U.S. patent application number 10/743054 was filed with the patent office on 2005-02-17 for methods for eradication of nanobacteria.
Invention is credited to Ciftcioglu, Neva, Kajander, Olavi E..
Application Number | 20050036904 10/743054 |
Document ID | / |
Family ID | 31949690 |
Filed Date | 2005-02-17 |
United States Patent
Application |
20050036904 |
Kind Code |
A1 |
Kajander, Olavi E. ; et
al. |
February 17, 2005 |
Methods for eradication of nanobacteria
Abstract
Nanobacteria contribute to pathological calcification in the
human and animal body, including diseases such as kidney stones,
salivary gland stones, dental pulp stones and atherosclerosis. The
present invention provides methods for sterilizing articles
contaminated with nanobacteria. The present invention also provides
methods of treating patients infected with nanobacteria. In
particular, the present invention provides a method for preventing
the recurrence of kidney stones in a patient that has suffered from
kidney stones, comprising administration of an antibiotic, a
bisphosphonate, or a calcium chelator, either alone or in
combination, in an amount effective to inhibit or prevent the
growth and development of nanobacteria.
Inventors: |
Kajander, Olavi E.; (Kuopio,
FI) ; Ciftcioglu, Neva; (Kuopio, FI) |
Correspondence
Address: |
BURNS DOANE SWECKER & MATHIS L L P
POST OFFICE BOX 1404
ALEXANDRIA
VA
22313-1404
US
|
Family ID: |
31949690 |
Appl. No.: |
10/743054 |
Filed: |
December 23, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10743054 |
Dec 23, 2003 |
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09347189 |
Jul 2, 1999 |
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6706290 |
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60091716 |
Jul 6, 1998 |
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Current U.S.
Class: |
422/28 ; 422/22;
422/24 |
Current CPC
Class: |
A01N 37/44 20130101;
A01N 57/18 20130101; A61L 2202/23 20130101; A01N 37/36 20130101;
A61K 31/00 20130101; A01N 59/02 20130101; Y10S 424/06 20130101;
A01N 59/00 20130101; A61L 2/0088 20130101; A61L 2/081 20130101;
A01N 35/02 20130101; A01N 61/00 20130101; A61L 2/10 20130101; A61L
2/07 20130101; A61L 2/12 20130101; A61L 2/186 20130101; A61L 2/0035
20130101; A61L 2202/24 20130101; A61K 45/06 20130101; A01N 59/02
20130101; A01N 59/02 20130101; A01N 41/04 20130101; A01N 35/02
20130101; A01N 35/02 20130101; A01N 37/42 20130101; A01N 33/12
20130101; A01N 35/02 20130101; A01N 2300/00 20130101; A01N 59/02
20130101; A01N 2300/00 20130101 |
Class at
Publication: |
422/028 ;
422/024; 422/022 |
International
Class: |
A61L 002/00 |
Claims
1. A method for disinfecting an article contaminated with
nanobacteria, comprising exposing the nanobacteria to a
disinfectant mixture.
2. A method according to claim 1, wherein the disinfectant mixture
is a 1% mixture of 50% potassium persulfate and 5% sulfaminoic acid
in distilled water.
3. A method according to claim 1, wherein the disinfectant mixture
is a 3% mixture of 4.5% formaldehyde, 6.8% glyoxal, 1.5% glyoxylic
acid, and 6% dimethylaurylbenzyl-ammonium chloride.
4. A method according to claim 1, wherein the disinfectant mixture
is a low molecular weight organic acid that binds or chelates
calcium.
5. A method according to claim 4, wherein the acid is selected from
the group consisting of citric acid, acetic acid, lactic acid,
ascorbic acid, and and salicylic acid and its acetyl and
aminoacetyl derivatives.
6. A method for disinfecting an article contaminated with
nanobacteria by demineralizing the nanobacteria, and then exposing
the article to a disinfectant mixture.
7. A method according to claim 6, wherein demineralization is
accomplished by exposing the article to a mixture with low pH.
8. A method according to claim 7, wherein the mixture with low pH
is a strong acid.
9. A method according to claim 8, wherein the strong acid is
hydrochloric acid.
10. A method according to claim 6, wherein demineralization is
accomplished by exposing the article to an effective amount of a
calcium chelator.
11. A method according to claim 10, wherein the calcium chelator is
EDTA.
12. A method according to claim 6, wherein the disinfectant
chemical is selected from the group consisting of ethanol,
glutaraldehyde, formaldehyde, hypochlorite, hydrogen peroxide,
hydrochloric acid, sodium hydroxide, SDS, Tween 80, Triton X-100,
guanidium-hydrochloride, urea, Virkon.RTM., Erifenol.RTM.,
Klorilli.RTM., Buraton.RTM., and mixtures thereof.
13. A method according to claim 6, wherein the disinfectant
chemical is selected from the group consisting of ethanol (at least
a 70% solution); glutaraldehyde (at least a 2% solution);
formaldehyde (at least a 4% solution); hypochlorite (at least an
0.5% solution); hydrogen peroxide (at least a 3% solution);
hydrochloric acid (at least a 1M solution); sodium hydroxide (at
least a 1M solution); sodium dodecyl sulfate(at least a 1%
solution); Tween 80 (at least a 1% solution); Triton X-100 (at
least a 1% solution); guanidium-hydrochloride (at least a 3M
solution); urea (at least a 3M solution); Virkon.RTM. (at least a
1% solution); Erifenol.RTM. (at least a 1.5% solution);
Klorilli.RTM. (at least a 1% solution); Buraton.RTM. (at least a 3%
solution); and mixtures thereof.
14. A method according to claim 6, further comprising autoclaving
the article at a temperature of at least 121.degree. C. for at
least 20 minutes.
15. A method for disinfecting an article contaminated with
nanobacteria by demineralizing the nanobacteria, and then
autoclaving the article at a temperature of at least 121.degree. C.
for at least 20 minutes.
16. A method according to claim 6, further comprising exposing the
article to ultraviolet radiation, by exposing the article to a UV-C
lamp of at least 15 W at a distance of 60 cm or less for at least 1
hour.
17. A method for disinfecting an article contaminated with
nanobacteria by demineralizing the nanobacteria, and then exposing
the article to ultraviolet radiation, by exposing the article to a
UV-C lamp of at least 15 W at a distance of 60 cm or less for at
least 1 hour.
18. A method for disinfecting an article contaminated with
nanobacteria comprising exposing the article to at least three
megarads of gamma radiation.
19. A method for disinfecting an article contaminated with
nanobacteria by heating the article for at least one hour at a
temperature of at least 100.degree. C.
20. A method for disinfecting an article contaminated with
nanobacteria by demineralizing the nanobacteria, and then heating
the article for at least 15 minutes, at a temperature of at least
60.degree. C.
21. A method for disinfecting an article contaminated with
nanobacteria by demineralizing the nanobacteria, and then heating
the article for at least 30 minutes at a temperature of at least
100.degree. C.
22. A tissue culture medium formulated to be free of nanobacteria,
wherein said tissue culture medium comprising a
nanobacteria-antibiotic-effective amount of one or more antibiotics
selected from the group consisting of .beta.-lactam antibiotics,
aminoglycoside antibiotics, and mixtures thereof.
23. A tissue culture medium according to claim 22, wherein the
.mu.-lactam antibiotics are selected from the group consisting of
penicillin, phenethicillin, ampicillin, azlocillin, bacmpicillin,
carbenicillin, cylclacillin, mezlocillin, piperacillin, epicillin,
hetacillin, cloxacillin, dicloxacillin, methicillin, nafcillin,
oxacillin, and salts thereof.
24. A tissue culture medium according to claim 23, wherein the
aminoglycoside antibiotics are selected from the group consisting
of streptomycin, kanamycin, gentamycin, amikacin, neomycin,
pardomycin, tobramycin, viomycin, and salts thereof.
25. A method for preventing or treating the development of
calcifications in vivo in a patient in need of such prevention or
treatment, comprising administering an antibiotic to the patient in
an amount sufficient to inhibit or prevent the growth of
nanobacteria.
26. A method according to claim 25, wherein the antibiotic is
selected from the group consisting of .beta.-lactam antibiotics,
aminoglycoside antibiotics, tetracycline antibiotics, and
pharmaceutically acceptable salts thereof, and mixtures
thereof.
27. A method according to claim 26, wherein the .beta.-lactam
antibiotics are selected from the group consisting of penicillin,
phenethicillin, ampicillin, azlocillin, bacmpicillin,
carbenicillin, cylclacillin, mezlocillin, piperacillin, epicillin,
hetacillin, cloxacillin, dicloxacillin, methicillin, nafcillin,
oxacillin, and pharmaceutically acceptable salts thereof.
28. A method according to claim 26, wherein the aminoglycoside
antibiotics are selected from the group consisting of streptomycin,
kanamycin, gentamycin, amikacin, neomycin, pardomycin, tobramycin,
viomycin, and pharmaceutically acceptable salts thereof.
29. A method according to claim 26, wherein the tetracycline
antibiotics are selected from the group consisting of tetracycline,
chlortetracycline, demeclocycline, doxycycline, methacycline,
oxytetracycline, rolitetracycline, minocycline, sancycline, and
pharmaceutically acceptable salts thereof.
30. A method according to claim 26, wherein the antibiotic is
coadministered with a citrate compound.
31. A method for preventing or treating the development of
calcifications in vivo in a patient in need of such prevention or
treatment, comprising administering a bisphosphonate to the patient
in an amount sufficient to inhibit or prevent the growth of
nanobacteria.
32. A method according to claim 31, wherein the bisphosphonate is
selected from the group consisting of alendronic acid, etidronic
acid, clodronic acid, oxidronic acid, and pharmaceutically
acceptable salts thereof.
33. A method according to claim 31, wherein the bisphosphonates are
administered at a dose of approximately 5-20 mg/kg/day.
34. A method according to claim 31, wherein the bisphosphonates are
coadministered with an antibiotic.
35. A method according to claim 34, wherein the antibiotic is a
tetracycline antibiotic, selected from the group consisting of
tetracycline, chlortetracycline, demeclocycline, doxycycline,
methacycline, oxytetracycline, rolitetracycline, minocycline,
sancycline, and pharmaceutically acceptable salts thereof.
36. A method for preventing the development of kidney stones in a
patient that has previously suffered from kidney stones, comprising
administering an antibiotic to the patient in an amount effective
to inhibit or prevent the growth of nanobacteria.
37. A method for the prevention or treatment of any condition or
disease state caused by nanobacteria, said method comprising
administering an effective amount to prevent or treat a condition
or disease state caused by nanobacteria to a patient in need of
such prevention or treatment.
38. A method according to claim 37, wherein said condition or
disease state is chronic fatigue syndrome.
39. A method for eradicating nanobacteria from liquid materials,
comprising sonicating said liquid.
40. A method according to claim 39, wherein said liquid is cooled
during sonication.
41. A method according to claim 39, wherein said liquid is
sonicated for at least about five minutes.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention relates to methods of disinfecting
articles infected with nanobacteria, and methods of treating
patients infected with nanobacteria.
[0003] 2. Description of the Related Art
[0004] The formation of discrete and organized inorganic
crystalline structures within macromolecular extracellular matrices
is a widespread biological phenomenon generally referred to as
biomineralization. Mammalian bone and dental enamel are examples of
biomineralization involving apatite minerals. Environmental apatite
stones have almost the same chemical composition as in bone and
dentine. Recently, bacteria have been implicated as factors in
biogeochemical cycles for mineral formation in aqueous sediments.
The principal constituent of modern authigenic phosphate minerals
in marine sediments is carbonate (hydroxy)fluorapatite
Ca.sub.10(PO.sub.4).sub.6-x(CO.sub.3).sub.x(F,OH).sub.2+x.
Microorganisms are capable of depositing apatite outside
thermodynamic equilibrium in sea water. They can segregate Ca from
Mg, and actively nucleate carbonate apatite by means of specific
oligopeptides under conditions pH<8.5 and [Mg]:[Ca]>0.1. Such
conditions are also present in the human body.
[0005] Nanobacteria approach the theoretical limit of the
self-replicating life with a size of only one hundredth of that of
usual bacteria. Nanobacteria can be isolated from mammalian blood
and blood products (see, U.S. Pat. No. 5,135,851 to Kajander, the
contents of which are incorporated herein by reference).
Energy-dispersive X-ray microanalysis and chemical analysis reveals
that nanobacteria produce biogenic apatite on their cell envelope.
The thickness of the apatite depends mostly on the culture
conditions of the nanobacteria. Nanobacteria are the smallest cell
walled, apatite forming bacteria isolated from mammalian blood and
blood products. Their small size (0.05-0.5 .mu.m), and unique
properties make their detection difficult with conventional
microbiological methods. In nanobacteria-infected mammalian cells,
electron microscopy revealed intra- and extracellular acicular
crystal deposits, stainable with von Kossa staining and resembling
calcospherules found in pathological calcification.
