Topographic heterogeneity of lung microbiota in end-stage idiopathic pulmonary fibrosis: the Microbiome in Lung Explants-2 (MiLEs-2) study.

BACKGROUND: Lung microbiota profiles in patients with early idiopathic pulmonary fibrosis (IPF) have been associated with disease progression; however, the topographic heterogeneity of lung microbiota and their roles in advanced IPF are unknown. METHODS: We performed a retrospective, case-control study of explanted lung tissue obtained at the time of lung transplantation or rapid autopsy from patients with IPF and other chronic lung diseases (connective tissue disease-associated interstitial lung disease (CTD-ILD), cystic fibrosis (CF), COPD and donor lungs unsuitable for transplant from Center for Organ Recovery and Education (CORE)). We sampled subpleural tissue and airway-based specimens (bronchial washings and airway tissue) and quantified bacterial load and profiled communities by amplification and sequencing of the 16S rRNA gene. FINDINGS: Explants from 62 patients with IPF, 15 patients with CTD-ILD, 20 patients with CF, 20 patients with COPD and 20 CORE patients were included. Airway-based samples had higher bacterial load compared with distal parenchymal tissue. IPF basilar tissue had much lower bacterial load compared with CF and CORE lungs (p<0.001). No microbial community differences were found between parenchymal tissue samples from different IPF lobes. Dirichlet multinomial models revealed an IPF cluster (29%) with distinct composition, high bacterial load and low alpha diversity, exhibiting higher odds for acute exacerbation or death. INTERPRETATION: IPF explants had low biomass in the distal parenchyma of all three lobes with higher bacterial load in the airways. The discovery of a distinct subgroup of patients with IPF with higher bacterial load and worse clinical outcomes supports investigation of personalised medicine approaches for microbiome-targeted interventions.

Alterations in Oral Microbiota in HIV Are Related to Decreased Pulmonary Function.

Rationale: Mechanisms of HIV-associated chronic obstructive pulmonary disease (COPD) are poorly understood. The oral microbiome shapes the lung microbiome, and gut dysbiosis can affect lung diseases; however, relationships of the oral and gut microbiome to COPD in HIV have not been explored.Objectives: To examine alterations in the oral and gut microbiome associated with pulmonary disease in persons with HIV (PWH).Methods: Seventy-five PWH and 93 HIV-uninfected men from the MACS (Multicenter AIDS Cohort Study) performed pulmonary function testing. Sequencing of bacterial 16S ribosomal RNA in saliva and stool was performed. We used nonmetric multidimensional scaling, permutational multivariate ANOVA, and linear discriminant analysis to analyze communities by HIV and lung function.Measurements and Main Results: Oral microbiome composition differed by HIV and smoking status. Alterations of oral microbial communities were observed in PWH with abnormal lung function with increases in relative abundance of Veillonella, Streptococcus, and Lactobacillus. There were no significant associations between the oral microbiome and lung function in HIV-uninfected individuals. No associations with HIV status or lung function were seen with the gut microbiome.Conclusions: Alterations of oral microbiota in PWH were related to impaired pulmonary function and to systemic inflammation. These results suggest that the oral microbiome may serve as a biomarker of lung function in HIV and that its disruption may contribute to COPD pathogenesis.

Respiratory Tract Dysbiosis Is Associated with Worse Outcomes in Mechanically Ventilated Patients.

Rationale: Host inflammatory responses have been strongly associated with adverse outcomes in critically ill patients, but the biologic underpinnings of such heterogeneous responses have not been defined.Objectives: We examined whether respiratory tract microbiome profiles are associated with host inflammation and clinical outcomes of acute respiratory failure.Methods: We collected oral swabs, endotracheal aspirates (ETAs), and plasma samples from mechanically ventilated patients. We performed 16S ribosomal RNA gene sequencing to characterize upper and lower respiratory tract microbiota and classified patients into host-response subphenotypes on the basis of clinical variables and plasma biomarkers of innate immunity and inflammation. We derived diversity metrics and composition clusters with Dirichlet multinomial models and examined our data for associations with subphenotypes and clinical outcomes.Measurements and Main Results: Oral and ETA microbial communities from 301 mechanically ventilated subjects had substantial heterogeneity in α and ß diversity. Dirichlet multinomial models revealed a cluster with low α diversity and enrichment for pathogens (e.g., high Staphylococcus or Pseudomonadaceae relative abundance) in 35% of ETA samples, associated with a hyperinflammatory subphenotype, worse 30-day survival, and longer time to liberation from mechanical ventilation (adjusted P < 0.05), compared with patients with higher α diversity and relative abundance of typical oral microbiota. Patients with evidence of dysbiosis (low α diversity and low relative abundance of "protective" oral-origin commensal bacteria) in both oral and ETA samples (17%, combined dysbiosis) had significantly worse 30-day survival and longer time to liberation from mechanical ventilation than patients without dysbiosis (55%; adjusted P < 0.05).Conclusions: Respiratory tract dysbiosis may represent an important, modifiable contributor to patient-level heterogeneity in systemic inflammatory responses and clinical outcomes.

