Unpredictability of the Fitness Effects of Antimicrobial Resistance Mutations Across Environments in Escherichia coli.

The evolution of antimicrobial resistance (AMR) in bacteria is a major public health concern, and antibiotic restriction is often implemented to reduce the spread of resistance. These measures rely on the existence of deleterious fitness effects (i.e. costs) imposed by AMR mutations during growth in the absence of antibiotics. According to this assumption, resistant strains will be outcompeted by susceptible strains that do not pay the cost during the period of restriction. The fitness effects of AMR mutations are generally studied in laboratory reference strains grown in standard growth environments; however, the genetic and environmental context can influence the magnitude and direction of a mutation's fitness effects. In this study, we measure how three sources of variation impact the fitness effects of Escherichia coli AMR mutations: the type of resistance mutation, the genetic background of the host, and the growth environment. We demonstrate that while AMR mutations are generally costly in antibiotic-free environments, their fitness effects vary widely and depend on complex interactions between the mutation, genetic background, and environment. We test the ability of the Rough Mount Fuji fitness landscape model to reproduce the empirical data in simulation. We identify model parameters that reasonably capture the variation in fitness effects due to genetic variation. However, the model fails to accommodate the observed variation when considering multiple growth environments. Overall, this study reveals a wealth of variation in the fitness effects of resistance mutations owing to genetic background and environmental conditions, which will ultimately impact their persistence in natural populations.

On the De Novo Emergence of Ecological Interactions during Evolutionary Diversification: A Conceptual Framework and Experimental Test.

AbstractEcological interactions are crucial to the structure and function of biological communities, but we lack a causal understanding of the forces shaping their emergence during evolutionary diversification. Here we provide a conceptual framework linking different modes of diversification (e.g., ecological diversification), which depend on environmental characteristics, to the evolution of different forms of ecological interactions (e.g., resource partitioning) in asexual lineages. We tested the framework by examining the net interactions in communities of Pseudomonas aeruginosa produced via experimental evolution in nutritionally simple (SIM) or complex (COM) environments by contrasting the productivity and competitive fitness of whole evolved communities relative to their component isolates. As expected, we found that nutritional complexity drove the evolution of communities with net positive interactions whereas SIM communities had similar performance as their component isolates. A follow-up experiment revealed that high fitness in two COM communities was driven by rare variants (frequency <0.1%) that antagonized PA14, the ancestral strain and common competitor used in fitness assays. Our study suggests that the evolution of de novo ecological interactions in asexual lineages is predictable at a broad scale from environmental conditions. Further, our work demonstrates that rare variants can disproportionately impact the function of relatively simple microbial communities.

Evolutionary Genomics of Niche-Specific Adaptation to the Cystic Fibrosis Lung in Pseudomonas aeruginosa.

The comparative genomics of the transition of the opportunistic pathogen Pseudomonas aeruginosa from a free-living environmental strain to one that causes chronic infection in the airways of cystic fibrosis (CF) patients remain poorly studied. Chronic infections are thought to originate from colonization by a single strain sampled from a diverse, globally distributed population, followed by adaptive evolution to the novel, stressful conditions of the CF lung. However, we do not know whether certain clades are more likely to form chronic infections than others and we lack a comprehensive view of the suite of genes under positive selection in the CF lung. We analyzed whole-genome sequence data from 1,000 P. aeruginosa strains with diverse ecological provenances including the CF lung. CF isolates were distributed across the phylogeny, indicating little genetic predisposition for any one clade to cause chronic infection. Isolates from the CF niche experienced stronger positive selection on core genes than those derived from environmental or acute infection sources, consistent with recent adaptation to the lung environment. Genes with the greatest differential positive selection in the CF niche include those involved in core cellular processes such as metabolism, energy production, and stress response as well as those linked to patho-adaptive processes such as antibiotic resistance, cell wall and membrane modification, quorum sensing, biofilms, mucoidy, motility, and iron homeostasis. Many genes under CF-specific differential positive selection had regulatory functions, consistent with the idea that regulatory mutations play an important role in rapid adaptation to novel environments.

Rapid diversification of Pseudomonas aeruginosa in cystic fibrosis lung-like conditions.