[0006] The present inventors have discovered nanobacteria in human
and cow blood that are cytotoxic in vitro and in vivo. They have
been deposited in DSM, Braunschweig, Germany at accession No.
5819-5821. Human and bovine nanobacteria grow similarly, share the
same surface antigens, and other special features. They both
produce carbonate apatite as well. Nanobacteria possess unusual
properties making their detection difficult with standard
microbiological methods. Although they typically have diameters of
0.2-0.5 .mu.m, they also exist in tiny forms (0.05-0.2 .mu.m) as
observed using transmission electron microscopy (TEM). Thus
nanobacteria manage to pass through 0.1 .mu.m filters. Nanobacteria
are poorly disruptable, stainable, fixable and exceptionally
resistant to heat. Their doubling time is about 3 days. High doses
of y-irradiation or aminoglycoside antibiotics prevented their
multiplication. According to the 16S rRNA gene sequence (EMBL
X98418 and X98419), nanobacteria fall within the .alpha.-2 subgroup
of Proteobacteria, which also includes Brucella and Bartonella
species. The latter genera include human and animal pathogens that
share similarities with nanobacteria, e.g., some of the same
antigens and cytopathic effects.
[0007] Competition for nutrients necessary for life is enormous in
natural environments and thus clever adaptations and survival
strategies for unfavorable conditions are needed. Bacteria can form
spores, cysts and biofilm, which help them survive unfavorable
periods of time. Bacteria in such forms have significantly slower
metabolic functions, but vegetative cells can slow down their
metabolism as well. The increased resistance of bacteria in biofilm
or as spores is not only because of the slower metabolic rate. The
impermeable structures around the organism serve as mechanical
barriers blocking the entrance of potentially harmful compounds.
Some additional mechanisms are also known which help in the
survival of bacteria. The heat resistance of bacterial spores can
be attributed to three main factors, these are protoplast
dehydration, mineralization and thermal adaptation. Radiation
resistance is commonly associated with sophisticated DNA repair
systems. Minimizing metabolic rate and multiplication are obviously
the main preconditions for bacterial survival, allowing time for
the repair of DNA and other damaged cellular components. Very slow
metabolism, and ability to form biofilm are also characteristics of
nanobacteria. Because of their minimal size, the presence of
complicated systems for nucleic acid repair in nanobacteria seems
very unlikely. A possible explanation for the observed gamma
irradiation resistance may be their very small size, and the
peculiarities in their nucleic acid structure.
[0008] Apatite may play a key role in the formation of kidney
stones. The crystalline components of urinary tract stones are
calcium oxalate, calcium phosphate, struvite, purines, or cystine.
The majority of urinary stones are admixtures of two or more
components, with the primary admixture being calcium oxalate and
apatite. Furthermore, fermentor model studies have shown that
calcium phosphate nidi are always formed initially, and may
subsequently become coated with calcium oxalate or other
components. Urinary tract infection, causing struvite and carbonate
apatite formation, is a common cause of kidney stones. Conventional
therapy has usually consisted of surgical removal of the stone,
combined with a short course of antimicrobial therapy. Such
treatment is curative in about 50% of cases. Recurrent stone
formation and progressive pyelonephritis occur in those who are not
cured. The morbidity and expense that result from this disease is
significant.
[0009] Tissue calcification of carbonate apatite in nature is
common in other diseases, e.g., atherosclerotic plaques accumulate
calcium phosphate. 25% of atherosclerotic plaques in human aorta
specimens were found to contain nanobacterial by immunoassay and
immunohistochemical staining. Hemodialysis patients can develop
extensive metastatic and tumoral calcification. Acute periarthritis
is apatite arthropathy related to intratendinous calcifications.
Apatite crystals also cause inflammation when injected into the
synovial space. Tissue calcification is also found in several kinds
of cancer.
[0010] Pulp stones or denticles are polymorphous mineralized bodies
of various sizes occasionally found in the pulpal connective tissue
of human teeth. Their etiology remains unclear although they have
been frequently associated with aging or pathology of the pulp.
They may also be present in permanent teeth that are impacted free
of pathology for a long time. Although pulp stones have been
extensively studied morphologically, their origin is still obscure
and little is known about their chemical composition. An
histochemical study of pulpal calcifications has shown that the
organic matrix consists of reticular connective tissue fibers and a
ground substance containing glycoproteins and acid polysaccharides.
The mineral phase of pulp calcification has been studied with X-ray
energy dispersive spectrometry and chemical analysis, and proven
that calcium salts are deposited in the form of apatite, possibly
carbonate containing apatite. In fact, there is not much difference
between the chemical structure of a tooth and denticles. Bone and
tooth formation in the body have similar mechanisms, leaving many
unanswered questions. Apatite formation in the body (except in
tooth and bone) is called pathologic biomineralization, e.g.,
dental pulp stones, kidney stones, and joint calcifications.
[0011] Malacoplakia is a rare chronic inflammatory disease of
unknown cause, but a bacterial factor has been strongly implicated.
It may be fatal. The disease is characterized by von Kossa staining
positive, calcified laminated or target-shaped bodies termed
Michaelis-Gutmann bodies which are composed of apatite. The
structure of these calcospherules closely resembles calcified
nanobacteria.
[0012] Tissue calcifications are found in several diseases such as
ovarian serous tumor, papillary adenocarcinoma of the endometrium,
breast carcinoma, papillary carcinoma of the thyroid, duodenal
carcinoid tumor, and craniopharyngioma. In many malignant tumors,
needle-shaped crystals are found in epithelial cells. To detect
this kind of calcification it is necessary to use electron
microscopy, since the crystals are too small to be seen with the
light microscope, and their origin is unknown. Many malignant cells
have receptors for nanobacterial adherence. They could introduce
nanobacteria into the tumor with subsequent calcification.
Furthermore, some dividing cells under inflammatory stimuli may
have receptors for adherence, e.g., in atherosclerotic plaques
known to have calcium phosphate accumulation. In this disease,
although electron probe analysis showed that the surface and
interior of the mineral deposit had the same chemical composition,
SEM revealed different kinds of structures such as spherical
particles and fibers which resemble nanobacteria. Similarly, acute
periarthritis has been associated with the presence of
hydroxyapatite crystals in the joints.
[0013] Alzheimer plaques may be labeled with anti-nanobacterial
polyclonal antibodies. These polyclonal antibodies contain some
autoantibodies, and the present inventors have also obtained some
monoclonal autoantibodies in nanobacterial immunizations. Slow
bacterial infection has been suggested to play a role in autoimmune
diseases. Tissue calcification is often present in these diseases.
Nanobacteria are a new example of slowly growing organisms,
infecting man for long periods of time. The apatite structure and
anomalous nucleic acids may contribute to abnormalities in immune
response to this infection.
[0014] Several aspects of biogenic apatite nucleation, crystal
growth and morphology have been determined both in vivo and in
vitro. However, many details remain unresolved, including the
specific nature of the initial precipitating phases, the mechanism
and factors which control the incorporation of ionic impurities
into the crystal lattice, details of the crystallographic
ultrastructure and morphology in mineralized tissues (bone,
dentine), and the relationship of the inorganic components with the
complex collagen based matrix. The reason behind the calcium
phosphate deposition in many diseases remain speculative. It has
been shown that an accumulation of calcium in mitochondria, which
is presumably dependent upon residual substrate for energy
production, appeared to cause calcification. Amorphous calcium
phosphate in the form of spheroids, and possibly fine fibrils and
granules, also appears to play a role in calcification by their
transformation into apatite.
SUMMARY OF THE INVENTION
[0015] The present invention provides methods for sterilizing
articles contaminated with nanobacteria. Such methods according to
the present invention will be particularly useful for disinfecting
and/or sterilizing medical equipment and solutions used in patient
treatment and diagnosis.
[0016] The present invention also provides methods of preventing
nanobacterial infection, and treating patients infected with
nanobacteria. In particular, the present invention provides a
method for preventing the recurrence of kidney stones in a patient
that has suffered from kidney stones, comprising administration of
an antibiotic in an amount effective to inhibit or prevent the
growth and development of nanobacteria.
[0017] With the foregoing and other objects, advantages and
features of the invention that will become hereinafter apparent,
the nature of the invention may be more clearly understood by
reference to the following detailed description of the preferred
embodiments of the invention and to the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1. Light and electron microscopic images of
nanobacteria and their analyses with energy-dispersive X-ray
microanalysis (EDX). (A) Differential interference constrast image
of bottom-attached nanobacteria after a 2-month culture period. (B)
DNA staining of the same area (.times.1600) with the modified
Hoechst method. (C) Negative staining of nanobacteria isolated
directly from fetal bovine serum (Bar=200 nm). (D) SEM micrograph
showing their variable size (Bar=1 .mu.m). (E) A dividing
nanobacterium covered with a `hairy` apatite layer (Bar=100 nm).
(F) TEM micrograph of nanobacteria buried in an apatite layer after
a 3-month-long culture period (Bar=1 .mu.m), (G) at higher
magnification (Bar=200 nm). White central areas in F are artefacts
due to loss of the mineral layer in sectioning. (H)
Energy-dispersive X-ray microanalysis. in SEM of nanobacteria
showing Ca and P peaks similar to hydroxyapatite (I).
[0019] FIG. 2. Nanobacterial stony colonies, and comparison with
hydroxyapatite.(A) Colonies on modified Loeffler medium in a 10 cm
plate. The colonies penetrated through the medium forming stony
pillars. Arrow shows one typical greyish-brown colony depicted in B
(.times.40). (C) Needle-like crystal deposits in the pillar
revealed by TEM (Bar=200 nm). (D) TEM image of reference apatite
crystals (Bar=100 nm).
[0020] FIG. 3. Nanobacteria cultured under SF conditions, and their
interaction with cells. (A) Light microscopic micrograph, (B) DNA
staining of the same area with the modified Hoechst staining
method. (C) Differential interference contrast images of
nanobacteria inside a common apatite shelter, and (D) a partly
demineralized nanobacterial group (A-D, .times.860). (E and F) SEM
micrographs of nanobacterial dwellings detached from the culture
vessel (Bars=1 .mu.m). (G) IIFS of internalized mineralized
nanobacteria (white arrows) in 3T6 cells. (H) DNA staining of the
same area with standard Hoechst method (.times.540). (I-L) TEM
micrographs of intracellular calcifications in 3T6 cells caused by
SF-nanobacteria (Bars, I and K=2 .mu.m, J=500 nm, L=200 nm).
[0021] FIG. 4. Examples of extra- and intracellular calcification
by nanobacteria. (A) TEM micrograph of cultured nanobacteria
(Bar=20 nm) from fetal bovine serum, and (B) a bacterium in a
kidney stone after demineralization (Bar=50 nm). (C) IIFS of the
same kidney stone with anti-nanobacteria mAb. (D and E) von Kossa
staining results of 3T6 cells exposed to SF-nanobacteria for 24 hr,
and (F) negative control (.times.270).
[0022] FIG. 5. Graphic showing the effect of heat on the growth of
serum nanobacteria. Nanobacteria were exposed for heat in PBS and
cultured for 16 days. Only thirty minutes boiling resulted in the
inactivation of nanobacteria. Exponential growth was observed with
all other treatments. The medium containing 10% gamma irradiated
serum (Negative control) did not show any grow.
[0023] FIG. 6. Graphic presenting the effect of antibiotics on
nanobacterial growth. The growth is compared to that of
nanobacteria cultured without antibiotics. Doses of antibiotics ten
times higher than recommended for use in cell culture were needed
to prevent the growth of nanobacteria.
[0024] FIG. 7. SEM images of nanobacteria cultured with and without
antibiotics for one month in medium containing 10% fetal bovine
serum. Bars=1 .mu.m. (A) Nanobacteria cultured without antibiotics.
(B) Nanobacteria cultured with 100 .mu.g/ml gentamycin. Arrows show
changes in the morphology.
[0025] FIG. 8. SEM images of teeth with (A and B), and without (C
and D) dental calculi. The tooth shown in (A) was extracted because
of periodontal problems, and bone desorption caused by severe
dental pulp stone formation. Higher magnification from the area
shown by arrow depicts round, and fibrous calcification (B). The
tooth shown in (C) was extracted because of an orthodontic problem.
This tooth was autoclaved and exposed to DMEM culture medium for
one month, in a cell culture condition. No crystallization on the
surface was observed (D) Shows the higher magnification of the area
marked with an arrow in (C). The vertically cut other half of the
same tooth was used for the experiment described in FIG. 9.
[0026] FIG. 9. SEM micrographs of the healthy tooth shown in FIGS.
