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Alternative View on the Putative Organism, Nanobacterium sanguineum
Professor Charles F.A. Bryce, Braids Education Consultants, Edinburgh EH10 6NZ, Scotland
1. Background
Professor George Kindness introduced the author to the
nature and biological rôle of the putative organism Nanobacterium sanguineum,
particularly in relation to its involvement with intra- and extracellular
calcification and stone formation. As a result of more detailed discussions and
consideration of a range of diverse yet inter-related areas of science, it
quickly became clear that the evidence base for the existence of this
microorganism was cast in doubt and that an alternative interpretation was more
likely. The issues which we felt required further clarification were:
- The
physical size of the Nanobacterium sanguineum in relation to other
microorganisms and in relation to the theoretical minimum size for a
free-living organism
- The
biological properties of Nanobacterium
sanguineum
- The
chemical properties of hydroxyapatite in relation to its binding of
nucleic acids and proteins
- The
physical properties of hydroxyapatite, particularly with respect to its
crystallisation
- The role of
EDTA in clinical treatment regimen for atherosclerotic plaque, kidney disease and other diseased states
The approach taken was to undertake an extensive
literature and internet search for each of these specific areas of study with a
view to arriving at a robust interpretation on the most likely interpretation
on the biological nature and properties of nanobacterium sanguineum. The Report
is structured as an Executive Summary, followed by a summary of key evidence
for and against the existence of such an organism (Appendix 1) together with a
series of key bullet points relevant to the evidence base (Appendix 2). A
CD-ROM copy of the Report is supplied in order to make effective use of the
numerous cited references which have been hyperlinked to the internet.
2. Executive
Summary
At the present time the genus nanobacterium
and the type species Nanobacterium sanguineum have no formal inclusion
in the List of Bacterial Names with
Standing in Nomenclature[1].
The two scientists who first studied Nanobacterium sanguineum submitted
the species to the German Collection of Microorganisms (DSM No. 5819-5821)
where their phylogenetic position was located in the α-2 subgroup of
proteobacteria[2]. From
the present study it is concluded by the author that the putative organism Nanobacterium
sanguineum does not represent a free-living biological entity but is,
instead, a microcrystalline form of hydroxyapatite complexed with exogeneous
biological macromolecules, including DNA and protein.
The first problem which I
identified was the very small size of the organism. Its size is only about
1/100th to 1/1000th the size of conventional bacteria at
20nm [3].
It is worth noting that this happens also to be the standard size of
commercially produced hydroxyapatite nanocrystals[4].
In looking at survival strategies of bacteria in the natural environment,
Roszak and Cowell[5]
concluded that ultramicrobacteria (e.g.
Spirillum, Leucothrix, Flavobacterium, Cytophaga and Vibrio spp) are representative of the autochthonous bacterial
communities in the marine and estuarine environment. What is important to note
is that these ultramicrobacteria are still of the order of 200-300nm. At a
recent Workshop it was suggested that the theoretical minimum size for a
free-living organism (capable of holding the minimal molecular complement of
~250-450 proteins, genes and ribosomes would be 250-300 nm in diameter, a
figure which matches well that described for the ultramicrobacteria. Indeed,
even a single ribosome, if surrounded by membrane and wall, would occupy a
sphere of 57 nm in diameter[6].
The latter study was further supported by the study of the gene complement of Mycoplasma genitalium[7]
(with just a 0.58 megabase genome this has been proclaimed the minimal gene
complement). Through comparison with the gene set for Haemophilus influenzae[8]
(both represent ancient bacterial lineages with one being Gram-positive and the
other Gram-negative it was possible to identify 256 genes which the authors
felt represented the minimal gene set necessary and sufficient to sustain
translation, replication, recombination and repair, transcription, chaperone
functions, nucleotide metabolism, amino acid metabolism, lipid metabolism,
energy roles, coenzyme metabolism and utilisation, polysaccharides, uptake of
inorganic ions, secretion, receptors and other conserved functions.
Observations on Archaea indicate that, in general, they have size limits
similar to those for conventional bacteria. In an interesting theoretical paper
by Moore[9]
he argues that to “design” a cell of the order of 60nm then we would have to
invoke a supporting biochemistry unlike any known in modern cellular life and
that a cell even smaller would require even more radical departures, which he
lacks the imagination to consider, a view which is shared by many of the other
contributors to this Workshop. It is concluded from such work that the much
smaller bodies (~50nm) called Nanobacteria may not, themselves, be
viable living organisms.
