Microbial genetics: Exploiting genomics, genetics and chemistry to combat antibiotic resistance

© 2003 Nature Publishing Group

432 | JUNE 2003 | VOLUME 4 www.nature.com/reviews/genetics

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Antibiotics are of immense value for combating infec-tious diseases and are estimated to have extended thelifespan of the average citizen of the United States by tenyears1. In recent decades, their effectiveness has beenthreatened by an inexorable rise in the prevalence ofmicrobial drug resistance. Because microbial infectionscan generally be treated with several different anti-biotics, resistance to an antibiotic does not translatedirectly into therapeutic failure. Resistance and the riskof inappropriate therapy, however, increasingly limittherapeutic options. For some important pathogens, inwhich serious resistance problems exist, few effectiveantibiotics now remain.

Staphylococcus aureus is perhaps the most significantof these pathogens. It causes community and hospitalacquired infections and is associated with high morbid-ity and mortality rates2.Vancomycin has been used as theantibiotic of last resort to treat methicillin-resistant S. aureus infections (MRSA) with multiple resistance,since the 1980s. Strains with some level of resistance to vancomycin (vancomycin-intermediate-resistant S. aureus; VISA) have been known since at least 1996(REF. 3), but the newly identified highly-resistant strain(vancomycin-resistant S. aureus; VRSA) heralds a newstage in our battle with this pathogen4,5. It might be onlya matter of time before the gene cluster that confers this

resistance (FIG. 1) is naturally transferred into strains withmultiple resistance, such as MRSA. So, the arrival ofVRSA is a significant marker of the end of the initialperiod in the antibiotic era. Other serious treatmentproblems include multidrug resistance in tuberculosis6,7,vancomycin-resistant enterococci (VRE)8, resistanceowing to extended spectrum β-lactamases (ESBLs) inEnterobacteriaceae and Pseudomonas aeruginosa9, andpenicillin resistance in Streptococcus pneumoniae10.

In the context of the increasing number of problemswith antibiotic resistance, it is important to considerhow to maintain our ability to deal effectively withbacterial infectious diseases. Antibiotics are also usedin agriculture and the development of antibiotic resis-tance in agricultural systems is a problem in itself. Thisreview, however, focuses on combating resistance inhuman pathogens. The options for combating anti-biotic resistance that are discussed in this review includedeveloping new antibiotics, containing or reversingantibiotic resistance and developing alternatives to anti-biotic therapy, such as vaccines. Research in genomics isproviding a catalogue of potential new targets forantibiotic and vaccine therapy. Techniques in moleculargenetics are facilitating the rapid evaluation of theessentiality of these targets on a genomic scale. Finally,COMBINATORIAL-CHEMISTRY techniques are making feasible

EXPLOITING GENOMICS, GENETICSAND CHEMISTRY TO COMBATANTIBIOTIC RESISTANCEDiarmaid Hughes

To address the worsening problem of antibiotic-resistant bacteria there is an urgent need to developnew antibiotics. Comparative genomics and molecular genetics are being applied to produce listsof essential new targets for compound screening programmes. Combinatorial chemistry andstructural biology are being applied to rapidly explore and optimize the interactions between leadcompounds and their biological targets. Several compounds that have been identified from target-based screens are now in development, but technical and economic constraints might result in atrickle, rather than a flood, of new antibiotics onto the market in the near future.

COMBINATORIAL CHEMISTRY

Methods that can, in acontrolled manner, result in thesynthesis of numerous chemicalvariants of a molecule.

Department of Cell andMolecular Biology, Box 596,The Biomedical Center,Uppsala University,S-751 24 Uppsala, Sweden.e-mail:[email protected]:10.1038/nrg1084

M I C R O B I A L G E N E T I C S


NATURE REVIEWS | GENETICS VOLUME 4 | JUNE 2003 | 433

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TWO-COMPONENT

REGULATORY SYSTEM

A signal-transduction systemthat consists of a sensor proteinthat senses and responds to anexternal signal, and which actson a response-regulator proteinthat transmits the signal to othercomponents of the cell.

ENTEROCOCCI

Gram-positive cocci bacteria,which include Enterococcusfaecium and Enterococcusfaecalis.

on resistance in humans19 and the BIOLOGICAL FITNESS coststhat are associated with resistance20–22.Also, the success ofa few clones of S. pneumoniae23 and S. aureus24 with mul-tiple resistance is intriguing. It indicates that clonalgenetic features other than resistance determinants —for example, the success of particular bacterial clones at establishing an invasive infection — can contributesignificantly to the success and spread of resistant strains.

Genomics as a tool to combat resistanceIdentifying new targets. With the advent of genomics,the emphasis in drug-discovery programmes haschanged from screening compound libraries to firstseeking targets. The basic strategy for target-basedantibiotic discovery and development is outlined inFIG. 2. Some recent comparative genome analyses showthat there might be fewer than 300 broad-spectrumessential potential target genes25,26. Genome analysiscan also be used to identify narrow-spectrum targetsthat might be of special interest in problem organismssuch as Mycobacterium tuberculosis. Another potentialuse is to identify ‘Achilles heels’ that are associated withvirulent or resistant isolates and are not shared by bac-terial species in general. For example, MOLECULAR-TYPING

studies on drug-resistant S. pneumoniae and S. aureusshow that there is limited genetic diversity amongthese important pathogenic groups23,24,27,28. This lack of genetic diversity among successful clones showsthat genomic analysis might be used to identify targetsthat are specific to these problematic clones.

