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

Abstract

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...

Purported nanobacteria in human blood as calcium carbonate nanoparticles

“…Larger so-called ''shelter-like forms'' of several micrometers were also observed among these particles, albeit in much smaller number ( Fig. 1 C and D), again consistent with earlier reports (2). Although these structures were initially described as shelters for actively growing nanobacteria (2), they were seen in our own studies as being porous structures with partial crystallization inside.…”

“…To distinguish our findings from those in the literature, we refer to our own material as nanobacteria-like particles (NLP). The criteria used to confirm that we reproduced the phenomenology of putative nanobacteria were based on earlier publications (2,20). They included the typical bacterialike morphology seen by electron microscopy, slow doubling-time of 3 days, detection of HAP, and staining with nanobacterial antibodies.…”

“…The intriguing characteristics of human nanobacteria include extremely small size from 80 to 500 nm (12), 0.2-m filterability (3), widespread abundance in animals and humans (11), as well as nucleation of HAP (2). These unusual characteristics actually lead to the suggestions that nanobacteria may represent an overlooked primitive form of life (13) with the smallest cellular dimension known on Earth (14).…”

“…The hypothesis that nanobacteria might represent living microorganisms is based on an unusual set of microbiological characteristics that include their slow growth in liquid cell culture media (20), expression of specific antigens (3), and typical bacterial appearance (2). However, observations of abiotic substrates would caution against assigning any life activity to unusual biological entities because certain chemical substrates may adopt the appearance of simple microorganisms (22).…”

Diverse uncultivated ultra-small bacterial cells in groundwater

“…Over the following years, various publications proposed the existence of 'nannobacteria' (or nanobes) in terrestrial samples 2 , sometimes with implied medical 3 and environmental 2 significance. However, a lack of solid biological evidence for the existence of cells considered to be too small to accommodate sufficient genomic DNA, RNA, proteins and solvent for life consigned nanobacteria to the scientific fringe ('the cold fusion of microbiology' 4 ).…”