Integron activity accelerates the evolution of antibiotic resistance. - Related Documents




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926601.0000Integron activity accelerates the evolution of antibiotic resistance. Mobile integrons are widespread genetic platforms that allow bacteria to modulate the expression of antibiotic resistance cassettes by shuffling their position from a common promoter. Antibiotic stress induces the expression of an integrase that excises and integrates cassettes, and this unique recombination and expression system is thought to allow bacteria to 'evolve on demand' in response to antibiotic pressure. To test this hypothesis, we inserted a custom three-cassette integron into Pseudomonas aeruginosa and used experimental evolution to measure the impact of integrase activity on adaptation to gentamicin. Crucially, integrase activity accelerated evolution by increasing the expression of a gentamicin resistance cassette through duplications and by eliminating redundant cassettes. Importantly, we found no evidence of deleterious off-target effects of integrase activity. In summary, integrons accelerate resistance evolution by rapidly generating combinatorial variation in cassette composition while maintaining genomic integrity.202133634790
926710.9999Off-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 β-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.202336840554
926920.9999The Stringent Response Promotes Antibiotic Resistance Dissemination by Regulating Integron Integrase Expression in Biofilms. Class 1 integrons are genetic systems that enable bacteria to capture and express gene cassettes. These integrons, when isolated in clinical contexts, most often carry antibiotic resistance gene cassettes. They play a major role in the dissemination of antibiotic resistance among Gram-negative bacteria. The key element of integrons is the integrase, which allows gene cassettes to be acquired and shuffled. Planktonic culture experiments have shown that integrase expression is regulated by the bacterial SOS response. In natural settings, however, bacteria generally live in biofilms, which are characterized by strong antibiotic resilience and by increased expression of stress-related genes. Here, we report that under biofilm conditions, the stringent response, which is induced upon starvation, (i) increases basal integrase and SOS regulon gene expression via induction of the SOS response and (ii) exerts biofilm-specific regulation of the integrase via the Lon protease. This indicates that biofilm environments favor integron-mediated acquisition of antibiotic resistance and other adaptive functions encoded by gene cassettes. IMPORTANCE: Multidrug-resistant bacteria are becoming a worldwide health problem. Integrons are bacterial genetic platforms that allow the bacteria to capture and express gene cassettes. In clinical settings, integrons play a major role in the dissemination of antibiotic resistance gene cassettes among Gram-negative bacteria. Cassette capture is catalyzed by the integron integrase, whose expression is induced by DNA damage and controlled by the bacterial SOS response in laboratory planktonic cultures. In natural settings, bacteria usually grow in heterogeneous environments known as biofilms, which have very different conditions than planktonic cultures. Integrase regulation has not been investigated in biofilms. Our results showed that in addition to the SOS response, the stringent response (induced upon starvation) is specifically involved in the regulation of class 1 integron integrases in biofilms. This study shows that biofilms are favorable environments for integron-mediated acquisition/exchange of antibiotic resistance genes by bacteria and for the emergence of multidrug-resistant bacteria.201627531906
926830.9998The expression of integron arrays is shaped by the translation rate of cassettes. Integrons are key elements in the rise and spread of multidrug resistance in Gram-negative bacteria. These genetic platforms capture cassettes containing promoterless genes and stockpile them in arrays of variable length. In the current integron model, expression of cassettes is granted by the P(c) promoter in the platform and is assumed to decrease as a function of its distance. Here we explored this model using a large collection of 136 antibiotic resistance cassettes and show the effect of distance is in fact negligible. Instead, cassettes have a strong impact in the expression of downstream genes because their translation rate affects the stability of the whole polycistronic mRNA molecule. Hence, cassettes with reduced translation rates decrease the expression and resistance phenotype of cassettes downstream. Our data puts forward an integron model in which expression is contingent on the translation of cassettes upstream, rather than on the distance to the P(c).202439455579
