All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional (Ethical clearance approval number YUEC.022/16), national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The study was approved by the Institutional Ethics Committee (IEC) and Scientific Review Board (SRB) of Yenepoya (Deemed to be University), India. Written informed consents were obtained from each participating patient prior to specimen collection.

All chemicals used were purchased from commercial sources. Sodium metasilicate (Na₂SiO₃), ammonium dihydrogenphosphate (NH₄H₂PO₄) and magnesium acetate solution (C₄H₆MgO₄) were of analytical grade. These chemicals were dissolved in Milli-Q Type 1 water. The synthetic urine used for biogenic crystallization was prepared according to Griffith et al. [15]: CaCl₂. 2H₂O, 0.651; MgCl₂. 6H₂O, 0.651; NaCl, 4.6; Na₂SO₄, 2.3; KH₂PO₄, 2.8; KCl, 1.6; NH₄Cl, 1.0; sodium citrate, 0.65; sodium oxalate, 0.02; urea, 25.0; creatine, 1.1; and tryptic soy broth, 10.0 (g/L). The content of the mineral components in synthetic urine corresponds to mean concentration found in 24 h period in normal human urine.

Surgically removed struvite stones were collected from patients who had undergone percutaneous nephrolithotomy (PCNL). Presence of struvite composition was confirmed by Fourier transform infrared spectroscopy (FT-IR) and X-ray powder diffraction (XRD). The collected stones were washed with sterile distilled water, homogenized and plated onto MacConkey agar, and Nutrient agar (Hi-media, Mumbai). The plates were incubated aerobically at 37°C for 24 h. Growth of bacteria was observed and total numbers of colonies were counted.

For bacteriology, kidney stone homogenates were transferred to sterile distilled water and further inoculated to MacConkey agar and Nutrient agar and incubated for 24 h at 37°C. Following incubation, each colony was counted using a digital colony counter and further processed for Gram staining. The bacteria were taxonomically identified using 16S rRNA gene sequencing. Briefly, genomic DNA was extracted from the bacterial cells harvested from tryptic soy broth by phenol: chloroform extraction method. The concentration of resulting DNA was measured using Nanodrop-1000 (Thermo Scientific, USA). The genomic DNA was amplified in polymerase chain reaction using standard primers namely 27F (5′-CCAGAGTTTGATCMTGGCTCAG-3′) and 1490R (5′-GGTTACCTTGTTACGACTT-3′) [16]. Amplifications were performed in a Thermal Cycler under the conditions: initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation at 94°C for 1 min, primer annealing for 1 min at 55°C, and primer extension for 1 min at 72°C with a final extension period at 72°C for 10 min. The amplified product was purified by precipitation using 20% Polyethylene glycol (using 2.5 M NaCl) and sequenced both using forward and reverse primers using the BigDye Terminator v3.1 Cycle Sequencing Kit and ABI 3730 XL DNA Analyzer (Applied Biosystems Inc, USA).

Initial early stage crystallization was carried out directly and observed using light microscopy. Single diffusion gel growth technique was used to grow the struvite crystals as described elsewhere [3, 10, 17]. Briefly, sodium metasilicate solution of specific gravity 1.05 was used to prepare the gel. An aqueous solution of ammonium di-hydrogen phosphate (0.5 M) was mixed with sodium metasilicate solution in an appropriate amount to set the pH of the gel at 7.0. Bacteria cultured in trypticase soy broth overnight at 37°C were suspended in synthetic urine to a concentration of 5x10⁵ CFU/mL. Prior to gelation experiments, bacterial growth in the synthetic urine was assessed to check the viability. After the gelation took place, 20 mL supernatant solutions of 0.5 M magnesium acetate was gently poured on the gels in the test tubes (140 mm length and 25 mm diameter) without disturbing the gel. The same compositions were also used for control without bacteria in synthetic urine. All experiments were performed in triplicate under aseptic conditions incubated at a temperature of 37 ± 0.5°C. During the experiment, pH of the samples was measured using a digital pH meter and samples from different stages of the crystallization process were harvested for investigations. Structural attributes of the in vitro generated struvite crystals and patient-derived struvite were studied by FT-IR, XRD, and FESEM as explained elsewhere [18].

Struvite crystals grown in the presence and absence of bacteria were studied by X-ray micro-tomography experiments conducted at X-ray Imaging Beamline (BL-04) on Indus-2 Synchrotron Source RRCAT, Indore (Fig 1) [19]. The 2D projection images for all the samples were acquired at 15 keV beam energy obtained from fixed exist Si (111) double crystal monochromator (DCM) at 150 mm sample to detector distance. The projection images were acquired with rotation step size of 0.2^o for total 180^o. The exposure time taken was 500 m sec. for each projection including the bright and dark field images. High-resolution CCD detector with pixel size 4.5 and an active area of 4008 x 2670 pixels was used to acquire the experimental images. The data acquired was preprocessed, reconstructed and 3D volume rendering was done by Drishti visualization software [20]. Quantitative analysis of the SR-μCT data on porosity variations was carried out using ImageJ software. For calculation of porosity, the 3D volume was first binarized using Otsu method of threshold followed by noise removal using the median filter. Patient derived stones were also similarly investigated.

