Background: Prenatal opioid exposure (POE) induces long-term neurodevelopmental, behavioral and cognitive deficits for which no targeted treatments are available. The mechanisms underlying POE deficits are poorly understood, but have been linked to a range of central, peripheral, and enteric nervous system changes. Emerging evidence indicates that maternal microbiota changes may also contribute to these long-term offspring deficits. Here we test the efficacy of the short-chain fatty acid sodium butyrate (NaB) to mitigate POE-induced deficits in a rat model. Method: 26 pregnant rat dams received methadone via mini-osmotic pump and/or NaB via drinking water in a 2 x 2 design. We then performed a battery of developmental, behavioral, microbiome and neurobiological assessments across offspring infancy, adolescence and adulthood. Results: Both methadone and sodium butyrate treatments altered dam microbiota composition and function: notably methadone disrupted microbial gene expression of enzymes critical for butyrate production and reduced faecal butyric acid levels. In pups, methadone increased infant gut barrier permeability that was reversed with NaB, and induced infant microbial enzymatic disruption that resolved in adulthood. POE induced anxiety-like behavior in adolescence, and adulthood working spatial memory and attentional processing deficits that were partially rescued by prenatal NaB treatment. POE decreased myelination in the hippocampus, and this was partially reversed by NaB. Conclusions: Together these results highlight for the first time the link between the gut-brain axis in animal models of POE. Furthermore, they provide evidence for NaB as a simple yet effective treatment to significantly improve the outcomes of children born with POE.

Obstructive sleep apnea (OSA) and primary snoring (PS) represent a spectrum of sleep-related breathing conditions. Emerging evidence links OSA to perturbations in the oropharyngeal and gut microbiomes and the pathogenesis of OSA-related comorbidities. However, the microbiome composition and factors driving dysbiosis in children remain unresolved. Two pediatric cohorts were examined to assess the microbiome associated with sleep-disordered breathing in the airway (Cohort A) and gut (Cohort B) using 16S rRNA gene profiling. Oropharyngeal swabs were collected from participants with OSA, PS, and healthy controls (HC) (n = 60). Cohort B participants (OSA and HC, n = 46) provided stool samples for microbiome and fecal calprotectin measurements and completed a dietary survey. Oropharyngeal microbial diversity differed between OSA and HC, characterized by an increase in gastrointestinal specific taxa and reduced oral commensals. Similar shifts were observed between PS and HC, with few taxa differing between OSA and PS. In the gut, children with OSA showed an imbalance marked by an increase in opportunistic pathogens and reduced beneficial organisms. However, diversity assessments did not show any indication of dysbiosis or inflammation, and there were no overall differences in dietary intake. Dysbiosis in the oropharyngeal microbiomes of OSA and PS points to shared pathophysiological factors (e.g., snoring) as possible drivers of microbiome disruption across the spectrum of sleep-disordered breathing. The gastrointestinal microbiome of children with OSA indicates a mild microbial imbalance that may elicit harmful outcomes or manifest as dysbiosis if left untreated. Together, these findings support a role of the microbiome as a possible mediator of comorbidities across the spectrum of sleep-disordered breathing. Current knowledge/study rationale: Obstructive sleep apnea (OSA) and primary snoring (PS) represent a continuum of sleep-related breathing disorders. While adult and animal studies suggest OSA-induced microbiome disruptions contribute to the onset of comorbidities, the factors driving dysbiosis in children remain unresolved. This study investigated the airway and gut microbiomes in pediatric OSA, PS, and healthy controls to identify microbial alterations linked to these conditions. Study impact: This study revealed that children with obstructive sleep apnea and primary snoring exhibit similar altered oropharyngeal microbiomes, distinct from that of healthy controls, suggesting common underlying pathophysiological factors.

