Perturbation and resilience of the gut microbiome up to 3 months after ß-lactams exposure in healthy volunteers suggest an important role of microbial ß-lactamases.

BACKGROUND: Antibiotics notoriously perturb the gut microbiota. We treated healthy volunteers either with cefotaxime or ceftriaxone for 3 days, and collected in each subject 12 faecal samples up to day 90. Using untargeted and targeted phenotypic and genotypic approaches, we studied the changes in the bacterial, phage and fungal components of the microbiota as well as the metabolome and the ß-lactamase activity of the stools. This allowed assessing their degrees of perturbation and resilience. RESULTS: While only two subjects had detectable concentrations of antibiotics in their faeces, suggesting important antibiotic degradation in the gut, the intravenous treatment perturbed very significantly the bacterial and phage microbiota, as well as the composition of the metabolome. In contrast, treatment impact was relatively low on the fungal microbiota. At the end of the surveillance period, we found evidence of resilience across the gut system since most components returned to a state like the initial one, even if the structure of the bacterial microbiota changed and the dynamics of the different components over time were rarely correlated. The observed richness of the antibiotic resistance genes repertoire was significantly reduced up to day 30, while a significant increase in the relative abundance of ß-lactamase encoding genes was observed up to day 10, consistent with a concomitant increase in the ß-lactamase activity of the microbiota. The level of ß-lactamase activity at baseline was positively associated with the resilience of the metabolome content of the stools. CONCLUSIONS: In healthy adults, antibiotics perturb many components of the microbiota, which return close to the baseline state within 30 days. These data suggest an important role of endogenous ß-lactamase-producing anaerobes in protecting the functions of the microbiota by de-activating the antibiotics reaching the colon. Video Abstract.

Changing fitness effects of mutations through long-term bacterial evolution.

The distribution of fitness effects of new mutations shapes evolution, but it is challenging to observe how it changes as organisms adapt. Using Escherichia coli lineages spanning 50,000 generations of evolution, we quantify the fitness effects of insertion mutations in every gene. Macroscopically, the fraction of deleterious mutations changed little over time whereas the beneficial tail declined sharply, approaching an exponential distribution. Microscopically, changes in individual gene essentiality and deleterious effects often occurred in parallel; altered essentiality is only partly explained by structural variation. The identity and effect sizes of beneficial mutations changed rapidly over time, but many targets of selection remained predictable because of the importance of loss-of-function mutations. Taken together, these results reveal the dynamic-but statistically predictable-nature of mutational fitness effects.

Deep mutational scanning reveals the molecular determinants of RNA polymerase-mediated adaptation and tradeoffs.

RNA polymerase (RNAP) is emblematic of complex biological systems that control multiple traits involving trade-offs such as growth versus maintenance. Laboratory evolution has revealed that mutations in RNAP subunits, including RpoB, are frequently selected. However, we lack a systems view of how mutations alter the RNAP molecular functions to promote adaptation. We, therefore, measured the fitness of thousands of mutations within a region of rpoB under multiple conditions and genetic backgrounds, to find that adaptive mutations cluster in two modules. Mutations in one module favor growth over maintenance through a partial loss of an interaction associated with faster elongation. Mutations in the other favor maintenance over growth through a destabilized RNAP-DNA complex. The two molecular handles capture the versatile RNAP-mediated adaptations. Combining both interaction losses simultaneously improved maintenance and growth, challenging the idea that growth-maintenance tradeoff resorts only from limited resources, and revealing how compensatory evolution operates within RNAP.

Predicting the effect of mutations to investigate recent events of selection across 60,472 Escherichia coli strains.

