Localized delivery of therapeutics impact laryngeal mechanics, local inflammatory response, and respiratory microbiome following upper airway intubation injury in swine.

BACKGROUND: Laryngeal injury associated with traumatic or prolonged intubation may lead to voice, swallow, and airway complications. The interplay between inflammation and microbial population shifts induced by intubation may relate to clinical outcomes. The objective of this study was to investigate laryngeal mechanics, tissue inflammatory response, and local microbiome changes with laryngotracheal injury and localized delivery of therapeutics via drug-eluting endotracheal tube. METHODS: A simulated traumatic intubation injury was created in Yorkshire crossbreed swine under direct laryngoscopy. Endotracheal tubes electrospun with roxadustat or valacyclovir- loaded polycaprolactone (PCL) fibers were placed in the injured airway for 3, 7, or 14 days (n = 3 per group/time and ETT type). Vocal fold stiffness was then evaluated with normal indentation and laryngeal tissue sections were histologically examined. Immunohistochemistry and inflammatory marker profiling were conducted to evaluate the inflammatory response associated with injury and ETT placement. Additionally, ETT biofilm formation was visualized using scanning electron microscopy and micro-computed tomography, while changes in the airway microbiome were profiled through 16S rRNA sequencing. RESULTS: Laryngeal tissue with roxadustat ETT placement had increasing localized stiffness outcomes over time and histological assessment indicated minimal epithelial ulceration and fibrosis, while inflammation remained severe across all timepoints. In contrast, vocal fold tissue with valacyclovir ETT placement showed no significant changes in stiffness over time; histological analysis presented a reduction in epithelial ulceration and inflammation scores along with increased fibrosis observed at 14 days. Immunohistochemistry revealed a decline in M1 and M2 macrophage markers over time for both ETT types. Among the cytokines, IL-8 levels differed significantly between the roxadustat and valacyclovir ETT groups, while no other cytokines showed statistically significant differences. Additionally, increased biofilm formation was observed in the coated ETTs with notable alterations in microbiota distinctive to each ETT type and across time. CONCLUSION: The injured and intubated airway resulted in increased laryngeal stiffness. Local inflammation and the type of therapeutic administered impacted the bacterial composition within the upper respiratory microbiome, which in turn mediated local tissue healing and recovery.

Microbiome-host interactions in the pathogenesis of acute exacerbation of chronic obstructive pulmonary disease.

Objective: To explore the underlying mechanisms the airway microbiome contributes to Acute Exacerbation of Chronic Obstructive Pulmonary Disease(AECOPD). Methods: We enrolled 31 AECOPD patients and 26 stable COPD patients, their sputum samples were collected for metagenomic and RNA sequencing, and then subjected to bioinformatic analyses. The expression of host genes was validated by Quantitative Real-time PCR(qPCR) using the same batch of specimens. Results: Our results indicated a higher expression of Rothia mucilaginosa(p=0.015) in the AECOPD group and Haemophilus influenzae(p=0.005) in the COPD group. The Different expressed genes(DEGs) detected were significantly enriched in "type I interferon signaling pathway"(p<0.001, q=0.001) in gene function annotation, and "Cytosolic DNA-sensing pathway"(p=0.002, q=0.024), "Toll-like receptor signaling pathway"(p=0.006, q=0.045), and "TNF signaling pathway"(p=0.006, q=0.045) in KEGG enrichment analysis. qPCR amplification experiment verified that the expression of OASL and IL6 increased significantly in the AECOPD group. Conclusion: Pulmonary bacteria dysbiosis may regulate the pathogenesis of AECOPD through innate immune system pathways like type I interferon signaling pathway and Toll-like receptor signaling pathway.

The Role of the Airway and Gut Microbiome in the Development of Chronic Lung Disease of Prematurity.

