Antibiotic Resistance and Efflux Pumps

Abstract

The main purpose of this manuscript is to review the resistance against antibiotics and efflux pumps, one of the mechanisms important in resistance against antibiotics. As a definition, the resistance against antibiotics is accepted as the capability of a microorganism to resist the activity of antimicrobials, which were successfully used to kill the microorganism once. Antibiotic resistance is characterized by several antibiotic susceptibility tests. The wide consumption of antibiotics; the over prescription of antimicrobial drugs by medical doctors; unnecessary, incorrect and inadequate self-medication by the patient and use of several antimicrobial agents either to support a healthy growth or therapeutic purposes in animals consumed as food triggered severe antibiotic resistance. Therefore, the resistance against antimicrobials became a considerable, wide-spread issue in all around the world and the studies have been initiated to overcome the resistance against antibiotics. There are several different mechanisms, which could lead bacteria to be resistant overtime. One of the mechanism of action, which leads to antibiotic resistance, is efflux pumps. Several efflux pump inhibitors were discovered until now, but since some of them are highly cytotoxic, they have very limited use. Understanding efflux pumps and discovering new inhibitors against these pumps could probably save the future of human beings.

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Cite this article Altınöz E, Altuner EM. Antibiotic Resistance and Efflux Pumps. International Journal of Innovative Research and Reviews
(INJIRR) (2019) 3(2) 1-9
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International Journal of Innovative Research and Reviews, 3(2) 1-9
International Journal of Innovative Research and Reviews
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Research paper, Short communication, Review, Technical paper
RE V I E W AR T I C L E
Antibiotic Resistance and Efflux Pumps
Eda ALTINÖZ1,*, Ergin Murat ALTUNER1
1Kastamonu University, Faculty of Science and Arts, Department of Biology, Kuzeykent, Kastamonu,- TURKEY
*Corresponding author E-mail: altinozedaa@gmail.com
HI G H L I G H T S
 The wide consumption of antibiotics; the over prescription of antimicrobial drugs by medical doctors; unnecessary,
incorrect and inadequate self-medication by the patient and use of several antimicrobial agents either to support a healthy
growth or therapeutic purposes in animals consumed as food triggered severe antibiotic resistance.
 One of the mechanism of action, which leads to antibiotic resistance, is efflux pumps.
 Understanding efflux pumps and discovering new inhibitors against these pumps could probably save the future of
human beings.
AR T I C L E IN F O
Received : 07.29.2019
Accepted : 09.26.2019
Published : 12.15.2019
Keywords:
Antibiotic resistance
Efflux pumps
Inhibitors
AB S T R A C T
The main purpose of this manuscript is to review the resistance against antibiotics and efflux
pumps, one of the mechanisms important in resistance against antibiotics. As a definition,
the resistance against antibiotics is accepted as the capability of a microorganism to resist the
activity of antimicrobials, which were successfully used to kill the microorganism once.
Antibiotic resistance is characterized by several antibiotic susceptibility tests. The wide
consumption of antibiotics; the over prescription of antimicrobial drugs by medical doctors;
unnecessary, incorrect and inadequate self-medication by the patient and use of several
antimicrobial agents either to support a healthy growth or therapeutic purposes in animals
consumed as food triggered severe antibiotic resistance. Therefore, the resistance against
antimicrobials became a considerable, wide-spread issue in all around the world and the
studies have been initiated to overcome the resistance against antibiotics. There are several
different mechanisms, which could lead bacteria to be resistant overtime. One of the
mechanism of action, which leads to antibiotic resistance, is efflux pumps. Several efflux
pump inhibitors were discovered until now, but since some of them are highly cytotoxic, they
have very limited use. Understanding efflux pumps and discovering new inhibitors against
these pumps could probably save the future of human beings.
Contents
1. Introduction ................................................................................................................................................................... 2
2. Mechanism of Action in Antibiotics ............................................................................................................................. 2
2.1. Antibiotics Targeting Cell Wall Synthesis ............................................................................................................... 2
2.2. Antibiotics Targeting Cell Membrane ..................................................................................................................... 3
2.3. Antibiotics Targeting Nucleic Acid Synthesis ......................................................................................................... 3
2.4. Antibiotics Targeting Protein Synthesis .................................................................................................................. 3
2.5. Antibiotics Targeting Folic Acid Metabolism ......................................................................................................... 3
3. Antibacterial Drug Resistance ....................................................................................................................................... 3
4. Efflux Pumps ................................................................................................................................................................. 4
4.1. The Classification of Efflux Pump Systems ............................................................................................................ 5
4.1.1. Major Facilitator (MF) Superfamily ................................................................................................................ 5
4.1.2. Multidrug and Toxic Compound Extrusion (MATE) Superfamily ................................................................. 5

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4.1.3. Resistance-Nodulation-Division (RND) Superfamily ..................................................................................... 5
4.1.4. Small Multidrug Resistance (SMR) Superfamily ............................................................................................ 5
4.1.5. ATP Binding Cassette (ABC) Superfamily ..................................................................................................... 5
4.2. The Structure of Efflux Pumps ................................................................................................................................ 5
4.3. Some Bacteria Having Clinical Importance with Antibiotic Resistance.................................................................. 6
4.4. Some Efflux Pump Inhibitors .................................................................................................................................. 6
4.4.1. Peptidomimetics .............................................................................................................................................. 6
4.4.2. Ionophore and Proton Motive Force Uncouplers ............................................................................................ 6
4.4.3. Alkaloids ......................................................................................................................................................... 6
4.4.4. Piperazine Derivatives ..................................................................................................................................... 6
4.4.5. Calcium Ion (Ca+) Antagonists ........................................................................................................................ 6
4.4.6. Phenothiazines ................................................................................................................................................. 6
4.4.7. Phenylpiperidine Selective Serotonin Re-uptake Inhibitors ............................................................................ 6
4.4.8. Proton Pump Inhibitors ................................................................................................................................... 6
4.4.9. Macrolide Analogs .......................................................................................................................................... 6
4.4.10. Piperidine-carboxylic Acid Derivatives .......................................................................................................... 7
5. Conclusion ..................................................................................................................................................................... 7
References ................................................................................................................................................................................ 7
1. Introduction
Infectious diseases are diseases, which are persistent for long
years and even more cause high morbidity and mortality
throughout the world [1]. With an historical perspective,
smallpox, tuberculosis, cholera and plague can be given as
instances of diseases, which are infectious that transmitted
worldwide with catastrophic effects [2]. Throughout the
history, several actions were taken against these diseases and
not only natural remedies, such as plants, natural paints and
incenses, but also synthetic therapeutics were invented [3, 4].
By using disinfectants and antiseptic substances, some
surgical infections and childbed fever (puerperal fever) have
been diminished to a certain degree. However, antibiotics are
known to be invented in the late 19th and early 20th century.
Penicillin is the first antibiotic known to be invented [3].
Antibiotics are the substances either existing in natural
resources, such as plants and fungi, or are produced
artificially to inhibit and/or destroy the development of
microorganisms. The word “antibiotics” is originated by two
words from Ancient Greek, namely “anti” and “bios”, which
mean “against” and “life” respectively [5].
With the invention of the first antibiotic, “The Era of
Antibiotics” has started and a great number of synthetic,
semi-synthetic and natural antimicrobial drugs were
developed and used against infectious diseases [3]. About the
mid of 1900’s in addition to penicillin, several new antibiotic
classes were discovered [6]. These new antibiotics took great
attention between the late 1960s and 1970s, even some
scientists started to believe that such diseases could defeated
forever. But sadly, in the beginning of 1990s, scientists
realized a new challenge of an exceptional number of
infectious diseases either new or raised again, although
previously defeated [7]. The very first data, which clearly
present the resistance to antimicrobial agents were collected
by Paul Ehrlich, who is accepted as the father of modern
chemotherapy [8].
