Polymicrobial lung infections are common in individuals with cystic fibrosis (CF). Pathogen communities typically follow an ecological succession in which early colonizers such as Haemophilus influenzae and Staphylococcus aureus are later joined by Pseudomonas aeruginosa . Although adaptation to the lung environment is well described for single pathogens in adult people with CF, much less is known about the role of pathogen interactions and how changes in individual pathogens influence community dynamics at the early stages of disease. To address these questions, we combined genome sequencing with phenotypic screens and pathogen interaction assays using longitudinal clinical isolates (19 P. aeruginosa , 44 S. aureus , and 21 H. influenzae ) collected from 23 children with CF (0–7.9 years of age) enrolled in the SCILD (Swiss CF Infant Lung Development) cohort. Our analysis revealed that early pathogen communities are characterized by a combination of strain turnover and persistence of isolates undergoing first steps of within–host evolution. Most notably, quorum–sensing–deficient P. aeruginosa variants repeatedly emerged, showing reduced protease production and diminished inhibition of S. aureus and H. influenzae . These changes indicate that P. aeruginosa becomes less antagonistic towards co–occurring pathogens, possibly promoting community stability. Together, our results show that ecological and evolutionary dynamics between pathobiome members may play an underappreciated role in shaping CF lung disease during early childhood. ### Competing Interest Statement The authors have declared no competing interest. Swiss National Science Foundation, 212266 University of Zurich Research Priority Program (URPP) Evolution in Action PhD studentship from the Biotechnology and Biological Sciences Research Council Midlands Integrative Biosciences Training Partnership, BB/M01116X/1

It has been reported that the introduction of thirteen-valent pneumococcal conjugate vaccine (PCV13) had an influence on antibiotic resistance rates of invasive pneumococcal disease (IPD). Recent data about antibiotic resistance in IPD are scarce. The aim of the current study was to analyse the impact of PCV13 introduction and COVID-19 pandemic on antibiotic resistance in IPD and the effects of antibiotic resistance on mortality. Furthermore, we correlate vaccine and non-vaccine serotypes with antibiotic resistance. We included 8747 IPD cases from a Swiss nationwide IPD surveillance database for 2012–2022. Regression analyses were performed to examine significant trends over time and to identify serotypes and patient characteristics associated with antibiotic resistance. The proportion of non-susceptibility to erythromycin (IRR: 0.9, p < 0.001) and cotrimoxazole (IRR: 0.9, p < 0.001) decreased from 2012 to 2022. Penicillin non-susceptibility remained constant with a dip in 2022 (2012: 9.2

Pig farming is one of the most intensive human-animal interfaces, and farm workers carry gut microbiotas that differ from those of non-farmers. Whether this overlap reflects genuine strain transmission or shared environmental exposure is unclear. Using shotgun metagenomics, we profiled the gut microbiomes of pigs at three growth stages (suckling, weaning, fattening), their farmers, and non-farmer controls, at species and strain resolution, and characterized the resistome in parallel. Farmers shared more species with pigs than controls, with Prevotellaceae the most consistently enriched family. Strain-level analysis of 137 shared species showed that most (71%) maintain host-specific sub-species clades. Strain sharing ran an order of magnitude below species sharing and was confined to early growth stages; its direction could not be determined. Pigs and farmers shared a farm-associated resistome signature, including the beta-lactamase cfxA4 and the methyltransferases ermF and cfrE. ermF and tet(X) were carried together on the same clonal Tn4351-family transposon, present in 86% of farmers and 80% of pigs but only 19% of controls. We found no evidence for transmission of mobile genetic elements between pigs and farmers; the shared resistome is best explained by carriage within shared gut taxa. Species sharing, strain transmission, and resistance gene carriage thus reflect different scales of microbial exchange at the livestock-human interface. Our results argue against ongoing strain or mobile genetic element transmission from handled animals: the shared species and resistome are best explained by shared environmental exposure and by carriage of resistance genes within shared gut taxa.

