2007_molecular mechanisms of antibacterial multidrug resistance
TRANSCRIPT
-
7/24/2019 2007_Molecular Mechanisms of Antibacterial Multidrug Resistance
1/14
Leading Edge
Review
Cell 128, March 23, 2007 2007 Elsevier Inc. 1037
Efforts aimed at identifying new antibiotics were once a
top research and development priority among pharma-
ceutical companies. The potent broad spectrum drugs
that emerged from these endeavors provided extraordi-
nary clinical efficacy. Success, however, has been com-
promised. We are now faced with a long list of microbes
that have found ways to circumvent different structural
classes of drugs and are no longer susceptible to most,
if not all, therapeutic regimens.
The means that microbes use to evade antibiot-
ics certainly predate and outnumber the therapeutic
interventions themselves. In a recent collection of soil-
dwelling Streptomyces (the producers of many clini-
cal therapeutic agents), every organism was multidrug
resistant. Most were resistant to at least seven different
antibiotics, and the phenotype of some included resist-
ance to 1521 different drugs (DCosta et al., 2006).
Moreover, many isolates were resistant to daptomycin,
quinupristin-dalfopristin, and telithromycinall drugs
approved by the United States Food and Drug Admin-
istration (FDA) within the last decadeas well as purely
synthetic agents such as ciprofloxacin. These data not
only suggest that our surroundings can act as a res-
ervoir for new (and old) resistance mechanisms, but
that the drugs we use to treat infectious diseases have
long-lasting effects outside of the hospital. Many anti-
microbial molecules exist for millennia stably within the
environment (Cook et al., 1989), where they select and
promote growth of resistant strains.
Resistance to single antibiotics became prominent in
organisms that encountered the first commercially pro-
duced antibiotics. The most notable example is resist-
ance to penicillin among staphylococci, specified by an
enzyme (penicillinase) that degraded the
antibiotic (Barber, 1947). Over the years,
continued selective pressure by different
drugs has resulted in organisms bearing
additional kinds of resistance mechanisms
that led to multidrug resistance (MDR)
novel penicillin-binding proteins (PBPs),
enzymatic mechanisms of drug modifica-
tion, mutated drug targets, enhanced efflux
pump expression, and altered membrane permeability.
Some of the most problematic MDR organisms that are
encountered currently include Pseudomonas aeruginosa
(another microbe of soil origin),Acinetobacter bauman-
nii, Escherichia coliand Klebsiella pneumoniaebearing
extended-spectrum -lactamases (ESBL), vancomycin-
resistant enterococci (VRE), methicillin-resistant Staphy-
lococcus aureus(MRSA), vancomycin-resistant MRSA,
and extensively drug-resistant (XDR) Mycobacterium
tuberculosis (Table 1). Some like methicillin-resistant S.
aureuscouple MDR with exceptional virulence capabili-
ties (Miller et al., 2005). Others, including some strains of
P. aeruginosa,A. baumannii, and K. pneumoniae, man-
age to evade every drug within the physicians arsenal
(Levin et al., 1999).
Molecular Mechanisms of Antibacterial
Multidrug ResistanceMichael N. Alekshun1,* and Stuart B. Levy2,*1Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, NJ 07033, USA2Center for Adaptation Genetics and Drug Resistance, Department of Molecular Biology & Microbiology and Department of
Medicine, Tufts University School of Medicine, Boston, MA 02111, USA
*Correspondence: [email protected] (M.N.A), [email protected] (S.B.L.)
DOI 10.1016/j.cell.2007.03.004
Treatment of infections is compromised worldwide by the emergence of bacteria that are
resistant to multiple antibiotics. Although classically attributed to chromosomal muta-
tions, resistance is most commonly associated with extrachromosomal elements acquired
from other bacteria in the environment. These include different types of mobile DNA
segments, such as plasmids, transposons, and integrons. However, intrinsic mechanisms
not commonly specified by mobile elementssuch as efflux pumps that expel multiple
kinds of antibioticsare now recognized as major contributors to multidrug resistance in
bacteria. Once established, multidrug-resistant organisms persist and spread worldwide,
causing clinical failures in the treatment of infections and public health crises.
There is probably no chemotherapeutic drug to which
in suitable circumstances the bacteria cannot react
by in some way acquiring fastness [resistance].
Alexander Fleming, 1946
-
7/24/2019 2007_Molecular Mechanisms of Antibacterial Multidrug Resistance
2/14
1038 Cell 128, March 23, 2007 2007 Elsevier Inc.
Rather than providing an extensive list of antibiotic
resistance mechanisms (which can be found elsewhere
[Alekshun and Levy, 2000]), the aim of this Review is to
highlight common genetic and molecular mechanisms
of resistance as they relate to important pathogens and
to drugs used by physicians today.
Genetics of Multidrug Resistance
Bacterial antibiotic resistance can be attained through
intrinsic or acquired mechanisms (Figure 1). Intrinsic
mechanisms are those specified by naturally occurringgenes found on the hosts chromosome, such as, AmpC
-lactamase of gram-negative bacteria and many MDR
efflux systems (see below). Acquired
mechanisms involve mutations in
genes targeted by the antibiotic and
the transfer of resistance determinantsborne on plasmids, bacteriophages,
transposons, and other mobile genetic
material (Figure 1 and Table 2). In general, this exchange
is accomplished through the processes of transduc-
tion (via bacteriophages), conjugation (via plasmids and
conjugative transposons), and transformation (via incor-
poration into the chromosome of chromosomal DNA,
plasmids, and other DNAs from dying organisms) (Levy
and Marshall, 2004). Although gene transfer among
organisms within the same genus is common, this proc-
ess has also been observed between very different
genera, including transfer between such evolutionarily
distant organisms as gram-positive and gram-negativebacteria (Courvalin, 1994). Plasmids contain genes for
resistance and many other traits; they replicate inde-
Table 1. General Characteristics of Multidrug-Resistant Organisms
Organism Common Infections Key Antibiotic Resistances
Drugs Considered for
Treatment of MDRa
P. aeruginosa Lung, wound -lactams, fluoroquinolones, aminoglycosides Colistin
Acinetobacterspp. Lung, wound,
bone, blood
-lactams, fluoroquinolones, aminoglycosides Colistin, tigecycline
E. coliand K.
pneumoniaebear-
ing extended-spec-trum -lactamases
Urinary, biliary,
gastrointestinal
tracts, lung, blood
-lactams, fluoroquinolones, aminoglycosides Colistin (for K. pneumoniae),
tigecycline
Vancomycin-resist-
ant enterococci
Blood, heart,
intra-abdominal
Vancomycin Quinupristin-dalfopristin,
linezolid, daptomycin
Methicillin-resistant
S. aureus
Skin and soft tis-
sue, respiratory
tract, blood
-lactams, fluoroquinolones, macrolides Quinupristin-dalfopristin,
daptomycin, linezolid,
tigecycline, vancomycin
Multidrug-resistant
S. pneumoniae
Ear, lung, blood,
cerebrospinal fluid
-lactams, macrolides, tetracyclines, co-trimoxazole Fluoroquinolones, tigecycline
Extensively
drug-resistant M.
tuberculosis
Lung Rifampin, isoniazid, and three of the following:
aminoglycosides, polypeptides, fluoroquinolones,
thioamides, cycloserine, or para-aminosalicylic acid
3rdline agents, drug
combinations
aAgents either have been approved for use by a regulatory agency (e.g., FDA), have shown usefulness in treating infection, or
exhibit promising in vitro activity and await a determination of clinical efficacy.
Figure 1. Acquisition of Antibiotic ResistanceBacteria can become antibiotic resistant (Abr)
by mutation of the target gene in the chromo-
some. They can acquire foreign genetic material
by incorporating free DNA segments into their
chromosome (transformation). Genes are alsotransferred following infection by bacteriophage
(transduction) and through plasmids and con-
jugative transposons during conjugation. The
general term transposable element has been
used to designate (1) an insertion sequence, (2)
composite (compound), complex, and conjuga-
tive transposon, (3) transposing bacteriophage,
or (4) integron.
-
7/24/2019 2007_Molecular Mechanisms of Antibacterial Multidrug Resistance
3/14
Cell 128, March 23, 2007 2007 Elsevier Inc. 1039
pendently of the host chromosome and can be distin-guished by their origins of replication. Multiple plasmids
can exist within a single bacterium, where their genes
add to the total genetics of the organism. Transposons
are mobile genetic elements that can exist on plasmids
or integrate into other transposons or the hosts chro-
mosome. In general, these pieces of DNA contain termi-
nal regions that participate in recombination and specify
a protein(s) (e.g., transposase or recombinase) that
facilitates incorporation into and from specific genomic
regions. Conjugative transposons are unique in having
qualities of plasmids and can facilitate the transfer of
endogenous plasmids from one organism to another.
Integrons contain collections of genes (gene cassettes)
that are generally classified according to the sequence
of the protein (integrase) that imparts the recombination
function (Mazel, 2006) (Figure 2A). They have the abil-
ity to integrate stably into regions of other DNAs where
they deliver, in a single exchange, multiple new genes,
particularly for drug resistance. The super-integron, one
which contains hundreds of gene cassettes ( represent-
ing about ?3% of the hosts genome), is distinct from
other integrons; it was first identified in Vibrio cholerae
(Mazel et al., 1998).
