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PubMed2025

Impact of RNA extraction on respiratory microbiome analysis using third-generation sequencing.

BACKGROUND: The respiratory microbiome, which comprises bacteria, fungi, and viruses, plays a crucial role in respiratory health and disease. However, its study is limited by the low microbial biomass in respiratory samples and the dominance of host RNA. Metatranscriptomics offers comprehensive insights into active microbial communities and their interactions with the host but requires optimized RNA extraction protocols for robust and unbiased analysis. This study evaluated two RNA extraction kits—one employing chemical lysis (CL) and another combining chemical and mechanical lysis (CML)—to determine their effectiveness for metatranscriptomic analysis of respiratory samples. RESULTS: The CML protocol significantly increased double-stranded DNA (dsDNA) library yields, leading to higher sequencing read counts for both sample types (p < 0.0001). The read length was unaffected by the lysis protocol for the BAL and NPS samples. Taxonomic profiling revealed that CML enhanced the detection of robust microorganisms, such as gram-positive bacteria and fungi, without compromising viral detection. CONCLUSIONS: The CML protocol demonstrated superior recovery of genetic material, particularly for fungi and gram-positive bacteria, making it better suited for comprehensive metatranscriptomic analyses. These findings underscore the need for tailored RNA extraction strategies on the basis of sample type and research objectives. Optimized metatranscriptomic protocols are pivotal for advancing our understanding of the respiratory microbiome and its role in health and disease.

Microbiota↗

PubMed2026

Longitudinal dynamics of respiratory microbiome composition in infants after new tracheostomy placement.

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.

Humans↗

PubMed2025

Controlled human infection model of Neisseria lactamica in late pregnancy investigating mother-to-infant transmission in the UK: a single-arm pilot trial.

BACKGROUND: The infant respiratory microbiome is derived largely from the mother and is associated with downstream health and disease. Manipulating maternal respiratory flora peripartum to influence the infant microbiome has not previously been investigated. Neisseria lactamica is a harmless pharyngeal commensal that correlates inversely with Neisseria meningitidis carriage and disease. Intranasal N lactamica inoculation is a safe and well characterised controlled human infection model (CHIM) in non-pregnant healthy adults. We hypothesised that N lactamica inoculation in pregnancy induces mother-to-infant N lactamica transmission postnatally. METHODS: In this single-arm trial, 21 healthy pregnant female participants aged 18 years or older were inoculated at 36-38 weeks' gestation with 105 colony-forming units of N lactamica Y92-1009 at University Hospital Southampton Clinical Research Facility, Southampton, UK. N lactamica selective culture, genome sequencing, and serological testing were performed on maternal and infant oral, nasopharyngeal, breastmilk, and serum samples over 15 weeks postpartum. Seven female participants naturally colonised with N lactamica at baseline were followed up, but not inoculated. Oral samples were obtained from 12 cohabiting siblings younger than 5 years. The primary endpoint was infant N lactamica colonisation. This study was registered with ClinicalTrials.gov, NCT04784845, and is now complete. FINDINGS: Between Oct 25, 2021, and March 7, 2022, 31 adult female participants (median age 33·5 years [range 23·1-39·9]; 26 [84%] were White, British) were screened and enrolled, of whom seven were already colonised with N lactamica. After exclusion of three participants, 21 participants were inoculated, of whom 15 (71%) became N lactamica-colonised, and no sustained N lactamica Y92-1009 transmission to their infants was observed. Conversely, non-Y92-1009 N lactamica strain sharing was observed in four (57%) of seven uninoculated mother-sibling pairs, and Moraxella catarrhalis strain sharing in nine (38%) of 24 mother-infant pairs completing the study. Anti-N lactamica serum IgG titres increased in seven (88%) of eight N lactamica Y92-1009-colonised female participants, but none of their infants (where paired sera were available). There were no serious adverse reactions to the inoculum. INTERPRETATION: As the world's first perinatal CHIM, this trial demonstrates that this model in pregnancy is feasible, and that N lactamica Y92-1009 can safely and efficiently colonise pregnant individuals. Lack of sustained mother-to-infant N lactamica transmission, despite evidence supporting mother-to-infant M catarrhalis and sibling-to-mother N lactamica transmission, challenges conventional perceptions of infants as passive recipients of maternal microbes, suggesting that respiratory commensal transmission is selective and microbe-specific. FUNDING: Medical Research Council and National Institute for Health Research Southampton Biomedical Research Centre.