Functional studies of how early-life interventions shape the airway microbiome remain scarce. Here, we performed metagenomic sequencing of 704 longitudinal nasal swabs from infants with and without cystic fibrosis (CF) to construct and characterize a non-redundant gene atlas of the infant nasal microbiome. We aimed to determine how the nasal microbiome is perturbed by early therapies, as CF is commonly treated with inhaled hypertonic saline to improve mucociliary clearance. We found functional and compositional microbiome changes linked to inhalation therapy, including an expansion of salt-associated transporter genes and a community shift toward CF-associated microbial opportunists, including Haemophilus influenzae and fungi, carrying the identified salt-associated transporter genes with high sequence and structural identity. Hypertonic, compared with isotonic, saline accelerates H. influenzae growth and induces efflux pumps linked to antibiotic tolerance in vitro. This study establishes a reference framework for functional airway microbiome research, enabling the examination of therapeutic perturbations and their impact on microbial adaptation.

Despite the rising challenge of antibiotic resistance, current approaches to eradicate nasal pathobionts Staphylococcus aureus and Streptococcus pneumoniae rely on antibacterials. An alternative is the artificial inoculation of commensal bacteria, i.e., probiotic treatment, supported by the increasing evidence for commensal-mediated inhibition of pathogens. To systematically investigate the potential of this approach, we developed a quantitative framework simulating the nasal microbiome dynamics by combining mathematical modeling with longitudinal microbiota data. By inferring community parameters using 16S ribosomal RNA (rRNA) amplicon sequencing data and simulating the nasal microbial dynamics of patients colonized with S. aureus, we compared the decolonization performance of probiotic and antibiotic treatments under different assumptions on patients' community composition and susceptibility profile. To further compare the robustness of these treatments, we simulated an S. aureus challenge and quantified the recolonization probability. Through in vitro experiments using nasal swabs of adults colonized with S. aureus, we confirmed that after antibiotic treatment, recolonization of S. aureus was inhibited in samples treated with a probiotic mixture compared to the nontreated control. Our results suggest that probiotic treatment outperforms antibiotics in terms of decolonization performance, recolonization robustness, and leads to less collateral reduction in the microbiome diversity. Thus, probiotic treatment may provide a promising alternative to combat antibiotic resistance, with the additional advantage of personalized treatment options via using the patient's own metagenomic data. The combination of an in silico framework with in vitro experiments using clinical samples reported in this work is an important step forward to further investigate this alternative in clinical trials.

BACKGROUND:Nasal microbiota composition of patients with diffuse type 2 chronic rhinosinusitis with nasal polyps (CRSwNP) is altered compared to healthy individuals. Dupilumab, an anti-IL-4Rα-mAb, modulates type 2 inflammation, but the effect on microbiota composition in CRSwNP is unknown. The aim of this study was to investigate longitudinal effects of dupilumab on the nasal passage and gastrointestinal microbiota in patients with diffuse type 2 CRSwNP. METHODS:Twenty-seven patients with diffuse type 2 CRSwNP treated with dupilumab 300 mg subcutaneously every 2 weeks, 10 untreated patients with CRSwNP, and 11 healthy controls were included. Nasal and stool samples were collected at Days 0, 28, 90, and 180 posttreatment of the treated CRSwNP group and at Days 0 and 28 of untreated CRSwNP and healthy controls. The samples were analyzed using 16S rRNA gene amplicon sequencing (V3/V4). RESULTS:In CRSwNP patients, the most abundant genera in nasal passage microbiota were Corynebacterium and Staphylococcus. Cutibacterium and Lawsonella were less abundant in CRSwNP at baseline compared to healthy controls. Dupilumab treatment was associated with increased relative abundances in the nasal passage of genera such as Lawsonella, Corynebacterium, and Dolosigranulum. Microbial diversity of the gastrointestinal microbiota in CRSwNP at baseline was significantly higher than in healthy controls. There were no changes in gastrointestinal microbiota during dupilumab treatment. CONCLUSION:Dupilumab treatment was associated with a shift in the nasal passage bacterial microbiota toward that of healthy controls, whereas the composition of gastrointestinal microbiota did not change. These findings suggest that nasal passage microbiota composition is influenced by the underlying inflammatory endotype.

