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SEPSIS AND SEPTIC SHOCK: PATHOGENESIS AND TREATMENT PERSPECTIVES

Hayk Minasyan
Address for correspondence:
Mamikonyanz 38-38, Yerevan, Armenia, 0014
Tel: [+374] 77255295
E-mail address: haykminasyan@rambler.ru

Abstract
The majority of bacteremias do not develop to sepsis. Bacteria usually are cleared from the
bloodstream. Humoral immunity and oxygen released from erythrocytes are the main bactericidal
factors in the bloodstream. Sepsis begins when bacteria are resistant to oxidation and start to
proliferate in erythrocytes. Abundant release of oxygen from erythrocyte to the plasma triggers a
cascade of events that cause: 1. oxygen delivery failure to cells and hypoxia; 2. oxidation of
plasma components and impairment of hormonal regulatory mechanisms. 3. Hypoxia and
hormonal dysregulation cause multiple organs’ failure. Bacterial reservoir inside erythrocyte
provides long-term survival of bacteria and ineffectiveness of antibiotics and host immune
reactions. Treatment perspectives that include different aspects of sepsis development are
discussed.

Keywords: bacteremia, sepsis, septic shock, pathogenesis, treatment.

Introduction
Sepsis is both best known yet most poorly understood medical disorders [1]. Sepsis leads to
shock, multiple organ failure and death if not recognized early and treated promptly [2]. It is a
serious clinical condition that represents a patient’s response to infection and has a high mortality
rate [3]. Sepsis remains the dominant challenge in the care of critically ill patients [4]. Up to 19
million cases of sepsis worldwide per annum is estimated. The true incidence is higher [1].
Sepsis is associated with a mortality rate of 25 - 30% and mortality due to septic shock is 50-85%
[6-8]. Patients with sepsis requiring intensive care unit (ICU) admission had high rates of ICU
and overall hospital mortality, ranging from 18 to 50% [9-12]. The most common sites of
infection are the lungs (40%), abdomen (30%) and urinary tract (10%) [13]. Gram-positive and
poly microbial infection accounted for 30%-50% and 25% of cases respectively [14]. In another
research, gram-negative bacteria were isolated in 62% of patients with sepsis [15]. Gram-
negative infection most often occurs in the lung [16]. Staphylococcus aureus and Streptococcus
pneumoniae are gram-positive isolates, whereas Escherichia coli, Klebsiella species, and
Pseudomonas aeruginosa predominate among gram-negative isolates [17, 18]. Gram-positive
organisms cause sepsis by at least two mechanisms: by producing exotoxins that act as
superantigens and by their cell wall components that can stimulate immune cells [19].
Lipopolysaccharide (LPS, endotoxin) is a component of the membrane of Gram-negative bacteria
that causes sepsis [20]. Bacterial toxins play a pivotal role in the pathophysiology of sepsis and
mechanisms of multiple organ system dysfunction. However, the literature illustrates that no
single mediator/system/pathway/pathogen drives the pathophysiology of sepsis [21]. Current
knowledge of sepsis pathogenesis [1, 22-26] includes infection development and interaction with
the host before bacteria enter the bloodstream. Actually, the mechanisms of host defense in the
extravasal compartment (local immunity) is different from the mechanisms of intravasal
(bloodstream) defenense because extravasal defense is provided mainly by leukocytes whereas
intravasal cellular defense is fulfilled by erythrocytes. The humoral immunity events of pre-septic
stage affect bacteremic (septic) stage and interfere with the study of sepsis “per se.” As a result,
the pathogenesis and pathophysiology of some pivotal aspects of sepsis remain unclear.

1. Pre-septic (local) and septic (bloodstream) stages of infection

Not all bacteremias lead to sepsis. People have everyday bacteremia, particularly, from oral
cavity, but sepsis develops rarely [27-31]. It occurs when the infection is resistant to host
antibacterial defense. The latter is different in the bloodstream and tissues. If the infection
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develops locally (tissue, cavity, etc.) and then enters the bloodstream, there are two stages of
sepsis: pre-septic (local) and septic (generalized). If infection enters the bloodstream directly
from an external source (intravenous injection, bite, etc.), the pre-septic stage is absent. Local
antibacterial defense is provided by phagocytosis (leukocytes and their local versions: resident
macrophages), immune reactions and bactericidal humoral factors, NETs, etc., whereas in the
bloodstream bacteria are killed by humoral factors and oxygen released from erythrocytes [32,
33]. Blood natural resistance factors (complement, lysozym, etc.) are not effective enough if
infection enters the blood from the tissues. Proliferating in the tissues, bacteria become resistant
to serum bactericidal factors (they are available both in serum and the tissues). Sepsis develops
when bacteria in the bloodstream survive oxidation on the surface of erythrocytes [32-36].

