The cost of multiple drug resistance in Pseudomonas aeruginosa

The spread of bacterial antibiotic resistance mutations is thought to be constrained by their pleiotropic fitness costs. Here we investigate the fitness costs of resistance in the context of the evolution of multiple drug resistance (MDR), by measuring the cost of acquiring streptomycin resistance mutations (StrepR) in independent strains of the bacterium Pseudomonas aeruginosa carrying different rifampicin resistance (RifR) mutations. In the absence of antibiotics, StrepR mutations are associated with similar fitness costs in different RifR genetic backgrounds. The cost of StrepR mutations is greater in a rifampicin‐sensitive (RifS) background, directly demonstrating antagonistic epistasis between resistance mutations. In the presence of rifampicin, StrepR mutations have contrasting effects in different RifR backgrounds: StrepR mutations have no detectable costs in some RifR backgrounds and massive fitness costs in others. Our results clearly demonstrate the importance of epistasis and genotype‐by‐environment interactions for the evolution of MDR.     

Epistasis buffers the fitness effects of rifampicin- resistance mutations in Pseudomonas aeruginosa

Epistatic interactions between resistance mutations in antibiotic-free environments potentially play a crucial role in the spread of resistance in pathogen populations by determining the fitness cost associated with resistance. We used an experimental evolution approach to test for epistatic interactions between 14 different pairs of rifampicin mutations in the pathogenic bacterium Pseudomonas aeruginosa in 42 different rifampicin-free environments. First, we show that epistasis between rifampicin-resistance mutations tends to be antagonistic: the fitness effect of having two mutations is generally smaller than that predicted from the effects of individual mutations on the wild-type. Second, we show that sign epistasis between resistance mutations is both common and strong; most notably, pairs of deleterious resistance mutations often partially or completely compensate for each others' costs, revealing a novel mechanism for compensatory adaptation. These results suggest that antagonistic epistasis between intragenic resistance mutations may be a key determinant of the cost of antibiotic resistance and compensatory adaptation in pathogen populations.     

Initial mutations direct alternative pathways of protein evolution

Whether evolution is erratic due to random historical details, or is repeatedly directed along similar paths by certain constraints, remains unclear. Epistasis (i.e. non-additive interaction between mutations that affect fitness) is a mechanism that can contribute to both scenarios. Epistasis can constrain the type and order of selected mutations, but it can also make adaptive trajectories contingent upon the first random substitution. This effect is particularly strong under sign epistasis, when the sign of the fitness effects of a mutation depends on its genetic background. In the current study, we examine how epistatic interactions between mutations determine alternative evolutionary pathways, using in vitro evolution of the antibiotic resistance enzyme TEM-1 β-lactamase. First, we describe the diversity of adaptive pathways among replicate lines during evolution for resistance to a novel antibiotic (cefotaxime). Consistent with the prediction of epistatic constraints, most lines increased resistance by acquiring three mutations in a fixed order. However, a few lines deviated from this pattern. Next, to test whether negative interactions between alternative initial substitutions drive this divergence, alleles containing initial substitutions from the deviating lines were evolved under identical conditions. Indeed, these alternative initial substitutions consistently led to lower adaptive peaks, involving more and other substitutions than those observed in the common pathway. We found that a combination of decreased enzymatic activity and lower folding cooperativity underlies negative sign epistasis in the clash between key mutations in the common and deviating lines (Gly238Ser and Arg164Ser, respectively). Our results demonstrate that epistasis contributes to contingency in protein evolution by amplifying the selective consequences of random mutations.     

Pervasive sign epistasis between conjugative plasmids and drug-resistance chromosomal mutations

Multidrug-resistant bacteria arise mostly by the accumulation of plasmids and chromosomal mutations. Typically, these resistant determinants are costly to the bacterial cell. Yet, recently, it has been found that, in Escherichia coli bacterial cells, a mutation conferring resistance to an antibiotic can be advantageous to the bacterial cell if another antibiotic-resistance mutation is already present, a phenomenon called sign epistasis. Here we study the interaction between antibiotic-resistance chromosomal mutations and conjugative (i.e., self-transmissible) plasmids and find many cases of sign epistasis (40%)--including one of reciprocal sign epistasis where the strain carrying both resistance determinants is fitter than the two strains carrying only one of the determinants. This implies that the acquisition of an additional resistance plasmid or of a resistance mutation often increases the fitness of a bacterial strain already resistant to antibiotics. We further show that there is an overall antagonistic interaction between mutations and plasmids (52%). These results further complicate expectations of resistance reversal by interdiction of antibiotic use.     

