BACKGROUND:Relatively little is known about Pseudomonas aeruginosa lineages during early infection in individuals with cystic fibrosis (CF). The Early Pseudomonas Infection Control Clinical Trial (EPIC-CT), which treated individuals with CF and newly detected P. aeruginosa infections, presents an ideal opportunity to study this phenomenon. METHODS:We performed whole-genome sequencing on 572 P. aeruginosa isolates from 190 EPIC-CT subjects. RESULTS:We identified 203 P. aeruginosa lineages causing newly detected infections near the time of enrollment of these subjects. Twelve subjects were initially infected with more than one P. aeruginosa lineage, and 20 subjects had different PA lineages detected during the follow-up period of the EPIC-CT. Of the 203 lineages causing initial infections, 144 were not detected again by culture and 59 were detected again despite antibiotic therapy. Multilocus sequence typing was as accurate as whole genome single nucleotide variant (SNV) typing in discriminating lineages, but pulsed-field gel electrophoresis inaccurately classified 17 genetically related isolates as distinct. Although subjects were infected for a relatively short time, 23 P. aeruginosa genes acquired nonsynonymous SNVs in at least 2 subjects. Of these, 14 had been previously identified as pathoadaptive, confirming that such mutations can emerge early in CF infections. CONCLUSIONS:Our study demonstrates the complexity of early P. aeruginosa infections in people with CF and the potential of these complexities to confound interpretation of antibiotic efficacy studies.

Objectives This prospective longitudinal study characterised respiratory microbiome dynamics following new tracheostomy placement among infants. Setting A tertiary care paediatric hospital system in the United States. Participants Fifteen infants ≤12 months of age contributed 84 tracheal aspirate samples collected from day 1 through 3–4 months post-placement. Primary and secondary outcome measures Bacterial composition, including abundance, from 16S rRNA gene sequencing; alpha and beta diversity measures over time. Results 16S rRNA gene sequencing revealed immediate and sustained bacterial community shifts. Staphylococcus abundance increased and alpha diversity decreased in the first 30 days post-tracheostomy (p<0.05) before returning to baseline. Beta diversity demonstrated compositional changes immediately and with ongoing divergence through 3–4 months. Time and clinical factors (prematurity, ventilation and neurologic impairment) were significantly associated with microbiome structure (p=0.001). Conclusions This study provides novel evidence that new tracheostomy placement induces rapid and prolonged airway microbiome disruption in infants, highlighting a previously uncharacterised window of vulnerability with implications for respiratory health.

BACKGROUND:Low bone mineral density (BMD) and increased fracture risk are common in individuals with cystic fibrosis (CF). The extent to which the CF transmembrane conductance regulator (CFTR) modulator elexacaftor-tezacaftor-ivacaftor (ETI) benefits BMD was a focus of the endocrine sub-study of PROMISE, a multicenter observational study of clinically prescribed ETI. We examined changes in whole-body (WB), lumbar spine (LS), total hip (TH), and femoral neck (FN) areal BMD (aBMD, g/cm2) in the 24-30 months (mos) following ETI initiation. METHODS:Participants had CF, ≥1 F508del mutation, and were aged ≥12 years (y). Dual-energy X-ray absorptiometry (DXA) scans of the WB, LS, TH, and FN were collected before and following 12-18 mos and 24-30 mos of ETI therapy. Changes in aBMD Z-scores (aBMDZ) were examined with longitudinal mixed effects models. RESULTS:Baseline aBMDZ was below-average at all skeletal sites in youth and adults (aBMDZ <0). Mixed model results for youth [n = 60 at baseline; average age 15y (range: 12-19.8); 48 % female] revealed decreases in WB (less head) (β-coefficient=-0.27; 95 %CI: -0.46, -0.09), LS (β=-0.26; 95 %CI: -0.42, -0.10), TH (β=-0.29; 95 %CI: -0.45, -0.13), and FN (β=-0.37; 95 %CI: -0.57, -0.17) aBMDZ between baseline and 12-18 mos. These changes persisted but did not worsen at 24-30 mos. Changes in adult [n = 73 at baseline; average age 28y (range: 20-58.8); 51 % female] aBMDZ were negative but modest compared to youth (no β-coefficient >-0.11). CONCLUSIONS:Youth aBMDZ was lower at multiple skeletal sites 12-18 mos after ETI initiation, and these changes persisted at 24-30 mos. Adult aBMDZ generally remained unchanged.