Although Alexander Fleming selected and described
mutants resistant to penicillin soon after he had dis-
covered the antibiotic, no one could have predicted the
speed with which bacteria would acquire the capacity
for dealing with multiple antibacterial agents. Plasmids,specifying a collection of individual antibiotic resistance
determinants (originally termed resistance factors, R
factors), were initially described as the vehicle used to
rapidly spread resistance traits among bacteria. Strains
of Shigellabearing the self-replicating and self-transfer-
able plasmids were easily selected and propagated dur-
ing a period of considerable sulfonamide use in Japan
after World War II (Watanabe, 1963). Antibiotics such
as streptomycin, chloramphenicol, and tetracycline
were subsequently introduced for the treatment of the
sulphonamide-resistant organisms. Shigellaand E. coli
strains bearing resistance to all four agents, however,
were recognized in 1955 (Watanabe, 1963). Now, more
than 60 years after antibiotics were first introduced into
clinical practice, the prescience of Fleming has come to
fruition as the infectious disease community has yet to
identify an antibiotic that has managed to circumvent the
development of resistance.
Resistance through Chromosomal Mutation
Fluoroquinolones
Virtually all clinically important fluoroquinolone resistance
can be attributed to mutations within the drugs targets,
DNA gyrase and topoisomerase IV (Drlica and Malik,
2003). These multisubunit complexes perform critical
ATP-dependent functions during DNA replication; each
is comprised of two subunits: GyrA and GyrB for DNA
Table 2. Mobile Genetic Elements
Genetic Element General Characteristics Resistance Determinant(s) Specified/Examplesa
Plasmid Variable size (1- >100 kb), conjugative, and
mobilizable
R factor: multiple resistances
Insertion sequence Small (5 kb), flanked by short terminal
inverted repeats, and specifies a
transposase and recombinase
Tn1 and Tn3: -lactamase
Tn7: Tmp, Str, Spc
Tn1546: glycopeptides
Conjugative transposon Promotes self-transfer Tn916: Tet and Mino
Tn1545: Tet, Mino, Ery, and Kan
Transposable bacteriophage A bacterial virus that can insert into the
chromosome
Mu
Other transposable elements Other than composite, complex, andconjugative transposons
Tn4: Amp, Str, Sul, and HgTn1691: Gen, Str, Sul, Cm, and Hg
Integron Facilitates acquisition and dissemination
of gene cassettes; specifies an integrase,
attachments sites, and transcriptional
elements to drive expression of multiple
resistance genes
Class 1: Multiple single determinants and MDR
efflux pump (Qac)b
Class 2: Tmp, Strp, Str, and Spc (Tn7)
Class 3: carbapenems
Class 4: Vibriospp. super-integron
See Figure 1.aAbbreviations: Amp, ampicillin; Bleo, bleomycin; Cm, chloramphenicol; Ery, erythromycin; Fus, fusidic acid; Gen, gentamicin;
Hg, mercury; Kan, kanamycin; Mino, minocycline; Spc, spectinomycin; Str, streptomycin; Strp, streptothricin; Sul, sulfonamide;
Tet, tetracycline; Tmp, trimethoprim; Van, vancomycin.bSee Figure 2.
-
7/24/2019 2007_Molecular Mechanisms of Antibacterial Multidrug Resistance
4/14
1040 Cell 128, March 23, 2007 2007 Elsevier Inc.
gyrase and ParC/GrlA and ParE/GrlB for topoisomerase
IV. The GyrA and ParC/GrlA proteins contain the DNA-
binding functions and are targeted by the fluoroquinolo-
nes (which are purely synthetic antibiotics), whereas
GyrB and ParE/GrlB perform the roles of ATP binding and
hydrolysis and are inhibited by coumarin antibiotics. In
gram-negative bacteria, mutations in DNA gyrase occur
first, whereas in gram-positive organisms mutations in
topoisomerase IV arise initially in the stepwise movement
to clinically relevant fluoroquinolone resistance.
Mutations that give rise to fluoroquinolone resistanceare predominantly found in the quinolone-resistance-
determining-region (QRDR) of GyrA and ParC/GrlA.
Mutations occur more frequently at particular amino
acids than at others. Multiple mutational events can be
selected in a stepwise manner so as to train resist-
ance in a bacterium; successive mutations have additive
effects. Isolates that are highly fluoroquinolone resist-
ant bear multiple QRDR mutations and generally other
mechanisms of fluoroquinolone resistance, such as
drug efflux (see below).
Rifamycins
Rifamycins are bactericidal drugs that arrest transcrip-
tion by interacting with RpoB, the subunit of RNA
polymerase (RNAP). Although combination regimens
that include rifampin or rifapentine and isoniazid, pyrazi-
namide, ethambutol, or streptomycin remain primary
choices for front-line therapy of M. tuberculosisinfection,
resistance through point mutations in the rifampin-bind-
ing region ofrpoBoccur at a frequency of 1 108and
are widespread. M. tuberculosisresistant to rifampin and
isoniazid at a minimum are defined as multidrug resist-
ant and impede successful therapy among patients in all
regions of the world (Sharma and Mohan, 2006).
Sulfonamide and Trimethoprim Antibiotics
The sulfonamides, the first antimicrobials developed forlarge-scale introduction into clinical practice (in 1935),
target dihydropteroate synthase. Their serendipitous
discovery (the antibacterial activity was seen initially in
vivo when the active compound was released as part of
a dye) pales only in comparison with that of Flemings
chance discovery of penicillin (Levy, 2002). Trimetho-
prim, introduced in 1968, inhibits dihydrofolate reductase
and was the last structurally unique antibiotic approved
prior to the release of linezolid in 2000. Mutations in the
gene specifying dihydropteroate synthase decrease the
enzymes affinity for the sulfonamides and have been
found in laboratory experiments using E. coliand Strep-
tococcus pneumoniaeand in clinical isolates of Campy-
lobacter jejuni and Haemophilus influenzae (Skold,
Figure 2. Transposable Elements that Confer Antibiotic Resistance(A) Organization of a class 1 integron.int1specifies the integrase (the recombination protein),att1is the primary recombination site, attCare the
59 base pair elements that are substrates of the integrase, and Pc is a common promoter that drives high-level expression of antibiotic resistance
determinants (Abr) such as those for aminoglycosides, -lactams, chloramphenicol, and trimethoprim inserted downstream of this element. qacE
andsul1specify genes for resistance to quaternary ammonium compounds (partially deleted) and sulfonamides, respectively; these two genes are
found specifically in class 1 integrons.
(B) The vanAgene cluster (adapted from Courvalin, 2006). Orf1 and Orf2 impart the transposition features. VanR (response regulator) and VanS
(histidine kinase) comprise a two-component system that regulates expression of vanHAXYZ. VanA (ligase) and VanH (dehydrogenase) are respon-
sible for the synthesis of the modified depsipeptide (D-Ala-D-Lac, see text) while VanX (D,D-dipeptidase) and VanY (D,D-carboxypeptidase) cleave
normal peptidoglycan substrates. VanZ is of unknown function. PRand PHare promoters that control expression of two separate transcriptionalunits, and the entire element is flanked by imperfect inverted repeats.
-
7/24/2019 2007_Molecular Mechanisms of Antibacterial Multidrug Resistance
5/14
Cell 128, March 23, 2007 2007 Elsevier Inc. 1041
2001). Mutations in the chromosomal gene specifying
dihydrofolate reductase can result in overexpression of
an enzyme with a reduced affinity for trimethoprim and
thereby offer very high-level trimethoprim resistance in
E. coliand H. influenzae(Skold, 2001).
Tetracycline, Aminoglycoside, and MLS Antibiotics
Agents within the tetracycline, aminoglycoside and mac-
rolide-lincosamide-streptogramin (MLS) classes of anti-
biotics target the ribosome to inhibit protein translation.
As a consequence, resistance via chromosomal muta-
tion is uncommon. Tetracyclines and aminoglycosides
interact with 16S rRNA (rrs) and the macrolide-lincosa-
mide-streptogramin (MLS) family binds to 23S rRNA
(rrl). In most bacteria, multiple rrs and rrloperons are
present and susceptibility mediated by any one of these
targets can be dominant, making resistance difficult to
attain without a mutation in all or a majority of the other
operons. However, in organisms with low rRNA (rrn) copynumbers, chromosomal mutations conferring resistance
have appeared. Tetracycline resistance via a point muta-
tion in Propionibacterium acnes (3 rRNA operons) and
Helicobacter pylori(2 copies ofrrn) (Gerrits et a l., 2002;
Ross et al., 1998) has been documented. Mutations in
rrs conferring resistance to amikacin and kanamycin
and alterations in small ribosome protein S12 (rpsL) or
rrs affecting streptomycin (all aminoglycoside drugs)
susceptibility in clinical M. tuberculosis (1 rrn operon)
have been described (Alangaden et al., 1998). The emer-
gence of resistance to erythromycin (a macrolide) dur-
ing therapy caused by mutant rrl in S. pneumoniae (4
copies of rrn) has been reported (Musher et al., 2002).Moreover, mutations in the large ribosome protein L4
(rplD) have also been shown to alter MLS susceptibil-
ity (Tait-Kamradt et al., 2000). The ketolides, recently
introduced into clinical practice, were designed to cir-
cumvent macrolide resistance; nonetheless decreased
susceptibility and resistance to telithromycin (a ketolide)
have been found in S. pneumoniaewith mutations inrrl,
rplD, and large ribosome protein L22 (rplV) (Hisanaga
et al., 2005). Mutations in L22 also affect quinupris-
tin-dalfopristin (streptogramins that act individually in
a bacteriostatic manner) susceptibility by affecting the
synergistic relationship important to the combinations
bactericidal mechanism of action (references in Hersh-berger et al., 2004).