Introduction The introduction of CFTR modulators such as elexacaftor/tezacaftor/ivacaftor (ETI) has significantly altered the nutritional trajectory of children with cystic fibrosis (cwCF). While most eligible children in high-resource countries have commenced ETI, the rollout has outpaced the development of nutritional guidance. This study aimed to: (1) systematically review the literature on ETI and nutrition (2) develop expert consensus-based statements and (3) highlight existing research gaps and priorities. Methods A systematic review and guideline development process was registered on PROSPERO (ID: CRD42024587618). An expert panel of 22 professionals, including dietitians, gastroenterologists, respirologists, endocrinologists, and a parent representative was convened with attention to global representation. Research questions were developed using the Population, Intervention, Comparison, Outcome (PICO) framework. A comprehensive literature search was conducted across Medline, Embase, CINAHL, and Cochrane (January 2010 to April 2025). Studies were independently screened, assessed for quality using Scottish Intercollegiate Guidelines Network (SIGN) criteria, and extracted into evidence tables. Draft statements were developed and refined through iterative feedback and online discussion. Final consensus was achieved using real-time Delphi, with ≥80% agreement required for endorsement. Reporting followed Accurate Consensus Reporting Document (ACCORD) guidelines. Results 67 consensus-based statements were developed. The top research priorities were identified based on the greatest proportion of “strongly agree” responses. Conclusion ETI has altered nutritional requirements in cwCF, yet evidence remains limited. This study presents the first consensus-based guidance for the nutritional care of cwCF receiving ETI, providing interim, expert informed recommendations and highlighting research priorities, particularly the need to re-evaluate traditional BMI targets.

Background Gut dysbiosis is a hallmark of cystic fibrosis (CF), typically characterised using non-specific diversity metrics and study-specific taxonomic lists, limiting comparability across studies. We aimed to develop a metagenomic, species-level CF Gut Microbial Dysbiosis Index (CF-GMDI) to standardise measurement of gut microbial imbalance in children with CF (cwCF) Methods CF-GMDI was derived using stool metagenomic data from the PEARL-CF study (70 cwCF; 67 healthy controls (HC); 0-6 years). Differentially abundant taxa were identified between cwCF and HC using MaAsLin2. The index was calculated as the log10 ratio of the summed relative abundances of taxa enriched in CF vs HC to those depleted in CF vs HC. Reproducibility was assessed in the independent EARTH cohort (56 cwCF; 56 HC; 0-18 years). Responsiveness to therapy was evaluated using publicly available metagenomic data from an Elexacaftor/Tezacaftor/Ivacaftor (ETI) study (39 cwCF; 6-18 years). Results CF-GMDI was significantly higher in cwCF than HC (p < 0.001), inversely correlated with species richness (ρ = -0.74, p < 0.001), and higher in pancreatic-insufficient vs pancreatic-sufficient cwCF in the PEARL-CF cohort (p = 0.01). Key ecological and clinical associations were replicated in the EARTH cohort. In the ETI study, CF-GMDI decreased significantly at 6 and 12 months post-treatment, whereas alpha diversity remained unchanged. Conclusions CF-GMDI is a CF-associated metric that captures clinically relevant gut microbiome restructuring not detected by standard diversity measures in cwCF (0-18 years). It differentiates disease and pancreatic status and tracks therapeutic modulation, supporting its use as a novel endpoint in CF intervention studies.

BACKGROUND:Polyketide synthase island-positive (pks+) Escherichia coli is a genotoxic gut bacterium linked to colorectal cancer (CRC) tumorigenesis via the genotoxin colibactin. In adults with Cystic Fibrosis (CF), there is an increased incidence and earlier development of CRC but the biological mechanisms underlying this increased risk remain incompletely understood. We aimed to determine the prevalence of pks+ E. coli in adults with CF. METHODS:Stool samples and DNA were analyzed from the SCREENCF study cohort. Metagenomic libraries were sequenced on the NovaSeq X Plus platform, using Illumina protocols. Detection of the pks island was assessed with polymerase chain reaction (PCR) targeting the clbB gene. RESULTS:Of the 49 CF participants; pks+ E. coli was detected in 1/35 (3%) of the no pathology (NORMAL) group, 5/12 (42%) in the adenomatous polyps (AP) group, and 2/2 (100%) in the ileocolonic cancer (ICC) group. Individuals with any ileocolonic neoplasia were 34 times more likely to harbor pks+ E. coli than those with NORMAL colonoscopy findings (OR = 34.0, 95% CI 5.00-691, p = 0.002). The presence of pks+ E. coli correlated with higher overall E. coli burden (p = 0.0009), but not with fecal inflammation, other genotoxic bacterial species or overall bacterial composition. CONCLUSION:pks+ E. coli is infrequently detected among adults with CF, but its presence is associated with ileocolonic neoplasia, indicating a potential role in pathogenesis. If validated in larger cohorts, pks+ E. coli could provide a clinically meaningful biomarker for early detection, risk stratification and a potential target for precision intervention.