Rapid Decline of Ceftazidime Resistance in Antibiotic-Free and Sublethal Environments Is Contingent on Genetic Background

Trade-offs of antibiotic resistance evolution, such as fitness cost and collateral sensitivity (CS), could be exploited to drive evolution toward antibiotic susceptibility. Decline of resistance may occur when resistance to other drug leads to CS to the first one and when compensatory mutations, or genetic reversion of the original ones, reduce fitness cost. Here we describe the impact of antibiotic-free and sublethal environments on declining ceftazidime resistance in different Pseudomonas aeruginosa resistant mutants. We determined that decline of ceftazidime resistance occurs within 450 generations, which is caused by newly acquired mutations and not by reversion of the original ones, and that the original CS of these mutants is preserved. In addition, we observed that the frequency and degree of this decline is contingent on genetic background. Our results are relevant to implement evolution-based therapeutic approaches, as well as to redefine global policies of antibiotic use, such as drug cycling.     

Bacterial evolution of antibiotic hypersensitivity

The evolution of resistance to a single antibiotic is frequently accompanied by increased resistance to multiple other antimicrobial agents. In sharp contrast, very little is known about the frequency and mechanisms underlying collateral sensitivity. In this case, genetic adaptation under antibiotic stress yields enhanced sensitivity to other antibiotics. Using large‐scale laboratory evolutionary experiments with Escherichia coli, we demonstrate that collateral sensitivity occurs frequently during the evolution of antibiotic resistance. Specifically, populations adapted to aminoglycosides have an especially low fitness in the presence of several other antibiotics. Whole‐genome sequencing of laboratory‐evolved strains revealed multiple mechanisms underlying aminoglycoside resistance, including a reduction in the proton‐motive force (PMF) across the inner membrane. We propose that as a side effect, these mutations diminish the activity of PMF‐dependent major efflux pumps (including the AcrAB transporter), leading to hypersensitivity to several other antibiotics. More generally, our work offers an insight into the mechanisms that drive the evolution of negative trade‐offs under antibiotic selection.     

Adaptation to the cost of resistance: a model of compensation, recombination, and selection in a haploid organism

Populations of pathogenic organisms often evolve resistance in response to the use of pesticides or antibiotics. This rise of resistance may be followed by a fall when chemical control is suspended and resistance alleles carry a fitness cost. Another possibility is that mutations at secondary loci compensate for the cost, usually without loss of resistance. This enables resistant types to withstand invasion by the susceptible wild-type; resistance then persists in the population, which reduces the efficacy of future pesticide or antibiotic use. We examined a two-locus model of a haploid organism that adapts to the cost of resistance by a single compensatory mutation. We addressed the question how different combinations of cost and compensation and different levels of recombination affect the consequences of a single pesticide application. Resistance will become fixed in the population when the fraction of the population exposed to pesticide exceeds the cost of resistance. Compensatory mutations reduce the cost of resistance and therefore this threshold level of pesticide use. In the absence of pesticide, recombination promotes stability of equilibria. In the presence of pesticide, recombination accelerates the fixation of resistance and compensating alleles; recombination may also enable the persistence of compensated resistant types after pesticide use.     

