Review TRENDS in Molecular Medicine
New strategies for combating
multidrug-resistant bacteria
Gerard D. Wright and Arlene D. Sutherland
Antimicrobial Research Centre, Department of Biochemistry and Biomedical Sciences, DeGroote School of Medicine,
McMaster University, 1200 Main St W, Hamilton, Ontario, L8N 3Z5, Canada
Antibiotic resistance is a problem that continues to
challenge the healthcare sector. In particular, multidrug
resistance is now common in familiar pathogens such as
Staphylococcus aureus and Mycobacterium tuberculosis,
as well as emerging pathogens such as Acinetobacter
baumannii. New antibiotics and new therapeutic strat-
egies are needed to address this challenge. Advances in
identifying new sources of antibiotic natural products and
expanding antibiotic chemical diversity are providing
chemical leads for new drugs. Inhibitors of resistance
mechanisms and microbial virulence are orthogonal strat-
egies that are also generating new chemicals that can
extend the life of existing antibiotics. This new chemistry,
coupled with a growing understanding of the mechan-
isms, origins and distribution of antibiotic resistance,
position us to tackle the challenges of antibiotic resist-
ance in the 21st century.
The problem of antibiotic resistance
One of the foremost challenges in the management of
infectious diseases is antimicrobial drug resistance. This
is an issue that impacts the treatment of all infections,
whether they are caused by viruses, parasites, fungi or
bacteria. However, it is the manifestation of multidrug
resistance in bacteria over the past several decades that
is resulting in one of the most pressing clinical problems in
modern medicine: Where does drug resistance come from,
and how can we address it?
Antibiotic resistance in bacteria is the product of innate
genetics and physiology, which are vertically transmitted
through species, and the remarkable propensity of bacteria
to exchange genetic material horizontally across species and
genera. This combinatorial genetic strategy has resulted in
the accumulation of multidrug-resistance (MDR) pheno-
types in many species of bacteria. The Infectious Disease
Society of America has identified methicillin-resistant
Staphylococcus aureus (MRSA), vancomycin-resistant
Enterococcus faecium, extended-spectrum b-lactamase-pro-
ducing Enterobacteriaceae and MDR Acinetobacter bau-
mannii and Pseudomonas aeruginosa as bacterial
pathogens that are especially challenging for the manage-
ment of infectious diseases [1]. This list will surely grow in
the future; witness the recent emergence of extensively
drug-resistant tuberculosis (XDR-TB) [2] and MDR Clostri-
dium difficile [3], for example. If the drug-discovery pipeline
Corresponding author: Wright, G.D. (wrightge@mcmaster.ca).
Available online 9 May 2007.
www.sciencedirect.com 1471-4914/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.molmed.2007.04.004
Vol.13 No.
does not produce new antibiotics for confronting the increas-
ing problem of bacterial MDR, clinical options for treating
infections caused by these pathogens will be very limited.
Combating MDR requires: (i) a detailed understanding
of the molecular basis, evolution and dissemination of
resistance; (ii) new chemicals with antibiotic properties
to fill the traditional antibiotic pipeline; and (iii) innovative
strategies that can extend the life of antibiotic molecules or
that can uncover new approaches for controlling pathogen
growth. Recent whole-genome sequence data for bacteria
as well as exploration of the resistance burden in environ-
mental bacteria suggest resistance is much more wide-
spread than previously thought. Could MDR be the natural
state of microorganisms? If so, is resistance inevitable?
Where will new antibiotics come from, and how can the
emergence of resistance be countered? A renaissance in
natural-product discovery, based on an expanding effort to
explore non-traditional sources of microbial biodiversity
and molecular tools that can exploit natural-product bio-
synthesis, is revealing new chemical classes with the
potential to prime the antibiotic pipeline. Furthermore,
non-traditional anti-infection targets, such as inhibitors of
resistance mechanisms and microbial virulence, are emer-
ging as alternative strategies. Recent progress in each of
these areas will be discussed in this review.
Antibiotics and resistance
The major mechanisms of antibiotic resistance include
enzymatic transformation, modification of the molecular
target, sequestration of the drug, active efflux from the cell
interior and, conversely, prevention of entry of the com-
pound into the cell (reviewed in Refs [4–6]). Resistance
arises either passively as a result of pre-existing innate
mechanisms or actively via the acquisition of new genetic
material by means of mobile genetic elements such as
plasmids or transposons [7,8].
Innate resistance includes physiological barriers such
as the outer membrane of Gram-negative bacteria, the
expression of intrinsic efflux pumps and natural mutations
in antibiotic targets. Some bacteria are replete with such
innate defense systems, for example, P. aeruginosa [9].
This opportunistic pathogen is widely distributed in
aquatic and soil environments and can express a stunning
variety of efficient efflux-pumps as well as antibiotic-inac-
tivating enzymes. These have probably evolved as a means
for the organism’s survival in numerous environments,
each with its own antibacterial chemical challenges to
overcome. When associated with infection, P. aeruginosa
 Review TRENDS in Molecular Medicine
is therefore very challenging to eradicate as a result of this
innate, combinatorial protection-system.
By contrast, the often commensal organisms, such as
Escherichia coli and S. aureus, that are commonly associ-
ated with infections in the community and hospitals
readily acquire antibiotic-resistance gene clusters through
resistance plasmids and other mobile genetic elements.
Such gene clusters frequently confer resistance to several
antibiotics at once, and as a result individual resistance
genes can be maintained even without direct selection.
