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Molecular mechanisms of antibiotic resistance

Gerard D. Wright*
DOI: 10.1039/c0cc05111j
Published on 01 February 2011 on http://pubs.rsc.org | doi:10.1039/C0CC05111J

Over the past decade, resistance to antibiotics has emerged as a crisis of global
proportion. Microbes resistant to many and even all clinically approved antibiotics are
increasingly common and easily spread across continents. At the same time there are
fewer new antibiotic drugs coming to market. We are reaching a point where we are no
longer able to confidently treat a growing number of bacterial infections. The molecular
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mechanisms of drug resistance provide the essential knowledge on new drug

development and clinical use. These mechanisms include enzyme catalyzed antibiotic
modifications, bypass of antibiotic targets and active efflux of drugs from the cell.
Understanding the chemical rationale and underpinnings of resistance is an essential
component of our response to this clinical challenge.

Antibiotics and resistance: a discovered by Ehrlich at the turn of the
20th century and the sulfonamide inhibitors
delicate balance
of folate metabolism developed by
Antibiotics are arguably the most Domagz in the 1930s. With the develop-
successful drugs deployed over the past ment of penicillin in the 1940s however,
100 years, responsible not only for saving the field turned to microbially derived
countless lives but also enabling modern natural products as a rich source of
medical procedures that otherwise would antibiotics. Indeed the majority of
be unthinkable. The first antibiotics successful chemical scaffolds that
were synthetically prepared chemicals, continue to find use in treating microbial
notably the arsenicals such as Salvarsan infections today are dominated by these
compounds.1 As a class of pharmaceuticals,
antibiotics have been economic
M.G. DeGroote Institute for Infectious powerhouses, representing 5% of the
Disease Research, McMaster University, global drug market and resulting in
1200 Main St W, Hamilton, ON, Canada.
$42B USD in sales in 2009.2
E-mail: wrightge@mcmaster.ca;
Fax: +1 905 528 5330; Despite this success, new antibiotic
Tel: +1 905-525-9140 discovery is perhaps at its nadir since
Gerry Wright has over 20 years of experience in
antibiotics and resistance research. Areas of emphasis
include resistance to glycopeptides, aminoglycosides,
lincosamides, streptogramins, tetracyclines, lipo-
peptides, macrolides, and b-lactams. He is the co-
founder of the McMaster High Throughput Screening
Laboratory, holds a Tier 1 Canada Research Chair in
Antibiotic Chemical Biology and is the Director of the
Michael G. DeGroote Institute for Infectious Disease
Research.
Gerard D. Wright
the pre-antibiotic era. Among the many
reasons for this state of affairs is the fact
that all antibiotics, whether they originate
as products of microbial secondary
metabolism or are completely synthetic,
are subject to resistance. Unlike any
other class of drugs, antibiotics have a
limited lifespan of utility. With the use
and misuse of antibiotics selected for
multi-drug resistant organisms, the fact
that many resistance elements are
readily passed on both vertically to
microbial progeny and horizontally to
their neighbors, and the advent of
affordable global travel, resistance is
inevitable and often rapidly spread
through microbial populations across
the world.3 Resistance is therefore a
significant barrier to antibiotic develop-
ment and deployment.
Paradoxically, the past decade has
seen significant advances in our under-
standing of the mechanisms and chemistry
of antibiotic action and resistance that
can be leveraged in new drug develop-
ment and antibiotic stewardship. In this
review, I outline some of the highlights
of our understanding of the chemical
mechanisms of resistance and its impor-
tance in generating the needed new anti-
biotics over the next decade as well as
advances in the antibiotic target and
compound discovery.

