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Leading Edge

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Leading Edge Review Molecular Mechanisms of Antibacterial Multidrug Resistance Michael N. Alekshun * and Stuart B.

Molecular Mechanisms of Antibacterial Multidrug Resistance

Michael N. Alekshun 1, * and Stuart B. Levy 2, *

1 Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA 2 Center 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: michael.alekshun@spcorp.com (M.N.A), stuart.levy@tufts.edu (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 elements—such as efflux pumps that expel multiple kinds of antibiotics—are 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.

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.

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

“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

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),

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 15–21 different drugs (D’Costa et al., 2006). Moreover, many isolates were resistant to daptomycin, quinupristin-dalfopristin, and telithromycin—all drugs approved by the United States Food and Drug Admin - istration (FDA) within the last decade—as 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

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 coli and Klebsiella pneumoniae bearing extended-spectrum β -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. aureus couple 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 physician’s arsenal (Levin et al., 1999).

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Rather than providing an extensive list of antibiotic resistance mechanisms (which can be found elsewhere [Alekshun
Rather than providing an extensive list of antibiotic resistance mechanisms (which can be found elsewhere [Alekshun

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 occurring genes found on the host’s chromosome, such as, AmpC β -lactamase of gram-negative bacteria and many MDR

Figure 1. Acquisition of Antibiotic Resistance

Bacteria can become antibiotic resistant (Ab r ) 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 also transferred 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.

efflux systems (see below). Acquired mechanisms involve mutations in genes targeted by the antibiotic and the transfer of resistance determinants borne 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-negative bacteria (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 MDR a

P.

aeruginosa

Lung, wound

β-lactams, fluoroquinolones, aminoglycosides

Colistin

Acinetobacter spp.

Lung, wound,

β-lactams, fluoroquinolones, aminoglycosides

Colistin, tigecycline

bone, blood

E.

coli and K.

Urinary, biliary,

β-lactams, fluoroquinolones, aminoglycosides

Colistin (for K. pneumoniae),

pneumoniae bear-

gastrointestinal

tigecycline

ing extended-spec-

tracts, lung, blood

trum β-lactamases

Vancomycin-resist-

Blood, heart,

Vancomycin

Quinupristin-dalfopristin,

ant enterococci

intra-abdominal

linezolid, daptomycin

Methicillin-resistant

Skin and soft tis-

β-lactams, fluoroquinolones, macrolides

Quinupristin-dalfopristin,

S.

aureus

sue, respiratory tract, blood

daptomycin, linezolid, tigecycline, vancomycin

Multidrug-resistant

Ear, lung, blood,

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

S.

pneumoniae

cerebrospinal fluid

Extensively

Lung

Rifampin, isoniazid, and three of the following:

3 rd line agents, drug

drug-resistant M.

aminoglycosides, polypeptides, fluoroquinolones,

combinations

tuberculosis

thioamides, cycloserine, or para-aminosalicylic acid

a Agents 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.

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Table 2. Mobile Genetic Elements Genetic Element General Characteristics Resistance Determinant(s) Specified/Examples Plasmid Variable size (1-

Table 2. Mobile Genetic Elements

Genetic Element

General Characteristics

Resistance Determinant(s) Specified/Examples a

Plasmid

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

R factor: multiple resistances

Insertion sequence

Small (<2.5 kb), contains terminal inverted repeats, and specifies a transposase

IS1, IS3, IS4, etc.

Composite (compound) transposon

Flanked by insertion sequences and/or inverted repeats

Tn5: Kan, Bleo, and Str

Complex transposon

Large (>5 kb), flanked by short terminal inverted repeats, and specifies a transposase and recombinase

Tn1 and Tn3: β-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, and conjugative transposons

Tn4: Amp, Str, Sul, and Hg Tn1691: 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: Vibrio spp. super-integron

See Figure 1. a Abbreviations: 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. b See Figure 2.

