Restriction-modification systems have shaped the evolution and distribution of plasmids across bacteria.

Many novel traits such as antibiotic resistance are spread by plasmids between species. Yet plasmids have different host ranges. Restriction-modification systems (R-M systems) are by far the most abundant bacterial defense system and therefore represent one of the key barriers to plasmid spread. However, their effect on plasmid evolution and host range has been neglected. Here we analyse the avoidance of targets of the most abundant R-M systems (Type II) for complete genomes and plasmids across bacterial diversity. For the most common target length (6 bp) we show that target avoidance is strongly correlated with the taxonomic distribution of R-M systems and is greater in plasmid genes than core genes. We find stronger avoidance of R-M targets in plasmids which are smaller and have a broader host range. Our results suggest two different evolutionary strategies for plasmids: small plasmids primarily adapt to R-M systems by tuning their sequence composition, and large plasmids primarily adapt through the carriage of additional genes protecting from restriction. Our work provides systematic evidence that R-M systems are important barriers to plasmid transfer and have left their mark on plasmids over long evolutionary time.

Warming alters life-history traits and competition in a phage community.

Host-parasite interactions are highly susceptible to changes in temperature due to mismatches in species thermal responses. In nature, parasites often exist in communities, and responses to temperature are expected to vary between host-parasite pairs. Temperature change thus has consequences for both host-parasite dynamics and parasite-parasite interactions. Here, we investigate the impact of warming (37°C, 40°C, and 42°C) on parasite life-history traits and competition using the opportunistic bacterial pathogen Pseudomonas aeruginosa (host) and a panel of three genetically diverse lytic bacteriophages (parasites). We show that phages vary in their responses to temperature. While 37°C and 40°C did not have a major effect on phage infectivity, infection by two phages was restricted at 42°C. This outcome was attributed to disruption of different phage life-history traits including host attachment and replication inside hosts. Furthermore, we show that temperature mediates competition between phages by altering their competitiveness. These results highlight phage trait variation across thermal regimes with the potential to drive community dynamics. Our results have important implications for eukaryotic viromes and the design of phage cocktail therapies.IMPORTANCEMammalian hosts often elevate their body temperatures through fevers to restrict the growth of bacterial infections. However, the extent to which fever temperatures affect the communities of phages with the ability to parasitize those bacteria remains unclear. In this study, we investigate the impact of warming across a fever temperature range (37°C, 40°C, and 42°C) on phage life-history traits and competition using a bacterium (host) and bacteriophage (parasite) system. We show that phages vary in their responses to temperature due to disruption of different phage life-history traits. Furthermore, we show that temperature can alter phage competitiveness and shape phage-phage competition outcomes. These results suggest that fever temperatures have the potential to restrict phage infectivity and drive phage community dynamics. We discuss implications for the role of temperature in shaping host-parasite interactions more widely.

Stochastic bacterial population dynamics restrict the establishment of antibiotic resistance from single cells.

A better understanding of how antibiotic exposure impacts the evolution of resistance in bacterial populations is crucial for designing more sustainable treatment strategies. The conventional approach to this question is to measure the range of concentrations over which resistant strain(s) are selectively favored over a sensitive strain. Here, we instead investigate how antibiotic concentration impacts the initial establishment of resistance from single cells, mimicking the clonal expansion of a resistant lineage following mutation or horizontal gene transfer. Using two Pseudomonas aeruginosa strains carrying resistance plasmids, we show that single resistant cells have <5% probability of detectable outgrowth at antibiotic concentrations as low as one-eighth of the resistant strain's minimum inhibitory concentration (MIC). This low probability of establishment is due to detrimental effects of antibiotics on resistant cells, coupled with the inherently stochastic nature of cell division and death on the single-cell level, which leads to loss of many nascent resistant lineages. Our findings suggest that moderate doses of antibiotics, well below the MIC of resistant strains, may effectively restrict de novo emergence of resistance even though they cannot clear already-large resistant populations.

Susceptibility profiles and resistance genomics of Pseudomonas aeruginosa isolates from European ICUs participating in the ASPIRE-ICU trial.

