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Phage diversity in cell-free DNA identifies bacterial pathogens in human sepsis cases

Nature Microbiology volume 8, pages 1495–1507 (2023)Cite this article

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Abstract

Bacteriophages, viruses that infect bacteria, have great specificity for their bacterial hosts at the strain and species level. However, the relationship between the phageome and associated bacterial population dynamics is unclear. Here we generated a computational pipeline to identify sequences associated with bacteriophages and their bacterial hosts in cell-free DNA from plasma samples. Analysis of two independent cohorts, including a Stanford Cohort of 61 septic patients and 10 controls and the SeqStudy cohort of 224 septic patients and 167 controls, reveals a circulating phageome in the plasma of all sampled individuals. Moreover, infection is associated with overrepresentation of pathogen-specific phages, allowing for identification of bacterial pathogens. We find that information on phage diversity enables identification of the bacteria that produced these phages, including pathovariant strains of Escherichia coli. Phage sequences can likewise be used to distinguish between closely related bacterial species such as Staphylococcus aureus, a frequent pathogen, and coagulase-negative Staphylococcus, a frequent contaminant. Phage cell-free DNA may have utility in studying bacterial infections.

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Fig. 1: The circulating phageome reflects infection aetiology.
Fig. 2: cfDNA phageomes reflect pathogens in another large study.
Fig. 3: E. coli phages reflect host strain characteristics.
Fig. 4: Phages specify bacterial species in Staphylococcus infections.

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Data availability

Sequencing data with human reads removed have been deposited into NCBI SRA under bioproject PRJNA860730. Publicly available data utilized: the SepSeq study data have been previously published under bioproject PRJNA507824. No new computational tools were developed as part of this study. The INPHARED v1.7 database was downloaded and used for analyses in this study (https://github.com/RyanCook94/inphared). Infection aetiology metadata associated with samples sequenced for this study are included in the Stanford sepsis cohort sheet of Supplementary Data. The CPD FASTA file used for creating the Blast database is publicly available at https://doi.org/10.5281/zenodo.7154236. The Phage dictionary and Coliphage dictionary are additionally available as sheets in Supplementary Data. All associated supplementary files have additionally been made publicly available at https://doi.org/10.5281/zenodo.7644125.

Code availability

The R code used to summarize BLAST phageome annotations with the CPD has been made publicly available at https://doi.org/10.5281/zenodo.7734114. This includes an R markdown file detailing processing of BLAST outputs to create phage hit tables across all samples, and subsequent use of the CPD to summarize representation of phage taxonomic families and known bacterial host characteristics. A phage hit table for our sequenced samples is available along with this R code and can be used to re-create phage summary tables as well as for calculation of diversity using the R package ‘vegan’. Processing of raw data, removal of human reads, and BLAST annotations were done using existing software and are described in the relevant Methods sections.

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Acknowledgements

We thank T. Blauwkamp (Karius Inc.), S. Bercovici (Karius Inc.) and N. Noll (Karius Inc.) for their assistance providing additional metadata for the SepSeq dataset. We thank the funding sources supporting this work: NIH R01HL148184-01 (P.L.B.), NIH R01AI12492093 (P.L.B.), NIH R01DC019965 (P.L.B.), Cystic Fibrosis Foundation (P.L.B.), grant from the Emerson Collective (P.L.B.), NSF GRFP (N.L.H.), NIH T32HL129970-06 (L.J.B.), NIH R01AI148623 (A.S.B.), NIH R01AI143757 (A.S.B.), Stand Up 2 Cancer grant (A.S.B.), the Allen Distinguished Investigator Award (A.S.B.), NIH R21GM147838 (S.Y. and P.L.B.), NIH R01AI153133 (S.Y.), NIH R01AI137272 (S.Y.) and NIH R01AI138978 (S.Y.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Author information

Author notes

  1. These authors contributed equally: Naomi L. Haddock, Layla J. Barkal.

Authors and Affiliations

  1. Immunology Program, School of Medicine, Stanford University, Stanford, CA, USA

    Naomi L. Haddock

  2. Division of Pulmonary, Allergy, and Critical Care Medicine, School of Medicine, Stanford University, Stanford, CA, USA

    Layla J. Barkal

  3. Department of Emergency Medicine, School of Medicine, Stanford University, Stanford, CA, USA

    Nikhil Ram-Mohan & Samuel Yang

  4. Department of Pediatrics, School of Medicine, Stanford University, Stanford, CA, USA

    Gernot Kaber

  5. Department of Medicine, Division of Infectious Diseases, University of California San Francisco, San Francisco, CA, USA

    Charles Y. Chiu

  6. Division of Hematology, School of Medicine, Stanford University, Stanford, CA, USA

    Ami S. Bhatt

  7. Department of Genetics, Stanford University, Stanford, CA, USA

    Ami S. Bhatt

  8. Division of Infectious Diseases and Geographic Medicine, School of Medicine, Stanford University, Stanford, CA, USA

    Paul L. Bollyky

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Contributions

N.L.H., L.J.B., N.R.-M., G.K., S.Y. and P.L.B. designed the study. N.L.H., L.J.B. and N.R.-M. performed experiments. N.L.H., L.J.B., G.K., N.R.-M., S.Y., A.S.B., C.Y.C. and P.L.B. analysed data. N.L.H., L.J.B. and P.L.B. wrote the manuscript.

