U.S. patent application number 09/894467 was filed with the patent office on 2002-10-24 for human blood bacterium.
This patent application is currently assigned to Pathobiotek Diagnostics Inc.. Invention is credited to Lindner, Luther E., MacPhee, Kathleen.
Application Number | 20020155519 09/894467 |
Document ID | / |
Family ID | 22056229 |
Filed Date | 2002-10-24 |
United States Patent
Application |
20020155519 |
Kind Code |
A1 |
Lindner, Luther E. ; et
al. |
October 24, 2002 |
Human blood bacterium
Abstract
The present invention reports a newly-identified human blood
bacterium (HBB), provides characterization, culturing and
diagnostic methodologies therefor and methods for the treatment of
pathophysiological states caused by the bacterium. The bacterium is
apparently present in the bloodstream of all humans in very low
numbers, and appears to be directly or indirectly associated with
several diseases such as chronic fatigue syndrome, multiple
sclerosis and other "autoimmune" diseases. Also provided are uses
of engineered HBB.
Inventors: |
Lindner, Luther E.; (College
Station, TX) ; MacPhee, Kathleen; (Spring,
TX) |
Correspondence
Address: |
Dr. Benjamin Adler
Adler & Associates
8011 Candle Lane
Houston
TX
77071
US
|
Assignee: |
Pathobiotek Diagnostics
Inc.
|
Family ID: |
22056229 |
Appl. No.: |
09/894467 |
Filed: |
June 28, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09894467 |
Jun 28, 2001 |
|
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09187946 |
Nov 2, 1998 |
|
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60064472 |
Nov 6, 1997 |
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Current U.S.
Class: |
435/34 ;
435/252.1 |
Current CPC
Class: |
C12N 1/205 20210501;
A61P 31/04 20180101; C12R 2001/01 20210501; C07K 14/195 20130101;
A61K 38/00 20130101 |
Class at
Publication: |
435/34 ;
435/252.1 |
International
Class: |
C12Q 001/04; C12N
001/20 |
Claims
What is claimed-is:
1. A method for culturing human blood bacterium, comprising the
steps of: isolating human blood bacterium from a sample; adding a
medium comprising CaCl.sub.2; MgCl.sub.2 (anhydrous); KCl; NaCl;
NaH.sub.2PO.sub.4 (monobasic); lactalbumin hydrolysate; yeast
extract, lactose; manganese chloride; and a buffer selected from
the group consisting of sodium bicarbonate, Tris, and HEPES; and
incubating said human blood bacterium at a temperature which allows
for growth of said human blood bacterium.
2. The method of claim 1, wherein said medium further contains
sodium arachidonate and lipoxidase.
3. A method for culturing human blood bacterium, comprising the
steps of: isolating HBB from a sample; adding a medium comprising
CaCl.sub.2; MgCl.sub.2 (anhydrous); KCl; NaCl; NaH.sub.2PO.sub.4
(monobasic); lactalbumin hydrolysate; yeast extract; manganese
chloride; and a sugar selected from the group consisting of
glucose, fructose, and sucrose; and incubating said human blood
bacterium at a temperature which allows for growth of said human
blood bacterium.
4. A method for diagnosing a pathophysiological state in an
individual resulting from an imbalance in a presence of human blood
bacterium in the blood of said individual, comprising the steps of:
determining a count of human blood bacterium in said blood;
comparing said count from the test individual with a count obtained
from a control individual known to be in a healthy state, wherein
if said count from said test individual is greater than said count
from said control individual, said test individual has abnormal
levels of said human blood bacterium, if said count from said test
individual is not greater than said count from said control
individual, said test individual has normal levels of said human
blood bacterium.
5. The method of claim 4, wherein the count from said control
individual is not greater than 400/HPF (High power field) after 1
week's growth of blood from said control individual in medium O or
modified RPMI medium.
6. The method of claim 4, wherein said count is determined by
performing a technique selected from the group consisting of
quantification by a solid or liquid culture; ELISA assay; flow
cytometry; TAQ man; Western blot hybridization; antibody-based
tests and nucleic acid-probe based tests.
7. The method of claim 6, wherein said nucleic acid-probe based
test is selected from the group consisting of in situ hybridization
and PCR.
8. The method of claim 7, wherein probes used for said in situ
hybridization are selected from the group consisting of SEQ ID
No:19 and SEQ ID No:20.
9. The method of claim 7, wherein primers used for said PCR are
selected from the group consisting of primers specific for human
blood bacterium intergenic spacer region, primers specific for
human blood bacterium 16S rRNA, primers specific for human blood
bacterium 23S rRNA and primers specific for human blood bacterium
drug resistant protein gene.
10. The method of claim 9, wherein said primers specific for human
blood bacterium intergenic spacer region are selected from the
group consisting of SEQ ID No:15 and SEQ ID No:16.
11. The method of claim 9, wherein said primers specific for human
blood bacterium 16S rRNA are selected from the group consisting of
SEQ ID No: 7 and SEQ ID No: 8.
12. The method of claim 9, wherein said primers specific for human
blood bacterium 23S rRNA are selected from the group consisting of
SEQ ID No: 9, SEQ ID No:10, SEQ ID No:11, SEQ ID No:12, SEQ ID
No:13 and SEQ ID No:14.
13. The method of claim 9, wherein said primers specific for human
blood bacterium drug resistant protein gene are selected from the
group consisting of SEQ ID No:17 and SEQ ID No:18.
14. A method of monitoring treatment of human blood
bacterium-related disease in an individual, comprising the step of:
determining quantification of human blood bacterium in the blood of
said individual at various stages of treatment, wherein a decrease
in said quantification of said human blood bacterium indicates
effective treatment, and an increase in said quantification of said
human blood bacterium indicates ineffective treatment.
15. The method of claim 14, wherein said quantification is
determined by performing a technique selected from the group
consisting of quantification by a solid or liquid culture; ELISA
assay; flow cytometry; TAQ man; Western blot hybridization;
antibody-based tests and nucleic acid-probe based tests.
16. The method of claim 15, wherein said nucleic acid-probe based
test is selected from the group consisting of in situ hybridization
and PCR.
17. The method of claim 14, wherein said disease is selected from
the group consisting of chronic fatigue syndrome, multiple
sclerosis, lupus erythematosis, rheumatoid arthritis and
fibromyalgia.
18. A method for treating a pathophysiological state in an
individual having human blood bacterium in the blood, comprising
the step of: administering to said individual a therapeutically
effective amount of at least one antibiotic selected from the group
consisting of penicillin G, penicillin V, probenecid, Augmentin,
dicloxacillin, Ciprofloxacin, Isoniazid, third-generation
cephalosporins, azithromycin, clarithromycin, chloroquin,
hydroxychloroquin, minocycline, doxycycline and primaquin.
19. The method of claim 18, wherein said antibiotic is administered
with a therapeutically effective amount of one or more substances
selected from the group consisting of Nystatin, Nizoral, Diflucan,
steroids, vitamin B-6, vitamin C, folic acid, vitamin E, niacin,
chromium, zinc, sulfhydryl compounds, steroids and ibuprofin.
20. A method for culturing human blood bacterium, comprising the
steps of: isolating human blood bacterium from a sample; adding a
medium comprising salts, at least one sugar, and lactalbumin
hydrolysate; and incubating said human blood bacterium at a
temperature which allows for growth of said human blood
bacterium.
21. A vaccine generated from human blood bacterium or components
thereof.
22. A method of treating a diseased individual, comprising the step
of administering engineered HBB to said individual.
23. The method of claim 22, wherein said disease has the condition
of toxic metabolite being accumulated in plasma or serum of said
individual.
24. The method of claim 22, wherein said engineered HBB expresses
therapeutical gene products selected from the group consisting of
hormones, growth regulators, antitumor antigens, antibodies and
interleukins.
25. DNA encoding a human blood bacterium in a normal individual's
blood having a sequence selected from the group consisting of SEQ
ID No: 1 and 3.
26. DNA encoding a human blood bacterium in a diseased individual's
blood having having a sequence selected from the group consisting
of SEQ ID No: 2 and 4.
27. The DNA of claim 26, wherein said individual having a disease
selected from the group consisting of chronic fatigue syndrome,
multiple sclerosis, lupus erythematosis, rheumatoid arthritis and
fibromyalgia.
28. DNA encoding a human blood bacterium having an intergenic
spacer region sequence shown in SEQ ID No: 5.
29. Human blood bacterium of claim 28, wherein primers specific for
said intergenic spacer region having a sequence shown in SEQ ID
No:15 or SEQ ID No:16.
30. DNA encoding a human blood bacterium having a drug resistant
protein gene sequence shown in SEQ ID No: 6.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This patent application claims benefit of provisional patent
application U.S. Serial No. 60/064,472, filed Nov. 6, 1997, now
abandoned.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates generally to bacteriology and
human pathology. More specifically, the present invention relates
to a bacterium present in the human blood, the characterization,
culturing and diagnostic methodologies therefor and the treatment
of pathophysiological states caused by this bacterium.
[0004] 2. Description of the Related Art
[0005] Infectious agents are a main cause of human disease and the
leading cause of death worldwide. Bacterial infections are cause
more deaths than any other class of infectious organisms. The
single leading cause of death via infectious organisms worldwide
remains tuberculosis, caused by the bacterium Mycobacterium
tuberculosis; however scores of diseases or disorders caused by
infectious agents are present in both developed and undeveloped
countries.
[0006] Examples of infectious disorders include chronic fatigue
syndrome and conditions such as fibromyalgia. These disorders
affect about 1,000,000 Americans. There is no known cause or
effective treatment for these conditions, nor is there any
definitive diagnostic laboratory test. Epidemiologic evidence
suggests an infectious agent as the cause; however, no causative
infectious agent has been identified.
[0007] Multiple sclerosis produces disability to variable degrees
and occasional death in about 400,000 Americans. Multiple sclerosis
is a disease of unknown cause and has considerable variability,
with minimal disability in some patients and almost total
disability in others. As with chronic fatigue syndrome, there is no
definitive diagnostic laboratory test, and clinical diagnosis is
based on a constellation of symptoms and tests. Epidemiologic data
suggests that multiple sclerosis has an underlying infectious cause
and that the infectious cause is acquired years prior to the
development of symptoms.
[0008] Another loosely-defined group of diseases are those in the
"autoimmune" category such as lupus erythematosis and rheumatoid
arthritis. These relatively common diseases are often disabling and
the underlying cause of these diseases is unknown. Rheumatoid
arthritis is rarely fatal, but lupus erythematosis has a
significant mortality rate. Laboratory diagnostic tests for these
conditions are satisfactory, but no test is definitive, so a
variety of tests in combination with the correlation of clinical
symptoms is necessary. A variety of medications are available to
control symptoms to some degree and slow progression of the
diseases, but no treatment is curative. Certain antibiotics are
currently accepted as one therapeutic option for rheumatoid
arthritis, but no specific infectious cause has been
demonstrated.
[0009] Thus, the prior art is deficient in the identification and
characterization of the specific bacterium from human blood
described herein, methods for culturing it and for diagnosing and
treating diseases that result from an increase in the presence of
the bacterium in an individual's blood. The present invention
fulfills this long-standing need and desire in the art.
SUMMARY OF THE INVENTION
[0010] One object of the present invention is to provide a culture
system for the human blood bacterium (HBB) described herein. One
approach to this culture system is to provide a method for
culturing HBB, comprising the steps of: isolating HBB from a
sample; adding a medium comprising salts, at least one sugar, and
lactalbumin hydrolysate; and incubating said HBB at a temperature
which allows for growth of said HBB.
[0011] In one embodiment of the present invention, there is
provided a method for culturing HBB, comprising the steps of:
isolating HBB from a sample; adding a medium comprising CaCl.sub.2;
MgCl.sub.2 (anhydrous); KCl; NaCl; NaH.sub.2PO.sub.4 (monobasic);
lactalbumin hydrolysate; yeast extract, lactose; manganese
chloride; and a buffer selected from the group of sodium
bicarbonate, Tris, and HEPES; and incubating said HBB at a
temperature which allows for growth of said HBB. In a preferred
embodiment, the culture medium further contains sodium arachidonate
and lipoxidase.
[0012] In yet another embodiment of the present invention, there is
provided a method for culturing HBB, comprising the steps of:
isolating HBB from a sample; adding a medium comprising CaCl.sub.2;
MgCl.sub.2 (anhydrous); KCl; NaCl; NaH.sub.2PO.sub.4 (monobasic);
lactalbumin hydrolysate; yeast extract; manganese chloride; and a
sugar selected from the group of glucose, fructose, and sucrose;
and incubating said HBB at a temperature which allows for growth of
said HBB.
[0013] An additional object of the present invention is to provide
methods of diagnosing a pathophysiological state in an individual
that results from an increase in the presence of the human blood
bacterium (HBB) in an individual's blood. Additionally, the methods
can be used to identify individuals at risk for developing a
pathophysiological state or for monitoring progress of treatment
for disease. The methods make use of antibodies capable of reacting
with HBB and polynucleotides capable of duplexing with the HBB
genome. Infection may be detected by various techniques,
particularly nucleic acid hybridization and immunoassays.
[0014] In another embodiment of the present invention, there is
provided a method for diagnosing a pathophysiological state in an
individual resulting from an imbalance in a presence of HBB in the
individual's blood, comprising the step of determining a count of
HBB from blood of the individual, and comparing the counts between
the test and control individuals, wherein the control individual is
known to be healthy. If the count of test individual is greater
than that of control individual, the test individual has abnormal
levels of HBB in its blood. Otherwise, the test individual has
normal levels of HBB. Preferably, the count from control individual
is no greater than 400/HPF (High power field) after growing its
blood for 1 week in medium O or modified RPMI medium. The methods
for making this determination include, but are not limited to, a
technique selected from the group of quantification by a solid or
liquid culture; ELISA assay; flow cytometry; TAQ man; Western blot
hybridization; antibody-based tests and nucleic acid-probe based
tests including PCR and in situ hybridization. Specifically, probes
for in situ hybridization are selected from the group consisting of
SEQ ID No:19 and SEQ ID No:20. Primers used for PCR are selected
from the group consisting of SEQ ID Nos: 7-18.
[0015] A further object of the present invention is to treat a
pathophysiological state that results from an increase in the
presence of the bacterium in an individual's blood. In one
embodiment of the present invention, there is provided a method for
treating a pathophysiological state that results from an imbalance
in a presence of HBB in an individual's blood, comprising the step
of administering to said individual a therapeutically effective
amount of at least one antibiotic from the group consisting of
penicillin G, penicillin V, primaquine, Augmentin, dicloxacillin,
Ciprofloxacin, Isoniazid, third-generation cephalosporins,
azithromycin, clarithromycin, chloroquin, hydroxychloroquin,
minocycline, doxycycline. In a preferred embodiment, the antibiotic
is administered with a therapeutically effective amount of one or
more substances from the group consisting of probecid, Nystatin,
Nizoral, Diflucan, steroids, vitamin B-6, vitamin C, folic acid,
vitamin E, niacin, chromium, zinc, sulfhydryl compounds, steroids,
and ibuprofen.
[0016] In yet another embodiment of the present invention, there is
provided a vaccine generated from HBB or components thereof.
