Evolution of the Pseudomonas aeruginosa mutational resistome in an international Cystic Fibrosis clone

López-Causapé, Carla; Sommer, Lea Mette Madsen; Cabot, Gabriel; Rubio, Rosa; Ocampo-Sosa, Alain A.; Johansen, Helle Krogh; Figuerola, Joan; Cantón, Rafael; Kidd, Timothy J.; Molin, Søren; Oliver, Antonio

doi:10.1038/s41598-017-05621-5

Cited by 129 publications

(191 citation statements)

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“…To examine if the precise mutations found in our in vitro study were also found in clinical isolates, we searched published genomes of P. aeruginosa clinical isolates from cystic fibrosis patients who had likely been treated with aminoglycosides like TOB (Bolard et al, 2017; Chung et al, 2012; López-Causapé et al, 2017, 2018; Markussen et al, 2014). We identified fusA1 and ptsP mutations in these genomes, suggesting that these mutations evolve during infections.…”

Section: Resultsmentioning

confidence: 99%

Parallel evolution of tobramycin resistance across species and environments

Scribner

Santos-López

Marshall

et al. 2019

Preprint

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words)13 An important problem in evolution is identifying the genetic basis of how different species adapt 14 to similar environments. Understanding how various bacterial pathogens evolve in response to 15 antimicrobial treatment is a pressing example of this problem, where discovery of molecular 16 parallelism could lead to clinically useful predictions. Evolution experiments with pathogens in 17 environments containing antibiotics combined with periodic whole population genome 18 sequencing can be used to characterize the evolutionary dynamics of the pathways to 19 antimicrobial resistance. We separately propagated two clinically relevant Gram-negative 20 pathogens, Pseudomonas aeruginosa and Acinetobacter baumannii, in increasing 21 concentrations of tobramycin in two different environments each: planktonic and biofilm. 22Independent of the pathogen, populations adapted to tobramycin selection by parallel evolution 23 of mutations in fusA1, encoding elongation factor G, and ptsP, encoding phosphoenolpyruvate 24 phosphotransferase. As neither gene is a direct target of this aminoglycoside, both are relatively 25 novel and underreported causes of resistance. Additionally, both species acquired antibiotic-26 associated mutations that were more prevalent in the biofilm lifestyle than planktonic, in electron 27 transport chain components in A. baumannii and LPS biosynthesis enzymes in P. aeruginosa 28 populations. Using existing databases, we discovered both fusA1 and ptsP mutations to be 29 prevalent in antibiotic resistant clinical isolates. Additionally, we report site-specific parallelism of 30 fusA1 mutations that extend across several bacterial phyla. This study suggests that strong 31 selective pressures such as antibiotic treatment may result in high levels of predictability in 32 molecular targets of evolution despite differences between organisms' genetic background and 33 environment.The notion that evolution can be forecasted at the level of phenotype, gene, or even 36 amino acid is no longer a fantasy in the post-genomic era (Lässig et al., 2017). If we 37 acknowledge that most forecasting efforts rely on history to anticipate the future, the explosive 38 growth of whole-genome sequencing (WGS) sets the stage to resolve evolutionary phenomena 39 in action and suggest the next selected path. Among the best examples, bacterial populations 40 exposed to strong selection like antibiotics and analyzed by WGS are likely to identify gene 41 regions that produce resistance (Ahmed et al., 2018a; Cooper, 2018; Feng et al., 2016; Palmer 42 and Kishony, 2013). Repeated instances of the same antibiotic selection may enrich the same 43 types of mutations and ultimately enable some measure of predictability (Ibacache-Quiroga et 44 al., 2018; Wong et al., 2012). For instance, we can be confident that exposure of many bacteria 45 to high doses of fluoroquinolones like ciprofloxacin may select for substitutions in residues 83 or 46 87 of the drug target, DNA gyrase A (Fàbrega et al., 2009; Wong and Kassen, 2011).47 Furt...

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Section: Resultsmentioning

confidence: 99%

Parallel evolution of tobramycin resistance across species and environments

Scribner

Santos-López

Marshall

et al. 2019

Preprint

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“…Interestingly, in contrast to the 1995 isolate, the 2002 and all the 2011 isolates showed susceptibility to piperacillin-tazobactam, resistance to which seems to have been lost after the acquisition of resistance to tobramycin, thus suggesting collateral sensitivity to penicillin-type-β-lactams as previously described by Barbosa et al (29). On the other hand, even though colistin was used from 2004, all isolates showed MIC values ranging from 8 to 32 µg/ml, which are relatively high compared to other data sets from clinical isolates (27, 28, 30, 31) (Fig. 2).…”

Section: Resultsmentioning

confidence: 68%

“…In order to investigate the molecular bases of the antimicrobial resistance observed in CFD isolates, we explored the acquisition of mutations in a set of 168 chromosomal genes, here defined as the resistome, which have been described to be involved in P. aeruginosa antibiotic resistance mechanisms (27, 28, 32). Thus, using the 1991 genome as reference, we analyzed the distribution of a total of 5710 SNPs and 1078 indels accumulated in a period of 20 years of infection, which we have previously detected in the collection of isolates by WGS analysis (24).…”

