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  • 7/24/2019 2007_Molecular Mechanisms of Antibacterial Multidrug Resistance

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    Leading Edge

    Review

    Cell 128, March 23, 2007 2007 Elsevier Inc. 1037

    Efforts aimed at identifying new antibiotics were once a

    top research and development priority among pharma-

    ceutical companies. The potent broad spectrum drugs

    that emerged from these endeavors provided extraordi-

    nary clinical efficacy. Success, however, has been com-

    promised. We are now faced with a long list of microbes

    that have found ways to circumvent different structural

    classes of drugs and are no longer susceptible to most,

    if not all, therapeutic regimens.

    The means that microbes use to evade antibiot-

    ics certainly predate and outnumber the therapeutic

    interventions themselves. In a recent collection of soil-

    dwelling Streptomyces (the producers of many clini-

    cal therapeutic agents), every organism was multidrug

    resistant. Most were resistant to at least seven different

    antibiotics, and the phenotype of some included resist-

    ance to 1521 different drugs (DCosta et al., 2006).

    Moreover, many isolates were resistant to daptomycin,

    quinupristin-dalfopristin, and telithromycinall drugs

    approved by the United States Food and Drug Admin-

    istration (FDA) within the last decadeas well as purely

    synthetic agents such as ciprofloxacin. These data not

    only suggest that our surroundings can act as a res-

    ervoir for new (and old) resistance mechanisms, but

    that the drugs we use to treat infectious diseases have

    long-lasting effects outside of the hospital. Many anti-

    microbial molecules exist for millennia stably within the

    environment (Cook et al., 1989), where they select and

    promote growth of resistant strains.

    Resistance to single antibiotics became prominent in

    organisms that encountered the first commercially pro-

    duced antibiotics. The most notable example is resist-

    ance to penicillin among staphylococci, specified by an

    enzyme (penicillinase) that degraded the

    antibiotic (Barber, 1947). Over the years,

    continued selective pressure by different

    drugs has resulted in organisms bearing

    additional kinds of resistance mechanisms

    that led to multidrug resistance (MDR)

    novel penicillin-binding proteins (PBPs),

    enzymatic mechanisms of drug modifica-

    tion, mutated drug targets, enhanced efflux

    pump expression, and altered membrane permeability.

    Some of the most problematic MDR organisms that are

    encountered currently include Pseudomonas aeruginosa

    (another microbe of soil origin),Acinetobacter bauman-

    nii, Escherichia coliand Klebsiella pneumoniaebearing

    extended-spectrum -lactamases (ESBL), vancomycin-

    resistant enterococci (VRE), methicillin-resistant Staphy-

    lococcus aureus(MRSA), vancomycin-resistant MRSA,

    and extensively drug-resistant (XDR) Mycobacterium

    tuberculosis (Table 1). Some like methicillin-resistant S.

    aureuscouple MDR with exceptional virulence capabili-

    ties (Miller et al., 2005). Others, including some strains of

    P. aeruginosa,A. baumannii, and K. pneumoniae, man-

    age to evade every drug within the physicians arsenal

    (Levin et al., 1999).

    Molecular Mechanisms of Antibacterial

    Multidrug ResistanceMichael N. Alekshun1,* and Stuart B. Levy2,*1Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA2Center for Adaptation Genetics and Drug Resistance, Department of Molecular Biology & Microbiology and Department of

    Medicine, Tufts University School of Medicine, Boston, MA 02111, USA

    *Correspondence: [email protected] (M.N.A), [email protected] (S.B.L.)

    DOI 10.1016/j.cell.2007.03.004

    Treatment of infections is compromised worldwide by the emergence of bacteria that are

    resistant to multiple antibiotics. Although classically attributed to chromosomal muta-

    tions, resistance is most commonly associated with extrachromosomal elements acquired

    from other bacteria in the environment. These include different types of mobile DNA

    segments, such as plasmids, transposons, and integrons. However, intrinsic mechanisms

    not commonly specified by mobile elementssuch as efflux pumps that expel multiple

    kinds of antibioticsare now recognized as major contributors to multidrug resistance in

    bacteria. Once established, multidrug-resistant organisms persist and spread worldwide,

    causing clinical failures in the treatment of infections and public health crises.

    There is probably no chemotherapeutic drug to which

    in suitable circumstances the bacteria cannot react

    by in some way acquiring fastness [resistance].

    Alexander Fleming, 1946

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    1038 Cell 128, March 23, 2007 2007 Elsevier Inc.

    Rather than providing an extensive list of antibiotic

    resistance mechanisms (which can be found elsewhere

    [Alekshun and Levy, 2000]), the aim of this Review is to

    highlight common genetic and molecular mechanisms

    of resistance as they relate to important pathogens and

    to drugs used by physicians today.

    Genetics of Multidrug Resistance

    Bacterial antibiotic resistance can be attained through

    intrinsic or acquired mechanisms (Figure 1). Intrinsic

    mechanisms are those specified by naturally occurringgenes found on the hosts chromosome, such as, AmpC

    -lactamase of gram-negative bacteria and many MDR

    efflux systems (see below). Acquired

    mechanisms involve mutations in

    genes targeted by the antibiotic and

    the transfer of resistance determinantsborne on plasmids, bacteriophages,

    transposons, and other mobile genetic

    material (Figure 1 and Table 2). In general, this exchange

    is accomplished through the processes of transduc-

    tion (via bacteriophages), conjugation (via plasmids and

    conjugative transposons), and transformation (via incor-

    poration into the chromosome of chromosomal DNA,

    plasmids, and other DNAs from dying organisms) (Levy

    and Marshall, 2004). Although gene transfer among

    organisms within the same genus is common, this proc-

    ess has also been observed between very different

    genera, including transfer between such evolutionarily

    distant organisms as gram-positive and gram-negativebacteria (Courvalin, 1994). Plasmids contain genes for

    resistance and many other traits; they replicate inde-

    Table 1. General Characteristics of Multidrug-Resistant Organisms

    Organism Common Infections Key Antibiotic Resistances

    Drugs Considered for

    Treatment of MDRa

    P. aeruginosa Lung, wound -lactams, fluoroquinolones, aminoglycosides Colistin

    Acinetobacterspp. Lung, wound,

    bone, blood

    -lactams, fluoroquinolones, aminoglycosides Colistin, tigecycline

    E. coliand K.

    pneumoniaebear-

    ing extended-spec-trum -lactamases

    Urinary, biliary,

    gastrointestinal

    tracts, lung, blood

    -lactams, fluoroquinolones, aminoglycosides Colistin (for K. pneumoniae),

    tigecycline

    Vancomycin-resist-

    ant enterococci

    Blood, heart,

    intra-abdominal

    Vancomycin Quinupristin-dalfopristin,

    linezolid, daptomycin

    Methicillin-resistant

    S. aureus

    Skin and soft tis-

    sue, respiratory

    tract, blood

    -lactams, fluoroquinolones, macrolides Quinupristin-dalfopristin,

    daptomycin, linezolid,

    tigecycline, vancomycin

    Multidrug-resistant

    S. pneumoniae

    Ear, lung, blood,

    cerebrospinal fluid

    -lactams, macrolides, tetracyclines, co-trimoxazole Fluoroquinolones, tigecycline

    Extensively

    drug-resistant M.

    tuberculosis

    Lung Rifampin, isoniazid, and three of the following:

    aminoglycosides, polypeptides, fluoroquinolones,

    thioamides, cycloserine, or para-aminosalicylic acid

    3rdline agents, drug

    combinations

    aAgents either have been approved for use by a regulatory agency (e.g., FDA), have shown usefulness in treating infection, or

    exhibit promising in vitro activity and await a determination of clinical efficacy.

