Microbial

pathogens and strategies for combating them: science, technology and education (A. Mndez-Vilas, Ed.)
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Current advances on bacterial pathogenesis inhibition and treatment

strategies
K. Ivanova, M. M. Fernandes and T. Tzanov*
Group of Molecular and Industrial Biotechnology, Department of Chemical Engineering, Universitat Poltecnica de
Catalunya, Ernest Lluch s/n, 08222 Terrassa, Spain
Bacterial pathogenesis is a multi-factorial process that is regulated by the production of virulence factors, enabling bacteria
to cause various infectious diseases. For many years, antibiotics have been successfully applied for fast treatment of
bacteria-mediated diseases, offering an effective infection transmission control. However, it has become clear in recent
years that the overuse of antibiotics leads to an increased emergence and spread of multi-drug resistant microorganisms.
As a consequence, bacteria cause life threatening infections and increased mortality, morbidity, length of hospitalization
and health care costs. There is a need to decrease the antibiotic usage and to develop new effective antimicrobial strategies
to prevent and treat certain infections. This review summarizes recent advances in attenuation of bacterial virulence and
treatment of infections. Prevention strategies that provide minimal evolutionary stress and no resistance development such
as interference with bacterial cell-to-cell signalling (quorum sensing) pathways and virulence mechanisms are reviewed.
Moreover, new advances on antimicrobial agents such as antimicrobial peptides, bacteriophages, nanoantibiotics and
natural polyphenols as well as a new strategy of resistance genes disruption using the bacterial adaptive immune system as
a potential therapeutic tool, are also considered.
Keywords:bacteria virulence; multi-drug resistance; virulence factors; anti-virulence strategies; emerging therapeutics

1. Introduction
Among the 100 trillion cells that constitute the human body, only 1 in 10 is actually human. The remaining cells are
microorganisms such as bacteria and viruses [1]. These microorganisms are harmless and live in perfect balance with
human body, playing an important role in supporting and maintaining vital functions such as our immune and digestive
systems [1]. However, when this balance is broken and the delicate ecosystems that bacteria carefully construct in
different parts of human body are disrupted, bacteria become pathogenic, causing infection diseases. The introduction
of antibiotics in the early 20th century initiated a new era in the treatment of microbial infections. They were the most
successful drug ever introduced saving countless lives, extending life span and permitting previously deadly medical
procedures. To kill bacteria, antibiotics target different cell components and use different mechanisms of action such as
the inhibition of cell wall synthesis (lactams and glycopeptides), protein production (macrolides, aminoglycosides,
tetracyclines) and nucleic acids synthesis (fluoroquinolones, rifampin) [2, 3]. Antimicrobial agents such as sulfonamides
and folic analogues also disrupt essential metabolic pathway for folic acid synthesis followed by the inhibition of DNA
synthesis [3].
Although antibiotic strategies are highly effective in the treatment of bacterial infections, they have been responsible
for a substantial evolutionary stress caused on bacterial population and the emergence of drug and multi-drug resistance
[2, 3]. Dangerous bacterial species such as the methicillin-resistant Staphylococcus aureus (MRSA) and vancomycinresistant enterococci (VRE) have emerged due to antibiotic overuse. This resistance development placed the bacterial
infections as a leading cause of death worldwide and is now one of the greatest challenges of the twenty-first century.
Nevertheless, the increasing understanding of bacterial pathogenesis and intercellular communication has been a
valuable tool to develop new strategies in the treatment of bacteria-mediated diseases. Alternative approaches through
inhibition of bacterial pathogenesis thus causing less evolutionary stress on bacteria population have been also studied.
This review provides a brief overview of bacterial virulence as a new target for attenuation of bacterial pathogenesis
and treatment of the acquired infections. Promising anti-virulence strategies based on the interference with the virulence
factors disruption are outlined. As alternative therapeutic approaches special focus is given to the antimicrobial
peptides, bacteriophages, natural polyphenols and nanoantibiotics as promising antimicrobial agents, in addition to a
novel strategy that targets resistance gene disruption in bacteria.

2. Bacterial pathogenesis and the virulence mechanisms

During pathogenesis bacteria use an array of virulence factors to cause disease in the human host, acting individually or
simultaneously at different stages of the infection [4]. This is a multi-factorial process that depends on the immune
status of the host, the nature of the species or strains (virulence factors) and the number of organisms in the initial
exposure [5]. Once bacteria find the suitable site for colonization they initiate the expression of target genes followed by
the production of virulence factors that invade the host cells and trigger the infection process [2]. These virulence
factors include: i) membrane proteins that play an important role in adhesion and binding to host cells, facilitating the

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colonization, biofilm formation and intercellular communication [6]; ii) secretory proteins such as toxins, which kill or
change signal transduction in mammalian cells and are responsible for some host cell-bacteria communication [7]; iii)
specialized secretion systems that resemble a syringe and are used by bacteria to inject toxins (effectors) into the host
cell [8]; iv) polysaccharide capsules that surround the bacterial cell and have anti-phagocytic properties [9]; v) cell wall
and outer membrane components, like peptidoglycan layer and lipopolysaccharide, mainly in Gram-negative bacteria
that protect against complement-mediated lysis and are potent inducer of inflammation [10]; and vi) a group of other
virulence factors that include proteins involved in biofilm formation and siderophores [4]. Biofilm formation confers
pathogenic bacteria increased resistance to convectional antibiotics and host defences mechanisms [11]. Pathogenic
bacteria, such as P. aeruginosa, E. coli, Staphylococci and Mycobacterium are able to form biofilms on living (lungs,
burn wounds and urinary tract) or nonliving surfaces, such as distinct medical devices (indwelling catheters, artificial
hips and contact lenses) causing intractable infections [11-13]. Understanding how pathogenic bacteria use virulence
factors to interact with their hosts and originate the disease is a prerequisite to define new targets for vaccines and drug
development.