927040.9998Activation of class 1 integron integrase is promoted in the intestinal environment. Class 1 integrons are widespread genetic elements playing a major role in the dissemination of antibiotic resistance. They allow bacteria to capture, express and exchange antibiotic resistance genes embedded within gene cassettes. Acquisition of gene cassettes is catalysed by the class 1 integron integrase, a site-specific recombinase playing a key role in the integron system. In in vitro planktonic culture, expression of intI1 is controlled by the SOS response, a regulatory network which mediates the repair of DNA damage caused by a wide range of bacterial stress, including antibiotics. However, in vitro experimental conditions are far from the real lifestyle of bacteria in natural environments such as the intestinal tract which is known to be a reservoir of integrons. In this study, we developed an in vivo model of intestinal colonization in gnotobiotic mice and used a recombination assay and quantitative real-time PCR, to investigate the induction of the SOS response and expression and activity of the class 1 integron integrase, IntI1. We found that the basal activity of IntI1 was higher in vivo than in vitro. In addition, we demonstrated that administration of a subinhibitory concentration of ciprofloxacin rapidly induced both the SOS response and intI1 expression that was correlated with an increase of the activity of IntI1. Our findings show that the gut is an environment in which the class 1 integron integrase is induced and active, and they highlight the potential role of integrons in the acquisition and/or expression of resistance genes in the gut, particularly during antibiotic therapy.202235482826
928450.9998The population and evolutionary dynamics of homologous gene recombination in bacterial populations. In bacteria, recombination is a rare event, not a part of the reproductive process. Nevertheless, recombination -- broadly defined to include the acquisition of genes from external sources, i.e., horizontal gene transfer (HGT) -- plays a central role as a source of variation for adaptive evolution in many species of bacteria. Much of niche expansion, resistance to antibiotics and other environmental stresses, virulence, and other characteristics that make bacteria interesting and problematic, is achieved through the expression of genes and genetic elements obtained from other populations of bacteria of the same and different species, as well as from eukaryotes and archaea. While recombination of homologous genes among members of the same species has played a central role in the development of the genetics and molecular biology of bacteria, the contribution of homologous gene recombination (HGR) to bacterial evolution is not at all clear. Also, not so clear are the selective pressures responsible for the evolution and maintenance of transformation, the only bacteria-encoded form of HGR. Using a semi-stochastic simulation of mutation, recombination, and selection within bacterial populations and competition between populations, we explore (1) the contribution of HGR to the rate of adaptive evolution in these populations and (2) the conditions under which HGR will provide a bacterial population a selective advantage over non-recombining or more slowly recombining populations. The results of our simulation indicate that, under broad conditions: (1) HGR occurring at rates in the range anticipated for bacteria like Streptococcus pneumoniae, Escherichia coli, Haemophilus influenzae, and Bacillus subtilis will accelerate the rate at which a population adapts to environmental conditions; (2) once established in a population, selection for this capacity to increase rates of adaptive evolution can maintain bacteria-encoded mechanisms of recombination and prevent invasion of non-recombining populations, even when recombination engenders a modest fitness cost; and (3) because of the density- and frequency-dependent nature of HGR in bacteria, this capacity to increase rates of adaptive evolution is not sufficient as a selective force to provide a recombining population a selective advantage when it is rare. Under realistic conditions, homologous gene recombination will increase the rate of adaptive evolution in bacterial populations and, once established, selection for higher rates of evolution will promote the maintenance of bacteria-encoded mechanisms for HGR. On the other hand, increasing rates of adaptive evolution by HGR is unlikely to be the sole or even a dominant selective pressure responsible for the original evolution of transformation.200919680442
