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YMPAT1582_proof 9 February 2015 1/7

Microbial Pathogenesis xxx (2015) 1e7

Contents lists available at ScienceDirect

Microbial Pathogenesis
journal homepage: www.elsevier.com/locate/micpath

Review

Surviving sepsis in the era of antibiotic resistance: Are there any


alternative approaches to antibiotic therapy?
Q5

Divakara S.S.M. Uppu, Chandradhish Ghosh, Jayanta Haldar*


Chemical Biology and Medicinal Chemistry Laboratory, New Chemistry Unit, Jawaharlal Nehru Centre for Advanced Scientic Research, Jakkur,
Bengaluru 560064, Karnataka, India

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 12 December 2014
Received in revised form
4 February 2015
Accepted 6 February 2015
Available online xxx

Sepsis, a serious cause of morbidity in humans, has no proper single medication dedicated to it. In this
review, we look at the current treatment modalities, the different approaches attempted towards
treating it and alternative approaches that could be implemented to counter this neglected disease
condition. The use of antibiotics towards treatment of sepsis, use of combinations and strategies derived
from natural antimicrobial peptides has been dealt with in details. The social and technical difculties
associated with treating sepsis and the possible ways of combating them have also been discussed.
2015 Published by Elsevier Ltd.

Keywords:
Sepsis
Antibiotics
Antimicrobial peptides
TNF-a

1. Introduction
Globally, an estimated 18 million cases of sepsis occur each year
which means that 3 in a 1000 people get sepsis [1]. Every hour,
about 36 people die from sepsis [1]. It causes more deaths than
prostate cancer, breast cancer and HIV/AIDS combined [1]. One
million children die in the rst four weeks of life every year in India
and out of these deaths, 190,000 are caused by sepsis [1].
Sepsis: Sepsis is the systemic response to infection. It is dened
as the presence of several clinical, hematologic, biochemical and
immunologic variables associated with an infection. The clinical
parameters which are considered as symptomatic for sepsis are
furnished in Table 1. Severe sepsis is complicated by organ
dysfunction. Severe Sepsis and septicemia are the syndromes of
overwhelming response of the body to severe infections. At times,
it can be protective to body but at times it can lead to sequential
multi-organ failure leading to death. Septic shock refers to a state
of acute circulatory failure characterized by arterial hypotension
despite adequate uid resuscitation so that vasopressor therapy is
necessary to restore a minimally acceptable arterial pressure.
Hypotension is dened by a systolic arterial pressure below
90 mm Hg or a reduction of more than 40 mm Hg from baseline,

* Corresponding author.
E-mail address: jayanta@jncasr.ac.in (J. Haldar).

and it is associated with signs of altered tissue perfusion such as


oliguria, altered mental status, or altered skin perfusion and a
metabolic marker (i.e., increased blood lactate levels) [2]. The
clinical denition of sepsis has been clearly understood to diagnose the condition with appropriate variables [2]. Apart from
diagnosis using the clinical variables, Blood Transcriptional Signatures is a relatively new approach in the biomarker development. Blood transcription signatures refer to analysis of whole
genome RNA expression in human clinical samples and this
pattern of RNA expression (transcriptional signature) provides a
biological snapshot of a disease. By comparing the transcriptional
signatures of healthy individuals and the patients affected by a
particular disease or by sequentially sampling over a course of
time, it is possible to delineate a comparative transcriptional
response to a given disease. Blood transcriptional proling has
improved diagnosis and understanding of disease pathogenesis.
This approach has been shown to be effective in deciphering the
immune response to infections and has the potential to be
developed as a marker for diagnosing the sepsis disease condition
[3]. One study showed that whole genome studies of blood from
patients suffering from sepsis caused by Burkholderia pseudomallei
and sepsis caused by other bacteria have different signals [4].
Neutrophil reporter assay in combination with whole transcriptome readout have also been reported to predict sepsis
severity [5]. These approaches bear much promise as diagnostic
tools for rapid and timely detection of sepsis.

http://dx.doi.org/10.1016/j.micpath.2015.02.001
0882-4010/ 2015 Published by Elsevier Ltd.

Please cite this article in press as: D.S.S.M. Uppu, et al., Surviving sepsis in the era of antibiotic resistance: Are there any alternative approaches to
antibiotic therapy?, Microbial Pathogenesis (2015), http://dx.doi.org/10.1016/j.micpath.2015.02.001

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Table 1
Clinical features for diagnosis of sepsis.
Clinical parameters

Parameter values

1.
2.
3.
4.
5.
6.
7.
8.

