scholarly journals Quantification of Plasmid-Mediated Antibiotic Resistance in an Experimental Evolution Approach

10.3791/60749 ◽  
2019 ◽  
Author(s):  
Tanita Wein ◽  
Fenna T. Stücker ◽  
Nils F. Hülter ◽  
Tal Dagan
Keyword(s):  
Antibiotic Resistance ◽  
Experimental Evolution ◽  
Evolution Approach

scholarly journals Evolution of Cost-Free Resistance under Fluctuating Drug Selection in Pseudomonas aeruginosa

mSphere ◽  
2017 ◽  
Vol 2 (4) ◽  
Author(s):  
Anita H. Melnyk ◽  
Nicholas McCloskey ◽  
Aaron J. Hinz ◽  
Jeremy Dettman ◽  
Rees Kassen
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Antibiotic Treatment ◽  
Experimental Evolution ◽  
Public Health Problem ◽  
Content Type ◽  
Compensatory Evolution ◽  
High Fitness ◽  
Treatment Conditions ◽  
Resistant Strains

ABSTRACT Antibiotic resistance is a global problem that greatly impacts human health. How resistance persists, even in the absence of antibiotic treatment, is thus a public health problem of utmost importance. In this study, we explored the antibiotic treatment conditions under which cost-free resistance arises, using experimental evolution of the bacterium Pseudomonas aeruginosa and the quinolone antibiotic ciprofloxacin. We found that intermittent antibiotic treatment led to the evolution of cost-free resistance and demonstrate that compensatory evolution is the mechanism responsible for cost-free resistance. Our results suggest that discontinuous administration of antibiotic may be contributing to the high levels of antibiotic resistance currently found worldwide. Antibiotic resistance evolves rapidly in response to drug selection, but it can also persist at appreciable levels even after the removal of the antibiotic. This suggests that many resistant strains can both be resistant and have high fitness in the absence of antibiotics. To explore the conditions under which high-fitness, resistant strains evolve and the genetic changes responsible, we used a combination of experimental evolution and whole-genome sequencing to track the acquisition of ciprofloxacin resistance in the opportunistic pathogen Pseudomonas aeruginosa under conditions of constant and fluctuating antibiotic delivery patterns. We found that high-fitness, resistant strains evolved readily under fluctuating but not constant antibiotic conditions and that their evolution was underlain by a trade-off between resistance and fitness. Whole-genome sequencing of evolved isolates revealed that resistance was gained through mutations in known resistance genes and that second-site mutations generally compensated for costs associated with resistance in the fluctuating treatment, leading to the evolution of cost-free resistance. Our results suggest that current therapies involving intermittent administration of antibiotics are contributing to the maintenance of antibiotic resistance at high levels in clinical settings. IMPORTANCE Antibiotic resistance is a global problem that greatly impacts human health. How resistance persists, even in the absence of antibiotic treatment, is thus a public health problem of utmost importance. In this study, we explored the antibiotic treatment conditions under which cost-free resistance arises, using experimental evolution of the bacterium Pseudomonas aeruginosa and the quinolone antibiotic ciprofloxacin. We found that intermittent antibiotic treatment led to the evolution of cost-free resistance and demonstrate that compensatory evolution is the mechanism responsible for cost-free resistance. Our results suggest that discontinuous administration of antibiotic may be contributing to the high levels of antibiotic resistance currently found worldwide.

scholarly journals Genomic Adaptations to the Loss of a Conserved Bacterial DNA Methyltransferase

mBio ◽  
2015 ◽  
Vol 6 (4) ◽  
Author(s):  
Diego Gonzalez ◽  
Justine Collier
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Experimental Evolution ◽  
Dna Methyltransferase ◽  
Metabolic Regulation ◽  
Caulobacter Crescentus ◽  
Point Mutations ◽  
Rich Medium ◽  
Content Type ◽  
Evolution Approach