Microbial genomics studies focusing on the dynamics of selection have often used a small number of distant genomes. As a result, they could only analyze mutations that had become fixed during the divergence between species. However, thousands of genomes of some species are now available in public databases, thanks to high-throughput sequencing. These data provide a more complete picture of the polymorphisms segregating within a species, offering a unique insight into the processes that shape the recent evolution of a species. In this study, we present GLASS (Gene-Level Amino-acid Score Shift), a selection test that is based on the predicted effects of amino acid changes. By comparing the distribution of effects of mutations observed in a gene to the expectation in the absence of selection, GLASS can quantify the intensity of selection. We applied GLASS to a dataset of 60,472 Escherichia coli strains and used this to reexamine the longstanding debate about the role of essentiality versus expression level in the rate of protein evolution. We found that selection has contrasting short-term and long-term dynamics, with essential genes being subject to strong purifying selection in the short term, while expression level determines the rate of gene evolution in the long term. GLASS also found an overrepresentation of inactivating mutations in specific transcription factors, such as efflux pump repressors, which is consistent with selection for antibiotic resistance. These gene-inactivating polymorphisms do not reach fixation, suggesting another contrast between short-term fitness gains and long-term counterselection.

Towards evolutionary predictions: Current promises and challenges.

Evolution has traditionally been a historical and descriptive science, and predicting future evolutionary processes has long been considered impossible. However, evolutionary predictions are increasingly being developed and used in medicine, agriculture, biotechnology and conservation biology. Evolutionary predictions may be used for different purposes, such as to prepare for the future, to try and change the course of evolution or to determine how well we understand evolutionary processes. Similarly, the exact aspect of the evolved population that we want to predict may also differ. For example, we could try to predict which genotype will dominate, the fitness of the population or the extinction probability of a population. In addition, there are many uses of evolutionary predictions that may not always be recognized as such. The main goal of this review is to increase awareness of methods and data in different research fields by showing the breadth of situations in which evolutionary predictions are made. We describe how diverse evolutionary predictions share a common structure described by the predictive scope, time scale and precision. Then, by using examples ranging from SARS-CoV2 and influenza to CRISPR-based gene drives and sustainable product formation in biotechnology, we discuss the methods for predicting evolution, the factors that affect predictability and how predictions can be used to prevent evolution in undesirable directions or to promote beneficial evolution (i.e. evolutionary control). We hope that this review will stimulate collaboration between fields by establishing a common language for evolutionary predictions.

[The impact of neutral mutations on genome evolvability]. / L'impact des mutations neutres sur l'évolvabilité et l'évolution des génomes.

Beneficial mutations with strong effects are rare and deleterious mutations are purged by natural selection. Therefore, the majority of mutations that accumulate in genomes have very weak or no selective effects, being then called neutral mutations. Over the last two decades, it has been shown that mutations, even when they are neutral, affect evolvability by providing access to new phenotypes through later-occurring mutations that would not have been available otherwise. We propose here that in addition to this effect, many mutations -independent of their selective effects- can affect the mutability of neighboring DNA sequences and modulate the efficiency of homologous recombination. Such mutations do not alter the spectrum of accessible phenotypes, but rather the rate at which new phenotypes will be produced, a process that has long-term but also potentially short-term consequences for cancer emergence.

Title: L'impact des mutations neutres sur l'évolvabilité et l'évolution des génomes. Abstract: Les mutations bénéfiques à forts effets sont rares et les mutations délétères sont éliminées par la sélection naturelle. La majorité des mutations qui s'accumulent dans les génomes ont donc des effets sélectifs très faibles, voire nuls ; elles sont alors appelées mutations neutres. Au cours des deux dernières décennies, il a été montré que les mutations, même en l'absence d'effet sur la valeur sélective des organismes, affectent leur évolvabilité1, en donnant accès à de nouveaux phénotypes par le biais de mutations apparaissant ultérieurement, et qui n'auraient pas été disponibles autrement. En plus de cet effet, de nombreuses mutations neutres ­ indépendamment de leurs effets sélectifs ­ peuvent affecter la mutabilité de séquences d'ADN voisines, et moduler l'efficacité de la recombinaison homologue. De telles mutations ne modifient pas le spectre des phénotypes accessibles, mais plutôt la vitesse à laquelle de nouveaux phénotypes seront produits, un processus qui a des conséquences à long terme mais aussi potentiellement à court terme, en lien avec l'émergence de cancers.

Klebsiella pneumoniae Carbapenemase Variants Resistant to Ceftazidime-Avibactam: an Evolutionary Overview.