2.The features of sepsis causing bacteria

Relatively few pathogens can cause sepsis. For causing sepsis bacteria should have certain
features that provide their survival, proliferation and dissemination in human body. The
characteristics of the pathogens, that most frequently cause sepsis, may or may not be common
for all of them (Table 1).
Sepsis causing bacteria are both gram positive and gram negative. Gram-positive organisms are
better suited to invade host tissues and elicit, in general, a brisker phagocytic response than gram-
negative organisms [37]. The lack of endotoxin in the outer cell wall is compensated for by the
presence of exposed peptidoglycan and a range of other toxic secreted products. It appears that
cell wall components of gram-positive bacteria may signal via the same receptor as gram-
negative endotoxin, although the type of signal and coreceptor may differ [37]. Gram-negative
organisms are associated with poorer outcomes in first-hit infections; an inverse relationship
between Gram status and mortality is observed in second-hit infections [38].
The majority of sepsis causing bacteria is facultative anaerobes. These organisms can switch
between aerobic and anaerobic types of metabolism: they make ATP by aerobic respiration if
oxygen is present, but are capable of switching to fermentation or anaerobic respiration if oxygen
is absent [39]. This type of respiration is the most flexible and it facilitates pathogen survival,
proliferation and dissemination in the variety of environmental conditions. The pathogens that are
not facultative anaerobes, may express additional respiratory mechanisms that make their
respiration close to facultative anaerobes. For example, Neisseria meningitides is usually treated
as a strict aerobe and is cultured under fully aerobic conditions in the laboratory. Although N.
meningitidis fails to grow under strictly anaerobic conditions, under oxygen limitation the
bacterium expresses a denitrification pathway (reduction of nitrite to nitrous oxide via nitric
oxide) and this pathway supplements growth. The expression of the gene aniA, which encodes
nitrite reductase, is regulated by oxygen depletion and nitrite availability via transcriptional
regulator FNR and two-component sensor-regulator NarQ/NarP respectively [40, 41] .
All sepsis causing bacteria produce superoxide dismutase (SOD), catalase and glutathione
peroxidases, that protect them against oxidative stress caused by reactive oxygen species. The
primary source of oxidative stress for sepsis causing bacteria is the attack by host phagocytic
cells. The generation and release of toxic reactive oxygen species by phagocytic cells is thought
to be an important component of the host’s immunity against bacterial infections. In response, all
successful pathogens, including sepsis causing bacteria, have evolved effective systems for
defense against oxidative stress that include combinations of reducing enzymes, molecular
scavengers, and protein and DNA repair enzymes [42]. Phagocytes utilize the cytotoxic effects of
the reactive oxygen species, such as superoxide, hydrogen peroxide, and the highly toxic
hydroxyl radical. These reactive oxygen species can damage the nucleic acids, proteins, and cell
membranes of pathogens. Sepsis causing bacteria have evolved effective enzymatic pathways of
oxidant inactivation, including those catalyzed by superoxide dismutase (SOD),
catalase/peroxidase, and glutathione in combination with glutathione peroxidase and glutathione
reductase [43]. The same pathways may protect sepsis causing bacteria from oxidation and
killing on the surface of erythrocytes [34].
Sepsis causing bacteria may be either oxidase positive or oxidase-negative. The production of
cytochrome c oxidase has no critical role in causing sepsis.
Certain structures of bacteria is indispensable for causing sepsis. All sepsis causing bacteria have
S-layer and produce capsules, slime layer and biofilm. These structures protect the bacteria in
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the tissues against phagocytosis, ROS, lytic enzymes, immune complexes, etc., whereas in the
bloodstream capsule and slime layer protect the bacteria against triboelectric charging, attraction
and fixation on the surface of erythrocytes, oxidation and killing by the oxygen released from
erythrocytes [33].
Sepsis causing bacteria produce hemolysins . Erythrocytes are thought to be the main bactericidal
cells in the bloodstream and hemolysins are necessary for bacterial survival in the blood. If the
speed of bacterial growth in the tissue is limited by host immune reactions, bacteria have enough
time to produce a capsule, slime layer and biofilm for surviving host attacks. After entering the
bloodstream, bacterial capsule and slime layer prevent triboelectric charging and fixation on the
surface of erythrocytes. In case of bacterial rapid proliferation in the tissue they are short of time
to produce a capsule and slime layer and after entering the bloodstream, they are triboelectrically
charged and then are electrically caught and fixed on the surface of erythrocytes. If bacteria
survive oxidation on the surface of erythrocytes, they produce hemolysins that destroy
erythrocytes or provide bacterial penetration into the inner space of erythrocytes that is a rich
source of protein and iron for bacterial growth. Hemolysins are important for the development of
sepsis to advanced stages.
Motility is not a crucial factor for causing sepsis. Sepsis causing bacteria may be either motile or
not motile organisms.

3.Bacteria killing in the tissues

In the tissues the main bactericidal cells of innate immunity are neutrophils, monocytes and their
local versions – resident macrophages. Upon encountering bacteria, neutrophils engulf these
microbes into a phagosome, which fuses with intracellular granules to form a phagolysosome [44
]. In the phagolysosome the bacteria are killed after exposure to enzymes, antimicrobial peptides
and reactive oxygen species (ROS). The arsenal of cytotoxic agents has been traditionally
divided into either oxygen- independent or -dependent mechanisms. Both of these systems
collaborate in killing microbes [45]. The second mechanism of neutrophil killing is oxygen-
dependent [46]. Phagocytosing neutrophils undergo an ‘oxidative burst’ during which the
NADPH oxidase complex assembles at the phagosomal membrane and produces O 2-, which is
rapidly converted to hydrogen peroxide by the enzyme superoxide dismutase. In turn, a
constituent of the azurophil granules, myeloperoxidase, generates hypochlorous acid (HOCl)
from hydrogen peroxide. HOCL is the most effective bacterial killer.
Besides killing bacteria inside the phagolysosomes, neutrophils can also degranulate and release
antimicrobial factors into the extracellular space [47]. The cells can also generate neutrophil
extracellular traps (NETs), which are composed of granule and nuclear constituents that kill
bacteria extracellularly [48].
Neutrophil extracellular traps (NETs) are networks of extracellular fibers, primarily composed of
DNA from neutrophils, which bind pathogens. NETs disarm pathogens with antimicrobial
proteins such as neutrophil elastase, cathepsin G and histones that have a high affinity for DNA
[49]. NETs provide for a high local concentration of antimicrobial components and bind, disarm,
and kill microbes extracellularly independent of phagocytic uptake. In addition to their
antimicrobial properties, NETs may serve as a physical barrier that prevents further spread of the
pathogens. Furthermore, delivering the granule proteins into NETs may keep potentially injurious
proteins like proteases from diffusing away and inducing damage in tissue adjacent to the site of
inflammation [50].
Platelets activate neutrophils to trap bacteria. Platelets rapidly localize to sites of injury and
infection [51]. Both platelets and neutrophils have the potential to trap microbial pathogens
independently of each other, however, together platelet-neutrophil interactions induce
transcellular synthesis and hyperactivation of neutrophils to produce increased pro-inflammatory
molecules [50, 52]. Platelets have the ability to bind and internalize bacteria and viruses through
engulfing endosome-like vacuoles that fuse with the α-granules of the platelet and allow the
granular proteins to have access to the pathogen [53]. As a result, thrombocytopenia correlates
with the severity of the sepsis and the rate of mortality [54].

4.Survival of sepsis causing bacteria in phagocytes

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After phagocytosis by macrophages, bacteria are located in a membrane-bound vacuole

(phagosome), but the ensuing trafficking of this vacuole and subsequent bacterial survival
strategies vary considerably [55]. If the ingested bacteria have no intracellular survival
mechanisms, the bacteria-containing phagosomes fuse with the lysosomal compartment, and
bacteria are digested within 15–30 min. The metabolic burst in activated phagocytes results in
production of nitric oxide and reactive oxygen species, such as chloramines, hydroxyl radicals,
and hydrogen peroxide, which are usually converted into the potent oxidant hypochlorous acid
[56]. The cascade of these events is the following (Fig.1). After phagocytosis lysosomes fuse
with the phagosome, forming a phagolysosome and proteases are introduced into the phagosome
in this way. In addition, a membrane protein phagocyte oxidase(NADPH oxidase) winds up in
the membrane of the phagolysosome.
Phagocyte oxidase takes an electron from NADPH and transfers it to O2, forming the superoxide
radical, O2-. . The superoxide radical is moderately reactive and it is soon converted to hydrogen
peroxide by superoxide dismutase. Hydrogen peroxide can damage microbes, but it is converted
to more effective bactericidal (HOCl) by myeloperoxidase. Hypochlorite is the most effective
intracellular bactericidal.
Sepsis causing bacteria protect themselves against the oxygen-dependent bactericidal mechanism
of phagocytes by producing superoxide dismutase (SOD), catalase and glutathione peroxidase
(Fig.1). Bacterial superoxide dismutase accelerates the conversion of superoxide (02) to
hydrogen peroxide (H202), while bacterial catalase and glutathione peroxidase accelerate the
conversion of hydrogen peroxide (H202) to water and oxygen (02) that is relatively non toxic for
facultative anaerobes. This conversion rapidly depletes all converted hydrogen peroxide to
innocuous water and oxygen and prevents formation of extremely harmful for bacteria
hydrochloride.

