Development and Initial Characterization of Pigs with DNAI1 Mutations and Primary Ciliary Dyskinesia.

Mutations in more than 50 different genes cause primary ciliary dyskinesia (PCD) by disrupting the activity of motile cilia that facilitate mucociliary transport (MCT). Knowledge of PCD has come from studies identifying disease-causing mutations, characterizing structural cilia abnormalities, finding genotype-phenotype relationships, and studying the cell biology of cilia. Despite these important findings, we still lack effective treatments and people with PCD have significant pulmonary impairment. As with many other diseases, a better understanding of pathogenic mechanisms may lead to effective treatments. To pursue disease mechanisms, we used CRISPR-Cas9 to develop a PCD pig with a disrupted DNAI1 gene. PCD pig airway cilia lacked the outer dynein arm and had impaired beating. MCT was impaired under both baseline conditions and after cholinergic stimulation in PCD pigs. Neonatal PCD pigs developed neonatal respiratory distress with evidence of atelectasis, air trapping, and airway mucus obstruction. Despite airway mucus accumulation, lung bacterial counts were similar between neonatal wild-type and PCD pigs. Sinonasal disease was present in all neonatal PCD pigs. Older PCD pigs developed worsening airway mucus obstruction, inflammation, and bacterial infection. This pig model closely mimics the disease phenotype seen in people with PCD and can be used to better understand the pathophysiology of PCD airway disease.

Pancreatic enzymes digest obstructive meconium from cystic fibrosis pig intestines.

Introduction: Meconium ileus (MI) is a life-threatening obstruction of the intestines affecting ∼15% of newborns with cystic fibrosis (CF). Current medical treatments for MI often fail, requiring surgical intervention. MI typically occurs in newborns with pancreatic insufficiency from CF. Meconium contains mucin glycoprotein, a potential substrate for pancreatic enzymes or mucolytics. Our study aim was to determine whether pancreatic enzymes in combination with mucolytic treatments dissolve obstructive meconium using the CF pig model. Methods: We collected meconium from CF pigs at birth and submerged it in solutions with and without pancreatic enzymes, including normal saline, 7% hypertonic saline, and the reducing agents N-acetylcysteine (NAC) and dithiothreitol (DTT). We digested meconium at 37 °C with agitation, and measured meconium pigment release by spectrophotometry and residual meconium solids by filtration. Results and discussion: In CF pigs, meconium appeared as a solid pigmented mass obstructing the ileum. Meconium microscopically contained mucus glycoprotein, cellular debris, and bile pigments. Meconium fragments released pigments with maximal absorption at 405 nm after submersion in saline over approximately 8 h. Pancreatic enzymes significantly increased pigment release and decreased residual meconium solids. DTT did not improve meconium digestion and the acidic reducing agent NAC worsened digestion. Pancreatic enzymes digested CF meconium best at neutral pH in isotonic saline. We conclude that pancreatic enzymes digest obstructive meconium from CF pigs, while hydrating or reducing agents alone were less effective. This work suggests a potential role for pancreatic enzymes in relieving obstruction due to MI in newborns with CF.

Parallel evolution of linezolid-resistant Staphylococcus aureus in patients with cystic fibrosis.