Two urease positive strains namely, YU22S and YU27S isolated from the stone culture of the patients with struvite stones were used for in vitro struvite crystallization. Based on 16S rRNA gene sequencing, the isolates YU22S and YU27S were identified respectively as P. aeruginosa and E. cloacae. The NCBI GenBank accession numbers for the sequences are MH021663 and MH021673 respectively. The cultures were deposited at Microbial Culture Collection Center (NCMR, Pune) with culture collection numbers; YU22S as MCC 3101 and YU27S as MCC 3111.

The early stage (10 min- 48 h) crystallization of struvite revealed by the initial experiment is given in Fig 2. During this, the size varied from a few microns to more than 10 microns. Struvite crystals in both control and bacteria induced sets exhibited a coffin-like habit with well-defined planes growing to X-shaped dendrite crystal (Fig 2iv–2vi). The bacteria induced struvite subsequently formed twins (Fig 2viii–2xi).

The time-resolved experiments revealed the growth of small dendritic type struvite crystals immediately after the addition of supernatant solution, at the gel-liquid interface. The initially grown pinpoint crystal tubes tend to nucleate randomly throughout the test tube and on the gel surface (S1 Fig). In the presence of bacteria, the pH of the supernatant solution at this stage increased gradually.

At the end of third-day, the crystals grown in all the conditions appeared as single crystals exhibiting coffin-like habit with well-defined planes. In the presence of P. aeruginosa and E. cloacae X-shaped dendrite crystals were observed the end of the fifth day, with high accumulation of crystals compared to control. The aggregation of crystals was rapid in the presence of bacteria compared to the control sets. At pH greater than 9, the hopper like crystals were also observed coexisting with dendrite-like crystals.

At the initial stages of struvite mineralization, crystals displayed well-defined faces (Fig 3). The arrowhead-shaped crystals grown in the presence of bacteria showed preferential growth in (001), (101), (Ῑ01) and (012) faces (Fig 3). The SR- μCT of struvite crystals is presented in Fig 4. At the second stage, morphology of the crystals formed in the bacterial set was distinct presenting an intermediate form between the single and dendrite-like crystals (Fig 4iii). Distinct X-shaped structures developed further in the presence of bacteria (Fig 4iv). Growth of these X-shaped crystals occurred along the (111) direction of every assembled subunit. The crystals formed in the control also showed the X- shaped morphology and the general morphology of the X- shaped crystals displayed differences between the control and the bacterial induced crystallization. The patient derived stones presented layered pattern and exhibited void spaces with cracks inwards from the outer surfaces (S2 Fig).

The percentage porosity showed a distinct variation at different stages of struvite formation in the presence and absence of bacteria (S1 Table). The mean porosity of bacterially induced struvite crystal increased from initial 0.0098% to final stage 11.45%. But, in the control the porosity decreased from 12.17% to 8.43% from initial to final stage.

The FESEM images from single struvite crystals showed well-defined faces, and the porous nature of struvite was distinctly seen in all the struvite crystals (Fig 5). The pore sizes of bacterially induced struvite crystals were in the range from tens to hundreds of nanometers and the middle of the cross section in the mineral architecture displayed rougher and high porous appearance compared to control (Fig 5). Aggregates of bacteria in the struvite crystals were observed resembling a biofilm architecture (Fig 6). Naturally derived struvite stones consisted of layered pattern and exhibited void spaces with cracks inwards from the outer surfaces.

The crystals showed characteristic IR spectrum with a strong peak at 1010 cm^-1 due to the absorption of PO4^3- (v3 mode) antisymmetric stretch. The absorption peaks at 1469, 1435 and 1400 cm^-1 attributed to (v4) NH₄⁺ antisymmetric bending were also present.

The XRD pattern revealed the appearance of struvite crystallizes in the orthorhombic Pmn21 space group (unit cell parameters, a = 6.955 Å, b = 11.2 Å, c = 6.142 Å). Representative XRD patterns for the crystals obtained with and without bacteria are shown in Fig 7. All the diffraction peaks were well indexed as struvite without any traces of other impurity phases. The bacteria treated samples showed higher intensity in the peak corresponding to the (221) plane as compared to the control sample.

In conclusion, a series of time-course experiments with and without bacteria revealed that urease-producing bacteria such as P. aeruginosa and E. cloacae play a major role in the specific morphogenesis of struvite and these results demonstrate that the bacteria can control the nucleation, modulate crystalline phases and shape of the growing crystal. The biogenic struvite initially displayed a small arrowhead-shaped morphology subsequently evolving into large X-shaped tabular structures. Presence of bacteria can also induce transformations in crystal morphology.