Longitudinal profiling of the intestinal microbiome in children with cystic fibrosis treated with elexacaftor-tezacaftor-ivacaftor

ABSTRACT The intestinal microbiome influences growth and disease progression in children with cystic fibrosis (CF). Elexacaftor-tezacaftor-ivacaftor (ELX/TEZ/IVA), the newest pharmaceutical modulator for CF, restores the function of the pathogenic mutated CF transmembrane conductance regulator (CFTR) channel. We performed a single-center longitudinal analysis of the effect of ELX/TEZ/IVA on the intestinal microbiome, intestinal inflammation, and clinical parameters in children with CF. Following ELX/TEZ/IVA, children with CF had significant improvements in body mass index and percent predicted forced expiratory volume in one second, and required fewer antibiotics for respiratory infections. Intestinal microbiome diversity increased following ELX/TEZ/IVA coupled with a decrease in the intestinal carriage of Staphylococcus aureus, the predominant respiratory pathogen in children with CF. There was a reduced abundance of microbiome-encoded antibiotic resistance genes. Microbial pathways for aerobic respiration were reduced after ELX/TEZ/IVA. The abundance of microbial acid tolerance genes was reduced, indicating microbial adaptation to increased CFTR function. In all, this study represents the first comprehensive analysis of the intestinal microbiome in children with CF receiving ELX/TEZ/IVA. IMPORTANCE Cystic fibrosis (CF) is an autosomal recessive disease with significant gastrointestinal symptoms in addition to pulmonary complications. Recently approved treatments for CF, CF transmembrane conductance regulator (CFTR) modulators, are anticipated to substantially improve the care of people with CF and extend their lifespans. Prior work has shown that the intestinal microbiome correlates with health outcomes in CF, particularly in children. Here, we study the intestinal microbiome of children with CF before and after the CFTR modulator, ELX/TEZ/IVA. We identify promising improvements in microbiome diversity, reduced measures of intestinal inflammation, and reduced antibiotic resistance genes. We present specific bacterial taxa and protein groups which change following ELX/TEZ/IVA. These results will inform future mechanistic studies to understand the microbial improvements associated with CFTR modulator treatment. This study demonstrates how the microbiome can change in response to a targeted medication that corrects a genetic disease.

Over the past decade, small-molecule therapies which address the primary defect in the CFTR protein and rescue CFTR function in select genotypes have been developed.These "CFTR modulators" have dramatically changed the trajectory of CF patient care, yielding remarkable improvements in lung function, growth, and projected lifespan (27).The most recent CFTR modulator formulation approved for clinical care, elexacaftortezacaftor-ivacaftor (ELX/TEZ/IVA), includes two CFTR correctors (ELX and IVA) and one CFTR potentiator (TEZ ) (28).The approval of ELX/TEZ/IVA is anticipated to be the most important advancement in CF therapy since CFTR was identified over 30 years ago (27,28).
Given the progressive nature of CF, initiation of therapy in early childhood is critical to stall disease progression.ELX/TEZ/IVA was approved by the FDA for pediatric patients in 2019 (ages 12 years and older), 2021 (ages 6-11 years old), and 2023 (ages 2-5 years old).The impact of ELX/TEZ/IVA on clinical outcomes and the intestinal microbiome in children with CF requires targeted study.To date, studies examining the effects of CFTR modulators on the intestinal microbiome have been limited by the use of single modulator formulations or small cohort sizes (29)(30)(31).Importantly, no studies have characterized the effect of ELX/TEZ/IVA on the intestinal microbiome of children with CF.We, therefore, undertook a longitudinal study to identify changes in clinical outcomes and the intestinal microbiomes of pediatric patients following the initiation of ELX/TEZ/ IVA.To our knowledge, this study represents the first comprehensive study of the highly effective CFTR modulator formulation ELX/TEZ/IVA on the intestinal microbiome of children with CF.

