Journal of Cystic Fibrosis-Vol. 24 - Suppl.2

October 2025 Volume 24, S2 October 22–25, Seattle, Washington Abstracts of the 2025 North American Cystic Fibrosis Conference Journal of Cystic Fibrosis The Official Journal of the European Cystic Fibrosis Society www.ECFS.eu ISSN 1569-1993 6.0

Editorial Board Editor-in-Chief: Patrick Flume, Medical University of South Carolina, Charleston, South Carolina, USA Deputy Editors: Carlo Castellani, Giannina Gaslini Institute, Genova, Italy Harriet Corvol, Sorbonne University, Paris, France Editorial Board: Editor-in-Chief, Cystic Fibrosis Research News: Harry Heijerman, University Medical Centre Utrecht, Utrecht, Netherlands Previous Editors-in-Chief: Harry Heijerman, University Medical Centre Utrecht, Utrecht, Netherlands Gerd Döring, University of Tübingen, Tübingen, Germany Scott Bell, The Prince Charles Hospital, Queensland, Australia Scott Blackman, USA Pierre-Régis Burgel, France Rebecca Dobra, UK Scott Donaldson, USA Aleksander Edelman, France Pascale Fanen, France Carlos Farinha, Portugal Sonya Heltshe, USA Niels Høiby, Denmark Dominic Hughes, UK Andrea Kelly, USA Susannah King, Australia Paul McCray, USA Marianne Muhlebach, USA Chee Yee Ooi, Australia Nicoletta Pedemonte, Italy Gerald Pier, USA Felix Ratjen, Canada Kristin A. Riekert, USA Dorota Sands, Poland Michal Schteinberg, USA Anne Stephenson, Canada Rhonda Szczesniak, USA Cliff Taggart, UK Jennifer Taylor-Cousar, USA Daan Touw, The Netherlands Michael Tunney, UK Donald Van Devanter, USA Valerie Waters, Canada Michael Wilschanski, Israel Jeffrey Wine, USA Amsterdam — Boston — London —New York — Oxford —Paris — Philadelphia —San Diego —St. Louis — Tokyo

Volume 21 (2022) Publication of this Abstract Supplement is supported by The Cystic Fibrosis Foundation. Abstracts of the North American Cystic Fibrosis Conference November 3–5, 2022 Volume 21, Supplement 2 (2022) 2025 North American Cystic Fibrosis Conference October 22–25, Seattle, Washington Volume 24, Supplement 2 (2025)

Contents, Volume 17 (2018) Supplement Abstracts of the 40th European Cystic Fibrosis Conference Seville, Spain, 7–10 June 2017 Workshops S1 Workshop 1. Old and new thoughts on antibiotics ............................................................................ S1 Workshop 2. Nutrition: from daily practice to enzymes for the future ............................................ S3 Workshop 3. New therapies targeting the airway surface ................................................................. S4 Workshop 4. New insights from inflammation and immunology...................................................... S6 Workshop 5. Anxiety and depression: a family affair ........................................................................ S7 Workshop 6. CFTR dysfunction: what happens where? ..................................................................... S9 Workshop 7. Microbiome analysis in CF: what’s new in lung and gut?............................................ S11 Workshop 8. CT and MRI, where are we? Ready yet for the clinics? ................................................ S12 Workshop 9. Exercise and correlations with other outcomes............................................................ S14 Workshop 10. The impact of CF: high mountains – deep valleys ...................................................... S16 Workshop 11. Newborn screening and diagnostic advances .............................................................. S17 Workshop 12. Bone health, glucose metabolism and CFRD ............................................................... S19 Workshop 13. Rescuing CFTR: new developments ............................................................................. S20 Workshop 14. Basic pathogenesis: Pseudomonas, microbiota interaction and viruses...................... S22 Workshop 15. New lung function methods to monitor disease and treatment ................................ S24 Workshop 16. Understanding and teaching, a knowledge network................................................... S26 Workshop 17. CF related liver disease and pancreatic insufficiency: can we do better? .................. S28 Workshop 18. CFTR: Functional tests for therapeutic interventions.................................................. S29 Workshop 20. Evolving epidemiology and risk factors for lung infection......................................... S31 Workshop 21. How to personalise chest physiotherapy? ................................................................... S32 Workshop 23. Insights from registries and cohorts............................................................................ S34 ePS01. Screening and Diagnosis ........................................................................................................... S36 ePS02. The CF team in development ................................................................................................... S39 ePS03. New treatments ........................................................................................................................ S41 ePS04. Multi-tasking physiotherapists: managing hygiene, pain, exercise, . . . .................................. S43 2 21 2022 Abstracts of the North American Cystic Fibrosis Conference November 3–5, 2022 Poster Categories ��S1 Endocrine/Bone ��S1 Epidemiology & Population-based Research��S12 Quality Improvement�� S35 Pulmonary �� S66 Transplantation �� S84 Clinical Genetics �� S87 Clinical Trials & Outcome Measures�� S89 GI/Nutrition �� S110 Nursing Issues �� S142 Pharmacy �� S146 Physical & Respiratory Therapy �� S157 Psychosocial/Behavioral �� S166 Education �� S199 Health Equity, Care, Delivery & Access to Care ��S207 Airways Physiology, Pathophysiology & Airways Defense �� S223 Infection/Microbiology �� S260 Extrapulmonary Physiology & Pathophysiology ��S317 Path to a Cure ��S327 CFTR �� S352 This abstract book has been produced electronically by Elsevier B�V�, and is also available on USB� Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds or experiments described herein� Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made� To the fullest extent of the law, no responsibility is assumed by Elsevier or the European Cystic Fibrosis Society for any injury and/ or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein� 24 (2025) Supplement 2 2025 North American Cystic Fibrosis Conference October 22–25, 2025 Infection/Microbiology �� S1 Clinical Trials & Outcome Measures ��S53 Airways Physiology, Pathophysiology & Airways Defense ��S84 Path to a Cure ��S122 CFTR �� S160 Clinical Genetics ��S181 Extrapulmonary Physiology & Pathophysiology ��S184 Health Equity, Care, Delivery, & Access to Care ��S205 Education �� S239 Nursing Issues ��S250 Pharmacy/Pharmacology �� S260 Physical & Respiratory Therapy ��S281 Psychosocial/Behavioral �� S293 GI/Nutrition �� S323 Transplantation �� S374 Endocrine/Bone �� S401 Epidemiology & Population-Based Research ��S425 Quality Improvement ��S461 This abstract book has been produced electronically by Elsevier B.V., and is also available on USB. experiments described herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. To the fullest extent of the law, no responsibility is assumed by Elsevier or the European Cystic Fibrosis Society for any injury and/ instructions, or ideas contained in the material herein.

INFECTION/MICROBIOLOGY 1 Amine oxidases mediate fungal adaptation to host-relevant stressors in Aspergillus fumigatus E. Reinhardt1, S. Dolan2. 1Genetics and Biochemistry, Clemson University, Clemson, SC, USA; 2Eukaryotic Pathogens Innovation Center, Clemson University, Clemson, SC, USA Background: Aspergillus fumigatus is a filamentous fungus and opportunistic pathogen that frequently co-colonizes the cystic fibrosis (CF) lung alongside bacterial species such as Pseudomonas aeruginosa. These polymicrobial interactions significantly reshape fungal physiology and virulence, yet the underlying molecular mechanisms remain poorly defined. This project investigates the role of amine oxidases—enzymes that catalyze the oxidative deamination of amines, producing aldehydes, ammonia, and hydrogen peroxide—in fungal adaptation to complex microbial environments. While extensively studied in bacteria and higher eukaryotes, the function of amine oxidases in filamentous fungi remains largely unexplored. Methods: Through genome-wide analysis, we identified 17 putative amine oxidases in A. fumigatus, several of which are transcriptionally upregulated in response to CF-associated bacterial pathogens, implicating them in interkingdom adaptation. To define their roles, we constructed a complete gene deletion library encompassing all 17 oxidases. Using a combination of phenotypic profiling, fluorescence microscopy with mNeonGreen-tagged fusion proteins, and co-culture assays with P. aeruginosa, we are systematically characterizing the contribution of each oxidase to fungal growth, stress tolerance, and bacterial interactions. Results: Preliminary results reveal that specific deletions impact fungal fitness during bacterial co-culture or upon exposure to associated stressors, highlighting a potential role in microbial competition. Conclusions: This work will advance our understanding of fungal metabolic adaptation in polymicrobial infections and may uncover novel targets for antifungal intervention. Acknowledgements: SKD is supported by grant K22AI174009 from the National Institute of Allergy and Infectious Diseases and the Cystic Fibrosis Foundation (DOLAN24A0). 