African swine fever virus (ASFV) is a major threat for pig health and meat production in many countries. The development and commercialization of vaccine candidates are complicated by efficacy and safety concerns. Improved vaccine design requires further studies to identify factors that regulate immune responses to vaccines leading to protective immunity against a virulent challenge. In a previous study, we reported that infection with the moderately virulent ASFV field strain Estonia 2014 was less severe in specific pathogen-free (SPF) pigs than in conventional farm pigs, which differ in their gut microbiome and their basal immune activation status. As shown previously using intramuscular infection, SPF pigs were more resilient to oronasal infection with the ASFV Estonia 2014 strain compared to farm pigs, which showed increased fever and clinical signs. All SPF and farm pigs nevertheless survived the infection and remained viremic for approximately 4 months. When all animals had no detectable viremia, both groups were rechallenged with the virulent ASFV Armenia 2008 strain. SPF pigs were fully protected against disease and showed little or no viremia upon re-challenge. In contrast, farm pigs developed high viremia, high proinflammatory cytokine responses, severe clinical signs, and 40% (2 of 5 pigs) reached humane endpoints. Our findings suggest that limited prior immune exposure to other pathogens and/or the microbiome composition of SPF pigs promotes resilience to infection with a moderately virulent strain such as Estonia 2014, and importantly promotes the development of a strong protective immune response against a second challenge with a virulent ASFV strain. In conclusion, testing safety and efficacy of live attenuated vaccine candidates should take into account the specific hygiene conditions and the associated changes of general immune status of pigs in clinical trials.

In Switzerland, thirteen-valent pneumococcal conjugate vaccine (PCV13) has been introduced in 2011. During the COVID-19 pandemic, cases of invasive pneumococcal disease (IPD) have decreased but consequences on the serotype epidemiology are less clear. The objective of the study has been to analyse the impact of PCV13 introduction and COVID-19 pandemic on the IPD epidemiology, and investigate the changes of the case fatality risk (CFR).We analysed data from a nationwide surveillance for the period 2012-2022. Poisson and logistic regression analyses were performed allowing to inspect trends over time and to define serotypes that are associated with case fatality.In total, 8747 IPD cases were included from 2012 to 2022. IPD incidence dropped in the years 2020 (6.0/100'000) and 2021 (5.5/100'000) but recovered in 2022 (9.1/100'000). While the incidence numbers of patients >65 years did not reach the pre-pandemic level, numbers significantly increased in infants <1 year in 2022 (IRR 1.08, 95%CI: 1.01 to 1.16). The incidence of PCV13 serotypes among all IPD cases decreased until 2019 before increasing again during the pandemic (in 2022). Logistic regression analyses revealed that the PCV20 serotype 11A (OR: 1.76, 95%CI: 1.14-2.64), and the PCV13 serotypes 3 (OR: 1.26, 95% CI: 1.04-1.53) and 19F (OR: 1.76, 95%CI: 1.14-2.65) were significantly associated with increased CFR.In conclusion, the COVID-19 pandemic has had only minor temporary effects on the serotype distribution. Continued use of vaccines with extended serotype coverage may further reduce IPD disease burden and mortality.

Humans and animals are ubiquitously colonized by Enterobacteriaceae, a bacterial family that contains both commensals and clinically significant pathogens. Here, we report Enterobacteriaceae megaplasmids of up to 1.58 Mbp in length in infant and adult guts, and other microbiomes. Of 19 complete plasmid genomes, one was reconstructed from an E. coli isolate; others were linked to species of Citrobacter and Enterobacter via analysis of genome modification patterns. The detection of related plasmids in different Enterobacteriaceae, conjugation machinery, and more diverse modified motifs in certain plasmids compared to hosts suggests that these elements are self-transmissible, with a broad host range. The plasmids encode multi-drug efflux systems and potential secreted effectors. Up to 208 tRNAs are encoded and include sequence variants that may counter tRNA-centric defense mechanisms. Overall, the vast megaplasmid coding capacity may broaden host range, increase competitiveness, control invasion by other elements, and counter programmed cell death.

Streptococcus pneumoniae colonizes human airways, where it acquires sugars from glycosylated mucins using glycoside hydrolases and sugar transport systems. This study identifies widespread nucleotide sequence variation in the promoter of a pneumococcal operon encoding a glycan scavenging system. We identify 78 promoter sequence patterns across 21,155 genomes, with variation clustered within a stretch of adenines, where mutations accumulate via strand slippage during DNA replication. Promoter mutations influence operon transcription, and multiple promoter patterns are co-identified during single-carriage episodes, suggesting that heterogeneous gene expression provides population-level benefits. In a mouse nasopharyngeal colonization model, promoter mutations arise and undergo selection, with nucleotide insertion promoting gene expression and prolonging carriage longevity. Pre-existing immunity confers resistance to colonization by strains carrying single promoter patterns but does not protect against mixed infections with otherwise isogenic strains differing in promoter sequence. Promoter region sequence variation offers an evolutionary strategy for exploration of phenotypic space to maximize fitness within-host.