3. Inhibition of bacterial pathogenesis - anti-virulence strategies

A novel approach for the inhibition of bacterial pathogenesis and treatment of bacterial infections involves targeting the
virulence factors. These anti-virulence strategies interfere with the process of infection before the host damage occurs.
Bacterial virulence is blocked specifically, without killing or inhibiting bacterial growth, causing less than the
traditional antibiotics evolutionary pressure for the development of resistant genes. Moreover, by preventing the
expression or activity of virulence traits, the bacteria are less prone to colonize the host. Among the main anti-virulence
strategies are these targeting the inhibition of: i) toxins; ii) bacterial adhesion to the host cell; iii) specialized bacterial
secretory systems; iv) organism-specific virulence gene expression; and v) organism-specific cell-to-cell signalling (i.e.
quorum-sensing) (Table 1). By targeting these virulence factors the infection progression is interrupted and the host
immune system is able to eliminate the pathogen. These strategies are discussed below.
Table 1 Anti-virulence strategies

Targets

Anti-virulence agents

Mode of action

Reference

Toxins production

Antibodies
Toxin analogues
Pilicides
Mono- and oligosaccharides

Neutralize anthrax toxin

Block toxin activation
Interfere with pilus formation
Block carbohydrate - specific site
of pili/fimbrae
Inhibit toxin secretion, interaction
with host cells or functional
assembly

[14]
[15]
[16]
[17, 18]

Block AHLs production

Inactivation of AHLs
Target transcriptional receptor
inhibition

[20]
[21-25]
[26-28]

Inactivate AIPs
Block membrane associated
receptors

[29]
[30, 31]

Bacterial adhesion
Specialized secretory
systems

Inhibitory molecules

Quorum sensing

Gram negative bacteria:

SAM analogues
Enzymes
Antagonists
Gram positive:
Apolipoprotein B
AIP analogues

[19]

3.1. Inhibition of bacterial toxins

Toxins are usually identified as the first virulent factors to be secreted during bacterial pathogenesis acting directly or
indirectly on the host cells [2, 4, 10]. The most studied toxin systems are those of B. anthracis (anthrax toxin) and E.
coli (shiga toxin). They are known as AB toxins and consist of two subunits A and B with distinct functions in infection
development. B-binding subunit is crucial for bacterial adhesion and binds to a specific host receptor, while A-active
subunit exhibits selective enzymatic activity and injures the host cells [2, 10]. Understanding the mechanisms that
toxins use to interact with the host has been a valuable tool to define several inhibition strategies and combat bacterial
pathogenesis. For example, the neutralization of the toxins using monoclonal antibodies has been considered as an
attractive treatment approach providing rapid and extensive protection [19, 32-34]. Monoclonal antibodies isolated from
chimpanzees were shown to inhibit the binding subunit of anthrax toxin therefore preventing and treating the anthrax
infection [14]. Moreover, analogues of the toxin binding subunits successfully blocked the activation of A subunit and