Adult↗

PubMed2024

Longitudinal effects of elexacaftor/tezacaftor/ivacaftor on the oropharyngeal metagenome in adolescents with cystic fibrosis.

BACKGROUND: Triple modulator therapy elexacaftor/tezacaftor/ivacaftor (ETI) improves lung function and impacts upon the respiratory microbiome in people with Cystic fibrosis (pwCF) with advanced lung disease. However, adolescents with cystic fibrosis (CF) are less colonized with bacterial pathogens than adult pwCF but their microbiota already differs from healthy individuals. The aim of this study was to longitudinally analyze the impact of ETI on the respiratory metagenome in adolescents with predominantly mild CF lung disease. METHODS: In this prospective observational study, we included pwCF aged 12-20 years with at least one F508del mutation, who collected oropharyngeal swabs before and after initiation of ETI therapy twice per week to biweekly over three months. We performed whole metagenome shotgun sequencing, followed by host DNA filtering and taxonomic profiling. We used linear and additive mixed effects models adjusted for known confounders and corrected for multiple testing to study longitudinal development of the microbiome. We analyzed bacterial diversity, abundance, and strain-level phylogeny. RESULTS: We analyzed the metagenomic data of 297 swabs of 20 pwCF. Microbiome composition changed after initiation of ETI therapy. We observed a slight diversification of the microbiome over time (Inv Simpson, Coef 0.085, 95 %CI 0.003, 0.17, p = 0.04). Strain-level analysis and clustering showed that strain retention of the most frequent bacterial species is predominant even during ETI therapy. CONCLUSIONS: During three months of ETI therapy, commensal bacteria increased, which may help to prevent overgrowth of bacterial pathogens.

Humans↗

PubMed2026

Mannheimia haemolytica strain-level diversity in cattle populations.

High-resolution genomic characterization is essential for understanding diversity, pathogenicity, and transmission dynamics of bacterial pathogens. Mannheimia haemolytica (Mh) is the most consequential bacterial agent associated with bovine respiratory disease (BRD) in cattle, as a leading cause of morbidity, mortality, and antimicrobial use. Historically, BRD pathogens, including Mh, have been studied using culture or PCR approaches that provided limited ability to characterize fine-scale genomic variation across communities. Here, we evaluated target-enriched (TE) shotgun sequencing, a culture-independent method capable of strain-level resolution within metagenomic data, for detecting and characterizing Mh in comparison with qPCR and 16S rRNA gene sequencing. Nasal swabs (10 individual and 2 composited DNA samples per pen) and environmental samples (three ropes hung on pen rails and three water bowl swabs per pen) were collected from four pens in each of five distinct cattle populations. DNA was extracted for TE sequencing to identify Mh at both species and genomic sequence variant (GSV) levels, and to characterize antimicrobial resistance genes across the bacterial communities. qPCR was performed to quantify Mh genome copies, and 16S rRNA gene sequencing was used to assess the broader respiratory microbiome. TE sequencing identified Mh in 100% of TE-tested samples and classified multiple GSVs in all but 3 of 121 samples. GSV profiles clustered within housing groups and varied across cattle populations, indicating structured strain-level diversity. In contrast, Mannheimia spp. were detected in only 47.7% of samples by 16S rRNA sequencing. These findings demonstrate that TE sequencing enables sensitive, strain-level characterization of Mh in cattle and environmental samples and reveals substantial within-population genomic diversity not captured by conventional approaches.IMPORTANCETarget-enriched shotgun sequencing enabled sensitive, strain-level detection of Mannheimia haemolytica (Mh), revealing multiple co-circulating genomic sequence variants (GSVs) within and among cattle groups. This demonstrates greater genetic variability of Mh populations in beef cattle than has been previously recognized. The clustering of GSVs within housing groups, together with the overlap between respiratory and environmental samples, is consistent with the hypothesis that contagious transmission contributes to Mh ecology. These results highlight the potential utility of composite nasal swab and environmental samples for future studies evaluating relationships between Mh genomic variation and disease risk.

