REVIEWS

Evolutionary consequences of drug

resistance: shared principles across
diverse targets and organisms
Diarmaid Hughes and Dan I. Andersson
Abstract | Drug therapy has a crucial role in the treatment of viral, bacterial, fungal and
protozoan infections, as well as the control of human cancer. The success of therapy is
being threatened by the increasing prevalence of resistance. We examine and compare
mechanisms of drug resistance in these diverse biological systems (using HIV and
Plasmodium falciparum as examples of viral and protozoan pathogens, respectively) and
discuss how factors — such as mutation rates, fitness effects of resistance, epistasis
and clonal interference — influence the evolutionary trajectories of drug-resistant
clones. We describe commonalities and differences related to resistance development
that could guide strategies to improve therapeutic effectiveness and the development of
a new generation of drugs.

Uppsala University,
Department of Medical
Biochemistry and
Microbiology, Biomedical
Center, BOX 582S‑75123,
Uppsala, Sweden.
Correspondence to D.H.  choices were made for their importance in human health
e-mail: diarmaid.hughes@
imbim.uu.se
doi:10.1038/nrg3922
Published online 7 July 2015
The success of modern medicine is mostly based on the
use of drug therapy to control or cure infections that are
caused by various pathogens, as well as other diseases
such as cancer 1. This legacy is currently threatened by
the development and evolution of resistance to the drugs
on which these therapies rely.
There are multifaceted aspects of drug resistance
that are being actively studied, such as the emergence
and molecular nature of the resistance mutations, the
fitness consequences of resistance (in the presence and
absence of drugs) and how these aspects influence the
evolutionary trajectories of the resistant viruses or cells.
A more complete understanding of these features will be
crucial to predict resistance and to combat it through the
optimal use of existing drugs (concentrations, periodic
drug removal or switching, and drug combinations) and
to guide the design of new drugs.
In this Review, we discuss diverse aspects of drug
resistance, including mechanisms, evolutionary conse-
quences and therapeutic implications. We broadly cover
viral (using HIV as an example), bacterial and fungal
infections, vector-borne parasites (using Plasmodium
falciparum as an example), and the uncontrolled cell
growth that is associated with human cancers. These
and the quality of data relating to the mechanisms and
dynamics of drug-resistance development. We are aware
that other systems — for example, the hepatitis and
influenza viruses — are also extremely clinically impor-
tant, but we have limited the scope of this Review in the
interests of space and because lessons learned from HIV
can be applied to other human viruses. Each system has
particular features that affect the trajectories of resist-
ance evolution (TABLE 1). We highlight the features that
they have in common and the problems that are unique
to each system.
Systems and their drug-resistance mechanisms
We ask below to what extent mechanisms of resistance
development are common across systems and which, if
any, are unique.
HIV. HIV, the RNA virus that causes AIDS, has very high
spontaneous mutation rates of 10−4–10−5 per nucleo­tide
per genome replication2,3, and it can also undergo inter-
viral recombination within the human host to generate
novel virus variants4. Despite its high mutation rate,
genetic variation in HIV may be ultimately constrained
by the need of the virus to successfully transmit to a new
human host.
Three major classes of drugs are used to combat
AIDS caused by HIV. Nucleoside reverse transcriptase
inhibitors (NRTIs) and non-nucleoside reverse tran-
scriptase inhibitors (NNRTIs) result in the termination of
reverse transcription and hence block viral replication5,6,
whereas viral protease inhibitors affect viral maturation7.

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REVIEWS

Table 1 | Distinctions and similarities of biological systems treated with drugs to control growth and transmission
HIV Pathogenic bacteria Pathogenic fungi Plasmodium Human cancer
falciparum
Host Human Human, animal, plant and Human, animal, plant and Human and Human
environmental environmental mosquito
Infection Intracellular Extracellular (either on Extracellular (either on Extracellular and Tumour cell growth and
lifestyle the host’s body surface the host’s body surface or intracellular metastasis
or an internal infection) an internal infection), and
and/or intracellular intracellular (microsporidia)
Genome size 9.7 kb, ssRNA 1.8 Mb (Haemophilus Varied: ≥2.5 Mb 22.9–26.8 Mb 3.2 Gb (only a subset of
genome influenzae) to 6.3 Mb (microsporidia), 14.9 Mb sequence is associated
(Pseudomonas (Candida albicans) and with cancer)
aeruginosa), dsDNA 29.4 Mb (Aspergillus
genome fumigatus)
Mutation rate ≈10−5 per nt ≈10−10 per nt per ≈10−10 per nt per ≈10−9 per bp per Mutation is an initiating
per genome generation11. Gene generation (Saccharomyces generation164 event; subsequently huge
replication2,3 amplification 10−2–10−5 cerevisiae)163 variation in mutation rate
per gene per generation13 as cancer develops
Mechanisms Mutation Mutation, genomic Mutation and genomic Mutation Mutation and genomic
of genetic and interviral rearrangements and rearrangements15 and genomic rearrangements29,32
diversification recombination4 horizontal gene transfer19 rearrangements24
In‑host 107–108 Highly variable Complicated by many fungi 103–4 × 104 Very varied: from 1 to 10
population productively depending on the having a dimorphic lifestyle merozoites (the form circulating tumour cells
size estimates infected human pathogen and infection (yeast and hyphal forms). that subsequently per 10 mL blood169, up
cells165 generating site. Up to 1011 per Candida albicans, ≥108 invades red blood to ~108 cells per gram
up to 1010 virions bladder in urinary tract cfu per gram caecum in a cells) per infected of tumour tissue67 (with
per day2,3 infections166 murine infection model167 liver cell168 tumours potentially
growing up to over 1 kg
in weight)
Major causes HIV viral growth Tissue damage as a result Tissue damage as a result Growth of Growth of cancer cells can
of disease leads to the of the immune system of the immune system Plasmodium exert pressure on various
symptoms destruction of CD4 response to bacterial response to fungal growth. falciparum within organs and tissues, reduce
human immune growth. Many pathogenic Some pathogenic fungi human red blood available energy supply,
system T cells, bacteria also release also release toxins that cells causes their release substances that
causing increased toxins that directly directly damage human destruction, leading change physiology and
susceptibility to damage human tissue tissue to the symptoms of affect the activity of the
other infections malaria immune system
Transmission Person to person Multiple routes: person Multiple routes: person to Vector-borne No transmission, but
routes via sexual contact to person (touch, sexual person; water; fomites; and (human to mosquito cancer may be induced
or exposure to intercourse, saliva and opportunistic infections to human) by infectious agents such
body fluids air); food; water; insects; in immunosuppressed as Helicobacter pylori or
and fomites individuals human papilloma virus
cfu, colony-forming unit; dsDNA, double-stranded DNA; nt, nucleotide; ssRNA, single-stranded RNA.

