Challenges and future prospects of antibiotic therapy: from peptides to phages utilization

Abstract

Bacterial infections are raising serious concern across the globe. The effectiveness of conventional antibiotics is decreasing due to global emergence of multi-drug-resistant (MDR) bacterial pathogens. This process seems to be primarily caused by an indiscriminate and inappropriate use of antibiotics in non-infected patients and in the food industry. New classes of antibiotics with different actions against MDR pathogens need to be developed urgently. In this context, this review focuses on several ways and future directions to search for the next generation of safe and effective antibiotics compounds including antimicrobial peptides, phage therapy, phytochemicals, metalloantibiotics, lipopolysaccharide, and efflux pump inhibitors to control the infections caused by MDR pathogens.

Full text

REVIEW ARTICLE
published: 13 May 2014
doi: 10.3389/fphar.2014.00105
Challenges and future prospects of antibiotic therapy: from
peptides to phages utilization
Santi M. Mandal 1*†, Anupam Roy 1†, Ananta K. Ghosh1,Tapas K. Hazra2, Amit Basak1
and Octavio L. Franco3*
1Central Research Facility, Department of Chemistr y and Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, India
2Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Texas Medical Branch at Galveston, Galveston,TX, USA
3Centro de Análises Proteômicas e Bioquímicas, Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, Brasilia, Brazil
Edited by:
Miguel Castanho, University of
Lisbon, Portugal
Reviewed by:
Frederico Nuno C. Aires Da Silva,
University of Lisbon, Portugal
Margarida Bastos, University of Porto,
Portugal
Marta Planas, University of Girona,
Spain
*Correspondence:
Santi M. Mandal, Central Research
Facility, Indian Institute ofTechnology
Kharagpur, Kharagpur - 721302, India
e-mail: mandalsm@gmail.com;
Octavio L. Franco, Centro de Análises
Proteômicas e Bioquímicas,
Pós-Graduação em Ciências
Genômicas e Biotecnologia,
Universidade Católica de Brasília,
SGAN 916N, Modulo C, Avenue W5,
Asa Norte, Sala 219, 70790-160
Brasilia, D. F., Brazil
e-mail: ocfranco@gmail.com
†Santi M. Mandal and Anupam Roy
have contributed equally to this work.
Bacterial infections are raising serious concern across the globe. The effectiveness of
conventional antibiotics is decreasing due to global emergence of multi-drug-resistant
(MDR) bacterial pathogens.This process seems to be primarily caused by an indiscriminate
and inappropriate use of antibiotics in non-infected patients and in the food industry. New
classes of antibiotics with different actions against MDR pathogens need to be developed
urgently. In this context, this review focuses on several ways and future directions to search
for the next generation of safe and effective antibiotics compounds including antimicrobial
peptides, phage therapy, phytochemicals, metalloantibiotics, lipopolysaccharide, and efflux
pump inhibitors to control the infections caused by MDR pathogens.
Keywords: antibiotics, multi-drug-resistant pathogens, infection control
INTRODUCTION
Antibiotics are essential therapeutics, commonly used to con-
trol bacterial infections. They are one of the most significant
contributions to modern science and have proved to be of vital
importance in the dramatic rise in average life expectancy. Nev-
ertheless, antimicrobial resistance is clearly ready to jeopardize
this development now and in the near future. Four years after
the successful introduction of penicillin, the first appearance of
an antibiotic-resistant strain was reported during World War II
(Levy, 2002). Maurois (1959) warned about the deadly fact of
antibiotic resistance, stating that the inappropriate use of peni-
cillin could lead to the selection of resistant “mutant forms” of
Staphylococcus aureus causing serious infections in the host. Since
then, acquired bacterial resistance has caused nosocomial infec-
tions with morbidity and mortality in hospitalized patients, and
to general alarm these infections have been observed spreading
to immune depressed patients (Michael et al., 2013). Each year
in the United States of America, at least two million persons
become infected with antibiotic-resistant bacteria and at least
23,000 people die every year as a direct result of such infec-
tions (Antibiotic resistance threats in the United States, 2013).
Many examples of resistant strains could be cited. Between 1987
and 2004, high levels of penicillin resistance in Streptococcus
pneumoniae were observed, reaching almost 20%. At the same
time, a 50% increase in methicillin-resistant in Staphylococcus
aureus (MRSA) was also observed (Herrmann and Laxminarayan,
2010). Additionally, very frequent and inappropriate use of antibi-
otics, lack of educational awareness and regulatory authority
regarding antibiotic usage, production, and marketing as well the
lack of infection control in hospitals and inadequate water and
sanitation in the community makes the situation worse. Spread of
Gram-negative bacilli resistance is an emerging problem of Asian
countries.
Surveillance study on the resistance on Salmonella enter-
ica serotype Typhi (Salmonella typhi) and Paratyphi (Salmonella
paratyphi) conducted in seven Asian countries (Korea, Taiwan,
Vietnam, Philippines, Singapore, Hong Kong, and Sri Lanka) from
2002 to 2004 emerged high rates of resistance against normally
used antibiotics. In Vietnam, the proportion of multi-drug-
resistant (MDR) strains was 30% higher than in the other six
countries (Chuang et al., 2008).
