Deciphering the Resistome of the Widespread Pseudomonas aeruginosa Sequence Type 175 International High-Risk Clone through Whole-Genome Sequencing

Cabot, Gabriel; López-Causapé, Carla; Ocampo-Sosa, Alain A.; Sommer, Lea Mette Madsen; Domı́nguez, M.A.; Zamorano, Laura; Juan, Carlos; Tubau, Fé; Rodríguez, C.; Moyá, Bartolomé; Peña, Carmen; Martínez-Martínez, Luis; Plésiat, Patrick

doi:10.1128/aac.01720-16

Cited by 104 publications

(96 citation statements)

References 52 publications

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“…Briefly, indexed paired-end libraries were generated from genomic DNA using a commercial library preparation kit (Nextera XT, Illumina, USA) and sequenced on an Illumina MiSeq benchtop sequencer with a MiSeq reagent kit (version 2). Obtained pairedended reads were aligned to the P. aeruginosa PAO1 reference genome, and sequence variation was further analyzed for the 146 chromosomal genes related to antimicrobial resistance (13). The presence of horizontally acquired resistance determinants was further explored using online databases (https://cge.cbs.dtu.dk//services/ResFinder/).…”

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confidence: 99%

“…Moreover, sequence variation on 146 chromosomal genes related to antimicrobial resistance was evaluated as previously described (13). Briefly, indexed paired-end libraries were generated from genomic DNA using a commercial library preparation kit (Nextera XT, Illumina, USA) and sequenced on an Illumina MiSeq benchtop sequencer with a MiSeq reagent kit (version 2).…”

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confidence: 99%

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In Vivo Emergence of Resistance to Novel Cephalosporin–β-Lactamase Inhibitor Combinations through the Duplication of Amino Acid D149 from OXA-2 β-Lactamase (OXA-539) in Sequence Type 235 Pseudomonas aeruginosa

Fraile-Ribot

Mulet

Cabot

et al. 2017

Antimicrob Agents Chemother

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Resistance development to novel cephalosporin-␤-lactamase inhibitor combinations during ceftazidime treatment of a surgical infection by Pseudomonas aeruginosa was investigated. Both initial (97C2) and final (98G1) isolates belonged to the high-risk clone sequence type (ST) 235 and were resistant to carbapenems (oprD), fluoroquinolones (GyrA-T83I, ParC-S87L), and aminoglycosides (aacA7/aacA8/aadA6). 98G1 also showed resistance to ceftazidime, ceftazidime-avibactam, and ceftolozanetazobactam. Sequencing identified bla OXA-2 in 97C2, but 98G1 contained a 3-bp insertion leading to the duplication of the key residue D149 (designated OXA-539). Evaluation of PAO1 transformants producing cloned OXA-2 or OXA-539 confirmed that D149 duplication was the cause of resistance. Active surveillance of the emergence of resistance to these new valuable agents is warranted.KEYWORDS extended-spectrum OXA, Pseudomonas aeruginosa, multidrug resistance, ceftolozane-tazobactam, ceftazidime-avibactam T he increasing prevalence of nosocomial infections produced by multidrug-resistant (MDR) or extensively drug-resistant (XDR) Pseudomonas aeruginosa strains severely compromises the selection of appropriate treatments and is therefore associated with significant morbidity and mortality (1-3). This growing threat results from the extraordinary capacity of this pathogen to develop resistance to nearly all available antibiotics by the selection of mutations in chromosomal genes and from the increasing prevalence of transferable resistance determinants, particularly those encoding class B carbapenemases (metallo-␤-lactamases [MBLs]) or extended-spectrum ␤-lactamases, frequently cotransferred with genes encoding aminoglycoside-modifying enzymes (4, 5). The emergence of MDR/XDR global clones, deemed high-risk clones, disseminated in several hospitals worldwide adds further concern (6, 7).The recent introduction of novel ␤-lactam-␤-lactamase inhibitor combinations, namely, ceftolozane-tazobactam and ceftazidime-avibactam, which are stable against most mutational resistance mechanisms, including the overexpression of the chromosomal cephalosporinase AmpC, partially alleviates the urgent clinical need for new agents that combat infections by MDR/XDR P. aeruginosa (8-10). Thus, the potential emergence of resistance to these antibiotics is of particular concern. Therefore, we report here the characterization of the resistance mechanisms of a P. aeruginosa clinical In vivo emergence of resistance to novel cephalosporin-β-lactamase inhibitor combinations through the duplication of amino acid D149 from OXA-2 β-lactamase (OXA-539) in sequence type 235 Pseudomonas aeruginosa. Antimicrob Agents Chemother

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confidence: 99%

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In Vivo Emergence of Resistance to Novel Cephalosporin–β-Lactamase Inhibitor Combinations through the Duplication of Amino Acid D149 from OXA-2 β-Lactamase (OXA-539) in Sequence Type 235 Pseudomonas aeruginosa

Fraile-Ribot

Mulet

Cabot

et al. 2017

Antimicrob Agents Chemother

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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%

“…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%

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

Preprint

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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

Preprint

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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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Deciphering the Resistome of the Widespread Pseudomonas aeruginosa Sequence Type 175 International High-Risk Clone through Whole-Genome Sequencing

Cited by 104 publications

References 52 publications

In Vivo Emergence of Resistance to Novel Cephalosporin–β-Lactamase Inhibitor Combinations through the Duplication of Amino Acid D149 from OXA-2 β-Lactamase (OXA-539) in Sequence Type 235 Pseudomonas aeruginosa

In Vivo Emergence of Resistance to Novel Cephalosporin–β-Lactamase Inhibitor Combinations through the Duplication of Amino Acid D149 from OXA-2 β-Lactamase (OXA-539) in Sequence Type 235 Pseudomonas aeruginosa

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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