IBEROAMERICAN JOURNAL OF MEDICINE 01 (2022) 60-74
Review
IBEROAMERICAN
JOURNAL OF
MEDICINE
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Reducing bacterial antibiotic resistance by targeting bacterial
metabolic pathways and disrupting RND efflux pump activity
Tatiana Hillman %*\®
“Independent Research, USA
ARTICLE INFO
ABSTRACT
Article history:
Received 04 November 2021
Received in revised form 15
December 2021
Accepted 02 January 2022
Keywords:
Efflux pump
Antibiotic resistance
Bacterial metabolic pathways
Antibiotic resistance is a significant issue for the medical community, worldwide. Many
bacteria develop drug resistance by utilizing multidrug resistant or MDR efflux pumps that can
export antibiotics from bacterial cells. Antibiotics are expelled from bacteria by efflux pumps a
part of the resistance nodulation division (RND) family. Types of RND efflux pumps include the
AcrAB-TolC tripartite protein pump. There are an excessive number of antibiotic compounds
that have been discovered; however, only a few antibiotics are effective against MDR bacteria.
Many bacteria become drug resistant when sharing genes that encode MDR efflux pump
expression. MDR efflux pump encoding genes are incorporated into plasmids and then shared
among bacteria. As a consequence, advancements in genetic engineering can sufficiently target
and edit pathogenic bacterial genomes for perturbing drug resistance mechanisms. In this
perspective and review, support will be provided for utilizing genetic modifications as an
antimicrobial approach and tool that may effectively combat bacterial MDR. Ayhan et al. found
that deleting acrB, acrA, and tolC increased the levels of antibiotic sensitivity in Escherichia coli.
Researchers also found that glucose, glutamate, and fructose all induced the absorption of
antibiotics by upregulating the gene expression of maeA and maeB that is a part of the MAL-
pyruvate pathway. Therefore, the current perspective and review will discuss the potential
efficacy of reducing antibiotic resistance by inhibiting genes that encode efflux protein pump
expression while simultaneously upregulating metabolic genes for increased antibiotic uptake.
© 2022 The Authors. Published by Iberoamerican Journal of Medicine. This is an open access article under
the CC BY license (http://creativecommons. org/licenses/by/4.0/).
* Corresponding author.
E-mail address: thillman @thel4binc.org
ISSN: 2695-5075 / © 2022 The Authors. Published by Iberoamerican Journal of Medicine. This is an open access article under the CC BY license
(http://creativecommons. org/licenses/by/4.0/).
https://doi.org/10.53986/ibjm.2022.0008
IBEROAMERICAN JOURNAL OF MEDICINE 01 (2022) 60-74
61
Reducir la resistencia a los antibidticos bacterianos al dirigirse a las vias metabolicas
bacterianas e interrumpir la actividad de la bomba de salida de RND
INFO. ARTICULO RESUMEN
Historia del articulo:
Recibido 04 Noviembre 2021
Recibido en forma revisada 15
Diciembre 2021
Aceptado 02 Enero 2022
La resistencia a los antibidticos es un problema importante para la comunidad médica en todo
el mundo. Muchas bacterias desarrollan resistencia a los farmacos mediante el uso de bombas
de eflujo MDR o resistentes a multiples farmacos que pueden exportar antibidticos de las
células bacterianas. Los antibidticos se expulsan de las bacterias mediante bombas de eflujo
que forman parte de la familia de la divisidn de nodulacion de resistencia (RND). Los tipos de
bombas de eflujo RND incluyen la bomba de proteinas tripartita AcrAB-TolC. Hay un nimero
Palabras clave:
Bomba de flujo
Resistencia antibidtica
Vias metabdlicas bacterianas
excesivo de compuestos antibidticos que se han descubierto; sin embargo, solo unos pocos
antibidticos son eficaces contra la bacteria MDR. Muchas bacterias se vuelven resistentes a los
farmacos cuando comparten genes que codifican la expresion de la bomba de eflujo MDR. Los
genes que codifican la bomba de eflujo MDR se incorporan a los plasmidos y luego se
comparten entre las bacterias. Como consecuencia, los avances en la ingenierfa genética
pueden apuntar y editar suficientemente los genomas bacterianos patégenos para perturbar
los mecanismos de resistencia a los medicamentos. En esta perspectiva y revision, se brindara
apoyo para utilizar modificaciones genéticas como un enfoque y una _ herramienta
antimicrobianos que pueden combatir eficazmente la MDR bacteriana. Ayhan y col.
encontraron que la eliminacion de acrB, acrA y tolC aumentaba los niveles de sensibilidad alos
antibidticos en Escherichia coli. Los investigadores también encontraron que la glucosa, el
glutamato y la fructosa inducfan la absorcién de antibidticos al regular al alza la expresion
génica de maeA y maeB que es parte de la via MAL-piruvato. Por lo tanto, la perspectiva actual
y la revision discutiran la eficacia potencial de reducir la resistencia a los antibioticos al inhibir
los genes que codifican la expresion de la bomba de proteinas de salida y, al mismo tiempo,
regular al alza los genes metab6licos para una mayor absorci6on de antibidticos.
© 2022 Los Autores. Publicado por Iberoamerican Journal of Medicine. Este es un articulo en acceso abierto
bajo licencia CC BY (http://creativecommons. org/licenses/by/4.0/).
HOWTO CITE THIS ARTICLE: Hillman T. Reducing bacterial antibiotic resistance by targeting bacterial metabolic pathways and
disrupting RND efflux pump activity. Iberoam J Med. 2022;4(1):60-74. doi: 10.53986/ibjm.2022.0008.
1. INTRODUCTION
Multiple drug resistant (MDR) bacteria present an
enormous challenge for the medical community, in
which bacteria develop resistance against more than one
antibiotic drug agent. The World Economic Forum
Global Risks present data uncovers the greatest growing
danger to our health, currently, is antibiotic resistance
[1]. The World Health Organization (WHO) and the
Centers for Disease Control (CDC) have communicated
their immense concerns for the rising rate of antibiotic
resistance [2]. In the 2019 Centers of Disease Control
and Prevention Antibiotic Resistant Threats Report, it
was confirmed that antimicrobial resistant pathogens
caused 2.8 million infections with more than 35,000
deaths each year in the United states between the years
of 2012 and 2017 [3]. Infections due to antibiotic
resistance cause approximately 600 deaths annually
from the exposure to bacterial pathogens in many
healthcare centers such as hospitals [4]. However,
multiple drug resistant (MDR) Gram-negative bacteria
cause a majority of bacterial infections. The Centers for
Disease Control have confirmed that Acinetobacter
baumanii, a Gram-negative bacterium, causes a majority
of hospital infections [5].
hospitals and are highly contagious pathogenic bacteria.
