Journal of Applied Microbiology Symposium Supplement 2002, 92, 65S–71S
Active efflux, a common mechanism for biocide
and antibiotic resistance
S.B. Levy
Centre for Adaptation Genetics and Drug Resistance and the Departments of Molecular Biology and
Microbiology and of Medicine, Tufts University School of Medicine, Boston, MA, USA
1. Summary, 65S
2. Introduction, 65S
3. Efflux, 65S
3.1. Tetracycline-resistant bacteria, 66S
3.2. Tet proteins, 66S
1. SUMMARY
Energy-driven drug efflux systems are increasingly recog-
nized as mechanisms of antibiotic resistance. Chromo-
somally located or acquired by bacteria, they can either be
activated by environmental signals or by a mutation in a
regulatory gene. Two major categories exist: those systems
energized by proton motive force and those dependent on
ATP. The pumps may have limited or broad substrates, the
so-called multiple drug resistance pumps, which themselves
form a number of related families. The multiple antibiotic
resistance (mar) locus and mar regulon in Escherichia coli and
other members of the Enterobacteriaceae is a paradigm for a
generalized response locus leading to increased expression of
efflux pumps. One such pump, the AcrAB pump extrudes
biocides such as triclosan, chlorhexidine and quaternary
ammonium compounds as well as multiple antibiotics. In
Pseudomonas aeruginosa, a number of multidrug efflux
pumps export a broad range of substrates. Since bacteria
expressing these pumps thwart the efficacy of both kinds of
therapeutic agents which control infectious diseases –
biocides which prevent transmission of infectious disease
agents and antibiotics which treat and cure infectious
diseases – they are of particular concern. The prudent use
of antibiotics and biocides will guard against the selection
and propagation of drug – resistant mutants and preserve
the efficacy of these valuable anti-infective agents.
Correspondence to: Dr S.B. Levy, Centre for Adaptation Genetics and Drug
Resistance and the Departments of Molecular Biology and Microbiology and of
Medicine, Tufts University School of Medicine, Boston, MA 02111, USA
(e-mail: stuart.levy@tufts.edu).
ª 2002 The Society for Applied Microbiology
3.3. Efflux blocking, 67S
4. Regulation of multidrug efflux, 68S
5. Resistance to biocides, 69S
6. Conclusions, 70S
7. References, 70S
2. INTRODUCTION
Bacteria respond to the widespread use of growth inhib-
itory agents, such as antibiotics, by emerging with progeny
resistant to these substances. While some bacteria acquire
resistance traits from other bacteria, many become resistant
following mutations in the chromosomal gene coding for
the target of the growth inhibitory compound. Increasingly
recognized are energy-driven drug efflux systems intrinsic
to bacteria which are either activated in response to
environmental signals or by a mutation in a gene which
regulates their expression. Regulatory loci, such as the
multiple antibiotic resistance (mar) locus in E. coli and
other members of the Enterobacteriaceae, are also subject to
activation or mutation and affect the expression of genes
which mediate multidrug resistance. When MarA of the
locus is activated, either through mutation in the regulator
MarR or in response to external stimuli, the expression of
over 60 genes is altered, including the upregulation of the
AcrAB multidrug efflux pump. The latter exports biocides
(e.g. triclosan, chlorhexidine and QACs) as well as multiple
antibiotics as do endogenous multidrug efflux pumps in
Ps. aeruginosa. These kinds of pumps raise special public
health concern since bacteria expressing them will resist
biocides designed to prevent transmission of infectious
disease agents and antibiotics aimed at curing infectious
diseases.
3. EFFLUX
Efflux as a mechanism of drug resistance has clearly come of
age. Initially considered a curiosity when first described for
the tetracyclines (McMurry et al. 1980), today a large
number of integral membrane and membrane-associated
proteins are involved in pumping antibiotics, biocides and
66S S . B . L E V Y
other substances out of the microbial cell. Efflux pumps
come in a variety of structures (Nikaido 1996; Paulsen et al.