>38.2  C
>120 beats min1
<110 mm Hg
<150  109 L1
<4  109 L1
>145 mmol L1
>30 mmol L1
>15 mmol L1

Temperature
Heart rate
Static blood pressure
Platelet count
Leukocyte count
Sodium ion
Bilirubin
Urea

2. Current treatment modalities for sepsis


As far as the management modalities are concerned there is no
specic treatment available to control the inammation. SALVAGE
therapy has been recommended in India for all patients with severe
sepsis and septic shock, wherein SALVAGE stands for, Steroids,
Antibiotics, Low-dose heparin, Ventilation with lung protective
strategy, activated protein C, Glucose homeostasis, early goal
directed therapy. Drotrecogin alfa (activated) (Xigris, marketed by
Eli Lilly and Company) is a recombinant form of human activated
protein C that has anti-thrombotic, anti-inammatory, and probrinolytic properties. Drotrecogin alpha (activated) belongs to the
class of serine proteases [6]. It is used mainly in intensive care
medicine as a treatment for severe sepsis. Activated Protein C was
withdrawn from the market due to its exorbitant prices and associated adverse events [6]. The Sepsis Six is a set of interventions
which can be delivered during the treatment of severe sepsis
condition. (1) Administer high ow oxygen. (2) Take blood cultures.
(3) Give broad spectrum antibiotics. (4) Give intravenous uid
challenges. (5) Measure serum lactate and haemoglobin. (6) Measure accurate hourly urine output [7].
Available therapies are directed to achieve the secondary goals
of sepsis management where antibiotics are prescribed to control
the infection and uid therapy (intravenous uids) to stabilize the
patient's condition. There is no primary therapy available to control
the series of inammatory events induced by multiple infections.
Unfortunately, it is not known in the sepsis treatment whether the
use of broad spectrum antibiotics is a boon or a bane. The use of
antibiotics can have unknown adverse effects on the sepsis
treatment.
3. Antibiotics in the treatment of sepsis
Antibiotics are greatest tools in combating the attack of microorganisms, provided they are used wisely and timely. Antibiotics
alone are not the magic bullets which will revert sepsis syndrome.
They have to be used expeditiously along with other early interventions for sepsis. However, the use of antibiotics is known to
trigger the release of bacterial cell wall components that contribute
to the severe inammation in the body that leads to sepsis [8].
First report: The spontaneous and penicillin-stimulated release
of water-soluble, glycerol-labeled polymers was compared in
Streptococcus sanguis. In contrast to the spontaneous release
occurring in exponentially growing or stationary-phase bacteria,
penicillin-treated cells released the bulk of these polymers, and
they were not replenished by synthesis during antibiotic treatment.
Furthermore, a major portion of the extracellular polymers was
characterized as acylated lipoteichoic acid [9].
Recent research on septic shock has increased the awareness
that this syndrome is caused by a host response to bacterial cell
wall components [10,11]. In the case of gram-negative bacteria,
lipopolysaccharide (LPS), a part of the outer membrane, has been