Non-tuberculous mycobacteria (NTM) represent a significant disease burden in the cystic fibrosis (CF) population. Whilst the acquisition of NTM to the respiratory tract is predominantly attributed to environmental exposures, the contribution of the gut-lung axis to NTM pulmonary disease remains poorly understood. In this study, we retrospectively examined 1285 stool microbiomes from 121 children and 52 adults with CF, and 135 healthy control (HC) children, in order to assess the prevalence, abundance and clinical significance of NTM in the gut. We detected various species of low clinical concern in the stool of 2% of people with CF and 1% of HC sampled, with longitudinal sampling revealing their occurrence to be sporadic and transient. Of the participants studied, only 11 had a history of prior clinical NTM respiratory colonisation or infection, but there was limited evidence to support ongoing persistence in the intestinal tract. This study suggests that the gut is not a meaningful reservoir for pulmonary NTM infections and it is unlikely to serve as a transmission route within the CF population.

BACKGROUND:The depletion of short chain fatty acid (SCFA) producing bacterial species in cystic fibrosis (CF) is hypothesized to reduce overall SCFA production, contributing to the onset and persistence of gastrointestinal inflammation. METHODS:In a prospective longitudinal observational study, we compared children with CFto age-matched healthy controls (HC). Participants completed a validated food frequency questionnaire and provided fecal samples for SCFA analysis (targeted liquid chromatography-mass spectrometry (LC-MS)), fecal calprotectin, and microbial community composition (16S rRNA gene sequencing). RESULTS:64 children with CF (cwCF) [median age (IQR) = 8.4 (4-11.9)] and 64 HC [median age (IQR) = 7.8 (3.4-13.4)] were recruited. Valerate and isobutyrate were significantly reduced and more variable over time in cwCF compared to HC. Age-associated increases in butyrate, valerate, isobutyrate and isovalerate observed in HC, were absent in CF. In CF, butyrate was positively correlated with alpha diversity (Richness = 0.3, p = 0.0005; Shannon diversity = 0.3, p = 0.0005) and inversely correlated with elevated calprotectin concentrations (-0.34, p = 0.01). CwCF had higher relative intake of fats, predominated by trans and saturated fats, alongside reduced relative intake of fibre, wholegrains and resistant starch. CONCLUSIONS:Our findings indicate that reduced microbial diversity, depletion of key SCFA-producing taxa, and limited dietary fibre intake may promote alternative, less efficient pathways of butyrate synthesis in cwCF. When butyrate is reduced, heightened inflammation is more likely. Further investigation into the physiological roles of valerate and isobutyrate is needed to understand the implications of their depletion in CF.

Cystic fibrosis (CF) patients experience higher risks of colorectal cancer but the pathogenesis is unclear. In the general population, polyketide synthase-positive (pks+) E. coli is implicated in intestinal carcinogenesis via the production of colibactin; however, the relevance in CF is unknown. In this study, we investigate pks+E. coli prevalence in CF and potential associations between pks+E. coli, gastrointestinal inflammation, and microbiome dynamics with fecal calprotectin and 16SrRNA gene taxonomic data. Cross-sectional analysis demonstrated no difference in pks+E. coli carriage between CF patients and healthy controls, 21/55 (38%) vs. 26/55 (47%), p = 0.32. Pks+E. coli was not associated with significant differences in mean (SD) calprotectin concentration (124 (154) vs. 158 (268) mg/kg; p = 0.60), microbial richness (159 (76.5) vs. 147 (70.4); p = 0.50) or Shannon diversity index (2.78 (0.77) vs. 2.65 (0.74); p = 0.50) in CF. Additionally, there was no association with exocrine pancreatic status (p = 0.2) or overall antibiotic use (p = 0.6). Longitudinally, CF subjects demonstrated intra-individual variation in pks+E. coli presence but no significant difference in overall prevalence. Future investigation into the effects of repeat exposure on risk profile and analysis of older CF cohorts is necessary to identify if associations with colorectal cancer exist.

