Tackling antibiotic resistance: a dose of common antisense?
Neil Woodford1* and David W. Wareham2on behalf of the UK Antibacterial
Antisense Study Group
1Antibiotic Resistance Monitoring and Reference Laboratory, Centre for Infections, Health Protection Agency,
London NW9 5EQ, UK;2Centre for Infectious Disease, Institute of Cell and Molecular Science, Barts
and The London School of Medicine and Dentistry, London E1 2AT, UK
Resistance to antimicrobial agents undermines our ability to treat bacterial infections. It attracts
intense media and political interest and impacts on personal health and costs to health infrastructures.
Bacteria have developed resistance to all licensed antibacterial agents, and their ability to become
resistant to unlicensed agents is often demonstrated during the development process. Conventional
approaches to antimicrobial development, involving modification of existing agents or production of
synthetic derivatives, are unlikely to deliver the range or type of drugs that will be needed to meet all
future requirements. Although many companies are seeking novel targets, further radical approaches
to both antimicrobial design and the reversal of resistance are now urgently required. In this article, we
discuss ‘antisense’ (or ‘antigene’) strategies to inhibit resistance mechanisms at the genetic level.
These offer an innovative approach to a global problem and could be used to restore the efficacy of
clinically proven agents. Moreover, this strategy has the potential to overcome critical resistances, not
only in the so-called ‘superbugs’ (methicillin-resistant Staphylococcus aureus, glycopeptide-resistant
enterococci and multidrug-resistant strains of Acinetobacter baumannii, and Pseudomonas aeruginosa),
but in resistant strains of any bacterial species.
Keywords: resistance inhibitors/modulators, oligonucleotides, modified nucleic acids, bacteriophage,
Antibiotic resistance is a global public health concern that
impacts to varying extents on the efficacy of all licensed antibac-
terial agents. Resistance limits therapeutic options and drives
clinicians to use newer and more expensive agents. In extreme
cases, multiresistance leaves no treatment options. A continuing
supply of new antibiotics offers an obvious way to overcome
resistance, but the pipeline of agents in development by the
pharmaceutical industry is very limited; the number of compa-
nies investing in new antibacterials is falling, partly due to
mergers, but also because of the costs involved and the return
on investment relative to the development of other therapeutic
classes of drugs.1,2There is a pressing need to develop and
evaluate novel alternative strategies for combating a worsening
clinical situation, to overcome resistance and reduce the
morbidity and mortality associated with infections caused by
One strategy would be to use ‘antisense’ or ‘antigene’ agents
to inhibit resistance mechanisms at the nucleic acid level.
Strictly, ‘antisense’ and‘antigene’
collectively as antisense) oligonucleotides bind mRNA to
prevent translation or bind DNA to prevent gene transcription,
(hereafter referred to
respectively. Interrupting expression of resistance genes in this
manner could restore susceptibility to key antibiotics, which
would be co-administered with the antisense compound. This
would extend the lifespan of existing antibiotics, which offer
clinically proven therapies, and are often cheaper, more effective
or less toxic than the alternatives. Antisense molecules that bind
complementary mRNA sequences are a well-established means
of modifying gene expression in mammalian systems.3Indeed,
the manipulation of eukaryotic RNA processing pathways with
small interfering RNAs (siRNAs) has revolutionized research in
mammalian cell biology, with libraries of custom-made mol-
ecules spanning entire genomes now commercially available.
Antisense strategies have been used therapeutically in the treat-
ment of human genetic disorders, such as muscular dystrophy
and familial hypercholesterolaemia,4or viral diseases (see http://
www.avibio.com and http://www.isispharm.com), with some
clinical trials ongoing and with a small number of agents already
licensed for clinical use.5
However, antisense strategies have been considered as anti-
bacterial agents by relatively few researchers, and usually as bac-
tericidal agents targeting essential genes.6–10A complementary,
*Corresponding author. Tel: þ44-20-8327-7255; Fax: þ44-20-8327-6264; E-mail: firstname.lastname@example.org
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Journal of Antimicrobial Chemotherapy (2009) 63, 225–229
Advance Access publication 11 November 2008
at St Bartholomew's & the Royal on March 25, 2011
but equally valid, research path involves the use of antisense
oligonucleotides to target the genes responsible for antibiotic
resistance. There is limited proof-of-principle evidence for resist-
ance modulation by antisense agents; the approach has been
applied successfully in vitro to reverse, for example, amikacin
resistance,11,12chloromycetin resistance13and multidrug efflux in
Escherichia coli,14and glycopeptide resistance in enterococci.15
Developing resistance inhibitors is a sound, well-validated
strategy, which complements the development of directly anti-
bacterial agents. For example, b-lactamase inhibitors, such as
clavulanic acid, tazobactam and sulbactam, are widely used
clinicallyto restorethe susceptibility
co-administered b-lactam antibiotics. The economic and clinical
value of this rationale is demonstrated by efforts to market new
combinations (for example, cefixime/clavulanate; http://www.