Resistance to NRTIs and NNRTIs arises by mutations
that affect reverse transcriptase8. Resistance to protease
inhibitors also arises by mutation, usually by reducing the
catalytic activity of the protease and conferring a fitness
cost for resistance9 (see BOX 1 for a summary of meth-
ods for measuring fitness). To reduce the probability of
resistance development, combination therapy with three
or more drugs is used to suppress HIV replication rates
to below the level of detection10.
Bacteria and fungi. Bacteria and fungi have much lower
mutation rates than RNA viruses, of up to ~10−10 per
nucleotide per replication cycle. These less frequent
mutations limit the rate of resistance development by
mutation11, although subpopulations of cells with muta-
tor phenotypes have rates that are 1,000‑fold higher 12.
An important class of resistance-associated mutation in
bacteria is duplication or amplification of parts of the
genome, which is mediated by recombination between
repeated sequences in the genome13. Amplification
occurs at a rate of >10−2–10−5 per gene per replication
and can cause resistance, for example, by amplifying a
gene encoding a β‑lactamase with low intrinsic activity
against a particular drug substrate14. Bacteria can also
become resistant by horizontal gene transfer (HGT)
from a very extensive gene pool (via bacteriophage
transduction, conjugation or transformation), whereas
genetic exchange in fungi is limited to recombination
within species15. HGT processes in bacteria can cause
multidrug resistance in a single genetic event 16. Most
bacterial and fungal pathogens are not dependent on
their ability to infect humans for survival. As a con-
sequence, the selective forces that drive bacterial and
fungal pathogen evolution include survival in patients
undergoing drug therapy and survival in environments
in which drug exposure may be at a very low level.