There are several factors, which are the reasons of emerging
resistance against antimicrobials. Probably the most
remarkable factor is either the over prescription of
antimicrobial drugs by medical doctors as a result of
misevaluation of susceptibility tests or without any
significant indications, or unnecessary, incorrect and
inadequate self-medication by the patient, which may
usually noncompliant with proper treatments. Other
important factor regarding medication is failing in full-
course therapy by discontinuing the medication right after
feeling better due to a decrease in the symptoms. In addition,
some common practices applied in the hospitals against
nosocomial infections are also known contribute to the
antimicrobial resistance too. An additional factor, which is
also responsible in resistance against antibiotics is the usage
of several antimicrobials either to support a healthy growth
or therapeutic purposes in animals consumed as food [9].
2. Mechanism of Action in Antibiotics
Antibiotics act against bacteria in two different ways, namely
as a bacteriostatic or a bactericidal agent. The meaning of
being bacteriostatic or bactericidal agent seem to be very
clear to microbiologists. The agents, which prevent the
bacterial growth, in other words that keep bacteria in the
stationary phase of growth, known as bacteriostatic, where
the one kill the bacteria stated as bactericidal [10]. The
reaction of antibiotics killing the bacterial cells mainly based
on inhibiting some cellular functions through a target-drug
reaction. The main specific targets of antibiotics are the
synthesis of the cell wall, cell membrane, nucleic acid (DNA
and RNA), protein and folate synthesis [11–13].
2.1. Antibiotics Targeting Cell Wall Synthesis
Bacteria are enclosed by a cell wall, which protects them
from harsh and unpredictable environmental changes.
Bacteria containing these structures are categorized as gram-
positive and gram-negative [14]. The cell walls of gram-
negative bacteria are built by a thin layer of peptidoglycan,
and an outer membrane surrounds this structure too. But
outer membranes are not observed in gram-positive bacteria
and contain only a thicker peptidoglycan layer than the one
found in the gram-negative bacteria [15, 16]. Peptidoglycan
is a long sugar polymer, which presents cross-linking
between glycan strands and the peptide chains projecting
from the sugars form cross-links from one peptide to another

Altınöz and Altuner / Antibiotic Resistance and Efflux Pumps
3
[17]. These cross-links, which support the cell wall, are
formed between the D-alanyl-alanine section of peptides and
glycine residues as penicillin binding proteins (PBP) are
present [18].
Β-lactams and the glycopeptides, cause an inhibition in
synthesis of the cell walls. Key target of the β-lactams, such
as cephalosporin, penicillin, monobactam and carbapenem,
are the PBP. Since the β-lactam ring have similar structure
with the D-alanyl D-alanine section of peptides it can easily
bind to PBP, so that PBP can’t be available for a new
peptidoglycan synthesis. Thus the disorder in the
peptidoglycan layer causes the lysis of bacteria [19]. In
addition to β-lactams, glycopeptides (vancomycin,
bacitracin and etc.) also prevent synthesis of the cell wall
[12]. It is known that the glycopeptides bind to D-alanyl D-
alanine section of peptide side chain of the precursor
peptidoglycan subunit. As a result, a large antibiotic agent
vancomycin inhibits forming of a bond between the PBP and
D-alanyl subunit, therefore synthesis of the cell wall is also
inhibited [20].
2.2. Antibiotics Targeting Cell Membrane
Polymyxins disrupt the structure of either outer or inner
bacterial cell membrane by interacting with
lipopolysaccharide (LPS) or phospholipids respectively. As
polymyxins bind to LPS or phospholipids, they modify the
membrane structure, so the membrane become more
permeable. As a result, osmotic balance is disrupted, cellular
molecules are leaked, respiration is inhibited and water
uptake is increased, which induce cell death [21].
2.3. Antibiotics Targeting Nucleic Acid Synthesis
During processes called transcription or replication DNA
separation is essential, in which bacterial DNA gyrase has an
important role. It is known that this enzyme is inhibited by
fluoroquinolones [22–24].
Rifampicin, one of the rifamycins, inhibits the initiation of
RNA synthesis by blocking bacterial RNA polymerase [25].
As DNA gyrase and RNA polymerase inhibited, nucleic acid
synthesis is blocked.
2.4. Antibiotics Targeting Protein Synthesis
Ribosomes take an important role in protein synthesis. The
bacterial 70S ribosome is composed of 30S and 50S subunits
[22]. Antimicrobials, which target the 30S or 50S subunit,
inhibit protein biosynthesis [26, 27]. Tetracyclines and
aminoglycosides are known to target 30 S, where
macrolides, clindamycin, linezolid, chloramphenicol and
streptogramin are targeting 50S subunit [12]. Thus,
antibiotics targeting either 30S or 50S subunits inhibit
protein synthesis.
2.5. Antibiotics Targeting Folic Acid Metabolism
Sulfonamides and trimethoprim inhibits different steps in
folic acid metabolism [12].
3. Antibacterial Drug Resistance
As a definition, the resistance against antibiotics is accepted
as the capability of a microorganism to resist the activity of
antimicrobials, which were successfully used to kill the
microorganism once [28]. As it was mentioned previously,
the extensive use, especially the misuse of antibacterial drugs
will cause antibacterial drug resistance, so that conventional
treatments are failed to be successful against that resistant
microorganism [29]. On the other hand, using an incomplete
or a low dose of antibiotic will led to a slow selection of high
level resistance to antibiotics, where the regular dose used
before cannot be sufficient later [30].
Drug resistance may occur through several mechanisms,
such as intrinsic resistance, mutation, enzymatic damaging
of the drugs having antimicrobial properties through enzyme
catalyzed reactions, modifications in the proteins that are key
targets of antimicrobials, horizontal gene transfer, efflux
pumps, an alteration of membrane permeability for
antimicrobial agents, biofilm resistance and quorum sensing
[31, 32].
As it was mentioned previously β-lactams inhibit the
synthesis of the bacterial cell wall. Bacterial resistance
against β-lactams can be generated due to acquiring
plasmids, which encode β-lactamases. As a result, the
resistance occurs through modification in porins, which are
barriers for the permeability. It is known that β-lactamases
cut antibiotics’ β-lactam rings. Modification of the target for
the drug by the production of β-lactamases and inhibiting the
release of autolytic enzymes, causes lower attraction of PBP
for β-lactams. Thus, this cause them to be inactive [33, 34].
Researchers produced carbapenems and cephalosporins,
which are based on the β-lactams’ structure. But it was
observed later that carbapenemases cleaved carbapenems
and extended spectrum β-lactamases (ESBL) cleaved
cephalosporins [34–36]. Moreover, efflux pump
overexpression such as in Pseudomonas aeruginosa and
Escherichia coli with MexA-MexB-OprM and AcrA-AcrB-
TolC pumps respectively caused resistance against
cephalosporins and β-lactams [36]. Overexpression of these
efflux pumps also causes a multidrug resistance against
tetracyclines, rifamycin, oxazolidinones, chloramphenicol,
macrolides and fluoroquinolones [37].
As it was mentioned previously, glycopeptides prevent
transpeptidases to recognise their substrate as they bind to
the peptidoglycan chain by D-alanyl-D-alanine terminal
[38]. The resistance against glycopeptides is developed by
changing the terminal D-alanyl-D-alanine of the
peptidoglycan chain either to D-alanyl-D-lactic acid or D-
alanyl D-serine. However, glycopeptides can still bind to D-
alanyl-D-lactic acid and D-alanyl D-serine, but the affinity is
not as much as the one against D-alanyl-D-alanine [34, 39].
Lipopeptides are antibiotics, which are targeting especially
the membranes of gram-positive bacteria. It is proposed that
these antibiotics are inserted into the membrane irreversibly,
so that they create pores in the bacterial membrane through
oligomerisation, which causes leakage of the cellular
biomolecules that will disrupt bacterial homeostasis [40]. An
antibacterial resistance has been proposed against
lipopeptides and the mechanism of resistance is suggested to

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International Journal of Innovative Research and Reviews 3(2) 1-9
be due to ring-opening esterases, removing fatty acyl tails by
lipases or proteases [41].