    Figure 1. Acquisition of Antibiotic ResistanceBacteria can become antibiotic resistant (Abr)

    by mutation of the target gene in the chromo-

    some. They can acquire foreign genetic material

    by incorporating free DNA segments into their

    chromosome (transformation). Genes are alsotransferred following infection by bacteriophage

    (transduction) and through plasmids and con-

    jugative transposons during conjugation. The

    general term transposable element has been

    used to designate (1) an insertion sequence, (2)

    composite (compound), complex, and conjuga-

    tive transposon, (3) transposing bacteriophage,

    or (4) integron.

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    Cell 128, March 23, 2007 2007 Elsevier Inc. 1039

    pendently of the host chromosome and can be distin-guished by their origins of replication. Multiple plasmids

    can exist within a single bacterium, where their genes

    add to the total genetics of the organism. Transposons

    are mobile genetic elements that can exist on plasmids

    or integrate into other transposons or the hosts chro-

    mosome. In general, these pieces of DNA contain termi-

    nal regions that participate in recombination and specify

    a protein(s) (e.g., transposase or recombinase) that

    facilitates incorporation into and from specific genomic

    regions. Conjugative transposons are unique in having

    qualities of plasmids and can facilitate the transfer of

    endogenous plasmids from one organism to another.

    Integrons contain collections of genes (gene cassettes)

    that are generally classified according to the sequence

    of the protein (integrase) that imparts the recombination

    function (Mazel, 2006) (Figure 2A). They have the abil-

    ity to integrate stably into regions of other DNAs where

    they deliver, in a single exchange, multiple new genes,

    particularly for drug resistance. The super-integron, one

    which contains hundreds of gene cassettes ( represent-

    ing about ?3% of the hosts genome), is distinct from

    other integrons; it was first identified in Vibrio cholerae

    (Mazel et al., 1998).

    Although Alexander Fleming selected and described

    mutants resistant to penicillin soon after he had dis-

    covered the antibiotic, no one could have predicted the

    speed with which bacteria would acquire the capacity

    for dealing with multiple antibacterial agents. Plasmids,specifying a collection of individual antibiotic resistance

    determinants (originally termed resistance factors, R

    factors), were initially described as the vehicle used to

    rapidly spread resistance traits among bacteria. Strains

    of Shigellabearing the self-replicating and self-transfer-

    able plasmids were easily selected and propagated dur-

    ing a period of considerable sulfonamide use in Japan

    after World War II (Watanabe, 1963). Antibiotics such

    as streptomycin, chloramphenicol, and tetracycline

    were subsequently introduced for the treatment of the

    sulphonamide-resistant organisms. Shigellaand E. coli

    strains bearing resistance to all four agents, however,

    were recognized in 1955 (Watanabe, 1963). Now, more

    than 60 years after antibiotics were first introduced into

    clinical practice, the prescience of Fleming has come to

    fruition as the infectious disease community has yet to

    identify an antibiotic that has managed to circumvent the

    development of resistance.

    Resistance through Chromosomal Mutation

    Fluoroquinolones

    Virtually all clinically important fluoroquinolone resistance

    can be attributed to mutations within the drugs targets,

    DNA gyrase and topoisomerase IV (Drlica and Malik,

    2003). These multisubunit complexes perform critical

    ATP-dependent functions during DNA replication; each

    is comprised of two subunits: GyrA and GyrB for DNA

    Table 2. Mobile Genetic Elements

    Genetic Element General Characteristics Resistance Determinant(s) Specified/Examplesa

    Plasmid Variable size (1- >100 kb), conjugative, and

    mobilizable

    R factor: multiple resistances

    Insertion sequence Small (5 kb), flanked by short terminal

    inverted repeats, and specifies a

    transposase and recombinase

    Tn1 and Tn3: -lactamase

    Tn7: Tmp, Str, Spc

    Tn1546: glycopeptides

    Conjugative transposon Promotes self-transfer Tn916: Tet and Mino

    Tn1545: Tet, Mino, Ery, and Kan

    Transposable bacteriophage A bacterial virus that can insert into the

    chromosome

    Mu

    Other transposable elements Other than composite, complex, andconjugative transposons

    Tn4: Amp, Str, Sul, and HgTn1691: Gen, Str, Sul, Cm, and Hg

    Integron Facilitates acquisition and dissemination

    of gene cassettes; specifies an integrase,

    attachments sites, and transcriptional

    elements to drive expression of multiple

    resistance genes

    Class 1: Multiple single determinants and MDR

    efflux pump (Qac)b

    Class 2: Tmp, Strp, Str, and Spc (Tn7)

    Class 3: carbapenems

    Class 4: Vibriospp. super-integron

    See Figure 1.aAbbreviations: Amp, ampicillin; Bleo, bleomycin; Cm, chloramphenicol; Ery, erythromycin; Fus, fusidic acid; Gen, gentamicin;

    Hg, mercury; Kan, kanamycin; Mino, minocycline; Spc, spectinomycin; Str, streptomycin; Strp, streptothricin; Sul, sulfonamide;

    Tet, tetracycline; Tmp, trimethoprim; Van, vancomycin.bSee Figure 2.

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    1040 Cell 128, March 23, 2007 2007 Elsevier Inc.

    gyrase and ParC/GrlA and ParE/GrlB for topoisomerase

    IV. The GyrA and ParC/GrlA proteins contain the DNA-

    binding functions and are targeted by the fluoroquinolo-

    nes (which are purely synthetic antibiotics), whereas

    GyrB and ParE/GrlB perform the roles of ATP binding and

    hydrolysis and are inhibited by coumarin antibiotics. In

    gram-negative bacteria, mutations in DNA gyrase occur

    first, whereas in gram-positive organisms mutations in

    topoisomerase IV arise initially in the stepwise movement

    to clinically relevant fluoroquinolone resistance.

    Mutations that give rise to fluoroquinolone resistanceare predominantly found in the quinolone-resistance-

    determining-region (QRDR) of GyrA and ParC/GrlA.

    Mutations occur more frequently at particular amino

    acids than at others. Multiple mutational events can be

    selected in a stepwise manner so as to train resist-

    ance in a bacterium; successive mutations have additive

    effects. Isolates that are highly fluoroquinolone resist-

    ant bear multiple QRDR mutations and generally other

    mechanisms of fluoroquinolone resistance, such as

    drug efflux (see below).

