We propose that kidney stone formation is a nanobacterial disease analogous to Helicobacter pylori infection and peptic ulcer disease. Both diseases are initiated by bacterial infection and subsequently endogenous and dietary factors influence their progression.

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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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Nanobacteria or its antigens were present in PKD kidney, liver, and urine. The identification of candidate microbial pathogens is the first step in ascertaining their contribution, if any, to human disease.

Compounds from 16 classes of antimicrobial drugs were tested for their abilities to inhibit the in vitro multiplication of nanobacteria (NB), a newly discovered infectious agent found in human kidney stones and kidney cyst fluids from patients with polycystic kidney disease (PKD). Because NB form surface calcifications at physiologic levels of calcium and phosphate, they have been hypothesized to mediate the formation of tissue calcifications. We describe a modified microdilution inhibitory test that accommodates the unique growth conditions and long multiplication times of NB. This modified microdilution method included inoculation of 96-well plates and determination of inhibition by periodic measurement of the absorbance for 14 days in cell culture medium under cell culture conditions. Bactericidal or bacteriostatic drug effects were distinguished by subsequent subculture in drug-free media and monitoring for increasing absorbance. NB isolated from fetal bovine serum (FBS) were inhibited by tetracycline HCl, nitrofurantoin, trimethoprim, trimethoprim-sulfamethoxazole, and ampicillin at levels achievable in serum and urine; all drugs except ampicillin were cidal. Tetracycline also inhibited multiplication of isolates of NB from human kidney stones and kidney cyst fluids from patients with PKD. The other antibiotics tested against FBS-derived NB either had no effect or exhibited an inhibitory concentration above clinically achievable levels; the aminoglycosides and vancomycin were bacteriostatic. Antibiotic-induced morphological changes to NB were observed by electron microscopy. Bisphosphonates, aminocaproic acid, potassium citrate-citric acid solutions, and 5-fluorouracil also inhibited the multiplication of NB in a cidal manner. Insights into the nature of NB, the action(s) of these drugs, and the role of NB in calcifying diseases may be gained by exploiting this in vitro inhibition test system.

Nanobacteria, also known as calcifying nanoparticles (CNP), are controversial infectious agents not matching the current criteria for ‘living organism’. Despite the controversy of their classification, they propagate and cause cell death in vitro and are associated or found in many human diseases. Thus, more efforts should be focussed on research on pathogenicity of CNP.

Life on Earth and Mars could have started with self-assembled nanovesicles similar to the present nanobacteria (NB). To resist extreme environmental stress situations and periods of nutritional deprivation, nanovesicles would have had a chemical composition protected by a closed mineralized compartment, facilitating their development in a primordial soup, or other early wet environment. Their survivability would have been enhanced if they had mechanisms for metabolic communication, and an ability to collect primordially available energy forms. Here, we establish an irreducible model system for life formation starting with NB.