Genomic and phenotypic characterization of myxoma virus from Great Britain reveals multiple evolutionary pathways distinct from those in Australia.

The co-evolution of myxoma virus (MYXV) and the European rabbit occurred independently in Australia and Europe from different progenitor viruses. Although this is the canonical study of the evolution of virulence, whether the genomic and phenotypic outcomes of MYXV evolution in Europe mirror those observed in Australia is unknown. We addressed this question using viruses isolated in the United Kingdom early in the MYXV epizootic (1954-1955) and between 2008-2013. The later UK viruses fell into three distinct lineages indicative of a long period of separation and independent evolution. Although rates of evolutionary change were almost identical to those previously described for MYXV in Australia and strongly clock-like, genome evolution in the UK and Australia showed little convergence. The phenotypes of eight UK viruses from three lineages were characterized in laboratory rabbits and compared to the progenitor (release) Lausanne strain. Inferred virulence ranged from highly virulent (grade 1) to highly attenuated (grade 5). Two broad disease types were seen: cutaneous nodular myxomatosis characterized by multiple raised secondary cutaneous lesions, or an amyxomatous phenotype with few or no secondary lesions. A novel clinical outcome was acute death with pulmonary oedema and haemorrhage, often associated with bacteria in many tissues but an absence of inflammatory cells. Notably, reading frame disruptions in genes defined as essential for virulence in the progenitor Lausanne strain were compatible with the acquisition of high virulence. Combined, these data support a model of ongoing host-pathogen co-evolution in which multiple genetic pathways can produce successful outcomes in the field that involve both different virulence grades and disease phenotypes, with alterations in tissue tropism and disease mechanisms.

Metagenomic identification of severe pneumonia pathogens in mechanically-ventilated patients: a feasibility and clinical validity study.

BACKGROUND: Metagenomic sequencing of respiratory microbial communities for pathogen identification in pneumonia may help overcome the limitations of culture-based methods. We examined the feasibility and clinical validity of rapid-turnaround metagenomics with Nanopore™ sequencing of clinical respiratory specimens. METHODS: We conducted a case-control study of mechanically-ventilated patients with pneumonia (nine culture-positive and five culture-negative) and without pneumonia (eight controls). We collected endotracheal aspirates and applied a microbial DNA enrichment method prior to metagenomic sequencing with the Oxford Nanopore MinION device. For reference, we compared Nanopore results against clinical microbiologic cultures and bacterial 16S rRNA gene sequencing. RESULTS: Human DNA depletion enabled in depth sequencing of microbial communities. In culture-positive cases, Nanopore revealed communities with high abundance of the bacterial or fungal species isolated by cultures. In four cases with resistant clinical isolates, Nanopore detected antibiotic resistance genes corresponding to the phenotypic resistance in antibiograms. In culture-negative pneumonia, Nanopore revealed probable bacterial pathogens in 1/5 cases and Candida colonization in 3/5 cases. In controls, Nanopore showed high abundance of oral bacteria in 5/8 subjects, and identified colonizing respiratory pathogens in other subjects. Nanopore and 16S sequencing showed excellent concordance for the most abundant bacterial taxa. CONCLUSIONS: We demonstrated technical feasibility and proof-of-concept clinical validity of Nanopore metagenomics for severe pneumonia diagnosis, with striking concordance with positive microbiologic cultures, and clinically actionable information obtained from sequencing in culture-negative samples. Prospective studies with real-time metagenomics are warranted to examine the impact on antimicrobial decision-making and clinical outcomes.

Microbiome in lung explants of idiopathic pulmonary fibrosis: a case-control study in patients with end-stage fibrosis.