Oxazolidinones
Linezolid (another protein synthesis inhibitor) was
approved recently as an agent to treat methicillin-resist-
ant S. aureus and vancomycin-resistant enterococci
infections. Its availability in both intravenous and oral
formulations makes it useful for treating infections in
both the hospital and community. Resistance to linezolid
in laboratory studies has been linked to point mutations
inrrlin S. aureusand E. faecalis. Now, clinical isolates of
Staphylococcus epidermidis, S. aureus, Streptococcus
oralis,Enterococcus faecium, and E. faecalisresistant to
linezolid have been documented and many of the strains
bear rrlmutations (Meka and Gold, 2004). As with the
fluoroquinolones, the level of resistance in S. aureus
increases with mutations in multiplerrlalleles (Wilson et
al., 2003) leading to clinically relevant resistance.
Lipopeptides
Daptomycin (a drug acting on bacterial membranes) was
approved by the FDA in 2003 and while it has been suc-
cessfully used for treating infections caused by methicil-
lin-resistant S. aureusand other gram-positive bacteria,
treatment failures have been noted (Hayden et al., 2005).
Laboratory experiments have established that mutations
in multiple chromosomal loci (i.e., mprF, yycG, rpoB,
andrpoC) affect daptomycin susceptibility (Friedman et
al., 2006). With regard to mprF, which specifies a lysyl-
phosphatidylglycerol synthetase, similar mutations have
been identified in nonsusceptible clinical isolates as well
(Friedman et al., 2006).
Genomic DuplicationsA mechanism for drug resistance common in eukaryotic
cells (e.g., certain mammalian cancers and parasites)
is gene amplification, which leads to overexpression
of multidrug transporters and drug targets (Albertson,
2006). Recently, a large tandem duplication of the E.
coli acrABlocus in a mutant isolated in the presence of
tetracycline was found to overexpress the AcrAB drug
efflux pump, producing an MDR phenotype (Nicoloff
et al., 2006). The mutants, however, were unstable and
reverted to the wild-type phenotype in the absence of
drug (Nicoloff et al., 2007). Genomic amplification has
also been shown to affect susceptibility to methicillin in
S. aureus(Matthews and Stewart, 1988). It is expectedthat examples of gene duplication as a mechanism of
resistance, not mutation, will increase in recognition
among bacterial isolates. The likely phenotype will be an
unstable form of resistance.
Acquired Resistance: Enzymatic Drug Modification
Enzymes that modify antibacterial drugs are divided into
two general classes: those such as -lactamases that
degrade antibiotics and others (including the macrolide
and aminoglycoside-modifying proteins) that perform
chemical transformations.
-Lactam Antibiotics
There are hundreds of -lactamases that have been dis-
covered and characterized, and a more complete over-
view of the individual enzymes within this protein family
is available elsewhere (Jacoby and Munoz-Price, 2005).
Most of these resistances are specified by genes located
on plasmids and transposons; others are chromosomal
and provide intrinsic resistance.
-lactamases are classified using schemes based
on function (the system of Bush-Jacoby-Medeiros)
or structure (Ambler classification) but in general are
broadly separated into enzymes with a serine active site
and those that require a metal ion cofactor (references
in Jacoby and Munoz-Price, 2005). The classification
scheme of Ambler divides the -lactamases into four
groups: class A, C, and D enzymes are proteins with a
-
7/24/2019 2007_Molecular Mechanisms of Antibacterial Multidrug Resistance
6/14
1042 Cell 128, March 23, 2007 2007 Elsevier Inc.
serine residue at their active sites and the class B pro-
teins are zinc-dependent metalloenzymes. Some class
A proteins function as extended-spectrum -lactamases
and as carbapenemases. The class B metallo-enzymes
that hydrolyze carbapenems are susceptible to inhibi-
tion by EDTA, whereas they are not susceptible to inhi-
bition by clavulanate (a -lactamase inhibitor). AmpC,
an inducible and usually chromosomal enzyme found in
many species of the Enterobacteriaceaeand P. aerugi-
nosa, is the prototype class C enzyme. Recent reports
have described plasmid-borne ampC genes that can
be transferred among E. coli, Klebsiella spp., and Sal-
monella spp. (references in (Jacoby and Munoz-Price,
2005). Class D enzymes have been found in only a few
species such as P. aeruginosa,Acinetobacterspp., and
Aeromonasspp.
Aminoglycosides
A large number of aminoglycoside-modif ying enzymesspecified by genes on transferable elements are wide-
spread in clinically relevant organisms (Davies and
Wright, 1997). Here, resistance is accomplished with
proteins that N-acetylate (acetyltransferases), phos-
phorylate (phosphotransferases), and adenylate (nucle-
otidyltransferases) the aminoglycosides. The acetyl-
transferases are capable of modifying tobramycin,
gentamicin, netilmicin, and amikacin; the nucleotidyl-
transferase proteins alter the activity of tobramycin; and
the phosphotransferases affect amikacin susceptibility.
Many of the aminoglycoside-modifying enzymes are
found on integrons and other mobile genetic elements
where they associate with additional resistance determi-nants. For example, three acetyltransferase genes were
found on a class 1 integron from P. aeruginosathat also
specified resistance to carbapenems and sulfonamides
(Poirel et al., 2001).
MLS Antibiotics
There are a number of inactivating enzymes that act on
the MLS antibiotics. The genes encode esterases, hydro-
lases, glycosylases, phosphotransferases, nucleotidyl-
transferases, and acetyltransferases and are found less
frequently than efflux and ribosome-modifying genes in
clinical isolates (Weisblum, 1998). Esterases act on 14-
(e.g., erythromycin) and 15- (e.g., azithromycin) mem-
bered macrolides; the hydrolases affect streptogramin
B drugs; the acetyltransferases inactivate streptogramin
A antibiotics; and the nucleotidyltransferases confer
resistance to the lincosamides (e.g., clindamycin). Phos-
photransferases modify 14-, 15-, and 16-membered
macrolides with varying specificities and telithromycin
(Matsuoka and Sasaki, 2004).
Chloramphenicol
Acetyltransferases that inactivate chloramphenicol (a
generally bacteriostatic inhibitor of protein synthesis)
are the most common resistance mechanisms for this
antibiotic and are separated into type A and B proteins
(Schwarz et al., 2004). Both types of enzymes func-
tion as homotrimers but are unrelated based on amino
acid sequence analyses. The type B enzymes are also
termed xenobiotic acetyltransferases and appear to
share an evolutionary lineage that includes some strep-
togramin-inactivating enzymes found in the enterococci
and staphylococci. Constitutive expression as well as
translational attenuation underlie the regulation of type
A and B proteins.
Tetracyclines
Recently, a flavin-dependent monooxygenase, desig-
nated tet(X), originally identified in Bacteroides fragilis,
that acts on older tetracyclines (such as tetracycline,
oxytetracycline, and chlortetracycline) as well as newer
compounds (such as doxycycline, minocycline, and
tigecycline) has been characterized (Moore et al., 2005).
The enzyme catalyzes regioselective hydroxylation to
inactivate its substrates, but the modification products
produced by TetX are not stable at physiological pH.
The tet(X) determinant has not yet been found in com-
mon, clinically relevant tetracycline-resistant isolates,although a related tet(X)-like gene in P. aeruginosahas
been reported (A. Nakamura et al., 2002, paper pre-
sented at Interscience Conference on Antimicrobial
Agents and Chemotherapy, San Diego, CA).
Altered, Substituted, and Protected Drug Targets
-Lactam Antibiotics
The first penicillin-resistant S. aureus identified in the
mid-1940s expressed a -lactamase (termed PCI) that
afforded resistance. A penicillin derivative, methicillin,
that was resistant to this -lactamase was subsequently
introduced in 1959 to treat the penicillin-resistant iso-
lates, but methicillin-resistant S. aureuswere identifiedshortly thereafter (Barber, 1961). Here, the -lactam
resistance was linked to the acquisition of a gene for
an altered PBP. Resistance in the staphylococci (Fuda
et al., 2005) and streptococci (Jacobs, 1999) frequently
occurs following the acquisition of genes for PBPs that
are not sensitive to -lactam inhibition. The altered PBP
of methicillin-resistant S. aureus, PBP2a, is specified
by mecA and is transported on a mobile genetic ele-
ment called the staphylococcal cassette chromosome
(SCCmec) (Fuda et al., 2005). In addition to mecA,
SCCmec contains the mecR1-mecIregulatory loci and
encodes the enzymes that are involved in site-specific
recombination.
Under normal circumstances, S. aureus uses multi-
ple PBPs during cell wall biosynthesis. One, PBP2, is a
bifunctional enzyme with transpeptidase and transgly-
cosylase activities. When methicillin-resistant S. aureus
is exposed to methicillin, PBP2 functions as the transg-
lycosylase whereas PBP2a contributes the transpepti-
dase activity in order to confer resistance to nearly all
-lactam antibiotics. Removal of the transglycosylase
function of PBP2 imparts -lactam susceptibility and
demonstrates the importance of both attributes of the
enzyme (reviewed in Fuda et al., 2005).