Corresponding author

Correspondence to Paul L. Bollyky.

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Competing interests

A.S.B. has consulted for biomX and is on the scientific advisory boards of ArcBio and Caribou Biosciences. The remaining authors declare no competing interests.

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Nature Microbiology thanks Bryan Kraft, Evelien Adriaenssens, Jeremy Barr, Paul Turner and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Non-Human reads in Asymptomatic and Septic individuals.

(A) Average proportion of bacterial hit genus in asymptomatic (N = 10) and septic (N = 61) nonhuman cfDNA as identified by BLAST search. (B) Violin plots of proportions of non-human read identities by BLAST search from both asymptomatic (N = 10) and septic (N = 61) individuals. Descriptive statistics are available in Extended Data Table 1. (C) Proportions of bacteriophage hits removed in secondary human sequence homolog removal step (mean 0.042 SD 0.038, N = 71). (D) Average distribution of unique phages by bacterial host genus with and without secondary human sequence homology removal (N = 71). Uncleaned Hits, mean proportions and SD (Pseudomonas: mean 0.029 SD 0.30, Enterobacter mean 0.021 SD 0.015, Escherichia mean 0.042 SD 0.038, Klebsiella mean 0.015 SD 0.020, Salmonella mean 0.013 SD 0.009, Not Annotated mean 0.603 SD 0.070, Staphylococcus mean 0.009 SD 0.011, Streptococcus mean 0.014 SD 0.036, Enterococcus mean 0.001 SD 0.002, Bacillus mean 0.006 SD 0.007, Other mean 0.245 SD 0.052), Cleaned Hits, mean proportions and SD (Pseudomonas: mean 0.047 SD 0.058, Enterobacter mean 0.050 SD 0.058, Escherichia mean 0.076 SD 0.065, Klebsiella mean 0.023 SD 0.036, Salmonella mean 0.014 SD 0.016, Not Annotated mean 0.35 SD 0.100, Staphylococcus mean 0.020 SD 0.040, Streptococcus mean 0.032 SD 0.076, Enterococcus mean 0.002 SD 0.004, Bacillus mean 0.012 SD 0.015, Other mean 0.373 SD 0.123). All violin plots are shown with individual data points with median and quartiles shown by dashed lines.

Extended Data Fig. 2 Bacterial host distribution does not change in sepsis, though individual variation remains.

(A) Violin plot of bacteriophage host genus proportions between Asymptomatic (N = 10) and Septic (N = 61) patient samples, associated statistics are in Extended Data Table 2. (B) Heatmap of Pearson dissimilarity matrix between patients with sepsis (N = 61). (C) Heatmap of Pearson dissimilarity matrix between asymptomatic controls (N = 10). (D) Histogram of prevalence across sequenced samples of each phage. (E) Phage bacterial host genus proportions per asymptomatic patient (N = 10). (F) Phage bacterial host genus proportions per septic patient (N = 61).

Extended Data Fig. 3 Proportion of ‘Not Annotated’ Phages from CHVD Gut Metagenome Phages.

(A) Proportion of ‘Not Annotated’ Phages from Gut Metagenome Phages in Stanford Sepsis Cohort (Asymptomatic mean: 0.413 SD: 0.060, N = 10. Sepsis mean: 0.300 SD:0.119, N = 61. Unpaired two-sided t test, P = 0.0046) (B) Proportion of ‘Not Annotated’ Phages from Gut Metagenome Phages in SepSeq cohort (Asymptomatic mean: 0.464 SD:0.271 N = 10. Sepsis mean: 0.418, SD:0.29, N = 61. Unpaired two sided t test, P = 0.101).

Extended Data Fig. 4 Number of E. coli phages by genetically classified taxonomic phage family.

(A) Sankey diagram of taxonomic family classifications from NCBI Taxonomy classification (Left) to genetically classified family (Right) (B) Number of E. coli phages by genetically classified taxonomic phage family tested by Brown-Forsythe and Welch Anova Test with two sided Dunnet’s T3 test multiple comparisons for Asymptomatic (N = 166), SIRS (N = 95), Other Sepsis (N = 55) and E. coli Sepsis (N = 36).