First variants of the Klebsiella pneumoniae carbapenemase (KPC), KPC-2 and KPC-3, have encountered a worldwide success, particularly in K. pneumoniae isolates. These beta-lactamases conferred resistance to most beta-lactams including carbapenems but remained susceptible to new beta-lactam/beta-lactamase inhibitors, such as ceftazidime-avibactam. After the marketing of ceftazidime-avibactam, numerous variants of KPC resistant to this association have been described among isolates recovered from clinical samples or derived from experimental studies. In KPC variants resistant to ceftazidime-avibactam, point mutations, insertions and/or deletions have been described in various hot spots. Deciphering the impact of these mutations is crucial, not only from a therapeutic point of view, but also to follow the evolution in time and space of KPC variants resistant to ceftazidime-avibactam. In this review, we describe the mutational landscape of the KPC beta-lactamase toward ceftazidime-avibactam resistance based on a multidisciplinary approach including epidemiology, microbiology, enzymology, and thermodynamics. We show that resistance is associated with three hot spots, with a high representation of insertions and deletions compared with other class A beta-lactamases. Moreover, extension of resistance to ceftazidime-avibactam is associated with a trade-off in the resistance to other beta-lactams and a decrease in enzyme stability. Nevertheless, the high natural stability of KPC could underlay the propensity of this enzyme to acquire in vivo mutations conferring resistance to ceftazidime-avibactam (CAZavi), particularly via insertions and deletions.

The Population Genomics of Increased Virulence and Antibiotic Resistance in Human Commensal Escherichia coli over 30 Years in France.

Escherichia coli is a commensal species of the lower intestine but is also a major pathogen causing intestinal and extraintestinal infections that is increasingly prevalent and resistant to antibiotics. Most studies on genomic evolution of E. coli used isolates from infections. Here, instead, we whole-genome sequenced a collection of 403 commensal E. coli isolates from fecal samples of healthy adult volunteers in France (1980 to 2010). These isolates were distributed mainly in phylogroups A and B2 (30% each) and belonged to 152 sequence types (STs), the five most frequent being ST10 (phylogroup A; 16.3%), ST73 and ST95 (phylogroup B2; 6.3 and 5.0%, respectively), ST69 (phylogroup D; 4.2%), and ST59 (phylogroup F; 3.9%), and 224 O:H serotypes. ST and serotype diversity increased over time. The O1, O2, O6, and O25 groups used in bioconjugate O-antigen vaccine against extraintestinal infections were found in 23% of the strains of our collection. The increase in frequency of virulence-associated genes and antibiotic resistance was driven by two evolutionary mechanisms. Evolution of virulence gene frequency was driven by both clonal expansion of STs with more virulence genes ("ST-driven") and increases in gene frequency within STs independent of changes in ST frequencies ("gene-driven"). In contrast, the evolution of resistance was dominated by increases in frequency within STs ("gene-driven"). This study provides a unique picture of the phylogenomic evolution of E. coli in its human commensal habitat over 30 years and will have implications for the development of preventive strategies. IMPORTANCE Escherichia coli is an opportunistic pathogen with the greatest burden of antibiotic resistance, one of the main causes of bacterial infections and an increasing concern in an aging population. Deciphering the evolutionary dynamics of virulence and antibiotic resistance in commensal E. coli is important to understand adaptation and anticipate future changes. The gut of vertebrates is the primary habitat of E. coli and probably where selection for virulence and resistance takes place. Unfortunately, most whole-genome-sequenced strains are isolated from pathogenic conditions. Here, we whole-genome sequenced 403 E. coli commensals isolated from healthy French subjects over a 30-year period. Virulence genes increased in frequency by both clonal expansion of clones carrying them and increases in frequency within clones, whereas resistance genes increased by within-clone increased frequency. Prospective studies of E. coli commensals should be performed worldwide to have a broader picture of evolution and adaptation of this species.

Deciphering polymorphism in 61,157 Escherichia coli genomes via epistatic sequence landscapes.

Characterizing the effect of mutations is key to understand the evolution of protein sequences and to separate neutral amino-acid changes from deleterious ones. Epistatic interactions between residues can lead to a context dependence of mutation effects. Context dependence constrains the amino-acid changes that can contribute to polymorphism in the short term, and the ones that can accumulate between species in the long term. We use computational approaches to accurately predict the polymorphisms segregating in a panel of 61,157 Escherichia coli genomes from the analysis of distant homologues. By comparing a context-aware Direct-Coupling Analysis modelling to a non-epistatic approach, we show that the genetic context strongly constrains the tolerable amino acids in 30% to 50% of amino-acid sites. The study of more distant species suggests the gradual build-up of genetic context over long evolutionary timescales by the accumulation of small epistatic contributions.

