Horizontal gene transfer facilitates the molecular reverse-evolution of antibiotic sensitivity in experimental populations of H. pylori.

Reversing the evolution of traits harmful to humans, such as antimicrobial resistance, is a key ambition of applied evolutionary biology. A major impediment to reverse evolution is the relatively low spontaneous mutation rates that revert evolved genotypes back to their ancestral state. However, the repeated re-introduction of ancestral alleles by horizontal gene transfer (HGT) could make reverse evolution likely. Here we evolve populations of an antibiotic-resistant strain of Helicobacter pylori in growth conditions without antibiotics while introducing an ancestral antibiotic-sensitive allele by HGT. We evaluate reverse evolution using DNA sequencing and find that HGT facilitates the molecular reverse evolution of the antibiotic resistance allele, and that selection for high rates of HGT drives the evolution of increased HGT rates in low-HGT treatment populations. Finally, we use a theoretical model and carry out simulations to infer how the fitness costs of antibiotic resistance, rates of HGT and effects of genetic drift interact to determine the probability and predictability of reverse evolution.

Leveraging the MMV Pathogen Box to Engineer an Antifungal Compound with Improved Efficacy and Selectivity against Candida auris.

Fungal infections pose a significant and increasing threat to human health, but the current arsenal of antifungal drugs is inadequate. We screened the Medicines for Malaria Venture (MMV) Pathogen Box for new antifungal agents against three of the most critical Candida species (Candida albicans, Candida auris, and Candida glabrata). Of the 14 identified hit compounds, most were active against C. albicans and C. auris. We selected the pyrazolo-pyrimidine MMV022478 for chemical modifications to build structure-activity relationships and study their antifungal properties. Two analogues, 7a and 8g, with distinct fluorine substitutions, greatly improved the efficacy against C. auris and inhibited fungal replication inside immune cells. Additionally, analogue 7a had improved selectivity toward fungal killing compared to mammalian cytotoxicity. Evolution experiments generating MMV022478-resistant isolates revealed a change in morphology from oblong to round cells. Most notably, the resistant isolates blocked the uptake of the fluorescent dye rhodamine 6G and showed reduced susceptibility toward fluconazole, indicative of structural changes in the yeast cell surface. In summary, our study identified a promising antifungal compound with activity against high-priority fungal pathogens. Additionally, we demonstrated how structure-activity relationship studies of known and publicly available compounds can expand the repertoire of molecules with antifungal efficacy and reduced cytotoxicity to drive the development of novel therapeutics.

The evolutionary mechanism of non-carbapenemase carbapenem-resistant phenotypes in Klebsiella spp.

Antibiotic resistance is driven by selection, but the degree to which a bacterial strain's evolutionary history shapes the mechanism and strength of resistance remains an open question. Here, we reconstruct the genetic and evolutionary mechanisms of carbapenem resistance in a clinical isolate of Klebsiella quasipneumoniae. A combination of short- and long-read sequencing, machine learning, and genetic and enzymatic analyses established that this carbapenem-resistant strain carries no carbapenemase-encoding genes. Genetic reconstruction of the resistance phenotype confirmed that two distinct genetic loci are necessary in order for the strain to acquire carbapenem resistance. Experimental evolution of the carbapenem-resistant strains in growth conditions without the antibiotic revealed that both loci confer a significant cost and are readily lost by de novo mutations resulting in the rapid evolution of a carbapenem-sensitive phenotype. To explain how carbapenem resistance evolves via multiple, low-fitness single-locus intermediates, we hypothesised that one of these loci had previously conferred adaptation to another antibiotic. Fitness assays in a range of drug concentrations show how selection in the antibiotic ceftazidime can select for one gene (blaDHA-1) potentiating the evolution of carbapenem resistance by a single mutation in a second gene (ompK36). These results show how a patient's treatment history might shape the evolution of antibiotic resistance and could explain the genetic basis of carbapenem-resistance found in many enteric-pathogens.

Horizontal Gene Transfer, Fitness Costs and Mobility Shape the Spread of Antibiotic Resistance Genes into Experimental Populations of Acinetobacter Baylyi.