Linezolid is an antibiotic used to treat serious Staphylococcus aureus infections. Resistance to linezolid is considered rare but could emerge with repeated dosing. We recently reported widespread prescription of linezolid for a cohort of patients with cystic fibrosis (CF). The goals of this study were to determine the incidence of linezolid-resistant methicillin-resistant Staphylococcus aureus (MRSA) in CF and identify molecular mechanisms for linezolid resistance. We identified patients who cultured S. aureus resistant to linezolid with minimum inhibitory concentration (MIC) >4 at the University of Iowa CF Center between 2008 and 2018. We obtained isolates from these patients and retested susceptibility to linezolid using broth microdilution. We used whole genome sequencing to perform phylogenetic analysis of linezolid-resistant isolates and examine sequences for mutations or accessory genes that confer linezolid resistance. Between 2008 and 2018, 111 patients received linezolid, and 4 of these patients cultured linezolid-resistant S. aureus. We sequenced 11 resistant and 21 susceptible isolates from these 4 subjects. Phylogenetic analysis indicated that linezolid resistance developed in ST5 or ST105 backgrounds. Three individuals had linezolid-resistant S. aureus with a G2576T mutation in 23S rRNA. One of these subjects additionally had a mutS- mutL- hypermutating S. aureus that produced five resistant isolates with multiple ribosomal subunit mutations. In one subject, the genetic basis for linezolid resistance was unclear. We conclude that linezolid resistant S. aureus can occur through multiple genetic mechanisms in patients with repeated exposure to this antibiotic. IMPORTANCE Patients with cystic fibrosis have persistent lung infections with Staphylococcus aureus that require extensive antibiotic treatments. Linezolid, an antibiotic given by oral or intravenous route, is prescribed repeatedly for patients whose lung disease has progressed. After treatment with linezolid, S. aureus strains can evolve antibiotic resistance through multiple genetic mechanisms. In addition to a common mutation in the 23S ribosomal RNA known to confer linezolid resistance, S. aureus strains can evolve novel resistance based on a combination of mutations affecting the bacterial ribosome. This combination of mutations was observed in a strain that exhibited hypermutation owing to the loss of the DNA repair genes mutS and mutL. In this cohort of patients with cystic fibrosis, linezolid resistance was transient, possibly due to the growth disadvantage of resistant strains. However, ongoing chronic exposure to linezolid may create optimal conditions for the future emergence of resistance to this critical antibiotic.

Patient and Caregiver Perceptions of Airway Clearance Methods Used for Cystic Fibrosis.

Introduction: Cystic Fibrosis Foundation guidelines recommend people with CF perform daily airway clearance. This can be difficult for patients, as some find it time consuming or uncomfortable. Data comparing airway clearance methods are limited. We surveyed patients and their families to understand which methods are preferred and identify obstacles to performing airway clearance. Methods: We designed a REDCap survey and enrolled participants in 2021. Respondents reported information on airway clearance usage, time commitment, and medication use. They rated airway clearance methods for effectiveness, comfort, time commitment, importance, and compatibility with other treatments. The analysis included descriptive statistics and clustering. Results: 60 respondents started and 52 completed the survey. The median patient age was 20 years. Respondents experienced a median of four airway clearance methods in their lifetime, including chest wall oscillation (vest, 92%), manual chest physical therapy (CPT, 88%), forced expiration technique (huff or cough, 77%), and exercise (75%). Past 30-day use was highest for exercise (62%) and vest (57%). The time commitment was generally less than 2 hours daily. Of those eligible for CFTR modulators, 53% reported decreased time commitment to airway clearance after starting treatment. On a scale of 0-100, respondents rated CFTR modulators as their most important treatment (median 99.5), followed by exercise (88). Discussion. Patients and caregivers are familiar with several methods of airway clearance for CF. They report distinct strengths and limitations of each method. Exercise and vest are the most common methods of airway clearance. The use of CFTR modulators may reduce patient-reported time commitment to airway clearance.

Parallel Evolution of Linezolid Resistant Staphylococcus aureus in Patients with Cystic Fibrosis.

Background: Linezolid is an antibiotic used to treat serious Staphylococcus aureus infections. Resistance to linezolid is considered rare but could emerge with repeated dosing. We recently reported widespread prescription of linezolid for a cohort of patients with cystic fibrosis (CF). Objectives: The goals of this study were to determine the incidence of linezolid resistance in CF and identify molecular mechanisms for linezolid resistance. Methods: We identified patients with S. aureus resistant to linezolid (MIC > 4) at the University of Iowa CF Center between 2008 and 2018. We obtained isolates from these patients and retested susceptibility to linezolid using broth microdilution. We used whole genome sequencing to perform phylogenetic analysis of linezolid resistant isolates and examine sequences for mutations or accessory genes that confer linezolid resistance. Main Results: Between 2008 and 2018, 111 patients received linezolid and 4 of these patients cultured linezolid resistant S. aureus . We sequenced 11 resistant and 21 susceptible isolates from these 4 subjects. Phylogenetic analysis indicated that linezolid resistance developed in ST5 or ST105 backgrounds. Three individuals had linezolid resistant S. aureus with a G2576T mutation in 23S rRNA. One of these subjects additionally had a mutS - mutL - hypermutating S. aureus that produced 5 resistant isolates with multiple ribosomal subunit mutations. In one subject, the genetic basis for linezolid resistance was unclear. Conclusions: Linezolid resistance evolved in 4 of 111 patients in this study. Linezolid resistance occurred by multiple genetic mechanisms. All resistant strains developed in ST5 or ST105 MRSA backgrounds. Key Point: Linezolid resistance arises through multiple genetic mechanisms and could be facilitated by mutator phenotypes. Linezolid resistance was transient, possibly due to growth disadvantage.