Streptococcus pneumoniae (the pneumococcus) is a leading cause of community-acquired pneumonia. Pneumococci are categorised into serotypes, based on the type of capsular polysaccharide produced, which has important implications for virulence, vaccine impact and global surveillance. Recently, we identified a novel serotype, which we named 33G, that is comprised of an O-acetylated hexasaccharide repeat unit. In this study, we report and describe variants of 33G, designated 33G-like, which we isolated from the nasopharynx of two adults hospitalised with pneumonia in Mongolia. Serological comparison of 33G and 33G-like pneumococci were conducted by Quellung serotyping. Genetic analysis of the capsular polysaccharide loci was performed using whole genome sequencing. Polysaccharide composition was determined using 1H nuclear magnetic resonance. By Quellung serotyping, 33G pneumococci type as both 10B and 33B whereas 33G-like pneumococci type as both 10B and 33F. Genomic analysis of the capsular polysaccharide locus revealed 33G-like loci are identical to 33G, except for frameshift mutations in the wciG gene which encodes an acetyltransferase responsible for the O-acetylation of beta-galactofuranose (β-Galf) in the capsular polysaccharide repeat unit. We constructed an artificial 33G-like by deleting wciG in a 33G strain and confirmed this gene was responsible for the serological differences between 33G and 33G-like pneumococci. Lastly, 1H nuclear magnetic resonance confirmed the O-acetylation present in the 33G polysaccharide is absent in the 33G-like polysaccharide. Here, we have provided serological, genetic and biochemical evidence that the 33G-like capsule differs to 33G and all other pneumococcal serotypes, meeting the requirements to be designated as a new serotype, which we have named 33H.

In Switzerland, thirteen-valent pneumococcal conjugate vaccine (PCV13) has been introduced in 2011. During the COVID-19 pandemic, cases of invasive pneumococcal disease (IPD) have decreased but consequences on the serotype epidemiology are less clear. The objective of the study has been to analyse the impact of PCV13 introduction and the COVID-19 pandemic on the IPD epidemiology and investigate the changes in the case fatality risk (CFR). We analysed data from the Swiss nationwide surveillance for the period 2012-2022. Poisson and logistic regression analyses were performed allowing us to inspect trends over time and to define serotypes that are associated with case fatality. In total, 8747 IPD cases were included from 2012 to 2022. IPD incidence dropped in the years 2020 (6.0/100,000) and 2021 (5.5/100,000) but recovered in 2022 (9.1/100,000). While the incidence numbers of patients >65 years did not reach the pre-pandemic level, numbers significantly increased in infants <1 year in 2022 (IRR 1.08, 95%CI: 1.01-1.16). The incidence of PCV13 serotypes among all IPD cases decreased until 2019 before increasing again during the pandemic (in 2022). Logistic regression analyses revealed that the PCV20 serotype 11A (OR: 1.76, 95%CI: 1.14-2.64), and the PCV13 serotypes 3 (OR: 1.26, 95% CI: 1.04-1.53) and 19F (OR: 1.76, 95%CI: 1.14-2.65) were significantly associated with increased CFR. In conclusion, the COVID-19 pandemic has had only minor temporary effects on the serotype distribution. Continued use of vaccines with extended serotype coverage may further reduce IPD disease burden and mortality.

This editorial piece co-authored by the Senior Editors at Microbiome aims to highlight current challenges in the field of environmental and host-associated microbiome research. We also take the opportunity to clarify our expectations for the articles submitted to the journal. At Microbiome, we are seeking studies that provide either new mechanistic insights into the role of microbiomes in health and environmental systems or substantial conceptual or technical advances. Manuscripts need to meet high standards of language accuracy, quality of microbiome analyses, and data and protocol availability, including detailed reporting of wet-lab and in silico protocols, all of which can critically enhance transparency and reproducibility. We think that such efforts are essential to push the boundaries of our knowledge on microbiomes in a concerted, international effort.