Recruitment of subjects
Pediatric patients with a diagnosis of CF at Monroe Carell Jr. Children's Hospital at Vanderbilt (MCJCHV) were recruited beginning in July 2017 with follow-up until October 2022.Patients with the Phe508 CFTR mutation who were deemed eligible for ELX/TEZ/IVA by their pulmonologist were eligible for this study.Patients previously treated with other CFTR modulator regimens were permitted.Patients with pre-exist ing non-CF gastrointestinal disease were excluded.A total of 39 participants were recruited (Table 1).Informed consent was obtained from parental guardians, and assent was obtained from pediatric subjects in accordance with institutional research ethics guidelines.This study was approved by the MCJCHV Institutional Review Board (IRB # 200396).The lead investigators of this study had no direct role in the patients' routine medical care.All study data were stored in a Research Electronic Data Capture database per institutional guidelines (32).

Stool sample collection and DNA extraction
Stool samples were collected prior to ELX/TEZ/IVA initiation either through targeted collection as part of this study or from our center's biobank of CF stool samples (33,34).Subsequent stool samples were collected at approximately 6-and 12-month intervals after initiation of ELX/TEZ/IVA (Fig. 1A).Fecal samples were collected in sterile collection cups and refrigerated until transport to the laboratory.Patients who were unable to provide a stool sample in clinic were provided with an OMNIgene•GUT Stool Collection Kit for at-home collection and stabilization, returned via overnight shipping, and stored according to manufacturer specifications.Aliquots of stool samples were aseptically aliquoted into cryovials in a laminar flow biosafety cabinet to minimize aerosols and stored at −80°C until processing.Percent predicted forced expiratory volume in one second (ppFEV 1 ) values were obtained in the outpatient setting at the time of stool sample collection; the highest spirometry value of three attempts was recorded.Clinical data including body mass index (BMI, percentiles), ppFEV 1 , medications, and laboratory values were recorded for visits when a stool sample was collected.Total DNA was extracted from 114 stool samples using QIAamp PowerFecal Pro DNA Kits, according to the manufacturer's instructions.Bead beating for efficient lysis was conducted for 10 minutes.All steps, excluding bead beating and centrifugation, were conducted in a laminar flow biosafety cabinet.No human DNA depletion or enrichment of microbial DNA was performed.DNA yield was estimated by spectrophotometry (NanoDrop 2000c) in parallel with ensuring satisfactory A260/A280 ratio for DNA purity.

Fecal calprotectin measurements
Fecal calprotectin was measured using the Calprotectin ELISA Assay Kit (Eagle Bioscien ces).Duplicate portions of stool (50-100 mg) were weighed and processed according to the manufacturer's instructions.Absorbance was measured with a SpectraMax i3x (Molecular Devices).Fecal calprotectin (µg/g feces) was calculated using a seven-point standard curve.The assay's reported normal cutoff is <43.2 µg/g.

Shotgun metagenomic sequencing
Sequencing libraries were prepared using Illumina reagents as described elsewhere (35).
Pooled libraries were sequenced on NextSeq2000 to generate 150-bp paired-end reads.An average of 13.9 million reads were produced per sample (range 9.1-30.1 million).

Taxonomic and functional profiling of metagenomic sequence data
Species abundances were determined with MetaPhlAn4 following read alignment to the MetaPhlan4 database (37).Diversity metrics were calculated with the vegan R package (38) and plotted with ggplot2.A weighted UniFrac distance matrix was constructed with the MetaPhlAn R script "calculate_unifrac." Functional profiling was conducted with HUMAnN 3.0 with mapping to UniRef90 gene families and MetaCyc metabolic pathways (39)(40)(41).UniRef90 gene families were reformatted into KEGG orthology (KO) groups using the HUMAnN command "humann_regroup_table." In total, we identified 722 species, 526 MetaCyc pathways, and 8,634 distinct KO group annotations in the 114 samples.All functional annotations were normalized using the HUMAnN command "humann_renorm_table --units cpm." Antibiotic resistance genes (ARGs) were identified by ShortBRED (42) with the reference Comprehensive Antibiotic Resistance Database (CARD version 2017) (43).ARG abundance was calculated as reads per kilobase of reference sequence per million sample reads (RPKM).