8C and D after autoclaving, and incubating with SF-nanobacteria for
one month in cell culture conditions. (A) General image showing the
surface of the tooth, higher magnification to an area shown by the
arrow is seen in (B). (C) Nanobacteria cultured for 3 months, and
adhered to cell culture vessel; bar is 1 .mu.m. (D) An area in the
same tooth having voluminous pulp stone that appeared after
SF-nanobacteria exposure for one month. (E) Higher magnification of
the same area shown with big arrow in (D). Small arrows show the
growth of SF-nanobacteria on the surface of calculi.
[0027] FIG. 10. SEM images of SF-nanobacteria growing on a piece of
dolomite in the culture medium.
[0028] FIG. 11. Energy dispersive X-ray microanalysis of human
dental calculi (O) and SF-nanobacteria (B).
[0029] FIG. 12. Interaction of SF-nanobacteria with fibroblasts
(3T6 cells). (A) SF-nanobacteria internalized by a fibroblast
(arrow head shows the nanobacteria inside a vacuole), (B) higher
magnification showing the needle-like apatite structure of the
internalized SF-nanobacteria, (C) von Kossa staining result of the
nanobacteria infected fibroblasts, and (D) negative control.
Magnification in (C and D) is 270.times.. Arrow in (C) shows
stained nanobacteria after staining with the von Kossa method which
is a standard calcification detection method used in pathology.
[0030] FIG. 13. TEM micrographs of a carbonate apatite human kidney
stone and nanobacteria. (A) A kidney stone before demineralization,
(B) SF-nanobacteria cultured for one month, (C) the same kidney
stone after demineralization, (D) nanobacteria cultured in serum
containing medium for 2 months. The kidney stone (C) was
demineralized by incubating the smashed stone in 1N HCl for 10 min
at room temperature, neutralized with NaOH and potassium phosphate
buffer, and epon embedded. Both cultures of nanobacteria (B and D)
adhered to the bottom of their culture vessels.
[0031] FIG. 14. TEM (A), and FITC images (B) of demineralized
nanobacteria and immunofluorescence positivity in different kind of
kidney stones (C-E). Kidney stones and nanobacteria were stained by
using specific anti-nanobacteria monoclonal antibodies, after
demineralization of the samples as described in FIG. 13. Thick
arrows show immuno-fluorescence-positive individual coccoid
particles. Immunopositivity on the surface of the small units
composing the stone is shown with the small arrows. Magnifications:
(B-D) 1600.times.; (E) 640.times..
[0032] FIG. 15. Graphics showing the nanobacterial growth in the
subculture of the demineralized, neutralized and sterile-filtered
20 different human kidney stones, and nanobacteria. * Indicates no
growth in the first 6 days.
[0033] FIG. 16: Trimethoprim effect on nanobacteria in 12 days,
expressed as minimum inhibitory concentration (MIC).
[0034] FIG. 17: Tetracycline effect on nanobacteria in 12 days,
expressed as minimum inhibitory concentration (MIC).
[0035] FIG. 18: Nitrofurantoin effect on nanobacteria in 12 days,
expressed as minimum inhibitory concentration (MIC).
[0036] FIG. 19: Doxycyline effect on nanobacteria in 12 days,
expressed as minimum inhibitory concentration (MIC).
[0037] FIG. 20: Gentamycin effect on nanobacteria in 12 days,
expressed as minimum inhibitory concentration (MIC).
[0038] FIG. 21: Neomycin effect on nanobacteria in 12 days,
expressed as minimum inhibitory concentration (MIC).
[0039] FIG. 22: Kanamycin effect on nanobacteria in 12 days,
expressed as minimum inhibitory concentration (MIC).
[0040] FIG. 23: Vancomycin effect on nanobacteria in 12 days,
expressed as minimum inhibitory concentration (MIC).
[0041] FIG. 24: Time course of antibiotic effects of 4 .mu.g/ml of
tetracycline, trimethoprim, trimethoprim+sulph, nitrofurantoinh,
doxycycline, and positive and negative controls.
[0042] FIG. 25: Effect of tetracyline on human nanobacteria.
Tetracycline is effective for human nanobacteria isolates similarly
compared to the bovine nanobacteria standard strain.
[0043] FIG. 26: Dose/response and time course for ampicillin.
[0044] FIG. 27: Dose/response and time course for citrate.
[0045] FIG. 28: Dose/response and time course for EDTA.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE
INVENTION
[0046] The present inventors have surprisingly discovered the first
mineral-coated organism where the mineral constitutes a part of the
cell wall essential for survival strategy of the organism. In
nanobacteria this mineral is carbonate apatite. As a result, the
present inventors have found that any therapeutic agent that is
targeted to the apatite may be useful in antinanobacterial
therapy.
[0047] More particularly, the present invention relates to methods
for disinfecting an article contaminated with nanobacteria. As used
in the context of the present invention, an article to be
disinfected is any article for which complete sterility is desired,
including medical devices, surgical tools, and medical and surgical
supplies including needles, syringes, tubing, and the like. Also
included within the scope of the present invention are solutions
for use in medical treatment, and for drug formulation, including
sterile water, saline, Ringer's, and other solutions. Any type of
mixture, e.g., solution or suspension, can be sterilized by the
practice including any formulations for medical treatment or
diagnosis, including, but not limited to, any pharmaceutical
intended for administration to a patient, including a human or
animal.
[0048] In a preferred embodiment, the present invention provides a
method for disinfecting an article contaminated with nanobacteria
comprising exposing the nanobacteria to a disinfectant solution. A
disinfectant mixture according to the present invention is
preferably a mixture of potassium persulfate and sulfaminoic acid
in water, preferably distilled water. The mixture can be a
solution, suspension, or the like. In a preferred embodiment, the
mixture will be a solution of from about 35% to about 70% potassium
persulfate and about 1% to about 15% sulfaminoic acid. In a
particularly preferred embodiment, the mixture will be about 50%
potassium persulfate and about 5% sulfaminoic acid. This mixture
may be used at full strength or may be diluted for use in
physiological conditions, preferably to a concentration of about
0.1% to about 10%, most preferably at a concentration of about
1%.
[0049] In an alternative embodiment, a disinfectant mixture
according to the present invention is a mixture of formaldhyde,
glyoxal, glyoxylic acid, and dimethylaurylbenzyl-ammonium chloride
in water, preferably distilled water. The mixture can be a
solution, suspension, or the like. In a preferred embodiment, the
mixture will be a solution of from about 1% to about 10%
formaldehyde, about 5 to about 10% glyoxal, about 0.1% to about 5%
glyoxylic acid, and about 3% to about 12%
dimethylaurylbenzyl-ammonium chloride. In a particularly preferred
embodiment, the mixture will be about 4.5% formaldehyde, about 6.8%
glyoxal, about 1.5% glyoxylic acid, and about 6%
dimethylaurylbenzyl-ammo- nium chloride. This mixture may be used
at full strength or may be diluted for use in physiological
conditions, preferably to a concentration of about 0.1% to about
10%, most preferably at a concentration of about 3%.
[0050] Alternatively, an article may be decontaminated by
demineralization of the nanobacteria followed by exposure to a
disinfectant chemical. Demineralization may be accomplished by
exposing the nanobacteria to low pH, preferably with a strong acid,
preferably hydrochloric acid. Alternatively, demineralization may
be accomplished by exposure to a calcium chelator. Suitable calcium
chelators for use in the present invention include
ethylenediaminetetraacetic acid (EDTA), citric acid, and citrate
compounds. In connection with demineralization, any of a broad
spectrum of disinfectant chemicals may suitably employed. A
particularly preferred disinfectant mixture to be used in
connection with demineralization comprise one or more of the
following:
1 Acceptable Preferred AGENT concentration concentration ethanol
>50% >70% glutaraldehyde >0.1% >2% formaldehyde >1%
>4% hypochlorite >0.1% >0.5% hydrogen peroxide >0.1%
>3% hydrochloric acid >0.1 M >1 M sodium hydroxide >0.1
M >1 M sodium dodecyl sulfate (SDS) >0.1% >1% Tween 80
>0.1% >1% Triton X-100 >0.1% >1% guanidium HCl >1 M
>3 M urea >1 M >3 M Virkon .RTM. >0.1% >1% Erifenol
.RTM. >0.1% >1.5% Klorilli .RTM. >0.1% >1% Buraton
.RTM. >0.1% >3%
[0051] Following demineralization, as either an alternative to use
of a disinfectant solution, or as an adjunct to the use of
disinfectant solution, the article may optionally be autoclaved,
preferably at a temperature of at least 121.degree. C., preferably
for at least 20 minutes.
[0052] Altematively, following demineralization, as either an
alternative to use of a disinfectant solution, or as an adjunct to
the use of disinfectant solution, the article may be exposed to
ultraviolet radiation, for example, by exposing the article to
ultraviolet light approximately equivalent to exposure to a UV-C
lamp of at least about 15 W at a distance of about 60 cm or less
for at least about 1 hour, preferably for at least about 3 hours,
still more preferably, at least overnight. Alternatively, the
article may be exposed to at least about three megarads of gamma
radiation.
[0053] Nanobacteria may also be eradicated from liquids by using
sbnication. Any conventional sonicator may be used; the sonication
times will vary with the power of the sonicator and the volume and
characteristics of the liquid to be disinfected. Adequate
sonication times may easily be determined by those of ordinary
skill in the art without the need for undue experimentation.
Typically, sonication of samples from 5-10 minutes will be
sufficient to eradicate nanobacteria from most solutions;
Sonication can be applied to any sample scale if the ultrasound
power and the sample container have suitable ratios to each other.
High-power ultrasound sources allow for continuous flow
applications as well. The method is applicable in the treatment of
any solution. It is gentle for proteins and other subcellular
components of the sample if excessive heating is prevented by
cooling the solution, either continuously (e.g., by placing the
container in which the solution to be disinfected is held into a
cold water bath), or by periodically interrupting the sonication
process to cool the solution (e.g., by placing the container in
which the solution to be disinfected is held into an ice bath).
[0054] In a particularly preferred embodiment of the present
invention, the nanobacteria are destroyed by drying the article to
be disinfected by heating for at least about one hour at a
temperature of at least about 100.degree. C. Alternatively,
following demineralization as discussed above, the article may be
heated for at least about 15 minutes, preferably at least about 30
minutes, to temperatures of at least about 60.degree. C.,
preferably at least about 100.degree. C.
[0055] The present invention further provides tissue culture media
formulated to be free of nanobacteria. Said nanobacteria-free
tissue culture media may be any standard tissue culture media
available to the skilled artisan, additionally comprising a
nanobacteria-antibiotic-effect- ive amount of one or more
antibiotics selected from the group consisting of .beta.-lactam
antibiotics, aminoglycoside antibiotics, tetracycline antibiotics,
and mixtures thereof. Suitable .beta.-lactam antibiotics for use in
the present invention include, but are not limited to, penicillin,
phenethicillin, ampicillin, azlocillin, bacmpicillin,
carbenicillin, cylclacillin, mezlocillin, piperacillin, epicillin,
hetacillin, cloxacillin, dicloxacillin, methicillin, nafcillin,
oxacillin, and salts thereof. Suitable aminoglycoside antibiotics
for use in the present invention include, but are not limited to,
streptomycin, kanamycin, gentamycin, amikacin, neomycin,
pardomycin, tobramycin, viomycin, and salts thereof. Suitable
tetracyclines include tetracycline, chlortetracycline,
demeclocycline, doxycycline, methacycline, oxytatracycline,
rolitetracycline, minocycline, sancycline, and pharmaceutically
acceptable salts thereof. Additionally, prior to use, a culture
medium according to the present invention is preferably sterilized
according to one of the methods set forth above. The ordinary
skilled artisan may select the method of sterilization most
suitable to the particular culture medium.
[0056] The present invention also provides a method for preventing
the development of calcifications in vivo, i.e., in a patient in
need of such treatment, comprising administering an antibiotic to
the patient in an amount effective to inhibit or prevent the growth
of nanobacteria. In the context of the present invention, in vivo
calcifications includes, but is not limited to, kidney stones,
atherosclerosis, acute periarthritis, dental pulp stones or
denticles, malacoplakia, Alzheimer's disease, autoimmune disease
including scleroderma, and metastatic and tumoral calcification
found in hemodialysis patients and calciphylaxis, malignant tumors
including ovarian serous tumor, papillary adenocarcinoma of the
endometrium, breast carcinoma, papillary carcinoma of the thyroid,
duodenal carcinoid tumor, and craniopharyngioma. In the context of
the present invention, a "patient" is any mammal, preferably a
human, suffering from tissue calcification, especially in
connection with one of the disorders listed above.