Gram-negative bacteria have a higher rate of antibiotic
A. baumanii resides in
resistance because of the Gram-negative bacterial outer
membrane that acts as a natural physiological barrier of
resistance against antibiotics. Gram-negative bacteria
have a cell envelope that consists of an outer membrane,
a periplasmic space, and an inner membrane. The three
layers of the cell envelope allows Gram-negative
bacteria to resist a broader range of antimicrobial drug
agents and compounds. For instance, any changes in the
outer membrane of Gram-negative bacteria, such as
altering its hydrophobic components or mutations of
genes required for the protein expression of porins, can
develop into resistance [6]. Gram-negative bacteria also
62 IBEROAMERICAN JOURNAL OF MEDICINE 01 (2022) 60-74
have many additional mechanisms of resistance, such as
DNA mutations of antimicrobial targets; degrading
antimicrobials and modifying antimicrobials; acquiring
genetic components such as plasmids, transposons or
integrons; altering the components of the cell wall and
modifying lipopolysaccharides; decreasing _ the
expression of porins, which reduces the uptake of
antimicrobials; and the overexpression of efflux pumps
[2]. Therefore, selecting effective antibiotics to treat
bacterial infections, generated by resistant and
pathogenic bacteria, has been immensely challenging
[3].
Treating MDR bacterial infections is arduously
challenging because many MDR bacteria overexpress
efflux pumps [2]. Overexpression of multiple drug
resistant efflux pumps or MDR efflux pumps allows the
massive export of a broad range of antibiotic compounds
[2]. Gram-negative bacteria utilize all the classified
families of efflux pumps, such as the ABC superfamily;
the major facilitator superfamily (MFS); the multidrop
and TolC compound extrusion (MATE) family; the
small multidrug resistance pumps (SMR) family and the
Resistance Nodulation Division (RND) family [2].
However, Gram-negative bacteria mainly use RND
efflux pumps for MDR efflux activity [Anes]. RND
pumps are accountable for the expulsion of a broad range
of agents and compounds. RND pumps_ include
homotrimers termed as the AcrAB-TolC efflux pump
[7]. The AcrAB-TolC subunits are transmembrane
proteins that are oriented into the inner membrane, outer
membrane, and through the periplasmic space. Its
structure is a triplex with protein subunits termed AcrA
located in the periplasmic space, the TolC is oriented in
the bacterial outer membrane, and AcrB resides in the
inner membrane region of a bacterial cell [7] (Figure 1).
The binding sites of AcrAB-TolC, a homotrimer efflux
pump, can bind to and attach to many antibiotics with
different sizes and chemical properties [8]. MDR efflux
pumps can expel a wide range of antibiotics, which
contributes to the continued proliferation of MDR
Gram-negative bacteria.
In contrast, manipulating the metabolism of bacteria can
increase the uptake of antibiotics. Metabolites in high
concentrations increase bacterial antibiotic resistance.
Metabolites such as indole, ammonia, nitric oxide, and
hydrogen sulfide can amplify antibiotic resistance [9].
However, carbon in higher amounts eradicates more
pathogenic bacteria by increasing antibiotic sensitivity.
The high influx of carbon from the environment
surrounding bacteria controls antibiotic sensitivity by
ANTIOBIOTIC EFFLUX PUMP
PERIPLASM
INNER
MEMBRANE
® DRUG
CYTOPLASM
Figure 1: The AcrAB-TolC Efflux Pump, RND antibiotic
efflux pump, ex-ports antibiotic drugs from the cytoplasmic
space of a bacterial cell, which traverses through the inner
membrane, into the periplasm, and then exits the bacterium’s
outer membrane via the AcrB, AcrA, and TolC subunits of
AcrAB-TolC efflux pump, respectively. AcrB is located in the
inner membrane and periplasm while AcrA is implanted in
the periplasm. TolC is oriented into the periplasm and Outer
membrane. The presence of antibiotics triggers the AcrAB
complex to alter its conformation in order to retrieve and
recruit the TolC subunit. The opening of TolC remains closed
until the antibiotic expulsion in order to isolate the periplasm
from the external environment. Lastly, the fully assembled
AcrAB-TolC pump contracts and opens to expel the antibiotic
drug through its chamber. The AcrAB-TolC then closes
following the expulsion of the antibiotic drug molecule.
RND: resistance nodulation division.
raising nicotinamide adenine dinucleotide (NAD) +
hydrogen (H) or NADH activity and increasing the
proton motive force (PMF) [10]. The rise of NADH
levels and the rapid activity of PMF can both activate the
TCA cycle of bacterial carbon-dependent metabolism
[10]. Upregulating the expression of proteins as a part of
bacterial metabolic pathways can amplify the rate of the
PMF, which can intensify antibiotic sensitivity. PMF
also powers the efflux activity of many RND pumps. By
inhibiting or deleting the genes that code for the
expression of efflux pump proteins, the PMF can
decrease, thereby disrupting efflux activity.
Although many novel antibiotics have been developed
to treat resistant bacterial infections, many pathogenic
bacterial strains persistently resist many of these novel
agents [3]. Designing novel and alternative antibiotics
that are gene-based could combine novel genetic
IBEROAMERICAN JOURNAL OF MEDICINE 01 (2022) 60-74
63
engineering techniques with antibiotic development to
combat antibiotic resistance. However, to overcome the
antibiotic resistance caused by drug efflux, the current
literature available needs a more succinct elucidation of
the fundamentals of drug efflux mechanisms [2]. An
understanding of drug efflux mechanisms, such as the
regulation and response to antimicrobials, is urgently
required for combating antibiotic resistance [2]. For
these reasons, this review attempts to accurately describe
the drug efflux mechanisms of the RND AcrAB-TolC
efflux pump through a genomic and metabolomic
perspective of antibiotic efflux and influx. This review
and perspective will discuss the reduction of antibiotic
resistance by inhibiting drug efflux protein pump
expression and stimulating bacterial metabolic activity
to increase antibiotic influx. The overall purpose of this
review is to present an alternative gene-based
antimicrobial strategy against resistant bacteria that may
increase the antibiotic retention in bacterial cells by
inhibiting antibiotic efflux and metabolically amplifying
antibiotic influx.
