Engineered matrices reveal stiffness-mediated chemoresistance in patient-derived pancreatic cancer organoids.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by its fibrotic and stiff extracellular matrix. However, how the altered cell/extracellular-matrix signalling contributes to the PDAC tumour phenotype has been difficult to dissect. Here we design and engineer matrices that recapitulate the key hallmarks of the PDAC tumour extracellular matrix to address this knowledge gap. We show that patient-derived PDAC organoids from three patients develop resistance to several clinically relevant chemotherapies when cultured within high-stiffness matrices mechanically matched to in vivo tumours. Using genetic barcoding, we find that while matrix-specific clonal selection occurs, cellular heterogeneity is not the main driver of chemoresistance. Instead, matrix-induced chemoresistance occurs within a stiff environment due to the increased expression of drug efflux transporters mediated by CD44 receptor interactions with hyaluronan. Moreover, PDAC chemoresistance is reversible following transfer from high- to low-stiffness matrices, suggesting that targeting the fibrotic extracellular matrix may sensitize chemoresistant tumours. Overall, our findings support the potential of engineered matrices and patient-derived organoids for elucidating extracellular matrix contributions to human disease pathophysiology.

Using Genomic Tools to Predict Antimicrobial Resistance and Markers in Clinical Bacterial Samples.

Antimicrobial resistance (AMR) poses a critical threat to hospital infections particularly in the context of hospital-acquired infections (HAIs). This study leverages genomic tools to predict AMR and identify resistance markers in clinical bacterial samples associated with HAIs. Using comprehensive genomic and phenotypic analyses, we evaluated the genetic profiles of Pseudomonas aeruginosa and Staphylococcus aureus to uncover resistance mechanisms. Our results demonstrate that genomic tools, such as CARD-RGI and the Solu platform, can accurately identify resistance genes and predict AMR phenotypes in nosocomial pathogens. These findings underscore the potential of integrating genomic approaches into clinical practice to enhance the management of resistant infections in hospital settings and inform the development of novel antimicrobial strategies. Importance: This study investigates the impact of prophages on antibiotic resistance in two clinically significant bacteria, Pseudomonas aeruginosa and Staphylococcus aureus. Understanding how prophages influence resistance mechanisms in these pathogens is crucial, as Pseudomonas aeruginosa is known for its role in chronic infections in cystic fibrosis patients, while Staphylococcus aureus, including MRSA strains, is a leading cause of hospital-acquired infections. By exploring the relationship between prophage presence and resistance, this research provides insights that could inform the development of more effective treatment strategies and enhance our ability to combat antibiotic-resistant infections, ultimately improving patient outcomes and public health.

Pf bacteriophages hinder sputum antibiotic diffusion via electrostatic binding.

Despite great progress in the field, chronic Pseudomonas aeruginosa (Pa) infections remain a major cause of mortality in patients with cystic fibrosis (pwCF), necessitating treatment with antibiotics. Pf is a filamentous bacteriophage produced by Pa and acts as a structural element in Pa biofilms. Pf presence has been associated with antibiotic resistance and poor outcomes in pwCF, although the underlying mechanisms are unclear. We have investigated how Pf and sputum biopolymers impede antibiotic diffusion using pwCF sputum and fluorescent recovery after photobleaching. We demonstrate that tobramycin interacts with Pf and sputum polymers through electrostatic interactions. We also developed a set of mathematical models to analyze the complex observations. Our analysis suggests that Pf in sputum reduces the diffusion of charged antibiotics due to a greater binding constant associated with organized liquid crystalline structures formed between Pf and sputum polymers. This study provides insights into antibiotic tolerance mechanisms in chronic Pa infections and may offer potential strategies for novel therapeutic approaches.

Hydrogels for Local and Sustained Delivery of Bacteriophages to Treat Multidrug-Resistant Wound Infections.