Currently, application of antibiotics seems to be the main
anti-infective solution for patients in major trauma or in inten-
sive care. Furthermore, similar antibiotic therapies are generally
applied to prevent post-surgery infections or in the treatment
of life-threatening infection in patients with various kinds of
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Mandal et al. Challenges and future prospects of antibiotic therapy
cancer. These treatments, however, have become more difficult
due to pathogen resistance. Antibiotic resistance has led a string of
researchers to work on alternative strategies to“reset the clock” for
resistance levels in particular pathogens. Although some promis-
ing antibiotics have reached phase three trials, and many of
them are under phase two, the continuous development of new
compounds is extremely important, as will be described below.
In this context, this review article sheds some light on future
directions to search for the next generation of antimicrobial
compounds and examines strategies like antimicrobial peptides
(AMPs), phage therapy, phytochemicals, metallo-antibiotics,
lipopolysaccharide (LPS) inhibitors, and efflux pump inhibitors
to control the infections caused by MDR bacterial pathogens
(Table 1 ).
OVERVIEW OF MECHANISMS OF ANTIBIOTIC RESISTANCE
Antibiotic resistances are commonly relatedto bacterial mutations.
Such mutations could occur due to the selection pressure exerted
by the random and inappropriate use of bactericidal or bacte-
riostatic agents. Under continued selection pressure, the selected
bacteria may become resistant to antibiotics and spread to other
bacteria by transferring the resistance gene (Levy and Marshall,
2004). These unique resistance capabilities are generally subdi-
vided into four major issues. First is enzymatic drug inactivation,
as observed in the case of beta-lactamases (Davies, 1994). Sec-
ond, resistance could be related to alteration of specific target
sites (Spratt, 1994), as observed in the case of penicillin-binding
proteins (PBPs) in MRSA. Third, bacteria may acquire several
genes for a metabolic pathway. This alters bacterial cell walls and
thus makes antimicrobial agents incapable of binding to a bac-
terial target. Finally, the fourth issue is the reduction in drugs’
cellular uptake (Smith, 2004). In this case, para-amino benzoic
acid (PABA) is an important precursor for bacterial folic acid and
nucleic acid synthesis. Some sulphonamide-resistant bacteria do
not require PABA, instead using preformed folic acid as observed
in mammalian cells. As a result, a decrease in drug permeability
or an increase in active efflux of the drug across the cell surface
causes a decrease in drug accumulation in cellular compart-
ments (Nakaido, 1994). Bacteria may also acquire efflux pumps
that extrude the antibacterial agent from the cell before it can
reach its target site and exert its deleterious effect. This resistance
mechanism plays a vital role in reducing the clinical efficacy of
antibiotics. Moreover, the overproduction of efflux pumps is gen-
erally accompanied by a resistance improvement of two or more
Table 1 |Major types of antimicrobial compounds with their mechanisms of action.
Future therapy Mechanism Contemporary strategies to improve activity
Antimicrobial peptides Attach and insert into membrane bilayers to form
pores by “barrel-stave,” “carpet,” or
“toroidal-pore” mechanisms. DNA and
macromolecule synthesis inhibitors.
Optimization of peptide length and content of their sequences.
Conversion into peptidomimetics.
Generation of targeted antimicrobial peptides (Peptide antibiotic
conjugation).
Generation of antimicrobial peptides as prodrug candidates.
Antimicrobial peptides loaded into nanoparticle or micelles for sustained
release.
Phage therapy Bacteriophages are viruses that act as pathogens
against bacteria and completely lyse the bacteria.
Genetically engineered phages.
Genetically engineered phase as antibiotic delivery.
Engineered bacteriophage for phage targeted drug delivery.
Scale up of endolysin production.
Phytochemicals Multiple actions. Search for novel compounds and cost-effective methods of extraction
and purification of phytochemical.
Transgenic production in plant and microbial system to enhance number
of novel compounds.
Search for endophytic fungal metabolomics for the production of novel
compound of host.
Synthesis and modification of natural structure and analogs.
Metalloantibiotic Increased spectrum of conventional antibiotic
action.
Synthetic or semi-synthetic antimicrobial compound development
attaching metal to its structure.
In situ reducing and capping of metal nanoparticle with enhanced
antimicrobial activity.
Efflux pump inhibitor Molecules to inhibit the active protein pump in the
bacterial cell.
Chemical synthesis of effective efflux pumps inhibitor.
Screening of efflux pump inhibitors from natural origin and modifying
this compound synthetically.
Rationally designed transmembrane peptide mimics.