Quinolones and fluoroquinolones are antibiotics, which
target DNA synthesis. The resistance mechanisms against
quinolones and fluoroquinolones are proposed to be grouped
into three main groups, namely target-mediated, plasmid-
mediated and chromosome-mediated resistance [42]. The
type of resistance, which is target-mediated is the generally
well-known resistance observed against quinolones and
fluoroquinolones. This type of resistance is appeared as a
result of alterations in DNA gyrase and topoisomerase IV,
which are the target enzymes and due to the alterations in the
efflux and entry of the drug [43]. Due to mutations in the
enzymes, the interactions between these enzymes and
quinolones were deteriorated. Extrachromosomal elements
affect another type of resistance, which is plasmid-mediated.
This type of resistance is end up with encoding several
proteins those can block the interactions between the enzyme
and quinolone, which may increase quinolone efflux or
modify drug metabolism. Chromosome-mediated resistance
may be observed due to overexpression of efflux pumps or
under expression of porins, which lowers the quinolone
concentration within the bacterial cell [42].
As it was mentioned previously, rifamycin inhibits the
initiation of RNA synthesis by blocking bacterial RNA
polymerase. It was shown that rifamycin tightly binds to
RNA polymerase from its β-subunit. A mutation observed in
a gene, namely rpoB, which is responsible to encode RNA
polymerase’s β-subunit causes a reduced affinity between
rifamycin and the subunit itself [34].
The resistance against aminoglycosides, which target protein
synthesis can be achieved by aminoglycoside-modifying
enzymes [34], where the resistance for tetracycline, another
antibiotic targeting 30S subunit of ribosome, is achieved by
tetracycline efflux [34, 44].
Chloramphenicol and macrolides also target protein
synthesis by preventing elongation of peptide chains [34].
There are several mechanisms observed in the resistance
against chloramphenicol, which are mutations in the 50S
subunit of ribosomes and reduction in membrane
permeability [45]. Chloramphenicol efflux is also effective
and mainly chloramphenicol acetyltransferases (CAT) plays
an important role in chloramphenicol resistance by attaching
an acetyl group to chloramphenicol, which could affect the
antibiotic to bind to 50S ribosomal subunit [34]. The
resistance against macrolides is observed due to peptide-
mediated resistance and inducible expression of Erm
methyltransferases [46]. Also, an efflux pump for macrolide,
which is encoded by the mef genes are responsible in
macrolide resistance too [47].
As it was mentioned previously sulfonamides and
trimethoprim inhibits different steps in folic acid
metabolism. It is known that bacteria need to synthesise folic
acid to grow. They convert folic acid to tetrahydrofolate,
which is required for nucleotide biosynthesis. Sulfonamides
and trimethoprim block tetrahydrofolate synthesis together.
Sul1 and sul2, which are drug-resistant dihydropteroate
synthase genes, accepted as the reason of most sulfonamide
resistance, where several dfr genes are the reason of
trimethoprim resistance [34, 48].
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4. Efflux Pumps
Efflux pumps are known to be transport proteins, which are
active pump systems those are important in discharging of
toxic substances from cells to extracellular environment.
These pumps exist not only in gram-negative and gram-
positive bacteria, but in eukaryotic cells too [49].
Overexpression in these pumps are accepted to be linked to
a resistance against drugs [50]. Efflux pumps reduces the
drug concentration without modification of the antibiotic
itself [51]. A decrease in the permeability of the outer
membrane cause a decrease in influx of antimicrobial agents.
Therefore, this causes resistance in several important clinical
microorganisms [52].
The first discovered efflux pump system was tetracycline
efflux pump by Stuart Levy et al. in Escherichia coli [44, 53–
55]. The tetracycline pump is a secondary active transporter
that is activated by membrane proton gradient [55, 56]. This
type of resistance is controlled by plasmids or chromosomes
[57].
Efflux pumps are the resistance mechanisms used in several
bacteria for some antibiotics from different classes, such as
tetracyclines, β-lactams, macrolides, aminoglycosides,
streptogramins, lincosamides, phenicols, oxazolidinones,
pyrimidines, quinolones, rifamycins, sulphonamides and
cationic peptides [58].
For example, the multidrug efflux pump NorA that was
firstly recognised in Staphylococcus aureus isolates, which
are fluoroquinolone-resistant, isolated from a hospital in
Japan in 1986 and is known to export several antimicrobials,
such as acriflavine, benzalkonium chloride,
tetraphenylphosphonium bromide, cetrimide, ethidium
bromide and fluoroquinolones [59–65]. With the same
mechanism Escherichia coli exhibited resistance against the
hydrophilic quinolone norfloxacin [65, 66].
Tetracycline resistance is achieved by several types of
tetracycline resistance genes, such as tetA, tetB, tetC, tetD,
tetE, tetG, tetK, tetL, tetM, tetO, tetS, tetA(P), tetQ and tetX,
which are exist in gram-negative and gram-positive bacteria
[67, 68]. MarRAB, another tetracycline resistance related
operon was observed to be widely distributed among enteric
bacteria, such as Salmonella, Shigella and Escherichia coli
[69].
Although the resistance to a range of antimicrobial agents in
gram-negative bacteria was previously attributed to their
outer membrane structure and function [70], today it is clear
that efflux pumps have a vital position for antimicrobial
resistance in these microorganisms [71–73].
Efflux pumps are known to be specialised for merely to one
compound or lead resistance to a wide-ranging chemicals,
for example antibiotics, antimicrobial peptides, biocides,
detergents, cancer chemotherapeutic agents, colourants and

Altınöz and Altuner / Antibiotic Resistance and Efflux Pumps
5
heavy metals by discharging them from the bacterial cell,
which could lead to a multiple drug resistance (MDR) [74,
75].
The efflux pump mechanisms are triggered by mutations in
the regulatory genes or environmental signals and this
requires energy [75, 76]. The resistant cells use ATP-driven
transporters and/or proton-driven antiporters to discharge
toxic compounds, which can generally flow into the cell by
passive diffusion [77].
One of the reasons that cause bacterial resistance is the low
concentration of antibiotics in the cell, which may arise the
probability of resistance mutations [78]. There are two major
types of mechanisms that cause low antibiotic concentration
in the cell, which are due to the efflux pumps and
modifications in the surfaces of the cells such as reduction in
the number of the entry channels, like porins, namely,
adaptive and mutational types of resistance. These two
factors have a great importance in the acceleration of the
resistance against antimicrobials in microorganisms, which
are pathogenic [79].
The influx and efflux of endogenous or exogenous
compounds are regulated by the membrane transporter
proteins [80]. Approximately 5-10% of all bacterial genes
are related to the transport, where a majority of these genes
code efflux pumps [81–83].
4.1. The Classification of Efflux Pump Systems
Efflux proteins defined until now have been classified into
five different superfamilies: Major Facilitator (MF),
Multidrug and Toxic Compound Extrusion (MATE),
Resistance-Nodulation-Division (RND), Small Multidrug
Resistance (SMR) and ATP Binding Cassette (ABC) [84].
4.1.1. Major Facilitator (MF) Superfamily
Major facilitator superfamily (MFS) is one of the two largest
families of membrane transporter proteins [85]. MF
transmitters contain approximately 400 amino acids [77].
Typically MFS permeases have either 12 or 14
transmembrane α-helical segment [85] with a large
cytoplasmic loop between helices six and seven. The MFS
and ATP-binding cassette (ABC) superfamily [86–89] are
two superfamilies, which are found universally in all living
organisms. They regulate uniport, symport and antiport
processes [90–95]. MFS transport sugar [96, 97], drugs and
Krebs cycle metabolites [93, 98]. This type of efflux pumps
transport aminoglycosides, tetracyclines, rifampicin,
fluoroquinolone, macrolides, chloramphenicols,
lincosamides and pristinamycins to the outside of the cell
[99].