ABSTRACTCcrM is an orphan DNA methyltransferase nearly universally conserved in a vast group ofAlphaproteobacteria.InCaulobacter crescentus, it controls the expression of key genes involved in the regulation of the cell cycle and cell division. Here, we demonstrate, using an experimental evolution approach, thatC. crescentuscan significantly compensate, through easily accessible genetic changes like point mutations, the severe loss in fitness due to the absence of CcrM, quickly improving its growth rate and cell morphology in rich medium. By analyzing the compensatory mutations genome-wide in 12 clones sampled from independent ΔccrMpopulations evolved for ~300 generations, we demonstrated that each of the twelve clones carried at least one mutation that potentially stimulatedftsZexpression, suggesting that the low intracellular levels of FtsZ are the major burden of ΔccrMmutants. In addition, we demonstrate that the phosphoenolpyruvate-carbohydrate phosphotransfer system (PTS) actually modulatesftsZandmipZtranscription, uncovering a previously unsuspected link between metabolic regulation and cell division inAlphaproteobacteria. We present evidence that point mutations found in genes encoding proteins of the PTS provide the strongest fitness advantage to ΔccrMcells cultivated in rich medium despite being disadvantageous in minimal medium. This environmental sign epistasis might prevent such mutations from getting fixed under changing natural conditions, adding a plausible explanation for the broad conservation of CcrM.IMPORTANCEIn bacteria, DNA methylation has a variety of functions, including the control of DNA replication and/or gene expression. The cell cycle-regulated DNA methyltransferase CcrM modulates the transcription of many genes and is critical for fitness inCaulobacter crescentus. Here, we used an original experimental evolution approach to determine which of its many targets make CcrM so important physiologically. We show that populations lacking CcrM evolve quickly, accumulating an excess of mutations affecting, directly or indirectly, the expression of theftsZcell division gene. This finding suggests that the most critical function of CcrM inC. crescentusis to promote cell division by enhancing FtsZ intracellular levels. During this work, we also discovered an unexpected link between metabolic regulation and cell division that might extend to otherAlphaproteobacteria.

Experimental evolution as an efficient tool to dissect adaptive paths to antibiotic resistance

2013 ◽  
Vol 16 (6) ◽  
pp. 96-107 ◽  
Author(s):  
Gunther Jansen ◽  
Camilo Barbosa ◽  
Hinrich Schulenburg
Keyword(s):  
Antibiotic Resistance ◽  
Experimental Evolution ◽  
Efficient Tool

scholarly journals A large-scale comparison shows that genetic changes causing antibiotic resistance in experimentally evolved Pseudomonas aeruginosa predict those in naturally evolved bacteria

2019 ◽  
Author(s):  
Samuel J. T. Wardell ◽  
Attika Rehman ◽  
Lois W. Martin ◽  
Craig Winstanley ◽  
Wayne M. Patrick ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Antibiotic Resistance ◽  
Experimental Evolution ◽  
Large Scale ◽  
Genetic Basis ◽  
Clinical Isolates ◽  
Resistant Bacteria ◽  
Antibiotic Resistant ◽  
Health Crisis ◽  
Wide Range

AbstractPseudomonas aeruginosa is an opportunistic pathogen that causes a wide range of acute and chronic infections. An increasing number of isolates have acquired mutations that make them antibiotic resistant, making treatment more difficult. To identify resistance-associated mutations we experimentally evolved the antibiotic sensitive strain P. aeruginosa PAO1 to become resistant to three widely used anti-pseudomonal antibiotics, ciprofloxacin, meropenem and tobramycin. Mutants were able to tolerate up to 2048-fold higher concentrations of antibiotic than strain PAO1. Genome sequences were determined for thirteen mutants for each antibiotic. Each mutant had between 2 and 8 mutations. There were at least 8 genes mutated in more than one mutant per antibiotic, demonstrating the complexity of the genetic basis of resistance. Additionally, large deletions of up to 479kb arose in multiple meropenem resistant mutants. For all three antibiotics mutations arose in genes known to be associated with resistance, but also in genes not previously associated with resistance. To determine the clinical relevance of mutations uncovered in experimentally-evolved mutants we analysed the corresponding genes in 457 isolates of P. aeruginosa from patients with cystic fibrosis or bronchiectasis as well as 172 isolates from the general environment. Many of the genes identified through experimental evolution had changes predicted to be function-altering in clinical isolates but not in isolates from the general environment, showing that mutated genes in experimentally evolved bacteria can predict those that undergo mutation during infection. These findings expand understanding of the genetic basis of antibiotic resistance in P. aeruginosa as well as demonstrating the validity of experimental evolution in identifying clinically-relevant resistance-associated mutations.ImportanceThe rise in antibiotic resistant bacteria represents an impending global health crisis. As such, understanding the genetic mechanisms underpinning this resistance can be a crucial piece of the puzzle to combatting it. The importance of this research is that by experimentally evolving P. aeruginosa to three clinically relevant antibiotics, we have generated a catalogue of genes that can contribute to resistance in vitro. We show that many (but not all) of these genes are clinically relevant, by identifying variants in clinical isolates of P. aeruginosa. This research furthers our understanding of the genetics leading to resistance in P. aeruginosa and provides tangible evidence that these genes can play a role clinically, potentially leading to new druggable targets or inform therapies.