The microbiome has been proposed to play a role in the progression of idiopathic pulmonary fibrosis (IPF) based on bronchoalveolar lavage analyses, but the microbiome of lung tissue in IPF has not been explored. In a case-control study of lung explants analysed by 16S rRNA gene sequencing, we could not reliably detect bacterial DNA in basilar tissue samples from patients with either chronic or acute exacerbations of IPF, in contrast to control candidate-donor lungs or cystic fibrosis explants. Thus, our data do not indicate microbiome alterations in regions of IPF lung with advanced fibrosis.

Characterization of five unclassified orthobunyaviruses (Bunyaviridae) from Africa and the Americas.

The Bunyaviridae family is made up of a diverse range of viruses, some of which cause disease and are a cause for concern in human and veterinary health. Here, we report the genomic and antigenic characterization of five previously uncharacterized bunyaviruses. Based on their ultrastructure, antigenic relationships and phylogenomic relationships, the five viruses are classified as members of the Orthobunyavirus genus. Three are viruses in the California encephalitis virus serogroup and are related to Trivittatus virus; the two others are most similar to the Mermet virus in the Simbu serogroup, and to the Tataguine virus, which is not currently assigned to a serogroup. Each of these five viruses was pathogenic to newborn mice, indicating their potential to cause illness in humans and other animals.

Topographic diversity of the respiratory tract mycobiome and alteration in HIV and lung disease.

RATIONALE: Microbiome studies typically focus on bacteria, but fungal species are common in many body sites and can have profound effects on the host. Wide gaps exist in the understanding of the fungal microbiome (mycobiome) and its relationship to lung disease. OBJECTIVES: To characterize the mycobiome at different respiratory tract levels in persons with and without HIV infection and in HIV-infected individuals with chronic obstructive pulmonary disease (COPD). METHODS: Oral washes (OW), induced sputa (IS), and bronchoalveolar lavages (BAL) were collected from 56 participants. We performed 18S and internal transcribed spacer sequencing and used the neutral model to identify fungal species that are likely residents of the lung. We used ubiquity-ubiquity plots, random forest, logistic regression, and metastats to compare fungal communities by HIV status and presence of COPD. MEASUREMENTS AND MAIN RESULTS: Mycobiomes of OW, IS, and BAL shared common organisms, but each also had distinct members. Candida was dominant in OW and IS, but BAL had 39 fungal species that were disproportionately more abundant than in the OW. Fungal communities in BAL differed significantly by HIV status and by COPD, with Pneumocystis jirovecii significantly overrepresented in both groups. Other fungal species were also identified as differing in HIV and COPD. CONCLUSIONS: This study systematically examined the respiratory tract mycobiome in a relatively large group. By identifying Pneumocystis and other fungal species as overrepresented in the lung in HIV and in COPD, it is the first to determine alterations in fungal communities associated with lung dysfunction and/or HIV, highlighting the clinical relevance of these findings. Clinical trial registered with www.clinicaltrials.gov (NCT00870857).

Comparative analysis of the complete genome sequence of the California MSW strain of myxoma virus reveals potential host adaptations.

Myxomatosis is a rapidly lethal disease of European rabbits that is caused by myxoma virus (MYXV). The introduction of a South American strain of MYXV into the European rabbit population of Australia is the classic case of host-pathogen coevolution following cross-species transmission. The most virulent strains of MYXV for European rabbits are the Californian viruses, found in the Pacific states of the United States and the Baja Peninsula, Mexico. The natural host of Californian MYXV is the brush rabbit, Sylvilagus bachmani. We determined the complete sequence of the MSW strain of Californian MYXV and performed a comparative analysis with other MYXV genomes. The MSW genome is larger than that of the South American Lausanne (type) strain of MYXV due to an expansion of the terminal inverted repeats (TIRs) of the genome, with duplication of the M156R, M154L, M153R, M152R, and M151R genes and part of the M150R gene from the right-hand (RH) end of the genome at the left-hand (LH) TIR. Despite the extreme virulence of MSW, no novel genes were identified; five genes were disrupted by multiple indels or mutations to the ATG start codon, including two genes, M008.1L/R and M152R, with major virulence functions in European rabbits, and a sixth gene, M000.5L/R, was absent. The loss of these gene functions suggests that S. bachmani is a relatively recent host for MYXV and that duplication of virulence genes in the TIRs, gene loss, or sequence variation in other genes can compensate for the loss of M008.1L/R and M152R in infections of European rabbits.