This journal is c The Royal Society of Chemistry 2011 Chem. Commun., 2011, 47, 4055–4061 4055
 View Online

Molecular mechanisms of
antibiotic resistance
Resistance to antibiotics can occur by
modification of a drug target, molecular
bypass, active efflux (or decreased entry),
and chemical modification of the
compound. All of these mechanisms are
clinically important and most antibiotics
are subject to several mechanisms
(Fig. 1). Frequently bacteria can
harbor several distinct resistance
Published on 01 February 2011 on http://pubs.rsc.org | doi:10.1039/C0CC05111J
amino acid with a bulkier side chain
(Leu, Trp, Ile) at position 83, or Asp87
to Asn, Tyr, or Gly is common in
fluoroquinolone resistant GyrA.
Iterative mutations in the same gene
can increase the level of resistance.6
These relatively small changes in an
amino acid sequence alter the protein
structure sufficiently to impede anti-
biotic binding and action as shown
by a recent crystal structure analysis
of the Mycobacterium tuberculosis
Target modification can also arise
through highly efficient and regio-
selective modification catalyzed by
enzymes. Ribosome methyltransferases
are an example of this catalytic resistance
strategy (Fig. 2). The Erm enzymes
modify the 23S rRNA of the large sub-
unit of the ribosome at position A2058
(E. coli numbering) conferring resistance
to three structurally distinct classes of
antibiotics: the macrolides such as
erythromycin, lincosamides such as

elements with different mechanisms that wild type and quinolone resistant clindamycin, and type B streptogramins
impact a single class of antibiotics. The GyrA protein.7 like quinupristin. While greatly differing
genome sequence of a single strain of
Acinetobacter baumannii, a Gram
negative opportunistic pathogen,
revealed an 86 kb resistance ‘island’ in
the chromosome comprised of 45 resistance
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genes including multiple b-lactamases,

aminoglycoside modifying enzymes
and efflux systems.4 On the other hand,
specific mechanisms can dominate across
the globe, for example the molecular
bypass mechanism of vancomycin
resistance is the prevailing (though not
exclusive) mode of resistance to glyco-
peptide antibiotics.5

Target modification
A prominent mechanism of resistance is
modification of the molecular target.
Often, this can arise through point
mutations in selected genes resulting in
relatively rapid and facile resistance
where such changes have a minimal
impact on microbe fitness. For example,
the synthetic fluoroquinolone antibiotics
such as ciprofloxacin target the type IIA
topoisomerases required for relaxing
supercoiled DNA at replication forks
and decatenation of daughter strands
during bacterial DNA replication.
Single mutations in target genes such as Fig. 2 Erm methyltransferases chemically modify A2058 sterically and electronically blocking
gyrA provide high level resistance. vital interactions with antibiotics resulting in resistance. The methyl donor SAM is converted to
For example, mutation of Ser to an SAH (S-adenosylhomoserine).

Fig. 1 Antibiotics are targeted by multiple mechanisms of resistance as exemplified by the macrolide erythromycin.

