Leading Edge

Review
Molecular Mechanisms of Antibacterial
Multidrug Resistance
Michael N. Alekshun1,* and Stuart B. Levy2,*

Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA
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
1
2

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

resistant to multiple antibiotics. Although classically attributed to chromosomal mutations, 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.
Efforts aimed at identifying new antibiotics were once a
top research and development priority among pharmaceutical companies. The potent broad spectrum drugs
that emerged from these endeavors provided extraordinary clinical efficacy. Success, however, has been compromised. 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 antimicrobial 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 produced antibiotics. The most notable example is resistance to penicillin among staphylococci, specified by an
enzyme (penicillinase) that degraded the
antibiotic (Barber, 1947). Over the years,
There is probably no chemotherapeutic drug to which
continued selective pressure by different
in suitable circumstances the bacteria cannot react
drugs has resulted in organisms bearing
by in some way acquiring fastness [resistance].
additional kinds of resistance mechanisms
that led to multidrug resistance (MDR)
novel penicillin-binding proteins (PBPs),
Alexander Fleming, 1946
enzymatic mechanisms of drug modification, mutated drug targets, enhanced efflux
The means that microbes use to evade antibiotpump expression, and altered membrane permeability.
ics certainly predate and outnumber the therapeutic
Some of the most problematic MDR organisms that are
interventions themselves. In a recent collection of soilencountered currently include Pseudomonas aeruginosa
dwelling Streptomyces (the producers of many clini(another microbe of soil origin), Acinetobacter baumancal therapeutic agents), every organism was multidrug
nii, Escherichia coli and Klebsiella pneumoniae bearing
resistant. Most were resistant to at least seven different
extended-spectrum -lactamases (ESBL), vancomycinantibiotics, and the phenotype of some included resistresistant enterococci (VRE), methicillin-resistant Staphyance to 1521 different drugs (DCosta et al., 2006).
lococcus aureus (MRSA), vancomycin-resistant MRSA,
Moreover, many isolates were resistant to daptomycin,
and extensively drug-resistant (XDR) Mycobacterium
quinupristin-dalfopristin, and telithromycinall drugs
tuberculosis (Table 1). Some like methicillin-resistant S.
approved by the United States Food and Drug Adminaureus couple MDR with exceptional virulence capabiliistration (FDA) within the last decadeas well as purely
ties (Miller et al., 2005). Others, including some strains of
synthetic agents such as ciprofloxacin. These data not
P. aeruginosa, A. baumannii, and K. pneumoniae, manonly suggest that our surroundings can act as a resage to evade every drug within the physicians arsenal
ervoir for new (and old) resistance mechanisms, but
(Levin et al., 1999).
Cell 128, March 23, 2007 2007 Elsevier Inc. 1037

Figure 1. Acquisition of Antibiotic Resistance

Bacteria can become antibiotic resistant (Abr)
by mutation of the target gene in the chromosome. 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 conjugative transposons during conjugation. The
general term transposable element has been
used to designate (1) an insertion sequence, (2)
composite (compound), complex, and conjugative transposon, (3) transposing bacteriophage,
or (4) integron.

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 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 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 transduction (via bacteriophages), conjugation (via plasmids and
conjugative transposons), and transformation (via incorporation 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 process 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

Drugs Considered for
Treatment of MDRa

Organism

Common Infections Key Antibiotic Resistances

P. aeruginosa

Lung, wound

-lactams, fluoroquinolones, aminoglycosides

Colistin

Acinetobacter spp.

Lung, wound,
bone, blood

-lactams, fluoroquinolones, aminoglycosides

Colistin, tigecycline

E. coli and K.
Urinary, biliary,
neumoniae bearp
gastrointestinal
ing extended-spec- tracts, lung, blood
trum -lactamases

-lactams, fluoroquinolones, aminoglycosides

Colistin (for K. pneumoniae),

tigecycline

Vancomycin-resistant enterococci

Vancomycin

Quinupristin-dalfopristin,
linezolid, daptomycin

Methicillin-resistant Skin and soft tisS. aureus

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

3rd line agents, drug

combinations

Blood, heart,
intra-abdominal

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.
a

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

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 (<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: -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 distinguished 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 chromosome. In general, these pieces of DNA contain terminal 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 ability 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 (representing 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 discovered 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-transferable plasmids were easily selected and propagated during 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 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
Cell 128, March 23, 2007 2007 Elsevier Inc. 1039

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 (Abr) such as those for aminoglycosides, b-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 responsible 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. PR and PH 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 DNAbinding functions and are targeted by the fluoroquinolones (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-resistancedetermining-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 resistance in a bacterium; successive mutations have additive
effects. Isolates that are highly fluoroquinolone resistant bear multiple QRDR mutations and generally other
mechanisms of fluoroquinolone resistance, such as
drug efflux (see below).
Rifamycins
Rifamycins are bactericidal drugs that arrest transcription by interacting with RpoB, the subunit of RNA
polymerase (RNAP). Although combination regimens
1040 Cell 128, March 23, 2007 2007 Elsevier Inc.

