NANOBACTERIA, THEIR EXISTENCE AND EFFECTS ON HUMAN HEALTH

Even though I know practice as a software engineer, I studied Laboratory Medicine in university, and specialized in medical microbiology.
I stumbled on this article which I wrote for uni in 2016 and figured there’s no harm sharing. The older I get, the more I yearn to work on the intersection between Technology and medicine. But that’s a journey we will get to. Enjoy :)

12/12/2016

Abstract

Nanobacteria are cell walled microorganisms with a size much smaller than the generally accepted lower limit for life (200nm). Nanobacteria produce a rock-hard calcium phosphate substance that is chemically identical to the substance found in hardening arteries, prostate disease, kidney disease, periodontal disease and breast cancer. But even though it is able to replicate and grow, scientists still do not understand how it achieves these feats, hence resulting in a controversy in the Scientific community, as to whether Nanobacteria are alive or not. This work examined secondary datasources, specifically publications in the Nanobacteria space, between 1988 and 2010, primarily from the American National Center for Biotechnology Information (NCBI) database. The question of Nanobacteria being living or non-living, has multiple good arguments from both factions, but this question remains secondary to the fact that nanobacteria produce real fatal conditions, and so, needs cheap and efficient methods of diagnosis and treatment, especially since popular methods like the Polymerase Chain Reaction (PCR) has proved less effective.

Chapter One

1.0 Introduction

Nanobacteria which were first described by Kajander and Ciftçioğlu in 1988, are members of a proposed class of living organisms, specifically cell-walled microorganisms with a size much smaller than the generally accepted lower limit for life (about 200 nm for bacteria, like mycoplasma) (Kajander et al, 1998). Nanobacteria is generally a very controversial topic, as a faction of scientists believe them to be classified as non living organisms, while another faction believes that they are living organisms, and might even be the source of life on earth (Martel et al, 2008).

Since their discovery in 1998, they have been associated with multiple disease conditions, including Heart Disease, Kidney Stones and Cancer, (Ciftcioglu et al, 1999) due to their unique property of biomineralization, nanobacteria have also been found in meteorite chipped from the surface of the Red Planet indicated life had once existed on Mars (McKay et al, 1996). Geologic research shows they are from the very dawn of life could have shaped Earth’s early terrain. They also have been discovered in sedimentary rocks and in hot-springs (Kajander et al, 1998).

1.1 Structure and size

According to initial researchers, nanobes are biological structures that are composed of Carbon, Oxygen and Nitrogen. According to the 16S rRNA gene sequence, nanobacteria fall within the α-2 subgroup of Proteobacteria, which also includes human pathogens like Brucella and Bartonella species (Ciftcioglu, 1999).

Although they typically have diameters of 0.2–0.5 μm, they can pass through 0.1-μm filters. Ultra thin sections of nanobes show the existence of an outer layer or membrane that may represent a cell wall. This outer layer surrounds an electron dense region interpreted to be the cytoplasm and a less electron dense central region that may represent a nuclear area. Nanobes show a positive reaction to three DNA stains [49, 6-diamidino-2 phenylindole (DAPI), Acridine Orange and Feulgen] which strongly suggests that nanobes contain DNA (Uwins, 1998).

The particle has a special habit no other blood particle has been known to possess: it forms a rock-hard calcium phosphate shell that is chemically identical to the substance found in hardening of the arteries, prostate disease, kidney disease, periodontal disease and breast cancer. The problem is, the particle is so small that it apparently can’t accommodate nucleic acid strings that, according to commonly accepted wisdom, would let it replicate on its own and be alive. So scientists are still confused over how it manages to self-replicate. (Ciftcioglu et al, 2005).

Chapter 2

2.0 History of self replicating nanoparticles

Man has for many decades tried to find a yard stick at identifying and classifying organisms on earth, and around the universe, especially with mans quest to find life around the universe. Until recently, every life form was known to have a nucleic acid sequence (RNA and DNA), that aided both identification and categorisation of organisms in relation to each other. Even old tiny bits of genetic material could be amplified using a technique known as Polymerase Chain Reaction (PCR), their nucleic acid sequence compared with sequences of other known living organisms. This was done by making primers of subsequences known as 16S rRNA, and amplifying the DNA that codes for the 16s rRNAs.

