REVIEW ARTICLE
A global view of antibiotic resistance
Jose Luis Martinez1,2, Alicia Fajardo1,2, Leonor Garmendia1,2, Alvaro Hernandez1,2, Juan Francisco
Linares1,2, Laura Martı́nez-Solano1,2 & Marı́a Blanca Sánchez1,2
1
Departamento de Biotecnologı́a Microbiana, Centro Nacional de Biotecnologı́a, CSIC, Cantoblanco, Madrid, Spain; and 2CIBERESP, Spain
Correspondence: Jose Luis Martinez,
Departamento de Biotecnologı́a Microbiana,
Centro Nacional de Biotecnologı́a, CSIC,
Darwin 3, Cantoblanco, 28049-Madrid,
Spain. Tel.: +34 915854542; fax: +34
915854506; e-mail: jlmtnez@cnb.csic.es
Present address: Juan Francisco Linares,
Department of Molecular Oncogenesis,
Genome Research Institute, University of
Cincinnati College of Medicine, Cincinnati,
OH 45229, USA.
Received 3 July 2008; revised 10 October 2008;
accepted 10 October 2008.
First published online 19 November 2008.
DOI:10.1111/j.1574-6976.2008.00142.x
Editor: Victor de Lorenzo
Keywords
antibiotic resistance; bacterial evolution; fitness
cost; hypermutation; horizontal gene transfer;
HGT.
Introduction
Bacterial pathogens, once susceptible to antimicrobial
agents, are becoming increasingly resistant. In addition,
hospitals are now facing problems caused by new opportu-
nistic pathogens (from nonclinical environments) that show
little susceptibility to antibiotics (Quinn, 1998; Gaynes &
Edwards, 2005). Increasing antibiotic resistance poses im-
portant risks to human health (Cohen, 1992; Levy, 1998;
Levy & Marshall, 2004); it can affect the course of infectious
disease (World Health Organization, 1999) and increase the
danger associated with immunosuppression (e.g. in trans-
plantation and anticancer chemotherapy), intubation, ca-
theterization and other common procedures, all of which
rely on antibiotics to overcome the infections with which
they are commonly associated. As stated by the World
Health Organization, the spread of antibiotic-resistant
bacteria at hospitals ‘means that commonplace medical


c 2008 Federation of European Microbiological Societies
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Abstract
Antibiotic resistance is one of the few examples of evolution that can be addressed
experimentally. The present review analyses this resistance, focusing on the
networks that regulate its acquisition and its effect on bacterial physiology. It is
widely accepted that antibiotics and antibiotic resistance genes play fundamental
ecological roles – as weapons and shields, respectively – in shaping the structures of
microbial communities. Although this Darwinian view of the role of antibiotics is
still valid, recent work indicates that antibiotics and resistance mechanisms may
play other ecological roles and strongly influence bacterial physiology. The
expression of antibiotic resistance determinants must therefore be tightly regulated
and their activity forms part of global metabolic networks. In addition, certain
bacterial modes of life can trigger transient phenotypic antibiotic resistance under
some circumstances. Understanding resistance thus requires the analysis of the
regulatory networks controlling bacterial evolvability, the physiological webs
affected and the metabolic rewiring it incurs.
procedures once previously taken for granted could be
conceivably consigned to medical limbo. The repercussions
are almost unimaginable’ (World Health Organization,
2000).
Nonetheless, antibiotic resistance is one of the few
examples of evolution that can be studied in real time
(Martinez et al., 2007; Courvalin, 2008); this is the focus of
the present review [the danger to health and the basic
mechanisms of resistance have been reviewed elsewhere
(Cohen, 1992; Levy, 1998; Levy & Marshall, 2004)]. This
paper does not, therefore, use the clinical definition of
resistance based on minimal inhibitory concentration
breakpoints (Turnidge & Paterson, 2007), but rather adopts
the wider view that it is represented by any mechanism that
reduces susceptibility to antibiotics. Antibiotic resistance
can come about either as the consequence of genetic change
(mutations) (Martinez & Baquero, 2000) or by the acquisi-
tion of antibiotic resistance genes (Davies, 1997) through
FEMS Microbiol Rev 33 (2009) 44–65
Antibiotic resistance
horizontal gene transfer (HGT). Some organisms have a
characteristic phenotype of low susceptibility to antibiotics
(Bonomo & Szabo, 2006; Fajardo et al., 2008), acquired
before the use of antibiotics in medicine.
A global analysis of the phenomenon of antibiotic resis-
tance, understood as an example of bacterial adaptation to
stressful conditions (Hamilton-Miller, 2004), requires that
the different features of this process be addressed. These
include the physiological role of resistance determinants, the
regulatory networks that modulate genetic events leading to
resistance (namely mutation, recombination and HGT), the
overall effect on the bacterial physiology of the acquisition of
resistance and the existence of global metabolic networks
that mediate bacterial susceptibility to antibiotics. Non-
pathogenic bacteria may contain many determinants that
can afford resistance, and yet just a few are encountered in
pathogen populations (Martinez et al., 2007). Understand-
ing the evolutionary process behind resistance requires a
global comprehension of the genetic causes and physiologi-
cal consequences of its acquisition (Martinez & Baquero,
2002; Martinez et al., 2007).
Functional role of antibiotics and
antibiotic resistance genes in natural
environments
Although all environments may be said to be equally
natural, this review uses the terms ‘natural environments’
or ‘environmental bacteria’ to describe those with no hu-
man-associated (infective or commensal) evolutionary link.
Rapid evolution towards resistance to antibiotics (the con-
sequence of their use in the treatment of human infections)
is unlikely in environmental bacteria; this contrasts sharply
with the situation for human pathogens.
Antibiotics as signals
The search for antibiotics in natural ecosystems was origin-
ally based on the idea that the latter contained compounds
that inhibited the growth of pathogens; certainly, pathogens
are rarely found in the environment (Waksman & Woodruff,
1940). The extraordinary success of this approach, plus the
fact that several of the antibiotics used for treating infections
are synthesized by soil microorganisms, led to the proposi-
tion that, in natural environments, antibiotics have the
function of inhibiting the growth of competitors – an
example of the Darwinian idea of ‘the struggle for life’.
Conversely, antibiotic resistance genes must be elements that
evolved to avoid the activity of antibiotics; work on the
molecular basis of antibiotic production has clearly shown
that antibiotic producers usually possess determinants that
afford them resistance to the antimicrobial agents they
produce (Benveniste & Davies, 1973). The acquisition by
FEMS Microbiol Rev 33 (2009) 44–65
45
pathogens of resistance genes that originated in antibiotic-
producing microorganisms [perhaps even via DNA contam-
ination of antibiotic preparations (Webb & Davies, 1993)]
still affords a compelling explanation for the rise of anti-
biotic resistance.
If the only roles of antibiotics and their resistance
elements are to serve as weapons and shields, respectively,
the study of antibiotic resistance would not require a global
approach, an idea this review reiterates throughout. Most of
the several thousand works so far published on antibiotic
resistance are based on the implicit belief that resistance is a
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very specific phenomenon, in which any given mechanism
of resistance is simply the defence response to the presence
of a given antibiotic. However, recent work indicates that the
situation is more complex. Whereas on some occasions the
weapon/shield role is still a reasonable explanation for the
function of antibiotics and resistance determinants in nature
(Pang et al., 1994), alternative functional roles for resistance
elements are now being proposed.
Antibiotics have been sought as compounds capable of
inhibiting the growth of microbial pathogens (Waksman &
Woodruff, 1940), and on some occasions they likely play this
role in natural environments (de Lorenzo et al., 1984;
Raaijmakers et al., 2002; Haas & Defago, 2005). However, it
should be remembered that nearly any compound can be
toxic when in a sufficiently high concentration. Because the
concentrations of antibiotics used for therapy can be much
higher than those encountered in natural environments, it
may be that, like other toxic compounds, they have a
hormetic effect (Fig. 1), i.e. at low concentrations they
induce responses in their target organisms that are actually
beneficial, but become toxic at high concentrations.
It has been suggested that antibiotics might serve as
signalling molecules at the low concentrations in which they
Fig. 1. Hormesis and the effect of antibiotics on bacterial physiology.
Hormesis has been defined as the property of a compound that is
beneficial at low concentrations and toxic at high concentrations. At
low concentrations, antibiotics can trigger the expression of a specific set
of genes that may be beneficial to bacteria. However, at higher
concentrations, stress responses are induced and bacterial growth is
inhibited. The white box indicates the region of beneficial, adaptive
responses and the grey box indicates the region of harmful effects.


