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Accepted Manuscript

Sepsis and septic shock: Pathogenesis and treatment perspectives

Hayk Minasyan

PII: S0883-9441(16)31053-X
DOI: doi: 10.1016/j.jcrc.2017.04.015
Reference: YJCRC 52482
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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 are cleared from the bloodstream.
Oxygen released from erythrocytes and humoral immunity kill bacteria in the bloodstream.

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Sepsis develops if bacteria are resistant to oxidation and proliferate in erythrocytes. Bacteria

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provoke oxygen release from erythrocytes to arterial blood. Abundant release of oxygen to the
plasma triggers a cascade of events that cause: 1. oxygen delivery failure to cells; 2. oxidation of
plasma components that impairs humoral regulation and inactivates immune complexes; 3.

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disseminated intravascular coagulation and multiple organs failure. Bacterial reservoir inside
erythrocytes provides the long-term survival of bacteria and is the cause of ineffectiveness of
antibiotics and host immune reactions. Treatment perspectives that include different aspects of
sepsis development are discussed.

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

Introduction
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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 patients response to infection and has a high mortality
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rate [3]. Sepsis remains the dominant challenge in the care of critically ill patients [4]. More than
30 million cases of sepsis worldwide per annum are estimated. The incidence of sepsis increases
9-13% annually, a mortality rate is 33-35% (Tab. 1). The most common sites of infection are the
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lungs (40%), abdomen (30%) and urinary tract (10%) [13]. Sepsis may be caused by gram-
positive, gram-negative and poly microbial infection [14, 15]. Gram-negative infection most
often occurs in the lungs [16]. Staphylococcus aureus and Streptococcus pneumoniae are gram-
positive isolates, whereas Escherichia coli, Klebsiella species, and Pseudomonas aeruginosa
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predominate among gram-negative isolates [17, 18]. Gram-positive organisms cause sepsis by
producing exotoxins and by their cell wall components [19]. Gram-negative bacteria cause
sepsis by their membrane lipopolysaccharides (endotoxins) [20]. Bacterial toxins play a pivotal
role in the pathophysiology of sepsis, however, the literature illustrates that no single
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mediator/system/pathway/pathogen drives the pathophysiology of sepsis [21]. Current knowledge


of sepsis pathogenesis includes infection interaction with the host before bacteria enter the
bloodstream. Actually, the mechanisms of host defense in the tissues differ from the mechanisms
of intravasal (bloodstream) defense because extravasal defense is provided mainly by leukocytes
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whereas intravasal defense is fulfilled by erythrocytes. The humoral immunity events take place
in pre-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 rarely develops [27-31]. It occurs when the infection is resistant to host
antibacterial defense. The latter is different in the tissues and the bloodstream. If the infection
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 (contaminated intravenous injection, bite, etc.), the pre-septic stage is
absent. Local antibacterial defense is provided by phagocytosis (leukocytes and their local
versions: resident macrophages), complement, NETs, etc., whereas in the bloodstream bacteria
are killed by bactericidal humoral factors and oxygen that is released from erythrocytes [32, 33].
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Bacteria proliferate in the tissues being resistant to complement. Blood natural resistance factors
(complement, lysozym, etc.) are not effective if infection enters the blood from 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 (see tab. 2).
Sepsis causing bacteria are both gram positive and gram negative. Gram-positive organisms are
better suited to invade host tissues than gram-negative organisms [37]. The lack of endotoxin in

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the outer cell wall is compensated for by the presence of exposed peptidoglycan and a range of

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other toxic secreted products. Cell wall components of gram-positive bacteria may signal via the
same receptor as gram-negative endotoxin [37]. Gram-negative organisms are associated with
poorer outcomes in first-hit infections; an inverse relationship between Gram status and mortality

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is observed in second-hit infections [38].
The majority of sepsis causing bacteria is facultative anaerobes [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