    Rifamycins

    Rifamycins are bactericidal drugs that arrest transcrip-

    tion by interacting with RpoB, the subunit of RNA

    polymerase (RNAP). Although combination regimens

    that include rifampin or rifapentine and isoniazid, pyrazi-

    namide, ethambutol, or streptomycin remain primary

    choices for front-line therapy of M. tuberculosisinfection,

    resistance through point mutations in the rifampin-bind-

    ing region ofrpoBoccur at a frequency of 1 108and

    are widespread. M. tuberculosisresistant to rifampin and

    isoniazid at a minimum are defined as multidrug resist-

    ant and impede successful therapy among patients in all

    regions of the world (Sharma and Mohan, 2006).

    Sulfonamide and Trimethoprim Antibiotics

    The sulfonamides, the first antimicrobials developed forlarge-scale introduction into clinical practice (in 1935),

    target dihydropteroate synthase. Their serendipitous

    discovery (the antibacterial activity was seen initially in

    vivo when the active compound was released as part of

    a dye) pales only in comparison with that of Flemings

    chance discovery of penicillin (Levy, 2002). Trimetho-

    prim, introduced in 1968, inhibits dihydrofolate reductase

    and was the last structurally unique antibiotic approved

    prior to the release of linezolid in 2000. Mutations in the

    gene specifying dihydropteroate synthase decrease the

    enzymes affinity for the sulfonamides and have been

    found in laboratory experiments using E. coliand Strep-

    tococcus pneumoniaeand in clinical isolates of Campy-

    lobacter jejuni and Haemophilus influenzae (Skold,

    Figure 2. Transposable Elements that Confer Antibiotic Resistance(A) Organization of a class 1 integron.int1specifies the integrase (the recombination protein),att1is the primary recombination site, attCare the

    59 base pair elements that are substrates of the integrase, and Pc is a common promoter that drives high-level expression of antibiotic resistance

    determinants (Abr) such as those for aminoglycosides, -lactams, chloramphenicol, and trimethoprim inserted downstream of this element. qacE

    andsul1specify genes for resistance to quaternary ammonium compounds (partially deleted) and sulfonamides, respectively; these two genes are

    found specifically in class 1 integrons.

    (B) The vanAgene cluster (adapted from Courvalin, 2006). Orf1 and Orf2 impart the transposition features. VanR (response regulator) and VanS

    (histidine kinase) comprise a two-component system that regulates expression of vanHAXYZ. VanA (ligase) and VanH (dehydrogenase) are respon-

    sible for the synthesis of the modified depsipeptide (D-Ala-D-Lac, see text) while VanX (D,D-dipeptidase) and VanY (D,D-carboxypeptidase) cleave

    normal peptidoglycan substrates. VanZ is of unknown function. PRand PHare promoters that control expression of two separate transcriptionalunits, and the entire element is flanked by imperfect inverted repeats.

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    Cell 128, March 23, 2007 2007 Elsevier Inc. 1041

    2001). Mutations in the chromosomal gene specifying

    dihydrofolate reductase can result in overexpression of

    an enzyme with a reduced affinity for trimethoprim and

    thereby offer very high-level trimethoprim resistance in

    E. coliand H. influenzae(Skold, 2001).

    Tetracycline, Aminoglycoside, and MLS Antibiotics

    Agents within the tetracycline, aminoglycoside and mac-

    rolide-lincosamide-streptogramin (MLS) classes of anti-

    biotics target the ribosome to inhibit protein translation.

    As a consequence, resistance via chromosomal muta-

    tion is uncommon. Tetracyclines and aminoglycosides

    interact with 16S rRNA (rrs) and the macrolide-lincosa-

    mide-streptogramin (MLS) family binds to 23S rRNA

    (rrl). In most bacteria, multiple rrs and rrloperons are

    present and susceptibility mediated by any one of these

    targets can be dominant, making resistance difficult to

    attain without a mutation in all or a majority of the other

    operons. However, in organisms with low rRNA (rrn) copynumbers, chromosomal mutations conferring resistance

    have appeared. Tetracycline resistance via a point muta-

    tion in Propionibacterium acnes (3 rRNA operons) and

    Helicobacter pylori(2 copies ofrrn) (Gerrits et a l., 2002;

    Ross et al., 1998) has been documented. Mutations in

    rrs conferring resistance to amikacin and kanamycin

    and alterations in small ribosome protein S12 (rpsL) or

    rrs affecting streptomycin (all aminoglycoside drugs)

    susceptibility in clinical M. tuberculosis (1 rrn operon)

    have been described (Alangaden et al., 1998). The emer-

    gence of resistance to erythromycin (a macrolide) dur-

    ing therapy caused by mutant rrl in S. pneumoniae (4

    copies of rrn) has been reported (Musher et al., 2002).Moreover, mutations in the large ribosome protein L4

    (rplD) have also been shown to alter MLS susceptibil-

    ity (Tait-Kamradt et al., 2000). The ketolides, recently

    introduced into clinical practice, were designed to cir-

    cumvent macrolide resistance; nonetheless decreased

    susceptibility and resistance to telithromycin (a ketolide)

    have been found in S. pneumoniaewith mutations inrrl,

    rplD, and large ribosome protein L22 (rplV) (Hisanaga

    et al., 2005). Mutations in L22 also affect quinupris-

    tin-dalfopristin (streptogramins that act individually in

    a bacteriostatic manner) susceptibility by affecting the

    synergistic relationship important to the combinations

    bactericidal mechanism of action (references in Hersh-berger et al., 2004).

    Oxazolidinones

    Linezolid (another protein synthesis inhibitor) was

    approved recently as an agent to treat methicillin-resist-

    ant S. aureus and vancomycin-resistant enterococci

    infections. Its availability in both intravenous and oral

    formulations makes it useful for treating infections in

    both the hospital and community. Resistance to linezolid

    in laboratory studies has been linked to point mutations

    inrrlin S. aureusand E. faecalis. Now, clinical isolates of

    Staphylococcus epidermidis, S. aureus, Streptococcus

    oralis,Enterococcus faecium, and E. faecalisresistant to

    linezolid have been documented and many of the strains

    bear rrlmutations (Meka and Gold, 2004). As with the

    fluoroquinolones, the level of resistance in S. aureus

    increases with mutations in multiplerrlalleles (Wilson et

    al., 2003) leading to clinically relevant resistance.

    Lipopeptides

    Daptomycin (a drug acting on bacterial membranes) was

    approved by the FDA in 2003 and while it has been suc-

    cessfully used for treating infections caused by methicil-

    lin-resistant S. aureusand other gram-positive bacteria,

    treatment failures have been noted (Hayden et al., 2005).

    Laboratory experiments have established that mutations

    in multiple chromosomal loci (i.e., mprF, yycG, rpoB,

    andrpoC) affect daptomycin susceptibility (Friedman et

    al., 2006). With regard to mprF, which specifies a lysyl-

    phosphatidylglycerol synthetase, similar mutations have

    been identified in nonsusceptible clinical isolates as well

    (Friedman et al., 2006).