Microbial dysbiosis index
The microbial dysbiosis index (MD-index) was calculated as the log10 of the ratio of the relative abundance of taxa that were previously positively and negatively associated with newly diagnosed pediatric Crohn's disease (44).Specifically, the numerator includes Enterobacteriaceae, Pasteurellaceae, Fusobacteriaceae, Neisseriaceae, Veillonellaceae, and Gemellaceae; the denominator includes Bacteroidales, Clostridiales (excluding Veillonellaceae), Erysipelotrichaceae, and Bifidobacteriaceae.Higher indices correspond to a greater inflammatory taxonomic profile.The MD-index has been previously applied to stool samples of children with CF (14,45).

Differential abundance testing
We used MaAsLin2 (Microbiome Multivariable Association with Linear Models, Maaslin2 R package) to identify differentially abundant features (46).We included ELX/TEZ/IVA, age, and recent antibiotic exposure as fixed effects.Subject ID was specified as a random effect due to multiple samples from the same subject.Species, KEGG orthologs, and MetaCyc pathways detected at least 10% of samples were tested (i.e., prevalence = 0.1); no minimum abundance was specified.Abundances were log transformed within the MaAsLin2 function.The general linear "LM" model was used.MaAsLin2 coefficients are equivalent to log2(fold change).The Benjamini-Hochberg procedure was used to correct P values, and corrected P values are reported as false discovery rates (FDR).

Statistical analyses
Statistical analyses were conducted using GraphPad Prism 9 and R (version 4.2.1)software.Details of the statistical tests used and the significance thresholds are presented in the figure legends.All boxplot graphs are defined as follows: center line -median; box limits-upper and lower quartiles; whiskers-1.5×interquartile range.

Study cohort and stool sample collection
Stool samples were collected from a total of 39 children with CF (

Microbiome diversity increases after ELX/TEZ/IVA
From shotgun metagenomic sequencing on 114 stool samples, we compared measures of microbiome taxonomy and diversity.In contrast to previously published CF micro biome data sets of infants (8,22), our data set of older children was dominated by the phylum Firmicutes with minimal Proteobacteria (Fig. 2A; Data Set S1).Alpha diversity, as measured by the Shannon index and richness (observed species), significantly increased following ELX/TEZ/IVA (Fig. 2B and C, P = 0.021 and P = 0.026, respectively).The number of species observed increased from a median (IQR) of 83 (62,115) species before ELX/TEZ/IVA to 109 (82, 125) species after ELX/TEZ/IVA (Fig. 2B).The cumulative increases in alpha diversity were not significantly different when comparing between the four timepoints individually (Fig. S2A and B), and there were no differences in overall community composition (beta diversity) before and after ELX/TEZ/IVA (Fig. S2C and D).
To determine the effect of prior CFTR modulator treatment besides ELX/TEZ/IVA, we compared diversity metrics between modulator-naïve samples (samples = 45) and samples collected while subjects were receiving another CFTR modulator (samples = 8) before ELX/TEZ/IVA.Samples collected while subjects were receiving another CFTR modulator had similar diversity to modulator-naïve samples (Fig. S3A and B).Following ELX/TEZ/IVA, samples from subjects who had previously received another modulator (samples = 20) had similar improvements in microbiome diversity to samples from subjects who had not previously received another modulator (samples = 41), indicating that the residual effects of another modulator did not influence microbiome diversity improvement on ELX/TEZ/IVA (Fig. S3C and D).
To determine the influence of antibiotic exposure on microbiome diversity meas ures regardless of ELX/TEZ/IVA status, we categorized patient stool samples as having received antibiotics within 6 months prior to stool sample collection (samples = 62) or no antibiotic receipt (samples = 52).Recent antibiotic exposure significantly impacted alpha diversity measures reducing Shannon diversity and richness (Fig. 2D and E).Likewise, population structure (beta diversity) was significantly different between samples with and without recent antibiotic exposure (Fig. S2E, permutational multivariate analysis of variance = 0.003).

