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 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

 

 

 

Key “evidence” relating to the biological status of Nanobacterium sanguineum

 

 

 

 

 

 

 

“Evidence” for the existence of the Nanobacterium

 

 

“Evidence” against the existence of the Nanobacterium

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

 

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

 

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

The novel organism has a cell wall and division septa that clearly indicate it as a living organism. Typically coccoid or coccobacillar in shape.

http://www.uku.fi/~kajander/comparison.html

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

 

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

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

 

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

 

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)

 

 

Neither nucleic acid nor extensive proteins were found on washed biofilm material.

http://www.doctorshealthsupply.com/dentists/an_alternative_interpretation.htm

 

 

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

 

 

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

 

 

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

 

 

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:

 

1

Nanobacteria are cytotoxic to fibroblasts and kidney cells in vitro.

http://www.nanobaclabs.com/events/events.asp

 

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

 

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

 

4

Nanoisolates were excreted into urine more effectively than control substances (hydroxyapatite and commercial nanocolloid).

http://www.nanobaclabs.com/events/events.asp

 

5

Nanobacterium sanguineum cannot be killed by Penicillin, cephalosporins, macrolides and most other antibiotics.

http://www.nanobaclabs.com/nanobaclabs-nanobacteria-what-are-nanobacteria.asp

 

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

 

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

 

8

Its uniform layers of calcification, called a biofilm, with which it encapsulates and protects itself.

http://home.swipnet.se/isop/biofilms.htm

 

9

It has recently been shown to give a false positive test for Chlamydia.

http://home.swipnet.se/isop/biofilms.htm

 

10

It requires culturing on living cell cultures and not on artificial media agar like common bacteria.

http://home.swipnet.se/isop/biofilms.htm

 

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

 

12

Several scanning electron micrographs of Nanobacterium and clusters.

http://www.lifescore.com/nanoscore.htm

 

13

Calcific atherosclerosis is a result of infection with Nanobacterium.

http://www.heartfixer.com/indexNB.htm

 

14

Properties of microcrystalline hydroxyapatite.

http://www.villaparkpharmacy.com/abs04.htm

 

15

Leading manufacturers of microcrystalline hydroxyapatite complex.

http://www.indiamart.com/clarion/

 

16

Supplier of microcrystalline hydroxyapatite complex in Ohio, USA.

http://store.yahoo.com/vitanet/calmichyd180.html

 

17

Detection of hydroxyapatite using FT-Raman spectroscopy.

http://www.s-a-s.org/journal/00/November2000_ea2.htm

 

18

Direct synthesis of nanocrystalline hydroxyapatite with particle size ~ 100nm

http://www.ul.ie/~cost523/poster1/Jaroslav.htm

 

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

 

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                                               

 



[1] http://www.bacterio.cict.fr/nonvalid.html

[2] Ciftcioglu, N. and Kajander, E. O. Pathophysiology, 4, 259-270, (1998)

[3] http://www.nanobaclabs.com/nanobaclabs-nanobacteria-what-are-nanobacteria.asp

[4] http://www.hydroxyapatite.com/crystals.html

[5] Roszak, D.B. and Colwell, R.R. Microbiological Reviews, 51, 365-379, (1987)

[6] http://www.nationalacademies.org/ssb/nanooverview.htm

[7] Himmelreich, R.H., Hilbert, H., Plagens, H., Pirkl, E., Li, B-C and Herrmann, R. Nucleic Acid Research, 24, 4420-4449, (1996)

[8] Mushegian, A.R. and Koonin, E.V. Proc. Nat. acad. Sci., 93, 10268-10273 (1996)

[9] Moore, P.B. Size Limits of Very Small Microorganisms, Proceedings of a Workshop organised by the Space Studies Board [ISBN 0-309-06634-4] pages 16-20

[10] http://www.thedoctorsdoctor.com/diseases2/nanobacteria.htm

[11] http://www.nationalacademies.org/ssb/nanopanel4.htm

[12] Maniloff, J. Science, 276, 1776, (1997)

[13] Nealson, K.H. Science, 276, 1776, (1997)

[14] Psenner, R. and Loferer, M. Science, 276, 1776-1777, (1997)

[15] Ruzicka, F. Mycoplasmen-Kommensalen oder Kofaktor bei der Onkogenese? Fa Reprotext Wien (1983) [http://home.pages.at/ruzicka/Preface.htm]

[16] Cisar, J.O., Xu De-Qi, Thompson, J., Swaim, W., Hu, Lan and Kopecko, D.J. Proc. Natl. Acad. Sci., 97, 11511-11515, 2000 [http://www.doctorshealthsupply.com/dentists/an_alternative_interpretation.htm]

[17] Kajander, E.O. and Ciftcioglu, N. Proc. Natl. Acad. Sci., 95, 8274-8279, 1998

[18] Cranton, E.M. in http://www.drcranton.com/nanobacteria.htm

[19] http://www.gla.ac.uk/leadingedge/6/4.html

[20] http://www.ioi.com/patient/ha.html, http://www.villaparkpharmacy.com/abs04.htm, http://store.yahoo.com/vitanet/calmichyd180.html, http://www.hydroxyapatite.com/main.html, http://www.bioteck.com/ing/rigenerazione_i.htm and http://www.indiamart.com/clarion/

[21] Holden, C., NIH Trial to Test Chelation Therapy. Science, 297, 1109 (2002) [http://www.sciencemag.org/cgi/content/full/297/5584/1109a]