BACKGROUND:Ventricular reservoir infections and cerebrospinal fluid (CSF) shunt infections are diagnosed when bacteria are recovered from microbiological cultures of CSF samples from these devices. We applied high throughput sequencing (HTS) to understand the course of changes in ventricular reservoir and shunt infection microbiota. OBJECTIVES:Evaluate the utility of monitoring microbiota in CSF (1) from ventricular reservoirs to detect development of an infection and (2) during treatment of CSF shunt infections to assess treatment response. METHODS:Study populations included (1) neonates with temporizing ventricular reservoirs who developed reservoir infection and (2) children undergoing treatment for conventional culture-confirmed CSF shunt infection. The V4 region of the 16S ribosomal RNA gene was amplified and sequenced. Comparison of taxonomic results of HTS with standard microbiological culture results (when available) was described for each CSF sample. A robust HTS signal was defined by a microbial load of ≥1e5 microbial genome equivalents/mL. RESULTS:In none of the five ventricular reservoir infection cases was there a robust HTS signal for the responsible bacteria immediately prior to infection. In six of the seven CSF shunt infection cases, there was a robust HTS signal for the genus of the responsible bacteria in the sample at the time of positive CSF culture. The proportion of sequences from the genus associated with the responsible bacteria decreased during infection treatment. CONCLUSIONS:These pilot data suggest limited utility in using HTS for surveillance for ventricular reservoir infections, as they emerge abruptly. In CSF shunt infection, HTS demonstrates a return to heterogeneous microbiota when bacterial cultures become negative.

BACKGROUND:For children with Crohn's disease (CD), dietary therapy with exclusive enteral nutrition (EEN) is effective in achieving clinical and biochemical remission. We investigated changes in metabolites in multiple clinical sample types from children with CD following a whole foods-blended ("reverse-engineered") EEN formula and defined associations between these changes and remission. METHODS:Stool, urine, serum, and plasma from a prospective study of newly diagnosed pediatric patients with CD enrolled in a 4-week trial of reverse-engineered exclusive enteral nutrition (RE-EEN) were analyzed using mass spectrometry targeting aqueous metabolites, bile acids, and short-chain fatty acids. Principal component analysis, mixed-effects models, and exploratory multivariate approaches including random forest and partial least-squares discriminant analysis were used to identify patterns associated with diet and metabolite abundances. RESULTS:Fecal, urine, serum, and plasma metabolomes changed significantly during RE-EEN treatment from baseline. These global changes were largely driven by increases in amino acids and related metabolites and decreases in different amino acids and metabolites reflecting carbohydrate and purine metabolism, in all sample types. While global bile acid profiles changed with the RE-EEN diet, no changes in specific bile acid levels reached significance, and no changes were found in short-chain fatty acid concentrations. CONCLUSION:Metabolomic profiles for pediatric patients with CD changed broadly during RE-EEN, indicating that this therapy may modulate key systemic and gut-associated metabolic processes. The findings further suggest that the gut metabolic processes influenced by RE-EEN, and that contribute to clinical improvement, may differ from those impacted by commercial EEN diets. Further research is crucial to validate these findings, uncover causal relationships, and optimize dietary protocols to enhance therapeutic outcomes in CD.

Current approaches for computationally analyzing viruses within human microbiomes often rely on databases largely composed of fragmented viral genomes from gastrointestinal samples, limiting identification of viruses exclusively found outside the gastrointestinal tract and analyses requiring high-quality genomes. To address these issues, we created the Unified Human Virome Database (UHVDB), comprising 575,497 high-quality, annotated viral genomes from human gastrointestinal, airway, skin, and urogenital sample metagenomes. We developed an associated toolkit that uses UHVDB to characterize viruses and their potential activity from metagenomes, then applied this toolkit to 1,983 airway sample metagenomes from people with cystic fibrosis. Over half of detected viruses lacked evidence of potential activity and were detected transiently. UHVDB is nearly three times larger than prior viral databases and its ability to identify likely active viruses enables rigorous analysis of viruses from diverse human sample types, expanding the capacity to define virus contributions to health and disease.

