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Mechanisms of Antimicrobial Resistance (AMR) and Alternative Approaches to

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Current Drug Discovery Technologies, 2019, 16, 00-00 1

REVIEW ARTICLE

Mechanisms of Antimicrobial Resistance (AMR) and Alternative

Approaches to Overcome AMR

Chew-Li Mooa, Shun-Kai Yanga, Khatijah Yusoffb, Mokrish Ajatc, Warren Thomasd, Aisha Abushelaibie,
Swee-Hua-Erin Limd, e and Kok-Song Laia,*

a
Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Ma-
laysia, 43400 Serdang, Selangor, Malaysia, bDepartment of Microbiology, Faculty of Biotechnology and Biomolecular
Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia, cDepartment of Veterinary Pre Clinical Sci-
ences, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia, dPerdana
University-Royal College of Surgeons in Ireland School of Medicine, Perdana University, MAEPS Building, Serdang,
Selangor, Malaysia, eHealth Sciences Division, Abu Dhabi Women’s College, Higher Colleges of Technology, 41012
Abu Dhabi, United Arab Emirates.

  Abstract: Antimicrobials are useful compounds intended to eradicate or stop the growth of harm-
A R T I C L E H I S T O R Y
Received: August 09, 2018
Revised: January 30, 2019
Accepted: January 30, 2019
DOI:
10.2174/1570163816666190304122219
ful microorganisms. The sustained increase in the rates of antimicrobial resistance (AMR) world-
wide is worrying and poses a major public health threat. The development of new antimicrobial
agents is one of the critical approaches to overcome AMR. However, in the race towards develop-
ing alternative approaches to combat AMR, it appears that the scientific community is falling be-
hind when pitched against the evolutionary capacity of multi-drug resistant (MDR) bacteria. Alt-
hough the “pioneering strategy” of discovering completely new drugs is a rational approach, the time
and effort taken are considerable, the process of drug development could instead be expedited if ef-
forts were concentrated on enhancing the efficacy of existing antimicrobials through: combination
therapies; bacteriophage therapy; antimicrobial adjuvants therapy or the application of
nanotechnology. This review will briefly detail the causes and mechanisms of AMR as background,
and then provide insights into a novel, future emerging or evolving strategies that are currently being
evaluated and which may be developed in the future to tackle the progression of AMR.

Keywords: Antimicrobial resistance (AMR), current approach, combination therapy, nanotechnology.

1. INTRODUCTION resistance (AMR) rapidly accelerated and spread among dif-

Antimicrobials represent a broad spectrum of compounds
that may act against a wide repertoire of disease-causing
microorganisms such as bacteria, viruses, parasites, fungi
and protozoa. These compounds have been used since the
early 20th century to treat infected patients and this has
helped significantly in lowering the morbidity and mortality
rates of most infectious diseases. In 1928, Alexander Flem-
ing discovered penicillin and in the 1940s, it came into clini-
cal use just in time for World War II [1]. After only four
years of use, however, the first penicillin-resistant strains of
bacteria emerged; this phenomenon of evolved antimicrobial
*Address correspondence to this author at the Department of Cell and Mo-
lecular Biology, Faculty of Biotechnology and Biomolecular Sciences,
Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia,
Tel: +603-8946 8021; E-mail: laikoksong@upm.edu.my
ferent pathogenic species as a result of persistent exposure
and the non-targetted use of antimicrobials in the clinical as
well as in agricultural settings. The advancement of AMR
has rendered many current antimicrobials ineffective and it is
believed that the non-judicious use and overdosing of antibi-
otics are the primary factors causing the increase in the de-
tection of drug-resistant bacteria [2].
Over the last few decades, research on AMR has been fo-
cused on understanding the resistance mechanisms and also
on the development of new alternative drugs. Nevertheless,
pharmaceutical companies are investing less than ever in
antibiotic development because of the projected amount of
time and the scale of resources required to demonstrate safe-
ty and to gain approval for the use of novel drugs including
antibiotics [3]. It is a daunting task to identify new potential
drugs, determine their efficacy in appropriate models and
then to demonstrate their safety and efficacy in humans. The
scale of investment required and large lead-in time mean that
only a limited number of novel antimicrobials are at an ad-