Animals↗

PubMed2026

Lpb. plantarum inhalation powders for the reduction of lung inflammation and S. aureus growth control in non-CF bronchiectasis.

INTRODUCTION: The reduced diversity of the lung microbiome in respiratory conditions, including bronchiectasis, promotes bacterial infection and inflammation, contributing to worsening clinical outcomes. Traditional treatments often fail to address both infection and inflammation. Because of their potential dual-action, lactic acid bacteria (LAB) directly administered to the lungs could represent an innovative therapeutic approach for these diseases. RESEARCH DESIGN AND METHODS: Two powders for inhalation containing Lpb. plantarum, lactose, l-leucine with and without raffinose, a prebiotic, were produced by spray drying and in vitro tested. The focus was on investigating their potential in vitro anti-inflammatory and anti-microbial activities against S. aureus. RESULTS: The powders showed a fine particle fraction (<5 µm) of >40% and allowed the maintenance of anti -inflammatory activity in vitro for both treatment and prevention. Moreover, both powders led to a significant reduction in S. aureus growth. The stability study of the powders in capsules at different storage conditions showed the preservation of the LAB up to 90 days in refrigerated conditions (4°C/-20°C). CONCLUSIONS: This proof-of-concept study shows that inhalable spray-dried live LABs retain biological activity and are suitable for pulmonary delivery, supporting their potential as a microbiota-modulating therapy, which, however, requires further preclinical validation.

Inhalation↗

PubMed2026

Dual-transcriptomic analysis of human nasal transcriptome and microbiome reveals host-bacteria associations in symptomatic respiratory infection.

BACKGROUND: The human nasopharynx is colonized by a diverse community of commensal microbiota linked to many respiratory diseases, yet their associations with the host remain unclear. RESULTS: In this study, we introduced a dual-transcriptomics analysis strategy, which can characterize the host transcriptome and microbiome from nasal samples simultaneously. We applied this workflow to a local SARS-CoV-2 cohort with 76 asymptomatic infected patients, among whom 52 (68.42%) developed symptomatic infection during a 1-week follow-up period. Nasal swabs were collected from all 76 patients at enrollment and from 73 patients at one-week later follow-up. We detected a median of 8.94% reads that did not map to the human genome across all 149 samples, among which around half (median 49.68%) were successfully mapped to microbiome genome. Meta-transcriptomic analysis detected significantly higher SARS-related coronavirus loads in samples from the symptomatic group at enrollment (P = 0.004), and both groups showed decreased loads one week later (symptomatic, P = 0.001; asymptomatic, P = 0.035). Compared with benchmarking 16 S rRNA sequencing on 53 samples, our computational strategy showed high correlation of relative abundance in all top 20 genera (median Rho = 0.90, Pmax < 0.001). A total of 670 bacteria species were identified to show a relative abundance ≥ 0.01% in at least 10% samples. Differential abundance analysis identified 76 species (DASs) from six phyla with significantly decreased abundance in samples from the symptomatic group (log2(fold change or FC) < -1 and adjusted P < 0.05) compared to the asymptomatic group at enrollment. Integrating these symptom-associated DASs with host's gene expression using an expression quantitative trait bacteria (eQTB) model, we found 45 symptom-associated DASs identified at enrollment were significantly associated with one to 14 genes (adjusted P < 0.05). GSEA showed a series of symptom-associated DASs were significantly correlated with pathways related to olfactory function, keratinocyte differentiation, and DNA methylation. CONCLUSIONS: In summary, our dual-transcriptomic analysis strategy effectively characterized host-microbiome associations, offering insights into microbial contributions to respiratory diseases.

Humans↗

PubMed2025

Transitions in lung microbiota landscape associate with distinct patterns of pneumonia progression.