Abnormal respiratory microbiomes are reported in children with artificial airways, yet the timing and persistence of these disruptions have not been defined in infants following new tracheostomy placement. We conducted a prospective longitudinal study to characterize airway microbiome dynamics following new tracheostomy placement during early life, a critical period for microbiome development. Fifteen hospitalized infants <=12 months contributed 84 tracheal aspirate samples collected from day 1 through 3 to 4 months post-procedure. 16S rRNA sequencing revealed immediate and sustained community shifts. Staphylococcus abundance increased after tracheostomy, peaking at 40 days (mean 27%) before declining, with a more pronounced bloom in infants without home mechanical ventilation (HMV). Alpha diversity decreased significantly in the first 30 days (p<0.05) and returned to baseline by 61 to 90 days. Beta diversity analysis demonstrated marked compositional changes immediately post-tracheostomy and ongoing divergence through 3 to 4 months. Time since tracheostomy and clinical factors (gestational age, HMV, neurologic impairment) were significantly associated with microbiome structure (p=0.001). These findings provide novel evidence that tracheostomy induces rapid and prolonged airway microbiome disruption in infants, highlighting a previously uncharacterized window of vulnerability with implications for respiratory health and individualized care.

OBJECTIVES:Evaluate if early therapeutic initiation may begin ameliorating olfactory dysfunction in addition to chronic rhinosinusitis in young children with cystic fibrosis. METHODS:Participants ≤ 10 years were enrolled across six U.S. cystic fibrosis centers (May 2023-December 2024). Data were collected at baseline and one year following therapeutic initiation. Mixed-effects models were adjusted for age, sex, and nasal steroid use. MEASUREMENTS:Sinus magnetic resonance imaging analysis included sinus volume, sinus opacification, olfactory bulb volume, olfactory cleft opacification, Lund-Mackay, and CRS-MRI scores. Psychophysical olfactory testing and quality of life assessments included the Pediatric Smell Wheel, Brief Questionnaire of Olfactory Disorders, EuroQol-5-Dimensions-Youth, and Sinonasal-5.Main Results: 31 participants (mean age 4.8 years) enrolled, 27 initiated treatment, and 26 completed follow-up (mean treatment duration 1.0 years). Following therapy, three olfactory measures improved: odor identification scores increased by 1.3 points (p = 0.03), olfactory bulb volume increased by 7.4mm3 (p = 0.02), and olfactory cleft opacification decreased by 14.4% (p = 0.004). Sinus opacification, Lund-Mackay, and CRS-MRI scores improved by 25.5% (p = 0.001), 5.0 points (p = 0.002) and 7.0 points (p = 0.005), respectively. Quality of life scores remained at near-normal levels. CONCLUSIONS:Children receiving therapy demonstrated improvements in olfactory function, olfactory structure, and sinonasal opacification. Predominantly stable, near-normal quality of life metrics preliminarily suggest symptom-based instruments alone may be insufficient to identify sinonasal disease and treatment response at this age.

Abnormal respiratory microbiomes are reported in children with artificial airways, yet the timing and persistence of these disruptions have not been defined in infants following new tracheostomy placement. We conducted a prospective longitudinal study to characterize airway microbiome dynamics following new tracheostomy placement during early life, a critical period for microbiome development. Fifteen hospitalized infants <=12 months contributed 84 tracheal aspirate samples collected from day 1 through 3 to 4 months post-procedure. 16S rRNA sequencing revealed immediate and sustained community shifts. Staphylococcus abundance increased after tracheostomy, peaking at 40 days (mean 27%) before declining, with a more pronounced bloom in infants without home mechanical ventilation (HMV). Alpha diversity decreased significantly in the first 30 days (p<0.05) and returned to baseline by 61 to 90 days. Beta diversity analysis demonstrated marked compositional changes immediately post-tracheostomy and ongoing divergence through 3 to 4 months. Time since tracheostomy and clinical factors (gestational age, HMV, neurologic impairment) were significantly associated with microbiome structure (p=0.001). These findings provide novel evidence that tracheostomy induces rapid and prolonged airway microbiome disruption in infants, highlighting a previously uncharacterized window of vulnerability with implications for respiratory health and individualized care. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This project was funded by the Gerber Foundation grant number 7264. Dr. Steuart was supported by the Childrens Research Institute at Childrens Wisconsin via a KL2 award and the National Center for Advancing Translational Sciences, National Institutes of Health, Award Number UL1 TR001436. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Institutional Review Boards at Children's Hospital Los Angeles and Stanford University's reviewed and approved the study protocol under expedited review (CHLA-20-00074, approved 3/26/2020; Stanford ID: 71998, approved 10/16/2024). Parents provided written informed consent for infants to participate. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors.