[0017] In still yet another embodiment of the present invention,
there is provided a method of treating an individual with a disease
where toxic metabolites are accumulated in its plasma or serum by
administering engineered HBB to the individual. Preferably, the
engineered HBB expresses therapeutical gene products selected from
the group consisting of hormones, growth regulators, antitumor
antigens, antibodies, interleukins and other therapeutical
antigens.
[0018] Still another project of the present invention is to
characterize this new human blood bacterium.
[0019] In one embodiment of the present invention, there is
provided human blood bacterium in a normal individual's blood
having a 16S rRNA sequence shown in SEQ ID No: 1.
[0020] In another embodiment of the present invention, there is
provided human blood bacterium in a diseased individual's blood
having a 16S rRNA sequence shown in SEQ ID No: 2. Preferably, the
individual has a disease selected from the group consisting of
chronic fatigue syndrome, multiple sclerosis, lupus erythematosis,
rheumatoid arthritis and fibromyalgia.
[0021] In still another embodiment of the present invention, there
is provided human blood bacterium in a normal individual's blood
having a 23S rRNA sequence shown in SEQ ID No: 3.
[0022] In still yet another embodiment of the present invention,
there is human blood bacterium in a diseased individual's blood
having a 23S rRNA sequence shown in SEQ ID No: 4. Preferably, the
individual has a disease selected from the group consisting of
chronic fatigue syndrome, multiple sclerosis, lupus erythematosis,
rheumatoid arthritis and fibromyalgia.
[0023] In yet another embodiment of the present invention, there is
provided human blood bacterium having an intergenic spacer region
sequence shown in SEQ ID No: 5. Preferably, primers specific for
the intergenic spacer region having a sequence shown in SEQ ID
No:15 or SEQ ID No:16.
[0024] In still yet another embodiment of the present invention,
there is provided human blood bacterium having a drug resistant
protein gene sequence shown in SEQ ID No: 6.
[0025] Other and further aspects, features, and advantages of the
present invention will be apparent from the following description
of the presently preferred embodiments of the invention. These
embodiments are given for the purpose of disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] So that the matter in which the above-recited features,
advantages and objects of the invention, as well as others which
will become clear, are attained and can be understood in detail,
more particular descriptions of the invention briefly summarized
above may be had by reference to certain embodiments thereof which
are illustrated in the appended drawings. These drawings form a
part of the specification. It is to be noted, however, that the
appended drawings illustrate preferred embodiments of the invention
and therefore are not to be considered limiting in their scope.
[0027] FIG. 1 shows a culture from serum in medium L after one
week's growth. A relatively large number of bacteria is present,
ranging in appearance from cocci to short bacilli. Most are
isolated, but some clumps of organisms are noted. Magnification
approx. 600.times., phase contrast.
[0028] FIG. 2 is a photomicrograph of a culture from serum in
medium L. In contrast to FIG. 1, a much lower concentration of
bacteria is present. In this culture, atypical forms dominate, with
marked variation in size and refractility. Many are round and many
show budding-like division. Most are phase-dark, but some contain
phase-bright inclusions and crystal-like phase-bright bodies are
also present. The background contains many red blood cell ghosts
which are uniform in size and low in contrast; the majority are out
of focus. Magnification approx. 600.times., phase contrast.
[0029] FIG. 3 is a transmission electron micrograph of human blood
bacterium isolated from a patient with chronic fatigue syndrome. A
single organism is shown, demonstrating a gram negative wall
structure with a wavy outer membrane. The nucleoid is seen
centrally and there is some extracellular material that presumably
represents the slime material. Magnification approx.
150,000.times..
[0030] FIG. 4 is a transmission electron micrograph of human blood
bacterium isolated from a patient with chronic fatigue syndrome.
Two organisms are shown, one demonstrating features similar to the
organism pictured in FIG. 3, the other showing transparent vacuoles
and surface projections that could represent pili, conjugation
tubes, or filamentous phages. Both contain small dense inclusions.
There is some extracellular material that presumably represents the
slime material. Magnification approx. 75,000.times..
[0031] FIG. 5 shows human blood bacterium isolated from a culture
of a patient with multiple scerlosis. The bacterium is hybridized
with a Methylobacterium-specific rhodamine-labeled probe. The probe
is bound to a diploid rod apparently in division. Magnification
approx. 1,250.times..
[0032] FIG. 6 is a photograph of the same microscopic field as the
one shown in FIG. 5. It shows that the diploid rod is positive for
the counterstain 4', 6'-diamidino-2-phenylindole.2HCl (DAPI). DAPI
binds only to nucleic acids and confirms the presence of bacteria.
Magnification approx. 1,250.times..
DETAILED DESCRIPTION OF THE INVENTION
[0033] As used herein, the term "human blood bacterium" or "HBB"
refers to a bacterium described herein that is present in the blood
of essentially all humans which can be grown in the culture system
described herein. Elevated levels of HBB indicate the presence of
or likelihood of developing certain diseases such a s chronic
fatigue syndrome, multiple sclerosis and other autoimmune diseases.
Further, there is a 100% correlation between a decrease in the
presence of HBB in the blood and a diminishing or complete
elimination of symptoms of these diseases.
[0034] As used herein, the term "antibiotic" refers to a chemical
or drug which inhibits selectively the growth of a bacterium or
kills it completely.
[0035] As used herein, the term "autoimmune disease" refers to a
disease characterized by an immune reaction created by the host
that is directed against specific components of the host's
body.
[0036] As used herein, the term "bacterial growth" refers to an
increase in the number of bacteria as a result of division of a
bacterium into two or more progeny.
[0037] As used herein, the term "bacterial culture" refers to a
collection of bacteria growing in or on a nutritive mixture of
materials in vitro.
[0038] As used herein, the term "exudate" or "slime" refers to a
complex material produced by bacteria that is semi-solid in
consistency.
[0039] As used herein, the term "improved" in the context of
disease treatment refers to a decrease in the severity of reported
symptoms or in other objective signs which characterize a
particular disease.
[0040] The present invention is directed to methods of culturing a
human blood bacterium (HBB) useful in diagnosing pathophysiological
states in individuals that result from an increase in the presence
of this bacterium in an individual's blood. It is contemplated
additionally that the methods of the present invention can be used
to identify individuals at risk for developing such a
pathophysiological state or for monitoring progress of treatment.
Further, the methods of the present invention, drawn to controlling
the human blood bacteria by antibiotics or through genetic
engineering, may be used treat a pathophysiological state that
results from an increase in the presence of the bacterium in an
individual's blood.
[0041] In accordance with the present invention there may be
employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g.,
"Current Protocols in Molecular Biology", Volumes 1-3 (Ausubel et
al., Ed, 1994-1997, John Wiley and Sons); Sambrook, Fritsch &
Maniatis, "Molecular Cloning: A Laboratory Manual (1989); "DNA
Cloning: A Practical Approach," Volumes I and II (D. N. Glover ed.
1985); "Oligonucleotide Synthesis" (M. J. Gait ed. 1984); "Nucleic
Acid Hybridization" (B. D. Hames & S. J. Higgins eds. (1985));
"Transcription and Translation" (B. D. Hames & S. J. Higgins
eds. (1984)); "Animal Cell Culture" (R. I. Freshney, ed. (1986));
"Immobilized Cells And Enzymes" (IRL Press, (1986)) and "Current
Protocols in Immunology", Volumes 1-3 (Ausubel et al., Ed,
1994-1997, John Wiley and Sons).
[0042] A "DNA molecule" refers to the polymeric form of
deoxyribonucleotides (adenine, guanine, thymine, or cytosine) in
either single stranded form, or a double-stranded helix. This term
refers only to the primary and secondary structure of the molecule,
and does not limit it to any particular tertiary forms. Thus, this
term includes double-stranded DNA found, inter alia, in linear DNA
molecules (e.g., restriction fragments), viruses, plasmids, and
chromosomes. In discussing the structure herein, only the sequence
in the 5' to 3' direction along the nontranscribed strand of DNA is
given (i.e., the strand having a sequence homologous to the
mRNA).
[0043] A DNA "coding sequence" is a double-stranded DNA sequence
which is transcribed and translated into a polypeptide in vivo when
placed under the control of appropriate regulatory sequences. The
boundaries of the coding sequence are determined by a start codon
at the 5' (amino) terminus and a translation stop codon at the 3'
(carboxyl) terminus. A coding sequence can include, but is not
limited to, prokaryotic sequences, cDNA from eukaryotic mRNA,
genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and
even synthetic DNA sequences. A polyadenylation signal and
transcription termination sequence will usually be located 3' to
the coding sequence.
[0044] The term "oligonucleotide" or "probe" as used herein, refers
to a molecule comprised of ribonucleotides or deoxyribonucleotides.
The exact size of the oligonucleotide or probe will depend upon
many factors which, in turn, depend upon the ultimate function and
use of the oligonucleotide. Diagnostic tests for detecting the
presence of HBB in biological samples may be performed using
polynucleotide probes. Such polynucleotide probes may be prepared
based on the sequence of the HBB genome. The length of the probe is
not critical, but will usually comprise at least about 12 bases,
more usually comprising at least about 16 bases, such that the
probe is substantially complementary to a portion of the bacterial
genome; however, the probe need not have perfect complementarity
with the HBB genome. The probes may be prepared synthetically, with
suitable synthetic techniques, and most likely include a detectable
label. Usually, the synthetic sequences are expanded in common,
publicly-available cloning vectors and suitable hosts in order to
obtain large quantities of the probe. The expanded vectors may
themselves be labeled for use as probes, or shorter fragments
containing complementary strands may be excised and labeled.
Methods for the preparation and utilization of nucleotide probes
for diagnostic testing are described in the references listed
above, supra, and in U.S. Pat. No. 4,358,535 to Falkow, et al.
[0045] The term "primer" as used herein refers to an
oligonucleotide, whether occurring naturally as in a purified
restriction digest or produced synthetically, which is capable of
acting as a point of initiation of synthesis when placed under
conditions in which synthesis of a primer extension product, which
is complementary to a nucleic acid strand, is induced, i.e., in the
presence of nucleotides and an inducing agent such as a DNA
polymerase and at a suitable temperature and pH. The primer may be
either single-stranded or double-stranded and must be sufficiently
long to prime the synthesis of the desired extension product in the
presence of the inducing agent. The exact length of the primer will
depend upon many factors, including temperature, the source of
primer and the method used. For example, for diagnostic
applications, depending on the complexity of the target sequence,
the oligonucleotide primer typically contains 15-25 or more
nucleotides, although it may contain fewer nucleotides.
[0046] The primers herein are selected to be "substantially"
complementary to different strands of a particular target DNA
sequence. This means that the primers must be sufficiently
complementary to hybridize with their respective strands.
Therefore, the primer sequence need not reflect the exact sequence
of the template. For example, a non-complementary nucleotide
fragment may be attached to the 5' end of the primer, with the
remainder of the primer sequence being complementary to the strand.
Alternatively, non-complementary bases or longer sequences can be
interspersed into the primer, provided that the primer sequence has
sufficient complementarity with the sequence or hybridize therewith
and thereby form the template for the synthesis of the extension
product.
[0047] A "heterologous" region of the DNA construct is an
identifiable segment of DNA within a larger DNA molecule that is
not found in association with the larger molecule in nature. Thus,
when the heterologous region encodes a mammalian gene, the gene
will usually be flanked by DNA that does not flank the mammalian
genomic DNA in the genome of the source organism. In another
example, coding sequence is a construct where the coding sequence
itself is not found in nature (e.g., a cDNA where the genomic
coding sequence contains introns, or synthetic sequences having
codons different than the native gene). Allelic variations or
naturally-occurring mutational events do not give rise to a
heterologous region of DNA as defined herein.
[0048] Polypeptides used for detection and diagnosis of methods of
the present invention are either haptenic or antigenic, include at
least six amino acids, and include usually at least twelve or more
amino acids found contiguously within one of the natural HBB
proteins. Polypeptides generally correspond to at least one
epitopic site which is characteristic of HBB, preferably to
epitopes associated with B and/or T cells. The term
"characteristic" in this context means that the epitopic site
allows immunologic detection of HBB in a physiological sample with
reasonable assurance. Usually, it is desirable that the epitopic
site be immunologically distinct from bacteria other than HBB. The
HBB polypeptides may be natural; i.e., including an entire HBB
protein or fragments thereof isolated from a natural source, or the
polypeptides may be synthetic. Natural polypeptides may be isolated
and used to prepare an affinity column by techniques known in the
art. Such techniques are taught, for example, in Hudson and Hay,
Chapter 8, Practical Immunology, Blackwell Scientific Publications,
Oxford (1980).
[0049] Synthetic polypeptides which are immunologically
cross-reactive with a natural HBB protein may be produced by two
general methods. First, polypeptides having fewer than about 80
amino acids may be synthesized by the Merrifield solid-phase
synthesis method where amino acids are sequentially added to a
growing chain. An alternative method involves expression in
cultured cells of recombinant DNA molecules encoding a desired
portion of the HBB genome. The portion of the HBB genome may itself
be natural or synthetic, with natural genes obtainable from the
isolated bacterium by conventional techniques.
[0050] To be useful in the detection and diagnostic methods of the
present invention, the polypeptides are obtained in a substantially
pure form. The proteins may be purified by use of the antibodies
described hereinafter using immunoabsorbant affinity columns. Once
a sufficient quantity of HBB polypeptides has been obtained,
polyclonal antibodies specific for HBB may be produced by in vitro
or in vivo techniques. In vitro techniques involve in vitro
exposure of lymphocytes to the antigenic polypeptides, while in
vivo techniques require the injection of the polypeptides into a
wide variety of vertebrates. Suitable vertebrates are non-human,
and include mice, rats, rabbits, sheep, goats, donkeys and the
like. Polypeptides having more than about thirty amino acids,
usually more than about fifty amino acids, may serve directly as
the immunogen. If the polypeptide is smaller than about 10 kD,
particularly less then about 6 kD, it may be necessary to join the
polypeptide to a larger molecule to elicit the desired immune
response. The immunogens are then injected into the animal, and the
animal is bled periodically with successive bleeds having improved
titer and specificity. Injections may be made intramuscularly,
subcutaneously, and an adjuvant, such as incomplete Freund's
adjuvant, will usually be employed.
[0051] If desired, monoclonal antibodies can be obtained by
preparing immortalized cell lines capable of producing antibodies
having the desired specificity. Such immortalized cell lines may be
produced in a variety of ways. Conveniently, a small vertebrate,
such as a mouse, is hyperimmunized with the desired antigen by the
method just described. The vertebrate is then killed, usually
several days after the final immunization, the spleen removed, and
the spleen cells immortalized. Presently, the most common technique
is fusion with a myeloma cell fusion partner, as first described by
Kohler and Milstein, Eur. J. Immunol., 6:511-19 (1976). Other
techniques include EBV transformation, transformation with
oncogenes, and transformation with retroviruses, or any other
method which provides for stable maintenance of the cell line and
production of monoclonal antibodies.
[0052] When employing fusion with a fusion partner, the manner of
fusion is not critical and various techniques may be employed.