2. BACTERIAL ANTIBIOTIC RESISTANCE
Bacteria can adapt well too many _ different
environmental conditions partially because of its ability
to select advantageous DNA mutations that allows
bacterial cells to survive, reproduce, and dominate its
population. Antibiotic genes
transferred between bacterial cells and limit the bacterial
resistance can be
susceptibility of antibiotics. Bacteria become multidrug
resistant through
resistance genes that are transferred by plasmids or
transposons [11]. Each resistant gene may code for the
inhibition of a drug agent or encode multidrug efflux
pump activity [11]. Bacteria develop
resistance by the transference of antibiotic resistance
genes through a horizontal gene transfer [12]. Bacteria
have three modes of horizontal gene transfer such as
the accumulation of antibiotic
antibiotic
conjugation sexual
transformation
(bacterial reproduction),
(incorporating naked DNA), and
transduction (mediated by phages) [13]. Bacterial cells
receive antibiotic resistant genes and after gene
expression the diffusion of antibiotics through bacterial
cell pores is prevented, causing antibiotic efflux; the
antibiotics’ binding to antimicrobial target sites is
blocked; the antibiotics are targeted for hydrolysis and
inactivation; and changes in metabolic pathways can
occur and alter regulatory networks [13].
The addition of antibiotics to a bactertum’s environment
forces the bacterium to select gene mutations that give it
an advantage. Bacteria can acquire these DNA
mutations, which allows the bacterial cells to continue
reproducing in the presence of antibiotic drugs. Bacteria
carrying advantageous DNA mutations of antibiotic
resistance can reproduce without much competition
from the dead bacteria that lacked the advantageous
mutations. After the exposure to bactericide antibiotics,
the population of bacteria that survived are called
persister cells. The persister cells not only survive but
also are key determinants for further initiation of
antibiotic resistance [14]. Consequently,
selection is the central component and the driving force
for the development of antibiotic resistance in bacteria
[15] where exposure to antibiotics impels the selection
of antibiotic resistance genes that then become rapidly
disseminated between many bacterial cells.
Pathogenic bacteria can become resistant to antibiotics
through acquiring antibiotic resistant genes that code for
degrading antibiotics, using efflux pumps, and shielding
natural
targets from binding to antibiotics. For example, bacteria
can acquire DNA from its environment and transfer
antibiotic resistant genes called ‘mosaic’ genes [16].
Streptococcus develops
resistance to penicillin by sharing mosaic penicillin-
binding protein genes or pbp genes between bacteria
[17]. The mutations in pbp genes alter proteins such as
enzymes to exhibit less affinity for penicillin. The
mosaic pbp genes originated from the continued mixture
and rearrangements of DNA by the bacteria named
pneumoniae antibiotic
Streptococcus mitis [18]. The influx of a class of
antibiotics known as cefiximes, which are third-
generation cephalosporins, has also been blocked after
mosaicism and point mutations in penA genes that code
for Pbp expression in N. gonorrhoeae [19]. Additionally,
A. baumanii becomes antibiotic resistant by obtaining
resistant genes from conjugation, which is mostly a
horizontal transfer of genes, and via mutations in its
DNA. The enzymes of acetyltransferase and the
nucleotidyltransferases modify the antibiotics called
aminoglycosides that form resistant A. baumannii
bacterial strains [5]. A. baumanii inserts mutations into
the genes gyrA and parC. Then, enzymes become
overexpressed to alter the antibiotic target called 16S
rRNA and prevents the aminoglycoside to bind to 16S
rRNA, inhibiting 16S rRNA activity. Another example
of antibiotic tolerance includes propionate, a volatile
fatty acid (VFA), which may become toxic for
Escherichia coli cells, yet E. coli can develop tolerance
of propionate by utilizing propionate as a carbon source
64 IBEROAMERICAN JOURNAL OF MEDICINE 01 (2022) 60-74
for generating energy. The volatile fatty acid or VFA
called propionate can inhibit many metabolic processes
when the VFAs amass in high concentrations within a
bacterial cell and can assist with eliminating antibiotic
resistant bacteria [20]. However, E. coli cells modify the
mRNA transcripts of genes needed for metabolizing
propionate, and then the anaerobic metabolism of
propionate is amplified.
The bacterial antibiotic resistance of quinolone is an
example of antibiotic resistance caused by the
destruction of antibiotics and through blocking the
binding of antibiotics to bacterial targets. Quinolone can
effectively block the expulsion of antibiotic drugs from
bacterial cells, and copies of quinolone antibiotics have
been derived to inhibit the efflux pump called NorA in
S. aureus [21-30]. However, the resistance to quinolone
antibiotic drugs is propagated through the gene
expression of gnr. The gnr mRNA transcript is
translated into QnR proteins that consist of pentapeptide
repeat proteins or PRPs [10]. The PRPs binding to DNA
gyrase and to topoisomerase IV restricts the quinolones
inhibition of DNA gyrase and topoisomerase IV. When
the quinolone antibiotic attempts to attach, the PRPs
begin to bind to the DNA and inhibits the quinolones’
binding to the enzyme-to-DNA interfaces of
topoisomerase IV and of the DNA gyrase [31]. The
PRPs prevent the quinolones from converting DNA
gyrase and the topoisomerase IV into toxic enzymes that
degrade bacterial chromosomes, allowing the
topoisomerases to continue completing the activities of
rebuilding dsDNA breaches, eliminating knots in
chromosomes, and relaxing torsional stress [31].
Furthermore, many bacterial enzymes can also
hydrolyze and destroy antibiotics, such as Beta-lactams,
aminoglycosides, phenicols, and macrolides. Therefore,
the continued overuse of antibiotics will only increase
the proliferation of resistant bacterial strains [32]. For
these reasons, elucidating the functions that establish
and proliferate antibiotic resistant bacteria is required for
advancing the pharmaceutical design of novel antibiotics
that can accurately target antibiotic resistant genes and
the cellular properties of drug resistant bacteria.
3. COMBATING BACTERIAL ANTIBIOTIC
RESISTANCE
Additional alternative strategies that can effectively
combat antibiotic resistance are still presently and
urgently needed. A possible alternative method to
combat antibiotic resistance was developed by Ayhan et
al. that constructed antisense RNA (asRNA) molecules
attached to the mRNA transcripts of antibiotic resistant
genes. Ayhan et al. designed phosphorodiamidate
morpholino rings connected by a phosphorodiamidate
(PMO) backbone [33]. Each ring of the PMO was bound
to an antisense nucleotide base and blocked the
translation of antibiotic resistant genes via steric
hindrance [33]. The site of steric hindrance was tightly
adjacent to the ribosome binding sequence of the
bacteria that obstructed the entrance of bacterial mRNA
into ribosomes. Adding peptides to the Peptide-
Conjugated Phosphorodiamidate Morpholino Oligomer
or PPMOs enhanced its entrance into bacterial cells
during conjugation [33]. The phosphorodiamidate, a
PMO, backbone impedes the nuclease activity that
would disassemble the PPMO. PPMOs present a few
issues, which need more research in the area of
pharmacokinetics for effectively combining PPMOs
with antibiotics [33]. There are issues of mismatched
base pairing, determining the position and size of the
oligomer, and the effectiveness of PPMOs requires
further examination.
