Skip to main content

Full text of "Reducing bacterial antibiotic resistance by targeting bacterial metabolic pathways and disrupting RND efflux pump activity"

See other formats





Journal homepage: 

Reducing bacterial antibiotic resistance by targeting bacterial 
metabolic pathways and disrupting RND efflux pump activity 

Tatiana Hillman %*\® 

“Independent Research, USA 



Article history: 

Received 04 November 2021 
Received in revised form 15 
December 2021 

Accepted 02 January 2022 

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 

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/). 



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 


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. 


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 


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 

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 





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 



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. 


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 


conjugation sexual 


(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 


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 


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. 



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- 



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. 


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 

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 



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]. 


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 



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). 


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 



AcrAB-TolC Efflux Pump 

A) Conformations 
Closed * Open 
ToiC > TolC 
B) Conformations 

Loose Zee 

I f 
Li o-Li 


1 7] 


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 
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. 


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 



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]. 


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 


activity of PMF, protons flow inwardly into the 



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]. 


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. 


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. 


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. 


The authors declare no conflict of interest. This research 
received no external funding. 


1. Osman K, Badr J, Al-Maary KS, Moussa IM, Hessain AM, Girah ZM, et al. 
Prevalence of the Antibiotic Resistance Genes in Coagulase-Positive-and 
Negative-Staphylococcus in Chicken Meat Retailed to Consumers. Front 
Microbiol. 2016;7:1846. doi: 10.3389/fmicb.2016.01846. 

2. Anes J, McCusker MP, Fanning S, Martins M. The ins and outs of RND 
efflux pumps in Escherichia coli. Front Microbiol. 2015;6:587. doi: 

3. Tamma PD, Aitken SL, Bonomo RA, Mathers AJ, van Duin D, Clancy CJ. 
Infectious Diseases Society of America Guidance on the Treatment of 
Extended-Spectrum B-lactamase Producing Enterobacterales (ESBL-E), 
Carbapenem-Resistant Enterobacterales (CRE), and Pseudomonas aeruginosa 
with Difficult-to-Treat Resistance (DTR-P. aeruginosa). Clin Infect Dis. 

2021 ;72(7):e169-e183. doi: 10.1093/cid/ciaal478. 

4. Friedman ND, Temkin E, Carmeli Y. The negative impact of antibiotic 
resistance. Clin Microbiol Infect. 2016;22(5):416-22. doi: 

5. Eichenberger EM, Thaden JT. Epidemiology and Mechanisms of Resistance 
of Extensively Drug Resistant Gram-Negative Bacteria. Antibiotics (Basel). 
2019;8(2):37. doi: 10.3390/antibiotics8020037. 

6. Breijyeh Z, Jubeh B, Karaman R. Resistance of Gram-Negative Bacteria to 
Current Antibacterial Agents and Approaches to Resolve It. Molecules. 
2020;25(6):1340. doi: 10.3390/molecules2506 1340. 

7. Shi X, Chen M, Yu Z, Bell JM, Wang H, Forrester I, et al. In situ structure 
and assembly of the multidrug efflux pump AcrAB-TolC. Nat Commun. 
2019;10(1):2635. doi: 10.1038/s41467-019-10512-6. 

8. Yu EW, Aires JR, Nikaido H. AcrB multidrug efflux pump of Escherichia 
coli: composite substrate-binding cavity of exceptional flexibility generates its 
extremely wide substrate specificity. J Bacteriol. 2003 ;185(19):5657-64. doi: 

9. Thomas-Lopez D, Carrilero L, Matrat S, Montero N, Claverol S, Filipovic 
MR, et al. H2S mediates interbacterial communication through the air 
reverting intrinsic antibiotic resistance. bioRxiv. 2017:202804. doi: 

10. Crabbé A, Ostyn L, Staelens S, Rigauts C, Risseeuw M, Dhaenens M, et al. 
Host metabolites stimulate the bacterial proton motive force to enhance the 
activity of aminoglycoside antibiotics. PLoS Pathog. 2019; 15(4):e1007697. 
doi: 10.1371/journal.ppat.1007697. 

