In Vivo Entombment of Bacteria and Fungi during Calcium Oxalate, Brushite, and Struvite Urolithiasis

Abstract

Background: Human kidney stones form via repeated events of mineral precipitation, partial dissolution and reprecipitation, which are directly analogous to similar processes in other natural and man-made environments where resident microbiomes strongly influence biomineralization. High-resolution microscopy and high-fidelity metagenomic (microscopy-to-omics) analyses, applicable to all forms of biomineralization, have been applied to assemble definitive evidence of in vivo microbiome entombment during urolithiasis. Methods: Stone fragments were collected from a randomly chosen cohort of 20 patients using standard percutaneous nephrolithotomy (PCNL). Fourier transform infrared (FTIR) spectroscopy indicated that 18 of these patients were calcium oxalate (CaOx) stone formers, while one patient each formed brushite and struvite stones. This apportionment is consistent with global stone mineralogy distributions. Stone fragments from 7 of these 20 patients (5 CaOx, 1 brushite and 1 struvite) were thin sectioned and analyzed using brightfield (BF), polarization (POL), confocal, superresolution autofluorescence (SRAF) and Raman techniques. DNA from remaining fragments, grouped according to each of the 20 patients, were analyzed with amplicon sequencing of 16S rRNA gene sequences (V1-V3, V3-V5) and internal transcribed spacer (ITS1, ITS2) regions. Results: Bulk entombed DNA was sequenced from stone fragments in 11 of the 18 CaOx patients, as well as the brushite and struvite patients. These analyses confirmed the presence of an entombed low-diversity community of bacteria and fungi, including Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria, and Aspergillus niger. Bacterial cells ~1 µm in diameter were also optically observed to be entombed and well-preserved in amorphous hydroxyapatite spherules and fans of needle-like crystals of brushite and struvite. Conclusions: These results indicate a microbiome is entombed during in vivo CaOx stone formation. Similar processes are implied for brushite and struvite stones. This evidence lays the groundwork for future in vitro and in vivo experimentation to determine how the microbiome may actively and/or passively influence kidney stone biomineralization.

Size Frequency Distributions, Fracture Patterns, and Reactive Surface Area of Shock Wave Lithotripsy-Derived Particles: A GeoBioMed Perspective on Kidney Stone Recurrence

“…Crystalline Architecture of PCNL-Derived Fragments. The high-and super-resolution optical, laser, x-ray microscopy, and Raman spectroscopy conducted in the present study indicate that the original PCNLderived fragments have a crystalline architecture that is consistent with those previously observed in CaOx kidney stones [38][39][40][41][42] . Following the approaches presented in these previous studies, the term "crystalline architecture" is herein used to refer to stone structure (crystal size, shape, intergrown morphologies), stratigraphy (crystal and organic matter layering), diagenetic phase transitions (postdepositional dissolution and recrystallization), and paragenesis (historical sequence of formational events).…”

“…Following the approaches presented in these previous studies, the term "crystalline architecture" is herein used to refer to stone structure (crystal size, shape, intergrown morphologies), stratigraphy (crystal and organic matter layering), diagenetic phase transitions (postdepositional dissolution and recrystallization), and paragenesis (historical sequence of formational events). Therefore, the approaches and terminology of GeoBioMed [38][39][40][41][42] have been directly adopted and applied in the present study. RL microscopy and x-ray micro-computed tomography (micro-CT) indicate that the original PCNL-derived fragments are irregular 4-12mm-diameter crystalline aggregates that exhibit SPL probe impressions and breakage patterns (Figs.…”

“…Individual crystal faces (sector zones) within COM C are common and form as the result of disequilibrium precipitation, during which ions and organic matter are differentially incorporated on age-equivalent crystal growth faces (Figs. 3B, C, H) [38][39][40][41][42]51 . Free-oating COM crystals (COM FF ) are entombed, either individually or in clusters, on growing COM C concentric surfaces that seed the growth of radiating crystal bundles (COM B ) that either truncate or redirect the growth of sector zones (Fig.…”

An apatite for kidney stones

“…More than 70% of kidney stones worldwide are primarily composed of calcium oxalate -a calcium salt of oxalic acid and one of the most commonly occurring organic minerals in nature 6 . However, virtually all calcium oxalate stones are composed of multiple minerals that include major to trace amounts of phosphate-rich apatites, brushite and struvite, as well as carbonates, uric acid and several others 7,8 . The complex multi-mineralogy of the stones results from interactions between the same factors in people that operate in all other natural systems 4 , such as temperature, flow rate, pH, ionic saturation, living cell activity (physiology, metabolism, biochemistry) and mineral growth and diagenesis.…”

Induced mineralization in Escherichia coli biofilms: the key role of bacterial alkaline phosphatase

“…8,16,17 This observation encouraged scientists to reconsider the role of bacteria in kidney stone formation where bacteria may not only become trapped, but could also play a significant role in stone growth. 8,18,19…”