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ORIGINAL ARTICLE
Small molecules originating from microbes (SMOM)
and their role in microbes – host relationship
N. V. Beloborodova and G. A. Osipov
From the Research group of Academician Yu.Isakov, Russian Academy of Medical Sciences,
Sadovo-Kudrinskaya St., 15, 103001 Moscow, Russia
Correspondence to: N. V. Beloborodova, Research group of Academician Yu.Isakov, Russian Academy of
Medical Sciences, Sadovo-Kudrinskaya St., 15, 103001 Moscow, Russia. Tel: +095 254 1039; Fax: +095 255
9300; E-mail: [email protected]
Microbial Ecology in Health and Disease 2000; 12: 12–21
A hypothesis proposed, according to which non-specific purulent-inflammatory processes are mainly caused by disturbances at
low-molecular level —i.e. by small molecules originating from microbes (SMOM), while SMOM homeostasis decompensation takes the
leading role in complex polyfactorial pathogenesis of sepsis. The hypothesis is logically justified by understanding the reasonableness for
creation within the ontogenesis the system of signal molecules, which would serve as mediators for informational exchange between
microorganisms and host’s cells. To check this hypothesis we’ve studied blood of 25 healthy donors and 161 patients with various
conditions (peritonitis, endocarditis, infections of urinary tract etc.) using Gas chromatography-Mass spectrometry. In all cases we
detected SMOMs-fatty acids (hydroxy-acids, branched, unsaturated and cyclopropanoic acids), aldehydes, alcohol’s and phenyl-carbonic
substances, that are never normally synthesized by mammals, but are structural components or metabolites of microorganisms. The
quantitative and qualitative characteristics of SMOMs varied significantly in patients from those of healthy people. Substances of low
molecular weight — that are the structural components of normal endogenous microflora and dominate in blood of healthy people — have
disappeared or were present in very low quantities. Meanwhile concentration of bacterial molecules that are rarely found in healthy
persons, has increased by 10–100 times. Other SMOMs, never present in healthy people, were also found. The data obtained stand pro
the hypothesis on constant presence of SMOMs in blood of healthy and ill individuals and allow to formulate the Concept on
Homeostasis of small molecules originating from microbes; serious break up of this homeostasis (decompensation of SMOM homeostasis)
serves as inductor of general inflammatory response, septic shock and multisystem organ failure. Key words: microbial markers,
phenylcarbonic compounds, gas chromatography–mass spectrometry, fatty acids, homeostasis, human blood, non-antibiotics, sepsis.
INTRODUCTION
Sterile human organism comes into the world teemed with
various microorganisms (air, water, soil etc.). Life-threatening specific infections (diphtheria, plague, cholera,
botulism, tetanus, and others) are caused by pathogenic
bacteria, possessing large variety of pathogenic factors.
Pathogenic bacteria life cycles are characterized by production of highly toxic substances, that are intended to kill the
host, whose death leads finally to death of bacteria themselves, i.e. from biological sense this rout of evolution of
micro-universe comes out as a dead lock. Coming true
nowadays and based on this approach is the forecast,
dating to 40 years ago: with time life threatening infections
would be of secondary importance in comparison with
those caused by host’s own opportunistic flora (autochthonous infection) (1).
In the end of the 1990s sepsis has become the leading
cause of lethality among ICU patients from high-risk
groups with different pathologies, such as surgical or
oncologic-hematological diseases, trauma, burns, premature birth etc. It’s well known nowadays that it is not
© Taylor & Francis 2000. ISSN 0891-060X
pathogenic bacteria, that cause sepsis, but the conflict,
arising from inadequate immune reactions to organism’s
own opportunistic microflora, colonizing opened
biocenoses.
There’s still serious lack of knowledge on basic relationship between the superior-host’s and inferior-microbe’s
organisms. Better knowledge of this relationship will
provide physicians with new tools of regulating host’s
relationship with it’s own microflora in different pathologic conditions.
Let’s refer to a number of well known and the most
recent data on the issue. The most important is considered
the discovery of cytokine signal system, which is responsible for informational exchange between host’s cells and
allows to explain a number of phenomena. For example,
cytokines TNF, IL-1, IL-6 are the inductors of acute
inflammatory phase of bodily response an integral part of
comprehensive human internal protective system against
all foreign substances and pathogenic microorganisms.
