Parallel evolution of Pseudomonas aeruginosa phage resistance and virulence loss in response to phage treatment in vivo and in vitro
- University of Exeter, United Kingdom
- University of York, United Kingdom
- Queen Astrid Military Hospital, Belgium
Abstract
With rising antibiotic resistance, there has been increasing interest in treating pathogenic bacteria with bacteriophages (phage therapy). One limitation of phage therapy is the ease at which bacteria can evolve resistance. Negative effects of resistance may be mitigated when resistance results in reduced bacterial growth and virulence, or when phage coevolve to overcome resistance. Resistance evolution and its consequences are contingent on the bacteria-phage combination and their environmental context, making therapeutic outcomes hard to predict. One solution might be to conduct 'in vitro evolutionary simulations' using bacteria-phage combinations from the therapeutic context. Overall, our aim was to investigate parallels between in vitro experiments and in vivo dynamics in a human participant. Evolutionary dynamics were similar, with high levels of resistance evolving quickly with limited evidence of phage evolution. Resistant bacteria - evolved in vitro and in vivo - had lower virulence. In vivo, this was linked to lower growth rates of resistant isolates, whereas in vitro phage resistant isolates evolved greater biofilm production. Population sequencing suggests resistance resulted from selection on de novo mutations rather than sorting of existing variants. These results highlight the speed at which phage resistance can evolve in vivo, and how in vitro experiments may give useful insights for clinical evolutionary outcomes.
Data availability
All data and R script files can be found at the following GitHub repository. This repository outlines how the files may be used for analysis and Figure production. A full description of data file meanings and annotated R scripts is provided.https://github.com/mcastledine96/Parallel_evolution_phage_resistance_virulence_trade-offs_invivo_invitroRaw sequencing files have been archived on the European Nucleotide Archive with the project accession number PRJEB47945https://www.ebi.ac.uk/ena/browser/view/PRJEB47945?show=reads
Article and author information
Author details
Meaghan Castledine
Funding
Medical Research Council (MR/N0137941/1)
- Meaghan Castledine
Royal Society (CH160068)
- Angus Buckling
Natural Environment Research Council (NE/S000771/1)
- Angus Buckling
Royal Higher Institute for Defence (HFM 19-12)
- Maya Merabishvili
Biotechnology and Biological Sciences Research Council (BB/T014342/1)
- Ville-Petri Friman
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Human subjects: The decolonisation study protocol was approved by the Leading Ethics Committee of the "Université Catholique de Louvain" (Avis N{degree sign}: B-403201111110). The study was performed in accordance with the ethical standards as laid down in the Declaration of Helsinki and as revised in 2013. The patients gave informed consent and their anonymity was preserved.
Copyright
© 2022, Castledine et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Parallel evolution of Pseudomonas aeruginosa phage resistance and virulence loss in response to phage treatment in vivo and in vitro
eLife 11:e73679.
https://doi.org/10.7554/eLife.73679
Further reading
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Gene regulation is essential for life and controlled by regulatory DNA. Mutations can modify the activity of regulatory DNA, and also create new regulatory DNA, a process called regulatory emergence. Non-regulatory and regulatory DNA contain motifs to which transcription factors may bind. In prokaryotes, gene expression requires a stretch of DNA called a promoter, which contains two motifs called –10 and –35 boxes. However, these motifs may occur in both promoters and non-promoter DNA in multiple copies. They have been implicated in some studies to improve promoter activity, and in others to repress it. Here, we ask whether the presence of such motifs in different genetic sequences influences promoter evolution and emergence. To understand whether and how promoter motifs influence promoter emergence and evolution, we start from 50 ‘promoter islands’, DNA sequences enriched with –10 and –35 boxes. We mutagenize these starting ‘parent’ sequences, and measure gene expression driven by 240,000 of the resulting mutants. We find that the probability that mutations create an active promoter varies more than 200-fold, and is not correlated with the number of promoter motifs. For parent sequences without promoter activity, mutations created over 1500 new –10 and –35 boxes at unique positions in the library, but only ~0.3% of these resulted in de-novo promoter activity. Only ~13% of all –10 and –35 boxes contribute to de-novo promoter activity. For parent sequences with promoter activity, mutations created new –10 and –35 boxes in 11 specific positions that partially overlap with preexisting ones to modulate expression. We also find that –10 and –35 boxes do not repress promoter activity. Overall, our work demonstrates how promoter motifs influence promoter emergence and evolution. It has implications for predicting and understanding regulatory evolution, de novo genes, and phenotypic evolution.