This fact can be an impediment to antibiotic-cycling strat-
egies for overcoming antibiotic resistance in health-care
settings. Resistance gene cassettes are also associated with
transposons and integrons [10]. The former enable the
mobilization of genes from plasmids into bacterial chromo-
somes and vice versa. Integrons, whether on the chromo-
some or a plasmid, enable the continued accretion of
resistance genes resulting in an expansion of MDR [7,8,11].
Whether the antibiotic is of natural origin or synthetic,
resistance will eventually emerge (although perhaps not in
all bacterial species) and may already be circulating in the
environment. Evidence for this assertion comes from a
recent study that sampled spore-forming soil bacteria in
several different environments and found all of these
microbes to be multidrug resistant [12]. On average, each
strain of non-pathogenic bacteria was found to be resistant
to 7–8 antibiotics from a panel of 21 sampled. Resistance
was not restricted to antibiotics of natural origin, as may
have been predicted from such a screen. Rather, significant
levels of resistance were encountered even to new synthetic
antibiotics, such as linezolid and ciprofloxacin, as well as to
semi-synthetic natural-antibiotic derivatives designed to
overcome known resistance mechanisms; one example of
such a drug is telithromycin (Ketek1). This work, along
with the identification of antibiotic-resistance genes in
metagenomic screens of environmental bacteria [13],
shows that the collection of all resistance genes, the anti-
biotic resistome [7], is genetically extensive and can
respond to a broad range of chemical diversity.
Over the past 40 years, it has been well established in
both the clinic and the farm that antibiotic use selects for
the horizontal spread of acquired resistance elements on
mobile genetic elements. Resistance can also emerge as a
result of mutation in target genes and persist as a result of
second-site mutations. Furthermore, the presence of anti-
biotics can accelerate the development of innate resistance
Figure 1. New natural-product antibiotics. Platensimycin (soil bacterium), abyssomicin C (marine bacterium), horminone (plant).
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Vol.13 No.6 261
through the SOS response [14]. Antibiotic resistance,
therefore, is far reaching and highly adaptable, can emerge
rapidly and can progress through bacterial populations
vertically and horizontally with relative ease.
New antibiotics
One of the major problems facing the antibiotic-drug-
discovery sector is the difficulty in identifying new
drug-like compounds with suitable antibacterial activity.
Historically, the first successful antibacterial compounds
were synthetic in origin. These include salvarsan, which
was identified at the turn of the last century for the
treatment of syphilis, and the sulfonamides that target
p-aminobenzoic acid biosynthesis. Despite the initial
supremacy of synthetic antimicrobial drugs, it has been
natural-product antibiotics, largely of bacterial origin, that
have dominated as the preferred chemical scaffolds of
antibacterial drugs since the middle of the last century.
However, the frequency of identification of new antibacter-
ial compounds from this source has dropped since the
1970s. Because many soil bacteria (the traditional source
of antibiotics in current clinical use) produce a similar
range of antibiotics identifying new antibiotics from these
sources becomes ever more difficult. Richard Baltz has
discussed this problem [15] and has noted that the anti-
biotic daptomycin (approved in 2003 as Cubicin1) was only
found in 2 of 107 soil actinomycetes. By contrast, the
frequency of identification of a streptothricin producer is
2 in 10. This frequency range of 1067 for identifying
producers of new antibiotic scaffolds is daunting. Never-
theless, promising new antibiotics such as platensimycin
[16] (Figure 1) continue to be identified from soil bacteria.
Despite the challenges of identifying new natural-pro-
duct antibiotics, recent work has proven that there are
untapped sources of antibiotic natural products. These
include microorganisms from new ecological niches,
natural products from plants, natural-product biosynthetic
clusters in known organisms and previously discovered
compounds that are re-evaluated. Furthermore, synthetic
antimicrobials must not be forgotten and will continue to
have their place in the future of antibiotics.
New sources of antimicrobial chemicals
Identifying new ecological niches for antibiotic-producing
bacteria is providing access to new organisms and new
bioactive chemicals. The recent realization that there are
262 Review TRENDS in Molecular Medicine
bona fide marine actinomycetes and the discovery of
laboratory growth conditions for their culture has resulted
in the identification of several new groups of actinomy-
cetes, including the Salinispora and Marinispora (revie-
wed in Ref. [17]). These marine bacteria have proven to be
sources of several new antitumor and antibiotic natural
products. For example, abyssomicin C (Figure 1) is a novel
inhibitor of bacterial p-aminobenzoic acid biosynthesis
[18,19].
Another source of new antibiotic-producing bacteria are
bacterial symbionts of insects [20,21] and other arthropods
[22]. These bacteria have evolved to exist in highly specific
niches and as a result have their own specific antimicrobial
chemical diversity tuned to their complex interactions with
the host. These probably represent just a small portion of
the available antibiotic-producing biodiversity available
for exploration. Baltz has estimated that, even in the soil,
only 2% of the antibiotics produced by Streptomyces have
been identified [15].
Plants [23] and the engineering of known biosynthetic
pathways to expand antibiotic chemical diversity [24,25]
are additional sources of new antibiotics. Medicinal plants
have been used for millennia in the treatment of infection.
Efforts to isolate active agents in plants have led to the
identification of several antibacterial agents such as hor-
minone (reviewed in ref. [26]) (Figure 1).
The recent ability to modify antibiotic chemical scaffolds
in combinatorial fashion via the manipulation of biosyn-
thetic genes has greatly expanded the chemical diversity of
several classes of antibiotics, including the polyketides and
cyclic peptides. This approach makes it possible to explore
chemical space on antibiotic scaffolds that in many cases
are out of reach for synthetic chemistry. So far no new
antibiotics have been brought to market from plants or
from biosynthetic-engineering studies, but these are early
days for these sources of antimicrobial agents and there is
reason to be optimistic that they will contribute clinically-
useful agents in the future.
