The natural microbiota of the urinary tract includes Lactobacillus spp., which secrete molecules with antimicrobial properties and have antagonistic activity against many pathogens. This paper focuses on the antibacterial effect of Lactobacillus strains isolated from urine against clinical strains of Proteus mirabilis isolated from kidney stones and from urine with coexisting urolithiasis. The study involved analyzing the main antimicrobial molecules secreted by Lactobacillus. In order to indicate which agent had the strongest antimicrobial effect, the supernatants were made alkaline and treated with catalase and high temperature. Both treated and untreated supernatants were analyzed for their activity. Exposing uropathogens to all untreated cell-free supernatants of Lactobacillus significantly reduced their growth, and it was established that these properties were related to organic acid secretion by these strains. Using LC–MS/MS and spectrophotometric techniques, lactic, citric, and succinic acids were determined qualitatively and quantitatively. The influence of these acids on the P. mirabilis growth and biofilm formation and their influence on membrane permeability were also investigated. The results indicate that organic acids secreted by Lactobacillus strains have a high antibacterial potential and could be used as novel agents in the treatment of urinary tract infections caused by P. mirabilis.

Proteus mirabilis urinary tract infections can lead to serious complications such as development of urinary stones. Lactobacillus spp., belonging to the natural microbiota of the urinary tract, exhibit a number of antagonistic mechanisms against uropathogens, including the secretion of organic acids. In this study, we determined the anti-adhesion, anti-cytotoxicity and anti-crystallization properties of the substances secreted by Lactobacillus. For this purpose, membrane inserts with a pore diameter 0.4 μm were used, which prevent mixing of cultured cells, simultaneously enabling the diffusion of metabolic products. The intensity of crystallization was assessed by measuring the levels of Ca²⁺, Mg²⁺ and NH₃ and by observing crystals using microscopic methods. The cytotoxicity of the HCV-29 cell line was determined using the LDH and MTT assays, and the impact of lactobacilli on P. mirabilis adhesion to the bladder epithelium was assessed by establishing CFU/mL after cell lysis. It was shown that in the presence of L. gasseri the adhesion of P. mirabilis and the cytotoxicity of the cells decreased. The degree of crystallization was also inhibited in all experimental models. Moreover, it was demonstrated that L. gasseri is characterized by the secretion of a high concentration of L-lactic acid. These results indicate that L-lactic acid secreted by L. gasseri has a significant impact on the crystallization process and pathogenicity of P. mirabilis.

Proteus mirabilisis a common cause of infectious urolithiasis. The first stage in the formation of urinary stones is the crystallization of mineral salts in the urine induced by urease activity of this microorganism.Lactobacillusspp.are an important component of the human microbiota and in large quantities occur in foods. Regardless of their origin, those with probiotic properties are proposed as an alternative to antibiotic therapy in the treatment of urinary tract infections. The aim of the study was to check the effect of selectedLactobacillus plantarumandLactobacillus brevisstrains on crystallization caused byP. mirabilisin an in vitro experiment. It has been confirmed that selectedLactobacillusstrains have antibacterial properties and colonize the urinary tract epithelium. During 24-h incubation of bacterial cultures, containingP. mirabilisand individualLactobacillusstrains, in synthetic urine, bacterial viability (CFU/mL), pH, and crystallization were determined. Crystallization was assessed quantitatively and qualitatively using AAS and XRD techniques as well as phase-contrast microscopy. It has been shown that in the presence of selectedLactobacillusstrains, the culture pH increases faster, especially after 8 h of incubation, compared with the pureP. mirabilisculture. An increase in pH reduces the viability ofP. mirabilis; however, in the presence of some lactobacilli, the uropathogen grows more intensively. The presence ofLactobacillusalso affected crystallization by increasing its intensity, and the resulting crystals were larger in size. TestedL. plantarumandL. brevisstrains could therefore accelerate the formation of urinary stones and development of infection.