Horizontal gene transfer (HGT) is important for microbial evolution, but how evolutionary forces shape the frequencies of horizontally transferred genetic variants in the absence of strong selection remains an open question. In this study, we evolve laboratory populations of Acinetobacter baylyi (ADP1) with HGT from two clinically relevant strains of multidrug-resistant Acinetobacter baumannii (AB5075 and A9844). We find that DNA can cross the species barrier, even without strong selection, and despite substantial DNA sequence divergence between the two species. Our results confirm previous findings that HGT can drive the spread of antibiotic resistance genes (ARGs) without selection for that antibiotic, but not for all of the resistance genes present in the donor genome. We quantify the costs and benefits of horizontally transferred variants and use whole population sequencing to track the spread of ARGs from HGT donors into antibiotic-sensitive recipients. We find that even though most ARGs are taken up by populations of A. baylyi, the long-term fate of an individual gene depends both on its fitness cost and on the type of genetic element that carries the gene. Interestingly, we also found that an integron, but not its host plasmid, is able to spread in A. baylyi populations despite its strong deleterious effect. Altogether, our results show how HGT provides an evolutionary advantage to evolving populations by facilitating the spread of non-selected genetic variation including costly ARGs.

Bacteriophages evolve enhanced persistence to a mucosal surface.

The majority of viruses within the gut are obligate bacterial viruses known as bacteriophages (phages). Their bacteriotropism underscores the study of phage ecology in the gut, where they modulate and coevolve with gut bacterial communities. Traditionally, these ecological and evolutionary questions were investigated empirically via in vitro experimental evolution and, more recently, in vivo models were adopted to account for physiologically relevant conditions of the gut. Here, we probed beyond conventional phage-bacteria coevolution to investigate potential tripartite evolutionary interactions between phages, their bacterial hosts, and the mammalian gut mucosa. To capture the role of the mammalian gut, we recapitulated a life-like gut mucosal layer using in vitro lab-on-a-chip devices (to wit, the gut-on-a-chip) and showed that the mucosal environment supports stable phage-bacteria coexistence. Next, we experimentally coevolved lytic phage populations within the gut-on-a-chip devices alongside their bacterial hosts. We found that while phages adapt to the mucosal environment via de novo mutations, genetic recombination was the key evolutionary force in driving mutational fitness. A single mutation in the phage capsid protein Hoc-known to facilitate phage adherence to mucus-caused altered phage binding to fucosylated mucin glycans. We demonstrated that the altered glycan-binding phenotype provided the evolved mutant phage a competitive fitness advantage over its ancestral wild-type phage in the gut-on-a-chip mucosal environment. Collectively, our findings revealed that phages-in addition to their evolutionary relationship with bacteria-are able to evolve in response to a mammalian-derived mucosal environment.

Long-term experimental evolution decouples size and production costs in Escherichia coli.

Body size covaries with population dynamics across life's domains. Metabolism may impose fundamental constraints on the coevolution of size and demography, but experimental tests of the causal links remain elusive. We leverage a 60,000-generation experiment in which Escherichia coli populations evolved larger cells to examine intraspecific metabolic scaling and correlations with demographic parameters. Over the course of their evolution, the cells have roughly doubled in size relative to their ancestors. These larger cells have metabolic rates that are absolutely higher, but relative to their size, they are lower. Metabolic theory successfully predicted the relations between size, metabolism, and maximum population density, including support for Damuth's law of energy equivalence, such that populations of larger cells achieved lower maximum densities but higher maximum biomasses than populations of smaller cells. The scaling of metabolism with cell size thus predicted the scaling of size with maximum population density. In stark contrast to standard theory, however, populations of larger cells grew faster than those of smaller cells, contradicting the fundamental and intuitive assumption that the costs of building new individuals should scale directly with their size. The finding that the costs of production can be decoupled from size necessitates a reevaluation of the evolutionary drivers and ecological consequences of biological size more generally.

Recombination resolves the cost of horizontal gene transfer in experimental populations of Helicobacter pylori.