Additionally, targeting quorum sense signalling between
bacterial cells could provide another highly effective
method for reducing the antibiotic resistance of
Salmonella typhimurium. When comparing mutant
Salmonella typhimurium versus its wild type, the wild-
type showed activation of secondary molecules that
continued the expression of antibiotic resistant genes
under stress and increased pressure. However, the
mutant form was not as regulated and did not activate
secondary molecules. S. Typhimurim passes through the
M cells of the epithelial lining of the intestines, leading
to a more systemic infection. The systemic infection
caused by S. Typhimurium depends on the virulent
genomic region called the S. Typhimurium pathogenicity
island or SPI-1 and SPI-4. The SPI-1 and SPI-4 are less
regulated in its mutant form, which lessens the
frequency of bacteria crossing the M cells of the
gastrointestinal tract (GIT) [34]. SPI-2 acts to fortify
bacterial cells to evade phagocytosis by macrophages. A
mutated gene that encodes the PhoPQ protein decreases
expression of the LsrACDB protein complex, which
then decreases quorum sensing (QS). When
upregulating the gene expression of SPI-1, the gene
expression of JsrACDB gradually increases [34]. The
quorum sense signaling of a S$. Typhimurium infection is
completely eradicated after mutation of the gene that
encodes the PhoPQ protein. A possible strategy could
include an attenuated S. Typhimurium can become re-
IBEROAMERICAN JOURNAL OF MEDICINE 01 (2022) 60-74
65
engineered by adding a QS detecting promoter site with
a small interfering RNA (siRNA) or asRNA sequences
of IsrACDB to a plasmid vector. The plasmid vector
would consist of a pluxR promoter site to detect QS
molecules and then release the small interfering RNAs
(siRNAs) or asRNA for blocking the transcription of
IsrACDB where LsrACDB expression can be reduced,
interrupting the quorum sense signalling between S.
Typhimurium bacterial cells.
The phytonutrients of plants can potentially reduce
antibiotic resistance. Possible alternatives to reduce
antibiotic resistance may include the use of plants that
release cytotoxic phytonutrients and provide immunity
against foreign bacterial cells. For example, reserpine is
a plant alkaloid extracted from the roots of the petitiva
plant. Reserpine inhibits the efflux pump of Gram-
positive bacteria known as Bacillus subtilis. Reserpine
binds to the pumps that express three amino acids with
the side chain residue groups of phenylalanine and
valine. Reserpine causes a 4-fold decrease in the
antibiotic resistance of methicillin-resistant
Staphylococcus aureus or MRSA caused by S. Aureus.
Berberine has a MIC of 256 mg/L, but when the NorA
pump is active, the berberine MIC is reduced to 16 mg/L
[4]. Other examples of plant phytonutrients include
polyphenol blockers found in green tea extracts that can
inhibit MRSA resistance. Adding 20 mg/L of
polyphenols to the antibiotics norfloxacin resulted in a
4-fold decrease in MIC [4]. However, there are not many
studies that focus on the medicinal properties and
benefits of plant alkaloids [4].
Furthermore, novel synthetic antibiotics have also been
developed to decrease antibiotic resistance. Novel
antibiotics such as capistruin and malleilactone block
bacterial cell transformation. Capistruin blocks bacterial
RNA polymerase activity. Malleilactone interacts with
quorum sensing molecules. However, Staphylococcus
aureus has a lysine at position 164, resulting in antibiotic
resistance of thailandamide [35]. The antibiotic of
bactobulin can connect to the LuxI-LuxR proteins
responsible for quorum sensing [35-38]. The LuxI-R
linkage to bactobulin stimulates a molecule that inhibits
bacterial translation in the ribosome. In addition, a list of
criteria and properties are needed for establishing a
database to help identify and examine the environment
for highly pathogenic resistant bacterial types [37-48].
Additional procedures that avoid and prevent bacterial
antibiotic resistance include the following: regulate the
antibiotics given to animals, perform future research that
elucidates the mechanisms of antibiotic resistance and
the monitoring of resistance [49], improve the laws and
regulations to reduce the prevalence of antibiotic
resistance, amplify the research of more novel therapies
[50], and provide more aid to developing and low-
income countries to more effectively regulate issues of
antibiotic resistance [50]. Currently, WHO has an
antimicrobial surveillance system that collects data to
help reduce the rate of antibiotic resistance [CHokshi].
Reducing the use of plastic items may also block the
interaction between microplastics and antibiotic
resistance genes in aquatic environments [51]. However,
Hall et al. concluded that antibiotic resistance originates
from a genetic source [52]; therefore, the solution for
eliminating multidrug resistant bacteria may require a
gene-based approach. Modifying the genes that encode
the binding sites of MDR efflux pumps such as the RND
AcrAB-ToIC efflux pumps can limit efflux activity and
reduce antibiotic resistance.
RESISTANCE
Many genes coding for the protein synthesis of RND
pumps can be shared between bacterial cells through
conjugation [53]. The mutations of genes encoding for
increased efflux activity are found in the regulatory
portion of DNA sequences that modulate the translation
and synthesis of efflux pump complexes. The expression
of efflux pumps on the bacterial cell surface is altered by
the increased presence of gene mutations in an adjacent
promoter site. Many DNA mutations in an adjacent
promoter site can affect the production of transcription
factors that control the cell surface expression of efflux
pumps. For example, a point mutation approximately 10
base pairs upstream of the mtrC gene of Neisseria
gonorrhoeae increases the amount of activity at the
promoter, resulting in amplified expression of efflux
pumps to rapidly remove antibiotic drugs [54]. Genes
encoding the expression of MDR efflux pumps can also
become incorporated into plasmids and then transferred
among bacteria. For example, Resistance Nodulation
Division (RND) pumps are transported via IncH1
plasmids isolated from the Citrobacter freundii bacteria,
which transmits the New Delhi metallo-Beta-lactamase
1 or NDM1-34 for enzymatic decay of antibiotic drugs
[55].
