Journal of Cystic Fibrosis - Vol. 23 - Suppl.2

September 2024 Volume 23, S2 September 26–28, Boston, Massachusetts Abstracts of the 2024 North American Cystic Fibrosis Conference

Editorial Board Editor-in-Chief: Patrick Flume, Medical University of South Carolina, Charleston, South Carolina, USA Deputy Editors: Carlo Castellani, IRCCS Instituto Giannina Gaslini, Cystic Fibrosis Centre, Genoa, Italy Jane Davies, National Heart & Lung Institute, Imperial College London and Royal Brompton and Harefield NHS Foundation Trust, London, UK Editorial Board: Scott Blackman, USA Pierre-Régis Burgel, France 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 KeithOoi, Australia Nicoletta Pedemonte, Italy Gerald Pier, USA Felix Ratjen, Canada Kristin A. Riekert, USA Dorota Sands, Poland Rhonda Szczesniak, USA Anne Stephenson, Canada Cliff Taggart, UK Jennifer Taylor-Cousar, USA DaanTouw, The Netherlands Michael Tunney, N. Ireland Donald Van Devanter, USA Valerie Waters, Canada Michael Wilschanski, Israel Jeffrey Wine, USA Editor In Chief, Cystic Fibrosis Research News: Harry Heijerman, University Medical Center Utrecht, The Netherlands Previous Editors-in-Chief: Harry Heijerman, University Medical Center Utrecht, The Netherlands Gerd Döring, University of Tübingen, Tübingen, Germany Scott Bell, The Prince Charles Hospital, Queensland, Australia Amsterdam — Boston — London — New York — Oxford — Paris — Philadelphia — San Diego — St. Louis — Tokyo Harriet Corvol, Hospital Armand Trousseau, Sorbonne University, Paris, France Editorial Board Editor-in-Chief: Patrick Flume, Medical University of South Carolina, Charleston, South Carolina, USA Deputy Editors: Carlo Castellani, IRCCS Instituto Giannina Gaslini, Cystic Fibrosis Centre, Genoa, Italy Jane Davies, National Heart & Lung Institute, Imperial College London and Royal Brompton and Harefield NHS Foundation Trust, London, UK Editorial Board: Scott Blackman, USA Pierre-Régis Burgel, France 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 KeithOoi, Australia Nicoletta Pedemonte, Italy Gerald Pier, USA Felix Ratjen, Canada Kristin A. Riekert, USA Dorota Sands, Poland Rhonda Szczesniak, USA Anne Stephenson, Canada Cliff Taggart, UK Jennifer Taylor-Cousar, USA DaanTouw, The Netherlands Michael Tunney, N. Ireland Donald Van Devanter, USA Valerie Waters, Canada Michael Wilschanski, Israel Jeffrey Wine, USA Editor In Chief, Cystic Fibrosis Research News: Harry Heijerman, University Medical Center Utrecht, The Netherlands Previous Editors-in-Chief: Harry Heijerman, University Medical Center Utrecht, The Netherlands Gerd Döring, University of Tübingen, Tübingen, Germany Scott Bell, The Prince Charles Hospital, Queensland, Australia Amsterdam — Boston — London — New York — Oxford — Paris — Philadelphia — San Diego — St. Louis — Tokyo

The Official Journal of the European Cystic Fibrosis Society Description The Journal of Cystic Fibrosis is the official journal of the European Cystic Fibrosis Society. The journal is devoted to promoting the research and treatment of cystic fibrosis. To this end the journal publishes original scientific articles, editorials, case reports, short communications and other information relevant to cystic fibrosis. The journal also publishes news and articles concerning the activities and policies of the ECFS as well as those of other societies related to the ECFS. Audience Pediatricians, pulmonologists, gastroenterologists, internists, microbiologists, pharmacologists, immunologists, psychologists, basic scientists, physiotherapists, dieticians and nurses dealing with the investigation and treatment of cystic fibrosis. Publication Information: Journal of Cystic Fibrosis (ISSN 1569-1993). For 2023 volume 22/1 to 22/6 is scheduled for publication. 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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) 2024 North American Cystic Fibrosis Conference September 26–28, Boston, Massachusetts Volume 23, Supplement 2 (2024)

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� 23 (2024) Supplement 2 2024 North American Cystic Fibrosis Conference September 26–28, 2024 Infection/Microbiology ���������������������������������������������������������������������������������������������������������������������������������������������������������������� S1 Clinical Trials & Outcome Measures �������������������������������������������������������������������������������������������������������������������������������������S63 Airways Physiology, Pathophysiology & Airways Defense ��������������������������������������������������������������������������������������������S95 Path to a Cure ����������������������������������������������������������������������������������������������������������������������������������������������������������������������������S136 CFTR ���������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� S164 Clinical Genetics �����������������������������������������������������������������������������������������������������������������������������������������������������������������������S191 Extrapulmonary Physiology & Pathophysiology ������������������������������������������������������������������������������������������������������������S201 Education ������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������ S212 Health Equity, Care, Delivery, & Access to Care ��������������������������������������������������������������������������������������������������������������S221 Nursing Issues ���������������������������������������������������������������������������������������������������������������������������������������������������������������������������S261 Pharmacy ������������������������������������������������������������������������������������������������������������������������������������������������������������������������������������ S267 Physical & Respiratory Therapy �������������������������������������������������������������������������������������������������������������������������������������������S285 Psychosocial/Behavioral ��������������������������������������������������������������������������������������������������������������������������������������������������������� S298 GI/Nutrition �������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� S329 Endocrine/Bone ������������������������������������������������������������������������������������������������������������������������������������������������������������������������ S364 Epidemiology & Population-Based Research �������������������������������������������������������������������������������������������������������������������S391 Quality Improvement ���������������������������������������������������������������������������������������������������������������������������������������������������������������S418 Pulmonary ���������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� S480 Transplantation ������������������������������������������������������������������������������������������������������������������������������������������������������������������������� S499 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 Pseudomonas aeruginosa-phage interactions are influenced by polymicrobial biofilms P. Zamora1, Y. Hilliam1, R.Martin2, J. Bomberger1. 1Department of Microbiology and Immunology, Geisel School of Medicine, Dartmouth College, Hanover, NH; 2Molecular and Cellular Biology Program, Dartmouth College, Hanover, NH Background: Phage therapy is a promising therapeutic alternative for the treatment of multidrug-resistant (MDR) organisms in people with cystic fibrosis (PwCF). Despite the positive results of this therapeutic in vitro, the clearing of Pseudomonas aeruginosa in the lungs of PwCF has not been as successful as predicted in clinical trials. One factor that is often not considered when choosing phages for therapeutics is that P. aeruginosa is part of a complex polymicrobial environment characterized by interspecies interactions. The presence of other organisms affects P. aeruginosa physiology, influencing its susceptibility to be killed by phages. Consequently, models that recapitulate the CF airway microbiome are necessary for the effective development and optimization of phage therapy for PwCF. Methods: From CF sputum 16S rRNA sequencing and metabolic comparisons, we modeled a CF microbial community composed of P. aeruginosa, Staphylococcus aureus, Streptococcus sanguinis, and Prevotella melaninogenicathat we grew in synthetic CF sputum media. To model phage therapy in CF,weusedP. aeruginosalytic phages that have been used in clinical trials to treat MDR infections in PwCF. Results: To determine the biogeography of polymicrobial biofilms, we labeled each bacterial species with unique fluorophores and imaged by confocal microscopy. We observed that bacteria aggregate in multispecies communities, which recapitulates results from CF sputum, where multiple species reside in close physical proximity. When we treated P. aeruginosa with phages, we discovered that the killing of P. aeruginosa in a polymicrobial community was impaired when compared to lysis in single-species biofilms, suggesting that other microbial members of the community affect bacterial susceptibility to phages. This observation varied with the phage used. Interestingly, phage treatments also affected the growth of other organisms