that include rifampin or rifapentine and isoniazid, pyrazinamide, ethambutol, or streptomycin remain primary
choices for front-line therapy of M. tuberculosis infection,
resistance through point mutations in the rifampin-binding 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 resistant 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 Flemings
chance discovery of penicillin (Levy, 2002). Trimethoprim, 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. coli and Streptococcus pneumoniae and in clinical isolates of Campylobacter jejuni and Haemophilus influenzae (Skold,

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 macrolide-lincosamide-streptogramin (MLS) classes of antibiotics target the ribosome to inhibit protein translation.
As a consequence, resistance via chromosomal mutation is uncommon. Tetracyclines and aminoglycosides
interact with 16S rRNA (rrs) and the macrolide-lincosamide-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 mutation 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 emergence of resistance to erythromycin (a macrolide) during 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 susceptibility (Tait-Kamradt et al., 2000). The ketolides, recently
introduced into clinical practice, were designed to circumvent 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 quinupristin-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 Hershberger et al., 2004).
Oxazolidinones
Linezolid (another protein synthesis inhibitor) was
approved recently as an agent to treat methicillin-resistant 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 successfully used for treating infections caused by methicillin-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 lysylphosphatidylglycerol 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 -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 discovered and characterized, and a more complete overview 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
Cell 128, March 23, 2007 2007 Elsevier Inc. 1041

serine residue at their active sites and the class B proteins 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 inhibition by EDTA, whereas they are not susceptible to inhibition by clavulanate (a -lactamase inhibitor). AmpC,
an inducible and usually chromosomal enzyme found in
many species of the Enterobacteriaceae and P. aeruginosa, is the prototype class C enzyme. Recent reports
have described plasmid-borne ampC genes that can
be transferred among E. coli, Klebsiella spp., and Salmonella 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 widespread in clinically relevant organisms (Davies and
Wright, 1997). Here, resistance is accomplished with
proteins that N-acetylate (acetyltransferases), phosphorylate (phosphotransferases), and adenylate (nucleotidyltransferases) the aminoglycosides. The acetyltransferases are capable of modifying tobramycin,
gentamicin, netilmicin, and amikacin; the nucleotidyltransferase 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 determinants. 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, hydrolases, glycosylases, phosphotransferases, nucleotidyltransferases, 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) membered macrolides; the hydrolases affect streptogramin
B drugs; the acetyltransferases inactivate streptogramin
A antibiotics; and the nucleotidyltransferases confer
resistance to the lincosamides (e.g., clindamycin). Phosphotransferases 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 function as homotrimers but are unrelated based on amino
acid sequence analyses. The type B enzymes are also
1042 Cell 128, March 23, 2007 2007 Elsevier Inc.

termed xenobiotic acetyltransferases and appear to

share an evolutionary lineage that includes some streptogramin-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, designated 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 common, clinically relevant tetracycline-resistant isolates,
although a related tet(X)-like gene in P. aeruginosa has
been reported (A. Nakamura et al., 2002, paper presented 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 isolates, but methicillin-resistant S. aureus were identified
shortly 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 element called the staphylococcal cassette chromosome
(SCCmec) (Fuda et al., 2005). In addition to mecA,
SCCmec contains the mecR1-mecI regulatory loci and
encodes the enzymes that are involved in site-specific
recombination.
Under normal circumstances, S. aureus uses multiple PBPs during cell wall biosynthesis. One, PBP2, is a
bifunctional enzyme with transpeptidase and transglycosylase activities. When methicillin-resistant S. aureus
is exposed to methicillin, PBP2 functions as the transglycosylase whereas PBP2a contributes the transpeptidase 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. aureus that
have emerged recently outside of hospitals in the community share some features with hospital-associated

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

complete -lactam resistance phenotype and the ability to cause a range of serious life-threatening infections. Although many community-associated strains are
multidrug susceptible (except to -lactam antibiotics),
the genome of an epidemic clone distributed throughout the US (USA300) contains chromosomal mutations
that confer fluoroquinolone resistance and plasmids that
mediate resistance to tetracycline, erythromycin, clindamycin, streptogramin B agents, and mupirocin (Diep
et al., 2006).
Glycopeptides
Glycopeptides (including vancomycin and teicoplanin)
interact with peptidoglycan precursors to exert bactericidal activity. Resistance to the glycopeptides in grampositive 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 phenotype is accomplished using multiple proteins specified in gene clusters and each result in the production
of a modified peptidoglycan. Of the many drug-resistance 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 resistance (Figure 2B). Either a racemase or dehydrogenase
results in the production of serine (VanC, E, or G) or lactate 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 machinery. 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, imparting 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. Vancomycin-resistant E. faecalis and methicillin-resistant S.
aureus were also obtained from the Michigan patient