Gene-engineering technique allowed discovering a number
of other cytokines (IL-2, IL-10) with anti-inflammatory
Microbial Ecology in Health and Disease
SMOM and microbe – host relationships
13
Table I
Cytokine-inducing bacterial modulines-from published materials (2–4)
Target
LPS and other bacterial modulines
Monocytes/macrophages
Epithelial cells
Fibroblasts
Osteoclasts
Lymphocytes
functions, that are responsible for blocking inflammatory
reactions to residential microflora. Bacterial modulins — is
a definition, offered to outline a new type of virulent factors
of those bacterial structures, which possess cytokine-inducing potential (2).
Lipopolysaccharides (LPS) are the focus of attention
during the last 30–40 years. LPS as well as other proteins
and phospholipids are built into macromolecular endotoxin
complex of gram-negative bacteria (3, 4). It’s clear today,
that a number of other bacterial complexes, besides LPS,
are also capable of inducing cytokine secretion. During the
last 5–10 years the components, stimulating synthesis of
cytokine—such as carbohydrates, proteins and lipids — are
the subject of thorough investigation (Table I).
Medical literature of last years is the evidence of active
search for other bacterial modulines. For gram-negative
bacteria these are the external membrane proteins: porines,
lipid A-bound proteins (LAP) or endotoxin-bound
proteins, fimbrial (pili) proteins (5). Among other membrane-bound bacterial modulines are non-identified superficial proteins, protein A and heat-shock proteins of some
microorganisms (6).
Because lipoproteins of cytoplasmatic membrane act as
bacterial modulines in both gram-negative and gram-positive bacteria, some researchers got involved in studying
interleukin-stimulating effects of synthetic analogues and
structural derivatives of lipoproteins (7). There’s a growing
number of publications resulting from studies of bacterial
membranous glycoprotein, which stimulates not only
macrophagal secretion of TNF, but its synthesis as well (8).
Monocytal and epithelial cell synthesis and secretion of
cytokines is influenced by extracellular proteins—such as
proteases, exotoxins and others, at the same time some
lipids and polysaccharides also have cytokine-stimulating
properties. For example, protein-free membranous poliol –
lipid of Mycoplasma fermentans stimulates macrophagal
secretion of IL-6 and TNF-a (9). As to polysaccharides—
there’s not only in vitro, but also experimental evidence of
increasing TNF-a concentration—as it was demonstrated
for Streptococcus B group-specific polysaccharide in newborn rats (10).
Results
Cell reaction modifications
Secretion of eicosanoids
and cytokines
Chemotaxis
Metabolic activity
Proliferation
Inhibition of proliferation
Differentiation
Apoptosis and others
Teichoic and lipoteichoic acids — principal components
of cellular wall in gram-positive bacteria (Streptococcus
pyogenes, Staphylococcus aureus, S. epidermidis) — together
with peptidoglycan initiate development of cytokine induced septic shock, caused by gram-positive bacteria.
Host’s enzymes quickly destroy peptidoglycan, but its
fragments have cytokine-inducing properties (11).
It’s important to point out, that according to experimental findings at least one of the bacterial modulines (membranous protein 39-kDa of Proteus mirablis) is capable of
inhibiting LPS-induced synthesis of interleukin IL-1 and
macrophagal production of substances with free oxygen
radicals (12).
Data of a number of experiments show potential capability of microbial derivatives either to fuel inflammatory
process or to inhibit it — in particular through regulation of
macrophagal activity. This is an important detail within
widely accepted role of macrophages as key factor in
development of polyorganic failure in sepsis (13). Cumulative results of SMOM studies are not giving yet full picture
of human – autochthonous bacteria relationship.
This relationship with bacteria, colonising human’s intestines, skin, mucous membranes of respiratory and urinary
tracts, etc., is symbiotic. Bacterial concentration is maximal
on marginal surfaces which restrict inner media from the
outer world: skin concentration reaches 105 – 6 CFU/g,
intestinal mucous concentration exceeds 1011 – 12 CFU/g.
The contacting surface of sterile inner media in human
organism is huge: it’s length is 4 – 6 m, while smaller
intestines total surface reaches 180 – 200 m2. It is already
proved that invasion of saprophyte bacteria or their fragments from mucous surfaces into internal media happens
more often, than it was thought before. Numerous data on
penetrability of mucous membranes for microorganisms
and large molecules, on continued migration of incorporated into macrophages microbes into the bloodstream, on
direct bacterial translocation into inner media under certain
conditions (like stress, shock, haemodynamic disorders,
endotoxemia, etc.), on quite widely spread short-term
episodes of transient bacteremia, that is observed, for
example, after tooth extraction and even after brushing
teeth — has been accumulated.
