Coping with antibiotic resistance: contributions from genomics.

Abstract

Antibiotic resistance is a public health issue of global dimensions with a significant impact on morbidity, mortality and healthcare-associated costs. The problem has recently been worsened by the steady increase in multiresistant strains and by the restriction of antibiotic discovery and development programs. Recent advances in the field of bacterial genomics will further current knowledge on antibiotic resistance and help to tackle the problem. Bacterial genomics and transcriptomics can inform our understanding of resistance mechanisms, and comparative genomic analysis can provide relevant information on the evolution of resistant strains and on resistance genes and cognate genetic elements. Moreover, bacterial genomics, including functional and structural genomics, is also proving to be instrumental in the identification of new targets, which is a crucial step in new antibiotic discovery programs.

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Antibiotic resistance as a global public health problem
The advent of antibiotics for treating bacterial infections
is considered one of the major advances in modern
medicine. However, compared with other drugs, the life­
time of antibiotics for clinical use has been substantially
limited by the phenomenon of antibiotic resistance.
Failures of antimicrobial chemotherapy due to resistance
were observed soon after the introduction of penicillin in
clinical practice and, thereafter, the emergence of resis­
tance has always followed the release of new antibiotics,
posing a major clinical challenge and at the same time
acting as a major driver for drug discovery and develop­
ment in the field of antimicrobial agents.
Antibiotic resistance is now recognized as a major
public health problem because of its magnitude and
global scale. In fact, resistance does not affect only
industrialized countries (where this is expected given the
unlimited access to the antibiotic market) but also low­
income settings, where resistance rates have been found
to be even higher because of less well regulated anti­
microbial policies and worse hygiene conditions and
infection­control practices [1]. It is also well documented
that resistance has a significant impact in terms of
morbidity, mortality and healthcare­associated costs [2].
For instance, infections caused by methicillin­resistant
Staphylococcus aureus (MRSA, one of the most wide­
spread and challenging resistant superbugs) were shown to
be associated with an increased risk of mortality (almost
twofold for bacteremias; more than threefold for surgical­
site infections) in comparison with infections of the same
type caused by methicillin­susceptible S. aureus (MSSA).
There is also a positive correlation with the length of stay
in hospital and the healthcare­associated costs of handling
MRSA infections compared with MSSA infections [2].
Although antibiotic resistance continues to evolve at a
steady pace and the spreading of resistant strains has
been facilitated by increasing international travel and
migration, most pharmaceutical companies have also
recently chosen to restrict programs aimed at the
discovery and development of new antibiotics. This has
plunged the phenomenon of antibiotic resistance into a
major crisis, with the increasing emergence of strains
that are resistant to most or all antibiotics currently
available for clinical use [3], which threatens to turn the
clock back to the pre­antibiotic era.
Here, we briefly review and discuss how the recent
advance ment of bacterial genomic sciences can contri­
bute to further current knowledge on resistance and to
find new solutions to address this important problem.
The genetics of antibiotic resistance
Acquired antibiotic resistance can be due to a plethora of
mechanisms (Table 1). These include, first, drug inactiva­
tion, for example by β­lactamases and aminoglycoside­
modifying enzymes, which confer resistance to β­lactams
and aminoglycosides, respectively; second, drug target
Abstract
Antibiotic resistance is a public health issue of global
dimensions with a significant impact on morbidity,
mortality and healthcare-associated costs. The
problem has recently been worsened by the steady
increase in multiresistant strains and by the restriction
of antibiotic discovery and development programs.
Recent advances in the field of bacterial genomics will
further current knowledge on antibiotic resistance
and help to tackle the problem. Bacterial genomics
and transcriptomics can inform our understanding of
resistance mechanisms, and comparative genomic
analysis can provide relevant information on the
evolution of resistant strains and on resistance genes
and cognate genetic elements. Moreover, bacterial
genomics, including functional and structural
genomics, is also proving to be instrumental in the
identification of new targets, which is a crucial step in
new antibiotic discovery programs.
© 2010 BioMed Central Ltd
Coping with antibiotic resistance: contributions
from genomics
Gian Maria Rossolini1* and Maria Cristina Thaller2
REVIEW
*Correspondence: rossolini@unisi.it
1Department of Molecular Biology, Section of Microbiology, University of Siena,
and Clinical Microbiology Unit, Siena University Hospital, Policlinico Santa Maria
alle Scotte, Viale Bracci, 53100 Siena, Italy
Full list of author information is available at the end of the article
Rossolini and Thaller Genome Medicine 2010, 2:15
http://genomemedicine.com/content/2/2/15
© 2010 BioMed Central Ltd