Background Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis are leading causes of invasive bacterial disease worldwide. The aims of this study were to measure post-COVID-19 pandemic changes in the incidence of disease caused by these bacterial pathogens and assess the evidence for any age- or serotype/group-specific changes. Methods Prospectively, cases of invasive disease from 2018-2023 were submitted to national and/or regional microbiology reference laboratories in each of 27 countries/territories. An effective pandemic period was estimated, and interrupted time series analyses quantified the effect of the introduction and withdrawal of pandemic containment measures. Disease incidence rates for S pneumoniae, H influenzae and N meningitidis were calculated, stratified by age and serotype/group. Streptococcus agalactiae was investigated as a comparator invasive pathogen not transmitted via the respiratory route. Findings The median duration of pandemic containment measures among all 27 countries was 22 months (range, 14-26 months). Withdrawal of containment measures led to significant increases in invasive disease for S. pneumoniae (42%, incidence rate ratio [IRR] 1.42, 1.30-1.55) and H influenzae (68%, IRR 1.68, 1.47-1.91), but not N meningitidis (15%, IRR 1.15, 0.92-1.44). In the post-pandemic period, the risk of H influenzae disease (risk ratio [RR] 1.02, 0.91-1.14) returned to the pre-pandemic risk. Conversely, for S pneumoniae (RR 0.91, 0.85-0.97) and N meningitidis (RR 0.61, 0.54-0.69) the risk of invasive disease was significantly below pre-pandemic risk. There was no evidence for changed rates of S. agalactiae disease. The distribution of invasive disease by age was broadly as expected, and age-stratified disease incidence by serotype/group was similar to pre-pandemic rates, but with some notable changes. Interpretation By 2023, while the risk of invasive disease caused by H influenzae returned to pre-pandemic levels, the risk of S pneumoniae and N meningitidis disease remained below pre-pandemic levels. Changes were observed in the post-pandemic incidence and age-stratified distributions of some serotypes/groups. Evidence before this study We searched PubMed, bioRxiv, and medRxiv for articles published up to 31 Dec 2019 (before the COVID-19 pandemic) that reported on the effects of containment measures implemented in response to a pandemic. We identified 262 papers by searching for ‘pandemic’ AND ‘microbial transmission’ OR ‘transmission’ AND ‘containment’ but none of these described the effects of implementing large-scale containment measures during a pandemic. The Invasive Respiratory Infection Surveillance (IRIS) Consortium previously reported that the incidence of invasive bacterial disease due to S pneumoniae, H influenzae and N meningitidis was significantly reduced when COVID-19 pandemic containment measures were implemented to control the transmission of SARS-CoV-2. Added value of this study We extended the previous analyses to estimate the effective containment measure period within each of 27 countries across six continents, and calculated rates of invasive bacterial disease after the pandemic containment measures were withdrawn. This demonstrated that invasive bacterial disease caused by all three pathogens increased once pandemic containment measures were removed and incidence rates exceeded the rates observed before the pandemic. Subsequently, disease caused by H influenzae generally returned to pre-pandemic rates, whilst disease caused by S pneumoniae and N meningitidis remained below pre-pandemic rates. Some serotype/group and age-specific changes were also observed for each of the pathogens. Implications of all the available evidence The COVID-19 pandemic had profound direct and indirect effects on global public health. The IRIS Consortium has shown that invasive bacterial disease caused by S pneumoniae, H influenzae and N meningitidis was also altered, and that changes in the circulating serotypes/groups were observed among countries participating in the IRIS Consortium. Whether or not these epidemiological changes persist, and to what extent they might affect bacterial vaccination coverage and disease rates, will be revealed over time and thus invasive disease due to these pathogens needs to be closely monitored. ### Competing Interest Statement CHI de Creteil, France received research grants from the French Public Health Agency, Pfizer, and MSD. University Hospitals Leuven, Belgium received consulting fees and payment for lectures from MSD. MH participated on a Data Safety Monitoring Board or Advisory Board for both Pfizer and MSD. MH also holds investigator-initiated grants from Pfizer and MSD paid to his institution. However, the sponsors had no role in the data analysis and content of the manuscript. The National Medicines Institute, Warsaw, Poland received funding from the National Science Centre, MSD, and Pfizer, and received equipment from The Great Orchestra of Christmas Charity Foundation, and the Clinical Microbiology Center Foundation. AK received payments from Pfizer for lectures. AS received payments from MSD and Pfizer for lectures and support for attending meetings and/or travel, and from MSD, Pfizer, and Sanofi Pasteur for participation in advisory boards. ABB received funding from MSD for IRIS pneumococcal genome sequencing. ABB was an unpaid advisor