5.Bacteria killing in the bloodstream

Leukocytes cannot recognize and engulf pathogens in high velocity liquids, and erythrocytes are
the main bactericidal cells in the bloodstream [32, 33]. Bacteria are attracted and fixed on the
surface of erythrocytes by electrical charge interaction force. Bacteria activate erythrocyte
membrane receptors, causing the oxygen release (from oxyhemoglobin) that kills bacteria by “per
contact” oxidation. If this mechanism is effective, bacteria are killed on the surface of
erythrocytes and then are disintegrated and digested in the reticuloendothelial system (Fig.2,
scenario 1). If bacteria enter the bloodstream, having thick capsules that prevent triboelectric
charging, they may avoid attraction, oxidation and killing on the surface of erythrocytes and, as a
result, bacteria are filtered in the liver and the spleen, and their killing and decomposition is
provided by Kuppfer cells and spleen macrophages. In case of bacterial overloading, the liver and
the spleen may be damaged by bacteria and their toxins (Fig.2, scenario 2). If the bacterial
capsule is not thick enough for preventing bacterial attraction and fixation on the surface of
erythrocytes, but bacteria survive oxidation on the membrane of the erythrocytes, they may enter
erythrocytes by making a hole in the membrane. Inside erythrocyte the bacterium is exposed
higher concentrations of oxygen. Released oxygen kills the bacterium by oxidation and then
killed bacterium is released back to plasma. However, bacteria may survive inside erythrocyte if
they are resistant to oxidation inside erythrocytes and/or there is the lack of oxygen (Fif.2,
scenario 3). As a result, bacteria proliferate in erythrocyte and the latter from the bacteria killer
becomes bacterial incubator and reservoir [34, 35]. Bacteria inside erythrocytes have all
necessary nutrients for fast growing and proliferation, besides, they are out of reach of
antibiotics, immune complexes and serum antibacterial factors. Bacterial proliferation tears
erythrocyte membrane and bacteria are released back into the plasma and then are
triboelectrically charged, attracted and fixed on the surface of erythrocytes and the cycle of
entering erythrocytes, proliferating, releasing and contaminating new erythrocytes occurs again
and again.

6.Sepsis and septic shock pathogenesis

Host response to infection is a complex process that localizes and controls bacterial invasion,
while initiating the repair of injured tissue. Sepsis develops when the response to infection
becomes generalized and involves normal tissues remote from the site of injury or infection.
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Oxidation of the components of the blood is one of causes that provide distant tissues injury and
disregulation in sepsis.
6.1.Oxidation of blood plasma components
The oxidation of blood plasma components, including regulatory hormones, proteins, peptides
and other active substance is one of ignored factors of sepsis and septic shock. Oxidation of
plasma components is caused by oxygen release to plasma, and it destroys humoral regulation of
different cells, tissues and organs. Human body comprises two separate but interacting
compartments: (a) compartment of blood circulation (pulmonary and systemic circulation); (b)
compartment out of blood circulation. In the majority of cases, bacterial infection proliferates in
the compartment out of blood circulation and then enters into the compartment of blood
circulation. Bacteria blood invasion has different consequences from innocuous bacteremia to
fatal septic shock.
Bacteremia, sepsis, severe sepsis, septic shock may be interpreted as different segments of a
continuum determined by the amount of oxygen released by erythrocytes into plasma. Bacterial
stimulation of surface receptors of erythrocytes causes the oxygen release. The more oxygen is
released from erythrocyte to the arterial blood, the more severe is sepsis. The consequences of the
abundant release of oxygen are multiple. First, erythrocytes become unable to supply oxygen and
perform their respiratory (oxygen transportation) function. As a result, general multi-organ
hypoxia develops. Second, released oxygen is highly reactive and destroys and transforms
plasma proteins, peptides, immune complexes, hormones, amino acids, fatty acids, vitamins and
many other substances necessary for cell nutrition, proliferation, protection, energy production,
functioning, etc. Proteins, like lipids and DNA, are substrates for biological oxidation
[57].Oxidative changes to proteins can lead to diverse functional consequences, such as
inhibition of enzymatic and binding activities, increased susceptibility to aggregation and
proteolysis, increased or decreased uptake by cells, and altered immunogenicity [58].The most
important aspect of this oxidation is inactivation of regulatory substances, in particular, hormones
(including pituitary gland hormones). Metal-catalyzed oxidation (MCO) represents a prominent
pathway of hGH degradation [59]. The Growth Hormone and Insulin-like Growth Factor-1
(IGF-1) axis play a pivotal role in critical illness, with a derangement leading to profound
changes in metabolism. Protein wasting with skeletal muscle loss, delayed wound healing, and
impaired recovery of organ systems are some of the most feared consequences [60]. Growth
hormone administration reduces nitrogen production and improves nitrogen balance in patients
with severe sepsis [61]. Oxidative inactivation of other proteins, for example, insulin, impairs
the ability of cells to uptake glucose, amino acids and other essential substances. Dityrosine
formation and other oxidative chemical changes of insulin due to its oxidation decrease and
abolish its biological activity [62]. Insulin is necessary for glucose entering many kinds of cells,
so deactivation of insulin causes hyperglycemia - one of the metabolic derangements that
influence sepsis outcome [63-66].
The oxidation of blood components by the oxygen released from erythrocytes may cause
hypothalamic-pituitary-adrenal insufficiency [67]. Primary and secondary adrenal insufficiency
occurs in patients with sepsis and is associated with a poor outcome [68 - 71]. Blood oxidation
affects the hypothalamo-pituitary-thyroidal axis, inactivating thyrotropine , thyroid gland
hormones (triiodothyronine(TT3), thyroxine (TT4) and their binding proteins. Thyroid
hormone regulates metabolism and has an impact on sepsis prognosis. The level of TT4 is lower
in patients with septic shock than in patients without septic shock [72-74]. De-iodinations of
iodothyronines play key roles in metabolic regulation [75, 76].
Vasopressin (Antidiuretic hormone) also is oxidized by the oxygen released from erythrocytes.
Vasopressin plays a role in circulatory homeostasis and serum osmolality. Oxytocin and
vasopressin are oxidized with the formation of dityrosine [77]. Its oxidation and depletion
cause vasodilatory shock - a syndrome with high mortality [78-81]. Low expression levels of
Angiotensin II and ACE (angiotensin converting enzyme) are valuable in predicting the
mortality of patients with severe sepsis [82]. Systemic vasodilatation and arterial hypotension
are landmarks of septic shock.
Albumin oxidation causes hypoalbuminemia in sepsis [83, 84]. Albumin, a 66.5 kDa protein, is
quantitatively the most important plasma protein [85]. Albumin is the main determinant of
plasma oncotic pressure, it plays a pivotal role in modulating the distribution of fluids between
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compartments, and exhibits many other biological functions, such as transport of endogenous
and exogenous compounds, modulation of capillary permeability, neutrophil adhesion and
activation, hemostasis and free radical scavenging (for review see 86]. Even mild oxidation of
human serum albumin (HAS) impairs HSA functional properties including protease
susceptibility, ligand-binding affinity and antioxidant activity [87]. The major structural change
in oxidized HSA is a disulfide-bonded cysteine at the thiol of Cys34 of reduced HAS [88].
HAS exerts important antioxidant activities against oxidative damage. Oxidative damage results
in protein modification. The antioxidant properties of HSA are largely dependent on Cys34 and
its contribution to the maintenance of intravascular homeostasis, including protecting the
vascular endothelium under disease conditions related to oxidative stress [89]. Hypoalbuminemia
is an independent mortality predictor [90]. Albumin is recommended as the resuscitation fluid in
sepsis [91,92], although it is still unclear whether the use of albumin decreases mortality or not
[93,94].
Oxygen released from erythrocyte destroys also plasma other proteins, including immune
complexes and immunoglobulins, particularly IgG and IgM . The initial steps of oxidation may
change the specificity and avidity of immunoglobulins due to chemical alteration of the
hypervariable region. The oxidation of antibodies increases the hydrophilic nature of the
paratopes and makes them more susceptible for the binding to cationic surfaces, even without the
strong surface-to-surface fitting. The oxidation of IgG significantly changes the
immunoreactivity and specificity of IgG fractions [95]. Oxidation of the histidine residue in a
human immunoglobulin gamma (IgG) 1 molecule was discovered by mass spectrometry [96].
Oxidized immunoglobulins have autoimmune and proinflammatory activity [97, 98].
Low levels of immunoglobulins are frequent in severe sepsis and septic shock [99-101].
However, intravenous immunoglobulins (IVIG) as adjunctive therapy for sepsis have not shown
the benefit for the treatment of sepsis [102, 103]. It may be explained by the destruction
(oxidation) of injected immunoglobulins, besides, bacteria inside erythrocytes are out of reach of
immunoglobulins.
Thus, oxygen release to blood plasma from erythrocytes destroys humoral regulation. This may
be one of the causes of the development of multiple organ dysfunction syndrome (known as
multiple organ failure or multisystem organ failure) [104-107].
6.2. Anemia, cell hypoxia and lactate production
The release of oxygen from erythrocytes to plasma in arterial blood (before erythrocytes enter to
capillaries) causes failure of oxygen delivery to cells and hypoxia [33-36]. Another co-factor of
hypoxia is anemia.
Many factors contribute to the development of anemia in sepsis, including blood sampling,
decreased erythrocyte synthesis, bone-marrow suppression, low production of erythropoetin,
increased erythrocyte uptake, etc. [108-111]. The main factor of anemia in sepsis is increased
destruction of erythrocytes caused by: a) erythrocyte membrane injury caused by bacteria
located on the surface of erythrocyte; b) erythrocyte membrane holes and hemoglobin pouring
out as a result of bacterial penetration into the inner space of erythrocyte; c) increased destruction
of injured erythrocytes and bacteria containing erythrocytes in blood plasma and in the RES,
particularly, in the spleen [33-36].
Diminished availability of oxygen at the cellular level determines general dysfunction of cells.
Tissue-related hypoxic injury results from hypoxemia and hypoperfusion and cytokine-mediated
mitochondrial dysfunction termed cytopathic hypoxia [112-116]. The lack of oxygen transforms
cell metabolism from aerobic to anaerobic. As a result, Krebs cycle is suppressed and anaerobe
metabolism with lactic acid accumulation occurs. Elevated lactic acid is a marker for the
suboptimal supply of oxygen to the tissues and is associated with increased mortality in sepsis
[117-127]. Lack of oxygen delivery to the tissues results in decreased cellular metabolism and
increase in cellular lactate production [117-121]. High levels of lactic acid are associated with
increased mortality [118-127]. This association is independent of organ dysfunction [118-122].
Lactate clearance is more useful parameter for guiding therapy (the initial lactate - subsequent
lactate/initial lactate × 100) [117-120]. Lactate non clearance in sepsis is a significant
independent predictor of death [117,119].

