Observations of the Ultrastructure of Infected Kidney Stones

McLean, Robert; Nickel, J. Curtis; Beveridge, Terrance; Costerton, J. W.

doi:10.1099/00222615-29-1-1

“…Only a few models have been published for studying apatite mineralization in vitro (19)(20)(21), e.g.,: (i) Bacterial models of (kidney) stones using bacteria having urease and often AP activity (22,23). (ii) Osteoblastic cell cultures for studying bone formation.…”

Section: Discussionmentioning

confidence: 99%

Nanobacteria: An Alternative Mechanism for Pathogenic Intra- and Extracellular Calcification and Stone Formation

Kajander

¹

,

Çiftçioğlu

²

1999

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Calcium phosphate is deposited in many diseases, but formation mechanisms remain speculative. Nanobacteria are the smallest cell-walled bacteria, only recently discovered in human and cow blood and commercial cell culture serum. In this study, we identified with energydispersive x-ray microanalysis and chemical analysis that all growth phases of nanobacteria produce biogenic apatite on their cell envelope. Fourier transform IR spectroscopy revealed the mineral as carbonate apatite. The biomineralization in cell culture media resulted in biofilms and mineral aggregates closely resembling those found in tissue calcification and kidney stones. In nanobacteria-infected fibroblasts, electron microscopy revealed intra-and extracellular acicular crystal deposits, stainable with von Kossa staining and resembling calcospherules found in pathological calcification. Previous models for stone formation have led to an hypothesis that elevated pH due to urease and͞or alkaline phosphatase activity is a lithogenic factor. Our results indicate that carbonate apatite can be formed without these factors at pH 7.4, at physiological phosphate and calcium concentrations. Nanobacteria can produce apatite in media mimicking tissue f luids and glomerular filtrate and provide a unique model for in vitro studies on calcification.The formation of discrete and organized inorganic crystalline structures within macromolecular extracellular matrices is a widespread biological phenomenon generally referred to as biomineralization. Mammalian bone and dental enamel are examples of biomineralization involving apatite minerals. The molecular basis of mineralization remains largely unknown (1). Recently, bacteria have been implicated as factors in biogeochemical cycles for mineral formation in aqueous sediments (2, 3). The principal constituent of modern authigenic phosphate minerals in marine sediments is carbonate (hydroxy)fluorapatite Ca 10 (PO 4 ) 6-x (CO 3 ) x (F,OH) 2ϩx . Microorganisms are capable of depositing apatite outside thermodynamic equilibrium in sea water. They can segregate Ca from Mg and actively nucleate carbonate apatite by means of specific oligopeptides under conditions pH Ͻ 8.5 and [Mg]:[Ca] Ͼ 0.1 (4). Such conditions are also present in the human body.We have discovered nanobacteria in human and cow blood that are cytotoxic in vitro (5) and in vivo (6). They have been deposited in DSM (no. 5819-5821; Braunschweig, Germany). Nanobacteria possess unusual properties, making their detection difficult with standard microbiological methods. Although they typically had diameters of 0.2-0.5 m, they passed through 0.1-m filters probably because tiny forms (0.05-0.2 m) were also observed in transmission electron microscopy (TEM) (7). Nanobacteria were poorly disruptable, stainable, fixable, and exceptionally resistant to heat (8, 9). Their doubling time was about 3 days. High doses of ␥-irradiation or aminoglycoside antibiotics prevented their multiplication. According to the 16S rRNA gene sequence (EMBL X98418 and X98419), na...

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“…Only a few models have been published for studying apatite mineralization in vitro (19)(20)(21), e.g.,: (i) Bacterial models of (kidney) stones using bacteria having urease and often AP activity (22,23). (ii) Osteoblastic cell cultures for studying bone formation.…”

Section: Discussionmentioning

confidence: 99%

Nanobacteria: An alternative mechanism for pathogenic intra- and extracellular calcification and stone formation

Eo

¹

,

Çiftçioğlu

²

1998

Proc. Natl. Acad. Sci. U.S.A.

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Calcium phosphate is deposited in many diseases, but formation mechanisms remain speculative. Nanobacteria are the smallest cell-walled bacteria, only recently discovered in human and cow blood and commercial cell culture serum. In this study, we identified with energydispersive x-ray microanalysis and chemical analysis that all growth phases of nanobacteria produce biogenic apatite on their cell envelope. Fourier transform IR spectroscopy revealed the mineral as carbonate apatite. The biomineralization in cell culture media resulted in biofilms and mineral aggregates closely resembling those found in tissue calcification and kidney stones. In nanobacteria-infected fibroblasts, electron microscopy revealed intra-and extracellular acicular crystal deposits, stainable with von Kossa staining and resembling calcospherules found in pathological calcification. Previous models for stone formation have led to an hypothesis that elevated pH due to urease and͞or alkaline phosphatase activity is a lithogenic factor. Our results indicate that carbonate apatite can be formed without these factors at pH 7.4, at physiological phosphate and calcium concentrations. Nanobacteria can produce apatite in media mimicking tissue f luids and glomerular filtrate and provide a unique model for in vitro studies on calcification.The formation of discrete and organized inorganic crystalline structures within macromolecular extracellular matrices is a widespread biological phenomenon generally referred to as biomineralization. Mammalian bone and dental enamel are examples of biomineralization involving apatite minerals. The molecular basis of mineralization remains largely unknown (1). Recently, bacteria have been implicated as factors in biogeochemical cycles for mineral formation in aqueous sediments (2, 3). The principal constituent of modern authigenic phosphate minerals in marine sediments is carbonate (hydroxy)fluorapatite Ca 10 (PO 4 ) 6-x (CO 3 ) x (F,OH) 2ϩx . Microorganisms are capable of depositing apatite outside thermodynamic equilibrium in sea water. They can segregate Ca from Mg and actively nucleate carbonate apatite by means of specific oligopeptides under conditions pH Ͻ 8.5 and [Mg]:[Ca] Ͼ 0.1 (4). Such conditions are also present in the human body.We have discovered nanobacteria in human and cow blood that are cytotoxic in vitro (5) and in vivo (6). They have been deposited in DSM (no. 5819-5821; Braunschweig, Germany). Nanobacteria possess unusual properties, making their detection difficult with standard microbiological methods. Although they typically had diameters of 0.2-0.5 m, they passed through 0.1-m filters probably because tiny forms (0.05-0.2 m) were also observed in transmission electron microscopy (TEM) (7). Nanobacteria were poorly disruptable, stainable, fixable, and exceptionally resistant to heat (8, 9). Their doubling time was about 3 days. High doses of ␥-irradiation or aminoglycoside antibiotics prevented their multiplication. According to the 16S rRNA gene sequence (EMBL X98418 and X98419), na...

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“…which has the ability to form crystals inside the host cells (7), but other bacterial species such as Pseudomonas spp., Escherichia coli or Staphylococcus aureus are also involved in nephrolithiasis (8). After calculi development, the germs can survive in the inorganic matrix, where the antibiotics do not penetrate, thus the persistent infection can determine the growth of calculi in all the kidney cavities (calyx, pelvis) in time of weeks or months (9). Several studies showed that the urinary lithiasis can be induced by chronic or persistent UTI (so-called "infection-induced stones").…”

Section: Introductionmentioning

confidence: 99%