[0057] In a preferred embodiment, the present invention provides a
method for preventing the development of calcifications in vivo in
a patient in need of such treatment comprising administering an
antibiotic nanobacteria-antibiotic-effective amount of one or more
antibiotics selected from the group consisting of .beta.-lactam
antibiotics, aminoglycoside antibiotics, tetracyclines, and
pharmaceutically acceptable salts thereof, and mixtures thereof.
Suitable .beta.-lactam antibiotics for use in the present invention
include, but are not limited to, penicillin, phenethicillin,
ampicillin, aziocillin, bacmpicillin, carbenicillin, cylclacillin,
mezlocillin, piperacillin, epicillin, hetacillin, cloxacillin,
dicloxacillin, methicillin, nafcillin, oxacillin, and
pharmaceutically acceptable salts thereof. Suitable aminoglycoside
antibiotics for use in the present invention include, but are not
limited to, streptomycin, kanamycin, gentamycin, amikacin,
neomycin, pardomycin, tobramycin, viomycin, and pharmaceutically
acceptable salts thereof. Suitable tetracyclines include
tetracycline, chlortetracycline, demeclocycline, doxycycline,
methacycline, oxytatracycline, rolitetracycline, minocycline,
sancycline, and pharmaceutically acceptable salts thereof. In a
particularly preferred embodiment, antibiotics are coadministered
with citrate compounds.
[0058] In an alternative embodiment, Vitamin K, and/or its analogs,
may be employed in the methods of the present invention. Suitable
Vitamin K analogs for use in the present invention include, but are
not limited to, menadione, phytonadione (vitamin K.sub.1), and
pharmaceutically acceptable salts thereof. For use in the present
invention, Vitamin K is preferably employed in a concentration of
at least 1 .mu.g/ml.
[0059] In a further embodiment, p-amino salicylic acid, as well as
other salicylic acid derivatives may be used in the method of the
present invnetion. Particularly preferred is acetylsalicylic acid
(i.e., Aspirin).
[0060] In a further embodiment, bisphosphonates may be employed in
the methods of the present invention. As a family, bisphosphonates
are characterized pharmacologically by their ability to inhibit
bone resorption, whereas, pharmacokinetically, they are classified
by their similarity in absorption, distribution, and
elimination.
[0061] Although all bisphosphonates have similar physicochemical
properties, their antiresorbing activities differ. Activity is
dramatically increased when the amino group is contained in the
aliphatic carbon chain. For example, alendronate, an
aminobisphosphonate, is approximately 700-fold more potent than
etidronate, both in vitro and in vivo. In general, bisphosphonates
are poorly absorbed from the gastrointestinal tract as a result of
their poor lipophilicity. In vitro and in vivo studies have shown
that bisphosphonates are absorbed from the gastrointestinal tract
via paracellular transport. Systemically available bisphosphonates
disappear very rapidly from plasma, and are partly taken up by the
bone and partly excreted by the kidney. The relative contribution
of these two processes to overall plasma elimination differs among
bisphosphonates. To date, all bisphosphonates studied show no
evidence of metabolism. Renal excretion is the only route of
elimination. Studies with alendronate in rats indicate that the
drug is actively secreted by an uncharacterized renal transport
system, and not by the anionic or cationic renal transport
systems.
[0062] Bisphosphonates have a P-C-P bond instead of the P-O-P bond
of inorganic pyrophosphate that makes them resistant to enzymatic
degradation and gives them a high affinity for hydroxyapatite. They
are potent blockers of osteoclastic bone resorption and have been
successfully used to treat metabolic bone diseases that involve
increased bone resorption. It is possible to synthesize a variety
of bisphosphonates by substituting the hydrogen on the carbon atom.
Suitable bisphosphonates for use in the present invention include,
but are not limited to, alendronic acid, etidronic acid, clodronic
acid, oxidronic acid, and pharmaceutically acceptable salts
thereof. For use in the present invention, bisphosphonates are
administered preferably at a dose of approximately 5-20
mg/kg/day.
[0063] A still further part of this invention is a pharmaceutical
composition of matter suitable for prevention of calcifications in
vivo that comprises at least one or more of the compounds set forth
above, mixtures thereof, and/or pharmaceutical salts thereof, and a
pharmaceutically-acceptable carrier therefor. Such compositions are
prepared in accordance with accepted pharmaceutical procedures, for
example, as described in Remington's Pharmaceutical Sciences,
seventeenth edition, ed. Alfonso R. Gennaro, Mack Publishing
Company, Easton, Pa. (1985).
[0064] For therapeutic use in the method of the present invention,
an antibiotic, or its salt, can be conveniently administered in the
form of a pharmaceutical composition containing one or more
antibiotics, or salts thereof, and a pharmaceutically acceptable
carrier therefor. Suitable carriers are well known in the art and
vary with the desired form and mode of administration of the
pharmaceutical composition. For example, they may include diluents
or excipients such as fillers, binders, wetting agents,
disintegrators, surface-active agents, lubricants, and the like.
Typically, the carrier may be a solid, liquid, or vaporizable
carrier, or combinations thereof. Each carrier must be "acceptable"
in the sense of being compatible with the other ingredients in the
composition and not injurious to the patient. The carrier must be
biologically acceptable and inert, i.e., it must permit the
antibiotic compound(s) to inhibit the development of nanobacteria
and, particularly, the apatite crystals associated with
nanobacteria.
[0065] Antibiotic compounds for use in the method of the present
invention, or salts thereof, may be formulated together with the
carrier into any desired unit dosage form. Typical unit dosage
forms include tablets, pills, powders, solutions, suspensions,
emulsions, granules, capsules, and suppositories; tablets and
capsules are particularly preferred. Formulations include those
suitable for oral, rectal, nasal, topical (including buccal and
sublingual), vaginal and parenteral (including subcutaneous,
intramuscular, intravenous, intradermal, and transdermal)
administration, with formulations appropriate for oral
administration being preferred.
[0066] For example, to prepare formulations suitable for injection,
solutions and suspensions are sterilized and are preferably
isotonic to blood. In making injectable preparations, carriers
which are commonly used in this field can also be used, for
example, water, ethyl alcohol, propylene glycol, ethoxylated
isostearyl alcohol, polyoxylated isostearyl alcohol,
polyoxyethylene sorbitol, sorbitate esters, and the like. In these
instances, adequate amounts of isotonicity adjusters such as sodium
chloride, glucose or glycerin can be added to make the preparations
isotonic. The aqueous sterile injection solutions may further
contain anti-oxidants, buffers, bacteriostats, and like additions
acceptable for parenteral formulations.
[0067] The formulations may conveniently be presented in unit
dosage form and may be prepared by any method known in the art of
pharmacy. Such methods include the step of bringing into
association the active ingredient with the carrier which may
encompass one or more accessory ingredients. In general, the
formulations are prepared by uniformly and intimately bringing into
association the active ingredient with liquid carriers or finely
divided solid carriers or both, and then if necessary shaping the
product. Various unit dose and multidose containers, e.g., sealed
ampules and vials, may be used, as is well known in the art.
[0068] In addition to the ingredients particularly mentioned above,
the formulations of this invention may also include other agents
conventional in the art for this type of pharmaceutical
formulation.
[0069] A compound for use in the present invention may be present
in the composition in an broad proportion to the carrier. For
instance, the compound may be present in the amount of 0.01 to 99.9
wt %, and more preferably in about 0.1 to 99 wt %. Still more
preferably, the compound may be present in an amount of about 1 to
70 wt % of the composition.
[0070] The dosage of the antibiotics, pharmaceutically acceptable
salts thereof, or mixtures thereof, administered to a patient
according to the present invention will vary depending on several
factors, including, but not limited to, the age, weight, and
species of the patient, the general health of the patient, the
severity of the symptoms, whether the composition is being
administered alone or in combination with other therapeutic agents,
the incidence of side effects and the like.
[0071] In general, a dose suitable for application in-the method of
the present invention is about 0.001 to 100 mg/kg body weight/dose,
preferably about 0.01 to 60 mg/kg body weight/dose, and still more
preferably about 0.1 to 40 mg/kg body weight/dose per day. The
desired dose may be administered as 1 to 6 or more subdoses
administered at appropriate intervals throughout the day. The
antibiotic compounds may be administered repeatedly over a period
of months or years, or it may be slowly and constantly infused to
the patient. Higher and lower doses may also be administered.
[0072] The daily dose may be adjusted taking into account, for
example, the above-identified variety of parameters. Typically, the
present compositions may be administered in an amount of about
0.001 to 100 mg/kg body weight/day. However, other amounts may also
be administered. To achieve good plasma concentrations,
theantibiotics may be administered, for instance, by intravenous
injection of an approximate 0.1 to 1% solution of the antibiotics,
optionally in saline, or orally administered as a bolus.
[0073] The active ingredient may be administered for therapy by any
suitable routes, including topical, oral, rectal, nasal, vaginal
and parenteral (including intraperitoneal, subcutaneous,
intramuscular, intravenous, intradermal, and transdermal) routes.
It will be appreciated that the preferred route will vary with the
condition and age of the patient, the nature of the disorder and
the chosen active ingredient including other therapeutic agents.
Preferred is the oral route. Also preferred is the intravenous
route. However, other routes may also be utilized depending on the
conditions of the patient and how long-lasting the treatment
is.
[0074] While it is possible for the antibiotic(s) to be
administered alone, it is preferably present as a pharmaceutical
formulation. The formulations of the present invention comprise at
least one antibiotic, as defined above, together with one or more
acceptable carriers thereof and optionally other therapeutic
agents.
[0075] The above method may be practiced by administration of the
compounds by themselves or in a combination with other antibiotics,
including other therapeutic agents in a pharmaceutical composition.
Other therapeutic agents suitable for use herein are any compatible
drugs that are effective by the same or other mechanisms for the
intended purpose, or drugs that are complementary to those of the
antibiotics listed above. The compounds utilized in combination
therapy may be administered simultaneously, in either separate or
combined formulations, or at different times than the present
compounds, e.g., sequentially, such that a combined effect is
achieved. The amounts and regime of administration will be adjusted
by the practitioner, by preferably initially lowering their
standard doses and then titrating the results obtained. The
therapeutic method of the invention may be used in conjunction with
other therapies as determined by the practitioner.
[0076] Having now generally described this invention, the same will
be better understood by reference to certain specific examples,
which are included herein for purposes of illustration only and are
not intended to be limiting of the invention or any embodiment
thereof, unless so specified.
EXAMPLE 1
[0077] Mineralization by Nanobacteria
[0078] In this study, the present inventors provide evidence that
nanobacteria can act as crystallization centers (nidi) for the
formation of biogenic apatite structures. The mineralization
process was studied in vitro with one bovine isolate from
commercial fetal bovine serum and with a human isolate. These
findings are of concern in medicine because nanobacterial
bacteraemia occurs in humans, and nanobacterial nidi might initiate
pathological calcification.
[0079] Materials and Methods
[0080] Culture Methods for Nanobacteria.
[0081] Nanobacteria were cultured in DMEM (GIBCO) under mammalian
cell culture conditions (37.degree. C.; 5-10% CO.sub.2/90-95% air).
Serum was used at 10% final concentration as the supplement and
source of nanobacteria, which were fetal bovine serum (Sera Lab,
lot 901045), or human serum from a 29-years-old Finnish male. The
cultures were prepared using strict aseptic techniques in a cell
culture facility. Nanobacterial samples were filtered through 0.2
.mu.m filters before culturing. Subcultures were made using either
the same serum or y-irradiated fetal bovine serum (y-FBS) as a
culture supplement. Fetal bovine serum and nanobacteria were
y-irradiated, when indicated, at a minimum dose of 30 kGy given at
room temperature during about 16 hr by Kolmi-Set (llomantsi,
Finland).
[0082] Subculturing of nanobacteria in serum-free (SF) DMEM was
performed with monthly passages 1:11 for five years.
SF-nanobacteria attach firmly to the bottom of the culture vessel.
These cultures were passage or harvested with a rubber scraper.
Cultures were established on Loeffler medium supplemented with 10%
conditioned medium from nanobacterial culture, and DMEM replaced
water in the formula. The incubation period was 6 weeks under cell
culture conditions.
[0083] Only pure nanobacterial cultures were used. Positive
identification of nanobacteria involved typical growth rates and
optical properties, specific stainability with Hoechst 33258 and
with indirect immunofluorescence staining (IIFS), as described
below. Control experiments were performed to determine whether
spontaneous crystallization could occur in a culture medium. The
medium was incubated with or without y-FBS or y-irradiated
nanobacteria. Neither mineralization nor nanobacteria
multiplication was observed even during the 6-month follow-up.
[0084] Preparation and Infection of 3T6 Cells.
[0085] 3T6 cells (ATCC CCL 96) were cultured on coverslips.