11. Nikaido H. Multidrug resistance in bacteria. Annu Rev Biochem. 
2009;78:119-46. doi: 10.1146/annurev. biochem. 78.082907.145923. 

12. Headd B, Bradford SA. Physicochemical Factors That Favor Conjugation 
of an Antibiotic Resistant Plasmid in Non-growing Bacterial Cultures in the 
Absence and Presence of Antibiotics. Front Microbiol. 2018;9:2122. doi: 

13. Munita JM, Arias CA. Mechanisms of Antibiotic Resistance. Microbiol 
Spectr. 2016;4(2):10.1128/microbiolspec. VMBF-0016-2015. doi: 
10.1128/microbiolspec. VMBF-0016-2015. 

14. Verstraeten N, Knapen W, Fauvart M, Michiels J. A Historical Perspective 
on Bacterial Persistence. Methods Mol Biol. 2016; 1333:3-13. doi: 

15. Ventola CL. The antibiotic resistance crisis: part 1: causes and threats. P 
T. 2015;40(4):277-83. 

16. Chewapreecha C, Marttinen P, Croucher NJ, Salter SJ, Harris SR, Mather 
AE, et al. Comprehensive identification of single nucleotide polymorphisms 


associated with beta-lactam resistance within pneumococcal mosaic genes. 
PLoS Genet. 2014; 10(8):e1004547. doi: 10.1371/journal.pgen. 1004547. 

17. Li Y, Metcalf BJ, Chochua S, Li Z, Gertz RE Jr, Walker H, et al. Validation 
of B-lactam minimum inhibitory concentration predictions for pneumococcal 
isolates with newly encountered penicillin binding protein (PBP) sequences. 
BMC Genomics. 2017;18(1):621. doi: 10.1186/s12864-017-4017-7. 

18. Pimenta F, Gertz RE Jr, Park SH, Kim E, Moura I, Milucky J, et 

al. Streptococcus infantis, Streptococcus mitis, and Streptococcus 

oralis Strains With Highly Similar cps5 Loci and Antigenic Relatedness to 
Serotype 5 Pneumococci. Front Microbiol. 2019;9:3199. doi: 

19. Deng X, Allan-Blitz LT, Klausner JD. Using the genetic characteristics of 
Neisseria gonorrhoeae strains with decreased susceptibility to cefixime to 
develop a molecular assay to predict cefixime susceptibility. Sex Health. 
2019; 16(5):488-99. doi: 10.1071/SH18227. 

20. Simonte FM, Détsch A, Galego L, Arraiano C, Gescher J. Investigation on 
the anaerobic propionate degradation by Escherichia coli K12. Mol Microbiol. 
2017;103(1):55-66. doi: 10.111 1/mmi.13541. 

21. Mahmood HY, Jamshidi S, Sutton JM, Rahman KM. Current Advances in 
Developing Inhibitors of Bacterial Multidrug Efflux Pumps. Curr Med Chem. 
2016;23(10):1062-81. doi: 10.2174/0929867323666160304150522. 

22. Stavri M, Piddock LJ, Gibbons S. Bacterial efflux pump inhibitors from 
natural sources. J Antimicrob Chemother. 2007;59(6): 1247-60. doi: 

23. Aghayan SS, Kalalian Mogadam H, Fazli M, Darban-Sarokhalil D, 
Khoramrooz SS, Jabalameli F, et al. The Effects of Berberine and Palmatine 
on Efflux Pumps Inhibition with Different Gene Patterns in Pseudomonas 
aeruginosa Isolated from Burn Infections. Avicenna J Med Biotechnol. 