2 Shared evolutionary trajectories of Pseudomonas aeruginosain fungal co-culture and chronic lung infections C. Kennedy1, S. Dolan2. 1Genetics and Biochemistry, Clemson University, Clemson, SC, USA; 2Eukaryotic Pathogens Innovation Center, Clemson University, Clemson, SC, USA Background: Pseudomonas aeruginosa (Pa) is a Gram-negative opportunistic pathogen associated with a broad spectrum of infections, including sepsis, pneumonia, urinary tract infections, and chronic respiratory disease in individuals with cystic fibrosis (CF). In the CF lung, Pa frequently coexists with the filamentous fungus Aspergillus fumigatus (Af), forming polymicrobial biofilms that drive microbial evolution through complex and dynamic selective pressures. Methods: To investigate how Pa adapts in these polymicrobial environments, we conducted an experimental evolution study using synthetic CF sputum medium (SCFM2) to simulate the CF airway. Pa populations were serially passaged over 15 days in monoculture and in co-culture with Af. Phenotypic variation and adaptive changes were assessed through longitudinal growth analysis and whole-genome sequencing of pooled populations from 10 independent lineages per condition. Results: Sequencing data were mapped to the Pa reference genome and analyzed using the breseq pipeline to identify mutations arising under each condition. Strikingly, co-culture conditions consistently selected for loss-of-function mutations in genes which are also frequently observed in Pa isolates from chronically infected CF lungs, suggesting that fungal coculture recapitulates key evolutionary pressures of the host environment. Conclusions: Follow-up validation using reverse genetics and in vitro assays confirmed that these adaptations have functional consequences for Pa physiology during co-culture. This study highlights the importance of interkingdom interactions in shaping bacterial evolution and provides a tractable model for understanding microbial adaptation during chronic CF infection. Acknowledgements: SKD is supported by grant K22AI174009 from the National Institute of Allergy and Infectious Diseases and the Cystic Fibrosis Foundation (DOLAN24A0). 3 Quorum sensing-linked metabolic rewiring governs Pseudomonas aeruginosacompetition with fungal pathogens E. Kazi1, S. Dolan2. 1Genetics and Biochemistry, Clemson University, Clemson, SC, USA; 2Eukaryotic Pathogens Innovation Center, Clemson University, Clemson, SC, USA Background: Pseudomonas aeruginosa (Pa) frequently co-colonizes the cystic fibrosis (CF) lung with the filamentous fungus Aspergillus fumigatus (Af), where it exhibits a competitive advantage and inhibits fungal growth. However, the molecular arsenal and regulatory circuits Pa employs to antagonize Af within polymicrobial biofilms remain incompletely understood. Methods: To identify the secondary metabolites and genetic networks underlying this antagonism, we screened culture supernatants from a panel of Pa mutants—each deficient in quorum sensing or toxin biosynthesis—for their ability to inhibit Af growth and disrupt preformed Af biofilms. In parallel, we performed dual-species transcriptomic profiling during co-culture to capture context-dependent regulatory changes and to uncover uncharacterized genes that contribute to fungal antagonism. Results: Our results reveal that Pa utilizes a diverse suite of quorum sensing-regulated factors to inhibit Af. Strikingly, we identified a previously uncharacterized S-adenosylmethionine-dependent methyltransferase that plays a central role in coordinating these antifungal responses under nutrient-limited conditions. Deletion of this gene results in extensive transcriptional rewiring, including impaired expression of key antagonistic metabolites. Comparative transcriptomics of the wild-type and mutant strains revealed disruptions across both canonical and noncanonical quorum sensing circuits, along with major changes in genes involved in lipopolysaccharide biosynthesis and modification. Conclusions: Together, these findings define a new regulatory node that links quorum sensing to metabolic adaptation and antifungal activity, shedding light on the molecular logic Pa uses to dominate fungal competitors in polymicrobial infections. Journal of Cystic Fibrosis 24S2 (2025) S1–S530