SF-nanobacterial cultures were scraped and 100 .mu.l portions were
added to the cell cultures and incubated for 24 hr in the
incubator. Only DMEM was added to the control experiments.
Transmission electron microscopy (TEM), IIFS, and DNA and von Kossa
staining were used for the observation of the cell-SF nanobacteria
interaction.
[0086] Kidney Stones.
[0087] Thirty randomly collected kidney stones (K-SKS, Stone
Analysis Central Laboratory, Finland) were demineralized in 1N HCl
and then neutralized, centrifuged at 14,000.times.g for 15 min, and
the pellets were used for IIFS and TEM. Part of the pellets were
suspended in DMEM, sterile-filtered and cultured in DMEM
supplemented with y-FBS under nanobacterial culture conditions.
[0088] Staining Methods.
[0089] DNA staining with Hoechst 33258 fluorochrome was carried out
as described in Hoechst Stain Kit, Flow Laboratories, except, where
indicated, increasing the stain concentration from 0.5 .mu.g/ml to
5 .mu.g/ml. IgG1 class anti-nanobacterial monoclonal antibodies
(mAb), Nb 8/0 and Nb 5/2, were used in IIFS. The epitope of the
latter mAb was inactivated by incubating it in sodium borohydrate
(3.times.1 min; 0.5 mg/ml in PBS), when indicated, to test
specificity of the binding. The samples were viewed under a Nikon
Microphot-FXA microscope with fluorescence and differential
interference contrast (DIC) optics. Specific calcification
detection was performed with von Kossa staining. 3T6 cells exposed
to SF-nanobacteria for 48 hr were used as samples.
[0090] Electron Microscopy and Energy Dispersive X-ray
Microanalysis.
[0091] For negative staining, nanobacteria were isolated by
centrifugation at 40000 g for 1 hr directly from fetal bovine serum
diluted 1:5 in PBS. A carbon-coated 400 mesh copper grid was placed
on a drop of the suspension of nanobacteria in PBS for 1 min,
washed with water, and stained on a drop of 1% phosphotungstic acid
for 90 sec. Scanning electron microscopy (SEM) and TEM were
performed. The topographic features of the nanobacteria were
investigated with a SEM equipped with energy-dispersive X-ray
microanalysis (EDX). Hydroxyapatite (Sigma, No-H-0252, St. Louis,
Mo.) was used as a reference.
[0092] Fourier Transform IR Spectroscopy (FTIR), Chemical Analysis,
and Enzyme Assays.
[0093] Hydroxyl and carbonate groups in the apatite minerals were
detected using FTIR by K-SKS, Stone Analysis Central Laboratory,
Finland, following standard methods. Chemical analysis of
nanobacteria was carried out by analyzing urease enzyme activity
and alkaline phosphatase(AP) with p-nitrophenylphosphate as
substrate at pH 9.5.
[0094] Results
[0095] Culture Properties, Morphology, and Apatite Formation by
Nanobacteria in Serum-containing Media.
[0096] Light microscopy with DIC revealed barely detectable
nanobacteria near the bottom of the culture vessel after about a
one week culture period. In two weeks, nanobacteria appeared as
groups easily visible in microscopy. After one month, many were in
clumps and started to attach to the bottom of the culture vessel,
and by the end of two months, most were in a white-colored biofilm
visible to the naked eye. The criteria for pure nanobacterial
culture were refractile aggregates of typical coccoid-shaped
particles (FIG. 1A), showing DNA stainability (FIG. 1B) only with
the modified method, a negative culture result on sheep-blood agar
and IIFS positivity with anti-nanobacteria mAbs.
[0097] Negative-staining of nanobacteria in uncultured fetal bovine
serum revealed 0.2-0.3 .mu.m coccoid particles (FIG. 1C). After a
one-month culture period, SEM revealed similar coccoid shape with a
diameter of 0.2 to 0.5 .mu.m (FIG. 1D). Their rough surfaces
resembled those seen in TEM (FIG. 1E-G). During longer culture
periods, they were mostly attached to the culture vessel and
finally were in a mineral layer (FIGS. 1F and G). Chemical analysis
using EDX gave similar Ca and P peaks as detected for
hydroxyapatite (FIGS. 1H and I). Cultures of the human isolate gave
identical results (not shown). Chemical analysis of nanobacteria
harvested after a 3-month culture period revealed a high content of
inorganic material. The pellet dry weight varied from 23% to 39%
and consisted of: N (1-1.3%); P (12.3-14.6%); Ca (23.4-23.5%); Mg
(1.4-1.9%); K (0.1%); and Na (1.2-1.4%). FTIR revealed that
carbonate apatite was present in samples from all culture ages
between 7-180 days in both human and bovine nanobacteria. Control
hydroxyapatite was correctly identified in the test. The analytical
methods do not exclude the possible presence of minor quantities of
other mineral phases. To exlude that possibility, crystallographic
analysis are needed. Nanobacteria did not produce urease or AP
activity, and their culture medium remained at pH 7.4.
[0098] Apatite Formation by Nanobacteria in Loeffler Medium.
[0099] Macroscopic nanobacterial colonies on modified Loeffler
medium (FIGS. 2A and B) were stony, greyish-brown, passagable and
penetrated the medium layer and attached to the bottom of the
culture vessel after 6 weeks of culture. IIFS with
anti-nanobacteria mAbs (data not shown), and TEM revealed
nanobacteria coated in needle-like apatite crystals (FIG. 2C),
similar to the hydroxyapatite crystals (FIG. 2D).
[0100] Apatite Formation by Nanobacteria in SF-medium.
[0101] When washed nanobacterial pellets or SF-nanobacteria were
subcultured in SF-DMEM, bottom-attached coccoid organisms were
observed within one day. Differential interference contrast
microscopy revealed a several-micrometer-thick mineral layer around
each nanobacteria reaching a yeast-size within one week (FIG. 3A).
Their morphology differed extensively from the coccoid
nanobacteria, but similar DNA stainability was observed (FIG. 3B).
They produced biomass at about half the rate observed in serum
containing cultures. The. metabolic incorporation of
[.sup.35S]methionine and [5-.sup.3H]uridine is proof that they were
replicating. Differential interference contrast microscopy revealed
nanobacterial multiplication inside the mineral formations (FIG.
3C). These apatite shelters, were shown in SEM to have a hollow
interiors, were apparently the dwelling-place of the organisms
(FIGS. 3E and F). The size of the cavity is probably dependent on
the number of nanobacteria it contains (FIG. 3F ). Apparently, the
openings of the cavities were facing the bottom of the culture
vessel before scraping. Thus, the apatite shelters provided
complete protection for the organisms. The cultures could be
passage monthly for over 5 years and always followed a similar
growth pattern. After addition of y-FBS, these nanobacterial
formations returned to the forms found in serum cultures (see FIG.
3D). That the shelters were apatite in nature was proven by EDX.
FTIR determined that it was carbonate apatite. The human isolate
produced similar formations.
[0102] Intra- and Extracellular Calcification in Fibroblast
Cultures.
[0103] 3T6 cells infected for 48 hr with SF-nanobacteria showed
altered cell morphology, e.g., large vacuolization with
internalized SF-nanobacteria (FIG. 3G). Control cells were negative
(not shown). Standard DNA staining of the nanobacteria-infected
cells revealed no ordinary contamination (FIG. 3H). TEM
occasionally revealed SF-nanobacteria adhering to the cell surface,
but mostly they were in various compartments within the cells (FIG.
3I-L), including nucleus (not shown). von Kossa staining revealed
intra- and extracellular calcification in these cells (FIGS. 4D and
E): Heavily infected cells showed nuclear abnormalities, e.g.,
macronucleus, as shown in FIG. 4E, and abnormal nuclear shape
(FIGS. 3G, H, K and L). Control cells were von Kossa negative and
did not have nuclear abnormalities (FIG. 4F).
[0104] Detection of Nanobacterial Antigens in Kidney Stones.
[0105] The present inventors supervised a survey on 30 human kidney
stones in an attempt to detect the presence of nanobacteria.
Nanobacteria-specific mAb revealed positive, nanobacteria-sized
cocci at various concentrations in all 30 demineralized stones
using IIFS. An image of a relevant sample is seen in FIG. 4C. The
results were repeated with another nanobacteria-specific mAb, Nb
5/2, that detects a carbohydrate epitope, and antibody binding
could be abolished with sodium borohydrate treatment, which
destroys carbohydrate antigens. Specificity was further proven with
negative staining results with 4 different mAbs (IgG1 class)
detecting nonrelevant antigens (data not shown). Bacteria of
similar size and morphology (FIG. 4B) as nanobacteria (FIG. 4A)
were found with TEM in strongly positive stones (FIGS. 4B and C).
In nanobacterial culture conditions, sterile-filtered extracts of
all the stones revealed microorganisms having the growth rate,
morphology, mineralization, and staining properties of
nanobacteria.
[0106] Discussion
[0107] the present inventors have found nanobacterial culture
systems that allow for reproducible production of apatite
calcification in vitro. Depending on culture conditions, tiny
nanocolloid-sized particles covered with apatite, or biofilm, sand,
stones and tumor-like growths of apatite could be produced (Table
1).
2TABLE 1 Culturability of nanobacteria and apatite formation
Culture condition Replication Size Apaptite and its form Serum + S
+, nanocolloid 10-50% serum in DMEM + S ++, nanocolloid DMEM + L
+++, sand 50% DMEM - 50% urine + L +++, sand Urine +/- n.e. n.e.
Modified Loeffler medium + L +++, tumor-like S, small size (200-400
nm); L, large size (1 .mu.m to 1 mm, including the mineral); n.e.,
not evaluated because of crystal formation. Pluses in the last
column refer to amount.
[0108] The principal precondition for mineralization was low levels
of intact serum in the culture medium. Serum contains powerful
proteinaceous inhibitors of apatite crystal formation, osteopontin,
osteocalcin and fetuin, which may account for the observed
inhibition and even dissolution of the formed minerals after
replenishment of fetal bovine serum. In cases of nanobacterial
cultures in serum containing medium, the inhibitors permitted only
marginal mineralization. Mineralization increased in parallel with
the dilution of the serum in cell culture medium. Finally, in
SF-medium, apatite formation was extensive and rapid. Although
modified Loeffler medium contains 75% serum, the serum proteins
were denatured during the sterilization steps. Thus, apatite
formation was not inhibited resulting in solid apatite colonies
about 1-5 mm in diameter in 6 weeks. Living nanobacteria are needed
to produce apatite in the nanobacterial model. y-lrradiated
nanobacteria did not multiply and, although they could gather
apatite on them, no sizable calcification was produced even after
6-month long incubations.
[0109] Chemical analysis revealed that the overall composition of
biofilm and solid mineral formation was similar to that of bone,
except carbonate apatite was formed, as in most extraskeletal
tissue calcification and stones whereas in bone, hydroxyapatite is
the prevalent form. In the nanobacterial model, apatite was formed
at [Ca] 1.8 mM and [P.sub.i] 0.9 mM or less, without replenishment
of the medium.
[0110] Nanobacteria were found in all 30 human kidney stones that
thes inventors have screened. Previously, only struvite stones
(4-15% of all kidney stones) composed of magnesium ammonium
phosphate and small amounts of apatite have been regarded as
deriving from bacteria. They are formed in vitro and probably in
vivo by Proteus, Staphylococci and E. coli that produce urease,
elevating the local pH to more lithogenic levels. Alkaline
phosphatase may augment the lithogenicity. Nanobacteria do not
produce urease or AP, but nucleate carbonate apatite directly on
their surfaces at pH 7.4 suggesting the presence of nucleating
molecules. Since nanobacteria are culturable under physiological
conditions in media similar in composition to glomerular filtrate,
nanobacteria offer a unique model for kidney stone formation.
EXAMPLE 2
[0111] Eradication of Nanobacteria
[0112] The selection of an appropriate test for nanobacterial
disinfection is not straightforward, and accurate comparisons of
the results obtained from different tests are problematic, due to
the number of factors affecting disinfection. These factors include
duration of exposure, presence of organic load, type, age,
concentration and diluent of the disinfectant, and number, age,
growth form of the microorganisms present, and the temperature.
Currently there are several types of disinfection tests, but these
are mainly suitable for rapidly growing bacteria. The disinfection
tests of slowly growing Mycobacteria, some of which are extremely
resistant, have long suffered from lack of appropriate, reliable
standardization. Typically, centrifugation or very high dilution
have been used to eliminate the effect of residual concentrations
of disinfectants. Subsequently, plating on agar medium for colony
count is done for evaluating the reduction in viability. For
nanobacteria such assays are not suitable. Recovery of nanobacteria
by centrifugation generally results in unpredictable losses. Due to
their slow growth rate, high dilutions result in very long
incubation times, and extremely poor culturability on solid media
makes the evaluation of a nanobacteria count impossible.