in the polymicrobial community, as we detected lower biomass forP. melaninogenicainP. aeruginosa-phage treated communities. To study the effect of lytic P. aeruginosa phages in airway epithelial barrier function, we treated CF airway epithelial cells (AECs) and evaluated innate immune responses. We found that phages elicited proinflammatory cytokine secretion that segregated with specific phage families. In experiments where we treated single-species P. aeruginosa biofilms grown on AECs with phages, we determined that proinflammatory cytokine secretion decreased in the presence of phages. Conclusions: Our studies revealed that the dynamics of phage-bacterial interactions is affected by other microbial members in multispecies communities. The composition of the microbial community affected the ability of phage to reduce P. aeruginosa burden. Our future work will continue to examine the molecular mechanisms that govern microbemicrobe interactions during phage therapy and how modulating the airway environment could improve phage outcomes in PwCF. Acknowledgements: This work was supported by a CFF Postdoctoral Fellowship ZAMORA20F0 and CFF grant BOMBER21P0. 2 Multi-lobe bronchoscopy reveals ETI’s effects on regional lung infection and inflammation S.Durfey1, S. Kapnadak2,M.Teresi3,T.Gambol2, M. Willmering4, J.Godwin2, L. Boyken5,M. Stroik6, S. Singh7, C. Steele8, C. Esther9, J.Woods10, D. Stoltz11,12, T. Pena6,7, J. Clancy13,M. Aitken2, P. Singh1,2. 1Department of Microbiology, School of Medicine, University of Washington, Seattle, WA; 2Department of Medicine, School of Medicine, University of Washington, Seattle, WA; 3University of Iowa Hospitals and Clinics, Iowa City, IA; 4Center for Pulmonary Imaging Research, Cincinnati Children’s Hospital Medical, Cincinnati, OH; 5Department of Pathology, University of Iowa, Iowa City, IA; 6Department of Medicine, University of Iowa, Iowa City, IA; 7Department of Pediatrics, University of Iowa, Iowa City, IA; 8Department of Microbiology and Immunology, Tulane University, New Orleans, LA; 9Marsico Lung Institute, University of North Carolina at Chapel Hill, NC; Pediatrics, University of North Carolina at Chapel Hill, NC; 10Department of Pulmonary Medicine, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; 11Department of Internal Medicine, Pappajohn Biomedical Institute, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA; 12Department of Molecular Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, University of Iowa, Iowa City, IA; 13Cystic Fibrosis Foundation, Bethesda, MD Background: Most people with CF (PwCF) infected with Pseudomonas aeruginosa remain infected after elexacaftor/tezacaftor/ivacaftor (ETI) treatment. A leading hypothesis is that infections persist because lung regions with severe disease fail to clear bacteria. This hypothesis is attractive because the lungs of PwCF exhibit marked heterogeneity in conditions that could affect infection outcomes, but the hypothesis has not been tested. We used bronchoscopy sampling to address three main questions. How heterogenous are regional lung conditions before treatment? Does ETI produce similar effects in lung segments within an individual, or do some regions clear while others remain infected? What conditions are associated with persistent infection and inflammation after ETI? Methods: We sampled five lung segments per subject in each ofnine9 subjects before ETI, selecting segments with mild and severe disease, and re-sampled the same segments after 18 months of ETI. We measured P. aeruginosadensity, neutrophil elastase (NE), structural lung damage, sialic acid (reflects mucus concentration), two of P. aeruginosa’s preferred carbon sources (proline and arginine, called“nutrients” below), and 63 cytokines. Results: Bronchoscopy revealed marked within-lung heterogeneity in conditions that could affect infection outcomes, including P. aeruginosa density, NE, structural lung damage, mucus, and nutrients. All of these parameters were positively correlated (p < 0.05 for all), meaning that adverse conditions tended to co-localize with each other within the same regions. Despite these marked regional differences, P. aeruginosaresponses in lung segments within individuals were generally similar. P. aeruginosa became undetectable after ETI in all sampled segments in 3 of 9 subjects. In the 6 subjects remaining P. aeruginosa-infected, P. aeruginosa persisted in all but 2 sampled lung segments. A comparison of segments that did and did not clear P. aeruginosa independent of subject origin showed that segments remaining infected had higher baseline levels of structural Journal of Cystic Fibrosis 23S2 (2024) S1–S505