Alterations to specific bacterial taxa following ELX/TEZ/IVA
To determine whether specific bacterial taxa change following ELX/TEZ/IVA treatment, we used MaAsLin2 to identify differentially abundant microbial taxa (46).No phyla were differentially abundant following ELX/TEZ/IVA (Fig. 2A; Table S4).At a granular taxonomic level, 17 species were differentially abundant between samples before and after ELX/TEZ/IVA (FDR <0.1, Fig. 4A; Table S3), with 11 species increasing in abundance after ELX/TEZ/IVA, and 6 species decreasing.The three species with the lowest FDR and, therefore, highest reliability were Butyricicoccus SGB14985, S. aureus, and Roseburia faecis (Fig. 4A and B).Butyricicoccus SGB14985, an uncultivated species, was decreased in abundance following ELX/TEZ/IVA (fold change 0.16, FDR = 0.022).Roseburia faecis was increased in abundance following ELX/TEZ/IVA (fold change 7.35, FDR = 0.025) and negatively correlated with recent antibiotic exposure (Fig. S5A, fold change 0.20, FDR = 0.086).The intestinal tract has immunological cross-talk between the separate but similarly structured mucosal environment of the lung, deemed the gut-lung axis (20).Since the intestinal tract can harbor similar respiratory pathogens in patients with CF (15,48,49), we performed focused analysis of CF pathogens.S. aureus, the predominant respiratory pathogen in children with CF (50), was decreased in abundance after ELX/TEZ/IVA (Fig. 4A and B, fold change 0.40, FDR = 0.042).Other members of the respiratory microbiota which are frequently detected in the intestines (14, 15) -specifically the genera Haemophilus, Prevotella, Streptococcus, Veillonella-did not significantly change after ELX/TEZ/IVA initiation (Fig. S5B).Pseudomonas aeruginosa, an additional respiratory pathogen of importance in CF, was detected in only 2 of the 114

Intestinal inflammation decreases after ELX/TEZ/IVA
Fecal calprotectin was measured across the four timepoints and markedly decreased following ELX/TEZ/IVA ( Fig. 5A; Fig. S5A).Overall, mean fecal calprotectin decreased from 109 µg/g before ELX/TEZ/IVA to 44.8 µg/g for a mean decrease of 64.2 µg/g (95% CI −16.1, -112.3,Fig. 5A).We then computed the MD-index, a ratio of bacterial taxa positively and negatively associated with newly diagnosed pediatric Crohn's disease, to assess the relationship between intestinal inflammation and specific microbial signatures (44).We noted a nominal reduction in the MD-index following ELX/TEZ/IVA (Fig. 5B, P = 0.068), which was less pronounced when comparing samples from patients with recent antibiotic exposure and those without (Fig. 5C, P = 0.77).