[0113] Mineralization is the most characteristic property of
nanobacteria, and possibly the main mechanism of pathology caused
by the organism. The mineral formed under standard culture
conditions is hydroxyl or carbonate apatite as revealed by several
methods, including energy dispersive X-ray microanalysis and
Fourier transform IR spectroscopy. One of the primary functions of
the mineral may be protection against harsh environmental
conditions. The apatite can prevent the penetration of harmful
compounds to the interior of the organism. Depending on the culture
time and culture conditions, various degrees of mineralization has
been observed. Mineralization by nanobacteria cultured without
serum (SF-nanobacteria) is much more extensive than that observed
in nanobacteria cultured with serum containing medium. The doubling
time of serum nanobacteria and SF-nanobacteria, are about three
days and six days respectively, measured by amino acid
incorporation.
[0114] Disinfecting chemicals at concentrations generally used have
now been tested against cultured nanobacteria. The chemicals
selected represent a wide variety of mechanisms which are known to
affect biological systems. Survival of nanobacteria at high
temperature, in drying and under UV-C irradiation was also tested.
There are several mechanisms for antibiotic resistance in bacteria
which have not been discussed here. The present inventors evaluated
the effect of four antibiotics against nanobacteria. The
antibiotics are those commonly used in cell culture.
[0115] Experimental Design
[0116] Nanobacteria Culture in Serum Containing Medium
[0117] Nanobacteria were cultured with 10% fetal bovine serum in
DMEM medium (serum nanobacteria) for one month at 37.degree. C. in
an atmosphere of 5% CO.sub.2-95% air. The cultures were harvested
by centrifugation. For the autoclaving, UV, microwave, heating and
drying treatments, the harvested nanobacteria were suspended in
phosphate buffered saline (pH 7.4; PBS). After treatments,
subculturing of the nanobacteria was made in 10% gamma irradiated
fetal bovine serum in DMEM medium. The growth of serum nanobacteria
was followed by light microscopy and absorbance measurement with a
spectrophotometer at 650 nm.
[0118] Nanobacteria Culture without Serum
[0119] SF-nanobacteria were cultured in DMEM medium for one week at
37.degree. C. in an atmosphere of 5% CO.sub.2-95% air, and all the
cultures firmly adhered to the culture vessel. The cultures were
exposed to the disinfectants after removal of the culture medium.
For the autoclaving, UV, microwave, and drying treatments, the
medium was removed and an equal amount of PBS used instead. For the
heat treatments, the SF-nanobacteria were harvested by scraping the
culture vessel followed by centrifugation of the medium. The
obtained pellet was suspended in PBS and used in the test. After
treatments the SF-nanobacteria were subcultured in DMEM medium and
the growth followed by light microscopy to see the adherence and
typical mineralization.
[0120] Chemical Disinfection for Sf-nanobacteria
[0121] The concentrations of the chemicals used were those commonly
used for disinfection or as instructed by the manufacturer. The
chemicals included 70% ethanol, 2% glutaraldehyde, 4% formaldehyde,
0.5% hypochlorite, 3% hydrogen peroxide, 1M hydrochloric acid
(HCl), 1M sodium hydroxide (NaOH), 1% sodium dodecyl sulfate (SDS),
1% Tween 80, 1% Triton X-100, 3M guanidium-hydrochloride, 3M urea,
1% Virkon.RTM. (Antec International Ltd., Suffolk, England; 100%
product contains 50% potassium persulfate, 5% sulfaminoic acid),
1.5% Erifenol.RTM. (Orion OY, Finland; 100% product contains <5%
NaOH, <5% o-benzyl-p-chlorophenol, 5-15% p-chloro-m-cresol), 1%
Klorilli.RTM. (Orion OY, Finland; 100% product contains sodium
metasilicate, sodium N-chloro-p-toluenesulfonamide-3-hydr- ate and
20,000 ppm active chlorine), and 3% Buraton.RTM. (Schulke &
Mayr, Germany; 100% product contains 4.5% formaldehyde, 6.8%
glyoxal, 1.5% glyoxylic acid, 6% dimethylaurylbenzyl-ammonium
chloride). The dilutions to be used were freshly prepared on the
day of exposure in sterile distilled water. As a positive control,
only diluent was used. Negative control contained only culture
medium.
[0122] The SF-nanobacteria were exposed to the chemicals for 10 and
30 minutes at room temperature after removal of the culture medium.
After exposure, the disinfectant solution was removed and fresh
medium added (with a neutralization step in the case of HCl and
NaOH). If any significant deattachment occurred, nanobacteria were
recovered by centrifugation, and subcultured. The exposed
serum-free cultures were passage 1:10 after 48 hours and the growth
was followed by light microscopy for three weeks.
[0123] Autoclaving, UV, and Drying Treatments
[0124] Serum and SF-nanobacteria were autoclaved in a small volume
of phosphate buffered saline (PBS), pH 7.4 at 121.degree. C. for 20
minutes. UV treatment was given to both nanobacteria in PBS in a
laminar hood under Philips 15 W UV-C lamp for periods of 1 and 3
hours and overnightly in petri dishes with the lids removed. The
distance of the cultures from the lamp was about 60 cm. Drying
treatments were carried out by drying nanobacteria overnightly at
room temperature or by heating for one hour at 100.degree. C.
SF-nanobacteria was dried only overnightly at room temperature.
Microwave treatment was given by bringing the samples ten times to
boiling point (100.degree. C.)in a 1400 W microwave oven.
[0125] Heating of Nanobacteria
[0126] Heat effect on survival of the nanobacteria was determined
by exposing nanobacteria as pellets in PBS for 15 and 30 minutes,
with temperatures varying between 60.degree. C. and 100.degree. C.
Exposed SF-nanobacteria were cultured in DMEM medium and the growth
followed by microscopy as above. The growth of serum nanobacteria
cultures was followed by light microscopy and absorbance
measurement with a spectrophotometer at 650 nm.
[0127] Antibiotic Sensitivity Tests
[0128] Antibiotic sensitivity of serum nanobacteria was tested with
a mixture of penicillin (.beta.-lactam) and streptomycin
(aminoglycoside) (PS) at 1.times. and 10.times. concentration (100
IU penicillin, 100 .mu.g/ml streptomycin=1.times.), kanamycin
(aminoglycoside) at 1.times. and 10.times. concentration (100
.mu.g/ml=1.times.) and gentamycin (aminoglycoside) at 1.times.
concentration (100 .mu.g/ml). The 1.times. concentrations are those
recommended for cell culture. After 10 days culture in 10% serum
containing DMEM with the antibiotic, growth was compared to that of
nanobacteria cultures without antibiotics present.
[0129] Results
[0130] Chemical Disinfection
[0131] SF-nanobacteria showed a wide resistance to the
disinfectants used. Only Virkon was effective in killing
SF-nanobacteria after thirty minutes. Hydrochloric acid treatment
dissolved the apatite layer of nanobacteria, but remineralization
was observed after addition of culture medium. The
guanidium-hydrochloride and Buraton treatments. resulted in the
deattachment of the SF-nanobacteria, but the disinfection efficacy
of Buraton was slightly less than that of guanidium-hydrochloride.
Results of the chemical treatments are presented in Table 2.
Survival was determined after subculture by comparison to the
treatment with only diluent.
[0132] Autoclaving, UV, and Drying Treatments
[0133] Drying at a temperature of 100.degree. C. killed serum
nanobacteria, but drying at room temperature did not. Autoclaving
was not detrimental to the SF-nanobacteria, but a marked reduction
in the survival of serum nanobacteria was observed. SF-nanobacteria
tolerated UV light with no
3TABLE 2 Resistance of SF-nanobacteria to chemical disinfectants.
Exposure time Chemical 10 min 30 min 70% ethanol +++ +++ 2%
glutaraldehyde +++ +++ 4% formaldehyde +++ +++ 0.5% hypochlorite
+++ +++ 3% H.sub.2O.sub.2 +++ +++ 1M HCl n.d. ++* 1M NaOH +++ +++
1% SDS +++ +++ 1% Tween 80 +++ +++ 1% Triton X-100 +++ +++ 3M
Guanidium HCl n.d. 0 3M Urea +++ +++ 1% Virkon .RTM. n.d. -* 1.5%
Erifenol .RTM. +++ +++ 1% Klorilli .RTM. +++ +++ 3% Buraton .RTM.
n.d. ++* +++: No effect; ++: Reduced survival; +: Markedly reduced
survival; -: No survival; *= partial or total detachment on
exposure; n.d. = not determined
[0134] effect on growth, but serum nanobacteria was significantly
inactivated. Nanobacteria samples dried during the overnight UV
treatment, and thus there became an additional stress for the
organisms. Drying obviously had little or no effect to the result,
since the survival of nanobacteria with all the UV treatments was
similar. Because of lack of an UV radiometer, no UV dosage could be
calculated, and more accurate tests with nanobacteria in culture
medium should be conducted. Microwave treatment was more like a
heat shock treatment than a sterilization step, short boilings
being completely ineffective. Results of the follow-up of the
nanobacteria survival after autoclaving, UV, microwave and drying
treatments are presented in Table 3. SF-nanobacteria was much more
resistant than nanobacteria cultured with serum. SF-nanobacteria
survived all test conditions without a marked reduction in
viability. Serum nanobacteria were killed by drying for one hour at
100.degree. C., and survival was markedly reduced in all other test
conditions.
4TABLE 3 Survival of nanobacteria after physical exposure. Survival
of Survival of serum serum-free Treatment nanobacteria nanobacteria
Autoclave + +++ UV irradiation (1 h) + +++ UV irradiation (3 h) +
+++ UV irradiation (overnight) + +++ Microwaves +++ +++ Drying (RT)
+ +++ Drying (100.degree. C.) - n.d. +++: No effect; ++: Reduced
survival; +: Markedly reduced survival; -: No survival; * = partial
or total detachment on exposure; n.d. = not determined
[0135] Heat Resistance of Nanobacteria
[0136] Nanobacteria were very heat resistant. Fifteen minutes
boiling was not enough for killing serum nanobacteria, but thirty
minutes inactivated them. Growth curves of serum nanobacteria after
heat treatment are presented in FIG. 5. Importantly, the growth of
serum nanobacteria was very similar, with no observed lag period,
even after the fifteen minute boiling. Microscopical observations
of the SF-nanobacteria cultures after heat treatment revealed that
they had survived all the tested conditions including boiling at
100.degree. C. for 30 minutes. Initially, reduction in the amount
of viable SF-nanobacteria was observed with the higher
temperatures, but after two weeks there was no difference in the
test culture results as compared to the non-heated control.
[0137] Antibiotic Resistance of Nanobacteria
[0138] High resistance to the tested antibiotics was observed. Ten
times higher concentrations than normally used in cell culture were
needed to prevent the growth of nanobacteria. FIG. 6 shows the
effect of antibiotics on growth of nanobacteria cultured with serum
containing medium. Interestingly, at concentrations of antibiotics
with no effect on growth, there was a profound effect in the
morphology of nanobacteria as seen in SEM (FIGS. 7A and 7B). This
suggests that nanobacteria have adaptive ways for protecting
themselves for detrimental attacks, e.g., by secreting slimy
layers.
[0139] Conclusions
[0140] Nanobacteria can tolerate harsh conditions extremely well.
SF-nanobacteria were much more resistant than the nanobacteria
cultured in serum containing medium. Extremes in pH, oxidizing
agents, free chlorine, and chemicals affecting the proteins as well
as irradiation, heat and drying have very little effect on
SF-nanobacteria. This indicates that the mineral layer offers extra
protection to the organism. Exceptional survival of nanobacteria
has also been observed in association with human kidney stones.
Viable nanobacteria were recovered from almost all kidney stones by
demineralizing the stones with hydrochloric acid (see below).
[0141] An effective way to eradicate nanobacteria with disinfecting
chemicals, should include a demineralization step. Apatite can be
dissolved at low pH or by means of calcium chelators such as
ethylenediaminetetraacetic acid (EDTA). A second step should be
then included to kill the organism by another mechanism. Virkon,
composed of peroxygen compounds, surfactant, organic acids and an
inorganic buffer system, proved to be effective against
nanobacteria most likely because of the acidity (1% solution in
water has pH 2.6) combined with other disinfection mechanisms.
[0142] Doses of three megarads gamma irradiation are needed to
ensure destruction of nanobacteria. Gamma irradiation is probably
the best and most reliable method for killing nanobacteria. Drying
at elevated temperatures or boiling for extended periods, can also
be used in eradicating nanobacteria. Boiling for 30 minutes is
effective against almost all living organisms, except some
endospores, especially the spores of Bacillus stearothermophilus
and hyperthermophilic archae having 90.degree. C. or more as
optimum temperature for growth. This treatment is also not enough
to kill SF-nanobacteria. Importantly, normal autoclaving procedure
(121.degree. C. for 20 min) was also insufficient to eradicate
nanobacteria.