1570-1638/19 $58.00+.00 © 2019 Bentham Science Publishers

2 Current Drug Discovery Technologies, 2019, Vol. 16, No. 0
vanced stage of development at the current time [2]. Howev-
er, the development of AMR is a very pressing clinical prob-
lem that is expected to significantly worsen in the coming
years. In order to mitigate this effectively, clinical diagnostic
processes and treatment practices that are both rapid and yet
highly specific are required This should minimize unneces-
sary prescriptions and lead to a general improvement in the
quality of prescribing practice. Greater control needs to be
exercised in the use of existing effective antibiotics and pa-
tients need to be educated in the need to adhere to specific
prescription regimes.
Since the development of novel antimicrobials is going to
take a considerable amount of time, a stop-gap measure is
required. Research resources should be invested in strategies
that focus on lowering the resistance of microorganisms to-
wards existing, approved antimicrobial agents, or at least, de-
celerating the emergence and sharing of AMR- related genes
between pathogenic organisms. Here we briefly detail the
causes and mechanisms of AMR, and aim to provide insights
into the strategies presently being applied to mitigate the
spread of AMR.
2. CAUSES OF ANTIMICROBIAL RESISTANCE
2.1. Inappropriate Use and Prescribing
The inappropriate use of antibiotics is one of the
principal factors that will continue to contribute to the emer-
gence of AMR in bacterial pathogens. Several studies have
shown instances where either antibiotics have been inappro-
priately administered, an incorrect agent was chosen or an
inappropriate course duration was defined in 30% to 50% of
all prescriptions [4]. It was also found that approximately
30% to 60% of the antibiotics specifically prescribed for
patients in hospital intensive care units are unnecessary, in-
appropriate or are not at the optimal in dosage form [4]. In-
deed antibiotic prescriptions are sometimes dispensed by the
primary healthcare providers just to placate a persistent pa-
tient who has a viral infection, with no therapeutic benefit
whatsoever for the patient. Over time, the unnecessary use of
antibiotics will increase exposure and the frequency with
which AMR emerges in exposed bacterial populations. Anti-
biotic concentrations that are sub-inhibitory and sub-
therapeutic will encourage bacteria to become resistant to the
antibiotic by driving the natural selection of bacteria that have
only a slightly better resistance to the antibiotic. In time, muta-
tions result in altered resistance gene sequences or changes in
gene expression levels that allow resistance to higher, therapeu-
tic doses of antibiotics to develop. Horizontal gene transfer
(HGT) allows the exchange of resistance gene cassettes be-
tween strains, species and even different genera of bacteria [5].
Bacteria with enhanced gene expression or gain of gene func-
tion mutations that are driven by antibiotic exposure can in-
crease the virulence of pathogens, whereas HGT and increased
mutagenesis may cause antibiotic resistance genes to spread
through a mixed bacterial population, for example in the gut [3].
A lack of strict legislation and its ineffective implementa-
tion on the sale of antimicrobials also lead to the misuse of
antimicrobials by the general public [6, 7]. In some countries,
antimicrobials can be obtained over-the-counter without prop-
er medical prescription or advice, and even from an
unqualified vendor. For some members of the public pharma-
Moo et al.
cies operating without a license may seem more accessible as
the waiting time is shorter, and also provide services at a lower
cost without consultation fees [8]. Some pharmacies may be
willing to provide a spectrum of treatment options to accom-
modate the financial resources of the patient, and which may
not be the most appropriate for treating the presenting illness.
2.2. Extensive Non-Human Use of Antimicrobials
In agriculture, antibiotics are regularly administered to
livestock as growth supplements. It has been estimated that
in the United States alone, 80% of antibiotics sold are used
in animal husbandry, mainly to boost growth and to prophy-
lactically prevent infection [9]. It is believed that antimicro-
bial treated livestock has better overall health, produces larg-
er yields and higher quality products. Nevertheless, contact
with treated animals or consumption of food from animals
treated with antimicrobials promotes the transfer of resistant
bacteria to humans. There is a particular risk when food is
improperly prepared. This issue was first noted in 1980
where high rates of antibiotic resistance were found in or-
ganisms from the microbiome of farmers and this was linked
to the detection or resistance in the intestinal flora of their
farm animals [10]. Molecular detection methods have re-
vealed that consumers come into contact with resistant bacte-
ria from the farms through meat consumption [9]. It is be-
lieved that such a transfer may lead to significant human
health implications. Antibiotics used in agriculture can also
affect the environmental microbial ecosystem since as much
as 90% of antibiotics given to livestock are excreted in the
stool and urine. These excretions enter the environment, then
spread through groundwater, fertilizer and surface runoff
into rivers exposing environmental microorganisms to low
doses of clinically important antibiotics.
2.3. Lack of Surveillance on The Susceptibility of Antimi-
crobials
Epidemiological information allows the distribution of
AMR to be monitored among the members of a population
and geographically. The collection of community-based an-
timicrobial susceptibility data will help health care centers in
specific communities to correctly treat infections in a timely
manner. Susceptibility testing of antimicrobials has to be done
routinely for surveillance and to better understand the local
epidemiology of resistance, these data are vital for effective
clinical practice in order to develop appropriate responses to
AMR. However, due to the lack of human resources, cost and
available infrastructure, locality-based susceptibility testing of
antimicrobials is rarely performed in remote rural settings due
to resourcing issues. Since resistance rates in a particular re-
gion of a country may change dramatically over time, ideally
the collection of surveillance data should be performed regu-
larly in order to have an accurate epidemiological model [7].
3. ANTIBIOTICS CLASSIFICATION AND MODE
OF ACTIONS
Antibiotics can be classified according to their mecha-
nism(s) of action and the effects that they exert on target
microorganisms, causing either bacteriostatic or bacteri-
cidal-type activity. Nowadays, there are many different
types of antibiotics available, but the most widely accept-
Mechanisms of Antimicrobial Resistance (AMR) and Alternative Approaches
ed classification based on chemical structure identifies the
following groups: beta-lactams; tetracyclines; macrolides;
aminoglycosides; peptides antibiotics; lincosamides and
streptogramins. Antibiotics can be further grouped ac-
cording to their mode of action, which may be inhibition
of bacterial cell wall synthesis; inhibition of ribosomal
function; inhibition of nucleic synthesis or inhibition of
other metabolic processes.
3.1. Inhibitors of Cell Wall Synthesis
The biosynthesis of the bacterial cell wall is separated
into several stages. The first stage is the cytosolic phase,
which is followed by the membrane synthesis phase and,
subsequently, by the cell wall synthesis phase.
3.1.1. The Cytosolic Phase of Synthesis
The cytosolic phase of cell wall generation coincides
with the synthesis of the nucleotide precursors UDP-N-
acetylmuramyl (UDP-MurNAc)-pentapeptide and UDP-
N-acetylglucosamine (UDP-NAG [11]. Fosfomycin is a
broad-spectrum antibiotic that acts on common Gram-
positive (Staphylococcus aureus (S. aureus), Enterococ-
cus), and Gram-negative (Klebsiella pneumoniae and
Pseudomonas aeruginosa) bacteria by inhibition of the
enzyme, pyruvyl transferase, which is also known as UDP-
N-acetylglucosamine enolpyruvyl transferase, or MurA
[12, 13]. This enzyme catalyzes the first step in e cell wall
biosynthesis, by transferring an enolpyruvyl group from
PEP to UDP-NAG [14]. Fosfomycin acting as an analogue
of PEP is able to form a covalent bond due to the presence
of the cysteine amino acid at the active site of the MurA
enzyme [15]. This is similar to the molecular interaction
between UDP-NAG and PEP. However, the formation of a
covalent bond occurs when the epoxide ring of fosfomycin
reacts with the thiol group of the cysteine resulting in per-
manent inactivation of the enzyme [16].
3.1.2. The Membrane Phase of Synthesis
At this stage, the processes of cell wall synthesis are
localized to the cytoplasmic membrane and are catalyzed
by membrane-bound enzymes [17]. The previously
formed non-nucleotide portion of the precursor molecules
is transferred to a carrier in the cytoplasmic membrane. A
UDP-MurNAc-pentapeptide precursor is attached to un-
decaprenyl pyrophosphate, a transport lipid. Lipid I
[MurNAc-(pentapeptide)-pyrophosphoryl-undecaprenol]
is then formed. Subsequently, the formation of lipid II
[GlcNAc-β-(1,4)-MurNAc-(pentapeptide)-
pyrophosphoryl-undecaprenol] occurs by adding glu-
cosamine from UDP-NAG [18]. The peptide cross-bridge
is added at the third amino acid residue where peptidogly-
can is indirectly cross-linked. The flipping of lipid II to
the external surface of the cell membrane occurs and pen-
icillin-binding proteins (PBPs) incorporate it into the nas-
cent peptidoglycan layer [19]. One of the critical antibiot-
ics that inhibit the cell wall synthesis during this phase is
bacitracin, which acts most effectively on S. aureus [20].
3.1.3. The Cell Wall Phase of Synthesis
At the cell wall phase, polymerization of the wall sub-
units occurs by transferring new peptidoglycan to the non-
Current Drug Discovery Technologies, 2019, Vol. 16, No. 0 3
reducing N-acetylglucosamine of the new saccharide-
peptide. Peptidoglycan is linked to the cell wall and the
new peptidoglycan attaches to the existing peptidoglycan
of the cell wall. The attachment is catalyzed by a trans-
peptidase reaction, involving the peptide chains of both
molecules [21]. One of the polymers must contain a D-
alanyl-D-alanine terminus. The peptide bond connecting
two D-alanyl residues in the pentapeptide is then cleaved
by transpeptidase. This is followed by acylation through
the carbonyl group of the second to the last D-alanine
residue. Antibiotics that are responsible for inhibiting the
cell wall phase synthesis are the β-lactams like penicillin,
which contain a four-membered ring that undergoes
acylation reaction with the transpeptidases [22].
3.2. Inhibitors of Cell Membrane Function
The small antimicrobial peptides (AMPs), are de-
scribed as cell membrane acting antibiotics and the bacte-
rial membrane is usually their target. The action of AMPs
is initiated by the binding of the peptide to the cell mem-
brane. The binding causes perturbation of the structure of
the membrane and hence disrupts the membrane function-
ality. Disruption of membrane structure and function can
disturb events that are associated with the membrane,
such as the biosynthesis of the cell wall. AMPs can also
be transported across the membrane, enabling the peptides