Finally, we must not forget that chemists have the
ability to synthesize de novo new classes of antibiotics.
The first new class of antibiotics in clinical use in more
than 25 years is the oxazolidinones, represented by line-
zolid (Zyvox1). Unlike most other classes of antibiotics, the
oxazolidinones do not have their origins in natural-product
chemistry. This group of compounds emerged from syn-
thetic-chemistry laboratories first at DuPont and then at
Pharmacia and Pfizer more than 15 years ago. The more
structurally complex diversity-oriented synthetic-chemical
libraries inspired by natural products might be tremen-
dous sources of new chemistry and provide the opportunity
to exhibit antimicrobial activity [27].
Mining natural-product biosynthetic clusters
uncovered in bacterial genomes
One of the many surprises of the growing availability of
bacterial-genome sequences is the number of unknown
natural-product biosynthetic gene clusters that have
emerged even from very well-studied organisms. The
genes encoding natural products (including antibiotics)
produced by bacteria are generally clustered in a contig-
uous fashion along the genome. This greatly facilitates
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the identification of the number and type of natural
products that an organism is predicted to be able to
biosynthesize. For example, it was well established that
Bacillus subtilis, which has been studied for more than a
century, produced several antibiotics, such as surfactin
and fengycin. However, the complete genome sequence of
B. subtilis 168 revealed a new 80 kb predicted natural-
product biosynthetic cluster thought to be non-functional
[28]. This cluster has now been shown to produce the
polyketide-peptide hybrid compound bacillaene [29]. Sim-
ilarly, Streptomyces coelicolor, which has been studied for
more than 50 years, was known to produce 5–6 natural
products. Its genome sequence, however, revealed poten-
tial gene clusters for 20 natural products [30]. Recently,
one of these was shown to produce a new siderophore,
coelochelin [31].
The ability to predict not only the possibility of natural
product biosynthesis from the presence of a biosynthetic
gene cluster but also the chemical structure of the metab-
olite has been improved dramatically by the refinement of
bioinformatics tools. The structure of coelochelin [31] is one
example where a mixture of bioinformatic prediction fol-
lowed by experimental analysis resulted in the structural
elucidation of a new natural product. Another example is a
novel cyclic lipopeptide from Pseudomonas syringae [32].
The group at Ecopia Biosciences has also pioneered a new
informatics approach called DECIPHER1 to successfully
mine bacterial genomes for natural-product biosynthesis
clusters and predict the structure of the metabolites
[33,34].
Old antibiotics – new medicines
It is instructive to recall that all of the three new classes of
antibiotics brought to market in the past decade, Syner-
cid1 (streptogramin combination), linezolid (oxazolidi-
none), and daptomycin (lipopeptide), were discovered
and then discounted as viable antibiotics. The streptogra-
mins, a combination of two natural-product antibiotics
produced by the same species of bacteria, were first ident-
ified in the 1950s. They were used primarily as animal-feed
additives until medicinal chemists at Rhone-Poulenc
improved the solubility of both components of the antibiotic
to make them suitable for human therapy. The oxazolidi-
nones were first discovered in the mid-1980s by chemists at
Dupont, and daptomycin was identified at roughly the
same time at Lilly. Both antibiotic classes were dropped
as clinical candidates because of toxicity concerns. Sub-
sequent revisiting of these agents with new chemistry or,
in the case of daptomycin, new therapeutic dosing regi-
mens has rescued promising agents for clinical use. Other
examples of the revisiting of ‘old’ antibiotic scaffolds are the
mannopeptimycins, which block lipid II [35], and ever-
ninomycin, which inhibits protein translation [36]. Neither
of these has emerged from early-stage clinical trials, but
they illustrate the point that historical antibiotic collec-
tions are worthy of re-investigation. Since the first screens
of soil organisms for antibiotic activity, several thousand
antibiotics have been reported [37]. Are there blockbuster
drugs awaiting rediscovery in this historical literature?
The examples of daptomycin and the streptogramins
suggest there are.
 Review TRENDS in Molecular Medicine
Certainly, there are leads for new antimicrobial
chemicals and associated targets in the thousands of
known compounds with antimicrobial activity. Modern
biochemical and genomic tools enable rapid identification
of the mode of action of antibiotics. These tools, coupled
with powerful synthetic-chemical-library methods for
rapidly sampling and expanding chemical diversity around
an active scaffold, allow us to gain new and extensive
information on target-compound pairs in relatively short
periods of time. For example, a family of acyldepsipeptides
produced by Streptomyces hawaiiensis and reported in
1985 has now been shown to be potent inhibitors of the
regulation of the ClpP protease, which leads to bacterial
cell death, and this finding has led to the identification of a
new target-inhibitor pair that can be exploited in down-
stream drug design [38].
New strategies for extending the life of antibiotics
Inhibitors of resistance enzymes
The concept of utilizing cognate pairs of antibiotics and
inhibitors of antibiotic-resistance proteins is clinically pro-
ven in the b-lactamase field. There are hundreds of known
b-lactamases, and these have been classified by Ambler
et al. [39] as serine (Ser) dependent (classes A, C and D) or
metal dependent (class B). Clavulanic acid (Figure 2),
produced by Streptomyces clavuligerus, is a b-lactam with
weak antibacterial activity but that potently inhibits
Figure 2. Inhibitors of b-lactamases. Clavulanic acid, identified in 1976, is in wide clinical use in combination with b-lactam antibiotics and inhibits type A b-lactamases.
a–Vinylidene-b-lactams such as compound A and 3-substituted cephalosporins such as compound B are representative synthetic molecules that have expanded class
A, C, and D b-lactamase inhibition activity. The 6-methylidene penem compound C has broad-range anti-b-lactamase activity including activity against extended-spectrum
b–lactamases. Bulgecin A is a natural-product inhibitor of type B metallo-b-lactamases.