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express additional respiratory mechanisms that make their respiration close to facultative
anaerobes [40, 41].
All sepsis causing bacteria produce superoxide dismutase (SOD), catalase and glutathione
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peroxidases (tab.2), 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. All successful pathogens have evolved effective systems for defense against oxidative
stress [42]. Phagocytes utilize the cytotoxic effects of the reactive oxygen species, such as
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superoxide, hydrogen peroxide, and the highly toxic hydroxyl radical. 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
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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.
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Certain structures of bacteria are indispensable for causing sepsis. All sepsis causing bacteria
have S-layer and produce capsules, slime layer and biofilm (tab. 2). These structures protect the
bacteria in the tissues against phagocytosis, ROS, lytic enzymes, immune complexes, etc.,
whereas in the bloodstream capsule and slime layer prevent triboelectric charging, attraction and
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fixation on the surface of erythrocytes, oxidation and killing by the oxygen released from
erythrocytes [33 ].
Sepsis causing bacteria produce hemolysins . Erythrocytes are the main bactericidal cells in the
bloodstream and hemolysins are necessary for bacterial survival in the bloodstream. If the speed
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of bacterial growth in the tissue is limited by host immune reactions, bacteria produce a capsule,
slime layer and biofilm for surviving. After entering the bloodstream, bacterial capsule and slime
layer prevent triboelectric charging and fixation on the surface of erythrocytes. If bacteria
rapidly proliferate in the tissues, they are short of time to produce a capsule and slime layer and
after entering the bloodstream, they are 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. Hemolysins are
important for the development of sepsis to advanced stages.
Motility is not a crucial factor for causing sepsis (tab 2.). Sepsis causing bacteria may be either
motile or not motile organisms.

3.Bacteria killing in the tissues


Neutrophils, monocytes and resident macrophages are the main bactericidal cells in the tissues.
Upon encountering bacteria, neutrophils engulf them into a phagosome, which fuses with
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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) [45].
Neutrophils undergo an oxidative burst during which the NADPH oxidase complex assembles
at the phagosomal membrane and produces O2-, 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 [46].
Neutrophils can degranulate and release antimicrobial factors into the extracellular space [47].
They can also generate neutrophil extracellular traps (NETs), which are composed of granule and
nuclear constituents that kill bacteria extracellularly [48]. NETs disarm pathogens with elastase,
cathepsin G and histones that have a high affinity for DNA [49]. NETs may serve as a physical
barrier that prevents further spread of the pathogens [50].

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Platelets activate neutrophils to trap bacteria. Platelets rapidly localize to sites of injury and

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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

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molecules [50, 52]. Platelets have the ability to bind and internalize bacteria through engulfing
endosome-like vacuoles that fuse with the -granules of the platelet and allow the granular
proteins to have access to the pathogens [53]. As a result, thrombocytopenia correlates with the
severity of the sepsis and the rate of mortality [54].

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4.Survival of sepsis causing bacteria in phagocytes
After phagocytosis by macrophages, bacteria are located in a membrane-bound vacuole
(phagosome), but the ensuing trafficking of this vacuole and subsequent bacterial survival
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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 1530 min. The metabolic burst in activated phagocytes results in
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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 (see fig. 1). After phagocytosis lysosomes fuse
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with the phagosome, forming a phagolysosome and proteases are introduced into the phagosome.
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 O 2,
forming the superoxide radical, O2-. . The superoxide radical is converted to hydrogen peroxide
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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
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of phagocytes by their capsule (chemical insulator) and by producing superoxide dismutase


(SOD), catalase and glutathione peroxidase (figure 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
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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 [32, 33]. In the
bloodstream bacteria are attracted and fixed on the surface of erythrocytes by electrical charge
interaction force. Bacteria activate erythrocyte membrane receptors, stimulating 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 have a thick capsule that
prevents 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. Bacteria may
overload the liver and the spleen and damage them (Fig. 2, scenario 2). If bacteria survive
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oxidation on the membrane of erythrocytes, they enter erythrocytes by making holes in the
membrane. Inside erythrocytes bacteria may be killed by higher concentration of oxygen.
However, bacteria may survive inside erythrocytes if there is lack of oxygen or bacteria are
resistant to oxidation (Fig. 2, scenario 3). Surviving, bacteria proliferate in erythrocyte and the
latter bacterial incubator and reservoir [34, 35]. Bacteria inside erythrocytes have nutrients for
proliferation, besides, they are out of reach of antibiotics, immune complexes and other
antibacterial factors. Bacterial proliferation tears erythrocyte membrane and bacteria, being
released back into the plasma, infect new erythrocytes.