scholarly journals Mammalian ANP32A and ANP32B proteins drive alternative avian influenza virus polymerase adaptations

2020 ◽  
Author(s):  
Thomas. P. Peacock ◽  
Carol M. Sheppard ◽  
Ecco Staller ◽  
Rebecca Frise ◽  
Olivia C. Swann ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Avian Influenza ◽  
Avian Influenza Virus ◽  
Experimental Evolution ◽  
Human Cells ◽  
Influenza Viruses ◽  
Avian Viruses ◽  
Evolution Approach ◽  
Influenza Polymerase

AbstractANP32 proteins, which act as influenza polymerase co-factors, vary between birds and mammals. The well-known mammalian adaptation, PB2-E627K, enables influenza polymerase to use mammalian ANP32 proteins. However, some mammalian-adapted influenza viruses do not harbour this adaptation. Here, we show that alternative PB2 adaptations, Q591R and D701N also allow influenza polymerase to use mammalian ANP32 proteins. PB2-E627K strongly favours use of mammalian ANP32B proteins, whereas D701N shows no such bias. Accordingly, PB2-E627K adaptation emerges in species with strong pro-viral ANP32B proteins, such as humans and mice, while D701N is more commonly seen in isolates from swine, dogs and horses where ANP32A proteins are more strongly pro-viral. In an experimental evolution approach, passage of avian viruses in human cells drives acquisition of PB2-E627K, but not when ANP32B is ablated. The strong pro-viral support of ANP32B for PB2-E627K maps to the LCAR region of ANP32B.

Experimental Evolution of Antibiotic Resistance in Bacteria

2007 ◽  
Vol 69 (2) ◽  
pp. 94-97 ◽  
Author(s):  
Amy C. Krist ◽  
Sasha A. Showsh
Keyword(s):  
Antibiotic Resistance ◽  
Experimental Evolution

scholarly journals Complex interactions with females and rival males limit the evolution of sperm offence and defence

2007 ◽  
Vol 274 (1619) ◽  
pp. 1779-1788 ◽  
Author(s):  
Adam Bjork ◽  
William T Starmer ◽  
Dawn M Higginson ◽  
Christopher J Rhodes ◽  
Scott Pitnick
Keyword(s):  
Genetic Variation ◽  
Experimental Evolution ◽  
Complex Nature ◽  
Sperm Precedence ◽  
Response To Selection ◽  
Outbred Population ◽  
Complex Interactions ◽  
Component Traits ◽  
Rival Male ◽  
Evolution Approach

Postcopulatory sexual selection favours males which are strong offensive and defensive sperm competitors. As a means of identifying component traits comprising each strategy, we used an experimental evolution approach. Separate populations of Drosophila melanogaster were selected for enhanced sperm offence and defence. Despite using a large outbred population and evidence of substantive genetic variation for each strategy, neither trait responded to selection in the two replicates of this experiment. Recent work with fixed chromosome lines of D. melanogaster suggests that complex genotypic interactions between females and competing males contribute to the maintenance of this variation. To determine whether such interactions could explain our lack of response to selection on sperm offence and defence, we quantified sperm precedence across multiple sperm competition bouts using an outbred D. melanogaster population exhibiting continuous genetic variation. Both offensive and defensive sperm competitive abilities were found to be significantly repeatable only across matings involving ejaculates of the same pair of males competing within the same female. These repeatabilities decreased when the rival male stayed the same but the female changed, and they disappeared when both the rival male and the female changed. Our results are discussed with a focus on the complex nature of sperm precedence and the maintenance of genetic variation in ejaculate characteristics.