c2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
46
are usually found in natural habitats (Davies, 2006; Yim
et al., 2006a, b, 2007; Fajardo & Martinez, 2008). This
suggestion is based on the fact that low concentrations of
antibiotics trigger specific transcriptional changes (Tsui
et al., 2004; Yim et al., 2006a, b; Fajardo & Martinez, 2008)
that are independent of the microbial networks involved in
the general stress response (Goh et al., 2002), that these
responses can be beneficial for the microorganisms involved
(Linares et al., 2006; Dietrich et al., 2008; Fajardo &
Martinez, 2008; Gerber et al., 2008) and that some com-
pounds previously characterized as bona fide signalling
molecules also have antimicrobial activity (Ji et al., 1997;
Deziel et al., 2004; Kaufmann et al., 2005).
Functional roles of antibiotic resistance genes in
their original hosts
If antibiotics have functions other than the killing of
competitors, antibiotic resistance genes may have functions
other than providing resistance. Even in antibiotic produ-
cers, the presence of an antibiotic resistance gene does not
necessarily imply that its original role was to help resist the
action of the antibiotic produced by the host (Nodwell,
2007). For instance, Streptomyces coelicolor encodes proteins
similar in sequence and indeed in mode of action to those
involved in resistance to vancomycin in human pathogens
(Hong et al., 2002), even though this microorganism does
not produce glycopeptide antibiotics.
One example of antibiotic resistance determinants with a
functional role other than affording resistance is provided by
multidrug resistance (MDR) efflux pumps (Fig. 2). These
transporters are present in all organisms (Saier & Paulsen,
2001; Piddock, 2006a, b; Lubelski et al., 2007), and the same
MDR pumps are present in all strains of a given species
Fig. 2. Structure of a tripartite efflux pump. The tripartite efflux pumps
of Gram-negative bacteria are formed by an integral membrane protein,
whose activity is commonly linked to the membrane proton motive force,
an outer membrane protein and a periplasmic protein. IM, inner
membrane; OM, outer membrane.


c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
J.L. Martinez et al.
(Alonso et al., 1999b; Alonso & Martinez, 2001; Sanchez
et al., 2004). In bacteria, it has been shown that besides
offering resistance to antibiotics and other toxic compounds
(Silver & Phung, 1996; Hernandez et al., 1998; Ramos et al.,
2002; Sanchez et al., 2005), they contribute to virulence
(Piddock, 2006a, b), to maintaining homeostasis (Lewinson
& Bibi, 2001) and to the detoxification of intracellular
metabolites (Aendekerk et al., 2005), among other func-
tions. Mutations leading to the constitutive expression of
these transporters afford antibiotic resistance (Alonso &
Martinez, 1997; Kohler et al., 1997; Ziha-Zarifi et al., 1999;
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Sanchez et al., 2002a, b) and produce global changes in
bacterial metabolism specific for each MDR pump. It is
noteworthy that some of these MDR pumps can efflux signal
compounds (Evans et al., 1998; Kohler et al., 2001), indicat-
ing that signalling networks may be important in triggering
antibiotic resistance.
Any enzyme capable of modifying a metabolite with a
structure similar to a given antibiotic might modify the
antibiotic itself and confer resistance. This is the case of
chromosomal 2 0 -N-acetyltransferase in Providencia stuartii.
This enzyme, which modifies bacterial peptidoglycan, can
inactivate gentamicin because the latter’s structure is similar
to that of the enzyme’s original substrate (Macinga &
Rather, 1999). The fact that elements involved in bacterial
metabolism can be important in antibiotic resistance is
further shown in that a large number of Pseudomonas
aeruginosa genes contribute to its characteristic low-suscept-
ibility phenotype (Fajardo et al., 2008). It has also been
suggested that b-lactamases, which have a structure similar
to penicillin-binding proteins (PBPs, the targets of b-lactam
antibiotics), might have originally been PBPs themselves,
involved in the synthesis of peptidoglycan. Their activity
against b-lactam antibiotics may have been a side effect
of their original function (Massova & Mobashery, 1998;
Meroueh et al., 2003).
In summary, the fact that antibiotics trigger specific
transcriptional responses, together with the role that deter-
minants involved in signal trafficking or bacterial metabo-
lism might have in resistance, supports the idea that
antibiotic resistance should be examined from a more global
viewpoint. Only then can the bacterial networks involved in
resistance, as well as the changes in bacterial physiology
associated with a phenotype, be addressed. These changes
might be important in the context of human infections and
in the survival and dissemination of resistant bacteria.
Regulation of evolvability and acquired
antibiotic resistance
Resistance to antibiotics can be broadly classified as either
intrinsic (Hogan & Kolter, 2002; Fajardo et al., 2008; Tamae
et al., 2008) (due to the mechanisms present in all the strains
FEMS Microbiol Rev 33 (2009) 44–65
Antibiotic resistance
of a given bacterial species) or acquired (Davies, 1994;
Alekshun & Levy, 2007) (due to the acquisition of a specific
mechanism of resistance, the consequence of mutation,
recombination or HGT). Studies of experimental evolution
using Metazoans as models are virtually impossible because
of the time and population sizes required (Navas et al.,
2007), but the acquisition of antibiotic resistance by bacteria
provides an example of an evolutionary process that can be
readily addressed experimentally. Learning about the me-
chanisms that lead to resistance has implications for under-
standing evolution as a whole. Because genome changes are
the basis of any evolutionary process, the probability of such
events occurring represents the first point of control in the
development of resistance. In recent years, it has become
evident that the ability to evolve [i.e. evolvability – see
Kirschner & Gerhart (1998) for an in-depth review of this
concept] is a regulated phenomenon that must be under-
stood from a global viewpoint (Pepper, 2003; Colegrave &
Collins, 2008; Isalan et al., 2008; Pigliucci, 2008; Schlichting,
2008). Some aspects of the networks regulating evolvability
that can lead to the acquisition of antibiotic resistance are
discussed below.
Regulation of mutation rates
Bacterial populations tend to have low mutation rates,
which helps preserve their genomes and avoid lethal muta-
tions. DNA error correction mechanisms bring bacterial
evolution speeds down by around 1000-fold below their
theoretical natural speed limit (Zeldovich et al., 2007). Some
authors report, however, that the mutation rate (and thus
the speed of evolution) of some bacterial subpopulations is
higher than normal (LeClerc et al., 1996; Matic et al., 1997;
Oliver et al., 2000; Baquero et al., 2004, 2005), mainly due to
defects in the DNA repair systems (Oliver et al., 2002).
Bacterial hypermutators are more commonly found in popu-
lations under stress, including chronic infections (Oliver
et al., 2000), or in the presence of strong selective elements
such as antibiotics (Macia et al., 2005) or bacteriophages (Pal
et al., 2007). The presence of hypermutators increases the
probability of acquiring resistance (Macia et al., 2005; Hen-
richfreise et al., 2007), and antibiotics select for hypermuta-
tors (Mao et al., 1997) in such a way that they enhance the
probability of resistance appearing (Blazquez et al., 2002).
The modulation of mutation frequencies may also be
important in the development of resistance. Some works
report increased numbers of antibiotic-resistant mutants in
bacterial populations under stress (Alonso et al., 1999a), and
that the number of mutants in colonies of nongrowing
bacteria can increase. Two mechanisms have been proposed
to explain this (Roth et al., 2006): one based on gene
amplification (Andersson et al., 1998; Pettersson et al.,
2005) and the other on the regulated increase of mutation
FEMS Microbiol Rev 33 (2009) 44–65
47
rates (Gomez-Gomez et al., 1997; Bjedov et al., 2003; Foster,
2005; Saint-Ruf & Matic, 2006). Recently, the faster growth
of resistant mutants has been suggested to be the cause of
increased mutation frequencies with respect to rifampicin
resistance in ‘ageing’ bacterial colonies (Wrande et al.,
2008).
Key components of the mismatch repair system are
downregulated under the control of the general-stress sigma
factor RpoS (Tsui et al., 1997). In addition, the SOS response
to DNA damage (Erill et al., 2007) triggers the transcription
of several genes, one of which is dinB, which codes for the
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error-prone DNA polymerase IV (a Y family DNA polymer-
ase) (Kim et al., 1997; Godoy et al., 2007). This enzyme is
able to bypass DNA lesions that block chain elongation by
replicative DNA polymerase. In addition, PolIV shows less
fidelity than proofreading polymerases when operating on
undamaged DNA (Wagner et al., 1999). When PolIV is
overproduced, it introduces mutations (which may afford
antibiotic resistance) at a high rate (Kim et al., 1997). It has
been shown that dinB expression forms part of a large
regulatory network that includes the LexA-controlled SOS
regulon (Kim et al., 1997; Godoy et al., 2007), RpoS
polymerase (Layton & Foster, 2003), the histone-like protein
HU (Williams & Foster, 2007) and the polyphosphate kinase
(Stumpf & Foster, 2005), among others.
Bacterial mutation rates (and thus the probability of
mutation-driven resistance) are therefore subject to com-
plex regulation that we are now just beginning to under-
stand. Because this regulation can be triggered by
environmental or metabolic signals (Bjedov et al., 2003), it
is important to know which situations might increase the
mutation rate and thus the likelihood of acquiring resis-
tance. Quinolones (Phillips et al., 1987) and b-lactams
(Miller et al., 2004) induce the SOS response, and could
therefore increase the mutation rate. Recent work has shown
that PBP3 inhibition by ceftazidime induces transcription of
the error-prone polymerase PolIV and induces mutagenesis
in P. aeruginosa (Blazquez et al., 2006). Although the SOS
system regulates the expression of dinB, other elements are
also involved. For instance, studies on Escherichia coli have
shown that the induction of dinB by b-lactams occurs via a
pathway independent of the SOS response (Perez-Capilla
et al., 2005), highlighting that different regulatory networks
can modulate bacterial mutability. This view is further
supported by recent work showing that subinhibitory con-
centrations of quinolones and aminoglycosides may result
in increased mutability in Streptococcus pneumoniae, inde-
pendent of DinB activity (Henderson-Begg et al., 2006).
Regulation of recombination
Recombination plays a major role in HGT (Thomas &
Nielsen, 2005). Both the acquisition of DNA through