ranbaxy.com) or to develop novel b-lactamase inhibitors (for
example, NXL104; http://www.novexel.com).16
b-lactamases, efflux pump inhibitors offer a tantalizing and
much-explored route whereby bacterial susceptibility could be
restored simultaneously to multiple antibiotic classes.17The
principle of using antisense therapeutics as modulators of
bacterial resistance is broadly applicable and could be used
to overcome resistance, potentially, in any pathogenic species.
Furthermore, in contrast to agents targeting essential genes, it
may be possible to target only antibiotic-resistant bacteria, limit-
ing disruption of the normal flora, particularly if the antisense
allows the co-administration of a narrow-spectrum agent.
Toxicity would also be anticipated to be minimal because:
(i) antibiotic resistance genes have virtually no homology to
human genes, and (ii) humans are continually exposed to bac-
terial nucleic acids. However, many obstacles must be overcome
if these innovative technologies are to be harnessed to reduce
the burden of antibiotic resistance for the benefit of patients.
Inhibition of prokaryotic protein synthesis by antisense mol-
ecules may occur by at least four mechanisms, most of which
rely on the activity of intracellular nucleases. The degradation
of RNA:mRNA duplexes by dsRNA-specific RNAses is one
example of a natural means of transcriptional regulation
controlling membrane transport in E. coli18and virulence in
Staphylococcus aureus.19When antisense RNA is artificially gen-
erated from transfected plasmid vectors, short-lived inhibition of
bacterial gene expression can also be observed.20The introduction
of antisense oligonucleotides able to form stable DNA:RNA inter-
actions leads to degradation of the resulting heterodimers through
the activity of RNAse H. If external guide sequences are coupled
to the oligonucleotide, degradation occurs via the action of
RNAse P. Alternatively, undegraded oligonucleotide:RNA hetero-
duplexes are able to inhibit translation by a steric block of riboso-
mal maturation and polypeptide elongation processes (Figure 1).
However, the steric block is not efficient in the coding region of
the genes and is restricted to RNA sites where translation is
initiated or where other RNA processing events occur. There are
myriad chemistries for modified nucleic acids that could be
developed as antisense therapeutics, including phosphorothioate
oligonucleotides (PS-ODNs), locked nucleic acids (LNAs),
20-O-methyloligoribonucleotides (20OMes), phosphorodiamidate
morpholino oligonucleotides (PMOs) and peptide nucleic acids
(PNAs).21–24All offer advantages of increased nuclease stability
and markedly increased antisense activity, but at hugely inflated
synthetic cost.2520OMes, LNAs, PMOs and PNAs all bind more
tightly to RNA than oligonucleotides or PS-ODNs and therefore
can be used at shorter lengths and at lower concentrations when
used in the steric block mode.26
Although conceptually simple, translating the antisense
approach into tangible therapeutic agents is hampered by two
formidable obstacles. First, how can the most appropriate anti-
sense molecule be identified? Secondly, how can it be delivered:
(i) to the site of infection in the patient, and (ii) to its site of
action within the bacterial cell? Clinically useful antibacterial
antisense agents require research far beyond proving their effi-
cacy in in vitro cell-free translation systems, or after electropora-
tion of recombinant DNA into antibiotic-resistant bacterial cells.
OligodeoxynucleotideModified Oligodeoxynucleotide External Guide Sequence
RNAse H Steric Block RNAse P
Figure 1. Mechanisms of ‘knock down’ of gene expression by various antisense molecules (adapted from Figure 1 in Rasmussen et al. with permission from
at St Bartholomew's & the Royal on March 25, 2011
Another potential hurdle could be how the regulatory authorities
would handle such agents; early consideration may serve to
encourage those seeking to develop this type of approach.
Design of the antisense oligonucleotide is crucial but, in con-
trast to work in eukaryotes, very little has been undertaken to
design oligonucleotides for the inhibition of bacterial genes.
A search of the AOBase database (http://www.bioit.org.cn/ao/
aobase/), which catalogues sequences known to be effective,
returns results for only a single bacterial gene; E. coli K12 23S
rRNA. In order to form stable DNA:RNA heteroduplexes, anti-
sense molecules must bind accessible regions of the target
mRNA. However, as very few crystal structures are available,
sites are usually selected using bioinformatic algorithms that
predict the secondary structure of the molecule using calcu-
lations of minimal free energy. Although these algorithms (for
example mfold27) have been useful in modelling structural
RNAs, they have been less successful when applied to mRNAs.