to interact with targets in the cytoplasm [23]. The interac-
tion between AMPs and the intracellular target structure
causes the inhibition of the microorganism's normal biol-
ogy. The precise mechanisms by which AMPs cause cel-
lular dysfunction and mortality are now being studied by
many researchers. One of the ways in which the AMPs
work is through the disruption of the biochemical struc-
ture and composition of the membrane [24]. AMPs can
also form pores in some membranes causing the leakage
of metabolites, membrane depolarization and ultimately
cell death [24]. In addition, disturbance in the normal cell
membrane structure affects peptidoglycan precursor syn-
thesis and translocation. Examples of natural antimicrobi-
al peptides include defensins and protegrins, which exhib-
it antibiotic properties against Gram-positive and Gram-
negative bacteria such as Streptococcus spp., S. aureus, K.
pneumoniae and Escherichia coli [25]
3.3. Inhibitors of Ribosome Function
The ribosomes of bacteria consist of the 50S and 30S
subunits [26]. Antibiotics are able to target one or both
subunits simultaneously. Aminoglycoside antibiotics are
complex sugars with glycosidic linkages that allow them
to bind to particular ribosomal subunits [27]. Aminogly-
cosides are differentiated by the structure of their molecu-
lar nucleus, with either of the aminohexoses , 2-
deoxystreptamine or streptamine linked to the nucleus.
NH4 and OH groups are essential for the interaction of
aminoglycosides with particular ribosomal proteins [28].
The first aminoglycoside studied was streptomycin. Strep-
tomycin causes the misreading of DNA genetic code by
binding to a specific S12 protein in the 30S ribosomal
subunit (Fig. 1). The tetracycline family antibiotics also
work by binding to the 30S ribosomal subunit (Fig. 1).
4 Current Drug Discovery Technologies, 2019, Vol. 16, No. 0
Fig. (1) Inhibition of sites of protein biosynthesis sites by various antibiotics that bind to the 30S and 50S ribosome.
This binding obstructs the interaction of the aminoacyl-
tRNA with site A of the bacterial ribosome [29]. The
bacteriostatic action of tetracyclines inhibits a wide range
of bacteria including Mycoplasma sp. and Chlamydia sp.
[30]. Drugs such as chloramphenicol and the macrolides
inhibit the 50S ribosomal subunit. The formation of pep-
tide bonds is inhibited by chloramphenicol through at-
tachment to a peptidyltransferase enzyme. This enzyme is
located on the 50S ribosomal subunit of both Gram-
positive and Gram-negative bacteria [31]. The structure of
macrolides comprises a large lactone ring that binds to 50S
subunits and impairs the activity or the translocation (or
both) of peptidyltransferase. Erythromycin is considered
to be the most important macrolide and inhibits some
Gram-negative species belonging to the genera Chlamyd-
ia, Haemophilus, Legionella and Mycoplasma as well as
Gram-positive bacteria [13].
3.4. Inhibitors of Nucleic Acid Synthesis
Antimicrobial compounds interfering with the synthe-
sis of nucleic acids can act at several different stages of
the process, either by inhibiting: nucleotide synthesis;
DNA-directed DNA polymerase activity or the replication
of the DNA.
3.4.1. Interference With Nucleotide Synthesis
Most antimicrobial compounds, that interfere with nu-
cleic acid synthesis, either interfere with the synthesis of
Moo et al.
pyrimidine and purine bases or with the incorporation of
the nucleotides into polymers. Some agents that act as
analogues are able to incorporate themselves into polynu-
cleotides to interfere with accurate nucleic acid transcrip-
tion or replication. For example, flucytosine (5-
fluorocytosine) is an antifungal agent. Flucytosine is con-
verted into 5-fluorouracil in the target cell. Fluorouracil
will then be integrated into RNA resulting in premature
chain termination. Premature chain termination inhibits
the action of thymidylate synthetase, halting the synthesis
of thymine nucleotides [32]. This, in turn, result in im-
paired DNA synthesis. Arabinoside analogues are phos-
phorylated by kinases in the same manner as deoxynucle-
osides and inhibit DNA synthesis through DNA polymer-
ase antagonism. Arabinosides can be considered as
deoxyucleoside analogues [33], and adenosine arabino-
side can be integrated into DNA in competition with, the
integration of dATP. Clinically the nucleoside analog,
acyclovir, inhibits DNA polymerase and also the thymi-
dine kinase of herpes viruses once the drug is converted
into a triphosphate [34].
3.4.2. Inhibition of DNA-Directed DNA Polymerase
Rifamycins are in a group of antibiotics that inhibit the
DNA-directed RNA polymerase [35]. Promoter sites that
initiate DNA transcription will be bound by the RNA pol-
ymerase. The RNA polymerase attaches to a factor that
confers specificity for recognition of specific promoter
sites. Although rifampin binds to a subunit of RNA non-
Mechanisms of Antimicrobial Resistance (AMR) and Alternative Approaches
covalently, the binding is still strong enough to interfere
with the transcription initiation process (Fig. 1), and there
will be no effect of the antibiotic on RNA synthesis if it is
administered after a cycle of polymerization has com-
menced [13]. Rifamycins bind to RNA polymerase tightly
and, hence, only small amounts of these antibiotics are
needed in order to inhibit RNA synthesis. In addition, the
binding does not affect DNA polymerase activity, as it is
specific for the RNA polymerase.
3.4.3. Inhibition of DNA Replication
DNA gyrase and topoisomerase I act together to sus-
tain the DNA double helix in the supercoiled state [36].
During chromosomal replication in bacteria, DNA gyrase,
a tetrameric protein comprising two A and two B subunits
is essential in relieving the molecular torsion. The catalyt-
ic action of gyrase gives rise to a temporary covalent bond
between the DNA and the A subunit in what is termed as
“the double strand passage reaction”. Quinolones can an-
tagonize DNA replication. During strand passage, quin-
olones bind to the cleavage intermediate; and this causes a
deleterious effect on the normal process of DNA replica-
tion. Quinolones bind to the cut ends of the DNA in the
enzyme-DNA complex to prevent the rotation and religa-
tion of the cut ends of DNA [36]. Quinolones can either be
bacteriostatic or bactericidal depending on the concentra-
tion. At low concentrations, the antibiotic remains bound to
the cut ends of DNA. Replication and transcription are in-
hibited as DNA remains supercoiled which stops the repli-
cation fork or the transcription complex from further pro-
gression (Fig. 1). The attachment of quinolone to the gyrase
enzyme complexed with the cut ends of the DNA is re-
versible, and so explains how quinolones have bacteriostat-
ic properties. At high concentrations of a quinolone, the
DNA ends will be freed from the complex, chromosomal
DNA is cleaved and this eventually leads to cell death [37].
3.5. Inhibitors of Other Metabolic Processes
One mechanism by which drugs can inhibit the critical
metabolism of bacterial cells is by blocking the biosyn-
thesis of tetrahydrofolate. Tetrahydrofolate is a co-factor
in the process of de novo synthesis of thymidine mono-
phosphate, which is needed for the biosynthesis of bacte-
rial DNA and RNA [38]. When tetrahydrofolate is defi-
cient, there is no synthesis of thymidine and hence the
bacteria is unable to produce new DNA or RNA. Tetrahy-
drofolate is synthesised from dihydropteroate diphosphate
and p-aminobenzoic acid (PABA). Bacteria produce
dihydropteroic acid from these two precursor compounds,
a reaction catalyzed by dihydropteroate synthetase. Sul-
fonamides block the synthesis of dihydropteroate by com-
peting with PABA for binding to the enzyme as a struc-
tural mimic [39]. Consequently, sulfonamides act as com-
petitive inhibitors of dihydropteroate synthetase to block
the synthesis of dihydropteroate. This leads to tetrahydro-
folate deficiency. Deficiency in tetrahydrofolate results in
the inability to synthesize thymidine, which will eventual-
ly cause cell death.
Current Drug Discovery Technologies, 2019, Vol. 16, No. 0 5
4. MECHANISMS OF ANTIBIOTIC RESISTANCE
Concurrent with the emergence of new resistance
mechanisms and novel resistance-associated genes, new
transmission vectors are also being discovered. Therefore,
we will now focus on current discoveries relating to the
mechanisms by which AMR is conferred to bacteria via
intrinsic, naturally acquired or externally acquired mecha-
nisms. The mechanisms discussed will include how the
pathogen prevents access to drug targets, changes in the
structure of antibiotic targets, as well as the modification
or inactivation of antibiotics that can be affected.
Resistance in bacteria to some antibiotics can either be
intrinsic or acquired through gene mutations or via HGT.
Bacterial species with intrinsic resistance have the capa-
bility to survive the actions of certain antibiotics due to
innate structural or functional features [40]. Intrinsic re-
sistance is achieved via inherent structural or functional
characteristics of the molecular target. The antibiotic dap-
tomycin is active against Gram-positive bacteria but inef-
fective against Gram-negative bacteria; this is the result of
innate differences in the cytoplasmic membrane composi-
tion between the Gram-positives and Gram-negatives. The
Gram-negative cell membrane is comprised of less
anionic phospholipids than the cytoplasmic membrane of
Gram-positive bacteria [40]. This reduction in the fraction
of anionic phospholipids affects the Ca2+ -dependent in-
sertion efficiency of daptomycin into the cytoplasmic
membrane, which is needed for its antibacterial activity.
Recent studies have determined that several genes are
responsible for the innate resistance that exists towards
certain classes of antibiotics, such as fluoroquinolones, β-
lactams and aminoglycosides. The key resistance genes are
thioredoxin reductase (trxB), thioredoxin A (Trx A), SapC,
DacA, FabI and D-Ala-D-Ala carboxypeptidase. So, com-
bined antagonism of these gene products could help to
boost the activity of existing drugs such as rifampin, ami-
noglycosides as well as some β-lactams in the treatment of
infection [41]. The genes responsible for innate resistance
were identified from high-density genome mutant libraries
and high-throughput screening studies. To create the librar-
ies, either mutagenesis by random transposon insertion or
targeted insertion was carried out in bacteria such as Staph-
ylococcus aureus, Escherichia coli and Pseudomonas ae-
ruginosa [41, 42]. This library screening approach is par-
ticularly effective in discovering possible novel drug com-
binations that will enable inhibition of intrinsic resistance
mechanisms [40]. Consequently the spectrum of activity of
other antibiotics can be extended to other pathogens be-
yond their common target species.
In addition to intrinsic resistance, there are several
other resistant mechanisms that can be acquired by bacte-
ria, all of which fall into three primary groups: prevention of
access to target; mutational changes in antibiotic targets and
finally, modification of targets [40]. These modes of action
have been reviewed extensively over the past decades. Thus,
this review will provide an update on the most recent studies
for each type of antibiotic resistance mechanism.
6 Current Drug Discovery Technologies, 2019, Vol. 16, No. 0 Moo et al.