Small Colonies, Bigger Problems? New Evidence That Staphylococcus aureus Small Colony Variants Can Worsen Lung Inflammation in Cystic Fibrosis Rats.

Staphylococcus aureus is the most prevalent cystic fibrosis (CF) pathogen. During chronic airway infections, S. aureus adaptation to antibiotics includes evolving small colony variants (SCVs). Observational studies correlate SCVs with deteriorating lung function in CF, but it is unclear whether SCVs cause disease progression or if they are markers of intensified treatment. G. E. Bollar, J. D. Keith, A. M. Oden, M. R. Kiedrowski, and S. E. Birket (Infect Immun 90:e00237-22, 2022, https://doi.org/10.1128/iai.00237-22) provide intriguing new experimental evidence that an SCV elicits greater inflammation than its normal colony progenitor strain in CF rats.

Elastic mucus strands impair mucociliary clearance in cystic fibrosis pigs.

SignificanceIn many lung diseases, increased amounts of and/or abnormal mucus impair mucociliary clearance, a key defense against inhaled and aspirated material. Submucosal glands lining cartilaginous airways secrete mucus strands that are pulled by cilia until they break free from the duct and sweep upward toward the larynx, carrying particulates. In cystic fibrosis (CF) pigs, progressive clearance of insufflated microdisks was repeatedly interrupted as microdisks abruptly recoiled. Aerosolizing a reducing agent to break disulfide bonds linking mucins ruptured mucus strands, freeing them from submucosal gland ducts and allowing cilia to propel them up the airways. These findings highlight the abnormally increased elasticity of CF mucus and suggest that agents that break disulfide bonds might have value in lung diseases with increased mucus.

MRSA strains with distinct accessory genes predominate at different ages in cystic fibrosis.

RATIONALE: Methicillin resistant Staphylococcus aureus (MRSA) is prevalent and consequential in cystic fibrosis (CF). Whole genome sequencing (WGS) could reveal genomic differences in MRSA associated with poorer outcomes or detect MRSA transmission. OBJECTIVES: To identify MRSA genes associated with low lung function and potential MRSA transmission in CF. METHODS: We collected 97 MRSA isolates from 74 individuals with CF from 2017 and performed short-read WGS. We determined sequence type (ST) and the phylogenetic relationship between isolates. We aligned accessory genes from 25 reference genomes to genome assemblies, classified isolates by accessory gene content, and correlated the accessory genome to clinical outcomes. RESULTS: The most prevalent ST were ST5 (N = 55), ST8 (N = 15), and ST105 (N = 14). Closely related MRSA strains were shared by family members with CF, but rarely between unrelated individuals. Three clusters of MRSA were identified by accessory genome content. Cluster A, including ST5 and ST105, was highly prevalent at all ages. Cluster B, including ST8, was more limited to younger patients. Cluster C included 6 distantly related strains. Patients 20 years old and younger infected with Cluster A had lower forced expiratory volume in the first second (FEV1 ) and higher sputum biomass compared to similar-aged patients with Cluster B. CONCLUSIONS: In this CF cohort, we identified MRSA subtypes that predominate at different ages and differ by accessory gene content. The most prevalent cluster of MRSA, including ST5 and ST105, was associated with lower FEV1 . ST8 MRSA was more common in younger patients and thus has the potential to rise in prevalence as these patients age.

Sustained Coinfections with Staphylococcus aureus and Pseudomonas aeruginosa in Cystic Fibrosis.

