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Until recently, Cryptococcus gattii infections occurred mainly in tropical and subtropical climate zones. However, during the past decade, C. gattii infections in humans and animals in Europe have increased. To [...]

Summary Biofilms—communities of bacteria encased in a polymer-rich matrix—confer bacteria with the ability to persist in pathologic host contexts, such as the cystic fibrosis (CF) airways. How bacteria assemble polymers into biofilms is largely unknown. We find that the extracellular matrix produced by Pseudomonas aeruginosa self-assembles into a liquid crystal through entropic interactions between polymers and filamentous Pf bacteriophages, which are long, negatively charged filaments. This liquid crystalline structure enhances biofilm function by increasing adhesion and tolerance to desiccation and antibiotics. Pf bacteriophages are prevalent among P. aeruginosa clinical isolates and were detected in CF sputum. The addition of Pf bacteriophage to sputum polymers or serum was sufficient to drive their rapid assembly into viscous liquid crystals. Fd, a related bacteriophage of Escherichia coli , has similar biofilm-building capabilities. Targeting filamentous bacteriophage or the liquid crystalline organization of the biofilm matrix may represent antibacterial strategies. [ABSTRACT FROM AUTHOR]

Pseudomonas strains that produce filamentous bacteriophage are associated with chronic infection and increased antibiotic resistance in patients with cystic fibrosis. Infection-boosting phage: Chronic Pseudomonas aeruginosa infection is common in patients with cystic fibrosis (CF). Filamentous bacteriophage (Pf phage) can infect P. aeruginosa and has been shown to contribute to the virulence of infection in animal models. However, whether Pf phage plays a role in the pathogenicity of P. aeruginosa in CF is unknown. Now, Burgener et al. showed that Pf phage was abundantly expressed in sputum samples from two large cohorts of patients with CF. The presence of Pf phage was associated with increased antibiotic resistance and reduced lung function. The results suggest that Pf phage might play a role in the pathogenicity of P. aeruginosa infection in CF. Filamentous bacteriophage (Pf phage) contribute to the virulence of Pseudomonas aeruginosa infections in animal models, but their relevance to human disease is unclear. We sought to interrogate the prevalence and clinical relevance of Pf phage in patients with cystic fibrosis (CF) using sputum samples from two well-characterized patient cohorts. Bacterial genomic analysis in a Danish longitudinal cohort of 34 patients with CF revealed that 26.5% (n = 9) were consistently Pf phage positive. In the second cohort, a prospective cross-sectional cohort of 58 patients with CF at Stanford, sputum qPCR analysis showed that 36.2% (n = 21) of patients were Pf phage positive. In both cohorts, patients positive for Pf phage were older, and in the Stanford CF cohort, patients positive for Pf phage were more likely to have chronic P. aeruginosa infection and had greater declines in pulmonary function during exacerbations than patients negative for Pf phage presence in the sputum. Last, P. aeruginosa strains carrying Pf phage exhibited increased resistance to antipseudomonal antibiotics. Mechanistically, in vitro analysis showed that Pf phage sequesters these same antibiotics, suggesting that this mechanism may thereby contribute to the selection of antibiotic resistance over time. These data provide evidence that Pf phage may contribute to clinical outcomes in P. aeruginosa infection in CF. [ABSTRACT FROM AUTHOR]

A summary is presented of an article published online at http://dx.doi.org/10.1126/science.aat9691 on pathogenic roles that bacteriophage virions play in bacterial infections. It states that Pseudomonas aeruginosa (Pa) and the filamentous bacteriophage Pf produced by Pa was studied to determine the impact Pf played in Pa wound infections in animal models and humans. It considers the possible therapeutic benefits of vaccination against bacteriophage virions to reduce virulence.