DISCUSSION
ELX/TEZ/IVA has changed the trajectory of CF patient care and dramatically improved clinical outcomes.Nutritional status and components of the intestinal microbiome are strong predictors of future clinical outcomes, particularly in children with CF (6)(7)(8).We therefore sought to determine the effects of ELX/TEZ/IVA on intestinal inflammation and the intestinal microbiome in children with CF.Herein, we present a comprehensive longitudinal characterization of the intestinal microbiome in children with CF treated with ELX/TEZ/IVA.We identified widespread changes to the intestinal microbiome after ELX/TEZ/IVA, including taxonomic composition, reduced carriage of ARGs, and altered microbiota metabolic functions.
Although randomized controlled trials demonstrated that ELX/TEZ/IVA was safe and effective, there are limited post-approval clinical data, particularly in children over 2 years old (52)(53)(54)(55)(56). BMI and ppFEV 1 are useful markers of clinical response to CFTR modula tor treatment in children (57).Our cohort demonstrated a significant improvement in ppFEV 1 and BMI percentile within 6 months of starting ELX/TEZ/IVA, with no additional differences at later timepoints (Fig. S1A and B).This is consistent with prior clinical trial and real-world data showing the greatest impact on nutritional status and anthropomet ric parameters immediately after ELX/TEX/IVA therapy initiation with subsequent plateau without regression in children (52,56,58).Prior baseline BMI, particularly underweight status, has been predicted to be a major determinant of increase in weight gain in patients with CF treated with ELX/TEZ/IVA (59).Furthermore, between the timepoint immediately before ELX/TEZ/IVA initiation (T2) and post-ELX/TEZ/IVA timepoints (T3 and T4), there was a significant increase in height percentile (Fig. S1D).This suggests that CFTR modulator therapy may improve linear growth in children with CF.In sum, these results support the clinical responsiveness of children with CF to ELX/TEZ/IVA.
Direct comparison of our results to studies containing healthy controls is difficult, given different sampling and sequencing methodologies.Notably, although our study identified subtle changes in the intestinal microbiome after initiation of ELX/TEZ/IVA in children with CF, the post-ELX/TEZ/IVA intestinal microbiome remains significantly altered from what has been described in healthy children.Compared to healthy children, the intestinal microbiome of children with CF is delayed in development and exhibits decreased microbial diversity relative to healthy controls throughout childhood and adolescence (22,24,63).Among the most consistent differences between healthy controls and patients with CF is reduced abundance of the phylum Bacteroidetes in patients with CF (13,20,61,63,66).In our data set, Bacteroidetes remained depleted post-ELX/TEZ/IVA (Fig. 2A; Table S4).Likewise, Shannon diversity remained below that of similarly aged healthy controls (24,63), as discussed henceforth.
In our cohort, alpha diversity, as measured by the Shannon index and richness (observed species), significantly increased following ELX/TEZ/IVA (Fig. 2B and C).These results demonstrate modest but statistically significant differences in an older cohort of children with CF, whose microbiome resembles that of more of a stable "adult-like" microbiome.The difference in the median Shannon diversity was increased 0.29 after ELX/TEZ/IVA (Fig. 2B).In contrast, a similarly aged cohort of children with CF exhibited a median of ~1.0 reduced Shannon index compared to healthy controls in the same study (24).Thus, while our cohort exhibited significant increases in Shannon diversity, it is unlikely that this magnitude of increase restores the diversity to that of similarly aged healthy controls.This is consistent with the notion that "adult-like" intestinal microbiota are remarkably stable and resilient to intervention (67)(68)(69).As CFTR modulator formula tions are approved for younger children, particularly infants for whom the microbiome is still developing, studying the effects of CFTR modulators on the developing intestinal microbiome will be an important research endeavor and may show more substantial differences.
Existing studies regarding the impact of CFTR modulators on the intestinal micro biome have been limited by small sample sizes and have exhibited mixed results, with some studies reporting no differences (31,70) and others reporting modest improvements (29).We identified no differences in diversity metrics in patients receiving another CFTR modulator formulation before ELX/TEZ/IVA (Fig. S3A).These prior formulations included both CFTR correctors and potentiators (Table 1).As described above, ELX/TEZ/IVA consistently improved microbiome diversity metrics in our study, suggesting the ELX/TEZ/IVA is unique among existing CFTR modulator formulations.In vitro and animal studies may elucidate the relative contributions of the individual CFTR modulators to improvements in gastrointestinal physiology and microbiome diversity metrics.