Bacteriophages (phages), viruses that specifically target and kill bacteria, represent a promising strategy to combat multidrug-resistant (MDR) pathogens such as Pseudomonas aeruginosa (Pa). However, delivering sufficient concentrations of active phages directly to the infection site remains challenging, with current methods having variable success. Here we present "HydroPhage", an innovative hydrogel system for the sustained release of high-titer phages to effectively treat infections caused by MDR pathogens. Our injectable hydrogels, featuring dual-crosslinking of hyaluronic acid and PEG-based hydrogels through static covalent thioether bonds and dynamic covalent hemithioacetal crosslinks (DCC), encapsulate phages at concentration up to 1011 PFU/mL, and achieves controlled release of 109 PFU daily over a week, surpassing levels of current clinical dosages, with more than 60% total phage recovery. In a preclinical mouse model of extended wound infection, compared to intravenous treatment, we demonstrate enhanced bacterial clearance by localized, high-dose, and repeated phage dosing despite the emergence of bacterial resistance to phages. This work advances the development of clinically practical wound dressings tailored for resistant infections.

Phage Therapy for Respiratory Infections: Opportunities and Challenges.

We are entering the post-antibiotic era. Antimicrobial resistance (AMR) is a critical problem in chronic lung infections resulting in progressive respiratory failure and increased mortality. In the absence of emerging novel antibiotics to counter AMR infections, bacteriophages (phages), viruses that infect bacteria, have become a promising option for chronic respiratory infections. However, while personalized phage therapy is associated with improved outcomes in individual cases, clinical trials demonstrating treatment efficacy are lacking, limiting the therapeutic potential of this approach for respiratory infections. In this review, we address the current state of phage therapy for managing chronic respiratory diseases. We then discuss how phage therapy may address major microbiologic obstacles which hinder disease resolution of chronic lung infections with current antibiotic-based treatment practices. Finally, we highlight the challenges that must be addressed for successful phage therapy clinical trials. Through this discussion, we hope to expand on the potential of phages as an adjuvant therapy in chronic lung infections, as well as the microbiologic challenges that need to be addressed for phage therapy to expand beyond personalized salvage therapy.

A review of recent advances in the use of complex metal nanostructures for biomedical applications from diagnosis to treatment.

Complex metal nanostructures represent an exceptional category of materials characterized by distinct morphologies and physicochemical properties. Nanostructures with shape anisotropies, such as nanorods, nanostars, nanocages, and nanoprisms, are particularly appealing due to their tunable surface plasmon resonances, controllable surface chemistries, and effective targeting capabilities. These complex nanostructures can absorb light in the near-infrared, enabling noteworthy applications in nanomedicine, molecular imaging, and biology. The engineering of targeting abilities through surface modifications involving ligands, antibodies, peptides, and other agents potentiates their effects. Recent years have witnessed the development of innovative structures with diverse compositions, expanding their applications in biomedicine. These applications encompass targeted imaging, surface-enhanced Raman spectroscopy, near-infrared II imaging, catalytic therapy, photothermal therapy, and cancer treatment. This review seeks to provide the nanomedicine community with a thorough and informative overview of the evolving landscape of complex metal nanoparticle research, with a specific emphasis on their roles in imaging, cancer therapy, infectious diseases, and biofilm treatment. This article is categorized under: Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease Diagnostic Tools > Diagnostic Nanodevices.

Targeted deletion of Pf prophages from diverse Pseudomonas aeruginosa isolates has differential impacts on quorum sensing and virulence traits.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that commonly causes medical hardware, wound, and respiratory infections. Temperate filamentous Pf phages that infect P. aeruginosa impact numerous virulence phenotypes. Most work on Pf phages has focused on Pf4 and its host P. aeruginosa PAO1. Expanding from Pf4 and PAO1, this study explores diverse Pf phages infecting P. aeruginosa clinical isolates. We describe a simple technique targeting the Pf lysogeny maintenance gene, pflM (PA0718), that enables the effective elimination of Pf prophages from diverse P. aeruginosa hosts. The pflM gene shows diversity among different Pf phage isolates; however, all examined pflM alleles encode the DUF5447 domain. We demonstrate that pflM deletion results in prophage excision but not replication, leading to total prophage loss, indicating a role for lysis/lysogeny decisions for the DUF5447 domain. This study also assesses the effects different Pf phages have on host quorum sensing, biofilm formation, pigment production, and virulence against the bacterivorous nematode Caenorhabditis elegans. We find that Pf phages have strain-specific impacts on quorum sensing and biofilm formation, but nearly all suppress pigment production and increase C. elegans avoidance behavior. Collectively, this research not only introduces a valuable tool for Pf prophage elimination from diverse P. aeruginosa isolates but also advances our understanding of the complex relationship between P. aeruginosa and filamentous Pf phages.IMPORTANCEPseudomonas aeruginosa is an opportunistic bacterial pathogen that is frequently infected by filamentous Pf phages (viruses) that integrate into its chromosome, affecting behavior. Although prior work has focused on Pf4 and PAO1, this study investigates diverse Pf in clinical isolates. A simple method targeting the deletion of the Pf lysogeny maintenance gene pflM (PA0718) effectively eliminates Pf prophages from clinical isolates. The research evaluates the impact Pf prophages have on bacterial quorum sensing, biofilm formation, and virulence phenotypes. This work introduces a valuable tool to eliminate Pf prophages from clinical isolates and advances our understanding of P. aeruginosa and filamentous Pf phage interactions.