scholarly journals Phenotypic Suppression of Streptomycin Resistance by Mutations in Multiple Components of the Translation Apparatus

2015 ◽  
Vol 197 (18) ◽  
pp. 2981-2988 ◽  
Author(s):  
Jennifer F. Carr ◽  
Hannah J. Lee ◽  
Joshua B. Jaspers ◽  
Albert E. Dahlberg ◽  
Gerwald Jogl ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Long Range ◽  
Experimental Evolution ◽  
Thermus Thermophilus ◽  
Thermophilic Bacterium ◽  
Streptomycin Resistance ◽  
Resistance Mutations ◽  
Content Type ◽  
Translation Apparatus ◽  
Antibiotic Resistance Mutations

ABSTRACTThe bacterial ribosome and its associated translation factors are frequent targets of antibiotics, and antibiotic resistance mutations have been found in a number of these components. Such mutations can potentially interact with one another in unpredictable ways, including the phenotypic suppression of one mutation by another. These phenotypic interactions can provide evidence of long-range functional interactions throughout the ribosome and its functional complexes and potentially give insights into antibiotic resistance mechanisms. In this study, we used genetics and experimental evolution of the thermophilic bacteriumThermus thermophilusto examine the ability of mutations in various components of the protein synthesis apparatus to suppress the streptomycin resistance phenotypes of mutations in ribosomal protein S12, specifically those located distant from the streptomycin binding site. With genetic selections and strain constructions, we identified suppressor mutations in EF-Tu or in ribosomal protein L11. Using experimental evolution, we identified amino acid substitutions in EF-Tu or in ribosomal proteins S4, S5, L14, or L19, some of which were found to also relieve streptomycin resistance. The wide dispersal of these mutations is consistent with long-range functional interactions among components of the translational machinery and indicates that streptomycin resistance can result from the modulation of long-range conformational signals.IMPORTANCEThe thermophilic bacteriumThermus thermophilushas become a model system for high-resolution structural studies of macromolecular complexes, such as the ribosome, while its natural competence for transformation facilitates genetic approaches. Genetic studies ofT. thermophilusribosomes can take advantage of existing high-resolution crystallographic information to allow a structural interpretation of phenotypic interactions among mutations. Using a combination of genetic selections, strain constructions, and experimental evolution, we find that certain mutations in the translation apparatus can suppress the phenotype of certain antibiotic resistance mutations. Suppression of resistance can occur by mutations located distant in the ribosome or in a translation factor. These observations suggest the existence of long-range conformational signals in the translating ribosome, particularly during the decoding of mRNA.

scholarly journals Shared and Unique Evolutionary Trajectories to Ciprofloxacin Resistance in Gram-Negative Bacterial Pathogens

mBio ◽  
2021 ◽  
Author(s):  
Jaime E. Zlamal ◽  
Semen A. Leyn ◽  
Mallika Iyer ◽  
Marinela L. Elane ◽  
Nicholas A. Wong ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Experimental Evolution ◽  
Bacterial Pathogens ◽  
Genome Rearrangements ◽  
Microbial Populations ◽  
Gram Negative ◽  
Evolutionary Trajectories ◽  
Ciprofloxacin Resistance

The challenge of spreading antibiotic resistance calls for systematic efforts to develop more “irresistible” drugs based on a deeper understanding of dynamics and mechanisms of antibiotic resistance acquisition. To address this challenge, we have established a comparative resistomics approach which combines experimental evolution in a continuous-culturing device, the morbidostat, with ultradeep sequencing of evolving microbial populations to identify evolutionary trajectories (mutations and genome rearrangements) leading to antibiotic resistance over a range of target pathogens.

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