931160.9998Various plasmid strategies limit the effect of bacterial restriction-modification systems against conjugation. In bacteria, genes conferring antibiotic resistance are mostly carried on conjugative plasmids, mobile genetic elements that spread horizontally between bacterial hosts. Bacteria carry defence systems that defend them against genetic parasites, but how effective these are against plasmid conjugation is poorly understood. Here, we study to what extent restriction-modification (RM) systems-by far the most prevalent bacterial defence systems-act as a barrier against plasmids. Using 10 different RM systems and 13 natural plasmids conferring antibiotic resistance in Escherichia coli, we uncovered variation in defence efficiency ranging from none to 105-fold protection. Further analysis revealed genetic features of plasmids that explain the observed variation in defence levels. First, the number of RM recognition sites present on the plasmids generally correlates with defence levels, with higher numbers of sites being associated with stronger defence. Second, some plasmids encode methylases that protect against restriction activity. Finally, we show that a high number of plasmids in our collection encode anti-restriction genes that provide protection against several types of RM systems. Overall, our results show that it is common for plasmids to encode anti-RM strategies, and that, as a consequence, RM systems form only a weak barrier for plasmid transfer by conjugation.202439413206
931270.9998Why There Are No Essential Genes on Plasmids. Mobile genetic elements such as plasmids are important for the evolution of prokaryotes. It has been suggested that there are differences between functions coded for by mobile genes and those in the "core" genome and that these differences can be seen between plasmids and chromosomes. In particular, it has been suggested that essential genes, such as those involved in the formation of structural proteins or in basic metabolic functions, are rarely located on plasmids. We model competition between genotypically varying bacteria within a single population to investigate whether selection favors a chromosomal location for essential genes. We find that in general, chromosomal locations for essential genes are indeed favored. This is because the inheritance of chromosomes is more stable than that for plasmids. We define the "degradation" rate as the rate at which chance genetic processes, for example, mutation, deletion, or translocation, render essential genes nonfunctioning. The only way in which plasmids can be a location for functioning essential genes is if chromosomal genes degrade faster than plasmid genes. If the two degradation rates are equal, or if plasmid genes degrade faster than chromosomal genes, functioning essential genes will be found only on chromosomes.201525540453
930680.9998Establishment Genes Present on pLS20 Family of Conjugative Plasmids Are Regulated in Two Different Ways. During conjugation, a conjugative DNA element is transferred from a donor to a recipient cell via a connecting channel. Conjugation has clinical relevance because it is the major route for spreading antibiotic resistance and virulence genes. The conjugation process can be divided into different steps. The initial steps carried out in the donor cell culminate in the transfer of a single DNA strand (ssDNA) of the conjugative element into the recipient cell. However, stable settlement of the conjugative element in the new host requires at least two additional events: conversion of the transferred ssDNA into double-stranded DNA and inhibition of the hosts' defence mechanisms to prevent degradation of the transferred DNA. The genes involved in this late step are historically referred to as establishment genes. The defence mechanisms of the host must be inactivated rapidly and-importantly-transiently, because prolonged inactivation would make the cell vulnerable to the attack of other foreign DNA, such as those of phages. Therefore, expression of the establishment genes in the recipient cell has to be rapid but transient. Here, we studied regulation of the establishment genes present on the four clades of the pLS20 family of conjugative plasmids harboured by different Bacillus species. Evidence is presented that two fundamentally different mechanisms regulate the establishment genes present on these plasmids. Identification of the regulatory sequences were critical in revealing the establishment regulons. Remarkably, whereas the conjugation genes involved in the early steps of the conjugation process are conserved and are located in a single large operon, the establishment genes are highly variable and organised in multiple operons. We propose that the mosaical distribution of establishment genes in multiple operons is directly related to the variability of defence genes encoded by the host bacterial chromosomes.202134946067