24. Barreto HM, Coelho KM, Ferreira JH, Dos Santos BH, de Abreu AP, 
Coutinho HD, et al. Enhancement of the antibiotic activity of aminoglycosides 
by extracts from Anadenanthera colubrine (Vell.) Brenan var. cebil against 
multi-drug resistant bacteria. Nat Prod Res. 2016;30(11):1289-92. doi: 

25. Bohnert JA, Schuster S, Kern WV, Karcz T, Olejarz A, Kaczor A, et al. 
Novel Piperazine Arylideneimidazolones Inhibit the AcrAB-TolC Pump in 
Escherichia coli and Simultaneously Act as Fluorescent Membrane Probes ina 
Combined Real-Time Influx and Efflux Assay. Antimicrob Agents Chemother. 
2016;60(4):1974-83. doi: 10.1128/AAC.01995-15. 

26. Amaral L, Spengler G, Martins A, Armada A, Handzlik J, Kiec- 
Kononowicz K, et al. Inhibitors of bacterial efflux pumps that also inhibit efflux 
pumps of cancer cells. Anticancer Res. 2012;32(7):2947-57. 

27. Bag A, Chattopadhyay RR. Efflux-pump inhibitory activity of a gallotannin 
from Terminalia chebula fruit against multidrug -resistant uropathogenic 
Escherichia coli. Nat Prod Res. 2014;28(16):1280-3. doi: 

28. Chovanova R, Mezovska J, Vaverkova 8, Mikulasova M. The inhibition the 
Tet(K) efflux pump of tetracycline resistant Staphylococcus epidermidis by 
essential oils from three Salvia species. Lett App! Microbiol. 2015;61(1):58- 
62. doi: 10.111 1/am.12424. 

29. Opperman TJ, Kwasny SM, Kim HS, Nguyen ST, Houseweart C, D'Souza 
S, et al. Characterization of a novel pyranopyridine inhibitor of the AcrAB 
efflux pump of Escherichia coli. Antimicrob Agents Chemother. 
2014;58(2):722-33. doi: 10.1128/AAC.01866-13. 

30. Bhattacharyya T, Sharma A, Akhter J, Pathania R. The small molecule 
IITRO8027 restores the antibacterial activity of fluoroquinolones against 
multidrug-resistant Acinetobacter baumannii by efflux inhibition. Int J 
Antimicrob Agents. 2017;50(2):219-26. doi: 

31. Aldred KJ, Kerns RJ, Osheroff N. Mechanism of quinolone action and 
resistance. Biochemistry. 2014;53(10):1565-74. doi: 10.1021/bi5000564. 

32. Exner M, Bhattacharya S, Christiansen B, Gebel J, Goroncy-Bermes P, 
Hartemann P, et al. Antibiotic resistance: What is so special about multidrug - 
resistant Gram-negative bacteria? GMS Hyg Infect Control. 2017;12:Doc05. 
doi: 10.3205/dgkh000290. 

33. Ayhan DH, Tamer YT, Akbar M, Bailey SM, Wong M, Daly SM, et al. 
Sequence-Specific Targeting of Bacterial Resistance Genes Increases 

Antibiotic Efficacy. PLoS Biol. 2016; 14(9):e 1002552. doi: 
10.137 1/journal.pbio. 1002552. 

34. Wang-Kan X, Blair JMA, Chirullo B, Betts J, La Ragione RM, Ivens A, et 
al. Lack of AcrB Efflux Function Confers Loss of Virulence on Salmonella 
enterica Serovar Typhimurium. mBio. 2017;8(4):e00968-17. doi: 

35. Wozniak CE, Lin Z, Schmidt EW, Hughes KT, Liou TG. Thailandamide, a 
Fatty Acid Synthesis Antibiotic That Is Coexpressed with a Resistant Target 
Gene. Antimicrob Agents Chemother. 2018;62(9):e00463-18. doi: 

36. Wu Y, Seyedsayamdost MR. The Polyene Natural Product Thailandamide 
A Inhibits Fatty Acid Biosynthesis in Gram-Positive and Gram-Negative 
Bacteria. Biochemistry. 2018;57(29):4247-51. doi: 

10.102 I/acs.biochem.8b00678. 

37. Evans AL. The Distinctive Regulatory Mechanisms of Bacterial Acetyl-CoA 
Carboxylase. LSU Doctoral Dissertations. 4701. 2018. Available from: 

38. Kénanian G, Morvan C, Weckel A, Pathania A, Anba-Mondoloni J, 
Halpern D, et al. Permissive Fatty Acid Incorporation Promotes 
Staphylococcal Adaptation to FASII Antibiotics in Host Environments. Cell 
Rep. 2019;29(12):3974-82.e4. doi: 10.1016/j.celrep.2019.11.071. 