Validating and prioritizing new targets. The ability to rapidly identify essential genes in which loss offunction coincides with loss of viability or virulenceattenuation is the driving force behind genomics-based target validation. One form of target validationis to identify genes that are essential for bacterialgrowth under all conditions. So, high-density randomTRANSPOSON MUTAGENESIS has been used to identify essen-tial genes in particular species29,30. In this way, 478essential genes were identified in Haemophilusinfluenzae, 259 of which had no known function30.Alternatively, mutagenesis can be targeted to a selectedlist of conserved genes. For example, in S. pneumoniae,347 genes that are conserved in Bacillus subtilis,Enterococcus faecalis, Escherichia coli and S. aureus weredesignated as potential targets. Target-gene disruptionwas achieved by allelic exchange using PCR-generatedDNA fragments from each target, which were clonedinto a SUICIDE VECTOR. The resulting analysis identified113 essential genes among the 347 S. pneumoniae can-didates26. A third method to identify essential genes isby expressing antisense RNA. This has been done in S. aureus, in which transformation with a plasmidlibrary that contained staphylococcal DNA fragmentsbehind a tetracycline-regulated promoter led to theidentification of ~150 essential S. aureus genes31.

An alternative approach to choosing new drug targets is to identify gene products that are involved invirulence. Theoretically, this approach has the advantagethat it can identify relatively narrow-spectrum targets

the rapid synthesis of large numbers of drug candidatesfor evaluation in high-throughput screens.

Resistance as a clinical problemThere are several molecular mechanisms by which bacteria can become antibiotic resistant (BOX 1), all ofwhich result from the reduced concentration or reducedactivity of antibiotics in the bacterial cell11. However,regardless of the underlying molecular mechanism,several other factors influence how soon a particularresistant strain becomes a clinical problem12. These fac-tors include rates of mutation13,14, the frequency of theLATERAL GENE TRANSFER of resistance determinants15,16, theintensity of antibiotic usage in hospitals and the com-munity17,18, the impact of antibiotic use in agriculture

Box 1 | The molecular basis of antibiotic resistance

Sensitive bacteria can become antibiotic resistant in several ways:• Import of an antibiotic can be inhibited by mutations that downregulate, delete ormodify outer-membrane porins136.

• Mutations that upregulate the expression of transmembrane efflux pumps can reducethe concentration of antibiotic in the bacterial cell. In many cases, efflux pumps canpump out several different antibiotics causing a multidrug-resistance phenotype137,138.

• Enzymes that modify, cleave or otherwise inactivate an antibiotic are another commoncause of resistance. The most important example clinically is the widespread occurrenceof β-lactamases that cleave the β-lactam ring of β-lactam antibiotics9.

• Finally, the antibiotic target can be altered (by mutation139,140, recombination141 orreplacement with an alternative target as happens with vancomycin resistance inenterococci142) such that the affinity of the antibiotic for its target is reduced.

D-AlaVancomycinresistance

vanR vanS vanH vanA vanX vanY vanZ

P P

Pyruvate

Activator

D-Lac D-Lac

Cleavage ofD-Ala-D-Ala

D-Ala-D-Ala pentapeptide → tetrapeptideVancomycin

sensitivity

Vancomycin

L-Ala-D-Glu-L-Lys-D-Ala-D-Ala

Sensor and regulatoractivates vanHAX operon

D-Ala-D-Lac synthesisvancomycin resistant

Figure 1 | The VanA gene cluster that confers vancomycin resistance. A TWO-COMPONENT

REGULATORY SYSTEM VanR–VanS regulates vancomycin resistance in vancomycin-resistantENTEROCOCCI (VRE) and vancomycin-resistant Staphylococcus aureus (VRSA) strains. VanS isa membrane-associated sensor (of vancomycin) that controls the level of phosphorylation ofVanR. VanR is a transcriptional activator of the operon encoding VanH, VanA and VanX. VanH is a dehydrogenase that reduces pyruvate to D-Lac, whereas VanA is a ligase thatcatalyses the formation of an ester bond between D-Ala and D-Lac. Vancomycin does not bindto D-Ala-D-Lac, which leads to vancomycin resistance. VanX is a dipeptidase that hydrolysesthe normal peptidoglycan component D-Ala-D-Ala, which prevents it from causing vancomycinsensitivity. VanY is a D,D-carboxypeptidase that hydrolyses the terminal D-Ala residue of latepeptidoglycan precursors that are produced if elimination of D-Ala-D-Ala by VanX is notcomplete. So, D-Ala-D-Lac replaces the normal dipeptide D-Ala-D-Ala in peptidoglycansynthesis resulting in vancomycin resistance150. VanZ confers resistance to teicoplanin by anunknown mechanism.



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Another way in which genome-scale analysis mightinform the selection of new targets is to analyse gene-expression profiles during exposure to an antibiotic.In this approach, microarrays of DNA fragments thatcorrespond to every open reading frame (ORF) in agenome are used to obtain the signature of a specificinhibitor. Potentially, this can identify a number ofgenes that are involved in the biosynthetic pathway thatthe inhibitor targets. This has been applied in the tran-scriptional response of M. tuberculosis to the antibioticisoniazid49 to identify a set of genes that are induced byisoniazid and to indicate a number of potential newtargets for antimicrobial drugs.