that bore vancomycin-resistant S. aureus and each contained plasmids that were identical, except for the presence 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 tetracyclines in the clinic are drug efflux (see below) and ribosome protection. Determinants of ribosome protection
(Connell et al., 2003) have sequence similarity to bacterial elongation factors (EF-G an EF-Tu). They also possess GTPase activity and function to facilitate release
of tetracycline from the ribosome in an energy-dependent 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 protection 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 overlapping genetic unit (Sloan et al., 1994).
Binding of drugs within the MLSK (macrolide-lincosamide-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 represent the predominant macrolide resistance mechanisms
in Europe and South Africa. Currently, there are 34 different 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 position A2058). Methylation results in the MLSB phenotype,
which encompasses resistance to 14-, 15-, and 16-membered 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 expression, 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 dihydropteroate synthases are present on class 1 integrons
(sul1) or plasmids (sul2), whereas dfr variants (with dfr1
Cell 128, March 23, 2007 2007 Elsevier Inc. 1043

Figure 3. Antibiotic Efflux Systems

For simplicity, the illustration represents a
gram-negative bacterium. Some substrates
of AcrAB (e.g., aminoglycosides) are presumably recognized by a portion of AcrB that
faces the cytoplasm while others (e.g., tetracycline) 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 transporters 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 systems (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 integrons. 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 susceptibility. 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 mediates resistance presumably by protecting DNA gyrase
and topoisomerase IV from the inhibitory action of the
fluoroquinolones (Tran and Jacoby, 2002). Another fluoroquinolone 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 ciprofloxacin. 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 resistance may be favored in organisms bearing Qnr or MfpA
if they survive the presence of low fluoroquinolone concentrations.
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 distinct protein families: the resistance-nodulation-cell
division (RND), major facilitator (MF), staphylococcal/
small multidrug resistance (SMR), ATP-binding cas1044 Cell 128, March 23, 2007 2007 Elsevier Inc.

sette (ABC), and multidrug and toxic compound extrusion (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) transporters (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 proteins 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 bacteria, 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 transmembrane-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 translation 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 manner [references in Sapunaric et al., 2005]).

MLS Antibiotics
MsrA (macrolide-streptogramin resistance) is an ABC
efflux protein that produces resistance to 14- and 15membered 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 resistance to streptogramin A antibiotics and lincosamides,
and VgaB affects pristinamycin (a mixture of streptogramin 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-membered 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 resistant 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 mutations 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-spectrum 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 general, 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 mechanism 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., Stenotrophomonas maltophilia, and Burkholderia cepacia, intrinsic
resistance is attributed to the expression of the RND
efflux system(s) (Figure 3). This mechanism is an effective means for dealing with different antibiotic classes
using a single resistance determinant. The natural function 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 Neisseria 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 tigecycline 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 substrates. 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 systems 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 hydrophobic and hydrophilic compounds. In order to circumvent 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 contribute 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
Cell 128, March 23, 2007 2007 Elsevier Inc. 1045

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

4th 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. 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

PmrA

FQ, Acr, and EtBr

S. pneumoniae

Abbreviations: 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-dalfopristin; TET, tetracycline family; Tet, tetracycline; Tig, tigecycline; Tmp, trimethoprim; TPP, tetraphenylphosphonium bromide;
Tri, triclosan.
a

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 intermediate-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 bacterial cell envelope that do not involve porins (references
in Falagas and Kasiakou, 2005). In Salmonella enterica
serovar Typhimurium, PmrAB (a two-component regulatory system) regulates resistance to polymyxin by modi-

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

fying lipopolysaccharide and lipid A (references in Falagas and Kasiakou, 2005). The RosAB efflux system of
Yersinia enterocolitica also affects susceptibility to polymyxin 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 Enterobacteriaceae, 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 susceptibility (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 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 chromosomal 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 resistance 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) methyltransferases in a variety of pathogens are single proteins 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 (amikacin, 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 resistance, and the two determinants together functioned in
an additive manner to yield clinical levels of fluoroquinolone resistance.
Reduced susceptibility to the macrolides, chloramphenicol, and linezolid in clinical S. pneumoniae isolates 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 chloramphenicol frequently occurs following the acquisition of
a modifying enzyme (see above). For linezolid, previous
resistance in the enterococci and S. aureus was attributed 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 antibiotics 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 characterized 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. Resistance 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 antimicrobial 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 drugresistant microbes (Levy et al., 1976). There are compelling 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 emergence 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 Institutes of Health). The authors thank Bonnie Marshall for her help in the
preparation of the manuscript and figures.
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