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prevented the invasion of host cells [15, 35]. Current strategies to attenuate pathogenesis involve the blocking of the
intracellular uptake of the active part of the toxin or the inhibition of its active site [36].
3.2. Inhibition of bacterial adhesion
Many pathogenic bacteria use specific structures such as pilus, fimbrae and flagella to attach and colonize the host cells
[37-39]. For instance, uropathogenic E.coli, which cause lifethreatening urinary tract infections, use a long
multisubunit appendage (pilus) to attach more efficiently to the urogenital epithelia and to prevent its washing by the
urine flow [40]. This mechanism of action indicates that by affecting the bacterial adhesion a decrease of the risk of
infections development may be attained. Several compounds, called pilicides were shown to interfere with pilus
formation resulting in a non-functional structure that fails to bind the host and consequently decrease the bacterial
colonization and prevent the pathogenesis [16]. Mono or oligosaccharides that interact specifically with the
carbohydratespecific site on the pili/fimbrae structures were also able to block the bacterial adhesion [17, 18].
3.3. Inhibition of bacterial secretory systems
During pathogenesis bacteria use secretory systems to transport and inject the toxins (effectors) into target cells. These
systems have been considered as potential targets for novel anti-virulence therapeutic agents [2, 17]. Inhibition of the
secretory systems has been accomplished using small molecules that prevent their functional assembly, toxins secretion
or interaction with the host cells [2, 41].Various studies revealed that some compounds were capable of inhibiting the
secretion systems and attenuate the virulence of S. typhimurium, Pseudomonas, Francisella and reduce their
pathogenesis [42]. For example, acylated hydrazones of salicylaldehydes were found to efficiently inhibit the secretory
system of Chlamydia and prevent pathogenesis [43].
3.4. Inhibition of organism-specific virulence gene expression
Targeting the virulence gene expression has been mainly researched on V. cholerae. This bacterium is responsible for
some of the largest episodes of diarrhoeal disease pandemics and different approaches to treat the infection that it
causes have been extensively studied. During infection, V. cholerae secretes two virulence factors: the cholera toxin, a
protein that causes watery diarrhoea and the toxin-coregulated pilus (TCP), a thin, flexible, filamentous appendage on
the surface of bacterial cells that colonize the small intestine [44]. A small molecule 4-[N-(1,8-naphthalimide)]-nbutyric acid (virstatin) was shown to efficiently inhibit virulence regulation in V. cholerae [45]. This was the first study
showing that a targeted anti-virulence approach could prevent cholera disease in animal models and the mechanism by
which this inhibition occurs has been since then studied extensively [46].
3.5. Quorum sensing inhibition strategies
The expression of the virulence factors in important human and plant pathogens such as P. aeruginosa, S.aureus, E.coli,
S. typhimurium, Erwinia, and A. tumefaciens, among others is regulated by a process called quorum sensing (QS) [4749]. QS allows bacteria to regulate community-wide behaviours including biofilm formation, virulence, conjugation,
sporulation, and swarming motility [50]. This is possible due to a mechanism of cell-to-cell communication that is
based on the production, secretion, and detection of small signalling molecules, called autoinducers (AIs). The QS
systems used by Gram-negative and Gram-positive bacteria differ in the type of QS signalling molecules they use and
in the signal transduction systems (Fig. 1). The most intensively studied AIs used by Gramnegative bacteria are acyl
homoserine lactones (AHLs) (Table 2). The AHLs signalling molecules are produced by AHLs synthases, which use
adenosyl-methionine (SAM) as a source for lactone ring formation and acyl-carrier proteins (ACP) as a source for the
side fatty acid chain of AHLs. On the other hand, Gram-positive bacteria use linear or cyclic oligopeptide signals, called
autoinducing peptides (AIPs) (Table 2) [12]. These AIPs are produced as precursor propeptides in the intracellular
compartment, further processed by a membrane-bound endopeptidase (Fig.1-green circle) and secreted to the
extracellular environment as mature AIPs [51].
Targeting QS systems in Gram-negative and Gram-positive bacteria constitutes a novel pharmacological approach to
control bacteria virulence and biofilm formation. In recent years, the development of new anti-quorum sensing drugs
that have the advantage to affect bacterial behaviours, but do not kill or inhibit their growth has been gaining ground
[50, 52-54]. This would allow the host defense system to eliminate attenuated bacteria or substantially increase the
effect of co-administered antibiotics. Different strategies aiming the QS signalling interruption in Gram-negative and
Gram-positive are discussed below.

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Fig. 1 Quorum sensing systems in bacteria. Gram-negative bacteria (left) secrete AHLs (red triangles) that in threshold
concentrations penetrate into the cells and activate the cognate AHL receptor and induce the QS regulated genes expression. Grampositive bacteria (right) produce mature AIPs (red circles) that further interact with a transmembrane histidine kinase receptor
activating the target gene expression via autophosphorylation of the trascriptional regulator.

3.5.1. Gram-negative bacteria

Blockage of AIs synthesis
A potential target for inhibiting QS pathway is the blockage of AHL production in Gram-negative bacteria. This
inhibition strategy is the least investigated and relies on the development of structural analogues of the substrates for
AHL synthases - S-adenosyl methionine (SAM) and acyl-carrier protein (ACP) [55]. Structural analogues of SAM such
as L/D-S-adenosylhomocysteine and sinefungin have been found to suppress the AHL synthesis and inhibit the first
step in QS signalling [20]. The inhibitory activity of these analogues has been proved only in vitro and they have not
been tested in vivo yet. Other enzymes in living systems, however, also use SAM and its analogues could potentially
cause side effects [53, 55].
Inactivation of AIs in Gram-negative bacteria
The inactivation of AHLs by degrading them has been regarded as a promising strategy for pathogenesis inhibition.
AHLs degradation does not allow signal accumulation in extracellular environment and consequently QS-regulated
virulent genes are not expressed [47]. Altering AIs in extracellular surrounding provides minimal evolutionary stress on
pathogens and would eliminate the risk of resistance development. Enzymes such as acylase, lactonase, and
oxidoreductases are known to selectively inactivate AHLs involved in Gram-negative bacteria QS communication
systems (Fig. 2).
First evidences that these enzymes were able to quench QS signals was attained with lactonase- and acylaseproducing bacterial species such as Bacillus sp. 240B1, Bacillus strain COT1, A. tumefaciens, B. thuringiensis,
Arthrobacter sp. IBN110, B. thuringiensis, B. cereus, B. mycoides, V. paradox, Anabaena, Ralstonia, Rhodococcus. The
enzymes extracted from these bacteria were capable of degrading AHL molecules [21, 54, 56] and have been also found
to interrupt the QS signalling of Gram-negative bacteria in a process called quorum quenching (QQ) [21, 57]. Dong et
al. reported an AHL-lactonase produced by Bacillus sp. and encoded by the aiiA gene, that was able to degrade the ester
bond in the AHL lactone ring [21]. In fact, the expression of aiiA gene encoded in plants has shown to attenuate the
virulence of the plant pathogen E. carotovora. In vitro studies demonstrated that lactonase effectively degraded a range
of AHL derivatives and could be used to quench QS and prevent pathogenesis [21, 58]. Moreover, there is evidence that
lactonase and antibiotics applied together can be used to block not only the pathogenesis but also the multi-drug
resistance in clinical isolates such as P. aeruginosa [59]. By cleaving the amide bond between the homoserine lactone
and the fatty acid chain the acylase-producing bacteria V. paradoxusm was also able to degrade AHL signals [22, 56].
Lin et al. have demonstrated that the expression of acylase encoded by genes from Ralstonia in pathogenic P.
aeruginosa PAO1 efficiently suppressed the production of QS regulated virulence factors (elastase and pyocianin),
decreased its swarming motility and attenuated the pathogenesis to C. elegans [22]. Lately, Huang et al. demonstrated
that the soil P. aeruginosa PAI-A are also capable to degrade long AHLs as a sole energy source for growth in selective
conditions [60]. Acylase I isolated from kidney was also shown to be capable to deacylate in vitro model AHL
molecules including N-butyryl- and N-octanoyl-l-homoserine lactones, and thereby inhibit the QS pathway in P.
aeruginosa and reduce the formation of pathogenic biofilms on a polystyrene surface [23].