Pf bacteriophages hinder sputum antibiotic diffusion via electrostatic binding.

Despite great progress in the field, chronic Pseudomonas aeruginosa (Pa) infections remain a major cause of morbidity and mortality in patients with cystic fibrosis, necessitating treatment with inhaled antibiotics. Pf phage is a filamentous bacteriophage produced by Pa that has been reported to act as a structural element in Pa biofilms. Pf presence has been associated with resistance to antibiotics and poor outcomes in cystic fibrosis, though the underlying mechanisms are unclear. Here, we have investigated how Pf phages and sputum biopolymers impede antibiotic diffusion using human sputum samples and fluorescent recovery after photobleaching. We demonstrate that tobramycin interacts with Pf phages and sputum polymers through electrostatic interactions. We also developed a set of mathematical models to analyze the complex observations. Our analysis suggests that Pf phages in sputum reduce the diffusion of charged antibiotics due to a greater binding constant associated with organized liquid crystalline structures formed between Pf phages and sputum polymers. This study provides insights into antibiotic tolerance mechanisms in chronic Pa infections and may offer potential strategies for novel therapeutic approaches.

The contribution of neutrophils to bacteriophage clearance and pharmacokinetics in vivo.

With the increasing prevalence of antimicrobial-resistant bacterial infections, there is great interest in using lytic bacteriophages (phages) to treat such infections. However, the factors that govern bacteriophage pharmacokinetics in vivo remain poorly understood. Here, we have examined the contribution of neutrophils, the most abundant phagocytes in the body, to the pharmacokinetics of intravenously administered bacteriophage in uninfected mice. A single dose of LPS-5, an antipseudomonal bacteriophage recently used in human clinical trials, was administered intravenously to both wild-type BALB/c and neutropenic ICR mice. Phage concentrations were assessed in peripheral blood and spleen at 0.5, 1, 2, 4, 8, 12, and 24 hours after administration by plaque assay and qPCR. We observed that the phage clearance is only minimally affected by neutropenia. Indeed, the half-life of phages in blood in BALB/c and ICR mice is 3.45 and 3.66 hours, respectively. These data suggest that neutrophil-mediated phagocytosis is not a major determinant of phage clearance. Conversely, we observed a substantial discrepancy in circulating phage levels over time when measured by qPCR versus plaque assay, suggesting that substantial functional inactivation of circulating phages occurs over time. These data indicate that circulating factors, but not neutrophils, inactivate intravenously administered phages.

Regulatory T cells use heparanase to access IL-2 bound to extracellular matrix in inflamed tissue.

Although FOXP3+ regulatory T cells (Treg) depend on IL-2 produced by other cells for their survival and function, the levels of IL-2 in inflamed tissue are low, making it unclear how Treg access this critical resource. Here, we show that Treg use heparanase (HPSE) to access IL-2 sequestered by heparan sulfate (HS) within the extracellular matrix (ECM) of inflamed central nervous system tissue. HPSE expression distinguishes human and murine Treg from conventional T cells and is regulated by the availability of IL-2. HPSE-/- Treg have impaired stability and function in vivo, including in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Conversely, endowing monoclonal antibody-directed chimeric antigen receptor (mAbCAR) Treg with HPSE enhances their ability to access HS-sequestered IL-2 and their ability to suppress neuroinflammation in vivo. Together, these data identify a role for HPSE and the ECM in immune tolerance, providing new avenues for improving Treg-based therapy of autoimmunity.