A Microbiota-Dependent Response to Anticancer Treatment in an In Vitro Human Microbiota Model: A Pilot Study With Hydroxycarbamide and Daunorubicin.

Background: Anticancer drug efficacy is linked to the gut microbiota's composition, and there is a dire need to better understand these interactions for personalized medicine. In vitro microbiota models are promising tools for studies requiring controlled and repeatable conditions. We evaluated the impact of two anticancer drugs on human feces in the MiniBioReactor Array (MBRA) in vitro microbiota system. Methods: The MBRA is a single-stage continuous-flow culture model, hosted in an anaerobic chamber. We evaluated the effect of a 5-day treatment with hydroxycarbamide or daunorubicine on the fecal bacterial communities of two healthy donors. 16S microbiome profiling allowed analysis of microbial richness, diversity, and taxonomic changes. Results: In this host-free setting, anticancer drugs diversely affect gut microbiota composition. Daunorubicin was associated with significant changes in alpha- and beta-diversity as well as in the ratio of Firmicutes/Bacteroidetes in a donor-dependent manner. The impact of hydroxycarbamide on microbiota composition was not significant. Conclusion: We demonstrated, for the first time, the impact of anticancer drugs on human microbiota composition, in a donor- and molecule-dependent manner in an in vitro human microbiota model. We confirm the importance of personalized studies to better predict drug-associated-dysbiosis in vivo, linked to the host's response to treatment.

rRNA operon multiplicity as a bacterial genome stability insurance policy.

Quick growth restart after upon encountering favourable environmental conditions is a major fitness contributor in natural environment. It is widely assumed that the time required to restart growth after nutritional upshift is determined by how long it takes for cells to synthesize enough ribosomes to produce the proteins required to reinitiate growth. Here we show that a reduction in the capacity to synthesize ribosomes by reducing number of ribosomal RNA (rRNA) operons (rrn) causes a longer transition from stationary phase to growth of Escherichia coli primarily due to high mortality rates. Cell death results from DNA replication blockage and massive DNA breakage at the sites of the remaining rrn operons that become overloaded with RNA polymerases (RNAPs). Mortality rates and growth restart duration can be reduced by preventing R-loop formation and improving DNA repair capacity. The same molecular mechanisms determine the duration of the recovery phase after ribosome-damaging stresses, such as antibiotics, exposure to bile salts or high temperature. Our study therefore suggests that a major function of rrn operon multiplicity is to ensure that individual rrn operons are not saturated by RNAPs, which can result in catastrophic chromosome replication failure and cell death during adaptation to environmental fluctuations.

The ability to modulate translation capacity, which resides greatly on a number of ribosomes, provides robustness in fluctuating environments. Because translation is energetically the most expensive process in cells, cells must constantly adapt the rate of ribosome production to resource availability. This is primarily achieved by regulating ribosomal RNA (rRNA) synthesis, to which ribosomal proteins synthesis is adjusted. The multiplicity of rRNA encoding operons per bacterial genome exceeds requirements for the maximal growth rates in non-stress conditions. In this study, the authors provide evidence that a major function of rRNA operon multiplicity is to ensure that individual operons are not saturated by RNA polymerases during adaptation to environmental fluctuations, which can result in catastrophic chromosome replication failure and cell death.

Local and Global Protein Interactions Contribute to Residue Entrenchment in Beta-Lactamase TEM-1.

Due to their rapid evolution and their impact on healthcare, beta-lactamases, protein degrading beta-lactam antibiotics, are used as generic models of protein evolution. Therefore, we investigated the mutation effects in two distant beta-lactamases, TEM-1 and CTX-M-15. Interestingly, we found a site with a complex pattern of genetic interactions. Mutation G251W in TEM-1 inactivates the protein's function, just as the reciprocal mutation, W251G, does in CTX-M-15. The phylogenetic analysis revealed that mutation G has been entrenched in TEM-1's background: while rarely observed throughout the phylogeny, it is essential in TEM-1. Using a rescue experiment, in the TEM-1 G251W mutant, we identified sites that alleviate the deviation from G to W. While few of these mutations could potentially involve local interactions, most of them were found on distant residues in the 3D structure. Many well-known mutations that have an impact on protein stability, such as M182T, were recovered. Our results therefore suggest that entrenchment of an amino acid may rely on diffuse interactions among multiple sites, with a major impact on protein stability.

