Alejandro Couce

The rule of declining adaptability in microbial evolution experiments.

One of the most recurrent observations after two decades of microbial evolution experiments regards the dynamics of fitness change. In a given environment, low-fitness genotypes are recurrently observed to adapt faster than their more fit counterparts. Since adaptation is the main macroscopic outcome of Darwinian evolution, studying its patterns of change could potentially provide insight into key issues of evolutionary theory, from fixation dynamics to the genetic architecture of organisms. Here, we re-analyze several published datasets from experimental evolution with microbes and show that, despite large differences in the origin of the data, a pattern of inverse dependence of adaptability with fitness clearly emerges. In quantitative terms, it is remarkable to observe little if any degree of idiosyncrasy across systems as diverse as virus, bacteria and yeast. The universality of this phenomenon suggests that its emergence might be understood from general principles, giving rise to the exciting prospect that evolution might be statistically predictable at the macroscopic level. We discuss these possibilities in the light of the various theories of adaptation that have been proposed and delineate future directions of research.

Genomewide Overexpression Screen for Fosfomycin Resistance in Escherichia coli: MurA Confers Clinical Resistance at Low Fitness Cost

ABSTRACT To determine whether the overexpression of chromosomal genes can confer fosfomycin resistance, genomewide screening of a complete set of 5,272 plasmid-expressed open reading frames of Escherichia coli (ASKA collection) was performed. Major results are that (i) no clinical level of resistance is achieved by overexpressing chromosomal genes, except murA; (ii) this level is reached at a low fitness cost; and (iii) this cost is much lower than that imposed by other mutations conferring fosfomycin resistance.

Frequency of Spontaneous Resistance to Fosfomycin Combined with Different Antibiotics in Pseudomonas aeruginosa

Chronic infections caused by Pseudomonas aeruginosa are probably the most recurrent clinical situation where antibiotic treatment fails (5). The presence of hypermutable strains exacerbates this phenomenon and appears to be an important factor for the development of multiple-antimicrobial resistance (6, 8). The shortage of new antimicrobials led to reconsideration of old antibiotics, including fosfomycin, as appealing alternatives for treatments (2). The good effectiveness of fosfomycin combined with other antibiotics has been reported (2, 3, 7, 9, 11, 15). However, P. aeruginosa has a very high mutant frequency for fosfomycin resistance in vitro (12) and in vivo (13), suggesting an elevated risk of resistance to combined treatments. We analyzed the frequency of mutants resistant to fosfomycin in combination with other antimicrobials currently used in P. aeruginosa infections, including tobramycin, amikacin, imipenem, meropenem, ceftazidime, ciprofloxacin, and colistin. PA14 and its hypermutable mutS::MAR2xT7 derivative (4) were used as model strains. Experiments were performed in quintuplicate in all cases as described previously (12). Antibiotic concentrations were chosen according to each drug clinical breakpoint, as established by EUCAST (www.eucast.org/clinical_breakpoints/), except for fosfomycin, which was used at 128 μg/ml to avoid the background on plates (no significant differences were found in mutant frequencies at 32, 64, and 128 μg/ml [not shown]). The mutant frequencies of PA14 for individual antibiotics were very high for fosfomycin, high for imipenem and meropenem, moderate for ceftazidime, and relatively low for ciprofloxacin, tobramycin, and amikacin (Table ​(Table1).1). The frequencies of the hypermutable strain were, as expected, 100- to 1,000-fold higher. However, the mutant frequencies of the wild-type (WT) strain for the combinations were below the limit of detection (<1 × 10−10) for all antibiotics, except for imipenem plus fosfomycin (1.1 × 10−9). For the hypermutable strain, the mutant frequencies for combinations of fosfomycin with tobramycin, amikacin, meropenem, ciprofloxacin, and colistin were below the limit of detection. However, combinations with ceftazidime or imipenem yielded a higher-than-expected number of mutants resistant to both antibiotics (higher than the product of the frequencies for each single antibiotic), with values of 1.0 × 10−8 and 1.1 × 10−7, respectively. These interesting results remain to be explained, although antagonism between fosfomycin and these antibiotics in vitro could not be demonstrated (data not shown). These results suggest that the combinations of fosfomycin with ceftazidime or imipenem are less appropriate, in terms of probability of mutant occurrence, than those with tobramycin or ciprofloxacin. TABLE 1. Frequency of mutants of P. aeruginosa PA14 and its hypermutable mutS derivative resistant to single antibiotics and their combinations with fosfomycin In cases of chronic infection, such as that involving cystic fibrosis, the bacterial load of P. aeruginosa can be as high as 107 to 109 CFU per ml of mucus secretion (14), with a high frequency of hypermutable strains (10). According to our results, the probability of finding mutants resistant to the combination of fosfomycin with ceftazidime or imipenem is dangerously high. Antibiotic combinations must be carefully considered to minimize the selection of strains with double resistance. Further studies on combinations need to be done considering different criteria, including pharmacological activity and the possibility of emergence of resistant mutants.