Acknowledgements: SKD is supported by grant K22AI174009 from the National Institute of Allergy and Infectious Diseases and the Cystic Fibrosis Foundation (DOLAN24A0). 4 Cystic fibrosis mouse has altered response to gut infection Clostridioides difficile E. Gill1, C. Kaple2, B. Lampert3, B. Hausman2, J. Cadnum2, J. Feczko4, I. Bederman5,M. Drumm6, C. Donskey2, C. Hodges6. 1School of Medicine, Dept. Genetic and Genome Science, Case Western Reserve University, Cleveland, OH, USA; 2Louis Stokes Veterans Affairs Hospital, Case Western Reserve University, Cleveland, OH, USA; 3School of Medicine, Case Western Reserve University, Cleveland, OH, USA; 4Case Western Reserve University, Cleveland, OH, USA; 5School of Medicine, Dept. Genetic and Genome Science, Case Western Reserve University, Cleveland, OH, USA; 6Genetics and Genome Sciences, Case Western Reserve University, Cleveland, OH, USA Background: CFTR dysfunction is associated with many changes in the intestinal microenvironment including changes in pH, microbiome, and cellular alterations including those associated with oxidative stress and endosomal processing. Gut infections in people with CF are rarely studied, and thus our understanding of the role of CFTR dysfunction in gut infections is limited. Clostridioides difficile is a Gram-positive, toxinproducing, anaerobic bacteria that is the most common cause of antibiotic associated diarrhea. We sought to characterize the CF mouse response toC. difficile infection to explore the influence of CFTR dysfunction on the gut’s response to infection. Methods: CFmice (Cftr G542X/G542X) (N = 11) and wildtype mice (Cftr+/+) (N = 13) were treated with the antibiotic cefoperazone for 5 days then 2 days of sterile drinking water before being challenged with 100 μl of 104 colony forming units (CFU) of liveC. difficilevegetative cells (ribotypes 027) via oral gavage. Stool and symptoms were collected on days 1, 3, 6, 10, and 14. Stool was plated on selective media and incubated anaerobically for 48 hours. Mice (n = 6) were sacrificed on day 3 and bile acid, plasma, liver, and colon tissue were collected. Results: The first 7 days after C. difficile challenge CF mice gained weight while WT all lost weight (p < 0.01). On days 1 and 3 no CF mouse experienced diarrhea while 92% (n = 11) WT mice experienced moderate to severe diarrhea (p < 0.01) despite similar levels of toxin present (p > 0.05). CF mice had similar levels of C. difficile CFU’s on day 1 but cleared the infection significantly faster than WT (p < 0.001) (fig. 1) and had almost no detectable toxin by day 10 and none on day 14, while all WT had moderate to heavy toxin presence. CF mice had little to no colon damage, while WT had moderate to severe on pathology. Both primary and secondary bile acids were markedly decreased (primary, WT: 0.11 ± 0.01 vs. CF: 0.07 ± 0.01 μmols/g, secondary WT: 0.21 ± 0.04 vs. CF 0.04 ± 0.01 μmols/g). Lipidomics analyses of stool revealed significant steatorrhea (total lipids, WT: 2.4 ± 0.3 vs. CF: 5.8 ± 0.5 μmols/g). Metabolomics analyses of stool revealed significantly lower excretion of many amino acids and succinate (WT: 1.7 ± 0.03 vs. CF: 0.2 ± 0.01 μmols/g). Conclusions: CF mice present with a remarkable response to the gut bacteriaC. difficile. Diarrhea is a hallmark of C. difficile infection, and while 92% of WT mice exhibited this symptom no CF mouse did, even though both groups had similar levels of toxin present. This was supported by pathology where CF mice had little to no damage to the colon epithelium, while WT had moderate to severe, highlighting that toxin had less impact in CF mice. CF mice also cleared the infection significantly faster than WT mice and had little to no evidence of toxin by day 10 and none by day 14. This may be explained by the significantly lower bile acids present in CF mice. Of note, C. difficile exploits increased succinate levels in antibiotic disturbed biomes to increase colonization. CF mice had significantly lower levels of succinate despite antibiotic treatment than WT mice, which may explain the CF mice’s ability to clear the infection significantly faster. In conclusion, CF mice have a remarkable and unique response to the gut infectionC. difficile that we theorize is due to CFTR dysfunction in the gut. 5 Cefiderocol treatment for Gram-negative bacterial infections in patients with cystic fibrosis: results from the multinational observational PROVE study J. Pearson1, F. Parquin2, K. Gill3, S. Verardi3, A. Santerre Henriksen3, S. Nguyen4. 