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Box 1 | Methods for measuring fitness
Biological fitness, defined as replicative success relative to competing organisms in
a particular environment, comprises survival, reproduction and transmission (see
the figure, which summarizes how each component of fitness can be measured).
Organisms in nature must often be capable of surviving in multiple spatial and
temporal environments. Although fitness measurements in a laboratory cannot
capture the full complexity of real life, they can be a useful proxy to predict relative
fitness in the real world19. Assays of fitness in the laboratory include growth rate
and competitiveness in vitro, as well as survival, virulence and competitiveness in
animal or cell infection models. These methods are complemented by
deep-sequencing approaches that increase the possibility of examining relative
fitness in complex populations, including in natural and host environments.
Additionally, animal models and epidemiological studies, including population
surveillance, can provide insights into real-life fitness over longer time periods in
clinical and natural environments and can be used to estimate the effects of
resistance on transmission to new hosts21,150,151.
The gold standard for measuring relative fitness in vitro and in vivo is growth
competition of isogenic variants using genetically tagged strains19,80. For example,
the relative fitness of isogenic HIV variants was recently measured using
synonymous nucleotide substitutions as tags36,152. Competition assays may need
to be complemented with whole-genome sequencing or deep sequencing to
overcome the potential complications that are caused by high frequencies of
genetic amplification (in bacteria) or point mutations (in viruses). In bacteria the
steady-state frequency of amplification is in the range of 10–2–10–4 per gene, and
they are frequently selectively maintained by antibiotics13,153. In RNA viruses,
extremely high mutation rates can generate a quasi-species of competing viral
genomes within the host154,155. Deep sequencing can reportedly detect viral variants
at frequencies as low as 0.1% in a heterogeneous population156–158. Another very
promising method is lineage tracking, which uses sequencing of random genetic
barcodes to observe evolutionary dynamics in populations, as was recently applied
in yeast159.
Deep sequencing can also be used to predict drug efficacy and resistance
development. A recent study of domain 1 of the NS5A protein in hepatitis C virus,
targeted by daclatasvir, used mutagenesis to generate every possible amino acid
substitution, and then applied deep sequencing to quantify the effects of individual
mutations on replication fitness and drug sensitivity160. Such data could be used to
quantify genetic barriers to resistance and to predict clinical outcomes using
mathematical models.
Deep sequencing that is focused on a subset of genes also has an important role
in high-resolution studies of cancer evolution161. Heterogeneity within tumours
creates an environment for the selection of the fittest clones (based on resource
competition), and understanding these processes provides a basis for optimizing
drug therapy and for predicting tumour development. However, it is difficult to
achieve sufficient sequence depth and resolution to detect small subpopulations
before they acquire additional critical mutations that facilitate expansion and
proliferation162. Currently, deep sequencing is the methodology that provides
the best information on cancer cell fitness, but a caveat to interpretation is that the
most prevalent cell type may not necessarily be the most important cell type that
leads to patient death.
Survival Reproduction
• Viability in vitro over time at different • Exponential growth rates, lag
drug concentrations phases and yields for single strains
• Survival assays in vitro (e.g. stationary cultured in different media
phase, serum resistance and survival during • Competition between strains
exposure to reactive oxygen species, in different media during
low pH, and so on) exponential growth and complete
• Survival assays in cell cultures growth cycles
(e.g. phagocytic cells) • Deep-sequencing techniques to
• Survival assays in hosts (animals or humans) monitor population dynamics
Transmission
• Epidemiological data
• Animal and human experimental
models of transmission
Nature Reviews | Genetics
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The most important classes of antibacterial drugs in
terms of quantity used are the β‑lactams, fluoroquinolo-
nes and macrolides. Mechanisms of resistance include
spontaneous mutation — in particular, mutations that
alter the drug target or that upregulate drug efflux — and
the acquisition of resistance genes by HGT17. The relative
importance of mutation versus HGT depends on drug
class and bacterial species. In Mycobacterium tuberculosis,
resistance occurs exclusively by spontaneous muta-
tion18. In Gram-negative and Gram-positive bacteria,
resistance to β-lactams occurs almost exclusively by the
acquisition of β‑lactamases by HGT, whereas resistance
to fluoroquinolones is primarily associated with muta-
tions, although HGT also contributes17,19. HGT is very
important clinically because the genetic linkage of resist-
ance genes on mobile elements causes multidrug resist-
ance16. Finally, a resistance mechanism that is sometimes
overlooked is the ability of bacteria to form biofilms dur-
ing infection, thus providing physical and physiological
protection against drug therapy 20.
The most important classes of antifungal drugs
are the azoles, the echinocandins and the polyenes15.
Resistance is caused by mutations that increase efflux,
alter the drug target or change cell metabolism to reduce
the accumulation of toxic products in the presence
of the drug 15. Fungi do not have access to a wide gene
pool for HGT, and resistance development is dependent
on mutation rates.
Plasmodium falciparum. The vector-borne pathogen
P. falciparum must passage between an invertebrate
host — the mosquito — and a vertebrate host — the
human — typically encountering drug selection only in
the human host 21. Successful transmission is essential,
and any evolution of drug resistance is only possible to
the extent that it does not break the complex cycle of
transmission.
The major drug classes used in the treatment of
malaria are chloroquine, sulfadoxine–pyrimethamine
and artemisinin22. Chloroquine inhibits haemoglobin
degradation, causing haem to accumulate to toxic levels
in P. falciparum, and sulfadoxine–pyrimethamine inhib-
its DNA and RNA synthesis by blocking the folate bio-
synthesis pathway 22. The mode of action of artemisinin
is still controversial, but recent research suggests that it
is activated by interaction with ferrous iron and that an
Fe–artemisinin adduct then interacts with a calcium
pump enzyme, PfATP6, which leads to the loss of
function of the calcium pump and to the death of the
P. falciparum23. Resistance to chloroquine and the anti-
folates caused by mutation is widespread, and the current
standard of care is to use artemisinin-based combination
therapies as the first-line treatment for malaria caused by
P. falciparum24. However, artemisinin-resistant malaria,
with a phenotype of delayed clearance after therapy, has
recently been identified in South-East Asia25,26, putting
the efficacy of this essential drug therapy at risk27,28. In
conclusion, resistance to antimalarial drugs arises by
point mutations, and there is no evidence that it is asso-
ciated with a high mutation rate or HGT. The slow rate
of global resistance development, despite the very heavy
REVIEWS
Isogenic variants
Genetic variants that are
derived from a single cell
or genotype.
Epistatic
Refers to the phenomenon
of epistasis, which involves
interactions (genetic,
regulatory, and physiological)
between or within genes and
results in non-additive effects
with regard to phenotype.
462 | AUGUST 2015 | VOLUME 16 www.nature.com/reviews/genetics
use of only a few antimalarial drugs over several decades,
may be a consequence of the small mutation supply rate
and the difficulty in developing low-cost resistance by
drug-target mutations.
Human cancer. Human cancer cells are subjected to
intense drug selection pressure, but as cancer develops
de novo in each individual, the only equivalent to trans-
mission is within the individual in the form of metastasis
— the movement of cancer cells to establish tumours
at other body sites. Thus, cancer differs from infectious
diseases in that drug resistance and the relative fitness
of cancer cells are not properties that are transmitted
between individuals.
The targets of anticancer drugs include thymidylate
synthase, DNA replication, topoisomerases I and II,
and tubulin, and resistance mechanisms include point
mutations, deletions and genetic rearrangements or
amplifications29. Chromosomal instability is a common
feature of cancers (amplifications, deletions and other
rearrangements) and is associated with tumour hetero-
geneity and drug resistance30,31. This association is analo-
gous to the situation in bacteria in which amplifications
can protect against drug therapy and function as a step
Potential costs
• Metabolic costs of activity
• Reduced uptake or increased
loss of essential compounds
b Concentration
regulation
c Enzymatic
a Target alteration action
Potential costs Potential costs
• Reduced kinetic • Metabolic cost of expression
efficiency • Toxic breakdown products
• Downstream effects • Transport cost of enzyme
Figure 1 | The nature of fitness costs in different
systems.  The diagram of a cell illustratesNature Reviews | Genetics
the three major
mechanisms of drug resistance: reducing interaction
with the drug target by mutation, modification or
protection8,12,15,17,19,24,41,48 (part a); restricting the internal
concentration of the drug by altering efflux or influx16,17,19
(part b); and reducing the concentration of the active drug
by enzymatic activity that either degrades or fails to
activate the drug17,19 (part c). Each alteration can incur
general metabolic costs in addition to mechanism-specific
costs that negatively affect physiology19,52.
© 2015 Macmillan Publishers Limited. All rights reserved
in the evolution of stable resistance13. Some resistance
mechanisms are specific to cancer, including the pro-
motion of the epithelial-to-mesenchymal transition by
chemotherapy 32. This transition is an important step in
cancer metastasis, as cells develop a morphology that is
associated with increased motility and invasive capac-
ity 29. The growth environment of cancer cells in a solid
tumour is itself associated with an increased probability
of surviving drug therapy 33. This protection bears com-
parison to the resistance shown by bacteria growing as
biofilms, combining elements of physical protection with
altered physiological states. Finally, epigenetic changes
(reversible DNA methylation patterns and histone modi-
fication patterns) may influence cancer development 34
and resistance to anticancer drugs35 by changing gene
expression patterns.
Fitness costs of drug resistance
Common mechanisms by which drug resistance arises
are target alterations, reductions in internal drug con-
centration and inactivation of (or failure to activate)
the drug (FIG. 1). Because drugs target important cel-
lular mechanisms, mutational resistance is frequently
associated with reduced replicative fitness. There are
many documented examples of the fitness costs that
are associated with drug resistance in different systems.
For example, a dual-infection competition assay
with sequence-tagged HIV36 (BOX 1) measured replica-
tive fitness costs caused by mutations leading to resist-
ance to NNRTI drugs37. The relative magnitude of the
fitness costs depended on the HIV subtype (subtype B or
subtype C), suggesting epistatic effects on fitness38.
The fitness costs of resistance in bacteria have been
extensively reviewed19,39,40. Briefly, most mutations and
HGT that cause resistance in bacteria are initially asso-
ciated with fitness costs. In most cases, the cost can be
ameliorated by the acquisition of compensatory muta-
tions. However, there are examples in which mutational
resistance is apparently cost-free, with clinical data sug-
gesting that there is no significant fitness cost in infected
individuals41,42.
There is also evidence that fitness costs have a role
in limiting the establishment of resistance to one of the
major antifungal drug classes, the polyenes. Resistance to
the most commonly used polyene, amphotericin B, which
is a drug that has been in use for more than 50 years, is
very rare clinically. Analyses in vitro and in animal mod-
els showed that every resistance mutation imposed huge
fitness costs on fungal pathogenicity, making the fungi
hypersensitive to the host immune system and unable
to invade or damage host tissue43. This may be an exam-
ple of a drug that is effective against the target pathogen
and for which the mechanism of resistance is strongly
associated with reduced fitness. However, a downside of
this drug is frequent treatment failure due to poor tissue
penetration and dose-limiting renal toxicity 43.
In malaria therapy, the evidence suggests that resist-
ance to all classes of antimalarial drugs, including arte-
misinin, is associated with significant fitness costs to
P. falciparum in the absence of drug selection44, possibly
explaining the slow development of resistance.