14
N. V. Beloborodo6a and G. A. Osipo6
Phagocytosis and natural autolysis of microbial cells are
the principal sources of microbial fragments— lipids,
amino acids, peptides, carbohydrates, and small molecules
(monomers of lipids, proteins and carbohydrates), which
are the products of microbial cells disintegration. Theoretically justified is the bloodstream bacterial molecules’ role
as regulators of pro- and anti-inflammatory activity of
cells and providers of homeostasis, rather than as cytokine-inducing factors of virulence.
We present the hypothesis on constant SMOM circulation in the bloodstream of both healthy and ill people. It is
coming out of ontogenetically justified necessity in special
signal molecules to exchange information between host
and microorganisms. In other words, we are offering the
direction where the ABC of the ancient dialogue language
between macro- and micro-universe should be looked for.
Gas chromatography–mass spectrometry (GC-MS)
method was used (14–17).
AIMS OF THE STUDY
To detect simple by chemical structure non-protein
molecules originating from microbes with molecular
weight up to 500 [small molecules originating from microbes—SMOM] in human blood under different health
conditions.
MATERIAL AND METHODS
Two groups were studied. The first one was comprised of
25 healthy volunteer-donors that were examined and underwent standard laboratory tests. Blood was collected
from 25 donors. The second group was comprised of
patients with different pathologies: n =20 with peritonitis;
n=32 with endocarditis; n= 109 with local inflammatory
and post-operative infectious processes. Their blood was
taken on the pick of clinical and laboratory manifestations
of infectious process during diagnostic or curative
venipunctures. The blood was conserved in heparinised
test-tube and frozen under −5°C.
Gas chromatography–mass spectrometry (GC-MS) was
used to identify bacterial chemical substances in patient’s
blood: fatty acids, alcohol and phenylcarbones. Whole
blood (50 ml) was dried and subjected to acid methanolysis
during 1 h, using 0.4 ml of 1M HCl in methanol under the
temperature of 80°C. At this stage fatty acids are extracted
from complex glycerol and cholesterol ethers in the form
of methyl ethers. Fraction of fatty acids’ methyl ethers
with other lipid components was extracted twice from
reactive mixture with 200 ml of hexane, dried and derivated
with 20 ml of N,O-bis (trimethylsilyl)-trifluoracethamide
(BSTFA) for 15 min at 80°C to form trimethylsilyl derivatives of hydroxy acids, alcohol’s and sterols. 1–2 ml of
reactive mixture was injected into GC-MS system (QP2000 Shimadzu, Japan). Chromatographic separation of
the sample was performed in a capillary column with
methyl-silicone phase Ultra-1 (Hewlett-Packard); length 25
m, inner diameter 0.2 mm, film thickness 0.25mm. Temperature program starting at 120°C with further increase up
to 300 – 320°C, 5°C per min. Principal lipid components of
the sample were identified on the basis of their spectra
obtained by quadrupole mass spectrometer (electron ionization 70 eV), operating at full scan mode. The same
pattern was used for identification of carbonic and phenylcarbonic acids and spirits. For detection of specific markers of bacteria high sensitivity mode of selected ion
monitoring (mass-fragmentography) was used. Five groups
of ions were switched in course of gas chromatographic
separation. To detect small quantities of C10 – C20 fatty
acids prominent m/z 87 ion was used; chosen group intervals allowed to avoid peaks of dominating in microbial
lipid acid’s profile of C16 and C18 acids. For verification
of b-hydroxy-acids two types of ions were used: characteristic for homologue row ion m/z 175 and acid-specific ion
of M-15 type, for verification of a-acids-specific ion of
M-59 type. Specific ions were used also to identify fatty
alcohol’s and sterols. Additional identification criteria
were the following: chromatographic relative retention
time and selected ions’ peak ratios. Carbonic and phenylcarbonic acids and alcohol’s were extracted from acidified
whole blood (pH 2) with diethyl ether. Ether extract was
taken to dryness at the temperature up to 40°C, the dry
residue was treated with 20 ml of BSTFA for 15 min at
80°C to form trimethylsilyl derivatives of acids and alcohol’s. Two microliters of mixture was injected. Full scan
spectrum, similar chromatographic column (as in fatty
acid analysis) was used. Temperature program: 7 min at
80°C with further ramp up to 250°C. Eluted substances
were identified by its mass spectra using standard library
search program (NBS library).