BackgroundChronic rhinosinusitis (CRS) and olfactory dysfunction (OD) are prevalent disease complications in people with cystic fibrosis. These understudied comorbidities significantly impact quality of life. The impact of highly effective modulator therapy (HEMT) in young children with cystic fibrosis (YCwCF) on these disease complications is unknown. This proposed study aims to characterise CRS and OD in YCwCF and assess the efficacy of HEMT in improving sinus and olfactory health in this young age group.MethodsThis six-centre, prospective, observational study will enrol 80 YCwCF aged 2–8 years. Patients are divided into two groups: those receiving HEMT and those not on HEMT based on clinical indication. Both groups undergo sinus magnetic resonance imaging, psychophysical olfactory tests, and complete patient- or parent-reported quality of life surveys over 2 years. Outcomes will be compared before and after initiation of HEMT and between groups. Ethical approval has been obtained for all sites, and this study has been registered on ClinicalTrials.gov (NCT06191640).ResultsEnrolment began in April 2023. 21 participants have been enrolled as of October 2023 with ongoing enrolment at all sites.ConclusionThis investigation is expected to provide critical insights into the potential benefits of early HEMT initiation in managing CRS and OD in YCwCF. It will assist in developing targeted interventions and contribute to the understanding of HEMT's role in altering the disease course in this demographic.

BACKGROUND:People with cystic fibrosis (PwCF) often have fecal dysbioses relative to those without CF, characterized by increased pro-inflammatory microbiota and gastrointestinal (GI) inflammation as measured by fecal calprotectin, suggesting that inflammation contributes to CF GI disease. The multicenter observational PROMISE study (NCT04038047) found that calprotectin decreased in PwCF treated with elexacaftor/tezacaftor/ivacaftor (ETI). To better understand the dynamics between fecal dysbiosis and GI inflammation, we characterized the microbiomes of fecal samples from PROMISE and the relationships with calprotectin before, 1-month post, and 6-months post ETI. METHODS:Fecal microbiota from participants ≥12 y/o were determined by shotgun metagenomic sequencing with random forest modeling and multivariate linear regression analysis to define relationships between microbiota, calprotectin, and deltaF508 genotype before and after ETI. RESULTS:We analyzed 345 samples from 124 participants. At baseline, we observed community-level differences in the fecal microbiota among participants with abnormal compared to normal calprotectin. With ETI, the relative abundances of 7 bacterial species - Escherichia coli, Staphylococcus aureus, Clostridium scindens, Enterocloster clostridioformis, Clostridium butyricum, Anaeroglobus geminatus, and Ruminococcus gnavus - decreased significantly, correlating with calprotectin decrease. We detected community-level differences in the fecal microbiota based on CFTR genotype and a distinct pattern of microbiota change in F508del homozygous compared to heterozygous participants after ETI. CONCLUSIONS:We identified 7 species for which fecal abundances decreased with ETI and correlated with calprotectin decrease, supporting a close relationship between fecal microbiota and inflammation in PwCF. Future work will define these relationships with metabolites and GI symptoms during long-term ETI therapy.