    Genomic DuplicationsA mechanism for drug resistance common in eukaryotic

    cells (e.g., certain mammalian cancers and parasites)

    is gene amplification, which leads to overexpression

    of multidrug transporters and drug targets (Albertson,

    2006). Recently, a large tandem duplication of the E.

    coli acrABlocus in a mutant isolated in the presence of

    tetracycline was found to overexpress the AcrAB drug

    efflux pump, producing an MDR phenotype (Nicoloff

    et al., 2006). The mutants, however, were unstable and

    reverted to the wild-type phenotype in the absence of

    drug (Nicoloff et al., 2007). Genomic amplification has

    also been shown to affect susceptibility to methicillin in

    S. aureus(Matthews and Stewart, 1988). It is expectedthat examples of gene duplication as a mechanism of

    resistance, not mutation, will increase in recognition

    among bacterial isolates. The likely phenotype will be an

    unstable form of resistance.

    Acquired Resistance: Enzymatic Drug Modification

    Enzymes that modify antibacterial drugs are divided into

    two general classes: those such as -lactamases that

    degrade antibiotics and others (including the macrolide

    and aminoglycoside-modifying proteins) that perform

    chemical transformations.

    -Lactam Antibiotics

    There are hundreds of -lactamases that have been dis-

    covered and characterized, and a more complete over-

    view of the individual enzymes within this protein family

    is available elsewhere (Jacoby and Munoz-Price, 2005).

    Most of these resistances are specified by genes located

    on plasmids and transposons; others are chromosomal

    and provide intrinsic resistance.

    -lactamases are classified using schemes based

    on function (the system of Bush-Jacoby-Medeiros)

    or structure (Ambler classification) but in general are

    broadly separated into enzymes with a serine active site

    and those that require a metal ion cofactor (references

    in Jacoby and Munoz-Price, 2005). The classification

    scheme of Ambler divides the -lactamases into four

    groups: class A, C, and D enzymes are proteins with a

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    serine residue at their active sites and the class B pro-

    teins are zinc-dependent metalloenzymes. Some class

    A proteins function as extended-spectrum -lactamases

    and as carbapenemases. The class B metallo-enzymes

    that hydrolyze carbapenems are susceptible to inhibi-

    tion by EDTA, whereas they are not susceptible to inhi-

    bition by clavulanate (a -lactamase inhibitor). AmpC,

    an inducible and usually chromosomal enzyme found in

    many species of the Enterobacteriaceaeand P. aerugi-

    nosa, is the prototype class C enzyme. Recent reports

    have described plasmid-borne ampC genes that can

    be transferred among E. coli, Klebsiella spp., and Sal-

    monella spp. (references in (Jacoby and Munoz-Price,

    2005). Class D enzymes have been found in only a few

    species such as P. aeruginosa,Acinetobacterspp., and

    Aeromonasspp.

    Aminoglycosides

    A large number of aminoglycoside-modif ying enzymesspecified by genes on transferable elements are wide-

    spread in clinically relevant organisms (Davies and

    Wright, 1997). Here, resistance is accomplished with

    proteins that N-acetylate (acetyltransferases), phos-

    phorylate (phosphotransferases), and adenylate (nucle-

    otidyltransferases) the aminoglycosides. The acetyl-

    transferases are capable of modifying tobramycin,

    gentamicin, netilmicin, and amikacin; the nucleotidyl-

    transferase proteins alter the activity of tobramycin; and

    the phosphotransferases affect amikacin susceptibility.

    Many of the aminoglycoside-modifying enzymes are

    found on integrons and other mobile genetic elements

    where they associate with additional resistance determi-nants. For example, three acetyltransferase genes were

    found on a class 1 integron from P. aeruginosathat also

    specified resistance to carbapenems and sulfonamides

    (Poirel et al., 2001).

    MLS Antibiotics

    There are a number of inactivating enzymes that act on

    the MLS antibiotics. The genes encode esterases, hydro-

    lases, glycosylases, phosphotransferases, nucleotidyl-

    transferases, and acetyltransferases and are found less

    frequently than efflux and ribosome-modifying genes in

    clinical isolates (Weisblum, 1998). Esterases act on 14-

    (e.g., erythromycin) and 15- (e.g., azithromycin) mem-

    bered macrolides; the hydrolases affect streptogramin

    B drugs; the acetyltransferases inactivate streptogramin

    A antibiotics; and the nucleotidyltransferases confer

    resistance to the lincosamides (e.g., clindamycin). Phos-

    photransferases modify 14-, 15-, and 16-membered

    macrolides with varying specificities and telithromycin

    (Matsuoka and Sasaki, 2004).

    Chloramphenicol

    Acetyltransferases that inactivate chloramphenicol (a

    generally bacteriostatic inhibitor of protein synthesis)

    are the most common resistance mechanisms for this

    antibiotic and are separated into type A and B proteins

    (Schwarz et al., 2004). Both types of enzymes func-

    tion as homotrimers but are unrelated based on amino

    acid sequence analyses. The type B enzymes are also

    termed xenobiotic acetyltransferases and appear to

    share an evolutionary lineage that includes some strep-

    togramin-inactivating enzymes found in the enterococci

    and staphylococci. Constitutive expression as well as

    translational attenuation underlie the regulation of type

    A and B proteins.

    Tetracyclines

    Recently, a flavin-dependent monooxygenase, desig-

    nated tet(X), originally identified in Bacteroides fragilis,

    that acts on older tetracyclines (such as tetracycline,

    oxytetracycline, and chlortetracycline) as well as newer

    compounds (such as doxycycline, minocycline, and

    tigecycline) has been characterized (Moore et al., 2005).

    The enzyme catalyzes regioselective hydroxylation to

    inactivate its substrates, but the modification products

    produced by TetX are not stable at physiological pH.

    The tet(X) determinant has not yet been found in com-

    mon, clinically relevant tetracycline-resistant isolates,although a related tet(X)-like gene in P. aeruginosahas

    been reported (A. Nakamura et al., 2002, paper pre-

    sented at Interscience Conference on Antimicrobial

    Agents and Chemotherapy, San Diego, CA).

    Altered, Substituted, and Protected Drug Targets

    -Lactam Antibiotics

    The first penicillin-resistant S. aureus identified in the

    mid-1940s expressed a -lactamase (termed PCI) that

    afforded resistance. A penicillin derivative, methicillin,

    that was resistant to this -lactamase was subsequently

    introduced in 1959 to treat the penicillin-resistant iso-

    lates, but methicillin-resistant S. aureuswere identifiedshortly thereafter (Barber, 1961). Here, the -lactam

    resistance was linked to the acquisition of a gene for

    an altered PBP. Resistance in the staphylococci (Fuda

    et al., 2005) and streptococci (Jacobs, 1999) frequently

    occurs following the acquisition of genes for PBPs that

    are not sensitive to -lactam inhibition. The altered PBP

    of methicillin-resistant S. aureus, PBP2a, is specified

    by mecA and is transported on a mobile genetic ele-

    ment called the staphylococcal cassette chromosome

    (SCCmec) (Fuda et al., 2005). In addition to mecA,

    SCCmec contains the mecR1-mecIregulatory loci and

    encodes the enzymes that are involved in site-specific

    recombination.