A team from NASA even lobbied to use this tried and trusted method: get a piece of RNA and amplify it. The team, lead by Dr. Gary Ruvkun at the Department of Genetics in Massachusetts General Hospital, Boston, got money from NASA to build a PCR machine that would automatically seek life in harsh environments such as those found on Mars. All these, was amidst arguments by other scientists that the PCR machine approach is a waste of money since, it is possible that organisms might have evolved differently, especially on harsh environments such as other planets. This argument lacked evidence, until in 2003, when a team lead by Professor Karl Stetter of the University of Regensburg, Germany published the genome of an extremophile known as Nanoarchaeum equitans, which was discovered in Volcanic vents at Iceland. (Waters et al, 2003)

N. equitans had the smallest genome known at that time, but not just that, the 16S rRNA sequence which was used as a yardstick for other living organisms was in a different form and location, and did not respond to the conventional PCR tests. The 16S rRNA sequence was different in areas addressed by the PCR primers, and did not amplify, so the so called universal probes that worked on humans, animals, plants, eukaryotes and bacteria, did not work on the nanoarchaeum members.(Waters et al, 2003)

How, then, was the discovery made if the organism couldn’t be sequenced in that way? Stetter had found that the organism’s sequence where the traditional “universal” primers are located was abnormal. This finding let him use other means to sequence the gene. In reporting their discovery, the Stetter team observed that the information-processing systems and simplicity of Nanoarchaeum’s metabolism suggests “an unanticipated world of organisms to be discovered”.

Stetter’s finding gave ammunition to scientists such as Neva Ciftcioglu who say they have found other extremophiles, including human nanobacteria, that cannot have their nucleic acids detected with standard PCR amplification.

One of the differences between Stetter’s N. equitans and the nanobacteria found by Ciftcioglu and Kajander’s team is that Nanoarchaeae need another organism to replicate, while nanobacteria seem to replicate by themselves. Another difference is that Nanoarchaeae are slightly wider: 400 nanometres compared to 100–250 for nanobacteria. The greater size allows for what conventional wisdom says is the smallest allowable space for life-replicating ribosomes.

2.1 How do nanobacteria replicate

Evidence for self-replicating nanoparticles has been around for years in everything from oil wells to heart disease, but failure to sequence them using regular PCR led some to dismiss them as contamination or mistakes. However, researchers have found characteristics that make the particles hard to explain away. They replicate on their own, and so cannot be classified as viruses. They resist high-level radiation, which suggests that they are not bacteria. They respond well to light, where non-living crystals don’t.(Ciftcioglu et al, 2005) So if they are not viruses, regular bacteria or crystals, what are they?

2.2 Nanobacteria infected vaccines
When Dr Olavi Kajander discovered nanobacteria in 1998, he was not looking for disease at all. He was looking for what was killing the cells that are used to develop vaccines(Kajander et al, 1998). Labs everywhere have a vexing and expensive problem with these widely used cell cultures: they stop reproducing or die after a few generations and have to be thrown out.
Kajander surmised that something invisible was killing them; and when he incubated supposedly sterile samples for more than a month under special conditions, he got a milky biofilm. That biofilm contained particles that he later named nanobacteria, unaware at the time that some of their characteristics made them quite distinct from bacteria.
The serum that Kajander used to grow the nanobacteria came from the blood of cow foetuses. Serum from the UK especially was full of nanobacteria, but a much later study also concluded they were present in some cow herds in the eastern US. In other words, nanobacteria are in cows, and cow blood is used to develop many vaccines. Kajander emphasizes that this should not stop people from using vaccines, because the immediate risk from diseases that the vaccines are intended to prevent is relatively higher than the calcification risk in the short term. Nonetheless, the potentially explosive implications of contaminated vaccines and cow by-products would be clear to everyone at government agencies who has examined the issue.

2.3 Nanobacteria and Kidney Stone Formation

An early enquiry into the role of Nanobacteria in kidney stone formation happened through the work of Dr. Neva Çiftçioglu et al in 1999 (Çiftçioglu et al, 1999 ), where Nanobacteria and kidney stone units were examined using a scanning electron microscope (SEM). Demineralized kidney stones were screened for nanobacteria using a double-staining method and a specific culture method. Isolated nanobacteria were analyzed for mineral formation in vitro with Ca and 85Sr incorporation tests. Through the experiment, it was noticed that the nanobacteria resembled the smallest units of the kidney stones. When the kidney stones were demineralised with 1N HCL, they showed presence of Nanobacterium antigens. The combined data of all the specimens showed that 70 of the 72 stones (that is, 97.2%), were nanobacteria positive, irrespective of the stone types. And it was still possible to create cultures from these nanobacteria, even after treatment with 1N HCL.

Dr Çiftçioglu’s conclusion was that kidney stone formation is a nanobacterial disease analogous to Helicobacter pylori infection and peptic ulcer disease. And that the progression is basically influenced by endogenous and dietary factors. Her conclusion was based on the following findings:

1. 97.2% of the analyzed kidney stones contained nanobacteria.
2. Almost all kidney stones have apatite as a component .
3. Nanobacteria are the only known organisms in the human body that produce apatite and accumulate in the kidney .
4. Nanobacteria isolated from human kidney stones produced stones in culture.
5. An organism cytotoxic to mammalian cells in vitro and causing apoptosis in kidney tissue (our unpublished data) is unlikely to be a mere bystander in the development of kidney stones.
6. Contamination was ruled out because the control cultures remained negative.

