Antibacterial properties and urease suppression ability of Lactobacillus inhibit the development of infectious urinary stones caused by Proteus mirabilis

Abstract

Infectious urolithiasis is a type of urolithiasis, that is caused by infections of the urinary tract by bacteria producing urease such as Proteus mirabilis. Lactobacillus spp. have an antagonistic effect against many pathogens by secreting molecules, including organic acids. The aim of the study was to determine the impact of Lactobacillus strains isolated from human urine on crystallization of urine components caused by P. mirabilis by measuring bacterial viability (CFU/mL), pH, ammonia release, concentration of crystallized salts and by observing crystals by phase contrast microscopy. Moreover, the effect of lactic acid on the activity of urease was examined by the kinetic method and in silico study. In the presence of selected Lactobacillus strains, the crystallization process was inhibited. The results indicate that one of the mechanisms of this action was the antibacterial effect of Lactobacillus, especially in the presence of L. gasseri, where ten times less P. mirabilis bacteria was observed, compared to the control. It was also demonstrated that lactic acid inhibited urease activity by a competitive mechanism and had a higher binding affinity to the enzyme than urea. These results demonstrate that Lactobacillus and lactic acid have a great impact on the urinary stones development, which in the future may help to support the treatment of this health problem.

Figure 1

Percentage of ammonia release inhibition (bars) in mixed cultures in synthetic urine, where pure P. mirabilis samples were 100%, and changes in urinary pH (lines) in pure and mixed samples after 3, 6, 8 and 24 h of incubation. (A) Corresponds to P. mirabilis KP; (B) P. mirabilis K8/MC; (C) P. mirabilis 5628 and (D) P. mirabilis 608/221. The results are presented as mean ± standard deviation (SD) of three experiments; *p < 0.05 for comparison of the pH value and ammonia release of P. mirabilis pure culture vs. co-culture with Lactobacillus, Mann–Whitney U test.

Figure 2

P. mirabilis viability after 3, 6, 8, and 24 h of incubation in pure and mixed cultures in synthetic urine. (A) corresponds to P. mirabilis KP; (B) P. mirabilis K8/MC; (C) P. mirabilis 5628 and (D) P. mirabilis 608/221. The results are presented as mean ± standard deviation (SD) of three experiments, **p < 0.01, *p < 0.05 for P. mirabilis viability in mixed cultures vs. pure culture, Mann–Whitney U test.

Figure 3

Calcium and magnesium concentrations in the tested and control samples after 3, 6, 8 and 24 h of incubation in synthetic urine. (A) Corresponds to P. mirabilis KP; (B) P. mirabilis K8/MC; (C) P. mirabilis 5628 and (D) P. mirabilis 608/221. The results are presented as mean ± standard deviation (SD) of three experiments, **p < 0.01, *p < 0.05 for P. mirabilis viability in mixed cultures vs. pure culture, Mann–Whitney U test.

Figure 4

Carbonate apatite (A) and struvite (S) crystals in mixed and pure cultures after 6 h of incubation. (1) corresponds to P. mirabilis KP; (2) P. mirabilis K8/MC; (3) P. mirabilis 5628 and (4) P. mirabilis 608/221 and (A) control samples; (B) samples with L. crispatus 1.2; (C) L. crispatus 4; (D) L. jensenii 22.2 and (E) L. gasseri 35.3. The scale bar represents 20 µm.

Figure 5

(A) The effect of Lactobacillus strains on crystallization in synthetic urine with phenol red caused by four tested P. mirabilis strains after 5 h of incubation in the ratio 1:5. (B) The effect of Lactobacillus strains on crystallization in synthetic urine with phenol red caused by the Jack bean urease (lines) and % of ammonia release inhibition (bars) in samples with Lactobacillus compared to the control with urease alone. (C) The effect of different concentrations of lactic acid on crystallization in synthetic urine with phenol red. The results are presented as mean ± standard deviation (SD) of three experiments; **p < 0.01, *p < 0.05 for comparison of controls vs. tested samples, Kruskal–Wallis test.

Figure 6

The Lineweaver–Burk plot showing the competitive inhibition of urease-catalysed hydrolysis of urea by different concentrations of lactic acid (A). Influence of increased concentrations of urea on urease activity in the presence of 38 mM lactic acid; data are presented as inhibition percentage of ammonia release (B); zero value means no inhibition of urease activity. The Michaelis–Menten plot of the predicted reaction rate of urea hydrolysis by urease as a function of a substrate concentration is shown in Fig. S1 (Supplementary Material).

Figure 7

The three-subunit structure of P. mirabilis urease (homo-trimer) built using homology modeling methods; nickel ions in an active site of subunits are marked in red; three subunits are in different colors.

Figure 8

(A) Lactic acid (LA, in orange) docked to the catalytic center of subunit alpha of P. mirabilis urease located in the vicinity of two nickel ions (pink spheres). (B) The best docking pose of LA (orange) interacting with amino acids (blue tubes) in the active center of P. mirabilis urease, binding energy ΔG −8.22 kcal/mol (estimated Ki 0.94 μM); hydrogen bonds between LA and His219, Ala167 and Gly277 are visualized as solid lines; long-range interactions, salt bridges, between lactic acid carboxyl and imidazole groups of five histidine residues are marked as dashed yellow lines; yellow spheres are charge centers in imidazole rings and LA carboxyl; complexation of Ni ions (pink balls) are marked as dark dotted lines; hydrogen atoms are omitted for clarity. Structures were analyzed and visualized in the PLIP 2.2.0 and PyMOL 2.3.4 software. (C) A table with listed amino acids interacting with LA through hydrogen bond interaction and salt bridges; calculations in PLIP 2.2.0 software.