OBJECTIVES: To determine the susceptibility profiles and the resistome of Pseudomonas aeruginosa isolates from European ICUs during a prospective cohort study (ASPIRE-ICU). METHODS: 723 isolates from respiratory samples or perianal swabs of 402 patients from 29 sites in 11 countries were studied. MICs of 12 antibiotics were determined by broth microdilution. Horizontally acquired ß-lactamases were analysed through phenotypic and genetic assays. The first respiratory isolates from 105 patients providing such samples were analysed through WGS, including the analysis of the resistome and a previously defined genotypic resistance score. Spontaneous mutant frequencies and the genetic basis of hypermutation were assessed. RESULTS: All agents except colistin showed resistance rates above 20%, including ceftolozane/tazobactam and ceftazidime/avibactam. 24.9% of the isolates were XDR, with a wide intercountry variation (0%-62.5%). 13.2% of the isolates were classified as DTR (difficult-to-treat resistance). 21.4% of the isolates produced ESBLs (mostly PER-1) or carbapenemases (mostly NDM-1, VIM-1/2 and GES-5). WGS showed that these determinants were linked to high-risk clones (particularly ST235 and ST654). WGS revealed a wide repertoire of mutation-driven resistance mechanisms, with multiple lineage-specific mutations. The most frequently mutated genes were gyrA, parC, oprD, mexZ, nalD and parS, but only two of the isolates were hypermutable. Finally, a good accuracy of the genotypic score to predict susceptibility (91%-100%) and resistance (94%-100%) was documented. CONCLUSIONS: An overall high prevalence of resistance is documented European ICUs, but with a wide intercountry variability determined by the dissemination of XDR high-risk clones, arguing for the need to reinforce infection control measures.

Evaluating evolutionary models of stress-induced mutagenesis in bacteria.

Increased mutation rates under stress allow bacterial populations to adapt rapidly to stressors, including antibiotics. Here we evaluate existing models for the evolution of stress-induced mutagenesis and present a new model arguing that it evolves as a result of a complex interplay between direct selection for increased stress tolerance, second-order selection for increased evolvability and genetic drift. Further progress in our understanding of the evolutionary biology of stress and mutagenesis will require a more detailed understanding both of the patterns of stress encountered by bacteria in nature and of the mutations that are produced under stress.

The Genomic Basis of Evolutionary Innovation in Pseudomonas aeruginosa.

Novel traits play a key role in evolution, but their origins remain poorly understood. Here we address this problem by using experimental evolution to study bacterial innovation in real time. We allowed 380 populations of Pseudomonas aeruginosa to adapt to 95 different carbon sources that challenged bacteria with either evolving novel metabolic traits or optimizing existing traits. Whole genome sequencing of more than 80 clones revealed profound differences in the genetic basis of innovation and optimization. Innovation was associated with the rapid acquisition of mutations in genes involved in transcription and metabolism. Mutations in pre-existing duplicate genes in the P. aeruginosa genome were common during innovation, but not optimization. These duplicate genes may have been acquired by P. aeruginosa due to either spontaneous gene amplification or horizontal gene transfer. High throughput phenotype assays revealed that novelty was associated with increased pleiotropic costs that are likely to constrain innovation. However, mutations in duplicate genes with close homologs in the P. aeruginosa genome were associated with low pleiotropic costs compared to mutations in duplicate genes with distant homologs in the P. aeruginosa genome, suggesting that functional redundancy between duplicates facilitates innovation by buffering pleiotropic costs.

The genomic basis of adaptation to the fitness cost of rifampicin resistance in Pseudomonas aeruginosa.

Antibiotic resistance carries a fitness cost that must be overcome in order for resistance to persist over the long term. Compensatory mutations that recover the functional defects associated with resistance mutations have been argued to play a key role in overcoming the cost of resistance, but compensatory mutations are expected to be rare relative to generally beneficial mutations that increase fitness, irrespective of antibiotic resistance. Given this asymmetry, population genetics theory predicts that populations should adapt by compensatory mutations when the cost of resistance is large, whereas generally beneficial mutations should drive adaptation when the cost of resistance is small. We tested this prediction by determining the genomic mechanisms underpinning adaptation to antibiotic-free conditions in populations of the pathogenic bacterium Pseudomonas aeruginosa that carry costly antibiotic resistance mutations. Whole-genome sequencing revealed that populations founded by high-cost rifampicin-resistant mutants adapted via compensatory mutations in three genes of the RNA polymerase core enzyme, whereas populations founded by low-cost mutants adapted by generally beneficial mutations, predominantly in the quorum-sensing transcriptional regulator gene lasR. Even though the importance of compensatory evolution in maintaining resistance has been widely recognized, our study shows that the roles of general adaptation in maintaining resistance should not be underestimated and highlights the need to understand how selection at other sites in the genome influences the dynamics of resistance alleles in clinical settings.

