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Antibiotics are one
of the most successful treatment worldwide. Their use has aided to reduce
childhood mortality and increased life expectancy. They have successfully
prevented or treated infections in many patients such as those who have
received chemotherapy and those with complex surgeries. However, there has been
an increase in incidence of antibiotic resistance worldwide, which lead to a
rise in untreatable infections.5   Antibiotic
resistance infections has become an economic burden for the patients, their
families and health care system. It has been found that in Europe alone 25,000
people die each year due to multidrug-resistant bacterial infections and concurrent
cost to the European Union economy is roughly €1.5 billion annually 2. Antibiotic
resistance infections are found to be more common in hospitals due to the high
number of vulnerable patients who are admitted, the elevated use of antibiotic
and invasive surgeries that take place in these settings. The development of
resistance can delay the administration of antibiotic therapy which means
patients will require prolonged hospital stay (from 6.4 to 12.7 days) and
therefore greater hospital charge. It is challenging and takes massive amount of time to develop
new antibiotics. Thus it becomes essential to protect the current antibiotics
from developing new modes of resistance and finding ways to overcome resistance.
It is also vital to have coordinated efforts to come up with new guidelines,
implementation of these guideline’s and new research programmes in order to
overcome the spread of antibiotic resistance. 6


There are several mechanisms in which
gram-negative bacteria such as E.coli
can develop resistance. Resistance can occur due to; mutations involved in
specific antimicrobial targets, antimicrobial inactivation through production
of B-lactamase enzymes, acquisition of mobile genetic material via plasmids,
transposons, or integrons, alteration in the cell wall composition, reduced
number or porins in the cell wall, and over production of efflux pumps. 3 Out
of all these different mechanisms of resistance to antibiotics, efflux pumps
interact synergistically with other resistance mechanisms such as membrane
permeability and those that have been mentioned above. Efflux pumps, therefore plays
a huge role in antibiotic resistance and currently presents a major challenge during
development of antibiotics 7.   In E.coli
there are five different antibiotic efflux transporters (Fig 1). These include;
Small Multidrug Resistance (SMR) family, the Multidrug And Toxic compound
Extrusion (MATE) family, the Major Facilitator Superfamily (MFS), the
ATP-Binding Cassette (ABC) family and the Resistance-Nodulation-cell Division
(RND) family. Out of the five family of efflux pumps, the ABC pump require
energy released from the hydrolysis of ATP to remove antibiotic out of the
cell, whereas the other four efflux pumps use electrochemical gradient. 4 The
RND transporter is part of a tripartite complex, which includes three subunits;
acrB, tolC, and acrA (which links together acrB and tolC). The RND tripartite
complex span the inner membrane, the periplasm and the outer membrane channel. The
RND pump are much more efficient in creating intrinsic and acquired resistance
to antibiotics (in particular the AcrB subunit), because the pump actively
pumps out antibiotic out into the external medium, whereas the other pumps
excrete the antibiotic into the periplasm and therefore there is a rapid back
diffusion of drug back into the cytosol. The AcrB subunit, has two binding
pockets, which can bind to substrates of different sizes and properties. This
property is responsible for the resistance seen in large number of drugs such
as quinolones, tetracycline, macrolides, chloramphenicol, novobiocin and
B-lactams 11. However, it’s important to note, all three components of the RND
pump is needed for drug efflux property, the absence of just one subunit could
make the whole pump non-functional. For example the AcrA subunit is needed to stimulate the activity of the pump. 12

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Since efflux pumps plays a major role in
antibiotic resistance, inhibiting these pumps would be a promising strategy to
protect the efficiency of various antibiotics and possibly could help the re-introduction
of ineffective antibiotics back into use. There have been many efflux pump inhibitors
that have been studied in the past including natural products, synthetic
molecules and antibiotics. Inhibiting the efflux pump has many advantage. it
helps to increase the intracellular concentration of antibiotic within a
bacterial cell to a level that is required to show its activity and therefore
help to reduce the minimal inhibitory concentration that is needed for the
antibiotic to kill the resistant organism. It also helps to reduce the capability of
bacteria to acquire additional resistance and to re-establish the drug susceptibility
of resistant strains in clinics.


Natural products that have been obtained
by plants have shown weak antimicrobial effect and have very little or no
toxicity when used clinically. Therefore, natural products can be used
clinically to overcome resistance in bacteria by inhibiting efflux pumps. It
has been also known before that some antibiotics seems to exert synergy when
used in combination with Efflux Pump Inhibitors (EPI). Chequerboard assay have
been used in the past to identify such EPI 8. Currently there are no
EPI/antimicrobial drug combinations that are commercially available for
clinical use. However, research is still ongoing in finding a suitable EPI. Many
plant compounds that have been previously studied has shown to act synergistically
with antibiotics on gram-positive bacteria 9, 10.  In this study, two natural products were selected;
Reserpine and Quercetin both of which is isolated from plants to act as EPI. Quercetin is a typical flavonol-type flavonoid and
limited studies have been carried out to test its ability as efflux pump
inhibitor. Reserpine which is isolated from the
roots of Rauwolfia vomitoria Afz has
shown EPI activity against the efflux pump called Bmr of Bacillus subtilis. However, not many studies have been carried out
to show the EPI activity of Reserpine in gram-negative bacteria such as in E.coli strain. Gram-negative species are
the most problematic bacteria to treat in the future.8 New treatments against
gram-negative bacteria is needed due to the intrinsic high drug resistance and
the increased incidence of drug-resistant Gram-negative infections.


The aim of the study was to investigate
the synergic effects of natural products as efflux pump inhibitors with
antibiotics. Reserpine and Queceptin  were used in combination with two different
anitbiotics; tetracycline and chloramphenicol on both mutant (M843 ACRA- has a
non-functioning RND efflux pump) and wildtype(BW25113-has a functioning RND
efflux pump) strains of E.coli. Another
aim of the study was to determine the Minimal Bactericidal Concentration (MBC),
to see if natural product/ antimicrobial drug combinations had a bactericidal
or bacteriostatic effect. 

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