Modeling the bacterial dynamics in the gut microbiota following an antibiotic-induced perturbation.

Recent studies have highlighted the importance of ecological interactions in dysbiosis of gut microbiota, but few focused on their role in antibiotic-induced perturbations. We used the data from the CEREMI trial in which 22 healthy volunteers received a 3-day course of ceftriaxone or cefotaxime antibiotics. Fecal samples were analyzed by 16S rRNA gene profiling, and the total bacterial counts were determined in each sample by flux cytometry. As the gut exposure to antibiotics could not be experimentally measured despite a marked impact on the gut microbiota, it was reconstructed using the counts of susceptible Escherichia coli. The dynamics of absolute counts of bacterial families were analyzed using a generalized Lotka-Volterra equations and nonlinear mixed effect modeling. Bacterial interactions were studied using a stepwise approach. Two negative and three positive interactions were identified. Introducing bacterial interactions in the modeling approach better fitted the data, and provided different estimates of antibiotic effects on each bacterial family than a simple model without interaction. The time to return to 95% of the baseline counts was significantly longer in ceftriaxone-treated individuals than in cefotaxime-treated subjects for two bacterial families: Akkermansiaceae (median [range]: 11.3 days [0; 180.0] vs. 4.2 days [0; 25.6], p = 0.027) and Tannerellaceae (13.7 days [6.1; 180.0] vs. 6.2 days [5.4; 17.3], p = 0.003). Taking bacterial interaction as well as individual antibiotic exposure profile into account improves the analysis of antibiotic-induced dysbiosis.

The interplay between anticancer challenges and the microbial communities from the gut.

Cancer being an increasing burden on human health, the use of anticancer drugs has risen over the last decades. The physiological effects of these drugs are not only perceived by the host's cells but also by the microbial cells it harbors as commensals, notably the gut microbiota. Since the early '50 s, the cytotoxicity of anticancer chemotherapy was evaluated on bacteria revealing some antimicrobial activities that result in an established perturbation of the gut microbiota. This perturbation can affect the host's health through dysbiosis, which can lead to multiple complications, but has also been shown to have a direct effect on the treatment efficiency.We, therefore, conducted a review of literature focusing on this triangular relationship involving the microbial communities from the gut, the host's disease, and the anticancer treatment. We focused specifically on the antimicrobial effects of anticancer chemotherapy, their impact on mutagenesis in bacteria, and the perspectives of using bacteria-based tools to help in the diagnostic and treatment of cancer.

Dynamics of extended-spectrum beta-lactamase-producing Enterobacterales colonization in long-term carriers following travel abroad.

Travel to tropical regions is associated with high risk of acquiring extended-spectrum beta-lactamase-producing Enterobacterales (ESBL-E) that are typically cleared in less than 3 months following return. The conditions leading to persistent carriage that exceeds 3 months in some travellers require investigation. Whole-genome sequencing (Illumina MiSeq) was performed on the 82 ESBL-E isolates detected upon return and 1, 2, 3, 6 and 12 months later from the stools of 11 long-term (>3 months) ESBL-E carriers following travel abroad. One to five different ESBL Escherichia coli strains were detected per traveller upon return, and this diminished to one after 3 months. Long-term carriage was due to the presence of the same ESBL E. coli strain, for more than 3 months, in 9 out of 11 travellers, belonging to epidemic sequence type complexes (STc 10, 14, 38, 69, 131 and 648). The mean carriage duration of strains belonging to phylogroups B2/D/F, associated with extra-intestinal virulence, was higher than that for commensal-associated A/B1/E phylogroups (3.5 vs 0.5 months, P=0.021). Genes encoding iron capture systems (fyuA, irp), toxins (senB, sat), adhesins (flu, daaF, afa/nfaE, pap, ecpA) and colicin (cjrA) were more often present in persistent strains than in transient ones. Single-nucleotide polymorphism (SNP) analysis in persistent strains showed a maximum divergence of eight SNPs over 12 months without signs of adaptation. Genomic plasticity was observed during the follow-up with the loss or gain of mobile genetic elements such as plasmids, integrons and/or transposons that may contain resistance genes at different points in the follow-up. Long-term colonization of ESBL-E following travel is primarily due to the acquisition of E. coli strains belonging to epidemic clones and harbouring 'virulence genes', allowing good adaptation to the intestinal microbiota.