    Under normal circumstances, S. aureus uses multi-

    ple PBPs during cell wall biosynthesis. One, PBP2, is a

    bifunctional enzyme with transpeptidase and transgly-

    cosylase activities. When methicillin-resistant S. aureus

    is exposed to methicillin, PBP2 functions as the transg-

    lycosylase whereas PBP2a contributes the transpepti-

    dase activity in order to confer resistance to nearly all

    -lactam antibiotics. Removal of the transglycosylase

    function of PBP2 imparts -lactam susceptibility and

    demonstrates the importance of both attributes of the

    enzyme (reviewed in Fuda et al., 2005).

    The new strains of methicillin-resistant S. aureusthat

    have emerged recently outside of hospitals in the com-

    munity share some features with hospital-associated

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    strains (Naimi et al., 2003). These include the nearly

    complete -lactam resistance phenotype and the abil-

    ity to cause a range of serious life-threatening infec-

    tions. Although many community-associated strains are

    multidrug susceptible (except to -lactam antibiotics),

    the genome of an epidemic clone distributed through-

    out the US (USA300) contains chromosomal mutations

    that confer fluoroquinolone resistance and plasmids that

    mediate resistance to tetracycline, erythromycin, clin-

    damycin, streptogramin B agents, and mupirocin (Diep

    et al., 2006).

    Glycopeptides

    Glycopeptides (including vancomycin and teicoplanin)

    interact with peptidoglycan precursors to exert bacteri-

    cidal activity. Resistance to the glycopeptides in gram-

    positive cocci is another illustration of an altered drug

    target (Courvalin, 2006). In the enterococci, acquired

    glycopeptide resistance is attributable to VanA, B, D,E, and G phenotypes whereas VanC affords intrinsic

    resistance. VanA and VanD provide resistance to both

    vancomycin and teicoplanin, whereas the others provide

    resistance to vancomycin alone. The resistance phe-

    notype is accomplished using multiple proteins speci-

    fied in gene clusters and each result in the production

    of a modified peptidoglycan. Of the many drug-resist-

    ance determinants currently known, the determinant for

    glycopeptide resistance is probably the most complex

    (Figure 2B).

    The activity of many enzymes specified by the gene

    cluster are involved in conferring glycopeptide resist-

    ance (Figure 2B). Either a racemase or dehydrogenaseresults in the production of serine (VanC, E, or G) or lac-

    tate from pyruvate (VanA, B, or D), which a ligase uses

    to form a C-terminal D-Ala-D-Ser or D-Ala-D-Lac in the

    altered peptidoglycan. A two-component regulatory

    system controls expression of the biosynthetic machin-

    ery. Although the glycopeptides have a lower affinity

    for the D-Ala-D-Ser or D-Ala-D-Lac substrates, they

    can still bind and inhibit peptidoglycan biosynthesis if

    an intact D-Ala target remains. Two additional enzymes

    (or a single bi-functional protein for VanC) complete the

    phenotype by removing the antibiotics normal target:

    a dipeptidase cleaves the C-terminal D-Ala-D-Ala and

    a carboxypeptidase provides a redundant function of

    removing the terminal D-Ala in the absence of, or under

    circumstances when, the dipeptidase is less active.

    Recently, the enterococcal vanA gene cluster has

    made its way into methicillin-resistant S. aureus, impart-

    ing full-blown vancomycin resistance on this prominent

    pathogen (Weigel et al., 2003), although this molecular

    event had been demonstrated in laboratory experiments

    years before (Noble et al., 1992). Vancomycin-resistant

    S. aureushave now been identified in patients from three

    different US locales, Michigan, Pennsylvania, and New

    York. In all instances, the resistance determinant resides

    on a plasmid-specified transposon, Tn1546. Vanco-

    mycin-resistant E. faecalisand methicillin-resistant S.

    aureus were also obtained from the Michigan patient

    that bore vancomycin-resistant S. aureusand each con-

    tained plasmids that were identical, except for the pres-

    ence of Tn1546in the isolate of vancomycin-resistant E.

    faecalis. It is assumed that the plasmid from E. faecalis

    was the vehicle for vanA entry into S. aureus and that

    the vanAgene cluster was subsequently transferred by

    means of transposition into the S. aureusplasmid.

    Tetracyclines and MLS Antibiotics

    The most prevalent forms of resistance to the tetracy-

    clines in the clinic are drug efflux (see below) and ribos-

    ome protection. Determinants of ribosome protection

    (Connell et al., 2003) have sequence similarity to bacte-

    rial elongation factors (EF-G an EF-Tu). They also pos-

    sess GTPase activity and function to facilitate release

    of tetracycline from the ribosome in an energy-depend-

    ent manner (Connell et al., 2003). Bacteria that exhibit

    resistance to minocycline generally contain a ribosome

    protection determinant.In Megasphaera elsdenii (a rumen bacterium), a

    mosaic gene containing two unique ribosome protec-

    tion determinants (TetO and TetW) has been described

    (Stanton and Humphrey, 2003). Moreover, the tet(P)

    determinant of Clostridium perfringens specifies both

    ribosome protection and efflux mechanisms in an over-

    lapping genetic unit (Sloan et al., 1994).

    Binding of drugs within the MLSK (macrolide-lin-

    cosamide-streptogramin-ketolide) family to 23S rRNA

    is affected by erm(erythromycin resistance methylase

    or erythromycin ribosome methylation) specified gene

    products (Zhanel et al., 2001). These mechanisms repre-

    sent the predominant macrolide resistance mechanismsin Europe and South Africa. Currently, there are 34 dif-

    ferent classes of Erm proteins (http://faculty.washington.

    edu/marilynr/), and each functions by methylating a

    single adenine residue of the E. coli23S rRNA (at posi-

    tion A2058). Methylation results in the MLSBphenotype,

    which encompasses resistance to 14-, 15-, and 16-mem-

    bered macrolides, lincosamides, and streptogramin B

    drugs. Some of the resistance determinants are induced

    following exposure to MLS drugs, whereas others are

    constitutively synthesized. In the staphylococci, agents

    like erythromycin and azithromycin induce ermexpres-

    sion, but 16-membered macrolides do not (Zhanel et

    al., 2001). Constitutive ermexpression in some clinical

    and laboratory strains confers telithromycin resistance

    (Hisanaga et al., 2005).

    Sulfonamide and Trimethoprim Antibiotics

    The activities of the sulfonamides and trimethoprim are

    also affected by acquired genes specifying enzymes

    that are insensitive to drug inhibition (Skold, 2001). sul1

    andsul2are the main determinants of clinical resistance

    to sulfonamide, whereassul3was found to be prevalent

    in farm animals (Perreten and Boerlin, 2003). In contrast,

    more than 20 trimethoprim resistance determinants

    (numbered chronologically from dfr1) are documented.

    The genes specifying the sulfonamide-insensitive dihy-

    dropteroate synthases are present on class 1 integrons

    (sul1) or plasmids (sul2), whereas dfrvariants (with dfr1

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    being the most common in gram-negative bacteria)

    move from organism to organism on class 1 and 2 inte-

    grons. dfr1 is present on the Tn7transposon, thereby

    facilitating its integration into the E. colichromosome.