Chronic lung disease (CLD) of prematurity, a common cause of morbidity and mortality in preterm-born infants, has a multifactorial aetiology. This review summarizes the current evidence for the effect of the gut and airway microbiota on the development of CLD, highlighting the differences in the early colonisation patterns in preterm-born infants compared to term-born infants. Stool samples from preterm-born infants who develop CLD have less diversity than those who do not develop CLD. Pulmonary inflammation, which is a hallmark in the development of CLD, may potentially be influenced by gut bacteria. The respiratory microbiota is less abundant than the stool microbiota in preterm-born infants. There is a lack of clear evidence for the role of the respiratory microbiota in the development of CLD, with results from individual studies not replicated. A common finding is the presence of a single predominant bacterial genus in the lungs of preterm-born infants who develop CLD. Probiotic preparations have been proposed as a potential therapeutic strategy to modify the gut or lung microbiota with the aim of reducing rates of CLD but additional robust evidence is required before this treatment is introduced into routine clinical practice.

The alterations of oral, airway and intestine microbiota in chronic obstructive pulmonary disease: a systematic review and meta-analysis.

Background: Increasing evidence indicates the microbial ecology of chronic obstructive pulmonary disease (COPD) is intricately associated with the disease's status and severity, and distinct microbial ecological variations exist between COPD and healthy control (HC). This systematic review and meta-analysis aimed to summarize microbial diversity indices and taxa relative abundance of oral, airway, and intestine microbiota of different stages of COPD and HC to make comparisons. Methods: A comprehensive systematic literature search was conducted in PubMed, Embase, the Web of Science, and the Cochrane Library databases to identify relevant English articles on the oral, airway, and intestine microbiota in COPD published between 2003 and 8 May 2023. Information on microbial diversity indices and taxa relative abundance of oral, airway, and intestine microbiota was collected for comparison between different stages of COPD and HC. Results: A total of 20 studies were included in this review, involving a total of 337 HC participants, 511 COPD patients, and 154 AECOPD patients. We observed that no significant differences in alpha diversity between the participant groups, but beta diversity was significantly different in half of the included studies. Compared to HC, Prevotella, Streptococcus, Actinomyces, and Veillonella of oral microbiota in SCOPD were reduced at the genus level. Most studies supported that Haemophilus, Lactobacillus, and Pseudomonas were increased, but Veillonella, Prevotella, Actinomyces, Porphyromonas, and Atopobium were decreased at the genus level in the airway microbiota of SCOPD. However, the abundance of Haemophilus, Lactobacillus and Pseudomonas genera exhibited an increase, whereas Actinomyces and Porphyromonas showed a decrease in the airway microbiota of AECOPD compared to HC. And Lachnospira of intestine microbiota in SCOPD was reduced at the genus level. Conclusion: The majority of published research findings supported that COPD exhibited decreased alpha diversity compared to HC. However, our meta-analysis does not confirm it. In order to further investigate the characteristics and mechanisms of microbiome in the oral-airway- intestine axis of COPD patients, larger-scale and more rigorous studies are needed. Systematic review registration: PROSPERO (https://www.crd.york.ac.uk/prospero/), identifier CRD42023418726.

Comparison of the upper and lower airway microbiome in early postoperative lung transplant recipients.

