Morphological and immunological characteristics of nanobacteria from human renal stones of a north Indian population

Abstract

The aim of this study was to detect, isolate and characterize the nanobacteria from human renal stones from a north Indian population, and to determine their role in biomineralization. Renal stones retrieved from the kidneys of 65 patients were processed and subjected to mammalian cell culture conditions. The isolated bacteria were examined using scanning (SEM) and transmission electron microscopy (TEM). They were characterized for the presence of DNA, proteins and antigenicity. The role of these bacteria in biomineralization was studied by using the (14)C-oxalate based calcium oxalate monohydrate (COM) crystallization assay. We observed the presence of apatite forming, ultrafilterable gram negative, coccoid microorganisms in 62% of the renal stones. SEM studies revealed 60-200 nm sized organisms with a distinct cell wall and a capsule. TEM images showed needle like apatite structures both within and surrounding them. They were heat sensitive, showed antibiotic resistance and accelerated COM crystallization. A potent signal corresponding to the presence of DNA was observed in demineralized nanobacterial cells by flow cytometry. The protein profile showed the presence of several peptide bands of which those of 18 kDa and 39kDa were prominent. Apatite forming nanosized bacteria are present in human renal stones and may play a role in the pathophysiology of renal stone formation by facilitating crystallization and biomineralization. However, further studies are required to establish the exact mechanism by which nanobacteria are involved in the causation of renal stones.

The role of calcifying nanoparticles in biology and medicine

“…Bacterioscopic (DNA-specific dyes Hoechst 33258, propidium iodide, PicoGreen, staining after a demineralization is optimal) using scanning and transmission electron microscopy, [16][17][18][19][20][21][22][23][24] von Kossa staining, which is specific to calcium compounds, 16 staining by 2% uranyl acetate (possibly with lead citrate) to detect specific mucus on the hydroxyapatite shell, 1 staining by alizarin red S in the mineralized state, 26 staining by phosphotungstic acid, 16 after the long-term cultivation light microscopy with von Kossa staining is possible to be used for detection. 1,16 Bacteriological (cultivation in DMEM or RPMI-1640 without serum under 37°C for 4-6 weeks after filtration through 0.10-0.22 μm pores), 1,16 replication can be assessed by spectrophotometry (650 nm wavelength).…”

“…1,16 Bacteriological (cultivation in DMEM or RPMI-1640 without serum under 37°C for 4-6 weeks after filtration through 0.10-0.22 μm pores), 1,16 replication can be assessed by spectrophotometry (650 nm wavelength). 17,20 and 8D10 [to porin] 21,32,43 of NanoBiotech Pharma, Tampa, FL) -ELISA, 43 immunohistochemistry, 32 immunofluorescence reaction, 32 immunoblotting, Ouchterlony immunodiffusion.…”

“…Therefore, CNPs being life forms cannot be ruled out based on their extremely small size alone. Also, it is not possible to rule out the uniqueness of the replicative and metabolic mechanisms Susceptibility to a number of antimicrobial chemotherapeutics possessing antimetabolic activity and not possessing the chelating activity, 30 (nitrofurantoin, trimethoprim, trimethoprim-sulfamethoxasole), to 5-fluorouracil, cytosine arabinoside, 6-aminocaproic acid, 30 antimycin A, sodium azide, potassium cyanide 22 Morphological similarity to various mineral and mineralo-protein complexes containing inorganic calcium, phosphorus, and apatite compounds, and also proteins and phospholipids 2,6,[8][9][10] Acceleration of replication and reduction of formation of biofilms after photobiostimulation 29 Resistance to DNase and RNase activity 5 Presence of proteins, including bacterial ones, after a demineralization 16,20,21 (EF-Tu, 22,26 EF-G, 26 of CNPs requiring a smaller size for proper functioning, and these mechanisms can feasibly be characterized by a greater compactness compared with their analogs in other organisms.…”

HIV and nanobacteria

“…Cultured nanobacteria have been observed to respond to physiological and biomechanical stress by spontaneously releasing substantial amounts of a slime. This suggests that nanobacteria circulating in the blood would respond to stress in their environment similarly, and potentially induce kidney stones [3,4] by forming slime-interconnected conglomerates, or heart disease [5,6] by acting as thrombogenic nuclei. HIVinfected patients are extremely vulnerable to opportunistic infectious agents.…”

Association between dental pulp stones and calcifying nanoparticles

“…CNPs isolated in the present study were coccoid with a highly refractive shell, similar in size and morphology to the CNPs reported in other pathological calcification tissues in the human body. 22,38,39 The morphological changes could be observed during the culture period. From the barely detectable tiny coccoid or dumbbell particles with a diameter of 0.025-0.05 mm after 2-week culture, these particles grew gradually to an average diameter of about 0.10 mm after 1 month.…”