The new strains of methicillin-resistant S. aureusthat
have emerged recently outside of hospitals in the com-
munity share some features with hospital-associated
-
7/24/2019 2007_Molecular Mechanisms of Antibacterial Multidrug Resistance
7/14
Cell 128, March 23, 2007 2007 Elsevier Inc. 1043
strains (Naimi et al., 2003). These include the nearly
complete -lactam resistance phenotype and the abil-
ity to cause a range of serious life-threatening infec-
tions. Although many community-associated strains are
multidrug susceptible (except to -lactam antibiotics),
the genome of an epidemic clone distributed through-
out the US (USA300) contains chromosomal mutations
that confer fluoroquinolone resistance and plasmids that
mediate resistance to tetracycline, erythromycin, clin-
damycin, streptogramin B agents, and mupirocin (Diep
et al., 2006).
Glycopeptides
Glycopeptides (including vancomycin and teicoplanin)
interact with peptidoglycan precursors to exert bacteri-
cidal activity. Resistance to the glycopeptides in gram-
positive cocci is another illustration of an altered drug
target (Courvalin, 2006). In the enterococci, acquired
glycopeptide resistance is attributable to VanA, B, D,E, and G phenotypes whereas VanC affords intrinsic
resistance. VanA and VanD provide resistance to both
vancomycin and teicoplanin, whereas the others provide
resistance to vancomycin alone. The resistance phe-
notype is accomplished using multiple proteins speci-
fied in gene clusters and each result in the production
of a modified peptidoglycan. Of the many drug-resist-
ance determinants currently known, the determinant for
glycopeptide resistance is probably the most complex
(Figure 2B).
The activity of many enzymes specified by the gene
cluster are involved in conferring glycopeptide resist-
ance (Figure 2B). Either a racemase or dehydrogenaseresults in the production of serine (VanC, E, or G) or lac-
tate from pyruvate (VanA, B, or D), which a ligase uses
to form a C-terminal D-Ala-D-Ser or D-Ala-D-Lac in the
altered peptidoglycan. A two-component regulatory
system controls expression of the biosynthetic machin-
ery. Although the glycopeptides have a lower affinity
for the D-Ala-D-Ser or D-Ala-D-Lac substrates, they
can still bind and inhibit peptidoglycan biosynthesis if
an intact D-Ala target remains. Two additional enzymes
(or a single bi-functional protein for VanC) complete the
phenotype by removing the antibiotics normal target:
a dipeptidase cleaves the C-terminal D-Ala-D-Ala and
a carboxypeptidase provides a redundant function of
removing the terminal D-Ala in the absence of, or under
circumstances when, the dipeptidase is less active.
Recently, the enterococcal vanA gene cluster has
made its way into methicillin-resistant S. aureus, impart-
ing full-blown vancomycin resistance on this prominent
pathogen (Weigel et al., 2003), although this molecular
event had been demonstrated in laboratory experiments
years before (Noble et al., 1992). Vancomycin-resistant
S. aureushave now been identified in patients from three
different US locales, Michigan, Pennsylvania, and New
York. In all instances, the resistance determinant resides
on a plasmid-specified transposon, Tn1546. Vanco-
mycin-resistant E. faecalisand methicillin-resistant S.
aureus were also obtained from the Michigan patient
that bore vancomycin-resistant S. aureusand each con-
tained plasmids that were identical, except for the pres-
ence of Tn1546in the isolate of vancomycin-resistant E.
faecalis. It is assumed that the plasmid from E. faecalis
was the vehicle for vanA entry into S. aureus and that
the vanAgene cluster was subsequently transferred by
means of transposition into the S. aureusplasmid.
Tetracyclines and MLS Antibiotics
The most prevalent forms of resistance to the tetracy-
clines in the clinic are drug efflux (see below) and ribos-
ome protection. Determinants of ribosome protection
(Connell et al., 2003) have sequence similarity to bacte-
rial elongation factors (EF-G an EF-Tu). They also pos-
sess GTPase activity and function to facilitate release
of tetracycline from the ribosome in an energy-depend-
ent manner (Connell et al., 2003). Bacteria that exhibit
resistance to minocycline generally contain a ribosome
protection determinant.In Megasphaera elsdenii (a rumen bacterium), a
mosaic gene containing two unique ribosome protec-
tion determinants (TetO and TetW) has been described
(Stanton and Humphrey, 2003). Moreover, the tet(P)
determinant of Clostridium perfringens specifies both
ribosome protection and efflux mechanisms in an over-
lapping genetic unit (Sloan et al., 1994).
Binding of drugs within the MLSK (macrolide-lin-
cosamide-streptogramin-ketolide) family to 23S rRNA
is affected by erm(erythromycin resistance methylase
or erythromycin ribosome methylation) specified gene
products (Zhanel et al., 2001). These mechanisms repre-
sent the predominant macrolide resistance mechanismsin Europe and South Africa. Currently, there are 34 dif-
ferent classes of Erm proteins (http://faculty.washington.
edu/marilynr/), and each functions by methylating a
single adenine residue of the E. coli23S rRNA (at posi-
tion A2058). Methylation results in the MLSBphenotype,
which encompasses resistance to 14-, 15-, and 16-mem-
bered macrolides, lincosamides, and streptogramin B
drugs. Some of the resistance determinants are induced
following exposure to MLS drugs, whereas others are
constitutively synthesized. In the staphylococci, agents
like erythromycin and azithromycin induce ermexpres-
sion, but 16-membered macrolides do not (Zhanel et
al., 2001). Constitutive ermexpression in some clinical
and laboratory strains confers telithromycin resistance
(Hisanaga et al., 2005).
Sulfonamide and Trimethoprim Antibiotics
The activities of the sulfonamides and trimethoprim are
also affected by acquired genes specifying enzymes
that are insensitive to drug inhibition (Skold, 2001). sul1
andsul2are the main determinants of clinical resistance
to sulfonamide, whereassul3was found to be prevalent
in farm animals (Perreten and Boerlin, 2003). In contrast,
more than 20 trimethoprim resistance determinants
(numbered chronologically from dfr1) are documented.
The genes specifying the sulfonamide-insensitive dihy-
dropteroate synthases are present on class 1 integrons
(sul1) or plasmids (sul2), whereas dfrvariants (with dfr1
-
7/24/2019 2007_Molecular Mechanisms of Antibacterial Multidrug Resistance
8/14
1044 Cell 128, March 23, 2007 2007 Elsevier Inc.
being the most common in gram-negative bacteria)
move from organism to organism on class 1 and 2 inte-
grons. dfr1 is present on the Tn7transposon, thereby
facilitating its integration into the E. colichromosome.
Fluoroquinolones
The plasmid-specified qnr determinants provide an
unusual mechanism of decreased fluoroquinolone sus-
ceptibility. Variants of the qnrelement, first identified in
K. pneumoniae (Martinez-Martinez et al., 1998), have
been found in E. coli, Enterobacter cloacae, Providencia
stuarti i,Citrobacter freundii, C. koseri, Shigella flexneri
2b, and non-Typhi Salmonella enterica. Qnr belongs to
the pentapeptide repeat family of proteins and medi-ates resistance presumably by protecting DNA gyrase
and topoisomerase IV from the inhibitory action of the
fluoroquinolones (Tran and Jacoby, 2002). Another fluo-
roquinolone resistance mechanism, MfpA, has been
found in M. tuberculosis, which contains a single type II
topoisomerase (Hegde et al., 2005). It, too, is a member
of the pentapeptide repeat family of proteins but also
acts as an inhibitor of DNA gyrase from M. tuberculosis
by virtue of its ability to mimic the structure of B-form
DNA. The interaction of MfpA with DNA gyrase likely
interferes with the inhibitory action of agents like cipro-
floxacin. By themselves, Qnr and MfpA confer only low
levels of fluoroquinolone resistance but can augmentresistance when coupled with other mechanisms (see
below). Moreover, selection for fluoroquinolone resist-
ance may be favored in organisms bearing Qnr or MfpA
if they survive the presence of low fluoroquinolone con-
centrations.
Efflux Systems, Porins, and Altered Membranes
Efflux as a mechanism of antibiotic resistance was
originally described for tetracyclines (McMurry et al.,
1980) and is now well-appreciated and commonly
found. Most drug efflux proteins belong to five dis-
tinct protein families: the resistance-nodulation-cell
division (RND), major facilitator (MF), staphylococcal/
small multidrug resistance (SMR), ATP-binding cas-
sette (ABC), and multidrug and toxic compound extru-
sion (MATE) families (Li and Nikaido, 2004). Efflux by
proteins in the RND, SMR, MF, and MATE families is
driven by proton (and sodium) motive force and is
therefore called secondary transport (Figure 3). ATP
hydrolysis drives efflux in the primary (ABC) transport-
ers (Figure 3 ). Efflux proteins also come in two general
types. Some, like the tetracycline (Tet) (Levy, 1992)
and macrolide (Mef ) (Zhanel et al., 2001) transporters,
are single component efflux systems that have narrow
substrate profiles, acting on a few agents or multiple
agents within the same drug class. Others, such as
the RND family members, require two additional pro-teins to confer resistance but are capable of binding
multiple structurally unrelated compounds and confer
broad resistance phenotypes (Li and Nikaido, 2004).
The organizations of the RND-based efflux systems,
which are found in a number of gram-negative bacte-
ria, allow them to transport drugs from the cy toplasm
and across the inner and outer membranes of the cell
envelope (Figure 3).
Tetracyclines
There are now more than 20 different tetracycline efflux
proteins that have been allocated to six different groups
(Sapunaric et al., 2005). The proteins contain either 12-
(e.g., TetA-E in gram-negative bacteria) or 14- (e.g., TetKand TetL in gram-positive organisms) putative trans-
membrane-spanning segments. Expression of proteins
within group 1 is controlled by a transcription repressor
such as TetR. The antibiotic inactivates the repressor
and allows expression of the tetracycline efflux systems.