c2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
48
transformation and the combination of new elements (e.g.
antibiotic resistance genes) into transferable replicons (e.g.
plasmids) require recombination among DNA sequences.
Studies on E. coli have shown that efficient recombination
occurs in plasmids when homologous sequences of 25 bp are
present. Increasing the length of homology enhances re-
combination (Lovett et al., 2002). Recombination is there-
fore favoured among closely related organisms or elements
involved in HGT with similar structural features.
Some particularly important features that favour the
spread of antibiotic resistance should be mentioned at this
point. Firstly, gene-capture units exist that favour the
acquisition of exogenous DNA and its incorporation into
a single element. The clearest examples are the integrons
(Fig. 3), genetic platforms that contain small repetitive
sequences (typically 59 bp) that favour recombination and
gene recruitment (Hall & Collis, 1995; Rowe-Magnus &
Mazel, 2001; Holmes et al., 2003; Mazel, 2006). These are
very important in the dissemination of antibiotic resistance
genes (Carattoli, 2001; Rowe-Magnus & Mazel, 2002; Rowe-
Magnus et al., 2002). Secondly, the need for homologous
sequences to trigger recombination can be relaxed under
some circumstances. For instance, some hypermutator
strains, particularly those with defects in the methyl-direc-
ted DNA-repair genes (such as mutS), recombine at high
frequencies with divergent DNA (Matic et al., 2000).
Thirdly, antibiotics such as ciprofloxacin may stimulate
recombination among different sequences (Lopez et al.,
2007). All this indicates that recombination rates,
Fig. 3. Structure of an integron. Integrons are formed by a gene coding
for a site-specific recombinase known as integrase (intl1), an attachment
site (attI) and a strong promoter that drives the expression of the genes
located downstream from the attachment site (P). Some conserved
genes are present at the 3 0 -end of integrons of the same type, suggest-
ing a common origin. The figure shows a Type I integron containing qac,
which encodes for a protein involved in resistance to quaternary
ammonium disinfectants, sul1 that encodes sulphonamide resistance
and orf5, which has no known function. Antibiotic resistance genes can
be recruited by site-specific recombination (a) driven by the integrase
(Intl1). After gene recruitment (b) the recombination sites are recon-
structed, allowing the incorporation of new gene cassettes. This struc-
ture allows the formation of integrons containing arrangements of anti-
biotic resistance genes that can be acquired simultaneously by bacterial
pathogens.


c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
J.L. Martinez et al.
like mutation rates, change as a consequence of the
structure of microbial populations (hypermutators) and as
a response to external inputs, such as antibiotics (Hastings
et al., 2004).
Regulation of DNA uptake
Transformation and conjugation usually involve the trans-
location of single-stranded DNA through the bacterial
envelope (Chen & Dubnau, 2004; Chen et al., 2005). After
gaining entry, general mechanisms such as DNA modifica-
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tion/restriction (Tock & Dryden, 2005) or plasmid exclusion
(Garcillan-Barcia & de la Cruz, 2008) must be bypassed by
the exogenous DNA if it is to be maintained. The mechan-
isms that regulate bacterial DNA uptake and maintenance
have been reviewed elsewhere (Chen et al., 2005; Thomas &
Nielsen, 2005); only those aspects concerning resistance are
discussed here. Natural competence involves the induction
of a complex system (the com regulon) usually regulated by
quorum sensing (QS) (Pestova et al., 1996; Cheng et al.,
1997; Luo et al., 2003). Notably, it has recently been found
that the same peptides that induce competence in closely
related members of the streptococci family can kill other
members of the same genus, making large amounts of DNA
available and increasing the efficiency of lateral gene transfer
(Johnsborg et al., 2008). This provides an example of the
dual antimicrobial and signalling role of some metabolites
(discussed above) (Fajardo & Martinez, 2008).
The simultaneous induction of competence in microor-
ganisms and their predation in the same environment is
particularly important for S. pneumoniae, which acquires
resistance via HGT from DNA released by related commen-
sal species (Sibold et al., 1994; Reichmann et al., 1997;
Hakenbeck, 1998). Streptococcus pneumoniae lacks an SOS-
like system, but possesses the recA gene, whose induction by
DNA-damaging agents is strictly dependent on the ability of
these compounds to trigger competence. It has been sug-
gested that the com regulon of S. pneumoniae might have
features similar to those of the SOS response regulon in
E. coli (Prudhomme et al., 2006). Because the SOS system is
induced by some antibiotics, it might be possible for
antibiotics to induce competence in S. pneumoniae. Indeed,
it has been demonstrated that aminoglycosides and fluor-
oquinolones induce competence, and that an intact compe-
tence regulatory cascade is needed (induction is lost in comA
mutants) (Prudhomme et al., 2006).
Antibiotics can thus accelerate DNA acquisition in
S. pneumoniae by two different mechanisms: by killing
susceptible neighbouring cells and increasing genetic trans-
formability (Prudhomme et al., 2006). If the bacteria that
are resistant to a different type of antibiotic are killed, and
their DNA is released, this would increase the probability of
S. pneumoniae acquiring that resistance.
FEMS Microbiol Rev 33 (2009) 44–65
Antibiotic resistance
Building blocks in HGT-acquired antibiotic
resistance and the founder effect
The elements of bacterial mobilomes are reviewed in an-
other article of this special issue; this paper only analyses the
features concerned with antibiotic resistance. The acquisi-
tion and dissemination of resistance by HGT follows a
Chinese-box scheme. Inspection of the elements producing
resistance at hospitals (Baquero et al., 2002) shows that there
are predominant clones that may contain predominant
plasmids, which in turn contain predominant elements
(transposons, integrons, etc.) in which antibiotic resistance
genes are enclosed (Fig. 4). This hierarchical scheme of the
elements involved in resistance dissemination has been
discussed in detail elsewhere (Baquero, 2004).
Although the number and variability of the elements
present in natural bacterial populations that might contri-
bute to resistance is huge, only a few are present in human
Fig. 4. Building blocks in the acquisition of HGT-mediated antibiotic
resistance. The elements involved in HGT have common backbones that
can incorporate different modules. Antibiotic resistance can be trans-
ferred by plasmids that acquire transposons, which in turn can contain
integrons that recruit antibiotic resistance genes. The modular structure
of the elements involved in HGT allows for rapid evolution when under
antibiotic threat. IS, insertion sequences; tra, transference genes; psk,
postsegregational killing; mrs, multimer resolution; rep, replication; cop,
copy number control; par, partitioning. The figure of the plasmid is
modified from Thomas (2000).
FEMS Microbiol Rev 33 (2009) 44–65
49
pathogens. For instance, E. coli strains isolated before the era
of antibiotics contain the same groups of plasmids as those
isolated after the introduction of antibiotics into medicine,
the only difference between them being the recruitment of
resistance genes in the latter (Datta & Hughes, 1983).
Similarly, the diversity of transposons and integrons asso-
ciated with resistance is not as high as might be predicted
from the diversity of these elements in natural ecosystems.
Modularity is a common characteristic of biological
systems (Kashtan & Alon, 2005; Pereira-Leal et al., 2006).
The rules that govern the combination of modules (Stadler
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et al., 2001) have been analysed in different types of
biological networks (Force et al., 2005; Pereira-Leal et al.,
2006; Mete et al., 2008) and, more recently, in the structure
of bacterial genomes (Dagan et al., 2008). It has been
suggested that, like for other modular systems, there are
rules (grammar) governing the association of the blocks
required for building a transferable resistance element
(Baquero, 2004). This grammar has different components:
(1) functional elements: each replicon can make use of the
transcriptional and metabolic machineries of certain bacter-
ial groups but not of others; they therefore have host
specificity; (2) structural features: DNA structures that
favour the combination of some elements but not others.
On many occasions, homologous recombination is required,
so that only those elements with a high degree of sequence
identity are able to combine; (3) ecological aspects: first,
only those building blocks coexisting in the same environ-
ment can combine; second, the most abundant elements
may be favoured; and third, in the presence of strong
selection, the first element conferring a selective advantage
(antibiotic resistance) has the highest probability of spread-
ing and predominating (Fig. 5) in the bacterial population
under antibiotic threat.
The integrons (Stokes & Hall, 1989) are modular struc-
tures with a major role in the development of antibiotic
resistance. These gene-recruitment determinants (Fig. 3)
have a high impact in clustering antibiotic resistance genes,
which may later be transferred to pathogens. Although
integrase sequences in natural environments show wide
variability (Nield et al., 2001; Elsaied et al., 2007), that of
the different integrases involved in integrons containing
resistance genes in pathogenic bacteria is low. The Type 1
integrons are the most important (Walsh, 2006). If environ-
ment-driven selection of integrons occurs, it may be that
this type of integrase is the best adapted to infective
environments. However, Type 1 integrases are found in a
wide range of pathogens from E. coli to P. aeruginosa, which
come from different habitats. The founder effect explains
the dissemination of the most common types of integrons in
human pathogens. For instance, Type 1 integrons contain a
conserved gene coding for resistance to sulphonamides
(Stokes & Hall, 1989), a family of antimicrobial agents