    Fluoroquinolones

    The plasmid-specified qnr determinants provide an

    unusual mechanism of decreased fluoroquinolone sus-

    ceptibility. Variants of the qnrelement, first identified in

    K. pneumoniae (Martinez-Martinez et al., 1998), have

    been found in E. coli, Enterobacter cloacae, Providencia

    stuarti i,Citrobacter freundii, C. koseri, Shigella flexneri

    2b, and non-Typhi Salmonella enterica. Qnr belongs to

    the pentapeptide repeat family of proteins and medi-ates resistance presumably by protecting DNA gyrase

    and topoisomerase IV from the inhibitory action of the

    fluoroquinolones (Tran and Jacoby, 2002). Another fluo-

    roquinolone resistance mechanism, MfpA, has been

    found in M. tuberculosis, which contains a single type II

    topoisomerase (Hegde et al., 2005). It, too, is a member

    of the pentapeptide repeat family of proteins but also

    acts as an inhibitor of DNA gyrase from M. tuberculosis

    by virtue of its ability to mimic the structure of B-form

    DNA. The interaction of MfpA with DNA gyrase likely

    interferes with the inhibitory action of agents like cipro-

    floxacin. By themselves, Qnr and MfpA confer only low

    levels of fluoroquinolone resistance but can augmentresistance when coupled with other mechanisms (see

    below). Moreover, selection for fluoroquinolone resist-

    ance may be favored in organisms bearing Qnr or MfpA

    if they survive the presence of low fluoroquinolone con-

    centrations.

    Efflux Systems, Porins, and Altered Membranes

    Efflux as a mechanism of antibiotic resistance was

    originally described for tetracyclines (McMurry et al.,

    1980) and is now well-appreciated and commonly

    found. Most drug efflux proteins belong to five dis-

    tinct protein families: the resistance-nodulation-cell

    division (RND), major facilitator (MF), staphylococcal/

    small multidrug resistance (SMR), ATP-binding cas-

    sette (ABC), and multidrug and toxic compound extru-

    sion (MATE) families (Li and Nikaido, 2004). Efflux by

    proteins in the RND, SMR, MF, and MATE families is

    driven by proton (and sodium) motive force and is

    therefore called secondary transport (Figure 3). ATP

    hydrolysis drives efflux in the primary (ABC) transport-

    ers (Figure 3 ). Efflux proteins also come in two general

    types. Some, like the tetracycline (Tet) (Levy, 1992)

    and macrolide (Mef ) (Zhanel et al., 2001) transporters,

    are single component efflux systems that have narrow

    substrate profiles, acting on a few agents or multiple

    agents within the same drug class. Others, such as

    the RND family members, require two additional pro-teins to confer resistance but are capable of binding

    multiple structurally unrelated compounds and confer

    broad resistance phenotypes (Li and Nikaido, 2004).

    The organizations of the RND-based efflux systems,

    which are found in a number of gram-negative bacte-

    ria, allow them to transport drugs from the cy toplasm

    and across the inner and outer membranes of the cell

    envelope (Figure 3).

    Tetracyclines

    There are now more than 20 different tetracycline efflux

    proteins that have been allocated to six different groups

    (Sapunaric et al., 2005). The proteins contain either 12-

    (e.g., TetA-E in gram-negative bacteria) or 14- (e.g., TetKand TetL in gram-positive organisms) putative trans-

    membrane-spanning segments. Expression of proteins

    within group 1 is controlled by a transcription repressor

    such as TetR. The antibiotic inactivates the repressor

    and allows expression of the tetracycline efflux systems.

    Alternatively, synthesis of TetK and TetL is inducible by

    tetracyclines through mechanisms that involve trans-

    lation attenuation and reinitiation, respectively. Also,

    the mechanism of tetracycline efflux is not conserved

    among all members; some, such as Tet(B), engage in

    electroneutral reactions (that is, one tetracycline-Mg

    chelate exits the cell as a proton enters whereas others,

    such as TetL, accomplish efflux in an electrogenic man-

    ner [references in Sapunaric et al., 2005]).

    Figure 3. Antibiotic Efflux SystemsFor simplicity, the illustration represents a

    gram-negative bacterium. Some substrates

    of AcrAB (e.g., aminoglycosides) are presum-

    ably recognized by a portion of AcrB that

    faces the cytoplasm while others (e.g., tetra-cycline) migrate thorough the inner membrane

    to an AcrB-binding pocket(s) (Murakami and

    Yamaguchi, 2003). The E. coli AcrAB-TolC

    and Tet efflux systems are secondary trans-

    porters that use proton motive force (H+) while

    NorM (Vibriospp.) is a secondary transporter

    that exploits a gradient of sodium ions (Na+)

    to drive efflux (Li and Nikaido, 2004). MacAB

    is a primary transporter that uses the energy

    derived from ATP hydrolysis to drive efflux (Li

    and Nikaido, 2004). Single-component sys-

    tems (e.g., NorM, Tet) transport substrates

    across the inner membrane where they then

    diffuse through porins (e.g., OmpF) or the

    outer membrane into the extracellular medium (Li and Nikaido, 2004). Multicomponent systems like AcrAB-TolC and MacAB-TolC direct the

    transport of substrates to the extracellular medium. Abbreviations: LPS, lipopolysaccharide; Ab, antibiotic; Tc, tetracycline; Mac, macrolide.

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    MLS Antibiotics

    MsrA (macrolide-streptogramin resistance) is an ABC

    efflux protein that produces resistance to 14- and 15-

    membered macrolides and streptogramin B antibiotics in

    the streptococci and staphylococci, but susceptibility to

    clindamycin is not affected by this system (Li and Nikaido,

    2004). In the staphylococci, relatives of MsrA (VgaA and

    VgaB) are specified on plasmids. VgaA confers resist-

    ance to streptogramin A antibiotics and lincosamides,

    and VgaB affects pristinamycin (a mixture of strepto-

    gramin A and B antibiotics) susceptibility (Chesneau

    et al., 2005). Alternatively, the Mef efflux transporters,

    which are the predominant macrolide resistance proteins

    in the United States, effectively handle 14- and 15-mem-

    bered macrolides in the streptococci, but strains that

    express the proteins are susceptible to 16-membered

    macrolides, lincosamides, and streptogramin B drugs

    (Li and Nikaido, 2004). E. faecalis are naturally resist-ant to quinupristin-dalfopristin and this characteristic

    is attributed to the expression of lsa, which specifies a

    streptogramin efflux protein belonging to the ABC family,

    as mutational inactivation of Lsa confers susceptibility

    to quinupristin-dalfopristin in this organism (references

    in Hershberger et al., 2004). This scenario has been

    observed with other (MDR) efflux proteins where removal

    of an efflux system renders an organism susceptible to

    antibiotics, even in the presence of chromosomal muta-

    tions that reduce binding of the drug to its target and

    would otherwise confer clinical resistance (Lomovskaya

    et al., 1999; Oethinger et al., 2000).