Fig. (2). Mechanisms of antibiotic resistance. (a) β-lactamase breaks β-lactam antibiotics via hydrolysis and hence deactivating the
antibiotics. (b) Prevention of access to target due to increased efflux. The efflux pump of bacteria transport antibiotics actively out of
the cell. (c) Downregulation in the expression of porin proteins or the substitution of porins with more-selective channels. (d) Changes
in the structure of the target such as mutation makes the binding of antibiotics less efficient, without affecting the functionality of the
target itself, conferring antimicrobial resistance. (e) Post-translational modification of targets.

family (MATE), the major facilitator superfamily (MFS),

4.1. Prevention of Access to Target
Denial of access of an antibiotic to its target can be
exemplified by the resistance of Enterobacteriaceae to
carbapenems, which is due to reduced permeability of the
bacterial membrane (Fig. 2). Hydrophilic antibiotics dif-
fuse into the bacterial cell via the porin proteins in the
outer membrane. OmpC and OmpF of E. coli are typical
examples of the major porins found in most Enterobacte-
riaceae. Several studies have suggested that resistance
strategies employed by bacteria include the downregula-
tion in expression of porin proteins, or the substitution of
major porins with more-selective membrane channels
(Fig. 2). So the resistance to carbapenems in the Entero-
bacteriaceae in the clinical setting persists even without
carbapenemase production by the bacteria; instead critical
mutations reduce porin production or result in the expres-
sion of mutant porin alleles [43, 44].
The prevention of access to the antibiotic's target can
be achieved through increased efflux of the antibiotic. The
efflux pump of bacteria transports antibiotics actively out
of the cell in bulk. This contributes significantly to the
intrinsic resistance in Gram-negative bacteria to a variety
of drugs that are used in the treatment of bacterial infec-
tions. The five major types of efflux pumps that have been
identified include the ATP-binding cassette family
(ABC), the multidrug and toxic compound extrusion
the small multidrug resistance family (SMR) and the re-
sistance-nodulation-cell-division family (RND) [45].
Multidrug-resistant (MDR) efflux pumps transport a vast
range of structurally different substrates. There are several
genes encoding the MDR efflux pumps located on bacte-
rial chromosomes, however, some of those genes can be
transferred between bacteria, due to mobilization on the
genes onto plasmids [40]. Recently, it has been found out
that the IncH1 plasmid, isolated from Citrobacter freun-
dii, has a gene cassette encoding a novel resistance modu-
lation division (RND) pump together with the gene for
New Delhi metallo-β-lactamase 1 (NDM1) [46]. This de-
velopment is a major concern since this indicates that the-
se specific resistances can be transmitted between bacteria
on plasmids, which may facilitate the spread of novel re-
sistances to other bacterial pathogens that are clinically
relevant.
The most well-characterized RND family pump that
has clinical relevance is found in Gram-negative bacteria.
A broad range of substrates can be exported at an acceler-
ated rate when the RND is overexpressed [47]. RND
pumps are located in the inner membrane and a tripartite
complex is formed with an outer-membrane channel TolC
or OprM and a periplasmic adaptor protein AcrA or
MexA [40]. One of the identified RND pumps is AcrB,
which is a homotrimeric protein. Computational studies
Mechanisms of Antimicrobial Resistance (AMR) and Alternative Approaches
and co-crystallization of E. coli AcrB in complex with
transported substrates have identified two binding pockets
in AcrB. The binding pockets are able to accommodate
substrates of different sizes and chemical properties,
which establishes how the pumps confer resistance to a
wide range of different antibiotics [48, 49]. Enterobacteri-
aceae, S. aureus and P. aeruginosa overexpress the RND
efflux pump. Overexpression of the efflux pumps is regu-
lated by local regulators, such as EmrR from E. coli,
QacR from S. aureus and also by global regulators. Tran-
scription factors of the AraC-XyIS family are important
global expression regulators. MtrA enhances transcription
of mtrCDE in N. gonorrhoeae [50]. The regulator family,
Arac-XyIS is encoded together with a repressor of the
several antibiotic resistance protein MarR family [51].
4.2. Changes in Antibiotic Targets by Mutation
Antibiotics have evolved or are designed specifically
so that they are able to bind to their specific targets with
high affinity and consequently disrupt the normal activity
of the target. Alterations in the structure of the target can
make the binding of antibiotics less efficient, without af-
fecting the functionality of the target itself, conferring
antimicrobial resistance (Fig. 2). A single point mutation
in the gene encoding an antibiotic target can result in re-
sistance towards the given antibiotic [40]. One of the clas-
sic examples is the development of resistance towards
rifampin. This occurs due to single point mutations, caus-
ing an amino acid substitution in the rpoB gene. Conse-
quently, these mutations decreased the affinity of rifampin
for its target, allowing the transcription to continue [45].
Some organisms have multiple copies of genes that
encode the targets of some antibiotics. For example, the
23S rRNA ribosomal unit gene of Gram-positive bacteria
is encoded by multiple identical copies of the gene. Re-
sistance to the antibiotic linezolid is conferred to S.
pneumoiniae and S. aureus through multiple copies of the
23S rRNA gene. The gene copies have a high recombina-
tion rate that produces bacterial populations with resistant
mutant alleles. Eventually, the whole population becomes
resistant towards linezolid [52, 53]. Another example of a
mutation in a target that confers resistance is possession
of a gene that is homologous to the original target. This
can be seen in methicillin-resistant S. aureus (MRSA)
whereby the resistance is conferred by having the genetic
element known as staphylococcal cassette chromosome mec
(SCCmec). This element bears the mecA gene, which is re-
sponsible for the β-lactam insensitive protein penicillin-
binding protein PBP2a, the mutated penicillin-binding pro-
tein in this case, supports cell wall biosynthesis, despite the
fact that the native PBP is inhibited by antibiotics [54].
4.3. Modification (and Protection) of Targets
Post-translational modification of targets is another
way of achieving antibiotic resistance without a mutation-
al event occurring in the genes that encode target mole-
cules (Fig. 2). For instance, methylation of 16S rRNA and
the resulting alteration in the drug-binding sites is
achieved by the erythromycin ribosome methylase (erm)
family. This activity prevents the binding of lincosamides,
macrolides and streptogramin to the 16S rRNA [55]. An-
Current Drug Discovery Technologies, 2019, Vol. 16, No. 0 7
other example recently identified is the chloramphenicol-
florfenicol resistance gene (cfr) product which methylates
the A2503 residue in the 23S rRNA. This causes the tar-
get to become resistant to an extensive range of antibiotics
that interacting with 23S rRNA near this methylation site.
These antibiotics include lincosamides, oxazolidinones,
pleuromutilins, phenicols and streptogramins [56]. Both
of the genes, erm and cfr are often found on plasmids
which drive the dissemination of AMR among bacteria
[57]. Another example of such a modification strategy is
the methylation of the ribosomal proteins which prevents
the binding of aminoglycosides, so inhibiting protein syn-
thesis. The armA and rmt genes, which encode different
methyltransferases, have been discovered in clinical iso-
lates of Enterobacteriaceae [58]. In addition, the re-
sistance genes for quinolone, the qnr gene families, have
also been found on plasmids. Pentapeptide repeat proteins
(PRPs) are encoded by the qnr genes. PRPs bind to topoi-
somerase IV and DNA gyrase to protect them from quino-
lone action. A recent discovery suggested that after drug
binding, the interaction of PRPs and the topoisomerase-
quinolone complex liberates the quinolone [59]. The lib-
eration of quinolone enables the topoisomerase to main-
tain its activity and re-ligate the DNA, preventing the
breakage of the double-stranded DNA [59].
4.4. Direct Modification of Antibiotics
In addition to preventing antibiotics from entering the
cell, bacteria can also modify the structure of antibiotics,
rendering them inactive. One of the mechanisms by which
bacteria inactivate antibiotics is through a hydrolysis reac-
tion (Fig. 2). To date, there are a collection of enzymes,
such as carbapenemases, chloramphenicol acetyltrans-
ferases, which can degrade or alter different classes of
antibiotics, such as macrolides, β-lactams, aminoglyco-
sides and phenicols. Over the years, the classes of antibi-
otics have been expanded in order to overcome the prob-
lems posed by hydrolytic enzymes. Both the early β-
lactamases and the extended-spectrum β-lactamases
(ESBLs) were active against β-lactams, while ESBLs also
have activity against oxyimino-cephalosporins. Imipen-
emase (IMP), New Delhi Metallo-β-lactamase and Vero-
na integron encoded Metallo β-lactamase (VIM) are car-
bapenemases and also ESBL carriers which give rise to
the emergence of β-lactam antibiotic-resistant isolates.
These have serious consequences for the treatment of se-
vere bacterial infections [60]. Due to the increase in
ESBL gene carrying bacteria, carbapenem antibiotic use
is increasing in clinic. Subsequently, this will cause an in-
crease in the number of β-lactamase expressing clinical
isolates that carry carbapenemases with carbapenem
hydrolyzing activity [61, 62]. Another mechanism by
which bacteria inactivate antibiotics is by addion of a
chemical group. Addition of a chemical group to the anti-
biotic molecules prevents its binding to the target mole-
cule due to steric hindrance. Chemical groups that are
transferable onto antibiotics include acyl, nucleotidyl,
phosphate and ribitoyl groups. There is a broad range of
enzymes responsible for these transferase reactions result-
ing in a substantial and diverse group of antibiotic-
resistance gene families [63].
8 Current Drug Discovery Technologies, 2019, Vol. 16, No. 0
The aminoglycosidic antibiotics are large molecules
carrying exposed amide and hydroxyl groups that are
prone to modification. The enzymes that modify amino-
glycosides confer a high level of resistance to the antibi-
otics [40]. Acetyltransferases, nucleotidyltransferases and
phosphotransferases modify the aminoglycoside family,
that include the commonly prescribed antibiotics gen-
tamicin, streptomycin and amikacin [64]. Recently, there
has been a worrying development, whereby a novel ge-
nomic island in Campylobacter coli has been discovered;
