5.rivas vivent etal 2006 (1)

The fallacies of hope:willwe discover newantibiotics to combat
pathogenic bacteria in time?
Miguel Vicente1, John Hodgson2, Orietta Massidda3, Tone Tonjum4, Birgitta Henriques-Normark5 &
Eliora Z. Ron6
1Centro Nacional de Biotecnolog´ıa, Consejo Superior de Investigaciones Cient´ıficas, Campus de Cantoblanco, Madrid, Spain; 2Novexel SA. Parc
Biocitech, Romainville, France; 3Dipartimento di Scienze e Tecnologie Biomediche, Sez. Microbiologia Medica, Universita` di Cagliari, Cagliari, Italy;
4Center for Molecular Biology and Neuroscience and Institute of Microbiology, University of Oslo, Oslo, Norway; 5Swedish Institute for Infectious
Disease Control and Microbiology and Tumorbiology Center, Karolinska Institutet, Stockholm, Sweden; and 6Department of Molecular Microbiology
and Biotechnology, Faculty of Life Sciences, Tel Aviv University, Israel
Correspondence: Miguel Vicente, Centro
Nacional de Biotecnolog´ıa, CSIC Campus de
Cantoblanco, c/Darwin 3, E-28049 Madrid,
Spain. Tel.: 134 91 585 46 99; fax: 134 91
585 45 06; e-mail: mvicente@cnb.uam.es
Received 14 February 2006; revised 10 May
2006; accepted 25 May 2006.
First published online 19 July 2006.
DOI:10.1111/j.1574-6976.2006.00038.x
Editor: Ramo´ n D´ıas Orejas
Keywords
antibiotics; antibiotic resistance; drug
development; antimicrobial targets; infectious
disease; genomics.
Abstract
While newly developed technologies have revolutionized the classical approaches
to combating infectious diseases, the difficulties associated with developing novel
antimicrobials mean that these technologies have not yet been used to introduce
new compounds into the market. The new technologies, including genomics and
structural biology, open up exciting possibilities for the discovery of antibiotics.
However, a substantial effort to pursue research, and moreover to incorporate the
results into the production chain, is required in order to bring new antimicrobials
to the final user. In the current scenario of emerging diseases and the rapid spread
of antibiotic resistance, an active policy to support these requirements is vital.
Otherwise, many valuable programmes may never be fully developed for lack of
‘‘interest’’ and funds (private and public). Will we react in time to avoid potential
disaster?
Introduction: the hope and the fallacies
In the 21st century affluent societies live under the impres-sion
that they are free fromthe attack of pathogenic bacteria,
and, moreover, that if they by any chance do suffer an
infection, there will be an antibiotic to cure the disease. In
this article we postulate that, if the present discovery
scenario does not change rapidly, this impression is false.
Furthermore, if we base our future health on the hope that
new antibiotics to combat infectious diseases will be avail-able
within a short time, we, as a society, and certainly as
individuals, may eventually be confronted by a catastrophic
event.
Marketed antibiotics are generally safe drugs that have
been successfully used to combat infectious diseases for the
past sixty years. They have been both wonderful medicines
and lousy consumer goods. As antibiotics can cure infec-tions
they have kept us free from many plagues that were the
scourges of humanity until the second half of the 20th
century (Armstrong et al., 1999). However, a paradox of
the effectiveness of antibiotics is their weak value as market-able
goods: patients stop buying them once their health
returns, after relatively short courses of treatment. In con-trast,
the drugs prescribed for chronic diseases have to be
taken for life. The production of antibiotics might be made
more appealing to the industry if they could be priced to
satisfy the need for adequate capital returns; however, this
would impose an extra burden on the public health budget.
Although it may seem obvious, it is essential to point out
that the antibiotics that were easy to discover have already
been found, and it is likely that the search for new members
of existing classes, and certainly for new classes of antibio-tics,
will involve a substantial amount of high-quality,
expensive and laborious research. Besides presenting the
need for research to find new antibiotics, we discuss how
new technologies may help in this search and briefly suggest
some strategies that should guide the implementation of
adequate research programmes.
The need for new antimicrobials
While a large battery of antibiotics to combat most bacterial
diseases is presently available, several alarms have recently
been raised on the need to develop new antimicrobials
FEMS Microbiol Rev 30 (2006) 841–852 c 2006 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved

842 M. Vicente et al.
(references are too numerous to be cited individually, but a
comprehensive collection of reviews can be found in Cour-valin
Davies, 2003). This need has arisen for several
reasons, among them the spread of antibiotic resistance, the
threat of emerging and re-emerging pathogens, and the
consequential high social and economic impact of infectious
diseases. Vaccination, a classical antimicrobial weapon, is
able to prevent the onset of infection, but it does not usually
cure it once it is established. It is for this reason that vaccines
must be considered as agents to prevent, rather than to heal,
infectious diseases. Moreover, despite the indisputable his-torical
success of vaccines to combat some important
bacterial pathogens, the prevalence of different serotypes,
the complexity and variability of virulence among the most
frequent pathogens, and the difficulties confronting their
development further restrict their utility. Nevertheless, when
available, besides preventing disease, vaccines, such as the
pneumococcal one, may also help to reduce the frequency of
antibiotic-resistant isolates (Schrag et al., 2004). This is,
however, controversial as some studies have failed to find a
reduction of resistance after pneumococcal vaccination,
concluding that, besides vaccination, a reduction in the
antibiotic pressure may be needed to reduce the resistance
frequency (Fraza˜o et al., 2005).
Despite the fact that the need for new antibiotics has been
felt for some time, at the moment it appears that many
clinicians are satisfied with the available ones. Thus an
informal enquiry of medical professionals working in Ma-drid
and Cagliari hospitals (J. Bl ´azquez O. Massidda, pers.
commun.) indicated that only about one-third of them
thought that the discovery of new antibiotics was urgently
required. The rest were satisfied that most ‘‘normal’’ cases
can be treated with one or a few available drugs, despite their
estimates that antibiotic therapy failure in compromised
patients could be as high as 15%. The opinion of many
clinicians, that new antibiotics are not required so urgently,
may be based on their relative abundance when compared
with the number of drugs available to treat other diseases
(viral infections, tumours, etc). Moreover, if treatment with
a single drug fails, the option to associate two or more
antibacterials that exert a synergistic action is frequently
successful. In contrast to laboratory testing, clinical practice
shows both that in vitro susceptibility of a pathogen to a
given antibiotic is not a full guarantee of therapeutic success
in the patient, and that therapeutic failure is not always
caused by antibiotic resistance (Greenwood, 1981; Sanders,
1991; Phillips, 2001; Varaldo, 2002). In addition, pathogens
resistant to a certain antibiotic in vitro can sometimes be
eradicated with that antibiotic, as is the case of some
infections caused by penicillin intermediate-resistant pneu-mococci
(Bishai, 2002). The belief that the need for new
antibiotics is not pressing may then appear as numerically
justified, but for cases involving elderly or immunocompro-mised
patients, for whom the prognosis is so dangerously
poor, the development of new treatments should be a matter
of priority.