Extended Data Fig. 5 E. coli Phage Host Characteristic Proportions.

(A)Proportion of E. coli phage host characteristics in violin plots with individual data points with median and quartiles shown by dashed lines. Analyzed by two-way ANOVA with Sidak’s multiple comparisons only in samples with E. coli phage for Asymptomatic (N = 100), SIRS (N = 62), Other Sepsis (N = 36) and E. coli Sepsis (N = 36) patients. Phage characteristic source of variation P = 7.93E-292, Patient category source of variation P > 0.99, Interaction of Phage category and patient category P = 1.75E-50. Lab Strain Associated Phage (Mean: Asymptomatic 0.69, SIRS 0.58, Other Sepsis 0.45, E. coli Sepsis 0.36. Multiple comparisons: E. coli Sepsis vs: Asymptomatic P = 4.37E-33, SIRS P = 4.45E-13, Other Sepsis P = 0.03. Other Sepsis vs: SIRS P = 5.88E-5, Asymptomatic P = 2.19E-18. Asymptomatic vs SIRS P = 1.80E-6), Unspecified Host Associated Phage (Mean: Asymptomatic 0.18, SIRS 0.17, Other Sepsis 0.27, E. coli Sepsis 0.17. Multiple comparisons: E. coli Sepsis vs: Asymptomatic P > 0.99, SIRS P > 0.99, Other Sepsis P = 0.01. Other Sepsis vs: SIRS P = 2E-3, Asymptomatic P = 3E-3. Asymptomatic vs SIRS P = 0.994), STEC Associated Phage (Mean: Asymptomatic 0.05, SIRS 0.14, Other Sepsis 0.13, E. coli Sepsis 0.31. Multiple comparisons: E. coli Sepsis vs: Asymptomatic P = 4.76E-22, SIRS P = 1.19E-8, Other Sepsis P = 5.22E-8. Other Sepsis vs: SIRS P = 0.998, Asymptomatic P = 0.02. Asymptomatic vs SIRS P = 1.79E-4), ETEC Associated Phage (Mean: Asymptomatic 0.03, SIRS 0.04, Other Sepsis 0.03, E. coli Sepsis 0.05. Multiple comparisons: E. coli Sepsis vs: Asymptomatic P = 0.85, SIRS P = 0.99, Other Sepsis P = 0.98. Other Sepsis vs: SIRS P > 0.99, Asymptomatic P > 0.99. Asymptomatic vs SIRS P > 0.99), EPEC Associated Phage Associated Phage (Mean: Asymptomatic 3.67E-3, SIRS 0.01, Other Sepsis 6.78E-4, E. coli Sepsis 0.01. Multiple comparisons: E. coli Sepsis vs: Asymptomatic P > 0.99, SIRS P > 0.99, Other Sepsis P > 0.99. Other Sepsis vs: SIRS P > 0.99, Asymptomatic P > 0.99. Asymptomatic vs SIRS P > 0.99), ExPEC Associated Phage (Mean: Asymptomatic 0.02, SIRS 0.04, Other Sepsis 0.04, E. coli Sepsis 0.05. Multiple comparisons: E. coli Sepsis vs: Asymptomatic P = 0.89, SIRS P > 0.99, Other Sepsis P > 0.99. Other Sepsis vs: SIRS P > 0.99, Asymptomatic P = 0.99. Asymptomatic vs SIRS P > 0.99), Sewage/Manure/Water Associated Phage (Mean: Asymptomatic 0.02, SIRS 0.03, Other Sepsis 0.07, E. coli Sepsis 0.03. Multiple comparisons: E. coli Sepsis vs: Asymptomatic P > 0.99, SIRS P > 0.99, Other Sepsis P = 0.73. Other Sepsis vs: SIRS P = 0.66, Asymptomatic P = 0.33. Asymptomatic vs SIRS P > 0.99).

Extended Data Table 1 Asymptomatic vs septic non-human cfDNA proportions
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Extended Data Table 2 Asymptomatic vs septic phage host proportion statistical summary
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Extended Data Table 3 Stanford sepsis cohort pathogen associated phage Mann-Whitney summary
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Extended Data Table 4 Stanford sepsis cohort coinfected sample summary
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Extended Data Table 5 SepSeq pathogen associated phage Dunn’s multiple comparison summary
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Supplementary information

Reporting Summary

Supplementary Data

This file contains the following sheets: Phage dictionary, Coliphage dictionary and Stanford sepsis cohort (infection metadata), negative control summary, PBS control BLAST hits and water control BLAST hits.

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Haddock, N.L., Barkal, L.J., Ram-Mohan, N. et al. Phage diversity in cell-free DNA identifies bacterial pathogens in human sepsis cases. Nat Microbiol 8, 1495–1507 (2023). https://doi.org/10.1038/s41564-023-01406-x

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