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Table 2 Chemical structure of natural autoinducers.

NH

Gram - negative
Acyl homoserine lactones

O
R

NH

R1

R1=C3H7
V.fischeri;E.carotovora;E. chrysanthemi [67,
69,70]
R1=C5H7
A.tumefaciens [71]
R1=C7H15
P.putida; Yersinia pestis [72, 73]

R=C3H7
P. aeruginosa; A. hydrophila; A. salmonicida [61, 62]

R=C3H6OH
V. harveyi [63]
R= C5H11
C.violaceum;P.aureofaciens; Y. pseudotuberculosis
[64-66]
R=C7H15
R1=C9H19
V. fischeri; B. cenocepacia [67, 68]
P. aeruginosa [61]
Gram - positive
Auto-inducing peptides
O
Met
Ile

Phe

Cys

Phe Asp

Thr

Ser

Leu

Tyr

AIP-I

Leu
Cys

Ser Ser

Asn
Ala

Val

Leu

Gly

Met

Cys

Phe Asp

AIP-II

Ile
Asn

Ile

Cys

Phe Tyr

AIP-III

Ser
Thr

AIP-IV

(S.aureus)
Ala

Asp
Ile
Arg
Trp* Asp
Pro
Thr
Gln
Gly

Glu

Met

Arg

Leu

Ser

ComX (B. subtilis) [74]

Lys

Ile
Gln
Lys
Phe
Asp
Arg
Lys
Arg
Phe
Leu
Phe

CSP (S. mutans) [75]

Another group of QQ enzymes that has been reported includes oxidoreductases produced by species as R.
erythropolis, Burkholderia sp. GG4 [56, 76]. Oxidoreductases confuse bacterial signalling pathways through the
modification of the 3C keto group of the acyl side chain of AHLs into hydroxyl group [76]. Upon modification, AHL
fails to bind to the cognate transcription regulator and further activation of QS regulated genes do not occur [56]. An
oxidoreductase produced by Burkholderia sp. GG4 has also been demonstrated to modulate the signalling molecule 3oxo-C6-HSL and attenuate E. carotovora virulence [24].
Another family of human enzymes called paraoxonases (PONs) was recently reported to exhibit lactonase activity
disrupting AHL-mediated QS systems [77]. Teiber et al. reported PONs activity on QS molecules produced by P.
aeruginosa and other species including Burkholderia, Yersinia, Serratia and Aeromonas [25].

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NH

Acyl homoserine lactone (AHL)

R = O or H, n = 1 - 9
Oxidoreductase

Lactonase
Acylase

O
HO
HO

NH
Acyl homoserine

O
NH2

Homoserine lactone

HO

n
Fatty acid

O
O

OH

NH

3 - hydroxy AHL

Fig. 2 Enzymatic degradation of AHL signals by AHL-lactonase and AHL-acylase. Oxidoreductases inactivate AHL by substituting
the oxo group at the C3 with hydroxyl group [21].