[0143] Resistance of nanobacteria to the tested antibiotics was
very high. Cell culture antibiotics used in this study are
effective only in very high concentrations. A possible resistance
mechanism is the production of a protective slime as revealed by
SEM. Modificating the cell wall is a common strategy for many
bacteria to acquire resistance to antibiotics. When a nanobacterium
faces unfavorable conditions it starts to secrete polymers and form
mineral upon them. The tested antibiotics were mainly
aminoglycosides.
[0144] Observed resistance of serum nanobacteria shows that it is
at least as resistant as Mycobacteria and Bacillus subtilis spores,
which are the model organisms for disinfection resistance. The
resistance of SF-nanobacteria is clearly superior to these.
[0145] The apatite mineral around the organism serves as a primary
defense shield against various chemicals and irradiation. The
survival of nanobacteria is clearly not only due to the mineral,
because treatment with 1M hydrochloric acid could not kill
nanobacteria, and remineralization could be observed later in the
culture. A double defense with the apatite layer and impermeable
membrane combined with a very slow metabolism is a likely
explanation for the observed resistance of nanobacteria. The
increased resistance of SF-nanobacteria is probably due to the
extensive mineralization, slower metabolism and adherence to
surfaces. Nanobacterial resistance mechanisms appear to be
multiplicative: thus, nanobacteria having an apatite coat,
impermeable cell wall, slow metabolism and possibly other still
unknown mechanisms, becomes extremely resistant to most
disinfecting methods.
EXAMPLE 3
[0146] Dental Pulp Stones Made by Nanobacteria
[0147] The purpose of the experiments conducted in this example was
to investigate if nanobacteria participate in the dental pulp stone
formation. The design of the study was to culture nanobacteria on a
healthy tooth, without dental pulp stone, and compare the results
with those obtained from a tooth having dental pulp stone. Mineral
formations were observed under SEM. Additionally, an
epidemiological screening was carried out on the possible
correlation between dental pulp stone and kidney stone disease, and
other bodily calcifications in 18 patients using a
questionnaire.
[0148] Correlation between Dental Pulp Stones and Other Stone
Formation in the Body.
[0149] 18 patients were randomly selected from a private dental
practice in Turkey based upon their periodontal problems caused by
severe pulp stone formation. Collected pulp stones were stored in
PBS containing 0.05% NaN.sub.3 at +4.degree. C. The samples were
demineralized in 1N HCl for 10 min at room temperature, neutralized
with NaOH and potassium phosphate buffer, and immunostained by
using anti-nanobacteria monoclonal antibodies. Treatment of the
samples with 1N HCl did not effect the epitopes recognized by the
monoclonal antibodies used in these experiments. Immunostaining
revealed positive, small cocci at various concentrations in all
samples. Specificity of the staining was further proven with
negative staining results with three different monoclonal
antibodies detecting nonrelevant antigens.
[0150] The results obtained from the patient questionnaire showed a
high incidence of kidney stones and gallstones in both patients and
their parents (Table 4).
5TABLE 4 The presence of calcification and stone formation in the
patients with dental pulp stones, and in their parents. Patients (9
M + 9 F) Mothers Fathers Kidney stones 5/18 (28%) 3/18 (17%) 6/18
(33%) Urinary sand 6/18 (33%) 1/18 (6%) 0/18 (0%) Gallstones 2/18
(11%) 7/18 (39%) 3/18 (17%) Tissue calcifications 1/18 (6%) 5/18
(28%) 1/18 (6%)
[0151] There is an increase in calculus formation on teeth among
the laboratory animals whenever common drinking water was given,
which suggests that flora is transferred from one animal to an
other. In addition, erythromycin strongly inhibits calculus
formation, whereas chloramphenicol, and penicillin do not. This
suggests that the organisms involved in calculus accumulation may
be very specific. These findings provide a possible explanation to
the results shown in Table 4, indicating high incidence for stone
formation and calcification in the family members.
[0152] Nanobacteria Cause Dental Calculi Formation in Vitro
[0153] In SEM observations of a tooth with dental pulp stones (FIG.
8A), at high magnification, mineralized fibers, and numerous small
globular bodies near them were observed (FIG. 8B). There were no
calcospherules observed in the control tooth (FIGS. 8C and D).
[0154] When the inventors exposed a healthy tooth to
SF-nanobacteria culture for one month, SEM revealed voluminous
mineral formation, resembling dental pulp stones, on the surface of
the tooth (FIGS. 9A, B, D, and E).
[0155] The cavity-like structure indicated by the large arrows in
FIG. 9 is a very typical structure for SF-nanobacteria (see above).
It is suggested that different structural features correspond to
various stages of mineralization of the pulp stones.
[0156] There are many ideas about the reason for dental calculi
formation, e.g., diet, and age. Animal experiments have proven that
addition of Ca and P to the diet increases the rate of dental stone
formation. The present inventors have shown that Ca is very
necessary element for production of apatite by nanobacteria.
Addition of a sterilized dolomite piece to SF-nanobacteria culture
increased their multiplication rate. SEM revealed adhered,
multiplying SF-nanobacteria on the dolomite surface (FIG. 10).
[0157] Chemical Composition of Dental Pulp Stones
[0158] The features of crystal components of human dental calculi
have been attributed to Ca/P molar ratio: i) calcified forms of
microorganisms including cocci and rods with a Ca/P ratio close to
1.7, carbonated hydroxyapatite; ii) calcophoritic calcifications
and dense calcifications with a Ca/P ratio close to 1.7, carbonated
hydroxyapatite; iii) aggregated plates or clusters of platelets and
fan-like aggregations with a Ca/P ratio close to 1.33, octacalcium
phosphate; iv) cuboidal forms of varying sizes with a Ca/P ratio
close to 1.4, whitlockite. The concentration of some other elements
in dental pulp stones is much lower than Ca and P (0.88% F; 0.75%
Na; 0.51% Mg). The other analyzed constituents (K, Cl, Mn, Zn, Fe)
are present at trace concentrations.
[0159] In accordance with the aim of this study, to clarify the
relationship between morphology, chemical composition of material
in dental calculi, and nanobacteria, the EDX results were matched
as seen in FIG. 11. Previously, the inventors identified with EDX
and chemical analysis that all growth phases of nanobacteria
produce biogenic apatite on their cell envelope. Fourier transform
IR spectroscopy revealed the mineral as carbonate apatite.
[0160] Conclusion
[0161] These data indicates that dental pulp stones are associated
with apatite forming nanobacteria.
EXAMPLE 4
[0162] Stone Formation and Calcification by Nanobacteria in Human
Body
[0163] In these experiments, the inventors provide further evidence
that nanobacteria can act as crystallization centers (nidi) for the
formation of biogenic apatite structures in the mammalian body, and
in environmental sources.
[0164] Calcification Caused by Nanobacteria in a Cell Culture
Model
[0165] Nanobacteria are cytotoxic in vitro and in vivo. 3T6
fibroblastoid cells infected for 48 hours with nanobacteria
(cultured in serum free condition, SF-nanobacteria), showed altered
cell morphology due to internalized SF-nanobacteria (FIGS. 12A and
B). von Kossa staining revealed intra- and extracellular
calcification in the infected cells (FIG. 12C). Heavily infected
cells showed nuclear abnormalities, e.g., macronucleus. There was
no calcification and nuclear abnormalities in the control cells
stained with the von Kossa method (FIG. 12D).
[0166] Nanobactena and Kidney Stones
[0167] Urinary tract stone crystalline components are of five
types: calcium oxalate, calcium phosphate, bacterial-related,
purines or cystine. The majority of urinary stones are admixtures
of two or more components, with the primary admixture being calcium
oxalate with apatite. The viability and location of bacteria within
infection stones (struvite [MgNH.sub.4PO.6H20] and/or carbonate
apatite [Ca.sub.10(PO.sub.4).sub.6CO.sub.3] stones) have been
investigated. It was found that large numbers of bacterial
impressions and bodies were existing in the interstices surrounded
by crystals of apatite and struvite from the nuclei to the
peripheral layers. The presence of bacterial colonies even in the
nuclear portion of the stones suggests that bacteria participate in
the initial stone formation, as well as in growth of infection
stones. In the invetors work, bacteria of similar size and
morphology (FIGS. 13A and C) as nanobacteria (FIGS. 13B and D) were
found with TEM in human kidney stones.
[0168] The present inventors screened 60 human kidney stones for
nanobacteria using immunofluorescence staining and culture methods.
Nanobacteria show a thick apatite envelope layer on their surface
in TEM (FIG. 14A). Demineralization under harsh conditions (e.g.,
incubation with 1N HCl) did not affect their epitopes recognized by
the monoclonal antibodies used in these experiments.
Nanobacteria-specific monoclonal antibodies revealed positive,
small cocci at various concentrations in all demineralized stone
samples (FIG. 14C-E) and nanobacteria (FIG. 14B). Different
distribution patterns of nanobacteria were observed in the stones,
e.g., central and/or peripheral location (FIG. 14E) in the small
stone units, or random distribution (FIG. 14D). Specificity of the
staining was further proven with negative staining results with
four different monoclonal antibodies detecting nonrelevant
antigens.
[0169] The demineralized, screened kidney stone samples were
sterile-filtered through a 0.2 .mu.m filter, and cultured under
nanobacterial culture conditions for three weeks as described
above. Gamma irradiated serum at 10% concentration was used as a
culture supplement. In each experiment, only gamma irradiated serum
culture was used as a negative control, and no growth was
observed.
[0170] Interestingly, the present inventors observed nanobacterial
growth in 90% of the stone samples despite the harsh
demineralization step. In addition, the stones had been stored at
room temperature for more than one month before screening.
Demineralized control nanobacteria, the positive controls,
multiplied well (FIGS. 15A and B). Nucleic acid staining by using
Hoechst (#33258) stain proved no other kind of bacterial growth was
present in the cultures. For further proof, 3T6 cells were infected
with the nanobacteria cultured from stone samples, and stained with
anti-nanobacteria monoclonal antibodies. Five different kind of
nanobacteria-cell interaction was observed (data not shown).
[0171] Conclusions
[0172] Nanobacteria are novel emerging pathogens and may be related
to small mineral forming bacteria found in sedimentary rocks,
linking medicine to geology. They produce biogenic apatite in vitro
and also seem to do so in vivo. Since apatite is considered to be
the main nidus initiating the formation of most kidney stones,
nanobacteria seem to be excellent candidates for triggering this
process. Nanobacteria injected to blood circulation of laboratory
animals were shown to penetrate through kidney cells and pass into
urine. In urine, apatite formation by nanobacteria is further
increased. Other minerals may thereafter bind onto this nidus.
EXAMPLE 5
[0173] Treatment of a Human Patient Infected with Nanobacteria.
[0174] The present inventors have now treated one 35-year-old
Finnish female suffering from chronic fatigue syndrome for
nanobacterial infection. The patient was nanobacteria-positive in
three urine and serum samples collected before tetracycline therapy
was commenced. She received 500 mg tetracycline HCl 4 times per day
for one month, followed by 500 mg twice a day for 5 months. The
patient was nanobacteria negative after one month therapy. Her
condition was improved simultaneously and she has remained negative
in monthly samples.
EXAMPLE 6
[0175] Antibiotic Susceptibility of Nanobacteria
[0176] Antibiotic sensitivity tests were carried out by measuring
Minimal Inhibition Concentration (MIC), a common practice in
clinical microbiology.
[0177] Methods
[0178] The tests were performed in 96-well plates. DMEM (commercial
cell culture medium) containing 10% gamma-irradiated fetal bovine
serum (FBS)(dose about 3 Mrads; this treatment inactivates
nanobacteria in the serum so that the basic medium is sterile) was
used as the basic culture medium. These components are commercially
available, e.g., from Gibco. The antibiotic stock solutions were
sterile filtered through 0.2 micrometer filters. The stock
solutions were then serially diluted (into the basal culture
medium) to provide a final starting concentration of 0.5 mg/ml and
1:2 dilutions therefrom, unless otherwise specified. For each
antibiotic three parallel tests were performed using all the
antibiotic dilutions and, additionally, positive and negative
control experiments. Positive controls had nanobacteria with no
antibiotic addition, while negative controls had only the basic
medium. Nanobacteria were cultured from FBS, human serum from a
patient having Polycystic Kidney Disease (PKD), and from human
kidney stones as described in our earlier work (Kajander and
Ciftcioglu, PNAS 95:8274-8279, 1998). 100 .mu.l of dilutions of the
nanobacteria inoculum were added to all wells except the negative
controls. The plates were incubated at mammalian cell culture
conditions (37.degree. Celsius, 5% carbon dioxide, 95% humid air).