The precise microbial determinants driving clinical outcomes in severe pneumonia are unknown. Competing ecological forces produce dynamic microbiota states in health and disease, and a more thorough understanding of these states has the potential to improve pneumonia therapy. Here, we leverage a large collection of bronchoscopic samples from patients with suspected pneumonia to determine lung microbial ecosystem dynamics throughout the course of pneumonia. We combine 16S rRNA gene, metagenomic, and metatranscriptomic sequencing with bacterial-load quantification to reveal clinically relevant drivers of pneumonia progression. Microbiota states are predictive of pneumonia subtypes and exhibit differential stability and pneumonia therapy response. Disruptive forces, such as aspiration, are associated with cohesive changes in gene expression and microbial community structure. In summary, we show that host and microbiota landscapes change in unison with clinical phenotypes and that microbiota state dynamics reflect pneumonia progression. We suggest that distinct pathways of lung microbial community succession mediate pneumonia progression.

Humans↗

PubMed2024

Effectiveness of stabilization methods for the immediate and short-term preservation of bovine fecal and upper respiratory tract genomic DNA.

Previous research on stabilization methods for microbiome investigations has largely focused on human fecal samples. There are a few studies using feces from other species, but no published studies investigating preservation of samples collected from cattle. Given that microbial taxa are differentially impacted during storage it is warranted to study impacts of preservation methods on microbial communities found in samples outside of human fecal samples. Here we tested methods of preserving bovine fecal respiratory specimens for up to 2 weeks at four temperatures (room temperature, 4°C, -20°C, and -80°C) by comparing microbial diversity and community composition to samples extracted immediately after collection. Importantly, fecal specimens preserved and analyzed were technical replicates, providing a look at the effects of preservation method in the absence of biological variation. We found that preservation with the OMNIgene®•GUT kit resulted in community structure most like that of fresh samples extracted immediately, even when stored at room temperature (~20°C). Samples that were flash-frozen without added preservation solution were the next most representative of original communities, while samples preserved with ethanol were the least representative. These results contradict previous reports that ethanol is effective in preserving fecal communities and suggest for studies investigating cattle either flash-freezing of samples without preservative or preservation with OMNIgene®•GUT will yield more representative microbial communities.

Cattle↗

PubMed2025

Investigation of associations between the neonatal gut microbiota and severe viral lower respiratory tract infections in the first 2 years of life: a birth cohort study with metagenomics.

BACKGROUND: Early-life gut microbiota affects immune system development, including the lung immune response (gut-lung axis). We aimed to investigate whether gut microbiota composition in neonates in the first week of life is associated with hospital admissions for viral lower respiratory tract infections (vLRTIs). METHODS: The Baby Biome Study (BBS) is a prospective birth cohort, which enrolled mother-baby pairs between Jan 1, 2016, and Dec 31, 2017, at three UK hospitals. In the present study, we only included BBS babies with a sequenced first-week stool sample and successful data linkage. Stool was collected in the first week of life for shotgun-metagenomic sequencing. We examined the following microbiota features: alpha diversity (Chao1, Shannon, and Simpson indices) and community structures (cluster-partitioning against medoids method). The participants were followed up through linkage to the Hospital Episode Statistics-Admitted Patient Care (HES-APC) database to determine vLRTI hospital admission incidence in the first 2 years of life. We used Poisson mixed-effects models for univariable and multivariable analyses to evaluate the association between microbiota features and vLRTI hospital admission incidence, adjusting for confounders identified through direct acyclic graphs. FINDINGS: 3305 (95%) of the 3476 BBS-enrolled babies for whom consent to data linkage was obtained were included in the present study. 1111 (34%) babies had a first-week sequenced stool sample, of whom 1082 (97%; 564 born vaginally and 518 born by caesarean section) were successfully linked to HES-APC, and had median follow-up of 2·0 years (IQR 1·4-2·9). Most babies were born at term (996 [92%] ≥37 weeks gestational age and 1070 [99%] >35 weeks gestational age) and healthy (1050 [97%] had no comorbidities), and 520 (48%) were female and 562 (52%) were male. Higher first-week gut microbiota alpha diversity was associated with reduced rates of vLRTI hospital admission (Chao1 Index adjusted hazard ratio [HR] 0·92 [95% CI 0·85-0·99]; Shannon Index adjusted HR 0·57 [0·33-0·98]; and Simpson Index adjusted HR 0·36 [0·11-1·20]). Three microbiota clusters were identified. Cluster 1 had a mixed composition and cluster 2 was dominated by Bifidobacterium breve, with both clusters observed in babies born vaginally and by caesarean section. Cluster 3 was found only in vaginally born babies and was dominated by Bifidobacterium longum. Having cluster 1 (mixed) or cluster 2 (B breve dominated) was independently associated with increased rates of vLRTI hospital admission compared with cluster 3 (B longum dominated; cluster 1 [mixed] 3·05 [1·25-7·41] and cluster 2 [B breve dominated] 2·80 [1·06-7·44]). INTERPRETATION: We report observational evidence that first-week gut microbiota differences are associated with clinically severe vLRTI in young children. This study identified bacterial species that could be of interest for vLRTI prevention. This finding has important implications for the design of future research and intervention strategies. FUNDING: The Wellcome Trust and Wellcome Sanger Institute core funding.