39. Berendonk TU, Manaia CM, Merlin C, Fatta-Kassinos D, Cytryn E, Walsh 
F, et al. Tackling antibiotic resistance: the environmental framework. Nat Rev 
Microbiol. 2015; 13(5):310-7. doi: 10.1038/nrmicro3439. 

40. Rizzo L, Manaia C, Merlin C, Schwartz T, Dagot C, Ploy MC, et al. Urban 
wastewater treatment plants as hotspots for antibiotic resistant bacteria and 
genes spread into the environment: a review. Sci Total Environ. 2013 ;447:345- 
60. doi: 10.1016/j.scitotenv.2013.01.032. 

41. Martinez JL, Coque TM, Baquero F. What is a resistance gene? Ranking 
risk in resistomes. Nat Rev Microbiol. 2015;13(2):116-23. doi: 

42. Gillings MR, Gaze WH, Pruden A, Smalla K, Tiedje JM, Zhu YG. Using the 
class I integron-integrase gene as a proxy for anthropogenic pollution. SME 
J. 2015;9(6):1269-79. doi: 10.1038/ismej.2014.226. 

43. Ashbolt NJ, Amézquita A, Backhaus T, Borriello P, Brandt KK, Collignon 
P, et al. Human Health Risk Assessment (HHRA) for environmental 
development and transfer of antibiotic resistance. Environ Health Perspect. 
2013;121(9):993-1001. doi: 10.1289/ehp. 1206316. 

44. Allen HK, Donato J, Wang HH, Cloud-Hansen KA, Davies J, Handelsman 
J. Call of the wild: antibiotic resistance genes in natural environments. Nat 
Rev Microbiol. 2010;8(4):251-9. doi: 10.1038/nrmicro23 12. 

45. Rodriguez-Mozaz S, Chamorro S, Marti E, Huerta B, Gros M, Sanchez- 
Melsio A, et al. Occurrence of antibiotics and antibiotic resistance genes in 
hospital and urban wastewaters and their impact on the receiving river. Water 
Res. 2015;69:234-42. doi: 10.1016/j.watres.2014.11.021. 

46. Pruden A, Larsson DG, Amézquita A, Collignon P, Brandt KK, Graham 
DW, et al. Management options for reducing the release of antibiotics and 
antibiotic resistance genes to the environment. Environ Health Perspect. 
2013;121(8):878-85. doi: 10.1289/ehp. 1206446. 

47. Czekalski N, Gascon Diez E, Biirgmann H. Wastewater as a point source of 
antibiotic-resistance genes in the sediment of a freshwater lake. ISME J. 
2014;8(7):1381-90. doi: 10.1038/ismej.2014.8. 

48. Li B, Yang Y, Ma L, Ju F, Guo F, Tiedje JM, et al. Metagenomic and 
network analysis reveal wide distribution and co-occurrence of environmental 
antibiotic resistance genes. ISME J. 2015;9(11):2490-502. doi: 

49. Cunningham CJ , Kuyukina MS, Ivshina IB , Konev Al, Peshkur TA , 
Knapp CW. Potential risks of antibiotic resistant bacteria and genes in 
bioremediation of petroleum hydrocarbon contaminated soils. Environ Sci 
Process Impacts. 2020;22(5):1110-24. doi: 10.1039/c9em00606k. 

50. Chokshi A, Sifri Z, Cennimo D, Horng H. Global Contributors to Antibiotic 
Resistance. J Glob Infect Dis. 2019;11(1):36-42. doi: 

51. Dong H, Chen Y, Wang J, Zhang Y, Zhang P, Li X, et al. Interactions of 
microplastics and antibiotic resistance genes and their effects on the 


aquaculture environments. J Hazard Mater. 2021 ;403:123961. doi: 

52. Hall CW, Mah TF. Molecular mechanisms of biofilm-based antibiotic 
resistance and tolerance in pathogenic bacteria. FEMS Microbiol Rev. 
2017;41(3):276-301. doi: 10.1093/femsre/fux010. 

