Parallel evolution of Pseudomonas aeruginosa phage resistance and virulence loss in response to phage treatment in vivo and in vitro
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Evaluation Summary:
With the increased interest in phage therapy to treat antibiotic resistant infections, there are questions about the ease at which bacteria evolve phage resistance. To examine this, Castledine et al. cultured a set of bacterial isolates from a patient pre- and during phage therapy and also experimentally evolved a mixture of the bacterial isolates from the patient in the absence or presence of phage in vitro. Overall, the authors observed similarities between the evolutionary outcomes (genomic and phenotypic) in the patient and in vitro.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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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 coevolves 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.
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Author response:
Reviewer #2 (Public Review):
This work by Castledine et al. addresses the important question of whether results from in vitro (laboratory-based) evolution studies may be useful for predicting evolution during phage therapy in a clinical setting. In order to explore this question, the authors cultured a set of bacterial isolates from a patient pre- and during phage therapy, as well as phages from several time points during therapy. They then experimentally evolved (in vitro) a mixture of the bacterial isolates from the patient in the absence of phage, or in the presence of phage using two different treatments (phage added once or added repeatedly). Overall, they observed similarities between the evolutionary outcomes (genomic and phenotypic) in vitro and in the patient. Resistance evolved rapidly in the patient and in …
Author response:
Reviewer #2 (Public Review):
This work by Castledine et al. addresses the important question of whether results from in vitro (laboratory-based) evolution studies may be useful for predicting evolution during phage therapy in a clinical setting. In order to explore this question, the authors cultured a set of bacterial isolates from a patient pre- and during phage therapy, as well as phages from several time points during therapy. They then experimentally evolved (in vitro) a mixture of the bacterial isolates from the patient in the absence of phage, or in the presence of phage using two different treatments (phage added once or added repeatedly). Overall, they observed similarities between the evolutionary outcomes (genomic and phenotypic) in vitro and in the patient. Resistance evolved rapidly in the patient and in vitro under phage selection, and similar genomic changes were observed in both environments. The approach of using bacterial isolates directly from the patient (as well as the phages used for therapy) in vitro is clever, and the observed similarities are compelling.
We thank the reviewer for appreciating the novelty in our results and methodology.
However, I think there are some limitations with the study that should be addressed in the text.
In particular, (1) While the similarities in vitro and in the patient are quite interesting, there are some differences that were dismissed as being minor without justification. Calling the results "highly parallel" is a bit subjective - in vitro in the repeated phage treatment (which is suggested to be most similar to the clinical context), there did appear to be phage coevolution that was not observed in vivo. The tradeoffs/relationships between traits (as shown in Fig. 3) also differed to some extent.
We agree this could have been more objectively phrased at the start of the discussion – this has been edited to reflect this. We have highlighted the differences between in vivo and in vitro treatments with respect to phage evolution. Moreover, we have also highlighted that the observed trade-offs had different underlying mechanisms which may not always result in parallel evolutionary changes between in vivo and in vitro environments.
Additionally, for the genomic results only a subset of variants were plotted (those in genes of known function), but there were far more significant variants in genes of unknown function that were not included. It is difficult to assess whether the genomic findings are truly similar across environments if only a fraction of those results were presented in the manuscript.
We chose to concentrate on genes of only known function so that we could better understand their potential significance, and also because the figures and analyses (Figures 4 and 5) would become extremely complex and large and uninterpretable with genes of unknown function included. This is especially true for Figure 5, which would have required us to show 284 rows if all genes would have been included. Ultimately, whichever way we do this exploratory analysis, it is going to be difficult to see if findings are truly similar across environments because we only have a single patient who had phage therapy.
However, we have redone the analysis with all of the significant genetic changes (SNPs and indels from both known and unknown genes) included.