Social and economic impacts of bacterial
infectious disease
Even now, in the antibiotic era, common infectious diseases
are major contributors to morbidity and mortality, in
particular in the developing world, but also in the developed
world (World Health Organization, 1996). In developing
countries, infectious diseases, many of them caused by
bacterial pathogens, cause over 60% of total deaths. They
are the third leading cause of death in Europe, mostly in
elderly and debilitated populations, and, despite existing
antibiotic therapies and vaccines, they remain the leading
cause of mortality and morbidity worldwide.
If we take pneumococcal disease as an example, in the
United States, despite access to antibiotics and intensive
care, the mortality rates in invasive pneumococcal infections
remain high: 5% of pneumonia cases, 20% of septicaemia
cases, and 30% of meningitis cases (Tomasz, 1997).
Although groups of any age may be affected, small children
and the elderly are at higher risk. It is estimated that about
20 million children contract pneumococcal pneumonia
every year and that over 1 million die from the disease
(Klein, 1999). The risk of contracting pneumonia, lower
than 1% at ages below 19, rises to 12% at 70, and therefore
the risk of death as a consequence of this disease is over one
in a thousand for individuals older than 50 (MacFarlane
et al., 1993; Bartlett et al., 1998). With the elderly becoming
a larger segment of the population as a consequence of
improved living standards in developed societies, there will
be a need to re-examine whether the toll caused by what is
commonly perceived as a problem of the past is socially
acceptable. Vaccination, although being a valuable proce-dure
to curb pneumonia, does not provide full protection
because not all individuals respond equally well to the
immunization and because the immunity provided by the
available capsular polysaccharide-based vaccines does not
cover all the possible serotype variants of the pathogen
(Bernatoniene Finn, 2005).
A similar question can be raised concerning those patients
who, for diverse medical reasons, are immunocompromised.
Their numbers, as medical procedures continue to improve
technically, are likely to rise, creating another segment of the
population with an increased risk of succumbing to infec-tions.
In developed countries, nosocomial infections, occur-ring
in 5–7% of patients hospitalised for other reasons,
increase the hospital stay by an average of four days, with an
increased cost per day of nearly 500 h. If patients are in an
Intensive Care Unit both the risk and the cost are more than
doubled; their additional stay can extend up to 19 days, with
c 2006 Federation of European Microbiological Societies FEMS Microbiol Rev 30 (2006) 841–852

The fallacies of hope 843
a concomitant higher mortality rate, often associated with
antibacterial therapeutic failure (Kollef, 2003).
Emerging and re-emerging diseases
The need for further research in antibiotic discovery is vital
when considering the threat posed by the emergence of
previously unknown or uncommon infectious diseases
(Morens et al., 2004). A contemporary example is provided
by the frequent outbreaks of Legionella, an organism that
only became a serious health threat when the extensive use
of large air-conditioning systems created a favourable en-vironment
both for the multiplication of the pathogen and
for its delivery as aerosols to the human respiratory system.
Predictive microbiology studies, based on metagenomics
(an approach that allows the identification of the gene pool
present in a particular environment regardless of whether
genes are present within easily cultivable or uncultivable
microorganisms), may contribute in the future to identify-ing
unexpected potential pathogens following the identifica-tion
of the ‘‘resistome’’, that is, all the virulence-related genes
present in an environment (D’Costa et al., 2006).
Tuberculosis, a disease that was once considered to be
disappearing, has made a return in the recent past. This is
not only as a consequence of its association with AIDS, but
also because of the prevalence of strains of Mycobacterium
tuberculosis that are resistant to several of the drugs used to
combat the disease (World Health Organization, 2000).
Multidrug-resistant Mycobacteria arise for a complex set of
reasons, an important one being the high failure rates for
completion of therapeutic courses, which are often asso-ciated
with a lack of resources required to observe compli-ance
to relatively long-term therapeutic regimens. It seems
clear that, besides research, additional social measurements
are urgently required to deal with the problem of infectious
diseases in a global scenario.
Diseases caused by pathogenic bacteria that were not
previously a cause for concern are now receiving more
attention. This is the case for the virulent Escherichia coli
strains causing extraintestinal infections (ExPEC, extrain-testinal
pathogenic E. coli) (Johnson Russo, 2002). These
bacteria are involved in a diverse spectrum of diseases,
including urinary tract infections (UTI), newborn meningi-tis
(NBM), and abdominal sepsis and septicaemia (Mokady
et al., 2005; Ron, 2006). ExPEC infections are an increasing
problem for human health, especially in patients who are
immunocompromised owing to disease, chemotherapy or
old age, and even in the community they are a leading cause
of bloodstream infections, especially in newborns. Combat-ing
ExPEC infections is difficult because of the high in-cidence
of drug resistance often transmissible by plasmids
(Siegman-Igra et al., 2002; Girardeau et al., 2003; Maslow
et al., 2004; Blomberg et al., 2005; Branger et al., 2005;
Jackson et al., 2005).
Antibiotic resistance and antibiotic use
Although resistance to an antibiotic is perceived as a
problem only when it is manifested as a clinical therapy
failure, the use of antibiotics has been closely followed by the
emergence of antibiotic-resistant microbial populations that
in some cases are prevalent (Bush, 2004; Levy Marshall,
2004). In contrast to other drugs, antibiotics can start to lose
their efficacy immediately after their clinical use begins
through the development of antibiotic resistance by bacter-ial
pathogens. Pathogens can become resistant to antibiotics
through acquisition of resistance genes from other bacteria
or by modification of some of their own genes. In the case of
acquisition of resistance genes by pathogens, antibiotic-producing
organisms can be envisaged as a potential source
of antibiotic resistance genes (Davies, 1994).
Resistance genes that encode systems to either expel or
inactivate antibiotics occur naturally because many antibio-tic-
producing organisms need them to avoid self-destruc-tion.
However, it is not only naturally occurring
mechanisms that contribute to the persistence of antibio-tic-
resistant microorganisms, even under natural condi-tions.
It has been proposed that both virulence and
antibiotic resistance are adaptive mechanisms selected to
survive under stress conditions (either host invasion or
antibiotic treatment) (Mart´ınez Baquero, 2002). Thus it
is well documented that antibiotic usage boosts the fre-quency
of resistant organisms. Bacterial pathogens mutate
frequently even during the course of a single treatment, and
therefore their target can be modified to confer resistance in
a very short time after the introduction of a new drug. In the
most puzzling cases (as was the case for penicillin and more
recently for linezolid, an oxazolidinone that interacts with
the peptidyl-tRNA binding P site at the 50S subunit), the
emergence of resistant microorganisms has even preceded
the clinical use of some antibiotics (Bush, 2004). The success
of the drug industry in introducing different classes of new,
effective antibiotics into medical use has been met by further
developments in antibiotic resistance such that multidrug-resistant
bacterial pathogens are now increasingly common.
Once a resistance gene is present in a bacterial population
it can be transferred to similar bacteria by natural processes.
One of these involves the transfer of antibiotic resistance
genes from plasmids. Such resistance plasmids are ubiqui-tous
and often carry a battery of resistance genes. They are
often conjugative and contain toxin–antitoxin addiction
systems that ensure their continuous presence in the popu-lation
(Jensen et al., 1995; Smith Rawlings, 1998; Rawl-ings,
1999; Camacho et al., 2002; Deane Rawlings, 2004;
Zielenkiewicz Ceglowski, 2005). In addition, there are also

chromosomal toxin–antitoxin systems, some of which may
be induced by antibiotics, resulting in interference with
bacterial proliferation and intensifying the effect of the drug.