Wild type Drug-resistant Drug-resistant mutant
mutant with fitness compensation
a ing why the most resistant variant is not necessarily
Intragenic
mutation
b selected to confer NRTI resistance. This triple mutant
Increased
dosage
c Genetic compensation of fitness costs for antibiotic
Intergenic
mutation
d enzyme. Stable fitness compensation can be conferred
Bypass
GGTCAATAATAGC GGTCAATAATAGC GGTCAATAATAGC mechanism
Figure 2 | Common fitness-compensatory mechanisms.  The figure represents
proteins in which drug-resistance mutations (red circles) arise that cause
Nature a reduction
Reviews | Genetics
in organism fitness, and illustrates several different mechanisms of fitness
compensation19. a | Compensation by an intragenic mutation (dark green circle) is
shown46,47,53. b | Compensation by increased dosage of a low-fitness mutant protein
is shown13,56. c | Compensation by an intergenic mutation (dark green circle) is shown,
which is relevant when protein complexes or protein interactions are involved in
physiological activity48,51. Different proteins are shown as light blue and light green
colours. d | A bypass mechanism whereby, for example, an additional regulatory factor
compensates for the reduced functionality of the mutant protein19,49.
The interplay between fitness costs and resistance
is complicated when there are mixed infections or, in
the case of cancers, mixed cell populations. In cancer
therapy, in which lineages within a tumour compete
for space and nutrients, a less aggressive chemotherapy
can sometimes improve patient survival, in compari-
son to overwhelming drug treatment that removes sus-
ceptible cell lineages, allowing drug-resistant lineages
to kill the patient 45.
The overall conclusion from all of these studies is
that fitness costs are a frequent consequence of primary
resistance mutations across all systems tested.
Mechanisms of fitness cost compensation
The potential mechanisms of fitness compensation for
the costs of drug resistance are outlined in FIG. 2. We
consider below whether these mechanisms are shared
across pathogens and systems.
Genetic analysis of recombinant HIV viruses car-
rying resistance mutations has shown that in some
cases compensatory mutations were selected within
the genes encoding the drug targets to improve their
function46,47 but in other cases that the compensation
involved mutations in other regions of the virus48.
An interesting example is that of drug resistance in
HIV that is caused by multiple mutations in the pro-
tease that reduce fitness: the fitness consequences can
be rescued by mutations in the vicinity of the viral
Gag proteolytic cleavage sites, leading to improved
processing of Gag by the highly mutated protease49.
NATURE REVIEWS | GENETICS VOLUME 16 | AUGUST 2015 | 463
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Frequencies of mutant variants in clinical isolates of
HIV correlate with relative replicative fitness, explain-
the most frequent 50. Interestingly, the fitness cost
of an NNRTI-resistant double mutant (K103N and
L100I in reverse transcriptase) was compensated by
the addition of a third mutation (L74V) that itself is
is found clinically 51, suggesting that it is selected as
a fitness-compensatory combination that confers
cross-resistance to different drug classes.
resistance has been extensively reviewed13,39,52. Briefly,
a temporal relief of fitness costs in bacteria is fre-
quently conferred by tandem amplification mutations,
increasing, for example, the activity of a rate-limiting
by point mutations that improve the activity of a target
protein or the flow through a biochemical pathway.
An additional mechanism of compensation is the
increase in resistance owing to mutations that cause
the increased catalytic efficiency of an enzyme but
that also decrease the stability of the enzyme to reduce
fitness. Mutations in the same gene that increase
thermo­dynamic stability of the enzyme can compen-
sate for such costs53. Analysis of compensation shows
that there are frequent epistatic interactions between
the drug target mutations and components of the
physiology of the cell54,55.
Evidence for fitness costs of resistance in fungal
systems and subsequent cost compensation is sparse15
except for the evidence that resistance to ampho-
tericin B is both costly and apparently very difficult to
reduce by compensatory evolution43.
Epistasis is also involved in fitness compensation
in drug-resistant P. falciparum 56. Mutations in dihy-
drofolate reductase (pfdhfr), which confer resistance
to pyrimethamine, reduce parasite fitness and can be
compensated for by the amplification of the first gene
in the folate synthesis pathway, GTP cyclohydrolase 1
(pfgch1). Because the amplification of pfgch1 itself
confers low-level resistance, this amplification is prob-
ably selected early by the drug and reduces the cost of
acquiring subsequent mutations in pfdhfr that further
increase resistance56.
In human cancers undergoing drug therapy, the
population of cells accumulates mutations that result
for some in increased growth rates and/or drug resist-
ance, but result for others in reduced growth rates
and/or associated drug resistance57,58. The variety of
genetic alterations within a cancer cell, and the range
of different cancer cell genotypes that compete for
growth within a tumour, make it difficult to defini-
tively identify individual mutations conferring fitness
costs or fitness compensation. We assume that the
interplay between mutational fitness costs and com-
pensatory mutations has a role in determining the
progress of a cancer and the emergence of a dominant
clone, but to the best of our knowledge there is no
definitive evidence that associates specific mutations
with fitness compensation.
REVIEWS

Population bottlenecks
The concept that only a limited
number of individuals (and
thus genotypes) act as
founders of the next generation
of cells or organisms.
Fixation
When there are at least
two variants of a gene in a
population, fixation refers
to the situation when,
owing to selection or chance
fluctuations, only one allele
remains.
Susceptible
‘wild type’
Evolutionary dynamics and mechanisms
The rate at which drug resistance emerges and spreads in
a population of organisms (or cells in the case of cancer)
is determined by the interplay of several different factors,
many of which are still poorly understood. Among the
factors that influence the rate and trajectory of resist-
ance evolution, we can identify the following: mutation
supply rate; relative fitness of the drug-resistant mutant
as a function of drug concentration; strength of selective
pressure; clonal interference; compensatory evolution;
and the presence of epistatic interactions between resist-
ance genes or interactions between drugs. We discuss
these in more detail below. Additionally, there are epide-
miological factors, including host population structure
and density, immunity status and transmission control,
that are beyond the scope of this Review.
A useful graphic illustration of resistance evolution
is to consider a mutational space: that is, all types of
mutations or HGT events that can generate resistance
Weak selection
Strong selection
to a particular drug (FIG. 3) and how a selective pressure
influences which mutants are selected. Which muta-
tions will be enriched depends on the factors discussed
below, and a key question is: how can we describe,
predict and slow down resistance evolution?
Mutation supply rate. The generation of genetic het-
erogeneity in a homogenous infecting population is
determined by mutation supply rate and population
dynamics within the host with regard to the occur-
rence of population bottlenecks59. Mutation supply rate
is in turn determined by population size and rates of
mutation and HGT.
Mutation and HGT rates have been measured in
both bacteria and viruses in vitro 60,61. Substitution
mutation rates span at least eight orders of magnitude
from 10−3 to 10−5 per nucleotide per genome replica-
tion across different types of RNA viruses62, from 10−6
to 10−8 in DNA viruses62, and ≤10−10 per nucleotide per
genome replication in bacteria and fungi11,63. Similarly,
HGT rates (most available data are for bacterial con-
jugation) vary extensively depending on the type of
transfer mechanism, organism, transfer conditions and
methodology used to assess the rates64.
Bacterial, fungal, viral and parasitic infections can
be caused by a single, or very few, infectious particles
(for example, see REF. 65). This makes the probability of
drug resistance at the time of infection highly depend-
ent on the frequency of resistance in the relevant com-