Commonly, the spleen and myeloma cells are combined in the presence
of a non-ionic detergent, usually polyethylene glycol, and other
additives such as Dulbecco's Modified Eagle's medium for a few
minutes. At the end of the fusion, the non-ionic detergent is
removed by washing the cells. The fused cells are promptly
dispensed in small culture wells (usually in a microtiter plate at
relatively low density, at about 5.times.10.sup.6 cells/well) in a
selective medium chosen to support growth of the hybrid cells while
being lethal to the myeloma cells. Usually, the myeloma cell line
has been mutated to be sensitive, and the medium includes a HAT
concentration sufficient to inhibit the proliferation of the
unfused myeloma cells. After sufficient time, usually from about
one to two weeks, colonies of hybrids are observed and plates
containing hyperpositive wells are identified. The plates and wells
having only one colony per well are selected, and supernatants from
these wells are tested for binding activity against HBB or a
particular HBB protein. Once positive hybridomas are identified,
the cell line can be maintained as a viable culture.
[0053] Further screening of the hybridomas may be desirable,
depending on the desired use for the antibodies. For use in
immunodiagnostic assays, antibodies having very high specificity
and affinity for the antigenic site of an HBB polypeptide of this
invention are desirable. Thus, once the desired hybridomas have
been selected, monoclonal antibodies may be isolated from
supernatants of the growing colonies. The yield of antibodies
obtained, however, is usually low, but may be enhanced by various
techniques such as injection of the hybridoma cell line into the
peritoneal cavity of a vertebrate host. Most antibodies may then be
harvested from the ascites fluid or from the blood. Proteins and
other contaminants are removed from the monoclonal antibodies prior
to use by conventional techniques; e.g., chromatography, gel
filtration, precipitation or extraction. Alternatively cDNAs
encoding the antibody chains can be obtained by known methods and
host cells can be transformed with vectors containing the cDNA to
produce the anti-HBB antibodies recombinantly.
[0054] Polypeptides and antibodies of the present invention may be
used with or without modification for the detection of HBB.
Frequently, the polypeptides and antibodies are labeled by either
covalently or non-covalently combining the polypeptide with a
second substance which provides for detectable signal. A wide
variety of labels and conjugation techniques are known and are
reported extensively in both the scientific and patent literature.
Some of the labels include radionuclides, enzymes, substrates,
cofactors, inhibitors, fluorescers, chemiluminescers, magnetic
particles and the like.
[0055] Antibodies and polypeptides prepared as described above can
be used in various immunological techniques for detecting HBB and
anti-HBB antibodies in physiological specimens, particularly body
fluid samples including blood, blood serum, and cerebrospinal
fluid. Depending on the nature of the sample, both immunoassays and
immunohistochemical staining techniques may be used. Liquid phase
immunoassays and Western blot analysis are used in detection of HBB
in blood and CFS. The use of antibodies in protein binding assays
is well established. Numerous competitive and noncompetitive
protein binding assays have been described in the scientific and
patent literature, and a large number of such assays are
commercially available. Detailed methods for detecting the presence
of HBB from blood or CFS is set out in the Experimental Section.
Additionally, a general protocol for enzyme linked immunosorbent
assays (ELISAs) for detecting presence of antibodies to HBB in
blood are also set forth in Example 9.
[0056] It is specifically contemplated that pharmaceutical
compositions may be prepared using the novel engineered HBB of the
present invention. In such a case, the pharmaceutical composition
comprises the novel engineered HBB and a pharmaceutically
acceptable carrier. In addition, it is contemplated that various
antibiotics known to the art may be used to treat the autoimmune
conditions and other diseases associated with HBB. A person having
ordinary skill in the art of molecular pharmacology would be able
to determine readily, without undue experimentation, the
appropriate dosages and routes of administration of either the HBB
of the current invention, or the antibiotics used in the methods of
treatment of disease of the present invention. When used in vivo
for therapy, the engineered HBB or the antibiotic is administered
to the patient or to an animal in therapeutically effective
amounts; i.e., amounts that reduce or eliminate the bacteria in the
bloodstream in the case of treatment of autoimmune disease. The
dose and dosage regimen will depend upon the nature of the disease,
the stage of infection, the characteristics of the particular
pharmacologic agent--e.g., its therapeutic index, the patient, the
patient's history and other factors. The schedule will be continued
to optimize effectiveness, balanced against negative effects of
treatment. See Remington's Pharmaceutical Science, 17th Ed. (1990)
Mark Publishing Co., Easton, Pa.; and Goodman and Gilman's: The
Pharmacological Basis of Therapeutics 8th Ed (1990) Pergamon
Press.
[0057] Antibiotic treatment generally will not be via parenteral
administration due to the very slow growth cycle of HBB and
probable existence of spores or a spore-like state. The existence
of a spore-like state requires that any antibiotic therapy be
carried out over a long time span, compared to more, typical
antibiotic therapy. Parenteral administration over a long period of
time is inconvenient; intravenous administration in particular over
a long time carries serious risks of infection, embolism, and other
complications.
[0058] The engineered HBB of the present invention may be
administered parenterally. For parenteral administration, the
engineered HBB will most typically be formulated in a unit dosage
injectable form (solution, suspension, emulsion) in association
with a pharmaceutically acceptable parenteral vehicle. Such
vehicles are preferably non-toxic and non-therapeutic. Examples of
such vehicles are water, saline, Ringer's solution, dextrose
solution, and 5% human serum albumin. Nonaqueous vehicles such as
fixed oils and ethyl oleate may also be used. Liposomes may be used
as carriers. The vehicle may contain minor amounts of additives
such as substances that enhance isotonicity and chemical stability,
e.g., buffers and preservatives.
[0059] The present invention reports a newly-identified and
characterized human blood bacterium (HBB). The bacterium is present
in the bloodstream of all humans in very low numbers--too low to
recognize when examining blood smears--although it has been seen on
direct observation of blood. There have been sporadic observations
over the years that may have been HBB, but HBB has never been
successfully cultured or characterized in the prior art. The
bacterium appears to be associated with several diseases, either
directly or indirectly. It is almost certainly the underlying
(indirect) cause of the chronic fatigue syndrome. Further, it is
also associated with multiple sclerosis. Further observations
indicate that it is related to other "autoimmune" diseases.
[0060] One of the most notable features of HBB is its very slow and
limited growth. Levels of 10.sup.6 to 10.sup.8 bacteria per culture
flask can be recovered from 1 ml of serum after one week's growth.
The number of organisms in blood samples prior to culture is still
not known precisely, but is at least one to two orders of magnitude
lower. The slow growth restricts characterization and requires
running experiments for longer time than is usual for bacteriologic
studies, typically from days up to a month.
[0061] Blood culture is done normally by inoculating whole
anticoagulated blood into a complex medium that provides a variety
of nutrients for bacterial growth. Different media and culture
conditions are typically required to grow different bacteria;
nevertheless most bacteria that are cultured from the blood have a
rapid growth rate and grow essentially without limit as long as
nutrients are supplied, so very high numbers are eventually
achieved. It is routine to culture blood specimens for no more than
one week. However, HBB cannot be cultured in this way because
growth is slow and the ultimate level of growth is
limited--sufficient growth is not achieved to identify the bacteria
in the presence of large numbers of red blood cells. In addition,
most ordinary culture media does not supply the necessary nutrients
for HBB and contains materials that actively inhibit growth.
Further, the observation that HBB could be recovered from serum is
counterintuitive, as organisms are generally trapped in the clot.
The actual composition of the culture medium used in the
experiments leading to the present invention was developed from a
long series of trial and error experiments extending over several
years, typically running each experiment over a month or more. The
single most important component of the medium, the lactalbumin
hydrolysate, is occasionally used as a bacterial culture medium
component, but is relatively unusual.
[0062] The techniques utilized in the present invention and
described herein were run using standard bacteriologic techniques,
but with the periods of time for culture and chemical analysis
extended. Where the experiments compare growth, controls without
additives were run parallel to the experimental cultures. For
biochemical tests, uninoculated medium was used as a control.
[0063] The following examples are given for the purpose of
illustrating various embodiments of the invention and are not meant
to limit the present invention in any fashion.
EXAMPLE 1
[0064] Characterization
[0065] HBB has only been recovered from "sterile" human sources,
i.e., human blood and cerebrospinal fluid (CSF). Because of the
very slow growth of HBB, other organisms would be expected to
overgrow HBB rapidly in cultures from nonsterile sources. To
culture HBB from blood, the blood was allowed to clot, retract and
draw off the serum containing HBB. A remarkable feature of the
bacterium is that it does not get trapped in the clot. The easiest
and most reproducible approach to isolate HBB is to use a sterile
vacutainer-type clot tube with a separator gel. The serum was then
centrifuged so as to obtain the bacteria, or the serum was
aspirated in a sterile manner for culture. Alternatively, blood can
be drawn in a vacutainer-type clot tube without a separator gel and
allowed to clot and retract, and prior to culturing, the tube is
inverted to resuspend organisms. The red cells are allowed to
settle, and the cell-free serum above the cell layer is aspirated
for culture. Cerebrospinal fluid can be cultured via lumbar
puncture in a sealed sterile tube.
[0066] HBB can be isolated from the bloodstream of any individual,
although the degree of infection varies considerably. It has been
recovered from at least 21 normal subjects, over 75 subjects with
"chronic fatigue syndrome" and over 100 subjects with multiple
sclerosis. There is some overlap between the levels of bacteria in
the normal population and those with the chronic fatigue syndrome
and multiple sclerosis, but there is a correlation between elevated
levels of the bacterium and the presence of active symptoms of
these diseases. It has been recovered from the cerebrospinal fluid
of multiple sclerosis patients.
[0067] The fact that HBB is always present in the bloodstream
implies first that the body lacks effective defense against it, and
second, that something critical to its growth is available in a
restricted amount, thus preventing its overgrowth.
EXAMPLE 2
[0068] Culture Method
[0069] Routine culture was done in broth cultures in T-25 cell
culture flasks using either medium L or medium O, or 1/2 strength
medium O or RPMI-1640 cell culture medium with or without
modification. Normally 1 ml of serum was added to 9 ml of medium.
Larger flasks with a proportionately larger amount of serum and
medium are used to produce bacteria in quantity. Modulators might
be added to some flasks in 10.times. or higher concentration
(corresponding to addition of 1 ml or less). When examining the
effect of modulators, control flasks were prepared similarly
without the addition of the modulator. Levels were observed
microscopically (inverted phase microscope, observing the number of
organisms per field crudely or by actual count) after incubation,
typically at several points in time.
[0070] When multiple comparisons were done at once, they were done
in 12 well or 24 well cell culture plates, most often the latter.
With the latter, typically 1 ml of medium inoculated with serum is
added to each well, with modulators added at 10.times. or higher
concentration (0.1 ml or less). When comparisons are done in well
plates, control wells without any additions are included. Levels
were observed microscopically (inverted phase microscope) after
incubation, typically at several points in time. The well plate
cultures were also used for antibiotic sensitivity testing.
[0071] Solid medium with agarose added to the formulations has been
used for specific studies, but it is generally less satisfactory.
The bacteria penetrate 1.5% agarose; at 2% and above they will
remain on the surface, but colonies are not formed due to their
movement over the surface. This type of medium has only been used
for attempts to determine antibiotic sensitivity by Kirby-Bauer
technique. Ordinary bacteriologic agar, as opposed to agarose,
strongly inhibits growth, partly explaining the failure to isolate
the bacterium with conventional media.
EXAMPLE 3
[0072] Culture Conditions
[0073] The bacterium is strictly aerobic, and requires no CO.sub.2.
HBB is temperature tolerant with an optimum growth around
33-37.degree. C., but survives for long periods of time
refrigerated, and can withstand temperatures up to 50.degree. C. in
culture; however, both high and low temperatures retard growth of
HBB (1). HBB can be recovered successfully from serum in vacutainer
clot tubes that have been refrigerated for several weeks. Typically
there is good growth of HBB in culture for a few weeks, then growth
slows and eventually stops, often with degeneration. Subculture of
HBB stimulates further growth; however this process ceases after a
few subcultures. The growth rate of HBB is very slow in comparison
to most bacteria, with a doubling time typically ranging from a few
days to over a week. HBB can remain viable in a sealed culture
flasks for up to about a year, although if the culture system is
modified to stimulate growth, degeneration is more likely to
develop.
[0074] HBB apparently can enter a metabolically inactive state
which is likely a true spore state. The yield of organisms from the
blood varies with the individual and the individual's health, but
typically 1 ml of serum yields 10.sup.6 to 10.sup.8 organisms after
a week's growth using medium O or L; corresponding to about
25.fwdarw.1000 bacteria per high power field in a Corning T-25.TM.
flask using approximately 9 ml of medium.
[0075] Serum from several patients was submitted to a local
bacterial reference laboratory, where no growth was obtained either
aerobic or anaerobic on the various media typically used to grow
bacterial strains from patient samples, although same serum grew
well in above described culture media.
EXAMPLE 4
[0076] Growth Requirements and Modulators
[0077] Bacterial growth requires lactalbumin hydrolysate and yeast
extract. Division of the bacterium is inhibited slightly by
brain-heart infusion, which produces longer bacilli. A sugar is
apparently required, and it has been determined that the following
sugars support growth: glucose, sucrose, fructose, lactose,
dextran, raffinose and meilibose. A combination of glucose,
sucrose, and fructose and/or of lactose and glucose has permitted
isolation of HBB in 100% of subjects tested (>300 isolations).
The addition of pyruvate to growth media produces longer bacilli
with increased motility and increases the amount of slime produced
by the bacteria. In contrast, pyruvate stimulates growth in
1/2-strength medium O or RPMI-1640 when exposed to a grow-light and
especially when in flasks with gas-permeable caps. There are no
known vitamin requirements other than those supplied by yeast
extract. There appears to be no inhibition by methotrexate in the
isolates tested.
[0078] Simple salts are required for growth and growth is
stimulated by several trace metal ions, including manganese (1
.mu.g/ml optimum), copper, tin, iron, mercury, and silver. Growth
appears to be inhibited by zinc, but is or may be stimulated by
stannous tartrate (14 .mu.g/ml), tin ethyl hexanoate (14 .mu.g/ml),
tin caproate (<14 .mu.g/ml), or dibutyltindilaurate (1.4
.mu.g/ml). Growth might also be stimulated by hydrogen peroxide and
(oxidized) arachidonate (2-10 .mu.g/ml).
[0079] There appears to be a slight stimulation with titanium
ethoxide ({fraction (1/14)} saturated), benzophenone ({fraction
(1/14)} saturated), diphenylamine ({fraction (1/14)} saturated),
tetraethylmethylenediamine (14 .mu.g/ml), morpholine (1.4 .mu.g/ml)
and ubiquinone (Q.sub.10) (1/5 saturated). Little or no stimulation
or inhibition was observed by chromium picolinate, silver
proteinate, amantadine, potassium iodide, picolinic acid, borax (14
.mu.g/ml), dichlorophenylenediamine ({fraction (1/14)} saturated),
spermine (7-10 .mu.g/ml), zirconium chloride (1.4 .mu.g/ml),
titanium butoxide ({fraction (1/14)} saturated), resazurin (14
.mu.g/ml), methylene blue (14 .mu.g/ml), fluorescein (14 .mu.g/ml),
L-carnitine (2 .mu.g/ml), phosphorylethanolamine (3 .mu.g/ml),
orotic acid (4 .mu.g/ml), cysteine (20 .mu.g/ml), sodium sulfide
(20 .mu.g/ml), N-acetyl cysteine (20 .mu.g/ml), ammonium sulfate
(20 mg/ml), 2-mercaptoethanol (10 .mu.g/ml), dithiotheitol (10
.mu.g/ml), taurine 20 .mu.g/ml). No effect of light level (1) was
observed when using medium L or medium O and ordinary sunlight or
fluorescent light, but stimulation of growth is observed with
grow-lights or special blue incandescent lights or special bluish
fluorescent lights, particularly with 1/2 strength O or RPMI-1640.