c2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
50
Fig. 5. The founder effect in the acquisition of antibiotic resistance
genes. Natural environments (grey box) contain a large number of
antibiotic resistance genes (letters). Some (Greek letters) cannot be
transferred to bacterial pathogens (white box) because they do not share
the same ecosystem or because they have not been incorporated into
gene-transfer elements compatible with human pathogens (a). Although
several resistance genes (Roman letters) can be transferred, only a few
are currently present in infectious bacteria (darker grey box in b). If the
selective pressure does not change, the same antibiotic resistance genes
are maintained and spread in the population (c), impeding the entrance
of others that could play a similar role (founder effect). If the selective
forces change (e.g. the appearance of new antibiotics belonging to the
same family, darkest grey box in d), the original resistance gene evolves
into more efficient variants (white ‘a’ letters in d) with different
specificities.
widely used in the first half of the 20th century. It might be
suggested that, under strong selective pressure, the first
integron containing a sulphonamide resistance gene was
selected, and that this became the ancestor of the integrons
now found in clinical settings. It is important to note that
sulphonamide resistance is still widespread in the United
Kingdom despite national prescribing restrictions (Enne
et al., 2001), probably because of the success of these
integrons in recruiting other antibiotic resistance genes.
The full sequences of different plasmids of the same
family can provide new clues regarding the evolution of the
elements involved in the HGT of antibiotic resistance genes.
The sequences of five IncW plasmids show that two different
pathways were involved in the acquisition of resistance genes
by the common plasmid backbone. Three plasmids contain
a single Integron I platform, in the same position in each
plasmid, with different gene cassettes. In the other two
plasmids, resistance arose because of the insertion of several
transposons containing antibiotic resistance genes (Revilla
et al., 2008). The best explanation for this is that, in the
former three plasmids, the integron was inserted once into
the IncW backbone, followed by diversification of the
integron arrangements, while it evolved independently from
an IncW ancestor in the other two plasmids (Revilla et al.,
2008).
The number of antibiotic resistance genes in nonclinical
environments is huge (Alonso et al., 2001; D’Acosta et al.,
2006; Wright, 2007; Martinez, 2008; Sanchez et al., 2008),