    PhenicolsChloramphenicol and florfenicol are broad-spec-

    trum agents that have utility in human clinical practice

    (chloramphenicol) and animal husbandry (florfenicol).

    Because of toxicity (aplastic anemia) seen with the

    former, the current use of chloramphenicol has been

    limited to the treatment of some severe life-threatening

    infections such as bacterial meningitis in patients with

    allergies to the penicillins. Efflux proteins specific for the

    phenicols have been described in a number of clinically

    important bacteria and are classified into eight different

    groups (E-1 through E-8) (Schwarz et al., 2004). In gen-

    eral, these proteins provide levels of resistance greater

    than that of the multidrug efflux proteins described below,

    and members of groups E-3 and E-4 confer resistance

    to both phenicols. For some chloramphenicol-specific

    efflux proteins like CmlA, expression of the resistance

    determinant is mediated through an inducible mecha-

    nism of translational attenuation similar to that used for

    particular chloramphenicol acetyltransferase resistance

    genes (see above).

    Multidrug Resistance Efflux Systems

    Historically, the gram-negative cell envelope was thought

    to affect antibiotic susceptibility by greatly restricting

    drug penetration (Li and Nikaido, 2004). Contemporary

    studies, however, have shown that most antibacterial

    agents effectively penetrate gram-negative organisms

    (Li and Nikaido, 2004) but fail to reach intracellular tar-

    gets because of efflux (Levy, 1992). In gram-negative

    bacteria, including E. coliand nonfermenting organisms

    such as P. aeruginosa,Acinetobacterspp., Stenotropho-

    monas maltophilia, and Burkholderia cepacia, intrinsic

    resistance is attributed to the expression of the RND

    efflux system(s) (Figure 3). This mechanism is an effec-

    tive means for dealing with different antibiotic classes

    using a single resistance determinant. The natural func-

    tion of the E. coliAcrAB efflux system is thought to have

    evolved to protect the cell from the inhibitory activity

    of toxic substances such as bile salts (Li and Nikaido,

    2004). Other related systems function similarly in Neis-

    seria gonorrhoeae (Hagman et al., 1995) and export

    molecules involved in quorum sensing in P. aeruginosa

    (Pesci et al., 1999).

    Tigecycline, which gained FDA approval in 2005, has

    poor activity against P. aeruginosa, Proteus mirabilis,

    Morganella morganii, and Klebsiella pneumonia; this isattributed to RND efflux systems (references in Stein

    and Craig, 2006). Elimination of an AcrA ortholog in M.

    morganii, MexXY-OprM in P. aeruginosa, and an AcrB

    ortholog in P. mirabilisincreased susceptibility to tige-

    cycline 16- to 133-fold (references in Stein and Craig,

    2006), whereas deletion in E. coliof AcrAB and AcrEF

    had a more modest (4-fold) effect (Hirata et al., 2004).

    Bacillus subtilis Bmr (Bacillus multidrug resistance)

    (Neyfakh et al., 1991) and S. aureus Qac (quaternary

    ammonium compound) (Tennent et al., 1989) are two

    MDR efflux proteins (members of the MF superfamily)

    that were first characterized in gram-positive cells. Like

    many members of the RND family in gram-negativebacteria, Bmr is constitutively expressed and therefore

    engenders intrinsic resistance to chloramphenicol and

    fluoroquinolones. Another MDR efflux pump from B.

    subtilis, Blt, includes spermidine among its list of sub-

    strates. It is now thought that the natural function of Blt

    is to facilitate the removal of polyamines from the cell

    (Woolridge et al., 1997). The staphylococcal Qac sys-

    tems provide resistance to antiseptics and disinfectants

    (e.g., quaternary ammonium compounds, chlorhexidine,

    and diamidines). Unlike most other MDR efflux proteins,

    these are specified on plasmids, a feature that facilitates

    their dissemination.

    Permeability

    As alluded to previously, the outer membrane of the

    gram-negative cell envelope is a barrier to both hydro-

    phobic and hydrophilic compounds. In order to circum-

    vent this permeability barrier, these organisms have

    evolved porin proteins (e.g., OmpF in E. coliand OprD

    in P. aeruginosa) that function as nonspecific entry

    and exit points for antibiotics and other small-molecule

    organic chemicals. Imipenem (and to a lesser extent

    meropenem) and basic amino acids pass through OprD;

    mutations that decrease expression of the porin con-

    tribute to clinical imipenem resistance. Moreover, the

    expression of OprD and the MexEF-OprM efflux system

    is coregulated (Ochs et al., 1999), and in this manner

    resistance to the carbapenems and other substrates of

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    MexEF-OprM (Table 3) can develop in mutants where

    the expression of OprD and the efflux pump has been

    altered.

    In 1996, Hiramatsu and colleagues identified clinical S.

    aureus isolates in Japan that exhibited an intermediate

    level of resistance to vancomycin (the vancomycin inter-

    mediate-resistant S. aureus [VISA] strains) (Hiramatsu

    et al., 1997), and shortly thereafter additional organisms

    with a glycopeptide-insensitive phenotype appeared in

    the United States (the GISA isolates). A prominent feature

    of the VISA isolates is the presence of a thickened cell

    wall. It is presumed that this property traps vancomycinand prevents the antibiotic from reaching its target (Cui

    et al., 2006a). More recently, the same group performed

    a preliminary characterization of the VISA isolates and

    found reduced susceptibility to daptomycin (Cui et al.,

    2006b). Although additional molecular and biochemical

    characterizations are certainly needed, the findings do

    carry a cautionary note.

    Mutations that offer resistance to polymyxin B in P.

    aeruginosa presumably involve changes in the bacte-

    rial cell envelope that do not involve porins (references

    in Falagas and Kasiakou, 2005). In Salmonella enterica

    serovar Typhimurium, PmrAB (a two-component regula-

    tory system) regulates resistance to polymyxin by modi-

    fying lipopolysaccharide and lipid A (references in Fala-

    gas and Kasiakou, 2005). The RosAB efflux system of

    Yersinia enterocoliticaalso af fects susceptibility to poly-

    myxin B (references in Falagas and Kasiakou, 2005).