1996). A single protein may act alone to perform efflux.
Some of these single protein systems use ATP as an energy
source, e.g. the Lmr protein in Lactobacillus (van Veen
1999). Other forms of a single polypeptide use proton
motive force to energize transport of the drug. Tet proteins,
which deal with tetracyclines, are prototype examples of
proton motive force-dependent single polypeptide efflux
pumps (Levy 1992; McMurry and Levy 2000). Alternat-
ively, three-component systems, as exemplified by the
MexAB/OmpM protein complex in Pseudomonas (Li et al.
1995) and AcrAB-TolC in E. coli (Fralich 1996), involve an
integral membrane protein and an outer membrane porin
connected by a cytoplasmic fusion protein.
The expression of all tetracycline resistance efflux systems
described to date is regulated (Levy 1989; McMurry and
Levy 2000). In fact, all variants of the TetA type Gram-
negative efflux pump have a similar genetic organization,
consisting of a repressor gene upstream and transcribed in
the opposite direction to the gene for the structural efflux
protein. The repressor acts in a classic negative fashion to
prevent transcription of the efflux gene. Tet proteins appear
to have evolved specifically to handle tetracyclines or very
related molecules since the affinity of tetracyclines for the
protein is high (Km 6–20 lmol l)1; McMurry et al. 1980;
Yamaguchi 1990). In contrast, the multidrug efflux pumps,
e.g. MexAB and AcrAB, are not saturable by the known
substrates; the affinity for any of the substrates is very low.
3.1 Tetracycline-resistant bacteria
In the author’s initial studies of tetracycline-resistant
bacteria, tetracycline accumulation in tetracycline-resistant
and -susceptible isogenic strains with and without energy
was compared (McMurry et al. 1980). The drug was
actively accumulated in susceptible cells only when energy
(e.g. lactate) was supplied. When the cells were
de-energized, in the presence of the ionophore dinitrophenol
(DNP), accumulation was greatly reduced and represented
that occurring by passive diffusion. Provided with lactate,
the same bacterium, bearing a plasmid which specified
resistance to tetracycline by an efflux pump, accumulated
tetracycline at a level below that observed in the
de-energized cell, i.e. that achieved by passive diffusion.
This finding was the first indication that cells were actively
keeping the drug out of the cell by some means. Before it
could be called efflux, it had to be demonstrated that
tetracycline was being actively removed from the cell. A
system initially described by Barry Rosen to demonstrate
calcium efflux was adapted (Tsuchiya and Rosen 1976).
After the outer membrane was removed, E. coli were
ruptured by passing them through a French pressure cell. In
ª 2002 The Society for Applied Microbiology, Journal of Applied Microbiology Symposium Supplement, 92, 65S–71S
the process, the inner membrane was flipped inside out to
produce everted inner membrane vesicles (McMurry et al.
1980). Radioactive tetracycline entered the inverted vesicles
in the presence of lactate as an energy source and was
assayed when the vesicles were trapped on filters. In this
assay, the tetracycline being pumped into the vesicles
actually represented what would normally be pumped out of
whole cells. This technique demonstrated very clearly that,
in the presence of an energy source, tetracycline accumu-
lated in the everted membrane vesicles (to 300 lmol l)1),
significantly above the external concentration (5 lmol l)1).
When an energy inhibitor (such as DNP or carbonyl cyanide
m-chlorophenol hydrazone (CCCP)) was added, the accu-
mulated tetracycline came streaming out of the vesicles.
This finding showed that the drug was not accumulating in
the everted membrane vesicles because of precipitation, but
was clearly being pumped into the vesicle; in the absence of
energy, this accumulation did not occur. This transport
could then be described as an ‘active efflux’ system, the first
described for an antibiotic (McMurry et al. 1980; Levy
1992). Further studies by the author’s group (McMurry
et al. 1980) and also by Yamaguchi and his group (Kaneko
et al. 1985) demonstrated that this efflux by Tet proteins
involved an antiport system exporting a tetracycline : cation
complex in exchange for a proton in an electroneutral
exchange (Fig. 1).