munity or hospital environment. For many pathogens,

Mutant fitness

High-risk space
we have a poor knowledge of the transmission bottle-
neck population or of census population sizes within
infected hosts, but for some infections we know that the
total pathogen population can be so large that, with
the mutation rates described above, resistant mutants
are likely to arise during the infection. For example,
in respiratory and wound infections, the number of
bacteria typically exceeds 10 10 per gram of tissue66.
Low-risk space Virus population sizes are also very large. During an
untreated HIV infection, population sizes are in the
Resistance level order of 1010 (REF. 2) and, with mutation rates of 10−4 to
10−5 per nucleotide per genome replication, all poten-
Figure 3 | The relationship between fitness, degree of resistance and rate| Genetics
Nature Reviews of tial single, double and triple mutants could arise in the
formation of drug-resistant mutants.  The relationship between relative fitness, population, implying that the mutation supply rate is
resistance and rate of formation is shown for a hypothetical set of all possible unlikely to be rate limiting for the emergence of resist-
drug-resistant mutant variants. Each circle represents one specific resistant mutant, and
ance after the infection is established. In human cancer,
the size of each circle represents its rate of formation. The probability of fixation of a
resistant variant is dependent on the interplay between these three variables: that is, the if a tumour is nourished with a blood supply, it can
rate of formation, level of drug selection and relative fitness153. The negative correlation grow to weigh between several grams and several kilo-
shown between rate of formation and resistance level is intentional: there are more grams, with approximately 108 cells per gram of tissue67.
mutational events that are capable of generating small decreases in drug susceptibility The total cell population size will ensure that there is
than there are events capable of causing high-level resistance. These high-frequency little or no limitation in the supply of potential drug-
events include gene-knockout mutations and genetic duplications13,153. The solid line resistance mutations within the tumour, and methods
boxes indicate mutant types that would be selected at weak selection pressures (low are now available to measure the absolute numbers and
drug concentrations, <minimal inhibitory concentration (MIC)) and strong selection types of mutations in developing tumours68.
pressures (high drug concentrations, >MIC). The dashed line boxes indicate mutational
spaces in which the risk of resistance development is either high or low. In particular, a
Fitness. The relative fitness of a drug-resistant pathogen
high risk of sustained drug resistance is provided by mutations that confer substantial
drug resistance accompanied by negligible or minor detrimental effects on fitness80,82,83. (in the absence or presence of the drug) is a key param-
It is important to note that there is no necessary correlation between level of resistance eter in determining success within the host and trans-
and relative fitness, as shown by a recent comprehensive analysis of rifampicin-resistance mission between hosts for bacteria19,69–71, viruses72–75
mutations78. The solid line boxes, as illustrated in the figure, can in practice overlap, so and parasites44. Thus, the rate of emergence and fixation
that a low-cost mutation can confer high-level resistance42,80. of resistant mutants at a given mutation supply rate is

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© 2015 Macmillan Publishers Limited. All rights reserved