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modification by mutation, such as DNA topoisomerase
or RNA polymerase modifications, conferring resistance
to quinolones and rifampicin, respectively; third, drug
target protection, for example ribosomal methylation
confer ring resistance to aminoglycosides or macrolides,
or topoisomerase protection by Qnr proteins conferring
resistance to quinolones; fourth, drug target bypass, for
example peptidoglycan synthesis by a novel penicillin­
binding­protein, PBP2a, conferring resistance to β­lactams;
fifth, impermeability, for example by loss of outer
membrane porin channels, conferring resistance to
carba penems; and finally drug efflux, for example by Tet
major facilitator superfamily (MFS)­type pumps, confer­
ring resistance to tetracyclines, or by tripartite resistance­
nodulation­cell division superfamily (RND)­type pumps,
conferring a multidrug resistance phenotype. This diver­
sity of resistance mechanisms reflects the broad reper­
toire of available agents, the multiplicity of their mecha­
nisms of action, the biological diversity of bacteria, and
the plasticity of their genomes. From a genetic stand­
point, resistance mechanisms can arise either by muta­
tion or by the acquisition and expression of heterologous
genes encoding the resistance determinant through
horizontal gene transfer mechanisms such as conjugation,
transformation or phage transduction.
Once acquired, the resistance determinants confer a
selective advantage to the host when it is exposed to
antibiotics, which can result in an expansion of the
resistant clone relative to susceptible ones; this expansion
can assume epidemic or even pandemic dimensions.
Typical examples of resistant clones of Gram­positive
patho gens that underwent an international dissemina­
tion, with remarkable clinical and epidemiological impact,
include the MRSA strains belonging to clonal complexes
(CCs) 5 and 8 [4], the glycopeptide­resistant Enterococcus
faecium strains of CC17 [5] and the several penicillin­
nonsusceptible pneumococcal clones spread ing worldwide
[6]. Similar examples of resistant clones of Gram­negative
pathogens with high spreading potential include the
Escherichia coli clone O25:H4­ST­131, which produces
the CTX­M­15 extended­spectrum β­lacta mase (ESBL)
[7], the Klebsiella pneumoniae clone (KPC) ST­258, which
produces acquired KPC­type carbapene mases [8], and the
Acinetobacter baumannii derivatives of major European
clone II, which produces oxacillin (OXA)­type carbapene­
mases [9].
Table 1. Examples of clinically relevant resistance mechanisms in major bacterial pathogens
Resistance mechanism Molecular basis Antibiotics affected Relevant clinical pathogens
Drug inactivation

β-Lactamases

β-Lactams (variable spectrum,
depending on the enzyme type)
Many Gram-positive and Gram-negative
pathogens



Aminoglycoside-modifying
enzymes

Aminoglycosides (variable
spectrum, depending on the
enzyme type)
Many Gram-positive and Gram-negative
pathogens
Target modification

Mutated DNA topoisomerases

Quinolones and fluoroquinolones

Many Gram-positive and Gram-negative
pathogens


Mutated RNA polymerase

Rifampin

Staphylococcus aureus,
Mycobacterium tuberculosis



Altered peptidoglycan
(presence of d-Ala-d-Lac
depsipeptide)
Glycopeptides Enterococci
Target protection

23S ribosomal RNA methylation
by Erm methylases
Macrolides Streptococci, Staphylococci, anaerobes


16S ribosomal RNA methylation
by Arm/Rmt-like methylases
Aminoglycosides Enterobacteriaceae
Ribosomal protection by Tet proteins Tetracyclines Many Gram-positive pathogens


Topoisomerase protection by
Qnr proteins
Quinolones and fluoroquinolones Enterobacteriaceae
Bypass of target function