3.2 Tet proteins
Most Tet proteins in Gram-negative bacteria consist of a
six-plus-six organization of 12 transmembrane regions. The
author and colleagues demonstrated some time ago that the
Cell
H+
TET
OUT PROTEIN IN
Tc:Mg2+ Membrane
Fig. 1 Tetracycline efflux by the inner membrane Tet protein
involves the exchange of one proton for a tetracycline : cation complex
in an electroneutral antiport reaction

two halves of the protein were highly homologous and
appeared to have arisen by gene duplication (Rubin et al.
1990). In fact, by looking at other members of the major
facilitator family, a strong similarity can be seen between the
alpha (N-terminal) and beta (C-terminal) halves suggesting,
as for Tet protein, a common evolution by duplication of
one domain and linkage of the two by a cytoplasmic string of
amino acids. If either the alpha or beta domain of the Tet
protein is deleted or mutated, resistance is not mediated,
both domains being needed for resistance (Curiale and Levy
1982; Curiale et al. 1984). If inactive Tet proteins with a
single mutation in either the alpha or beta domain are placed
in the same bacterial cell, resistance is restored (Curiale et al.
1984). If an alpha domain from a Class C determinant (there
are now over 30 classes of tetracycline resistance determi-
nants) is fused with the beta domain from a Class B
determinant, resistance is not achieved (Rubin and Levy
1990). There is a necessary ‘cross-talk’ between the alpha
and beta domains of the same class in order to provide
resistance, i.e. the alpha and beta halves have evolved
together to be able to interact. However, suppressor
mutations in one or the other half of a TetC/B hybrid
allow the domains to ‘talk’ again, providing tetracycline
resistance (Saraceni-Richards and Levy 2000a, 2000b). Such
studies have suggested a model for the Tet protein. These
findings complement much more comprehensive site-direc-
ted mutational studies by Yamaguchi and his group
(Tamura et al. 2001).
Data were sought to understand how the Tet protein
existed in the membrane. The amino acid sequence would
suggest that there are 12 alpha-helical transmembrane
structures. Tet protein was purified using a method that
had previously been successful for cytoplasmic proteins. A
polyhistidine tail was placed on the Tet protein and then
purified on a nickel column. Greater than 90% pure Tet-his
protein was obtained after washing the column with low
concentrations of imidazole and then elution with high-dose
imidazole. Circular dichroism (CD) spectral analysis in vitro
was performed with the Tet protein preparation in dode-
cylmaltoside. The analysis at wavelengths of 180–260 and
200–260 nm provided information on the alpha-helical
content of the purified protein (Aldema et al. 1996). These
data correlated well with the theoretical value of alpha-
helical structure and made it clear that the protein contained
about 65% alpha-helical structure.
In other studies, binding to a nickel column was used to
investigate the interaction between the alpha and beta
domains as suggested by genetic studies (Curiale and Levy
1982; Curiale et al. 1984). The results obtained were not
those expected. Purified alpha or beta domain polypeptides
or full-length Tet protein were passed over nickel columns
bearing either the alpha or beta domain. No Tet protein or
half Tet protein bound to the column bearing the beta
ª 2002 The Society for Applied Microbiology, Journal of Applied Microbiology Symposium Supplement, 92, 65S–71S
ACTIVE EFFLUX 67S
domain. In contrast, the column to which the alpha domain
was attached retained alpha domain polypeptides or full-
length Tet proteins but no beta domain polypeptide
(McMurry and Levy 1995). This result allowed the design
of a model for Tet as a dimer in the membrane based on
alpha–alpha domain interaction (McMurry and Levy 1995).
The structure agreed with prior genetic complementation
data that implied a multimer structure (Hickman and Levy
1988). It was concluded that the most likely structure for the
protein in the cell was a multimer, at least a dimer.