This shortcoming may be attributable to the existence of mul-
tiple heterogeneous secondary structures within a given popu-
lation of mRNA molecules.28Other approaches, such as random
screening of oligonucleotide libraries or scanning arrays, rep-
resent little more than costly ‘fishing’ exercises with limited
chances of success. ‘Sfold’ (http://sfold.wadsworth.org/)29offers
improved mRNA structural prediction, and has parameters
specific for prokaryotic RNA. Sfold-designed oligonucleotides
have been evaluated in cell-free expression experiments and
shown to be highly potent inhibitors of the E. coli lacZ gene.30
Importantly, Sfold assesses the maximum length of antisense
sequences, which must be considered to reduce the costs associ-
ated with modified nucleic acids.
Following identification, candidate sequences have to be
delivered into bacterial cells. Aside from the pharmacokinetic
and pharmacodynamic parameters that would need to be
addressed in the development of an antisense drug, the mol-
ecules will have to traverse the bacterial membrane(s) and resist
degradation by intracellular nucleases to exert their effect.
Stability of the antisense message can be improved by using
modified nucleic acids, but these molecules are too large to
enter the cell by passive diffusion. An unmodified 10-mer oligo-
nucleotide is 2-3 kDa, and the various chemical modifications
outlined above add further to this size. In short, antisense thera-
peutics are likely to be considerably larger than vancomycin, for
example, and therefore require a delivery system. A schematic
diagram of the processes involved in the delivery of an antisense
molecule targeting a b-lactamase is shown in Figure 2.
Entry into bacterial cells can be improved by attaching the
antisense agent to a cell-permeabilizing peptide (CPP) carrier,
which enables uptake through peptide permeases in the cyto-
plasmic membrane.31–33Such peptides can be attached readily
to PNAs or PMOs, which are electrically neutral nucleic
acid analogues,34and may then facilitate better delivery than
when coupled to unmodified oligonucleotides. Indeed, it is
peptide-PMO conjugates that are being taken to clinical trials
for other disease indications, including Duchenne muscular
Figure 2. Schematic to illustrate modulation of a resistance mechanism (in this case, b-lactamase production) using an antisense strategy. 1: mRNA encoding
a b-lactamase is translated into a functional polypeptide by the bacterial ribosome and is transported across the cytoplasmic membrane by type II secretion.
2: b-Lactams traverse the bacterial outer membrane via specific porins. 3: b-Lactams are bound to b-lactamases in the periplasm and hydrolysed. 4: The peptide
component of a CPP-antisense oligonucleotide conjugate causes local disruption of the outer membrane allowing entry to the periplasm. The conjugate traverses
the cytoplasmic membrane aided by peptide permeases and enters the cytoplasm. 5: The antisense oligonucleotide then binds to the complementary sequence on
the mRNA and inhibits translation of the b-lactamase. 6: b-Lactams are not hydrolysed in the periplasm, and are able to bind to peptidoglycan-synthesizing
enzymes (the PBPs), leading to cell lysis.
at St Bartholomew's & the Royal on March 25, 2011
dystrophy (see, for example, http://www.avibio.com). Many CPP
carriers are known, but few have been evaluated, in part due to
the complete lack of data on the appropriate antisense molecule
with which to couple them, and the lack of any standardized
way of quantifying their efficiency. A PMO directed against a
critical target (acpP, encoding acyl carrier protein, which is
involved in fatty acid biosynthesis) and linked to the decapeptide
KFFKFFKFFK had activity in a mouse model of E. coli
considerable haemolytic activity36and so is unlikely to be a
strong candidate for clinical development. Other variants, such
as RFFRFFRFFRXB (X is 6-aminohexanoic acid and B is
b-alanine), look significantly better.37The ability of any CPP
considered for clinical development to transport antisense agents
across bacterial membranes would have to be assessed not just
in E. coli, but also in relatively ‘impermeable’ genera, such as
Acinetobacter and Pseudomonas.
Delivery of antisense molecules packaged in bacteriophage
has not been widely considered, but offers the conceptual advan-
tage over CPP-oligonucleotide conjugates of tailored delivery
only to the bacterial species of interest. Such specificity might
be expected to reduce collateral damage to commensal species
coincidentally carrying the targeted resistance gene, and thus
potentially to reduce the speed of development of resistance by
reducing non-species-specific selection pressure. Liposomes
might also be considered as delivery vehicles.