Production of PBP2a, which takes over
the functions of other PBPs
Most β-lactams

Methicillin-resistant Staphylococcus aureus
(MRSA)
Impermeability Loss of OprD porin Carbapenems
Pseudomonas aeruginosa
Drug efflux Tet MFS-type pumps Tetracyclines Enterobacteriaceae, Staphylococci


Upregulation of MexAB RND-type
pump
Fluoroquinolones and most
anti-pseudomonal β-lactams
Pseudomonas aeruginosa


Upregulation of MexXY RND type pump

Fluoroquinolones, aminoglycosides,
cefepime and meropenem
Pseudomonas aeruginosa
PBP: penicillin-binding-protein.
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Mobile genetic units, such as conjugative plasmids or
transposons and lysogenic bacteriophages, can also be
very successful in transferring across different strains,
leading to a multi­clonal emergence of resistant strains. A
typical example is represented by several conjugative
plasmids encoding CTX­M­type ESBLs that were respon­
sible for rapid and efficient dissemination of these resis­
tance determinants, conferring resistance to the expanded­
spectrum cephalosporins, among E. coli [10]. Other
examples of mobile genetic elements that contributed
significantly to horizontal dissemination of resistance
genes are the large conjugative transposons and phage­like
elements carrying macrolide resistance determinants of
the erm and mef type, responsible for the dissemination of
these resistance determinants among different clones of
pneumococci and group A strepto cocci [11].
The role of genomics in surveillance and control of
resistance
Tracing the epidemiology of resistant strains and resis­
tance genes is of paramount importance for surveillance
and control of antibiotic resistance, which can no longer
rely on the simple phenotypic characterization of
bacterial isolates. Molecular epidemiology is the disci­
pline that studies the epidemiology of resistant strains
and resistance genes by characterizing them at the
molecular level, and it has provided major breakthroughs
in the understanding of this phenomenon, with practical
implications for resistance control strategies. The infor­
ma tion provided by molecular analysis has a variable
degree of resolution depending on the analytical tools,
with genetic ones being the most versatile and of highest
resolution. From this perspective, full genomic analysis of
major resistant clones can be very informative for
understanding their lifestyle and evolution and would be
the golden standard for their comparison, as it was clearly
shown with MRSA [12].
Genomic knowledge can also be instrumental to the
development of sets of molecular probes for easy and
specific identification of resistant clones of high spread­
ing propensity and clinical impact, such as those
described above, which is crucial in infection control
practices. These probes can be used by reference labora­
tories or even by the largest diagnostic laboratories, in
multiplexed amplification or DNA microarray technolo­
gies, for identification of such ‘high risk’ resistant clones
involved in hospital or community outbreaks.
The importance of identifying high risk resistant clones
and understanding their evolution explains the con­
siderable effort that has recently been undertaken to
sequence the genomes of multiresistant strains of
bacterial pathogens [13], such as MRSA (eight projects),
Acinetobacter baumannii (five projects), Mycobacterium
tuberculosis (three projects) and Pseudomonas aeruginosa
(two projects), and also to sequence plasmids involved in
the dissemination of important resistance determinants,
such as ESBLs, AmpC­type β­lactamases or (quinolone
resistance) Qnr proteins [14].
Genomics can inform our understanding of
resistance mechanisms
Understanding resistance mechanisms to novel drugs is
crucial in the process of discovery and development of
antimicrobial drugs. Knowledge on newly emerging
resistance mechanisms to antibiotics already available for
clinical use is also of paramount importance for the
prediction of resistance evolution, antibiotic policies and