Deleterious
A mutation that reduces
relative fitness under a
particular condition.
Sweep
A selective sweep is the
reduction or elimination of
variation within a population as
a result of an increase in the
proportion of one ‘successful’
variant.
NATURE REVIEWS | GENETICS VOLUME 16 | AUGUST 2015 | 465
influenced by mutant fitness (FIG. 3). In addition, the
steady-state frequency of resistance at a given selective
pressure, and the rate of reversibility when the selec-
tive pressure is reduced, is generally determined by the
fitness costs of the resistance mechanism.
The influence of pathogen fitness on evolutionary
success in clinical settings with regard to emergence
and transmission has been demonstrated in different
studies. That is not to say that we know which par-
ticular fitness components are most relevant in clinical
settings or during epidemics (and this probably differs
between pathogens), or which components influence
growth within the host and which affect transmission
between hosts. There is a dearth of knowledge in this
important area. However, laboratory assays of relative
growth fitness (BOX 1) together with epidemiological
studies of genetic allele prevalence in clinical isolates
suggest that fitness measures made in vitro can have
clinical relevance. An illustrative example is given by
the studies of fitness and resistance in M. tuberculosis.
It was shown that for aminoglycoside-resistant bacte-
ria the fitter bacteria (as determined by growth rate
in vitro) were also the most frequent types found in
patients41,42. The level of resistance influenced success
such that the most common clinical mutants not only
had high fitness but also had a sufficiently high level of
resistance76. Similarly, in clinical isolates of M. tuber-
culosis and Staphylococcus aureus, the spectrum of
rifampicin-resistance mutations is biased in favour
of low-cost mutations77,78. Similar observations have
been made for P. falciparum44, and for HIV79 for which
low fitness cost variants have spread on a global scale,
whereas more costly mutations have remained local or
waned over time.
Selective pressures. In natural and clinical settings, bac-
terial, viral and parasitic pathogens are exposed to a
wide range of drug concentrations, generating a vari-
able range of selective pressures. When drug concen-
trations are high enough to prevent pathogen growth,
resistant mutants need to pre-exist and the rate of
enrichment is determined by the number of mutants
in the population and their fitness. By contrast, dur-
ing a non-lethal selection, mutants may emerge and
their rate of enrichment is determined by the fitness
difference between susceptible and resistant cells. As
shown by recent studies in bacteria, the rate of emer-
gence and the type of mutants selected differ between
weak and strong selective pressures39,80–82. At low drug
concentrations, mutant phenotypes usually result from
many mutations of small effect, whereas lethal selec-
tions typically enrich pre-existing mutations of large
effect. Thus, mutants with low fitness cost are selec-
tively enriched at low antibiotic concentrations, and the
smaller the fitness differential relative to the susceptible
wild-type or other mutants the lower the selective drug
concentration that is required for enrichment (FIG. 3).
Furthermore, the stepwise selection of successive
small-effect mutations enriches for mutator strains that
have an increased probability of acquiring resistance by
mutation or HGT83.
© 2015 Macmillan Publishers Limited. All rights reserved
REVIEWS
Clonal interference. Depending on the number of
potential mutants available, and their respective rates
of emergence, one may predict that many types of resist-
ant mutants are often simultaneously present in a given
population. This is especially true for viruses with high
mutation rates for which the number of variants might be
very high at any given point during infection84,85, but also
for bacteria, as several types of mutants can emerge and
be simultaneously present in the same population86–88. In
this situation, clonal interference (competition between
asexual clones) influences the evolutionary trajectory
(FIG. 4). Thus, when different beneficial (for example,
resistance) mutations arise independently in different
individuals, they compete against each other, typically
leading to the loss of most clones and the appearance of a
‘dominant clone’ (REFS 70,89). Large population sizes and
high mutation rates increase the number of competing
resistance mutations, thereby increasing the potential for
clonal interference. Clonal interference has been dem-
onstrated during the adaptation of antibiotic resistance
plasmids to their bacterial hosts90, adaptation of bacte-
ria to the fitness cost of resistance91, and adaptation to
various selective pressures in RNA viruses92, bacteria93
and yeast 94,95.
Compensatory evolution. Most mutations that occur in
any organism are deleterious. Their fate in the absence
of selection may be extinction, persistence at low fre-
quency, reversion to the susceptible state or compen-
satory evolution whereby the costs are reduced or
eliminated, thus increasing the probability of mainte-
nance. Which process dominates depends on the rela-
tive rates of reversion and compensation, the relative
fitness of the reverted and compensated mutants, and
the population size (FIG. 4). Generally, the compensatory
mutation rate is considerably higher than the reversion
rate because the mutational target for compensation is
larger than the reversion target. Thus, the compensatory
mutation rate versus the reversion rate is usually in the
range of 10–100:1 (REFS 96–100). The revertant mutant
is often the fittest variant (in the absence of drugs), but
there are examples in which the compensated mutant
reaches the same fitness level as the revertant 96,101.
Whether a revertant or compensatory mutation evolves
from a resistant population also depends on the popula-
tion size such that the more frequently occurring com-
pensatory mutation will dominate in small populations,
whereas in large populations, with both revertants and
compensatory mutations present simultaneously, the
fitter revertants dominate96,102. Experimental and clini-
cal studies show that the fitness cost of resistance can
be reduced by compensatory mutations and that com-
pensated mutants can rapidly sweep a drug-resistant
population of bacteria19,103, viruses104–106 and parasites44
(see the mechanisms of fitness cost compensation
section above).
Interactions between resistance mutations. Recent data
from bacterial, viral and eukaryotic systems show that
epistasis is pervasive and that it might have an impact
on resistance evolution107–109.
 REVIEWS

Epistatic interactions with regard to fitness (FIG. 5)
are observed in bacteria, including Escherichia
coli 110–113, Salmonella enterica subsp. enterica serovar
Typhimurium55,96 and Pseudomonas aeruginosa114. These
studies showed that combinations of fitness-reducing
chromosomal resistance mutations and/or resist-
ance plasmids can generate either no epistasis, posi-
tive epistasis (a double mutant has a higher fitness
than expected from the sum of the costs of individual
mutations) or negative epistasis (a double mutant has a
lower fitness than expected from the sum of the costs
of individual mutations), with positive epistasis being
especially pervasive. In some cases, reciprocal sign
epistasis was observed in which the fitness of the dou-
ble mutant was higher than that of either of the single
mutants. This implies that the acquisition of additional
resistance mutations or plasmids can increase the fit-
ness of a resistant strain and that reversal of resist-
ance might not be achievable merely by reducing or
withdrawing drug use.
Epistasis can also occur within a gene. Studies of evo-
lution of β‑lactam resistance in vitro and quantification
of fitness landscapes suggest that mutations within a
gene show strong epistatic interactions that constrain
the type and order of selected mutations and make the
evolutionary trajectory contingent on the first random
mutation115,116.
Interactions between drugs. Another type of interaction
involves the effect of resistance to one drug on the sus-
ceptibility to other drugs117. An early study by Szybalski
and Bryson 118 and several subsequent studies 119–123
have shown that resistance to one drug class might either
increase resistance to another drug or result in increased
susceptibility (so‑called collateral sensitivity). In most
cases, the mechanisms for collateral sensitivity are poorly
understood, but an exception is vancomycin-resistant
strains of methicillin-resistant S. aureus (MRSA) that are
susceptible to a combination of vancomycin and oxacil-
lin but resistant to each drug when given individually 122.
This synergy occurs because the oxacillin-resistant
penicillin-binding protein 2A (PBP2A) enzyme can-
not process peptidoglycan precursors ending in
d‑Ala–d‑Lac (which are present owing to glycopeptide

Susceptible Resistant cells or Compensatory Clonal interference Epistatic interactions Dominant cells
cells or organisms evolution Competition between In a multidrug- or organisms
organisms Different resistance Fitness is restored several different clones resistant cell or
genotypes emerging at to different degrees organism interactions
different rates and with by compensatory between different
varying effects on fitness mutations types of resistance
genotypes
influence fitness
Fitness

Time

Figure 4 | Evolutionary trajectories of drug-resistance development.  Nature

Evolutionary progression from a wild-type pathogen or cell type undergoing
drug treatment to a drug-resistant variant is shown, displaying the effects
of fitness costs of resistance, compensatory evolution, clonal interference
and epistatic interactions on the trajectory of evolution, leading
to a dominant clone (cell or organism). Each arrow represents a specific
resistant mutant, and the thickness of the arrow represents
the relative rate of emergence. At the top of the figure are examples of the
relevant cells or organisms that are present at each stage, with mutations
shown as different coloured stars. From the left-hand side of the figure:
resistant genetic variants generally have lower fitness than the susceptible
parent, and in the absence of drug selection or of compensatory evolution,
will gradually be lost from the population39,40 (the Reviews
ordering Genetics
of the| thick and
thin arrows is not important). Multiple different mutant variants will
frequently arise in a population, and the enrichment of specific successful
variants will be determined by their relative fitness, the effects of clonal
interference on competition between variants91,92,95 and the effects of
epistasis on the expression of mutant phenotypes107,109. The multiple parallel
arrows indicate that the population of competing cells or organisms may
contain many genetic variants, with some being driven to extinction and
newer variants arising, before one or a few dominant cells or organisms
finally approach fixation in the population70,89. In the final panel on the
right-hand side of the figure, the emerging dominant clones are those that
provide sufficient resistance without substantially impairing fitness.