Current data suggest that the structure is more compli-
cated. With collaborators in the UK, the author’s group has
obtained two-dimensional crystals of Tet protein in lipid
membranes. Both multiple and single lipid-embedded
crystals were analysed. A trimer picture emerged at 17 Å
resolution, involving three alpha domains forming a central
core with beta domains in the periphery (Yin et al. 2000).
There are a number of related membrane proteins which
confer tetracycline resistance bearing 12 or 14 transmem-
brane alpha-helical structures (Levy 1992). Besides Tet
proteins, there are also those which mediate the efflux of
chloramphenicol, ethidium bromide, norfloxacin, as well as
14 trans-membrane proteins which efflux tetracycline and
others which efflux the QACs (Levy 1992). Each is
energized by proton motive force. The Tet protein can
serve as a model for understanding how these other related
proteins function.
3.3 Efflux blocking
Work that began in the author’s laboratory at Tufts
University and is now being continued by Paratek
Pharmaceuticals (Boston, MA, USA) aims to identify
new tetracyclines which either block the efflux pump or get
around it. If the ability of Tet protein to pump tetracy-
clines out of the cells could somehow be interrupted, the
classic tetracyclines would be returned to power. Mark
Nelson, in the author’s group, began to design and
synthesize new tetracyclines. In the initial work, very
simple changes made on the molecule provided effective
efflux blockers (Nelson and Levy 1999). One contained a
13-cyclopentyl on the sixth carbon of the tetracycline
molecule. The studies used the everted vesicle assay to
identify the blocking tetracycline derivatives. When the
potential inhibitor was isotetracycline, there was no effect
on tetracycline uptake (Fig. 2) but, with 13-cyclopentyl
tetracycline (CPTC), there was significant inhibition of the
tetracycline uptake at less than 1 lmol l)1. Further studies
showed that CPTC competitively inhibited tetracycline
uptake in everted vehicles (Levy and Nelson 1998). If
everted vesicles were pretreated with 13-cyclopentyl, the
efflux of tetracycline was inhibited producing a synergistic
drug combination (Nelson and Levy 1999).
68S S . B . L E V Y
CH3
OH
O
OH
OH O OH O
(a)
H-Tc mM, internal concentration
14
12
10
8
6
4
2
3
0 0
0 1
Time (min)
Fig. 2 Competition by tetracycline derivatives for tetracycline uptake in everted membrane vesicles: (a) isotetracycline and (b) cyclopentyl
tetracycline (CPTC). Accumulation of 3H-tetracycline was assayed in energized everted membrane vesicles in the presence and absence of two
different tetracycline derivatives. CPTC showed strong competition with the uptake of 3H-tetracycline. h, Control; r, 100 lg ml)1; j, 200 lg ml)1
Using additional chemistry at the C13 position, Nelson
was able to derive structure–activity relationships, providing
a length and width relationship of a proposed hydrophobic
pocket in the Tet protein (Levy and Nelson 1998). This
information has helped to define potential efflux blockers as
well as new tetracyclines which are not subject to efflux.
Fortunately, these same tetracycline derivatives were not
subject to the second mechanism of tetracycline resistance,
ribosomal protection.
4. REGULATION OF MULTIDRUG EFFLUX
Some time ago, the author discovered a multiple antibiotic
resistance (mar) operon was discovered in E. coli which
consisted of genes for three proteins: a regulator MarR, a
transcriptional activator MarA and a small protein MarB,
for which a function is not yet known. A second transcript
in the other direction which overlaps the operator-promoter
site (marO) specifies a six transmembrane integral mem-
brane protein designated MarC, the function of which is
unknown (Alekshun and Levy 1997). The mar locus
controls the cell’s response to multiple different toxic
substances. While named the ‘multiple antibiotic resistance’
locus, it is probably best described as a ‘multiple adapta-
tional response’ locus because it deals not only with
antibiotic resistance but also cell metabolism, DNA repair,
ª 2002 The Society for Applied Microbiology, Journal of Applied Microbiology Symposium Supplement, 92, 65S–71S
S
N(CH3)2 13CH
OH N(CH3)2
2
OH OH
NH2 NH2
OH
O OH O O
OH O
12 (b)
10
8
6
4
2
2 3 0 1 2 3
Time (min)
superoxide stress and other physiological systems linked to
cell danger (Barbosa and Levy 2000; Okusu et al. 1996).