Finally, an interesting new approach is illustrated bythe validation of prolyl-tRNA synthetase as an essentialtarget. Initially, a specific peptide binder of the targetenzyme was selected in vitro50. Subsequently, inducedexpression of the inhibitory peptide in an animal-infection model rescued the animal, thereby showingthe essentiality of the target to the disease process in vivo. The advantage of this approach is that once thetarget, the peptide and the peptide binding site werevalidated by these methods, screening for small mole-cules that displaced the peptide was used as a directway to obtain new drug leads50. This approach hasfacilitated the discovery of lead compounds that targetdifferent tRNA synthetases51–53.

Optimizing new antibioticsTraditionally, new antimicrobial agents have been iden-tified from biological sources and from large collec-tions of individual compounds that have been gatheredfrom various laboratories. Classical biological sourcesare the actinomycetes and fungi, which continue to beused in the search for new antibiotics54. A successfulantibiotic must satisfy a bewildering number ofdemands. It should be able to cross bacterial cell mem-branes, avoid bacterial efflux pumps, not be a target forbacterial modifying or hydrolyzing enzymes that couldinactivate it, and reach its target at a sufficiently highconcentration so as to inhibit a vital bacterial function.Also, for economic and diagnostic reasons, it shouldhave a broad spectrum of antibacterial activity. Finally,it should reach inhibitory concentrations at the site ofinfection, have little or no toxicity and have minimalside effects in humans. This set of limitations means, inpractice, that almost any new chemical that is initiallyidentified as having an interesting antimicrobial activ-ity in a screening process will undergo many chemicalmodifications before being approved and marketed as an antibiotic. Several different technologies havecrucial roles in this process. Medicinal chemistry, andin particular combinatorial chemistry, is crucial bothfor the generation of new and expanded chemicallibraries, and for the optimization of lead compounds.Combinatorial biology also has an important role as anapproach to producing new antibiotics, in particularthose with large complex structures. Biochemistry iscrucial in the development of appropriate and sensitivein vitro assays to rapidly screen for target inhibitionusing chemical libraries.

that would reduce the probability of resistance emergingbecause they reduce the impact of antibiotic selection onthe resident flora32. Techniques that are useful for thispurpose identify genes that are specifically switched onduring infection, and include in vivo expression technol-ogy (IVET)33, differential fluorescence induction (DFI)34

and signature-tagged mutagenesis (STM)35,36 (BOX 2). Adrawback with the first two techniques is that expressionin vivo does not necessarily mean that the gene productis essential in vivo. By contrast, STM identifies therequirement for a gene product during infection inwhich inactivation of the gene results in attenuation ofvirulence35,36. STM has been successfully applied to sev-eral pathogens37–46, including S. pneumoniae47,48 in whichalmost 400 mutants were identified as attenuated forinfection in a murine model. Most of the genes identifiedhad not previously been associated with virulence andthe list includes many of unknown function47.

LATERAL GENE TRANSFER

The transfer of DNA, frequentlycassettes of genes, betweenorganisms.

BIOLOGICAL FITNESS

A relative measure of the abilityof a particular group of bacteriato compete successfully in aparticular environment.

MOLECULAR TYPING

The use of molecular genetictechniques — for example,multiplex PCR, pulse-field gelelectrophoresis, Southernblotting and multilocussequence typing — to geneticallycompare and characterizebacterial genomes.

DNA sequencing

Bioinformatics

Molecular genetics

Structural biology Biochemistry

Biochemistry

Compound library High-throughput screening

Discovering a lead compound (‘hit’)

Interesting bacteria

Genome sequences

Potential target list

Validated target list

Target structure and function

Inhibition/binding assay

Initial hits

Developing a lead compound

Whole-cell target-specific activity

In vivo activity

Combinatorial chemistryStructure-based design

Whole-cell assayCombinatorial chemistry

Animal-model assayCombinatorial chemistry

Clinical trials

Initial hits

Improved hits

Approval and marketing

Figure 2 | Target-based antibiotic discovery and development — the basic strategy.a | The presence of the target in all organisms of interest is established before the expensivedevelopment programme begins. Initially, sequences are required for the genomes of thoseorganisms of interest. This information is already available for the most important pathogenicbacteria (see The Institute for Genomic Research in online links box). Genomes are thencompared and analysed using bioinformatics tools to identify genes and biosynthetic pathwaysthat are conserved and common to a broad spectrum of pathogens, but which are absent in thehuman genome. Molecular genetic techniques (for example, transposon mutagenesis) are thenapplied to test the essentiality of the potential target genes and pathways. For targets that arevalidated as essential, the structure and function should ideally be determined. This informationwill then inform the development of biochemical assays and high-throughput screens for inhibitionof function, and the rational design of inhibitor molecules. b | ‘Hits’ can be structurally analysedand optimized for improved inhibition by traditional and combinatorial chemistry. The process ofchemical modification and improvement continues, because most hits that are initially identified intarget-based assays do not have significant activity in whole-cell assays or in vivo. At this stage,some assessment of the potential for resistance problems can be made. The most promisinglead compounds then enter the long and expensive process of clinical trials.