As a result, MDR efflux is responsible for amplified
antibiotic resistance, which exports antibiotics from
bacteria. The antibiotics are pumped and exported from
the cytoplasm of the bacteria. The AcrAB-ToIC efflux
4. RND-EFFLUX PUMPS AND ANTIBIOTC-
66 IBEROAMERICAN JOURNAL OF MEDICINE 01 (2022) 60-74
pumps are a part of the RND family of efflux pumps and
consist of three protein subunits. The three subunits
include AcrA, the mid-transmembrane part of the pump,
AcrB located in the bacterial inner membrane, and TolC
is positioned in the region of the outer membrane (Figure
1). The AcrAB-TolC pump is assembled in a sequential
order [7]. The first step of the AcrAB-TolC assembly
includes the interaction between AcrB and AcrA, which
then forms the AcrAB bipartite complex [7]. Secondly,
the conformation of AcrA changes to then engage and
recruit TolC [7]. When the TolC becomes bound to the
AcrAB complex, the completely assembled AcrAB-
TolC pump enters and remains in a resting state. Thirdly,
when AcrB engages a drug, the fully assembled pump
opens its conformation and then a constraction expels
the drug from the channel into the external environment
of the cell [7]. Through observation of tomography
results and data collected in vivo, it was found that AcrB
and AcrA interact to form into a bipartite complex [7].
The AcrAB bipartite complex stabilizes the AcrAB-
TolC efflux pump’s orientation into the bacterial cell
envelope [7].
The membrane fusion protein of AcrA connects the
pump of the inner membrane to the pump located in the
external lipid membrane region where the TolC channel
resides. The N-terminus of AcrA anchors itself into the
inner membrane through its a-hairpin interaction with
the peptidoglycan layer [7]. The hairpin of AcrA is
helical and forms an alpha-helical barrel during the shift
from no ligand-bound to a ligand-bound state during the
assembly of AcrAB-TolC [7] (Figure 1). The AcrAB-
TolIC protein channel pump does not require substrates
with a specific molecular weight, net charge, or surface
area. However, mutations in the AcrA gene can weaken
the antibiotic resistance of bacteria, which become more
sensitive to kanamycin and ampicillin. The genes
encoding the AcrAB-TolC protein complex are a part of
an operon, and a change in the DNA sequence of the
acrA gene decreases the efflux of antibiotics. For
example, AcrS is a protein that inhibits AcrA gene
expression. The acrS gene extends 663 base pairs and is
implanted in the acrEF operon [56]. AcrS attaches to the
DNA promoter site of acrA required for initiating
transcription, and then the bound AcrS inhibits the
transcription of acrA.
Blocking the expression of AcrS increases antibiotic
resistance in E. coli cells. After culturing EF. coli in
kanamycin at sub-inhibitory levels and deleting acrS in
E. coli, increased levels of acrE transcription were
exhibited. It was concluded that increasing AcrS
expression increases the inhibition of acrE and this then
decreases E. coli resistance to kanamycin [57-62]. AcrS
represses efflux pump structures and complexes.
However, the effect of AcrS on controlling acrD mRNA
transcription is not known [57-62]. AcrD is an efflux
pump for aminoglycosides related to other RND drug
efflux pumps in E. coli. AcrD has been proven to induce
adaptive resistance to kanamycin after culturing in
kanamycin at sub-inhibitory levels [57-62]. Cultures of
E. coli, lacking acrS, showed more kanamycin resistance
than the wild-type strain.
AcrS was determined to be an inhibitor of acrD
expression. Therefore, inhibiting acrS can export more
aminoglycosides through alternative and other RND
efflux pumps [49-54]. AcrD is a cytoplasmic membrane
drug efflux exporter. AcrD is known to be a part of the
generation of resistance to aminoglycoside antibiotics.
Because AcrD is similar to AcrB, it is argued that AcrD
forms a protein efflux complex with AcrA, which is a
fusion protein located in the periplasm, and with TolC
oriented in the outer membrane [63-68]. After deleting
acrD, the mutant acrD bacterial sample strain continued
to develop antibiotic resistance and was not susceptible
to four antibiotics when compared with the wild type
strain [63-68]. As a consequence, further investigation
of the effects of acrD is required. However, efflux pump
blockers are increasingly toxic for human cells, but a
nontoxic method of applying phosphorodiamidate
morpholino rings can provide less toxicity via an
antisense inhibition of antibiotic resistant mRNA
transcription [56].
5. THE BINDING CAPACITY AND SPECIFICITY
OF SUBSTRATES FOR ACRAB-TOLC EFFLUX
Four families of export pumps accompany the pumps
classified as resistance-nodulation division (RND). The
four other families are the major facilitator superfamily
(MFS), the multidrop and TolC compound extrusion
(MATE), the small multidrug resistance pumps (SMR),
and the ATP-binding cassette (ABC) [69]. The ABC
pump depends on the energy generated from ATP
hydrolysis. RND pumps remove drugs and other toxic
cations. RND pumps are a triplex of proteins that reside
in the bacterial inner membrane region, in the outer
membrane channel, and extend through the middle
periplasm. The triplex releases drugs and toxins into the
outer environment of the bacterial cell. After the toxins
are removed, it is arduous for the antibiotics to regain re-
entry into the cytosol through the bacterial outer
membrane. The MFS and SMR pumps are less effective.
The drugs are only secreted into the middle periplasm
IBEROAMERICAN JOURNAL OF MEDICINE 01 (2022) 60-74
67
and not into the outer membrane by SMR and MFS
pumps [70]. However, the RND pumps operate within
the periplasm to expel many antibiotics remaining after
MEFS and SMR drug exportation. Because RND efflux
pumps have a broad range of specificity for many types
of substrates, this stagnates the discovery and design of
novel antibiotic drugs [71-76]. However, pathogenic
bacteria must also overcome the oxidative activity of
phagocytosis by immune cells. For pathogenic bacteria
to survive phagocytosis and oxidative stress, the
pathogenic bacteria increases RND pump expression to
expel phagocytic oxidative compounds and particles
from reactive oxygen species (ROS) and reactive
nitrogen species (RNS) [77].
The substrates or antibiotics bind to the AcrB segment
of the AcrAB-TolC protein pump. The nonpolar and
highly hydrophobic substrates interact with the water
molecules external to the bacterial cell. The antibiotics
connect and form hydrogen bonds with the water
molecules. The enzyme AcrAB-TolC binds the
hydrophilic region of the antibiotics, which is then
hydrogen-bonded to water [78]. The hydrophilic
domains surrounding the antibiotics allow the facile
removal and export of drugs from the bacteria. The
multidrug pumps do not need specific residues or small
enough binding sites for capturing and removing
antibiotics [79]. Because of the water molecules forming
hydrogen bonds with less stable antibiotic drugs, the
binding site can be as large as possible for export, and
this helps the pump overcome the initial threshold of
binding free energy [79-80]. Antibiotic resistance via
efflux pump activity is observed only when the substrate
is bound in the periplasmic cell region of the tripartite
efflux pump. The substrate cannot remain in the cytosol
of the bacterial cells [81-85]. For example, adding
magnesium to AcrA rapidly increased the export of
phospholipid-like substrates. The AcrA connects to two
vesicles for transporting the phospholipids.