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Mandal et al. Challenges and future prospects of antibiotic therapy
structurally unrelated antibiotics and significantly contributes to
the emergence of MDR pathogens. There are five major families
of efflux transporters, MFS (major facilitator superfamily), MATE
(multi-drug and toxic compound extrusion), RND (resistance
nodulation cell division) superfamily, SMR (small multi-drug-
resistance), and ABC (ATP-binding cassette) transporters (Eda
et al., 2011;Spellberg et al., 2013). All these mechanisms of resis-
tance have been targeted by the scientific community finding the
search for novel antibiotics with multiple functions, as described
above.
ANTIMICROBIAL PEPTIDES
Over the last few decades, several AMPs have been identified
(Figure 1) and rigorously investigated as alternatives to antibiotics.
They have been widely tested on the antibiotic-resistant bacte-
rial infections (Hancock and Lehrer, 1998;Ganz, 2003). AMPs
are the first line of defense in various organisms including plants
(Mandal et al., 2013;Roy et al., 2013), humans, insects and other
invertebrates, amphibians, birds, fish, and mammals (Martin et al.,
1995;Wang and Wang, 2004). Most AMPs are cationic in nature
and generally possess a specific amphipathic conformation. These
key players in defense systems have attracted extensive research
attention worldwide. AMPs are generally short (<100 amino acid
residues), positively charged and amphiphilic in nature. This
allows them to bind and insert themselves into membrane bilay-
ers to form pores by “barrel-stave,” “carpet,” or “toroidal-pore”
mechanisms (Matsuzaki et al., 1996;Oren and Shai, 1998;Yang
et al., 2001). Several data previously provided suggest that translo-
cate peptides may alter cytoplasmic membrane septum formation
(Salomon and Farias, 1992;Shi et al., 1996), inhibiting cell wall
synthesis (Brotz et al., 1998), bind to nucleic acids (Yonezawa et al.,
1992;Brotz et al., 1998;Park et al., 1998), inhibit nucleic acid syn-
thesis (Yonezawa et al., 1992;Boman et al., 1993;Silvestro et al.,
1997;Subbalakshmi and Sitaram, 1998), impede protein syn-
thesis (Yonezawa et al., 1992;Boman et al., 1993;Silvestro et al.,
1997;Subbalakshmi and Sitaram, 1998), or inhibit enzymatic
activity (Andreu and Rivas, 1998;Brogden, 2005). These fea-
tures make some AMPs most acceptable as a novel antibiotic class
and they can complement conventional antibiotic therapy (Mar-
shall and Arenas, 2003;Hancock and Sahl, 2006;Jenssen et al.,
2006).
The activity of AMPs may vary by switching amino acid com-
position, amphipathicity, cationic charges and size. In this context,
AMPs can be improved through the amalgamation of hydropho-
bic or charged amino acids, which has been revealed to modify
the selectivity for fungal and bacterial membranes (Davies, 1994;
Levy and Marshall, 2004). In this approach, different strategies in
designing novel peptides have been pursued, as described in sev-
eral studies (Tamamura et al., 1994;Pini et al., 2005). There are
descriptions of potential AMPs from natural sources helping to
design “tailor-made”AMPs owing to their easy availability through
solid-phase peptide synthesis (SPPS; Martin et al., 1995;Wang and
Wang, 2004). Several synthetic analogs of a number of naturally
occurring AMPs have been made, in an effort to identify signifi-
cant structural features that contribute to their enhanced activities
in vitro against Gram-positive and Gram-negative bacteria, fungi
as well as some enveloped viruses (Jenssen et al., 2006).
Cationic peptides contain cationic residues including arginines
and lysines. These residues are involved in the attraction of the neg-
atively charged bacterial cell surface. Structure–function studies of
FIGURE 1 |Some representative structure of antimicrobial peptides.
Structure represents beta defensin peptide from avian (AvBD2); an
amphipathic alpha-helical peptide from skin mucous of Pleuronectes
americanus (Pleurocidin); an antimicrobial hemolytic peptide from skin
of Bombina variegata (bombinin H2); skin secretion of Rana
temporaria (temporin); another antimicrobial peptide frog Xenopus
laevis (magainin) and peptide with helix-turn-helix motif from Rana
cascadae (ranatuerin).
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Mandal et al. Challenges and future prospects of antibiotic therapy
host defense α-helical peptides have been carried out, with the aim
of designing diverse engineered cationic antimicrobial peptides
(eCAPs; Tencza et al., 1999;Jing et al., 2003;Phadke et al., 2003;
Deslouches et al., 2005a,b;Chan et al., 2006;Andrushchenko et al.,
2007,2008;Nguyen et al., 2010). Their focus was on structure–
function relationships of host-derived synthetic AMPs. A series
of eCAPs, called a lytic base unit (LBU) series, formed of only
Arg and Val, has been engineered to fold onto a flawless amphi-
pathic helical motifs in the occurrence of lipid membranes or
membrane mimitope solvents. Furthermore, Rozek et al. (2000)
explored the structure of the bovine AMP indolicidin linked
to dodecylphosphocholine and sodium dodecyl sulfate micelles.