Divergent evolution peaks under intermediate population bottlenecks during bacterial experimental evolution.

There is growing evidence that parallel molecular evolution is common, but its causes remain poorly understood. Demographic parameters such as population bottlenecks are predicted to be major determinants of parallelism. Here, we test the hypothesis that bottleneck intensity shapes parallel evolution by elucidating the genomic basis of adaptation to antibiotic-supplemented media in hundreds of populations of the bacterium Pseudomonas fluorescens Pf0-1. As expected, bottlenecking decreased the rate of phenotypic and molecular adaptation. Surprisingly, bottlenecking had no impact on the likelihood of parallel adaptive molecular evolution at a genome-wide scale. However, bottlenecking had a profound impact on the genes involved in antibiotic resistance. Specifically, under either intense or weak bottlenecking, resistance predominantly evolved by strongly beneficial mutations which provide high levels of antibiotic resistance. In contrast with intermediate bottlenecking regimes, resistance evolved by a greater diversity of genetic mechanisms, significantly reducing the observed levels of parallel genetic evolution. Our results demonstrate that population bottlenecking can be a major predictor of parallel evolution, but precisely how may be more complex than many simple theoretical predictions.

The population genetics of antibiotic resistance: integrating molecular mechanisms and treatment contexts.

Despite efforts from a range of disciplines, our ability to predict and combat the evolution of antibiotic resistance in pathogenic bacteria is limited. This is because resistance evolution involves a complex interplay between the specific drug, bacterial genetics and both natural and treatment ecology. Incorporating details of the molecular mechanisms of drug resistance and ecology into evolutionary models has proved useful in predicting the dynamics of resistance evolution. However, putting these models to practical use will require extensive collaboration between mathematicians, molecular biologists, evolutionary ecologists and clinicians.

Testing the role of genetic background in parallel evolution using the comparative experimental evolution of antibiotic resistance.

Parallel evolution is the independent evolution of the same phenotype or genotype in response to the same selection pressure. There are examples of parallel molecular evolution across divergent genetic backgrounds, suggesting that genetic background may not play an important role in determining the outcome of adaptation. Here, we measure the influence of genetic background on phenotypic and molecular adaptation by combining experimental evolution with comparative analysis. We selected for resistance to the antibiotic rifampicin in eight strains of bacteria from the genus Pseudomonas using a short term selection experiment. Adaptation occurred by 47 mutations at conserved sites in rpoB, the target of rifampicin, and due to the high diversity of possible mutations the probability of within-strain parallel evolution was low. The probability of between-strain parallel evolution was only marginally lower, because different strains substituted similar rpoB mutations. In contrast, we found that more than 30% of the phenotypic variation in the growth rate of evolved clones was attributable to among-strain differences. Parallel molecular evolution across strains resulted in divergent phenotypic evolution because rpoB mutations had different effects on growth rate in different strains. This study shows that genetic divergence between strains constrains parallel phenotypic evolution, but had little detectable impact on the molecular basis of adaptation in this system.

Here's to the losers: evolvable residents accelerate the evolution of high-fitness invaders.

Recent work has shown that evolvability plays a key role in determining the long-term population dynamics of asexual clones. However, simple considerations suggest that the evolvability of a focal lineage of bacteria should also be influenced by the evolvability of its competitors. First, evolvable competitors should accelerate evolution by impeding the fixation of the focal lineage through a clonal interference-like mechanism. Second, evolvable competitors should increase the strength of selection by rapidly degrading the environment, increasing selection for adaptive mutations. Here we tested these ideas by allowing a high-fitness clone of the bacterium Pseudomonas aeruginosa to invade populations of two low-fitness resident clones that differ in their evolvability. Both competition from mutations in the resident lineage and environmental degradation lead to faster adaptation in the invader through fixing single mutations with a greater fitness advantage. The results suggest that competition from mutations in both the successful invader and the unsuccessful resident shapes the adaptive trajectory of the invader through both direct competition and indirect environmental effects. Therefore, to predict evolutionary outcomes, it will be necessary to consider the evolvability of all members of the community and the effects of adaptation on the quality of the environment. This is particularly relevant to mixed microbial communities where lineages differ in their adaptive potential, a common feature of chronic infections.