It has been stated that Nanobacterium are
unique in that they can develop a calcium apatite cell wall, forming an
enclosure around the organism[10].
Considering the size of the hydroxyapatite ‘wall’ it is even more difficult to
see how a living organism can be within such a small structure. Whilst it may
be possible to hypothesise a minimal cell size of a 50nm sphere for a living,
replicating single biopolymer system (i.e. one in which the nucleic acid
is both catalytic and genetic), a two biopolymer system (i.e. one with
nucleic acid together with proteins/enzymes) would have to be 5-10 times the
volume. Clearly, on this basis, Nanobacterium sanguineum could not
reasonably represent a living, replicating organism, a view also shared by
Ferris[11]
and others[12],[13],[14].
Interestingly, an earlier study by Ruzicka[15]
in 1983 identified what he believed to be a very small bacteria isolated from
peripheral blood which he called Basoplasma sanguineum. These very small
organisms had a mean diameter of 0.25µm, some of which he purported to have a
cell wall whilst others were cell wall deficient. He now believes that his
organism and the putative Nanobacterium are one and the same.
Late in the review process two key published documents
were identified which also cast very serious doubts on the interpretation that
these microcrystalline bodies are indeed living organisms. Thus, Cisar et al [16]published
a critical paper in October 2000 in Proceedings of the National Academy of
Sciences in which they dispute the earlier findings of Kajander and Ciftcioglu[17].
One of the key scientific findings from this study was that the 16S rDNA
sequences previously ascribed to Nanobacterium sanguineum were found to
be indistinguishable from those of an environmental microorganism, Phyllobacterium
mysinascearum. More recently, Cranton[18]
published an Internet article in which he demonstrated that the alleged Nanobacteria
do not cause calcification of arterial plaque.
This leads to the obvious conclusion that the
particles identified as the living organism Nanobacterium sanguineum are
in fact non-living but self-generating inorganic particles of hydroxyapatite
which have been complexed with nucleic acids, proteins and other ionic
biomolecules. It has been demonstrated that organic materials have key roles as
nucleating surfaces, so triggering crystal growth in the biomineralisation of
apatite, in addition to modulating and finally inhibiting the process[19].
In this context it is worth noting that crystal growth is enhanced in low
gravitational environments and this may help to explain why astronauts
returning to earth are prone to calcific atherosclerosis.
Microcrystalline hydroxyapatite complex (MCHC) is
currently widely used to prevent osteoporosis, to help remineralisation during
lactation, to stimulate fracture healing, for rapid growth of children, general
orthopaedic surgery and for ocular implants[20].
For the most part these are derived from an organic source (young bovine or
ovine bone) and represent a complex of microcrystalline hydroxyapatite,
minerals, protein, and carbohydrates etc.
As the complex resembles natural bone it has been shown to lack toxicity. The
antigenic properties of the structures previously identified as Nanobacterium
sanguineum could be explained as humeral antibody response to a number of
biomolecules which readily adsorb onto the surface of the hydroxyapatite
microcrystals.
Cranton10 is quite scathing in relation to
the NanoBac use of EDTA as ‘treatment’ for nanobacterial ‘infection’,
concluding that this putative microorganism is a myth. It is interesting to
note the recent announcement that the National Institutes of Health (NIH) is
putting $30 million into a major clinical trial of the efficacy of chelation
therapy for sufferers from heart disease. As intimated in this study,
supporters of chelation offer two major theories for its action. The first
relates to the binding of calcium ions to EDTA (which removes the metal ions
from arterial plaques, so facilitating their dissolution) whilst the second
relates to the role of EDTA as a powerful antioxidant[21].
It would make good sense for us to monitor the progress and outcomes from this
large study as it may have beneficial impact on existing and future clinical
strategies for AmScot Medical Laboratories.
It is my considered opinion,
in light of all the evidence presented, that there is little or no conclusive
evidence to support the view that the putative microorganism Nanobacterium
sanguineum truly exists and that the weight of scientific evidence from a
range of diverse sources leads to the view that the more credible conclusion is
that this represents self-propagating microcrystalline hydroxyapatite.