    Regulatory Genes in Multidrug Resistance

    Chromosomal loci specifying global regulatory proteins

    that control MDR have been characterized in bacteria

    including E. coli, K. pneumoniae, and other Enterobac-

    teriaceae, Neisseria gonorrhoeae, Bacillus subtilis, and

    S. aureus (Grkovic et al., 2002). In most instances, the

    regulatory proteins act as activators or repressors oftranscription and contain either a single DNA-binding

    domain or two separate regions responsible for DNA

    binding and interacting with small-molecule substrates

    (Schumacher and Brennan, 2002). In the case of E. coli

    MarA, the protein regulates the expression of a large

    number of other chromosome genes (the Mar regulon),

    including those specifying an MDR efflux pump and other

    proteins (e.g., porins) that mediate antibiotic susceptibil-

    ity (Barbosa and Levy, 2000). In a clinical K. pneumoniae

    isolate exhibiting reduced susceptibility to tigecycline

    and an MDR phenotype, transposon mutagenesis was

    used to find that inactivation of ramA, which specifies a

    MarA ortholog, produced a 16-fold increase in suscep-

    Table 3. MDR Efflux Systems of Clinically Important Bacteria

    Bacterial Organism Efflux System(s) Representative Antibiotic Resistancea

    P. aeruginosa MExAB-OprM BLA and FQ

    MexCD-OprJ 4thgen ceph

    MexEF-OprN FQ, Cm, Tmp, and Tri

    MexHI-OprD EtBr, Nor, and Acr

    MexJK-OprM Cip, Tet, Ery, and Tri

    MexVW-OprM FQ, Cm, Tet, Ery, EtBr, and Acr

    MexXY-OprM AG and Tig

    A. baumannii AdeABC AG, FQ, TET, Ctx, Cm, Ery, and Tmp

    S. maltophilia SmeABC AG, BLA, and FQ

    SmeDEF MC, TET, FQ, CAR, Cm, and Ery

    B. cepacia CeoAB-OpcM Cm, Cip, and Tmp

    B. pseudomallei AmrAB-AprA MAC and AG

    E. coli AcrAB-TolC FQ, BLA, TET, Cm, Acr, Tri

    K. pneumoniae AcrAB-TolC FQ, BLA, TET, and Cm

    S. aureus MepA Tig, Mino, Tet, Cip, Nor EtBr, and TPP

    E. faecalis EmeA Nor, EtBr, Clind, Ery, and Nov

    Lsa Clind and QD

    S. pneumoniae PmrA FQ, Acr, and EtBr

    aAbbreviations: 4th gen ceph, fourth-generation cephalosporins; Acr, acriflavin; AG, aminoglycosides; BLA, -lactams; CAR,

    carbapenems; Cip, ciprofloxacin; Clind, clindamycin; Cm, chloramphenicol; Ctx, cefotaxime; Ery, erythromycin; EtBr, ethidium

    bromide; FQ, fluoroquinolones; MC, macrolides; Mino, minocycline; Nor, norfloxacin; Nov, novobiocin; QD, quinupristin-dalfo-

    pristin; TET, tetracycline family; Tet, tetracycline; Tig, tigecycline; Tmp, trimethoprim; TPP, tetraphenylphosphonium bromide;

    Tri, triclosan.

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    tibility to tigecycline and abrogated the MDR phenotype

    (Ruzin et al., 2005). Although other clinical isolates that

    overexpress MarA or a MarA ortholog and exhibit a MDR

    phenotype have been described previously (Schneiders

    et al., 2003 and references therein), the studies that

    involved K. pneumoniae offer further proof that chro-

    mosomal transcription factors that regulate MDR efflux

    systems can produce clinical drug resistance.

    Single Determinants of Multidrug Resistance

    Except for the case of MDR efflux pumps, a single resist-

    ance mechanism commonly affords protection against

    one antibiotic or, at the most, drugs within the same

    general class, e.g., PBP2 of MRSA. Still, resistance

    determinants that specify erythromycin (Erm) methyl-

    transferases in a variety of pathogens are single pro-

    teins that give rise to macrolide, lincosamide, and type

    B streptogramin resistance: structurally unique agentsthat share a common target and mechanism of action

    (Roberts, 2004).

    A mutant aminoglycoside acetyltransferase (specified

    byaac(6 )- Ib-cr) that gave rise to aminoglycoside (ami-

    kacin, kanamycin, and tobramycin) and fluoroquinolone

    (ciprofloxacin and norfloxacin) resistance was identified

    recently (Robicsek et al., 2006). Although the level of

    resistance conferred was low,aac(6 )- Ib-crwas located

    on a plasmid bearing another unusual mechanism (the

    qnrdeterminant [see above]) of fluoroquinolone resist-

    ance, and the two determinants together functioned in

    an additive manner to yield clinical levels of fluoroqui-

    nolone resistance.Reduced susceptibility to the macrolides, chloram-

    phenicol, and linezolid in clinical S. pneumoniae iso-

    lates has been attributed to mutations in large ribosomal

    protein (L4) (Wolter et al., 2005). Although mutations in

    the genes that specify 23S rRNA, L4, or L22 commonly

    lead to macrolide resistance, susceptibility to chloram-

    phenicol frequently occurs following the acquisition of

    a modifying enzyme (see above). For linezolid, previous

    resistance in the enterococci and S. aureuswas attrib-

    uted solely to mutations in the locus that encodes 23S

    rRNA (see above).

    What the Past Can Teach Us about the FutureMore than half a century has passed since the first anti-

    biotics were introduced commercially. It did not take

    long for microbes to develop drug fastness to the

    original magic bullets, and widespread use of many

    antibacterial drugs provides ideal conditions for the

    spread of MDR organisms. Many early researchers were

    expertly skilled in identifying the means used to evade

    the inhibitory effects of these drugsfrom investigators

    such as Esther and Joshua Lederberg, who character-

    ized the random nature of mutational events conferring

    resistance to streptomycin, to others such as Tsutomu

    Watanabe, who surmised that mutation alone would not

    be sufficient for explaining the MDR phenotype. Resist-

    ance transfer factors (RTFs), later called resistance (R)

    factors and then plasmids, would provide the basis for

    multiple drug resistance caused by infective heredity

    (reviewed in Watanabe, 1963).

    For bacteria, there is more than one way to evade a

    drug class, and organisms unresponsive to all antimi-

    crobial agents, even ones not encountered previously,

    are now familiar. Microbes have means for collecting

    single resistance traits. Theoretically, one needs only a

    single (broad-spectrum) agent that ef fectively deals with

    resistances affecting just one class of drugs. The MDR

    efflux pumps that render multiple classes ineffective,

    however, have greatly confounded efforts in finding new

    agents.

    Multidrug resistance is a worldwide problem that does

    not obey international borders and can indiscriminately

    affect members of all socioeconomic classes. A slew of

    epidemiological studies have documented the clonal,

    worldwide spread of infectious disease entities suchas MDR S. pneumoniae from specific regions of the

    world. Also, the use of antibiotics in animal husbandry

    has been shown to foster human colonization with drug-

    resistant microbes (Levy et al., 1976). There are com-

    pelling data that link use of agents like virginiamycin (a

    streptogramin) and avoparcin (a glycopeptide) in animal

    husbandry to clinical drug-resistant strains.

    Bacteria adopt intricate strategies to avoid the lethal

    effects of antibiotics. Awareness of these mechanisms

    of resistance can help in the design of new drugs. As

    we face this critical problem, we need to be aware of

    the fluidity of the microbial genome and the relative ease

    with which resistance can emerge by mutation or geneacquisition. Recognizing the potential for the emer-

    gence of resistance should garner newfound respect for

    the discovery of new agents so urgently needed to cure

    infectious diseases.

    ACKNOWLEDGMENTS

    S.B.L. is supported by the US Public Health Service (National Insti-

    tutes of Health). The authors thank Bonnie Marshall for her help in the

    preparation of the manuscript and figures.

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