Areas of selected ion trace peaks were integrated automatically or manually and fixed in the protocol. Afterwards all data were processed by based in EXCEL tables
evaluation program. Pure component’s calibration data
were used by adding of known quantity of tridecanoic acid
or 12-OH-octadecanoic acid. Blood concentration of heptadecanic, i.e. margarinic acid were used in routine analysis. Its content is approximately constant in humans and
equal to 20 9 5mg/ml (unpublished, our own measurements). This approach allowed to avoid repeated
calibration.
RESULTS
In blood of healthy donors were identified foreign for
human body low-molecular chemical substances: hydroxy-,
branched, non-saturated and cyclopropanoic fatty acids
(Table II).
Hard to ignore the relative qualitative and quantitative
stability of definite SMOMs: for example, h18, i14 and a19
were found in 88 – 94% blood samples of healthy donors,
SMOM and microbe – host relationships
15
Table II
Detection frequency and concentration range (ng/ml) of fatty acids–structural components of bacterial cells,
and metabolic sterols identified in blood of healthy donors (n = 18)
Name
Abbreviation*
Detection frequency
Range (ng/ml)
No. of persons
% of total
Hydroxy-acids
Hydroxy-decanoic
Hydroxy-lauric
2-Hydroxy-lauric
Hydroxy-myristic
2-Hydroxy-myristic
Hydroxy-iso-pentadecanoic
Hydroxy-palmitic
Hydroxy-iso-heptadecanoic
Hydroxy-stearic
10-Hydroxy-stearic
Hydroxy-iso-eicozanoic
h10**
h12
2h12
h14
2h14
hi15
h16
hi17
h18
10h18
hi20
10
11
11
18
13
3
18
13
16
18
2
55
61
61
100
72
17
100
72
88
100
11
0–50
0–40
0–30
0–45
0–100
0–10
10–590
0–70
7–150
1–77
0–10
Branched fatty acids
anteiso-Tridecanoic
iso-Myristic
iso-Pentadecanoic
anteiso-Pentadecanoic
iso-Palmitic
iso-Heptadecanoic
anteiso-Heptadecanoic
anteiso-Nonadecanoic
Tuberculostearic
a13
i14
i15
a15
i16
i17
A17
a19
10Me18
11
17
18
18
18
18
18
16
18
61
94
100
100
100
100
100
88
100
0–20
0–110
60–380
50–470
90–720
150–2000
630–3600
0–15
3–25
Unsaturated fatty acids
9,10-Tetradecenoic
iso-Heptadecenoic
Heptadecenoic
cis-Vaccenic
14:1
i17:1
17:1
18:1D11
8
6
18
14
44
33
100
78
0–120
0–10
60–310
0–620
Cyclopropanoic acids
cyclo-Heptadecanoic
cyclo-Nonadecanoic
17cyc
19cyc
9
18
50
100
0–40
2–60
14
3
4
78
17
23
0–150
0–30
0–100
Alcohols
Coprostanol
Cholestendiol
Metabolite 428
* Marking: 17:1–17- is the number of carbon atoms, the figure after colon denotes the number of double
bonds; h-hydroxy-acid; a,i-indicates methyl-branching; cyc-means cyclopropanoic acid. For example,
2hi15 means 2 -hydroxy-iso-pentadecanoic acid.
** 3-hydroxy-acids—if position of hydroxyl is not indicated.
while such molecules as h14, h16, 10h18, i15,a15, i16, i17,
a17, 10Me18, 17:1, 19cyc were found in all 100% cases.
In the second group—i.e. in patients with manifested
infectious processes—were revealed accentuated qualitative and quantitative deviations in the spectrum of low
molecular SMOMs (Table III).