Additionally, adding streptomycin, an antibiotic, to a
more acidic mid-periplasm bacterial region of the
AcrAB-TolC pump channel increased the frequency of
pumping and efflux. Therefore, the removal of
substrates or antibiotics can only occur via the AcrA
subunit that is located in the periplasmic space [86].
The AcrB acts as a regulator when the concentration of
antibiotics lessens the transmembrane pH gradient in the
pump’s channel. The rate of hydrolysis in AcrAB-TolC
affects the rate of efflux when calculating the Vmax and
Km of enzymatic activity. The graphical analysis after
comparing the rate of nitrocefin efflux to the periplasmic
concentration of nitrocefin in the AcrAB-TolC pump
yielded a Michaelis-Menten curve [81]. The Michaelis-
Menten curve displayed a Vmax of 0.024 nmol/mg/s and
a nitrocefin Km _ concentration of 5M, which
demonstrated less competition and more cooperativity
[81]. There was more cooperativity between the rate of
efflux and the pump’s hydrolysis rate that was affected
by the high concentration of nitrocefin. More antibiotics
contained in bacteria lead to a higher velocity of efflux
with rapid antibiotic removal, in which, the AcrB of E.
coli increases the minimum inhibitory and minimal
bactericidal concentration (MICs) of antibiotics [87].
The binding protomer of the AcrAB-TolC pump is
mainly hydrophobic and lies centered within the
bacterial periplasm. There are three different protomer
activities of the AcrAB-TolC pump: 1) access, 2)
binding, and 3) exportation. Each of the three subunit
proteins of the AcrAB-TolC pump rotates as each
protomer alters their orientation and conformations
(Figure 2). The conformational changes are a result of
the movements from the disulfide crosslinks and the
adjacent side chain residue groups. The three subunits
simultaneously rotate at least 1 or 2 subunits of the
AcrAB-TolC protein complex at alternate times [81].
When antibiotics are present, the AcrAB complex begins
to re-orient its conformation to allocate and recruit TolC
[7]. The opening of TolC closes in the outer membrane
to maintain the isolation of the periplasm from the
external environment [7] (Figure 2). Then, the AcrAB-
TolC pump opens by contracting in order to expel a
substrate through its chamber and then rapidly closes
after the expulsion of the drug molecule [7] (Figure 1).
ACTIVITY
Li et al. reported the effects of site-directed mutagenesis
of genes that encode the replacement of nonpolar
phenylalanine R-groups within the AcrAB-TolC binding
site [80]. Without phenylalanine, the efflux of antibiotics
was blocked, and the phenylalanine was altered to
express Phe610Ala. After the mutagenesis, the substrate
and antibiotic doxorubicin remained bound to the
binding site because the gene mutation of acrB caused
the obstruction of the substrates’ separation from the
binding site of the outer AcrAB-TolC pocket [88]. The
acrB mutation caused the binding site to eliminate
expression of the glycine loop, which removed the
separation between the distal and proximal binding sites
of AcrAB-TolC [88]. A mutation of acrB caused the
efflux through the AcrB pump and channel to cease and
this lessened the intensity of virulence. After the
mutation of acrB, AcrB function is disrupted, and AcrB
is aided by the transmembrane protein called D408. The
D408 transports protons similar to AcrB. The mutation
of the acrB gene incorporates a point mutation that
affects the translation of the D408A transmembrane
domain in AcrAB-TolC [89]. After the mutagenesis of
the acrB gene, the movement of protons within the pump
is decelerated and antibiotic efflux is inhibited.
Salmonella Typhimurium SL1344 in mice, Galleria
6. THE INHIBITION OF ACRAB-TOLC EFFLUX
68 IBEROAMERICAN JOURNAL OF MEDICINE 01 (2022) 60-74
AcrAB-TolC Efflux Pump
A) Conformations
Closed * Open
ToiC > TolC
AcrB
B) Conformations
Loose
Loose Zee
I f
Li o-Li
=
80
ii
rt
We
1 7]
oT
AcrA
Conformations
Closed Open
Contraction Contraction
Figure 2: The Conformational changes of AcrAB-TolC Efflux
Pumps (a) TolC rotates counterclockwise and clockwise.
Counter clockwise closes the TolC channel while clockwise
rotation opens TolC. (b) AcrB conformations include loose,
tight, and open orientations. A drug substrate (s) enters the
initial loose form of AcrB and then changes into a tight
position after a hydrogen proton enters AcrB. AcrB alters
into an open adjustment and then returns to a loose assort-
ment that forcefully exports the drug substrate. (c) AcrA
contracts in a closed state and loosens with an opened
contraction.
mellonella, and in tissue cultures all displayed less efflux
following an acrB gene mutation [81]. The elimination
of efflux for each organism was confirmed through RNA
sequencing [89]. The D408A mutant also forced more
regulation of gene expression for the flagella synthesis
that is important for initiating the early stages of
systemic infection.
The mutations of genes that encode the efflux pumps
also inhibit the production of autoinducers (AJ), which
are quorum-sensing molecules, when there is less
accumulation of AI molecules [89]. As a result, quorum
sensing is not initiated, and virulent gene expression is
decreased. Lux-S, which mediates and induces quorum
sensing between bacterial cells, can be monitored for
controlling the process of transcription. The
upregulation of Lux-S causes metabolites to accumulate
in the mutated bacterial strain where the high
concentrations of metabolites triggers the upregulation
of the EmrAB-MDR efflux pump [89]. EmrAB interacts
with the TolC of the AcrAB-TolC pump, which removes
antibiotics, toxins, and free fatty acids from bacteria.
Therefore, inhibiting AcrAB-TolC activity can trigger
EmrAB expression as a substitute, under pressure, and
use available energy to remove the accumulated toxins.
Nevertheless, the gene inhibition of acrB can increase
antibiotic susceptibility; thus, Ayhan et al. also inhibited
the acrA and tolC genes in E. coli.
In the Ayhan et al. study, inhibiting acrB, acrA, and tolC
increased the levels of antibiotic sensitivity in E. coli. E.
coli exhibited less antibiotic resistance after inhibiting
these three genes [30]. Ayhan et al. targeted the mRNA
for each of the three genes with PPMOs. The PPMO of
acrA produced the most impactful antisense molecules
where E. coli treated with acrA-PPMOs increased the
antibiotic effectiveness from 2 to 40-fold [33]. The AcrA
protein was not further translated, and the efflux of
antibiotics was diminished without any toxicity to
human cells [33]. The inhibition of acrA may have
yielded the most significant reduction of antibiotic
resistance because the AcrA protein subunit mediates
communication between AcrB and the TolC for the
purpose of controlling the pump’s closure and opening
[7]. The AcrA hexamer is pertinent because it guides the
opening of the TolC, which then allows the construction
and activation of the AcrAB-TolIC tripartite complex [7].