1Department of Pharmacy, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA; 2Thoracic Intensive Care Unit, Foch Hospital, Suresnes, France; 3Shionogi BV, London, UK; 4Medical Affairs, Shionogi Inc, Florham Park, NJ, USA Background: Pseudomonas aeruginosa, Burkholderia cepacia complex, Stenotrophomonas maltophilia, and Achromobacter xylosoxidans are among the most common Gram-negative pathogens associated with cystic fibrosis (CF) lung infections, and chronic lung infections are the primary cause of morbidity and mortality in CF [1]. Treatment options are limited for multidrug-resistant infections. The clinical outcomes for 44 cefiderocol-treated patients with CF and serious Gram-negative bacterial infections enrolled in the PROVE study were assessed. Methods: PROVE was a multinational, observational medical chart review study [2] conducted between November 2020 and July 2024. Data were analyzed from hospitalized patients with CF (reported as a comorbidity) and confirmed Gram-negative bacterial infections, who were treated with cefiderocol for the first time for ≥72 hours. Information about baseline demographics, clinical characteristics, cefiderocol use, clinical response at the end of treatment (EOT), and all-cause mortality (ACM) rates were collected. Results: The median age of the CF cohort was 31 years, 47.7% were male, and 52.3% were enrolled in the USA (Table 1). Cefiderocol was given for a median (interquartile range) of 13.0 (7.5–17.5) days and as combination therapy in 59.1% of patients. The majority of the cohort (79.5%) had monomicrobial infections, mostly with P. aeruginosa (63.6%). Other identified monomicrobial pathogens included Burkholderia spp. (n=4) and Achromobacter spp. (n = 3). Rare non-fermenters in polymicrobial infections includedAchromobacter spp. (n = 4), B. multivorans (n=1), andS. maltophilia (n = 1). Most patients (86.4%) received cefiderocol for the treatment of respiratory tract infections. Overall, the clinical response rate at EOT was 72.7%: 71.4% for monomicrobial and 77.8% for polymicrobial infections. The overall 30-day ACM rate was 18.2%. For those with P. aeruginosa infections, clinical response and 30-day ACM rates were 71.4% and 21.4%, respectively. The clinical response and 30-day ACM rates in patients with monomicrobial infections other thanP. aeruginosawere71.4% and 14.3%, respectively. Conclusions: Cefiderocol may play an important role in the treatment of difficult-to-treat Gram-negative infections in patients with CF. Acknowledgements: The study was funded by Shionogi & Co., Ltd., Osaka, Japan. References [1] Françoise A, Héry-Arnaud G. The microbiome in cystic fibrosis pulmonary disease. Genes (Basel). 2020;11:536. [2] Clancy CJ, Cornely OA, Marcella SWet al. Effectiveness and safety of cefiderocol in clinical practice for treatment of patients with Gramnegative bacterial infections: US interim results of the PROVE study. Infect Drug Resist. 2024;17:4427–43. Journal of Cystic Fibrosis 24S2 (2025) S1–S530 S2

Table 1(abstract 5). Journal of Cystic Fibrosis 24S2 (2025) S1–S530 S3

6 Hidden in plain sight: rediscovering mast cells in cystic fibrosis I.Miralda1, J.Moran2, M. Shrestha2,H. Ozuna2, S. Durfey2, B. Kopp3. 1Center for Cystic Fibrosis and Airways Disease Research (CF-AIR), Division of Pulmonology, Asthma, Cystic Fibrosis, and Sleep, Emory University School of Medicine, Atlanta, GA, USA; 2Center for Cystic Fibrosis and Airways Disease Research (CF-AIR), Division of Pulmonology, Asthma, Cystic Fibrosis, and Sleep, Department of Pediatrics, School of Medicine, Emory University, Atlanta, GA, USA; 3Center for Cystic Fibrosis and Airways Disease Research (CF-AIR), Division of Pulmonology, Asthma, Cystic Fibrosis, and Sleep, Department of Pediatrics, School of Medicine, Emory University, Atlanta, GA, USA; Children’s Healthcare of Atlanta, Atlanta, GA, USA Background: CFTR modulator therapy has significantly improved clinical outcomes and quality of life for people with Cystic Fibrosis (pwCF); however, persistent immune dysfunction and bacterial infections remain significant challenges in managing CF disease. The role of dysfunctional CFTR in epithelial cell biology is well understood; however, its function in immune cells, which also express CFTR, is less characterized. Without a comprehensive understanding of the contributors to chronic infections and inflammation, it will be challenging to develop targeted therapies for immune dysfunction in people with CF (pwCF). Mast cells (MC) are tissueresident immune cells with critical roles in lung homeostasis and disease. Despite their low abundance, dysregulated MC activation in the airways is linked to asthma, chronic obstructive pulmonary disease (COPD), fibrotic lung diseases, and can exacerbate respiratory infections. The role of MC in CF pathology is not well defined, and it remains unclear whether CFTR mutations lead to intrinsic dysregulation of MC function or how CFTR modulator treatments affect MC activity. Methods: Peripheral blood CD34+ progenitors from healthy controls (HC) and people with CF (pwCF) were differentiated into MC in vitro. Using primary HC MC and CF MC ± culture with elexacaftor/tezacaftor/ivacaftor (ETI), we determined CFTR expression and function, assessed changes in MC markers and phenotypes, and quantified changes in MC activation using CF-relevant