damage (measured by Brody scores of CT scans: p = 0.03) and nutrients (proline: p = 0.04; arginine: p = 0.02). We then investigated conditions associated with residual lung inflammation after ETI and found that NE became undetectable in lung segments that cleared P. aeruginosa but remained elevated in persistently infected segments (p < 0.001). Additional markers of inflammation, including IL-1β, IL-8, TNF-α, and 25 others, were also significantly higher in segments that remained persistently P. aeruginosa-infected than in those that cleared P. aeruginosa (false discovery rate p< 0.05). The coefficient of determination between P. aeruginosa density and NE and 22 of these 28 P. aeruginosaassociated inflammatory markers increased after treatment, indicating that P. aeruginosa density as an individual variable better explained inflammation levels after than before ETI. Conclusions: Despite marked regional lung heterogeneity in many key conditions, post-ETI P. aeruginosa infections were not limited to severely diseased lung segments. Instead, people remaining infected continued to harbor P. aeruginosa throughout their lungs, including in lung segments with multiple favorable conditions. Across subjects, regions clearing P. aeruginosa had less baseline structural damage and lower nutrient concentration. Our data suggest persistent P. aeruginosa infection may be the primary driver of lung inflammation after ETI. Acknowledgements: This work was supported by CFF grants SINGH19K0 and ESTHER22Y2-SVC]. 3 Antibiotics used to treat Pseudomonas infections may enhance Mycobacterium abscessus persistence in the CF lung J. Corley1, J. Congel1, K. Haist1, A. Ochoa1, K. Malcolm1,2, J. Nick1, K. Hisert1. 1Department of Medicine, National Jewish Health, Denver, CO; 2Department of Medicine, Anschutz Medical Center, University of Colorado, Aurora, CO Background: Nontuberculous mycobacterial (NTM) infections are increasing globally and infect a significant portion of people with cystic fibrosis (PwCF). Thus, a critical need exists to understand determinants of susceptibility to NTM. Co-infection with Pseudomonas aeruginosa has been found by some to be a risk factor associated with acquisition of NTM pulmonary infection in PwCF. NTM species like Mycobacterium abscessus (Mabsc) are resistant to antibiotics used to treat P. aeruginosa; thus, it is generally thought that these antibiotics do not affect NTM viability in the lung. Because previous work demonstrated that subinhibitory concentrations of the anti-Mabsc aminoglycoside amikacin activates expression of the transcription factor WhiB7 and increases survival of Mabsc in macrophages, we hypothesized that antibiotics with similar mechanisms of action used to kill P. aeruginosawould also inducewhiB7expression. We demonstrate that tobramycin, an aminoglycoside antibiotic (like amikacin) frequently administered to PwCF with P. aeruginosa during exacerbations and as maintenance therapy, also activates whiB7 expression. As follows, we hypothesize that tobramycin used to kill P. aeruginosa in the CF airway enhances Mabsc persistence in macrophages by inducing whiB7 expression, thus increasing the chance of Mabsc becoming a chronic infection in the lung. Methods: Gene expression of Mabsc whiB7after tobramycin, amikacin, and levofloxacin treatment was measured using qRT-PCR. Growth of Mabsc wild type (WT) and whiB7 deletion strains (ΔwhiB7) were measured in human monocyte-derived macrophage (MDM) and alveolar macrophages (AlvMac) after 4 days. Reactive oxygen species (ROS) resistance of Mabsc +/−antibiotic treatment andΔwhiB7was evaluated using H2O2MIC assays. To determine if tobramycin increasedMabscpersistence in vivo, mice were administered tobramycin one day before and during infection with WT Mabsc, and bacterial burden was assessed after 7 days of infection. Results: We demonstrated that tobramycin-mediated induction of Mabsc whiB7expression was dose dependent and temporal. Maximal expression of whiB7 occurred after 24-hour treatment with 25 mg/mL tobramycin. Induction of whiB7did not occur with the fluoroquinolone levofloxacin, an oral antibiotic used to treat P. aeruginosainfections in PwCF. TreatingMabsc with amikacin before infection in MDMs and AlvMacs promoted Mabsc survival and amikacin and tobramycin enhance resistance to H2O2. Furthermore, compared to WT, ΔwhiB7 Mabsc demonstrated decreased survival in macrophages and a lower H2O2 MIC. Treatment of mice with parenteral tobramycin resulted in increased lung Mabsc burden at 1 week than in placebo-treated mice. Conclusions: Our data suggest that tobramycin used to treat P. aeruginosa changes Mabsc gene expression to promote Mabsc survival in the host. Similar gene expression changes are not seen after exposure of Mabsc to levofloxacin, suggesting that the antibiotic effects on Mabsc survival may be class specific. Future tobramycin-treated Mabsc RNAseq studies will determine which WhiB7-regulated Mabsc genes are activated by tobramycin. We will also assess clinical relevance by evaluating whiB7 expression in clinical Mabsc isolates after tobramycin exposure using qRT-PCR studies. This study addresses mechanisms by which Mabsc infections may persist in some PwCF and suggests that current antibiotic therapies for other CF pathogens may affect Mabsc infection. 