Moreover, these toxins are able to interfere with basic and
general biological processes such as bacterial DNA and
protein synthesis, and, being present in most free-living
prokaryotes, including many bacterial pathogens, but absent
from eukaryotes, they could serve to develop new antimi-crobials
(Jensen et al., 1995; Smith Rawlings, 1998;
Rawlings, 1999; Camacho et al., 2002; Deane Rawlings,
2004; Engelberg-Kulka et al., 2004; Gerdes et al., 2005;
Pandey Gerdes, 2005; Zielenkiewicz Ceglowski, 2005).
Although the acquisition of a resistance gene often
imposes a toll on the fitness of the resistant microorganism,
compensatory mutations may alleviate it, and, when suc-cessful,
may block the reversion to the sensitive phenotype
(Fig. 1) (Andersson, 2003). These naturally occurring me-chanisms
contribute to the persistence of antibiotic-resistant
microorganisms, even under natural conditions. In addition
to the inherited or acquired resistances, diverse conditions
associated with the physiology of bacteria may also play an
important role in antibiotic resistance (Mart´ınez Baquero,
2002). An interesting example of antibiotic resistance asso-ciated
with a behavioural change is observed for bacteria
growing in biofilms. Not only are bacteria in biofilms more
resistant to antibiotic treatment, but also, in certain cases,
the antibiotic itself may induce biofilm formation (Hoffman
et al., 2005).
The spread of resistance in clinical and
community settings
Nowhere is the problem of bacterial resistance to conven-tional
antibacterial therapy more apparent and critical than
in the hospital environment (Farr et al., 2001; Cant ´on et al.,
2003). In industrialized countries, over half of hospital-acquired
infections are caused by drug-resistant microor-ganisms.
Most bacterial species that are capable of causing
infections have acquired resistance to at least one antibiotic,
and many have resistance to multiple drugs. This complex
problem is related, as we have discussed, to the degree of
exposure to antibiotics, and is exacerbated by inappropriate
use in both developed and developing regions. In conse-quence,
antibiotic resistance poses one of the greatest
challenges facing public health officials today, because the
increased resistance of bacteria to many antimicrobials
results in significant increases in health-care costs. For
example, the emergence of multidrug-resistant M. tubercu-losis
has forced the use of drugs that are one hundred times
more expensive than traditional therapy (Murray, 2006).
The resistant pathogens, including methicillin-resistant
Staphylococcus aureus (MRSA), and vancomycin-resistant S.
aureus (VRSA) and Enterococci (VRE), are no longer con-fined
to hospitals, but are also found in community settings
(Kourbatova et al., 2005; Stevenson et al., 2005; Weber,
2005).
Bacterial resistance is increasing not only in those bacteria
that have always been poorly susceptible to antimicrobial
therapy, but also in those that for years have been considered
exquisitely sensitive to antimicrobial drugs. For example, in
Spain and France more than 50% of the Streptococcus
pneumoniae strains in 2000–2001 were not susceptible to
penicillin (Jones et al., 2003). In the USA, penicillin resis-tance
in S. pneumoniae is as high as 33% (Felmingham et al.,
2002). Furthermore, multidrug resistance (resistance to
more than two classes of antibiotics) has been observed in
more than 50% of pneumococcal isolates in Hong Kong,
Taiwan and South Korea (Felmingham, 2004). In Southeast
Asia, combined resistance (chromosomal- or plasmid-borne)
to penicillins among gonococcal isolates ranges from
48% in Vietnam to 98% in Korea, invalidating the use of
cheap therapies to fight against the disease (The WHO
Western Pacific Gonococcal Antimicrobial Surveillance Pro-gramme,
2001). In a worst-case scenario, the emergence of
resistance towards a variety of antibiotics may lead to
treatment failure in all patient classes, not only the elderly
and the immunocompromised. As it takes a long time to
develop a new antibiotic for clinical use, in the future we
Fig. 1. Compensatory mutations may help to fix antibiotic resistance in
bacteria. In many instances pathogens gain resistance to an antibiotic at
the expense of a decrease in fitness. This may be as a result of the burden
imposed by the need to express an extra set of genetic information in the
presence of the antibiotic. In the absence of other pressure, antibiotic-resistant
strains should be overridden by sensitive strains once the
antibiotic is withdrawn from the environment. Compensatory mutations
ameliorate the cost of resistance and may then work to fix the antibiotic-resistant
microbial population even in the absence of antibiotic selective
pressure (Andersson, 2003; this figure is adapted from D. Andersson,
pers. commun.).

may be faced with bacterial infections that may be resistant
to all available drugs and find that it is too late to react.
The cost of drug development: impact on
anti-infectives
A significant number of pharmaceutical (Pharma) compa-nies
have abandoned their anti-infectives research pro-gramme
in the recent past. This trend can be highlighted by
the observation that it is quicker to name the few that still
retain a programme, even if it is not prioritized, than to
enumerate those who have abandoned their anti-infective
research. This change in strategy is driven by ‘‘return on
investment’’ considerations.
The cost of bringing a new drug to market is estimated to
be more than 800 million h. Costs have spiralled upwards
owing to more stringent regulatory requirements in safety,
efficacy and manufacturing, and will probably continue to
increase as a result of the fall off in ‘‘large Pharma’’
productivity, as measured by the number of new drugs
approved in recent years (Tufts Center for the Study of Drug
Development, 2003). Substantially increased costs have
therefore focused the minds of many large Pharma company
executives on the development of the so-called ‘‘blockbus-ter’’
drugs that produce annual returns greater than 900
million h, with the outcome that there has been a significant
reallocation of resources to those therapeutic areas in which
the medical need is for therapies for chronic conditions.
As large pharmaceutical companies have headed for the
exit (Projan, 2003), concerns increase that the industry will
no longer be able to meet future needs for new and effective
antibacterial therapies. These concerns are supported both
by the steady decrease in the number of approved new
antibacterial agents since the mid-1980s and by the failure
to bring new class agents, with the recent exceptions of
ZyvoxTM (linezolid) and CubicinTM (daptomycin, a cyclic
lipopeptide antibiotic showing a unique mechanism of
action that results in destruction of the membrane poten-tial),
to the marketplace. However, there may be cause for
some optimism in that anti-infective research and design
has expanded in biotechnological (Biotech) organizations
and small Pharma companies. Success in delivering new
anti-infectives to market will depend in large measure on the
ability of the Biotechs to attract the investment required to
move novel compounds through clinical trials. This may
require some rethinking of business models (Barrett, 2005).
Historically, antibiotics have been discovered by screening
natural products for antibiotic activity and subsequently
chemically modifying these structures to incorporate addi-tional
desirable pharmacological properties. This approach
fuelled antibiotic drug discovery in the mid to late 20th
century. However, after the realization by the pharmaceu-tical
industry that not all infectious disease problems had
been solved and that drug resistance was a serious issue,
this ‘‘classical’’ approach was no longer considered to be
sufficient to provide the novel antibiotics required to
meet the new medical needs. So, what might be of value to
novel antibiotic discovery from the array of new technolo-gies
emerging from academia and the pharmaceutical
industry?