Alternatively, synthesis of TetK and TetL is inducible by
tetracyclines through mechanisms that involve trans-
lation attenuation and reinitiation, respectively. Also,
the mechanism of tetracycline efflux is not conserved
among all members; some, such as Tet(B), engage in
electroneutral reactions (that is, one tetracycline-Mg
chelate exits the cell as a proton enters whereas others,
such as TetL, accomplish efflux in an electrogenic man-
ner [references in Sapunaric et al., 2005]).
Figure 3. Antibiotic Efflux SystemsFor simplicity, the illustration represents a
gram-negative bacterium. Some substrates
of AcrAB (e.g., aminoglycosides) are presum-
ably recognized by a portion of AcrB that
faces the cytoplasm while others (e.g., tetra-cycline) migrate thorough the inner membrane
to an AcrB-binding pocket(s) (Murakami and
Yamaguchi, 2003). The E. coli AcrAB-TolC
and Tet efflux systems are secondary trans-
porters that use proton motive force (H+) while
NorM (Vibriospp.) is a secondary transporter
that exploits a gradient of sodium ions (Na+)
to drive efflux (Li and Nikaido, 2004). MacAB
is a primary transporter that uses the energy
derived from ATP hydrolysis to drive efflux (Li
and Nikaido, 2004). Single-component sys-
tems (e.g., NorM, Tet) transport substrates
across the inner membrane where they then
diffuse through porins (e.g., OmpF) or the
outer membrane into the extracellular medium (Li and Nikaido, 2004). Multicomponent systems like AcrAB-TolC and MacAB-TolC direct the
transport of substrates to the extracellular medium. Abbreviations: LPS, lipopolysaccharide; Ab, antibiotic; Tc, tetracycline; Mac, macrolide.
-
7/24/2019 2007_Molecular Mechanisms of Antibacterial Multidrug Resistance
9/14
Cell 128, March 23, 2007 2007 Elsevier Inc. 1045
MLS Antibiotics
MsrA (macrolide-streptogramin resistance) is an ABC
efflux protein that produces resistance to 14- and 15-
membered macrolides and streptogramin B antibiotics in
the streptococci and staphylococci, but susceptibility to
clindamycin is not affected by this system (Li and Nikaido,
2004). In the staphylococci, relatives of MsrA (VgaA and
VgaB) are specified on plasmids. VgaA confers resist-
ance to streptogramin A antibiotics and lincosamides,
and VgaB affects pristinamycin (a mixture of strepto-
gramin A and B antibiotics) susceptibility (Chesneau
et al., 2005). Alternatively, the Mef efflux transporters,
which are the predominant macrolide resistance proteins
in the United States, effectively handle 14- and 15-mem-
bered macrolides in the streptococci, but strains that
express the proteins are susceptible to 16-membered
macrolides, lincosamides, and streptogramin B drugs
(Li and Nikaido, 2004). E. faecalis are naturally resist-ant to quinupristin-dalfopristin and this characteristic
is attributed to the expression of lsa, which specifies a
streptogramin efflux protein belonging to the ABC family,
as mutational inactivation of Lsa confers susceptibility
to quinupristin-dalfopristin in this organism (references
in Hershberger et al., 2004). This scenario has been
observed with other (MDR) efflux proteins where removal
of an efflux system renders an organism susceptible to
antibiotics, even in the presence of chromosomal muta-
tions that reduce binding of the drug to its target and
would otherwise confer clinical resistance (Lomovskaya
et al., 1999; Oethinger et al., 2000).
PhenicolsChloramphenicol and florfenicol are broad-spec-
trum agents that have utility in human clinical practice
(chloramphenicol) and animal husbandry (florfenicol).
Because of toxicity (aplastic anemia) seen with the
former, the current use of chloramphenicol has been
limited to the treatment of some severe life-threatening
infections such as bacterial meningitis in patients with
allergies to the penicillins. Efflux proteins specific for the
phenicols have been described in a number of clinically
important bacteria and are classified into eight different
groups (E-1 through E-8) (Schwarz et al., 2004). In gen-
eral, these proteins provide levels of resistance greater
than that of the multidrug efflux proteins described below,
and members of groups E-3 and E-4 confer resistance
to both phenicols. For some chloramphenicol-specific
efflux proteins like CmlA, expression of the resistance
determinant is mediated through an inducible mecha-
nism of translational attenuation similar to that used for
particular chloramphenicol acetyltransferase resistance
genes (see above).
Multidrug Resistance Efflux Systems
Historically, the gram-negative cell envelope was thought
to affect antibiotic susceptibility by greatly restricting
drug penetration (Li and Nikaido, 2004). Contemporary
studies, however, have shown that most antibacterial
agents effectively penetrate gram-negative organisms
(Li and Nikaido, 2004) but fail to reach intracellular tar-
gets because of efflux (Levy, 1992). In gram-negative
bacteria, including E. coliand nonfermenting organisms
such as P. aeruginosa,Acinetobacterspp., Stenotropho-
monas maltophilia, and Burkholderia cepacia, intrinsic
resistance is attributed to the expression of the RND
efflux system(s) (Figure 3). This mechanism is an effec-
tive means for dealing with different antibiotic classes
using a single resistance determinant. The natural func-
tion of the E. coliAcrAB efflux system is thought to have
evolved to protect the cell from the inhibitory activity
of toxic substances such as bile salts (Li and Nikaido,
2004). Other related systems function similarly in Neis-
seria gonorrhoeae (Hagman et al., 1995) and export
molecules involved in quorum sensing in P. aeruginosa
(Pesci et al., 1999).
Tigecycline, which gained FDA approval in 2005, has
poor activity against P. aeruginosa, Proteus mirabilis,
Morganella morganii, and Klebsiella pneumonia; this isattributed to RND efflux systems (references in Stein
and Craig, 2006). Elimination of an AcrA ortholog in M.
morganii, MexXY-OprM in P. aeruginosa, and an AcrB
ortholog in P. mirabilisincreased susceptibility to tige-
cycline 16- to 133-fold (references in Stein and Craig,
2006), whereas deletion in E. coliof AcrAB and AcrEF
had a more modest (4-fold) effect (Hirata et al., 2004).
Bacillus subtilis Bmr (Bacillus multidrug resistance)
(Neyfakh et al., 1991) and S. aureus Qac (quaternary
ammonium compound) (Tennent et al., 1989) are two
MDR efflux proteins (members of the MF superfamily)
that were first characterized in gram-positive cells. Like
many members of the RND family in gram-negativebacteria, Bmr is constitutively expressed and therefore
engenders intrinsic resistance to chloramphenicol and
fluoroquinolones. Another MDR efflux pump from B.
subtilis, Blt, includes spermidine among its list of sub-
strates. It is now thought that the natural function of Blt
is to facilitate the removal of polyamines from the cell
(Woolridge et al., 1997). The staphylococcal Qac sys-
tems provide resistance to antiseptics and disinfectants
(e.g., quaternary ammonium compounds, chlorhexidine,
and diamidines). Unlike most other MDR efflux proteins,
these are specified on plasmids, a feature that facilitates
their dissemination.
Permeability
As alluded to previously, the outer membrane of the
gram-negative cell envelope is a barrier to both hydro-
phobic and hydrophilic compounds. In order to circum-
vent this permeability barrier, these organisms have
evolved porin proteins (e.g., OmpF in E. coliand OprD
in P. aeruginosa) that function as nonspecific entry
and exit points for antibiotics and other small-molecule
organic chemicals. Imipenem (and to a lesser extent
meropenem) and basic amino acids pass through OprD;
mutations that decrease expression of the porin con-
tribute to clinical imipenem resistance. Moreover, the
expression of OprD and the MexEF-OprM efflux system
is coregulated (Ochs et al., 1999), and in this manner
resistance to the carbapenems and other substrates of
-
7/24/2019 2007_Molecular Mechanisms of Antibacterial Multidrug Resistance
10/14
1046 Cell 128, March 23, 2007 2007 Elsevier Inc.
MexEF-OprM (Table 3) can develop in mutants where
the expression of OprD and the efflux pump has been
altered.
In 1996, Hiramatsu and colleagues identified clinical S.
aureus isolates in Japan that exhibited an intermediate
level of resistance to vancomycin (the vancomycin inter-
mediate-resistant S. aureus [VISA] strains) (Hiramatsu
et al., 1997), and shortly thereafter additional organisms
with a glycopeptide-insensitive phenotype appeared in
the United States (the GISA isolates). A prominent feature
of the VISA isolates is the presence of a thickened cell
wall. It is presumed that this property traps vancomycinand prevents the antibiotic from reaching its target (Cui
et al., 2006a). More recently, the same group performed
a preliminary characterization of the VISA isolates and
found reduced susceptibility to daptomycin (Cui et al.,
2006b). Although additional molecular and biochemical
characterizations are certainly needed, the findings do
carry a cautionary note.
Mutations that offer resistance to polymyxin B in P.
aeruginosa presumably involve changes in the bacte-
rial cell envelope that do not involve porins (references
in Falagas and Kasiakou, 2005). In Salmonella enterica
serovar Typhimurium, PmrAB (a two-component regula-
tory system) regulates resistance to polymyxin by modi-
fying lipopolysaccharide and lipid A (references in Fala-
gas and Kasiakou, 2005). The RosAB efflux system of
Yersinia enterocoliticaalso af fects susceptibility to poly-
myxin B (references in Falagas and Kasiakou, 2005).