4056 Chem. Commun., 2011, 47, 4055–4061 This journal is c The Royal Society of Chemistry 2011

in molecular structure, these antibiotics
all bind to the peptide exit tunnel region
of the large subunit of the ribosome.
This has been revealed through the
determination of the 3D-structures of
bacterial ribosomes in complex with anti-
biotics, which have revolutionized our
understanding of antibiotic action and
resistance.8–12 A2058 makes direct or
indirect contacts with these antibiotics
through the exocyclic amine of the purine
ring, N6. Erm methyltransferases
Published on 01 February 2011 on http://pubs.rsc.org | doi:10.1039/C0CC05111J
catalyze S-adenosylmethionine (SAM)
dependent mono- and di-methylation of
N6 resulting in loss of H-bond properties
and steric block of the antibiotic
binding site.
Using similar chemistry and molecular
logic, methyltransferases that modify
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A1408 or G1405 of the 16S rRNA of
the small ribosomal subunit have
emerged over the past decade as formidable
agents of broad-spectrum aminoglyco-
side antibiotic resistance.13 A1408 and
G1405 are critical residues for amino-
glycoside–antibiotic interaction with the
ribosome, again revealed by atomic
resolution structures of antibiotic–
ribosome complexes.12
These resistance methyltransferases
demonstrate remarkable substrate
specificity and emphasize the challenge
that relatively small molecular changes
can have on antibiotic activity.
Molecular bypass
Microbes have also evolved mechanisms
that circumvent antibiotic action by
evolving ‘work arounds’ that are not
antibiotic sensitive. The resistance to
glycopeptide antibiotics such as
vancomycin is an elegant example of this
strategy. Vancomycin binds to the
acyl-D-Alanyl-D-Alanine terminus of the
growing peptidoglycan component of
the bacterial cell wall. This terminal
dipeptide is the substrate for enzymes
that crosslink adjacent chains of peptido-
glycan through a transpeptidation
reaction. The importance of these
enzymes is underscored by the fact that
they are the targets of the b-lactam
antibiotics such as the penicillins and
cephalosporins. The crosslinked peptido-
glycan strands are required to strengthen
the cell wall and avoid cell lysis. Since
vancomycin binds to a near ubiquitous
small molecule substrate and not a
This journal is c The Royal Society of Chemistry 2011
protein, it would seem to be impervious
to resistance arising from spontaneous
mutations or other similar mechanisms.
Indeed, vancomycin was used in the
clinic for several decades before
resistance emerged in disease-causing
enterococci in the late 1980s.14
Vancomycin forms a non-covalent
complex with acyl-D-Alanyl-D-Alanine
through a series of five hydrogen bonds
(Fig. 3).15 Resistance comes about
through the substitution of acyl-D-
Alanyl-D-Alanine with the isosteric
depsipeptide acyl-D-Alanyl-D-Lactate.
The replacement of the amide bond of
D-Ala-D-Ala with an ester linkage
eliminates a key H-bond donor and
introduces electronic repulsion that
prevents productive binding of the anti-
biotic (Fig. 3).
This sophisticated resistance
mechanism requires the action of five
new genes. Two genes encode a regula-
tory two-component pathway consisting
of a membrane bound His-kinase, VanS,
and a transcriptional control element,
VanR, that sense the presence/activity
of vancomycin and induce the expression
of three genes encoding enzymes that
reprogram peptidoglycan synthesis:
VanH, VanA, and VanX. VanH is a
D-lactate dehydrogenase that generates
D-Lactate and VanA is a ligase that
catalyzes synthesis of D-Alanyl-D-Lactate
that together provide the depsipeptide
substrate for incorporation into new cell
wall synthesis. Normal D-Alanine-D-
Alanine production continues in the
cell despite the presence of vancomycin.
The VanX protein is a highly specific
Fig. 3 Vancomycin and other glycopeptide antibiotics interact through a network of five hydrogen
bonds with the acyl-D-Ala-D-Ala terminus of growing peptidoglycan chains and their precursors.
Resistance arises from isosteric replacement of the amide with an ester (acyl-D-Ala-D-Lactate)
resulting in loss of one hydrogen bond.
Chem. Commun., 2011, 47, 4055–4061
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DD-dipeptidase that cleaves D-Ala-D-
Ala but remarkably not D-Ala-D-Lactate,
thereby ensuring enrichment of peptido-
glycan with the depsipeptide and high-
level drug resistance. The origins of this
elegant mechanism are likely in the
glycopeptide producing organisms,
which also use the five gene cluster to avoid
suicide during antibiotic production.16
The vast reservoir of antibiotic resistance
elements within producing organisms
and other environmental bacteria is
now well established and has been
termed the antibiotic resistome.17–19
The precise molecular trigger that
induces the VanS–VanR system has been
debated for several years. We recently
designed and synthesized a photoaffinity
probe based on the vancomycin scaffold
and showed that VanS is a receptor
capable of binding the antibiotic.20
Efflux
The active removal of antibiotics from
within the cell is a highly efficient
mechanism of resistance. There are five
families of membrane-spanning efflux
proteins that accomplish this task, and
over the past decade X-ray structures
have been determined for representative
members of each group. Perhaps the
most intriguing and important to
resistance in Gram negative bacteria are
the tripartite resistance-nodulation-cell
division (RND) class. Among the most
studied of the RND family members are
AcrAB-TolC from Escherichia coli, and
MexAB-OprM from Pseudomonas
aeruginosa. Crystal structures of all
4057