For antibiotic and biocide resistance, mar upregulates
expression of the multidrug efflux pump AcrAB and its
outer membrane TolC component, a functional homologue
of the Mex pumps in Pseudomonas (Li et al. 1995). The mar
locus, through MarA, increases expression of micF, an
RNA molecule which causes reduced production of the
principal outer membrane porin OmpF (Cohen et al. 1988).
The MarR protein is affected by a number of structurally
unrelated compounds (Seoane and Levy 1995). By exam-
ining the fold increase in LacZ activity when fused to marO
in the presence of MarR, different inducers of the mar
operon were identified, including uncouplers, such as
CCCP and DNP, and redux-cycling agents, such as
menadione. In collaboration with Lee Rosner at the
National Institutes of Health, salicylate and acetometaphen
were found to be inducers (Cohen et al. 1993). Many of the
inducers can directly affect MarR in vitro (Alekshun and
Levy 1999). When the cell goes into a so-called ‘Mar state’,
numerous changes occur. Quinolones lose their bacterio-
cidal activity and become bacteriostatic agents at relatively
low levels of drug resistance: two- to four-fold resistance to
multiple drugs (Goldman et al. 1996). Mar does not
initially provide high-level resistance; it is often the first
step to higher levels of resistance, secondary to drug target

mutations (Oethinger et al. 1998). In collaboration with
colleagues at Boston University, the author’s group resolved
the crystal structure of a dimer of MarR at 2Æ3 Å (Alekshun
et al. 2001). It has a winged helix form with two likely
DNA-binding domains. The protein is now being crystal-
lized with different substrates as well as with DNA to
determine its substrate-binding site and to confirm the
putative DNA-binding sites. There are over 50 known
homologues of MarR, one of which is MexR which controls
the MexAB efflux system in Ps. aeruginosa (Li et al. 1995;
Nikaido 1996).
The author’s group recently published an examination of
the mar regulon using macroassays (Barbosa and Levy
2000). Overexpression of MarA affected the expression of
more than 60 genes. In addition to genes whose functions
were known, the work identified genes for which no
function has been assigned. Some are conserved among
other bacterial genomes. While of unknown function, they
presumably have something to do with stress because they
are affected by this stress locus. Through the MarA
activity, it was further noted that combining decreased
outer membrane permeability (by decreasing porins) with
indigenous efflux systems produced resistance much higher
than expected from diffusion or efflux alone (Levy 1990).
The combination of outer membrane impermeability and
inner membrane efflux enhanced the effect of the two
changes in drug transport. If resistance is linked to a
cytoplasmic target mutation, such as mutated topoisomer-
ase for fluoroquinolone resistance, efflux amplifies the
effect of the mutation (Kern et al. 2000).
5. RESISTANCE TO BIOCIDES
The recognition by the author’s group of the relationship
between household products and antibiotic resistance
bilesalts
Bile salts
organic
Organic
solvents
solvents
Fig. 3 MarA from the mar locus in Escherichia
coli causes upregulation of the AcrAB/TolC
complex leading to the efflux of antibiotics,
organic solvents, antiseptics, disinfectants and
other diverse structural compounds
ª 2002 The Society for Applied Microbiology, Journal of Applied Microbiology Symposium Supplement, 92, 65S–71S
ACTIVE EFFLUX 69S
initially emerged from work with Merri Moken, a New
Jersey high school student. She selected E. coli mutants
resistant to pine oils and noted that they were also resistant
to antibiotics. She shared her unexpected findings and it was
determined that the mutants constitutively expressed MarA
(Moken et al. 1997). The mutations were in MarR which
allowed MarA expression and activation of the multidrug
efflux pump AcrAB, which was able to efflux pine oils as
well as antibiotics.