Further research by Dr Çiftçioglu et al in 2005 (Çiftçioglu et al,2005) examined nanobacterial cultures in High Aspect Rotating Vessels (HARVs) which are built to simulate some aspects of microgravity, as a way to inquire into the higher incidence of Kidney stone formation in astronauts after long durations in space. The growth curves for the nanobacteria showed that Nanobacteria cultured in HARVs showed 4.5% faster growth than cultures in normal gravity. Dr Çiftçioglu was also able to show that Nanobacteria was inhibited in vitro at clinically achievable levels in serum and urine by ampicillin, trimethoprim, trimethoprim-sulfamethoxazole, nitrofurantoin (a urinary antiseptic), and tetracycline (Ciftcioglu et al, 2002). But then again, some bacteria (Escherichia coli and Staphylococcus aureus) have been found to be more resistant to antibiotics in space than on Earth.(Tixado et al, 1985).

2.4 Nanobacteria and Heart Disease

While nanobacteria has been associated with calcification in kidney stones, there is another form of calcification that causes major issues in humans. That is pathologic calcification of cardiac valves present in rheumatic heart disease. Ye-Rong Hu et al attempted to detect, isolate, culture, and characterize nanobacteria-like material from human calcified cardiac valves with rheumatic heart disease in 2010 (Hu YR et al, 2010). For the study, Normal and calcified cardiac valve groups, as well as positive (nanobacteria strain Se90) and negative (serum radiated with gamma rays) control groups, were examined. Part of each valve was immunostained with nanobacterial antibody 8D10, and the remaining parts were homogenized, filtered, and maintained in culture. The cultures were checked with a microscope weekly. Culture medium at different time points was analyzed with a spectrophotometer. The cultures maintained for 3 weeks were further examined with immunofluorescence double staining and transmission electron microscopy. The result was the isolation of nanobacteria like particles similar to those found in kidney stone conditions.

Chapter 3

3.0 Discussion

The discovery of nanobacteria introduced a new paradigm shift in the field of science. The idea that organisms exist which defy the basic genetic rules that other organisms follow, is quite preposterous. Nanobacteria, however gave an explanation for occurrences that had no previous explanations. For example, the almost spontaneous calcification of organs in the human body. With this paradigm shift, it becomes apparent that there is need of newer ideas or approaches towards diagnosis.

3.1 Conclusion

Nanobacteria are as small or smaller than viruses, even though they do not require host cells for replication like viruses do. This nature makes them unable to be detected by common procedures like regular microscopy and even Polymerase Chain reactions since their genetic materials do not possess the 16S marker other living organisms possess. Efficient diagnosis of nanobacteria-based conditions are important, especially now that these organisms have been associated with multiple potentially fatal cases.

References

Ciftcioglu N, Bjorklund M, Kuorikoski K, Kajander E.(1999). Nanobacteria: an infectious cause for kidney stone formation. Kidney International, 56(5): 1893–1898

Ciftcioglu N, Miller-Hjelle M, Hjelle J, Kajander E. (2002).Inhibition of nanobacteria by antimicrobial drugs as measured by modified microdilution method. Antimicrobial Agents and Chemotherapy, 46: 2077–2086

Ciftçioglu N, Haddad R, Golden D, Morrison D, McKay D.(2005). “A potential cause for kidney stone formation during space flights: Enhanced growth of nanobacteria in microgravity”, Kidney International, 67: 1–9

Hu YR, Zhao Y, Sun YW, Lü WD, Liu ZL, et al.(2010) Detection of nanobacteria-like material from calcified cardiac valves with rheumatic heart disease. Cardiovascular Pathology, 5: 286–292

Kajander E and Ciftcioglu N. (1998).Nanobacteria: an alternative mechanism for pathogenic intra-and extracellular calcification and stone formation. Proceedings of the National Academy of Sciences, 95(14):8274–8279.

Martel J. & Young J. (2008). Purported nanobacteria in human blood as calcium carbonate nanoparticles. Proceedings of the National Academy of Sciences,105: 5549–5554.

McKay D., et al. (1996). “Search for Past Life on Mars: Possible Relic Biogenic Activity in Martian Meteorite ALH84001”. Science 273 (5277): 924–930

R. Tixado, G. Richoilley, G. Gasset, et al.(1985). Study of minimal inhibitory concentration of antibiotics on bacteria cultivated in vitro in space (Cytos 2 experiment). Aviation, Space, and Environmental Medicine 56, :748–751

Uwins P, Webb R and Taylor A.(1998) Novel nano-organisms from Australian sandstones. American Mineralogist, 83: 1541–1550

Waters E, Hohn M., Ahel I, Graham D., Adams M., Barnstead M,Stetter K. et al. (2003).The Genome of Nanoarchaeum equitans: Insights into early archaeal evolution and derived parasitism. Proceedings of the National Academy of Sciences, 100(22):12984–12988.