identied as the major immunostimulatory component. LPS is


released during bacterial growth as well as after lysis of the bacterial cells, for instance, by the actions of antibiotics, although antibiotics may differ in their capacity to cause lysis. Analogous to the
LPS in gram-negative bacteria, two cell wall components of the
gram-positive microorganism Staphylococcus aureus, i.e., peptidoglycan (PG) and lipoteichoic acid (LTA), are able to induce the
production of proinammatory cytokines by monocytes in vitro.
LTA and PG are released spontaneously into the culture medium
during growth of gram-positive bacteria. Moreover, incubation
with antibiotics was found to enhance the release of LTA and PG.
LPS and LTA have long been known to be the culprits in triggering
the inammation by acing as the TLR4 and TLR2 agonists respectively of human immune cells like macrophages. Both PG and LTA
have been shown to stimulate inammatory responses in a number
of in vivo and in vitro experimental models. Gram-positive bacteria
also produce the membrane bound lipopeptides and some secrete
exotoxins, such as staphylococcal enterotoxin B (SEB) and toxic
shock syndrome toxin (TSST-1). These components are important
in the pathophysiological conditions associated with specic infections [10,11].
Antibiotics with different mechanisms of action may vary with
respect to their effects on the release and immunostimulatory activities of cell wall fragments from gram-positive bacteria. S. aureus
was cultured in the absence of antibiotics (control) and in the
presence of b-lactam antibiotics (imipenem, ucloxacillin, or cefamandole) and protein synthesis-inhibiting antibiotics (erythromycin, clindamycin, or gentamicin). LTA and PG levels in the
bacterial supernatants were measured. b-Lactam antibiotics greatly
enhanced the release of LTA and PG (4- to 9-fold and 60- to 85-fold,
respectively), whereas protein synthesis inhibitors did not affect PG
release and even inhibited the release of LTA compared to the
amount of LTA released in control cultures. The capacity of b-lactam
supernatants to stimulate the production of tumor necrosis factor
alpha and interleukin-10 in human whole blood was signicantly
higher than that of protein synthesis inhibitor or control supernatants; the amounts of these cytokines released were directly proportional to the concentrations of PG and LTA in the supernatants
[12].
Because appropriate antibiotic treatment nevertheless signicantly reduces mortality in sepsis, the question arises of whether
equally effective antibiotics have a differential effect on the increase
in endotoxin exposure following treatment. The concentration and
accessibility of endotoxin can increase following antibiotic killing of
gram-negative bacteria. There are indications that antibiotics may
differ in this respect. Endotoxin levels, tumor necrosis factor alpha
(TNF-a) and interleukin-6 production in whole blood ex-vivo were
measured after exposure of log-phase Escherichia coli to antibiotics
belonging to different classes [13]. Ceftazidime and ciprooxacin
treatment resulted in levels of endotoxin, TNF-a, and interleukin-6
signicantly higher than those of imipenem and gentamicin
(P < 0.001). Polymyxin B and, to a lesser degree, recombinant
bactericidal/permeability-increasing protein 21 (rBPI-21) were
found to be potent inhibitors of TNF-a release, supporting the
concept that the differences between the antibiotics in cytokine
production were indeed due to differences in amounts of biologically active endotoxin [13].
At concentrations approximating serum Cmax, tigecycline
decreased IL-6 by 52%e57% and IFN-g production by 43%e53%
compared with toxin alone (P  0.05) and linezolid inhibited TNF-a
by 12%e35% and IL-8 by 25%e42% (P  0.02). However, trimethoprim/sulfamethoxazole increased TNF-a and IL-8 production.
Clindamycin, daptomycin, vancomycin and azithromycin had no
consistent signicant effect at approximate serum Cmax concentrations. All antibiotics had a concentration-dependent effect on

Please cite this article in press as: D.S.S.M. Uppu, et al., Surviving sepsis in the era of antibiotic resistance: Are there any alternative approaches to
antibiotic therapy?, Microbial Pathogenesis (2015), http://dx.doi.org/10.1016/j.micpath.2015.02.001

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Table 2
Pathogen associated molecular patterns that stimulate the immune cells.
Pathogen associated molecular
patterns (PAMPs)

Target receptors on immune cells

1.
2.
3.
4.
5.

Toll-like receptor-2 (TLR-2)


Toll-like receptor-2 (TLR-2)

Lipopolysaccharide (LPS)
Lipoteichoic acid (LTA)
Peptidoglycan (PG)
Staphylococcal enterotoxin B (SEB)
Toxic shock syndrome toxin (TSS-1)

Unknown
Unknown

cytokine production, with tigecycline, clindamycin and trimethoprim/sulfamethoxazole being the most potent inhibitors of cytokine production at concentrations exceeding 25 mg/L [14].
The effect of trovaoxacin, ciprooxacin and ceftriaxone on
cytokine production of human peripheral blood mononuclear cells
(PBMCs) was examined [15]. PBMC responses were measured after
stimulation with lipopolysaccharide (LPS), lipoteichoic acid (LTA) or
killed or viable Streptococcus pneumoniae and Haemophilus inuenzae. Trovaoxacin inhibited the production of TNF-a, interleukin1b (IL-1b), IL-6 and IL-8 by PBMCs after stimulation with either LPS
or LTA by 83%. Similar inhibition occurred in PBMCs incubated with
killed or live bacteria and trovaoxacin, but not with ciprooxacin
or ceftriaxone. The relevance of this in vitro observation was
explored by examining TNF-a and IL-6 responses in trovaoxacintreated mice. Serum concentrations of both cytokines 1 h after
LPS challenge were 95% less than serum concentrations in mice that
were not given trovaoxacin. Reverse transcriptione polymerase
chain reaction studies of the mechanisms determining cytokine
down-regulation demonstrated that trovaoxacin reduced TNF-a,
IL-1b and IL-6 mRNA to levels similar to those of unstimulated cells.
These observations indicate that trovaoxacin can consistently and
signicantly reduce production of cytokines that play an important
role in sepsis. In vitro, this effect can occur in the presence of
bacteriolysis and is associated with inhibition of transcription of
cytokine genes [15].
Telithromycin had immunomodulatory effects as a result of
alteration of secretion of cytokines by monocytes. The antibiotic
telithromycin was examined for its effect on secretion of interleukins, IL-6, IL-10, and tumor necrosis factor alpha (TNF-a) by
lipopolysaccharide (LPS)-stimulated monocytes of eight human
donors. Secretion of each cytokine was signicantly increased by
LPS alone, whereas treatment with telithromycin signicantly
inhibited secretion of TNF-a but not secretion of IL-6, and IL-10 [16].