7.Diagnostic problems
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Sepsis diagnosis relies on nonspecific physiological criteria (including changes in temperature

and heart and respiration rates) and culture-based pathogen detection. This results in diagnostic
uncertainty, therapeutic delays, the mis- and overuse of antibiotics and many other problems that
increase mortality [128-131]. Blood cultures are used to identify the pathogens and are the gold
standard for the diagnosis of bacteremic patients. Blood cultures provide unambiguous etiology
of the infection and (following subculture) purified colonies for antimicrobial susceptibility
testing. However, getting the colonies takes two–three days and this approach is slow and leads
to delayed and inappropriate treatment [132-134]. Moreover, sepsis may be culture negative
[135-137] and culture false-positive [138-140]. The accurate and timely detection of sepsis
remains a challenge [141]. For early detection of sepsis different markers are used, for example,
acute-phase protein biomarkers [142-144], procalcitonin [145-147], pentraxins [148-149],
cytokine/chemokine biomarkers (IL-6, IL-8, IL-10, TNF-α, etc.)[150-151], macrophage
migration inhibitory factor [152-153], high-mobility-group box 1 (HMGB1) [154,155],
coagulation biomarkers [156, 157], triggering receptor expressed on myeloid cells 1 (TREM-1)
[158-160], midregional proadrenomedullin [161], polymorphonuclear CD64 index [162,163], etc.
Taking into account that positive blood cultures can be found in only 30% of sepsis patients
[164] and low sensitivity of the blood culture method for many slow-growing and fastidious
organisms [165], several molecular approaches (including PCR) have been suggested to improve
the conventional culture-based identification [166], however, a broader clinical evaluation of this
approach is still missing [141]. Another strategy is the extraction and amplification of microbial
nucleic acids from a blood culture and subsequent hybridization on a microarray platform to
detect the gyrB, parE, and mecA genes of 50 bacterial species, which has recently been evaluated
in an observational multicenter design with blood culture as the comparator [167]. Systems
biology approaches such as transcriptomics, proteomics, and metabolomics have been tested as
sepsis biomarkers [168,169]. However, despite decades of research and attempts of sepsis early
diagnostics, improvements in the treatment of sepsis have been modest [170].
Vital phase-contrast microscopy of the blood and microscopy of stained blood samples may be
informative for detection of sepsis. Revealing of living bacteria in erythrocytes shows that sepsis
is developing to more advanced stages. Bacteria may also be seen on the surface of erythrocyte
but as soon as blood is taken from a vessel erythrocytes lose triboelectric charge, and bacteria are
released to plasma [32-35]. Out of the bloodstream (in vitro) bacteria are not triboelectrically
charged and can proliferate [33, 36]. The refractive index of some pathogens is close to the index
of erythrocyte inner media and so these bacteria may be invisible in erythrocytes [32]. Phase-
contrast microscopy and differential interference-contrast microscopy (DIC) are effective and
simple methods for immediate revealing bacteremia and making predictions regarding its course.
Dark field microscopy is an additional microscopic technique, however, it has disadvantages –
artifacts and image distortions. The microscopy of the blood plasma precipitate after plasma
centrifugation and supernatant removal increases the chance of bacteria detection.
Bacterial motion differs from Brownian motion and motile bacteria are easily detected. Non-
motile bacteria, particularly, Staphylococcus and Streptococcus species, are identified by their
microscopic appearance in stained samples of plasma precipitate. However, optical microscopy
of stained blood samples is less informative for detection of bacteria in erythrocytes. Standard
staining of blood samples with methylene blue, eosin, azure cannot reveal bacteria in
erythrocytes. Gram stains of the plasma precipitate after centrifugation may reveal whether
bacteria are gram positive or gram negative [32]. Simultaneous use of different methods of
microscopy (phase-contrast, dark field, microscopy of stained samples, etc.) increase the
effectiveness of bacteremia and sepsis prognosis by revealing whether pathogens have penetrated
erythrocytes or not.