The upper and lower respiratory tract may share microbiome because they are directly continuous, and the nasal microbiome contributes partially to the composition of the lung microbiome. But little is known about the upper and lower airway microbiome of early postoperative lung transplant recipients (LTRs). Using 16S rRNA gene sequencing, we compared paired nasal swab (NS) and bronchoalveolar lavage fluid (BALF) microbiome from 17 early postoperative LTRs. The microbiome between the two compartments were significantly different in Shannon diversity and beta diversity. Four and eight core NS-associated and BALF-associated microbiome were identified, respectively. NS samples harbored more Corynebacterium, Acinetobacter, and Pseudomonas, while BALF contained more Ralstonia, Stenotrophomonas, Enterococcus, and Pedobacter. The within-subject dissimilarity was higher than the between-subject dissimilarity, indicating a greater impact of sampling sites than sampling individuals on microbial difference. There were both difference and homogeneity between NS and BALF microbiome in early postoperative LTRs. High levels of pathogens were detected in both samples, suggesting that both of them can reflect the diseases characteristics of transplanted lung. The differences between upper and lower airway microbiome mainly come from sampling sites instead of sampling individuals. IMPORTANCE: Lung transplantation is the only therapeutic option for patients with end-stage lung disease, but its outcome is much worse than other solid organ transplants. Little is known about the NS and BALF microbiome of early postoperative LTRs. Here, we compared paired samples of the nasal and lung microbiome from 17 early postoperative LTRs and showed both difference and homogeneity between the two samples. Most of the "core" microbiome in both NS and BALF samples were recognized respiratory pathogens, suggesting that both samples can reflect the diseases characteristics of transplanted lung. We also found that the differences between upper and lower airway microbiome in early postoperative LTRs mainly come from sampling sites instead of sampling individuals.

Human genetic associations of the airway microbiome in chronic obstructive pulmonary disease.

Little is known about the relationships between human genetics and the airway microbiome. Deeply sequenced airway metagenomics, by simultaneously characterizing the microbiome and host genetics, provide a unique opportunity to assess the microbiome-host genetic associations. Here we performed a co-profiling of microbiome and host genetics with the identification of over 5 million single nucleotide polymorphisms (SNPs) through deep metagenomic sequencing in sputum of 99 chronic obstructive pulmonary disease (COPD) and 36 healthy individuals. Host genetic variation was the most significant factor associated with the microbiome except for geography and disease status, with its top 5 principal components accounting for 12.11% of the microbiome variability. Within COPD individuals, 113 SNPs mapped to candidate genes reported as genetically associated with COPD exhibited associations with 29 microbial species and 48 functional modules (P < 1 × 10-5), where Streptococcus salivarius exhibits the strongest association to SNP rs6917641 in TBC1D32 (P = 9.54 × 10-8). Integration of concurrent host transcriptomic data identified correlations between the expression of host genes and their genetically-linked microbiome features, including NUDT1, MAD1L1 and Veillonella parvula, TTLL9 and Stenotrophomonas maltophilia, and LTA4H and Haemophilus influenzae. Mendelian randomization analyses revealed a potential causal link between PARK7 expression and microbial type III secretion system, and a genetically-mediated association between COPD and increased relative abundance of airway Streptococcus intermedius. These results suggest a previously underappreciated role of host genetics in shaping the airway microbiome and provide fresh hypotheses for genetic-based host-microbiome interactions in COPD.

Influence of the gut and airway microbiome on asthma development and disease.

There are ample data to suggest that early-life dysbiosis of both the gut and/or airway microbiome can predispose a child to develop along a trajectory toward asthma. Although individual studies show clear associations between dysbiosis and asthma development, it is less clear what (collection of) bacterial species is mechanistically responsible for the observed effects. This is partly due to issues related to the asthma diagnosis and the broad spectrum of anatomical sites, sample techniques, and analysis protocols that are used in different studies. Moreover, there is limited attention for potential differences in the genetics of individuals that would affect the outcome of the interaction between the environment and that individual. Despite these challenges, the first bacterial components were identified that are able to affect the transcriptional state of human cells, ergo the immune system. Such molecules could in the future be the basis for intervention studies that are now (necessarily) restricted to a limited number of bacterial species. For this transition, it might be prudent to develop an ex vivo human model of a local mucosal immune system to better and safer explore the impact of such molecules. With this approach, we might move beyond association toward understanding of causality.

Airway commensal bacteria in cystic fibrosis inhibit the growth of P. aeruginosa via a released metabolite.