4.1.2. Multidrug and Toxic Compound Extrusion
(MATE) Superfamily
MATE transporters have quite similar size to MFS
transporters, which contain approximately 450 amino acids
and have 12 α-helical segment [77]. Firstly they defined as a
bacterial drug transporter family, but today it is known that
they are found nearly in all eukaryotes and prokaryotes
[100]. MATE family cause multidrug resistance by carrying
wide-ranging therapeutic compounds across the membrane
[101].
4.1.3. Resistance-Nodulation-Division (RND)
Superfamily
Resistance-Nodulation-Division (RND) transporters have
larger size than MFS transporters, which contain
approximately 1000 amino acids and have 12 α-helical
segment [77]. RND pumps are key factors for resistance
against multidrug especially in gram-negative bacteria [84].
The first inhibitor discovered, which inhibits efflux pumps
of RND types was phenylalanine-arginine β-naphthylamide
(PAβN) [102–104]. These types of efflux pumps transport β-
lactams, fucidic acid and sulphonamide to the outside of the
cell [99].
4.1.4. Small Multidrug Resistance (SMR) Superfamily
SMR protein family is composed of proteins, which are
bacterial multidrug transporters. As their name implies they
are small proteins, which contain about 100 to 140 amino
acids and have 4 transmembrane α-helical segment. The best
defined SMR pump is EmrE, which exists in E coli that
contributes resistance against EtBr (Ethidium Bromide) and
methyl viologen [103]. This type of efflux pumps transport
tetracycline, sulfadiazine and erythromycin to the outside of
the cell [99].
4.1.5. ATP Binding Cassette (ABC) Superfamily
ABC type efflux pumps composed of proteins, which uses
substrates, such as various drugs, xenobiotics (including
dietary toxins and drugs) and endogenous compounds to
transport them through the membranes [105]. While ABC
superfamilies of membrane transporters are pumping their
substrates through cell membrane, since they are primary
active transporters, they supply the energy required for
transportation from the hydrolysis of ATP [77, 106]. As it is
true for microorganisms, ABC efflux transporters, which
facilitate transportation of both endogenous and exogenous
compounds through membranes are commonly expressed in
membranes of several organs of the human body, such as
testis, mammary gland, uterus, placenta, lungs, heart, brain,
intestine, kidney and liver too [107]. Some important
members of ABC superfamily, such as breast cancer
resistance protein (BCRP), multidrug resistance associated
proteins (MRPs) and P-glycoprotein (P-gp), have an
important function in detoxification and pharmacokinetics of
drugs and drug metabolites promoting excretion of drugs
into urine in kidneys and intestinal secretion into the bile in
liver [108]. ABC proteins are expressed both in healthy cells
and cancer cells. Since ABC type efflux pumps transport
drugs through the membranes, they not only support the
cancer cell survival, but also the cancer progression [109].
The transported compounds are either antibiotics or cancer
drugs, the resistance shown against multiple drugs is known
as multidrug resistance (MDR) [37, 109]. This type of efflux
pumps transport aminoglycosides, tetracyclines, rifampicin,
fluoroquinolone, macrolides, chloramphenicols and
lincosamides to extracellular environment [99].
4.2. The Structure of Efflux Pumps
Transporters can be categorized according to the substrate
specificity, the phylogenic relationship and the energy
source. The primary active transporters are the drug efflux
pumps, which use energy produced by the hydrolysis of
ATP. They belong to ABC superfamily. The secondary
active transporters, are the drug pumps, which employ the
proton motive force (PMF) or sodium motive force (SMF) in

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International Journal of Innovative Research and Reviews 3(2) 1-9
order to discharge drugs. This system work as antiporters of
H+/drug or Na+/drug. Secondary active transporters belong
to several families, such as MF, SMR, RND and MATE
superfamily [110–113].
Structures for efflux systems present differences as a result
of the bacterial cell wall type. While a single pump protein
facilitates efflux in gram-positive bacteria, in gram-negative
bacteria efflux is facilitated either by a single pump protein
or a system of a pump composed of three protein parts [99,
114, 115]. This three-part system consists of a transport
efflux pump protein located in the cell membrane; a channel
protein as an outer membrane factor (OMF) or outer
membrane channel (OMC) and a membrane fusion protein
(MFP) that provides continuous connection between these
two proteins [99, 116].
4.3. Some Bacteria Having Clinical Importance with
Antibiotic Resistance
It is a well-known issue that gram-negative bacteria present
more resistance compared to gram-positive [117]. Efflux
systems, which cause resistance against antimicrobials were
defined in various types of bacteria having clinical
importance, such as Compylobacter jejuni (CmeABC),
Pseudomonas aeruginosa (MexAB-OprM, Mex-CD-Oprj,
MexXY-OprM), Streptococcus pneumoniae (PrmA),
Staphylococcus aureus (NorA), Escherichia coli (AcrAB-
TolC, AcrEF-TolC, EmrB, EmrD) and Salmonella
typhimurium (AcrB) [49].
4.4. Some Efflux Pump Inhibitors
There are several compounds discovered, which could
inhibit efflux pumps, known as efflux pump inhibitors,
where phenyl-arginine, INF271, β-naphthylamide, carbonyl
cyanide m-chlorophenyl hydrozon, bicodar (incel),
reserpine, timkodar, milbemycin, verapamil, paroxetine,
chlorpromazine and omeprazole can be given as examples.
However, pump inhibitors with clinical importance have
very limited use due to their toxicity problems [99].
Efflux pump inhibitors can be classified under several
classes.
4.4.1. Peptidomimetics
As a result of the studies regarding the efflux systems acting
on gram-negative resistant Pseudomonas aeruginosa several
efflux pump inhibitors (EPI) was discovered and classified
as peptidomimetics. PAβN (phenylalanine arginyl β-
naphthylamide) (MC-207 110), INF271 (BLT-4) and INF55
are examples of peptidomimetic EPIs. Substrates are
determined as quinolones, chloramphenicol, macrolides,
carbenicillin, tetracycline and they can potentially be used in
several microorganisms, such as P. aeruginosa,
Campylobacter jejuni, K. pneumoniae, E. aerogenes, E. coli
and Acinetobacter baumannii [118, 119]. PAβN, INF271
and INF55 affect efflux pumps such as gram-negative
(RND), gram-positive (MFS) and gram-positive (MFS)
respectively [99].
4.4.2. Ionophore and Proton Motive Force Uncouplers
These compounds have serious effects on the bacterial
membrane energy level. Carbonyl cyanide m-
chlorophenylhydrazone (CCCP) is an example for this type
of EPIs. Since it causes damage in the PMF of the membrane,
it leads to cell death. There are still debates regarding the
activity of CCCP, whether it acts as EPI to kill the bacteria
or the alteration of in the PMF. Several ionophore and proton
motive force uncouplers, for example
chlorophenylhydrazone (CCCP), are accepted as extremely
harmful and having high cytotoxicity. Because of that they
have nearly no clinical use [118–122]. CCCP affects pumps
found in gram-negative bacteria, such as MFS, RND and
MATE and mycobacteria [99].
4.4.3. Alkaloids
Reserpine is an example for this type of EPIs. Reserpine is
known to inhibit efflux pumps, such as Bmr and NorA,
present in gram-positive bacteria. It changes the generation
of the membrane PMF, which is essential for the activity of
MDR efflux pumps. It can also inhibit the ABC efflux
pumps, but the concentration needed to block this efflux
pump was founded to be neurotoxic [116–118]. Reserpine
affects pumps found in gram-positive bacteria, such as ABC
and MFS [99].