Humans↗

PubMed2022

Cystic Fibrosis Airway Mucus Hyperconcentration Produces a Vicious Cycle of Mucin, Pathogen, and Inflammatory Interactions that Promotes Disease Persistence.

The dynamics describing the vicious cycle characteristic of cystic fibrosis (CF) lung disease, initiated by stagnant mucus and perpetuated by infection and inflammation, remain unclear. Here we determine the effect of the CF airway milieu, with persistent mucoobstruction, resident pathogens, and inflammation, on the mucin quantity and quality that govern lung disease pathogenesis and progression. The concentrations of MUC5AC and MUC5B were measured and characterized in sputum samples from subjects with CF (N = 44) and healthy subjects (N = 29) with respect to their macromolecular properties, degree of proteolysis, and glycomics diversity. These parameters were related to quantitative microbiome and clinical data. MUC5AC and MUC5B concentrations were elevated, 30- and 8-fold, respectively, in CF as compared with control sputum. Mucin parameters did not correlate with hypertonic saline, inhaled corticosteroids, or antibiotics use. No differences in mucin parameters were detected at baseline versus during exacerbations. Mucin concentrations significantly correlated with the age and sputum human neutrophil elastase activity. Although significantly more proteolytic cleavages were detected in CF mucins, their macromolecular properties (e.g., size and molecular weight) were not significantly different than control mucins, likely reflecting the role of S-S bonds in maintaining multimeric structures. No evidence of giant mucin macromolecule reflecting oxidative stress-induced cross-linking was found. Mucin glycomic analysis revealed significantly more sialylated glycans in CF, and the total abundance of nonsulfated O-glycans correlated with the relative abundance of pathogens. Collectively, the interaction of mucins, pathogens, epithelium, and inflammatory cells promotes proteomic and glycomic changes that reflect a persistent mucoobstructive, infectious, and inflammatory state.

Cystic Fibrosis↗

PubMed2026

Gut microbiome composition and predicted functions relate to growth and behavior in a Japanese preschool cohort.

Early childhood is a period of rapid brain maturation and gut microbiome assembly, when emerging behavioral difficulties can shape later mental health and learning trajectories. Microbiota-gut-brain communication has been implicated in neurodevelopment through microbial metabolites and immune signaling. However, most pediatric evidence comes from high-risk or clinically referred cohorts, and gut microbiome-related correlates of typical behavioral variation in community-based preschool children remain poorly defined. In a cross-sectional sample of typically developing Japanese preschool children, we observed exploratory nominal associations between behavioral variation within normative ranges and gut microbiome composition and predicted functions. Internalizing domains showed candidate links with taxa and predicted pathways related to inflammatory potential and nucleotide biosynthesis, whereas somatic complaints and withdrawn behavior showed nominal associations with lower predicted respiratory and fermentative activity. Sleep-related difficulties showed multiple representative nominal pathway-level associations, including pathways related to methyl-donor and heme biosynthesis, while externalizing domains showed candidate links with predicted cell-envelope and carbohydrate-remodeling pathways. In contrast, age, height, and weight tracked expected maturation-related microbiome features, indicating that behavioral associations were not simple proxies of growth. Together, these findings provide an exploratory profile of microbiome-behavior correlations in a low-risk Japanese preschool cohort and highlight pathway-level candidates that may interface with neurodevelopment.