In cystic fibrosis (CF), Pseudomonas aeruginosa infection plays a critical role in disease progression. Although multiple studies suggest that airway commensals might be able to interfere with pathogenic bacteria, the role of the distinct commensals in the polymicrobial lung infections is largely unknown. In this study, we aimed to identify airway commensal bacteria that may inhibit the growth of P. aeruginosa. Through a screening study with more than 80 CF commensal strains across 21 species, more than 30 commensal strains from various species have been identified to be able to inhibit the growth of P. aeruginosa. The underlying mechanisms were investigated via genomic, metabolic and functional analysis, revealing that the inhibitory commensals may affect the growth of P. aeruginosa by releasing a large amount of acetic acid. The data provide information about the distinct roles of airway commensals and provide insights into novel strategies for controlling airway infections.

Preterm Infants' Airway Microbiome: A Scoping Review of the Current Evidence.

The aim of this scoping review was to investigate and synthesize existing evidence on the airway microbiome of preterm infants to outline the prognostic and therapeutic significance of these microbiomes within the preterm population and identify gaps in current knowledge, proposing avenues for future research. We performed a scoping review of the literature following the Arskey and O'Malley framework. In accordance with our inclusion criteria and the intended purpose of this scoping review, we identified a total of 21 articles. The investigation of the airway microbiome in preterm infants has revealed new insights into its unique characteristics, highlighting distinct dynamics when compared to term infants. Perinatal factors, such as the mode of delivery, chorioamnionitis, the respiratory support, and antibiotic treatment, could impact the composition of the airway microbiome. The 'gut-lung axis', examining the link between the lung and gut microbiome as well as modifications in respiratory microbiome across different sites and over time, has also been explored. Furthermore, correlations between the airway microbiome and adverse outcomes, such as bronchopulmonary dysplasia (BPD), have been established. Additional research in neonatal care is essential to understand the early colonization of infants' airways and explore methods for its optimization. The critical opportunity to shape long-term health through microbiome-mediated effects likely lies within the neonatal period.

Inhaler Steroid Use Changes Oral and Airway Bacterial and Fungal Microbiome Profile in Asthma Patients.

INTRODUCTION: The full spectrum of bacterial and fungal species in adult asthma and the effect of inhaled corticosteroid use is not well described. The aim was to collect mouthwash and induced sputum samples from newly diagnosed asthma patients in the pretreatment period and in chronic asthma patients while undergoing regular maintenance inhaled corticosteroid therapy, in order to demonstrate the bacterial and fungal microbiome profile. METHODS: The study included 28 asthmatic patients on inhaler steroid therapy, 25 steroid-naive asthmatics, and 24 healthy controls. Genomic DNA was isolated from induced sputum and mouthwash samples. Analyses were performed using bacterial primers selected from the 16S rRNA region for the bacterial genome and "panfungal" primers selected from the 5.8S rRNA region for the fungal genome. RESULTS: Dominant genera in mouthwash samples of steroid-naive asthmatics were Neisseria, Haemophilus, and Rothia. The oral microbiota of asthmatic patients on inhaler steroid treatment included Neisseria, Rothia, and Veillonella species. Abundant genera in induced sputum samples of steroid-naive asthma patients were Actinomyces, Granulicatella, Fusobacterium, Peptostreptococcus, and Atopobium. Sputum microbiota of asthma patients taking inhaler steroids were dominated by Prevotella and Porphyromonas. Mucor plumbeus and Malassezia restricta species were abundant in the airways of steroid-naive asthma patients. Choanephora infundibulifera and Malassezia restricta became dominant in asthma patients taking inhaled steroids. CONCLUSION: The oral and airway microbiota consist of different bacterial and fungal communities in healthy and asthmatic patients. Inhaler steroid use may influence the composition of the oral and airway microbiota.

Airway microbiome: environmental exposure-respiratory health nexus.

Toxicants such as smoke, biofuel, and pollutants constantly challenge our respiratory health, but little is known about the pathophysiological processes involved. In a new report, Lin et al. provide evidence that our bacterial and fungal lung populations orchestrate the interplay between environmental exposure and lung functions, thereby conditioning health outcomes.

Characterization of upper airway microbiome across severity of COVID-19 during hospitalization and treatment.