Optimized preparation pipeline for emergency phage therapy against Pseudomonas aeruginosa at Yale University.

Bacteriophage therapy is one potential strategy to treat antimicrobial resistant or persistent bacterial infections, and the year 2021 marked the centennial of Felix d'Hérelle's first publication on the clinical applications of phages. At the Center for Phage Biology & Therapy at Yale University, a preparatory modular approach has been established to offer safe and potent phages for single-patient investigational new drug applications while recognizing the time constraints imposed by infection(s). This study provides a practical walkthrough of the pipeline with an Autographiviridae phage targeting Pseudomonas aeruginosa (phage vB_PaeA_SB, abbreviated to ΦSB). Notably, a thorough phage characterization and the evolutionary selection pressure exerted on bacteria by phages, analogous to antibiotics, are incorporated into the pipeline.

The Inovirus Pf4 Triggers Antiviral Responses and Disrupts the Proliferation of Airway Basal Epithelial Cells.

BACKGROUND: The inovirus Pf4 is a lysogenic bacteriophage of Pseudomonas aeruginosa (Pa). People with Cystic Fibrosis (pwCF) experience chronic airway infection with Pa and a significant proportion have high numbers of Pf4 in their airway secretions. Given the known severe damage in the airways of Pa-infected pwCF, we hypothesized a high Pf4 burden can affect airway healing and inflammatory responses. In the airway, basal epithelial cells (BCs) are a multipotent stem cell population critical to epithelium homeostasis and repair. We sought to investigate the transcriptional responses of BCs under conditions that emulate infection with Pa and exposure to high Pf4 burden. METHODS: Primary BCs isolated from pwCF and wild-type (WT) donors were cultured in vitro and exposed to Pf4 or bacterial Lipopolysaccharide (LPS) followed by transcriptomic and functional assays. RESULTS: We found that BCs internalized Pf4 and this elicits a strong antiviral response as well as neutrophil chemokine production. Further, we found that BCs that take up Pf4 demonstrate defective migration and proliferation. CONCLUSIONS: Our findings are highly suggestive of Pf4 playing a role in the pathogenicity of Pa in the airways. These findings provide additional evidence for the ability of inoviruses to interact with mammalian cells and disrupt cell function.

Rapid assessment of changes in phage bioactivity using dynamic light scattering.

Extensive efforts are underway to develop bacteriophages as therapies against antibiotic-resistant bacteria. However, these efforts are confounded by the instability of phage preparations and a lack of suitable tools to assess active phage concentrations over time. In this study, we use dynamic light scattering (DLS) to measure changes in phage physical state in response to environmental factors and time, finding that phages tend to decay and form aggregates and that the degree of aggregation can be used to predict phage bioactivity. We then use DLS to optimize phage storage conditions for phages from human clinical trials, predict bioactivity in 50-y-old archival stocks, and evaluate phage samples for use in a phage therapy/wound infection model. We also provide a web application (Phage-Estimator of Lytic Function) to facilitate DLS studies of phages. We conclude that DLS provides a rapid, convenient, and nondestructive tool for quality control of phage preparations in academic and commercial settings.

Bacterial outer membrane vesicles bound to bacteriophages modulate neutrophil responses to bacterial infection.