Cross-resistance to cefiderocol and ceftazidime-avibactam in KPC ß-lactamase mutants and the inoculum effect.

OBJECTIVES: Ceftazidime-avibactam (CZA) and cefiderocol are recently commercialized molecules active against highly drug-resistant bacteria, including carbapenem-resistant members of the Enterobacteriaceae. Mutants resistant to CZA have been described, notably in Klebsiella pneumoniae carbapenemase (KPC) producers. Considering the structural similarities between ceftazidime and cefiderocol, we hypothesized that resistance to CZA in KPC-producing members of the Enterobacterales may lead to cross-resistance to cefiderocol. METHODS: CZA-resistant mutants from three clinical isolates of the Enterobacterales carrying either blaKPC-2 or blaKPC-3 were selected in vitro. Mutants with increased MIC to CZA compared to the ancestral allele were cloned in a pBR322 plasmid and expressed in Escherichia coli TOP10. We evaluated the impact of these mutations on cefiderocol MICs and minimal bactericidal concentrations (MBCs), and we assessed the impact of bacterial inoculum size on cefiderocol MICs. RESULTS: We used 37 KPC mutants with increased CZA MICs. Of these, six have been described previously in clinical isolates. Compared to the wild-type alleles, increases in the cefiderocol MICs of 4- to 32-fold were observed for 75.6% of tested mutants (28/37), MICs reaching up to 4 mg/L in E. coli TOP10 for KPC-31 (D179Y-H274Y mutations). MBCs and MICs of cefiderocol were similar, confirming the bactericidal activity of this drug. Finally, when using higher inocula (107 CFU/mL), a large increase in cefiderocol MIC was observed, and all isolates were categorized as resistant. CONCLUSION: We observed that most of the CZA-resistant KPC variants have a possible impact on cefiderocol by increasing the cefiderocol MICs. In addition, cefiderocol is greatly impacted by the inoculum effect, suggesting that precautions should be taken when treating infections with a suspected high inoculum.

Success of prophylactic antiviral therapy for SARS-CoV-2: Predicted critical efficacies and impact of different drug-specific mechanisms of action.

Repurposed drugs that are safe and immediately available constitute a first line of defense against new viral infections. Despite limited antiviral activity against SARS-CoV-2, several drugs are being tested as medication or as prophylaxis to prevent infection. Using a stochastic model of early phase infection, we evaluate the success of prophylactic treatment with different drug types to prevent viral infection. We find that there exists a critical efficacy that a treatment must reach in order to block viral establishment. Treatment by a combination of drugs reduces the critical efficacy, most effectively by the combination of a drug blocking viral entry into cells and a drug increasing viral clearance. Below the critical efficacy, the risk of infection can nonetheless be reduced. Drugs blocking viral entry into cells or enhancing viral clearance reduce the risk of infection more than drugs that reduce viral production in infected cells. The larger the initial inoculum of infectious virus, the less likely is prevention of an infection. In our model, we find that as long as the viral inoculum is smaller than 10 infectious virus particles, viral infection can be prevented almost certainly with drugs of 90% efficacy (or more). Even when a viral infection cannot be prevented, antivirals delay the time to detectable viral loads. The largest delay of viral infection is achieved by drugs reducing viral production in infected cells. A delay of virus infection flattens the within-host viral dynamic curve, possibly reducing transmission and symptom severity. Thus, antiviral prophylaxis, even with reduced efficacy, could be efficiently used to prevent or alleviate infection in people at high risk.

Insertion-sequence-mediated mutations both promote and constrain evolvability during a long-term experiment with bacteria.