Q3

Table 3
The different classes of antibiotics that aggravate or reduce the sepsis disease
condition.
Antibiotics that aggravate sepsis

Antibiotics that reduce sepsis

Cell-wall biosynthesis inhibitors


b-lactams
 Penicillin
 Flucloxacillin
 Cefamandole
 Oxacillin
 Ceftazidime
 Ceftriaxone
Glycopeptides
 Vancomycin

Protein synthesis inhibitors


Aminoglycosides
 Gentamicin
Lincosamides
 Clindamycin

DNA biosynthesis inhibitors


 Ciprooxacin
Folic acid biosynthesis inhibitors
 Trimethoprim/Sulfamethoxazole

Macrolides
 Erythromycin
 Telithromycin
Tetracyclines
 Tigecycline
Oxazolidinone
 Linezolid
Membrane-active antibiotics
 Polymyxin
 Daptomycin
DNA biosynthesis inhibitors
 Trovaoxacin

The mechanism of how the release of endotoxins upon antibiotic treatment causes deleterious effects on organ function of
the human body are more or less clearly understood [10,11].
Endotoxin, the lipopolysaccharide (LPS) constituent of the outer
membrane of the cell wall of gram-negative bacteria, is considered
to be the most important bacterial factor in the pathogenesis of
the gram-negative septic syndrome. It induces the release of the
cytokines TNF-a, IL-1 and IL-6. These cytokines activate a cascade
of secondary inammatory mediators, eventually leading to
endothelial damage and hemodynamic and metabolic derangements. Adequate antibiotic treatment is thought to be pivotal
in the therapy of severe gram-negative infections, but many invitro as well as animal and clinical studies have indicated that the
endotoxin concentration may increase following bacterial death
caused by exposure to antibiotics. Three mechanisms can account
for this increase: (i) accumulation of bacterial biomass following
antibiotic treatment, for instance because of lament formation;
(ii) an increase in the accessibility of endotoxin that remains
bound to the bacteria, which was demonstrated even when subMIC levels of antibiotics were used; or (iii) release of unbound
(free) endotoxin [10,11]. To avoid confusion, the overall increase in
the amount of biologically active endotoxin is best described as an
increase in endotoxin exposure.
Table 3 lists the different classes of antibiotics which either
aggravate or reduce sepsis. Overall, the use of antibiotics instead of
causing benecial effects can lead to adverse conditions of aggravating the sepsis disease condition rather than treating it. Unfortunately, the current crisis of antibiotic resistance makes even the
little benecial effects of antibiotics unavailable for the sepsis
treatment. Over 30% of neonatal sepsis deaths are attributed to
antibiotic resistance. There is an urgent need for developing alternative approaches for the treatment of sepsis in the era of antibiotic
resistance [1].

4. Alternative approaches for sepsis treatment


Now that we have established the problems caused by sepsis,
the reason for occurrence of sepsis and current treatment for
septic condition, we focus the next part of the review on potential alternative approaches towards treatment of sepsis.
Before going into the details of how the eld has been directed
towards alternative strategies for curbing sepsis, let us rst
consider the possible approaches that can control sepsis. First, is
there a way of treating bacterial infection without treatment
with antibiotics? This approach deals with enhancing the immune system to tackle infections in the natural way. The infection can be cured if proper enhancement of innate immunity is
achieved. Second, even if antibiotics are used, are there agents
which can suppress the ability of toxins to induce proinammatory responses in the body? In this approach, both
bacterial infections and the problem of sepsis can be controlled.
Search for agents which can have such anti-endotoxin properties
is the challenging task in this approach. Third, are their alternative antibiotics, which, either do not lead to sepsis, or have
both antibacterial and anti-endotoxin properties? The interesting
part about this approach is that a single agent will be able to take
care of both the problems.
Understanding the body's rst line of defense against invading
pathogens at this juncture is crucial in order to understand how
newer alternatives toward prevention of sepsis could be designed.
Let us rst consider the body's rst line of defense. Antimicrobial
peptides form the body's rst line of defense against invading
pathogens [17]. We will briey discuss antimicrobial peptides and
subsequently lead into the alternative approaches.