Pseudomonas aeruginosa is a major human pathogen, particularly effective at colonizing the airways of patients with cystic fibrosis. Bacteriophages are highly abundant at infection sites, but their impact on mammalian immunity remains unclear. We previously showed that Pf4, a temperate filamentous bacteriophage produced by P. aeruginosa, modifies the innate immune response to P. aeruginosa infections via TLR3 signaling, but the underlying mechanisms remained unclear. Notably, Pf4 is a single-stranded DNA and lysogenic phage, and its production does not typically result in lysis of its bacterial host. We identified previously that internalization of Pf4 by human or murine immune cells triggers maladaptive viral pattern recognition receptors and resulted in bacterial persistence based on the presence of phage RNA. We report now that Pf4 phage dampens inflammatory responses to bacterial endotoxin and that this is mediated in part via bacterial vesicles attached to phage particles. Outer membrane vesicles (OMVs) are produced by Gram-negative bacteria and play a key role in host pathogen interaction. Recently, evidence has emerged that OMVs differentially package small RNAs. In this study, we show that Pf4 are decorated with OMVs that remain affixed to Pf4 despite of purification steps. These phages are endocytosed by human cells and delivered to endosomal vesicles. We demonstrate that short RNAs within the OMVs form hairpin structures that trigger TLR3-dependent type I interferon production and antagonize production of antibacterial cytokines and chemokines. In particular, Pf4 phages inhibit CXCL5, preventing efficient neutrophil chemotaxis in response to endotoxin. Moreover, blocking IFNAR or TLR3 signaling abrogates the effect of Pf4 bound to OMVs on macrophage activation. In a murine acute pneumonia model, mice treated with Pf4 associated with OMVs show significantly less neutrophil infiltration in BAL fluid than mice treated with purified Pf4. These changes in macrophage phenotype are functionally relevant: conditioned media from cells exposed to Pf4 decorated with OMVs are significantly less effective at inducing neutrophil migration in vitro and in vivo. These results suggest that Pf4 phages alter innate immunity to bacterial endotoxin and OMVs, potentially dampening inflammation at sites of bacterial colonization or infection.

Targeted deletion of Pf prophages from diverse Pseudomonas aeruginosa isolates impacts quorum sensing and virulence traits.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that commonly causes medical hardware, wound, and respiratory infections. Temperate filamentous Pf phages that infect P. aeruginosa impact numerous bacterial virulence phenotypes. Most work on Pf phages has focused on strain Pf4 and its host P. aeruginosa PAO1. Expanding from Pf4 and PAO1, this study explores diverse Pf strains infecting P. aeruginosa clinical isolates. We describe a simple technique targeting the Pf lysogeny maintenance gene, pflM (PA0718), that enables the effective elimination of Pf prophages from diverse P. aeruginosa hosts. This study also assesses the effects different Pf phages have on host quorum sensing, biofilm formation, virulence factor production, and virulence. Collectively, this research not only introduces a valuable tool for Pf prophage elimination from diverse P. aeruginosa isolates, but also advances our understanding of the complex relationship between P. aeruginosa and filamentous Pf phages.

Inhibition of hyaluronan synthesis prevents ß-cell loss in obesity-associated type 2 diabetes.

Pancreatic ß-cell dysfunction and death are central to the pathogenesis of type 2 diabetes (T2D). We identified a novel role for the inflammatory extracellular matrix polymer hyaluronan (HA) in this pathophysiology. Low concentrations of HA were present in healthy pancreatic islets. However, HA substantially accumulated in cadaveric islets of T2D patients and islets of the db/db mouse model of T2D in response to hyperglycemia. Treatment with 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis, or the deletion of the main HA receptor CD44, preserved glycemic control and insulin concentrations in db/db mice despite ongoing weight gain, indicating a critical role for this pathway in T2D pathogenesis. 4-MU treatment and the deletion of CD44 likewise preserved glycemic control in other settings of ß-cell injury including streptozotocin treatment and islet transplantation. Mechanistically, we found that 4-MU increased the expression of the apoptosis inhibitor survivin, a downstream transcriptional target of CD44 dependent on HA/CD44 signaling, on ß-cells such that caspase 3 activation did not result in ß-cell apoptosis. These data indicated a role for HA accumulation in diabetes pathogenesis and suggested that it may be a viable target to ameliorate ß-cell loss in T2D. These data are particularly exciting, because 4-MU is already an approved drug (also known as hymecromone), which could accelerate translation of these findings to clinical studies.

Theranostic gold-in-gold cage nanoparticles enable photothermal ablation and photoacoustic imaging in biofilm-associated infection models.