Longitudinal studies on upper respiratory tract microbiome in coronavirus disease 2019 (COVID-19) without potential confounders such as antimicrobial therapy are limited. The objective of this study is to assess for longitudinal changes in the upper respiratory microbiome, its association with disease severity, and potential confounders in adult hospitalized patients with COVID-19. Serial nasopharyngeal and throat swabs (NPSTSs) were taken for 16S rRNA gene amplicon sequencing from adults hospitalized for COVID-19. Alpha and beta diversity was assessed between different groups. Principal coordinate analysis was used to assess beta diversity between groups. Linear discriminant analysis was used to identify discriminative bacterial taxa in NPSTS taken early during hospitalization on need for intensive care unit (ICU) admission. A total of 314 NPSTS samples from 197 subjects (asymptomatic = 14, mild/moderate = 106, and severe/critical = 51 patients with COVID-19; non-COVID-19 mechanically ventilated ICU patients = 11; and healthy volunteers = 15) were sequenced. Among all covariates, antibiotic treatment had the largest effect on upper airway microbiota. When samples taken after antibiotics were excluded, alpha diversity (Shannon, Simpson, richness, and evenness) was similar across severity of COVID-19, whereas beta diversity (weighted GUniFrac and Bray-Curtis distance) remained different. Thirteen bacterial genera from NPSTS taken within the first week of hospitalization were associated with a need for ICU admission (area under the receiver operating characteristic curve, 0.96; 95% CI, 0.91-0.99). Longitudinal analysis showed that the upper respiratory microbiota alpha and beta diversity was unchanged during hospitalization in the absence of antimicrobial therapy.

Air pollution-associated shifts in the human airway microbiome and exposure-associated molecular events.

Publications addressing air pollution-induced human respiratory microbiome shifts are reviewed in this article. The healthy respiratory microbiota is characterized by a low density of bacteria, fungi and viruses with high diversity, and usually consists of Bacteroidetes, Firmicutes, Proteobacteria, Actinobacteria, Fusobacteria, viruses and fungi. The air's microbiome is highly dependent on air pollution levels and is directly reflected within the human respiratory microbiome. In addition, pollutants indirectly modify the local environment in human respiratory organs by reducing antioxidant capacity, misbalancing proteolysis and modulating inflammation, all of which regulate local microbiomes. Improving air quality leads to more diverse and healthy microbiomes of the local air and, subsequently, residents' airways.

The community of bacteria, viruses and fungi in the human body, known as the microbiome, plays an important role in human health. These communities vary in different locations in the body, for example in the gut, airways and skin. The microbiome within our airways is affected by air pollution because pollutants cause changes in the microbiome that may result in illness. In this article we review the available information on the effect of air pollution on the airway microbiome. We conclude that improving air quality is important to promoting healthy microbiomes and general human health.

The microbial composition of larval airways from Drosophila melanogaster differ between specimens from laboratory and natural habitats.

BACKGROUND: The fruit fly Drosophila melanogaster lives in natural habitats and has also long been used as a model organism in biological research. In this study, we used a molecular barcoding approach to analyse the airways microbiome of larvae of D. melanogaster, which were obtained from eggs of flies of the laboratory strain w1118 and from immune deficient flies (NF-kB-K), and from wild-caught flies. To assess intergenerational transmission of microbes, all eggs were incubated under the same semi-sterile conditions. RESULTS: The airway microbiome of larvae from both lab-strains was dominated by the two families Acetobacteraceae and Lactobacillaceae, while larvae from wild-caught flies were dominated by Lactobacillaceae, Anaplasmataceae and Leuconostocaceae. Barcodes linked to Anaplasmataceae could be further assigned to Wolbachia sp., which is a widespread intracellular pathogen in arthropods. For Leuconostoceae, the most abundant reads were assigned to Weissella sp. Both Wolbachia and Weissella affect the development of the insects. Finally, a relative high abundance of Serratia sp. was found in larvae from immune deficient relish-/- compared to w1118 and wild-caught fly airways. CONCLUSIONS: Our results show for the first time that larvae from D. melanogaster harbor an airway microbiome, which is of low complexity and strongly influenced by the environmental conditions and to a lesser extent by the immune status. Furthermore, our data indicate an intergenerational transmission of the microbiome as shaped by the environment.