4 Within-lung interspecies gene transfer causes rapid evolution of extreme antibiotic resistance in cystic fibrosis S. Karash1, H. Betts1,M. Radey1, R. Hernandez2,3,W. Ni1, B. Waddell4, M. Parkins5,6, C.Manoil7, P. Singh1,8. 1Department of Microbiology, University of Washington, Seattle, WA; 2Department of Pediatrics, University of Washington, Seattle, WA; 3Seattle Children’s Research Institute, Seattle, WA; 4Microbiology, Immunology, and Infectious Diseases, University of Calgary, Calgary, Canada; 5Infectious Diseases, University of Calgary, Calgary, Canada; 6Calgary Adult CF Clinic, Calgary, Canada; 7Genome Sciences, University of Washington, Seattle, WA; 8Department of Medicine, School of Medicine, University of Washington, Seattle, WA Background: Antibiotic resistance is linked to poor outcomes in people with CF (PwCF) infected with Pseudomonas aeruginosa. We analyzed longitudinal antibiotic resistance profiles from PwCF and found a substantial number in whom sensitive P. aeruginosa suddenly became extremely tobramycin (Tob) resistant. Resistance developed rapidly (sensitive and extreme-resistant isolates detected weeks apart), was not explained by strain switching (isolates were clonally related), and was highly durable after emerging. This extreme resistance is likely to compromise the high Tob levels achieved by aerosol delivery, because isolate MICs were ∼100 to 1,000 times the Clinical and Laboratory Standards Institute resistance breakpoint. We sought to identify the mechanism of extreme Tob resistance in CF P. aeruginosa that develops in vivo. Methods: We identified extremely resistant P. aeruginosa from 13 chronically infected PwCF from two CF biobanks in the United State and Canada. We generated complete genome sequences from 250P. aeruginosa and other co-infecting bacteria and identified putative resistance genes. Resistance genes were integrated into the chromosome of a P. aeruginosa reference strain for further analysis using allelic exchange. Genetic stabilities of plasmids carrying resistance genes were assessed in laboratory cultures in clinical host strains. Results: We studied 13 PwCF developing extreme Tob resistance. We focused first on five who had preexisting Tob-sensitive clonally related isolates banked. Genomic comparisons of clonally related sensitive and extremely resistant pairs demonstrated no shared genes that became mutated to explain extreme resistance, but the extremely resistant P. aeruginosaisolate pairs contained a novel plasmid-borne transposon (that we named Tn-CF1) encoding a previously undescribed aminoglycoside acetylase aac(3) subtype resistance gene. Transformation of the sensitive clinical isolates and the PAO1 reference strain with plasmids carrying TnCF1 and insertion of a single chromosomal copy of aac(3) was sufficient to produce extreme resistance. Long-duration passaging experiments found plasmid carriage was stable and without apparent fitness cost in laboratory conditions. Plasmid-borne Tn-CF1 was also found inP. aeruginosafrom10 of 13 PwCF with extreme resistance. We also found Tn-CF1 in public genome databases in pathogens from other PwCF in North America and Europe. Our finding that plasmid-borne Tn-CF1 was independently acquired by the P. aeruginosa lineages infecting multiple PwCF led us to hypothesize that co-infecting pathogens transferred the plasmids toP. aeruginosawithinthe lungs of PwCF. In two PwCF, co-infecting species had been banked, and we identified Achromobacter, Stenotrophomonas maltophilia, and P. putida species carrying the identical plasmid (all contained Tn-CF1) that was Journal of Cystic Fibrosis 23S2 (2024) S1–S505 S2