Thus, inhibiting the genes that code for efflux pump
protein synthesis can reduce the expression of efflux
pumps and decrease antibiotic expulsion. Inhibiting the
expression of MDR efflux protein pumps can increase
the bacterial sensitivity of antibiotics.
7. BACTERIAL METABOLISM AND
ANTIBIOTIC RESISTANCE
Stimulating the metabolic pathways of bacteria can
increase drug uptake in antibiotic tolerant bacteria [90-
92]. For example, cellular respiration regulates
tobramycin antibiotic potency by increasing drug import
and through elevating bacterial cell death. The citric acid
IBEROAMERICAN JOURNAL OF MEDICINE 01 (2022) 60-74
69
cycle (TCA cycle) intermediate of fumarate can amplify
tobramycin susceptibility [90].
cultured alanine and glucose with the bacteria known as
Edwardsiella tarda, which is a bacterium resistant to
multiple tetracycline,
chloramphenicol, streptomycin, and sulfonamide. They
examined the potential of alanine and glucose to increase
the kanamycin sensitivity of E. tarda. Their predictions
and hypotheses were confirmed, adding alanine and
glucose with kanamycin to the E. tarda bacterial cultures
Researchers also
antibiotics such as
eliminated many bacteria. The E. tarda cultured with
only 1000mg of kanamycin, 40mM of alanine, and with
10mM of glucose decreased the number of bacterial cells
by 101, to 3228, and then to 276,000-fold, respectively
[93]. Therefore, it has been concluded that glucose
elevates the uptake of antibiotics by increasing the
activity of the proton motive force or PMF. Glucose
becomes converted into pyruvate and vice versa via the
glycolysis/gluconeogenesis pathway. Metabolism of
pyruvate is the final stage of the TCA cycle that
increases NADH levels, which then amplifies PMF. The
increased PMF can increase the antibiotic uptake of
aminoglycosides [94, 95]
Certain metabolic stimuli can induce aminoglycoside
eradication of Gram-negative and Gram-positive
persisters. For example, after
amplification of PMF, the uptake of aminoglycosides is
advanced and elevated [96-98]. Culturing EF. tarda with
alanine and/or glucose resulted in E. tarda cell induced
apoptosis [93]. Pyruvate increased proportionally to the
activation and
elevated levels of alanine and/or glucose [93]. The
concentrations of NADH and PMF also were amplified.
It was concluded that alanine and glucose increased and
induced the absorption of antibiotics [85]. The
kanamycin inside of the FE. tarda cells were in high
concentration after exposure to alanine and glucose.
Bacterial samples without alanine or glucose exhibited a
kanamycin uptake of 9.5 ng/mL, however, the cultures
with alanine and glucose raised the absorption of
kanamycin to 65-123 ng/ml and then to 113-231 ng/ml
[93]. The alanine and glucose cultures of E. tarda
trounced the activity of the multidrug efflux pump
removal of antibiotics.
Stimulating the metabolism of E. coli cells can also
increase its uptake of antibiotics. Through a Michaelis-
Menten kinetics experiment, researchers found E. coli
K12 to develop an enhanced sensitivity to the
gentamicin antibiotic when cultured in Luria-Bertani
broth supplemented with glutamate and acetate [99]. The
cycle of NADH plus PMF was significantly increased
after culturing E. coli cells with glutamate [99]. The
increase in the gene expression of maeA and maeB
produced more pyruvate in the presence of glucose,
which indicates that the TCA cycle is dependent on the
pyruvate cycle, termed the P-cycle [88]. Manipulating
the P-cycle through the OAA-PEP-pyruvate-AcCoACIT
pathway and the TCA cycle may be a favorable target
for inhibiting antibiotic resistant genes [96]. The P-cycle
depends on the abundance of exogenous metabolites, so
as a result, antibiotic resistance to aminoglycosides
could continue to decrease after genes of the P-cycle are
also silenced [96]. In striking contrast, bacteria rapidly
become more resistant to antibiotics when sources of
carbon and nutrients are less available. For instance,
bacterial species detect and respond to the unavailability
and limitation of nutrients via a regulatory mechanism
termed the stringent response (SR) [100]. Less access or
starvation of carbon, amino acids, and iron stimulates the
SR by activating the expression of re/A and spoT gene
components to construct the alarmone known as
(p)ppGpp. SR was inactivated by inhibiting re/A and
spoT gene expression in Pseudomonas aeruginosa that
causes infections and is used as a model to study biofilm
formation. After inactivating SR, (p)ppGpp synthesis
was eliminated through the starvation-induced serine
analog called serine hydroxamate (SHX) [100].
ACRAB-TOLC EFFLUX PUMP
Additionally, AcrAB-TolC pump is
inactivated or inhibited, metabolites begin to accumulate
that then inactivate AcrR. Ruiz and Levy reported that
the accumulation of toxic metabolites affect the
when the
expression of transcriptional controllers of acrAB such
as AcrR [93]. Thus, Ruiz and Levy confirmed that the
AcrAB-TolC pump controls the expression of the acrAB
operon by responding to changes in cellular metabolism
[101]. For example, AcrAB-TolC efflux is dependent on
the PMF acquired through the electron transport chain of
bacterial metabolism and generated by the electro-
chemical gradient of protons formed across the cell
membrane. AcrB is a component of the multidrug efflux
pump that functions as a drug/proton transport system
[102]. The PMF provides energy for multidrug resistant
AcrAB-TolC efflux pumps. The functions of PMF
include coupling membrane-associated
transporting through the membrane, or
maintaining the pH of the cytoplasm [103]. During the
enzymes,
solutes
activity of PMF, protons flow inwardly into the
8. BACTERIAL METABOLISM AND THE
70 IBEROAMERICAN JOURNAL OF MEDICINE 01 (2022) 60-74
cytoplasm that energizes efflux activity [102]. A proton
is transferred from the periplasmic space and pool to the
D407-D408 pair of AcrB, and then the positive charge
of a side chain in the AcrB protomer returns to the D407-
D408 pair for deprotonation. After the proton is released,
the proton continues to cross the membrane, and then the
remaining energy from PMF is released [63-68].
Inhibiting efflux activity and increasing antibiotic
susceptibility are possible through altering the levels of
PMF in the AcrB protomer. Using plating assays
revealed that these acrAB mutants were more
susceptible to polymyxin B by 10,000-fold [103].