c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
J.L. Martinez et al.
whereas the variability of HGT-acquired resistance determi-
nants in human pathogens is low. A founder effect (Fig. 5)
also explains the presence of some antibiotic resistance genes
but not of others in bacterial pathogens. Under stable,
strong selective pressure, the first determinant conferring
resistance to this pressure would predominate and spread in
the population. Only after the selective forces change does
diversification of the antibiotic resistance determinants
usually occur. This is likely the situation with plasmid-
encoded b-lactamases. The first b-lactamase described in
bacterial pathogens was TEM-1. This enzyme, which inacti-
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vates the first class of b-lactams, has been prevalent for
several years in resistant enteric Gram-negative bacilli (Roy
et al., 1983), whereas another b-lactamase, SHV-1, is pro-
duced by the vast majority of resistant Klebsiella pneumoniae
strains (Roy et al., 1983; Livermore, 1995). After the
introduction of b-lactamase inhibitors and new generations
of b-lactams, a burst in TEM-1- and SHV-1-derived en-
zymes occurred (Paterson & Bonomo, 2005) – an example
of accelerated evolution under stress (Medeiros, 1997). In
addition, novel families of b-lactamases (Paterson & Bono-
mo, 2005) began to be acquired by human pathogens,
indicating that a change in selective force is required for the
diversification of antibiotic resistance determinants (Fig. 5).
Effect of resistance in bacterial physiology
It is generally accepted that the acquisition of antibiotic
resistance produces a global burden on bacterial metabo-
lism (fitness cost), making resistant bacteria less competitive
than their susceptible partners (Andersson & Levin, 1999;
Andersson, 2006). This idea is based on the fact that muta-
tions leading to resistance occur in genes that are important
for bacterial physiology (lethal targets, transporters, etc.). A
modification of the target proteins should cause deadapta-
tion from their normal functional role, leading to less
efficient bacterial metabolism. Similarly, acquisition of a
resistance gene by HGT, for instance due to plasmid transfer,
should be associated with a metabolic cost, the consequence
of the replication, transcription and translation of the genes
present in the new replicon (Bouma & Lenski, 1988). These
fitness costs will be reflected in a reduced growth rate. Thus,
most studies on fitness costs have involved in vitro analyses
using defined growth media.
Nevertheless, some authors indicate that the effect of
antibiotic resistance on bacterial physiology is more com-
plex and more specific than previously thought. For in-
stance, it has been shown that the mutations that
compensate for fitness defects are different in vivo and in
vitro (Bjorkman et al., 2000), and that the same antibiotic
resistance mutation can produce opposite effects in two
different strains of the same bacterial species (Luo et al.,
2005). To understand in depth the effect of resistance on
FEMS Microbiol Rev 33 (2009) 44–65
Antibiotic resistance
bacterial physiology, the concepts and tools used in the field
of system biology must be used. Current models for analys-
ing bacterial infections are based on the use of bacterial
strains that are susceptible to antibiotics. With the increase
in resistance in bacterial populations, models using resistant
strains – if their physiological properties (including viru-
lence) are different – should be used as well.
Alterations in metabolic networks as the
consequence of antibiotic resistance mutations
In this section, and for the rest of this review, the term
‘mutation’ is used in its broadest sense, and includes point
mutations, deletions, insertions and intrachromosomal re-
combination. As stated above, mutations that produce
resistance to antibiotics occur in genes that usually play
important roles in bacterial physiology. The products of
those genes form part of complex and highly integrated
metabolic and regulatory networks. Alterations in their
activities likely alter the cell’s entire metabolism. Recent
work has shown, however, that the rewiring of E. coli
metabolic networks is usually well tolerated (Isalan et al.,
2008). Thus, metabolic alterations arising from antibiotic
resistance are not necessarily manifested as generally re-
duced bacterial fitness for all possible environments. On the
contrary, a certain degree of specificity and even gain of
fitness might be expected. For instance, a Stenotrophomonas
maltophilia mutant that overproduces the MDR efflux
pump SmeDEF (Alonso & Martinez, 2000) shows impaired
virulence in a Dyctiostelium discoideum model, but is more
proficient in the utilization of sugars such as gentibiose,
dextrin or mannose as well as the C1 carbon source formic
acid (Alonso et al., 2004). This indicates that the rewiring of
metabolism has occurred rather than the appearance of any
general burden (fitness cost). Some recent data on the
metabolic profiling of antibiotic-resistant mutants suggest
that this may be common. For example, resistance to
rifampin is due to mutations in the gene rpoB that encodes
the RNA polymerase b subunit (Trinh et al., 2006). Some
authors indicate that changes in the rifampin-binding
pocket result in alterations in the interactions of the RNA
polymerase of Bacillus subtilis with promoters and tran-
scriptional regulators, leading to important changes in
phenotypes for sporulation, germination and competence
in DNA-mediated transformation (Maughan et al., 2004). In
the latter work, some rifampin-resistant mutants, but not
others, showed reduced growth rates in a rich medium.
However, the mutants were found to be capable of using
b-glucosides, nutrients present in soil [produced from de-
composing plant matter through the breakdown of celluloses
and hemicelluloses (Zhang et al., 2007)], more effectively
than their susceptible counterparts (Perkins & Nicholson,
2008). Therefore, the metabolic rewiring produced by
FEMS Microbiol Rev 33 (2009) 44–65
51
rifampin resistance likely led to greater fitness of the
resistant mutants (compared with the wild-type strains) for
the soil environment.
Enhanced fitness of antibiotic-resistant mutants for a
given environment is not as rare as previously thought.
Bacteria can acquire compensatory mutations that reduce
fitness costs (Bjorkman et al., 2000; Andersson, 2006), but
sometimes resistance confers an advantage, even in the
absence of selective pressure. For example, high-level quino-
lone resistance is mainly due to mutations in the topoi-
somerase genes, which encode the targets of this family of
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antibiotics (Martinez et al., 1998; Piddock, 1999). Because
topoisomerases regulate DNA supercoiling, and DNA super-
coiling may regulate gene expression (Galan & Curtiss, 1990;
Aleixandre et al., 1991; Dorman, 1991), it is likely that
mutations in the topoisomerases lead to changes in the
bacterial transcriptome. Some mutations in S. pneumoniae
topoisomerases are associated with fitness costs when mea-
sured in in vitro tests, whereas other mutations have no
associated costs (Johnson et al., 2005; Rozen et al., 2007;
Balsalobre & de la Campa, 2008). This is consistent with the
fact that different mutations in the topoisomerases alter
their activity by different degrees, changing global bacterial
metabolism in different ways. It might be predicted that only
those mutants with low costs should predominate, which is
often the case. However, the situation is generally more
complex. Work involving an in vivo model of chicken
colonization by Campylobacter jejuni has shown that a
single-point quinolone-resistance mutation in the gyrA gene
can produce enhanced persistence in the avian host (Luo
et al., 2005). Although the basis for this increased fitness has
not been studied in depth, it is conceivable that transcrip-
tional changes caused by alterations in the activity of the
topoisomerase GyrA affect the expression of the C. jejuni
genes involved in chicken colonization. This enhanced
fitness is host specific. In fact, the same mutation in two
different C. jejuni strains belonging to the same clonal
complex had opposite effects: enhanced persistence for one
and reduced fitness for the other (Luo et al., 2005). Because
the metabolic and regulatory networks of different strains of
the same bacterial species can have subtle differences with
effects on their interaction with the host (Carilla-Latorre et al.,
2008), network rewiring might also have different conse-
quences. This type of strain-specific response to an external
input is manifested by the very different transcriptomic
changes shown by two strains of P. aeruginosa in response to
the same type of oxidative stress (Salunkhe et al., 2005).
Two conclusions can be drawn from the above studies: (1)
the effect of high-level quinolone resistance on bacterial
physiology is specific for the mutation and strain involved
and (2) small genetic changes, such as single-point muta-
tions leading to antibiotic resistance, may produce dramatic,
yet specific, changes in bacterial physiology.


c2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
52
The specificity of bacterial responses to mutations leading
to resistance is perhaps best explained by studying mutants
that overproduce MDR efflux pumps. Pseudomonas aerugi-
nosa has several genes coding for them. The expression of
MDR pumps is commonly downregulated (Grkovic et al.,
2002), but mutations in their transcriptional regulators may
lead to their constitutive expression and hence reduced
susceptibility to several antibiotics. Because MDR pumps
efflux a wide range of substrates it is conceivable that their
constitutive expression might produce a global, nonspecific
metabolic burden – the consequence of the noncontrolled
release of metabolites from bacterial cytoplasm. Some
authors report, however, that the effect of MDR overpro-
duction is specific for each MDR pump. For instance,
constitutive overexpression of MexAB-OprM or MexEF-
OprN is associated with defects in P. aeruginosa QS because
these MDR pumps efflux QS autoinducers (Evans et al.,
1998; Kohler et al., 2001). On the other hand, overexpres-
sion of either MexEF-OprN or MexCD-OprJ, but not of
MexXY or MexAB-OprN, reduces P. aeruginosa Type 3
secretion (T3S) and cytotoxicity independent of the QS
response (Linares et al., 2005). Finally, mutants overexpres-
sing either MexAB-OprM or MexCD-OprJ produce more
biofilm in in vitro static assays than their parental wild-type
strain (Sanchez et al., 2002b).
The above results show that the effect of overproducing
one MDR pump or another is rather specific and not the
consequence of a general metabolic burden. Work at our
laboratory (J.F. Linares et al., unpublished data) indicates
that the overexpression of MDR pumps can lead to global
changes in the P. aeruginosa transcriptome, and that these
changes show a high degree of specificity. An important
question to address is the effect of such changes on
P. aeruginosa virulence. It was earlier stated that overpro-
duction of MDR pumps reduces this (Cosson et al., 2002;
Hirakata et al., 2002), at least in models that resemble acute
infection. However, P. aeruginosa is a common cause of
chronic infection and the virulence factors required for
producing chronic and acute infections differ (Goodman
et al., 2004; Ventre et al., 2006). Isolates from acute infec-
tions are commonly cytotoxic and produce large amounts of
proteases and siderophores but little in the way of biofilms.
In contrast, the evolution of P. aeruginosa during chronic
infection renders strains with the opposite phenotype
(Martinez-Solano et al., in press). It should be noted that a
mutant overproducing MexCD-OprJ has low cytotoxicity,
produces only small amounts of protease and is impaired in
T3S (and thus cytotoxicity) – the phenotypes observed in
P. aeruginosa strains obtained from chronic infections. It has
been shown that the causes of quinolone resistance in
P. aeruginosa involved in acute infections usually involve
mutations in the topoisomerase genes, whereas overproduc-
tion of MDR pumps is common in isolates from chronic