4.4.4. Piperazine Derivatives
1-(1-Naphthylmethyl)-piperazine (NMP) is an example for
this type of EPIs. It was shown that could reverse the MDR,
especially found in E. coli, by affecting pumps of RND type
[99, 118, 123].
4.4.5. Calcium Ion (Ca+) Antagonists
These types of efflux pump inhibitors are blockers of
transmembrane Ca+ influx, which are also known as Ca+
antagonists. Verapamil is an example for this type of EPIs.
Verapamil affects pumps, such as ABC and MFS found in
gram-negative bacteria [99, 118].
4.4.6. Phenothiazines
Phenothiazines proved to block several energy dependent
systems in bacteria, such as the function of some MDR efflux
pumps [124]. Chlorpromazine is an example for this type of
EPIs. Chlorpromazine is known to affect potassium flux
across the membrane in the yeast Saccharomyces cerevisiae
and S. aureus [125–127]. ABC and SMR types of pumps,
which are found in gram-positive bacteria are affected by
chlorpromazine [99].
4.4.7. Phenylpiperidine Selective Serotonin Re-uptake
Inhibitors
Phenylpiperidine selective serotonin re-uptake inhibitors are
proved to inhibit the function of two S. aureus multidrug
efflux pumps and also affect moderately the activity of the
AcrAB-TolC pump in E. coli [127]. Paroxetine is an example
for this type of EPIs. It was one of the first defined
phenylpiperidine selective serotonin re-uptake inhibitor that
inhibits MepA (MATE) and NorA (MFS) efflux pumps
[128]. Paroxetine affects MFS and RND efflux pumps in
gram-positive bacteria [99].
4.4.8. Proton Pump Inhibitors
Proton pump inhibitors, such as pantoprazole, esomeprazole
and omeprazole are known to be P-glycoprotein inhibitors
[129]. Omeprazole affects MFS efflux pumps in gram-
positive bacteria [99].
4.4.9. Macrolide Analogs
Milbemycin is an example for this type of EPIs. Milbemycin
affects ABC efflux pumps in gram-positive and gram-

Altınöz and Altuner / Antibiotic Resistance and Efflux Pumps
7
negative bacteria and bricodar affects MFS and ABC efflux
pumps in gram-positive and gram-negative bacteria [99].
4.4.10. Piperidine-carboxylic Acid Derivatives
Timcodar and bricodar are two examples for this type of
EPIs. Both timcodar and bricodar affect MFS and ABC
efflux pumps in gram-positive and gram-negative bacteria
[99].
5. Conclusion
The wide consumption of antibiotics; the over prescription
of antimicrobial drugs by medical doctors; unnecessary,
incorrect and inadequate self-medication by the patient and
use of several antimicrobial agents either to support a healthy
growth or therapeutic purposes in animals consumed as food
triggered severe antibiotic resistance. Therefore, the
antibiotic resistance has become a major, wide-spread issue
in all around the world and the studies have been initiated to
overcome the resistance against antibiotics.
There are several different mechanisms, which could lead
bacteria to be resistant overtime. One of the mechanism of
action, which leads to antibiotic resistance is efflux pumps.
Several efflux pump inhibitors were discovered until now,
but since some of them are highly cytotoxic they have very
limited use. Understanding efflux pumps and discovering
new efflux pump inhibitors could probably save the future of
human beings.
References
[1] Drexler M. What You Need to Know about Infectious Disease.
Institute of Medicine (US): National Academies Press (US) (2010).
[2] Epstein PR. Commentary: Pestilence and Poverty—Historical
Transitions and the Great Pandemics. American Journal of
Preventive Medicine (1992) 8(4):263–265.
[3] Töreci K. Antibiyotik kullanimi ve direnç iliskisi [The relationship
between antibiotic use and resistance]. Flora (2003) 8(2):89–110.
[4] Çiftçi A, Aksoy A. Acquired Resistance Mechanisms Against
Antibiotics. Turkiye Klinikleri Journal of Veterinary Sciences-
Pharmacology and Toxicology-Special Topics (2015) 1(2):1–10.
[5] Gökçe T. Birinci basamak saglik kurulusuna basvuran hastalarin
antibiyotik kullanimi konusundaki davranis ve bilgi düzeylerinin
arastirilmasi [Research on habits and awareness levels on the
antibiotic use of the patients who consult primary care health
services]. Pamukkale University.
[6] Infectious Diseases Society of America. Bad Drugs, No Drugs:
Infectious Diseases Society of America (2010).
[7] Conly JM, Johnston BL. Where are all the new antibiotics? The new
antibiotic paradox. Canadian Journal of Infectious Diseases and
Medical Microbiology (2005) 16(3):159–160.
[8] Jacoby G. History of Drug-Resistant Microbes. In: Mayers D, Sobel
J, Ouellette M, Marchaim D, editors. Antimicrobial Drug
Resistance: Humana Press (2009).
[9] Knobler SL, Lemon SM, Najafi M, Burroughs T. The Resistance
Phenomenon in Microbes and Infectious Disease Vectors:
Implications for Human Health and Strategies for Containment:
Workshop Summary: The National Academies Press (2003).
[10] Pankey G, Sabath L. Clinical Relevance of Bacteriostatic versus
Bactericidal Activity in the Treatment of Gram-Positive Bacterial
Infections. Clinical Infectious Diseases (2004) 38(6):864–870.
[11] Kohanski M, Dwyer D, Collins J. How antibiotics kill bacteria: from
targets to networks. Nature Reviews Microbiology (2010) 8(6):423–
435.
[12] Kapoor G, Saigal S, Elongavan A. Action and resistance
mechanisms of antibiotics: A guide for clinicians. Journal of
Anaesthesiology Clinical Pharmacology (2017) 33(3):300–305.
[13] Walsh C. Antibiotics: Actions, Origins, Resistance: ASM Press
(2003).
[14] Silhavy T, Kahne D, Walker S. The Bacterial Cell Envelope. Cold
Spring Harbor Perspectives in Biology (2010) 2(5):a000414-
a000414.
[15] Vollmer W, Blanot D, Pedro M de. Peptidoglycan structure and
architecture. FEMS Microbiology Reviews (2008) 32(2):149–167.
[16] Gan L, Chen S, Jensen G. Molecular organization of Gram-negative
peptidoglycan. Proceedings of the National Academy of Sciences
(2008) 105(48):18953–18957.
[17] Kahne D, Leimkuhler C, Lu W, Walsh C. Glycopeptide and
Lipoglycopeptide Antibiotics. Chemical Reviews (2005)
105(2):425–448.
[18] Reynolds P. Structure, biochemistry and mechanism of action of
glycopeptide antibiotics. European Journal of Clinical Microbiology
& Infectious Diseases (1989) 8(11):943–950.
[19] Dzidic S, Šuškovic J, Kos B. Antibiotic Resistance Mechanisms in
Bacteria: Biochemical and Genetic Aspects. Food Technology &
Biotechnology (2008) 46(1):11–21.
[20] Grundmann H, Aires-de-Sousa M, Boyce J, Tiemersma E.
Emergence and resurgence of meticillin-resistant Staphylococcus
aureus as a public-health threat. The Lancet (2006) 368(9538):874–
885.
[21] Trimble M, Mlynárcik P, Kolár, Mand Hancock, RE. Polymyxin:
Alternative Mechanisms of Action and Resistance. Cold Spring
Harbor Perspectives in Medicine (2016) 6(10):a025288.
[22] YONEYAMA H, KATSUMATA R. Antibiotic Resistance in
Bacteria and Its Future for Novel Antibiotic Development.
Bioscience, Biotechnology, and Biochemistry (2006) 70(5):1060–
1075.
[23] Wise R. A Review of the Mechanisms of Action and Resistance of
Antimicrobial Agents. Canadian Respiratory Journal (1999)
6(SupplA):20A-2A.
[24] Higgins P, Fluit A, Schmitz FJ. Fluoroquinolones: Structure and
Target Sites. Current Drug Targets (2003) 4(2):181–190.