Genomics, a recent tool for the discovery
of new targets -- advantages and pitfalls
Comparative genomics yields information on the univers-ality
of targets in important pathogens. A naive view
predicted that simple criteria would allow the identification
of the ‘‘ideal’’ bacterial targets; for example, those bacterial
gene products that are absent in humans would be expected
to be less likely to cause safety issues (Moir et al., 1999;
Rosamond Allsop, 2000).
A recent genomic search comparing the genomes of three
important pathogens, Haemophilus influenzae, S. pneumo-niae
and S. aureus, indicated that more than 350 bacterial
genes are possible targets (Payne, 2004). After identifying a
target at the genome level, substantial additional work is
required in order to obtain sufficient information on its
properties to confirm its suitability for exploitation in anti-infective
drug discovery. Exploitation of the target requires
further work to set up an assay and validate it for high-throughput
screening (HTS). While genomic technologies
are amenable to large-scale analysis, being so useful for the
initial stages of target identification, most of the subsequent
work required to characterize fully and exploit an already
identified target is not so easily scalable.
In addition to target identification, genomics can help to
refine and validate targets by analysing changes in the
expression of genes that take place in the microorganisms
when they are subject to stressful conditions that mimic the
environment confronted during the process of infection.
However, despite the plethora of genomics-derived data for
microbial pathogens, the world still awaits the first marketed
antibiotic that has been spawned from the genomics revolu-tion.
It should be stressed, however, that many of the available
genome annotations suffer from several defects or are
altogether wrong or misleading. Mistakes are usually intro-duced
as a result of lack of knowledge on gene function and
cell physiology, a problem that was more serious in the
genomes that were annotated earliest. Not a minor problem
of incorrect annotation is that the errors spread expo-nentially,
as new genomes may be annotated, almost
automatically, just by sequence comparison. A serious effort
that should be as enthusiastic as the impetus devoted to the
sequencing of new genomes should be directed at correcting
these errors. A periodic procedure to update genome

annotation (Riley et al., 2006) is needed if we wish fully to
develop the potential of these new technologies to extend
the number of potential inhibitable targets.
Targeting the bacterial essential
functions: the advantages and the risks of
inhibiting protein activity
Many antibiotics act by inhibiting protein activity. Some
antibacterial drugs are active through the inhibition of
critical enzymes that are present only in bacteria. For
example, sulphonamides are analogues of p-amino-benzoic
acid that inhibit folic acid synthesis. Although humans
require folic acid, we do not have the enzymatic machinery
to synthesize folic acid, and obtain it from food intake. Thus
human metabolism is not affected by treatment with
sulphonamides (Hitchings, 1971; Bardos, 1974). Similarly,
the most widely used class of antibacterials, the b-lactams,
which include penicillins, cephalosporins and carbapenems,
target proteins that are not found in humans.
The b-lactam antibiotics inhibit the transpeptidase activ-ity
of enzymes known as penicillin-binding proteins, which
are involved in the biosynthesis of bacterial cell walls causing
the death of bacteria. While b-lactams are generally con-sidered
bactericidal drugs, other antibiotics are bacterio-static
and inhibit but do not kill bacteria. However,
inhibition of growth usually suffices to overcome the
bacterial infection, as the bacteria do not increase in number
or in activity (such as toxin formation etc.) and are dealt
with by the immune system of the host.
In contrast to the above, many proteins that could be used
as inhibitable targets contain binding sites that are widely
distributed in proteins of eukaryotic cells. Thus, among the
cell-division proteins that are essential for bacterial prolif-eration,
FtsZ, a homologue of Tubulin (Löwe Amos,
1998), is a GTPase (de Boer et al., 1992; RayChaudhuri
Park, 1992; Mukherjee et al., 1993), and FtsA, belonging to
the actin family, has an ATP binding site (Sánchez et al.,
1994; van den Ent Löwe, 2000) (Fig. 2). In these cases
(many of them underexplored) it would be necessary to
include protocols that could identify and discard potential
inhibitors that inhibit both the bacterial and human protein
activities and might therefore be expected to be toxic, while
retaining those that selectively block the prokaryotic target.
Similar care has to be taken when developing antibacterial
drugs that inhibit unexploited targets in the bacterial ribo-some,
bacterial DNA replication or DNA repair activities.
Although bacteria are distinct from humans by having 70S
ribosomes, it should be noted that mitochondria contain the
bacterial-type ribosomes, and therefore can be affected by
ribosome inhibitors. Likewise, topoisomerases and other
enzymes involved in DNA metabolism are conserved in
both prokaryotic and eukaryotic cells. These factors make
the development of antibiotics that inhibit bacterial protein
or DNA metabolism more complex.
The underexplored territory of
protein--protein interactions
Assays based on protein interactions are considered less
likely to yield hits in screening assays than those based on
biochemical activity. The choice targets used for screening of
potential inhibitors are therefore those based on biochem-ical
reactions, while those that involve protein–protein
interactions are considered unlikely to yield useful hits.
However, many proteins that participate in the proliferation
of bacteria, and that would be choice targets to inhibit
infection (Fig. 3), form complexes with other proteins (for a
recent review see Vicente et al., 2006). Antibiotics could be
designed in the future as molecules that interfere with
protein interactions by overlaying the surface of the loops
involved in protein–protein interaction. In this way toxicity
problems would be circumvented, and, moreover, the in-hibitors
would constitute a fully new class of molecules that
are unlikely to have pre-existed in nature. Unfortunately, we
do not yet have sufficient knowledge and technology to
address this question realistically.
For example, although the three-dimensional structures
of several proteins essential for bacteria are known, the
published structures for some of them (e.g. FtsA and FtsZ)
correspond to proteins found in thermophilic microorgan-isms,
either bacteria or archea, as many of their mesophilic
counterparts have proven refractory to yield crystals useful
for structural determination (J. Löwe, pers. commun.).
Modelling on the structures obtained from thermophilic
microorganisms yields reasonable predictions for binding
sites, but the predictions are not so good for other regions,
in particular for the external loops, the regions that are more
likely to establish interactions with other proteins.
In consequence, virtual screening, the use of protein
structure data to predict the structure of molecules that are
most likely to interact with inhibitable proteins, is presently
more a desire than a reality. Although some initial steps
exploiting the interactions between FtsZ and ZipA, two
proteins that assemble together into the divisome of many
bacteria, have recently been reported (Jennings et al., 2004;
Rush et al., 2005), we need to improve substantially our
knowledge of structural biology and bacterial physiology in
order to realistically address projects that take advantage of
the available powerful informatics tools. Progress in mole-cular
modelling and in the synthetic skills needed for
mimicking protein surfaces is still required in order for the
knowledge derived from the study of the full set of protein
interactions within a microorganism (the interactome) to be
fully exploited. In the future, it is to be hoped that the study
of the interactome will identify those domains involved in

establishing the essential molecular interactions required for
the survival of the pathogen and for its interaction with the
host. If suitable mimics able to block the interacting surfaces
are then synthesized they can be used as scaffolds to build an
altogether new class of bacterial inhibitors.