935590.9998Conjugative type IV secretion systems enable bacterial antagonism that operates independently of plasmid transfer. Bacterial cooperation and antagonism mediated by secretion systems are among the ways in which bacteria interact with one another. Here we report the discovery of an antagonistic property of a type IV secretion system (T4SS) sourced from a conjugative plasmid, RP4, using engineering approaches. We scrutinized the genetic determinants and suggested that this antagonistic activity is independent of molecular cargos, while we also elucidated the resistance genes. We further showed that a range of Gram-negative bacteria and a mixed bacterial population can be eliminated by this T4SS-dependent antagonism. Finally, we showed that such an antagonistic property is not limited to T4SS sourced from RP4, rather it can also be observed in a T4SS originated from another conjugative plasmid, namely R388. Our results are the first demonstration of conjugative T4SS-dependent antagonism between Gram-negative bacteria on the genetic level and provide the foundation for future mechanistic studies.202438664513
9307100.9998Integrons. Integrons are genetic elements able to acquire and rearrange open reading frames (ORFs) embedded in gene cassette units and convert them to functional genes by ensuring their correct expression. They were originally identified as a mechanism used by Gram-negative bacteria to collect antibiotic resistance genes and express multiple resistance phenotypes in synergy with transposons. More recently, their role has been broadened with the discovery of chromosomal integron (CI) structures in the genomes of hundreds of bacterial species. This review focuses on the resources carried in these elements, on their unique recombination mechanisms, and on the different mechanisms controlling the cassette dynamics. We discuss the role of the toxin/antitoxin (TA) cassettes for the stabilization of the large cassette arrays carried in the larger CIs, known as superintegrons. Finally, we explore the central role played by single-stranded DNA in the integron cassette dynamics in light of the recent discovery that the integron integrase expression is controlled by the SOS response.201020707672
3836110.9998Bacterial recombination promotes the evolution of multi-drug-resistance in functionally diverse populations. Bacterial recombination is believed to be a major factor explaining the prevalence of multi-drug-resistance (MDR) among pathogenic bacteria. Despite extensive evidence for exchange of resistance genes from retrospective sequence analyses, experimental evidence for the evolutionary benefits of bacterial recombination is scarce. We compared the evolution of MDR between populations of Acinetobacter baylyi in which we manipulated both the recombination rate and the initial diversity of strains with resistance to single drugs. In populations lacking recombination, the initial presence of multiple strains resistant to different antibiotics inhibits the evolution of MDR. However, in populations with recombination, the inhibitory effect of standing diversity is alleviated and MDR evolves rapidly. Moreover, only the presence of DNA harbouring resistance genes promotes the evolution of resistance, ruling out other proposed benefits for recombination. Together, these results provide direct evidence for the fitness benefits of bacterial recombination and show that this occurs by mitigation of functional interference between genotypes resistant to single antibiotics. Although analogous to previously described mechanisms of clonal interference among alternative beneficial mutations, our results actually highlight a different mechanism by which interactions among co-occurring strains determine the benefits of recombination for bacterial evolution.201222048956
4168120.9998Various pathways leading to the acquisition of antibiotic resistance by natural transformation. Natural transformation can lead to exchange of DNA between taxonomically diverse bacteria. In the case of chromosomal DNA, homology-based recombination with the recipient genome is usually necessary for heritable stability. In our recent study, we have shown that natural transformation can promote the transfer of transposons, IS elements, and integrons and gene cassettes, largely independent of the genetic relationship between the donor and recipient bacteria. Additional results from our study suggest that natural transformation with species-foreign DNA might result in the uptake of a wide range of DNA fragments; leading to changes in the antimicrobial susceptibility profile and contributing to the generation of antimicrobial resistance in bacteria.201223482877