Background: Immune-related adverse events (irAEs) are major barriers of clinical management and further development of immune checkpoint inhibitors (ICIs) for cancer therapy. Therefore, biomarkers associated with the onset of severe irAEs are needed. In this study, we aimed to identify immune features detectable in peripheral blood and associated with the development of severe irAEs that required clinical intervention., Methods: We used a 43-marker mass cytometry panel to characterize peripheral blood mononuclear cells from 28 unique patients with melanoma across 29 lines of ICI therapy before treatment (baseline), before the onset of irAEs (pre-irAE) and at the peak of irAEs (irAE-max). In the 29 lines of ICI therapy, 18 resulted in severe irAEs and 11 did not., Results: Unsupervised and gated population analysis showed that patients with severe irAEs had a higher frequency of CD4 + naïve T cells and lower frequency of CD16 + natural killer (NK) cells at all time points. Gated population analysis additionally showed that patients with severe irAEs had fewer T cell immunoreceptor with Ig and ITIM domain (TIGIT + ) regulatory T cells at baseline and more activated CD38 + CD4 + central memory T cells (TCM) and CD39 + and Human Leukocyte Antigen-DR Isotype (HLA-DR) + CD8 + TCM at peak of irAEs. The differentiating immune features at baseline were predominantly seen in patients with gastrointestinal and cutaneous irAEs and type 1 diabetes. Higher frequencies of CD4 + naïve T cells and lower frequencies of CD16 + NK cells were also associated with clinical benefit to ICI therapy., Conclusions: This study demonstrates that high-dimensional immune profiling can reveal novel blood-based immune signatures associated with risk and mechanism of severe irAEs. Development of severe irAEs in melanoma could be the result of reduced immune inhibitory capacity pre-ICI treatment, resulting in more activated TCM cells after treatment., Competing Interests: Competing interests: SH-L has been scientific advisor/consultant for: Amgen, Ascendis, Astellas, BMS, Genmab, Merck, Nektar, Neon Therapeutics, Novartis, Regeneron, Vaccinex, Xencor; and has done contracted research through her affiliated institutions with Astellas, BioAtla, BMS, Boehringer Ingelheim, Checkmate, Dragonfly, F Star, Genentech, Kite Pharma, Merck, Neon Therapeutics, OncoC4, Pfizer, Plexxikon, Vaccinex, Vedanta, Xencor. US reports consultancy to Astellas, Exelixis, Seattle Genetics, Imvax, Sanofi, AstraZeneca and Gilead and research funding to institute from Janssen, Exelixis and Astellas/Seattle Genetics. JH conducts clinical trials from the following entities: BMS, Merck, Amgen, Philogen, Lyell, Lovance, NCI, Takara, Natera, Skyline. JM, AE-W, MK-B, ALE, JPM : None. JMS, NS, L-CC, WDC, COM and NN are currently employed by Teiko Bio and are shareholders. SAE and CPH was formerly employed by Teiko Bio and is a current shareholder. MHS and RS are founders, shareholders and board members of Teiko.bio. MHS has received a speaking honorarium from Standard BioTools. and Kumquat Bio, has been a paid consultant for Five Prime, Ono, January, Earli, Astellas, and Indaptus, and has received research funding from Roche/Genentech, Pfizer, Valitor, and Bristol Myers Squibb. WDC was employed by Fate Therapeutics within the last 24 months. NN was employed by Atreca within the last 24 months., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: There is increasing interest in the pulmonary microbiome's bacterial and viral communities, particularly in the context of chronic airway infections in cystic fibrosis (CF). However, the isolation of microbial DNA from the sputum from patients with CF is technically challenging and the optimal protocols for the analysis of viral species, including bacteriophage, from clinical samples remains difficult. Materials and Methods: In this study, we evaluate a set of methods developed for processing and analyzing sputum from patients with CF with the goal of detecting Pf bacteriophage virion-derived nucleic acid. We evaluate the impact of bead beating, deoxyribonuclease digestion, and heating steps in these protocols focusing on the quantitative assessment of Pseudomonas aeruginosa and Pf bacteriophage in sputum. Results: Based on these comparative data, we describe an optimized protocol for processing sputum from patients with CF and isolating DNA for polymerase chain reaction or sequencing-based studies. Conclusion: These studies demonstrate the assessment of a specific bacteriophage and bacteria in sputum from patients with CF., Competing Interests: C.E.M. reports grants from Cystic Fibrosis Foundation, clinical trial support from Vertex Pharmaceuticals, Proteostasis and Parion, and consulting fees from Vertex. P.L.B. reports grants from the Cystic Fibrosis Foundation. No competing financial interests exist for other authors., (Copyright 2020, Mary Ann Liebert, Inc., publishers.)

