Nanobacteria or so called calcifying nanoparticles, are self-replicating
bacteria and the smallest described bacteria to date, with dimensions of 20-200
nm in length (Kajander and Ciftcioglu, 1998). Furthermore,
these organisms were found to produce a biofilm containing hydroxyl apatite
or carbonate, preventing their effective staining. The stimuli for calcium salt
deposition in patients with these conditions are unclear but nidi (meaning that
biomineralization is taking place out of chemical equilibrium) for precipitation
and crystallization are needed even under supersaturation conditions (Carson,
1998). Nanobacteria are carbonate apatite forming, cytotoxic bacteria recently
discovered in human and bovine blood and blood products (Kajander
et al., 1997; Ciftcioglu et al., 1997;
Kajander and Ciftcioglu, 1998). Calcific kidney stones
in humans are located on renal papillary surfaces and consist of an organic
matrix and crystals of calcium oxalate and/or calcium phosphate (Khan,
1997). It has been stated that the core of 67% of calcium oxalate stones
contains calcium phosphate (Abraham and Smith, 1987).
Nanobacteria are thought to play an important role in extraskeletal calcifying
diseases including stones formation, urolithiasis and polycystic kidney disease
(Kajander et al., 2003). Nanobacteria are phylogenetically
close relatives of mineral forming bacteria (Kajander et
al., 1997). These particles have been isolated from kidney stones and
urine of patients with renal lithiasis (Ciftcioglu et
al., 1999), renal fluid taken from patients with polycystic kidneys.
It has been speculated that nanobacteria may be the spherical deposits found
in the kidneys of patients who suffer from kidney stones (Vogel,
1998; Bradbury, 1998). Two strains, one of Nanobacterium
sanguineum and the other of Nanobacterium sp., were isolated from
kidney stones and human and bovine sera, respectively. Phylogenetic analysis
based on comparison of 16S ribosomal DNA (rDNA) sequences has placed the nanobacteria
isolated from fetal calf serum into the α2 subgroup of Proteobacteria
(Kajander et al., 1997), closely related to
Thiobacillus, a water contaminant and Agrobacterium and Rhizobium
which are plantassociated bacteria. In this study, we provide evidence that
nanobacteria can act as crystallization centers (nidi) for the formation of
biogenic apatite structures. The mineralization process was studied in vitro
with one bovine isolate from commercial Fetal Bovine Serum (FBS) and with
a human isolate. Present findings are of concern in medicine because nanobacterial
bacteremia occurs in humans and nanobacterial nidi might initiate pathological
calcification. The aims of our study were to confirm the presence of nanoparticles
in urinary tract stones by morphological evidence with Scanning Electron Microscopy
(SEM) and Transmission Electronic Microscopy (TEM) of inoculated cells. Moreover,
the chemical analysis was detected by with energy dispersive X-ray (EDX) spectra.
MATERIALS AND METHODS
Stones: Eight urinary tract stones were collected from male and female patients
hospitalized in the Kasr El Aini, Cairo University, Egypt. Stones were demineralized
in 1M HCl and then neutralized (Folk, 1993), centrifuged
at 14,000 X g for 15 min and the pellets used for Immuno-fluorescence Staining
(IIFS) and Transmission Electronic Microscopy (TEM). Part of the pellets were
suspended in Dulbecco-Vogts modification of Eagles medium DMEM,
sterile-filtered and cultured in (DMEM) supplemented with γ-FBS (Sera-Lab,
Crawley Down, Sussex, U.K.) under nanobacterial culture conditions. One fragment
of each stone was preserved for culture analysis and the other fragment was
used to determine its chemical structure by with Energy Dispersive X-ray (EDX)
spectra (Blijenberg et al., 1997). Hydroxyapatite
(No-H-0252; Sigma) was used as a reference. Hydroxyl and carbonate groups in
the apatite minerals were detected (National Research Institute, Dokki-Egypt)
following the standard method. Chemical analysis of demineralized stone was
carried out as described previously by Hyvonen et al.