Regulatory Genes in Multidrug Resistance
Chromosomal loci specifying global regulatory proteins
that control MDR have been characterized in bacteria
including E. coli, K. pneumoniae, and other Enterobac-
teriaceae, Neisseria gonorrhoeae, Bacillus subtilis, and
S. aureus (Grkovic et al., 2002). In most instances, the
regulatory proteins act as activators or repressors oftranscription and contain either a single DNA-binding
domain or two separate regions responsible for DNA
binding and interacting with small-molecule substrates
(Schumacher and Brennan, 2002). In the case of E. coli
MarA, the protein regulates the expression of a large
number of other chromosome genes (the Mar regulon),
including those specifying an MDR efflux pump and other
proteins (e.g., porins) that mediate antibiotic susceptibil-
ity (Barbosa and Levy, 2000). In a clinical K. pneumoniae
isolate exhibiting reduced susceptibility to tigecycline
and an MDR phenotype, transposon mutagenesis was
used to find that inactivation of ramA, which specifies a
MarA ortholog, produced a 16-fold increase in suscep-
Table 3. MDR Efflux Systems of Clinically Important Bacteria
Bacterial Organism Efflux System(s) Representative Antibiotic Resistancea
P. aeruginosa MExAB-OprM BLA and FQ
MexCD-OprJ 4thgen ceph
MexEF-OprN FQ, Cm, Tmp, and Tri
MexHI-OprD EtBr, Nor, and Acr
MexJK-OprM Cip, Tet, Ery, and Tri
MexVW-OprM FQ, Cm, Tet, Ery, EtBr, and Acr
MexXY-OprM AG and Tig
A. baumannii AdeABC AG, FQ, TET, Ctx, Cm, Ery, and Tmp
S. maltophilia SmeABC AG, BLA, and FQ
SmeDEF MC, TET, FQ, CAR, Cm, and Ery
B. cepacia CeoAB-OpcM Cm, Cip, and Tmp
B. pseudomallei AmrAB-AprA MAC and AG
E. coli AcrAB-TolC FQ, BLA, TET, Cm, Acr, Tri
K. pneumoniae AcrAB-TolC FQ, BLA, TET, and Cm
S. aureus MepA Tig, Mino, Tet, Cip, Nor EtBr, and TPP
E. faecalis EmeA Nor, EtBr, Clind, Ery, and Nov
Lsa Clind and QD
S. pneumoniae PmrA FQ, Acr, and EtBr
aAbbreviations: 4th gen ceph, fourth-generation cephalosporins; Acr, acriflavin; AG, aminoglycosides; BLA, -lactams; CAR,
carbapenems; Cip, ciprofloxacin; Clind, clindamycin; Cm, chloramphenicol; Ctx, cefotaxime; Ery, erythromycin; EtBr, ethidium
bromide; FQ, fluoroquinolones; MC, macrolides; Mino, minocycline; Nor, norfloxacin; Nov, novobiocin; QD, quinupristin-dalfo-
pristin; TET, tetracycline family; Tet, tetracycline; Tig, tigecycline; Tmp, trimethoprim; TPP, tetraphenylphosphonium bromide;
Tri, triclosan.
-
7/24/2019 2007_Molecular Mechanisms of Antibacterial Multidrug Resistance
11/14
Cell 128, March 23, 2007 2007 Elsevier Inc. 1047
tibility to tigecycline and abrogated the MDR phenotype
(Ruzin et al., 2005). Although other clinical isolates that
overexpress MarA or a MarA ortholog and exhibit a MDR
phenotype have been described previously (Schneiders
et al., 2003 and references therein), the studies that
involved K. pneumoniae offer further proof that chro-
mosomal transcription factors that regulate MDR efflux
systems can produce clinical drug resistance.
Single Determinants of Multidrug Resistance
Except for the case of MDR efflux pumps, a single resist-
ance mechanism commonly affords protection against
one antibiotic or, at the most, drugs within the same
general class, e.g., PBP2 of MRSA. Still, resistance
determinants that specify erythromycin (Erm) methyl-
transferases in a variety of pathogens are single pro-
teins that give rise to macrolide, lincosamide, and type
B streptogramin resistance: structurally unique agentsthat share a common target and mechanism of action
(Roberts, 2004).
A mutant aminoglycoside acetyltransferase (specified
byaac(6 )- Ib-cr) that gave rise to aminoglycoside (ami-
kacin, kanamycin, and tobramycin) and fluoroquinolone
(ciprofloxacin and norfloxacin) resistance was identified
recently (Robicsek et al., 2006). Although the level of
resistance conferred was low,aac(6 )- Ib-crwas located
on a plasmid bearing another unusual mechanism (the
qnrdeterminant [see above]) of fluoroquinolone resist-
ance, and the two determinants together functioned in
an additive manner to yield clinical levels of fluoroqui-
nolone resistance.Reduced susceptibility to the macrolides, chloram-
phenicol, and linezolid in clinical S. pneumoniae iso-
lates has been attributed to mutations in large ribosomal
protein (L4) (Wolter et al., 2005). Although mutations in
the genes that specify 23S rRNA, L4, or L22 commonly
lead to macrolide resistance, susceptibility to chloram-
phenicol frequently occurs following the acquisition of
a modifying enzyme (see above). For linezolid, previous
resistance in the enterococci and S. aureuswas attrib-
uted solely to mutations in the locus that encodes 23S
rRNA (see above).
What the Past Can Teach Us about the FutureMore than half a century has passed since the first anti-
biotics were introduced commercially. It did not take
long for microbes to develop drug fastness to the
original magic bullets, and widespread use of many
antibacterial drugs provides ideal conditions for the
spread of MDR organisms. Many early researchers were
expertly skilled in identifying the means used to evade
the inhibitory effects of these drugsfrom investigators
such as Esther and Joshua Lederberg, who character-
ized the random nature of mutational events conferring
resistance to streptomycin, to others such as Tsutomu
Watanabe, who surmised that mutation alone would not
be sufficient for explaining the MDR phenotype. Resist-
ance transfer factors (RTFs), later called resistance (R)
factors and then plasmids, would provide the basis for
multiple drug resistance caused by infective heredity
(reviewed in Watanabe, 1963).
For bacteria, there is more than one way to evade a
drug class, and organisms unresponsive to all antimi-
crobial agents, even ones not encountered previously,
are now familiar. Microbes have means for collecting
single resistance traits. Theoretically, one needs only a
single (broad-spectrum) agent that ef fectively deals with
resistances affecting just one class of drugs. The MDR
efflux pumps that render multiple classes ineffective,
however, have greatly confounded efforts in finding new
agents.
Multidrug resistance is a worldwide problem that does
not obey international borders and can indiscriminately
affect members of all socioeconomic classes. A slew of
epidemiological studies have documented the clonal,
worldwide spread of infectious disease entities suchas MDR S. pneumoniae from specific regions of the
world. Also, the use of antibiotics in animal husbandry
has been shown to foster human colonization with drug-
resistant microbes (Levy et al., 1976). There are com-
pelling data that link use of agents like virginiamycin (a
streptogramin) and avoparcin (a glycopeptide) in animal
husbandry to clinical drug-resistant strains.
Bacteria adopt intricate strategies to avoid the lethal
effects of antibiotics. Awareness of these mechanisms
of resistance can help in the design of new drugs. As
we face this critical problem, we need to be aware of
the fluidity of the microbial genome and the relative ease
with which resistance can emerge by mutation or geneacquisition. Recognizing the potential for the emer-
gence of resistance should garner newfound respect for
the discovery of new agents so urgently needed to cure
infectious diseases.
ACKNOWLEDGMENTS
S.B.L. is supported by the US Public Health Service (National Insti-
tutes of Health). The authors thank Bonnie Marshall for her help in the
preparation of the manuscript and figures.
REFERENCES
Alangaden, G.J., Kreiswirth, B.N., Aouad, A., Khetarpal, M., Igno,
F.R., Moghazeh, S.L., Manavathu, E.K., and Lerner, S.A. (1998).
Mechanism of resistance to amikacin and kanamycin in Mycobacte-
rium tubercu losis. Antimicrob. Agents Chemother.42, 12951297.
Albertson, D.G. (2006). Gene amplification in cancer. Trends Genet .
22, 447455.
Alekshun, M.N., and Levy, S.B. (2000) . Bacter ial drug resistance:
response to survival threats. In Bacterial Stress Responses, G.
Storz, and R. Hengge-Aronis, eds. (Washington, D.C.: ASM Press),
pp. 323-366.
Barber, M. (1947). Staphylococci infection due to penicillin-resis-
tant strains. BMJ2, 863865.
Barber, M. (1961). Methicillin-resistant staphylococci. J. Clin.
Pathol. 14, 385393.
Barbosa, T.M., and Levy, S.B. (2000). Differential expression of over
-
7/24/2019 2007_Molecular Mechanisms of Antibacterial Multidrug Resistance
12/14
1048 Cell 128, March 23, 2007 2007 Elsevier Inc.
60 chromosomal genes in Escherichia coliby constitutive expres-
sion of MarA. J. Bacteriol. 182, 34673474.
Chesneau, O., Ligeret, H., Hosan-Aghaie, N., Morvan, A., and Das-
sa, E. (2005). Molecular analysis of resistance to streptogramin A
compounds conferred by the Vga proteins of staphylococci. Anti-microb. Agents Chemother.49, 973980.