this genomic island was originally isolated from broiler
chickens in China. The genomic island encoded all three
classes of the six aminoglycoside-modifying enzymes. This
cassette confers resistance to all the aminoglycoside antibi-
otics, which are currently in use to treat Campylobacter
infections. Of particular concern is resistance to gentami-
cin, which is considered an antibiotic of last resort in severe
bacterial infections with highly resistant strains [65].
5. STRATEGIES TO COMBAT AMR
AMR is a global threat that is escalating and is a cause
of increased morbidity and mortality in the community
from previously controllable infections. The clinical set-
ting is increasingly associated with the development of
AMR due to the high level of antibiotic use. The spread of
antibiotic resistance to other environmental reservoirs fur-
ther complicates effectual strategies in combating AMR.
The process of developing new antimicrobials needs to be
expedited as the speed of AMR development has exceeded
the developmental rate of antimicrobials. Unfortunately,
identification of new and effective pharmaceuticals is not a
straightforward task. Thus, researchers have embraced alter-
native strategies in order to combat AMR. In the subsequent
sections, of this review, we will discuss current and future
approaches to combating AMR.
5.1. Combination Drug Therapy
Combination drug therapy is defined as the use of two
or more medications or therapies to address the same con-
dition. Combination drug therapy is used rather than indi-
vidual therapy (monotherapy) on microorganisms because
microorganisms are more prone to resistance development
in monotherapy. Although drug-drug interactions may be
a stumbling block in this strategy and needs to be taken
into consideration during the development of drugs, evi-
dence points to this therapeutic approach greatly lessening
the probability of primary resistance to the components of
the combination course (Kaur, 2016). The combination
drug regimens are divided into 3 mechanistic strategies:
1) Combination of drugs acting on different targets in dif-
ferent pathways. 2) Combination drugs acting on different
targets in the same pathway. 3) Combination drugs acting
on a single target, but in different dimensions [66].
One classical example of the combination of drugs act-
ing on different targets in different pathways, is the treat-
ment used for Mycobacterium tuberculosis infections. The
four combined drugs are: an inhibitor of arabinosyltrans-
ferases involved in cell wall biosynthesis (ethambutol); an
inhibitor of the enoyl reductase subunit of the fatty acid
synthase (isoniazid) and twoRNA polymerase inhibitors
Moo et al.
(pyrazinamide and rifampicin [66]. The principle behind
this therapy anticipates that the bacterium will modify the
individual antibiotic targets in order to become resistant. If all
strategies of resistance are targetted simultaneously this ena-
bles the drug combination to remain effective, thus, keeping
the likelihood of bacterial propagation at the lowest level.
Combination therapy has also proved effective for the
treatment of Plasmodium falciparum, malaria pathogen.
The efficacy of chloroquine has reduced due to the spread
of the resistant P. Falciparum strains. Hence, artemisinin
is used to treat malaria by inhibiting SERCA-family Ca2+-
ATPase PfATP6. Artesunate or artemether, derived from
artemisinin is usually administered in combination with
lumefantrine, amodiaquine or mefloquine in order to ex-
tend the efficacy of artemisinin.
A study reported that tunicamycin interacts synergisti-
cally with β-lactam antibiotics. Tunicamycin is an inhibi-
tor of an enzyme involved in the biosynthesis pathway
for cell wall teichoic acid, the N-acetylglucosamine-1-
phosphate transferase TarO, in S. aureus. Synergistic in-
teraction between tunicamycin and β-lactam antibiotics
greatly decreased the minimum inhibitory concentration
(MIC) of oxacillin against one MRSA clinical isolate
from 50 µg/mL to 0.4 µg/mL at a tunicamycin concentra-
tion of only 0.08 µg/mL [67]. This is an example of the
inhibition of different targets within the same pathway.
Furthermore, β-lactam antibiotics also showed significant
synergy with early cell wall synthesis inhibitors, such as
β-chloro-D-alanine, bacitracin, D-cycloserine, fosfomy-
cin, vancomycin and teicoplanin at sub-inhibitory concen-
trations. The synergistic interaction is able to lower the
resistance of S. aureus strain towards methicillin by two-
fold for β-chloro-D-alanine, and up to 128-fold for
fosfomycin, as measured by the reduction in MIC [68]. A
combination of drugs acting on different targets in the
same pathway has less breadth of efficacy in comparison
to a combination of drugs acting on different targets in
different pathways. For instance, inhibiting two enzymes
that are involved in the biosynthesis pathway of folate will
be effectual as the target cell requires folate to synthesize
dTMP for DNA synthesis. However, if a subpopulation of
target cells can avoid the need of folate, then this combina-
tion of drugs would become less effective. An example of a
combination of drugs acting on different targets in unrelat-
ed pathways, is the combination of amoxicillin, the β-
lactam antibiotic with clavulanic acid [69]. Amoxicillin
inhibits cell-wall synthesis of bacteria but is often de-
stroyed by β-lactamase, on the other hand, clavulanate acid
is an inhibitor of β-lactamase. So both of the compounds
work synergistically, wherein clavulanate prevents β-
lactamase from degrading amoxicillin at bacterial cell wall,
so maintaining the level of amoxicillin, thereby enhancing
its antibacterial activity. Streptogramins are another group
of agents used in combination therapy. They act on a single
target via different mechanisms as they are made up of two
active molecules: a non-ribosomal peptide and a polypep-
tide non-ribosomal peptide hybrid. Streptogramins attach at
two adjacent sites on the 50S subunit, near to the peptidyl
transferase center [70]. The potency is 10 to 100 fold high-
er when combined together as compared to either one mol-
ecule acting as a single agent.
Mechanisms of Antimicrobial Resistance (AMR) and Alternative Approaches
Many debates have arisen regarding the prospective
benefits of combination therapy versus monotherapy. One
of the advantages of combination therapy is that it em-
ploys the synergistic interaction between two drugs result-
ing in enhanced bacterial killing. These synergies are of-
ten initially identified through in vitro studies. This can be
seen for example in the case of the combination of colistin
and the glycopeptide vancomycin or teicoplanin. In vitro
studies showed synergistic interactions between these
antibiotics against multidrug-resistant isolates of Acineto-
bacter baumannii that may have clinical relevance [71].
Combination therapy may also give a greater overall spec-
trum of activity of specific antibiotics against different
pathogens. Despite the benefits of this combination ap-
proach, some adverse effects, such as aminoglycoside
nephrotoxicity, have been documented. In a study involv-
ing 876 patients, ceftazidime was compared with gen-
tamicin and piperacillin, the renal toxicity was notably
higher in the combination therapy group (P < 0.001),
whereby 5 of the patients required hemodialysis and one
died due to renal insufficiency [72]. Roughly 85% of
aminoglycosides are found in the renal cortex [73], and
when cytosolic concentrations become high, apoptosis is
triggered [74]. In addition, several meta-analyses of com-
bination therapies have shown that renal toxicity occurs
more often in patients who receive aminoglycosides ther-
apy than those who do not [75].
5.2. Bacteriophage Therapy
The rise in the number of infections caused by drug-
resistant bacteria within the clinical setting has propelled
the research community to explore alternatives to conven-
tional antibiotics. Bacteriophages are abundant [76], and
most likely every bacterium has their own specific viruses
that could be used as antibacterial agents [77]. Phages
were first used experimentally as therapeutic agents in the
early 20th century. The main challenges regarding phage
therapy are the strict regulatory guidelines that needed to
be complied with, as well as developing effective thera-
peutic practices [78]. In spite of this, phage therapy could
potentially be used as an alternative to conventional anti-
biotics as long as the special requirements of phage-based
medicines are met.
A bacteriophage is a virus that lyses and disrupts the
metabolism of bacteria following cellular invasion. Phage
therapy suppresses pathogenic bacterial infections during
the lytic cycle [40]. The lytic cycle begins with phage
attachment to a host cell. The tail fibers of the virion then
penetrate the bacterial cell wall and inject the viral ge-
nome into the cytoplasm. Immediately after penetration,
phage DNA is transcribed and begins early protein syn-
thesis. Some of the synthesized proteins break down the
host (bacterial) DNA whereas some are enzymes required
for the replication of the phage DNA. The newly synthe-
sized phage DNA produces late proteins, which are the
subunits of phage capsid. Capsid proteins assemble to
form a head with condensed viral DNA packed inside.
The tail then joins to head forming progeny virions. Late
proteins lyse the host cell, which then releases viral prog-
eny into the surrounding environment [70]. The fact that
phages infect bacterial hosts very selectively, without
Current Drug Discovery Technologies, 2019, Vol. 16, No. 0 9
harming commensal bacterial flora is an advantage over
conventional antibiotics. Nevertheless, bacteria can de-
velop resistance to phages in a brief period of time inter-
rupting the antibacterial effect [79]. In order to reduce the
development of resistance to phages, mixtures of phages
are often used. However, obtaining a mixture of phages
which are sufficiently effective against pathogens and
their variants, is a difficult task [80]. One problem that
can arise is that when a mixture of phages is used in an
effort to increase the host range the effectiveness of spe-
cific phages in the mixture against certain strains of bacte-
ria may decrease. To circumvent this, a cocktail of phages
should be made specifically for each patient so that only
phages that are effective against the bacterial strains re-
sponsible for the infection will be used.
Antibiotics are non-specific, promoting the destruction
of pathogens and commensal bacteria with equivalent
potency, this is a significant problem, as loss of the nor-
mal microflora can result in a novel pathogenic state. This
can be observed with the use of fluoroquinolones, which
can lead on to superinfections with Clostrodium difficile,
following the loss of the normal gut bacteria. Unlike anti-
biotics, bacteriophages have high specificity in killing
bacteria without harming or altering gut microbiota. High-