Nanobacterial culture: The cultures were prepared using strict aseptic
techniques in a cell culture facility. Nanobacterial samples were filtered through
0.2 mm filters before culturing. Subcultures were made using γ-FBS as a
culture supplement. FBS and nanobacteria were γ-irradiated, when indicated;
at a minimum dose of 30 kGy given at room temperature during about 16 h. Subculturing
of nanobacteria in Serum-free (SF) DMEM was performed with monthly passages.
SF nanobacteria attach firmly to the bottom of the culture vessel. These cultures
were passaged or harvested with a rubber scraper. Cultures were established
on Loeffler medium supplemented with 10% conditioned medium from nanobacterial
culture and DMEM replaced water in the formula (Nash and
Krenz, 1991). The incubation period was 6 weeks under cell culture conditions.
Only pure nanobacterial cultures were used. Control experiments were performed
to determine whether spontaneous crystallization could occur in a culture medium.
The samples were viewed under Light microscopy with Differential Interference
Contrast (DIC) optics.
Preparation and Infection of 3T6 Cells: 3T6 cells (ATCC CCL 96) were
cultured on coverslips as described before (Ciftcioglu
and Kajander, 1998). SF nanobacterial cultures were scraped and 100-mL portions
were added to the cell cultures and incubated for 24 h in theincubator. Only
DMEM was added to the control experiments. TEM and IIFS were used for the observation
of the cell-SF nanobacteria interaction.
Electron microscopy and Energy Dispersive X-ray microanalysis (EDX):
For negative staining, nanobacteria were isolated by centrifugation at 40,000
X g for 1 h directly from FBS diluted 1:5 in PBS. A carbon-coated 400 mesh copper
grid was placed on a drop of the suspension of nanobacteria in Phosphate Buffered
Saline (PBS) for 1 min, washed with water and stained on a drop of 1% phosphotungstic
acid for 90 sec. Scanning Electron Microscopy (SEM) and Transmission Electronic
Microscopy (TEM) were performed by a BX-51 optical microscope (Olympus) and
dark-field condenser (CERBE). Images were acquired by using a KY-F55 color camera
(JVC). The topographic features of the nanobacteria were investigated with a
scanning electron microscope equipped with EDX as described previously (Suzuki
et al., 1997).
The clinical specimens of kidney stones, were cultured in DMEM medium for
six weeks and examined macroscopically once a week for the appearance of a white
biofilm or floccules adhering to the culture flask. After incubation, the mineralized
biofilm was visible to the naked eye in 60% of the sample studied. Macroscopic
nanobacterial colonies on DMEM medium were stony, grayish brown and penetrated
the medium layer and attached to the bottom of the culture vessel after 6 wk
of culture. Light microscopy with DIC optics revealed revealed nanobacteria
coated in needle-like apatite crystals similar to the hydroxyapatite crystals
(Fig. 1a, b).
Scanning electron microscopy images of the spherical units in the carbonate
apatite kidney stones were clearly similar in size and morphology to the mineralized
forms of nanobacteria cultured under SF conditionsshowed similar characteristics
(Fig. 2a, b). To visualize the presence
of nanobacteria all the mineralized cultures were scraped off,
harvested by centrifugation and reexamined by SEM. As expected, SEM analysis
revealed clusters of spherical particles displaying morphological properties
typical of nanobacteria. Spherical coccoid particles were observed which were
grouped in coarse clusters and bound together to a mineral structure (Fig.
3a, b and 4a, b).
|Fig. 1: (a, b) Nanobacterial stony colonies penetrated through
the medium forming stony needle-like crystal deposits
|Fig. 2: Details from a fractured renal stone (hydroxyaptite)
from SEM analysis Magnification, X 15,000
|Fig. 3: SEM of (a) negative culture derived from control FBS
diluted to 10% in DMEM (b) Nanobacteria obtained by incubating FBS diluted
to 10% in DMEM showing colonies of coccoid bacteria-like formations grouped
together in coarse clusters. Their diameter was between 200 and 300 nm.
Bar, 1 μm. Magnification, X 40,000
These spherical units were similar in size and morphology. The size of these
particles varied between 200 and 300 nm and they appeared to have developed
in stone cavities. Their rough surfaces were illustrated with hairy apatite
layer surrounding a central cavity. TEM of inoculated 3T6 cell monolayers has
shown transient intracytoplasmic vacuolar formations containing 200 to 300 nm
particles in cell cultures (Fig. 5a, b).