[25] Clancy C, Yu Y, Lewin A, Nguyen MH. Inhibition of RNA
Synthesis as a Therapeutic Strategy against Aspergillus and
Fusarium: Demonstration of In Vitro Synergy between Rifabutin
and Amphotericin B. Antimicrobial Agents and Chemotherapy
(1998) 42(3):509–513.
[26] Vannuffel P, Cocito C. Mechanism of Action of Streptogramins and
Macrolides. Drugs (1996) 51(Supplement 1):20–30.
[27] Johnston N, Mukhtar T, Wright G. Streptogramin Antibiotics: Mode
of Action and Resistance. Current Drug Targets (2002) 3(4):335–
344.
[28] Prestinaci F, Pezzotti P, Pantosti A. Antimicrobial resistance: a
global multifaceted phenomenon. Pathogens and Global Health
(2015) 109(7):309–318.
[29] Li B, Webster TJ. Bacteria antibiotic resistance: New challenges and
opportunities for implant-associated orthopedic infections. Journal
of Orthopaedic Research (2017) 36(1):22–32.
[30] Bhattacharjee MK. Development of Resistance to Antibiotics. In:
Bhattacharjee MK, editor. Chemistry of Antibiotics and Related
Drugs: Springer (2016).
[31] Ali J, Rafiq Q, Ratcliffe E. Antimicrobial resistance mechanisms
and potential synthetic treatments. Future Science OA (2018)
4(4):FSO290.
[32] Dever L, Dermody T. Mechanisms of bacterial resistance to
antibiotics. Archives of Internal Medicine (1991) 151(5):886–895.
[33] Heesemann J. Mechanisms of Resistance to Beta-Lactam
Antibiotics. Infection (1993) 21(Suppement 1):S4-S9.
[34] Carlson-Banning KM, Zechiedrich L. Antibiotic Classes and
Mechanisms of Resistance. In: Highlander SK, Rodriguez-Valera F,
White BA, editors. Encyclopedia of Metagenomics, Environmental
Metagenomics: Springer (2015).
[35] Boucher H, Talbot G, Bradley J, Edwards J, Gilbert D, Rice L, et al.
Bad Bugs, No Drugs: No ESKAPE! An Update from the Infectious
Diseases Society of America. Clinical Infectious Diseases (2009)
48(1):1–12.
[36] Wilke MS, Lovering AL, Strynadka NC. ß-Lactam antibiotic
resistance: a current structural perspective. Current Opinion in
Microbiology (2005) 8(5):525–533.
[37] Nikaido H. Multidrug Resistance in Bacteria. Annual Review of
Biochemistry (2009) 78(1):119–146.
[38] Pootoolal J, Neu J, Wright GD. Glycopeptide Antibiotic Resistance.
Annual Review of Pharmacology and Toxicology (2002) 42(1):381–
408.
[39] Méndez-Álvarez S, Pérez-Hernández X, Claverie-Martín F.
Glycopeptide Resistance in Enterococci. International Microbiology
(2000) 3(2):71–80.

8
International Journal of Innovative Research and Reviews 3(2) 1-9
[40] Beiras-Fernandez A, Vogt F, Sodian R, Weis F. Daptomycin: a
novel lipopeptide antibiotic against Gram-positive pathogens.
Infection and Drug Resistance (2010) 3:95–101.
[41] D’Costa V, Mukhtar T, Patel T, Koteva K, Waglechner N, Hughes
D, et al. Inactivation of the Lipopeptide Antibiotic Daptomycin by
Hydrolytic Mechanisms. Antimicrobial Agents and Chemotherapy
(2012) 56(2):757–764.
[42] Aldred K, Kerns R, Osheroff N. Mechanism of Quinolone Action
and Resistance. Biochemistry (2014) 53(10):1565–1574.
[43] Jacoby G. Mechanisms of Resistance to Quinolones. Clinical
Infectious Diseases (2005) 41(Supplement_2):S120-S126.
[44] Nelson M, Levy S. The history of the tetracyclines. Annals of the
New York Academy of Sciences (2011) 1241(1):17–32.
[45] Das B, Patra S. Nanostructures for Antimicrobial Therapy. In: Anton
F, Grumezescu A, editors. Antimicrobials: Meeting the Challenges
of Antibiotic Resistance through Nanotechnology: Elsevier (2017).
[46] Gaynor M, Mankin A. Macrolide Antibiotics: Binding Site,
Mechanism of Action, Resistance. Frontiers in Medicinal Chemistry
- Online (2005) 2(1):21–35.
[47] Schlünzen F, Zarivach R, Harms J, Bashan A, Tocilj A, Albrecht R,
et al. Structural basis for the interaction of antibiotics with the
peptidyl transferase centre in eubacteria. Nature (2001)
413(6858):814–821.
[48] Sköld O. Resistance to trimethoprim and sulfonamides. Veterinary
Research (2001) 32(3/4):261–273.
[49] Webber MA, Piddock LJV. The importance of efflux pumps in
bacterial antibiotic resistance. Journal of Antimicrobial
Chemotherapy (2003) 51(1):9–11.
[50] Sun, Jand Deng, Zand Yan, A. Bacterial multidrug efflux pumps:
Mechanisms, physiology and pharmacological exploitations.
Biochemical and Biophysical Research Communications (2014)
453(2):254–267.
[51] Fraqueza MJ. Antibiotic resistance of lactic acid bacteria isolated
from dry-fermented sausages. International Journal of Food
Microbiology (2015) 212:76–88.
[52] Denyer S, Maillard J. Cellular impermeability and uptake of
biocides and antibiotics in Gram-negative bacteria. Journal of
Applied Microbiology (2002) 92(s1):35S-45S.
[53] McMurry L, Petrucci RE, Levy SB. Active efflux of tetracycline
encoded by four genetically different tetracycline resistance
determinants in Escherichia coli. Proceedings of the National
Academy of Sciences (1980) 77(7):3974–3977.
[54] McMurry L, Levy S. Two Transport Systems for Tetracycline in
Sensitive Escherichia coli: Critical Role for an Initial Rapid Uptake
System Insensitive to Energy Inhibitors. Antimicrobial Agents and
Chemotherapy (1978) 14(2):201–209.
[55] Kumar S, Varela MF. Molecular mechanisms of bacterial resistance
to antimicrobial agents. In: Méndez-Vilas A, editor. Microbial
Pathogens and Strategies for Combating Them: Science, Technology
and Education: Formatex Research Center (2013). p. 522–534.
[56] McMurry LM, Cullinane JC, Petrucci RE, Levy SB. Active uptake
of tetracycline by membrane vesicles from susceptible Escherichia
coli. Antimicrobial Agents and Chemotherapy (1981) 20(3):307–
313.
[57] Balassiano IT, Bastos M, Madureira DJ, Silva I, Freitas-Almeida Â,
Oliveira S. The involvement of tetA and tetE tetracycline resistance
genes in plasmid and chromosomal resistance of Aeromonas in
Brazilian strains. Memórias do Instituto Oswaldo Cruz (2007)
102(7):861–866.
[58] Sikri N, Dalal S, Taneja R. Efflux Pumps: An Overview.
International Journal of Pharmaceutical Sciences and Research
(2018) 9(3):854–861.
[59] Deng X, Sun F, Ji Q, Liang H, Missiakas D, Lan L, et al. Expression
of Multidrug Resistance Efflux Pump Gene norA Is Iron Responsive
in Staphylococcus aureus. Journal of Bacteriology (2012)
194(7):1753–1762.
[60] Costa SS, Viveiros M, Amaral L, Couto I. Multidrug Efflux Pumps
in Staphylococcus aureus: an Update. The Open Microbiology
Journal (2013) 7(1):59–71.
[61] Ubukata K, Itoh-Yamashita N, Konno M. Cloning and expression of
the norA gene for fluoroquinolone resistance in Staphylococcus
aureus. Antimicrobial Agents and Chemotherapy (1989) 33(9):1535–
1539.