Please cite this article in press as: D.S.S.M. Uppu, et al., Surviving sepsis in the era of antibiotic resistance: Are there any alternative approaches to
antibiotic therapy?, Microbial Pathogenesis (2015), http://dx.doi.org/10.1016/j.micpath.2015.02.001

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D.S.S.M. Uppu et al. / Microbial Pathogenesis xxx (2015) 1e7

4.1. Natural and synthetic antimicrobial peptides


Antimicrobial peptides or host defense peptides are considered
to be antibiotics for the future. These small proteins, found ubiquitously in nature, are generally cationic and segregate into hydrophobic and hydrophilic domains in solution [17,18]. This
amphipathic nature allows these peptides to interact with microbial cell membranes, which eventually leads to disruption of the
membrane and microbial cell death. Apart from direct killing, host
defense peptides also possess immunomodulatory properties
[17,18]. In other words, these peptides stimulate the immune system to act against invading pathogens. This immunomodulatory
activities encompass alteration of host gene expression,
chemokine-like action and/or induction of chemokine production,
inhibition of pro-inammatory cytokine production caused by
lipopolysaccharide, promotion of wound healing and so on [9].
4.2. Agents with only immunomodulatory activities
Development of cationic antimicrobial peptides with no antibacterial activity but with antiendotoxin and immunomodulatory
activities is an interesting concept. Direct antimicrobial activity
poses a chance of development of resistance against the peptides.
Additionally, once bacteria are killed, antimicrobial agents induce
release of bacterial toxins which is detrimental to the body. However, activation of the immune system to counter the threat of
invading pathogens would also lead to recovery. Moreover, several
antimicrobial peptides such as LL-37 are known to lose their antimicrobial activity in different physiological conditions. Studies with
LL-37 have improved the knowledge of immunomodulatory properties of Antimicrobial peptides by a great extent. The various
properties of LL-37 and its molecular mechanisms have been discussed elsewhere [20]. In an effort to dislodge direct antimicrobial
activity from Anti-endotoxin activity novel IDR peptides were
prepared which had excellent immunomodulatory activities but no
antimicrobial activity. It was shown that these peptides were able
to bring down the expression of TNF-a and IL-6 at the site of
infection. IDR-1 was believed to act on monocytes and macrophages and not on neutrophils or B- and T-lymphocytes. It was
shown that IDR peptides protect mice from succumbing to severe
bacterial infection, without having any direct antimicrobial activity
[21]. IDR peptides in combination with rst line malarial drugs
were also able to improve the survival of P. berghei infected mice
(cerebral malaria). In this model too, the pro-inammatory factors
were considerably suppressed [22]. IDR peptides have completed
Phase I clinical trials for treatment against infections caused by
Gram-positive and Gram negative bacteria.
4.3. Combination of antibacterial agents
Combination of one or more antimicrobial agents can be a potential treatment for the treatment of sepsis. Exposure of any of six
clinical isolates of S. aureus to daptomycin alone or in combination
with vancomycin or oxacillin (compared with vancomycin or
oxacillin alone) led to a dampened macrophage inammatory
response with diminished tumor necrosis factor secretion and
reduced accumulation of inducible nitric oxide synthase protein
[23].
The efcacy of imipenem, piperacillin combined with cecropin B
was investigated in the prevention of lethality in rat models of
septic shock. All compounds reduced the lethality when compared
with controls. Piperacillin and imipenem signicantly reduced the
lethality and the number of E. coli in abdominal uid compared
with saline treatment. On the other hand, each b-lactam caused an
increase of plasma endotoxin and TNF-a concentration.