Chronic infection of the cystic fibrosis (CF) airway by the opportunistic pathogen Pseudomonas aeruginosa is the leading cause of morbidity and mortality for adult CF patients. Prolonged infections are accompanied by adaptation of P. aeruginosa to the unique conditions of the CF lung environment, as well as marked diversification of the pathogen into phenotypically and genetically distinct strains that can coexist for years within a patient. Little is known, however, about the causes of this diversification and its impact on patient health. Here, we show experimentally that, consistent with ecological theory of diversification, the nutritional conditions of the CF airway can cause rapid and extensive diversification of P. aeruginosa Mucin, the substance responsible for the increased viscosity associated with the thick mucus layer in the CF airway, had little impact on within-population diversification but did promote divergence among populations. Furthermore, in vitro evolution recapitulated traits thought to be hallmarks of chronic infection, including reduced motility and increased biofilm formation, and the range of phenotypes observed in a collection of clinical isolates. Our results suggest that nutritional complexity and reduced dispersal can drive evolutionary diversification of P. aeruginosa independent of other features of the CF lung such as an active immune system or the presence of competing microbial species. We suggest that diversification, by generating extensive phenotypic and genetic variation on which selection can act, may be a key first step in the development of chronic infections.

The evolution and fate of diversity under hard and soft selection.

How genetic variation arises and persists over evolutionary time despite the depleting effects of natural selection remains a long-standing question. Here, we investigate the impacts of two extreme forms of population regulation-at the level of the total, mixed population (hard selection) and at the level of local, spatially distinct patches (soft selection)-on the emergence and fate of diversity under strong divergent selection. We find that while the form of population regulation has little effect on rates of diversification, it can modulate the long-term fate of genetic variation, diversity being more readily maintained under soft selection compared to hard selection. The mechanism responsible for coexistence is negative frequency-dependent selection which, while present initially under both forms of population regulation, persists over the long-term only under soft selection. Importantly, coexistence is robust to continued evolution of niche specialist types under soft selection but not hard selection. These results suggest that soft selection could be a general mechanism for the maintenance of ecological diversity over evolutionary time scales.

Anaerobically Grown Escherichia coli Has an Enhanced Mutation Rate and Distinct Mutational Spectra.

Oxidative stress is a major cause of mutation but little is known about how growth in the absence of oxygen impacts the rate and spectrum of mutations. We employed long-term mutation accumulation experiments to directly measure the rates and spectra of spontaneous mutation events in Escherichia coli populations propagated under aerobic and anaerobic conditions. To detect mutations, whole genome sequencing was coupled with methods of analysis sufficient to identify a broad range of mutational classes, including structural variants (SVs) generated by movement of repetitive elements. The anaerobically grown populations displayed a mutation rate nearly twice that of the aerobic populations, showed distinct asymmetric mutational strand biases, and greater insertion element activity. Consistent with mutation rate and spectra observations, genes for transposition and recombination repair associated with SVs were up-regulated during anaerobic growth. Together, these results define differences in mutational spectra affecting the evolution of facultative anaerobes.

What drives parallel evolution?: How population size and mutational variation contribute to repeated evolution.

Parallel evolution is the repeated evolution of the same phenotype or genotype in evolutionarily independent populations. Here, we use evolve-and-resequence experiments with bacteria and yeast to dissect the drivers of parallel evolution at the gene level. A meta-analysis shows that parallel evolution is often rare, but there is a positive relationship between population size and the probability of parallelism. We present a modeling approach to estimate the contributions of mutational and selective heterogeneity across a genome to parallel evolution. We show that, for two experiments, mutation contributes between ∼10 and 45%, respectively, of the variation associated with selection. Parallel evolution cannot, therefore, be interpreted as a phenomenon driven by selection alone; it must also incorporate information on heterogeneity in mutation rates along the genome. More broadly, the work discussed here helps lay the groundwork for a more sophisticated, empirically grounded theory of parallel evolution.

The properties of spontaneous mutations in the opportunistic pathogen Pseudomonas aeruginosa.

