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Journal of Clinical Microbiology, July 2003, p. 3460-3461, Vol. 41, No. 7
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.7.3460-3461.2003

LETTER TO THE EDITOR

Pitfalls in Detection of Novel Nanoorganisms


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Drancourt el al. have reported on their attempts to isolate nanobacteria from upper urinary tract stones (3). Their findings and opinions are valuable for the nanobacteria research. However, we want to point out difficulties that any researcher will face when working with nanobacteria: lack of published data and working instructions, lack of tested commercial culture media and identification tools, and lack of readily available positive and negative controls. Novel paradigms are difficult to publish. Manuscripts on nanobacteria have so far been returned from Nature, Science, etc. Lack of publications on basic findings and methods used leads to two important consequences. (i) Scientists will waste their time trying to work with well-established routine methods, which unfortunately need modifications or must be replaced by new technologies. (ii) Negative results are obtained and accepted as such. Although the results were not properly controlled (culture media were neither pretested for growth promotion of nanobacteria nor controlled by positive test cultures), people and journals may choose the easiest way.

Any microbiological classification of tentative nanoorganisms, such as nanobacteria proposed by Kajander and Ciftcioglu (6) and nanobes proposed by Uwins et al. (8), is difficult because they are not typical bacteria. They have also virus-, fungus-, and prion-like characteristics and thus cannot fit into any existing class of microorganisms (Table 1). They should be considered as their own entity. Research tools and techniques for nanoorganisms require new attitudes and ideas. Recent findings have indicated that there are many surprises to come (4). Isolation of Nanoarchaeum equitans, a symbiont of hyperthermophilic bacteria, required extra efforts in characterization because standard PCR techniques failed in detecting the organism's genetic material, the presence of which was revealed with DNA stains (5). Interestingly, both nanobacteria and nanobes contain nucleic acid material detectable with DNA and RNA stains (6, 8).


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  		TABLE 1. Unique properties of nanobacteria (7) with respect to those of viruses, prions, and bacteriaa

 
It is of utmost importance to realize the limits of our current methodologies with respect to detection and culture of novel nanoorganisms, as exemplified by nanobacteria. Many so-called negative reports have been able to repeat the morphological finding of calcium phosphate self-propagating units (1, 2, 3). Nanobacteria-like organisms have been found in human atherosclerotic plaques (T. E. Rasmussen, B. L. Kirkland, J. Charlesworth, G. Rodgers, S. R. Severson, J. Rodgers, R. L. Folk, and V. M. Miller, 51st Annual Meeting of the American College of Cardiology, J. Am. Coll. Cardiol. 39[Suppl. 1]:206, 2002). Atherosclerosis is a burden to billions of people. Clinical and microbiological laboratories should not take the easiest way and judge the calcium phosphate particles as artifacts. Who would like to carry self-propagating nanocrystalline apatite in their blood, blood vessels, stones, and tissues? Evaluation of nanobacteria phenomena should not be based just on routine bacteriological criteria but rather on multidisciplinary efforts by innovative and open-minded scientists.


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  1. Barr, S. C., R. A. Linke, D. Janssen, C. L. Guard, M. C. Smith, C. S. Daugherty, and J. M. Scarlett. 2003. Detection of biofilm formation and nanobacteria under long-term cell culture conditions in serum samples of cattle, goats, cats and dogs. Am. J. Vet. Res. 64:176-182.[CrossRef][Medline]
  2. Cisar, J. O., D.-Q. Xu, J. Thompson, W. Swaim, L. Hu, and D. J. Kopecko. 2000. An alternative explanation of nanobacteria-induced biomineralization. Proc. Natl. Acad. Sci. USA 97:11511-11515.[Abstract/Free Full Text]
  3. Drancourt, M., V. Jacomo, H. Lepidi, E. Lechevallier, V. Grisoni, C. Coulange, E. Ragni, C. Alasia, B. Dussol, Y. Berland, and D. Raoult. 2003. Attempted isolation of Nanobacterium sp. microorganisms from upper urinary tract stones. J. Clin. Microbiol. 41:368-372.[Abstract/Free Full Text]
  4. Hohn, M. J., B. P. Hedlund, and H. Huber. 2002. Detection of 16S rRNA sequences representing the novel phylum "Nanoarchaeota": indication for a wide distribution in high temperature biotopes. Syst. Appl. Microbiol. 25:551-554.[CrossRef][Medline]
  5. Huber, H., M. J. Hohn, R. Rachel, T. Fuchs, V. C. Wimmer, and K. O. Stetter. 2002. A new phylum of Archaea represented by a nanosized hyperthermophilic symbiont. Nature 417:63-67.[CrossRef][Medline]
  6. Kajander, E. O., and N. Ciftcioglu. 1998. Nanobacteria: an alternative mechanism for pathogenic intra- and extracellular calcification and stone formation. Proc. Natl. Acad. Sci. USA 95:8274-8279.[Abstract/Free Full Text]
  7. Kajander, E. O., N. Ciftcioglu, K. Aho, and E. Garcia-Cuerpo. Characteristics of nanobacteria and their possible role in stone formation. Urol. Res. 31:47-54.
  8. Uwins, P. J. R., R. I. Webb, and A. P. Taylor. 1998. Novel nano-organisms from Australian sandstones. Am. Mineral. 83:1541-1550.[Abstract]
Katja Aho*
E. Olavi Kajander
Department of Biochemistry
University of Kuopio
P.O. Box 1627
70211 Kuopio, Finland