8.Treatment problems:
Sepsis is a systemic infection. Empiric antimicrobial therapy is the cornerstone of the treatment
[171,172]. Current guidelines recommend starting antibiotic therapy within one hour of
identification of septic shock [173]. Every hour delay is associated with a 6% rise in mortality
[174-176]. Survival rates dropped when antimicrobial treatment was delayed to within the sixth
hour [131]. There are no prospective data that early broad-spectrum antibiotic therapy reduces
mortality in severe sepsis [177] but prompt initiation of antimicrobial therapy remains important
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for suspected infections [179]. If the pathogen is resistant to antibiotic, early or late initiation of
antibiotic therapy cannot improve the outcome. Inappropriateness of empirical antibiotic therapy
can contribute to high level of mortality [180]. The crisis emerges of antibiotic resistance for
microbial pathogens [181-183]. Without new and effective antibiotics, the problem will escalate.
However, it is not excluded, that even new antibiotics cannot increase the effectiveness of sepsis
therapy. One of the possible causes is that in sepsis erythrocytes may be a reservoir for bacteria
that proliferate there and are protected inside erythrocytes from antibiotics, immune complexes
and other bactericidal factors [34]. Antibiotics can kill bacteria in blood plasma, but cannot
penetrate erythrocytes and kill bacteria there. Bacterial proliferation tears the membrane of
erythrocytes and bacteria are released into plasma. In the bloodstream released bacteria become
triboelectrically charged and attracted by erythrocytes, fixed on their surface and then penetrate
inside erythrocytes again. The cycle of erythrocyte penetration, proliferation and release to the
plasma is perpetual. The turnaround time of the cycle depends upon bacteria size and
proliferation speed. Constant bacterial reservoirs in erythrocytes decreases antibacterial and
immune therapy effectiveness and may be one of many other factors that make sepsis therapy so
problematic.

9.Treatment perspectives
Although improvements in supportive care of patients with sepsis (more effective and less
damaging mechanical ventilation, improved fluid resuscitation, and broad-spectrum antibiotic
coverage) have improved survival rates, sepsis remains a condition with high mortality. Despite
many clinical trials, to date, no FDA-approved drug is available for use in sepsis, a lack that
underscores the importance of future sepsis research [184].
The biology of sepsis is complex and not specific to infection. The systemic inflammatory
response is biologically complex, redundant, and activated by both infectious and noninfectious
triggers. Its manipulation can cause both benefit and harm. More than 100 randomized clinical
trials have tested the hypothesis that modulating the septic response to infection can improve
survival. With one short-lived exception, none of these have resulted in new treatments. The
current challenge of sepsis research lies in a failure of concept and reluctance to abandon a
demonstrably ineffectual research model [185].
The treatment of sepsis should be based on the understanding of its pathogenesis. The
pathogenesis of sepsis is not fully understood. Bacteria from external or local sources enter
bloodstream causing bacteremia. Taking into account that phagocytosis in the bloodstream is
impossible, blood humoral bactericidal factors and erythrocytes are probably the main
antibacterial forces in the blood circulation. The humoral factors are more effective against
bacteria from external source. Bacteria from a local source may be more resistant to the
bactericidal action of plasma because blood humoral factors also present in the tissues and before
entering the bloodstream from the tissues, bacteria should have overcome their action
Both erythrocytes and bacteria are triboelectrically charged in the bloodstream. The charge of
erythrocytes attracts the charge of bacteria and keeps bacteria on the surface of erythrocytes. The
interaction of the charges at the surface of erythrocyte stimulates the release of oxygen from
oxyhemoglobin. The oxygen is released from hemoglobin to the surface of erythrocytes as a
mixture of different allotropes of oxygen (monatomic oxygen O(3P), dioxygen, singlet oxygen,
triatomic oxygen O3, etc.) that are very reactive and kill bacteria by oxidation. This auspicious
scenario is accompanied with no or mild clinical signs and ends without complications. Bacteria
can survive oxidation having thick capsule, slime layer and producing catalase, SOD and GPX.
If bacteria survive oxidation on the surface of erythrocyte, they may: 1. cause an intense release
of oxygen from erythrocytes to the plasma without penetrating erythrocytes; 2. Cause the oxygen
release and penetrate erythrocytes. If bacteria do not penetrate erythrocytes, immune complexes
and antibiotics may eliminate the infection. If bacteria are killed inside erythrocyte, bacteremia
becomes self-limited and does not develop to sepsis. Released oxygen oxidizes components of
plasma and causes mild hypoxia and functional problems in organs and tissues. Sepsis starts
when bacteria survive and proliferate in erythrocytes. As a result, erythrocytes become a constant
source of bacterial dissemination, simultaneously, the number of erythrocytes decreases.
Antibiotics and immune complexes cannot kill bacteria in erythrocytes and the infection becomes
persistent and uncontrollable. Massive release of oxygen from erythrocytes causes oxidation of
 9

plasma components (hormones, peptides, cytokines, etc.) and interfering oxygen delivery to cells,
lead to multiple organ failure. The treatment of sepsis should tear the links of this chain of sepsis
development. The following is promising:

9.1. Suppression of bacterial antioxidant defense:

9.1.1. Inhibition of bacterial catalase production. The resistance of bacteria to oxidation on
the surface and inside erythrocytes depends upon several factors including catalase production.
Inhibition of bacterial catalase production increases the effectiveness of bacteria killing by
erythrocytes. However, available bacterial catalase inhibitors are not safe [186-189] and new
inhibitors are needed.
9.1.2. Inhibition of bacterial superoxide dismutase production. The manganese and zinc
binding protein calprotectin (CP) reduces bacterial superoxide dismutase activity resulting in the
accumulation of intracellular superoxide in leukocytes and increased sensitivity to oxidative
stress. The inhibition of superoxide defenses by CP increases bacterial sensitivity to neutrophil-
mediated killing [190, 191]. Bacterial MnSOD phosphorylation on serine and threonine residues
decreases the bacteria capacity to counteract ROS [192].