these systems have been reported
(reviewed in ref. 21). The RND systems
are comprised of a cell membrane
spanning pump (AcrB, MexB), an
outermembrane pore (TolC, OprM),
and a periplasmic adapter protein
(AcrA, MexA) that bridges the two.
The crystal structures of RND systems
along with biochemical studies have
provided unparalleled detail on the mode
of action of these proteins. Antibiotic
efflux is coupled with vectorial proton
Published on 01 February 2011 on http://pubs.rsc.org | doi:10.1039/C0CC05111J
influx into the cell. The AcrB/MexB
pumps are trimers that recognize a broad
array of small molecules in at least two
cavities (‘‘cave’’ and ‘‘groove’’).22
Biochemical23 and crystallographic24,25
evidence support a trimer rotation
mechanism for passage of molecules
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through the outermembrane pore. These
molecular machines are remarkable in
function and especially challenging to
overcome, spurring the search for broad
spectrum small molecule inhibitors.21,26
Chemical modification
Enzyme-catalyzed inactivation is arguably
the apogee of the bacterial antibiotic
countermeasures.27 The evolution of
such highly efficient catalysts is evidence
of the strong and sustained selective
pressure of antibiotic action and consistent
with the concept of an antibiotic
resistome.18 The first evidence for such
enzymes was reported in 1940 with the
discovery of the penicillin inactivating
b-lactamase activity,28 several years
before penicillin was available for
Fig. 4 General mechanism of Ser and metallo-b-lactamases.
4058 Chem. Commun., 2011, 47, 4055–4061
widespread clinical use. Consistent with
the primacy of b-lactam antibiotics in
medicine (penicillins, cephalosporins,
carbapenems and monobactams)
b-lactamases are among the most
widespread and clinically important
resistance enzymes.29
Two distinct chemical mechanisms are
known: formation of a covalent enzyme
intermediate followed by hydrolysis, or
metal-activation of a nucleophilic water
molecule (Fig. 4).30 The former operates
through a catalytic Ser and is functionally
analogous to Ser proteinases. The
hydroxyl group of the catalytic Ser
attacks the electrophilic carbonyl carbon
of the b-lactam ring resulting in a
covalent enzyme intermediate, mimicking
the modification of peptidoglycan trans-
peptidases that are the antibiotic targets.
Subsequent fast (>103 s1) hydrolysis
releases the ring opened and inactive
antibiotic to the solvent. The metallo-b-
lactamases, on the other hand, operate
through 1–2 active site Zn2+ atoms that
activate a water molecule for direct
attack on the b-lactam centre.
While the general chemical mechanisms
of b-lactamases have not changed over
the past decade, the expansion of anti-
biotic substrate specificity through
evolution of known enzymes and the
emergence of completely new ones has
been dramatic. Chief among these are the
ESBLs (extended spectrum b-lactamases)
so-called because of their ability to
inactivate newer cephalosporins with
broad spectrum antibiotic activity.31
These have arisen either by mutation
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of well known lactamase scaffolds
(TEM, SHV) or by mobilization of
new elements from non-pathogenic
environmental bacteria into pathogens
(e.g. CTX-M).32 The movement of chromo-
somal AmpC b-lactmases onto mobile
genetic elements has also emerged as a
clinical problem as these hydrolyze many
important cephalosporins and are not
susceptible to inhibition by clinically used
b-lactamase inhibitors (see below).33
The distribution of ESBLs and AmpC
b-lactamases has been countered by the
increased use of carbapenems such as
imipenem and meropenem. However this
has given rise to the emergence of the KPC
group of Ser-carbapenemases, creating
an additional clinical challenge.34,35
Finally, metallo-b-lactamases such as
NDM-1, IMP, and VIM, which readily
cleave carbapenems and most other
b-lactams and were rare or undiscovered
in the 20th century, have recently
emerged as a significant clinical threat.36
Scaffold ring opening mechanisms are
also known for other antibiotics including
macrolides and type B streptogramins.
The enzyme Vgb, which acts on type B
streptogramin antibiotics such as the
semi-synthetic quinupristin component
of the combination antibiotic Synercids,
has been determined to have a novel
mechanism (Fig. 5). Type B streptogra-
min antibiotics are depsipeptides cyclized
through a C-terminal carboxylate of a
phenylglycine residue and the secondary
hydroxyl group of a Thr residue. Unlike
the b-lactamases that catalyze ring
opening inactivation through hydrolysis,
c The Royal Society of Chemistry 2011