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Vol.13 No.6 263
several class A Ser-b-lactamases [40]. These resistance
enzymes act by catalyzing a two-step hydrolytic reaction
to inactivate the antibiotic. First, an active-site Ser residue
forms a covalent bond with the antibiotic by cleaving the
b-lactam ring. This is followed by a second step where a
molecule of water hydrolyzes the antibiotic-enzyme com-
plex and thus regenerates active b-lactamase and releases
the inactive antibiotic. Clavulanic acid inhibits b-lacta-
mases by forming a highly stable enzyme-Ser-antibiotic
complex in the first catalytic step, but it is unable to
undergo the second hydrolytic step to release the free
enzyme, and this failure effectively inactivates the anti-
biotic-resistance enzyme. When certain drugs are pack-
aged with a b-lactam antibiotic such as amoxicillin,
clavulanic acid has led to the development of potent drug
combinations for clinical use; one such drug is Augmen-
tin1. Similarly, combinations of the b-lactamase inhibitors
sulbactam and tazobactam with b-lactam antibiotics are in
clinical use.
These combinations have been most effective against
the Ambler class A [39] Ser-b-lactamases; however, resist-
ant isozymes and the increasing frequency of non-suscept-
ible class B, C and D enzymes is a growing problem. This
has spurred efforts to synthesize and evaluate broad-spec-
trum inhibitors of b-lactamases. For example, Buynak and
colleagues have reported several potent inhibitors of class
A, C and D b-lactamase inhibitors based on both penicillin
264 Review TRENDS in Molecular Medicine
and cephalosporin scaffolds (e.g. Figure 2, reviewed in Ref.
[41]), and Wyeth has reported on a series of 6-methylidene
penems with activity against extended-spectrum b-lacta-
mases (Figure 2) [42].
The emergence of class B metallo-b-lactamases in the
clinic is a growing problem [43]. These enzymes use a
different catalytic mechanism than the Ser-b-lactamase
to inactivate the antibiotic. Instead of a covalent enzyme-
antibiotic intermediate, an active-site Zn2+ ion (1–2 atoms
per active site) generates a reactive water molecule that is
poised for cleavage of the b-lactam ring (reviewed in Ref.
[44]). b-Lactam-based inhibitors of the Ser-enzymes are in
fact substrates for these enzymes and therefore have no
inhibitory effect. As a result, new classes of small-molecule
inhibitors specific to metallo-b-lactamases are being
sought. Recent studies have identified several such inhibi-
tors that chelate the active site Zn2+; these include thiols
and tetrazoles (e.g. Refs. [45,46] and reviewed in Ref. [43])
as well as the sulfated glycopeptide natural product bul-
gecin A [47] (Figure 2).
Inhibitors of aminoglycoside-resistance enzymes have
been described (reviewed in Ref. [48]). More recently, inhi-
bition of an aminoglycoside kinase, APH(30 )-IIIa, by a
rationally designed ankyrin-repeat protein that traps the
enzyme in a non-productive complex has been reported [49].
This ankyrin-repeat protein is unlikely to be clinically use-
ful. Nevertheless, this inhibition strategy, with a focus on
non-active site-directed compounds, could be emulated with
small molecules. Bisubstrate inhibitors of aminoglycoside
acetyltransferase, AAC(60 )-Ii, that reverse resistance in
bacterial culture have also been recently published [50].
One difficulty with targeting aminoglycoside resistance is
that many clinically resistant bacteria harbor more than one
resistance enzyme, often with completely different inacti-
vation chemistry (kinase, acetyltransferase and adenylyl-
transferase). This makes small-molecule targeting of
aminoglycoside resistance a challenge. However, cationic
peptides have shown in vitro ability to inhibit multiple
enzymes, including kinases and acetyltransferases [51],
suggesting that these compounds could form the basis for
an expanded search for a pan-aminoglycoside-resistance
inhibitor.
Inhibitors of antibiotic efflux
Gram-negative and Gram-positive bacteria use efflux
proteins to pump antibiotics from the periplasm or cytosol
to the extracellular medium. These membrane-spanning
proteins use energy, by performing ATP hydrolysis, by
coupling efflux to ion transport or by harnessing the pro-
ton-motive force, to pump antibiotic out of the cell against a
concentration gradient. There are five classes of antibiotic
efflux proteins, each with a distinctive topology, quatern-
ary structure and primary sequence; these include the
ABC-transporter, major-facilitator, MATE, SMR and
RND families (reviewed in Refs [52,53]). The importance
of these resistance proteins, in particular members of the
RND and major-facilitator classes, in clinical antibiotic-
resistance has prompted the search for inhibitors of these
pumps [54].
Inhibitors of efflux pumps include synthetic and
natural-product molecules. Plants in particular have been
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shown to produce several inhibitors of antibiotic efflux
proteins; these include reserpine, one of the first pump
inhibitors shown to potentiate antibiotics in Gram-positive
bacteria (reviewed in Ref. [55]). The Lewis group has
shown that in the case of the antibiotic berberine, species
of plants simultaneously produce 50 -methoxyhydnocarpin,
an inhibitor of major-facilitator efflux proteins [56], pre-
sumably to overcome the problem of antibiotic resistance.