Combination of cecropin B and betalactams showed to be the most


effective treatment in reducing all variables measured [24].
Combining biocides with different types of antibiotics prevented macrophage activation in the presence of bacteria. Studies
demonstrated the potential of chlorhexidine and alexidine to suppress inammatory responses caused by activation of TLRs. Alexidine and chlorhexidine bind not only to LPS but also to LTA from
Gram-positive bacteria. Alexidine has a higher afnity than chlorhexidine for both compounds. Calorimetric titration shows an
initial endothermic contribution indicating participation of hydrophobic interactions in LPS binding, while binding to LTA displayed initial exothermic contribution. Both compounds prevent
cell activation of TLR4 and TLR2 by LPS and LTA, respectively. The
addition of both compounds suppressed NO production by macrophages in the presence of bacteria treated with different types of
antibiotics. Chlorhexidine and alexidine suppress bacterial
membrane-induced cell activation at concentrations two orders of
magnitude lower than that used in topical applications [25].
4.4. Agents with both antibacterial and anti-endotoxin properties
Antimicrobial Lipopeptides: Polycationic peptides like Polymyxin
B have been known to bind to LPS strongly and thus do not promote
the production of pro-inammatory cytokines, thereby preventing
sepsis [26]. Host defense peptides being polycationic are also expected to be able to bind to LPS and inhibit the production of proinammatory cytokines. Defensins isolated from rabbits (polycationic rabbit alveolar macrophage cationic proteins MCP-1 and
MCP-2) were shown to displace Dansyl-polymyxin bound to puried LPS or whole cells competitively [27]. In a separate study, a
cecropin/melittin hybrid peptide called CEMA, which exhibited
excellent antibacterial activity against Pseudomonas aeruginosa
(MIC: 2.4 ug/ml) both in vitro and in vivo, was also found to bind to
LPS at the negatively charged sites, which are normally lled by
divalent cations under physiologicai conditions [28]. The afnity of
these peptides were found to be equal to that of Polymyxin B for
LPS. Further research into the properties of these compounds
revealed that they prevented the induction of endotoxic shock
mediator, tumor necrosis factor (TNF), when incubated with RAW
264.7 murine macrophage cell line. These peptides also protected
mice, charged with 10 mg of E. coli O111:B4 LPS, from lethal
endotoxic shock [29].
A series of peptides were prepared in an exemplary work, in
order to the study the structure-activity-relationships both with
respect to antibacterial and anti-endotoxin abilities. In this study,
the number of amino acids in the peptides varied from 18 to 30
while the number of positive charges varied from 4 to 9. Most of
the peptides showed moderate antibacterial activity against E. coli,
P. aeruginosa and S. typhmurium. Antimicrobial activities of the
compounds could not be signicantly correlated with respect to
charge, length and hydrophobicity. There was signicant correlation between antimicrobial activity and ability of the compounds to
bind to LPS. This correlation was also reected in the ability of the
peptides to suppress macrophage induced production of cytokines
like TNF-a and IL-6. But the degree of suppression was found to vary
proving that the peptides had roles beyond just interacting with
LPS, to prevent binding to macrophage cell lines [30].
To study the effect of length with respect to antiendotoxin
ability of peptides, two small cationic peptides, one 13-mer indolicidin (derived from bovine neutrophils) and another 12-mer
Bac2A (derived from Bactenecin) were prepared. LL-37, the human
cathelicidin was used as a comparator in the study. The two small
peptides exhibited somewhat contrasting immunomodulatory
properties. While indolicidin was able to suppress the secretion of
LPS induced TNF-alpha at concentrations comparable to LL-37,

Please cite this article in press as: D.S.S.M. Uppu, et al., Surviving sepsis in the era of antibiotic resistance: Are there any alternative approaches to
antibiotic therapy?, Microbial Pathogenesis (2015), http://dx.doi.org/10.1016/j.micpath.2015.02.001

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Bac2A was unable to do so. Bac2A, unlike indolicidin was unable to