Mammalian adaptation in the PB2 gene of avian H5N1 influenza virus.

The substitution of glutamic acid (E) for lysine (K) at position 627 of the PB2 protein of avian H5N1 viruses has been identified as a virulence and host range determinant for infection of mammals. Here, we report that the E-to-K host-adaptive mutation in the PB2 gene appeared from day 4 and 5 along the respiratory tracts of mice and was complete by day 6 postinoculation. This mutation correlated with efficient replication of the virus in mice.

Genome scale evolution of myxoma virus reveals host-pathogen adaptation and rapid geographic spread.

The evolutionary interplay between myxoma virus (MYXV) and the European rabbit (Oryctolagus cuniculus) following release of the virus in Australia in 1950 as a biological control is a classic example of host-pathogen coevolution. We present a detailed genomic and phylogeographic analysis of 30 strains of MYXV, including the Australian progenitor strain Standard Laboratory Strain (SLS), 24 Australian viruses isolated from 1951 to 1999, and three isolates from the early radiation in Britain from 1954 and 1955. We show that in Australia MYXV has spread rapidly on a spatial scale, with multiple lineages cocirculating within individual localities, and that both highly virulent and attenuated viruses were still present in the field through the 1990s. In addition, the detection of closely related virus lineages at sites 1,000 km apart suggests that MYXV moves freely in geographic space, with mosquitoes, fleas, and rabbit migration all providing means of transport. Strikingly, despite multiple introductions, all modern viruses appear to be ultimately derived from the original introductions of SLS. The rapidity of MYXV evolution was also apparent at the genomic scale, with gene duplications documented in a number of viruses. Duplication of potential virulence genes may be important in increasing the expression of virulence proteins and provides the basis for the evolution of novel functions. Mutations leading to loss of open reading frames were surprisingly frequent and in some cases may explain attenuation, but no common mutations that correlated with virulence or attenuation were identified.

Evolutionary history and attenuation of myxoma virus on two continents.

The attenuation of myxoma virus (MYXV) following its introduction as a biological control into the European rabbit populations of Australia and Europe is the canonical study of the evolution of virulence. However, the evolutionary genetics of this profound change in host-pathogen relationship is unknown. We describe the genome-scale evolution of MYXV covering a range of virulence grades sampled over 49 years from the parallel Australian and European epidemics, including the high-virulence progenitor strains released in the early 1950s. MYXV evolved rapidly over the sampling period, exhibiting one of the highest nucleotide substitution rates ever reported for a double-stranded DNA virus, and indicative of a relatively high mutation rate and/or a continually changing selective environment. Our comparative sequence data reveal that changes in virulence involved multiple genes, likely losses of gene function due to insertion-deletion events, and no mutations common to specific virulence grades. Hence, despite the similarity in selection pressures there are multiple genetic routes to attain either highly virulent or attenuated phenotypes in MYXV, resulting in convergence for phenotype but not genotype.

Longitudinal multicompartment characterization of host-microbiota interactions in patients with acute respiratory failure.

Critical illness can significantly alter the composition and function of the human microbiome, but few studies have examined these changes over time. Here, we conduct a comprehensive analysis of the oral, lung, and gut microbiota in 479 mechanically ventilated patients (223 females, 256 males) with acute respiratory failure. We use advanced DNA sequencing technologies, including Illumina amplicon sequencing (utilizing 16S and ITS rRNA genes for bacteria and fungi, respectively, in all sample types) and Nanopore metagenomics for lung microbiota. Our results reveal a progressive dysbiosis in all three body compartments, characterized by a reduction in microbial diversity, a decrease in beneficial anaerobes, and an increase in pathogens. We find that clinical factors, such as chronic obstructive pulmonary disease, immunosuppression, and antibiotic exposure, are associated with specific patterns of dysbiosis. Interestingly, unsupervised clustering of lung microbiota diversity and composition by 16S independently predicted survival and performed better than traditional clinical and host-response predictors. These observations are validated in two separate cohorts of COVID-19 patients, highlighting the potential of lung microbiota as valuable prognostic biomarkers in critical care. Understanding these microbiome changes during critical illness points to new opportunities for microbiota-targeted precision medicine interventions.

Kolente virus, a rhabdovirus species isolated from ticks and bats in the Republic of Guinea.