9296130.9998Genome plasticity: insertion sequence elements, transposons and integrons, and DNA rearrangement. Living organisms are defined by the genes they possess. Control of expression of this gene set, both temporally and in response to the environment, determines whether an organism can survive changing conditions and can compete for the resources it needs to reproduce. Bacteria are no exception; changes to the genome will, in general, threaten the ability of the microbe to survive, but acquisition of new genes may enhance its chances of survival by allowing growth in a previously hostile environment. For example, acquisition of an antibiotic resistance gene by a bacterial pathogen can permit it to thrive in the presence of an antibiotic that would otherwise kill it; this may compromise clinical treatments. Many forces, chemical and genetic, can alter the genetic content of DNA by locally changing its nucleotide sequence. Notable for genetic change in bacteria are transposable elements and site-specific recombination systems such as integrons. Many of the former can mobilize genes from one replicon to another, including chromosome-plasmid translocation, thus establishing conditions for interspecies gene transfer. Balancing this, transposition activity can result in loss or rearrangement of DNA sequences. This chapter discusses bacterial DNA transfer systems, transposable elements and integrons, and the contributions each makes towards the evolution of bacterial genomes, particularly in relation to bacterial pathogenesis. It highlights the variety of phylogenetically distinct transposable elements, the variety of transposition mechanisms, and some of the implications of rearranging DNA, and addresses the effects of genetic change on the fitness of the microbe.200415148416
9282140.9998Could DNA uptake be a side effect of bacterial adhesion and twitching motility? DNA acquisition promotes the spread of resistance to antibiotics and virulence among bacteria. It is also linked to several natural phenomena including recombination, genome dynamics, adaptation and speciation. Horizontal DNA transfer between bacteria occurs via conjugation, transduction or competence for natural transformation by DNA uptake. Among these, competence is the only mechanism of transformation initiated and entirely controlled by the chromosome of the recipient bacteria. While the molecular mechanisms allowing the uptake of extracellular DNA are increasingly characterized, the function of competence for natural transformation by DNA uptake, the selective advantage maintaining it and the reasons why bacteria take up DNA in the first place are still debated. In this synthesis, I review some of the literature and discuss the four hypotheses on how and why do bacteria take up DNA. I argue that DNA uptake by bacteria is an accidental by-product of bacterial adhesion and twitching motility. Adhesion and motility are generally increased in stressful conditions, which may explain why bacteria increase DNA uptake in these conditions. In addition to its fundamental scientific relevance, the new hypothesis suggested here has significant clinical implications and finds further support from the fact that antibiotics sometimes fail to eliminate the targeted bacterium while inevitably causing stress to others. The widespread misuse of antibiotics may thus not only be selecting for resistant strains, but may also be causing bacteria to take up more DNA with the consequent increase in the chances of acquiring drug resistance and virulence-a scenario in full concordance with the previously reported induction of competence genes by antibiotics in Streptococcus pneumoniae and Legionella pneumophila.201323381940
9615150.9998Persistence and resistance as complementary bacterial adaptations to antibiotics. Bacterial persistence represents a simple of phenotypic heterogeneity, whereby a proportion of cells in an isogenic bacterial population can survive exposure to lethal stresses such as antibiotics. In contrast, genetically based antibiotic resistance allows for continued growth in the presence of antibiotics. It is unclear, however, whether resistance and persistence are complementary or alternative evolutionary adaptations to antibiotics. Here, we investigate the co-evolution of resistance and persistence across the genus Pseudomonas using comparative methods that correct for phylogenetic nonindependence. We find that strains of Pseudomonas vary extensively in both their intrinsic resistance to antibiotics (ciprofloxacin and rifampicin) and persistence following exposure to these antibiotics. Crucially, we find that persistence correlates positively to antibiotic resistance across strains. However, we find that different genes control resistance and persistence implying that they are independent traits. Specifically, we find that the number of type II toxin-antitoxin systems (TAs) in the genome of a strain is correlated to persistence, but not resistance. Our study shows that persistence and antibiotic resistance are complementary, but independent, evolutionary adaptations to stress and it highlights the key role played by TAs in the evolution of persistence.201626999656