resistance surveillance and control strategies. Genome­
scale investigations may provide relevant insights into
unknown mechanisms of antibiotic resistance.
For instance, a comparative genomic analysis between
S. aureus strains showing heterogeneous or homogeneous
intermediate resistance to vancomycin (hVISA or VISA
strains) and the susceptible parent strain has recently
revealed the role of mutations of the genes encoding the
vraSR and graSR two­component regulatory system in
conferring this resistance phenotype, which is associated
with clinical failures of glycopeptides [15]. A similar
comparative genomic approach, carried out using array­
based methodology, provided insights into mutational
mechanisms that, in S. aureus, could be responsible for
the emergence of reduced susceptibility to daptomycin, a
new anti­Gram­positive antibiotic recently released for
the treatment of staphylococcal infections [16]. Another
genome­scale investigation that advanced our knowledge
of resistance mechanisms was that by Breidenstein et al.
[17], who, using a comprehensive P. aeruginosa mutant
library, identified a number of new genes that, when
inactivated, are associated with decreased susceptibility
to fluoroquinolones in this species. These genes include
PA2047 and PA3574, which encode transcriptional
regulators, the nuo genes, which encode NADH dehydro­
genase I subunits, and several other genes encoding
hypo thetical proteins.
Transcriptomics at a genome­wide scale has also been
used successfully to investigate resistance mechanisms.
For example, transcription profiling following cipro­
floxacin exposure has revealed an important role of the
mfd gene, which encodes a transcription­repair coupling
factor involved in strand­specific DNA repair, in the
development of fluoroquinolone resistance in Campylo­
bacter jejuni [18]. Transcriptome analysis of S. aureus
treated with sublethal concentrations of cationic anti­
microbial peptides has revealed a role for several genes,
including the vraSR cell wall regulon, in the bacterial
response to these agents, as well as a role of the VraDE
putative ATP­binding cassette (ABC) transporter in
confer ring resistance to bacitracin [19].
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Genomic knowledge and antibacterial drug
discovery
Knowledge on bacterial genomes has progressed at a fast
pace during recent years, with almost 1,500 completed
bacterial genomes and more than 600 additional genome
projects in progress at the beginning of 2010 [13]. Indeed,
since its very beginning, the advent of bacterial genomics
was not only regarded as a fascinating scientific tool, but
also raised great hopes for renewing the golden era of
antimicrobial discovery at a time when this is sorely
needed because of the growing impact of bacterial
resistance. The rationale behind this expectation was that
comparative genomic analysis could reveal valuable
information on bacterial genes that presumably encode
proteins that are essential to survival or fitness of
bacterial pathogens and do not have close eukaryotic
counterparts; these proteins could then be potential
targets for new antimicrobial agents.
This approach has been extensively pursued and has
returned some potential targets for new antimicrobial
agents that fulfilled the above criteria, including enzymes
involved in biosynthetic pathways (for example coenzyme
A, chorismate, lipid A and fatty acids), protein synthesis
(such as aminoacyl tRNA synthetases and peptide defor­
mylase), protein secretion (such as signal peptidase 1)
and DNA replication (such as FtsZ/FtsA) [20,21]. Some
two­component signal transduction systems have also
been considered [20].
However, it was soon evident that, although identifi­
cation of potential targets is important, a comprehensive
understanding of bacterial biochemistry, physiology and
pathogenicity are essential for exploiting this information
for antimicrobial drug discovery. After the identification
of the potential targets, there are several bottlenecks to
