Evolved Aztreonam Resistance Is Multifactorial and Can Produce Hypervirulence in
            <i>Pseudomonas aeruginosa</i>

Jorth, Peter; McLean, Kathryn; Ratjen, Anina; Secor, Patrick R.; Bautista, Gilbert E.; Ravishankar, Sumedha; Rezayat, Arash Akhavan; Garudathri, Jayanthi; Harrison, Joe J.; Harwood, Rachel A; Penewit, Kelsi; Waalkes, Adam; Singh, Pradeep Kumar; Salipante, Stephen J.

doi:10.1128/mbio.00517-17

Cited by 75 publications

(121 citation statements)

References 81 publications

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“…Two other elements that are selected for in both treatments are ftsI and clpA. The first encodes PBP3, the target of different β-lactams (28, 29), which has been already found to be mutated in numerous resistant P. aeruginosa isolates. Indeed, the mutations R504C/H found in populations 1 and 5 are also present among isolates from widespread nosocomial P. aeruginosa clones (29-31).…”

Section: Resultsmentioning

confidence: 99%

Mutation-driven evolution of Pseudomonas aeruginosa in the presence of either ceftazidime or ceftazidime/avibactam

Sanz-García

Hernando-Amado

Martínez

2018

Preprint

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12Ceftazidime/avibactam is a combination of beta-lactam/beta-lactamases inhibitor, which 13 use is restricted to some clinical cases including cystic fibrosis patients infected with 14 multidrug resistant Pseudomonas aeruginosa, in which mutation is the main driver of 15 resistance. This study aims to predict the mechanisms of mutation-driven resistance that 16 are selected for when P. aeruginosa is challenged with either ceftazidime or 17 ceftazidime/avibactam. For this purpose, P. aeruginosa PA14 was submitted to 18 experimental evolution in the absence of antibiotics and in the presence of increasing 19 concentrations of ceftazidime or ceftazidime/avibactam for 30 consecutive days. Final 20 populations were analysed by whole-genome sequencing. All evolved populations 21 reached similar levels of ceftazidime resistance. Besides, all of them were more 22 susceptible to amikacin and produced pyomelanin. A first event in the evolution was the 23 selection of large chromosomal deletions containing hmgA (involved in pyomelanin 24 production), galU (involved in β-lactams resistance) and mexXY-oprM (involved in 25 aminoglycoside resistance). Besides mutations in mpl and dacB that regulate β-26 lactamase expression, mutations related to MexAB-OprM overexpression were 27 prevalent. Ceftazidime/avibactam challenge selected mutants in the putative efflux 28 pump PA14_45890-45910 and in a two-component system (PA14_45870-45880), likely 29 regulating its expression. All populations produce pyomelanin and were more 30 susceptible to aminoglycosides likely due to the selection of large chromosomal 31 deletions. Since pyomelanin-producing mutants, presenting similar deletions are 32 regularly isolated from infections, the potential aminoglycosides hyper-susceptiblity and 33 reduced β-lactams susceptibility of pyomelanin-producing P. aeruginosa should be 34 taken into consideration for treating infections by these isolates. 35 42 including those encoding multidrug (MDR) efflux pumps (4) and beta-lactamases. In 43 addition, an increasing number of P. aeruginosa isolates has acquired several resistance 44 genes through horizontal gene transfer (HGT), including different classes of 45carbapenemases. Finally, P. aeruginosa is able to develop resistance through mutation, 46 particularly when causing chronic infections, to nearly any available antibiotic. Under 47 this situation, the emergence and spread of MDR resistant global clones is of special 48 concern (5). 49The use of β-lactam/β-lactamase inhibitor combinations, such as amoxicillin/clavulanic 50 acid or ceftolozane/tazobactam, has proven to be effective against class A β-lactamases 51 (which include narrow and extended-spectrum β-lactamases and some 52 carbapenemases); whereas effective combinations against class B, C (extended 53 spectrum cephalosporinases) and D β-lactamases (6-8) have not been available until 54 recently. One of them is the ceftazidime/avibactam combination, whose use was 55 approved in 2015 by the FDA (9). 56 Avibactam, formerly known as NXL104, be...

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Section: Resultsmentioning

confidence: 99%

Mutation-driven evolution of Pseudomonas aeruginosa in the presence of either ceftazidime or ceftazidime/avibactam

Sanz-García

Hernando-Amado

Martínez

2018

Preprint

View full text Add to dashboard Cite

show abstract

“…Some of these mutations (Y367C, H394R, N427S, Q458R, Q475R, R504L, V523A, V523M and F533L) are located in the transpeptidase β-lactam binding site of the protein, and the latter mutation has been shown to play a key role in β-lactam recognition (42). Importantly, these mutations have been documented to emerge among P. aeruginosa CF collections (28, 43, 44) and upon aztreonam exposure in vitro (45), whereas other mutations are described for the first time in this work (Table S1). The rest of the PBP encoding genes showed few mutations among the CFD collection.…”

Section: Resultsmentioning

confidence: 74%

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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“…Ten of the mutants had mutations in nalC, nalD or mexR, with mutations in these genes causing upregulation of the efflux pumps and being associated with β-lactam resistance (28,(40)(41)(42). Mutations in ftsI, that encodes the meropenem binding protein PBP3A, were present in 3 mutants consistent with the known role of such mutations in resistance (24,43). Mutations were also present in genes not commonly associated with meropenem resistance.…”

Section: Meropenem-selected Mutantsmentioning

confidence: 71%

“…Importantly, experimentally evolved mutants in this study also had mutations in genes not usually associated with resistance. These include PA3491 and pilin-encoding genes in ciprofloxacin-selected mutants; aroB and tRNA ligases in meropenem-selected mutants (39,43); and PA1767, cco and wbpL in tobramycin-selected mutants. The occurrence of mutations in these genes in multiple independently-evolved lines ( Table 1, Table S1) strongly suggests that the mutations increase antibiotic tolerance in our selection system.…”

Section: Discussionmentioning

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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Evolved Aztreonam Resistance Is Multifactorial and Can Produce Hypervirulence in Pseudomonas aeruginosa

Cited by 75 publications

References 81 publications

Mutation-driven evolution of Pseudomonas aeruginosa in the presence of either ceftazidime or ceftazidime/avibactam

Mutation-driven evolution of Pseudomonas aeruginosa in the presence of either ceftazidime or ceftazidime/avibactam

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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