsbA). The matrix in the center (with filled dots; black) indicates specific gene combinations observed across strains. Those filled dots connected by lines to represent co-occurrence. The vertical bar charts (purple) display the number of strains exhibiting each unique combinations of virulence genes

Fig. 2

Antimicrobial susceptibility of P. mirabilis strains isolated from (a) urine and (b) non-urine specimens

Fig. 3

Gene expression differences among (A) urine-derived and (B) non-urine-derived P. mirabilis strains