BACKGROUND: Natural genetic variation ultimately arises from the process of mutation. Knowledge of how the raw material for evolution is produced is necessary for a full understanding of several fundamental evolutionary concepts. We performed a mutation accumulation experiment with wild-type and mismatch-repair deficient, mutator lines of the pathogenic bacterium Pseudomonas aeruginosa, and used whole-genome sequencing to reveal the genome-wide rate, spectrum, distribution, leading/lagging bias, and context-dependency of spontaneous mutations. RESULTS: Wild-type base-pair mutation and indel rates were ~10(-10) and ~10(-11) per nucleotide per generation, respectively, and deficiencies in the mismatch-repair system caused rates to increase by over two orders of magnitude. A universal bias towards AT was observed in wild-type lines, but was reversed in mutator lines to a bias towards GC. Biases for which types of mutations occurred during replication of the leading versus lagging strand were detected reciprocally in both replichores. The distribution of mutations along the chromosome was non-random, with peaks near the terminus of replication and at positions intermediate to the replication origin and terminus. A similar distribution bias was observed along the chromosome in natural populations of P. aeruginosa. Site-specific mutation rates were higher when the focal nucleotide was immediately flanked by C:G pairings. CONCLUSIONS: Whole-genome sequencing of mutation accumulation lines allowed the comprehensive identification of mutations and revealed what factors of molecular and genomic architecture affect the mutational process. Our study provides a more complete view of how several mechanisms of mutation, mutation repair, and bias act simultaneously to produce the raw material for evolution.

The effect of selection environment on the probability of parallel evolution.

Across the great diversity of life, there are many compelling examples of parallel and convergent evolution-similar evolutionary changes arising in independently evolving populations. Parallel evolution is often taken to be strong evidence of adaptation occurring in populations that are highly constrained in their genetic variation. Theoretical models suggest a few potential factors driving the probability of parallel evolution, but experimental tests are needed. In this study, we quantify the degree of parallel evolution in 15 replicate populations of Pseudomonas fluorescens evolved in five different environments that varied in resource type and arrangement. We identified repeat changes across multiple levels of biological organization from phenotype, to gene, to nucleotide, and tested the impact of 1) selection environment, 2) the degree of adaptation, and 3) the degree of heterogeneity in the environment on the degree of parallel evolution at the gene-level. We saw, as expected, that parallel evolution occurred more often between populations evolved in the same environment; however, the extent of parallel evolution varied widely. The degree of adaptation did not significantly explain variation in the extent of parallelism in our system but number of available beneficial mutations correlated negatively with parallel evolution. In addition, degree of parallel evolution was significantly higher in populations evolved in a spatially structured, multiresource environment, suggesting that environmental heterogeneity may be an important factor constraining adaptation. Overall, our results stress the importance of environment in driving parallel evolutionary changes and point to a number of avenues for future work for understanding when evolution is predictable.

Temporal patterns of local adaptation in soil pseudomonads.

Strong divergent selection leading to local adaptation is often invoked to explain the staggering diversity of bacteria in microbial ecosystems. However, examples of specialization by bacterial clones to alternative niches in nature are rare. Here, we investigate the extent of local adaptation in natural isolates of pseudomonads and their relatives to their soil environments across both space and time. Though most isolates grew well in most environments, patchily distributed low-quality environments were found to drive specialization. In contrast to experimental evolution work on microbial adaptation, temporal adaptation was stronger than spatial adaptation among the isolates and environments we sampled. Time-shift analysis of fitness across two seasons of growth revealed an unexpectedly strong effect of preadaptation. This pattern of apparent future adaptation may be caused by unknown abiotic properties of these environments, phages, bacterial competitors or general mechanisms of ecological niche release, and warrants future study.

Evolutionary genomics of epidemic and nonepidemic strains of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen of humans and is a major cause of morbidity and mortality in patients with cystic fibrosis (CF). Prolonged infection of the respiratory tract can lead to adaptation of the pathogen to the CF lung environment. To examine the general patterns of adaptation associated with chronic infection, we obtained genome sequences from a collection of P. aeruginosa isolated from airways of patients with CF. Our analyses support a nonclonal epidemic population structure, with a background of unique, recombining genotypes, and the rare occurrence of successful epidemic clones. We present unique genome sequence evidence for the intercontinental spread of an epidemic strain shared between CF clinics in the United Kingdom and North America. Analyses of core and accessory genomes identified candidate genes and important functional pathways associated with adaptive evolution. Many genes of interest were involved in biological functions with obvious roles in this pathosystem, such as biofilm formation, antibiotic metabolism, pathogenesis, transport, reduction/oxidation, and secretion. Key factors driving the adaptive evolution of this pathogen within the host appear to be the presence of oxidative stressors and antibiotics. Regions of the accessory genome unique to the epidemic strain were enriched for genes in transporter families that efflux heavy metals and antibiotics. The epidemic strain was significantly more resistant than nonepidemic strains to three different antibiotics. Multiple lines of evidence suggest that selection imposed by the CF lung environment has a major influence on genomic evolution and the genetic characteristics of P. aeruginosa isolates causing contemporary infection.