Rationale: Elexacaftor/tezacaftor/ivacaftor (ETI) provided substantial health benefits to children with cystic fibrosis (CF) in clinical trials; there is less information about its effectiveness in a "real world" setting. Objectives: The aim of the PROMISE (A Prospective Study to Evaluate Biological and Clinical Effects of Significantly Corrected CFTR Function) Pediatric substudy is to determine the long-term (four years) impact of clinically prescribed ETI among children 6-11 years of age at enrollment. The primary outcome measure is the lung clearance index at a 2.5% (LCI2.5). Methods: The PROMISE Pediatric substudy enrolled children with CF 6 to <12 years of age starting ETI. Outcomes measured at baseline (before ETI) and 1, 3, 6, and 12 months after ETI initiation included LCI2.5, percentage predicted forced expiratory volume in 1 second, Cystic Fibrosis Questionnaire-Revised (CFQ-R) respiratory domain symptom score, height, weight, oropharyngeal cultures, and culture and deoxyribonucleic acid-based analysis of sputum microbiology (when sputum was available). Sweat chloride was assessed at baseline and at 1 and 6 months. Results: One hundred twenty-five participants were enrolled at 20 U.S. CF centers. Lung function improvement after ETI initiation was rapid and sustained through 12 months, with a mean decrease in LCI2.5 of -0.79 (95% confidence interval [CI], -1.04 to 0.55) and a mean increase in percentage predicted forced expiratory volume in 1 second of 5.6% (95% CI, 3.4% to 7.7%). Respiratory symptoms also diminished significantly (mean change in CFQ-R respiratory domain symptom score, 4.1 [95% CI, 1.94 to 6.24]). Sweat chloride decreased significantly at 6 months (mean change, -47.2 mmol/L [95% CI, -51.99 to -43.8 mmol/L]). Weight, body mass index, and height z-scores were not different from baseline at 12 months. Staphylococcus aureus prevalence in oropharyngeal or sputum cultures did not change, but its density in sputum cultures decreased a mean of 1.47 log10 colony-forming units/g (95% CI, -2.37 to -0.58 colony-forming units/g) at 12 months. Conclusions: Initiation of ETI in a real-world setting was associated with clinically significant improvements in lung function and symptoms and decreased S. aureus sputum density at one year; lung function improvements were smaller than those reported in clinical trials. Clinical trial registered with www.clinicaltrials.gov (NCT04038047).

"Sticky Staph: A New Story About Mucoidy and Cystic Fibrosis." American Journal of Respiratory and Critical Care Medicine, 0(ja), pp.

BACKGROUND:Elexacaftor/tezacaftor/ivacaftor (ETI) is a highly effective therapy that improves lung disease in people with cystic fibrosis (pwCF), but its effect on glucose tolerance and insulin secretion is unclear. METHODS:PROMISE is a multicenter prospective, observational study of ETI in pwCF ≥12 years and at least one F508del allele. The PROMISE Endocrine substudy (PROMISE-ENDO) enrolled participants at 10 CF Centers where hemoglobin A1c (HbA1c) was collected and 3-hour oral glucose tolerance tests (OGTT) conducted to examine glucose tolerance, glucose excursions, and insulin secretory rates (deconvolution of C-peptide) and sensitivity (oral minimal model) prior to ETI and 12 to 18 months and 24-30 months following ETI initiation. Longitudinal mixed effects models were used to test within-subject ETI effects. RESULTS:At baseline, 79 participants completed OGTTs (39 [49%] male, median [IQR] age 19.6 [14.7, 27.3] years, BMI z-score 0.12 [-0.51, 0.65]). At 12-18 months n = 68 and at 24-30 months n = 58 completed OGTTs. At 24-30 months, fasting glucose (mg/dL) decreased (94 [92, 96] to 90 [88, 93], P = .02) in the subset not on insulin therapy (n = 61), but no differences in 1-hour or 2-hour glucose were found. HbA1c decreased from 5.8% (5.6%, 5.9%) to 5.5% (5.4%, 5.6%), P < .001 by 24-30 months. Although insulin sensitivity (mU/L-1 min-1) decreased (8.4 [7.2, 9.5] vs 6.8 [5.8, 7.9], P = .03), no changes in oral disposition index were found, P = .14. CONCLUSION:After 2 years of ETI, fasting glucose and HbA1c showed modest decreases. Glucose tolerance varied, and overall measures of insulin secretion did not deteriorate.