The main statistics are presented at Figs. 1 and 2
where distribution of microbial markers values in blood
of donors and patients is shown. As far as concentrations
of markers differ substantially, their values were normalized to interval for optimization of comparison: minimal
value was subtracted from each measurement, and residue
was divided into interval. Data on 25 donors and 161
patients frequency distributions are shown as ‘box and
whiskers’. The box is the part of distribution that lies
between the upper and lower quartiles (25 and 75% of
distribution), and whiskers correspond to 9 1.5 quartile
distance. Data between + 1.5 and + 3 quartile distance
are designated by circles while asterisks indicate figures
exceeding 3 quartile. Distribution diagrams of patients
show configurations with extra right sided skewness and
left-sided tailing when compared with symmetrical diagrams of donors. Extreme values exceeding +1.5 quartile
distance are rarely found in donors, but common in patients. Such values probably indicate inflammation,
caused by corresponding microbes.
16
N. V. Beloborodo6a and G. A. Osipo6
The markers form two distinct groups, termed as ‘zero’
and ‘nonzero’. Markers showing generally zero level are
included in the ‘zero’ group. For instance hi20 (Chlamydia), hi17 (B. fragilis), hi15 (Pre6otella melaninogenica),
E242 (Corynebacterium urealyticum), h10 (Pseudomonas)
and other markers are assigned to pathogens or to rarely
detected commensals. ‘Nonzero’ are the markers which
show distribution with nonzero mode and are found both
in patients and in donors. Statistical comparison between
the controls and study patients could be applied only to
‘nonzero’ group. Altogether thirteen markers were found
corresponding to the criteria of ‘Nonzero’ group: 10h18,
a19, i14, i15, 19cyc, h14, a15, 17:1, i16, 18:1D11, h16, a17,
h18. The Mann – Whitney test has shown valuable differences at p B0.03 for five markers: a19, i14, i15, h14, a15.
But this test should preferably be applied to normal,
Gaussian frequency distribution. Because of real right-side
tailing of distribution, we apply also Kolmogorov-Smirnov
test as more convenient one. According to this test two
more markers 19cyc and 18:1D11 have shown valuable
difference at pB 0.05 between the patient and control
groups. As a result of these tests six following markers,
i.e. 10h18, 17:1, i16, h16, a17 and h18 are remaining out
of statistical confirmation, that are assigned mainly
Table III
Concentration (ng/ml) of fatty acids— structural components of bacterial cells — and metabolic sterols
identified in blood of patients (n = 59)
Name
Abbreviation*
Detection frequency
No. of persons
% of total
Hydroxy-acids
Hydroxy-decanoic
Hydroxy-lauric
2-Hydroxy-lauric
Hydroxy-myristic
2-Hydroxy-myristic
Hydroxy-iso-pentadecanoic
Hydroxy-palmitic
Hydroxy-iso-heptadecanoic
Hydroxy-stearic
10-Hydroxy-stearic
Hydroxy-iso-eicozanoic
h10**
h12
2h12
h14
2h14
hi15
h16
hi17
h18
10h18
hi20
15
37
17
48
11
30
59
28
59
59
8
25
63
29
81
19
51
100
47
100
100
13
0–96
0–120
0–90
0–114
0–90
0–120
6–300
0–90
6–1800
6–1380
0–120
Branched fatty acids
anteiso-Tridecanoic
iso-Myristic
iso-Pentadecanoic
anteiso-Pentadecanoic
iso-Palmitic
iso-Heptadecanoic
anteiso-Heptadecanoic
anteiso-Nonadecanoic
Tuberculostearic
a13
i14
i15
a15
i16
i17
a17
a19
10Me18
54
59
59
59
59
59
59
59
54
91
100
100
100
100
100
100
100
91
0–126
50–540
60–1380
120–1620
12–3000
40–5200
102–6900
6–2040
0–108
Unsaturated fatty acids
9,10-Tetradecenoic
iso-Heptadecenoic
Heptadecenoic
cis-Vaccenic
14:1
i17:1
17:1
18:1D11
51
33
59
34
86
56
100
58
0–2160
0–60
60–2460
0–1440
Cyclopropanoic acids
cyclo-Heptadecanoic
cyclo-Nonadecanoic
17cyc
19cyc
29
57
49
97
0–60
6–120
26
20
10
44
33
16
0–540
0–6160
0–360
Alcohols
Coprostanol
Cholestendiol
Metabolite 428
Range
* Marking: 17:1–17- is the number of carbon atoms, the figure after colon denotes the number of
double bonds; h-hydroxy-acid; a,i-indicates methyl-branching; cyc-means cyclopropanoic acid. For
example, 2hi15 means 2 -hydroxy-iso-pentadecanoic acid.
