Directed mutagenesis of Mycobacterium smegmatis 16S rRNA to reconstruct the in vivo evolution of aminoglycoside resistance in Mycobacterium tuberculosis

Drug resistance in Mycobacterium tuberculosis is a global problem, with major consequences for treatment and public health systems. As the emergence and spread of drug‐resistant tuberculosis epidemics is largely influenced by the impact of the resistance mechanism on bacterial fitness, we wished to investigate whether compensatory evolution occurs in drug‐resistant clinical isolates of M. tuberculosis. By combining information from molecular epidemiology studies of drug‐resistant clinical M. tuberculosis isolates with genetic reconstructions and measurements of aminoglycoside susceptibility and fitness in Mycobacterium smegmatis, we have reconstructed a plausible pathway for how aminoglycoside resistance develops in clinical isolates of M. tuberculosis. Thus, we show by reconstruction experiments that base changes in the highly conserved A‐site of 16S rRNA that: (i) cause aminoglycoside resistance, (ii) confer a high fitness cost and (iii) destabilize a stem‐loop structure, are associated with a particular compensatory point mutation that restores rRNA secondary structure and bacterial fitness, while maintaining to a large extent the drug‐resistant phenotype. The same types of resistance and associated mutations can be found in M. tuberculosis in clinical isolates, suggesting that compensatory evolution contributes to the spread of drug‐resistant tuberculosis disease.     

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.     

耐药结核分枝杆菌的适应性代价与补偿性进化

结核病耐药率的攀升是目前全球结核病防控面临的重大挑战。结核分枝杆菌主要通过其基因组中耐药相关基因发生点突变而获得耐药性。由于耐药相关基因通常具有重要的生理功能,其突变往往会导致结核分枝杆菌自身适应性下降,即产生“适应性代价”。然而,耐药结核分枝杆菌可通过进一步积累其他特定突变来回复其适应性,这种能使其适应性上升的突变称为“补偿性突变”。耐药结核分枝杆菌的补偿性进化被认为是耐药结核病广泛传播与流行的生物学基础。近年来,在结核病分子流行病学和基础研究领域,针对耐药结核分枝杆菌的补偿性进化开展了大量研究。本文从结核分枝杆菌的耐药分子机制、耐药突变的适应性代价与补偿性进化,以及补偿性进化如何影响耐药结核病传播等方面,综述耐药结核分枝杆菌补偿性进化的研究进展。     

The interplay between drug resistance and fitness in malaria parasites

Controlling the spread of antimalarial drug resistance, especially resistance of Plasmodium falciparum to artemisinin‐based combination therapies, is a high priority. Available data indicate that, as with other microorganisms, the spread of drug‐resistant malaria parasites is limited by fitness costs that frequently accompany resistance. Resistance‐mediating polymorphisms in malaria parasites have been identified in putative drug transporters and in target enzymes. The impacts of these polymorphisms on parasite fitness have been characterized in vitro and in animal models. Additional insights have come from analyses of samples from clinical studies, both evaluating parasites under different selective pressures and determining the clinical consequences of infection with different parasites. With some exceptions, resistance‐mediating polymorphisms lead to malaria parasites that, compared with wild type, grow less well in culture and in animals, and are replaced by wild type when drug pressure diminishes in the clinical setting. In some cases, the fitness costs of resistance may be offset by compensatory mutations that increase virulence or changes that enhance malaria transmission. However, not enough is known about effects of resistance mediators on parasite fitness. A better appreciation of the costs of fitness‐mediating mutations will facilitate the development of optimal guidelines for the treatment and prevention of malaria.     

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.     

Evolution of drug-resistant viral populations during interruption of antiretroviral therapy

Analysis of a large number of HIV-1 genomes at multiple time points after antiretroviral treatment (ART) interruption allows determination of the evolution of drug-resistant viruses and viral fitness in vivo in the absence of drug selection pressure. Using a parallel allele-specific sequencing (PASS) assay, potential primary drug-resistant mutations in five individual patients were studied by analyzing over 18,000 viral genomes. A three-phase evolution of drug-resistant viruses was observed after termination of ART. In the first phase, viruses carrying various combinations of multiple-drug-resistant (MDR) mutations predominated with each mutation persisting in relatively stable proportions while the overall number of resistant viruses gradually increased. In the second phase, viruses with linked MDR mutations rapidly became undetectable and single-drug-resistant (SDR) viruses emerged as minority populations while wild-type viruses quickly predominated. In the third phase, low-frequency SDR viruses remained detectable as long as 59 weeks after treatment interruption. Mathematical modeling showed that the loss in relative fitness increased with the number of mutations in each viral genome and that viruses with MDR mutations had lower fitness than viruses with SDR mutations. No single viral genome had seven or more drug resistance mutations, suggesting that such severely mutated viruses were too unfit to be detected or that the resistance gain offered by the seventh mutation did not outweigh its contribution to the overall fitness loss of the virus. These data provide a more comprehensive understanding of evolution and fitness of drug-resistant viruses in vivo and may lead to improved treatment strategies for ART-experienced patients.     