6.Sepsis and septic shock pathogenesis


In bacteremia, two events are critical for the development of sepsis: a. infection resistance to
oxidation; b. provoked by bacteria premature release of oxygen from erythrocytes. The

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consequences of these two events determine the course of sepsis and its deterioration to septic

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shock. Oxidation of blood plasma components and lack of oxygen in erythrocytes cause distant
injury of the tissues.
6.1.Oxidation of blood plasma components

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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 destroys humoral regulation. Human body comprises two separate but
interacting compartments: (a) compartment of blood circulation (pulmonary and systemic

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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. It has different consequences: from innocuous bacteremia to fatal septic shock.
Bacteremia, sepsis, severe sepsis and septic shock may be interpreted as a continuum different
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amount of oxygen released by erythrocytes into plasma. Bacterial stimulation of the surface
receptors of erythrocytes causes the release of oxygen. The more oxygen is released from
erythrocytes to the arterial blood, the more severe is sepsis. The consequences of oxygen release
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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 activates platelets and causes disseminated intravascular coagulation. Third,
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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 are substrates for biological oxidation [57]. Oxidative changes to proteins lead to
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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
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pathway of hGH degradation [59]. The Growth Hormone and Insulin-like Growth Factor-1
(IGF-1) axis play a pivotal role in critical illness. 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
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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]. Deactivation of insulin causes
hyperglycemia - one of the metabolic derangements that influence sepsis outcome [63-66].
The oxidation of blood components 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 also affects the hypothalamo-pituitary-thyroidal axis,
inactivating thyrotropin, 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) is oxidized as well. Oxytocin and vasopressin are
oxidized with the formation of dityrosine [77]. Its oxidation and depletion cause vasodilatory
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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. Systemic vasodilatation and arterial hypotension are landmarks of septic shock
[82].
Albumin oxidation causes hypoalbuminemia in sepsis [83,84]. Even mild oxidation of human
serum albumin (HAS) impairs HSA functional properties including protease susceptibility,
ligand-binding affinity and antioxidant activity [85-87]. The major structural change in oxidized
HSA is a disulfide-bonded cysteine at the thiol of Cys34 of reduced HAS [88].
Oxidative damage results in protein modification. [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 immune complexes and immunoglobulins,

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particularly IgG and IgM . The oxidation of IgG significantly changes the immunoreactivity and

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specificity of IgG fractions [95]. Oxidized immunoglobulins have autoimmune and
proinflammatory activity [96-98].
Low levels of immunoglobulins are frequent in severe sepsis and septic shock [99-101].

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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.

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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].
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6.2. Anemia, cell hypoxia and lactate production
The release of oxygen to arterial blood (before erythrocytes enter to capillaries) causes failure of
oxygen delivery to cells and hypoxia [33-36]. Another co-factors of hypoxia is anemia caused by
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intensive destruction of erythrocytes, suppression of bone-marrow, low production of


erythropoietin, etc. [108-111]. The main factor of anemia in sepsis is increased destruction of
erythrocytes by infection. Making holes in the membranes of erythrocytes, bacteria cause
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hemoglobin pouring out, besides, they penetrate inside erythrocytes. The liver and, especially, the
spleen actively destroy injured and bacteria-containing erythrocytes [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
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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
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[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
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lactate/initial lactate 100) [117-120]. Lactate non clearance in sepsis is a significant


independent predictor of death [117,119].

7.Diagnostic problems
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 twothree 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,
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acute-phase protein biomarkers [142-144], procalcitonin [145-147], pentraxins [148-149],


cytokine/chemokine biomarkers (IL-6, IL-8, IL-10, TNF-, 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

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in an observational multicenter design with blood culture as the comparator [167]. Systems

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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].

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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

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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 optically invisible in erythrocytes [32].
Phase-contrast microscopy and differential interference-contrast microscopy are effective and
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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
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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
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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
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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.
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8.Treatment problems:
Sepsis is a systemic infection. Empiric antimicrobial therapy is the base of the treatment
[171,172]. Current guidelines recommend starting antibiotic therapy within one hour of
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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
for suspected infections [179,179]. 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, new antibiotics cannot increase the effectiveness of sepsis therapy if
pathogens proliferate inside erythrocytes. [34]. Antibiotics kill bacteria in blood plasma, but
insufficiently penetrate erythrocytes for killing bacteria there. Constant bacterial reservoirs in
erythrocytes decreases antibacterial and immune therapy effectiveness and may be one of the
factors that make sepsis therapy so problematic.
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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, no FDA-approved drug is available for use in sepsis [184].
The biology of sepsis is complex and not specific to infection. More than 100 randomized
clinical trials have tested the hypothesis that modulating the septic response to infection can
improve survival. None of these have resulted in new treatments [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. Phagocytosis in the bloodstream is impossible and blood
humoral bactericidal factors and erythrocytes are the main antibacterial forces in the bloodstream.