Figure 5 has been recreated and is now included as "Figure 5 - Figure supplement 1". All of the statistical analysis on (a) the number of SNP/indels seen (b) genetic distance from ancestor and (c) alpha diversity give quantitatively similar results. That is, although all the estimates are generally much higher after including many more genetic variants, all of the significant results from both the overall model fit and post-hoc multiple comparisons remain the same. One interesting result that came out of looking at all the genetic changes was that for genetic variants occurring in a gene of known function, 56% (28 out of 50) were de novo mutations, whereas this value was only 42% (98 out of 234) for variants in genes of unknown function.
We then looked at the proportion of genetic variants (both in known and unknown genes) found in vitro that were also found in vivo. For genes of known function, 62% of genetic variants were found in vivo (31 of 50) and this was comparable to the 65% of genetic variants in genes of unknown function (153 of 234). Of the 26 genes of known function with differences identified in the in vitro analysis, 16 (61%) were also found to have genetic changes in vivo. The equivalent metric for genes of unknown function was 86% (85 of 99). Similar to in vitro, variants occurring in a gene of known function were more likely to be de novo mutations (77%) compared to variants occurring in a gene of unknown function (46%).
While these patterns and exploratory analyses are interesting, they have extremely limited statistical power and therefore do not alter the conclusions or results of the work presented. For these reasons, we have chosen not to include these results in the already long manuscript. We have added a line to say we have done it both way:
“We performed all downstream statistical analyses on (a) only genetic variants in genes of known function and (b) all genetic variants.”
And we also added a line at the beginning of the genomic analysis results section:
“Results were not affected whether we included only genetic variants occurring in genes of known function or all genetic variants (Figure 5-Figure supplement 1). As we were interested in attributing potential functions to the variants identified, we only present the results for genetic variants occurring in genes of known function.”
(2) Much of the text is framed around whether in vitro outcomes are predictive of those in vivo, but this study only included results from a single patient. Thus, it is impossible to know whether these findings are by chance or representative of a more general relationship between in vitro and in vivo evolution.
We agree that having a single patient for our in vivo comparison limits the generalisability of our results. We have highlighted this in the revised manuscript. However, that our replicated in vitro experiments agreed broadly with our in vivo results and that of other studies (finding resistance-virulence trade-offs) suggests that at least in some circumstance in vitro dynamics are predictive of in vivo dynamics. Further studies are clearly needed (and hopefully will arise as a consequence of this work) to determine the generalisability of this finding and the circumstances where this parallelism might break down.
(3) Although the evolutionary outcomes appear to be similar, the pathogen was successfully cleared from the patient but persisted throughout experimental evolution. Whether the pathogen is successfully eliminated or not is presumably the most important clinical outcome, and while this difference is not surprising, it is an important one to point out to the reader. Essentially, evolution was similar to some extent but the consequences of evolution for bacterial persistence in each environment were quite different.
We have now highlighted this difference to the reader in the revised manuscript.
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Evaluation Summary:
With the increased interest in phage therapy to treat antibiotic resistant infections, there are questions about the ease at which bacteria evolve phage resistance. To examine this, Castledine et al. cultured a set of bacterial isolates from a patient pre- and during phage therapy and also experimentally evolved a mixture of the bacterial isolates from the patient in the absence or presence of phage in vitro. Overall, the authors observed similarities between the evolutionary outcomes (genomic and phenotypic) in the patient and in vitro.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
The study tests the validity of using in vitro models to recapitulate (and thus potentially predict) the outcome of human phage therapy. To do this they sample a human volunteer undergoing nasal decolonisation by phages, in vivo isolated bacteria and phages and characterised phenotypically and genetically, and also used to establish in vitro experimental treatments which are then similarly characterised.
The key strength and novelty of the study is the ability to directly compare the evolutionary response of the same bacteria to phage therapy both in vivo and in vitro. To my knowledge this has not been achieved before. An understandable weakness is that there is only one human subject, but this is compensated for by the ability to perform well replicated in vitro studies which also allow testing of …
Reviewer #1 (Public Review):
The study tests the validity of using in vitro models to recapitulate (and thus potentially predict) the outcome of human phage therapy. To do this they sample a human volunteer undergoing nasal decolonisation by phages, in vivo isolated bacteria and phages and characterised phenotypically and genetically, and also used to establish in vitro experimental treatments which are then similarly characterised.