Pseudomonas aeruginosa (P. aeruginosa) is a major nosocomial pathogen of increasing relevance to human health and disease, particularly in the setting of chronic wound infections in diabetic and hospitalized patients. There is an urgent need for chronic infection models to aid in the investigation of wound pathogenesis and the development of new therapies against this pathogen. Here, we describe a protocol that uses delayed inoculation 24 hours after full-thickness excisional wounding. The infection of the provisional wound matrix present at this time forestalls either rapid clearance or dissemination of infection and instead establishes chronic infection lasting 7-10 days without the need for implantation of foreign materials or immune suppression. This protocol mimics a typical temporal course of post-operative infection in humans. The use of a luminescent P. aeruginosa strain (PAO1:lux) allows for quantitative daily assessment of bacterial burden for P. aeruginosa wound infections. This novel model may be a useful tool in the investigation of bacterial pathogenesis and the development of new therapies for chronic P. aeruginosa wound infections.

Pseudomonas aeruginosa (Pa) and Candida albicans (Ca) are major bacterial and fungal pathogens in immunocompromised hosts, and notably in the airways of cystic fibrosis patients. The bacteriophages of Pa physically alter biofilms, and were recently shown to inhibit the biofilms of Aspergillus fumigatus. To understand the range of this viral-fungal interaction, we studied Pa phages Pf4 and Pf1, and their interactions with Ca biofilm formation and preformed Ca biofilm. Both forms of Ca biofilm development, as well as planktonic Ca growth, were inhibited by either phage. The inhibition of biofilm was reversed by the addition of iron, suggesting that the mechanism of phage action on Ca involves denial of iron. Birefringence studies on added phage showed an ordered structure of binding to Ca. Electron microscopic observations indicated phage aggregation in the biofilm extracellular matrix. Bacteriophage-fungal interactions may be a general feature with several pathogens in the fungal kingdom.

Pseudomonas aeruginosa (Pa) and Aspergillus fumigatus (Af) are major human pathogens known to interact in a variety of disease settings, including airway infections in cystic fibrosis. We recently reported that clinical CF isolates of Pa inhibit the formation and growth of Af biofilms. Here, we report that the bacteriophage Pf4, produced by Pa, can inhibit the metabolic activity of Af biofilms. This phage-mediated inhibition was dose dependent, ablated by phage denaturation, and was more pronounced against preformed Af biofilm rather than biofilm formation. In contrast, planktonic conidial growth was unaffected. Two other phages, Pf1 and fd, did not inhibit Af, nor did supernatant from a Pa strain incapable of producing Pf4. Pf4, but not Pf1, attaches to Af hyphae in an avid and prolonged manner, suggesting that Pf4-mediated inhibition of Af may occur at the biofilm surface. We show that Pf4 binds iron, thus denying Af a crucial resource. Consistent with this, the inhibition of Af metabolism by Pf4 could be overcome with supplemental ferric iron, with preformed biofilm more resistant to reversal. To our knowledge, this is the first report of a bacterium producing a phage that inhibits the growth of a fungus and the first description of a phage behaving as an iron chelator in a biological system.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen associated with many types of chronic infection. At sites of chronic infection, such as the airways of people with cystic fibrosis (CF), P. aeruginosa forms biofilm-like aggregates. These are clusters of bacterial cells encased in a polymer-rich matrix that shields bacteria from environmental stresses and antibiotic treatment. When P. aeruginosa forms a biofilm, large amounts of filamentous Pf bacteriophage (phage) are produced. Unlike most phage that typically lyse and kill their bacterial hosts, filamentous phage of the genus Inovirus, which includes Pf phage, often do not, and instead are continuously extruded from the bacteria. Here, we discuss the implications of the accumulation of filamentous Pf phage in the biofilm matrix, where they interact with matrix polymers to organize the biofilm into a highly ordered liquid crystal. This structural configuration promotes bacterial adhesion, desiccation survival, and antibiotic tolerance - all features typically associated with biofilms. We propose that Pf phage make structural contributions to P. aeruginosa biofilms and that this constitutes a novel form of symbiosis between bacteria and bacteriophage., Competing Interests: Conflict of interest: The authors declare no conflict of interest.