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Both erythrocytes and bacteria are triboelectrically charged in the bloodstream. The charge of

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erythrocytes attracts the charge of bacteria and keeps bacteria on the surface of erythrocytes. The
interaction of the charges at the surface of erythrocytes stimulates the release of oxygen from
oxyhemoglobin. The oxygen is released from hemoglobin as a mixture of different allotropes of

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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

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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 then penetrate
erythrocytes. If bacteria do not penetrate erythrocytes, immune complexes and antibiotics may
eliminate the infection. If bacteria are killed inside erythrocytes, bacteremia becomes self-limited
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and does not develop to sepsis. Released oxygen oxidizes components of plasma and causes
hypoxia and functional problems in organs and tissues. Sepsis starts when bacteria survive in
erythrocytes, becoming a constant source of bacterial proliferation and dissemination. Antibiotics
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and immune complexes cannot kill bacteria inside erythrocytes and the infection becomes
persistent and uncontrollable. Massive release of oxygen from erythrocytes causes disseminated
intravascular coagulation, oxidation of plasma components (hormones, proteins, peptides,
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cytokines, etc.), makes impossible oxygen delivery to cells, and leads to multiple organ failure.
The cornerstone of sepsis therapy should be: a. arterial blood clearing from pathogens; b.
prevention of premature release of oxygen from erythrocytes; c. prevention of bacterial reservoir
forming in erythrocytes. The following is promising:
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9.1. Suppression of bacterial antioxidant mechanisms:


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.
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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
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binding protein calprotectin (CP) reduces bacterial superoxide dismutase activity [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.) 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 is regulated by tyrosine
phosphatase (PTP) and a protein tyrosine kinase [200,201]. Inhibition of these proteins may stop
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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. A potential antibiofilm drug
that can either facilitate the dispersion of preformed biofilms or inhibit the formation of new
biofilms is needed [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, D-amino acids,

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monomeric trimethylsilane (TMS), ionic liquids, particularly, 1-alkylquinolinium bromide ionic

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liquids exhibit promising antimicrobial and antibiofilm properties [213-217].
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].

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Unfortunately, till now no antibiofilm drug has been registered and used in clinical practice [219,
220].

9.3. Bloodstream bacteria removal by technical devices.

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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). The data indicated that the
bacteria adhered to the ACAC, but that the charcoal was not bactericidal.
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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
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plasma in an extracorporeal perfusion system. 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].
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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 opsoninmannose-binding lectin (MBL)that
captures a broad range of pathogens and toxins without activating complement factors or
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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 biospleen unit. A mechanical devices has been developed to remove a variety of
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cytokines, lipopolysaccharide, or C5a from plasma [225]. It is unclear 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 250 nm [226]. Porosity was
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characterized by SEM and porosimetry. Removal of inflammatory cytokines TNF, IL-6, IL-1
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-1
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. Presumably, dialysis
like device with electric trap for bacteria may attract and remove bacteria from the
bloodstream.
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9.4. Development of new antimicrobials


Despite advances in medicine, sepsis continues to account for an increasing number of deaths
[229]. The cause is 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. It is also 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

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(and also inside erythrocytes) versus the amount bound to cellular debris or otherwise inactivated

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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

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bacterial reservoir. High concentration of antibacterial drugs in erythrocyte is indispensable for
infection elimination.

9.5. The use of ozone and hyperbaric oxygen therapies

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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. The latter may
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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, increasing the oxidative potential of erythrocytes in arterial blood, facilitate bacteria
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killing by oxidation, but in case of bacterial resistance to oxidation and premature release of
oxygen from erythrocytes, ozone and hyperbaric oxygen therapies may provoke disseminated
intravascular coagulation and intensify blood plasma oxidation.
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9.6. Replacement therapy