466 | AUGUST 2015 | VOLUME 16 www.nature.com/reviews/genetics

© 2015 Macmillan Publishers Limited. All rights reserved

 REVIEWS

induction of the vanA operon), and therefore cell wall
biosynthesis is inhibited.
Recent large-scale studies have systematically exam-
ined the extent of cross-resistance and collateral sen-
sitivity 124–127 and demonstrated that both are common
phenomena. Applying these findings to drug-cycling
experiments shows that, under in vitro conditions, an
alternating drug-cycling protocol may slow the rate of
resistance development124,125. Such interactions are also rel-
evant to drug combinations because using synergistic drug
combinations below the minimal inhibitory concentration
(MIC) could result in faster resistance development
than antagonistic drug combinations128,129. These find-
ings imply that antagonistic drug combinations (which
are usually avoided in clinical settings) might be better
than synergistic combinations in slowing resistance
development. Whether collateral sensitivity and antago-
nistic interactions can be applied to clinical situations
to reduce the rate of resistance evolution remains to be
determined.
Therapeutic implications
Treatment. In principle, detailed knowledge of fitness
costs and interactions between resistance mutations
and between drugs could be applicable to clinical set-
tings to reduce resistance development. For example,
single drugs or drug combinations could be rationally
designed such that the drug itself or the resulting resist-
ance mechanism confers a reduction in pathogen fitness
or virulence. Alternatively, identification of epistatic
interactions between resistance mutations could allow
Relative fitness
catastrophe’ whereby the virus is mutagenized to
death131,132. Lethal mutagenesis has been demonstrated in
experiments with the drug ribavirin for several viruses,
including hepatitis C133, poliovirus134 and foot-and-
mouth disease virus135, and has been widely discussed
as a more general approach to treat viral infections such
as HIV136,137. A concern is the extent to which resist-
ance can develop to mutagenic drugs138–140. However,
these resistance mutations may also be associated with
reduced fitness and virulence141 and susceptibility to
another mutagen, implying that cycling of mutagenic
compounds might be a feasible approach to achieve virus
extinction142.
Combinations of different classes of drugs have been
widely used in clinical settings, in particular for HIV,
tuberculosis (TB), malaria and cancer, for which com-
binations are standard therapy to increase efficacy and
to reduce resistance development 24,143–145, and they also
show promise in potentially creating fitness trade-offs
that might limit resistance development when used in
antifungal therapy 146. An extension of drug combination
use is the search for collateral sensitivity networks in
1.0
0.8
0.6

the combined use of the corresponding drugs to reduce

resistance emergence.
Resistance will almost always develop to any drug 0.48
used in clinical settings, but there are exceptions in
which resistance is absent or slow to appear. For exam-
ple, in Streptococcus pyogenes, no cases of penicillin
resistance are known despite penicillin being the stand-
ard treatment of S. pyogenes infections for more than
60 years, and, in fungi, resistance to the polyene drug
amphotericin B has remained very low even after dec-
pe

ve

ive

ive

sig eci sis

is
ist al
nt

nt

as
iti
ty

ades of widespread use43. A similar example of a lack of

ep c
a
t

sit

n pro
a

ist
ga
d
ild

ut

ut

Po
Ad

ep
Ne
M

resistance development, but with a much shorter clinical

W

gn

R
Si

use, is the HIV integrase inhibitor dolutegravir (DTG),

Minimal inhibitory
concentration
(MIC). The lowest
concentration of an
antimicrobial drug that,
under a set of agreed
conditions, inhibits the visible
growth of a microorganism
after overnight incubation.
which was recently introduced as a part of first-line HIV
therapy. In these cases, it is likely that the lack of resist-
ance development is due to high fitness costs (reduced
replication capacity and growth rates, or hypersensitivity
to the host immune system43) associated with resistance
and the absence of efficient compensatory mutations.
Thus, DTG-resistant mutants with point mutations in
HIV integrase were dramatically impaired in replication
and the ability to acquire resistance against RTIs79,130.
These examples suggest that certain types of drug targets
or drugs are less prone to resistance development and
that this is strongly associated with a high fitness cost of
resistance (see below).
An interesting concept regarding the treatment
of RNA viruses with intrinsically high mutation rates
is to increase the mutation rate beyond the level that is
compatible with virus survival by inducing an ‘error
Epistasis
Single mutants Double mutants
Figure 5 | Epistasis and relativeNature
fitness.  The relative
Reviews | Genetics
fitness of two drug-resistant mutants (mutant 1 with
a fitness of 0.8 and mutant 2 with a fitness of 0.6) is used to
illustrate different possible types of epistatic interaction
between mutations. With no epistasis (additive effect), the
fitness of the double mutant is predictable from the fitness
values of the individual single mutants (0.8 × 0.6 = 0.48).
Epistasis is illustrated for the double mutant as negative
(the double mutant has a lower fitness than that predicted
by an additive effect), positive (the double mutant has a
higher fitness than that predicted by an additive effect but
a lower fitness than the single mutants), sign epistasis (the
double mutant has a higher fitness than one of the single
mutants) and reciprocal sign epistasis (the double mutant
has a higher fitness than each of the single mutants).