Recently, this group has shown that chemically linking an
antibiotic and efflux-pump inhibitor makes it possible to
obtain successful hybrid compounds with activity against
resistant bacteria [57].
Novel antibiotic strategies
In addition to new antibiotic compounds and inhibitors of
resistance, new strategies to prolong the effectiveness of
the current arsenal of antibiotic drugs as well as agents
that interfere with bacterial virulence and block infection
are being explored. For example, bacterial exposure to
certain classes of antibiotics has been demonstrated to
actually accelerate the emergence of resistance by inducing
horizontal gene transfer [58] and mutation via the SOS
response [14]. Preventing the SOS response, for example
by blocking the protease LexA [14] or by inhibiting RecA
[59], are novel strategies for preventing the emergence of
resistance during antibiotic chemotherapy and could be
used in combination with existing antibiotics.
Another new strategy aimed at interfering with
bacterial physiology is the blockade of intercellular com-
munication. Bacteria use quorum-sensing molecules such
as acyl homoserine lactones during infection to promote
both colonization and cell-type modulation (e.g. the for-
mation of biofilm [60]). Therefore, interfering with this
process could be a target for novel antimicrobial agents
that do not necessarily kill the cell but rather prevent or
attenuate infection by targeting virulence. A recent high-
throughput screen of 200 000 small molecules identified
cell-permeable inhibitors of the P. aeruginosa LasR-de-
pendent quorum-sensing system (Figure 3) that could
serve as leads for new anti-virulence compounds [61].
Other recent examples of new anti-virulence strategies
include inhibitors of Chlamydia trachomatis type III
secretion [62], ToxT-dependent gene expression in Vibrio
cholerae [63] (Figure 3) and sortase-dependent protein
modification of Gram-positive cell wall [64]. This approach
does not inhibit cell growth, and as a result, targeting
virulence might be less prone to selection for resistance
because the organisms are not in ‘adapt or die’ mode where
the presence of an antibiotic can powerfully select for a
rare resistant mutant.
Another important virulence factor is the outer
membrane of Gram-negative bacteria. This asymmetric
bilayer includes a lipopolysaccharide layer that has highly
conserved and highly variable polymer components. Cells
that are deficient in the synthesis of the conserved lipid
and carbohydrate components of lipopolysaccharide are
either non-viable (e.g. LpxC) or avirulent (e.g. heptose
biosynthesis). Furthermore, viable mutants impaired in
heptose biosynthesis are hyper-susceptible to many anti-
biotics [65]. This has resulted in the exploration of lipopo-
lysaccharide biosynthesis as a target for either novel
 Review TRENDS in Molecular Medicine
Figure 3. Small-molecule inhibitors of bacterial virulence. (a) Inhibitors of quorum sensing in P. aeruginosa [61]. (b) Virstatin is an inhibitor of ToxT-dependent gene
expression in V. cholerae [63], and INP0400 blocks C. trachomatis type III secretion [62].
antibacterials or antibiotic adjuvants, that is, compounds
that synergize with antibiotics. For example, inhibitors
of LpxC have been shown to have narrow-spectrum
but potent antibacterial activity [66], and a recent high-
throughput screen of heptose biosynthesis identified the
first small-molecule inhibitors of this pathway [67]. Sim-
ilarly, in Gram-positive bacteria inhibiting the biosyn-
thesis of cell-wall polymers teichoic and lipoteichoic acid
represent both novel antibacterial and antibiotic-sensitiz-
ing targets [68,69].
The expanded concept of antibiotic adjuvants is worthy of
exploration. These are compounds that may not have sig-
nificant antibiotic activity themselves but that improve
their biological activity when combined with antibiotics.
Inhibitors of resistance mechanisms and inhibitors of lipo-
polysaccharide biosynthesis, discussed above, are examples
of such adjuvants. Synergistic combinations of antibiotics
are another example that is well precedented in infectious-
disease medicine. Given the number of predicted natural-
product biosynthetic clusters in sequenced actinomycetes
(20 in Streptomyces coelicolor and 30 in Streptomyces
avermitilis), Challis and Hopwood hypothesized that
natural-product synergy may be a normal state for anti-
biotic-producing bacteria [70]. In other words, the unex-
pected capacity for bacteria to produce multiple natural
products may reflect an ecological advantage for co-pro-
duction of secondary metabolites. Co-production of the
synergistic combination of chemically unrelated type A
and type B streptogramin antibiotics are a proven example
of genetically programmed synergy [71]. Analysis of the
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Vol.13 No.6 265
systems biology of antibiotic combinations is beginning to
allow exploration of this concept in a rigorous fashion [72]
and may yield new avenues for antibiotic therapy.
Conclusions
After a half century of clinical antibiotic use, resistance,
and in particular MDR, has emerged as a formidable
problem with no signs of abating. In fact, MDR may be a
natural state for many bacteria. Addressing this issue will
require the collaboration of many disciplines and inno-
vation in the identification of new therapeutics and com-
plimentary antimicrobial strategies.
There are several challenges in identifying new
antibiotics with the potential to make it through clinical
trials and into medical practice. Among these is the fact
that discovering new chemically distinct antibiotics from
traditional sources (such as soil bacteria and existing
synthetic-chemical collections) is increasingly difficult.
Sampling the biological and chemical diversity of atypical
sources such as marine bacteria is beginning to identify
novel antibiotic scaffolds. Whether any of these will be
suitable to be developed into new drugs remains to be seen.
However, given the precedent of the previous 50 years, it
seems highly likely that this new chemistry will eventually
yield new drugs.
The focus of many antimicrobial drug-discovery
programs has been, and continues to be, on broad-spec-
trum antibiotics that target as many bacteria as possible.