53. Nolivos S, Cayron J, Dedieu A, Page A, Delolme F, Lesterlin C. Role of 
AcrAB-TolC multidrug efflux pump in drug-resistance acquisition by plasmid 
transfer. Science. 2019;364(6442):778-82. doi: 10.1126/science.aav6390. 

54. Ohneck EA, Zalucki YM, Johnson PJ, Dhulipala V, Golparian D, Unemo 
M, et al. A novel mechanism of high-level, broad-spectrum antibiotic 
resistance caused by a single base pair change in Neisseria gonorrhoeae. 
mBio. 2011;2(5):e00187-11. doi: 10.1128/mBio.00187-11. 

55. Blair JM, Webber MA, Baylay AJ, Ogbolu DO, Piddock LJ. Molecular 
mechanisms of antibiotic resistance. Nat Rev Microbiol. 2015; 13(1):42-51. 
doi: 10.1038/nrmicro3380. 

56. Belmans G, Liu E, Tsui J, Zhou B. AcrS is a potential repressor of acrA 
expression in Escherichia coli and its deletion confers increased kanamycin 
resistance in E. coli BW25113. J Exp Microbiol Immunol. 2016;20:12-7. 

57. Emami M, Xu S, Chan T. AcrS is an Activator of acrD Expression in 
Escherichia coli K-12 Following Exposure to Sub-inhibitory Concentration of 
Kanamycin Pretreatment. J Exp Microbiol Immunol. 2014;18:7-11. 

58. Sidhu K, Talbot M, Van Mil K, Verstraete M. Treatment with sub-inhibitory 
kanamycin induces adaptive resistance to aminoglycoside antibiotics via the 
AcrD multidrug efflux pump in Escherichia coli K-12. J Exp Microbiol 
Immunol. 2012; 16:11-6. 

59. Usui M, Nagai H, Hiki M, Tamura Y, Asai T. Effect of Antimicrobial 
Exposure on AcrAB Expression in Salmonella enterica Subspecies enterica 
Serovar Choleraesuis. Front Microbiol. 2013;4:53. doi: 

60. Besse S, Raff D, Thejomayen M, Ting P. Sub-inhibitory concentrations of 
kanamycin may induce expression of the aminoglycoside efflux pump acrD 
through the two-component systems CpxAR and BaeSR in Escherichia coli K- 
12. J Exp Microbiol Immunol. 2014; 18:1-6. 

61. Lee K, Lin Q, LowA, Luo L. Short-term Adaptive Resistance in E. coli K- 
12 is not dependent on acrD, acrA and tolC. J Exp Microbiol Immunol. 

62. Chu W, Fallavollita A, Lau WB, Park JJ. BaeR, EvgA and CpxR differ- 
entially regulate the expression of acrD in Escherichia coli K-12 but increased 
acrD transcription alone does not demonstrate a substantial increase in 
adaptive re- sistance against kanamycin. J Exp Microbiol Immunol. 

63. Alian S, Qazi U, Sou J. AcrA and TolC are important efflux components in 
the development of low level adaptive amino glycoside resistance in 
Escherichia coli K-12 following sub-inhibitory kanamycin pre-treatment. J Exp 
Microbiol Immunol. 2013;17:1-7. 

64. Kafilzadeh F, Farsimadan F. Investigating multidrug efflux pumps in 
relation to the antibiotic resistance pattern in Escherichia coli strains from 
patients in Iran. Biomed Res. 2016;27(4):1130-5. 

65. Zhang CZ, Chang MX, Yang L, Liu YY, Chen PX, Jiang HX. Upregulation 
of AcrEF in Quinolone Resistance Development in Escherichia coli When 
AcrAB-TolC Function Is Impaired. Microb Drug Resist. 2018;24(1):18-23. 
doi: 10.1089/mdr.2016.0207. 