The effect is enhanced when flasks with gas-permeable caps are
used. One isolate tested grew very slowly on a Sabouraud's dextrose
medium (Difco) slant over six month's time at room temperature. No
anaerobical growth was observed in Difco Anaerobic Culture System,
while more than 300 isolates grew well without CO.sub.2.
[0080] HBB has been found to be moderately pH tolerant, growing
over a tested range of 5.8-9.0, with the optimum pH varying with
the strain. There is no significant effect of sodium sulfide (20
.mu.g/ml), n-acetyl cysteine, mercaptoethanol, or dithiothreitol.
Cysteine (20 .mu.g/ml) appears to inhibit growth in some isolates,
methionine (20 .mu.g/ml) appears to do so less consistently. There
is no effect of ammonium sulfate or taurine on the growth. Some
stimulation of growth was observed when alcohols were added (3.3%
ethanol, 3.3% n-butanol, 1.67% isoamyl alcohol). Additionally,
there was no effect on reduction of cell wall-defective forms from
addition of cell wall precursors N-acetylglucosamine,
N-acetyllactoasamine, muramic acid, N-acetylmuramic acid,
diaminopimelic acid, although some stimulation of culture growth
was observed with several of these, particularly
N-acetylactosamine. Oxidative inhibitors had little effect on
growth, such as azide, cyanide and dinitrophenol.
EXAMPLE 5
[0081] Culture Medium
[0082] Dozens of formulae have been tested and several formulae
have been used, based on the medium used for the initial,
successful isolation and subsequent experiments. The following
formula--referred to as medium L--has been developed and found to
give much better performance: 1L of Mitsuhashi-Maramorosch insect
cell culture medium (Sigma) or its components minus the glucose;
namely, CaCl.sub.2: 0.15102 g; MgCl.sub.2 (anhydrous): 0.04695 g;
KCl: 0.2 g; NaCl: 7.0 g; NaH.sub.2PO.sub.4 (monobasic): 0.1739 g;
lactalbumin hydrolysate: 6.5 g; yeast extract: 5.0 g, lactose: 10
g, manganese chloride: 1 mg; plus sodium bicarbonate or another
buffer such as Tris or HEPES. To these components is added 1 L
water, and, while stirring, a small amount of xanthone and
phenothiazine powder should be added. Growth may be improved by
adding sodium arachidonate and lipoxidase (Sigma, soybean origin).
Once dissolved, the medium should be adjusted to pH 7.5, though it
has been found that the optimum pH for differing isolates varies
slightly. The pH-adjusted medium should be sterile filtered or
autoclaved and then stored at 4.degree. C. This medium allows for
growth and maintenance of HBB in culture for at least six months at
4.degree. C.
[0083] An alternative medium, referred to as medium O, replaces the
sugars in medium L, above, with 4 g glucose, 4 g fructose, 4 g
sucrose, eliminating the buffer, xanthone, phenothiazine,
arachidonate and lipoxidase. Medium L generally allows for better
growth of HBB, but with some strains medium O has been better.
[0084] Dilution of medium L or medium O to a half concentration
with water occasionally allows for better growth, with an enhanced
visibility of organisms due to their swelling to a larger size.
However, a problem of precipitate formation developed when using
these diluted media. It was found that diluting the medium with
normal saline instead of water reduced the precipitation but did
not entirely eliminate it. Another alternative culture medium is
RPMI-1640 cell culture medium, as well as several related media
such as Dulbecco's, Iscove's and McCoy's. Greater growth is
achieved with the addition of 5-10 mM pyruvate when grown under a
grow-light.
EXAMPLE 6
[0085] Physical Characteristics
[0086] Morphology: HBB has been found to be extremely pleomorphic
by phase contrast microscopy (1). When not dividing--i.e., under
relatively static conditions including when viewed directly in
blood--HBB appears as a long, flexible, often serpiginous rod, up
to 15 microns or more in length. The ends of the rod sometimes
appear swollen. When dividing, the bacterium is much shorter,
typically becoming a coccus (FIG. 1). The diameter of the cells
vary during the lifecycle, tending to be smaller when actively
growing. At the smallest, they barely can be resolved with a
40.times. objective. HBB is gram variable, not acid fast. No
capsules or flagellae have been observed by normal staining methods
(1), although the Hiss stain does demonstrate extracellular
material that is presumed to be a polysaccharide or
polysaccharide-rich material (slime). There appear to be no
included pigments. The presence of nucleic acids has been confirmed
by staining of fixed organisms with acridine orange, with 4,6
diamidino-2-phenylindole or with the Bac-Light stain (Molecular
Probes, Inc.) and viewing with a fluorescence microscope. The
Bac-Light stain also confirms viability of the organisms.
Transmission electron microscopy (1) of multiple isolates clearly
demonstrates a gram negative wall structure, with a wavy outer
membrane (FIG. 3), possibly with an S-layer, with stainable
granular material between the inner and outer membranes, absence of
flagellae or an outer sheath, with a clearly demonstrated nucleoid
and several types of inclusions, including clear, lipid-like
inclusions that probably represent poly-betahydroxybutyrate and
electron-dense inclusions that are currently not further
characterized. Some micrographs demonstrate linear protrusions from
the surface that could represent pili, conjugation tubes, or linear
phages (FIG. 4). Extracellular material is seen that probably
represents polysaccharide or glycoprotein, but a true capsule was
not seen. Although a spore-like state appears to exist, no true
spores have been demonstrated by electron microscopy. In cultures
grown in RPMI-1640, some of the bacteria seem to lack cell walls,
while in 1/2 strength medium O the wall thickness varies in
specific isolates and is sometimes thicker than normal.
[0087] What appear to be cell-wall-defective forms are often
prominent in culture, and can appear as anything from swollen cocci
to irregularly budding forms resembling budding yeasts to large
spheroids. The tendency of HBB to form defective forms depends in
part on the patient from which the HBB were isolated and in part on
subsequent culture conditions. Defective forms vary over time in
the patient as changes are made in the patient's nutritional status
or as antibiotics are administered, suggesting that the defective
forms may be a function of nutritional limitations.
[0088] Refractile inclusions are often present in HBB, observed by
light microscopy, especially with phase contrast. These inclusions,
like the cell wall-defective forms, also depend on the culture
conditions and the patient source. The inclusions have not yet been
fully characterized. Staining with Loeffler's methylene blue and
toluidine blue shows some metachromasia (2). Alcoholic or glycolic
sudan black stain (3) suggests that the inclusions contain both
polybetahydroxybutyrate and pyrophosphate. Extracellular crystals
are sometimes seen that are bipyramidal and acid soluble,
suggesting calcium oxalate and that some of the intracellular
inclusions could be calcium oxalate. There is apparent spore
formation as demonstrated by Wirtz and Conklin stain (2). No true
capsules are seen by crystal violet-copper sulfate staining (2).
The cell wall defective forms in particular often contain large
highly refractile inclusions. Some of the large, spherical,
highly-refractive forms appear to have thick walls when viewed by
phase contrast microscopy, which again suggests spore formation.
Other evidence suggesting a spore-like state includes relative heat
and radiation resistance (e.g., at 50.degree. C. for seven days)
and persistence of viable organisms that can be subcultured from
flasks that have been in an incubator up to two years without
adding fresh medium.
[0089] Large quantities of an extracellular "slime" or exudate are
produced under some culture conditions. Microscopically, the
exudate can form a fibrillar meshwork that completely obscures the
organisms. The material binds the HBB cells together in clumps.
[0090] Though the HBB cells at times assume a yeast-like
morphology, experiments suggest that the yeast-like cells are
cell-wall defective bacterium, not yeast. For example, there is no
growth of yeast-like forms in Sabouraud's medium (seven isolates,
six months at room temperature), and no suppression by amphotericin
B, miconazole, clotrimazole, sodium undecylate, sodium benzoate,
sodium propionate, or nystatin. There appears to be no formation of
hyphae. Additionally, the organism is not present in culture
immediately after filtration through a 1.2 micron membrane filter,
but "returns" after subsequent incubation. Further, the
"yeast-like" cells are uniformly present and identifiable in all
HBB isolates from blood, although in varying number. Moreover,
there is an apparent transitional form between typical bacteria and
the yeast-like forms. Staining with calcofluor white (0.01% in
water) is weak and inconsistent; similarly, poor staining is
obtained with periodic acid-Schiff's stain and with methenamine
silver (3-4).
[0091] Motility: The motility of HBB in culture is partially
dependent on the culture conditions. Under conditions where growth
is limited but activity is stimulated by pyruvate or when freshly
isolated from the blood, the rods show continuous active
bending--definitely not the type of motility seen with ordinary
flagellae or with spirochetes. Motility appears to be stimulated by
pyruvate. It has not been possible to grow colonies as HBB actively
penetrate 1.5% agarose, medium O for substantial distances. Higher
agar concentrations cause the HBB to spread on the agar surface.
This spreading indicates a possible gliding motility. The fact of
this active motility, coupled with the glycocalyx, may explain why
the HBB do not get trapped in clots during isolation from
blood.
[0092] A few cultures viewed by vital staining of live organisms
with Bac-Light stain (Molecular Probes, Inc.) have shown a
different type of motility in a small percentage of the organisms.
These show much more rapid movement of the type that is expected
with flagellae. The presence of flagellae has not been confirmed by
electron microscopy, but it is possible that the bacterium may be
able to produce flagellae under appropriate conditions.
EXAMPLE 7
[0093] Chemical Reactions
[0094] There appears to be a remarkable paucity of identifiable
chemical reactions. Despite the apparent use of sugars, very little
pH shift occurs over time, and the shift may be either to a
slightly acid or slightly basic. The only reactions that have been
demonstrated consistently are hippurate hydrolysis, a relatively
nonspecific phosphatase/DNAse activity and acetate esterase. A blue
color change is produced in the presence of gold chloride, the
chemistry of which is unknown; a weaker, brown color change is seen
with silver nitrate. HBB make an extracellular exudate or gel. The
exudate appears to be composed largely or entirely of complex
carbohydrate. It is assumed that the extracellular material is very
important to the pathogenicity of HBB in vivo, and that some of the
immune reactions that lead to the diseases observed and associated
with HBB may be against the exudate rather than against the HBB
organism proper. Alternatively, the exudate may protect the
bacteria from serum factors such as complement and antibody.
[0095] The amount of the exudate produced is strongly influenced by
or dependent upon the composition of the culture medium. The
current medium formulae (L and O) have been designed to reduce
slime production. In the original, crude medium, a centrifuged
culture may consist of more than 10% of this gel by volume. In the
L and O media, older cultures of HBB may develop a thin layer of
gel that traps the cells and makes them adherent to the culture
flask. It has been observed that lectins that bind sialic acid,
including wheat germ agglutinin, Maackia amurensis I, and Sambucus
nigra, bind to this material and the bacteria; however, most
lectins binding other sugars, including Maackia II, soybean,
Dolichos bifloris, peanut agglutinin, Phaseolus vulgaris
leukoagglutinin and erythroagglutinin, succinylated wheat germ
agglutinin, Sophora japonicum, Bandeirea (Griffonia) simplicifolia
I and II, Erythrina cristagalli, Pisum sativa, Lens culinaris,
Datura stramonium, Jacalin, Lycopersicon esculentum, Solanum
tuberosum, and Vicia villosa, do not appear to bind.
[0096] The following data summarize observed chemical reactions
(referred to the methods in Refs.1-2). There was no significant
acid or base production over a long time scale (>40 isolates),
no color change with FeCl.sub.3 addition (negative for deamination
of several amino acids (9 strains)), Voges-Proskauer weakly
positive (7 of 8 strains), Urease-deamination (by Nessler's
reagent)-trace positive (7 of 8 strains), Esculin not hydrolysed (9
strains), Hippurate hydrolysed (9 strains), Nitrate not reduced (9
strains), no hydrolysis of ONPG (2 mg/ml) (4 strains),
p-nitrophenyl acetate (1/6 saturated) hydrolysed (4 strains),
though weak reaction seen with control, o-nitrophenyl caprate (1/6
saturated) not hydrolysed (4 strains), p-nitrophenyl phosphate (2
mg/ml) hydrolysed (4 strains), bis-p-nitrophenyl phosphate (2
mg/ml) hydrolysed (4 strains), DNA-methyl green (50 .mu.g/ml)
hydrolysed (4 strains), p-nitrophenylphosphoryl choline (2 mg/ml)
hydrolysed (4 strains), Oxidase positive (3 of 5 strains,
relatively weak), catalase (2 of 2 strains, weakly positive) and no
reaction with triphenyltetrazolium chloride (500 .mu.g/ml) (10
isolates)
EXAMPLE 8
[0097] Antibiotic Sensitivities
[0098] Antibiotic sensitivity is a problem characteristic with HBB:
very simply, HBB are resistant to almost everything, making the
development of a treatment regimen extremely difficult. Some
strains seem to be relatively sensitive to -penicillin G or V, and
are even more sensitive when supplemented with clavulanic
acid-containing Augmentin in vivo, likely due to an inducible
beta-lactamase. Some positive clinical results have been observed
with a combination of penicillin, probenecid, and Augmentin; the
Augmentin is added to provide a beta-lactamase inhibitor. There is
less sensitivity to amoxicillin and generally to the oxacillin
group; however occasional isolates show higher sensitivity to
oxacillin or cloxacillin. Some strains are sensitive to
ciprofloxacin. Relatively little testing has been done with
relatives of cipro, and HBB uniformly has shown resistance to
aminoglycosides, sulfas, and trimethoprim. HBB is resistant to
erythromycin, but some strains show sensitivity to clarithromycin
and/or azithromycin. In addition, HBB appears to be resistant to
most cephalosporins, but a few isolates show sensitivity to third
generation cephalosporins only. A few of the HBB strains have shown
limited sensitivity to doxycycline/minocycline.
[0099] An interesting feature is that some isolates are sensitive
to isoniazid, though there is no reason to think that HBB is a
mycobacterium, which is the only bacterium with which isoniazid is
normally used. The isoniazid sensitivity may be related to the drug
binding to a catalase/peroxidase-like enzyme in HBB as it has been
shown to do in M. tuberculosis; thus, there may be a critical
oxidative enzyme associated with the HBB that is similar to the M.
tuberculosis catalase/peroxidase.
[0100] Sulfhydryl agents sometimes inhibit growth of HBB,
consistent with the suggestion of a critical oxidative feature, but
consistent with metal ion requirements as well. Treatment of
patients with the antibiotics found to be effective on HBB does not
eliminate the organism from the bloodstream; however levels of the
HBB in blood can be suppressed in some patients. These suppressed
levels of HBB correlate with the suppression of pathophysiologic
symptoms. From the data, it appears that antibiotic sensitivity is
not a simple phenomenon but that other factors in the patient,
including nutritional factors, play a role.