The replacement of hormones, peptides and other active substances in sepsis is indispensable
Corticosteroids were the first anti-inflammatory drugs tested in randomized controlled trials
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[238-242], then catecholamines, anti-diuretic hormone, thyroxin, insulin, adrenocorticotropin,


growth hormone, estrogens, androgens, etc. were also tested [243-250]. The results of separate
and combined use of hormones are controversial. Hormonal replacement therapy (protocol)
should include a combination of hormones that takes into account their synergism and
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antagonism, anabolic and catabolic properties, half-life, resistance to oxidation,


pharmacokinetics, pharmacodynamics, etc. The profile and proportions of most important
hormones and regulatory substances for support of vital functions should be established. Injected
components may be oxidized and inactivated so constant control of their concentrations should
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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
facilitates bacterial penetration as well; 2. in-bag hemolysis increases free hemoglobin (protein
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and iron) in patients 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 is an important 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

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following. After attraction and fixation of bacteria on the surface of erythrocyte direct physical

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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. If bacteria are

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resistant to oxidation they continue to stimulate oxygen release from erythrocytes. Released
oxygen oxidizes plasma components and causes disseminated intravascular coagulation.
Bacterial toxins injure erythrocyte membrane and form pores that provide bacteria penetration.
The inactivation of bacterial toxins may prevent oxygen release and the penetration of bacteria to

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erythrocytes. 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.
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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
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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
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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
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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
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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
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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,
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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 m, 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.

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9.9.3. Saccharomyces therapy

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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

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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

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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
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maintenance of epithelial barrier integrity during bacterial infection [283]. SB affects the immune
response of host cells and stimulates the secretion of secretory immunoglobulin A [284,285]. SB
inhibits the growth of Candida albicans [286]. Probably, the antimicrobial and antifungal
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products, produced by SB may be studied as a possible therapeutic option in sepsis.

9.10. Acceleration of blood flow


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Bacteria cannot proliferate in big vessels (arteries, veins) because blood high velocity causes
triboelectric charging that inhibits bacterial transmembrane metabolism. In arterial blood bacteria
are attracted by electric charge of erythrocytes and killed by released oxygen. In capillaries
bacteria are killed by being squeezed between erythrocytes, besides, during oxygen/CO 2
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exchange between erythrocytes and the tissues, bacteria are killed by oxidation. 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
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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
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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 bacteremia progression to sepsis and its deterioration to septic shock.
Infection resistance to oxidation provides bacterial survival on the surface of erythrocytes,
penetration of pathogens to erythrocytes, forming infection reservoir inside erythrocytes,
destruction of erythrocytes and anemia, infection dissemination to all organs and tissues.
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Premature release of oxygen from erythrocytes causes inactivation of plasma components,


disseminated intravascular coagulation and generalized hypoxia.
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 capsule, wall components and toxins; 5. Bacteria survive inside erythrocyte and
proliferate forming infectious reservoir; 6. Released from erythrocyte oxygen oxidizes plasma
components (proteins, hormones, peptides, amino acids, fatty acids, etc.), causing hormonal
regulation disarrangement, humoral immunity inactivation and nutrient delivery failure;
Premature release of oxygen from erythrocytes causes tissue hypoxia; All these factors cause
multiple organ failure.
Treatment of sepsis and septic shock after bacterial penetration to erythrocytes and premature

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release of oxygen from oxyhemoglobin is very problematic. The most perspective approach to

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sepsis and septic shock is prevention of bacterial penetration to erythrocytes and premature
release of oxygen to arterial blood. It may be achieved by suppression of bacterial antioxidant
mechanisms and inactivation of bacterial endotoxins and exotoxins. Development of new

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antimicrobials is a temporary and less effective approach. Bloodstream bacteria removal by
technical devices, adequate replacement therapy and the use of biological weapon against
sepsis causing bacteria may be useful as addition to sepsis and septic shock combined therapy.

No funding US
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There is no conflict of interest
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Table 1
THE DYNAMICS OF SEPSIS AND SEPTIC SHOCK INCIDENCE AND
MORTALITY.

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- Global estimates of sepsis (cases a year)
**
- US Census included 308,745,538 individuals
***
- 28 day mortality

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Table 2
THE COMMON FEATURES OF SEPSIS CAUSING BACTERIA.

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SOD superoxide dismutase


GPX glutathione peroxidase
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A aerobic bacteria
FAN facultative anaerobic bacteria
MA micro aerobic bacteria
OANA obligate anaerobic bacteria
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Highlights
Bacteria in the bloodstream are killed by oxygen released from
erythrocytes.
Sepsis development depends upon bacterial resistance to oxidation .
Released oxygen oxidizes plasma components and destroys humoral
regulation.
Oxygen release in arterial blood causes hypoxia in the tissues.
The severity of sepsis correlates with the amount of released oxygen.

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