The SOS response increases bacterial fitness, but not evolvability, under a sublethal dose of antibiotic.

Exposure to antibiotics induces the expression of mutagenic bacterial stress-response pathways, but the evolutionary benefits of these responses remain unclear. One possibility is that stress-response pathways provide a short-term advantage by protecting bacteria against the toxic effects of antibiotics. Second, it is possible that stress-induced mutagenesis provides a long-term advantage by accelerating the evolution of resistance. Here, we directly measure the contribution of the Pseudomonas aeruginosa SOS pathway to bacterial fitness and evolvability in the presence of sublethal doses of ciprofloxacin. Using short-term competition experiments, we demonstrate that the SOS pathway increases competitive fitness in the presence of ciprofloxacin. Continued exposure to ciprofloxacin results in the rapid evolution of increased fitness and antibiotic resistance, but we find no evidence that SOS-induced mutagenesis accelerates the rate of adaptation to ciprofloxacin during a 200 generation selection experiment. Intriguingly, we find that the expression of the SOS pathway decreases during adaptation to ciprofloxacin, and this helps to explain why this pathway does not increase long-term evolvability. Furthermore, we argue that the SOS pathway fails to accelerate adaptation to ciprofloxacin because the modest increase in the mutation rate associated with SOS mutagenesis is offset by a decrease in the effective strength of selection for increased resistance at a population level. Our findings suggest that the primary evolutionary benefit of the SOS response is to increase bacterial competitive ability, and that stress-induced mutagenesis is an unwanted side effect, and not a selected attribute, of this pathway.

Sequencing of plasmids pAMBL1 and pAMBL2 from Pseudomonas aeruginosa reveals a blaVIM-1 amplification causing high-level carbapenem resistance.

BACKGROUND: Carbapenemases are a major concern for the treatment of infectious diseases caused by Gram-negative bacteria. Although plasmids are responsible for the spread of resistance genes among these pathogens, there is limited information on the nature of the mobile genetic elements carrying carbapenemases in Pseudomonas aeruginosa. METHODS: We combined data from two different next-generation sequencing platforms, Illumina HiSeq2000 and PacBio RSII, to obtain the complete nucleotide sequences of two blaVIM-1-carrying plasmids (pAMBL1 and pAMBL2) isolated from P. aeruginosa clinical isolates. RESULTS: Plasmid pAMBL1 has 26 440 bp and carries a RepA_C family replication protein. pAMBL1 is similar to plasmids pNOR-2000 and pKLC102 from P. aeruginosa and pAX22 from Achromobacter xylosoxidans, which also carry VIM-type carbapenemases. pAMBL2 is a 24 133 bp plasmid with a replication protein that belongs to the Rep_3 family. It shows a high degree of homology with a fragment of the blaVIM-1-bearing plasmid pPC9 from Pseudomonas putida. Plasmid pAMBL2 carries three copies of the blaVIM-1 cassette in an In70 class 1 integron conferring, unlike pAMBL1, high-level resistance to carbapenems. CONCLUSIONS: We present two new plasmids coding for VIM-1 carbapenemase from P. aeruginosa and report that the presence of three copies of blaVIM-1 in pAMBL2 produces high-level resistance to carbapenems.

Antibiotic resistance alters the ability of Pseudomonas aeruginosa to invade bacteria from the respiratory microbiome.