9614160.9998Antibiotic-Independent Adaptive Effects of Antibiotic Resistance Mutations. Antibiotic usage selects for the accumulation and spread of antibiotic-resistant bacteria. However, resistance can also accumulate in the absence of antibiotic exposure. Antibiotics are often designed to target widely distributed regulatory housekeeping genes. The targeting of such genes enables these antibiotics to be useful against a wider variety of pathogens. This review highlights work suggesting that regulatory housekeeping genes of the type targeted by many antibiotics function as hubs of adaptation to conditions unrelated to antibiotic exposure. As a result of this, some mutations to the regulatory housekeeping gene targets of antibiotics confer both antibiotic resistance and an adaptive effect unrelated to antibiotic exposure. Such antibiotic-independent adaptive effects of resistance mutations may substantially affect the dynamics of antibiotic resistance accumulation and spread.201728629950
9259170.9998Static recipient cells as reservoirs of antibiotic resistance during antibiotic therapy. How does taking the full course of antibiotics prevent antibiotic resistant bacteria establishing in patients? We address this question by testing the possibility that horizontal/lateral gene transfer (HGT) is critical for the accumulation of the antibiotic-resistance phenotype while bacteria are under antibiotic stress. Most antibiotics prevent bacterial reproduction, some by preventing de novo gene expression. Nevertheless, in some cases and at some concentrations, the effects of most antibiotics on gene expression may not be irreversible. If the stress is removed before the bacteria are cleared from the patients by normal turnover, gene expression restarts, converting the residual population to phenotypic resistance. Using mathematical models we investigate how static recipients of resistance genes carried by plasmids accumulate resistance genes, and how specifically an environment cycling between presence and absence of the antibiotic uniquely favors the evolution of horizontally mobile resistance genes. We found that the presence of static recipients can substantially increase the persistence of the plasmid and that this effect is most pronounced when the cost of carriage of the plasmid decreases the cell's growth rate by as much as a half or more. In addition, plasmid persistence can be enhanced even when conjugation rates are as low as half the rate required for the plasmid to persist as a parasite on its own.200616723146
9283180.9998Vibrio cholerae: Measuring Natural Transformation Frequency. Many bacteria can become naturally competent to take up extracellular DNA across their outer and inner membranes by a dedicated competence apparatus. Whereas some studies show that the DNA delivered to the cytoplasm may be used for genome repair or for nutrition, it can also be recombined onto the chromosome by homologous recombination: a process called natural transformation. Along with conjugation and transduction, natural transformation represents a mechanism for horizontal transfer of genetic material, e.g., antibiotic resistance genes, which can confer new beneficial characteristics onto the recipient bacteria. Described here are protocols for quantifying the frequency of transformation for the human pathogen Vibrio cholerae, one of several Vibrio species recently shown to be capable of natural transformation.201425367272
3837190.9998Evolutionary Paths That Expand Plasmid Host-Range: Implications for Spread of Antibiotic Resistance. The World Health Organization has declared the emergence of antibiotic resistance to be a global threat to human health. Broad-host-range plasmids have a key role in causing this health crisis because they transfer multiple resistance genes to a wide range of bacteria. To limit the spread of antibiotic resistance, we need to gain insight into the mechanisms by which the host range of plasmids evolves. Although initially unstable plasmids have been shown to improve their persistence through evolution of the plasmid, the host, or both, the means by which this occurs are poorly understood. Here, we sought to identify the underlying genetic basis of expanded plasmid host-range and increased persistence of an antibiotic resistance plasmid using a combined experimental-modeling approach that included whole-genome resequencing, molecular genetics and a plasmid population dynamics model. In nine of the ten previously evolved clones, changes in host and plasmid each slightly improved plasmid persistence, but their combination resulted in a much larger improvement, which indicated positive epistasis. The only genetic change in the plasmid was the acquisition of a transposable element from a plasmid native to the Pseudomonas host used in these studies. The analysis of genetic deletions showed that the critical genes on this transposon encode a putative toxin-antitoxin (TA) and a cointegrate resolution system. As evolved plasmids were able to persist longer in multiple naïve hosts, acquisition of this transposon also expanded the plasmid's host range, which has important implications for the spread of antibiotic resistance.201626668183