Biofilms are structured communities of microbial cells embedded in a self-produced matrix of extracellular polymeric substances. Biofilms are associated with many health issues in humans, including chronic wound infections and tooth decay. Current antimicrobials are often incapable of disrupting the polymeric biofilm matrix and reaching the bacteria within. Alternative approaches are needed. Here, we described a complex structure of a dextran-coated gold-in-gold cage nanoparticle that enabled photoacoustic and photothermal properties for biofilm detection and treatment. Activation of these nanoparticles with a near infrared laser could selectively detect and kill biofilm bacteria with precise spatial control and in a short timeframe. We observed a strong biocidal effect against both Streptococcus mutans and Staphylococcus aureus biofilms in mouse models of oral plaque and wound infections, respectively. These effects were over 100 times greater than those seen with chlorhexidine, a conventional antimicrobial agent. Moreover, this approach did not adversely affect surrounding tissues. We concluded that photothermal ablation using theranostic nanoparticles is a rapid, precise, and nontoxic method to detect and treat biofilm-associated infections.

Rapid assessment of changes in phage bioactivity using dynamic light scattering.

Extensive efforts are underway to develop bacteriophages as therapies against antibiotic-resistant bacteria. However, these efforts are confounded by the instability of phage preparations and a lack of suitable tools to assess active phage concentrations over time. Here, we use Dynamic Light Scattering (DLS) to measure changes in phage physical state in response to environmental factors and time, finding that phages tend to decay and form aggregates and that the degree of aggregation can be used to predict phage bioactivity. We then use DLS to optimize phage storage conditions for phages from human clinical trials, predict bioactivity in 50-year-old archival stocks, and evaluate phage samples for use in a phage therapy/wound infection model. We also provide a web-application (Phage-ELF) to facilitate DLS studies of phages. We conclude that DLS provides a rapid, convenient, and non-destructive tool for quality control of phage preparations in academic and commercial settings.

Monocytes use protrusive forces to generate migration paths in viscoelastic collagen-based extracellular matrices.

Circulating monocytes are recruited to the tumor microenvironment, where they can differentiate into macrophages that mediate tumor progression. To reach the tumor microenvironment, monocytes must first extravasate and migrate through the type-1 collagen rich stromal matrix. The viscoelastic stromal matrix around tumors not only stiffens relative to normal stromal matrix, but often exhibits enhanced viscous characteristics, as indicated by a higher loss tangent or faster stress relaxation rate. Here, we studied how changes in matrix stiffness and viscoelasticity, impact the three-dimensional migration of monocytes through stromal-like matrices. Interpenetrating networks of type-1 collagen and alginate, which enable independent tunability of stiffness and stress relaxation over physiologically relevant ranges, were used as confining matrices for three-dimensional culture of monocytes. Increased stiffness and faster stress relaxation independently enhanced the 3D migration of monocytes. Migrating monocytes have an ellipsoidal or rounded wedge-like morphology, reminiscent of amoeboid migration, with accumulation of actin at the trailing edge. Matrix adhesions and Rho-mediated contractility were dispensable for monocyte migration in 3D, but migration did require actin polymerization and myosin contractility. Mechanistic studies indicate that actin polymerization at the leading edge generates protrusive forces that open a path for the monocytes to migrate through in the confining viscoelastic matrices. Taken together, our findings implicate matrix stiffness and stress relaxation as key mediators of monocyte migration and reveal how monocytes use pushing forces at the leading edge mediated by actin polymerization to generate migration paths in confining viscoelastic matrices. Significance Statement: Cell migration is essential for numerous biological processes in health and disease, including for immune cell trafficking. Monocyte immune cells migrate through extracellular matrix to the tumor microenvironment where they can play a role in regulating cancer progression. Increased extracellular matrix (ECM) stiffness and viscoelasticity have been implicated in cancer progression, but the impact of these changes in the ECM on monocyte migration remains unknown. Here, we find that increased ECM stiffness and viscoelasticity promote monocyte migration. Interestingly, we reveal a previously undescribed adhesion-independent mode of migration whereby monocytes generate a path to migrate through pushing forces at the leading edge. These findings help elucidate how changes in the tumor microenvironment impact monocyte trafficking and thereby disease progression.

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

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.