[0101] The antibiotic resistance of HBB may be exploited for
selective isolation from nonsterile sites. In addition, HBB resist
all detergents tested; namely, 0.1% bovine bile, 0.1% Brij-35, 0.1%
n-laurylsarcosine, 0.1% NP-40, 0.1% Triton X, 0.1% Tween-20, with
the exception of 0.1% SDS, which does cause lysis. Additionally,
HBB is resistant to 1:1000 benzalkonium chloride saturated
hexachlorophene and 0.05% saponin.
[0102] The following data summarize observed antibiotic and
antifungal sensitivities: amikacin (2 .mu.g/ml)-no effect (1
isolate); 5-aminosalicylic acid (20 .mu.g/ml)-no effect (4
isolates); amphoptericin (0.25 .mu.g/ml)-no effect (1 isolate);
azothromycin (0.25 .mu.g/ml)-no effect (6 isolates), sensitive (7
isolates); cephalexin (2 .mu.g/ml)-no effect (1 isolate);
cefadroxil (4 .mu.g/ml)-no effect (1 isolate); cefixime (2
.mu.g/ml)-no effect (8 isolates), sensitive (5 isolates);
ciprofloxacin (1-2 .mu.g/ml)-no effect (5 isolates), partially
sensitive (1 isolate), sensitive (1 isolate); clindamycin (2.5
.mu.g/ml)-no effect (1 isolate); clotrimazol ({fraction (1/10)}
saturated)-no effect (1 isolate); cloxacillin (2 .mu.g/ml)-no
effect (16 isolates), sensitive (5 isolates), growth stimulated (6
isolates); dapsone ({fraction (1/20)}saturated)-no effect (1
isolate); furazolidone ({fraction (1/20)}saturated)-no effect (1
isolate); gentamycin (2 .mu.g/ml)-no effect (1 isolate); isoniazid
(2 .mu.g/ml)-no effect (24 isolates), sensitive (5 isolates),
growth stimulated (6 isolates); miconazol ({fraction (1/10)}
saturated)-no effect (1 isolate); minocycline (1-2 .mu.g/ml)-no
effect (29 isolates), sensitive (12 isolates), growth stimulated (4
isolates); nalidixic acid (4 .mu.g/ml)-no effect (1 isolate);
nystatin (saturated)-no effect (1 isolate); ofloxacin ({fraction
(1/20)} saturated)-no effect (1 isolate); penicillin V (1-2
.mu.g/ml)-no effect (27 isolates), sensitive (17 isolates), growth
stimulated (4 isolates); primaquine (2 .mu.g/ml)-no effect (31
isolates), sensitive (15 isolates), growth stimulated (1 isolate);
quinacrine (2 .mu.g/ml)-no effect (1 isolate); rifampicin
({fraction (1/200)}-{fraction (1/10)} saturated)-no effect (18
isolates), sensitive (7 isolates); sodium benzoate (50 .mu.g/ml)-no
effect (1 effect); sodium propionate (50-250 .mu.g/ml)-no effect (2
isolates); .sodium undecylate (50 .mu.g/ml)-no effect (1 isolate);
spectinomycin (4 .mu.g/ml)-(1 isolate); sulfamethoxazole-no effect
(1 isolate); sulfapyridine ({fraction (1/10)} saturated)-no effect
(6 isolates), no effect (1 isolate); sulfasalazine ({fraction
(1/10)} saturated)-no effect (6 isolates), no effect (isolate);
tolnaftate (2 .mu.g/ml)-no effect (1 isolate); trimethoprim
({fraction (1/20)} saturated)-no effect (1 isolate); vancomycin (2
.mu.g/ml)-no effect (1 isolate).
EXAMPLE 9
[0103] Sequence Analysis and Classification
[0104] The entire 16S rRNA gene (Rb 16S rRNA: SEQ ID NO: 1 and 58
16S rRNA: SEQ ID NO: 2), most of the 23S rRNA gene (Rb 23S rRNA:
SEQ ID NO: 3 and 58 23S rRNA: SEQ ID NO: 4) and most of the
intergenic spacer region between 16S and 23S (SEQ ID NO: 5) have
been collected. Preliminary sequence data of a gene encoding a
putative multidrug resistance protein is also shown (SEQ ID NO: 6).
These data were produced from DNA isolated from cells cultured
using either {fraction (1/20)} O medium or modified RPMI
medium.
[0105] For the 16S sequence data, DNA was amplified using both 16S
rRNA universal and specific primers designed from literature
searches (GAGTTTGATCCTGCTCAGAACGAACGCTGG, SEQ ID No: 7 and
CATCGGCATCCCCTTGGACGCCG- ACCTAGTG, SEQ ID No: 8). The 16S sequence
of 1208 base pairs (bp) was obtained from a control sample and has
been completed in both directions with overlaps (SEQ ID NO: 1).
Different isolates produce DNA that aligns with different strains
of Methylobacterium. Methylobacterium is a chlorine-resistant,
pink-pigmented bacteria isolated from a number of sources including
water and soil. This gram-negative rod was identified as a member
of the genus Pseudomonas on the basis of physiological traits but
has been reclassified. Recently there have been a number of
Methylobacterium isolated from clinical specimens.
[0106] The strain having the most prevalent homology (99%) compared
to the presently disclosed Rb 16S rRNA sequence is Methylobacterium
organophilium with a difference by only 5 bases (1184 bp out of
1189 bp are the same). The remaining 19 bp of the 1208 bp are not
found in the published sequence from GenBank. Other strains
sequenced from different patients have been similar, however, not
identical to Methylobacterium organophilium. These sequences are
similar to strains 05, strain 15 and strain 73. The second 16S rRNA
sequence obtained from a patient (namely 58 16S rRNA, SEQ ID NO: 2)
contains 1502 bp and is most similar to Methylobacterium sp.
(strain F05). The published sequence for Methylobacterium sp.
(strain F05) is 1405 bp long or approximately 100 bp shorter than
the presently disclosed 58 16S rRNA sequence, wherein the sequence
of 58 16S rRNA of HBB is 99% identical to strain F05 (1398 bp out
of 1403 bp are identical). The two sequences Rb and 58 for 16S rRNA
gene differ from each other by 4% with different base pairs from
1167 to 1208.
[0107] The significance of this strain variation between control
samples and patients has not yet been established. It is possible
that some strains are pathogenic and others are not. Whether the
sequenced HBB is a modified form of these listed strains is
unknown. However, HBB appears to be different morphologically and
in terms of growth requirements from a strain obtained from ATCC.
The fact that HBB is able to survive in the blood stream would
require some modification from the wild-type strain. Analysis of
the genome may demonstrate the presence of a pathogenicity
island(s) or other transmissible genetic element(s). These elements
may have been acquired by Methylobacterium through horizontal gene
transfer during microbial evolution leading to the creation of
HBB.
[0108] The 23S rRNA sequence obtained to date includes 2542 bp (Rb
23S rRNA: SEQ ID NO: 3). About 3/4's of the length has been
sequenced in both directions, however there are areas where the
sequence abuts without overlap. This sequence, aligns with about
90% similarity to the 23S rRNA gene for Rhodopseudomonas palustris,
which is the most closely related alpha proteobacterium with
published data on the 23S gene. Sequence data from a control
sample, Rb 23S rRNA (SEQ ID NO: 3) and from a patient sample, 58
23S rRNA (SEQ ID NO: 4) were obtained. The two sequences Rb and 58
for 23S rRNA differ from each other by 5% (1903 bp out of 1996 bp
are identical) with 6 gaps. Primer pairs used for 23S rRNA PCR
reactions were constructed: CCGAATGGGGVAACCC (SEQ ID No: 9) and
TCGACCAAGAGRRGCTTT (SEQ ID No: 10); TAGCTGGTTCTCYYCGAA (SEQ ID
No:11) and GGCATTGAAGCCCTCTTCC (SEQ ID No:12); AAACCGACACAGGTRG
(SEQ ID No:13) and CGACTTYCGTAGATTC (SEQ ID No:14) (Ludwig et
al.).
[0109] The intergenic spacer region (IGS) found between the 16S and
the 23S gene was obtained from a control sample and sequenced (Rb
IGS: SEQ ID NO: 5) using primers designed from the two above
sequences (ACGGTAGGGTCAGCGAC, SEQ ID No:15 and CCTCCCAGCTTCCACCC,
SEQ ID No:16). It contains 836 bps that align poorly to a number of
IGS genes. There are very few homologies in the database with
Bradyrhizobium japonicum, another alpha proteobacterium being the
most closely related. The area of the bacterial genome is not well
conserved between species and should provide ample opportunities
for the design of very specific primers for DNA based diagnostics
including PCR and in situ hybrizations.
[0110] A sequence produced from a set of PCR primers specific for a
family of Methylobacterium genes was obtained from a control sample
(namely drug resistance protein gene, SEQ ID NO: 6). The primers
used are ATGTCCTGCGTGTCTGCA (SEQ ID No:17) and GTACTAGTCCAGCGTGTC
(SEQ ID No:18). This sequence does not match any known sequence in
the database, but when translated to protein is a close match for a
multidrug resistant protein from Bradyrhizobium japonicum.
[0111] The 16S rRNA sequence (SEQ ID NO: 1) establishes the
bacterium as a member of the genus Methylobacterium. The reported
members of this genus are able to grow on media which provide the
carbon source from methyl compounds such as methanol, formaldehyde,
formate, methylamine, etc. They are facultative in their carbon
sources, and can utilize sugars and a variety of nitrogen sources.
Significant growth of HBB has not been obtained using the various
media used for continuous culture of the defined Methylobacterium
species. Addition of various methyl compounds such as methanol,
methyl acetate, trimethylamine and trimethyl phosphate to 1/2
strength medium O or RPMI has produced minimal or no stimulation of
growth. Methylobacterium has on rare occasions been reported from
immunosuppressed humans, but is not ordinarily isolated from human
or mammalian sources. Instead, it is ordinarily isolated from
environmental sources such as bodies of water. Thus HBB
significantly differs from any known Methylobacterium isolates
[0112] Very little is known about the strain differences of HBB.
There appear to be consistent morphologic differences from
different patients. Antigenic characterization has not been
examined. Patterns of antibiotic sensitivity suggest that certain
antibiotic sensitivities may be more frequent with certain
diagnoses.
EXAMPLE 10
[0113] In situ Hybridization
[0114] In situ hybridization was performed using probes specific
for Rb 16S rRNA of Methylobacterium. Two probes were synthesized
(TCGCAGTTCCACCAAC, SEQ ID No:19 and CTGTGGTTGAGCCACA, SEQ ID
No:20), each of which are labeled on the 5' end with the
fluorescent dye, rhodamine. Two other probes from the set of
universal probes designed to bind all bacterial species were
labeled with the fluorescent dye, fluorescein. When hybridized with
cells from HBB culture, bacteria were bound by the probes from Rb
16S rRNA and were fluorescent orange indicating they were bound by
the rhodamine-labeled probe (FIG. 5). E. coli was used as a control
bacteria and did not bind the Methylobacterium-specific probe. A
third dye, 4', 6'-diamidino-2-phenylindole.2HCl (DAPI), was used to
detect all cells containing DNA (FIG. 6). These results were
consistant with PCR sequencing results and indicate that HBB is
closely related to Methylobacterium.
EXAMPLE 11
[0115] Clinical Indications
[0116] Experiments have been conducted investigating the
correlation of the presence of or increased presence of HBB with
the clinical symptoms of three autoimmune diseases, and the results
of instituting an antibiotic regimen in patients. Eighty patients
were seen, 66 of whom received antibiotics long enough to allow
clinical evaluation. The clinical evaluation was based principally
on reported symptoms, although there was also objective evidence of
improved function for some multiple sclerosis patients. All
patients were cultured repeatedly to determine levels of HBB. The
antibiotic treatment regimen was based on antibiotic sensitivities
done on a pilot series of cultures and on the known properties of
the organism, which clearly requires long-term, continuous
treatment to address its very slow growth rate. This approach
differs from usual treatment regimens for other bacterial
infections. Neither the subjects nor the investigators were blind,
and there was no untreated or placebo-treated control group.
[0117] Antibiotic treatment consisted of one or more of the
following three antibiotic regimens: (1) penicillin V, 500 mg four
times a day, plus probenecid, 500 mg four times a day, plus
Augmentin 500 mg four times a day; (2) ciprofloxacin, 500 mg three
times a day; or (3) isoniazid, 100 mg three times a day. Most
patients received a combination of two of the regimens
concurrently. Further, if a certain regimen did not appear to be
effective or efficacy declined over the treatment period, the
patient was often switched to a different regimen. None of the
three regimens appeared to be significantly better or worse than
the other two.
[0118] The 66 patients for whom follow-up was adequate are
summarized below by diagnosis: Chronic fatigue syndrome: 41
patients total; 30 patients improved, 11 patients did not improve.
Reduced levels of HBB were seen in all improved patients and HBB
levels were not reduced in unimproved patients. Multiple sclerosis
(typical): 7 patients total; 4 patients improved; 3 patients did
not improve. Two of the four improved patients had marked
functional improvement, for example, were able to walk with a cane
where a wheelchair had been previously required. There were reduced
levels of HBB in three of the improved patients, with an
insufficient culture follow-up in the remaining improved patient.
There was no reduction in HBB in the patients that remained
unimproved. Atypical multiple sclerosis: One patient was tested and
was markedly and objectively improved for five months before
developing antibiotic resistance (resistance later documented with
sensitivity testing). There was a reduced level of HBB during the
period of improvement and a marked increase in HBB during relapse.
Borderline diagnosis (either chronic fatigue syndrome or multiple
sclerosis: Three patients were tested. All three patients improved,
with reduced levels of HBB. Fibromyalgia (pure or associated with
chronic fatigue): Three patients were tested, and all patients
improved, with corresponding, reduced levels of HBB in the blood.
Arthritis (rheumatoid or other): Five patients were tested; four
improved and one did not improve. All improved patients
demonstrated reduced levels of HBB in their blood, with no
reduction in the level of HBB in the unimproved patient. Behcet's
syndrome: One patient was tested and did not improve, and there was
n o reduction in the level of HBB in this patient's blood.
[0119] The striking feature of the treatment of these patients was
that there was a 100% correlation between clinical improvement in
the patient and reduction of the levels of HBB in the patient's
bloodstream. This was true even though the improvement resulted
from treatment with antibiotics--despite these illnesses never
having been associated with a bacterial cause in the prior art.
[0120] Subsequent to this study, laboratory support has been
supplied to various physicians treating patients. Results have been
similar, except that the percentage of responding multiple
sclerosis patients has appeared to be lower than the level that was
seen in the small, initial group. Furthermore, one collaborator who
has been treating chronic fatigue syndrome patients with penicillin
and probenecid has reported about 100 patients who are in complete
remission from symptoms. This correlates to about a 20% cure
rate.
[0121] Clearly, the improvement in symptoms correlates with
reduction in HBB in the bloodstream. Long-term follow-up has shown
that for some of the responding patients of the original study,
antibiotic resistance has developed, though each patient benefited
greatly overall from treatment. Lately, study has also demonstrated
an important phenomenon, that is, frequently when antibiotic
resistance is present, the antibiotics can actually stimulate the
growth of the bacteria. The mechanism for this effect is unknown,
but it has been seen repeatedly both in culture and in patients.
The increases in HBB when resistance is present have correlated
with increased symptoms.