Section: Resultsmentioning

confidence: 99%

“…Recent advances in whole-genome sequencing (WGS) techniques have provided insights into the evolutionary trajectories of adaptation of P. aeruginosa to the CF environment, particularly with regard to patho-adaptive mutations, such as those associated with antibiotic resistance (11, 21-26). In this sense, “the mutational resistome” was recently defined as the set of mutations involved in modulation of antibiotic resistance levels in absence of horizontal gene transfer (27, 28).…”

Section: Introductionmentioning

confidence: 99%

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HypermutatorPseudomonas aeruginosaexploits multiple genetic pathways to develop multidrug resistance during long-term infections in the airways of cystic fibrosis patients

Colque

Orio

Feliziani

et al. 2019

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21 cystic fibrosis 22 WORD COUNT ABSTRACT: Abstract, 249 words. Importance, 147 words 23 WORD COUNT TEXT: 4995 words.24 2 25 ABSTRACT 26 Pseudomonas aeruginosa exploits intrinsic and acquired resistance mechanisms to resist almost 27 every antibiotic used in chemotherapy. Antimicrobial resistance in P. aeruginosa isolated from 28 cystic fibrosis (CF) patients is further enhanced by the occurrence of hypermutator strains, a 29 hallmark of chronic CF infections. However, the within-patient genetic diversity of P. aeruginosa 30 populations related to antibiotic resistance remains unexplored. Here, we show the evolution of 31 the mutational resistome profile of a P. aeruginosa hypermutator lineage by performing 32 longitudinal and transversal analyses of isolates collected from a CF patient throughout 20 years 33 of chronic infection. Our results show the accumulation of thousands of mutations with an overall 34 evolutionary history characterized by purifying selection. However, mutations in antibiotic 35resistance genes appear to be positively selected, driven by antibiotic treatment. Antibiotic 36 resistance increased as infection progressed towards the establishment of a population constituted 37 by genotypically diversified coexisting sub-lineages, all of which converged to multi-drug 38 resistance. These sub-lineages emerged by parallel evolution through distinct evolutionary 39 pathways, which affected genes of the same functional categories. Interestingly, ampC and fstI, 40 encoding the β-lactamase and penicillin-binding protein 3, respectively, were found among the 41 most frequently mutated genes. In fact, both genes were targeted by multiple independent 42 mutational events, which led to a wide diversity of coexisting alleles underlying β-lactam 43 resistance. Our findings indicate that hypermutators, apart from boosting antibiotic resistance 44 evolution by simultaneously targeting several genes, favor the emergence of adaptive innovative 45 alleles by clustering beneficial/compensatory mutations in the same gene, hence expanding P. 46 aeruginosa strategies for persistence. 47 IMPORTANCE 483 By increasing mutation rates, hypermutators boost antibiotic resistance evolution by enabling 49 bacterial pathogens to fully exploit their genetic potential and achieve resistance mechanisms for 50 almost every known antimicrobial agent. Here, we show how co-existing clones from a P. 51 aeruginosa hypermutator lineage that evolved during 20 years of chronic infection and antibiotic 52 chemotherapy, converged to multidrug resistance by targeting genes from alternative genetic 53 pathways that are part of the broad P. aeruginosa resistome. Within this complex assembly of 54 combinatorial genetic changes, in some specific cases, multiple mutations are needed in the same 55 gene to reach a fine tuned resistance phenotype. Hypermutability enables this genetic edition 56 towards higher resistance profiles by recurrently targeting these genes, thus promoting new 57 epistatic relationships and the emergence of innovativ...

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“…Studies have confirmed the involvement of genes previously proposed to be associated with resistance in P. aeruginosa. These include the fusA1 gene that encodes the EFG-1 protein and is associated with aminoglycoside resistance (27,29,30) and the ftsI gene that encodes a penicillin binding protein and is associated with carbapenem resistance (30)(31)(32)(33)(34). This approach has high potential for identifying previously unknown antibiotic resistanceassociated mutations and genes, but some key issues remain to be addressed.…”

Section: Introductionmentioning

confidence: 99%

A large-scale comparison shows that genetic changes causing antibiotic resistance in experimentally evolvedPseudomonas aeruginosapredict those in naturally evolved bacteria

Wardell¹,

Rehman²,

Martin³

et al. 2019

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Pseudomonas 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 experimentallyevolved 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.

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Evolution of the Pseudomonas aeruginosa mutational resistome in an international Cystic Fibrosis clone

Cited by 129 publications

References 65 publications

Parallel evolution of tobramycin resistance across species and environments

Parallel evolution of tobramycin resistance across species and environments

HypermutatorPseudomonas aeruginosaexploits multiple genetic pathways to develop multidrug resistance during long-term infections in the airways of cystic fibrosis patients

A large-scale comparison shows that genetic changes causing antibiotic resistance in experimentally evolvedPseudomonas aeruginosapredict those in naturally evolved bacteria

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