Connell, S.R., Tracz, D.M., Nierhaus, K.H., and Taylor, D.E. (2003).
Ribosomal protection proteins and their mechanism of tetracycline
resistance. Antimicrob. Agents Chemother.47, 36753681.
Cook, M., Molto, E., and Anderson, C. (1989). Fluorochrome label-
ing in Roman period skeletons from Dakhleh Oasis, Egypt. Am. J.
Phys. Anthropol. 80, 137143.
Courvalin, P. (1994). Transfer of antibiotic resistance genes be-
tween gram-positive and gram-negative bacteria. Antimicrob.
Agents Chemother. 38, 14471451.
Courvalin, P. (2006). Vancomycin resistance in gram-positive cocci.
Clin. Infect. Dis.42(Suppl 1), S25S34.
Cui, L., Iwamoto, A., Lian, J.Q., Neoh, H.M., Maruyama, T., Hori-kawa, Y., and Hiramatsu, K. (2006a) . Novel mechanism of antibiotic
resistance originating in vancomycin-intermediate Staphylococcus
aureus.Antimicrob. Agents Chemother. 50, 428438.
Cui, L., Tominaga, E., Neoh, H.M., and Hiramatsu, K. ( 2006b). Cor-
relation between reduced daptomycin susceptibility and vancomy-
cin resistance in vancomycin-intermediate Staphylococcus aureus.
Antimicrob. Agents Chemother. 50, 10791082.
Davies, J., and Wright, G.D. (1997). Bacterial resistance to amino-
glycoside antibiotics. Trends Microbiol. 5, 234240.
DCosta, V.M., McGrann, K.M., Hughes, D.W., and Wright, G.D.
(2006). Sampling the antibiotic resistome. Science 311, 374377.
Diep, B.A., Gill, S.R., Chang, R.F., Phan, T.H., Chen, J.H., Davidson,
M.G., Lin, F., Lin, J., Carleton, H.A., Mongodin, E.F., et al. (2006).
Complete genome sequence of USA300, an epidemic clone ofcommunity-acquired methicillin-resistant Staphylococcus aureus.
Lancet 367, 731739.
Drlica, K., and Malik, M. (2003). Fluoroquinolones: act ion and resis-
tance. Curr. Top. Med. Chem. 3, 249282.
Falagas, M.E., and Kasiakou, S.K. (2005). Colistin: the revival of
polymyxins for the management of multidrug-resistant gram-nega-
tive bacterial infections. Clin. Infect. Dis. 40, 13331341.
Friedman, L., Alder, J.D., and Silverman, J.A. (2006). Genetic
changes that correlate with reduced susceptibility to daptomycin
in Staphylococcus aureus. Antimicrob. Agents Chemother. 50,
21372145.
Fuda, C.C., Fisher, J.F., and Mobashery, S. (2005). Beta-lactam re-
sistance in Staphylococcus aureus: the adaptive resistance of a
plastic genome. Cell. Mol. Life Sci. 62, 26172633.
Gerrits, M.M., de Zoete, M.R., Arents, N.L., Kuipers, E.J., and
Kusters, J.G. (2002). 16S rRNA mutation-mediated tetracycline
resistance in Helicobacter pylori.Antimicrob. Agents Chemother.
46, 29963000.
Grkovic, S., Brown, M.H., and Skurray, R.A. (2002). Regulation of
bacterial drug export systems. Microbiol. Mol. Biol. Rev. 66, 671
701.
Hagman, K.E., Pan, W., Spratt, B.G., Balthazar, J.T., Judd, R.C.,
and Shafer, W.M. (1995). Resistance of Neisseria gonorrhoeae to
antimicrobial hydrophobic agents is modulated by the mtrRCDE
efflux system. Microbiol. 141, 611622.
Hayden, M.K., Rezai, K., Hayes, R.A., Lolans, K., Quinn, J.P., and
Weinstein, R.A. (2005). Development of daptomycin resistance in
vivoin methicillin-resistant Staphylococcus aureus.J. Clin. Micro-biol.43, 52855287.
Hegde, S.S., Vetting, M.W., Roderick, S.L., Mitchenall, L.A., Max-
well, A., Takiff, H.E., and Blanchard, J.S. (2005). A fluoroquinolone
resistance protein from Mycobacterium tuberculosis that mimics
DNA. Science 308, 14801483.
Hershberger, E., Donabedian, S., Konstantinou, K., and Zervos,M.J. (2004). Quinupristin-dalfopristin resistance in gram-positive
bacteria: mechanism of resistance and epidemiology. Clin. Infect.
Dis. 38, 9298.
Hiramatsu, K., Aritaka, N., Hanaki, H., Kawasaki, S., Hosoda, Y.,
Hori, S., Fukuchi, Y., and Kobayashi, I. (1997). Dissemination in
Japanese hospitals of strains of Staphylococcus aureusheteroge-
neously resistant to vancomycin. Lancet 350, 16701673.
Hirata, T., Saito, A., Nishino, K., Tamura, N., and Yamaguchi, A.
(2004). Effects of efflux transporter genes on susceptibility of Esch-
erichia colito tigecycline (GAR-936). Antimicrob. Agents Chemoth-
er.48, 21792184.
Hisanaga, T., Hoban, D.J., and Zhanel, G.G. (2005). Mechanisms of
resistance to telithromycin in Streptococcus pneumoniae.J. Anti-
microb. Chemother. 56, 447450.
Jacobs, M.R. (1999). Drug-resistant Streptococcus pneumoniae:
rational antibiotic choices. Am. J. Med. 106, 19S25S.
Jacoby, G.A., and Munoz-Price, L.S. (2005). The new beta-lac-
tamases. N. Engl. J. Med. 352, 380391.
Levin, A.S., Barone, A.A., Penco, J., Santos, M.V., Marinho, I.S.,
Arruda, E.A., Manrique, E.I., and Costa, S.F. (1999). Intravenous
colistin as therapy for nosocomial infections caused by multidrug-
resistant Pseudomonas aeruginosaand Acinetobacter baumannii.
Clin. Infect. Dis.28, 10081011.
Levy, S.B. (1992). Active efflux mechanisms for antimicrobial resis-
tance. Antimicrob. Agents Chemother. 36, 695703.
Levy, S.B. (2002). The Antibiotic Paradox: How the Misuse of Anti-
biotics Destroys Their Curative Powers (Cambridge, MA: Perseus
Publishing).
Levy, S.B., and Marshall, B. (2004). Antibacterial resistance world-
wide: causes, challenges and responses. Nat. Med. 10, S122
S129.
Levy, S.B., Fitzgerald, G.B., and Macone, A.B. (1976). Spread of
antibiotic-resistance plasmids from chicken to chicken and from
chicken to man. Nature260, 4042.
Li, X.Z., and Nikaido, H. (2004). Efflux-mediated drug resistance in
bacteria. Drugs 64, 159204.
Lomovskaya, O., Lee, A., Hoshino, K., Ishida, H., Mistry, A., War-
ren, M.S., Boyer, E., Chamberland, S., and Lee, V.J. (1999). Use
of a genetic approach to evaluate the consequences of inhibition
of efflux pumps in Pseudomonas aeruginosa.Antimicrob. Agents
Chemother.43, 13401346.
Martinez-Martinez, L., Pascual, A., and Jacoby, G.A. (1998). Quino-
lone resistance from a transferable plasmid. Lancet 351, 797799.
Matsuoka, M., and Sasaki, T. (2004). Inactivation of macrolides
by producers and pathogens. Curr. Drug Targets Infect. Disord. 4,
217240.
Matthews, P.R., and Stewart, P.R. (1988). Amplification of a section
of chromosomal DNA in methicillin-resistant Staphylococcus au-
reus following growth in high concentrations of methicillin. J. Gen.
Microbiol. 134, 14551464.
Mazel, D. (2006). Integrons: agents of bacterial evolution. Nat. Rev.
Microbiol.4, 608620.
Mazel, D., Dychinco, B., Webb, V.A., and Davies, J. (1998). A dis-
tinctive class of integron in the Vibrio cholerae genome. Science280, 605608.
-
7/24/2019 2007_Molecular Mechanisms of Antibacterial Multidrug Resistance
13/14
Cell 128, March 23, 2007 2007 Elsevier Inc. 1049
McMurry, L., Petrucci, R.E., Jr., and Levy, S.B. (1980). Active efflux
of tetracycline encoded by four genetically different tetracycline
resistance determinants in Escherichia coli.Proc. Natl. Acad. Sci.
USA 77, 39743977.
Meka, V.G., and Gold, H.S. (2004). Antimicrobial resistance to line-zolid. Clin. Infect. Dis. 39, 10101015.
Miller, L.G., Perdreau-Remington, F., Rieg, G., Mehdi, S., Perlroth,
J., Bayer, A.S., Tang, A.W., Phung, T.O., and Spellberg, B. (2005).
Necrotizing fasciitis caused by community-associated methicillin-
resistant Staphylococcus aureusin Los Angeles. N. Engl. J. Med.
352, 14451453.
Moore, I.F., Hughes, D.W., and Wright, G.D. (2005). Tigecycline is
modified by the flavin-dependent monooxygenase TetX. Biochem-
istry44, 1182911835.
Murakami, S., and Yamaguchi, A. (2003). Multidrug-exporting sec-
ondary transporters. Curr. Opin. Struct. Biol. 13, 443452.