ly specific bacteriophages, however, only infect a single
species of bacteria. Bacteriophages attach to highly spe-
cific receptors available on the host cell surface, in order
to enter a bacterial cell. These receptors are absent from
non-target species so avoiding disruption of the normal
microflora [78]. Moreover, bacteriophages are only need-
ed in small doses for the treatment of bacterial infections
since they are able to replicate in vivo. Bacteriophages are
consequently less immunogenic because a reduced dose
of a foreign substance is being administered into the body
[67]. So a single bacteriophage has the capability to kill a
single bacterial strain, unlike antimicrobials, which work
via the concept of sub-lethal dosing that can affect the
whole gut microbiome.
A study has demonstrated that the overall effect of the
antibiotic and phage could be increased synergistically.
Through manipulating the structures of the bacterial cell
by genetic engineering, the effect of the drug was en-
hanced increasing the susceptibility of E. coli towards
antibiotic therapy. Phage-encoded genes that repress the
SOS response of E. coli resulted in reduced selection
pressure. This eventually led to a decrease in antibiotic
resistance. Following the treatment, there was no sign of
bacterial recovery after a period of either 12 or 24 hours
and killing efficacy increased tremendously by several
orders of magnitude in comparison with conventional
therapy. Nonetheless, strains of bacteria were not com-
pletely eliminated by this treatment, so some bacteria
must have arisen that were resistant to the phage and anti-
biotic [81]. In the year 2016, in vivo studies investigating
how Enterococcus faecalis acquired resistance to bacteri-
ophages were reported. In the experiments, it was found
out that the pivotal role of a phage infection protein from
Enterococcus faecalis (PIPEF) was a factor in phage tro-
pism [82]. The in vivo studies on a gnotobiotic mouse
model showed that E. faecalis acquired phage resistance
through mutations in PIPEF. Phage therapy may also have
10 Current Drug Discovery Technologies, 2019, Vol. 16, No. 0
a prophylactic application. Administering phages orally
can eliminate pathogens such as Salmonella spp., E. coli
and Clostridium difficile which can cause diarrhea. Bacte-
riophages could be designed to regulate the gastrointestinal
microbiota composition, ultimately benefitting the host in
different ways given the growing interest in understanding
how the microbiome affects overall metabolism [83].
Bacteriophages are active against both Gram-positive,
as well as Gram-negative bacteria [84]. Bacteriophages
function by lysis, which is totally different from the
mechanism of how antibiotics work. As mentioned be-
fore, bacteriophages are specific. This specificity enables
phage therapy to narrow the antibacterial spectrum, limit-
ing its effect to either single species or even single strain
within a species, without damaging other non-targeted
bacteria. Moreover, in terms of the economic aspects of
phage therapy, this approach appears to be promising.
Although the duration of phage therapy is longer than
conventional antibiotic treatment, the cost is lower. This
was shown in phage treatment study on 6 patients infected
with various Staphylococcus bacterial strains including
methicillin-resistant S. aureus [85].
Despite the fact that phage therapy has several ad-
vantages, studies still need to be done to determine the
capability of phages to treat infections in vivo. The admin-
istration route, the optimal dose, the duration and fre-
quency of treatment all have to be determined for specific
pathogens and phages. The specificity of phage therapy
being specific to a pathogen is an advantage, but it is also
a liability. A clinical sample has to be isolated, cultured
and the pathogen identified before a bacteriophage specif-
ic to that particular sample can be defined and given to the
patient for administration. Innovations in rapid diagnostic
methods may help. However, all of these are time-
consuming, and most clinical microbiology laboratories
and health care centers have limited resources. Besides
that, the possibility of phage transferring DNA from a
bacterium to another is also a concern of phage therapy.
The chances of developing a new microbe or resistant
bacteria are high if the genetic material is transferred [86].
Fortunately, the frequency of resistance in vivo during
phage therapy, thus far, is reportedly low, as compared to
in vitro resistance [87].
5.3. Antimicrobial Adjuvants
Antimicrobial adjuvants are compounds that generally
do not possess any intrinsic antimicrobial activity. The
primary aim of using antimicrobial adjuvants is to en-
hance the efficacy of existing antibiotics, as well as to
suppress the emergence of resistant strains. Adjuvants
usually act by reversing the existing resistance mecha-
nisms of microbes. The functions of adjuvants are: 1) In-
hibition of antimicrobial resistance elements, 2) Enhance-
ment the uptake of antimicrobial in target cells, 3) Nullifi-
cation of the effect of the efflux pumps, 4) Disruption of
biofilms and 5) Promotion of bacterial oxidative stress.
Antimicrobial adjuvants may act to inhibit antimicro-
bial resistance elements. For example, adjuvants may act
as β-lactamase inhibitors and so can be added to a β-
lactam antibiotic to protect theβ-lactam ring from distor-
Moo et al.
tion as well as maintaining the efficacy of the antibiotic
against β-lactamase expressing Gram-positive bacteria.
One remarkable example is the addition of the adjuvant
cilastatin to inhibit dehydropeptidase, the enzyme that
degrades the β-lactam antibiotic imipenem. Cilastatin pro-
tects imipenem from degradation and prolongs its antimi-
crobial effects when given in combination [88]. Adjuvants
also enhance the uptake of antimicrobial agents by the
targets organisms. Colistin (Polymyxin B) is a poly-
cationic molecule containing both hydrophilic and lipo-
philic ends. Its use is limited due to the nephrotoxicity of
the drug. Despite this limitation, in low doses, colistin can
be used as an adjuvant because it acts as a membrane de-
tergent and so enhances the penetration of simultaneously
administered hydrophilic antibiotics, such as carbapenems,
glycopeptides, rifampicin and tetracyclines [66].
Some adjuvants also nullify the effect of the efflux
pump in exporting antibiotics from the bacterial cells. For
example, the primary mechanism of tetracycline resistance
is through its expulsion from bacterial cells by efflux. An
adjuvant that is structurally similar to tetracycline and
competes with tetracycline for binding to the efflux pumps
will reduce the efflux of active tetracycline [89].
This could suppress the development of resistance by
maintaining the effective intracellular concentration of the
antibiotic. Examples of derivatives that act as tetracycline
efflux inhibitors include the 6-(alkythio)methyl-
doxycycline analogues. These derivatives are more potent
inhibitors of Gram-negative bacterial growth compared to
Gram-positive bacteria. Therefore, the derivatives pro-
duce synergistic effects with tetracyclines in Gram-
negative but have an additive effect in Gram-positive bac-
teria where they support the activity of tetracycline by
competing for access to the efflux pump. Another exam-
ple of a compound which has the ability to inhibit efflux
pump is reserpine. Reserpine is a known mammalian
MDR pump inhibitor. It has the ability to suppress the
growth of S. aureus and Streptococcus pneumoniae
strains that have become resistant to ciprofloxacin [89].
Through a screening program for inhibitors of the efflux
pump, Phe-Arg-β-naphthylamine (PAβN), MC-207,110 ,
showed an ability to inhibit the 4 clinically relevant
pumps in P. Aeruginosa: MexCD-OprJ; MexAB-OprM;
MexEF-OprN and MexXYOprM, and also similar pumps
in other multidrug-resistant Gram-negative bacteria.
PAβN lowered the resistance of wild-type strains of
P.aeruginosa towards levofloxacin by eight-fold, while in
efflux pump overexpressing strains the resistance was
decreased by up to 6-fold [90].
Adjuvants may also disrupt biofilm stability. Bacteria
form biofilms to protect themselves from antimicrobial
agents and to overcome mechanical stress. Bacillus sub-
tilis produces a mixture of D-amino acids, preventing bio-
film formation as well as disassembling the existing bio-
film of other organisms. The mixture of D-amino acids
consists of D-leucine, D-tyrosine, D-methionine and D-
tryptophan. Treatment of biofilms with D-amino acids
causes the detachment of amyloid fibers that link bacterial
cells together in the biofilm. It is now known that D-
amino acids are released by many bacteria, and can be co-
Mechanisms of Antimicrobial Resistance (AMR) and Alternative Approaches
administrated therapeutically with broad range antibiotics
to disperse biofilms and regain antibiotic sensitivity.
Some adjuvants promote oxidative stress. Tellurium oxy-
anion tellurite (tellurite) causes high levels of oxidative
stress in bacterial cells. When tellurite is used at sub-
lethal concentrations, together with antibiotics, it inter-
feres with cell wall and protein synthesis, enhancing the
bactericidal action of the antibiotics by many folds. In addi-
tion to direct bactericidal activity, the generation of reactive
oxygen species results in damage to metabolic enzymes,
lipid peroxidation and glutathione depletion [66].
5.4. Nanotechnology for Targeted Drug Delivery to
Combat Antibiotic Resistance
Nanotechnology is the design and application of nano-
sized materials (1-100 nm). Nano-sized structures are
small in size, have a high surface-to-volume ratio and are
amenable to surface modification. They may be exploited
as a means of delivering therapeutic or diagnostic com-
pounds across physiological barriers, gaining access to
biological molecules and intended targets. Nanoparticles
may be used to combat microbial resistance and multi-
drug-resistant mutants. Nanoparticle-based materials have
been investigated in combating microbial resistance re-
cently, since they may facilitate the delivery and action of
conventional antibiotics at the site of the membrane [91].
It has been proposed that nanoparticles may have inherent
bactericidal action based on their physiochemical proper-
ties and also that the nanoparticles are able to prevent bio-
film formation [92]. Biofilm structure is one of the factors
that promote bacterial resistance by protecting the bacteria
from high antibiotic concentrations while, at the same
time, exposing the bacterial population to sufficient anti-
biotic to drive mutation of potential resistance genes,
among different bacterial cells. Studies have identified
gold-based, silver-based and magnesium-based nanoparti-
cles that prevent biofilm formation [93]. This activity is
achieved by modifying the shape and in size of the parti-
cles in order to maximize the surface-area-to-mass-ratio.