** 3-hydroxy-acids—if position of hydroxyl is not indicated.
SMOM and microbe – host relationships
17
Fig. 1. Distribution of microbial
markers values (blood, donors n =
25). For better comparison of
markers which concentration are
differ substantially in scale their
values were normalized to interval:
minimal value was subtracted from
each measurement, and residue was
divided to interval.
to C. perfringens, C. albicans, actinomycetes, B. urealyticum, corineforms and H. pylori, respectively. But we
have no doubt about microbial origin of this substances.
Three of them are hydroxy acids and come from gramnegative microbes’ LPS. Heptadecenoic acid 17:1 shows
much enough circle and asterisk points (Fig. 2, patient), as
well as a17, 10h18, h16 and i16. More data are needed for
statistically reliable results for this markers. Blood levels of
H. pylori h18 markers show no difference between patients
with stomach ulcer and donors, for this marker is known
to accumulate rather in urine, than in blood (our unpublished data).
Thorough search for volatile bacterial phenyl derivatives
in healthy people’s blood gave the following results (Table
IV). Four acids from this list—phenylacetic, phenylpropi-
onic and their hydroxy-derivatives– are known as anaerobic bacteria metabolites (18). The table shows that blood
of healthy people contains benzoic acid, benzaldehyde and
two phenol alcohols — phenylmethanol and phenoxyethanol. These substances are rarely found in unhealthy, while phenylacetic and phenylpropionic acids, as
well as their hydroxy-derivatives and phenylpentadecan,
are characteristic for the disease.
Concentration of phenylcarbonic substances in blood of
healthy people seems to be individual, for it fluctuates
from barely detectable quantities (10 ng/ml) to 10 mg/ml.
We are far from considering the details of phenol substances quantitative analysis as faultless as it is with fatty
acids, that is why explanations for these fluctuations are
not offered.
18
N. V. Beloborodo6a and G. A. Osipo6
DISCUSSION
In summary Table V certain SMOMs, found in human
blood, are related with maximal probability to definite
groups of microorganisms. These data are based on the
results of our investigation on chemical composition of
bacterial cell fatty acids (16, 17), as well as on digest of
scientific literature on the issue, in particular on chemodifferentiation of bacteria (15).
Presence of SMOMs in healthy people could be explained
by natural mechanisms of phagocyte digestion up to eventual products of bacteria, normally colonising mucous
membranes of respiratory and gastrointestinal tracts. It is
well known, that symbiotic microflora plays an important
role in health maintenance, which is explained by a number
of factors, such as colonization resistance mechanisms,
synthesis of vitamins, etc. Bacterial cells are also destroyed
by such mechanisms as autolysis, or enzymatic hydrolysis
by blood protein complement, etc. Cells are cleaved up to
monomers of biologic polymers. It has been estimated, that
every minute bloodstream/interstitial space exchange of
plasma reaches up to 70% of total plasma volume, including SMOMs. That is why small fragments of microbial
biopolymers, secreted into interstitial space, practically
instantly get into bloodstream. Taken into account, that
phagocytes stay mostly extravascularly— i.e. interstitially,
easily migrating through vascular wall, it is natural to
presume, that microbial markers as well as phagocytes
and transporting proteins get continuously and quickly
Fig. 2. Distribution of microbial
markers values (blood, patients
n =161). See Fig. 1 comments.
SMOM and microbe – host relationships
19
Table IV
Phenyl-carbonic substances in blood of healthy donors and patients
Name
Phenylmethanol
Benzoic acid
Benzaldehyde
Phenoxyethanol
Phenylacetic acid
Phenylpropionic acid
Hydroxy-phenylacetic acid
Hydroxy-phenylpropionic acid
Phenyl-pentadecane
Number of positives
Healthy donors (n = 18)
Patients (n =10)
No. of persons
%
No. of persons
%
18
16
9
14
0
0
0
0
0
100
88
50
78
0
0
0
0
0
2
5
0
0
6
4
3
3
3
20
50
0
0
60
40
30
30
30
into bloodstream independent of inflammatory site. I.e.
range of microbial markers reflects the spectrum of microbial species within the human body, independent of their
habitat or inflammatory process location.