induce the production IL-8, in any cell type. However, it was
chemotactic for THP-1 cells, whereas indolicin and LL-37 were not.
Understanding of the properties of such small peptides was vital for
the development of further alternatively directed therapeutic
agents [31].
Understanding the parameters that determine antibacterial and
antiendotoxin activities of antimicrobial peptides require thorough
analysis. In an excellent study, a series of peptides, all bearing the
same amino acid composition K6L9 were prepared. However,
depending upon the sequence and the structure of the amino acids,
variation in the properties was observed Specically, three different
sets of peptides were prepared, one formed an a-helical structure;
in another one, the sequence of amino acids were scrambled and in
the third type the hydrophobic and cationic amino acids were
segregated. Additionally, the D analogues were also incorporated
into the designs [32]. Almost all of these peptides exhibited good to
moderate antibacterial activities. The major conclusion was that the
biophysical parameters required for potent antibacterial activity
were not the same as those required to have LPS detoxication
ability. Broadly, the biophysical characteristic necessary for the
peptides to have potent LPS neutralizing activity was the stability in
the structure of the peptide both at the outer membrane and inner
membrane of bacteria. These peptides are also known to have weak
effect on the phosphate groups on the LPS. Peptides which had
potent antimicrobial activity were characterized to have unstable
structures at both the inner and outer membrane. These also had a
strong effect on the LPS phosphate groups. But the peptides with
both antimicrobial and LPS neutralizing activities had both dened
and exible structures, they had the ability to disintegrate LPS
micelles and had strong effect on the LPS.
The same group further studied the effect of hydrophobicity to
net positive charge ratio towards antibacterial and anti-endotoxin
activities of structurally similar AMPs. In their study they had
prepared 12 peptides (based on D/L leucine and lysine in varying
ratios) and their fatty acid conjugates [33]. The important conclusion of the study was that upon increasing the ratio of hydrophobicity/net positive charges, both antimicrobial and LPS
neutralization activities were increased, although with different
modes of contribution.
Synthetic Mimics of AMPs: The rst report of synthetic membrane active agents designed from principles governing the activity of Antimicrobial Peptides was of compounds that were
based on aryl scaffolds; these were decorated with pendant amine
groups to bring about amphiphillic structure. These compounds
demonstrated potent antibacterial activity and excellent selectivity. One of the compounds SMAMP-4, which contained a central
naphthalene ring and six positive charges, had high selectivities
towards S. aureus and E. coli compared to human erythrocytes.
SMAMP-4, which was also potent in inducing the production of
TNF-a on its own, did not down-regulate the activity of LPS. This
activity is different from the immunomodulatory activities of
natural peptides and the synthetic IDR peptide. Moreover,
SMAMP-4 could induce the production of neutrophil chemoattractant, murine KC, in mouse primary cells. This was the rst
report of a non-peptidic membrane active agent which displayed
both potent antimicrobial activity and immunomodulatory activity [34].
Aryl amide foldamers are potent antibacterial agents which are
undergoing Phase-II clinical trials as topical antibacterial agents
[35]. Some compounds bearing structural similarity to aryl amide
foldamers were able to bind to LTA of S. aureus and prevented the
induction of TNF, IL-6 and IL-10. They synergistically exhibited
potent antibacterial activity as well as inhibited LTA induced sepsis
in vivo [36].

In another study, a series of 20 compounds were created, based


on a peptide/peptoid trimer which had lipophilic appendages
varying from C-11 to C-20. Fourteen of these compounds were able
to induce the production of Gro-a and IL-8. Compounds containing
LysGlyLys and NLysGlyNLys sequences were not able to induce
cytokine production, but the compounds containing LysLysLys and
NLysNLysNLys were active only when a lipid tail at least sixteen
carbons long was appended to it. Three lipopeptides were able to
induce the production of IL-8 at amounts higher than that produced by LL-37 at same concentrations. None were able to induce
the production of TNF-a below 100 mM. Peptoids C16OH-NLysNLysNLys and C16OHNHarNHarNHar selectively induced IL-8 production but did not induce the production of TNF-a or IL-1 [37]. Due
to their protease resistant designs, these compounds were
considered to be benecial over existing in-vivo strategies to
counter sepsis.
AA-peptides were potent antibacterial agents with protease
resistant design [38]. Lipidated cyclic g-AApeptides were potent
membrane active agents which also possess immunomodulating
properties. These derivatives were effective in inhibiting the LPS
induced NF-kB signaling response and also in suppressing the
release of harmful pro-inammatory cytokines including tumor
necrosis factor-a (TNF-a). These were novel antimicrobial agents
which not only killed Gram-positive and Gram-negative bacteria
but also suppressed harmful pro-inammatory response caused by
such pathogens [39].
Histidine-derived antimicrobial peptides were another group
peptidomimetic molecules which showed efcient and broad
spectrum activity. These compounds were composed dimers or
trimers histidine (His) and arginines with hydrophobic alkyl tails as
appendages. Other than potent antibacterial activity these compounds also showed potent anti-inammatory activity. One of the
compounds, a trimer with two arginines anking a central histidine, appended to lipophilic tail, was able to suppress LPS induced
NO production and TNF-a production at efciency similar to LL-37
[37].
All the molecules presented in these sections possess excellent
proles to be used clinically. Although extensive in-vivo proles of
these compounds are yet to determined, their design principles will
denitely lead to development of much more efcient drugs in the
future.
4.5. Tumor necrosis factor (TNF) antagonists for controlling
inammation
Apart from targeting the PAMPs like LPS to reduce the inammation as described above, targeting the cytokines like TNF can be
another potential approach for the treatment of sepsis. The release
of pathogen associated molecular patterns (PAMPs) (Table 2) during an infection stimulates the immune cells in the body to produce
pro-inammatory cytokines like TNF-a, IL-6 etc. As previously
mentioned, this excessive and uncontrolled harmful inammation
leads to sepsis. Developing the antagonists for TNF would be a
promising approach to control the inammation. The role of the
antagonist is to reduce the bioavailability of TNF so that the amount
of inammation can be reduced. Biologic therapies that target the
proinammatory cytokine tumor necrosis factor (TNF) have been
shown to be efcacious in the treatment of several autoimmune
diseases, including moderate to severe plaque psoriasis, moderate
to severe rheumatoid arthritis, and psoriatic arthritis [40e43].
There are currently two classes of marketed biologic drugs that
reduce TNF bioavailability: soluble TNF receptor-Fc fusion proteins
(etanercept) and anti-TNF mAbs (adalimumab and iniximab)
[40e43]. Small molecular antagonists of TNF have also been reported and represent promising alternatives to macromolecular