agonists (Pseudomonas aeruginosa, and Staphylococcus aureus). MC intracellular calcium influx, degranulation, and cytokine production were used as readouts for MC activation. To determine transcriptional changes in MC with dysfunctional CFTR compared to healthy control MC or other innate immune cells, we analyzed published single-cell RNA sequencing data from nasal brushings of healthy controls and pwCF before and after ETI treatment [1]. Results: Primary human HC MC express CFTR, whose expression can be partially restored in CF MC with ETI treatment. Flow cytometry analysis showed that CF MC are smaller (FSC-A), less complex (SSC-A), and expressed more c-kit (Stem Cell Factor receptor) but fewer FCεRIα (High affinity IgE receptor) than HC MC, which have strong functional implications. This is supported by the result that CF MC have elevated intracellular calcium at baseline, but upon activation, the calcium influx is less robust compared to HC MC. Additionally, CF MC were less efficient at controlling bacterial growth as HC MC and reduced macrophage phagocytosis and bacterial killing. Finally, while ETI drives monocytes and neutrophils toward a transcriptional state closer to homeostasis, ETI induced a distinct transcriptional signature in MC that remains divergent from healthy controls. Conclusions: MC are dysfunctional in CF and play an underappreciated role in inflammation and innate defenses in CF. MC represent a new immunotherapeutic target in CF. Reference [1] Loske J, Völler M, Lukassen S, Stahl M, Thürmann L, Seegebarth A, et al. Pharmacological Improvement of Cystic Fibrosis Transmembrane Conductance Regulator Function Rescues Airway Epithelial Homeostasis and Host Defense in Children with Cystic Fibrosis. Am J Respir Crit Care Med. 2024 Jun 1;209(11):1338–50. 7 Laboratory glimpses of P. aeruginosa living in the cystic fibrosis mix: methods matter D. Chance1, T. Mawhinney2. 1Molecular Microbiology & Immunology, University of Missouri School of Medicine, Columbia, MO, USA; Pediatrics, University of Missouri School of Medicine, Columbia, MO, USA; 2Biochemistry, University of Missouri, Columbia, MO, USA; Pediatrics, University of Missouri School of Medicine, Columbia, MO, USA Background: Cystic fibrosis (CF) respiratory infections are commonly frequent and persistent, often progress to chronic colonization, and are potentially polymicrobial. P. aeruginosa(PA), S. aureus (SA), and early onH. influenza (HI) are key players in CF. These adaptive opportunists demonstrate various morphological and physiological phenotypes, yet data on frequency of occurring together and potential for co-localization is limited. Toward greater understanding of variables impacting effective study and treatment of CF infections, we present glimpses of host-adapted PA observed in conditions pertinent to the CF airway, including presence of other organisms. Methods: CF sputum and throat culture isolates were emphasized. Cocultures of PA, SA, HI, or C. albicans (CA) were often of 2 or more patient coisolates or 1 isolate and reference strain. Data collection included (a) extraction of data from clinical database; (b) culture of isolates under various growth conditions; and (c) comparisons of growth characteristics via various detection means. Results: Sputum cultured on rich media revealed multiple organisms and colony types (i.e. polymicrobial culture, PMc), indicative of polymicrobial colonization. Small scale local data base review of CF airway cultures suggested >70% were positive for ≥2 species +/−normal respiratory flora. SA, PA, CA, and other yeasts were the most frequent PMc organisms appearing in >50% of PMcs, and HI in∼13% of PMcs. Liquid co-cultures of CF PA, +/−SA, and HI showed patient dependent results: 1) a patient’sHIwere more likely to co-exist with their mucoid PA (mPA) than non-mucoid PA (nmPA); 2) co-isolated PA + SA or SA + HI grew together readily; and 3) SA seemingly improved survival of HI with PA. Liquid co-cultures of wild type PA with CF patients’ HI showed no surviving HI after 20 h. With CF sputum co-isolates in mixed plate cultures: 1) CA with mPA under anaerobic then aerobic conditions revealed PA survival under reduced O2 and healthy coexistence under more optimum conditions; and 2) SA grown aerobically with nmPA or mPA showed more SA co-localization with mPA than with nmPAwhere nmPAvisually dominated. Liquid culture feeding experiments with GFP-PA in minimal media (MM) plus test additives found GFP detection was limited by fluorescence quenching and the need for adequate phosphate. CFU plate counts without regard to fluorescence provided more suitable enumeration. TEM visualization of characteristics of CF isolates going into assays requiring MM growth revealed