Decreased concentration of some SMOMs under certain
inflammatory conditions, and especially in case of treated
(by broad spectrum antibiotics) sepsis, might reflect ‘micro- ecological’ breaks in affected human organism. This
did not affect the patient distribution curves against
donors in its left ‘healthy’ branch, but results in frequency
of simultaneous decreasing several markers in patients.
Although, this phenomenon could be interpreted differently, i.e. microbial–immunologic homeostasis of a
healthy human organism is provided through balanced
concentration of various SMOMs; some of them are responsible for activation of macrophages, others—for their
inhibition. It is likely, that SMOMs, that are found in
maximum concentrations in blood of healthy people, inhibit macrophage reaction to all foreign agents. Results of
the research allow to presume inhibiting properties in
branched fatty acids and some phenyl-carbonic substances.
Hypothesis on existence of microbe-induced inflammatory process inhibitors looks natural, for such a mechanism is really needed to preserve immunologic homeostasis
under constant inflow of bacteria into human organism, as
for example, bacterial translocation from mucous membranes. Clinically manifested inflammation develops only
when pro-inflammatory SMOMs significantly prevail over
anti-inflammatory SMOMs.
Results of this research enabled us to formulate the
Concept of homeostasis of small molecules originating
from microbes (SMOM) in human organism, which is an
important regulating mechanism of the relationship between host’s organism and endogenous microflora.
It should be pointed out, that in a healthy person
markers of normally predominating intestinal bacteria —
such as Bacteroides, Eubacterium and Bifidobacterium—
are absent or barely found (traces), which favours the
theory on immunology tolerance towards indigenous
anaerobes and, consequently, low probability of their
phagocytosis.
Quite obvious, that presence of markers in the blood
does not indicate bacteriemia: small molecules can penetrate into bloodstream from all sites of natural replication — i.e. from microflora loci in any part of the body.
Apparently high concentration of SMOMs is explained
by their accumulation in monocytes, macrophages and
neutrophils. It is well known, that neutrophils are natural
accumulators of different microbial markers, for each cell
is capable to ‘digest’ hundreds of bacteria during its short
life, which lasts for 6 – 8 h in bloodstream and up to two
days in interstitial space or on mucous membranes.
As it is seen in Table V, SMOMs prevailing in blood of
healthy people are the markers of such bacteria, as Bacteroides, Bacillus, Candida, Enterococcus, Clostridium, and
diphteroids i.e. basic representatives of endogenous human
microflora. But these SMOMs might be as well crossmarkers, for are found in other microbes. Markers of
potentially pathogenic microbes, such as Peptostreptococcus anaerobius, Clostridium perfringens, Staphylococcus,
Candida, Pseudomonas, Eubacterium, Klebsiella and others
were dominating in blood of patients with infectious manifestations. In this research no markers of absolutely
pathogenic bacteria (such as tuberculostearinic acid —of
Mycobacterium tuberculosis; hydroxydodecanoic acid —of
Neisseria, Moraxella; 2,3-dihydroxy-iso-myristinic acid—
of Legionella, and also markers Brucella, Francisella, Treponema) were found in either group of healthy people, or
patients with non-specific purulent-septic conditions.
Quantitative analysis of SMOMs revealed significant
deviations in case of disease. For a number of SMOMs
deviations were fixed in both directions — towards elevation and decrease. That is especially characteristic for
branched and unsaturated acids, components of bacilli and
lactobacilli cells, corine- and propionic bacteria, streptococci and candida.
20
N. V. Beloborodo6a and G. A. Osipo6
Basic postulates of the Concept of SMOM homeostasis
could be formulated as follows:
1. Molecules of microbial origin are always present in
blood of healthy and ill people. Some molecules activate host’s cells (first of all monocytes, macrophages),
thus inducing put out of eicosanoids (prostaglandines,
leucotriens, tromboxans), interleikins and other cytokines, while other inhibit cell’s activity; admitted, that
some molecules could stay indifferent.
2. In healthy organism normal symbiotic microflora is
considered to be the source of SMOMs.
3. Healthy organism preserves balance between SMOMs,
having pro- and anti-inflammatory activity. Stability of
this balance is reached thanks to functional interchangeability of functionally identical SMOMs.
Healthy state is provided by domination of inflammation inhibitors over inflammation inductors. Excess of
inhibitors allows to avoid inflammatory reactions in
case of accidental bacterial intrusion — as it is seen in
translocation.