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by small molecules. A recent example of the successfulapplication of this approach is the screening of high-density microarrays that carry 3,780 small molecules,generated by diversity-orientated synthesis, with thefluorescently-labelled yeast protein Ure262. The smallmolecules that are synthesized are structurally complexand unbiased towards any particular protein target.Before the screening, no small molecules that werecapable of binding Ure2 were known. Eight binderswere identified and one in particular, uretupamine, wasanalysed by whole-genome transcription profiling, andwas shown to bind to the protein of interest and tomodulate its specific functions62.

Optimizing known inhibitors. Combinatorial chemistrycan generate completely new compounds, but it is stillmost frequently used to build new diversity that is basedon antibiotics in clinical use, including tetracyclines63,macrolides64 and oxadolidinones65. An alternativeapproach is to re-investigate old hits that were neverdeveloped for a variety of reasons (narrow spectrum ofactivity, toxicity, weak ligand binding and so on). Forexample, a combinatorial library strategy applied to amoenomycin A disaccharide template identified a newclass of bacterial cell-wall biosynthesis inhibitors thatalso have potent antibacterial activity66–68.

Rational drug design. When a new biological target has been well defined structurally and functionally it ispossible to use rational design to define an inhibitor

Combinatorial chemistry approach. Combinatorialchemistry facilitates the rapid synthesis of a large num-ber of distinct compounds that can then be assayed forantimicrobial activity55. A total of 305 small-moleculelibraries were published in 2001 (REF. 56). The presentlimitations of chemical synthesis mean that, in practice,most libraries are used to explore a specific pharma-cophore (a structural motif with biological activity). Forscreening these large libraries, it has been necessary todevelop high-throughput screens that are suitable for thesmall quantities of individual compounds in the library57.In such cases, an assay that can identify weak ligands isbest for the discovery of new classes of antibacterials.Subsequent combinatorial synthesis of a large number ofanalogues of an initial ‘hit’ can then be used to identifycompounds with better properties58. It is frequently diffi-cult to optimize complex high-molecular-weight leadcompounds59,60, which underscores the desirability ofinitiating screening programmes with libraries of simplelow-molecular-weight structures.

The most commonly applied chemical approach togenerate variation in small molecules is referred to as‘target-orientated synthesis’, in which the targets are spe-cific drug candidates. An exciting alternative approachnow under development is ‘diversity-orientated synthe-sis’61. The aim of the latter method is to synthesize collections of structurally diverse and complex smallmolecules. These would then be screened for biologicalactivity, leading eventually to the identification of thera-peutic protein targets that are capable of being modulated

TRANSPOSON MUTAGENESIS

The use of transposons togenerate (knock-out) mutations.

SUICIDE VECTOR

A vector (plasmid) that is unable to replicate in a particularhost and is maintained only ifit recombines into the hostgenome.

Box 2 | Molecular techniques for identifying virulence genes

In vivo expression technology (IVET)33

This method identifies genes that bacteria infecting a host express. First, IVET requires the identification of a gene(geneX) the activity of which is essential in vivo. A promoterless version of geneX is then introduced at random into thechromosome of a bacterial strain in which the resident geneX has been deleted. The aim is to create fusions of thepromoterless geneX to different bacterial promoters. Such a bacterial population is then introduced into the animalmodel and surviving clones are isolated. The bacteria that survive in vivo probably have geneX fused to a promoter thatwas active in vivo.

Differential fluorescence induction (DFI)34

This method involves the insertion of random pieces of bacterial DNA upstream of a green fluorescent protein (GFP)reporter gene (gfp) on a plasmid. Bacteria that harbour fusions are pooled and used to infect a mammalian cell culture.Individual bacteria that express GFP are identified and sorted by the use of flow cytometry and fluorescence-activatedcell sorting (FACS). An advantage over IVET is that DFI can identify promoters that are only weakly or transientlyinduced during infection.

Signature-tagged mutagenesis (STM)35

This method requires a mixture of transposons, each carrying a unique DNA sequence (signature) tag, on a suicidevector that can be maintained as a plasmid in Escherichia coli. Transformation and selection of the tagged pool into arecipient bacterial population results in near-random transposition into the chromosome. So, each mutant will have aunique signature by being associated with a uniquely tagged transposon. A bank of tagged mutants is arrayed inmicrotitre dishes and DNA colony blots that represent individual members of the bank are prepared by replica platingfrom the microtitre dishes. The 96 mutants from each microtitre dish are pooled and an aliquot is removed for DNAextraction (INPUT pool). The remaining pooled bacterial cells are injected into the animal and, after an appropriatetime, organs are removed and those bacteria that have reached the organs and multiplied are recovered. The recoveredcolonies are pooled and DNA is extracted (OUTPUT pool). The tags from the INPUT and OUTPUT pools are amplified,radiolabelled and used, separately, to probe the replica colony blots from the original microtitre dish. Colonies thathybridize to the probe from the INPUT pool, but not to the probe from the OUTPUT pool, represent mutants withattenuated virulence in the animal model. The known sequence, or antibiotic resistance, of the transposon is used toclone and sequence the affected gene.