[62] Hsieh P, Siegel S, Rogers B, Davis D, Lewis K. Bacteria lacking a
multidrug pump: A sensitive tool for drug discovery. Proceedings of
the National Academy of Sciences (1998) 95(12):6602–6606.
[63] Kaatz G, Seo S. Inducible NorA-mediated multidrug resistance in
Staphylococcus aureus. Antimicrobial Agents and Chemotherapy
(1995) 39(12):2650–2655.
[64] Neyfakh AA, Borsch CM, Kaatz GW. Fluoroquinolone resistance
protein NorA of Staphylococcus aureus is a multidrug efflux
transporter. Antimicrobial Agents and Chemotherapy (1993)
37(1):128–129.
[65] Ng EY, Trucksis M, Hooper DC. Quinolone resistance mediated by
norA: physiologic characterization and relationship to flqB, a
quinolone resistance locus on the Staphylococcus aureus
chromosome. Antimicrobial Agents and Chemotherapy (1994)
38(6):1345–1355.
[66] Yu J, Grinius L, Hooper D. NorA Functions as a Multidrug Efflux
Protein in both Cytoplasmic Membrane Vesicles and Reconstituted
Proteoliposomes. Journal of Bacteriology (2002) 184(5):1370–1377.
[67] Bryan A, Shapir N, Sadowsky M. Frequency and Distribution of
Tetracycline Resistance Genes in Genetically Diverse, Nonselected,
and Nonclinical Escherichia coli Strains Isolated from Diverse
Human and Animal Sources. Applied and Environmental
Microbiology (2004) 70(4):2503–2507.
[68] Roberts MC. Tetracycline resistance determinants: mechanisms of
action, regulation of expression, genetic mobility, and distribution.
FEMS Microbiology Reviews (1996) 19(1):1–24.
[69] Cohen S, Yan W, Levy S. A Multidrug Resistance Regulatory
Chromosomal Locus Is Widespread among Enteric Bacteria.
Journal of Infectious Diseases (1993) 168(2):484–488.
[70] Nikaido H. Outer membrane barrier as a mechanism of antimicrobial
resistance. Antimicrobial Agents and Chemotherapy (1989)
33(11):1831–1836.
[71] Ma D, Cook D, Hearst J, Nikaido H. Efflux pumps and drug
resistance in Gram-negative bacteria. Trends in Microbiology (1994)
2(12):489–493.
[72] Poole K, Krebes K, McNally C, Neshat S. Multiple antibiotic
resistance in Pseudomonas aeruginosa: evidence for involvement of
an efflux operon. Journal of Bacteriology (1993) 175(22):7363–
7372.
[73] Zhao Q, Li X-Z, Srikumar R, Poole K. Contribution of Outer
Membrane Efflux Protein OprM to Antibiotic Resistance in
Pseudomonas aeruginosa Independent of MexAB. Antimicrobial
Agents and Chemotherapy (1998) 42(7):1682–1688.
[74] Anes J, McCusker M, Fanning S, Martins M. The ins and outs of
RND efflux pumps in Escherichia coli. Frontiers in Microbiology
(2015) 6:587.
[75] Zarakolu P. Mikroorganizmalarda direnç mekanizmasi olarak aktif
pompa sistemleri [Active Pump Systems as Resistance Mechanism
in Microorganisms]. Hastane Enfeksiyonlari Dergisi [Turkish
Journal of Hospital Infections] (2003) 7(3):131–136.
[76] Blanco P, Hernando-Amado S, Reales-Calderon J, Corona F, Lira F,
Alcalde-Rico M, et al. Bacterial Multidrug Efflux Pumps: Much
More Than Antibiotic Resistance Determinants. Microorganisms
(2016) 4(1):14.
[77] BORGES-WALMSLEY MI, McKEEGAN K, WALMSLEY A.
Structure and function of efflux pumps that confer resistance to
drugs. Biochemical Journal (2003) 376(2):313–338.
[78] Sandegren L. Selection of antibiotic resistance at very low antibiotic
concentrations. Upsala Journal of Medical Sciences (2014)
119(2):103–107.
[79] Fernández L, Hancock R. Adaptive and Mutational Resistance: Role
of Porins and Efflux Pumps in Drug Resistance. Clinical
Microbiology Reviews (2012) 25(4):661–681.
[80] Girardin F. Membrane Transporter Proteins: A Challenge for CNS
Drug Development. Dialogues in Clinical Neuroscience (2006)
8(3):311–321.
[81] Piddock L. Clinically Relevant Chromosomally Encoded Multidrug
Resistance Efflux Pumps in Bacteria. Clinical Microbiology Reviews
(2006) 19(2):382–402.
[82] Lomovskaya O, Warren M, Lee A, Galazzo J, Fronko R, Lee M, et
al. Identification and Characterization of Inhibitors of Multidrug
Resistance Efflux Pumps in Pseudomonas aeruginosa: Novel Agents
for Combination Therapy. Antimicrobial Agents and Chemotherapy
(2001) 45(1):105–116.
[83] Saier, Jr, Milton H, Paulsen IT. Phylogeny of multidrug transporters.
Seminars in Cell & Developmental Biology (2001) 12(3):205–213.
[84] Fernando D, Kumar A. Resistance-Nodulation-Division Multidrug
Efflux Pumps in Gram-Negative Bacteria: Role in Virulence.
Antibiotics (2013) 2(1):163–181.
[85] Pao SS, Paulsen IT, Saier MH. Major Facilitator Superfamily.
Microbiology and Molecular Biology Reviews (1998) 62(1):1–34.
[86] Dean M, Allikmets R. Evolution of ATP-binding cassette transporter
genes. Current Opinion in Genetics & Development (1995)
5(6):779–785.

Altınöz and Altuner / Antibiotic Resistance and Efflux Pumps
9
[87] Fath MJ, Kolter R. ABC Transporters: Bacterial Exporters.
Microbiology and Molecular Biology Reviews (1993) 8(1):67–113.
[88] Higgins CF. ABC Transporters: From Microorganisms to Man.
Annual Review of Cell Biology (1992) 8(1):67–113.
[89] Kuan G, Dassa E, Saurin W, Hofnung M, Saier MH. Phylogenetic
analyses of the ATP-binding constituents of bacterial
extracytoplasmic receptor-dependent ABC-type nutrient uptake
permeases. Research in Microbiology (1995) 146(4):271–278.
[90] Saier Jr MH, Beatty JT, Goffeau A, Harley KT, Heijne WH, Huang
SC, et al. The Major Facilitator Superfamily. Journal of Molecular
Microbiology and Biotechnology (1999) 1(2):257–279.
[91] Baldwin SA. Mammalian passive glucose transporters: members of
an ubiquitous family of active and passive transport proteins.
Biochimica et Biophysica Acta (BBA) - Reviews on Biomembranes
(1993) 1154(1):17–49.
[92] Goswitz VC, Brooker RJ. Structural Features of the
Uniporter/Symporter/Antiporter Superfamily. Protein Science
(1995) 4(3):534–537.
[93] Griffith J, Baker M, Rouch D, Page M, Skurray R, Paulsen I, et al.
Membrane transport proteins: implications of sequence comparisons.
Current Opinion in Cell Biology (1992) 4(4):684–695.
[94] Henderson P. Sugar transport proteins. Current Opinion in
Structural Biology (1991) 1(4):590–601.
[95] Marger MD, Saier MH. A major superfamily of transmembrane
facilitators that catalyse uniport, symport and antiport. Trends in
Biochemical Sciences (1993) 18(1):13–20.
[96] Henderson P, Maiden M. Homologous Sugar Transport Proteins in
Escherichia coli and Their Relatives in Both Prokaryotes and
Eukaryotes. Philosophical Transactions of the Royal Society B:
Biological Sciences (1990) 326(1236):391–410.