Blocking of the receptors in QS pathways

Although the mechanism of AHLs binding to the receptors is not clear, numerous compounds have been reported to
block the binding of these QS molecules to the cognate receptor in QS signalling [53]. Some examples of different
strategies used to block the receptors include the development of antagonists that are based on: i) AHL analogues, ii)
structurally unrelated antagonists, and iii) natural QS inhibitors.
Several reports have been published concerning the AHL analogues ability to block the receptors and therefore act as
receptor antagonists. The development of these analogues is usually achieved through modification of the AHL acyl
side chain, lactone ring or both the lactone ring and the side chain [55]. Strategies such as: the replacement of the C3
atom by a sulphur atom [78], the introduction of aromatic side chains such as phenylpropionyl and phenoxyacetyl
groups or of a bulky group [79], the replacement of the AHL carboxamide bond with sulfonamide bond, or even the
introduction of sulfone or sulfoxide group have been regarded as effective approaches to induce antagonistic activity
[53, 80]. Development of AHL antagonists through lactone ring modifications have been less reported. By changing the
lactone functional group with ketones, alcohols and amines has shown to provide antagonistic effect against the
transcriptional regulators in P. aerugionosa [53]. Ishida et al. synthesized acyl cycloalkylamide analogues of AIs and
observed that N-octanoyl cyclopentylamide and its structural analogue N-decanoyl cyclopentylamide interfered with
AHL-mediated QS systems in P. aeruginosa [26].
Other compounds structurally unrelated to AHL were screened using bioassay reporter strains for their capability to
inhibit bacterial quorum sensing. Several chemical compounds including 4-nitro-pyridine-N-oxide (4-NPO), Pbenzoquinone, 2,4,5-tri-bromo-imidazole and 3-nitro-benzen-sulfonamide were found to successfully interfere with QS.
Among these chemical antagonists, the most effective was 4-NPO [55]. The 4-NPO significantly reduced the expression
of virulence genes regulated by QS systems in P. aeruginosa and also decreased its biofilm formation [27, 55].
Numerous quorum sensing inhibitors (QSI) have been extracted from natural sources such as plants, herbs and fungi
[55]. Plants and fungi naturally produce QS inhibitors as their first line of defence against pathogenic bacteria,
inhibiting their colonisation and attenuating their virulence [55]. The screening of QSI produced by Penicillium species
revealed that penicillic acid and patulin produced by Pe. radicicola and Pe. coprobium, are the most effective
antagonists of LasR and RhlR receptors of P. aeruginosa QS systems [55]. Moreover, in vitro study of P. aeruginosa
biofilm formation in presence of patulin and tobramycin, showed increased sensitivity to antibiotics [78]. A number of
QS inhibitors extracted from fruits have proved to interfere with bacterial QS and considered as alternative antiinfective agents. For instance, studies performed in vitro using V. harveyi screening bioassay have shown that the
grapefruit juice inhibited AHLs signalling [81]. Further investigation showed that furocoumarins, presented in
grapefruit could also prevented the biofilm formation of E. coli O157:H7, S. typhimurium and P. aeruginosa [81].
Rasmussen et al. reported a number of plants and herbs extracts including garlic, carrot, bean, water lily, chamomile,
habanero and propolis capable to suppress the QS regulated virulence genes. The most effective was found to be the
garlic extract containing at least three different inhibitors of the QS pathway. Moreover, a synergistic effect of the garlic
extract and tobramycin was also reported to reduce biofilm formation [28]. Adonizio et al. demonstrated that extracts of
several south Florida medical plant species including B. buceras C. erectus, C. viminalis attenuated P. aerugionsa
PAO1 pathogenesis inhibiting the virulence factors production [82]. Moreover, plant-derived polyphenols have been
also shown to affect the QS and biofilm formation. The inhibition mechanism of QS by these compounds is still not
well understood. However, there are evidences that (-)-epigallocatechingalate (EGCG), ellagic acid, tannic acid, and

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pyrogallol interfere with AHL-dependent QS systems [78]. EGCG showed significant inhibitory effect on pathogenic E.
coli O157:H7 suppressing the QS-regulated genes to express the virulence factors [83]. Another extracts form Camellia
sinensis (Green tea) demonstrated anti-QS activity modulating the expression of virulence factors in P.aeruginosa
PAO1[84].
The halogenated furanone compounds (or fimbrolides) are a largely investigated group of QS inhibitors isolated
from red macroalga D. pulchra [53, 78]. This alga produces more than 30 furanones as secondary metabolites that were
shown to interfere with AHL-based QS signalling systems by inhibiting the swarming motility of S. liquefaciens and P.
mirabilis [78]. Indeed, natural furanones have been shown to target QS systems also in V. fischeri, V. harveyi, S. ficaria
and other bacteria [53]. The furanone - (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H) produced by D. pulchra, in
particular, inhibited pathogenic phenotypes of E. coli such as swarming motility and biofilm formation [85].
Nevertheless, natural furanones with significant activity over a number of bacterial species failed against P. aeruginosa.
In contrast, synthetic derivatives of naturally occurring furanones (i.e. halogenated furanones) increased the survival
time of mice infected with lethal P. aeruginosa [86]. It has to be pointed out, however, that the halogenated furanones
are too reactive and might be toxic to the human cells [87].
QSIs (antagonists) are not intrinsically antimicrobial agents, but rather cause the bacteria to be more susceptible to
antimicrobials and raise the immune responses of the host. Moreover, their combination with classical antibiotics and/or
novel antimicrobials is a possible strategy to generate new hybrid therapeutics with complimentary modes of action.
3.5.2. Gram-positive bacteria
The interference with AIP-mediated quorum sensing systems in Gram-positive bacteria relies on different targets.
Gram-positive bacteria possess a twocomponent QS system consisting of a membrane-bounded histidine kinase
receptor and a responsive regulator (Fig. 1). Other components of AIP-mediated signalling systems including AIP
synthases, activators, efflux AIP transport systems, transcriptional regulators are also considered as targets [53].
Understanding the mechanisms of receptor activation by QS molecules have led to the development of new QSI that
shut down the entire QS pathway and attenuate bacterial pathogenesis.
Peterson et al. reported apolipoprotein B as a sequester of AIPs from S. aureus preventing the activation of the
receptor and therefore the expression of virulence genes [29]. Several AIP-mediated QS systems have been extensively
studied including the agr system of pathogenic S. aureus and the agr-like system of E. faecalis, both possessing
structurally similar AIP signals. The agrquorum sensing system of S. aureus uses various thiolactone containing
peptides to control the pathogenesis [2, 53]. This system consists of an AgrC histidine sensor kinase receptor that
selectively interact with AIP, which results in the activation of the AgrA transcriptional regulator via phosphorylation
[53].
Based on the discovery that the AIP protein side chain (also called tail) is crucial for receptor binding and activation,
it is reasonable to think that its synthesized analogues might inhibit bacterial signalling [53]. Otto et al. reported
different AIPs derivatives of S. epidermidis that successfully suppressed the agrcontrolled production of the virulence
factor -toxin and toxin, and it did not affect the bacterial growth [30]. S. aureus uses second signalling system that
regulates the activation of agr-system, known as RNAIII activating peptide (RAP) mediated system [53, 55]. The
(RAP)mediated pathway was successfully disrupted by the RNA III inhibiting peptide (RIP), a heptapeptide originally
isolated from S. xylosus [31, 88]. In vivo studies have demonstrated the inhibitory activity of synthetic RIP (amide form
of the originally isolated one) by reducing S. aureus infections such as cellulitis, septic arthritis, keratitis, osteomyelitis
and mastitis [88, 89]. A synergistic effect of RIP and antibiotics has been also reported to act against biofilm formation
of S. aureus [88]. There is evidence that a non peptide analogue of RIP, known as 2,5-di-O-galloyl-D-hamamelose
(hamamelitannin), a natural product of Hamamelis virginiana (witch hazel) also interferes with the QS pathway in S.
aureus and S. epidermidis, and reduces the risk of infection [90].
The greatest advantage of the discussed anti-virulence strategies targeting virulence factors and signalling pathways
in pathogenic bacteria is the reduced evolutionary pressure for emergence of resistance. Those strategies affect only the
pathogens and reduce the risk for harmful to the mammalian cells side effects, unlike the conventional therapeutics. An
interaction, however, with beneficial microbiota in human gastrointestinal tract should not be excluded. It is also likely
that bacteria may develop resistance to anti-virulence agents using alternative routes. Therefore, the understanding of
bacterial virulence is a key issue for the rationale design of such therapeutic agents. Creating anti-virulence drugs that
are efficient against broad spectrum of bacteria without adverse side effects is highly challenging.