differing growth patterns and apparent health, and structurally saw the presence of flagella on non-motile isolates, reinforcing the concept that observed plate culture phenotypes do not necessarily reflect features present during experiments. Conclusions: Assay methods specifics, including use of CF co-adapted isolates in mixed cultures, do affect research outcomes. CF isolates variability makes generalization difficult and caution emphasized if assuming characteristics in the airway may be comparable. Detection and visualization of clinically relevant organisms by multiple means provides a more solid understanding of actual status during experiments. Glimpses of laboratory life of isolates from the CF mix suggest that adaptation to coexistence in the host is possible and highly variable with host conditions. Acknowledgements: This work was supported by the Cystic Fibrosis Associations of Missouri and West Plains; Leda J. Sears Trust; & Univ. of Missouri Experiment Station Chemical Laboratories. Journal of Cystic Fibrosis 24S2 (2025) S1–S530 S4

8 Co-morbidities alter immune response and drive increased Pseudomonas abundance during viral infection in the airways of people with cystic fibrosis Y. Hilliam1, C. Armbruster2, S. Atteih3, G. Rapsinski4, J.Moore5, J. Koirala4, J. Gaston4, L. Krainz4, J. Williams6, V. Cooper4, S. Lee7, J. Bomberger1. 1Department of Microbiology and Immunology, Geisel School of Medicine at Dartmouth, Hanover, NH, USA; 2Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA; 3Eudowood Division of Pediatric Respiratory Sciences, Johns Hopkins School of Medicine, Baltimore, MD, USA; 4Department of Microbiology and Molecular Genetics, University of Pittsburgh, PA, USA; 5University of Pittsburgh Medical Center, Pittsburgh, PA, USA; 6Division of Infectious Diseases, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA; 7Division of Otolaryngology, Brigham and Women’s Hospital, Boston, MA, USA Background: Viral-bacterial co-infection is a driver of worsening disease and chronic infection in the airways of people with cystic fibrosis (pwCF). The host antiviral response to airway infection increases inflammation in a chronically inflamed environment and changes the milieu in which bacteria reside. We have shown previously that respiratory viral infections cause metabolic reprogramming in CF bronchial epithelial cells, which increases Pseudomonas aeruginosa(Pa) biofilm biogenesis. We hypothesize that changes to the airway environment during viral infection allow for expansion of Pseudomonas populations and that repeated viral infections allowfor Pato become the dominant airway organism. Methods: We carried out a prospective longitudinal observational study of 38 adults with CF and chronic rhinosinusitis attending the UPMC Adult CF Clinic. Samples for microbiome and viral panel testing were collected from the sinus by swabbing of the maxillary sinus under endoscopic observation. Sputum samples were collected by spontaneous expectoration during the visit. Sinus aspirates were also collected for host biomarker analysis. DNA was extracted from swabs and sputum samples and 16S rRNAvariable region sequencing was performed to determine the airway microbiota. Viral infection at time of sampling was determined by qPCR panel. Cytokine biomarker levels were quantified by Luminex MAGPIX system. Results: Significant disruption to bacterial community structure was observed by 16S rRNA sequencings in samples with a co-occurring viral infection, with a significant increase in Pseudomonas relative abundance. We observed significant differences in the relationship between concentrations of Chitinase-3 like-protein 1, interleukin (IL)-10, IL-22, and IL-29 (IFN-l) andPseudomonas relative abundance during viral infection. Greater cytokine concentrations were associated with higher relative abundance during viral infection. Correlation analysis of subject demographics revealed that increased Pseudomonas abundance during viral infection was associated with allergic rhinitis (AR). Interestingly, the positive association between Pseudomonas relative abundance with IL-22 and IL29 (IFN-l) during viral co-infection was significantly associated with AR, suggesting that AR was a risk factor for increased abundance of Pseudomonas during viral co-infection. Conclusions: We have previously demonstrated that viral-bacterial coinfection leads to increases in IFN-mediated antiviral immunity in the airway, which causes rewiring of the airway epithelial metabolism and subsequently promotes Pa biofilm growth. Our observations of increased Pseudomonas abundance and cytokine concentrations in the CF airways during viral infection confirm these findings in a clinical population. We identified a