66. Jabar RM, Hassoon AH. The expression of efflux pump AcrAB in MDR 
Klebsiella pneumoniae isolated from Iraqi patients. J Pharm Sci Res. 
2019; 11(2):423-8. 

67. Krishnamoorthy G, Tikhonova EB, Zgurskaya HI. Fitting periplasmic 
membrane fusion proteins to inner membrane transporters: mutations that 
enable Escherichia coli AcrA to function with Pseudomonas aeruginosa MexB. 
J Bacteriol. 2008; 190(2):691-8. doi: 10.1128/JB.01276-07. 

68. Rampioni G, Pillai CR, Longo F, Bondi R, Baldelli V, Messina M, et al. 
Effect of efflux pump inhibition on Pseudomonas aeruginosa transcriptome and 
virulence. Sci Rep. 2017;7(1):11392. doi: 10.1038/s41598-017-11892-9. 

69. Pérez-Varela M, Corral J, Aranda J, Barbé J. Roles of Efflux Pumps from 
Different Superfamilies in the Surface-Associated Motility and Virulence 

of Acinetobacter baumannii ATCC 17978. Antimicrob Agents Chemother. 
2019;63(3):e02190-18. doi: 10.1128/AAC.02190-18. 

70. Nikaido H. Structure and mechanism of RND-type multidrug efflux pumps. 
Adv Enzymol Relat Areas Mol Biol. 2011;77:1-60. doi: 

71, Fernando DM, Kumar A. Resistance-Nodulation-Division Multidrug Efflux 
Pumps in Gram-Negative Bacteria: Role in Virulence. Antibiotics (Basel). 
2013;2(1):163-81. doi: 10.3390/antibiotics2010163. 

72. Delmar JA, Su CC, Yu EW. Bacterial multidrug efflux transporters. Annu 
Rev Biophys. 2014;43:93-117. doi: 10.1146/annurev-biophys-051013-022855. 

73. Leus IV, Weeks JW, Bonifay V, Smith L, Richardson S, Zgurskaya HI. 
Substrate Specificities and Efflux Efficiencies of RND Efflux Pumps of 
Acinetobacter baumannii. J Bacteriol. 2018;200(13):e00049-18. doi: 

74. Puzari M, Chetia P. RND efflux pump mediated antibiotic resistance in 
Gram-negative bacteria Escherichia coli and Pseudomonas aeruginosa: a 
major issue worldwide. World J Microbiol Biotechnol. 2017;33(2):24. doi: 

75. Kuete V, Ngameni B, Tangmouo JG, Bolla JM, Alibert-Franco S, Ngadjui 
BT, et al. Efflux pumps are involved in the defense of Gram-negative bacteria 
against the natural products isobavachalcone and diospyrone. Antimicrob 
Agents Chemother. 2010,;54(5):1749-52. doi: 10.1128/AAC.01533-09. 

76. Reygaert WC. An overview of the antimicrobial resistance mechanisms of 
bacteria. AIMS Microbiol. 2018 ;4(3):482-501. doi: 

77. Trastoy R, Manso T, Ferndndez-Garcia L, Blasco L, Ambroa A, Pérez Del 
Molino ML, et al. Mechanisms of Bacterial Tolerance and Persistence in the 

Gastrointestinal and Respiratory Environments. Clin Microbiol Rev. 
2018;31(4):e00023-18. doi: 10.1128/CMR.00023-18. 

78. Elkins CA, Nikaido H. Substrate specificity of the RND-type multidrug 
efflux pumps AcrB and AcrD of Escherichia coli is determined predominantly 
by two large periplasmic loops. J Bacteriol. 2002; 184(23):6490-8. doi: 

79. Li XZ, Plésiat P, Nikaido H. The challenge of efflux-mediated antibiotic 
resistance in Gram-negative bacteria. Clin Microbiol Rev. 2015;28(2):337- 
418. doi: 10.1128/CMR.001 17-14. 