This reflects our woefully poor ability to rapidly identify
the causative agent of infection in most hospital and
266 Review TRENDS in Molecular Medicine
community settings as well as the fact that in many cases
infection may be the result of more than one microbe. This
‘carpet bombing’ strategy of infection control has surely
contributed to the problem of antibiotic resistance, as exem-
plified by the rise in highly virulent infections caused by
drug-resistant C. difficile [73]. Leveraging of improved diag-
nostic technology and microbial-genome sequencing has the
potential to be applied in the rapid identification of the
pathogens involved in infection. As a result, there is a
growing opportunity to explore organism-specific anti-
biotics. This will require new paradigms in infection control,
regulatory approval of new diagnostics and, importantly,
confidence among physicians that the strategy will work.
However, the benefits of preventing the eradication of
beneficial flora as collateral damage and the broad selective
pressure associated with antibiotic resistance merit rese-
arch efforts in this area.
Antibiotic adjuvants, including novel compound
combinations and inhibitors of resistance mechanisms,
as well as targeting of microbial virulence, are approaches
that can extend the life of successful antibiotic drugs.
Because these antibiotics have known pharmacology and
sometimes decades of clinical history, the time to market
may be relatively short. The problem of multiple methods
of drug resistance is significant; however, the success of the
b-lactamase inhibitors demonstrates that there is a mar-
ket for well-designed antibiotic adjuvants. The regulatory
and pharmacological challenges in this approach are not
trivial (see Box 1): Compound combinations must have
similar pharmacological profiles, and designing successful
clinical trials for drug combinations or anti-virulence com-
pounds and the massive expense of these are major hurdles
to be overcome.
Despite these challenges, in the 21st Century we are in a
better position to address the problem of resistance than
ever before. Exploration of new biological diversity is
revealing new antimicrobial chemical diversity, our un-
derstanding of the molecular mechanisms of resistance
and its evolution is improving and the genomics revolution
has resulted not only in a remarkable and growing number
of whole-genome sequences, but also in the stunning ability
to generate atomic-resolution structures of a myriad of
antibiotic targets, from the ribosome to cell-wall synthetic
machinery. In many ways we are entering into what should
be a new ‘Golden era’ of antibiotics that builds on the first
Box 1. Outstanding questions
1. What are the best natural-product and synthetic-chemical
sources of new antibiotic chemical scaffolds? How can we enrich
compound collections and natural-product extracts with anti-
microbial compounds?
2. Can we identify new highly effective inhibitors of antibiotic-
resistance mechanisms with the potential to reverse resistance
during infection?
3. Can novel drug combinations extend the life of ‘old’ antibiotics?
4. Are inhibitors of microbial virulence good candidates for new
prophylactic drugs?
5. Can rapid diagnostics coupled with organism-specific antibiotics
help to reduce antibiotic resistance in microbial populations and
off-target killing of beneficial microbial species?
6. Will the regulatory community support new antibiotics ap-
proaches such as drug combinations and anti-virulence drugs?
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Vol.13 No.6
‘Golden era’ from 1945–1960, when the majority of
antibiotic scaffolds in current use were identified. The
new technologies and innovative strategies discussed
above have the potential to answer the challenge of multi-
drug-resistant bacteria in the coming years.
Acknowledgements
This work is supported by the Canadian Institutes of Health Research,
the Natural Sciences and Engineering Research Council of Canada and a
Canada Research Chair in Antibiotic Biochemistry.
References
1 Talbot, G.H. et al. (2006) Bad bugs need drugs: an update on the
development pipeline from the Antimicrobial Availability Task
Force of the Infectious Diseases Society of America. Clin. Infect. Dis.
42, 657–668
2 Dorman, S.E. and Chaisson, R.E. (2007) From magic bullets back to the
Magic Mountain: the rise of extensively drug-resistant tuberculosis.
Nat. Med. 13, 295–298
3 Sebaihia, M. et al. (2006) The multidrug-resistant human pathogen
Clostridium difficile has a highly mobile, mosaic genome. Nat. Genet.
38, 779–786
4 Wright, G.D. (2005) Bacterial resistance to antibiotics: enzymatic
degradation and modification. Adv. Drug Deliv. Rev. 57, 1451–1470
5 Walsh, C.T. (2003) Antibiotics: Actions, Origins, Resistance, ASM Press
6 Levy, S.B. and Marshall, B. (2004) Antibacterial resistance worldwide:
causes, challenges and responses. Nat. Med. 10, S122–S129
7 Wright, G.D. (2007) The antibiotic resistome: the nexus of chemical and
genetic diversity. Nat. Rev. Microbiol. 5, 175–186
8 Summers, A.O. (2006) Genetic linkage and horizontal gene transfer,
the roots of the antibiotic multi-resistance problem. Anim. Biotechnol.