significant disruption to the airway microbiota in pwCF during viral infection via increased abundance of Pseudomonas. We also demonstrated correlations between increased Pseudomonas abundance and greater concentrations of several antiviral cytokines. The relationship between increased cytokines and Pseudomonas relative abundance were further mediated by AR, a common comorbidity in pwCF. Viral infections are an important driver of disease progression in pwCF and we propose that comorbidities introduce variation in the antiviral response that may play a role in long-term disease outcomes. Further work is needed to understand the pathways that cause Pseudomonas proliferation as a result of antiviral immune responses. 9 Evolution and pathoadaptation of Pseudomonas aeruginosa through the gut-lung axis in cystic fibrosis R. Valls1, R. Christine2, C. Armbruster3, Y. Hilliam1, J. Baker1, A. Kohan2, J. Bomberger1. 1Department of Microbiology and Immunology, Dartmouth’s Geisel School of Medicine, Hanover, NH, USA; 2Department of Endocrinology and Metabolism, University of Pittsburg, Pittsburg, PA, USA; 3Department of Biological Sciences at Carnegie Mellon University, Pittsburgh, PA, USA Background: People with cystic fibrosis (pwCF) face significant challenges with fat malabsorption linked to chronic lung complications from an early age. The“gut-lung axis” suggests that gut microbiome changes profoundly affect lung health, with GI dysbiosis being a predictor of pulmonary exacerbations in pwCF. Dietary fatty acids are converted into chylomicrons in the small intestine and enter the blood via lymph before reaching the heart and lungs. Gut microbiota influence bile acids, cholesterol, and lipid metabolism. Despite advances with highly effective modulator therapy (HEMT), pathogens like Pseudomonas aeruginosa (Pa) persist, and gut dysbiosis does not revert to a non-CF state. The effects of gut dysbiosis on chylomicron transport andPa adaptation in the lungs remain unclear. Methods: To investigate gut-derived lipids’ impact on the nutritional environment of the lungs and their effects onPa populations, we gavaged mice with 3H-oleate and collected bronchoalveolar lavage fluid (BALF), whole-genome sequenced clinical Pa isolates from the airways of pwCF before and after HEMT, developed a gut-lung axis evolution of adapted (a) Pa model, where we passage Pa (lab strain) on CFBEs exposed to gutderived chylomicrons, performed biofilm assays, macrophage killing assays, and competitive fitness assessments of aPa. Results: Measurement of 3H-labeled dietary lipids revealed increase in gut-derived lipids in plasma, BALF, and lungs. Analysis of chylomicronevolvedPa’sfunctional pathways revealed enrichment of mutations in lipid metabolism pathways in post-HEMT Pa populations from the respiratory tract of pwCF. In the chylomicron-evolved Pa populations, we observed several mutations in the fatty acid degradation (fad) pathway. Mutations were found infadA, fadD, fadE, andfadJ. Interestingly, fad pathway variants in the same genes were also observed in clinical isolates of Papost-ETI from pwCF. 48-houraPabiofilms demonstrated greater resistance to tobramycin compared to WT MPAO1, even at concentrations below physiologically relevant levels. In the macrophage killing assay, tnFadE MPAO1 showed increased survival when primed with LCFAs, suggesting that mutations in chylomicron-evolved populations confer an adaptive advantage under specific nutritional conditions. We see dramatic shifts in a model CF respiratory microbiome (MCFRM), whereby at 24hrs PatnFadE abundance increased relative to WT, while other microbiota depleted in its presence. Conclusions: The increase in3H-labeled dietary lipids in plasma and lungs supports fatty acid trafficking from the gut to the lungs. Analysis of functional pathway categories reveals significant increases in lipid pathway mutations in post-HEMT clinical Pa isolates, which were confirmed in our gut-lung axis evolution of Pamodel, supporting that chylomicron-derived lipids are an important selective pressure for Pa. A representative transposon mutant in fadE (tnFadE) showed increased resistance to tobramycin and macrophage killing when primed with chylomicronderived nutrients. Preliminary findings suggest that chylomicron-aPawill outcompete other members of the MCFRM. Together, these results provide an explanation for Pa persistence in the post-HEMT CF lung. Furthermore, we propose a novel paradigm in the gut-lung axis that explores how gutderived lipids can shape the nutritional environment of the lung, and drive adaptation of pathogens that cause chronic infections. Journal of Cystic Fibrosis 24S2 (2025) S1–S530 S5

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