Seasonal Dynamics of the Upper Respiratory Tract Microbiome in Chronic Obstructive Pulmonary Disease.

Introduction: Increasing evidence suggests that seasonal changes can trigger the alternation of airway microbiome. However, the dynamics of the upper airway bacterial ecology of chronic obstructive pulmonary disease (COPD) patients across different seasons remains unclear. Methods: In this study, we present a 16S ribosomal RNA survey of the airway microbiome on 72 swab samples collected in different months (March, May, July, September, and November) in 2019 from 18 COPD patients and from six resampled patients in November in 2020. Results: Our study uncovered a dynamic airway microbiota where changes appeared to be associated with seasonal alternation in COPD patients. Twelve clusters of temporal patterns were displayed by differential and clustering analysis along the time course, systematically revealing distinct microbial taxa that prefer to grow in cool and warm seasons, respectively. Moreover, the upper airway microbiome composition was relatively stable in the same season in different years. Discussion: Given the tight association between airway microbiome and COPD disease progression, this study can provide useful information for clinically understanding the seasonal trend of disease phenotypes in COPD patients.

Human genetics influences microbiome composition involved in asthma exacerbations despite inhaled corticosteroid treatment.

BACKGROUND: The upper-airway microbiome is involved in asthma exacerbations despite inhaled corticosteroid (ICS) treatment. Although human genetics regulates microbiome composition, its influence on asthma-related airway bacteria remains unknown. OBJECTIVE: We sought to identify genes and biological pathways regulating airway-microbiome traits involved in asthma exacerbations and ICS response. METHODS: Saliva, nasal, and pharyngeal samples from 257 European patients with asthma were analyzed. The association of 6,296,951 genetic variants with exacerbation-related microbiome traits despite ICS treatment was tested through microbiome genome-wide association studies. Variants with 1 × 10-4 

Airway dysbiosis accelerates lung function decline in chronic obstructive pulmonary disease.

Progressive lung function decline is a hallmark of chronic obstructive pulmonary disease (COPD). Airway dysbiosis occurs in COPD, but whether it contributes to disease progression remains unknown. Here, we show, through a longitudinal analysis of two cohorts involving four UK centers, that baseline airway dysbiosis in COPD patients, characterized by the enrichment of opportunistic pathogenic taxa, associates with a rapid forced expiratory volume in 1 s (FEV1) decline over 2 years. Dysbiosis associates with exacerbation-related FEV1 fall and sudden FEV1 fall at stability, contributing to long-term FEV1 decline. A third cohort in China further validates the microbiota-FEV1-decline association. Human multi-omics and murine studies show that airway Staphylococcus aureus colonization promotes lung function decline through homocysteine, which elicits a neutrophil apoptosis-to-NETosis shift via the AKT1-S100A8/A9 axis. S. aureus depletion via bacteriophages restores lung function in emphysema mice, providing a fresh approach to slow COPD progression by targeting the airway microbiome.

The upper airway microbiome in Hispanic children with cystic fibrosis.