The Escherichia coli SOS Gene dinF Protects against Oxidative Stress and Bile Salts

DNA is constantly damaged by physical and chemical factors, including reactive oxygen species (ROS), such as superoxide radical (O2 −), hydrogen peroxide (H2O2) and hydroxyl radical (•OH). Specific mechanisms to protect and repair DNA lesions produced by ROS have been developed in living beings. In Escherichia coli the SOS system, an inducible response activated to rescue cells from severe DNA damage, is a network that regulates the expression of more than 40 genes in response to this damage, many of them playing important roles in DNA damage tolerance mechanisms. Although the function of most of these genes has been elucidated, the activity of some others, such as dinF, remains unknown. The DinF deduced polypeptide sequence shows a high homology with membrane proteins of the multidrug and toxic compound extrusion (MATE) family. We describe here that expression of dinF protects against bile salts, probably by decreasing the effects of ROS, which is consistent with the observed decrease in H2O2-killing and protein carbonylation. These results, together with its ability to decrease the level of intracellular ROS, suggests that DinF can detoxify, either direct or indirectly, oxidizing molecules that can damage DNA and proteins from both the bacterial metabolism and the environment. Although the exact mechanism of DinF activity remains to be identified, we describe for the first time a role for dinF.

Bypass of genetic constraints during mutator evolution to antibiotic resistance

Genetic constraints can block many mutational pathways to optimal genotypes in real fitness landscapes, yet the extent to which this can limit evolution remains to be determined. Interestingly, mutator bacteria elevate only specific types of mutations, and therefore could be very sensitive to genetic constraints. Testing this possibility is not only clinically relevant, but can also inform about the general impact of genetic constraints in adaptation. Here, we evolved 576 populations of two mutator and one wild-type Escherichia coli to doubling concentrations of the antibiotic cefotaxime. All strains carried TEM-1, a β-lactamase enzyme well known by its low availability of mutational pathways. Crucially, one of the mutators does not elevate any of the relevant first-step mutations known to improve cefatoximase activity. Despite this, both mutators displayed a similar ability to evolve more than 1000-fold resistance. Initial adaptation proceeded in parallel through general multi-drug resistance mechanisms. High-level resistance, in contrast, was achieved through divergent paths; with the a priori inferior mutator exploiting alternative mutational pathways in PBP3, the target of the antibiotic. These results have implications for mutator management in clinical infections and, more generally, illustrate that limits to natural selection in real organisms are alleviated by the existence of multiple loci contributing to fitness.

Mutator genomes decay, despite sustained fitness gains, in a long-term experiment with bacteria

Significance Bacterial genomes are extremely diverse in size and composition. Biologists have long sought to explain such variability based on present-day selective and mutational forces. However, mutation rates can change dramatically over time, and experiments with hypermutable bacteria show that their genomes rapidly decay when propagated under the near absence of selection. Whether selection can prevent this decay is unclear. Here, we document the rapid genome decay of hypermutable bacteria even during tens of thousands of generations of sustained adaptation to a laboratory environment. These findings suggest the need to reexamine current ideas about the evolution of bacterial genomes, and they have implications for other hypermutable systems such as viruses and cancer cells. Understanding the extreme variation among bacterial genomes remains an unsolved challenge in evolutionary biology, despite long-standing debate about the relative importance of natural selection, mutation, and random drift. A potentially important confounding factor is the variation in mutation rates between lineages and over evolutionary history, which has been documented in several species. Mutation accumulation experiments have shown that hypermutability can erode genomes over short timescales. These results, however, were obtained under conditions of extremely weak selection, casting doubt on their general relevance. Here, we circumvent this limitation by analyzing genomes from mutator populations that arose during a long-term experiment with Escherichia coli, in which populations have been adaptively evolving for >50,000 generations. We develop an analytical framework to quantify the relative contributions of mutation and selection in shaping genomic characteristics, and we validate it using genomes evolved under regimes of high mutation rates with weak selection (mutation accumulation experiments) and low mutation rates with strong selection (natural isolates). Our results show that, despite sustained adaptive evolution in the long-term experiment, the signature of selection is much weaker than that of mutational biases in mutator genomes. This finding suggests that relatively brief periods of hypermutability can play an outsized role in shaping extant bacterial genomes. Overall, these results highlight the importance of genomic draft, in which strong linkage limits the ability of selection to purge deleterious mutations. These insights are also relevant to other biological systems evolving under strong linkage and high mutation rates, including viruses and cancer cells.

