Struvite (MgNH₄PO₄·6H₂O) and carbonate apatite (Ca₁₀(PO₄)₆CO₃; CA) are the major components of infectious urinary stones. Urine of a healthy person is undersaturated with regard to struvite and CA formation. However, infectious stones are formed in the case of urinary tract infection by urease-producing bacteria. Urease – bacterially produced enzyme – splits urea, natural component of urine – into ammonia and carbon dioxide. This in turn raises the pH of the urine. Under alkaline conditions, an increase in the concentration of the ammonium, carbonate and phosphate ions occurs. These ions together with the ions of calcium and magnesium present in the urine lead to the crystallization of CA and then of struvite.

In the present study we describe the effect of disodium EDTA on the mineralization of CA and struvite in the solution of artificial urine in the absence and presence of Proteus mirabilis and the comparative study of the growth processes. The aim of this study is to determine and understand the role of Proteus mirabilis in the formation of CA and struvite with the presence of disodium EDTA. The present study is a continuation of the investigation of an influence of disodium EDTA on the growth of struvite and carbonate apatite in the absence of bacteria [1]. 

Microorganisms from Proteus species are isolated in the case of 70% of infectious stones [2,3]. Proteus includes, among others, Proteus mirabilis responsible for urinary tract infections and was chosen to our study. Crystallization occurs after addition of the suspension of bacteria to the solution of artificial urine and incubation at 37^o C. The experiment designed in such a manner guarantees that the crystallization process occurs at conditions emulating the natural conditions existing in human body during the infection by Proteus mirabilis. Control experiments without bacteria were performed using artificial urine of the same composition. In this case the crystallization process occurs after consecutive addition of aqueous ammonia solution. Such an addition causes an increase in pH value and the concentration of the ammonium ions. In other words the addition of aqueous ammonia solution mimics urease activity and therefore emulates real urinary tract infection [4]. 

The results demonstrate that in both cases – in the presence and in the absence of bacteria – disodium EDTA reduces the mass of struvite and CA formed in the solution of artificial urine. The addition of disodium EDTA to the urine does not affect the morphology and habit of single crystals. However, in the presence of bacteria, when pH value increases, the presence of single crystals vanishes progressively and the majority of the crystals are hopper and dendrite-like. The evolution of dendrite-like crystals is mainly influenced by the rate of change in pH. Kinetics of pH changes is faster in the presence of bacteria compared to the absence of bacteria. Therefore, in the case of absence of bacteria, dendrite-like crystals are observed very rarely. In both cases (with and without bacteria), increasing disodium EDTA concentration causes the induction time increase compared to baseline (without disodium EDTA). Therefore, the pH level of urine increases much more slowly in the presence of disodium EDTA compared to baseline. For this reason urine is undersaturated with respect to struvite and CA formation for longer time compared with the control test (without disodium EDTA).

The results demonstrate also that in the presence of bacteria the number of formed nuclei decreases compared with the absence of bacteria. This means that the nucleation rate decreases in the presence of bacteria. The nucleation rate is defined as the number of nuclei generated per unit volume and per unit time and is correlated with surface energy crystal-solution denoted by γ. Our results suggest that the surface energy γ is enlarged in the presence of bacteria. Increasing the surface energy leads to decreasing in the nucleation rate. Consequently, the number of formed nuclei decreases. These nuclei grow to larger crystals compared with the crystals which grow in the case of absence of bacteria. Consequently, the struvite crystals which grow in the presence of bacteria are larger and the number of crystals is less compared with the absence of bacteria. To sum up, the results demonstrate that the growth process is modified in the presence of bacteria and provide evidence for the importance of biological regulation in crystallization process.

References

[1] Prywer, J., Olszynski, M. J. Crystal Growth  375 (2013) 108–114.

[2] Lerner, SP, Gleeson MJ, Griffith DP. J. Urol.  141 (1989) 753–758.

[3] Kramer G, Klinger HC, Steiner GE. Curr. Opin Urol.  10 (2000) 35–38.

[4] Prywer, J.; Torzewska, A. Cryst. Res. Technol.  45 (2010) 1283–1289.

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1. In vitro studies on disodium EDTA effect on mineralization of struvite and carbonate apatite
2. Influence of Proteus mirabilis on the growth process and morphology of struvite crystals
3. The theoretical analysis of growth habits of sodium fluorosilicate ice-analogues crystals grown from solution and gel
4. Growth morphology of crystals from kinetic and geometric perspective