[97] Maiden MC, Davis E, Baldwin S, Moore D, Henderson P.
Mammalian and bacterial sugar transport proteins are homologous.
Nature (1987) 325(6105):641–643.
[98] Paulsen I, Skurray R. The POT family of transport proteins. Trends
in Biochemical Sciences (1994) 19(10):404.
[99] Aygül A. Antibiyotik Direncinde Disa Atim Sistemlerinin ve
Dirençle Mücadelede Disa Atim Pompa Inhibitörlerinin Önemi [The
Importance of Efflux Systems in Antibiotic Resistance and Efflux
Pump Inhibitors in the Management of Resistance]. Mikrobiyoloji
Bülteni [Bulletin of Microbiology] (2015) 49(2):278–291.
[100] Moriyama Y, Hiasa M, Matsumoto T, Omote H. Multidrug and
toxic compound extrusion (MATE)-type proteins as anchor
transporters for the excretion of metabolic waste products and
xenobiotics. Xenobiotica (2008) 38(7-8):1107–1118.
[101] Lu M. Structures of multidrug and toxic compound extrusion
transporters and their mechanistic implications. Channels (2016)
10(2):88–100.
[102] Bay D, Rommens K, Turner R. Small multidrug resistance proteins:
A multidrug transporter family that continues to grow. Biochimica et
Biophysica Acta (BBA) - Biomembranes (2008) 1778(9):1814–1838.
[103] Renau T, Léger R, Flamme E, Sangalang J, She M, Yen R, et al.
Inhibitors of Efflux Pumps inPseudomonasaeruginosaPotentiate the
Activity of the Fluoroquinolone Antibacterial Levofloxacin. Journal
of Medicinal Chemistry (1999) 42(24):4928–4931.
[104] Opperman T, St. Nguyen. Recent advances toward a molecular
mechanism of efflux pump inhibition. Frontiers in Microbiology
(2015) 6:421.
[105] Kettenmann H, Ransom BR. The Concept of Neuroglia: A Historical
Perspective. In: Kettenmann H, Ransom BR, editors. Neuroglia:
Oxford University Press (2005). p. 1–16.
[106] Venter H, Shilling RA, Velamakanni S, Balakrishnan L, van Veen
HW. An ABC transporter with a secondary-active multidrug
translocator domain. Nature (2003) 426(6968):866–870.
[107] Xie W. Drug Metabolism in Diseases: Academic Press (2016).
[108] Kara ZP, Öztürk N, Öztürk D, Okyar A. ABC Tasiyici Proteinleri:
Sirkadiyan Ritimler vee Cinsiyete Bagli Farkliliklar [ABC Carrier
Proteins: Circadian Rhythms and Gender Differences]. MÜSBED
(2013) 3(1):1–13.
[109] Begicevic RR, Falasca M. ABC Transporters in Cancer Stem Cells:
Beyond Chemoresistance. International Journal of Molecular
Sciences (2017) 18(11):2362.
[110] van Bambeke F, Balzi E, Tulkens PM. Antibiotic efflux pumps.
Biochemical Pharmacology (2000) 60(4):457–470.
[111] Paulsen IT, Sliwinski MK, Nelissen B, Goffeau A, Saier MH.
Unified inventory of established and putative transporters encoded
within the complete genome of Saccharomyces cerevisiae. FEBS
Letters (1998) 430(1-2):116–125.
[112] Paulsen IT, Sliwinski MK, Saier MH. Microbial genome analyses:
global comparisons of transport capabilities based on phylogenies,
bioenergetics and substrate specificities 1 1Edited by G. Von Heijne.
Journal of Molecular Biology (1998) 277(3):573–592.
[113] Lubelski J, Konings WN, Driessen AJM. Distribution and
Physiology of ABC-Type Transporters Contributing to Multidrug
Resistance in Bacteria. Microbiology and Molecular Biology
Reviews (2007) 71(3):463–476.
[114] Levy SB. Active efflux, a common mechanism for biocide and
antibiotic resistance. Journal of Applied Microbiology (2002)
92(s1):65S-71S.
[115] Hasdemir U. Çoklu Ilaç Direncinde Bakteri Hücre Duvari
Organizasyonu ve Aktif Pompa Sistemlerinin Rolü [The Role of
Cell Wall Organization and Active Efflux Pump Systems in
Multidrug Re-sistance of Bacteria]. Mikrobiyoloji Bülteni [Bulletin
of Microbiology] (2007) 41:309–327.
[116] Li XZ, Nikaido H. Efflux-Mediated Drug Resistance in Bacteria.
Drugs (2009) 69(12):1555–1623.
[117] Wada A, Kono M, Kawauchi S, Takagi Y, Morikawa T, Funakoshi
K. Rapid Discrimination of Gram-Positive and Gram-Negative
Bacteria in Liquid Samples by Using NaOH-Sodium Dodecyl
Sulfate Solution and Flow Cytometry. PLoS ONE (2012)
7(10):e47093.
[118] Mahamoud A, Chevalier J, Alibert-Franco S, Kern W, Pagès JM.
Antibiotic efflux pumps in Gram-negative bacteria: the inhibitor
response strategy. Journal of Antimicrobial Chemotherapy (2007)
59(6):1223–1229.
[119] Lomovskaya O, Bostian K. Practical applications and feasibility of
efflux pump inhibitors in the clinic—A vision for applied use.
Biochemical Pharmacology (2006) 71(7):910–918.
[120] Pagès JM, Masi M, Barbe J. Inhibitors of efflux pumps in Gram-
negative bacteria. Trends in Molecular Medicine (2005) 11(8):382–
389.
[121] Mallea M, Chevalier J, Bornet C, Eyraud A, Davin-Regli A, Bollet
C, et al. Porin alteration and active efflux: two in vivo drug
resistance strategies used by Enterobacter aerogenes. Microbiology
(1998) 144(11):3003–3009.
[122] Thanassi DG, Cheng LW, Nikaido H. Active efflux of bile salts by
Escherichia coli. Journal of Bacteriology (1997) 179(8):2512–2518.
[123] Kern W, Steinke P, Schumacher A, Schuster S, Baum H, Bohnert J.
Effect of 1-(1-naphthylmethyl)-piperazine, a novel putative efflux
pump inhibitor, on antimicrobial drug susceptibility in clinical
isolates of Escherichia coli. Journal of Antimicrobial Chemotherapy
(2005) 57(2):339–343.
[124] Amaral L, Martins A, Spengler G, Molnar J. Efflux pumps of Gram-
negative bacteria: what they do, how they do it, with what and how
to deal with them. Frontiers in Pharmacology (2014) 4:168.
[125] Eilam Y. Membrane effects of phenothiazines in yeasts. I.
Stimulation of calcium and potassium fluxes. Biochimica et
Biophysica Acta (BBA) - Biomembranes (1983) 733(2):242–248.
[126] Kristiansen J, Mortensen I, Nissen B. Membrane stabilizers inhibit
potassium efflux from Staphylococcus aureus strain No. U2275.
Biochimica et Biophysica Acta (BBA) - Biomembranes (1982)
685(3):379–382.
[127] Kaatz G, Moudgal V, Seo S, Kristiansen J. Phenothiazines and
Thioxanthenes Inhibit Multidrug Efflux Pump Activity in
Staphylococcus aureus. Antimicrobial Agents and Chemotherapy
(2003) 47(2):719–726.
[128] Handzlik J, Matys A, Kiec-Kononowicz K. Recent Advances in
Multi-Drug Resistance (MDR) Efflux Pump Inhibitors of Gram-
Positive Bacteria S. aureus. Antibiotics (2013) 2(1):28–45.
[129] Omar MS, Damanhuri NS, Kumolosasi E. Influences of proton
pump inhibitor on Helicobacter pylori adherence to the
gastrointestinal cell lines. The Turkish Journal of Gastroenterology
(2017) 28(1):53–59.