be overcome in the process of developing new drugs; in
particular, the need to set up high­throughput screening
of banks of small molecules to obtain potential hits,
which has prompted great efforts in the fields of
functional and structural genomics [22]. Moreover, many
screenings have turned out to be unproductive, or it was
discovered that effective drugs could not be developed
from the potential inhibitors [20].
This accounts for the overall dearth of new drugs
discovered using genomic approaches that have made it
into advanced clinical phases of the antibiotic pipeline
after almost 15 years of efforts in this area. However, the
natural products platensimycin and platenmycin,
produced by Streptomyces platensis, have been dis­
covered to be inhibitors of FabF and/or FabH (enzymes
involved in bacterial type II fatty­acid biosynthesis) [23]
and are now awaiting further preclinical studies, and
some peptide deformylase inhibitors have already entered
clinical trials [20,21]. Therefore, the potential of microbial
genomics for discovery of new drugs should not be
underestimated. Indeed, a novel screening technique,
combining chemical genomics with a yeast­based assay,
has enabled the discovery of both a P. aeruginosa
virulence­associated target (ExoS) and its inhibitor [24].
Finally, cloning and sequencing metagenomes from
various ecological niches in which antibiotic­producing
bacteria are expected to be present could reveal new
antimicrobial synthetic gene clusters from unknown or
uncultured bacteria. This strategy has already yielded
some results such as the gene clusters for synthesis of
turbomycins A and B, new glycopeptides and other
compounds with antimicrobial activity cloned from
environmental DNA (eDNA) libraries [25].
Concluding remarks
Antibiotic resistance is one of the greatest clinical
challenges in the treatment of infectious diseases, given
that it is a complex phenomenon whose emergence and
evolution is still only partially understood. Bacterial
genomics provides an invaluable contribution to
furthering current knowledge on antibiotic resistance
mecha nisms and evolution, and on molecular epidemio­
logy of resistance, which is instrumental to infection
control practices. Moreover, information from functional
and structural genomics has a key role in the identi­
fication of novel antimicrobial targets. The number of
new agents discovered following this approach that are
found in the advanced stages of the antibiotic pipeline is
still very limited; however, the exploitation of bacterial
genomics has been a major breakthrough for discovery
and development of new antibiotics active against
multiresistant pathogens.
Abbreviations
CC, clonal complex; ESBL, extended-spectrum β-lactamase; MRSA, methicillin-
resistant S. aureus; MSSA, methicillin-susceptible S. aureus.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Both authors contributed to reviewing the relevant literature. MCT analyzed
microbial genome databases. GMR wrote the article.
Author details
1Department of Molecular Biology, Section of Microbiology, University of
Siena, and Clinical Microbiology Unit, Siena University Hospital, Policlinico
Santa Maria alle Scotte, Viale Bracci, 53100 Siena, Italy
2Department of Biology, University of Rome ‘Tor Vergata’, Viale della Ricerca
Scientifica s.n.c. 00133 Rome, Italy
Published: 25 February 2010
References
1. Okeke IN, Laxminarayan R, Bhutta ZA, Duse AG, Jenkins P, O’Brien TF, Pablos-
Mendez A, Klugman KP: Antimicrobial resistance in developing countries.
Part I: recent trends and current status. Lancet Infect Dis 2005, 1:481-493.
2. Maragakis LL, Perencevich EN, Cosgrove SE: Clinical and economic burden
of antimicrobial resistance. Expert Rev Anti Infect Ther 2008, 6:751-763.
3. Boucher HW, Talbot GH, Bradley JS, Edwards JE, Gilbert D, Rice LB, Scheld M,
Spellberg B, Bartlett J: Bad bugs, no drugs: no ESKAPE! An update from the
Rossolini and Thaller Genome Medicine 2010, 2:15
http://genomemedicine.com/content/2/2/15
Page 4 of 5