Appendix 1
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Key “evidence” relating to
the biological status of Nanobacterium sanguineum
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“Evidence” for the existence of the Nanobacterium |
“Evidence” against the existence of the Nanobacterium |
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DNA, RNA and lipopolysaccharide profiles have been accurately mapped
by multiple scientific researchers at many universities worldwide. http://www.nanobaclabs.com/nanobaclabs-nanobacteria-what-are-nanobacteria.asp |
Its size is only about 1/100th to 1/1000th the
size of conventional bacteria at 20nm http://www.nanobaclabs.com/nanobaclabs-nanobacteria-what-are-nanobacteria.asp This happens also to be the standard size of commercially produced
hydroxyapatite nanocrystals. http://www.hydroxyapatite.com/crystals.html At a recent Workshop it was suggested that the theoretical minimum
size for a free-living organism (capable of holding the minimal molecular
complement of ~250-450 proteins, genes and ribosomes would be 250-300 nm in
diameter. The much smaller bodies (~50nm) called Nanobacteria may not,
themselves, be viable organisms. Observations on Archaea indicate that, in
general, they have size limits similar to those for bacteria. http://www.nationalacademies.org/ssb/nanooverview.htm |
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Our body does not recognise calcified Nanobacteria as a
foreign substance or pathogen. http://www.nanobaclabs.com/nanobaclabs-nanobacteria-what-are-nanobacteria.asp |
Replication time of 3–6 days and culture times of 2-4 weeks versus
minutes/hours for conventional bacteria. http://www.nanobaclabs.com/events/events.asp http://www.nanobaclabs.com/nanobaclabs-nanobacteria-what-are-nanobacteria.asp |
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Sensitive to high doses of gamma irradiation and some antibiotics e.g.
tetracycline. http://www.nanobaclabs.com/events/events.asp http://www.uku.fi/~kajander/comparison.html |
Cannot be grown in standard culture media. http://www.nanobaclabs.com/nanobaclabs-nanobacteria-what-are-nanobacteria.asp |
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The novel organism has a cell wall and division septa that clearly
indicate it as a living organism. Typically coccoid or coccobacillar in
shape. |
One form secretes a calcific biofilm around itself that provides
protection as well as allowing for multiple bacteria to connect as a colony. http://www.nanobaclabs.com/nanobaclabs-nanobacteria-what-are-nanobacteria.asp |
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Their multiplication could be detected by specific ELISA, optical
density, microscopic counting, SDS-PAGE or methionine and uridine
incorporation. http://www.uku.fi/~kajander/threat.html |
Conventional methods of sterilization do not work. http://www.nanobaclabs.com/nanobaclabs-nanobacteria-what-are-nanobacteria.asp |
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Nanobacteria contain novel proteins and “tough”
polysaccharides, one of the former being a functional porin protein (a
hallmark of gram negative bacteria). http://www.nationalacademies.org/ssb/nanopanel2kajander.htm |
DNA cannot be isolated – it is associated with binding molecules and
seems to have an aberrant structure. http://www.nanobaclabs.com/events/events.asp http://www.uku.fi/~kajander/comparison.html |
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Nanobacteria use large amounts of Gln, Asn and Arg from
medium/environment. http://www.nationalacademies.org/ssb/nanopanel2kajander.htm Interestingly, these all represent basic amino acids which could
easily bind to the Ca+ ions. |
Even a single ribosome, if surrounded by membrane and wall, would
occupy a sphere of 57 nm in diameter. http://www.nationalacademies.org/ssb/nanooverview.htm |
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Nanobacteria have DNA amounts between that of mycoplasmas and
mitochondrion. http://www.nationalacademies.org/ssb/nanopanel2kajander.htm |
Sequence analysis of the DNA from Nanobacterium has been shown
to be indistinguishable from that of a known contaminating environmental
microorganism (Phyllobacterium mysinacearum) in culture media and
laboratory reagents,. http://www.doctorshealthsupply.com/dentists/an_alternative_interpretation.htm Other
investigators have also recently suggested that the 16S rDNA sequences of
nanobacteria could be PCR artefacts. Pitcher, D.G. and Fry,
N.K. J. Infectious Diseases, 40, 116-120, (2000) |
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Neither nucleic acid nor extensive proteins were found on washed
biofilm material. http://www.doctorshealthsupply.com/dentists/an_alternative_interpretation.htm |
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Phospholipids, proteins etc.