Kolente virus (KOLEV) is a rhabdovirus originally isolated from ticks and a bat in Guinea, West Africa, in 1985. Although tests at the time of isolation suggested that KOLEV is a novel rhabdovirus, it has remained largely uncharacterized. We assembled the complete genome sequence of the prototype strain DakAr K7292, which was found to encode the five canonical rhabdovirus structural proteins (N, P, M, G and L) with alternative ORFs (>180 nt) in the P and L genes. Serologically, KOLEV exhibited a weak antigenic relationship with Barur and Fukuoka viruses in the Kern Canyon group. Phylogenetic analysis revealed that KOLEV represents a distinct and divergent lineage that shows no clear relationship to any rhabdovirus except Oita virus, although with limited phylogenetic resolution. In summary, KOLEV represents a novel species in the family Rhabdoviridae.

Presence of oseltamivir-resistant pandemic A/H1N1 minor variants before drug therapy with subsequent selection and transmission.

A small proportion (1%-1.5%) of 2009 pandemic influenza A/H1N1 virus strains (A[H1N1]pdm09) are oseltamivir resistant, almost exclusively because of a H275Y mutation in the neuraminidase protein. However, many individuals infected with resistant strains had not received antivirals. Whether drug-resistant viruses are initially present as minor variants in untreated individuals before they emerge as the dominant strain in a virus population is of great importance for predicting the speed at which resistance will arise. To address this issue, we used ultra-deep sequencing of viral populations from serial nasopharyngeal specimens from an immunocompromised child and from 2 individuals in a household outbreak. We observed that the Y275 mutation was present as a minor variant in infected hosts before the onset of therapy. We also found evidence for the transmission of this drug-resistant variant with drug-susceptible viruses. These observations provide important information on the relative fitness of the Y275 mutation in the absence of oseltamivir treatment.

Magnetic-activated cell sorting identifies a unique lung microbiome community.

BACKGROUND: The advent of culture-independent, next-generation DNA sequencing has led to the discovery of distinct lung bacterial communities. Studies of lung microbiome taxonomy often reveal only subtle differences between health and disease, but host recognition and response may distinguish the members of similar bacterial communities in different populations. Magnetic-activated cell sorting has been applied to the gut microbiome to identify the numbers and types of bacteria eliciting a humoral response. We adapted this technique to examine the populations of immunoglobulin-bound bacteria in the lung. METHODS: Sixty-four individuals underwent bronchoalveolar lavage (BAL). We separated immunoglobulin G-bound bacteria using magnetic-activated cell sorting and sequenced the 16S rRNA gene on the Illumina MiSeq platform. We compared microbial sequencing data in IgG-bound bacterial communities compared to raw BAL then examined the differences in individuals with and without HIV as a representative disease state. RESULTS: Immunoglobulin G-bound bacteria were identified in all individuals. The community structure differed when compared to raw BAL, and there was a greater abundance of Pseudomonas and fewer oral bacteria in IgG-bound BAL. Examination of IgG-bound communities in individuals with HIV demonstrated the differences in Ig-bound bacteria by HIV status that were not seen in a comparison of raw BAL, and greater numbers of immunoglobulin-bound bacteria were associated with higher pulmonary cytokine levels. CONCLUSIONS: We report a novel application of magnetic-activated cell sorting to identify immunoglobulin G-bound bacteria in the lung. This technique identified distinct bacterial communities which differed in composition from raw bronchoalveolar lavage, revealing the differences not detected by traditional analyses. Cytokine response was also associated with differential immunoglobulin binding of lung bacteria, suggesting the functional importance of these communities. Video Abstract.

Respiratory Fungal Communities are Associated with Systemic Inflammation and Predict Survival in Patients with Acute Respiratory Failure.