Functional genomics and proteomics
Functional genomics includes the analysis of the genome
and its expression (transcriptomics using microarrays, and
proteomics using proteome analysis either by two-dimen-sional
gels or by gel-free separation methods). These tools
enable the study of the expression of an individual gene as a
function of specific environmental conditions, and can
reveal the existence of gene expression networks (stimulons
and regulons). Functional genomics provides new tools for
gaining valuable information on the physiology of pathogens,
their interaction with the host, and their response to drug
treatments, particularly those that may trigger the acquisition
of resistance. However, these novel tools still need to be fully
validated in model systems before they can be applied with
confidence to the search for new antimicrobials.
Genomics can also yield valuable information on the
spread of an inhibitable target among different pathogens,
a desirable property for the development of new drugs as
pharmaceutical companies prefer to market drugs that are
effective on a broad spectrum of bacteria. Functional
genomics will allow us, in the future, to discover if the
expression pattern of a given target is similar in all the
microorganisms that carry it.
Functional genomics has already provided data proving
the existence of a variety of genes that are differentially
expressed in the pathogen and in the host. Many of these
genes are unique to bacteria, and include some of the genes
associated with virulence. As an example, many bacteria
carry genes coding for aggregative curly fibres, shown in
septicaemic E. coli strains to be important for bacterial
internalization into epithelial cells. In nonvirulent E. coli
Fig. 2. Bacterial septation proteins that have
been considered as sources for new inhibitable
targets. The structure of two septation proteins
is shown together with that of their eukaryotic
counterparts. FtsZ is phylogenetically ubiquitous
both in the septation machinery of bacteria and
in some organelles and has a strong structural
resemblance to tubulin, both showing a GTPase
activity. Several details pertaining to the binding
of the nucleotide (Mingorance et al., 2001;
Romberg Mitchison, 2004) are nevertheless
different between FtsZ and Tubulin and may be
a promising source of targets to identify inhibi-tors
of the bacterial protein that do not impair
the eukaryotic homologue. In the case of FtsA,
an ATP-binding protein belonging to the actin
family, its structure differs from that of actin in
the orientation of a complete domain. Although
FtsA is not as phylogenetically widespread as
FtsZ, this structural difference makes it an at-tractive
source of potential inhibitable targets.
The prokaryotic structures shown belong to the
archea Methanococcus janascchii for FtsZ and to
the thermophilic bacterium Thermotoga mari-tima
for FtsA. The eukaryotic structures are boar
tubulin and yeast actin.

DNA
strains these fibres are expressed only at low temperatures
and low osmolarity, while in septicaemic strains they are
expressed at high temperatures and high osmolarity, i.e. for
the conditions found in the host (Gophna et al., 2001, 2002).
Exploiting RN-omics to discover novel
drug targets
Even though noncoding RNA (ncRNA) genes are involved
in many important biological processes, they have been
largely ignored until recently. A variety of systematic screens
have identified a large number of ncRNA genes (other than
tRNAs and rRNAs) in E. coli as well as in Caenorhabditis
elegans and in the human and other genomes (Mattick,
2005). Currently over 60 ncRNAs have been verified in E.
coli, while many more have been predicted. These genes have
many important roles, ranging from degradation of prema-turely
terminated translation products (tmRNA, ssrA) to
antisense regulation of other genes (microRNAs). The
assessment, through RN-omics, of transcribed intergenic
regions will probably reveal novel ncRNAs as drug targets.
Active compounds identified by screening RNA targets are
completely different from those classes that have been
picked up by screening protein targets (Zaman et al., 2003).
Thus, when targetting RNA, a completely different chemis-try
may be found, full of new challenges, but also of new
prospects, for the development of new drugs.
In addition, targetting mRNA is another challenging new
approach that is complementary to traditional drug discov-ery
focused on proteins. The assessment at the RNA level of
well-established protein targets that have failed to yield
useful leads is economical, as it does not require the long
and expensive functional genomic studies needed for en-tirely
new targets but can build on biological knowledge that
has been gathered over many years. In addition, targetting
mRNA creates new strategies for drug discovery, such as
protein upregulation by increasing the stability of a parti-cular
mRNA.
Targeting the virulence functions of
pathogens
Traditionally, antibiotics have been obtained as compounds
that prevent the proliferation of both pathogens and non-pathogens.
Consequently, most of our available antibacter-ials
do not distinguish between members of the healthy
human flora and the disease-causing pathogens, a fact that
has contributed to the development of resistance. The use of
inhibitors specifically targetted against pathogens could
therefore be a safer treatment for patients and contribute to
alleviating the spread of resistance.
Many bacterial pathogens possess a number of virulence
traits (obviously missing in commensal microorganisms)
that are required to cause disease, and that, if blocked, could
allow the selective inhibition of the pathogens without
affecting other bacteria. These include the ability to attach
to mucosal surfaces, to penetrate deeper into tissues, to
modulate the innate immune responses, to avoid eradica-tion,
and to produce a large number of toxic products. Some
of these virulence genes code for products that are them-selves
responsible for the secretion of other virulence factors
upon specific host contact (reviewed in Mahan et al., 2000).
Recently a small molecule, virstatin, was shown to block two
Vibrio cholerae virulence factors, the toxin production and
the toxin co-regulated pilus, by inhibiting the transcription
factor ToxT (Hung et al., 2005).
transcription
ribosome
assembly
septation
translation
replication
proteins
mRNA
processing
partition division ring
Fig. 3. Several essential bacterial processes remain as underexplored
sources of inhibitable targets. Fluoroquinolones, topoisomerase inhibi-tors,
remain as the main inhibitors used to block DNA replication, a
biochemical reaction in which a complex set of proteins need to interact,
together with nucleic acids, to effect duplication of the genetic informa-tion.
Partition of the genome (chromosome and plasmids), often invol-ving
suicide systems, is another process in which inhibitors can be
identified. The mechanisms of ribosome assembly and mRNA processing
have also received little attention when compared with the more
traditional inhibitors of the ribosomal stages of protein synthesis and
RNA polymerase. Septation, an underexplored process in itself, may also
yield unsuspected possibilities for the finding of inhibitors, as alterations
in other essential processes usually cause a septation block; for example,
triggering of the SOS response to repair DNA damages leads to cell-division
arrest mediated by SulA, a protein that prevents the interaction
of FtsZ with GTP, therefore blocking FtsZ ring assembly (Dai et al., 1994).

Another potential antivirulence target is the type III
secretion system that delivers effector proteins into host
cells. This dedicated system is indispensable for the viru-lence
of Salmonella, Shigella, Yersinia (Rosqvist et al., 1995)
and many pathogenic E. coli strains (e.g. enterohaemorragic
O157:H7), and it is also present in opportunistic pathogens
such as Pseudomonas aeruginosa and the obligate intracel-lular
common sexually transmitted pathogen Chlamydia
trachomatis (Fields and Hackstadt, 2000). Type III secretion
inhibitors have recently been identified and may constitute a
novel approach to treating diseases caused by these patho-gens.
Although they have usually been associated with
Gram-negative bacteria, it is likely that Gram-positive
bacteria may also contain similar systems involved in caus-ing
disease (Madden et al., 2001), in which case they would
be an attractive target to search for wider spectrum inhibi-tors.