Horizontal gene transfer (HGT) is important for microbial evolution, yet we know little about the fitness effects and dynamics of horizontally transferred genetic variants. In this study, we evolve laboratory populations of Helicobacter pylori, which take up DNA from their environment by natural transformation, and measure the fitness effects of thousands of transferred genetic variants. We find that natural transformation increases the rate of adaptation but comes at the cost of significant genetic load. We show that this cost is circumvented by recombination, which increases the efficiency of selection by decoupling deleterious and beneficial genetic variants. Our results show that adaptation with HGT, pervasive in natural microbial populations, is shaped by a combination of selection, recombination, and genetic drift not accounted for in existing models of evolution.

Polymyxin dose tunes the evolutionary dynamics of resistance in multidrug-resistant Acinetobacter baumannii.

OBJECTIVES: Evolutionary principles have informed the design of strategies that slow or prevent antibiotic resistance. However, how antibiotic treatment regimens shape the evolutionary dynamics of resistance mutations remains an open question. Here, we investigate varying concentrations of the last-resort polymyxins on the evolution of resistance in Acinetobacter baumannii. METHODS: Polymyxin resistance was measured in 18 multidrug-resistant A. baumannii AB5075 populations treated over 14 days with concentrations of polymyxin B informed by human pharmacokinetics. Time-resolved whole-population sequencing was conducted to track the genetics and population dynamics of susceptible and resistant subpopulations. RESULTS: A critical threshold concentration of polymyxin B (1 mg/L; i.e. 4 × MIC) was identified. Below this threshold concentration, low levels of resistance repeatedly evolved, but no mutations were fixed, and this resistance was reversed upon removal of the antibiotic. This contrasted with evolution at super-MIC levels (≥4 × MIC) of polymyxin B, which drove the evolution of irreversible resistance, with higher levels of antibiotic correlating with greater rates of molecular evolution. Polymyxin-resistant subpopulations carried mutations in a variety of genes, most commonly pmrB, ompA, glmU/glmS, and wecB/wecC, which contributed to membrane remodelling and virulence in A. baumannii. CONCLUSIONS: Our results show that the strength of the selective pressure applied by polymyxin tunes the dynamics of genetic variants within the population, leading to different evolutionary outcomes for the degree, cost and reversibility of resistance. Our study highlights the critical role of integrating evolutionary findings into pharmacokinetics/pharmacodynamics to optimise antibiotic use in patients.

Species interactions constrain adaptation and preserve ecological stability in an experimental microbial community.

Species loss within a microbial community can increase resource availability and spur adaptive evolution. Environmental shifts that cause species loss or fluctuations in community composition are expected to become more common, so it is important to understand the evolutionary forces that shape the stability and function of the emergent community. Here we study experimental cultures of a simple, ecologically stable community of Saccharomyces cerevisiae and Lactobacillus plantarum, in order to understand how the presence or absence of a species impacts coexistence over evolutionary timescales. We found that evolution in coculture led to drastically altered evolutionary outcomes for L. plantarum, but not S. cerevisiae. Both monoculture- and co-culture-evolved L. plantarum evolved dozens of mutations over 925 generations of evolution, but only L. plantarum that had evolved in isolation from S. cerevisiae lost the capacity to coexist with S. cerevisiae. We find that the evolutionary loss of ecological stability corresponds with fitness differences between monoculture-evolved L. plantarum and S. cerevisiae and genetic changes that repeatedly evolve across the replicate populations of L. plantarum. This work shows how coevolution within a community can prevent destabilising evolution in individual species, thereby preserving ecological diversity and stability, despite rapid adaptation.

Host diversity slows bacteriophage adaptation by selecting generalists over specialists.