17, 125–135
9 Lee, D.G. et al. (2006) Genomic analysis reveals that Pseudomonas
aeruginosa virulence is combinatorial. Genome Biol. 7, R90
10 Roy, P.H. (1999) Horizontal transfer of genes in bacteria. Microbiol.
Today 26, 168–170
11 Fluit, A.C. and Schmitz, F.J. (2004) Resistance integrons and super-
integrons. Clin. Microbiol. Infect. 10, 272–288
12 D’Costa, V.M. et al. (2006) Sampling the antibiotic resistome. Science
311, 374–377
13 Riesenfeld, C.S. et al. (2004) Uncultured soil bacteria are a reservoir of
new antibiotic resistance genes. Environ. Microbiol. 6, 981–989
14 Cirz, R.T. et al. (2005) Inhibition of mutation and combating the
evolution of antibiotic resistance. PLoS Biol. 3, e176
15 Baltz, R.H. (2005) Antibiotic discovery from actinomycetes: will a
renaisssance follow the decline and fall? SIM News 55, 186–196
16 Wang, J. et al. (2006) Platensimycin is a selective FabF inhibitor with
potent antibiotic properties. Nature 441, 358–361
17 Fenical, W. and Jensen, P.R. (2006) Developing a new resource for drug
discovery: marine actinomycete bacteria. Nat. Chem. Biol. 2, 666–673
18 Riedlinger, J. et al. (2004) Abyssomicins, inhibitors of the para-
aminobenzoic acid pathway produced by the marine Verrucosispora
strain AB-18-032. J. Antibiot. (Tokyo) 57, 271–279
19 Bister, B. et al. (2004) Abyssomicin C-A polycyclic antibiotic from a
marine Verrucosispora strain as an inhibitor of the p-aminobenzoic
acid/tetrahydrofolate biosynthesis pathway. Angew. Chem. Int. Ed.
Engl. 43, 2574–2576
20 Kaltenpoth, M. et al. (2005) Symbiotic bacteria protect wasp larvae
from fungal infestation. Curr. Biol. 15, 475–479
21 Currie, C.R. et al. (1999) Fungus-growing ants use antibiotic-producing
bacteria to control garden parasites. Nature 398, 701–704
22 Gebhardt, K. et al. (2002) Screening for biologically active metabolites
with endosymbiotic bacilli isolated from arthropods. FEMS Microbiol.
Lett. 217, 199–205
23 Lewis, K. and Ausubel, F.M. (2006) Prospects for plant-derived
antibacterials. Nat. Biotechnol. 24, 1504–1507
24 Lamb, S.S. and Wright, G.D. (2005) Accessorizing natural products:
adding to nature’s toolbox. Proc. Natl. Acad. Sci. U. S. A. 102, 519–520
25 Baltz, R.H. (2006) Molecular engineering approaches to peptide,
polyketide and other antibiotics. Nat. Biotechnol. 24, 1533–1540
26 Gibbons, S. (2004) Anti-staphylococcal plant natural products. Nat.
Prod. Rep. 21, 263–277
 Review TRENDS in Molecular Medicine
27 Thomas, G.L. et al. (2006) Enriching chemical space with diversity-
oriented synthesis. Curr. Opin. Drug Discov. Devel. 9, 700–712
28 Kunst, F. et al. (1997) The complete genome sequence of the gram-
positive bacterium Bacillus subtilis. Nature 390, 249–256
29 Straight, P.D. et al. (2007) A singular enzymatic megacomplex from
Bacillus subtilis. Proc. Natl. Acad. Sci. U. S. A. 104, 305–310
30 Bentley, S.D. et al. (2002) Complete genome sequence of the model
actinomycete Streptomyces coelicolor A3(2). Nature 417, 141–147
31 Lautru, S. et al. (2005) Discovery of a new peptide natural product by
Streptomyces coelicolor genome mining. Nat. Chem. Biol. 1, 265–269
32 de Bruijn, I. et al. (2007) Genome-bases discovery, structure prediction
and functional analysis of cyclic lipopeptide antibiotics in
Pseudomonas species. Mol. Microbiol. 63, 417–428
33 Zazopoulos, E. et al. (2003) A genomics-guided approach for discovering
and expressing cryptic metabolic pathways. Nat. Biotechnol. 21, 187–
190
34 McAlpine, J.B. et al. (2005) Microbial genomics as a guide to drug
discovery and structural elucidation: ECO-02301, a novel antifungal
agent, as an example. J. Nat. Prod. 68, 493–496
35 He, H. (2005) Mannopeptimycins, a novel class of glycopeptide
antibiotics active against gram-positive bacteria. Appl. Microbiol.
Biotechnol. 67, 444–452
36 Boucher, H.W. et al. (2001) In vivo activity of evernimicin (SCH 27899)
against methicillin-resistant Staphylococcus aureus in experimental
infective endocarditis. Antimicrob. Agents Chemother. 45, 208–211
37 Berdy, J. (2005) Bioactive microbial metabolites. J. Antibiot. (Tokyo)
58, 1–26
38 Brotz-Oesterhelt, H. et al. (2005) Dysregulation of bacterial proteolytic
machinery by a new class of antibiotics. Nat. Med. 11, 1082–1087
39 Ambler, R.P. et al. (1991) A standard numbering scheme for the class A
beta-lactamases. Biochem. J. 276, 269–270
40 Neu, H.C. and Fu, K.P. (1978) Clavulanic acid, a novel inhibitor of beta-
lactamases. Antimicrob. Agents Chemother. 14, 650–655
41 Buynak, J.D. (2004) The discovery and development of modified
penicillin- and cephalosporin-derived beta-lactamase inhibitors.