Haney et al. (2012a) showed how specific amino acid side chains
influence the antimicrobial activity and structure of bovine lacto-
ferrampin. All the data have revealed the membrane perturbation
properties of Arg and Trp. Further research on the optimiza-
tion of the amphipathic helix has also been carried out. An
unusual series of eCAPs (6–18 residues long) has recently been
reported (Deslouches et al., 2013)tohavebroadandpotentin
vitro activity against MDR pathogens. These eCAPs consist exclu-
sively of Arg on the hydrophilic face and Trp on the hydrophobic
face.
Peptide modification, formulation, and delivery technologies
have also been explored to overcome the shortcomings of pharma-
cokinetics, bioavailability, and toxicity (Cole et al., 2003;Devocelle,
2012;Haney et al., 2012b). In this regard, several strategies have
been used, such as the optimization of peptide length and content,
offering an increase in selective antibacterial activity. Optimization
is generally done by minimizing the peptide length or switching
the peptide surface properties and systematically substituting each
residue. For these cases, computer-assisted AMP design is very
useful for an accurate estimation or prediction of the desired bio-
logical activity from the primary peptide structure (Fjell et al.,
2012). Moreover, conversion into peptidomimetics techniques is
able to improve the pharmacokinetic properties of AMPs, since
peptidomimetic structures are resistant to proteolysis.
Other strategies have also been applied to discovering novel
antimicrobials. Recently classical antibiotics were conjugated for
host defense peptide sequence, thereby increasing selectivity and
effectiveness against bacteria (Pokrovskaya and Baasov, 2010;
Arnusch et al., 2012). Additionally, the generation of AMP pro-
drug candidates has also been focused (Hancock, 2001;Stella,
2004;Gordon et al., 2005;Desgranges et al., 2012). Last but not
least, AMPs have been nanoencapsulated by strategies including
self-assembly, liposomes, polymeric structures, hydrogels, den-
dritic polymers, nanospheres, nanocapsules, carbon nanotubes,
and DNA cages. These strategies offer enhanced antimicrobial
activity, a reduction in collateral effects and also a clear protec-
tion from metabolic degradation (Urbán et al., 2012;Roy et al.,
2013).
The problem regarding the costs of synthesis and screening, sys-
temic, high manufacturing costs, and local toxicity, susceptibility
to proteolysis, sensitization, and allergic responses after repeated
uses of cationic membranolytic AMPs is the key barrier in success-
ful clinical application (Gordon et al., 2005). But AMPs, with their
unique multidirectional mode of action (Figure 2) and broad-
spectrum activities, rapid onset of killing, potentially low levels
FIGURE 2 |Schematic diagram of antibacterial peptides with
mechanism of action.
of induced resistance seem to represent one of the most promis-
ing future strategies to overcome increasing antibiotic-resistant
pathogens. These desirable and remarkable compounds are now
being studied extensively, and attempts to create them syntheti-
cally are being made by both academics and industries. Preclinical
and clinical studies of AMPs are being focused more in order to
overcome the problems. Efforts to produceAMPs on an industrial
large scale are now also in progress (Giuliani et al., 2007). A good
number of synthetic AMPs and at least 15 peptides or mimetics
are undergoing advanced clinical trials or have completed trials
as antimicrobial or immunomodulatory agents (Fjell et al., 2012).
Antimicrobial cationic lipopeptides like polymyxin, gramicidin S,
bracitracin, and cationic lantibiotic nisin have offered clinical effi-
cacy and are used widely (Laverty et al., 2010). In the future, the
inappropriate use of AMPs may lead to more resistant forms of
microorganisms that produce deadly infections. In this context,
innovative computer-assisted design strategies can strengthen the
ongoing development of next-generation therapeutic peptides and
peptide mimetics.
PHAGE THERAPY
The use of bacteriophages in controlling bacterial infections is
also a promising therapeutic option. Bacteriophages are bacterial
viruses that act as pathogens against bacteria. They show the abil-
ity of specifically attacking and killing only host bacterial cells at
the end of infection process (Sulakvelidze et al., 2001). After the
first isolation of bacteriophage in 1917, an oral phage prepara-
tion to treat bacterial dysentery was used (d’Herelle, 1917). Phages
are then extensively used and developed mainly in former Soviet
Union countries. Several commercial laboratories and compa-
nies in the USA, France and Germany developed phase products
(Hausler, 2006). The golden age in use of phage was in the 1930s,
but with antibiotics discovery, the progress of phage research and
use was reduced.
Bacteriophages have the ability to interfere between two cycles
lysogenic or lytic (Temperateness). In the lytic phage, the viral
DNA exists as a separate molecule within the bacterial cell, and
replicates separately from the host bacterial DNA. Each phage fol-
lows a unique pathway to control bacteria. Some of them show
a lytic infection cycle upon infecting their bacterial host. In this
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Mandal et al. Challenges and future prospects of antibiotic therapy
case, they grow in high numbers in bacterial cells, leading to cel-
lular lyses. At the end of the cycle, a release of newly formed
phage particles is observed (Figure 3). Using the lysogenic path-
way, the phage genome integrates as part of the host genome. It
stays in a dormant state as a prophase for extended periods of
time. Adverse environmental conditions for the host bacterium
may activate the prophase, turning on the lytic cycle. At the end,
the newly formed phage particles are ready to lyse the host cell
(Skurnika and Strauch, 2006).