Page 5

Infectious Diseases Society of America. Clin Infect Dis 2009, 48:1-12.
Deurenberg RH, Vink C, Kalenic S, Friedrich AW, Bruggeman CA, Stobberingh
EE: The molecular evolution of methicillin-resistant Staphylococcus aureus.
Clin Microbiol Infect 2007, 13:222-235.
Willems RJ, Top J, Santen Mv, Robinson DA, Coque TM, Baquero F,
Grundmann H, Bonten MJ: Global spread of vancomycin-resistant
Enterococcus faecium from distinct nosocomial genetic complex. Emerg
Infect Dis 2005, 11:821-828.
Woodford N, Livermore DM: Infections caused by Gram-positive bacteria:
a review of the global challenge. J Infect 2009, 59:S4-S16.
Nicolas-Chanoine M-H, Blanco J, Leflon-Guibout V, Demarty R, Alonso MP,
Caniça MM, Park YJ, Lavigne JP, Pitout J, Johnson JR: Intercontinental
emergence of Escherichia coli clone O25:H4-ST131 producing CTX-M-15.
J Antimicrob Chemother 2008, 61:273-281.
Nordmann P, Cuzon G, Naas T: The real threat of Klebsiella pneumoniae
carbapenemase-producing bacteria. Lancet Infect Dis 2009, 9:228-236.
Higgins PG, Dammhayn C, Hackel M, Seifert H: Global spread of
carbapenem-resistant Acinetobacter baumannii. J Antimicrob Chemother
2010, 65:233-238.
10. Valverde A, Cantón R, Garcillán-Barcia MP, Novais A, Galán JC, Alvarado A, de
la Cruz F, Baquero F, Coque T: Spread of bla(CTX-M-14) is driven mainly by
IncK plasmids disseminated among Escherichia coli phylogroups A, B1,
and D in Spain. Antimicrob Agents Chemother 2009, 53:5204-5212.
11. Varaldo P, Montanari MP, Giovanetti E: Genetic elements responsible for
erythromycin resistance in streptococci. Antimicrob Agents Chemother 2009,
53:343-353.
12. Harris SR, Feil EJ, Holden MTG, Quail MA, Nickerson EK, Chantratita N, Gardete
S, Tavares A, Day N, Lindsay JA, Edgeworth JD, de Lencastre H, Parkhill J,
Peacock SJ, Bentley SD: Evolution of MRSA during hospital transmission
and intercontinental spread. Science 2010, 327:469-474.
13. NCBI Genome Project [http://www.ncbi.nlm.nih.gov/genomeprj]
14. Carattoli A: Resistance plasmid families in Enterobacteriaceae. Antimicrob
Agents Chemother 2009, 53:2227-2238.
15. Cui L, Neoh HM, Shoji MKH: Contribution of vraSR and graSR point
mutations to vancomycin resistance in vancomycin-intermediate
Staphylococcus aureus. Antimicrob Agents Chemother 2009, 53:1231-1234.
16. Friedman L, Alder JD, Silverman JA: Genetic changes that correlate with
reduced susceptibility to daptomycin in Staphylococcus aureus. Antimicrob
Agents Chemother 2006, 50:2137-2145.
4.
5.
6.
7.
8.
9.
17. Breidenstein EBM, Khaira BK, Wiegand I, Overhage J, Hancock RE: Complex
ciprofloxacin resistome revealed by screening a Pseudomonas aeruginosa
mutant library for altered susceptibility. Antimicrob Agents Chemother 2008,
52:4486-4491.
18. Han J, Sahin O, Barton YW, Zhang Q: Key role of Mfd in the development of
fluoroquinolone resistance in Campylobacter jejuni. PLoS Pathog 2008,
4:e1000083.
19. Pietiäinen M, François P, Hyyryläinen HL, Tangomo M, Sass V, Sahl HG,
Schrenzel J, Kontinen VP: Transcriptome analysis of the responses of
Staphylococcus aureus to antimicrobial peptides and characterization of
the roles of vraDE and vraSR in antimicrobial resistance. BMC Genomics
2009, 10:429.
20. Payne DJ, Gwynn MN, Holmes DJ, Pompliano DL: Drugs for bad bugs:
confronting the challenges of antibacterial discovery. Nat Rev Drug Discov
2007, 6:29-40.
21. Falconer SB, Brown ED: New screens and targets in antibacterial drug
discovery. Curr Opin Microbiol 2009, 12:497-504.
22. Monaghan RL, Barrett JF: Antibacterial drug discovery - then, now and the
genomics future. Biochem Pharmacol 2006, 71:901-909.
23. Wang J, Kodali S, Lee SH, Galgoci A, Painter R, Dorso K, Racine F, Motyl M,
Hernandez L, Tinney E, Colletti SL, Herath K, Cummings R, Salazar O, Gonzalez
I, Basilio A, Vicente F, Genilloud O, Pelaez F, Jayasuriya H, Young K, Cully DF,
Singh SB: Discovery of platencin, a dual FabF and FabH inhibitor with in
vivo antibiotic properties. Proc Natl Acad Sci USA 2007, 104:7612-7616.
24. Arnoldo A, Curak J, Kittanakom S, Chevelev, Lee VT, Sahebol-Amri M, Koscik B,
Ljuma L, Roy PJ, Bedalov A Giaever G, Nislow C, Merrill RA, Lory S, Stagljar I:
Identification of small molecule inhibitors of Pseudomonas aeruginosa
exoenzyme S using a yeast phenotypic screen. PLoS Genet 2008,
4:e1000005.
25. Brady SF, Simmons L, Kim JH, Schmidt EW: Metagenomic approaches to
natural products from free-living and symbiotic organisms. Nat Prod Rep
2009, 26:1488-1503.
doi:10.1186/gm136
Cite this article as: Rossolini GM, Thaller MC: Coping with antibiotic
resistance: contributions from genomics. Genome Medicine 2010, 2:15.
Rossolini and Thaller Genome Medicine 2010, 2:15
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