acquired by the individual’s sensitive P. aeruginosa. In both people, coinfecting species carrying plasmid-borne Tn-CF1 were present in sputum months before the plasmids appeared in the P. aeruginosa lineage. Ex vivo conjugation experiments showed that the plasmid-containing Ac, Sm, and Pt clinical isolates could transfer plasmid-borne Tn-CF1 to the sensitive P. aeruginosa clinical isolates from corresponding subjects, resulting in extreme Tob resistance. Conclusions: Co-infecting pathogens can transfer antibiotic resistance genes to highly sensitiveP. aeruginosawithin the lungs of PwCF, causing the sudden onset of extreme, durable, low fitness-cost resistance. Acknowledgements: The first author is supported by CFF KARASH23F0 postdoc fellowship. 5 Reduction in positive cultures for nontuberculous mycobacteria and pulmonary disease in people with CF in the era of highly effective modulator therapy in longitudinal PREDICT study participants S. Martiniano1,2, A. Magaret3,V. Lovell4,A.Keck2, J. VanDalfsen3, S. Heltshe3, N.Midamba3, C. Chapdu3, J. Nick5. 1Department of Pediatrics, University of Colorado, Aurora, CO; 2Breathing Institute, Children’s Hospital Colorado, Aurora, CO; 3Cystic Fibrosis Foundation Therapeutics Development Network Coordinating Center, Seattle Children’s Research Institute, Seattle, WA; 4Division of Pulmonary, Critical Care, and Sleep Medicine, National Jewish Health, Denver, CO; 5Department of Medicine, National Jewish Health, Denver, CO Background: Since approval of the highly effective modulator therapies (HEMT) ivacaftor and most recently elexacaftor/tezacaftor/ivacaftor (ETI) in October 2019, Cystic Fibrosis Foundation Patient Registry data and other reports have described reduced sputum production and decreased burden and prevalence of certain CF pathogens, including nontuberculous mycobacteria (NTM) in people with CF (PwCF). We aimed to determine impact of HEMT on NTM in our saturated population with a recent history of a positive NTM culture. Methods: The PRospective Evaluation of NTM Disease In CysTic fibrosis trial (PREDICT, NCT02073409) is a prospective, multicenter (19 sites), observational study of PwCF and NTM that began in 2013 and is ongoing. Included are PwCF, aged 6 and older who are sputum producing and have a recent a positive NTM culture of a species that has never been treated. Participants are followed longitudinally and follow a standardized approach to NTM pulmonary disease (NTM-PD) diagnosis, including microbiologic, radiographic, and clinical assessments and optimization of CF care. Sputum collection for NTM culture is attempted at each clinical visit, and clinical data are obtained. Since 2023, a home sputum culture kit has been provided annually for an additional NTM culture. NTM culture results were evaluated and summarized for all enrolled participants. For those starting HEMT (ivacaftor or ETI) during the study with at least 1 day of follow-up, prevalence of positive cultures from the first positive culture recorded (before enrollment) to initiation of HEMT was compared with prevalence of positive from the start of HEMT to the present (or until diagnosis of NTM-PD). Results: A total of 274 participants have been enrolled since 2013. There was a slowing of enrollment in 2020 and 2021, but enrollment rates increased in 2022 and 2023. All newly enrolled participants have had a recent positive NTM culture as per inclusion criteria. In 2023, 70% of participants had at least one sputum culture obtained and sent for NTM culture. Before ETI availability between 2013 and 2020, NTM culture positivity rate for all participants was 37%. In 72 participants with data available before and after starting HEMT, culture positivity rates decreased from 39% to 11% (median difference–27% (p < 0.001)). The overall NTM-PD diagnosis rate for the entirety of the study is 23%. There are similar rates of NTM-PD in those with Mycobacterium aviumcomplex and M. abscessus complex infection. As the number of participants in PREDICT were started on HEMT increased, the proportion diagnosed with NTM-PD decreased (Figure 1). Of those not on HEMT, the overall NTM-PD rate is 25%, compared with 6% in those on HEMT. Conclusions: In the era of HEMT, PwCF are still producing sputum and being infected with NTM, but culture positivity rates are significantly lower. NTM continues to be a pathogen in this population, but NTM-PD rates have also decreased. We suspect that NTM is harder to detect because of lower sputum production frequency and volumes and because the NTMPD clinical syndrome is more subtle, which contributes to an apparent decline in NTM-positive cultures and NTM-PD. Continued partnerships between basic-translational scientists and existing clinical studies and the NTM National Resource Centers are critical for the development of moresensitive NTM diagnostic and outcome measures. Acknowledgements: Funded by CFF NTM-OB-17 and MARTIN22K0. Figure 1(abstract 5): Proportion of PREDICT participants with NTM developing NTM-PD by year among those at risk. Journal of Cystic Fibrosis 23S2 (2024) S1–S505 S3

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