80. Vergalli J, Atzori A, Pajovic J, Dumont E, Malloci G, Masi M, et al. The 
challenge of intracellular antibiotic accumulation, a function of 

fluoroquinolone influx versus bacterial efflux. Commun Biol. 2020;3(1):198. 

doi: 10.1038/s42003-020-0929-x. 

81. Palmer M. Efflux of cytoplasmically acting antibiotics from gram-negative 
bacteria: periplasmic substrate capture by multicomponent efflux pumps 
inferred from their cooperative action with single-component transporters. J 
Bacteriol. 2003;185(17):5287-9. doi: 10.1128/JB.185.17.5287-5289.2003. 

82. Alav I, Sutton JM, Rahman KM. Role of bacterial efflux pumps in biofilm 

formation. J Antimicrob Chemother. 2018;73(8):2003-20. doi: 


83. Lomovskaya O, Totrov M. Vacuuming the periplasm. J Bacteriol. 
2005;187(6):1879-83. doi: 10.1128/JB.187.6.1879-1883.2005. 

84. Aires JR, Nikaido H. Aminoglycosides are captured from both periplasm 
and cytoplasm by the AcrD multidrug efflux transporter of Escherichia coli. J 
Bacteriol. 2005;187(6):1923-9. doi: 10.1128/JB.187.6.1923-1929.2005. 

85. Nichols WW. Modeling the Kinetics of the Permeation of Antibacterial 
Agents into Growing Bacteria and Its Interplay with Efflux. Antimicrob Agents 
Chemother. 2017;61(10):e02576-16. doi: 10.1128/AAC.02576-16. 

86. Merdanovic M, Clausen T, Kaiser M, Huber R, Ehrmann M. Protein 
quality control in the bacterial periplasm. Annu Rev Microbiol. 2011;65:149- 
68. doi: 10.1146/annurev-micro-090110-102925. 

87. Assadian O, Wehse K, Hiibner NO, Koburger T, Bagel S, Jethon F, et al. 
Minimum inhibitory (MIC) and minimum microbicidal concentration (MMC) 
of polihexanide and triclosan against antibiotic sensitive and resistant 
Staphylococcus aureus and Escherichia coli strains. GMS Krankenhhyg 
Interdiszip. 2011;6(1):Doc06. doi: 10.3205/dgkh000163. 

88. Zgurskaya HI, Walker JK, Parks JM, Rybenkov VV. Multidrug Efflux 
Pumps and the Two-Faced Janus of Substrates and Inhibitors. Acc Chem Res. 
2021;54(4):930-9. doi: 10.1021/acs.accounts.0c00843. 

89. Meylan S, Porter CBM, Yang JH, Belenky P, Gutierrez A, Lobritz MA, et 
al. Carbon Sources Tune Antibiotic Susceptibility in Pseudomonas aeruginosa 


via Tricarboxylic Acid Cycle Control. Cell Chem Biol. 2017;24(2):195-206. 
doi: 10.1016/j.chembiol.2016.12.015. 

90. Peng B, Su YB, Li H, Han Y, Guo C, Tian YM, et al. Exogenous alanine 
and/or glucose plus kanamycin kills antibiotic-resistant bacteria. Cell Metab. 
2015;21(2):249-62. doi: 10.1016/j.cmet.2015.01.008. 

91. Baeva ME, Golin AP, Mysuria S, Suresh P. Plasmid-mediated overex- 
pression of AcrS may decrease kanamycin resistance in Escherichia coli. J Exp 
Microbiol Immunol. 2018;4:1-10. 

92. Ahn S, Jung J, Jang IA, Madsen EL, Park W. Role of Glyoxylate Shunt in 
Oxidative Stress Response. J Biol Chem. 2016;291(22):11928-38. doi: 

93. Lin X, Kang L, Li H, Peng X. Fluctuation of multiple metabolic pathways is 
required for Escherichia coli in response to chlortetracycline stress. Mol 
Biosyst. 2014; 10(4):901-8. doi: 10.1039/c3mb70522f. 