Determinants of Genetic Diversity of Spontaneous Drug-Resistance in Bacteria

Any pathogen population sufficiently large is expected to harbor spontaneous drug-resistant mutants, often responsible for disease relapse after antibiotic therapy. It is seldom appreciated, however, that while larger populations harbor more mutants, the abundance distribution of these mutants is expected to be markedly uneven. This is because a larger population size allows early mutants to expand for longer, exacerbating their predominance in the final mutant subpopulation. Here, we investigate the extent to which this reduction in evenness can constrain the genetic diversity of spontaneous drug resistance in bacteria. Combining theory and experiments, we show that even small variations in growth rate between resistant mutants and the wild type result in orders-of-magnitude differences in genetic diversity. Indeed, only a slight fitness advantage for the mutant is enough to keep diversity low and independent of population size. These results have important clinical implications. Genetic diversity at antibiotic resistance loci can determine a population’s capacity to cope with future challenges (i.e., second-line therapy). We thus revealed an unanticipated way in which the fitness effects of antibiotic resistance can affect the evolvability of pathogens surviving a drug-induced bottleneck. This insight will assist in the fight against multidrug-resistant microbes, as well as contribute to theories aimed at predicting cancer evolution.

Parallel Evolution of High-Level Aminoglycoside Resistance in Escherichia coli Under Low and High Mutation Supply Rates

Antibiotic resistance is a major concern in public health worldwide, thus there is much interest in characterizing the mutational pathways through which susceptible bacteria evolve resistance. Here we use experimental evolution to explore the mutational pathways toward aminoglycoside resistance, using gentamicin as a model, under low and high mutation supply rates. Our results show that both normo and hypermutable strains of Escherichia coli are able to develop resistance to drug dosages > 1,000-fold higher than the minimal inhibitory concentration for their ancestors. Interestingly, such level of resistance was often associated with changes in susceptibility to other antibiotics, most prominently with increased resistance to fosfomycin. Whole-genome sequencing revealed that all resistant derivatives presented diverse mutations in five common genetic elements: fhuA, fusA and the atpIBEFHAGDC, cyoABCDE, and potABCD operons. Despite the large number of mutations acquired, hypermutable strains did not pay, apparently, fitness cost. In contrast to recent studies, we found that the mutation supply rate mainly affected the speed (tempo) but not the pattern (mode) of evolution: both backgrounds acquired the mutations in the same order, although the hypermutator strain did it faster. This observation is compatible with the adaptive landscape for high-level gentamicin resistance being relatively smooth, with few local maxima; which might be a common feature among antibiotics for which resistance involves multiple loci.

Intrapopulation variability in mutator prevalence among urinary tract infection isolates of Escherichia coli.

Bacteria with elevated mutation rates represent a risk factor for treatment failure and are often found with high frequency in clinical isolates from different sources. How this frequency reflects the among-population and within-population proportion of hypermutators is unknown, despite its importance to the choice of antibiotic therapies that minimize the likelihood of resistance development. Here we screened for hypermutators among the urine of 80 patients with urinary tract infections, at an unprecedented resolution of 24 isolates per sample. We found hypermutators in four patients (5%), at frequencies ranging from 4.2% to 62.5%. Molecular characterization revealed alterations in the oxidized guanine (GO) and methly-directed mistmatch repair (MMR) systems as the genetic basis of hypermutability. These observations suggest that mutators may be present in more patients than previously anticipated, at frequencies that are difficult to detect but still sufficient to impact on adaptation to antibiotics or the host environment.

The molecular basis of genetic interaction diversity in a metabolic pathway

Metabolic imbalances underlie a large spectrum of diseases, spanning congenital and chronic conditions and cancer. Our ability to explain and predict such imbalances remains severely limited by the diversity of underlying mutation effects and their dependence on the genetic background and environment, but it is unclear whether these complicating factors can be reduced to simple quantitative rules. Here, we characterise their interplay in determining cell physiology and fitness by systematically quantifying almost 4,000 interactions between expression variants of two genes from a classical sugar-utilisation pathway containing a toxic metabolite in the model bacterium, Escherichia coli, in different environments. We detect a remarkable variety of types and trends of intergenic interaction in this linear pathway, which cannot be reliably predicted from the effects of each variant in isolation, along with a dependence of this epistasis on the environment. Despite such apparent complexity, the fitness consequences of interactions between alleles and environment are explained by a mechanistic model accounting for catabolic flux and toxic metabolite concentration. Our findings reveal how, contrary to a common assumption, the nature of fitness interactions is governed by more than just the topology of the molecular network underlying a selected trait. Our prospects of predicting disease and evolution will therefore improve by expanding our knowledge of the links among proteome, metabolome and physiology.Our ability to predict the impact of mutations on traits relevant for disease and evolution remains severely limited by the dependence of their effects on the genetic background and environment. Even when molecular interactions between genes are known, it is unclear how these translate to organism-level interactions between alleles. We therefore characterized the interplay of genetic and environmental dependencies in determining fitness by quantifying ~4,000 fitness interactions between expression variants of two metabolic genes, in different environments. We detect a remarkable variety of environment-dependent interactions, and demonstrate they can be quantitatively explained by a mechanistic model accounting for catabolic flux, metabolite toxicity and expression costs. Complex fitness interactions between mutations can therefore be predicted simply from their simultaneous impact on a few connected molecular phenotypes.