In children with CF, markers of intestinal inflammation correlate with growth failure (11,24).Moreover, intestinal inflammation in patients with CF is linked to higher rates of IBD and colorectal cancer in patients with CF (16,17,71).Fecal calprotectin, a laboratory marker of intestinal inflammation (72)(73)(74), significantly decreased following ELX/TEZ/IVA (Fig. 5A), corroborating prior reports in the PROMISE and RECOVER cohorts (75,76).Using a dysbiosis index of bacterial taxa correlated with pediatric Crohn's disease (44), we identified that this dysbiosis index nominally decreased following ELX/TEZ/IVA initiation (Fig. 5B).The decrease in the median MD-index was 0.166 before and after ELX/TEZ/IVA.The median post-ELX/TEZ/IVA MD-index was lower (i.e., reduced inflammatory taxa) than that of healthy controls in the original publication describing the MD-index (44).This suggests that the magnitude of decrease in the MD-index in our study may be biologi cally meaningful.Furthermore, the difference between pre-and post-ELX/TEZ/IVA (Fig. 5B) was more pronounced than in samples with and without recent antibiotic exposure (Fig. 5C).This suggests that this effect may be specific to ELX/TEZ/IVA treatment and not due to reduced antibiotic use.At the species level, the butyrate-producing species Roseburia faecis was significantly enriched following ELX/TEZ/IVA (Fig. 4A and B).Prior studies have identified R. faecis as reduced in abundance in patients with CF (20,(77)(78)(79).In parallel, we detected reduced abundance of genes involved in oxygen-dependent metabolism following ELX/TEZ/IVA (Fig. 5D and E).Intestinal inflammation is character ized by a shift toward oxygen-dependent microbiota metabolism, perpetuating a cycle of inflammatory damage (80)(81)(82).From multiple lines of evidence, our results suggest that ELX/TEZ/IVA reduces intestinal inflammation in children with CF.
Respiratory infections require frequent antibiotics in patients with CF.Some respiratory pathogens also colonize the intestinal tract and temporally correlate with respiratory colonization (15,48,49).S. aureus is among the first pathogens to colonize the respiratory tract and cause infections in children with CF (50).We detected reduced intestinal abundance of S. aureus following ELX/TEZ/IVA (Fig. 4A and B).Antibiotic exposure in patients with CF has been associated with increased intestinal carriage of antibiotic-resistant bacteria compared to healthy controls which are a poor prog nostic factor (83)(84)(85).Following ELX/TEZ/IVA, patients in our cohort required far fewer antibiotics (Fig. 1E).In turn, we detected reduced intestinal abundance of ARGs (Fig. 2E).These results indicate disease-relevant taxonomic changes to the intestinal microbiome following ELX/TEZ/IVA, as well as reduced ARGs.
The CFTR channel permits transepithelial movement of bicarbonate (HCO3 − ) and chloride (Cl − ) (5).Intestinal pH is lower in patients with CF due to the lack of neutral izing bicarbonate (86) yet increases with CFTR modulator treatment, consistent with increased CFTR activity in the intestinal tract (87).We observed a reduced abundance of microbial protein groups associated with acid tolerance (Fig. 5D), consistent with microbial adaptation to increased CFTR channel function in the intestines.These results display physiologically intuitive functional changes to the microbiome post-ELX/TEZ/IVA.As CFTR modulators become the mainstay of treatment for CF, further research will be necessary to mechanistically describe the effects of CFTR modulators on gastrointestinal function.
Our study is strengthened by longitudinal sampling over a 5-year period com bined with the real-world use of ELX/TEZ/IVA.Using shotgun metagenomic sequenc ing, we comprehensively compared microbiota differences before and after ELX/TEZ/ IVA.Sampling of the intestinal microbiome over extended periods will be important to uncover additional long-term improvements to the microbiome.Similarly, further research is necessary to delineate the effects of ELX/TEX/IVA on the interconnected respiratory and intestinal microbiomes.Although our study was limited by the singlecenter focus, our cohort mirrors the overall CF pediatric population in clinical and demographic factors (Table 1).Our study lacks untreated CF controls or healthy controls, which would allow for additional comparisons.
In summary, our results indicate that the CFTR modulator ELX/TEZ/IVA alters the intestinal microbiome in children with CF.We identified taxonomic and functional changes to the intestinal microbiome that represent improvements to CF intestinal microbiome structure and function.Our results also support that CFTR modulators reduce intestinal inflammation in children with CF.