Patterns of local adaptation in space and time among soil bacteria.

Our understanding of microbial biogeography has been governed by the dictum "Everything is everywhere, but the environment selects." In other words, the distribution of microbes is thought to occur in a regime of extensive dispersal and strong selection, generating local adaptation. However, direct tests of these assumptions are rare. Here, we investigate the extent of local adaptation in space and time of a collection of soil-derived microbial isolates, most belonging to the genus Pseudomonas, across a growing season from a deciduous forest in western Quebec, Canada, using a reciprocal transplant design. Average performance of all clones varied substantially in both space and time, in line with the expectation of strong selection in both dimensions. The behavior of genotype-by-environment variance in fitness and its components, responsiveness and inconsistency, in space and through time suggests that the strength of divergent selection increases as sites become more distant from each other in both dimensions. However, divergent selection was not strong enough to maintain different specialized types across the environments studied, which suggests that Pseudomonas and their close relatives are not locally adapted to the prevailing conditions of growth.

Evolution of Fitness Trade-Offs in Locally Adapted Populations of Pseudomonas fluorescens.

Local adaptation seems to be common in natural systems, but the genetic causes of its evolution remain poorly understood. Here we characterize the genetic causes of trade-offs generating local adaptation in populations of Pseudomonas fluorescens that had previously been evolved for specialization on three different carbon resources. We measured the fitness effects of mutations that arose during selection in that environment and in alternative environments to quantify the degree of specialization. We find that all mutations are beneficial in the environment of selection and that those arising later during adaptation are associated with increasingly antagonistic effects in alternative environments compared with those arising earlier, consistent with a multioptima version of Fisher's geometric model of adaptation. We also find that fitness of pairs of beneficial mutations are consistently less than additive in selection environments, producing a pattern of diminishing returns, but are more variable in alternative environments, being either positive or negative. Finally, we find that mutations in genes associated with loss of motility are beneficial across all environments, whereas mutations involving other functions, such as gene regulation, had more variable effects, being more environment specific. Taken together, these results provide a detailed account of the genetics of specialization and suggest that the evolution of trade-offs associated with local adaptation may often result from the antagonistic effects of beneficial mutations substituted later in adaptation.

Genomics of adaptation during experimental evolution of the opportunistic pathogen Pseudomonas aeruginosa.

Adaptation is likely to be an important determinant of the success of many pathogens, for example when colonizing a new host species, when challenged by antibiotic treatment, or in governing the establishment and progress of long-term chronic infection. Yet, the genomic basis of adaptation is poorly understood in general, and for pathogens in particular. We investigated the genetics of adaptation to cystic fibrosis-like culture conditions in the presence and absence of fluoroquinolone antibiotics using the opportunistic pathogen Pseudomonas aeruginosa. Whole-genome sequencing of experimentally evolved isolates revealed parallel evolution at a handful of known antibiotic resistance genes. While the level of antibiotic resistance was largely determined by these known resistance genes, the costs of resistance were instead attributable to a number of mutations that were specific to individual experimental isolates. Notably, stereotypical quinolone resistance mutations in DNA gyrase often co-occurred with other mutations that, together, conferred high levels of resistance but no consistent cost of resistance. This result may explain why these mutations are so prevalent in clinical quinolone-resistant isolates. In addition, genes involved in cyclic-di-GMP signalling were repeatedly mutated in populations evolved in viscous culture media, suggesting a shared mechanism of adaptation to this CF-like growth environment. Experimental evolutionary approaches to understanding pathogen adaptation should provide an important complement to studies of the evolution of clinical isolates.

Distribution of fitness effects among beneficial mutations before selection in experimental populations of bacteria.

The extent to which a population diverges from its ancestor through adaptive evolution depends on variation supplied by novel beneficial mutations. Extending earlier work, recent theory makes two predictions that seem to be robust to biological details: the distribution of fitness effects among beneficial mutations before selection should be (i) exponential and (ii) invariant, meaning it is always exponential regardless of the fitness rank of the wild-type allele. Here we test these predictions by assaying the fitness of 665 independently derived single-step mutations in the bacterium Pseudomonas fluorescens across a range of environments. We show that the distribution of fitness effects among beneficial mutations is indistinguishable from an exponential despite marked variation in the fitness rank of the wild type across environments. These results suggest that the initial step in adaptive evolution--the production of novel beneficial mutants from which selection sorts--is very general, being characterized by an approximately exponential distribution with many mutations of small effect and few of large effect. We also document substantial variation in the pleiotropic costs of antibiotic resistance, a result that may have implications for strategies aimed at eliminating resistant pathogens in animal and human populations.