BACKGROUND: Hispanic people with cystic fibrosis (CF) have decreased life expectancy and earlier acquisition of Pseudomonas aeruginosa compared to non-Hispanic white individuals with CF. Racial and ethnic differences in the airway microbiome of CF may contribute to known health disparity, but have not been studied. The objective was to describe differences in the upper airway microbial community in Hispanic and non-Hispanic white children with CF. METHODS: This prospective, observational cohort study of 59 Hispanic and non-Hispanic white children with CF, ages 2-10 years old, was performed at Texas Children's Hospital (TCH) from February 2019 to January 2020. Oropharyngeal swabs were collected from the cohort during clinic visit. Swab samples underwent sequencing (16S V4 rRNA), diversity analysis, and taxonomic profiling. Key demographic and clinical data were collected from the electronic medical record and the CF Foundation Patient Registry (CFFPR). Statistical analysis compared sequencing, demographic, and clinical data. RESULTS: We found no significant difference in Shannon diversity or relative abundance of bacterial phyla between Hispanic and non-Hispanic children with CF. However, a low abundant taxa- "uncultured bacterium" belonging to the order Saccharimonadales was significantly higher in Hispanic children (mean relative abundance = 0.13%) compared to the non-Hispanic children (0.03%). Hispanic children had increased incidence of P. aeruginosa (p = 0.045) compared to non-Hispanic children. CONCLUSION: We did not find a significant difference in the airway microbial diversity between Hispanic and non-Hispanic white children with CF. However, we found a greater relative abundance of Saccharimonadales and higher incidence of P. aeruginosa in Hispanic children with CF.

Chronic Obstructive Pulmonary Disease and the Airway Microbiome: What Respirologists Need to Know.

Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality worldwide. The lower airways contain a rich and diverse microbiome, which may play a significant regulatory role in both health and disease. In COPD, the microbiome becomes perturbed, causing dysbiosis. Increased representation of members in the Proteobacteria phylum and certain members in the Firmicutes phylum has been associated with increased risk of exacerbations and mortality. Therapies such as inhaled corticosteroids and azithromycin may modulate the airway microbiome or its metabolites in patients with COPD. This paper provides an up-to-date overview of the airway microbiome and its importance in the pathophysiology of COPD and as potential therapeutic target in the future.

Effects of Lumacaftor-Ivacaftor on Airway Microbiota-Mycobiota and Inflammation in Patients with Cystic Fibrosis Appear To Be Linked to Pseudomonas aeruginosa Chronic Colonization.

Lumacaftor-ivacaftor is a cystic fibrosis transmembrane conductance regulator (CFTR) modulator combination approved for patients with cystic fibrosis (CF) who are homozygous for the F508del allele. This treatment showed significant clinical improvement; however, few studies have addressed the evolution of the airway microbiota-mycobiota and inflammation in patients receiving lumacaftor-ivacaftor treatment. Seventy-five patients with CF aged 12 years or older were enrolled at the initiation of lumacaftor-ivacaftor therapy. Among them, 41 had spontaneously produced sputa collected before and 6 months after treatment initiation. Airway microbiota and mycobiota analyses were performed via high-throughput sequencing. Airway inflammation was assessed by measuring the calprotectin levels in sputum; the microbial biomass was evaluated via quantitative PCR (qPCR). At baseline (n = 75), bacterial alpha-diversity was correlated with pulmonary function. After 6 months of lumacaftor-ivacaftor treatment, a significant improvement in the body mass index and a decreased number of intravenous antibiotic courses were noted. No significant changes in bacterial and fungal alpha- and beta-diversities, pathogen abundances, or calprotectin levels were observed. However, for patients not chronically colonized with Pseudomonas aeruginosa at treatment initiation, calprotectin levels were lower, and a significant increase in bacterial alpha-diversity was observed at 6 months. This study shows that the evolution of the airway microbiota-mycobiota in CF patients depends on the patient's characteristics at lumacaftor-ivacaftor treatment initiation, notably chronic colonization with P. aeruginosa. IMPORTANCE The management of cystic fibrosis has been transformed recently by the advent of CFTR modulators, including lumacaftor-ivacaftor. However, the effects of such therapies on the airway ecosystem, particularly on the microbiota-mycobiota and local inflammation, which are involved in the evolution of pulmonary damage, are unclear. This multicenter study of the evolution of the microbiota under protein therapy supports the notion that CFTR modulators should be started as soon as possible, ideally before the patient is chronically colonized with P. aeruginosa. (This study has been registered at ClinicalTrials.gov under identifier NCT03565692).