c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
J.L. Martinez et al.
infections (Jalal et al., 2000; Henrichfreise et al., 2007).
Further, experimental antibiotic therapy with ciprofloxacin
in P. aeruginosa-infected mice selects MexCD-OprJ over-
producers (Macia et al., 2006). Although these results are
not fully conclusive, they suggest that strains of P. aeruginosa
overproducing MexCD-OprJ might be more fit than other
antibiotic-resistant mutants with respect to the production
of chronic infection.
Because antibiotic resistance may be linked to global
bacterial metabolism, genome-scale reconstructions of me-
tabolic networks can provide insights into resistance. This
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has been addressed in the small-colony-variants (SCVs) of
Staphylococcus aureus. SCVs are a type of mutant produced
by different bacterial species (Proctor et al., 2006) and are
characterized by slow growth, changes in their susceptibility
to antibiotics and sometimes changes in their virulence
properties. SCVs are frequently found in biofilms (Haussler,
2004; Allegrucci & Sauer, 2007) and in chronic infections
(Haussler et al., 1999; von Gotz et al., 2004). The genetic
causes of SCVs are variable, but on some occasions, the
phenotype is linked to the interruption of the electron
transport chain. To analyse the effect of the acquisition of
an SCV phenotype, an S. aureus mutant lacking the haeme-
requiring cytochrome-b oxidase was constructed and its
phenotype was analysed in the context of the reconstructed
S. aureus metabolic network (Heinemann et al., 2005).
Under anaerobic conditions, wild-type S. aureus produces
energy from glucose via fermentation, whereas under aero-
biosis it uses glycolysis, the pentose phosphate pathway and
the triacarboxylic acid cycle. The predictions of the model,
confirmed by experimental data, were that the SCV hemB
mutant would have an essentially fermentative metabolism
even in the presence of oxygen. Some amino acids such as
glutamate, arginine or glutamine enhanced growth by
affording additional energy production, while isoleucine,
leucine, valine and lysine enhance growth because they can
be used directly in protein synthesis. A third group, includ-
ing glycine and alanine, is not used at all by these bacteria
(Heinemann et al., 2005). This evidence supports the idea
that resistance in SCV isolates is associated with the rewiring
of S. aureus metabolism.
Alterations in metabolic networks as the
consequence of acquiring foreign antibiotic
resistance genes
Whereas chromosomal mutations leading to resistance
likely alter bacterial metabolism, the situation may not be
the same with respect to acquired genes. Although on some
occasions the expression of HGT-acquired antibiotic resis-
tance genes is regulated (Depardieu et al., 2007), they are
sometimes under the control of constitutive promoters
(Fig. 3). In other words, these genes do not take part in the
FEMS Microbiol Rev 33 (2009) 44–65
Antibiotic resistance
same regulatory networks as in their original hosts. Further,
the proteins they encode find themselves in a different
metabolic context, sometimes without their original bio-
chemical substrates or without the protein partners required
for their activity. Under such circumstances, their only
function is resistance (if the corresponding antibiotic is
present); such situations are said to represent ‘functional
shifting’ (Martinez, 2008). Under such circumstances, a
global metabolic burden can be expected; in fact, it has been
shown that the acquisition of a plasmid produces a meta-
bolic burden that is rapidly compensated by mutations in
both the plasmid and the chromosome (Bouma & Lenski,
1988).
However, on some occasions, HGT-acquired antibiotic
resistance determinants may encounter a metabolic sub-
strate/protein partner in the new host, so that specific
changes in the host’s metabolism become possible. For
instance, it has been found that the expression of the b-
lactamase AmpC by a plasmid leads to a reduction in the
growth rate and reduced invasiveness in Salmonella enterica
serotype Typhimurium. Changes in colony morphology are
also seen (Morosini et al., 2000). Such changes are not
observed when the plasmid also contains the ampC repres-
sor AmpR, indicating that they are not the consequence of
the metabolic load caused by plasmid replication. Nor are
such alterations seen when another b-lactamase is expressed,
indicating that the observed effects are not the consequence
of metabolic load caused by plasmid translation. It is thus
clear that AmpC production reduces the invasiveness of
S. enterica in a specific manner. Although not reported, the
fact that AmpC may be involved in peptidoglycan synthesis,
together with the evidence that protein–peptidoglycan in-
teractions modulate the assembly of the T3S translocon in
Salmonella (Pucciarelli & Garcia-del Portillo, 2003), suggests
that AmpC overproduction might produce subtle altera-
tions in the bacterial envelope that might alter T3S.
Resistance to glycopeptides in Gram-positive bacteria,
mediated by the vanA gene cluster, is a well-studied example
of the integration of a resistance determinant into bacterial
metabolism (for a review, see Mainardi et al., 2008). This
cluster, present in the transposon Tn1546 (Arthur et al.,
1993), encodes VanH dehydrogenase, which reduces pyru-
vate to form D-Lac, and VanA ligase, an enzyme that
catalyses the formation of an ester bond between D-Ala and
D-Lac. This pathway forms the depsipeptide D-Ala-D-Lac,
which is used for the biosynthesis of peptidoglycan with a
structure that ends in D-Lac instead of D-Ala (Arthur et al.,
1992; Handwerger et al., 1992). The resistance mechanisms
are therefore integrated into the physiology of the bacteria.
Nevertheless, this integration somehow alters microbial
metabolism. The substitution of D-Ala by D-Lac is well
tolerated by the enzymes involved in the peptidoglycan
assembly, but the modified precursor challenges the in vivo
FEMS Microbiol Rev 33 (2009) 44–65
53
activities of PBPs. For instance, PBP5 is a low-affinity PBP
responsible for resistance to ampicillin and third-generation
cephalosporins. The induction of VanA-mediated glycopep-
tide resistance increases susceptibility to b-lactam antibio-
tics, indicating that the D,D-transpeptidase PBP5 does not
function properly with peptidoglycan ending in D-Lac.
Antibiotic-dependent bacterial growth
The existence of bacterial mutants dependent on the pre-
sence of an antibiotic for their growth was recognized soon
after antibiotics became available in medicine (Gocke &
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Finland, 1950; Barber, 1953; Worthington et al., 1999). Such
dependence does not mean that bacteria use antibiotics as a
food resource (Dantas et al., 2008). Because dependence is
usually linked to resistance, the underlying reason might be
the metabolic rewiring produced by the resistance mechan-
ism now only allowing the growth of the bacterium in the
presence of the antibiotic. The causes of antibiotic-depen-
dent growth have been studied in vancomycin-resistant
enterococci (Baptista et al., 1997; Sifaoui & Gutmann,
1997; Van Bambeke et al., 1999). As stated above, resistance
to glycopeptides involves the formation of a D-Ala-D-Lac
depsipeptide. Under strong selective pressure and in the
presence of the vancomycin-resistance cluster, the activity of
the host D-Ala : D-Ala ligase is not required because the
bacteria make use of an alternative ligase involved in
resistance (VanA or VanB) that ligates D-Lac to D-Ala. Thus,
the ddl gene encoding the host D-Ala : D-Ala ligase accumu-
lates mutations and bacteria can only grow in the presence
of the antibiotic because it induces expression of the
alternative, antibiotic-resistance-linked ligase (Baptista
et al., 1997; Sifaoui & Gutmann, 1997; Van Bambeke et al.,
1999).
Remodelling bacterial structure as the
consequence of antibiotic resistance
The first careful biochemical studies on the changes in
bacterial structure as the consequence of antibiotic resis-
tance examined the effect of penicillin resistance on the
structure of pneumococcal cell walls (Garcia-Bustos et al.,
1988; Garcia-Bustos & Tomasz, 1990). Penicillin resistance
in this bacterial species is due to the molecular remodelling
of the cell wall synthesis enzymes (PBPs), and consequently
a different biosynthetic activity of the cell wall. It is reported
that, while the peptidoglycan of antibiotic-susceptible
strains contains monomeric and oligomeric forms of pri-
marily linear stem peptides with the sequence L-Ala-D-iGln-
L-Lys-D-Ala (where iGln is isoglutamine), the major peptide
species of the cell walls of resistant bacteria are branched-
stem peptides carrying Ala–Ser or Ala–Ala dipeptides on the
E-amino groups of lysine residues (Garcia-Bustos & Tomasz,
1990). This remodelling of the cell wall structure has been


c2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
54 J.L. Martinez et al.

observed for other antibiotics targeting peptidoglycan bio-
synthesis and has been recently reviewed in Mainardi et al.
(2008). Resistance to two classes of antibiotics, namely
glycopeptides and b-lactams, strongly modifies the structure
of the peptidoglycan in Gram-positive bacteria.
The main conclusion of these studies is that antibiotic
resistance, rather than being a general metabolic burden,
produces specific changes in the metabolism and in the
structure of bacterial pathogens that can only be fully
understood from a global perspective.
best-studied bacterial networks involved in antibiotic resis-
tance. Since its first description, the mar system has been
identified in several bacterial genera, mainly belonging to
Enterobacteriaceae (Cohen et al., 1993a, b; Sulavik et al.,
1997; Kunonga et al., 2000; Udani & Levy, 2006). The
operon marRAB encodes MarR, a repressor that down-
regulates marRAB expression, MarA, a global activator that
regulates the expression of a large number of genes includ-
ing marA, and marB, which encodes MarB, a small protein
for which an independent effect has not been demonstrated
but that increases antibiotic resistance when present along-