Most viruses can infect multiple hosts, yet the selective mechanisms that maintain multi-host generalists over single-host specialists remain an open question. Here we propagate populations of the newly identified bacteriophage øJB01 in coculture with many host genotypes and find that while phage can adapt to infect any of the new hosts, increasing the number of hosts slows the rate of adaptation. We quantify trade-offs in the capacity for individual phage to infect different hosts and find that phage from evolved populations with more hosts are more likely to be generalists. Sequencing of evolved phage reveals strong selection and the genetic basis of adaptation, supporting a model that shows how the addition of more potential hosts to a community can select for low-fitness generalists over high-fitness specialists. Our results show how evolution with multiple hosts alters the rate of viral adaptation and provides empirical support for an evolutionary mechanism that promotes generalists over specialists.

Phenotypic and molecular evolution across 10,000 generations in laboratory budding yeast populations.

Laboratory experimental evolution provides a window into the details of the evolutionary process. To investigate the consequences of long-term adaptation, we evolved 205 Saccharomyces cerevisiae populations (124 haploid and 81 diploid) for ~10,000,000 generations in three environments. We measured the dynamics of fitness changes over time, finding repeatable patterns of declining adaptability. Sequencing revealed that this phenotypic adaptation is coupled with a steady accumulation of mutations, widespread genetic parallelism, and historical contingency. In contrast to long-term evolution in E. coli, we do not observe long-term coexistence or populations with highly elevated mutation rates. We find that evolution in diploid populations involves both fixation of heterozygous mutations and frequent loss-of-heterozygosity events. Together, these results help distinguish aspects of evolutionary dynamics that are likely to be general features of adaptation across many systems from those that are specific to individual organisms and environmental conditions.

The evolution of coexistence from competition in experimental co-cultures of Escherichia coli and Saccharomyces cerevisiae.

Microbial communities are comprised of many species that coexist on small spatial scales. This is difficult to explain because many interspecies interactions are competitive, and ecological theory predicts that one species will drive the extinction of another species that competes for the same resource. Conversely, evolutionary theory proposes that natural selection can lead to coexistence by driving competing species to use non-overlapping resources. However, evolutionary escape from extinction may be slow compared to the rate of competitive exclusion. Here, we use experimental co-cultures of Escherichia coli and Saccharomyces cerevisiae to study the evolution of coexistence in species that compete for resources. We find that while E. coli usually outcompetes S. cerevisiae in co-culture, a few populations evolved stable coexistence after ~1000 generations of coevolution. We sequenced S. cerevisiae and E. coli populations, identified multi-hit genes, and engineered alleles from these genes into several genetic backgrounds, finding that some mutations modified interactions between E. coli and S. cerevisiae. Together, our data demonstrate that coexistence can evolve, de novo, from intense competition between two species with no history of coevolution.

Horizontal gene transfer potentiates adaptation by reducing selective constraints on the spread of genetic variation.

Horizontal gene transfer (HGT) confers the rapid acquisition of novel traits and is pervasive throughout microbial evolution. Despite the central role of HGT, the evolutionary forces that drive the dynamics of HGT alleles in evolving populations are poorly understood. Here, we show that HGT alters the evolutionary dynamics of genetic variation, so that deleterious genetic variants, including antibiotic resistance genes, can establish in populations without selection. We evolve antibiotic-sensitive populations of the human pathogen Helicobacter pylori in an environment without antibiotic but with HGT from an antibiotic-resistant isolate of H. pylori We find that HGT increases the rate of adaptation, with most horizontally transferred genetic variants establishing at a low frequency in the population. When challenged with antibiotic, this low-level variation potentiates adaptation, with HGT populations flourishing in conditions where nonpotentiated populations go extinct. By extending previous models of evolution under HGT, we evaluated the conditions for the establishment and spread of HGT-acquired alleles into recipient populations. We then used our model to estimate parameters of HGT and selection from our experimental evolution data. Together, our findings show how HGT can act as an evolutionary force that facilitates the spread of nonselected genetic variation and expands the adaptive potential of microbial populations.

Sex alters molecular evolution in diploid experimental populations of S. cerevisiae.