Proteus mirabilis-mediated CAUTIs are usually initiated by the adherence of bacteria to a urinary catheter surface. In this paper, three isolates of different origin and exhibiting different adhesion abilities were investigated in search of any changes in lipidome components which might contribute to P. mirabilis adhesion to catheters. Using GC-MS and LC-MS/MS techniques, 21 fatty acids and 27 phospholipids were identified in the examined cells. The comparison of the profiles of phospholipids and fatty acids obtained for catheter-attached cells and planktonic cells of the pathogens indicated C11:0 and PE 37:2 levels as values which could be related to P. mirabilis adhesion to a catheter, as well as cis C16:1, PE 32:0, PE 33:0, PE 38:2, PG 33:1, PG 34:0, PE 30:1, PE 32:1 and PG 30:2 levels as values which could be associated with cell hydrophobicity. Based on DiBAC₄ (3) fluorescence intensity and an affinity to p-xylene, it was found that the inner membrane depolarization, as well as strong cell-surface hydrophobicity, were important for P. mirabilis adhesion to a silicone catheter. A generalized polarization of Laurdan showed lower values for P. mirabilis cells attached to the catheter surface than for planktonic cells, suggesting lower packing density of membrane components of the adherent cells compared with tightly packed, stiffened membranes of the planktonic cells. Taken together, these data indicate that high surface hydrophobicity, fluidization and depolarization of P. mirabilis cell membranes enable colonization of a silicone urinary catheter surface.

Outer membrane vesicles (OMVs) are extracellular structures, ranging in size from 10 to 300 nm, produced by Gram-negative bacteria. They can be incorporated into the outer membrane of a recipient’s cells, which may enable the transfer of substances with lytic properties. Due to the scarce information regarding the OMVs produced by Proteus mirabilis, the aim of this study was to test the blebbing abilities of the clinical P. mirabilis O77 and O78 strains and to determine the blebs’ interactions with bacterial cells, including their possible bactericidal activities. The production of OMVs was visualised by Transmission electron microscopy (TEM). The presence of OMVs in the obtained samples as well as the phenomenon of OMV fusion to recipient cells were confirmed by Enzyme-Linked ImmunoSorbent Assay (ELISA) and Western blotting assays. The bacteriolytic activity of the OMVs was examined against P. mirabilis clinical strains and reference Staphylococcus aureus and Escherichia coli strains. It was shown that each of the two tested P. mirabilis strains could produce OMVs which were able to fuse into the cells of the other strain. The lytic properties of the O78 OMVs against another P. mirabilis O78 strain were also demonstrated. This promising result may help in the future to better understand the mechanisms of the pathogenesis and to treat the infections caused by P. mirabilis.

Infection urinary stones account for approximately 10–15% of all urinary stones worldwide, with a rising incidence observed in recent decades, particularly in countries with a high Socio-Demographic Index (SDI). This trend has been partially attributed to dietary changes, including increased consumption of processed foods. Heavy metals belong to a group of substances, the source of which can be both food and the human environment. Among many heavy metals, in this study, we focus on copper and investigate its influence on the nucleation and growth of struvite crystals, the primary component of infection urinary stones. Experiments were conducted in artificial urine, both in the presence and absence of Proteus mirabilis, a urease-producing bacterium commonly associated with infection urinary stones. In a bacteria-free system, bacterial urease activity was mimicked by the addition of aqueous ammonia solution. Our results demonstrate that the presence of copper in artificial urine induces a slight shift in the struvite crystallization toward lower pH values, indicating that crystal formation initiates earlier compared to a control test. Additionally, the amount of precipitated struvite increases modestly in the presence of copper. Struvite crystals formed in copper-containing artificial urine are larger and exhibit altered habit and morphology. Energy-dispersive X-ray spectroscopy (EDS) and X-ray photoelectron spectroscopy (XPS) analyses confirm that copper does not incorporate into either the bulk or surface structure of the struvite crystals. X-ray diffraction (XRD) data show that struvite remains the sole crystalline phase, consistent with the control samples. Microbiological assays reveal that copper, at the concentrations tested, does not affect the viability of P. mirabilis, indicating an absence of bacteriostatic or bactericidal effects. To elucidate the physicochemical mechanisms underlying copper’s influence on nucleation and growth of struvite, speciation analysis of chemical complexes was performed. This revealed the formation of various copper complexes in artificial urine, including Cu(OH)⁺, CuCit⁻, CuC₂O₄, Cu(OH)₂, CuHPO₄, Cu(NH₃)²⁺, Cu(NH₃)₂²⁺, and Cu(NH₃)₃²⁺. These chemical complexes modulate the equilibrium and formation of complexes with Mg²⁺ and PO₄³⁻ (e.g., MgHCit, MgCit⁻, MgOH⁺, MgC₂O₄, MgSO₄, MgHPO₄), contributing to the observed shift in struvite crystallization to lower pH values.