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Global regulatory networks and antibiotic side MarA (White et al., 1997). MarA regulates, directly or
resistance indirectly, the expression of several genes (Barbosa & Levy,
2000) that encode proteins with different functions in
Intrinsic antibiotic resistance is a specific feature of any
general metabolism, DNA repair and translation, pH and
given bacterial species. It is thus conceivable that, like other
superoxide stress responses, and low-level resistance to
characteristic phenotypes, it is integrated into global regula-
antibiotics, disinfectants and organic solvents (Cohen et al.,
tory networks. A good example of the need to study
1993a, b; Martin et al., 1996; Alekshun & Levy, 1997, 1999;
antibiotic resistance from a global perspective is that pro-
Pomposiello et al., 2001). Two other regulatory proteins
vided by the protein IgaA. A study of mucoid mutants of
homologous to MarA, SoxS and Rob are able to activate the
S. enterica resistant to mecillinam revealed that mutations in
Mar regulon directly or indirectly.
the gene mucM, leading to RcsCDB overexpression, pro-
The use of bioinformatics, genomics and molecular
duced mucoidity and antibiotic resistance (Costa & Anton,
genetics in the analysis of transcriptomic data led to the
2001). Independent work undertaken by another group
conclusion that these three transcriptional activators di-
(Cano et al., 2001) characterized the protein IgaA as an
rectly activate a common set of 40 promoters in E. coli
attenuator of intracellular growth required for virulence in
(Martin & Rosner, 2002). Each activator is regulated by
S. enterica. It has since been shown (Mariscotti & Garcia-Del
different signals: aromatic weak acids such as salicylate
Portillo, 2008) that mucM and igaA are the same gene, and
increase marRAB transcription (Cohen et al., 1993a, b),
that it is simultaneously involved in resistance and virulence
superoxides trigger soxS transcription (Amabile-Cuevas &
in S. enterica. Further work showed that mutations leading
Demple, 1991; Wu & Weiss, 1991) and bile salts, decanoate
to unstable forms of IgaA induce the RcsCDB system,
and bipyridyl activate Rob (Rosenberg et al., 2003).
leading to overproduction of colanic acid (Mariscotti &
The marA/soxS/rob regulon therefore encompasses a
Garcia-Del Portillo, 2008), and that RcsCDB overactivation
complex network that produces a global bacterial response
in IgaA-defective strains attenuates the acute virulence of
to external signals. The upregulation of the MarA, SoxS and
S. enterica (Tierrez & Garcia-del Portillo, 2004). Later it was
Rob activators increases antibiotic efflux (acrAB-tolC), DNA
shown that RcsCDB provides a phospho-relay system con-
repair (nfo) and superoxide resistance (zwf, fpr and sodA),
sisting of a hybrid sensor kinase, a phosphotransferase and a
and reduces outer membrane permeability (ompF via micF).
response regulator. RcsCDB regulates a large number of
It is important to note here that the main resistance system
bacterial genes (Majdalani & Gottesman, 2007), including
regulated by MarA is the efflux pump AcrAB-TolC. This
those involved in capsule production, which are important
MDR pump can efflux antibiotics and bile salts (Ma et al.,
in intrinsic resistance to b-lactams such as mecillinam
1995; Thanassi et al., 1997; Prouty et al., 2004). Because bile
(Laubacher & Ades, 2008). This example shows how two
salts are common compounds in the natural habitat of
independent but convergent studies, one focusing on viru-
Enterobacteriaceae it is likely that the original function of
lence and the other on resistance, concluded the involve-
AcrAB-TolC was the resistance of these detergents. It has
ment of the same elements, illustrating the need to make
now been shown that AcrAB-TolC is required for chicken
studies on these topics more integrated. Below, two exam-
colonization and virulence by S. enterica (Buckley et al.,
ples of networks that simultaneously modulate several
2006), highlighting that this MDR determinant has other
different bacterial phenotypes, including resistance, are
functions besides resistance.
discussed.
The rules that govern the differential expression of the
members of this network are beginning to be understood. It
The mar regulon
has been shown recently that both the abundance and the
The mar (from multiple antibiotic resistance) regulon type of activator trigger different responses in the network
(George & Levy, 1983; Cohen et al., 1989) is one of the (Martin et al., 2008). For instance, both MarA and SoxS can

c 2008 Federation of European Microbiological Societies FEMS Microbiol Rev 33 (2009) 44–65
Published by Blackwell Publishing Ltd. All rights reserved
Antibiotic resistance
saturate in vivo the marRAB and sodA promoters. However,
saturation with MarA leads to more marRAB and less sodA
expression than does SoxS saturation. The extent to which
genes of this operon are activated is a function of the MarA
concentration, a phenomenon termed ‘commensurable reg-
ulon activation,’ because it allows E. coli to elicit a propor-
tional response to stress signals (the activation of the
minimum number of genes required to evade this signal)
(Martin et al., 2008). It was further suggested that this type
of activation allows bacteria to mount different defensive
responses depending on the type of signal received, the
amplitude of the signal and its duration. This regulon thus
provides a flexible response to different kinds and level of
threat, including the presence of antibiotics.
The mgrA regulon
MgrA was simultaneously described by different groups as a
regulator of the expression of the MDR pump NorA of
S. aureus (Truong-Bolduc et al., 2003), of the autolysis
process in this bacterial species (Ingavale et al., 2003) and
as a general regulator affecting the transcription of S. aureus
a-toxin, nuclease and protein A, among others (Luong et al.,
2003). Since then, it has been shown that around 350 genes
are regulated by MgrA (Luong et al., 2006). These genes
include several MDR pumps (Truong-Bolduc et al., 2003,
2005) and virulence factors (Ingavale et al., 2005). Notably,
the expression of several metabolic genes is regulated by
MgrA, highlighting the crossconnection between resistance,
virulence and the global metabolism of bacterial pathogens.
Although it has been shown that MgrA binds some
promoter regions (Truong-Bolduc et al., 2003), several of
the observed effects are likely indirect. Moreover, the fact
that some genes are upregulated by MgrA in one growth
phase while they are downregulated in another indicates that
there must be different pathways of regulation of the mgrA
regulon (Luong et al., 2006). This indirect effect can be
mediated by the activity of MgrA in the regulation of other
regulators, as seen in S. aureus autolysis.
Murein hydrolases in staphylococci have important roles
in cell division, including daughter cell separation and
peptidoglycan recycling. Their expression is tightly con-
trolled because, if they are expressed at too high a level, they
destroy the cell wall and cell lysis ensues. It has been shown
that MgrA regulates expression of the autolytic regulators
lysSR, lgrAB and arlRS (Ingavale et al., 2005). Mutations in
mgrA result in enhanced autolysis of S. aureus and increased
susceptibility to detergents and penicillin.
As mentioned above, MgrA regulates the expression of
several MDR pumps in S. aureus. However, its effect on
susceptibility to penicillin is also linked to its role in
regulating penicillin-induced autolysis, indicating that mul-
tiple layers exist in the regulation of resistance by MgrA.
FEMS Microbiol Rev 33 (2009) 44–65
55
Altogether, this indicates that the antibiotic resistance of
S. aureus is under the control of global regulation networks
that involve virulence determinants as well as basal meta-
bolic or structural genes.
Metabolic control of antibiotic resistance
It has already been shown that antibiotic resistance can
produce specific changes in bacterial metabolism and that
resistance is integrated into global regulatory networks. It is
thus conceivable that resistance might be controlled by the
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metabolic condition of bacteria. The clearest example of this
noninherited phenotypic resistance (Martinez et al., 1994;
Levin & Rozen, 2006) is the lack of effect of several
antibiotics against cells that are not actively dividing (dor-
mant cells). Known as ‘antibiotic indifference,’ this phenom-
enon was recognized soon after the first use of antibiotics
(Lee et al., 1944; McDermott, 1958). It is of no importance
in infections produced by actively growing bacteria, but
reservoirs of nongrowing bacteria can be important in the
persistence and reappearance of some infections. The ex-
istence of dormant cells helps to explain the presence of
persistent subpopulations in antibiotic-susceptible bacterial
populations, and in the phenotypic resistance shown by
bacterial biofilms. The global regulation shown by bacteria
in response to particular modes of life is also important in
noninherited resistance (Levin & Rozen, 2006). This is
discussed below.
Swarming motility and global regulation
Swarming is a specific type of motility across a semisolid
surface commonly demonstrated by flagellated bacteria
(Fraser & Hughes, 1999; Jarrell & McBride, 2008). It is a
multicellular phenomenon dependent on bacterial cell den-
sity, nutrients conditions and surface moistness.
Swarmer cells show structural changes, including an
increase in their number of flagellae or in cell length. They
also show substantial alterations in their metabolism, in-
dicating that swarming is a global lifestyle adaptation in
response to specific conditions rather than just a form of
locomotion. In Salmonella, swarming is associated with
phenotypic antibiotic resistance (Kim & Surette, 2003).
Recent work has shown that swarming in P. aeruginosa is
influenced by a large number of cooperating genes (Overhage
et al., 2007), and that swarmer P. aeruginosa cells show
altered levels of expression for 407 genes. Several virulence
determinants and one MDR efflux pump are also upregu-
lated in swarmer cells, indicating that this phenomenon is a
global response that might be useful in host colonization in
the presence of several antibiotics. One characteristic feature
of swarmer cells is their enhanced resistance to polymyxin B,
gentamicin and ciprofloxacin; it has been suggested that this