Virulence factors are therefore of considerable interest,
even if their potential inhibitors are not conventional
antimicrobials in a strict sense because, although they may
prevent disease by blocking some function required for the
pathogen to attack the host, they do not block proliferation
of the pathogen in vitro and may ormay not inhibit it during
infection. In consequence, simple microbial growth and
viability determinations, such as those already used for
antibiotics, are not suitable for quantifying the effectiveness
of virulence inhibitors and more elaborate assays in reliable
living models must be used. In many cases virulence is
multifactorial and species-specific, and as a consequence
virulence inhibitors may be neither totally effective nor
wide-spectrum drugs.
Many virulence factors are dispensable and are therefore
encoded by variable sets of genes. When comparing the
entire genomes of different bacterial isolates belonging to
the same species, the contents of dispensable genes have
recently been observed to possess significant variability
(Medini et al., 2005). Even a complete genome sequence
may not be fully indicative of the infectivity profiles found
within a bacterial species, which further complicates the
genome-wide screening for virulence genes as antimicrobial
targets, and, although to a lesser extent, their use as vaccine
candidates (Maione et al., 2005; Tettelin et al., 2005).
Other novel ways to inhibit bacterial
proliferation
The translation of advances in new target discovery and
drug delivery into clinical practice is dependent on over-coming
two major barriers, namely the effective delivery of
classical drugs to new target families and the effective
delivery of new classes of biomolecular drugs to classical
targets. Sophisticated and molecularly engineered delivery
systems are needed to meet these challenges for topical, local
and systemic applications. If these problems can be solved
then new therapeutics such as inhibitory RNA (antisense)
and inhibitory antibodies, which are potential antimicrobial
tools, might become available for combating pathogenic
microorganisms.
Cells in their natural environment are often exposed to
considerable stress and mechanical force fields. Emerging
molecular and nanotechnology tools are for the first time
enabling exploration of how stress and mechanical forces
acting on single biomolecules can change their conforma-tional
state. Most high-resolution structures of proteins are
derived from crystal structures, thus representing equili-brium
states, and we have little high-resolution information
on the functional states of proteins. There is a need to
develop an understanding, at atomic resolution, of how
nature uses chemical cues in synergy with mechanical cues
to regulate the exposure or the conformation of molecular
recognition sites, thereby regulating cell adhesion, cell
signalling and gene expression. Once these principles are
understood, their application will open new avenues to
designing strategies to combat bacterial disease by interfer-ing
with early processes in the establishment of infection.
Concluding remarks: strategies
From what we already know, it seems that new antibiotics
are certainly needed in order to confront present issues
better, for example to resist resistance, and their need will be
even more pressing to combat as yet unsuspected emerging
diseases in the future. If we are so persuaded that infectious
diseases are still a serious threat for our health, it is unwise to
rely on a single procedure, source or target to supply our
future medicines. To bring new chemical-class antibiotics,
with activity against drug-resistant pathogens, into use it is
necessary to engage both biological and chemical technolo-gies,
more than has hitherto generally been the case. Knowl-edge
of the mechanisms of antibiotic resistance, ways to
circumvent them, and, more importantly, of new ways to
combat infectious diseases are likely to emerge from a
number of scientific research areas. In many instances, as
for example genomic technologies, these areas have seen
recent developments that are improving constantly, and
therefore economic returns should not be envisaged in the
short term.
Unfortunately, scientists will not be able to meet the
demand for new antibiotics that may be effective against
drug-resistant pathogens and against emerging diseases
if the clinicians and society in general do not demand
them and urge the financial agents to fund research on the
topic.
Funding agencies should consider that the provision of
sufficient and continuous funds to develop research along
several of the scientific lines summarized in this review is

likely to be beneficial. It may be that the ensuing scientific
discoveries might not provide the platform for the discovery
of broad-spectrum antibacterials with sufficient blockbuster
potential to attract large pharmaceutical companies. How-ever
a greater scientific understanding could be expected to
provide a sound basis for the discovery and development of
‘targetted’ antibiotics with commercial returns attractive
enough for small pharmaceutical and biotechnology com-panies.
Certainly, a failure to fund microbiological research
means that we may fail to yield vital new drugs in time, and
society will face a return to the preantibiotic era for
infections caused both by drug-resistant pathogens and by
new ones that may produce a disease as a result of environ-mental
or social changes. The final issue to be examined is
whether the research needed to find new antibacterials will
have sufficient continuity within the pharmaceutical and
biotechnological industries. If this should prove not to be
the case, strategic reasons should perhaps motivate the
public sector to devote a more sustained effort, at least in
the initial stages of discovery, to obtain new antimicrobials.
Acknowledgements
Part of the title of this study derives from an unfinished
poem written by Joseph Mallord William Turner
(1775–1851) to provide themes for the titles of several of
his paintings.
Work was funded by projects QLK3-2000-00079 (SANI-TAS)
Framework Programme 5 (to MV and OM), LSSM-CT-
2003-502801 (micro-MATRIX) (to MV), PREVIS Fra-mework
Programme 6 (to BHN) and COLIRISK Frame-work
Programme 6 (to EZR) from the European
Commission; and BIO2000-0451-P4-02, BIO2001-1542
and GEN2003-20234-C06-02 from Ministerio de Ciencia y
Tecnolog´ıa (to MV); BIO2005-02194 from Ministerio de
Educaci ´on y Ciencia (to MV); and GR/SAL/0642/2004 from
Comunidad de Madrid (to MV).
References
Andersson DI (2003) Persistence of antibiotic resistant bacteria.
Curr Opin Microbiol 6: 452–456.
Armstrong GL, Conn LA Pinner RW (1999) Trends in
infectious disease mortality in the United States during the
20th century. J Am Med Assoc 281: 61–66.
Bardos TJ (1974) Antimetabolites: molecular design and mode of
action. Top Curr Chem 52: 63–98.
Barrett JF (2005) Can biotech deliver new antibiotics? Curr Opin
Microbiol 8: 498–503.
Bartlett JG, Breiman RF, Mandell LA File TM Jr (1998)
Community-acquired pneumonia in adults: guidelines for
management. The infectious diseases society of America. Clin
Infect Dis 26: 811–838.
Bernatoniene J Finn A (2005) Advances in pneumococcal
vaccines: advantages for infants and children. Drugs 65:
229–255.
Bishai W (2002) The in vivo–in vitro paradox in pneumococcal
respiratory tract infections. J Antimicrob Chemother 49:
433–436.
Blomberg B, Jureen R, Manji KP et al. (2005) High rate of fatal
cases of pediatric septicemia caused by gram-negative bacteria
with extended-spectrum beta-lactamases in Dar es Salaam,
Tanzania. J Clin Microbiol 43: 745–749.
Branger C, Zamfir O, Geoffroy S, Laurans G, Arlet G, Thien HV,
Gouriou S, Picard B Denamur E (2005) Genetic background
of Escherichia coli and extended-spectrum beta-lactamase type.
Emerg Infect Dis 11: 54–61.
Bush K (2004)Why it is important to continue antibacterial drug
discovery. ASM News 70: 282–287.