Perspective: Sign epistasis and genetic constraint on evolutionary trajectories

Epistasis for fitness means that the selective effect of a mutation is conditional on the genetic background in which it appears. Although epistasis is widely observed in nature, our understanding of its consequences for evolution by natural selection remains incomplete. In particular, much attention focuses only on its influence on the instantaneous rate of changes in frequency of selected alleles via epistatic contribution to the additive genetic variance for fitness. Thus, in this framework epistasis only has evolutionary importance if the interacting loci are simultaneously segregating in the population. However, the selective accessibility of mutational trajectories to high fitness genotypes may depend on the genetic background in which novel mutations appear, and this effect is independent of population polymorphism at other loci. Here we explore this second influence of epistasis on evolution by natural selection. We show that it is the consequence of a particular form of epistasis, which we designate sign epistasis. Sign epistasis means that the sign of the fitness effect of a mutation is under epistatic control; thus, such a mutation is beneficial on some genetic backgrounds and deleterious on others. Recent experimental innovations in microbial systems now permit assessment of the fitness effects of individual mutations on multiple genetic backgrounds. We review this literature and identify many examples of sign epistasis, and we suggest that the implications of these results may generalize to other organisms. These theoretical and empirical considerations imply that strong genetic constraint on the selective accessibility of trajectories to high fitness genotypes may exist and suggest specific areas of investigation for future research.     

Fitness landscapes emerging from pharmacodynamic functions in the evolution of multidrug resistance

Adaptive evolution often involves beneficial mutations at more than one locus. In this case, the trajectory and rate of adaptation is determined by the underlying fitness landscape, that is, the fitness values and mutational connectivity of all genotypes under consideration. Drug resistance, especially resistance to multiple drugs simultaneously, is also often conferred by mutations at several loci so that the concept of fitness landscapes becomes important. However, fitness landscapes underlying drug resistance are not static but dependent on drug concentrations, which means they are influenced by the pharmacodynamics of the drugs administered. Here, I present a mathematical framework for fitness landscapes of multidrug resistance based on Hill functions describing how drug concentrations affect fitness. I demonstrate that these ‘pharmacodynamic fitness landscapes’ are characterized by pervasive epistasis that arises through (i) fitness costs of resistance (even when these costs are additive), (ii) nonspecificity of resistance mutations to drugs, in particular cross‐resistance, and (iii) drug interactions (both synergistic and antagonistic). In the latter case, reciprocal drug suppression may even lead to reciprocal sign epistasis, so that the doubly resistant genotype occupies a local fitness peak that may be difficult to access by evolution. Simulations exploring the evolutionary dynamics on some pharmacodynamic fitness landscapes with both constant and changing drug concentrations confirm the crucial role of epistasis in determining the rate of multidrug resistance evolution.     

EVOLUTIONARY REVERSALS OF ANTIBIOTIC RESISTANCE IN EXPERIMENTAL POPULATIONS OF PSEUDOMONAS AERUGINOSA

Antibiotic resistance mutations are accompanied by a fitness cost, and two mechanisms allow bacteria to adapt to this cost once antibiotic use is halted. First, it is possible for resistance to revert; second, it is possible for bacteria to adapt to the cost of resistance by compensatory mutations. Unfortunately, reversion to antibiotic sensitivity is rare, but the underlying factors that prevent reversion remain obscure. Here, we directly study the evolutionary dynamics of reversion by experimentally mimicking reversion mutations—sensitives—in populations of rifampicin‐resistant Pseudomonas aeruginosa. We show that, in our populations, most sensitives are lost due to genetic drift when they are rare. However, clonal interference from lineages carrying compensatory mutations causes a dramatic increase in the time to fixation of sensitives that escape genetic drift, and mutations surpassing the sensitives’ fitness are capable of driving transiently common sensitive lineages to extinction. Crucially, we show that the constraints on reversion arising from clonal interference are determined by the potential for compensatory adaptation of the resistant population. Although the cost of resistance provides the incentive for reversion, our study demonstrates that both the cost of resistance and the intrinsic evolvability of resistant populations interact to determine the rate and likelihood of reversion.     

Persistence of antibiotic resistant bacteria

Bacterial adaptation to antibiotics has been very successful and over the past decade the increase in antibiotic resistance has generated considerable medical problems. Even though many drug resistances confer a fitness cost, suggesting that they might disappear by reducing the volume of antibiotic use, increasing evidence obtained from laboratory and epidemiological studies indicate that several processes will act to cause long-term persistence of resistant bacteria. Compensatory evolution that ameliorates the costs of resistance, the occurrence of cost-free resistances and genetic linkage between non-selected and selected resistances will confer a stabilization of the resistant bacteria. Thus, it is of importance that we forcefully implement strategies to reduce the rate of appearance and spread of resistant bacteria to allow new drug discovery to catch up with bacterial resistance development.     

