Journal of Culture Collections National Bank for Industrial Microorganisms and Cell Cultures ISSN: 1310-8360 Vol. 2, Num. 1, 1998, pp. 77-82

Emil Kalfin

State University Hospital of Pulmonary Diseases, 19 D. Nestorov blvd, Sofia, Bulgaria or 2 Grivitza, 1202 Sofia, Bulgaria,

Code Number: cc98013

Summary  

An erythrocyte – like microorganism (ELM) was isolated in pure culture from 8 samples of blood taken from newborn umbilical cord, 50 samples of human blood for transfusion, and also in blood samples from 4 professors and 6 associate professors, who wanted to assure themselves in the discovery of new resident microbial flora, this time in human erythrocytes, about which there was no information in the medical literature.

The size of ELM varied ¹between 0.3 µm and 2.6 µm and because of this (with a size of 3.5 µm to 7.5 µm) 2 to 12 ELM cells could be seen (like in nest) in one erythrocyte after cultivation for 14 days at a temperature of 43°C in brain and heart liquid media. The electron microscope examination showed that ELM expulsed something like a cell nucleus being outside the human erythrocyte and it remained nucleus – free just like human red blood cells. This mimicry explained why ELM was able to multiply as a resident microbial flora in the human erythrocyte.

ELM formed a unique red coating in the brain and heart media and very small grey colonies (which are almost invisible) on human blood agar after cultivation for 21 days at temperature 37°C, but not on sheep blood agar. Lysol as well as heat (70°C for 10 minutes) killed the ELM. The urease, lysine decarboxylase and catalase tests were positive. ELM produced acid from glucose and maltose, but not from sucrose and lactose. DNA could be demonstrated by the use of SDS polyacrilamide gel electrophoresis, but not by fluorescent stain because there was no nucleus in the ELM outside the erythrocytes. Sodium polyanetholsulphonate was bacteriostatic for ELM and due to this for the ELM isolation from blood the media should be prepared using sodium citrate.

Introduction

There is no information in the available literature about the existence of resident microbial flora in human erythrocytes (RMHE) [ 1, 2, 3, 4] . The only representative of RMHE is an erythrocyte – like microorganism (ELM) which can not multiply in the nutrient media unless native human blood is added. ELM was isolated in a small microbiological laboratory (without a grant). Because of financial difficulties this laboratory uses outdated human blood, unlike the other laboratories, where sheep blood is used for the preparation of nutrient media. The laboratory receives free supplies of such outdated blood. The laboratories in the developed countries do not use human blood for the preparation of nutrient media, and this fact explains why they have not discovered RMHE.

The aim of this study was to announce the discovery of RMHE, to describe a method of ELM isolation in pure culture, and to describe the basic characteristics of ELM which make possible the differentiation of the new microorganism in small laboratories from all the described in literature microorganisms.

Materials and Methods

Liquid medium. The original brain and heart nutrient media produced by Merck or BBL^® (100 ml), sodium citrate (0.25 g), gentamicine (2 mg), and chlornitromycine (5 mg) were used. The medium was dissolved and distributed in sterile test tubes, 4 ml in each, and was sterilised in autoclave at 121°C for 15 minutes.

Human blood agar. Noble Difco agar (1.2%) was added to the liquid medium, which then was sterilized, cooled down to approximately 50°C and 10% of the outdated native human blood was added.

Isolation of the microorganism. Sodium polyanetholsulphonate (SPS) was toxic for ELM and could not be used. Human blood (0.5 ml) was inoculated in a tube with 4 ml of liquid medium and wascultivated at 37°C or at 43°C. Once per week the cultures were exposed to air by vigorous manual shaking for 10 sec, so that foam to be formed on the surface of the medium. The growth was assessed using Gram stained slides after 14, 21 and 30 days for the samples cultivated at 37°C and after 14 days for the ones cultivated at 43°C.

Patients. The samples from blood taken from the umbilical cord of 8 newborn infants, and from 50 blood samples for transfusion in patients, without selection, were cultivated for ELM isolation. As a control we used blood samples from 4 professors and 6 associate professors in medicine, who wanted to assure themselves that ELM multiplied as resident flora in their own erythrocytes.

Results

Growth assessment. The liquid nutrient medium remained transparent during the first week of the cultivation and there was no hemolysis. When the multiplication had began a unique coating was formed inside the liquid medium, on the glass wall and not on the surface like in the cases with known microorganisms. The coating was whitish after 3 weeks of cultivation. In about 4 weeks it became pink, and in 2 months it was red (Fig. 1A). This specific coating could differentiate ELM from all the other microorganisms described in literature. No coating was formed at room temperature (Fig. 1A). The ELM formed tiny gray colonies on human blood agar (Fig. 1B) (nearly invisible) after 21 days incubation at 37°C. These tiny gray colonies, formed only on human blood agar, but not on sheep blood agar, again could differentiate ELM from all the microorganisms described in literature (Fig. 1B). The ELM could be visualised on Gram stained slides as round, yeast – like cells. Their size varied between 0.3 µm and 2.6 µm, when compared to human erythrocytes which ranged between 3.5 µm and 7.5 µm (Fig. 1, C and D). No capsule, spores or micelles could be seen (Fig. 1C).

The ELM could multiply inside the human erythrocytes and in one erythrocyte 2 to 12 cells might be seen (Fig. 1D). ¹These unique nests were specific for ELM only and could differentiate it from all the other micro-organisms described in literature (Fig. 1D).