Humans↗

PubMed2026

Airway microbiome diversity, intramucosal bacteria, and spatial immunity in asthmatic adults and controls.

RATIONALE: Asthma is characterized by disruption of the thoracic airway mucosae and loss of microbial diversity. Spatial profiling of the mucosal transcriptome may systematically discover mechanisms for microbial influences on immunity. OBJECTIVES: We investigated relationships between clinical measures, microbial communities, and the host mucosal transcriptome within different strata of bronchial biopsies in subjects with and without asthma. METHODS: We performed bronchoscopy in 65 asthmatic adults and 44 healthy controls, quantifying bacterial operational taxonomic units (OTUs) in bronchial brushings by 16S ribosomal RNA (rRNA) gene amplicon sequences. Biopsy histologic features were scored blind to diagnosis. Following 16S rRNA in situ hybridization of 44 biopsies, bacterial foci were scored in epithelium, basement membrane, and stroma. Global human gene expression was quantified in epithelial and stromal compartments using digital spatial profiling. MEASUREMENTS AND MAIN RESULTS: Clinical asthma was independently predicted by basement membrane abnormalities (BaseMA), endobronchial bacterial diversity, and circulating eosinophil counts, but not by specific OTU abundances. 16S rRNA staining revealed bacteria within epithelium and mucosa of all biopsies. Intramucosal bacteria counts correlated negatively with spatially organized coexpression networks encoding antigen-specific immunity, neutrophil functions, and matrix activation, whereas BaseMA correlated positively with the adaptive immunity module. Eosinophil counts correlated with epithelial bacterial counts and senescence pathways. Clinical asthma was accompanied by upregulation of a regulatory T-cell network. CONCLUSIONS: Asthma and its related phenotypes are accompanied by complex mucosal events that extend beyond eosinophilic pathways. Components of diverse airway microbiota may modify immunity by beneficial interactions within the mucosa.

Humans↗

PubMed2024

Design of the SPIROMICS Study of Early COPD Progression: SOURCE Study.

BACKGROUND: The biological mechanisms leading some tobacco-exposed individuals to develop early-stage chronic obstructive pulmonary disease (COPD) are poorly understood. This knowledge gap hampers development of disease-modifying agents for this prevalent condition. OBJECTIVES: Accordingly, with National Heart, Lung and Blood Institute support, we initiated the SubPopulations and InteRmediate Outcome Measures In COPD Study (SPIROMICS) Study of Early COPD Progression (SOURCE), a multicenter observational cohort study of younger individuals with a history of cigarette smoking and thus at-risk for, or with, early-stage COPD. Our overall objectives are to identify those who will develop COPD earlier in life, characterize them thoroughly, and by contrasting them to those not developing COPD, define mechanisms of disease progression. METHODS/DISCUSSION: SOURCE utilizes the established SPIROMICS clinical network. Its goal is to enroll n=649 participants, ages 30-55 years, all races/ethnicities, with ≥10 pack-years cigarette smoking, in either Global initiative for chronic Obstructive Lung Disease (GOLD) groups 0-2 or with preserved ratio-impaired spirometry; and an additional n=40 never-smoker controls. Participants undergo baseline and 3-year follow-up visits, each including high-resolution computed tomography, respiratory oscillometry and spirometry (pre- and postbronchodilator administration), exhaled breath condensate (baseline only), and extensive biospecimen collection, including sputum induction. Symptoms, interim health care utilization, and exacerbations are captured every 6 months via follow-up phone calls. An embedded bronchoscopy substudy involving n=100 participants (including all never-smokers) will allow collection of lower airway samples for genetic, epigenetic, genomic, immunological, microbiome, mucin analyses, and basal cell culture. CONCLUSION: SOURCE should provide novel insights into the natural history of lung disease in younger individuals with a smoking history, and its biological basis.

SPIROMICS↗

PubMed2026

DNA sequencing for microbial surveillance in cystic fibrosis airways: advances, challenges, and clinical translation.