Impact of mutation rate on the adaptation of gut bacteria

To study the role of mutator bacteria in the evolution of bacterial populations, we followed the impact of the mutation rate of Escherichia coli strains in the colonisation of the gut of axenic mice and the evolution of the mutation rate of bacterial populations living in the gut. We show that mutator bacteria have an advantage during the colonization. This adaptive advantage comes from their ability to generate adaptive mutations faster than wild type strains, mutations that allow their maintenance in the ecosystem. However, while mutator bacteria are becoming specialised to the environment they are living in, they accumulate mutations that may be deleterious or lethal in secondary environments. By following the evolution of the mutation rate of bacterial populations living in the gut of mice receiving antibiotics, we show that this therapy selects not only for antibiotic resistant mutants but also for mutator alleles that enhance mutation rates and are responsible for the appearance of the resistance. The costs of a high mutation rate, due to the accumulation of mutations, is seen in environments where changes are recurrent. In an ever-changing situation where every change is new, mutator bacteria might help the evolution of bacterial populations.     

Source-sink dynamics shape the evolution of antibiotic resistance and its pleiotropic fitness cost

Understanding the conditions that favour the evolution and maintenance of antibiotic resistance is the central goal of epidemiology. A crucial feature explaining the adaptation to harsh, or 'sink', environments is the supply of beneficial mutations via migration from a 'source' population. Given that antibiotic resistance is frequently associated with antagonistic pleiotropic fitness costs, increased migration rate is predicted not only to increase the rate of resistance evolution but also to increase the probability of fixation of resistance mutations with minimal fitness costs. Here we report in vitro experiments using the nosocomial pathogenic bacterium Pseudomonas aeruginosa that support these predictions: increasing rate of migration into environments containing antibiotics increased the rate of resistance evolution and decreased the associated costs of resistance. Consistent with previous theoretical work, we found that resistance evolution arose more rapidly in the presence of a single antibiotic than two. Evolution of resistance was also more rapid when bacteria were subjected to sequential exposure with two antibiotics (cycling therapy) compared with simultaneous exposure (bi-therapy). Furthermore, pleiotropic fitness costs of resistance to two antibiotics were higher than for one antibiotic, and were also higher under bi-therapy than cycling therapy, although the cost of resistance depended on the order of the antibiotics through time. These results may be relevant to the clinical setting where immigration is known to be important between chemotherapeutically treated patients, and demonstrate the importance of ecological and evolutionary dynamics in the control of antibiotic resistance.     

Evolutionary rescue by compensatory mutations is constrained by genomic and environmental backgrounds

Since deleterious mutations may be rescued by secondary mutations during evolution, compensatory evolution could identify genetic solutions leading to therapeutic targets. Here, we tested this hypothesis and examined whether these solutions would be universal or would need to be adapted to one's genetic and environmental makeups. We performed experimental evolutionary rescue in a yeast disease model for the Wiskott–Aldrich syndrome in two genetic backgrounds and carbon sources. We found that multiple aspects of the evolutionary rescue outcome depend on the genotype, the environment, or a combination thereof. Specifically, the compensatory mutation rate and type, the molecular rescue mechanism, the genetic target, and the associated fitness cost varied across contexts. The course of compensatory evolution is therefore highly contingent on the initial conditions in which the deleterious mutation occurs. In addition, these results reveal biologically favored therapeutic targets for the Wiskott–Aldrich syndrome, including the target of an unrelated clinically approved drug. Our results experimentally illustrate the importance of epistasis and environmental evolutionary constraints that shape the adaptive landscape and evolutionary rate of molecular networks.     