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 host’s 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 host’s 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 Shigella bearing 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. Shigella and 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 drug’s 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

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Figure 2. Transposable Elements that Confer Antibiotic Resistance (A) Organization of a class 1 integron. int1

Figure 2. Transposable Elements that Confer Antibiotic Resistance

  • (A) Organization of a class 1 integron. int1 specifies the integrase (the recombination protein), att1 is the primary recombination site, attC are 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 (Ab r ) such as those for aminoglycosides, β-lactams, chloramphenicol, and trimethoprim inserted downstream of this element. qacE

and sul1 specify genes for resistance to quaternary ammonium compounds (partially deleted) and sulfonamides, respectively; these two genes are found specifically in class 1 integrons.

  • (B) The vanA gene 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. P R and P H are promoters that control expression of two separate transcriptional units, and the entire element is flanked by imperfect inverted repeats.

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 resistance are 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 β subunit of RNA polymerase (RNAP). Although combination regimens

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that include rifampin or rifapentine and isoniazid, pyrazi- namide, ethambutol, or streptomycin remain primary choices for front-line therapy of M. tuberculosis infection,

resistance through point mutations in the rifampin-bind - ing region of rpoB occur at a frequency of 1 × 10 8 and are widespread. M. tuberculosis resistant 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 for large-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 Fleming’s 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 enzyme’s affinity for the sulfonamides and have been found in laboratory experiments using E. coli and Strep - tococcus pneumoniae and in clinical isolates of Campy- lobacter jejuni and Haemophilus influenzae (Skold,

2001). Mutations in the chromosomal gene specifying dihydrofolate reductase can result in overexpression of an enzyme

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. coli and 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 rrl operons 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 ) copy numbers, chromosomal mutations conferring resistance have appeared. Tetracycline resistance via a point muta - tion in Propionibacterium acnes (3 rRNA operons) and Helicobacter pylori (2 copies of rrn ) (Gerrits et al., 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. pneumoniae with mutations in rrl, 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 combination’s 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 in rrl in S. aureus and E. faecalis. Now, clinical isolates of Staphylococcus epidermidis, S. aureus, Streptococcus oralis, Enterococcus faecium, and E. faecalis resistant to linezolid have been documented and many of the strains bear rrl mutations (Meka and Gold, 2004). As with the

fluoroquinolones, the level of resistance in S. aureus increases with mutations in multiple rrl alleles (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. aureus and 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, and rpoC ) 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 Duplications A 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 acrAB locus 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 expected that 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 β -lactamases that degrade antibiotics and others (including the macrolide and aminoglycoside-modifying proteins) that perform chemical transformations. β-Lactam Antibiotics There are hundreds of β -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. β -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 β -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.

serine residue at their active sites and the class B pro - teins are zinc-dependent metalloenzymes. Some class A proteins function as extended-spectrum β -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 β -lactamase inhibitor). AmpC, an inducible and usually chromosomal enzyme found in many species of the Enterobacteriaceae and 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, Acinetobacter spp., and Aeromonas spp. Aminoglycosides

A large number of aminoglycoside-modifying enzymes specified 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. aeruginosa that 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

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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. aeruginosa has 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

β-Lactam Antibiotics The first penicillin-resistant S. aureus identified in the mid-1940s expressed a β -lactamase (termed PCI) that afforded resistance. A penicillin derivative, methicillin, that was resistant to this β -lactamase was subsequently introduced in 1959 to treat the penicillin-resistant iso - lates, but methicillin-resistant S. aureus were identified shortly thereafter (Barber, 1961). Here, the β -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 β -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” (SCC mec ) (Fuda et al., 2005). In addition to mecA, SCC mec contains the mecR1-mecI regulatory 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 β -lactam antibiotics. Removal of the transglycosylase function of PBP2 imparts β -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. aureus that have emerged recently outside of hospitals in the com - munity share some features with hospital-associated

strains (Naimi et al., 2003). These include the nearly complete β -lactam resistance phenotype and the

strains (Naimi et al., 2003). These include the nearly complete β -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 β -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 dehydrogenase results 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 antibiotic’s 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. aureus have 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. faecalis and methicillin-resistant S. aureus were also obtained from the Michigan patient

that bore vancomycin-resistant S. aureus and each con - tained plasmids that were identical, except for the pres -

ence of Tn1546 in 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 vanA gene cluster was subsequently transferred by means of transposition into the S. aureus plasmid. 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 mechanisms in 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. coli 23S rRNA (at posi- tion A2058). Methylation results in the MLS B phenotype, 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 erm expres - sion, but 16-membered macrolides do not (Zhanel et al., 2001). Constitutive erm expression 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 and sul2 are the main determinants of clinical resistance to sulfonamide, whereas sul3 was 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 dfr variants (with dfr1