The emergence and spread of antibiotic resistance in bacterial pathogens is a global health threat. One important unanswered question is how antibiotic resistance influences the ability of a pathogen to invade the host-associated microbiome. Here we investigate how antibiotic resistance impacts the ability of a bacterial pathogen to invade bacteria from the microbiome, using the opportunistic bacterial pathogen Pseudomonas aeruginosa and the respiratory microbiome as our model system. We measure the ability of P. aeruginosa spontaneous antibiotic-resistant mutants to invade pre-established cultures of commensal respiratory microbes in an assay that allows us to link specific resistance mutations with changes in invasion ability. While commensal respiratory microbes tend to provide some degree of resistance to P. aeruginosa invasion, antibiotic resistance is a double-edged sword that can either help or hinder the ability of P. aeruginosa to invade. The directionality of this help or hindrance depends on both P. aeruginosa genotype and respiratory microbe identity. Specific resistance mutations in genes involved in multidrug efflux pump regulation are shown to facilitate the invasion of P. aeruginosa into Staphylococcus lugdunensis, yet impair invasion into Rothia mucilaginosa and Staphylococcus epidermidis. Streptococcus species provide the strongest resistance to P. aeruginosa invasion, and this is maintained regardless of antibiotic resistance genotype. Our study demonstrates how the cost of mutations that provide enhanced antibiotic resistance in P. aeruginosa can crucially depend on community context. We suggest that attempts to manipulate the microbiome should focus on promoting the growth of commensals that can increase the fitness costs associated with antibiotic resistance and provide robust inhibition of both wildtype and antibiotic-resistant pathogen strains.

Plasmid-mediated phenotypic noise leads to transient antibiotic resistance in bacteria.

The rise of antibiotic resistance is a critical public health concern, requiring an understanding of mechanisms that enable bacteria to tolerate antimicrobial agents. Bacteria use diverse strategies, including the amplification of drug-resistance genes. In this paper, we showed that multicopy plasmids, often carrying antibiotic resistance genes in clinical bacteria, can rapidly amplify genes, leading to plasmid-mediated phenotypic noise and transient antibiotic resistance. By combining stochastic simulations of a computational model with high-throughput single-cell measurements of blaTEM-1 expression in Escherichia coli MG1655, we showed that plasmid copy number variability stably maintains populations composed of cells with both low and high plasmid copy numbers. This diversity in plasmid copy number enhances the probability of bacterial survival in the presence of antibiotics, while also rapidly reducing the burden of carrying multiple plasmids in drug-free environments. Our results further support the tenet that multicopy plasmids not only act as vehicles for the horizontal transfer of genetic information between cells but also as drivers of bacterial adaptation, enabling rapid modulation of gene copy numbers. Understanding the role of multicopy plasmids in antibiotic resistance is critical, and our study provides insights into how bacteria can transiently survive lethal concentrations of antibiotics.

A trade-off between oxidative stress resistance and DNA repair plays a role in the evolution of elevated mutation rates in bacteria.

The dominant paradigm for the evolution of mutator alleles in bacterial populations is that they spread by indirect selection for linked beneficial mutations when bacteria are poorly adapted. In this paper, we challenge the ubiquity of this paradigm by demonstrating that a clinically important stressor, hydrogen peroxide, generates direct selection for an elevated mutation rate in the pathogenic bacterium Pseudomonas aeruginosa as a consequence of a trade-off between the fidelity of DNA repair and hydrogen peroxide resistance. We demonstrate that the biochemical mechanism underlying this trade-off in the case of mutS is the elevated secretion of catalase by the mutator strain. Our results provide, to our knowledge, the first experimental evidence that direct selection can favour mutator alleles in bacterial populations, and pave the way for future studies to understand how mutation and DNA repair are linked to stress responses and how this affects the evolution of bacterial mutation rates.

A mixture of "cheats" and "co-operators" can enable maximal group benefit.

Is a group best off if everyone co-operates? Theory often considers this to be so (e.g. the "conspiracy of doves"), this understanding underpinning social and economic policy. We observe, however, that after competition between "cheat" and "co-operator" strains of yeast, population fitness is maximized under co-existence. To address whether this might just be a peculiarity of our experimental system or a result with broader applicability, we assemble, benchmark, dissect, and test a systems model. This reveals the conditions necessary to recover the unexpected result. These are 3-fold: (a) that resources are used inefficiently when they are abundant, (b) that the amount of co-operation needed cannot be accurately assessed, and (c) the population is structured, such that co-operators receive more of the resource than the cheats. Relaxing any of the assumptions can lead to population fitness being maximized when cheats are absent, which we experimentally demonstrate. These three conditions will often be relevant, and hence in order to understand the trajectory of social interactions, understanding the dynamics of the efficiency of resource utilization and accuracy of information will be necessary.