Musher, D.M., Dowell, M.E., Shortridge, V.D., Flamm, R.K., Jor-
gensen, J.H., Le Magueres, P., and Krause, K.L. (2002). Emergence
of macrolide resistance during treatment of pneumococcal pneu-monia. N. Engl. J. Med. 346, 630631.
Naimi, T.S., LeDell, K.H., Como-Sabetti, K., Borchardt, S.M.,
Boxrud, D.J., Etienne, J., Johnson, S.K., Vandenesch, F., Fridkin,
S., OBoyle, C., et al. (2003). Comparison of community- and health
care-associated methicillin-resistant Staphylococcus aureusinfec-
tion. JAMA290, 29762984.
Neyfakh, A.A., Bidnenko, V., and Chen, L.B. (1991). Efflux-medi-
ated multidrug resistance in Bacillus subtilis: similarities and dis-
similarities with mammalian systems. Proc. Natl. Acad. Sci. USA
88, 47814785.
Nicoloff, H., Perreten, V., McMurry, L.M., and Levy, S.B. (2006).
Role for tandem duplication and lon protease in AcrAB-TolC- de-
pendent multiple antibiotic resistance (Mar) in an Escherichia coli
mutant without mutations in marRAB or acrRAB.J. Bacteriol. 188,
44134423.
Nicoloff, H., Perreten, V., and Levy, S.B. (2007). Increased genome
instability in Escherichia coli lon mutants: relation to emergence
of multiple antibiotic resistant (Mar) mutants caused by insertion
sequence elements and large tandem genomic amplifications. An-
timicrob. Agents Chemother. Published online January 12, 2007.
10.1128/AAC.01128-06.
Noble, W.C., Virani, Z., and Cree, R.G. (1992). Co-transfer of van-
comycin and other resistance genes from Enterococcus faecalis
NCTC 12201 to Staphylococcus aureus.FEMS Microbiol. Lett. 72,
195198.
Ochs, M.M., McCusker, M.P., Bains, M., and Hancock, R.E.W.
(1999). Negative regulation of the Pseudomonas aeruginosaouter
membrane porin OprD selective for imipenem and basic amino ac-
ids. Antimicrob. Agents Chemother.43, 10851090.
Oethinger, M., Kern, W.V., Jellen-Ritter, A.S., McMurry, L.M., and
Levy, S.B. (2000). Ineffectiveness of topoisomerase mutations in
mediating clinically significant fluoroquinolone resistance in Esch-
erichia coli in the absence of the AcrAB efflux pump. Antimicrob.
Agents Chemother.44, 1013.
Perreten, V., and Boerlin, P. (2003). A new sulfonamide resistance
gene (sul3) in Escherichia coliis widespread in the pig population of
Switzerland. Antimicrob. Agents Chemother.47, 11691172.
Pesci, E.C., Milbank, J.B.J., Pearson, J.P., McKnight, S., Kende,
A.S., Greenberg, E.P., and Ig lewski , B.H. (1999). Quinolone signal-
ing in the cell-to-cell communication system of Pseudomonas ae-
ruginosa.Proc. Natl. Acad. Sci. USA 96, 1122911234.
Poirel, L., Lambert, T., Turkoglu, S., Ronco, E., Gaillard, J., and
Nordmann, P. (2001). Characterization of Class 1 integrons fromPseudomonas aeruginosathat contain the bla(VIM-2) carbapenem-
hydrolyzing beta-lactamase gene and of two novel aminoglycoside
resistance gene cassettes. Antimicrob. Agents Chemother. 45,
546552.
Roberts, M.C. (2004). Resistance to macrolide, lincosamide, strep-
togramin, ketolide, and oxazolidinone antibiotics. Mol. Biotechnol.28, 4762.
Robicsek, A., Strahilevitz, J., Jacoby, G.A., Macielag, M., Abbanat,
D., Park, C.H., Bush, K., and Hooper, D.C. (2006). Fluoroquinolone-
modifying enzyme: a new adaptation of a common aminoglycoside
acetyltransferase. Nat. Med. 12, 8388.
Ross, J.I., Eady, E.A., Cove, J.H., and Cunliffe, W.J. (1998). 16S rRNA
mutation associated with tetracycline resistance is a gram-positive
bacterium. Antimicrob. Agents Chemother. 52, 17021705.
Ruzin, A., Visalli, M.A., Keeney, D., and Bradford, P.A. (2005). Influ-
ence of transcriptional activator RamA on expression of multidrug
efflux pump AcrAB and tigecycline susceptibility in Klebsiella pneu-
moniae.Antimicrob. Agents Chemother.49, 10171022.
Sapunaric, F.M., Aldema-Ramos, M., and McMurry, L.M. (2005).
Tetracycline resistance: efflux, mutation, and other mechanisms. InFrontiers in Antimicrobial Resistance: A Tribute to Stuart B. Levy,
D.G. White, M.N. Alekshun, and P.F. McDermott, eds. (Washington,
DC: ASM Press), pp. 3-18.
Schneiders, T., Amyes, S.G., and Levy, S.B. (2003). Role of AcrR
andramA in fluoroquinolone resistance in clinical Klebsiella pneu-
moniae isolates from Singapore. Antimicrob. Agents Chemother.
47, 28312837.
Schumacher, M.A., and Brennan, R.G. (2002). Structural mecha-
nisms of multidrug recognition and regulation by bacterial multi-
drug transcription factors. Mol. Microbiol.45, 885893.
Schwarz, S., Kehrenberg, C., Doublet, B., and Cloeckaert, A.
(2004). Molecular basis of bacterial resistance to chloramphenicol
and florfenicol. FEMS Microbiol. Rev.28, 519542.
Sharma, S.K., and Mohan, A. (2006). Multidrug-resistant tubercu-losis: A menace that threatens to destabilize tuberculosis control.
Chest 130, 261272.
Skold, O. (2001). Resistance to trimethoprim and sulfonamides.
Vet. Res. 32, 261273.
Sloan, J., McMurry, L.M., Lyras, D., Levy, S.B., and Rood, J.I.
(1994). The Clostridium perfringensTet P determinant comprises
two overlapping genes: tetA(P), which mediates active tetracycline
efflux, and tetB(P), which is related to the ribosomal protection
family of tetracycline-resistance determinants. Mol. Microbiol. 11,
403415.
Stanton, T.B., and Humphrey, S.B. (2003). Isolation of tetracycline-
resistant Megasphaera elsdenii strains with novel mosaic gene
combinations of tet(O) and tet(W) from swine. Appl. Environ. Mi-
crobiol. 69, 38743882.
Stein, G.E., and Craig, W.A. (2006). Tigecycline: A critical analysis.
Clin. Infect. Dis.43, 518524.
Tait-Kamradt, A., Davies, T., Appelbaum, P.C., Depardieu, F., Cour-
valin, P., Petitpas, J., Wondrack, L., Walker, A., Jacobs, M.R., and
Sutcliffe, J. (2000). Two new mechanisms of macrolide resistance
in clinical strains of Streptococcus pneumoniae from Eastern Eu-
rope and North America. Antimicrob. Agents Chemother.44, 3395
3401.
Tennent, J.M., Lyon, B.R., Midgley, M., Jones, I.G., Purewal, A.S.,
and Skurray, R.A. (1989). Physical and biochemical characteriza-
tion of the qacAgene encoding antiseptic and disinfectant resis-
tance in Staphylococcus aureus.J. Gen. Microbiol. 135, 110.
Tran, J.H., and Jacoby, G.A. (2002). Mechanism of plasmid-medi-
ated quinolone resistance. Proc. Natl. Acad. Sci. USA 99, 56385642.
-
7/24/2019 2007_Molecular Mechanisms of Antibacterial Multidrug Resistance
14/14
Watanabe, T. (1963). Infective heredity of multiple drug resistance
in bacteria. Bacteriol. Rev.27, 87115.
Weigel, L.M., Clewell, D.B., Gill, S.R., Clark, N.C., McDougal, L.K.,
Flannagan, S.E., Kolonay, J.F., Shetty, J., Killgore, G.E., and Tenover,
F.C. (2003). Genetic analysis of a high-level vancomycin-resistantisolate of Staphylococcus aureus.Science 302, 15691571.
Weisblum, B. (1998). Macrolide resistance. Drug Resist. Updat. 1,
2941.
Wilson, P., Andrews, J.A., Charlesworth, R., Walesby, R., Singer, M., Far-
rell, D.J., and Robbins, M. (2003). Linezolid resistance in clinical isolates
of Staphylococcus aureus.J. Antimicrob. Chemother. 51, 186188.
Wolter, N., Smith, A.M., Farrell, D.J., Schaffner, W., Moore, M.,
Whitney, C.G., Jorgensen, J.H., and Klugman, K.P. (2005). Novel
mechanism of resistance to oxazolidinones, macrolides, and chlor-
amphenicol in ribosomal protein L4 of the pneumococcus. Antimi-
crob. Agents Chemother.49, 35543557.
Woolridge, D.P., Vazquez-Laslop, N., Markham, P.N., Chevalier,
M.S., Gerner, E.W., and Neyfakh, A.A. (1997). Efflux of the natural
polyamine spermidine facilitated by the Bacillus subtilismultidrug
transporter Blt. J. Biol. Chem. 272, 88648866.
Zhanel, G.G., Dueck, M., Hoban, D .J., Vercaigne, L.M., Embil, J.M.,
Gin, A.S., and Karlowsky, J.A. (2001). Review of macrolides and ke-
tolides: focus on respiratory tract infections. Drugs 61, 443498.