Insertion sequences (IS) are ubiquitous bacterial mobile genetic elements, and the mutations they cause can be deleterious, neutral, or beneficial. The long-term dynamics of IS elements and their effects on bacteria are poorly understood, including whether they are primarily genomic parasites or important drivers of adaptation by natural selection. Here, we investigate the dynamics of IS elements and their contribution to genomic evolution and fitness during a long-term experiment with Escherichia coli. IS elements account for ~35% of the mutations that reached high frequency through 50,000 generations in those populations that retained the ancestral point-mutation rate. In mutator populations, IS-mediated mutations are only half as frequent in absolute numbers. In one population, an exceptionally high ~8-fold increase in IS150 copy number is associated with the beneficial effects of early insertion mutations; however, this expansion later slowed down owing to reduced IS150 activity. This population also achieves the lowest fitness, suggesting that some avenues for further adaptation are precluded by the IS150-mediated mutations. More generally, across all populations, we find that higher IS activity becomes detrimental to adaptation over evolutionary time. Therefore, IS-mediated mutations can both promote and constrain evolvability.

Escherichia coli Genomic Diversity within Extraintestinal Acute Infections Argues for Adaptive Evolution at Play.

Adaptive processes in chronic bacterial infections are well described, but much less is known about the processes at play during acute infections. Here, by sequencing seven randomly selected isolates per patient, we analyzed Escherichia coli populations from three acute extraintestinal infections in adults (meningitis, pyelonephritis, and peritonitis), in which a high-mutation-rate isolate or mutator isolate was found. The isolates of single patients displayed between a few dozen and more than 200 independent mutations, with up to half being specific to the mutator isolate. Multiple signs of positive selection were evidenced: a high ratio of nonsynonymous to synonymous mutations (Ka /Ks ratio) and strong mutational convergence within and between patients, some of them at loci well known for their adaptive potential, such as rpoS, rbsR, fimH, and fliC For all patients, the mutator isolate was likely due to a large deletion of a methyl-directed mismatch repair gene, and in two instances, the deletion extended to genes involved in some genetic convergence, suggesting potential coselection. Intrinsic extraintestinal virulence assessed in a mouse model of sepsis showed variable patterns of virulence ranging from non-mouse killer to mouse killer for the isolates from single patients. However, genomic signature and gene inactivation experiments did not establish a link between a single gene and the capacity to kill mice, highlighting the complex and multifactorial nature of the virulence. Altogether, these data indicate that E. coli isolates are adapting under strong selective pressure when colonizing an extraintestinal site.IMPORTANCE Little is known about the dynamics of adaptation in acute bacterial infections. By sequencing multiple isolates from monoclonal extraintestinal Escherichia coli infections in several patients, we were able to uncover traces of selection taking place at short time scales compared to chronic infection. High genomic diversity was observed in the patient isolates, with an excess of nonsynonymous mutations, and the comparison within and between different infections showed patterns of convergence at the gene level, both constituting strong signs of adaptation. The genes targeted were coding mostly for proteins involved in global regulation, metabolism, and adhesion/motility. Moreover, virulence assessed in a mouse model of sepsis was variable among the isolates of single patients, but this difference was left unexplained at the molecular level. This work gives us clues about the E. coli lifestyle transition between commensalism and pathogenicity.

Primary and promiscuous functions coexist during evolutionary innovation through whole protein domain acquisitions.

Molecular examples of evolutionary innovation are scarce and generally involve point mutations. Innovation can occur through larger rearrangements, but here experimental data is extremely limited. Integron integrases innovated from double-strand- toward single-strand-DNA recombination through the acquisition of the I2 α-helix. To investigate how this transition was possible, we have evolved integrase IntI1 to what should correspond to an early innovation state by selecting for its ancestral activity. Using synonymous alleles to enlarge sequence space exploration, we have retrieved 13 mutations affecting both I2 and the multimerization domains of IntI1. We circumvented epistasis constraints among them using a combinatorial library that revealed their individual and collective fitness effects. We obtained up to 104-fold increases in ancestral activity with various asymmetrical trade-offs in single-strand-DNA recombination. We show that high levels of primary and promiscuous functions could have initially coexisted following I2 acquisition, paving the way for a gradual evolution toward innovation.