4. Bacterial molecules — markers of pathogenic bacteria — are missing in blood of healthy people and also in
Table V
Database of chemical substances, related to those microorganisms, in which they are found the most often
1
Code*
Chemical name
Characteristic microorganism
1
2
3
4
5
6
7
8
9
10
11
11
12
13
14
15
16
17
10:0
a13
i14
14:1D9
i15
à15
i16:0
i17:1
i17:0
a17:0
17:1
17cyc
17:0
18:1D11
i18
19cyc
a19
10Me18
Decanoic
anteiso-Tridecanoic
iso-Myristic
9,10-Tetradecanoic
iso-Pentadecanoic
anteiso-Pentadecanoic
iso-Palmitic
iso-Pentadecenoic
iso-Heptadecanoic
anteiso-Heptadecanoic
Heptadecenoic
cyclo-Heptadecanoic
Heptadecanoic
cis-Vaccenic
iso-Octadecanoic
cyclo-Nonadecanoic
anteiso-Nonadecanoic
10-Methyl-octadecanoic
(tuberculostearic) hydroxyl-acids
Streptococcus
Bacillus cereus
Peptostreptococcus anaerobius, Streptomyces, Bacillus
Streptococcus pneumonia, Clostrydium
Propionibacterium, Bacteroides, Staphylococcus
Staphylococcus, Bacillus, corynebacteria
Bacillus, Streptomyces, Bacteroides, Bre6ibacterium
Fla6obacterium
Bacillus, Pre6otella, Propionibacterium
Corynebacterium (diphteroids)
Candida albicans
Enterobacteriaceae
Human cells, invariant
Serratia, Enterobacteriaceae, Pseudomonas,
Bacillus subtilis, Peptostreptococcus, Bifidobacterium,
Enterococcus, Lactobacillus
Staphylococcus
Mycobacterium, Corynebacterium xerosis group, C. urealyticum
Hydroxy-decanoic
Hydroxy-dodecanoic
Hydroxy-lauric
Hydroxy-tridecanoic
Hydroxy-myristic
Hydroxy-acids
18
h10
19
h12:1
20
h12
21
h13
22
h14
23
24
hi15
h16
Hydroxy-iso-pentadecanoic
Hydroxy-palmitic
25
26
27
28
29
30
31
32
hi17
h18
10h18
hi20
h22**
2h12
2h14
2hi15
Hydrohy-iso-heptadecanoic
Hydroxy-stearic
10-Hydroxy-stearic
Hydroxy-iso-eicosanoic
3-Hydroxy-docosanoic
2-Hydroxy-lauric
2-Hydroxy-myristic
2-Hydroxy-iso-pentadecanoic
Pseudomonas
Pseudomonas aeruginosa
Pseudomonas, Neisseria, Vibrio, Acinetobacter
E. coli, Bacteroides hypermegas, Selenomonas
Enterobacteriaceae, Fusobacterium, Neisseria, Haemophilus,
Moraxella, Campylobacter
Pre6otela
Bacteroides urealyticum, Brucella, Wolinella, Fusobacterium,
Bordetella, Campylobacter
Bacteroides fragilis
Helicobacter pylori, Francisella, Brucella
Clostridium perfringens
Chlamydia trachomatis
Chlamydia trachomatis
Pseudomonas aeruginosa, Acinetobacter
Klebsiella
Fla6obacterium
Coprostanol (cholestanol)
Ergosterol
Eubacterium
Candida, Aspergillus
Sterols
33
34
* Marking: 17:1–17- is the number of carbon atoms, the figure after colon denotes the number of double bonds; h-hydroxy-acid;
a,i-indicates methyl-branching; cyc-means cyclopropanoic acid. For example, 2hi15 means 2 -hydroxy-iso-pentadecanoic acid.
** 3-hydroxy-acids—if position of hydroxyl is not indicated.
SMOM and microbe – host relationships
purulent-septic conditions. Broken homeostasis of
SMOM serves as the basis for development of non-specific infectious diseases.
5. Adequate and optimal is the therapy which is aimed at
and provides rebalancing of SMOMs, activators and
inhibitors of phagocytes.
6. Volatile aromatic ‘microbial’ substances have the most
accentuated inhibiting properties Anaerobic intestinal
flora stays as the main source of these substances.
Provision and restoration of normobiocenosis guarantees SMOM homeostasis.
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