* Phone: 358 17 16 3078
Fax: 358 17 2811 510
E-mail: kmaho{at}hytti.uku.fi


Author's Reply


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K. Aho and O. Kajander are commenting on the negative attempts in the previous study by my laboratory to grow nanobacteria. My coworkers and I tried to reproduce their technique without success. Since 1998, we have tried to obtain the strain from Kajander. Here we failed to confirm their work. The putative "Nanobacterium" strain is protected and not available, but they sell products to detect nanobacteria. To the best of my knowledge, nobody has reproduced this work. I would be happy to test their strain and change my mind if the data are convincing. The authors cite references on Nanoarchaea (1, 2), which have no correlation with this topic but the name. The main problem is that in science, the exact method and the obtained strains should be exchanged to allow other investigators to reproduce and confirm the work. Regarding my alleged reluctance to find new microorganisms, I suggest that the authors consider previous studies from my laboratory, including reports of the culture of the biggest virus (3), that of Tropheryma whipplei (5), and that of other microorganisms, including Rickettsia species, which are small bacteria (4, 6).


   REFERENCES 
Top
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 Letter 
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  1. Hohn, M. J., B. P. Hedlund, and H. Huber. 2002. Detection of 16S rRNA sequences representing the novel phylum "Nanoarchaeota": indication for a wide distribution in high temperature biotopes. Syst. Appl. Microbiol. 25:551-554.
  2. Huber, H., M. J. Hohn, R. Rachel, T. Fuchs, V. C. Wimmer, and K. O. Stetter. 2002. A new phylum of Archaea represented by a nanosized hyperthermophilic symbiont. Nature 417:63-67.
  3. La Scola, B., S. Audic, C. Robert, L. Jungang, X. de Lamballerie, M. Drancourt, R. Birtles, J. M. Claverie, and D. Raoult. 2003. A giant virus in amoebae. Science 299:2033.[Free Full Text]
  4. Ogata, H., S. Audic, P. Renesto-Audiffren, P. E. Fournier, V. Barbe, D. Samson, V. Roux, P. Cossart, J. Weissenbach, J. M. Claverie, and D. Raoult. 2001. Mechanisms of evolution in Rickettsia conorii and R. prowazekii. Science 293:2093-2098.[Abstract/Free Full Text]
  5. Raoult, D., M. L. Birg, B. La Scola, P. E. Fournier, M. Enea, H. Lepidi, V. Roux, J. C. Piette, F. Vandenesch, D. Vital-Durand, and T. J. Marrie. 2000. Cultivation of the bacillus of Whipple's disease. New Engl. J. Med. 342:620-625.[Abstract/Free Full Text]
  6. Raoult, D., P. E. Fournier, F. Fenollar, M. Jensenius, T. Prioe, J. J. de Pina, G. Caruso, N. Jones, H. Laferl, J. E. Rosenblatt, and T. J. Marrie. 2001. Rickettsia africae, a tick-borne pathogen in travelers to sub-Saharan Africa. N. Engl. J. Med. 344:1504-1510.[Abstract/Free Full Text]
Didier Raoult*
Unité des Rickettsies
Faculté de Médecine
27 Boulevard Jean Moulin
13385 Marseille Cedex 5, France

* Phone: 33 (0)4 91 83 55 17
Fax: 33 (0)4 91 83 03 90
E-mail: Didier.Raoult{at}medecine.univ-mrs.fr

Journal of Clinical Microbiology, July 2003, p. 3460-3461, Vol. 41, No. 7
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.7.3460-3461.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.




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