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heptapeptide GLYCOPEPTIDES, the 14- and 16-memberedmacrolides, and the 13-amino-acid lipopeptide dapto-mycin. It is not economical to synthesize such complexmolecules chemically. The goal of combinatorial biol-ogy is to harness and direct the evolution of complexbiosynthetic pathways to expand the repertoire of sec-ondary metabolites, such as antibiotics74. The main tar-gets of genetic manipulations in combinatorial biology,at present, are the modular type I polyketide synthases(PKSs) and, to a lesser extent, the non-ribosomal peptidesynthases (NRPs).

PKSs and NRPs are organized into repeated units or‘modules’, each of which is responsible for the catalysis ofone complete cycle of polyketide or polypeptide chainelongation and the associated chain modifications75,76.Using molecular genetic technology, it is possible to alterthe number, content and order of these modules and, inso doing, to alter the structure of the resulting moleculesand create ‘unnatural’ natural products (BOX 3). A limita-tion with this approach, so far, is that it delivers deriva-tives of known antibiotics, such as erythromycin77 andvancomycin78, rather than new classes of antibiotic.However, this technology is in the early developmentstage, as illustrated by the recent discovery of C–O bondformation (normally C–C bonds are formed) by poly-ketide synthases79, and has great potential to create newcompounds with enormous structural complexity.

structure69. Combinatorial chemistry can then be usedto explore this structure space and identify lead com-pounds for optimization. In this way, new PEPTIDE-

DEFORMYLASE inhibitors have been identified and are nowbeing developed70,71. This new class of antibiotics thatare active in vivo was the first to be discovered usingmechanism-based drug design and combinatorialchemistry.

A future target for rational drug design is PBP2A. Thisprotein is an important determinant of broad-spectrumβ-lactam resistance in MRSA and its structure wasdetermined recently at high resolution, both alone andin complex with penicillin G and methicillin72. Thesestructures provide a physical explanation for the lowbinding affinity of PBP2A towards most β-lactamantibiotics. The structural information shows in detailhow to design new classes of drug to overcome MRSAβ-lactam resistance. This is also an example of how mol-ecular knowledge could facilitate the direct targeting ofproblematic pathogens such as MRSA.

Combinatorial biology. The principle of combinatorialbiology is to use the enzymes from antibiotic biosyn-thetic pathways to create new chemical structures73,74.Of the antibiotics in clinical use, those with the mostcomplex chemical structures are all based on secondarymetabolites of microorganisms. These include the

PEPTIDE DEFORMYLASE

An essential bacterialmetalloenzyme thatdeformylates the N-formylmethionine of newlysynthesized bacterialpolypeptides.

PBP2A

A penicillin-resistanttranspeptidase that is encodedby mecA in methicillin-resistantS. aureus strains.

GLYCOPEPTIDES

The antibiotic class to whichvancomycin belongs.

Loading domain

Module 1 Module 3 Module 2

AT ACP KS AT KR ACP KS AT KR ACP KS AT ACP KS AT DH ER KR ACP KS AT KR ACP KS AT KR ACP TE

Module 5 Module 4 Module 6

Linker peptides Linker peptides

Direction of polyketide chain growth

eryAI (DEBS1) eryAII (DEBS2) eryAIII (DEBS3)

Box 3 | Combinatorial biology — the example of modular type I polyketide synthase

This technology is based on the biosynthetic machinery that produces antibiotics in microorganisms, in particular themodular polyketide synthases (PKSs) the products of which include erythromycin. PKSs are enzymes that polymerize fattyacids into a variety of chemical structures called polyketides. The erythromycin PKS 6-deoxyerythonolide B synthase(DEBS) from Saccharopolyspora erythraea143 has served as a model system for discovering ways to engineer PKSs and toexplore their structural plasticity and substrate tolerance. The DEBS PKS consists of three >280-kDa multifunctionalprotein subunits that are assembled into the complete PKS complex. The DEBS subunits are encoded by three contiguouserythromycin A (eryA) genes. The figure shows the genetic information in the three genes, which are organized into sixmodules (in which each module contains multiple functional domains) that specify how each two-carbon unit of the finalproduct is assembled. These functional domains catalyse chain elongation (ketosynthase, KS; acyltransferase,AT; and acylcarrier protein,ACP), alteration of oxidation states (ketoreductase, KR; dehydratase, DH; and enoyl reductase, ER) andmacrocyclization (thioesterase, TE). The modules in a polypeptide chain are separated by short peptide linkers thatfacilitate genetic engineering to switch modules. The polypeptide chains interact with one another in the correct sequenceby protein–protein interactions that are partially mediated by intermodular linkers and small interpolypeptide linkerpeptides144. The last functional unit, TE, catalyses the release and cyclization of the polyketide chain.

Importantly, PKSs are processive enzymes and the genetic template is collinear with the polyketide product. So, thelinear order and composition of catalytic sites in the PKS represents a ‘code’ that determines the identity of the polyketideproduct. Genetic engineering of DEBS, cloned into a Streptomyces host on either single or multiple compatible plasmids,has now reached the level of sophistication that multiple changes can be successfully introduced in a combinatorialfashion77,145. So, engineered DEBS were produced carrying single, double and triple domain substitutions, in which theDEBS domains were substituted with functional homologues from the rapamycin PKS, which produced >100 newmacrolides77,145. Recent work that explores the tolerance of these enzymes to non-natural substrates shows that thediversity of products can be greatly expanded146.