The absorbance values at 650 nm were recorded by using an ELISA
reader at the start, and at 4, 8, 12, 14 days. Final MIC values
(50% and 90% inhibition) were calculated at the 12 day time point.
Calculations were based on absorbance curves where absorbance was
plotted against concentration of the antibiotic.
[0179] Results
[0180] The results of these experiments are summarized in Table
5.
6TABLE 5 Summary of MIC 50 and MIC 90 values for selected
antibiotics (mg/l): Compounds MIC 50 MIC 90
Trimethoprim-Sulphamethaxazole 15 >500 Trimethoprim 0.7 5
Tetracycline 0.3 1.3 Doxycyclin 50 80 Nitrofurantoin 0.6 1.5
Gentamycin 60 250 Neomycin 16 30 Kanamycin 50 200 Vancomycin 130
250 Ampicillin 500 Cefuroxim >500 Pyrazinamide >500
Ethambutol >500 Metronidasole >500 Ciprofloxacine >500
Rifampicin >500 Clarithromycin >500 Clindamycin >500
Spectinomycin >500 Streptomycin >500 Cephalothin >500
Erythromycin >500 Lincomycin >500 Chloramphenicol >500
Penicillin >500 Polymyxin B >500
[0181] As can been seen from the results presented in Table 5, the
trimethoprim-sulphamethaxazole combination was not as effective as
trimethoprim alone (FIG. 24). This was somewhat surprising in view
of the fact that this combination is widely used in the treatment
of urinary tract infections. Trimethoprim is highly effective but
was only bacteriostatic in the in vitro test (FIG. 16).
Trimethoprim is a very potential antinanobacteria drug for human
and animal therapy.
[0182] Tetracycline is highly effective and was bactericidal in the
in vitro test (FIGS. 17, 25). Kidney stone patients treated with
4.times.500 mg/day initially had nanobacteria-positive urine
culture results, but began to have negative urine cultures during
the treatment. This indicates that tetracycline treatment can be
effective in human and animal treatments for nanobacteria
eradication. As stated previously, tetracycline is bound and
concentrated to the mineral surface of nanobacteria. This may
explain why tetracycline has bactericidal effect on nanobacteria.
This bactericidal effect is unique to nanobacteria: other bacteria
show only bacteriostatic effect. Tetracyclines are thus potential
drugs for eliminating nanobacteria from cell cultures and
biological products. Because the drug is bound to nanobacteria,
even short exposure periods have substantial antinanobacterial
effect.
[0183] Nitrofurantoin is highly effective but was bacteriostatic in
vitro (FIG. 18). This compound is used for urinary tract infections
only because of its rapid elimination into urine. In addition to
human and animal antinanobacterial therapies, nitrofurantoin can be
useful in eliminating or reducing the number of nanobacteria in
cell cultures and biological fluids and products.
[0184] Doxycycline, which is also a tetracycline compound, was
effective on inhibiting nanobacterial growth (FIG. 19). This
compound was not stable under the test conditions. MIC values
calculated from the 8 day results indicated effectiveness at around
1 mg per liter. Thus doxycycline would also be a good candidate for
the human and animal therapies. It should be noted that
tetracyclines have been used in the treatment of pathological
calcifications and autoimmune diseases with often remarkably good
results.
[0185] The aminoglycoside antibiotics gentamycin, neomycin,
kanamycin (FIG. 20-22), and streptomycin all show antinanobacterial
bacteriostatic effects at high antibiotic concentrations. Such
concentrations are present in local drug forms, such as skin cream,
ointment, plasters or washing solutions and in ear and eye drops.
Their antinanobacterial effect offers a novel explanation for the
known efficacy of gentamycin and streptomycin in the treatment of
inner ear problems involving pathological calcification, such as
Menier's disease.
[0186] Vancomycin is an effective bacteriostat against nanobacteria
at relatively high concentrations (FIG. 23). Such concentrations
can be present in local therapies with this drug. Vancomycin is not
absorbed from the gastrointestinal tract or other mucosal surfaces,
but can be used to treat mucosal bacterial infestation. Thus
vancomycin can be effective in eradication of gastrointestinal
nanobacteria or in other local applications.
[0187] Ampicillin is a wide-spectrum penicillin group antibiotic.
Ampicillin shows a weak bacteriostatic effect on nanobacteria (FIG.
26). Since ampicillin and related drugs are administered at very
high doses and are concentrated into urine at levels exceeding the
observed MIC values, they can be useful in the treatment of urinary
tract nanobacterial infection.
[0188] Other tested antibiotics were found to have MIC values
exceeding 500 mg per liter. These antibiotics are thus unlikely to
be effective in eradication of nanobacteria when used in monodrug
therapy, although they may be effectively employed in conjunction
with other antibiotics in a multidrug treatment regimen.
[0189] As shown in Table 2, bisphosphonates, as exemplified here by
chlodronate and ethidronate, are extremely effective
antinanobacterial agents that exert a bacteriocidic effect on
nanobacteria at concentrations much smaller than those found in
patients treated with the drug. This is a novel finding in
microbiology, since these drugs have not been used for
antibacterial therapies. These drugs are in medical use because of
their effects on bone resorption in cancer or osteoporosis. Recent
publications indicate that bisphosphonates can reduce pathological
calcification, an opposite reaction to their accepted use, in
atherosclerosis. Atherosclerosis involves calcification, the nature
of which has not been understood. We suggest that atherosclerosis
may be partly an infectious disease that involves nanobacteria as
copathogens with Chlamydia pneumoniae or other agents, including
local viral infections.
7TABLE 6 Summary of MIC 50 and MIC 90 values for selected Ca
chelators and other compounds: Compounds MIC 50 MIC 90 Chlodronate
0.1 mg/l 0.5 mg/l Ethidronate 0.1 mg/l 0.5 mg/l Citrate 0.2 mM 1 mM
EDTA 0.3 mM 2.5 mM Vitamin K (Menadion) 2 mg/l Vitamin D >0.025
mM Acetylsalicylic acid 0.5 g/l
[0190] Bisphosphonates can be used for the elimination of
nanobacteria in cell cultures, biological fluids and products. They
are concentrated on the nanobacterial apatite and kill the
nanobacteria relatively rapidly, even after a single exposure. Thus
they can be useful in industrial nanobacteria elimination, e.g.,
from FBS. FBS or other sera can be exposed to low levels, e.g.
micrograms to grams per liter, of a bisphosphonate that will
inactivate nanobacteria and prevent their multiplication when the
serum is used in industrial or research purposes (e.g., in tissue
or cell culture). A similar approach can be used to treat human
blood and blood derived products, vaccines, cell culture products
and biotechnological products. Treatment time can be from minutes
to days and treatment temperature from 0-100.degree. Celsius.
Treatment can be carried out in solutions having pH 3-10. The drug
is harmless to humans or animals at low levels. If necessary, the
drug can be removed from the serum or product after the exposure,
e.g., by using dialysis, or reduced to low levels by absorption on
calcium phosphate mineral surfaces (apatite and other calcium
minerals will do it), e.g., by using apatite filters or particles.
Because bisphosphonates are excreted and highly concentrated in
urine, they are highly potent drugs to treat nanobacterial diseases
like kidney stones.
[0191] Citrate is an effective antinanobacterial agent at
concentrations that can be reach by oral or intravenous or local
treatments in humans or animals. Citrate chelates calcium. Calcium
is a key element for nanobacterial cell wall integrity. Similarly
other short chain organic acids are weak calcium chelators and can
be useful in nanobacterial eradication. Such acids include lactic
acid, acetic acid and natural products containing such acids, e.g.,
cranberry juice. The latter has been used to treat urinary tract
infections. However, the mechanism of action against nanobacteria
appears to be a weakening of the apatite cell wall, whereas
cranberry juice is thought to prevent adhesion of common bacteria
in the urinary tract. Similarly trials with citrate for treatment
of kidney stones have been made because citrate lowers free calcium
levels. However, the present inventors have discovered that these
acids exert antinanobacterial effect (FIG. 27). Most importantly,
citrate is much more effective than strong acids, e.g., HCl, in
killing nanobacteria. Other short chain organic acids have been
tested later: acetic acid, lactic acid, and ascorbic acid all
showed inhibitory effect on nanobacteria in culture tests. Their
MIC50 values were 10-50 mM indicating potential for
antinanobacterial therapy.
[0192] EDTA and EGTA are calcium chelators that exert
antinanobacterial effects (FIG. 28). They can be used in patient
treatment as exemplified by the use in divalent cation, e.g., lead,
poisoning. Furthermore, such agents can be used in drug and
biotechnology preparations to prevent nanobacterial growth or
inactivate them.
[0193] Vitamin K is toxic to nanobacteria at concentrations that
are not harmful to mammalian cells. The compound is adsorbed by
apatite and thus is concentrated by nanobacteria. Vitamin K may be
used as an anti-nanobacterial agent in the treatment of serum or
biotechnological products. After treatment excessive amounts can
removed by extraction with organic solvents or lipophilic filters,
hydrophobic chromatography or affinity chromatographic techniques
using affinity matrixes binding vitamin K or similar methods.
[0194] Vitamin D modifies calcium metabolism and has a very weak
inhibitory effect on nanobacteria.
[0195] Acetylsalicylic acid is structurally rather similar to
para-amino salicylic acid, which is used as antituberculosis agent.
These compounds affect the cell wall or other targets in
nanobacteria. The effect is weak but such agents can be used at
high concentrations which makes them as potential drugs against
nanobacteria. Imporantly, para-aminosalicylic acid used in
tuberculosis treatment was found to have similar effect: its MIC
value wasa identical with acetylsalicylic acid. This may apply for
other anti-inflammatory drugs having a short-chain acid moiety.
[0196] Dental pulp stones contain apatite mineral in a biomatrix.
We have shown that nanobacteria can be found from human dental pulp
stones and that nanobacteria grow on human teeth producing
identical stones than the natural dental pulp stones. Fluoride was
found to inhibit nanobacterial stone formation on human teeth under
in vitro culture model using the fluoride concentrations typically
present in toothpastes.
[0197] Nanobacteria also absorb some heavy metals. Nanobacteria can
be stained well with silver or copper compounds. Moreover, silver
and copper ions added to nanobacterial culture media at
submillimolar levels prevented nanobacterial growth. Silver and
copper parts or platings on surfaces, such as catheters or stents,
can provide an antinanobacterial effect by preventing nanobacterial
biofilm formation. In in vitro tests nanobacteria avidly formed
biofilm on two different commercial stents made of a plastic type
of material. This biofilm formation was prevented by the addition
of silver or copper salts to the culture medium.
[0198] Drug Combinations
[0199] The most potent antibiotic, tetracycline, was tested in
combination with the most potent nonantibiotic drug, ethidronate.
It was found that combinations of these drugs, at concentrations
that are ineffective when administered alone, produced a marked
antinanobacterial effect. The MIC 90 value was about 0.01 mg per
liter for the combination, whereas individual compounds had MIC
values much higher. Thus, a synergistic effect was present. Such an
effect may be useful in designing drug combinations for
antinanobacterial effect. Specifically, therapy with combination of
an antibiotic together with a bisphosphonate, a calcium chelators,
a weak acid (such as citric, lactic, or acetic acid), or an
anti-inflammatory acidic drug (such as aspirin) is useful in the
treatment of nanobacterial infection, especially pathological
calcification and stones, such as kidney stones and salivary
stones. For treatment of nanobacteria in dental pulp stones
fluoride may be included into the combination. The drugs can be
administered in the form of a toothpaste, mouth wash or dental
adhesive coating.
EXAMPLE 6
[0200] Susceptibility of Nanobacteria to Sonication
[0201] Eradication of nanobacteria in solutions was tested also by
using sonication with B.Braun Labsonic 2000 sonicator. The sample,
nanobactena-contaminated commercial FBS (fetal bovine serum), was
subjected to sonication at full power in 100 ml portions using
round conical container (175 ml nominal volume, Nalgene Cat. No
3143). The procedure followed the sonicator manufacture's
instructions. The sound tip was selected to produce high power
sonication, and was introduced 1 cm above the bottom of the tube.
Ultrasound was given at 1 min pulses with 1 min pauses on ice bath
to prevent heating of the sample. The real sonication times were 0,
1, 2, 3, 5 and 10 min. Thereafter, the samples were subjected to
standard nanobacteria culture. Samples from 5 and 10 min sonication
revealed no culturable organisms.
[0202] While the invention has been described and illustrated
herein by references to various specific material, procedures and
examples, it is understood that the invention is not restricted to
the particular material, combinations of material, and procedures
selected for that purpose. Numerous variations of such details can
be implied and will be appreciated by those skilled in the art.
* * * * *