Additionally, treating the E. coli acrAB mutant cultures
with the PMF uncoupler termed carbonyl cyanide m-
chlorophenyl hydrazone or CCCP increased polymyxin
susceptibility [104]. A possible explanation for the
increased antibiotic susceptibility of acrAB mutants after
CCCP treatment includes that a lesser frequency of
protonation and deprotonation in AcrB lowers the PMF
activity needed for powering AcrAB-TolIC efflux. For
instance, the three amino acid residues contained in
AcrB such as the acidic Asp407 and Asp408 residues,
located in the transmembrane (TM) helix termed TM4,
with one basic side chain residue of Lys940 in TM10,
form salt bridges and hydrogen bonds (H-bonds) that
participate in the transfer and transport of protons [102].
Because AcrB consists of two acidic residues with one
basic side chain residue, replacing one of these residues
with an alanine, within the D407-K940-T978-D408
complex of AcrB, suppresses the salt-bridge and H-
bonding interactions [99]. The salt bridges and H-bonds
are perturbed because the alanine cannot become
protonated or deprotonated [99]. Mutations of acrB
reduce protonation and deprotonation reactions required
for triggering the PMF essential for AcrAB-TolIC efflux
[99]. In addition, by blocking the cellular assembly of
protein efflux pumps, the antibiotic transport through the
protein pump channel can become reduced and impede
the energy garnered through ATP hydrolysis required
for powering pump activity. However, currently, the
effects of PMF uncouplers and blockers on MDR efflux
pump activity are not sufficiently understood [104].
9. CONCLUSIONS AND PERSPECTIVES
Developing novel antibiotics to combat bacterial
antibiotic resistance has become stagnant because of
many economic and governmental obstacles. In 2013,
according to the Infectious Diseases Society of America
(IDSA), not many antibacterial agents passed into phase
2 or phase 3 of development [105]. Approximately, 15
of 18 pharmaceutical companies have relinquished
developing antibiotics [105]. Academia has also
dwindled its discovery of new antibiotics due to less
availability of funding [105]. The development and
discovery of new antibiotics is presently not considered
a savvy economic investment in the pharmaceutical
industry [105]. Since antibiotics can quickly cure
infections, antibiotics are not as expensive and cannot
produce a profit as amply large as drugs used for chronic
conditions such as asthma or diabetes. Pharmaceutical
companies have a greater propensity to invest in
developing medicines used for chronic conditions
because these types of medicines are immensely
profitable [105]. An additional factor that has lessened
the development of antibiotics includes a deficiency of
financial attraction because antibiotics are sold at lower
prices. Antibiotics that are new have a price of 1,000
USD to 3,000 USD that is significantly lower than the
10,000 dollars or more drugs used for cancer
chemotherapy [105]. Additionally, many
microbiologists have argued for lesser use of antibiotics
and this has caused many physicians to reduce their
prescribing of many antibiotics in fear of their
proliferation of bacterial drug resistance [105].
Therefore, because there is an urgent need to develop
new antibiotics, this review attempted to provide an
alternative perspective in support of discovering novel
antimicrobials that are gene-based. In this review, an
alternative antimicrobial design was discussed that can
target the genes responsible for expressing specific
subcellular and molecular components, which may
induce the antibiotic sensitivity of multidrug resistant
bacteria. The susceptibility of bacteria to develop
increased sensitivity of antibiotics depends on a few
conditions, such as the bacterial metabolism of carbon,
exposure to metabolites, its horizontal transfer of
antibiotic resistant genes, efflux pumps, and a
bacterium’s direct hydrolysis of antibiotic drugs. In this
review, it was noted that a mutated acrB gene could alter
the AcrAB-TolC binding site by substituting a nonpolar
phenylalanine amino acid residue with an alanine. The
mutagenesis of the acrB gene, encoding the efflux
pumps’ binding sites for AcrAB-TolIC, inhibited the exit
of antibiotics through the AcrAB-TolC efflux pumps.
Gene inhibition of the acrA and the acrB genes in E. coli
lowered the level of virulence and increased antibiotic
efficacy. Moreover, altering the metabolic output of an
MDR bacterial cell by exposure to glucose increased the
antibiotic sensitivity of E. tarda. Researchers cultured E.
IBEROAMERICAN JOURNAL OF MEDICINE 01 (2022) 60-74 71
tarda with glucose and alanine, and the uptake of
kanamycin increased, eliminating approximately 3,000
times the number of MDR bacteria compared to the cells
only treated with kanamycin. A mutation or deletion of
acrB can reduce the levels of protonation and
deprotonation, which then limits the energy produced by
PMF for AcrAB-ToIC efflux.
Novel synthetic biological strategies can be used for re-
engineering complex microbial pathways and networks,
which is currently becoming more simplistic and
increasingly practical [106]. Accordingly, through re-
engineering plasmids, bacterial gene circuits may be
rewired to upregulate metabolic genes and inhibit genes
that encode efflux protein pump expression. Plasmids
can become genetically engineered to induce
transcription of metabolic genes such as maeA and
maeB, thereby, amplifying the pyruvate production that
increases PMF. The higher levels of PMF activity
intensifies antibiotic uptake. Simultaneously, plasmids
could be synthetically engineered to produce the
clustered regularly interspaced short palindromic repeats
and CRISPR-associated protein 9 (CRISPR-Cas9)
nuclease deletion of genes such as acrA and acrB or
small interfering RNAs of these genes can reduce MDR
drug efflux. The advantage of the CRISPR-Cas system
is that it offers more weapons of warfare against
antibiotic resistant pathogens. Research scientists using
the CRISPR-Cas system can find alternative solutions
for extracellular infections such as MRSA and other
intracellular antibiotic resistant bacteria such as
Burkholderia pseudomallei. However, presently, there
remains an intense challenge when applying CRISPR-
Cas9 antibacterials against resistant bacteria external to
the laboratory because there may exist many ethical
concerns for changing or modifying bacterial genomes.
Additionally, the delivery of antimicrobial gene
therapies is presently a major challenge [107]. Bacterial
metabolism of multi-drug resistant bacteria can alter
efflux pump activity. Moreover, stagnating efflux pump
drug export can affect the metabolism of drug-resistant
bacteria. Perhaps, targeting the genes that regulate
bacterial metabolism and generate efflux pump activity
may reduce antibiotic resistance.
10. ACKNOWLEDGEMETS
Many thanks are given to my mentors who provided
lectures, workshops, seminars, and research readings,
which helped to establish my understanding of the
fundamentals for microbiology. I also received hands-on
experience in applying common and current novel
microbiological techniques.
11. CONFLICT OF INTERESTS
The authors declare no conflict of interest. This research
received no external funding.
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