Pathogen clonal expansion underlies multiorgan dissemination and organ-specific outcomes during murine systemic infection

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    Evaluation Summary:

    Monitoring changes in pathogenic bacterial populations after bloodstream infection are poorly understood and in this work the authors develop new genetic analytic tools to dissect bacterial population dynamics at sites of infection. By harvesting bacteria from different sites and times of infection and performing deep sequencing to define the distribution of specific tagged-strains the authors provide a highly detailed snapshot of populations, with discriminatory power superior to prior studies. The work paves the way to future such studies in other organisms that cause persistent infection in humans.

    (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

The dissemination of pathogens through blood and their establishment within organs lead to severe clinical outcomes. However, the within-host dynamics that underlie pathogen spread to and clearance from systemic organs remain largely uncharacterized. In animal models of infection, the observed pathogen population results from the combined contributions of bacterial replication, persistence, death, and dissemination, each of which can vary across organs. Quantifying the contribution of each these processes is required to interpret and understand experimental phenotypes. Here, we leveraged STAMPR, a new barcoding framework, to investigate the population dynamics of extraintestinal pathogenic Escherichia coli , a common cause of bacteremia, during murine systemic infection. We show that while bacteria are largely cleared by most organs, organ-specific clearance failures are pervasive and result from dramatic expansions of clones representing less than 0.0001% of the inoculum. Clonal expansion underlies the variability in bacterial burden between animals, and stochastic dissemination of clones profoundly alters the pathogen population structure within organs. Despite variable pathogen expansion events, host bottlenecks are consistent yet highly sensitive to infection variables, including inoculum size and macrophage depletion. We adapted our barcoding methodology to facilitate multiplexed validation of bacterial fitness determinants identified with transposon mutagenesis and confirmed the importance of bacterial hexose metabolism and cell envelope homeostasis pathways for organ-specific pathogen survival. Collectively, our findings provide a comprehensive map of the population biology that underlies bacterial systemic infection and a framework for barcode-based high-resolution mapping of infection dynamics.

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  1. Evaluation Summary:

    Monitoring changes in pathogenic bacterial populations after bloodstream infection are poorly understood and in this work the authors develop new genetic analytic tools to dissect bacterial population dynamics at sites of infection. By harvesting bacteria from different sites and times of infection and performing deep sequencing to define the distribution of specific tagged-strains the authors provide a highly detailed snapshot of populations, with discriminatory power superior to prior studies. The work paves the way to future such studies in other organisms that cause persistent infection in humans.

    (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.)

  2. Reviewer #1 (Public Review):

    The authors used a high-resolution barcoding approach to probe the complex infection dynamics of systemic ExPEC infection. They identified organ-specific population dynamics and discovered that clearance failures are pervasive, as the results of dramatic expansions of very few bacterial cells from the inoculum. These expansions may not be easily identified by routine CFU-based metrics, but could be readily detected and quantified by their methods. In general, this study is well performed and the manuscript is well written. Additional details on how the parameters (e.g. Nb, Nr, GD, RD, FRD) were calculated should be added in order to help the readers to understand the models.

  3. Reviewer #2 (Public Review):

    This is a very interesting study analyzing the pathogen population trajectories during systemic infections. Utilizing the extra-intestinal pathogenic Escherichia coli CFT073, the authors analyzed the trajectories and mechanisms used by this bacterium to persist in particular niches. The authors found that although most of the inoculum was eradicated after injection, some bacteria were able to persist (in some tissues/organs while not in others), mainly because the ability of some individual clones to expand. The authors also analyzed the consequences of this clonal expansion in the ability of the bacterium to disseminate to other locations. While some of the conclusions obtained in this study are not completely novel, no previous studies have analyzed these processes with the detail and depth shown here. And therefore, this work represents an impressive tour de force analyzing these important processes in vivo. Since the quantity of work that this manuscript already has is impressive, more than asking for additional experiments I would like to ask a few general questions, with the hope that they will better connect the results obtained here with what is observed in clinical infections:

    1. The authors have shown in their study that changes in different parameters (i.e. inoculum) impact the trajectories followed by the pathogens. One can imagine that in a normal scenario, the number of bacterial cells that will arrive to the blood will be much lower than the inoculum used in these experiments. Similarly, I do not know if additional host factors will also influence which people will be infected or not. I would like the authors to explain more clearly why the model used is relevant to understand what is happening during normal infections.

    2. The authors clearly show that E. coli CFT073 colonizes the liver much better than other organs. Is there any evidence that liver infections are over-represented in patients suffering with E. coli bacteremia?

    3. In the last part of the paper, the authors identified several genes involved in pathogen survival after infection (early stages). I'm not sure this part is related with the previous part of the manuscript, because my feeling is that the observed clonal expansion was a stochastic process, not driven by mutations in specific genes. I would suggest that some of the clones that expanded were sequenced, to clearly show if this process of clonal expansion was random (or conversely, was driven in some cases by mutations in specific genes).

    4. Finally, there is an interesting paper from Fitzgerald's group (PMID: 31807698), analyzing both intra and inter-host dissemination that should be referenced here.

  4. Reviewer #3 (Public Review):

    Changes in the population of bacteria that cause systemic infections after bloodstream infection are poorly understood. New genetic analytic tools, such as those developed and employed by the investigators here, afford opportunities to dissect bacterial populations at sites of infection, and understand the kinetics of pathogenesis. The authors of this study infected mice intravenously with a library of extra-intestinal E coli strains that were genetically identical except for unique DNA tags that did not affect their growth. By harvesting bacteria from different sites and times of infection, and performing deep sequencing to define the distribution of specific tagged-strains, investigators provide a highly detailed snapshot of populations, with discriminatory power superior to prior studies. Strengths of the project include logical and straightforward study design, and the novelty of the molecular and analytic tools employed. The paper is cogently written, which is essential given the attention to detail necessary to follow the work and results clearly. Weaknesses are that this is a preliminary, proof of concept type of project, and reproducibility of findings, mechanistic studies, and deeper scientific investigation of temporal-spatial patterns will require future study.

    It is also unclear if results are specific to the strain of extra-intestinal E coli or strain of mice used here. However, this study and the methods used provide a necessary foundation for such future studies. The results are presented in descriptive, straightforward fashion, and interpretations and conclusions are supported by the data. The study will have impact on future studies to better understand pathogenesis, and in providing methods, tools and experimental templates for future studies. As such, the paper is a solid advance for the field.