It has been noted that the bacterial mechanism of resistance
to phage seems to be lower when compared to antibiotics, which
are prone to bacterial resistance. The exponentially higher growth
kinetics generally overcomes bacterial growth (López et al., 1997).
Moreover, phages seem to show an extra advantage over com-
mon antibiotics, which are generally reduced by metabolism
and excretion, with several repeated administrations being neces-
sary (Inal, 2003). In the case of phages, increasing titers during
different periods removes the need for repeated doses. Addi-
tionally, high specificity for a particular bacterium does not
disturb the host-organism, so that phages do not affect com-
mensal intestine micro-flora, which is generally a side-effect in
the case of antibiotic ingestion (Inal, 2003). Although phages
may carry a virulence factor or toxic genes (Mesyanzhinov
et al., 2002;Brüssow et al., 2004;McGrath et al., 2004), a full
knowledge of phage genome sequences can address the possi-
ble complications during phage therapy (Skurnika and Strauch,
2006). Specific and non-toxic phages with high therapeutic
index can be applied to reduce the chances of opportunistic
pathogens.
Generally whole virulent phages are used as antibacterials.
Genetically modified phages are now also being studied, and have
been reported as useful in delivering antimicrobial agents to bac-
teria. Westwater et al. (2003) documented the use of a non-lytic
phage to precisely target and deliver DNA encoding bactericidal
proteins to target. Engineered bacteriophages can also enhance the
killing of antibiotic-resistant bacteria, persistent cells and biofilm
cells. They reduce the number of antibiotic-resistant bacteria
that ascend from an antibiotic-treated population, and act as a
robust adjuvant for other bactericidal antibiotics (Lu and Collins,
2009;Kaur et al., 2012). Moreover, a study on endolysins, which
are hydrolytic enzymes secreted by bacteriophage, has revealed
potential antimicrobial activity (Nelson et al., 2012).
FIGURE 3 |Mechanism of phage therapy. Image represents the schematic diagram of developmental cycle of lytic bacteriophage.
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Mandal et al. Challenges and future prospects of antibiotic therapy
Treatment using the phage is not approved yet in countries
other than Russia and Georgia. Phages are currently being used
therapeutically only in the Russia and Georgia to treat extreme bac-
terial infections where conventional to antibiotics do not respond
(Karl, 2004;Parfitt, 2005). The use of phage technology for bacte-
rial control could also be applied in veterinary products focusing
on animal health. Bacterial resistance to antibiotics is a serious
concern for animal production, which could also be addressed by
phage therapy in the near future. Based on several features of bac-
teriophages, it has even been proposed that they be used in food
to prevent bacterial foodborne infections in food products and
on food contact surfaces (Borysowski et al., 2011). The example
includes LMP-102, which was regarded safe for use as food addi-
tive in meat and poultry products as an antibacterial agent against
Listeria monocytogenes (Daniells, 2006).
Archaebacteria-specific viruses or archeophages are the most
recent discoveries to show successful results in controlling bacte-
rial spread. Some bacteriophages have been used as anti-infective
agents, such as bacteriophage lysins and bacteriophage tail-like
bacteriocins (Schuch et al., 2002;Huff et al., 2004). For exam-
ple, a G phage lysine, PlyG, can effectively control Bacillus
anthraces in mice models (Schuch et al., 2002). Phages are recently
being commercialized in several areas of biological applica-
tions. Companies throughout the world like Intralytix (Baltimore,
MD, USA -product based on food safety), Phage Biotech Ltd
have (Rehovot, Israel -Anti-Pseudomonas infectives), BioCon-
trol (Southampton, UK -Pseudomonas infections of the ear),
EBI Food Safety (Wageningen, Netherlands -product based on
Food Safety. cocktail of phage against Listeria), JSC Biochim-
pharm (Tbilisi, Republic of Georgia -mixture of phage lysates),
Gangagen (India -phage against Staphylococcus aureus), Omni-
lytics (Salt Lake City, UT, USA -Agricultural use) etc., (Housby
and Mann, 2009). Despite the clear benefits of phage therapy,
some problems like development of antibodies after repeated
treatment with phages, rapid uptake and inactivation of phages
by spleen, contamination of therapeutic phage preparations
with endotoxins from bacterial debris, limited host range, reg-
ulation, bacterial resistance to phages, engineering, bacterial
lysis side effects and delivery should be scrutinized more thor-
oughly to make these potent therapeutics in the near future
(Inal, 2003;Lu and Koeris, 2011). In summary the phage
theraphy contribution in terms of continued investments in
research, development, and clinical trials from the public and
private sectors are needed to overcome regulatory and technical
hurdles.