4. Emerging therapeutic strategies

In contrast to the anti-virulence strategies that interfere with the process of infection before the host damage occurs,
therapeutic strategies are intended to act when the infection is already established. Alternatives to the antibiotic
treatments are presented below.

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4.1. Antimicrobial peptides

Antimicrobial peptides (AMPs) have long been considered as efficient alternatives to conventional antibiotics to fight
multi-drug resistant pathogens and to prevent pathogenic biofilm formation [91]. These compounds were initially
identified as host defence modulators in organisms such as frogs and insects and further isolated from tissues and cell
types in many other living organisms [91-93]. AMPs share common properties including: a net positive charge under
physiological conditions, amphiphilicity containing cationic and hydrophobic amino acids [91, 94] and efficiency in
low concentrations [95]. Despite their mode of action is not fully understood, it is known that these compounds possess
different mechanisms of action towards mammalian and microbial cells [93]. It is believed that upon contact with
microbial cell membrane, AMPs form a secondary structure, allowing insertion of hydrophobic components into the
membrane lipid domains, which results in disruption of the membrane structure. They are extremely rapid in killing
bacteria and decrease the risk of resistance development compared to conventional antibiotics. Disruption of cell
membrane can be achieved by different pores-forming mechanisms such as barrel-stave, toroidal pore, and carpet
models [93, 96]. AMPs are also reported to interfere with other intracellular mechanisms including inhibition of cell
wall synthesis, protein inhibition or action on DNA or RNA [96].
Protegrins, a natural AMP isolated from porcine leucocytes, was shown to possess broad-spectrum antibacterial
activity against E. coli, S. aureus, P.aeruginosa, C. trachomatis, N. gonorrhoeae, yeasts (C. albicans) and viruses (HIV1) by interacting with their cell membranes [94]. Magainins found in the skin of Xenopus laevis, also exhibited broadspectrum selective antibacterial activity against Gram-negative and Gram-positive bacteria while analogues of
magainins were active against malignant melanoma in vivo and effectively treated E. coli infections in mice [97].
Arenicins, AMPs produced by marine polychaeta Arenicola marina showed bactericidal properties against pathogenic
Gram-positive bacteria such as S. aureus, L. monocytogenes, B. megaterium, and Gram-negative bacteria such E. coli
and A. tumefaciens and against fungus C. albicans [98]. The combination of AMPs with conventional antibiotics have
been shown to induce a synergistic effect preventing drug-resistance development and reduce the treatment antibiotic
dosage [91]. Anantharaman et al. demonstrated the synergistic effect of designed antibacterial peptides with nonpeptide antibiotics such as rifampin and kanamycin against pathogenic E. coli [99]. In fact, the number of studies that
focus on antibiotic and AMPs combinations as an alternative approach to control multi-drug resistance has been
increasing. Enhanced antibacterial effect against opportunistic human pathogen P. aeruginosa was observed in vitro
when tachyplesin III and colistin were applied together, and in vivo when alpha-helical antimicrobial peptides and
rifampicin were combined [100, 101].
AMPs have also been reported to act upon bacterial biofilm. Wang et al. showed that chrysophsin-1, a cationic
antimicrobial peptide, could be used as an alternative drug in preventing and treating pathogenic S. mutans biofilms,
the major causative agent for dental caries [102]. Natural AMP, called LL-37 and several of its synthetic fragments
successfully inhibited biofilm formation of P. aeruginosa PAO1 strain [103]. Moreover, several bovine AMPs at subinhibitory concentration did not exhibit bactericidal effect against bacteria, but were able to inhibit biofilm formation.
These peptides were shown to be efficient agents to eradicate pre-formed biofilm and for the treatment of cystic fibrosis
diseases [104].
There are evidences that bacteria can also develop resistance to AMPs by proteolytic inactivation or changes in the
cell membrane surfaces. It is believed, however, that the process is longer and less common when compared with
conventional antibiotics [91]. Nevertheless, the mechanisms of AMPs interaction and the potential resistance
development have to be well investigated [92].
4.2. Polyphenols protective agents and therapeutic potential
Polyphenols are bioactive compounds extracted from plants that have numerous pharmacological properties, such as
antimicrobial, antioxidant, antiviral and anticancer activities [105]. Epidemiological studies suggests that increased
dietary intake of polyphenol-rich fruits and vegetables promote human health [106].
The antibacterial activity of phenolic compounds is thought to be due to a direct interference with bacterial growth or
through the inhibition of virulence factors production, resulting in attenuated pathogenesis [107]. For instance,
Panduratin A is a natural chalcone compound derived from the rhizomes of fingerroot (Boesenbergia rotunda) that was
shown to exhibit antimicrobial activity against clinical isolates of Staphylococcus and Enterococcus (E. faecalis and E.
faecium), with a MIC about 2 g/mL [108]. Epigallocatechin-3-gallate, a derivative of non-flavonoid gallic acid, was
shown to possess a broad spectrum of antibacterial activity against both Grampositive (meticillin-resistant S.aureus
and S. mutans) and Gramnegative bacteria [109, 110]. Polyphenolic compounds have also been shown to act
synergistically with antibiotics. Flavonoids combined with antibiotics have been shown to be effective against drugresistant pathogens by decreasing the antibiotic concentration dosage and eliminating the target pathogen [111].
Moreover, evidences that polyhenolic extracts are also promising anti-biofilm agent due to their ability to inhibit
bacterial growth and virulence factors production have been arising. Cranberry polyphenolic extracts were shown to
prevent initial adhesion and further maturation of E. coli, E. faecalis and specific uropathogenic isolates of P-fimbriated
E.coli [112, 113]. Non-flavonoid gallic acid, in particular, eradicated E. coli, P.aeruginosa, S. aureus and L.