EXAMPLE 12
[0122] Treatment of HBB-Associated Disease
[0123] Most treatment information has been developed primarily from
uncontrolled patient trials; however, in vitro sensitivity testing
is routinely being done. One skilled in the art, o n the other
hand, recognizes that, as with all pharmaceutical preparations, in
vitro and in vivo results do not correlate completely and dose and
dosage regimens depend upon the nature of the disease, the stage of
infection, the characteristics of the particular pharmacologic
agent--e.g., its therapeutic index, the patient, the patient's
history and other factors. A regime or schedule is continued to
optimize effectiveness, balanced against negative effects of
treatment. Sensitivity tests against the agents that show promise
at this time are run routinely, and it is urged that this practice
be continued in further testing, as patients have had contrary
therapeutic results when treated with an antibiotic to which their
isolate was resistant.
[0124] Several treatment regimens have been thoroughly tested and
have shown value both when testing in vitro and in trials on
patients in vivo. These regimens produced actual complete relief
from the symptoms of chronic fatigue syndrome patients in a few
months in a substantial minority of patients. In these trials, many
patients experienced a significant improvement in symptoms but did
not experience complete relief from symptoms, at least over the
testing period of a few months. Additionally, there is long-term
testing data with a few patients that suggests that long-term cure
may occur after withdrawal of antibiotics even though organisms
remain. Many patients do develop antibiotic resistance with time,
and a proportion of patients do not respond at all--presumably a
result of antibiotic resistance or of a rate of growth of the
bacteria in these patients that outstrips the antibiotics.
[0125] With multiple sclerosis patients, less than 50% have
responded positively to treatment. Further, complete recovery
cannot be expected as some nervous system damage may have accrued
prior to treatment and cannot be reversed; however, about 20% of
patients had a definite improvement in motor function. It is yet
undetermined whether the responsive individuals will remain
improved indefinitely, or whether progression of the disease is
altered after cessation of therapy, though several patients have
maintained the improved level of function for several years.
[0126] With HBB, "true" cure--i.e., elimination of all
bacteria--has not been achieved with the antibiotics that are
available; and indeed, may not need to be as HBB appears to be
present at some level in all human beings. However, control of HBB
for a period of time long enough to allow healing to occur and/or
the immune system to recover is clearly possible.
[0127] The following antibiotics have shown value in vivo to date;
however the present invention should not be limited to the
following treatment regimes as one skilled in the art of
pharmacology would recognize a host of alternatives Penicillins
with probenecid: Clinical responses and culture tests suggest that
response is best to the penicillin G/penicillin V spectrum.
Clinical responses and failures indicate that HBB makes a
penicillinase or general beta-lactamase, likely slowly inducible.
Thus, penicillin alone is not effective for a sustained response in
most patients, although it gives short-term response in many. The
penicillinase can be inhibited by simultaneous administration of
Augmentin (Clavulin; amoxicillin+clavulanic acid) or a
penicillinase-resistant penicillin. The current recommendation for
adults is a combination of oral penicillin V 500-1000 mg.,
Augmentin 500 mg., and probenecid 500 mg., all taken four times a
day. A few patients have done better with dicloxacillin, but the
majority of HBB isolates shows poorer sensitivity to this group of
penicillins.
[0128] An alternative therapy to penicillins with probenecid,
above, is administration of Ciprofloxacin in divided doses of 1-2
grams/day. The suggested dose for most adult patients is 500 mg
three times a day. The effect of adding probencid to the regimen
has not been tested; the probencid may reduce excretion and
increase penetration of the CNS. If probenecid is given, it should
be given at an approximate level of 500 mg three or four times
daily. Since probenecid slightly raises blood and appears to raise
the cerebrospinal fluid (CSF) level of ciprofloxacin, the dosage of
ciprofloxacin may be reduced if probencid is used, perhaps to 250
mg three or four times daily. The effect of giving higher doses
less often, e.g., 750 mg twice a day has not been tested. In
theory, with HBB, it is better to give smaller, more frequent doses
to maintain continuous blood levels. At a minimum, ciprofloxacin
administration should be continued for at least two months, with
continuous assessment of HBB levels and clinical response. Such
treatment is very expensive and it is contraindicated in children.
For patients who do not tolerate ciprofloxacin, another drug of the
same group may be a viable alternative.
[0129] Yet a third alternative for treatment is administration of
Isoniazid, 100 mg given three times a day. It is unclear why HBB is
occasionally sensitive to Isoniazid, as it is not related to
Mycobacterium. However, this anomaly is advantageous as Isoniazid
is inexpensive and well-tolerated. Isoniazid must be administered
with vitamin B-6 (pyridoxine) as it binds the vitamin in vivo and
can lead to vitamin deficiency with peripheral neuropathy.
[0130] Third-generation cephalosporins appear to be an alternative;
however, HBB is resistant to first- and second-generation
cephalosporins. At present, sensitivity testing has been done with
cefixime, and the normal maximum sustainable dosage is recommended.
CNS penetration may be unreliable, and at least to this point,
third-generation cephalosporins appear to be less efficacious than
other drugs. Further, some data indicates that third-generation
cephalosporins may actually stimulate growth in culture.
[0131] Azithromycin and clarithromycin are additional alternate
antibiotics. HBB is resistant to erythromycin. Data is limited on
the results of treatment with azithromycin and clarithromycin, so
an in vivo dosage has not been established. Clarithromycin appears
more effective in culture, but in vivo, the tissue penetration
capabilities of azithromycin appear to be important. However, as
with third-generation cephalosporins, some data collected to date
suggests that azithromycin and clarithromycin often stimulate
growth. There appears to be a synergistic effect with a few
isolates upon treatment with chloroquin and hydroxychloroquin.
[0132] Minocycline or doxycycline are alternate antibiotics;
however, sensitivity to these drugs is less common than to the
drugs listed above, and minocycline or doxycycline do not kill the
bacteria so much as slow their growth. Doses have not been
established; although, the normal maximum sustainable dosage is
recommended.
[0133] Sporadic sensitivity to other drugs has been noted, but so
far results do not appear to be consistent enough for general use,
though routine experimentation is likely to result in the
identification of additional effective antibiotics. Aminoglycosides
and sulfa-trimethoprim have no effect on HBB in culture. Due to the
requirement for long-term treatment, drugs that cannot be
administered orally have not been studied. Moreover, use of
nutritional adjustments in combination with the antibiotics may
generally improve results.
[0134] As a rule of thumb, therapy should be continued for a
minimum of two months, with constant monitoring of HBB levels and
clinical response. Longer treatment likely is needed in the
majority of patients, particularly for MS patients. Treatment is
complicated by the fact that the organism has so-called "resting
forms" or possibly true spores and makes cell wall defective-forms
which resist antibiotics in normal treatment schedules, requiring
continuous high-dose antibiotics over a long period of time.
Cautious withdrawal of drugs after several months after improvement
has leveled off is recommended. Any treatment regimen should take
into consideration drug risks known and published in the art, as
well a s in vitro sensitivities, clinical tolerance and economic
considerations.
[0135] A factor in the long-term effect of treatment appears to be
due to the restoration of efficacy of the patient's immune system.
Symptoms likely are the result of immune reaction directed against
HBB or a product thereof. The above regimens and others are likely
to produce Candida overgrowth. These yeasts can usually be
controlled with oral nystatin 500,000U three or four times a day,
Nizoral 200 mg once a day, or Diflucan 100 mg once a day. Nystatin
only treats the GI tract, is appropriate for most males, and only
may need to be prescribed for a few weeks. Nizoral produces a
general effect and is suggested for women. Diflucan is one
alternative to Nizoral and is safer, possibly more effective, but
is more expensive.
[0136] Most responding patients have been found to have a
pronounced Jarisch-Herxheimer reaction for the first few days or
weeks of therapy. In theory, the Jarisch-Herxheimer reaction is due
to the rapid death of organisms releasing internal contents that
stimulates a short-term hypersensitivity response, and is
presumably a sign of antibiotic sensitivity. The reaction should
not be mistaken for a drug reaction or for an adverse response to
therapy. The patients have been found to be more comfortable if a
steroid is co-administered for a few days. Indeed, steroids may
have an additional benefit as they may inhibit production of
compounds that may stimulate growth of the bacteria. During the
initial phase of treatment, depression may be a problem, and a few
patients have complained of muscle cramping.
[0137] All drugs may produce allergic reactions, and some patients
with increased levels of HBB have a much higher than usual
incidence of drug allergies. Ciprofloxacin has been linked with
rare but severe reactions such as liver or marrow failure and can
be a problem to the kidney due to dehydration. Ciprofloxacin also
produces photosensitivities and produces necrosis of cartilage in
children and is contraindicated in children for this reason; there
is a risk of damage to cartilage with long-term use in adults as
well.
[0138] Probenecid is contraindicated in patients with kidney
problems due to problems with dehydration. Likewise, Isoniazid
produces rare severe hepatitis or very rare marrow failure, more
often in older patients. GI complaints are occasionally seen with
any antibiotic and can be severe enough to require a change of
therapy. Pseudomembranous enterocolitis is a remote possibility. It
is recommended that the CBC and liver, hepatic and renal functions
be monitored periodically, initially every 3-4 weeks and whenever
there are adverse symptoms. A few patients have shown unpredictable
symptoms such as chest pain and tachycardia. It is likely that
there is a low-grade myocarditis present in a few patients that is
exacerbated with the antibiotics. Propranolol may help these
symptoms as well as lessen the headaches that some patients
experience. Some patients who do not tolerate therapy well have
shown positive response utilizing a regime which starts with low
dosage of the drug, continuing with gradual increases in dosage.
When multiple drugs are being given, it may help to identify
adverse and positive drug reactions if the drugs are started
sequentially. If a patient does not respond after a reasonable
trial (1-2 months) or if the patient's condition declines, the
regime should be discontinued. There have been rare cases where
patients did not respond until more than a month of treatment, but
studies thus far show overwhelmingly that patients respond within
weeks of treatment.
[0139] Ongoing inflammation and/or stress promotes the growth of
HBB, related in part to release of arachidonate derivatives. NSAIDs
have a limited role; particularly ibuprofen which produces
symptomatic improvement and some reduction in bacterial levels.
Control of stress and other infections is an adjunct to treatment.
It is likely that a major site of infection with HBB is the mouth,
especially the gingiva. Many patients give a history of symptoms
beginning shortly after dental problems, especially abscesses and
root canals, or of flareups when dental problems developed. It is
important that the patient be surveyed to be sure that there are no
lurking dental infections and that any problems that are present be
corrected.
[0140] An important aspect of the present invention is that HBB is
present in all "normal" persons and appears in increased levels in
persons with nonspecific symptoms or individuals having any one of
a number of clinical disorders including chronic fatigue and immune
dysfunction syndrome (CFIDS), fibromyalgia, and several autoimmune
disorders such as multiple sclerosis, lupus erythematosis, and
rheumatoid arthritis. There is a definite positive correlation
between reduction in HBB counts in patients on an appropriate
antibiotic regimen and significant improvement in symptoms.
[0141] For many patients, antibiotic therapy may not be appropriate
or may not be possible, but it may be possible to partially control
the level of the bacteria by nutritional measures. As is known
generally, persons who are ill should attempt to maintain good
nutrition. Some of the following guidelines are suggestions that
have been previously made in the CFIDS literature; and many have
been derived from direct experiments in culture.
[0142] Many patients have observed that high-dose vitamins make
them feel better. There is no evidence that the bacteria are
dependent on externally-provided vitamins; thus, there is no reason
not to take them. A good multivitamin containing higher than normal
levels of the B vitamins, especially vitamin B-6 and folic acid,
vitamin C, and possibly vitamin E is recommended. Vitamins A and D
can produce toxicity at significantly higher than normal levels so
an excess should be avoided, but some supplementation may be good.
Of course, very large doses of certain of the B vitamins, vitamin C
and vitamin E also can be toxic, so there is a limit to how much of
any of the vitamins should be taken. It is recommended that
vitamins with multiple minerals be avoided, as certain of the
minerals discussed below may be a problem. Some patients have
reported improvement in symptoms from vitamin B-12 injections.
These are usually administered by a physician. Supplementation with
niacin (or nicotinic acid, not niacinamide or nicotinamide), B-6
(pyridoxine), B-12 and folic acid may be particularly important in
persons using cysteine (cystine) supplements.
[0143] The CFIDS literature suggests that there may be improvement
from injections of magnesium sulfate and oral zinc supplements.
Results obtained in studies in the present invention indicate that
increased levels of magnesium have little effect on the growth of
the bacteria. Presumably the reported effects of magnesium are due
to a secondary effect on symptoms. Supplementation with oral
magnesium is a possibility, but dosage should be monitored,
particularly if the magnesium is not taken in combination with
calcium. Magnesium compounds are laxatives at oral doses over a few
hundred milligrams at a time.
[0144] Testing indicates that the growth of HBB is stimulated by
compounds of copper, manganese, tin, iron and possibly other metals
including aluminum, silver and mercury, suggesting that mineral
supplements containing these minerals, as well as foods that are
high in these minerals, should be avoided. It is likely that copper
and iron are the most significant.
[0145] Both stress in general and inflammatory processes in
particular stimulate the growth of the bacteria. Inflammation
results in the release of several copper and manganese-containing
enzymes from white blood cells and raises blood copper levels.
Inflammation, stress, and certain hormones all elevate blood levels
of copper, mostly bound to a specific, protein; thus a reduced
intake of foods high in copper, particularly shrimp, lobster, crab,
crayfish, and liver is indicated. On the other hand, an increased
intake of zinc is recommended, as zinc suppresses the growth of
these bacteria in culture and increased zinc intake competes with
some of the other minerals that have been indicated as being a
problem. Zinc also has a beneficial effect on the immune system in
general, which is why it was originally recommended. The
recommended amount is 25 mg a day of zinc as zinc sulfate or an
equivalent zinc compound for the average adult. Amounts over that
can lead to overdosage. Overdosage of zinc produces anemia and
weakness because excessive reduction of available copper interferes
with the body's ability to make enzymes that supply the bulk of the
energy to cells in the body. Further, zinc can interfere with the
incorporation of iron into the hemoglobin in red blood. Again, zinc
levels, as well as iron levels in the blood, should be monitored
closely. An additional reason for monitoring iron levels is that
there is a possibility that iron stimulates bacterial growth. The
best test for total body iron stores is the serum ferritin, and it
is recommended that the iron level be kept at the lower end of the
normal range.
[0146] Another trace metal that has been suggested as beneficial in
the CFIDS literature is chromium. Testing indicated no inhibition
of bacterial growth by chromium, and, in fact, indicated that
chromium possibly stimulates some strain. Chromium is known to be
needed for insulin activity. Further, many CFIDS patients have
sugar cravings and increasing fatigue several hours after eating.
Chromium antagonizes this state. Since increased zinc will probably
antagonize absorbtion of chromium, some chromium supplementation
along with zinc is probably needed, at a dosage of 100-200 mg per
day. Chromium also reduces the risk of arteriosclerosis.
[0147] Further, CFIDS literature suggests that certain
sulfur-containing compounds may be beneficial. Garlic in particular
has been mentioned, and one patient consumed large amounts and was
convinced that it helped his symptoms. The compound that gives
garlic its characteristic odor is one of these sulfur-containing
compounds. These compounds react with several metals within the
body, including copper, and therefore may be complementing zinc.
The active compounds have a substantial odor; however, the garlic
powders and oils that have been deodorized may lack the active
sulfur compounds. Onions and other relatives of garlic may have the
same benefits.