c2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
56
is due to metabolic differentiation during swarming and due
to changes in the cell envelope (Overhage et al., 2008).
Persistence
Persistence is a phenomenon by which bacterial subpopula-
tions enter a refractory state in the presence of different
antibiotics (Balaban et al., 2004; Levin, 2004), and has been
described in several species. Around one in every million of
cells in the exponential growth phase, and around one in
every hundred in the stationary growth phase possess a
persistence phenotype. Persistent cells can grow when the
antibiotic disappears, but, like the susceptible wild-type
population, they are killed if the antibiotic is added again
(Fig. 6). This indicates that persistence is not the conse-
quence of mutations. It has been suggested that persistence
provides a means of survival in changing environments
(Kussell et al., 2005).
It was first suggested that, in asynchronous cultures,
persistence was the consequence of the presence of a
bacterial subpopulation still in the lag period. These cells
are in a dormant state and thus refractory to antibiotics.
Single-cell analysis using microfluidic devices allowed two
types of persisters to be defined. Type I is represented by the
preexisting population of growing cells generated during the
stationary phase that did not resume growth. Type II
persisters are the consequence of a switch between normal
Fig. 6. Bacterial persistence to antibiotics. Persistence in the presence of
an antibiotic is characterized by the presence of a small bacterial
subpopulation refractory to the antimicrobial agent. If the antibiotic is
removed (arrow), this population resumes growth. If it is added again,
growing bacteria are killed, indicating that persistence is a noninherited
resistance phenotype.


c 2008 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
J.L. Martinez et al.
and persister cells that occurred during growth. This switch
can be mathematically described in the framework of a
simple two-state model (Balaban et al., 2004). Recent work
indicates that the dormancy shown by Type I persistence is
not completely attributable to a stationary-phase state.
Single-cell analysis has shown that protein production takes
place over a small time window (around 1.5 h for E. coli) in
Type I persisters following their exit from the stationary
phase (Gefen et al., 2008). This suggests that the dormancy
that protects persisters from antibiotics does not occur
during the stationary phase but rather develops after this
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phase is over. Notably, during this time window, persistent
cells are as susceptible as normal cells to antibiotics, indicat-
ing that, even for Type I persisters, this phenotype implies a
metabolic switch in a subset of the bacterial population
(Gefen et al., 2008).
Compelling evidence supports the notion that persistence
is highly regulated. Persistence might well be explained by
the activity of toxin/antitoxin systems (Keren et al., 2004;
Lewis, 2008), whose actions maintain a fraction of the
population in a nongrowing state. Recent work confirms
this, and indicates that persistence is due to several mechan-
isms involving the participation of a number of global
regulators and metabolic enzymes (Hansen et al., 2008).
Biofilms
Most studies in microbiology are performed with bacteria
growing planktonically, but bacterial populations frequently
grow attached to surfaces where they form biofilms (Cost-
erton et al., 1999; Hall-Stoodley et al., 2004; Kolter &
Greenberg, 2006; Hansen et al., 2007). There is still some
debate as to whether a specific program exists for triggering
biofilm formation (O’Toole et al., 2000; Stoodley et al.,
2002) or whether growth in biofilms is simply due to the
physicochemical characteristics of the cell envelopes and
substrate, and due to the fluid dynamics (including bacterial
motility) of the system (van Loosdrecht et al., 2002).
However, it is clear that bacteria growing in biofilms
experience changes in microbial metabolism (Whiteley
et al., 2001; Sauer et al., 2002; Waite et al., 2005, 2006),
including the development of greater resistance to antibio-
tics than planktonically growing cells (Drenkard & Ausubel,
2002; Davies, 2003). Several hypotheses have been proposed
to explain this more resilient phenotype (Stewart, 2002),
some of them involving the structure of the biofilm and
others involving the metabolic state of biofilm-growing
bacteria. For instance, the structure of the biofilm itself
might preclude the entrance of antibiotics through the
matrices (e.g. oligosaccharides or DNA) that form part of
biofilms (Suci et al., 1994). Because biofilms are structured
environments (Lawrence et al., 1991) containing regions
with different amounts of oxygen and nutrients, the
FEMS Microbiol Rev 33 (2009) 44–65
Antibiotic resistance
metabolic activity in different parts of the biofilm is likely
different (Huang et al., 1995; Sternberg et al., 1999); thus,
biofilms are home to bacterial populations with different
metabolic statuses. Owing to the scarcity of nutrients, it has
been suggested that subpopulations of bacteria forming
biofilms enter a dormant state, similar to that seen in
antibiotic persisters.
The study of the response of mature, preformed biofilms
to antibiotics may provide more insight into noninherited
resistance. Resistance in biofilms is dependent on the
structure of the biofilm and on the type of antibiotic used
(Folkesson et al., 2008). Escherichia coli in mature biofilms
are resistant to the antibiotic peptide colistin but not to the
fluoroquinolone ciprofloxacin. Increased survival in the
presence of polymyxin is linked to modifications in bacterial
lipopolysaccharide that favour sequestration of the antibio-
tic. These modifications are the consequence of upregula-
tion of the yfbE operon mediated by the two-component
system basR/basS. This specific response is produced before
polymyxin is added to the culture. Thus, it is not a classically
induced resistance mechanism but the consequence of the
metabolic changes experienced by a bacterial subpopulation
in response to as yet ignored signals in given locations of the
biofilm structure (Folkesson et al., 2008). It is important to
note that the Salmonella typhimurium operon pmrH, which
is homologous to yjbE, is induced during swarming (Kim &
Surette, 2003), a phenomenon during which reduced sus-
ceptibility to antibiotics is also shown.
Recent studies have shown that MDR efflux pumps can
specifically contribute to phenotypic resistance in biofilms
(Zhang & Mah, 2008). The metabolically heterogeneous
nature of biofilms is highlighted by a study showing that
P. aeruginosa growing in biofilms is tolerant to ciprofloxacin
or tetracycline in the deeper areas, whereas tolerance to
colistin is restricted to the upper regions (Pamp et al., 2008).
Cells in the deepest regions of biofilms show low metabolic
activity, consistent with their refractory state. However, the
upper layer of a biofilm contains metabolically active
bacteria; thus, resistance to colistin cannot be due to
dormancy. It is reported that tolerance to colistin in
P. aeruginosa biofilms is dependent on the pmr operon,
which mediates lipopolysaccharide modification, and on the
MDR pump MexAB-OprM (Pamp et al., 2008). Notably,
this efflux pump does not contribute to intrinsic resistance
to colistin in planktonic P. aeruginosa cells, although it seems
to be important to subpopulations surviving the presence of
colistin within P. aeruginosa biofilms (Pamp et al., 2008). All
this indicates that the tolerance of biofilms to antibiotics is
mediated by several different elements in bacterial subpo-
pulations with different metabolic signatures.
It is thus clear that different bacterial ways of life can
trigger the expression of specific sets of genes that reduce
susceptibility to antibiotics. Understanding this type of
FEMS Microbiol Rev 33 (2009) 44–65
57
process will help in the fight against noninherited antibiotic
resistance.
Acknowledgements
Work in our laboratory is supported by grants BIO2005-
04278 and BIO2008-00090 from the Spanish Ministerio de
Educacion y Ciencia, and LSHM-CT-2005-518152 and
LSHM-CT-2005-018705 from the European Union. A.F. is
the recipient of a fellowship from the Spanish Ministerio de
Downloaded from https://academic.oup.com/femsre/article/33/1/44/2683937 by guest on 09 December 2020
Educacion y Ciencia. We thank Adrian Burton for help with
the English manuscript.
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