Off-Target Integron Activity Leads to Rapid Plasmid Compensatory Evolution in Response to Antibiotic Selection Pressure.

Integrons are mobile genetic elements that have played an important role in the dissemination of antibiotic resistance. Under stress, the integron can generate combinatorial variation in resistance cassette expression by cassette reshuffling, accelerating the evolution of resistance. However, the flexibility of the integron integrase site recognition motif hints at potential off-target effects of the integrase on the rest of the genome that may have important evolutionary consequences. Here, we test this hypothesis by selecting for increased-piperacillin-resistance populations of Pseudomonas aeruginosa with a mobile integron containing a difficult-to-mobilize ß-lactamase cassette to minimize the potential for adaptive cassette reshuffling. We found that integron activity can decrease the overall survival rate but also improve the fitness of the surviving populations. Off-target inversions mediated by the integron accelerated plasmid adaptation by disrupting costly conjugative genes otherwise mutated in control populations lacking a functional integrase. Plasmids containing integron-mediated inversions were associated with lower plasmid costs and higher stability than plasmids carrying mutations albeit at the cost of a reduced conjugative ability. These findings highlight the potential for integrons to create structural variation that can drive bacterial evolution, and they provide an interesting example showing how antibiotic pressure can drive the loss of conjugative genes. IMPORTANCE Tackling the public health challenge created by antibiotic resistance requires understanding the mechanisms driving its evolution. Mobile integrons are widespread genetic platforms heavily involved in the spread of antibiotic resistance. Through the action of the integrase enzyme, integrons allow bacteria to capture, excise, and shuffle antibiotic resistance gene cassettes. This integrase enzyme is characterized by its ability to recognize a wide range of recombination sites, which allows it to easily capture diverse resistance cassettes but which may also lead to off-target reactions with the rest of the genome. Using experimental evolution, we tested the off-target impact of integron activity. We found that integrons increased the fitness of the surviving bacteria through extensive genomic rearrangements of the plasmids carrying the integrons, reducing their ability to spread horizontally. These results show that integrons not only accelerate resistance evolution but also can generate extensive structural variation, driving bacterial evolution beyond antibiotic resistance.

Interkingdom interactions between Pseudomonas aeruginosa and Candida albicans affect clinical outcomes and antimicrobial responses.

Infections that involve interkingdom microbial communities, such as those between bacteria and yeast pathogens, are difficult to treat, associated with worse patient outcomes, and may be a source of antimicrobial resistance. In this review, we address co-occurrence and co-infections of Candida albicans and Pseudomonas aeruginosa, two pathogens that occupy multiple infection niches in the human body, especially in immunocompromised patients. The interaction between the pathogen species influences microbe-host interactions, the effectiveness of antimicrobials and even infection outcomes, and may thus require adapted treatment strategies. However, the molecular details of bacteria-fungal interactions both inside and outside the infection sites, are insufficiently characterised. We argue that comprehensively understanding the P. aeruginosa-C. albicans interaction network through integrated systems biology approaches will capture the highly dynamic and complex nature of these polymicrobial infections and lead to a more comprehensive understanding of clinical observations such as reshaped immune defences and low antimicrobial treatment efficacy.

Mixed strain pathogen populations accelerate the evolution of antibiotic resistance in patients.

Antibiotic resistance poses a global health threat, but the within-host drivers of resistance remain poorly understood. Pathogen populations are often assumed to be clonal within hosts, and resistance is thought to emerge due to selection for de novo variants. Here we show that mixed strain populations are common in the opportunistic pathogen P. aeruginosa. Crucially, resistance evolves rapidly in patients colonized by multiple strains through selection for pre-existing resistant strains. In contrast, resistance evolves sporadically in patients colonized by single strains due to selection for novel resistance mutations. However, strong trade-offs between resistance and growth rate occur in mixed strain populations, suggesting that within-host diversity can also drive the loss of resistance in the absence of antibiotic treatment. In summary, we show that the within-host diversity of pathogen populations plays a key role in shaping the emergence of resistance in response to treatment.