NATURE REVIEWS | GENETICS VOLUME 16 | AUGUST 2015 | 467

© 2015 Macmillan Publishers Limited. All rights reserved

REVIEWS
Pleiotropic
When one gene influences
multiple phenotypic traits.
Resistome
The collection of all of the
antibiotic resistance genes
and their precursors in
both pathogenic and
non-pathogenic bacteria.
468 | AUGUST 2015 | VOLUME 16 www.nature.com/reviews/genetics
which resistance to one drug confers increased suscepti-
bility to a second drug 123–126,147. In such a case, two drugs
could be alternated to select against resistance to either
drug. Experimental data suggest that this approach can
slow the rate of resistance development 124,125, but further
work is needed to determine whether it can impede
resistance evolution in patients. It should be noted that
this discussion about drug combinations is applicable to
mutationally acquired resistance but not necessarily
to horizontally transferred resistance, which is the most
common mode of acquiring clinically relevant antibiotic
resistance in bacteria. Thus, using drug combinations
for bacteria for which the corresponding resistance is
typically carried on a mobile element could speed up
resistance development compared with monotherapy,
adding to the complexity of the cost–benefit analysis of
drug combination use.
Drug design. Can we use our knowledge of the rates
and fitness costs of resistance development to design
drugs with a low propensity for resistance development?
A priori there are several predictions that could be made
regarding the characteristics of a low-propensity resist-
ance mechanism: the rate of mutation or HGT should
be low; fitness costs associated with resistance should be
high; and the rate and efficiency of compensatory
evolution should be low.
Which resistance mutation or mechanisms would
be associated with high fitness costs? Drug targets with
complex downstream effects — for example, RNA poly-
merase, the target of rifampins — are attractive because
resistance mutations typically have pleiotropic effects
on pathogen physiology and reduce bacterial fitness.
However, evidence from both in vitro measurements and
clinical prevalence shows that compensatory second-site
mutations in RNA polymerase occur with a high fre-
quency and restore function sufficiently so that resist-
ance is widespread and established98,148. Accordingly,
fitness costs of resistance alone are not a good predictor
of subsequent clinical resistance development.
A low rate of penetrance (the probability of a muta-
tion being expressed as a drug-resistance phenotype)
is expected if the target molecule is encoded by several
genes (for example, drugs that target ribosomal RNA
encoded by several rRNA operons in bacteria and where
susceptibility is dominant). In bacteria, resistance to
this type of targeting can be mediated by HGT of genes
encoding enzymes that modify all of the rRNAs or that
reduce the activity of the drug itself, but this type of tar-
get could still be potentially useful in fungi and other
eukaryotic pathogens for which HGT is not a substantial
issue. HGT events are hard to predict but depend on
the following four factors: first, the presence of a resist-
ance gene in the total resistome; second, the ecological
opportunity for HGT (frequency of pathogen–donor
interaction); third, the efficiency of transfer versus
genetic barriers (restriction systems or clustered regu-
larly interspaced short palindromic repeat (CRISPR)
systems); and fourth, the fitness effects of the resistance
mechanism on the recipient. Currently, we have only a
very limited knowledge of these factors.
© 2015 Macmillan Publishers Limited. All rights reserved
It was also thought that resistance development
would be severely reduced if a drug acted on multiple
targets149. However, experience with fluoroquinolones,
an antibiotic class that acts on two different essential
and conserved bacterial targets (DNA gyrase and DNA
topoisomerase IV) is not encouraging in this respect.
Resistance to fluoroquinolones developed easily and
became clinically established, probably because the
relative activity of the drugs is not perfectly balanced
between the two targets, allowing resistance to develop
first in one target and then in the other.
An alternative strategy to developing one drug that
hits multiple targets is to develop and use multiple drugs
to hit multiple targets. This is routine practice in HIV10,
TB18 and cancer therapy 29. This is an area of research that
is now receiving increased interest for the development of
effective antibacterial and antifungal therapies146, using
available drugs in combination, or drug–potentiator
combinations, and which will probably strongly
influence many future drug discovery and development
programmes against pathogens.
Concluding remarks
As discussed in this Review, resistance development
to toxic drugs in viruses, bacteria, fungi, parasites and
cancers shows both important similarities and differ-
ences that allow us to make certain generalizations, but
that also emphasize the need to sometimes describe and
analyse resistance evolution in a manner that is specific
to particular pathogens or resistance mechanisms. With
regard to similarities, it is striking that the mechanisms of
resistance are often genetically and functionally similar
between these different organisms and cells. For exam-
ple, resistance by mutational target alteration occurs in
all systems, and upregulation of drug efflux systems is
widespread, except in viruses. Furthermore, with regard
to the dynamics of resistance evolution, the effect of fac-
tors such as mutation supply rates and fitness effects of
the resistance mechanisms are very similar between
these systems. Likewise, epistatic interactions between
resistance mechanisms and interactions between drugs
seem to be shared and pervasive phenomena.
However, there are also important differences, and
one major difference concerns the extent of constraints
on resistance mechanisms with regard to their effects on
organism fitness and transmission. Cancer cells and
associated resistance mechanisms can be considered the
least constrained, as there is no selection for cancer cells
to transmit between different hosts, and evolution can
be short-sighted and local. By contrast, the long-term
evolutionary success of viruses, bacteria and parasites
depends not only on survival and growth within the
infected host but also on the ability to transmit to new
hosts. Furthermore, many bacteria and parasites spend
time outside the host in the natural environment, where
they need to respond to different problems and selec-
tion pressures, creating adaptive conflicts that must be
resolved. Another important difference between bacteria
versus viruses, fungi, parasites and cancers is that the
major resistance mechanisms in bacteria involve HGT
that has different characteristics and dynamics from
 REVIEWS

those of mutational mechanisms. HGT events are cur-
rently less predictable at the population level. Moreover,
in contrast to mutational mechanisms, resistance to mul-
tiple classes of drugs can be acquired in a single genetic
event, suggesting that resistance evolution in bacteria
may proceed in great leaps compared with more gradual
changes in other organisms.
Looking to the future, what are the major challenges,
where do we next go to solve them, and are there lessons
to be learned from one system that can inform studies
in any other system? The major practical challenge that
we face is to discover, develop and bring into clinical
practice a new generation of drugs to regenerate effec-
tive therapy for each of these biological systems, and to
ensure that a continuous pipeline of drug discovery and
development is maintained. This will involve learning
how to access new areas of chemical space, including
natural products, to increase the probability of discov-
ering novel drugs. It is important that as far as possible
all new drugs and targets should combine a propensity
for a low frequency of resistance selection, high fitness
costs associated with resistance and low probability of
fitness compensation. One of the major academic chal-
lenges that we face is to deepen our understanding of the
resistome and the dynamics of HGT that constitute one
of the major causes of antibacterial drug resistance. A
second academic challenge is to better understand drug
structures and how their chemical properties relate to
combining efficacy with a low level of toxic liabilities
against the patient. The single most important lesson
from comparing different biological systems is that the
use of drugs in combination should become standard
practice to help to minimize resistance development in
all systems in which we use drug therapy.

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Acknowledgements
D.H. and D.I.A. acknowledge funding from the Innovative
Medicines Initiative Joint Undertaking under grant agree‑
ment number 115583, resources of which are composed of
financial contribution from the European Union’s Seventh
Framework Programme (FP7/2007‑2013) and EFPIA compa‑
nies’ in kind contribution. D.H. and D.I.A. also acknowledge
support from Vetenskapsrådet (Swedish Science Council), SSF
(Swedish Strategic Science Foundation), Vinnova (Swedish
Innovation Science), and the Knut and Alice Wallenberg
Foundation (RiboCore Project). D.I.A. acknowledges support
from the EU (EvoTar project).
Competing interests statement
The authors declare no competing interests.