to the World Health Organisation providing expertise related to vaccines and antimicrobial resistance. ABB is an unpaid General Assembly member (2022 onwards), Board member from 2016-2022, and Secretary from 2018-2022 for the ISPPD Society. MD has received financial support to attend national scientific meetings. HH received a grant from Pfizer for molecular aspects of invasive pneumococcal disease. HH received consulting fees from Bons Secours Hospital Group (Ireland) to provide advice on hospital infection and control issues with a new build. HH received payment from Scottish Hospitals Enquiry for expert testimony related to healthcare ventilation and healthcare-associated infections. KAJ received personal royalties from GSK. TTL is an unpaid Board member for the European Society for Meningococcal and Haemophilus influenzae disease (EMGM), and the German Society for Hygiene and Microbiology (DGHM), committee for microbial systematics, population genetics and infection epidemiology (FG MIP). HCS received funding from Pfizer for a pneumococcal carriage project. HCS received funding for consultations on a Data Safety Monitoring Board or Advisory Board for MSD. MvdL received payment or honoraria from Pfizer and Merck and is a member of advisory boards for Pfizer, Merck and GSK. AvG is the chairperson for national NiTAG (NAGI) for South Africa. NvS received consulting fees from MSD, GSK, and Pfizer, and research funding from MSD, GSK, Pfizer, the Dutch Health Counsel, Amsterdam UMC, Argenx, and the Leducq Foundation, which are all directly paid to the institution. NvS holds a patent (WO 2013/020090 A3) on vaccine development against Streptococcus pyogenes. NvS is an unpaid scientific advisor to the ItsME foundation, and a scientific advisor to Rapua te me ngaro ka tau, but fees are paid to the University of Amsterdam. NvS holds personal stock in Genmab BV and Bank of America. JY received grant support from grants from MSD-USA (MISP Call), Pfizer and MEIJI; payment for travel expenses and meeting fees from MSD and Pfizer; and participated in MSD and Pfizer advisory boards. JS participated in an advisory board for MSD. JAV performs contract work for the Institute of Health Carlos III funded by Pfizer, and receives consulting fees from Pfizer, GSK and Sanofi Pasteur. MC received an Investigator Initiated Research grant from Pfizer (W1243730) which has been paid to the institution (Childrens Health Ireland). MC is part of a working group for The National Immunisation Advisory Committee (NIAC) in Ireland. KGK received funding from the European Society for Clinical Microbiology and Infectious Diseases to attend the ESCMID Global meeting in Barcelona April 2024 as a member of the Professional Affairs Committee. KGK is a member of the lcelandic State Communicable Disease and Prevention Committee. KGK has stocks in a start-up innovation company ArcanaBio that is developing novel diagnostic tests. CMA received payments for lectures from MSD and Sanofi-Pasteur. CMA received support from MSD, Pfizer and Sanofi-Pasteur to attend meetings. LC received support from MSD for attendance and travel to international symposium ISPPD-13, organized in South Africa in March 2024. ### Funding Statement The infrastructure for the IRIS Consortium was funded by a Wellcome Trust Investigator Award to ABB (grant number 206394/Z/17/Z). The IRIS databases are part of PubMLST, which is funded by a Wellcome Trust Biomedical Resource Grant awarded to MJCM, ABB, and KAJ (grant number 218205/Z/19/Z). DS received an Oxford Clarendon Scholarship covering University fees plus a stipend, and a fellowship from the Nuffield Department of Population Health. This work was also partially supported by: research funding from the Polish Ministry of Health and the Polish Ministry of Science and Higher Education to the National Medicines Institute, Poland; The Swiss National Reference Center for Invasive Pneumococci (NZPn) received funding from the Federal Office of Public Health; the Seoul National University College of Medicine received funding from Pfizer (grant number 69765907); the Robert Koch-Institute with funds of the German Federal Ministry of Health (funding code 1369-237) to the National Reference Centre for Meningococci and Haemophilus influenzae; the Instituto Nacional de Salud de Colombia (National Reference Laboratory); research funding from Pfizer and the European Centre for Disease Prevention and Control (ECDC) for pneumococcal surveillance work at the Irish Meningitis and Sepsis Reference Laboratory; financial support from Pfizer and Merck for invasive pneumococcal disease surveillance to the University Hospital RWTH Aachen. The funders had no role in data collection, analysis, interpretation, writing of the manuscript or the decision to submit. All authors agreed to be accountable for all aspects of the work. All authors had full access to all data in the study and the corresponding author (ABB) had final responsibility for the decision to submit this work for publication. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics committee/IRB (OxTREC) of the University of Oxford waived ethical approval for this work. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes It is not possible to share the study data because doing so would risk identifying individual cases of invasive disease in countries with small numbers of cases.