Curr. Med. Chem. 11, 1951–1964
42 Weiss, W.J. et al. (2004) In vitro and in vivo activities of novel 6-
methylidene penems as beta-lactamase inhibitors. Antimicrob. Agents
Chemother. 48, 4589–4596
43 Walsh, T.R. et al. (2005) Metallo-beta-lactamases: the quiet before the
storm? Clin. Microbiol. Rev. 18, 306–325
44 Crowder, M.W. et al. (2006) Metallo-beta-lactamases: novel weaponry
for antibiotic resistance in bacteria. Acc. Chem. Res. 39, 721–728
45 Toney, J.H. et al. (1998) Antibiotic sensitization using biphenyl
tetrazoles as potent inhibitors of Bacteroides fragilis metallo-beta-
lactamase. Chem. Biol. 5, 185–196
46 Concha, N.O. et al. (2000) Crystal structure of the IMP-1 metallo beta-
lactamase from Pseudomonas aeruginosa and its complex with a
mercaptocarboxylate inhibitor: binding determinants of a potent,
broad-spectrum inhibitor. Biochemistry 39, 4288–4298
47 Simm, A.M. et al. (2005) Bulgecin A: a novel inhibitor of binuclear
metallo-beta-lactamases. Biochem. J. 387, 585–590
48 Wright, G.D. (2000) Resisting resistance: new chemical strategies for
battling superbugs. Chem. Biol. 7, R127–R132
49 Kohl, A. et al. (2005) Allosteric inhibition of aminoglycoside
phosphotransferase by a designed ankyrin repeat protein. Structure
13, 1131–1141
50 Gao, F. et al. (2006) Synthesis and structure-activity relationships of
truncated bisubstrate inhibitors of aminoglycoside 60 -N-acetyltran-
sferases. J. Med. Chem. 49, 5273–5281
www.sciencedirect.com
Vol.13 No.6 267
51 Boehr, D.D. et al. (2003) Broad-spectrum peptide inhibitors of
aminoglycoside antibiotic resistance enzymes. Chem. Biol. 10, 189–196
52 Piddock, L.J. (2006) Clinically relevant chromosomally encoded
multidrug resistance efflux pumps in bacteria. Clin. Microbiol. Rev.
19, 382–402
53 Poole, K. (2005) Efflux-mediated antimicrobial resistance. J.
Antimicrob. Chemother. 56, 20–51
54 Lomovskaya, O. and Bostian, K.A. (2006) Practical applications and
feasibility of efflux pump inhibitors in the clinic–a vision for applied
use. Biochem. Pharmacol. 71, 910–918
55 Stavri, M. et al. (2006) Bacterial efflux pump inhibitors from natural
sources. J Antimicrob Chemother
56 Stermitz, F.R. et al. (2000) Synergy in a medicinal plant: antimicrobial
action of berberine potentiated by 50 -methoxyhydnocarpin, a multidrug
pump inhibitor. Proc. Natl. Acad. Sci. U. S. A. 97, 1433–1437
57 Ball, A.R. et al. (2006) Conjugating berberine to a multidrug efflux
pump inhibitor creates an effective antimicrobial. ACS Chem Biol 1,
594–600
58 Hastings, P.J. et al. (2004) Antibiotic-induced lateral transfer of
antibiotic resistance. Trends Microbiol. 12, 401–404
59 Lee, A.M. et al. (2005) A molecular target for suppression of the
evolution of antibiotic resistance: inhibition of the Escherichia coli
RecA protein by N(6)-(1-naphthyl)-ADP. J. Med. Chem. 48, 5408–5411
60 Gonzalez, J.E. and Keshavan, N.D. (2006) Messing with bacterial
quorum sensing. Microbiol. Mol. Biol. Rev. 70, 859–875
61 Muh, U. et al. (2006) Novel Pseudomonas aeruginosa quorum-sensing
inhibitors identified in an ultra-high-throughput screen. Antimicrob.
Agents Chemother. 50, 3674–3679
62 Muschiol, S. et al. (2006) A small-molecule inhibitor of type III secretion
inhibits different stages of the infectious cycle of Chlamydia
trachomatis. Proc. Natl. Acad. Sci. U. S. A. 103, 14566–14571
63 Hung, D.T. et al. (2005) Small-molecule inhibitor of Vibrio cholerae
virulence and intestinal colonization. Science 310, 670–674
64 Frankel, B.A. et al. (2004) Vinyl sulfones: inhibitors of SrtA, a
transpeptidase required for cell wall protein anchoring and
virulence in Staphylococcus aureus. J. Am. Chem. Soc. 126, 3404–3405
65 Valvano, M.A. et al. (2002) Novel pathways for biosynthesis of
nucleotide-activated glycero-manno-heptose precursors of bacterial
glycoproteins and cell surface polysaccharides. Microbiology 148,
1979–1989
66 Onishi, H.R. et al. (1996) Antibacterial agents that inhibit lipid A
biosynthesis. Science 274, 980–982
67 De Leon, G.P. et al. (2006) An in vitro screen of bacterial
lipopolysaccharide biosynthetic enzymes identifies an inhibitor of
ADP-heptose biosynthesis. Chem. Biol. 13, 437–441
68 May, J.J. et al. (2005) Inhibition of the D-alanine:D-alanyl carrier protein
ligase from Bacillus subtilis increases the bacterium’s susceptibility to
antibiotics that target the cell wall. FEBS J. 272, 2993–3003
69 Brown, E.D. and Wright, G.D. (2005) New targets and screening
approaches in antimicrobial drug discovery. Chem. Rev. 105, 759–774
70 Challis, G.L. and Hopwood, D.A. (2003) Synergy and contingency as
driving forces for the evolution of multiple secondary metabolite
production by Streptomyces species. Proc. Natl. Acad. Sci. U. S. A.
100 (Suppl 2), 14555–14561
71 Cocito, C. (1979) Antibiotics of the virginiamycin family, inhibitors
which contain synergistic components. Microbiol. Rev. 43, 145–192
72 Yeh, P. et al. (2006) Functional classification of drugs by properties of
their pairwise interactions. Nat. Genet. 38, 489–494
73 Bartlett, J.G. (2006) Narrative review: the new epidemic of Clostridium
difficile-associated enteric disease. Ann. Intern. Med. 145, 758–764