[0148] Experiments on the effect of sulfhydryl reagents suggest
other compounds may have at least as much value as garlic, but also
suggest that some sulfhydryl compounds may sometimes be
contraindicated. The effects of 1-cysteine (cystine), an amino acid
that is one of the basic building blocks of proteins, has been
studied. The amount of cysteine in the diet affects the
availability of other amino acids through various metabolic
interactions. Increased cysteine clearly suppresses growth of some
strains of HBB in cultures, but stimulates other strains. Cysteine
is a minor component of almost all proteins, but is present in
quantity in only a few sources. A daily cysteine (cystine)
supplementation of 500 mg/day is suggested--not higher--and only in
those patients whose isolate shows inhibition by cysteine in
culture. Additionally, cysteine (cystine) should not be taken at
the same time as zinc, calcium, or magnesium supplements, as that
might interfere with the absorbtion of one of the metals.
[0149] In rare individuals the kidney has a problem handling
cystine, and it is excreted in amounts so high that the cysteine
crystallizes in the urine. Thus, supplementation could lead to the
formation of kidney stones. Persons with a diagnosis of cystinuria
should not take supplemental amounts, and microscopic urinalysis is
recommended after treatment with supplements. Maintenance of good
hydration will help with this potential problem.
[0150] The CFIDS literature suggests that taurine may be
beneficial. In the testing to date, taurine has no effect on
cultures. In the body cysteine is converted to taurine. Taurine may
be inhibiting the breakdown of cysteine.
[0151] HBB growth is somehow tied directly to the inflammatory
process. Isolates of the bacteria from patients who have been
stressed, are ill, or who have active inflammatory processes, show
not only greater numbers of organisms, but also display organisms
with a much greater growth potential in culture which subsides over
time. Apparently the growth of the bacteria is affected by the
availability of derivatives of unsaturated fatty acids that are
produced as part of inflammation.
[0152] The critical material(s) for HBB growth are presumably
derived from an unsaturated fatty acid known as arachidonic acid
that is released as one of the primary products in the inflammatory
process. Arachidonic acid is a normal and necessary component of
the body and can be derived from the diet directly or synthesized
in the body from other unsaturated omega-6 fatty acids. Many
antiinflammatory drugs such as aspirin and its relatives block the
conversion of arachidonic acid to certain compounds that are
effective mediators of inflammation. Unfortunately, these drugs
block some of the possible conversions but not all, and
administration of most drugs in this class has not produced much
effect on HBB or the symptoms of the patients.
[0153] Steroids partially block the release of free arachidonic
acid. Steroids have been used for a long time in the treatment of
various "autoimmune" disorders including lupus erythematosis,
rheumatoid arthritis, and multiple sclerosis, all of which are
related to HBB. However, steroids are not a cure-all, and there are
many complications and side effects associated with their use, nor
do they block the conversion to arachidonic acid completely. It is
possible that the beneficial effect of the steroids is related
directly to suppression of growth of the bacteria, rather than
merely suppressing inflammation.
[0154] Ibuprofen may partially block this process and its use at a
moderate level should be considered, taking into consideration the
risks of long-term use. In any case, obviously, the first
consideration in treatment should be to control any treatable
inflammatory processes that exist in the body and to control
infection and inflammation in general, no matter what the source.
This includes a general control of stress. It has been
well-documented that CFIDS and MS patients tend to worsen in
general as physical or mental stress or illness occurs.
[0155] Another dietary adjustment that can be made to decrease the
amount of arachidonic acid that is available to be released in the
body is to restrict the dietary intake of fatty acids. Fat intake
in general should be reduced, particularly the intake of most
cooking oils and margarines. Additionally, CFIDS literature
suggests that supplementation with carnitine helps some patients.
Carnitine plays a role in the metabolism of fatty acids, so it may
affect the availability of arachidonic acid. At this time,
carnitine supplements are not recommended.
[0156] A large portion of the patients tested are partially or
completely resistant to the available antibiotics. In general,
these patients have the highest levels of bacteria and their
bacteria show the greatest growth potential in culture. Nutritional
manipulation to slow the growth of bacteria based on dietary
supplementation with vitamins (increased B vitamins, C, and E but
not A and D), zinc 25-50 mg/day, chromium 100-200 mcg/day,
sulfhydryl compounds such as cystine 500 mg/day and garlic,
appropriate sources of non omega-6 unsaturated fatty acids (at
least 5000 mg per day), elimination of undesirable foods,
particularly those containing high levels of copper and omega-6
unsaturated fatty acids, and reduction of iron levels may convert
some of these untreatable patients into responders or at least will
reduce the severity of their clinical problems.
EXAMPLE 13
[0157] Detection and Diagnosis
[0158] HBB can be quantified to diagnose disease and evaluate
efficacy of course of antibiotic treatment. These procedures are
those known in the art and include uses of antibodies and DNA
probes, Western and Southern blots, PCR and the diagnostic kits
therefor. First, organisms can be quantified by culture. This has
been done successfully by microscopic counting of organisms within
a field after growth. If a means is found to immobilize the
bacteria to grow isolated colonies, counts will be more accurate.
Quantification is used to follow the progress of treatment and to
correlate HBB levels with specific disease syndromes.
Quantification is used also for antibiotic sensitivity
measurements, comparing the level of growth with antibiotic
treatment to the level of growth without treatment. Such antibiotic
sensitivity measurements establish the basis for the clinical
choice of antibiotic drugs.
[0159] In addition, ELISA may be performed with at least two
purposes in mind--ELISA for detection of serum antibodies and an
ELISA for quantification of organisms. ELISAs may be performed
using whole, intact or disrupted HBB or one or more specific
components of HBB prepared by purification or by recombinant
production. Such components are bound to the wells of a plate or to
another solid support. A dilution of the serum is incubated with
the immobilized component attached to the solid support. The solid
phase is washed and incubated with one or more secondary reagents
ultimately linked to an enzyme that binds to the antibodies
(immunoglobulins) in the serum. The solid support is treated with
an enzyme substrate to produce a detectible and quantifiable
reaction that is read with a spectrophotometer or similar
instrument.
[0160] For quantitation, a solid support would be coated with
disrupted bacteria or bacterial components including recombinant
components, unmodified or modified, incubated with serum and then
with a tagged anti-human immunoglobulin. An enzyme reaction would
produce a quantifiable product. The level of antibodies in general,
or the level of antibodies against specific bacterial components
that correlate with specific disease entities are used to establish
diagnosis for entities that are currently defined on the basis of
their clinical presentations. For detection of serum antibodies,
solid supports are coated with a specific antibacterial antibody
and incubated with serum. This binds bacteria to the solid phase.
The plate is incubated with tagged antibacterial antibodies and an
enzyme reaction produces a quantifiable product.
[0161] Polyclonal and monoclonal antibodies are used in multiple
ways. Antibodies are tagged in a variety of ways and used to
identify organisms in tissues and to quantify directly organisms in
fluids--including blood--for instance via laser flow cytometry,
allowing faster and more accurate quantitation.
[0162] An additional technique well known in the art is the use of
Western blots. In this procedure, disrupted bacteria are
electrophoresed in a system such as SDS-PAGE, separating bacterial
protein components in a reproducible manner. The proteins are
blotted onto a suitable support, such as nitrocellulose or a nylon
membrane, and incubated with serum. A tagged antihuman
immunoglobulin is reacted with the blot and an enzyme reaction
produces a visible product in areas of serum binding. The pattern
of reaction is anticipated to correlate with specific
diagnoses.
[0163] Probe-based tests directed against specific DNA or RNA
sequences can be used both qualitatively and quantitatively. DNA or
RNA probes are tagged and hybridized directly with nucleic acids
extracted from samples, analogous to the use of antibodies above.
DNA amplification methods such as PCR may be used to increase the
sensitivity of the test to identify the presence of very low
numbers of organisms. Southern and Northern blots are used
routinely, and the probes may be attached to solid phases for
automated screening. Specific probes and mutational analysis are
used to identify specific strains that may have particular disease
associations. Finally, any of the above methods can be packaged
into a diagnostic kit for convenient use.
EXAMPLE 14
[0164] Uses of Engineered HBB
[0165] The whole HBB bacterium or components thereof also are used
to create a vaccine, with modifications to make the bacterium
sufficiently immunogenic. The procedures in Example 13, as well as
a vaccine are employed using cloned or modified (engineered)
proteins, DNA or RNA. As is known in the art, certain modifications
have particular advantages in particular situations. Further,
modified organisms are used to replace pathogenic strains in the
body with strains that do not elicit disease-causing reactions, and
to deliver a variety of genes or gene products (proteins) to an
individual--both genes known to be defective or vaccine components,
for immunogenic proteins associated with viruses.
[0166] Additionally, HBB is engineered to contain and express genes
for therapeutically important gene products, including but not
limited to hormones, growth regulators, antitumor antigens,
antibodies, other therapeutic antigens, and interleukins. In so
doing, HBB is engineered to transcribe, translate and process
heterologous proteins. HBB also is engineered to respond to human
effectors such as, e.g., serum or plasma glucose, alanine, and
various hormones. Specifically, diseases where the toxic metabolite
is accumulated in the plasma or serum are very effectively treated
by engineered HBB. Further, suicide genes are inserted into HBB so
that the bacterium may be destroyed when therapeutically effective
to do so. One further facet of immunological use of HBB is to
engineer HBB to produce the antigen of choice on its cell surface
so as to provide continual boosting for the immune system.
[0167] The following references were cited herein.
[0168] 1. Gerhardt, ed in chief, Methods of General and Molecular
Bacteriology, American Society for Microbiology 1994.
[0169] 2. Sonnenwirth, et al., Gradwohl's Clinical Laboratory
Methods and Diagnosis, eighth edition. C. V. Mosby Co. 1980.
[0170] 3. Lennette, Edwin H., ed in chief, Manual of Clinical
Microbiology, 3rd ed. American Society for Microbiology, 1980.
[0171] 4. Prophet, et al., eds, Laboratory Methods in
Histotechnology. American Registry of Pathology 1992.
[0172] 5. Hacker, et al., Molecular Microbiology (1997) 23 (6)
10.sup.89-1097. Nucleic Acid Techniques in Bacterial Systematics,
Edited by E. Stackebrandt and M Goodfellow, 1991, John Wiley and
Sons.
[0173] 6. Clinical and Pathogenic Microbiology, Howard, B. (Ed.)
1994, A.C.V. Mosby-Year Book, Inc.
[0174] 7. Ludwig, et al., 1992, System. Appl. Microbiol. 15:
487-501.
[0175] Any patents or publications mentioned in this specification
are indicative of the levels of those skilled in the art to which
the invention pertains. Further, these patents and publications are
incorporated by reference herein to the same extent as if each
individual publication was specifically and individually indicated
to be incorporated by reference.
[0176] One skilled in the art will appreciate readily that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those objects,
ends and advantages inherent herein. The present examples, along
with the methods, procedures, treatments, molecules, and specific
compounds described herein are presently representative of
preferred embodiments, are exemplary, and are not intended as
limitations on the scope of the invention. Changes therein and
other uses will occur to those skilled in the art which are
encompassed within the spirit of the invention as defined by the
scope of the claims.