The impact of genetic background during laboratory evolution of Pseudomonas aeruginosa in a cystic fibrosis-like environment.

Genetic background has the potential to influence both the tempo and trajectory of adaptive change: Different genotypes of a given species may adopt varied solutions to the same environmental challenge, or they may approach the same solution at different rates. Laboratory selection has been widely used to experimentally examine the evolutionary consequences of variation in genetic background, although largely using genotypes differing by only a few mutations. Here, we leverage natural variation in the bacterium Pseudomonas aeruginosa to investigate whether different adaptive solutions are accessible from distant points of departure on an adaptive landscape. We evolved 17 diverse genotypes in a laboratory medium that partially mimics the lung sputum of cystic fibrosis patients, and we measured changes in 10 phenotypes as well as in fitness. Using phylogenetically informed analyses, we found that genetic background impacted the tempo, but not the trajectory, of phenotypic evolution: Different starting genotypes converged toward similar phenotypes, but at varying rates. Our findings add to a growing body of evidence supporting widespread diminishing return epistasis during adaptation. The importance of genetic background toward the trajectory of adaptation remains inconsistent across experimental systems and conditions.

Detection of SARS-CoV-2 in schools using built environment testing in Ottawa, Canada: A multi-facility prospective surveillance study.

Classroom and staffroom floor swabs across six elementary schools in Ottawa, Canada were tested for SARS-CoV-2. Environmental test positivity did not correlate with student grade groups, school-level absenteeism, pediatric COVID-19-related hospitalizations, or community SARS-CoV-2 wastewater levels. Schools in neighbourhoods with historically elevated COVID-19 burden showed a negative but non-significant association with lower swab positivity.

A link between evolution and society fostering the UN sustainable development goals.

Given the multitude of challenges Earth is facing, sustainability science is of key importance to our continued existence. Evolution is the fundamental biological process underlying the origin of all biodiversity. This phylogenetic diversity fosters the resilience of ecosystems to environmental change, and provides numerous resources to society, and options for the future. Genetic diversity within species is also key to the ability of populations to evolve and adapt to environmental change. Yet, the value of evolutionary processes and the consequences of their impairment have not generally been considered in sustainability research. We argue that biological evolution is important for sustainability and that the concepts, theory, data, and methodological approaches used in evolutionary biology can, in crucial ways, contribute to achieving the UN Sustainable Development Goals (SDGs). We discuss how evolutionary principles are relevant to understanding, maintaining, and improving Nature Contributions to People (NCP) and how they contribute to the SDGs. We highlight specific applications of evolution, evolutionary theory, and evolutionary biology's diverse toolbox, grouped into four major routes through which evolution and evolutionary insights can impact sustainability. We argue that information on both within-species evolutionary potential and among-species phylogenetic diversity is necessary to predict population, community, and ecosystem responses to global change and to make informed decisions on sustainable production, health, and well-being. We provide examples of how evolutionary insights and the tools developed by evolutionary biology can not only inspire and enhance progress on the trajectory to sustainability, but also highlight some obstacles that hitherto seem to have impeded an efficient uptake of evolutionary insights in sustainability research and actions to sustain SDGs. We call for enhanced collaboration between sustainability science and evolutionary biology to understand how integrating these disciplines can help achieve the sustainable future envisioned by the UN SDGs.

Competition both drives and impedes diversification in a model adaptive radiation.

Competitors are known to be important in governing the outcome of evolutionary diversification during an adaptive radiation, but the precise mechanisms by which they exert their effects remain elusive. Using the model adaptive radiation of Pseudomonas fluorescens, we show experimentally that the effect of competition on diversification of a focal lineage depends on both the strength of competition and the ability of the competitors to diversify. We provide evidence that the extent of diversification in the absence of interspecific competitors depends on the strength of resource competition. We also show that the presence of competitors can actually increase diversity by increasing interspecific resource competition. Competitors that themselves are able to diversify prevent diversification of the focal lineage by removing otherwise available ecological opportunities. These results suggest that the progress of an adaptive radiation depends ultimately on the strength of resource competition, an effect that can be exaggerated or impeded by the presence of competitors.