94. Allison KR, Brynildsen MP, Collins JJ. Metabolite-enabled eradication of 
bacterial persisters by aminoglycosides. Nature. 201 1 ;473(7346):216-20. doi: 

95. Cheng ZX, Yang MJ, Peng B, Peng XX, Lin XM, Li H. The depressed 
central carbon and energy metabolisms is associated to the acquisition of 
levofloxacin resistance in Vibrio alginolyticus. J Proteomics. 2018;181:83-91. 
doi: 10.1016/j.jprot.2018.04.002. 

96. Su YB, Peng B, Li H, Cheng ZX, Zhang TT, Zhu JX, et al. Pyruvate cycle 
increases aminoglycoside efficacy and provides respiratory energy in bacteria. 
Proc Natl Acad Sci U S A. 2018;115(7):E1578-E1587. doi: 

10.1073/pnas. 1714645115. 

97. Collins JJ, Meylan S, Moskowitz S, inventors; Boston University, General 
Hospital Corp, assignee. Intermediate metabolism products to potentiate 
aminoglycoside antibiotics in bacterial infections. United States patent 
application US 14/914,516. 2016 Jul 14. Available from: 

https://patentimages. storage. 

98. Hay M, Li YM, Ma Y. Deletion of AcrS Results in Increased Expression of 
acrE and Confers an Increase in Kanamycin Resistance in Escherichia coli 
BW25113. J Exp Microbiol Immunol. 2017;3:63-9. 

99. Su CC, Li M, Gu R, Takatsuka Y, McDermott G, Nikaido H, Yu EW. 
Conformation of the AcrB multidrug efflux pump in mutants of the putative 

proton relay pathway. J Bacteriol. 2006; 188(20):7290-6. doi: 

100. Nguyen D, Joshi-Datar A, Lepine F, Bauerle E, Olakanmi O, Beer K, et 
al. Active starvation responses mediate antibiotic tolerance in biofilms and 
nutrient-limited bacteria. Science. 2011 ;334(6058):982-6. doi: 
10.1126/science. 1211037. 

101. Ruiz C, Levy SB. Regulation of acrAB expression by cellular metabolites 
in Escherichia coli. J Antimicrob Chemother. 2014;69(2):390-9. doi: 

102. Domenech A, Brochado AR, Sender V, Hentrich K, Henriques-Normark 
B, Typas A, et al. Proton Motive Force Disruptors Block Bacterial Competence 
and Horizontal Gene Transfer. Cell Host Microbe. 2020;27(4):544-55.e3. doi: 

103. Warner DM, Levy SB. Different effects of transcriptional regulators 
MarA, SoxS and Rob on susceptibility of Escherichia coli to cationic 
antimicrobial peptides (CAMPs): Rob-dependent CAMP induction of the 
marRAB operon. Microbiology (Reading). 2010;156(Pt 2):570-8. doi: 

104. Griffith JM, Basting PJ, Bischof KM, Wrona EP, Kunka KS, Tancredi AC, 
et al. Experimental Evolution of Escherichia coli K-12 in the Presence of 
Proton Motive Force (PMF) Uncoupler Carbonyl Cyanide m- 
Chlorophenylhydrazone Selects for Mutations Affecting PMF-Driven Drug 
Efflux Pumps. Appl Environ Microbiol. 2019;85(5):e02792-18. doi: 

105. Morehead MS, Scarbrough C. Emergence of Global Antibiotic 
Resistance. Prim Care. 2018;45(3):467-84. doi: 10.1016/j.pop.2018.05.006. 

106. Stokes JM, Lopatkin AJ, Lobritz MA, Collins JJ. Bacterial Metabolism 
and Antibiotic Efficacy. Cell Metab. 2019;30(2):251-9. doi: 

107. Shabbir MAB, Shabbir MZ, Wu Q, Mahmood S, Sajid A, Maan MK, et al. 
CRISPR-cas system: biological function in microbes and its use to treat 
antimicrobial resistant pathogens. Ann Clin Microbiol Antimicrob. 
2019;18(1):21. doi: 10.1186/s12941-019-0317-x.