PHYTOCHEMICALS
Phytochemicals are the tremendous gift of nature. They are the
secondary metabolites basically found in plants for specific func-
tional purposes. In most cases, these substances act as a plant
defense mechanism against microorganisms, insects and herbi-
vores. At the dawn of civilization, phytochemicals, in the form
of plants, were the only weapon in a struggle between man and
microbes. In recent times they have stimulated the same inter-
est both as fundamental sources of new chemical diversity and
integral components of today’s pharmaceutical compendium. But
with ever more incidents of MDR pathogens, the search for new
chemicals from plants offering a wide range of activity is the recent
focus of researchers.
At the moment thousands of compounds derived from plants
have been listed as antimicrobials. Phytochemicals within the
group of phenolics, terpenoids, essential oils, alkaloids, proteins
and peptides, possess potent antimicrobial potential with varying
mode of action (Cowan, 1999;Figure 4). Modern research is not
only confined to searching for antimicrobial compounds but has
also found several enzymatic inhibitors. Berberine is a hydropho-
bic cation found in common barberry (Berberis species) plants and
the medicinal plant goldenseal (Hydrastis canadensis). Although
the immutable targets and positive charge (facilitating active accu-
mulation in bacterial cells) makes berberine an efficient antibac-
terial, the fact that it is readily extruded by pathogen-encoded
MDR pumps rendered it ineffective. This limitation was overcome
by finding and applying another barberry-isolated compound,
5-methoxyhydnocarpin, which acts by blocking so-called major
facilitator MDRs of Gram-positive bacteria. In combination, the
two act as potent antimicrobials (Hsieh et al., 1998;Stermitz et al.,
2000;Lewis, 2001;Boucher et al., 2009). Furthermore, a newly
identified compound, 4-[N-(1,8-naphthalimide)]-n-butyric acid,
showed inhibition activity of the Vibrio cholerae transcriptional
regulator ToxT. Cholera toxin and the toxin-co-regulated pilus are
regulated by ToxT. This compound was tested in an animal model,
with infant mice, and was also reported to protect intestinal col-
onization by V. c h o l e r a (Hung et al., 2005;Lewis and Ausubel,
2006).
Strategies like study the combo effects of antibiotic and phyto-
chemical are recently been studied. Recently interaction studies are
drawn to study the associated effects of antibiotic and phytochemi-
cals (Sakharkar et al., 2009;Jayaraman et al., 2013). A current study
revealed that phytochemical-antibiotic conjugates have multitar-
get inhibitors of Pseudomononas aeruginosa GyrB/ParE and DHFR
was extremely effective (Jayaraman et al., 2010).
In the search for novel compounds and cost-effective methods
of extraction, purification of phytochemicals is a major concern.
FIGURE 4 |Representative chemical structures of some antimicrobial
phytochemicals. Phytochemicals like phenolics, saponins, essential oils,
terpenoids, alkaloids, and flavonids are the major classes showed
antibacterial activity.
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Mandal et al. Challenges and future prospects of antibiotic therapy
But the mode of action derived from the structure may lead new
potent antibiotic and precursor molecule. For this, genetically
modified plant and microbial systems are also now being tried
to explore an enhanced number of novel compounds. Addition-
ally, novel antimicrobial structures have been created synthetically.
Synthetic analogs and modification of the structure may lead
to the discovery of novel structures with a broader spectrum of
activity.
METALLOANTIBIOTICS
Among different strategies to discover novel antibiotics,the incor-
poration of metal ions in antimicrobial compounds seems to be
promising. Metal ions perform an essential role in the functions
of synthetic and natural metalloantibiotics, being involved in very
specific interactions of such antibiotics with membranes, pro-
teins, nucleic acids, and several other biomolecules. This makes
metal ions effective in antibiotic structure or as an operative link-
age offering unique and specific bioactivities. Although several
antibiotics do not possess any metal ions in their structure, oth-
ers require metal ions for their proper functional activities. In
some cases metal ions are bound tightly to the antibiotic struc-
ture and regulate its action (Ming, 2003). Bacitracin, bleomycin,
streptonigrin, and albomycin are examples of such antibiotics.
In some cases metal ions are attached to the antibiotic molecule
without causing a major change in antibiotic structure. Tetra-
cyclines, aureolic acids, and quinolones are frequently used
in these strategies (Chohan et al., 2005). Synthetic or semi-
synthetic antimicrobial compounds also possess metal ions in their
structure.
In general, antimicrobials which contain metal compounds in
their structure in a natural form (nature-occurring) or which
have metal compounds incorporated synthetically are termed
metalloantibiotics. Transition metals are generally preferred in
metalloantibiotics and are present in very low concentration in
vivo. The ligand environment of transition metal ions can gener-
ally change considerably upon administration of a therapeutically
effective dose of an antibacterial drug (Sekhon, 2010). Some
strategies are followed to synthesize metal nanoparticles using
antibiotics as in situ reducing and capping agent. Here antibiotics
actasanin situ reducing and capping agent, thus offering potent
antimicrobial activities as well their application in antimicrobial
coatings (Jagannathan et al., 2007;Rai et al., 2010). Although
interactions with essential metal ions make more controllable
conditions for bacterial infections, the unwanted side effects like
toxicity and hemolytic activity sometimes increase simultaneously
(Heidenau et al., 2005). So the potential for oral administration or
internal use may be hampered.