Published on 01 February 2011 on http://pubs.rsc.org | doi:10.1039/C0CC05111J
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Fig. 5 Predicted mechanism of Vgb lyase.
Vgb is a lyase that operates through an
elimination mechanism.37 Determination
of the 3D-structure of Vgb enabled
exploration of this novel resistance
mechanism.37 C–O bond cleavage requires
abstraction of the proton on the b-carbon,
with predicted pKa > 25. The N-terminal
residue is a 3-hydroxypicolinic acid that
binds a Mg2+ that is required for catalysis.
The 3D-structure of Vgb predicts that the
metal–antibiotic–Vgb complex withdraws
electrons through the 3-hydroxypicolinic
acid setting up the Thr Ca-proton for
deprotonation by an active site His
imidazole base. This predicted mechanism
is supported by mutation and substrate
specificity studies.
Group transfer is a common mode of
antibiotic inactivation. Kinases, adenyly-
transferases, and acetyltransferases
dominate in the clinic, however glycosyl-
transferases and ribosyltransferases are
also known in environmental bacteria.27
These enzymes often share structure and
mechanism with known enzymes of
metabolism and other cellular functions.
For example, aminoglycoside and
Ser/Thr/Tyr protein kinases have a
common protein fold and general catalytic
mechanism. The most studied aminoglyco-
side antibiotic kinase is APH(30 )-IIIa from
Gram positive bacteria. It was the first to
be shown to have the protein kinase fold38
and has been demonstrated to operate
by a dissociative metaphosphate-like
mechanism39 analogous to some Tyr
protein kinases such as Csk.40 Structures
This journal is c The Royal Society of Chemistry 2011
and steady state kinetic studies of other
aminoglycoside kinases have been
reported demonstrating significant
diversity among these enzymes41–44 and it
remains to be seen if the dissociative
mechanism holds for all of them.
Several structures and mechanistic
studies of aminoglycoside antibiotic
acetyltransferases have been reported
and these place the enzymes in the large
GNAT superfamily of acyltransferases
(e.g. ref. 45). The Blanchard group in
particular has been at the forefront of
this body of work over the past
decade.46–50 This group of enzymes has
provided a fascinating example of very
recent evolution of drug resistance in the
selection of an aminoglycoside acetyl-
transferase that also can inactivate members
of the synthetic fluoroquinolone class of
antibiotics such as ciprofloxacin.51 The
enzyme in question, AAC(6 0 )-Ib-cr, is an
aminoglycoside acetyltransferase that
has gained the ability to acylate
unmodified piperazine ring-containing
fluoroquinolone antibiotics.51,52 The
gene encoding this enzyme is now widely
distributed across the globe and is a
Fig. 6 Predicted mechanism of Ser-b-lactamase inhibition by NXL104.
Chem. Commun., 2011, 47, 4055–4061
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sobering example of the rapidity of
evolution of drug resistance by natural
selection.53
Inhibition of resistance
Blocking resistance by inhibition of
resistance elements is a proven therapeutic
strategy. Combinations of b-lactamase
inhibitors clavulanic acid, sulbactam
and tazobactam and various b-lactam anti-
biotics have been in use for >25 years
and have been highly successful and
profitable drugs.30 Several new b-lacta-
mase inhibitors have been reported.30
One of the most interesting is NXL104
from Novexel, which is in Phase II
clinical trials (Fig. 6). NXL104 represents
a new family of diaza-bicyclo-octane
inhibitors of Ser b-lactamases with
broader specificity than the b-lactamase
inhibitors currently in clinical use.
NXL104 forms a covalent acyl–enzyme
complex with b-lactamases with very
slow deacylation rates (h).54 No clinical
trial calibre inhibitors of metallo-b-lacta-
mases have yet been reported. As the
clinical incidence of these enzymes is on
the increase, identifying such molecules
should be a top priority.
This strategy of inhibiting resistance
has so far been limited to the b-lacta-
mases. This reflects the frequency of
b-lactam antibiotic use in the clinic, the
relatively uncomplicated number of
catalytic mechanisms to target
(dominated by the Ser-based mechanism),
and the ‘drugability’ of b-lactamases by
b-lactam-based compounds. In principle
this strategy should be applicable to all
classes of resistance elements, however
the plethora of mechanisms that target
some antibiotics (e.g. aminoglycosides
can be modified by acylation, phosphoryl-
ation, or adenylylation, are subject to
efflux, and can be blocked by ribosomal
methylation) can be daunting. Several
efforts have been made to block efflux
systems with some success (reviewed in
ref. 55), however the fact that many
4059