Rationale: Staphylococcus aureus and Pseudomonas aeruginosa often infect the airways in cystic fibrosis (CF). Because registry studies show higher prevalence of P. aeruginosa versus S. aureus in older patients with CF, a common assumption is that P. aeruginosa replaces S. aureus over time. In vitro, P. aeruginosa can outgrow and kill S. aureus. However, it is unknown how rapidly P. aeruginosa replaces S. aureus in patients with CF.Methods: We studied a longitudinal cohort of children and adults with CF who had quantitative sputum cultures. We determined the abundance of P. aeruginosa and S. aureus in cfu/ml. We determined the duration and persistence of infections and measured longitudinal changes in culture positivity and abundance for each organism.Measurements and Main Results: Between 2004 and 2017, 134 patients had ≥10 quantitative cultures, with median observation time of 10.15 years. One hundred twenty-four patients had at least one positive culture for P. aeruginosa, and 123 had at least one positive culture for S. aureus. Both species had median abundance of >106 cfu/ml. Culture abundance was stable over time for both organisms. There was an increase in the prevalence of S. aureus/P. aeruginosa coinfection but no decrease in S. aureus prevalence within individuals over time.Conclusions: S. aureus and P. aeruginosa are abundant in CF sputum cultures. Contrary to common assumption, we found no pattern of replacement of S. aureus by P. aeruginosa. Many patients with CF have durable long-term coinfection with these organisms. New strategies are needed to prevent and treat these infections.

Lack of airway submucosal glands impairs respiratory host defenses.

Submucosal glands (SMGs) are a prominent structure that lines human cartilaginous airways. Although it has been assumed that SMGs contribute to respiratory defense, that hypothesis has gone without a direct test. Therefore, we studied pigs, which have lungs like humans, and disrupted the gene for ectodysplasin (EDA-KO), which initiates SMG development. EDA-KO pigs lacked SMGs throughout the airways. Their airway surface liquid had a reduced ability to kill bacteria, consistent with SMG production of antimicrobials. In wild-type pigs, SMGs secrete mucus that emerges onto the airway surface as strands. Lack of SMGs and mucus strands disrupted mucociliary transport in EDA-KO pigs. Consequently, EDA-KO pigs failed to eradicate a bacterial challenge in lung regions normally populated by SMGs. These in vivo and ex vivo results indicate that SMGs are required for normal antimicrobial activity and mucociliary transport, two key host defenses that protect the lung.

High Prevalence of Staphylococcus aureus Enterotoxin Gene Cluster Superantigens in Cystic Fibrosis Clinical Isolates.

BACKGROUND: Staphylococcus aureus is a highly prevalent respiratory pathogen in cystic fibrosis (CF). It is unclear how this organism establishes chronic infections in CF airways. We hypothesized that S. aureus isolates from patients with CF would share common virulence properties that enable chronic infection. METHODS: 77 S. aureus isolates were obtained from 45 de-identified patients with CF at the University of Iowa. We assessed isolates phenotypically and used genotyping assays to determine the presence or absence of 18 superantigens (SAgs). RESULTS: We observed phenotypic diversity among S. aureus isolates from patients with CF. Genotypic analysis for SAgs revealed 79.8% of CF clinical isolates carried all six members of the enterotoxin gene cluster (EGC). MRSA and MSSA isolates had similar prevalence of SAgs. We additionally observed that EGC SAgs were prevalent in S. aureus isolated from two geographically distinct CF centers. CONCLUSIONS: S. aureus SAgs belonging to the EGC are highly prevalent in CF clinical isolates. The greater prevalence in these SAgs in CF airway specimens compared to skin isolates suggests that these toxins confer selective advantage in the CF airway.

Pathogen acquisition in patients with cystic fibrosis receiving ivacaftor or lumacaftor/ivacaftor.