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monocytogenes biofilms in more than 70 % as well as inhibited the bacterial motility [114]. In vitro experiments using
biofilm-producing enterococcal strains showed strong anti-biofilm activity of Panduratin A [108].
4.3. Nanoantibiotics a new trend for treating infections
Nanotechnology is another approach for the development of novel non-traditional antimicrobial agents. This new
paradigm for the effective treatment of infectious diseases designs new antimicrobial drugs, called nanoantibiotics, that
possesses many advantages over other antimicrobial agents including increasing effectiveness against drug resistant
species, lack of adverse effects and overcoming resistance development interfering with multiple biological pathways
[115]. These nanoantibiotics either show antimicrobial activity by themselves or elevate the effectiveness and safety of
conventional antibiotics administration creating high local concentrations [116, 117]. Antimicrobial NPs could cause
mechanical perturbation of the bacterial membrane and even access the cell unrecognized as a treat by the defense
system of the bacteria.
Metal and metal oxides nanoparticles (NPs) such as zinc, silver, gold, aluminum, copper, zinc oxide, titanium oxide,
nitric-oxide have been studied for their antibacterial ability [118-120]. The mechanisms of action by which these NPs
kill bacteria involve: i) production of reactive oxygen species e.g. OH, H2O2, and O2(ROS), ii) disturbance of the
bacterial cell wall membrane, iii) inhibition of intracellular enzymes activity and DNA synthesis, and iv) interruption of
energy transduction [121, 122]. Silver has always been considered as a potent antimicrobial agent and in the form of
nanoparticles its unique properties are magnified [123]. Silver particles were capable to inhibit the bacterial growth and
biofilm formation of pathogenic E. coli and P. aeruginosa PAO1[124]. With a broad-spectrum of antibacterial activity
ZnO NPs were highly effective against medically relevant bacteria species. The mechanism of antibacterial action of
ZnO is thought to be due to ionic zinc or reactive oxygen species (ROS) generation upon contact with bacteria [125].
Apart from bearing antimicrobial activity on its own, NPs are also promising platform for efficient antibiotic delivery
due to their unique physicochemical properties including small and controllable size, functionalizable structure and
increased surface to volume ratio, which allow high drug loading and better interaction with bacterial and host cells
[122]. Moreover, nanocarriers induce targeted delivery, better solubility of water insoluble drugs, prolonged drug
circulation and longer therapeutic effect [126]. Different NP platforms such as liposomes, polymeric NPs, solid lipid
NPs and dendrimers, among others, have been used to facilitate the delivery of antimicrobials to the infection site [127].
For instance, when antibiotics such as penicillin, gentamicin, streptomycin, or ciprofloxacin are encapsulated in
liposomes, an improved antibacterial activity against multi-drug resistant bacteria is observed in comparison to their
free forms. Gentamicin encapsulated in polyethyleneglycol (PEG) liposomes had an enhanced therapeutic effect against
resistant K. pneumonia in vitro. Liposome-encapsulated tobramycin showed antibacterial activity at sub-MIC
concentrations against several bacteria including P. aeruginosa, E. coli and S.aureus [128]. Although the
nanoantibiotics constitute promising strategies to overcome antibiotic resistance and treat the infectious diseases the
clinical use of NPs in recent years appears to be limited by their potential toxicity [129].
4.4. Disruption of resistant genes
Recently, systems called clustered regularly interspaced short palindromic repeats (CRISPR) have been identified in
prokaryotes. CRISPR function as a bacteria prokaryotic immune system, playing an important role in their resistance to
exogenous genetic elements such as phages and other invaders (plasmids) [130, 131]. The resistance development
occurs when the short sequence of an invader genetic material is inserted in CRISPR sequence array. The RNA
transcribed from the CRISPR array is then processed by Cas proteins into RNA-based spacers in a mechanism known
as interference stage of action of CRISPR/Cas sytem [132, 133]. The spacers are often acquired from plasmid and
phage DNA sequences. This acquisition of spacers into the bacterial CRISPR array guides the system to constantly
cleave nucleic acid molecules harboring these sequences. Thus, the system is competent in adaptively and specifically
targeting invaders. CRISPR/Cas adaptive immune system can also target antibiotic resistant genes. In recent years this
system has been studied in order to decrease the acquirement of antibiotic resistant genes after its transfer in certain
species [134]. There are some evidences that rational designing of the system based on the insertion of short DNA
sequences may be used to specifically target desired DNA molecules, such as those encoding resistance determinants
and therefore eliminate the spread of antibiotic resistance [132, 135]. Marraffini et al.have shown that interfering with
CRISPR prevents the routes of plasmids carrying the resistant genes and eliminates the spread of resistance in
pathogenic staphylococci [136].
4.5. Bacteriophage therapy to treat infections
The discovery of bacterial viruses, called bacteriophages or phages, by Frederick and Flix dHrelle in 1915 and 1917,
marked the beginning of a treatment strategy of bacterial infections. The early clinical studies using phage therapy have
only been carried out in Eastern Europe and the results published mainly in Russian, Georgian, and Polish journals
[137]. In these countries, phage therapy has been successfully applied to cure bacteria-mediated infections since the