9.2. Suppression of bacterial capsule and biofilm production.

Capsule polysaccharides (CPS) are not only fundamental virulence factors for a wide range of
Gram-negative (e.g. Klebsiella pneumonia, Escherichia coli and others) and Gram-positive
(e.g.Streptococcus pneumonia, Staphylococcus aureus, etc.) bacterial pathogens [193-195], but
they also inhibit complement activity and phagocytosis [196], provide bacterial resistance to
antimicrobials [197], immune recognition by antigen-specific antibodies [198], killing by human
antibody [199] and, being bacterial cell “insulator”, bacterial capsule decreases attraction,
fixation and killing of bacteria by erythrocytes [33-36]. The biosynthesis of bacterial capsules in
both Gram-negative and Gram–positive bacteria is regulated by a system involving a protein
tyrosine phosphatase (PTP) and a protein tyrosine kinase [200, 201]. Inhibition of these proteins
may stop capsule production. As a result, bacterial virulence decreases and bacteria killing by
oxidation increases. Capsule inhibitory drugs may become an important addition to anti-sepsis
therapies.
In the biofilm form, bacteria are more resistant to various antimicrobial treatments, can survive
harsh conditions and withstand the host's immune system [202, 203] Biofilm-associated
infections are very difficult to treat with conventional antibiotics, Therefore, the development of
antibiofilm agents is indispensable. A potential antibiofilm drug that can either facilitate the
dispersion of preformed biofilms or inhibit the formation of new biofilms in vivo is needed. So
far, a plethora of potential antibiofilm agents with unique structures, mainly inspired by natural
products, have been developed and shown great promise in dispersing existing biofilms or
preventing bacteria from forming biofilms in vitro. In contrast to conventional antibiotics, the
majority of the recently developed antibiofilm molecules do not directly affect bacterial survival
and thus the expectation is that resistance to these molecules will not readily occur. In the coming
years, it is hoped that some of these lead compounds would be translated into antibiofilm
drugs.[204]. To date, many antibiofilm compounds have been identified from diverse natural
sources, for example, brominated furanones [205], ursine triterpenes [206], corosolic acid and
asiatic acid [207], ginseng [208] and 3-indolylacetonitrile [209].
Indole, which is generated by the degradation of tryptophan by tryptophanase [210] is an
intercellular signal molecule that can affect multiple aspects of some bacterial species [211]
inhibiting biofilm formation and motility [212]. N-acyl homoserine lactones, cationic molecules
that contain an excess of lysine and arginine residues, D-amino acids, monomeric trimethylsilane
(TMS), ionic liquids, particularly, 1-alkylquinolinium bromide ionic liquids exhibit promising
antimicrobial and antibiofilm properties [213-217].
The important biological messenger, nitric oxide (NO) is a signal for biofilm dispersal, inducing
the transition from the biofilm mode of growth to the free swimming planktonic state [218].
Unfortunately, till now no antibiofilm drug has been registered and used in clinical practice. As a
result, the treatment of biofilm-related infections is hard. Antibiotics, which usually are
ineffective in biofilm infections, should be combined with antibiofilm agents. Antibiofilm agents
 10

that can both disperse and kill biofilm bacteria could have some useful applications, but remain
rare [219, 220].

9.3.Bloodstream bacteria removal by technical devices.

The idea of bacteria removal from the bloodstream was offered more than 25 years ago [221]. E.
coli bacteria were successfully removed from contaminated RBC/plasma by using a special
matrix of micro-encapsulated albumin activated charcoal (ACAC). Efficacy of removing the
bacteria was directly related to the amount of time the contaminated blood was in contact with
the charcoal. The data indicated that the bacteria adhered to the ACAC, but that the charcoal was
not bactericidal.
Another device for removing bacterial toxins from blood useful for treating sepsis was patented
10 years ago [222]. The device includes hollow fiber material for selective binding of the toxins
and removes bacterial lipopolysaccharides (LPS) and lipoteichoic acids (LTA) from blood or
plasma in an extracorporeal perfusion system. The device is arranged so that the liquid that enters
the housing through a first opening must pass through the hollow fibers before leaving through a
second opening and passing through the rest of the perfusion circuit.
Some years ago, for bacteria and endotoxin removing from the blood magnetic nanoparticles
(MNPs) modified with bis-Zn-DPA, a synthetic ligand that binds to both Gram-positive and
Gram-negative bacteria, was used [223].
An external device that mimics the structure of a spleen and cleanses the blood in acute sepsis
has been tested recently[224]. Blood flowing from an infected individual is mixed with magnetic
nanobeads coated with an engineered human opsonin—mannose-binding lectin (MBL)—that
captures a broad range of pathogens and toxins without activating complement factors or
coagulation. Magnets pull the opsonin-bound pathogens and toxins from the blood; the cleansed
blood is then returned back to the individual. The biospleen efficiently removes multiple Gram-
negative and Gram-positive bacteria, fungi and endotoxins from whole human blood flowing
through a single biospleen unit at up to 1.25 liters per h in vitro. In rats infected with
Staphylococcus aureus or Escherichia coli, the biospleen cleared >90% of bacteria from blood,
reduced pathogen and immune cell infiltration in multiple organs and decreased inflammatory
cytokine levels. In a model of endotoxemic shock, the biospleen increased survival rates after a
5-h treatment.
A mechanical devices has been developed to remove a variety of cytokines, lipopolysaccharide,
or C5a from plasma of septic animals [225]. Although there is limited evidence that this
technology will significantly and persistently reduce mediator levels in plasma, it is too early to
know whether such devices would be clinically efficacious for sepsis in humans.
A prototype in-line filtration/adsorption device has been developed using novel synthetic
pyrolysed carbon monoliths with controlled mesoporous domains of 2–50 nm [226]. Porosity was
characterized by SEM and porosimetry. Removal of inflammatory cytokines TNF, IL-6, IL-1β
and IL-8 was assessed by filtering cytokine spiked human plasma through the walls of the carbon
modules under pressure. The effect of carbon filtration on the plasma clotting response and total
plasma protein concentration was also assessed. Significant removal of the cytokines IL-6, IL-1β
and IL-8 was observed
A cytokine adsorption device (CAD) filled with porous polymer beads which efficiently depletes
middle-molecular weight cytokines from a circulating solution has been also developed [227].
Continuous venovenous hemofiltration (CVVHF) combined with plasmapheresis (TPE) reduced
mortality in single- and double-organ failure as high as 28 % in septic patients with combined
extracorporeal detoxification [228].
Bacteria and erythrocytes are triboelctrically charged in the bloodstream and the interaction of
electric charge attracts and fixes bacteria on the surface of erythrocytes. Presumably, dialysis like
device with “electric trap” for bacteria may attract and remove bacteria from the bloodstream.