Sex is common among eukaryotes, but entails considerable costs. The selective conditions that drive the evolutionary maintenance of sexual reproduction remain an open question. One long-standing explanation is that sex and recombination facilitate adaptation to fluctuating environmental conditions, although the genetic mechanisms that underlie such a benefit have not been empirically observed. In this study, we compare the dynamics and fitness effects of mutations in sexual and asexual diploid populations of the yeast Saccharomyces cerevisiae during adaptation to a fluctuating environment. While we find no detectable difference in the rate of adaptation between sexual and asexual populations, only the former evolve high fitness mutations in parallel, a genetic signature of adaptation. Using genetic reconstructions and fitness assays, we demonstrate that evolved, overdominant mutations can be beneficial in asexual populations, but maintained at lower frequencies in sexual populations due to segregation load. Overall these data show that sex alters the molecular basis of adaptation in diploids, and confers both costs and benefits.

Microbial Experimental Evolution - a proving ground for evolutionary theory and a tool for discovery.

Microbial experimental evolution uses controlled laboratory populations to study the mechanisms of evolution. The molecular analysis of evolved populations enables empirical tests that can confirm the predictions of evolutionary theory, but can also lead to surprising discoveries. As with other fields in the life sciences, microbial experimental evolution has become a tool, deployed as part of the suite of techniques available to the molecular biologist. Here, I provide a review of the general findings of microbial experimental evolution, especially those relevant to molecular microbiologists that are new to the field. I also relate these results to design considerations for an evolution experiment and suggest future directions for those working at the intersection of experimental evolution and molecular biology.

Diverse hydrogen production and consumption pathways influence methane production in ruminants.

Farmed ruminants are the largest source of anthropogenic methane emissions globally. The methanogenic archaea responsible for these emissions use molecular hydrogen (H2), produced during bacterial and eukaryotic carbohydrate fermentation, as their primary energy source. In this work, we used comparative genomic, metatranscriptomic and co-culture-based approaches to gain a system-wide understanding of the organisms and pathways responsible for ruminal H2 metabolism. Two-thirds of sequenced rumen bacterial and archaeal genomes encode enzymes that catalyse H2 production or consumption, including 26 distinct hydrogenase subgroups. Metatranscriptomic analysis confirmed that these hydrogenases are differentially expressed in sheep rumen. Electron-bifurcating [FeFe]-hydrogenases from carbohydrate-fermenting Clostridia (e.g., Ruminococcus) accounted for half of all hydrogenase transcripts. Various H2 uptake pathways were also expressed, including methanogenesis (Methanobrevibacter), fumarate and nitrite reduction (Selenomonas), and acetogenesis (Blautia). Whereas methanogenesis-related transcripts predominated in high methane yield sheep, alternative uptake pathways were significantly upregulated in low methane yield sheep. Complementing these findings, we observed significant differential expression and activity of the hydrogenases of the hydrogenogenic cellulose fermenter Ruminococcus albus and the hydrogenotrophic fumarate reducer Wolinella succinogenes in co-culture compared with pure culture. We conclude that H2 metabolism is a more complex and widespread trait among rumen microorganisms than previously recognised. There is evidence that alternative hydrogenotrophs, including acetogenic and respiratory bacteria, can prosper in the rumen and effectively compete with methanogens for H2. These findings may help to inform ongoing strategies to mitigate methane emissions by increasing flux through alternative H2 uptake pathways, including through animal selection, dietary supplementation and methanogenesis inhibitors.

Fitness cost of mcr-1-mediated polymyxin resistance in Klebsiella pneumoniae.

Objectives: The discovery of mobile colistin resistance mcr-1, a plasmid-borne polymyxin resistance gene, highlights the potential for widespread resistance to the last-line polymyxins. In the present study, we investigated the impact of mcr-1 acquisition on polymyxin resistance and biological fitness in Klebsiella pneumoniae. Methods: K. pneumoniae B5055 was used as the parental strain for the construction of strains carrying vector only (pBBR1MCS-5) and mcr-1 recombinant plasmids (pmcr-1). Plasmid stability was determined by serial passaging for 10 consecutive days in antibiotic-free LB broth, followed by patching on gentamicin-containing and antibiotic-free LB agar plates. Lipid A was analysed using LC-MS. The biological fitness was examined using an in vitro competition assay analysed with flow cytometry. The in vivo fitness cost of mcr-1 was evaluated in a neutropenic mouse thigh infection model. Results: Increased polymyxin resistance was observed following acquisition of mcr-1 in K. pneumoniae B5055. The modification of lipid A with phosphoethanolamine following mcr-1 addition was demonstrated by lipid A profiling. The plasmid stability assay revealed the instability of the plasmid after acquiring mcr-1. Reduced in vitro biological fitness and in vivo growth were observed with the mcr-1-carrying K. pneumoniae strain. Conclusions: Although mcr-1 confers a moderate level of polymyxin resistance, it is associated with a significant biological fitness cost in K. pneumoniae. This indicates that mcr-1-mediated resistance in K. pneumoniae could be attenuated by limiting the usage of polymyxins.