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beginning of their discovery [138]. To treat various infections, formulations including tablet for administration, liquids
for spraying or injection have been developed [139].
The appearance of antibiotic resistant bacteria and subsequent severe consequences on humans health renewed the
interest in bacteriophages as promising alternative antibacterial agents [140, 141]. Bacteriophages possess a polyhedral
protein coat, called capsid, that encapsulates nucleic acids either DNA or RNA, and a tail that is required for the
attachment and invasion of the host cells [141, 142]. The phages recognise specific receptors on bacterial surface inject
their genetic material into the cells, start to multiply and at the end of its growth cycle kill bacteria via lysis [13, 142].
Following cell lysis, virions (e.g. virus particles) are released that can infect bacteria located on other sites in the body
[141]. This means that the phages can increase their number in the presence of the target, an interesting phenomenon
not observed among antimicrobial agents [141]. Other advantage of phage therapy is the specific phage-bacteria
interaction targeting only harmful bacteria without affecting beneficial and/or human cells. However, major drawback is
that phage therapy needs correct identification of target bacterial strain [139, 140]. Nevertheless, with the development
of better diagnostic approaches, the identification of the pathogens nowadays could be achieved faster and more
accurately making the selection of phage for specific treatment easier [140].
Recent studied have shown that phage therapy was successfully used to treat infections caused by resistant bacteria
such as K.pneumonia and P. aeruginosa [138, 143] and in combination with appropriate antibiotic treatment was
shown to act synergistically on biofilms-associated infections of S. aureus and P. aeruginosa species [144]. The
combination of phage therapy and antibiotics is regarded as a possible strategy to decrease resistance development
[145].The possibility that bacteria develop resistance to phages through mutation of the specific receptor on the
bacterial cell surface or inactivation of phage nucleic acid using CRISPR adaptive immune system should be also
considered [13, 131]. Moreover, phages can induce immune response in the human body and release upon bacterial
lysis toxins causing a toxic shock [142]. Recently, the immunogenicity of phages was shown to be mitigated when
phages with reduced immunogenicity and lacking infectivity are used for targeted drug delivery. Drug-carrying phages
were shown to be not toxic in mice, and the unique drug loading via an aminoglycoside linker greatly reduced the
immunogenicity of the phages [146].

5. Conclusions
For more than 50 years antibiotics have been saving lives from many infectious diseases, being one of the most
successful drugs ever introduced. However, their overuse resulted in increased emergence of multi-drug resistant
bacteria and is now a major threat for human beings. New alternative approaches to prevent and cure bacteria-mediated
infections using novel anti-virulence and therapeutic agents are under investigation. Understanding the mechanisms of
bacterial pathogenesis and drug resistance development provided new insights in the field of drug discovery. Novel
strategies that control the initial stages of bacterial pathogenesis using molecules capable to attenuate virulence
mechanisms have the potential to prevent infections and overcome the antibiotics resistance. On the evidence that the
anti-virulence agents do not kill bacteria but control bacterial virulence, it is believed that the host immune system will
be capable of overcoming any infection without the need of antibiotic treatment. In the case when the human organism
cannot overcome the infection, non-conventional treatments including AMPs, polyphenols, bacteriophages, and
nanoantibiotics may be applied. Combination of these therapeutics using different types/ratios could also result in a
synergistic effect in the treatment of multi-drug resistant bacteria. Non-conventional therapeutics kill bacteria via
different mechanism and do not induce evolutionary stress which decreases the opportunity of resistance development.
Despite of some drawbacks of these approaches such as the high cost, low bioavailability, insufficient pharmacokinetic
data, possibility to affect the beneficial microbiota and opportunity to induce host immune response it is believed that
continued investigations will lead to a deeper understanding of bacterial virulence, antibacterial strategies and
therapeutics that will ultimately result in efficient pathogenesis inhibition and treatment.

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