BACKGROUND: The cystic fibrosis transmembrane conductance regulator (CFTR) modulators ivacaftor and lumacaftor/ivacaftor improve the status of existing infections in patients with cystic fibrosis (CF). It is unknown how well these drugs protect patients against incident infections. We hypothesized that CFTR modulator treatment would decrease new infections with Pseudomonas aeruginosa or Staphylococcus aureus. METHODS: We retrospectively studied a single-center cohort of patients with CF during two time periods (2008-2011, Era 1) and (2012-2015, Era 2) based on the January 2012 approval of ivacaftor. Using Kaplan-Meier analysis, we compared the time to any new infection with P. aeruginosa, methicillin-resistant S. aureus (MRSA), or methicillin-sensitive S. aureus (MSSA) that was absent during a 2-year baseline. We stratified the analysis based on whether patients received ivacaftor or lumacaftor/ivacaftor during Era 2. We used the log-rank test and considered P < 0.05 statistically significant. RESULTS: For patients receiving ivacaftor or lumacaftor/ivacaftor in Era 2, there was a statistically significant delay in the time to new bacterial acquisition in Era 2 vs. Era 1 ( P = 0.008). For patients who did not receive CFTR modulators, there was a trend toward slower acquisition of new bacterial infections in Era 2 compared to Era 1, but this was not statistically significant ( P = 0.10). CONCLUSIONS: Patients receiving ivacaftor or lumacaftor/ivacaftor for CF had significantly delayed acquisition of P. aeruginosa and S. aureus after these drugs were released. This method for analyzing incident infections may be useful for future studies of CFTR modulators and bacterial acquisition in CF registry cohorts.

Mucus strands from submucosal glands initiate mucociliary transport of large particles.

Mucus produced by submucosal glands is a key component of respiratory mucociliary transport (MCT). When it emerges from submucosal gland ducts, mucus forms long strands on the airway surface. However, the function of those strands is uncertain. To test the hypothesis that mucus strands facilitate transport of large particles, we studied newborn pigs. In ex vivo experiments, interconnected mucus strands moved over the airway surface, attached to immobile spheres, and initiated their movement by pulling them. Stimulating submucosal gland secretion with methacholine increased the percentage of spheres that moved and shortened the delay until mucus strands began moving spheres. To disrupt mucus strands, we applied reducing agents tris-(2-carboxyethyl)phosphine and dithiothreitol. They decreased the fraction of moving spheres and delayed initiation of movement for spheres that did move. We obtained similar in vivo results with CT-based tracking of microdisks in spontaneously breathing pigs. Methacholine increased the percentage of microdisks moving and reduced the delay until they were propelled up airways. Aerosolized tris-(2-carboxyethyl)phosphine prevented those effects. Once particles started moving, reducing agents did not alter their speed either ex vivo or in vivo. These findings indicate that submucosal glands produce mucus in the form of strands and that the strands initiate movement of large particles, facilitating their removal from airways.

Mucociliary Transport in Healthy and Cystic Fibrosis Pig Airways.

Cystic fibrosis (CF) lung disease is the major cause of morbidity and mortality in people with CF. Abnormal mucociliary transport has been the leading hypothesis for the underlying pathogenesis of CF airway disease. However, this has been difficult to investigate at very early time points. A porcine CF model, which recapitulates many features of CF disease in humans, enables studies to be performed in non-CF and CF pigs on the day that they are born. In newborn CF pigs, we found that under basal conditions, mucociliary transport rates in non-CF and CF pigs are similar. However, after cholinergic stimulation, which stimulates submucosal gland secretion, particles become stuck in the CF airways owing to a failure of mucus strands to release from submucosal glands. In this review, we summarize these recent discoveries and also discuss the morphology, composition, and function of mucins in the porcine lung.

Surfactant deficiency syndrome in an infant with a C-terminal frame shift in ABCA3: A case report.

Deficiency in ATP binding cassette A3 (ABCA3) causes neonatal respiratory distress, hypoxemic respiratory failure, and interstitial lung disease. ABCA3 transports phospholipids into the lamellar bodies of type II alveolar cells, a critical step in alveolar surfactant production. We report a term infant with ABCA3 surfactant deficiency syndrome with the E292V (c.875A>T; p.Glu292Val) mutation in trans with a novel C-terminal frame shift mutation (c.4938delC; p.Met1647fs). This mutation removes the final 58 amino acids and substitutes 33 incorrect amino acids. The frame shift spares membrane spanning and nucleotide binding domains, but disrupts a highly conserved C-terminal domain, which includes sequence motifs necessary for the function of human paralogs ABCA1, ABCA4, and the bacterial homolog DrrA. This observation suggests the C-terminal domain is also required for normal function of ABCA3.