Camacho AG, Misselwitz R, Behlke J, Ayora S,Welfle K,Meinhart
A, Lara B, Saenger W, Welfle H Alonso JC (2002) In vitro
and in vivo stability of the epsilon2zeta2 protein complex of
the broad host-range Streptococcus pyogenes pSM19035
addiction system. Biol Chem 383: 1701–1713.
Cant ´on R, Coque TM Baquero F (2003) Multi-resistant gram-negative
bacilli: from epidemics to endemics. Curr Opin Infect
Dis 16: 315–325.
Courvalin P Davies J (2003) Antimicrobials. Curr Opin
Dai K, Mukherjee A, Xu Y Lutkenhaus J (1994) Mutations in
ftsZ that confer resistance to SulA affect the interaction of FtsZ
with GTP. J Bacteriol 176: 130–136.
Davies J (1994) Inactivation of antibiotics and the dissemination
of resistance genes. Science 264: 375–382.
D’Costa VM, McGrann KM, Hughes DW Wright GD (2006)
Sampling the antibiotic resistome. Science 311: 374–377.
Deane SM Rawlings DE (2004) Plasmid evolution and
interaction between the plasmid addiction stability systems of
two related broad-host-range IncQ-like plasmids. J Bacteriol
186: 2123–2133.
de Boer P, Crossley R Rothfield L (1992) The essential bacterial
cell division protein FtsZ is a GTPase. Nature 359: 254–256.
Engelberg-Kulka H, Sat B, Reches M, Amitai S Hazan R (2004)
Bacterial programmed cell death systems as targets for
antibiotics. Trends Microbiol 12: 66–71.
Farr BM, Salgado CD, Karchmer TB Sherertz RJ (2001) Can
antibiotic-resistant nosocomial infections be controlled?
Lancet Infect Dis 1: 38–45.
Felmingham D (2004) Comparative antimicrobial susceptibility
of respiratory tract pathogens. Chemotherapy 50(Suppl 1):
3–10.
Felmingham D, Feldman C, HryniewiczW, Klugman K, Kohno S,
Low DE, Mendes C Rodloff AC (2002) Surveillance of
resistance in bacteria causing community-acquired respiratory
tract infections. Clin Microbiol Infect 8(Suppl 2): 12–42.
Fields KA Hackstadt T (2000) Evidence for the secretion of
Chlamydia trachomatis CopN by a type III secretion
mechanism. Mol Microbiol 38: 1048–1060.

Frazaõ N, Brito-Avoˆ A, Simas C et al. (2005) Effect of the seven-valent
conjugate pneumococcal vaccine on carriage and drug
resistance of Streptococcus pneumoniae in healthy children
attending day-care centers in Lisbon. Pediatr Infect Dis J 24:
243–252.
Gerdes K, Christensen KS Lobner-Olensen A (2005)
Prokaryotic toxin-antitoxin stress response loci. Nat Rev
Girardeau JP, Lalioui L, Said AM, De Champs C Le Bouguenec
C (2003) Extended virulence genotype of pathogenic
Escherichia coli isolates carrying the afa-8 operon: evidence of
similarities between isolates from humans and animals with
extraintestinal infections. J Clin Microbiol 41: 218–226.
Gophna U, Barlev M, Seijffers R, Oelschlager TA, Hacker J Ron
EZ (2001) Curli fibers mediate internalization of Escherichia
coli by eukaryotic cells. Infect Immun 69: 2659–2665.
Gophna U, Oelschlaeger TA, Hacker J Ron EZ (2002) Role of
fibronectin in curli-mediated internalization. FEMS Microbiol
Lett 212: 55–58.
Greenwood D (1981) In vitro veritas? Antimicrobial susceptibility
tests and their clinical relevance. J Infect Dis 144: 380–385.
Hitchings GH (1971) Folate antagonists as antibacterial and
antiprotozoal agents. Ann N Y Acad Sci 186: 444–451.
Hoffman LR, D’Argenio DA, MacCoss MJ, Zhang Z, Jones RA
Miller SI (2005) Aminoglycoside antibiotics induce bacterial
biofilm formation. Nature 436: 1171–1175.
Hung DT, Shakhnovich EA, Pierson E Mekalanso JJ (2005)
Small-molecule inhibitor of Vibrio cholerae virulence and
intestinal colonization. Science 310: 670–674.
Jackson LA, Benson P, Neuzil KM, Grandjean M Marino JL
(2005) Burden of community-onset Escherichia coli
bacteremia in seniors. J Infect Dis 191: 1523–1529.
Jennings LD, Foreman KW, Rush TS III et al. (2004)
Combinatorial synthesis of substituted 3-(2-
indolyl)piperidines and 2-phenyl indoles as inhibitors of
ZipA-FtsZ interaction. Bioorg Med Chem 12: 5115–5131.
Jensen RB, Grohmann E, Schwab H, D´ıaz-Orejas R Gerdes K
(1995) Comparison of ccd of F, parDE of RP4, and parD of R1
using a novel conditional replication control system of
plasmid R1. Mol Microbiol 17: 211–220.
Johnson JR Russo TA (2002) Extraintestinal pathogenic
Escherichia coli: ‘‘the other bad E coli’’. J Lab Clin Med 139:
155–162.
Jones ME, Blosser-Middleton RS, Critchley IA, Karlowsky JA,
Thornsberry C Sahm DF (2003) In vitro susceptibility of
Streptococcus pneumoniae, Haemophilus influenzae and
Moraxella catarrhalis: a European multicenter study during
2000–2001. Clin Microbiol Infect 9: 590–599.
Klein DL (1999) Pneumococcal disease and the role of the
conjugate vaccines. Microb Drug Resist 5: 147–157.
Kollef MH (2003) The importance of appropriate initial
antibiotic therapy for hospital-acquired infections. Am J Med
115: 582–584.
Kourbatova EV, Halvosa JS, King MD, Ray SM, White N
Blumberg HM (2005) Emergence of community-associated
methicillin-resistant Staphylococcus aureus USA 300 clone as a
cause of health care-associated infections among patients with
prosthetic joint infections. Am J Infect Control 33: 385–391.
Levy SB Marshall B (2004) Antibacterial resistance worldwide:
causes, challenges and responses. Nat Med 10: S122–S129.
Löwe J Amos LA (1998) Crystal structure of the bacterial cell-division
protein FtsZ. Nature 391: 203–206.
Macfarlane JT, Colville A, Guion A, Macfarlane RM Rose DH
(1993) Prospective study of aetiology and outcome of adult
lower-respiratory-tract infections in the community. Lancet
341: 511–514.
Madden JC, Ruiz N Caparon M (2001) Cytolysin-mediated
translocation (CMT): a functional equivalent of type III
secretion in gram-positive bacteria. Cell 104: 143–152.
Mahan MJ, Heithoff DM, Sinsheimer RL Low DA (2000)
Assessment bacterial pathogenesis analysis of gene expression
in the host. Annu Rev Genet 34: 139–164.
Maione D, Margarit I, Rinaudo CD et al. (2005) Identification of
a universal Group B streptococcus vaccine by multiple genome
screen. Science 309: 148–150.
Mart´ınez JL Baquero F (2002) Interactions among strategies
associated with bacterial infection: pathogenicity, epidemicity,
and antibiotic resistance. Clin Microbiol Rev 15: 647–679.