Fitness of antimicrobial-resistant Campylobacter and Salmonella

Campylobacter and Salmonella are the most commonly reported bacterial causes of human foodborne infections, and increasing proportions of these pathogens become resistant to medically important antimicrobial agents, imposing a burden on public health. Acquisition of resistance to antibiotics affects the adaptation and evolution of Salmonella and Campylobacter in various environments. Many resistance-conferring mutations entail a biological fitness cost, while others (e.g. fluoroquinolone resistance in Campylobacter) have no cost or even enhanced fitness. In Salmonella, the fitness disadvantage due to antimicrobial resistance can be restored by acquired compensatory mutations, which occur both in vitro and in vivo. The compensated or even enhanced fitness associated with antibiotic resistance may facilitate the spread and persistence of antimicrobial-resistant Salmonella and Campylobacter in the absence of selection pressure, creating a significant barrier for controlling antibiotic-resistant foodborne pathogens.     

Evolution can favor antagonistic epistasis

The accumulation of deleterious mutations plays a major role in evolution, and key to this are the interactions between their fitness effects, known as epistasis. Whether mutations tend to interact synergistically (with multiple mutations being more deleterious than would be expected from their individual fitness effects) or antagonistically is important for a variety of evolutionary questions, particularly the evolution of sex. Unfortunately, the experimental evidence on the prevalence and strength of epistasis is mixed and inconclusive. Here we study theoretically whether synergistic or antagonistic epistasis is likely to be favored by evolution and by how much. We find that in the presence of recombination, evolution favors less synergistic or more antagonistic epistasis whenever mutations that change the epistasis in this direction are possible. This is because evolution favors increased buffering against the effects of deleterious mutations. This suggests that we should not expect synergistic epistasis to be widespread in nature and hence that the mutational deterministic hypothesis for the advantage of sex may not apply widely.     

Genotype‐by‐environment interactions due to antibiotic resistance and adaptation in Escherichia coli

Mutations that are beneficial in one environment can have different fitness effects in other environments. In the context of antibiotic resistance, the resulting genotype‐by‐environment interactions potentially make selection on resistance unpredictable in heterogeneous environments. Furthermore, resistant bacteria frequently fix additional mutations during evolution in the absence of antibiotics. How do these two types of mutations interact to determine the bacterial phenotype across different environments? To address this, I used Escherichia coli as a model system, measuring the effects of nine different rifampicin resistance mutations on bacterial growth in 31 antibiotic‐free environments. I did this both before and after approximately 200 generations of experimental evolution in antibiotic‐free conditions (LB medium), and did the same for the antibiotic‐sensitive wild type after adaptation to the same environment. The following results were observed: (i) bacteria with and without costly resistance mutations adapted to experimental conditions and reached similar levels of competitive fitness; (ii) rifampicin resistance mutations and adaptation to LB both indirectly altered growth in other environments; and (iii) resistant‐evolved genotypes were more phenotypically different from the ancestor and from each other than resistant‐nonevolved and sensitive‐evolved genotypes. This suggests genotype‐by‐environment interactions generated by antibiotic resistance mutations, observed previously in short‐term experiments, are more pronounced after adaptation to other types of environmental variation, making it difficult to predict long‐term selection on resistance mutations from fitness effects in a single environment.     

EFFECTS OF EPISTASIS ON INFECTIVITY RANGE DURING HOST‐PARASITE COEVOLUTION

Understanding how parasites adapt to changes in host resistance is crucial to evolutionary epidemiology. Experimental studies have demonstrated that parasites are more capable of adapting to gradual, rather than sudden changes in host phenotype, as the latter may require multiple mutations that are unlikely to arise simultaneously. A key, but as yet unexplored factor is precisely how interactions between mutations (epistasis) affect parasite evolution. Here, we investigate this phenomenon in the context of infectivity range, where parasites may experience selection to infect broader sets of genotypes. When epistasis is strongly positive, we find that parasites are unlikely to evolve broader infectivity ranges if hosts exhibit sudden, rather than gradual changes in phenotype, in close agreement with empirical observations. This is due to a low probability of fixing multiple mutations that individually confer no immediate advantage. When epistasis is weaker, parasites are more likely to evolve broader infectivity ranges if hosts make sudden changes in phenotype, which can be explained by a balance between mutation supply and selection. Thus, we demonstrate that both the rate of phenotypic change in hosts and the form of epistasis between mutations in parasites are crucial in shaping the evolution of infectivity range.