ELM in parrot. The ELM was a microbe of birds, because it multiplied faster at 43°C, than at 37°C, but it could not exist like in nests inside the bird erythrocytes (Fig. 2, A and B), because the last ones were almost fraught by a nucleus (Fig. 2C).

ELM on electron microscope (EM) photographs. The EM photographs revealed an unbelievable discovery in microbiology. The ELM had no visible nucleus on the EM photographs (Fig. 3, A and B). The cells were erythrocyte - like, but compared to the erythrocytes they were bushy.

ELM mimicry. The ELM used mimicry to deceive human granulocytes. Inside the human erythrocytes ELM possessed something which was not a classic nucleus (Fig. 3C), but outside them it expulsed the nucleus and remained nucleus free – an erythrocyte – like cell (Fig. 3, A, B and C).

Fig. 4 demonstrates ELM in the volunteers blood.

ELM biochemical characteristics.The ELM was aerobe and nonmotile. Lysol (1.5 %) as well as heat (70°C for 10 min) killed the ELM. Bidistilled water and acetic acid destroyed human erythrocytes, but the ELM remained intact, because of being a living microorganism. ELM produced acid from glucose and maltose, but not from sucrose and lactose. The urease, lysine decarboxylase and catalase tests were positive, but the ornitine decarboxylase, indole and hydrogen sulfide tests were negative. SPS inhibited the ELM growth in blood culture media. However, after the removal of the medium, ELM could again multiply in media without SPS. The in vitro ELM multiplication at 37°C lead to a decrease in the mean corpuscular hemoglobin to 26 pg after 21 days. The conclusion could be made that fast growth of ELM in some individuals might produce some degree of iron deficiency anaemi.

ELM possessed no nucleus and this was the reason for the negative acridinorange fluorescent staining, but it possessed DNA (determined by SDS polyacrilamide gel electrophoresis).

ELM had exhibited pathogenic properties both in birds and humans, but intraperitoneal inoculations of pure cultures in white mice and guinea pigs were negative (prof. I. Raichev).

Discussion

Microorganisms live on the Earth for millions of years, but Microbiology is younger than 200 years and as a rule microbiologists have less than 40 years of experience. These facts mean that today an Ephemera vulgata must decide what the living microorganisms are.

Here are two examples of Ephemera vulgata judgements: "The viruses are not living microorganisms, because they are too small to be living microorganisms" and "ELM is not a living microorganism, because ELM has no nucleus and there is no microorganism without a nucleus described in literature".

It is ridiculous to say that ELM is an erythrocyte or some blood artifact after the formation of colonies, which are observed on human blood agar (Fig. 1B). It is a waste of time and money to repeat that ELM produces acid from glucose and maltose, but not from sucrose and lactose, that ELM has DNA, but human erythrocytes possess no DNA, etc. (see results). Our hypothesis is that ELM exists in human erythrocytes from an early stage of the human evolution, because it is an acme of perfection: the colonies are small, nearly invisible (Fig. 1B) to obstruct the blood vessels. ELM does not multiply every 20 minutes in order to assure balance to the greatest possible extent between the normal death of human erythrocytes and the introduction of new erythrocytes in the circulation. ELM lives in human erythrocytes like in nests, because this is the best possible solution for the microorganisms (which avoid passing from one erythrocyte to another) and for the human (not loosing a great number of erythrocytes).

Finally, ELM has no nucleus, because using mimicry to deceive human granulocytes it can exist as RMHE.

In summary, a bird microbe can not multiply in the bird erythrocytes like in nests (Fig. 2, A and B), because they possess nucleus, which occupies almost the whole erythrocyte (Fig. 2C). There is no nucleus in the human erythrocyte (Fig. 3C) and the bird microbe can multiply in it like in a nest (Fig. 1D). Human granulocytes do not destroy the bird microbe, because it uses mimicry to deceive them (Fig. 3C) and has erythrocyte – like appearance (Fig. 3, A and B). As a conclusion, the discovery of RMHE is more surprising for the scientists on the Earth, than the discovery of some forms of life on Mars, which is imminent. The discovery of RMHE is interesting not only for the patients with some forms of anemia, but also for the healthy people, because everybody has RMHE by birth. Medicine is an ever changing science and the physicians and the microbiologists are responsible to study every new discovery in time and also to inform their patients about the new problems.

Acknowledgements

The author thanks to prof. N. Alexiev, prof. M. Milchev, prof. A. Petrov, prof. K. Milenkov, assoc. prof. R. Petkov, assoc. prof. A. Kujumdjiev, assoc. prof. P. Vachkov, assoc. prof. M. Markova, assoc. prof. J. Petrovska which volunteered to isolate ELM from their own blood, and also to assoc. prof. R. Petkov for the EM photographs.

References

1. Bernard, M.B., P.S Thimas, 1990. Hematology. A pathophysiological approach, sec. edn, New York: Churchill Livingstone.
2. Petkov, R., E. Kalfin, 1998. An electron microscopy study of resident microbial flora in human erythrocytes. IX Congress of Bulgarian Microbiologists, 15 – 17 Oct., 1998, Sofia (unpublished data).
3. Sleigh, J.D., M.C Timbury, 1998. Notes of medical Bacteriology, fifth edn., New York: Chuchill Livingstone.
4. Willet, J., A. Wilfert, 1996. Zinsser Microbiology, International edn., Prentice Hel International Inc.

This article is published without any corrections regarding the insistence of a depositor of ours (NBIMCC 3300 and 3301).

Copyright 1998 - National Bank for Industrial Microorganisms and Cell Cultures - Bulgaria

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