Mutational Spectrum Drives the Rise of Mutator Bacteria

Understanding how mutator strains emerge in bacterial populations is relevant both to evolutionary theory and to reduce the threat they pose in clinical settings. The rise of mutator alleles is understood as a result of their hitchhiking with linked beneficial mutations, although the factors that govern this process remain unclear. A prominent but underappreciated fact is that each mutator allele increases only a specific spectrum of mutational changes. This spectrum has been speculated to alter the distribution of fitness effects of beneficial mutations, potentially affecting hitchhiking. To study this possibility, we analyzed the fitness distribution of beneficial mutations generated from different mutator and wild-type Escherichia coli strains. Using antibiotic resistance as a model system, we show that mutational spectra can alter these distributions substantially, ultimately determining the competitive ability of each strain across environments. Computer simulation showed that the effect of mutational spectrum on hitchhiking dynamics follows a non-linear function, implying that even slight spectrum-dependent fitness differences are sufficient to alter mutator success frequency by several orders of magnitude. These results indicate an unanticipated central role for the mutational spectrum in the evolution of bacterial mutation rates. At a practical level, this study indicates that knowledge of the molecular details of resistance determinants is crucial for minimizing mutator evolution during antibiotic therapy.     

Evolutionary dynamics of Candida albicans during in vitro evolution

While mechanisms of resistance to major antifungal agents have been characterized in Candida albicans, little is known about the evolutionary trajectories during the emergence of drug resistance. Here, we examined the evolutionary dynamics of C. albicans that evolved in vitro in the presence or absence of fluconazole using the visualizing evolution in real-time (VERT) method, a novel experimental approach that facilitates the systematic isolation of adaptive mutants that arise in the population. We found an increase in the frequency of adaptive events in the presence of fluconazole compared to the no-drug controls. Analysis of the evolutionary dynamics revealed that mutations that led to increased drug resistance appeared frequently and that mutants with increased levels of resistance arose in independent lineages. Interestingly, most adaptive mutants with increased fitness in the presence of the drug did not exhibit a significant fitness decrease in the absence of the drug, supporting the idea that rapid resistance can arise from mutations in strains maintained in the population prior to exposure to the drug.     

Research that influences policy and practice – characteristics of operational research to improve malaria control in Mpumalanga Province, South Africa

Background

Results from numerous studies point convincingly to correlations between mutations at selected genes and phenotypic resistance to antimalarials in Plasmodium falciparum isolates. In order to move molecular assays for point mutations on resistance-related genes into the realm of applied tools for surveillance, we investigated a selection of P. falciparum isolates that were imported during the year 2001 into Europe to study the prevalence of resistance-associated point mutations at relevant codons. In particular, we tested for parasites which were developing resistance to antifolates and chloroquine. The screening results were used to map the prevalence of mutations and, thus, levels of potential drug resistance in endemic areas world-wide.

Results

337 isolates have been tested so far. Prevalence of mutations that are associated with resistance to chloroquine on the pfcrt and pfmdr genes of P. falciparum was demonstrated at high levels. However, the prevalence of mutations associated with resistance to antifolates at the DHFR and DHPS genes was unexpectedly low, rarely exceeding 60% in endemic areas.

Conclusions

Constant screening of imported isolates will enable TropNetEurop to establish a screening tool for emerging resistance in endemic areas.     

The role of compensatory mutations in the emergence of drug resistance

Pathogens that evolve resistance to drugs usually have reduced fitness. However, mutations that largely compensate for this reduction in fitness often arise. We investigate how these compensatory mutations affect population-wide resistance emergence as a function of drug treatment. Using a model of gonorrhea transmission dynamics, we obtain generally applicable, qualitative results that show how compensatory mutations lead to more likely and faster resistance emergence. We further show that resistance emergence depends on the level of drug use in a strongly nonlinear fashion. We also discuss what data need to be obtained to allow future quantitative predictions of resistance emergence.     