Rationale: Tracheostomy aspirate (TA) samples from children with tracheostomies have abnormal microbiology. Because these samples drive clinical antibiotic choices, it is important to define tracheostomy microbiome variability over time following new tracheostomy placement, and whether microbiome profiles evolve in predictable patterns. Objective: To identify TA sample microbiome changes longitudinally following new tracheostomy placement in children, and variation in these patterns by time, individual, and ventilator status. Methods: A prospective study of children 0-36 months old undergoing new tracheostomy placement. Tracheostomy aspirates (TA) were collected on the day of tracheostomy placement (day 0), at first tracheostomy change (day 7) and immediately after tracheostomy tube changes, approximately every 2 weeks during the initial and subsequent hospitalizations. Sample DNA was analyzed using 16S rRNA gene sequencing to identify and quantify bacteria. Results were analyzed for taxonomy, relative abundances, and within-sample (alpha) diversity, and microbiome profiles compared (beta diversity) by Principal Coordinate Analyses (PCoA) using Bray-Curtis dissimilarity and Permutational Multivariate Analysis of Variance (PERMANOVA). Results: We collected 163 TA samples from 21 children (median 6 samples per child, IQR: 4-9) with median age of 6.5 months (IQR: 3-13.75) at tracheostomy placement. Half were born prematurely (14% early preterm, 38% late preterm), and 65% discharged using chronic mechanical ventilation. The most abundant TA taxa differed between children, but included Pseudomonas, Staphylococcus, and Streptococcus. Overall sample bacterial community richness was stable across sampling points, but evenness decreased after tracheostomy placement. PCoA plots demonstrated that TA sample microbiomes were more similar between children at tracheostomy placement than at later timepoints (Figure). Within-subject microbiome profiles clustered relatively tightly for eight children, indicating their microbiomes were relatively stable over time compared with the others. Microbiome profiles for samples from children without chronic ventilation clustered more tightly than did those from children using ventilation. Seven children with microbiome stability (88%) were born prematurely, and 88% used a ventilator chronically. Most (75%) had persistently identified Pseudomonas (>10% relative abundance). Predominant taxa and relative abundances stabilized by day 7 after tracheostomy placement for this subgroup. Conclusions: Respiratory microbiomes changed rapidly in the first months after new tracheostomy placement in young children, stabilizing for only a subset while changing frequently over time for most children. TA samples collected within the first week largely did not represent future bacteriology. For most children with tracheostomies, past TA samples may not be reflective of current microbiology or antibiotic needs.

BACKGROUND:Systemic antibiotics can impact all microbes inhabiting patients, regardless of the intended target organism(s). We studied the simultaneous effects on respiratory and fecal microbiomes of β-lactam antibiotics administered for respiratory symptoms in infants with cystic fibrosis (IWCF). OBJECTIVE:To compare the magnitude and duration of intended (respiratory) and unintended (fecal) antimicrobial action by analyzing oropharyngeal (OP) and fecal microbiota in IWCF. DESIGN:Shotgun metagenomic sequencing and qPCR were performed on OP and fecal samples collected longitudinally from 14 IWCF (ages 1-17 months) during ("On Antibiotics") and after ("Off Antibiotics") β-lactam therapy, and from 5 IWCF (3-16 months) never treated with antibiotics. RESULTS:Total bacterial loads (TBL) for On Antibiotics samples were lower than for both Never (OP and fecal) and Off Antibiotics samples (fecal only). α-diversities (within-sample) for OP On Antibiotics samples were lower than for Never and Off Antibiotics samples but did not differ between fecal sample groups. β-diversity (between-sample) differed between all OP sample groups and between fecal On and Never Antibiotics and Off and Never antibiotics samples; however, fecal On and Off Antibiotics sample β-diversities did not differ. Patterns of change in antibiotic resistance gene abundances reflected shifts in microbial community composition. CONCLUSIONS:β-lactam antibiotic exposure was followed by marked alterations in both OP and fecal microbiota. While microbiota appeared to rebound after treatment in both sample types, our results suggest that fecal microbiota recovered less than OP. The clinical consequences of these findings should be studied in IWCF and other populations frequently treated with antibiotics.