SUMMARYDNA sequencing has revolutionized microbial surveillance in cystic fibrosis (CF), transforming pathogen identification from culture-dependent to total microbial community identification using molecular-based approaches. Techniques such as 16S rRNA gene sequencing have uncovered the complexity of the CF airway microbiome, while shotgun metagenomics, metatranscriptomics, and viromics now provide strain-level, functional, and viral insights beyond bacterial identification. Despite these advances, key technical and logistical challenges remain, including the processing of high-viscosity sputum samples, overwhelming host DNA contamination, managing large data sets, and the integration of complex bioinformatic outputs into clinical workflows. Emerging innovations such as host DNA depletion protocols, targeted enrichment panels, and adaptive sampling on Oxford Nanopore platforms are helping to overcome these barriers, improving microbial recovery and sequencing efficiency. As cystic fibrosis transmembrane conductance regulator (CFTR) modulator therapies are changing the lives of people with cystic fibrosis (pwCF), sequencing offers an unprecedented opportunity to track potential microbial adaptation in response. This review investigates current advances, limitations, and translational opportunities in DNA sequencing for CF airway microbiome surveillance, highlighting how these technologies can help reshape research and clinical microbiology in the post-modulator era.

Cystic Fibrosis↗

PubMed2025

ITIH4 alleviates OVA-induced asthma by regulating lung-gut microbiota.

BACKGROUND: Inter-alpha-trypsin inhibitor heavy chain 4 (ITIH4), a Type 2 acute phase protein, is critical for resolving inflammation and promoting tissue repair. While its role in chronic respiratory diseases is recognized, its effects on asthma remain unclear. This study investigated the effects of ITIH4 on the modulation of lung and gut microbiota, the attenuation of allergic inflammation, and the improvement of respiratory outcomes in an asthma mouse model. METHODS: Six-week-old male Balb/c mice were divided into five groups: control, ITIH4, ovalbumin (OVA), and two OVA + ITIH4 treatment groups at different doses. Lung function and oxygen saturation were measured, and bronchoalveolar lavage fluid (BALF) was analyzed for white blood cell counts and cytokines. Lung and gut microbiota were profiled using 16 S rRNA gene sequencing, and short-chain fatty acids (SCFAs) were measured using gas chromatography-mass spectrometry (GC-MS). Proteomic profiling of intestinal tissues was conducted to identify ITIH4-associated signaling pathways. RESULTS: ITIH4 administration significantly mitigated OVA-induced asthma symptoms by reducing weight loss, airway resistance, and tissue damping (p < 0.05). Histological analysis showed decreased airway wall thickening and lung injury scores (p < 0.05). ITIH4 also lowered BALF eosinophils and lymphocytes, IgE, and Th2 cytokines (IL-4, IL-5, and IL-13) (p < 0.05). ITIH4 treatment modulated microbiome composition, enriching Gram-positive taxa (Nocardioidaceae and Acholeplasmataceae) and depleting Gram-negative Helicobacteraceae (p < 0.05). SCFAs correlated with microbiome alterations, notably reduced 4-methylpentanoic acid levels (p < 0.05). Proteomic analysis revealed a dose-dependent activation of granzyme A signaling and suppression of metabolic and solute transport pathways. CONCLUSIONS: ITIH4 ameliorates asthma symptoms by modulating lung and gut microbiota, dampening Th2-driven inflammation, and restoring mucosal immune balance. These findings support ITIH4 as a potential candidate for microbiome-targeted asthma therapy.

Animals↗

PubMed2026

Increasing gut short-chain fatty acids protects intestinal barrier function but does not spare muscle glycogen or impact aerobic performance.