in plasma act as nucleators of biomineralisation. http://www.drcranton.com/nanobacteria.htm Tetracycline also has calcium binding properties, thus halting
calcific propagation. http://www.drcranton.com/nanobacteria.htm |
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The current market “treatment” for nanobacterial infection relates
little to targeting a living organism but more to chelating calcium ions. http://www.drcranton.com/nanobacteria.htm |
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Nanobacterium when compared with another bacterium was found to
have a twentieth part of DNA, a tenth of RNA and equal amounts of protein. http://www.uku.fi/~kajander/comparison.html |
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No DNA could be isolated from Nanobacteria with the standard
DNA isolation methods http://www.uku.fi/~kajander/comparison.html |
Appendix 2
Key points to be noted:
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1 |
Nanobacteria are cytotoxic to fibroblasts and kidney cells in
vitro. http://www.nanobaclabs.com/events/events.asp |
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2 |
16S rRNA gene sequence results showed the bacterium to be in the alpha-2 subgroup of Proteobacteria. http://www.nanobaclabs.com/events/events.asp |
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3 |
Nanobacteria are novel apatite mineral-forming agents found in
human and animal blood and tissues, and arouse an antibody response. http://www.nanobaclabs.com/events/events.asp |
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4 |
Nanoisolates were excreted into urine more effectively than control
substances (hydroxyapatite and commercial nanocolloid). http://www.nanobaclabs.com/events/events.asp |
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5 |
Nanobacterium sanguineum cannot be killed by Penicillin,
cephalosporins, macrolides and most other antibiotics. http://www.nanobaclabs.com/nanobaclabs-nanobacteria-what-are-nanobacteria.asp |
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6 |
Nanobacteria are extremophiles and have been found to be the
most resistant of all bacterial SuperBugs to destruction. http://www.nanobaclabs.com/nanobaclabs-nanobacteria-what-are-nanobacteria.asp |
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7 |
Some controversy remains surrounding the characteristics and
pathogenicity of this ultra-small and difficult to detect bacterium. http://home.swipnet.se/isop/biofilms.htm |
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8 |
Its uniform layers of calcification, called a biofilm, with which it
encapsulates and protects itself. http://home.swipnet.se/isop/biofilms.htm |
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9 |
It has recently been shown to give a false positive test for
Chlamydia. http://home.swipnet.se/isop/biofilms.htm |
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10 |
It requires culturing on living cell cultures and not on artificial
media agar like common bacteria. http://home.swipnet.se/isop/biofilms.htm |
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11 |
The Nanobac treatment regimen involves rectal suppositories of EDTA,
oral enzymes, vitamins that augment EDTA and a nightly dosage of
tetracycline. http://home.swipnet.se/isop/biofilms.htm |
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12 |
Several scanning electron micrographs of Nanobacterium and
clusters. http://www.lifescore.com/nanoscore.htm |
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13 |
Calcific atherosclerosis is a result of infection with Nanobacterium. http://www.heartfixer.com/indexNB.htm |
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14 |
Properties of microcrystalline hydroxyapatite. http://www.villaparkpharmacy.com/abs04.htm |
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15 |
Leading manufacturers of microcrystalline hydroxyapatite complex. http://www.indiamart.com/clarion/ |
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16 |
Supplier of microcrystalline hydroxyapatite complex in Ohio, USA. http://store.yahoo.com/vitanet/calmichyd180.html |
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17 |
Detection of hydroxyapatite using FT-Raman spectroscopy. http://www.s-a-s.org/journal/00/November2000_ea2.htm |
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18 |
Direct synthesis of nanocrystalline hydroxyapatite with particle size
~ 100nm http://www.ul.ie/~cost523/poster1/Jaroslav.htm |
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19 |
Protein binding to hydroxyapatite is through ionic interactions as
well as adsorption effects. Acidic proteins bind to calcium ions whereas
basic proteins bind to phosphate groups. http://www.chromatography.co.uk/techniqs/bio/bio_011.htm |
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20 |
According to Ciftcioglu the nanobacterial DNA seems to have an
aberrant structure. http://www.nanobaclabs.com/events/events.asp |
Professor C.F.A. Bryce
Braids Education Consultants
206 Braid Road
Braids
Edinburgh EH10 6NZ
Scotland
Tel : +44 131 447 4488
E-mail: cfabryce@hotmail.com
WWW: http://www.cfab.org