Sequence CWU 1
1
20 1 1208 DNA Unknown gene nt40; nt60 Entire Rb 16S rRNA gene
sequence of a new human blood bacterium (HBB). The identification
of nucleotide n located at positions 40 and 67 is unknown. 1
cgacgatcag tagctggtct gagaggatga tcagccacan tgggactgag acacggccca
60 gactccnacg ggaggcagca gtggggaata ttggacaatg ggcgcaagcc
tgatccagcc 120 atgccgcgtg agtgatgaag gccttagggt tgtaaagctc
ttttgtccgg gacgataatg 180 acggtaccgg aagaataagc cccggctaac
ttcgtgccag cagccgcggt aatacgaagg 240 gggctagcgt tgctcggaat
cactgggcgt aaagggcgcg taggcggcca tttaagtcgr 300 gggtgaaagc
ctgtggctca accacagaat tgccttcgat actgggtggc ttgagtccgg 360
aagaggttgg tggaactgcg agtgtagagg tgaaattcgt agatattcgc aagaacaccg
420 gtggcgaagg cggccaactg gtccggaact gacgctgagg cgcgaaagcg
tggggagcaa 480 acaggattag ataccctggt agtccacgcc gtaaacgatg
aatgccagcc gttgggcagc 540 ttgctgctca gtggcgcagc caacgctttg
agcattccgc ctggggagta cggtcgcaag 600 attaaaactc aaaggaattg
acggggggcc cgcacaagcg gtggagcatg tggtttaatt 660 cgaagcaacg
cgcagaacct taccatccct tgacatggca tgttacccag agagatttgg 720
ggtcctcttc ggaggcgtgc acacaggtgc tgcatggctg tcgtcagctc gtgtcgtgag
780 atgttgggtt aagtcccgca acgagcgcaa cccacgtcct tagttgccat
cattcagttg 840 ggcactctag ggagactgcc ggtgataagc cgcgaggaag
gtgtggatga cgtcaagtcc 900 tcatggccct tacgggatgg gctacacacg
tgctacaatg gcggtgacag agggacgcga 960 aggggcgacc tggagcaaat
cccgaaaaac cgtctcagtt cggattgcac tctgcaactc 1020 gggtgcatga
aggcggaatc gctagtaatc gtggatcagc atgccacggt gaatacgttc 1080
ccgggccttg tacacaccgc ccgtcacacc atgggagttg gtcttacccg acggcgctgc
1140 gccaaccgca aggaggcagg cgaccacggt agggtcagcg actggggtga
agtcgtaaca 1200 aggtagcc 1208 2 1502 DNA Unknown 58 rRNA sequence
of a new human blood bacterium 2 gctcggtacc acgcatgctg cagacgcgtt
acgtatcgga tccagaattc gtgatgtgtc 60 cagccgcagg ttcccctacg
gctaccttgt tacgacttca ccccagtcgc tgaccctacc 120 gtggtcgcct
gcctccttgc ggttggcgca gcgccgtcgg gtaagaccaa gtcccatggt 180
gtgacgggcg gtgtgtacaa ggcccgggaa cgtattcacc gtggcatgct gatccacgat
240 tactagcgat tccgccttca tgcactcgag ttgcagagtg caatccgaac
tgagacggct 300 tttggggatt tgctccagat cgctccttcg cgtcccactg
tcaccgccat tgtagcacgt 360 gtgtagccca tcccgtaagg gccatgagga
cttgacgtca tccacacctt cctcgcggct 420 tatcaccggc agtctcccta
gagtgcccaa ctgaatgatg gcaactaagg acgtgggttg 480 cgctcgttgc
gggacttaac ccaacatctc acgacacgag ctgacgacag ccatgcagca 540
cctgtgtgcg cgccaccgaa gtggacccca aatctctctg ggtaacacgc catgtcaaag
600 gatggtaagg ttctgcgcgt tgcttcgaat taaaccacat gctccaccgc
ttgtgcgggc 660 ccccgtcaat tcctttgagt tttaatcttg cgaccgtact
ccccaggcgg aatgctcaaa 720 gcgttagctg cgctactgcg gtgcaagcac
cccaacagct ggcattcatc gtttacggcg 780 tggactacca gggtatctaa
tcctgtttgc tccccacgct ttcgcgcctc agcgtcagta 840 atggtccagt
tggccgcctt cgccaccggt gttcttgcga atatctacga atttcacctc 900
tacactcgca gttccaccaa cctctaccat actcwagcgt cccagtatcg aaggccattc
960 tgtggttgag ccacaggctt tcacccccga cttaaaacgc cgcatacgcg
ccctttacgc 1020 ccagtgattc cgagcaacgc tagccccctt cgtattaccg
cggctgctgg cacgaagtta 1080 gccggggcta attcctccgg taccgtcatt
atcgtcccgg ataaaagagc tttacaaccc 1140 taaggccttc atcactcacg
cggcatggct ggatcaggct tgcgcccatt gtccaatatt 1200 ccccactgct
gcctcccgta ggagtctggg ccgtgtctca gtcccagtgt ggctgatcat 1260
cctctcagac cagctactga tcgtcgcctt ggtaggccgt taccccacca actagctaat
1320 cagacgcggg ccgatcttcc ggcagtaaac ctttccccat aagggcgtat
ccggtattag 1380 ccctagtttc ccagggttat tccgaaccgg aaggcacgtt
cccacgcgtt actcacccgt 1440 ccgccgctga ccccgaaagg tccgctcgac
ttgcatgtgt taagcctgcc gccagcgttc 1500 gt 1502 3 2542 DNA Unknown Rb
23S rRNA sequence of a new human blood bacterium 3 gatggggaaa
cccaccttcg accctccgta tggtggtctc acgggcgact gtgagattgc 60
catacggttg gtcagatgaa ggtatcaagc cctgaataca ataggggttt gaagcgaacc
120 cggggaactg aaacatctca gtacccggag gaaaggacat caacgagact
ccgtcagtag 180 tggcgagcga acgcggatca ggccagtgct tgtgtcgaga
ttaccggaac ggtctggaaa 240 ggccggcgcg aagggtgaca gccccgtacg
ggacggtcga gacacaagac tcgagtaggg 300 cgggacacgt gaaatcctgt
ctgaacatgg ggggaccacc ctccaagcct aagtactcct 360 cagcgaccga
tagtgaacca gtaccgtgag ggaaaggtga aaagcacccc gacgagggga 420
gtgaaacagc acctgaaacc ggatgcttac aaacagtggg agcccaaggt tcgtcctggg
480 tgaccgcgta ccttttgtat aatgggtcag cgacttaaag ttacgagcaa
gcttaagccg 540 gtaggtggag gcgtagcgaa agcgagtctg aacagggcgt
tcagttcgtg gctttagacc 600 cgaaaccgag tgatctagcc atgtgcagga
tgaaggtggg gtaacaccca ctggaggtcc 660 gaaccagtgc ccgttgaaaa
ggtcttggat gacgtgtggc taggggtgaa aggccaatca 720 aactcggaaa
tagctggttc tccgcgaaag ctatttaggt agcgcctcgt gtgaatgcct 780
tgcggggtag agcactggat gggctagggc cgcccacagc ggtaccgcac tcaaccaaac
840 tccgaatacg caagagcact gcacgggaga cacacggcgg gtgctaacgt
ccgtcgtgga 900 gagggaaaca accctgaccg acagctaagg cccccaattc
gtggctaagt gggaaaggat 960 gtgggaatcc caaaacaacc aggaggttgg
cttagaagca gccatccttt aaagaaagcg 1020 taacagctca ctggtctaaa
caagggttcc tgcgccgaaa atgtaacggg gctcaagcca 1080 cgagccgaag
cttcggtgca tcgcaagatg cgcggtagcg gagcgttccc taggcctgcg 1140
aagggagacc cgtgagggct cctggaggta tgggaagtgc gaatgctgac atgagtaacg
1200 acaaagagtg tgaaagacac tctcgccgaa agtccaaggg ttcctgcgta
aagttaatct 1260 gcgcagggtt agccggcccc taaggcgagg ccgaaaggcg
tagtcgatgg gaacggggcg 1320 aatattcccc ggccagtgga tggtgacgga
tcccgtgtgt tgttcggcct taacggattg 1380 gtcgggcagc gaaggggtcc
caggaaagag cctccacgtg agaccgtacc cgaaaccgac 1440 acaggtggac
tggtagagta taccaaggcg cttgagagaa cgatgctgaa ggaactcggc 1500
aatttgcctc cgtaacttcg ggataaggag gcctcgtatg cgggcaaccg tgtgcgaggg
1560 gcacagacca gggggtggcg actgtttatc taaaacacag ggctctgcga
agtctgtaag 1620 acgacgtata gggcctgacg cctgcccggt gccggaaggt
taagaggaga ggtgagagcc 1680 ttgaattgaa gccccggtaa acggcggccg
taactataac ggtcctaagg tagcgaaatt 1740 ccttgtcggg taagttccga
cctgcacgaa tggcgtaacg atctccccgc tgtctccagc 1800 atcggctcag
tgaaattgaa ttccccgtga agatgcgggg ttcctgcggt cagacggaaa 1860
gaccccgtgc acctttactg tagctttgcg ctggccttcg tgtcggcatg tgtaggatag
1920 gtggtaggct ttgaagttcg ggcgccagcc tggatggagc cacccttgaa
ataccaccct 1980 tgacgatatg gtggtctaac cgcgcgccct gatcgggcgc
cgggaccgcg catggcaggc 2040 agtttgactg gggcggtcgc ctcccaaagc
gtaacggagg cgtacgaagg tgggctcaga 2100 gcggtcggaa atcgctcgtc
gcgtgcaatg gcataagccc gcttgactgc gagacggaca 2160 tgtcgagcag
agacgaaagt cggtcatagt gatccggtgg tcccgcgtgg gtgggccatc 2220
gctcaacgga taaaaggtac gccggggata acaggctgat gacccccaag agtccatatc
2280 gacggggtcg tttggcacct cgatgtcggc tcatcacatc ctggggctgg
agaaggtccc 2340 aagggttcgg ctgttcgccg attaaagtgg tacgtgagct
gggttcagaa cgtcgtgaga 2400 cagttcggtc cctatctgcc gtgggtgtaa
ggagacttga gaggatttgt ccctagtacg 2460 agaggaccgg gatgaacgta
cctctggtgg agctgttgtg gcgccagccg cagtgcagcg 2520 tagctacgta
cggacgggat aa 2542 4 2061 DNA Unknown 58 23S rRNA sequence of a new
human blood bacterium 4 gaaaccaacc ttcgaccttc cgtattgcgg gaccaggcgc
gagcctggtt cctcaatacg 60 gttggtcaca tgaaggtatc aaatcctgaa
tccatagggg tttgaagcga acccggggaa 120 ctgaaacatc tcagtacccg
gaggaaagga catcaacgag actccgtcag tagtggcgag 180 cgaacgcgga
tcaggccagt gcctgtgttg agtttaccgg aacggtctgg aaaggccggc 240
gcgatgggtg acagccccgt acgggacgga cgacacacag gactcgagta gggcgggaca
300 cgtgaaatcc tgtctgaaca tggggggacc accctccaag cctaagtact
cctcagcgac 360 cgatagcgaa ccagtaccgt gagggaaagg tgaaaagcac
cccgacgagg ggagtgaaac 420 agcacctgaa accggatgct tacaaacagt
gggagcccaa ggttcgtcct gggtgaccgc 480 gtaccttttg tataatgggt
cagcgactta aagttacgag cgagcttaag ccgataggtg 540 gaggcgcagc
gaaagcgagt ctgaacaggg cgttcagttc gtggctttag acccgaaacc 600
gagtgatcta gccatgtgca ggatgaaggt ggggtaacac ccactggagg tccgaaccag
660 tgcccgttga aaaggtcttg gatgacgtgt ggctaggggt gaaaggccaa
tcaaactcgg 720 aaatagctgg ttgtccccga aagctattta ggtagcgcct
cgagtgaata cctcacgggg 780 tagagcactg gatgggctag ggccgcccac
agcggtacca aacccaacca aactccgaat 840 acgtgagagt actgctcggg
agacacacgg cgggtgctaa cgtccgtcgt ggagagggaa 900 acaaccctga
ccgacagcta aggcccccaa ttcgtggcta agtgggaaag gatgtgggac 960
tcccaaaaca accaggaggt tggcttagaa gcagccatcc tttaaagaaa gcgtaacagc
1020 tcactggtct aaataagggg tcctgcgccg aaaatgtaac ggggctcaag
ccacgagccg 1080 aagcttcgga tgcactcctt cgggggtgcg tggtagcgga
gcgttcccta ggcctgtgaa 1140 gcggtacctg tgaggggccg tggaggtatg
ggaagtgcga atgctgacat gagtaacgac 1200 aaagagtgtg aaagacactc
tcgccgaaag tccaagggtt cctgcgtaaa gttaatctgc 1260 gcagggttag
ccggccccta aggcgaggcc gaaaggcgta gtcgatggga acggggcgaa 1320
cattccccgg ccagcggatg gtgacggatg ccgtgtatcg tttgacctta tcggattggt
1380 caggcggtga aggggtccca ggaaatagcc tccgcgtaag accgtacccg
aaaccgacac 1440 aggtggactg gtagagtata ccaaggcgct tgagagaacg
atgctgaagg aactcggcaa 1500 tttgcctccg taacttcggg ataaggaggc
ctctgtcttg ggcaaccagg gcagaggggc 1560 acagaccagg gggtggcgac
tgtttatcta aaacacaggg ctctgcgaag tctgtaagac 1620 gacgtatagg
gcctgacgcc tgcccggtgc cggaaggtta agaggagagg tgagagcttt 1680
gaatcgaagc cccggtaaac ggcggccgta actataacgg tcctaaggta gcgaaattcc
1740 ttgtcgggta agttccgacc tgcacgaatg gcgtaacgat ctccccgctg
tctccagcat 1800 cggctcagtg aaattgaatt ccccgtgaag atgcggggtt
cctgcggtca gacggaaaga 1860 ccccgtgcac ctttactgta gctttgcgct
ggccttcgtg tcggcatgtg taggataggt 1920 ggtaggcttt gaagcggggg
cgtcagcctt cgtggagcca tccttgaaat accacccttg 1980 gcgatatggt
ggtctaaccg cgacccttga tcggggtccg ggaccgcgca tggcaggcag 2040
tttgactggg cgtcgcctcc c 2061 5 836 DNA Unknown intergenic spacer
region (IGS) sequence of a new human blood bacterium 5 ttctaaggat
gttgctggca ggatgatcgg ccggtcttcg gacccggtcc ggtccgctcc 60
tcgtgcgacg tcattggaat atgggctcag tcagagcccc tcatgagcgg gacgctcgtt
120 aagagcggag ccgtcctcgt ttctctttct catccggaca atagcgggat
cgccgaggcg 180 gcgtcttcgg acgcctggct tctcggacga ccggctcggg
cctgtagctc aggtggttag 240 agcgcacccc tgataagggt gaggtcggac
gttcgagtcg tcccaggccc accagcttca 300 gtggtcgcaa cgccccgagc
cgacggctcg ccgggccgag cggcgcgcgc ggccatctcc 360 gaggaaaacg
gggctgtagc tcagttggga gagcggttgc tttgcaagca tcaggtcgtc 420
ggttcgatcc cgtccagctc caccagcgcc ctcgaggctc gcaagaacct cgggcagcgg
480 gtgacgtgag agaagtccgg agagagaggg caagagtttg ccatccgtga
gcgcggtgcg 540 cggcgggtcg gcagtgatat cgaacatcgt gaagagggaa
tgtggccgtt tggttcttcg 600 aaccatccaa ggtcatgttc ggcaagcatg
tgatcgaacc gaaaggttcg gtcactggtc 660 tttatcgtga ccgtggctgg
gtgatcggcg acagcgtagc tgctgccgat cgcgccggac 720 atcgatcatg
agagcgatca agtgccttaa gagcattcgg tggatgcctt ggcgctgaga 780
ggcgatgaag gacgtggtac gctgcgataa gccttgggga gctgcgaacg agcttt 836 6
592 DNA Unknown a multidrug resistant protein gene of a new human
blood bacterium 6 acgtatcgga tccagaattc gtgatgtatt cgcgcaacac
cgacaaggac ctcgaggtcg 60 cccggcgcat cgaggcgcgg ctgaagcgga
ccaagggcct cgtcgacgtg catctccacc 120 agatcgtcga cgtgccgcaa
ttcttcgtgg acgtggaccg gcgcctcgcc tccgagctcg 180 gcctgaccca
gcagcagatc gcccagagcc tcaacgtctc gctctcgggc tccttccagg 240
tcaccccgaa cttctggacc gacccgaaaa ccggcatccc ctaccagctc tgggtgcaga
300 cacgcaggac atatctgaat tcgtcgacaa gcttctcgag cctaggctag
ctctagacca 360 cacgtgtggg ggcccgagct cgcggccgca caattcactg
gccgtcgttt tacaacgtcg 420 tgactgggaa aaccctggcg ttacccaact
taatcgcctt gcagcacatc cccctttcgc 480 cagctggcgt aatagcgaag
aggccgcacc gatcgccctt cccaacagtt gcgcagcctg 540 aatggcgaat
ggaaattgta agcgttaata ttttgttaaa attcgcgtta aa 592 7 31 DNA
artificial sequence primer specific for 16S rRNA of a new human
blood bacterium 7 gagtttgatc ctggctcaga acgaacgctg g 31 8 31 DNA
artificial sequence primer specific for 16S rRNA of a new human
blood bacterium 8 catcggcatc cccttggacg ccgacctagt g 31 9 16 DNA
artificial sequence primer_bind primer specific for 23S rRNA of a
new human blood bacterium 9 ccgaatgggg vaaccc 16 10 18 DNA
artificial sequence primer_bind primer specific for 23S rRNA of a
new human blood bacterium 10 tcgaccaaga grrgcttt 18 11 18 DNA
artificial sequence primer_bind primer specific for 23S rRNA of a
new human blood bacterium 11 tagctggttc tcyycgaa 18 12 19 DNA
artificial sequence primer_bind primer specific for 23S rRNA of a
new human blood bacterium 12 ggcattgaag ccctcttcc 19 13 16 DNA
artificial sequence primer_bind primer specific for 23S rRNA of a
new human blood bacterium 13 aaaccgacac aggtrg 16 14 16 DNA
artificial sequence primer_bind primer specific for 23S rRNA of a
new human blood bacterium 14 cgacttycgt agattc 16 15 17 DNA
artificial sequence primer_bind primer specific for intergenic
spacer region (IGS) sequence of a new human blood bacterium 15
acggtagggt cagcgac 17 16 17 DNA artificial sequence primer_bind
primer specific for intergenic spacer region (IGS) sequence of a
new human blood bacterium 16 cctcccagct tccaccc 17 17 18 DNA
artificial sequence primer_bind synthesized primer 17 atgtcctgcg
tgtctgca 18 18 18 DNA artificial sequence primer_bind primer
specific for drug resistant protein gene of a new human blood
bacterium 18 gtactagtcc agcgtgtc 18 19 16 DNA artificial sequence
primer_bind probe for in situ hybridization 19 tcgcagttcc accaac 16
20 16 DNA artificial sequence primer_bind probe for in situ
hybridization 20 ctgtggttga gccaca 16
* * * * *