EFFLUX PUMP INHIBITORS
One of the most important strategies to combat bacterial resis-
tance to antibiotics seems to be the efflux pump. Bacteria may
pump the drug out of the cell after its entrance, and among the
transporters involved in this pumping process are plasma mem-
brane translocases. Being non-specific in nature such transporters
are known as multi-drug-resistance pumps, being main determi-
nants of the antibiotic concentration inside a bacterial cell. Many
of them also act as drug/proton antiporters (protons enter the cell
as the drug leaves). This is a very common resistance mechanism
found in Escherichia coli, P. aeruginosa, Mycobacterium smegmatis,
and Staphylococcus aureus (Takiff et al., 1996). Depending upon
their varying structure and function, efflux pumps are subdivided
into five classes: SMR pumps of the drug/metabolite transporters
(DMTs) superfamily, ABC, RND, MFS, and MATE transporters
of the multi-drug/oligosaccharidyl-lipid/polysaccharide flippases
(MOP) superfamily (Piddock, 2006;Bhardwaj and Mohanty,
2012).
In order to restore the activity of antibiotics, an obvious
strategy consists of developing a compound that inhibits the
effects of efflux pump. Such molecules are named efflux pump
inhibitors (Figure 5). In order to get effective results, sev-
eral strategies have been taken including rational design of
efflux pump inhibitors, their chemical synthesis and potential-
ity as combination with commercial antibiotics (Marquez, 2005;
Delmar et al., 2014).
Although, there are few efflux pump inhibitors are available
in market and research in progress raises the hope that they
may be found in the near future. Screening of efflux pump
inhibitors from natural origins or under synthetic production
has attracted remarkable attention (Hudson et al., 2003). Struc-
tural modifications of such compounds may lead to an increase
in the spectrum of activities A wide number of effective chemical
compounds belonging to various chemical families have already
showed efflux pump inhibition (Brincat et al., 2011;Holler et al.,
2012a,b). Moreover, some plant-derived NorA EPIs and their
chemical modifications are carried out effectively (Thota et al.,
2008;Sabatini et al., 2011;Kalia et al., 2012). Recent rationally
designed transmembrane peptide mimics may work in efflux
pump inhibition. Maurya etal. (2013) have reported rationally
designed transmembrane peptide mimics of the multi-drug trans-
porter protein Cdr1. This acts as an antagonist to selectively
block drug efflux and chemosensitize azole-resistant Candida albi-
cans clinical isolates (Maurya et al., 2013). However, the major
advantages of efflux pump inhibitors are that the possibilities
on slower development of resistance by the target bacteria. Sev-
eral disadvantages are also documented including their chemical
synthesis due to bulky structure, solubility or permeability prob-
lems, required at higher concentration and chance of decreased the
activity at one or both target sites for steric or electronic config-
urations, unless these molecules are carefully designed (Bremner,
2007).
LPS INHIBITORS
The LPS layer or LPS in Gram-negative bacteria acts as a protec-
tive barrier. It prevents or slows down the entry of antibiotics
and another toxic compound that could kill or injure bacte-
ria (Miki and Hardt, 2013). LPS inhibitors are compounds that
generally work by inhibiting 3 deoxy-D-manno-octulosonic acid
8-phosphate synthase (KDO 8-p synthase), an important enzyme
in the LPS pathway. There have been several reports describ-
ing inhibitors, including PD404182 and polymyxin B (PMB),
which are compounds that have been applied with antibiotic
therapy, allowing the antibiotic to pass through the bacte-
rial cell wall (Palmer and Rifkind, 1974;Lindemann, 1988;
Birck et al., 2000).
www.frontiersin.org May 2014 |Volume 5 |Article 105 |7

Mandal et al. Challenges and future prospects of antibiotic therapy
FIGURE 5 |Few representative chemical structures of efflux pump inhibitors.
Among the strategies for exploiting LPS inhibitors more fully,
studies on bacterial enzymes that are essential for growth have
recently provoked much interest and will certainly attract more
attention in the near future in controlling bacterial resistance.
Some recently discovered molecules like pedopeptins are promis-
ing LPS inhibitor candidates, preventing bacterial growth (Cheung
et al., 2013;Kozuma et al., 2013). Some bacterial strains can
control other LPS functions, such as the ability of various Lac-
tobacillus strains to prevent Salmonella LPS-induced damage to
the epithelial barrier function (Yeung et al., 2013). This drug is
yet to be clinically validated with sufficient data. However, it is
likely that this and other small molecules of animal or plant ori-
gin or synthetically engineered may be found and developed soon
for use in inhibiting the translation machinery with an in vitro
system.