It would be shortsighted to assume that antisense agents
will be ‘immune’ to the emergence of resistance; rather, its
emergence is an evolutionary inevitability. However, by giving
careful consideration to the plethora of potential strategies avail-
able, and by pursuing those considered to pose the least risk for
the selection of resistant variants, we could develop antisense
agents as both effective antibacterial agents and resistance
inhibitors. It may well be possible to design synergistically
acting antisense agents, targeting either more than one resistance
gene, or a resistance gene(s) in combination with a critical bac-
terial target. It is exciting to consider a future where clinicians
treating infections caused by an antibiotic-resistant bacterial
strain might be able to co-administer a molecular inhibitor of
the critical resistance gene together with the relevant antibiotic,
such as a mecA inhibitor plus flucloxacillin for methicillin-
resistant S. aureus. Such an approach would be of enormous
benefit to patients and healthcare systems. The concept should
attract funding and the developmental powers of the pharma-
ceutical industry which could find new niches for long-
abandoned generic drugs or agents still under patent but now
compromised by resistance.
Members of the UK Antibacterial Antisense Study Group: Ian
Chopra, University of Leeds; Matthew Ellington, HPA Centre
for Infections; Virve I. Enne, University of Bristol; Heather
Fairhead, Phico Therapeutics; William Fraser, Aston University;
Michael J. Gait, Medical Research Council, Laboratory of
Molecular Biology, Cambridge; Peter A. Lambert, Aston
University; David M. Livermore, HPA Centre for Infections;
Alex J. O’Neill, University of Leeds; Thamarai Schneiders,
University of Edinburgh; David W. Wareham, Barts and The
London School of Medicine and Dentistry, London; Neil
Woodford, HPA Centre for Infections; Davis E. Yakubu,
University of Sunderland.
Discussions of the UK Antibacterial Antisense Study Group that
resulted in this article were supported by AstraZeneca and the
BSAC. D. W. W.’s work on antisense antibacterials has been
supported by a Royal College of Pathologists Pilot Research
N. W. has received research grants and accepted speaking
engagements/conference invitations from various companies.
None of these constitute a conflict of interest with the content of
the current paper. D. W. W. has none to declare.
1. FoxJL. Thebusiness ofdeveloping antibacterials.Nat
Biotechnol 2006; 24: 1521–8.
2. Projan SJ, Shlaes DM. Antibacterial drug discovery: is it all
downhill from here? Clin Microbiol Infect 2004; 10 Suppl 4: 18–22.
3. Wall NR, Shi Y. Small RNA: can RNA interference be exploited
for therapy? Lancet 2003; 362: 1401–3.
4. van Ommen GJ, van Deutekom J, Aartsma-Rus A. The thera-
peutic potential of antisense-mediated exon skipping. Curr Opin Mol
Ther 2008; 10: 140–9.
5. Wacheck V. Oligonucleotide therapeutics—an emerging novel
class of compounds. Wien Med Wochenschr 2006; 156: 481–7.
6. Chopra I. Prospects for antisense agents in the therapy of
bacterial infections. Expert Opin Investig Drugs 1999; 8: 1203–8.
7. Good L. Antisense antibacterials. Expert Opin Ther Patents
2002; 12: 1173–9.
8. Geller BL. Antibacterial antisense. Curr Opin Mol Ther 2005; 7:
9. Kurupati P, Tan KSW, Kumarasinghe G et al. Inhibition of gene
expression and growth by antisense peptide nucleic acids in a multire-
Antimicrob Agents Chemother 2007; 51: 805–11.
10. Nekhotiaeva N, Awasthi SK, Nielsen PE et al. Inhibition of
Staphylococcus aureus gene expression and growth using antisense
peptide nucleic acids. Mol Ther 2004; 10: 652–9.
11. Sarno R, Ha H, Weinsetel N et al. Inhibition of aminoglycoside
60-N-acetyltransferase type Ib-mediated amikacin resistance by anti-
sense oligodeoxynucleotides. Antimicrob Agents Chemother 2003; 47:
12. Soler Bistue ´ AJC, Ha H, Sarno R et al. External guide
sequences targeting the aac(60)-Ib mRNA induce inhibition of amikacin
resistance. Antimicrob Agents Chemother 2007; 51: 1918–25.
13. Gao MY, Xu CR, Chen R et al. Chloromycetin resistance of clini-
cally isolated E coli is conversed by using EGS technique to repress
the chloromycetin acetyl transferase. World J Gastroenterol 2005; 11:
14. White DG, Maneewannakul K, von Hofe E et al. Inhibition of
the multiple antibiotic resistance (mar) operon in Escherichia coli by
antisense DNA analogs. Antimicrob Agents Chemother 1997; 41:
at St Bartholomew's & the Royal on March 25, 2011
15. Torres VC, Tsiodras S, Gold HS et al. Restoration of vancomycin Download full-text
susceptibility in Enterococcus faecalis by antiresistance determinant
gene transfer. Antimicrob Agents Chemother 2001; 45: 973–5.