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This compound — MC-207,110 — is effective inreversing efflux-mediated fluoroquinolone resistanceand reduces the emergence of resistant strains88.

Recently, the first high-resolution structure of amultidrug-resistance pump (MDR), AcrB from E. coli,has been solved89. The selection and location of MDR-pump mutants with altered substrate specificity90 showsthat the large periplasmic loops of the pump proteincontain multiple sites of interaction for various struc-turally diverse compounds. Taken together, the struc-tural detail and the genetic data should enhance thedevelopment of new pump inhibitors, and of antibioticcompounds that are capable of evading efflux pumps.

Alternative strategiesOptimizing antibiotic use. Restricting antibiotic con-sumption has been indicated as one way to reduce theprevalence of resistant bacteria. The basis of this idea isthat a biological fitness cost is usually associated withantibiotic resistance and this cost might lead to the lossof resistant strains in the absence of selection by antibi-otics21. However, some resistance mutations have littleor no cost20,91,92, and fitness costs, if they do occur, can beameliorated by the accumulation of further compen-satory mutations22,91,93–97 or by expressing the resistancephenotype only in response to the antibiotic98,99. In twocommunity studies, in which specific antibiotics wererestricted for several years, only small reductions in theproportion of resistant strains were detected17,100–102.Greater success has been reported in a hospital environ-ment103,104, in which control is easier and the entry ofnew patients dilutes the prevalence of resistant bacteriatowards the level found in the community. The conclu-sion is that, although commendable for many reasons, astrategy to reverse the level of antibiotic resistance onthe basis of decreasing levels of antibiotic consumptionwill probably have limited success.

If rapid molecular diagnostic methods were avail-able, it would be feasible to use narrow-spectrum drugswith safety. This would reduce the selective pressure forthe spread of resistance. The development of rapidmolecular diagnostics would also be beneficial forincreasing the efficacy of dosing with traditional BROAD-

SPECTRUM ANTIBIOTICS. A successful diagnostic test wouldneed to combine rapidity, sensitivity, specificity andubiquity105, and would probably be based on nucleic-acid amplification106. Research into the development ofsuch techniques is now underway107,108.

The options discussed previously aim to reduce theantibiotic selective pressure for the emergence of resis-tance and are, therefore, passive in their approach. Moreproactive approaches include antibiotic combinationtherapy and the use of MUTANT-PREVENTIVE CONCENTRATIONS

(MPCs) of antibiotics. The rationale for combinationtherapy is that if antibiotic action is more effective(faster killing, less survival and so on) this will reducethe probability of selecting resistance among survivingbacteria in a treated population109. This approach isparticularly relevant for the development of more effec-tive treatments of multidrug-resistant M. tuberculosis inwhich treatment is lengthy110. The MPC is the MINIMAL

Developing resistance inhibitorsβ-lactamase inhibitors. Traditionally, new antibiotics witha greater potency or broader spectrum have been derivedfrom antibiotics that are already successful. In the case ofthe β-lactam antibiotics, this has been necessary to com-bat the main resistance mechanism — β-lactamaseenzymes that destroy the β-lactam ring of the antibiotics.This approach is exemplified by the development ofβ-lactam antibiotics into second and third generationcephalosporins with better resistance to β-lactamases.An alternative approach is to use secondary inhibitors tocounteract the resistance mechanism80.

Inhibitors extend the effective spectrum of a β-lactamantibiotic by conferring potency against β-lactamaseproducing pathogens. The β-lactamase enzyme inhibitorclavulanic acid is used in combination with the β-lactamantibiotic amoxicillin (augmentin) and continues to beeffective after 20 years of clinical use. However, the con-tinued evolution of β-lactamases — there are over 250different types — necessitates the development of a sec-ond generation of β-lactamase inhibitor molecule. Thepresent need is for effective inhibitors to encompass classA and C β-lactamases and combat emerging resistance80.An argument against investing in the discovery anddevelopment of secondary agents, such as β-lactamaseinhibitors, is that their discovery is as resource intensiveas the discovery of new classes of antibiotic, whereastheir benefit is restricted to one class of antibiotic andresistance mechanism. However, given the clinical popu-larity of β-lactam antibiotics, and our extensive knowl-edge of their activity, structure and inhibition, thisinvestment is probably justified80.

Efflux inhibitors. The intrinsic resistance of Gram-negative bacteria to many antibiotics requires constitutively expressed pumps of the resistance-nodulation-division (RND) type, as well as the outermembrane barrier81. So, the inactivation of the MexAB-OprM pump of P. aeruginosa, or the AcrAB pump ofE. coli, makes these bacteria susceptible to many anti-biotics against which they are normally resistant. It hasbecome clear that the problem of multidrug resistancein bacteria is often caused by the overexpression ofMULTIDRUG EFFLUX systems82. Although increased effluxalone might not be sufficient to create a clinically rele-vant level of resistance, such effects can enhance survivaluntil further changes occur that result in high-levelresistance. This problem has led to an interest inunderstanding how efflux pumps work. One possibil-ity is to design drugs that are less efficiently pumpedout. An alternative is to find inhibitors of efflux pumps.The aims are, on the one hand, to restore antibioticsusceptibility to resistant mutants, and on the otherhand, to make intrinsically resistant species susceptibleto a wider range of antibiotics.