The key strength and novelty of the study is the ability to directly compare the evolutionary response of the same bacteria to phage therapy both in vivo and in vitro. To my knowledge this has not been achieved before. An understandable weakness is that there is only one human subject, but this is compensated for by the ability to perform well replicated in vitro studies which also allow testing of alternative treatment regimens.
The aims of the study are achieved. The results are compelling, showing that resistance readily evolves both in vivo and in vitro, and results in similar alterations in the ability of phage resistant bacteria to establish acute infections, reduced growth rate, and changes in biofilm formation. In hindsight and in light of the genomic data there are several additional phenotypes that it would be interesting to test (in particular motility) but these additional phenotypes would be unlikely to alter the overall message of the study. The results support the conclusions.
The work is likely to have an impact on the field. There are still relatively few studies of resistance evolution against cocktails, and even fewer of this process in vivo. The validation of using in vitro systems could dramatically improve the ability to test phage cocktails for robustness to resistance prior to clinical use, thus leading to translational clinical impacts of the work.
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Reviewer #2 (Public Review):
This work by Castledine et al. addresses the important question of whether results from in vitro (laboratory-based) evolution studies may be useful for predicting evolution during phage therapy in a clinical setting. In order to explore this question, the authors cultured a set of bacterial isolates from a patient pre- and during phage therapy, as well as phages from several time points during therapy. They then experimentally evolved (in vitro) a mixture of the bacterial isolates from the patient in the absence of phage, or in the presence of phage using two different treatments (phage added once or added repeatedly). Overall, they observed similarities between the evolutionary outcomes (genomic and phenotypic) in vitro and in the patient. Resistance evolved rapidly in the patient and in vitro under phage …
Reviewer #2 (Public Review):
This work by Castledine et al. addresses the important question of whether results from in vitro (laboratory-based) evolution studies may be useful for predicting evolution during phage therapy in a clinical setting. In order to explore this question, the authors cultured a set of bacterial isolates from a patient pre- and during phage therapy, as well as phages from several time points during therapy. They then experimentally evolved (in vitro) a mixture of the bacterial isolates from the patient in the absence of phage, or in the presence of phage using two different treatments (phage added once or added repeatedly). Overall, they observed similarities between the evolutionary outcomes (genomic and phenotypic) in vitro and in the patient. Resistance evolved rapidly in the patient and in vitro under phage selection, and similar genomic changes were observed in both environments. The approach of using bacterial isolates directly from the patient (as well as the phages used for therapy) in vitro is clever, and the observed similarities are compelling. However, I think there are some limitations with the study that should be addressed in the text.
In particular,
(1) While the similarities in vitro and in the patient are quite interesting, there are some differences that were dismissed as being minor without justification. Calling the results "highly parallel" is a bit subjective - in vitro in the repeated phage treatment (which is suggested to be most similar to the clinical context), there did appear to be phage coevolution that was not observed in vivo. The tradeoffs/relationships between traits (as shown in Fig. 3) also differed to some extent. Additionally, for the genomic results only a subset of variants were plotted (those in genes of known function), but there were far more significant variants in genes of unknown function that were not included. It is difficult to assess whether the genomic findings are truly similar across environments if only a fraction of those results were presented in the manuscript.(2) Much of the text is framed around whether in vitro outcomes are predictive of those in vivo, but this study only included results from a single patient. Thus, it is impossible to know whether these findings are by chance or representative of a more general relationship between in vitro and in vivo evolution.
(3) Although the evolutionary outcomes appear to be similar, the pathogen was successfully cleared from the patient but persisted throughout experimental evolution. Whether the pathogen is successfully eliminated or not is presumably the most important clinical outcome, and while this difference is not surprising, it is an important one to point out to the reader. Essentially, evolution was similar to some extent but the consequences of evolution for bacterial persistence in each environment were quite different.
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