The chemical compositions and their percentage of distribution among the screened
kidney stones and the Energy-dispersive X-ray EDX spectrum of nanobacteria cultured
from the liquid fraction of the stones revealed a simple profile of carbon,
oxygen and sodium at atomic C:O:Na ratios of 42:30:0.3 (Fig. 6).
No phosphorus was detected that might indicate the presence of hydroxyapatite.
Therefore, hydroxyapatite may not be a necessary component of nanobacteria because
it appears to deposit onto nanobacteria only under particular circumstances.
|Fig. 4: TEM of (a) Negative culture and (b) Nanobacteria with
an electron-dense core after incubation in DMEM for 1 month
|Fig. 5: TEM analysis of renal stone-inoculated 3T6 monolayers.
(a) Free 3T6 cells and (b) Nanoparticles inside vesicles in 3T6 cells
|Fig. 6: EDX spectrum analysis in SEM of nanobacteria obtained
by incubating the soluble liquid fraction of the stones reveals the absence
of calcium and phosphate peaks
We have tried to demonstrate the presence of nanobacteria in renal stones
by using SEM and TEM of inoculated 3T6 cell monolayers. These approaches were
previously reported with success (Kajander and Ciftcioglu,
1998; Ciftcioglu et al., 1999). We observed
spherical nanoparticles grouped in clusters binding to the mineral surface and
cavities of renal stones. Two mechanisms have been proposed for calculi formation:
the development of calculi attached to papillary epithelium and the development
of calculi in cavities without any attachment to urothelium (Grases
et al., 1998). The small apatite units were observed in all kidney
stones in different proportions. We also observed similar formations in in
vitro nanobacteria culture under SF conditions. Nanobacteria first adhere
to the surface of the culture vessel and then create "cave-like" apatite fortresses
with a concave face (Kajander and Ciftcioglu, 1998).
Additionally, SEM images proved that nanobacterial mineralization takes place
via the formation of several thin mineral layers the same as in kidney stone
formation. There have been several studies on the possible mechanisms of crystal
aggregate formation following the initial nucleation of crystals from supersaturated
urine (Mandel, 1996). Renal tubular fluid in the distal
nephron is supersaturated with calcium and oxalate ions that nucleate to form
the most common crystal, calcium oxalate monohydrate, in renal stones. Urine
supersaturation values correlated well with stone composition (Asplin
et al., 1998). Intracellular assemblies similar to nanoparticles
were transiently observed by TEM in 3T6 cells inoculated with material derived
from renal stones (Kajander and Ciftcioglu, 1998; Kajander
et al., 1997). The structures observed have been routinely considered
as artifacts, as are the structures we observed when using TEM on renal stone
material-inoculated 3T6 cells. The apatite produced by nanobacteria is biogenic
because it is formed in a carbon-containing biomatrix, forms small spherical
units of apatite in nanoscale crystal size (that are very resistant to acid
hydrolysis) and can be formed at non saturating concentrations of calcium and
phosphate. In this respect, Ciftcioglu et al. (1997)
has proven that all kinds of nanobacterial forms are internalized by many types
of mammalian cells and once internalized, they are cytotoxic. In a rabbit experiment,
Akerman et al. (1997) showed that 99mTc-labeled
nanobacteria, injected intravenously, had a tissue specific distribution with
a major accumulation in the kidneys and subsequently in urine. The presence
of live excreted nanobacteria in urine is proof that these organisms may be
involved in the kidney stone formation. Ciftcioglu et
al. (1999) propose a new theory to explain the formation of human kidney
stones: Nanobacteria may act as nidi for kidney stone formation.
This theory is supported by the following findings:
||97.2% of the analyzed kidney stones contained nanobacteria
||Almost all kidney stones have apatite as a component (Mandel,
||Nanobacteria are the only known organisms in the human body that produce
apatite and accumulate in the kidney (Akerman et
||Nanobacteria isolated from human kidney stones produced stones in culture.
The availability of the nanobacterial strain for scientific investigators
would be an important step in making progress in our research on nanobacteria
This study was funded by Cairo university, Project No, 3/5 2009 Application
of nanobacteria in the new millennium.
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