Animal studies suggest gut microbiota-derived short-chain fatty acids (SCFA) provide an intestinal barrier-protecting, glycogen-sparing energy source that increases aerobic endurance performance, but confirmation in humans is needed. This study aimed to determine whether increasing colonic SCFA availability impacts intestinal barrier function, substrate metabolism, muscle glycogen and aerobic performance in healthy adults. Using a randomized, double-blind, crossover design 12 active men (age 18-30 years;40.0 ± 7.1 mL/kg/min) performed prescribed exercise and consumed a provided diet supplemented with acetylated and butyrylated high-amylose maize starch engineered to deliver SCFA to the colon (HAMS-A/B) or low-amylose maize starch (LAMS) for 7 days, separated by a 2 week washout. Indirect calorimetry, stable isotopes and blood, muscle and urine biomarkers were measured on intervention day 8 while participants completed 90 min of steady-state cycle ergometry (ExSS; 60 ± 5%) followed by a 5 km treadmill time trial. HAMS-A/B, relative to LAMS, increased faecal and serum SCFA. Multiple markers of intestinal barrier damage and permeability were lower, and the respiratory exchange ratio during ExSS was higher (0.02 [95% confidence interval (CI): 0.01, 0.03], Ptreatment < 0.001) following HAMS-A/B versus LAMS. However no between-treatment difference in glucose turnover, muscle glycogen depletion (14 µmol/kg/g dry wt. [95% CI: -116, 143], Pinteractio n = 0.613) or TT performance (5 s [95%CI: -44, 54], Ptreatment = 0.816) was observed. Increasing colonic and circulating SCFA modestly altered substrate oxidation and preserved intestinal barrier function during endurance exercise. However effects were not sufficient to spare muscle glycogen or increase aerobic endurance performance, leaving the practical relevance unclear and underscoring challenges inherent in translating promising preclinical findings to humans. KEY POINTS: Animal studies suggest gut microbiota-derived short-chain fatty acids (SCFA) provide an intestinal barrier-protecting, glycogen-sparing energy source that increases aerobic endurance performance, but confirmation in humans is lacking. A gut microbiota-targeted dietary supplementation strategy was used to deliver SCFA to the colon and successfully increased colonic and systemic SCFA concentrations in healthy, physically active adults before and during an endurance exercise bout and aerobic performance test. Increasing colonic and systemic SCFA availability preserved intestinal barrier function but did not impact glucose turnover, alter protein expression in muscle or spare muscle glycogen during endurance exercise. Increasing colonic and systemic SCFA availability did not impact aerobic endurance performance.

Humans↗

PubMed2026

Gut microbiota dynamics and metabolic pathways associated with bleomycin-induced pulmonary fibrosis progression.

BACKGROUND: Pulmonary fibrosis (PF) is a progressive respiratory disease characterized by epithelial injury, aberrant repair and excessive extracellular matrix deposition. Although the gut-lung axis is increasingly implicated in respiratory disorders, stage-resolved characterization of gut microbiota taxonomic and functional potential during PF development is limited. METHODS: We established a bleomycin-induced murine PF model and performed cross-sectional shotgun metagenomic sequencing of fecal samples from separate cohorts at three defined stages: baseline (control), day 7 (early fibrosis; M7), and day 14 (established fibrosis; M14). Microbial taxonomy, alpha/beta diversity, and predicted functional capacity were inferred using Kyoto Encyclopedia of Genes and Genomes (KEGG) and Carbohydrate-Active enZymes (CAZy) annotations; associations were assessed using Procrustes and Spearman correlation analyses. RESULTS: Histopathology and immunohistochemistry confirmed progressive fibrogenesis with increased TGF-β1 and α-SMA expression. Compared with baseline, bleomycin-treated groups exhibited stage-specific shifts in gut microbial composition, including depletion of mucin-associated taxa (e.g., Prevotella, Akkermansia muciniphila) and expansion of Muribaculaceae- and Clostridiaceae-affiliated taxa. Alpha and beta diversity metrics differed across groups. KEGG/CAZy-based annotations revealed predicted, stage-dependent changes in microbial metabolic potential, including early reductions in pathways related to amino acid and glycan metabolism (M7) and later increases in predicted starch/sucrose catabolism, phosphotransferase system (PTS) representation, and secondary bile acid biosynthesis (M14). Correlation analyses linked compositional shifts to these predicted functional changes. CONCLUSION: In a stage-resolved, cross-sectional study, bleomycin-associated pulmonary fibrosis was accompanied by compositional and predicted functional alterations in the gut microbiota. These data identify candidate taxa and predicted pathways for follow-up mechanistic testing, but functional (metabolomic) and causality experiments are required to confirm whether and how microbial changes contribute to PF pathogenesis.

Animals↗