Please cite this article in press as: D.S.S.M. Uppu, et al., Surviving sepsis in the era of antibiotic resistance: Are there any alternative approaches to
antibiotic therapy?, Microbial Pathogenesis (2015), http://dx.doi.org/10.1016/j.micpath.2015.02.001

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inhibitors [40e43]. This approach of developing antagonists to TNF


can be a looked over as a novel approach to target the sepsis related
diseases and has immense potential for the treatment of sepsis.
5. Conclusions and future directions

the federal government is needed to tackle this ever-growing disease condition of sepsis.
Uncited reference
[19].

Sepsis, a dangerous medical condition, is a result of after-effects


of an infection by microorganisms. Treatment options for sepsis are
very sparse in the clinics and antibiotics remain to be the major
contenders in the current treatment modalities. However, the
in vitro data and in vivo animal studies suggest that few antibiotics
upon treatment make the sepsis disease condition even worse.
These data suggest that few antibiotics promote the production of
endotoxins from bacteria leading to increased production of cytokines. Thereby, the over-stimulation of immune system aggravating
the sepsis results in fatal conditions. b-lactam antibiotics have been
shown to be the causative agents of aggravating the sepsis. However, the current global threat of antibiotic-resistant superbugs
leaves us with no treatment options available for sepsis. In this
light, novel alternative strategies have been developed for the
treatment of sepsis. Bacterial cell membrane targeting molecules
like antimicrobial peptides (AMPs) and lipopeptides offer us the
hope as they have been shown to have low propensity for the
development of bacterial resistance. A variety of natural AMPs and
their mimics have been shown to reduce the production of cytokines on stimulation of the immune cells with bacterial endotoxins
like LPS, LTA or PG. Combination of antibiotics and cationic peptides
have also shown some promise as anti-inammatory options for
sepsis treatment. Development of TNF antagonists can be another
promising approach to reduce the inammation for the treatment
of sepsis.
Early diagnosis of sepsis is an extremely important point that
should not be neglected. The limitation of incomplete understanding of the entire process of sepsis has made clinical symptoms as the primary mode of diagnosis of sepsis. However, delay
in treatment can have fatal consequences. Hence, it is important to
identify novel tools for diagnosing sepsis. In this regard identication of biomarkers related to sepsis can be considered as diagnostic tools. We have already describe three different approaches
taken in this direction [3e5]. More studies towards the diagnosis
of sepsis are required to facilitate cure of sepsis in shorter time
frames.
There is an urgent need to increase awareness among clinicians
to be careful while prescribing the antibiotics which can cause
deleterious effects during the treatment of sepsis. There is an absolute lack of clinical data on the effect of antibiotics in aggravating
sepsis disease conditions and clinical data collection is needed for
the proper monitoring of disease pathology, diagnosis and progression at all settings. Unfortunately, this data is only available for
in-vitro and animal studies. Moreover, the global research initiatives related to develop alternative strategies for the treatment of
sepsis are sparse. All the available data in developing alternative
strategies for the treatment of sepsis is at a very preliminary level
and is far from seeing the light of clinical settings. More importantly, lack of animal models that properly mimic the human inammatory diseases prevent the development of new drug
molecules for the treatment of sepsis [44]. To date, there have been
nearly 150 clinical trials testing candidate agents intended to block
the inammatory response in critically ill patients, and every one of
those trials failed [45].
In summary, signicant amount of research needs to be directed
towards sepsis to completely understand and treat the disease. Use
of antibiotics towards the treatment backres in several cases, thus
the search for alternative medicines is extremely important. A
consensus effort of clinicians, researchers, policy makers and also

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antibiotic therapy?, Microbial Pathogenesis (2015), http://dx.doi.org/10.1016/j.micpath.2015.02.001

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Please cite this article in press as: D.S.S.M. Uppu, et al., Surviving sepsis in the era of antibiotic resistance: Are there any alternative approaches to
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