The role of probiotics in children with cystic fibrosis (CwCF) remains unclear. The PEARL-CF study was an international, double-blind, randomized, placebo-controlled study involving CwCF (0–6 years), and assessed the effects of a probiotic on intestinal microbiota and clinical outcomes. A multi-strain probiotic (15 Lactobacillus/Bifidobacterium strains; ∼2-3×10 10 CFU daily) or placebo was administered for 12-months and participants followed a further 12-months post-intervention. Among 77 CwCF (38 probiotic, 39 placebo), bacterial alpha diversity did not differ between groups. Participants ≥4 years on probiotic demonstrated higher bacterial richness (post-hoc analysis). Haemophilus influenzae was less prevalent in clinically indicated respiratory swabs in the probiotic cohort during the intervention (27% vs 56%; p=0.03), especially when commenced prior to 4 years old. Fecal M2-pyruvate kinase decreased in the probiotic cohort from baseline to 12 months. This study provides support for multi-strain probiotics in CwCF, even in the era of modulator therapy.

Gastrointestinal symptoms often persist in children with Hirschsprung’s disease (HD) even after “corrective” pull-through surgery. Alteration of the gut microbiota (“dysbiosis”) has emerged as a potential contributing factor. Animal studies show gut ecosystem changes that are both intrinsic to HD and caused by bowel resection itself, but human studies comparing the intestinal microbiota of children with HD and healthy children are limited. We collected food frequency dietary surveys, clinical and symptom data, and stool samples from 15 post-operative children with HD and 15 healthy controls (HCs). We performed 16S rRNA gene sequencing from the stool samples and quantified faecal calprotectin as a measure of gastrointestinal inflammation. Despite no global changes in the microbiota between HD and HC cohorts and no differences between individuals with and without a history of HD-associated enterocolitis (HAEC), we identified evidence of altered microbiota development and inflammatory trajectories in HD. In HCs, alpha diversity increased with age (r = 0.83, p < 0.001), while calprotectin levels declined (Spearman’s ρ = −0.53, p = 0.04). These age-related patterns were absent in HD. Across the combined cohort, lower alpha diversity was associated with higher faecal calprotectin (Spearman’s ρ = −0.47, p = 0.01). In HD, Fusobacteria abundance showed a strong positive correlation with calprotectin (Spearman’s ρ = 0.76, adjusted p = 0.02). Pediatric Quality of Life (PedsQL) and gastrointestinal disease-specific symptom scores were lower in HD compared to HC but were not directly linked to microbial diversity or inflammation. Overall, we observed a divergence from healthy peers in the typical developmental trajectory of gut microbial communities and inflammation in children with HD that may involve Fusobacteria. Children with HD reported reduced health-related quality of life compared with HC, consistent with ongoing gastrointestinal symptoms. No microbiota differences were associated with HAEC history, though this may reflect limited sample size.

BACKGROUND:People with cystic fibrosis (pwCF) have increased colorectal cancer (CRC) risk. Colonoscopy is recommended, yet CF comorbidities increase complexity and risk. METHODS:We conducted a prospective, observational study of pwCF meeting colonoscopy screening guidelines at an Australian centre (2019 - 2023). Immunochemical faecal occult blood test (iFOBT), faecal calprotectin (FC), and faecal tumour pyruvate kinase isoenzyme type M2 (TuM2-PK) were evaluated for detecting adenomatous polyps and malignant ileocolonic lesions in pwCF. Stools were collected within 3 months of colonoscopy. Diagnostic performance and optimal cut-offs were calculated. RESULTS:Among 49 participants [mean (SD) age 47.8 (8.2) years; 53 % female], 12 (24.5 %) were post-solid organ transplant, 10 (20.4 %) had > 3 months of triple modulator therapy at stool testing, 12 (24.5 %) had adenomatous polyps and 2 (4 %) had ileocolonic malignancy. Malignancies were in non-transplanted individuals, in the terminal ileum (age 43) and hepatic flexure/ascending colon (age 48). Higher BMI (>23.5 kg/m²) was associated with abnormal colonoscopy (p = 0.03). iFOBT, FC and TuM2PK demonstrated excellent predictive performance for malignancy (AUC 0.93, 1.00, 0.83; all p < 0.05). Only FC had acceptable predictive performance for pre-malignant lesions (AUC 0.73; p = 0.008). For adenomatous polyps, FC ≤100 µg/g achieved a sensitivity of 91.7 % and an NPV of 95.5 %. For ileocolonic malignancy, FC ≥1000 µg/g showed 100 % sensitivity and specificity (p = 0.0009). CONCLUSION:CRC screening in pwCF is critical given the high prevalence of neoplasia. Alternative non-invasive screening may support risk stratification among individuals with comorbidities, or reluctance, though performance could be influenced by CFTR modulator therapy.