target pathogens have redundant
efflux systems that can be recruited to
overcome such a blockade makes this
strategy challenging.
Conclusions: the future of
new antibiotics and targets
New antibiotics will be required to over-
come the pressing crisis in antibiotic
resistance. The Infectious Diseases
Published on 01 February 2011 on http://pubs.rsc.org | doi:10.1039/C0CC05111J
Society of America has proposed a
10  20 challenge: delivering 10 new
antibiotics by the year 2020.56 To meet
this ambitious goal new targets and new
strategies will need to be pursued with
vigor. One of the challenges is knowing
which chemical matter to focus on. The
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large libraries of small molecules that
adhere to well established properties of
orally available drugs (e.g. Lipinski’s
rules), which dominate the chemical
libraries of most large pharmaceutical
companies, have so far failed to deliver
any new antibiotics.57 This is likely
because antibiotics occupy a unique
chemical space not well sampled by such
collections.58 Natural products represent
privileged structures with antibiotic
activity, however discovering new
chemical entities has proven so difficult
that the majority of the pharmaceutical
industry has abandoned this strategy.59
Nevertheless, we are in a remarkably
prolific period of discovery of natural
products and understanding of their
biosynthesis from bacterial sources, and
this offers hope for the delivery of new
antibiotic structures.60 Furthermore, the
ability to rapidly clone, characterize, and
manipulate natural product biosynthetic
genes is opening up new opportunities to
deliver complex bioactive chemical
matter for antibiotic screening. The
application of synthetic biology strategies
to natural product synthesis has been
already delivered in the area of anti-
malarials61,62 and could be marshalled
for new antibiotic discovery.
The antimicrobial target field has
never been more exciting. We have high
resolution structures of key targets
including the ribosome, peptidoglycan
assembly proteins, DNA and RNA, as
well as most of the essential proteins in
the bacterial cell. We have genome scale
tools for drug discovery in the form of
gene deletion and overexpression sets.
4060 Chem. Commun., 2011, 47, 4055–4061
We are beginning to understand that
inhibition of single targets vastly under-
estimates the antibiotic drug landscape
of the cell, opening up the possibility for
screening of new drug combinations.
Finally, our understanding of resistance
at the molecular and population levels is
now enabling us to understand the
details and scope of the resistance
problem. Identifying new antibiotics
and ways to bring them to the clinic
under current economic models will not
be easy, but we have unprecedented
tools, infrastructure, and a growing
understanding of the underlying
chemical properties of antibiotics and
resistance to guide the next phase of
antibiotic discovery.
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