16. Livermore DM, Mushtaq S, Warner M et al. NXL104 combi-
nations versus Enterobacteriaceae with CTX-M extended-spectrum
b-lactamases and carbapenemases. J Antimicrob Chemother 2008;
17. Stavri M, Piddock LJV, Gibbons S. Bacterial efflux pump
inhibitors from natural sources. J Antimicrob Chemother 2007; 59:
18. Delihas N. Regulation of gene expression by trans-encoded anti-
sense RNAs. Mol Microbiol 1995; 15: 411–4.
19. Romby P, Vandenesch F, Wagner EG. The role of RNAs in the
regulation of virulence-gene expression. Curr Opin Microbiol 2006; 9:
20. Chen G, Patten CL, Schellhorn HE. Controlled expression of an
rpoS antisense RNA can inhibit RpoS function in Escherichia coli.
Antimicrob Agents Chemother 2003; 47: 3485–93.
21. Ecker DJ, Freier SM. PNA, antisense, and antimicrobials. Nat
Biotechnol 1998; 16: 332.
22. Mojzisek M. Triplex forming oligonucleotides—tool for gene
targeting. Acta Medica (Hradec Kralove) 2004; 47: 151–6.
23. Hojland T, Kumar S, Babu BR et al. LNA (locked nucleic acid)
and analogs as triplex-forming oligonucleotides. Org Biomol Chem
2007; 5: 2375–9.
24. Lebleu B, Moulton HM, Abes R et al. Cell penetrating peptide
conjugates of steric block oligonucleotides. Adv Drug Deliv Rev 2008;
25. Warfield KL, Panchal RG, Aman MJ et al. Antisense treatments
for biothreat agents. Curr Opin Mol Ther 2006; 8: 93–103.
26. Kurreck J. Antisense technologies. Improvement through novel
chemical modifications. Eur J Biochem 2003; 270: 1628–44.
27. Zuker M. Mfold web server for nucleic acid folding and hybridiz-
ation prediction. Nucleic Acids Res 2003; 31: 3406–15.
28. Ding Y, Lawrence CE. A statistical sampling algorithm for
RNA secondary structure prediction. Nucleic Acids Res 2003; 31:
29. Ding Y, Chan CY, Lawrence CE. Sfold web server for statistical
folding and rational design of nucleic acids. Nucl Acids Res 2004; 32:
30. Shao Y, Wu Y, Chan CY et al. Rational design and rapid screen-
ing of antisense oligonucleotides for prokaryotic gene modulation.
Nucleic Acids Res 2006; 34: 5660–9.
31. Hammond SM, Claesson A, Jansson AM et al. A new class of
synthetic antibacterials acting on lipopolysaccharide biosynthesis.
Nature 1987; 327: 730–2.
32. Dryselius R, Nekhotiaeva N, Good L. Antimicrobial synergy
between mRNA- and protein-level inhibitors. J Antimicrob Chemother
2005; 56: 97–103.
33. Geller BL, Deere JD, Stein DA et al. Inhibition of gene
expression in Escherichia coli by antisense phosphorodiamidate mor-
pholino oligomers. Antimicrob Agents Chemother 2003; 47: 3233–9.
34. Nikravesh A, Dryselius R, Faridani OR et al. Antisense PNA
accumulates in Escherichia coli and mediates a long post-antibiotic
effect. Mol Ther 2007; 15: 1537–42.
35. Tan XX, Actor JK, Chen Y. Peptide nucleic acid antisense
oligomer as a therapeutic strategy against bacterial infection: proof of
principle using mouse intraperitoneal infection. Antimicrob Agents
Chemother 2005; 49: 3203–7.
36. Vaara M, Porro M. Group of peptides that act synergistically with
Antimicrob Agents Chemother 1996; 40: 1801–5.
37. Tilley LD, Mellbye BL, Puckett SE et al. Antisense peptide-
phosphorodiamidate morpholino oligomer conjugate: dose–response
in mice infected with Escherichia coli. J Antimicrob Chemother 2007;
38. Rasmussen L, Sperling-Petersen H, Mortensen K. Hitting bac-
teria at the heart of the central dogma: sequence-specific inhibition.
Microb Cell Factories 2007; 6: 24.
at St Bartholomew's & the Royal on March 25, 2011