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Figure 3. Antibiotic Efflux Systems For simplicity, the illustration represents a gram-negative bacterium. Some substrates of
Figure 3. Antibiotic Efflux Systems For simplicity, the illustration represents a gram-negative bacterium. Some substrates of

Figure 3. Antibiotic Efflux Systems

For 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 (Vibrio spp.) 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.

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 Tn7 transposon, thereby facilitating its integration into the E. coli chromosome. Fluoroquinolones The plasmid-specified qnr determinants provide an unusual mechanism of decreased fluoroquinolone sus - ceptibility. Variants of the qnr element, first identified in K. pneumoniae (Martinez-Martinez et al., 1998), have been found in E. coli, Enterobacter cloacae, Providencia stuartii, 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 augment resistance 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 cytoplasm 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., TetK and 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]).

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MLS Antibiotics MsrA (macrolide-streptogramin resistance) is an ABC efflux protein that produces resistance to 14- and

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).

Phenicols

Chloramphenicol 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. coli and nonfermenting organisms such as P. aeruginosa, Acinetobacter spp., 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. coli AcrAB 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 is attributed 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. mirabilis increased susceptibility to tige - cycline 16- to 133-fold (references in Stein and Craig, 2006), whereas deletion in E. coli of 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-negative bacteria, 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. coli and 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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Table 3. MDR Efflux Systems of Clinically Important Bacteria Bacterial Organism Efflux System(s) Representative Antibiotic Resistance

Table 3. MDR Efflux Systems of Clinically Important Bacteria

Bacterial Organism

Efflux System(s)

Representative Antibiotic Resistance a

  • P. MExAB-OprM

aeruginosa

BLA and FQ

MexCD-OprJ

4 th gen 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. AdeABC

baumannii

AG, FQ, TET, Ctx, Cm, Ery, and Tmp

  • S. SmeABC

maltophilia

AG, BLA, and FQ

SmeDEF

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

  • B. CeoAB-OpcM

cepacia

Cm, Cip, and Tmp

  • B. AmrAB-AprA

pseudomallei

MAC and AG

  • E. AcrAB-TolC

coli

FQ, BLA, TET, Cm, Acr, Tri

  • K. AcrAB-TolC

pneumoniae

FQ, BLA, TET, and Cm

  • S. MepA

aureus

Tig, Mino, Tet, Cip, Nor EtBr, and TPP

faecalis

  • E. EmeA

Nor, EtBr, Clind, Ery, and Nov

Lsa

Clind and QD

  • S. PmrA

pneumoniae

FQ, Acr, and EtBr

a Abbreviations: 4 th gen ceph, fourth-generation cephalosporins; Acr, acriflavin; AG, aminoglycosides; BLA, β-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.

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 vancomycin and 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-

  • 1046 Cell 128, March 23, 2007 ©2007 Elsevier Inc.

fying lipopolysaccharide and lipid A (references in Fala- gas and Kasiakou, 2005). The RosAB efflux system of Yersinia enterocolitica also affects 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 of transcription 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 -

tibility to tigecycline and abrogated the MDR phenotype (Ruzin et al., 2005). Although other clinical isolates

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 agents that share a common target and mechanism of action (Roberts, 2004). A mutant aminoglycoside acetyltransferase (specified by aac(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-cr was located on a plasmid bearing another unusual mechanism (the qnr determinant [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. aureus was attrib - uted solely to mutations in the locus that encodes 23S rRNA (see above).

What the Past Can Teach Us about the Future

More 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 drugs—from 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 effectively 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 such as 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 gene acquisition. 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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