Rationale: Disruption of respiratory bacterial communities predicts poor clinical outcomes in critical illness; however, the role of respiratory fungal communities (mycobiome) is poorly understood. Objectives: We investigated whether mycobiota variation in the respiratory tract is associated with host-response and clinical outcomes in critically ill patients. Methods: To characterize the upper and lower respiratory tract mycobiota, we performed rRNA gene sequencing (internal transcribed spacer) of oral swabs and endotracheal aspirates (ETA) from 316 mechanically-ventilated patients. We examined associations of mycobiome profiles (diversity and composition) with clinical variables, host-response biomarkers, and outcomes. Measurements and Main Results: ETA samples with >50% relative abundance for C. albicans (51%) were associated with elevated plasma IL-8 and pentraxin-3 (p=0.05), longer time-to-liberation from mechanical ventilation (p=0.04) and worse 30-day survival (adjusted hazards ratio (adjHR): 1.96 [1.04-3.81], p=0.05). Using unsupervised clustering, we derived two clusters in ETA samples, with Cluster 2 (39%) showing lower alpha diversity (p<0.001) and higher abundance of C. albicans (p<0.001). Cluster 2 was significantly associated with the prognostically adverse hyperinflammatory subphenotype (odds ratio 2.07 [1.03-4.18], p=0.04) and predicted worse survival (adjHR: 1.81 [1.03-3.19], p=0.03). C. albicans abundance in oral swabs was also associated with the hyperinflammatory subphenotype and mortality. Conclusions: Variation in respiratory mycobiota was significantly associated with systemic inflammation and clinical outcomes. C. albicans abundance emerged as a negative predictor in both the upper and lower respiratory tract. The lung mycobiome may play an important role in the biological and clinical heterogeneity among critically ill patients and represent a potential therapeutic target for lung injury in critical illness.

Meta-omics profiling of the gut-lung axis illuminates metabolic networks and host-microbial interactions associated with elevated lung elastance in a murine model of obese allergic asthma.

Obesity and associated changes to the gut microbiome worsen airway inflammation and hyperresponsiveness in asthma. Obesogenic host-microbial metabolomes have altered production of metabolites that may influence lung function and inflammatory responses in asthma. To understand the interplay of the gut microbiome, metabolism, and host inflammation in obesity-associated asthma, we used a multi-omics approach to profile the gut-lung axis in the setting of allergic airway disease and diet-induced obesity. We evaluated an immunomodulator, nitro-oleic acid (NO2-OA), as a host- and microbial-targeted treatment intervention for obesity-associated allergic asthma. Allergic airway disease was induced using house dust mite and cholera toxin adjuvant in C57BL6/J mice with diet-induced obesity to model obesity-associated asthma. Lung function was measured by flexiVent following a week of NO2-OA treatment and allergen challenge. 16S rRNA gene (from DNA, taxa presence) and 16S rRNA (from RNA, taxa activity) sequencing, metabolomics, and host gene expression were paired with a Treatment-Measured-Response model as a data integration framework for identifying latent/hidden relationships with linear regression among variables identified from high-dimensional meta-omics datasets. Targeting both the host and gut microbiota, NO2-OA attenuated airway inflammation, improved lung elastance, and modified the gut microbiome. Meta-omics data integration and modeling determined that gut-associated inflammation, metabolites, and functionally active gut microbiota were linked to lung function outcomes. Using Treatment-Measured-Response modeling and meta-omics profiling of the gut-lung axis, we uncovered a previously hidden network of interactions between gut levels of amino acid metabolites involved in elastin and collagen synthesis, gut microbiota, NO2-OA, and lung elastance. Further targeted metabolomics analyses revealed that obese mice with allergic airway disease had higher levels of proline and hydroxyproline in the lungs. NO2-OA treatment reduced proline biosynthesis by downregulation of pyrroline-5-carboxylate reductase 1 (PYCR1) expression. These findings are relevant to human disease: adults with mild-moderate asthma and BMI ≥ 25 had higher plasma hydroxyproline levels. Our results suggest that changes to structural proteins in the lung airways and parenchyma may contribute to heightened lung elastance and serve as a potential therapeutic target for obese allergic asthma.

Prognostic Insights from Longitudinal Multicompartment Study of Host-Microbiota Interactions in Critically Ill Patients.

Critical illness can disrupt the composition and function of the microbiome, yet comprehensive longitudinal studies are lacking. We conducted a longitudinal analysis of oral, lung, and gut microbiota in a large cohort of 479 mechanically ventilated patients with acute respiratory failure. Progressive dysbiosis emerged in all three body compartments, characterized by reduced alpha diversity, depletion of obligate anaerobe bacteria, and pathogen enrichment. Clinical variables, including chronic obstructive pulmonary disease, immunosuppression, and antibiotic exposure, shaped dysbiosis. Notably, of the three body compartments, unsupervised clusters of lung microbiota diversity and composition independently predicted survival, transcending clinical predictors, organ dysfunction severity, and host-response sub-phenotypes. These independent associations of lung microbiota may serve as valuable biomarkers for prognostication and treatment decisions in critically ill patients. Insights into the dynamics of the microbiome during critical illness highlight the potential for microbiota-targeted interventions in precision medicine.