Adaptive evolution of genomically recoded Escherichia coli.

Efforts are underway to construct several recoded genomes anticipated to exhibit multivirus resistance, enhanced nonstandard amino acid (nsAA) incorporation, and capability for synthetic biocontainment. Although our laboratory pioneered the first genomically recoded organism (Escherichia coli strain C321.∆A), its fitness is far lower than that of its nonrecoded ancestor, particularly in defined media. This fitness deficit severely limits its utility for nsAA-linked applications requiring defined media, such as live cell imaging, metabolic engineering, and industrial-scale protein production. Here, we report adaptive evolution of C321.∆A for more than 1,000 generations in independent replicate populations grown in glucose minimal media. Evolved recoded populations significantly exceeded the growth rates of both the ancestral C321.∆A and nonrecoded strains. We used next-generation sequencing to identify genes mutated in multiple independent populations, and we reconstructed individual alleles in ancestral strains via multiplex automatable genome engineering (MAGE) to quantify their effects on fitness. Several selective mutations occurred only in recoded evolved populations, some of which are associated with altering the translation apparatus in response to recoding, whereas others are not apparently associated with recoding, but instead correct for off-target mutations that occurred during initial genome engineering. This report demonstrates that laboratory evolution can be applied after engineering of recoded genomes to streamline fitness recovery compared with application of additional targeted engineering strategies that may introduce further unintended mutations. In doing so, we provide the most comprehensive insight to date into the physiology of the commonly used C321.∆A strain.

Beneficial Mutations from Evolution Experiments Increase Rates of Growth and Fermentation.

A major goal of evolutionary biology is to understand how beneficial mutations translate into increased fitness. Here, we study beneficial mutations that arise in experimental populations of yeast evolved in glucose-rich media. We find that fitness increases are caused by enhanced maximum growth rate (R) that come at the cost of reduced yield (K). We show that for some of these mutants, high R coincides with higher rates of ethanol secretion, suggesting that higher growth rates are due to an increased preference to utilize glucose through the fermentation pathway, instead of respiration. We examine the performance of mutants across gradients of glucose and nitrogen concentrations and show that the preference for fermentation over respiration is influenced by the availability of glucose and nitrogen. Overall, our data show that selection for high growth rates can lead to an enhanced Crabtree phenotype by the way of beneficial mutations that permit aerobic fermentation at a greater range of glucose concentrations.

The dynamics of molecular evolution over 60,000 generations.

The outcomes of evolution are determined by a stochastic dynamical process that governs how mutations arise and spread through a population. However, it is difficult to observe these dynamics directly over long periods and across entire genomes. Here we analyse the dynamics of molecular evolution in twelve experimental populations of Escherichia coli, using whole-genome metagenomic sequencing at five hundred-generation intervals through sixty thousand generations. Although the rate of fitness gain declines over time, molecular evolution is characterized by signatures of rapid adaptation throughout the duration of the experiment, with multiple beneficial variants simultaneously competing for dominance in each population. Interactions between ecological and evolutionary processes play an important role, as long-term quasi-stable coexistence arises spontaneously in most populations, and evolution continues within each clade. We also present evidence that the targets of natural selection change over time, as epistasis and historical contingency alter the strength of selection on different genes. Together, these results show that long-term adaptation to a constant environment can be a more complex and dynamic process than is often assumed.