Pseudomonas eradication and clinical effectivness of Ivacaftor in four Hispanic patients with S549N.

Ivacaftor was approved for rarer class-III CFTR mutations including S549N in 2014. Since these mutations are uncommon, ongoing reports of patient experiences with Ivacaftor and these mutations are important. This case series describes the clinical effectiveness (including airway infection status, lung function, and growth) of Ivacaftor therapy in four pediatric Hispanic patients with S549N and F508del over 24 months. In these patients, Ivacaftor was highly efficacious with no further Pseudomonas-positive cultures despite prior chronic colonization in three patients as well as notable improvements in lung function and growth. The remarkable improvements in lung function and growth were similar to G551D patients with more striking changes in airway infection status. Pediatr Pulmonol 2017;52:E37-E39. © 2017 Wiley Periodicals, Inc.

Acute administration of ivacaftor to people with cystic fibrosis and a G551D-CFTR mutation reveals smooth muscle abnormalities.

BACKGROUND: Airflow obstruction is common in cystic fibrosis (CF), yet the underlying pathogenesis remains incompletely understood. People with CF often exhibit airway hyperresponsiveness, CF transmembrane conductance regulator (CFTR) is present in airway smooth muscle (ASM), and ASM from newborn CF pigs has increased contractile tone, suggesting that loss of CFTR causes a primary defect in ASM function. We hypothesized that restoring CFTR activity would decrease smooth muscle tone in people with CF. METHODS: To increase or potentiate CFTR function, we administered ivacaftor to 12 adults with CF with the G551D-CFTR mutation; ivacaftor stimulates G551D-CFTR function. We studied people before and immediately after initiation of ivacaftor (48 hours) to minimize secondary consequences of CFTR restoration. We tested smooth muscle function by investigating spirometry, airway distensibility, and vascular tone. RESULTS: Ivacaftor rapidly restored CFTR function, indicated by reduced sweat chloride concentration. Airflow obstruction and air trapping also improved. Airway distensibility increased in airways less than 4.5 mm but not in larger-sized airways. To assess smooth muscle function in a tissue outside the lung, we measured vascular pulse wave velocity (PWV) and augmentation index, which both decreased following CFTR potentiation. Finally, change in distensibility of <4.5-mm airways correlated with changes in PWV. CONCLUSIONS: Acute CFTR potentiation provided a unique opportunity to investigate CFTR-dependent mechanisms of CF pathogenesis. The rapid effects of ivacaftor on airway distensibility and vascular tone suggest that CFTR dysfunction may directly cause increased smooth muscle tone in people with CF and that ivacaftor may relax smooth muscle. FUNDING: This work was funded in part from an unrestricted grant from the Vertex Investigator-Initiated Studies Program.

Sonographic evidence of abnormal tracheal cartilage ring structure in cystic fibrosis.

OBJECTIVES/HYPOTHESIS: Tracheal cartilage ring structural abnormalities have been reported in cystic fibrosis (CF) mice and pigs. Whether similar findings are present in humans with CF is unknown. We hypothesized that tracheal cartilage ring shape and size would be different in people with CF. STUDY DESIGN: Tracheal cartilage ring size and shape were measured in adults with (n = 21) and without CF (n = 18). METHODS: Ultrasonography was used in human subjects to noninvasively assess tracheal cartilage ring structure in both the sagittal and the transverse planes. Tracheal cartilage ring thickness was also determined from histological sections obtained from newborn non-CF and CF pigs. These values were compared with human data. RESULTS: Human CF tracheas had a greater width and were less circular in shape compared to non-CF subjects. CF tracheal cartilage rings had a greater midline cross-sectional area and were thicker compared to non-CF rings. Maximal tracheal cartilage ring thickness was also greater in both newborn CF pigs and human adults with CF, compared to non-CF controls. CONCLUSIONS: Our findings demonstrate that structural differences exist in tracheal cartilage rings in adults with CF. Comparison with newborn CF pig data suggests that some of these changes may be congenital in nature. LEVEL OF EVIDENCE: 3b