Nanoparticles may also be used as an antibiotic carrier
system. There are several types of nanoparticles that are
currently used for drug delivery, such as liposomal and
dendrimer nanoparticles and also carbon nanomaterials,
just to name a few. The advantages of nanoparticles for
drug delivery, in comparison with the conventional
approaches, are in terms of size, protection of the cargo
and precision in the site of delivery [93]. In particular,
nanoparticles are suitable to be used in the treatment of
intracellular bacterial infections. Antibiotics have poor
trans-membrane transport properties due to their large
molecular size, whereas nanoparticles can be loaded with
a drug and used to transfer the drug across a membrane.
The drug enters host cells through endocytosis and is re-
leased intracellularly to achieve a high intracellular con-
centration [93]. In addition, drugs incorporated into a na-
noparticle carrier are protected from chemical reactions
that may be detrimental to their activity, hence, the drugs’
potency can be maintained for a longer period of time.
Researchers have shown that nanoparticles can overcome
the resistance mechanisms of bacteria such as increased
Current Drug Discovery Technologies, 2019, Vol. 16, No. 0 11
efflux pump activity and decreased uptake in bacteria
such as P. aeruginosa and E. coli [94].
Nanoparticle-based targeted drug delivery is divided
into passive or active targeting. Passive targeting enhanc-
es the permeability and retention at the infection site,
whereas active targeting is directed by factors, such as
receptor interactions or and local activation temperatures.
Active targeting is achieved through modifying the sur-
face of nanoparticles which allow them to recognize spe-
cific ligands on the cells at the infection site. Vancomycin
has high efficacy in inhibiting Gram-positive bacteria.
However, vancomycin has high toxicity towards the ear
and kidney. Vancomycin mesoporous silica nanoparticles
make the specific killing of pathogenic Gram-positive
bacteria selectively over macrophage-like cells [95]. De-
spite the fact that nanoparticles can be beneficial as a drug
delivery system, they may, however, promote drug re-
sistance. This was identified in one study, whereby plas-
mids were transferred at a higher frequency between bac-
terial cells with nanoparticle treatment. It was assumed
that aluminum nanoparticles promoted the conjugative
transfer of plasmids and that this led to the spread of mul-
tidrug resistance between bacteria [96]. The three factors
that promoted this effect are probably: Cell membrane
damage due to oxidative stress caused by aluminum na-
noparticles, temperature and pH of water which affects
the transfer and lastly, the selectively promoted expres-
sion of specific genes that is crucial for replication and
transfer of plasmids. The high surface-to-volume ratio
increases the possibility of nano-materials interacting with
pathogens and membranes [97]. Therefore, nano-
technology has great potential uses for medical applica-
tions such as specific drug targeting, cell labeling and
gene therapy. Some examples are discussed below.
5.5. Chitosan
Chitosan is a linear polysaccharide, derived from chi-
tin. It interacts well with negatively charged microbial cell
walls and cytoplasmic membranes due to its polycationic
properties. This membrane interaction causes decreased
osmotic stability, subsequently leading to disruption of
the cellular membrane and eventually leakage of intracel-
lular contents. Nano-sized chitosan has a high surface area
to volume ratio. It is able to enter the bacterial cells and
the nuclei of fungal cells to inhibit mRNA and protein
synthesis when microbial DNA is bound by it. In addition,
the high surface area to volume ratio of chitosan
nanoparticles results in higher surface charge density, en-
hancing the affinity of chitosan for bacterial and fungal
cells and so conferring greater antimicrobial activity [97].
Wound infections are caused acutely by microorganisms
and, most chronic infection is accompanied by infections
with by multiple AMR bacteria. Nanoparticles have a
broad-spectrum antimicrobial activity that is able to inhib-
it the growth of different pathogenic bacteria. Nanosilver,
polyvinyl alcohol and chitosan combinations have been
studied for antiseptic activity, and an impregnated fiber
mat with a combination of these agents has been evaluat-
ed in wound healing studies [98].
12 Current Drug Discovery Technologies, 2019, Vol. 16, No. 0
5.6. Carbon Nano-tubes
Carbon nanotubes (CNTs) are new nano-vectors used
for the delivery of a variety of therapeutic molecules.
CNTs can be loaded with macromolecules, such as pro-
teins, oligosaccharides, proteins and antibiotics. Organi-
cally functional end groups and side walls of f-CNTs can
be covalently modified. Further modification can render f-
CNTs more soluble in a range of solvents, including wa-
ter, and the interaction between water-soluble carbon
nanotubes and mammalian cells leads to cytoplasmic
translocation of the nanotubes into mammalian cells [97].
5.7. Dendrimers
Dendrimers are highly branched polymers with low
polydispersity and defined nano-architecture. Their size
can be precisely controlled, and drug conjugation capabil-
ities are considerable due to the multiple branching points
in the structure. Dendrimers have a high surface function-
ality as their synthetic process takes place in a layer-by-
layer manner, around a central unit allowing for sequen-
tial modifications [99]. The highly branched characteris-
tics of dendrimers provide a large surface area to the ratio
for microorganisms to interact in vivo. The functional
groups achieve a high surface density and allow the den-
drimers to be synthesized with high binding affinity for a
wide variety of viral and bacterial receptors [100]. Hydro-
philic or hydrophobic drugs can be inserted into the empty
hollow in the central unit and on the multivalent surfaces
of the dendrimers. Increased efflux and decreased uptake
of antibiotics in bacterial cells are the most common
mechanism for AMR to develop. Studies showed that na-
noparticles are able to overcome the resistance mecha-
nisms and, hence, suppress drug resistance. For instance,
nanoparticle dendrimers inhibit the P-glycoprotein medi-
ated efflux in the gastrointestinal tract microflora [101]
5.8. Liposomes
Liposomes are spherical vesicles made up of one or
more phospholipid bilayers surrounding a water-filled
cavity. In order to change the physiochemical properties
of liposomes, the proportions and composition of different
lipids in the liposome formulation can be adjusted to mod-
ify the size of the liposome; surface charge; pH sensitivi-
ty; temperature sensitivity and liposomal membrane fluid-
ity [97]. The liposome vesicles can act as carriers with
hydrophilic or hydrophobic properties. Drug distribution
is highly influenced by the size of the liposomal vesicle,
and encapsulation of antibiotics in precisely designed li-
pid vesicles can achieve the optimal pharmacokinetic and
pharmacodynamic properties for delivery and activity
[101]. By encapsulating antibiotics in lipid vesicles, bio-
distribution and pharmacokinetics can be improved within
the host, toxicity is decreased, and activity against intra-
cellular and extracellular pathogens is enhanced, particu-
larly in helping to overcome antibiotic resistance. The
biodistribution of Klebsiella pneumoniae phage particles
incorporated into liposomes in the different organs of
BALB/c mice was assessed. From this study, it was
shown that liposome-entrapped phages were more stable
than free phages in the bloodstream and also in the organs
Moo et al.
of the body. It was concluded that liposome-entrapped
phages had longer bio-retentivity rates in the lungs and in
the kidney, making this approach an appropriate treatment
for respiratory tract and urinary tract infections caused by
K. pneumoniae [102].
5.9. Metal Ions
The properties of nanoparticles can be altered by
changing their size. This was shown in E. coli and S. au-
reus which are highly susceptible to CuO and ZnO nano-
particles of a specific size range. Since the size of bacteria
is usually in the micrometer range, the nanoparticles have
to have the ability to enter the bacterial cells. The activity
of nanoparticles also depends on their stability and con-
centration in the growth medium [103]
Synthetic CuO, ZnO and iron (III) oxide (Fe2O3) na-
noparticles were tested for bactericidal activity against E.
coli, S. aureus, B. subtilis,and P.aeruginosa in a well-
diffusion based assay. CuO and ZnO were more potent
than Fe2O3 nanoparticles, with ZnO having the greatest
bactericidal activity. In addition, the studies showed that
with a larger surface-area-to-volume ratio, the antibacteri-
al activity of the nanoparticles increased [104]. J.S. Möh-
ler et al., found that silver exhibited a synergistic interac-
tion with beta-lactam antibiotics. The structure and inter-
action between Zn(II), Ag(I), or Cu(II), and β-lactam an-
tibiotics were examined using isothermal titration calo-
rimetry (ITR) and nuclear magnetic resonance (NMR).
ITC and NMR revealed that the antibiotic activity was
increased in the presence of metal ions which could be
due to the inhibition of β-lactamase activity. Moreover,
kinetic assays conducted demonstrated that IMP-1enzyme
activity was inhibited in the presence of an Ag(I)-
cefuroxime complex. These findings indicated that the
synergistic interaction between Ag(I) and β-lactam antibi-
otics inhibited the β-lactamase activity, enabling the anti-
biotics to act on bacteria with greater efficacy [105]. Ex-
posing bacterial cells to silver causes puncturing of the
cell wall and consequently leakage of the cytoplasmic
contents. The bacteria become more susceptible to the
influx of silver ions, disrupting the production of cellular
protein and interfering with cellular metabolism. Eventu-
ally, cell growth is affected because silver ions cause ge-
netic disturbance and enzymatic inhibition [104].
Gold nanoparticles also exhibit antibacterial activity
by interfering with protein synthesis in prokaryotes. Col-
loidal gold reduced the affinity of the ribosome for tRNA
andgold nanoparticles also affected bacterial metabolism
by lowering the ATP synthase activity required for ATP
production. In the bacterial respiratory chain, gold nano-
particles suppressed nicotinamide adenine dinucleotide
(NADH) dehydrogenase activity by binding irreversibly
onto the thiol groups on NADH dehydrogenase. This
affects the reduction-oxidation balance in the cell which
results in the generation of oxidative stress [106]
5.10. Molecular Docking
Molecular docking is a computational tool that can be
used to discover drugs. This method assesses the affinity of
binding between two molecules, such as small molecules
Mechanisms of Antimicrobial Resistance (AMR) and Alternative Approaches Current Drug Discovery Technologies, 2019, Vol. 16, No. 0 13