The screening of small-molecule compound librarieshas led to the identification of inhibitors of several multi-drug transport systems in S. aureus 83–85 and E. coli 86. Abroad-spectrum efflux-pump inhibitor, active against all three Mex pumps in P. aeruginosa and against theAcrAB-TolC pump in E. coli 87, is now under development.

MULTIDRUG EFFLUX

The process by which effluxpumps bind and efflux a varietyof structurally dissimilarcompounds from the cell.

BROAD-SPECTRUM ANTIBIOTICS

Antibiotics that kill or inhibit thegrowth of phylogeneticallydistant bacterial species, forexample, Gram-negative andGram-positive bacteria.

MUTANT-PREVENTIVE

CONCENTRATION

(MPC). The antibioticconcentration that prevents thegrowth of bacteria carryingsingle mutations to resistance.



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The failure to develop new classes of antibiotics inthe decades before the launch of linezolid, in 2001, wasnot the result of a lack of knowledge about potentialtargets, but rather a lack of interest in investing in thedevelopment of new antibiotics. In effect, by the middleof the 1960s, with ~20 different classes of antibioticavailable for clinical use, the market was saturated andprogrammes to discover new antibiotics were no longera priority. Development programmes continued, butthese concentrated on finding analogues of existingproven antibiotics.

Analogue development is driven by several overlap-ping and complementary desires. One is the desire toimprove or alter the activity spectrum of an antibioticsuch that its clinical usefulness is enhanced. A goodexample is the continuing development of new fluoro-quinolone antibiotics with improved activity againstGram-positive bacteria130. Other reasons for analoguedevelopment include the need to keep ahead of evolv-ing antibiotic-resistance problems, the perception thatit is less costly to develop analogues than to embark ona new discovery programme and, not least, the need tohave patent rights on an antibiotic that are current.Pharmaceutical company interest in discovering anddeveloping new antibiotics was reawakened in the 1990s.In part, there was a perception that there now existed amarket for new antibiotics (profit motive), and, in part, abelief that new technologies in genomics, molecularbiology and chemistry would lead to the rapid discoveryof new drug candidates (success motive).

Present disappointment at the low level of success in generating new antibiotics from target-based screen-ing131,132 is, at least in part, a reflection of inflated initialexpectations. However, genomics and molecular genet-ics techniques have delivered on their promise in thesense that we now have access to, or can generate, vali-dated target lists for any pathogen of interest (FIG. 2a). Asignificant limiting factor, however, is successfully con-verting lead compounds that inhibit a target in vitro intobiologically active compounds that have target-specificwhole-cell antimicrobial activity132 (FIG. 2b). This limita-tion might be intrinsic to the chemistry of the (mostlysmall and natural) compounds that are used in screen-ing programmes. Developing chemistry methods thatcan generate and manipulate large and new complexmolecules, or developing combinatorial biology meth-ods, would open up the range of compounds that areavailable for screening programmes and might lead togreater success in identifying new antimicrobials intarget-based screening. However, the immediate aim todevelop and launch a few new antibiotics is likely to beachieved, even if the number of successful screeningprogrammes is low. The hunt for efflux-pump inhibitorsis also being aided by genomics, and successful develop-ments in this area will provide useful complementarydrugs that extend the life and spectrum of importantantibiotics. The overall conclusion is that we have tech-nological solutions, at least in the medium term, to theantibiotic-resistance problem. In the longer term, limi-tations on the production and chemical manipulationof complex molecules with antibiotic activity will need

INHIBITORY CONCENTRATION (MIC) of the least susceptiblesingle-step mutant111. The concept is that by dosing witha sufficiently high amount of antibiotic it should bepossible to prevent the growth of any strains thatbecome resistant by a single-step resistance mutation,as can happen, for example, with fluoroquinoloneantibiotics112,113. Whether this concept can be appliedclinically with success remains to be tested.

Non-antibiotic strategies. Vaccines are a serious alter-native, and a complement, to the antibiotic therapy of bacterial pathogens. Successful CONJUGATE VACCINES

have been introduced in the past decade against H. influenzae SEROTYPE b (REF. 114), Neisseria meningitidis115,and S. pneumoniae116. A new conjugate vaccine against S. aureus, StaphVax, is showing promising results inclinical trials117. Bioinformatics programmes are nowavailable to help predict from the analysis of genomesequences which gene products are likely to be locatedon the surface of the bacterial cell118,119. This approachhas led to the identification of new vaccine candidatesagainst S. pneumoniae120, N. meningitidis121,122 and M. tuberculosis123. Furthermore, new vaccine candidatesthat have been identified from the meningococcal Bgenome sequencing project are now being evaluatedand several are in clinical trials124.

