Multilocus amplicon sequencing of Pseudomonas aeruginosa cystic fibrosis airways isolates collected prior to and after early antipseudomonal chemotherapy

Fischer, Sebastian; Greipel, Leonie; Klockgether, Jens; Dorda, Marie; Wiehlmann, Lutz; Cramer, Nina; Tümmler, Burkhard

doi:10.1016/j.jcf.2016.10.013

Cited by 6 publications

(4 citation statements)

References 31 publications

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“…Amplicon sequencing can also capture mutations in a subset of the genome with deep coverage. We used hybridization-based enrichment, which did not require any experimental optimization for different targets and is less likely to introduce biases in inferring the frequencies of mutations, like IS insertions, that change amplicon sizes [64]. With enough input DNA and enough sequencing, our approach could be scaled to more genes or the whole genome.…”

Section: Discussionmentioning

confidence: 99%

“…We tracked spontaneous beneficial mutations within targeted genome regions, or a portion of what one could more specifically describe as the adaptome (Ryall et al, 2012). Amplicon sequencing can also capture mutations in a subset of the genome with deep coverage (Chubiz et al, 2012;Fischer et al, 2017;Hong et al, 2018). We used hybridization-based enrichment, which did not require any experimental optimization for different targets and is less likely to introduce biases in inferring the frequencies of mutations when IS insertions and large indels that change amplicon sizes are present.…”

Section: Discussionmentioning

confidence: 99%

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Profiling the initial burst of beneficial genetic diversity in clonal cell populations to anticipate evolution

Deatherage

Barrick

2020

Preprint

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Clonal populations of cells continuously evolve new genetic diversity, but it takes a significant amount of time for the progeny of a single cell with a new beneficial mutation to outstrip both its ancestor and competitors to fully dominate a population. If genotypes with these driver mutations can be discovered earlier—while they are still extremely rare—it may be possible to anticipate the future evolution of these populations. For example, one could diagnose the likely course of incipient diseases, such as cancer and bacterial infections, and better judge which treatments will be effective, by tracking rare drug-resistant variants. To test this approach, we replayed the first 500 generations of a >70,000-generation Escherichia coli experiment and examined the trajectories of new mutations in eight genes known to be under positive selection in this environment in six populations. By employing a deep sequencing procedure using molecular indexes and target enrichment we were able to track 236 beneficial mutations at frequencies as low as 0.01% and infer selection coefficients for 180 of these. Distinct molecular signatures of selection on protein structure and function were evident for the three genes in which beneficial mutations were most common (nadR, pykF, and topA). We detected mutations hundreds of generations before they became dominant and tracked beneficial alleles in genes that were not mutated in the long-term experiment until thousands of generations had passed. Therefore, this targeted adaptome sequencing approach can function as an early warning system to inform interventions that aim to prevent undesirable evolution.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Profiling the initial burst of beneficial genetic diversity in clonal cell populations to anticipate evolution

Deatherage

Barrick

2020

Preprint

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“…Due to accessibility and reduced costs of next-generation sequencing, re-sequencing tools, such as amplicon population sequencing, are gaining popularity in the publication of other adaptive evolution studies ( Ostman et al, 2012 ; Wong and Seguin, 2015 ; Feng et al, 2016 ) and the analyses of clinical and environmental samples ( Johnning et al, 2015 ; Fischer et al, 2016 ; Greipel et al, 2016 ). While this method is powerful, it is limited in a number of ways.…”

Section: Discussionmentioning

confidence: 99%

Time-Resolved Tracking of Mutations Reveals Diverse Allele Dynamics during Escherichia coli Antimicrobial Adaptive Evolution to Single Drugs and Drug Pairs

2017

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Understanding the evolutionary processes that lead to antibiotic resistance can help to achieve better treatment strategies. Yet, little is known about the dynamics of the resistance alleles during adaptation. Here, we use population sequencing to monitor genetic changes in putative resistance loci at several time-points during adaptive evolution experiments involving five different antibiotic conditions. We monitor the mutational spectra in lineages evolved to be resistant to single antibiotics [amikacin (AMK), chloramphenicol (CHL), and ciprofloxacin (CIP)], as well as antibiotic combinations (AMK + CHL and CHL + CIP). We find that lineages evolved to antibiotic combinations exhibit different resistance allele dynamics compared with those of single-drug evolved lineages, especially for a drug pair with reciprocal collateral sensitivity. During adaptation, we observed interfering, superimposing and fixation allele dynamics. To further understand the selective forces driving specific allele dynamics, a subset of mutations were introduced into the ancestral wild type enabling differentiation between clonal interference and negative epistasis.

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“…Two distinct studies provided evidence supporting this notion, as the lasR gene of some P. aeruginosa isolates already exhibited mutations at initial detection in CF respiratory samples, consistent with a direct acquisition from environmental sources ( 40 , 68 ). While P. aeruginosa lineages have been observed to lose LasR function over time during an infection, the discussion above indicates that such events are not necessarily specifically selected by the CF lung environment ( 30 , 69 – 72 ).…”

Section: Perspectivementioning

confidence: 99%

The end of the reign of a “master regulator’’? A defect in function of the LasR quorum sensing regulator is a common feature of Pseudomonas aeruginosa isolates

Trottier,

de Oliveira Pereira,

Groleau

et al. 2024

mBio

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Pseudomonas aeruginosa , a bacterium causing infections in immunocompromised individuals, regulates several of its virulence functions using three interlinked quorum sensing (QS) systems ( las , rhl , and pqs ). Despite its presumed importance in regulating virulence, dysfunction of the las system regulator LasR occurs frequently in strains isolated from various environments, including clinical infections. This newfound abundance of LasR-defective strains calls into question existing hypotheses regarding their selection. Indeed, current assumptions concerning factors driving the emergence of LasR-deficient isolates and the role of LasR in the QS hierarchy must be reconsidered. Here, we propose that LasR is not the primary master regulator of QS in all P. aeruginosa genetic backgrounds, even though it remains ecologically significant. We also revisit and complement current knowledge on the ecology of LasR-dependent QS in P. aeruginosa , discuss the hypotheses explaining the putative adaptive benefits of selecting against LasR function, and consider the implications of this renewed understanding.

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Multilocus amplicon sequencing of Pseudomonas aeruginosa cystic fibrosis airways isolates collected prior to and after early antipseudomonal chemotherapy

Abstract: Early treatment selects pathoadaptive mutations in P. aeruginosa that are typical for chronic infections of CF lungs.

Cited by 6 publications

References 31 publications

Profiling the initial burst of beneficial genetic diversity in clonal cell populations to anticipate evolution

Profiling the initial burst of beneficial genetic diversity in clonal cell populations to anticipate evolution

Time-Resolved Tracking of Mutations Reveals Diverse Allele Dynamics during Escherichia coli Antimicrobial Adaptive Evolution to Single Drugs and Drug Pairs

The end of the reign of a “master regulator’’? A defect in function of the LasR quorum sensing regulator is a common feature of Pseudomonas aeruginosa isolates

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