Table 1. Examples of studies done using a few alternatives for combat of AMR.

Types of Therapy Strategy Outcome Reference

Tunicamycin synergized with oxacillin, lowering the mini-

Tunicamycin and oxacillin [66]
mal inhibitory concentration against S. aureus

Combinatory Therapy Plasmodium falciparum is resistant to chloroquine alone.

Artesunate/ artemether (artemisinin
However, the combination of an artemisinin derivative and
derivative) and amodiaquine/ meflo- [109]
amodiaquine/ mefloquine successfully inhibited the SER-
quine/ lumefantrine
CA-family Ca2+-ATPase PfATP6

The combination of cinnamon bark oil and meropenem had

successfully reduced the effective dosage of cinnamon bark
- Cinnamon bark oil and meropenem [110]
essential oil from 0.16% to 0.08% and meropenem from 32
µg/ml to 16 µg/ml when applied on K. pneumoniae.

Minimum inhibitory concentration (MIC) of peppermint

- Peppermint essential oil and meropenem
essential oil (PEO) was 8% v/v and meropenem was 4
µg/mL when used singly on KPC-3 producing E. coli
pMG309. In combinatorial treatment, PEO and meropenem [111]
reacted synergistically against KPC-3 producing E. coli
pMG309, significantly lowered the MIC of PEO to 1% v/v;
and meropenem to 0.5 µg/mL.

Combination of staphylococcal phage and antibiotics cured

Staphylococcal phage and antibiotics [112]
78 % of patients with sepsis
Bacteriophage Therapy Phage application decreased the bacterial titre in all patients'
Phage preparations against secondary
sputum specimens and the general health of patients im- [112]
infections in cystic fibrosis patients
proved.

Celecoxib is a non-steroidal drug used to treat arthritis by

inhibiting cyclooxygenase-2 (COX-2). Suppression of drug
resistance has been shown in cancers because of the inhibi-
tion of the MDR1 efflux pump. It has also been determined
Celecoxib [113]
to increase the sensitivity of S. aureus to several antibiotics,
such as ciproflaxin, chloramphenicol, ampicillin ciproflaxin
Antimicrobial Adjuvants and kannamycin by causing the bacteria to accumulate
drugs inside their cells.

Chitosan enhanced the antimicrobial efficacy of silver-

loaded membranes up to 70% which resulted in larger zones
Chitosan of inhibition on E. coli and S. Aureus cultures. Antimicrobi- [114]
al activity was enhanced by 70 % by increasing the chitosan
concentrations,

Liposomes were used as antibiotic carrier systems to coun-

ter extracellular pathogens. Studies found that with proper
lipid formulations, drug distribution, and vesicle-bacterium
Liposome [115]
interactions, antimicrobial activity against bacteria such as
Nano-particles E. coli, P. aeruginosa, Acinetobacter sp., K. pneumoniae,
`and S. aureus can be enhanced.

The uptake of silver ions and the resulting cascade of intra-

Silver cellular reactions in teicoplanin resistant S. Pneumoniae [116]
overcame resistance.

Colloidal gold caused damage to DNA and the bacterial cell

Gold [117]
wall in cefotaxime resistant E. coli and K. pneumoniae

Predicted the binding mechanism of S-1360, one of the

Molecular Docking Docking studies first beta-diketoacid integrase inhibitors to enter clinical [107]
studies.
14 Current Drug Discovery Technologies, 2019, Vol. 16, No. 0
and protein targets to identify potential inhibitory com-
pounds based on the binding affinity [107]. This method
can maximize the chances of success in producing new
antibiotics which reduces the cost of developing new an-
tibiotics empirically. Ligands can be developed with dif-
ferent degrees of selectivity. For instance, if the binding
sites of the bacterial target and human protein are availa-
ble, they can be superimposed in order to identify features
that are distinctive to the bacterial binding site. This can
be used to favour the ligand toward the bacterial target
and minimize the side effects of the antibiotic. Further-
more, resistance in bacteria sometimes arises from the
mutation of the bacterial target that results in weakened
affinity for the antibiotic. In such a situation, ligands that
target both the mutant and wild type binding sites can be
developed by focusing on conserved structures in the
binding sites of the two targets [108].
Studies undertaken using each of the alternatives de-
scribed with examples are summarized in Table 1.
CONCLUSION
Focused efforts and sound research are needed to over-
come the challenges that will be posed by the continuous
advancement of AMR. Since the development of new anti-
microbials takes time, in the immediate future, it is more
practical to focus on improving and augmenting existing
antimicrobials. Important strategies include combination
therapies which help to circumvent the problems that cause
existing antimicrobials to fail, in effect, by supplementing
them with the missing link in their effective lytic action. Re-
search also needs to be streamlined on understanding the
toxicity of potential adjuvant therapies in animal and human
cells. The mechanisms by which resistance to common anti-
biotics is developed needs to be fully elucidated, so that ap-
proaches to overcoming resistance can be designed. The
primary challenge of combination therapy is the ability to
develop customized techniques that are optimized for the
isolation and purification of safer and newer natural antimi-
crobials against MDR, as well as having a better understand-
ing of the structure, mechanism and function of AMPs,
which will lead to better design to mitigate the rise in MDR
pathogens [118]. Moreover, a number of antibiotics have
been put to one side due to their high intrinsic resistance
rates such as ADEPs. By incorporating these antibiotics in
combination therapy strategies, new therapies with novel
resistance profiles that do not overlap, could be introduced
into clinical use. The main challenges that will be encoun-
tered are the specific toxicity, pharmacokinetic and pharma-
codynamic properties that arise out of combination therapies
that are not seen when the same drugs are used in monother-
apy [105]. Another key question relevant to combination
therapy is whether there is a way to identify molecules that
can act as specific adjuvants for individual antimicrobial
agents. Furthermore, can adjuvants which do not possess
any intrinsic antimicrobial activity not only enhance the
effect of co-administered antibiotic but also prevent the
emergence of resistance? There may be any such adjuvants
which have yet to be identified; some phages have greater
diversity in their mechanisms of action in comparison with
other antibacterial agents, and. phage therapy shows increas-
ing promise in the treatment of infections. Over an extended
Moo et al.
period of time, disturbance of the human microbiome due to
the use of broad-spectrum antibacterial agents may result in a
drive for the use of phage therapy as an alternative narrow-
spectrum antibacterial treatment. In such a situation, it will
be important to design a cocktail of phages that can be made
specifically for each bacterial species or even strain, so that
only phages that are effective against the bacterial strains
responsible for the infection will be used.
In addition to combination therapy and phage therapy,
there is also increasing interest in nano-technology for medi-
cal applications such as specific drug targeting, cell labeling
and gene therapy. However, the full spectrum of the antibac-
terial mechanisms elicited by nanoparticles (NPs) is still un-
certain and requires further research. For example, antibacte-
rial activity is attributed to oxidative stress, this may not be
the case for all nanoparticle formulations and some of the
antibacterial mechanism may not be related to oxidative
stress. It is challenging to study the antibacterial actions of
nanoparticles as there is no single analytical method which
fulfills the criterias for obtaining all the necessary infor-
mation. Experience with chemotherapeutic drugs to treat
cancer has taught us that, in vitro studies seldom mimic in
vivo conditions. Thus, it is impossible to anticipate the spe-
cific antibacterial action of NPs by depending on in vitro
bacterial cell culture alone. The mechanism and safety of
membrane crossing NPs have yet to be fully studied. Moving
forward, researchers have proposed that endocytosis by bac-
teria, which resembles what is observed in eukaryotic cells,
could be a mechanical movement of the nanoparticles into
bacterial cells. Nevertheless, no findings have yet been re-
ported regarding this topic. The current most reasonable
mechanism is that bacteria cells exposed to the lower con-
centration of nanoparticles experience some damage to their
structure including the removal of the LPS layer. With the
LPS removed, the inner cell membrane protrudes in the form
of vesicular structures from the cell surface. The vesicles
bind to nanoparticles that later enter the cell by electrostatic
attraction [93]. Furthermore, it is also worth addressing the
intracellular inhibitory mechanisms. Some studies have also
considered the action of nanoparticles on gene expression,
metabolism of the bacterial cells and protein synthesis.
However, studies addressing this issue remain limited.
Although there are still many unanswered questions and
hurdles to be overcome in tackling AMR, the future pro-
spects for these approaches (combination therapy, phage
therapy nanotechnology and docking studies) for targeted
drug delivery seem promising. Combination therapy presents
several advantages compared to the development of new
antibiotics, such as decreasing the chance of developing re-
sistance that may assist us in the development of novel and
effective biomarkers [119], although there are issues with
drug-drug interactions. In phage therapy, the manipulation of
phages to overcome pathogens is not as simple as it seems
and it is unlikely that there is a definitive approach that is
able to treat all bacterial infections. Nanoparticle develop-
ment is another viable alternative to antibiotics which seems
to have high potential to solve the problems encountered in
using conventional antibiotics such as the emergence of
multidrug-resistant bacteria. In-depth studies of how nano-
particles exhibit their antibacterial actions could contribute to
developing effectual antibacterial nanoparticles and prevent
Mechanisms of Antimicrobial Resistance (AMR) and Alternative Approaches
nanoparticle cytotoxicity. Computational methods have the
capacity to enable the design of new antibiotics to combat
resistant organisms. In order to maximize its potential, this
approach needs to be integrated into systems biology
modelling where potential off-target effects on non-
microbial processes can also be identified at an early stage.
Modelling the potential for resistance to lead compounds
allows for compounds with estimated high resistance poten-
tial to be excluded from investigation at earlier stages in or-
der to focus resources more efficiently[108]. To effectively
overcome the challenges of AMR, antimicrobial compounds
with new mechanisms of action need to be discovered by the
scientific community and only through methodical experi-
mental design and perseverance can we fully exploit the po-
tential of the existing approaches to AMR described in this
review.
CONSENT FOR PUBLICATION
Not applicable.
CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or
otherwise.
Acknowledgements
This work was funded by the Fundamental Research
Grant Scheme (FRGS/1/2018/SKK11/PERDANA/02/1) by
the Ministry of Education Malaysia and Malaysia Medical
Association (MMA). The authors would like to thank all the
members of Floral Biotechnology Laboratory, UPM. C.L.M
wrote the review with assistance from S.K.Y. K.S.L,
S.H.E.L, K. Y, M.A and A. A. W. T. and S.H.E.L signifi-
cantly refined the manuscript. All authors read and ap-
proved the final manuscript.
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