9.4. Development of new antimicrobials

Search for new antibacterials is indispensable. New antibiotics (or modification of available
antibiotics) and antibacterial chemotherapeutics that better penetrate erythrocytes are needed.
Despite advances in public health, sanitation, vaccines, and antituberculosis chemotherapy and
other antimicrobial agents, sepsis continues to account for an increasing number of deaths in
 11

critically ill patients [229]. One of the possible causes may be the fact, that until now erythrocyte
has been a neglected compartment in antibiotics pharmacokinetics and pharmacodynamics.
While it is generally agreed that antibiotic serum concentrations should be above the minimal
inhibitory concentration for the infecting organism, it is also true that most infections are not in
serum but are found in one or more sequestered tissues, which may have entirely different
antibiotic penetration [230]. This is true also regarding the inner space of erythrocytes that may
become a bacterial reservoir in sepsis. Secondly, it is generally stated that only free antibiotic
molecules will inhibit bacteria. The importance of this concept is clear, but the widely quoted
free and bound antibiotic concentrations are actually derived from in vitro studies of binding to
serum proteins, as opposed to study of infection site binding factors. Thus, it is seldom apparent
what amount of antibiotic is actually available at an infection site (and also inside erythrocytes)
versus the amount bound to cellular debris or otherwise inactivated by local condition [230].
Intraerythrocytic concentrations of antibiotics is higher for lipid-soluble compounds, besides,
plasma proteins binds antibiotics [231]. Sepsis treatment is impossible without antibacterial drug
penetration to erythrocyte. In sepsis the erythrocyte is a long-term bacterial reservoir. High
concentration of antibacterial drugs in erythrocyte is indispensable for infection elimination.

9.5.The use of ozone and hyperbaric oxygen therapies

Alternative use of these treatments in sepsis patients may give controversial results because their
positive potential may be carried out in case of adequate use only. Both hyperbaric oxygen
therapy [232-235] and ozone therapy [236, 237] were studied in experimental and clinical sepsis,
but there are no recommendations regarding adequate use of these therapies. These therapies may
improve or deteriorate the condition of sepsis patients depending upon the stage of sepsis,
infection resistance to oxidation, severity of hypoxia, etc. Ozone and hyperbaric oxygen therapies
increase blood oxygenation in the lungs and it may have different consequences, for example,
increasing of oxidative potential of erythrocytes in arterial blood facilitates bacteria killing by
oxidation, but in case of bacterial resistance to oxidation, the oxygen released into plasma
destroys humoral regulation. Proven recommendations regarding when and how to use ozone and
hyperbaric oxygen therapies are needed.

9.6. Replacement therapy

The replacement of hormones, peptides and other active substances is indispensable in restoring
homeostasis. Corticosteroids were the first anti-inflammatory drugs tested in randomized
controlled trials [238-242]. Some other hormones (catecholamines, anti-diuretic hormone,
thyroxin, insulin, adrenocorticotropin, growth hormone, estrogens, androgens, etc.) were also
tested [243-250]. The results of separate use of hormones are controversial and the positive effect
is not convincing. Hormonal replacement therapy (protocol) should include simultaneous use of
a combination of hormones that takes into account synergism and antagonism, anabolic and
catabolic properties, half-life, resistance to oxidation, pharmacokinetics and pharmacodynamics
of the hormones. The profile and proportions of most important hormones and regulatory
substances for support of vital functions should be established and the replacement of all
indispensable hormonal and other regulatory components should be performed. Injected
components may be oxidized and inactivated so constant control of their concentrations should
be performed.

9.7.Search for optimal blood transfusion triggers for sepsis patients

Approximately 40-50% of patients admitted to the ICU are transfused at least 1 RBC unit. RBC
transfusion in sepsis does not improve oxygen delivery and consumption, mixed venous oxygen
saturation or lactate levels [251, 252]. RBC transfusions in sepsis are not associated with an
improvement in tissue oxygenation in spite of a significant increase in hemoglobin levels [253].
The existing evidence supports the use of restrictive transfusion triggers in most patients [254].
Optimal transfusion triggers in sepsis patients are not known. RBC transfusions cause
complications, such as infection, acute lung injury (TRALI), circulatory overload (TACO),
immunomodulation (TRIM), multiorgan failure and increased mortality [255]. Performing RBC
transfusion in sepsis the following should be taken into account: 1. the blood should be as fresh
as possible. Bacteria easily penetrate old erythrocytes. The lack of oxygen in erythrocytes
 12

facilitates bacterial penetration as well; 2. in-bag hemolysis increases free hemoglobin (protein
and iron) in patient’s plasma stimulating bacterial growth and proliferation; 3. The more massive
is bacteremia, the less effective is blood transfusion; 4. Sepsis patients are sensitive to even
minimal number of bacteria in transfused blood. Before blood transfusion a sterility test is
necessary.

9.8.Inactivation of endotoxins and exotoxins

The systemic spread of microbial toxins, rather than bacteremia itself, is the crucial event in the
pathogenesis of sepsis [256, 257]. Human-specific bacterial toxins make pores in erythrocyte
membrane [258]. The pores cause hemolysis [259]. One of the complications of sepsis is the
rapid development of anemia caused by hemolysis. Free hemoglobin is an important predictor of
survival in sepsis. In non-survivors, free hemoglobin concentration was twice the concentration
compared to survivors [260]. Bigger size bacteria enter erythrocytes through the pores [34-36].
The scenario is the following. After attraction and fixation of bacteria on the surface of
erythrocyte direct physical contact of bacterial body and erythrocyte membrane occurs. Electric
charge interaction causes the release of bacterial toxins. High local concentration of toxins on the
surface of erythrocyte irritates the membrane of erythrocyte causing oxygen release from
erythrocyte (this is an evolutionary mechanism for killing bacteria in the bloodstream). If bacteria
are resistant to oxidation they continue to stimulate oxygen release from erythrocytes. Released
oxygen oxidizes plasma components. Bacterial toxins injure erythrocyte membrane and form
pores that provide bacteria penetration. The inactivation of bacterial toxins may prevent the
penetration of bacteria to erythrocyte. The following is promising: 1. toxin production inhibition
by means of bacterial protein inhibition; 2. toxin inactivation by binding with synthetic polymers,
natural or synthetic antibodies, different toxin-inactivating compounds; 3. toxin inactivation by
modulation of target cell membrane characteristics.