While removing the mar locus did not affect triclosan
resistance, overexpression of the MarA protein via a MarR
mutation led to a three- to four-fold increased triclosan
resistance – exactly as occurs with antibiotics (McMurry
et al. 1998a). The same phenomenon is seen with a SoxR
mutation leading to overexpression of the related SoxS
protein. Similarly, overexpression of the AcrAB efflux pump
itself will provide resistance (Wang et al. 2001). Mar is able
to turn on efflux systems, and perhaps other mechanisms,
for drug resistance.
Given the two types of effect of triclosan on bacterial cells,
bacteriostatic at low and bacteriocidal at high levels, the
effect of the AcrAB pump on these two activities was
investigated (Levy 2001). At low levels, triclosan inhibited
growth in the wild type cell (50% at 0Æ15 lg ml)1; up to
90% at 0Æ6 lg ml)1); the amount causing lysis (bactericidal)
was 8 lg ml)1. When the acrAB gene complex, and thus the
constitutive expression of the AcrAB pump, was removed
the growth inhibitory level of triclosan was reduced 10-fold
in the wild type cell. Deletion of the pump also affected cell
lysis; the amount of triclosan needed to cause lysis dropped
from 8 to 3–4 lg ml)1. This result was unexpected since
lysis had been considered a chemically-modified membrane
disruption.
In one of the high level triclosan E. coli mutants with a
mutation in the FabI protein (the target for triclosan
antibiotics
Antibiotics antiseptics
Antiseptics
Disinfectants
disinfectants
pineoils
Pine oils
MarA AcrAB/TolC
70S S . B . L E V Y
(McMurry et al. 1998b)) 50% growth inhibition was at
13 lg ml)1, a 100-fold increase over the wild type cell. It
was not possible to find sufficiently soluble triclosan, above
32 lg ml)1, to give 90% inhibition. Similarly, lysis did not
occur within the solubility limits of triclosan. When the
AcrAB pump was eliminated, growth inhibition dropped by
10-fold with the MIC 90% at 2Æ1 lg ml)1. Remarkably, the
amount needed for cell lysis became the same as that for the
wild type cell, i.e. 3–4 lg ml)1. These data strongly imply
that triclosan does not cause lysis by a non-specific chemical
reaction; it has to get into the cell and its action is impeded
by the AcrAB efflux pump. The pump somehow protects
the cell by keeping the drug from another target independ-
ent of FabI. If the multidrug resistance pumps can be
inhibited, the activity of triclosan and other drugs against
E. coli and other bacteria can be greatly increased.
In summary, the MarA protein of the mar operon
upregulates AcrAB and the TolC outer membrane protein
to produce resistance to antibiotics, organic solvents (e.g.
cyclohexane) pine oils, bile salts and antiseptics and
disinfectants, such as triclosan, QACs and chlorhexidine
(Fig. 3). This regulatory protein protects the cell from
biocides and antibiotics through at least one known mech-
anism – antibiotic efflux pumps.
6. CONCLUSIONS
As with antibiotics, given enough time and dose, biocide
resistance will emerge. The continued effect of the biocides
and the increasing number of resistance determinants (target
gene or otherwise) will produce widespread biocide resist-
ance. If biocides are spread all over households, diluted down
to less than their growth inhibitory concentrations, bacteria
with resistance mutations will emerge (McMurry et al.
1998b). The same phenomenon has occurred for antibiotics,
as illustrated by the newly emerging resistance to vancomy-
cin among Staphylococcus aureus and to fluoroquinolones
among E. coli. Some of the resistance mechanisms, such as
efflux, can provide cross-resistance to other drugs. As urged
in the use of antibiotics, prudent use of antibacterial agents,
namely those which leave residues, should be followed in
order to curb resistance and preserve efficacy of these
products when needed to protect the vulnerable patient.
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