Long-term adaptation of epistatic genetic networks

Gene networks are likely to govern most traits in nature. Mutations at these genes often show functional epistatic interactions that lead to complex genetic architectures and variable fitness effects in different genetic backgrounds. Understanding how epistatic genetic systems evolve in nature remains one of the great challenges in evolutionary biology. Here we combine an analytical framework with individual-based simulations to generate novel predictions about long-term adaptation of epistatic networks. We find that relative to traits governed by independently evolving genes, adaptation with epistatic gene networks is often characterized by longer waiting times to selective sweeps, lower standing genetic variation, and larger fitness effects of adaptive mutations. This may cause epistatic networks to either adapt more slowly or more quickly relative to a nonepistatic system. Interestingly, epistatic networks may adapt faster even when epistatic effects of mutations are on average deleterious. Further, we study the evolution of epistatic properties of adaptive mutations in gene networks. Our results show that adaptive mutations with small fitness effects typically evolve positive synergistic interactions, whereas adaptive mutations with large fitness effects evolve positive synergistic and negative antagonistic interactions at approximately equal frequencies. These results provide testable predictions for adaptation of traits governed by epistatic networks and the evolution of epistasis within networks.     

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.     

Escaping an evolutionary lobster trap: drug resistance and compensatory mutation in a fluctuating environment

The fitness landscape concept aids intuition on adaptive evolution through low fitness genotypes. Evolutionary processes become complex when environments and therefore fitnesses fluctuate. Antibiotic resistance evolution in bacteria is an important example of such dynamics. Resistance bears a cost in the drug-free environment, but compensatory mutation can lower this cost, creating a fitness valley. With the drug present, the valley becomes a hill that is easily climbed. Once a population is dominated by resistant-compensated genotypes, reversion to sensitivity is difficult: this phenomenon has been described as an evolutionary lobster trap. With increasing frequencies of drug resistance among pathogenic bacteria, it is critical to understand how this trap can be escaped. Here, we develop stochastic models to investigate these dynamics. The residual fitness cost (the cost remaining after compensatory mutation has occurred) is a key parameter. Reversion to sensitivity is favored when the time spent in the absence of the drug relative to its presence is high compared to the residual fitness cost. Population sizes are also important: in large populations, resistant-compensated mutants appear in resistant-uncompensated or sensitive-compensated genotypes without fixation of these intermediates. This stochastic tunneling effect occurs when sufficient time is allowed by the rates of environmental fluctuation.     

Fitness-compensatory mutations in rifampicin-resistant RNA polymerase

Mutations in rpoB (RNA polymerase β-subunit) can cause high-level resistance to rifampicin, an important first-line drug against tuberculosis. Most rifampicin-resistant (Rif(R)) mutants selected in vitro have reduced fitness, and resistant clinical isolates of M. tuberculosis frequently carry multiple mutations in RNA polymerase genes. This supports a role for compensatory evolution in global epidemics of drug-resistant tuberculosis but the significance of secondary mutations outside rpoB has not been demonstrated or quantified. Using Salmonella as a model organism, and a previously characterized Rif(R) mutation (rpoB R529C) as a starting point, independent lineages were evolved with selection for improved growth in the presence and absence of rifampicin. Compensatory mutations were identified in every lineage and were distributed between rpoA, rpoB and rpoC. Resistance was maintained in all strains showing that increased fitness by compensatory mutation was more likely than reversion. Genetic reconstructions demonstrated that the secondary mutations were responsible for increasing growth rate. Many of the compensatory mutations in rpoA and rpoC individually caused small but significant reductions in susceptibility to rifampicin, and some compensatory mutations in rpoB individually caused high-level resistance. These findings show that mutations in different components of RNA polymerase are responsible for fitness compensation of a Rif(R) mutant.     

Recombination in HIV and the evolution of drug resistance: for better or for worse?

The rapid evolution of drug resistance remains a major obstacle for HIV therapy. The capacity of the virus for recombination is widely believed to facilitate the evolution of drug resistance. Here, we challenge this intuitive view. We develop a population genetic model of HIV replication that incorporates the processes of mutation, cellular superinfection, and recombination. We show that cellular superinfection increases the abundance of low fitness viruses at the expense of the fittest strains due to the mixing of viral proteins during virion assembly. Moreover, we argue that whether recombination facilitates the evolution of drug resistance depends critically on how resistance mutations interact to determine viral fitness. Contrary to the commonly held belief, we find that, under the most plausible biological assumptions, recombination is expected to slow down the rate of evolution of multi-drug-resistant virus during therapy.     

Biological cost and compensatory evolution in fusidic acid-resistant Staphylococcus aureus

Fusidic acid resistance resulting from mutations in elongation factor G (EF-G) of Staphylococcus aureus is associated with fitness costs during growth in vivo and in vitro. In both environments, these costs can be partly or fully compensated by the acquisition of secondary intragenic mutations. Among clinical isolates of S. aureus, fusidic acid-resistant strains have been identified that carry multiple mutations in EF-G at positions similar to those shown experimentally to cause resistance and fitness compensation. This observation suggests that fitness-compensatory mutations may be an important aspect of the evolution of antibiotic resistance in the clinical environment, and may contribute to a stabilization of the resistant bacteria present in a bacterial population.