9. 9. “Biological weapon” against sepsis causing bacteria

Predation and antagonism is persistent at all levels of life, found in all walks of life and possibly
in all environments. Predation and antagonism between microorganisms has been known for a
long time [261]. Antibiotics are the best illustration of antagonism between fungi and bacteria.
Antagonism and predation against sepsis causing pathogens are very promising. The use of
following therapies may be effective.
9.9.1 Bacteriophage therapy. Bacteriophages may be useful in the treatment of sepsis caused by
antibiotic resistant bacterial infections. They have some theoretical advantages over antibiotics
being more effective in treating certain infections in humans [262-265]. Bacterial isolates from
septicemia patients spontaneously secrete phages active against other isolates of the same
bacterial strain, but not to the strain causing the disease [266]. Such phages were also detected in
the initial blood cultures, indicating that phages are circulating in the blood at the onset of sepsis.
The fact that most of the septicemic bacterial isolates carry functional prophages suggests an
active role of phages in bacterial infections [266]. Prophages present in sepsis-causing bacterial
clones play a role in clonal selection during bacterial invasion. The use of phages is an attractive
option to battle antibiotic resistant bacteria in certain bacterial infections, but the role of phage
ecology in bacterial infections is obscure [266].
9.9.2. Therapy by Bdellovibrio like organisms
Bdellovibrio and like organisms (BALOs) are small, predatory, Deltaproteobacteria that prey on
other bacteria. Many authors have unfolded the possible use of BALOs as biological control
agents in environmental as well as medical microbiological settings [267, 268].
BALOs, particularly, Bdellovibrio bacteriovorus is a solitary hunter that attacks a wide range of
pathogens: Escherichia coli , Salmonella enteric, Pseudomonas aeruginosa , S. aureus and
others [269-271].
One of the methods used by bacteria for predation is periplasmic invasion. The predator cell
invades and grows within a specific compartment found in Gram-negative cells, the periplasm.
This group of predators is unique in the fact that the predator is a bacterium that is clearly a living
organism, as opposed to viruses and phages and is smaller than the prey. They were named
B.bacteriovorus, the name describing the morphology and the supposed way of life of the
bacteria; they were curved and seemed to stick to their prey and to absorb the prey cell content,
 13

reminiscent of a leech (“bdella” in Greek). Robert E. Buzzzchanan coined the term

B.bacteriovorus [272]. Bdellovibrio are highly motile, flagellated, tiny measuring about 0.25 ×
1.0 μm, Gram-negative Deltaproteobacteria [273].
Bdellovibrio bacteriovorus uses a single polar flagellum to stalk other bacteria. Using
appendages located at the nonflagellated pole, this tiny predator binds its prey tightly. Secreted
enzymes now permit the predator to burrow through the surface of its prey, where it wedges
between the outer membrane and the peptidoglycan wall. Here, it begins to reprogram both itself
and its prey. This includes the partial degradation of the prey peptidoglycan wall, which causes
the prey to round up into a structure called the bdelloplast. Nestled within the confines of this
bdelloplast, the predator consumes its host from the inside out [274, 275]. Bdellovibrio
bacteriovorus has dual probiotic and antibiotic nature [276] and it is reasonable to try it in the
therapy of sepsis.
9.9.3.Saccharomyces therapy
Saccharomyces boulardii (SB) is a non-pathogenic, thermophilic yeast, used as a probiotic strain
in the prevention or the treatment of intestinal diseases, mainly diarrheas [277, 278]. SB directly
inhibits the growth of several pathogens (Candida albicans, E. coli, Shigella, Pseudomonas
aeruginosa, Staphylococcus aureus, Entamoeba hystolitica), and cell invasion by Salmonella
typhimurium and Yersinia enterocolitica [279-281]. SB exerts several anti-microbial activities
that could be divided in two groups: direct anti-toxin effects and inhibition of growth and
invasion of pathogens. The anti-toxin action elicited by SB is mainly due to small peptides
produced by the yeast. A 54kDa serine protease is able to inhibit enterotoxin and cyto-toxic
activities of C. difficile by degradation of toxin A and B [281].
Sb produces a phosphatase able to dephosphorylate endotoxins (such as lipopolysaccharide of E.
coli 055B5) and inactivates its cytotoxic effects [282]. SB also has a positive effect on the
maintenance of epithelial barrier integrity during bacterial infection [283]. S. boulardii affects the
immune response of host cells and stimulates the secretion of secretory immunoglobulin A [284,
285]. S. boulardii inhibits the growth of Candida albicans [286]. Probably, the antimicrobial and
antifungal products, produced by SB may be studied as a possible therapeutic option in sepsis.

9.10. Acceleration of blood flow

Bacteria cannot proliferate in big vessels (arteries, veins). High blood velocity causes
triboelectric charging that inhibits bacterial transmembrane metabolism. In arterioles and
capillaries the velocity of blood flow decreases and bacterial growth inhibition occurs by high
concentrations of oxygen in plasma and squeezing of bacteria between erythrocytes in capillaries.
In venules and small veins, slow blood flow and deoxygenation are auspicious for the
proliferation of bacteria. Infection may grow in vessels with slow blood flow (systemic veins,
skin venous plexuses). Decreased blood velocity increases the risk of thrombosis. Blood viscosity
in venules increases 100% when blood flow reduces 60% [287]. Coagulation abnormalities in
sepsis range from a small decrease in platelet count and subclinical prolongation of global
clotting times to fulminant disseminated intravascular coagulation (DIC), characterized by
simultaneous widespread microvascular thrombosis and profuse bleeding from various sites
[288]. Blood clots become a source of iron and protein for bacterial proliferation. The
acceleration of blood flow velocity increases turboelectric charging of bacteria and decreases the
risk of thrombosis. Blood circulation may be accelerated by different ways: muscular exercises
and physical rehabilitation [289, 290], special physiotherapy [291], massage [292], etc.
Unfortunately, these procedures give short-term effect.

Conclusion
In bacteremia, two events are critical for the development of sepsis: infection resistance to
oxidation and intensive release of oxygen to arterial blood. The consequences of these two events
determine the course of sepsis and its deterioration to septic shock.
In sepsis, the following consecutive events occur: 1. Bacteria enter the bloodstream; 2. Bacteria
are attracted and fixed on the surface of erythrocytes; 3. Bacteria irritate erythrocyte membrane,
and erythrocyte releases oxygen; 4. Survived bacteria enter erythrocyte through pores formed by
bacterial toxins; 5. Bacteria survive inside erythrocyte and proliferate forming infectious
reservoir; 6. Released from erythrocyte oxygen oxidizes plasma components (proteins,
 14

hormones, peptides, amino acids, fatty acids, etc.), causing hormonal regulation disarrangement,
humoral immunity inactivation and nutrients delivery failure; 7. Premature release of oxygen
before erythrocytes entering capillaries causes tissue hypoxia; 8. Inactivation of hormones and
hypoxia cause dysfunction of multiple organs; 9. Increased destruction of bacteria containing
erythrocytes in the plasma and in the spleen causes anemia, free hemoglobin concentration
increasing in blood, overload of RES by killed and alive bacteria; 9. Bacteria and their toxins
injure the liver and the spleen. The treatment of sepsis and septic shock should affect different
aspects of pathogen-host interaction and restore structure and normal physiological functions of
the human body.

There is no conflict of interest

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