ABSTRACTStaphylococcus aureusis one of the most common pathogens isolated from the lungs of people with cystic fibrosis (CF), but little is known about its ability to colonize this niche. We performed a Tn-seq screen to identify genes necessary forS. aureusgrowth in media prepared fromex vivoCF sputum. We identified 19 genes that were required for growth in all sputum media tested and dozens more that were required for growth in at least one sputum medium. Depleted mutants of interest included insertions in many genes important for surviving metal starvation as well as the primary regulator of cysteine metabolismcymR. To investigate the mechanisms by which these genes contribute toS. aureusgrowth in sputum, we quantified low-molecular-weight thiols, nutrient transition metals, and the host metal-sequestration protein calprotectin in sputum from 11 individuals with CF. In all samples, the abundance of calprotectin exceeded nutrient metal concentration, explaining theS. aureusrequirement for metal-starvation genes. Further, all samples contain potentially toxic quantities of cysteine and sufficient glutathione to satisfy the organic sulfur requirements ofS. aureus. Deletion of the cysteine importer genestcyAandtcyPin the ΔcymRbackground restored growth to wild-type levels in CF sputum, suggesting that the mechanism by whichcymRis required for growth in sputum is to prevent uncontrolled import of cysteine or cystine from this environment. Overall, this work demonstrates that calprotectin and cysteine limitS. aureusgrowth in CF sputum.IMPORTANCEStaphylococcus aureusis a major cause of lung infections in people with cystic fibrosis (CF). This work identifies genes required forS. aureusgrowth in this niche, which represent potential targets for anti-Staphylococcal treatments. We show that genes involved in surviving metal starvation are required for growth in CF sputum. We also found that the primary regulator of cysteine metabolism, CymR, plays a critical role in preventing cysteine intoxication during growth in CF sputum. To support these models, we analyzed sputum from 11 individuals with CF to determine concentrations of calprotectin, nutrient metals, and low-molecular-weight thiols, which have not previously been quantified together in the same samples.

SUMMARYThis guidance presents recommendations for clinical microbiology laboratories for processing respiratory samples from people with cystic fibrosis (pwCF). Appropriate processing of respiratory samples is crucial to detect bacterial and fungal pathogens, guide treatment, monitor the epidemiology of cystic fibrosis (CF) pathogens, and assess therapeutic interventions. Thanks to CF transmembrane conductance regulator modulator therapy, the health of pwCF has improved, but as a result, fewer pwCF spontaneously expectorate sputum. Thus, the collection of sputum samples has decreased, while the collection of other types of respiratory samples such as oropharyngeal and bronchoalveolar lavage samples has increased. To optimize the detection of microorganisms, including Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, and Burkholderia cepacia complex; other less common non-lactose fermenting Gram-negative bacilli, e.g., Stenotrophomonas maltophilia, Inquilinus, Achromobacter, Ralstonia, and Pandoraea species; and yeasts and filamentous fungi, non-selective and selective culture media are recommended for all types of respiratory samples, including samples obtained from pwCF after lung transplantation. There are no consensus recommendations for laboratory practices to detect, characterize, and report small colony variants (SCVs) of S. aureus, although studies are ongoing to address the potential clinical impact of SCVs. Accurate identification of less common Gram-negative bacilli, e.g., S. maltophilia, Inquilinus, Achromobacter, Ralstonia, and Pandoraea species, as well as yeasts and filamentous fungi, is recommended to understand their epidemiology and clinical importance in pwCF. However, conventional biochemical tests and automated platforms may not accurately identify CF pathogens. MALDI-TOF MS provides excellent genus-level identification, but databases may lack representation of CF pathogens to the species-level. Thus, DNA sequence analysis should be routinely available to laboratories for selected clinical circumstances. Antimicrobial susceptibility testing (AST) is not recommended for every routine surveillance culture obtained from pwCF, although selective AST may be helpful, e.g., for unusual pathogens or exacerbations unresponsive to initial therapy. While this guidance reflects current care paradigms for pwCF, recommendations will continue to evolve as CF research expands the evidence base for laboratory practices.