FIG 1
FIG 1 Study schematic, timeline, and clinical improvement after ELX/TEZ/IVA.(A) Overview of study procedure and analyses.(B) Timeline illustrating the four timepoints.Day 0 is depicted as the day of ELX/TEZ/IVA initiation.A total of 114 samples were collected from 39 unique patients, of which 53 samples were before ELX/TEZ/IVA treatment and 61 samples after treatment.(C-E) Depictions of clinical data before and after ELX/TEZ/IVA; (C) percent predicted forced expiratory volume in one second (ppFEV 1 ), (D) body mass index (BMI) percentile, and (E) antibiotic days per 6 months.Each dot represents the clinical data associated with a stool sample.P values calculated by Wilcoxon rank-sum test.

FIG 2
FIG 2 Microbiome diversity and intestinal carriage of antibiotic resistance genes.(A) Phylum-level relative abundance for samples collected before (samples = 53) and after (samples = 61) ELX/TEZ/IVA.Samples are organized by the relative abundance of the phylum Firmicutes.(B and C) Alpha diversity before and after ELX/TEZ/IVA.Shannon index calculated with the R package vegan using species abundance table from Metaphlan4.Microbial richness represents the number of unique species per sample.Each dot represents a stool sample (samples = 114).P values calculated by Wilcoxon rank-sum test.(D and E) Alpha diversity between samples with and without recent antibiotic exposure.Recent antibiotic exposure categorized as any systemic antibiotic within the past 6 months.Shannon index calculated with the R package vegan using species abundance table from MetaPhlAn4.Microbial richness represents the number of unique species per sample.Each dot represents a stool sample (samples = 114).P values calculated by Wilcoxon rank-sum test.

FIG 3
FIG 3 (A) Antibiotic resistance gene abundance (RPKM) before and after ELX/TEZ/IVA.ARGs were profiled using ShortBRED and CARD.Each dot represents a stool sample (samples = 114).P values calculated by Wilcoxon rank-sum test.(B) ARG abundance (RPKM) by class of antibiotic to which they confer resistance.Abundance values of zero are plotted on the vertical axis.P values calculated by Wilcoxon signed-rank test.

FIG 5
FIG 5 Intestinal inflammation decreases after ELX/TEZ/IVA and microbiome functional changes.(A) Fecal calprotectin before and after ELX/TEZ/IVA.Each dot represents a stool sample (samples = 114).P values calculated by Wilcoxon rank-sum test.(B and C) Microbial dysbiosis index before and after ELX/TEZ/IVA (B) or between samples with and without recent antibiotic exposure (C).MD-index calculated as the log10 ratio of species positively/negatively correlated with new onset pediatric Crohn's disease (44).Each dot represents a stool sample (samples = 114).P values calculated by Wilcoxon rank-sum test.(D) Differentially abundant KEGG orthologs following MaAsLin2 multivariable association modeling, using ELX/TEZ/IVA status, age, and recent antibiotics as fixed effects in the model.Participant ID was used as a random effect.Horizontal dashed line depicts FDR = 0.25.(E) Subset of differentially abundant MetaCyc pathways following MaAsLin2 multivariable association modeling, using ELX/TEZ/IVA status, age, and recent antibiotics as fixed effects in the model.Only pathways with FDR <0.25 are depicted.

TABLE 1
Cohort details a a Table includes patient characteristics at the time of recruitment prior to initiation of ELX/TEZ/IVA.Categorical and dichotomous variables are expressed as n and as a proportion of the total for the group.Respiratory culture history refers to isolation of the microbial species from sputum or bronchoalveolar lavage within 3 years prior to enrollment.IQR, interquartile range; CFLD, CF-related liver disease; CFRD, CF-related diabetes; TEZ/IVA, tezacaftor/ivacaftor, Symdeko; LUM/IVA, lumacaftor/ivacaftor, Orkambi; IVA, ivacaftor, Kalydeco.