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.

BACKGROUND:Little is known about the mediating role of nasal microbiome on the association between pre- and postnatal air pollution exposure and subsequent respiratory morbidity in infancy. We aimed to examine the impact of air pollution on microbiome and respiratory symptoms, and whether microbiome mediates the association between air pollution and symptoms. METHODS:Nasal swabs from 270 infants in the prospective Basel-Bern Infant Lung Development cohort were analyzed by 16S ribosomal RNA gene sequencing. We investigated the association of pre- and postnatal nitrogen dioxide (NO2) and particulate matter ≤2.5 μm (PM2.5) with microbiome at 4-6 weeks and with respiratory symptoms during the first year of life. Hierarchical clustering and generalized structural equation modeling were used. RESULTS:Mean prenatal air pollution levels were 21.54 μg/m3 (NO2) and 13.84 μg/m3 (PM2.5) (WHO guideline limits: NO2: 40 μg/m3 (2005), 10 μg/m3 (2021); PM2.5: 10 μg/m3 (2005), 5 μg/m3 (2021)). We identified two distinct microbiome clusters, characterized by high Corynebacterium/Dolosigranulum and high Staphylococcus abundance. Higher pre- and postnatal air pollution exposure was associated with Staphylococcus cluster (e.g., per 10 μg/m3 increase of prenatal NO2: odds ratio 1.58, 95% confidence interval 1.08; 2.29, padj = 0.034). Pre- and postnatal PM2.5 was associated with increased risk of severe respiratory symptoms. This association was not mediated by nasal microbiome. CONCLUSION:Pre- and postnatal air pollution was associated with microbiome and respiratory symptoms in infancy. The microbiome did not mediate the association of air pollution with respiratory symptoms, which may indicate that other mechanisms are more relevant at this age.

BackgroundRespiratory tract infections (RTIs) drive lung function decline in children with cystic fibrosis (CF). While the respiratory microbiota is clearly associated with RTI pathogenesis in infants without CF, data on infants with CF is scarce. We compared nasal microbiota development between infants with CF and controls and assessed associations between early-life nasal microbiota, RTIs, and antibiotic treatment in infants with CF.MethodsWe included 50 infants with CF and 30 controls from two prospective birth cohorts followed throughout the first year of life. We collected 1511 biweekly nasal swabs and analyzed the microbiota after amplifying the V3-V4 region of the 16S rRNA gene. We conducted structured weekly interviews to assess respiratory symptoms and antibiotic treatment. We calculated generalized additive mixed models and permutational analysis of variance.ResultsHere, we show that the nasal microbiota is already altered before the first RTI or antibiotic treatment in infants with CF. Microbiota diversity differs between infants with CF and controls following RTIs and/or antibiotic treatment. CF infants with lower alpha-diversity have a higher number of subsequent RTIs.ConclusionsEarly nasal microbiota alterations may reflect predisposition or predispose to RTIs in infants with CF, and further change after RTIs and antibiotic treatment. This highlights the potential of targeting the nasal microbiota in CF-related RTI management, while also questioning current practices in the era of novel modulator therapies. Cystic fibrosis (CF) is an inherited condition which can increase the risk of developing respiratory tract infections (RTIs). We investigated the microorganisms present in the respiratory tract of infants from birth to the age of one. We found that infants with CF had differences in the microorganisms present immediately after birth compared to infants without CF. These differences increased after development of RTIs and following antibiotic treatment. Our results suggest that infants with CF could potentially benefit from treatments that modify microorganisms present in their respiratory tract prior to development of any RTI, or from different antibiotics to those used by infants without CF. Steinberg et al. explore the associations between the nasal microbiota, respiratory tract infections (RTIs) and antibiotics in infants with cystic fibrosis (CF) and controls during the first year of life. Infants with CF have a different microbiota before their first RTI or antibiotic treatment, with lower diversity linked to higher number of RTIs.