The increased problem of antibiotic resistance hasre-awakened interest in the use of BACTERIOPHAGE as anantibacterial therapy125,126 (see accompanying review byAllan Campbell on p471 in this issue). The rationale isthat bacteriophage that target and kill specific bacterialpathogens in vitro might also be able to achieve this in vivo. Typical problems encountered are narrow hostrange, the emergence of bacterial resistance and a fail-ure to achieve high phage concentrations at the site ofinfection. However, these problems can be overcome byselection and optimization of the phage used in ther-apy. An example of a recent successful experiment is areport showing that bacteriophage could rescue micewith a vancomycin-resistant Enterococcus faecium bac-teremia127. Bacteriophage therapy is a potentially usefulapproach in infection control, but it is in the early stagesof development, and must be tested and evaluated, likeantibiotic drugs, in rigorously controlled experiments.

ConclusionGenomics, combinatorial chemistry and target-basedscreening programmes have received much attention inrecent years as the methods of choice in the search fornew antibiotics. However, it is worth remembering thatthe goal is to have effective antibiotic therapies, inde-pendent of how this is achieved. Genomics is not theonly game in town and the traditional approach ofdeveloping new analogues of successful antibioticclasses continues128 (see accompanying review by LynnMiesel and colleagues on p442 in this issue). Amongthese is a semi-synthetic derivative of a natural ana-logue of vancomycin — oritavancin. Developed by EliLilly, oritavancin is bacteriocidal against glycopeptide-susceptible and -resistant Gram-positive bacteria, andis now reaching the end of PHASE III CLINICAL TRIALS129.

MINIMAL INHIBITORY

CONCENTRATION

(MIC). The minimal amount ofantibiotic that is required toprevent the growth of a bacterialstrain under defined conditions.

CONJUGATE VACCINE

A vaccine in which the bacterialpolysaccharide antigen iscoupled to a protein carrier thatcan be processed and presentedto T cells bearing specificreceptors for the proteincomplex.

SEROTYPE

A bacterial strain that isclassified on the basis of itssurface antigens.

BACTERIOPHAGE

A virus that infects, grows onand can kill specific strains ofbacteria.

PHASE III CLINICAL TRIALS

The assessment of efficacy andside-effects; generally involveshundreds of patients enrolled atdifferent clinics nationwide orworldwide.



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policies of increased regulation and restriction, andthat might be a disincentive to new investments inantibiotic development. Also, going beyond normalregulation, government threats to break patents — ashappened with Bayer and their drug ciprofloxacin in2001 (REF. 133) — do not create an environment that isconducive to large investments in new antibiotic devel-opment. Finally, as the potential dangers to humanmedicine of antibiotic usage in agriculture continue tobe debated134, it is clear that restricting their use in agri-culture also impacts negatively on the total size of themarket for antibiotics135. The solutions to these com-peting interests lie in the arena of politics rather thanscience and technology.

to be solved if we are to fully realize the potential ofthe validated target lists resulting from genomics andmolecular genetics.

An issue not addressed in this review is whether theexpense of new antibiotic development, coupled withchanging rules on clinical trials, political decisions onpatenting and medical pressure to restrict antibioticusage, will cause abandonment or delay in the comple-tion of these projects (BOX 4). The public clearly wantseffective antibiotics for use in medicine, and the phar-maceutical companies have the technology to developthem, but will only do so if they believe it is a profitableenterprise. However, the need to maintain the long-termeffectiveness of antimicrobial therapies is promoting

Box 4 | Reasons not to invest

It has been reported that several large pharmaceutical companies, including Roche, Lilly and Bristol-Myers Squibb, havewithdrawn from research and development into antimicrobials, and others, such as Abbott and Glaxo-SmithKline, havescaled back their efforts132,147. Given the present concern over the lack of new antibiotics, this apparent move away fromthe field is both surprising and worrying.Among the reasons that have been indicated are disappointment with thesuccess rate of target-based screening strategies in finding new compounds with antimicrobial activity132. However, asoutlined in this review, the genomic target-based approach has produced interesting leads, and as a technology still in itsinfancy it will undoubtedly continue to do so131. Perhaps of greater concern are proposed changes in the Food and DrugAdministration (FDA) rules that govern clinical tests, which are reported to have led to the abandonment of severalprojects147–149. Regulatory changes to increase the stringency of clinical tests (to show the ‘non-inferiority’of a test drugrelative to an existing drug) could require that many more patients be tested in clinical trials. These changes, if applied,will greatly increase the cost and time it takes to develop a drug, and increase the probability that a test drug will be refusedapproval late in development148. These factors can translate into an increased risk and a reduced profit for thepharmaceutical companies. Because antibiotic development generally takes about 10–12 years (out of a total patentperiod of 20 years), uncertainty surrounding the rules that will apply might be as great a discouragement to starting newprojects as the changes themselves.

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AcknowledgementsThe author is supported by grants from the Swedish ResearchCouncil (Medicine and Natural Sciences), the European Union andLeo Pharma. I thank the anonymous reviewers for their positive andhelpful suggestions to improve the manuscript. This paper is dedi-cated to the late Pat Hughes.

Online links

DATABASESThe following terms in this article are linked online to:Entrez: http://www.ncbi.nlm.nih.gov/entrez/query.fcgiPBP2ASaccharomyces Genome Database:http://genome-www.stanford.edu/SaccharomycesUre2

FURTHER INFORMATION The Institute for Genomic Research: http://www.tigr.org/tdbAccess to this interactive links box is free online.

Microbial genetics: Exploiting genomics, genetics and chemistry to combat antibiotic resistance

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