MYXOPYRONIN AND ARCHAEOCINS
Regulated gene expression is important in changing environ-
ments, stresses, and gene developmental programs. The activity
of transcriptional factors enables RNA polymerases (RNAPs)
to catalyze the transcription of DNA into RNA (Birck et al.,
2000;Wiesler et al., 2012). Myxopyronin is an α-pyrone antibi-
otic produced by Myxococcus fulvus offering broad-spectrum
antimicrobial activities for most Gram-positive species and some
Gram-negative bacteria [E. coli D21f2tolC (rfa tolC), Moraxella
catarrhalis ATCC25238]. It acts as inhibiting or binding bacterial
RNAP by changing the structure of the RNAP switch region of the
β-subunit of the enzyme. That renders the reading and transmit-
ting DNA code inactive, resulting in bacterial control (Irschik et al.,
1983;Campbell et al., 2001;Mukhopadhyay et al., 2008;Belogurov
et al., 2009;Ho et al., 2009;Srivastava et al., 2011). Rifampin, an
RNAP inhibitor in clinical utilization is capable of binds to the
β-subunit of RNAP within the DNA/RNA channel and blocks
the RNA elongation when the transcript converts two to three
nucleotides in length (Campbell et al., 2001). It is a broad spectrum
antimicrobial and is particularly active against M. tuberculosis.But,
the major problem in treatment failure and fatal clinical outcome
is due to the resistance to rifampin. The development of resistance
to rifampin is due to mutations in 81 base pair (27 codons) of the
β-subunit of RNAP (rpoB).
Archaea also contain potent antimicrobial compounds known
as archaeocins, which are archaeal proteinaceous antimicrobials.
Eight archaeocins from this family, among them halocins and
sulfolobicins, have been partially or fully characterized showing
antibacterial activity (O’Connor and Shand, 2002). The unique
mode of action offered by these groups of antimicrobials draws
remarkable attention as future antibiotic research. Besides, there
are lot of unknown bacterial and archaeal sources yet to be
explored. Thus discovery of new Myxopyronin and archaeocins
hinges on recovery and cultivation of bacterial and archaeal organ-
isms from the environment. Synthetic modification may lead this
compound to become a future potent drug.
CONCLUSION AND PROSPECTS
Growing concern about antibiotic resistance is propelling the
urgent modification of existing antibiotics and parallel devel-
opment of newer antibiotics. There are generally three inherent
pipelines available to fight against antibiotic resistance; i.e.,
antimicrobial chemical weaponry from natural products, syn-
thetic chemical compounds turned into antibiotics, and phages.
Antimicrobial compounds from natural product (AMPs, phyto-
chemicals, efflux pump inhibitors, LPS inhibitors myxopyronin,
Frontiers in Pharmacology |Experimental Pharmacology and Drug Discovery May 2014 |Volume 5 |Article 105 |8

Mandal et al. Challenges and future prospects of antibiotic therapy
and Archaeocins) have drawbacks regarding their isolation and
purification. The cost of production can be reduced by isolating
potent compounds from natural origins and then synthesizing
them, or by rationally modifying derived compounds. Phyto-
chemicals isolated from natural sources and then chemically
synthesized via modifications are likely to provide the most
effective antimicrobial drugs in the near future.
New compounds that target bacterial virulence can be devel-
oped to control the enormous threat posed by multi-drug-
resistance. Antibiotic structural modifications can be carried out
by synthesizing potent structures from already existing antibiotics.
Here the metalloantibiotics can play a great role.
In parallel, further research into toxicity against animal or
human cells, mechanisms of action, in vivo effects, and nega-
tive and positive interactions with common antibiotics should be
incorporated. The main challenge is to find the most effective
techniques for isolating and purifying newer and safer naturally
occurring antimicrobials against MDR pathogens. A better under-
standing of the structure, function and action mechanism of
existing and newly identified AMPs will lead to their being fine-
tuned by proper design to work against MDR pathogens. Phages
may also play a major role in treating bacterial infections in
humans. Combined treatment of phages with antibiotics is likely
to be a future choice. The problem regarding expansion of phages
can only be solved if large-scale clinical trials are carried out by
major pharmaceutical companies.
In summary, it is imperative to develop new classes of antibi-
otic or antimicrobial agents with different modes of action against
MDR pathogens. Combinational drug use is extensively used to
treat bacterial infection, but even this combinational dose pattern
may lead to resistance among pathogens. To overcome the chal-
lenges of antibiotic resistance, antimicrobial compounds with a
new mechanistic approach should be urgently sought.
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Conflict of Interest Statement: The authors declare that the research was conducted
in the absence of any commercial or financial relationships that could be construed
as a potential conflict of interest.
Received: 10 March 2014; accepted: 22 April 2014; published online: 13 May 2014.
Citation: Mandal SM, Roy A, Ghosh AK, Hazra TK, Basak A and Franco OL
(2014) Challenges and future prospects of antibiotic therapy: from peptides to phages
utilization. Front. Pharmacol. 5:105. doi: 10.3389/fphar.2014.00105
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