INTRODUCTION:Disorders of gut-brain interaction (DGBI), such as irritable bowel syndrome, occur at a higher prevalence in adults with cystic fibrosis (CF), compared to the general population. DGBI are associated with impaired quality of life and significant health-system costs. This study aimed to assess the proportion of cwCF with DGBI, compared to non-CF controls. METHODS:Validated for the assessment of DGBI, ROME IV surveys were distributed to cwCF and non-CF controls aged 0-18 years as part of the Evaluating the Alimentary Tracts in Health and Disease (EARTH) observational study. CF participants were recruited from the Sydney Children's Hospital (SCH) CF outpatient clinic between 2018 and 2022. Non-CF controls were recruited from outpatient clinics, advertisements, and word-of-mouth. RESULTS:Forty-four cwCF (female = 22 [50%], median [IQR] age = 7.04 [2.25-11.06] years) and 48 non-CF controls (female = 22 [45.83%], median (IQR) age = 8.04 [3.57-12.77] years) completed baseline surveys. Symptoms consistent with at least one DGBI were observed more frequently in cwCF compared to non-CF controls (36.36% vs. 10.42%%, p = 0.01). Functional abdominal pain was experienced at a higher prevalence in cwCF compared to non-CF controls (13.79% vs. 0%, p = 0.02). No significant differences in the prevalence of other specific disorders were observed between cwCF and non-CF controls. CONCLUSION:Compared to non-CF, cwCF were significantly more likely to experience functional abdominal pain, and at least one DGBI collectively. Further large-scale studies are needed to validate our findings and ascertain the role of routine screening of DGBI in pediatric CF cohorts.

Hydrocarbon contaminated soils resulting from human activities pose a risk to the natural environment, including in the Arctic and Antarctic. Engineered biopiles constructed at Casey Station, Antarctica, have proven to be an effective strategy for remediating hydrocarbon contaminated soils, with active ex-situ remediation resulting in significant reductions in hydrocarbons, even in the extreme Antarctic climate. However, the use of urea-based fertilisers, whilst providing a nitrogen source for bioremediation, has also altered the natural soil chemistry leading to increases in pH, ammonium and nitrite. Monitoring of the urea amended biopiles identified rising levels of nitrite to be of particular interest, which misaligns with the long term goal of reducing contaminant levels and returning soil communities to a ‘healthy’ state.Here, we combine amplicon sequencing, microfluidic qPCR on field samples and laboratory soil microcosms to assess the impact of persistent nitrite accumulation (up to 60 months) on nitrifier abundances observed within the Antarctic biopiles. Differential inhibition of ammonia oxidizing bacteria (AOB) and nitrite oxidizing bacteria (NOB) Nitrobacter and Nitrospira in the cold, urea treated, alkaline soils (pH 8.1) was associated with extensive nitrite accumulation (76 ± 57 mg N/kg at 60 months). When the ratio of Nitrospira:AOB dropped below ∼1:1, Nitrobacter was completely inhibited or absent from the biopiles, and nitrite accumulated. Laboratory soil microcosms (incubated at 7 °C and 15 °C for 9 weeks) reproduced the pattern of nitrite accumulation in urea fertilized soil at the lower temperature, consistent with our longer-term observations from the Antarctic biopiles, and with other temperature-controlled microcosm studies. Diammonium phosphate amended soil did not exhibit nitrite accumulation, and could be a suitable alternative biostimulant to avoid excessive nitrite build-up.