Maslow JN, Lautenbach E, Glaze T, BilkerW Johnson JR (2004)
Colonization with extraintestinal pathogenic Escherichia coli
among nursing home residents and its relationship to
fluoroquinolone resistance. Antimicrob Agents Chemother 48:
3618–3620.
Mattick JS (2005) The functional genomics of noncoding RNA.
Science 309: 1527–1528.
Medini D, Donati C, Tettelin H, Masignani V Rappuoli R
(2005) The microbial pan-genome. Curr Opin Genet Dev 15:
589–594.
Mingorance J, Rueda S, Gómez-Puertas P, Valencia A VicenteM
(2001) Escherichia coli FtsZ polymers contain mostly GTP and
have a high nucleotide turnover. Mol Microbiol 41: 83–91.
Moir DT, Shaw KJ, Hare RS Vovis GF (1999) Genomics and
antimicrobial drug discovery. Antimicrob Agents Chemother
43: 439–446.
Mokady D, Gophna U Ron EZ (2005) Virulence factors of
septicemic Escherichia coli strains. Int J Med Microbiol 295:
455–462.
Morens DM, Folkers GK Fauci AS (2004) The challenge of
emerging and re-emerging infectious diseases. Nature 430:
242–249.
Mukherjee A, Dai K Lutkenhaus J (1993) Escherichia coli cell
division protein FtsZ is a guanine nucleotide binding protein.
Proc Natl Acad Sci USA 90: 1053–1057.
Murray S (2006) Challenges of tuberculosis control. Can Med
Assoc J 174: 33–34.
Pandey DP Gerdes K (2005) Toxin–antitoxin loci are highly
abundant in free-living but lost from host-associated
prokaryotes. Nucleic Acids Res 33: 966–976.
Payne DJ (2004) Antimicrobials – where next? Microbiol today 31:
55–57.

Phillips I (2001) Reevaluation of antibiotic breakpoints. Clin
Infect Dis 33(Suppl 3): 230–232.
Projan SJ (2003) Why is big Pharma getting out of antibacterial
drug discovery? Curr Opin Microbiol 6: 427–430.
Rawlings DE (1999) Proteic toxin–antitoxin, bacterial plasmid
addiction systems and their evolution with special reference to
the pas system of pTF-FC2. FEMS Microbiol Lett 176: 269–277.
RayChaudhuri D Park JT (1992) Escherichia coli cell division
gene ftsZ encodes a novel GTP-binding protein. Nature 359:
251–254.
Riley M, Abe T, Arnaud MB et al. (2006) Escherichia coli K-12: a
cooperatively developed annotation snapshot–2005. Nucleic
Acids Res 34: 1–9.
Romberg L Mitchison TJ (2004) Rate-limiting guanosine 50-
triphosphate hydrolysis during nucleotide turnover by FtsZ, a
prokaryotic tubulin homologue involved in bacterial cell
division. Biochemistry 43: 282–288.
Ron EZ (2006) Host specificity of Escherichia coli: human and
avian pathogens. Curr Opin Microbiol 9: 28–32.
Rosamond J Allsop A (2000) Harnessing the power of the
genome in the search for new antibiotics. Science 287:
1973–1976.
Rosqvist R, Hakansson S, Forsberg A Wolf-Watz H (1995)
Functional conservation of the secretion and translocation
machinery for virulence proteins of yersiniae, salmonellae and
shigellae. EMBO J 14: 4187–4195.
Rush TS III, Grant JA, Mosyak L Nicholls A (2005) A shape-based
3-D scaffold hopping method and its application to a
bacterial protein–protein interaction. J Med Chem 48:
1489–1495.
Sánchez M, Valencia A, Ferrándiz MJ, Sander C Vicente M
(1994) Correlation between the structure and biochemical
activities of FtsA, an essential cell division protein of the actin
family. EMBO J 13: 4919–4925.
Sanders CC (1991) A problem with antimicrobial susceptibility
tests. ASM News 57: 187–190.
Schrag SJ, McGee L, Whitney CG, Beall B, Craig AS, Choate ME,
Jorgensen JH, Facklam RR, Klugman KP the Active Bacterial
Core Surveillance Team (2004) Emergence of Streptococcus
pneumoniae with very-high-level resistance to penicillin.
Antimicrob Agents Chemother 48: 3016–3023.
Siegman-Igra Y, Fourer B, Orni-Wasserlauf R, Golan Y, Noy A,
Schwartz D Giladi M (2002) Reappraisal of community-acquired
bacteremia: a proposal of a new classification for the
spectrum of acquisition of bacteremia. Clin Infect Dis 34:
1431–1439.
Smith AS Rawlings DE (1998) Autoregulation of the pTF-FC2
proteic poison-antidote plasmid addiction system (pas)
is essential for plasmid stabilization. J Bacteriol 180:
5463–5465.
Stevenson KB, Searle K, Stoddard GJ Samore M (2005)
Methicillin-resistant Staphylococcus aureus and vancomycin-resistant
enterococci in rural communities, western United
States. Emerg Infect Dis 11: 895–903.
Tettelin H, Masignani V, Cieslewicz MJ et al. (2005) Genome
analysis of multiple pathogenic isolates of Streptococcus
agalactiae: implications for the microbial ‘‘pan-genome’’.
Proc Natl Acad Sci USA 102: 13950–13955 (Erratum in:
102:16530).
The WHOWestern Pacific Gonococcal Antimicrobial
Surveillance Programme (2001) Surveillance of antibiotic
resistance in Neisseria gonorrhoeae in the WHOWestern
Pacific Region, 2000. Commun Dis Intell 25: 274–277.
Tomasz A (1997) Antibiotic resistance in Streptococcus
pneumoniae. Clin Infect Dis 24(Suppl 1): S85–S88.
Tufts Center for the Study of Drug Development (2003) Post-approval
RD raises total drug development costs to $897
million. Impact Report, Vol 5, no. 3.
van den Ent F Löwe J (2000) Crystal structure of the cell
division protein FtsA from Thermotoga maritima. EMBO J 19:
5300–5307.
Varaldo PE (2002) Antimicrobial resistance and susceptibility
testing: an evergreen topic. J Antimicrob Chemother 50: 1–4.
Vicente M, Rico AI, Mart´ınez-Arteaga R Mingorance J (2006)
Septum enlightenment: the assembly of the bacterial division
proteins. J Bacteriol 188: 19–27.
Weber JT (2005) Community-associated methicillin-resistant
Staphylococcus aureus. Clin Infect Dis 41(Suppl 4):
S269–S272.
World Health Organization (1996) The World Health Report 1996
– Fighting Disease, Fostering Development. World Health
Organization, Geneva.
World Health Organization (2000) Anti-tuberculosis Drug
Resistance in the World. Report no. 2: Prevalence and Trends.
WHO/CDS/TB/2000/.278 The WHO/IUATLD Global Project
on Anti-Tuberculosis Drug Resistance Surveillance. World
Health Organization, Geneva.
Zaman GJ, Michiels PJ van Boeckel CA (2003) Targeting RNA:
new opportunities to address drugless targets. Drug Discov
Today 8: 297–306.
Zielenkiewicz U Ceglowski P (2005) The toxin–antitoxin
system of the streptococcal plasmid pSM19035. J Bacteriol 187:
6094–6105.

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